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Phylogeography and evolution of the Florida crown conch (Melongena corona)

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Title:
Phylogeography and evolution of the Florida crown conch (Melongena corona)
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Book
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English
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Hayes, Kenneth A., 1970-
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University of South Florida
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Tampa, Fla.
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Subjects / Keywords:
biogeography
population genetics
gastropoda
microsatellite dna
mitochondrial dna
Dissertations, Academic -- Biology -- Masters -- USF   ( lcsh )
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government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Summary:
ABSTRACT: Melongena corona and closely related congeners are a conspicuous part of the marine intertidal benthic communities of Florida and southeastern Alabama. Significant genetic differentiation among adjacent populations has been conjectured based on variation in shell morphology, habitat discontinuity, low levels of adult motility, and the presence of an aplanic lecithotrophic larval stage. Furthermore, studies of the highly variable shell morphology often have resulted in confusing specific and subspecific definitions of these gastropods, which are often referred to as the "corona complex". Variation in shell morphology may indicate local adaptation or environmentally induced phenotypic plasticity. In this study I utilized mitochondrial DNA sequences in order to reconstruct the phylogenetic relationships of crown conchs, and nuclear microsatellite loci to investigate the patterns of relatedness within and among populations inhabiting the southeastern United States. Approximately 500 individuals from 20 populations throughout the known range of the Crown Conch were genotyped at eight microsatellite loci. Additionally, a 1200bp portion of the cytochrome oxidase subunit I gene was sequenced along with a 490bp fragment of the 16s ribosomal gene from individuals representing all known species and subspecies of the genus Melongena. Phylogenetic analyses completed with these data provide no support for current taxonomic designations within this group and these genetic data indicate that the corona complex is composed of a single polymorphic species. Furthermore, microsatellite data reveal population structure consistent with restricted gene flow between extant populations and phylogeography heavily influenced by historical sea-level fluctuations during the Late Pleistocene.
Thesis:
Thesis (M.S.)--University of South Florida, 2003.
Bibliography:
Includes bibliographical references.
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by Kenneth A. Hayes.
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Title from PDF of title page.
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Document formatted into pages; contains 201 pages.

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PHYLOGEOGRAPHY AND EVOLUTION OF THE FLORIDA CROWN CONCH ( MELONGENA CORONA ) by KENNETH A. HAYES A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Biology College of Arts and Sciences University of South Florida Major Professor: Stephen A. Karl, Ph.D. James R. Garey, Ph.D. Gordon A. Fox, Ph.D. Date of Approval: November 20, 2003 Keywords: population genetics, microsate llite dna, mitochondrial dna, gastropoda, biogeography Copyright 2003, Kenneth A. Hayes

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This thesis is dedicated to my wife Pam. Without her unwavering support, patience, love, and companionship this jour ney would not have been possible or worthwhile. In the voice of seashells, in the echo of blood rushing through our veins, the waters of life, the sea, are singing. Deborah Cramer 2001

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Acknowledgements There are a number of people to whom I ow e my sincere gratitude for making the last four years a memorable and wort hwhile educational experience. First, my thesis advisor Dr. Stephen Karl for allowing me to work in his lab, his patience, always challenging me to strive toward ex cellence and a more t horough understanding of the world around me, and providing guidance an d support in both academic and personal pursuits. I hope that the e xperience was as enriching for him as it was for me. Dr. James Garey who in addition to serving on my comm ittee and acting as a dive buddy also gave me my initial introduction to the world of molecular genetics when I was an undergraduate. Dr. Gordon Fox for serving on my committee and providing thoughtful discussions and insightful comm ents concerning my thesis. Dr. Joseph Simon, as a mentor and a friend, in spired and encouraged me in his unique way to pursue excellence in science and in life. My extended Karl lab family; Anna Bass, Tonia Schwartz, Caitlin Curtis, Cecilia Puchulutegui, Emily Severance, Maria Cattell, Stefan Schulze, Kevin Jansen, and Andrey Castro, all of whom have contributed in unique ways to enriching my thesis and my life. I will miss the Bahamas “research” cruises. Matt Moody, my undergraduate assistant, for isolating DNA from a large number of snails. Members of the Garey lab; Brent Nichol s, Mike Roberson, Heather Hamilton, Terry Campbell, David Karlen for providing se quencing facility support and for numerous conversations about science, invertebrates, gr aduate school, and life in general. Special thanks to a good friend and exceptional dive buddy Brent Nichols, who was one of the few people I could convince to go diving at 5:00 am. The collecting trip to Alabama would not have been as memorable without him. The USF Department of Biology support st aff who make everyt hing run smoothly and without them very few graduate students would ever finish.

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Phil Poland for providing samples, photos and enlightening discussions about morphological variation in populations of Melongena corona Dr. Jennifer Walker for samples and conversations about the ecology and natural declines in populations of crown conchs. Emilia Gonzalez Vallejo and Dr. Harris Lessios for samples of M. bispinosa and M. patula respectively. Dr. Stuart Berlocher for insights and advi ce about the evolution of Melongenidae and speciation in marine gastropods. Funding for this thesis was provided by Amer ican Malacological Society, Conchologist of America, Sigma Xi, American Museum of Natural History, Florida Association of Benthologists, University of South Florid a Dept of Biology, Biol ogy graduate student organization, and a National Science Foundati on Grant in Systematics to Dr. Stephan Karl. Finally, I owe my overwhelming gratitude to my family. My parents who’s encouragement, support, and beli ef in me have allowed me to pursue my dreams of being a biologist. My sister Re na, who throughout my life has always been there when I needed her, and my brother Tony who has remained an ever present friend despite the fact that I tortured him endlessly as a ch ild. His generosity and creativity are an inspiration. My wonderful wife to whom I dedicate this thesis.

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i Table of Contents List of Tables iii List of Figures iv Abstract v Chapter One – Natural History of Crown Conchs & Thesis Overview 1 Introduction to the Family Melongenidae 1 Paleontological Perspec tives and Geologic History 1 Taxonomy and Distribution 6 Ecology 10 Reproduction and Development 13 Thesis Overview – Objectives & Organization 15 References 16 Chapter Two – Mitochondrial Phylogeny of Crown Conchs: The Corona Complex Simplified 27 Introduction 27 Mitochondrial DNA 30 Methods 32 Sample Collection & Tissue Storage 32 DNA Isolation 33 mtDNA Amplification & Sequencing 34 Phylogenetic Analysis 37 Results 38 Sequence Characteristics COI 38 Sequence Characteristics 16S 40 Sequence Diversity 40

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ii Phylogenetic Analysis 42 Discussion 43 Divergence Timing 49 Other Intertidal Invertebrates 54 Taxonomy 56 Conclusions 57 References 58 Chapter Three – Microsatellite Analysis of Crow n Conchs: Population Structure and Evolution of an Intertidal Snail. 72 Introduction 72 Methods Sample Collection & Library Development 79 Cloning 81 DNA Isolation 82 Genotyping 82 Statistical Analyses 83 Results 86 Microsatellite Diversity 86 Disequilibrium Tests 87 Intra-Population Measures 87 Population Structure 88 Discussion 91 Conclusions 104 References 107 Appendices 131 Appendix A.1 – COI Variable Sites 132 Appendix A.2 – 16S Variable Sites 138 Appendix B – Sample Data 139 Appendix C – Allelic Frequencies For Each Locus Across All Populations 179 Appendix D – Allelic Frequencies By Locus Per Population 187

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iii List of Tables Table 2.1 COI primers 69 Table 2.2 Sequence divergence D xy 70 Table 2.3 COI distance matrix 71 Table 2.4 16S distance matrix 71 Table 3.1 Genescan Mixes 119 Table 3.2 AMOVA groupings 120 Table 3.3 Microsatelli te characteristics 121 Table 3.4 Populations statistics 122 Table 3.5 Results from test for Bottleneck 127 Table 3.6 Pairwise FST & RST estimates 128 Table 3.7 Pairwise ( )2 & geographic distances 129 Table 3.8 AMOVA results 132

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iv List of Figures Figure 1.1 Distribution of the genus Melongena 21 Figure 1.2 Shell morphology w ithin the corona complex 23 Figure 1.3 Distribution of taxa within the corona complex 24 Figure 1.4 Live Melongena corona in their natural habitat 25 Figure 1.5 Egg Capsules from Melongena corona 26 Figure 1.6 Newly emerged juvenile Melongena corona 26 Figure 2.1 Map of sample sites with numbers sequenced for COI & 16S 66 Figure 2.2 COI bootstrapped maximum likelihood tree 67 Figure 2.3 16S bootstrapped ma ximum likelihood tree 68 Figure 3.1 Map of sample sites with number of indi viduals genotyped 115 Figure 3.2 Neighbor-joining tree from FST estimates 116 Figure 3.3 Neighbor-joining tree from RST estimates 117 Figure 3.4 Neighbor-joining tree from ( )2 estimates 128

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v Phylogeography and Evolution of the Florida Crown Conch ( Melongena corona ). Kenneth A. Hayes ABSTRACT Melongena corona and closely related congeners ar e a conspicuous part of the marine intertidal benthic communities of Flor ida and southeastern Alabama. Significant genetic differentiation among adjacent popul ations has been conjectured based on variation in shell morphology, habitat discontin uity, low levels of adult motility, and the presence of an aplanic lecithotrophic larval stage. Furthermore, studies of the highly variable shell morphology often have result ed in confusing specific and subspecific definitions of these gastropods, which are of ten referred to as the “corona complex”. Variation in shell morphology may indicate lo cal adaptation or environmentally induced phenotypic plasticity. In this study I utilized mitochondria l DNA sequences in order to reconstruct the phylogenetic relationships of crown conchs, and nucle ar microsatellite loci to investigate the patte rns of relatedness within and among populations inhabiting the southeastern United States. Approximately 500 individuals from 20 populations throughout the known range of the crown conch were genotyped at eigh t microsatellite loci Additionally, a 1200bp portion of the cytochrome oxidase subun it I gene was sequenced along with a 490bp fragment of the 16s ribosomal gene from individuals representi ng all known species and subspecies of the genus Melongena Phylogenetic analyses co mpleted with these data provide no support for current taxonomic design ations within this group and these genetic data indicate that the corona complex is composed of a single polymorphic species. Furthermore, microsatellite data reveal popul ation structure consiste nt with restricted gene flow between extant populations and phylogeography heavily influenced by historical sea-level fluctuati ons during the Late Pleistocene.

