Developing a model of orchid seed germination

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Title:
Developing a model of orchid seed germination in vitro studies of the threatened Florida species Bletia purpurea
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english
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Johnson,Timothy R
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University of Florida
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Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Horticultural Science
Committee Chair:
Kane, Michael E
Committee Members:
Reinhardt Adams, Carrie H.
Gray, Dennis J
Perez, Hector
Miller, Deborah L

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Subjects / Keywords:
conservation -- development -- germinate -- germination -- orchid -- orchidaceae -- physiology -- rhizoid -- seed -- seedling
Horticultural Science -- Dissertations, Academic -- UF
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Horticultural Science thesis, Ph.D.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract:
The orchid family is one of the most diverse and most threatened plant families on Earth. Threats vary by region, but include loss of habitat, habitat degradation and illegal collection. These threats limit what conservationists can do with purely in situ conservation strategies and often leading to the incorporation of ex situ conservation methods including seed propagation. Unfortunately, very little is known about the physiology of orchid seed germination as most research on orchid seed propagation relies on media screens to develop adequate propagation protocols for individual species. Because of this, several studies were carried out to elucidate the nutritional and environmental factors that regulate orchid seed germination using the Florida native orchid, Bletia purpurea, as a model system. The breeding system and population habitat preferences of this species were also studied. Bletia purpurea seeds were able to germinate under a wide range of seasonally simulated temperature regimes. Rhizoid production, germination and development were most delayed by simulated winter temperatures. This effect was more pronounced when seeds were exposed to light suggesting a frost-detection system whereby growth and development are delayed when seed are at the soil surface and exposed to low temperatures. Complex interactions among illumination, nutrient availability and sucrose were detected. Germination was inhibited by illumination when seeds were cultured on water agar containing sucrose. When sucrose was excluded from media, germination was significantly enhanced by illumination. When seeds were cultured on mineral salt medium, development was enhanced by illumination though germination was not affected. Though seeds were not able to germinate when cultured with mannitol alone, mannitol was found to enhance germination, seedling development and rhizoid production when sucrose or mannitol were also available, indicating a role of this sugar as an osmolant. Abscisic acid inhibited germination and seedling development. Gibberellic acid was not able to overcome this inhibition. Instead, gibberellic acid also inhibited germination and exacerbated the inhibitory effects of abscisic acid. Chlormequat had little to no effect on germination in the absence of exogenous gibberellic acid. However, development and rhizoid production were significantly reduced in the presence of chlormequat. Supplementing gibberellic acid in the presence of chlormequat increased development and rhizoid production at some levels of gibberellic acid. These results indicate that germination and subsequent development is mainly energy limited, though certain carbohydrates, osmolants, illumination and plant growth regulators play a role in regulating these responses. Pollinator exclusion studies revealed that while Bletia purpurea plants on the Florida Panther National Wildlife Refuge sometimes produce flowers that appear to be chasmogamous, pollination is exclusively, or near exclusively, autogamous. Autogamy is thought to be the result of a reduced rostellum allowing the pollinia to develop in close proximity (if not in contact with) the stigma. Non-metric multidimensional scaling analysis of co-occurring plant species, dendrograms of Jaccard similarity indexes and soil mineral analysis indicated that B. purpurea populations can be found in a wide range of habitats. Some evidence for distinct habitat clusters were found, though it seems more likely that B. purpurea is able to grow along a fairly wide range of soils and plant communities.
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In the series University of Florida Digital Collections.
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Includes vita.
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Statement of Responsibility:
by Timothy R Johnson.
Thesis:
Thesis (Ph.D.)--University of Florida, 2011.
Local:
Adviser: Kane, Michael E.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-02-29

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1 DEVELOPING A MODEL OF ORCHID SEED GERMINATION: IN VITRO STUDIES OF THE THREATENED FLORIDA SPECIES B LETIA PURPUREA By TIMOTHY R. JOHNSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIA L FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Timothy R. Johnson

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3

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4 ACKNOWLEDGMENTS First, I thank my colleagues and lab mate s Dr. Phil Kauth, Dr. Scott Stewart, Nancy Philman Daniela Dutra Jonathan Jasinski J ames J Sadler and Nguyen Hoang for years of help designing an d carrying out experiments in the laboratory and in the field. I also thank Larry Richardson, Wade Gurly an d the staff of the Florida Panther National Wildlife Refuge for assistance with field work for protecting experiments from wildfires and for making the refuge feel like home. Plant identification would not have been possible without the help of Lucas Maju re from the Laboratory of Molecular Systematics and Evolutionary Genetics at the Florida Museum of Natural History (University of Florida) I also w ish to recognize Dr. Mike Kane, advisor and mentor who has gui ded me through this proces s and taught me how to be a teacher, researcher and mentor. Additionally, Dr. Wil Taylor gave me the keys to his electron microscopy lab when I was an undergraduate and told me to go look at stuff. I cannot seem to stop. My committee also deserves recognit ion for making this chall e nging process fun and exciting: t hank you Drs. Dennis Gray, Deborah Miller, Hector P re z and Carrie Reinhardt Adams I thank my friends and family for humoring my desire to stay in school for so long and supporting me all along t he way. My chief emotional supporter has been my wife, Danielle, who has gone so far as to help me collect data in June in south Florida simply because I asked for her help; I love you more for it. Much of this work was made possible by grants from the U.S Fish and Wildlife Service, as well as from the University of Florida Alumni Fellowship program

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 11 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 REVIEW OF LITERATURE ................................ ................................ .................... 17 Plant Conservation ................................ ................................ ................................ 17 The Orchidaceae ................................ ................................ ................................ .... 19 Flower Morphology and Pollination Biology ................................ ............................ 20 Orchid Seed Physiology ................................ ................................ .......................... 22 In Situ Germination ................................ ................................ ........................... 24 Orchid Seed Dormancy ................................ ................................ .................... 25 The Orchid Fungi Symbiosis ................................ ................................ ............ 28 Orchid fungi ................................ ................................ ................................ 28 Anatomy of orchid mycorrhizae ................................ ................................ .. 29 Fungal specificity ................................ ................................ ....................... 30 Physiology and ecology of mycotroph y ................................ ...................... 31 Restoration of Orchid Populations ................................ ................................ .......... 32 Conservation Genetics ................................ ................................ ............................ 34 Species of Study ................................ ................................ ................................ ..... 37 Project Description ................................ ................................ ................................ .. 39 Project Objectives ................................ ................................ ............................ 39 Rationale and Significance ................................ ................................ ............... 40 2 THE EFFECTS OF TEMPERATURE AND ILLUMINATION ON GERMINATION AND EARLY SEEDLING DEVELOPMENT ................................ ............................ 45 Background ................................ ................................ ................................ ............. 45 Methods ................................ ................................ ................................ .................. 46 Seed Collection and Storage ................................ ................................ ............ 46 Seed Sowing and Culture Conditions ................................ ............................... 47 Data Collection and Statistical Analysis ................................ ........................... 48 Results ................................ ................................ ................................ .................... 49 Discussion ................................ ................................ ................................ .............. 50 3 GERMINATION AND SEEDLING DEVELOPMENT IS ENHANCED BY ILLUMINATION, MINERAL SALT NUTRIENTS AND NON ALCOHOL SUGARS .. 58

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6 Background ................................ ................................ ................................ ............. 58 Materials and M ethods ................................ ................................ ............................ 60 Seed Collection ................................ ................................ ................................ 60 Estimate of Seed Viability ................................ ................................ ................. 60 Germination and Early Seedling Development in the Presence of Sucrose ..... 61 Germination and Early Seedling Development in the Presence of Sucrose and Mineral Salts ................................ ................................ .......................... 62 Germination and Early Seedling Development in the Presence of Various Carbohydrates ................................ ................................ ............................... 63 Statistical Analysis ................................ ................................ ............................ 63 Results ................................ ................................ ................................ .................... 65 Effects of Sucrose and Illumination on Germination and Early Seedling Development on Water Agar ................................ ................................ ......... 65 Effects of Sucrose and Illumination on Germination and Early Seedling Development on Mineral Nutrient Agar ................................ ......................... 67 Germination and Development of Seeds Cultured in the Presence of Various Carbohydrates ................................ ................................ ................. 69 Discussion ................................ ................................ ................................ .............. 70 Carbohydrate Utilization by Germinating Seeds and Developing Seedlings .... 71 Role of Nutrients in Regulating Germination and Seedling Development ........ 73 Effect of Light on Germination and Seedli ng Development .............................. 74 Germination in the Absence of Nutrients and Carbohydrates ........................... 76 4 EFFECTS OF SUGAR ALCOHOLS ON GERMINATION AND SEEDLING DEVELOPMENT IN THE PRESENCE OF THE GERMINATION PROMOTING CARBOHYDRATES FRUCTOSE AND SUCROSE ................................ ................ 91 Background ................................ ................................ ................................ ............. 91 Materials and Methods ................................ ................................ ............................ 93 Seed Collection, Sterilization and Viability ................................ ........................ 93 Asymbiotic Culture and Experimental Treatments ................................ ............ 94 Data Collection and Statistical Analysis ................................ ........................... 94 Results ................................ ................................ ................................ .................... 96 Interactions Between Sucrose and Sorbitol ................................ ...................... 96 Interactions Between Sucrose and Mannitol ................................ .................... 96 Interactions Between Fructose and Sorbitol ................................ ..................... 98 Interaction s Between Fructose and Mannitol ................................ .................... 99 Discussion ................................ ................................ ................................ .............. 99 5 EFFECTS OF GIBBERELLIC ACID AND ABSCISIC ACID ON GERMINATION AND SEEDLING DEVELOPMENT ................................ ................................ ....... 110 Background ................................ ................................ ................................ ........... 110 Materials and Methods ................................ ................................ .......................... 112 Seed Collection ................................ ................................ .............................. 112 Estimating Viability ................................ ................................ ......................... 112 Seed Sowing and Culture Conditions ................................ ............................. 113

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7 ABA Experiment ................................ ................................ ............................. 113 GA 3 Experiment ................................ ................................ .............................. 114 ABA GA 3 Experiment ................................ ................................ .................. 114 G A Isomers Experiment ................................ ................................ ................. 115 Experimental Design, Data Collection and Statistical Analysis ....................... 115 Results ................................ ................................ ................................ .................. 117 Effect of ABA on Seed Germination and Early Seedling Development .......... 117 Effect of GA 3 on Seed Germination and Early Seedling Development ........... 118 Effect of GA 3 on ABA Induced Inhibition of Germination and Development ... 119 Comparative Effects of GA 3 and GA 4+7 on Germination, Development and Rhizoid Production ................................ ................................ ...................... 120 Discussion ................................ ................................ ................................ ............ 121 6 EFFECT OF THE GIBBERELLIN BIOSYNTHESIS INHIBITORS PACLOBUTRAZOL AND CHLORMEQUAT ON GERMINATION AND EARLY SEEDLING DEVELOPMENT ................................ ................................ ................ 141 Background ................................ ................................ ................................ ........... 141 Materials and Methods ................................ ................................ .......................... 142 Seed Collection ................................ ................................ .............................. 142 Estimating Viability ................................ ................................ ......................... 142 Seed Sowing ................................ ................................ ................................ .. 143 Paclobutrazol Experiment ................................ ................................ ............... 143 GA 3 Chlormequat Experiment ................................ ................................ ..... 143 Experimental Design and Statistical Analysis ................................ ................. 144 Results ................................ ................................ ................................ .................. 144 Effects of Paclobutrazol on Germination, Development and Rhizoid Production ................................ ................................ ................................ ... 144 Effects of CCC on Germination, Development and Rhiz oid Production ......... 145 Discussion ................................ ................................ ................................ ............ 146 7 BREEDING SYSTEM OF POPULATIONS ON THE FLORIDA PANTHER NATIONAL WILDLIFE REFUGE ................................ ................................ .......... 153 Background ................................ ................................ ................................ ........... 153 Methods ................................ ................................ ................................ ................ 155 Experimental Design ................................ ................................ ...................... 155 Data Collection and Statistical Analysis ................................ ......................... 156 Results ................................ ................................ ................................ .................. 158 Discussion ................................ ................................ ................................ ............ 160 8 SUMMARY ................................ ................................ ................................ ........... 170 APPENDIX A EFFECTS OF BRIEF LIGHT EXPOSURE ON SEED GERMINATION AND EARLY SEEDLING DEVELOPMENT ................................ ................................ ... 176

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8 Background ................................ ................................ ................................ ........... 176 Materials and Methods ................................ ................................ .......................... 177 Seed Collection, Surface Sterilization and Media Preparation ....................... 177 Light Treatments, Experimental Design and Data Analysis ............................ 178 Results ................................ ................................ ................................ .................. 179 Discussion ................................ ................................ ................................ ............ 180 B EFFECT OF ETHANOL SOLVENT ON SEED GERMINATION AND DEVELOPMENT ................................ ................................ ................................ ... 184 Background ................................ ................................ ................................ ........... 184 Methods ................................ ................................ ................................ ................ 184 Results and Discussion ................................ ................................ ......................... 185 C ULTRASTRUCTURE OF BLETIA PURPUREA EMBRYOS ................................ 188 Background ................................ ................................ ................................ ........... 188 Methods ................................ ................................ ................................ ................ 188 Transmission Electron Microscopy ................................ ................................ 188 Light Microscopy ................................ ................................ ............................ 189 Results ................................ ................................ ................................ .................. 191 Discussion ................................ ................................ ................................ ............ 192 D HABITAT CHARACTERIZATION OF BLETIA PURPUREA POPULATIONS ON THE FLORIDA PANTHER NATIONAL WILDLIFE REFUGE ................................ 195 Background ................................ ................................ ................................ ........... 195 Materials and Methods ................................ ................................ .......................... 196 Sampling Methods ................................ ................................ .......................... 196 Habitat and Soil Characterization ................................ ................................ ... 197 Results and Discussion ................................ ................................ ......................... 199 LIST OF REFERENCES ................................ ................................ ............................. 215 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 245

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9 LIST OF TABLES Table page 2 1 Average monthly and seasonal t emperature minima and maxima for Immokalee, FL (1971 2000) ................................ ................................ .............. 53 2 2 Description of stages used to assess Bletia purpurea germina tion and seedling development ................................ ................................ ......................... 54 2 3 ANOVA results for the effects of temperature and illumination on Bletia purpurea germination and seedling development ................................ ............... 55 3 1 Description of stages used to asses s Bletia purpurea germination and seedling development ................................ ................................ ......................... 80 3 2 ANOVA results and class comparisons for the effects of seed source, media, sucrose and light on Bletia purpurea seed germination an d seedling development ................................ ................................ ................................ ....... 81 3 3 Effect of illumination and sucrose on germination index and developmental index of three Bletia purpurea seed sources ................................ ...................... 83 3 4 ANOVA results and class comparisons for the effects of carbohydrate source on Bletia purpurea germination, development and other parameters ................. 84 4 1 Results of ANOVA and cla ss comparisons for sugar alcohol experiments ....... 103 5 1 ANOVA results for the effect of abscisic aci d illumination and sucrose on Bletia purpurea germi nation and seedling development ................................ ... 124 5 2 ANOVA results for th e effect of gibberellic acid illumination and sucrose on Bletia purpurea germi nation and seedling development ................................ ... 125 5 3 ANOVA results for th e effect of gibberillic acid and abscisic acid on Bletia purpurea germination and seedling development ................................ ............. 126 5 4 ANOVA results for the effect of brief exposures to lig ht during observation on resp onses in a factorial experiment ................................ ................................ .. 127 5 5 ANOVA results of the effects of gibberellic acid isomers on germination, development, rhizoid production and seedling elonga tion ................................ 128 6 1 ANOVA results of the effects of paclobutrazol on germination, development and rhizoid production ................................ ................................ ...................... 149 6 2 ANOVA result s of the effects of chlormequat on germination, development and rhizoid production ................................ ................................ ...................... 150

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10 7 1 ANOVA results for the effect of pollinator exclusion on the number of Bletia purpurea flowers per infl orescence, capsule fo rmation and capsule development ................................ ................................ ................................ ..... 164 A 1 ANOVA results for the effect of sucrose and brief exposures to light on Bletia purpurea seed germination, seedling dev elopment and r hizoid production ...... 182 D 1 Co occurrence and abundance of plant species, and bare ground cover at Florida Panther National Wildlife Refuge and Fakah atchee Strand State Park sites where Bletia pu rpurea is found ................................ ................................ 202 D 2 Soil and vegetation parameters of Bletia purpurea habitats ............................. 210 D 3 Correlation between NMS axes and site v ariables ................................ ........... 211

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11 LIST OF FIGURES Figure page 1 1 Anatomy of orchid flowers and sexual structures. ................................ .............. 43 1 2 Morphology of Bletia purpurea plant ................................ ................................ ... 44 2 1 Bletia purpurea seeds and seedlings cultured under 6 combinations of illumination and seasonal temperatures ................................ ............................. 56 2 2 Effect of temperature and illumination on Bletia purpurea germination and seedling development. ................................ ................................ ........................ 57 3 1 Comparative growth and development of Bletia pu rpurea seeds and seedlings cultured on water agar under 6 combinations of illumination and sucrose. ................................ ................................ ................................ .............. 85 3 2 Effects of seed source, illumination and sucrose on Bletia purpurea seed germination a nd seedling development on water agar. ................................ ...... 86 3 3 Comparative growth and development of Bletia purpurea seeds and seedlings cultured on mineral nutrient agar under 6 combinations of illumination an d sucrose. ................................ ................................ .................... 87 3 4 Effects of seed sources, illumination and sucrose on Bletia purpurea seed germination and early seedling development on mineral nutrient agar. .............. 88 3 5 Comparative growth and development of Bletia purpurea seeds and seedlings cultured with 6 different carbohydrates. ................................ .............. 89 3 6 Effect of carbohydrate source and molarity on Bletia purpurea seed germination and early seedling development. ................................ .................... 90 4 1 Effects and interactions of sucrose and sorbitol on Bletia purpurea germination, mean germination time seed ling development and rhizoid production ................................ ................................ ................................ ......... 105 4 2 Comparative growth and development of Bletia purpurea seeds and seedlings germinated in the presence of sucrose and/or mannito l .................. 106 4 3 Effects and interactions of sucrose and mannitol on Bletia purpurea germination mean germination time, seedling development and rhizoid production. ................................ ................................ ................................ ........ 107 4 4 Effects and interactions of fructose and sorbitol on Bletia purpurea germinati on, mean germination time, seedling development and rhizoid production ................................ ................................ ................................ ......... 108

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12 4 5 Effects and inte ractions of sucrose and mannitol on Bletia purpurea germination, mean germination time, seedling development and rhizoid production ................................ ................................ ................................ ......... 109 5 1 Comparative growth and development of Bletia purpurea seeds and seedlings cultured with combinations of abscisic acid, sucrose and illumination. ................................ ................................ ................................ ....... 129 5 2 Effect of abscisic acid illumination and sucrose on Bletia purpurea seed germinat ion, mean g ermination time seedling development, and rhizoid production ................................ ................................ ................................ ......... 130 5 3 Comparative growth and development of Bletia purpurea seeds and seedlings cultured with comb inations of gibberellic acid s ucrose and illumination ................................ ................................ ................................ ........ 131 5 4 Effect of gibberellic acid illumination and sucrose on Bletia purpurea seed germination, mean germination time seedling development, and rhizoid production. ................................ ................................ ................................ ........ 132 5 5 Bletia purpurea seedling elongation after treatment with gibberellic acid. ........ 133 5 6 Effect of gibberellic acid illumination and sucrose on Bletia purpurea embryo elongation. ................................ ................................ ................................ ........ 134 5 7 Bletia purpurea seedling elongation after eight weeks c ulture with gibberellic acid under lighted conditions. ................................ ................................ ........... 135 5 8 Eff ect of gibberellic acid and abscisic acid on Bletia purpurea germination and seedling development. ................................ ................................ ............... 136 5 9 Comparative growth and development of Bletia purpurea seeds and seedlings treated with 6 comb inations of gibberellic acid and abscisic acid. .... 137 5 10 Comparative growth and development of Bletia purpurea seedling elongation after treatmen t with two gibberellic acid isomers. ................................ ............. 138 5 11 Effect of gibberellic acid isomers on germination, development and rhizoid production. ................................ ................................ ................................ ........ 139 5 12 Effect of gibberellic acid isomers on seedling elongation. ................................ 140 6 1 Effect of gibberellin biosynthesis inhibition by paclobutrazol on germination, development and rhizoid pro duction. ................................ ................................ 151 6 2 Effect of gibberellin biosynthesis inhibition by chlormequat on germination, development and rhizoid production. ................................ ................................ 152

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13 7 1 Bletia purpurea infructescence and capsule ................................ ..................... 165 7 2 Pollinator exclusion bags on Bletia purpurea inflorescences in situ. ................. 166 7 3 Effect of pollinator exclusion on Bletia purpurea flower production, capsule formation and capsule dehiscence. ................................ ................................ .. 167 7 4 Time course of the effect of pollinator exclusion on Bletia purpurea capsule formation and capsule dimensions. ................................ ................................ .. 168 7 5 Development of flowers and mode of autopollination in Bletia purpurea .......... 169 8 1 M odel of orchid seed germination and early seedling development. ................ 175 A 1 Effects of brief exposure to light on germination and early seedling development. ................................ ................................ ................................ .... 183 B 1 Effects of ethanol on Bletia purpurea germination and seedling development. 187 C 1 Morphology and ultrastructure of Bletia purpurea seeds. ................................ 194 D 1 Locations of Bletia purpurea sites sampled for habitat characterization. .......... 212 D 2 Dendrogram of Jaccard distances for sampled Bletia purpurea si tes. .............. 213 D 3 Non metric multidimensional scaling analysis of sampled terrestrial Bletia purpurea sites. ................................ ................................ ................................ .. 214

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14 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 DEVELOPING A MODEL OF ORCHID SEED GERMINATION: IN VITRO STUDIES OF THE THREATENED FLORIDA SPECIES B LETIA PURPUREA By Tim othy R. Johnson August 2011 Chair: Michael E. Kane Major: Hortic ultural Science The orchid family is one of the most diverse and most threatened plant families on Earth. Threats vary by region, but include loss of habitat habitat degradation and illega l collection. These threats limit what conservationists can do with purely in situ conservation strategies and often leading to the incorporation of ex situ conservation methods including seed propagation. Unfortunately very little is known about the phys iology of orchid seed germination as most research on orchid seed propagation relies on media screens to develop adequate propagation protocols for individual species. Because of this, several studies were carried out to elucidate the nutritional and envir onmental factors that regulate orchid seed germination using the Florida native orchid, Bletia purpurea as a model system. The breeding system and population habitat preferences of this species were also studied. Bletia purpurea seeds were able to germina te under a wide range of seasonally simulated temperature regimes. Rhizoid production, germination and development were most delayed by simulated winter temperatures. This effect was more pronounced when seeds were exposed to light s uggesting a frost detec tion system whereby growth and development are delayed when seed are at the soil surface and exposed to low

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15 temperatures. Complex interactions among illumination, nutrient availability and sucrose were detected. Germination was inhibi ted by illumination wh en seeds were cultured on water agar containing sucrose. When sucrose was excluded from media, germination was significantly enhanced by illumination. When seeds were cultured on mineral salt medium, development was enhanced by illumination though germinat ion was not affected. Though seeds were not able to germinate when cultured with mannitol alone mannitol was found to enhance germination, seedling development and rhizoid production when sucrose or mannitol were also available, indicating a role of this sugar as an osmolant. A bscisic a cid inhibited germination and seedling development. Gibberellic acid was not able to overcome this inhibition. Instead, gibberellic acid also inhibited germination and exacerbated the inhibitory effects of abscisic acid. Chl ormequat had little to no effect on germination in the absence of exogenous gibberellic acid. However, development and rhizoid production were significantly reduced in the presence of chlormequat. Supplementing gibberellic acid in the presence of chlormequ at increased development and rhizoid production at some levels of gibberellic acid. These results indicate that germination and subsequent development is mainly energy limited, though certain carbohydrates, osmolants, illumination and plant growth regulato rs play a role i n regulating these responses. Pollinator exclusion studies revealed that while Bletia purpurea plants on the Florida Panther National Wildlife Refuge sometimes produce fl owers that appear to be chasmoga mous, pollination is exclusively, or n ear exclusively, autogamous. Autogamy is thought to be the result of a reduced rostellum allowing the pollinia to develop in close proximity (if not in contact with) the stigma. Non metric multidimensional scaling

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16 analysis of co occurring plant species, de ndrograms of Jaccard similarity indexes and soil mineral analysis indicated that B. purpurea populations can be found in a wide range of habitats. Some evidence for distinct habitat clusters were found, though it seems more likely that B. purpurea is able to grow along a fairly wide range of soils and plant communities.

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17 CHAPTER 1 REVIEW OF LITERATURE Plant Conservation Conservation biologists strive to sustain, p rotect and repair the diversity of natural ecosystems in the face of anthropogenic degradatio n and destruction (Coates and Dixon, 2007) The scientific discipline of conservation biology wa s historically comprised of the disciplines of ecology, genetics, evolution and systematics. However, conservation biology has grown now to include conservation genetics, landscape ecology, res toration ecology, land planning and economics (Coates and Dixon, 2007) When possible, plant co mmunities should be protected and repaired in situ However, in many situations, it is not possible to pro tect plants in the wild. In these situations, ex situ conservation methods can be used in conjunction with in situ efforts to protect diversity. E x situ plant conservation must be more than simply cul tivating and growing plants in artificial environments like botanic gardens. Practitioners must be aware that a pplying unmodified agricultural and horticultural practices for conservation purposes could result in populations that are adapted for container culture, but maladapted for survival in situ (Knapp and Dyer, 1997) E very effort should be made first to maintain in situ populations that can serve as sources for restoration material. Then, when needed, ex situ popul ations can be established and cultivated in such a way as to preserve the genetic diversity and local adaptations of a species or population. The Orchidaceae is one of the largest and most diverse plant families in the world. Since their discovery, orchid s have been sought by collectors for their showy flowers and exotic air. Sadly, wild collection of orchids has led to the total destruction of

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18 some wild populations and habitats (Arditti, 1992) Even in recent years, the willingness of collectors to pay for rare orchids has led to the near extinction of wi ld populations. All known plants of a species described in 2002, Phragmi pedium kovachii were poached from the wild within three days of its discovery (Atwood et al., 2002; Yoon, 2002; Shapira, 2003) Luckily more populations were subsequently located (Decker 2007) In addition to poaching and commercial exploitation, orchids in many areas face the threat of habitat loss and fragmentation due to habi tat mismanagement, urbanization and land use conversion (Brundrett, 2007) The diversity of the O rchidaceae may also hinder the conservation of orchids Since species are the common target of restoration efforts, accurate delineation of species lines is critical. This is often difficult within the Orchidaceae, especially at the species level (Dressler, 1981; Flanagan et al., 2007) Successful orchid conservation depends upon both preventative and active conservation methods. Preventative measures may include the protection of suitable habitat, protection of existing populatio ns, deli neation of evolutionary species and accurate assignment of conservation priorities (Dixon et al., 2003; Pierce et al., 2006; Flanagan et al., 2007) Active conservation requires an understanding of many facets of requires an understanding o f plant fungus interactions, pollinat ion biology, population biology and habitat requirements. This information can then be used to develop appropri ate in situ and ex situ conservation techniques (Dixon, 1994; Brundrett, 2007; Swarts et al., 2007) It is likely that even relatively closely related taxa will require different conservation strategies (Batty et al., 2006b)

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19 Of the approximately 250 orchid species found in the United States, 118 are found in Florida making it a hot spot of orchid diversity in the North America Urbanization of Florida has steadily increased since the 1930s when only 2% of the land area was Urbanized land increased 10 % by 1986 and a gricultural land increased from 17% to 30% while marsh and forest lands declined 12% and 11%, respe ctively during this time (Kautz, 1993) Between 1985 1989 and 2003 an additional 13% of natural and semi natural lands were urbanized, developed or converted to agricultural usage (Kautz e t al., 1997) listed as threatened or endangered in the state at present It is e ssential that steps be taken to protect native populations in the context of an ever expanding human population in Florida. The O rchidacea e The Orchidaceae is comprised of approximately 20,000 species and over 8 00 genera (Cronquist, 1981; Dressler, 1993a; Cameron et al., 1999) Recent evidence indicates that the origins of the Orchidaceae may have begun as early as 84 million years ago (Ramirez et al., 2007) Members of the family are highly variable in both growth habit and plant form. The vast majority of orchid species are epiphytic, but 4,000 known species of orchids belong to primarily terrestrial genera, and as many as 25% of orchids are terrestrial (Dressler, 1981) The diversification of the Orchidaceae appears to have been driven by three key adaptations. The evolution of ep iphytism allowed for the exploitation of the intra canopy environment and likely lead to the high degree of epiphytic radiation (Gravendeel et al., 2004) Additionally, p ollination strategies that attract specific pollinators likely promot e d speciation and prevent ed hybridization (Peakall and Beattie, 1996) Finally, specialized

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20 symbiotic relationships with fungi and degrees of mycotrophy may hav e promote d speciation and bolstered species boundaries (Taylor et al., 2003; Taylor et al., 2004; Hollick et al., 2005) Rasmussen (1995) argues that the specialization seen in the Orchidaceae is due almost entirel y t o the evolution of mycotrophy. This dependence upon fungi likely lead to the reductions in seed storage organs se en in the Orchidaceae F ungal distributions are heterogeneous and the chances that any one seed will be dispersed to soils containing approp riate fungi are small. This likely lead to increased seed numbers at the expense of seed size. These microseeds have the added advantage of greater potential for long distance dispersal, thus improving the chances that seeds will be dispersed to soils with suitable fungi. The highly evolved mechanisms for avoiding inbreeding (Pollinator specificity, precision pollination and bundled packets of pollen [pollinium]) can also be interpreted as a consequence of this evolutionary trajectory set off by mycotrophy and culminating in microseeds. First, the potential benefits of outbreeding (hybrid vigor) may be especially important when the chances of offspring survival are so low and each flower is likely to mate with a single partner. Secondly, fertilization of tho usands of seeds requires pollination with thousands of pollen grains, as are delivered in a single pollinium. S mall seeds may have also been the catalyst for the evolution of epiphytism as they are easily lifted into the tree canopy where plants could esta blish epiphytically with very small amounts of wind energy Flower Morphology and Pollination B iology The flower morphology of orchids is as variable as the growth forms. Dressler (1993a) attributed this to the idea that the family has not yet undergone the degree o f natural selection and extinction that older angiosperm families have naturally

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21 experienced over time The idea that there are many extant evolutionary lineages may contribute to the taxonomical complexity of this family. O rchid flower s (Figure 1 1 A, B ) are typically bilaterally symmetrical and trimerous with inferior ovaries (Dressler, 1993a) O ne of the petals is typically larger and more ornate than the others and termed the lip or labellum (Arditti, 1992; Dressler, 1993a) Orchid flowe rs usually express resupination twi sting or bending of the pedicel during development. Because of this the labellum, which is adaxial in the flower bud, co mes to rest in an abaxial position (Arditti, 1992; Dressler, 1993a) Another defining characteristic of orchids is that their stamens are all located on one side of the flower rather than distributed radially like those of most angiosperms (Dressler, 1981) In additio n the style and filaments of orchid s are fused to form a column (Figure 1 1A, B, C and D) (Arditti, 1992; Dressler, 1993a) The rostellum (Figure 1 1D) a modification to the column that separates the stigmatic surface from th e anthers, is located near the end of the column a n d prevents the pollinia from contacting the stigma during flower development and anthesis (Dressler, 1981; Ardi tti, 1992; Dressler, 1993a) While evolutionary basal orchids still have powdery pollen, the pollen of more derived orchids is conta ined in discrete, hard packets called pollinia that are attached to a sticky disc termed the viscidium and held behind the anther cap (Figure 1 1D, E, F, G) It is the viscidium that attaches to pollinators for transport and subsequent pollination of other flowers (Arditti, 1992; Dressler, 1993a) R ecent fossil evidence suggests that the orchid pollinator interaction is an ancient relationship (Ramirez et al., 2007) Charles Darwin (1885) wrote extensively about the orchi d pollination mechanisms, and while he studied and presented instances of highly

