Source Population, Introduction History, and Genetic Diversity in the Pantropical Yam Dioscorea bulbifera L.

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Source Population, Introduction History, and Genetic Diversity in the Pantropical Yam Dioscorea bulbifera L. An Invasive Vine in Florida
Croxton, Matthew
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[Gainesville, Fla.]
University of Florida
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1 online resource (46 p.)

Thesis/Dissertation Information

Master's ( M.S.)
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University of Florida
Degree Disciplines:
Forest Resources and Conservation
Committee Chair:
Andreu, Michael Gardner
Committee Members:
Overholt, William A.
Smith, Jason A.
Williams, Dean
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Chloroplast DNA ( jstor )
DNA ( jstor )
Ecology ( jstor )
Genetic diversity ( jstor )
Genotypes ( jstor )
Haplotypes ( jstor )
Population genetics ( jstor )
Species ( jstor )
Vines ( jstor )
Yams ( jstor )
Forest Resources and Conservation -- Dissertations, Academic -- UF
biocontrol, bulbil, clonal, dioscoreaceae, florida, genetics, invasive, pantropical, vine, yam
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Forest Resources and Conservation thesis, M.S.


The air potato vine, Dioscorea bulbifera L., was imported into Florida from a number of locations in the tropics near the beginning of the 20th century and became recognized as a pest weed within a short time. Established control methods (chemical treatment and manual removal) are expensive, and herbicides may have damaging off-target effects. Because it has a variable phenotype and high genetic diversity, analyses using plastid and genomic markers were conducted in order to determine the source population of the vine. Locating the source population is the accepted method for identifying the most closely co-adapted herbivores of the host plant, and may be essential to the implementation of a host-specific biological control strategy for this species. I found no genetic diversity of D. bulbifera in Florida, affirming the clonal reproduction of the species in Florida through propagation from aerial bulbils, and suggesting that the source population may be localized to a relatively small geographic region once the source genotype is identified from the native range. Results from the source population study are emphatic that invasive D. bulbifera in Florida is not African in origin, and host specific biocontrols to the invasive are likely to be discovered from Southeast Asia or Oceania. ( en )
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Thesis (M.S.)--University of Florida, 2009.
Adviser: Andreu, Michael Gardner.
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by Matthew Croxton.

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2 2009 Matthew David Croxton


3 To my mom and dad--you chose me and have always believed in me.


4 ACKNOWLEDGMENTS For funding, I wish to acknowledge the Florida Departm ent of Environmental Protection and William Haller University of Florida (UF) Institute of Food and Agricultural Sciences (IFAS) Center for Aquatic and Invasive Plants. For their advisement, I wish to thank Michael Andreu (UF), Dean Williams (Texas Christian University), William Overholt (UF), and Jason Smith (UF). Their input, enthusiasm, and encourag ement have truly made this work possible. For laboratory use and assistance I wish to thank Dean Williams (TCU), Jason Smith (Forest Pathology), Claire Anderson for assistance with PAL primer design, the UF Forest Genetics Group, Natalia Peres, Aliya Momotaz; the UF Interdisciplinary Center for Biotechnology Research (ICBR) for DNA sequenc ing and fragment analysis services, and Texas Christian University (TCU) for DNA sequencing and fragment analysis. For collection of plant material that made this study possible I wish to thank th e following individuals, listed by the location of their contribution: Benin: Georg Goergen; Bu rundi: William Overholt; China: Fangyuan Hua, Xiaolan Wang, Huiping Chon; Ghana: Brandford Mochiah; India: Abhishek Mukherjee; Madagascar: Rasabotsy Franois (via Joseph and Frances Bachman) Togo: Georg Goergen; Uganda: William Overholt; United States: Puerto Rico: Lauren Raz; Florida: John Pipoly, Audrey Norman, David and Linda Croxton, Eleanor Foerste, Blo ssom Williams, Maria Couper, Dale Watson, Vicki Johnson, Guy Williams, Ra lph Mitchell, Christ ine Kelly-Begazo, Pat Beckwith, Larry Figart, Larry Markle, Barbra Gardener, Fred Koonce, Joan Bradshaw, Lois McNamara, James Mallory, Ann Berendzen, Cynthia Sterchele, Judi DeLisle, Jerome Carris, Alice Mikkleson, Edmund Thralls, Daniel Culber t, Rodrigo Diaz, Willia m Overholt, Michael Andreu, Dean Williams, and Kim Gabel.


5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES ...........................................................................................................................6LIST OF FIGURES .........................................................................................................................7ABSTRACT ...................................................................................................................... ...............8CHAPTER 1 INTRODUCTION .................................................................................................................. ..9Invasion Biology of Study Species ...........................................................................................9Management, Identification, and Invasive Ecology ...............................................................11Taxonomy and Distribution ....................................................................................................13Introduction History .......................................................................................................... ......14Previous Work ........................................................................................................................162 METHODS ....................................................................................................................... ......19Collection .................................................................................................................... ............19DNA Extraction ................................................................................................................ ......19Chloroplast DNA Sequencing ................................................................................................ 20Microsatellite Analysis ...........................................................................................................213 RESULTS ....................................................................................................................... ........22Sampling ...................................................................................................................... ...........22Chloroplast DNA Analysis ..................................................................................................... 22Microsatellite Analysis ...........................................................................................................234 DISCUSSION AND FUTURE WORK ................................................................................. 28Interpretation of Results .........................................................................................................28Varietal Classification in D. bulbifera L. ...............................................................................29Biocontrol Implications ..........................................................................................................31Future Needs and Concluding Thoughts ................................................................................ 32APPENDIX SUPPLEMENTAL DATA .................................................................................... 34REFERENCES .................................................................................................................... ..........41BIOGRAPHICAL SKETCH .........................................................................................................46


6 LIST OF TABLES Table page 1-1 Introductions of D. bulbifera to Florida and adjacent areas: 1500s to 1919... ...................183-1 Chloroplast DNA haplotypes. ............................................................................................ 243-2 Microsatellite genotypes of D bulbifera at loci Da1F08 and Da1A01. ............................27A-1 Accession Data and Summary of Results.. ........................................................................ 35


7 LIST OF FIGURES Figure page 3-1 Map of 99 D. bulbifera L. accessio ns in Florida, USA ..................................................... 253-2 Parsimonious haplotype network from chloroplast DNA alignments ............................... 263-3 Neighbor-joining tree of ch loroplast DNA alignments ..................................................... 263-4 Principal coordinate analysis of codominant microsatellite markers Da1A01 and Da1F08 ..............................................................................................................................27A-1 Morphological classification of va rieties and geographic locations in D. bulbifera L. .....40


8 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 of Science SOURCE POPULATION, INTRODUCTION HI STORY, AND GENETIC DIVERSITY IN THE PANTROPICAL YAM Dioscorea bulbifera L.: AN INVASIVE VINE IN FLORIDA By Matthew David Croxton May 2009 Chair: Michael G. Andreu Major: Forest Resources and Conservation The air potato vine, Dioscorea bulbifera L., was imported into Florida from a number of locations in the tropics n ear the beginning of the 20th century and became recognized as a pest weed within a short time. Established control methods (chemical treatment and manual removal) are expensive, and herbicides may have damaging off-target effects. Because it has a variable phenotype and high genetic diversity, analyses using plastid and genomic markers were conducted in order to determine the source population of the vine. Locating the source population is the accepted method fo r identifying the most closely co-adapted herbivores of the host plant, and may be essentia l to the implementation of a ho st-specific biological control strategy for this species. I found no genetic diversity of D. bulbifera in Florida, affirming th e clonal reproduction of the species in Florida through propagation from aerial bulbils, and suggesting that the source population may be localized to a relatively sma ll geographic region once the source genotype is identified from the native range. Results fr om the source population study are emphatic that invasive D. bulbifera in Florida is not African in origin, and host specific biocontrols to the invasive are likely to be discovere d from Southeast Asia or Oceania.


