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Integrated Conservation of Florida Orchidaceae in the Genera Habenaria and Spiranthes

Permanent Link: http://ufdc.ufl.edu/UFE0021367/00001

Material Information

Title: Integrated Conservation of Florida Orchidaceae in the Genera Habenaria and Spiranthes Model Orchid Conservation Systems for the Americas
Physical Description: 1 online resource (226 p.)
Language: english
Creator: Stewart, Scott Lynn
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: aflp, asymbiotic, ceratorhiza, conservation, diversity, epulorhiza, florida, fungus, germination, habenaria, integrated, mycobiont, orchid, orchidaceae, pollination, seed, spiranthes, symbiotic
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Rapid loss of native orchid habitat throughout ecologically-important areas (e.g., Florida) has prompted researchers to develop appropriate conservation systems for the preservation and recovery of native orchid species. These conservation systems must incorporate more than habitat preservation and plant propagation, they must integrate the study of native orchid ecology, mycology, propagation, pollination biology, and population genetic diversity in a combined conservation effort. The current study examines the ecology and demography, mycology, asymbiotic and symbiotic propagation, pollination biology, and population genetic diversity of orchids in the genera Habenaria and Spiranthes. The specific Florida native orchid included in this study are: Habenaria macroceratitis, Spiranthes floridana, S. brevilabris, and the Deep South race of S. cernua. The application of integrated conservation to the preservation of these native orchids is discussed, and integrated conservation steps are proposed that will help ensure the long-term population viability of these species. Furthermore, these integrated conservation systems could be applicable outside the United States and Florida.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Scott Lynn Stewart.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Kane, Michael E.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021367:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021367/00001

Material Information

Title: Integrated Conservation of Florida Orchidaceae in the Genera Habenaria and Spiranthes Model Orchid Conservation Systems for the Americas
Physical Description: 1 online resource (226 p.)
Language: english
Creator: Stewart, Scott Lynn
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: aflp, asymbiotic, ceratorhiza, conservation, diversity, epulorhiza, florida, fungus, germination, habenaria, integrated, mycobiont, orchid, orchidaceae, pollination, seed, spiranthes, symbiotic
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Rapid loss of native orchid habitat throughout ecologically-important areas (e.g., Florida) has prompted researchers to develop appropriate conservation systems for the preservation and recovery of native orchid species. These conservation systems must incorporate more than habitat preservation and plant propagation, they must integrate the study of native orchid ecology, mycology, propagation, pollination biology, and population genetic diversity in a combined conservation effort. The current study examines the ecology and demography, mycology, asymbiotic and symbiotic propagation, pollination biology, and population genetic diversity of orchids in the genera Habenaria and Spiranthes. The specific Florida native orchid included in this study are: Habenaria macroceratitis, Spiranthes floridana, S. brevilabris, and the Deep South race of S. cernua. The application of integrated conservation to the preservation of these native orchids is discussed, and integrated conservation steps are proposed that will help ensure the long-term population viability of these species. Furthermore, these integrated conservation systems could be applicable outside the United States and Florida.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Scott Lynn Stewart.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Kane, Michael E.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021367:00001


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INTEGRATED CONSERVATION OF FLORIDA ORCHIDACEAE INT THE GENERA
Habenaria AND Splitantherl~ MODEL ORCHID CONSERVATION SYSTEMS FOR THE
AMERICAS




















By

SCOTT L. STEWART


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007
































O 2007 Scott L. Stewart


































To all those who have a true passion in life









ACKNOWLEDGMENTS

I thank Dr. Michael Kane for his support, patience, dedication, and encouragement during

my research, and for the many conversations about plants and more. I also thank Dr. Charles

Guy, Dr. Thomas Sheehan, Dr. Doria Gordon, and Dr. James Kimbrough for serving on my

supervisory committee. The members of my supervisory committee have shown me what it

means to be true professionals and academicians.

Much of this work could never have been undertaken without a great deal of help from

many people, including Paul Martin Brown (Ocala, Florida), Dr. Lawrence Zettler (Illinois

College), Larry Richardson (USFWS-FPNWR), James Durwachter (USFWS-FPNWR), Layne

Hamilton (USFWS-FPNWR), and Ben Nottingham (USFWS-FPNWR). Their guidance and

assistance made many experiments and field research sessions possible.

I also thank the fellow graduate students with whom I have worked over the years: Carmen

Valero-Aracama, Philip Kauth, Daniela Dutra, Timothy Johnson, and Xiliu Shen. Brainstorming

sessions, research conversations, and field work would have not been possible without their

assistance.

I thank the Socash family (Brooskville, Florida) for generously allowing me to use their

population of Habenaria macroceratitis as a research site, as well as the Rayonier Corporation

(Starke, Florida) for allowing me access to the population of Splilanthes,~ floridana.d~~~~~ddddd~~~~ The City of

Brooksville (Florida), Marj orie Harris Carr Cross Florida Greenway, National Fish and Wildlife

Foundation, U. S. Fish and Wildlife Service, Florida Division of Forestry, Florida Division of

Environmental Protection, Florida Division of Plant Industry, and the San Diego County Orchid

Society all contributed to the completion of this work. The assistance of Ginger Clark and the

University of Florida ICBR Genetic Analysis laboratory with the preparation and analysis of

AFLP data is greatly appreciated.









I also thank my parents, Duane and Victoria Stewart; and my sister, Leslie Stewart, for

their unwavering support of my educational goals despite not fully understanding what I was

doing. Finally, I thank my wife, Angela O'Donnell, for her unflinching love, support, grudging

interest, and immense degree of patience during my work. Without her love and understanding

none of this would have been possible.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S ............ ...... ._ ...............4....


LIST OF TABLES ............_...... ._ ...............9....


LIST OF FIGURES .............. ...............10....


AB S TRAC T ............._. .......... ..............._ 13...


CHAPTERS


1 LITERATURE REVIEW ................. ...............14...............


Introduction............... ..............1

Study Rationale............... .. ......... .........1
Conservation Ethic and Modes ................. ...............15......... .....

Integrated Orchid Conservation .............. ...............19....
Plants of Interest ................ ........... ........ ........ ....._.. ....._.. .........20
S el section Criteria ................. ...............20.......... ......
Plants of Study ................. ............ ...............21......
Habenaria macroceratitis .............. ...............2 1....

.l'iiaiderl~ florid anadddd~~~~~~dddddd ............ ..... .. ...............22.
.l'ii ainhe a~ brevilabris ........._.._.. ...._... ...............23..
.lit ainhesl~ cernua Deep South race ........._._ ...... .__ ...............24
Overview of Pertinent Literature ........._._ ...... .... ...............24..
Orchid Ecology .........._.. ........ .. ...............24....
Orchid-Mycobiont Association .............. ...............27....
History and background .............. ...............27....
Infection and digestion ............ ...... ._._ ...............28...
Photomycotrophic balance .............. ...............30....
Orchid mycobiont taxonomy ................. ...............3.. 1......... ....
Orchid Propagation and Culture ................. ...............35................
Asymbiotic seed culture ................ ...............35........... ....
Symbiotic seed culture .............. ...............40....
Orchid Pollination Biology ................. ........... ........... ...... .........4
Molecular Genetics and Genetic Diversity in Orchids............... ...............50


2 POLLINATION BIOLOGY AND GENETIC DIVERSITY OF Habenaria
macroceratitis ............ ......._ ...............63...


Introduction................ .............6
Materials and Methods .............. ...............65....

Study Sites ........._.. _..... ._ ...............65...
Plant Demography ........._.. _..... .__ ...............65...
Pollinator Observations .............. ...............66....












Pollination Mechanism, Seed Viability, and Asymbiotic Seed Germination .................67
Sampling, DNA Extraction and Amplified Fragment Length Polymorphism
(A FLP) .............. ...............70....
Re sults ................ ...............72.................

Study Sites ................. ...............72.......... .....
Plant Demography ................. ...............72.......... .....
Pollinator Observations ........................ ... .. .. ... ...................7
Pollination Mechanism, Seed Viability, and Asymbiotic Seed Germination .................74
AFLP Data ................. ...............75.................
D iscussion............... ..... .. .................7

Implications for Integrated Conservation Planning ................. ...............83........... ...

3 SEED CULTURE AND IN VITRO SEEDLING DEVELOPMENT OF Habenaria
macroceratitis ............ ...... ._ ...............96...


Introduction................ .............9
Materials and Methods .............. ...............99....

Asymbiotic Seed Germination .............. ...............99....
Seed source and sterilization ................. .....___ ...............99. ....
Asymbiotic media survey ..........._.._.. ...... ....... .. ......._. ............9
Effects of carbohydrate source on asymbiotic seed germination.............._._. ........100
Effects of exogenous cytokinins on asymbiotic seed germination ........................ 101
Effects of photoperiod on asymbiotic seed germination............. ..._........._._ ..101
Effects of photoperiod on in vitro seedling development ........._.._.. ......._.._.....102
Symbiotic Seed Germination............... ..............10
Seed source and sterilization ................. .....___ ...............102 ....

Mycobiont isolation and identification .............. ...............103....
Symbiotic co-culture ................. .. ........... .........0
Effects of photoperiod on symbiotic co-culture ........................... ...............105
Re sults.................. .. ... ....... .. ...............106.....

Asymbiotic Seed Germination .............. ...............106....
Asymbiotic media survey ................. ...... ....... .. .......... ............10
Effects of carbohydrate source on asymbiotic seed germination ................... ........107
Effects of exogenous cytokinins on asymbiotic seed germination ........................ 108
Effects of photoperiod on asymbiotic seed germination ................. ............._..108
Effects of photoperiod on in vitro seedling development ........._.._.. ......._.._.....109
Symbiotic Seed Germination ........._.._.. ........_. ...............110....
Mycobiont isolation and identification ................. ...............110...............
Sy mbiotic co-culture ................... .. ........... ...............110......
Effects of photoperiod on symbiotic co-culture ........................... ...............11 1
Discussion................... ................11

Asymbiotic Seed Germination ................. ......... ...............111 ....
Symbiotic Seed Germination................ .............12
Implications for Integrated Conservation Planning ................. .............. ......... .....123












4 POLLINATION BIOLOGY AND GENETIC DIVERSITY OF .gitaidesr,~ floridanadddd~~~~~~dddddd .......143

Introduction................ .............14
Materials and Methods .............. ...............145....

Study Sites .........__.. ..... .__ ...............145...
Plant Demography ........._.___..... .__. ...............145....
Pollinator Observations .............. ...............145....
Pollination M echanism ........._._.... ... .... ... ._._ .. .. .._ ... ..... ........4

Sampling, DNA Extraction, and Amplified Fragment Length Polymorphism
(AFLP) ................. ...............147......... ......
Re sults ................ ...............148................

Study Site............... ...............148.
Plant Demography .............. .... .. .... ........ ............ ............14
Pollinator Observations and Pollination Mechanism .............. ...............149....
AFLP Data ................. ...............150................
D iscussion............... ....... .................15

Implications for Integrated Conservation ................. ...............157...............

5 SEED CULTURE OF .gitaidesrl~ brevilabris AND DEEP SOUTH S. cernua .................163

Introduction................ .............16
Materials and Methods ............... ... ...............165..

Fungal Isolation and Identification............... ............16
Seed Collection and Sterilization .............. ...............166....

Symbiotic Co-Culture............... ..............16
Re sults.................... .. .. ...... .. ...._ __ ...... ..........6

Fungal Mycobionts: .gitainhesl~ brevilabris ................ ...............168........... ...
Fungal Mycobionts: Deep South .gitainhesl~ cernua ......___ ........___ ...............169
Symbiotic Co-Culture: .git ainhesl~ brevilabris ........._.._.. ....._.. ........._.._......169
Symbiotic Co-Culture: Deep South .gitainhesl~ cernua. ........._._ ..... .._._...........170
D iscussion............... ..... .. .. ...............17

Implications for Integrated Conservation Planning ....._____ ..... ... ._ .. ........_......178


APPENDICES


A MORPHOLOGICAL VARIATION IN Habenaria macroceratitis .............. ................1 88


B AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP)
SUPPLEMENTARY DATA FOR Habenaria macroceratitis............._._ ........_._......193


LIST OF REFERENCES ........._.._ ..... ._._ ...............200...


BIOGRAPHICAL SKETCH .............. ...............226....










LIST OF TABLES


Table page

1-1. Anamorphic and corresponding teleomorph species of common orchid mycobionts in
the Rhizoctonia complex. ........... ..... .... ...............55...

2-1. Experimental pollination conditions applied to flowers of Habenaria macroceratitis. ........86

2-2. Thermal cycler parameters for preselective and selective AFLP amplification of
prepared genomic DNA ................ ...............87........... ....

2-3. Results of the genetic analysis for each population of Habenaria macroceratitis. ..............88

3-1. Comparative mineral salt, vitamin, and amino acid content of asymbiotic orchid seed
germination media used in the asymbiotic germination of Habenaria macroceratitis ...126

3-2. Seed germination and protocorm development stages in Habenaria macroceratitis .........127

3-3. Sources of mycobionts used in the in vitro symbiotic co-culture of Habenaria
macroceratitis ........._._ ..... ._ ...............128...

5-1. Sources of mycobionts used in the in vitro symbiotic co-culture of .gitanthes,~
brevilabris ........._..._. ...._ ... ...............18 1....

5-2. Sources of mycobionts used in the in vitro co-culture of the Deep South race of
.@lii au th' \ e a erua ................. ................. 18......... 2..











LIST OF FIGURES


Figure page

1-1. Diagrammatic representation of the steps and connections of integrated orchid
conservation ....._ ................ ...............56.......

1-2. Habenaria macroceratitis inflorescence and flower ................. ..............................57

1-3. .gitaidesrl~ brevilabris and S. floridanadddd~~~~~~dddddd inflorescences ................. ............... 58...........

1-4. Deep South race of .g'itaidesr, cernua inflorescence and flower. ................ .............. .....59

1-5. Example of mycobiont pelotons in the cortical tissues of .gitaidesr,~ brevilabris ................60

1-6. Cellulase assay............... ...............61.

1-7. Polyphenol oxidase assay .............. ...............62....

2-1. Study sites for Habenaria macroceratitis in Florida............... ...............89

2-2. Average height and spur length ofHabenaria macroceratitis plants at four study sites
in west central Florida............... ...............90

2-3. Average leaf number and flower number of Habenaria macroceratitis plants at four
study sites in west central Florida............... ...............91

2-4. Growth cycle ofHabenaria macroceratitis under field conditions .............. ...................92

2-5. Cocytius antaeus captured during pollinator observation of Habenaria macroceratitis ......93

2-6. Temperature and relative humidity profiles during Habenaria macroceratitis pollinator
observations .............. ...............94....

2-7. Effects of pollination condition on percent germination and protocorm development of
Habenaria macroceratitis ........._.. _...... .__ ...............95...

3-1. Seed germination and protocorm development stages in Habenaria macroceratitis .........129

3-2. Effects of culture media on percent germination and protocorm development of
Habenaria macroceratitis. ........... _...... __ ...............130..

3-3. Effects of carbohydrate type and banana powder on percent germination and protocorm
development of Habenaria macroceratitis after 7 weeks ........._._....... ._.............1 3 1

3-4. Effects of carbohydrate type and banana powder on percent germination and protocorm
development of Habenaria macroceratitis after 2 1 weeks..........._._... ......_._........13 2











3-5. Effects of four cytokinins and four concentrations on percent seed germination of
Habenaria macroceratitis ........._._ ...... .._ ...............133..

3-6. Effects of three photoperiods on in vitro asymbiotic seed germination and protocorm
development of Habenaria macroceratitis ......_......._.__........._ ...........13

3-7. Morphological effects of three photoperiods on asymbiotically germinated Habenaria
macroceratitis ........._._ ..... ._ ..............1 5....

3-8. Effects of three photoperiods on tuber and leaf production per in vitro Habenaria
macroceratitis seedlings .............. ...............136....

3-9. Effects of three photoperiods on tuber and shoot biomass of in vitro Habenaria
macroceratitis seedlings .............. ...............137....

3-10. Effects of three photoperiods on tuber diameter and length and leaf length and width
of in vitro Habenaria macroceratitis seedlings ......___ .........._ ....._.._........3

3-11. Examples of mycobionts isolated from Habenaria macroceratitis. ........._.... ...............139

3-12. Effects of six mycobionts on percent germination and protocorm development of
Habenaria macroceratitis ........._._ ...... .._ ...............140..

3-13. Photoperiodic effect on in vitro symbiotic seed germination and protocorm
development of Habenaria macroceratitis ......_......._.__........._ ...........14

3-14. Photoperiodic effects on the in vitro symbiotic protocorm development of Habenaria
macroceratitis ........._._ ..... ._ ...............142...

4-1. Rayonier study site for .git anthei florid anadddd~~~~~~dddddd ..........._.._ ........ ...............15

4-2. Temperature and relative humidity profiles at Rayonier site on 7 April 2003 during
.lit anthe frr~ lorid anadd~~~~~ddddd~~~~ pollinator ob servati ons ................. ........._..._._....... ..6

4-3. Temperature and relative humidity profiles at Rayonier site on 13 April 2003 during
.lit anthe frr~ lorid anadd~~~~~ddddd~~~~ pollinator ob servati ons ................. ........._..._._....... ..6

4-4. Lack of pollinia in 9'iianthes f lorid anadddd~~~~~~dddddd ..........._.... ...............162...._..__

5-1. Mycobionts isolated from .9'iianthesr l floridanadddd~~~~~~ddddd and S. brevilabris. ........._.._... ................1 83

5-2. Mycobionts used in the symbiotic co-culture of the Deep South race of .git anther,~
cernua .............. ...............184....

5-3. Effects of four mycobionts on percent germination and protocorm development of
.lit an the s\ brevilabris ........._._. ._......_.. ...............18 5..

5-4. Effect of mycobiont Sbrev-266 on percent germination and protocorm development of
.l'ii au the a brevilabris ................. ...............186........ ....











5-5. Effects of two mycobionts on percent germination and protocorm development of the
Deep South race of .g'it anthe,~ cernua. ........._..__......_ .. ...............18

A-1i. Lateral striping variant of Habenaria macroceratitis. ......_.__ .... ... ._ .................19 1

A-2. Plants of Habenaria macroceratitis at the Socash study site showing upright leaf
growth habit ........... ..... ._ ...............192...

B-1. AFLP fingerprint of genomic DNA from the Socash site of Habenaria macroceratitis ...193

B-2. AFLP fingerprint of genomic DNA from the Old Dade Highway site of Habenaria
macroceratitis ........._._ ..... ._ ...............194...

B-3. AFLP fingerprint of genomic DNA from the Rayonier site of .git anthe f\ loridana.d~~~~dddd~~~~ddd ........195

B-4. Correlation dendrogram of genotypic diversity within the Socash site of Habenaria
macroceratitis ........._._ ..... ._ ...............196...

B-5. Correlation dendrogram of genotypic diversity within the Old Dade Highway site of
Habenaria macroceratitis ........._._ ........_ ...............197..

B-6. Correlation dendrogram of genotypic diversity between the Socash and Old Dade
Highway sites of Habenaria macroceratitis ......_.__ .... ....__ ......_ ...........9

B-7. Correlation dendrogram of genotypic diversity within the Rayonier site of .@ilmathes~
florid anadddd~~~~~~dddddd ..........._...__........ ...............199.....









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

INTEGRATED CONSERVATION OF FLORIDA ORCHIDACEAE INT THE GENERA
Habenaria AND @lilmathesl MODEL ORCHID CONSERVATION SYSTEMS FOR THE
AMERICAS

By

Scott L. Stewart

August 2007

Chair: Michael Kane
Major: Horticultural Science

Rapid loss of native orchid habitat throughout ecologically-important areas (e.g., Florida)

has prompted researchers to develop appropriate conservation systems for the preservation and

recovery of native orchid species. These conservation systems must incorporate more than

habitat preservation and plant propagation, they must integrate the study of native orchid

ecology, mycology, propagation, pollination biology, and population genetic diversity in a

combined conservation effort. The current studies examine the ecology and demography,

mycology, asymbiotic and symbiotic propagation, pollination biology, and population genetic

diversity of orchids in the genera Habenaria and .@i' athesl The specific Florida native orchid

included in this study are: Habenaria macroceratitis, .gitanther f~\loridddanaa S. brevilabris, and

the Deep South race of S. cernua. The application of integrated conservation to the preservation

of these native orchids is discussed, and integrated conservation steps are proposed that will help

ensure the long-term population viability of these species. Furthermore, these integrated

conservation systems could be applicable outside the United States and Florida.









CHAPTER 1
LITERATURE REVIEW

Introduction

The appeal and curiosity surrounding orchids throughout the ages have far surpassed that

of any plant. This interest has not been limited to just the hobbyist and commercial plant venues,

but there has been an untiring pursuit of scientific knowledge on this large and complex plant

family. Hundreds of scientific and popular texts are published each year concerning some aspect

of orchid biology. Both the floristic and propagation complexities of the Orchidaceae have

pleasure and plagued researchers, hobbyists, and commercial growers for decades.

The Orchidaceae comprise approximately 10% of all known angiosperms, has a holarctic

distribution, and is considered the largest family of flowering plants with approximately 35,000

species (Cronquist 1981; Dressler 1981). Furthermore, the orchid family has evolved highly

specialized associations with both pollinators and mycorrhizal fungi, the latter actually being a

parasitic relationship where the orchid consumes fungi as a source of nutrients and water (Zettler

1997a; Tremblay 1992). The size and diversity of the Orchidaceae has been linked to this

specialization in pollination and mycorrhizal association (Benzing 1981; Williams 1947; Stewart

and Kane 2007a; Cozzolino and Widmer 2005; Taylor et al. 2003). This unique

endomycorrhizal association allows Corallorhiza trifida Chatelain to survive in the tundra of

northern Canada, Platanthera lacera (Michaux) G. Don to live in the tallgrass prairies of the

Midwest, and Cattleya skinneri Bateman to live in the tropics of Costa Rica.

This almost primordial curiosity with orchids can be traced back to ancient Greek times,

where one orchid creation myth claims that a young, handsome Greek prince, named Orkhis, fell

in love with a beautiful priestess of the god Bakkhos. Pursuing his love for the priestess, Orkhis

journeyed to her temple where he encountered wild animals guarding the priestess. The animals









tore love-struck Orkhis limb from limb, scattering his tattered body over the land. In the places

where Orkhis' body parts landed flowers grew, which were named for him--orchises (Berliocchi

1996; Hansen 2001). This story helps to explain both the origin of the temperate orchid genus

Orchis, and the entire family Orchidaceae.

Unfortunately, the great beauty and mysticism that surrounds orchids have also directly

contributed to their mass decline as wild plants. Unique mycorrhizal associations have enabled

orchids to survive in diverse habitats around the world. In evolving to exist in these disparate

habitats, orchids have evolved fascinating flower forms and colors. These often spectacular

flowers and the intimate relationship orchids have with their habitats have led directly to the

family's decline in the wild due to mass over-collecting, habitat conversion to urban uses, habitat

conservation to agricultural lands, and habitat mismanagement. For this reason, a large amount

of scientific information exists concerning the propagation of orchids for conservation and

restoration purposes. Orchids were among the first plants to be successfully cultured under in

vitro conditions--from seed (Knudson 1922) and by micropropagation (Rotor 1949; Arditti

1984). Numerous studies have focused on asymbiotic propagation of orchids from seed (for

reviews see Arditti 1967; Sheehan 1983). Less research has been conducted on the symbiotic

propagation and restoration of orchids, despite a nearly universal acknowledgement of the need

for such work (for review see Rasmussen 1995). Scientific information on the symbiotic seed

germination and restoration of orchids lags far behind that of both systematic studies and

asymbiotic culture of orchids.

Study Rationale

Conservation Ethic and Modes

The American conservation ethic began to take shape during the mid-1800's era of societal

prosperity and urbanization, spearheaded by the publication of George Perkins Marsh's (1864)









"Man and Nature". Partly in response to Marsh's book and environmental movement, the

government of the United States realized a need to conserve a national heritage and developed

the national park system beginning in 1872. John Muir fixed a national conservation

consciousness in the mind of Americans by organizing the first private national conservation

organization, The Sierra Club. By the mid-1900's, Aldo Leopold solidified the notions of

conservationism and environmental protection in America through the publication of "A Sand

County Almanac" (1949). The next big move forward in American conservationism came in

1962 with the publication of Rachel Carson' s book Silent Spring", which is often credited with

igniting the modern environmental movement. The capstone to the influence these authors had

on the American environmental ethic came in 1973 with the passing of the Federal Endangered

Species Act. Additionally, most states afford both plants and animals some degree of

hierarchical protection and management. Orchids have traditionally been the focus of national

and international protection efforts, particularly focusing on the management of the international

trade of orchid plants and their parts (CITES 1973).

Classically, orchid conservation has taken three traditional modes: 1) the development of

action plans, 2) the assessment of a species' or genus' population or conservation status in the

world, 3) or plant propagation and reintroduction. A fourth, and often overlooked, orchid

conservation mode is the implementation and study of management regimes. Orchid

conservation action plans often contain a mixed bag of personal opinions concerning orchid

conservation (Lapiner 1970; Moir 1970; Lynch 1970) and a review of the application of state,

national, and international conservation regulations to the field of orchid conservation (Campbell

1979; Campbell and Tarr 1980a, 1980b). An ever-present and recently reemerging field in the

development of orchid conservation action plans is the establishment of long-term orchid









monitoring studies (M.H.S. Light personal communication). While discussions of orchid

conservation and the establishment of long-term monitoring programs are both valuable in

conservation planning, taken alone this mode of orchid conservation is limited in its

effectiveness in protecting current populations of orchids or restoring declining or extirpated

populations.

The assessment of a species' or genus' conservation status is another common mode of

orchid conservation. These assessments typically examine an orchid's ecological status

throughout its native range, the primary threats to the orchid's continued existence in its natural

setting, and any current conservation programs that apply to the orchid. The work of Cribb and

Sandison (1998) on the conservation status of Cypripedium species worldwide represents a

genus-wide application of this assessment notion; others have chosen a species-by-species

approach to the assessment of orchid conservation (Gulliver et al. 2004a, 2004b). Recovery

plans represent another type of orchid conservation assessment, and can be focused on single-

species (COSEWIC 2003; Brownell 1986; Seevers and Lang 1998a, 1998b; Ramstetter 2001;

Bowles et al. 1998) or multi-species (Coates et al. 2003). Furthermore, others have taken the

approach of public education and general species diversity assessment as a population extension

of scientific conservation assessment (Jost 2006). Again, all these assessment approaches are

valuable to orchid conservation planning as tools in understanding the need for orchid

conservation. However, the impact of these conservation assessments remain low if no further

conservation work comes from their publication.

The propagation and reintroduction of orchids represents another classic mode of orchid

conservation. A great deal of attention has been given to the seed culture of orchids, although

the maj ority of this research has focused on the propagation efficiency of media types or culture










systems and not propagation for conservation purposes. Stewart (2002) and Stewart (2003),

working with the rare Florida terrestrial orchid Splitaidesrl~ brevilabris Lindl., are recent examples

of the integration of orchid seed culture and plant reintroduction for conservation purposes.

Glicenstein (2006) presented a similar integration of seed culture and plant reintroduction in

working with Cypripedium acaule Aiton. Zettler (1994) and Zettler (1996) reported, in a popular

publication format, on efforts to conserve Platanthera integrilabia (Correll) Luer in Tennessee

through propagation and ecological studies. Surprisingly, few other examples exist of the

successful propagation and reintroduction of North American native orchids. While this mode of

orchid conservation has the highest impact for natural populations, considerations of seed source

and planting location remain understudied and underappreciated by most orchid conservation

biologists.

A final mode of orchid conservation is the examination of management techniques on

orchid population viability and sustainability. Despite producing the most valuable orchid

conservation information, few reports exist concerning the effects of orchid or orchid habitat

management. Curtis (1946) reported on the use of mowing as a management technique for

Cypripedium candidum Muhl. ex Willd. More recently, Jane~kova et al. (2006) reported on the

effect of management techniques and environmental conditions on the population survival of

Dactylorhiza majalis (Rchb.) P.F. Hunt & Summerhayes. In both these cases, mowing was used

as a typical management technique for both of these temperate terrestrial orchid species.

However, little information exists on orchid management in tropical regions or for epiphytic

species (S.L. Stewart, personal observation). A new emerging and exciting area of orchid

management is the reporting of orchid refuges in man-made settings such as coffee or tree

plantations (Solis-Montero et al. 2005). As before, the reporting of orchid management









techniques is an important part of orchid conservation; however, taken as an individual effort the

development of a management technique without incorporating other aspects of orchid

conservation biology limits the long-term effectiveness of this method.

The main weakness of all the aforementioned orchid conservation modes is the lack of

integration among all aspects of each mode. This lack of integration often leads to an incomplete

or skewed orchid conservation system. For example, focusing exclusively on the propagation of

an orchid species could detract from the planning for plant reintroduction or long-term

management of the species. For this reason, integrated orchid conservation techniques must be

utilized by orchid conservation programs worldwide.

Integrated Orchid Conservation

Effective plant conservation programs involve a mode of conservation that allows careful

consideration of conservation-oriented questions and structured criteria in making difficult

choices when investing limited resources. This intense focus has not allowed those interested in

orchid conservation the flexibility to make well-informed choices when confronted with

questions of population-, genus-, state-, or regional-level orchid conservation systems (S.L.

Stewart personal observation). Orchid conservation practices must integrate an understanding of

existing and future threats, taxonomic distinction, population size fluctuation, pollination and

reproductive biology, in vitro and in situ propagation, and the maintenance of population genetic

diversity (for reviews see Cribb et al. 2003; Koopowitz et al. 2003). For this to be possible,

conservation practices should involve experimentation in both in situ ecological contexts and ex

situ laboratory-based contexts (Brundrett 2007; Swarts et al. 2007).

Therefore, orchid conservation systems must maintain a balance between the need for

urgent action to avoid immediate loss of species diversity and long-term landscape- or species-

level actions that yield valuable ecological information (Spring et al. 2007). As outlined by










Hopper (1997), the integrated conservation strategy focuses on the study of interactions among

all trophic levels of plant conservation biology--basic ecological criteria, mycological

interactions, propagation science technologies, pollination biology and breeding systems,

population genetic diversity measures, and species reintroduction and recovery methods.

Integrated conservation systems vary according to which species they are being applied;

however, the basic concept remains the same (Figure 1-1).

The present studies investigated aspects of the integrated conservation of several Florida

terrestrial orchid species. These species include: Habenaria macroceratitis, Splitanthesl~

floridanad~~~~~ddddd~~~~ S. brevilabris, and S. cernua. Each of these species represents an understudied and

critically-imperiled native orchid in Florida. The development of integrated conservation

methods for these orchid species will help insure their long-term sustainability in Florida, along

with demonstrating the applicability of integrated conservation methods to orchids throughout

the world.

Plants of Interest

Selection Criteria

Species included in the present studies were chosen based upon several conservation-

oriented criteria, including state listing status, number of extant populations, presently known

conservation information on the species, past or present conservation attention given to the

species, and immediacy of current threats. Historic and modern accounts of the status of

candidate species were also considered (Brown 2002, 2005; Luer 1972; Ames-Plimpton 1979;

Hawkes 1948). Species for which there were few (<10) known populations, little-to-no presently

known conservation information, and little-to-no past or present conservation attention were

considered as viable candidates for the present studies. Furthermore, species with an immediate

conservation threat were also considered for the present studies.









The four species included in the current studies are: Habenaria macroceratitis, Splitainhesl~

floridanad~~~~~ddddd~~~~ S. brevilabris, and the Deep South race of S. cernua. These species represent four rare

and understudied Florida native terrestrial orchids.

Plants of Study

Habenaria macroceratitis

The subtribe Habenariinae is a large subtribe with approximately 930 species in 23 genera

(Dressler 1993). Habenaria is the largest genus in the subtribe Habenariinae. The distribution

of Habenaria is mainly throughout the tropical Americas, tropical Africa, India, southeastern

Asia, northern Australia, and extending to eastern China and Japan (Pridgeon et al. 2001). This

large distribution is reflected in the infrageneric sectioning of the genus into 37 sections (Batista

et al. 2006). The genus includes both common and rare species throughout its worldwide range.

Habenaria macroceratitis Willdenow (section Macroceratitae), the long-horned rein

orchis, is a rare terrestrial orchid that grows in the mesic hammocks of west-central Florida

(Figure 1-2). This species is known from outside the United States, occurring in Mexico, the

West Indies, and Central America (Brown 2002, 2005). A recent report of this species from

Cuba is not surprising, but requires careful verification since the close relative of H

macroceratitis, H. quinqueseta (Michaux) Swartz, is known from the island nation (Llamacho

and Larramendi 2005). Plants are known to grow up to 75 cm in height, typically possess 3-7

elliptic blue-green leaves, and can have 15-25 flowers arranged in a loose raceme (Brown 2002,

2005).

Habenaria macroceratitis has historically been segregated as a variety of H. quinqueseta

(e.g., H. quinqueseta var. macroceratitis; Hawkes 1948, 1950; Luer 1972); however, a recent

treatment of the genus suggests the two are separate species (Brown 2000b; FNA 1993+).

Brown (2000b) cites that the primary distinguishing character between H. quinqueseta and H.









macroceratitis is the proportional length of the segments of the lateral petals, where the distal

division of the lateral petals in H. quinqueseta is less than two-times the length of the proximal

division and is more than two-times the length of the proximal division in H. macroceratitis

(FNA 1993+). Other minor distinguishing characters can be noted after extensive field work

with both species (S.L. Stewart personal observation), such as the square flower profile ofH.

quinqueseta versus the rectangular flower profile ofH. macroceratitis. Additionally, the length

of the nectar spur is somewhat helpful in combination with other morphological traits (P.M.

Brown personal communication). Further study on the taxonomic clarity, possibly using a

combination of morphological- and molecular-based techniques, would help to elucidate the true

placement of these two species.

Spiranthes floridana

The Spiranthinae is a large and diverse subtribe in the subfamily Spiranthoideae, with

approximately 400 species in 30-40 genera (Dressler 1993). .gitaidesrl~ contains about 40

species and reaches its maximum diversity in North America (Pridgeon et al. 2003). Generally,

the genus is distributed throughout North America, Mexico, Europe, Asia, and eastern Australia.

.gli tainhesr' is one of the most recognizable orchid genera; however, it is also one of the

most taxonomically-confusing for those new to the field. Species delimitation in the genus has

long been problematic due to natural hybridization and polyploidy (Luer 1975; Sheviak 1982;

Dueck and Cameron 2007). Phylogenetic analysis has shown the genus to be sister to Mexican

and Mesoamerican genera such as M~esadenus, Dichromanthus, and Deiregyne (Pridegon et al.

2003).

.litainhesr l floridanadddd~~~~~~ddddd (Wherry) Cory, the Florida ladies'-tresses, is a rare terrestrial orchid

native to pine flatwoods of north-central Florida and persists in semi-disturbed edges such as

roadsides and cemeteries (Figure 1-3). This species was historically known from Texas, east









through Florida, and north to North Carolina; however, due to a combination of taxonomic

clarification from the closely-related S. brevilabris and extripation from other areas the species is

now considered endemic to Florida (Brown 2002, 2005; FNA 1993+; Luer 1972). Two sites are

presently know for this species: Bradford County, discovered in 1998 and Duval County,

discovered in 2004. Plants are known to grow between 20-40 cm in height, typically possess 3-5

ovate yellow-green leaves that are withering at flowering, and can have 10-35 flowers arranged

in a single-ranked spiraled or second raceme (Brown 2002, 2005).

.litanthesr l floridanadddd~~~~~~ddddd has historically been segregated as a variety of S. brevilabris (e.g., S.

brevilabris var. floridana;d~~~~~ddddd~~~~ Luer 1972); however both Brown (2002, 2005) and FNA (1993+)

recognize S. floridanadd~~~~~ddddd~~~~ as a separate species from S. brevilabris. This separation is based on the

presence of dense pubescence on flowers and stems of S. brevilabris and the lack of this

pubescence on flowers and stems of S. floridana. The validity of this species-pair separation was

confirmed by the phylogenetic treatment of glitanthes,~ by Dueck and Cameron (2007).

Spiranthes brevilabris

.litanthesl~ brevilabris, the short-lipped ladies'-tresses, is a rare terrestrial orchid native to

pine flatwoods of north-central Florida and persists in semi-disturbed edges such as roadsides

and cemeteries (Figure 1-3). Historically, the species is historically known from Alabama,

Georgia, Louisiana, Mississippi, Texas, and Florida; although it is currently known only from

Florida and Texas (Brown 2002, 2005; E.L. Keith personal communication). The species current

status is likely due to taxonomic confusion with allied spring-flowering .gitantherl~ species in the

southeastern coastal plain and population extripation. .9'iianthesl~ brevilaris is presently known

from only two populations in Levy County, Florida. Plants are known to grow 20-40 cm in

height, possess 3-6 ovate yellow-green leaves that are typically withered at flowering, and can

have 10-35 flowers arranged in a single rank spiraled or second raceme (Brown 2002, 2005).









Spiranthes cernua Deep South race

Splitaidesl' cernZua (L.) Richard, the nodding ladies'-tresses, is a facultatively

agamospermic polyploidy compilospeices known from the eastern half of the United States,

extending from Maine in the north to the panhandle of Florida in the south (FNA 1993+). This

compilospecies is made up of many different geographi cally-restricted races with unidirectional

gene flow from related diploids (Sheviak 1991, 1982). At present, the Deep South race of S

cernua is known to persist in only from one semi-disturbed roadside site in the panhandle of

Florida in Apalachicola National Forest (Liberty County; Figure 1-4; S.L. Stewart personal

observation). The natural habitat of the Deep South race of S cernua is not know, although the

species likely is native to mesic prairies along the edges of small streams. Plants are known to

grow 10-35 cm in height, possess 3-5 linear-oblanceolate leaves that are usually withered at

flowering, and can have 10-50 flowers arranged in a tightly or loosely spiraled spike (Brown

2005).

Overview of Pertinent Literature

Orchid Ecology

The ecology and distribution of the Orchidaceae has been of interest to researchers since

Lindley's taxonomic and ecological descriptions of orchids in the 1800's (as reported in Arditti

1984). Significantly less new information has been generated since the 1800's on the basic

ecology and conservation of orchids, in comparison to the information generated in the study of

orchid taxonomy and propagation. Nonetheless, the study of orchid ecology is a foundational

step in the integrated conservation of orchid species and populations.

Plant distribution and life history studies represent important information that can be used

by conservation planners in designing orchid recovery plans. Unfortunately, little information on

either of these topics exists. Tremblay (1997) reported on the distribution and dispersion









patterns in nine species of f epaides,~ in Puerto Rico. Based on the study of orchid patch size and

number of reproductive versus vegetative individuals, Tremblay (1997) suggested that most

epiphytic orchids may exist as isolated patches with great distances between patches. This

would indicate that a small effective population size is common and that gene flow may be

restricted, depending on pollinator and pollination mechanism. In a similar study, Willems and

Melser (1998) studied the population dynamics of the terrestrial orchid Coeloglossum viride (L.)

Hartm. In this study, C. viride was found to be a short-lived terrestrial orchid with an average

flowering percentage per population of 50%. Vegetative or asexual propagation was found to be

minimal in this species. Willems and Melser (1998) suggested that based on their life-history

and population dynamics data populations of C. viride in the Netherlands were in a "healthy

state" with "vigorous age structure". Similar life-history and population dynamics studies have

been conducted on Cleistes bifaria (Fernald) Catling & Gregg (Mery and Gregg 2003) and

Cypripedium calceolus (L.) var. parviflorum (Salisb.) Hulten (Shefferson et al. 2003).

Trej o-Torres and Ackerman (2001) conducted an extensive biogeographic study of the

orchid distributions throughout the Antilles. This study reported a strong affinity among orchid

distributions on islands at considerable distances from one another, relating the congruency

among orchid flora to the dust-like character of orchid seeds (Trej o-Torres and Ackerman 2001;

Rasmussen and Whigham 1993). In general, this study suggests that similarities in orchid floras

were determined by ecological factors on individual islands rather than distance between islands

as a barrier to plant dispersal. The application of this type of flori stic/ecological study to orchid

flora distributions could be applied to non-island situations, particularly as a modeling system in

examining changes in distributions of orchids in a landscape.









An often understudied area of orchid ecology is the examination of habitat factors that

affect orchid plant distributions. Frei and Dodson (1972) examined the effects of bark substrates

on the seed germination and early growth of epiphytic orchids. This study identified chemical

substances in the bark of some trees that were inhibitory to the growth of orchids. Furthermore,

the presence of epiphytic orchids on certain trees was correlated with production of toxic

chemicals by those trees. In a similar study, Boland and Scott (1992) reported on the effects of

habitat on the presence or absence of three terrestrial orchids (Arethusa bulbosa L., Calopogon

tuberosus (L.) Britton, Sterns, & Poggenberg, Pogonia ophioglossoides (L.) Ker-Gawler).

Species distributions of the three orchids reflected differences in study site hydrology and

microhabitat partitioning, with P. ophioglossoides preferring slightly wetter habitats and

Arethusa and Calopogon preferring dryer habitats.

Allen et al. (2004), in a study on the vegetation surrounding Cypripedium kentuckiense

C.F. Reed sites throughout Louisiana, reported sites to be dominated by American beech (Fagus

grandifolia Ehrh.), eastern hophornbeam (Ostrya virginiana (P. Mill) K. Koch, white oak

(Quercus alba L.), horse-sugar (Symplocos tinctoria L.), and witch hazel (Hamamnelis virginiana

L.). Habitat characterization data such as these play an important role in the conservation

planning for orchid species recovery and in identifying potential orchid reintroduction sites.

Collins et al. (2005), in a similar study, reported on the soil and ecological features of

Hexalectris sites in Texas. Again, knowing the ecological profile of orchid sites will aid in both

the conservation of current sites and in identifying potential orchid reintroduction sites.

In order to better understand the conservation needs of orchid species, it is important to

understand the ecology, distribution, and factors affecting the growth of plants in the wild. The

study of field-based orchid ecology is often overlooked and underappreciated in light of









propagation or molecular-based studies; however, more valuable conservation information can

be gained from the study of current orchid populations in their natural settings. Further study on

the ecology and distribution of orchids in the wild is necessary in order to ensure their long-term

sustainability and conservation.

Orchid-Mycobiont Association

History and background

The Orchidaceae have long been known to possess a unique association with fungal

partners (i.e., mycobionts). Bernard, in 1899, was the first to recognize the role of these

mycobionts in orchid seed germination and attempt the co-culture of orchid seed with

mycobionts isolated from a parent plant (as reported in Arditti 1967). Bernard's early attempts at

symbiotic seed germination were problematic, but nonetheless demonstrated the role mycobionts

played in orchid seed germination. Following the work of Bernard, Burgeff continued to study

the role orchid mycobionts played in the germination of orchids seeds, as well as studying the

two organisms in association with one another (Arditti 1984). Despite early difficulties, Bernard

and Burgeff laid the foundation of the study of symbiotic seed germination in the Orchidaceae.

Their work, as well as the work of other practitioners of the symbiotic germination technique,

would remain in the background during the ensuing 60 years due to the discovery of asymbiotic

orchid seed germination techniques by Knudson (1922, 1946). The study of orchid-mycobiont

associations has received a renewed interest in recent years (for reviews see Warcup 1975;

Hadley 1982).

In nature, orchids digest endomycorrhizal mycobionts as sources of minerals,

carbohydrates, water, and vitamins in an action termed mycotrophy. Mycotrophic consumption

of mycobionts provides the non-endospermic orchid seed a two-fold advantage during

germination: 1) infection followed by digestion initiates seed germination by providing nearly all









the necessary nutrients, moisture, and other organic compounds to the proembryo and 2)

infection and digestion contributes entirely to the development of the non-photosynthetic

protocorm life stages of orchids. In particular, the digestion of these mycobionts have been

shown to provide simple carbohydrates (Smith 1966, 1967; Gebauer and Meyer 2003; Tsutsui

and Tomita 1990; Hadley and Purves 1974; Cameron et al. 2006; McKendrick et al. 2000),

cytokinins (Crafts and Miller 1974), auxins (Withner 1959), phosphate (Alexander et al. 1984),

phosphorous (Cameron et al. 2007), nitrogen (Lucas 1977; Gebauer and Meyer 2003; Cameron

et al. 2006), vitamins (Hijner and Arditti 1973), and water (Yoder et al. 2000). These

compounds either directly aid in the germination process or in the mobilization of other

germination factors. Additionally, these nutrients and other organic compounds can be used as

stored energy sources by the orchid seedling and plant during later life stages. In recent years

some researchers have questioned the necessity of orchid mycobionts (Bayman et al. 2002);

however, the dependence of orchids on their mycobionts for at least germination should be

considered as universally accepted (Rasmussen 1995).

Infection and digestion

The orchid-mycobiont association begins with the infection of the orchid seed by fungal

hyphae, typically via the suspensor region of the seed (Clements 1988). Infection allows for

direct contact between the fungal hyphae and the orchid embryo, as well as allows for the

introduction of water via the mechanical breaking of the typically hydrophobic orchid testa

(Curtiss 1893; Davis 1948; Stoutamire 1964; Yoder et al. 2000). The exact cascade of

mechanisms that immediately follow mycobiont infection of the orchid embryo remains a

mystery; however, it is assumed that upon infection mycobiont proliferation within the orchid

embryo occurs and is soon followed the formation of pelotons within the cells of the embryo

(Currah and Zelmer 1992; Zettler 1997a). In adult orchid root tissues, pelotons are restricted to










the cortical regions of the roots and typically exclude from storage organs such as tubers or

corms.

The infection, sequestering, and subsequent digestion of these mycobionts was first

described by Burgeff (as reported in Hadley 1982). Two forms of infection and digestion are

common in the Orchidaceae: tolypophagy, in the photomycotrophic orchids, and ptyophagy, in

the achlorophyllous orchids. Tolypophagy is the intercellular digestion of fungal coils, or

peletons (Figure 1-5), by the cortical cells of an orchid root; whereas, in ptyophagy there is no

digestion of fungal coils, but the formation of haustoria or haustoria-like structures that act as

nutrient exchange areas (Burgeff 1959). Here, the discussion is limited to tolypophagy. In adult

orchid plants, mycobionts typically infect the root through root hairs on the epidermis which lead

to host cell layers. It is in these host cell layers that fungal hyphae are allowed to proliferate,

eventually entering a layer of digestion cells. Pelotons, intercellular coils of fungal hyphae, are

formed in these digestion cells, and are subsequently broken-down by intercellular enzymatic

degradation (Figure 1-5). The proximity of the digestion cell layer to the vasculature of the

orchid root allows for easy translocation of nutrients for either immediate use in plant

metabolism or storage.

At no time during the infection and digestion cycle does the orchid harm its mycobiont to

the point of death. This "farming" of the mycobiont has lead some researchers to refer to orchids

as "efficient 'fungus managers'" (Zettler 1997a). While one portion of the orchid mycobiont

continues its natural function of cellulitic decay, the "farmed" portion of the mycobiont is being

digested within an orchid root (Hadley 1969; Midgley et al. 2006). Additionally, orchid

mycobionts (and orchid plants themselves) may associate with other soil microorganisms,









including bacteria and cyanobacteria (Wilkinson et al. 1989; Tsavkelova et al. 2001; Tsavkelova

et al. 2007).

Once an orchid plant produces photosynthetically-capable parts, the role of the mycobiont

becomes less clear. Adult vegetative and flowering orchid plants are known to harbor

mycobionts and pelotons, and the pelotons are known to be digested; however, the degree of

reliance upon the mycotrophic food source is not know (for review see Rasmussen 1995).

Gaddy (1983) and Gill (1996) proposed that at least some temperate terrestrial orchids use the

mycobionts and mycotrophy as a source of nutrients during over-wintering dormancy, or as a

source of nutrients and water during extreme stress conditions (i.e., drought, grazing). While this

hypothesis appears plausible for temperate orchids, the role of mycobionts in the adult life stages

of tropical epiphytic orchids is grossly understudied. Yoder et al. (2000) suggested that the

mycobiont of an epiphytic orchid may provide the photomycotrophic plant with mainly a source

of water. This conclusion, while based on experimental data, needs further verification. Thus,

our knowledge of the orchid-mycobiont association remains poor, especially when compared to

other aspects of orchid biology (i.e., taxonomy).

Photomycotrophic balance

The reliance of orchid seed on a mycobiont to support germination is well understood, as is

the reliance of the orchid protocorm on a mycobiont as a source of energy and water (for review

see Rasmussen 1995). However, once the protocorm produces photosynthetic organs, the orchid

must now manage energy acquisition from both photosynthesis and mycotrophy. At this life

stage, little is known about how the orchid establishes a balance between its Rhizoctonia

mycobiont and photosynthetic gain of nutrients. Alexander et al. (1984) and Alexander and

Hadley (1985), working with the evergreen terrestrial orchid Goodyera repens (L.) R. Brown,

both reported little-to-no movement of nutrients from the mycobiont to orchid tissues in adult










plants using radioactively-labeled CO2 and phosphate. While these studies demonstrate a

minimal input of nutrients by a mycobiont to its adult host orchid, these two studies represent

work based on one evergreen orchid species that is not representative of the maj ority of orchid

species present worldwide. Furthermore, these studies do not take into account outliers such as

albino mutants of orchids that are occasionally seen growing alongside photomycotrophic

counterparts (O'Brien 1953; S.L. Stewart personal observation).

Recently, Cameron et al. (2006) and Cameron et al. (2007) demonstrated both the

movement of nutrients (carbon and phosphorus, respectively) from mycobiont to orchid plant

and the potential for mutualistic nutrient transfer between orchid plant and mycobiont using the

evergreen terrestrial orchid G. repens. These reports indicate that the orchid plant may have a

high degree of photomycotrophic control and be able to switch between a one- and two-way

nutrient cycle with its mycobiont. Lending a genetic basis to this notion of orchid plant-based

control of the orchid-mycobiont association, Watkinson (2002) reported that the terrestrial orchid

Cypripedium parviflorum Salisbury var. pubescence (Willd.) Knight, when grown with an

appropriate mycobiont (Thanatephorus pennatus Currah), demonstrated upregulation of

trehalose-6-phosphate synthase/phosphatase and downrergulation of a nucleotide binding

protein. Plants grown in the absence of a mycobiont demonstrated no effect on the regulation of

either gene. However, while many researchers agree on a genetic basis for the regulation of

mycobiont infection and digestion, most disagree on the mutualistic potential (e.g., Cameron et

al. 2006; Cameron et al. 2007) of some orchid-mycobiont associations (H.N. Rasmussen

personal communication).

Orchid mycobiont taxonomy

Surprisingly, surveys of orchid mycobiont diversity worldwide from both epiphytic and

terrestrial species have yielded little mycobiont diversity. Most mycobionts isolated from









orchids throughout the world have been assignable to the anamorphic form-genus Rhizoctonia

(for review see Zettler et al. 2003). A number of non-Rhizoctonia fungi have been isolated from

orchids, including vesicular-arbusclar mycorrhizae (Raja et al. 1996), ascomycetes (Sharma et al.

2007; Bayman et al. 1997; S.L. Stewart unpublished data), and conidia-producing hyphomycetes

(Currah et al. 1990; Currah et al. 1987; Richardson et al. 1993). However, the role of these fungi

in the mycorrhizae of orchids is not clear.

The use of traditional techniques in the identification of species within the Rhizoctonia

complex is problematic due to the lack of stable, diagnostic morphological features in this group

of fungi. Most Rhizoctonia strains exist as sterile mycelia in pure culture, yielding no definitive

morphological or taxonomic characters. The induction of teleomorphic states from these sterile

isolates have been previously reported, and when accomplished do yield assignable characters.

Warcup and Talbot (1967, 1971, 1980) studied the mycobiont diversity in Australian and British

orchids by inducing perfect states (i.e., teleomorphs) and assigning mycobionts to known

teleomorphs. Many other researchers have studied the Rhizoctonia complex from both a purely

mycological standpoint through the use of septal pore ultrastructures, sporulation, and

cytochemistry (Tu and Kimbrough 1975, 1978; Tu et al. 1969, 1977; Sneh et al. 1991), as well as

from a functional standpoint in relation to the Orchidaceae (Moore 1987; Richardson et al.

1993). All of these approaches are applicable to the identification and taxonomic placement of

orchid mycobionts, particularly for those interested in the symbiotic propagation and

conservation of orchid plants.

Although mostly unpopular with plant pathologists and mycologists, Moore (1987)

segregated Rhizoctonia into functional orchid-related fungal groups based on anamorphic

character differences. Sneh et al. (1991) considers Moore's system to be taxonomically sound









because it is based on conservative characters such as septal pore ultrastructure, cell nuclear

number, and teleomorphic affinities. For example, the anamorphs Epulorhiza, Ceratorhiza, and

Moniliopsis are assignable to the teleomorphs Tula;snella/lSebacina, Ceratoba~sidium, and

Thanatephorus/Waitea (Moore 1987; Rasmussen 1995; Zettler et al. 2003; Table 1-1). Of

particular utility for the non-mycologist studying orchid mycobionts is that these three

anamorphic genera are often distinguished by simple light microscopy and examination of

hyphal characteristics and monilioid cell morphology (L.W. Zettler personal communication).

Furthermore, enzyme assays can play an important role in the easy identification of orchid

mycobionts at the generic level. Cellulase and polyphenol oxidase are two enzymes that can

distinguish among anamorphic genera with relative ease. In the cellulase assay, test tubes

containing a lower 1/5 PDA layer and an upper layer of cellulose azure stain medium are

inoculated with the mycobiont to be surveyed (Smith 1977). The tubes are incubated for 5 days

in continual light (24/0 h L/D) at 250C. A positive cellulase reaction is indicated by the diffusion

of blue dye from the cellulose azure medium layer into the lower V/2 PDA layer (Figure 1-6a). A

weak reaction is recorded if the interface between the two media layers remained visible, while a

negative reaction is indicated as no diffusion of dye (Figure 1-6b-c). In the polyphenol oxidase

assay, Petri plates containing tannic acid medium (TAM) are inoculated with the mycobiont to

be surveyed and incubated for 5 days in continual darkness (0/24 h L/D) at 250C (Davidson et al.

193 8). A positive polyphenol oxidase reaction is indicated by a dark brown discoloration of the

TAM surrounding the point of mycobiont inoculation as viewed from both the top and bottom of

the plate (Figure 1-7a). A weak reaction is recorded as the discoloration being visible from only

the top of the plate, while no color change indicates a negative reaction (Figure 1-7b).









Moore's anamorphic taxonomy of orchid-inhabiting mycobionts also has a great degree of

practical merit, since the maj ority of all orchid mycobionts isolated worldwide are assignable to

one of these anamorphic genera. However, Eberhardt et al. (1999) highlight the instability of

vegetative characters in these anamorphic genera and discussed the possibility of genetic

differences among morphologically similar strains. Given this, the use of sensitive molecular-

based identification techniques is beginning to become standard in the study of orchid

mycobionts, particularly when studying ecological, diversity, and evolutionary aspects of orchid

mycobionts.

The advantages of molecular marker systems in identifying and assessing orchid

mycobiont diversity is only recently being applied. McKendrick et al. (2000) used internal

transcribed spacer restriction fragment length polymorphiam (ITS-RFLP) and ITS sequencing to

identify mycobionts of a number of Corallorhiza species as belonging exclusively to the

Thelephora-Tomentella complex of the Thelephoraceae. Kristiansen et al. (2001) utilized single-

stranded conformation polymorphism (SSCP) and mitochondrial ribosomal DNA sequences to

identify single-peloton isolations from the terrestrial orchid Dactylorhiza majalis. Random

amplified polymorphic DNA (RAPD) analysis and cleaved amplified polymorphic sequences

(CAPS) have been combined with cultural morphology, nuclear number, and septal pore

ultrastructure in a comparative study of taxonomic power in the study of Rhizoctonia isolates

from a number of orchid speices (Shan et al. 2002). In their study, the traditional morphological

methods used in this study separated the 21 Rhizoctonia isolates in the same manner as did the

molecular methods, although the molecular methods were able to directly assign a teleomorph to

each isolate. The advantage of using molecular-based marker system in the identification of

orchid mycobionts is obvious--speed of identification and nearly instantaneous assignment of









teleomorphs. Future fine-scale study of orchid mycobiont diversity and orchid-mycobiont

evolution will require the continued use of these molecular-based identification methods.

Orchid Propagation and Culture

Asymbiotic seed culture

Knudson (1922) was the first person to demonstrate the germination of orchid seeds

without a mycorrhizal fungus. Originally, Knudson was interested in the effects of sugars on

plants (Steward 1958; Arditti 1967) and enzyme production in fungi (Knudson 1913). Based on

these interests, Knudson realized the role of orchid mycobionts--as carbohydrate hydrolyzers,

breaking complex molecules, such as starch and cellulose, into simple sugars that an orchid plant

could utlize. In testing this hypothesis, Knudson sowed seeds of Cattleya mossiae Hooker on

Pfeffer' s mineral salt solution supplemented with 1% sucrose and within 7 months seeds

germinated. To further test his hypothesis, Knudson then sowed seeds of the hybrid C.

intermedia Graham x C. Imrrenceana Rchenb. on Knudson Solution B supplemented with either

2% sucrose or 2% glucose. Within one year, seeds of this hybrid also germinated (Knudson

1922). Eventually, Knudson refined his Solution B to Solution C (Knudson 1946), which is

currently known as Knudson C and widely used as a standard asymbiotic orchid seed

germination medium.

Since the publication of Knusdon' s asymbiotic germination medium and method in 1922,

the vast maj ority of orchid seed germination research has focused on using these methods.

Symbiotic seed germination requires expertise in both mycology and plant propagation; whereas

asymbiotic seed germination techniques require the researcher to be only a plant propagation

specialist (L.W. Zettler personal communication). Asymbiotic germination techniques have

been applied to a number of tropical, temperate, epiphytic, and terrestrial orchid taxa. Vanilla is

one orchid genus that has received attention from those interested in the mass propagation of this









commercially important crop from seed (Knudson 1950; Withner 1955). While asymbiotic seed

germination of Vanilla is possible, the use of these seed-born seedlings in commercial production

of Vanilla simply never became attractive since this genus is easily propagated clonally by

cuttmngs.

A number of reports exist concerning the asymbiotic seed germination of orchid species in

an attempt to optimize germination and production conditions. Knudson (1951) reported on the

comparative effects of asymbiotic germination of Cattleya skinneri seeds sown on Knudson C

versus Vacin and Went medium. Not surprising, Kundson (1951) demonstrated a higher percent

germination and better in vitro seedling growth on his Knudson C medium. Ernst(1975)

reported on attempts to enhance the asymbiotic germination and in vitro seedling growth of

Phalaenopsis. In particular, Ernst (1975) was interested in examining the effects of activated

charcoal and banana additives on asymbiotic seed germination. This study reported a 174%

increase in seedling fresh weight (over control) from seedlings in the Knudson C + charcoal

treatment and a 481% increase in seedling fresh weight (over control) from seedlings in the

Knudson C + charcoal + banana treatments. These data demonstrate a common theme among

practitioners of asymbiotic orchid seed germination--basal medium modification for the

enhancement of germination and in vitro seedling growth.

Others have demonstrated the use of asymbiotic germination techniques in the

conservation of rare or medicinally-important orchids. Light and MacConaill (2003) reported on

the asymbiotic germination of Galeandra batemanii Rolfe and G. greemroodiana Warford as a

means of plant production for conservation purposes. Shimada et al. (2001) reported the

asymbiotic germination of Habenaria radiata Thumb. and the induction of in vitro tubers by

these asymbiotically-propagation plants. The in vitro tubers where used as planting material in









the reintroduction of this species in Japan. Lo et al. (2004) recently reported on the asymbiotic

germination of the medicinally important orchid Dendrobium tosaense Makino in Taiwan.

Furthermore, in vitro asymbiotic germination has been used to study the germination

physiology of orchid seeds. Harrison and Arditti (1978) used asymbiotic germination methods to

investigate the role of carbohydrate source in the germination of Cattleya aurantiaca (Batem. ex

Lindl.) P.N. Don. Ernst et al. (1971) and Ernst and Arditti (1990) demonstrated the usefulness of

asymbiotic orchid seed germination methods in the study of carbohydrate usage in in vitro

seedlings of several Phalaenopsis hybrids. By growing seedlings on media containing different

carbohydrate sources, they were able to demonstrate the utilization of simple sugars by orchid

seedlings versus complex carbohydrates. Similarly, asymbiotic seed culture has been used to

demonstrate the rapid uptake of simple sugars (i.e., fructose) by both differentiated and

undifferentiated Dendrobium tissues (Hew and Mah 1989). Zeigler et al. (1967) used asymbiotic

seed culture to study the effects of amino acid supplementation of media with Edamin on the

germination of the hybrid Cattleya Enid. alba x Laelia anceps Lindl. var. veitchii. In media

treatments with the addition of Edamin, seeds of C. Enid. alba x L. ancepes var. veitchii required

10 to 13 days less to germinate and show signs of greening.

The orchid genus Cypripedium has received a disproportionate amount of attention from

asymbiotic practitioners. Because of their reasonably attractive flowers and hardy growth habit,

species of Cypripedium are highly sought after as horticultural specimens and research plants.

However, seeds of this temperate orchid genus have been notoriously difficult to germinate using

asymbiotic techniques due to strong seed dormancy issues (Liddell 1952; Chu and Mudge 1996).

A great deal of research has focused on media selection and modification for this genus (De

Pauw and Remphrey 1993), seed germination scale-up procedures (Malmgren 1992), the use of









immature seed (Hoshi et al. 1994; St-Arnaud et al. 1992), and the effects of seed chilling and

chemical pretreatments (Miyoshi and Mii 1998). Research focusing on the asymbiotic

germination of Cypripedium species worldwide has shed new light on the usefulness and power

of asymbiotic seed germination techniques.

In examining the role of asymbiotic media effects and media supplementation, Nakamura

(1982) investigated the nutritional conditions required for the asymbiotic germination of seeds of

the achlorophyllous orchid Galeola septentrionalis Reichb. These studies found that nitrogen

source played a large role in the support of germination, with organic sources of nitrogen

supporting high germination percentages and inorganic sources supporting minimal germination

of G. septentrionalis. This implies that G. septentrionalis likely obtains organic forms of

nitrogen from its mycobiont, instead of inorganic forms of nitrogen from the soil. Additionally,

simple sugars supported higher germination percentages than did more complex sugars. The

presence or absence of vitamins was found to have no affect on seed germination or in vitro plant

growth. Finally, auxins were found to improve seedling growth post-germination and cytokinins

were found to have no effect on seedling growth. In studying the asymbiotic seed culture of G.

septentrionalis, Nakamura (1982) was able to demonstrate that this achlorophyllous orchid

species possesses the same nutritional requirements as chlorophyllous orchids and likely gains

these nutrients through its mycobiont. Thompson et al. (2006) conducted a similar survey of

asymbiotic germination requirements in the South African orchid genus Disa. Interestingly,

Thompson et al. (2006) combined a survey of asymbiotic germination requirements (i.e., media,

media supplementation) with conservation by developing germination protocols for a number of

Disa species.









An emerging use of asymbiotic seed culture techniques has come in the form of asymbiotic

media screens as a way to optimize germination efficiency prior to further physiological

experimentation. Stenberg and Kane (1998), studying the Florida epiphytic orchid Prosthechea

boothiana (Lindl.) W.E. Higgins var. 'i~ phirl ninitk's (Small) W.E. Higgins (syn. = Encyclia

boothiana (Lindl.) Dressler var. ('i rl y~ib~itonitsik (Small) Luer), reported variable asymbiotic

germination in a screen of four commonly-available asymbiotic media. Kauth et al. (2006)

conducted a similar asymbiotic media screen in studies on the germination and in vitro seedling

development of Calopogon tuberosus var. tuberosus. They reported significant differences in

seed germination on three different asymbiotic media, as well as differences in in vitro seedling

growth and development. In the case of Kauth et al. (2006), asymbiotic media screen research

laid the foundation for further investigations of the effects of photoperiod and temperature on

ecotypic delimitation of C. tuberosus var. tuberosus by identifying an optimal medium to support

the rapid germination and seedling growth of the species (P. Kauth personal communication). A

similar approach was taken by Stewart and Kane (2006a) in investigations on the asymbiotic

seed germination and effects of photoperiod, exogenous cytokinins, and carbohydrate source on

the germination of the Florida terrestrial orchid Habenaria macroceratitis.

The continued focus on asymbiotic orchid seed culture methods is an important part of

orchid conservation biology, especially for the study of the physiological effects of mineral

nutrition, photoperiod, and the effects of exogenous growth regulators on orchid seed

germination and seedling development. Furthermore, the use of asymbiotic media screens as a

means to optimize asymbiotic germination before conducting further in vitro physiological or

ecological studies should receive continued attention. Although asymbiotic orchid seed

germination methods may represent an efficient means to propagate species for conservation










purposes, this method does not account for the role of the mycobiont. Without these mycobionts,

orchid seed germination and plant development ex situ would not occur. Symbiotic seed culture

methods should be explored as a more viable means of species conservation.

Symbiotic seed culture

The early orchid mycobiont work of Bernard and Burgeff centered around not only the

descriptive aspects of the orchid-mycobiont association, but also the use of isolated mycobionts

in seed-mycobiont co-culture experiments. Both Bernard and Burgeff successfully germinated

seeds of orchids under co-culture conditions with appropriate mycobionts (as reported in Arditti

1984). Little new research on the symbiotic seed germination of orchids continued after

Knudson' s (1922) report of asymbiotic orchid seed germination.

Not until the 1940's, was interest in symbiotic seed germination techniques resurrected.

Downie (1940) published one of the first modern reports of symbiotic germination. Working

with the terrestrial orchid Goodyera repens, Downie reported increased germination efficiency

and an increase in protocorm size using the symbiotic technique in comparison to the asymbiotic

technique. Later, Hadley (1970) reported on the symbiotic germination of a number of orchid

species, including Epidendrum radicans Paven ex Lindl., Sp ath' ,~lrighni plicata Blume,

Dacty/lorhiza pupurpurlla (T. Stephenson & T.A. Stephenson) So6,, and Goodyera repens. While

reporting on the symbiotic germination of these species, Hadley focused his research on the

question of orchid-mycobiont preference by demonstrating that some mycobionts are nearly

universal in their ability to support symbiotic germination while others appear to support the

germination of only a narrow range of orchid taxa. Warcup (1973) reported on the symbiotic

germination of several Australian Pterostylis, Diuris, and Thelymitra species using several

species of Tulasnella and Ceratoba~sidium mycobionts. He reported that the various isolates of

Tulasnella supported variable germination among the different orchid genera tested. Warcup









(1973) was one of the first researchers to report that mycobionts have different capacities to

support symbiotic seed germination, and that the mycobionts that support the most efficient

symbiotic germination in vitro may not originate from the orchid taxa from which seed were

collected. This orchid-mycobiont preference debate still rages today (Taylor and Bruns 1999;

Stewart and Kane 2007a; Otero et al. 2005; Shefferson et al. 2005; Taylor et al. 2003), and has

serious ecological and orchid conservation consequences now being recognized (Stewart and

Zettler 2002; Stewart and Kane 2006b, 2007).