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22 specific plant pollinator interactions to illustrate his theories about co evolution, these instances of extreme specificity may be the exceptions rather than the rules in Orc hidaceae (Dressler, 1993a) Orchid species o ften demonstrate so called deceptive pollinator attraction strategies modes of attracting pollinators without rewarding them (Dressler, 1981; Arditti, 1992; Cozzolino and Widmer, 2005) Deception takes many forms in the Orchidaceae including simulation of prey, simulation of substrata and (Arditti, 1992) As many as 400 species of orchids are sexually deceptive, offering some resemblance (color, texture, shape, fragrance) to the sexual partners of pollinato r s pecies. O ne third of all orchids are food deceptive (Cozzolino an d Widmer, 2005) Attraction without reward is believed to be primitive in orchids as it is prevalent in the family and may be a long term adaptive strategy that limits inbreeding depression and excessive fruit se t, both of which result in decreased offspr ing fitness (Cozzolino and Widmer, 2005) Still other species or ec otypes of species have evolved means of auto pollination known as spontaneous autogamy, during which the pollinia of a flower are deposited on the stigma without the aid of a pollinator Autogamy can occur when species fail to form a rostellum, when polli nia crumble onto the stigma or when the caudicle bends around the rostellum and pollinates the flower (Gonzalez Diaz and Ackerman, 1988; Catling and Lefkovitch, 1989; Catling, 1990; Gale, 2007; Micheneau et al., 2008; Peter and Johnson, 2009) Orchid Seed Physiology Orchid seeds are minute, contain an undifferentiated embryo and lack endosperm. Because of this, in situ germination of orchid seeds appears to be dependent upon infection by a compatible fungus (Rasmussen, 1995; Baskin and Baskin, 2001) Prior to the 1920s it was believed that orchid seeds could not germinate without the aid of a

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23 co mpatible mycobiont. Then Lewis Knudson (1922) demonstrated that orchid seeds could be germinated asymbiotically in vitro on sterile nutrient media. This method subsequently made germination of many genera possible and is at present the preferred method for commercia l orchid production. Relatively little is known about the mechanisms and physiology that governs orchid seed germination or how various abiotic and biotic fac tors promote and inhibit germination. In par t this is due to the vast number of species and high d egree of variability within the Orchidaceae. However, this void in knowledge is also likely an artifact of the and the difficult h istory of propagat ing o rchids from seed. Because of the se challenges most orchid s eed germination studies have focused on production (i.e. media screens) rather than physiology Authors commonly es po u se on the p hysiolog ical or ecological implications of such work by drawing conclusions about photoblasticity and germination phenology, bu t basic aspects of orchid seed physiology, such as the effects and interactions of light, temperature, nutrients, carbohydrate s and plant growth regulators, have rarely been rigorously tested In general, in vitro germination of orchids is highly variable and observed germination rates are often low. For example, Lindn (1980) reported that fo r 29 temperate orchid species studied, 20 had observed germination of less than 10% and eight species germinated to 1% or less. Variability in germination in vitro may be due to a number of factors including maternal effects (Light and MacConaill, 1998) pollen source (Johnson et al., 2009) fungal strain (Zettler et al., 2005; Stewart, 2006; Stewart and Kane, 2006b; Johnson et al., 2007; Stewart and Kane, 2007) media (Stewart and

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24 Kane, 2006a; Johnson and Kane, 2007; Dutra et al., 2008; Kauth et al., 2008a; Dutra et al., 2009b) culture conditions (Rasmussen et al., 1990b, 1990a; Rasmussen and Rasmussen, 1991; Fukai et al., 1997) and seed dormancy (Van der Kinderen, 1987; Ras mussen, 1992; Miyoshi and Mii, 1998; Lee et al., 2005; Lee et al., 2007) The key discovery that allowed Knudson to successfully germinate orchid seeds without fungi was the incorporation of sucrose into the growth medium (Knudson, 1922) Orchids are able to utilize a wide range of carbohydrates in asymbiotic culture and during later seedling development including sucrose, glucose, fructo se, mannose, maltose, raffinose trehalose and xy lose (Yates and Curtis, 1949; Grushvitsky, 1967; Smith, 1973; Stewart and Kane, 2010) but not more complex sugars such as galactose, arabinose and rhamnose (Wynd, 1933; Ernst et al., 1971) In S itu G ermination An understandi most integrated conservation efforts. For restoration practitioners, direct see ding is often the most favorable option for restoring a species (Guerrant Jr. and Kaye, 2007) When compared to planting of transplants, direct seeding is typi cally less expensive and less time consuming, even though seeding survival rates are often low. However, d irect seeding of orchids among existing populations has been shown to promote seedling recruitment of only a few species (Huber, 2002; Wright et al., 2007) The development of a method for the retri (Rasmussen and Whigham, 1993; Van der Kinderen, 1995; Brundrett et al., 2003) has been helpful in isolating germination promoting mycobionts and studying orchid seed ecology. For this method, seeds are incased in fine mesh packets plac ed in native soils and attached to a stake for easy retrieval. This method has revealed that

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25 in situ germination is effected by a number of factors including dominant vegetation, adu lt plant proximity burial depth, availability of compatible mycobionts an d organic matter composition (Van der Kinderen, 1995; Rasmussen and Whigham, 1998; McKendrick et al., 2000; Batty et al., 2006a; Whigham et al., 2006; Diez, 2007) Orchid S eed D ormancy Orchid seed germination may be delayed by a number of dormancy mechanisms. The re is evidence for phy siological dormancy in some difficult to germinate orchid species. Cold stratification, a common dormancy relieving treatment, has been shown to im prove the germinability of some orchid species (de Pauw and Remphrey, 1993; Tomita and Tomita, 1997; Miyoshi and Mii, 1998; Kauth et al., 2011) In addition, minute quantities of abscisic acid (ABA) have been isolated from the embryos and seeds of Calanthe tricarinata Dactylorhiza maculata and Epipactis helleborine (Van der Kinderen, 1987; Lee et al., 2007) For C. tricarinata embryos, endogenous ABA levels 1 FW at 90 days after pollination ( DAP ) to approxim 1 FW at 210 DAP. This coincided with a decline in germination from 30% at 150 DAP to almost 0% at 210 DAP (Lee et al., 2007) supporting the idea that germination is prevented by physiological dormancy Physical dormancy caused by an impermeable seed covering which prevents imbibition (Baskin an d Baskin, 2001) may inhibit germination of several Cypripedium species (St Arnaud et al., 1992; de Pauw and Remphrey, 1993; Light, 1994; Lo et al., 2004; Lee et al., 2005) Declining germination of Cephalanthera falcat a beginning 70 DAP and Cypripedium formosanum beginning 90 DAP has been linked to the gradual lignification or cutinization of the inner integument surrounding the embryos (Lee et al., 2005; Yamazaki and Miyoshi, 2006) These layers may pose a considerable barrier to

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26 imbibition, but can be overcome by sowing immature seeds or scarifying mature seeds with oxidative treatments, sonication drying or freezing with liquid nitrogen (Van Waes and Debergh, 1986; Miyoshi and Mii, 1988, 1998; Batty et al., 2001; Pezzani and Montana, 2006) Seed germination may also be delayed by changes in different phytochrome proteins between the red light absorbing inactive form s and the far red light absorbing active form s (Casal and Sanchez, 1998; Seo et al., 2009) This phenomenon, sometimes referred to as p hotodormancy or photoinhibition (Leubner Metzger, 2001; Moy o et al., 2009) is regulated by the ratio of red (R) and far red (FR) light that seeds are exposed to Seeds which germinate better under dark or FR light and which are inhibited by R light are said to be negatively photoblastic. Seeds that are inhibited from germinating under dark and FR light are said to be positively photoblastic (Baskin and Baskin, 2001) Van Waes and Debergh (1986) tested the effect of light on germination of 11 European species and found that germination for all species tested was highest under dark conditions. Low intensity lig ht (14/10 h our light/dark [L/D] photoperiod at 1.2 2 s 1 ) reduced germination of all species tested and completely inhibited germination in five species, while moderate intensity light (14/10 h our L/D photoperiod M m 2 s 1 ) inhibited germination of all 11 species. Dark in cubation has also been found to improve germination of Calanthe Satsuma, Calanthe falcate Cyrtopodium punctatum and symbiotic cultures of Dactylorhiza majalis and Habenaria macroceratitis (Rasmussen and Rasmussen, 1991; Light, 1994; Fukai et al., 1997; Stewart and Kane, 2006b; Yamazaki and Miyoshi, 2 006; Dutra et al., 2009b) However, light improve s germination and/or development of Bletia purpurea Calopogon

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27 tuberosus and Goodyera repens var. op h ioides (McKinley and Cam per, 1997; Kauth et al., 2006; Dutra et al., 2008) It is worth mentioning that in all of these studies, seeds were either provided exogenous carbohydrates such as sucrose, or were co cultured with symbiotic fungi. It is thus unclear how or if carbohydrat e availability and illumination interact regarding the regulation of orchid seed ger mination and early development. While the preceding studies exemplify certain types of dormancies, the inability of most orchid species to germinate when cultured without e ither a compatible fungus or mineral salt media containing carbohydrates indicate that a significant germination barr ier exists even in non dormant seeds. As previously discussed, this phenomenon is thought to be caused by the absence of limiting amount s o f energy reserves in orchid seed s (Rasmussen, 1995; Baskin and Baskin, 2001) However, Manning and van Staden (1987) concluded that Disa polygonoides Disperi s fanniniae and Huttonaea pulchra embryos contained relatively large quantities of lipids and proteins, as well as several different soluble sugars. They found that imbi bed seeds did no t mobilize protein or possibly provided by fungal infection Little is known about what specific nutrients are needed to allow germina tion to proceed in non dormant orchid seeds or more generally whether germination is limited by nutrient availability A better understanding of exactly which compounds in asymbiotic media allow germination to proceed may provide clues as to how symbiosis promotes germination as well. Microcosm studies have shown that mycobionts can enhance accum ulation of phosphorus, nitrogen and carbon in symbiotic seedlings of Goodyera repens and Dactylorhiza purp u rella (Smith, 1966, 1967; Hadley and Purves,

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28 1974; Purves and Hadley, 1976; Alexander et al., 1984; Cameron et al., 2006) Any one of these may function at least in part as a germination cue The O rchid F ungi Sy mbiosis The importance of fungi in terrestrial ecosystems is just beginning to be fully appreciated It is estimated that 80% of terrestrial land plants form symbiotic relationships with fungi (Smith and Read, 2008) As previously mentioned, orchi ds are reliant upon germinatio n promoting fungi for seedling establishment. Natural orchid stands are only found where compatible fungi exist (Brundrett, 2007) Orchids also utilize fungi as a nutrient source throughout their lifespan, as evidenced by year round infection of Corallorhiza odontorhiza Galearis spectabilis Goodyera pubescens Liparis lilifolia and Tipularia dis color (Rasmussen and Whigham, 2002) Interestingly, the photosynthet ic strategies of these five species range from evergreen ( G. pubescens ) to non photosynthetic ( C. odontorhiza ). Carbon acquisition strategies can even vary within a species; achlorophyllous forms of Cephalanthera damasonium are entirely mycotrophic while g reen plants acquire half of their carbon from mycotrophy and half from photo synthesis (Julou et al., 2005) The evolution of the non photosynthetic or minimally photosynthetic orchid lineages Corallorhiza Hexalectris and Rhizant hella is a testament to the effectiveness of this strategy. Orchid fung i Most o rchid fungi belong to the subdivision Basidiomycota (club fungi) and the imperfect form genus Rhizoctonia (Currah et al., 1997) Rhizoctonia are characterized by intracellular connections with continuous parenthesomes (dolipores), cream to light brown hyphae, laminated walls, constrictions ne ar hyphal branches, aggregates of swollen moniloid or monilifo rm cells and the lack of clamp connections (Currah, 1991)

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29 Rhizoctonia are further divided into the teleo morph based subgenera Ceratorhiza Epulorhiza Moniliopsis and Rhizoctonia Ana morphs of these genera are respectively Ceratobasidium Tulasnella Thanatephorus and Helicobasidium (Currah, 1991; Dijk et al., 1997) Ceratorhiza and Moniliopsis are characte rized by rapid growth, the ability to utilize many different nitrogen sources and binucleate cells. Epulorhiza are slower growing, unable to utilize nitrate or ammonium and multinucleate (Dijk et al., 1997) Morphology and polyphenol oxidase assay s are used to distinguish between polyphenol oxidase negative Epulorhiza species and typically positive Ce ratorhiza species (Currah et al., 1997) Anatomy of o rchid m ycorrhiza e Orchid mycorrhizal are unique in that the hyph ae are degraded within the plant, either by the host or due to autolysis (Currah, 1991) The orchid appears to have a high degree of control over the symbiosis. There is debate as to whether infection occurs via rhizoids exclusively, or through the suspensor region (Hadley, 1982; Clements, 1988; Rasmussen, 1990; Dijk et al., 1997) though both seem likely Afte r infection, hyphal bundles (pelotons) form either between the cell wall and the plasma membrane without penetrating the plasma membrane (Hadley and William son, 1971; Currah, 1991) or within the cell cytoplasm (Rasmussen, 1990) I n normal symbiosis, i nf ection is restricted to the subepidermal cortex of seedling s and roots. While pelotons are routinely digested in this area, an outer layer of infected cells host li ving hyphae which recolonize within the digestion zone (Currah, 1991; Dijk et al., 1997) Parasitic fungal infection and infection of tubers may be prevented by the production of the phytoalexins orcinol and hirconol (Fisch et al., 1973) Uncontrolled infection through the epidermis, or

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30 uncontrolled spreading of a fungus following infection results in seedling death due to fungal parasitism (Clements, 1988) Fungal s pecifici ty Some researchers have argued that orchid species, and perhaps even populations of species, are only able to utilize a narrow range of fungi for germination or in later life stag es The concept of fungal specificity is supported by evidence that a) some species germinate better when co cultured with fungi originating from seed source populations than from other populations of the same species b) closely allied species do not share mycobionts and c) some species are consistently found to have specific end ophytes (Masuhara and Kat suya, 1994; Zettler and Hofer, 1998; Zettler et al., 2005; Stewart and Kane, 2006b; Johnson et al., 2007) O rchid species may also be adapted to utilize different fungi throughout their range (McKendrick et al., 2002) In contrast, there are reports of fungal strains that are able to promote germi nation in many different orchid taxa (Hadley, 1970; Clements, 1988; Shan et al., 2002; Stewart and Zettler, 2002) Similarly some orchids can ger minate with fungal isolates originating from other orchid species (Zettler and McInnis, 1992; Zett ler et al., 1999; Zettler et al., 2005; Stewart and Kane, 2007) An in vitro study of 6 Australian species found that two disturbance tolerant species, Disa bractea t a and Microtis media were able to utilize a wider range of fungi originating from other s pecies and locations than other studied species (Bonnardeaux et al., 2007) indicating that fungal specificity may be linked to plant ecology. It is not clear whether the degree of observed fungal specificity is correlated more with characteristics of the fungi or the orchid. Hadley (1982) has suggested that many in vitro fungal compatibility screens likely expose orchid seeds to a wider range of fungal

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31 taxa than they encounter under field conditions. This may inflate estimates of ecologically relevant specificity. Recent molecular reports support this theory. Studie s of non photosynthetic orchids have revealed that some of these species associate with a small number of closely related endophytes, but that orchid populations may have evolved specificity for regionally distributed endophyte strains (Taylor and Bruns, 1997; Taylor et al., 2003; Taylor et al., 2004) Physiology and ecology of m ycotrophy While dependence upon symbiotic fungi for germination may be a risky strategy, it is also an effective means of utilizin g the digestive abilities of fungi. Orchid fungi are able to utilize a wide range of nitrogen and c arbon sources, which are then made available to the host orchid following infection (Hadley and Ong, 1978) Wynd (1933) convert complex carbohydrat es like the polymer mannan into water soluble forms like mannose. As previously mentioned, a few studies have shown that mycobionts can enhance accum ulation of phosphorus, nitrogen and carbon in symbiotic seedlings of G. repens and D. purp u rella (Smith, 1966, 1967; Hadley and Purves, 1974; Purves and Hadley, 1976; Alexander et al., 1984; Cameron et al., 2006) Undefined fungal extracts have also been shown to promote germination of G. repens in vitro (Downie and Orothy, 1949) Rasmussen (1990) noted that stimulation of growth in Dacty l orhiza majalis seeds was initiated in the presence of a compatible fungus prior to contact supporting the hypothesis that extra fungal chemical signals may prepare the seed fo r germination. It seems possible that fungi stimulate germination by providing chemical germination signals, pr oducing plant growth regulators or by enhancing water uptake as suggested by Yoder et al. (2000)

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32 Restoration of Orchid Populations Reintrod uction can be defined as population level r estoration involving the (Maunder, 1992) Reintroduct ions are distinguished from introductions in that introductions may be intentional or accidental releases of species outside their natural range (IUCN 1987) The goal of a reintroduction is to reduce extinction risk for species in the wild by establishing self sustaining populations that have the genetic di versity and ecosystem function of natural populations (Guerrant Jr. and Kaye, 2007) Guerrant and Kaye (2007) distinguish between two types of success criteria for evaluating restoration projects: biological success and project success. Biological success relates to the goals of all restorations, which is to establish self sufficient and integrated populations. Project success criteria are used to evaluate the knowledge gained from a scientific exploration of the factors that affect restoration success and failure. Commonly tested reintroduction hypotheses include the effect of propag ule type, source, breeding, receptor site (i.e. home site advantage studies), geographic location, habitat manipulation and seasonality on project success (Guerrant Jr. and Kaye, 2007) Orchid reintroductions could contribute to a rest oration project by adding biodiversity and functionality of an ecosys tem, by providing a symbol of c onservation for a given project or by ameliorating collection pressure placed on other natural populations (Maunder, 1992) There have now been many efforts to establish or acclimatize artificially propagated orchids in natural and semi natural environments. Many documented orchid reintroductions have not been used to generate scientific data (Anderson, 1996; Ramsey and Stewart, 1998; Huber, 2002; Camara Neto et al., 2007; Zettler et al., 2007) Of those orchid reintroduction efforts that have involved

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33 experimentation, very few factors that might affect the restoration of orch id populations have been studied Survival of orchid seedling transplants in situ and establishment of populations using direct seeding has been generally low. Fatality of 6 Western Australia terrestrial species was 40 80% in the first 14 weeks post transplant (Scade et al., 2006) Translocated symbiotic seedlings of two other Australian species, Caladenia arenicola and Pterostylis sanguinea fared well in the fir st year after transplant (approximately 50% and 90% survival, respectively). However, C. arenicola transplants did not reemerge and P. sanguinea plants only persisted for two growing seasons. Only 10% of Diuris magnifica plants persistence into the third g rowing season (Batty et al., 2006a) A range of factors that might affect orchid transplant success include physiol ogical state of transplants, pest damage and both abiotic and biotic site variables. Work with Australian tuber forming species indicates that plant s transplanted as dormant tubers do not survive as well as actively growing symbiotic seedlings (Batty et al., 2006a; Smith et al., 2007) The survival of the European Dactylorhiza praetermissa was improved from less than 10% to 40% when plants were protected from grazing and slug herbivory (McKendrick, 1995) Other species may benefit from weed control (Scade et al., 2006) Soil cultivation impro ved the survival of Diuris fragrantissima symbiotic seedlings, possibly by decreasing competition or by stimulating fungal activity (Smith et al., 2007) Finally, researchers in Western Australia have suggested that rainfall during the dry, hot summer months may play a significant role in promoting reintroduction success (Batty et al., 2006a)

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34 In concert, these studies illustrate that very few species and very few aspects of orchid reintroduction methodology have bee n studied. Those variables that have been studie d requ ire a dditional inquiry utilizing additional species and geographic regions Due to the targeting of Orchidaceae for collection and the unabated rate of orchid habitat loss across the globe, the immediate need for such studies cannot be overstated. Conserva tion Genetics All restoration project managers must consider how the techniques they utilize affect the genetic structure of constructed populations and surrounding populations since genetic pitfalls can limit project su ccess. An artificial bottleneck lead ing to strong founder effect s caused by restricted seed sampling of Mauna Kea silversword ( Argyroxiphium sandwicense ssp. sandwicense ) w as blamed for the population crash at a reintroduction site (Friar et al., 2000) Genetic diversity of reintroduced plants may be limited at several stages of plant procurement or production At the time of collection, donor sites may have low inherent div ersity, collection methods may be non random o r a small sample may be collected Artificial selection during cultivation may limit the genetic diversity o f restored populations as well. Finally diversity of outplanted population s may be decreased if donor plants have low genetic diversity (Williams, 2001) It is important to note that even small natural populations can have significant genetic diversity and high frequencies of rare alleles (Friar et al., 2000) Therefore, even small restoration proj ects should attempt to restore adequate genetic divers ity in created populations. The concept of seed transfer zones has been used to suggest that restoration propagules originate in close proximity to a restoration site. Seed transfer zones

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35 assume that plants are more likely to share life histories and adapt ations with plants growing near them than with plants growing further away. An understanding of breeding structure is paramount to delimiting transfer zones and protecting local adaptation (Ledig, 1992; Hufford and Mazer, 2003) Other genetic considerations for restoration ecologists include how restoration methods may affect mating and fitness within and around restoration sites. Restored population s with low genetic diversity may be prone to bottlenecks, in breeding depression and local extinction (Saccheri et al., 1998; Hufford and Mazer, 2003; Reusch et al., 2005) I f reintroduced ecotypes are not adapted for local habitat s the fitness of surrounding native populations could be lowered by genetic swamping and outbreeding depression (Fenster and Galloway, 2000; Hufford and Mazer, 2003) S tudies of various orchid species have revealed that population and subpopulation level genetic diversity is highly varia ble (Forrest et al., 2004) Some studies indicate that breeding system may be a good predictor of genetic diversity (Wallace, 2006) while other s suggest otherwise A comparison of the genetic diversity of 20 allozymes in three co lonizing Asian species with different breeding systems found that all three species had low observed heterozygosity (H o ). H o of Zeuxine strateumatica (apomictic), Spiranthes hongkongensis (selfing) and Eulophia sinensis (outbreeding) was 0, 0 and 0.00012, respectively (Sun, 1997) Conversely, a study of the endangered Liparis loeselii found amplified fragment length polymorphisms (AFLP) estimates of allelic diversity were moderately high in at least some European and North American

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36 diversity (Pillon et al., 2007) One important finding in this study was that larger popula tions tended to have greater genotypic and a llelic diversity throughout the g events from large, genetically diverse populations to smaller populations are important in maintaining sustainable levels of diversity in these small populations. Rare or unlikely mating events may be important in maintaining genetic diversity and preventing genetic isolation of distant populations as well. Evidence of g ene flow based on 11 alloz yme markers indicates that Cypri pedium calceolus gene flow is possible between island populations separated by as much as two kilometers (Brzosko et al., 2002) Large subpopulations may also allow for sympatric differentiation of patches, thus increasing local genetic diversity (Alexandersson and gren, 2000) In some orchids, considerable evidence for spatial structure has been found at short distances. Forest et al. (2004) reported ST = 0.892 for 86 polymorphic AFLP loci for 10 populations of Spiranthes romanzoffiana indicating a high degree of the total diversity was held within subpopulations Statistically significant spatial structure was detected at 4 12 m scale in populations of Cremastra appendiculata may be the result of limited seed dispersal and vegetative propagation (Chung et al., 2004) Liparis loeselii populations did not exhibit evide nce of spatial structure within populations but populations in similar habitats (dune slacks compared to fens) were found to be si gnificantly different (Pillon et al., 2007) While molecular data can be useful for population genetics, confident interpretation of such data can be difficult. For example, a Random Amp lification of Polymorphic DNA ( RAPD ) study of Platanthera le ucophaea demonstrates the difficulties

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37 interpreting the results of population genetic studies without additional biological information. In a survey of 192 P. leucophaea plants in seven population s, all individuals were found to have unique genotypes. A high percentage of polymorphic loci were also observed (36 62% of 58 loci were variable in each population ) and geographic distance was found not to predict genetic distance, indicating a high degre e of gene flow across fragmented populations (Wallace, 2002) While these are indicators that there is a high degree of geneti c diversity between and among populations the high degree of observed genetic diversity may be an artifact of a once healthy population that has only recently been fragmented. Finally, because of the array of methods available for estimating population ge netic parameters, making direct comparisons between studies that do not use identical methods is likely inappropriate (Powell et al., 1996; Sonstebo et al., 2007) Species of Study Bletia purpurea (Figure 1 2) is a terrestrial orchid with tropical affinities. This species occupies several different habitats throughout it s range. It can be found in dry or mesic habitats, in or along scrub lands or pinelands, on floating logs or stumps in cypress swamps and along highly disturbed lake edges and cliffs (Correll, 1978; Dressler, 1993b; Williams and Allen, 1998; Brown, 2002) In the United States, B. purpurea is only found in south Florida (Figure 1 3) where it is listed as a state threatened species (Coile and Garland, 2003) Interestingly, this species (syn. Helleborine a mericana ) was one of the first, if not the first, tropical orchid flowered in Europe circa 1732 (Arditti, 1992) The genus Bletia is comprised of approximately 40 species of terrestrial and semi terrestr ial species found throughout the New World tropics and subtropics (Sosa and

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38 Daz Dumas, 1997; Palestina and Sosa, 2002) Dressler placed Bletia in the largest orchid subfamily Epidendroid, the tribe Arethuseae, and the subtribe Bletiinae (Dressler, 1993a) subfamily concept h as been well supported by various molecular sequence analyse s (Cameron et al., 1999; Chase et al., 2003; Grniak et al., 2010) However, molecular a nalysis has led to reorganization of Arethuseae The subtribe Bletiinae has been removed from Arethuseae and placed into the tribe Epidendreae with an assortment of terrestrial and epiphytic species (Cameron et al., 1999; Chase et al., 2003; Grniak et al., 2010) Pollin ation by autogamy and/ or cleistogamy has been p u r ported in Florida possibly facilitated by the lack of a rostellum in at least some flowers (Stoutamire, 1974; Arditti, 1992; Brown, 2002) It has been suggested that autogamy evolved following long distance dispersal to Florida from the Caribbean without co migration of suita ble pollinators (Arditti, 1992) R eports of autogam y and cleistogamy within more contiguous portions of B. purpurea range including Panama and Costa Rica (Dressler, 1993b) indicates that cleistogamy could be wide spread for this species. Rates of cleistogamy have not been previously quantified and there is evidence of a mixe d breeding system in Florida as some flowers appear chasmogamous, often on the same inflorescence as cleistogamous flowers (personal obs ervation ). Reports of pollinati on by an orchid bee ( Euglossa sp. ) in Ecuador (Dodson and Frymire, 1961; Dodson, 1962) also indicate that B. purpurea breeding systems vary among populations Bletia purpurea is easily propagated from seed using asymbiotic techniques attaining nearly 100% germination in 6 weeks (Dutra et al., 2008) The apparent lack of physiological dormancy, combined with the ease of germination and reliably high

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39 viability of f ield collected seeds over many growing seasons (personal observation ) make it an ideal species for examining seed physiolo gy of orchids. Isolation of fungi from adult plants was attempted twice however pelotons were not observed in roots, few fungi resemb ling typical mycobionts were isolated and no isolates support ed seed germination (personal observation ; S. Stewa rt personal communication ). Given the mentioned issues with and concerns for orchid conservation efforts worldwide and specific concerns about the preservation of B. purpurea in North America, a series of studies was car ried out to examine the seed physiology, breeding system and population biology of B. purpurea The specific objectives and project rational follow. Project Description Project Ob jectives 1. Determine how temperature and illumination effect seed germination and early seedling development of Bletia purpurea 2. Determine whether mineral nutrients and/or sucrose act as germination signals for Bletia purpurea seeds 2.1. Determine whether the ef fects of mineral nutrients and sucrose are altered by illumination 3. Determine whether Bletia purpurea seeds are able to utilize a wide range carbohydrates commonly used in asymbiotic cultures 4. Determine whether abscisic acid (ABA) or gibberellic acid (GA) i nfluence germination and early seedling development of Bletia purpurea 4.1. Determine whether the effects of ABA and GA on germination and early seedling development are altered by illumination and/or sucrose

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40 4.2. Determine whether GA isomers have different effects on germination and early development 4.3. Determine whether the effects of ABA and GA act antagonistically in regulating seed germination 4.4. Determine whether the inhibition germination by GAs is caused by supraoptimal levels of GA 5. Determ ine the degree of cleist ogamy expressed by Bletia purpurea plants at the Florida Panther National Wildlife Refuge 6. Assess the genetic diversity and population structure of Bletia purpurea populations within the Florida Panther National Wildlife Refuge Rationale and Significance O rchids face many threats in the wild, most of which are anthropogenic. While a considerable amount of research on orchid restoration was been conducted in Australia and Europe, the methods developed there may not apply to the subtropical environment in Flo threatened or endangered. Since the acreage of natural areas in Florida is also declining, steps should be taken now to develop restoration protocols for threatened native orchids including Bletia purpurea O rchid seed germination is known to be affected by temperature. However, most of the studies on this subject have examined temperate species, some of which have confounding dormancies. Since seasonal temperature changes in the subtropics and tropics are no t as drastic as in temperate climates, it is unclear if

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41 temperature plays an important role in regulating germination phenology in this environment. It is generally thought that orchid seeds cannot germinate in situ without infection by a germination promo ting fungus. However, Bletia purpurea seeds contain enough stored nutrients for the embryo to swell, green and survive on sterile water when cultured under lighted conditions (pers onal observation ). This indicates that at least some orchid species are able to germinate and grow under a wider range of environmental conditions than commonly recognized. Gaining a better u nderstand ing of the environmental and chemical signals that regulate orchid seed germination could lead to better natural resource management of this family through enhanced understanding of the factors that influence germination and stand establishment. Bletia purpurea may be an ideal species for investigating these variables as it grows readily in vitro. Pollination is a limiting event in the life cycle of orchids. Because of this, effective in situ orchid conservation relies in part on our ability to recognize and protect a rchid species with a high degree of pollinator specificity may be in part lim ited by pollinator distribution (Brundrett, 2007) Some evidence suggests that orchid pollina tor s in Florida may be declining due to a number of anthropogenic causes (Dutra et al., 2009a) Since pollination is a critical life cycle stage for orchids, it is important that the pollinators and pollination mechanism of Bletia purpurea be iden tif ied to facilitate the persistence of this threatened species.

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42 Understanding the existing genetic diversity of populations is vital to rare species conservation. Such data may provide clues to the history of populations elucidate population structure an d corroborate mating system data. Observed genetic diversity can also be used to guide ex situ conservation efforts. Population genetic analysis of Bletia purpurea could be helpful in determining both the degree and effect s of cleistogamy and vegetative pr opagation in populations. In addition, genetic markers can be used to study popul ation structure, heterozygosity and extinction risk ; these invisible indicators of threatene d

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43 Figure 1 1 A natomy of orchid flowers and sexual structures A) Spathoglottis flower with three sepals, three petals (one mod ified into the labellum) and a column B) Side view of flower with labellum removed to show column more clearly. C) C olumn i n profile. Scale bar = 2.5 mm. D) Ventral side view of column showing anther cap, rostellum and the location of the stigma (hidden). Scale bar = 2.5 mm. E) Distal view of anther cap. Scale bar = 0.5 mm. F) Proximal view of anther cap showing some pollinia still lodged in the cap and some attached to a dissecting pin. Scale bar = 0.5 mm. G) Pollinia attached to dissecting pin via sticky viscidia.

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44 Figure 1 2 Morphology of Bletia purpurea plant. A) Vegetative plant. Scale bar = 5 cm. B) Inflorescence and flower development sequence. Scale bar = 3 cm. C) Flowers. Sale b ar = 1 mm. D) Corms with shoots forming. Scale bar = 2 cm.

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45 CHAPTER 2 THE EFFECTS OF TEMPE RATURE AND ILLUMINAT ION ON GERMINATION A ND EARLY SEEDLING DEVEL OPMENT 1 Background The importance of temperature in promoting and inhibiting seed germination is well documented and responses are highly variable among species. Heat stress plays a role in delaying germination of some species (Kepczynska et al., 2006; Norsworthy and Oliveira, 2006) and long exposures to high temperatures can induce secondary dormancy (Nascimento et al., 2000) For other species, warm stratification breaks various dormancies or promotes germination (Leon and Owen, 2003; Turner et al., 2006) Cold stratification (Baskin and Baskin, 2001; Moyo et al., 2009; Han and Long, 2010) and oscillating temperatures (Baskin and Baskin, 2001, 2003) have also been shown to break dormancy and/or promote germination. Temperature can have profound effects on the light sensitivity of seeds (Hilton, 1984) and may play an important role in regulating seasonal germination responses (Heschel et al., 2007) Fluctuating temperatures can also overcome FR light induced inhibition of germination (Benvenuti et al., 2001; Honda and Katoh, 2007) This response likely functions as an ecologically important gap sensing mechanism by which seeds sense opening in the vegetative canopy; bare soil surfaces are expected to be less insulated from tem perature fluctuations than a patch of soil covered with dense vegetation. Since seeds that are dispersed to a low competition safe site (e.g. bare soil) may still be exposed to FR rich filtered light from tree canopies, fluctuating temperatures may be a mo re reliable gap indicator. 1 Significant portions of this chapter were originally accepted for publication in Plant Species Biology and are published here in accordance with John Willey and Sons, Ltd. author permissions.

