Intergeneric and intrageneric phylogenetic relationships of Encyclia (Orchidaceae) based upon holomorphology

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Intergeneric and intrageneric phylogenetic relationships of Encyclia (Orchidaceae) based upon holomorphology
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Thesis:
Thesis (Ph. D.)--University of Florida, 2000.
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Includes bibliographical references (leaves 284-295).
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by Wesley Ervin Higgins.
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INTERGENERIC AND INTRAGENERIC PHYLOGENETIC RELATIONSHIPS
OF ENCYCLIA (ORCHIDACEAE) BASED UPON HOLOMORPHOLOGY









By

WESLEY ERVIN HIGGINS


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


2000








































UNIVERSITY OF FLORIDA
3 1262 08554 463I2il I II11 I
3 1262 08554 4632



























Copyright 2000

by

Wesley E. Higgins












This work is dedicated to all the orchidologists that have struggled throughout the

centuries to understand the relationships of Orchidaceae.













ACKNOWLEDGMENTS

I thank my committee, Bijan Dehgan, Tom Sheehan, Mark Whitten, Walter Judd,

and Charlie Guy, for the guidance that each provided in their own area of expertise. I

also thank numerous professors at the University of Florida who were willing to discuss

various aspects of this research.

I thank the following agencies that provided financial support for this project. The

American Orchid Society provided a doctoral fellowship and a research grant. The

College of Agriculture and the Graduate School of the University of Florida provided

grant matching funds. Funding was also received from the 11 World Orchid Congress

Fellowship, the Florida Department of Agriculture Scholastic Achievement Award, and

the following scholarships: Sidney B. Meadows Endowment Fund, Muriel Rumsey, Dr. H.

Harold Humes, Garden Club of Halifax Country, Donnell Fund, and the Action Chapter

and Royal Palm Chapter of FNGA. Travel was partially funded by an AABGA Student

Travel Award, Graduate Student Government Association, Institute of Food and

Agriculture Sciences (IFAS), Jim Ackerman, and Bijan Dehgan.

Plant material was donated by Jim Ackerman, Weyman Bussey, Bill Toms, Eric

Hagsater, Bijan Dehgan, Mary Ragan, Phill White, Tom Sheehan, Jerry Sellers, John

Atwood, Gerardus Staal, Miguel Soto Arenas, Mary Jean Poetz, Bob Porgorski, Irv

Quitmyer, Woody Phillips, Bob Dressler, Claude Hamilton, German Camevali, Missouri

Botanical Gardens, Royal Botanic Garden Kew, and Marie Selby Botanical Gardens.

Thanks to Cassio van den Berg, Alec Pridgeon and Mark Whitten for donating DNA

sequences. I am grateful to Mark Chase for funding the laboratory expenses during my

practicum at the Jodrell Laboratory, and to Norris Williams for the loan of computer






equipment and the use of the molecular systematic laboratory in the Florida Museum of

Natural History.

I acknowledge the following herbaria and personnel whose support was

invaluable in obtaining permits for field work: University of Florida herbarium (FLAS),

Kent Perkins and Trudy Lindler; Herbario AMO, Eric Hagsater, Miguel Soto Arenas,

Rolando Jimenez, and Javier Garcia Cruz; and the Herbario del Jardin Botanico

Nacional (JBSD), Dominican Republic, Francisco Jimenez.

I recognize the following people for their generous help: Lena Landherr for her

mothering and editorial assistance, Scott Sheaffer for PageMaker drawings, Fe Almira

for her assistance in the plant morphology lab, and Sherrie Higgins for all her patience

and understanding. This project could not have become a reality without the

cooperation and gracious assistance from numerous persons and agencies.

















TABLE OF CONTENTS


ACKNOWLEDGMENTS................................................................................................ iv

ABSTRAC T .................... ................ ... .............................................................................. x

1. INTRODUCTION .................................................................................................... 1

Background ... ................................... .......................................................... ............. 1
Problem Statem ent ................................... .. .................. ................ ..... .................... 2
Approach ................................................................................................. ....................3
Ingroup Selection ..............................................................................................3.
O utgroup Selection........ ................ ............ ..................... ........................................ 5

2. MORPHOLOGY ............................ ................... .................................................... 7

Introduction.................... ......... ........................................................................... 7
M materials and M ethods........................................................ ..................... .................. 8
Morphological Characters...........................................................................................9
Vegetative Morphology....................................................................................9
W hole plant.. ....................... ............................................... ..... .................... 10
Orchid pseudobulbs .............................................................. .............10
Orchid leaves........................................................... ............ ..... ..... 11
O rchid roots ............................................................................................ ..... 12
Reproductive Morphology........................ ....................... .......................13
Plant inflorescence ............................ ........................................... ............ ....13
O rchid flowers...................................................................... ..................... ......... 14
Seed capsule.... ..................................................... ..... ................ 18
Secondary Plant Compounds......................................... ....................................19
Morphological Phylogenetic Analyses ........... .................................. ............20
Equal-Weighted Analysis............................................................................. 21
Equal-weighted tree search ............................................................. 21
Equal-weighted decay analysis............................... ....................... 22
Equal-weighted bootstrap analysis....................................................................22
Weighted Analysis ..... ............. .................. .................................... 23
Weighted tree search........................................................................................23
Weighted decay analysis .......................................... .....................................23
Weighted bootstrap analysis..................................... ................... ..24



vi






Morphological Results.......... ............................... ..................................................24
Equal-weighted Results......... .................................. .........................................24
W weighted Results............................................ .......................................... .........24
Morphological Discussion ...................................................................................... 25

3. MOLECULAR Studies ........................................................................................... 81

Introduction........................................................................................................ ....... 81
Research in Orchidaceae ....................................................................................... 82
Nuclear Genome................................................................................................ 82
Plastid Genome................... ..............................................................................83
tm L-F region .................................................................................................. 84
m afK gene..........................................................................................................85
Materials and Methods............ ............................................................................... 85
DNA Extraction.................................................................................................. 85
DNA Amplification .......................................................................... ........................87
ITS region ...................................................... ............................ ....................... 90
tm L-F region.................................................................................................... 91
m atK gene ........................................... ................... .................................... 92
PCR cleaning................................................................... ................................92
Cycle Sequencing ..... ........ ... ......................... ......... ........................ ...... .......... 93
Sequencing protocol..........................................................................................95
Sequencing primers ........... ............................. ............................................... 95
Cycle sequencing cleaning .................................................... ........................ 96
Automated sequencing ................................................................................... 97
Data Processing ................................................... .............................................. 98
Sequence editing .................... ........................... ................... ................ ....... 98
Sequence assembly.................................... ..................................................... 99
Data matrix ........................................................................................................ 99
DNA Analysis................ ...................................................................................... 100
Parsimony Analysis................................................... ........ ............................101
Bootstrap Analysis............................................... ......... ................................... 103
Decay Analysis................................................................................................ 104
Indel Matrix...................................................................................................... 104
Molecular Results ............................................................................................... 105
Nuclear Results.............................................. ..................................................... 105
Plastid Results ................................................................................................ 106
trnL results ............................................................................... ..................... 106
matK results..................... ....... ......................... ............................................... 106
Combined plastid results...................................... ..................... ..................... 107
DNA Discussion...... .................................................................................... ........... 107
ITS Discussion ................................................................................................ 107
Plastid Discussion ......................................................................................... 109
tm L-F discussion .......................................................................................... .. 109
m atK discussion....................... ................................................................... 110
Indel discussion ............................................................................................ 110

4. COM BINED ANALYSES ..................................................................................... 135

Introduction................................... ................. ......................................................1.. 35
Matrix Methods ................................................................................................... 136


vii








Combined Analyses.................. .................................. ................. ... .... 137
Parsim ony Analyses............................................................ .............................. 137
Bootstrap Analyses .......... ............................................. .137
Decay Analyses ................................................................................................... 138
Combined Results .... ................. ................... ..................................................... 138
Combined DNA Results.................................................................................... .. 138
Holomorphology Results .................................................................................. 139
Combined Discussion .......................................................................................... 140
Tree Topology ................................................................................. .................. 141
Character Evolution..................Evolution .................... .......... ........................142
Molecular Evolution ....................................................................................... 146

5. APPLICATIONS AND CONCLUSIONS.................................................. ......... 158

Introduction ..... ............................................. ... ........ ................... ................ 158
Application of Results ....... .................................................................................. 158
Taxonom ic History............. ................... ........................................... ................ 159
General overview ............ .................... .... ........................... ............. .... 159
Specific histories............................... ...... ....................... .................... 160
Phylogenetic Classifications ................................................ .. .............. 164
Subtribal classification ............................................. ................................ ....164
Generic classification................................................... ..... ........165
Subgeneric classification ............................. .................. ........ 165
Sectional classification............................................... ...... .......................... 165
New classification .............................................. ....................... 166
Specific classification...... ...... ..................................................................... .. 168
Rejected classifications...... ......... ....... ............................................... .... ... 169
Project Summary .......... .................. ..................................................... ...............169
Continued Research............. ....................................................... 170

APPENDIX A SPECIMEN VOUCHER NUMBERS............................................... 173


APPENDIX B CHARACTER STATE DELIMITATION.............................................. 175


APPENDIX C USEFUL FORMULAE........................................... 178


APPENDIX D ITS DNA COMPOSITION ANALYSIS ........................................ .. 181


APPENDIX E tmL DNA COMPOSITION ANALYSIS....... .. ....................... 183


APPENDIX F matK DNA COMPOSITION ANALYSIS .................................. 185


APPENDIX G COMBINED DNA MATRIX ..................... ............................. 188


APPENDIX H HOLOMORPHOLOGY WEIGHT SET.............................................................271






APPENDIX I PATRISTIC DISTANCE MATRIX .......................................................274


APPENDIX J PAIRWISE HOMOPLASY MATRIX .......................................................280


LIST OF REFERENCES ........................................................................................ 284


BIOGRAPHICAL SKETCH ........................................................................................ 296












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



INTERGENERIC AND INTRAGENERIC PHYLOGENETIC RELATIONSHIPS
OF ENCYCLIA (ORCHIDACEAE) BASED UPON HOLOMORPHOLOGY
by

Wesley Ervin Higgins

May 2000


Chairperson: Bijan Dehgan
Cochairperson: Thomas J. Sheehan
Major Department: Horticultural Science

The goal of taxonomy is to provide a classification system that is natural and

predictive. This project examines the classification of the genus Encyclia (Orchidaceae)

sensu Dressier. The objectives of this research are to determine the position of Encyclia

within the subtribe Laeliinae, and to resolve the phylogeny of Encyclia to the sectional

level. A holomorphological approach, which combines characters from several

disciplines, was used to develop a total-evidence hypothesis of the phylogenetic

relationships. Characters from floral and vegetative morphology, secondary glucoside

chemistry, and DNA sequences from the plastid and nuclear genomes were utilized.

The molecular data is derived from sequencing the region of the Internal Transcribed

Spacers (ITS) and the 5.8S ribosomal gene from the nuclear genome, and two regions

from the plastid genome, the tmL-F region (transfer RNA of leucine) and the matK gene

(a RNA maturase). The data matrix was analyzed using a parsimony algorithm.

Homoplasious characters were down-weighted by successive reweighting based on their






individual rescaled consistency indexes. The final weighted holomorphology analysis

produced one tree of 3242 steps. This analysis shows that Encyclia is not monophyletic

as circumscribed by Dressier. The results imply that classification schemes based solely

on floral morphology may be misleading. The taxonomic consequences of this research

are that five of the six sections of Encyclia have been raised to generic status. Two

have new generic names, Euchile and Ostlundia, and three have reverted to older

names, Prosthechea, Dinema, and Encyclia. One sectional name, Hormidium, has been

abandoned.














CHAPTER 1
INTRODUCTION



Background


Floral diversity of the orchid family has intrigued botanists for centuries (Marden,

1971). The floral form within the family is extremely plastic and produces a variety of

eye-captivating shapes that mimic bees, wasps, butterflies, or moths (Simon, 1975).

The flowers invoke images that can resemble doves, swans, frogs, lizards or miniature

men (Arditti, 1992; Senghas, 1993). Orchidaceae are the largest flowering plant family

with 800-900 genera and 25,000-35,000 species (Sheehan and Sheehan, 1994). The

family is divided into 5 subfamilies, 20 tribes, and 74 subtribes (Dressier, 1993). The

members of subtribe Laeliinae are among the most commonly cultivated and frequently

hybridized orchids (Withner, 1988). Encyclia is included in the subtribe Laeliinae, which

consists of 43 genera (Dressler, 1993). The taxonomy of this subtribe appears artificial

due to its reliance on pollinia number (Dressler, 1993). For example, the presence of

eight pollinia has been used to group plants from Mexico and Brazil which have very

different vegetative morphology, into the same genus, Laelia.

Encyclia is a diverse genus encompassing about 200 mostly epiphytic species.

The range of Encyclia extends from Mexico to the West Indies, including Florida, and

southward throughout most of Central America and tropical South America. There are

two apparent centers of speciation, one in southern Mexico and the other in southern

Brazil. The genus is characterized by: (1) pseudobulbs with 1-4 leaves, (2) labellum free






or partially adnate to the column, and (3) the terminal anther containing four waxy

pollinia with caudicles (Hooker, 1828; Lindley, 1831; Luer, 1972). The shape of the

column is the most consistent feature by which specimens may be placed to subgenus

level (Dressier and Pollard, 1976). The classification of Encyclia by Dressier and Pollard

will be used as the starting point for this investigation (Table 1-1). Note that the species

in section Osmophytum have recently been transferred to Prosthechea (Higgins, 1997)

and the species in section Euchile have been transferred to Euchile (Withner, 1998).