Modern symbiotic seed germination techniques were first popularized and applied to the

conservation of orchids in the 1980's. Muir (1987) reported on the symbiotic germination of a

cultivated native orchid in Europe, Orchis laxiflora Lam. 'Jersey Girl'. Unidentified strains of

the mycobiont Ceratoba~sidium were used in these studies. The symbiotic germination of another

European terrestrial orchid, Dactylorhiza maculata (L.) So6, using unidentified species of

mycobionts was reported by Mitchell (1988). This study used a modified oats medium

containing only powdered rolled oats, sucrose, and agar. This simple oat-based symbiotic

medium proposed by Mitchell (1988) remains a standard symbiotic media today. Clements et al.

(1986) reported the symbiotic germination of five European orchid genera (23 species) using

mycobionts isolated from these same taxa. Most importantly, Clements et al. (1986) reported on

the use of modified oats medium in their symbiotic experiments. This medium--containing

minor mineral salts, sucrose, agar, and powdered rolled oats--is still used today as a basic media

for symbiotic seed germination studies.

The maj ority of symbiotic germination reports since the early 1990's have focused on the

use of this propagation system in the seed germination and conservation of various orchid taxa.

A number of epiphytic orchids have been germinated using symbiotic seed germination









techniques, including Salrcochilus (Markovina and McGee 2000), Epidendrum magnoliae

Mithlenberg var. magnoliae (syn. = E. conopseum R. Brown; Zettler et al. 1998), Encyclia

ttttttttttttttttampensis (Lindl.) Small (Zettler et al. 1999), and Epidendrum nocturnum Jacquin (Zettler et al.

2007). The majority of these reports focus on the conservation-driven symbiotic propagation of

epiphytic orchids (Zettler et al. 2007). However, both Zettler et al. (1999) and Zettler et al.

(2007) contribute important information concerning mycobiont preference in epiphytic orchids, a

generally understudied topic. In both cases, seeds of epiphytic orchids were germinated using

mycobionts originating from different orchid taxa--mycobiont from Epidendrum magnoha~e var.

magnoliae in the case of Zettler et al. (1999) and mycobiont from .9'i' illbes' brevilabris in the

case of Zettler et al. (2007). As seen with the reports from Hadley (1970) and Warcup (1973),

mycobiont preference in epiphytic orchid taxa appears to be variable and species-dependant.

Continued research in this area of symbiotic orchid seed germination is necessary.

The orchid genera Platanthera and Habenaria have received considerable attention from

practitioners of symbiotic seed germination in recent years, especially by researchers in North

America. Zettler and McInnis (1992) reported on the symbiotic propagation of the rare

terrestrial orchid Platanthera integrilabia using mycobionts originating from P. ciliaris (L.)

Lindl., P. clavellata (Michaux) Luer, P. cristat (Michaux) Lindl., P. integrilabia, and

.lit ainhe s~ cernua. The highest percent seed germination reported was supported not by a

mycobiont originating from P. integrilabia, but from a mycobiont originating from P. ciliaris.

However, the highest percentage of soil established seedlings were supported by the mycobiont

originating from P. integrilabia. Further studies on the affects of light during the symbiotic

germination of P. integrilabia were reported by Zettler and McInnis (1994). Interestingly, the

highest percent symbiotic seed germination was supported when seeds were exposed to seven










days of 16/8 h L/D photoperiod before being subj ected to darkness for the remainder of the

study. Zettler and McInnis (1994) represent one of the first reports on the affects of photoperiod

on the symbiotic germination of an orchid. Zettler et al. (2001) and Zettler et al. (2005) both

reported on the development of symbiotic seed germination techniques for the North American

temperate terrestrial orchid Platanthera leucophaea (Nutt.) Lindl.. Furthermore, Zettler et al.

(2005) proposed an intermediate acclimatization step between in vitro and greenhouse conditions

for symbiotic seedlings. This intermediate step appears to not only help in the acclimatization of

seedlings, but also in the acclimatization of the mycobiont to a new growing environment.

Working with Habenaria radiata, an allied species to the genus Platanthera, Takahashi et al

(2000) reported on the species' symbiotic seed germination and the effects of seed age, culture

media, period, and mycobiont preference. More recently, Stewart and Zettler (2002) and Stewart

and Kane (2006b) reported on the symbiotic seed germination of the subtropical terrestrial orchid

Habenaria macroceratitis.

Another orchid genus that has received considerable attention from those interested in

symbiotic seed germination and conservation is gitainhes.,~\ Anderson (1991) first reported on

the symbiotic germination of the terrestrial orchid .Swit aider l \ magnicamporum Sheviak using the

mycobiont Epulorhiza repens (Bernard) Moore isolated from the same species. Furthermore,

Anderson (1991) also detailed the growth and development of S. magnicamporum in symbiotic

culture both in vitro and in the greenhouse. Zettler and McInnis (1993) reported on the

symbiotic seed germination of .9'ilmiubes,~ cernua and another Spiranthoideae orchid, Goodyera

pubescens (Will.) R. Brown. This study represents another example of the exploration of orchid-

mycobiont preference in the Orchidaceae, by using mycobionts isolated from S. cernua,

Platanthera integrilabia, and P. ciliaris. Zelmer and Currah (1997) investigated the symbiotic










germination and mycobiont diversity of S lacera Rafinesque, finding a mixture of strains of

Ceratorhiza and Epulorhiza in the roots of this species. In their study, only the mycobiont strain

Ceratorhiza goodyerae-repentis (Costantin & Dufour) Moore supported the in vitro symbiotic

germination ofS. lacera. Zettler and Hofer (1997) used the symbiotic germination of the

terrestrial orchid S. odoara to experimentally explore the effects of light on symbiotic

germination. They reported the highest final percent germination in 0/24 h L/D treatments,

followed by 8/16 h and 14/10 h L/D photoperiods. Stewart and Kane (2006b) described a similar

germination response in the symbiotic germination of Habenaria macroceratitis with 0/24 h,

16/8 h, and 24/0 h L/D photoperiods. Stewart et al. (2003) reported on the conservation-driven

symbiotic propagation and reintroduction of the endangered Florida terrestrial orchid S.

brevilabris. Again, this study highlighted the degree of mycobiont preference some orchid taxa

exhibit for their mycobionts-mycobionts originating from both the study species and the

Florida epiphytic species Epidendrum magnohiae var. magnoliae supported in vitro symbiotic

germination. Stewart et al. (2003) was the first describe the successful reintroduction of in vitro-

grown symbiotic seedlings of a North American terrestrial orchid. Finally, Stewart and Kane

(2007a) investigated the symbiotic germination and degree of mycobiont preference in the orchid

species pair S. brevilabris and S. floridana.d~~~~~ddddd~~~~ In this report, a high degree of mycobiont preference

was suggested for both S. brevilabris and S. floridanadd~~~~~ddddd~~~~ based on mycobiont isolation and in vitro

symbiotic seed germination studies, despite a high degree of genetic relatedness between these

two orchid taxa.

Seed of a number of other orchid taxa have been successfully germinated using symbiotic

techniques. Jorrgensen (1995) reported on the symbiotic culture of the hardy terrestrial orchid

Dactylorhiza majalis. Tan et al. (1998) discussed the symbiotic seed germination of the









commercially-important terrestrial orchid Spathoglottis plicata. An interesting modification

proposed in Tan et al. (1998) to the standard symbiotic technique is the encapsulation of orchid

seed and mycobiont within an alginate bead. This bead would then be planted directly into the

greenhouse or field, transferring both the seed and an appropriate mycobiont in one unit. The

symbiotic germination of another orchid of commercial interest, Bletilla striata (Thunberg)

Rchb., was reported by Johnson (1994). Finally, Yagame et al. (2007) reported the symbiotic

germination from seed to flowering plant of the achlorophyllous orchid Epipogium roseum (D.

Don) Lindl. Few achlorophyllous orchids have been successfully germinated using symbiotic

techniques, and none have been reported as flowering from symbiotic culture conditions.

Symbiotic seed germination techniques with orchids have a long and storied history. The

techniques originally developed by Bernard and Burgeff in their studies of the orchid-mycobiont

association remain applicable through to today. Of particular interest is the application of

modern symbiotic germination techniques in the propagation and conservation of orchid species.

This mode of propagation allows for orchids to be grown in conjunction with their mycobiont

associate, unlike in asymbiotic germination methods where the mycobiont is not physiologically

accounted for. For conservation and species recovery purposes, symbiotic seed germination

should represent the primary mode of plant propagation.

Orchid Pollination Biology

The evolutionary relationship between orchids and their pollinators has resulted in a great

diversity of flower morphologies and pollination mechanisms in the Orchidaceae (Tremblay

1992; Cozzolino and Widmer 2005). Pollinator specialization and unique pollination

mechanisms should, in theory, enhance the fitness of the Orchidaceae by reducing the costs

associated with reproduction. However, obligate pollinators, such as those orchids are likely to

rely on, are often scarce in the landscape (Herrera 1989) and possibly susceptible to population









decline due to environmental changes because of their general scarcity. The identification of

both pollinators and pollination mechanisms is an important part of the integrated conservation

of orchid species, and can often lead to the direct conservation and management of pollinators in

habitats supporting them.

Despite the volumes of literature concerning the pollination biology of orchids, few species

have been carefully studied, their pollinators identified, and their pollination mechanism

determined. Catling and Catling (1991) reported that only basic pollination information is

known for only 40% of all North American orchid species, and that detailed information is

available on only about 15% of species. It is doubtful that these percentages have improved in

the intervening years since this report.

Tremblay (1992) suggests a reduction in the number of pollinators per orchid species

beginning with the Cypripedioideae (6.3 pollinators/species) and moving to the Epidendroideae

(1.5 pollinators/species), with the Orchidoideae between the two (4.0 pollinators/species). From

an evolutionary standpoint, these data suggest a significant reduction in pollinators per species

from the most ancestral to the more recently derived. To best understand this large, ever-

evolving plant family, the study of the pollination biology of the Epidendroideae should be

highlighted. However, the maj ority of pollination biology research has focused on the more

ancestral subfamilies of the Cypripedioideae and the Orchidoideae (Catling and Catling 1991).

The specialization of pollinators and diversity of pollination mechanisms is quite evident

in the Orchidoideae. As a result, pollinators and pollination mechanisms have been used for a

variety of purposes, from clarifying the identity of taxonomically confusing species to

identifying pollination ecotypes within the distribution of a particular species. Sheviak and

Bowles (1986) used differences in pollination mechanism and pollinia placement on pollinators









to taxonomically distinguish Platanthera leucophaea and P. praeclara Sheviak & Bowles. By

determining these pollination differences and relating them to evolutionary forces in the

speciation of these species, Sheviak and Bowles (1986) again made the link between orchid

pollination and orchid evolution. Smith and Snow (1976) identified the different pollinators of

P. cilaris and P. blephariglottis (Willd.) Lindl., two closely-related species. In their studies, the

colorful P. cilaris was pollinated by spicebush swallowtail butterflies (Papilio troihts L.),

whereas the white-colored P. blephariglottis was found to be pollinated by a variety of moth

species. Smith and Snow (1976) surmised that color served as the main attractant for pollinators

of P. cilaris and scent served as the attractant for P. blephariglottis pollinators. Despite their

taxonomic relatedness and often sympatric growth habits, these two Platanthera species had

evolved two distinct systems to attract different pollinators. In a reassessment of the pollination

biology ofP. blephariglottis, Cole and Firmage (1984) confirmed that this species is pollinated

by moths. A related Platanthera species, P. strict Lindl., was reported as being pollinated by

both moths and bees (Patt et al., 1989). This demonstrates a degree of plasticity that can be seen

in some orchid pollination systems, although this plasticity is not typical.

An interesting application of the study of pollination systems in the Orchidoideae is the

report of pollination ecotypes based on altitude in the orchid P. ciliaris (Robertson and Wyatt

1990a, 1990b). Plants of this species growing in mountainous regions were found to possess a

significantly shorter nectar spur when compared to plants growing in coastal plain areas. This

environmental adaptation was further shown to target two different pollinators--Papilio troihts

in the mountains and P. palamnedes in the coastal plain regions. This pollination mechanism

adaptation is an example of how closely tied orchids and their pollinators are.









In an unique case of auto-pollination versus pollinator-mediated pollination, Johnson et al.

(1994) and Johnson (1994) reported on differential pollination systems in the South African

terrestrial orchid genus Disa. Auto-pollination was reported for three Disa species--D. vaginata

Hary. ex Lindl., D. glan2dulosa Burch. ex Lindl., and D. rosea Lindl. (Johnson et al. 1994),

whereas an anthophorid bee was reported as the pollinator of D. versicolor Rchb. (Johnson

1994). A change in flower color was also reported during pollination studies on D. versicolor,

and this color change was suggested as an orientation cue for the bee pollinator to locate newly

opened flowers versus old or previously-pollinated flowers. Johnson et al. (1994) surmise that

auto-pollination in Disa likely evolved in four independent lineages and that, and that habitat and

lack of pollinators were likely driving forces behind this evolution.

The higher average number of pollinators per species in the Cypripedioideae, relative to

the Orchioideae, appears to be accompanied by lower rates of auto-pollination or agamospermy

in the Cypripedioideae (Catling and Catling 1991). Recent reports on the pollination biology of

Cypripedium species indicate that bees and bee allies are the primary pollinators in this genus

(Banziger et al. 2005; Li et al. 2006).

Orchids are known to possess a number of other floral characteristics that promote

pollination. Trapnell and Hamrick (2006) reported on the importance of floral display and

effective population sizes in the epiphytic orchid Laelia rubescens Lindl. Because effective

population sizes for this species were found to be small, effective floral display was found to be a

maj or contributing factor to the pollination success of L. rubescens.

Sexual deception as a means to insure pollination occurs only among the Orchidaceae

(Wiens 1978; Nilsson 1992). Alcock (2000) reported on the use of sexual deception as a

pollination mechanism in the Australian terrestrial orchid Spiculaea ciliata Lindl. This species is










pollinated by male Thynnoturneria wasps, and was shown to attract males in as little as two

minutes of flower presentation. However, Alcock (2000) demonstrated that fewer than half of

the male wasps attempting pseudocopulation with flowers of S. ciliata came in contact with

pollinia and affected pollination.

In the case of the species pair Cleistes divaracata (L.) Ames and C. bifaria, pollen

abundance, not floral display, played an important role in the success of pollination (Gregg

1991). Bumblebees (Bombus), the pollinators of both Cleistes species, were repeatedly observed

collecting Cleistes pollen as a food source while affecting pollination of individual plants. This

report represents a unique use of food-attraction in the Orchidaceae as a means to insure

pollination.

Nectar production is another common pollinator attractant in the Orchidaceae. Orchid

species that produce nectar as a reward or attractant for pollinators typically target members of

the Lepidoptera. Zettler et al. (1996) reported that the nectar producing terrestrial orchid

Platanthera integrilabia used nectar as a probable attractant or reward for its observed

pollinators-Epa~rgyreus clarus Cramer, Papilio glaucus L., and P. troilus. Luyt and Johnson

(2001) reported the similar use of nectar as a reward or pollinator attractant in the African

epiphytic orchid Mystacidium venosum Hary. ex Rolfe. Jacquemyn et al. (2005) attempted to use

orchid nectar production as an assessment tool predicting species rarity and extinction

probabilities. They concluded that nectar production does not serve as a highly reliable measure

of extinction probability, and that habitat loss and fragmentation are better measures.

The specialization among pollinators, pollination mechanisms, and species in the

Orchidaceae is directly correlated to the diversity seen in the family. In order to preserve this

high level of species diversity, careful study of orchid pollination biology is necessary to insure









the long-term viability and fitness of species and populations. As suggested by Catling and

Catling (1991), the continued investigation of the pollinators and pollination mechanisms of

orchids will greatly aid in the integrated conservation of this family.

Molecular Genetics and Genetic Diversity in Orchids

The use of molecular-based techniques in the study of the Orchidaceae has traditionally

focused on two main areas: 1) phylogenetics and 2) conservation genetics/genetic diversity

studies. Both of these molecular approaches to the study of the Orchidaceae have revealed new

insight into the evolution, relations, and conservation of orchids worldwide. However, there has

been a traditional disconnect between those interested in the molecular phylogenetics of orchids

and those interested in the conservation of orchids. Recently, a number of researchers have

begun to couple the study of phylogenetics and conservation biology to the benefit of worldwide

orchid conservation (Dueck and Cameron 2007; Pillon and Chase 2007; Pillon et al. 2006).

The widest application of molecular biology in the Orchidaceae has come in the form of

molecular phylogenetics and molecular evolution studies. Traditionally, taxonomy in the

Orchidaceae focused on the study of varying morphology, habitat preference, and chromosome

numbers (Dressler 1981, 1992; Sheviak 1982). However, the movement toward molecular-based

techniques in the study of orchid taxonomy has elucidated a number of interesting cryptic

relationships within particular genera and between species. Hedren et al. (2001) and Hedren et

al. (2007) utilized both amplified fragment length polymorphism (AFLP) and plastid DNA

techniques to investigate the origins of and relations between Dactylorhiza species. Traditional

taxonomists struggled to understand relationships within this genus, especially due to the

existence of several geographically- and habitat-specific forms, varieties, and ecotypes. The use

of molecular phylogenetics in this genus has allowed a more precise understanding of within-

genus species relations; therefore, allowing identification of rare or endemic species or forms and









better conservation planning for this genus throughout Europe. A more traditional use of

molecular phylogenetics is in the determination of species. Examples of this use include the

revised delimitation of Orchidinae and selected Habenariinae (Bateman et al. 2003) and the

molecular delimitation of Cranichideae and Spiranthinae (Salazar et al. 2003).

Molecular phylogenetic research has also been used to investigate the evolution and

differentiation of a number of orchid species. In a molecular-based study of the North American

terrestrial genus Cleistes, no support for the traditional separation of C. divaracata and C. bifaria

was found (Smith et al., 2004), although these two species are morphologically distinct.

Similarly, Carlsward et al. (2003) reported a revised broad definition of the orchid genus

Dendrophylax to include the genera Harrisella, Polyradicion, and Camnpylocentrum; again,

despite distinct morphological characters to the contrary. Similarly, Higgins et al. (2003)

reported on a combined molecular phylogeny of the epiphytic orchid genus Encyclia, with the

redelimitation of a number of species within this genus. In a novel use of molecular-based

taxonomy, Carlsward et al. (2006) investigated the evolution of leaflessness in the orchid tribe

Vandeae. They determined the gross morphological changes in this tribe, driven by genetic

changes, resulted in the reduction of leaves, typically to a simple scale, and the evolution of gas

exchange complexes in photosynthetically-capable roots lead to the development of leafless

morphologies. Finally, Goldman et al. (2004) used both molecular and cytological methods to

more clearly define speciation in the North American terrestrial orchid genus Calopogon. This

study reinforced traditional species concepts in this genus without redefining new species.

The second-most common application of molecular-based technologies to the Orchidaceae

is in the study of conservation genetics and population genetic diversity. Gaining knowledge of

genetic diversity in plants is a key step in understanding the long-term conservation needs of









individual species and populations. Genetic diversity plays an important role in the persistence

of individuals in a changing environment and the ability of those individuals to adapt (Frankel

and Soule 1981; Lande and Barrowclough 1987). There are a number of ways to measure

genetic diversity in orchid populations, including morphological approaches, protein markers,

and DNA markers (Qamaruz-Zaman et al. 1998b). This brief review will focus on only DNA

markers, with a particular emphasis on the AFLP technology.

Protein-based marker systems remain quite prevalent in the study of orchid conservation

genetics despite advances in DNA-based systems (Sun 1997; Sun and Wong 2001; Wallace and

Case 2000; Sharma et al. 2000; Sharma et al. 2003; Sharma et al. 2001; Case et al. 1998). This is

in spite of their functional drawbacks, such as difficulty in keeping sampled materials fresh or

appropriately frozen from the field to the laboratory, the difficulty in detecting new alleles, and

issues surrounding polyploid speices (Qamaruz-Zaman et al. 1998b). Nonetheless, protein-based

marker systems continue to be used to provide conservation genetics data to those interested in

orchid conservation.

A number of DNA-based markers have been used to investigate the conservation genetics

and population genetic diversity of orchids. Bush et al. (1999) used random amplified

polymorphic DNA (RAPD) techniques to investigate the genetic variation in the epiphytic orchid

Epidendrunt nagnoliae (syn. = E. conopseunt), as well as the co-occurring epiphytic fern

Pleopeltis polypodioides (L.) E.G. Andrews & Windham. Studying the conservation genetics of

Splitainhesl~ diluvialis Sheviak, Szalanski et al. (2001) used random fragment length

polymorphism (RFLP) to reveal no genetic variation within or among populations of this species.

Gustafsson and Sjoigren-Gulve (2002) used an improved microsatellite technique to compare

genetic diversity between the rare Gymnandenia odoratissinta (L.) L.C.M. Richard and the more









common G. conopsea (L.) R. Brown, reporting a distinction between the mainland and island

populations of G. odoratissima. Wallace (2002a, 2002b) utilized RAPD methods to examine the

effects of habitat fragmentation on genetic variation in the Midwestern terrestrial orchid

Platanthera leucophaea. Smith et al. (2002) used intersimple sequence repeat (ISSR) to

examine genetic variation in Tipularia discolor (Prush) Nutt., reporting gene flow among four

distinct populations of the species despite a number of field-based reports of clonal growth of

populations. Cozzolino et al. (2003) reported both severe habitat fragmentation and genetic

bottlenecks in the rare orchid Anacamptis palustris using minisatellite methods. Finally,

Tsukaya (2005) reported on the genetic variation in populations of Spit il ellr,\ sinensis (Pers.)

Ames, as well as systematic information on this cryptic species using plastid sequencing data.

The variety of molecular-based tools at the disposal of those interested in the conservation

genetics of orchids is staggering. However, one method has been shown to be the most powerful

in the investigation of population genetic diversity and conservation genetics--amplified

fragment length polymorphism (AFLP).

The AFLP technique, as developed by Vos et al. (1995), is a powerful tool to assess

within- and between-population genetic diversity, and has been used widely in the study of plant

conservation genetics (Scariot et al. 2007; Arcade et al. 2000; Ranamukhaarachchi et al. 2000;

Roldan-Ruiz et al. 2000; DeRiek et al. 1999). The value of the AFLP technique over other

currently-available techniques lies in its overall simplicity. Five main advantages have been

identified: 1) no development time, 2) large numbers of loci and samples can be quickly assayed

due to automation, 3) provides 10-100 times more markers per reaction than other techniques, 4)

technique is highly reproducible, and 5) combines the reliability of RFLP with the simplicity of









PCR (Qamaruz-Zaman et al. 1998a). These factors make the use of AFLP in the study of orchid

conservation genetics highly valuable.

Because of these many advantages, the AFLP technique is increasingly being applied to

orchid conservation genetics. Qamaruz-Zaman et al. (1998a) used AFLP to investigate the

conservation genetics of Orchis simia Lam., reporting the direct use of the resulting data in the

nation-wide management of the remaining populations of this species in the United Kingdom. In

an attempt to develop parental identification methods for Vanda breeding in Singapore, Chen et

al. (1999) used the AFLP technique to develop genetic fingerprints of properly identified species

and hybrids that could be used as standards against which to measure unknown species and/or

hybrid combination. Forrest et al. (2004) identified two distinct meta-populations of the

terrestrial orchid Splitanthesl~ romanzoffiana in Europe based on AFLP data and breeding system

determinations. Pillon et al. (2007) reported on the genetic diversity and ecological

differentiation in the widespread, but rare, terrestrial orchid Liparis loeselii (L.) Richard. This

species has a disjunct distribution in both Europe and the United States this is reflected in a high

degree of genetic separation between the two continents. Finally, Hedren et al. (2001) and

Hedren et al. (2007) reported on the use of AFLP methods in elucidating the polyploidy

evolution of Dactylorhiza species, varieties, and ecotypes. The power of the AFLP technique in

studying the conservation genetics of orchids is exceptional, as the aforementioned examples

highlight. AFLP should be considered a crucial technique in the integrated conservation of

orchids worldwide (Stewart and Kane 2007b).










Table 1-1. Anamorphic and corresponding teleomorph species of common orchid mycobionts in
the Rhizoctonia complex, after Zettler et al. (2003).
Anamorph Teleomorph
Ceratorhiza pernacatena unknown
Ceratorhiza goodyerae-repentis Ceratoba~sidium cornigerum
unknown Ceratoba~sidium angustisporum
unknown Ceratoba~sidium globisporum
unknown Ceratoba~sidium sphaerosporum
unknown Ceratoba~sidium stevensii
unknown Ceratoba~sidium papillatum
Epulorhiza albertaensis unknown
Epulorhiza anaticula unknown
Epulorhiza calendulina unknown
Epulorhiza inquilina unknown
unknown Tula~snella allantospora~~11~~~11~~~11
unknown Tula~snella asymmetrica
Epulorhiza repens Tula~snella deliquescens
unknown Tula~snella cruciate
unknown Tula~snella irregularis
unknown Tula~snella violacea
M~oniliopsis anomala unknown
M~oniliopsis solanzi Thanatephorus cucumeris
M~oniliopsis zeae Waitea circinata
unknown Ma~natephorus obscurum
unknown Ma~natephorus orchidicola
unknown Ma~natephorus pennatus
unknown Ma~natephorus sterigmaticus






































Figure 1-1. Diagrammatic representation of the steps and connections of integrated orchid
conservation.





























Figure 1-2. Habenaria macroceratitis. A) Habenaria macroceratitis inflorescence in habitat.
B) Greenhouse grown H. macroceratitis plant prior to anthesis. C) Habenaria
macroceratitis flower. Scale bars = 1 cm.









































Figure 1-3. .$>iianikrl~ brevilabris and S. florida~na. A) .$>i' iauthea brevilabris inflorescence in
habitat. B) .\?ilmathes florid anad~~~ddd inflorescence in habitat. Scale bars = 1 cm.




























































Figure 1-4. Deep South race of .git ainites~ cernua. A) .git ainites~ cernua inflorescence, scale
bar = 1 cm. B) .git ainites~ cernua lip detail, scale bar = 1 mm. C) .9'i' iaidict~ cernua
flower profile, scale bar = 1 cm.





















FigureI 1-.Eapeo yoin eoosi h oria ise fSiaie rvlbi.A
Grup of peotn (400x; scl a 0p) )Ioae eoo 10x cl a
= 1 pm)



























Figure 1-6. Cellulase assay. A) Positive reaction. B) Weak reaction. C) No reaction.























Figure 1-7. Polyphenol oxidase assay. A) Positive reaction. B) No reaction.









CHAPTER 2
POLLINATION BIOLOGY AND GENETIC DIVERSITY OF Habenaria macroceratitis

Introduction

A great deal of attention has been given to the study of orchid plant growth and

development in the wild (for reviews see Dressler 1981, 1993; Hutchings 1987a, 1987b),

although few studies have followed individual plants or populations for more than one or two

years (Willems 1982; Wells 1967; Light and MacConaill 2007; Jacquemyn et al. 2007; Pfeifer et

al. 2006). This lack of in-depth or long-term life history trait studies has lead to a general lack of

biological understanding of many orchid genera. Long-term study of life history traits would

facilitate a better understanding of the vegetative and reproductive dynamics of individual orchid

genera and species, as well as populations of orchids. Only a few long-term studies of orchid life

history traits exist (Wells 1967; Tamm 1972; Hutchings 1987a, 1987b; Calvo 1990), and the

value of these reports are only now being fully recognized (M.H. S. Light, personal

communication).

Pollination biology represents an essential component of the study of plant life history

traits. The understanding of a species' mode of reproduction-asexual, sexual, pollinator-

dependant--can be a critical aspect in its conservation (Whigham and McWethy 1980; Sipes and

Tepedino 1995). Understanding pollination biology is particularly important in the Orchidaceae

since most orchid species have developed highly specific plant-pollinator relationships that often

limit the number of possible pollinators per orchid species to less than two insects (Tremblay

1992). Compounding the problem of specific plant-pollinator relationships in orchid pollination

biology, is the evolutionary development of specific pollination mechanisms in many orchid

species, often requiring outcrossing between flowers from different inflorescences to achieve

seed set (Catling and Catling 1991; Bowles et al. 2002). By combining a basic ecological









understanding of orchid species and populations with pollinator identification and pollination

mechanism determination, a more complete ecological understanding of orchids in natural

settings can be achieved (Catling 1987; Wong and Sun 1999).

A final component to the understanding of the ecology of orchids is an evaluation of the

genetic diversity within and between populations. The loss of genetic diversity can result in the

limiting of adaptive and evolutionary potential and be a critical factor in the long-term

persistence of plants in a changing environment (Frankel and Soule 1981; Lande and

Barrowclough 1987; Qamaruz-Zaman et al. 1998b). Developing an understanding of within- and

between-population genetic diversity is key in the conservation of plant species (Lande 1988),

particularly orchids (Stewart and Kane 2007b).

Investigating genetic diversity requires the use of molecular markers. Amplified fragment

length polymorphisms (AFLPs) represent a multilocus marker system (Vos et al. 1995) that has

been employed to investigate genetic diversity in a number of orchids (Pillon et al. 2007; Hedren

et al. 2001; Chen et al. 1999; Qamaruz-Zaman et al. 1998a) and non-orchids (Juan et al. 2004;

Arcade et al. 2000; Ranamukhaarachchi et al. 2000; Travis et al. 1996). The AFLP method is an

attractive genetic diversity analysis method in plant systems because of its reproducibility, its

requirement for small amounts of genomic DNA, and its ability to resolve multiple polymorphic

bands per AFLP reaction (Mueller and Wolfenbarger 1999; Ridout and Donini 1999; Chen et al.

1999; Lin et al. 1996).

The terrestrial orchid Habenaria macroceratitis was chosen as the species in these studies

because no information exists on the ecology, reproductive biology, or genetic diversity of this

species (Figure 1-2). Moreover, no reports exist concerning the ecology, reproductive biology,

or genetic diversity of any Habenaria species. However, a number of reports exist on the









ecology (Sing-Chi 1983; Stoutamire 1996; Maad and Alexandersson 2004; Sheviak and Bowles

1986), reproductive biology (Wallace 2003; Thien 1969; Hapeman 1997; Cole and Firmage

1984; Robertson and Wyatt 1990a, 1990b; Smith and Snow 1976; Little et al. 2005; Bowles et al.

2002; Zettler et al. 1996; Patt et al. 1989), and genetic diversity (Wallace 2006; Gustafsson 2000;

Gustafsson and Sjoigren-Gulve 2002) of the closely-allied orchid genus Platanthera. In the

present study, data are presented concerning the general plant demography, above-ground and

below-ground vegetative development, and ecological habitat profile ofH. macroceratitis in

Florida. Data are also presented concerning the pollinator and reproductive biology of this

species. Finally, the within- and between-population genetic diversity of H macroceratitis is

also described. These studies represent a first step leading to an integrated conservation and

species-level recovery of H macroceratitis in Florida (Figure 1-1).

Materials and Methods

Study Sites

Four sites were chosen for these studies: Socash (Hernando County), Old Dade Highway

(Hernando County), Battle Slough (Sumter County), and Cross Florida Greenway (Marion

County; Figure 2-1). The Socash and Old Dade Highway sites were located within 8.8 km and

0.8 km of Brooksville, Florida, respectively, at elevations above sea level between 50-55 m. The

Battle Slough site was located within 1 km of Wahoo, Florida at an elevation above sea level of

40-45 m. The Cross Florida Greenway site was located within 15.7 km of Ocala, Florida at an

elevation above sea level of 20 m. Data on associated plant species were recorded at each site

yearly from 2002-2005.

Plant Demography

In 2002, one monitoring site per study site was established. From 2002-2005 these four

monitoring sites were visited every three weeks during the active growth cycle of H









macroceratitis (1 August-3 1 December). Data on number of vegetative and flowering plants,

plant height (cm), nectar spur length (cm), number of leaves per plant, and number of flowers per

plant was taken during each monitoring site visit. These data were pooled within site per

category per data collection week and averaged per data collection year. These yearly data were

then averaged over all data collection years and analyzed for year-to-year variance using general

linear model procedures and Waller-Duncan mean separation at a = 0.05 (SAS 1999). Plant

development in the field was also documented.

Pollinator Observations

Preliminary pollinator observations were conducted at the following study sites during

peak flowering: Socash site, 26-27 August 2003 from 1100 to 0000 hours; Cross Florida

Greenway site, 28-29 August 2003 from 1800 to 0300 hours; and Old Dade Highway, 1-2

September 2003 from 1800 to 0300 hours.

Non-destructive pollinator observations were conducted at the Socash site from 28-29

August 2004. These observations were repeated 27-28 August 2005. The entire Socash

population of H. macroceratitis was selected for observation activities due to its compact size

and high flowering plant density. Observation methods followed those of Zettler et al. (1996) for

pollinator observations of Platanthera integrilabia (Correll) Luer, an allied taxon to H.

macroceratitis. A circular path around the entire Socash population was established that allowed

easy observation of 183 individual inflorescences. Observations occurred over a 24-hour period

beginning 28 August 2004 at 1100 hours and ending 29 August 2004 at 1100 hours, without

interruption. All data were recorded in fair weather, and no insect repellant or insect attractants

were used within the study area. Observations were recorded during the last 20 minutes of each

hour by continuously walking the preestablished path in a counterclockwise direction around the

flowering plants. Nocturnal observations were aided using a small, head-mounted flashlight









fitted with a red fi1ter, and dark clothing was worn to minimize possible insect disturbance due to

moonlight reflectance. Pollinator observations were recorded as those insects carrying at least

one pollinium after a flower visit, while visitors were recorded as those insects lacking pollinia.

Insects were identified using Carter (2002). Relative humidity and temperature were recorded at

the Socash site during pollinator observations using two HOBO H18 data loggers (Onset

Computer Corporation, Bourne, Massachusetts). One data logger was placed at ground level,

while the second was placed 30 cm above ground level. This height was chosen to parallel the

average inflorescence height ofH. macroceratitis at this site.

Nectar volume and sugar concentration sampling was undertaken 27-28 August 2004 at the

Socash site for a 12 hour period beginning at 1800 hours and ending at 0600 hours without

interruption. Sampling followed the procedure of Zettler et al. (1996). Samples were taken by

microcapillary pipette (Drummond Scientifie Company, Broowall, Pennsylvania). Preliminary

investigation found H. macroceratitis nectar to be restricted to the bottom 1/6th of the nectar

spur. Nectar was withdrawn from the base of each spur and its volume recorded to the nearest

0.1 Cl. Sugar concentration was analyzed using a pocket refractometer (0-62% range; Atago

USA Inc., Bellevue, Washington), as outlined in Zettler et al. (1996). Nectar volume and sugar

concentration samples were taken from three randomly selected flowers on three separate

randomly selected inflorescences during the first 15 minutes of each hour. A total of 39 flowers

were sampled during the sampling period.

Pollination Mechanism, Seed Viability, and Asymbiotic Seed Germination

A pollination mechanism study was designed to investigate the breeding system of H

macroceratitis following the procedures of Wong and Sun (1999), modified by the inclusion of a

seventh pollination condition-self-pollination (Table 2-1). The mechanism experiment was

conducted at the Socash site during both 2004 and 2005 flowering periods. Twelve plants, two









per experimental pollination condition, with pre-anthesis inflorescences were bagged with a fine

plastic mesh (1 mm2 meSh size) stretched over a 1 m tall wire frame. The mesh covered wire

frame allowed the tall H. macroceratitis inflorescences to develop normally while excluding

pollination events from occurring prior to the initiation of pollination mechanism studies.

Experimental pollination treatments were applied to two inflorescences, each with five

flowers per inflorescence (Table 2-1). A total of 60 flowers were used in the pollination

mechanism determination study. Emasculation was accomplished by the removal of pollinia

from individual flowers without allowing pollinia contact with the stigmatic surfaces. Hand

pollinations were conducted by placing pollinia onto stigmas using a pair of micro-forceps.

Fresh pollinia from the Old Dade Highway site were used in testing all outcrossing experimental

pollination conditions. The mesh-covered wire frames were replaced after each pollination

condition was applied to the plants, and remained covering the plants until the conclusion of the

study .

Resulting capsules from the pollination mechanism study were allowed to mature on the

plants and collected on 18 October 2004 and 24 October 2005. Prior to collection, capsule size

(width at mid-point x length, cm) was recorded. Capsules were removed from the parent plant,

placed in a paper envelope, placed in a plastic bag over silica gel desiccant, stored in darkness,

and transported to the laboratory (< 6 hours). Seed capsules were then stored over silica gel

desiccant at 250 C for two weeks, after which time capsules dehisced and mature seeds were

collected. Seeds from different plants within the same pollination treatments were pooled and

stored in glass vials over silica gel desiccant at -100 C until use in tetrazolium viability staining

and asymbiotic seed germination studies (< 1 week).









Tetrazolium (2,3,5-triphenyltetrazolium chloride; Sigma-Aldrich Chemical Company, St.

Louis, Missouri) viability staining of the resulting mature seed from each experimental

pollination mechanism condition followed the methods of Lakon (1949) modified by Ramsay

and Dixon (2003). Between 100-120 seeds from each experimental pollination mechanism

condition were placed in 1.5 mL microcentrifuge tubes (USA Scientific Inc., Ocala, Florida) and

pretreated with a 5% calcium hypochlorite (w/v) solution for 1.5 hours. Pretreated seeds were

then rinsed in sterile deionized water (DI) once and soaked in sterile DI water for 24 hours.

Seeds were then soaked in 1% tetrazolium solution (pH = 7.0) for 24 hours at 300 C in darkness

(0/24 h L/D). After exposure to the tetrazolium solution, seeds were rinsed for 5 minutes three

times in sterile DI water. Seeds were then suspended in sterile DI water, transferred to a Petri

plate using a Pasture pipette, and scored as viable or non-viable with the assistance of a

stereomicroscope. Viable embryos were scored as those embryos showing any degree of red or

pink staining, whereas non-viable embryos were scored as those showing no degree of staining.

The percentage of viable seeds was calculated by dividing the number of stained seeds by the

number of total seeds in the sample.

Asymbiotic seed germination was undertaken to test the vigor of seeds resulting from each

experimental pollination mechanism condition. Seed germination methods followed those of

Stewart and Kane (2006a) for H. macroceratitis with minor modifications: 1) seeds were only

cultured on Malmgren Modified Terrestrial Orchid Medium (PhytoTechnology Laboratories

LCC, Shawnee Mission, Kansas) supplemented with 20 g 10 sucrose, 2) between 60-100 seeds

were placed on each Petri plate, 3) plates were incubated in darkness (0/24 h L/D) for 8 weeks

and scored only once, and 4) 15 replicate plates per pollination condition (Table 2-1) were

prepared for this experiment. Seeds were scored on a scale of 0-5 (Stewart and Kane 2006a).









Germination percentages were calculated by dividing the number of seeds in each germination

and development stage by the number of seeds in each sample. Data was analyzed using general

linear model procedures and Waller-Duncan mean separation at a = 0.05 (SAS 1999).

Germination percentages were arcsine transformed to minimize variation and normalize

variation.

Sampling, DNA Extraction and Amplified Fragment Length Polymorphism (AFLP)

Fresh, green leaves of H. macroceratitis were collected from both the Socash and Old

Dade Highway sites in 2003 and placed in 50 mL conical bottom plastic tubes (BD Biosciences,

San Jose, California) containing silica gel desiccant (Chase and Hills 1991; W.M. Whitten

personal communication). Leaf samples were removed from each plant using scissors, which

were washed with 95% ethanol and allowed to air dry between each sample to minimize sample

cross contamination (M.W. Whitten personal communication). Twenty-two samples from the

Socash and 21 samples from the Old Dade Highway sites were collected. Samples were stored

at room temperature (ca. 250 C) until used in DNA extraction protocols.

Genomic DNA was extracted using the DNeasy" Plant Mini Kit (Qiagen, Valencia,

California). Manufacturer' s instructions were followed with the following modification: eluates

were not pooled and the second elution was retained only as a precaution. The DNA was

quantified using an Agilent Technologies NanoDrop (Wilmington, Delaware)

spectrophotometer.

As reported in Herden et al. (2001), the Applied Biosystems (Foster City, California)

AFLP" Plant Mapping protocol was followed in order to take advantage of automated

sequencing and computer-based analysis of fragment data. This kit-based system consists of two

modules, a ligation and preselective amplification module and a selective amplification module.

Manufacturer' s instructions were followed for each step in each module (ABI 2005). Sample









DNA was restricted with EcoRI and Msel endonucleases and ligated to suitable double-stranded

adapters per the manufacturer' s recommendations. Following this, a two step amplification

process followed: 1) preselective amplification using a 1 base pair (bp) extension and 2) selective

amplification using a 3 bp extension. Four primer combinations were chosen: -ACT/-CAG, -

AGC/-CAG, -ACT/-CTA, and -AGC/-CTA, and polymerase chair reaction (PCR) conducted.

Thermal cycle parameters for both the preselective amplification and the selective amplification

followed those suggested by the manufacturer (Table 2-2). After amplification, samples were

then prepared for fragment reading by combining 9.9 CIL formamide, 0.1 CIL Liz-600 size

standard (Applied Biosystems), and 1.5 CIL PCR product. Samples were sequenced using a

3730xl Automated DNA Sequencer (Applied Biosystems).

A matrix containing all AFLP fragment data ranging from 50 to 500 bp was compiled.

Fragment data were analyzed by the computer software GeneMarker version 1.6 (SoftGenetics,

State College, Pennsylvania), using the preselected AFLP settings. Fragments with a low signal

(< 2% full detection level) were excluded. Recognized bands were scored as present (1) or

absent (0).

AFLP data were analyzed using computer-aided processes. Genetic diversity within and

between populations of H. macroceratitis were estimated using the Nei and Shannon diversity

indices (based on allele frequencies), as calculated with POPGENE 1.31 (Yeh et al. 1999).

Within and between population genetic structure (i.e., genetic differentiation; FST) WAS estimated

using a combination of results generated with POPGENE 1.3 1 and equation calculations with

1000 permutations. Dendrograms of genotypic correlations were constructed used GeneMarker

version 1.6 cluster analysis tool.












Study Sites

Both the Old Dade Highway and Battle Slough study sites for H. macroceratitis were

classified as mesic hammocks (Chafin 1990). These sites have a dense canopy dominated by

live oak (Quercus virginiana Mill), sabal palm (Sabal palmetto (Walt.) Lodd.), southern

magnolia (Magnolia grandiflora L.), and very few slash pine (Pinus elliottii Engelm.). The

understory of the Old Dade Highway site was dominated by the exotic invasive plant air potato

(Dioscorea bulbifera L.), while the understory of the Battle Slough site was dominated by

partridge berry (M~itchella repens L.). Both sites had an open mid-story. The Socash site was

classified as a slope forest/mesic hammock (Chafin 1990), with an open canopy dominated by

sweet gum (Liquidambar~~~dddd~~~~ddd styraciflua L.), sabal palm, southern magnolia, and very few slash pine.

Live oak was a minor component of this site. This site slopes moderately to steeply (max. 200)

westward toward a forested wetland. Erosion caused by moving water heavily impacts this site.

The Cross Florida Greenway site was classified as a pine dominated slope forest (Chafin 1990)

surrounded by higher elevation sandhills. This site represents a portion of the manmade,

unfinished cross Florida barge canal. Slash pine and a few young oaks (Quercus chapmanii

Sarg., Q. laevis Walt.) dominate the canopy of this site. The understory of this site was

dominated by numerous grasses and herbs.

Plant Demography

From 2002-2005 the average number of vegetative and flowering plants at each H.

macroceratitis study site varied among sites: Socash, 250 145.7 plants per year (33% flowering

per year), Old Dade Highway, 61 18.6 plants per year (31% flowering per year), Battle Slough,

12 +4.6 plants per year (23% flowering per year), and Cross Florida Greenway, 16 12.5 plants

per year (25% flowering per year). No significant variation in plant height or spur length across


Results









all observation years was found among sites ofH. nacroceratitis in Florida (Figure 2-2). A

significantly lower number of leaves were found only at the Old Dade Highway site across all

observation years (Figure 2-3). All other sites showed no significant variation in leaf number

(Figure 2-3). Flower number was not significantly different across all observation years for all

sites of H. nacroceratitis in Florida (Figure 2-3). The average height of plants ranged between

35 cm (Socash) and 23.2 cm (Old Dade Highway). Average length of the nectar spur per flower

per plant ranged between 15.2 cm (Socash) and 13.9 cm (Socash). Leaves per plant averaged

between 7 (Socash) and 5 (Battle Slough). The average number of flowers per plant ranged

between 9 (Socash) and 4 (Cross Florida Greenway).

In the field, plants regularly produce tubers with a single growth point and remain dormant

during winter months (November-June; Figure 2-4). In the late spring (June), plants initiate

shoot growth from dormant tubers and produce leaves directly from these shoots (Figure 2-4).

Flowering typically begins in mid-summer (August), although plants may remain vegetative

during these months (Figure 2-4).

Pollinator Observations

Only one visitor to H. nacroceratitis flowers was noted during the entire 24 hour

pollinator observation study on 28-29 August 2004. The giant sphinx moth (Cocytius antaeus

Drury; Sphingidae) was noted probing individual flowers at 0000 hours on 29 August 2004, and

subsequently captured for identification (Figure 2-5). This same sphinx moth species was noted

visiting flowers ofH. nacroceratitis on a previous pollinator observation study conducted at the

Old Dade Highway site on 1-2 September 2003 and a subsequent pollinatior study at the Socash

site on 27-28 August 2005, although the moth was not captured in either case.

The flowers ofH. nacroceratitis at the Socash site were found to have a sweet, lemon

night scent starting at approximately 1900 hours and continuing until approximately 0600 hours.









Site temperature ranged between 20.80C-32.80C (ave. = 28.50C 4.60C; Figure 2-6 a). Site

relative humidity ranged between 59%-96% (ave. = 80.4% 14.3%; Figure 2-6 b). Habenaria

macroceratitis nectar was found to contain an average of 17.5% soluble sugars with an average

volume of 4.25 Cl.

Pollination Mechanism, Seed Viability, and Asymbiotic Seed Germination

Habenaria macroceratitis reproduction appears to rely on the movement of pollen between

flowers on the same or different inflorescences. In the pollination mechanism study, seed

capsules were set in only four of the seven (Table 2-1) pollination conditions--open pollination

(control), induced autogamy, artificial geitonogamy, and artificial xenogamy. Capsules were not

set in the spontaneous autogamy, agamospermy, or self-pollination conditions. The largest

capsules were produced in the artificial geitonogamy pollination condition (ave. 5.65 x 28.15

cm), followed by capsules in the artificial xenogamy condition (ave. 5.12 x 27.42 cm), the open

pollination condition (ave. 4.23 x 23.28 cm), and the induced autogamy pollination condition

(ave. 2.58 x 23.66 cm). Tetrazolium staining of samples of the mature seed from each capsule-

producing pollination condition revealed significant differences in embryo viability based on

pollination condition. Seeds from the open pollination (control) condition yielded 91.0% viable

embryos, while seeds from the artificial geitonogamy condition yielded 86.3% viable embryos,

seeds from the induced autogamy condition yielded 76.8% viable embryos, and seeds from the

artificial xenogamy yielded 50.7% viable embryos.

Asymbiotic seed germination percentages paralleled tetrazolium embryo viabilities, with

seeds originating from the open pollination (control) condition having both the highest overall

percent germination (54.2%) and highest percent protocorm development to an advanced stage

(Stage 3; 3.2%; Figure 2-7). Seeds originating from the artificial geitonogamy condition had a

maximum germination of 19.0% (Stage 1) after 8 weeks dark (0/24 h L/D) incubation, while









seeds from the induced autogamy and artificial xenogamy conditions had a maximum

germination of 53.9% (Stage 2) and 9.0% (Stage 1), respectively (Figure 2-7).

AFLP Data

Amplification of all four primer pairs tested was successful in all 43 individuals sampled.

Output gel images are given in Figures B-1 and B-2. However, only the -ACT/-CAG and -

AGC/-CAG primer pairs resulted in the highest polymorphic band resolution, averaging 52 and

45 bands per individual, respectively. Bands that were either present or absent in a single sample

were excluded from analysis as likely being artefactual (Pillon et al., 2007). From both primer

pairs, a total of 24 unambiguous polymorphic bands were selected. When combining these 24

bands, 10 genotypes could be distinguished. The numbers of genotypes observed, number of

polymorphic loci, Nei's diversity index, Shannon's diversity index, and FST are given in Table 2-

3. Genotypic correlation dendrograms are given in Figures B-4, B-5, and B-6.

Discussion

The ecology, pollination biology, and population genetic diversity of H. macroceratitis has

been previously unknown. Interestingly, very few reports on North American native orchid

ecology exist, particularly in the tribe Orchideae (subtribes Orchidinae and Habenariinae). The

maj ority of field-based ecological reports on the Orchidaceae come from the subfamily

Cypripedioideae, primarily in the genus Cypripedium (Sheviak 1983; Stoutamire 1983, 1989;

Light and MacConaill 2007, 2005). A few reports on the field ecology ofPlatanthera and allied

species (i.e., Himantoglossum) do exist. These Orchidinae genera are evolutionary cousins to the

Habenaria.

Bowles (1983) and Stoutamire (1996) reported on the ecology and habitat of the Federally-

threatened Platanthera leucophaea (Nutt.) L., a terrestrial orchid native to the Midwestern

United States. Unlike H. macroceratitis, which grows in shaded hardwood hammocks in central









Florida, P. leucophaea is reported as growing in mesic and wet prairie, bog, and fen-like habitats

in its native range of eastern North America. Of interest is the parallel between the requirement

for P. leucophaea sites to be wet during active growth phases and dry during dormant periods,

and the need for H. macroceratitis sites to be moist during the active growth phase and dry

during plant dormancy. This wet-to-dry life history trait appears to be consistent in many

orchids, particularly species in the Orchideae (Dressler 1993).

The importance of such field-based ecological information (i.e., Bowles 1983; Stoutamire

1996) and long-term studies (i.e., Light and MacConaill 2005) in the conservation and recovery

of orchids is just now being appreciated. Ecological data such as these are important parts of

integrated conservation planning and species recovery in the Orchidaceae. Unfortunately, only a

small number of orchid species restricted to a smaller number of genera have been studied for

such field-based ecological information. Furthermore, even a smaller number of orchid species

have been studied for more than a year or two at one time (Light and MacConaill 2005). Pfeifer

et al. (2006) reported on the long-term (ca. 26 years) demographic fluctuations of the

Platanthera-allied orchid species Himantoglossum hircinum (L.) Spreng. They reported that life

history traits, such as flowering, presence/absence of plants from year-to-year, and height of

plant, remained variable over the 26-year study period. Also, they report that overall population

size of H. hircinum in the study area increased exponentially, but no plant density effects were

observed. Data such as these provide valuable insight into not only the life history of orchids,

but also shed light on predictive models for orchid population management decisions.

While the current study with Habenaria macroceratitis did not attempt to use predictive

modeling or extensive long-term study to gain insight into the life history of the species (i.e.,

Pfeifer et al. 2006), data were produced that revealed a degree of demographic and










morphological stability within and between populations of the species. This stability in

morphological character could be indicative of a life history strategy based on small- or

moderately-sized populations existing within isolated hardwood hammock habitats spread

throughout central Florida. In this model, each population would act as an individual or isolated

population with little connection (i.e., gene flow) between populations. Further morphological,

genetic, and long-term study of H macroceratitis populations in central Florida is needed to

better elucidate this life history model.

An interesting note to a discussion on the ecology of the Orchideae is the similarity in

ecology, habitat, and distribution between Platanthera and Habenaria species native to eastern

Asia and North America. These two geographic areas share a high diversity of Orchideae, at the

generic level, and show both connected and disjunctive distributions of many Orchideae (Sing-

Chi 1983). On a morphological level, the Habenaria native to the southeastern United States

show a high degree of similarity to Asian Habenaria species, particularly H. radiate (Thumb.)

Spring. and H. rostelliifera Rchb. (S.L. Stewart personal observation). Further study on the

genetic and morphological similarities between the Orchideae flora of these two areas should be

conducted.

Despite many decades of reproductive and pollination biology study in a number of plant

species (Schmid 1975), the pollination biology of the Orchidaceae remains mostly understudied.

Tropical orchids have received the most pollination biology study in recent years (Blanco and

Barboza 2005; Trapnell and Hamrick 2006, 2005; Singer and Koehler 2003; Borba et al. 2001).

North American orchid species have, classically, received little pollination biology study--

breeding system information is available for only approximately 40% of North American

species, and detailed studies have been conducted for only approximately 15% of North









American orchids (Catling and Catling 1991). Two North American genera have garnered the

maj ority of pollination biology research: Platanthera and Cypripedium. Reflecting the interest

in the ecology of North America Cypripedium species, this genus has drawn a great deal of

pollination biology attention (e.g., Vogt 1990; Stoutamire 1967; Covell and Medley 1986;

Catling and Knerer 1980; Klier et al. 1991). Platanthera has drawn the greatest interest from

those studying the pollination biology of North American Orchidaceae (e.g., Hapeman 1997;

Folsom 1984; Stoutamire 1974; Thien 1969; Robertson and Wyatt 1990a, 1990b; Sheviak and

Bowles 1986; Smith and Snow 1976; Little et al. 2005; Zettler et al. 1996; Patt et al. 1989).

Interestingly, no data exist concerning the reproductive or pollination biology of any North

American Habenaria species (Catling and Catling 1991).

A common theme among members of the Orchideae appears to be their reliance on

pollination by butterflies and/or moths (Lepidoptera; Catling and Catling 1991). In the current

study, H. macroceratitis is suspected as pollinated by the giant sphinx moth (Cocytius antaeus).

While no data exists for a comparative discussion of the pollinators and pollination biology of

Habenaria in North America, data on similar pollinators do exist in the genus Platanthera.

Sheviak and Bowles (1986) reported several species of2anduca (Sphingidae) as pollinators for

both P. leucophaea and P. praeclara Sheviak & Bowles. Both of these aforementioned

Platanthera species possess white flowers with a nocturnal scent, similar to H. macroceratitis

(Sheviak and Bowles 1986). Furthermore, the flowers of P. leucophaea, P. praeclara, and H.

macroceratitis share similar long nectar spurs, although the spur of H. macroceratitis is much

longer than the spur of either Midwestern Platanthera species. Due to these pollination ecology

similarities, it is not surprising to find that these two Platanthera species and H. macroceratitis

are all pollinated by long-proboscis sphinx moths. However, the sphinx moth pollinators of P.









leucophaea and P. praeclara are more temperate in distribution, as is the distribution of the

orchids these moths pollinate. Cocytius antaeus is known to be distributed in more tropical

regions, paralleling the distribution of Habenaria species in North, Central, and South Americas

(Carter 2002).

A number ofPlatanthera species are known to be pollinated by Lepidoptera other than

moths, mainly butterflies. Zettler et al. (1996) reported the pollination ofP. integrilabia, a rare

terrestrial orchid from Tennessee, by the day-flying butterflies Epargyreus clarus Cramer and

Papilio glaucus L. No day-flying pollinators were observed visiting or pollinating flowers ofH.

macroceratitis in the current study.

Few reports exist on the nectar volume and nectar sugar concentration of orchid flowers as

they relate to pollination biology. In the current study, flowers ofH. macroceratitis were found

to contain an average of 4.25 Cll of nectar at an average of 17.5% soluble sugars. Cole and

Firmage (1984) reported a lower average nectar volume (1.55 CIl) for the allied species

Platanthera blephariglottis (Willd.) L. Robertson and Wyatt (1990) reported a range of nectar

volumes (3.25-6 CIl) and sugar concentrations (19-23%) for several ecotypes ofP. ciliaris (L.) L.

Platanthera integrilabia nectar volume and concentrated ranged from 2.9-6.8 Cll and 17.2-20.8%/,

respectively (Zettler et al., 1996). Hapeman (1997) reported a nectar volume and concentration

of 1.5 Cll and 19.0%, respectively, for the terrestrial orchid P. peramnoena (Gray) Gray. Nectar

amount appears to not be a function of nectar spur length, with P. blephariglottis, P. ciliaris, and

H. macroceratitis having long nectar spurs, P. integrilabia having a moderately long nectar spur,

and P. peramnoena having a short spur. Nectar sugar concentration appears to also not be

correlated with spur length. Supporting this conclusion, Zettler et al. (1996) reported no

significant difference in nectar sugar concentration ofP. integrilabia. In all these Orchideae










species, nectar volume or sugar concentration does not appear to serve as a primary mode of

pollinator attraction. Given the strong evening scent reported for H. macroceratitis, it is

suspected that this scent serves as the primary pollinator attractant for this species. Zettler et al.

(1996) reported a similar scent-driven attractant system in P. integrilabia, as did Huber et al.

(2005) in Gymnadenia conopsea and G. odoratissima.

A number of reports exist concerning the connection between pollination mechanism and

seed set in orchids (Chung and Chung 2005; Kropf and Renner 2005; Whigham and McWethy

1980). These reports indicate the success of a particular pollination mechanism based on seed

capsule formation. For example, Chung and Chung (2005) reported that self-pollination and

artificial geitonogamy pollination conditions resulted in nearly 90% seed capsule set in Bletilla

striate (Thumb.) Richenb. However, seed capsule set is not necessarily a good measure of

reproductive fitness in orchids. Seed viability and germination (i.e., seed vigor) represent better

measures of the success of experimental pollination mechanisms on reproductive fitness in the

Orchidaceae.

In the present study, H. macroceratitis set seed capsules in only four of seven experimental

pollination mechanism conditions--open pollination (control), induced autogamy, artificial

geitonogamy, and artificial xenogamy. Seed viability and asymbiotic germination (Figure 2-6)

demonstrated that both the open-pollination (control) and artificial geitonogamy pollination

conditions supported the highest seed vigor of the four capsule-producing pollination conditions.

These data indicate that H. macroceratitis likely relies on insect-mediated pollen movement

between flowers on the same or different inflorescence within the same population. Bowles et

al. (2002) reported a similar effect on seed viability in Platanthera leucophaea. In their study,

seeds originating from between-population and within-population crosses produced the highest










percent viable seeds when compared to self-pollinated plants. Furthermore, Borba et al. (2001)

reported no seed capsule formation in flowers of Hyve Brazilian PlainI har~lli\ species that were

self-pollinated or subjected to agamospermic pollination conditions. In all these cases, including

H. macroceratitis, the need for pollinators to achieve not only successful seed capsule set, but

also achieve high seed vigor (i.e., reproductive fitness) was demonstrated. Only the current

study with H. macroceratitis attempts to relate experimental pollination mechanism condition,

seed viability, and seed germination as a combined measure of reproductive fitness in the

Orchidaceae.

The AFLP technique represents a powerful tool in developing an understanding of plant

population genetic diversity within and between populations (Meudt and Clarke 2007). The

present study represents the first investigation of the genetic diversity within and between

populations of H macroceratitis, or any Habenaria species, using AFLP technology. In the

present study, a moderate level of within-population genetic differentiation was found (Socash

FST = 0.11, Old Dade Highway FST = 0.06). Four genotypes were identified in the Socash

population, while 6 genotypes were identified from the Old Dade Highway population. In spite

of the moderate within-population genetic differentiation, low between-population genetic

differentiation was found (overall FST = 0.02).

A number of reports of population genetic diversity in Platanthera, an allied genus to

Habenaria, exist. Wallace (2002a, 2002b) used the RAPD technique to investigate the

population genetic diversity and effects of habitat fragmentation on the Midwestern terrestrial

orchid P. leucophaea. Despite a moderate level of population differentiation, genetic and

geographic distances between populations were found to be not significant. This suggested a

lack of interpopulation gene flow among geographically-i isolated and fragmented P. leucophaea










populations. A similar trend of between population differentiation and potential interpopulation

gene flow help explain the moderate levels of within-population differentiation measured in H.

macroceratitis as compared to the between population genetic differentiation.

A similar genetic structure as seen in H. macroceratitis was reported by Wallace (2004) in

an investigation of the genetic structure of P. huronensis (Nutt.) Linl. P. aquilonis Sheviak, and

P. dilatat (Prush) Lindl. using intersimple sequence repeat (ISSR) markers. In the study, a

significant amount of the species-level genetic diversity in P. huronensis and P. dilatata was

found to reside within populations of these two terrestrial orchids, and not between populations.

Wallace (2004) suggested that the low between population genetic variation seen in P.

huronensis may be due to a limited number of origins, genetic bottlenecks, or low among

population gene flow.

The interpretation of these AFLP population genetic diversity data in a context of species

management and conservation planning is critical. The measure of population and species

genetic diversity, and any subsequent change in that diversity is an important step in the overall

conservation planning for a species. To understand the genetic diversity present within and

between populations of this orchid species, is to begin to understand the ability of those

populations to respond to natural selection, speciation, and other evolutionary pressures

(Qamaruz-Zaman et al. 1998a). Preservation of genetic diversity is crucial in the long-term

conservation of plant species (Crozier 1992), particularly when little other information is known

concerning the ecology, propagation, or pollination biology of the species. The demonstration of

moderate levels of within-population genetic differentiation in H. macroceratitis populations

would suggest that moderate to large isolated populations should be maintained in order to

preserve this degree of differentiation within populations. Data from pollination biology and










asymbiotic seed germination studies support this conclusion. However, the low between

population genetic differentiation measured using AFLP suggests that the two populations

sampled have some degree of gene flow between them. This suspected gene flow could be due

to several factors: 1) occasional across-population pollination events, 2) historic meta-population

now fragmented by urbanization, and/or 3) founder population and founded population

relationship. Higher-order data analysis and further population sampling is necessary in order to

properly investigate these potential relationships between these two populations of H

nzacroceratitis.

Furthermore, careful molecular- and morphological-based investigations of the overall

differences, gene flow, potential hybridization, and population structure of both H.

nzacroceratitis and its sister species, H. quinqueseta, should be conducted to better understand

the past, current, and future relationships between these two species (Dueck and Cameron 2007;

Gustafsson and Sjoigren-Gulve 2002; Wallace and Case 2000; Trapnell et al. 2004; Bateman et

al. 2003; Case et al. 1998). These data would greatly help in the long-term conservation

planning and species-recovery efforts for H. nacroceratitis both in Florida and throughout the

species' range.

Implications for Integrated Conservation Planning

The present studies on the plant demography, pollination biology, and genetic diversity of

the Florida terrestrial orchid H. nacroceratitis have supported the need to understand these

important ecological factors of orchid biology before implementing conservation and recovery

plans. Studies such as these highlight the need to integrate ecological, reproductive biology, and

molecular-genetic studies to produce ecologically functional data on the species-level

conservation of plants. For example, recognizing the relationship between the pollination

mechanism of H. nacroceratitis and the species population genetic structure has allowed these









studies to suggest that moderate to large isolated populations of H macroceratitis be maintained

and managed throughout the range of the species in Florida (Stewart and Kane 2006c).

Studies on the pollination biology ofH. macroceratitis have revealed the need for insect-

mediated cross pollination to support high reproductive fitness in this species. In identifying a

potential pollination of this orchid species, not only does useful information concerning the role

of this pollinator in the reproductive biology of H. macroceratitis come to light, but also the need

for pollinator conservation and management become important. As demonstrated in the

integration of the pollinator observation studies with the pollination mechanism studies, the need

for a strong-flying pollinator, such as Cocytius antaeus, to repeatedly visit flowers ofH.

macroceratitis on the same inflorescence or within the same population is vital to this orchid's

reproduction. Pollination events where C. antaeus transfers pollinia between populations appear

to be the exception. Furthermore, the larval stage of C. antaeus is known to feed from the pond-

apple tree (Annona glabra L.), a tree typical of wet and swampy habitats in south Florida and

throughout the tropics. To insure the continued reproductive fitness ofH. macroceratitis in

Florida, the species' pollinator (C. antaeus) must be conserved and managed, meaning that A.

glabra trees throughout south Florida and the habitats that support them must also be conserved

and managed properly. Without this integrated multilevel conservation and management system

in place, the continued existence ofH. macroceratitis in Florida may in jeopardy. Unfortunately,

no such system currently exists.

The current studies concerning the plant demography, pollination biology, and population

genetic diversity of H macroceratitis represent another step toward the species-level integrated

conservation of this rare terrestrial orchid in Florida (Figure 1-1). By adding to the body of

knowledge on the conservation biology of this orchid species, the present studies not only










promote the conservation biology of H. macroceratitis, but also promote the conservation and

management of a number of other plant, animal, and insect species throughout Florida and the

tropics.











Table 2-1. Experimental pollination conditions applied to flowers of~labenaria macroceratitis,


after Wong and Sun (1999).
Condition Bagging Treatment
Control Unbagged Untreated


Pollen Source
Open pollination

No pollination


Objective
Evaluate fruit set under
natural conditions
Evaluate the rate of non-
sexual reproduction
Measure the need for
pollinators
Evaluate
self-compatibility

Evaluate
self-compatibility
Evaluate outbreeding at
long distance
Evaluate outbreeding at
short distance


Agamospermy

Spontaneous autoganw

Induced autoganw

Artificial
genitonoganw

Artificial xenoganw

Induced xenoganw


Bagged Emasculated


Bagged


Untreated The same flower


Bagged Emasculated


Bagged Emasculated

Bagged Emasculated

Bagged Emasculated


The same flower


Different flower on
same plant
Flower from a
distant population
Flower from same
population, distant plant










Table 2-2. Thermal cycler parameters for preselective (top) and selective (bottom) AFLP
amplification of prepared genomic DNA, following ABI (2005).
Preselective Parameters


Hold


Cycle
20 Cycles of Each


Hold


Hold


720C 940C 560C
2 min 20 sec 30 sec


720C 600C
2 min 30 min


40C
continuous


Selective Parameters
Hold


Cycle

66 C
30 sec
65 C
30 sec
64 C
30 sec
63 C
30 sec
62 C
30 sec
61 C
30 sec
60 C
30 sec
59 C
30 sec
58 C
30 sec
57 C
30 sec
56 C
30 sec


Number
of Cycles


94 C
2 min


94 C
20 sec
94 C
20 sec
94 C
20 sec
94 C
20 sec
94 C
20 sec
94 C
20 sec
94 C
20 sec
94 C
20 sec
94 C
20 sec
94 C
20 sec
94 C
20 sec


72, C
2 min
720 C
2 min
720 C
2 min
720 C
2 min
720 C
2 min
720 C
2 min
720 C
2 min
720 C
2 min
720 C
2 min
720 C
2 min
720 C
2 min


600 C
30 min
40 C
continuous










Table 2-3. Results of the genetic analysis for each population of Habenaria macroceratitis.
Socash = Socash study site (Hernando County, Florida). Old Dade = Old Dade
Highway study site (Hernando County, Florida).
Population Estimated Number of Number of Number of Nei's Shannon's FST
Size Samples Genotypes Polymorphic Index Index
Loci
Socash >200 (211114) 22 4 13 0.184 0.309 0.11
Old Dade ca 0 li1)21 6 11 0.161 0.27 0.06

Overall 43 10 24 0.176 0.302 0.02































Figure 2-1. Study sites for Habenaria macroceratitis in Florida: Marion County, Sumter
County, and Hernando County.









I


Spur Length


SSocash
SBattle Slough
I Cross Florida Greenway
I Old Dade Highway







a a


a
a


30 -


25 -


20 -


15 -


10 -
5-


01


Height


Demographic Measurement


Figure 2-2. Average height (cm) and spur length (cm) of Habenaria macroceratitis plants at
four study sites in west central Florida. Data from 2002-2005 observations pooled
within site and across years. Histobars with the same letter are not significantly
different within demographic measurement (a = 0.05). Error bar = SE.



