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46 Seasonal regulation of germination timing may also be influenced by conditional dormancy. Conditionally dormant seeds are able to germinate under some temperatures, but not under the widest possible range of temperatures (Baskin and Baskin, 2001) resulting in enhanced germination in some seasons or simulated seasonal temperatures (Kettenring and Galatowitsch, 2007) Thus conditional dormancy can have a profound effect on the timing of seedling emergence. Though the Orchidaceae is the largest plant fam ily and accounts for a large proportion of the biodiversity of some ecosystems, little is known about the role temperature plays in controlling orchid seed germination as very few species have been studied. Such investigations have important implications f or the propagation of species for both ex situ and in situ conservation programs and as a means of revealing something about the poorly understood nature of orchid seed germination in situ. The objectives of this study were to determine the effect of simul ated South Florida seasonal temperatures on Bletia purpurea seed germination and early seedling development and to determine whether oscillating temperatures promote germination under asymbiotic culture conditions. An additional objective was to determine whether there was an interaction between the effects of temperature and illumination (continual darkness compared to 12 hour photoperiod) on germination and development. Methods S eed C ollection and S torage Five undehisced, nearly mature Bletia purpurea cap sules were collected from a population at the Florida Panther National Wildlife Refuge (FPNWR ; Collier County ) in Burn Unit 6 (Figure D 1) on 23 May 2007. Capsules were stored over silica gel

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47 dehisced. Seeds were removed from all capsules, pooled and homogenized, then placed into cold storage at Orchid S eed Bank. Seed S owing and C ulture C onditions Approximately 5.0 mg of seed was surface sterilized for 60 seconds in a solution of 6% NaOCl:100% ethanol:sterile distilled deionized (dd) water (1:1:18), then rinsed three times in sterile distilled water. Ster ilized seeds were sown onto 9.0 cm diameter Petri plates containing ~25 mL sterile Vacin and Went Modified Orchid Media (PhytoTechnology Laboratories, Vacin and Went, 1949) amended with 1 g L 1 activated charcoal (Dutra et al., 2008) Approximately 30 50 seeds (41 12; average standard deviation ) were sown onto each plate. Plates were sealed with a single layer of NescoFilm (Karlan Research Products Corporation) and randomly assigned a temperature and illumin ation treatment. Petri plates were cultured in growth chambers under one of five different temperature regiments. Four of these treatments were alternating 12/12 hour light/dark (L/D) temperature regimes designed to approximate seasonal temperature fluctua tions in south Florida (Table 2 1). National Oceanic and Atmospheric Administration NOWData on maximum and minimum monthly temperatures for Immokalee, FL between 1971 and 2000 was obtained from the National Weather Service ( http://www.weather.gov/ ) and used to estimate seasonal air temperature fluctuations. Actual temperatures tested were slightly different than weather data estimates as incubators were used concurrently for seed germination studies of species found i n other parts of Florida. Experimental t C (winter, spring, summer and fall temperature treatments) and a

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48 consta C C temperature treatment was chosen a s the control. Constant, or near constant, temperatures in the range of 20 25 are often used for orchid seed germination studies (see for example Pedroza Manrique and Mican Gutierrez, 2006; Stewart and Kane, 2006a; Yamazaki and Miyoshi, 2006; Lauzer et al., 2007; Dutra et al., 2009b; Stewart and Kane, 2010) In additi on to temperature treatments, seeds were also treated with either continual darkness or a 12/12 hour L/D photoperiod. Illumination was provided by cool white fluorescent lights at 2 s 1 Oscillating temperature treatments were performed in Percival 130VL incubators with side lighting and 25 C treatments were carried ou t in a Percival I36LL with overhead lighting. A comparison of the effects of these chambers on germination and development at 25 C and 16 hour photoperiod revealed no statistically significant differences in germination (F 1, 13 = 0.26, p = 0.62) or seedlin g development (F 1, 13 = 0.95, p = 0.35). Data C ollection and S tatistical A nalysis A completely randomized design (CRD) was used for this experiment with four replicate plates performed for all treatments. This experiment was repeated once (n = 8). Seeds an d seedlings were observed after three and 6 weeks of culture. At these times, data was collected on germination and subsequent seedling development as outlined in Table 2 2. Percent germination was calculated by dividing the number of seeds and seedlings a t stage 2 or greater by the total number of seeds in each replicate. These data were arcsine transformed to normalize data prior to statistical analysis. Untransformed means were graphed. Average developmental stage (D) was calculated using the equation where for each replicate plate, is the

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49 number of seeds in stage i multiplied by the stage number ( i ) and S is the total number of seeds in each replicate. ANOVA and least square (LS) mean separation at = 0.05 wa s performed using PROC MIXED in SAS software v 9.1.3 (SAS Institute Inc., 2003) LS mean separation utilizes multiple t tests (also called the least significant difference procedure) and is more liberal in identifying significant differences among means than corrected multiple comparison procedures as it d oes not maintain an experiment wise Type I error rate (i.e. LS mean separation does not correct for the number of treatments being compared). Instead each comparison is tested at a designated level. That being said, the main benefit of this method is that the ability to detect differences among means is not altered by the number of treatments tested Given this caveat, it has been recommended that it be viewed as a hypothesis generating proce dure rather than a simultaneous hypothesis generating and testing procedure (Saville, 1990) Results Seeds exhibited high germinat ion percentages (nearly 100%) within 6 weeks regardless of treatment. However, low temperatures and illumination delayed both germination and development (Figure 2 1 and 2 2). At week three, seeds cultured under illumin ation exhibited significantly lower germination than seeds cultured in darkness (Table 2 3; Figure 2 2). Interestingly, there was no significant difference between illumination treatments at constant 25 C or 33 /24 C Temperature and the interaction betwee n the main effects temperature and illumination also had a significant effect on germination at week three (Table 2 3; Figure 2 2) with lowest germination observed at simulated winter temperatures (22 /11 C ) After 6 weeks culture, germination was not sign ificantly affected by illumination, temperature or the interaction of main effects (Table 2 3).

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50 As with germination, seedling development at week three was significantly affected by illumination with a pronounced developmental lag observed in light treate d seeds at lower oscillating temperatures (Table 2 3 ; Figure 2 2). Temperature and the interaction of temperature and illumination also had significant effects on development at this time with lower seedling development observed at simulated winter tempera tures being compounded by light (Table 2 3 ; Figure 2 2). After 6 weeks culture main effects of temperature and illumination, as well as the interaction of these main effects had a significant effect on development (Table 2 3 ). At this time illumination no longer had a negative effect on germination as seedlings in most temperature treatments had developed to a significantly greater average stage than dark treated seeds (Figure 2 2). Seeds and seedlings in simulated winter temperatures still had lower avera ge development than other treatments regardless of illumination treatment. Gre ater development was observed 25 , 29/19 and 33/24 C when seeds were exposed to light ( Figure 2 2). Though not quantified, rhizoid production appeared to be suppressed under i lluminated conditions, as well as under simulated summer temperatures ( Figure 2 1 ) Discussion T emperature can have a profound effect on dormancy, germination and seedling emergence, thus it is of paramount importance to propagating and understanding the seed ecology of a species. Bletia purpurea seeds were able to germinate under a wide range of temperatures that they might encounter in Florida. Flowering is somewhat erratic, but concentrated between March and May with capsules maturing between Ap ril and July on the FPNWR (personal observation ). Seeds are likely shed in summer when daytime highs can reach 33 C (Table 2 1). There is no indication from this study that

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51 such temperatures would be inhibitory for germination. Because B. purpurea seeds were able to germinate under a wide range of seasonal temperatures, water, light and/or fungal infection, not temperature, are likely the limiting factors regulating germination and seedling emergence There are v ery few reports on the effect of temperature on orchi d seed germination. Therefore more studies of this kind are needed in order to identify response trends for terrestrial versus epiphytic or tropical versus temperate orchid species. Though B. purpurea seeds were capable of germinating under a wide range of temperatures including relatively high temperatures, such high temperatures have been reported to be inhibitory for other orchid species. Optimal incubation temperature for the symbiotic germination of Dactylorhiza majalis was between 20 and teep declines in germination observed above 25 C (Rasmussen et a l., 1990b; Rasmussen and Rasmussen, 1991) However, seeds of the tropical species Vanilla fragrans have been shown to germinate to 20% when cultured at 32 C but only 3% at 28 C and 0% at 25 C (Knudson, 1950) These temperatures are high compared to the 20 25 C range that is typically used for both asymbiotic an d symbiotic orchid seed propagation of orchids. Low temperatures may also affect germination of some orchid species. Temperatures below 10 C drastically reduced symbiotic germination of D. majalis (Rasmussen et al., 1990b) and reductions in germination were also observed for symbiotically cultured seeds of Dactylorhiza purpurella (syn. Orchis purpurella ) cultured at 11 C and 17C (Harvais and Hadley, 1967) It is not clear from these studies whether germ ination was inhibited because of some constraints on the seed or if symbiosis is

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52 affected by low temperatures. Germination and development of B. purpurea was slower in simulated winter temperatures, but final germination was not affected The vast majority of orchid seed germination studies have tested constant temperatures. However, as previously discussed, oscillating temperatures may provide important environmental cues that enhance germination under some conditions (Ho nda and Katoh, 2007) There is evidence that germination and development of B. purpurea seedlings is impaired by oscillating temperatures in illuminated conditions when compared to continual dark treatments ( Figure 2 2). It is possible that this is due to differences in phytochrome activity at different temperatures as has been reported in Arabidopsis (Heschel et al., 2007) A cold induced negative photo blastic response may protect seeds from germinating and developing at the soil surface when temperatures are low and frost is likely.

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53 Table 2 1 Average monthly a nd seasonal temperature minima and maxima for Immokalee, FL (1971 2000). Season Winter Spring Summer Fall Parameter Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec. Annual Max. Temp. ( C ) 24.7 25.5 27.5 31.1 31.7 32.7 33.1 33.0 32.2 30.2 27.6 25.3 29.4 Min. Temp. ( C ) 11.2 11.4 13.4 15.1 18.2 21.1 22.0 22.4 22.1 19.1 15.7 12.3 17.0 Ave. Temp. ( C ) 17.9 18.5 20.5 22.2 24.9 26.9 27.6 27.7 27.2 24.6 21.6 18.8 23.3 Seasonal Max. ( C ) 25.9 31.8 32.8 27.7 Seasonal Min. ( C ) 12.0 18.1 22.2 15.7 Seasonal Ave. ( C ) 19.0 24.7 27.5 21.7

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54 Table 2 2. Description of stages used to assess Bletia purpurea germination and seedling development. Modified from Dutra et al. (2008) Stage Description 0 Hyaline embryo, testa intact 1 Embryo swollen, rhizoids may be present 2 Testa ruptured by enlarged embryo (= germination) 3 Emerged embryo with conical apex 4 Differentiation of first leaf 5 Elongation of firs t leaf 6 Emergence of second leaf

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55 Table 2 3. ANOVA results for the effects of temperature and illumination on Bletia purpurea germination and seedling development. Effects considered significant and these F values are bolded. Germi nation Development Week 3 Week 6 Week 3 Week 6 Effect df F p F p F p F p Illumination (I) 1 83.40 < 0.01 1.44 0.23 57.57 < 0.01 22.26 < 0.01 Temperature (T) 4 36.37 < 0.01 0.36 0.84 42.74 < 0.01 89.25 < 0.01 I T 4 7.49 < 0.01 0.57 0. 69 5.97 < 0.01 7.72 < 0.01

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56 Figure 2 1. Bletia purpurea seeds and s eedlings cultured under 6 combinations of illumination and seasonal temperatures for 6 weeks. Arrows point to rhizoids. A) 25 /25 C dark. B) 25 /25 C

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57 Figure 2 2 Effect of temperature and ill umination on Bletia purpurea germination and seedling development after three and 6 weeks. Grey filled bars = dark treatment. White hatch marked bars = 12 /12 hour photoperiod. Error bars represent positive standard error of means. Within each graph, bars w ith the

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58 CHAPTER 3 GERMINAT ION AND SEEDLING DEV ELOPMENT IS ENHANCED BY ILLUMINA TION, MINERAL SALT NUTRIEN TS AND NON ALCOHOL SUGARS 2 Background In nature, germination of orchid seeds is depend ent upon the formation of a symbiotic relationship with fungi, which supply nutrients and carbohydrates to the minute, non endospermic seeds and rudimentary embryo (Alexander et al., 1984; Alexander and Hadley, 1985; Manning and van Staden, 1987; Rasmussen, 1995; McKendrick et al., 2000; Yoder et al., 2000) This relationship can be replicated in vitro by co culturing orchid seeds with compatible fu ngi (symbiotic seed germination) and has been demonstrated with many different orchid species (Shimura and Koda, 2005; Zettler et al., 20 05; Batty et al., 2006b; Stewart, 2006; Johnson et al., 2007; Stewart and Kane, 2007; Yagame et al., 2007; Stewart and Kane, 2010) Alternatively, asymbiotic seed germination, in which seeds are cultured without fungi, but with nutrient rich media contain ing mineral nutrients, carbohydrates (typically sucrose) and organic compounds can be used. While symbiotic seed culture is useful for studying fungal specificity and nutrient flow from between symbionts, and important for reintroductions (Zettler, 1997) asymbiotic culture allows scientists to manipulation and to study the effects of specific compounds and environmental conditions on seed germination and seedlin g development. The aim of many orchid asymbiotic seed germination studies has been plant production. In these papers, conclusions are often drawn about the light and nutritional requirements of orchid seeds, but the impact of exogenous carbohydrates 2 Significant portions of this chapter were originally published in Plant Growth Regulation (2011, 63:89 99) and are published here in accordance with author permissions outlined in the copyright transfer with Springer Science+Business The entirety Media B.V.

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59 and mi neral nutrients on photoblasticity, for example, is often overlooked. The objective of such studies is to develop propagation protocols, which is important, necessary and often challenging in itself. However, such studies reveal very little about the ecolo gical and physiological requirements for germination. Carbohydrate utilization by seeds serves a number of functions during germination. Primarily, stored carbohydrates serve as an energy source that supports germination and seedling growth (Grecki et al., 1996; Young et al., 1997; Bonfil, 1998; Kitajima, 2003; Obendorf et al., 2009) Carbohydrates may also act as signaling molecules involved in the regulation and integration of several important biochemical pat hways that affect germination, seed dormancy and seed reserve mobilization (Karrer a nd Rodriguez, 1992; Perata et al., 1997; Finkelstein and Lynch, 2000) Most scientific knowledge about the effects of carbohydrates on regulating germination comes from work with mutant Arabidopsis plants. This work has revealed that germination is the cu lmination of an array of complex and well choreographed physiological processes (see reviews by Yuan and Wysocka Diller, 2006; Penfield and King, 2009) That exogenous carbohydrates are required for in vitro orchid seed propagation is almost universally accepted by researchers in this field. Even species that are able to germi nate without carbohydrates show limited post germination development (Downie, 1941; Vermeulen, 1947; Stoutamire, 1964; Smith, 1973; Stoutamire, 1974) However, the degree to which light and nutrients alter the germination promoting effects of soluble sugars on orchid seed germination and seedling development is not clear. The same lack of knowledge is true regarding the critical environmental cues orchid seeds require for the completion of germination and subsequent development. In t his study,

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60 asymbiotic seed culture was used to assess several hypotheses in order to answer the question: how is germination and early seedling development affected by genotype, nutrient availability, light and carbohyd rates ? A critical examination of poss ible germination stimuli in the absence of mycorrhizal fungi may illuminate the specific mechanisms by which carbohydrates (supplied by fungi in situ) and environmental conditions regulate orchid seed germination and advance the knowledge about orchid seed physiology and germination ecology. Materials and Methods Seed C ollection Bletia purpurea seeds from the Florida Panther National Wildlife Refuge (FPNWR ) were used for all experiments. Three to 6 browning, undehisced capsules were collected from three loc ations on 24 April 2008 and stored over silica gel desiccant at room temperature (~22C) for four weeks. Dry seed from each site was then removed from capsules, pooled by site and stored at C over silica gel desiccant until experimentation (Pritchard et al., 1999) Collections were made from three distinct habitats within the FPNWR (Figure D 1) : a pine flat woods (6 capsules; Burn Unit 9, seed source code 15 9), a lake margin (5 capsules; Burn Unit 50, seed source code 162), and a cypress swamp where B. purpurea was found growing on floating logs (3 capsules; Burn Unit 33 seed source code 164). Estimate of S eed V iability To assess seed viability, all seed sourc es were subjected to triphenyltetrazolium chloride (TZ) staining. A small volume of seed (approximately 100 200 seeds) was placed in 1.5 mL centrifuge tubes and treated with 5% Ca ( O C l ) 2 (w/v) for 30 minutes to weaken the testa and facilitate staining. Seed s were then rins ed three times in distilled

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61 water before seeds being resuspended in water and in cubated at room temperature for 23.5 hours. Water was then replaced with 1% TZ (pH 7.0) and seeds were incubated for 24 hours at 30 C Seeds were examined with a dissecting microscope; seeds containing embryos with any degree of pink to red staining throughout the embryos were considered viable while wholly unstained embryos were considered non viable. Five replicates were performed for all genotypes each consis ting of 50 200 seeds. P ercent viability was calculated by dividing the number of seeds with viable embryos by the total number of seeds with embryos. Germination and E arly Seedling D evelopment in the P resence of S ucrose The objective of this experiment was to test the hypothesis that germination and early seedling development of B. purpurea would increase when cultured in the presence of sucrose and light. Three seed sources were used for this experiment: 159, 162 and 164. A 2 (illumination) 3 (sucrose co ncentration) factorial was us ed to test this hypothesis with all three seed collections. Water agar (WA; distilled deionized [dd] water with 7 g l 1 TC agar [ Phyto Techn ology Laboratories ] ) was amended with 0, 10 or 50 m M sucrose, adjusted to pH 5.8 and aut oclaved for 40 minutes at 117.7 kPa and 121 C Mineral salts were not added to the media. Sterilized media were dispensed as 25 mL aliquots into 9 cm Petri dishes and allowed to solidify. Seeds were surface sterilized in a solution of 6.0% NaOCl :100% ethan ol: sterile dd water (1:1:18) for 60 seconds, then rinsed three times in sterile distilled water. A n average of 48 13 (mean standard deviation [SD] ) seeds were sown onto each plate with a sterile inoculating loop before plates were sealed with a single layer of NescoFilm (Karlan Research Products Corporation) For all experiments, plates were incubated in a growth chamber at 25C under 16/8 hour (light/dark) photoperiod provided by cool white fluorescent

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62 lights (Phillips F20T12/CW) at approximately 50 10 M m 2 s 1 for 6 weeks. Continual dark was provided by wrapping plates in heavy duty aluminum foil and only exposing seeds to brief periods of light (< 20 minutes) during observation. For all experiments seeds and seedlings were examined after two, four and 6 weeks for signs of germination and development. Seeds and seedlings were scored on a scale of 0 6 ( Table 3 1 ). For each seed source plates were randomized in the incubator and within aluminum foil sleeves. Four replicate plates were used for each t reatment. All experiments were repeated once. Germination and E arly S eedling D evelopment in the P resence of S ucrose and M ineral S alts Because of concerns that the lack of nutrients was limiting germination and development of seeds cultured without sucrose in Experiment 1, a second 2 (illumination) 3 (sucrose concentration) factorial experiment that incorporated mineral sal t nutrients in the culture medium (mineral nutrient agar; MNA) was performed. Again, the hypothesis was tested that germination and ear ly development of B. purpurea seeds would increase when cultured in the presence of sucrose and light. Bletia purpurea seed sources 159, S161 and 162 were all tested. Basal medium consisted of strength Murashige and Skoog medium (Murashige and Skoog, 1962) modified strength FeSO 4 7H 2 O and Na 2 EDTA. Medium was gelled with 7 g L 1 TC agar Medium was then amended with 0 (control), 10 or 50 m M sucrose, adjusted to pH 5.8 and autoclaved as previously described. An average of 37 7 (mean SD) seeds were sown onto each plate. Plates were cultured in dark or 16/8 h light/dark photo period at 25 C as previously described.

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63 Germination and E arly S eedling D evelopment in the P resence of V arious C arbohydrate s The objective of this experiment was to examine the ability of several different carbohydrates to support seed germination, seedling development and rhizoid production of B. purpurea under continual darkness. The hypothesis tested was that carbohydrate source and carbohydrate mol arity would affect seed germination, germination index (an estimate of the rate of germination), seedling de velopment, developmental index (an estimate of the rate of development) and the percentage of seedlings producing rhizoids. Basal medium was the same as in experiment 2. Carbohydrates used for this study were sucrose, D fructose, D glucose, D trehalose, D mannitol and D sorbitol at 10 and 50 mM A control was also tested. Carbohydrates were filter sterilized with nylon 0.2 m pore size syringe filters ( Nalgene catalogue number 195 2520 ) and added to media after it was autoclaved because of concerns that au toclaving carbohydrates may differentially alter their biological activ ity and/or composition. Seed source 164 was used for this experiment. An average of 36 7 (mean SD) seeds were sown onto each plate. All plates were maintained in continual darkness at 25 C as previously described. S tatistical A nalysis Percentage of germinated seeds and average stage of development were calculated for each replicate at two, four and 6 weeks after seeds were sown. During scoring, dark treated seeds were exposed to sho rt periods of light (< 20 min). These brief exposures were found to have little effect on germination (Appendix A). The percentage of seedlings producing rhizoids after 6 weeks in culture was examined during experiment three. Average stage of development a t week 6 ( D ) was calculated

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64 using the equation where for each replicate plate, is the number of seeds in stage i multiplied by the stage number ( i ) and S is the total number of seeds in each replicate. Melville et (1980) germination index (GI) was modified as described in Ranal and de Santana (2006) GI was calculat ed using the equation where i is the observation week, 7 is the dur ation of the study (6 weeks) + 1, G i is the number of seeds germinated on the i th observation, and S is the total number of seeds in each replicate. GI incorporates both total germination and germination rate into a single value. A greater GI represents a faster rate of germination and/or greater overall germination. Maximum possible GI in this study is 5.0 for a hypothetical treatment in which all seeds germinate by the first observation at week two. index (DI) using the equation where D i is the average developmental stage for each replicate at the i th observ ation. Factorial experiments were analyzed using a two way ANOVA to assess the effects of main factors and interactions on arcsine transformed percent germination data and average developmental stag e using PROC MIXED in SAS (SAS Institute Inc., 2003) treating repeat as a random variable. For the experimen t examining the effects of carbohydrate sources and molarity and germination and development, a one way ANOVA was used to assess the effects of treatment on the previously mentioned response variables as well as on the percentage of seedlings producing rhi zoids, GI and DI using PROC MIXED. Percent rhizoid production data was arcsine transformed, but

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65 germination percentages were not as the residuals of non transformed data were normally distributed and arcsine transformation resulted in bimodal distribution of the residuals. Class comparisons were used to test specific hypotheses about the effects of different carbohydrates on germination and development as illustrated in Table 3 2 and 3 3. Least squares (LS) mean separation was used to compare treatment mean 0.05. Results Effects of S ucrose and I llumination on G ermination and E arly S eedling D evelopment on W ater A gar Germination in seed source s 159 and 162 was < 40% in almost all treatments (Figure 3 2). Yet maximum germination surpassed 40% in most t reatments for seed source 164 (Figure 3 1). Observed germination was much lower than estimated viability (90.3% 1.1, 77.0% 4.8% and 89.1 2.3% [mean SD ] for seed source s 159, 162 and 164, respectively) for nearly all treatments and seed source s. Alt hough there were differences among seed source s, germination improved significantly over control treatments when seeds were cultured with sucrose regardless of light treatment or seed source (Figure 3 2 A, C, E; Table 3 2 ). Results of ANOVA indicate that i llumination had a significant effect on germination of seed source s 164, but not 159 or 162 However, subsequent LS mean separation analysis of lot 159 indicated that there were significant differences between illumina t ion treatments at 0 and 50 m M sucrose treatments. Class comparisons indicated that culture with 10 and 50 m M sucrose significantly enhanced germination over control for all seed source s. The interaction between illumination and sucrose had a significant effect on germination in all seed acces sions. In general, the effect of sucrose on germination was more pronounced when seeds were cultured in

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66 darkness. Additionally, the additive effects on germination of sucrose were not as pronounced for seeds cultured in light since control treated seeds we re able to germinate to a higher percentage compared to dark treated seeds. Sucrose and illumination both had a significant effect on GI for all seed source s tested, as did the interaction of these main effects ( Table 3 2 ). Lowest values were observed whe n seeds were cultured in darkness without sucrose and greatest when seeds were cultured in darkness with 50 m M sucrose ( Table 3 3 ). Increased sucrose levels corresponded to increased GI in both light and dark treatments. However, when sucrose was incorpora ted into WA, GI was consistently greater in darkness than in illuminated treatments indicat ing faster germination rates. As was observed with germination, seeds of lot 164 developed to a more advanced stage than 159 and 162. Seeds cultured in darkness swel led, but rarely ruptured their testas resulting in average developmental stages close to 1. For all seed source s, sucrose was found to have a significant effect on seed/seedling development and incorporation of sucrose into culture media enhanced seedling development of all tested lots (Figure 3 2 B, D, F; Table 3 2 ). ANOVA results indicated that illumination had a significant effect on development of seed source s 162 and 164, but not 159. However, subsequent LS mean separation analysis of lot 159 indicated that there were significant differences between illumination treatments at 0 and 50 m M sucrose treatments. Class comparisons indicated that 10 and 50 m M sucrose treatments significantly enhanced development over control for all seed source s. The interacti on between sucrose and illumination on development was significant for all seed source s as well. In general, development in control treatments was greater in light while development of seeds in 50

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67 m M sucrose treatments was greater in darkness. After 6 week s culture, seedlings grown on 50 m M sucrose treatments showed some signs of stress as indicated by necrosis and/or chlorosis. DI values were less variable and had fewer significant detectable differences between treatment means than other variables examine d ( Table 3 3 ). However, sucrose control treatments tended to have lower DI values than 10 and 50 m M treatments. ANOVA results for DI were less consistent among seed sources than other response variables that were examined. Sucrose and illumination had a si gnificant effect on the DI of seed source s 159 and 164, but not 162 ( Table 3 2 ). A significant interaction between sucrose and illumination was onl y detected for seed source 165. Effects of S ucrose and I llumination on G ermination and E arly S eedling D evelop ment on M ineral N utrient A gar In general, seed germination and development was much greater when seeds were cultured on mineral nutrient agar (MNA) than on WA ( Figure 3 3): most treatments yielding germination > 60% when nutrients were incorporated into cu lture media. Sucrose again had a significant effect on germination of all seed source s and treatment wit h sucrose significantly enhancing germination over control treatments (Figure 3 4 A, C, E; Table 3 2 ). Germination of all seed source s exceeded estimate d viability when seeds were treated with 10 m M sucrose. A clear correlation between estimated viability and germination was not apparent; as maximum germination was comparable between seed source s 159 and 162 even though viability estimates were considerab ly different (90.3% and 77.0%, respectively). Illumination had a significant effect on germination of all seed source s; the effect of light on germination was pronounced when seeds were cultured without sucrose, but not when seeds were cultured on media co ntaining 10 or

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68 50 m M sucrose. Greatest germination was observed when seeds were cultured on medium with 10 m M sucrose. A significant interaction of main effects was also detected. Greatest GI values were consistently observed when seeds were treated with 50 m M sucrose in darkness ( Table 3 3 ). Sucrose levels significantly affected GI of all seeds lots and class comparisons detected significant differences between control and 10 m M sucrose treatments for all seed sources ( Table 3 2 ). Illumination also had a significant effect on DI of all seed sources tested. Significant differences were also detected between control and 50 m M treatments of seeds source 164, but not 159 or 162. Interestingly increasing sucrose from 10 to 50 m M under both illuminated and cont inually dark conditions resulted in significantly lower GI values in seed source 159 and 162. Increasing sucrose molarity from 10 to 50 m M did not significantly affect GI of seed source 164 in light, but did significantly increase in dark treatments. A sig nificant interaction between sucrose and illumination was also detected for all seed sources. Under both illumination treatments, development was greater when seeds were treated with 10 m M sucrose compared to control and 50 m M sucrose levels (Figure 3 4). At all levels of sucrose, germination was consistently greater when seeds were exposed to light compared to dark treatments. Sucrose had a significant effect on development of seeds and sucrose treatments significantly enhanced development over control wh en seeds were cultured on MNA (Figure 3 4 B, D, F; Table 3 2 ). The effects of illumination treatment on development were also significant as light enhanced development at all sucrose treatments. A significant interaction between main effects was also detec ted. Greatest observed development was obtained for seeds cultured under illuminated conditions with 10 m M sucrose.

