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Reproductive Biology and Asymbiotic Seed Germination of Cyrtopodium punctatum, an Endangered Florida Orchid

Permanent Link: http://ufdc.ufl.edu/UFE0022692/00001

Material Information

Title: Reproductive Biology and Asymbiotic Seed Germination of Cyrtopodium punctatum, an Endangered Florida Orchid
Physical Description: 1 online resource (78 p.)
Language: english
Creator: Dutra, Daniela
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: breeding, cigar, conservation, florida, native, orchidaceae, pollination, seed, xylocopa
Environmental Horticulture -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Cyrtopodium punctatum Lindley is an endangered epiphytic orchid restricted in the United States to southern Florida. Due to its ornamental value, the species was extensively collected from the wild during the past 100 years. Today, only a few plants remain in protected areas. Over the past 10 years, careful observations of plant populations in the Florida Panther Wildlife Refuge indicated limited seed production in the remaining plants. Consequently, the long-term sustainability of remaining populations is in question. As part of a conservation plan for the species, a reproductive biology study was conducted and procedures for asymbiotic seed germination were developed. Information regarding the breeding strategies and pollination mechanisms in C. punctatum Florida populations is critical to develop strategies for recovery of the species. C. punctatum is self-compatible and not autogamous. A a pollinator is needed for seed production in the wild. C. punctatum relies on a deceit pollination system using aromatic compounds to attract insect pollinators. An effective protocol for the asymbiotic production of C. punctatum seedlings was also developed. Seed germination was promoted in dark and seedling development under a 16/8 L/D photoperiod. The asymbiotic seed germination protocol for C. punctatum will facilitate future reintroduction projects involving this endangered species.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Daniela Dutra.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Kane, Michael E.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022692:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022692/00001

Material Information

Title: Reproductive Biology and Asymbiotic Seed Germination of Cyrtopodium punctatum, an Endangered Florida Orchid
Physical Description: 1 online resource (78 p.)
Language: english
Creator: Dutra, Daniela
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: breeding, cigar, conservation, florida, native, orchidaceae, pollination, seed, xylocopa
Environmental Horticulture -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Cyrtopodium punctatum Lindley is an endangered epiphytic orchid restricted in the United States to southern Florida. Due to its ornamental value, the species was extensively collected from the wild during the past 100 years. Today, only a few plants remain in protected areas. Over the past 10 years, careful observations of plant populations in the Florida Panther Wildlife Refuge indicated limited seed production in the remaining plants. Consequently, the long-term sustainability of remaining populations is in question. As part of a conservation plan for the species, a reproductive biology study was conducted and procedures for asymbiotic seed germination were developed. Information regarding the breeding strategies and pollination mechanisms in C. punctatum Florida populations is critical to develop strategies for recovery of the species. C. punctatum is self-compatible and not autogamous. A a pollinator is needed for seed production in the wild. C. punctatum relies on a deceit pollination system using aromatic compounds to attract insect pollinators. An effective protocol for the asymbiotic production of C. punctatum seedlings was also developed. Seed germination was promoted in dark and seedling development under a 16/8 L/D photoperiod. The asymbiotic seed germination protocol for C. punctatum will facilitate future reintroduction projects involving this endangered species.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Daniela Dutra.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Kane, Michael E.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022692:00001


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REPRODUCTIVE BIOLOGY AND ASYMBIOT IC SEED GERMINATION OF Cyrtopodium punctatum AN ENDANGERED FLORIDA ORCHID By DANIELA DUTRA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008 1

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2008 Daniela Dutra 2

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To Larry Richardson, whose dedication and passi on for conservation inspired me along the way 3

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ACKNOWLEDGMENTS I thank my major professor, Dr. Michael Kane, for his generous time and commitment .I also thank Dr. Carrie Adams Reinhardt and Dr. Doria Gordon for serving at my committee and giving me excellent feedback during the writing process. I thank my fellow lab-workers and graduate students, Nancy Philman, Tim Johnson, and Scott Stewart. I also thank Phil Kauth for his valuable help with al l computer programs, statistical analysis, cool headedness and friendship. I thank the American Orchid Society, U.S. Fi sh and Wildlife Services the Florida Panther National Wildlife Refuge for valuable support an d funding during the course of this project. The completion of this project was possible wi th the support of many friends and family. I thank my family for understanding the distance and the time it takes to accomplish a dream. Finally, I thank my father, Frankl in Dutra, for his passion for plan ts and nature that influenced me to pursue this field. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 CHAPTER 1 LITERATURE REVIEW .......................................................................................................10 Introduction .................................................................................................................. ...........10 Study Species Information ..............................................................................................11 Orchid Pollination Biology ..............................................................................................14 Orchid Seed Propagation .................................................................................................18 Asymbiotic seed propagation ...................................................................................18 Symbiotic seed propagation .....................................................................................20 2 REPRODUCTIVE BIOLOGY OF Cyrtopodium punctatum .................................................24 Introduction .................................................................................................................. ...........24 Materials and Methods ...........................................................................................................27 Breeding system determination .......................................................................................27 Tetrazolium Seed Viability Test ......................................................................................28 Asymbiotic Seed Germination Test .................................................................................28 Pollinator Identification ...................................................................................................29 Fragrance Analysis ..........................................................................................................30 Results .....................................................................................................................................30 Breeding System Determination ......................................................................................30 Viability and Asymbiotic Seed Germination Tests .........................................................31 Pollinator Identification ...................................................................................................31 Fragrance Analysis ..........................................................................................................32 Discussion .................................................................................................................... ...........32 Breeding System ..............................................................................................................32 Seed Viability and Asymbiotic Germination ..................................................................34 Pollinator Identification ...................................................................................................35 Fragrance Analysis ..........................................................................................................35 Conservation Implications ...............................................................................................36 3 ASYMBIOTIC SEED GERMINAT ION AND IN VITRO SEEDLING DEVELOPMENT OF Cyrtopodium punctatum .....................................................................49 Introduction .................................................................................................................. ...........49 Materials and Methods ...........................................................................................................50 Seed Source and Ster ilization Procedure .........................................................................50 5

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Asymbiotic Seed Germination ........................................................................................51 Influence of Photoperiod on Seedling Growth and Development ...................................52 Greenhouse acclimatization .............................................................................................53 Statistical Analysis .......................................................................................................... 53 Results .....................................................................................................................................54 Asymbiotic Seed Germination ........................................................................................54 Role of Photoperiod on Gr owth and Development .........................................................55 Greenhouse acclimatization .............................................................................................55 Discussion .................................................................................................................... ...........56 4 SUMMARY ..................................................................................................................... .......67 LIST OF REFERENCES ...............................................................................................................70 BIOGRAPHICAL SKETCH .........................................................................................................78 6

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LIST OF TABLES Table page 1-1. Pollination treatments used to determine orchid breeding systems ...................................23 2-1. Pollination treatments used to dete rmine orchid breeding systems. ..................................38 2-2. Seed developm ental stages of Cyrtopodium punctatum ....................................................39 2-3. Seed viability, germination and development stage percenta ges generated from C. punctatum pollination treatments of the 2006-2007 flowering season ..............................40 2-4. Floral visitors of Cyrtopodium punctatum .........................................................................41 3-1. Nutrient composition of germination media used for the asymbiotic seed germination of Cyrtopodium punctatum ................................................................................................58 3-2. Seedling developmental stages of Cyrtopodium punctatum ..............................................59 3-3. Photoperiodic effects on in vitro seed germination and protocorm development of Cyrtopodium punctatum over 10 weeks culture on P723 medium ....................................60 3-4. Photoperiodic effects on seedling development of Cyrtopodium punctatum after 25 weeks culture on P723 medium .........................................................................................61 7

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LIST OF FIGURES Figure page 2-1. Breeding system experiment ..............................................................................................4 2 2-2. Seed viability and asymbiotic germination ........................................................................43 2-3. Percent capsule set among seve n breeding system treatments ..........................................44 2-4. Effect of breeding system treatme nt on mean capsule length (+ S.E.) ..............................45 2-5. Effect of breeding system treatment on mean capsule width (+ S.E.). ..............................46 2-6. Bees observed visiting flowers ..........................................................................................47 2-7. Gas chromatogram of the floral fragrance of Cyrtopodium punctatum ............................48 3-1. Comparative effects of culture media and photoperiod on germination of C. punctatum seeds after ten weeks asymbiotic culture .........................................................62 3-2. Comparative effects of culture media and photoperiod on in vitro seedling development of C. punctatum after ten weeks asymbiotic culture ....................................63 3-3. Photoperiodic effect on germina tion and protocorm development of C. punctatum after 8 weeks culture on P723 medium ..............................................................................64 3-4. Protocorm and progression of seedling development of C. punctatum cultured on P723 medium ................................................................................................................... ..65 3-5. In vitro seedling development stages of C. punctatum seeds cultured in P723 medium under 0/24 h and 16/8 h L/D ..............................................................................................66 8

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master in Science REPRODUCTIVE BIOLOGY AND ASYMBIOT IC SEED GERMINATION OF Cyrtopodium punctatum AN ENDANGERED FLORIDA ORCHID By Daniela Dutra August 2008 Chair: Michael Kane Major: Horticultural Science Cyrtopodium punctatum Lindley is an endangered epiphytic orch id restricted in the United States to southern Florida. Over the past 10 years, careful obse rvations of plant populations in the Florida Panther Wildlife Ref uge indicated limited seed production in the remaining plants. Consequently, the long-term sustainability of remaining populations is in question. As part of a conservation plan for the species, a reproductive biology study was conducted and procedures for asymbiotic seed germination were developed. In formation regarding the breeding strategies and pollination mechanisms in C. punctatum Florida populations is critical to develop strategies for recovery of the species. C. punctatum is self-compatible and not autogamous. A pollinator is needed for seed production in the wild. C. punctatum relies on a deceit pollination system using aromatic compounds to attract insect pollinators. An effective protocol for the asymbiotic production of C. punctatum seedlings was also developed. Seed germination was promoted in dark on P723 medium and seedling development under a 16/8 L/D photoperiod. The asymbiotic seed germination protocol for C. punctatum will facilitate future reintroduction projects involving this endangered species.