Flower Number


8-


6-




6



E 4-
Z



2-


M Socash
SBattle Slough
I Cross Florida Greenway
I Old Dade Highway


a a
a


a


01


Leaf Number


Demographic Measurement

Figure 2-3. Average leaf number and flower number of Habenaria macroceratitis plants at four
study sites in west central Florida. Data from 2002-2005 observations pooled within
site and among years. Histobars with the same letter are not significantly different
within demographic measurement (a = 0.05). Error bar = SE.





































Figure 2-4. Growth cycle ofHabenaria macroceratitis under field conditions. D = dormant
tuber stage. SG = shoot growth stage. VG = vegetative growth stage. Scale bar = 1
cm.

































Figure 2-5. Cocytius antaeus (giant sphinx moth) captured during pollinator observation of
Habenaria macroceratitis at the Socash site (Hernando County, Florida) 28-29
August 2004. Scale bar = 1 cm.











34


32-


30-


S28-


0- 26-


24-


22-


20

100-



90-








70 -7



60-



5 0 l l i i l s l u l s l s



RR~~RRRRRRRDate/TimeRR

Figure 2-6 Tepeatr and reltiv humdit profesa Socas sie(HranoCony
Flria 2-9 ugs 20 drng Hab nai mac oertitsoliaoobevtns




Figur 2A) Temperature (C and B relative humidity (RH;le %).cshst Henno ony















80 -b I Artificial Xenogamy



601 -1 a a



40-



20-
Ib

a bbb

Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

Germination Stage

Figure 2-7. Effects of pollination condition on percent germination and protocorm development
of Habenaria macroceratitis after 8 weeks in vitro asymbiotic culture on Malmgren
Modified Terrestrial Orchid Medium. Histobars with the same letter are not
significantly different within stage (a = 0.05). Error bar = SE.









CHAPTER 3
SEED CULTURE AND IN VITRO SEEDLING DEVELOPMENT OF Habenaria
macroceratitis

Introduction

The ongoing loss of suitable orchid habitat throughout the southeastern United States (e.g.,

Florida) has prompted interest in the preservation and restoration of these critical habitats. These

habitats are considered highly productive for both animal (Machr and Cox 1995) and plant

(Sprott and Mazzotti 2001) species. This loss, due mostly to urbanization, conversion for

agricultural purposes, and habitat mismanagement, has greatly impacted populations of many

rare and endangered herbaceous understory plants throughout Florida (Sprott and Mazzotti

2001), including many native orchids. Many native Florida terrestrial orchids inhabit threatened

habitats such as hardwood hammocks and pine flatwoods (S.L. Stewart personal observation)

and are at risk of population decline or extinction unless an effective method of propagation can

be developed to provide plants for restoration purposes.

Seed germination represents the most efficient method of native terrestrial orchid

propagation for conservation purposes (Stewart and Kane 2006a, 2006b, 2007b; Kauth et al.

2006), as well as one step in the integrated conservation of orchid species (Stewart and Kane

2007b; Figure 1-1). However, orchid seed germination studies are often viewed as unreliable or

unrealistic since little is known concerning the factors affecting in vitro germination and in vitro

seedling development for many North American native orchids (Arditti et al. 1981; Stewart and

Kane 2006a). Compounding this problem, Stoutamire (1974, 1989) found that many North

American native terrestrial orchids require up to eight years of ex vitro growth before reaching

reproductive maturity. This means that potential biochemical and physiological carry-over

effects of in vitro culture on plant growth, development, and fitness may not be evident until

many years after propagation and reintroduction. To overcome these problems some have










suggested the development of optimized seed germination methods for entire genera or

individual species (Stewart and Kane 2006a; Kauth et al. 2006). This approach shows great

promise and is employed here.

Asymbiotic seed germination represents a direct method for orchid seed germination on a

defined agar-solidified medium, typically supplemented with sucrose, various amino acids,

vitamins, and undefined compounds (i.e., banana powder, coconut water). The advantage of

asymbiotic orchid seed germination is that orchid fungi (mycobionts) need not be isolated from

field-collected seedling or adult plant material in order to germinate seed of orchid taxa.

However, asymbiotic orchid seed germination does have its disadvantages. Orchids that are

established using asymbiotic seedlings may not be able to capture and digest mycobionts once

introduced to a field setting (W. Stoutamire, personal communication). Furthermore,

populations of orchids that are established using asymbiotic seedlings will remain dependant

upon naturally-occurring mycobionts for seedling recruitment (Zettler 1997b). These

mycobionts may not be present at a reintroduction site if not already in association with the

reintroduced orchid plants, especially if the orchid species was not present at the site prior to

plant reintroduction. This lack of mycobiont may not allow for seedling recruitment at the

reintroduction site, thus limiting the long-term sustainability and fitness of the propagated orchid

taxon.

In nature, orchids digest endophytic mycorrhizal fungi as a source of nutrition

(mycotrophy) in a parasitic association that is known to support seed germination, as well as

protocorm and seedling development (Arditti 1966; Clements 1988; Rasmussen 1995;

Rasmussen and Rasmussen 2007). Therefore, the long-term survival and fitness of orchids in

managed or restored habitats requires the presence of appropriate mycobionts for proper plant









nutritional support and seedling recruitment (Zettler 1997a). The most efficient way to promote

this process is through the use of in vitro symbiotic co-culture methods (Dixon 1987; Zettler

1997a, 1997b; Clements et al. 1986). Symbiotic seed co-culture involves the combining of

orchid seed and a compatible mycobiont under in vitro conditions on an undefined agar-

solidified medium consisting of only finely pulverized rolled whole oats, and occasionally

undefined additives such as yeast extract. Unfortunately, few North American native orchids

have been cultured using this method; mostly species restricted to the genera Splitanihesl~

(Anderson 1991; Stewart and Kane 2007b; Zelmer and Currah 1997; Zettler and McInnis 1993;

Stewart et al. 2003), Platanthera (Anderson 1996; Zettler and Hofer 1998; Zettler and McInnis

1992; Sharma et al. 2003; Zettler et al. 2001; Zettler et al. 2005), and Habenaria (Stewart and

Kane 2006b; 2007b; Stewart and Zettler 2002). Moreover, little is known about the identity and

ecology of these mycobionts in pure culture or in nature.

The terrestrial orchid Habenaria macroceratitis was chosen as the species in this study

because little information exists on the asymbiotic and symbiotic seed culture, as well as the in

vitro seedling development of this species (Figure 1-2). Previously, Stewart and Zettler (2002)

proposed a symbiotic co-culture protocol for H. macroceratitis, although protocorms did not

develop to advanced leaf-bearing stages in their study. In this study, efficient asymbiotic and

symbiotic seed germination methods for H. macroceratitis are defined. These methods facilitate

protocorm development through a leaf-bearing stage. The effects of asymbiotic germination

medium, carbohydrate type and concentration, exogenous cytokinin application, photoperiod,

and in vitro mycobiont preference on the seed germination of H macroceratitis are presented.

Additionally, the effect of photoperiod on asymbiotic in vitro seedling development of H.









nzacroceratitis is also presented. This work represents one step in the integrated conservation

and species-level recovery of H. nzacroceratitis in Florida.

Materials and Methods

Asymbiotic Seed Germination

Seed source and sterilization

Seeds of were obtained from mature capsules prior to dehiscence on 26 October 2003.

Habenaria nzacroceratitis seeds were collected from a privately-owned site near Brooksville,

Florida (Hernando County). Immediately after collection, capsules were dried over silica gel

desiccant for 2 weeks at 25 + 50C, followed by storage in darkness at -180C for 52 days. Seeds

were surface disinfected for 1 min in a solution containing 5 mL ethanol (100%), 5 mL 6.00%

NaOC1, and 90 mL sterile deionized (DI) water. Following surface disinfection, seeds were

rinsed three times for 1 min each in sterile DI water. Solutions were removed from the surface

disinfection vial using a sterile Pasture pipette that was replaced after each use. Sterile DI water

was used to suspend the disinfected seed, and a sterile bacterial inoculating loop was used to sow

the seed. An average of 124 seeds per Petri plate were sown.

Asymbiotic media survey

The effects of six basal media (Table 3-1) on asymbiotic seed germination of H

nzacroceratitis were assessed. Three of the media were commercially prepared and modified by

PhytoTechnology Laboratories LCC (Shawnee Mission, Kansas): Murashige & Skoog (MS),

Malmgren Modified Terrestrial Orchid Medium (MM; Malmgren, 1996), and Modified Kundson

C (KC). Two of the media were commercially prepared and modified by Sigma Chemical

Company (St. Louis, MO): Vacin & Went (VW; Vacin and Went, 1949) and Lindemann (LM;

Lindemann et al., 1970). The Einal medium, Modified Lucke (ML), was prepared according to

Anderson (1996). MS, VW, and LM were further modified by the addition of 2% sucrose and









0.8% TC@ agar (PhytoTechnology Laboratories LCC, Shawnee Mission, Kansas) and ML

modified by the addition of 0.8% TC@ agar to be consistent with MM and KC. Media were

adjusted to pH 5.8 with 0. 1 N KOH after the addition of carbohydrate source and agar, and were

dispensed into 1 1 flasks prior to autoclaving for 40 min at 117.7 kPa and 1210C.

Sterile media were dispensed as 25 mL aliquots into 9 cm diameter Petri plates (Fisher

Scientific, Pittsburg, Pennsylvania). Surface sterilized seed were placed into the center of each

plate and the seed evenly spread on the medium. Ten replicate plates were inoculated per

medium type. Petri plates were sealed with a single layer ofNescofilm (Karlan Research

Products, Santa Rosa, California) before being cultured in continual darkness at 25 + 30C for 7

weeks without light interruption. At 7 and 16 weeks germination and protocorm development

were assessed by use of a dissection stereoscope. Germination and protocorm development were

scored on a scale of 0-5 (Table 3-2; Figure 3-1; Stewart and Kane 2006a).

Germination percentages were calculated by dividing the number of seeds in each

individual germination and development stage by the total number of viable seeds in the sample.

Data were analyzed using general linear model procedures and Waller-Duncan mean separation

at a=0.05 by SAS v 8.02 (SAS 1999). Germination counts were arcsine transformed to

normalize variation.

Effects of carbohydrate source on asymbiotic seed germination

Effects of three carbohydrate sources (fructose, sucrose, and dextrose) at 50 mM on the

asymbiotic seed germination of H. macroceratitis were examined. One asymbiotic germination

basal medium (MM) was used in treatment combinations with carbohydrate source and the

presence or absence of banana powder (BP). Medium was prepared as previously described and

supplemented with a 50 mM carbohydrate source (9.008 g 1-1 fructose, 17.115 g 1-1 sucrose, 9.008

g 1-1 dextrose). To those treatments containing BP, 15 g 1-1 BP was added prior to sterilization.









Sterile medium was dispensed (ca. 25 mL) into 9 cm diameter Petri plates. Ten replicate plates

were inoculated with seed. Plates were sealed with one layer ofNescofilm. Seed germination

and protocorm development were scored after 7 and 21 weeks dark incubation as previously

described. Germination percentages and statistical analyses were completed using general linear

model procedures and least square means at a=0.05 (SAS 1999). Seed germination percentages

were arcsine transformed prior to analysis using least square means.

Effects of exogenous cytokinins on asymbiotic seed germination

Effects of four cytokinins-benzyladenine (BA), 6-(y, y-dimethylallylamino) purine (2-iP),

zeatin (Zea), and kinetin (Kin)--at 0, 1, 3, and 10 CIM on the asymbiotic seed germination of H.

macroceratitis were examined. Basal medium MM was used in all treatment combinations.

Medium was prepared as previously described, with the exception that filter-sterilized (0.2 Clm

pore size) cytokinins were added to the molten (ca. 400C) pressure sterilized MM prior to

dispensing and solidification at the concentrations previously listed. Sterile medium was

dispensed (ca. 25 mL) into 9 cm diameter Petri plates. Ten replicate plates were inoculated.

Plates were sealed with one layer ofNescofilm. Seed germination and protocorm development

were rated after 14 weeks dark incubation as previously described. Germination percentages and

statistical analyses were completed as previously outlined.

Effects of photoperiod on asymbiotic seed germination

The effects of three photoperiod treatments (0/24, 16/8, 24/0 h L/D) on asymbiotic seed

germination of H. macroceratitis incubated at 25 + 30C were evaluated. Illumination was

provided by General Electric F96T12 cool white fluorescent tubes at 60.5 Clmol m-2S-1, as

measured at culture level. Plates in continual darkness were wrapped in two layers of aluminum

foil to fully exclude light. Seeds were cultured on sterile MM basal medium contained in 9 cm

diameter Petri plates. Plates were sealed with one layer ofNescofilm. Eleven replications per










photoperiod treatment were used, and seed germination and protocorm development were rated

as previously mentioned. Seed germination and protocorm development were scored after 14

weeks after medium inoculation. Germination percentage and statistical analyses were

completed as previously outlined for the asymbiotic media survey.

Effects of photoperiod on in vitro seedling development

The effects of three photoperiod treatments (8/16, 12/12, 16/8 h L/D) on in vitro seedling

development of H. macroceratitis were evaluated. Illumination source was as previously

described. Seeds were germinated on MM basal medium contained in 9 cm diameter Petri

plates, and 20 week old protocorms transferred to Magenta GA-7 vessels (Magenta Corporation,

Chicago, Illinois) containing 50 mL MM medium. Nine protocorms were transferred per vessel,

with ten replications per photoperiod. Vessels were sealed with one layer ofNescofilm.

Incubation and illumination conditions were as mentioned previously.

In vitro seedling development was scored after 30 weeks incubation. Effects of

photoperiods on tuber and leaf number, tuber and shoot fresh weight (fwt) and dry weight (dwt),

and leaf length and width were recorded. All developmental data were statistically analyzed

using general linear model procedures and Waller-Duncan mean separation at a=0.05 in SAS v

8.02 (SAS 1999).

Symbiotic Seed Germination

Seed source and sterilization

Seeds were obtained prior to dehiscence from mature capsules on 26 September 2003.

Seeds were collected from a large (>200 flowering and vegetative plants) population of H

macroceratitis occurring on privately-owned land in Hernando County, Florida. Immediately

following collection, capsules were dried over silica gel desiccant for 2 weeks at 250C, followed










by storage at -100C in darkness for 142 days. Prior to the initiation of symbiotic co-cultures, a

tetrazolium test (Lakon 1949) was conducted to assess H. macroceratitis seed viability.

Mycobiont isolation and identification

All mycobionts were recovered from the roots of the study species. Mycobionts were

isolated following the protocols outlined by Stewart and Zettler (2002) for Florida Habenaria

species. Adult flowering and leaf-bearing vegetative plants with intact root systems were

collected, the root systems were wrapped in paper towels moistened with sterile deionized water,

placed in plastic bags, stored in darkness at ca. 100C, and transported to the laboratory (<4 hrs).

Root segments were detached, rinsed with cold tap water to remove debris, and surface cleansed

1 minute in a solution containing 5 mL ethanol (100%), 5 mL 6.00% NaOC1, and 90 mL sterile

DI water. Clumps of cortical cells containing fungal pelotons were removed, placed on corn

meal agar (CMA; Sigma-Aldrich, St. Louis, Missouri) supplemented with 50 mg 1Y novobiocin

sodium salt (Sigma-Aldrich, St. Louis, Missouri), and incubated at 250C for 5 days. Hyphal tips

were excised from actively-growing pelotons and subcultured onto 1/5th-strength potato dextrose

agar (1/5 PDA): 6.8 g PDA (BD Company, Sparks, Maryland), 6.0 g granulated agar (BD

Company, Sparks, Maryland), 1 1 distilled deionized (dd) water.

Fungal isolates showing cultural characteristics similar to those orchid mycobionts

previously described in the literature (Moore 1987; Zettler 1997b; Currah et al. 1997; Currah et

al. 1987; Richardson et al. 1993; Stewart et al. 2003; Zelmer et al. 1996) were assigned a

reference number and stored at 100C on modified oat meal agar (MOMA): 3.0 g pulverized

rolled oats (Quaker Oats, Chicago, Illinois), 7.0 g granulated agar, 100 mg yeast extract (BD

Company, Sparks, Maryland), and 1 1 dd water (Clements et al. 1986). Mycobiont isolates were

stored until use in symbiotic co-culture experiments. Two representative mycobiont isolates









were accessioned into the University of Alberta Microfungus Herbarium (UAMH) as UAMH

10801 and UAMH 10802.

Mycobiont characterization and identification followed methods outlined by Zelmer and

Currah (1995), Currah et al. (1987, 1990, 1997), and Zelmer et al. (1996). Hyphal and monilioid

cell characteristics were assessed from cultures growing on both CMA and 1/5 PDA cultured in

continual darkness at 250C using a Nikon Labophat-2 light microscope (Nikon USA, Melville,

New York) fitted with a Nikon Coolpix 4500 digital camera (Nikon USA, Melville, New York).

Staining procedures followed those outlined by Phillips and Hayman (1970) modified by the use

of acid fuchsin as the mycobiont stain (Stevens 1974; J. Kimbrough personal communication).

Culture growth rates were determined from isolates growing on PDA incubated in continual

darkness at 250C as measured in three directions every 24 hours from the bottom of each Petri

plate. Cellulase production was determined by the cellulose azure method of Smith (1977)

modified by the use of 1/5 PDA as the basal medium (Figure 1-6). Polyphenol oxidase

production was detected by using the tannic acid medium (TAM) method of Davidson et al.

(1938) (Figure 1-7).

Symbiotic co-culture

The effects of six mycobionts (Table 3-3) on the in vitro symbiotic co-culture of II

macroceratitis were evaluated. Seeds were sown according to the procedures outlined by

Stewart and Zettler (2002) for Florida Habenaria species. Seeds were removed from cold-dark

storage, allowed to warm to room temperature (ca. 250C), surface disinfected for 1 min in the

same solution used during asymbiotic seed disinfection, and placed over the surface of a 1 cm x

4 cm sterile filter paper strip (Whatman No. 4, Whatman International, Maidstone, United

Kingdom) within a 9 cm diameter Petri plate containing 25 mL oat meal agar (OMA): 3.0 g

pulverized rolled oats, 7.0 g bacto-agar, and 1 1 dd water. Medium pH was adjusted to 5.8 with









0.1 N HCI prior to autoclaving at 117.7 kPa and 1210C for 40 min. Seeds were sown using a

sterile bacterial inoculating loop. Between 10 and 40 seeds were sown per plate. Each plate was

inoculated with a 1 cm3 block of mycobiont inoculum, one mycobiont per plate, and a total of 8

replicate plates per mycobiont. Eight uninoculated plates served as the control. Plates were

sealed with Nescofilm, wrapped in aluminum foil to exclude light, and maintained in darkness

(0/24 h L/D) for 58 days at 25 & 20C. Plates were examined weekly during dark maintenance for

signs of germination or contamination, exposing the seeds to brief (<10 min) periods of

illumination. Plates were returned to experimental conditions after visual inspection.

After 58 days dark culture, seed germination and protocorm development was assessed

using a dissecting stereomicroscope. Germination and seedling growth and development were

scored on a scale of 0-5 (Stewart and Kane 2006b; Table 3-2; Figure 3-1). Seed germination

percentages were based on viable seeds determined by visual inspection with the aid of a

dissection microscope. Viable seeds were considered those seeds containing a distinct, rounded

and hyaline embryo.

Germination percentages were calculated by dividing the number of seeds in each

germination and development stage by the total number of viable seeds in the sample. Data were

analyzed using general linear model procedures and Waller-Duncan mean separation at a=0.05

by SAS v 8.02 (SAS 1999). Germination counts were arcsine transformed to normalize

variation.

Effects of photoperiod on symbiotic co-culture

The effects of three photoperiod treatments (0/24 h, 16/8 h, 24/0 h L/D) on in vitro

symbiotic co-culture of H. macroceratitis maintained at 25 & 30C were evaluated. Seeds were

sown as previously described; with the exception that only one mycobiont was used in all three

photoperiod treatments. Mycobiont Sbrev-266, previously identified as a strain of Epulorhiza










repens (Bernard) Moore and originating from the roots of the Florida terrestrial orchid

Splitanthesl~ brevilabris Lind. (Stewart et al. 2003), was chosen because of its effectiveness at

germinating seeds of H. macroceratitis in a previous study, as well as seeds of other Florida

terrestrial and epiphytic orchids (Stewart and Zettler 2002; Stewart et al. 2003; Zettler et al.

2007; S.L. Stewart unpublished data). Illumination was provided by General Electric F96T12

cool white fluorescent tubes at 60.5 Clmol m-2S-1, as measured at culture level. Plates in continual

darkness were wrapped in aluminum foil to exclude light. Seeds were cultured on OMA in 9 cm

diameter Petri plates (ca. 25 mL). Plates were sealed with one layer ofNescofilm. Eleven

replications per photoperiod treatment were used. Seed germination and protocorm development

were scored after a 96 day culture period. Germination percentage and statistical analyses were

completed as previously outlined.

Results

Asymbiotic Seed Germination

Asymbiotic media survey

Seeds began swelling within three weeks after medium inoculation, and germination

commenced within six weeks after inoculation. Visual contamination rate of cultures was 5%.

A tetrazolium test revealed H. macroceratitis seeds collected from the Brooksville, Florida

(Hernando Co., Florida) site to be 41.4% viable, while visual inspection of seed revealed 52.6%

viability from the same site. Seeds of this species were monoembryonic.

Seed germination at week 7 was highest on both LM and KC, 89.1% and 89.2%

respectively (Figure 3-2). However, these germinated seed had only developed to Stage 1 by this

time. Maximum protocorm development at week 7 (Stage 2) was supported by ML and MM,

61.2% and 83.9% respectively (Figure 3-2). After 7 weeks incubation, seed germination was

minimal on MS, LM, and VW although supporting Stage 2 protocorm development. No










germinated seeds on KC developed beyond Stage 1. Seed germination and seedling

development progressed to at least Stage 1 on all media surveyed.

Seed germination percentages and protocorm development did not remain constant to week

16. Seed germination and protocorm development at week 16 was highest on VW, KC, and

MM, 98.8% (Stage 2), 95.3% (Stage 2), and 98.6% (Stage 4), respectively (Figure 3-2).

However, only MM supported protocorm development to a leaf-bearing (Stage 4) after 16 weeks

incubation, while VW and KC supported seed development to only Stage 2 during the same

incubation period. Paralleling week 7 data, maximum protocorm development in week 16 (Stage

4) was supported on ML and MM. After 16 weeks incubation, no seeds developed beyond Stage

2 on LM, VW and KC. No seeds developed beyond Stage 3 on MS.

Effects of carbohydrate source on asymbiotic seed germination

After 7 weeks dark incubation, all carbohydrate sources tested supported at least minimal

asymbiotic germination (i.e., Stage 1) of H. macroceratitis seeds. Stage 3 germination was

supported at this time by both fructose without banana powder and basal medium only control

(0.2% and 0.4%, respectively; Figure 3-2). However, the majority of germinating seeds were

observed in Stage 2 development, with the basal medium only control (83.9%), basal medium

with banana powder control (81.0%), dextrose without banana powder (80.9%), and fructose

without banana powder (79.1%) supporting the highest germination percentages in this

developmental stage (Figure 3-3). Only the fructose with banana powder treatment supported a

significantly lower germination percentage (57.6%) than did all other treatments. No significant

difference in germination percentages were found for Stage 1 germination in all treatments after

7 weeks.

All carbohydrate sources supported through Stage 5 germination and development after 21

weeks of in vitro culture under dark incubation conditions. Both the basal medium only control









and fructose without banana powder supported 100% Stage 5 development after 21 weeks

(Figure 3-4). Only dextrose with banana powder supported a significantly lower germination

percentage in Stage 5 (59.8%) than did all other treatments. Interestingly, this same treatment

supported a significantly higher percentage of Stage 4 protocorms (28. 1%) than did all other

treatments tested. Furthermore, only the dextrose with banana powder and fructose with banana

powder treatments supported Stage 3 development after 21 weeks (1.9% and 5.0%, respectively;

Figure 3-4).

Effects of exogenous cytokinins on asymbiotic seed germination

After 14 weeks dark incubation, all cytokinins tested demonstrated some effect on the

asymbiotic seed germination of H. macroceratitis. With the exception of Kin at 3 CIM, all other

cytokinins tested at both the 3 CIM and 10 CIM concentrations demonstrated no significant

difference in germination percentage from the control. Cytokinins Zea and Kin at 1 CIM had a

pronounced positive effect on seed germination, resulting in an increased germination percentage

(Zea = 58.1% and Kin = 47.2%) when compared to control conditions (Control = 14.2%; Figure

3-4). BA at 1 CIM also had a significant positive effect on seed germination percentage, but

significantly less that than either Zea or Kin (BA = 33.7%; Figure 3-5). Asymbiotic germination

was suppressed by all cytokinins tested at 10 CIM, as well as BA at 3 CIM.

Effects of photoperiod on asymbiotic seed germination

Seeds began germinating after four weeks regardless of photoperiodic condition. No

significant effect of photoperiod was found on the initial seed germination (e.g. Stage 1) of H.

macroceratitis. Those seeds incubated in continual darkness (0/24 h L/D) exhibited both the

highest Einal percent germination (91.7%) and most advanced protocorm developmental stage

(Stage 4; Figure 3-6). Protocorm development under both the 16/8 h L/D and 24/0 h L/D

treatments was supported to Stage 4, but displayed statistically lower seed germination









percentages, 17.5% and 54.6% respectively (Figure 3-6). Interestingly, while maximum

protocorm development under the 16/8 h L/D photoperiod was achieved at Stage 3, maximum

protocorm development achevied under the 0/24 h L/D and 24/0 h L/D photoperiods was Stage

4. Asymbiotic seed germination was optimal under the 0/24 h photoperiod in both seed

germination percentage and advanced developmental stage.

Pronounced differences in protocorm morphology were seen under all three photoperiods.

Those seed germinated in both the 16/8 h L/D and 24/0 h L/D photoperiods produced protocorms

that lacked rhizoids throughout their development to Stage 5 (Figure 3-7b-c). Conversely, those

seed germinated in the 0/24 h L/D photoperiod produced protocorms possessing numerous

rhizoids from Stage 1 though Stage 5, and beyond (Figure 3-7a).

Effects of photoperiod on in vitro seedling development

Photoperiod had a pronounced effect on several growth and development responses in in

vitro seedlings of H. macroceratitis after 30 weeks incubation. The number of tubers produced

per in vitro seedling was highest under the 8/16 h L/D photoperiod (1.06 tubers) versus under

either the 12/12 h L/D photoperiod (1.00 tubers) or 16/8 h L/D photoperiod (1.00 tubers; Figure

3-7). Tuber fresh weight (42.7 Gig) and dry weight (6.5 Gig) were highest on seedlings cultivated

under an 8/16 h L/D photoperiod, while tuber fresh and dry weights were statistically similar

under 12/12 h L/D and 16/8 h L/D photoperiods (Figure 3-9). Tuber size (diameter and length)

was also influenced by photoperiod, with tuber diameter being greatest under the 8/16 h L/D

photoperiod (3.1 mm) and smaller under all other photoperiod conditions. Tuber length

remained statistically similar under all three photoperiod conditions (Figure 3-10).

Shoot fresh weight was highest in seedlings incubated under the 8/16 h L/D photoperiod

(69.5 Gig), whereas shoot dry weight was similar for plants incubated under both the 16/8 h L/D

and 12/12 h L/D photoperiods (39.1 Clg and 50.1 Gig, respectively; Figure 3-8). The lowest









number of leaves per plant was produced on those seedlings incubated in the 8/16 h L/D

photoperiod, but those leaves were the longest and widest (Figures 3-7, 3-10). In fact, leaf length

and width per seedling significantly decreased from short-day (SD) to long-day (LD)

photoperiod, while the total number of leaves per seedling increased (Figures 3-8, 3-10).

Symbiotic Seed Germination

Mycobiont isolation and identification

Six fungal mycobionts were recovered from pelotons within the roots of flowering and

vegetative plants of H macroceratitis (Table 3-3; Figure 3-11) collected at two sites in Florida.

All six mycobionts were identified as members of the anamorphic fungal genus Epulorhiza

(Moore, 1987). Only superficial differences in cultural morphology were identified among the

group of six mycobionts. Isolates Hmac-309 and Hmac-310 were cream in color after 25 days

on 1/5 PDA, whereas all other isolates were ivory. These differences were determined as

inconsequential to taxonomic determinations. No differences in cellulase or polyphenol oxidase

activity were detected among the six isolates.

Symbiotic co-culture

Seeds began to swell within two weeks after sowing, and germination commenced within

five weeks. Visual contamination of cultures from bacteria and non-mycorrhizal fungi was 2%.

A tetrazolium test revealed H. macroceratitis seeds to be 41.4% viable, while visual inspection

revealed 52.6% viability from the same seed lot.

All inoculated seed germinated by 58 days. An effect of mycobiont preference was found

during the in vitro symbiotic co-culture of H macroceratitis. Germination after 58 days was

highest when seeds were inoculated with mycobiont Hmac-310 (65.7%; Figure 3-12). This

isolate not only promoted the highest final percent germination, but also promoted Stage 2

development. However, no significant difference in seed germination and protocorm









development was demonstrated by Stage 2 among Hmac-310, Hmac-312, or control treatments

(65.7%, 51.0%, 51.5% respectively; Figure 3-12).

Effects of photoperiod on symbiotic co-culture

Seeds began to swell within two-and-a-half weeks after sowing, and germination

commenced within four weeks. Visual contamination rate of cultures was 4%. Seed viabilities

remained as described previously.

Seeds began germinating after four weeks regardless of photoperiod condition. After 96

days culture a significant effect of photoperiod was found on the initial in vitro symbiotic co-

culture (e.g. Stage 1) of H macroceratitis. Seeds cultured under continual darkness (0/24 h L/D)

exhibited a lower initial seed germination percentage (17.1%) than seeds cultured under either

the 16/8 h L/D or 24/0 h L/D photoperiods (37.4% and 34.4%, respectively; Figure 3-13).

However, protocorm development to Stage 2 was stimulated under 0/24 h L/D conditions

(53.5%; Figure 3-12), whereas development was less under both 16/8 h L/D and 24/0 h L/D

(34.6% and 34.5%, respectively; Figures 3-13, 3-14). Symbiotic seed germination was highest

under 16/8 h L/D photoperiod, but protocorm development was most advanced under a 0/24 h

L/D photoperiod.

Discussion

Asymbiotic Seed Germination

The successful in vitro asymbiotic seed germination of H macroceratitis has not been

previously reported. Stewart and Zettler (2002) reported the successful in vitro symbiotic seed

germination of this species; however, this aspect of the propagation of H. macroceratitis will be

discussed in the following section. Only one report exists of the asymbiotic seed germination of

a Habenaria species. Working with Habenaria radiata, a related species to H. macroceratitis,

Takahashi et al. (2000) reported an asymbiotic seed germination percentage of 48.8% on










Hyponex medium under continual light incubation (24/0 h L/D) after 28 days. While in this

previously published study, the asymbiotic seed germination of a Habenaria species was

examined, the authors failed to address factors that may effect efficient in vitro seed germination

and in vitro seedling development. In the present study, an efficient protocol for the asymbiotic

seed germination of H macroceratitis is provided, as well as an examination of factors that may

affect the in vitro seed germination of this species. The effects of photoperiod on in vitro

seedling development are also addressed, providing a special emphasis on tuber development.

While all media used in the current study contain a nitrogen source, the form and

concentration of that source differ among media (Table 3-1). Knudson C, MS and VW contain

only inorganic sources of nitrogen, while MM contains only an amino acid (i.e., organic source)

as the sole source of nitrogen in the media. Conversely, LN and ML contain a mixture of

inorganic nitrogen sources and an amino acid. Modified Lucke contained the lowest

concentration of nitrogen (4.98 mM), as prepared in this study. All media supported asymbiotic

seed germination and protocorm development to at least Stage 2, suggesting that nitrogen in any

form and concentration will support asymbiotic seed germination in H. macroceratitis.

However, ML contained the lowest concentration of nitrogen and still supported seed

germination and protocorm development to Stage 4. This suggests that H. macroceratitis may

require an overall low concentration of nitrogen for the initiation of seed germination and early

(
moisture to support seed germination. Only ML and MM initiated seed germination and

supported advanced protocorm development (Stage 4). Anderson (1996), Malmgren (1992,

1996), and Van Waes and Debergh (1986) reported that seed germination of terrestrial orchids

could be improved by the addition of amino acids to the germination medium and a reduction in










the concentration of inorganic nitrogen sources. These researchers have suggested that organic

sources of nitrogen (i.e., amino acids) may be more readily available to a seed or plant than an

analogous inorganic nitrogen source. The germination of seeds and development of protocorms

cultured in the presence of inorganic nitrogen sources may be delayed due to a delay in the

production of nitrate reductase, which has been shown to be produced only several months after

imbibition in Cattleya (Raghaven and Torrey 1964). Both ML and MM contain an amino acid as

a nitrogen source--with glycine being the sole nitrogen source found in MM and glutamine,

along with potassium nitrate and magnesium nitrate, being the sources of nitrogen in ML. It is

conceivable that seeds of H. macroceratitis more readily utilize the organic source of nitrogen

available in MM to support germination and protocorm development than nitrogen sources in all

other media tested. Little reliable information exists on the absolute role of organic nitrogen

sources in asymbiotic orchid seed germination media. Further investigation should be

undertaken.

The role carbohydrates play in orchid seed germination has received some attention,

although little recently. Early researchers realized some carbohydrates were better suited to

support asymbiotic orchid seed germination than other, and that this response may be species-

specific (for review see Arditti and Ernst 1984). While not testing the effects of carbohydrate

source on seed germination, Ernst et al. (1971) did demonstrate that a number of carbohydrates,

such as glucose, fructose, and oligosaccharides containing these sugars, can support the growth

and development of Phalaenopsis seedlings. Furthermore, Ernst and Arditti (1990)

demonstrated that seeds of both Phalaenopsis Habsburg and Phalaenopsis Ruth Burton x

(Phalaenopsis Abendrot x Phalaenopsis Abendrot) can utilize a wide variety of carbohydrate

sources--glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and









maltoheptaose--to support asymbiotic seed germination on Knudson C medium. A similar

result was found in the current study using simple carbohydrates in treatment combination with

MM to support the asymbiotic seed germination of the Florida terrestrial orchid H.

macroceratitis.

In the current study, the simple carbohydrates fructose, sucrose, and dextrose all supported

advanced (>Stage 3) germination and development of H. macroceratitis. Similarly, Ernst and

Arditti (1990) reported that seeds of the two previously mentioned Phalaenopsis hybrids

germinated rapidly on Knudson C asymbiotic medium supplemented with glucose, maltose, or

maltotriose, which are all simple carbohydrates. When larger maltooligosaccharides where used,

seed germination percentages decreased, likely due to the inability of the germinating orchid

embryo to synthesize the enzymes necessary to hydrolyze these more complex carbohydrates

(Ernst and Arditti 1990). A more recent report indicated that carbohydrate hydrolysis by

extracellular hydrolytic enzymes is possible, as demonstrated with protocorm-like bodies of

Dendrobium (Hew and Mah 1989). However, this extracellular hydrolysis of complex

carbohydrates (i.e., maltooligosaccharides) was not reported. A similar trend in decreased seed

germination percentage would be expected if larger, complex carbohydrates would be surveyed

for their ability to support the asymbiotic germination of H. macroceratitis.

While all carbohydrate treatments in the current study supported asymbiotic seed

germination through Stage 5, both controls (basal medium with and without BP) also supported

seed germination through Stage 5. A similar trend has not been previously reported in earlier

studies concerning the effects of carbohydrate type and concentration on asymbiotic seed

germination and in vitro seedling development. While a minimal, but unknown, amount of

carbohydrate is present in BP, the advanced seed germination observed in the basal medium with









BP treatment could be explained as being supported by this undefined carbohydrate source.

However, Stage 5 germination was also observed in the basal medium without BP control

treatment. The basal medium (MM) contains 0.05 mg 1-1 biotin and 100 mg 1-1 nyo-inositol, and

these supplements could serve as minimal sources of carbohydrate nutrition. Arditti (1979)

reported that biotin enhanced the growth of Cattleya, Odontoglossum, Phaphiopediums, and

Cymbidium; and that inositol stimulated the germination of Cattleya. Both of these supplements

in MM could be supporting the germination through Stage 5 of H. macroceratitis in the basal

medium without BP control.

Asymbiotic orchid seed germination media are typically supplemented with simple

carbohydrates (i.e., sucrose). While orchid seeds germinate and develop readily on these simple

carbohydrates, free simple carbohydrates, such as sucrose, can be considered ecologically

unimportant since carbohydrates rarely exist in this simple form in nature (Harley 1969).

Germinating seed and developing plants in the wild are dependant upon their mycobionts for the

breakdown of complex carbohydrates and transport of the resulting simple compounds into plant

tissues (Harley 1969; Smith 1966, 1967). Therefore, future studies on the effects of simple and

complex carbohydrates on the germination of orchid seed should be conducted under both

asymbiotic and symbiotic culture conditions.

Previous work on the role of various hormones, including cytokinins, during asymbiotic

seed germination have yielded inconclusive results--sometimes enhancing germination, other

times inhibiting, and still other times showing no apparent effect. This inconsistent response to

exogenous hormone application has even been shown to vary from genus-to-genus and species-

to-species (for reviews see Arditti 1967; Withner 1959; Arditti and Ernst 1984). The addition of

exogenous cytokinins are of particular interest in asymbiotic orchid seed germination because









several mycorrhizal fungi have been shown to produce trace amounts of cytokinins (Crafts and

Miller 1974), thus we can assume that orchid seed germination and development in the wild may

be assisted by these fungal-based cytokinins. In the current study, Kin, Zea and BA enhanced

asymbiotic seed germination of H macroceratitis. However, this positive response was only

seen at the 1 CIM level for BA and Zea, and at both the 3 CIM and 10 CIM levels for Kin. These

results indicate that H. macroceratitis seed germination is promoted to a greater extent in the

presence of Kin and Zea at relatively low concentrations than in the presence of BA (Figure 3-7).

Miyoshi and Mii (1998) found a similar response for Kin at 1 CIM in the asymbiotic seed

germination of Cypripedium muiulu ssllr, Sw.

The increase in asymbiotic seed germination percentage in response to the application of

low concentrations of exogenous cytokinins reported in both C. mattl ainlessr l (Miyoshi and Mii

1998) and H. macroceratitis (present study) is not surprising. While Crafts and Miller (1974)

reported the production of cytokinins by mycorrhizal fungi, the concentration of cytokinins

within fungal bodies was considered as trace amounts. If orchid mycobionts do indeed provide

cytokinins to germinating orchid seeds in situ, the amount of these cytokinins would be minimal.

This notion appears to be supported by the present in vitro studies with H. macroceratitis where

germination percentage was increased over the control only in the presence of low

concentrations of exogenous cytokinins.

The current study also demonstrated a reduction in germination in the presence of higher (3

CIM and 10 CIM) BA, Zea and Kin concentrations. Arditti et al. (1981) demonstrated a similar

repression of germination in Epipactis gigan2tea Dougl. ex Hook. in the presence of BA. Eighty

percent germination was noted on full-strength Curtis medium for E. gigan2tea, while full-

strength Curtis medium with the addition of 4.44 CIM BA produced only 10% germination.









Furthermore, white seedlings of E gigan2tea remained white and small (2-3 cm height) when

transferred from a medium containing no cytokinins to media containing various concentrations

of BA (Arditti et al. 1981). Conversely, preliminary study has demonstrated that seedlings of H.

macroceratitis cultured on medium containing Kin, Zea or BA show no abnormal coloration or

developmental morphologies when transferred to 16/8 h L/D conditions (S.L. Stewart

unpublished data). Obviously, the role of plant growth regulators, especially cytokinins, in

asymbiotic orchid seed germination is not well understood, and may be genus or species specific.

The role of photoperiod in orchid seed germination is often overlooked and is also not well

understood, especially in terrestrial species. In vitro seed germination of many terrestrial orchids

has been found to be inhibited by incubation in light (Van Waes and Deberg 1986; Arditti et al.

1981; Takahashi et al. 2000). Light also plays an important role in in situ seed germination and

seedling recruitment. Availability of light was found to be a limiting factor for Cypripedium

calceolus L. seedling recruitment in Europe (Kull 1998). In the current study, no effect of

photoperiod on initial (Stage 1) asymbiotic seed germination of H. macroceratitis was

demonstrated. However, a significant effect on subsequent protocorm growth and development

(>Stage 2), as well as morphology, among the photoperiods was shown.

Habenaria macroceratitis typically inhabits heavily shaded floors of hammock habitats

throughout central Florida. Rasmussen and Rasmussen (1991) surmised that the seeds of orchids

inhabiting shaded forest floors were not exposed to large quantities of red light, but in fact

exposed to far-red light. Seeds exposed to far-red light would convert phytochrome into the Pr

form, thus inhibiting germination (Kendrick 1976). Given that far-red light is known to inhibit

seed germination (Kendrick 1976) and cool white fluorescent tubes emit large amounts of red

light (Toole 1963), it is surprising that the current study found the highest percent seed









germination and most advanced (>Stage 4) protocorm development in a 0/24 h L/D photoperiod

(complete darkness). It is interesting that either the total absence of light or the total absence of

darkness stimulated seed germination to relatively high percentages (91.7% and 54.6%), while a

16/8 h L/D photoperiod actually suppressed seed germination (17.5%). A similar photoperiodic

response has not been reported in any terrestrial or epiphytic orchid species.

Seeds germinated in continual darkness produced numerous rhizoids over the surface of

the developing protocorm, while those seeds germinated in either light-including photoperiods

did not produce rhizoids (Figure 3-6). This is the first report of such a finding for any Habenaria

species. Arditti et al. (1981) reported that 'absorbing hair' (i.e., rhizoid) production was not

effected by photoperiod or movement from dark to light in a number of terrestrial orchid species,

including species of Platanthera and Piperia, which are both related to Habenaria. Under

natural conditions, seeds of terrestrial orchids are known to produce copious rhizoids

(Rasmussen 1995). These structures are used as entry points during infection by myconionts,

and would therefore be necessary for the prolonged existence of protocorms in nature.

The current study has demonstrated the effects photoperiod on both asymbiotic seed

germination and subsequent in vitro seedling development in H. macroceratitis. Tuber number

and size appear to be influenced by photoperiod, where culture under SD resulted in more tubers

per in vitro seedling, higher fresh and dry weights, and greatest tuber diameter when compared to

seedlings cultured under LD conditions. This represents the first report describing the

photoperiodic control of tuberization in a North American terrestrial orchid. The photoperiodic

control of tuberization is known from several important agronomic crops. Omokolo et al. (2003)

found that tuber formation in .Gllnthousina,~ sagittifolium (L.) Schott could be controlled through

the manipulation of photoperiod, in addition to sucrose and BA concentrations. The most










important of these controls was found to be photoperiod, where both SD and SD-dark conditions

produced the highest number of tubers per plant at both the 50 g 1-1 and 80 g 1-1 sucrose levels.

This photoperiod control of tuberization has also been demonstrated in Solan2um tuberosum L..

Seabrook et al. (1993) reported that the potato cultivars 'Katahdin' and 'Russet Burbank' formed

more microtubers under LD-SD, SD-SD and SD-LD conditions than they did under LD-LD

conditions. More importantly, the diameters and fresh weights of those tubers produced under

LD-SD conditions were significantly higher than all other photoperiod conditions. Therefore, it

is not surprising to find that tuber number and size is influenced by photoperiod in H.

macroceratitis.

Machaikova et al. (1998) found that tuberization in S. tuberosum was mediated by

photoperiod through control of hormone levels, most notably the ABA/GA ratio. Under SD

conditions, the ABA/GA ratio appears to be highly in favor of ABA, resulting in the initiation of

tuberization. Similarly, the application of exogenous ABA to in vitro S. tuberosum plantlets

stimulated tuberization (Xu et al. 1998). A similar photoperiod control of the ABA/GA ratio in

H. macroceratitis may be responsible for greater tuber number and size under SD conditions.

Further study is needed to elucidate this possible interaction.

Leaf production on in vitro cultured seedlings of H. macroceratitis was reduced under a

SD photoperiod; however, those leaves were longer and wider. Moreover, leaf size significantly

decreased from SD to LD conditions while the total number of leaves per seedling increased.

This trend of few but larger leaves under SD conditions and more but smaller leaves under LD

conditions may indicate the ability of H macroceratitis to optimize photosynthetic capacity for

either tuber storage allocation or vegetative growth, respectively. A similar trend of carbon

allocation toward a primary storage organ with increasing SD conditions was reported in the










long-lived perennial orchid Tipularia discolor (Pursh) Nutt. (Tissue et al. 1995). As the growing

season progressed, higher amounts of carbon were allocated to the primary corm as a means of

over wintering storage. This trend has previously been reported for 7: discolor (Whigham 1984;

Zimmerman and Whigham 1992), but never reported for the sub-tropical terrestrial orchid H.

macroceratitis. Further investigation is warranted into the carbon allocation strategy of H

macroceratitis in relation to photoperiod condition based on the current findings of more tubers,

larger tubers, and larger leaves under SD conditions.

The current study presents a first look at the asymbiotic seed germination requirements of

a rare sub-tropical terrestrial orchid from Florida, H. macroceratitis. Data are also presented

concerning the growth and development of in vitro seedlings. Given the rare status of this orchid

in the wild and the critically threatened status of its natural habitat, these data present critical

information on the previously unknown early life-history stages of this orchid. Although

asymbiotic seed germination ofH. macroceratitis represents an efficient means to propagate the

species, it does not account for the role of the species' naturally-occurring mycobionts. Without

these mycobionts seed germination and plant development ex situ would not occur. For this

reason, the in vitro symbiotic co-culture of H macroceratitis was explored.

Symbiotic Seed Germination

In vitro symbiotic co-culture is a powerful method for both the production of mycobiont-

infected seedlings for use in plant reintroduction and the study of mycobiont preference within

and among Orchidaceae taxa. Few reports exist concerning the in vitro symbiotic co-culture of

North American terrestrial orchid species, especially sub-tropical terrestrial species. This is only

the second report describing the successful symbiotic co-culture of a North American Habenaria

species, and a first report of a photoperiodic effect on the in vitro symbiotic seed germination of









H. macroceratitis. This report also represents the first description of possible in vitro fungal

preference displayed by H. macroceratitis.

Stewart and Zettler (2002) have previously reported the in vitro symbiotic co-culture of H

macroceratitis. In their study, seeds were cultured on OMA with a mycobiont originating from

the roots of H. quinqueseta (Michaux) Eaton (Hque-29 1), a closely related taxon to H.

macroceratitis, resulting in a maximum of 63.7% germination after 83 days. However,

maximum protocorm development (Stage 4) was reported from treatments that were cultured

with a mycobiont originating from Splitainhesl~ brevilabris (Sbrev-266; UAMH 9824). In Stewart

and Zettler (2002), the mycobiont isolate originating from H. quinqueseta was identified as

belonging to the anamorphic genus Ceratorhiza Moore, while the isolate from S. brevilabris was

identified as belonging to the anamorphic genus Epulorhiza. In the present study a similar seed

germination percentage (65.7%; Figure 3-13) was achieved; however, this percentage was

achieved in less time (58 days) than that reported in Stewart and Zettler (2002).

Additionally, all mycobionts isolated in the current study were assignable to the

anamorphic genus Epulorhiza. All six mycobionts closely resembled E. repens in having

comparable average growth rates and ovoid monilioid cells. The slight variations in culture

color seen among the isolates were not considered highly differential among all mycobionts and

likely due to inconsequential nutrient differences in the 1/5 PDA culture medium from Petri

plate-to-Petri plate (Currah et al. 1997). A degree of preference by H. macroceratitis for its

mycobiont, as demonstrated through mycobiont isolations, is likely responsible for the rapid in

vitro germination seen in some mycobiont-seed co-cultures in the current study. Stewart and

Zettler (2002) suggested that H. macroceratitis was non-preferential for mycobionts, whereas the

present study suggests that mycobionts isolated from vegetative, and therefore possibly younger,









plants of H macroceratitis better support in vitro symbiotic co-culture. Fungal preference in the

Orchidaceae has been considered controversial for many years (Curtis 1939; Hadley 1970;

Rasmussen and Rasmussen 2007). Differences in orchid fungal preference have been identified

under in vitro versus in situ conditions (Bidartondo and Bruns 2005; Masuhara and Katsuya

1994; Taylor and Bruns 1999; Taylor et al. 2003), and these differences have led some to

consider orchid fungal preference as generally low (Hadley 1970; Stewart and Zettler 2002).

Nonetheless, the present study demonstrates H. macroceratitis does possess a degree of

mycobiont preference under in vitro conditions. This species appears to be preferential for

mycobionts isolated from plants existing in the same population where seed was collected, since

mycobionts isolated from a distant population supported statistically lower seed germination

percentages. A more complete study testing mycobionts isolated from both vegetative and

flowering plants at multiple geographic sites throughout the entire range ofH. macroceratitis is

suggested to further elucidate the apparent mycobiont preference found in this species.

Few reports exist concerning photoperiodic effects on the in vitro symbiotic co-culture of

terrestrial orchids. Typically, the seeds of terrestrial orchids germinate while buried in the soil,

but are initially exposed to a short period of illumination upon capsule dehiscence (Rasmussen et

al. 1990). Stewart and Zettler (2002) reported no increase in initial symbiotic seed germination

(Stage 1) when cultures of H macroceratitis were transferred from continuous dark conditions to

a 12/12 h L/D photoperiod; however, an increase in early protocorm development was reported.

Rasmussen and Rasmussen (1991) and Zettler and McInnis (1994) both reported a reduction in

asymbiotic and symbiotic seed germination percentage of Dactylorhiza majalis (Rchb.) P.R.

Hunt & Summerhayes and Platanthera integralabia, respectively, under conditions of a dark

pretreatment of seed before light exposure. A similar response was found in the current study.









Initial seed germination (Stage 1) was significantly lower under the 0/24 h L/D photoperiod than

either the 16/8 h L/D or 24/0 h L/D photoperiods (Figure 3-12). However, protocorm

development was most advanced (Stage 2) under the 0/24 h L/D photoperiod (Figure 3-11). As

mentioned previously, this reduction in initial seed germination percentage may be due to the

lack of a light pretreatment of the seed prior to sowing. Orchid seeds typically possess a

hydrophobic testa that allows a seed to remain above the soil surface where they can be exposed

to sunlight (Rasmussen and Rasmussen 1991). This light exposure may only initiate nutrient

mobilization or assist in imbibition and not lead directly to seed germination (Rasmussen and

Rasmussen 1991). Interestingly, Takahashi et al. (2000) reported no significant difference in in

vitro symbiotic seed germination percentages when seeds of Habenaria radiata were cultured

under continual darkness (0/24 h L/D) or 24/0 h L/D conditions. This may indicate that

terrestrial orchid seed germination response to photoperiod may be genus or species specific.

The current study presents new findings on the in vitro fungal preference and effects of

photoperiod on the in vitro symbiotic co-culture of a rare sub-tropical terrestrial orchid from

Florida, H. macroceratitis. The rare status of this orchid in the wild and the threatened status of

its natural habitat necessitate the development of efficient symbiotic seed germination protocols,

otherwise the species may not exist as an independent organism in its natural habitat for long.

These data present invaluable information concerning a previously unknown fungal preference

within populations ofH. macroceratitis. This information will be critical to future plant

production and reintroduction efforts aimed at the conservation of H macroceratitis in its natural

habitat.

Implications for Integrated Conservation Planning

The present studies on the in vitro seed culture of the Florida terrestrial orchid H.

macroceratitis have demonstrated the benefits of a careful study of the propagation science--










asymbiotic media selection, carbohydrate preference, application of exogenous cytokinins, dark

incubation during seed germination, and symbiotic co-culture requirements--of an orchid

species before implementing conservation and recovery plans. The usefulness of asymbiotic

culture techniques in the study of orchid seedling growth and development was also

demonstrated. These results show the importance of understanding an orchid's germination

requirements, in addition to a species' early growth and development stages, before attempting

further integrated conservation and species recovery efforts. For example, by demonstrating that

tuber formation in H. macroceratitis can be induced in vitro by incubation of plants under SD

conditions, an efficient means of tuber production as a source of plant material for reintroduction

purposes can now be investigated and applied to the management of this terrestrial orchid species

(Stewart and Kane 2006a).

Furthermore, the present studies on the in vitro symbiotic co-culture of H macroceratitis

demonstrated not only that the symbiotic co-culture of this species was possible, but also

elucidated a potential habitat-limited mycobiont preference within this taxon. Identifying an

apparent mycobiont preference that may be locally specific within the range ofH. macroceratitis

in Florida has a number of integrated conservation and species management implications. Of

primary interest is the result that H. macroceratitis is likely not preferential for only one or two

strains of Epulorhiza mycobiont throughout Florida, but that the species demonstrates a

preference for mycobionts that may be locally or site limited (Stewart and Kane 2006b). This

has a number of implications for the integrated conservation of H. macroceratitis in Florida.

Specifically, existence of a site limited mycobiont preference within the species means that

strains of H macroceratitis mycobionts from each known site in Florida will need to be isolated

and maintained in pure culture in order to insure the long-term conservation of the species.









Furthermore, seed from each known site must also be collected and stored if future in vitro

symbiotic co-culture production is to be considered as a viable tool in the long-term integrated

conservation of H macroceratitis.

The degree of mycobiont preference shown in the symbiotic co-culture of H

macroceratitis demonstrates the need for proper management for both plant and mycobiont

populations. While managing habitats for plant population sustainability can be accomplished

by maintaining the basic plant and soil structure historically and presently found in north and

central Florida hardwood hammocks containing populations of H macroceratitis, managing sites

for mycobiont diversity and sustainability is an unexplored area of orchid integrated conservation

biology. Further study is necessary before mycobiont management within orchid habitats is a

useful tool to insure the long-term sustainability of native orchid populations, including

populations of H. macroceratitis. However, to secure the long-term sustainability of native

orchid populations, entire habitats must be thought of as refugia for orchid mycobionts and

should be managed accordingly.

The current studies concerning the propagation science of H macroceratitis represent one

step in the species-level integrated conservation of this rare terrestrial orchid species in Florida

(Figure 1-1). In developing a more complete understanding of the asymbiotic and symbiotic

propagation requirements of H macroceratitis in Florida than that discussed by Stewart and

Zettler (2002), the present studies have suggested efficient means of both the asymbiotic and

symbiotic propagation of the species that will lead to the production of plant material for species

reintroductions.










Table 3-1. Comparative mineral salt, vitamin, and amino acid content of asymbiotic orchid seed
germination media used in the asymbiotic germination of Habenaria macroceratitis:


Knudson C (KC), Malmgren Modified Terrestrial
& Skoog (MS), Vacin & Went (VW), Lindemann
present (-).
KC MM MS


Orchid Medium (MM), Murashige
(LM), Modified Lucke's (ML), not


VW


3.78
0.4


1.01
5.19
7.03
2.24
5.3
0.1


LM


Macronutrients (mM)
Ammonium
Calcium
Chlorine
Magnesium
Nitrate
Potassium
Phosphate
Sulfate
Sodium
Micronutrients (CLM)


13.82
2.12
3.35
1.01
10.49
5.19
1.84
4.91


20.61
3
3
1.5
39.4
20.05
1.25
1.84
0.1


15.14
2.12
14.08
0.49
2.12
15.07
0.99
8.1


0.09



0.43
4.89
203.76
199.8


0.24


0.81


0.55
0.71
0.92
0.2


Boron -
Cobalt -
Copper -
Iron 90
Iodine -
Manganese 30
Molybdenum -
Nickel -
Zinc -
Vitamtttttttt~~~~~~~~~in & Amino Acids (mg/1)
Biotin -
Casein hydrolysate -


-100.27
0.19
~0.16
100 183


10 111.91
1.54


-53.26


16.4


-0.1
183 17.96
-0.6
33.7 37.63


94.3


0.24
3.5


0.05
400
0.5
2
100


Folic acid
Glycine
myo-Inositol
Nicotinic acid
Peptone
Pyridoxine
Thiamine
Total N (mM)

NH4' :NO3-


24.31 unpublished

1.32 unpublished


60.01


8.97 17.26 4.98


0.52 0.73


7.14 0.02









Table 3-2. Seed germination and protocorm development stages in Habenaria macroceratitis,
adapted from Stewart and Zettler (2002) and Stewart et al. (2003).
Stage Description
0 No germination, viable embryo
1 Swelled embryo, production of rhizoid(s) (=germination)
2 Continued embryo enlargement, rupture of testa
3 Appearance of protomeristem
4 Emergence of first leaf
5 Elonnation of first leaf










Table 3-3. Sources of mycobionts used in the in vitro symbiotic co-culture of Habenaria
macroceratitis.
Isolate Host Collection Information Identification
Hmac-309 Vegetative plant 27 September 2003, Hemnando Co., FL Epulorhiza sp.
Hmac-310 Vegetative plant 27 September 2003, Hemnando Co., FL Epulorhiza sp.
Hmac-311 Vegetative plant 27 September 2003, Hemnando Co., FL Epulorhiza sp.
Hmac-312 Flowering plant 30 September 2003, Sumter Co., FL Epulorhiza sp.
Hmac-313 Flowering plant 30 September 2003, Sumter Co., FL Epulorhiza sp.
Hmac-314 Flowering plant 30 September 2003, Sumter Co., FL Epulorhiza sp.























































Figure 3-1. Seed germination and protocorm development stages in Habenaria macroceratitis,
adapted from Stewart and Zettler (2002). Stage 0 = no germination, viable embryo.
Stage 1 = swelled embryo, production of rhizoids (arrow; = germination). Stage 2 =
continued embryo enlargement, rupture of testa. Stage 3 = appearance of
protomeristem. Stage 4 = emergence of first leaf. Stage 5 = elongation of first leaf.
Scale bars = 1 mm.











M ML b b 16 weeks
100 -MM
I ILM


80 -MK



o 60-



a, 40 I I


a a I b
20 -( La a




7 weeks
100-
b 't


80-






aba





20- c
c c d bcd



Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

Germination Stage


Figure 3-2. Effects of culture media on percent germination and protocorm development of
Habenaria macroceratitis after 7 and 16 weeks in vitro asymbiotic culture. Histobars
with the same letter are not significantly different within stage (a = 0.05). Error bar =
SE. Modified Lucke (ML), Murashige & Skoog (MS), Lindemann (LM), Vacin &
Went (VW), Malmgren Modified (MM), and Knudson C (KC).








































































Figure 3-3. Effects of carbohydrate type (fructose, sucrose, and dextrose) and presence or
absence of banana powder (+/- BP) on percent germination and protocorm
development of Habenaria macroceratitis after 7 weeks in vitro asymbiotic culture
on Malmgren Modified Terrestrial Orchid Medium. Histobars with the same letter
within each stage are not significantly different (a = 0.05). Error bar = SE.














131


4~e 4~e si


4~ ~SI "'


I


_ I


I


I


Stage 0


Stage 1


S+ BP
I -BP


Stage 2


Stage 3


ab


Stage 4


Stage 5


Fructose Sucrose Dextrose


Control


Fructose Sucrose Dextrose Control


Carbohydrate Treatment


Carbohydrate Treatment













I


Stage 0


Stage 1


S+ BP
I -BP


Stage 2


Stage


Stage 4


abc


Fructose Sucrose Dextrose

Carbohydrate Treatment


0


bc" bc bb


Fructose Sucrose Dextrose

Carbohydrate Treatment


Control


Control


Figure 3-4. Effects of carbohydrate type (fructose, sucrose, and dextrose) and presence or
absence of banana powder (+/- BP) on percent germination and protocorm

development of Habenaria macroceratitis after 2 1 weeks in vitro asymbiotic culture

on Malmgren Modified Terrestrial Orchid Medium. Histobars with the same letter

within each stage are not significantly different (a = 0.05). Error bar = SE.

















5 0


4 0


~~30




20



SBCA O 0




Figure 3-5. Effects of four cytokinins (BA, Zea, Kin, 2-iP) and four concentrations (0, 1, 3, 10
CIM) on percent seed germination of Habenaria macroceratitis after 14 weeks in vitro
asymbiotic culture on Malmgren Modified Terrestrial Orchid Medium. Histobars
with the same letter are not significantly different (a = 0.05). Benzyladenine (BA),
zeatin (Zea), kinetin (Kin), and 6-(y,y-dimethylallylamino) purine (2-iP).










S0/24 h L/D Photoperiod
S16/8 h L/D Photoperiod a
I 24/0 h L/D Photoperiod T


100 -


80 -


60 -


40 -


Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5
Developmental Stage

Figure 3-6. Effects of three photoperiods (0/24, 16/8, 24/0 h L/D) on in vitro asymbiotic seed
germination and protocorm development of Habenaria macroceratitis cultured on
Malmgren Modified Terrestrial Orchid Medium after 14 weeks. Histobars with the
same letter are not significantly different within stage (a = 0.05). Error bar = SE.


a a
a

ii ii


aa


































Figure 3-7. Morphological effects of three photoperiods (0/24, 16/8, 24/0 h L/D) on
asymbiotically germinated Habenaria macroceratitis seeds cultured on Malmgren
Modified Terrestrial Orchid Medium after 14 weeks. A) Protocorm development and
morphology under 0/24 h L/D, note numerous rhizoids. B) Protocorm development
and morphology under 16/8 h L/D, note lack of rhizoids. C) Protocorm development
and morphology under 24/0 h L/D, note lack of rhizoids. Scale bar = 1 mm.











S8/16 h L/D Photoperiod
S12/12 h L/D Photoperiod
S16/8 h L/D Photoperiod











gb b


b

8r


2.5 -



2.0 -


1.5 -


1.0 -



0.5 -



0.0 '


Tubers Leaves


Figure 3-8. Effects of three photoperiods (8/16, 12/12, 16/8 h L/D) on tuber and leaf production
per in vitro Habenaria macroceratitis seedling cultured on Malmgren Modified
Terrestrial Orchid Medium after 20 weeks. Histobars with the same letter are not
significantly different within group (a = 0.05). Error bar = SE.











S8/16 h L/D Photoperiod
S12/12 h L/D Photoperiod
I 16/8 h L/D Photoperiod


60




-
.c 40





20 -


Shoot fwt


Shoot dwt


b b


01


Tuber fwt


Tuber dwt


Tuber and Shoot Biomass


Figure 3-9. Effects of three photoperiods (8/16, 12/12, 16/8 h L/D) on tuber and shoot biomass
of in vitro Habenaria macroceratitis seedlings cultured on Malmgren Modified
Terrestrial Orchid Medium after 20 weeks. Histobars with same letter are not
significantly different within group (a = 0.05). Error bar = SE.





S8/16 h L/D Photoperiod
S12/12 h L/D Photoperiod
I 16/8 h L/D Photoperiod


a a


Tuber Diameter Tuber Length Leaf Length


Figure 3-10. Effects of three photoperiods (8/16, 12/12, 16/8 h L/D) on tuber diameter and
length and leaf length and width of in vitro Habenaria macroceratitis seedlings
cultured on Malmgren Modified Terrestrial Orchid Medium after 20 weeks.
Histobars with the same letter are not significantly different within group (a = 0.05).
Error bar = SE.





















138


Leaf Width














































Figure 3-11. Examples of mycobionts isolated from Habenaria macroceratitis (Hmac). A)
Hmac-309 whole culture morphology at 3 weeks, scale bar = 1 cm. B) Hmac-309
monilioid cells stained with acid fuchsin at 4 weeks (400 x), scale bar = 10 lm. C)
Hmac-312 whole culture morphology at 3 weeks, scale bar = 1 cm. D) Hmac-312
monilioid cells stained as above at 4 weeks (400x), scale bar = 10 lm.










00

M Hmac-309 a
I Hmac-310
I Hmac-311
SHmac-312
60 Hmac-313 ab
0 Hmac-314 ab b
b
Sa b b
c ab a
o ab
40 -ab

r a
t Iab
ab
20 ab







Stage 0 Stage 1 Stage 2

Developmental Stage


Figure 3-12. Effects of six mycobionts on percent germination and protocorm development of
Habenaria macroceratitis cultured on oat meal agar (OMA) after 8 weeks symbiotic
in vitro culture. Histobars with the same letter are not significantly different within
stage (a = 0.05). Error bar = SE.








60


50 -


40 -


30


20


b


b


i


Stage 1


Stage 0


Stage 2


Developmental Stage


Figure 3-13. Photoperiodic effect (0/24, 16/8, 24/0 h L/D) on in vitro symbiotic seed
germination and protocorm development of Habenaria macroceratitis cultured on oat
meal agar (OMA) with mycobiont Sbrev-266 after 14 weeks. Histobars with the
same letter are not significantly different within stage (a = 0.05). Error bar = SE.


M 0/24 h L/D

I 24/0 h L/D




























Figure ~ ~ ~ ~ ~ ~ 3-4 htproi fet /4 68 40hLD ntein ir yboi rtcr
deeopet fHaearamarceaitsusn mcbin Srv-6 clurdo
oat: melaa fe 4wes )Pooomdvlpmetudr /4hL/,nt
rhzod eelpmn. B Prtcr developetudr1/ /,nt ako
rhzod eelpmn. C rtcomdvlomn ndr2/ hLD ot ako
rhizod devlopmnt. Sale ars =1 mm









CHAPTER 4
POLLINATION BIOLOGY AND GENETIC DIVERSITY OF Split anthe f\ loridanadd~~~~~ddddd~~~~

Introduction

Knowledge of a rare plant' s reproductive biology and genetic diversity are critical factors

in its conservation and management. In understanding a species mode of reproduction--sexual

or asexual--attention can be drawn to ecological factors and conservation needs for that species.

Since reproduction is both an important means of increasing the number of individuals within a

population and the primary way to maintain genetic diversity (Sipes and Tepedino 1995), an

understanding of both pollination biology and genetic diversity is necessary in measuring the

fitness of rare plants. While a number of studies exist concerning the pollination biology and

genetic diversity of orchid species, these two factors are usually treated as separate from one

another. Few studies exist concerning the integration of pollination biology and genetic diversity

in orchids (Sun 1997, 1996; Sun and Wong 2001; Wong and Sun 1999; Sipes and Tepedine

1995).

Pollination biology represents an essential part in the conservation and species recovery

planning for rare plants. This is particularly important in the Orchidaceae, since most orchid

species have developed highly specialized pollination mechanisms (Tremblay 1992; Catling and

Catling 1991; Bowles et al. 2002). Members of the Orchidaceae are known to possess a variety

of pollination mechanisms and pollinators (Catling 1983a, 1983b, 1982; Dodson and Frymire

1961; Stoutamire 1975; Darwin 1869); however, pollinators are often species-specific (Tremblay

1992). Due to pollination mechanism variety and species-specific pollinators, careful study of

the pollination biology of orchids is crucial in developing a more complete ecological and

conservation understanding of individual species.









Population genetic diversity represents another component in developing a better

understanding of the conservation biology needs of individual orchid species and their

populations. Qamaruz-Zaman et al. (1998a) suggests that the unchecked loss of genetic diversity

within and between populations can result in the limiting of adaptive potential in orchid

populations. Furthermore, the maintenance of genetic diversity can be a critical factor in the

long-term persistence of plants in a changing environment (Frankel and Soule 1981; Lande and

Barrowclough 1987). An understanding of within- and between-population genetic diversity is a

critical step in the conservation of plant species (Lande 1988), particularly orchid species.