9 CHAPTER 1 INTRODUCTION Invasion Biology of Study Species Air potato, Dioscorea bulbifera L. is a twining, monocotyledonous vine that belongs to the taxonom ically complex yam family, the Dioscoreaceae. Both conventional and molecular treatments of this species have ascribed or applied numerous varietal designations for this pantropical plant. In Florida, USA, D. bulbifera is considered a Category I exotic pest plant (FLEPPC, 2007). While this designa tion is given due to demonstrat ed ecological impact, rather than economic impact or geographical extent, others have also established that it is both widespread and costly to manage. Expense of manual removal and chemical control, along with significant off-target effects (especia lly of the latter), have galvani zed research efforts to seek out host-specific biocontrols of this dioecious vine from its indigenous range in the tropics of the Old World (Wheeler et al. 2007). I present the results of gene tic analyses undertaken to determine the source population and genetic diversity in invasive Florida D. bulbifera A key weed risk assessment that has been adapted to Florida unequivocally identifies D. bulbifera post hoc as a risk for becoming a major invader (Gordon et al. 2008). Florida and Alabama are states where it is formally recogniz ed as a noxious weed with significant ecological impact, but it is also reported in the United St ates from Puerto Rico, and from the states of Hawaii (Hawaii, Maui, Molokai, Oahu, and Kauai Islands), Texas, Louisiana, Georgia, and Mississippi (Wester 1992; Zomlefer et al. 2008; USDA, Natural Re sources Conservation Service, 2009). It is also present (sometimes in cultivation) in the West Indies and Central America, but there is little data to verify eco logical or economic impacts in those regions (AlShehbaz & Schubert, 1989).


10 Invasive plant species can exert significant negative effects on ecosystems where they encroach, translating into costly management expens es incurred in the cour se of control activities (Pimentel et al. 2005). If left unchecked, invasive plants may change the trajectory of natural communities so significantly that native species are unable to maintain established functional roles, and are endangered with population declines if unable to adapt rapidly to new roles. Impacts of invasive species introductions often include: decrease in long-term biodiversity of native species, landscape fragmentation, abiotic m odifications to soil moisture and chemistry, changes in fire regime, light availability, disp lacement of native species, vectoring pathogens of economically important species, and conversion of diverse habitats into monocultures. Anthropogenic, long distance dispersal is frequently cited as the primary mode for invasive plant introductions. Often, following anthropogenic in troduction, invasive plants do not initially exhibit weedy tendencies. This 'lag phase' is long and well described for some invasive taxa, but may be very short in other cases. Two prevalent hypotheses are often used to expl ain the success of inva sive species outside their native range: enemy release and evolution of increased competitive ability (EICA). The first asserts that invasive plants thrive when introduced into new environments because reduced selective pressure from natural enemies allows them to thri ve (Liu & Stilling, 32006), while the second states that invasive species may evolve upon introduction to a novel environment in ways that allow them to better compete agains t native species (Blossey & Notzold, 1995). Classical weed biocontrol is premised upon in troducing a plant's na tural enemy into the invasive range where the plant lacks such natu ral enemies. Lack of natural enemies in the introduced range illustrates the importance of host specificity in preventing unchecked proliferation. Because no herbivore is adapted to the abundant resource, the plant continues to


11 spread, maximizing the amount of resources it can allocate to reproduction, dispersal, and growth, within the limits of the plant's vegeta tive architecture. Host specific natural enemies have maximized their success in phytophagy by adapting to their hos t over a significant time long enough to co-evolve with th e plant taxon and to diverge in concert with changes to the original host. When allopatry generates many unique host/herbivore relationships across a large geographic area, then locating the correct herbivore most closely co-evolved to the invasive is greatly enhanced by identifying the source population. Host sp ecificity is the primary qualification for potential biocontr ol agents, especially when nativ e, non-target taxa are closely related to the invasive, and could be impacted by host switching of the agent (Goolsby et al. 2006). Management, Identification, and Invasive Ecology The costs of m anaging D. bulbifera can be high; in one example using multiple herbicides to treat an area affected by ai r potato and other invasive plants, more than $1700 per ha/year is the expenditure recorded (Wheeler et al. 2007). Existing management activities include chemical control and manual removal programs (S tern & DiMarco, 2002). These activities are not sufficient or cost-effective enough to give land managers the desired management impact, and exert significant off-target effects on native vegetation. Air potato is readily identified in Florida by si mple, alternate, cordate, net-veined leaves, sinistrorse twining habit, and namesake aerial bu lbils. Only pistillate flowers, and just one observation of fruiting in Florida have been documented for this dioecious plant, suggesting the potential for rare sexual reproduction of the vine (Hammer, 1998). Hermaphrodite flowers were reported from D. bulbifera var. sativa a variety of air potato prev alent in Oceania (Prain & Burkill, 1936), but no hermaphrodite flowers have been reported from Florida. Vegetative propagation from aerial bulbils is the only mode of reproduction that can be readily verified for


12 invasive D. bulbifera and establishes the possibility that the entire Florida population may be clonal. However, two bulbil morphologies can be found in Florida: a smooth skinned bulbil having gilvous (faintly yellowish when viewed in bright light) periderm, and a warty, rough skinned bulbil with coffee-colored periderm. It is not established whether their presence indicates two or more genetic ally distinct varieties. In addition to disturbed areas, air potato invades a variety of natural areas in Florida, including pinelands, mesic hardwood hammocks, and occasionally intruding into xeric uplands (Hutchinson & Menges, 2006; Morisawa 1999). Although not salt tolerant, D. bulbifera can be found in the Florida Keys, as well as other lo cations closely abutting the coastline. The monocotyledonous stem of D. bulbifera has a near-obligate associa tion with upright vegetation, on which it relies for support, protection from desi ccation, and access to enriched organic soils. It rapidly grows to the tops of tree canopies and fo rms a vine mat that weighs down and shades out native vegetation during the growing season. Recr uitment and maintenance of native, latesuccessional growth is decreased in areas that are overtopped and shaded out by D. bulbifera with a preliminary study suggesting that this leads to decreases in canopy height diversity (Odom et al. 2008) and changes in function of the plant community. Once ai r potato invades an area, it is difficult to eliminate due to the prolific produ ction of persistent aeri al bulbils. In addition, invasiveness of D. bulbifera is facilitated by canopy disturba nce events such as hurricanes (Horvitz et al. 1998). Following disturbance events, the combination of rapid twining growth and sprouting from vegetative bulbils triggers preferential establishment of the species, displacing slower-growing woody vines that are de pendent on bark adherence to advance into the canopy (Horvitz & Koop, 2001; Gordon, 1998).