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1 Chapter 1: Natural History of Crown Conchs & Thesis Overview Introduction to the Family Melongenidae The family Melongenidae (Gill, 1867) consists of seven genera of in tertidal, carnivorous gastropods that are globally distributed in tropical and subtropical habitats. The two largest genera, Busycon and Melongena along with genus Hemifusus contain several species that are commercially harvested for their meat (Di Cosimo 1986; Kaplowitz 2001; FA 2003). Snails from this family are commonly referred to as whelks, melon conchs, or crown conchs; however, the latter ve rnacular is more specific to snails in the genus Melongena the family’s namesake. A few species within the genera Busycon and Melongena have been the subject of several studies and reviews in an attempt to explain the evolution, speciation, and phylogenetic relationships with in these genera (Clench and Turner 1956; Paine 1962; Harasewych 1982; Edwards 1988 Tucker 1994; Berlocher 2000). The present study focuses on the evolutio nary relationships of the extant members of the genus Melongena primarily those occupying the southeastern United States. Paleontological Perspectives & Geologic History In order to understand the relatio nships of extant species it is first necessary to review the evolutionary history of such ta xa. In addition to a number of extant species in the genus

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2 Melongena various fossil species have been de scribed over the years (Heilprin 1887; Dall 1890; Petuch 1994). Although the paleontologi cal history of the genus dates back to the Early Eocene, it first appears in the North American fossil record as Melongena crassicornuta (Conrad,1853). This species lived during a warmer period in North America (ca. 35 mybp) and was first desc ribed from material found in Vicksburg limestone north of present day Florida. M. sculpturata (Dall, 1890), a species that closely resembles present day forms, first appears in the Florida fossil record 10 million years (my) later (Miocene Ballast Point silex-beds near Tampa) (Dall 1890). The presence of crown conchs in the fossil record indicates th at the lineage has endured through considerable climatic variab ility during the last 35 my, much of which should have significantly influenced curre nt distribution patterns and genetic structure. During the Upper Eocene and Oligocene epochs the climate was warmer than present and Florida was a shallow subtropical carbonate ba nk similar to the Bahama Banks of today. Early in the Oligocene epoch the Appalach ian Mountains were dramatically uplifted resulting in increased erosion of sands, silt and clay sediments. As a result of the erosional uplift during the Oligocene, siliciclastic sediments from the Appalachians began to settle over the carbonate platform gradually producing the surface soils of Florida. By the Miocene (25 mybp) land biota begin to populate the once submerged areas of Florida. At the same time, in creased erosion combined with a sea level regression allowed the filling of the Gulf Trough eventually blocking the flow of the Suwannee current by the Early Pliocene ( ca. 4.1 mybp). The Suwannee current, which was at its deepest during the Oligocene, ha d a northeasterly flow passing over the area

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3 that is now the panhandle of Florida and sout hern Georgia. Prior to the Pliocene this seaway connected the Gulf of Mexico and the Western Atlantic Ocean, and swept away sediments preventing their deposition ove r the Florida Platform (Stuckey 1965; Huddlestun 1993; Scott 1997). A number of author s have also asserted that the former current provided a pathway for gene flow between the northern Gulf of Mexico and Atlantic populations of several marine taxa, and the genetic similarities among populations separated across this region are a testament to this gene flow. (Bert 1986; Cunningham et al. 1991; Avise 1994; McMillen-Jackson et al 1994). Throughout the Miocene the climate cooled due to growth of the Antarctic ice sheet, yet the global climate remained warmer than pr esent day conditions. As the Florida land mass grew, more shallow marine habitats b ecame available, and some have speculated that Melongena spp along with other invertebrate s expanded southward into newly formed shallow water regions (Petuch 1988; 1994; Jones 1997). Approximately 5 – 10 mybp the cooling began to slow and by th e beginning of the Pliocene the climate remained warmer and sea level was approxi mately 100 m higher than at present. Florida is rich with fossil records of now extinct Melongena spp from the Pliocene and Pleistocene. Many of these species occurr ed sympatrically, and their designation as species is based on often minor differences in shell size and sculpture (spination). For example, Petuch (1994) describes ten new sp ecies and a single new subspecies based primarily on shell size and spination. It is possible that these may be different forms that warrant separate species definitions; howev er, many appear to ha ve only slight and possibly insignificant deviations from shell morphologies found in present day species.

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4 Formation (biomineralization), sculpture, and coloration of mollu sc shells may be strongly influenced by the biotic (i.e. predators) and abiotic parameters (i.e. temperature, pH, and salinity) of the surrounding envi ronment (Strushaker 1968; Mitton 1977; Newkirk and Doyle 1975; Berger 1983; Kat & Davis 1983; Katoh & Foltz 1994; Trussell 2000; Chiu et al. 2002; Trussell 2002). Give n the extreme climatic instability of the time period, and the possibility of environm ental influences on shell morphology, an alternative explanation for the variations witnessed in Petu ch’s species and many extant species is that they are merely the products of such forces. Glacial events during the Pliocene and Pleist ocene drastically changed climates, altered habitats, and rearranged coastlin es in various regions of th e world resulting in complex patterns of evolutionary history. This is abundantly apparent in southeastern North America, specifically Florida’s coastal habitats. Florida, with its latitude, low relief and recent emergence is intimately linked with the sea. During the Pliocene peninsular Florida was completely submerged repeated ly as glaciers waxed and waned causing sea level to fall and rise. Pleistocene seas were similarly erratic; although, peninsular Florida was not completely inundated during any poi nt in this period (Webb 1990; Davis 1997; Scott 1997). For example, during the last glacial maximum (Wisconsinan ca. 20,000 ybp) Florida’s peninsular land mass was appr oximately twice its current size, which would put Miami Beach approximately 6 m iles inland (Webb 1990). Lowered sea level coincided with the onset of a drier and significantly cooler climate throughout the region resulting in mass extinctions of regional fauna, including numerous mollusc species (Jones 1997; Webb 1990; Petuch 1994, 1995; Muhs et al. 2002). In contrast, during

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5 Pleistocene glacial minima average temperat ures increased and mo st of the Florida platform was submerged leaving only small ridges of exposed land surrounded by warm shallow waters (Webb 1990; Muhs et al 2002). During these warmer periods many marine species proliferated a nd expanded their ranges, especia lly intertidal species that survived in the warmer southern waters (Webb 1990; Petuch 1994; Jones 1997). Recent studies indicate that over the last two million years there have been as many as two dozen such glacial episodes each assumed to have had similar impacts on Florida shorelines. Since the end of the last glaci al maximum sea level has con tinuously risen to its present day level (Davis 1997). The rate of sea-level rise ove r the last 12,000 years is responsib le for the coastal variation in Florida and has had a major impact on th e distribution of regional fauna, especially those intimately linked to intertidal habitats. For example, the rate of sea-level rise between 12,000 and 7,000 ybp has been estimated at approximately 1cm per year (Davis 1997). At this rate, af ter as little as 100 years, the Florida shoreline would have moved inland as much as 1,000 m (1 km) in some areas (Davis 1997). Approximately 7,000 ybp, the rate of sea-level rise began to slow to approximately 3 mm per year, which is still a substantial change in sea-level over a relatively short period. Then, about 3,000 ybp sea-level began to fluctuate around its current level (Fairbridge 1961; Davis 1997). This would have allowed substantial time for the formation of Florida’s present day coastal features, yet too little time would have passed to mask the influences of Florida’s dynamic past on the distribution and evol utionary history of intertidal fauna

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6 Taxonomy and Distribution The snails in the genus Melongena (Schumacher, 1817) are restrict ed to intertidal regions of the tropical and subtropical Western Atlan tic and Eastern Pacifi c (Figure 1.1) (Clench & Turner 1956). Presently, there are six extant species, three reported from the waters of Alabama and Florida and the other three o ccurring outside of the United States. Melongena melongena (Linne, 1758) and M. patula (Browerip & Sowerby, 1829) are closely related and considered geminate spec ies, speciating after the formation of the isthmus of Panama nearly 3.1 – 3.5 mybp (Bayer et al. 1970; Clench & Turner 1956; Keen 1971; Radwin 1969; Vermeij 1978; Collins 1989). M. melongena ranges from Tampico, Mexico through Central America, east along the northe rn coast of South America to Guyana, and islands of the An tilles (Clench & Turner 1956; Collins 1989). M. patula is found in the Eastern Paci fic from the northern part of the Gulf of California to Ecuador (Keen 1971; Abbott 1974). A third species, M. bispinosa (Philippi, 1844) is found only around the Yucatan Peninsula of Mexico and is believed to be a remnant of a once much wider distributed species since re legated to its current location. Based on fossil records from Pliocene beds in Florida it has been suggested that M. bispinosa may have closer affinities to the other species found in Florida and Alabama (Clench & Turner 1956). In North America, the first extant species described was Melongena corona (Gmelin, 1791). Gmelin asserted that this was the only species of Melongena in this region and that it was made up of a numbe r of varieties or morphotypes delineated by shell size and spination. Based on a few characters such as presence or absence of basal spines, number