The measurement of population genetic diversity requires the use of molecular marker

technologies. Recently, a number of molecular-based methods have been used in the

measurement of orchid population genetic diversity, including isozyme electrophoresis (Sun

1997, 1996), random amplified polymorphic DNA (RAPD; Sun and Wong 2001; Wong and Sun

1999), DNA sequencing (Szalanski et al. 2001), and amplified fragment length polymorphism

(AFLP; Forrest et al. 2004). The AFLP method represents one of the most attractive tools to

measure population genetic diversity because of the system's reproducibility, the small amount

of genomic DNA necessary, and its ability to resolve multiple polymorphic bands (Mueller and

Wolfenbarger 1999; Ridout and Donini 1999; Forrest et al. 2004; Chen et al. 1999; Lin et al.

1996).

The terrestrial orchid Splitatl fIlll\lori~ddana~ was chosen as the species in these studies

because no information exists on the reproductive biology or genetic diversity of this species

(Figure 1-3b). Several reports exist concerning the reproductive biology (Singer 2002; Sun

1997, 1996; Calvo 1990; Catling 1987; Sheviak 1982; Wong and Sun 1999; Antlfinger and

Wendel 1997; Sipes and Tepedino 1995; Schmidt and Antlfinger 1992) and population genetic










diversity (Sun 1997, 1996; Wong and Sun 1999; Sipes and Tepedino 1995) of other Splitanihesl~

and allied Spiranthinae species. In the present studies, data are presented concerning the

reproductive and pollination biology and within-population genetic diversity of S. foridana.

Reference is also made to the general plant demography and ecological habitat profile of this

species. These studies represent one step in the integrated conservation and species-level

recovery of S. foridanadd~~~~~ddddd~~~~ in Florida.

Materials and Methods

Study Sites

One site was chosen for these studies: Rayonier (Bradford County; Figure 4-1). At the

time of these studies, the Rayonier site was the only known S. foridanadd~~~~~ddddd~~~~ site in the southeastern

United States. The Rayonier site was located within 6 km of Starke, Florida at an elevation of 47

m above sea level. Data on associated plant species were recorded at this site yearly from 2003-

2005.

Plant Demography

In 2003, demographic monitoring of the entire Rayonier population was initiated. From

2003-2005 this monitoring site was visited weekly during the active growth cycle of S. Joridanadd~~~~~ddddd~~~~

(1 March-30 June). Data on number of vegetative and flowering plants, number of leaves per

plant, length of longest leaf per plant (cm), number of flowers per plant, height of inflorescence

per plant (cm), and number of flowers setting seed was collected and averaged per data

collection year. These yearly data were then averaged over all data collection years, and

analyzed using basic descriptive statistics.

Pollinator Observations

Non-destructive pollinator observations were conducted at the Rayonier site on 7 and 13

April 2003. The entire Rayonier population of S. foridanadd~~~~~ddddd~~~~ was selected for observation









activities because of its small, defined plant distribution at the site. Observation methods

followed those of Zettler et al. (1996) for Platanthera integrilabia modified for daytime-only

observations based on Catling (1987) for Sacoila lan2ceolata (Aublet) Garay var. lanceolata, a

Spiranthinae ally of S. floridana.d~~~~~ddddd~~~~ A circular path around the entire Rayonier population was

established that allowed easy observation of 21 individual inflorescences. Observations occurred

over a 6-hour period on 7 April 2003 beginning at 0600 hours and ending at 1800 hours.

Observations on 13 April 2003 occurred over a 6-hour period beginning at 0500 hours and

ending at 1700 hours. Both observation periods occurred without interruption. All data were

recorded in fair weather and no insect repellant or insect attractants were used within the study

area. Observations occurred in the first and last 20 minutes of each hour during each observation

period by walking the preestablished path in a counterclockwise direction around the population.

Pollinators were recorded as those insects carrying at least one pollinium after a flower visit,

while visitors were recorded as those insects lacking pollinia. Relative humidity and temperature

were recorded at the Rayonier site during pollinator observations using two HOBO H8 data

loggers. One data logger was placed at ground level, while the second was placed 25 cm above

ground level. This height was chosen to parallel the average inflorescence height of S. floridanadd~~~~~ddddd~~~~

at this site. Further, flower nectar volume and sugar concentration data was not taken for S.

floridanadd~~~~~ddddd~~~~ since preliminary studies demonstrated this species to not produce nectar as a

pollinator reward.

Pollination Mechanism

A pollination mechanism study was designed to investigate the breeding system of S

floridanadd~~~~~ddddd~~~~ following the procedures of Wong and Sun (1999), modified by the inclusion of a

seventh pollination condition-self-pollination (Table 2-1). The mechanism experiment was

conducted at the Rayonier site during the 2004 flowering period. Fourteen plants, two per









experimental pollination condition, with pre-anthesis inflorescences were bagged with a fine

plastic mesh (1 mm2 meSh size) stretched over a 1 m tall wire frame. The mesh covered wire

frame allowed the S. floridanadd~~~~~ddddd~~~~ inflorescences to develop normally while excluding pollination

events from occurring prior to the initiation of pollination mechanism studies.

Each experimental pollination treatment was applied to two flowers on each inflorescence

(Table 2-1). A total of 196 flowers were used in the pollination mechanism study. However,

during application of pollination treatments the lack of pollen production in flowers of S.

floridanadd~~~~~ddddd~~~~ was discovered and the pollination mechanism study terminated. Thereafter, the mesh-

covered wire frames were removed from all plants.

Sampling, DNA Extraction, and Amplified Fragment Length Polymorphism (AFLP)

Fresh, green leaves of S. floridanadd~~~~~ddddd~~~~ were collected from the Rayonier site in 2003 and

placed in 50 mL plastic tubes containing silica gel desiccant (Chase and Hills 1991; W.M.

Whitten personal communication). Leaf samples were removed from each plant using scissors,

which were washed with 95% ethanol and allowed to air dry between each sample to minimize

sample cross contamination (M.W. Whitten personal communication). All 23 plants present in

2003 were sampled. Samples were stored at room temperature (ca. 250C) until used in DNA

extraction protocols.

Once all samples were thoroughly dried, genomic DNA was extracted using the DNeasy"

Plant Mini Kit. Manufacturer's instructions were followed with the following modifications:

eluates were not pooled and the second elution was retained only as a precaution. The DNA was

quantified using an Agilent Technologies NanoDrop spectrophotometer.

Template DNA was prepared using the Applied Biosystems AFLP" Plant Mapping

protocol and kit-based system (ABI 2005). Preselective and selective amplification procedures

were identical to those discussed in Chapter 2 for Habenaria macroceratitis, and used the same









endonucleases, primer combinations, and PCR conditions (Table 2-2). AFLP reaction samples

were prepared for sequencing in the same manner as previously outlined, and sequencing

conditions were identical.

As with the AFLP fragment data from H. macroceratitis, a matrix containing all fragments

data from S. floridanadd~~~~~ddddd~~~~ ranging from 50 to 500 bp was compiled. These data were analyzed using

GeneMarker software. Fragements with a low signal (<2% full detection level) were excluded

from the analysis. Recognized bands were scored as present (1) or absent (0).

POPGENE 1.31 (Yeh et al. 1999) was used to estimate Nei and Shannon diversity indices.

Within and between population genetic structure (FST) WAS estimated using a combination of

results generated with POPGENE and equation calculations with 1000 permutations. Genotype

correlation dendrograms were constructed using GeneMarker version 1.6 software and the

included cluster analysis tool.

Results

Study Site

Before development into a rural home site, the Rayonier study site would have likely been

classified as a pine flatwood (Chafin 1990; S.L. Stewart personal observation). An adjacent pine

flatwood dominated by longleaf pine (Pinus palustris Mill) remains and provides the only

indication to the site's ecological history. Presently, the Rayonier site is treeless, with the

exception of one remnant longleaf pine, and dominated by Common Bermudagrass (Cynodon

dactylon (L.) Pers.). The site also consists of a small home, rock driveway and parking area, and

a moderately-sized barn.

Plant Demography

From 2003-2005, the number of S. floridanadd~~~~~ddddd~~~~ plants at the Rayonier study site ranged from

17 to 32 (ave. = 23.8 +2.52). In each study year, 100% of the plants flowered and no vegetative










plants were observed in any study year. No seedling plants were observed during the study

period. Plants ranged in height from 20.3 cm to 36.8 cm (ave. = 28.7 11.35). During study years

2003-2005, the number of flowers per plant ranged from 6 to 31 (ave. = 17.9 11.53), while the

number of flowers appearing to set seed ranged from 0 to 17 (ave. = 7.2 11.45). Leaf number

and length of longest leaf during study years 2003-2005 ranged from 0 to 4 (ave. = 1.6 10.22)

and 0.25 cm to 3.8 cm (ave. = 2.6 10.24), respectively.

Pollinator Observations and Pollination Mechanism

No pollinators or visitors of flowers of S. floridana were noted during either the 7 April

2003 or 13 April 2003 pollination observation periods at the Rayonier site. Furthermore, no

floral scent was noted at any time during the pollinator observations. Temperature at the site

during observations ranged between 21.50C-29.90C (ave. = 26.40C; 7 April; Figure 4-2a) and

9.40C-29.40C (ave. = 22.70C; 13 April; Figure 4-3b). Site relative humidity ranged between

54%-93% (ave. = 67.5%; 7 April; Figure 4-2a) and 23%-97% (ave. = 47.8%; 13 April; Figure 4-

3b).

During the application of experimental pollination treatments, it was noted that flowers of

S. floridanadd~~~~~ddddd~~~~ at the Rayonier study site lacked pollinia despite having a fully formed anther cap

and pollinial depression (Figure 4-4a-c). Further observations of 20 randomly selected flowers

in various stages of development (tight bud to fully open) from the Rayonier study site revealed

the same lack of pollinia in all flowers sampled. Splitatl fIlll\lori~ddana~ at the Rayonier site

appears to not produce pollinia, although ovaries do enlarge after each flower matures. Based on

these observations, it appears that S. floridanadd~~~~~ddddd~~~~ reproduces asexually by agamospermy; however,

immature seed capsules abort approximately three-fourths through the maturation process. This

phenomenon was observed in 100% of capsules (Figure 4-4d).









AFLP Data

Amplification of all four primer pairs tested was successful in all 23 individual samples.

The output gel image is given in Figure B-3. Tthe -ACT/-CAG and -AGC/-CAG primer pairs

resulted in the highest polymorphic band resolution, averaging 37 and 56 bands per individual,

respectively. Bands that were either present or absent in a single sample were excluded from

analysis as likely being artefactual (Pillon et al. 2007). From both primer pairs, a total of 36

unambiguous polymorphic bands were selected. When combining these 36 bands, 4 genotypes

could be distinguished. For this population of S. floridanad~~~~~ddddd~~~~ Nei's diversity index = 0.293 and

Shannon's index = 0.165. In examining the within population genetic differentiation, FST = 0.03,

suggesting very little differentiation within the sampled population. A genotype correlation

dendrogram is given in Figure B-7.

Discussion

Information concerning the demography, pollination biology, and population genetic

diversity of S. floridanadd~~~~~ddddd~~~~ has not been previously reported. Results from these studies suggest the

species is artificially imperiled due to the lack of a breeding system, low genotypic diversity, and

low genetic differentiation within the one population sampled. Reports concerning the

demography and ecology of Split anthe,~ species, or that of any other Spiranthinae species, are

limited (Calvo 1990; Hutchings 1987a, 1987b, Tamm 1972; Wells 1967; Willems and Dorland

2000; Jacquemyn et al. 2007). The majority of these ecological reports are in support of

taxonomic descriptions or revisions (Catling 1981, 1983; Sheviak 1982; Catling and Cruise

1974) or biosystematic studies of unique regional ecotypes (Sheviak 1982). Demographic and

life history trait data represent important factors that may contribute to the understanding of

evolutionary processes (Willems and Dorland 2000). This lack of basic demographic data on









.gliltaidesr' species, especially rare and endemic species such as S. floridanad~~~~~ddddd~~~~ has led to a lack of

understanding of life history traits in this orchid genus.

Jacquemyn et al. (2007) reported on the long-term population dynamics and viability of the

European terrestrial orchid S. spiralis (L.) Chevall., a related species to S. floridana (Dueck and

Cameron 2007) over a 24 year period. Like S. floridanadd~~~~~ddddd~~~~ at the Rayonier study site, S. spiralis is

known from open meadows where land-use is constant. Despite these similarities, S. spiralis

demonstrated extreme variability in the percentage of flowering individuals per year (0-100%)

and the total number of individuals per year (6-79). .gitaIllr~Joides lordana~ showed a high degree

of stability in both the total number of individuals per monitoring year (23.8 +2.52) and the

percentage of flowering plants per year (100%); however, this stability is somewhat irrelevant

since no reproduction or recruitment is present. Jacquemyn et al. (2007) suggest that the

plasticity seen in total individuals and flowering versus vegetative growth habits in S. spiralis is

advantageous to the long-term viability of S spiralis populations. According to their

calculations of extinction probabilities, S. spiralis has a 79% probability of surviving the next 20

years. While an extinction probability was not calculated for S. floridana in Florida, the lack of

population plasticity measured in the Rayonier population suggests that S. floridanadd~~~~~ddddd~~~~ would have

a low probability of surviving over the next 20 years.

Willems and Dorland (2000), studying the flowering frequency and plant performance of

S. spiralis, reported a decrease in the percentage of flowering plants every year over five

consecutive flowering years. They suggest that this decrease in flowering plant percentage is

due to the negative costs of repetitive reproduction of individual plants year-after-year. A

similar trend in generative reproduction costs has been reported in Cypripedium acaule Aiton

(Primack and Hall 1990; Primack et al. 1994) and Tipularia discolor (Snow and Whigham









1989). In the present studies with S. floridanad~~~~~ddddd~~~~ while the total number of plants per year

decreased at the Rayonier study site, the percentage of flowering plants never decreased.

Interestingly, those plants that reappeared in each study year flowered every study year. This

observation does not follow the trend of a high cost of successive generational reproduction from

year-to-year that is seen in most orchid species (Calvo 1993).

The number of S. floridanadd~~~~~ddddd~~~~ plants present at the Rayonier study site decreased by 54%

during the study period. This decrease could be attributed to plant mortality, in some cases;

however, it is possible that some plants transitioned to an underground dormant life stage.

Unfortunately, a more detailed life history of S. floridanadd~~~~~ddddd~~~~ is not available. Calvo (1990) reported

a similar phenomenon in the Spiranthinae orchid Cyclopogon cranichoides (Grisebach) Schl.,

where populations declined by an average of 20% each year and this decline was mostly

attributed to plant dormancy. Further study concerning the under- and above-ground life history

of S. floridanadd~~~~~ddddd~~~~ is necessary; however, without new populations these studies can not be afforded

without further damage to the existing populations.

Breeding system, and therefore pollination biology, has a profound effect on not only the

long-term population viability of plants, but also a profound effect on population genetic

diversity (Hamrick 1982). Unfortunately, pollination biology in the Orchidaceae is often

considered understudied despite a renewed interested over the past decade (Catling 1990;

Trapnell and Hamrick 2006). Tropical orchid species have received the most pollination biology

attention in recent years (Blanco and Barboza 2005; Trapnell and Hamrick 2006, 2005; Singer

and Koehler 2003; Borba et al. 2001). However, the temperate orchid subtribe Spiranthinae has

been the focus of a great deal of pollination biology research (Catling 1987, 1986, 1983, 1982;

Sipes and Tepedino 1995; Schmidt and Antlfinger 1992), particularly in the relationship between









breeding system and population genetic diversity (Sun 1997, 1996; Sun and Wong 2001; Wong

and Sun 1999).

A number of Spiranthinae species are known to require pollinia transfer to achieve

germination: Sauroglossum elatum Lind. (Singer 2002), .gitaidesrl~ diluvialis Sheviak (Sipes and

Tepedino 1995), and Goodyera procera (Ker-Gawl.) Hook (Wong and Sun 1999). However, the

greater number of Spiranthinae are known to be agamospermic: Sacoila lan2ceolata var.

lan2ceolata (Catling 1987), S. lan2ceolata var. paludicola (Luer) Sauleda (Catling 1987),

.litainhesl~ australis (R. Brown) L. (Ridley 1888), S. ca~sei Catling & Cruise (Catling 1982), S.

ca~sei var. novaescotiae Catling (Catling 1982), S. cernua (L.) L.C. Richard (Sheviak 1982;

Schmidt and Antlfinger 1992), S. hongkongensis Hu & Barr. (Sun 1997), S. magnicamporum

Sheviak (Catling 1982), S. ochroleuca (Rydb. Ex Britton) Rydb. (Catling 1982), S. odorata

(Nutt.) Lind. (Catling 1982), S. ovalis var. erostellata Catling (Catling 1983), S. parksii Correll

(Catling and McIntosh 1979), S. pra~sophylla Reichb. var. cleistogamna Ames & Correll (Ames

and Correll 1952). Therefore, the determination of agamospermy as the breeding system in S.

floridanadd~~~~~ddddd~~~~ was expected. Based on field observation of S. floridana plants at the Rayonier study

site, ovaries began to swell immediately upon flower maturation. When combined with the lack

of observed pollinators, these data suggest an agamospermic breeding system similar to that seen

in other North American .git ainhesl~ species (Sheviak 1982; Schmidt and Antlfinger 1992).

The observation of the lack of pollinia production in S. floridanadd~~~~~ddddd~~~~ is of great interest. A

similar report does not exist for any orchid species. Catling (1991) reports a number of orchid

species as being agamospermic, particularly in the Spirantheinae, with most agamospermic

species reproducing by adventitious embryony. However, there is no mention of the absence of

pollinia in these flowers. The root cause of this morphological and reproductive abnormality is









unknown at this time. Hypothetically, extreme inbreeding depression has been shown to

increase the probability of a population being homozygous and, therefore, less fit (Gillespie

1998). Examples of the effects of extreme inbreeding depression in plants are rare and

understudied; however, Morton et al. (1956) demonstrated the effects of inbreeding on the

reproductive viability of young children. In their study, the long-term viability of the study

subjects decreased as their inbreeding coefficient increased. Furthermore, in a study examining

the effects of mice inbreeding over 20 generations, Connor and Bellucci (1979) demonstrated a

significant decrease in both mice litter size and percent of mice progeny surviving over

generational time. The lack of pollinia seen in S. floridanadd~~~~~ddddd~~~~ could be attributed to a historically

high inbreeding coefficient for this population that resulted in a highly homozygous population

that is now incapable of purging deleterious alleles from its genome. The phenotypic,

morphologic, and reproductive result of this extreme inbreeding is the lack of pollinia production

and agamospermic capsule abortion during maturation. A more detailed molecular-based study

of the reproductive and molecular ecology of S. floridanadd~~~~~ddddd~~~~ is necessary to elucidate this

hypothesis.

AFLP technology has been demonstrated as a powerful tool in acquiring information

concerning within and between population genetic diversity in plants (for review see Meudt and

Clarke 2007). The present study represents the first investigation of the genetic diversity in a

population of the endemic Florida terrestrial orchid S. floridanad~~~~~ddddd~~~~ using AFLP technology. As a

genus, git iliesrl l~ species are often considered colonizing orchids whose presence is often

representative of a degree of habitat disturbance (S.L. Stewart personal observation). However,

orchids are poorly represented in the literature on colonizing plants and little is known about the

genetics of these so-called "colonizing" orchid taxa. .gitmilie fIlll\lori~ddana~ would likely not be









considered a colonizing orchid given the low total number of plants and populations despite

apparent suitable natural and disturbed habitat.

A number of reports exist concerning population genetic diversity measures in .9'i' iabel"

and other spiranthoid orchids. Sun (1997) reported a lack of allozyme diversity in Eulophia

sinensis Miq. (a non-spiranthoid orchid), .git aidesl~ hongkongensis, and Zeuxine stratemactica

(L.) Schl. (both spiranthoid orchids) both within and between populations of these native Hong

Kong orchids. However, all the orchids surveyed by Sun (1997) represent widespread species

within their respective ranges and are reproductively fit--E. sinensis being insect-mediated self-

compatible, S. hongkongensis being self-pollinating, and Z. stratemactica being apomictic--

unlike S. florid ana.d~~~~~ddddd~~~~

In a similar study, Sun and Wong (2001) reported high genetic diversity in the orchids Z.

gracilis Lindl. and E. sinensis using RAPD markers, and low genetic diversity in Z.

stratemactica. Differences among the breeding systems of these orchids are suggested as the

primary reason behind the observed differences in genetic diversity. For example, the low

genetic diversity observed in Z. stratematica may be attributable to its apomictic breeding

system--where no new allelic diversity is introduced from generation-to-generation. Over time,

this apomictic breeding system would lead to low levels of within-population genetic diversity,

but potentially high levels of between-population genetic diversity. In the present study, S.

floridanadd~~~~~ddddd~~~~ possesses low within-population genetic diversity; however, between population

diversity could not be measured since only one population was known at the time of sampling.

Inclusion of the newly-discovered Duval County (Florida) population in a study of the between-

population genetic diversity of this species would be helpful. It is possible that S. floridanadd~~~~~ddddd~~~~ may

have high between population diversity, but this diversity would likely be due to population










fragmentation and isolation and not due to the breeding system of the species, as suggested for Z.

stratemactica.

Forrest et al. (2004) reported on the population genetic structure of European populations

of the terrestrial orchid S. romanzoffiana Chamisso, finding differentiation between the northern

and southern populations of this species. Within the northern populations, genotypic diversity

was high and considered typical of sexually reproducing plants. However, the southern

populations showed only 12 genotypes among six populations. This low genotypic diversity was

suggested as being consistent with the agamospermous or autogamous breeding system of these

southern plants. Given that a number of Split anthe,~ species are known to be agamospermic, the

low genotypic diversity and low within population genetic differentiation measured in the

population of S. floridana could be a result of long-term agamospermic breeding system. The

agamospermic breeding system could have resulted in inbreeding depression, a genetic

bottleneck, and/or the maintenance of unwanted deleterious alleles within this one small, isolated

population of S. floridana (Ellstrand and Elam 1993). Over generations, the combination of

these factors could have resulted in the lack of breeding system presently seen in this species

(Nei 2005; King 1967; Kimura and Crow 1964; Nei et al. 1975). Higher-order molecular-based

analysis of these factors within S. floridanadd~~~~~ddddd~~~~ in Florida is necessary to better determine the root

cause of the apparent lack of breeding system. The use of functional molecular markers (i.e.,

gene targeted markers (GTMs)), as opposed to random markers (i.e., AFLP), would contribute a

great deal to the conservation genetics of S. floridanadd~~~~~ddddd~~~~ (Andersen and Ltibberstedt 2003).

The interpretation of AFLP population genetic diversity data in a context of species

management and conservation planning for S. floridanadd~~~~~ddddd~~~~ is problematic. The low genotypic

diversity and low genetic differentiation within the population of this species coupled with the









lack of a fit breeding system could be interpreted as factors leading to the extinction of S.

floridana.d~~~~~ddddd~~~~ Further searches of this species' historic habitat, the southeastern coastal plain of the

United States, should be conducted to determine the species absolute range status. Dueck and

Cameron (2007) validated the separation of S. brevilabris and S. floridanadd~~~~~ddddd~~~~ as two unique species,

as proposed by Brown (2001). Finally, further molecular-based population genetic diversity

studies should be conducted that include the newly discovered (2005) second Florida population

of S. floridanad~~~~~ddddd~~~~ as well as any other new populations.

Implications for Integrated Conservation

The present studies on the demography, pollination biology, and population genetic

diversity of the endemic Florida terrestrial orchid S. floridana have demonstrated the need to

understand these ecological factors and their relation to conservation and species recovery

planning. Studies such as these highlight the need to integrate field-based ecological data with

both reproductive biology and molecular-genetic data to determine ecologically functional

models of species-level plant conservation. This is particularly important when conserving an

endemic orchid species because most orchids have developed highly specific ecological

partnerships with pollinators, habitat, and fungi. For example, identifying the breeding system of

S. floridanadd~~~~~ddddd~~~~ as agamospermic, determining both the lack of pollinia production and capsule

abortion, and measuring low genotypic and within-population genetic differentiation could all

demonstrate a trend toward low population viability for S. floridanadd~~~~~ddddd~~~~ in Florida. This may lead

those interested in the conservation of this glitanther,~ species to consider the species as headed

toward extinction and in need of immediate conservation attention (Stewart and Kane 2006c).

Conservation efforts for most glitantherl~ species focuses on the preservation of suitable

habitat to help insure the long-term population reproductive viability of the species (S.L. Stewart

personal observation). However, this conservation model does not apply to an orchid such as S.









floridanadd~~~~~ddddd~~~~ that apparently produces no viable offspring. In identifying the lack of a reproductive

mode in S. floridanad~~~~~ddddd~~~~ the question arises, how best do we develop a conservation system for this

species? Previous studies have demonstrated that S. floridanadd~~~~~ddddd~~~~ is highly preferential for

Ceratorhiza mycobionts despite sharing a similar habitat type with and a genetic relation to S.

brevilabris, which prefers Epulorhiza mycobionts (Dueck and Cameron 2007; Stewart and Kane

2007a). Given that apparent reproductive sterility and high mycobiont preference shown in S.

floridanad~~~~~ddddd~~~~ simply preserving more suitable habitat will not insure the long-term viability of S

floridanadd~~~~~ddddd~~~~ populations in Florida.

The difficulty in conservation planning for S. floridanadd~~~~~ddddd~~~~ appears to be considerable.

However, due to the application of integrated conservation methods those interested in the long-

term conservation of this species now have a more complete understanding of the challenges

faced in conserving this species. Traditional approaches to the conservation of Splimathllr,\

species in North America would have overlooked the aforementioned details of the breeding

system, pollination biology, and population genetic diversity of S. floridana. These oversights

could have resulted in a misguided and misapplied conservation and species-recovery plan for S.

floridanadd~~~~~ddddd~~~~ in Florida.

The current studies concerning the demography, pollination biology, and genetic diversity

of S. floridanadd~~~~~ddddd~~~~ in Florida represent on step in the species-level integrated conservation of this

rare, endemic terrestrial orchid (Figure 1-1). In adding to the body of knowledge concerning the

conservation biology of this species, the present studies not only promote the conservation of S.

floridanad~~~~~ddddd~~~~ but also promote sound conservation and management planning of other plant species

throughout Florida.































Figure 4-1. Rayonier study site for Split anthe f~\lori~ddana~ in Bradford County, Florida.















30-



28-



0 26-



R 24-



22-



20

100-



90-







70-



60-



50





D ate/Trim e


Figure 4-2. Temperature and relative humidity profiles at Rayonier site (Bradford County,
Florida) 7 April 2003 during Splitantherr f\loridnan pollinator observations. A)
Temperature (oC) and B) relative humidity (RH; %).















30-



25-



0 20-


S 15-



10-




100-A



80-



o 60-



40-





20








Date/Time


Figure 4-3. Temperature and relative humidity profiles at Rayonier site (Bradford County,
Florida) 13 April 2003 during Splitantherr f\lorid~dhnan pollinator observations. A)
Temperature (oC) and B) relative humidity (RH; %).








































Figure 4-4. Lack of pollinia in 9>l'i' "'le floridana.d~~~~~ddddd~~~~ A) Flower of .gitmil'lies\ floridanadd~~~~~ddddd~~~~ before
dissection, scale bar = 1 mm. B) Frontal view of column from S. floridanadd~~~~~ddddd~~~~ showing
anther cap (AC), pollinial depression (PD), and column tip (CT) with no pollinia
present, scale bar = 0. 1 mm. C) Column of S. floridanadd~~~~~ddddd~~~~ showing anther cap (AC) and
column tip (CT) with no pollinia present, scale bar = 0.1 mm. D) Aborted
capsule/ovary (AO) and dried flower (FL) of S. floridanad~~~~~ddddd~~~~ scale bar = 1 mm.









CHAPTER 5
SEED CULTURE OF Splitanihesl~ brevilabris AND DEEP SOUTH S. cernua

Introduction

In nature, orchids consume naturally-occurring endophytic mycorrhizal fungi as sources of

carbohydrates, nutrients, and water through the action of mycotrophy. The digestion of these

mycobionts and subsequent uptake of nutrients by the immature orchid embryo stimulates seed

germination, protocorm development, and seedling growth (Arditti 1966; Clements 1988;

Rasmussen 1995). For this reason, the survival of orchids in managed or restored habitats may

require the presence of appropriate mycobionts to support plant development and subsequent

seedling recruitment (Zettler 1997b). Symbiotic seed co-culture techniques represent an efficient

way to promote the orchid-fungus parasitism under in vitro conditions, as well as to study in

vitro orchid-mycobiont preference (Zettler 1997a, 1997b; Stewart and Kane 2006b, 2007a,

2007b). While a number of symbiotic co-culture protocols exist for terrestrial orchid taxa, their

germination efficiency is often lower than expected (Anderson 1991, 1996; Stewart and Kane

2006b, 2007a; Stewart and Zettler 2002; Zelmer and Currah 1997; Zettler and Hofer 1998;

Zettler and McInnis 1992; Sharma et al. 2003; Stewart et al. 2003; Zettler et al. 2001; Zettler et

al. 2005), especially when compared to asymbiotic germination studies with the same taxa. This

low seed germination efficiency is likely due to a degree of preference many terrestrial orchids

appear to have for mycobionts at the time of germination versus during later life stages.

However, this preference has apparently been mostly overlooked by previous symbiotic co-

culture practitioners. Mycobiont preference has been shown to play an important role in

symbiotic orchid propagation, and is thought to play a critical function in the establishment of

orchids into field sites (Zettler 1997a, 1997b; Stewart et al. 2003; Batty et al. 2006a, 2006b).









Orchid-mycobiont preference has been considered controversial for many years. Many

researchers have considered the orchid-fungus relationship to be opportunistic and non-specific

both in vitro and in situ (Knudson 1922; Curtis 1939; Hadley 1970; Masuhara and Katsuya,

1989; Masuhara et al. 1993). Differences in orchid-fungal preference have been identified under

in vitro versus in situ conditions (Bidartondo and Bruns 2005; Masuhara and Katsuya 1994;

Taylor and Bruns 1999; Taylor et al. 2003), and these differences have led some to consider

orchid-mycobiont preference as generally low (Hadley 1970; Stewart and Zettler 2002).

However, others have suggested that preference, especially under in vitro conditions, is

surprisingly high (Clements 1988; Rasmussen and Rasmussen 2007; Smreciu and Currah 1989;

Stewart and Kane 2006b, 2007a, 2007b; Taylor and Bruns 1997; McKendrick et al. 2002;

Selosse et al. 2002; McCormick et al. 2006). In the most general sense, it appears that orchid-

mycobiont preference may be genus or species specific.

The terrestrial orchids .git aidesl~ brevilabris and S. cernua (Deep South race) were

chosen for this study because little information exists on the in vitro symbiotic co-culture of

these taxa, and these species represent the rarest members of the .gita(illrr, genus in Florida

(Figures 1-3, 1-4). Previously, Stewart et al. (2003) proposed a symbiotic co-culture protocol for

S. brevilabris using mycobionts isolated from the study species and the Florida epiphytic orchid

Epidendrum magnoliae Mithlenberg var. magnoliae (syn. = E. conopseum). While investigating

a successful co-culture protocol, Stewart et al. (2003) suggested that S. brevilabris is non-

preferential for mycobionts based on in vitro germination tests. However, their study only

utilized a mycobiont from a distantly-related epiphytic species to investigate mycobiont

preference; while the use of mycobionts from a closely-related taxon would have better

represented the dynamics of mycobiont species in S. brevilabris.









In the present studies, the in vitro symbiotic co-culture and potential for mycobiont

preference by S. brevilabris and the Deep South race of S cernua are investigated. A description

and tentative identification of all mycobionts utilized during these studies are provided. Finally,

the role of mycobiont specifieity in the distribution, current ecological status, and long-term

conservation of both S. brevilabris and S. cernua is given consideration.

Materials and Methods

Fungal Isolation and Identification

Four mycobionts were chosen for in vitro symbiotic co-culture and mycobiont preference

trials in S. brevilabris and two mycobionts for S. cernua (Tables 5-1, 5-2). Mycobiont Sbrev-

266 was previously isolated by Stewart et al. (2003) on 30 April 1999 from S. brevilabris, while

mycobiont Econ-242 was previously isolated by Zettler et al. (1999) on 7 June 1995 from

Epidendrum magnoliae (syn. = E. conopseum). .9'imibe fIlll\lori~ddana~ mycobionts were isolated

on 28 April 2004 following the procedure outlined by Stewart et al. (2003) for Florida .git aidesl~

species, modified by taking only root sections and not entire flowering plants due to the rarity of

the species. Only Hyve plants, representing 20% of the total population, were sampled because of

the small size of this population. The Duval County (Florida) population was not sampled

because, at the time of sampling, only two plants were known from this site. Mycobionts were

not isolated from the roots of the Deep South race of S. cernua because of the small size of the

known population (<5 plants) and permitting limitations at the time of this study.

Root systems of five adult flowering plants of S. floridanadd~~~~~ddddd~~~~ at the Bradford County, Florida

population were carefully excavated and root sections (<10 cm) were removed. The root

sections were wrapped in paper towels moistened with sterile deionized water, placed in plastic

bags, stored in darkness at ca. 100C, and transported to the laboratory (<4 h). Root sections were

rinsed with cold tap water to remove surface debris, and surface cleansed 1 min in a solution










containing 5 mL ethanol (100%), 5 mL 6.00% NaOC1, and 90 mL sterile DI water. Clumps of

cortical cells containing fungal pelotons were removed, placed on CMA supplemented with 50

mg 1Y novobiocin sodium salt and incubated at 250C for 4 days. Hyphal tips were excised from

actively growing pelotons and subcultured onto 1/5 PDA. The pH of all previously mentioned

media was adjusted to 5.8 with 0.1 N KOH prior to autoclaving at 117.7 kPa and 1210C for 20

mmn.

Mycobionts showing cultural characteristics similar to those orchid endophytes previously

described in the literature (Zettler 1997b; Currah et al. 1987, 1997; Richardson and Currah 1993;

Stewart et al. 2003; Zelmer et al. 1996) were assigned a reference number and stored at 100C on

OMA. Isolates were also stored on 1/5 PDA in continual darkness at 25 +20C until use in seed

germination experiments.

Mycobiont characterization and identification followed methods described by Davidson

(1938), Smith (1977), Zelmer and Currah (1995), Currah et al. (1987, 1990, 1997), Zelmer et al.

(1996), and previously in this work for cultural morphology, polyphenol oxidase production, and

celluase activity. Hyphal and monilioid cell characteristics were assessed using a Nikon

Labophat-2 light microscope fitted with a Nikon Coolpix 990 digital camera. Fungal staining

procedures followed those outlined by Phillips and Hayman (1970) modified by the use of acid

fuchsin as the stain (Stevens 1974; J. Kimbrough personal communication).

Seed Collection and Sterilization

Seeds of S. brevilabris were collected prior to capsule dehiscence from mature fruits on 17

April 2005. Seeds were collected from a roadside population in Levy County, Florida.

Immediately following collection, capsules were dried over silica gel desiccant for 2 weeks at

250C, followed by storage at -100C in darkness for 192 days. Prior to the initiation of in vitro

cultures, seed viability was visually assessed using the methods of Stewart et al. (2003). Viable










seeds were considered those seeds containing a distinct, rounded, and hyaline embryo. Seeds of

S. floridanadd~~~~~ddddd~~~~ could not be obtained because the species apparently aborts seed capsules soon after

pollination (S.L. Stewart personal observation; Chapter 4).

Seeds of the Deep South race of S. cernua were obtained from mature capsules prior to

dehiscence on 12 December 2004. Because of the small size of individual S. cernua seed

capsules, one entire inflorescence was collected from a roadside site in Apalachicola National

Forest (Liberty County, Florida). Immediately after collection, capsules were dried over silica

gel desiccant for two weeks at 250C, separated from capsule material, pooled, and immediately

used in in vitro experiments.

In preparation for in vitro germination experiments, seeds of both species were surface

disinfected for 45 seconds in a solution containing 5 mL ethanol (100%), 5 mL 6.0% NaOC1, and

90 mL sterile DI water. Following surface disinfection, seeds were rinsed three times for 30

seconds each in sterile DI water. Solutions were removed from the seed surface disinfection vial

using a sterile Pasture pipette that was replaced after each use. Sterile DI water was used to

suspend the surface disinfected seed, and a sterile inoculating loop was used to sow the seed.

Symbiotic Co-Culture

Seeds of both S. brevilabris and Deep South S. cernua were sown according to the

procedures described by Stewart et al. (2003) for Florida Splitaidesrl~ species. Seeds were

removed from cold-dark storage, allowed to warm to room temperature (ca. 250C), surface

disinfected for 1 minute in the solution described previously, and placed on the surface of a

sterile 1 cm x 4 cm filter paper strip within a 9 cm diameter Petri plate containing 25 mL OMA.

Germination medium pH was adjusted to 5.8 with 0.1 N HCI prior to autoclaving at 117.7 kPa

and 1210C for 40 min. Seeds were transferred to the filter paper strips using a sterile bacterial

inoculating loop. An average of 80 (S. brevilabris) and 107 (S. cernua) seeds per Petri plate









were sown. Each plate was inoculated with a 1 cm3 block of fungal inoculum and only one

mycobiont per plate. Ten replicate plates per mycobiont were inoculated with S. brevilabris

seed, while five replicate plates per mycobiont were inoculated for S. cernua. Plates without

mycobiont served as controls. Plates were sealed with one layer ofNescofilm and maintained in

darkness (0/24 h L/D) for 86 days (S. brevilabris) and 27 days (S. cernua) at 25 +20C. Plates

were examined weekly during dark incubation for signs of germination or contamination,

exposing the seeds to brief (<10 min) periods of illumination. Plates were returned to

experimental conditions after visual inspection. Seed germination and protocorm development

were assessed every weekly after the start of dark incubation using a dissecting

stereomicroscope.

Germination and seedling growth and development were scored on a scale of 0-5 (Table 3-

2; Figure 3-1; Stewart et al. 2003). Seed germination percentages were based on viable seeds

determined by visual inspection with the aid of a dissecting stereomicroscope. Germination

percentages for each developmental stage were calculated by dividing the number of seeds in

that particular germination and development stage by the total number of viable seeds in the

sample. Data was analyzed using general linear model procedures and Waller-Duncan mean

separation at a=0.05 by SAS v 8.02 (SAS 1999). Germination counts were arcsine transformed

to normalize variation.

Results

Fungal Mycobionts: Spiranthes brevilabris

Three mycobionts were recovered from root sections of flowering plants of S. floridanadd~~~~~ddddd~~~~

(Table 5-1; Figure 5-1). All three mycobionts were identified as members of the anamorphic

genus Ceratorhiza. Isolates Sflo-305 and Sflo-306 tested cellulase-negative, which is a typical

Ceratorhiza-like response (Zelmer and Currah 1997). Isolate Sflo-308 tested cellulase-positive.









All three isolates tested polyphenol oxidase-negative, which typically is indicative of orchid

mycobionts from the form genus Epulorhiza. However, a rapid average daily growth rate (Sflo-

305 = 11.3 mm d- Sflo-306 = 10.6 mm d- Sflo-308 = 12.1 mm d- ) and the production of large,

broadly-connected barrel-shaped monilioid cells (Sflo-305 = 36 x 24.8 Clm, Sflo-306 = 39.6 x

21.6 Clm, Sflo-308 = 39.2 x 29.2 Clm) support the tentative identification of these isolates as

Ceratorhiza species (Figure 5-3). Only superficial differences in cultural morphology were

identified among the group of three mycobionts. Isolate Sflo-305 formed smaller and more

numerous loose aerial sclerotia than either isolate Sflo-306 or Sflo-308, whereas isolate Sflo-306

formed more irregularly-shaped aerial sclerotia. Isolate Sbrev-266 was previously recovered

from the roots of S. brevilabris and identified as a strain of Epulorhiza repens (Bernard) Moore

(Table 5-1; Figure 5-1; Stewart et al. 2003).

Fungal Mycobionts: Deep South Spiranthes cernua

The two mycobionts (Econ-242 and Srev-266) utilized in the study of the in vitro co-

culture of the Deep South race of S. cernua were previously isolated and identified by Zettler et

al. (1998) and Stewart et al. (2003). Both mycobionts were also identified as strains in the

anamorphic fungal genus Epulorhiza (Table 5-2; Figure 5-2). Isolate Sbrev-266 has been

identified as a strain of E repens, a ubiquitous global orchid endophyte species complex

(Stewart 2007; Zelmer 2001; Stewart et al. 2003; Currah et al. 1987). Mycobiont Econ-242 has

been tentatively identified as a strain of Epulorhiza, likely closely allied with the E. repens

species complex (Stewart et al. 2003).

Symbiotic Co-Culture: Spiranthes brevilabris

Seeds in all mycobiont treatments began to swell within 2 weeks after sowing, and

germination commenced within 3 weeks. The visual contamination rate of cultures from









bacterial and non-mycorrhizal fungi was 1%. Visual inspection revealed S. brevilabris seeds to

be 94.6% viable. Seeds of this species were monoembryonic.

An effect of mycobiont strain was found on the in vitro symbiotic co-culture of S.

brevilabris. Germination after 3 weeks was highest when seeds were inoculated with

mycobionts Sflo-305, Sflo-306, and Sflo-308 (99.5%, 99.5%, 89.9% respectively; Figure 5-3).

However, these mycobionts only promoted seed germination to Stage 1, while isolate Sbrev-266

supported Stage 2 germination (32.4%) after 3 weeks dark incubation (Figure 5-3). Mycobionts

Sflo-305 and Sflo-306 did support Stage 2 germination after 10 weeks dark incubation, but only

to a minimal percentage (10.6%, 0.6% respectively; Figure 5-3). In contrast, mycobiont Sbrev-

266 supported a maximum of Stage 5 germination (46.2%) after 10 weeks dark incubation

(Figure 5-5). After a total of 12 weeks dark incubation, only mycobiont Sbrev-266 supported

germination to an advanced developmental stage (i.e., Stage 3 or greater; Figures 5-3, 5-4).

Control treatments supported only Stage 1 germination after a total of 12 weeks dark incubation

(Figure 5-3).

Symbiotic Co-Culture: Deep South Spiranthes cernua

Seeds in each mycobiont treatment began to swell within one week after sowing, and

germination commenced within one-and-a-half weeks. No visual contamination was evident in

any cultures. Visual inspection revealed Deep South S. cernua seeds to be 63.2% viable. Seeds

of this species were monoembryonic.

An effect of mycobiont strain was found on the in vitro symbiotic germination of Deep

South S. cernua. Germination (Stage 2+) occurred in both the Econ-242 and Sbrev-266

treatments after 4 weeks dark incubation (0.2% and 3.9%, respectively; Figure 5-5). However,

protocorm development after 4 weeks was highest when seeds were inoculated with mycobiont

Econ-242 (11.2%; Stage 5; Figure 5-5). Isolate Sbrev-266 supported germination and









development only through Stage 3 (1.2%) after 4 weeks. After 6 weeks dark incubation, both

isolates Econ-242 and Sbrev-266 supported protocorm development to Stage 5 (16.8% and 6.3%,

respectively; Figure 5-5). However, isolate Econ-242 supported a significantly higher

percentage germination and development after 6 weeks, continuing the trend seen after 4 weeks

of incubation. The control treatment supported germination only through Stage 1 after both 4

and 6 weeks incubation (Figure 5-5).

Discussion

The conservation of rare, endangered, and endemic Splitantherl~ species in Florida depends

on an understanding of not only the requirements for in vitro symbiotic co-culture, but also the

degree of mycobiont preference exhibited by each species within the genus. Developing an

understanding of both these factors represent vital steps in the development of integrated

conservation procedures for orchid species. Most studies on orchid-mycobiont preference

examine the topic at either the generic level within the Orchidaceae or among species

representing extremes in the family (i.e., terrestrial versus epiphytic; nonphotosynthetic), without

examining mycobiont preference effects on in vitro seed culture. These present studies represent

not only the first report of in vitro mycobiont preference in either S. brevilabris or Deep South S.

cernua, but also greatly expands the understanding of the in vitro co-culture requirements

leading to the conservation of both species.

Successful in vitro symbiotic co-culture of S brevilabris has been previously reported

(Stewart et al. 2003). Using mycobionts isolated from S. brevilabris (Sbrev-266) and the Florida

epiphytic species Epidendrum magnoliae var. magnoliae (syn. = E. conopseum; Econ-242),

Stewart et al. (2003) reported a maximum percent germination of 40.8% and 49.8%,

respectively, on modified OMA after 55 days in vitro culture. This was a similar final percent

germination as found in the present study for those seeds inoculated with isolate Sbrev-266 on










OMA after 12 weeks (53.1% Stage 5; Table 3-2; Figures 3-1, 5-3). Stewart et al. (2003)

concluded that S. brevilabris is likely non-preferential for mycobionts since comparable

germination percentages were supported by mycobionts originating from the study species and

an epiphytic Florida species. Stewart et al. (2003) tested mycobiont preference using two

mycobionts, none of which originated from closely-related Splitaidesrl~ taxa in Florida, such as S.

floridana.d~~~~~ddddd~~~~ To better demonstrate any orchid-mycobiont preference by S. brevilabris, mycobionts

from both S. brevilabris and its close relatives (i.e., S. floridana),~~dddd~~~ddd~~~ should have been tested.

The successful in vitro symbiotic co-culture of the Deep South race of S. cernua has not

been previously reported. However, Zettler and McInnis (1993) previously reported the

symbiotic co-culture of S. cernua (no race defined) from northwestern South Carolina using

mycobionts isolated from S. cernua (no race defined; Rabun Co., Georgia), Platanthera

integrilabia (Greenville Co., South Carolina), and P. ciliaris (Van Buren Co., Tennessee). In

this report, only the mycobionts from P. ciliaris and S. cernua supported germination to an

advanced stage (>Stage 4). Furthermore, only the mycobiont originating from P. ciliaris

supported S. cernua co-culture to a leaf-bearing stage followed by subsequent establishment and

flowering of plants under greenhouse conditions. These data lead Zettler and McInnis (1993) to

suggest that S. cernua exhibits a non-preferential characteristic for mycobionts during its entire

life cycle. In a similar test of mycobiont preference carried out in the current study, but using

seed from the Deep South race of S. cernua and mycobionts from S. brevilabris (Sbrev-266) and

Epidendrum magnohiae var. magnoliae (Econ-242), a similar non-preferential characteristic for

mycobionts was found. In light of the current data concerning the symbiotic co-culture and

mycobiont preference of the Deep South race of S. cernua, the conclusion of low mycobiont

preference in S. cernua by Zettler and McInnis (1993) appears to be supported. However, further










symbiotic co-culture data from the geographically wide-ranging S. cernua species complex is

necessary before a definitive conclusion about mycobiont preference in this group can be

reached.

Moreover, the use of a mycobiont originating from P. ciliaris in the symbiotic co-culture

of S cernua seeds presents an ethical dilemma for those interested in S. cernua conservation--

what are the potential ecological impacts of releasing a mycobiont not originating from S. cernua

into S. cernua habitats? A closer examination of mycobiont preference in the S. cernua species

complex may reveal a higher-than-expected degree of mycobiont specificity during in vitro

symbiotic co-culture. The current study of mycobiont preference in the S. brevilabris-S.

floridanadd~~~~~ddddd~~~~ species pair demonstrates this trend, with mycobionts isolated from S. floridanadd~~~~~ddddd~~~~

supporting no advanced stage symbiotic seed germination when co-cultured with seed of S.

brevilabris (Stewart and Kane 2007a). These two closely-related species appear to not share

mycobionts based on mycobiont isolations and in vitro symbiotic co-culture trials.

Unfortunately, symbiotic seed germination trials were not possible using seed of S. floridanadd~~~~~ddddd~~~~

because it appears the species aborts seed capsules soon after pollination and fertilization (S.L.

Stewart personal observation). Identifying geographic or regional mycobiont preference in any

orchid species pair or species complex (i.e., S. cernua species complex) would allow those

interested in the integrated conservation of a regionally-specific race of the complex to recognize

the importance of isolating the regionally-specific mycobionts to support the symbiotic co-

culture of the particular race.

Similarly to the co-culture study of the Deep South race of S. cernua, Zettler et al. (1999)

used mycobiont Econ-242 to germinate seeds of the Florida epiphytic orchid Encyclia ttttttttttttttttampensis

(Lind.) Small. While mycobiont Econ-242 did support the in vitro symbiotic seed germination









of~ E. ~~tttt~~~~ttttampensis to a final percent germination of 0.3% (Stage 5) after 13 weeks, it is unlikely that

this represented a true test of mycobiont preference in E. ttttttttttttttttampensis because only one mycobiont

was tested (Econ-242) and no mycobionts from E. ttttttttttttttttampensis were incorporated. While of some

basic interest, mycobiont preference across widely divergent genera does not elucidate orchid-

mycobiont preference at the generic level, thus circumventing questions of orchid-mycobiont

preference and possible mycobiont sharing within closely-related species pairs or species

complexes, such as S. brevilabris and S. floridanadd~~~~~ddddd~~~~ or the Deep South race of S. cernua in Florida.

Moreover, the use of widely diverse mycobionts in the in vitro symbiotic co-culture of widely

diverse genera yields little practical information on the symbiotic co-culture or conservation of

orchid species. As previously stated, the conservation of orchid species by symbiotic co-culture

relies on an understanding of not only mycobiont diversity and preference, but also the

physiological role specific mycobionts may provide to their orchid hosts (i.e., seed germination).

This can only be investigated once a through understanding of generic or species level

mycobiont preference has been achieved.

Otero et al. (2005) reported varied performance of mycobionts during in vitro symbiotic

co-culture of the tropical epiphytic species Tolumnia variegata (Swartz) Braem. They go on to

hypothesize that given the presumed geographic heterogeneous distribution of orchid

mycobionts, that these mycobionts may affect orchid distribution and population size. This

conclusion appears valid based on our current findings and may help to explain the current

distribution and rarity of both S. brevilabris and the Deep South race of S cernua in Florida.

The isolation of the mycobiont Epulorhiza repens from the roots of S. brevilabris was

previously reported (Stewart et al. 2003). Subsequent mycobiont isolations from other plants

within the only known S. brevilabris population have consistently yielded isolates identifiable as









strains of the species E. repens (S.L. Stewart unpublished data). Epulorhiza repens is considered

a ubiquitous global orchid endophyte found throughout many orchid habitats worldwide

(Anderson 1991; Stewart 2007; Zelmer 2001; Stewart et al. 2003; Currah et al. 1987). Thus its

repeated isolation from S. brevilabris is not surprising and its isolation from S. floridanadd~~~~~ddddd~~~~ was

expected. Moreover, given the taxonomic relatedness between S. brevilabris and S. floridanadd~~~~~ddddd~~~~

one would suspect the two species may share related mycobionts. However, repeated mycobiont

isolations from the roots of S. floridanadd~~~~~ddddd~~~~ at the Bradford County site consistently yielded isolates

identifiable as strains of the anamorphic fungal genus Ceratorhiza. Furthermore, mycobiont

isolations from S. floridanadd~~~~~ddddd~~~~ plants collected at the newly-discovered Duval County (Florida) site

also consistently yielded strains of the same Ceratorhiza mycobiont as was isolated from the

Bradford County (Florida) plants (S.L. Stewart, unpublished data). This apparent species level

orchid-mycobiont preference was surprising given the previously mentioned taxonomic

relatedness of the two orchid species.

Mycobiont isolations from the roots of the Deep South race of S. cernua were not possible

due to permitting difficulties. However, it is suspected that this race of S cernua would have

yielded isolates assignable to the anamorphic fungal genus Epulorhiza based on the previous

works of Zettler and McInnis (1993) with S. cernua from South Carolina, Zettler et al. (1995)

with S. odorata, a closely-related species to S. cernua, from Delaware, and S.L. Stewart

(unpublished data) with S. odorata from Florida. Further investigations into the identity of

mycobi onts from thi s geographi cally-restri cted S. cernua race should b e undertaken.

High mycobiont preference on the species level, as is hypothesized between S. brevilabris

and S. floridanad~~~~~ddddd~~~~ has been previously reported. Shefferson et al. (2005) reported high mycobiont

preference at the generic level in a study of the terrestrial orchid genus Cypripedium. Of the









seven Cypripedium species surveyed, five of the species shared mycobionts in the

Tulansnellaceae (i.e., Epulorhiza-like fungi). Two species surveyed, C. californicum Gray and

C. parviflorum Salisbury, demonstrated a higher degree of mycobiont diversity than all other

surveyed species. Cypripedium californicum Gray and C. parvillorum Salisb. demonstrated high

mycobiont preference at the generic level in comparison to other five Cypripedium species

studied, especially for mycobionts not commonly associated with the other five species surveyed.

This trend is similar to the mycobiont preference apparently demonstrated between S. brevilabris

and S. florid ana.d~~~~~ddddd~~~~

Likewise, Taylor et al. (2003) reported a high degree of preference between two closely

related nonphotosynthetic orchids in the genus Hexalectris. Four distinct types of fungi were

identified from samples of the common H. spicata (Walter) Barnhardt var. spicata, while only

one type was identified from samples of the rare H. spicata var. arizonica (S. Watson) Catling

and Engel. Taylor et al. (2003) hypothesized that the divergence seen in mycobiont preference

between these two varieties of the same orchid species represents evidence for the contribution

of mycobiont specificity to the evolutionary diversification of the Orchidaceae. High mycobiont

preference between closely-related varieties or races may require special consideration for the in

vitro symbiotic propagation and conservation of those species, but these factors were not

examined by Taylor et al. (2003). Moreover, Taylor and Bruns (1999) reported that two species

of the nonphotosynthetic orchid genus Corallorhiza each associated with several species of

Russula, but never shared fungi despite the plants growing sympatrically. Again, these types of

data can prove to be crucial for those interested in the symbiotic propagation or conservation of

orchid species. A similar preference trend may be occurring between S. brevilabris and S.










floridana;d~~~~~ddddd~~~~ however, further experimentation is necessary to properly hypothesize on the habitat

preference or selection pressures acting between these two species.

An opposite trend in mycobiont preference is hypothesized in the S. cernua species

complex, with particular emphasis on the Deep South race of the species. While Otero et al.

(2005), Taylor et al. (2003), and Taylor and Bruns (1999) hypothesize that high mycobiont

preference may be a maj or driving force behind orchid habitat preference and evolutionary

diversification, the apparent mycobiont non-specifieity demonstrated in the Deep South race of

S. cernua may be indicative of an inverse relationship between mycobiont preference and

geographic restriction and/or evolutionary diversification (Stewart 2007). If other members of

the S. cernua species complex exhibit mycobiont non-preference, this may help to explain the

wide geographic range of this species complex throughout the northeast, Midwest, mid-Atlantic,

and southeastern United States. Further study of mycobiont preference in the S. cernua complex

is necessary to elucidate the potential relationship among distribution, mycobiont preference, and

species rarity.

Of further interest is that the same mycobiont Stewart (2007), Stewart et al. (2003), and

Stewart and Kane (2007a) utilized in the symbiotic co-culture of S. brevilabris and Deep South

S. cernua (Sbrev-266; Table 5-2) continued to support in vitro symbiotic germination in the

present studies. Some authorities have suggested that the effectiveness of orchid mycobionts at

supporting symbiotic co-culture may lessen if the mycobionts are stored over long periods of

time and/or subjected to multiple subcultures (L.W. Zettler personal communication). These

present data suggest that mycobiont Sbrev-266 does not demonstrate any reduced symbiotic

germination capacity despite being routinely subcultured and stored at 250 C for 7 years (to date)

on both PDA and 1/5 PDA.









The current studies present a first look at orchid-mycobiont preference in the endangered

terrestrial orchids S. brevilabris and the Deep South race of S cernua in Florida. In the case of

S. brevilabris, preference was demonstrated through not only the isolation and identification of

different mycobionts from S. brevilabris and its Florida endemic congener S. floridanad~~~~~ddddd~~~~ but also

the use of those mycobionts in the in vitro symbiotic co-culture of S brevilabris. In the case of

the Deep South race of S cernua, mycobiont preference was demonstrated through the use of

mycobionts from similar and divergent Florida orchid taxa in the symbiotic co-culture of the

study species. Data such as reported here may prove invaluable in the integrated conservation of

S. brevilabris, S. floridanad~~~~~ddddd~~~~ and the Deep South race of S. cernua in Florida, especially given

their rare status within the state.

Implications for Integrated Conservation Planning

The present studies on the in vitro symbiotic co-culture and mycobiont preference of the

Florida terrestrial orchids S. brevilabris and Deep South S. cernua have demonstrated the

benefits of insightful studies of both the propagation science and mycology of orchid species

before implementing conservation or recovery plans. As seen in the asymbiotic and symbiotic

seed culture studies of Habenaria macroceratitis, the current results show the importance of

understanding an orchid's germination requirements-particularly mycobiont preference--

before attempting further integrated conservation and species recovery efforts. For example, we

now understand that the integrated conservation and recovery of S. brevilabris and S. floridanadd~~~~~ddddd~~~~

in Florida require the isolation and storage of mycobionts from individual S. brevilabris and S.

floridanadd~~~~~ddddd~~~~ sites throughout the state, not just a few isolations representing one or two populations.

The symbiotic co-culture, plant reintroduction, and long-term species recovery of both orchid

species is seriously questionable without the proper mycobiont diversity represented in plant

material collections (i.e., mycobiont and seed banks).









Furthermore, the high degree of mycobiont preference demonstrated through the in vitro

symbiotic co-culture of S. brevilabris raises a number of interesting questions concerning

conservation and recovery planning for this species. Specifically, is the distribution of S

brevilabris in Florida limited by the distribution of its preferred mycobiont? Split ainhesl~

brevilabris was historically distributed throughout the coastal plain of the southeastern United

States (Luer 1972); however, is currently only known from a few sites in Florida and Texas

(Brown 2002, 2005; P.M. Brown personal communication). Given the hypotheses of Otero et al.

(2005) and Taylor et al. (2003), it is conceivable that the decline in the range of S brevilabris is

due to not only habitat degradation and destruction, but also a decline in abundance of the

species' mycobiont due to ecological alteration of habitat. This means that to achieve the long-

term conservation and recovery of this species, more than simply targeted plant management

must be considered. Management of habitat for both plants and mycobionts will likely be

required if the recovery of S brevilabris throughout its historic range is to be successful. Further

investigations into the integrated conservation of S. brevilabris, as well as its historically

widespread congener S. floridanad~~~~~ddddd~~~~ will shed light on these aspects of species management.

Additionally, the low degree of mycobiont preference demonstrated through the symbiotic

co-culture of the Deep South race of S. cernua creates its own important questions concerning

the integrated conservation and management of both this particular race and the entire species

complex. Where the recovery of S. brevilabris and S. floridanadd~~~~~ddddd~~~~ may depend upon the

management of particular mycobionts in particular habitats, the recovery of Deep South S.

cernua may depend upon more upon habitat management and less upon mycobiont management.

Moreover, this general species recovery and management paradigm may be applicable on a

regional basis to the entire S. cernua species complex--where management of habitat for each










geographically defined race within the complex may prove to be of greater importance than

management for particular mycobionts. Of course, further investigation into not only the

mycobiont diversity and preference of the Deep South S. cernua, but also all other races of S.

cernua in the United States must be undertaken before such a wide-ranging species recovery and

management recommendation can be fully implemented.

The current studies concerning the propagation science and mycology of both S.

brevilabris and the Deep South race of S cernua represent two steps in the species-level

integrated conservation of these rare terrestrial orchid species in Florida (Figure 1-1). By

developing a more complete understanding of symbiotic co-culture requirements and mycobiont

preferences in these two orchid species, we are now better equipped to continue integrated

conservation efforts with both species that will lead to their recovery through habitat restoration

and plant reintroductions. Further studies concerning the population establishment, population

management, and long-term population sustainability of both S. brevilabris and the Deep South

S. cernua are encouraged.










Table 5-1. Sources of mycobionts used in the in vitro symbiotic co-culture of Spitanthes,~
brevilabris.
Isolate Host Collection information Identification
Sflo-305 S. floridanadd~~~~~ddddd~~~~ 28 April 2003; Bradford Co., FL Ceratorhiza sp.
Sflo-306 S. floridanadd~~~~~ddddd~~~~ 28 April 2003; Bradford Co., FL Ceratorhiza sp.
Sflo-308 S. floridanadd~~~~~ddddd~~~~ 25 April 2004; Bradford Co., FL Ceratorhiza sp.
Sbrev-266 S. brevilabris 30 April 1999; Levy Co., FL Epzulorhiza repzens










Table 5-2. Sources of mycobionts used in the in vitro co-culture of the Deep South race of
Splii~lll' ain e a erua.
Isolate Host Collection information Identification
Econ-242 Epi. magnoliae 7 June 1995; Alachua Co., FL Epulorhiza spp.
Sbrev-266 S. brevilabris 30 April 1999; Levy Co., FL Epulorhiza repens











































Figure 5-1. Mycobionts isolated from Splianhe fIlll\lori~ddana~ (Sflo) and S. brevilabris (Sbrev). A)
Sflo-305 whole culture morphology at 3 weeks, scale bar = 1 cm. B) Sflo-305
monilioid cells stained with acid fuchsin at 3 weeks (100 x), scale bar = 30 lm. C)
Sflo-306 whole culture morphology at 3 weeks, scale bar = 1 cm. D) Sflo-306
monilioid cells stained as above (100 x), scale bar = 30 lm. E) Sflo-308 whole
culture morphology at 3 weeks, scale bar = 1 cm. F) Sflo-308 monilioid cells stained
as above (100x), scale bar = 30 lm. G) Sbrev-266 whole culture morphology at 3
weeks, scale bar = 1 cm. H) Sbrev-266 monilioid cells stained as above (400x), scale
bar = 10 lm.














































Figure 5-2. Mycobionts used in the symbiotic co-culture of the Deep South race of Split anther,~
cernua. A) Sbrev-266 whole culture morphology at 3 weeks. B) Econ-242 whole
culture morphology at 3 weeks. Scale bars = 1 cm.
















80-
I 308


c 60- -1 a



E 40-
a, "a

20-a

aaa


100 b10 weeks



80-b


60-


S40-
a, a


aaa a



bb 3 weeks
100- b


80-


S60-

a, a






20


Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

Developmental Stage


Figure 5-3. Effects of four mycobionts on percent germination and protocorm development of
Spitant~hls brevilabris cultured on oat meal agar (OMA) after 3, 10, and 12 weeks in
vitro culture. Histobars with the same letter are not significantly different within
stage (a = 0.05). Error bar = SE.









70


60 -1 d Stg2
8I I Stage 3
a~ I Stage 4
50-


c 40-
I I ,Ca a
E 30 -1 i b

CD



20



3 wks 10 wks 12 wks

Development Time

Figure 5-4. Effect of mycobiont Sbrev-266 on percent germination and protocorm development
of Splitanthes,~ brevilabris cultured on oat meal agar (OMA) at 3, 10, and 12 weeks in
vitro culture. Histobars with the same letter are not significantly different within
development time (a = 0.05). Error bar = SE.












100 -1 b

abI I control

80-



o 60-



a, 40-



20 -1 a

b Ib


4 weeks
100 b



80-



o 60-



a, 40-



20-





Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

Germination Stage


Figure 5-5. Effects of two mycobionts on percent germination and protocorm development of
the Deep South race of Split anthe,~ cernua cultured on oat meal agar (OMA) after 4
and 6 weeks in vitro culture. Histobars with the same letter are not significantly
different within stage (a = 0.05). Error bar = SE.









APPENDIX A
MORPHOLOGICAL VARIATION IN Habenaria macroceratitis

Floral and vegetative structure variation is known to occur in the Orchidaceae. This

variation can occur both within and between populations of the same species, and is thought to

arise from microevolutionary pressures that may be site or population specific (Melendez-

Ackerman et al. 2005). Vallius et al. (2004) examined morphological variation within and

between populations of co-occurring varieties ofDactylorhiza incarnata (L.) So6. In a survey of

Hyve populations, they reported little morphological difference between populations of varieties;

however, varietal differences within populations were high. Vallius et al. (2004) suggest that

sympatric evolution is occurring among these co-occurring varieties.

Studying sympatric populations of the terrestrial orchid Gymnadenia conopsea (L.) R.

Brown, Soliva and Widmer (1999) identified a high degree of floral variation between early- and

late-flowering populations of this species. As before, this variation may represent

microevolutionary changes within metapopulations of G. conopsea to limit competition for

pollinators. A similar trend in competition-limiting morphologic variation has been documented

in the tropical epiphytic orchid Tolumnia variegata (Ackerman and Galarza-Perez 1991).

Within species or population morphological variation may be the result of

microevolutionary forces driving speciation in the Orchidaceae. To this end, many orchid

taxonomists have used this floral and/or vegetative variation to identify stable color and growth

forms of some orchid species. For example, Epidendrum amphistomum A. Richard forma

rubrifolium P.M. Brown has been identified as the red-leaf color form of the otherwise green-leaf

E. amphistomum (Brown 2000). Another example of a morphological variation resulting in a

unique taxonomic trait can be seen with Listera australis Lindl. forma scottii P.M. Brown

(Brown 2000). Typically, L. australis forms two leaves per inflorescence; however, forma









scottii regularly forms three leaves per inflorescence, and this trait is stable year-after-year in the

same plants. Finally, an example of floral color variation can be seen in the terrestrial orchid

Sacoila lan2ceolata var. paludicola (Luer) Sauleda, Wunderline, & Hansen forma auera P.M.

Brown (Brown 2001). This form differs in its yellow flowers, instead of its typical red flower

color. Based on these reports, floral and/or vegetative variations within the Orchidaceae appear

to be reasonably common.

During studies on the plant demography and pollination biology of Habenaria

nzacroceratitis at the Socash site (Hernando County, Florida), a unique leaf form and growth

habit of the species were noted. Typically, leaves of H. nacroceratitis possess a glossy bright

green appearance (Figure A-la). However, it was noted that leaves of particular plants of H.

nzacroceratitis within this population possess lateral parallel striping alternating between dull

green and yellow-green colors (Figure A-1b-c). This unique color pattern was seen on only a

few plants in the Socash population and remained stable during all observation years (2002-

2005). Plants possessing this lateral striping were not noted at any other H. nacroceratitis site

included in the present studies. It remains to be seen if this variation in leaf color is due to plant

nutrition, site variability, or some other microevolutionary force. Further study of the long-term

stability and reproduction of leaf color pattern from seed is needed before this color variant is

given further taxonomic consideration and form status.

A second unique growth habit was noted during the field study ofH. nacroceratitis at the

Socash site. Plants growing in high light conditions where no canopy shade was available

showed an upright growth form of their leaves (Figure A-2). Under normal conditions, the

leaves ofH. nacroceratitis grow perpendicular to the flowering stem and parallel with surface of

the soil. However, the leaves of these plants grew at an acute angle to the flowering stem and









nearly vertical from the soil surface (Figure A-2). This growth habit is likely due to exposure to

higher levels of light than what is typical for this species, and a morphological response to limit

light exposure of leaves and reduce water loss from transpiration. However, H. quinqueseta has

been observed growing in a similar high light situation in Levy County (Florida) without this

upright leaf growth (S.L. Stewart, personal observation). Further investigation into the

physiological and possible taxonomic bearing of this growth habit is needed.














































Figure A-1. Lateral striping variant of Habenaria macroceratitis at the Socash study site
(Hernando County, Florida). A) Comparison of striped leaf (top) and normal leaf
(bottom). B) Vegetative plants of H. macroceratitis showing lateral striping. C)
Flowering plant of H. macroceratitis showing an extreme example of lateral striping.