13 Taxonomy and Distribution A fam ily estimated by some to have as many as 900 species, the Dioscoreaceae are a diverse group of mostly tropi cal, climbing and herbaceous monocots belonging to the order Dioscoreales (Al-Shehbaz & Sc hubert, 1989). The dioecious genus, Dioscorea L., is the largest and most widely distribu ted, being comprised of about 450 species (Wilkin et al. 2005). Prior estimates of diversity in the genus judged th e number of species at 600 (Knuth, 1924), and have ranged as high as 850 (Al-Shehbaz & Schubert, 1989) A recent reconstruction of the molecular phylogeny for this group has done much to clarify intra-sectional relation ships in the genus, but certain clades have yet to be completely resolved (Wilkin et al. 2005). High morphological variability, dioecy, and sma ll flowers have been cited as reasons for the long-standing taxonomic diffi culties surrounding members of Dioscorea ; these mitigating factors continue to frustrate mo rphological treatments, all the way down to varietal classification. One taxon, as yet unresolved with genetic methods, is that of Dioscorea bulbifera L. (sampled from Madagascar), which is tentatively placed between a compound-leaved clade and a clade of Malagasy endemics in a combined plastid phylogeny (Wilkin et al. 2005). D. bulbifera has been assigned to the section Opsophyton (Uline), a group of five or six Eastern Hemisphere species, of which, D. bulbifera is the sole representative in China (Zhizun & Gilbert, 2000). Having the most extensive endemic range of any species in the family, and an even larger introduced range, D. bulbifera L. has long been studied, and under many different names. Prain and Burkill (1936) list more than 15 pre-Linn ean names and references between 1684 and 1750, while more than 25 binomial synonyms appear in late r works. This species is the only one in the family native to both Africa and Asia, with limits well defined by numerous authors and accounts (Burkill, 1960; Coursey, 1967). The native range in Africa extends from the West African coast, south of 10N lati tude, eastward to Ethiopia on the opposite end of the continent.


14 In locations where rainfall is sufficient, it ex tends through South and Ce ntral Africa almost to South Africa, at elevations between 200-1300 m (W ilkin, 2001). In Asia, air potato can be found from India and Nepal eastward to Southern Japan. It extends south through the Malay Archipelago and down to Queensland in Australi a. The adventive range of this species is extensive and includes many outlying Pacific isla nds, Madagascar, portions of Central and South America, the West Indies, and Florida. Disjunction in the range of D. bulbifera between Africa and Asia was caused by drying climatic conditions during the Pliocene; th e same environmental changes also disjoined the distributions of Dioscorea sections Lasiophyton and Enantiophyllum (except for D. alata), and the species of these sections are now considered endemic to either Africa or Asia, but not both (34Burkill, 441960). Introduction History Ya ms are among the earliest anthropogenic plant introductions to the New World by Europeans. Even earlier movements of D. bulbifera around the Old World and outlying areas are documented in association with cultiva tion. One example is the introduction of D. bulbifera to the islands of Hawaii by Polynesians as early as 1000 A.D. (Wester, 1992), and corroborated back to 1500 A.D. using radiocarbon dating at a well-preserved archaeological site (Burney et al. 2001). An introduction history of invasive D. bulbifera to North America and adjacent areas is incomplete and hindered somewhat by taxono mic complexity that resulted in numerous synonyms, and later, numerous varieties. Yam specialists have usually ascribed an African source for D. bulbifera in North America, rather than an Asian source (C oursey, 1967; Burkill, 1939, 1960; Prain & Burkill, 1936). The introduction of D. bulbifera var. anthropophagorum from Africa to America occurred as early as the 16th century. Accordi ng to Prain and Burkill (1936), this variety was found throughout the West Indies and Central Am erica during the 1930s (and various locations


15 in the Americas between latitudes 29N and 35 S), and was grown as a curiosity in northern Florida. A 1601 description of the Ycam intro duced to the West Indi es is likely the West African variety of D. bulbifera placing the introduction of the sp ecies to the New World early in the slave trade (Coursey, 1967). Yam bulbils and t ubers were transported across the Atlantic as durable foodstuffs during long sea voyages. Africa ns who were familiar with the cultivation and preparation of the yams plante d them upon their arrival in the New World, and most abundantly in the West Indies. Burkill maintains that the names given to the West Indian cultivars of D. bulbifera validate their introduction from the Elmin a Lagos coast (Burkill, 1939). Botanist William Bartram reported Discorea [sic] bulbifera from a Mobile garden in his Travels from 1777, but Bartram commentator Harper states that this species may not have been D. bulbifera L. (Harper, 1998). Comprehensive treatments of the Flor ida floras have asserted that D. bulbifera is of Asian origin, and was originally planted for ornament, but cite no references to support this assertion (Clewell, 1985; Hall, 1993; Wunderlin & Hansen 2003). Florida horticulturalist Nehrling, circa 1905, mentions the species in Florida, and was also the first to record the plants potential for weediness (Nehrling, 1933). Many yam species were introduced intentionally into Florida from Asia, Oceania, Africa, and the West Indies between 1840 and 1920. D. alata was introduced to Fort Myers during the 1840s by the United Stat es Department of Agriculture (USDA). The Office of Foreign Seed and Plan t Introductions was also responsi ble for bringing other yams into Florida and distributing them to plan t experimenters (Young, 1923). In documenting the introductions of D. bulbifera presented in this study, I encountered many more accounts of USDA yam introductions, often unidentified as to the species. Many of these introductions presumably did not become established, however, because weak demand and lack of knowledge


16 about cultivation practices made them a comm ercial failure (Young, 1923). As shown in Table 1-1, early 20th century introductions of D. bulbifera into Florida included material from Africa, Polynesia, the West Indies, and Southeast Asia. Previous Work Previous studies of intraspecific genetic diversity in D. bulbifera found high levels of differentiation between African a nd Asian types, and noted signifi cant variation am ong plants in Asia and Oceania. An analysis of chloroplast restriction fragment length polymorphisms (RFLP) indicated that certain chloroplast genotypes were distribute d over a wide geographic range (Thailand to Taiwan), while some locations had more than two types present in a small area (Taiwan). The same study concluded that Sout heast Asia was the di fferentiation center of D. bulbifera and found only a small amount of genetic variation present within Africa, based on a total of three collections from Ethiopi a, Tanzania, and Madagascar (Terauchi et al. 1991). A random amplified polymorphic DNA (RAPD) analysis (Ramser et al. 1996) added an additional three accessions from Ethiopia, but did not note signi ficant intraspecific vari ation relative to the amount present in Asia and Oceania. The first analysis of genetic diversity in D. bulbifera (Terauchi et al. 1991) suggested that the amount of variation present within the ge notypes of Asian and Oceanian accessions was equal to the amount of differentiation between the African and most closely related Asian samples. Subsequent analyses have bolstered the support for hi gh diversity in Oceania (Ramser et al. 1996), and in Asia (Ramser et al. 1996; Zheng et al. 2006), with the most recent study grouping a geographically diverse sample from main land China into 5 clusters using inter-simple sequence repeat (ISSR) markers. Uncertainty surrounding morphologica l treatments of varieties in D. bulbifera and demonstrable, intraspecific genetic diversity stru ctured across its broad geographical range, made


17 the decision straightforward to adopt a genetic methodology in iden tifying the source population of invasive D. bulbifera in Florida. To address the quest ion of source population, I collected samples of the target species in Florida (extensively) and across the native range (opportunistically). I sampled wide ly in Florida in order to in crease the likelihood of collecting from among the unique introductions documente d. By sequencing intragenic chloroplast DNA markers that are shown to possess intraspecific va riability across a variety of angiosperms (Shaw et al. 2005), I sought to identify the haplotype or type s most similar to those present in Florida. A second genetic method, microsatellite genoty ping, was used to evaluate intraspecific variability in Florida by using markers previously developed for this purpose in yams (Tostain et al. 2006).