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CHAPTER 1 LITERATURE REVIEW Introduction The Orchidaceae is the most diverse family of plants and can be found in almost every terrestrial ecosystem (Dressler 1981, 1993). The family is estimated to have between 20,00035,000 species (Dressler 1993) with new species of ten being described. Ma ny orchid species are threatened by extinction and orchid conservation is an important issue. The two major threats to orchid conservation are 1) habitat modification and destruction, 2) wild collecting (D ixon et al. 2003; Koopowitz et al. 2003). Habitat destruction due to logging is viewed as the most obvious cause of orchid diversity loss; however, habitat modification due to road construction, fire, urba nization, drainage, and other anthropogenic influences can also directly affect orchid habitats (Hgsater and Dumont 1996; Cribb et al. 2003). Over-colle ction of orchids is another issue in orchid conservation. The impact of orchid collecting depends on the spec ies and the type of co llecting being conducted (Cribb et al. 2003). Commercial co llection supplies plants to orch id hobbyists or for medicinal purposes (Cribb et al. 2003). The Convention on In ternational Trade in Endangered Species of Wild Fauna and Flora (CITES) only regulates or chid export and not wild collecting. In most countries, collecting can be done legally if permits are obtained (Cribb et al. 2003). Although the main causes of orch id diversity loss are well understood, effective orchid conservation can only be accomplished if biologists understand all aspects of species biology, habitat, and the processes th at effect species survival. In situ and e x situ conservation efforts are both needed in order to efficiently provide sound conservation plans for threatened species in the wild. For efficient and effective conservation to take place, knowledge of pollination biology, 10

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population genetics, and life-history strategy studies such as my corrhizal relationships, seed physiology and propagation is re quired (Cribb et al. 2003). Study Species Information The genus Cyrtopodium is comprised of approximately 42 species of Neotropical origin that can be found from southern Florida to Argentina (Romero-Gonzlez 1999; RomeroGonzlez and Fernndez-Concha 1999; Batista a nd Bianchetti 2004). Brazil is the center of diversity for the genus; the Cerrado region in the central Brazilian Plateau has an estimated 25 species (Batista and Bianchetti 2004). On ly two species are found in the U.S. ( C. polyphyllum and C. punctatum ), however, C. polyphyllum has been naturalized (Brown 2005). Cyrtopodium punctatum is found in southern Florida, Cuba, Hisp aniola, Puerto Rico, and the northwestern Caribbean coast of South America (Romero-G onzlez and Fernndez-Concha 1999). Very few species in the genus are epiphytic with C. punctatum being one of them. The terrestrial species of the genus show high adaptation to fire (Menezes 1994). Cyrtopodium punctatum also known as the cigar orch id, is listed as endangered in the state of Florida (Coile and Gardland 2003). The spec ies was over-collected during the past century and today only a few plants still exist in inacce ssible protected areas. Early accounts in the literature refer to C. punctatum as abundant throughout the so uth and southwest Florida, especially in the cypress swamps in the Big Cypress region (Ames 1904; Luer 1972). Photographs from the early 1900s show wagons loaded with plants being taken from the swamps of Florida. Florida populations were also depleted due to cypress logging since the species is mostly found growing epiphytically on cypress trees. Most old growth cypress was harvested from the state of Florida in th e first half of the 20th century. The Big Cypress region, what is now the Florida Panther Wildlife Refuge and the Fakaha tchee Strand State Preserve, was purchased by 11

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the Lee Tidewater Company in 1913. Logging in this area started in 1944 due to World War II needs and by 1957 all the old growth trees had been harvested. Following logging, fires damaged remaining ecosystems and plant populations (USFWS 2005). The remaining plants in Florida are found in small populations in protected areas such as the Everglades National Park, Bi g Cypress National Preserve, a nd the Florida Panther National Wildlife Refuge. Over the past 10 years, careful observations of plant popul ations in the Florida Panther Wildlife Refuge indicated the absence of capsule formation and subsequent seed production (Larry Richardson personal communicat ion). Insufficient information about breeding strategies and pollination in Florida C. punctatum populations has been av ailable to explain the apparent lack of sexual reproduction. Most of the pertinent literature on the pollination of Cyrtopodium punctatum refers to observations conducted in only one area of the species range (e.g., Puer to Rico; Ackerman, 1995). In different parts of its geographical range, a species may possess different breeding strategies, which is not uncomm on for tropical orchids species in Florida. For example, both Epidendrum nocturnum and Secoila lanceolata produce seed by agamospermy in Florida, but utilize animal cross-pollination in the more southern part of their range (Catling, 1987; Brown 2002). Pollinator species may also vary from one location in the geographical range to another. Pollination ecotypes have been report ed by Robertson and Wyatt (1990a) in Plantanthera ciliaris In this study, the pollination ecology of two disjunct populat ions was compared and the primary pollinators were f ound to differ between sites. C. punctatum is reported to be pollinated by Euglossa bees (van der Pijl and Dodson 1966; Jeffr ey et al. 1970; Luer 1972; Dressler 1993). Since Florida lies outside the range of euglossine bees, C. punctatum s pollinator (s) in Florida 12

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may vary (Chase and Hill s 1992). In Puerto Rico, C. punctatum is reported to be pollinated by Centris or Xylocopa bees (Ackerman 1995). Bees of the genus Centris are reported by Dodson and Adams (in Luer 1972) to visit flowers of C. punctatum in Florida. However, these observations have not been fu lly described or verified. Baskin and Bliss (1969) reported the presence of reducing sugars in floral exudates of Cyrtopodium punctatum ; however, others have argued that C. punctatum uses mimicry to attract food-seeking insects and offers no nectar as a reward for pollination (Chase and Hills 1992). Van Der Pijl (1966) argues that even though no food reward is produce d, this species is part of a group of advanced orchids that produce intoxi cating substances that attract male bees. A problem exists when relying on the present literature for an accurate picture of the breeding strategy of the species because mistaken identifications are frequent. A species in the genus Cyrtopodium that is often mistaken for C. punctatum is C. macrobulbon. For many years the two species were considered the same. More recently, Romero-Gonzlez and FernndezConcha (1999) differentiated the two species an d clarified the differences (Romero-Gonzlez and Fernndez-Concha 1999). C. punctatum is only found growing epiphytically, its roots grow upward forming trash-baskets, and the side l obes of the labellum arch towards the center. C. macrobulbon can be found growing as a terrestrial or epiphyte; however, its roots do not form trash-baskets (Romero-Gonzlez and Fernndez-C oncha 1999). The authors stated that there is no overlap on the geographical distributions of the species. However, in 2001, a picture of a C. malcrobulbon plant taken in southwest Florida wa s published in Brown (2001) as being C. punctatum In the later edition of Browns book, a ne w description of the species appeared (Brown 2005). Brown stated th at the current status of C. malcrobulbon in Florida is yet to be clarified (Brown 2005). The existing literature on C. punctatum should be critically analyzed 13

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since researchers may have been referring to C. malcrobulbon It is also important to notice the location of the study when taking information into consideration. For example, a breeding system study conducted in Venezuela listed C. punctatum as one of the species investigated (Jaimes and Ramrez 1999). However, C. punctatum is not found in Venezuel a. This is clearly a case of misidentification. Orchid Pollination Biology Pollination by animals is an important character istic of the Orchidaceae. Self-compatibility is common in the family; however, many out-crossing strategies have evolved in the Orchidaceae to promote cross-pollination mainly by insects (van der Cingel 1995). The diversity of orchid floral form and pollination syndromes may be attri buted to the evolutionary adaptation of orchid flowers to out-crossing insect pollinators (Jersko v et al. 2006). Pollination syndromes are sets of floral traits which favor attracting a particular type of pollinator (van der Cingel 1995). The study of pollination syndromes is of extreme importance in orchid conservation since cross-pollination can have a direct effect on the fitness of populations (Jerskov et al. 2006). Pollination aids on the long-term survival of most orchid species because the persistence of a population depends on the recruitment of new plants from seed by maintaining genetic diversity (Dixon et al. 2003). In order to adapt and surv ive the constant cha nges in the surrounding environment, sexual reproduction is of extrem e importance (Richards 1997). The subject of orchid pollination has been well studied and much information has been published (Darwin 1885; van der Pijl and Dodson 1966; van der Cingel 1995, 2001). However, in depth analysis of pollination and breeding systems have been stud ied in few orchid species. Comprehensive studies are only available for 15% of North Amer ican orchid species, and general information is presented for 40% of these species (Catling a nd Catling 1991). Although ot her studies regarding 14

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orchid pollination have been published since the Catling and Catling paper, the size of the orchid family dictates the need for more detailed studies. Most animal pollinated plants provide food re wards to their pollinators in exchange for their service. In the Orchidaceae, it is estimated that one third of the species achieve pollination by deception (van der Pijl and Dodson 1966; Dre ssler 1981; Ackerman 1986). There are seven types of reported deception modes in the family: 1) generalized food deception; 2) batesian floral mimicry; 3) brood-site imitati on; 4) shelter imitation; 5) pseudoantagonism; 6) redezvous attraction; and 7) sexual deception. However, generalized food deception is the most common deception mode in the family (Jerskov et al. 2006). Plants using generalized food deception mimic signals of food rewards by falsely advertis ing the presence of food using flower color, shape, and fragrances. Fragrances produced by plants draw pollinators and may function to select the type and number of visitors (Hills et al. 1972). High flor al variation has been associated with deception pollination systems based on generalized food de ception (Little 1983). Ther e are different types of floral fragrances and bees show learning behavior towards certain fragrances. Experienced bees make food choices based on fragrances when compared to inexperienced bees (van der Cingel 1995). It may be advantageous to have high floral variation in deception pollination syndromes because of the learning abilities of insect pollinators (Ackerman et al. 1997). Breeding systems are considered one of the mo st important determinants of plant genetic diversity since they have a significant eff ect on the genetic compos ition of the resultant populations (Hamrick 1982). For example, species that rely on self-fertilization have significantly fewer genotypes than out-crossing species (Brown 1979). Studies describing and evaluating orchid breeding systems are availa ble (Ackerman and Mesl er 1979; Robertson and 15

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Wyatt 1990b; Sipes and Tepedino 1995; Jaim es and Ramrez 1999; Wong and Sun 1999; Lehnebach and Robertson 2004; Gale 2007). These au thors use different va riations of a common method for determining breeding systems (Table 1-1). The pollination treatments used in these experiments were used to evaluate the reprodu ction strategy of the species in question. For example, the removal of pollinia with no further pollination (agamospermy) is used to evaluate asexual seed formation. In this case no pollen tr ansfer is required and the maternal tissue is responsible for generating seeds. Autogamy is defi ned as self-fertilization and this treatment is used to evaluate self-compatibility and the need for a pollinator. Most orchids are selfcompatible, but most species still need pollinators to make the pollen transfer occur (Catling and Catling 1991; Dressler 1993). Catling (1980) reported rain assisted pollination in Liparis loeselii in which water droplets push the anther cap dow nward causing it to stick to the stigma causing pollination to occur. Gale (2007) and Gandawidjaj a and Arditti (1982) also reported autogamy as the breeding system for two orchid species (Gandawidjaja and Arditt i 1982; Gale 2007). Treatments used to elucidate breeding syst ems include artificial and induced xenogamy and are used to assess the need for a pollinator. These treatments not only evaluate if pollen transfer between plants is neces sary for seed production, but also take into consideration the pollen source. In artificial xenogamy, pollen from a different population is us ed in the pollination treatment, therefore allowing evaluation of outbreeding depression (Wong and Sun 1999). If the species is not able to produ ce seeds through agamospermy or spontaneous autogamy, it must depend entirely on a pollinator for seed set. This is of major significance for conservation. If the insect becomes rare or disappears, then the plant species will be directly affected since it will not be able to sexually reproduce a nd consequently, seedling producti on will be negatively affected. 16

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In order to evaluate what pollination treatme nts worked, capsule formation is observed. Most studies consider capsule formation as the final result of the breedi ng system study without taking into consideration result ant seed viability (Wong and S un 1999; Lehnebach and Robertson 2004). Other authors use seed weight as a further measur ement for breeding system determination (Robertson and Wyatt 1990b). Howeve r, this type of measurement can produce uncertain results due to the difference in capsule maturity. Sipes and Te rpentino (1995) reported that fruit weight and fruit size was not correlated with seed number, and instead attempted to use seed number as another measurement for breedin g system (Sipes and Tepedino 1995). However, these authors found no significant differences am ong pollination treatments. Seed viability and germination experiments could be used to assi st in the breeding system determination as different modes of pollination may affect the percentage of viable seeds and their germinability. Lakon (1949) introduced the method of assessing viability by staining seeds with triphenyl tetrazolium chloride (TTC; Lakon 1949). The tetrazo lium seed viability test has been used to assess viability of European and North Ameri can temperate orchid species (Van Waes and Debergh 1986; Lauzer et al. 1994 ; Vujanovic et al. 2000; Bowles et al. 2002) and tropical epiphytic species (Singh 1981) Assessing viability and germinability is crucial for in vitro propagation of orchid species a nd their conservation. It may also be a helpful tool to more precisely determine pollination strategies of threatened and endangered orchid taxa. One of the consequences of the loss of biodi versity is the impact that those endangered organisms can have on the surrounding community, for example, the loss of pollination services. In these cases, other species within the community may suffe r reproductive losses that could ultimately disrupt community function (Kearns et al. 1998). From a conservation perspective, 17