191






































Figure A-2. Plants of Habenaria macroceratitis at the Socash study site (Hernando County,
Florida) showing upright leaf growth habit. A) Flowering plant showing acute angle
between leaf and flowering stem. B) Vegetative plant showing vertical leaves.












APPENDIX B
AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP) SUPPLEMENTARY DATA
FOR Habenaria macroceratitis


Hm a~l_1
Hm ~a l_1
Hm al_12
Hm al_13
Hm ala14
Hm al_15
Hm al_18
Hm alj17
Hm al_18
Hm al_19
Hm cal fil
Hm alL20
Hm al~_21
Hm alL22
Hm a 1112
Hm a $1j3
Hm sa 114
Hm a l5lo
Hm a 1116
Hm a 1317O
Hm alaLOB
Hm a 1319

0 100 200 300 400 500 600 700


Figure B-1. AFLP fingerprint of genomic DNA from the Socash site (Hernando County,
Florida) of Habenaria macroceratitis. Primer combination -ACT/-CAG used.
Molecular weight size range of fingerprints is 10-650 nucleotides.











Hmac~b)_10

Hmac~b)_ 11

Hmac~b)_12-

Hmac~b)_13-

Hmac~b)_14-
Hmac~b)_15.

Hmac~b)_16

Hmac~b)_17-

Hmac~b)_18

Hmac~b)_19
Hmac~b)_01

Hmac~b)_2

Hmac~b)_21
Hmac~b)_02.

Hmac~b) 03
Hmac~b)_04

Hmac~b)_05-

Hmac~b)_06

Hmac~b)_07-

Hmac~b)_08

Hmac~b)_09
0 50 100 150 200 250 300 350 400 450 500 550 600 650 700


Figure B-2. AFLP fingerprint of genomic DNA from the Old Dade Highway (Hernando County,
Florida) site of Habenaria macroceratitis. Primer combination -ACT/-CAG used.
Molecular weight size range of fingerprints is 10-650 nucleotides.













Sflo 10

Sflo_1

Sflo 12

Sflo 13

Sflo_14

Sflo_15

Sflo 16

Sflo 17

Sflo_18

Sflo_19

Sfe~lo1

Sflo_20

Sflo_21

Sflo_22

Sflo_23

Sflo_24

Sflo_02

Sflo 03

Sflo_04

Sflo 05

Sflo_06

Sflo 07

Sfl o_0

0 50 100 160 200 260 300 360 400 460 500 650 600 660 700



Figure B-3. AFLP fingerprint of genomic DNA from the Rayonier site (Bradford County,
Florida) of .Spit anthe floridana.n Primer combination -ACT/-CAG used. Molecular

weight size range of fingerprints is 10-750 nucleotides.















Correltllin 0.1 0.2 0.3 0.4 0.5 0.0 0.7 0.5 0.5 1.0


11Ue 200 lanl 41Illu 91100~Ul


HulOaC12
ftn l es,17

ll 1.5 C0*
* n P~~alo







Ruble EO1l

n4r15e.CN

m-a9lb_L*L




Anto I II'J


Ivat.* llg


Figure B-4. Correlation dendrogram of genotypic diversity within the Socash site of Habenaria macroceratitis. Four genotypes

present.











Cofrelalifor 0.1 0.2 0.3 0.4 0.5 0.6 P.7 0.5 0.9 1.0


IIucID 2510 minu 411110 eull


Hm~bal.'
CIrnIB roIa

Hnlieb EIIS
Inmllb 10

Ilml~b FIE


Hmlihb En
H nbb EUS

Ha~ll.. EQ
I Im.t. u 1


I

I~


~


Figure B-5. Correlation dendrogram of genotypic diversity within the Old Dade Highway site of Habenaria macroceratitis. Six
genotypes present.














IlUOJ 20131 3120 41111 U


?iMIU


Hm pl Clf.




Hml,DICI

I-m laD1


FII.T- .I








Hm_EES~

Hr~.Co4
Mm1a pt4
Mmae 011.






Hm Co OS
I r~ ~..r




1nItid F rl




I~BcFO2
i r?9 O?


HF~alkFi





Figure B-6. Correlation dendrogram of genotypic diversity between the Socash and Old Dade Highway sites of Habenaria

macroceratitis. Ten genotypes present.













O~rrelalori 0.1 0.2 0.9 0.4 0.5 0.6 0.7 0.8 0.9 1.0


10uO ?ilua H]is eUti i0051


Figure B-7. Correlation dendrogram of genotypic diversity within the Rayonier site of Split anthe f~\loridana.. Four genotypes present.









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BIOGRAPHICAL SKETCH

Scott Stewart was raised in Virden, Illinois. After high school, Scott attended Illinois

College in Jacksonville, Illinois. Starting his college career as a pre-engineering maj or, Scott

eventually switched his maj or to secondary education, then to English, and then to biology.

Early in his years as a biology maj or, Scott became involved in undergraduate research and

developed a passion for the native orchids of North America. After 6 years of a varied

undergraduate education, Scott decided to pursue a graduate-level education working on native

orchid conservation in the United States. After graduation from Illinois College in May 2002,

Scott began orchid conservation and plant ecology work with the U.S. Fish and Wildlife Service

on the Florida Panther National Wildlife Refuge (Naples, Florida) before joining the

Environmental Horticulture Department at the University of Florida in August 2002. Scott

continued his orchid conservation work by building a partnership between his research and the

USFWS in Florida. Upon graduating in August 2007 with a Doctor of Philosophy in

horticultural science from the University of Florida, Scott plans to purse an academic or industry

position that will allow him to continue his orchid conservation work. In his spare time Scott

enj oys nature photography, model building, and travel.





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INTEGRATED CONSERVATION OF FLORI DA ORCHIDACEAE IN THE GENERA Habenaria AND Spiranthes : MODEL ORCHID CONSERVA TION SYSTEMS FOR THE AMERICAS By SCOTT L. STEWART A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1

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2007 Scott L. Stewart 2

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To all those who have a true passion in life 3

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ACKNOWLEDGMENTS I thank Dr. Michael Kane for his support, pa tience, dedication, and encouragement during my research, and for the many conversations abou t plants and more. I also thank Dr. Charles Guy, Dr. Thomas Sheehan, Dr. Doria Gordon, a nd Dr. James Kimbrough for serving on my supervisory committee. The members of my s upervisory committee have shown me what it means to be true professionals and academicians. Much of this work could never have been unde rtaken without a great deal of help from many people, including Paul Martin Brown (O cala, Florida), Dr. La wrence Zettler (Illinois College), Larry Richardson (USFWS-FPNWR), James Durwachter (USFWS-FPNWR), Layne Hamilton (USFWS-FPNWR), and Ben Nottingham (USFWS-FPNWR). Their guidance and assistance made many experiments and field research sessions possible. I also thank the fellow graduate students with whom I have worked over the years: Carmen Valero-Aracama, Philip Kauth, Daniela Dutra, Timothy Johnson, and Xiliu Shen. Brainstorming sessions, research conversations, and field work would have not been possible without their assistance. I thank the Socash family (Brooskville, Florida) for generously allowing me to use their population of Habenaria macroceratitis as a research site, as we ll as the Rayonier Corporation (Starke, Florida) for allowing me access to the population of Spiranthes floridana The City of Brooksville (Florida), Marjorie Harris Carr Cross Florida Greenway, National Fish and Wildlife Foundation, U.S. Fish and Wildlife Service, Flor ida Division of Forestr y, Florida Division of Environmental Protection, Florida Division of Pl ant Industry, and the San Diego County Orchid Society all contributed to the completion of this work. The assistance of Ginger Clark and the University of Florida ICBR Genetic Analysis laboratory with the preparation and analysis of AFLP data is greatly appreciated. 4

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I also thank my parents, Duan e and Victoria Stewart; and my sister, Leslie Stewart, for their unwavering support of my educational goals despite not fully understanding what I was doing. Finally, I thank my wife, Angela ODonnell, for her unflinching love, support, grudging interest, and immense degree of patience during my work. Without her love and understanding none of this would have been possible. 5

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................9 LIST OF FIGURES .......................................................................................................................10 ABSTRACT ...................................................................................................................................13 CHAPTERS 1 LITERATURE REVIEW.......................................................................................................14 Introduction .............................................................................................................................14 Study Rationale .......................................................................................................................15 Conservation Ethic and Modes ........................................................................................15 Integrated Orchid Conservation ......................................................................................19 Plants of Interest .....................................................................................................................20 Selection Criteria .............................................................................................................20 Plants of Study .................................................................................................................21 Habenaria macroceratitis ........................................................................................21 Spiranthes floridana .................................................................................................22 Spiranthes brevilabris ..............................................................................................23 Spiranthes cernua Deep South race .........................................................................24 Overview of Pertinent Literature ............................................................................................24 Orchid Ecology ................................................................................................................24 Orchid-Mycobiont Association .......................................................................................27 History and background ...........................................................................................27 Infection and digestion .............................................................................................28 Photomycotrophic balance .......................................................................................30 Orchid mycobiont taxonomy ....................................................................................31 Orchid Propagation and Culture ......................................................................................35 Asymbiotic seed culture ...........................................................................................35 Symbiotic seed culture .............................................................................................40 Orchid Pollination Biology ..............................................................................................45 Molecular Genetics and Genetic Diversity in Orchids ....................................................50 2 POLLINATION BIOLOGY AND GENETIC DIVERSITY OF Habenaria macroceratitis .........................................................................................................................63 Introduction .............................................................................................................................63 Materials and Methods ...........................................................................................................65 Study Sites .......................................................................................................................65 Plant Demography ...........................................................................................................65 Pollinator Observations ...................................................................................................66 6

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Pollination Mechanism, Seed Viability, and Asymbiotic Seed Germination .................67 Sampling, DNA Extraction and Amplified Fragment Length Polymorphism (AFLP) .........................................................................................................................70 Results .....................................................................................................................................72 Study Sites .......................................................................................................................72 Plant Demography ...........................................................................................................72 Pollinator Observations ...................................................................................................73 Pollination Mechanism, Seed Viability, and Asymbiotic Seed Germination .................74 AFLP Data .......................................................................................................................75 Discussion ...............................................................................................................................75 Implications for Integrated Conservation Planning ................................................................83 3 SEED CULTURE AND IN VITRO SEEDLING DEVELOPMENT OF Habenaria macroceratitis .........................................................................................................................96 Introduction .............................................................................................................................96 Materials and Methods ...........................................................................................................99 Asymbiotic Seed Germination ........................................................................................99 Seed source and sterilization ....................................................................................99 Asymbiotic media survey .........................................................................................99 Effects of carbohydrate source on as ymbiotic seed germination ...........................100 Effects of exogenous cytokinins on asymbiotic seed germination ........................101 Effects of photoperiod on asymbiotic seed germination ........................................101 Effects of photoperiod on in vitro seedling development ......................................102 Symbiotic Seed Germination .........................................................................................102 Seed source and sterilization ..................................................................................102 Mycobiont isolation and identification ..................................................................103 Symbiotic co-culture ..............................................................................................104 Effects of photoperiod on symbiotic co-culture .....................................................105 Results ...................................................................................................................................106 Asymbiotic Seed Germination ......................................................................................106 Asymbiotic media survey .......................................................................................106 Effects of carbohydrate source on as ymbiotic seed germination ...........................107 Effects of exogenous cytokinins on asymbiotic seed germination ........................108 Effects of photoperiod on asymbiotic seed germination ........................................108 Effects of photoperiod on in vitro seedling development ......................................109 Symbiotic Seed Germination .........................................................................................110 Mycobiont isolation and identification ..................................................................110 Symbiotic co-culture ..............................................................................................110 Effects of photoperiod on symbiotic co-culture .....................................................111 Discussion .............................................................................................................................111 Asymbiotic Seed Germination ......................................................................................111 Symbiotic Seed Germination .........................................................................................120 Implications for Integrated Conservation Planning ..............................................................123 7

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4 POLLINATION BIOLOGY AND GENETIC DIVERSITY OF Spiranthes floridana .......143 Introduction ...........................................................................................................................143 Materials and Methods .........................................................................................................145 Study Sites .....................................................................................................................145 Plant Demography .........................................................................................................145 Pollinator Observations .................................................................................................145 Pollination Mechanism ..................................................................................................146 Sampling, DNA Extraction, and Amplified Fragment Length Polymorphism (AFLP) .......................................................................................................................147 Results ...................................................................................................................................148 Study Site .......................................................................................................................148 Plant Demography .........................................................................................................148 Pollinator Observations and Pollination Mechanism ....................................................149 AFLP Data .....................................................................................................................150 Discussion .............................................................................................................................150 Implications for Integrated Conservation .............................................................................157 5 SEED CULTURE OF Spiranthes brevilabris AND DEEP SOUTH S. cernua ...................163 Introduction ...........................................................................................................................163 Materials and Methods .........................................................................................................165 Fungal Isolation a nd Identification ................................................................................165 Seed Collection and Sterilization ..................................................................................166 Symbiotic Co-Culture ....................................................................................................167 Results ...................................................................................................................................168 Fungal Mycobionts: Spiranthes brevilabris ..................................................................168 Fungal Mycobionts: Deep South Spiranthes cernua .....................................................169 Symbiotic Co-Culture: Spiranthes brevilabris ..............................................................169 Symbiotic Co-Culture: Deep South Spiranthes cernua .................................................170 Discussion .............................................................................................................................171 Implications for Integrated Conservation Planning ..............................................................178 APPENDICES A MORPHOLOGICAL VARIATION IN Habenaria macroceratitis .....................................188 B AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP) SUPPLEMENTARY DATA FOR Habenaria macroceratitis .............................................193 LIST OF REFERENCES .............................................................................................................200 BIOGRAPHICAL SKETCH .......................................................................................................226 8

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LIST OF TABLES Table page 1-1. Anamorphic and corresponding teleomorph species of common orchid mycobionts in the Rhizoctonia complex. ...................................................................................................55 2-1. Experimental pollination cond itions applied to flowers of Habenaria macroceratitis .........86 2-2. Thermal cycler parameters for presel ective and selective AFLP amplification of prepared genomic DNA.....................................................................................................87 2-3. Results of the genetic analysis for each population of Habenaria macroceratitis ................88 3-1. Comparative mineral salt, vitamin, and am ino acid content of asym biotic orchid seed germination media used in the asymbiotic germination of Habenaria macroceratitis ...126 3-2. Seed germination and protocorm development stages in Habenaria macroceratitis .........127 3-3. Sources of mycobionts used in the in vitro symbiotic co-culture of Habenaria macroceratitis ..................................................................................................................128 5-1. Sources of mycobionts used in the in vitro symbiotic co-culture of Spiranthes brevilabris ........................................................................................................................181 5-2. Sources of mycobionts used in the in vitro co-culture of the Deep South race of Spiranthes cernua ............................................................................................................182 9

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LIST OF FIGURES Figure page 1-1. Diagrammatic representation of the step s and connections of integrated orchid conservation .......................................................................................................................56 1-2. Habenaria macroceratitis inflorescence and flower .............................................................57 1-3. Spiranthes brevilabris and S. floridana inflorescences .........................................................58 1-4. Deep South race of Spiranthes cernua inflorescence and flower ..........................................59 1-5. Example of mycobiont pelot ons in the cortical tissues of Spiranthes brevilabris ................60 1-6. Cellulase assay .......................................................................................................................61 1-7. Polyphenol oxidase assay......................................................................................................62 2-1. Study sites for Habenaria macroceratitis in Florida .............................................................89 2-2. Average height and spur length of Habenaria macroceratitis plants at four study sites in west central Florida ........................................................................................................90 2-3. Average leaf number and flower number of Habenaria macroceratitis plants at four study sites in west central Florida ......................................................................................91 2-4. Growth cycle of Habenaria macroceratitis under field conditions ......................................92 2-5. Cocytius antaeus captured during pollinator observation of Habenaria macroceratitis ......93 2-6. Temperature and relative humidity profiles during Habenaria macroceratitis pollinator observations .......................................................................................................................94 2-7. Effects of pollination condition on percen t germination and protocorm development of Habenaria macroceratitis ..................................................................................................95 3-1. Seed germination and protocorm development stages in Habenaria macroceratitis .........129 3-2. Effects of culture media on percent germination and protocorm development of Habenaria macroceratitis ................................................................................................130 3-3. Effects of carbohydrate type and banana powder on percent germination and protocorm development of Habenaria macroceratitis after 7 weeks ................................................131 3-4. Effects of carbohydrate type and banana powder on percent germination and protocorm development of Habenaria macroceratitis after 21 weeks ..............................................132 10

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3-5. Effects of four cytokinins and four c oncentrations on percent seed germination of Habenaria macroceratitis ................................................................................................133 3-6. Effects of three photoperiods on in vitro asymbiotic seed germination and protocorm development of Habenaria macroceratitis ......................................................................134 3-7. Morphological effects of three phot operiods on asymbiotically germinated Habenaria macroceratitis ..................................................................................................................135 3-8. Effects of three photoperiods on tuber and le af production per in vitro Habenaria macroceratitis seedlings ..................................................................................................136 3-9. Effects of three photoperiods on tuber and shoot biomass of in vitro Habenaria macroceratitis seedlings ..................................................................................................137 3-10. Effects of three photoperiods on tuber di ameter and length and leaf length and width of in vitro Habenaria macroceratitis seedlings ...............................................................138 3-11. Examples of mycobionts isolated from Habenaria macroceratitis ...................................139 3-12. Effects of six mycobionts on percent germination and protocorm development of Habenaria macroceratitis ................................................................................................140 3-13. Photoperiodic effect on in vitro symbiotic seed germination and protocorm development of Habenaria macroceratitis ......................................................................141 3-14. Photoperiodic effects on the in vitro symbiotic protocorm development of Habenaria macroceratitis ..................................................................................................................142 4-1. Rayonier study site for Spiranthes floridana .......................................................................159 4-2. Temperature and relative humidity prof iles at Rayonier site on 7 April 2003 during Spiranthes floridana pollinator observations ...................................................................160 4-3. Temperature and relative humidity prof iles at Rayonier site on 13 April 2003 during Spiranthes floridana pollinator observations ...................................................................161 4-4. Lack of pollinia in Spiranthes floridana ..............................................................................162 5-1. Mycobionts isolated from Spiranthes floridana and S. brevilabris .....................................183 5-2. Mycobionts used in the symbiotic co-culture of the Deep South race of Spiranthes cernua ..............................................................................................................................184 5-3. Effects of four mycobionts on percent germination and protocorm development of Spiranthes brevilabris ......................................................................................................185 5-4. Effect of mycobiont Sbrev-266 on percen t germination and protocorm development of Spiranthes brevilabris ......................................................................................................186 11

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5-5. Effects of two mycobionts on percent ge rmination and protocorm development of the Deep South race of Spiranthes cernua .............................................................................187 A-1. Lateral striping variant of Habenaria macroceratitis .........................................................191 A-2. Plants of Habenaria macroceratitis at the Socash study site showing upright leaf growth habit.....................................................................................................................192 B-1. AFLP fingerprint of genomi c DNA from the Socash site of Habenaria macroceratitis ...193 B-2. AFLP fingerprint of genomic DNA from the Ol d Dade Highway site of Habenaria macroceratitis ..................................................................................................................194 B-3. AFLP fingerprint of genomi c DNA from the Rayonier site of Spiranthes floridana .........195 B-4. Correlation dendrogram of genotypic diversity within the Socash site of Habenaria macroceratitis ..................................................................................................................196 B-5. Correlation dendrogram of genotypic diversity within the Old Dade Highway site of Habenaria macroceratitis ................................................................................................197 B-6. Correlation dendrogram of genotypic di versity between the Socash and Old Dade Highway sites of Habenaria macroceratitis ....................................................................198 B-7. Correlation dendrogram of genotypic diversity within the Rayonier site of Spiranthes floridana ...........................................................................................................................199 12

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INTEGRATED CONSERVATION OF FLORI DA ORCHIDACEAE IN THE GENERA Habenaria AND Spiranthes MODEL ORCHID CONSERVA TION SYSTEMS FOR THE AMERICAS By Scott L. Stewart August 2007 Chair: Michael Kane Major: Horticultural Science Rapid loss of native orchid ha bitat throughout ecologi cally-important areas (e.g., Florida) has prompted researchers to develop appropriate conservation systems for the preservation and recovery of native orchid species. These cons ervation systems must incorporate more than habitat preservation and plant propagation, they must integrat e the study of native orchid ecology, mycology, propagation, pollination biol ogy, and population gene tic diversity in a combined conservation effort. The current studies examine the ecology and demography, mycology, asymbiotic and symbiotic propagation, pollination biology, and population genetic diversity of orchid s in the genera Habenaria and Spiranthes. The specific Florida native orchid included in this study are: Habenaria macroceratitis Spiranthes floridana S. brevilabris and the Deep South race of S. cernua. The application of integrated conservation to the preservation of these native orchids is discussed, and integrated conservation steps are proposed that will help ensure the long-term population viability of th ese species. Furthermore, these integrated conservation systems could be applicable outside the United States and Florida. 13

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CHAPTER 1 LITERATURE REVIEW Introduction The appeal and curiosity surrounding orchids throughout the ages have far surpassed that of any plant. This interest has not been limite d to just the hobbyist a nd commercial plant venues, but there has been an untiring pursuit of scientific knowledge on this large and complex plant family. Hundreds of scientific and popular texts are published each year concerning some aspect of orchid biology. Both the floristic and propa gation complexities of the Orchidaceae have pleasured and plagued resear chers, hobbyists, and commercial growers for decades. The Orchidaceae comprise approximately 10% of all known angiosperms, has a holarctic distribution, and is considered the largest family of floweri ng plants with approximately 35,000 species (Cronquist 1981; Dressler 1981). Furthermore, the orch id family has evolved highly specialized associations with both pollinators and mycorrhizal f ungi, the latter actually being a parasitic relationship where the orchid consumes fungi as a source of nutrients and water (Zettler 1997a; Tremblay 1992). The size and diversity of the Orchidaceae has been linked to this specialization in pollination and mycorrhizal a ssociation (Benzing 1981; Williams 1947; Stewart and Kane 2007a; Cozzolino and Widmer 2005; Taylor et al. 2003). This unique endomycorrhizal association allows Corallorhiza trifida Chatelain to survive in the tundra of northern Canada, Platanthera lacera (Michaux) G. Don to live in the tallgrass prairies of the Midwest, and Cattleya skinneri Bateman to live in the tropics of Costa Rica. This almost primordial curiosity with orchid s can be traced back to ancient Greek times, where one orchid creation myth claims that a young, handsome Greek prince, named Orkhis, fell in love with a beautiful priestess of the god Bakkhos Pursuing his love for the priestess, Orkhis journeyed to her temple where he encountered w ild animals guarding the priestess. The animals 14

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tore love-struck Orkhis limb fr om limb, scattering his tattered body over the land. In the places where Orkhis body parts landed flowers grew, whic h were named for himorchises (Berliocchi 1996; Hansen 2001). This story helps to explain both the origin of the temperate orchid genus Orchis and the entire family Orchidaceae. Unfortunately, the great beauty and mysticism that surrounds orchids have also directly contributed to their mass decline as wild plants. Unique mycorrhizal asso ciations have enabled orchids to survive in diverse habitats around the world. In evolvi ng to exist in these disparate habitats, orchids have evolved fascinating flow er forms and colors. These often spectacular flowers and the intimate relationshi p orchids have with their habita ts have led directly to the familys decline in the wild due to mass over-co llecting, habitat conversion to urban uses, habitat conservation to agricultural lands, and habitat mismanagement. For this reason, a large amount of scientific information exists concerning the propagation of orchids for conservation and restoration purposes. Orchids were among the fi rst plants to be successfully cultured under in vitro conditionsfrom seed (Knudson 1922) and by micropropagation (Rotor 1949; Arditti 1984). Numerous studies have focused on asymbi otic propagation of orchids from seed (for reviews see Arditti 1967 ; Sheehan 1983). Less research ha s been conducted on the symbiotic propagation and restoration of or chids, despite a nearly universal acknowledgement of the need for such work (for review see Rasmussen 1995). Scientific information on the symbiotic seed germination and restoration of orchids lags fa r behind that of both sy stematic studies and asymbiotic culture of orchids. Study Rationale Conservation Ethic and Modes The American conservation ethic began to take shape during the mid-1800s era of societal prosperity and urbanization, spearheaded by the publication of George Perkins Marshs (1864) 15

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Man and Nature. Partly in response to Marshs book and environmental movement, the government of the United States realized a need to conserve a national heritage and developed the national park system beginning in 1872. John Muir fixed a national conservation consciousness in the mind of Americans by orga nizing the first privat e national conservation organization, The Sierra Club. By the mid1900s, Aldo Leopold solidified the notions of conservationism and environmental protection in America through the pu blication of A Sand County Almanac (1949). The next big move fo rward in American conservationism came in 1962 with the publication of Rachel Carsons book Silent Spri ng, which is often credited with igniting the modern environmental movement. Th e capstone to the influence these authors had on the American environmental ethic came in 1973 with the passing of the Federal Endangered Species Act. Additionally, mo st states afford both plants and animals some degree of hierarchical protection and manage ment. Orchids have traditionally been the focus of national and international protection efforts, particularly focusing on the management of the international trade of orchid plants an d their parts (CITES 1973). Classically, orchid conservation has taken three traditional modes: 1) the development of action plans, 2) the assessment of a species or genus population or conservation status in the world, 3) or plant propagati on and reintroduction. A fourt h, and often overlooked, orchid conservation mode is the implementation and study of management regimes. Orchid conservation action plans often contain a mixe d bag of personal opinions concerning orchid conservation (Lapiner 1970; Moir 1970; Lynch 1970) and a review of the application of state, national, and international conser vation regulations to the field of orchid conservation (Campbell 1979; Campbell and Tarr 1980a, 1980b). An ever-p resent and recently reemerging field in the development of orchid conservation action plan s is the establishment of long-term orchid 16

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monitoring studies (M.H.S. Light personal communication). While discussions of orchid conservation and the establishment of long-te rm monitoring programs are both valuable in conservation planning, taken alone this mode of orchid cons ervation is limited in its effectiveness in protecting current populations of orchids or restoring declining or extirpated populations. The assessment of a species or genus cons ervation status is a nother common mode of orchid conservation. These assessments typica lly examine an orchids ecological status throughout its native range, the primary threats to the orchids continued ex istence in its natural setting, and any current conservation programs that apply to the orchid. The work of Cribb and Sandison (1998) on the conservation status of Cypripedium species worldwide represents a genus-wide application of this assessment notion; others have chosen a species-by-species approach to the assessment of orchid conservation (Gulliver et al. 2004a, 2004b). Recovery plans represent another type of orchid conser vation assessment, and can be focused on singlespecies (COSEWIC 2003; Brownell 1986; S eevers and Lang 1998a, 1998b; Ramstetter 2001; Bowles et al. 1998) or multi-speci es (Coates et al. 2003). Furthe rmore, others have taken the approach of public education and general species diversity assessment as a population extension of scientific conservation asse ssment (Jost 2006). Again, all th ese assessment approaches are valuable to orchid conservation planning as tools in understanding the need for orchid conservation. However, the impact of these c onservation assessments remain low if no further conservation work comes from their publication. The propagation and reintroduction of orchids represents anothe r classic mode of orchid conservation. A great deal of attention has been given to the seed culture of orchids, although the majority of this research has focused on the propagation efficiency of media types or culture 17

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systems and not propagation for conservation purposes. Stewart (2002) and Stewart (2003), working with the rare Florida terrestrial orchid Spiranthes brevilabris Lindl., are recent examples of the integration of orchid seed culture and plan t reintroduction for conservation purposes. Glicenstein (2006) presented a similar integratio n of seed culture and plant reintroduction in working with Cypripedium acaule Aiton. Zettler (1994) and Zettler (1996) reported, in a popular publication format, on efforts to conserve Platanthera integrilabia (Correll) Luer in Tennessee through propagation and ecological studies. Surprisingly, few other examples exist of the successful propagation and reintroduc tion of North American native or chids. While this mode of orchid conservation has the highest impact for natu ral populations, considerations of seed source and planting location remain understudied and underappreciated by most orchid conservation biologists. A final mode of orchid conservation is the examination of management techniques on orchid population viability and sustainability. Despite producing the most valuable orchid conservation information, few reports exist concer ning the effects of orch id or orchid habitat management. Curtis (1946) reported on the us e of mowing as a management technique for Cypripedium candidum Muhl. ex Willd. More recently, Jane kov et al. (2006) reported on the effect of management techniques and environmen tal conditions on the po pulation survival of Dactylorhiza majalis (Rchb.) P.F. Hunt & Summerhayes. In both these cases, mowing was used as a typical management technique for both of these temperate terrestrial orchid species. However, little information exists on orchid ma nagement in tropical regions or for epiphytic species (S.L. Stewart, personal observation). A new emerging and exciting area of orchid management is the reporting of orchid refuges in man-made settings such as coffee or tree plantations (Solis-Montero et al. 2005). As before, the reporting of orchid management 18

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techniques is an important part of orchid conser vation; however, taken as an individual effort the development of a management technique wit hout incorporating othe r aspects of orchid conservation biology limits the long-ter m effectiveness of this method. The main weakness of all the aforementioned orchid conservation modes is the lack of integration among all aspects of each mode. This l ack of integration often leads to an incomplete or skewed orchid conservation system. For example, focusing exclusively on the propagation of an orchid species could detract from the pl anning for plant reintroduction or long-term management of the species. For this reason, inte grated orchid conservatio n techniques must be utilized by orchid conser vation programs worldwide. Integrated Orchid Conservation Effective plant conservation prog rams involve a mode of cons ervation that allows careful consideration of conservation-oriented questions and structured criter ia in making difficult choices when investing limited re sources. This intense focus has not allowed those interested in orchid conservation the flexibility to make well-informed choices when confronted with questions of population-, genus-, state-, or regional-level orchid conservation systems (S.L. Stewart personal observation). Orchid conservation practices must integrate an understanding of existing and future threats, taxonomic distin ction, population size fluctuation, pollination and reproductive biology, in vitro and in situ propagation, and the mainte nance of population genetic diversity (for reviews see Cribb et al. 2003; Koopowitz et al 2003). For this to be possible, conservation practices should i nvolve experimentation in both in situ ecological contexts and ex situ laboratory-based contexts (Bru ndrett 2007; Swar ts et al. 2007). Therefore, orchid conservation systems must maintain a balance between the need for urgent action to avoid immediate loss of species divers ity and long-term landscapeor specieslevel actions that yield valuable ecological in formation (Spring et al. 2007). As outlined by 19

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Hopper (1997), the integrated conservation stra tegy focuses on the study of interactions among all trophic levels of plant c onservation biologybasic ecolog ical criteria, mycological interactions, propagation science technologi es, pollination biology and breeding systems, population genetic diversity measures, and sp ecies reintroduction and recovery methods. Integrated conservation systems vary according to which species they are being applied; however, the basic concept rema ins the same (Figure 1-1). The present studies investigated aspects of th e integrated conservati on of several Florida terrestrial orchid species. These species include: Habenaria macroceratitis Spiranthes floridana, S. brevilabris and S. cernua. Each of these species represents an understudied and critically-imperiled native orchid in Florida. The development of integrated conservation methods for these orchid species will help insure their long-term sustainability in Florida, along with demonstrating the applicab ility of integrated conservati on methods to orchids throughout the world. Plants of Interest Selection Criteria Species included in the present studies we re chosen based upon several conservationoriented criteria, including state listing status, number of extant populations presently known conservation information on the species, past or present conservation a ttention given to the species, and immediacy of current threats. Hi storic and modern accounts of the status of candidate species were also considered (Brown 2002, 2005; Luer 1972; Ames-Plimpton 1979; Hawkes 1948). Species for which there were fe w (<10) known populations, little-to-no presently known conservation information, and little-to-no pa st or present conser vation attention were considered as viable candidates for the present studies. Furthermore, species with an immediate conservation threat were also consid ered for the present studies. 20

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The four species included in the current studies are: Habenaria macroceratitis Spiranthes floridana, S. brevilabris and the Deep South race of S. cernua. These species represent four rare and understudied Florida na tive terrestrial orchids. Plants of Study Habenaria macroceratitis The subtribe Habenariinae is a large subtribe with approxima tely 930 species in 23 genera (Dressler 1993). Habenaria is the largest genus in the subtri be Habenariinae. The distribution of Habenaria is mainly throughout the tropical Ameri cas, tropical Africa, India, southeastern Asia, northern Australia, and extending to eastern China and Japan (Pridgeon et al. 2001). This large distribution is reflected in the infrageneric sectioning of the genus into 37 sections (Batista et al. 2006). The genus include s both common and rare species throughout its worldwide range. Habenaria macroceratitis Willdenow (section Macroceratitae), the long-horned rein orchis, is a rare terrestrial or chid that grows in the mesic ha mmocks of west-central Florida (Figure 1-2). This species is known from outsi de the United States, occurring in Mexico, the West Indies, and Central America (Brown 2002, 2005). A recent report of this species from Cuba is not surprising, but re quires careful verification since the close relative of H. macroceratitis H. quinqueseta (Michaux) Swartz, is known from the island nation (Llamacho and Larramendi 2005). Plants are known to grow up to 75 cm in height, typically possess 3-7 elliptic blue-green leaves, and can have 15-25 flowers arranged in a loose raceme (Brown 2002, 2005). Habenaria macroceratitis has historically been segregated as a variety of H. quinqueseta (e.g., H. quinqueseta var. macroceratitis ; Hawkes 1948, 1950; Luer 1972); however, a recent treatment of the genus suggest s the two are separate specie s (Brown 2000b; FNA 1993+). Brown (2000b) cites that the primar y distinguishing character between H. quinqueseta and H. 21

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macroceratitis is the proportional length of the segments of the lateral petals, where the distal division of the lateral petals in H. quinqueseta is less than two-times the length of the proximal division and is more than two-times the length of the proximal division in H. macroceratitis (FNA 1993+). Other minor distinguishing characte rs can be noted after extensive field work with both species (S.L. Stewart personal observation), such as the square flower profile of H. quinqueseta versus the rectangular flower profile of H. macroceratitis. Additionally, the length of the nectar spur is somewhat helpful in co mbination with other morphological traits (P.M. Brown personal communication). Further st udy on the taxonomic clar ity, possibly using a combination of morphologicaland molecular-based techniques, would help to elucidate the true placement of these two species. Spiranthes floridana The Spiranthinae is a large and diverse subtri be in the subfamily Spiranthoideae, with approximately 400 species in 30-40 genera (Dressler 1993). Spiranthes contains about 40 species and reaches its maximum diversity in No rth America (Pridgeon et al. 2003). Generally, the genus is distributed throughout North America, Mexico, Europe Asia, and eastern Australia. Spiranthes is one of the most recognizable orchid genera; however, it is also one of the most taxonomically-confusing for those new to th e field. Species delimitation in the genus has long been problematic due to natural hybridization and polyploidy (Luer 1975; Sheviak 1982; Dueck and Cameron 2007). Phylogenetic analysis has shown the genus to be sister to Mexican and Mesoamerican genera such as Mesadenus Dichromanthus, and Deiregyne (Pridegon et al. 2003). Spiranthes floridana (Wherry) Cory, the Florida ladies-tres ses, is a rare terrestrial orchid native to pine flatwoods of north -central Florida and persists in semi-disturbed edges such as roadsides and cemeteries (Figure 1-3). This sp ecies was historically known from Texas, east 22

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through Florida, and north to North Carolina ; however, due to a combination of taxonomic clarification from the closely-related S. brevilabris and extripation from othe r areas the species is now considered endemic to Florida (Brown 2002, 2005; FNA 1993+; Luer 1972). Two sites are presently know for this species: Bradford County, discovered in 1998 and Duval County, discovered in 2004. Plants are known to grow between 20-40 cm in height, typically possess 3-5 ovate yellow-green leaves that are withering at flowering, and can have 10-35 flowers arranged in a single-ranked spiraled or secund raceme (Brown 2002, 2005). Spiranthes floridana has historically been se gregated as a variety of S. brevilabris (e.g., S. brevilabris var. floridana ; Luer 1972); however both Br own (2002, 2005) and FNA (1993+) recognize S. floridana as a separate species from S. brevilabris This separation is based on the presence of dense pubescence on flowers and stems of S. brevilabris and the lack of this pubescence on flowers and stems of S. floridana. The validity of this species-pair separation was confirmed by the phylogenetic treatment of Spiranthes by Dueck and Cameron (2007). Spiranthes brevilabris Spiranthes brevilabris the short-lipped ladies-tresses, is a rare terrestrial orchid native to pine flatwoods of north-central Florida and persis ts in semi-disturbed edges such as roadsides and cemeteries (Figure 1-3). Historically, the species is historically known from Alabama, Georgia, Louisiana, Mississippi, Texas, and Florida; although it is currently known only from Florida and Texas (Brown 2002, 2005; E.L. Keith personal communication). The species current status is likely due to taxonomic c onfusion with allied spring-flowering Spiranthes species in the southeastern coastal plain and population extripation. Spiranthes brevilaris is presently known from only two populations in Levy County, Florid a. Plants are known to grow 20-40 cm in height, possess 3-6 ovate yellow-gree n leaves that are typically withered at flowering, and can have 10-35 flowers arranged in a single ra nk spiraled or secund raceme (Brown 2002, 2005). 23

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Spiranthes cernua Deep South race Spiranthes cernua (L.) Richard, the nodding ladi es-tresses, is a facultatively agamospermic polyploidy compilospeices known fr om the eastern half of the United States, extending from Maine in the north to the panhandle of Florida in the south (FNA 1993+). This compilospecies is made up of many different geogr aphically-restricted races with unidirectional gene flow from related diploi ds (Sheviak 1991, 1982). At pres ent, the Deep South race of S. cernua is known to persist in only from one semi-dis turbed roadside site in the panhandle of Florida in Apalachicola National Forest (Liberty County; Figure 1-4; S.L. Stewart personal observation). The natural habita t of the Deep South race of S. cernua is not know, although the species likely is native to mesic prairies along th e edges of small streams. Plants are known to grow 10-35 cm in height, possess 3-5 linear-oblanceo late leaves that are usually withered at flowering, and can have 10-50 flow ers arranged in a tightly or l oosely spiraled spike (Brown 2005). Overview of Pertinent Literature Orchid Ecology The ecology and distribution of the Orchidaceae has been of interest to researchers since Lindleys taxonomic and ecological descriptions of orchids in th e 1800s (as reported in Arditti 1984). Significantly less new information has been generated since the 1800s on the basic ecology and conservation of orchids, in comparis on to the information generated in the study of orchid taxonomy and propagation. Nonetheless, the study of orchid ecology is a foundational step in the integrated conservation of orchid species and populations. Plant distribution and life histor y studies represent important information that can be used by conservation planners in design ing orchid recovery plans. Un fortunately, little information on either of these topics exists. Tremblay (1997) reported on the dist ribution and dispersion 24

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patterns in nine species of Lepanthes in Puerto Rico. Based on th e study of orchid patch size and number of reproductive versus vegetative individuals, Tremblay (1997) suggested that most epiphytic orchids may exist as isolated patches with great distances between patches. This would indicate that a small e ffective population size is common and that gene flow may be restricted, depending on pollinator and pollinatio n mechanism. In a similar study, Willems and Melser (1998) studied the population dynamics of the terrestrial orchid Coeloglossum viride (L.) Hartm. In this study, C. viride was found to be a short-lived terre strial orchid with an average flowering percentage per populati on of 50%. Vegetative or ase xual propagation was found to be minimal in this species. Willems and Melser ( 1998) suggested that based on their life-history and population dynamics data populations of C. viride in the Netherlands were in a healthy state with vigorous age struct ure. Similar life-history and population dynamics studies have been conducted on Cleistes bifaria (Fernald) Catling & Gre gg (Mry and Gregg 2003) and Cypripedium calceolus (L.) var. parviflorum (Salisb.) Hultn (S hefferson et al. 2003). Trejo-Torres and Ackerman (2001) conducted an extensive biogeographic study of the orchid distributions throughout the Antilles. This study reported a strong affinity among orchid distributions on islands at cons iderable distances from one a nother, relating the congruency among orchid flora to the dust-lik e character of orchid seeds (T rejo-Torres and Ackerman 2001; Rasmussen and Whigham 1993). In general, this study suggests that similariti es in orchid floras were determined by ecological factors on individual islands rather than distance between islands as a barrier to plant dispersal. The application of this type of floristic/ ecological study to orchid flora distributions could be applied to non-island situations, pa rticularly as a modeling system in examining changes in distributio ns of orchids in a landscape. 25

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An often understudied area of orchid ecology is the examination of habitat factors that affect orchid plant distributions. Frei and Dodson (1972) examined the effects of bark substrates on the seed germination and early growth of epiphytic orchids. This study identified chemical substances in the bark of some trees that were in hibitory to the growth of orchids. Furthermore, the presence of epiphytic orch ids on certain trees was correla ted with production of toxic chemicals by those trees. In a similar study, Bola nd and Scott (1992) reported on the effects of habitat on the presence or absence of three terrestrial orchids ( Arethusa bulbosa L., Calopogon tuberosus (L.) Britton, Sterns, & Poggenberg, Pogonia ophioglossoides (L.) Ker-Gawler). Species distributions of the three orchids re flected differences in study site hydrology and microhabitat partitioning, with P. ophioglossoides preferring slightly wetter habitats and Arethusa and Calopogon preferring dryer habitats. Allen et al. (2004), in a st udy on the vegetation surrounding Cypripedium kentuckiense C.F. Reed sites throughout Louisiana, reported sites to be dominated by American beech ( Fagus grandifolia Ehrh.), eastern hophornbeam ( Ostrya virginiana (P. Mill) K. Koch, white oak ( Quercus alba L.), horse-sugar (Symplocos tinctoria L.), and witch hazel ( Hamamelis virginiana L.). Habitat characterization data such as th ese play an important ro le in the conservation planning for orchid species recovery and in iden tifying potential orchid reintroduction sites. Collins et al. (2005), in a similar study, reporte d on the soil and ecological features of Hexalectris sites in Texas. Again, knowing the ecologica l profile of orchid sites will aid in both the conservation of current sites and in identi fying potential orchid reintroduction sites. In order to better understand the conservation ne eds of orchid species, it is important to understand the ecology, distribution, a nd factors affecting the growth of plants in the wild. The study of field-based orchid ecology is ofte n overlooked and underappreciated in light of 26

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propagation or molecular-based studies; however, more valuable conservation information can be gained from the study of current orchid popula tions in their natural settings. Further study on the ecology and distribution of orchids in the wild is necessary in order to ensure their long-term sustainability and conservation. Orchid-Mycobiont Association History and background The Orchidaceae have long been known to po ssess a unique association with fungal partners (i.e., mycobionts). Bernard, in 1899, was the first to recogni ze the role of these mycobionts in orchid seed germination and at tempt the co-culture of orchid seed with mycobionts isolated from a parent plant (as reported in Arditti 1967). Bernards early attempts at symbiotic seed germination were problematic, but nonetheless demonstrated the role mycobionts played in orchid seed germination. Following th e work of Bernard, Burgeff continued to study the role orchid mycobionts played in the germination of orchids seeds, as well as studying the two organisms in association with one another (A rditti 1984). Despite early difficulties, Bernard and Burgeff laid the foundation of the study of sy mbiotic seed germination in the Orchidaceae. Their work, as well as the work of other practi tioners of the symbiotic germination technique, would remain in the background during the ensuing 60 years due to the discovery of asymbiotic orchid seed germination techniques by K nudson (1922, 1946). The study of orchid-mycobiont associations has received a renewed interest in recent years (for reviews see Warcup 1975; Hadley 1982). In nature, orchids digest endomycorrhi zal mycobionts as sources of minerals, carbohydrates, water, and vitamins in an acti on termed mycotrophy. My cotrophic consumption of mycobionts provides the non-endospermic or chid seed a two-fold advantage during germination: 1) infection followed by digestion in itiates seed germination by providing nearly all 27

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the necessary nutrients, moisture, and other organic compounds to the proembryo and 2) infection and digestion contri butes entirely to the developm ent of the non-photosynthetic protocorm life stages of orchid s. In particular, the digesti on of these mycobionts have been shown to provide simple carbohydrates (Sm ith 1966, 1967; Gebauer and Meyer 2003; Tsutsui and Tomita 1990; Hadley and Purves 1974; Came ron et al. 2006; McKe ndrick et al. 2000), cytokinins (Crafts and Miller 1974), auxins (Withner 1959), phosphate (Alexander et al. 1984), phosphorous (Cameron et al. 2007), nitrogen (Lucas 1977; Gebauer and Meyer 2003; Cameron et al. 2006), vitamins (Hijne r and Arditti 1973), and water (Yoder et al. 2000). These compounds either directly aid in the germination process or in the mobilization of other germination factors. Additionally, these nutrien ts and other organic compounds can be used as stored energy sources by the orch id seedling and plant du ring later life stages. In recent years some researchers have questioned the necessity of orchid mycobionts (Bayman et al. 2002); however, the dependence of orch ids on their mycobionts for at least germination should be considered as universally accepted (Rasmussen 1995). Infection and digestion The orchid-mycobiont association begins with the infection of the orchid seed by fungal hyphae, typically via the suspensor region of the seed (Clements 1988). Infection allows for direct contact between the fungal hyphae and the orchid embryo, as well as allows for the introduction of water via the mechanical break ing of the typically hydrophobic orchid testa (Curtiss 1893; Davis 1948; Stoutamire 1964; Yoder et al. 200 0). The exact cascade of mechanisms that immediately follow mycobiont infection of the orchid embryo remains a mystery; however, it is assumed that upon infect ion mycobiont prolifera tion within the orchid embryo occurs and is soon followed the formation of pelotons within the cells of the embryo (Currah and Zelmer 1992; Zettler 1997a ). In adult orchid root tissu es, pelotons are restricted to 28

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the cortical regions of the root s and typically exclude from storage organs such as tubers or corms. The infection, sequestering, and subsequent digestion of these mycobionts was first described by Burgeff (as reporte d in Hadley 1982). Two forms of infection and digestion are common in the Orchidaceae: tolypophagy, in th e photomycotrophic orchids, and ptyophagy, in the achlorophyllous orchids. Tolypophagy is the inte rcellular digestion of fungal coils, or peletons (Figure 1-5), by the cortical cells of an orchid root; whereas, in ptyophagy there is no digestion of fungal coils, but the formation of haus toria or haustoria-like st ructures that act as nutrient exchange areas (Burgeff 1959). Here, the discussion is limited to tolypophagy. In adult orchid plants, mycobionts typically infect the ro ot through root hairs on the epidermis which lead to host cell layers. It is in these host cell laye rs that fungal hyphae are allowed to proliferate, eventually entering a layer of digestion cells. Pe lotons, intercellular coils of fungal hyphae, are formed in these digestion cells, and are subs equently broken-down by intercellular enzymatic degradation (Figure 1-5). The proximity of the digestion cell layer to the vasculature of the orchid root allows for easy translocation of nutrients for either immediate use in plant metabolism or storage. At no time during the infection and digestion cy cle does the orchid harm its mycobiont to the point of death. This farming of the mycobiont has lead some research ers to refer to orchids as efficient fungus managers (Zettler 1997a). While one portion of the orchid mycobiont continues its natural function of cellulitic decay, the farmed por tion of the mycobiont is being digested within an orchid root (Hadley 1969; Midgley et al. 2006). Additionally, orchid mycobionts (and orchid plants themselves) ma y associate with other soil microorganisms, 29

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including bacteria and cyanobact eria (Wilkinson et al. 1989; Tsa vkelova et al. 2001; Tsavkelova et al. 2007). Once an orchid plant produces photosynthetically -capable parts, the role of the mycobiont becomes less clear. Adult vegetative and flowering orchid plants are known to harbor mycobionts and pelotons, and the pelotons are kn own to be digested; however, the degree of reliance upon the mycotrophic food source is no t know (for review see Rasmussen 1995). Gaddy (1983) and Gill (1996) propose d that at least some temperate terrestrial orchids use the mycobionts and mycotrophy as a source of nutrients during over-wintering dormancy, or as a source of nutrients and water during extreme stress conditions (i.e., drought, grazing). While this hypothesis appears plausible for temp erate orchids, the role of mycobionts in the adult life stages of tropical epiphytic orchids is grossly understu died. Yoder et al. ( 2000) suggested that the mycobiont of an epiphytic orchid may provide the photomycotrophic plant with mainly a source of water. This conclusion, while based on experi mental data, needs further verification. Thus, our knowledge of the orchid-mycobiont association remains poor, especially when compared to other aspects of orchid biology (i.e., taxonomy). Photomycotrophic balance The reliance of orchid seed on a mycobiont to support germination is well understood, as is the reliance of the orchid protocorm on a mycobiont as a source of energy and wa ter (for review see Rasmussen 1995). However, once the protoc orm produces photosynthetic organs, the orchid must now manage energy acquisition from bot h photosynthesis and mycotrophy. At this life stage, little is known about how the orch id establishes a balance between its Rhizoctonia mycobiont and photosynthetic gain of nutrients Alexander et al. ( 1984) and Alexander and Hadley (1985), working with th e evergreen terrestrial orchid Goodyera repens (L.) R. Brown, both reported little-tono movement of nutrients from the mycobiont to orchid tissues in adult 30

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plants using radioa ctively-labeled CO 2 and phosphate. While these studies demonstrate a minimal input of nutrients by a mycobiont to its adult host orchid, these two studies represent work based on one evergreen orchid species that is not representative of the majority of orchid species present worldwide. Furthermore, these st udies do not take into acc ount outliers such as albino mutants of orchids that are occasiona lly seen growing alongside photomycotrophic counterparts (OBrien 1953; S.L. Stewart personal observation). Recently, Cameron et al. (2006) and Camer on et al. (2007) demonstrated both the movement of nutrients (carbon and phosphorus, re spectively) from mycobiont to orchid plant and the potential for mutualistic nutrient transfer between orchid plant and mycobiont using the evergreen terrestrial orchid G. repens These reports indicate that the orchid plant may have a high degree of photomycotrophic co ntrol and be able to switch between a oneand two-way nutrient cycle with its mycobiont. Lending a genetic basis to this notion of orchid plant-based control of the orchid-mycobiont a ssociation, Watkinson (2002) reporte d that the terrestrial orchid Cypripedium parviflorum Salisbury var. pubescence (Willd.) Knight, when grown with an appropriate mycobiont ( Thanatephorus pennatus Currah), demonstrated upregulation of trehalose-6-phosphate synthase/ phosphatase and downrergulati on of a nucleotide binding protein. Plants grown in the absence of a mycobiont demonstrated no effect on the regulation of either gene. However, while many researchers agree on a genetic basis for the regulation of mycobiont infection and digesti on, most disagree on the mutualis tic potential (e.g., Cameron et al. 2006; Cameron et al. 2007) of some orchid-mycobiont associati ons (H.N. Rasmussen personal communication). Orchid mycobiont taxonomy Surprisingly, surveys of orchid mycobiont diversity worldwide from both epiphytic and terrestrial species have yielded little mycobi ont diversity. Most mycobionts isolated from 31

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orchids throughout the world have been a ssignable to the anamorphic form-genus Rhizoctonia (for review see Zettler et al. 2003). A number of nonRhizoctonia fungi have been isolated from orchids, including vesicular-arbus clar mycorrhizae (Raja et al. 1996), ascomycetes (Sharma et al. 2007; Bayman et al. 1997; S.L. Stewart unpublished data), and conidi a-producing hyphomycetes (Currah et al. 1990; Currah et al. 1987; Richardson et al. 1993). However, the role of these fungi in the mycorrhizae of orchids is not clear. The use of traditional tec hniques in the identification of species within the Rhizoctonia complex is problematic due to the lack of stab le, diagnostic morphological features in this group of fungi. Most Rhizoctonia strains exist as sterile mycelia in pure culture, yielding no definitive morphological or taxonomic character s. The induction of teleomor phic states from these sterile isolates have been previously reported, and when accomplished do yield assignable characters. Warcup and Talbot (1967, 1971, 1980) studied the my cobiont diversity in Australian and British orchids by inducing perfect states (i.e., te leomorphs) and assigning mycobionts to known teleomorphs. Many other rese archers have studied the Rhizoctonia complex from both a purely mycological standpoint through the use of sept al pore ultrastructure s, sporulation, and cytochemistry (Tu and Kimbrough 1975, 1978; Tu et al. 1969, 1977; Sneh et al. 1991), as well as from a functional standpoint in relation to the Orchidaceae (Moore 1987; Richardson et al. 1993). All of these approaches are applicable to the identification a nd taxonomic placement of orchid mycobionts, particularly for those in terested in the symbiotic propagation and conservation of orchid plants. Although mostly unpopular with plant path ologists and mycologists, Moore (1987) segregated Rhizoctonia into functional orchid-related fungal groups based on anamorphic character differences. Sneh et al. (1991) considers Moores system to be taxonomically sound 32

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because it is based on conservative characters su ch as septal pore ultrastructure, cell nuclear number, and teleomorphic affinities. For example, the anamorphs Epulorhiza Ceratorhiza and Moniliopsis are assignable to the teleomorphs Tulasnella / Sebacina, Ceratobasidium and Thanatephorus / Waitea (Moore 1987; Rasmussen 1995; Zettle r et al. 2003; Table 1-1). Of particular utility for the non-mycologist studyi ng orchid mycobionts is that these three anamorphic genera are often distinguished by simple light microscopy and examination of hyphal characteristics and monilioid cell morphology (L.W. Zettler personal communication). Furthermore, enzyme assays can play an importa nt role in the easy id entification of orchid mycobionts at the generic level. Cellulase a nd polyphenol oxidase are two enzymes that can distinguish among anamorphic genera with relative ease. In the cellula se assay, test tubes containing a lower 1/5 PDA layer and an upper layer of cellulose az ure stain medium are inoculated with the mycobiont to be surveyed (Smith 1977). The tubes are incubated for 5 days in continual light (24/0 h L/D) at 25C. A positive cellulase reaction is indicated by the diffusion of blue dye from the cellulose azure medium laye r into the lower PDA la yer (Figure 1-6a). A weak reaction is recorded if th e interface between the two media la yers remained visible, while a negative reaction is indicated as no diffusion of dye (Figure 1-6bc). In the polyphenol oxidase assay, Petri plates containing ta nnic acid medium (TAM) are inoc ulated with the mycobiont to be surveyed and incubated for 5 days in continua l darkness (0/24 h L/D) at 25C (Davidson et al. 1938). A positive polyphenol oxidase reaction is indicated by a dark brown discoloration of the TAM surrounding the point of myco biont inoculation as viewed from both the top and bottom of the plate (Figure 1-7a). A weak reaction is recorded as the discoloration being visible from only the top of the plate, while no color change indicates a negative r eaction (Figure 1-7b). 33

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Moores anamorphic taxonomy of orchid-inhabi ting mycobionts also has a great degree of practical merit, since the majority of all orchid mycobionts isolated worl dwide are assignable to one of these anamorphic genera. However, Eberha rdt et al. (1999) highli ght the instability of vegetative characters in these anamorphic gene ra and discussed the possibility of genetic differences among morphologically similar strains. Given this, the use of sensitive molecularbased identification techniques is beginning to become standa rd in the study of orchid mycobionts, particularly when st udying ecological, diversity, and e volutionary aspects of orchid mycobionts. The advantages of molecular marker syst ems in identifying and assessing orchid mycobiont diversity is only rece ntly being applied. McKendrick et al. (2000) used internal transcribed spacer restriction fragment length polymorphiam (ITS-RFLP) and ITS sequencing to identify mycobionts of a number of Corallorhiza species as belonging exclusively to the Thelephora Tomentella complex of the Thelephoraceae. Kris tiansen et al. (2001) utilized singlestranded conformation polymorphism (SSCP) and mitochondrial ribosoma l DNA sequences to identify single-peloton isolations from the terrestrial orchid Dactylorhiza majalis Random amplified polymorphic DNA (RAPD) analysis an d cleaved amplified polymorphic sequences (CAPS) have been combined with cultural morphology, nuclear number, and septal pore ultrastructure in a comparative study of taxonomic power in the study of Rhizoctonia isolates from a number of orchid speices (Shan et al. 2002). In their study, the traditional morphological methods used in this study separated the 21 Rhizoctonia isolates in the same manner as did the molecular methods, although the molecular methods were able to directly assign a teleomorph to each isolate. The advantage of using molecularbased marker system in the identification of orchid mycobionts is obviousspeed of identific ation and nearly instantaneous assignment of 34

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teleomorphs. Future fine-scale study of or chid mycobiont diversity and orchid-mycobiont evolution will require the continued use of these molecular-based identification methods. Orchid Propagation and Culture Asymbiotic seed culture Knudson (1922) was the first person to demonstrate the germination of orchid seeds without a mycorrhizal fungus. Originally, Knudson was interested in the effects of sugars on plants (Steward 1958; Arditti 1967) and enzy me production in fungi (Knudson 1913). Based on these interests, Knudson realized the role of orchid mycobiont sas carbohydrate hydrolyzers, breaking complex molecules, such as starch and cellulose, into simple sugars that an orchid plant could utlize. In testing this hypothesis, Knudson sowed seeds of Cattleya mossiae Hooker on Pfeffers mineral salt solution supplemented with 1% sucrose and within 7 months seeds germinated. To further test his hypothesis, Knudson then sowed seeds of the hybrid C. intermedia Graham C. lawrenceana Rchenb. on Knudson Solution B supplemented with either 2% sucrose or 2% glucose. Within one year, seeds of this hybrid also germinated (Knudson 1922). Eventually, Knudson refined his Solution B to Solution C (Knudson 1946), which is currently known as Knudson C and widely used as a standard asymbiotic orchid seed germination medium. Since the publication of Knusdons asymbiotic germination medium and method in 1922, the vast majority of orchid seed germination research has focused on using these methods. Symbiotic seed germination requires expertise in both mycology and plant propagation; whereas asymbiotic seed germination techniques require the researcher to be only a plant propagation specialist (L.W. Zettler personal communication). Asymbiotic germination techniques have been applied to a number of tropical, temperat e, epiphytic, and terres trial orchid taxa. Vanilla is one orchid genus that has received attention from those interested in th e mass propagation of this 35

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commercially important crop from seed (Knudson 1950; Withner 1955). While asymbiotic seed germination of Vanilla is possible, the use of these seed-born seedlings in commercial production of Vanilla simply never became attractive since th is genus is easily propagated clonally by cuttings. A number of reports exist concerning the asymbi otic seed germination of orchid species in an attempt to optimize germination and produc tion conditions. Knudson (1951) reported on the comparative effects of asymbiotic germination of Cattleya skinneri seeds sown on Knudson C versus Vacin and Went medium. Not surprisi ng, Kundson (1951) demonstrated a higher percent germination and better in vitro seedling growth on his Knuds on C medium. Ernst (1975) reported on attempts to enhance the asymbiotic germination and in vitro seedling growth of Phalaenopsis In particular, Ernst (1975) was interested in examining the effects of activated charcoal and banana additives on asymbiotic se ed germination. This study reported a 174% increase in seedling fresh weight (over control) from seedlings in the Knudson C + charcoal treatment and a 481% increase in seedling fresh we ight (over control) fr om seedlings in the Knudson C + charcoal + banana treatments. These data demonstrate a common theme among practitioners of asymbiotic orchid seed germinationbasal medium modification for the enhancement of germination and in vitro seedling growth. Others have demonstrated the use of as ymbiotic germination techniques in the conservation of rare or medici nally-important orchids. Ligh t and MacConaill (2003) reported on the asymbiotic germination of Galeandra batemanii Rolfe and G. greenwoodiana Warford as a means of plant production for c onservation purposes. Shimada et al. (2001) reported the asymbiotic germination of Habenaria radiata Thumb. and the induction of in vitro tubers by these asymbiotically-propagation plants. The in vitro tubers where used as planting material in 36

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the reintroduction of this species in Japan. Lo et al. (2004) recently reported on the asymbiotic germination of the medicinally important orchid Dendrobium tosaense Makino in Taiwan. Furthermore, in vitro asymbiotic germination has been used to study the germination physiology of orchid seeds. Harrison and Arditti (1978) used asymbiotic germination methods to investigate the role of carbohydrat e source in the germination of Cattleya aurantiaca (Batem. ex Lindl.) P.N. Don. Ernst et al. (1971) and Ernst and Arditti (1990) demonstrated the usefulness of asymbiotic orchid seed germination me thods in the study of carbohydrate usage in in vitro seedlings of several Phalaenopsis hybrids. By growing seedlings on media containing different carbohydrate sources, they were able to demonstrate the utilization of simple sugars by orchid seedlings versus complex carbohydrates. Similarly, asymbiotic seed culture has been used to demonstrate the rapid uptake of simple suga rs (i.e., fructose) by both differentiated and undifferentiated Dendrobium tissues (Hew and Mah 1989). Zeigle r et al. (1967) used asymbiotic seed culture to study the effects of amino aci d supplementation of media with Edamin on the germination of the hybrid Cattleya Enid. alba Laelia anceps Lindl. var. veitchii In media treatments with the addition of Edamin, seeds of C. Enid. alba L. ancepes var. veitchii required 10 to 13 days less to germinate and show signs of greening. The orchid genus Cypripedium has received a disproportionate amount of attention from asymbiotic practitioners. Because of their reason ably attractive flowers and hardy growth habit, species of Cypripedium are highly sought after as horticultur al specimens and research plants. However, seeds of this temperate orchid genus ha ve been notoriously difficult to germinate using asymbiotic techniques due to strong seed dorma ncy issues (Liddell 1952; Chu and Mudge 1996). A great deal of research has focused on media selection and modification for this genus (De Pauw and Remphrey 1993), seed germination sc ale-up procedures (Malmgren 1992), the use of 37

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immature seed (Hoshi et al. 1994; St-Arnaud et al. 1992), and the effect s of seed chilling and chemical pretreatments (Miyoshi and Mii 1998). Research focusing on the asymbiotic germination of Cypripedium species worldwide has shed new light on the usefulness and power of asymbiotic seed germination techniques. In examining the role of asymbiotic media effects and media supplementation, Nakamura (1982) investigated the nutritional conditions requir ed for the asymbiotic germination of seeds of the achlorophyllous orchid Galeola septentrionalis Reichb. These studies found that nitrogen source played a large role in the support of germination, with organic sources of nitrogen supporting high germination percentages and inor ganic sources supporting minimal germination of G. septentrionalis This implies that G. septentrionalis likely obtains organic forms of nitrogen from its mycobiont, instead of inorganic forms of nitrogen from the soil. Additionally, simple sugars supported higher germination perc entages than did more complex sugars. The presence or absence of vitamins was found to have no affect on seed germination or in vitro plant growth. Finally, auxins were found to improve seedling growth post-germination and cytokinins were found to have no effect on seedling growth. In studying the asymbiotic seed culture of G. septentrionalis Nakamura (1982) was able to demonstr ate that this achlo rophyllous orchid species possesses the same nutritional requirements as chlorophyllous orchids and likely gains these nutrients through its mycobiont. Thompson et al. (2006) conducted a similar survey of asymbiotic germination requirements in the South African orchid genus Disa Interestingly, Thompson et al. (2006) combined a survey of asym biotic germination requirements (i.e., media, media supplementation) with conservation by deve loping germination protocols for a number of Disa species. 38

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An emerging use of asymbiotic seed culture te chniques has come in the form of asymbiotic media screens as a way to optimize germinati on efficiency prior to further physiological experimentation. Stenberg and Kane (1998) studying the Florida epiphytic orchid Prosthechea boothiana (Lindl.) W.E. Higgins var. erythronioides (Small) W.E. Higgins (syn. = Encyclia boothiana (Lindl.) Dressler var. erythronioides (Small) Luer), reporte d variable asymbiotic germination in a screen of four commonly-available asymbiotic media. Kauth et al. (2006) conducted a similar asymbiotic media scr een in studies on the germination and in vitro seedling development of Calopogon tuberosus var. tuberosus. They reported significant differences in seed germination on three different asymbio tic media, as well as differences in in vitro seedling growth and development. In th e case of Kauth et al. (2006), asym biotic media screen research laid the foundation for further investigations of the effects of photoperiod and temperature on ecotypic delimitation of C. tuberosus var. tuberosus by identifying an optimal medium to support the rapid germination and seedling growth of the species (P. Kauth person al communication). A similar approach was taken by Stewart and Kane (2006a) in investigations on the asymbiotic seed germination and effects of photoperiod, ex ogenous cytokinins, a nd carbohydrate source on the germination of the Florida terrestrial orchid Habenaria macroceratitis The continued focus on asymbiotic orchid seed culture methods is an important part of orchid conservation biology, especially for the study of the physiological effects of mineral nutrition, photoperiod, and the e ffects of exogenous growth regulators on orchid seed germination and seedling development. Furtherm ore, the use of asymbiotic media screens as a means to optimize asymbiotic germination before conducting further in vitro physiological or ecological studies should receive continued at tention. Although asymbiotic orchid seed germination methods may represent an efficien t means to propagate species for conservation 39

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purposes, this method does not account for the role of the mycobiont. Without these mycobionts, orchid seed germination and plant development ex situ would not occur. Symbiotic seed culture methods should be explored as a more viable means of species conservation. Symbiotic seed culture The early orchid mycobiont work of Bern ard and Burgeff centered around not only the descriptive aspects of the orchid-mycobiont associ ation, but also the use of isolated mycobionts in seed-mycobiont co-culture experiments. Bo th Bernard and Burgeff successfully germinated seeds of orchids under co-culture conditions with appropriate mycobionts (as reported in Arditti 1984). Little new research on the symbiotic seed germination of orchids continued after Knudsons (1922) report of asymbio tic orchid seed germination. Not until the 1940s, was interest in symbiotic seed germination techniques resurrected. Downie (1940) published one of the first modern reports of symbiotic germination. Working with the terrestrial orchid Goodyera repens Downie reported increased germination efficiency and an increase in protocorm size using the symbiotic technique in comparison to the asymbiotic technique. Later, Hadley (1970) reported on the symbiotic germin ation of a number of orchid species, including Epidendrum radicans Pavn ex Lindl., Spathoglottis plicata Blume, Dactylorhiza purpurella (T. Stephenson & T.A. Stephenson) So, and Goodyera repens While reporting on the symbiotic germination of these species, Hadley focused his research on the question of orchid-mycobiont preference by dem onstrating that some mycobionts are nearly universal in their ability to s upport symbiotic germination while others appear to support the germination of only a narrow range of orchid taxa. Warcup (1973) reported on the symbiotic germination of several Australian Pterostylis, Diuris and Thelymitra species using several species of Tulasnella and Ceratobasidium mycobionts. He reported that the various isolates of Tulasnella supported variable germination among the di fferent orchid gene ra tested. Warcup 40