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69 Incorporation of sucrose into MNA increased DI over control in both light and dark treatments ( Table 3 3 ). Sucrose had a significant effect on DI values of all seed sources tested ( Table 3 2 ). Class comparisons indicate that 0 and 10 m M sucrose treatments were significantly different for all seed sources, though a significant difference between 0 and 50 m M sucrose treatments was only detected for seed source 159. Illumination significantly affected DI of seed source 162, but not 159 or 164. Likewise, a significant interaction between sucrose and illumination was only detected with seed source 162. Germination and D evelopment of S eeds C ultured in the P resence of V arious C arbohydrate s Germination of seed source 164 depended upon carbohydrate source rather than carbohydrate concentration (Figure s 3 5, 3 6 A; Table 3 4 ). Less than 15% of seeds cultured without a carbohydrate germinated. Analysis o f different carbohydrate classes indicated that sugars (sucrose, fructose, glucose and trehalose) significantly enhanced germination compared to both control and polyol (sorbitol and mannitol) treatments. Polyols did not enhance germination compared to con trol. Carbohydrate source and molarity both had a significant effect on seedling development (Figure 3 6 B, Table 3 4 ). Development was least advanced when seeds were cultured on control medium or with polyols and much greater when treated with sugars. Cla ss comparisons revealed significant differences between control and both 10 and 50 m M carbohydrate treatments ( Table 3 4 ). Culture in the presence of 50 m M fructose resulted in less advanced development than 10 m M fructose treatment. For all other carbohyd rates tested, equal or greater development was observed with 50 m M treatments than with 10 m M treatments. As with germination response, sugars

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70 significantly enhanced development over control and polyol treatments. Greatest development was observed when see ds were cultured with 50 m M sucrose and 10 m M glucose. Greater molarity of carbohydrates resulted in greater GI and DI values when non alcohol sugars were incorporated into media, while control and sugar alcohol treatments yielded similar low GI and DI val ues (Figure 3 6 C and D, respectively). Treatment had a significant effect on both of these variables ( Table 3 4 ). Mannitol and sorbitol did not significantly enhance GI compared to control. Likewise sugar alcohols did not enhance DI compared to control tr eatment. Culture with sugars enhanced rhizoid production compared to control and polyol treatments (Figure 3 6 E; Table 3 4 ). The response to sucrose and fructose was enhanced by greater molarity. Interestingly, the number of seedlings producing rhizoids w hen treated with 10 m M glucose was greater than with 50 m M Treatments did have a significant effect on rhizoid production ( Table 3 4 ). Class comparison revealed that carbohydrates enhanced rhizoid production over control treatment. In addition, significan t differences were detected between control and sugars, as well as between sugars and polyols. However, no significant differences were detected between control treatments and polyols. Discussion Germination of Bletia purpurea seeds was possible in the abs ence of sucrose when seeds were cultured in light. In darkness, seed germination in the absence of sucrose or in the presence of polyols was limited. Early seedling development was similarly affected with limited development observed when seeds were cultur ed in darkness without sucrose or another suitable carbohydrate. The incorporation of mineral salts into culture media enhanced development compared to WA In experiment 2,

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71 increasing sucrose molarity from 10 to 50 m M resulted in decreased germination and development. This indicates either that seeds experienced osmotic stress at the higher molarity or that growth and development was inhibited by byproducts of sucrose hydrolysis during autoclaving (Schenk et al., 1991; Sawyer and Hsiao, 1992; Wang and Hsiao, 1995; Pan and van Staden, 1999) Inhibition by b yproduct s of sucrose hydrolysis seems more likely since in experiment 3, where carbohydrates were filter sterilized, germina tion was not inhibited and seedling development was more advanced in the presence of 50 m M sucrose compared to 10 m M treatments. Seeds were not able to utilize polyols for germination. Rhizoid production was influenced by both carbohydrate source and molar ity, but glucose was more effective at inducing rhizoid production at lower molarity than other carbohydrates tested. Seed viability was not a good indicator of germinability as maximum germinability consistently exceeded estimated viability and lower viab ility did not lead to relatively lower observed germinability or development. These results demonstrate the importance of corroborating viability estimates with germination essays when working with orchids. Carbohydrate U tilization by G erminating S eeds and D eveloping S eedlings Orchid seeds are small, have undifferentiated embryos and may not have suitable quantities of storage reserves for germination without infection by symbiotic mycorrhizal fungi (see discussion in Rasmussen, 1995) The few studies on orchid seed reserves reveal that storage materials are diverse and include lipids, proteins and sugars (arabinose, maltose, sucrose and rhamnose), though starch is generally lacking prior to germination (Manning and van Stade n, 1987; Richardson et al., 1992; Yeung and Law, 1992; Leroux et al., 1995) Under in vitro asymbiotic seed culture conditions, orchid seeds are able to utilize a wide range of oligosaccharides, disaccharides,

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72 monosaccharides and complex carbon containing plant extracts (Knudson, 1922; Wynd, 1933; Ernst, 1967; Ernst et al., 1971; Ernst and Arditti, 1990; Lo et al., 2004) Of the few reports of orc hid seed germination in the absence of carbohydrates or fungi (Downie, 1941; Vermeulen, 1947; Stoutamire, 1964; Smith, 1973; Stoutamire, 1974) it is not always clear from these studies if seeds were cultured in light or dark conditi ons or the criteria used for scoring germination (discussed below). As previously stated, a small fraction of B purpurea seeds were able to germinate in darkness without a carbohydrate present, though more were able to germinate under light. Development o f Dactylorhiza purpurella and Bletilla hyacinthina was likewise limited in darkness when seeds were cultured without carbohydrates and was enhanced in the presence of sucrose, glucose and trehalose (Smith, 1973) Similar results were obtained with Goodyera repens seeds, which were only able to germinate in darkness when supplied glucose, fructose, sucrose or trehalose (Purves and Hadley, 1976; Stewart and Kane, 2010) The se studies illustrate the important role exogenous carbohydrates play in promoting germination of B. purpurea and other orchid species, especially in the absence of light. It seems plausible that soluble carbohydrates serve to signal imbibed seeds of funga l infection in orchids, though further corroborative evidence is needed. While glucose is the universal source of energy and carbon in living cells, exogenous glucose has been shown to slow or stop seed germination of wild type Arabidopsis and seeds of oth er species by slowing the breakdown of ABA (Price et al., 2003; Dekkers et al., 2004; Yuan and Wysocka D iller, 2006; Zhao et al., 2009; Zhu et al., 2009) In the current study, higher doses of glucose did not decrease germination or development. However,

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73 we tested lower concentrations than have been found effective at inhibiting Arabidopsis seed germination (277.5 mM in Zhao et al., 2009) The sorbitol pathway has also been implicated as important during seed germination where it likely acts to modulate the buildup of fructose during reserve mobilization (Kuo et al., 1990) Neither sorbitol nor mannitol e nhanced B. purpurea seed germination or development over control treatment in the current study. However, sorbitol has been shown to support germination of the epiphytic orchid Epidendrum radicans (Gayatri and Kavyashree, 2007) The experimental design of the current study does not answer the question of whether polyols are simply unmetabolized by B. purpurea or if these compounds are inhibitory to germination and development. Additional study is needed to elucidate this question and determine if sorbitol metabolism is wide spread in the Orchidaceae. Role of N utrients in R egulating G ermination and S eedling D evelopment The impact of asymbiotic media nutrient composition on orchid seed germination and seedling development is well docume nted (Arditti et al., 1981; Znaniecka et al., 2005; Stewart and Kane, 2006a; Johnson et al., 2007; Dutra et al., 2008; Kauth et al., 2008a; Dutra et al., 2009b) In the current study, seed germination was possible in the absence of mineral nutrients if seeds were cultured with sucrose and/or under illumination, thoug h development was enhanced in the presence of mineral nutrients. This study indicates that B. purpurea seed germination is possible in the absence of nitrogen, phosphorus and other micro and macronutrients, though development is limited at least in part b y nutrient availability. With the exception of an apparent carbon limitation (i.e. the need for either exogenous sugars or autogenic photosynthates), germination is not unlike other plants that are able to germinate on moistened filter paper, but that do n ot survive long if nutrients are withheld.

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74 Effect of L ight on G ermination and S eedling D evelopment The effects of light on orchid seed germination has been studied extensively, albeit often in the presence of exogenous sucrose (see for example Rasmussen et al., 1990b; Fukai et al., 1997; McKinley and Camper, 1997; Kauth et al., 2006; Dutra et al., 2008; Dutra et al., 2009b) or mycorrhizal fungi that likely provide car bohydrates to developing embryos (see for example Rasmussen and Rasmussen, 1991; Johnson et al., 2007; Stewart and Kane, 2007; Zettler et al., 2007) However, in such studies possible interacti ons between exogenous sugars and photoperiod are not considered and it is not clear how sucrose or other carbohydrates may be interacting with other abiotic factors. Responses of orchid species to light are highly variable. Even low levels of light can g reatly inhibit germination of some terrestrial orchids (Van Waes and Debergh, 1986) For other species, light conditions have little or no effect on germination, though development may be markedly affected (Stewart and Kane, 2006a, 2006b; Dutra et al., 2008) In contrast, both germination and development of Cyrtopodium punctatum is profoundly enhanced under illuminated conditions (Dutra et al., 2009b) Development of Habena ria macroceratitis under symbiotic and asymbiotic conditions was sig nificantly greater after 14 weeks culture in darkness compared to continual light and 16/8 hour photoperiods (Stewart and Kane, 2006a, 2006b) Van Waes and Debergh (1986 ) reported increased germination of 11 European orchid species under continual darkness compared to even low levels (1.2 mol m 2 s 1 ) of presumably continual light. Symbiotic seed germination of Platanthera integrilabia was enhanced by exposing seeds to seven days of 16/8 hour photoperiod before moving them to continually dark conditions (Zettler and McInnis, 1994) while optimal Dactylorhiza majalis seed

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75 ge rmination requires imbibition in darkness for at least 14 d ays (Rasmussen et al., 1990a) Germination of Calopogon tuberosus was significantly reduced when seeds were initially cultured in darkne ss for two to 6 weeks before being moved to light compared to seeds cultured in continual darkness or 16/8 h photoperiod (Kauth et al., 2006) An interesting finding of the current study is that light can stimulate germination and development of B. purpurea seeds when carbohydrates are not present in culture media. This contradicts common opinion about orchid seed physiology which indicates that orchids rely on external energy sources for germination and early seedling development. However, a thorough review of the literature indicates that this hypothesis has rarely bee n tested (Downie, 1941; Vermeulen, 1947; Stoutamire, 1964; Smith, 1973; Harvais, 1974; Stoutamire, 1974) Culturing orchid seeds wi thout carbohydrates is not likely a commercially viable method of production given that some carbohydrates greatly enhance germination and development. However, conclusions drawn from studies that assume carbohydrates are required for germination may overl ook important ecologically significant interactions between orchids and their environment. An interaction between light and carbohydrate availability in regulating germination has not been previously reported in the Orchidaceae and indicates the possibilit y of two different pathways of germination: 1) buried seeds that are unable to photosynthesi ze sugars can germinate after receiving carbohydrates from infecting fungi or 2) seeds exposed to light can produce photosynthates that support germination. In the second scenario, infection may further enhance germination and development as exogenous sucrose enhanced development

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76 when seeds were exposed to light. Definitively testing this hypothesis is difficult and may rely more on corroboratory evidence from in vit ro studies than in situ or molecular studies. Under continual darkness, rhizoid production only occurred when seeds were cultured in the presence of carbohydrates that supported germination and development beyond Stage 2 (sucrose, fructose, glucose and tr ehalose). This indicates that rhizoid production di d not begin until a suitable carbohydrate was detected by the seed or until a carbohydrate was available for rhizoid production. Stewart and Kane (2010) made similar observations of Habenaria macroceratitis In contrast, Calopogon tuberosus seedlings were able to produce rhizoids in both light and dark conditions (Kauth et al., 2006) though rhizoid production was more abundant in dark ( P. Kauth, personal com munication ) Rhizoids were not observed on B. purpurea seeds and seedlings when seeds were cultured in light implicating light signaling inhibition, possibly via phytochrome or other photoreceptors, in the regulation of rhizoid production (for a review of phytochrome signaling see Wang and Deng, 2002) Inhibition or reduction of rhizoid production under illuminated conditions has been noted for other orchid species as well (Kauth et al., 2006; Stewart and Kane, 2006a) Interactions between light and sugar sensing have already been reported to regulate A rabidopsis germination, growth and development (Short, 1999; Finkelstein and Lynch, 2000) and may also play a role in regulating rhizoid production in orchids. Germination in the A bsence of N utrients and C arbohydrates Coeloglossum viride Gymnadenia conopsea Orchis macul at a var. elodes and Orchis purpurella on water agar and sterile water with glass wool substrate, though it is not clear from this report whether seeds were cultured i n light or dark. Orchis morio

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77 seeds have also been reported to germinate and produce rhizo ids when sown onto water agar (Verm eulen, 1947) Germination of Dactylorhiza purpurella and Bletilla hyacinthina on Knudson C mineral salts without a carbon source was reported by Smith (1973) though the published results are difficul t to interpret as only seedling dimensions were provided and photoperiod details are also missin g. Stoutamire (1964; 1974) reported that the embryos of numerous orchids ( Bletilla hyacinthina Calopogon tuberosus Disa unif lor a Habenaria radiata Microtis unifolia Sp iranthes cernua and Spiranthes sinensis ) become green upon imbibition in light Goodyera repens var. ophioid es Habenaria hyperborea and Habena ria obtusata all reported ly germinated on water agar, however the seedlings of these species did not became green or form leaves (Harvais, 1974) It is possible that the embryos of these species, like B. purpurea are able to imbibe enough water on carbohydrate free medium in darkness that the testa ruptures while no further development is observed. Downie wrote in 1941 point in any germination seed to germinate in the a bsence of exogenous carbon sources was commonly overlooked. This oversight has continued as few orchid seed germination studies have included a carbohydrate free control treatment in their experiments. There are two probable explanations for this; 1) there may be a general assumption that exogenous carbohydrates are required for orchid seed germination and 2) the objectives of many orchid seed germination studies is to propagate plants rather than to study the physiology of orchid seeds. Whatever the explan ation, the ability of orchid seeds to

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78 germinate in the absence of soluble carbohydrates may be underreported in the scientific literature. A more complete understanding of germination and early development within the Orchidaceae may require genetic and/or molecular approaches before theories about orchid seed germination codify However, soluble carbohydrates appear to play a key role in regulating germination and rhizoid production of B. purpurea Carbohydrates may thus play an important role in regulating the orchid fungus symbiosis by effecting rhizoid production and may serve as key signaling molecules in relieving seed dormancy or nutritional blocks to seed germination. However, advancing the scientific knowledge of orchid seed ecology and seed physiolo gy requires more careful experimentation. While the asymbiotic culture system allows for precise manipulation of nutrients, it has not often been used to study orchid seed physiology explicitly. There has been a tradition lack of separati on between the obj ectives of asymbiotically pro pagating plants from seed and studying the physiology of germination and development though researchers are often interested in discussing the ecological implications of such studies. Scientists that have used symbiotic appr oaches have recognized this dichotomy and many have focused on studying the biology of symbiosis. These in situ and in vitro studies of how symbiosis is affected by light quality, quantity and photoperiod (Rasmussen et al., 1990a; Rasmussen and Rasmussen, 1991; Zettler and McInnis, 1994; McKinley and Camper, 1997) substrate (Rasmussen and Whigham, 1998; Brundrett et al., 2003; Diez, 2007) nutrients (Tsutsui and Tomita, 1990; Zettler et al., 2005) temperature (ien et al., 2008) and fungal species (Stewart and Zet tler, 2002;

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79 Chou and Chang, 2004; Stewart and Kane, 2007; ien et al., 2008) can guide future efforts to unravel orchid seed physiology using asymbiotic techniques as well.

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80 Table 3 1. Description of stages used to assess Bletia purpurea germination and s eedling development. Modified from Dutra et al. (2008) and Table 2 2 Stage Description 0 Hyaline embryo, testa intact 1 Embryo swollen, rhizoids may be present 2 Testa ruptured by enlarged embryo (= germination) 3 Differentiation of first leaf 4 Differentiation of second leaf 5 Elongation of second leaf 6 Emergence of third leaf

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81 Table 3 2 ANOVA resu lts and class comparisons for the effects of seed source, media, sucrose and light on Bletia purpurea seed germination and seedling development after 6 weeks culture. Water agar (WA). Mineral nutrient agar (MNA). Effects with p icant and these F values are bolded. Germination Development GI DI Medium Seed Source Effect df F p F p F p F p WA 159 Sucrose 2 68.51 < 0.01 36.11 < 0.01 57.32 < 0.01 4.89 0.01 Sucrose 0 mM vs. 10 m M 1 76.25 < 0.01 28.32 < 0.01 93.62 < 0.01 90.8 < 0.01 Sucrose 0 mM vs. 50 m M 1 120.62 < 0.01 69.79 < 0.01 22.65 < 0.01 0.80 0.38 Illumination 1 00.11 0.74 2.39 0.13 20.89 < 0.01 0.82 0.37 Sucrose Illumination 2 18.42 < 0.01 11.67 < 0.01 18.41 < 0.01 0.05 0.95 162 Sucrose 2 120.46 < 0.01 50.92 < 0.01 106.06 < 0.01 1.95 0.15 Sucrose 0 mM vs. 10 m M 1 126.26 < 0.01 36.88 < 0.01 164.88 < 0.01 1.86 0.18 Sucrose 0 mM vs. 50 m M 1 221.75 < 0.01 100.34 < 0.01 47.23 < 0.01 2.04 0.16 Illumination 1 4.53 0.04 4.43 0.04 68.19 < 0.01 0.77 0.38 Sucrose Illumination 2 39.39 < 0.01 15.80 < 0.01 45.68 < 0.01 2.25 0.12 164 Sucrose 2 135.91 < 0.01 74.47 < 0.01 120.55 < 0.01 7.34 < 0.01 Sucrose 0 mM vs. 10 m M 1 176.53 < 0.01 82.36 < 0.01 208.39 < 0.01 11.30 < 0.01 Sucrose 0 mM vs. 50 m M 1 227.91 < 0.01 134.66 0.02 32.72 < 0.01 3.39 0.07 Illumination 1 5.53 0.02 10.52 < 0.01 5.80 0.02 0.38 0.54 Sucrose Illumination 2 32.47 < 0.01 16.61 < 0.01 30.37 < 0.01 6.11 < 0.0 1 MNA 159 Sucrose 2 579.79 < 0.01 467.88 < 0.01 368.53 < 0.01 62.11 < 0.01 Sucrose 0 mM vs. 10 m M 1 1031.3 < 0.01 934.20 < 0.01 736.70 < 0.01 116.64 < 0.01 Sucrose 0 mM vs. 50 m M 1 669.02 < 0.01 267.07 < 0.01 0.36 0.55 10.57 < 0.01 Illumination 1 25.13 < 0.01 134.97 < 0.01 7.01 0.01 3.84 0.06 Sucrose Illumination 2 37.33 < 0.01 40.40 < 0.01 33.33 < 0.01 0.79 0.46 162 Sucrose 2 449.29 < 0.01 587.37 < 0.01 314.97 < 0.01 14.62 < 0.01 Sucrose 0 mM vs. 10 m M 1 808.21 < 0.01 1157.60 < 0.01 629.00 < 0.01 28.69 < 0.01 Sucrose 0 mM vs. 50 m M 1 503.86 < 0.01 180.24 < 0.01 0.94 0.34 0.54 0.47 Illumination 1 56.71 < 0.01 162.26 < 0.01 9.10 < 0.01 5.08 0.03 Sucrose Illumination 2 95.46 < 0.01 35.72 < 0. 01 62.39 < 0.01 3.33 0.05

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82 Table 3 2 Continued Germination Development GI DI Medium Seed Source Effect df F p F p F p F p MNA 164 Sucrose 2 712.95 < 0.01 1015.23 < 0.01 424.46 < 0.01 24.37 < 0.01 Sucrose 0 mM vs. 10 m M 1 1134 .18 < 0.01 2029.98 < 0.01 836.56 < 0.01 48.67 < 0.01 Sucrose 0 mM vs. 50 m M 1 1000.46 0.05 480.92 < 0.01 12.39 < 0.01 0.07 0.79 Illumination 1 298.92 < 0.01 572.03 < 0.01 13.14 < 0.01 3.97 0.05 Sucrose Illumination 2 281.03 < 0.01 14 .30 < 0.01 113.01 < 0.01 2.48 0.10

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83 Table 3 3 Effect of illumination and sucrose on germination index ( GI ) and developmental index ( DI ) of three Bletia purpurea seed sources. Seed was cultured for 6 weeks on either water agar (WA) or mineral nutrient agar (MNA). For each parameter and seed source, mean values WA MNA Seed Source Illumination Sucrose ( m M ) GI DI GI DI 159 Dark 0 0.06a 0.20ab 0.23a 0.25a Dark 10 0.99c 0.26bc 2.78d 0.46b Dark 50 2.03d 0.28c 3.23e 0.41b Light 0 0.25a 0.19a 0.63b 0.28a Light 10 0.68b 0.24bc 2.77d 0.53c Light 50 0.80c 0.26bc 2.13c 0.43b 162 Dark 0 0.03a 0.17a 0.20a 0.26a Dark 10 1.08c 0.22ab 2.62d 0.38bc Dark 50 2.13d 0.31b 3.12e 0.42cd Light 0 0.25a 0.22ab 0.93b 0.31ab Light 10 0.54b 0.20a 2.51d 0.50d Light 50 0.68b 0.21a 1.84c 0.40c 164 Dark 0 0.17a 0.17a 0.38a 0.24a Dark 10 2.15c 0.25b 3.17c 0.47c Dark 50 3.55d 0.34c 3.92d 0.52c Light 0 1.02b 0.25b 2.01b 0.35b Light 10 1.86c 0.27b 3.18c 0.55c Light 50 2.13c 0.26b 3.03c 0.48c

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84 Table 3 4 ANOVA results and class comparisons for the effects of carbohydrate source on Bletia purpurea germination, development and other parameters after 6 weeks culture. Germination index ( GI). Developmental index (DI). Effect s with p Germination Development GI DI Rhizoid Effect df F p F p F P F p F p Treatment (T r ) 12 559.55 < 0.01 352.51 < 0.01 356.22 < 0.01 19.2 < 0.01 56.24 < 0.01 T r Control vs Carbohydrates 1 996.88 < 0.01 555.69 < 0.01 534.33 < 0.01 27.18 < 0.01 82.56 < 0.01 T r Control vs. Sugar alcohols 1 3.25 0.07 0.38 0.54 0.40 0.53 0.06 0.80 0.00 1.00 T r Control vs. Non alcohol sugars 1 2072.52 < 0.01 1181.46 < 0.01 1135.0 8 < 0.01 56.87 < 0.01 178.89 < 0.01 T r Alcohols vs. Non alcohol sugars 1 5709.53 < 0.01 3411.02 < 0.01 3271.45 < 0.01 158.79 < 0.01 536.66 < 0.01 T r 10 m M vs. 50 m M 1 0.03 0.87 29.29 < 0.01 250.37 < 0.01 23.28 < 0.01 8.38 < 0.01

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85 Figur e 3 1 Comparative growth and development of Bletia purpurea seeds and seedlings cultured on water agar under 6 combinations of illumination and sucrose for 6 weeks. Seed source 164 is shown. Seeds were cultured for 6 weeks under continually dar k (A, C, E) or 16/8 hour light/ dark (B, D, F) photoperiods with 0 (A, B), 10 (C, D), or 50 m M sucrose (E, F). Scale bar s = 0.5 mm.

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86 Figure 3 2 Effects of seed source illumination and sucrose on Bletia purpurea seed germination and seedling development after 6 weeks culture on water agar. Bars represent treatment means SD. Bars with different letters within each 159 (A, B), 162 (C, D) and 164 (E, F) Dashed lines (A, C, D) represent seed viability estimates.

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87 Figure 3 3. Comparative growth and development of Bletia purpurea seeds and seedlings cultured on mineral nutrient agar under 6 combinations of illumination and sucrose for 6 weeks Seed sou rce 164 is shown. Seeds were cultured for 6 weeks under continually dar k (A, C, E) or 16/8 hour light/ dark (B, D, F) photoperiods with 0 (A, B), 10 (C, D), or 50 m M sucrose (E, F). Scale bar = 0.5 mm.

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88 Figure 3 4. Effects of seed sources, illumination a nd sucrose on Bletia purpurea seed germination and early seedling development after 6 weeks culture on mineral nutrient agar. Bars represent treatment means SD. Bars with different sources were tested: 159 (A, B), 162 (C, D) and 164 (E, F). Dashed lines (A, C, D) represent seed viability estimates.

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89 Figure 3 5. Comparative growth and development of Bletia purpurea seeds and seedlings cultured with 6 different carbohydrates. Seeds were cultured for 6 weeks under continually dar k conditions on media containing no water soluble carbohydrate (A), sucrose (B, C), fructose (D, E), glucose (F, G), trehalose (H, I), mannitol (J, K) or sorbitol (L, M) at 10 (B, D, F, H, J) or 50 mM (C, E, G, I, K, M). Scale bars = 1 mm.

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90 Figure 3 6 Effect of carbohydrate source and mol arity on Bletia purpurea seed germination and early seedling development. Germination after 6 weeks culture (A). Development after 6 weeks culture (B). Germination index (C). Developmental index (C). Percent embryos producing rhizoid production after 6 weeks culture (E). Bars represent treatment means SD. Bars with

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91 CHAPTER 4 E FFECTS OF SUGAR ALCO HOLS ON GERMINATION AND SEEDLING DEVELOPMENT IN THE P RESENCE OF THE GERMI NATION PROMOTING CARBOHYDRATES FRUCTO SE AND SUCROSE 3 Background The impact of carbohydrates on germination, as well as carbohydrate utilization by germinating seeds, has long been an intriguing subject for orchid seed biologists (Wynd, 1933; Ernst et al., 1971; Ernst and Arditti, 1990; Stewart and Kane, 2010) Orchid seed morphology and germination is not prototypical as orchid seeds are minute, their embryos are usually undifferentiated, endosp erm is lacking and most (if not all) species require infection by compatible mycorrhizal fungi for germination in situ (Rasmussen, 1995) The seeds of most non orchid species require only moi sture, a favorable environment and possibly relief from dormancy for the completion of germination (Bewley and Black, 1994) Most orchid species are either unable to germinate or germinate and develop minimally without being infected with a compatible mycorrhizal fungi (Zettler and McInnis, 1992; Zettler and Hofer, 1998; Stewart and Kane, 2007) an exogenous carbohydrate supply (Stewart and Kane, 2010) or exposure to l ight (Johnson et al., 2011) These data combined with what is known about orchid seed morphology (i.e. the lack of endosperm) suggests that germination is energy limited at dispersal. Interestingly, embryos contain large quantities of lipids a nd proteins (Appendix C, Rasmussen, 1995) germination may be delayed by an inability to convert stored reserves to sugars (Harrison, 1977; Manning and van Staden, 1987) 3 Significant portions of this chapter were originally accepted for publication in Journal of Plant Nutrition and are published here in accordance with the schedule of author rights outlined by Taylor & Francis Group.

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92 Orchid seed physiol ogy and germination ecology are not well understood. In part this is due to the magnitude of diversity within the Orchidaceae and the lack of clear trends in orchid seed germinati on requirements. M any of the published reports on orchid seed germination hav e focused on problems associated with production and propagation rather than on physiology and ecology. Asymbiotic orchid seed germination involves growing seeds in sterile conditions on agar solidified media containing mineral nutrients, carbohydrates (ty pically sucrose) and additional growth promoting additives (Kauth et al., 2008b) This seed sowing method also allows for experimental investigations of how environment and nutrition effect orchid seed germination. Orchids are capable of utilizing a wide range of sugars including mono di and oligosac charides for germination and seedling development (S mith, 1973; Ernst and Arditti, 1990; Stewart and Kane, 2010) Interestingly, while mannitol has been found in over 100 plant species from a variety of families (Lewis, 1984; Stoop et al., 1996) and is a common fungal sugar that has been isolated from numerous pathogenic (Lowe et al., 2008; Dulermo et al., 2009) and symbiotic fungi (Hughes and Mitchell, 1995; Ceccaroli et al., 2007) including those that form orchid mycorrhizae (Smith, 1973; Purves and Hadley, 1976; Shachar Mill et al., 1995) mannitol does not appear to be utilized by orchids during germination (Smith, 1973; Purves and Hadley, 1976; Johnson et al., 2011) Even less is known about the ability of orchid seeds to utilize sorbitol during germination, though it does not enhance germination of Bletia purpurea (see Chapter 3) While these studies indicate that exogenous mannitol and sorbitol cannot be utilized during germination, it remains unknown whether these sugar alcohols inhibit germination in the presence of oth er germination promoting carbohydrates.

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93 Since sorbitol and mannitol do not support germination of orchid seeds, they may serve as useful osmotica for studying the effects of water stress as has been done with other model plants (Bargmann et al., 2009; Mhadhbi et al., 2009; Tunc Ozdemir et al., 2009; Zhou et al., 2009) The objective of this study was to examine whether mannitol and sorbitol are inhibitory to orchid seed germination using Bletia purpurea as a test organism. The specific hypotheses tested were that mannitol and sorbitol would inhibit germination and developmen t of seeds when asymbiotic culture media also contained the germination supporting carbohydrates sucrose and fructose. Materials and Methods Seed C ollection, S terilization and V iability Seeds were collected from the Florida Panther National Wild life Refuge Nine undehisced, browning capsules were collected from nine plants and transported to the University of Florida (Gainesville, FL) for further processing. Capsules were stored at room temperature (~22 C ) over silica gel desiccant for three weeks until cap sules dehisced. Capsules were then cut open and seeds were extracted. Seeds were pooled, homogenized and stored in 20 mL scintillation vials at 10 C over silica gel desiccant up to three months prior to experimentation. Viability of seed was estimated usi ng triphenyltetrazolium chloride (TZ) staining after all experiments had been completed or initiated. For TZ staining, seeds were pretreated with 5% Ca ( OCl ) 2 (w/v) for 30 minutes, rinsed three times in distilled water and soaked in distilled water for 23.5 hours at 22 C in darkness. Water was then replaced with 1% TZ (w/v) for 24 hours at 30 C in darkness. Seeds were examined with a dissecting microscope for signs of pink to red staining. Seeds with any degree of staining were considered viable. Percent via bility

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94 was calculated by dividing the number of seeds with stained embryos by the total number of seeds containing embryos. Three replicates of 200 250 seeds were scored. Asymbiotic C ulture and E xperimental T reatments Seeds were surface sterilized for 60 s econds in a 1:1:18 solution of 6% NaOCl:100% ethanol:sterilize dd water, then rinsed three times in sterile distilled water. Approximately 40 120 seeds (76 18; mean standard deviation [SD] ) were then sown onto 9 cm diameter Petri plates containing vari ous media and sealed with a single layer of NescoFilm (Karlan Research Products Corporation) Basal medium consisted of strength Murashige and Skoog basal salts (Murashige and Skoog, 1962) with strength FeSO 4 7H 2 O and Na 2 EDTA. Medium was gelled with 7 g L 1 TC agar ( Phyto Technology Laboratories ) and adjusted to pH 5.7 before autoclaving. Basal medium was then amended with various sugars (sucrose or fructose) and sugar alcohols (sorbitol or mannitol), which were dissolved in distilled deionized water and fi lter sterilized using a nylon 0.2 m pore size syringe filters ( Nalgene ). Media were then dispensed in 25 mL aliquots into 9 cm diameter Petri plates and allowed to solidify. Petri plates from all treatments in each experiment were randomized, sealed with two layers of aluminum foil to exclude light and incubated at 25 C for 6 weeks. Four 4 3 factorial experiments were conducted to examine the interactions of sugars and polyols on various aspects of Bletia purpurea seed development. Sucrose was tested at 0, 1, 10 and 50 m M with 0, 10 and 50 m M of sorbitol or mannitol. Likewi se fructose was tested at 0, 1, 10 and 50 m M with 0, 10 and 50 m M of sorbitol or mannitol. Data C ollection and S tatistical A nalysis All experiments were CRDs with four replicate plates per treatment. All experiments were repeated once. Observations on grow th and development were made

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95 at two week intervals. Seeds and seedlings were observed under a dissecting microscope and assigned a developmental stage ( Table 3 1 ). Plates were exposed to light for < 20 minutes and examined with the aid of a dissecting micr oscope. Seeds were considered to have completed germination when the embryo swelled to the point of testa rupture. These data were used to calculate percent germination. Mean germination time (MGT) was calculated for all replicate plates using the equation where G is the number of seeds germinated at a given time (T), T is the day at which the count was made and F is the number of seeds that germinated during the experiment. Seed/seedlings developmental stages were scored on a scale 0 6 (Table 1). Average stage of development was calculated using the equation where for each replicate plate, is the number of seeds in stage i multiplied by the stage number ( i ) and S is the total number of se eds in each replicate. The number of seeds and seedlings producing rhizoids was also counted and used to calculate the percentage producing rhizoids. SAS v9.1 .3 (SAS Institute Inc., 2003) was used to perform two way ANOVA on all parameters at p = 0.05 using PROC MIXED and treating repeat as a random factor Percent germination and percent rhizoid production were arcsine transformed prior to analysis to normalize data; true means and standard errors for these variables are presented in figures. When a main factor was found to be significant, class comparison s were used to compare control treatments to other levels at p = 0.05. Least square (LS) mean separation was used to compare means within

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96 Results Interactions B etween S ucrose and S orbitol Viability of seeds was estimated at 96.9 1.8% (mean SD ) based on TZ staining. Addition of sucrose int o media enhanced germination compared to controls (Fig ure 4 1A; 4 2). Germination of seeds cultured with 10 and 50 m M sucrose was greater than 90% irrespe ctive of sorbitol molarity (Figure 4 1A). ANOVA results indicate that sucrose had a significant effect on seed germination (Table 2) and all levels of sucrose concentration significantly enhanced germination compared to control. Sorbitol did not significantly affect germination, nor was there a significant interaction between factors. Sucrose also affected MGT ( Table 4 1 ; Fig ure 4 1B), which was shortest when seeds were cultured with 50 m M sucrose at all levels of sorbitol. As with germination, MGT was not significantly affected by sorbitol or the interaction of main factors. Development was enhanced by the addition of sucrose (Figure 4 1C). While ANOVA results indicate that sorbitol significantly affected development (Table 4 1 ), no difference was found between 0 and 10 m M level. However, at 50 m M sucrose, increasing sorbitol molarity significantly reduced development. When seeds were cultured without sucrose, seeds and seedlings did not produce rhizoids (Figure 4 1E). ANOVA results indicate that sorbitol did not have a significant effect on rhizoid production, though there was a significant interaction of m ain effects. This interaction is apparent at 10 and 50 m M levels of sucrose where 50 m M sorbitol reduced rhizoid production compared to 0 m M sorbitol treatments. Interactions B etween S ucrose and M annitol As with the previous experiment, 10 and 50 mM sucros e treatments resulted in >90% germination (Fig ure 4 2A), and ANOVA results indicate that sucrose had a

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97 significant effect on germination ( Table 4 1 ). All germination of seeds treated with 1, 10 and 50 m M sucrose resulted in significantly greater germinatio n than 0 m M treatments. Treatment with 50 m M mannitol significantly enhanced germination at 1 m M sucrose, but not at 0, 10 or 50 m M sucrose treatments. Unlike sorbitol, mannitol had a significant effect on germination ( Table 4 1 ). In addition, a significan t interaction between main factors was detected by ANOVA ( Table 4 1 ). MGT ( Figure 4 3B) was again found to be significantly affected by sucrose molarity, but not by mannitol or the interaction of main factors ( Table 4 1 ). Treatment with sucrose was again f ound to significantly enhance development ( Table 4 1 ; Figure 4 3C). Mannitol also had a significant effect on development ( Table 4 1 ). No differences were detected between 0 and 10 m M mannitol treatments, however a significant difference between 0 and 50 m M mannitol treatments was detected ( Table 4 1 ). A significant interaction between main factors was also detected ( Table 4 1 ). Unlike sorbitol, treatment with 50 m M mannitol at 1 and 10 m M sucrose levels enhanced development. However, addition of 10 and 50 m M mannitol to 50 m M sucrose treatments reduced development. As previously observed, sucrose had a significant effect on rhizoid production ( Table 4 1 ; Figure 4 3D) However, unlike sorbitol, mannitol had a significant effect on rhizoid production ( Table 4 1 ) and class comparisons revealed significant differences between 0 and 50 m M mannitol treatments. A significant interaction between main factors was also detected ( Table 4 1 ). At both 1 and 10 m M sucrose levels, addition of 50 m M mannitol significantly e nhanced rhizoid production compared to 0 and 10 m M mannitol treatments. At 50 m M sucrose, addition of mannitol decreased rhizoid production slightly with the greatest decline observed with 50 m M sorbitol treatment.