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7 of shoulder spine rows, shell coloration, and ove rall size, subsequent authors speculated that these varieties were actually species an d/or subspecies within a complex that has become known as the “ Melongena corona species complex” or simply the “corona complex” (Figure 1.2 & 1.3). Over the last century a number of species and subspecies descriptions have appeared ; although, current classifica tion only recognizes three nomenclaturally valid species in the comp lex and two or possibl y three subspecies (Clench & Turner 1956; Tucker 1994). Based on the literature and a qualitative assessment of shell variation (primarily spination, size, and color), Clench and Turner (1956) reviewed the family Melongenidae, and concluded that in the south eastern United States the genus Melongena consisted of two species; Melongena corona and Melongena bicolor (Say, 1827). M. corona, in turn, consists of a complex of three subspecies, M. c. johnstonei (Clench & Turner 1956), M. c. corona, and M. c. altispira (Pilsbry & Vanatta 1934), o ccurring along the coasts of Alabama and the panhandle of Florida, the gulf coast of Florida and in the Keys and Atlantic coasts of Florida re spectively. They referred to this group of species as the Melongena corona complex, and assumed, as others have, that M. bispinosa was a remnant of the Pliocene, since isolated from the rest of the species in the complex. Additionally, they confirmed the previously designated geminate species within the genus, one in the Western Atlantic, Melongena melongena and another on the Pacific side of the Isthmus of Panama, Melongena patula Hamilton (1980) using a statistical approach, ev aluated the use of shell spination as the primary character in differentiating subspecies in the complex. Specifically, he evaluated

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8 the relationship between presence of basal sp ines and snail size in 568 individuals from two populations, one consisting of M. corona johnstonei and the other of M. corona corona He concluded that basal spines of M. corona are acquired on togenetically, and their presence is positively related to size of th e snail. Consequentl y, he established that shell spination is not a valid trait by which s ubspecific distinctions can be made in this species. Tucker (1994), unaware of or unconvinced by Hamilton’s results, completed the first comprehensive statistical examination of th e species complex throughout its range in the southeastern United States. In his analysis he used meas urements of shell width and length along with spine counts and the ratio of shoulder spine counts divided by anterior (basal) spine counts. He concluded that the complex actually consisted of three species, M. bicolor, M. sprucecreekensis (Tucker 1994) and M. corona which could be further divided into two subspecies, M. c johnstonei and M. c corona In his comparison between his classification and that of Clench and Turner, he lists M. bispinosa as part of the complex, yet he does not include samples from this species in his analyses or comment on its relation to the other species. From this it appears that he believed, as others have, that this species is a relic of a once widely di stributed species that is now only found in the Yucatan. Much of the evid ence for this is provided by a number of fossil shells of this species recovered from Pliocene sediments in Florida (Petuch 1994). The currently used classification scheme places the extant taxa of the corona complex into geographically distin ct localities (Figure 1.3 ) Melongena sprucecreekensis and Melongena bicolor both occur along the Atla ntic coast of Florida. M. bicolor is

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9 distributed from Florida Keys northward along the Florida coast to Matanzas inlet while M. sprucecreekensis is limited to the Spruce Creek Estuary approximately five miles north of New Smyrna Beach, Florida. M. corona occupies the gulf coast and panhandle of Florida, the southeastern most coas t of Alabama, and possibly overlaps with M. bicolor on the east coast of Florida. M. corona is comprised of eith er two (Tucker 1994) or three (Clench & Turner 1956) subspecies depending on investigator. Both Clench & Turner and Tucker give the range of M. corona johnstonei as Little Lagoon, Alabama, eastward along the panhandle to Keaton’s Beach, Florida. They agree that M. c. corona occurs from Cape Flamingo, Florida in the south to Deckle Beach, Florida where it is speculated to form a species gradation into M. c. johnstonei to the north. Rounding out Clench and Turner’s review of the corona complex is M. corona altispira which overlaps in range with M. bicolor Tucker, however, did not rec ognize this final subspecies, and only considered there to be two valid s ubspecies in the complex (Figure 1.3). In the Gulf of Mexico, corona complex speci es reach as far northw est as Little Lagoon, Alabama (Levy 1979; Tucker 1994). Clench and Turner (1956) gave Matanzas Inlet, south of St. Augustine as the northern limit of the crown conch on Florida’s east coast; however, they indicated this r ecord came from a single empty shell. Subsequently, Loftin (1987), gave a location just a bit further south near Daytona, Florida as the northern limit. Early in this study, I tended to agree with Lof tin, as I had made several trips to Matanzas Inlet and only found empty shells. In the summer of 2001, after communications with Dr. Richard Gleeson at the Tolomato Matanzas National Estuarine Research Reserve, I was able to collect a number of snails in the Matanzas Inlet as far north as Flagler Beach,

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10 FL. The collection of these individuals and the presence of a number of empty shells further north in the Inlet lead to the specu lation that these snai ls expand their range during warmer years, migrating northward in the Intracoastal Waterway. The previous few winters had been fairly mild in Fl orida (NOAA 2003), which may have facilitated such an expansion. In subsequent years the snails that migrate northward are most likely extirpated by cold winters. Based solely on these anecdotal accounts, this speculation seems reasonable, especially considering th e fact that crown conchs often suffer high mortalities during low tides with near freezing temperatures (Loftin 1987; J. Walker pers. com.). Until future research evaluating th is scenario is completed it will remain speculative, and the northern limit of crown conchs should be considered as Matanzas Inlet south of St. Augustine. Ecology Mature crown conchs ( Melongena spp.) primarily occupy the middle to upper intertidal zone where they make up a considerable part of the epifaunal community. They inhabit shallow low-energy embayments, lagoons, salt marshes, mangrove swamps and oyster bars; however, they are noticeably absent from high-energy beaches exposed to wave action (Figure 1.4) (Pilsbry & Vanatta 1934; Clench & Turner 1956; Loftin 1987). Occupants of intertidal habitats are exposed to a unique set of ch allenges not faced by subtidal organisms. Some of these challenge s include dealing with the tides, exposure to air, waves, extreme temperatures, and c onstantly changing salinity (Cheung 1997; Chapman 2000). All of these factors have play ed a role in shaping the distribution and life history attributes of intertidal organism s, and crown conchs are no exception. The

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11 impacts of temperature fluctuation on crow n conchs have already been discussed; however, other influential parameters includ ing wave action, salin ity, and depth help shape such facets of crown conch life hist ory as feeding, reproduction, and behavior (Clench & Turner 1956; Hathaway & Woodburn 1961; Loftin 1987). In the southeastern United States, crown conchs can often be found feeding on a variety of prey including bivalves (i.e. Anomalocardia spp., Macrocallista spp., Polymesoda spp., Crassostrea virginica ), gastropods (i.e. Stramonita spp. Fasciolaria hunteria, Cerithidea spp. Batillaria spp., Melongena spp. ), horseshoe crabs ( Limulus polyphemus ), and other invertebrates (Gunter 1957; P. Poland pers com.; pers. obs.). Additionally, they are voracious scavengers and quickly consume any dead and dying organisms in the intertidal zone. As is often th e case for intertidal organisms, the availability of these food resources may be altered by wave intensity (Barnes 2002; Miller et al 2002). Crown conchs are typically found in low wave energy, high depositional areas, and constraint to this particular habitat may result from the influence of wave action on various aspects of the ecology of these snai ls. High wave intensity alters abiotic parameters such as deposition ra tes and substrate types (Miller et al 2002), which in turn influence such facets of snail biology as ease of movement, hunting proficiency, prey abundance and reproduction (Lofton 1987; Bowling 1994; Koch & Wolff 1996; Chapman 2000; Barnes 2002). Hathaway & Woodburn (1961) determined that adult Melongena may survive for long periods in salinities as low as 8 ppt, and in nature they ma y experience extreme changes

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12 in salinities over short periods. Although th e adults endure these extremes, normal activities, including re production, require a salinity ra nge of 20 – 30 ppt (Hathaway & Woodburn 1961; Albertson 1980). As expected, embryos and juveniles are much more sensitive to changes in salinity and require a range of 25 – 30 ppt. Embryos as young as three days old subjected to salinity of 21.5 ppt developed abnormally, and those exposed to lower salinities often died (Hathaway & Woodburn 1961). Caldwell (1959) and Dinetz ( 1982) report lack of adult c onch movement to deeper waters, and low levels of motility out side of their home range. Although Melongena typically inhabit the shallow intertidal zone commercial crabbers have reported finding them in crab traps in water as deep 2.5m (Gleeson pers. com.). Conchs move by a combination of ciliary movement and pedal waves and can crawl over a variety of substrates. Therefore, there does not appear to be a physical barrier to the movement of adults to deeper waters; however, a number of ecological factors may inhibit dispersal into deeper regions. Predation on healt hy adults under natura l conditions is an uncommon occurrence; although, in rare instance s and in aquaria kept crown conchs, the Florida Horse Conch ( Pleuroploca gigantea ) and the Lace Murex ( Chicoreus florifer ) will kill and consume living Melongena These predatory species, as well as others, usually inhabit subtidal ha bitats surrounding those of Melongena which may provide the necessary pressure to prevent the dispersa l of adult crown conchs. Resource abundance may also play a role in limiting their disper sal to deeper territories. For instance, Melongena often feed upon small bivalves and othe r gastropods that occur in higher