Table 1-1. Classification of Encyclia.
Subgenus Sections
Dinema (monotypic)

Encyclia Encyclia
Leptophyllum

Osmophytum Osmophytum
Hormidium
Euchile
(Dressier and Pollard, 1971)



Problem Statement


The objectives of this research were to determine the position of Encyclia within

the subtribe Laeliinae, and to resolve the phylogeny of Encyclia at the sectional level.

The classification of Encyclia has been problematic since it was described by Hooker in

1828. This genus provides good examples of unique pollination biology and convergent

floral morphology in two centers of speciation in disjunct xerophytic tropical forests. This

convergent morphology may have resulted in a misleading classification. Previously, the

mode of lip encircling the column has been used to suggest relationships within the

subtribe (Hooker, 1828). The use of this single morphological floral character is

unreliable because this is probably a homoplasious character in the subtribe. A







holomorphological approach, that combines characters from several disciplines, will be

used to develop a total-evidence hypothesis of phylogenetic relationships.



Approach


Hoiomorphology, i.e., the totality of characters (Hennig, 1966), was the basis of

this study of Encyclia. This study utilized characters from DNA sequences, a secondary

chemical character glucosidee crystals), and floral and vegetative morphology. The

molecular study included sequences from the ribosomal region of Internal Transcribes

Spacer (ITS) from the nuclear genome and two genes from the plastid genome, tmL-F

(transfer RNA for leucine) and the matK gene (a RNA maturase). Morphological and

molecular data was analyzed separately and then combined for a total-evidence

analysis. Sixty-one species were included in the analysis (represented by 66

specimens). The voucher numbers are listed in Appendix A.



Ingroup Selection


The ingroup taxa (30 species) were selected to represent all sections of Encyclia

(Table 1-2). The type species for each section were sequenced when possible;

however, the type for the genus (Encyclia section Encyclia), E. viridiflora, has never

been recollected and has been lost to science. Specimens have been chosen to include

as much geographic variation as possible from Florida, Mexico, Brazil and the

Caribbean. Variation in floral morphology and biology has also been accounted for by

including resupinate and non-resupinate flowers, as well as wasp and bee pollinated

species. Species resolution was tested by inclusion of two species that have been






placed in synonymy, Encyclia chimborazoensis (Schltr.) Dressier and E. fragrans (Sw.)

Lemee, and by using two specimens for several species (E. tampensis, E. mariae, E.

polybulbon, E. luteorosea, and E. subulatifolia).


Table 1-2. In
Subgenus
Encyclia


Osmophytum


Dinema


group Taxa.


I


Origin

Mexico
Mexico
Mexico
Mexico
Mexico
Mexico
Brazil
Ecuador
Mexico
Brazil
Florida


Taxon
Section Encyclia
Encyclia adenocaula (Llave and Lex.)Schitr.
Encyclia aromatica (Bateman) Schltr.
Encyclia asperula Dressier & Pollard
Encyclia bractescens (Lindl.) Hoehne
Encyclia candollei (Lindl.) Schltr.
Encyclia cordigera (H. B. K.) Dressier
Encyclia dichroma (Lindl.) Schltr. in Schlechter
Encyclia diuma Schltr. in Fedde
Encyclia kienastii(Rchb.f.) Dressier & Pollard
Encyclia randii (Barb. Rodr.) Porto & Brade
Encyclia tampensis (Lind.) Small
Section Leptophyllum Dressier & Pollard
Encyclia cyanocolumna (Ames, F.T. Hubb. & C. Schweinf.) Dressier
Encyclia luteorosea (Rich. and Gal.) Dressier & Pollard
Encyclia subulatifolia (A.Rich & Galeotti) Dressier
Encyclia tenuissima (Ames, Hubb. and Schweinf.) Dressier
Section Osmophytum (Lindl.) Dressier & Pollard
Encyclia aemula (Lindl.) Camevali & 1. Ramirez
Encyclia chimborazoensis (Schltr.) Dressier
Encyclia cochleata (L.) Lemee
Encyclia cretacea Dressier & Pollard
Encyclia fragrans (Sw.) Lemee
Encyclia glauca (Knowles and Westc.) Dressier & Pollard
Encyclia ionocentra Dressier
Encyclia ochracea (Lindl.) Dressier
Encyclia prismatocarpa (Rchb. f) Dressier
Encyclia vitellina (Lindl.) Dressier
Section Hormidium (Lindl.) Dressier & Pollard
Encyclia pseudopygmaea (Finet) Dressier and Pollard
Encyclia pygmaea (Hook.) Dressier
Section Euchile Dressier & Pollard
Encyclia citrina (Llave and Lex.) Dressier
Encyclia mariae (Ames) Hoehne
(Lindl.) Dressier & Pollard
Encyclia polybulbon (Sw.) Dressier


Ecuador
Peru
Mexico
Mexico
Mexico
Mexico
Costa Rica
Mexico
Costa Rica
Mexico


Mexico
Mexico


Mexico
Mexico

Mexico


Mexico
Mexico
Mexico
Mexico


--


----










Outgroup Selection


A comprehensive outgroup was required because Encyclia sensu lato may not

be monophyletic (Maddison, et al., 1984). The outgroup taxa (31 species) were selected

from the subtribe Laeliinae and sister subtribes within Epidendreae based on the

affinities proposed by Dressler (1993). Three taxa not in Laeliinae were used as an

outgroup for the subtribe. Meiracyllium trinasutum (subtribe Meiracylliinae) was chosen

as an outgroup because of a velamen type that suggests a close alliance to the

Laeliinae. Pleurothallis racemiflora and Restrepiella ophiocephala (subtribe

Pleurothallidinae) were selected because the presence of the Pleurothallis seed type in

Ponera, a member of Laeliinae (Dressler, 1993). Outgroup taxa (Table 1-3) were

chosen from the Cattleya alliance, within the subtribe Laeliinae in order to represent as

much variation as possible, and from the subfamily Epidendroideae to help delimit the

subtribe.





Table 1-3. Outgroup Taxa.
Taxon
Acrorchis roseola Dressier
Brassavola cucullata (L.) R. Br.
Broughtonia negrilensis Fowlie
Cattleya dowiana Bateman
Cattleya forbesii Lindl.
Cattleyopsis lindenii Cogn.
Domingoa kienastii (Rchb.f.) Dressier
Epidendrum ibaguense Pavon ex Lindi.
Epidendrum conopseum R. Br. in Ait.
Hagsatera brachycolumna (L.O. Williams) R.Gonzalez
Hexadesmia Brongn.
Hexisea imbricata (Lindl.) Rchb.f.
Homalopetalum pumilio (Rchb.f.) Schltr.
Isochilus major Cham. & Schltdl.
Jacquiniella teretifolia (Sw.) Britton & P. Wilson
Laelia purpurata Lindl. & Paxton
Laelia rubescens Lindl.
Meiracyllium trinasutum Rchb.f.
Myrmecophila tibicinis (Bateman) Rolfe
Nidema boothii (Lindl.) Schltr.
Pleurothallis racemiflora Lindl. ex Lodd.
Ponera striata Lindl.
Psychilis mcconnelliae Sauleda
Psychilis krugii (Bello) Sauleda
Reichenbachanthus cuniculatus (Schltr.) Pabst.
Restrepiella ophiocephala (Lndl.) Garay and Dunsterv.
Rhyncholaelia glauca (Lindl.) Schltr.
Scaphyglottis pulchella (Schltr.) L.O. Williams
Schomburgkia splendid Schltr.
Sophronitis cemua Lindl.
Tetramicra elegans (Hamilt.)Cogn.


_ l~ IIIII II


Subtribe
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Meiracylliinae
Laeliinae
Laeliinae
Pleurothallidinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Pleurothallidinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae
Laeliinae













CHAPTER 2
MORPHOLOGY



Introduction


Morphology has been the basis of plant classification since its inception.

Traditionally, plants have been grouped based on a subjective analysis of their overall

similarities. This phenetic approach does not distinguish between ancestral and derived

plant characteristics. The current cladistic paradigm i.e., a cladistic or phylogenetic

approach, groups plants based on their shared derived characters (synapomorphies)

(Wiley, et al., 1991). Phenetic studies have been useful for detecting terminal units in

difficult species complexes (Johnson and Linder, 1995). Morphological and anatomical

data have an important role in resolving relationships in Orchidaceae (Adams, 1959),

with floral morphology providing the primary source of characters in many taxonomic

studies of orchids. However, evidence from cpDNA analysis suggests a previously

unsuspected degree of plasticity in floral morphology, demonstrated by the convergence

of gross floral features (Chase and Palmer, 1997). Rapid changes or reversals in floral

morphology may have resulted in poor resolution of phylogenetic relationships in

traditional classifications. Chase and Palmer (Chase and Palmer, 1989) hypothesized

that gross floral morphology is deceptive and cannot be trusted to lead to accurate

phylogenetic relationships in Orchidaceae. Oncidium is an example of a paraphyletic

genus that resulted from an over reliance on gross floral morphology in its

circumscription. Molecular approaches do not supplant studies of other features (Hillis,






1987). Studies of vegetative and floral characteristics are needed to augment molecular

data since most orchid subtribes are relatively uniform in non-floral aspects (Chase, et

al., 1994; Pridgeon, et al., 1999). A new understanding of phylogenetic relationships in

orchids will emerge only through the syntheses of data from various scientific disciplines

(Chase, et al., 1994). However, any morphological study of Orchidaceae must fully

consider the homoplasious nature of these characters.



Materials and Methods


Plants were grown in the Plant Science Facility at the University of Florida.

These were collected during field expeditions to Mexico and Dominican Republic,

donated by individuals or institutions, or purchased from commercial vendors. The

plants were photographed and specimens collected at various stages of their

reproductive cycle. Representative vegetative and floral material was pressed and dried

in a "Blue M" electric oven (Model #OV-510A-2) at 530 C for use as herbarium vouchers.

Flowers and capsules were observed under a microscope and photographed using a

Zeiss Tesovar. Flowers were dissected, attached to a Kodak projector slide cover glass

using transparent double stick tape and scanned with a Sharp JX-330 scanner at 600

dpi. This procedure is a modification of a technique developed at the National Museum

of Brazil (Valka Alves, 1996). Capsules were allowed to dehisce on the plant to observe

the method of opening. Entire flowers and capsules were also preserved in 95%

ethanol.








Morphological Characters


The morphological features were selected to include the characteristics that

taxonomists have traditionally used to either group or segregate the species and genera

of Laeliinae. Specific characters were selected to distinguish Encyclia's subgenera and

sections. Characteristics that are useful in identifying species tend to be homoplasious

across the subtribe (Arditti, 1992). Additional characters were then selected based on

the definition of subtribes of Dressler (1993). Laeliinae have a specific velamen type,

seed testa, and lack a column foot. These characters were obtained through direct

observation of living or preserved specimens. Certain characters were obtained from

published descriptions when living or preserved plants were not available (Withner,

1988; 1990; 1993; 1996; 1998).

Characters included in the analysis included several from vegetative morphology,

reproductive structures, and one secondary plant chemistry. Discrete character states

(present/absent) were used wherever possible. Size characters were defined in relative

terms (ratios/comparisons) where possible. Other measurements were delimited by

gaps in the data (Appendix B). Certain character states were delimited based on values

traditionally used by orchid taxonomists. A summary of characters and their states is

found in Table 2-1.



Vegetative Morphology


Vegetative morphology refers to all parts of the plant except the reproductive

structures. These characteristics are intrinsic to the plants existence and not dependent

on reproductive cycles. The parts examined were the growth habit, pseudobulbs,

leaves, and roots.






Whole plant

There are two general growth forms found in Orchidaceae, monopodial and

sympodial. However, only the sympodial form is found in Laeliinae. The plant habit has

been shown to be taxonomically useful (Pridgeon, et al., 1999). It describes whether a

plant is stationary or "mobile" (Figure 2-1). A plant that tends to grow in a stationary tuft

was coded as caespitosee." Whereas, a plant that "moves" by growing across a surface

was coded as "creeping." The plant size is based on the arbitrary value of 25 cm

because this is the value traditional used by orchidologists to delimit plant height

(McLeish, et al., 1995). Plants whose height is 25 cm or greater were coded as "large",

while plants less than 25 cm were coded as "small" (Appendix B). The stem shape is a

description of a single sympodial growth as a unit. A pencil-like stem was coded as

"stem" while a cane-like stem was coded as "cane" (Figure 2-2).



Orchid pseudobulbs

Epiphytic orchids often have enlarged portions of the stem called pseudobulbs,

which are used for water and carbohydrate storage. These organs were coded as being

present or absent. The shape and composition of the pseudobulbs also were coded as

discrete characters. Pseudobulbs may be circular in cross section or flattened (Figure 2-

3). The pseudobulb may arise directly from the rhizome or have a stipe (stalk) between

it and the rhizome (American Orchid Society, 1974). Pseudobulbs with a stipe were

coded as "stipitate" (Figure 2-4). The pseudobulb may form in one intemode or it can

consist of several internodes (Dressier, 1993). This character has been used to

distinguish genera (Pridgeon, et al., 1999). A single-noded pseudobulb was coded

"heteroblastic" while a pseudobulb with multiple nodes was coded "homoblastic" (Figure

2-5). Typically, pseudobulbs have a solid interior, but they may have a hollow cavity








(Figure 2-6). These cavities are often associated with colonies of ants. The surface

texture of the pseudobulb was coded as "smooth," "sandy," "wrinkled," or

"ridged/grooved" (Figure 2-7). Pseudobulbils are a small secondary swelling above the

lowest leaf atop a pseudobulb (Figure 2-8). This previously undescribed feature is

analogous to bulbils (Harris and Harris, 1994). Pseudobulbs were coded as "large" if

they exceeded 7 cm in length and as "small" if 7 cm or less (Appendix B). The

pseudobulb shape was based on a vertical section and coded as follows (Arditti, 1992):

"ovoid" if the slice is oval; "conic-ovoid" if the slice is an inverted cone on an oval;

"ellipsoid" if the slice is a ellipse; "cylindrical" if the slice is rectangular; or "spindle-

shaped" if the slice is rectangular and swollen on one end (Figure 2-9).