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(1973) was one of the first researchers to repor t that mycobionts have different capacities to support symbiotic seed germination, and that th e mycobionts that support the most efficient symbiotic germination in vitro may not originate from the orchid taxa from which seed were collected. This orchid-mycobiont preference debate still rages today (Taylor and Bruns 1999; Stewart and Kane 2007a; Otero et al. 2005; Shefferson et al. 2005; Taylor et al. 2003), and has serious ecological and orchid conservation consequences now being recognized (Stewart and Zettler 2002; Stewart and Kane 2006b, 2007). Modern symbiotic seed germination techniques were first popularized and applied to the conservation of orchids in the 1980s. Muir ( 1987) reported on the symb iotic germination of a cultivated native orchid in Europe, Orchis laxiflora Lam. Jersey Girl. Unidentified strains of the mycobiont Ceratobasidium were used in these studies. Th e symbiotic germination of another European terrestrial orchid, Dactylorhiza maculata (L.) So, using unidentified species of mycobionts was reported by Mitc hell (1988). This study used a modified oats medium containing only powdered rolled oats, sucrose, and agar. This simple oat-based symbiotic medium proposed by Mitchell (1988) remains a sta ndard symbiotic media today. Clements et al. (1986) reported the symbiotic germination of fi ve European orchid genera (23 species) using mycobionts isolated from these sa me taxa. Most importantly, Cl ements et al. (1986) reported on the use of modified oats medium in their sy mbiotic experiments. This mediumcontaining minor mineral salts, sucrose, agar and powdered rolled oatsis still used today as a basic media for symbiotic seed germination studies. The majority of symbiotic germination reports since the early 1990s have focused on the use of this propagation system in the seed germin ation and conservation of various orchid taxa. A number of epiphytic orchids have been germinated using symbiotic seed germination 41

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techniques, including Sarcochilus (Markovina and McGee 2000), Epidendrum magnoliae Mhlenberg var. magnoliae (syn. = E. conopseum R. Brown; Zettler et al. 1998), Encyclia tampensis (Lindl.) Small (Zettler et al. 1999), and Epidendrum nocturnum Jacquin (Zettler et al. 2007). The majority of these reports focus on th e conservation-driven sy mbiotic propagation of epiphytic orchids (Zettler et al. 2007). However, both Zettler et al. ( 1999) and Zettler et al. (2007) contribute important information concerning mycobiont preference in epiphytic orchids, a generally understudied topic. In both cases, s eeds of epiphytic orchids were germinated using mycobionts originating from differ ent orchid taxamycobiont from Epidendrum magnoliae var. magnoliae in the case of Zettler et al. (1999) and mycobiont from Spiranthes brevilabris in the case of Zettler et al. (2007). As seen with the reports from Hadley (1970) and Warcup (1973), mycobiont preference in epiphytic orchid taxa ap pears to be variable and species-dependant. Continued research in this area of symbio tic orchid seed germ ination is necessary. The orchid genera Platanthera and Habenaria have received consid erable attention from practitioners of symbiotic seed germination in recent years, es pecially by researchers in North America. Zettler and McInni s (1992) reported on the symbio tic propagation of the rare terrestrial orchid Platanthera integrilabia using mycobionts originating from P. ciliaris (L.) Lindl., P. clavellata (Michaux) Luer, P. cristata (Michaux) Lindl., P. integrilabia and Spiranthes cernua The highest percent seed germin ation reported was supported not by a mycobiont originating from P. integrilabia but from a mycobiont originating from P. ciliaris However, the highest percentage of soil establ ished seedlings were supported by the mycobiont originating from P. integrilabia Further studies on the affect s of light during the symbiotic germination of P. integrilabia were reported by Zettler and McI nnis (1994). Interestingly, the highest percent symbiotic seed germination wa s supported when seeds were exposed to seven 42

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days of 16/8 h L/D photoperiod before being subj ected to darkness for the remainder of the study. Zettler and McInnis (1994) represent one of the first repor ts on the affects of photoperiod on the symbiotic germination of an orchid. Zettl er et al. (2001) and Ze ttler et al. (2005) both reported on the development of symbiotic seed germination techniques for the North American temperate terrestrial orchid Platanthera leucophaea (Nutt.) Lindl.. Furthermore, Zettler et al. (2005) proposed an intermediate acclimatization step between in vitro and greenhouse conditions for symbiotic seedlings. This intermediate step appears to not only help in the acclimatization of seedlings, but also in the acclimatization of th e mycobiont to a new growing environment. Working with Habenaria radiata an allied species to the genus Platanthera Takahashi et al (2000) reported on the species symb iotic seed germination and the effects of seed age, culture media, period, and mycobiont preference. More recently, Stewart and Zet tler (2002) and Stewart and Kane (2006b) reported on the symbiotic seed germination of the subtropical terrestrial orchid Habenaria macroceratitis Another orchid genus that has received considerable attention from those interested in symbiotic seed germination and conservation is Spiranthes. Anderson (1991) first reported on the symbiotic germination of the terrestrial orchid Spiranthes magnicamporum Sheviak using the mycobiont Epulorhiza repens (Bernard) Moore isolated from the same species. Furthermore, Anderson (1991) also detailed th e growth and development of S. magnicamporum in symbiotic culture both in vitro and in the greenhouse. Zettler and McInnis (1993) reported on the symbiotic seed germination of Spiranthes cernua and another Spiranthoideae orchid, Goodyera pubescens (Will.) R. Brown. This study represents another example of the exploration of orchidmycobiont preference in the Orchidaceae, by using mycobionts isolated from S. cernua, Platanthera integrilabia and P. ciliaris Zelmer and Currah (1997) investigated the symbiotic 43

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germination and mycobiont diversity of S. lacera Rafinesque, finding a mixture of strains of Ceratorhiza and Epulorhiza in the roots of this species. In their study, on ly the mycobiont strain Ceratorhiza goodyerae-repentis (Costantin & Dufou r) Moore supported the in vitro symbiotic germination of S. lacera Zettler and Hofer (1997) used the symbiotic germination of the terrestrial orchid S. odoara to experimentally explore th e effects of light on symbiotic germination. They reported the highest final percent germination in 0/24 h L/D treatments, followed by 8/16 h and 14/10 h L/D photoperiods. Stewart and Kane (2006b) described a similar germination response in the symbiotic germination of Habenaria macroceratitis with 0/24 h, 16/8 h, and 24/0 h L/D photoperiods. Stewart et al. (2003) repor ted on the conservation-driven symbiotic propagation and rein troduction of the endangered Florida terrestrial orchid S. brevilabris Again, this study highlighted the degree of mycobiont preference some orchid taxa exhibit for their mycobiontsmycobionts origin ating from both the study species and the Florida epiphytic species Epidendrum magnoliae var. magnoliae supported in vitro symbiotic germination. Stewart et al. (2003) was the first describe the succe ssful reintroduction of in vitro grown symbiotic seedlings of a North American terrestrial orch id. Finally, Stewart and Kane (2007a) investigated the symbiotic germination a nd degree of mycobiont preference in the orchid species pair S. brevilabris and S. floridana In this report, a high degree of mycobiont preference was suggested for both S. brevilabris and S. floridana based on mycobiont isolation and in vitro symbiotic seed germination studies, despite a hi gh degree of genetic re latedness between these two orchid taxa. Seed of a number of other orchid taxa have been successfully germinated using symbiotic techniques. Jrgensen (1995) re ported on the symbiotic culture of the hardy terrestrial orchid Dactylorhiza majalis Tan et al. (1998) discussed the symbiotic seed germination of the 44

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commercially-important terrestrial orchid Spathoglottis plicata An interesting modification proposed in Tan et al. (1998) to the standard sy mbiotic technique is the encapsulation of orchid seed and mycobiont within an alginate bead. This bead would then be pl anted directly into the greenhouse or field, transferring bot h the seed and an appropriate mycobiont in one unit. The symbiotic germination of another orchid of commercial interest, Bletilla striata (Thunberg) Rchb., was reported by Johnson (1994). Finally, Yagame et al. ( 2007) reported the symbiotic germination from seed to flowering plant of the achlorophyllous orchid Epipogium roseum (D. Don) Lindl. Few achlorophyllous orchids have been successfully germin ated using symbiotic techniques, and none have been reported as flowering from symbiotic culture conditions. Symbiotic seed germination techniques with orchids have a long and storied history. The techniques originally developed by Bernard and Burgeff in their studies of the orchid-mycobiont association remain applicable th rough to today. Of particular interest is the application of modern symbiotic germination techniques in the pr opagation and conservation of orchid species. This mode of propagation allows for orchids to be grown in conjunction with their mycobiont associate, unlike in asymbiotic germination me thods where the mycobiont is not physiologically accounted for. For conservation and species r ecovery purposes, symbiotic seed germination should represent the primary mode of plant propagation. Orchid Pollination Biology The evolutionary relationship between orchids and their pollin ators has resulted in a great diversity of flower morphologies and pollinati on mechanisms in the Orchidaceae (Tremblay 1992; Cozzolino and Widmer 2005). Pollinator specialization and unique pollination mechanisms should, in theory, enhance the f itness of the Orchidacea e by reducing the costs associated with reproduction. However, obligate po llinators, such as those orchids are likely to rely on, are often scarce in the landscape (Her rera 1989) and possibly su sceptible to population 45

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decline due to environmental changes because of their general scarcity. The identification of both pollinators and pollination mechanisms is an important part of the integrated conservation of orchid species, and ca n often lead to the direct conservatio n and management of pollinators in habitats supporting them. Despite the volumes of literature concerning the pollination biology of orchids, few species have been carefully studied, their pollinat ors identified, and their pollination mechanism determined. Catling and Catling (1991) reported that only basic pollination information is known for only 40% of all North American orchid species, and that detailed information is available on only about 15% of speci es. It is doubtful that thes e percentages have improved in the intervening years si nce this report. Tremblay (1992) suggests a reduction in the number of pollinators per orchid species beginning with the Cypripedioideae (6.3 pollinator s/species) and moving to the Epidendroideae (1.5 pollinators/species), with th e Orchidoideae between the two ( 4.0 pollinators/species). From an evolutionary standpoint, these data suggest a significant reduction in pollinators per species from the most ancestral to the more recently derived. To best understand this large, everevolving plant family, the study of the pollinati on biology of the Epidendroideae should be highlighted. However, the majority of pollin ation biology research has focused on the more ancestral subfamilies of the C ypripedioideae and the Orchidoideae (Catling and Catling 1991). The specialization of pollinators and diversity of pollination mechanisms is quite evident in the Orchidoideae. As a result, pollinators and pollination mechanisms have been used for a variety of purposes, from clarifying the iden tity of taxonomically confusing species to identifying pollination ecotypes within the distri bution of a particular species. Sheviak and Bowles (1986) used differences in pollination mechanism and pollinia placement on pollinators 46

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to taxonomically distinguish Platanthera leucophaea and P. praeclara Sheviak & Bowles. By determining these pollination differences and relating them to evolutionary forces in the speciation of these species, Sheviak and Bowl es (1986) again made the link between orchid pollination and orchid evolution. Smith and Snow (1976) identified the different pollinators of P. cilaris and P. blephariglottis (Willd.) Lindl., two closely-related species. In their studies, the colorful P. cilaris was pollinated by spicebush swallowtail butterflies ( Papilio troilus L.), whereas the white-colored P. blephariglottis was found to be pollinated by a variety of moth species. Smith and Snow (1976) surmised that colo r served as the main attractant for pollinators of P. cilaris and scent served as the attractant for P. blephariglottis pollinators. Despite their taxonomic relatedness and often sympatric growth habits, these two Platanthera species had evolved two distinct systems to attract different pollinators. In a reassessment of the pollination biology of P. blephariglottis Cole and Firmage (1984) confirmed that this species is pollinated by moths. A related Platanthera species, P. stricta Lindl., was reported as being pollinated by both moths and bees (Patt et al., 1989). This demons trates a degree of plasticity that can be seen in some orchid pollination systems, a lthough this plasticity is not typical. An interesting application of the study of pollination systems in the Orchidoideae is the report of pollination ecotypes base d on altitude in the orchid P. ciliaris (Robertson and Wyatt 1990a, 1990b). Plants of this species growing in mountainous regions were found to possess a significantly shorter nectar spur when compared to plants growing in coastal plain areas. This environmental adaptation was further s hown to target two different pollinators Papilio troilus in the mountains and P. palamedes in the coastal plain regions. This pollination mechanism adaptation is an example of how closely tied orchids and their pollinators are. 47

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In an unique case of auto-pollination versus pollinator-mediated pollination, Johnson et al. (1994) and Johnson (1994) reporte d on differential pollination sy stems in the South African terrestrial orchid genus Disa Auto-pollination was reported for three Disa speciesD. vaginata Harv. ex Lindl., D. glandulosa Burch. ex Lindl., and D. rosea Lindl. (Johnson et al. 1994), whereas an anthophorid bee was re ported as the pollinator of D. versicolor Rchb. (Johnson 1994). A change in flower color was also reported during pollination studies on D. versicolor and this color change was suggested as an orient ation cue for the bee polli nator to locate newly opened flowers versus old or previously-pollinated flowers. Johnson et al. (1994) surmise that auto-pollination in Disa likely evolved in four independent lineages and that, a nd that habitat and lack of pollinators were likely driv ing forces behind this evolution. The higher average number of pollinators per species in the Cypripedioideae, relative to the Orchioideae, appears to be accompanied by lower rates of auto-pollination or agamospermy in the Cypripedioideae (Catling and Catling 1991) Recent reports on the pollination biology of Cypripedium species indicate that bees and bee allies are the primary pollinators in this genus (Bnziger et al. 2005; Li et al. 2006). Orchids are known to possess a number of othe r floral characteri stics that promote pollination. Trapnell and Hamrick (2006) repor ted on the importance of floral display and effective population sizes in the epiphytic orchid Laelia rubescens Lindl. Because effective population sizes for this species were found to be small, effective floral display was found to be a major contributing factor to the pollination success of L. rubescens Sexual deception as a means to insure pol lination occurs only among the Orchidaceae (Wiens 1978; Nilsson 1992). Alcock (2000) reported on the use of sexual deception as a pollination mechanism in the Australian terrestrial orchid Spiculaea ciliata Lindl. This species is 48

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pollinated by male Thynnoturneria wasps, and was shown to attr act males in as little as two minutes of flower presentation. However, Alcock (2000) demonstrated that fewer than half of the male wasps attempting pseudocopulation with flowers of S. ciliata came in contact with pollinia and affected pollination. In the case of the species pair Cleistes divaracata (L.) Ames and C. bifaria, pollen abundance, not floral display, played an impor tant role in the success of pollination (Gregg 1991). Bumblebees ( Bombus ), the pollinators of both Cleistes species, were repeatedly observed collecting Cleistes pollen as a food source whil e affecting pollination of individual plants. This report represents a unique use of food-attrac tion in the Orchidaceae as a means to insure pollination. Nectar production is another common pollinat or attractant in the Orchidaceae. Orchid species that produce nectar as a reward or attractant for pollinato rs typically target members of the Lepidoptera. Zettler et al (1996) reported that the nectar producing terrestrial orchid Platanthera integrilabia used nectar as a probable attrac tant or reward for its observed pollinators Epargyreus clarus Cramer, Papilio glaucus L., and P. troilus Luyt and Johnson (2001) reported the similar use of nectar as a reward or pollinator attractant in the African epiphytic orchid Mystacidium venosum Harv. ex Rolfe. Jacquemyn et al. (2005) attempted to use orchid nectar production as an assessment tool predicting species rarity and extinction probabilities. They concluded that nectar produc tion does not serve as a highly reliable measure of extinction probability, and that habitat lo ss and fragmentation are better measures. The specialization among pollinators, pollin ation mechanisms, and species in the Orchidaceae is directly correlated to the diversity seen in the family. In order to preserve this high level of species diversity, car eful study of orchid pollination biology is necessary to insure 49

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the long-term viability and fitness of species and populations. As suggested by Catling and Catling (1991), the continued investigation of the pollinators and pollination mechanisms of orchids will greatly aid in the integr ated conservation of this family. Molecular Genetics and Genetic Diversity in Orchids The use of molecular-based techniques in the study of the Orchid aceae has traditionally focused on two main areas: 1) phylogenetics and 2) conservation gene tics/genetic diversity studies. Both of these molecular approaches to the study of the Orchida ceae have revealed new insight into the evolution, rela tions, and conservation of orchids worldwide. However, there has been a traditional disconnect between those intere sted in the molecular p hylogenetics of orchids and those interested in the conservation of or chids. Recently, a number of researchers have begun to couple the study of phyl ogenetics and conservation biology to the benefit of worldwide orchid conservation (Dueck and Cameron 2007; Pillon and Chase 2007; Pillon et al. 2006). The widest application of molecular biology in the Orchidaceae has come in the form of molecular phylogenetics and molecular evolut ion studies. Traditionally, taxonomy in the Orchidaceae focused on the study of varying mo rphology, habitat preference, and chromosome numbers (Dressler 1981, 1992; Shevia k 1982). However, the movement toward molecular-based techniques in the study of orchid taxonomy ha s elucidated a number of interesting cryptic relationships within particular genera and between species. Hedrn et al. (2001) and Hedrn et al. (2007) utilized both amplified fragment length polymorphism (AFLP) and plastid DNA techniques to investigate the or igins of and relations between Dactylorhiza species. Traditional taxonomists struggled to understa nd relationships within this genus, especially due to the existence of several geographicallyand habitat-specific forms, vari eties, and ecotypes. The use of molecular phylogenetics in this genus has al lowed a more precise unde rstanding of withingenus species relations; therefore, allowing identifi cation of rare or endemic species or forms and 50

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better conservation planning for this genus thro ughout Europe. A more traditional use of molecular phylogenetics is in the determination of species. Examples of this use include the revised delimitation of Orchidinae and selected Habenariinae (Bateman et al. 2003) and the molecular delimitation of Cranichideae a nd Spiranthinae (Salazar et al. 2003). Molecular phylogenetic research has also b een used to investigate the evolution and differentiation of a number of or chid species. In a molecular-based study of the North American terrestrial genus Cleistes no support for the trad itional separation of C. divaracata and C. bifaria was found (Smith et al., 2004), although these two species are morphologically distinct. Similarly, Carlsward et al. (2003) reported a revised broad de finition of the orchid genus Dendrophylax to include the genera Harrisella, Polyradicion and Campylocentrum ; again, despite distinct morphological ch aracters to the contrary. Si milarly, Higgins et al. (2003) reported on a combined molecular phyl ogeny of the epiphytic orchid genus Encyclia with the redelimitation of a number of species within this genus. In a novel use of molecular-based taxonomy, Carlsward et al. (2006) i nvestigated the evolution of l eaflessness in the orchid tribe Vandeae. They determined the gross morphologic al changes in this tribe, driven by genetic changes, resulted in the reduction of leaves, typically to a simple scale, and the evolution of gas exchange complexes in photosynt hetically-capable roots lead to the development of leafless morphologies. Finally, Goldman et al. (2004) used both molecular and cytological methods to more clearly define speciation in the No rth American terrestrial orchid genus Calopogon. This study reinforced traditional spec ies concepts in this genus wi thout redefining new species. The second-most common application of mol ecular-based technologies to the Orchidaceae is in the study of conservation genetics and popul ation genetic diversity. Gaining knowledge of genetic diversity in plants is a key step in understanding the long-term conservation needs of 51

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individual species and p opulations. Genetic divers ity plays an important role in the persistence of individuals in a changing environment and the ability of those individuals to adapt (Frankel and Soul 1981; Lande and Barrowclough 1987). There are a number of ways to measure genetic diversity in orchid populations, includ ing morphological approach es, protein markers, and DNA markers (Qamaruz-Zaman et al. 1998b). This brief review will focus on only DNA markers, with a particular emphasis on the AFLP technology. Protein-based marker systems remain quite pr evalent in the study of orchid conservation genetics despite advances in DNA-based sy stems (Sun 1997; Sun and Wong 2001; Wallace and Case 2000; Sharma et al. 2000; Shar ma et al. 2003; Sharma et al. 2001 ; Case et al. 1998). This is in spite of their functional draw backs, such as difficulty in keeping sampled materials fresh or appropriately frozen from the field to the labora tory, the difficulty in detecting new alleles, and issues surrounding polyploid speices (Qamaruz-Zaman et al. 1998b). Nonetheless, protein-based marker systems continue to be used to provide conservation genetics data to those interested in orchid conservation. A number of DNA-based markers have been us ed to investigate th e conservation genetics and population genetic diversity of orchids. Bush et al. (1999) used random amplified polymorphic DNA (RAPD) techniques to investigate the genetic variation in the epiphytic orchid Epidendrum magnoliae (syn. = E. conopseum ), as well as the co-occurring epiphytic fern Pleopeltis polypodioides (L.) E.G. Andrews & Windham. St udying the conservation genetics of Spiranthes diluvialis Sheviak, Szalanski et al. (2001) used random fragment length polymorphism (RFLP) to reveal no genetic variation within or among populations of this species. Gustafsson and Sjgren-Gulve (2002) used an improved microsatellite technique to compare genetic diversity between the rare Gymnadenia odoratissima (L.) L.C.M. Richard and the more 52

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common G. conopsea (L.) R. Brown, reporting a distinct ion between the mainland and island populations of G. odoratissima Wallace (2002a, 2002b) utilized RAPD methods to examine the effects of habitat fragmentation on genetic vari ation in the Midwestern terrestrial orchid Platanthera leucophaea Smith et al. (2002) used intersimple sequence repeat (ISSR) to examine genetic variation in Tipularia discolor (Prush) Nutt., reporting gene flow among four distinct populations of the species despite a number of field-base d reports of clonal growth of populations. Cozzolino et al. ( 2003) reported both severe habita t fragmentation and genetic bottlenecks in the rare orchid Anacamptis palustris using minisatellite methods. Finally, Tsukaya (2005) reported on the gene tic variation in populations of Spiranthes sinensis (Pers.) Ames, as well as systematic information on this cryptic species using plastid sequencing data. The variety of molecular-based to ols at the disposal of those interested in the conservation genetics of orchids is staggeri ng. However, one method has been shown to be the most powerful in the investigation of population genetic di versity and conservation geneticsamplified fragment length polymorphism (AFLP). The AFLP technique, as developed by Vos et al. (1995), is a power ful tool to assess withinand between-population gene tic diversity, and has been used widely in the study of plant conservation genetics (Scariot et al. 2007; Arcade et al. 2000; Ranam ukhaarachchi et al. 2000; Roldn-Ruiz et al. 2000; DeRiek et al. 1999). The value of the AFLP technique over other currently-available techniques lies in its overall simplicity. Five main advantages have been identified: 1) no development time, 2) large numbe rs of loci and samples can be quickly assayed due to automation, 3) provides 10 -100 times more markers per react ion than other techniques, 4) technique is highly reproducible, and 5) combines the reliability of RFLP with the simplicity of 53

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PCR (Qamaruz-Zaman et al. 1998a). These factors make the use of AFLP in the study of orchid conservation genetics highly valuable. Because of these many advantages, the AFLP technique is increasingly being applied to orchid conservation genetics. Qamaruz-Zaman et al. (1998a) used AFLP to investigate the conservation genetics of Orchis simia Lam., reporting the direct use of the resulting data in the nation-wide management of the remaining populations of this species in the United Kingdom. In an attempt to develop parental identification methods for Vanda breeding in Singapore, Chen et al. (1999) used the AFLP technique to develop gene tic fingerprints of proper ly identified species and hybrids that could be used as standards against which to measure unknown species and/or hybrid combination. Forrest et al. (2004) identified two distinct meta-populations of the terrestrial orchid Spiranthes romanzoffiana in Europe based on AFLP data and breeding system determinations. Pillon et al. (2007) repor ted on the genetic diversity and ecological differentiation in the widesprea d, but rare, terre strial orchid Liparis loeselii (L.) Richard. This species has a disjunct distribution in both Europe and the United Stat es this is reflected in a high degree of genetic separation between the two c ontinents. Finally, Hedrn et al. (2001) and Hedrn et al. (2007) reported on the use of AFLP methods in eluc idating the polyploidy evolution of Dactylorhiza species, varieties, and ecotypes. The power of the AFLP technique in studying the conservation genetics of orchids is exceptional, as the aforementioned examples highlight. AFLP should be considered a crucia l technique in the inte grated conservation of orchids worldwide (Stewart and Kane 2007b). 54

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Table 1-1. Anamorphic and corresponding teleomorph species of common orchid mycobionts in the Rhizoctonia complex, after Zettler et al. (2003). Anamorph Teleomorph Ceratorhiza pernacatena unknown Ceratorhiza goodyerae-repentis Ceratobasidium cornigerum unknown Ceratobasidium angustisporum unknown Ceratobasidium globisporum unknown Ceratobasidium sphaerosporum unknown Ceratobasidium stevensii unknown Ceratobasidium papillatum Epulorhiza albertaensis unknown Epulorhiza anaticula unknown Epulorhiza calendulina unknown Epulorhiza inquilina unknown unknown Tulasnella allantospora unknown Tulasnella asymmetrica Epulorhiza repens Tulasnella deliquescens unknown Tulasnella cruciata unknown Tulasnella irregularis unknown Tulasnella violacea Moniliopsis anomala unknown Moniliopsis solani T hanatephorus cucumeris Moniliopsis zeae Waitea circinata unknown Thanatephorus obscurum unknown Thanatephorus orchidicola unknown Thanatephorus pennatus unknown Thanatephorus sterigmaticus 55

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Figure 1-1. Diagrammatic representation of the steps and connections of integrated orchid conservation. 56

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Figure 1-2. Habenaria macroceratitis A) Habenaria macroceratitis inflorescence in habitat. B) Greenhouse grown H. macroceratitis plant prior to anthesis. C) Habenaria macroceratitis flower. Scale bars = 1 cm. 57

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Figure 1-3. Spiranthes brevilabris and S. floridana A) Spiranthes brevilabris inflorescence in habitat. B) Spiranthes floridana inflorescence in habitat. Scale bars = 1 cm. 58

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Figure 1-4. Deep South race of Spiranthes cernua A) Spiranthes cernua inflorescence, scale bar = 1 cm. B) Spiranthes cernua lip detail, scale bar = 1 mm. C) Spiranthes cernua flower profile, scale bar = 1 cm. 59

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Figure 1-5. Example of mycobiont pelo tons in the cortical tissues of Spiranthes brevilabris A) Groups of pelotons (400; sc ale bar = 50 m). B) Isolat ed peloton (1000; scale bar = 1 m). 60

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Figure 1-6. Cellulase assay. A) Positive reaction. B) Weak reaction. C) No reaction. 61

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Figure 1-7. Polyphenol oxidase assay. A) Positive reaction. B) No reaction. 62

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CHAPTER 2 POLLINATION BIOLOGY AND GENETIC DIVERSITY OF Habenaria macroceratitis Introduction A great deal of attention has been given to the study of orchid plant growth and development in the wild (f or reviews see Dressler 1981, 1993; Hutchings 1987a, 1987b), although few studies have followed individual plan ts or populations for more than one or two years (Willems 1982; Wells 1967; Light and MacCona ill 2007; Jacquemyn et al. 2007; Pfeifer et al. 2006). This lack of in-depth or long-term life history trait studies has lead to a general lack of biological understanding of many orchid genera. Long-term study of life history traits would facilitate a better understanding of the vegetative and re productive dynamics of individual orchid genera and species, as well as popula tions of orchids. Only a few long-term studies of orchid life history traits exist (Wells 1967; Tamm 1972; Hutchings 1987a, 1987b; Calvo 1990), and the value of these reports are only now bei ng fully recognized (M.H.S. Light, personal communication). Pollination biology represents an essential component of th e study of plant life history traits. The understanding of a species mode of reproductionasexual, sexual, pollinatordependantcan be a critical aspect in its conservation (Whigham and McWethy 1980; Sipes and Tepedino 1995). Understanding pollination biology is particularly important in the Orchidaceae since most orchid species have developed highly specific plant-pollinator relationships that often limit the number of possible pollinators per orchid species to less than two insects (Tremblay 1992). Compounding the problem of specific plant-pol linator relationships in orchid pollination biology, is the evolutionary development of sp ecific pollination mechanisms in many orchid species, often requiring outcrossi ng between flowers from differe nt inflorescences to achieve seed set (Catling and Catling 1991; Bowles et al. 2002). By combini ng a basic ecological 63

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understanding of orchid species and populations with pollinator identification a nd pollination mechanism determination, a more complete eco logical understanding of orchids in natural settings can be achieved (C atling 1987; Wong and Sun 1999). A final component to the unders tanding of the ecology of orch ids is an evaluation of the genetic diversity within and betw een populations. The loss of gene tic diversity can result in the limiting of adaptive and evoluti onary potential and be a criti cal factor in the long-term persistence of plants in a changing environment (Frankel and Soul 1981; Lande and Barrowclough 1987; Qamaruz-Zaman et al. 1998b). Developing an understanding of withinand between-population genetic diversity is key in the conservation of plant species (Lande 1988), particularly orchids (S tewart and Kane 2007b). Investigating genetic diversity requires the use of molecular markers. Amplified fragment length polymorphisms (AFLPs) represent a multilocus marker system (Vos et al. 1995) that has been employed to investigate genetic diversity in a number of orchids (Pillon et al. 2007; Hedrn et al. 2001; Chen et al. 1999; Qamaruz-Zaman et al. 1998a) and non-orch ids (Juan et al. 2004; Arcade et al. 2000; Ranamukhaarachch i et al. 2000; Travis et al. 1996). The AFLP method is an attractive genetic diversity analysis method in plant systems because of its reproducibility, its requirement for small amounts of genomic DNA, a nd its ability to resolv e multiple polymorphic bands per AFLP reaction (Mueller and Wolfenba rger 1999; Ridout and Doni ni 1999; Chen et al. 1999; Lin et al. 1996). The terrestrial orchid Habenaria macroceratitis was chosen as the species in these studies because no information exists on the ecology, repr oductive biology, or genetic diversity of this species (Figure 1-2). Moreove r, no reports exist concerning the ecology, reproductive biology, or genetic diversity of any Habenaria species. However, a number of reports exist on the 64

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ecology (Sing-Chi 1983; Stoutamire 1996; Maad a nd Alexandersson 2004; Sheviak and Bowles 1986), reproductive biology (Wallace 2003; Thie n 1969; Hapeman 1997; Cole and Firmage 1984; Robertson and Wyatt 1990a, 1990b; Smith and Snow 1976; Little et al. 2005; Bowles et al. 2002; Zettler et al. 1996; Patt et al. 1989), and genetic diversity (Wallace 2006; Gustafsson 2000; Gustafsson and Sjgren-Gulve 2002) of the closely-allied orchid genus Platanthera In the present study, data are presented concerning the general plant demography, above-ground and below-ground vegetative development, and ecological habitat profile of H. macroceratitis in Florida. Data are also presented concerning the pollinator and reproductive biology of this species. Finally, the withinand be tween-population genetic diversity of H. macroceratitis is also described. These studies represent a first step leading to an integrated conservation and species-level recovery of H. macroceratitis in Florida (Figure 1-1). Materials and Methods Study Sites Four sites were chosen for these studies: Socash (Hernando County), Old Dade Highway (Hernando County), Battle Slough (Sumter Count y), and Cross Florida Greenway (Marion County; Figure 2-1). The Socash and Old Dade Highway sites were loca ted within 8.8 km and 0.8 km of Brooksville, Florida, respectively, at el evations above sea level between 50-55 m. The Battle Slough site was located within 1 km of Wa hoo, Florida at an elevation above sea level of 40-45 m. The Cross Florida Greenway site was located within 15.7 km of Ocala, Florida at an elevation above sea level of 20 m. Data on associated plant sp ecies were recorded at each site yearly from 2002-2005. Plant Demography In 2002, one monitoring site per study site was established. From 2002-2005 these four monitoring sites were visite d every three weeks during the active growth cycle of H. 65

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macroceratitis (1 August-31 December). Data on numb er of vegetative and flowering plants, plant height (cm), nectar spur le ngth (cm), number of leaves per plant, and number of flowers per plant was taken during each monitoring site visi t. These data were pooled within site per category per data collection week and averaged per data collection year. Th ese yearly data were then averaged over all data collection years and analyzed for year-to-year variance using general linear model procedures and Wa ller-Duncan mean separation at = 0.05 (SAS 1999). Plant development in the field was also documented. Pollinator Observations Preliminary pollinator observations were c onducted at the following study sites during peak flowering: Socash site, 26-27 August 2003 from 1100 to 0000 hours; Cross Florida Greenway site, 28-29 August 2003 from 1800 to 0300 hours; and Old Dade Highway, 1-2 September 2003 from 1800 to 0300 hours. Non-destructive pollinator observations were conducted at the Socash site from 28-29 August 2004. These observations were repeated 27-28 August 2005. The entire Socash population of H. macroceratitis was selected for observation activ ities due to its compact size and high flowering plant density. Observation methods followed thos e of Zettler et al. (1996) for pollinator observations of Platanthera integrilabia (Correll) Luer, an allied taxon to H. macroceratitis A circular path around the entire So cash population was established that allowed easy observation of 183 individual inflorescences. Observations occurred over a 24-hour period beginning 28 August 2004 at 1100 hours and en ding 29 August 2004 at 1100 hours, without interruption. All data we re recorded in fair weather, and no in sect repellant or insect attractants were used within the study area. Observations were recorded during the last 20 minutes of each hour by continuously walking the preestablished path in a counterclockwise direction around the flowering plants. Nocturnal observations were aided using a small, head-mounted flashlight 66

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fitted with a red filter, and dark clothing was worn to minimize possible insect disturbance due to moonlight reflectance. Pollinator observations were recorded as those insects carrying at least one pollinium after a flower visit, while visitors were recorded as those insects lacking pollinia. Insects were identified using Carter (2002). Re lative humidity and temperature were recorded at the Socash site during pollinator observations using two HOBO H8 data loggers (Onset Computer Corporation, Bourne, Ma ssachusetts). One data logger was placed at ground level, while the second was placed 30 cm above ground level. This height was chosen to parallel the average inflorescence height of H. macroceratitis at this site. Nectar volume and sugar concentration sampling was undertaken 27-28 August 2004 at the Socash site for a 12 hour period beginning at 1800 hours and ending at 0600 hours without interruption. Sampling followed the procedure of Zettler et al (1996). Samples were taken by microcapillary pipette (Drummond Scientific Company, Broowall, Pennsylvania). Preliminary investigation found H. macroceratitis nectar to be restri cted to the bottom 1/6 th of the nectar spur. Nectar was withdrawn from the base of ea ch spur and its volume recorded to the nearest 0.1 l. Sugar concentration was analyzed usi ng a pocket refractometer (0-62% range; Atago USA Inc., Bellevue, Washington), as outlined in Ze ttler et al. (1996). Ne ctar volume and sugar concentration samples were taken from three randomly selected flowers on three separate randomly selected inflorescences during the first 15 minutes of each hour. A total of 39 flowers were sampled during the sampling period. Pollination Mechanism, Seed Viability, a nd Asymbiotic Seed Germination A pollination mechanism study was designed to investigate the breeding system of H. macroceratitis following the procedures of Wong and Sun (1999), modified by the inclusion of a seventh pollination conditionself-pollination (T able 2-1). The mechanism experiment was conducted at the Socash site during both 2004 and 2005 flowering periods. Twelve plants, two 67

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per experimental pollination condition, with pre-an thesis inflorescences were bagged with a fine plastic mesh (1 mm 2 mesh size) stretched over a 1 m tall wi re frame. The mesh covered wire frame allowed the tall H. macroceratitis inflorescences to deve lop normally while excluding pollination events from occurring prior to the initiation of pollination mechanism studies. Experimental pollination treatments were appl ied to two inflorescences, each with five flowers per inflorescence (Table 2-1). A total of 60 flowers were us ed in the pollination mechanism determination study. Emasculation was accomplished by the removal of pollinia from individual flowers without allowing pollin ia contact with the stigmatic surfaces. Hand pollinations were conducted by placing pollinia onto stigmas using a pair of micro-forceps. Fresh pollinia from the Old Dade Highway site were used in testing all ou tcrossing experimental pollination conditions. The mesh-covered wire frames were replaced after each pollination condition was applied to the plants, and remained covering the plants until the conclusion of the study. Resulting capsules from the pollination mech anism study were allowed to mature on the plants and collected on 18 Oct ober 2004 and 24 October 2005. Prio r to collection, capsule size (width at mid-point length, cm) was recorded. Capsules were removed from the parent plant, placed in a paper envelope, placed in a plastic ba g over silica gel desiccant, stored in darkness, and transported to the laboratory (< 6 hours). Seed capsules were then stored over silica gel desiccant at 25 C for two weeks, after which time capsules dehisced and mature seeds were collected. Seeds from different plants within the same pollination treatments were pooled and stored in glass vials over silica gel desiccant at -10 C until use in tetrazolium viability staining and asymbiotic seed germination studies (< 1 week). 68

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Tetrazolium (2,3,5-triphenyltetrazolium chloride; Sigma-Aldrich Chemical Company, St. Louis, Missouri) viability staining of the re sulting mature seed from each experimental pollination mechanism condition followed the me thods of Lakon (1949) modified by Ramsay and Dixon (2003). Between 100-120 seeds from each experimental pollination mechanism condition were placed in 1.5 mL microcentrifuge tube s (USA Scientific Inc., Ocala, Florida) and pretreated with a 5% calcium hypochlorite (w/v) solution for 1.5 hours. Pretreated seeds were then rinsed in sterile deionized water (DI) once and soaked in sterile DI water for 24 hours. Seeds were then soaked in 1% tetrazolium solu tion (pH = 7.0) for 24 hours at 30 C in darkness (0/24 h L/D). After exposure to the tetrazolium solution, seeds were rinsed for 5 minutes three times in sterile DI water. Seeds were then susp ended in sterile DI water, transferred to a Petri plate using a Pasture pipette, and scored as viable or non-viab le with the assistance of a stereomicroscope. Viable embryos were scored as those embryos showing any degree of red or pink staining, whereas non-viable embryos were scored as thos e showing no degree of staining. The percentage of viable seeds was calculated by dividing the number of stained seeds by the number of total seeds in the sample. Asymbiotic seed germination was undertaken to test the vigor of seeds resulting from each experimental pollination mechan ism condition. Seed germination methods followed those of Stewart and Kane (2006a) for H. macroceratitis with minor modificatio ns: 1) seeds were only cultured on Malmgren Modified Terrestrial Orchid Medium ( Phyto Technology Laboratories LCC, Shawnee Mission, Kansas) supplemented with 20 g l -1 sucrose, 2) between 60-100 seeds were placed on each Petri plate, 3) plates were incubated in darkness (0/24 h L/D) for 8 weeks and scored only once, and 4) 15 replicate plat es per pollination condition (Table 2-1) were prepared for this experiment. Seeds were scored on a scale of 0-5 (Stewart and Kane 2006a). 69

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Germination percentages were calculated by dividing the number of seeds in each germination and development stage by the number of seeds in each sample. Data was analyzed using general linear model procedures and Wa ller-Duncan mean separation at = 0.05 (SAS 1999). Germination percentages were arcsine tran sformed to minimize variation and normalize variation. Sampling, DNA Extraction and Amplified Fr agment Length Polymorphism (AFLP) Fresh, green leaves of H. macroceratitis were collected from both the Socash and Old Dade Highway sites in 2003 and placed in 50 mL conical bottom plastic tubes (BD Biosciences, San Jose, California) containing silica gel de siccant (Chase and Hills 1991; W.M. Whitten personal communication). Leaf samples were re moved from each plant using scissors, which were washed with 95% ethanol and allowed to air dry between each sample to minimize sample cross contamination (M.W. Whitten personal communication). Twenty-two samples from the Socash and 21 samples from the Old Dade Highway sites were collected. Samples were stored at room temperature (ca. 25 C) un til used in DNA extraction protocols. Genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia, California). Manufacturers instru ctions were followed with the following modification: eluates were not pooled and the second elution was retained only as a precaution. The DNA was quantified using an Agilent Technologies NanoDrop (Wilmington, Delaware) spectrophotometer. As reported in Herdn et al. (2001), the Applied Biosystems (Foster City, California) AFLP Plant Mapping protocol was followed in order to take advantage of automated sequencing and computer-based analysis of fragment data. This kit-based system consists of two modules, a ligation and preselectiv e amplification module and a sele ctive amplification module. Manufacturers instructions were followed for each step in each module (ABI 2005). Sample 70

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DNA was restricted with EcoRI and MseI endonucleases and ligated to suitable double-stranded adapters per the manufacturer s recommendations. Following th is, a two step amplification process followed: 1) preselective amplification usi ng a 1 base pair (bp) ex tension and 2) selective amplification using a 3 bp extension. Four pr imer combinations were chosen: -ACT/-CAG, AGC/-CAG, -ACT/-CTA, and -AGC/-CTA, and polymerase chair reaction (PCR) conducted. Thermal cycle parameters for both the preselective amplification and the selective amplification followed those suggested by the manufacturer (Tab le 2-2). After amplification, samples were then prepared for fragment reading by co mbining 9.9 L formamide, 0.1 L Liz-600 size standard (Applied Biosystems), and 1.5 L P CR product. Samples were sequenced using a 3730xl Automated DNA Sequencer (Applied Biosystems). A matrix containing all AFLP fragment data ranging from 50 to 500 bp was compiled. Fragment data were analyzed by the computer software GeneMarker version 1.6 (SoftGenetics, State College, Pennsylvania), using the preselec ted AFLP settings. Fragments with a low signal (< 2% full detection level) were excluded. R ecognized bands were scored as present (1) or absent (0). AFLP data were analyzed using computer-aided processes. Genetic diversity within and between populations of H. macroceratitis were estimated using the Nei and Shannon diversity indices (based on allele frequencies), as cal culated with POPGENE 1.31 (Yeh et al. 1999). Within and between population genetic st ructure (i.e., genetic differentiation; F ST ) was estimated using a combination of results generated with POPGENE 1.31 and equation calculations with 1000 permutations. Dendrograms of genotypic corre lations were construc ted used GeneMarker version 1.6 cluster analysis tool. 71

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Results Study Sites Both the Old Dade Highway and Battle Slough study sites for H. macroceratitis were classified as mesic hammocks (Chafin 1990). These sites have a dense canopy dominated by live oak ( Quercus virginiana Mill), sabal palm ( Sabal palmetto (Walt.) Lodd.), southern magnolia ( Magnolia grandiflora L.), and very few slash pine ( Pinus elliottii Engelm.). The understory of the Old Dade Highway site was dominated by the exotic invasive plant air potato ( Dioscorea bulbifera L.), while the understory of th e Battle Slough site was dominated by partridge berry ( Mitchella repens L.). Both sites had an open mid-story. The Socash site was classified as a slope forest/mesic hammock (Chafin 1990), with an open canopy dominated by sweet gum ( Liquidambar styraciflua L.), sabal palm, southern magnolia, and very few slash pine. Live oak was a minor component of this site. Th is site slopes moderately to steeply (max. 20) westward toward a forested wetland. Erosion ca used by moving water heavily impacts this site. The Cross Florida Greenway site was classified as a pine dominated slope forest (Chafin 1990) surrounded by higher elevation sandhills. This site represents a portion of the manmade, unfinished cross Florida barge canal. Slash pine and a few young oaks ( Quercus chapmanii Sarg., Q. laevis Walt.) dominate the canopy of this site The understory of this site was dominated by numerous grasses and herbs. Plant Demography From 2002-2005 the average number of vegetative and flowering plants at each H. macroceratitis study site varied among sites: Socash, 250 .7 plants per y ear (33% flowering per year), Old Dade Highway, 61 .6 plants pe r year (31% flowering per year), Battle Slough, 12 .6 plants per year (23% fl owering per year), and Cross Florida Greenway, 16 .5 plants per year (25% flowering per year). No significant vari ation in plant height or spur length across 72

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all observation years wa s found among sites of H. macroceratitis in Florida (Figure 2-2). A significantly lower number of leav es were found only at the Old Dade Highway site across all observation years (Figure 2-3). All other sites showed no significant variation in leaf number (Figure 2-3). Flower number wa s not significantly different acro ss all observation years for all sites of H. macroceratitis in Florida (Figure 2-3). The aver age height of plants ranged between 35 cm (Socash) and 23.2 cm (Old Dade Highway). Average length of the ne ctar spur per flower per plant ranged between 15.2 cm (Socash) and 13 .9 cm (Socash). Leaves per plant averaged between 7 (Socash) and 5 (Battle Slough). Th e average number of flowers per plant ranged between 9 (Socash) and 4 (Cross Florida Greenway). In the field, plants regularly produce tubers with a single gr owth point and remain dormant during winter months (November-June; Figure 2-4). In the late spring (June), plants initiate shoot growth from dormant tubers and produce leav es directly from these shoots (Figure 2-4). Flowering typically begins in mid-summer (Au gust), although plants may remain vegetative during these months (Figure 2-4). Pollinator Observations Only one visitor to H. macroceratitis flowers was noted during the entire 24 hour pollinator observation study on 28-29 August 2004. The giant sphinx moth ( Cocytius antaeus Drury; Sphingidae) was noted probing individual flowers at 0000 hours on 29 August 2004, and subsequently captured for identification (Figure 25). This same sphinx moth species was noted visiting flowers of H. macroceratitis on a previous pollinator observation study conducted at the Old Dade Highway site on 1-2 September 2003 a nd a subsequent pollinatior study at the Socash site on 27-28 August 2005, although the moth was not captured in either case. The flowers of H. macroceratitis at the Socash site were found to have a sweet, lemon night scent starting at approximately 1900 hours and continuing until approximately 0600 hours. 73

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Site temperature ranged between 20.8C-32.8C (a ve. = 28.5C .6C; Figure 2-6 a). Site relative humidity ranged between 59%-96% (ave. = 80.4% .3%; Figure 2-6 b). Habenaria macroceratitis nectar was found to contain an average of 17.5% soluble sugars with an average volume of 4.25 l. Pollination Mechanism, Seed Viability, and Asymbiotic Seed Germination Habenaria macroceratitis reproduction appears to rely on the movement of pollen between flowers on the same or different inflorescen ces. In the pollination mechanism study, seed capsules were set in only four of the seven (Table 2-1) pollination conditionsopen pollination (control), induced autogamy, artif icial geitonogamy, and artificial xenogamy. Capsules were not set in the spontaneous autogamy, agamospermy, or self-pollination cond itions. The largest capsules were produced in the artificial geitonogamy pollination condition (ave. 5.65 28.15 cm), followed by capsules in the artificial xenogamy condition (ave. 5.12 27.42 cm), the open pollination condition (ave. 4.23 23.28 cm), and the induced autogamy pollination condition (ave. 2.58 23.66 cm). Tetrazolium staining of samp les of the mature seed from each capsuleproducing pollination condition revealed significant differences in embryo viability based on pollination condition. Seeds from the open pollina tion (control) condition yielded 91.0% viable embryos, while seeds from the artificial geit onogamy condition yielded 86.3% viable embryos, seeds from the induced autogamy condition yiel ded 76.8% viable embryos, and seeds from the artificial xenogamy yielded 50.7% viable embryos. Asymbiotic seed germination pe rcentages paralleled tetrazolium embryo viabilities, with seeds originating from the open pollination (con trol) condition having bot h the highest overall percent germination (54.2%) and highest percent protocorm development to an advanced stage (Stage 3; 3.2%; Figure 2-7). Seeds originating from the artificial geitonogamy condition had a maximum germination of 19.0% (Stage 1) after 8 weeks dark (0/24 h L/D) incubation, while 74

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seeds from the induced autogamy and arti ficial xenogamy conditions had a maximum germination of 53.9% (Stage 2) and 9.0% (Stage 1), respectively (Figure 2-7). AFLP Data Amplification of all four primer pairs tested was successful in all 43 individuals sampled. Output gel images are given in Figures B-1 and B-2. However, onl y the -ACT/-CAG and AGC/-CAG primer pairs resulted in the highest polymorphic band resolution, averaging 52 and 45 bands per individual, respectively. Bands that were either present or absent in a single sample were excluded from analysis as likely being arte factual (Pillon et al., 200 7). From both primer pairs, a total of 24 unambiguous polymorphic bands were selected. When combining these 24 bands, 10 genotypes could be distinguished. Th e numbers of genotypes observed, number of polymorphic loci, Neis diversity in dex, Shannons diversity index, and F ST are given in Table 23. Genotypic correlation dendrograms are given in Figures B-4, B-5, and B-6. Discussion The ecology, pollination biology, and population genetic diversity of H. macroceratitis has been previously unknown. Intere stingly, very few reports on No rth American native orchid ecology exist, particularly in the tribe Orchideae (subtribes Orchidinae and Habenariinae). The majority of field-based ecological reports on the Orchidaceae come from the subfamily Cypripedioideae, primarily in the genus Cypripedium (Sheviak 1983; Stoutamire 1983, 1989; Light and MacConaill 2007, 2005). A fe w reports on the field ecology of Platanthera and allied species (i.e., Himantoglossum ) do exist. These Orchidinae genera are evolutionary cousins to the Habenaria Bowles (1983) and Stoutamire (1996) reported on the ecology and habitat of the Federallythreatened Platanthera leucophaea (Nutt.) L., a terrestrial orchid native to the Midwestern United States. Unlike H. macroceratitis which grows in shaded hardwood hammocks in central 75

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Florida, P. leucophaea is reported as growing in mesic and we t prairie, bog, and fen-like habitats in its native range of eastern North America. Of interest is the parallel between the requirement for P. leucophaea sites to be wet during active growth phases and dry during dormant periods, and the need for H. macroceratitis sites to be moist during the active growth phase and dry during plant dormancy. This wetto-dry life history trait appears to be consistent in many orchids, particularly species in the Orchideae (Dressler 1993). The importance of such field-based ecological information (i.e., Bo wles 1983; Stoutamire 1996) and long-term studies (i.e., Light and MacConaill 2005) in the conservation and recovery of orchids is just now being appreciated. Ecological data such as thes e are important parts of integrated conservation planning an d species recovery in the Orch idaceae. Unfortunately, only a small number of orchid species restricted to a smaller number of genera have been studied for such field-based ecological information. Furtherm ore, even a smaller numb er of orchid species have been studied for more than a year or tw o at one time (Light and MacConaill 2005). Pfeifer et al. (2006) reported on the l ong-term (ca. 26 years) demographic fluctuations of the Platanthera -allied orchid species Himantoglossum hircinum (L.) Spreng. They reported that life history traits, such as flowering, presence/absence of plants fro m year-to-year, and height of plant, remained variable over the 26-year study pe riod. Also, they report that overall population size of H. hircinum in the study area increase d exponentially, but no plan t density effects were observed. Data such as these provide valuable in sight into not only the life history of orchids, but also shed light on predictive models for orchid population management decisions. While the current study with Habenaria macroceratitis did not attempt to use predictive modeling or extensive long-term st udy to gain insight into the lif e history of the species (i.e., Pfeifer et al. 2006), data were produced th at revealed a degree of demographic and 76

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morphological stability within and between populations of the species. This stability in morphological character could be indicative of a life history strategy based on smallor moderately-sized populations ex isting within isolated hardwood hammock habitats spread throughout central Florida. In this model, each p opulation would act as an individual or isolated population with little co nnection (i.e., gene flow) between po pulations. Further morphological, genetic, and long-term study of H. macroceratitis populations in central Florida is needed to better elucidate this life history model. An interesting note to a discussion on the eco logy of the Orchideae is the similarity in ecology, habitat, and distribution between Platanthera and Habenaria species native to eastern Asia and North America. These two geographic ar eas share a high diversity of Orchideae, at the generic level, and show both connected and disj unctive distributions of many Orchideae (SingChi 1983). On a morphological level, the Habenaria native to the southeastern United States show a high degree of similarity to Asian Habenaria species, particularly H. radiata (Thumb.) Spring. and H. rostelliifera Rchb. (S.L. Stewart personal observ ation). Further study on the genetic and morphological similarities between th e Orchideae flora of th ese two areas should be conducted. Despite many decades of reproductive and polli nation biology study in a number of plant species (Schmid 1975), the pollination biology of the Orchidaceae remains mostly understudied. Tropical orchids have received the most pollina tion biology study in rece nt years (Blanco and Barboza 2005; Trapnell and Hamrick 2006, 2005; Si nger and Koehler 2003; Borba et al. 2001). North American orchid species have, classica lly, received little pol lination biology study breeding system information is available fo r only approximately 40% of North American species, and detailed studies have been c onducted for only approximately 15% of North 77

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American orchids (Catling and Catling 1991). Two North American genera have garnered the majority of pollination biology research: Platanthera and Cypripedium Reflecting the interest in the ecology of North America Cypripedium species, this genus has drawn a great deal of pollination biology attention (e.g., Vogt 1990; Stoutamire 1967; Covell and Medley 1986; Catling and Knerer 1980; Klier et al. 1991). Platanthera has drawn the great est interest from those studying the pollination biology of North American Orchidaceae (e.g., Hapeman 1997; Folsom 1984; Stoutamire 1974; Thien 1969; R obertson and Wyatt 1990a, 1990b; Sheviak and Bowles 1986; Smith and Snow 1976; Little et al 2005; Zettler et al. 1996; Patt et al. 1989). Interestingly, no data exist concerning the reproductive or pollination biology of any North American Habenaria species (Catling and Catling 1991). A common theme among members of the Orch ideae appears to be their reliance on pollination by butterflies and/or moths (Lepidopte ra; Catling and Catling 1991). In the current study, H. macroceratitis is suspected as pollinate d by the giant sphinx moth ( Cocytius antaeus ). While no data exists for a comparative discussi on of the pollinators and pollination biology of Habenaria in North America, data on similar pollinators do exist in the genus Platanthera Sheviak and Bowles (1986) reported several species of Manduca (Sphingidae) as pollinators for both P. leucophaea and P. praeclara Sheviak & Bowles. Both of these aforementioned Platanthera species possess white flowers with a nocturnal scent, similar to H. macroceratitis (Sheviak and Bowles 1986). Fu rthermore, the flowers of P. leucophaea P. praeclara and H. macroceratitis share similar long nectar spurs, although the spur of H. macroceratitis is much longer than the spur of either Midwestern Platanthera species. Due to these pollination ecology similarities, it is not surprising to find that these two Platanthera species and H. macroceratitis are all pollinated by long-proboscis sphinx moths. However, the sphinx moth pollinators of P. 78

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leucophaea and P. praeclara are more temperate in distributi on, as is the distribution of the orchids these moths pollinate. Cocytius antaeus is known to be distributed in more tropical regions, paralleling the distribution of Habenaria species in North, Central, and South Americas (Carter 2002). A number of Platanthera species are known to be pollinated by Lepidoptera other than moths, mainly butterflies. Zettler et al. (1996) reported the pollination of P. integrilabia a rare terrestrial orchid from Tenness ee, by the day-flying butterflies Epargyreus clarus Cramer and Papilio glaucus L. No day-flying pollinators were observed visiting or pollinating flowers of H. macroceratitis in the current study. Few reports exist on the nectar volume and nectar sugar concentration of orchid flowers as they relate to pollination biology. In the current study, flowers of H. macroceratitis were found to contain an average of 4.25 l of nectar at an average of 17.5% soluble sugars. Cole and Firmage (1984) reported a lower average nect ar volume (1.55 l) for the allied species Platanthera blephariglottis (Willd.) L. Robertson and Wyatt (1990) reported a range of nectar volumes (3.25-6 l) and sugar concentrations (19-23%) for several ecotypes of P. ciliaris (L.) L. Platanthera integrilabia nectar volume and concentrated ranged from 2.9-6.8 l and 17.2-20.8%, respectively (Zettler et al., 1996) Hapeman (1997) reported a nectar volume and concentration of 1.5 l and 19.0%, respectively, for the terrestrial orchid P. peramoena (Gray) Gray. Nectar amount appears to not be a functi on of nectar spur length, with P. blephariglottis P. ciliaris and H. macroceratitis having long nectar spurs, P. integrilabia having a moderately long nectar spur, and P. peramoena having a short spur. Nectar sugar c oncentration appears to also not be correlated with spur length. Supporting this conclusion, Zettler et al. (1996) reported no significant difference in nect ar sugar concentration of P. integrilabia In all these Orchideae 79

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species, nectar volume or sugar concentration doe s not appear to serve as a primary mode of pollinator attraction. Given the strong evening scent reported for H. macroceratitis it is suspected that this scent serves as the primary pollin ator attractant for this species. Zettler et al. (1996) reported a similar scent-driven attractant system in P. integrilabia as did Huber et al. (2005) in Gymnadenia conopsea and G. odoratissima A number of reports exist concerning the connection between pollination mechanism and seed set in orchids (Chung and Chung 2005; Kr opf and Renner 2005; Whigham and McWethy 1980). These reports indicate the success of a partic ular pollination mechanism based on seed capsule formation. For example, Chung and C hung (2005) reported that self-pollination and artificial geitonogamy pollination conditions re sulted in nearly 90% seed capsule set in Bletilla striata (Thumb.) Richenb. However, seed capsule set is not necessari ly a good measure of reproductive fitness in orchids. Seed viability and germination (i .e., seed vigor) represent better measures of the success of experimental pollin ation mechanisms on reproductive fitness in the Orchidaceae. In the present study, H. macroceratitis set seed capsules in only four of seven experimental pollination mechanism conditionsopen pollination (control), induced autogamy, artificial geitonogamy, and artificial xenogamy. Seed viabili ty and asymbiotic germination (Figure 2-6) demonstrated that both the open-pollination (c ontrol) and artificial geitonogamy pollination conditions supported the highest se ed vigor of the four capsule-producing pollination conditions. These data indicate that H. macroceratitis likely relies on insect-mediated pollen movement between flowers on the same or different inflor escence within the same population. Bowles et al. (2002) reported a similar e ffect on seed viability in Platanthera leucophaea In their study, seeds originating from between -population and within-populati on crosses produced the highest 80

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percent viable seeds when compared to self-pollinated plants. Furthermore, Borba et al. (2001) reported no seed capsule formation in flowers of five Brazilian Pleurothallis species that were self-pollinated or subjected to agamospermic polli nation conditions. In all these cases, including H. macroceratitis the need for pollinators to achieve not only successful seed capsule set, but also achieve high seed vigor (i .e., reproductive fitness) was de monstrated. Only the current study with H. macroceratitis attempts to relate experiment al pollination mechanism condition, seed viability, and seed germination as a combined measure of reproductive fitness in the Orchidaceae. The AFLP technique represents a powerful tool in developing an understanding of plant population genetic diversity with in and between populations (M eudt and Clarke 2007). The present study represents the first investigati on of the genetic diversity within and between populations of H. macroceratitis, or any Habenaria species, using AFLP technology. In the present study, a moderate level of within-population genetic di fferentiation was found (Socash F ST = 0.11, Old Dade Highway F ST = 0.06). Four genotypes were identified in the Socash population, while 6 genotypes were identified from the Old Dade Highway population. In spite of the moderate within-population genetic differentiation, lo w between-population genetic differentiation was found (overall F ST = 0.02). A number of reports of popul ation genetic diversity in Platanthera an allied genus to Habenaria exist. Wallace (2002a, 2002b) used the RAPD technique to investigate the population genetic diversity and effects of habita t fragmentation on the Mi dwestern terrestrial orchid P. leucophaea Despite a moderate level of population differentiation, genetic and geographic distances betw een populations were found to be not significant. This suggested a lack of interpopulation gene flow among geographically-isolated and fragmented P. leucophaea 81

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populations. A similar trend of between populati on differentiation and po tential interpopulation gene flow help explain the moderate levels of within-population differentiation measured in H. macroceratitis as compared to the between population genetic differentiation. A similar genetic structure as seen in H. macroceratitis was reported by Wallace (2004) in an investigation of th e genetic structure of P. huronensis (Nutt.) Linl. P. aquilonis Sheviak, and P. dilatata (Prush) Lindl. using intersimple sequence repeat (ISSR) markers. In the study, a significant amount of the species -level genetic diversity in P. huronensis and P. dilatata was found to reside within populations of these two terrestrial orchid s, and not between populations. Wallace (2004) suggested that the low betw een population genetic variation seen in P. huronensis may be due to a limited number of or igins, genetic bottlenecks, or low among population gene flow. The interpretation of these AFLP population gene tic diversity data in a context of species management and conservation planning is crit ical. The measure of population and species genetic diversity, and any subsequent change in that diversity is an important step in the overall conservation planning for a species. To unders tand the genetic diversity present within and between populations of this orchid species, is to begin to understand the ability of those populations to respond to natura l selection, speciation, and ot her evolutionary pressures (Qamaruz-Zaman et al. 1998a). Preservation of genetic diversity is cr ucial in the long-term conservation of plant species (Crozier 1992), part icularly when little other information is known concerning the ecology, propagation, or pollination biology of the species. The demonstration of moderate levels of within-popul ation genetic differentiation in H. macroceratitis populations would suggest that moderate to large isolated populations should be maintained in order to preserve this degree of differentiation within populations. Data from pollination biology and 82

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asymbiotic seed germination studies support this conclusion. However, the low between population genetic differentiation measured usin g AFLP suggests that the two populations sampled have some degree of gene flow between them. This suspected gene flow could be due to several factors: 1) occasional across-populatio n pollination events, 2) historic meta-population now fragmented by urbanization, and/or 3) founder population and founded population relationship. Higher-order data analysis and further population sampling is necessary in order to properly investigate these potential relationships between these two populations of H. macroceratitis Furthermore, careful molecularand morphologi cal-based investigations of the overall differences, gene flow, pot ential hybridization, and popul ation structure of both H. macroceratitis and its sister species, H. quinqueseta should be conducted to better understand the past, current, and future relationships be tween these two species (Dueck and Cameron 2007; Gustafsson and Sjgren-Gulve 2002; Wallace and Case 2000; Trapnell et al. 2004; Bateman et al. 2003; Case et al. 1998). These data would greatly help in the long-term conservation planning and species-recovery efforts for H. macroceratitis both in Florida and throughout the species range. Implications for Integrated Conservation Planning The present studies on the pl ant demography, pollination biology, and genetic diversity of the Florida terrestrial orchid H. macroceratitis have supported the need to understand these important ecological factors of orchid biology before implemen ting conservation and recovery plans. Studies such as these highlight the need to integrate ecological, reproductive biology, and molecular-genetic studies to produce ecologi cally functional data on the species-level conservation of plants. For example, rec ognizing the relationship between the pollination mechanism of H. macroceratitis and the species population geneti c structure has allowed these 83

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studies to suggest that moderate to large isolated populations of H. macroceratitis be maintained and managed throughout the range of the speci es in Florida (Stewart and Kane 2006c). Studies on the polli nation biology of H. macroceratitis have revealed the need for insectmediated cross pollination to support high reproductiv e fitness in this species. In identifying a potential pollination of this orchid species, not only does useful information concerning the role of this pollinator in th e reproductive biology of H. macroceratitis come to light, but also the need for pollinator conservation and management become important. As demonstrated in the integration of the pollinator observation studies with the pollination mechanism studies, the need for a strong-flying pollinator, such as Cocytius antaeus to repeatedly visit flowers of H. macroceratitis on the same inflorescence or within the same population is vital to this orchids reproduction. Pollination events where C. antaeus transfers pollinia betw een populations appear to be the exception. Furthe rmore, the larval stage of C. antaeus is known to feed from the pondapple tree ( Annona glabra L.), a tree typical of wet and sw ampy habitats in south Florida and throughout the tropics. To insure the continued reproductive fitness of H. macroceratitis in Florida, the species pollinator ( C. antaeus ) must be conserved and managed, meaning that A. glabra trees throughout south Florida a nd the habitats that support th em must also be conserved and managed properly. Without this integrated multilevel conservation and management system in place, the con tinued existence of H. macroceratitis in Florida may in jeopardy. Unfortunately, no such system currently exists. The current studies concerning the plant demography, pollination biology, and population genetic diversity of H. macroceratitis represent another step toward the species-level integrated conservation of this rare terrestrial orchid in Florida (Figure 1-1). By adding to the body of knowledge on the conservation biology of this orchid species, the present studies not only 84

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promote the conservation biology of H. macroceratitis but also promote the conservation and management of a number of other plant, animal and insect species th roughout Florida and the tropics. 85

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Table 2-1. Experimental pollination conditions a pplied to flowers of Habenaria macroceratitis after Wong and Sun (1999). Condition Bagging Treatment Pollen Source Objective Control Unbagged Untreated Open pollination Evaluate fruit set under natural conditions Agamospermy Bagged Emasculated No pollination Evaluate the rate of nonsexual reproduction Spontaneous autogamy Bagged Untreated Th e same flower Measure the need for pollinators Induced autogamy Bagged Emascula ted The same flower Evaluate self-compatibility Artificial genitonogamy Bagged Emasculated Different flower on Evaluate same plant self-compatibility Artificial xenogamy Bagged Emasculated Flower from a Evaluate outbreeding at distant population long distance Induced xenogamy Bagged Emasculated Flower from same Evaluate outbreeding at population, distant plant short distance 86

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Table 2-2. Thermal cycler parameters for pr eselective (top) and selective (bottom) AFLP amplification of prepared ge nomic DNA, following ABI (2005). Preselective Parameters Hold Cycle Hold Hold 20 Cycles of Each 72 C 94 C 56 C 72 C 60 C 4 C 2 min 20 sec 30 sec 2 min 30 min continuous Selective Parameters Hold Cycle Number of Cycles 94 C 94 C 66 C 72 C 1 2 min 20 sec 30 sec 2 min 94 C 65 C 72 C 1 20 sec 30 sec 2 min 94 C 64 C 72 C 1 20 sec 30 sec 2 min 94 C 63 C 72 C 1 20 sec 30 sec 2 min 94 C 62 C 72 C 1 20 sec 30 sec 2 min 94 C 61 C 72 C 1 20 sec 30 sec 2 min 94 C 60 C 72 C 1 20 sec 30 sec 2 min 94 C 59 C 72 C 1 20 sec 30 sec 2 min 94 C 58 C 72 C 1 20 sec 30 sec 2 min 94 C 57 C 72 C 1 20 sec 30 sec 2 min 94 C 56 C 72 C 20 20 sec 30 sec 2 min 60 C 1 30 min 4 C 1 continuous 87

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Table 2-3. Results of the geneti c analysis for each population of Habenaria macroceratitis Socash = Socash study site (Hernando Count y, Florida). Old Dade = Old Dade Highway study site (Her nando County, Florida). Population Estimated Number of Number of Number of Nei's Shannon's F ST Size Samples Genotypes Polymorphic Index Index Loci Socash >200 (2004) 22 4 13 0.184 0.309 0.11 Old Dade ca. 100 (2004) 21 6 11 0.161 0.27 0.06 Overall 43 10 24 0.176 0.302 0.02 88

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Figure 2-1. Study sites for Habenaria macroceratitis in Florida: Marion County, Sumter County, and Hernando County. 89

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Demographic Measurement Height Spur Length Length (cm) 0 5 10 15 20 25 30 35 Socash Battle Slough Cross Florida Greenway Old Dade Highway a a a a a a a a Figure 2-2. Average height (cm) and spur length (cm) of Habenaria macroceratitis plants at four study sites in west central Florid a. Data from 2002-2005 observations pooled within site and across years. Histobars with the same letter are not significantly different within demographic measurement ( = 0.05). Error bar = SE. 90

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Demographic Measurement Leaf Number Flower Number Number 0 2 4 6 8 Socash Battle Slough Cross Florida Greenway Old Dade Highway a a a a a a a b Figure 2-3. Average leaf nu mber and flower number of Habenaria macroceratitis plants at four study sites in west central Florida. Data from 2002-2005 observations pooled within site and among years. Histobars with the same letter are not significantly different within demographic measurement ( = 0.05). Error bar = SE. 91