18Table 1-1. Introductions of D. bulbifera to Florida and adjacent areas: 1500s to 1919. Author Datea Source Destination Notes Reference Clusius, C. 1500's West Africa ("Elmina Lagos") W. Indies; Americas Introduced by Portugese ships trafficking African slaves (Burkill, 1939) Bartram, W. 1777 ? Mobile (Alabama) Not Asiatic D. bulbifera according to Harper (Harper, 1998) Nehrling, H. 1905 ? Gotha, Florida Noted weedy behavior and propensity for escape (Nehrling, 1933) USDA #18656 1906, May Mayaguez, Puerto Rico Miami, Florida Gunda cultivar; large irregular shaped axillary bulbils (USDA, 1907) USDA #21775 1908, January French Guinea Florida, (?) Se nt by M. A. Chevalier (USDA, 1909) USDA #45994 1918, April Mayaguez, Puerto Rico Florida, (?) Received by R.A. Young; Aerial tubers better for food than ground tubers (USDA, 1922a) USDA #46218 1918, May Honolulu, Hawaii Florida, (?) Sent by J. E. Higgins, Hawaii Agricultural Station (USDA, 1922a) USDA #47493 1919, April Singapore, Straits settlements Florida, (?) Sent by I. H. Burkill; Specimens from either Singapore, India, or Bangladesh (USDA, 1922b) a denotes either date of in troduction or report of sighting


19 CHAPTER 2 METHODS Collection Volunteers through the F lorida Cooperative Extension and Master Gardener programs collected accessions of D. bulbifera from July to November of 2006. Collectors were given species identification guidelines, in order to prevent the sampling of D. alata, the only yam in Florida with which D. bulbifera is likely to be confused on the basis of morphology. Collectors were also requested to note the presence of flowers or fruits, as well as to categorize the bulbil morphology as either warty/dark or smooth/light Geospatial coordinates for sample locations were recorded using global positioning system (G PS) instruments, or approximated using either a street address or detailed descriptions. Leaves were desiccated in sili ca gel for preservation. Most non-Florida accessions were collected between April and September of 2007. A few samples were obtained at dates outside this range; these include an accession from Puerto Rico (November, 2005) and accessions from Chin a (July, 2004), Uganda (February, 2004 and September, 2006). The data gathered for all acce ssions is summarized in the supplementary data. Data about flowering, fruiting, and bulbil morphology were not requested from collectors of nonFlorida samples; in addition to geopositions by GPS, many also included photographs, and indicated whether the collection was from a wild or cultivated plan t. As with Florida accessions, leaf material was preserved by desiccation in silica gel. DNA Extraction DNA was extracted from dried leaf m aterial (1 to 2 cm2) using a modified method of Kim et al. (1997). In some cases, extracts were further cleaned using Promegas Wizard DNA cleanup kit (Promega, USA). Extracts were s ubsequently used for both chloroplast and microsatellite analyses.


20 Chloroplast DNA Sequencing Two chloroplast DNA (cpDNA) intergenic regions, spanning psbM-trnD and ycf6-psbM (Shaw et al. 2005), were amplified for 26 accessions using the following 10uL PCR reaction mix: 2.5mM MgCl2, 0.5mM primer, 200mM each dNTP, 0.2U Taq polymerase, 1uL of DNA. Reactions were run using either ABI 2720 or MJ Research PTC-200 thermalcyclers with the following conditions: 2 minutes at 94C, followe d by 30 cycles of [94C for 30 seconds, 55C for 30 seconds, 72C for 1 minute], and a final ex tension of 72C for 5 minutes. For troublesome samples, hot-start polymerase (Promega HotStart ) was substituted for the standard polymerase enzyme. Unincorporated nucleotides and excess primers were removed from PCR products using ExoI and Antarctic Phosphatase (New England Bi olabs) according to manufacturer protocols. A total of 1,690 bp were sequenced in both forw ard and reverse directions using BigDye Terminator Cycle Sequencing kit v3.1 and elec trophoresed on either an ABI 3730xl or ABI 3130 genetic analyzers. Sequences were trimmed and contiged in Sequencher v4.8 (Gene Codes Corp.) then aligned using ClustalW. Relationships among haplotypes were visualized using a 95% parsimony haplotype network constructed with TCS 1.21 using a total of 1690 bp (Clement et al. 2000). Alignment gaps of more than one base that could represent a single insertion/deletion event were collapsed and treate d as a fifth state. A neighbor-j oining tree was also constructed using PAUP* ver. 4.0b10 (Swofford, 2002). Accessions of D. alata from India (421, 428, and 205) were defined as outgroup and used to root the resulting tree. A 1000-replicate neighborjoining (NJ) search was run using the distance criterion, followed by a 1000-replicate NJ bootstrap analysis on this tree. Bootstrap percentages were indi cated at their respective nodes on the NJ tree.


21 Microsatellite Analysis Ya m accessions (n=133) of Florida and non-Flor ida origin were tested for polymorphism using microsatellite markers Da1A01 and Da1F08 (Tostain et al., 2006) fluorescently labeled with 6-FAM (Eurofins MWG Operon). The 20uL P CR reactions had final concentrations of 2mM MgCl2, 200mM each dNTPs, 0.6mM each primer pair, 2uL DNA, and 1uL Biolase enzyme (Bioline). PCR reactions for microsatel lite markers were run on either a PTC-200 (MJ Research) or ABI 2720 model thermal cycler unde r the following conditions: 1 minute at 94C, followed by 30 cycles each of [94C for 30 seconds, 59C for 30 seconds, and 72C for 30 seconds]. Reaction products were separated by capillary electrophoresis using an ABI 3130 with the LIZ600 size standard. Genotypes were calle d using GeneMapper ver. 4.0 (ABI). GenAlEx ver. 6.1 was used to generate pairwise genotypic distances which were used to cluster genotypes in a principal coordinate an alysis (Peakall & Smouse, 2006).


22 CHAPTER 3 RESULTS Sampling In all, 100 accession s of Florida D. bulbifera were used in the an alyses, representing 93 unique geopositions. Only one observation of flower ing (pistillate plant) was reported from the collections in Florida (Accession 085). For 68 accessions from Flor ida, collectors recorded bulbil morphology: 39 reported the Warty/Dark morp hotype, while 20 reported the Smooth/Gray morphotype. For seven accessions, a Smooth/Gr ay/Warty type was recorded, and for two additional accessions a Gray/Warty bulbil type was seen. These observations are summarized in Table A-1. Chloroplast DNA Analysis There were seven haplotypes of D. bulbifera which were grouped into two distinct clusters. One cluster includes all the haplot ypes of African origin and th e other cluster groups haplotypes from Florida and Chinese samples (Figure 3-2). Accession data for the samples represented in this network are summarized (Table 3-1). Th e neighbor-joining tree also clearly groups the Florida samples with the Chinese samples separate from the African samples (Figure 3-3). Despite distinct genetic differences between th e African and Chinese ha plotypes observed, there was relatively little genetic diversity observed within each of the haplotype groups, when compared to the significant amount of divergen ce separating them. No intraspecific variation was observed within Florida. Four African haplotypes (D, E, F, and G) di ffer at two loci on the DNA alignment. Singlebase length polymorphism in a homonucleotide re gion differentiates African haplotypes D, E, and F from each other, while a two bp deletion in G differentiates it from haplotype F. Haplotypes A and B are separated by the same length polymorphism that also differentiates D


23 from E, and haplotype C is defined by a single base substitution from ha plotype B. African and Florida/Asian haplotype groups we re separated from each other by a total of eight changes a combination of substitutions (four), insertions/d eletions (two), and le ngth polymorphisms (two). A minimum of eight inferred mu tations separate the two clus ters representing haplotypes from Africa and those from Florida/China. Confid ence in this separation is very high, and is corroborated by the bootstrap per centages indicated in the nei ghbor-joining tree. Bootstrap percentages indicate the freque ncy at which bifurcating branches of a node on the tree are resolved in the given topology, unlike other measures of confidence, more commonly encountered (such as the venera ble 95% interval). In the NJ tree generated for this study, separation of the Florida/China samples from th e African group is supported with a bootstrap value of 100%. It also differentiates between Florida and China samples with a 98% bootstrap percent. A 64% bootstrap value separates a Ghanaian accession (120) from the rest of the main African clade. This is cons istent with the parsimony ne twork, where 120 is the sole representative of haplotype D, and is closer by one inferred mutation to the Florida accessions than any other African haplotype. The NJ tree did not resolve (412), haplotype E from the parsimony network, and this shows up in the lo wered bootstrap support for this group, at 51%. Microsatellite Analysis For both m icrosatellite loci, the products for each locus were biallelic, with five unique allele patterns observed among accessions of D. bulbifera (Table 3-2). No genotype differentiated between cultivated and wild D. bulbifera in Africa. In many African samples, DA1A01 failed to amplify, even after multiple amplif ication attempts, suggesting the presence of a null allele. All 100 Florida samples had identical genotypes. In just five samples from Florida, no product was recorded from one of the two loci This occurrence was rare enough to suggest an experimental anomaly, rather than a null allele, was res ponsible for the results obtained in these