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13 abundances intertidally, provi ding further impetus for adults to remain in the midintertidal range (Gunter 1957; Hath away 1958; Woodbury 1986; Loftin 1987) Reproduction & Development Melongena are typical direct developing pros obranch gastropods with aplanic lecithotrophic larvae. They have separate sexes with the females on average being slightly larger than the ma les (Caldwell 1959; Loftin 1987). Female conchs appear to have a preference for depositing their egg capsu les in a limited portion of the intertidal zone (Loftin 1987), and beginni ng in late winter and con tinuing throughout the summer they imbed 15 560 fertilized eggs into an e gg capsule and attach these capsules to the substrate along a single ribbon-like base in a row consisting of between six and twenty capsules each (Figure 1.5; Ha thaway & Woodburn 1961). Capsules are deposited in the lower intertidal zone on a number of different substrates and have been found on rocks, shells, wood, polychaete tubes, sea grass, mangroves, bridge pilings, discarded bottles and cans, shoes, and even on the shells of living crown conchs (Clench & Turner 1956; Hathaway 1958; Loftin 1987; pers. obs.). Clen ch and Turner (1956) reported that eggs hatch approximately 27 days after deposition, and they stated that juveniles were incapable of swimming. Crawling behavior on the sides of the aquarium immediately following emergence from the capsule has been observed numerous times by other authors (Hathaway 1958; Gunter & Menzel 1958; Albertson 1980). Hathaway (1958) and Loftin (1987) reported hatching after only 20 days and Loftin described swimming behavior in newly emerged juve niles. During this study my observations were similar to those of Loftin’s with respect to the timi ng of hatching and behavior of newly emerged

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14 juveniles (Figure 1.6). Loftin speculated that Clench and Turner may not have observed swimming hatchlings as a result of the ex tended period spent in the egg capsules by embryos in their aquaria. Other authors ma y have missed these swimming juveniles as well because of their small size and the relativ ely high rate at which they aggregate on the glass sides of the aquaria (Loftin 1987). Although few details exists regarding the life of juveniles after hatc hing, previous studies have found that despite capsule deposition in the lower intertidal zone, the youngest snails are typically found in the high intertidal zone (Woodbury 1986; Dinetz 1982). Loftin (1987) speculated that newly emerge d snails may swim to the surface and are carried shoreward by surface currents, which ma y be further pressure to deposit the egg capsules as close to shore as possible while minimizing the risk of exposing the capsules during low tide. Once they arrive in the uppe r intertidal zone young sn ails are believed to bury themselves in the sediment and feed upon a variety of detrital ma terial and juvenile bivalves. After first emerging from the capsule juvenile crown conchs measure less than 1 mm, and after only a couple of months may grow to more than 6 mm (Loftin 1987). In growth studies Caldwell (1959) concl uded that growth primarily occurs during the warmer months, which corresponds to the period when snails are most active; however, that study focused on snails located around Cedar Key. Since Cedar Key is located north of the frost line in Florida and may experience substa ntially lower temperature than much of the region south of that point further studies are needed to determine if seasonal variation in growth rates is consistent across th e geographic range of crown conchs.

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15 Thesis Overview Thesis Objectives and Organization The primary of objective of this thesis was to investigate the systematic relationships within the corona complex using molecu lar genetic techniques. Phylogenetic reconstruction was carried out using mitochondrial DNA sequences, and further resolution of inter-population rela tionships were explored thr ough microsatellite analysis. Using both of the these data sets in conj unction with climatic records and geologic history an attempt was made to understand the forces that have intermingled to produce the contemporary corona complex The second chapter covers th e phylogenetic reco nstruction of the crown conchs and discusses the possible factors responsible for evolution of the complex. Using the mitochondrial data along with data from simila r studies I place divergences within this group in an approximate time frame. In the fina l chapter, analyses of microsatellite loci are used to estimate levels of population subdi vision and genetic diversity, which in turn are used in conjunction with climatic a nd geologic records to understand current biogeography of Melongena spp in the southeastern United States.

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16 References Abbott, T. 1974. American seashells. 2nd edition. New York, Van Nostrand Reinhold Co. Albertson, H. D. 1980. Long term effects of high temperatures and low salinities on specimens of Melongena corona and Nassarius vibex Ph.D. Dissertation, University of Miami, Coral Gable FL pp.222. Avise, J. C. 1994. Molecular Markers, Natural History and Evolution. New York, Chapman & Hall. Barnes, R. S. K. 2002. The occurrence and eco logy of a marine hydrobi id mudsnail in the southern hemisphere: the Knysna Estuary, South Africa. African Journal of Ecology 40(3): 289-294. Bayer, F. M., G. L. Voss and C. R. Robi ns 1970. Report on the marine fauna and benthic shelf slope communities of the isthmian region. Bioenvironmental and radiological safety studies Atlantic-Pacific Interoceanic Canal. Miami, University of Miami: 1-99 + Appendices. Berger, E. M. 1983. Population genetics of marine molluscs. In Russel-Hunter, W.D. (Ed) The Mollusca Volume 6 Ecology. London, Academic Press, Inc. 6: 563-596. Berlocher, S. H. 2000. Allozyme varia tion in Busycon whelks (Gastropoda : Melongenidae). Biochemical Genetics 38(9-10): 285-295. Bert, T. M. 1986. Speciation in west ern Atlantic stone crabs (genus Menippe ): the role of geological processes and climatic events in the formation and distribution of species. Marine Biology 93: 157-170. Bowling, C. 1994. Habitat and size of the Florida crown conch ( Melongena corona Gmelin): Why big snails hang out at bars. Journal of Experime ntal Marine Biology and Ecology 175: 181-195. Caldwell, D. K. 1959. Notes on the crown conch, Melongena corona Nautilus 72(4): 117-122.

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17 Chapman, M. G. 2000. A comparative study of differences among species and patches of habitat on movements of three species of intertidal gastropods. Journal of Experimental Marine Bi ology and Ecology 244(2): 181-201. Cheung, S. G. 1997. Physiological and behavioura l responses of the intertidal scavenging gastropod Nassarius festivus to salinity changes. Marine Biology 129(2): 301-307. Chiu, Y.-W., H.-C. Chen, S.-C. Lee and C. A. Chen 2002. Morphometric analysis of shell and operculum variations in the viviparid snail, Cipangopaludina chinensis (Mollusca: Gastropoda), in Taiw an. Zoological Studies 41(3): 321-331. Clench, W. J. and R. D. Turner 1956. The fa mily Melongenidae in the Western Atlantic. Johnsonia, 3(35): 61-187. Collins, T. M. 1989. Rates of mitochondrial DNA evolution in transisthmian geminate species., Ph.D. Dissertation, Yale University. New Haven, Conn. Conrad, T. A. 1853. Monograph of the genus Fulgur Proceedings of the Academy of Natural Science 6: 319. Cunningham, C. W., L. W. Buss and C. Anderson 1991. Molecular and geologic evidence of shared history between hermit crabs and the symbiotic genus Hydractinia Evolution 45(6): 1301-1316. Dall, W. 1890. The Tertiary fauna of Florida. Transactions of the Wagner Free Institute of Science; Philadelphia 3: 118-122. Davis, R. A. 1997. Geology of the Florida Coast. In A. F. Randazzo and D. S. Jones (Eds), The Geology of Florida. University Press of Florida, Gainesville, FL. 155-168. Di Cosimo, J. 1986. Biological review and co mmercial whelk fisheries analysis of Busycon carica with comments on B. canaliculatum and B. contrarium MS Thesis. Virginia College of William and Mary: 125. Dinetz, B. J. 1982. Intraspecific si ze distribution of the crown conch, Melongena corona Gmelin: zonation on a low energy beach. MS Thesis. University of Florida, Gainesville, FL 73. Edwards, A. L. 1988. Latitudinal cl ines in shell morphologies of Busycon carica (Gmelin, 1791). Journal of She llfish Research 7(3): 461-466. Fairbridge, R. W. 1961. Eustat ic changes in sea level. Physical, Chemical, & Earth Sciences 4: 99-185.

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18 Fisheries Agency, Republic of Chin a 1996. Fish & Shell in Taiwan (I) http://www.fa.gov.tw/eindex.htm Gunter, G. and R. W. Menzel 1957. The crown conch, Melongena corona as a predator upon the Virginia oyster. Nautilus 70(3): 84-87. Hamilton, P. V. 1980. Shell spination in Melongena corona : subspecies characteristic or size related? Malacol ogical Review 13: 84-86. Harasewych, M. G. 1982. The evolution and zoogeography of the subfamily Busyconinae (Gastropoda: Melongenidae).PhD Dissertation, University of Delaware, Deleware. Hathaway, R. R. 1958. The crown conch Melongena corona Gmelin; its habits, sex ratios, and possible relations to the oyst er. Proceedings of the National Shellfish Association 48: 189-194. Hathaway, R. R. and K. D. Woodbu rn 1961. Studies on the crown conch Melongena corona Gmelin. Bulletin of Marine Science of the Gulf and Caribbean 11: 45-65. Huddlestun, P. F. 1993. A revision of the lithostr atigraphic units of the coastal plain of Georgia The Oligocene., Georgi a Geological Survey Bulletin: 152. Jones, D. S. 1997. The marine invert ebrate fossil record of Florida. In A. F. Randazzo and D. S. Jones (Eds), The Geology of Fl orida. University Press of Florida, Gainesville, FL. 89-117. Kaplowitz, M. D. 2001. Uncovering economic benefits of Chivita ( Melongena melongena Linnaeus,1758 & Melongena corona bispinosa Philippi, 1844). Journal of Shellfish Research 20(1): 295-299. Kat, P. W. and G. M. Davis 1983. Speciati on of molluscs from Turkana Basin. Nature 304: 660-661. Katoh, M. and D. W. Foltz 1994. Genetic s ubdivision and morphologi cal variation in a freshwater snail species complex formerly referred to as Viviparus georgianus (Lea). Biological Journal of the Linnean Society 53: 73-95. Keen, A. M. 1971. Sea shells of tropical west America. Stanford, Stanford University Press. Koch, V. and M. Wolff 1996. The mangrove snail Thais kiosquiformis Duclos: A case of life history adaptation to an extreme envi ronment. Journal of Shellfish Research 15(2): 421-432.