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pollination and breeding system studies should be conducted locally since variations may occur across the geographical range. Orchid Seed Propagation Orchid seeds are unique in being among th e smallest produced by flowering plants (Stoutamire 1964). They are extremely light w ith large air spaces (Arditti and Ghani 2000). These physical adaptations allow orchid seeds to be readily dispersed by wind (Arditti and Ghani 2000). They are often referred to as naked seeds because of the minimal nutrient and carbohydrate reserves and consist of only a proe mbryo surrounded by a seed coat. Since orchid seeds lack endosperm, germination occurs in nature in association w ith a mycorrhizal fungus (Rasmussen 1995). The mycobiont provides the embryo with water, carbohydrates and mineral nutrients (Smith 1966) Asymbiotic Seed Propagation Seeds can also be germinated asymbiotically in vitro, in the absence of a mycobiont, on a defined medium that supplies the embryo all nutrients and carbohydrates that are normally supplied by the fungal partner in situ These media formulations are more complex than symbiotic media in order to fulfill all the nutritional needs of the orchid embryo not supplied by the mycobiont. Asymbiotic seed germination is a very effective method of plant production for conservation since large number of genetically diverse plants can be produced for reintroduction (Stenberg and Kane 1998). From more than 200 years, orchid seed germin ation has been of large interest to botanists, horticulturalists and collectors. In 1802 germination was first described by R. A. Salisbury (Arditti 1984). Asymbiotic seed germination proved to be extremely difficult, despite the many attempts in Europe in the 1800s (Arditti 1984). The method was successfully developed by Lewis Knudson in the U.S. with the development of Knudson B solution (Knudson 1921, 1922) 18

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and the subsequent modifications that pr oduced Knudson C germination medium (Knudson 1946). Since then, Knudsons medium has been modified many times to fit the nutritional requirements of different orchid genera and spec ies. Other defined asymbiotic media that are often used include: Orchid Seed Sowing Medium (P723; Phyto Technology Laboratories LCC, Shawnee Mission, Kansas), Malmgren Modified Terrestrial Orchid Medium (MM; Malmgren 1996), Vacin & Went Modified Orchid Medium (VW; Vacin and Went 1949) and Murashige & Skoog (MS; Murashige and Skoog 1962). Temperate terr estrial orchid species can be difficult to germinate asymbiotically, and may require spec ial media and culture conditions (Arditti 1984). However, Knudson C medium and its modificati ons are suitable to ge rminate seeds of many tropical epiphytic orchid species. Germination media are often modified to enha nce germination, growth and development of certain orchid species and hybrids and many researchers have studied the nutritional requirements that affect asymbiotic germination in vitro (Arditti 1984). The type and concentration of carbohydrate, nitrogen, and minera ls can significantly affect the germination and subsequent growth of orchids in vitro (Harvais 1972, 1973; Thompson 1974; Arditti et al. 1982; Harvais 1982). Asymbiotic propagation techniques have been applied to the conservation of endangered and threatened orchid taxa. Ste nberg and Kane (1998) developed an effective protocol for the asymbiotic production of the epiphytic orchid Prosthechea boothiana var. erythronioides (syn. = Encyclia boothiana ), an epiphytic orchid. Stewart and Kane (2006) developed an asymbiotic germination protocol for Hebanaria macroceratitis a rare terrestrial orch id (Stewart and Kane 2006). Other authors have successfully developed methods for asymbiotic seed propagation for 19

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the purpose of plant conservation (Shimada et al. 2001; Thompson et al. 2001; Light and MacConaill 2003). Symbiotic Seed Propagation The association between orchids and fungi was noticed in the 1800s and the term mycorrhiza arose during that time to describe th e general relationship be tween fungi and plants (Arditti 1984). The fungus supplies the orchid embryo with water, mineral nutrients, and carbohydrates that can be readily absorbed (Ard itti 1967, 1984). Orchids rely on this association in different stages of their life cycle (Dre ssler 1981; Rasmussen 1995). Terrestrial orchids depend heavily on the mycorrhizal association during initial underground (i .e., protocorm phase) seed germination and seedling development (R asmussen 1995). Adult plants may also use the mycorrhizal association concur rent with photosynthesis throughout their adulthood in order to acquire nutrients and water (Zettler 1997). Seeds of epiphytic orchid species may rely on this fungal association to a lesser ex tent, since the habit of grow ing on tree branches and trunks allows them to receive more light to be us ed in photosynthesis (Rasmussen 1995). However, epiphytes may require the mycorrhizal association in times of environmental stress, such as drought, in order to receive wa ter (Stewart personal commun ication). The relationship of epiphytic orchids and their myc obionts is still not fully underst ood. There is a need to isolate mycorrhizal fungi and study this relationship in both germinating seeds and adult plants. This knowledge is extremely important especially when producing plants to be used in reintroduction projects, since these relati onships may be critical for plant establishment. When orchids are produced in vitro using the asymbiotic seed germination procedure, the mycorrizal association is not established in vitro If there is a dependency on these fungi during the adulthood of this species, seedling survival may be negatively affected in the wild if plants are not able to establish this relationship ex vitro It has also been show n for some terrestrial 20

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orchid species, symbiotic seed germin ation results in a higher rate of ex vitro seedling survival, and shorter maturation and flowering time (Dixon a nd Batty 2003). This result may also apply to epiphytes; however, more resear ch is needed in this area. The first step of symbiotic seed germination is to isolate species specific fungal symbionts that are capable of germinating the seed and a ssisting in the growth and development of the seedling. Fungi are isolated from th e host plant roots and maintained in vitro on nutrient media (Stewart and Zettler 2002; Dixon and Batty 200 3). Another approach is to attempt seed germination using different fungi previously isolated from other orchid species (Zettler 1997). However, if the goal is to not only to germinate s eeds, but also to reintroduce seedlings into the wild, it would be preferred that mycorrhizal fungi from the species being studied be isolated and used in symbiotic germination procedures. The media used for symbiotic seed germination are formulated to provide the fungus with the nutrients necessary for growth and development. The fungus then infects the orchid seed and provides the embryo with water and other nutrie nts required for germination. Symbiotic media are relatively simple in comparison to asymbiotic media because they are formulated to support mycobiont growth only. Asymbiotic media, on the ot her hand, have to supply all the organic and mineral requirements for seed germination and seedling development. Five media are commonly used for symbiotic orchid seed germination: oa t meal agar (OMA), H 1, H2, H4, and W2. These media are simple modifications of each other. OMA is the most basic medium and contains pulverized oats, agar, and yeast extract. Other un defined contents that may be used in other media include coconut water and pulverized decay ing wood. The formulations of the other media differ in mineral salt content, carbohy drate source, and undefined additives. 21

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In vitro symbiotic seed germination has been used to propagate temperate terrestrial orchid species. European terrestrial orch ids were successfully propagated using this approach (Clements et al. 1986; Muir 1987; Mitchell 1988). In North America, symbiotic culture of the genera Platanthera, Habenaria, and Spiranthes have received attention (Anderson 1991; Zettler and McInnis 1992, 1993, 1994; Zelmer and Currah 1997; Zettler and Hofer 1997; Takahashi et al. 2000; Zettler et al. 2001; Stewart and Zettler 200 2; Stewart et al. 2003; Zettler et al. 2005; Stewart and Kane 2006). Few epiphytic species have been germinated using symbiotic protocols (Zettler et al. 1998; Zet tler et al. 1999; Markovina and McGe e 2000; Otero et al. 2005; Zettler et al. 2007). Conservation based resear ch with epiphytic species and their mycobionts is of extreme importance. Seedlings generated asymbiotically in vitro for conservation and reintroduction purposes lack mycobionts. If the growing seedling requires the mycobiont to enhanced survival in the wild, symbiotic propagation would be the best way to propagate seed lings for this purpose. The major objectives of this research are: 1) to study the breeding system and pollination biology for C. punctatum and 2) to develop an asymbiotic seed culture protocol for the propagation of the species. 22

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23 Table 1-1. Pollination treatments used to determine orchid breeding systems. A dapted from Wong and Sun (1999) and Stewart (2007) Breeding System Test Flowers Bagged Treatment Pollen Source Control No None Open pollination Agamospermy Yes Emasculate No pollination Spontaneous Autogamy Yes None Same flower Induced Autogamy Yes Emasculate Same flower Artificial Geitonogamy Yes Emasculate Different flower, same plant Artificial Xenogamy Yes Emasculate Different population Induced Xenogamy Yes Emasculate Same population, different plant

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CHAPTER 2 REPRODUCTIVE BIOLOGY OF Cyrtopodium punctatum Introduction Several types of plant ra rity exist, affected by natural processes that affect species density and distribution (Rabinowitz 1981; Fiedler and Ahouse 1992). Species may be considered rare based on habitat specific ity, local population size and/ or si ze of geographical range. However, plants that were once common in their range may be now rare due to anthropogenic influences (Partel et al. 2005). The factors infl uencing rarity need to be consid ered in order for an effective conservation plan to be designed for any target species. Pollination systems are often linked to plant rarity, particularly if pl ants are dependent on insect sp ecies for pollination, seed production and fruit set. Decreased abundance or loss of pollinators often result s in increased rarity of the plant. The Orchidaceae is a very diverse family, and pollination systems may range from being very specific, with a plant being pollinated by only one insect, to very generalized, with a plant being pollinated by many species (van der Pijl and Dodson 1966; Dressler 1981; van der Cingel 1995). A comprehensive investigation of the pollina tion system is a prerequisite to developing a conservation plan for any rare plant species. Cyrtopodium punctatum (L.) Lindl. is an epiphytic orchid native to southern Florida, Cuba, Hispaniola, Puerto Rico, and the northwester n Caribbean coast of South America (RomeroGonzlez and Fernndez-Concha 1999). In Florida, th e species is listed as endangered (Coile and Garland 2003); populations are depleted due to cypress logging and ove r-collecting during the past century. Early accounts in the literature refer to C. punctatum as abundant throughout the south and southwest Florida, especially in the cypress swamps in the Big Cypress region (Ames 1904; Luer 1972). Photographs from the early 1900s show wagons loaded with plants being taken from the wild. Consequently, only a few pl ants still exist in very small populations in 24