Orchid leaves

Orchid leaves have parallel venation like most other monocotyledonous plants.

The shapes of orchid leaves vary from typical elliptic, ovate, lanceolate or oblanceolate

leaves to terete or grass-like. The leaves of a plant are the primary photosynthetic

organs that are sometimes modified for water storage. Leaf vemation is systematically

useful (Pridgeon, et al., 1999). Leaf types were coded as "fleshy" for soft thick water

storage leaves, "intermediate" for typical coriaceous orchid leaves, and "grass-like" for

very thin narrow leaves. The leaf position can be either distichous along the stem or

terminal near the top of the pseudobulb (Figure 2-10). Leaf shapes were coded as:

"linear" for a long narrow leaf, "linear-elliptic" for a long leaf slightly swollen in the middle,

"oblong-elliptic" for a wider leaf that is slightly swollen, or teretee" for a leaf that is pencil-

like with a groove (Figure 2-11). The leaf width was coded as narrow if 2.5 cm or less,

or broad if greater than 2.5 cm. Leaves in Laeliinae are duplicate and usually emerge

folded (Dressler, 1993). The leaf surface posture was coded as "conduplicate" for






duplicate leaves that do not open entirely; "flat" for duplicate leaves that open so the

margins and midrib are in the same plane; or teretee" for circular leaves with only a

groove (Figure 2-12). The leaf posture was coded as "rigid" for leaves that do not bend

or "flexible" for leaves that bend under their own weight (Figure 2-13). The leaf margin

was coded "entire" for a smooth undisrupted edge or "erose-dentate" for a disrupted

(rough) margin (Figure 2-14). The typical number of leaves when a plant reached

reproductive maturity was coded as 1, 2, 3, or 4+. The leaf length is a relative

measurement made by comparing the leaf to the stem (pseudobulb) length and was

coded as being shorter or longer.


Orchid roots

Orchid roots function as a hold-fast (anchorage) for the plant, photosynthesis,

water and nutrient uptake and storage. These adventitious roots typically arise from the

rhizome. Root types were determined by cutting the root with a razor blade: thick soft

roots were coded as "fleshy," thin hard roots were coded as "sinewy," and roots with a

hard core surrounded by a fleshy covering were coded as "intermediate." Orchid roots

have a spongy layer of cells outside the exodermis known as the velamen that functions

for water storage (Figure 2-15). The velamen layers were counted under a light

microscope after hand sectioning of a living root or obtained from the literature

(Pridgeon, 1987; Arditti, 1992).

The Pleurothallis type velamen is characterized by one to three layers of cells

that are extended in radial direction (Porembski and Barthlott, 1988). Epidermal cells

were characteristically smaller if the velamen was multi-layered. The exodermal cells

are slightly thickened in the outer walls (Arditti, 1992).








The Epidendrum type velamen is characterized by 4-12 layers with endovelamen

cells that are extended in radial direction and thickenings that form composed ledges

(Porembski and Barthlott, 1988). Endovelamen cells are typically larger than the

epivelamen cells (Arditti, 1992).



Reproductive Morphology


Reproductive characters are harder to collect since the structures are transitory.

Nonetheless, reproductive characters are important in plant classification. However,

floral morphology in orchids is extremely plastic in evolutionary terms (Pridgeon, et al.,

1999). The structures examined were the inflorescence, the flower, the capsule

dehiscence, and the seed coat.


Plant inflorescence

The inflorescence is collectively the flowers and the flower-bearing branch (or

system of branches). If the inflorescence arises from a sheath, it was coded as having a

spathee" (Figure 2-16). Otherwise, the spathe was coded as absent. The form of the

inflorescence was coded as "simple" when the flowers were arranged along the

peduncle, "fasciculate" when the flowers are clustered near the end, or "scorpioid" if

coiled (Figure 2-17). The type of inflorescence was coded as "sessile" if the peduncle is

very short or absent, as a racemee" if the peduncle was unbranched, or as a paniclee" if

the peduncle was branched forming a rachis (Figure 2-18). The position of the

inflorescence was coded as "lateral" or "terminal." The inflorescence length was coded

as being "shorter" or "longer" based on the relative length in relation to the leaf length.






Certain species can flower on a previous year's inflorescence. This ability to re-flower

on old inflorescences was coded as "yes" or "no."


Orchid flowers

The reproductive structures of an angiosperm are collectively called a flower,

including the calyx, corolla, gynostemium, pollinarium, and ovary, which develop into a

fruit. Orchid flowers have several distinctive characteristics: bilateral symmetry

(zygomorphy), a labellum, a central column containing a rostellum and pollinia, and an

inferior ovary; the fruit is a capsule with minute seeds. Orchid flowers have an inferior

ovary located in the receptacle that does not develop unless the flower is pollinated.

The perianth consists of two alternating whorls, the sepals (calyx) and the petals

(corolla). The "male" and "female" structures (style, stigma, and filament) are fused into

a central column (gynostemium). The entire stem of the flower including both the inferior

ovary and the pedicel is typically called the pedicell" by orchidologists since the ovary

development is delayed until pollination. If there is an articulation (abscission zone)

between the ovary and the true pedicel that allows the flowers to fall off leaving a

persistent pedicle on the rachis, then the ovary was coded as "jointed." The number of

flowers was coded as "few" for 1-3 flowers and "many" for 4 or more flowers (Appendix

B). The orientation of the flower was coded as "resupinate" if the flower twists 1800

during opening, orienting the lip on the bottom, or as "non-resupinate" if the bud does not

rotate, leaving the lip uppermost (see Figure 2-19)(Emst and Arditti, 1994). Flower size

was coded as "small" for flowers with a natural spread of 2.5 cm or smaller and "large"

for flowers larger than 2.5 cm (Appendix B). If the veins in the flower have a different

color than the surrounding tissue, producing striations, the character of colored veins

was coded as "present" (Figure 2-20). The presence or absence of a floral nectary was








coded as such (Figure 2-21). Typically, the pseudobulb matures before an inflorescence

is produced in Laeliinae. However, some species produce the inflorescence before the

pseudobulb matures. The growth stage of the pseudobulb when flowers are produced

was coded as "mature" if the pseudobulb was fully formed before flowering or as

"immature" if the flowers are produced while the pseudobulb is forming (Figure 2-22).

Sepals and petals. The sepals and petals make up the perianth of the flower.

In orchids, the outer whorl consists of three sepals, while the inner whorl consists of two

petals and a third modified petal called the lip or labellum. The lateral sepals were

binary coded as being "free" or "fused." The amount of fusion was then coded as being

"none," connatee at base" or connatee" (Figure 2-23). The length of the sepals was

coded as a relative measurement in comparison to the petals and was coded as "longer"

or approximately "equal" (Figure 2-24). The sepal width is also a relative measurement

in comparison to the petals and was coded as being "narrower," "similar," or "wider"

(Figure 2-25). The sepal and petal margins were coded as "undulate" or "not undulate"

(Figure 2-26). The general appearance of the sepals and petals, i.e., color, markings,

etc., was coded as "similar" or "different."

Labellum. One of the petals of an orchid flower is highly modified to form a lip.

The lip is important adaptation to facilitate cross-pollination (Pridgeon, et al., 1999).

When the lip was fused to the column it was coded as "adnate," if the lip is attached to

the receptacle in same place as the column it was coded as "partially adnate," otherwise

the lip was coded as "free." The degree of lip adnation was separated into "partially

adnate," "basally adnate" if attached to base of column, adnate "less than of column,

or adnate "more than of the column (Figure 2-27). The general configuration of the

lip was coded as "tubular" if it encircles the column or "not tubular" (Figure 2-28). The

attachment of the lip was coded as "hinged" if it was flexible allowing movement or "not






hinged" (Figure 2-29). The transition of the labellum from the base to the blade was

coded as "gradual" for a smooth change in lip shape or "abrupt" for a rapid change

(Figure 2-30). The number of lip lobes was coded as, 1, 2, or 3 (Figure 2-31). The size

of the side lobes is a relative measurement in comparison to the mid-lobe and was

coded as "smaller," "equal," or "larger." The adnation of the side lobes to the column

was coded as "fused" or "free" (Figure 2-32). The side-lobe posture was coded as

"upturned" when they are perpendicular to the mid-lobe, "flat" when they are in the same

plane, "clasping" when they touch the column, "encircle" when they wrap around the

column and touch each other above, or "down-turned" when they are perpendicular in a

downward direction (Figure 2-33). The mid-lobe plane was coded as, "flat," reflexedd,"

recurvedd,' "cupped," or "tubular" (Figure 2-34). Calli adorn the upper surface of the

labellum near the anther cap. The callus shape was coded as "none" when it was

absent, as "platform," "1 ridge," "2 ridges," "3 plus keels," "transverse ridges," or

"papillate" when various structures occurred (Figure 2-35). The lip shape and callus can

be diagnostic in orchid classification (Pridgeon, et al., 1999).

Column. The gynostemium or column is formed through a complete fusion of

stigma, style, and filaments. The pollen masses, pollinia, are located in the anther cap,

which is near the apex of the column. The stigmatic surface is a sticky depression on

the lower side of the column. There is a wall of tissue between the stigma and the

pollinia, known as the rostellum, that prevents self-pollination (Figure 2-36). The column

foot is a ventral extension near the base of the column that was coded as "present" or

"absent" (Figure 2-37). The general posture of the column was coded as "straight" or

"curved" (Figure 2-38). Appendages on the lower side of the column are known as

wings. These wings may be "present" or "absent" (Figure 2-39). The column has three

teeth at the tip that surrounds the anther cap. If the top (mid) tooth has a ligulate








appendage, it was coded as "present" (Figure 2-40). The mid-tooth shape was coded as

"deltoid" if it is triangular in shape, "obtuse" if rounded, "lanceolate" if pointed, "truncate"

if square, or fimbriatee" if it has finger-like extensions (Figure 2-41). The mid-tooth size

is a relative measurement to column size and was coded as "small" or "large" (Figure 2-

42). The relative length of the mid-tooth to the lateral teeth was coded as "shorter,"

"equal," or "longer" (Figure 2-43). The lateral tooth shape was coded as "deltoid,"

"obtuse," "lanceolate," "truncate," fimbriatee," "wing-like," or "hooked" (Figure 2-44). The

column teeth are separated by sinuses that were coded as "shallow" or "deep." The

anther cap sits between the column teeth. If the mid-tooth presses down on the anther

cap, it was coded as appressedd" (Figure 2-45). The length of the anther cap is a

relative measurement in relation to the mid-tooth. This length was coded as "subequal"

unless the anther cap protrudes beyond the mid-tooth, a condition that was coded as

"protruding" (Figure 2-46). The anther position has considerable significance in orchid

classification (Pridgeon, et al., 1999). Typically, the anther cap is in a terminal position

in Laeliinae. However, it may rarely occur on top of the column in Epidendroideae

(Figure 2-47).

The pollinia form inside the anther cap. Pollinium morphology for the

Epidendreae was illustrated by Brieger (1975; 1976). However, developmental studies

suggest that pollinia number may be a misinterpreted character state (Freudenstein and

Rasmussen, 1999). The number of pollinia was coded as 2, 4, 6, 8, or 12. The shape of

the pollinia is ovoid. If these are compressed in one plane they were coded as

"flattened" (Figure 2-48). The relative size of the pollinia, to each other, was coded as

"equal" or "unequal." The pollinia can be free or attached by a stem at the base. This

stem is a caudicle if it is an extension of the pollinia, or a stipe if it is of stigmatic origin.

The wall of tissue (rostellum) separating the pollinia from the stigmatic surface can have






a "thin" or "thickened" center. If the pollinia stem is attached to the rostellum in such a

way that the rostellum tears when the pollinia are removed, then the pollinia is said to

have a viscidium. The present or absence of the viscidium was coded likewise (Figure

2-49). The relative position of the rostellum in the column was coded as "vertical"(I),

"horizontal"(-), or "inclined"(/) when the column is held in a horizontal position.



Seed capsule

The capsule of orchids can contain several million seeds (Arditti, 1992). The

seed consist of a tiny embryo and a net-like testa. The embryo lacks a cotyledon and

endosperm is also lacking. The general capsule shape is based on its cross section,

and was coded as being "uniform" or "triangular." The triangular shaped capsules were

grouped into "3-winged" or "unwinged" (Figure 2-50). Orchid capsules release seeds by

opening a suture along the midline of each carpel during dehiscence (Pridgeon, et al.,

1999). The mechanism of opening is either a suture that splits open or a suture that is

covered by a strap of tissue, which lifts to uncover a suture (Figure 2-51). This

previously unreported strap of tissue was coded as "present" or "absent." The ovary

may be located in the receptacle directly behind the perianth or near the attachment of

the receptacle to the pedicel. When the ovary is near the base of the receptacle the

capsule apex has a beak, which was coded as "present" or "absent" (Figure 2-52). The

surface texture of the capsule was coded as being "smooth", "warty", or "ribbed" (Figure

2-53).