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19 Levy, I. C. 1979. A re-evaluation of the northwestern range of the Melongena corona complex Veliger 22(2): 206-209. Loftin, J. L. 1987. The distribution of Melongena corona (Gmelin 1791) egg capsules in North Florida. Thesis Dept of Biology, Fl orida State University,Tallahasee, FL. McMillen-Jackson, A. L., T. M. Bert and P. Steele 1994. Population genetics of the blue crab Callinectes sapidus : modest population structuring in a background of high gene flow. Marine Biology 118: 53-65. Miller, D. C., C. L. Muir and O. A. Haus er 2002. Detrimental effects of sedimentation on marine benthos: what can be learned from natural processes a nd rates? Ecological Engineering 19(3): 211-232. Mitton, J. B. 1977. Shell color and pattern variation in Mytilus edulis and its adaptive significance. Chesapeake Science 18: 387-390. Muhs, D. R., K. R. Simmons and B. Steinke 2002. Timing and warmth of the Last Interglacial period: new U-series evid ence from Hawaii and Bermuda and a new fossil compilation for North America. Quaternary Science Reviews 21: 1355-1383. Newkirk, G. F. and R. W. Doyle 1975. Genetic analysis of shell-shape variation in Littorina saxatilis on an environmen tal cline. Marine Biology 30: 227-237. NOAA 2003. Florida Climatic Summary Se ptember 2003, National Climatic Data Center, Ashville, N.C. 2003. Paine, R. T. 1962. Ecological diversifica tion in sympatric gastropods of the genus Busycon. Evolution 16: 515-523. Petuch, E. J. 1988. Neogene history of tropica l American mollusks. Charlottesville, VA., The Coastal Education & Research Foundation (CERF). Petuch, E. J. 1994. Atlas of Florida fossil shells (Pliocene and Pleistocene Marine Gastropods). Chicago, Chicago Spectrum Press & Graves Museum of Archaeology and Natural History. Petuch, E. J. 1995. Molluscan di versity in the Late Neogene of Florida: Evidence for a two-staged mass extinction. Science 270: 275-277. Pilsbry, H. A. and E. G. Vanatta 1934. Melongena corona and its races. Nautilus 47(4): 116-121. Radwin, G. E. 1969. A recent molluscan fauna fr om the Caribbean coast of southeastern Panama. Transactions of the San Diego Society of Natural History 15: 229-236.

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20 Scott, T. M. 1997. Miocene to Holocene history of Florida. In A. F. Randazzo and D. S. Jones (Eds), The Geology of Florida. Univer sity Press of Florid a, Gainesville, FL. 57-68. Strushaker, J. W. 1968. Selection mechanisms associated with interspecific shell variation in Littorina picta (Prosobranchia: Neogastropoda). Evolution 22: 459-480. Stuckey, J. L. 1965. North Caro lina: Its geology and mineral resources., North Carolina Division of Mineral Resources: 550. Trussell, G. C. 2000. Phenotypi c clines, plasticity, and morp hological trade-offs in an intertidal snail. E volution 54(1): 151-166. Trussell, G. C. 2002. Evidence of countergradient variation in the growth of an intertidal snail in response to wate r velocity. Marine Ecology Progress Series 243: 123-131. Tucker, J. K. 1994. The crown conch ( Melongena : Melongenidae) in Florida and Alabama with the description of Melongena sprucecreekensis n. sp. Bulletin of the Florida Museum of Natural Histor y, Biological Sciences 36(7): 181-203. Vermeij, G. J. 1978. Biogeography and adapta tion. Patterns of marine life. Cambridge, Mass, Harvard University Press. Webb, S. D. 1990. Historical biogeography. In R. L. Myers and J. J. Ewel (Eds), Ecosystems of Florida. Orlando, FL, Univ ersity of Central Florida Press: 70-102. Wessel, P. and W. H. F. Smith 1995. New vers ion of the generic mapping tools released. Eos, Transactions, American Geophysic al Union, Electronic Supplement 76: 329. Woodbury, B. D. 1986. The role of growth, pr edation, and habitat selection in the population distribution of the crown conch, Melongena corona Gmelin. Journal of Experimental Marine Biology and Ecology 97: 1-12.

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21 FIGURE 1.1: Map showing current distribution of extant members of the genus Melongena Map created using Generic Mapping Tools Software (ver. 3.0; Wessel & Smith 1995) available online at http: //www.aquarius.geomar.de/omc_intro.html. M. melongena M. patula M. corona complex M bis p inosa

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22 FIGURE 1.2: Digital images of shells from taxa within corona complex showing variation in shell morphology over the entire range of the group. Nomenclature based on the shell morphology and geographic location (Clench & Turner 1956; Tucker 1994). (1-3) M corona johnstonei (1,2) Little Lagoon Alabama, (3 ) Grand Lagoon – Panama City, FL; (4-7) M. corona corona (4) Cedar Key, FL, (5) Clearwate r, FL, (6) Safety Harbor, (7) Tampa Bay, FL; (8-10) M. bicolor ( corona aspinosa ), (8) Lower Matecombe Key, FL, (9) Key Largo, FL, (10) Plantation Key, FL; (11-13) M. bicolor ( corona altispira ), (11) Sebastian Inlet, FL, (12) Miami, FL, (13) Lake Worth, FL; (14-16) M. bicolor ( corona altispira ), (14) Indian River La goon, FL, (15) Port Orange, FL, (16) Indian River Lagoon, FL; (17-18) M. sprucecreekensis Spruce Creek Es tuary, FL; (19) M. bicolor Flagler County, FL. (All scale bars = 10 mm; *-indicates photos provided by Phil Poland).

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23 ** * * * * * *13 12 8 9 10 11 16 15 19 1718 14 1 6 7 4 35 2 FIGURE 1.2: Digital images of shells from taxa within corona complex

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Kenneth A. Hayes Chapter 1 24 M. corona johnstonei M. corona corona M. corona altispira M. bicolor M. sprucecreekensis FIGURE 1.3: Distribution of current nomenclaturally valid species and subspecies of the genus Melongena that make up the corona comp lex. Map created using Generic Mapping Tools Software (ver. 3.0; Wesse l & Smith 1995) available online at http://www.aquarius.geoma r.de/omc_intro.html. Georgia Alabama Florida Gulf of Mexico Atlantic Ocean

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Kenneth A. Hayes Chapter 1 25 FIGURE 1.4: (A) Living crown conch crawling over natural sand habitat at Ft. DeSoto Park in St. Petersburg, FL (photo by K. Haye s). (B) Live specimen from south Florida crawling over rock. (Photo B courte sy of P. Poland – available at http://www.jaxshells.org/corona.htm) A B

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Kenneth A. Hayes Chapter 1 26 FIGURE 1.5: Egg capsules from M. corona collected intertidally at Ft. DeSoto Park, Pinellas County, Florid a by KAH. Metric ruler in photo for size reference is in centimeter increments. FIGURE 1.6: Newly emerged juvenile Melongena corona (ca. 3 days). (A) Young snail extending foot from shell, note lack of velar lobes (scale bar = 0.5 mm); (B) Seve ral snails withdrawn into their shells (scale bar = 0.25 mm); (C) Several swimming juveniles with well developed velar lobe s (scale bar = 0.25); (D) A single juvenile swimming with use of velar lobes (scale bar = 0.25 mm). ft=foot, ey=eye, pc = protoconch, vl=velar lobe, fg = food groove, cl = cilia. Photos by K. Hayes. (A) (B) (C) (D) cl fg pc ey ft vl

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27 Chapter 2: Mitochondrial Phylogeny of Crown C onchs: The Corona Complex Simplified Introduction Molluscs are among some of the most studied marine invert ebrates, especially when including the large num ber of amateur malacologists invol ved in shell co llecting. Both amateur and professional malacologists alik e have been engaged in alpha taxonomy for decades, often resulting in a gluttony of speci es names and confusion over the validity of many of them. This is primarily the conseque nce of the ease in which shells are collected and studied; however, varia tion in shell morphology often may be the product of phenotypic plasticity (Berger 1983; Ka toh & Foltz 1994; Trussell 2000; Chiu et al. 2002.) As a result, many conchological base d species descriptions may be unsupportable and in need of reevaluation. Reassessments, whether based on genetic evidence or a combination of other characters, sometimes result in synonymization of species (Adamkewicz & Harasewych 1996; Knowlton 2000); although, occasionally such studies have demonstrated that minor shell char acters may hold the key to evolutionary complexity (Borsa & Benzie 1993; Parsons & Ward 1994; Johnson & Cumming 1995; Thollesson 1998; Trussel 2000). One striking example of a group of species described, almost exclusively, using shell morphology is that of the Melongena corona (Gmelin,1791) species group found intertidally in southeastern North America (for taxonomic review see Chapter 1).

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28 Gmelin originally described Melongena corona and since that time the taxonomic designation of this species has been the subject of debate among professional and amateur malacologists. Gmelin asserted that in Florida we have a single species, Melongena corona with a number of morphotypes or varieties delineated according to shell variation. Subsequently, other authors pu t forth the idea that these are actually a complex of species and subspecies within the genus Melongena often referred to as the “corona complex” (Dall 1890; Pilsbry & Vanatta 1934; Clench & Turner 1956; Tucker 1994) Lack of gene flow among populations re sulting from low vagility of juveniles is often cited as the primary fact or contributing to speciation in this group. This follows from the idea that benthic species with direct development have a lower dispersal potential than those with pla nktonic larvae that may remain in the water column for days to weeks. Supporting this is the widely held generalization of marine speciation maintaining that species with planktotr ophic larval stages ar e more genetically homogeneous across a wider geographic range and less subject to speciation events (Crisp 1978; Hedgecock 1986; Hansen 1983; Palumbi 1994; Hoskin 1997; Collin 2001). A growing number of studies, however, have clearly shown that many species with widely dispersing planktonic la rvae do not actually realize their true dispersal potential and many show levels of genetic structure no t anticipated based on this potential (Bert 1986; Reeb & Avise 1990; Karl & Av ise 1992; Bhaud 1998; Cunningham & Collins 1998; Schulze et al 2000). Several studies have found that other processes, such as localized extinction and recol onization, may play a greater role in shaping contemporary genetic structure relative to larval disp ersal potential (Cunni ngham & Collins 1998; Wilke 2003).