24 samples. In addition, five Florida samples that la cked a geoposition or unique location data were also excluded from the genetic distance calculatio n for the PCA. Principal coordinate analysis clustered 90 Florida, with one Puerto Ri can, and one Chinese accession, while African accessions clustered into two separate groups (F igure 3-4). Principal c oordinates one and two explain 74.85% and 21.60% of the variation in the data, respectiv ely. Similar to the cpDNA, data from the nuclear microsatellite loci exhibited ve ry low genetic diversity and most alleles across Africa differed by only 1 to 3 repeat units. Table 3-1. Chloroplast DNA haplotypes, listed by locality; some localities are given with a more specific regional identifier in parentheses. Coordinates given in decimal degrees. Haplotype Locality Latitude Longitude A Florida (Palm Beach) 26.923036 -80.185042 Florida (Hillsborough) 27.76258 -82.14865 Florida (Martin) 27.1738 -80.27323 B China (Yunnan) 21.894586 101.027001 C China (Guangdong) 23.212917 113.421556 D Ghana (Tuna) 9.801 -1.97 E Benin (Serou) 9.663333 1.697222 F Uganda 0.399123 33.01079 Uganda 0.39787 33.01608 Ghana (Tamale) 9.346 -0.823 Ghana (Anyinamso) 6.65194 -1.89803 Burundi (Kigwena) -4.09835 29.50636 Ghana (Ayinasu) 6.94795 -2.08802 Ghana (Mfensi) 6.7834 -1.802 Ghana (Pakyi) 6.53294 -1.66884 Togo (Tove) 6.878055 0.651389 Benin (Savalou) 7.855833 1.980556 Uganda 0.39895 33.01087 Uganda 0.39895 33.01087 Togo (Misahohe) 6.950834 0.595556 G Ghana (Nkurakan) 6.107 -2.2786 Togo (Kuma Adame) 6.973611 0.595833 Uganda 0.39729 33.01719


25 Figure 3-1. Map of 99 D. bulbifera L. accessions in Florida, USA. Out of 100 Florida accessions that were genotyped, 93 are represented as unique points on the map. Six accessions, recorded from the same point as anothe r accession, represent collections from secondary or tertiary plants near the or iginal place of collection, and for which a unique position was not recorded. One accessi on from Broward County had no other coordinate or distance/bearing in formation associated with it.


26 Figure 3-2. Parsimonious haplotyp e network from chloroplast DNA alignments. Lettered circles represent unique haplotypes, while lines connecting haplotypes each represent an inferred intermediate separated by a single mutation. The two lines indicated by arrows represent eight inferred mutations. Figure 3-3. Neighbor-joining tree of chloroplast DNA alignments. Bootstrap percentages are given at each node. The country of origin is indicated at the terminus of each branch, along with the corresponding accession numbe r. Sequences from accessions 421, 428, and 205 (all from India) were designated as an outgroup for this analysis.


27 Table 3-2. Microsat ellite genotypes of D bulbifera at loci Da1F08 and Da1A01. Two alleles are listed for each locus by size of fragment in base pairs, and suspected null alleles are noted with zeros. The number of locati ons where the genotype was found, listed in the second column, represent unique ge opositions only, after the convention explained in Figure 3-1. If a genotype was unique to a single accession, then the accession number is noted parenthetically in the first column. Number of Accessions with Genotype Locations Found Locus Da1F08 Locus Da1A01 95 Florida (93), Puerto Rico (1), China (1) 152 154 205 205 1 (408) China (1) 152 152 207 207 6 Uganda (4), Togo (2) 152 152 198 198 1 (186) Ghana (1) 152 152 198 211 11 Benin (3), Ghana (6), Burundi (1), Togo (1) 152 152 0 0 Figure 3-4. Principal coordinate analysis of codominant micr osatellite markers Da1A01 and Da1F08 in D. bulbifera Each point is labeled with th e name that corresponds to the places where the samples were collected.


28 CHAPTER 4 DISCUSSION AND FUTURE WORK Interpretation of Results Both analyses showed that genetic variability is low in Africa. Low genetic diversity in Africa, with no trend between ge notype/haplotype and cultivation status is som ewhat surprising because these results represent the largest geneti c survey (in geographic breadth and number of accessions) of D. bulbifera on this continent. West Africa, where sampling was most intensive, has been identified as the prim ary center for yam culture and cultivation. Ayensu and Coursey (1972) maintained that, It is only in certain parts of We st Africa that the yam is, or appears ever to have been, of importance as a food crop. However, in support of low African diversity, no male flowers of D. bulbifera were confirmed from a survey of South-Central Africa (female specimens were found from Zambia, Zimbabwe, Malawi and Mozambique). Wilkin has suggested that it may not be nativ e there, and also raises the question of how female plants in the region are able to set fruit (Wilkin, 2001). Also, dur ing the course of extens ive travels in Africa, Overholt reports seeing no fruit on D. bulbifera (William Overholt, pers onal communication, 2009). Although selection occurs during the process of cultivation, Afri can yam cultivation is characterized by vegetative pr opagation, and the bulbils of D. bulbifera are optimized for this method of regeneration (Coursey, 1967). Because there was no genetic diversity dete cted among the Florida accessions tested, I infer that low diversity is the result of a popul ation bottleneck during the establishment period of D. bulbifera into Florida, and indicates that very low propagule pressure may have been necessary for establishment. No intraspecific va riation was observed within Florida, suggesting that clonal reproduction and vegeta tive spread have distributed the species throughout Florida. Ecologists have often noted how very narrow po pulation bottlenecks do not seem to adversely


29 affect the establishment of invasive species. One such study reports that 66% of the most invasive class of weeds in China reproduce clonally (Liu et al. 2006). Given the results of this genetic analysis, and the scarcity of evidence for sexual reproduction among Florida D. bulbifera it is reasonable to assert that multiple introductions from unique source populations did not play a role in the establishment of D. bulbifera Rather, it would seem that clonal propagation of the invasive genotype was sufficient to avoid genetic barriers to establishment. Varietal Classification in D. bulbif era L. Ten varieties of D. bulbifera were described by Prain and Burkill (41936), but are taxonomically insignifican t or even invalid according to others (4Al-Shehbaz & Schubert, 41989; Zhizun & Gilbert, 2000). In another case, Mige ( via Terauchi et al. 1991) named 12 varieties in Ivory Coast alone. Varietal designa tions continue to be amended (5Yifeng et al. 52008), underscoring the need for a better understanding of the intraspecific ta xonomy of this yam. Phylogenetic treatments of Dioscorea have elucidated some of the relationships between yams (5Wilkin et al. 52005), and marker-based genetic diversity in D. bulbifera has often supported varietal designations, although minor discrepancies have been noted in each study (5Zheng et al. 62006; 6Terauchi et al. 61991; 6Ramser et al. 61996). The ten varieties of D. bulbifera first treated by Knuth in Das Pflanzenreich (non-vide, 1924), were later reiterate d with very minor modifications by Prain and Burkill (1936). Varieties mentioned in recent treatments that are not duplicated by Prain and Burkill's nomenclature include vars. bulbifera ramiflorus albitubera and pauciflorum These names are mentioned in a Chinese publication, and the proceedings from the conference in which it appears has only an abstract available (Pro ceedings of the 2006 Systematic & Evolution Botany Conference in China, p. 27; http://www.syst-evol .cn/2006/syst-evol_ii.pdf ). A very recent publication identifying a new variety of D. bulbifera (var. albotuberosa ) from China is potentially relevant,