The seed type is defined on the basis of size and surface characteristics of the

testa (Molvray and Kores, 1995). The ornamentation of the seed coat is taxonomically

significant (Pridgeon, et al., 1999). The seed of the Pleurothallis type are 150-300 Lm

long and 2-3 testa cells in length. The testa cells are all of the same length with flat








marginal ridges that are topped with a distinct cell border and with the anticlinal walls

having prominent thickenings (Rauh, et al., 1975). The seed of the Elleanthus type are

about 200 um long (Barthlott, 1976). The medial testa cells are strongly elongate while

the basal and apical cells are slightly elongate. The cells of the testa are deeply trough-

like with cell-border ridges. The periclinal walls have longitudinal reticulate thickenings.

The seed of the Epidendrum type are elongate to 500-1000 um long (Barthlott, 1976).

All testa cells are similar with cell comers that are acute-angled. The cell border is not

visible and the anticlinal walls are narrow, high and sharp-angled (Figure 2-54). The

seed type was coded as "Elleanthus" type, "Pleurothallis" type or the "Epidendrum" type

(Dressler, 1993).



Secondary Plant Compounds


Secondary chemistry attributes important ecological concepts to floral biology.

Flowers of Encyclia subgenus Osmophytum precipitate glycoside crystals when fixed in

ethanol (Pabst, et al., 1981). This secondary chemistry character of glucoside crystals,

flavonoid aglycone structure and linked carbohydrate sidechain of glucorhamnose, is

easily observed by preserving flowers in ethanol with 5% sodium hydroxide (Ferreira, et

al., 1986). These crystals fluoresce under ultraviolet light, probably adding to the

visibility of flowers for insect pollinators in a dense forest. Flowers were preserved in

95% ethanol to precipitate glucoside crystals that can be observed in the glass specimen

jar. The presence of crystals in the flower can also be detected by a sandy feel when

cutting the column of a flower with a razor blade. These crystals were coded as

"present" or "absent" (Figure 2-55).






Morphological Phylogenetic Analyses


The morphological matrix (Table 2-2) was constructed using MacClade 3.08

(Maddison and Maddison, 1992). A parsimony analysis was conducted using PAUP"

4.0 (Swofford, 1998). Due to the size of the matrix, a heuristic algorithm was preformed.

This algorithm is not guaranteed to find the shortest tree. However, the search strategy

used was designed to locate the islands with the shortest trees. This was accomplished

by running a large number of replicates but only saving a minimum number of trees (10)

per replicate. Once the islands of shortest trees are located additional swapping

identifies all the equally parsimonious trees on those islands (Maddison, 1991).

Confidence in the results is measured using several statistical methods. The first

criterion is tree length, (i.e., number of steps) with the shortest trees being the most

parsimonious (Felsenstein, 1978b). The Consistency Index (CI) is a measure of how

well the data fits tree topology (Kluge and Farris, 1969). The Retention Index (RI) is a

measure of the preservation of synapomorphies on a tree (Farris, 1989a). The Rescaled

Consistency index (RC) is a combined index of CI and RI that allows comparison of fit

between characters that reaches zero when maximum homoplasy is present (Farris,

1989b). All of the above ensemble tree scores were reported. The morphological matrix

was analyzed using both equal-weighted and weighted characters. Confidence in tree

topology was measured using bootstrap and decay analyses. Bootstrap involves

resampling of the data matrix in each replication creating random pseudo-states for 50

percent of the characters (Felsenstein, 1985). Bootstrap provides an indication of the

degree of support for a particular clade where 70 to 75 percent bootstrap is considered

"good" (Sanderson, 1989). Decay (Bremmer support) is a method of analysis that seeks

to find the shortest tree that is incompatible with a clade. In other words, the decay

analysis determines when a clade "collapses" in longer trees (Bremer, 1988). The








decay index (d) is the number of steps required to find a tree that breaks apart a clade

(where the clade decays) (Bremer, 1994).



Equal-Weighted Analysis


The morphological matrix was first analyzed with every character having a weight

of "1." This equally weighted analysis is often referred to as an unweightedd" analysis.

The assumption is that all characters have equal complexity or importance.


Equal-weighted tree search

The initial equal-weighted heuristic search criterion was set for maximum

parsimony. All characters had weight of 1 and were unordered. Of the 82 characters in

the matrix, 81 are parsimony-informative and one was parsimony-uninformative. Non-

applicable (n/a) character states are treated as "missing." Multi-state taxa interpretation

depends on"(and)" versus "{or)" designation. The starting trees for the heuristic search

are obtained via stepwise addition using random addition sequence. One tree was held

at each step during stepwise addition for each of the 1000 replicates. The branch-

swapping algorithm selected was subtree-pruning-regrafting (SPR). The steepest

descent option not selected. No more than 10 trees of score (length) greater than or

equal to 631 were saved in each replicate. If maximum branch length was zero, then the

branches were collapsed creating polytomies. The MULTREES option was selected to

save all the most parsimonious trees. Topological constraints were not enforced during

the search and the trees were unrooted (the search criterion requires unrooted trees).

Since the initial search limited the number of trees saved to 10 per replicate,

additional branch swapping was required to find all the equally-parsimonious trees of






that length. The shortest trees saved in the first round of 1000 replicates were then

swapped to completion to find all the trees of that length. The starting trees were

arbitrarily dichotomized by PAUP before branch swapping.


Equal-weighted decay analysis

AutoDecay (Eriksson, 1998) was used to construct a PAUP command file of 63

constraint trees. The number of constraint trees is determined by AutoDecay based on

the number of taxa and the results of the previous tree search. The PAUP analysis was

run for 100 replicates for each constraint tree using the HSEARCH parameters

ADDSEQ=random, NREPS=100, RSEED=1, NCHUCK=10, and CHUCKSCORE=222.

The results of these searches are saved in a log file that is extracted by AutoDecay. The

output of the AutoDecay extraction is a text file of decay values and a tree file. Tree files

can be viewed and printed with the TreeView software package (Page, 1996).


Equal-weighted bootstrap analysis

A bootstrap analysis replaces 50 percent of the characters with character states

randomly selected from the matrix. A heuristic search follows each of the 1000

replicates of random replacement. The same parameters are used as the tree search

except the number repetitions of heuristic random addition is reduced to 10 and the

branch-swapping algorithm was changed to nearest-neighbor interchange (NNI). A

bootstrap analysis produces a majority rule consensus tree that indicates the percentage

that each clade was present following each round of replacement.








Weighted Analysis


A weighted analysis is used to reduce the effect of parallelisms and reversals by

estimating the phylogenetic value of each character. Homoplasious characters are

down-weighted from the base value. This technique is useful to reject some equally-

parsimonious trees but can result in longer trees.



Weighted tree search

The trees from the equal-weighted search were used to assign weights to each

character in the matrix (Table 2-2). These initial characters were reweighted using a

base weight of 1000 based on the maximum value of Rescaled Consistency (RC)

indices. This index is a combination of the Consistency Index (CI) and the Retention

Index (RI). The search parameters were the same as used for the equal-weighted tree

search. The weighted trees collected after the 1000 replicates were then swapped to

completion.


Weighted decay analysis

The weighted decay analysis used the same protocol as the equal-weighted

decay analysis. The base weight of 1000 was used to adjust the results for comparison.

Since a weighted decay analysis uses different values for each step, the decay values

are not whole numbers.






Weighted bootstrap analysis

A bootstrap analysis of 1000 replicates using a heuristic search was conducted

on the weighted matrix. The search parameters used were the same as for the equal-

weighted bootstrap except simple weighting was used for this analysis.



Morphological Results


The results of the morphological analysis are given as trees scores and tree

topologies. Only a strict consensus tree is presented for the equal-weighted analysis.

Both an individual tree and a strict consensus tree are presented for the weighted

analysis.



Equal-weighted Results


The initial tree search found 90 equally parsimonious trees. When these trees

were swapped to completion, 32,700 equally parsimonious trees with a length of 631

steps were identified. The parsimony tree scores for these topologies were: CI = 0.225,

RI = 0.619, and RC = 0.139. The strict consensus of these trees is found in Figure 2-56.



Weighted Results


The initial weighted tree search found 204 equally parsimonious trees. The

characters were successively reweighted until the weights stabilized (4 rounds). When

these trees were swapped to completion 20 equally parsimonious trees were identified.

The parsimony tree scores for these topologies were: Length (L) = 665 steps, Cl =








0.214, RI = 0.592, and RC = 0.126. The strict consensus of these trees is shown in

Figure 2-57. The support for these trees was measured using bootstrap, and decay

values. Figure 2-58 is a randomly selected tree showing individual branch lengths.



Morphological Discussion


Early classification schemes relied solely on floral morphology using a subjective

phenetic approach (Swartz, 1800; Richard, 1818; Lindley, 1826). Vegetative characters

were not used for classification until the work of Pfitzer (1819). Morphological characters

from pollen, seeds and anatomy have only recently been introduced (Dressier and

Dodson, 1960). Initial attempts at using parsimony analysis for morphological data

proved less than satisfactory (Bums-Balogh and Funk, 1986). This was partially caused

by misplacement and misinterpretation of character states (Dressier, 1987). There is a

large amount of convergence and parallelism in both floral and vegetative characteristics

in Orchidaceae (Pridgeon, et al., 1999). A recent cladistic study of Orchidaceae based

on morphology supported the recognized subfamilies as monophyletic but provided poor

resolution at tribal levels (Freudenstein and Rasmussen, 1999). Robert L. Dressier said,

"This is a bad time to offer hypotheses about orchid phylogeny based only on

morphology" (Pridgeon, et al., 1999). Although homoplasy itself is not bad, the pattern

of homoplasy in morphological characters may obscure relationships in the orchid family.

However, synthesis of morphological and DNA data sets is expected to yield a

maximally informative data set (Freudenstein and Rasmussen, 1999).

The equally-weighted analysis of the morphological matrix produced very little

resolution in the subtribe (Laeliinae). This may be due to a small number of characters

or the homoplasious nature of the characters at the specific level. Generic level studies






of Laeliinae using generalized characters produce better resolution (Higgins, 1997). The

weighted analysis produced good resolution (with support) for the five sections of

Encyclia (Figure 2-57). The two exceptions are the placement of Encyclia kienastii and

E. subulatifolia. Meiracyllium falls between Encyclia subulatifolia and the rest of Encyclia

section Leptophyllum. Encyclia kienastii is positioned sister to sections Osmophytum,

Euchile, Encyclia, and Dinema. Hagsatera is the sister group of section Osmophytum.

However, these placements have weak bootstrap and decay support. Resolution of

Encyclia sensu Dressler as a clade is not supported.

The weighted analysis produced trees that are 34 steps longer than equally

weighted analysis. Real data sets may contain unreliable characters that do not contain

phylogenetic information as well as cladistically reliable characters. Successive

weighting is expected to produce a good estimate of the true tree (Farris, 1969). Since

the equal-weighted analysis produced an excessive number of equally parsimonious

trees, the matrix must contain a number of unreliable and homoplasious characters.

Examination of the branch lengths on the individual tree (Figure 2-58) revealed that the

polytomies in the strict consensus tree are in areas with short branch lengths.

Previous classifications based on phenetic groupings, often using a very limited

number of characters and are not supported using modem techniques. It is easy to

document errors in traditional classification (Dressier, 1990). For example, Dressler's

(1961; 1971) circumscription of Encyclia is not supported using a parsimony analysis of

morphological data. The weak support for the morphological phylogeny is most likely

due to homoplasy in the character states and the relatively few characters used. The

morphological analysis was used to augment the DNA analysis to clarify phylogenetic

relationships.








Table 2-1. Morphological characters and character states used in cladistic analysis.
Character Character States
1 Plant Habit 0= caespitose; 1=creeping
2 Pseudobulb 0=absent; 1= present
3 Plant Size 0=small (<25cm); 1= large (>25cm)
4 Stem Shape 0=stem; 1=cane
5 Pseudobulb Spacing 0=clustered; 1 =spaced; 2=superposed
6 Pseudobulb Base 0=not stipitate; 1 =stipitate
7 Pseudobulb Surface 0=smooth; 1=wrinkled; 2=ridged or grooved; 3=sandy
8 Pseudobulb Circumference 0=not flattened; 1 =flattened
9 Pseudobulb Interior 0=soild; 1 =hollow
10 Pseudobulb Content 0=homoblastic; 1 =hetroblastic
11 Pseudobulb Shape 0=cylindrical; 1=spindle-shaped; 2=ellipsoid ;3=ovoid;
4=conic-ovoid
12 Pseudobulb Size O=small (<7cm); 1 =large (>7cm)
13 Pseudobulbils 0=absent; 1= present
14 Ovary 0=jointed; 1 =not jointed
15 Leaf Type -=fleshy; 1=intermediate; 2=grass-like
16 Leaf Position 0=distichous; 1=terminal
17 Leaf Shape 0=linear; 1 =oblong elliptic; 2=terete; 3=linear elliptic
18 Leaf Width 0=narrow (<2.5cm); 1=broad (>2.5cm)
19 Leaf Surface 0=conduplicate; 1= flat; 2=terete
20 Leaf Posture 0=flexible; 1= rigid
21 Leaf Margin 0=entire; 1=erose-dentate
22 Leaf Number 0=one; 1 =two; 2=three; 3=four+
23 Leaf Length to Stem 0=shorter; 1=longer
24 Flavonoid Crystals 0=absent; 1= present
25 General Capsule Shape 0=uniform; 1=-3-winged or triangular
26 Specific Capsule Shape 0=ellipsoid; 1 =ovoid; 2=triangular; 3=3-winged
27 Capsule Suture Strap 0=absent; 1=present
28 Capsule Apex 0=not beaked; 1=beaked
29 Capsule Surface 0=smooth; 1 =warty; 2=ribbed; 3=muricate
30 Inflorescence Form 0=simple; 1=fasciculate; 2=scorpoid
31 Inflorescence Type 0=raceme; 1=panicle; 2=sessile
32 Inflorescence Position 0=terminal; 1=lateral
33 Inflorescence Length 0=less than leaf; 1=more than leaf
34 Floral Spathe 0=present; 1=absent
35 Flower Position 0=nonresupinate; 1= resupinate
36 Flower Number 0=few: one to three; 1 =many: four or more
37 Flower Size 0=small (<2.5cm); 1=large (>2.5cm)
38 Flower Veins Colored 0=no; 1=yes
39 Flowering Pseudobulb Stage 0=mature; 1=immature
40 Reflower Old Inflorescence 0=no; 1=yes
41 Floral Nectary 0=absent; 1 = present
42 Column Foot 0=absent; 1= present
43 Column Posture 0=straight; 1= curved
44 Column Size 0=stout; 1 =enlongate
45 Column Wings 0=absent; 1=present
46 Column Midtooth Appendage 0=absent; 1 =present
47 Column Midtooth Shape 0=truncate; 1=obtuse; 2=deltoid; 3=lanceolate; 4=frimbrate
48 Column Midtooth Relative 0=small; 1=large
Size
49 Column Mid/lateral-tooth 0=shorter; 1--equal; 2=longer
Length






Table 2-1---continued.