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29 In the southeastern United States, studies of regional biota have sought to disentangle the influences of climatic and other natural proc esses shaping the divers ity of extant species in the region. To date, a number of such studi es have implicated vicariant events of the Pliocene and Pleistocene in shaping the evolu tion of a regional fa una, including tortoises (Schwartz 2003), corals ( Severance & Karl in review ) mammals (Avise et al. 1998), snakes (Jansen & Karl in prep. ), fishes (Avise 2000), polychaetes (Schulze et al 2000), arthropods (Saunders et al. 1986; McMillen-Jackson et al 1994) and molluscs. ( Reeb & Avise 1990). Among these studies, those involvi ng intertidal inverteb rates have focused almost exclusively on species that produce pl anktonic larvae. The interest in these particular animals stems from the expectati on of minimal genetic structure over a large geographic range in species with potentia lly wide dispersing larvae. Counter to expectations, all of these studies repo rt significant gene tic subdivision among assemblages in the southeastern United States often revealing closel y related sister areas separated into the Gulf of Me xico and the Atlantic north of Cape Canaveral. These genetic partitions are the result of the comple x evolutionary history of the Gulf of Mexico and the Atlantic (Avise 1994; Cunningham and Collins 1998). The finding of substantial genetic breaks among these potentially long di stance dispersers brings into question whether there is an implicit expectati on of greater genetic heterogeneity among populations with direct devel oping larvae that occupy a sim ilar range; however, few have sought to investigate this e xpectation. The corona complex, in addition to being a taxonomic quagmire, offers an ideal opportunity to investigate assumptions of genetic structuring in an intertidal animal with direct developing, demersal larvae. To accomplish this, mitochondrial DNA (mtDNA) in c onjunction with climatic and geologic

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30 records were used to recons truct the phylogeny of the genus Melongena and to evaluate three hypotheses regarding cr own conchs in the southeas tern United States; 1) The corona complex is composed of genetically differentiated species partitioned into geographically isolated local ities, 2) Populations of Melongena exhibit patterns of genetic structure at mtDNA loci that are concordant with other marine intertidal species inhabiting the same range, and 3) th e patterns of relatedness among populations of Melongena in the southeastern United States are pr imarily the result of restricted gene flow due to a non-dispersing larval stage in the life-cycle of crown conchs. Mitochondrial DNA Animal mtDNA is a double stranded, typically closed-circular mol ecule containing 37 genes, and usually a non-coding region responsib le for initiation of replication. With only a few exceptions, most notably some mo lluscs, a nematode and cnidarians, the mtDNA genes encode a total of 13 proteins, whic h play a role in electron transport and ATP synthesis, a small and large ribo somal RNA (rRNA), and 22 transfer RNAs (tRNAs) (Boore 1999). The popularity and utility of mtDNA as a molecular marker for evolutionary and population-ge netic studies stems from a combination of inheritance characteristics (primarily maternal), mo lecular properties (rate of intraspecific divergence, lack of recombination, etc), and the availability of various oligonucleotide primers suitable for amplification via P CR (Avise 1994, 2000; Hewitt 2001). In animals it evolves faster than typical single copy nuclear loci and of ten exhibits high levels of intraspecific polymorphisms. Inheritance is almost exclusively maternal (for exceptions see Kondo et al. 1990; Ladoukakis & Zouros 2001), and generally does not undergo

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31 molecular recombination. The flood of phylogenetic studies employing mtDNA markers in the last two decades is a testimonial to its applicab ility and contribution to our understanding of animal relationships. (Cunningham & Collins 1998; Boore 1999; Avise 2000; Grosberg & Cunningham 2001; Rocha et al. 2002). Despite its popularity as a molecular marker, mtDNA is not without problem s that must be taken into consideration when applied to evolu tionary studies. Regardless of the vast amount of research that has been published using mtDNA and the steady stream of information that we continue to collect using it, it is still only a single molecule primarily reflecting the matrilineal history of any group. This history may or may not reflect the true evolut ionary history of the population or species. Because it is inherited maternally, the eff ective population size of mtD NA is 0.25 of nuclear autosomal sequences. As such, alleles wi ll be purged from a lineage at a faster rate than those of nuclear DNA, which may result in an undere stimate of genetic diversity and possibly oversimplification of evolutionary relations hips (Zhang & Hewitt 2003). Furthermore, mitochondrial pseudogenes in the nuclear ge nome of some organisms may complicate studies utilizing this marker (Bensasson et al. 2001). This is especially true if one considers the evidence that pseudogenes may exist in extremely high copy numbers with little variation among populations (Zhang & Hew itt 1996). This is problematic because if pseudogenes, as opposed true mtDNA loci, are sequenced and compared the genetic differentiation from these analyses may not re flect the true evolutio nary history of the taxa in question. Even with these issues, mtDNA can still yield fruitful information as long as the inherent challenges of the mark er are considered and dealt with when

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32 analyzing and interpre ting data from this source. For example, comparisons between newly obtained sequences with published nucle otide and amino acid sequences may help in discriminating pseudogenes fr om true mtDNA loci (Bensasson et al. 2001). Methods Sample Collections & Tissue Storage From July 1999 through May 2003 ma ture crown conchs were co llected in th e intertidal zone throughout their range in Florida a nd Alabama (Figure 2.1 and Appendix B). At each location, an attempt was made to colle ct 40 mature snails for a population level study utilizing microsatellite markers deve loped specifically for crown conchs (See Chapter 3). Smaller subsets of these snails were used in conjunction with other members of the genus to reconstruct th e phylogeny of crown conchs. In some instances it was not possible to collect 40 individuals without removing a substantial portion of the population, and at other sites it was not possible to find 40 indi viduals due to either cold weather or poor visibility at collection sites. Samples of M. bispinosa were provided by Emilia Gonzalez Vallejo, and pedal tissue from M. patula was provided by Harris Lessios (Appendix B). Busycon sinistrum Busycotypus spiratum, and Fasciolaria hunteria were collected at Ft. DeSoto State Park in Pinell as County, Florida to be used as outgroups in phylogenetic analysis. All snails were anesthetized using 7% MgCl2 in filtered seawater and frozen at -20 C until DNA extraction was performed. Upon removing snails from the freezer they we re allowed to thaw at room temperature while submerged in normal seawater (35ppt). After thawing, snails were removed from

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33 the shell and visually sexed by presence of a penis or pedal gland. If neither a pedal gland nor a penal structure were visually obvious the sex was left undetermined. A portion of the foot tissu e was removed and stored in 95% EtOH and the remainder of the tissue stored at -20 C. Shells were cataloged along with the operculum for later reference. DNA Isolation Total cell DNA (tDNA) was isolated from all individuals using multiple protocols depending on the state of the tissue (i.e. fr ozen or ethanol preserved). DNA isolation from approximately 1 g of fresh or fro zen tissue was done using phenol/chloroform protocol modified from Ausubel et al (1993). After completion of the final 75% EtOH wash the pellet was allowed to air dry a nd 300l of sterile 1X TE (10mM Tris-HCl, pH 8.0; 1mM EDTA pH 8.0) was added to resuspend the purified DNA. The tDNA was then incubated at 65C for 1 hr and precipitated by incubation at 4C overnight after adding 150l of 8M LiCl. DNA was pelleted by cen trifugation for 45 min at 14,000 X g. The supernatant was removed and the pellet gent ly washed one time with 70% EtOH, and allowed to air dry. The purified tDNA was resuspended in 50l of 1X TLE (10mM TrisHCl, pH 8.0; 0.1mM EDTA pH 8.0) and stored at -20C until needed. PCR amplification was carried out using 1.5 – 2l of this template in a 50l reaction. Template DNA extracted from tissue previ ously stored in 95% EtOH would not yield consistent amplifications using the above prot ocol, therefore alternative approaches were evaluated. After attempting a variety of procedures refe renced in the literature,

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34 consistently amplifiable template was obtai ned most often by using either a modified non-boiling chelex protocol (Walsh et al. 1991) or in some instances a NaCl extraction protocol (Simison & Lindberg 1999). In the few instances when templates extracted using any of the above protocols would still not amplify, the Wizard Genomic DNA Purification Kit (cat.# A1120; Promega, WI), was used. Forty-four snails representing a small subs et of the sample sites were used for phylogenetic reconstruction. These samples we re chosen such that all putative taxa within the genus Melongena were included. Additional taxa within and outside of the family Melongenidae also were included in the phylogenetic reconstruction. Sequences generated from this study are availa ble on GenBank (Accession #s AY464660 – AY464686). MtDNA Amplification and Sequencing Initially, a 1302 bp fragment of the cytoch rome oxidase subunit one (COI) gene was amplified from four individuals (OB-30, OB-13, BH-13, CCES-3) using degenerate oligonucleotide primers provided by Dr. Ken Halanych (unpublished). Amplifications were carried out in reactions cont aining a final concentration of 1.5 U Taq polymerase in storage buffer B (20mM Tris-HCl (pH 8.0), 100mM KCl, 0.1mM EDTA, 1mM DTT, 50% glycerol, 0.5% Nonidet-P 40 and 0.5% Tween-20; Promega, WI), 6g of Bovine Serum Albumin, 1.5 – 2.5mM MgCl2, 1X reaction buffer (50mM KCl, 10mM Tris-HCl pH 9.0, 0.1 % Triton X-100; Promega, WI), 0.2mM each dNTP, 25 pmols of each primer, and 1 – 1.5 l of tDNA. Cycling was perfor med on a Hybaid Omni Gene thermocycler