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almost inaccessible protected areas such as Everglades National Park, Big Cypress National Preserve, and the Florida Panther National Wildlife Refuge (FPNWR). Over the past 10 years, observations of plant populations in the FPNWR indicated very low capsule formation and seed production in the remaining plants (Larry Richardson personal communication). Additional information regarding the breeding strategies and pollination mechanisms in C. punctatum Florida populations is critical to develop strategies for recovery of the species. Pollination and breeding systems also influence the genetic diversity of populations (Hamrick and Godt 1996). Pollination aids the lo ng-term survival of most species because population persistence is thought to depend on the recruitment of new plants from genetically diverse seeds (Hamrick and Godt 1996; Dixon et al. 2003). Most of the pert inent literature on the pollination of Cyrtopodium punctatum refers to observations c onducted in only one area of the species range (i.e., Puerto Rico; Ackerman 1995). In different parts of its geographical range, a species may possess different breeding strategies which is not uncommon for tropical orchids species in Florida. For example, both Epidendrum nocturnum and Secoila lanceolata produce seed by agamospermy in Florida, but utilize animal cross-pollination in the more southern part of their range (Catling 1987; Brown 2002). Pollinator species may also vary from one location in the geographical range to another. Pollination ecotypes have been report ed by Robertson and Wyatt (1990a) in Plantanthera ciliaris In this study, the pollination ecology of two disjunct populat ions was compared and the primary pollinators were f ound to differ between sites. C. punctatum is reported to be pollinated by Euglossa bees (van der Pijl and Dodson 1966; Jeffr ey et al. 1970; Luer 1972; Dressler 1993). Since Florida lies outside the range of euglossine bees, C. punctatum s pollinator (s) in Florida are different (Chase and H ills 1992). In Puerto Rico, C. punctatum is reported to be pollinated by 25

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Centris or Xylocopa bees (Ackerman 1995). Bees of the genus Centris are reported by Dodson and Adams (in Luer 1972) to visit flowers of C. punctatum in Florida. However, these observations have not been fu lly described or verified. Fragrances produced by plants attract pollinators and may fu nction to select the type and number of visitors (Hills et al 1972). Floral fragrances have been associated with deception pollination systems based on generalized food decepti on (Little 1983) and can be an extra tool in the determination of pollination systems. In many orchid pollination strategy studies, cap sule formation is commonly observed as the final result of the breeding system study wit hout taking into consideration resultant seed viability (Wong and Sun 1999; Lehnebach and Robe rtson 2004). Other researchers have used seed number and weight as a further measuremen t for breeding system determination (Robertson and Wyatt 1990b; Sipes and Tepedino 1995). However, this type of measurement produced uncertain results due to differences in capsule maturity. Sipes and Tepedino (1995) reported that fruit weight and fruit size were not correlated with seed number, and instead used seed number as another measurement for breeding system determination. Since capsules of Cyrtopodium punctatum require approximately 11 months to mature, repeated capsule measurement over the course of capsule developmen t may minimize this limitation. Another tool used to evaluate breeding system s is tetrazolium seed viability testing (TZ) which has been used to assess viability of European and North American temperate orchid species (Van Waes and De bergh 1986; Lauzer et al 1994; Vujanovic et al. 2000; Bowles et al. 2002) and tropical epiphytic species (Singh 1981). Assessing seed vi ability and germinability is crucial for in vitro propagation of orchid species and thei r conservation and may be a helpful tool 26

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to more precisely determine effective pollination strategies of threatened and endangered orchid taxa. As a prerequisite to developing a comprehensive conservation plan for C. punctatum integrated field and laboratory studies were completed with the objectives to: 1) determine the breeding system of C. punctatum through controlled pollination experiments and the effects on both capsule formation and seed viability; 2) determine the pollinator(s) of C. punctatum in Florida; and 3) determine volatile pote ntial attractant compounds present in C. punctatum flowers. Materials and Methods Breeding system determination One population with 15 plants located in the FPNWR (Collie r County, FL) served as the source of plants. Six plants were used for the breeding system experiment during two consecutive years (2006-2007 and 2007-2008). Seven breeding system treatments (Table 2-1) were applied to five flowers of each plant. A total of 420 flowers were used in this study. The methods used to determine the breedi ng system were adapted from Wong and Sun (1999). An extra treatment (induced xenogamy ) was added based on Stewart (2007). The breeding system treatments were as described in Table 2-1. Pollen used in the artificial xenogamy treatment were obtained from plants of from a different population at the FPNWR. Inflorescences were bagged before flowers opened with 95 micron nylon mesh to prevent pollination events prior to initiating the experime nt (Figure 2-1). Plants remained bagged until signs of capsule formation were observed. The percentage of flowers forming capsules was recorded and capsule development was recorded bimonthly for a year by recording capsule length, width, and abortion. Data were analyzed using general linear procedures and WallerDuncan mean separation ( = 0.05) with SAS v 9.1 (2003). 27

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Tetrazolium Seed Viability Test Seed capsules produced from the breeding sy stem study treatments (2006-2007 year) were collected on February 23, 2007 and dried ove r silica desiccant for 70 days at 23 2 C. Seeds harvested from each capsule collected were transf erred to 20 ml scintillation vials. Vials were then maintained in cold storage (-10 2 C). Three 5 mg seed subsamples (75-100 seeds) from each capsule of each treatment were dispensed into a 1.5 ml micro-centrifuge tube an d homogenized with the aid of a vortex shaker. Methods of tetrazolium viability testing follow Lakon (1949). A pretreatment test done prior to experimentation indicated that a 15 min treatment in the seed scarification solution was ideal for C. punctatum seeds. Percent seed viability data were collected for each of the three subsamples per capsule by placing seeds suspended in water droplets into Petri dishes and then scored with the aid of a dissecting microscope. Approxima tely 100 seeds were counted per subsample. Embryos that contained any degree of red or pink coloration were scored as viable while white embryos were scored as non-viable (Figure 2-2, c). Percent viability was calculated for each replicate by dividing the number of stained embryos by the total number of embryos. Data were analyzed using general li near procedures and Walle r-Duncan mean separation ( = 0.05) using SAS v 9.1. Asymbiotic Seed Germination Test Seeds from the capsules produced from each sp ecific breeding system treatment were sampled (5 mg subsamples of 75-100 seeds). Se eds from the same treatment were then pooled and dispensed into 1.5 ml micro-centrifuge tubes. Seeds were surface ster ilized in a solution containing 5 ml ethanol (100%), 5 ml 6.0% (v/v) sodium hypochlor ite, and 90 ml sterile distilled water for 3 minutes, followed by three repetitive 30 sec rinses in sterile distilled water. 28

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P723 Orchid Seed Sowing Medium was prepared from concentrated stock solutions using the formulation developed by Phyto Technology Laboratories, LLC (Shawnee Mission, KS) and adjusted to pH 5.8 with 0.1N KOH prior to autoclaving at 117.7 kPa for 40 min at 121C. Autoclaved medium (ca. 50 ml medium/plate) were dispersed into square 100 X 15 mm Petri plates (Falcon Integrid Petri Dishes, Becton Dickinson Woburn, MA). The bottom of each dish was divided into 36, 13 X 13 mm ce lls (Figure 2-2, d). Only the 16 cells in the middle of the plate were considered for inoculation since the ce lls in the outer edges of the plate were more susceptible to drying. Five of th e 16 interior cells were selected randomly for inoculation using a computerized random number generator. Surface disinfected seeds were inoculated onto the surface of sterile germination medium using a st erile bacterial inoculating loop. Plates were sealed with NescoFilm (Karlan Research Produc ts, Santa Rosa, CA) and incubated under 12/12 h L/D (60 mol m-2 s-1) photoperiod at 25C C. Approximate ly 82 seeds were sown onto each plate (average seeds/plate = 82 av erage seeds per cell = 16.4). Five replicate plates were used for each breeding system treatment. Seed germination and protocorm development stage percentages were recorded weekly for 10 weeks. Seedling de velopment was scored on a scale of 1-5 (Table 2-2; modified from Stewart et al 2003). Germination percentages we re calculated by dividing the number of seeds in each germination and develo pment stage by the total number of viable seeds in the subsample. Data were analyzed using general linear model proc edures and Waller-Duncan at =0.05 with SAS v 9.1. Percent germination count s were arcsine transformed to normalize variation. Pollinator Identification Pollinator observations were conducted dur ing the 2007 and 2008 flowering seasons on a population consisting of 15 plants. Observations took place on plants in the population that had the largest number of inflorescences during bot h flowering seasons (12 and 14 inflorescences in 29

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2007 and 2008 respectively). Observations took place from 7:00 am to 6:00 pm for the first two days (March 13 and 14, 2007). When peak visitati on time was identified, observations took place from 10:00 am to 4:00 pm for the remaining days (March 15, 23-25, 2008). In 2008, observations took place March 18-19 (10:00 am -3:00 pm) and March 26 (11am-3:00 pm). Flower visitors were photographe d, collected and identified, and their behavior in the flowers documented. Flower visitors capable of pollinating the flowers were determined either by direct observation or by detection of po llinaria on their bodies. Insects co llected were deposited at the Florida State Collection of Ar thropods (Gainesville, FL). Fragrance Analysis Two inflorescences from two Cyrtopodium punctatum plants were collected from FPNWR during the 2007 flowering season. Inflorescences were collected still attach ed to the pseudobulbs to preserve the flowers and taken to the USDA Chemistry Resear ch Unit (Gainesville, FL). Inflorescences were placed in a 15cm diameter x 40cm tall glass volatile collection chamber. Volatiles were collected during 3 time periods: 9:00 am to 12:00 am, 12:00 am to 5:00 pm and 5:00 pm to 7:00 am. The volatile traps were ex tracted with 150 l methylene chloride. Gas Chromatography-Mass Spectroscopy (GC/MS) analys es of the collected volatiles were carried out on an HP-6890 gas chromatograph coupled to an HP5973 mass spectrometer. GC/MS peaks of interest identified by comparing their mass spectral data with those in 3 mass spectral libraries. Results Breeding System Determination Capsule formation occurred in 4 out of 7 treatments tested during both 2006-2007 and 2007-2008 seasons (Figure 2-3). There was no evidence that agamospermy, spontaneous autogamy, or open pollination (cont rol) treatments resulted in capsule formation (Figure 2-3). 30

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Induced geitonogamy (pollen from the same flower) and artificial geitonogamy (pollen from the same plant but different flowers) treatments resulted in sign ificantly fewer capsules (14.6 and 27.1% capsule formation respectively) being produ ced than artificial xenogamy (pollen from a different population) and induced xenogamy ( pollen from a different plant in the same population) treatments (48.9 and 73.9% respectively) (Figure 2-3). Capsule widths and lengths were signifi cantly different among pollination treatments (Figures 2-4 and 2-5). During the 2006-2007 growi ng season, the length of capsules formed following induced autogamy was significantly less than those of capsules produced following the other treatments 240 and 300 days post polli nation (Figure 2-4). In the 2007-2008 season, capsule length from capsules formed following both induced autogamy (pollen from the same flower) and artificial geitonogamy treatments (pollen from the same plant, different flower) were significantly less than from capsules formed from other treatments (Figure 2-4). Also in the 2007-2008 season, the width of capsules formed fr om the induced autogamy treatment was significantly less than from all other treatments throughout the year (Figure 2-5). Viability and Asymbiotic Seed Germination Tests We found no differences among treatments fo r either tetrazolium seed viability or asymbiotic seed germination. In general, seed germination was very low. This was unexpected considering the high seed viability observed ac ross treatments that resulted in seed production, particularly induced autogamy (79.9 %), artificial geitonogamy (67.6%), artificial xenogamy (87.2%), and induced xenogamy (79.1%; Table 2-3). Pollinator Identification During the two flowering seasons (2007 and 2008), four bee species were observed visiting the flowers of Cyrtopodium punctatum (Figure 2-6). Apis melifera was the most frequent visitor in 2007 (53 visits in 46.5 hours, Table 2-4). However, Apis melifera and Megachile 31