Lip Lobes
Lip Configuration
Lip Attachment
Lip Transition
Side to Midlobe Size
Lip Side Lobes to Column
Lip Side Lobe Posture

Lip Midlobe Plane
Lip Callus Shape

Velamen Type
Velamen Layers
Seed Type
Root Type


Character
50 Column Lateral-tooth Shape

51 Column Sinuses
52 Column Midtooth on
Anthercap
53 Antercap Position
54 Anthercap to Midtooth Length
55 Pollinia Number
56 Pollinia Shape
57 Pollinia Size
58 Pollinia Attachment
59 Rostellum Center
60 Rostellum Position
61 Viscidium
62 Lateral Sepals Fusion
63 Lateral Sepal Fusion Amount
64 Sepal to Petal Length
65 Sepals and Petals Margin
66 Sepals and Petals Shape
67 Sepals to Petals Width
68 Lip Adnation to Column
69 Lip Adnation


Character States
0=truncate; 1=obtuse; 2=deltoid; 3=lanceolate;
4=frimbrate; 5=hooked; 6=wing-like
0=shallow; 1=deep
O=appressed; 1=not appressed

O=terminal; 1=top; 2=bottom
O=subequal; 1=protrudes
O=two; 1=four; 2=six; 3=eight; 4=twelve
O=not flattened; 1=flattened
O=equal; 1 =unequal
O=stipe; 1 =caudicle; 2=none
O=not thickened; 1 =thickened
O=horizontal; 1=inclined; 2=vertical
present; 1=absent
0=free; 1=fused
O=none; 1=connate at base; 2=connate
O=equal; 1=longer
O=not undulate; 1=undulate
O=not similar; 1 =similar
O=narrower; 1=similar; 2=wider
O=free; 1=partially adnate; 2=adnate
O=free; 1=partially adnate; 2=basally adnate; 3=less than
half; 4=more than half; 5=complete
O=one; 1=two; 2=three
O=not tubular; 1=tubular
0=hinged; 1=not hinged
0=abrupt; 1=gradual
0=equal or smaller; 1=larger
O=free; 1=fused
0=flat; 1=uptumed; 2=clasp column; 3=encircle column;
4=tumed down
0=flat; 1 =tubular; 2=recurved; 3=cupped; 4=reflexed
0=platform; 1 =one ridge; 2=two ridges; 3=three+ keels;
4=flat 5=transverse ridges; 6=papillae; 7=absent
0=Pluerothallus; 1=Epidendrum
O=one-two; 1 =three-four; 2=five-six; 3=seven-eight
0=Elleanthus; 1=Epidendrum; 2=Pleurothallis
0=fleshy; 1= intermediate; 2=sinewy


I II I







Table 2-2. Morphological Matrix.


Character States
Restrepiella ophiocephala
Pleurothallis racemillora
Ponera striata
Isochilus major
Epidendrum ibaguense
Epidendrum conopseum
Nidema boothii
Scaphyglottis pulchella
Hexisea imbricata
Reichenbachanthus species
Hexadesmia species
Acrorchis roseola
Jacquiniella teretifolia
Hagsatera brachycolumna
Homalopetalum pumilio
Meiracyllium trinasutum
Psychilis mcconnelliae
Psychilis krugii
Broughtonia negrilensis
Tetiamicra elegans
Domingoa kienastii
Cattleyopsis lindenii
Brassavola cucullata
Laelia rubescens
Myrmecophila tibicinis
Cattleya dowiana
Rhyncholaelia glauca
Cattleya forbesii
Sophronitis cerua
Laelia purpurata
Schomburgkia splendid


1 2


3 4 5 6 7 8 9 10 11 12 13 14
1 0 n/a n/a n/a n/a n/a n/a n/a n/a n/a 0
0 0 n/a n/a n/a n/a n/a n/a n/a n/a n/a 0
1 1 n/a n/a n/a n/a n/a n/a n/a n/a n/a 1
1 1 n/a n/a n/a n/a n/a n/a n/a n/a n/a 1
1 1 n/a n/a n/a n/a n/a n/a n/a n/a n/a 1
0 0 n/a n/a n/a n/a n/a n/a n/a n/a n/a 1
0 n/a 1 1 1 1 0 1 2 0 0 1
1 0 2 1 0 0 0 1 0 1 0 0
1 n/a 2 1 2 1 0 1 0 1 0 1
1 1 2 n/a n/a n/a n/a n/a n/a n/a n/a 1
1 n/a 0 1 0 0 0 0 0 0 0 1
0 1 1 n/a n/a n/a n/a n/a n/a n/a n/a 1
1 1 n/a n/a n/a n/a n/a n/a n/a n/a n/a 1
1 n/a 2 1 1 1 0 0 2 1 0 1
0 n/a 1 0 3 0 0 1 0 0 0 1
0 0 n/a n/a n/a n/a n/a n/a n/a n/a n/a 1
1 n/a 1 0 2 0 0 0 0 1 0 1
1 n/a 1 0 2 0 0 0 0 1 0 1
1 n/a 0 0 2 1 0 0 2 0 0 1
0 0 1 n/a n/a n/a n/a n/a n/a n/a n/a 1
0 n/a 1 0 3 0 0 1 0 0 0 1
0 n/a 0 0 2 1 0 0 0 0 0 1
1 0 ? ? ? ? ? ? ? 1 ? 1
0 n/a 1 0 2 1 0 0 (23) 0 0 1
1 n/a 1 0 2 0 1 1 0 1 0 1
1 n/a 1 0 0 0 0 0 0 1 0 1
1 n/a 1 1 0 0 0 1 1 1 0 1
1 n/a 1 0 0 0 0 0 0 1 0 1
0 n/a 1 0 0 1 0 1 0 0 0 1
1 n/a 1 0 0 0 0 0 0 1 0 1


1 1 1 n/a 1


1 2 0 0 0 0 1 0 1


15 16 17 18 19
1 1 1 1 1
1 1 1 1 1
2 0 0 0 0
2 0 0 0 0
1 0 1 1 1
1 0 1 0 0
1 1 0 0 0
1 1 1 0 0
1 1 3 0 0
0 1 2 0 2
2 1 0 0 0
0 0 3 0 1
0 0 2 0 2
1 1 3 0 1
0 1 1 0 0
0 1 1 1 1
11 0 0 0
11 0 0 0
1 1 1 1 0
0 0 2 0 2
0 1 1 0 2
1 1 1 0 0
0 1 2 0 2
1 1 1 0 0
1 1 1 1 0
1 1 1 1 1
1 1 1 1 0
1 1 1 1 1
1 1 1 1 1
1 1 1 1 0
1 1 1 1 0


20 21
1 0
1 0
0 0
0 0
1 0
0 0
0 0
0 0
1 0
1 0
0 0
1 0
1 0
1 0
1 0
1 0
1 1
1 1
1 1
1 1
1 1
1 1
1 0
1 0
1 0
1 0
1 0
1 0
1 0
1 0
1 0





Table 2-2--continued.
Character States
Restrepiella ophiocephala
Pleurothallis racemiflora
Ponera striata
Isochilus major
Epidendrum ibaguense
Epidendrum conopseum
Nidema boothii
Scaphyglottis pulchella
Hexisea imbricata
Reichenbachanthus species
Hexadesmia species
Acrorchis roseola
Jacquiniella teretifolia
Hagsatera brachycolumna
Homalopetalum pumilio
Meiracyllium trinasutum
Psychilis mcconnelliae
Psychilis krugii
Broughtonia negrilensis
Tetramicra elegans
Domingoa kienastii
Cattleyopsis lindenhi
Brassavola cucullata
Laelia rubescens
Myrmecophila tibicinis
Cattleya dowiana
Rhyncholaelia glauca
Cattleya forbesii
Sophronitis cernua
Laelia purpurata
Schomburgkla splendid


22 23
1 0
0 0
3 0
3 0
3 0
3 0
0 1
1 0
1 0
0 1
1 ?
3 ?
3 0
0 1
0 (01)
0 1
1 1
1 1
1 1
3 1
0 0
1 1
0 1
0 1
1 0
1 1
0 1
1 0
0 1
0 1
1 0


31 32 33
2 0 0
0 0 1
2 (01) 0
0 0 1
0 0 1
0 0 1
0 0 0
0 1 0
2 0 0
2 1 0
1 0 0
0 0 1
2 0 0
0 0 0
2 0 0
2 0 0
0 0 1
0 0 1
0 0 1
1 0
0 0
0 0 1
2 0 0
0 0 1
0 0 1
0 0 0
0 0 0
0 0 0
2 0 0
0 0 0
0 0 1
001


36 37
0 0
1 0
0 0
1 0
1 0
1 0
(01) 0
0 0
1 0
0 0
1 0
0 0
0 0
1 1
0 0
0 0
1 1
1 1
1 1
1 0
0 1
1 1
0 1
1 1
1 1
0 1
0 1
(01) 1
1 0
0 1
1 1


40 41
0 ?
0 1
0 1
0 1
0 1
0 1
0 0
0 1
0 1
0 1
0 1
0 1
0 1
0 0
0 1
0 0
1 0
1 0
0 1
0 0
1 1
0 1
0 1
0 0
0 0
0 1
0 1
0 1
0 1
0 1
0 0






Table 2-2-continued.
Character States 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
Restrepiella ophiocephala 0 0 0 0 1 0 1 1 0 0 0 0 1 0 0 1 0 2 7 1 2
Pleurothallis racemiflora 1 0 0 0 3 0 0 3 1 0 0 0 0 0 0 2 0 2 0 1 2
Ponerastriata 1 0 0 0 1 0 2 1 0 0 0 1 1 1 0 2 0 ? 0 1 1
Isochilus major 0 1 0 0 1 0 0 1 0 0 0 1 1 1 0 1 0 1 0 1 2
Epidendrum ibaguense 0 1 0 0 4 0 1 4 1 0 1 0 1 0 0 2 0 0 0 0 0
Epidendrum conopseum 1 1 0 0 2 0 1 2 0 0 0 0 1 0 0 2 0 0 0 0 0
Nidema boothii 1 1 0 0 2 0 1 6 1 0 0 1 1 1 1 1 0 2 1 0 0
Scaphyglottis pulchella 0 1 0 0 1 0 2 1 0 0 2 0 2 1 0 1 0 1 1 1 1
Hexisea imbricata 0 0 0 0 2 0 0 2 0 0 0 1 1 1 0 1 0 1 0 1 1
Reichenbachanthus species 0 1 0 0 1 0 1 1 0 0 0 0 1 0 0 1 0 1 0 1 1
Hexadesmia species 0 ? 1 0 ? 0 1 ? 0 0 0 0 2 ? 0 1 0 2 1 0 0
Acrorchis roseola 0 ? 0 0 0 0 2 1 0 0 0 1 1 1 0 1 0 2 1 1 1
Jacquiniella teretifolia 0 0 0 0 0 0 1 1 0 0 0 0 1 1 1 2 0 2 1 0 0
Hagsatera brachycolumna 0 0 0 0 1 0 1 1 0 0 0 0 3 1 0 1 1 2 0 0 0
Homalopetalum pumilio 0 1 1 0 2 1 2 0 1 0 0 0 1 1 1 1 0 0 1 1 1
Meiracyllium trinasutum 0 0 0 0 3 0 0 3 1 0 1 1 3 0 1 1 0 0 0 0 0
Psychilis mcconnelliae 0 1 0 0 2 0 1 1 0 0 0 0 1 1 0 1 1 2 1 0 0
Psychilis krugii 0 1 0 0 2 0 1 1 0 0 0 0 1 1 0 1 1 2 1 0 0
Broughtonia negrilensis 0 1 1 0 1 0 1 1 0 0 0 0 1 1 1 1 1 2 1 0 0
Tetramicra elegans 0 ? 1 0 1 0 0 3 0 0 0 0 3 1 1 1 1 2 1 0 0
Domingoa kienastii 1 ? 1 0 1 0 0 1 1 0 0 0 1 1 0 ? 1 2 1 0 0
Cattleyopsis lindenil 0 1 1 0 1 0 0 1 0 0 0 1 3 1 1 1 0 2 1 0 0
Brassavola cucullata 0 0 0 0 4 1 1 4 1 0 0 0 4 1 1 1 0 2 1 0 0
Laelia rubescens 0 1 0 0 3 1 2 2 1 0 0 0 3 1 0 1 1 2 1 0 0
Myrmecophila tibicinis 0 1 0 0 2 0 1 2 0 0 0 1 3 1 0 2 1 2 1 0 0
Cattleya dowiana 0 1 0 0 1 0 1 1 0 0 0 1 1 1 0 0 0 2 1 0 0
Rhyncholaelia glauca 0 1 0 0 4 1 2 4 1 0 0 0 3 1 1 1 1 2 1 0 0
Cattleya forbesil 0 1 0 0 1 0 1 1 0 0 0 1 1 1 0 0 0 2 1 0 0
Sophronitis cemua 1 0 1 0 2 1 2 5 1 0 0 0 3 1 ? 1 0 1 1 0 0
Laelia purpurata 0 1 0 0 1 0 1 1 0 0 0 1 3 1 0 1 0 2 1 0 0
Schomburgkia splendid 1 1 0 0 0 0 1 3 0 0 0 1 3 1 0 1 1 2 1 0 0