30 due to the genetic similarity demonstrated in the present study between samples from China and Florida (Yifeng et al. 2008). On the basis of existing varietal classi fications (viz. Prain & Burkill's), African D. bulbifera could apparently be excluded as the source population of Florida air potato, because the Florida population lacks the highl y angular bulbils that characterize D. bulbifera var. anthropophagorum (see also: Figure A-1). Hamon also describes the bulbils of wild, West African D. bulbifera as polyhedral, and only occasionally round (Hamon et al. 1995); this is consistent with an image of African bulbils (plate 12) presented by Coursey (1967). Hamon does not note the morphology of bulbils on cu ltivated plants in this region. It is difficult to apply the key to D. bulbifera varieties beyond bulbil morphology; documentation of the underground tuber morphology in Florida is lacking, descriptions of character sizes in the key are vague, and staminate flowers are not found in Florida. Also, Overholt et al. (2008) have hypothesized a biannual tuber regeneration phenology that, if proven, would seem to frustrate the diagnostic value of tuber characters (Prain & Burkill, 1936). With the exception of some observations a bout differences between greatly divergent populations (i.e. Africa/Asia), I in terpret the varietal key of Pr ain and Burkill as equivocal for determining the source population, or even the variety of Florida D. bulbifera Rarity, ambiguity, or outright lack of these descri ptive characters in the Florida population frustrates diagnosis using the key. When compared to the very recent key to the varieties of D. bulbifera in China, similar obstacles emerge, such as reliance on male flowers to differentiate some varieties (Yifeng et al. 2008). I am inclined to agree with Prain and Burkill's (1936) assessment that D. bulbifera herbarium material "rarely suffices for distinguishing varieties." However, even morphometric examination of wild plants may be unproduc tive for distinguishing among varieties. A


31 multivariate phenetic study on the variable, compound-leaved D. quartiniana evidenced continuous variation, providing no statistical support for intr aspecific divisions, despite longstanding assertions of their validity (Wilkin, 1999). No genetic study to date has thoroughly studied the genetic differentiation between East and West African D. bulbifera so it is difficult to compare the amount of diversity found in the present study to the divergence between the African samples obser ved in previous works that examined only East African locations. Additional sampling of D. bulbifera in Asia will be necessary in order to determine if a more si milar genotype to the Florida accessions can be found, and also to define the locations in Asia where this genotype is present. Genetic diversity of African D. bulbifera from this study reinforces the high degree of intraspecific divergence from Asian D. bulbifera formerly reported. The small number of Asian D. bulbifera accessions obtained for this study (n=3) do not provide enou gh context to make detailed comparisons of genetic diversity in this region with other studi es that sampled this area much more thoroughly. Biocontrol Implications The USDA/ Agricultural Research Service (A RS) Invasive Plant Research Laboratory recently submitted a petition to the USDA/Animal and Plant Health Inspection Service (APHIS) Technical Advisory Group (T AG) for field release of Lilioceris sp. near impressa (Coleoptera: Chrysomelidae), a herbivore of D. bulbifera in Nepal ( ealth/permits/tag/petitions.shtml ). Results from the present study suggest that these beetles c ould have a higher likelihood of maintaining host specificity to the invasive population in Florida than do potential candidates from Africa. Host switching is of some concern, as the two native Florida yams, D. floridana and D. villosa are likely to be the alternates, if such a switch ever occurred. Of less con cern, due to their geographic isolation from


32 Florida over water, are members of the West Indies endemic genus, Rajania soon to be subsumed in Dioscorea L. (Wheeler et al. 2007). The EICA hypothesis may not be relevant for D. bulbifera because reproduction within the invasive range is thought to be entirely vege tative, so that novel genetic configurations conferring a selective advantage remain in th e somatic line and are not exchanged via sexual reproduction. Despite the appa rently clonal nature of D. bulbifera in Florida, somatic mutations conferring herbicide resistance or other attribut es associated with increased competitiveness could arise, as observed in othe r weed species that reproduce ve getatively, such as hydrilla (Michel et al ., 2004). Because I have established the o ccurrence of multiple introductions, likely of genetically distinct D. bulbifera there may have been a period of selection between genotypes during the lag phase following introduction. A genetic bottleneck, due to selection between genotypes, would have reduced the genetic di versity below the level present at initial introductions. D. bulbifera s success in Florida may be attributable to release from natural enemies, so finding suitable biological control agents may help reduce the negative ecological impacts of this invasive vine. Future Needs and Concluding Thoughts Historical research into the extent an d sources of intenti onal introductions of D. bulbifera in Florid a will provide necessary context to the genetic work used to further characterize the geography of the source population. In addition, some of the important varietal keys and genetic studies of Asian D. bulbifera are not yet translated from Chinese into English; an international collaboration with Chinese res earchers may be extremely produc tive for procuring both plant material and access to updated varietal treatments. In conclusion, this collection of studi es has documented geographically diverse introductions of D. bulbifera to Florida, synthesized the hi storical and taxonomic diversity


33 relevant to its identification a nd invasiveness in Florida ecosyste ms, and used a pair of genetic methodologies to overturn the paradigm that invasive D. bulbifera in Florida is an African introduction. Despite convincing evidence from other sources that the African variety may be differentiated from all others on the basis of bulbil morphology, extensive sampling there was worthwhile for a number of reas ons. Historical data and noted yam experts have asserted the African origin of Florida D. bulbifera introduction records establis hed their import from West Africa, preliminary results suggested an Af rican source (Overholt & Hughes, 2004), and the genetic diversity of West African D. bulbifera was unknown. This study also establishes that Florida D. bulbifera has a low level of genetic diversit y: a finding that contributes to understanding the invasion dynamics of weeds, and can be applied in efforts to find and import effective biocontrols from the Asia/Pacific region.




35Table A-1. Accession Data and Summary of Resu lts. Coordinates are given in decimal degree s. Dashes indicate that data was not obtained, and the haplotype column headi ng is abbreviated. Italicized accessions indicate that the yam species examined was not D. bulbifera # Date Latitude Longitude Locality, County Nation, State Bulbil Type Da1F08 Da1A01 Hap. Status 208 14-May-07 7.855833 1.980556 Savalou Benin 152 152 F Wild 412 12-May-07 9.663333 1.697222 Serou Benin 152 152 E Wild 446 07-May-07 9.661667 1.696944 Serou Benin 152 152 Wild 161 06-Apr-07 -4.09835 29.50636 Kigwena Forest Burundi 152 152 F Wild 401 27-Jun-07 23.212917 113.421556 China, Guangdong 152 152 154 205 205 C Wild 408 Mid-2007 24.100267 113.219414 Yingde City China, Guangdong 152 152 207 207 Wild 316 06-Jul-04 21.894586 101.027001 Menglun China, Yunnan B 120 Sept. 2007 9.801 -1.97 Tuna Ghana 152 152 D Cult. 136 Sept. 2007 9.346 -0.823 Tamale Ghana F Cult. 148 Sept. 2007 6.107 -2.2786 Nkurakan Ghana 152 152 G Cult. 149 Sept. 2007 6.65194 -1.89803 Anyinamso Ghana 152 152 F Wild 186 Sept. 2007 6.94795 -2.08802 Ayinasu Ghana 152 152 198 211 F Wild 188 Sept. 2007 6.7834 -1.802 Mfensi Ghana 152 152 F Wild 194 Sept. 2007 6.53294 -1.66884 Pakyi No.1 Ghana 152 152 F Wild 198 Sept. 2007 7.63116 -0.13327 Adumadum Ghana 152 152 Cult. 202 26-May-07 6.878055 0.651389 Tove Togo 152 152 198 198 F Wild 407 24-May-07 6.973611 0.595833 Kuma Adame Togo 152 152 G Wild 436 23-May-07 6.950834 0.595556 Misahohe Togo 152 152 198 198 F Wild 006 01-Feb-04 0.399123 33.01079 Mabira Forest Uganda W/D 152 152 198 198 F Wild 130 28-Sep-07 0.39787 33.01608 Mabira Forest Uganda 152 152 198 198 F Wild 317 07-Sep-06 0.39895 33.01087 Mabira Forest Uganda 152 152 198 198 Wild 318 07-Sep-06 0.39895 33.01087 Mabira Forest Uganda 152 152 198 198 F Wild 319 07-Sep-06 0.39895 33.01087 Mabira Forest Uganda 152 152 198 198 F Wild