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xylocopoides were only seen during the 2007 flowering season (Table 2-4). Both Xylocopa micans and X. virginica were frequent visitors of the fl owers during both flowering seasons (Table 2-4). Flowers were observed without pollinia, however no visitors were observed carrying pollen to or from flowers. Fragrance Analysis Nine fragrance compounds were identified as being produced in C. punctatum flowers: benzaldehyde, myrcene, benzyl alcohol, Z--Oc imene, E-Ocimene, 1,3,8-para-menthatriene, methyl salicylate, Indole, and E,Efarnesene. The relative abundance of these compounds was recorded (Figure 2-7). Discussion Breeding System Our results show that Cyrtopodium punctatum is not capable of re producing autogamously and requires a pollinator for capsule set. Gi ven that no capsules were formed from the agamospermy or spontaneous autogamy treatment s implemented in the field, we demonstrate that no spontaneous self-fertilization occurs. Although a few capsule s were formed from induced autogamy (selfing with pollen from the same flower; 17.2%) and artifici al geitonogamy (selfing with pollen from the same plant but a different flower, 27.2%), significantly more capsules were produced following artificial and induced xenoga my (48.9 and 73.9% respectively; Figure 2-3). Species within the Orchidaceae are predominantl y self-compatible; however species normally require an insect vector to facilitate pollen movement (Dressler 1981). This is supported in C. punctatum where spontaneous autogamy treatments re sulted in no seed formation while both induced autogamy and artificial geitonogamy tr eatments, where pollen was manually moved to simulate the action of the pollinator, resulted in capsule formation. However, no capsules were formed in the open-pollinated control. 32

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Other studies with epiphytic orchid species show a degree of self-i ncompatibility and the need for pollinators (Ackerman 1989; Jaimes a nd Ramrez 1999; Borba et al. 2001; Lehnebach and Robertson 2004). Lehnebach and Robertson ( 2004) and Borba et al. (2001) reported that capsules were not produced following agamospe rmy and spontaneous autogamy treatments on the epiphytic orchid species studied, thus also showing dependence on a pollinator. Some degree of self-incompatibility was also f ound for these species (Borba et al 2001; Lehnebach and Robertson 2004). Significantly fewer capsules were formed from artificial selfing than from induced outcrossing. Similarly, some degr ee of self-incompatibility exists in C. punctatum as fewer capsules were formed from selfing than outcrossing treatments. C. punctatum uses deceit using aromatic compounds a nd visual signals to attract insect visitors. Our fragrance analysis and compound id entification show a fl oral bouquet strategy in which a diverse array of attractan t compounds are produced. However, C. punctatum flowers provide no food reward to floral visitors. There are implications of having a deceit pollination strategy. Neiland and Wilcock (1998) found that orchids that offer f ood rewards (e.g., nectar) had double the probability of setting capsule than non-rewarding species across all geographical areas. In North America, the rate of capsule set was 49.3% for species that offer no nectar reward compared to 19.5% for rewarding orchids (Neila nd and Wilcock 1998). A closely related species in Brazil, C. polyphyllum, also uses a deceit pollination system. This plant mimics yellow flowers that occur in the same habitat. This spec ies also has low capsule set due to low pollinator visitation (Pansarin et al. 2008) Pollinator limitation was also observed in other deceptive orchids such as Serapias vomeracea and Pogonia japonica (Matsui et al 2001; Pellegrino et al. 2005). 33

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Historically, populations of C. punctatum in Florida were numerous and plants were abundant. In this situation, a deceit pollination system with low capsule production was viable because the probability of capsule formation was higher due to plant abundance. However, populations are now small and fragmented, thus the deceit pollination system may minimize future reproductive success for the species since the likelihood of capsules being produced is so low. The open pollination treatment resulted in no capsule formation during both the 2006-2007 and 2007-2008 growing seasons in the population st udied. However, 2 capsules were formed on 2 different plants during the 20062007 season in inflorescences not used in the breeding system study, suggesting that natural pol lination does occur, albeit at very low levels. A deceit pollination system alone may not be to cause for the low capsule formation at the FPNWR. A decrease in pollinator populations may also be a ffecting natural capsule setting. A more detailed study on the insect populations of the area shou ld be conducted, especially because of the closeness of the populations to agricultural fields that continually have pe sticide applications. Capsule size measurements (length and wi dth) taken during the 11 months maturation period in both years indicated that induced autoga my and artificial geitogamy treatments resulted in capsules that were relatively smaller than cap sules produced from other treatments (except for capsule length in 2006-2007). Howe ver, capsule size may vary from year to year due to the significant variation in environm ental factors, such as rainfall and temperature, which can influence capsule formation and maturation over time. Our research suggests that breeding system studies should be conducted across multiple growing seasons. Seed Viability and Asymbiotic Germination Tetrazolium seed viability testing showed hi gh seed viability in all breeding system treatments that formed capsules in C. punctatum However, germination was very low across treatments. This may be due to sub-optimal in vitro cultural conditions for asymbiotic 34

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germination. Light may inhibit germination of C. punctatum seeds (Dutra unpublished data). By sowing seeds and initially incubating them in the dark, germination could be significantly improved for this species. Pollinator Identification During the 2007 flowering season, honey bees ( Apis melifera ) were the most common visitors to C. punctatum flowers. However, these bees were too small to remove pollinaria and efficiently act as efficient pollinators. In th e 2008 flowering season honey bees were not seen visiting flowers. Xylocopa bees ( X. micans and X. virginica) were observed visiting the flowers but removal or deposition of pollinia was not obs erved during the field observation period. These bees are large enough to fit inside the flowers a nd efficiently remove pollinia. In Puerto Rico, C. punctatum is reported to be pollinated by Xylocopa bees (Ackerman 1995). Pemberton and Hung (in press) observed two species of Centris bees, visiting the flowers of C. punctatum in southeast Florida. However, these pollinator observations were conducted at Fairchild Tropical Botanical Garden and at Fort Lauderdale, in artificial settings and w ith plants of unknown origins. Observations conducted in a garden setting do not accurately reflect the real ecological links since a mixture of exotic and na tive plants are planted in unnatur al arrangements causing insect pollinator densities to change. Pollination observations conducted in situ are more representative since it is where natural conditions prevail and where future plant reintroductions wi ll take place. Fragrance Analysis The compounds identified in the fragrance analysis of C. punctatum are considered relatively common pollinator attractants produced by many other orchid species (Kaiser 1993). However, two compounds identified, indole and methyl salicylate, can be associated with Euglossini pollination system. Although Florida is located outside the range of Euglossini bees, C. punctatum is reported to be pollinated by Euglossa bees in parts of its range (van der Pijl and 35

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Dodson 1966; Jeffrey et al. 1970; Lu er 1972; Dressler 1993). An in troduced species of Euglossa ( E. viridissima ) has recently naturalized in southern Florida (Skov and Wiley 2005). Pemberton and Wheeler (2006) extracted and identified compounds collected by E. viridissima in southern Florida. Although methyl salicylate and i ndole are found to be collected by many other Euglossini species in the tropics (Ramrez et al. 2002), these compounds were not found in the collection storage organs of E. viridissima in southern Florida (see Pemberton and Wheeler 2006). It is possible that even if E. viridissima was found in the same location as C. punctatum it would not be attracted by the type of com pounds the plant produces thereby not affecting pollination. Conservation Implications Low capsule set may be expected for species with deceit pollination strategies. Indeed, observations made during this study and by biol ogists over the past 10 years at the FPNWR indicate that very few capsules were formed by C. punctatum Pesticide use in nearby agricultural areas is another possible direct cause of low capsule formation by decreasing pollinator populations at the study site. Similarly, habitat degradation may be affecting insect populations. The area has been impacted by drai nage that has shortened the hydroperiod. Local insect population dynamics should be studied for conservation purposes and land management in areas were C. punctatum occurs. Our study indicates that sexual reproduction in C. punctatum is severely depressed, suggesting that recruitment is hi ghly unlikely. Given this, the longterm viability/persistence of the existing population is at ris k. In response to a likely need for a reintroduction program, additional studies to determine the population ge netic diversity and structure of the existing populations and develop efficient asymbiotic germination procedur es for reintroduction are being conducted. While genetic diversity studies ar e being conducted, manual pollination should be 36

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conducted within populations to ensure that seeds are being produced for in situ recruitment and ex situ propagation for future reintroductions. Pemberton and Hung (in press) s uggested that restoration of C. punctatum could be aided by planting of Brysonima lucida a rare species in the Malpighia ceae restricted to southern most Florida which attracts Centris bees. However, B. lucida is not naturally fo und growing in the Big Cypress region where natural populations of C. punctatum are found (Wunderlin and Hansen 2004). We suggest that local recovery plans for the species be developed given the high habitat variation (e.g., between Evergl ades National Park versus FP NWR) and fragmentation found in Florida. Recommendations can not be made solely from relatively few plants in cultivation and should be based on in depth studies that will likely affect the survival of the species in the wild. 37

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Table 2-1. Pollination treatments used to determine orchid breeding systems. Adapted from Wong and Sun (1999) and Stewart (2007) Breeding System Test Flowers Bagged Treatment Pollen Source Control No None Open pollination Agamospermy Yes Emasculate No pollination Spontaneous Autogamy Yes None Same flower Induced Autogamy Yes Emasculate Same flower Artificial Geitonogamy Yes Emasculate Different flower, same plant Artificial Xenogamy Yes Emasculate Different population Induced Xenogamy Yes Emasculate Same population, different plant 38

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Table 2-2. Seed developmental stages of Cyrtopodium punctatum (modified from Stewart et al. 2003) Stage Description 1 Intact testa 2 Embryo enlarged, testa ruptured (= germination) 3 Appearance of protomeristem 4 Emergence of two first leaf primordia 5 Elongation of shoot and further development 39

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Table 2-3. Seed viability, germ ination and development stag e percentages generated from C. punctatum pollination treatments of the 2006-2007 flowering season. Six plants were used and 30 flowers were used per treatment Seedling Development Stages (%)1 Pollination Treatment % Seed Viability Total Germination 1 2 3 4 Control* Agamospermy* Spontaneous Autogamy* Induced autogamy 79.9 ab 2.9 bc 17.9 ab 1.2 b 1.7 a 0.35 a Artificial geitonogamy 67.6 b 1.9 c 18.1 a 1.2 b 0.49 b 0.44 a Artificial xenogamy 87.2 a 4.3 ab 17.6 b 2.7 a 1.9 a 0.55 a Induced xenogamy 79.1 ab 4.7 a 17.5 b 4.0 a 1.1 ab 0.29 a Treatments produced no capsules. 1See Table 2-2 for descriptions of seedling developmental stages. 40

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Table 2-4. Floral visitors of Cyrtopodium punctatum Total number of observation hours for 2007 and 2008 were 46.5 hrs and 14.2 hrs, respectively Insect Visitors 2007 2008 Xylocopa micans 28 17 Xylocopa virginica 15 2 Apis melifera 53 0 Megachile xylocopoides 2 0 41

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Figure 2-1. Breeding system experiment. a) Flower b) Artificial removal of pollinia. c) Capsule measurement. d) Cyrtopodium. punctatum plant with pollination bag in natural habitat at the Florida Panther National Wildlife Refuge (C ollier County, FL). 42

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Figure 2-2. Seed viability and asymbiotic germ ination. a) Germinating seeds (Stage 2). b) Protocorm at Stage 4 of development show ing two leaves. c) Stained and unstained seeds from tetrazolium. d) Falcon Integrid Petri Dish es showing cells. 43

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Figure 2-3. Percent capsule set among seven breeding system tr eatments. Data from 2006 and 2007. Six plants and 30 flowers per treatment were used per year. Percentages sharing the same letter are not significantly different ( =0.05). Control (no treatment, open pollination); agamospermy (emasculated flower, no pollination); spontaneous autogamy (no treatment, pollen from the same flower); induced autogamy (selfing with pollen from the same flower); artificial geitonogamy (selfing with pollen from the same plant, different flower); arti ficial xenogamy (pollen from different population); induced xenogamy (pollen fr om a plant in the same population). 44

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Figure 2-4. Effect of breeding syst em treatment on mean capsule length (+ S.E.). The interaction between 2006-2007 and 2007-2008 floweri ng seasons was significant. 45

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Figure 2-5. Effect of breeding syst em treatment on mean capsule width (+ S.E.). The interaction between 2006-2007 and 2007-2208 floweri ng seasons was significant. 46

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Figure 2-6. Bees observed visiting flowers. a) Apis mellifera (honey bee). b) Xylocopa virginica. c) X. micans (female). d) Megachile xylocopoides.e) X. micans (male). 47

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48 Figure 2-7. Gas chromatogram of the floral fragrance of Cyrtopodium punctatum. Nine compounds were identified as ma jor fragrance components.