Table 2-2-continued.
Character States
Restrepiella ophiocephala
Pleurothallis racemiflora
Ponera striata
Isochilus major
Epidendrum ibaguense
Epidendrum conopseum
Nidema boothii
Scaphyglottis pulchella
Hexisea imbricata
Reichenbachanthus species
Hexadesmia species
Acrorchis roseola
Jacquiniella teretifolia
Hagsatera brachycolumna
Homalopetalum pumilio
Meiracyllium trinasutum
Psychilis mcconnelliae
Psychilis krugii
Broughtonia negrilensis
Tetramicra elegans
Domingoa kienastii
Cattleyopsis lindenii
Brassavola cucullata
Laelia rubescens
Myrmecophila tibicinis
Cattleya dowiana
Rhyncholaelia glauca
Catleya forbesii
Sophronitis cemua
Laelia purpurata
Schomburgkia splendid


64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82


1 0 0 ? 0 0 2 0 0


1 0 0 1 0
1 n/a n/a n/a 0
1 n/a 0 0 4
1 0 0 1 0
0 0 0 0 0
0 0 0 0 0
1 n/a 0 1 0
1 0 0 0 0
1 n/a 0 n/a 0
1 0 0 0 2
1 n/a 0 n/a 0
1 0 0 1 2
1 0 0 1 0
1 0 0 0 4
1 n/a n/a n/a 0
0 n/a 0 n/a 3
0 0 1 1 0
0 0 1 1 2
1 n/a n/a n/a 1
0 0 1 0 0
0 0 0 0 2
1 n/a n/a n/a 1
0 n/a 0 n/a 0
1 0 0 2 0
1 1 0 2 0
1 0 0 2 1
0 0 0 0 4
1 1 0 3 1
0 0 0 1 1
1 ? ? 3 1
0 1 0 2 0


2 0


1


1 2
1 2
1 2
? 1
(12) 1
0 1
1 1
? 1
2 1
? 1
? 1
? 1
2 1
? 1
? 1
? 0
2 1
2 1
? 1
0 1
? 1
? 1
2 1
? 1
3 1
? 1
1 1
1 1
1 1
2 1
? 1






Table 2-2--continued.


Table 2-2---continued.


Character States
Encyclia citrina
Encyclia mariae
Encyclia mariae
Encyclia polybulbon
Encyclia polybulbon
Encyclia adenocaula
Encyclia bractescens
Encyclia aromatica
Encyclia cordigera
Encyclia tampensis
Encyclia tampensis alba
Encyclia dichroma
Encyclia diuma
Encyclia asperula
Encyclia candollel
Encyclia randii
Encyclia kienastii
Encyclia chimborazoensis
Encyclia fragrans
Encyclia aemula
Encyclia cochleata
Encyclia pygmaea
Encyclia pseudopygmaea
Encyclia vitellina
Encyclia glauca
Encyclia ionocentra
Encyclia prismatocarpa
Encyclia ochracea
Encyclia cretacea
Encyclia luteorosea
Encyclia luteorosea
Encyclia subulatifolia


3 4 5
0 n/a 0
0 n/a 0
0 n/a 0
0 n/a 1
0 n/a 1
1 n/a 0
1 n/a 0
1 n/a 0
1 n/a 0
0 n/a 0
0 n/a 0
1 n/a 0
1 n/a 0
0 n/a 0
0 n/a 0
1 n/a 0
1 n/a 1
1 n/a 1
0 n/a 1
0 n/a 1
1 n/a 1
0 n/a 1
0 n/a 1
0 n/a 0
0 n/a 0
1 n/a 1
1 n/a 1
0 n/a 1
1 n/a 0
0 n/a 0
0 n/a 0


14 15
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 1
1 2
1 2
1 0


0 0 0 0 n/a n/a n/a n/a n/a n/a n/a n/a n/a






Table 2-2-continued.
Character States
Encyclia citrina
Encyclia mariae
Encyclia mariae
Encyclia polybulbon
Encyclia polybulbon
Encyclia adenocaula
Encyclia bractescens
Encyclia aromatica
Encyclia cordigera
Encyclia tampensis
Encyclia tampensis alba
Encyclia dichroma
Encyclia diuma
Encyclia asperula
Encyclia candollei
Encyclia randii
Encyclia kienastii
Encyclia chimborazoensis
Encyclia fragrans
Encyclia aemula
Encyclia cochleata
Encyclia pygmaea
Encyclia pseudopygmaea
Encyclia vitellina
Encyclia glauca
Encyclia ionocentra
Encyclia prismatocarpa
Encyclia ochracea
Encyclia cretacea
Encyclia luteorosea
Encyclia luteorosea
Encyclia subulatifolia


24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


22 23
2 1
1 1
1 1
1 1
1 1
1 1
2 1
(01) 1
2 1
0 1
0 1
1 1
1 1
0 1
0 1
1 1
(12) 1
1 1
1 1
1 1
1 1
1 1
1 1
(12) 1
0 1
2 1
1 1
2 1
(12) 1
1 1
1 1
3 0


0
(01)
(01)
0
0
1
(01)
1
1
1
1
1
1
(01)
1
1
1
1
1
1
1
0
0
1
1
1
1
1
1
1
1
0


0 1
0 1
0 1
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 1
0 0
0 0
0 0
0 0
0 ?
0 ?
0 1






Table 2-2--continued.
Character States 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
Encyclia citrina 0 0 0 0 0 1 1 0 1 1 0 0 1 1 0 1 1 2 1 0 0
Encyclia mariae 0 0 0 0 0 1 1 0 1 1 0 0 1 1 0 1 1 2 1 0 0
Encyclia mariae 0 0 0 0 0 1 1 0 1 1 0 0 1 1 0 1 1 2 1 0 0
Encyclia polybulbon 0 1 1 0 1 0 0 3 1 0 0 0 1 1 0 2 1 2 1 0 0
Encyclia polybulbon 0 1 1 0 1 0 0 3 1 0 0 0 1 1 0 2 1 2 1 0 0
Encyclia adenocaula 0 1 1 0 2 0 1 2 0 0 0 1 1 1 0 1 1 2 1 0 0
Encyclia bractescens 0 1 1 0 2 0 1 2 0 0 0 1 1 1 0 1 1 2 1 0 0
Encyclia aromatica 1 1 0 0 2 0 1 2 0 0 0 1 1 1 0 1 1 2 1 0 0
Encyclia cordigera 0 1 0 0 2 0 1 2 0 0 0 1 1 1 0 1 1 2 1 0 0
Encyclia tampensis 0 1 1 0 2 0 1 2 0 0 0 1 1 1 0 1 1 2 1 0 0
Encyclia tampensis alba 0 1 1 0 2 0 1 2 0 0 0 1 1 1 0 1 1 2 1 0 0
Encyclia dichroma 7 1 1 0 2 0 1 2 0 0 0 1 1 1 0 1 1 2 1 0 0
Encyclia diuma ? 1 1 0 2 0 1 2 0 0 0 1 1 1 0 1 1 2 1 0 0
Encyclia asperula 0 1 1 0 1 0 1 2 0 0 0 1 1 1 0 1 1 2 1 0 0
Encyclia candollei 1 1 0 0 2 0 1 2 0 0 0 1 1 1 0 1 1 2 1 0 0
Encyclia randii ? 1 1 0 1 0 1 2 0 0 0 1 1 1 0 1 1 2 1 0 0
Encyclia kienastii 1 1 0 0 1 0 0 2 0 0 0 0 1 1 0 1 1 2 1 0 0
Encyclia chimborazoensis 0 0 0 1 1 1 1 1 1 1 0 0 1 1 0 1 1 2 1 0 0
Encyclia fragrans 0 0 0 1 1 1 1 1 1 1 0 0 1 1 0 1 1 2 1 0 0
Encyclia aemula 0 0 0 1 1 1 1 1 1 1 0 0 1 1 0 0 1 2 1 0 0
Encyclia cochleata 0 0 0 1 1 1 0 1 1 1 0 0 1 1 0 1 1 2 1 0 0
Encyclia pygmaea 0 0 0 1 3 1 2 2 0 1 0 0 1 1 0 1 1 2 1 0 0
Encyclia pseudopygmaea 0 0 0 1 1 1 2 2 0 1 0 0 1 1 0 1 1 2 1 0 0
Encyclia vitellina 0 0 0 1 0 1 0 0 1 1 0 0 1 1 0 1 1 2 1 0 0
Encyclia glauca 1 0 0 1 0 1 2 1 1 1 0 0 1 1 0 1 1 0 1 0 0
Encyclia ionocentra 0 0 0 1 4 1 1 6 1 1 0 0 1 1 0 1 1 2 1 0 0
Encyclia prismatocarpa 0 0 0 1 4 1 1 4 1 1 0 0 1 1 0 1 1 2 1 0 0
Encyclia ochracea 0 0 0 1 4 1 2 4 1 1 0 0 1 1 0 1 1 2 1 0 0
Encyclia cretacea 0 0 0 1 1 0 1 1 0 1 0 0 1 1 0 1 1 2 1 0 0
Encyclia luteorosea 0 0 0 0 1 0 1 6 0 0 1 1 1 1 0 1 0 0 0 0 0
Encyclia luteorosea 0 0 0 0 1 0 1 6 0 0 1 1 1 1 0 1 0 0 0 0 0
Encyclia subulatifolia 0 0 1 0 1 0 0 1 0 1 1 1 1 1 1 1 0 0 0 0 0






Table 2-2-continued.
Character States
Encyclia citrina
Encyclia maria
Encyclia maria
Encyclia polybulbon
Encyclia polybulbon
Encyclia adenocaula
Encyclia bractescens
Encyclia aromatica
Encyclia cordigera
Encyclia tampensis
Encyclia fampensis alba
Encyclia dichroma
Encyclia diuma
Encyclia asperula
Encyclia candollei
Encyclia randii
Encyclia kienastii
Encyclia chimborazoensis
Encyclia fragrans
Encyclia aemula
Encyclia cochleata
Encyclia pygmaea
Encyclia pseudopygmaea
Encyclia vitellina
Encyclia glauca
Encyclia ionocentra
Encyclia prismatocarpa
Encyclia ochracea
Encyclia cretacea
Encyclia luteorosea
Encyclia luteorosea
Encyclia subulatifolia


64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82


1
1
1
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0


0 0
? 0
? 0
? 0
? 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
1 0
0 0
0 0
? ?
? ?
? ?
? ?
1 0
1 0
0 0
0 0
0 0
0 0
0 0
0 0
? ?
? ?
? ?


0 1 2 1
? 1 2 1
? 1 2 1
? 0 2 1
? 0 2 1
2 0 2 1
2 1 2 1
2 0 2 1
2 0 2 1
2 0 2 1
2 0 2 1
2 2 2 1
2 0 2 1
2 2 2 1
2 1 2 1
2 0 2 1
2 0 2 1
? 3 0 1
7 3 0 1
? 3 0 1
? 3 0 1
2 2 2 1
2 2 2 1
4 0 0 1
1 4 0 1
0 0 0 1
0 0 0 1
1 0 0 1
1 0 3 1
? 0 6 1
7 0 6 1
? 2 6 1
? 26 1


2
7
0
0
0


7
1
?
?

2
2
3
2
?
3
1
?
(12)
1
?
(12)











0
?
7

1
1
?
1
?
7
?
0


, IIIIIIIII






Table 2-2-continued.
Character States 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Encyclia subulatifolia 0 0 0 0 n/a n/a n/a n/a n/a n/a n/a n/a n/a 1 0 0 2 0 2 1 0
Encyclia cyanocolumna 1 1 0 n/a 0 0 0 0 0 1 3 0 0 1 2 1 0 0 1 0 0
Encyclia tenuissima 1 1 0 n/a 0 0 0 0 0 1 3 0 0 1 2 1 0 0 1 0 0


Table 2-2-continued.
Character States 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
Encyclia subulatifolia 3 0 0 0 0 0 0 ? 0 0 0 1 1 0 0 0 1 0 0 1 0
Encyclia cyanocolumna 1 1 0 0 0 0 0 0 0 1 0 1 1 1 1 0 0 0 0 1 0
Encycliatenuissima 1 1 0 0 0 0 0 ? 0 0 0 1 1 1 (01) 0 0 0 0 1 0


Table 2-2--continued.
Character States 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
Encyclia subulatifolia 0 0 1 0 1 0 0 1 0 1 1 1 1 1 1 1 0 0 0 0 0
Encyclia cyanocolumna 0 0 0 0 1 0 0 6 0 0 1 1 1 1 0 1 0 0 0 0 0
Encyclia tenuissima 0 0 0 0 1 0 0 6 0 1 1 1 1 1 0 1 0 0 0 0 0


Table 2-2--continued.
Character States 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82
Encyclia subulatifolia 0 0 1 1 2 4 0 0 1 1 ? ? ? 2 6 1 0 1 1
Encyclia cyanocolumna 0 0 1 1 2 3 0 0 1 1 ? ? ? 0 6 1 1 1 1
Encyclia tenuissima 0 0 1 1 2 4 0 0 1 1 ? ? ? 0 6 1 ? 1 1










i


'V /


I
1%


Figure 2-1. Plant Habit: A. caespitose habit of Jacquiniella teretifolia; B. creeping habit of
Rhyncholaelia glauca.

