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98 Interactions B etween F ructose and S orbito l Germination of seeds treated with 1 m M fructose was approximately 40%. Addition of 10 and 50 m M fructose enhanced germination of seeds to > 80% germination ( Figure 4 4A). ANOVA results indicate that fructose had a significant effect on germination and al l molarities of fructose significantly enhanced germination compared to control ( Table 4 1 ). Sorbitol and the interaction of main factors did not have a significant effect on germination ( Table 4 1 ), though mean separation indicates that addition of sucros e at 10 m M of sucrose significantly reduced germination. MGT was shortest when seeds were cultured with 50 m M fructose, regardless of sorbitol treatment ( Figure 4 4B). Fructose had a significant effect on MGT and class comparisons reveal significant differ ences between control and 50 m M fructose treatments, but not between control and other fructose molarities ( Table 4 1 ). Neither sorbitol nor the interaction of main factors significantly affected MGT. Development of seedlings was dependent upon fructose co ncentration. In general, increased fructose resulted in increased development (Fig ure 4 4C). Fructose had a significant effect on germination ( Table 4 1 ) with addition of 1, 10 and 50 m M fructose all significantly enhancing development over 0 m M fructose t reatments. Sorbitol and the interaction of sorbitol and fructose also had a significant effect on development ( Table 4 1 ). This was most obvious at 50 m M fructose where development was greater at lower sorbitol molarities (0 and 10 m M ) than at 50 m M Fruct ose enhanced rhizoid production and was > 50% when seeds were cultured with 50 m M fructose (Fi gure 4 4D). Fructose had a significant effect on rhizoid production ( Table 4 1 ), and addition of 1, 10 and 50 m M treatments were significantly greater than 0 m M t reatments. Neither sorbitol alone, nor the interaction of main effects significantly affected rhizoid production ( Table 4 1 ).

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99 Interactions Between Fructose and Mannitol As noted previously, fructose significantly enhanced germination of seeds (Table 2) wi th > 90% germination observed when seeds were treated with 10 and 50 m M fructose (Fig ure 4 5A). As observed in the sucrose mannitol experiment, ANOVA results indicate that mannitol had a significant effect on germination ( Table 4 1 ). At 1 m M fructose, ge rmination of seeds treated with 50 m M mannitol was significantly greater than 0 and 10 m M mannitol treatments. Fructose and mannitol were again found to significantly affect MGT ( Table 4 1 ; Figure 4 5B). A significant interaction was also detected ( Table 4 1 ). At 0 and 1 m M levels of fructose, MGT was slightly slower with 50 m M mannitol treatments. This delay was not observed at 10 and 50 m M fructose treatments. Development was significantly affected by both fructose and mannitol ( Table 4 1 ; Figure 4 5C). A gain, higher mol arities of fructose resulted in greater development and treatment with 10 and 50 m M mannitol enhanced development at 1 and 10 m M fructose treatments. At 50 m M fructose, treatment with 50 m M mannitol decreased development compared to 0 and 1 0 m M mannitol treatments. Rhizoid production was also significantly affected by both mannitol and fructose ( Table 4 1 ; Fig 4 5D). At 1 and 10 m M fructose, treatment with higher molarity of mannitol resulted in significantly greater rhizoid production. Disc ussion The key finding of this study was that Bletia purpurea seed germination, seedling development and rhizoid production was enhanced by mannitol at low levels of fructose and sucrose. However, mannitol alone was not able to support germination or devel opment better than control treatments. Sorbitol did not affect germination, but did reduce development at high levels in combination with 50 m M sucrose and fructose

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100 treatments. In addition, sorbitol inhibited rhizoid production at moderate and high levels of sucrose. The ability of Bletia purpurea seeds to utilize fructose and sucrose during germination, early development and for rhizoid production has been reported in a prior study (Johnson et al., 2011) and is in agreement with prior reports of carbohydrate utilization by orchid seeds (Downie, 1943; Smith, 1973; Stewart and Kane, 2010) However, evidence for an interaction between sugars and mannitol or sorbitol has not been previously reported. Utilization of exogen ous sucrose in asymbiotic culture systems likely involves extracellular hydrolysis by sucrose invertases (Griffith et al., 1987; Botha and O'Kennedy, 1998; Godt and Roitsch, 2006) prior to import by hexose transporters (see review of sugar transporters by Williams et al., 2000) Intact sucrose may also be transported across membranes by sucrose/H + cotransporters as has been documented in various cells including celery sink organs (Lemoine, 2000; Noiraud et al., 2000) or by diffusion at high concentrations when sucrose transporters are repressed (Noiraud et al., 2000) Carbohydrate transportation in orchid seeds remains unstudied, though such studies may help illuminate the regulation of symbiosis between orchids and their fungal partners. The e ffect of solute concentration on orchid seed germination and early development is another area yet unstudied. Sorbitol and mannitol have both been routinely used for inducing osmotic stress in germination studies (Dekkers et al., 2004; Vicento et al., 2004; Chen et al., 2006) There is evidence that some or chid species are able to utilize sugar alcohols, which would make them inappropriate osmoticum; for example, sorbitol has been reported to support germination of the achlorophyll o us

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101 orchid Galeola septentrionalis (Nakamura, 1982) Similarly Phalaenopsis and Dendrobium seedlings were able to survive for one year in the presence of sorbitol and mannitol (Ernst, 1967) Sorbitol is an important metabolite for som e plant families, most notable the Rosaceae (Maurel et al., 2004; Gao et al., 2005) Sorbitol also accumulates in the axis of germin ating soybean seeds (Kuo et al., 1990) In this study, sorbitol did not enhance germination or early development, indicating that it is not absorbed and/or not metabolized. Sorbitol is taken up by Bletilla hyacinthin a leaf sections, th ough more slowly than fructose (Smith and Smith, 1973) Differences in the duration of studies may account for differing reports of sorbitol utilization by orchid seeds and seedlings. It is possible that longer term studies with B. purpurea would reveal e nhanced germination if sorbitol uptake is slow. It does not seem likely that enhanced development and rhizoid production observed with B. purpurea grown with mannitol in the presence of sucrose or fructose is due to mannitol catabolism. The first step in mannitol breakdown is catalyzed by mannitol dehydrogenase (MTD) prior to conversion to fructose 7 phosphate (Stoop et al., 1995; Stoop et al., 1996) If B. purpurea embryos have MT D, it is expected that the seeds should be able to utilize exogenous mannitol during germination. That was not observed in this study indicating that the favorable interaction between mannitol and sugars is not due to increased available energy. Slow metab olism of mannitol can be the result of low levels of MTD (Pharr et al., 1995) though we expect that this would still result in increased germination and development in the absence of other sugars. The most likely explanation is that mannitol uptake by B. purpurea is passive as has been documented in the root cells of several non orchid genera and pea seed coats (Cram,

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102 1984; De Jong et al., 1996) If this is the case, mannitol could diffuse into the embryo cells along a concentration gradient. At relatively low media solute levels, mannit ol could diffuse into the cells and enhance the rate of water uptake. This could be especially facultative for rhizoid production as rhizoids are delicate structures that rapidly dehydrate when relative humidity is reduced ( personal observation ). The role of mannitol in balancing osmolarity could also account for the decrease in development and rhizoid production associated with mannitol addition at high sucrose and fructose molarities. At these solu te concentrations, media water potential could restrict wa ter uptake, seedling development and rhizoid production. While the advent of asymbiotic culture technique nearly 100 years ago simplified propagation of orchids from seed (Knudson, 1922) the physiology of orchid seed germination remains poorly understood. The ability or inability of various culture media and media components to promote germination and support seedling development has routinely been attributed to the metabolism of these compounds. Here, we report that media solute concentration can also impact germination of orchid seeds. Additionally, mannitol, a fungal sugar, may play a non nutritive role in enhancing development by promoting rhizoid production.

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103 Table 4 1 Results of A NOVA and class comparisons for sugar alcohol experiments F values that are significant ( p 0.05) are bolded. Mean germination time (MGT). Germination MGT Development Rhizoids Experiment Effect df F p F p F p F p Sorbitol Sucrose So rbitol 2 0.22 0.81 2.92 0.06 5.97 < 0.01 2.15 0.12 Sorbitol 0 m M vs. 10 m M 1 . . 1.35 0.25 . Sorbitol 0 m M vs. 50 m M 1 . . 11.58 < 0.01 . Sucrose 3 787.35 < 0.01 41.91 < 0.01 804.51 < 0.01 605.46 < 0.01 Sucrose 0 m M vs 1 m M 1 482.22 < 0.01 33.14 < 0.01 158.30 < 0.01 63.03 < 0.01 Sucrose 0 m M vs. 10 m M 1 1661.10 < 0.01 18.79 < 0.01 1287.39 < 0.01 867.00 < 0.01 Sucrose 0 m M vs. 50 m M 1 1793.12 < 0.01 19.99 < 0.01 1849.79 < 0.01 1349.73 < 0.01 Sorbitol Sucrose 6 1.29 0.27 0.76 0.61 5.92 < 0.01 2.79 0.02 Sorbitol Fructose Sorbitol 2 0.73 0.48 2.42 0.10 4.89 0.01 1.71 0.19 Sorbitol 0 m M vs. 10 m M 1 . . 1.22 0.27 . Sorbitol 0 m M vs. 50 m M 1 . . 3.85 0.05 . Fructose 3 597.51 < 0.01 47.38 < 0.01 644.78 < 0.01 626.31 < 0.01 Fructose 0 m M vs. 1 m M 1 171.83 < 0.01 1.32 0.25 56.46 < 0.01 16.33 < 0.01 Fructose 0 m M vs. 10 m M 1 1066.59 < 0.01 3.41 0.07 790.62 < 0.01 635.70 < 0.01 Fructose 0 m M vs. 50 m M 1 1401.45 < 0.01 76.17 < 0.01 1549.31 < 0.01 1446.66 < 0.01 Sorbitol Fructose 6 2.27 0.05 0.52 0.80 5.34 < 0.01 0.97 0.45

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104 Table 4 1 Continued Germination MGT Development Rhizoids Experiment Effect df F p F p F p F p Mannit ol Sucrose Mannitol 2 5.47 0.01 0.51 0.60 8.91 < 0.01 13.18 < 0.01 Mannitol 0 m M vs. 10 m M 1 0.51 0.48 . 0.82 0.37 0.01 0.93 Mannitol 0 m M vs. 50 m M 1 9.97 < 0.01 . 16.24 < 0.01 20.27 < 0.01 Sucrose 3 1056.80 < 0.01 90.95 < 0.0 1 2224.63 < 0.01 1049.21 < 0.01 Sucrose 0 m M vs. 1 m M 1 946.66 < 0.01 82.41 < 0.01 791.72 < 0.01 178.12 < 0.01 Sucrose 0 m M vs. 10 m M 1 2398.35 < 0.01 23.80 < 0.01 4082.47 < 0.01 1705.66 < 0.01 Sucrose 0 m M vs. 50 m M 1 2332.43 < 0.01 4 3.77 < 0.01 5173.59 < 0.01 2339.09 < 0.01 Mannitol Sucrose 6 3.84 < 0.01 0.42 0.87 34.74 < 0.01 9.54 < 0.01 Mannitol Fructose Mannitol 2 7.82 < 0.01 8.14 < 0.01 9.86 < 0.01 21.58 < 0.01 Mannitol 0 m M vs. 10 m M 1 0.53 0.47 0.01 0.94 2.14 0.15 7.19 0.01 Mannitol 0 m M vs. 50 m M 1 13.93 < 0.01 11.96 < 0.01 19.04 < 0.01 42.71 < 0.01 Fructose 3 5.63 < 0.01 73.57 < 0.01 805.64 < 0.01 1038.50 < 0.01 Fructose 0 m M vs. 1 m M 1 226.29 < 0.01 45.66 < 0.01 129.99 < 0.01 36.70 < 0.01 Fructose 0 m M vs. 10 m M 1 1063.56 < 0.01 39.30 < 0.01 1285.16 < 0.01 1202.55 < 0.01 Fructose 0 m M vs. 50 m M 1 1312.93 < 0.01 37.35 < 0.01 1808.05 < 0.01 2272.38 < 0.01 Mannitol Fructose 6 1.17 0.33 4.33 < 0.01 6.09 < 0.01 10. 29 < 0.01

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105 Figure 4 1. Effects and interactions of sucrose and sorbitol on Bletia purpurea germination (A), mean germination time (MGT; B), seedling development (C) and rhizoid production (D). Bars represent means standard error Means least square mean separation.

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106 Figure 4 2. Comparative growth and development of Bletia purpurea seeds and seedlings germinated in the presence of sucrose and/or man nitol after 6 weeks culture Scale bar = 0.5 m m.

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107 Figure 4 3. Effects and interactions of sucrose and mannitol on Bletia purpurea germination (A), mean germination time (MGT; B), seedling development (C) and rhizoid p roduction (D). Bars represent means standard error Means least square mean separation.

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108 Figure 4 4. Effects and interactions of fructose and sorbitol on Bletia purpurea germination (A), mean germination time (MGT; B), seedling development (C) and rhizoid production (D). Bars represent means standard error Means least square mean separation.

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109 Figure 4 5. Effects and interactions of sucrose and mannitol on Bletia purpurea germination (A), mean germination time (MGT; B), seedling development (C) and rhizoid production (D). Bars represent means standard error Means with least square mean separation. Scale bar = 0.5 mm.

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110 CHAPTER 5 EFFECTS OF GIBBERELL IC ACID AND ABSCISIC ACID ON GERMINATION AND SEEDLING DEVELOPMENT Background The phytohormones absci sic acid (ABA) and gibberellic acid (GA) are important regulators of numerous plant growth and developmental processes. ABA is involved in sugar sensing (Rognoni et al., 2007) regulating diurnal control of stomatal opening (Tallman, 2004) osmotic stress responses (Verslues and Bray, 2006) and altering morphology of roots, shoots leaves, stomata and storage organs (Xu et al., 1998; LeNoble et al., 2004; Lin et al., 2005; Chen et al., 2008; Arend et al., 2009; Rodrigues et al., 2009) GA s play a role in flower development (Ben Nissan and Weiss, 1996) fruit development (Ozga et al., 2002) and shoot elongation (Little and MacDonald, 2003) Both ABA and GA are also involved in the regulation of seed development (Nakashima et al., 2009b; Singh et al., 2009) dormancy and ge rmination. T hese two phytohormones act antagonistically, co regulating the processes of dormancy maintenance /relief and germination ABA maintains dormancy an d inhibits radicle emergence by limiting seed reserve metabolism maintaining low embryo growth potential and preventing endosperm weakening (Garciarrubio et al., 1997; da Silva et al., 2004; Muller et al., 2006) GA s counteract ABA by increasing embryo growth potential, stimulating seed reserve hydrolysis and reducing ABA levels in the seed (Debeaujon and Koornneef, 2000; da Silva et al., 2004) Little is known about how GA s and ABA effect orchid seed germination and early see dling developme nt. Few studies h ave examined the impact of exogenous GA s on orchid seed germination. Results from these studies are inconsistent reporting germination to be inhibited (Miyoshi and Mii, 1995; Znaniecka et al., 2005) unaffected

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111 (Hadley and Harvais, 1968; Van Waes and Debergh, 1986) or enhanced by GA s (Pedroza Manrique et al., 2005) Our understanding of ho w and why GA respons es vary among orchids is hindere d by significant differences in cultural c onditions used in these studies. For example, s ome researchers used basal media containing exogenous sugars for culturing seeds (Hadley and Harvais, 1968; Van Waes and Debergh, 1986; Pedroza Manrique et al., 2005) while others used sugar free culture medium (Miyoshi and Mii, 1995) ; s ome researchers maintained cultures in darkness (Van Waes and Debergh, 1986; Pedroza Manrique et al., 2005) while others exposed seeds to light (Hadley and Harvais, 1968; Miyoshi and Mii, 1995) Therefore it is still un clear whether the observed variability in orchid seed respons es to GA s is altered by illumination and the availability of metabolizable carbohydrate s Even less is known about the impact of ABA on orchid seed germination. ABA has been extracted from orchid seeds (Van der Kinderen, 1987; Lee et al., 2007) but studies on the effects of exogenous ABA on germination have not been published. Since ABA can by biosynthesized by fungi (Van der Kinderen, 1987) and reduce s plant disease resis tance (Lee et al., 2007) demonstrated ABA sensitivity in orc h id seeds might provide clues about the regulati on of orchid fungi symbiosis, the commonalities among orchid mycorrhizal fungi and fungal specificity. The objectives of this study were to determine whether ABA and G A s affected germination and early seedli ng development of Bletia purpurea seedlings under asymbiotic culture condit ions. The effects of exogenous sucrose and illumination on these parameters were also examined. Finally, a factorial experiment was performed to

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112 test the hypothesis that GA 3 can ove rcome the inhibitory effects of ABA on germination and seedling development Materials and Methods Seed C ollection Bletia purpurea seeds were collected from the Florida Panther National Wildlife Refuge. Ten mature, undehisced capsules were collected from t h e field and stored over silica gel desiccant at room temperature (~22C) until capsules dehisced. Seeds were then removed from capsules, pooled into 10 mL scintillation vials and stored at 10 C over desiccant prior to experimentation. Estimating Viabilit y To assess seed viability, seeds were subjected to triphenyltetrazolium chloride (TZ) staining first before the ABA, GA 3 and ABA GA 3 experiments, and again before the GA isomer experiment A small volume of seed (approximately 100 200 seeds) was placed in 1.5 mL centrifuge tubes and treated with 5% Ca ( OCl ) 2 (w/v) for 30 minutes to weaken the testa and facilitate staining. Seeds were then rinsed three times in water before seeds were resuspended in water and incubated at room temperature for 23.5 hours. Water was then replaced with 1% TZ (pH 7.0) and seeds were incubated for 24 hours at 30 C After staining, s eeds were examin ed with a dissecting microscope. S eeds containing embryos with any degree of pink to red staining throughout the embryos were consid ered viable while wholly unstained embryos were considered non viable. Three replicates of 190 220 seeds were performed to estimate percent viability by dividing the number of seeds with viable embryos by the total number of seeds with embryos.

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113 Seed S owin g and Culture Conditions F or all experiments, seeds were surface sterilized in a solution of 6.0% sodium hypochlorite :100% ethanol :sterile distilled deionized water (5:5:90) for 60 seconds followed by three 20 second rinses in sterile distilled water befor e sowing onto various media. Basal medium consisted of strength Murashige and Skoog medium (Murashige and Skoog, 1962) modified with strength FeSO 4 7H 2 O and Na 2 EDTA. Medium was gelled with 7 g L 1 TC agar ( Phyto Technology Laboratories) and adjusted to pH 5.8. Sterilized seeds were sown onto 9 cm Petri plates containing appro ximately 25 mL of media before plates were sealed with a single layer of NescoFilm (Karlan Research Products Corporation). Plates were stored in a growth chambers at 25 C under either a 16/8 h light/dark photoperiod at 50 2 s 1 or continual darkness. Dark treated plates were wrapped in two layers of aluminum foil to exclude light for 6 or eight weeks. ABA E xperiment A 2 2 4 (illumination sucrose ABA) factorial was conducted to assess whether ABA inhibits seed germination under a variety of conditions that normally allow germination. Seeds were sown onto media with or without 10 m M sucrose and 0, 1, 5 or 10 M ABA. Both sucrose and ABA were filter sterilized with nylon 0.2 m pore size syringe filters ( Nalgene ) and added to autoclaved medium. Sterile dd water was also added to maintain constant final volumes of media. Because ABA is weakly soluble in water, it was dissolved in a small volume of 95% ethanol before being diluted, filter sterilized and dispensed. To control for possible effects of ethanol, the same volume of ethanol was added to control treatments as w as used in 10 M ABA treatments ( 100 l 95% ethanol l 1 medium) ; however this volume of ethanol has little to no effect on seed

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114 germination and devel opment (Appendix B) Approximately 60 90 seeds (79 14 ; mean standard deviation [SD ]) were sown onto Petri plates Seeds were examined for signs of germination and development (Stages 0 6) as described previously ( Table 3 1 ) after two, four and 6 weeks culture. The number of embryos/seedlings producing rhizoids was also recorded. Dark treated seeds were exposed to short periods of light (< 20 minutes), which have little to no effect on germination and development (Appendix A). GA 3 E xperiment A 2 2 4 (illumination sucrose GA 3 ) factorial was conducted to assess whether G A 3 promotes seed germination under different sucrose and illumination treatments Seeds were sown onto media with or without 10 m M sucrose and 0, 1, 10 or 100 M GA 3 GA 3 and sucro se were filter sterilized and added to basal medium after autoclaving as previously described Approximately 40 70 seeds (59 12; mean SD) were sown onto each Petri plate Seed sowing, incubator conditions and observation procedure s followed the methods outlined above. Additionally, embryo/ seedling lengths were estima ted after eight weeks culture. For this, 20 embryos and/or seedlings from each replicate plate were transferred to a drop of water on a glass slide and image d with a dissecting microscope eq uipped with a Nikon Coolpix 990 digital camera Images were then opened in ImageJ (Audenaert et al., 2002) and max imum lengths of embryos /seedlings were measured. ABA GA 3 E xperiment The ability of GA 3 to overcome ABA induced seed imbibition was assessed in a 2 4 (ABA GA 3 ) factorial experiment. Seeds were sown onto basal medi um with 10 m M sucrose; 1 or 10 M ABA ; and 0, 1, 10 or 100 M GA 3 Media preparation followed

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115 previously described methods. Approximately 40 80 seeds (55 11; mean SD) were sown onto each Petri plate. Seeds were maintained in darkness for 6 weeks Observation for signs of germinatio n, deve lopment and rhizoid prod u c tion were made a t two week intervals Because of concerns that brief exposures to light might affect experimental results, a second set of all treatments was maintained in darkness without light exposure for 6 weeks. GA Isomers Ex periment A 2 5 (isomer molarity) factorial was conducted to determine whether seeds respond differently to two different GA isomers. Seeds were sown onto 9 cm Petri plates containing approximately 25 mL basal media amended with M of GA 3 or GA 4+7 Because GA 4+7 is weakly soluble in water, it was dissolved in a small volume of 95% ethanol. The maximum concentration of 95% ethanol in treatment media was 0.6% (v/v ). Treatment with up to 1% ethanol was found not t o a ffect germination or deve lopment, though there was some a ffect on rhizoid production (see Appendix B for detailed results). GAs were filter sterilized as previously described Approximately 30 90 seeds (57 10; mean SD ) were sown per plate. Se eds wer e observed for signs of germination, development and rhizoid production after 6 weeks. Additionally, embryo/seedling lengths were estimated after eight weeks culture as previously described. Experimental Design Data Collection and Statistical A nalysis A c ompletely randomized design was used for all experiments. Five replicates were performed for ea ch treatment in the GA isomers experiment and f our replicates were used for all other experiments. Each experiment was repeated once. Germination and development al data was used to calculate percent germination, mean germination

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116 time ( MGT; where data was collected at more than one time) average developmental stage, DI and percentage of embryos/seedlings producing rhizoids. Mean germination time (MGT) was calculat ed for all replicate plates using the equation where G is the number of seeds germinated at a given time (T), T is the day at which the count was made and F is the number of seeds that germinated during the experiment. Seed/seedling s developmental stages were scored on a scale 0 6 (Table 1). Average stage of development was calculated using the equation where for each replicate plate, is the number of seeds in stage i multiplied by the st age number ( i ) and S is the total number of seeds in each replicate. Embryo/seedling lengths were calculated by averaging the replicate means of twenty measured embryos/seedlings. A three way ANOVA was used for the ABA and GA 3 experiments to analyze the e ffects of main factors and interactions on parameters When three way interactions (illumination sucrose ABA and illumination sucrose GA 3 ) were found not to be significant, ANOVA results were sliced by illumination treatment to examine the two way interactions of sucrose ABA and sucrose GA 3 in illuminated and dark treatments separately. To assess whether exposing dark treated seeds to light had a significant effect on germination, development and/or rhizoid production, a three way ANOVA was used to assess differences between ABA GA 3 experiments (one in which seeds were exposed to brief light during scoring and another where they were only scored after 6 weeks) using F tests to examine the effects of experiment, experiment ABA, experiment GA 3 and experiment ABA GA 3 on germination, development and

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117 r hizoid production A two way ANOVA was used to assess the variance in the ABA GA 3 experiment A one way ANOVA was used to analyze the GA isomers experiment followed by contrast comparisons of interest. PROC MIXED in SAS v 9.1 (SAS Institute Inc ., 2003) was used to analyze all experiments treating experiment repeat as a random factor. Germination percentages and rhizoid production percentages were arcsine transformed for analysis. LS mean separation was used to compare treatment means at For all experiments, data was analyzed using Results E ffect of ABA on S eed G ermination and E arly S eedling D evelopment Seed viability was estimated at 98.1 0.5% (mean SD) at first test ing As in previous experiments, illumination, sucrose and the interaction of these ef fects significantly a ffected germination ( Table 5 1 ). ABA also had a significant e ffect on germination and MGT, inhibiting germination and germination rate in a dose depe ndent manner ( Table 5 1 ; Figure 5 1, 5 2). ANOVA indicated significant e ffects on germination and MGT caused by the interaction s of illumination and ABA, as well as the interactions of sucrose and ABA. A significant three way interaction between illuminati on, sucrose and ABA treatments was also detected. In dark cultured seeds on media lacking sucrose where germination is typically low, germination was significantly reduced by all levels of ABA compared to ABA control. The inhibitory effect of ABA was buffe r ed by sucrose as germination declined less sharply with increasing ABA levels when seeds were cultured with sucrose regardless of illumination treatment. As with germination, ABA and the two way interactions of ABA with sucrose and ABA with illumination b oth significant ly a ffect ed development ( Table 5 1 ). A significant three way interaction between illumination, sucrose and ABA treatments was also

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118 detected. Treatment with ABA reduced development when seeds were cultured in darkness without sucrose (Figure 5 1, 5 2), though increasing ABA concentration did not increase the magnitude of development al depression In all other combinations of illumination and sucrose treatments, ABA reduced development in a dose dependent manner. ABA had a significant effect on rhizoid production ( Table 5 1 ), reducing rhizoid production when seeds were cultured in darkness with sucrose (Figure 5 1, 5 2). As demonstrated in previous experiments, these conditions (darkness and sucrose) favor rhizoid production while rhizoid produc tion is inhibited in light. Effect of G A 3 on S eed G ermination and E arly S eedling D evelopment Under all combinations of sucrose and illumination levels, GA 3 significantly inhibited germination and increased MGT ( Table 5 2, Figure 5 3, 5 4). Significant two way interactions between GA 3 and both illumination and sucrose were also detected, as was a significant three way interaction between GA 3 illumination and sucrose. The response was dose dependent in all treatments with higher GA 3 molarity leading to sign ificantly greater inhibition. GA 3 tr eatment also had a significant e ffect on development ( Table 5 2 ). Significant two and three way interactions with GA 3 level s and both illumination and sucrose were also detected Mean separation did not indicate any dif ferences between GA 3 treatments when seeds were cultured in darkness without sucrose (Figure 5 4). S ignificant differences among means were detected within all other combinations of sucrose and illumination. Rhizoid production was significantly affected by GA 3 treatments ( Table 5 2 Figure 5 3). While significant two and three way interactions involving GA 3 level s w ere

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119 detected, < 5% of embryos or seedlings produced rhizoids in lighted conditions and in darkness without sucrose (Figure 5 4). When seeds wer e cultured in darkness with sucrose, GA 3 significantly inhibited rhizoid production in a dose dependent manner. T he e ffect of GA 3 on embryo elongation was studied after a preliminary experiment revealed changes in seedling morphology when embryos were cult ured with GA 3 for 14 weeks (Figure 5 5 A, B). The observed effect after eight weeks culture were not as extreme, however some seedlings showed signs of elongation (Figure 5 5 C, D). ANOVA results indicate that GA 3 treatment had a significant e ffect on mean embryo/seedling length ( Table 5 2 ; Figure 5 6). When seeds were cultured without sucrose, GA 3 had little discernable effect on the variance among treatments When sucrose was present and seeds were cultured in darkness, the lower bounds of observed length s decreased with increasing GA 3 molarity This was observed to a lesser extent in light treatments as well The upper bounds of observed lengths also increased as GA 3 molarity increased to 10 M. In light treatments there was then a dramatic decrease in th e observed 95% limit of data when GA 3 molarity was increased from 10 to 100 M. Under lighted conditions, GA 3 had a pronounced effect on leaf architecture and elongation (Figure 5 7). Because these leaves tended to curl back and because length was measured parallel to the plane of growth, the full effect of GA 3 on these seedlings is not easily conveyed by measurements alone. Effect of G A 3 on ABA Induced Inhibitio n of Germination and Development Germination, MGT, development and rhizoid production were all significantly affected by the interaction of ABA and GA 3 levels ( Table 5 3 ). GA 3 did not enhance germination, development or rhizoid production in the presence of ABA ; rather these

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120 respons es were more strongly inhibited by increasing levels of GA 3 in conce rt with both 1 and 10 M ABA treatments ( Figure 5 8 ). A comparison of effects from two experiments, one in which seeds were exposed to short periods of light during scoring at weeks two and four and another in which seeds were only exposed to light at th e completion of the experiment, indi cated no significant effect on germination or seedling development (Table 5 4). A significant e ffect of the interaction experiment ABA on rhizoid production was detected, though the e ffects of experiment, experiment G A 3 and experiment ABA GA 3 did not significantly affect rhizoid production Comparative Effects of GA 3 and GA 4+7 on Germination, Development and Rhizoid Production Seed viability was estimated at 98.7 0.3% at the second testing prior to the start of t his experiment Both GA 3 and GA 4+7 significantly inhibited germination, development and rhizoid production (Table 5 5; Figure 5 10 5 11 ). Germination responses varied by isomer. The effect of GA 3 on germination was not as pronounced as in the previous exp eriment and no significant difference was detected between 1 and 10 M treatments; however germination decreased in a dose dependent manner as was obs erved in the prior experiment. The c lass comparison of GA isomers indicated no significant difference ( Tab le 5 5) Treatment had a significant effect on embryo/seedling lengths and a significant difference was detected between isomers (Table 5 5). Although significant differences between control and GA treatments were only observed at the highest molarity test ed, both GA isomers increased the variance in observed lengths compared to control (Figure 5 12) Increasing molarity of both isomers resulted in declines in the lowest

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121 observed lengths The upper 95% limit of data for GA 3 did not follow a clear trend as i t did in the previous experiment. Increasing GA 4+7 molarity resulted in a decrease in the observed upper 95% limit of data. Discussion Contrary to the classic model of balance theory which states that ABA and GA 3 are antagonists acting respectively as dor mancy promoters and germination promoters both plant growth regulators inhibited germination and development of B purpurea seeds. Additionally, GA 3 enhanced ABA induced delays in germination and seedling dev elopment. ABA has been detected in several orch id species and may promote physiological dormancy (Van der Kinderen, 1987; Lee et al., 2007) The current study also indicates that no n dormant orchid species like B. purpurea are also able to sense ABA which likely plays a role in seed maturation as it does in other families (Frey et al., 2004; Nakashima et al., 2009a) Experimental investigation of the mode of inhibition within the Orchidaceae is still needed. ABA accumulation during orchid seed maturation has been correlated with decreased imbibition rates (Lee et al., 2007) which could be caused by alter ed gene expression, lower e d embryo pressure potential, inhibit ed biosynthe sis of wall loosening enzymes and reduced reactive oxygen specie s production (da Silva et al., 2004; da Silva et al., 2005; Cadman et al., 2006; Finch Savage and Leubner Metzger, 2006; Mller et al., 2009) Inhibition of orchid seed germination by exogenous GA s has been noted for some o rchid taxa (Hadley and Harvais, 1968; Znaniecka et al., 2005) while other studies have found germination to be promoted or unaffected by GA applications (Hadley and Harvais, 1968; Van Waes and Debergh, 1986; Pedroza Manrique et al., 2005) For most non orchid species, GAs typically promote germination or have no effect on

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122 germination. E xogenous GA 3 and GA 4+7 were both found to inhibit germination of Coffea arabica cv. Rubi causing cell death in the radical of intact seeds (da Silva et al., 2005) Similar results were found with B. purpurea where both isomers inhibited germination With C. arabica var. Rubi, i nhibition of GA biosynthesis by paclobutrazol also inhibited germination, though the normal pheno type was expressed when seeds were treated with both paclobutrazol and GA 4+7 The authors concluded that supraoptimal GA levels resulting from the addition of GA to the endogenous pool resulted in the release of unidentified chemicals during endosperm brea kdown which were lethal to cells. The possibility that supraoptimal levels of GA are inhibiting B. purpurea germination needs to be investigated (Chapter 6). Since endosperm is lacking in orchid seeds, the results of the curren t study suggest there may be another mode of inhibition in Coffea and possibly a homologous mechanism with orchids In this study and in past studies, GA 3 has been found to alter orchid seedling morphology by elongating seedlings (Hadley and Harvais, 1968; vila Daz et al., 2009) GAs are known to promote cell elongation and division in various plant tissues MacDonald, 2003) O rchid embryos typi cally form globular structures, expa nd ing outward in all directions. Therefore delays in testa rupture may be the result of redirected growth especially when there is a relatively large amount of air space within the seed allowing the elongating embryo to grow without p ressing through the t es ta. However, if a species produces seeds with little air space and the testa tightly apprised to the embryo, germination could conceivably be enhanced if pressure is focused on one or two points of the test a

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123 ity to inhibit germination is more complex than simply preventing the weakening of embryo coverings. However, contrary to work with non orchid taxa which shows GA stimulate s germination by increasing embryo growth potential or cell wall loosening (Kucera et al., 2005; Obroucheva, 2010) exogenous GA 3 and GA 4+7 is inhibitory to the germination of B purpurea as has been documented for other orchid species

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124 Table 5 1 ANOVA results f or the effect of abscisic acid (ABA), illumination and sucrose on Bletia purpurea germination and seedling development. Mean germination time (MGT). Effects with p these F values are bolded. Germination MGT Development Rhizoids Effect df F p F p F p F p Illumination (I) 1 789.83 < 0.01 35.89 < 0.01 1222.74 < 0.01 344.57 < 0.01 Sucrose (S) 1 1237.58 < 0.01 1.34 0.2 5 1206.44 < 0.01 750.88 < 0.01 ABA 3 257.44 < 0.01 133.51 < 0.01 477.80 < 0.01 40.93 < 0.01 I S 1 308.28 < 0.01 2.06 0.15 17.14 < 0.01 507.42 < 0.01 I ABA 3 4.49 0.01 15.89 < 0.01 22.31 < 0.01 20.59 < 0.01 S ABA 3 20.66 < 0.01 1.00 0. 39 68.24 < 0.01 30.69 < 0.01 I S ABA 3 15.64 < 0.01 1.15 0.33 44.63 < 0.01 29.60 < 0.01 I Dark S ABA 3 . 50.37 < 0.01 . . I Light S ABA 3 . 14.28 < 0.01 . .