36Table A-1. Continued # Date Latitude Longitude Locality, County Nation, State Bulbil Type Da1F08 Da1A01 Hap. Status 411 28-Sep-07 0.39729 33.01719 Mabira Forest Uganda 152 152 198 198 G Wild 001 05-Aug-06 27.00324 -82.10045 Charlotte USA, FL W/D 152 154 205 205 002 11-Aug-06 27.37555 -80.83504 Okeechobee USA, FL W/D 152 154 205 205 003 11-Aug-06 27.24741 -80.84855 Okeechobee USA, FL 152 154 205 205 005 18-Aug-06 28.28339 -81.45721 Osceola USA, FL W/D 152 154 205 205 007 05-Aug-06 26.95124 -81.99168 Charlotte USA, FL W/D 152 154 205 205 009 18-Aug-06 27.654417 -80.360833 Indian River USA, FL 152 154 205 205 011 17-Aug-06 28.882972 -82.527072 Citrus USA, FL 152 154 205 205 014 18-Aug-06 27.807194 -80.482306 Indian River USA, FL 152 154 205 205 016 05-Aug-06 26.95369 -82.14495 Charlotte USA, FL W/D 152 154 205 205 017 17-Aug-06 28.738219 -82.520722 Citrus USA, FL 152 154 205 205 018 19-Aug-06 28.28344 -81.45726 Osceola USA, FL W/D 152 154 205 205 021 05-Aug-06 26.97425 -82.14115 Charlotte USA, FL W/D 205 205 022 05-Aug-06 26.99754 -82.15312 Charlotte USA, FL W/D 152 154 205 205 023 11-Aug-06 27.2365 -80.81324 Okeechobee USA, FL W/D 152 154 205 205 024 03-Aug-06 26.915523 -82.031814 Charlotte USA, FL W/D 152 154 205 205 025 29-Aug-06 28.28741 -81.41007 Osceola USA, FL G/W 152 154 205 205 030 18-Aug-06 27.799167 -80.497972 Indian River USA, FL 152 154 205 205 031 11-Aug-06 27.24864 -80.82101 Okeechobee USA, FL 152 154 205 205 032 29-Aug-06 28.31311 -81.40195 Osceola USA, FL 152 154 205 205 033 05-Aug-06 27.00748 -82.09835 Charlotte USA, FL W/D 152 154 205 205 035 29-Aug-06 28.28344 -81.45726 Osceola USA, FL 152 154 205 205 036 11-Aug-06 27.25012 -80.83302 Okeechobee USA, FL W/D 152 154 205 205 038 11-Aug-06 27.32457 -81.02231 Okeechobee USA, FL S/G 152 154 205 205 043 11-Aug-06 27.24803 -80.80004 Okeechobee USA, FL S/G 152 154 205 205 049 18-Aug-06 27.759694 -80.484833 Indian River USA, FL 152 154 205 205 051 10-Aug-06 26.99566 -81.0781 Glades USA, FL W/D 152 154 205 205 052 08-Aug-06 28.495436 -81.339258 Orange USA, FL S/G 152 154 205 205


37Table A-1. Continued # Date Latitude Longitude Locality, County Nation, State Bulbil Type Da1F08 Da1A01 Hap. Status 054 17-Aug-06 28.857861 -82.273992 Citrus USA, FL 152 154 205 205 058 10-Aug-06 26.7564 -81.4388 Hendry USA, FL W/D 152 154 205 205 059 11-Aug-06 27.42936 -80.3847 St. Lucie USA, FL S/G 152 154 205 205 066 10-Aug-06 26.75637 -81.4412 Hendry USA, FL W/D 152 154 205 205 069 10-Nov-06 25.73913 -80.29503 Miami-Dade USA, FL W/D 152 154 205 205 070 10-Aug-06 26.99543 -81.0783 Okeechobee USA, FL W/D 152 154 205 205 072 11-Aug-06 27.40737 -80.3759 St. Lucie USA, FL W/D 152 154 205 205 073 01-Aug-06 29.5461 -82.3211 Alachua USA, FL W/D 152 154 205 205 074 11-Aug-06 27.38436 -80.3385 St. Lucie USA, FL W/D 205 205 077 15-Oct-06 25.72277 -80.28062 Miami-Dade USA, FL W/D 152 154 205 205 080 12-Aug-06 27.47337 -80.3692 St. Lucie USA, FL W/D 152 154 205 205 082 13-Aug-06 26.920383 -80. 185125 Palm Beach USA, FL 152 154 205 205 085 11-Aug-06 27.58733 -80.3693 Indian River USA, FL S/G 152 154 205 205 089 10-Aug-06 27.2465 -80.8039 Okeechobee USA, FL W/D 152 154 205 205 090 10-Aug-06 26.76524 -81.4348 Hendry USA, FL W/D 152 154 205 205 093 09-Aug-06 28.585247 -81.360867 Orange USA, FL W/D 152 154 205 205 095 17-Aug-06 28.429269 -81.349947 Orange USA, FL W/D 152 154 205 205 098 10-Nov-06 25.70318 -80.30293 Miami-Dade USA, FL W/D 152 154 205 205 099 18-Aug-06 24.559692 -81.795211 Monroe USA, FL W/D 152 154 205 205 101 03-Oct-06 30.314906 -81.655419 Duval USA, FL 152 154 205 205 102 10-Aug-06 27.24621 -80.804 Okeechobee USA, FL W/D 152 154 205 205 104 10-Aug-06 28.535344 -81.363708 Orange USA, FL W/D 152 154 205 205 105 22-Jul-06 28.03248 -81.9395 Polk USA, FL 152 154 205 205 107 12-Aug-06 28.012781 -82.471131 Hillsborough USA, FL 152 154 205 205 108 09-Aug-06 27.76258 -82.14865 Hillsborough USA, FL 152 154 205 205 A 109 14-Oct-06 30.095947 -83.58395 Taylor USA, FL S/G 152 154 205 205 110 14-Oct-06 30.233108 -84.233264 Wakulla USA, FL S/G 152 154 205 205 114 05-Oct-06 30.290286 -81.727608 Duval USA, FL 152 154 205 205