Figure 2-2. Stem Shape: A. pencil-like in Brassavola cucullata; B. cane-like in Epiden-
drum subulatifolium.






























Figure 2-3. Pseudobulb Circumference: A. round in Encyclia hanburir, B. flattened in
Prosthechea livida.


WW B -.


Figure 2-4. Pseudobulb Base: A. not stipitate in Encyclia tampensis; B. stipitate in
Prosthechea livida.




























Figure 2-5. Pseudobulb Intemode: A. homoblastic in Broughtonia negrilensis B. hetero-
blastic in Prosthechea livida.


Figure 2-6. Pseudobulb Interior: A. solid in Cattleya forbesii, B. hollow in Myrmecophila
tibicinis.




















































Figure 2-7. Pseudobulb Surface: A. smooth in Dinema polybulbon; B. wrinkled in
Encyclia randiir C. rough in Domingoa kienastit, D ribbed in Myrmechophila tibicinis.





























Figure 2-8. Pseudobulbil: Present in
Prosthechea livida.


Figure 2-9. Pseudobulb Shape:
A. cylindrical in Cattleya bowringiana.























































Figure 2-9. Pseudobulb Shape-continued: B. ellipsoid in Laelia speciosa; C. spindle-
shaped in Rhyncholaelia glauca; D. ovoid in Euchile citrina; E. conic-oviod in Encyclia
phoenicea.





























Figure 2-10. Leaf Position: A. distichous in Epidendrum ibaguense; B. Terminal in
Encvclia handurii


Figure 2-12. Leaf Surface: A. conduplicate in Myrmecophila tibicinis; B. flat in
Sophronitis cemua.





















































Figure 2-11. Leaf Shape: A. linear in Encyclia cyanocolumna; B. terete in Brassavola
cucullata; C. linear-elliptic in Prosthechea chimborazoensis- C. oblong-elliptic in
Pleurothallis racemiflora.




46



























EA4





























Figure 2-13. Leaf Posture: A. rigid in Encyclia steinbachir, B. flexible in Prosthechea
baculus.

































Figure 2-14. Leaf Margin: A. entire in Cattleya forbesii B. erose-dentate in Broughtonia
negrilensis.


+"~54


*"1(


-A.5
2. z* dL* I*-EIBI
*rla


. L$:

v t r'';*


Figure 2-15. Velamen: Epidendrum type in Encyclia amanda.























































Figure 2-16. Spathe: A. present in Prosthechea boothiana; B. absent in Encyclia
tampensis.






















































Figure 2-17. Inflorescence Form: A. simple in Prosthechea boothiana; B. scorpioid in
Isochilus linearis, C. fasciculate in Encyclia adenocaula.





















































Figure 2-18. Inflorescence Type: A. sessile in Dinema polybulbon; B. raceme in
Pleurothallis racemiflora; C. panicle in Encyclia profusa.






















































-- -u


Figure 2-19. Flower Orientation: A. resupinate in Encyclia bractescens; B. non-resupi-
nate in Prosthechea trulla.



















































Figure 2-20. Flower Striations: A. not visible in Prosthechea vitellina; B. visible in
Encyclia tenuissima.































Figure 2-21. Nectary: A. present in Euchile madrae; B. absent in Encyclia phoenicea.


Figure 2-22. Pseudobulb Maturity: A. immature in Nidema boothii; B. mature in
Sophronitis cemua.




























Figure 2-23. Sepal Fusion: A. free in Encyclia belizensis; B. fused in Pleurothallis
racemiflora.


Figure 2-24. Sepal Length: A. equal in Encyclia asperula; B. shorter in Pleurothallis
racemiflora.




















































Figure 2-25. Petal to Sepal Width Ratio: A. wider in Laelia purpurata; B. equal in
Cattleya forbesii.




















































Figure 2-26. Sepal and Petal Margins: A. not undulate in Euchile mariae; B. undulate in
Myrmecophila tibicinis.




















































Figure 2-27. Lip Adnation: A. partially fused () in Prosthechea tripunctata; B. com-
pletely fused in Epidendrum ibaguense.


















































Figure 2-28. Lip Configuation: A. not tubular in Encyclia steinbachii B. tubular in
Cattleyopsis lindenii.
























































Figure 2-29. Lip Attachment: A. hinged in Bulbophyllum putidum; B. unhinged in
Encyclia cyanocolumna.























O


O
Figure 2-30. Lip Transition: A. abrupt in Brassavola cucullata; B. gradual in Hexisea
imbricata.


~v~-























































Figure 2-31. Lip Lobes: A. one in Nidema boothii; B. two in Euchile maria, C. three in
Tetramicra elegans.





















































Figure 2-32. Side-lobe adnation: A. fused in Psychilis mcconnelliae; B. free in
Prosthechea concolor.





63











4b W


































b.~A
.1 '. d
0 -










So
? 1


















Figure 2-33. Sidelobe Posture: A. encircle in Encyclia canddlei; B. flat in Epidendrum
ibaguense; C. upturned in Encyclia bracteata; D. clasping in Encyclia asperula.





















































Figure 2-34. Lip Plane: A. flat in Encyclia randit, B. recurved in Encyclia dichroma; C.
reflexed in Epidendrum subulatifolium.






















































Figure 2-35. Callus Shape: A. platform in Prosthechea concolor, B. papillate in
Prosthechea livida; C. ridged in Encyclia tarumana.




























Drawn by Besvey Mear.


Figure 2-36. Rostellum: verticle in
Psychilis mcconnelliae.


Figure 2-37. Column Foot: present in
Ponera striata.


Figure 2-38. Column Posture: curved in Nidema boothii.
























































Figure 2-39. Column Wings: A. absent in Psychilis mcconnelliae; B. present in Encyclia
thienii.



















































Figure 2-40. Mid-tooth Appendage: A. absent in Encyclia asperula; B. present in
Prosthechea cochleata.

























































Figure 2-41. Midtooth Shape: A. deltoid in Encyclia cyperifolia; B. obtuse in
Prosthechea magnispatha; C. fimbriate in Brassavola cucullata; D. truncate in Euchile
mariae.




























Figure 2-42. Midtooth Size: A. large in Prosthechea glauca; B. small in Encyclia diuma.


Figure 2-43. Column Teeth Length: A. short in Encyclia bracteata; B. long in
Prosthechea tripunctata.





















































Figure 2-44. Lateral Teeth Shape: A. wing-like in Encyclia distantiflora; B. deltoid in
Encyclia tarumana; lanceolate in Dinema polybulbor, D. obtuse in Prosthechea vitellina.






























Figure 2-45. Anthercap Appression: appressed in Encyclia tarumana.


Figure 2-46. Anthercap Length: protruding in Encyclia randii.
























Figure 2-47. Anthercap Position: top in Meiracyllium trinasutum.


Figure 2-48. Pollinia Shape: flattened in
Brassavola cucullata.


Figure 2-49. Viscidium: present in
Epidendrum conopseum.


...gr




















































Figure 2-50. Capsule Shape: A. winged in Prosthechea cochleata; B. fusiform in
Dinema polybulborn C. 3-angled in Prosthechea radiata; D. ellipsoid in Cattleyopsis
lindenii.





























Figure 2-51. Capsule Suture: present in Figure 2-52. Ovary Apex: beaked in
Prosthechea livida. Brassavola cucullata.


Figure 2-53. Capsule Surface: A. warty in Encyclia adenocaula; B. smooth in
Prosthechea chondylobulbon.





















































Figure 2-54. Seed Type: Epidendrum type in A. Prosthechea cochleata; B. Prosthechea
chimborazoensis- C. Encyclia dichroma; D. Encyclia phoenicia.













































0


Figure 2-55. Druse-type Crystals: A. present in Prosthechea cochleata; B. absent in
Encyclia tampensis.





P-esuIepiellai,3phioccphia)a
Pleurothallis racemilora
Ponera striata
Isochilus major
Epidendrum iLaguense
Epidendrum conopseum
Nidema boothii
Homalopetalum pumilio
Meiracyllium trinasutum
Sophronitis cernua
Brassavola cucullata
Rhf, nchjolaeia glauca
Hagsatera brachycolumna
Psychilis mcconnelliae
PS) cihr s Irurij
Broughtonia nIgtensji
Cdaile :pss lindenii
-[Tetramicra elegans
Domingoa kienastii
Laelia rubescens
Myrmecophila tibicinis
Schomburgkia splendid
Cattleya dowiana
Cattleya forbesii
Laelia purpurata
En cii citrina
Encyclia mariae
Encycla mariae
Er'c;,.:l chimborazoensis
SE Encyclia fragrans
Encyclia cochleata
Encyclia aemula
Encyclia pygmaea
Encyclia pseudopygmaea
Encyclia vitellina
Encyclia glauca
Encyclia ionocentra
Encyclia prismatocarpa
Encyclia ochracea
Encyclia cretacea
Encyclia polybulbon
Encyclia polybulbon
Encyclia adenocaula
Encyclia bractescens
Encyclia aromatica
Encyclia cordigera
Encyclia tampensis
Encyclia tampensis alba
Encyclia dichroma
Encyclia diurna
Encyclia asperula
Encyclia candollei
Encyclia randii
Encyclia L ei-a ir'i
tncyclia iuteorosea
Encyclia luteorosea
Encyclia cyanocolumna
Encyclia tenuissima
Encyclia subulatifolia
[ Encyclia subulatifolia
Hexadesmia species
S apr, ~,~,iots pulchella
Hexisea imbricata
Reichenbachanthus cuniculatus
_IAcrorchis roseola
Jacquiniella teretifolia














Figure 2-56. Equally weighted morphological strict consensus tree for 32700 equally parsimonious trees with a length of 631 steps.
The parsimony tree scores were: CI = 0.225, RI = 0.619, and RC = 0.139.





99 Restrepiella ophiocephala
-9 Pleurothallis racemiflora
56 -- Ponera striata
Isochilus major
Epidendrum ibaguense
Meiracyllium trinasutum
-76 Encyclia luteorosea
671 Encyclia luteorosea
Enc:}c'i, cyanocolumna
51 056 Enciia tenuissima
100 Encyclia subularioha
Encyclia .ut'uliatIjira
Epidendrum conopseum
Nidema boothii
Hagsatera brachycolumna
77 Encutli3 chimborazoensis
70 58 Encyclia fragrans
60 Ericclha cochleata
75 E-.-- Enc.cha aemula
-5 Encyclia ochracea
S100 En,: ;3 pygmaea
0.93 Encyclia pseudopygmaea
90 Encyclia ionocentra
86 -- Encyclia prismatocarpa
1.97 71 Encyclia vitellina
0.82 Encyclia glauca
Enc)c: cretacea
99 Encyclia citrina
0.52 83-- Encyclia maria
S- Encyclia maria
Encyclia adenocaula
Encyclia bractescens
Encyclia asperula
Encyclia candollei
55 Encyclia tampensis
En, :i, tampensis alba
Encyclia ai.: hr,.ma
Encyclia diurna
Encyclia randii
2 Encyclia aromatica
Encyclia cordigera
_- O100 Encyclia polybulbon
0.81 00 Encyclia polybulbon
Encyclia kienastii
100 Psychilis mcconnelliae
.99 Psychilis krugii
64 4- Broughtonia negrilensis
Cattleyopsis lindenii
Tetramicra elegans
76 -- Brassavola cucullata
1.30 Rhyncholaelia glauca
Laelia rubescens
I77 Myrmecophila tibicinis
0.59 SchomDurgkia splendid
SSophronitis cernua
61 Laelia purpurata
9 93 Cattleya dowiana
Cattleya forbesii
Acrorchis roseola
Jacquiniella 'erelrioa
2.92 Hexadesmia species
Domingoa kienastli
Reichenbachanthus cuniculatus
Homalopetalum pumi ,,
Hexisea imbricata
Scaphyglottis pulchella











Figure 2-57. Weighted morphological strict consensus tree for 20 equally parsimonious trees. The tree scores were: Length (L) =
665 steps. Cl = 0.214, RI = 0.592, and RC = 0.126. Bootstrap percentages greater than 50 percent are given above the line. Decay
indices greater than 0.5 steps are indicated below the line.