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125 Table 5 2 ANOVA results for the effect of gibbere llic acid (GA 3 ), illumination and sucrose on Bletia purpurea germination and seedling development Effects with p t and these F values are bolded Mean germination time (MGT). Germination MGT Development Rhizoids Leng th Effect df F p F p F p F p F p Illumination (I) 1 1002.52 < 0.01 30.44 < 0.01 1817.24 < 0.01 330.41 < 0.01 0.01 0.90 Sucrose (S) 1 1305.91 < 0.01 3.68 0.06 1192.89 < 0.01 390.45 < 0.01 177.03 < 0.01 GA 3 3 195.05 < 0.01 6.08 < 0.01 226.2 0 < 0.01 32.45 < 0.01 4.30 0.01 I S 1 397.83 < 0.01 14.75 < 0.01 0.21 0.65 390.72 < 0.01 < 0.01 0.99 I GA 3 3 4.01 < 0.01 1.31 0.28 26.55 < 0.01 22.39 < 0.01 0.24 0.87 S GA 3 3 4.45 0.01 2.45 0.07 45.44 < 0.01 27.63 < 0.01 1.30 0.28 I S GA 3 3 13.52 < 0.01 2.00 0.11 11.12 < 0.01 26.59 < 0.01 0.20 0.90 I Dark S GA 3 3 . 4.54 < 0.01 . . 13.64 < 0.01 I Light S GA 3 3 . 2.02 0.06 . . 14.30 < 0.01

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126 Table 5 3 ANOVA results for the effect of gibberelli c acid (GA 3 ) and abscisic acid (ABA) on Bletia purpurea germination and seedling development. Mean germinati on time (MGT). Effects with p values are bolded. Germination MGT Development Rhizoids Effect df F p F p F p F p ABA 1 785.54 < 0.01 8.32 0.01 590.61 < 0.01 419.41 < 0.01 GA 3 3 201.52 < 0.01 5.79 < 0.01 188.63 < 0.01 103.93 < 0.01 ABA GA 3 3 60.63 < 0.01 10.82 < 0.01 117.00 < 0.01 63.72 < 0.01

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127 Table 5 4. ANOVA results for the effect of brief exposures to light during observation on responses in a factorial experiment. Abscisic acid (ABA). Gibberellic acid (GA 3 ). E ffects with p bolded. Germination Development Rhizoids Effect df F p F p F p Experiment 1 1.73 0.19 1.36 0.25 0.45 0.50 E xperiment ABA 1 0.07 0.79 0.27 0.61 5.91 0.02 E xperiment GA 3 3 1.24 0.30 0.94 0.42 0.94 0.42 E xperiment ABA GA 3 3 0.49 0.69 0.31 0.31 2.25 0.09

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128 Table 5 5. ANOVA results of the effects of gibberellic acid isomers (GA 3 and GA 4+7 ) on germination, development, rhizoid production and seedling elongation Effects with p 0.05 are considered significant and these F values are bolded. Germination Development Rhizoids Length Effect df F p F p F p F p Treatment 8 33.43 < 0.01 42.74 < 0.01 40.6 < 0.01 8.92 < 0.01 Treatment Control vs. GA3 1 97.58 < 0.01 172.0 9 < 0.01 16.76 < 0.01 0.00 0.97 Treatment Control vs. GA4+7 1 95.10 < 0.01 174.12 < 0.01 49.12 < 0.01 1.98 0.16 Treatment GA 3 vs. GA4+7 1 0.04 0.84 0.01 0.90 21.24 < 0.01 4.69 0.03

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129 Figure 5 1. Comparative growth and development of Bletia purpurea seeds and seedlings cultured w ith combinations of abscisic acid sucrose and illumination for 6 weeks Scale bars = 1 mm scale.

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130 Figure 5 2. Effect of abscisic acid (ABA), illumination and sucrose on Bletia purpurea seed germination, mean germination time (MGT), seedling development, and rhizoid production. Bars rep resent means standard error. Within each graph, b Dashed line in germinati on graph indicates estimated viability.

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131 Figure 5 3 Comparative growth and development of Bletia purpurea seeds and seedlings cultured with combinations of gibberellic acid (GA 3 ) sucrose and illumination for 6 weeks. Scale bars = 1 mm

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132 Figure 5 4. Effect of gibberellic acid ( GA 3 ), illumination and sucrose on Bletia purpurea seed germination, mean germination time (MGT), seedling development, and rhizoid production. Bars re present means standard error. Within ea ch graph, b Dashed line in germination graph indicates estimated viability.

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133 Figure 5 5. Bletia purpurea seedling elongation after treatment with gibberellic acid (GA 3 ). Elongation was first observed in a preliminary study where seeds were cultured for 14 weeks on media containing 10 g L 1 sucrose without (A) or with 10 M GA 3 (B) Elongation was also observed was also observed after eight weeks culture on media containing 10 m M sucrose w ithout (C ) or with 10 M GA 3 (D). A, B scale bars = 1 mm. C, D scale bars = 0.5 mm.

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134 Figure 5 6. Effect of gibberellic acid ( GA 3 ), illumination and sucrose on Bletia purpurea embryo elongation. Thick lines within boxe s represent mean values. Thin lines within boxes represent median value. Boxes represent upper and lower quartiles. Whiskers represent 1.5 interquartile range. Dots indicate lower 5% and upper 95% limit of data range. Within each graph, boxes with the sa me letter are not significantly d

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135 Figure 5 7. Bletia purpurea seedling elongation after eight weeks c ulture with gibberellic acid ( GA 3 ) under lighted conditions. Seeds were cultured under 16/8 hour photoperiod on mineral nutrient agar containing 10 m M sucrose an d 0 (A), 1 M GA 3 (D) Scale bars = 1 mm. Inset images are an additional 2 magnification.

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136 Figure 5 8 Effect of gibberellic acid (GA 3 ) and abscisic acid (ABA) on Bletia purpurea germination and seedling development. Bars represent means standard error. Within each graph, bars with the same letter are not significantly Dashed line in germination graph indicates estimated viability. Mean germination time (MGT). Asterisk (* ) indicates treatment for which germination was observe in only one replicate.

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137 Figure 5 9 Comparative growth and development of Bletia purpurea seeds and seedlings treated with 6 combinations of g ibberellic acid (GA 3 ) and abscisic acid for 6 weeks. S cale bars = 250 m

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138 Figure 5 10 Comparative growth and development of Bletia purpurea seedling elongation after treatment with two gibberellic acid (GA) isomers. Scale bars = 500 m.

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139 Figure 5 11 Effect of gib berellic acid isomers (GA 3 and GA 4+7 ) on germination, development and rhizoid production. Bars represent means standard error. Within each graph, b = 0.05 Dashed line in germination graph indi cates estimated viability.

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140 Figure 5 12 Effect of gibberellic acid isomers (GA 3 and GA 4+7 ) on seedling elongation. Thick lines within boxes represent mean values. Thin lines within boxes represent median value. Boxe s represent upper and lower quartiles. Whiskers represent 1.5 interquartile range. Dots indicate lower 5% and upper 95% limit of data range. Within each graph, boxes with the same letter are not

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14 1 CHAPTER 6 EFFECT OF THE GIBBERELLIN BIOSYNTHESIS INHIBITORS PACLOBUTRAZOL AND CHLORMEQUAT ON GERMINATION AND EARLY SEEDLING DEVELOPMENT Background In prior studies, exogenous gibberellic acid ( GA 3 and GA 4+7 ) were found to inhibit germination, slow leaf differentiation and promote embryo elongation in Bletia purpurea seeds and seedlings. In addition, exogenous GA 3 exacerbated ABA induced inhibition of germination and development similar to what has been observed wit h Coffea arabica cv. Rubi (da Silva et al., 2005) In the case of C. arabica germination was also inhibited when seeds were treated with the gibberellin biosynthesis inhibito r, paclobutrazol However, the inhibitory effects of paclobutrazol c ould be alleviated by low doses of GA 4+ 7 indicating that the inhibitory effect of exogenous GA 4+7 was the result of supraoptimal levels of GA. The objective of this study was to indirectly determine whether the observed inhibition of germination and development by exogenous GA 3 observed with B. purpurea like that observed with C. arabica is due to supraoptimal levels of GA 3 To tes t this hypothesis, seeds were cultured with the gibberelli n biosynthesis inhibitors pac lobutrazol and chlormequat (CCC) with or without supplemental GA 3 Both growt h retardants block the synthesis of gibberellins e arly in the metabolic pathway; p aclobutrazol blocks the o xidation of ent kaurene into ent kaurenoic acid by monooxygenase while CCC blocks the catabolism of ent kaurene by binding to CPP synthase (primarily) and ent kaurene synthase (Rademacher, 1991, 2000) If treating seeds with exogenous GA 3 adds to an e xtant pool of de novo synthesized gibberellins resulting in supr aoptimal levels of biologically active gibberellic acids then

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142 simultaneously blocking gibberellin biosynthesis and supplementing biologically active gibberellic acid pools with exogenous GA 3 is expected to enhance germination. Materials and Methods Seed C ollection Bletia purpurea seeds were collected from the Florida Panther National Wildlife Refuge (Figure D 1) Ten mature, undehisced capsules were collected from the field and stored over Drierite desiccant at room temperature (~22C) until capsules dehis ced. The same seed source was used for these experiments as was use d for experiments described in C hapter 6. Seeds were then removed from capsules, pooled into 10 mL scintillation vials and stored at 10C over desiccant prior to experimentation. Estimatin g Viability To assess seed viability, seeds were again subjected to TZ staining prior to experimentation. A small volume of seed (approximately 100 200 seeds) was placed in 1.5 mL centrifuge tubes and treated with 5% Ca(OCl) 2 (w/v) for 30 minutes to weaken the testa and faci litate staining. Seeds were then rinsed three times in sterile water before being resuspended in water and incuba ted at room temperature for 23.5 hours. Water was then replaced with 1% TZ (pH 7.0) and seeds were incubated for 24 hours at 30C. Seeds were then examined with a dissecting microscope; seeds containing embryos with any degree of pink to red staining throughout the embryos were considered viable while wholly unstained embryos were considered non viable. Three replicates were pe rformed to estimate percent viability by dividing the number of seeds with viable embryos by the total number of seeds with embryos.

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143 Seed Sowing F or all experiments, seeds were surface sterilized in a solution of 6.0% NaOCl :100% ethanol :sterile water (5:5: 90) for 60 seconds followed by three 20 second rinses in sterile distilled water before sowing onto various media. Basal medium consisted of strength Murashige and Skoog medium (Murashige and Skoog, 1962) modified with strength FeSO 4 7H 2 O and Na 2 EDTA. Medium was gelled with 8 g L 1 TC agar ( Phyto Technology Laboratories) and adjusted to pH 5.8 before autoclaving F ilter sterilized sucrose was added to media after autoclaving to yield a 10 mM final concentration Paclobutrazol Experiment A one factor CRD was conducted to determine whether inhibition of endogenous gibberellin bi osynthesis by paclobutrazol e ffected seed germination and seedling development of B. purpurea Seeds were sown onto 9 cm Petri plates containing approximately 25 mL basal media amended with 0, 10, 50, 100, 200, 300 or 400 M paclobutrazol. As paclobutrazol is weakly soluble in water, it was dissolved in a small volume of DMSO. The maximum concentration of DMSO in treatment media was 1.6% (v/v). Two controls were tested: a DMSO control (1.6% DMSO [v/v] ) and a paclobutrazol solution control containing neither DMSO nor paclobutrazol Paclobutrazol solution and DMSO were filter sterilized as previously described and added to autoclaved media. Approximately 30 90 seeds ( 57 10; mean standard deviation [SD] ) were sown per plate. Plates were incubated and scored as previously described GA 3 Chlormequat Experiment A two factor CRD was conducted to determine whether exogenous GA 3 could enhance germination and seedling development when endogenous GA production was

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144 inhibited by CCC Seeds were sown on to 9 cm Petri p lates containing approximately 25 mL basal media amended with 0 1 or 10 mM CCC as well as 0, 1, 10 or 100 M GA 3 CCC and GA 3 were filter sterilized and added to autoclaved media as previous described. A pproximately 40 70 seeds (51 8; mean SD ) were s own per plate. Plates were incubated in darkness and scored as previously described. Experimental Design and Statistical A nalysis A completely randomiz ed design was used for both experiments. Five replicate plates were used for each treatment and all exper iments were repeated once. Germination and developmental data were used to ca lculate percent germination average developmental stage, DI and percentage of embryos/seedlings producing rhizoids. Data were analyzed with proc mixed in SAS software v. 9.1.3 (SAS Institute Inc., 2003) treating repeat as a rando m factor. Parameters of the one factor paclobutrazol experiment were also analyzed with select paired contrasts. Tests for significant effects of CCC at each level of GA 3 were performed by using the slice command in SAS. Least significant mean separation a t was used for both experiments Germination percentages and rhizoid production percentages were arcsine transformed for analysis. Results Effects of Paclobutrazol on Germination, Development and Rhizoid Production Paclobutrazol significantly redu ced germination, development and rhizoid production (Table 6 1 Figure 6 1 ). DMSO solvent control also s ignificantly reduced these responses compared to control. Treatment with 300 and 400 M paclobutrazol significantly reduced germination compared to solv ent control indicating that paclobutrazol had an addit ional inhibitory effect over that caused by DMSO alone

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145 Similarly treatment with 100 400 M paclobutrazol inh ibited development more than solvent control. Percent rhizoid production was greater than th e solvent control in all paclobutrazol treatments accept 400 M. Effects of CCC on Germination, Development and Rhizoid Production CCC had a significant e ffect on germination at all levels of GA 3 ( Table 6 2, Figure 6 2 ). When seeds were treated with 0 and 1 M GA 3 germination was not significantly reduced by 1 m M CCC but was significant inhibited by 10 m M (Figure 6 2) When seeds were treated with 10 M GA 3 1 m M CCC increased germination over control. N o significant difference was detected between 0 and 10 m M CCC treatments. However at 100 M GA 3 treatment with 1 and 10 m M CCC both significantly increased germination. CCC had a significant e ffect on development at all GA 3 levels (Table 6 2, Figure 6 2) In the absence of GA 3 CCC significantly reduced d evelopment in a dose dependent m anner. T reatment with 1 m M CCC signi ficantly increased development at all other levels of GA 3 tested When seeds were treated with 1 M GA 3 10 m M CCC significantly reduced development compared to 0 m M CCC T reatment with 10 m M CCC resulted in similar average development values as CCC control treatments when seeds were treated with 1 0 and 100 M GA 3 Rhizoid responses to CCC were more pronounced than was observed for germination or development. As with development, CCC signif icantly reduced rhizoid production in the absence of GA 3 in a dose dependent manner (Table 6 2, Figure 6 2). Wh en seeds were treated with 1 or 10 M GA 3 rhizoid production was significantly reduced by 10 m M CCC compared to controls, but not by 1 m M CCC No significant differences were detected among CCC treatments at 100 M GA 3

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146 Discussion Paclobutrazol treatments significantly reduced B. purpurea germination, development and rhizoid production. T he paclobutrazol solvent, DMSO also significantly reduced these responses compared to control treatments indicating that the observed sup pressions were at least partially due to the solvent. The effect of DMSO on seeds is highly variable Germination of A rabidopsis and cress seeds is unaffected by low concentrations (less than 1 % [ v/v ]) of DMSO while a 1 hour soak in 100% DMSO completely inhibited germination of sweet corn and longer exposures were lethal t o seeds and seedlings (Berry and Smith, 1970; Gusta et al., 1992; Hung et al., 1992; Debeaujon and Koornneef, 2000) While it is likely that the inhibitory effects of DMSO vary by species, variability in observed responses are likely also affected by the use of different concentrations of DMSO in these studies. Because DMSO increases the solubility and uptake of compounds, it cannot be ruled out that the inhibitory effects observed in the current study are the results of increased uptake of components of basal media rather than direct effects of DMSO. Additionally even when DMSO solvent controls have no effect on the studied response this solvent may interact wi th treatment compounds that are absent in solvent controls, enhancing their promotive or inhibitory effects (Hogland, 1980) This may be the case with B. purpurea ; w hile the results of the paclobutraz ol study seem to indicate that inhibiting gibberellin biosynthesis is inhibitory to seed germination due to an additional inhibition of paclobutrazol over solvent control the results of the CCC study indicate the contrary. When Bletia purpurea seeds were cultured on basal medi um without GA 3 low levels of CCC did not inhibit germination and high levels had a slight, but significant negative effect (3% reduction) In other species shown to be sensitive to CCC

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147 maximum germination is typically reduced by at least 50% at much lower conc entrations than were tested in the current study (50, 100, 150 300, and 300 600 M for Coffea Arabica cv. Rubi, celery, Barbarea stricta and Barbarea vulgaris respectively) and completely inhibited by mol arities less than 1 m M (Hepher and Roberts, 1985; Hintikk, 1988; Pressman and Shaked, 1991; da Silva et al., 2005) These results indicate that de novo synthesis of gibberellins is not needed for germination of B. purpurea While the germination promoting and dormancy alleviating effects of gibberellic acids are well known (see for example Baskin and Baskin, 2004; Kucera et al., 2005; Finch Savage and Leubner Metzger, 2006; Nonogaki, 2006; Hilhorst et al., 2010) their synthesis within the seed may not always be necessary for germination Stored gibberellin pools, including biologically active isomers, may be available in dry see ds (Ogawa et al., 2003) The inhibitory effect on germination of exogenous GAs observed with several species of orchid s indicate that embryo s are sensitive to GAs and that supraoptimal levels are inhibit ory (Hadley and Harvais, 1968; Znaniecka et al., 2005) Further study is needed to determine whether extant GA pools facilitate germination of orchid seeds as de novo synthesis appears not to be important. The germination res ponse of seeds treated simultaneously with CCC and GA 3 is more difficult to explain As GA 3 molarity was increased, germination of seeds treated with 1 m M CCC also increased compared to control treatments Similarly, while 10 m M CCC was found to impair germination at 1 M GA 3 compared to CCC control, germination was enhanced by 10 m M CCC at the greatest molarity of GA 3 tested. The ability of lower molarities of CCC to partially overcome GA 3 inhibition would appear to (2005) hypothesis that the availability of both exogenously

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148 supplied and de novo synthesized gibberellins results in supraoptimal levels of gibberellins and inhibited germination if not for the observation that blocking de novo synthesis of gibberellins in the absence of GA 3 has little to no effect on germination of B. purpurea GAs have been shown to promote embryo elongation in B. purpurea which may be one reason for the observed delay in germination (see Chapter 5 for data and discussion). The ability of low doses of CCC to suppress cell elongation and division (Rademacher, 2000) may counteract GA induced elongation, redirecting embryo growth towards t he testa walls resulting in more rapid germination. The ability of CCC to inhibit development and rhizoid production in the absence of exogenous GA 3 and at concentrations that leave germination unaffected suggests that gibberellin b iosynthesis is involved in seedling differentiation and rhizoid production The inhibitory effect of GA 3 on B. purpurea development documented in the current and p rior experiments (Chapter 5) was partially overcome by 1 m M CCC while higher molarities further inhibited or had no det ecta ble effect on development. W hile CCC and paclobutrazol are often used to inhibit gibberellin biosynthesis, growth retardants can alter enzym e activity in other pathways directly or indirectly, especially at higher concentrations (Berry and Smith, 1970; Rademacher, 2000) Of particular releva nce to the current study is the abi lity of CCC to stimulate sucrose cleavage by sucrose synthase to fructose and UDP glucose (Sharma et al., 1998) This may account for the promotive effect of CCC on B. purpurea germination in the presence of GA 3 as increased sucrose synthase activity c ould facilitate cell loading with glycolysis precursors

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149 Table 6 1 ANOVA results of the effects of paclobutrazol on germination, development and rhizoid production. Effects with p these F values are bolded. Germi nation Development Rhizoids Effect df F p F p F p Treatment 7 39.41 < 0.01 70.21 < 0.01 38.67 < 0.01 Treatment Control vs DMSO control 1 44.05 < 0.01 136.04 < 0.01 136.52 < 0.01 Treatment Control vs Paclobutrazol 1 105.74 < 0.01 399.33 < 0 .01 211.21 < 0.01

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150 Table 6 2 ANOVA results of the effects of chlormequat on germination, development and rhizoid production. Effects with p these F values are bolded. Germination Development Rhizoids Effect df F p F p F p Chlormequat (CCC) 2 20.60 < 0.01 31.83 < 0.01 98.52 < 0.01 Gibberellic acid (GA) 3 295.01 < 0.01 224.58 < 0.01 79.30 < 0.01 CCC GA 6 5.20 < 0.01 20.89 < 0.01 15.62 < 0.01 CCC GA 0 M GA 3 3.62 0.03 59.82 < 0.01 72.71 < 0.01 CCC GA 1 M GA 3 18.60 < 0.01 18.50 < 0.01 50.18 < 0.01 CCC GA 10 M GA 3 5.38 0.01 7.88 < 0.01 22.19 < 0.01 CCC GA 100 M GA 3 8.60 < 0.01 8.30 < 0.01 0.31 0.74

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151 Figure 6 1 Effect of gibberellin biosynthesis inhibition by paclobutrazol on germination, development and rhizoid production. Bars represent means standard error. Within eac h graph, b ars with the same letter are not = 0.05 Dashed line in germination graph indicates estimated seed viability.

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152 Figure 6 2 Effect of gibberellin biosynthesis inhibition by chlormequat on germination development and rhi zoid production Bars represent means standard error. Within each graph, b = 0.05 Dashed line in germination graph indicates estimated seed viability. Chlormequat (CCC). Gibberellic acid (GA 3 )

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153 CHAPTER 7 B REEDING SYSTEM OF POPULATIONS ON THE FLORIDA PANTH ER NATIONAL WILDLIFE REFUGE Background of in situ conservation programs and for assigning conse rvation prior ity (Gross et al., 2003; Ghazoul, 2005; Swart and Dixon, 2009) F or example, if pollinator li mitation is found to be the cause of declining pla nt po pulation s, then autogenic repair hinges in large part on the ability of restorat ion an d conservation practitioners to repair pollinator services while simultaneously restoring extant plant populations (Nayak and Davidar, 2010) In this hypothetical situation, artificial propagation and reintroduction will only p roduce short term success ( apparent increased connectivity of po pulations, larger populations and more populations ) ; however over longer time scales, populations would be expected to decline due to low reproduction rates, little seed rain and poor recruitment I n an increasingly human modified landscape, the consequences of habitat fragmentation may be felt more strongly by self incompatible plant populations as fr agmen tation reduces pollinator visits limits seed set and may increase inbreeding depression (Lennartsson, 2002) In contrast self compatibility and high rates of clonal growth can resu lt in reproductive assurance even at the expense of genetic diversity in isolated populations (Hill et al., 2008) In situations where seed set is not limited or needed for population expansion, allocating resources to alternative con servation methods such as land acquisition, habitat protection/restoration, removal of invasive species w ould be more efficient than protecting plant pollinator interactions Thus

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154 knowledge about breeding systems can profoundly influence management of threatened plants and communities. As discussed in earlier chapters there is some evidence that Bletia purpurea undergoes cleistogamous pollination (e.g. cleistogamy) in Florida (Stoutamire, 1974; Brown, 2002; Lennartsson, 2002) and the Caribbean (Catling, 1990) The ability for vectorless self pollination (i.e. autopollination, Smithson, 2006) while likely increasing inbreeding, ma y have facilitated expansion of the species into geographically isolated regions that lacked suitable pollinators (Catling, 1987, 1990) However, there lingers some doubt as to whether these populations are exclusively autopollinated. Catling (1990) hypothesized that there was temporal variability in relative rates of cleistogamous autopollination, observing that chasmogamous, presumably outcrossing flowe rs were more abundant in some years than others in Caribbean populations. Similarly chasmogamous flowers have been occasionally observed on Florida Panther National Wildlife Refuge ( FPNWR ) plants ( Figure 1 2 C). While there is strong observational evidenc e that B. purpurea is autopollinated in Florida (flowers lacking rostella that do not fully open have been reported, Luer, 1972) populations may not be exclusively autopollinated. The one migrant per generation rule predicts that as few as one effective migration (i.e. one foreign individual per generation surviving to mate with the native population) is sufficient to prevent complete divergence of populations and dramatically reduce s inbreeding depression within a population (Mills and Allendorf, 1996; Wang, 2004; Lopez et al., 2009) In addition, e xperimental studies have demonstr ated that low migration rates can significantly increase both fitness a nd the adaptive potential of populations (Newman and Tallmon, 2002; Swindell and

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155 Bouzat, 2006) Because of this, even po pulations with high rates of self fertilization could be significantly influenced by rare outcrossing events. Facilitating and preserving such events sh ould be a high priority for conservation practitioners Autopollination may also be geographically varia ble and so assumption s of autopollination may be incorrect (J ohnson et al., 2009) Therefore it is important to quantify relative rates of capsule formation via autopollination and outcrossing. In order to test for autopollination and possible mixed breeding systems, pollinator exclusion experiments must be perform ed. If excluding pollinators does not reduce capsule formation, autopollination is conclusively demonstrated as the dominant, if not exclusive, mode of reproduction. If capsules form when pollinators are excluded, but more capsules form when pollinators ar e not excluded, a mixed chasmogamous autopollinated breeding system can be assumed. Coupling experimental analysis of breeding systems with pollinator observations and genetic analysis of population structure and diversity can provide a great deal of insig ht and direction for management of threatened species. The primary objective of this study was to assess the degree to which B. purpurea plants on the FPNWR are autopollinated as determined by pollinator exclusion experiment s Additionally, flowers from gr eenhouse grown plants were examined to determin e the likely mode of autopollination Evidence for insect mediated pollination was passively studied during data collection. Methods Experimental D esign Rates of autopollinated capsule formation were examined in two large B. purpurea populations at two distant populations on the FPNWR in Burn Unit s 6 and 50 (Figure 7 2) Inflorescences without open buds were selected as they were encountered and

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156 randomly assigned one of two treatments: bagged to e xclude pollin ators or unbagged to allow open pollination. To exclude pollinators and assess rates of cleistogamous capsule formation, 12 mm 75 mm rectangular pollinator exclusion bags were constructed from mesh cloth with ov al openings (approximate large diameter of 2 mm and small diameter of 1. 2 mm). Bags we re deployed before bud anthesis F ive foot long bamboo stakes (2007) or 6 foot long garden stakes (2008, 2009) were placed next to emerging inflorescences. Pol linator exclusion bags were placed over both the stake s and the inflorescences and then secured below the lowest inflorescence buds with twist ties (Figure 7 2 ). In order to examine the mode of autopollination in FPNWR populations, plants were collected from the field and cultured in greenhouses. Flowers at various stages of maturity from single inflorescences were removed and examined with a dissecting microscope. Images of these plants were t hen compared to images of flowers from Puerto Rican plants (provided by James Ackerman, University of Puerto Rico) an d a published illustration (Tan, 1969) Data C ollection and S tatistical A nalysis Each treatment was replicated ten times and the experiment was performed in three successive years: 2008, 2009 and 2010 Experiments were initiate d at the beginning of the B. purpurea flowe ring season in February of 2008 and in April of 2007 and 2009 when inflores cences were approximately 5 10 cm from ground level. In 2009 data was not collected from Burn Unit 9 This unit was burned i n the Big Cyp ress Fire in April; f ew inflorescences emerged following the fire likely due to drought conditions T he majority of inflorescences that emerged were browsed by deer resulting in too few inflorescences to perform the study

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157 The number of flowers per i nflore scence, capsule formation and c apsule size were recorded 6 10 and 14 weeks after experiments were init iated, or until all flowers dehisced or formed capsules. Bags were removed during scoring and replaced until all flowers on an inflorescence dehisced or formed capsules. No flowers were visited during these brief exposures. Flower number was estimated by counting flowers, flower buds and bud scars. Capsules were assumed to be formi ng when the petals and sepals wilted, ovaries darkened, c arpe ls began to sw ell and carpe l septa became more prominent C apsules that were damaged by deer br ows e or during experiment set up or that withered before week 6 were excluded from analysis. Capsule dimensions were measured with electronic calipers. Capsule size has been f ound to be significantly correlated with both total seed number and seed viability in some studies (Faast et al., 2011) but not in others (Smithson, 2006) In the current study, c apsul e dimension data was recorded to track fruit abortion and evidence of abnormal fruit formation via auto pollination not as a proxies for seed production or fitness estimates as there may be little correl ation between these variables (Smithson, 2006) In all treatments and years, a minimum of seven infloresce nces were included in analys is ANOVA for flowers per inflorescence, percent capsule formation, capsule length and capsule diameter were analyzed as a repeated measures experiment using general li near modeling in SAS software v. 9.1 (SAS Institute Inc., 2003) at 0.05 LS mean separation at = 0.05 was used to assess differences in capsule lengths and diameters among treatments at different observation t imes, as well as to assess differences in the total number of flowers per inflorescence and the total percentage of capsule formation at each location within each year.