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Figure 2-4. Growth cycle of Habenaria macroceratitis under field conditions. D = dormant tuber stage. SG = shoot growth stage. VG = vegetative growth stage. Scale bar = 1 cm. 92

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Figure 2-5. Cocytius antaeus (giant sphinx moth) captured during pollinator observation of Habenaria macroceratitis at the Socash site (Hernando County, Florida) 28-29 August 2004. Scale bar = 1 cm. 93

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Date/Time 2 8 A u g 1 1 0 0 2 8 A u g 1 2 0 0 2 8 A u g 1 3 0 0 2 8 A u g 1 4 0 0 2 8 A u g 1 5 0 0 2 8 A u g 1 6 0 0 2 8 A u g 1 7 0 0 2 8 A u g 1 8 0 0 2 8 A u g 1 9 0 0 2 8 A u g 2 0 0 0 2 8 A u g 2 1 0 0 2 8 A u g 2 2 0 0 2 8 A u g 2 3 0 0 2 9 A u g 0 0 0 0 2 9 A u g 0 1 0 0 2 9 A u g 0 2 0 0 2 9 A u g 0 3 0 0 2 9 A u g 0 4 0 0 2 9 A u g 0 5 0 0 2 9 A u g 0 6 0 0 2 9 A u g 0 7 0 0 2 9 A u g 0 8 0 0 2 9 A u g 0 9 0 0 2 9 A u g 1 0 0 0 2 9 A u g 1 1 0 0 Relative Humidity (%) 50 60 70 80 90 100 Temperature (C) 20 22 24 26 28 30 32 34 A B Figure 2-6. Temperature and re lative humidity profiles at Socash site (Hernando County, Florida) 28-29 August 2004 during Habenaria macroceratitis pollinator observations. A) Temperature (C) and B) relative humidity (RH; %). 94

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Germination Stage Stage 0Stage 1Stage 2Stage 3Stage 4Stage 5 Germination (%) 0 20 40 60 80 100 Open Pollination Artificial Geitonogamy Induced Autogamy Artificial Xenogamy a b a c a b b a a b a b a b bb Figure 2-7. Effects of pollination condition on percent germination and protocorm development of Habenaria macroceratitis after 8 weeks in vitro asymbiotic culture on Malmgren Modified Terrestrial Orchid Medium. Hi stobars with the same letter are not significantly different within stage ( = 0.05). Error bar = SE. 95

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CHAPTER 3 SEED CULTURE AND IN VITRO SEEDLING DEVELOPMENT OF Habenaria macroceratitis Introduction The ongoing loss of suitable orch id habitat throughout the sout heastern United States (e.g., Florida) has prompted interest in the preservation and restoration of these critical habitats. These habitats are considered highly productive for both animal (Maehr and Cox 1995) and plant (Sprott and Mazzotti 2001) speci es. This loss, due mostly to urbanization, conversion for agricultural purposes, an d habitat mismanagement, has greatly impacted populations of many rare and endangered herbaceous understory plants throughout Fl orida (Sprott and Mazzotti 2001), including many native orchids. Many native Florida terrestrial orch ids inhabit threatened habitats such as hardwood hammocks and pine flatwoods (S.L. Stewart personal observation) and are at risk of population decline or extincti on unless an effective method of propagation can be developed to provide plan ts for restoration purposes. Seed germination represents the most efficient method of native terrestrial orchid propagation for conservation purposes (Stewart and Kane 2006a, 2006b, 2007b; Kauth et al. 2006), as well as one step in the integrated conservation of orch id species (Stewart and Kane 2007b; Figure 1-1). However, orchid seed germina tion studies are often viewed as unreliable or unrealistic since little is known concerning the factors affecting in vitro germination and in vitro seedling development for many North American native orchids (Arditti et al. 1981; Stewart and Kane 2006a). Compounding th is problem, Stoutamire (1974, 1989) found that many North American native terrestrial orchids require up to eight years of ex vitro growth before reaching reproductive maturity. This means that poten tial biochemical and phys iological carry-over effects of in vitro culture on plant growth, development, and fitness may not be evident until many years after propagation and reintroduction. To overcome these problems some have 96

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suggested the development of optimized seed germination methods for entire genera or individual species (Stewart a nd Kane 2006a; Kauth et al. 2006). This approach shows great promise and is employed here. Asymbiotic seed germination represents a di rect method for orchid seed germination on a defined agar-solidified medium, typically supplemented with sucrose, various amino acids, vitamins, and undefined compounds (i.e., banana powder, coconut water). The advantage of asymbiotic orchid seed germination is that orch id fungi (mycobionts) need not be isolated from field-collected seedling or adult plant material in order to germ inate seed of orchid taxa. However, asymbiotic orchid seed germination does have its disadvantages. Orchids that are established using asymbiotic seedlings may not be able to ca pture and digest mycobionts once introduced to a field setting (W. Stoutami re, personal communication). Furthermore, populations of orchids that are established usi ng asymbiotic seedlings will remain dependant upon naturally-occurring mycobionts for seed ling recruitment (Zettler 1997b). These mycobionts may not be present at a reintroduction site if not already in a ssociation with the reintroduced orchid plants, especially if the orch id species was not present at the site prior to plant reintroduction. Th is lack of mycobiont may not allo w for seedling recruitment at the reintroduction site, thus limiting the long-term sustai nability and fitness of the propagated orchid taxon. In nature, orchids digest endophytic myco rrhizal fungi as a source of nutrition (mycotrophy) in a parasitic asso ciation that is known to support seed germination, as well as protocorm and seedling development (Arditti 1966; Clements 1988; Rasmussen 1995; Rasmussen and Rasmussen 2007). Therefore, the l ong-term survival and f itness of orchids in managed or restored habitats requires the presence of appropriate mycobionts for proper plant 97

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nutritional support and seedling recr uitment (Zettler 1997a). The mo st efficient way to promote this process is through the use of in vitro symbiotic co-culture methods (Dixon 1987; Zettler 1997a, 1997b; Clements et al. 1986). Symbiotic s eed co-culture involves the combining of orchid seed and a compatible mycobiont under in vitro conditions on an undefined agarsolidified medium consisting of only finely pul verized rolled whole oats, and occasionally undefined additives such as yeast extract. Un fortunately, few North American native orchids have been cultured using this method; mo stly species restricted to the genera Spiranthes (Anderson 1991; Stewart and Kane 2007b; Zelmer and Currah 1997; Zettler and McInnis 1993; Stewart et al. 2003), Platanthera (Anderson 1996; Zettler and Ho fer 1998; Zettler and McInnis 1992; Sharma et al. 2003; Zettler et al. 2001; Zettler et al. 2005), and Habenaria (Stewart and Kane 2006b; 2007b; Stewart and Zettler 2002). More over, little is known about the identity and ecology of these mycobionts in pure culture or in nature. The terrestrial orchid Habenaria macroceratitis was chosen as the species in this study because little information exists on the asymbiotic and symbiotic seed culture, as well as the in vitro seedling development of this species (Figur e 1-2). Previously, Stewart and Zettler (2002) proposed a symbiotic co-culture protocol for H. macroceratitis although protocorms did not develop to advanced leaf-bearing stages in their study. In this study, efficient asymbiotic and symbiotic seed germination methods for H. macroceratitis are defined. These methods facilitate protocorm development through a leaf-bearing stag e. The effects of asymbiotic germination medium, carbohydrate type and concentration, exogenous cytokinin application, photoperiod, and in vitro mycobiont preference on the seed germination of H. macroceratitis are presented. Additionally, the effect of photoperiod on asymbiotic in vitro seedling development of H. 98

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macroceratitis is also presented. This work represents one step in the integrated conservation and species-level recovery of H. macroceratitis in Florida. Materials and Methods Asymbiotic Seed Germination Seed source and sterilization Seeds of were obtained from mature capsu les prior to dehiscence on 26 October 2003. Habenaria macroceratitis seeds were collected from a privat ely-owned site near Brooksville, Florida (Hernando County). Imme diately after collection, capsul es were dried over silica gel desiccant for 2 weeks at 25 5C, followed by st orage in darkness at -18C for 52 days. Seeds were surface disinfected for 1 min in a solution containing 5 mL etha nol (100%), 5 mL 6.00% NaOCl, and 90 mL sterile deionized (DI) wate r. Following surface disinfection, seeds were rinsed three times for 1 min each in sterile DI wa ter. Solutions were removed from the surface disinfection vial using a st erile Pasture pipette that was replaced after each use. Sterile DI water was used to suspend the disinfected seed, and a sterile bacterial inoculating loop was used to sow the seed. An average of 124 seeds per Petri plate were sown. Asymbiotic media survey The effects of six basal media (Table 31) on asymbiotic seed germination of H. macroceratitis were assessed. Three of the media were commercially prepared and modified by Phyto Technology Laboratories LCC (Shawnee Mi ssion, Kansas): Murashige & Skoog (MS), Malmgren Modified Terrestrial Orchid Medi um (MM; Malmgren, 1996), and Modified Kundson C (KC). Two of the media were commercially prepared and modified by Sigma Chemical Company (St. Louis, MO): V acin & Went (VW; Vacin and Went, 1949) and Lindemann (LM; Lindemann et al., 1970). The final medium, Modi fied Lucke (ML), was prepared according to Anderson (1996). MS, VW, and LM were further modified by the addition of 2% sucrose and 99

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0.8% TC agar ( PhytoTechnology Laboratories LCC, Shaw nee Mission, Kansas) and ML modified by the addition of 0.8% TC agar to be consistent with MM and KC. Media were adjusted to pH 5.8 with 0.1 N KOH after the ad dition of carbohydrate sour ce and agar, and were dispensed into 1 l flasks prior to auto claving for 40 min at 117.7 kPa and 121C. Sterile media were dispensed as 25 mL aliquot s into 9 cm diameter Petri plates (Fisher Scientific, Pittsburg, Pennsylvania). Surface steri lized seed were placed into the center of each plate and the seed evenly spread on the medium Ten replicate plates were inoculated per medium type. Petri plates were sealed with a single layer of Nescofilm (Karlan Research Products, Santa Rosa, California) before being cultured in conti nual darkness at 25 3C for 7 weeks without light interruption. At 7 and 16 weeks germination and protocorm development were assessed by use of a dissection stereoscope. Germination and protocorm development were scored on a scale of 0-5 (Table 3-2; Figure 3-1; Stewar t and Kane 2006a). Germination percentages were calculated by dividing the number of seeds in each individual germination and development stage by the total number of viable seeds in the sample. Data were analyzed using general linear mode l procedures and Waller-Duncan mean separation at =0.05 by SAS v 8.02 (SAS 1999). Germination counts were arcsine transformed to normalize variation. Effects of carbohydrate source on asymbiotic seed germination Effects of three carbohydrate sources (fructose, sucrose, and dextrose) at 50 mM on the asymbiotic seed germination of H. macroceratitis were examined. One asymbiotic germination basal medium (MM) was used in treatment co mbinations with carbohydrate source and the presence or absence of banana powder (BP). Me dium was prepared as previously described and supplemented with a 50 mM carbohydrate source (9.008 g l -1 fructose, 17.115 g l -1 sucrose, 9.008 g l -1 dextrose). To those treatments containing BP, 15 g l -1 BP was added prior to sterilization. 100

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Sterile medium was dispensed (ca. 25 mL) into 9 cm diameter Petri plates. Ten replicate plates were inoculated with seed. Plates were sealed with one layer of Nescofilm. Seed germination and protocorm development were scored after 7 and 21 weeks dark incubation as previously described. Germination percentage s and statistical analyses were completed using general linear model procedures and least square means at =0.05 (SAS 1999). Seed germination percentages were arcsine transformed prior to an alysis using least square means. Effects of exogenous cytokinins on asymbiotic seed germination Effects of four cytokini nsbenzyladenine (BA), 6-( -dimethylallylamino) purine (2-iP), zeatin (Zea), and kinetin (Kin)at 0, 1, 3, and 10 M on the asymbiotic seed germination of H. macroceratitis were examined. Basal medium MM was used in all treatment combinations. Medium was prepared as previously described, with the exception that filter-sterilized (0.2 m pore size) cytokinins were added to the molten (ca. 40C) pressure st erilized MM prior to dispensing and solidification at the concentrations previously listed. Sterile medium was dispensed (ca. 25 mL) into 9 cm diameter Petri pl ates. Ten replicate plat es were inoculated. Plates were sealed with one la yer of Nescofilm. Seed germin ation and protocorm development were rated after 14 weeks dark incubation as previously described. Germination percentages and statistical analyses were completed as previously outlined. Effects of photoperiod on as ymbiotic seed germination The effects of three photoperiod treatments (0/24, 16/8, 24/0 h L/D) on asymbiotic seed germination of H. macroceratitis incubated at 25 3C were evaluated. Illumination was provided by General Electric F96T12 cool white fluorescent tube s at 60.5 mol m -2 s -1 as measured at culture level. Plates in continual darkness were wrapped in two layers of aluminum foil to fully exclude light. Seeds were cultured on sterile MM basal medium contained in 9 cm diameter Petri plates. Plates were sealed with one layer of Nescofilm. Eleven replications per 101

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photoperiod treatment were used, and seed germin ation and protocorm development were rated as previously mentioned. Seed germination and protocorm development were scored after 14 weeks after medium inoculation. Germination percentage and statis tical analyses were completed as previously outlined for the asymbiotic media survey. Effects of photoperiod on in vitro seedling development The effects of three photoperiod trea tments (8/16, 12/12, 16/8 h L/D) on in vitro seedling development of H. macroceratitis were evaluated. Illumination source was as previously described. Seeds were germinated on MM basal medium contained in 9 cm diameter Petri plates, and 20 week old protocorms transferred to Magenta GA-7 vessels (Magenta Corporation, Chicago, Illinois) containing 50 mL MM medium. Ni ne protocorms were transferred per vessel, with ten replications per photope riod. Vessels were sealed wi th one layer of Nescofilm. Incubation and illumination conditions were as mentioned previously. In vitro seedling development was scored afte r 30 weeks incubation. Effects of photoperiods on tuber and leaf number, tuber and shoot fresh weight (fwt) and dry weight (dwt), and leaf length and width were recorded. All developmental data were statistically analyzed using general linear model procedures and Waller-Duncan mean separation at =0.05 in SAS v 8.02 (SAS 1999). Symbiotic Seed Germination Seed source and sterilization Seeds were obtained prior to dehiscence fr om mature capsules on 26 September 2003. Seeds were collected from a large (>200 fl owering and vegetative plants) population of H. macroceratitis occurring on privately-owned land in Hernando County, Florida. Immediately following collection, capsules were dried over sili ca gel desiccant for 2 weeks at 25C, followed 102

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by storage at -10C in darkness for 142 days. Prio r to the initiation of symbiotic co-cultures, a tetrazolium test (Lakon 1 949) was conducted to assess H. macroceratitis seed viability. Mycobiont isolation and identification All mycobionts were recovered from the ro ots of the study species. Mycobionts were isolated following the protocols outlined by Stewart and Zettler (2002) for Florida Habenaria species. Adult flowering and l eaf-bearing vegetative plants with intact root systems were collected, the root systems were wrapped in paper towels moistened with sterile deionized water, placed in plastic bags, stored in darkness at ca. 10C, and transported to the laboratory (<4 hrs). Root segments were detached, rinsed with cold tap water to remove debris, and surface cleansed 1 minute in a solution containi ng 5 mL ethanol (100%), 5 mL 6.00% NaOCl, and 90 mL sterile DI water. Clumps of cortical cells containing funga l pelotons were removed, placed on corn meal agar (CMA; Sigma-Aldrich, St. Loui s, Missouri) supplemented with 50 mg l -1 novobiocin sodium salt (Sigma-Aldrich, St. Louis, Missouri) and incubated at 25C for 5 days. Hyphal tips were excised from actively-growin g pelotons and subcultured onto 1/5th-strength potato dextrose agar (1/5 PDA): 6.8 g PDA (BD Company, Spar ks, Maryland), 6.0 g gr anulated agar (BD Company, Sparks, Maryland), 1 l di stilled deionized (dd) water. Fungal isolates showing cultu ral characteristics similar to those orchid mycobionts previously described in the literature (Moore 1987; Zettler 1997b; Curra h et al. 1997; Currah et al. 1987; Richardson et al. 1993; Stewart et al. 2003; Zelmer et al. 1996) were assigned a reference number and stored at 10C on modi fied oat meal agar (MOMA): 3.0 g pulverized rolled oats (Quaker Oats, Chicago, Illinois), 7.0 g granulated agar, 100 mg yeast extract (BD Company, Sparks, Maryland), and 1 l dd water (Cle ments et al. 1986). Myc obiont isolates were stored until use in symbiotic co -culture experiments. Two repr esentative mycobiont isolates 103

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were accessioned into the University of Al berta Microfungus Herbarium (UAMH) as UAMH 10801 and UAMH 10802. Mycobiont characterization and identificati on followed methods outlined by Zelmer and Currah (1995), Currah et al. (1987, 1990, 1997), and Zelmer et al. (1996). Hyphal and monilioid cell characteristics were assessed from culture s growing on both CMA and 1/5 PDA cultured in continual darkness at 25C using a Nikon Labopha t-2 light microscope (Nikon USA, Melville, New York) fitted with a Nikon Coolpix 4500 digita l camera (Nikon USA, Melville, New York). Staining procedures followed those outlined by Ph illips and Hayman (1970) modified by the use of acid fuchsin as the mycobiont stain (Stevens 1974; J. Kimbrough personal communication). Culture growth rates were determined from is olates growing on PDA in cubated in continual darkness at 25C as measured in three directions every 24 hours from the bottom of each Petri plate. Cellulase production was determined by the cellulose azure method of Smith (1977) modified by the use of 1/5 PDA as the basal medium (Figure 1-6). Polyphenol oxidase production was detected by using the tannic aci d medium (TAM) method of Davidson et al. (1938) (Figure 1-7). Symbiotic co-culture The effects of six mycobionts (Table 3-3) on the in vitro symbiotic co-culture of H. macroceratitis were evaluated. Seeds were sown according to the procedures outlined by Stewart and Zettler (2002) for Florida Habenaria species. Seeds were removed from cold-dark storage, allowed to warm to room temperature (ca. 25C), surface disinfected for 1 min in the same solution used during asymbiotic seed disi nfection, and placed over th e surface of a 1 cm 4 cm sterile filter paper strip (Whatman No. 4, Whatman International, Maidstone, United Kingdom) within a 9 cm diameter Petri plate containing 25 mL oat meal agar (OMA): 3.0 g pulverized rolled oats, 7.0 g bacto-agar, and 1 l dd water. Medium pH was adjusted to 5.8 with 104

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0.1 N HCl prior to autoclaving at 117.7 kPa and 121C for 40 min. Seeds were sown using a sterile bacterial inoculating loop. Between 10 and 40 seeds were sown per plate. Each plate was inoculated with a 1 cm 3 block of mycobiont inoculum, one mycobiont per plate, and a total of 8 replicate plates per mycobiont. Eight uninoculated plates served as the control. Plates were sealed with Nescofilm, wrapped in aluminum fo il to exclude light, and maintained in darkness (0/24 h L/D) for 58 days at 25 2C. Plates we re examined weekly during dark maintenance for signs of germination or contamination, exposin g the seeds to brief (<10 min) periods of illumination. Plates were returned to expe rimental conditions after visual inspection. After 58 days dark culture, seed germinat ion and protocorm development was assessed using a dissecting stereomicroscope. Germina tion and seedling growth and development were scored on a scale of 0-5 (Stewa rt and Kane 2006b; Tabl e 3-2; Figure 3-1). Seed germination percentages were based on viable seeds determined by visual inspection with the aid of a dissection microscope. Viable seeds were considered those se eds containing a distinct, rounded and hyaline embryo. Germination percentages were calculated by dividing the number of seeds in each germination and development stage by the total number of viable seeds in the sample. Data were analyzed using general linear model proce dures and Waller-Duncan mean separation at =0.05 by SAS v 8.02 (SAS 1999). Germination counts were arcsine transformed to normalize variation. Effects of photoperiod on symbiotic co-culture The effects of three photoperiod treatm ents (0/24 h, 16/8 h, 24/0 h L/D) on in vitro symbiotic co-culture of H. macroceratitis maintained at 25 3C were evaluated. Seeds were sown as previously described; with the exception that only one mycobiont was used in all three photoperiod treatments. Mycobiont Sbrev-266, previously identified as a strain of Epulorhiza 105

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repens (Bernard) Moore and originating from the roots of the Florida terrestrial orchid Spiranthes brevilabris Lind. (Stewart et al. 2003), was chosen because of its effectiveness at germinating seeds of H. macroceratitis in a previous study, as well as seeds of other Florida terrestrial and epiphytic orchid s (Stewart and Zettler 2002; Stew art et al. 2003; Zettler et al. 2007; S.L. Stewart unpublished data). Illumi nation was provided by General Electric F96T12 cool white fluorescent tubes at 60.5 mol m -2 s -1 as measured at culture level. Plates in continual darkness were wrapped in aluminum foil to exclud e light. Seeds were cultured on OMA in 9 cm diameter Petri plates (ca. 25 mL). Plates were sealed with one layer of Nescofilm. Eleven replications per photoperiod treatme nt were used. Seed germination and protocorm development were scored after a 96 day culture period. Germin ation percentage and stat istical analyses were completed as previously outlined. Results Asymbiotic Seed Germination Asymbiotic media survey Seeds began swelling within three weeks af ter medium inoculation, and germination commenced within six weeks after inoculation. Visual contamina tion rate of cultures was 5%. A tetrazolium test revealed H. macroceratitis seeds collected from the Brooksville, Florida (Hernando Co., Florida) site to be 41.4% viable, while visual in spection of seed revealed 52.6% viability from the same site. Seeds of this species were monoembryonic. Seed germination at week 7 was hi ghest on both LM and KC, 89.1% and 89.2% respectively (Figure 3-2). However, these germinated seed had only developed to Stage 1 by this time. Maximum protocorm development at w eek 7 (Stage 2) was supported by ML and MM, 61.2% and 83.9% respectively (Figure 3-2). Af ter 7 weeks incubation, seed germination was minimal on MS, LM, and VW although supporting Stage 2 protocorm development. No 106

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germinated seeds on KC developed beyond Stage 1. Seed germination and seedling development progressed to at leas t Stage 1 on all media surveyed. Seed germination percentages and protocorm de velopment did not remain constant to week 16. Seed germination and protocorm development at week 16 was highest on VW, KC, and MM, 98.8% (Stage 2), 95.3% (Stage 2), and 98.6 % (Stage 4), respectively (Figure 3-2). However, only MM supported protocorm developmen t to a leaf-bearing (Sta ge 4) after 16 weeks incubation, while VW and KC supported seed development to only Stage 2 during the same incubation period. Paralleling w eek 7 data, maximum protocorm de velopment in week 16 (Stage 4) was supported on ML and MM. After 16 w eeks incubation, no seeds developed beyond Stage 2 on LM, VW and KC. No seeds developed beyond Stage 3 on MS. Effects of carbohydrate source on asymbiotic seed germination After 7 weeks dark incubation, all carbohydrate sources tested supported at least minimal asymbiotic germination (i.e., Stage 1) of H. macroceratitis seeds. Stage 3 germination was supported at this time by both fructose without banana powder and basa l medium only control (0.2% and 0.4%, respectively; Figu re 3-2). However, the majority of germinating seeds were observed in Stage 2 development, with the ba sal medium only control (83.9%), basal medium with banana powder control (81.0 %), dextrose without banana powder (80.9%), and fructose without banana powder (79.1%) supporting the highest germination percentages in this developmental stage (Figure 3-3). Only the fr uctose with banana powder treatment supported a significantly lower germination pe rcentage (57.6%) than did all ot her treatments. No significant difference in germination percentages were found fo r Stage 1 germination in all treatments after 7 weeks. All carbohydrate sources supported through Stag e 5 germination and development after 21 weeks of in vitro culture under dark incubation conditions. Both the basal medium only control 107

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and fructose without banana powder supporte d 100% Stage 5 development after 21 weeks (Figure 3-4). Only dextrose with banana powder supported a significan tly lower germination percentage in Stage 5 (59.8%) than did all other treatmen ts. Interestingly, this same treatment supported a significantly higher pe rcentage of Stage 4 protocor ms (28.1%) than did all other treatments tested. Furthermore, only the dextrose with banana powder and fructose with banana powder treatments supported Stage 3 developmen t after 21 weeks (1.9% and 5.0%, respectively; Figure 3-4). Effects of exogenous cytokinins on asymbiotic seed germination After 14 weeks dark incubation, all cytokinins tested demonstrated some effect on the asymbiotic seed germination of H. macroceratitis With the exception of Kin at 3 M, all other cytokinins tested at both the 3 M and 10 M concentrations demonstrated no significant difference in germination percentage from the control. Cytokinins Zea and Kin at 1 M had a pronounced positive effect on seed germination, resul ting in an increased germination percentage (Zea = 58.1% and Kin = 47.2%) when compared to control conditions (Control = 14.2%; Figure 3-4). BA at 1 M also had a significant positive effect on seed germ ination percentage, but significantly less that than either Zea or Kin (BA = 33.7%; Fi gure 3-5). Asymbiotic germination was suppressed by all cytokinins tested at 10 M, as well as BA at 3 M. Effects of photoperiod on as ymbiotic seed germination Seeds began germinating after four weeks regardless of photoperiodic condition. No significant effect of photoperiod was found on the initial seed ge rmination (e.g. Stage 1) of H. macroceratitis Those seeds incubated in continual da rkness (0/24 h L/D) exhibited both the highest final percent germina tion (91.7%) and most advanced protocorm developmental stage (Stage 4; Figure 3-6). Protocorm devel opment under both the 16/8 h L/D and 24/0 h L/D treatments was supported to Stage 4, but disp layed statistically lo wer seed germination 108

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percentages, 17.5% and 54.6% respectively (Fig ure 3-6). Interestingly, while maximum protocorm development under the 16/8 h L/D photoperiod was achieved at Stage 3, maximum protocorm development achevied under the 0/ 24 h L/D and 24/0 h L/D photoperiods was Stage 4. Asymbiotic seed germination was optim al under the 0/24 h photoperiod in both seed germination percentage and a dvanced developmental stage. Pronounced differences in protocorm morphol ogy were seen under al l three photoperiods. Those seed germinated in both the 16/8 h L/ D and 24/0 h L/D photoperiods produced protocorms that lacked rhizoids throughout their development to Stage 5 (Figure 3-7b -c). Conversely, those seed germinated in the 0/24 h L/D photope riod produced protocorms possessing numerous rhizoids from Stage 1 though Stage 5, and beyond (Figure 3-7a). Effects of photoperiod on in vitro seedling development Photoperiod had a pronounced effect on several growth and development responses in in vitro seedlings of H. macroceratitis after 30 weeks incubation. The number of tubers produced per in vitro seedling was highest under the 8/16 h L/D photoperiod (1.06 tubers) versus under either the 12/12 h L/D photoperiod (1.00 tubers) or 16/8 h L/D photoperiod (1.00 tubers; Figure 3-7). Tuber fresh weight (42.7 g) and dry weight (6.5 g) were highest on seedlings cultivated under an 8/16 h L/D photoperiod, while tuber fresh and dry weight s were statistically similar under 12/12 h L/D and 16/8 h L/D phot operiods (Figure 3-9). Tuber size (diameter and length) was also influenced by photoperiod, with tuber diameter being greates t under the 8/16 h L/D photoperiod (3.1 mm) and smaller under all ot her photoperiod conditions Tuber length remained statistically similar under all three photoperiod conditions (Figure 3-10). Shoot fresh weight was highest in seedlings incubated under the 8/16 h L/D photoperiod (69.5 g), whereas shoot dry weight was similar for plants incubated under both the 16/8 h L/D and 12/12 h L/D photoperiods (39.1 g and 50.1 g, respectively; Figure 3-8). The lowest 109

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number of leaves per plant was produced on those seedlings incubated in the 8/16 h L/D photoperiod, but those leaves were the longest and widest (Figures 3-7, 3-10). In fact, leaf length and width per seedling signifi cantly decreased from short-da y (SD) to long-day (LD) photoperiod, while the total number of leaves per seedling increased (Figures 3-8, 3-10). Symbiotic Seed Germination Mycobiont isolation and identification Six fungal mycobionts were recovered from pe lotons within the ro ots of flowering and vegetative plants of H. macroceratitis (Table 3-3; Figure 3-11) collect ed at two sites in Florida. All six mycobionts were identified as members of the anamorphic fungal genus Epulorhiza (Moore, 1987). Only superficial differences in cultural morphology were identified among the group of six mycobionts. Isolates Hmac-309 and Hmac-310 were cream in color after 25 days on 1/5 PDA, whereas all other is olates were ivory. These differences were determined as inconsequential to taxonomic dete rminations. No differences in cellulase or polyphenol oxidase activity were detected among the six isolates. Symbiotic co-culture Seeds began to swell within two weeks after sowing, and germination commenced within five weeks. Visual contamination of cultures from bacteria and non-mycorrhizal fungi was 2%. A tetrazolium test revealed H. macroceratitis seeds to be 41.4% viable, while visual inspection revealed 52.6% viability fr om the same seed lot. All inoculated seed germinated by 58 days. An effect of mycobiont preference was found during the in vitro symbiotic co-culture of H. macroceratitis Germination after 58 days was highest when seeds were inoculated with mycobiont Hmac-310 (65.7%; Figure 3-12). This isolate not only promoted the highest final percent germination, but also promoted Stage 2 development. However, no significant diffe rence in seed germination and protocorm 110

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development was demonstrated by Stage 2 among Hmac-310, Hmac-312, or control treatments (65.7%, 51.0%, 51.5% respect ively; Figure 3-12). Effects of photoperiod on symbiotic co-culture Seeds began to swell within two-and-a-ha lf weeks after sowing, and germination commenced within four weeks. Visual contamina tion rate of cultures was 4%. Seed viabilities remained as described previously. Seeds began germinating after four weeks regardless of photoperiod condition. After 96 days culture a significant effect of photoperiod was found on the initial in vitro symbiotic coculture (e.g. Stage 1) of H. macroceratitis Seeds cultured under conti nual darkness (0/24 h L/D) exhibited a lower initial seed ge rmination percentage (17.1%) th an seeds culture d under either the 16/8 h L/D or 24/0 h L/D photoperiods (37.4 % and 34.4%, respectively; Figure 3-13). However, protocorm development to Stage 2 was stimulated under 0/24 h L/D conditions (53.5%; Figure 3-12), whereas development wa s less under both 16/8 h L/D and 24/0 h L/D (34.6% and 34.5%, respectively; Figures 3-13, 3-14) Symbiotic seed germination was highest under 16/8 h L/D photoperiod, but protocorm deve lopment was most advanced under a 0/24 h L/D photoperiod. Discussion Asymbiotic Seed Germination The successful in vitro asymbiotic seed germination of H. macroceratitis has not been previously reported. Stewart and Zettler (2002) reported the successful in vitro symbiotic seed germination of this species; however this aspect of the propagation of H. macroceratitis will be discussed in the following section. Only one report exists of the asymbiotic seed germination of a Habenaria species. Working with Habenaria radiata, a related species to H. macroceratitis, Takahashi et al. (2000) reporte d an asymbiotic seed germin ation percentage of 48.8% on 111

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Hyponex medium under continual li ght incubation (24/0 h L/D) after 28 days. While in this previously published study, the asymbiotic seed germination of a Habenaria species was examined, the authors failed to addre ss factors that may effect efficient in vitro seed germination and in vitro seedling development. In the present study, an efficient protocol for the asymbiotic seed germination of H. macroceratitis is provided, as well as an ex amination of factors that may affect the in vitro seed germination of this species. The effects of photoperiod on in vitro seedling development are also addressed, providi ng a special emphasis on tuber development. While all media used in the current study contain a nitrogen source, the form and concentration of that source differ among media (Table 3-1). Knudson C, MS and VW contain only inorganic sources of nitrog en, while MM contains only an amino acid (i.e., organic source) as the sole source of nitrogen in the media. Conversely, LN and ML contain a mixture of inorganic nitrogen sources and an amino acid. Modified Lucke contained the lowest concentration of nitrogen (4.98 mM), as prepared in this study. All media supported asymbiotic seed germination and protocorm development to at least Stage 2, suggesting that nitrogen in any form and concentration will support asymbiotic seed germination in H. macroceratitis However, ML contained the lowest concentr ation of nitrogen and still supported seed germination and protocorm development to Stage 4. This suggests that H. macroceratitis may require an overall low concentration of nitrogen for the initiation of seed germination and early (
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the concentration of inorganic ni trogen sources. These researcher s have suggested that organic sources of nitrogen (i.e., amino acids) may be more readily available to a s eed or plant than an analogous inorganic nitrogen source. The germina tion of seeds and development of protocorms cultured in the presence of inor ganic nitrogen sources may be delayed due to a delay in the production of nitrate reductase, which has been s hown to be produced only several months after imbibition in Cattleya (Raghaven and Torrey 1964). Both ML and MM contain an amino acid as a nitrogen sourcewith glycine being the sole nitrogen source found in MM and glutamine, along with potassium nitrate and magnesium nitrate, being the sources of nitr ogen in ML. It is conceivable that seeds of H. macroceratitis more readily utilize the organic source of nitrogen available in MM to support germination and protoc orm development than n itrogen sources in all other media tested. Little reliable information exists on the absolute role of organic nitrogen sources in asymbiotic orchid seed germina tion media. Further investigation should be undertaken. The role carbohydrates play in orchid seed germination has received some attention, although little recently. Early re searchers realized some carbohydr ates were better suited to support asymbiotic orchid seed ge rmination than other, and that this response may be speciesspecific (for review see Arditt i and Ernst 1984). While not tes ting the effects of carbohydrate source on seed germination, Ernst et al. (1971) did demonstrate that a number of carbohydrates, such as glucose, fructose, and oligosaccharides containing these sugars can support the growth and development of Phalaenopsis seedlings. Furthermore, Ernst and Arditti (1990) demonstrated that seeds of both Phalaenopsis Habsburg and Phalaenopsis Ruth Burton ( Phalaenopsis Abendrot Phalaenopsis Abendrot) can utilize a wide variety of carbohydrate sourcesglucose, maltose, maltotriose, maltote traose, maltopentaose, maltohexaose, and 113

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maltoheptaoseto support asymbiotic seed germination on Knudson C medium. A similar result was found in the current study using simp le carbohydrates in treatment combination with MM to support the asymbiotic seed germination of the Florida terrestrial orchid H. macroceratitis In the current study, the simple carbohydrates fruc tose, sucrose, and dextrose all supported advanced (>Stage 3) germination and development of H. macroceratitis Similarly, Ernst and Arditti (1990) reported that seeds of the two previously mentioned Phalaenopsis hybrids germinated rapidly on Knudson C asymbiotic medium supplemented with glucose, maltose, or maltotriose, which are all simple carbohydrates. When larger maltooligosaccharides where used, seed germination percentages de creased, likely due to the inabili ty of the germinating orchid embryo to synthesize the enzymes necessary to hydrolyze these more complex carbohydrates (Ernst and Arditti 1990). A more recent report indicated that carbohydrate hydrolysis by extracellular hydrolytic enzymes is possible, as demonstrated with protocorm-like bodies of Dendrobium (Hew and Mah 1989). However, this extracellular hydrolysis of complex carbohydrates (i.e., maltooligosacchar ides) was not reported. A sim ilar trend in decreased seed germination percentage would be expected if larger, complex carbohydrates would be surveyed for their ability to support the asymbiotic germination of H. macroceratitis While all carbohydrate treatments in the cu rrent study supported asymbiotic seed germination through Stage 5, both controls (basal medium with and without BP) also supported seed germination through Stage 5. A similar tre nd has not been previous ly reported in earlier studies concerning the effects of carbohydrate type and concen tration on asymbiotic seed germination and in vitro seedling development. While a minimal, but unknown, amount of carbohydrate is present in BP, the advanced seed germination observed in the basal medium with 114

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BP treatment could be explai ned as being supported by this undefined carbohydrate source. However, Stage 5 germination was also observed in the basal medium without BP control treatment. The basal medium (MM) contains 0.05 mg l -1 biotin and 100 mg l -1 myo-inositol, and these supplements could serve as minimal sources of carbohydrate nutrition. Arditti (1979) reported that biotin enhanced the growth of Cattleya Odontoglossum Phaphiopedilum and Cymbidium ; and that inositol stimul ated the germination of Cattleya Both of these supplements in MM could be supporting the ge rmination through Stage 5 of H. macroceratitis in the basal medium without BP control. Asymbiotic orchid seed germination media are typically supplemented with simple carbohydrates (i.e., sucrose). While orchid seeds germinate and develop readily on these simple carbohydrates, free simple carbohydrat es, such as sucrose, can be considered ecologically unimportant since carbohydrates rarely exist in this simple form in nature (Harley 1969). Germinating seed and developing plants in the wild are dependant upon their mycobionts for the breakdown of complex carbohydrates and transport of the resulting simple compounds into plant tissues (Harley 1969; Smith 1966, 1967). Therefore, future studies on the effects of simple and complex carbohydrates on the germination of or chid seed should be conducted under both asymbiotic and symbiotic culture conditions. Previous work on the role of various horm ones, including cytokinins, during asymbiotic seed germination have yielded inconclusive resultssometimes enhancing germination, other times inhibiting, and still other times showing no a pparent effect. This in consistent response to exogenous hormone application has even been sh own to vary from genus-to-genus and speciesto-species (for reviews see Arditti 1967; Withne r 1959; Arditti and Ernst 1984). The addition of exogenous cytokinins are of particular interest in asymbiotic orchid seed germination because 115

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several mycorrhizal fungi have been shown to produce trace amounts of cytokinins (Crafts and Miller 1974), thus we can assume that orchid se ed germination and development in the wild may be assisted by these fungal-based cytokinins. In the current study, Kin, Zea and BA enhanced asymbiotic seed germination of H. macroceratitis However, this positive response was only seen at the 1 M level for BA and Zea, and at both the 3 M and 10 M levels for Kin. These results indicate that H. macroceratitis seed germination is promoted to a greater extent in the presence of Kin and Zea at relatively low concentr ations than in the presen ce of BA (Figure 3-7). Miyoshi and Mii (1998) found a similar response for Kin at 1 M in the asymbiotic seed germination of Cypripedium macranthos Sw. The increase in asymbiotic seed germination percentage in response to the application of low concentrations of exogenous cytokinins reported in both C. macranthos (Miyoshi and Mii 1998) and H. macroceratitis (present study) is not surprising. While Crafts a nd Miller (1974) reported the production of cytoki nins by mycorrhizal fungi, the concentration of cytokinins within fungal bodies was consider ed as trace amounts. If orch id mycobionts do indeed provide cytokinins to germinating orchid seeds in situ the amount of these cytoki nins would be minimal. This notion appears to be supported by the present in vitro studies with H. macroceratitis where germination percentage was increased over the control only in the presence of low concentrations of exogenous cytokinins. The current study also demonstrat ed a reduction in germination in the presence of higher (3 M and 10 M) BA, Zea and Kin concentrations. Arditti et al. (1981) de monstrated a similar repression of germination in Epipactis gigantea Dougl. ex Hook. in the presence of BA. Eighty percent germination was noted on full-strength Curtis medium for E. gigantea while fullstrength Curtis medium with the addition of 4.44 M BA produced only 10% germination. 116

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Furthermore, white seedlings of E. gigantea remained white and small (2-3 cm height) when transferred from a medium contai ning no cytokinins to media c ontaining various concentrations of BA (Arditti et al. 1981). Conversely, prelim inary study has demonstrated that seedlings of H. macroceratitis cultured on medium contai ning Kin, Zea or BA show no abnormal coloration or developmental morphologies when transferre d to 16/8 h L/D condi tions (S.L. Stewart unpublished data). Obviously, the role of plant growth regulators, esp ecially cytokinins, in asymbiotic orchid seed germination is not well understood, and may be genus or species specific. The role of photoperiod in orchid seed germina tion is often overlooked and is also not well understood, especially in terrestrial species. In vitro seed germination of ma ny terrestrial orchids has been found to be inhibited by incubation in light (Van Waes and Deberg 1986; Arditti et al. 1981; Takahashi et al. 2000). Light also plays an important role in in situ seed germination and seedling recruitment. Availability of light was found to be a limiting factor for Cypripedium calceolus L. seedling recruitment in Europe (Kul l 1998). In the current study, no effect of photoperiod on initial (Stage 1) as ymbiotic seed germination of H. macroceratitis was demonstrated. However, a significant effect on subsequent protocorm growth and development ( Stage 2), as well as morphology, among the photoperiods was shown. Habenaria macroceratitis typically inhabits heavily shad ed floors of hammock habitats throughout central Florida. Rasmussen and Rasmussen (1991) surmised that the seeds of orchids inhabiting shaded forest floors we re not exposed to large quantit ies of red light, but in fact exposed to far-red light. Seeds exposed to farred light would convert phytochrome into the Pr form, thus inhibiting germination (Kendrick 1976). Given that far-red light is known to inhibit seed germination (Kendrick 1976) and cool wh ite fluorescent tubes em it large amounts of red light (Toole 1963), it is surpri sing that the current study f ound the highest percent seed 117

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germination and most advanced (>Stage 4) pr otocorm development in a 0/24 h L/D photoperiod (complete darkness). It is interesting that either the total absence of light or the total absence of darkness stimulated seed germination to relati vely high percentages (91.7% and 54.6%), while a 16/8 h L/D photoperiod actually suppressed seed ge rmination (17.5%). A similar photoperiodic response has not been reported in any terr estrial or epiphytic orchid species. Seeds germinated in continual darkness produc ed numerous rhizoids over the surface of the developing protocorm, while those seeds germ inated in either light-including photoperiods did not produce rhizoids (Figur e 3-6). This is the first re port of such a finding for any Habenaria species. Arditti et al. (1981) reported that absorbing hair (i .e., rhizoid) production was not effected by photoperiod or movement from dark to li ght in a number of terr estrial orchid species, including species of Platanthera and Piperia which are both related to Habenaria Under natural conditions, seeds of terrestrial or chids are known to produce copious rhizoids (Rasmussen 1995). These structures are used as entry points during in fection by myconionts, and would therefore be necessary for the prol onged existence of protocorms in nature. The current study has demonstrated the e ffects photoperiod on both asymbiotic seed germination and subsequent in vitro seedling development in H. macroceratitis Tuber number and size appear to be influenced by photoperiod, where culture under SD resulted in more tubers per in vitro seedling, higher fresh and dry weights, and greatest tuber diameter when compared to seedlings cultured under LD conditions. This represents the first report describing the photoperiodic control of tuberizati on in a North American terrestr ial orchid. The photoperiodic control of tuberization is known from several important agronomic crops. Omokolo et al. (2003) found that tuber formation in Xanthosoma sagittifolium (L.) Schott could be controlled through the manipulation of photoperiod, in addition to sucrose and BA concentrations. The most 118

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important of these controls was found to be photoperiod, where both SD and SD-dark conditions produced the highest number of tube rs per plant at both the 50 g l -1 and 80 g l -1 sucrose levels. This photoperiod control of tuberizati on has also been demonstrated in Solanum tuberosum L.. Seabrook et al. (1993) reported th at the potato cultivars Katahdin and Russet Burbank formed more microtubers under LD-SD, SD-SD and SD -LD conditions than they did under LD-LD conditions. More importantly, the diameters a nd fresh weights of those tubers produced under LD-SD conditions were significantl y higher than all other photoperi od conditions. Therefore, it is not surprising to find that tuber number and size is influenced by photoperiod in H. macroceratitis Mach kov et al. (1998) found that tuberization in S. tuberosum was mediated by photoperiod through control of hormone levels, mo st notably the ABA/GA ratio. Under SD conditions, the ABA/GA ratio appear s to be highly in favor of ABA, resulting in the initiation of tuberization. Similarly, the a pplication of exogenous ABA to in vitro S. tuberosum plantlets stimulated tuberization (Xu et al. 1998). A si milar photoperiod control of the ABA/GA ratio in H. macroceratitis may be responsible for greater tuber number and size under SD conditions. Further study is needed to eluc idate this possible interaction. Leaf production on in vitro cultured seedlings of H. macroceratitis was reduced under a SD photoperiod; however, those leaves were longer and wider. Mo reover, leaf size significantly decreased from SD to LD conditions while the total number of leaves per seedling increased. This trend of few but larger leaves under SD conditions and more but smaller leaves under LD conditions may indicate the ability of H. macroceratitis to optimize photosynthetic capacity for either tuber storage allocation or vegetative growth, respecti vely. A similar trend of carbon allocation toward a primary storage organ with increasing SD conditions was reported in the 119

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long-lived perennial orchid Tipularia discolor (Pursh) Nutt. (Tissue et al. 1995). As the growing season progressed, higher amounts of carbon were allo cated to the primary corm as a means of over wintering storage. This trend has previously been reported for T. discolor (Whigham 1984; Zimmerman and Whigham 1992), but never reported for the sub-tr opical terrestrial orchid H. macroceratitis Further investigation is warranted into the carbon allo cation strategy of H. macroceratitis in relation to photoperiod condition based on the current findings of more tubers, larger tubers, and larger leaves under SD conditions. The current study presents a first look at the asymbiotic seed germination requirements of a rare sub-tropical terrestr ial orchid from Florida, H. macroceratitis Data are also presented concerning the growth and development of in vitro seedlings. Given the rare status of this orchid in the wild and the critically threatened status of its natural habitat, these data present critical information on the previously unknown early life-history stages of th is orchid. Although asymbiotic seed germination of H. macroceratitis represents an efficient means to propagate the species, it does not account for the role of the species naturally-o ccurring mycobionts. Without these mycobionts seed germination and plant development ex situ would not occur. For this reason, the in vitro symbiotic co-culture of H. macroceratitis was explored. Symbiotic Seed Germination In vitro symbiotic co-culture is a powerful me thod for both the production of mycobiontinfected seedlings for use in plant reintroduction and the study of mycobiont preference within and among Orchidaceae taxa. Fe w reports exist concerning the in vitro symbiotic co-culture of North American terrestrial orchid species, especially sub-tropical terrestrial species. This is only the second report describing the successful sy mbiotic co-culture of a North American Habenaria species, and a first report of a photoperiodic effect on the in vitro symbiotic seed germination of 120

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H. macroceratitis This report also represents the first description of possible in vitro fungal preference displayed by H. macroceratitis Stewart and Zettler (2002) have previously reported the in vitro symbiotic co-culture of H. macroceratitis In their study, seeds were cultured on OMA with a mycobiont originating from the roots of H. quinqueseta (Michaux) Eaton (Hque-291), a closely related taxon to H. macroceratitis resulting in a maximum of 63.7% germination after 83 days. However, maximum protocorm development (Stage 4) was reported from treatments that were cultured with a mycobiont originating from Spiranthes brevilabris (Sbrev-266; UAMH 9824). In Stewart and Zettler (2002), the mycobiont isolate originating from H. quinqueseta was identified as belonging to the anamorphic genus Ceratorhiza Moore, while the isolate from S. brevilabris was identified as belonging to the anamorphic genus Epulorhiza In the present study a similar seed germination percentage (65.7%; Figure 3-13) wa s achieved; however, this percentage was achieved in less time (58 days) than that reported in Stewart and Zettler (2002). Additionally, all mycobionts is olated in the current st udy were assignable to the anamorphic genus Epulorhiza All six mycobionts closely resembled E. repens in having comparable average growth rates and ovoid monilio id cells. The slight variations in culture color seen among the isolates were not consid ered highly differential among all mycobionts and likely due to inconsequential nutrient differences in the 1/5 PDA culture medium from Petri plate-to-Petri plate (Currah et al. 1997). A degree of preference by H. macroceratitis for its mycobiont, as demonstrated thr ough mycobiont isolations, is lik ely responsible for the rapid in vitro germination seen in some mycobiont-seed co -cultures in the current study. Stewart and Zettler (2002) suggested that H. macroceratitis was non-preferential for mycobionts, whereas the present study suggests that mycobionts isolated from vegetative, and therefore possibly younger, 121

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plants of H. macroceratitis better support in vitro symbiotic co-culture. Fungal preference in the Orchidaceae has been considered controvers ial for many years (Curtis 1939; Hadley 1970; Rasmussen and Rasmussen 2007). Differences in or chid fungal preference ha ve been identified under in vitro versus in situ conditions (Bidartondo and Bruns 2005; Masuhara and Katsuya 1994; Taylor and Bruns 1999; Tayl or et al. 2003), and these differences have led some to consider orchid fungal preference as generally low (Hadley 1970; Stew art and Zettler 2002). Nonetheless, the present study demonstrates H. macroceratitis does possess a degree of mycobiont preference under in vitro conditions. This species a ppears to be preferential for mycobionts isolated from plants existing in the same population where seed was collected, since mycobionts isolated from a di stant population supported statistically lower seed germination percentages. A more complete study testing mycobionts isolat ed from both vegetative and flowering plants at multiple geographi c sites throughout the entire range of H. macroceratitis is suggested to further elucidate the apparent mycobiont preference found in this species. Few reports exist concerning photoperiodic effects on the in vitro symbiotic co-culture of terrestrial orchids. Typically, the seeds of terres trial orchids germinate while buried in the soil, but are initially exposed to a short period of illumination upon capsule dehiscence (Rasmussen et al. 1990). Stewart and Zettler ( 2002) reported no increase in initi al symbiotic seed germination (Stage 1) when cultures of H. macroceratitis were transferred from c ontinuous dark conditions to a 12/12 h L/D photoperiod; however, an increase in early protocorm development was reported. Rasmussen and Rasmussen (1991) and Zettler an d McInnis (1994) both re ported a reduction in asymbiotic and symbiotic seed germination percentage of Dactylorhiza majalis (Rchb.) P.R. Hunt & Summerhayes and Platanthera integralabia respectively, under conditions of a dark pretreatment of seed before light exposure. A similar response was f ound in the current study. 122

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Initial seed germination (Stage 1) was signifi cantly lower under the 0/24 h L/D photoperiod than either the 16/8 h L/D or 24/0 h L/D photope riods (Figure 3-12). However, protocorm development was most advanced (Stage 2) unde r the 0/24 h L/D photoperiod (Figure 3-11). As mentioned previously, this reducti on in initial seed germination percentage may be due to the lack of a light pretreatment of the seed pr ior to sowing. Orchid seeds typically possess a hydrophobic testa that allows a seed to remain a bove the soil surface where they can be exposed to sunlight (Rasmussen and Rasmussen 1991). Th is light exposure may only initiate nutrient mobilization or assist in imbibition and not lead directly to seed germination (Rasmussen and Rasmussen 1991). Interestingly, Takahashi et al (2000) reported no significant difference in in vitro symbiotic seed germination percentages when seeds of Habenaria radiata were cultured under continual darkness (0/24 h L/D) or 24/0 h L/D conditions. This may indicate that terrestrial orchid seed germin ation response to photoperiod may be genus or species specific. The current study presents new findings on the in vitro fungal preference and effects of photoperiod on the in vitro symbiotic co-culture of a rare s ub-tropical terrestri al orchid from Florida, H. macroceratitis The rare status of this orchid in the wild and the threatened status of its natural habitat necessitate the development of efficient symbiotic seed germination protocols, otherwise the species may not exis t as an independent organism in its natural habitat for long. These data present invaluable information c oncerning a previously unknown fungal preference within populations of H. macroceratitis This information will be critical to future plant production and reintroduction effort s aimed at the conservation of H. macroceratitis in its natural habitat. Implications for Integrated Conservation Planning The present studies on the in vitro seed culture of the Florida terrestrial orchid H. macroceratitis have demonstrated the benefits of a careful study of the propagation science 123

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asymbiotic media selection, carbohydrate preference, application of exogenous cytokinins, dark incubation during seed germination, and symbio tic co-culture requirementsof an orchid species before implementing cons ervation and recovery plans. The usefulness of asymbiotic culture techniques in the st udy of orchid seedling growth and development was also demonstrated. These results show the importa nce of understanding an orchids germination requirements, in addition to a species early growth and development stages, before attempting further integrated conservation and species recovery efforts. For example, by demonstrating that tuber formation in H. macroceratitis can be induced in vitro by incubation of plants under SD conditions, an efficient means of tuber production as a source of plant ma terial for reintroduction purposes can now be investigated a nd applied to the management of this terrestrial orchid species (Stewart and Kane 2006a). Furthermore, the present studies on the in vitro symbiotic co-culture of H. macroceratitis demonstrated not only that the symbiotic co-cul ture of this species was possible, but also elucidated a potential habitatlimited mycobiont preference with in this taxon. Identifying an apparent mycobiont preferen ce that may be locally spec ific within the range of H. macroceratitis in Florida has a number of inte grated conservation and species management implications. Of primary interest is the result that H. macroceratitis is likely not preferen tial for only one or two strains of Epulorhiza mycobiont throughout Florida, but that the species demonstrates a preference for mycobionts that may be locally or site limited (Stewart and Kane 2006b). This has a number of implications for the integrated conservation of H. macroceratitis in Florida. Specifically, existence of a site limited mycobi ont preference within the species means that strains of H. macroceratitis mycobionts from each known site in Florida will need to be isolated and maintained in pure culture in order to insu re the long-term conservation of the species. 124

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Furthermore, seed from each known site must also be collected and stored if future in vitro symbiotic co-culture production is to be considered as a viable tool in the long-term integrated conservation of H. macroceratitis. The degree of mycobiont pr eference shown in the symbiotic co-culture of H. macroceratitis demonstrates the need for proper ma nagement for both plant and mycobiont populations. While managing habitats for plant population sustainability can be accomplished by maintaining the basic plant a nd soil structure historically and presently found in north and central Florida hardwood hammo cks containing populations of H. macroceratitis, managing sites for mycobiont diversity and sustaina bility is an unexplored area of orchid integrated conservation biology. Further study is necessary before mycobiont management within orchid habitats is a useful tool to insure the l ong-term sustainability of nativ e orchid populations, including populations of H. macroceratitis. However, to secure the longterm sustainability of native orchid populations, entire habitats must be t hought of as refugia for orchid mycobionts and should be managed accordingly. The current studies concerni ng the propagation science of H. macroceratitis represent one step in the species-level integrated conservation of this rare terrestrial orchid species in Florida (Figure 1-1). In developing a more complete understanding of the asymbiotic and symbiotic propagation requirements of H. macroceratitis in Florida than that discussed by Stewart and Zettler (2002), the present studies have suggest ed efficient means of both the asymbiotic and symbiotic propagation of the species that will lead to the production of plant material for species reintroductions. 125

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Table 3-1. Comparative mineral sa lt, vitamin, and amino acid content of asymbiotic orchid seed germination media used in the asymbiotic germination of Habenaria macroceratitis : Knudson C (KC), Malmgren Modified Terrest rial Orchid Medium (MM), Murashige & Skoog (MS), Vacin & Went (VW), Lindema nn (LM), Modified Luckes (ML), not present (). KC MM MS VW LM ML Macronutrients (mM) Ammonium 13.82 20.61 3.78 15.14 0.09 Calcium 2.12 0.24 3 0.4 2.12 Chlorine 3.35 3 14.08 Magnesium 1.01 0.81 1.5 1.01 0.49 0.43 Nitrate 10.49 39.4 5.19 2.12 4.89 Potassium 5.19 0.55 20.05 7.03 15.07 203.76 Phosphate 1.84 0.71 1.25 2.24 0.99 199.8 Sulfate 4.91 0.92 1.84 5.3 8.1 Sodium 0.2 0.1 0.1 Micronutrients ( M) Boron 100.27 16.4 Cobalt 0.19 Copper 0.16 0.1 Iron 90 100 183 183 17.96 94.3 Iodine 5 0.6 Manganese 30 10 111.91 33.7 37.63 Molybdenum 1.54 Nickel 0.24 Zinc 53.26 3.5 Vitamins & Amino Acids (mg/l) Biotin 0.05 Casein hydrolysate 400 Folic acid 0.5 Glycine 2 myo-Inositol 100 Nicotinic acid Peptone Pyridoxine Thiamine Total N (mM) 24.31 unpublished 60.01 8.97 17.26 4.98 NH 4 + :NO 3 1.32 unpublished 0.52 0.73 7.14 0.02 126

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Table 3-2. Seed germination and protocorm development stages in Habenaria macroceratitis adapted from Stewart and Zettler (2002) and Stewart et al. (2003). Stage Description 0 No germination, viable embryo 1 Swelled embryo, production of rhizoid(s) (=germination) 2 Continued embryo enlargement, rupture of testa 3 Appearance of protomeristem 4 Emergence of first leaf 5 Elongation of first leaf 127

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Table 3-3. Sources of mycobionts used in the in vitro symbiotic co-culture of Habenaria macroceratitis Isolate Host Collection Information Identification Hmac-309 Vegetative plant 27 September 2003, Hernando Co., FL Epulorhiza sp. Hmac-310 Vegetative plant 27 September 2003, Hernando Co., FL Epulorhiza sp. Hmac-311 Vegetative plant 27 September 2003, Hernando Co., FL Epulorhiza sp. Hmac-312 Flowering plant 30 September 2003, Sumter Co., FL Epulorhiza sp. Hmac-313 Flowering plant 30 September 2003, Sumter Co., FL Epulorhiza sp. Hmac-314 Flowering plant 30 September 2003, Sumter Co., FL Epulorhiza sp. 128

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Figure 3-1. Seed germination and protocorm development stages in Habenaria macroceratitis adapted from Stewart and Zettler (2002). Stage 0 = no germination, viable embryo. Stage 1 = swelled embryo, production of rhizoi ds (arrow; = germination). Stage 2 = continued embryo enlargement, rupture of testa. Stage 3 = appearance of protomeristem. Stage 4 = emergence of firs t leaf. Stage 5 = elongation of first leaf. Scale bars = 1 mm. 129

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Germination Stage Stage 0Stage 1Stage 2Stage 3Stage 4Stage 5 Germination (%) 0 20 40 60 80 100 Germination (%) 0 20 40 60 80 100 a bc c ab c c a bc b c b d a bc c d e 7 weeks 16 weeksML MS LM VW MM KC b a b b a b b ab a a a a a Figure 3-2. Effects of culture media on percent germination and protocorm development of Habenaria macroceratitis after 7 and 16 weeks in vitro asymbiotic culture. Histobars with the same letter are not significantly different within stage ( = 0.05). Error bar = SE. Modified Lucke (ML), Murashige & Skoog (MS), Lindemann (LM), Vacin & Went (VW), Malmgren Modified (MM), and Knudson C (KC). 130

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Germination (%) 0 20 40 60 80 100 + BP BP Stage 0 a a a a a a a a Carbohydrate Treatment FructoseSucroseDextroseControl Germination (%) 0 20 40 60 80 100 Stage 4 0 20 40 60 80 100 Stage 1 a a a a a a a a 0 20 40 60 80 100 Stage 3 a a Carbohydrate Treatment FructoseSucroseDextroseControl 0 20 40 60 80 100 Stage 5 Germination (%) 0 20 40 60 80 100 a b ab ab ab b b b Stage 2 Figure 3-3. Effects of carbohydrate type (fructose, sucrose, a nd dextrose) and presence or absence of banana powder (+/BP) on percent germination and protocorm development of Habenaria macroceratitis after 7 weeks in vitro asymbiotic culture on Malmgren Modified Terrest rial Orchid Medium. Histobars with the same letter within each stage are not significantly different ( = 0.05). Error bar = SE. 131

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Germination (%) 0 20 40 60 80 100 + BP BP Stage 0 0 20 40 60 80 100 Stage 1 Germination (%) 0 20 40 60 80 100 Stage 2 Carbohydrate Treatment FructoseSucroseDextroseControl Germination (%) 0 20 40 60 80 100 Stage 4 bc ab bc c b a Carbohydrate Treatment FructoseSucroseDextroseControl 0 20 40 60 80 100 Stage 5 ab c abc abc a bc bc c 0 20 40 60 80 100 a a Stage 3 Figure 3-4. Effects of carbohydrate type (fructose, sucrose, a nd dextrose) and presence or absence of banana powder (+/BP) on percent germination and protocorm development of Habenaria macroceratitis after 21 weeks in vitro asymbiotic culture on Malmgren Modified Terrest rial Orchid Medium. Histobars with the same letter within each stage are not significantly different ( = 0.05). Error bar = SE. 132

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0 10 20 30 40 50 60 10 3 1 0 Control 2-iP Kin Zea BA( % )C y t o k i n i n C o n c e n t r a t i o n ( M )C y t o k i n i n T y p ea a b bc bcd bcde cdef efg fg fg g g defg Figure 3-5. Effects of four cyto kinins (BA, Zea, Kin, 2-iP) and four concentrations (0, 1, 3, 10 M) on percent seed germination of Habenaria macroceratitis after 14 weeks in vitro asymbiotic culture on Malmgren Modified Terrestrial Orchid Medium. Histobars with the same letter are not significantly different ( = 0.05). Benzyladenine (BA), zeatin (Zea), kinetin (Kin), and 6-( -dimethylallylamino) purine (2-iP). 133

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Developmental Stage Stage 0Stage 1Stage 2Stage 3Stage 4Stage 5 Germination (%) 0 20 40 60 80 100 0/24 h L/D Photoperiod 16/8 h L/D Photoperiod 24/0 h L/D Photoperiod a a a a a a b a a b a b c a a a Figure 3-6. Effects of three photoperiods (0/24, 16/8, 24/0 h L/D) on in vitro asymbiotic seed germination and protocorm development of Habenaria macroceratitis cultured on Malmgren Modified Terrestrial Orchid Medi um after 14 weeks. Histobars with the same letter are not significantly different within stage ( = 0.05). Error bar = SE. 134

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Figure 3-7. Morphological effects of thr ee photoperiods (0/24, 16/8, 24/0 h L/D) on asymbiotically germinated Habenaria macroceratitis seeds cultured on Malmgren Modified Terrestrial Orchid Medium after 14 weeks. A) Protocorm development and morphology under 0/24 h L/D, not e numerous rhizoids. B) Protocorm development and morphology under 16/8 h L/D, note lack of rhizoids. C) Protocorm development and morphology under 24/0 h L/D, note lack of rhizoids. Scale bar = 1 mm. 135

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TubersLeaves Number 0.0 0.5 1.0 1.5 2.0 2.5 3.0 8/16 h L/D Photoperiod 12/12 h L/D Photoperiod 16/8 h L/D Photoperiod bb a a b a Figure 3-8. Effects of three phot operiods (8/16, 12/12, 16/8 h L/ D) on tuber and leaf production per in vitro Habenaria macroceratitis seedling cultured on Malmgren Modified Terrestrial Orchid Medium after 20 weeks. Histobars with the same letter are not significantly different within group ( = 0.05). Error bar = SE. 136

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Tuber and Shoot Biomass Tuber fwtTuber dwtShoot fwtShoot dwt Weight (g) 0 20 40 60 80 8/16 h L/D Photoperiod 12/12 h L/D Photoperiod 16/8 h L/D Photoperiod a b b a b b a b b a ab b Figure 3-9. Effects of three phot operiods (8/16, 12/12, 16/8 h L/ D) on tuber and shoot biomass of in vitro Habenaria macroceratitis seedlings cultured on Malmgren Modified Terrestrial Orchid Medium after 20 weeks. Histobars with same letter are not significantly different within group ( = 0.05). Error bar = SE. 137

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Tuber DiameterTuber LengthLeaf LengthLeaf Width Measurement (mm) 0 2 4 6 8 10 12 14 16 8/16 h L/D Photoperiod 12/12 h L/D Photoperiod 16/8 h L/D Photoperiod a b c a a a a b c a b c Figure 3-10. Effects of thr ee photoperiods (8/16, 12/12, 16/8 h L/D) on tuber diameter and length and leaf length and width of in vitro Habenaria macroceratitis seedlings cultured on Malmgren Modified Terrestri al Orchid Medium after 20 weeks. Histobars with the same letter are not significantly differe nt within group ( = 0.05). Error bar = SE. 138

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Figure 3-11. Examples of mycobionts isolated from Habenaria macroceratitis (Hmac). A) Hmac-309 whole culture morphology at 3 weeks, scale bar = 1 cm. B) Hmac-309 monilioid cells stained with acid fuchsin at 4 weeks (400), scale bar = 10 m. C) Hmac-312 whole culture morphology at 3 weeks, scale bar = 1 cm. D) Hmac-312 monilioid cells stained as above at 4 weeks (400), scale bar = 10 m. 139

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Developmental Stage Stage 0 Stage 1 Stage 2 Germination (%) 0 20 40 60 80 Control Hmac-309 Hmac-310 Hmac-311 Hmac-312 Hmac-313 Hmac-314 a a b ab ab ab ab a ab ab ab ab ab b a ab ab b b b b Figure 3-12. Effects of six mycobionts on perc ent germination and protocorm development of Habenaria macroceratitis cultured on oat meal agar (O MA) after 8 weeks symbiotic in vitro culture. Histobars with the same lette r are not significantly different within stage ( = 0.05). Error bar = SE. 140

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Developmental Stage Stage 0 Stage 1 Stage 2 Germination (%) 0 10 20 30 40 50 60 0/24 h L/D 16/8 h L/D 24/0 h L/D a b c a b b a b b Figure 3-13. Photoperiodic e ffect (0/24, 16/8, 24/0 h L/D) on in vitro symbiotic seed germination and protocorm development of Habenaria macroceratitis cultured on oat meal agar (OMA) with mycobiont Sbrev266 after 14 weeks. Histobars with the same letter are not significantly different within stage ( = 0.05). Error bar = SE. 141

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Figure 3-14. Photoperiodic effect s (0/24, 16/8, 24/0 h L/D) on the in vitro symbiotic protocorm development of Habenaria macroceratitis using mycobiont Sbrev-266 cultured on oat meal agar after 14 weeks. A) Protocorm development under 0/24 h L/D, note rhizoid development. B) Protocorm deve lopment under 16/8 h L/D, note lack of rhizoid development. C) Protocorm deve lopment under 24/0 h L/D, note lack of rhizoid development. Scale bars = 1 mm. 142

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CHAPTER 4 POLLINATION BIOLOGY AND GENETIC DIVERSITY OF Spiranthes floridana Introduction Knowledge of a rare plants reproductive biolog y and genetic diversity are critical factors in its conservation and management. In unde rstanding a species mode of reproductionsexual or asexualattention can be draw n to ecological factors and conser vation needs for that species. Since reproduction is both an important means of increasing the number of individuals within a population and the primary way to maintain gene tic diversity (Sipes and Tepedino 1995), an understanding of both pollination biology and genetic diversity is necessary in measuring the fitness of rare plants. While a number of studi es exist concerning the pollination biology and genetic diversity of orchid species, these two factors are usually treated as separate from one another. Few studies exist concerning the integration of pollinati on biology and genetic diversity in orchids (Sun 1997, 1996; Sun and Wong 2001; Wong and Sun 1999; Sipes and Tepedine 1995). Pollination biology represents an essential part in the conservation and species recovery planning for rare plants. This is particularly important in th e Orchidaceae, since most orchid species have developed highly specialized pollin ation mechanisms (Tremblay 1992; Catling and Catling 1991; Bowles et al. 2002). Members of the Orchidaceae are known to possess a variety of pollination mechanisms and pollinator s (Catling 1983a, 1983b, 1982; Dodson and Frymire 1961; Stoutamire 1975; Darwin 1869); however, pollinat ors are often speciesspecific (Tremblay 1992). Due to pollination mechanism variety an d species-specific pollinat ors, careful study of the pollination biology of orchids is crucial in developing a more complete ecological and conservation understanding of individual species. 143