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CHAPTER 3 ASYMBIOTIC SEED GERMINAT ION AND IN VITRO SEEDLING DEVELOPMENT OF Cyrtopodium punctatum Introduction The genus Cyrtopodium is comprised of approximate ly 42 species of Neotropical origin that can be found from southern Florid a to Argentina (Batista and Bianchetti 2004; Romero-Gonzlez 1999; Romero-Gonzl ez and Fernndez-Concha 1999). C. polyphyllum and C. punctatum are the only two species f ound in the United States; however, C. polyphyllum has been naturalized (Brown 2005). Cyrtopodium punctatum is found in southern Florida, as well as Cuba, Hispaniola, Puerto Rico, and the northwestern Caribbean coast of South America (Rom ero-Gonzlez and Fernndez-Concha 1999). Very few species in the genus are epiphytic with C. punctatum being one of them. Cyrtopodium punctatum also known as the cigar orch id, is listed as endangered in the state of Florida (Coile and Garland 2003). The species wa s over-collected during the past century and today only a few plants still exist in remote protected areas. Early accounts in the literature refer to C. punctatum as abundant throughout southern Florida, especially in cypress swamps of the Bi g Cypress Basin (Ames 1904; Luer 1972). The remaining plants in Florida are found in sm all populations in protected areas such as Everglades National Park, Big Cypress Na tional Preserve, and the Florida Panther National Wildlife Refuge. Over the past 10 years, careful observations of plant populations in the Florida Panther Wildlife Refuge indicated lim ited seed production in the remaining plants. Pollin ation biology observations of C. punctatum revealed that limited seed production is a consequence of low pollination (Chapter 2). Consequently, the long-term sustainability of rema ining populations is in question. 49

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Asymbiotic seed propagation techniques have been applied to the conservation of endangered and threatened orchid taxa and may be useful in the re-introduction of C punctatum Stenberg and Kane (1998) developed an effective protocol for the asymbiotic production of the epiphytic orchid Prosthechea boothiana var. erythronioides (syn. = Encyclia boothiana ), an epiphytic orchid. Stewar t and Kane (2006) developed an asymbiotic germination protocol for Habenaria macroceratitis a rare terrestrial orchid (Stewart and Kane 2006). Other authors ha ve successfully developed methods for asymbiotic seed propagation for the purpos e of producing material for reintroduction efforts (Dutra et al. 2008; Light and MacCona ill 2003; Shimada et al. 2001; Thompson et al. 2001). The objectives of this study were to: 1) determine the procedures for asymbiotic seed germination of C. punctatum including media selection fo r optimal seed germination and protocorm development and 2) determine the influence of photoperiod on growth and development of C. punctatum seedlings. Materials and Methods Seed Source and Sterilization Procedure Seeds were obtained from a naturally pollin ated capsule collected at the Florida Panther National Wildlife Refuge Unit 51 (Collier Co., Florida) on February 23, 2007. The capsule was dried over silica desiccan t for 70 days at 25C 3C after which dehisced seeds were collected and transferred to a 20 ml scintillation vial and placed in cold storage at -10C (May 4, 2007). Seeds were surface sterilized in a solution containing 5 ml ethanol (100%), 5 ml 6.0% sodium hypochlorite, and 90 ml sterile distilled water for 3 minutes, followed by three repetitive 30 sec rinses in sterile distilled water. 50

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Asymbiotic Seed Germination Five asymbiotic orchid seed germination media (Table 3-1) were examined for their effectiveness in promoting germinati on and subsequent protocorm development of C. punctatum seeds. With the exception of P723 medium, all media were purchased from Phyto Technology Laboratories, LLC (Shawnee Mission, KS). Phyto Technology Orchid Seed Sowing Medium (P723) was prepared usin g concentrated stock solutions according to the Phyto Technology Laboratories formulation. Th e five media screened were: KC (#K400; Knudson 1946), P723, MM (#M551; Ma lmgren 1996), VW (#V895; Vacin and Went 1949) and MS (#M5524; Murashig e and Skoog 1962). Basal media were modified to standardize the concentrations of agar, sucrose, and activated charcoal as follows: 0.8% TC agar was added to KC and MS, 2.0% sucrose was added to MM and MS, 0.1% activated charcoal was added to KC, VW and MS. All media were adjusted to pH 5.8 prior to autoclaving at 117.7 kPa fo r 40 min at 121C. Autoclaved medium (ca. 50 ml medium/plate) were dispersed into square 100 x 15 mm Petri plates (Falcon Integrid Petri Plates, Becton Dickinson W oburn, MA). The bottom of each plate was divided into 36, 13 X 13 mm cells. Only the 16 inte rior cells were used for inoculation to avoid uneven medium drying. Five of the 16 in terior cells were selected randomly for inoculation using a computer ized random number generato r. Surface sterilized seeds were inoculated onto the surface of sterile ge rmination medium using a sterile bacterial inoculating loop. Plates were sealed with one layer of NescoFilm (Karlan Research Products, Santa Rosa, CA) and in cubated under dark (0/24 h L/ D) or light (16/8 h L/D; 60 mol m-2 s-1) photoperiod at 25CC. Approximately 77 seeds were sown into each plate (average seeds/plate = 76.5 average seeds per cell = 15 .3). Eight replicate plates were used for each germination medium per photoperiod treatment. Seed germination and 51

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protocorm development stage percentages were recorded every other week for 10 weeks. Seedling development was scored on a scale of 1-5 (Table 3-2; modifi ed from Stewart et al. 2003). Influence of Photoperiod on Seedling Growth and Development Based on asymbiotic media screen responses, photoperiodic effects on in vitro seedling development were further examined on P723 medium. Follo wing inoculation of surface sterilized seed onto th e germination medium, plates were sealed with Nescofilm and incubated under a 16/8 h, 12/12 h, or 8/16 h L/D photoperiod at 25 C 3 C, following the same methods outlined for the media screening procedures. Approximately 111 seeds were sown into each Falcon Integri d Petri Plate (avera ge seeds/plate = 111.2 average seeds per cell = 22.3). Eight replicat e plates were used for each germination medium per photoperiod treatment. Seed germ ination data and protocorm development stages were measured starting at 2 weeks a nd continuing every other week for a total of 10 weeks. Germination and seedling de velopment were scored as above. After 10 wks, developing seedlings (Stages 4-5) were transferred to Sigma-Aldrich (St. Louis, MO) Phytotra ys with 100 mL P723 medi um for continued seedling development. Phytotrays were sealed with one layer of NescoFilm and returned to the seedlings corresponding photoperiod. Nine seedlings were transferred into each Phytotrays, with 10 Phytotrays per p hotoperiod (90 seedlings per photoperiod; 30 Phytotrays total). Seedlings developed for an additional 15 wks (10 wks in germination + 15 wks in seedling development = 25 total wks) After the total 25 week culture period, fresh and dry weight, leaf length and number, root length and number, and shoot number were recorded. 52

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Greenhouse acclimatization After 35 weeks culture, C punctatum seedlings, previously cultured on P723 medium, were rinsed to remove residual me dium, potted in coconut husk in 38-cell plug trays before being transferred to greenhous e conditions. Coconut husk was used since C punctatum is often found growing on tree bark and stumps. Plug trays were covered with clear vinyl humidity domes to prevent desiccation during early acclimatization and placed under shade (239 mol m-2 s-1) in the greenhouse. Afte r one week, the plastic domes were lifted slightly to lower the relative humidity in each plug tray, and completely removed one week later. After 4 weeks, seedlings were grown under increased light (1025 mol m-2 s-1 ). Seedlings were watered once daily and fertilized weekly with 150 ppm Peters 20-20-20 liqui d fertilizer (The Scotts Company, Marysville, OH). Statistical Analysis Seed germination and development data from the asymbiotic experiment were analyzed using general linear model procedur es and least square means. The percentage of seedlings in each stage was obtained by dividing the number of seeds in each germination and development stage by the total number of viable seeds in the subsample. Percent germination data were arcsine transformed to normalize variation. Germination and seedling development data from the photoperiod experiment were analyzed using general linear mode l procedures and Waller-Duncan at =0.05. SAS v 9.1.3 (SAS, 2003) was used in all data analysis. 53

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Results Asymbiotic Seed Germination Regardless of media, highest germinati on was achieved under complete darkness (0/24 h L/D, Figure 3-1). Germination (Stage 2= testa rupture) was first observed at week 2 under the dark treatment (0/24 h L/D, Figure 3-2). Protocorms produced in both photoperiod treatments were achlorophyllous but became chlorophylous in light (16/8 h L/D) when they developed to Stage 3. Since cultures maintained under dark (0/24 h L/D) were only exposed to light at week 8, the prot ocorms that had reached Stages 3-4 in dark were achlorophyllous with numerous rhizoids (Figure 3-3, b) but became chlorophyllous soon after exposure to light. Immediately after testa rupt ure (Stage 2), a protocorm formed (Stage 3) with the appearance of a protomeristem. Stage 4 protocorms possessed two leaf primordia (Figure 3-4, b) and by Stage 5, shoot elongation commenced (Figure 3-4, c). At Stage 5 roots were also ev ident on some seedlings (Figure 3-4, d). Seeds cultured on P723 and VW media ha d the highest percent germination among all media in light (Figure 3-1; 27.3 and 26.1% respectively) when compared to -MS (12.9 %), KC (10.0 %), and MM (12.5 %). Adva nced stages of development (Stages 4 and 5) were observed in li ght (16/8 h L/D) on seeds cu ltured only on P723, -MS, and VW (Stage 4 only; Figure 3-2). In darkness, seeds cultured on all media achieved high germination (Figure 3-1) a nd also achieved Stage 4 (Figure 3-2, -MS 44.4%, KC 29.3%, MM 37.6%, P723 80.3%, and VW 73.5%). Ho wever, Stage 5 protocorms in darkness were only produced on P723, -MS and VW. Of the media screened, the highest percentages of advanced seedling de velopment stages (Stages 4 and 5; Figure 32) were observed by week 10 on P723 me dium regardless of photoperiods. The 54