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158 Results Over the three flowering seasons, 1,378 flowers wer e formed on experimental plants: 768 in 2008, 197 in 2009 and 413 in 2010. Insects were never observed visiting plants a nd open flowers with missing pollinia were only observed on two occasions, al though intensive surveys for pollinators and pollinia removal were not carried out. Over all years, 44% of flowers formed capsules. Rate of fruit maturation was highly var iable. I n 2008, 63 .5 7.4 % (mean standard error ) and 91.8 3.8 % of capsules had dehisced after 14 weeks in Burn Unit 6 and 50, respectively In 2009, 96.8 2.4 % of all observed capsules dehisced after only 10 weeks of study Much slower maturation was observed in 2010 with an overall average of 7.4 3.8 % to 29.8 10.1 % of capsules dehiscing after 14 weeks in Burn Unit 6 and 50, respectively Bagging plants did not significantly a ffect flower production in any years tested (Table 7 1, Figure 7 2). Average num ber of f lowers per inflorescence was highly variable from year to year and between sites, ranging from approximately 10 in 2010 in Burn Unit 6 to greater tha n 25 in 2008 in Burn Unit 6 (Figure 7 3) S ignificant dif ferences in flower numbers between sites w ere not detected in either 2008 or 2010 (Table 7 1). When only the total number of flowers observed over 14 weeks were analyzed, a significant difference between sites was detected in 2008 (F 1, 34 = 4.40, p = 0.04 Figure 7 3) Significant differences in the percentage of flowers forming capsules were detected between sites in 2008 but not in other study years F inal capsule percentages (Figure 7 2) were not significantly different between sites in this year (F 1, 33 = 1.73; p = 0.20). Ther efore detection of significant variance in capsule formation by ANOVA was due to variability in rates of capsule formation (i.e. capsule formation was more rapid at

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159 one site than the other), but not overall capsule formation. Significant differences in percent capsule for mation were only detected among treatment s 6 and 10 weeks after experiments began in 2008 (Figure 7 3). At these times, no significant differences were detected within sites. Time, time treatment and time site treatment all had a significant effect o n percent capsule formation in 2008 In 2010, only time had a significant effect on capsule formation. Time had a significan t effect on capsule length s in all years This is expected as capsules increase in length as they mature. Similarly, time had a sig nificant effect on capsule width in 2008 and 2010 but not in 2009 (Table 7 1). This is likely due the faster rate of capsule maturation observed in 2009 compared to other years Significant differences in capsule dimensions were only detected between site s 2008 (Table 7 1 Figure 7 4 ) In this year, significant differences in capsule widths were observed betwee n treatments in Burn Unit 6 with pollinator exclusion resulting in significantly wider capsules. Examination of flowers at serial stages of developm ent (Figure 7 5 ) and comparison with images of flowers from Puerto Rico and a published illustration revealed two poss ible modes of autopollination. While a rostellum is present, it appears to be reduced and may not effectively segregate the stigmatic surf ace and pollinia during flower development (autopollination type 5a, Catling 1990) In addition, a functio nal stigmatic depression is abs ent. Instead the stigma appears to b e folded over forming a slit This modification may put the stigmatic surface in closer proximity to the developing pollinia compared to the wild type morpho logy. This mode of autopollination is not discussed by Catling (1990) and may be novel.

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160 Discussion Po llinator exclusion did not significantly reduce capsule formation in B. purpurea populations. Therefore these populations appear to be exclusively or near exclusively autopollinated. Capsule formation was lower than reported for other autopollinated orchid s; fruit formation in the absence of pollinators was 90% and 67 84% f or Nervilia nipponica and Jumellea stenophylla respectively (Gale, 2007; Micheneau et al., 2008) Reported c apsule formation rates for the se species may be uncharacteristically high as these species produce solitary flowers; therefore p erce nt capsule formation is equal to the percentage of inflorescences producing frui t The percentage of inflorescences producing fruit in B. purpurea was comparable to those reported for N. nipponica and J. stenophylla (97%, 82% and 92% in 2008, 2009 and 2010 across sites [data not presented]), thus lower average rates of capsule formation per inflorescence may be resource limited in B. purpurea at the study site. Pollinators were not observed visiting plants during the course of data collection. In Ecuador B purpurea is pollinated by Euglossa bees 4 and other unidentified bee species 5 (Dodson and Frymire, 1961; v an der Pijl and Dodson, 1966) Flowers in these populations have nectaries on the labellum, a feature not found on flowers at the current study site in Florida Plants at the FPNWR do produce extrafloral nectar along the inflorescenc e which attracts ants, which were never observed removing pollini a. In 4 There is confusion about the i dentity of the Euglossa species observed and reported. The species is identified as Euglossa viridissima in Dodson and Frymire (1961, page 145) and was later changed to Euglossa hemichora in van der Pijl and Dodson (1966, page 181) after consultation betwe en C. H. Dodson with R. L. Dressler (C. H. Dodson, personal communicaton). However, due to the rapid expansion of the genus Euglossa which expanded from 60 to 110 species since the 1980s, the true identity of this bee is uncertain at present. 5 The unide ntified bee species observed by Dodson and Frymire (1961) are identified as Melipona sp. and Thygater sp. in van der Pijl and Dodson (1966).

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161 addition, pollinia removal appears to be rarely possible because the pollinia contact the stigma early in flower development prior to flower anthesis. Thus cleistogamy is the likely mode of reproduction A ga mospermy was not ruled out in the current study, but was not tested because flowers could not be reliably emasculated due to early contact between pollinia and stigma Additionally pollinia often lack ed viscidia making att achment to pollinators unlikely. These pollinia morphological traits were discovered in 2007 when I attempted to perform a cross pollination study to assess the breeding system of Bletia purpurea P ollinia could not be removed because they were routinely fused to the stigma or, in rare ca ses where pollinia had not contacted the stigma, pollinia would not stick to a toothpick during attempted emasculation and pollen harvest. Furth ermore, a folded stig ma makes pollinia deposition by insect vectors unlikely and the lack of floral rewards like ly reduces pollinator attraction O n a few occasions, apparently functionally outcrossing chasmogamous flowers were observed in the field, some with pollinia removed and with a n ormal stigmatic depression. A slight possibility remains that rare, pollinato r mediated outcrossing events do occur However, a study of autopollinated Eulophia species that produce a small percentage of funct ionally outcrossing flowers revealed that these flowers do not form capsules under natural conditions (Peter and Johnson, 2009) Longer term studies are needed to confirm these results, but it is likely that autopollinated species like B. purpurea and Eulophia sp. lose their ability to attract pollinators as this selection pressure is lost when reproduction is assured via autopollination The disadvantages of i nbreeding are well documented including loss of genetic diversity and reduced fitness (Buza et al., 2000; Eckert, 2000; Hayes et al., 2005)

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162 Orchids are well known for having both unique floral structures (pollinia, rostella and motile stipes) and pollinator attraction strategies (deceptive pollination and mimicry) that enhance outbreeding (Darwin, 1885; Johnson and Edwards, 2000; Cozzolino and Widmer, 2005) That being said, autopollination is not un common in the Orchidaceae Over 350 species are known to have autopollinated populations, though by one estimate it could be as high as 5 20% (Catling, 1990) Autopollination may have distinct advantages when individuals find themselves dispersed beyond the range of effective pollinators including reproductive assurance In addition, there are several reports of autopollinated taxa having wider ranges than outcrossing sister taxa (Garay 1979; Catling, 1983a; Catling, 1983b) indicat ing that autopollination can aid in range expansion and exploitation of novel environments. Inbreeding can also protect local adaptations (Rice and Knapp, 2008) and s elfing can have favorable effects in populations by purging deleterious recessive alleles (Wright et al., 2008; Larsen et al., 2011) Rate of inbreeding may be an important factor in balancing the benefits of purging deleterious alleles and the risk of reduced fitness with more rapid inbreeding resulting in more rapid declines in fitness, evolutionary potential and phenotyp ic plasticity (Day et al., 2003) though this is a controversial hypothesis (see Mikkelsen et al., 2010 for a counter example) Based on this work, there is no need for of B. purpurea management plans at the FPNWR to include management and protection of pollinators However, i t is important not to overgeneralize and characterize the species as autopollinated throughout Florida ; it cannot be ruled out that outcrossing populations are established elsewhere in Florida or that outcrossing populations could become established in the future. This seems

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163 increasingly possibly due to the recent discovery in Broward County, Florida of the exotic orchid bee and known B. purpurea pollinator, Euglossa viridissima (Skov and Wiley, 2005)

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164 Table 7 1. ANOVA results for the effect of pollinator exclusion on the number of Bletia purpurea flo wers per inflorescence, capsule formation and capsule development. Experiments were executed at two sites over three flowering seasons Observations were made after 6 and 10 weeks in 2009 and after 6 10 and 14 weeks in 2008 and 2010. Two sites we re tested in 2008 and 2010 in Burn Unit s 6 and 50 of the Florida Panther National Wildlife Refuge In 200 9, only one site was tested: Burn Unit 50. Factors with p these F values are bolded. Flowers Capsules (#) Capsules (%) Lengths Widths Year Effect df F p F p F p F p F p 2008 Time 2 6.64 < 0.01 18.82 < 0. 01 25.77 < 0.01 280.90 < 0.01 254.37 < 0.01 Site 1 3.72 0.06 1.35 0.25 7.33 0.01 7.30 0.01 30.27 < 0.01 Treatment (Tr) 1 0.37 0.55 0.94 0.34 1.17 0.29 0.20 0.66 1.91 0.18 Time Tr 2 0.75 0.47 1.79 0.18 5.05 < 0.01 0.43 0.65 0.25 0.78 Site Tr 2 0.00 0.99 0.00 0.95 0.28 0.60 0.27 0.61 3.43 0.07 Time Site Tr 4 3.32 0.02 4.28 < 0.01 4.92 < 0.01 24.06 < 0.01 0.63 0.64 2009 Time 1 0.58 0.46 2.07 0.18 2.91 0.17 6.10 0.04 0.49 0.50 Tr 1 0.31 0.59 0.04 0.84 0.02 0.84 0.52 0.48 0.13 0.73 Time Tr 1 0.45 0.51 0.12 0.74 1.22 0.74 0.70 0.43 0.29 0.61 2010 Time 2 3.96 0.03 8.94 < 0.01 4.37 0.02 76.61 < 0.01 114.72 < 0.01 Site 1 0.36 0.55 0.29 0.59 0.04 0.84 0.23 0.63 0.59 0.45 Tr 1 0.59 0.46 0.96 0.33 0.08 0.79 0.04 0.85 0.06 0.80 Time Tr 2 0.02 0.98 0.16 0.85 0.13 0.88 0.12 0.89 0.02 0.98 Site Tr 2 0.97 0.34 4.48 0.04 0.16 0.69 0.04 0.85 0.15 0.70 Time Site Tr 4 0.61 0.66 0.22 0.93 0.37 0.83 1.18 0.34 0.88 0.49

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165 Figu re 7 1 Bletia purpurea infructescence and capsule ( inset; scale bar = 1 cm).

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166 Figure 7 2 Pollinator exclusion bags on Bletia purpurea inflorescences in situ. Bags are approximately 75 cm tall.

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167 Figure 7 3 Effect of pollinato r exclusion on Bletia purpurea flower production capsule formation and capsule dehiscence at two sites and over three flowering seasons on the Florida Panther National Wildlife Refuge. Flowers represent the cumulative total of flowers observe d after 10 (2009) or 14 weeks (2008 and 2010). Capsule formation represents the total number of flowers forming capsules in each year. Capsules dehiscence after 10 (2009) or 14 weeks (2008 and 2010). Bars represent means standard error. For all parameter s and within each year, no significant differences were detected between treatments at each site.

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168 Figure 7 4 Time course of the e ffect of pollinator exclusion on Bletia purpurea capsule formation and capsule dimensi ons at two sites and over three flowering seasons on the Florida Panther National Wildlife Refuge. Data was collected after 6 10 and 14 weeks. Capsule percentages are the total number of capsules divided by the cumulative number of flowers observed at eac h data collection time. At each observation date within each graph, letters are arranged in the same order as treatment means. Error bars represent standard error of treatment means. Means represented by the same letter are not significantly different at = 0.05.

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169 Figure 7 5 Development of flowers and m ode of autopollination in Bletia purpurea Images show flowers from a single inflorescence at various stages of development. Rostella (R) are reduced and do not sufficiently separate pollinia (P) from t he malformed stigmatic surface (S) leading to presumed pollination during flower development. Scale bar = 1 mm.

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170 CHAPTER 8 SUMMARY The development of a model of orchid seed germination (Figure 8 1) has many practical implications and uses. First, a thoro ugh understanding of the individual factors that promote germination will help researcher s and conservationists to propagate rare and protected orchid taxa more efficiently Second ly a comprehensive understanding of orchid seed physiology may shed some li ght on the complex relationship between orchids and their germination promoting symbionts. Finally, a better understanding of the chemical stimuli that promote orchid seed germination may help scientists distinguish mycobiont s from closely related parasiti c fungi It is often assumed that exogenous carbohydrates are needed for orchid seeds to germination. From studies with Bletia purpurea it appears that germinatio n is primarily energy limited. Bletia purpurea s eeds contain large amounts of energy reserve s in the form of oils and protein bodies, but germination is limited in darkness when seeds are cultured without exogenous carbohydrates It is likely that these rare germination events are the result of passive uptake of water resulting in sufficient embr yo pressure to rupture the thin testa, but not due to growth or development processes that require active metabolism Under these culture conditions, the few germinated seeds do not differentiate leaves. R hizoids a re not produced in continual darkness wit hout exo genous carbohydrates. This supports the observation of other researchers that rhizoid production does not occur until after infection in symbiotic cultures (Rasmussen, 1995) suggesting that their production is limited by an exogenous energy supply. While initial infect ion is through the suspensor, rhizoids become the primary conduits for infection once they are produced (Rasmussen, 1995) Given this role in regulating infection and

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171 the observation that rhizoids are r arely produced when seeds are germinated in the light a hypothesis can be constructed that contact between orchid seeds and germination promoting fungi is less likely at the soil surface and possibly due to less hospitable conditions for fungal growth. Surprisingly B. purpurea seeds are able to germinate under asymbiotic condit ions without exogenous carbohydrates when exposed to light Under these condition s embryos turn green, indicating that chlorophyll is being produced and that seeds are photosynthetic. The ability of embryos to differentiate leaves under these conditions wh en nutrients are not limiting shows that exogenous carbohydrates are not essential for germination and subsequent seedling development. The results of various experiments examining the effect of illumination on germination and development also suggest that orchids may have the ability to undergo two different developmental paths from seed to seedling in situ depending upon whether seeds are exposed to light or maintained in continual darkness ; when buried, seeds require infection by compatible fungi to init iate growth and development, but are able to develop slowly on the soil surface without infection. It is also clear that e xogenous s ucrose and other water soluble sugars including glucose, fructose and trehalose stimulate germination and promote seedling developme nt regardless of illumination treatment Thus while it is possible for orchid seeds to germinate without an exogenous carbohydrate supply when cultured under illuminated conditions it is not a practical method for propagation. Evidence for differ ent sensitivity thresholds to different carbohydrates does have some interesting implications. Given evidence that higher solute levels are inhibitory to germination,

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172 development and rhizoid production, there may be advantages to using lower concentrations of glucose and trehalose to obtain comparative germ ination and developmental responses of higher concentrations of sucrose and fructose. Lower levels of carbohydrates would be consumed more rapidly than higher levels requiring more frequent transfers of s eedlings to fresh media. This must be weighed against evidence that higher levels of media solutes can inhibit seedling development and rhizoid productio n. Research into the water relations of orchid seeds and seedlings offers a challenging and nearly unex plored research area that may provide insight into the potential costs and benefits of increasing the water potential in media by using lower concentrations of carbohydrates and nutrients. If rhizoids are vital to regulating infection as has been posited, symbiotic seed germination may also be effected by the water status of seeds me dia and fungi Not surprisingly, germination and seedling development are impa ired when nutrient availability is limited Nutrients alone appear to have little or no effect on germination when seeds are cultured in darkness without sucrose. Under these conditions, the rare germinated seedling does not differentiate leaves suggesting that they are not growing or undergoing cellular growth. Thus nutrients such as nitrogenous compo unds do not appear to stimulate germination as has been reported for some non orchids (Baskin and Baskin, 2001) That nutrient composition affects germination, seedling development and the quality of a propagation protocol is well known from as ymbiotic propagation studies (e.g. Johnson et al., 2007; Dutra et al., 2008; Kauth et al., 2008a; Dutra et al., 2009b; Stewart and Kane 2010) but the availability of a

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173 germination promoting carbohydra te, provided by either fungus or as a component of culture media is more critical for the completion of germination. The role of exogenous abscisic acid (ABA) and gibberellic acids (GAs) in regulating B. purpurea germination and seedling development suggests that both are inhibitory. That ABA is inhibitory to germination and development is not surprising given its known function in maintaining seed dormancy and inhibiting germination (Finch Savage and Leubner Metzger, 2006) However, the inhibitory effect of GAs is surprising given the known rol e of GAs in promoting seed germination, reducing seed dormancy and enhancing seed reserve catabolism (Finch S avage and Leubner Metzger, 2006) This phenomenon deserves further investigation to identify the reasons for the response, as well as to clarify an apparent discrepancy in the scientific understanding of the role GAs play in seed germination. Clearly, GAs can drastically alter the morphology of seedlings, though this response is delayed and may be in addition to an initial inhibition of embryo growth. Experiments with the GA biosynthesis inhibitor, chlormequat, indicate that de novo synthesis of GAs is not needed for germination, but that blocking synthesis reduces seedling development and rhizoid production. Studies of the evolution of GA pools in quiescent germinating and germinated seeds are needed to understand why exogenous GAs inhibit germination and development in orchid seeds. Whether the model of orchid seed germination developed with B. purpurea will be widely applicable and adaptable to other species must be tested. However, the observation that seeds from different populations of B. purpurea ha d similarly patterned responses to illumination, sucrose and nutrient treatments is a positive finding that

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174 supports the possibility of a generalized germination model. For B. purpurea on the Florida Panther National Wildlife Refuge (FPNWR) the dominant m ode of pollination is inbreeding via cleistogamy, and t he genetic diversity and genetic structure of these populations is currently under investigation. The results of this investigation should provide in sites as to whether the observed differences in the quality of seed among the tested populations are under genetic or environmental control. Given that plants are expected to be highly inbred it seems more likely that seed quality is under environmental control. If this is the case, the unique biology and resulting low genetic diversity of B. purpurea populations on the FPNWR may offer unique opportunities to better understand the role that environment plays in controlling seed quality, and ultimately stand persistence, dispersal success and stand establi shment of orchids in situ. In this way, B. purpurea may become a model organism for orchid seed physiology both in the laboratory and in the field.

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175 Figure 8 1. Model of orchid seed germination and early seedling development based on experimental result s with Bletia purpurea Abscisic acid (ABA). Gibberellic acids (GAs). Water potential of media ( media ). Emerged Embryo Seedling Seedling w/ Rhizoids

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176 APPENDIX A EFFECTS OF BRIEF LIG HT EXPOSURE ON SEED GERMINATION AND EARL Y SEEDLING DEVELOPMENT Background During seed germination studies with Bletia purpurea dark treated seeds are routinely exposed to short periods of illumination (< 20 minutes ) after two and four weeks of culture in darkness in order to examine seeds for signs of germination This is a common methodology among orchid seed researchers (e.g. Rasmussen et al., 1990a; Rasmussen and Rasmussen, 1991; de Pauw and Remphrey, 1993; Miyoshi and Mii, 1998; Zettler et al., 1998; Zettler et al., 1999; Takahashi et al., 2000; Stewart and Zettler, 2002; Stewart and Kane, 2006b; Lauzer et al., 2007) though t he effect of the se short exposures to light on germination and s eedling development is unknown. This challenge has been handled in several ways by non orchid researchers In many studies of the effect of light on seed germination, researchers observe repeated measurements on light treated seeds, but forgo time course data on dark treated seeds (e.g. Qu e t al., 2008; Castro Marn et al., 2011) In these instances, final germination can be compared between light and dark treatments, but measures of germination rate cannot be compared. Destructive sampling may be an alternative to using repeated measurement s of dark treatments, however this makes calculating indices of growth (i.e. germination index, developmental index) problematic given the inherent variability in germination studies and the imprecision associated with sowing orchid seeds. Destructive samp ling may also impose additional constraints on experimental design as there are added supply costs, time inputs and space requirements I n some instances seed availability or concerns about the impact of harvesting large numbers of seed s on

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177 natural populat ions may also restrict experiment al design in general and the use of destructive sampling in particular This last concern does not typically apply to orchids which can produce thousands of seeds per capsule (Arditti, 1992) Another alternative is to examine seeds under low intensity green light s (Baskin and Baskin, 2001; Kettenring et al., 2006) However, green light can ha ve a stimulatory e ffect on seed germination T he effects of g reen light s on orchid seeds are unknown at this time a nd deserve experimental study Low intensity lighting may not be suitable for observing orchid seed germination as testa rupture can be difficult to detect under the best of conditions (i.e. suit able magnification and high intensity, directional light) ; this is because both the testa and embryo reflect light wh en wetted, making it difficult to see testa cracks without careful inspection. Given the constraints described above, interrupting dark inc ubation may be necessary for some studies. Thus it is important to understand how these brief exposures to light a ffect germination and development. Th e objective of this experiment wa s to assess the effect of short exposures to light on seed germination a nd development. Materials and Methods Seed Collection, Surface S teriliza tion and Media Preparation Bletia purpurea s eeds ( seed source 164) were collected as outlined in Chapter 5 and stored at 10 C prior to experimentation. Seed was then surface sterilize d following previously descr ibed procedures before being sown onto 9 cm diameter Petri plates containing 25 mL of mineral salt media Approximately 30 8 0 sterilized seeds (51 8; mean standard deviation ) seeds were sown onto each plate. Plates were sea led with a single layer of NescoFilm (Karlan Research Products Corporation) and wrapped in two layers of aluminum foil to exclude light.

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178 B asal medium consisted of strength Murashige and Skoog medium (Murashige and Skoog, 1962) modified with strength FeSO 4 7H 2 O and Na 2 EDTA gelled with 7 g L 1 TC agar ( Phyto Technology Laborat ories) and adjusted to pH 5.8. Two media were tested: basal media without sucrose (control) and basal media with 10 m M sucrose. Sucrose was dissolved in distilled deionized water, filter sterilized using 0.2 m pore size nylon syringe filters (Nalgene) and added to media after autoclaving. An equal volume of sterile water was added to control media after autoclaving to maintain constant final volumes between media Adjustments were made to the initial concentration of mineral salts prior to autoclaving to e nsure proper final concentrations of mineral salts, agar and sucrose. Light Treatments, Experimental Design and Data A nalysis In addition to the two media treatments tested, four light exposure treatments were tested resulting in a 2 4 factorial experime nt : 1 ) continual darkness for 6 weeks, 2 ) 20 minute exposure to bench top light ( GE F32T9 SP41 ECO florescent bulb ; mol m 2 s 1 ; no detectable far red light) with a 90 second exposure to microscope light ( Osram EJA halogen bulb; mol m 2 s 1 a t 14 cm ; red/far red = 1.21 ) after two and four weeks culture, 3 ) 60 minute exposure to bench top light with 3 minutes exposure to microscope light after two and four weeks culture and 4 ) 20 minute exposure to growth chamber light ( GE F20T12 CW florescent bulb; mol m 2 s 1 ; no detectable far red light) Treatment 2 was designed to reflect typical light exposures seeds were exposed to during experiments. Treatment 3 was designed to treat seeds with 3 a typical light exposure during observation. Treatment 4 was designed for direct comparison with treatment 2 in order to compare the effects of different light intensities and qualities on response variables. Red/far red rati os were measured with a Field

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179 Scout Red/Far Red Meter (Spectrum Technologies, Inc.) Four replicate plates were used for all tr eatments and the experiment was repeated once. After 6 weeks, seeds were examined for signs of germination subsequent development (Stage 0 6 ) and rhizoid production as outlined in previous chapters. SAS v9.1 .3 (SAS Institute Inc., 2003) was used to perform two way ANOVA on all parameters at 0.05 using PROC MIXED and treating repeat as a random factor. The whether light treatment had an effect on each media treatment Percent germination and percent rhizoid production were arc sine transformed prior to analysis to normalize data; true means and standard errors for these variables are presented in figures. Least square (LS) mean separation was used to compa re means within experiments at 0.05. Results ANOVA results indicated t hat sucrose had a significant effect on germination, development and rhizoid production, while brief exposures to light did not have a significant effect on these parameters (Table A 1 ; Fig ure A 1). Likewise, t he interaction of main effects did not have a significant effect on germination, development or rhizoid production When the interaction was sliced by sucrose levels, ANOVA results indicated that light treatment did not have a significant effect on seed germination, development or rhizoid production H owever, significant effects on development and rhizoid production were detected when seeds were cultured with 10 mM sucrose Mean separation reinforced most ANOVA results. No significant differences were detected among light treatments when seeds were cul tured on control media (Figure A 1). As in previous experiments, seeds and seedlings did not produce rhizoids without sucrose in darkness and maximum observed development was limited to Stage 2.

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180 When seeds were cultured on media containing sucrose, some si gnificant differences in germination were detected among light treatments, though germination under continual darkness, 60 minutes light with 3 minutes high intensity light, and 20 minutes of growth chamber light was not significantly different. All brief exposures to light significantly enhanced development in the presence of sucrose compared to control due to a significantly greater proportion of seeds developing to S tage 3 ( F 3, 27 = 3.02, p = 0.05 ). Within these treatments, no significant differences wer e detected among the percentages of seedlings developing to Stages 0 1, 2 or 4 (data not shown). All brief exposures to light significantly enhanced rhizoid production when seeds were cultured with sucrose and not significant differences were detected amo ng light exposed seeds. Discussion The result s of several studies consistently indicate that Bletia purpu r ea seed germination and seedling development is energy limited (see Chapters 3 5) Exposure to 16 hours light can partially substitute for exogenous s ucrose and enhanc e germination and development compared to dark treatments H owever, short periods of exposure to light at two week interval s similar to what seeds experience during scorin g was not sufficient to stimulate germination. Evidence of enhanced seedling development with the shortest exposures of light was surprising, though the effect was slight and the significant difference may be due to the liberal nature of the LS mean separation procedure Also surprising was the enhancement of rhizoid produ ction when seeds were exposed briefly to light as previous experiments indicated that 16 hour photoperiod inhibited rhizoid production (Figure 3 6). These dynamic responses may be the result of complex regulation by a host of photoreceptors such as phytoch romes and cryptochromes t hough further study is needed S hort exposures to light similar to what

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181 seeds experience during scoring do not stimulate germination, though these exposures do enhance early seedling development in the presence of sucrose These re sults should be accounted for, acknowledged and/or avoided in future investigation of orchid seed physiolog y

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182 Table A 1. ANOVA results for the effect of sucrose and brief exposures to light on Bletia purpurea seed germination, seedling dev elopment and rhi zoid production Factors with p alues are bolded Sucrose (S). Light (L). Germination Development Rhizoids Effect df F p F p F p Sucrose (S ) 1 2384.00 < 0.01 3634.87 < 0.01 1705.54 < 0.01 Light (L) 3 0.96 0.42 2.33 0.06 1.94 0. 13 S L 3 0.26 0.85 0.23 0.87 0.00 1.00 S 0 m M sucrose L 3 0.25 0.86 0.12 0.95 0.00 1.00 S 10 m M sucrose L 3 1.54 0.21 4.10 0.01 3.89 0.01

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183 Figure A 1. Effects of brief exposure to light on germina tion and early seedling development. Two media (with and without sucrose) and four light treatments were tested. Bars represent means standard error. Bars with the same letter are not significantly different at = 0.05 based on LS mean analysis.

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184 APPENDIX B EFFECT OF ETHANOL SO LVENT ON SEED GERMIN ATION AND DEVELOPMEN T Background In order to assess the effe cts of gibberellic acid (GA) isomers on germination and development of Bletia purpurea an experiment was co nducted in which seeds were treated with exogenous GA 3 or GA 4+7 GA 3 is available as a potassium complex salt that is easily dissolve d in water by heating. GA 4+7 is available as a pure compound which is weakly soluble in water (approximately 5 g L 1 ) but can be dissolved in ethanol. U sing GA 4+7 dissolved in ethanol for experimentation raises concerns about the impact of the solvent on experimental results. In order to resolve any potential issues with the ethanol solvent, an experiment was conducted ex amin ing the effect of ethanol on seed germination and development. Methods Seed was surface sterilized as described in previous experiments before being sown onto 9 cm Petri plates containing mineral salt medium with 0, 0. 0 1, 0.1, or 1% ethanol ( 95% ; v/v) Min eral salt medium consisted of strength Murashige and Skoog basal salts (Mu rashige and Skoog, 1962) with strength FeSO 4 7H 2 O and Na 2 EDTA with 10 mM sucrose gelled with 7 g L 1 TC agar (PhytoTechnology Labora tories) and adjusted to pH 5.8 95% ethanol and sucrose solutions were filter sterilized with nylon 0.2 m pore size syr inge filters ( Nalgene ) and added to media after autoclaving. Because the amount of solution added to autoclaved media differed, an appropriate volume of sterile d istilled d eionized water was also added to maintain constant final volumes of media.

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185 Thirty t o 70 (49 8; mean standard deviation) seeds were sown onto each plate before plates were sealed with a layer of NescoFilm (Karlan Research Products Corporation) Plates were randomized, wrapped in two layers of aluminum foil to exclude light and maintai ned in darkness at 25 C for 6 weeks After 6 weeks, seeds were scored on a scale from 0 6 as described in previous experiments. The number of seeds and seedlings producing rhizoids was also counted. Percent germination and percent rhizoid production were a rcsine transformed before data analysis. Five replicates per treatment were used and the experiment was repeated once. Responses were analyzed with a general linear model using PROC MIXED in SAS (SAS Institute Inc., 2003) treating experiment repeat as a random variable. LS mean separation at = 0.05 was u sed for all pair wise comparisons of treatment means. Results and Discussion Overall, treatment did not have a significant effect on germination (F 3, 35 = 2.12, p = 0.12). LS mean separation indicated that there were significant differences between control and the lowest level of ethanol tested (Figure B 1) However, this is likely the result of the liberal nature of LS mean separation and low variance in the responses as the difference between means of these two treatments was less than 2% (control and 0. 0 1 % ethanol ; 97. 2 0.6% and 99.1 0.5% respectively ). Increased concentration of ethanol did not result in germination percentages that we re significantly different than control further supporting the idea that ethanol did not have a biologically signifi cant effect on germination. Development was not significantly affected by ethanol treatment (F 3 35 = 1.53, p = 0.22) and no significant differences were detected between control and ethanol treatments (Figure B 1). Rhizoid production was significantly aff ected by ethanol treatment (F 3 35 = 4.45, p = 0.01; Figure B 1) with low concentrations of ethanol

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186 promoting rhizoid production However, no significant difference was detected between control and treatment with 1% ethanol While ethanol did not have an e ffect on germination of Bletia purpurea it has been shown to promote germination and overcome dormancy in other p lants (Taylorson and Hendricks, 1979; Adkins et al., 1984; Larondelle et al., 1987) This may be due to induced chang es in membrane permeability (Taylorson and Hendricks, 1979) or due to changes in metabolism (Adkins et al., 1984; Larondelle et al., 1987) Improved germination following ethanol treatment has been shown to increase respirat io n at low levels (less than 0.02% of 100% ethanol ) and to increase accumulation of fructose 2,6 bis phosphate, which is thought to stimulate glycolysis (Adkins et al., 1984; Larondelle et al., 1987) The observed effect on rhizoid production could be due to these induced physiological changes though so little is known about the physiology of rhizoid production that genera ting hypothese s about mechanisms is difficult. These structures appe ar to be both more sensitive and more variable in their responses to various treatments than is observed for germination or development, which could make rhizoid production a good indicator of biological responses related to orchid seed and seedling physio logy.

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187 Figure B 1. Effects of ethanol on Bletia purpurea germination and seedling development. Bars represent means standard errors. Means with the same letter are not significantly different based on LS mean se paration ( = 0.05).