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35 (ThermoHybaid) with the following parameters ; 4 min at 95C, 1.5 min at 45C, 2 min at 72C, followed by 35 cycles of 95C for 30 sec, 50C for 30 sec, 72C for 30 sec, and a final extension of 7 min at 72C. Su ccessful amplification was verified by electrophoresis on a 1.5% agarose gel st ained with ethidium bromide. Fresh PCR product from one of these indivi duals (OB-30) was ligat ed overnight at 12 C into the pBSK+ vector (Stratagene, CA), wh ich previously had single thymidine added to the 5’end following the protocol of Marchuk et al. (1991). Recombinant vector was subsequently transformed into NovaBlue Singles Competent Cells (Novagen, WI) according to the manufacture’s protocol. Fi fty microliters of cells were plated on ampicillin and X-gal treated agar culture plates and allowed to grow at 37C overnight. White colonies were screened using 30l P CR reactions and standard M13 primers. Clones that appeared recombinan t were grown overnight at 37 C in 3ml of Lauria Broth (5% NaCl, 1% triptone, 0.5% yeast extract, 0.1% 1N NaOH) culture media containing ampicillin. Eight hundred fifty microliters of the culture were placed in a 1.5ml cryotube with 150l of sterile glycerol for storage at -80C. One and a half milliliters of the remaining culture were used for miniprep isolation of plasmid following the boiling protocol of Sambrook et al. (1989). Purified plasmid was st ored at -20C. One quarter microliter of purified plasmid was used as template in PCR amplifications containing the same concentrations and the same cycling c onditions as previously described; however, initial annealing and cycl e annealing temperatures were raised to 55C. The PCR products from the remaining indi viduals (BH-13, CCES-03, OB-13) and those obtained using purified plasmid were con centrated using 30,000 MW Amicon Ultrafree

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36 MC filters (Millipore, MA) and used as template for cy cle sequencing. Cycle sequencing was carried out in both direc tions in 10l reactions usi ng the DYEnamic ET-Terminator Cycle Sequencing Premix Kit (Amersham Ph armacia Biotech Inc., NJ) following the manufactures protocol. M13 in ternal primers were used in reactions containing purified plasmid template, and the COI primers used in the initial amplifications were used for the remaining templates. Sequences were electrophoresed and an alyzed on an ABI 310 automated sequencer (Perkin-Elmer Applied Bi osystems, Inc., Foster City, CA) equipped with Sequencing Analysis Software (ver. 3.10). Forward and reverse sequences were edited, assembled, and aligned using Sequenc her (ver. 4.1.4; GeneCodes Corp., MI) and adjusted visually to insure accuracy. Four primer pairs were designed from the unambiguous conserved regions of these four sequences, using Primer 3 (http://www-genome.wi.mit.edu/cgibin/primer/primer3_www.cgi ) and Oligo 4.06 Primer Analysis Software (National Biosciences, MN) (mcCOI4F & R, mcCOI1F & R, mcCOI2F & R, mcCOI3F & R; Table 2.1). Approximately 1250 bp of the COI gene were amplified using mcCOI4F & mcCOI4R. PCR reactions were carried out usin g similar concentrations of reagents listed earlier and an amplification regime that cons isted of: initial denatu ration (95C for 240s); initial annealing (50C for 90s); an initial extension (72C for 120s); 35 cycles of denaturation (95C for 45s); annealing (50C for 45s), extension (72C for 45s); and a final extension (72C for 420s). The amount a nd facility of amplification was verified via agarose electrophoresis and single pr oduct amplifications were purified and concentrated using 30,000 MW Amicon Ultrafr ee MC filters (Milli pore, MA) prior to

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37 cycle sequencing. Cycle sequencing amplificat ions were performed in each direction as described previously using all primer pairs designed specifically for Melongena spp. Electrophoresis, editing, alignm ent, and evaluation of sequences were carried out as previously explained. The possibility of pseudogenes was excluded based on the ability to align the sequences in an open readi ng frame and through comparison of the amino acid sequences to those curren tly published on GenBank. A fragment of the 16S ribosomal DNA gene was amplified using the oligonucleotide primers 16S1 (5’ CGC CTG TTT ATC AAA AAC AT 3’) and 16S2 (5’ CTC CGG TTT GAA CTC AGA TC 3’) (Garey et al. 1998) in reactions containi ng final concentrations of reagents as those previously discussed. Cycling parameters were the same as with COI except cycling times were reduce from 45s to 30s each. Sequences were generated as described previously and edited in the sa me manner prior to analysis. Additional 16S sequences obtained from GenBank were adde d to the alignments for use as outgroups prior to carrying out phylogenetic analysis. Phylogenetic Analysis The most appropriate model of evolution for the sequence data used here was determined using the hierarchical likeli hood ratio test implemented in the program Modeltest version 3.06 (Posada & Crandall 1998). Phylogenetic trees were constructed under maximum likelihood (ML) analysis (heuristic search with TBR branch-swapping) using the program PAUP* (ver. 4.0b10; Swofford 2003). Data st ability and the robustness of the tree topology were assessed using 100 b ootstrap replicates (Felsens tein 1985). Bootstrapping

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38 was carried out in PAUP* using the like lihood settings recommended by Modeltest using minimum evolution optimality criterion and TBR branch swapping. Haplotype and nucleotide diversity parame ters including number of haplotypes (Nei 1987), sequence diversity (average number of nucleotide differences per site within groups; ) and sequence divergence (average number of nucleotide differences per site between groups; dxy) were calculated using DnaSP 3.53 (Rozas & Rozas 1999). Average pairwise distances between individual se quences were calculated using PAUP*. Results Sequence Characteristics COI Molecular analysis yielded a total of 27 COI sequences from individuals representing all current species of the genus Melongena occurring in the southeastern United States, three representing Melongena bispinosa a single Melongena patula and one Busycon sinistrum Five of these sequences containe d only 524 bp and the other twenty-two contained an additional 683 nucleotides ( 1207 bp). These additional nucleotides were sequenced in an effort to uncover additional variation at COI within the corona complex taxa. The longer fragments of COI came from 18 snails in the corona complex, three M. bispinosa and the single M. patula specimen. All sequences were unambiguously aligned to one another and to additional gastropod CO I sequences obtained from GenBank. All sequences generated in th is study are available on GenBank under accession numbers AY464660 – AY464676.

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39 Sequence variability among Melongena spp. was high with 271 (22.5%) variable nucleotides, and 178 (14.7%) were parsimonious ly informative. Within the corona complex there were 15 (1.2%) variable sites an d just over half of these, 8 (0.66%), were parsimoniously informative. Because most of the COI sequences available on GenBank were only 500 – 700 bp in length, compar isons of sequence variability between Melongena spp. and all outgroup taxa were carried out using the smaller COI fragment. For comparative purposes the characteristics of the smaller COI fragment in all outgroup taxa in addition to those from Melongena spp. are presented separa tely from those of the larger fragment. Analysis using only the sm aller COI fragment from all taxa revealed higher variability with 217 (41.4%) variable sites, a nd 186 (35.5%) of these being parsimoniously informative. Within the genus Melongena the smaller COI sequences possessed 107 (20.4%) polymorphic sites and 84 (16.0%) were parsimoniously informative. These values are much larger when compared to those from the smaller fragments within the corona complex which had a total of six polymorphic nucleotide sites with three (0.57%) being parsimoniously informative (Appendix A.1). Translation of the 1207 bp COI sequence yields 402 amino acid (aa) residues, of which 99.0% (398 aa) were conserved within the comp lex. The variability at this level was contained in six individuals; one from East Cape Sable (E CS-16), one from Barnes Sound (BS-18), and four individuals from Big To rch Key (BT-01, 02, 03, & 04) all sharing the same mutation at site 402. There were also ten variable residues within M. bispinosa sequences and 11 variable sites found in the M. patula sequence when compared with those from the complex.

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40 Typically, the COI gene has been reported to be A-T rich in other taxa, and Melongena spp are no exception (Holland & Hadfield 2002) Analysis of the 1207 bp fragment from Melongena spp revealed a bias in mean nucleotide base frequencies toward adenine and thymine (A = 0.248, T = 0.372, C = 0.182, and G = 0.198). Sequence Characteristics 16S Twenty-three sequences from a 475 bp fragme nt of the mitochondrial 16S gene were resolved and deposited in GenBank under accession numbers AY464677 – AY464686. As with COI, 20 of these fragments were obtained from species within the genus Melongena ; 14 coming from the corona complex, two from M. bispinosa and four representing M. patula Of the 475 sites across all taxa analyzed, 281 were conserved, 198 (41.7%) were variable, and 147 (30.9%) were parsimoniously informative. In addition to the single base differences, the a ligned data set also included 14 presumptive indels of 1 – 4 bases in length. Within the Melongena corona species group there was only a single base substitution at sight 237 in two individuals (MIB-03 & LW-03; Appendix A.2). Sequences from within the genus Melongena contained 41 (8.6%) variable sites, of which 15 ( 3.1%) were variable. There were no indels present within the aligned sequences containing only taxa fr om the complex, however, the alignment containing only taxa within the genus Melongena contained 4 indels of 1 – 4 bases in length (Appendix A.2).