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percentage of advanced development seed ling development (Stage 4) on P723 medium was significantly higher in co mplete darkness (Figure 3-5). Role of Photoperiod on Growth and Development Seeds were germinated under three photoperi ods (8/16 h, 12/12 h and 16/8 h L/D) and allowed to develop for 10 weeks. The e ffect of photoperiod on seed germination was statistically significant and was observed as early as at week 2. The percentage of seeds that germinated (Stage 2) under the 8/16 h L/D photoperiod at week 2 (Table 3-3) was statistically higher than under 12/12 h or 16/8 h L/D photoperiods. Total germination under the 8/16 h L/D was significantly higher at all weeks. Advanced stages of seedling development (Stages 4 and 5) were also significantly higher under the 8/16 h L/D photoperiod than in the other treatments rega rdless of week. Stage 4 protocorms were first observed by week 4 and significantly higher percentages were observed at all subsequent weeks than on the 12/12 h L/ D and 16/8 h L/D photope riods (Table 3-3). Stage 5 protocorms were first observed at w eek 10 and also occur in significantly higher percentages under the 8/16 h L/D than under th e other photoperiods. Seedling growth and development was than assessed after an addi tional 15 weeks culture following transfer into Phytotrays culture vessels (Figure 3-4, f). Seedlings cultured under 16/8 h L/D had significantly greater root produc tion, fresh and dry root weight s, and total fresh and dry weights measurements than seedlings cultu red under 8/16 h or 12/12 h L/D photoperiods (Table 3-4). Greenhouse acclimatization After humidity domes were completely re moved after two weeks, seedlings started to loose their leaves; however the original shoots remained alive. By week 3, a new shoot started to form at the base of the old shoot of each seedling and af ter 4 weeks seedlings 55

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had produced new leaves. Seedlings exhibi ted a 90% survival after five weeks acclimatization in the greenhouse. Discussion Low seed production in Cyrtopodium punctatum natural populati ons threatens the long-term sustainability of these populations (Chapter 2). This study indicates that Cyrtopodium punctatum seedlings can be produced in vitro using asymbiotic seed germination techniques. The use of manual po llination to promote seed capsule formation combined with this asymbiotic seed culture protocol and the subse quent re-introduction of seedlings provides a means to increase C. punctatum populations (Chapter 2). Both germination rate and seedling deve lopment were affected by asymbiotic culture media and photoperiod. Although seeds ge rminated on all culture media screened, only P723 medium supported the highest germ ination percentages a nd advanced seedling development (Stages 4 and 5). Possibly peptone in the culture media may have promoted the growth and advanced development of C. punctatum seedlings since P723 medium is the only medium used that contained pept one. A positive effect of peptone on seedling development was documented by Kauth et al. (2006) in Calopogon tuberosus a terrestrial species. While KC suppor ted highest seed germination for C. tuberosus seedlings grown on P723 medium displa yed enhanced seedling development. The promotive effect of darkness on seed germination was remarkable. Epiphytic orchid species are thought to generally germ inate in either light or dark (Arditti 1967; Arditti and Ernst 1984). However, species sp ecific light and dark requirements for germination are often not examined. Our re sults show that highest germination was achieved in dark for this epiphytic species. Interestingly, inhibition of seed germination following light exposure has been demonstrated in many temperate terrestrial orchid 56

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species (Arditti et al. 1981; Ernst 1982; Van Waes and Debergh 1986; Yamazaki and Miyoshi 2006). The genus Cyrtopodium is mostly comprised of terrestrial species, however; a few species in the genus can be found growing epiphytically. C. punctatum is only found growing as an epiphyte on the trunks of trees or on stumps of logged cypress tress exposed to full sun. Although seed germination was improved in dark, subsequent seedling growth and development was enhanced in the 16/8 h L/D photoperiod. Root numb er, root fresh and dry weights, seedling fresh and dry weights were significant ly greater under 16/8 h L/D. In situ C. punctatum forms a root basket that is may be used to collect detritus and for water absorption (Dressler 1981) During the wet summer mont hs in Florida under longer natural photoperiods, C. punctatum plants produce roots. Enhanced root growth under 16/8 h L/D in vitro may reflect a similar plant devel opmental response to environmental conditions occurring in situ. Acclimatized seedlings showed a high surviv al rate after 5 weeks acclimatization to greenhouse conditions, however grea t care should be taken to control insect pests such as scales after acclimatization. C. punctatum seedlings proved highly susceptible to hard scale infestation and monthly pes ticide applications were needed. A reliable asymbiotic seed culture method for the plant conservation of C punctatum has been described. We recommend germinating seeds on P723 under 0/24 h L/D for 8 weeks followed by seedling development under a 16/8 h L/D photoperiod. Seedlings successfully acclimatized to greenhouse conditions can be used for reintroduction and conservation purposes. This should aid in incr easing the long term sustainability of remaining C. punctatum populations. 57

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Table 3-1. Nutrient composition of germination media used for the asymbiotic seed germination of Cyrtopodium punctatum KC Knudson C, MM Malmgren Modified Terrestrial Orchid Medium, P723 Phyto Technology Orchid Seed Sowing Media, MS half-strengt h Murashige & Skoog, VW Vacin & Went Orchid Medium. Modifi ed from Dutra et al. 2008 KC P723 MM VW -MS Macronutrients (mM) . Ammonium 13.82 5.15 7.57 10.31 Calcium 2.12 0.75 0.73 1.93 1.50 Chlorine 3.35 1.50 3.1 Magnesium 1.01 0.62 0.81 1.01 0.75 Nitrate 10.49 9.85 5.19 19.70 Potassium 5.19 5.62 0.55 7.03 10.89 Phosphate 1.84 0.31 1.03 3.77 0.63 Sulfate 8.69 0.71 0.92 8.71 0.86 Sodium 0.10 0.20 0.20 0.10 Micronutrients ( M) . Boron 26.7 50 Cobalt 0.026 0.053 Copper 0.025 0.5 Iron 90 50 100 100 50 Iodine 1.25 2.50 Manganese 30 25 10 30 50 Molybdenum 0.26 0.52 Zinc 9.22 14.95 Organics (mg/l) . D-Biotin 0.05 Casein hydrolysate 400 Folic acid 0.5 L-Glutamine . Glycine 2.0 myo-Inositol 100 100 Nicotinic acid 1.0 Peptone 2000 PyridoxineHCl 1.0 ThiamineHCl 10 Total mineral salt concentration (mM) 46.72 24.72 4.35 35.54 48.01 Total inorganic N (mM) 24.31 15.00 0 12.76 30.01 NH4:NO3 1.32 0.52 0 1.46 0.52 58

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59 Table 3-2. Seedling developmental stages of Cyrtopodium punctatum. Modified from Stewart et al. 2003 Stage Description 1 Intact testa 2 Embryo enlarged, testa ruptured (= germination) 3 Appearance of protomeristem 4 Emergence of two first leaf primordia 5 Elongation of shoot and further development

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Table 3-3. Photoperiodic effects on in vitro seed germination and prot ocorm development of Cyrtopodium punctatum over 10 weeks culture on P723 medium Photoperiod Week Stage TG* 1 2 3 4 5 8/16 h L/D 2 93.7b 6.1a 0.0a 0.0 0.0 6.1a 4 76.36c 17.8a 2.8a 3.1a 0.0 23.6a 6 70.6b 15.9a 5.8a 7.8a 0.0 29.4a 8 63.8b 10.0a 14.5a 11.6a 0.0 36.2a 10 62.7b 4.3b 16.5a 14.9a 1.7a 37.3a 12/12 h L/D 2 97.6a 2.8b 0.0a 0.0 0.0 2.8b 4 86.3a 11.9b 1.4b 0.5b 0.0 13.7b 6 79.5a 16.0a 3.4b 2.5b 0.0 20.5b 8 72.9a 10.6a 11.3a 5.1b 0.0 27.0b 10 71.1a 7.9ab 15.1a 5.8b 0.2b 28.9b 16/8 h L/D 2 97.2a 2.4b 0.14a 0.0 0.0 2.4b 4 81.5b 17.1a 1.2b 0.2b 0.0 18.5c 6 75.7a 18.8a 2.0b 2.1b 0.0 24.3b 8 71.1a 11.1a 13.3a 4.5b 0.0 28.9b 10 69.9a 6.8a 17.7a 5.6b 0.0b 30.0b 60 Measurements with the same letter within the same stage and week are not significantly different at = 0.05. Numbers 1-5= stages of development. TG= total germination (stages 2-5).

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61Table 3-4. Photoperiodic effect s on seedling development of Cyrtopodium punctatum after 25 weeks culture on P723 medium Photoperiod Shoot # Leaf # Shoot length (mm) Leaf Width (mm) Root # Root length (mm) Fresh wt (mg) Fresh shoot wt (mg) Fresh root wt (mg) Dry wt (mg) Dry shoot wt (mg) Dry root wt (mg) 8/16 h L/D 1.06b 4.51a 61.99a 2.26a 3.47b 67.19b 21.25b 7.29a 13.96b 2.16b 0.73a 1.39b 12/12 h L/D 1.23a 4.49a 59.31a 2.25a 3.63b 78.86a 22.31b 7.26a 15.06b 2.42b 0.72a 1.52b 16/8 h L/D 1.28a 4.33a 69.86a 2.15a 4.45a 87.07a 28.98a 9.26a 19.71a 3.31a 0.91a 1.97a Seeds were germinated in square Petri pl ates and seedlings transferred after 10 weeks to Sigma-Aldrich Culture Boxes for an additional 15 weeks. Measurements represent the mean of 90 seedlings per treatment. Measurements with the same letter are not significantly differen t at = 0.05.

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Figure 3-1. Comparative effect s of culture media and photoperiod on germination of C. punctatum seeds after ten weeks asymbiotic culture. Histobars with the same letter are not significantly different ( =0.05). KC Knudson C, MM Malmgren Modified Terrestrial Orchid Medium, P723 Phyt oTechnology Orchid Seed Sowing Medium, MS half-strength Murashige & Skoog, VW Vacin & Went Orchid Medium. 62

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Figure 3-2. Comparative effect s of culture media and photop eriod on in vitro seedling development of C. punctatum after ten weeks asymbiotic culture. Histobars with the same letter within each seedling st age are not significantly different ( =0.05). KC Knudson C, MM Malmgren Modified Te rrestrial Orchid Medium, P723 PhytoTechnology Orchid Seed Sowing Medium, MS half-strength Murashige & Skoog, VW Vacin & Went Orchid Medium. 63

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Figure 3-3. Photoperiodic effect on germinati on and protocorm development of C. punctatum after 8 weeks culture on P723 medium. a) Seeds cultured in 16/8 h L/D photoperiod. b) Seeds cultured in 0/24 h L/D photoperiod. Scale bars= 0.5cm. 64

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Figure 3-4. Protocorm and progression of seedli ng development of C. punctatum cultured on P723 medium. a) Stage 1 seed (intact test a) and Stage 2 prot ocorms (tr= testa ruptured). b) Stage 3 protocorm (pm=protom eristem) and Stage 4 protocorm (lp=leaf primordia). c) Stage 5 protocorm (elonga tion of shoot). d) Stage 5 protocorms (r=root). e) Seedling with expanded leaves (el). f) Seedlings at 25 weeks culture. Scale bars= 0.5cm (a-f). g) Seedlings after one week acclim atization in the greenhouse. Scale bar= 5cm. 65

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66 Figure 3-5. In vitro seedling development stages of C. punctatum seeds cultured in P723 medium under 0/24 h and 16/8 h L/D. Histobars in e ach stage with the same letter are not significantly different ( =0.05).