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Population genetic diversity represents a nother component in developing a better understanding of the conservation biology need s of individual orchid species and their populations. Qamaruz-Zaman et al. (1998a) suggests that the unchecked lo ss of genetic diversity within and between populations can result in th e limiting of adaptive potential in orchid populations. Furthermore, the maintenance of gene tic diversity can be a critical factor in the long-term persistence of plants in a changi ng environment (Frankel and Soul 1981; Lande and Barrowclough 1987). An understanding of withinand between-population ge netic diversity is a critical step in the conservation of plant spec ies (Lande 1988), particularly orchid species. The measurement of population genetic diversity requires the use of molecular marker technologies. Recently, a number of molecula r-based methods have been used in the measurement of orchid population genetic diversity, including isozyme electrophoresis (Sun 1997, 1996), random amplified polymorphic DNA (R APD; Sun and Wong 2001; Wong and Sun 1999), DNA sequencing (Szalanski et al. 2001), and amplified fragment length polymorphism (AFLP; Forrest et al. 2004). The AFLP method re presents one of the mo st attractive tools to measure population genetic divers ity because of the systems reproducibility, the small amount of genomic DNA necessary, and its ability to resolve multiple polymorphic bands (Mueller and Wolfenbarger 1999; Ridout and Donini 1999; Forrest et al. 2004; Chen et al. 1999; Lin et al. 1996). The terrestrial orchid Spiranthes floridana was chosen as the species in these studies because no information exists on the reproductive biology or genetic diversity of this species (Figure 1-3b). Several repor ts exist concerning the reproductive biol ogy (Singer 2002; Sun 1997, 1996; Calvo 1990; Catling 1987; Sheviak 1982; Wong and Sun 1999; Antlfinger and Wendel 1997; Sipes and Tepedino 1995; Schmidt and Antlfinger 1992) and population genetic 144

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diversity (Sun 1997, 1996; Wong and Sun 1999; Sipes and Tepedino 1995) of other Spiranthes and allied Spiranthinae species. In the pres ent studies, data are pr esented concerning the reproductive and pollination biology and within-populati on genetic diversity of S. floridana. Reference is also made to the general plant de mography and ecological ha bitat profile of this species. These studies represent one step in the integrated conservation and species-level recovery of S. floridana in Florida. Materials and Methods Study Sites One site was chosen for these studies: Rayoni er (Bradford County; Fi gure 4-1). At the time of these studies, the Rayoni er site was the only known S. floridana site in the southeastern United States. The Rayonier site was located within 6 km of Starke, Florida at an elevation of 47 m above sea level. Data on associated plant speci es were recorded at th is site yearly from 20032005. Plant Demography In 2003, demographic monitoring of the entire Rayonier popul ation was initiated. From 2003-2005 this monitoring site was visited weekly during the activ e growth cycle of S. floridana (1 March-30 June). Data on number of vegetative and flowering plants, number of leaves per plant, length of longest leaf per plant (cm), number of flowers per plant, height of inflorescence per plant (cm), and number of flowers setti ng seed was collected and averaged per data collection year. These yearly data were then averaged over all data collection years, and analyzed using basic descriptive statistics. Pollinator Observations Non-destructive pollinator observations were conducted at the Rayonier site on 7 and 13 April 2003. The entire Rayonier population of S. floridana was selected for observation 145

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activities because of its small, defined plant distribution at the site. Observation methods followed those of Zettler et al. (1996) for Platanthera integrilabia modified for daytime-only observations based on Catling (1987) for Sacoila lanceolata (Aublet) Garay var. lanceolata a Spiranthinae ally of S. floridana. A circular path around the entire Rayonier population was established that allowed easy observation of 21 individual inflorescen ces. Observations occurred over a 6-hour period on 7 April 2003 beginni ng at 0600 hours and ending at 1800 hours. Observations on 13 April 2003 occurred ove r a 6-hour period begi nning at 0500 hours and ending at 1700 hours. Both obser vation periods occurred without interruption. All data were recorded in fair weather and no insect repellant or insect attractants were used within the study area. Observations occurred in the first and last 20 minutes of each hour during each observation period by walking the preestablished path in a counterclockwise direction around the population. Pollinators were recorded as those insects carryi ng at least one pollinium after a flower visit, while visitors were recorded as those insects la cking pollinia. Relative humidity and temperature were recorded at the Rayonier site during pollinator observati ons using two HOBO H8 data loggers. One data logger was placed at ground level, while the second was placed 25 cm above ground level. This height was chosen to parallel the average inflorescence height of S. floridana at this site. Further, flower nectar volume and sugar concen tration data was not taken for S. floridana since preliminary studies demonstrated this species to not produce nectar as a pollinator reward. Pollination Mechanism A pollination mechanism study was designed to investigate the breeding system of S. floridana following the procedures of Wong and S un (1999), modified by the inclusion of a seventh pollination conditionself-pollination (T able 2-1). The mechanism experiment was conducted at the Rayonier site during the 2004 fl owering period. Four teen plants, two per 146

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experimental pollination condition, with pre-anthesis inflores cences were bagged with a fine plastic mesh (1 mm 2 mesh size) stretched over a 1 m tall wi re frame. The mesh covered wire frame allowed the S. floridana inflorescences to develop normally while excluding pollination events from occurring prior to the initiation of pollination mechanism studies. Each experimental pollination treatment was applied to two flowers on each inflorescence (Table 2-1). A total of 196 flowers were us ed in the pollination mechanism study. However, during application of pollination treatments the lack of pollen production in flowers of S. floridana was discovered and the pollination mechanis m study terminated. Thereafter, the meshcovered wire frames were removed from all plants. Sampling, DNA Extraction, and Amplified Fr agment Length Polymorphism (AFLP) Fresh, green leaves of S. floridana were collected from th e Rayonier site in 2003 and placed in 50 mL plastic tubes containing sili ca gel desiccant (Chase and Hills 1991; W.M. Whitten personal communication). Leaf samples we re removed from each plant using scissors, which were washed with 95% ethanol and allowe d to air dry between each sample to minimize sample cross contamination (M.W. Whitten persona l communication). All 23 plants present in 2003 were sampled. Samples were stored at ro om temperature (ca. 25 C) until used in DNA extraction protocols. Once all samples were thoroughly dried, ge nomic DNA was extracted using the DNeasy Plant Mini Kit. Manufacturers instructions were followed with the following modifications: eluates were not pooled and the second elution wa s retained only as a precaution. The DNA was quantified using an Agilent Technologies NanoDrop spectrophotometer. Template DNA was prepared usi ng the Applied Biosystems AFLP Plant Mapping protocol and kit-based system (A BI 2005). Preselective and sele ctive amplification procedures were identical to those discussed in Chapter 2 for Habenaria macroceratitis and used the same 147

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endonucleases, primer combinations, and PCR cond itions (Table 2-2). AFLP reaction samples were prepared for sequencing in the same manner as previously outlined, and sequencing conditions were identical. As with the AFLP fragment data from H. macroceratitis, a matrix containing all fragments data from S. floridana ranging from 50 to 500 bp was compiled. These data were analyzed using GeneMarker software. Fragements with a low si gnal (<2% full detecti on level) were excluded from the analysis. Recognized bands were scored as present (1 ) or absent (0). POPGENE 1.31 (Yeh et al. 1999) was used to estimate Nei and Shannon diversity indices. Within and between populat ion genetic structure ( F ST ) was estimated using a combination of results generated with POPGENE and equation calculations with 1000 pe rmutations. Genotype correlation dendrograms were constructed usi ng GeneMarker version 1.6 software and the included cluster analysis tool. Results Study Site Before development into a rural home site, th e Rayonier study site would have likely been classified as a pine flatwood (C hafin 1990; S.L. Stewart personal observation). An adjacent pine flatwood dominated by longleaf pine ( Pinus palustris Mill) remains and provides the only indication to the sites ecological history. Presently, the Rayo nier site is treeless, with the exception of one remnant longleaf pine, and dominated by Common Bermudagrass (Cynodon dactylon (L.) Pers.). The site also c onsists of a small home, rock driveway and parking area, and a moderately-sized barn. Plant Demography From 2003-2005, the number of S. floridana plants at the Rayonier study site ranged from 17 to 32 (ave. = 23.8 .52). In each study year, 100% of the plants flowered and no vegetative 148

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plants were observed in any study year. No seedling plants were observed during the study period. Plants ranged in height from 20.3 cm to 36.8 cm (ave. = 28.7 .35). During study years 2003-2005, the number of flowers per plant ranged from 6 to 31 (ave. = 17.9 .53), while the number of flowers appearing to set seed ranged from 0 to 17 (ave. = 7.2 .45). Leaf number and length of longest leaf during study years 2003-2005 ranged from 0 to 4 (ave. = 1.6 .22) and 0.25 cm to 3.8 cm (ave. = 2.6 .24), respectively. Pollinator Observations and Pollination Mechanism No pollinators or visitors of flowers of S. floridana were noted during either the 7 April 2003 or 13 April 2003 pollination observation periods at the Rayonier site. Furthermore, no floral scent was noted at any time during the pol linator observations. Te mperature at the site during observations ranged be tween 21.5C-29.9C (ave. = 26.4C; 7 April; Figure 4-2a) and 9.4C-29.4C (ave. = 22.7C; 13 April; Figure 43b). Site relative humidity ranged between 54%-93% (ave. = 67.5%; 7 April; Figure 4-2a) and 23%-97% (ave. = 47.8%; 13 April; Figure 43b). During the application of experimental pollina tion treatments, it was noted that flowers of S. floridana at the Rayonier study site l acked pollinia despite having a fully formed anther cap and pollinial depression (Figure 4-4a-c). Further observations of 20 randomly selected flowers in various stages of development (tight bud to fully open) from the Rayonier study site revealed the same lack of pollinia in all flowers sampled. Spiranthes floridana at the Rayonier site appears to not produce pollinia, although ovaries do enlarge after each flower matures. Based on these observations, it appears that S. floridana reproduces asexually by agamospermy; however, immature seed capsules abort approximately thre e-fourths through the maturation process. This phenomenon was observed in 100% of capsules (Figure 4-4d). 149

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AFLP Data Amplification of all four primer pairs tested was successful in all 23 individual samples. The output gel image is given in Figure B-3. Tthe -ACT/-CAG and AGC/-CAG primer pairs resulted in the highest polymorphic band resolution, averaging 37 and 56 bands per individual, respectively. Bands that were either present or absent in a single sample were excluded from analysis as likely being artef actual (Pillon et al. 2007). From both primer pairs, a total of 36 unambiguous polymorphic bands were selected. When combining these 36 bands, 4 genotypes could be distinguished. For this population of S. floridana, Neis diversity index = 0.293 and Shannons index = 0.165. In examining the within population genetic differentiation, F ST = 0.03, suggesting very little differentiation within the sampled population. A genotype correlation dendrogram is given in Figure B-7. Discussion Information concerning the demography, pollination biology, and population genetic diversity of S. floridana has not been previously reported. Re sults from these studies suggest the species is artificially imperiled due to the lack of a breeding system, lo w genotypic diversity, and low genetic differentiation w ithin the one population sample d. Reports concerning the demography and ecology of Spiranthes species, or that of any other Spiranthinae species, are limited (Calvo 1990; Hutchings 1987a, 1987b, Tamm 1972; Wells 1967; Willems and Dorland 2000; Jacquemyn et al. 2007). The majority of these ecological reports are in support of taxonomic descriptions or revisions (Catling 1981, 1983; Sheviak 1982; Catling and Cruise 1974) or biosystematic studies of unique region al ecotypes (Sheviak 1982). Demographic and life history trait data represent important factors that may cont ribute to the understanding of evolutionary processes (Willems and Dorland 2000) This lack of basic demographic data on 150

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Spiranthes species, especially rare a nd endemic species such as S. floridana has led to a lack of understanding of life history tr aits in this orchid genus. Jacquemyn et al. (2007) reporte d on the long-term population dynamics and viability of the European terrestrial orchid S. spiralis (L.) Chevall., a related species to S. floridana (Dueck and Cameron 2007) over a 24 year period. Like S. floridana at the Rayonier study site, S. spiralis is known from open meadows where land-use is constant. Despite these similarities, S. spiralis demonstrated extreme variability in the percentage of flowering individuals per year (0-100%) and the total number of indi viduals per year (6-79). Spiranthes floridana showed a high degree of stability in both the total number of individuals per monito ring year (23.8 .52) and the percentage of flowering plants per year (100%); however, this stability is somewhat irrelevant since no reproduction or recruitment is present. Jacquemyn et al. (2007) suggest that the plasticity seen in total indi viduals and flowering versus vegetative growth habits in S. spiralis is advantageous to the long-term viability of S. spiralis populations. According to their calculations of extin ction probabilities, S. spiralis has a 79% probability of surviving the next 20 years. While an extinction probability was not calculated for S. floridana in Florida, the lack of population plasticity measured in the Rayonier population suggests that S. floridana would have a low probability of surviving over the next 20 years. Willems and Dorland (2000), studying the flow ering frequency and plant performance of S. spiralis reported a decrease in the percentage of flowering pl ants every year over five consecutive flowering years. Th ey suggest that this decrease in flowering plant percentage is due to the negative costs of re petitive reproduction of individual plants year-after-year. A similar trend in generative reproduct ion costs has been reported in Cypripedium acaule Aiton (Primack and Hall 1990; Primack et al. 1994) and Tipularia discolor (Snow and Whigham 151

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1989). In the present studies with S. floridana, while the total number of plants per year decreased at the Rayonier study s ite, the percentage of flower ing plants never decreased. Interestingly, those plants that reappeared in each study year flowered every study year. This observation does not follow the trend of a high co st of successive generational reproduction from year-to-year that is seen in most orchid species (Calvo 1993). The number of S. floridana plants present at the Rayonier study site decreased by 54% during the study period. This decrease could be at tributed to plant mortality, in some cases; however, it is possible that some plants transitioned to an underground dormant life stage. Unfortunately, a more detailed life history of S. floridana is not available. Calvo (1990) reported a similar phenomenon in the Spiranthinae orchid Cyclopogon cranichoides (Grisebach) Schl., where populations declined by an average of 20% each year and this decline was mostly attributed to plant dormancy. Further study concerning the underand above-ground life history of S. floridana is necessary; however, without new populations these studies can not be afforded without further damage to the existing populations. Breeding system, and therefore pollination bi ology, has a profound effect on not only the long-term population viability of plants, but also a profound effect on population genetic diversity (Hamrick 1982). Unfortunately, poll ination biology in the Orchidaceae is often considered understudied despite a renewed in terested over the past decade (Catling 1990; Trapnell and Hamrick 2006). Tropical orchid species have received the most pollination biology attention in recent years (Blanco and Bar boza 2005; Trapnell and Hamrick 2006, 2005; Singer and Koehler 2003; Borba et al. 2001). However, the temperate orchid subtribe Spiranthinae has been the focus of a great deal of pollina tion biology research (Catling 1987, 1986, 1983, 1982; Sipes and Tepedino 1995; Schmidt and Antlfinger 1992), particularly in the relationship between 152

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breeding system and population genetic di versity (Sun 1997, 1996; Sun and Wong 2001; Wong and Sun 1999). A number of Spiranthinae species are known to require pollinia transfer to achieve germination: Sauroglossum elatum Lind. (Singer 2002), Spiranthes diluvialis Sheviak (Sipes and Tepedino 1995), and Goodyera procera (Ker-Gawl.) Hook (Wong and Sun 1999). However, the greater number of Spiranthinae are known to be agamospermic: Sacoila lanceolata var. lanceolata (Catling 1987), S. lanceolata var. paludicola (Luer) Sauleda (Catling 1987), Spiranthes australis (R. Brown) L. (Ridley 1888), S. casei Catling & Cruise (Catling 1982), S. casei var. novaescotiae Catling (Catling 1982), S. cernua (L.) L.C. Richard (Sheviak 1982; Schmidt and Antlfinger 1992), S. hongkongensis Hu & Barr. (Sun 1997), S. magnicamporum Sheviak (Catling 1982), S. ochroleuca (Rydb. Ex Britton) Rydb. (Catling 1982), S. odorata (Nutt.) Lind. (Catling 1982), S. ovalis var. erostellata Catling (Catling 1983), S. parksii Correll (Catling and McIntosh 1979), S. prasophylla Reichb. var. cleistogama Ames & Correll (Ames and Correll 1952). Therefore, the determination of agamospermy as the breeding system in S. floridana was expected. Based on field observation of S. floridana plants at the Rayonier study site, ovaries began to swell im mediately upon flower maturation. When combined with the lack of observed pollinators, these data suggest an agam ospermic breeding system similar to that seen in other North American Spiranthes species (Sheviak 1982; Schmidt and Antlfinger 1992). The observation of the lack of pollinia production in S. floridana is of great interest. A similar report does not exist for any orchid spec ies. Catling (1991) reports a number of orchid species as being agamospermic, particularly in the Spirantheinae, with most agamospermic species reproducing by adventitious embryony. However, there is no mention of the absence of pollinia in these flowers. The root cause of this morphological and reproductive abnormality is 153

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unknown at this time. Hypothetically, extreme inbreeding depression has been shown to increase the probability of a population being ho mozygous and, therefore, less fit (Gillespie 1998). Examples of the effects of extreme i nbreeding depression in plants are rare and understudied; however, Morton et al. (1956) dem onstrated the effects of inbreeding on the reproductive viability of young children. In thei r study, the long-term viability of the study subjects decreased as their inbr eeding coefficient increased. Fu rthermore, in a study examining the effects of mice inbreeding over 20 generatio ns, Connor and Bellucci (1979) demonstrated a significant decrease in both mice litter size and percent of mice progeny surviving over generational time. The lack of pollinia seen in S. floridana could be attributed to a historically high inbreeding coefficient for this population that resulted in a highly homozygous population that is now incapable of purging deleterious alleles from its genome. The phenotypic, morphologic, and reproductive result of this extreme inbreeding is the lack of pollinia production and agamospermic capsule abortion during matura tion. A more detailed molecular-based study of the reproductive a nd molecular ecology of S. floridana is necessary to elucidate this hypothesis. AFLP technology has been demonstrated as a powerful tool in acquiring information concerning within and between population genetic di versity in plants (for review see Meudt and Clarke 2007). The present study represents the fi rst investigation of the genetic diversity in a population of the endemic Fl orida terrestrial orchid S. floridana using AFLP technology. As a genus, Spiranthes species are often consid ered colonizing orchids w hose presence is often representative of a degree of ha bitat disturbance (S.L. Stewart personal observation). However, orchids are poorly represented in the literature on colonizing plan ts and little is known about the genetics of these so-called colonizing orchid taxa. Spiranthes floridana would likely not be 154

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considered a colonizing orchid given the low total number of plants a nd populations despite apparent suitable natura l and disturbed habitat. A number of reports exist concerning population genetic diversity measures in Spiranthes and other spiranthoid orchids. Sun (1997) reported a lack of allozyme diversity in Eulophia sinensis Miq. (a non-spiranthoid orchid), Spiranthes hongkongensis, and Zeuxine stratemactica (L.) Schl. (both spiranthoid or chids) both within and between populations of these native Hong Kong orchids. However, all the orchids survey ed by Sun (1997) represen t widespread species within their resp ective ranges and ar e reproductively fitE. sinensis being insect-mediated selfcompatible, S. hongkongensis being self-pollinating, and Z. stratemactica being apomictic unlike S. floridana. In a similar study, Sun and Wong (2001) reporte d high genetic diversity in the orchids Z. gracilis Lindl. and E. sinensis using RAPD markers, and low genetic diversity in Z. stratemactica Differences among the breeding systems of these orchids are suggested as the primary reason behind the observed differences in genetic diversity. For example, the low genetic diversity observed in Z. stratematica may be attributable to its apomictic breeding systemwhere no new allelic dive rsity is introduced from genera tion-to-generation. Over time, this apomictic breeding system would lead to lo w levels of within-population genetic diversity, but potentially high levels of between-populati on genetic diversity. In the present study, S. floridana possesses low within-population genetic diversity; however, between population diversity could not be measured since only one population was known at the time of sampling. Inclusion of the newly-discovered Duval County (Florida) population in a study of the betweenpopulation genetic diversity of this species would be helpfu l. It is possible that S. floridana may have high between population diversity, but this diversity would likely be due to population 155

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fragmentation and isolation and not due to the br eeding system of the species, as suggested for Z. stratemactica Forrest et al. (2004) reported on the population genetic stru cture of European populations of the terrestrial orchid S. romanzoffiana Chamisso, finding differentiation between the northern and southern populations of this species. Within the northern populations, genotypic diversity was high and considered typica l of sexually reproducing plants. However, the southern populations showed only 12 genotypes among six popul ations. This low genotypic diversity was suggested as being consistent with the agamospermous or autogamous breeding system of these southern plants. Given that a number of Spiranthes species are known to be agamospermic, the low genotypic diversity and lo w within population genetic diffe rentiation measured in the population of S. floridana could be a result of long-term agamospermic breeding system. The agamospermic breeding system could have re sulted in inbreeding de pression, a genetic bottleneck, and/or the maintenance of unwanted deleterious alleles within this one small, isolated population of S. floridana (Ellstrand and Elam 1993). Over generations, the combination of these factors could have resulted in the lack of breeding system presently seen in this species (Nei 2005; King 1967; Kimura and Crow 1964; Nei et al. 1975). Higher-order molecular-based analysis of these factors within S. floridana in Florida is necessary to better determine the root cause of the apparent lack of breeding system. The use of functional molecular markers (i.e., gene targeted markers (GTMs)), as opposed to random markers (i.e., AFLP), would contribute a great deal to the cons ervation genetics of S. floridana (Andersen and Lbberstedt 2003). The interpretation of AFLP population genetic diversity data in a context of species management and conservation planning for S. floridana is problematic. The low genotypic diversity and low genetic differentiation within the popul ation of this species coupled with the 156

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lack of a fit breeding system could be interpreted as factor s leading to the extinction of S. floridana. Further searches of this sp ecies historic habitat, the sout heastern coastal plain of the United States, should be conducted to determine th e species absolute range status. Dueck and Cameron (2007) validated the separation of S. brevilabris and S. floridana as two unique species, as proposed by Brown (2001). Finally, further molecular-based populatio n genetic diversity studies should be conducted that include the newly discovered (2005) s econd Florida population of S. floridana, as well as any other new populations. Implications for Integrated Conservation The present studies on the demography, pollination biology, and population genetic diversity of the endemic Florida terrestrial orchid S. floridana have demonstrated the need to understand these ecological factor s and their relation to conser vation and species recovery planning. Studies such as these highlight the need to integrate field-base d ecological data with both reproductive biology and molecular-geneti c data to determine ecologically functional models of species-level plant conservation. This is particularly important when conserving an endemic orchid species because most orchid s have developed highly specific ecological partnerships with pollinators, hab itat, and fungi. For example, identifying the breeding system of S. floridana as agamospermic, determining both the lack of pollinia production and capsule abortion, and measuring low genotypic and with in-population genetic di fferentiation could all demonstrate a trend toward low population viability for S. floridana in Florida. This may lead those interested in the conservation of this Spiranthes species to consider the species as headed toward extinction and in need of immediate c onservation attention (Stewart and Kane 2006c). Conservation efforts for most Spiranthes species focuses on the preservation of suitable habitat to help insure the long-term population re productive viability of the species (S.L. Stewart personal observation). However, this conservatio n model does not apply to an orchid such as S. 157

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floridana that apparently produces no viable offspri ng. In identifying the la ck of a reproductive mode in S. floridana, the question arises, how best do we de velop a conservation system for this species? Previous studies have demonstrated that S. floridana is highly preferential for Ceratorhiza mycobionts despite sharing a similar hab itat type with and a genetic relation to S. brevilabris which prefers Epulorhiza mycobionts (Dueck and Cameron 2007; Stewart and Kane 2007a). Given that apparent reproductive sterility and high mycobiont preference shown in S. floridana, simply preserving more suitable habitat will not insure the long-term viability of S. floridana populations in Florida. The difficulty in conservation planning for S. floridana appears to be considerable. However, due to the application of integrated conservation methods those interested in the longterm conservation of this species now have a more complete understand ing of the challenges faced in conserving this species. Traditio nal approaches to the conservation of Spiranthes species in North America woul d have overlooked the aforementioned details of the breeding system, pollination biology, and population genetic diversity of S. floridana. These oversights could have resulted in a misguided and misapplied conservation and species-recovery plan for S. floridana in Florida. The current studies concerning the demogra phy, pollination biology, and genetic diversity of S. floridana in Florida represent on step in the speci es-level integrated conservation of this rare, endemic terrestrial orchid (Figure 1-1). In adding to the body of knowledge concerning the conservation biology of this species, the presen t studies not only promote the conservation of S. floridana, but also promote sound conservation and ma nagement planning of other plant species throughout Florida. 158

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Figure 4-1. Rayonier study site for Spiranthes floridana in Bradford County, Florida. 159

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Date/Time 7 A p r 0 6 0 0 7 A p r 0 7 0 0 7 A p r 0 8 0 0 7 A p r 0 9 0 0 7 A p r 1 0 0 0 7 A p r 1 1 0 0 7 A p r 1 2 0 0 7 A p r 1 3 0 0 7 A p r 1 4 0 0 7 A p r 1 5 0 0 7 A p r 1 6 0 0 7 A p r 1 7 0 0 7 A p r 1 8 0 0 Relative Humidity (%) 50 60 70 80 90 100 Temperature (C) 20 22 24 26 28 30 32 A B Figure 4-2. Temperature and re lative humidity profiles at Ra yonier site (Bradford County, Florida) 7 April 2003 during Spiranthes floridana pollinator observations. A) Temperature (C) and B) relative humidity (RH; %). 160

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Date/Time 1 3 A p r 0 5 0 0 1 3 A p r 0 6 0 0 1 3 A p r 0 7 0 0 1 3 A p r 0 8 0 0 1 3 A p r 0 9 0 0 1 3 A p r 1 0 0 0 1 3 A p r 1 1 0 0 1 3 A p r 1 2 0 0 1 3 A p r 1 3 0 0 1 3 A p r 1 4 0 0 1 3 A p r 1 5 0 0 1 3 A p r 1 6 0 0 1 3 A p r 1 7 0 0 Relative Humidity (%) 0 20 40 60 80 100 Temperature (C) 5 10 15 20 25 30 35 B A Figure 4-3. Temperature and re lative humidity profiles at Ra yonier site (Bradford County, Florida) 13 April 2003 during Spiranthes floridana pollinator observations. A) Temperature (C) and B) relative humidity (RH; %). 161

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Figure 4-4. Lack of pollinia in Spiranthes floridana A) Flower of Spiranthes floridana before dissection, scale bar = 1 mm. B) Frontal view of column from S. floridana showing anther cap (AC), pollinial depression (PD), and column tip (CT) with no pollinia present, scale bar = 0.1 mm. C) Column of S. floridana showing anther cap (AC) and column tip (CT) with no pollinia present, scale bar = 0.1 mm. D) Aborted capsule/ovary (AO) and dried flower (FL) of S. floridana, scale bar = 1 mm. 162

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CHAPTER 5 SEED CULTURE OF Spiranthes brevilabris AND DEEP SOUTH S. cernua Introduction In nature, orchids consume naturally-occurring endophytic mycorrhizal fungi as sources of carbohydrates, nutrients, and water through the ac tion of mycotrophy. The digestion of these mycobionts and subsequent uptake of nutrients by the immature orchid embryo stimulates seed germination, protocorm development, and seedling growth (Arditt i 1966; Clements 1988; Rasmussen 1995). For this reason, the survival of orchids in managed or restored habitats may require the presence of appropriate mycobionts to support plan t development and subsequent seedling recruitment (Zettler 1997b). Symbiotic seed co-culture techniques represent an efficient way to promote the orchid-fungus parasitism under in vitro conditions, as well as to study in vitro orchid-mycobiont preference (Zettler 1997a, 1997b; Stewart and Kane 2006b, 2007a, 2007b). While a number of symbiotic co-culture prot ocols exist for terrestr ial orchid taxa, their germination efficiency is often lower than expected (Anderson 1991, 1996; Stewart and Kane 2006b, 2007a; Stewart and Zettler 2002; Zelmer and Currah 1997; Zettler and Hofer 1998; Zettler and McInnis 1992; Sharma et al. 2003; Stewart et al. 2003; Zettler et al. 2001; Zettler et al. 2005), especially when compared to asymbiotic germination studies with the same taxa. This low seed germination efficiency is likely due to a degree of preference many terrestrial orchids appear to have for mycobionts at the time of germination versus during later life stages. However, this preference has apparently been mostly overlooked by previous symbiotic coculture practitioners. Mycobiont preference has been shown to play an important role in symbiotic orchid propagation, and is thought to pl ay a critical function in the establishment of orchids into field sites (Zet tler 1997a, 1997b; Stewar t et al. 2003; Batty et al. 2006a, 2006b). 163

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Orchid-mycobiont preference has been considered controversial for many years. Many researchers have considered the orchid-fungus re lationship to be opportuni stic and non-specific both in vitro and in situ (Knudson 1922; Curtis 1939; Hadl ey 1970; Masuhara and Katsuya, 1989; Masuhara et al. 1993). Differences in orchid-fungal preference have been identified under in vitro versus in situ conditions (Bidartondo and Bruns 2005; Masuhara and Katsuya 1994; Taylor and Bruns 1999; Taylor et al. 2003), and these differences have led some to consider orchid-mycobiont preference as generally lo w (Hadley 1970; Stewart and Zettler 2002). However, others have suggested th at preference, especially under in vitro conditions, is surprisingly high (Clements 1988; Rasmussen and Rasmussen 2007; Smreciu and Currah 1989; Stewart and Kane 2006b, 2007a, 2007b; Taylor and Bruns 1997; Mc Kendrick et al. 2002; Selosse et al. 2002; McCormick et al. 2006). In th e most general sense, it appears that orchidmycobiont preference may be genus or species specific. The terrestrial orchids Spiranthes brevilabris and S. cernua (Deep South race) were chosen for this study because li ttle information exists on the in vitro symbiotic co-culture of these taxa, and these species represent the rarest members of the Spiranthes genus in Florida (Figures 1-3, 1-4). Previously, Stewart et al. (2003) proposed a symbiotic co-culture protocol for S. brevilabris using mycobionts isolated from the study species and the Florida epiphytic orchid Epidendrum magnoliae Mhlenberg var. magnoliae (syn. = E. conopseum ). While investigating a successful co-culture protocol, St ewart et al. (2003) suggested that S. brevilabris is nonpreferential for mycobionts based on in vitro germination tests. However, their study only utilized a mycobiont from a distantly-related epiphytic species to investigate mycobiont preference; while the use of mycobionts from a closely-rela ted taxon would have better represented the dynamics of mycobiont species in S. brevilabris 164

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In the present studies, the in vitro symbiotic co-culture a nd potential for mycobiont preference by S. brevilabris and the Deep South race of S. cernua are investigated. A description and tentative identification of all mycobionts utili zed during these studies are provided. Finally, the role of mycobiont specificity in the dist ribution, current ecologica l status, and long-term conservation of both S. brevilabris and S. cernua is given consideration. Materials and Methods Fungal Isolation and Identification Four mycobionts were chosen for in vitro symbiotic co-culture and mycobiont preference trials in S. brevilabris and two mycobionts for S. cernua (Tables 5-1, 5-2). Mycobiont Sbrev266 was previously isolated by Stewar t et al. (2003) on 30 April 1999 from S. brevilabris while mycobiont Econ-242 was previously isolated by Zettler et al. (1999) on 7 June 1995 from Epidendrum magnoliae (syn. = E. conopseum ). Spiranthes floridana mycobionts were isolated on 28 April 2004 following the procedure outlined by Stewart et al. (2003) for Florida Spiranthes species, modified by taking only root sections and not entire flowering plants due to the rarity of the species. Only five plants, representing 20% of the total population, were sampled because of the small size of this population. The D uval County (Florida) population was not sampled because, at the time of sampling, only two plants were known from this site. Mycobionts were not isolated from the roots of the Deep South race of S. cernua because of the small size of the known population (<5 plants) and permitting limitations at the time of this study. Root systems of five adult flowering plants of S. floridana at the Bradford County, Florida population were carefully excavated and root sections (<10 cm) were removed. The root sections were wrapped in paper towels moistened with sterile deionized wa ter, placed in plastic bags, stored in darkness at ca. 10C, and transported to the laborat ory (<4 h). Root sections were rinsed with cold tap water to remove surface debris, and surface cleansed 1 min in a solution 165

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containing 5 mL ethanol (100%), 5 mL 6.00% NaOCl, and 90 mL sterile DI water. Clumps of cortical cells containing fungal pelotons we re removed, placed on CMA supplemented with 50 mg l -1 novobiocin sodium salt and incubated at 25C for 4 days. Hyphal tips were excised from actively growing pelotons and s ubcultured onto 1/5 PDA. The pH of all previously mentioned media was adjusted to 5.8 with 0.1 N KOH prio r to autoclaving at 117.7 kPa and 121C for 20 min. Mycobionts showing cultural ch aracteristics similar to those orchid endophytes previously described in the litera ture (Zettler 1997b; Currah et al. 1987, 1997; Richardson and Currah 1993; Stewart et al. 2003; Zelmer et al. 1996) were assigned a reference number and stored at 10C on OMA. Isolates were also stored on 1/5 PDA in continual darkness at 25 C until use in seed germination experiments. Mycobiont characterization and identificati on followed methods described by Davidson (1938), Smith (1977), Zelmer and Currah (1995), Currah et al. (1987, 1990, 1997), Zelmer et al. (1996), and previously in this work for cult ural morphology, polyphenol oxidase production, and celluase activity. Hyphal and monilioid cell characteristics were assessed using a Nikon Labophat-2 light microscope fitted with a Nikon Coolpix 990 digital camera. Fungal staining procedures followed those outlined by Phillips and Hayman (1970) modified by the use of acid fuchsin as the stain (Stevens 1974; J. Kimbrough personal communication). Seed Collection and Sterilization Seeds of S. brevilabris were collected prior to capsule dehiscence from mature fruits on 17 April 2005. Seeds were collected from a ro adside population in Levy County, Florida. Immediately following collection, capsules were dried over silica gel de siccant for 2 weeks at 25C, followed by storage at -10C in darkness for 192 days. Prior to the initiation of in vitro cultures, seed viability was visually assessed using the methods of Stewart et al. (2003). Viable 166

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seeds were considered those s eeds containing a distinct, rounded, and hyaline embryo. Seeds of S. floridana could not be obtained because the species apparently abor ts seed capsules soon after pollination (S.L. Stewart personal observation; Chapter 4). Seeds of the Deep South race of S. cernua were obtained from matu re capsules prior to dehiscence on 12 December 2004. Because of the small size of individual S. cernua seed capsules, one entire inflorescence was collected from a roadside site in Apalachicola National Forest (Liberty County, Florida). Immediately af ter collection, capsules were dried over silica gel desiccant for two weeks at 25C, separated from capsule material, pooled, and immediately used in in vitro experiments. In preparation for in vitro germination experiments, seed s of both species were surface disinfected for 45 seconds in a solution containing 5 mL ethanol (100%), 5 mL 6.0% NaOCl, and 90 mL sterile DI water. Following surface disi nfection, seeds were rinsed three times for 30 seconds each in sterile DI water. Solutions we re removed from the seed surface disinfection vial using a sterile Pasture pipette that was replaced after each use. Sterile DI water was used to suspend the surface disinfected seed, and a steril e inoculating loop was used to sow the seed. Symbiotic Co-Culture Seeds of both S. brevilabris and Deep South S. cernua were sown according to the procedures described by Stew art et al. (2003) for Florida Spiranthes species. Seeds were removed from cold-dark storage, allowed to warm to room temperature (ca. 25C), surface disinfected for 1 minute in the solution describe d previously, and placed on the surface of a sterile 1 cm 4 cm filter paper strip within a 9 cm diameter Petri plate containing 25 mL OMA. Germination medium pH was adjusted to 5.8 w ith 0.1 N HCl prior to au toclaving at 117.7 kPa and 121C for 40 min. Seeds were transferred to the filter paper strips us ing a sterile bacterial inoculating loop. An average of 80 ( S. brevilabris ) and 107 ( S. cernua ) seeds per Petri plate 167

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were sown. Each plate was inoculated with a 1 cm 3 block of fungal inoculum and only one mycobiont per plate. Ten replicate plat es per mycobiont were inoculated with S. brevilabris seed, while five replicate plates per mycobiont were inoculated for S. cernua. Plates without mycobiont served as controls. Plates were sealed with one layer of Nescofilm and maintained in darkness (0/24 h L/D) for 86 days (S. brevilabris ) and 27 days ( S. cernua) at 25 C. Plates were examined weekly during dark incubation for signs of germination or contamination, exposing the seeds to brief (<10 min) periods of illumination. Plates were returned to experimental conditions after vi sual inspection. Seed germina tion and protocorm development were assessed every weekly after the start of dark incubation using a dissecting stereomicroscope. Germination and seedling growth and developm ent were scored on a scale of 0-5 (Table 32; Figure 3-1; Stewart et al. 2003). Seed germination percentages were based on viable seeds determined by visual inspection with the aid of a dissecting stereomicroscope. Germination percentages for each developmental stage were ca lculated by dividing the number of seeds in that particular germination and development st age by the total number of viable seeds in the sample. Data was analyzed using general lin ear model procedures and Waller-Duncan mean separation at =0.05 by SAS v 8.02 (SAS 1999). Germinati on counts were arcsine transformed to normalize variation. Results Fungal Mycobionts: Spiranthes brevilabris Three mycobionts were rec overed from root sections of flowering plants of S. floridana (Table 5-1; Figure 5-1). All three mycobionts were identified as members of the anamorphic genus Ceratorhiza. Isolates Sflo-305 and Sflo-306 tested cellulase-negative, which is a typical Ceratorhiza -like response (Zelmer and Cu rrah 1997). Isolate Sflo-308 te sted cellulase-positive. 168

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All three isolates tested polyphe nol oxidase-negative, which typi cally is indicative of orchid mycobionts from the form genus Epulorhiza However, a rapid averag e daily growth rate (Sflo305 = 11.3 mm d -1 Sflo-306 = 10.6 mm d -1 Sflo-308 = 12.1 mm d -1 ) and the production of large, broadly-connected barrel-shaped monilioid cells (Sflo-305 = 36 24.8 m, Sflo-306 = 39.6 21.6 m, Sflo-308 = 39.2 29.2 m) support the tenta tive identification of these isolates as Ceratorhiza species (Figure 5-3). Only superficia l differences in cult ural morphology were identified among the group of three mycobionts. Isolate Sflo-305 formed smaller and more numerous loose aerial scle rotia than either isolate Sflo-306 or Sflo-308, whereas isolate Sflo-306 formed more irregularly-shaped aerial sclerotia. Isolate Sbre v-266 was previously recovered from the roots of S. brevilabris and identified as a strain of Epulorhiza repens (Bernard) Moore (Table 5-1; Figure 5-1; Stewart et al. 2003). Fungal Mycobionts: Deep South Spiranthes cernua The two mycobionts (Econ-242 and Srev -266) utilized in the study of the in vitro coculture of the Deep South race of S. cernua were previously isolated and identified by Zettler et al. (1998) and Stewart et al. (2003). Both mycobionts were al so identified as strains in the anamorphic fungal genus Epulorhiza (Table 5-2; Figure 5-2). Isolate Sbrev-266 has been identified as a strain of E. repens a ubiquitous global orch id endophyte species complex (Stewart 2007; Zelmer 2001; Stewart et al. 2003; Currah et al. 1987). Mycobiont Econ-242 has been tentatively identified as a strain of Epulorhiza likely closely allied with the E. repens species complex (Stewart et al. 2003). Symbiotic Co-Culture: Spiranthes brevilabris Seeds in all mycobiont treatments began to swell within 2 we eks after sowing, and germination commenced within 3 weeks. The visual contamination rate of cultures from 169

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bacterial and non-mycorrhizal fungi wa s 1%. Visual inspection revealed S. brevilabris seeds to be 94.6% viable. Seeds of this species were monoembryonic. An effect of mycobiont strain was found on the in vitro symbiotic co-culture of S. brevilabris Germination after 3 weeks was highes t when seeds were inoculated with mycobionts Sflo-305, Sflo-306, and Sflo-308 (99.5%, 99.5%, 89.9% resp ectively; Figure 5-3). However, these mycobionts only promoted seed germination to Stage 1, while isolate Sbrev-266 supported Stage 2 germination ( 32.4%) after 3 weeks da rk incubation (Figure 5-3). Mycobionts Sflo-305 and Sflo-306 did support Stage 2 germin ation after 10 weeks da rk incubation, but only to a minimal percentage (10.6%, 0.6% respectively ; Figure 5-3). In contrast, mycobiont Sbrev266 supported a maximum of Stage 5 germination (46.2%) after 10 weeks dark incubation (Figure 5-5). After a total of 12 weeks dark incubation, only mycobiont Sbrev-266 supported germination to an advanced developmental stage (i.e., Stage 3 or greater; Figures 5-3, 5-4). Control treatments supported only Stage 1 germination after a total of 12 weeks dark incubation (Figure 5-3). Symbiotic Co-Culture: Deep South Spiranthes cernua Seeds in each mycobiont treatment began to swell within one w eek after sowing, and germination commenced within one -and-a-half weeks. No visual contamination was evident in any cultures. Visual insp ection revealed Deep South S. cernua seeds to be 63.2% viable. Seeds of this species were monoembryonic. An effect of mycobiont strain was found on the in vitro symbiotic germination of Deep South S. cernua. Germination (Stage 2+) occurred in both the Econ-242 and Sbrev-266 treatments after 4 weeks dark incubation (0.2% and 3.9%, respectively; Figure 5-5). However, protocorm development after 4 weeks was highest when seeds were inoculated with mycobiont Econ-242 (11.2%; Stage 5; Figure 5-5). Is olate Sbrev-266 supported germination and 170

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development only through Stage 3 (1.2%) after 4 weeks. Afte r 6 weeks dark incubation, both isolates Econ-242 and Sbrev-266 supported protoc orm development to Stage 5 (16.8% and 6.3%, respectively; Figure 5-5). However, isol ate Econ-242 supported a significantly higher percentage germination and development after 6 weeks, continuing the trend seen after 4 weeks of incubation. The control treatment supported germination only through Stage 1 after both 4 and 6 weeks incubation (Figure 5-5). Discussion The conservation of rare, endangered, and endemic Spiranthes species in Florida depends on an understanding of not only the requirements for in vitro symbiotic co-culture, but also the degree of mycobiont preference exhibited by each species within the genus. Developing an understanding of both these factor s represent vital steps in th e development of integrated conservation procedures for orchid species. Most studies on orchid -mycobiont preference examine the topic at either the generic level within the Orchidaceae or among species representing extremes in the family (i.e., terrestrial versus ep iphytic; nonphotosynthetic), without examining mycobiont preference effects on in vitro seed culture. These present studies represent not only the first report of in vitro mycobiont preference in either S. brevilabris or Deep South S. cernua, but also greatly expands the understanding of the in vitro co-culture requirements leading to the conservation of both species. Successful in vitro symbiotic co-culture of S. brevilabris has been previously reported (Stewart et al. 2003). Usi ng mycobionts isolated from S. brevilabris (Sbrev-266) and the Florida epiphytic species Epidendrum magnoliae var. magnoliae (syn. = E. conopseum ; Econ-242), Stewart et al. (2003) reported a maximu m percent germination of 40.8% and 49.8%, respectively, on modified OMA after 55 days in vitro culture. This was a similar final percent germination as found in the present study for t hose seeds inoculated with isolate Sbrev-266 on 171

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OMA after 12 weeks (53.1% Stage 5; Table 3-2; Figures 3-1, 5-3). St ewart et al. (2003) concluded that S. brevilabris is likely non-preferential for mycobionts since comparable germination percentages were supported by myco bionts originating from the study species and an epiphytic Florida species. Stewart et al (2003) tested mycobiont preference using two mycobionts, none of which orig inated from closely-related Spiranthes taxa in Florida, such as S. floridana. To better demonstrate any orchid-mycobiont preference by S. brevilabris mycobionts from both S. brevilabris and its close relatives (i.e., S. floridana ), should have been tested. The successful in vitro symbiotic co-culture of the Deep South race of S. cernua has not been previously reported. Ho wever, Zettler and McInnis (1 993) previously reported the symbiotic co-culture of S. cernua (no race defined) from northwestern South Carolina using mycobionts isolated from S. cernua (no race defined; Rabun Co., Georgia), Platanthera integrilabia (Greenville Co., South Carolina), and P. ciliaris (Van Buren Co., Tennessee). In this report, only the mycobionts from P. ciliaris and S. cernua supported germination to an advanced stage (>Stage 4). Furthermor e, only the mycobiont originating from P. ciliaris supported S. cernua co-culture to a leaf-b earing stage followed by subs equent establishment and flowering of plants under gree nhouse conditions. These data lead Zettler and McInnis (1993) to suggest that S. cernua exhibits a non-preferential characteri stic for mycobionts during its entire life cycle. In a similar test of mycobiont pr eference carried out in th e current study, but using seed from the Deep South race of S. cernua and mycobionts from S. brevilabris (Sbrev-266) and Epidendrum magnoliae var. magnoliae (Econ-242), a similar non-pr eferential characteristic for mycobionts was found. In light of the current data concerning the symbiotic co-culture and mycobiont preference of the Deep South race of S. cernua, the conclusion of low mycobiont preference in S. cernua by Zettler and McInnis (1993) appears to be supported. However, further 172

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symbiotic co-culture data from the geographically wide-ranging S. cernua species complex is necessary before a definitive conclusion about mycobiont preference in this group can be reached. Moreover, the use of a my cobiont originating from P. ciliaris in the symbiotic co-culture of S. cernua seeds presents an ethical dilemma for those interested in S. cernua conservation what are the potential ecological impacts of releasing a mycobiont not originating from S. cernua into S. cernua habitats? A closer examinati on of mycobiont preference in the S. cernua species complex may reveal a higher-than-expected degree of mycobiont specificity during in vitro symbiotic co-culture. The current st udy of mycobiont preference in the S. brevilabris S. floridana species pair demonstr ats this trend, with mycobionts isolated from S. floridana supporting no advanced stage symbiotic seed ge rmination when co-cultured with seed of S. brevilabris (Stewart and Kane 2007a). These two clos ely-related species appear to not share mycobionts based on myc obiont isolations and in vitro symbiotic co-culture trials. Unfortunately, symbiotic seed germination trials were not possible using seed of S. floridana because it appears the species aborts seed capsule s soon after pollination and fertilization (S.L. Stewart personal observation). Id entifying geographic or regional mycobiont preference in any orchid species pair or species complex (i.e., S. cernua species complex) would allow those interested in the integrated conservation of a regionally-specific race of the complex to recognize the importance of isolating the regionally-spe cific mycobionts to support the symbiotic coculture of the particular race. Similarly to the co-culture study of the Deep South race of S. cernua, Zettler et al. (1999) used mycobiont Econ-242 to germinate s eeds of the Florida epiphytic orchid Encyclia tampensis (Lind.) Small. While mycobiont Econ-242 did support the in vitro symbiotic seed germination 173

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of E. tampensis to a final percent germina tion of 0.3% (Stage 5) after 13 weeks, it is unlikely that this represented a true test of mycobiont preference in E. tampensis because only one mycobiont was tested (Econ-242) and no mycobionts from E. tampensis were incorporated. While of some basic interest, mycobiont prefer ence across widely divergent gene ra does not eluc idate orchidmycobiont preference at the generic level, thus circumventing questions of orchid-mycobiont preference and possible mycobiont sharing within closely-rela ted species pairs or species complexes, such as S. brevilabris and S. floridana or the Deep South race of S. cernua in Florida. Moreover, the use of widely diverse mycobionts in the in vitro symbiotic co-culture of widely diverse genera yields little practical information on the symbiotic co-culture or conservation of orchid species. As previously stated, the conser vation of orchid species by symbiotic co-culture relies on an understanding of not only mycobi ont diversity and preference, but also the physiological role specific mycobionts may provide to their orchid hosts (i.e., seed germination). This can only be investigated once a through understanding of generi c or species level mycobiont preference has been achieved. Otero et al. (2005) reported varied performance of mycobionts during in vitro symbiotic co-culture of the tropical epiphytic species Tolumnia variegata (Swartz) Braem. They go on to hypothesize that given the presumed geographi c heterogeneous distribution of orchid mycobionts, that these mycobionts may affect or chid distribution and pop ulation size. This conclusion appears valid based on our current fi ndings and may help to explain the current distribution and rarity of both S. brevilabris and the Deep South race of S. cernua in Florida. The isolation of the mycobiont Epulorhiza repens from the roots of S. brevilabris was previously reported (Stewart et al. 2003). Subsequent mycobiont isolations from other plants within the only known S. brevilabris population have consistently yi elded isolates identifiable as 174

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strains of the species E. repens (S.L. Stewart unpublished data). Epulorhiza repens is considered a ubiquitous global orchid endophyte found th roughout many orchid habitats worldwide (Anderson 1991; Stewart 2007; Zelmer 2001; Stewart et al. 2003; Cu rrah et al. 1987). Thus its repeated isolation from S. brevilabris is not surprising and its isolation from S. floridana was expected. Moreover, given th e taxonomic relatedness between S. brevilabris and S. floridana one would suspect the two species may share related mycobionts. However, repeated mycobiont isolations from the roots of S. floridana at the Bradford County site consistently yielded isolates identifiable as strains of the anamorphic fungal genus Ceratorhiza Furthermore, mycobiont isolations from S. floridana plants collected at the newly-di scovered Duval County (Florida) site also consistently yielded strains of the same Ceratorhiza mycobiont as was isolated from the Bradford County (Florida) plants (S.L. Stewart, unpublished data). This apparent species level orchid-mycobiont preference was surprising given the previously mentioned taxonomic relatedness of the two orchid species. Mycobiont isolations from the r oots of the Deep South race of S. cernua were not possible due to permitting difficulties. However, it is suspected that this race of S. cernua would have yielded isolates assignable to the anamorphic fungal genus Epulorhiza based on the previous works of Zettler and McInnis (1993) with S. cernua from South Carolina, Zettler et al. (1995) with S. odorata, a closely-related species to S. cernua, from Delaware, and S.L. Stewart (unpublished data) with S. odorata from Florida. Further inves tigations into the identity of mycobionts from this geographically-restricted S. cernua race should be undertaken. High mycobiont preference on the species level, as is hypothesized between S. brevilabris and S. floridana has been previously reported. Sheffe rson et al. (2005) re ported high mycobiont preference at the generic level in a study of the terrestrial orchid genus Cypripedium Of the 175

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seven Cypripedium species surveyed, five of the sp ecies shared mycobionts in the Tulansnellaceae (i.e., Epulorhiza -like fungi). Two species surveyed, C. californicum Gray and C. parviflorum Salisbury, demonstrated a higher degree of mycobiont diversity than all other surveyed species. Cypripedium californicum Gray and C. parviflorum Salisb. demonstrated high mycobiont preference at the generic level in comparison to other five Cypripedium species studied, especially for mycobionts not commonly asso ciated with the other five species surveyed. This trend is similar to the mycobiont pr eference apparently demonstrated between S. brevilabris and S. floridana Likewise, Taylor et al. (2003) reported a high degree of pr eference between two closely related nonphotosynthetic orchids in the genus Hexalectris. Four distinct types of fungi were identified from samples of the common H. spicata (Walter) Barnhardt var. spicata while only one type was identified from samples of the rare H. spicata var. arizonica (S. Watson) Catling and Engel. Taylor et al. (2003) hypothesized that the divergence seen in mycobiont preference between these two varieties of th e same orchid species represents evidence for the contribution of mycobiont specificity to the evolutionary diversification of the Orchidaceae. High mycobiont preference between closely-relate d varieties or races may require special consideration for the in vitro symbiotic propagation and conservation of t hose species, but these factors were not examined by Taylor et al. (2003). Moreover, Tayl or and Bruns (1999) repo rted that two species of the nonphotosynthetic orchid genus Corallorhiza each associated with several species of Russula but never shared fungi despite the plants growing sympatrically. Again, these types of data can prove to be crucial for those interested in the symbiotic propagation or conservation of orchid species. A similar prefer ence trend may be occurring between S. brevilabris and S. 176

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floridana ; however, further experimentation is nece ssary to properly hypot hesize on the habitat preference or selection pressures acting between these two species. An opposite trend in mycobiont pr eference is hypothesized in the S. cernua species complex, with particular emphasis on the Deep Sout h race of the species. While Otero et al. (2005), Taylor et al. (2003), and Taylor and Bruns (1999) hypothesize that high mycobiont preference may be a major driving force behind orchid habitat preference and evolutionary diversification, the apparent mycobiont non-specific ity demonstrated in the Deep South race of S. cernua may be indicative of an inverse rela tionship between mycobiont preference and geographic restriction and/or evolutionary diversification (Stewa rt 2007). If other members of the S. cernua species complex exhibit mycobiont non-pref erence, this may help to explain the wide geographic range of this species complex throughout the north east, Midwest, mid-Atlantic, and southeastern United States. Furt her study of mycobiont preference in the S. cernua complex is necessary to elucidate the potential relationship among distribution, mycobiont preference, and species rarity. Of further interest is that the same myc obiont Stewart (2007), Stew art et al. (2003), and Stewart and Kane (2007a) utilized in the symbiotic co-culture of S. brevilabris and Deep South S. cernua (Sbrev-266; Table 5-2) continued to support in vitro symbiotic germination in the present studies. Some authoritie s have suggested that the effec tiveness of orchid mycobionts at supporting symbiotic co-culture may lessen if the mycobionts are stored over long periods of time and/or subjected to multiple subculture s (L.W. Zettler personal communication). These present data suggest that my cobiont Sbrev-266 does not dem onstrate any reduced symbiotic germination capacity despite being routinely subcultured and stored at 25 C for 7 years (to date) on both PDA and 1/5 PDA. 177

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The current studies present a first look at orchid-mycobiont prefer ence in the endangered terrestrial orchids S. brevilabris and the Deep South race of S. cernua in Florida. In the case of S. brevilabris preference was demonstrated through not only the isolation and identification of different mycobionts from S. brevilabris and its Florida endemic congener S. floridana but also the use of those mycobionts in the in vitro symbiotic co-culture of S. brevilabris In the case of the Deep South race of S. cernua, mycobiont preference was dem onstrated through the use of mycobionts from similar and divergent Florida orch id taxa in the symbiotic co-culture of the study species. Data such as repor ted here may prove invaluable in the integrated conservation of S. brevilabris S. floridana and the Deep South race of S. cernua in Florida, especially given their rare status within the state. Implications for Integrated Conservation Planning The present studies on the in vitro symbiotic co-culture and mycobiont preference of the Florida terrestrial orchids S. brevilabris and Deep South S. cernua have demonstrated the benefits of insightful studies of both the propa gation science and mycology of orchid species before implementing conservation or recovery plans. As seen in the asymbiotic and symbiotic seed culture studies of Habenaria macroceratitis the current results show the importance of understanding an orchids germination requi rementsparticularly mycobiont preference before attempting further integrat ed conservation and species recove ry efforts. For example, we now understand that the integrated conservation and recovery of S. brevilabris and S. floridana in Florida require the isolation and storage of mycobionts from individual S. brevilabris and S. floridana sites throughout the state, not just a few isolations repres enting one or two populations. The symbiotic co-culture, plant reintroduction, and long-term speci es recovery of both orchid species is seriously questionabl e without the proper mycobiont diversity represented in plant material collections (i.e., mycobiont and seed banks). 178

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Furthermore, the high degree of mycobi ont preference demonstrated through the in vitro symbiotic co-culture of S. brevilabris raises a number of inte resting questions concerning conservation and recovery planning for this sp ecies. Specifically, is the distribution of S. brevilabris in Florida limited by the distribut ion of its preferred mycobiont? Spiranthes brevilabris was historically distributed throughout the coastal plain of the southeastern United States (Luer 1972); however, is currently only known from a fe w sites in Florida and Texas (Brown 2002, 2005; P.M. Brown personal communicatio n). Given the hypothese s of Otero et al. (2005) and Taylor et al. (2003) it is conceivable that th e decline in the range of S. brevilabris is due to not only habitat degradation and destru ction, but also a decline in abundance of the species mycobiont due to ecologica l alteration of habitat. This means that to achieve the longterm conservation and recovery of this species, more than simply targeted plant management must be considered. Management of habita t for both plants and mycobionts will likely be required if the recovery of S. brevilabris throughout its historic range is to be successful. Further investigations into the in tegrated conservation of S. brevilabris as well as its historically widespread congener S. floridana, will shed light on these aspects of species management. Additionally, the low degree of mycobiont pr eference demonstrated through the symbiotic co-culture of the Deep South race of S. cernua creates its own important questions concerning the integrated conservation and management of bo th this particular race and the entire species complex. Where the recovery of S. brevilabris and S. floridana may depend upon the management of particular mycobionts in partic ular habitats, the recovery of Deep South S. cernua may depend upon more upon habitat management and less upon mycobiont management. Moreover, this general species recovery and management paradigm may be applicable on a regional basis to the entire S. cernua species complexwhere mana gement of habitat for each 179

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geographically defined race within the complex may prove to be of greater importance than management for particular mycobionts. Of c ourse, further investiga tion into not only the mycobiont diversity and preference of the Deep South S. cernua, but also all other races of S. cernua in the United States must be undertaken before such a wide -ranging species recovery and management recommendation can be fully implemented. The current studies concerning the pr opagation science and mycology of both S. brevilabris and the Deep South race of S. cernua represent two steps in the species-level integrated conservation of these rare terrestrial orchid species in Florida (Figure 1-1). By developing a more complete under standing of symbiotic co-culture requirements and mycobiont preferences in these two orchid species, we are now better equipped to continue integrated conservation efforts with both species that will lead to their recovery thro ugh habitat restoration and plant reintroductions. Further studies c oncerning the population establishment, population management, and long-term population sustainability of both S. brevilabris and the Deep South S. cernua are encouraged. 180

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Table 5-1. Sources of mycobionts used in the in vitro symbiotic co-culture of Spiranthes brevilabris Isolate Host Collection information Identification Sflo-305 S. floridana 28 April 2003; Bradford Co., FL Ceratorhiza sp. Sflo-306 S. floridana 28 April 2003; Bradford Co., FL Ceratorhiza sp. Sflo-308 S. floridana 25 April 2004; Bradford Co., FL Ceratorhiza sp. Sbrev-266 S. brevilabris 30 April 1999; Levy Co., FL Epulorhiza repens 181

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Table 5-2. Sources of mycobionts used in the in vitro co-culture of the Deep South race of Spiranthes cernua Isolate Host Collection information Identification Econ-242 Epi. magnoliae 7 June 1995; Alachua Co., FL Epulorhiza spp. Sbrev-266 S. brevilabris 30 April 1999; Levy Co., FL Epulorhiza repens 182

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Figure 5-1. Mycobiont s isolated from Spiranthes floridana (Sflo) and S. brevilabris (Sbrev). A) Sflo-305 whole culture morphology at 3 w eeks, scale bar = 1 cm. B) Sflo-305 monilioid cells stained with acid fuchsin at 3 weeks (100), scale bar = 30 m. C) Sflo-306 whole culture morphology at 3 w eeks, scale bar = 1 cm. D) Sflo-306 monilioid cells stained as above (100), scale bar = 30 m. E) Sflo-308 whole culture morphology at 3 weeks, scale bar = 1 cm. F) Sflo-308 monilioid cells stained as above (100), scale bar = 30 m. G) Sbrev-266 whole culture morphology at 3 weeks, scale bar = 1 cm. H) Sbrev-266 monilioid cells stained as above (400), scale bar = 10 m. 183

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Figure 5-2. Mycobionts used in the symbiotic co-culture of the Deep South race of Spiranthes cernua. A) Sbrev-266 whole culture morphol ogy at 3 weeks. B) Econ-242 whole culture morphology at 3 weeks. Scale bars = 1 cm. 184

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Germination (%) 0 20 40 60 80 100 a a a a a a b b b b a a a a a a Germination (%) 0 20 40 60 80 100 266 305 306 308 Control a a a a a a a a a b b b b a a a Developmental Stage Stage 0Stage 1Stage 2Stage 3Stage 4Stage 5 Germination (%) 0 20 40 60 80 100 a b b b b a b b b a 12 weeks 10 weeks 3 weeks Figure 5-3. Effects of four my cobionts on percent germination and protocorm development of Spiranthes brevilabris cultured on oat meal agar (OMA) after 3, 10, and 12 weeks in vitro culture. Histobars with the same letter are not significantly different within stage ( = 0.05). Error bar = SE. 185

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Development Time 3 wks 10 wks 12 wks Germination (%) 0 10 20 30 40 50 60 70 Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 a b c a b c c c d a b c c c d Figure 5-4. Effect of mycobiont Sbrev-266 on percent germination and protocorm development of Spiranthes brevilabris cultured on oat meal agar (OMA) at 3, 10, and 12 weeks in vitro culture. Histobars with the same letter are not significantly different within development time ( = 0.05). Error bar = SE. 186

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Germination Stage Stage 0Stage 1Stage 2Stage 3Stage 4Stage 5 Germination (%) 0 20 40 60 80 100 4 weeks a ab b a b b a b a a Germination (%) 0 20 40 60 80 100 242 266 control 6 weeks a ab b a b a b Figure 5-5. Effects of two my cobionts on percent germination and protocorm development of the Deep South race of Spiranthes cernua cultured on oat meal agar (OMA) after 4 and 6 weeks in vitro culture. Histobars with the sa me letter are not significantly different within stage ( = 0.05). Error bar = SE. 187

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APPENDIX A MORPHOLOGICAL VARIATION IN Habenaria macroceratitis Floral and vegetative structure variation is known to occur in the Orchidaceae. This variation can occur both within and between popul ations of the same species, and is thought to arise from microevolutionary pr essures that may be site or population specific (MelndezAckerman et al. 2005). Vallius et al. (2004) examined morphological variation within and between populations of co -occurring varieties of Dactylorhiza incarnata (L.) So. In a survey of five populations, they reported lit tle morphological difference between populations of varieties; however, varietal differences within populations were high. Vallius et al. (2004) suggest that sympatric evolution is occurring among these co-occurring varieties. Studying sympatric populations of the terrestrial orchid Gymnadenia conopsea (L.) R. Brown, Soliva and Widmer (1999) identified a high de gree of floral varia tion between earlyand late-flowering populations of this species. As before this variation may represent microevolutionary changes within metapopulations of G. conopsea to limit competition for pollinators. A similar trend in competition-limiting morphologic variation has been documented in the tropical epiphytic orchid Tolumnia variegata (Ackerman and Galarza-Prez 1991). Within species or population morphologi cal variation may be the result of microevolutionary forces driving speciation in the Orchidaceae. To this end, many orchid taxonomists have used this floral and/or vegetativ e variation to identify stable color and growth forms of some orchid species. For example, Epidendrum amphistomum A. Richard forma rubrifolium P.M. Brown has been identified as the red-l eaf color form of the otherwise green-leaf E. amphistomum (Brown 2000). Another example of a morphological variation resulting in a unique taxonomic trait can be seen with Listera australis Lindl. forma scottii P.M. Brown (Brown 2000). Typically, L. australis forms two leaves per inflorescence; however, forma 188

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scottii regularly forms three leaves per inflorescence, and this trait is stable year-after-year in the same plants. Finally, an example of floral color variation can be seen in the terrestrial orchid Sacoila lanceolata var. paludicola (Luer) Sauleda, Wunderline, & Hansen forma auera P.M. Brown (Brown 2001). This form differs in its yellow flowers, instead of its typical red flower color. Based on these reports, floral and/or ve getative variations within the Orchidaceae appear to be reasonably common. During studies on the plant demogr aphy and pollination biology of Habenaria macroceratitis at the Socash site (Hernando County, Flor ida), a unique leaf form and growth habit of the species were not ed. Typically, leaves of H. macroceratitis possess a glossy bright green appearance (Figure A-1a). However, it was noted that leaves of particular plants of H. macroceratitis within this population possess lateral pa rallel striping alternating between dull green and yellow-green colors (Figure A-1b-c). This unique color pattern was seen on only a few plants in the Socash population and remain ed stable during all observation years (20022005). Plants possessing this lateral striping were not not ed at any other H. macroceratitis site included in the present studies. It remains to be seen if this variation in leaf color is due to plant nutrition, site variability, or some other microevolutionary force. Furthe r study of the long-term stability and reproduction of leaf color pattern from seed is needed before this color variant is given further taxonomic consideration and form status. A second unique growth habit was noted during the field study of H. macroceratitis at the Socash site. Plants growing in high light conditions where no canopy shade was available showed an upright growth form of their leaves (Figure A-2) Under normal conditions, the leaves of H. macroceratitis grow perpendicular to the flowering stem and parallel with surface of the soil. However, the leaves of these plants gr ew at an acute angle to the flowering stem and 189

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nearly vertical from the soil surface (Figure A-2). This growth habit is likely due to exposure to higher levels of light than what is typical for this species, and a morphol ogical response to limit light exposure of leaves and reduce wate r loss from transpiration. However, H. quinqueseta has been observed growing in a similar high light si tuation in Levy County (Florida) without this upright leaf growth (S.L. Stew art, personal observation). Fu rther investigation into the physiological and possible taxonomic bearing of this growth habit is needed. 190

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Figure A-1. Lateral striping variant of Habenaria macroceratitis at the Socash study site (Hernando County, Florida). A) Comparison of striped leaf (top) and normal leaf (bottom). B) Vegetative plants of H. macroceratitis showing lateral striping. C) Flowering plant of H. macroceratitis showing an extreme example of lateral striping. 191

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Figure A-2. Plants of Habenaria macroceratitis at the Socash study site (Hernando County, Florida) showing upright leaf growth habit. A) Flowering plant showing acute angle between leaf and flowering stem. B) Ve getative plant showing vertical leaves. 192

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APPENDIX B AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP) SUPPLEMENTARY DATA FOR Habenaria macroceratitis Figure B-1. AFLP fingerpri nt of genomic DNA from the Socash site (Hernando County, Florida) of Habenaria macroceratitis Primer combination -ACT/-CAG used. Molecular weight size range of fi ngerprints is 10-650 nucleotides. 193

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Figure B-2. AFLP fingerprint of genomic DNA from the Old Da de Highway (Hernando County, Florida) site of Habenaria macroceratitis Primer combination -ACT/-CAG used. Molecular weight size range of fi ngerprints is 10-650 nucleotides. 194

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195 Figure B-3. AFLP fingerprint of genomic DNA from the Rayonier site (Bradford County, Florida) of Spiranthes floridana Primer combination -A CT/-CAG used. Molecular weight size range of fingerp rints is 10-750 nucleotides.

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Figure B-4. Correlation dendrogr am of genotypic diversity within the Socash site of Habenaria macroceratitis Four genotypes present. 196

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Figure B-5. Correlation dendrogram of genotypic diversity within th e Old Dade Highway site of Habenaria macroceratitis Six genotypes present. 197

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198 Figure B-6. Correlation dendrogram of genotypic diversity between the Socash and Old Dade Highway sites of Habenaria macroceratitis Ten genotypes present.

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199 Figure B-7. Correlation dendrogr am of genotypic diversity wi thin the Rayonier site of Spiranthes floridana Four genotypes present.

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