38Table A-1. Continued # Date Latitude Longitude Locality, County Nation, State Bulbil Type Da1F08 Da1A01 Hap. Status 116 14-Oct-06 29.653953 -82.523161 Alachua USA, FL S/G 152 154 205 205 117 14-Oct-06 30.095947 -83.58395 Taylor USA, FL S/G 152 154 205 205 126 27-Sep-06 30.295189 -81.768047 Duval USA, FL 152 154 205 205 127 05-Nov-06 27.99127 -82.15105 Hillsborough USA, FL S/G 152 154 205 205 131 22-Nov-06 29.647069 -82.361469 Alachua USA, FL S/G 152 154 205 205 137 09-Nov-06 27.42825 -81.41372 Highlands USA, FL W/D 152 154 205 205 140 24-Nov-06 28.13878 -82.02323 Polk USA, FL S/G 152 154 205 205 141 09-Nov-06 27.27438 -82.26928 Sarasota USA, FL W/D 152 154 205 205 145 2006/2007 27.0675 -80.31985 Martin USA, FL W/D 205 205 146 11-Nov-06 28.07032 -82.36972 Hillsborough USA, FL 152 154 205 205 150 09-Nov-06 27.25386 -81.34084 Highlands USA, FL S/G 152 154 205 205 164 24-Nov-06 28.0922 -82.00665 Polk USA, FL S/G 152 154 205 205 165 11-Nov-06 28.05777 -82.29782 Hillsborough USA, FL S/G 152 154 205 205 166 24-Apr-07 Broward USA, FL 152 154 205 205 171 11-Nov-06 28.0545 -82.32455 Hillsborough USA, FL S/G 152 154 205 205 180 24-Nov-06 28.09173 -81.99553 Polk USA, FL S/G 152 154 205 205 193 09-Nov-06 27.33471 -82.31184 Sarasota USA, FL S/G 152 154 205 205 321 20-Oct-06 27.22512866 -81.88877737 DeSoto USA, FL 152 154 205 205 322 20-Oct-06 27.22512866 -81.88877737 DeSoto USA, FL 152 154 205 205 323 20-Oct-06 27.22512866 -81.88877737 DeSoto USA, FL 152 154 205 205 325 20-Oct-06 27.2158333 -81.86527778 DeSoto USA, FL 152 154 205 205 326 20-Oct-06 27.2158333 -81.86527778 DeSoto USA, FL 152 154 205 205 327 20-Oct-06 27.23611831 -80.98949571 Highlands USA, FL 152 154 205 205 328 20-Oct-06 27.23611831 -80.98949571 Highlands USA, FL 152 154 329 20-Oct-06 27.23611831 -80.98949571 Highlands USA, FL 152 154 205 205 330 20-Oct-06 27.1738 -80.27323 Martin USA, FL S/G/W 152 154 205 205 A 331 20-Oct-06 27.17631 -80.26935 Martin USA, FL S/G/W 152 154 205 205 332 20-Oct-06 27.16284 -80.21102 Martin USA, FL S/G/W 152 154 205 205


39Table A-1. Continued # Date Latitude Longitude Locality, County Nation, State Bulbil Type Da1F08 Da1A01 Hap. Status 333 22-Sep-06 29.71757 -82.41824 Alachua USA, FL W/D 152 154 205 205 335 26-Oct-06 27.76538 -80.60124 Indian River USA, FL G/W 152 154 205 205 336 26-Oct-06 28.0778 -80.68611 Brevard USA, FL W/D 152 154 205 205 337 26-Oct-06 28.08878 -80.68721 Brevard USA, FL W/D 152 154 205 205 338 26-Oct-06 28.61094 -81.09422 Orange USA, FL 152 154 339 26-Oct-06 28.6285 -81.12235 Seminole USA, FL S/G 152 154 205 205 341 26-Oct-06 28.87751 -81.31046 Volusia USA, FL S/G/W 152 154 205 205 342 26-Oct-06 29.01163 -81.30248 Volusia USA, FL S/G/W 152 154 205 205 343 26-Oct-06 29.13915 -81.35762 Volusia USA, FL S/G/W 152 154 205 205 344 26-Oct-06 29.30308 -81.48654 Volusia USA, FL S/G/W 152 154 205 205 347 28.15408588 -81.97388657 Polk USA, FL 152 154 205 205 348 28.07758927 -81.96321675 Polk USA, FL 152 154 205 205 349 27.93274462 -81.85666331 Polk USA, FL 152 154 205 205 351 09-Nov-06 27.22252 -81.85878 DeSoto USA, FL W/D 152 154 205 205 151 28-Nov-05 18.332183 -66.717767 Rio Abajo State Forest, Arecibo USA, Puerto Rico 152 154 205 205 138 06-Apr-07 -4.09835 29.50636 Kigwena Forest Burundi 161 161 211 211 Wild 413 Oct. 2007 22.438444 88.4005 Kolkata India 172 174 198 214 415 Oct. 2007 22.438444 88.4005 Kolkata India 172 174 198 214 421 Oct. 2007 22.438444 88.4005 Kolkata India 172 174 198 214 433 Oct. 2007 22.438444 88.4005 Kolkata India 172 174 198 214 439 Oct. 2007 22.438444 88.4005 Kolkata India 172 174 198 214 444 Oct. 2007 22.438444 88.4005 Kolkata India 172 174 198 214 500 Nov. 2007 -21.25 47.45 Ranomafana, Ifanadiana Madagascar 172 174 198 214 187 23-May-07 6.950556 0.586667 Misahohe Togo 197 197 Wild


40Tuber Tuber/Bulbils Bulbils Large Tuber Flowers Leaves D. bulbifera variety Acrid and nauseous Angula r Undevelope d White, smooth; gilvous bulbils Bulbils dark, warte d Short-stalked; few rootlets Elongate d Male sepals up to 4m m Elongate d Triangula r Short Locations recorded by Prain & Burkill (1936) Locations recorded from Terauchi et al ., 1991; Ramser et al. 1996 vera Yes No No No No No No Yes Malay Peninsula; Australia Taiwan; Japan deltoidea Yes No No No No No Yes Hong Kong; Guangdong Province ("Kwang-tung"); Phillipines heterophylla Yes No No No No Yes Malay Peninsula Thailand simbha Yes No No No Yes Northern India Himalayas elongata Yes No No Yes Australia Papua New Guinea kacheo No No No No No Yes Sikkim Himalayas suavior No No No No Yes India; Java; Madoera; Australia; Fiji; Samoa; Tahiti Taiwan; Australia birmanica No No No Yes Burma (near borders w/ Thailand and China) sativa No No Yes Sri Lanka; India; Bangladesh; Japan; Andaman Islands; Singapore; Java; Phillipines Tonga; Hawaii anthropophagorum No Yes Africa Africa Florida Yes1 No1 No2 ? ? ? ? ? ? No2 No2 Figure A-1. Morphological clas sification of varieties a nd geographic locations in D. bulbifera L. Morphology summarized from the key of Prain and Burkill (1936). 1(Ward, 1977) 2(Wheeler et al ., 2007)


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BIOGRAPHICAL SKETCH A native of Florida, Matthew has been fascinat ed with trees, and the things that grow on them since early childhood. A terrific high school science faculty helped grow his wonderment for biology in unique directions, and first expos ed him to the world of plant taxonomy. Field exercises in this subject on a Magnolia virginiana wetland adjacent to his school first brought him into direct contact with invasive Dioscorea bulbifera. During the course of air potato fights in this glade, he learned firsthand about the power and risks of long distance anthropogenic dispersal lessons not easily re plicated in the classroom. He al so flung a few good ones himself. These experiences left a lasting (sometimes purpl ish) impression on him, and he credits them for his enduring fascination with this unique vine. His mentors at Bryan College, where he earned his B. S. in biology, stoked his interest in biogeography, evolution, and mo lecular biology. Two internships administered by the Oak Ridge Institute for Science and Education helped him ga in hands-on research experience. As a research associate in the DNA core facility of a cancer research center, he gained many of the skills needed to take on projects independently. Looki ng forward, he hopes to continue studies in biogeography.