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CHAPTER 4 SUMMARY Given the continued trends in th e loss of global biodiversity, it is of extreme importance to develop plans to conserve, restore, and protec t the remaining habitats and living organisms. Florida is one of the most spec ies-rich states in the United St ates; however, it also ranks second in the nation behind California for numbers of e ndangered species. Accordi ng to the US Fish and Wildlife Service, there are 70 endangered species in Florida. For effective rare plant species conservation to take occur, it is essential to determine the types of biological information required to select and implement effective cons ervation strategies that are appropriate for a species. As a result of the extremely limited distri bution, lack of seed production and endangered status of C punctatum in Florida populations, integrated field and laboratory experiments were completed to elucidate the reproductive biology, including pollination mechanism, capsule set, resultant seed viability and germination (Chapter 2). In addition, an efficient in vitro seed culture protocol was developed for production of generically diverse C punctatum seedlings (Chapter 3). The data collected provides cr itical baseline information and a propagation protocol critical to formulate an effective conservation plan for C. punctatum Results from breeding system studies can help to elucidate what strategies are necessary to assist rare plant populations. Reproductive biol ogy studies have been used in developing recovery plans for rare plants such as Ziziphus celata (Weekley and Race 2001) and Spiranthes diluvialis (Sipes and Tepedino 1995). Results revealed that depressed sexual reproduction in C. punctatum is probably due to the lack of insect pollinators and/or visitation. Floral fragrance analysis suggest that low capsule set may also be attributed to the presence of a relic deceit pollination strategy in C. punctatum that was once effective for capsule formation when both 67

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plant populations and densities we re significantly larger in the past. However, the reliance on a deceit pollination strategy in the small and fragmented populations that now exist may minimize future reproductive success for the species since the likelihood of seed capsule formation is so low. Other factors that may be affecting pollina tion in the study area are of anthropogenic origin. Pesticide use in nearby agricultural areas may be the cause of low capsule formation by decreasing pollinator populations in nearby ar eas. Similarly, habitat degradation may be affecting insect populations. The area has been impacted by drainage th at has shortened the hydroperiod. Local insect population dynamics shoul d be studied for conservation purposes and land management in areas were C. punctatum occurs, especially regard ing carpenter bees biology ( Xylocopa virginica and X. micans ). Regardless of the causes for the loss of pollinators and/or reduced visitation, these results indicate that recruitment is highly un likely, and as such, the long-term viability/persistence of the existing C. punctatum populations is at risk. A reliable asymbiotic seed culture method for the plant conservation of C punctatum was described (Chapter 3). Seedlings successfully ac climatized to greenhouse conditions can be used for reintroduction and conservati on purposes. The availability of genetically diverse seedlings should aid in increasing the long te rm sustainability of remaining C. punctatum populations. However, before plant reintroductions occur, a detailed population genetic analysis should be conducted of the remaining populations to assu re that the introduc tion of inappropriate genotypes/ecotypes will not furthe r compromise the sustainab ility of these populations. A genetic analysis of the populati on genetic diversity and structure will be completed to further strengthen conservation protocol s developed for the species. 68

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69 One approach to increase genetic diversity in populations comprised of limited genotypes has been to generate and reintroduce new ge notypes by cross pollinating between populations. Until an assessment of the genetic diversity within and between the existing C. punctatum populations can be completed, prio rity should be given to using seedlings for introduction that are produced in vitro via seed culture and are derived from seed generated from individual populations by natural open pollination. Seedlings should only be reintroduced back into donor populations. Furthermore, since capsule formation via open pollination is very limited, but seeds within each capsule are numerous it is recommended that whol e capsules not be collected but rather the capsules should be left on the plants and only some seeds harv ested for seed culture. Remaining seeds should be left in the capsule to allow natural seedling recruitment to take place within populations. If natural capsule formati on does not occur, hand pollination crosses should be conducted. Over collection of C punctatum for commercial purposes and by hobbyists has historically had a negative impact on natural populations. It is not clear whether th e capacity to propagate large numbers of plants using this seed culture protocol will mitigate future demand for field collection of mature flowering specimens. Th e species requires up to 15 years to attain reproductive maturity when propagated from seed. Such a prolonged delay may limit the desirability to commercially produce this sp ecies via seed propagation. Consequently, development of culture practices that decrease the time to maturi ty of seed propagated plants may be prove beneficial.

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LIST OF REFERENCES Ackerman JD (1986) Mechanisms and evolution of food-deceptive pollination systems in orchids. Lindleyana 1:108-113 Ackerman JD (1989) Limitations to Sexual Reproduction in Encyclia krugii (Orchidaceae). Syst Bot 14:101-109 Ackerman JD (1995) An Orchid Flora of Puerto Rico and the Virgin Islands. The New York Botanical Garden, Bronx Ackerman JD, Mesler MR (1979) Pollination biology of Listera cordata (Orchidaceae). Am J Bot 66:820-824 Ackerman JD, Melendez-Ackerman EJ, Salguero -Faria J (1997) Variation in pollinator abundance and selection on fragrance phenotype s in an epiphytic orchid. Am J Bot 84:1383-1383 Ames O (1904) A Contribution to Our Knowledge of the Orchid Flora of Southern Florida. Contributions from the Ames Botanical La boratory I. E. W. Wheeler, Cambridge Anderson AB (1991) Symbiotic and asym biotic germination and growth of Spiranthes magnicamporum (Orchidaceae). Lindleyana 6:183-186 Arditti J (1967) Factors aff ecting the germination of orchid seeds. Bot Rev 33:1-197 Arditti J, Ernst R (1984) Physiology of germinat ing orchid seeds. In: Arditti J (ed) Orchid Biology: Reviews and Perspectives III. Co rnell University Press, Ithaca, pp 177-222 Arditti J, Ghani AKA (2000) Tansley Review N o. 110. Numerical and physical properties of orchid seeds and their biological im plications. New Phytologist 145:367-421 Arditti J, Michaud JD, Oliva AP (1981) Seed germination of North American orchids I: California and related species of Calypso, Epipactis Goodyera Piperia and Platanthera Bot Gaz 142:442-453 Arditti J, Clements G, Fast G, Hadley G, Nish imura G, Ernst R (1982) Orchid seed germination and seedling culture. In: Ard itti J (ed). Orchid Biology Revi ews and Perspectives. Cornell University Press, Ithaca, pp 243-370 Batista JAN, Bianchetti LB (2004) Three new taxa in Cyrtopodium (Orchidaceae) from central and southeastern Brazil. Brittonia 56: 260-274 Borba EL, Semir J, Shepherd GJ (2001) Self-i ncompatibility, inbreeding depression and crossing potential in five Brazilian Pleurothal lis (Orchidaceae) species. Ann Bot 88:89-99 70

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Thompson PA (1974) Orchids from seed: A new basal medium. Orchid Review 82:179-183 Thompson DI, Edwards TJ, van Staden J (2001) In vitro germination of several South African summer rainfall Disa (Orchidaceae) species: Is seed te sta structure a f unction of habitat and a determinant of germinability? Syst Geogr Pl 71: 597-606 U.S. Fish and Wildlife Services, USFWS (2005) Florida Panther National Wildlife Refuge. Available at http://www.fws.gov/floridapanth er/brochure/Florida_Panther_Brochure.pdf Accessed 22 Feb 2008 Vacin EF, Went FW (1949) Some pH change s in nutrient solutions. Bot Gaz 110: 605-613 van der Cingel NA (1995) An Atlas of Orchid Pollination: European Orchids. Balkema Publishers, Rotherdam van der Cingel NA (2001) An Atlas of Orchid Pollin ation: America, Africa, Asia and Australia. Balkema Publishers, Rotherdam van der Pijl L, Dodson CH (1966) Orchid Flowers: Their Pollination and Evolution. University of Miami Press, Coral Gables Van Waes JM, Debergh PC (1986) In vitro germination of some Western European orchids. Physiol Plant 67:253-261 Vujanovic V, St-Arnaud M, Barabe D, Thibeault G (2000) Viability testing of orchid seed and the promotion of colouration and germination. Ann Bot 86:79-86 Wong Wong KC, Sun M (1999) Reproductive biology and conservation genetics of Goodyera procera (Orchidaceae). Am J Bot 86:1406-1413 Weekley CW, Race T (2001) The breeding system of Ziziphus celata Judd and D. W. Hall (Rhamnaceae), a rare endemic plant of the Lake Wales Ridge, Florida, USA: implications for recovery. Biol Con 100: 207-213 Wunderlin RP, Hansen BF (2004) Atlas of Florida Vascular Plants (http: //www. plantatlas. usf. edu/). Institute for Systematic Botany. University of South Florida, Tampa Yamazaki J, Miyoshi K (2006) In vitro asymbiotic germination of immature seed and formation of protocorm by Cephalanthera falcata (Orchidaceae). Ann Bot 98: 1197-1206 Zelmer CD, Currah RS (1997) Symbiotic germination of Spiranthes lacera (Orchidaceae) with a naturally occurring endophyt e. Lindleyana 12:142-148 Zettler LW (1997) Terrestrial orchid conserva tion by symbiotic seed germination: techniques and perspectives. Selbyana 18:188-194 Zettler LW, Hofer CJ (1997) Sensitivity of Spiranthes odorata seeds to light during in vitro symbiotic seed germination. Lindleyana 12:26-29 76

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77 Zettler LW, McInnis TM (1992) Propagation of Platanthera integrilabia (Correll) Luer, an endangered terrestrial orchid, through symbiotic seed germin ation. Lindleyana 7: 154-161 Zettler LW, McInnis TM (1993) Symbiotic seed germination and development of Spiranthes cernua and Goodyera pubescens (Orchidaceae: Spiranthoideae). Lindleyana 8:155-162 Zettler LW, McInnis TM (1994) Light enhan cement of symbiotic seed germination and development of an endangered terrestrial orchid ( Platanthera integrilabia ). Plant Sci 102:133-138 Zettler LW, Burkhead JC, Marshall JA ( 1999) Use of a mycorrh izal fungus from Epidendrum conopseum to germinate seed of Encyclia tampensis in vitro Lindleyana 14:102-105 Zettler LW, Delaney TW, Sunley JA (1998) Seed propagation of the epiphy tic green-fly orchid, Epidendrum conopseum R. Brown, using its endophytic fungus. Selbyana 19:249-253 Zettler LW, Piskin KA, Stewart SL, Hartsock JJ, Bowles ML, Bell TJ (2005) Protocorm mycobionts of the Federally threaten ed eastern prairie fringed orchid, Platanthera leucophaea (Nutt.) Lindley, and a technique to pr ompt leaf elongation in seedlings. Stud Myco 53:163-171 Zettler LW, Poulter SB, McDonald KI, Stewart SL (2007) Conser vation-driven propagation of an epiphytic orchid (Epidendrum nocturnum ) with a mycorrhizal fungus. HortSci 42:135139 Zettler LW, Stewart SL, Bowles ML, Jacobs KA (2001) Mycorrhizal fungi and cold-assisted symbiotic germination of the Federally th reatened eastern prairie fringed orchid, Platanthera leucophaea (Nuttall) Lindley. Am Midl Nat 145:168-175

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BIOGRAPHICAL SKETCH Daniela Dutra research interest started during her undergraduate work at the Harriet Wilkes Honors College (Florida Atlantic University). She studied ants in myrmecophytic orchids in Trinidad under the supervision of Dr. James Wetterer. After graduation in December of 2005, Daniela continued to pursue her interest in pl ant conservation while at graduate school in the Environmental Horticulture Depa rtment, University of Florida. Since graduation in August of 2008, with a Master of Science in horticultural sciences, Daniela is pursuing a doctoral degree in Botany at the University of Hawaii. 78