6 Restrepiella ophiocephala
Pleurothallis racemiltora
8 10 Ponera striata
I- 'SOh,ilu major
67 Epidendrum ibaguense
16 Meiracyllium trinasutum
3 6 2 0 Encyclia luteorosea
8 Encyclia luteorosea
12 1 Encyclia cyanocolumna
12 1 Encyclia tenuissima
7 0 Encyclia subulatifolia
0
6 Encyclia subulatifolia
16Epidendrum conopseum
Nidema boothii
17 Hagsatera brachycolumna
3 EnrI ia chimborazoensis
8 1 En- c c,. fri3 r ins
Encyclia cochleata
1 Encyclia aemula
S2 ---Encyclia ochracea
4 2 Encyclia pygmaea
0 I Encyclia pseudopygmaea
5 8 3 Encyclia ionocentra
8 0 Encyclia prismatocarpa
5 1 5 Encyclia vitellina
5 9 Encyclia glauca
1 Encyclia cretacea
39 2 Encyclia citrina
0 Encyclia mariao
5_ 0 Encyclia maria
3 4 Encyclia adenocaula
12 4 Encyclia bractescens
3 Encyclia asperula
3 4 Encyclia candollei
3 00 Encyclia tampensis
3 -Encyclia tampensis alba
S11 3 0 Encyclia dichroma
1 3 Encyclia diurna
S1 Encyclia randii
5 1 Encyclia aromatica
2 Encyclia cordigera
5 10 0 Encyclia polybulbon
5 Encyclia polybulbon
8 5 Encyclia polybulbon
8- 5Encyclia kienastii
8 0 Psychilis mcconnelliae
4 Psychilis krugii
71 7 4 Broughtonia negrilensis
56 Cattleyopsis lindenii
14 Tetramicra elegans
11 13 Brassavola cucullata
5 L- Rhyncholaelia glauca
3 7 Laelia rubescens
3 1 --12 6 Myrmecophila tibicinis
Schomburgkia splendid
_2 12 Sophronitis cernua
4 6 8Laelia purpurata
7 Cattleya dowiana
-- Cattleya forbesii
Acrorchis roseola
L-1010 Jacquiniella teretifolia
0 Hexadesmia species
14 9 Domingoa kienastii
6 Reichenbachanthus cuniculatus
S12 Homalopetalum pumilio
12 Hexisea imbricata
7 Scaphyglottis pulchella












Figure 2-58. Randomly selected tree for weighted morphology. The branch lengths are indicated in number of steps. Note: The
morphological characters are mapped onto the tree resulting from the holomorphology analysis in Chapter 4.














CHAPTER 3
MOLECULAR STUDIES



Introduction


Genomic DNA provides an invaluable source of information for use in estimating

the phylogeny of all organisms. A molecular study consists of six phases: gene

selection, DNA acquisition, DNA amplification, DNA sequencing, data processing, and

data analysis. The gene selection phase starts with an online search of GenBank for

sequences from your taxonomic group and related taxa (Benson, et al., 1999). The data

from a few sequences for study taxa or their relatives can help detect presence of useful

variation. Different regions of the nuclear, chloroplast, or mitochondrial genomes can be

sequenced depending on the taxonomic level under study. A significant difference

among the genomes is that the nuclear genome arises from biparental inheritance,

whereas chloroplast and mitochondrial genomes are typically inherited from only one

parent. Different gene regions have different levels of mutation (variation). The

appropriate region must be chosen for the taxonomic level of the study. There are a

number of regions that have been sequenced and the usefulness for answering specific

taxonomic questions is shown in Figure 3-1 (Soltis, et al., 1998). A search of GenBank

can also provide the names of other systematists working on related taxa (or genes).

Other researchers can be an important resource for primer selection or design and

research protocols.






Research in Orchidaceae


Molecular plant systematic data can be analyzed with methods similar to

traditional morphology-based cladistics. Nucleotide changes in DNA sequences are

used as characters. Indels (insertions or deletions) in DNA sequences can also be

coded as characters in sequences that are relatively conserved. DNA sequences can

provide large number of characters that prove to be informative in parsimony analyses.

Molecular characters are not ordered or a prior polarized although polarization occurs

when tree is rooted with an outgroup. As with morphological characters, molecular

characters are subject to homoplasy because there are only four possible bases

(A,T,C,G).

The use of DNA sequencing for taxonomic studies is relatively new for

Orchidaceae. Current techniques with appropriate selection of DNA for the taxonomic

level being studied have proven successful (Soltis, et al., 1997). For example, ITS

sequences have been extremely valuable in evaluating monophyly at generic level and

below in Cypripedioideae (Cox, et al., 1997) and at the subtribal level and below in

Catasetinae (Pridgeon and Chase, 1998), Oncidiinae (Williams and Chase, unpubl.),

Stanhopeinae (Whitten, et al., 2000), Disinae (Douzery, et al., 1999), Gastrodieae and

Neottieae (Kores & Molvray, unpubl.), Pleurothallidinae (Pridgeon and Chase, unpubl.),

and Orchidinae (Pridgeon, et al., 1997).



Nuclear Genome


Sequencing ITS regions has provided a good source of nuclear DNA characters

for inferring intrageneric and intergeneric evolutionary relationships in many plant groups

(Baldwin, et al., 1995), and preliminary studies suggest it will also be useful in








Orchidaceae. The study of intrageneric relationships requires DNA sequences of

adequate size and fast evolutionary rate (nucleotide variation) (Nickrent, et al., 1994).

The ITS regions of rDNA have been shown to evolve at rates appropriate for examining

diverging lineages (Baldwin, 1992). The ubiquity of rDNA and available techniques for

rapid determination of the nucleotide sequence make rDNA a good tool for inferring

evolutionary relationships, except in cases of hybridization (Hamby and Zimmer, 1992).

In hybrids, the nuclear genome is a recombination of DNA from both parents. Thus,

hybrid ITS sequences can be very polymorphic. The nuclear genes that code for

ribosomal DNA are arranged in a tandemly repeated unit that is found in high and

variable copy number (Rogers and Bendich., 1987). The functioning regions are highly

conserved due to selective pressures while the spacer regions that do not code for a

functional RNA are not subject to the same selective pressures. The spacer regions are

not highly conserved and contain species-specific variation (Hamby and Zimmer, 1992).

In these internal transcribed spacer (ITS 1 & 2) regions, the number of substitutions is

typically twice as large between genera as within genera (Savard, et al., 1993). Thus,

ITS regions are valuable for taxonomic studies at lower subgeneric levels in some taxa.



Plastid Genome


Plastid DNA is a relatively abundant component of total plant DNA with a

conservative rate of nucleotide substitution (Palmer, et al., 1988). The chloroplast

genomes of photosynthetic land plants are circular DNA molecules ranging from 120 to

217 kilobase pairs. The genome contains two large inverted repeats that separate the

large and small copy regions (Palmer, 1986). Expansions or contractions of the inverted

repeat regions are largely responsible for variations in the molecular size of the genome.






Both strands of the chloroplast genome are actively expressed. Recombination does not

play a major role in cpDNA evolution, where biparental transmission is rare, and

intraspecific diversity is low. Chloroplast DNA provides uniparental (usually maternal)

phylogenetic markers (Soltis, et al., 1992). The types of mutations that are found in DNA

include: nucleotide rearrangements, point mutation substitutions, insertions, and

deletions. Studies of combined plastid DNA have been useful in cladistic analyses of

Amaryllidaceae, another petaloid monocotyledon (Meerow, et al., 1999). The tmL-F

region and matK gene were chosen for this study because they have appropriate levels

of variation (mutation).


trnL-F region

The DNA that encodes for the transfer RNA for leucine is designated as tmL.

The region of the chloroplast genome spanning the area from the tmL 5' exon to the tmF

5' exon is defined as the trnL-F(UAA) intron sequence (Taberlet, et al., 1991). This

non-coding region displays one of the highest frequency of mutation in the chloroplast

genome (Palmer, et al., 1988). Additionally, length mutations, indels

(insertions/deletions) provide parsimony-informative characters (McDade and Moody,

1999). The trL-Fsequences have proven useful in the phylogenetic analysis at the

generic level (Gielly, et al., 1996). Researchers at the Jodrell Laboratory, RBG Kew

have found the tmL-F region to be useful in the resolution of intrageneric relationships

(Molvray, et al., 1999). This region provided an intermediate level of resolution within

Laeliinae.







matK gene

The matK gene encodes an RNA maturase involved in splicing introns from

transcripts. This region is located between the 5' and 3' exons of the transfer RNA gene

for Lysine. The matK gene has proven useful in resolving relationships in Saxifragaceae

(Johnson and Soltis, 1995) and Ericaceae (Kron and Judd, 1997; Kron, et al., 1999).

Indels in matK sequence data provide additional support for clades in Saxifraga (Soltis,

et al., 1996). This region provided limited deeper resolution within the Laeliinae

phylogeny.



Materials and Methods


Many methods for plant DNA extraction and amplification have been published

(Soltis, et al., 1998). Any method should be considered a starting point since nearly all

the protocols must be optimized for the organisms under study. There are also a

number of computer programs available for processing and analyzing molecular data

(Platnick, 1988). The selection of these programs is often the personal preference of the

researcher. In the present study, fresh plant tissue was used when available and field

collected specimens were preserved in silica gel (Chase and Hills, 1991). Recipes for all

required solutions are found in Appendix C.



DNA Extraction


The process of DNA extraction requires the following phases: breaking cell walls,

rupturing membranes, separating water soluble components, precipitating DNA,

removing salts, and resuspending purified DNA in a buffer. The DNA extraction used






was a modification of a typical Cetyl TrimethylAmmonium Bromide (CTAB) method

(Doyle and Doyle, 1987). Fresh plant tissue (0.2 g) was ground in a mortar with 1000 pl

of CTAB (2X) buffer and 8 pl of mercaptoethanol, until completely homogenized.

Mercaptoethanol inhibits enzymes that cause browning which can degrade DNA. The

homogenate (800 pI) was placed in a 1.5 ml eppendorf tube and heated at 65C (Fisher

Scientific Dry Bath Incubator, 11-718) for 30 minutes. The CTAB is a detergent that

lyses nuclear and organelle membranes releasing the DNA. Next 500 pl of SEVAG

(chloroform/isoamyl alcohol 24:1) was added and the solution vortexed (Vortex-Genie,

12-812) until a milky suspension was obtained. The chloroform is used to remove

chlorophyll and other lipids from the mixture. The suspension was centrifuged at 8,000

rpm for 10 minutes to separate the phases. The green chloroform layer remained on the

bottom, plant debris in the middle, and the aqueous layer containing the DNA was on

top. (The chloroform extraction can be repeated if the aqueous layer is still green.) The

aqueous phase was transferred into a clean 1.5 ml tube and the total volume was

recorded. Sodium acetate (3M, pH 4.8) was added to the aqueous phase to a final

concentration of 1.0 M (0.04 x total volume). Then 100% isopropanol (0.65 x new total

volume) was added and placed at -200C overnight (several hours) to precipitate DNA.

The DNA was pelleted at 10,000 rpm for 20 minutes. The DNA was decanted and 1000

ul of 70% ethanol was added to wash impurities (salts) from the pellet (and tube), this

step was repeated once. The open tube was placed in a vacuum centrifuge (CentroVap

Concentrator, Labconco 78100) heated to 65C for 10 minutes or until the pellet was dry.

The DNA was redissolved in 75 pl of TE (1X) by incubating at 65C for 15 minutes (to

assure resuspension of the DNA; finger-flick to mix). The total DNA was stored at -200C.

DNA quality was verified by electrophoresis in a 1% agarose gel containing

Ethidium Bromide (EtBr) in a Tris-Borate EDTA Buffer (TBE). The ethidium bromide







intercalates with the DNA making it fluorescent under UV light. The DNA was prepared

for viewing by mixing 2 pl of total DNA with 4 pl of loading dye on a sheet of Parafilm

producing a blue droplet. This droplet was added to a well in the agarose gel and run at

94 volts for 10 minutes. The DNA was viewed on an UV illuminator (VWR Scientific M-

20E) and photographed with a Polaroid camera (FB-PDC-34), with hood (FB-PDH-

1314), using Polapan 667 film. The total DNA sample should have a band of high

molecular weight DNA with a smear of smaller fragments (Figure 3-3).



DNA Amplification


Polymerase chain reaction (PCR) was used to amplify DNA from a specific

region or gene. The desired region was selected using a pair of forward and reverse

primers that flank the region to be amplified. Since the total DNA extract is a mixture of

nuclear, plastid, and mitochondrial DNA, a selected portion can be amplified from any of

the genomes. The primers are short complementary pieces of DNA that are used to

initiate replication. The purpose is to obtain enough DNA of that specific gene so it can

be sequenced.

The process requires a mixture of template (total DNA extract), buffer, dNTPs

(nucleotides), magnesium chloride, primers (forward and reverse), PCR enhancer, and

Taq polymerase. This mixture is heated to separate the DNA template strands, then

cooled to allow the primers to anneal to the template, and warmed to allow the

polymerase to replicate the specific area of the template. This cycle is then repeated.

The amplification of the DNA is roughly geometric (doubles each cycle) so that there are

millions of identical copies produced after 30-35 cycles. The PCR product is then

cleaned to remove excess reagents, primers, and enzyme. This purified product serves






as the template for sequencing. A Biometra UNO thermal cycler was used for all the

PCR and cycle sequencing protocols.

The pH of the mix affects the amount of magnesium chloride available, thus

affecting the specificity of binding of the primers to the template. Betaine (N,N,N-

trimethylglycine), a naturally occurring cryoprotectant in plants, can be used to increase

the efficiency of amplification (a PCR enhancer). Betaine acts as an isostabilizing agent

by relaxing the secondary structure of the template equalizing the contribution of CG and

AT base pairing to the stability of the DNA duplex (Frackman, et al., 1998). The optimal

amounts of these two ingredients can vary among taxa DNA. Thus, optimization (trial

and error) is required for a particular template. The time required to setup a PCR is

reduced by preparing a 2X premix. This premix can be made in large batches and

stored in the refrigerator for weeks. The mix is made in multiples of the amounts listed in

Table 3-1.

Table 3-1. 2X PCR Premix.
Component Amount
10x PCR buffer 5 pl
MgCI2, 25 mM 6 pl
dNTPs 20 mM 1 pl
Betaine, 5 M 13 pl
Total 25 pl

This premix is then used to prepare the Master mix for PCR reactions. The

master mix contains the premix, molecular grade water, and the forward and reverse

primers for a specific region. Typically, a master mix (Table 3-2) will be made for extra

reactions to account for pipetting error.

Table 3-2. Master Mix per tube.
Component Amount
Premix 25 pl
Water 22 pl
Forward primer" 1 pl
Reverse primer' 1 pI
*10 pmol/pl




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