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188 APPENDIX C U LTRASTRUCTURE OF B LETIA PURPUREA EMBRYOS Background For most plant s non dormant seeds require only suitable environmental conditions and moisture for the completion of germination (Bewley and Black, 1994) Orchids either do n ot germinate in water alone, or germinate but do not undergo further development after imbibition as demonstrated with Bletia purpurea Orchid seeds are small, undifferentiated and lack specialized energy storage organs such as endosperm or cotyledons How ever, evidence from a small collection orchid seed reserve studies indicates that they are energy rich (Harvais, 1974; Harrison, 1977; Manning and van Staden, 1987) Thus the inability of B. purpurea embryos to germinate in the absence of light and/or exogenous sucrose is not expected to be energy limited. In order to assess this hypothesis e mbryo s of B. purpurea were examined with transmission electron microscopy ( TEM) and light microscopy to visually assess the presence of seed energy reserves Methods Transmission Electron Microscopy Fi xation and staining for TEM followed the methods of Lee et al. (2005; 2006) with modifications Seed testas were nicked and soaked for 24 hours in water but did not readily sink. Therefore they were subsequently soaked for 24 hours in a 1% sol ution of Tween 20 under a 20 kBar vacuum The same vacuum pressure was used for all subsequent vac uums unless stated otherwise Imbibed seeds were fixed in K solution (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer at pH 6.8) for 48 hours with vacuum at room temperature (~22C) then rinsed

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189 three times for 15 minutes per rinse in 0.2 M sodium phosphate buffer. Seeds were post fixed for four hours with 1% osmium tetroxide ( OsO 4 ) in sodium phosphate buffer at room temp erature. Aft er a one minute soak in OsO 4 seeds were microwaved in a Pelco Biowave Microwave (45 seconds w ith 68 kBar vacuum at 245 W ) to expedite post fixation. Seeds were again rinsed three times for 15 minutes in buffer solution before being dehydrated in a graded acetone series (25%, 50% and 75% ) at 12 hours per solution, then 2 100% at 6 hours intervals Fixed seeds were embedded in Low Viscosity resin (Cat. # 14300; Electron Microscopy Sciences) using a graded acetone series (acetone:resin; 3:1; 1:1, 1:3, pure resin for 12 hours/solution) and a modified formula for medium hard blocks (Ellis, 2006) before being placed in fresh resin and polym erized in aluminum trays at 63 C for 16 hours. Blocks were sectioned to 50 120 nm with a diamond knife and a Leica Ultra cut UCT ultra microtome. Thin sections were mounted on formvar backed 2 0.5 mm slotted gri ds, stained with uranyl acetate (five minutes) and lead citrate ( two minutes) and viewed on a JEOL 2010 TEM with a side mount Vieworks 5M16MC digital camera. Light Microscopy Whole mounts and squash mounts were used for histochemical analysis using light microscopy. Whole mounts of triphenyl tetrazolium stained seeds were prepared by staining as de scribed in previous chapters, mounting samples in depression slides and viewing samples with a Nikon SMZ100 dissecting microscope Squash preparations were used t o assay for the presence of starch by squashing seeds under a coverslip with 1% iodine potassium iodide (IKI) stain, and to assay for the presence of oils by soaking seeds in 70% ethanol for 20 minutes before squashing see ds with 0.7% Sudan

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190 b lack B in 70% ethanol Squas h mounts were viewed with a Nikon Labophot 2 Images were captured with a Nikon Coolpix 990 digital camera Seeds were also emb edded in paraplast T hough staining preparations were unsuccessful, the methods are reported here for posterity. S eeds were chemically scarified in a solution of 6% sodium hyopchlorite :100% ethanol: distilled water (1:1:18 ) to facilitate penetration of fixative, then rinsed three times in distilled water. Scarified seeds were soaked for 24 hours in 1% Twe en 20 to allo w seeds to imbibe, and then hours. Seeds were rinsed three times in phosphate buffer and mounted in 1% agar blocks. This was done to reduce seed loss during solution transfers and to allow thin sectioning of a small block face with numerous seeds in each section. Molds were made by cutting the tapered bottom off of 1.5 mL centrifuge tubes, capping the tubes and using the tubes in the inverting position. Seeds were transferred to the molds before molten agar was added. The molds were agitated to disperse seeds throughout the agar and then placed in the refrigerator at 5 C until agar solidified. Dehydration, paraplast embedding and sectioning procedures were sup plied by D.B. McConnell and F. Almira (University of Florida ; personal communications ). The agar blocks containing seeds were dehydrat ed with an ethanol and tert buta nol (TBA) series (ethanol:TBA:water; 1:0:1, 4:1:5, 5:2:3, 10:7:3, 8:11:10, 1:3:0, 0:1:0) w ith solution changes at 8 16 hour intervals. Agar blocks were then stained with safranin O in TBA for 16 hours to aid in locating samples during paraplast embedment, mounting and sectioning. Samples were soaked in TBA for an additional 8 hours to remove ex cess safranin O before infiltrated with paraplast. Samples in TBA were first placed near the

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191 vent on top of the heating oven which was set at set to 58 C. Three paraplast chips were added to the TBA and allowed to slowly saturate the TBA for 16 hours. Add itional paraplast chips were added to the s amples in the TBA and partially d issolved paraplast solution and then placed in the oven with the screw cap loosely attached in order to slowly increase the concentration of paraplast by evaporating the TBA After 24 hours the cap was removed to allow remaining TBA to evaporate At this time more paraplast chips were added. After 24 hours, of the solution was poured off and replaced with molten paraplast. This was repeated two more times at 8 and 24 hour intervals. After an additional 24 hours, samples were mounted in aluminum weigh boats using a gradient hot plate. Samples were sectioned at 5 10 nm with a steel blade using an American Optical model 820 Spenser microtome Section fixing and staining procedures followed Ruzin (1999) Sections were floated on Hau pt fixative subbed slides with 4 % formalin. Slides were allowed to fully cure for 48 hours at 42 C. Sections wer e deparaffinized with two 5 minute soaks in Hemo De. Slides were hydrated with an ethanol series (100%, 95%, 70%, 50%, 30%, distilled water) at 15 minute intervals. After samples were hydrated to 70%, they were stained for 2 hours in a 0.7% solution of fre shly prepared Sudan b lack B in 70% ethanol. Sections were differentiated with 70% ethanol for one minute before hydration was continued. Fully hydrated sections were mounted in glycerin and examined with a Nikon Labophot 2 at up to 1000 magnification Re sults As with most orchids, the embryos of Bletia purpurea are undifferentiated and minute (Figure C 1A). TEM analysis revealed embryo cells were densely filled with oil bodies and putative protein bodies (Figure C 1B ). The lack of starch granules i s

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192 notew orthy. These structures have a distinct lamellated pattern when examined under TEM which was not observed Additionally, squash mounts of seeds in IKI did not reveal any exuded starch granules The presence of high concentrations of oil reserves was confi rmed in Sudan Black B stained squash preparations (Figure C 1C). As stated in the methods section attempts to stain pa raplast embedded cross sections with Sudan b lack B were unsuccessful. Because visualization of lipids was a olution was used for fixing as ethanol containing fixatives like formalin acid alcohol (FAA) can solubilize lipids (Ruzin, 1999) However, the quality of microtechniques and histochemical staining depends on optimizing many other factors including fixative pH, temperature, buffer solution, osmolarity s taining duration and exposure (Ruzin, 1999) It is possible that one or more of these factors was insufficient to yield good r esults. Finally, the possibility that optics were limiting cannot be ruled out; i t is possible that conventional light microscopy techniques are not able to clearly delimit the l ipid bodies in this species due to their relatively small size and high density (Figure C 1B) In this situation small amounts of fringing around t hese structures could impede their detection Discussion For most plant taxa, seed reserves are concentrated in cotyledons or endosperm, however orchids lack these structures with rare exception (a few species do have cotyledons, Arditti, 1992) Seed reserves can be broa dly divided into proteins, carbohydrates and lipids, though relative composition, distribution and structure of these reserve classes vary widely across taxa (Bewley and Black, 1994) As with B. purpurea t he lack of starch in mature orch id embryos has been noted by other researchers (Harvais, 1974; Harrison, 1977; Manning and van Staden, 1987) Instead, orchids store

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193 large quantities of lipids, and to a lesser extent, proteins (Harvais, 1974; Harrison, 1977; Manning and van Staden, 1987) with some species also storing free sugars (Manning and van Staden, 1987) While the need for exogenous carbohydrates in situ or infection with symbiotic fungi for germination has been attributed to a lack of reserves in these minute seed s, orchid embryos are laden with reserve materials. Thus the inability to germinate without an exogenous energy source may be due to the inability to breakdown stored lipids, possibly due to a lack of key enzymes (Manning and van Staden, 1987)

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194 Figure C 1 Morphology and ultrastructure of Bletia purpurea seed s. (A) Red stained embryos showing a positive reaction to triphenyltetrazolium and covered by the translucent testa Scale bar = 200 m. (B) Transmission electron microscope image of an embryo cell with oil bodies (electron light structures) and putative p rotein bodies (electron dense structures). Scale bar = 2 m. (C ) Squash mount of seeds in Sudan Black B stain. Blue black staining indicates a positive reaction with oils and/or lipids. Scale bar = 100 m.

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195 APPENDIX D HABITAT CHARACTERIZA TION OF B LETIA PU RPUREA POPULATIONS ON THE FLORIDA PANTHER NATI ONAL WILDLIFE REFUGE Background In the United States, Bletia purpurea is only found in south Florida where it is listed as a state threatened species (Coile and Garland, 2003) As described in Chapter 1, B. purpurea can be found growing in several distinct habitats (Correll, 1978; Dressler, 1993b; Williams a nd Allen, 1998; Brown, 2002) On the Florida Panther National Wildlife Refuge (FPNWR), plants may be found in dry or seasonally flooded marl soils of hardwood scrub habitats, in grass and forb dominated clearings of pinelands, in highly unstable soils alo ng roadsides and eroding lake edges, and on floating logs or stumps in cypress swamps (Personal observation). Bletia purpurea plants are often found in clumps. These clumps or individual shoots may be widely dispersed across a landscape or found at much hi gher densities of several hundred plants within only a few square meters (personal observation). A major challenge to the conservation of rare species in general, and o rchids with their often patchy distributions in particular, is accurately identifying a ecological niche (Tsiftsis et al., 2008) Challenges aside, the ability to accurately identify ributions, assessing rarity and setting conservation priorities (Sattler et al., 2007; Tsiftsis et al., 2008) Additionally, this data could be combined with environmental remote sensing data to develop species distr ibution models to guide surveys for rare species and improve reintroduction success (Valverde and Silvertown, 1997; Tsiftsis et al., 2008; Bartel and Setxton, 2009; Gogol Prokurat, 2011; Shapcott and Powell, 2011) These models could also be used more dynamically to predict the effects of large scale (e.g. global climate change) and

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196 smaller scale (e.g. habitat loss, la nd use change, exotic invasion) environmental (Acevedo et al., 2007; Rios Munoz and Navarro Siguenza, 2009; Rompre et al., 2009; Carroll, 2010) Identifying the range of habitat conditions that an organism can occupy could lead to a better understanding of its ecology and rarity, thus leading to more informed management decisions. The objective of this study was to qualitatively and quantitatively describe the diversity of habitats occupied by B. purpurea on the Florida Pant her National Wildlife Refuge (FPNWR), including soil characteristics and co occurring plant communities. These data may facilitate the construction of species distribution models, the discovery of new populations in south Florida, and when combined with po pulation genetic analysis studies, important information about the population biology of this protected species. Materials and Methods Sampling Methods Because B. purpurea plants tend to cluster within populations, a stratified random sampling technique wa s employed. Strata were chosen based on geographic isolation, density of plants and ease of sampling. Strata contained 25 201 vegetative shoots. Nineteen strata were sampled on the FPNWR in three distinct regions (Burn Units 6, 50 and 33; Figure 7 1) and t hree strata were sampled in the Fakahatchee Strand Preserve State Park ( FSPSP ). Two strata (one each from the FPNWR and FSPSP ) consisted of plants growing on floating logs while all others were in terrestrial ecosystems. Three or four 6 cm diameter soil c ores were collected from all terrestrial strata to a depth of 5 cm in October of 2009 (19 strata) and 2010 (3 strata including FSPSP sites and FPNWR site S). Soil cores were haphazardly collected within each stratum and

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197 homogenized. Soils were not collecte d from floating log sites because of concerns over the environmental impact to these microsites: soils were non existent or shallow, aggregated in small crevices, and comprised mostly of decaying wood and accumulated leaf litter. Within each terrestrial st ratum, three 1 m 2 quadrats were sampled for plant species and species abundance. Becau se vegetation on floating logs wa s distributed i n a narrow band along the trunk, and because vegetation at these site s is often limited to only a small section of the log quadrat size was determined by log diameter and quadrats were delineated within vegetated areas of logs. The net result was rectangular quadrats that covered less than 1 m 2 In terrestrial sites, sample quadrats were placed at random compass points 25 cm from the center of each strata in a non overlapping pattern. In floating log sites, quadrats were distributed along occupied vegetation. Habitat and Soil Characterization Plant s pecies composition (live plants and identifiable, standing plant residue) and percent cover (an estimate of abundance) of all species encountered was recorded in each stratum Data were collected in May 2010 when species abundance was high and a majority of species were flowering. Percent cover of each species was estim ated using B raun Blanquet cover classes (1 4%, 5 25%, 26 50%, 51 75%, 76 95% and >95%, Dressler, 1993b) Abundance estimates for each species were calculated by averaging the cover class midpoints across subplots (i. e. 2 .5 %, 15%, 38%, 63%, 85.5% and 97.5%, respectively). Similarity in species composition (presence/absence) among strata was compared in PC ORD v5 (McCune and Mefford, 2006) using group averaging cluster analysis of Jaccard Similarit y Indices. For terrestrial sites, which were analyzed for soil properties, species abundance and soils data were subjected to joint plot n on metric

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198 multidimensional scaling (NMS) in order to visualize similarities among B. purpurea sites and to examine the effects of soil characteristics and bare soil on species composition (Mueller Bombois and Ellenberg, 1974) Rare species found in only one stratum were removed from the main matrix prior to these analyses. A 1/8 th power transformation was applied to the main ma trix in order to reduce skew and the S Curtis dissimilarity) and a random starting point. Analysis of soil pH, phosphorus, total Kjeldahl nitrogen ( TKN) and loss on ignition (LOI; an estimate of organic matter) were performed by the University of Florida Institute for Food and Agricultural Sciences Analytical Research Laboratory. Soils were stored at 10 C prior to analysis, and then dried at 30 C for two to five days before being sifted through a #20 mesh (0.85 mm) grid. Soil pH was measured from a 1:2 (v:v) mixture of soil and water with a pH meter. Phosphorus was quantified by mixing 4 cm 3 of soil with 20 mL of Mehlich 1 extracting solution. The slur ry was shaken for five minutes, then filtered through Whatman No. 42 filter paper. The filtrate was analyzed with an inductive coupled plasma spectrometer in combination with colorimetric analysis. TKN, an estimate of nitrogen in organic materials (amines, proteins, ammonium and other organic nitrogenous compounds ), was quantified. LOI was determined by drying 5 6 g of soil to 105 C for a minimum of two hours, allowing the dried sample to cool to room temperature in a desiccator (oven weight), then heating the sample to 350 C for a minimum of two hours in a muffle furnace. Following high temperature treatment, samples were allowed to cool to room

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199 temperature in a desiccator before weighing (furnace weight). LOI was then calculated using the equation Results and Discussion In the 22 strata examined, 137 plant species were identified. Of these, 94 were found in more than one stratum and subsequently used for analysis (Table D 1) Distinct differences in vegetation and soil characteristics were detected among geographically isolated sites. Terrestrial sites had greater species richness than floating log sites (Table D 1, D 2; Figure D 1). Cluster analysis of Jaccard distances reve aled two distinct assemblages: a large cluster comprising of FPNWR terrestrial strata (Burn Units 6 and 50) and a smaller cluster comprised of floating log sites and FSPSP terrestrial strata (Figure D 1). Within the large cluster, strata within Burn Unit 6 and 50 formed distinct groupings. Ordination revealed evidence for different plant assemblages at the sample sites. NMS analysis indicated that a three dimensional ordination had the least stress with axes 1, 2 and 3 accounting for 29.6%, 35.3% and 23.7% of variance within plots (Figure D 2). Axis 1, 2 and 3 were most highly correlated with pH (r 2 = 0.351), TKN (r 2 = 0.433) and bare ground (r 2 = 0.234), respectively (Table D 3). Soils at all sites were slightly basic (7.5 8.3). TKN varied substantially bet ween sites. Burn Unit 6 had the lowest average value (1151 mg kg 1 ) while Burn Unit 50 and terrestrial FSPSP sites had more than double the amount of TKN (2601 and 2586 mg kg 1 respectively; Table D 2). More bare ground was found at FSPSP sites than FPNWR sites. Interestingly, the greatest and least amount of bare ground was found in the two floating log sites. Phosphorus had little effect on ordination structure, however detectable levels of phosphorus were

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200 only found in two samples, those being located i n Burn Unit 6 (Table D 2). Differentiation in community composition between Burn Unit 50 and 6 was most apparent when graphing ordination axes 2 and 3 with Burn Unit 50 sites having greater TKN and LOI values. Differences in community structure were correl ated with differences in LOI and TKN values. FSPSP community structure was distinct from FPNWR sites based on graphing axes 1 and 3. The occurrence of B. purpurea in a wide range of habitats has been noted before (Correll, 1978; Dress ler, 1993b; Williams and Allen, 1998; Brown, 2002) Correll (1978) described B. purpurea though semilithophytic may be a more accurate description because of its ability to grow on rock ledges. He noted that while the roots ar e fibrous like other terrestrial orchids, they are also covered with a thick layer of velemen similar to that found with epiphytic orchids, which may make it possible for B. purpurea to exploit these diverse habitats. However, the ability to grow in divers e habitats may not be uncommon among orchids as similar results have been found in other regions (Tsiftsis et al., 2008) A similar underlying geography likely has a strong impact on the results of soil analysis. All sampled sites are located on the Southwest Slope geological feature, which is characterized by low elevation (8 meters above sea level or less) and sandy upper soils overlaying eroded limestone (Web Soil Survey, U.S. Department of Agriculture Natural Resources Conservation Service, http://websoilsurvey.nrcs.usda.gov/ ). The majority of sampled terrestrial sites were located on Hallandale Fine Sand or a mixture of Hollandale Fine Sand and other sandy soils with the exception stratum R in Burn Unit 6, which was on Ochopee Fine Sandy

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201 Loam, Low (Natural Resources Conservation Service) The slight alkalinity encounte red in these soils is not surprising given the limestone parent material. At these pH values, some nutrients are less soluble (e.g. boron, copper, iron, manganese and zinc) which could reduce competitive pressure from faster growing species (Van Auken and Bus h, 1997) Higher pH values have also been found to be weakly correlated with increased in situ germination (Diez, 2007) Adaptations that facilitate survival in low fertility environments may be a common (2001) stress tolerant species that have relatively slow growth rates and that utilize different strategies to retain nutrients (e.g. corms, long lived leaves). Phosphorus was below detectable levels at most sampled B. purpurea site s, which is expected to significantly limit productivity at these sites. Under such conditions, orchids likely benefit from their mycorrhizal partners, which have been shown to supply phosphorus to adult plants (Cameron et al., 2007) Additionally, high levels of phosphorus can be detrimental to orchids, likely due to the stimulatory effect increased availability has on competitors when it is available in excess (Dijke, 1994; Hejcman et al., 2010) Lower soil fertility may also facilitate recruitment as in creased nitrogen availability in symbiotic seed cultures has been shown to switch the symbiotic relationship between orchids and germination promoting fungi to a parasitism of the developing embryo by the fungus (Beyrle et al., 1991) Low levels of soil nitrogen have the additional benefit of preventing phosphorus extraction by competitors (Hejcman et al., 2010)

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202 Table D 1. Co occurrence and abundance of plant species, and bare ground cover at Florida Panther National Wi ldlife Refuge and Fakahatchee Strand State Park ( FSPSP ) sites where Bletia purpurea is found. Abundance was estimated with percent cover classes. Strata numbers are listed after each site (e.g. Burn Unit 6 (8)). Percentage of subplots with species present (%). Species are arranged in order of commonness among all plots. Range of coverage percentages when species is present (R). Average cover percent when species is present ( ) Not applicable (na). Species Total Site Burn Unit 6 ( 8 ) Burn Unit 33 (3) Burn Unit 5 0 (1 0) FS PSP terrestrial (2 ) FS P SP log (1 ) % % R % R % R % R % R Bare ground na 21 na 2.5 63 20 na 2.5 15 11 100 2.5 38 15 na 2.5 85.5 45 na 38 63 55 Bidens alba 49 10 38 2.5 38 15 63 2.5 38 8 100 2.5 38 11 Fimbristylis cymosa 45 32 29 2.5 15 10 80 2.5 85.5 38 Phyla nodiflora 43 6 50 2.5 15 7 60 2.5 15 5 Baccharis halimifolia 36 17 21 2.5 38 22 47 2.5 38 15 50 2.5 38 19 100 2.5 15 11 Crotolaria rotundifolia 36 3 25 na 3 63 na 3 Sida rhombifolia 36 4 63 2.5 15 4 27 na 3 33 na 3 Eustachys glauca 35 20 21 2.5 15 5 63 2.5 63 23 Schizachyrium scoparium 33 26 77 2.5 63 26 Spermacoce verticillata 33 4 13 2.5 15 7 47 2.5 15 4 100 na 3 Polygala grandiflora 32 3 50 na 3 23 na 3 50 na 3 Ambrosia artemisiifolia 30 8 50 2.5 38 10 100 na 3 Eremochloa ophiuroides 30 62 88 15 97.5 62 Toxicodendron radicans 30 7 29 2.5 15 4 37 2.5 15 6 50 2.5 38 14 Chamaecrista nictitans 29 3 33 na 3 40 na 3 Paspalum blodgettii 28 8 8 2.5 15 9 57 2.5 15 8

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203 Table D 1. Continued Species Total Site Burn Unit 6 ( 8 ) Burn Unit 33 (3) Burn Unit 5 0 (1 0) FS P SP terrestrial (2 ) FS P SP log (1 ) % % R % R % R % R % R Rhynchosia minima 28 7 29 2.5 38 13 40 2.5 15 4 Sabal palmetto 25 15 42 2.5 38 11 20 2.5 38 16 17 na 38 Paspalum setaceum 23 6 8 na 3 40 2.5 38 7 33 na 3 Plukea rosea 23 6 33 2.5 15 9 27 na 3 Acer rubrum 22 3 4 na 3 100 2.5 15 7 17 na 3 50 na 3 100 na 3 Cirsium nuttalii 20 9 38 2.5 15 8 17 2.5 25 12 Pinus elliottii 19 3 54 2.5 15 3 Pteridium aquilinum 19 38 50 15 85.5 38 17 38 38 38 Spartina c.f patens 19 22 21 2.5 38 19 27 15 63 24 Symphyotrichum sp. 19 6 43 2.5 15 6 Andropogon glomeratus 17 9 4 na 15 23 2.5 15 11 50 na 3 33 na 3 Chamaesyce blodgettii 17 3 8 na 3 33 na 3 Gaura angustifolia 17 5 8 na 15 33 na 3 Rhyncospora globularis 17 3 25 na 3 13 na 3 17 na 3 33 na 3 Ruellia caroliniensis 17 3 4 na 3 33 na 3 17 na 3 Galactia volubilis 16 5 20 2.5 15 5 83 2.5 15 5 Ipomoea sagi ttata 16 7 29 2.5 15 6 13 2.5 15 9 Solidago stricta 16 5 37 2.5 15 5 Erigeron quercifolius 14 4 13 na 3 23 2.5 15 4

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204 Table D 1. Continued Species Total Site Burn Unit 6 ( 8 ) Burn Unit 33 (3) Burn Unit 5 0 (1 0) FS P SP terrestrial (2 ) FS P SP log (1 ) % % R % R % R % R % R Physalis walteri 14 5 42 2.5 15 5 Cenchrus gracillimus 13 13 30 2.5 38 13 Desmodium incanum 13 4 8 na 3 23 2.5 15 4 Dichanthelium acuminatum 13 4 3 8 2.5 15 4 Mecardonia acuminata 13 5 8 na 3 23 2.5 15 6 Melanthera nivea 13 5 30 2.5 15 5 Parthenocissus quinquefolia 13 8 10 2.5 15 7 100 2.5 15 9 CoreopsIs floridana 12 3 17 na 3 13 na 3 Dyschoriste angusta 12 3 25 na 3 7 na 3 Phyllanthus caroliniensis 12 3 4 na 3 23 na 3 Rhynchospora colorata 12 3 21 na 3 10 na 3 Rubus trivialis 12 6 33 2.5 15 6 Thelypteris kunthii 12 28 100 2.5 38 32 67 na 15 Acalypha gracilens 10 3 29 na 3 Centella asiatica 10 3 8 na 3 17 na 3 Pteris vittata 10 18 7 2.5 15 9 33 na 15 100 2.5 38 26 Andropogon virginicus 9 15 20 2.5 38 15 Annona glabra 9 19 33 na 15 67 2.5 63 24 33 na 3 Boehmeria cylindrica 9 17 100 2.5 38 19 100 na 15 Buchnera americana 9 3 8 na 3 13 na 3

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205 Table D 1. Continued Species Total Site Burn Unit 6 ( 8 ) Burn Unit 33 (3) Burn Unit 5 0 (1 0) FS P SP terrestrial (2 ) FS P SP log (1 ) % % R % R % % % R % R Chamaesyce hirta 9 3 8 na 3 13 na 3 Eryngium baldwinii 9 7 25 2.5 15 7 Eupatorium capillifolium 9 9 4 na 15 13 2.5 15 6 33 na 15 Paspalum notatum 9 13 25 2.5 15 13 Solidago leavenworthii 9 9 20 2.5 15 9 Stenotaphrum secundatum 9 17 4 na 15 83 2.5 38 17 Vitis rotundifolia 9 13 25 2.5 15 13 Aristida p atula 7 8 21 2.5 15 8 Dichanthelium aciculare 7 3 17 na 3 3 na 3 Dichanthelium dichotimum 7 3 8 na 3 10 na 3 Ludwigia erecta 7 3 17 na 3 Melochia spicata 7 3 21 na 3 Myrica cerifera 7 8 7 2.5 15 9 100 2.5 15 7 Pectis linearifolia 7 3 17 na 3 Scleria ciliata 7 3 21 na 3 Stylisma abdita 7 3 13 na 3 7 na 3 Acacia pinetorum 6 6 17 2.5 15 6 Aristida purpurascens 6 15 8 15 38 27 7 na 3 Chamaesyce hyssopifolia 6 3 4 na 3 10 na 3 Dichanthelium strigosum 6 3 17 na 3

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206 Table D 1. Continued Species Total Site Burn Unit 6 ( 8 ) Burn Unit 33 (3) Burn Unit 5 0 (1 0) FS P SP terrestrial (2 ) FS P SP log (1 ) % % R % R % % % R % R Elephantopus elatus 6 9 17 2.5 15 9 Eragrostis hi rsuta 6 6 13 2.5 15 6 Fraxinus carolina 6 6 67 2.5 15 9 33 na 3 Rhynchospora microcarpa 6 9 8 2.5 15 9 33 2.5 15 9 Sarcostemma clausum 6 9 67 na 15 7 n a 3 Sporobolus indicus 6 6 4 na 3 50 2.5 15 7 Urena lobata 6 6 17 2.5 15 6 Ampelopsis arborea 4 7 50 2.5 15 7 Casytha filiformis 4 7 10 2.5 15 7 Eragrostis cf. elliottii 4 3 10 na 3 Ficus aurea 4 26 67 na 38 33 na 3 Galium tinctorium 4 7 100 2.5 15 7 Hypoxis j uncea 4 7 8 2.5 15 9 3 na 3 Ludwigia repens 4 19 100 2.5 38 19 Nephrolepis exaltata 4 71 100 63 85.5 71 Polypremum procumbens 4 3 10 na 3 Rhus copallinum 4 3 13 na 3 Sporobolus virginicus 4 3 10 na 3 Vicia acutifolia 4 3 4 na 3 3 na 3 17 na 3 Brickellia eupatorioides 3 3 8 na 3

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207 Table D 1. Continued Species Total Site Burn Unit 6 ( 8 ) Burn Unit 33 (3) Burn Unit 5 0 (1 0) FS P SP terrestrial (2 ) FS P SP log (1 ) % % R % R % % % R % R c.f Desmodium sp. 3 3 8 na 3 Cladium jamaicense 3 15 7 na 15 Desmodium triflorum 3 3 7 na 3 Eragrostis sp. 3 9 7 2.5 15 9 Eupatorium mikanioides 3 3 8 na 3 Eustachys petraea 3 3 7 na 3 Mikania scandens 3 3 17 na 3 33 na 3 Ophioglossum petiolatum 3 3 4 na 3 3 na 3 Passiflora suberosa 3 3 8 na 3 Piriqueta cistoides 3 3 7 na 3 Pityopsis graminif olia 3 9 8 2.5 15 9 Proserpinaca palustris 3 3 67 na 3 Rapanea punctata 3 51 33 38 63 51 Smilax auriculata 3 15 8 na 15 Smilax bona nox 3 9 8 2.5 15 9 Strophostyles umbellata 3 3 33 na 3 Andropogon sp. 1 3 4 na 3 Asplenium serratum 1 15 33 na 15 Axonopus fissifolius 1 3 17 na 3 Berchemia scandens 1 3 17 na 3

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208 Table D 1. Continued Species Total Site Burn Unit 6 ( 8 ) Burn Unit 33 (3) Burn Unit 5 0 (1 0) FS P SP terrestrial (2 ) FS P SP log (1 ) % % R % R % % % R % R Croton willdenowii 1 3 3 na 3 Dicanthium sericeum 1 3 17 na 3 Dichanthelium commutatum 1 3 4 na 3 Erechtites hieraciifolia 1 3 3 na 3 Euphorbia heterophylla 1 3 3 na 3 Heliotropium fruticosum 1 3 4 na 3 Hypericum hypericoides 1 3 3 na 3 Imperata cylindrica 1 15 3 na 15 Ipomia sp. 1 3 17 na 3 Lantana camara 1 3 4 na 3 Lythrum alatum 1 3 3 na 3 Mitreola petiolata 1 15 33 na 15 Paspalum urvillei 1 15 3 na 15 Pterocaulon pyncnostachium 1 3 4 na 3 Quercus laurifolia 1 3 17 na 3 Rhynchospora caduca 1 15 3 na 15 Rh ynchospora divergens 1 38 3 38 38 38 Sacchrum giganteum 1 15 3 na 15 Schinus terebinthifolius 1 15 3 na 15 Scleria reticularis 1 3 4 na 3

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209 Table D 1. Continued Species Total Site Burn Unit 6 ( 8 ) Burn Unit 33 (3) Burn Unit 5 0 (1 0) FS P SP terrestrial (2 ) FS P SP log (1 ) % % R % R % % % R % R Spiranthes vernalis 1 3 4 na 3 Sysyrinchum augustifolium 1 3 3 na 3 Taxodium disticum 1 38 17 na 38 Total species 13 7 79 9 81 34 15

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210 Table D 2. Soil and vegetation parameters of Bletia purpurea habitats Florida Panther National Wildlife Refuge (FPNWR). Fakahatchee Strand Preserve State Park (FS P SP). Phosphorus (P). Total Kjeldahl nitrogen (TKN). Loss on ignition (LOI). Not detected (nd). Location Site (n) Habitat pH P (mg/kg) TKN (mg/kg) LOI (%) Bare Soil (%) Species Richness FPNWR Burn Unit 6 (8) Terrestrial 7.5 1.5 1151 4.4 20 25.1 FPNWR Burn Unit 33 (1 ) Log . . 11 10.0 FPNWR Burn Unit 50 (10) Terrestrial 7.7 nd 2601 6.7 15 31.1 FS P SP Roadside (2) Terrestrial 8.2 nd 2586 11.1 48 22.7 FS P SP Aquatic (1) Log . . 55 16.0

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211 Table D 3. Correlation between NMS axes an d site variables. Axis 1 Axis 2 Axis 3 Parameter r r 2 r r 2 r r 2 Bare ground 0.291 0.085 0.181 0.330 0.483 0.234 Loss on ignition 0.434 0.189 0.553 0.306 0.302 0.091 Nitrogen 0.246 0.061 0.658 0.433 0.302 0.091 pH 0.592 0.351 0.118 0. 014 0.141 0.020 Phosphorus 0.513 0.263 0.141 0.020 0.051 0.003

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212 Figure D 1. Locations of Bletia purpurea sites sampled for habitat characterization.

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213 Figure D 2. Dendrogram of Jaccard distances for sampled Bletia purpurea sites. Fakahatchee Strand Preserve State Park (FSPSP).

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214 Figure D 3 Non metric multidimensional scaling analysis of sampled terrestrial Bletia purpurea sites. Florida Panther National Wildlife Refuge (FPNWR). Fakahatchee Strand Preserve State Park (FS P SP). Loss on ignition (LOI). Total Kjeldahl nitrogen (TKN).

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245 BIOGRAPHICAL SKETCH Timothy Johnson grew up in Eau Claire, WI where he attended the University of Wisconsin Eau Claire. He began college as an English major and dreamed of being a creative writer, but soon found a passion for biology. His interest was nurtured by diverse courses, excellent educators and a great research mentor, Dr. Wilson Taylor, pictures. 2003 with a B.S. in biology and a minor in anthropology. After a short hiatus from higher education, he attended the University of Florida where he was a research assistant i n Laboratory in the Environmental Horticulture Department. There, he studied the seed propagation of Vandaceous orchids and collaborated with labmates on various orchid conse rvation research projects. After earning his M.S. in Horticulture (2004), he was awarded an Alumni Fellowship to study Environmental Horticulture at the University of Florida and continued working with Dr. Kane. Timothy credits much of his academic success to his mentors who helped him nurture his teaching and research interests. Timothy and his wife Danielle have one son, Finley. They enjoy playing with blocks, reading books, going for walks, splashing in the water and snuggling. Timothy also enjoys portra it photography and watching movies. He is a 3:22 marathoner.