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41 Sequence Diversity Sequence divergence ( dxy) between the corona complex and other lineages within the genus Melongena range from 0.0908 (Complex – M. patula ) to 0.1569 (Complex – M. bispinosa ) for the COI gene. Although the values were lower, the pattern of sequence divergence was similar with the 16S gene ranging from 0.0246 (Complex – M. melongena ) to 0.0705 ( Complex – M. bispinosa ) (Table 2.2). Sequence diversity ( ) within the corona group taxa studied was 0.0022 based on the short COI fragment, 0.0025 with the longer COI sequence, and a much lower 0.0006 based on the 16S gene. Analysis revealed two 16S haplotypes with in the complex, compared to six and 11 haplotypes from the COI short and long fragment s respectively. Average pairwise distances among individuals within the corona group were lower than anticipated for presumed interspecific comp arisons. Distance variability ranged from 0.000 – 0.008 calculated from COI sequences (T able 2.3). Mean distances calculated from the 16S data were corrected for missing data (indels) and ranged from 0.000 – 0.002 within the complex (Table 2.4). These va lues were similar to those seen among individuals of M. patula for 16S data (0.00 – 0.002) and both 16S and COI data among M. bispinosa (0.000 – 0.005). Among groups within the genus Melongena distances calculated from the COI gene had ranges of 0.082 – 0.094 (Corona Complex – M. patula ) and 0.138 – 0.164 (Complex – M. bispinosa ; Table 2.3). Between M. bispinosa and the corona group 16S distances ranged fr om 0.065 – 0.067, where as distances between corona group taxa and M. patula and M. melongena were only 0.026 – 0.030 and 0.024 – 0.026 respectively (Table 2.4). Distances calcul ated from 16S data between the geminate

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42 pairs of M. melongena and M. patula ranged from 0.026 – 0.028, while distances between both of these species and M. bispinosa were 0.067 and 0.069 – 0.072 respectively (Table 2.4). Sequences of the COI gene from M. melongena were not determined; however, distances calculated between M. patula and M. bispinosa varied in range from 0.136 – 0.138 (Table 2.3). Phylogenetic Analysis A series of hierarchical l og likelihood ratio tests, implemented using Modeltest 3.06 indicated different substitution model parameters for each fragment; TVM+I+G and HKY85+G for the short and long COI fragme nts respectively, TVM+I+G for all COI sequences combined, and TVM+G for the 16S data set. Since the different size fragments of COI resulted in different mode ls of substitution phylogenetic analyses was carried out separately for each, and then on the combined data. Maximum likelihood trees produced under the various models di d not differ in overall topology and all analyses uncovered a monophyletic group composed of the genus Melongena The corona complex was resolved as a single monophyletic clade with bootstrap support of 100% and 98% within COI and 16S trees respectively (Figures 2.2 & 2.4). Although analysis included only three ( Melongena Busycon and Busycotypus ) of the proposed 7 genera within the family, both data sets produ ced polytomies that faile d to clearly resolve the monophyly of Melongeni dae (Figures 2.2 & 2.3). Analyses of COI and 16S sequences did not uncover any genetic structure within the corona complex (Figures 2.2 & 2.3). With the exception of only minor variations

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43 attributable to the different ta xa in each data set, tree topo logies were consistent using either locus. Unexpectedly, both data sets place M. patula and M. melongena as sister taxa to the corona complex, instead of the presumed sister species, M. bispinosa (Figures 2.2 & 2.3). Discussion Data generated from two regions of mt DNA were used to clarify the phylogenetic relationships within the corona complex, and to evaluate hypotheses regarding the genetic structure of crown conchs in the southeastern United States. Both 16S and COI data sets revealed congruent patterns of phylogenetic relationships within the complex, and both data sets show maximum levels of sequence divergence that are far below those found for homologous sequences within other taxa described as subspecies (Sarver et al. 1998; Efford et al 2002) or even species (Thomaz et al. 1996; Schulze et al. 2000; Holland & Hadfield 2002; Hasse et al 2003). There are several possible expl anations for the lack of sy stematic support for the taxa belonging to the corona complex. Assessm ent of hypothesis regarding relationships within the corona complex requires that the pl ausibility of each expl anation be explored. In phylogenetics, polytomies may result from two different situations. First, simultaneous cladogensis within a group will re sult in a “hard polytomy”, and bifurcation order can not be resolved even with an infinite amount of sequence data (Maddison 1989). The other possibility, a “soft polytomy” results from an insufficient amount of sequence data to resolve the sequential bi furcation order (Coddington & Scharff 1996).

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44 The polytomy formed by the corona complex ta xa is most likely not a hard polytomy (Figures 2.2 & 2.3) because any cladogenic even t that would have gi ven rise to extant lineages simultaneously would have taken place at least 3 mybp. Other studies of marine gastropods have found that taxa separated for this length of time have accumulated mtDNA sequence diverges of 5 – 13% (Colli ns 1989; Hellberg & Va cquier 1999; Wilke & Pfenninger 2002), yet maximum sequence dive rgence within the corona complex is only 0.8% (see discussion of divergence timing) Because there is always the possibility that more data will provide further resolution it is difficult to dismi ss the possibility of a soft polytomy; however, based on studies of ot her marine gastropods it is anticipated that increasing the amount of data analyzed will not reveal a larger amount of variation (Collins 1989; Wilke 2003). If taxa with in the corona group were good species or subspecies they should have accumulated a su fficient amount of vari ation at mtDNA loci to indicate this designation, ye t both loci exhibit levels of variability far below those listed for gastropods desc ribed as species (Thomaz et al. 1996; Holland & Hadfield 2002; Wilke 2003). Furthermore, Walsh et al. (1999), demonstrated, through a power analysis test, that the amount of sequence data re quired to resolve bifurcation order, of divergences as recent as 100,000 year s, was between 215 and 1200 bp of mtDNA nucleotide data. The present studied used a total of 1686 nucleotides from two mtDNA loci, which makes the possibility of insufficien t data to resolve the relationships within the complex unlikely. Inadequate sampling of extant taxa, resulting in the exclusion of evolutionary lineages, may create phylogenetic artifacts includi ng unresolved nodes, over-estimation of

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45 divergence times and exaggerated substitution rates (Emerson et al. 2000; Emerson 2002). Sampling of taxa within the comple x was carried out to encompass the entire geographic and taxonomic range of this group. Within the corona group, multiple individuals from each putative species or subspecies were sequenced for each locus. Furthermore, individuals from all extant members of the genus Melongena were included, in addition to members from the confamilial genus Busycon Given this level of inclusiveness it is unlikely that I have missed any unique evolutionary lineages within this group. The occurrence of nuclear pseudogenes presen t a real problem for phylogenetic studies using mtDNA, and may confound the patterns of relatedness. In general, mtDNA loci evolve at a much higher rate than nuclear markers, which results in nuclear pseudogenes having decreased levels of sequence variab ility when compared to functional mtDNA (Perna & Kocher et al 1996). Although this is generally true in mammals and birds, it may not always hold true in invertebrates, which means that pseudogenes may evolve faster, slower, or at the sa me rate as mtDNA loci (Sharp & Li 1989). The mtDNA loci used in this study show very little sequence variation am ong a number of putative species and subspecies, and because little is known a bout the evolution of nuclear pseudogenes in molluscs no assumptions regarding their rate of evolution can be made. It may not be possible to infer a rate of evolution for nuclear pseudogenes in the group studied here; however, since pseudogenes are no longer constrained to prod uce functional proteins or ribosomal RNA the characteristics of their evol ution are expected to be different than their true, functional mtDNA counterparts. B ecause they lose thei r functionality as a

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46 result of transfer to the nuclear genome their substitution rates should be equal across codon positions, where as mtDNA sequences w ould be expected to have a bias for variation at the third position. In the abse nce of purifying selection, nuclear pseudogenes often accumulate frameshift mutations that re sulting in the inability to obtain an open reading frame using these sequences. The truncated amino acid sequences of pseudogenes will not produce functional gene products, yet the COI sequences determined in this study were all unambiguous ly aligned in an open reading frame with other published COI sequences. Furthermore, amino acid sequences were compared to published amino acid sequences for COI from other gastropods. Despite taking such measures to verify the integrity of these data, there still exists the possibility that these loci are nuclear pseudogenes. If the duplication and transf er of the mtDNA copy to the nuclear genome were a recent event there may not have been enough time for mutations to accumulate, which may explain the overall lack of variability within the complex. However, in order for this to be a plausibl e explanation the event would have had to be fairly recent and either occurred multiple times in all the taxa sequenced or prior to the separation of extant populations. Given that sequences used in the present study were obtained from multiple species within the genus Melongena and different genera ( Busycon & Fasciolaria ), which based on fossil data are believed have diverged from on another more than 35 mybp (Harasewych 1982; Pe tuch 1994), it is much less likely that this is a possible explanation. An unusually slow rate of mtDNA evolution so lely within taxa from the corona complex is difficult to negate, yet, I know of no pl ausible explanation be yond selection that can

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47 satisfactorily explain such a process. Natu ral selection may exer t constraint over the amino acid sequence, effectively maintaining ge netic homogeneity of a particular locus in a population through the elimination of delete rious mutations via background selection. Furthermore, natural selection may act to homogenize homologous loci in different populations through processes such as “sel ective sweeps” (Rand 2001). Essentially, selective sweeps occur when an allele of single locus under selection goes to fixation and causes the same to occur at neutral alleles of a linked loci, which in turn makes the neutral loci appear to be under selection (Kaplan et al. 1989). Since the mtDNA is effectively a single linked supergene, strong se lection at one gene will act on the entire genome. It is possible, since Melongena spp. occupy similar hab itats throughout their range in the SE U.S., that strong selection in those environments may account for the overall lack of mtDNA vari ation in this group. This seems unlikely because the frequency of mutations in large populations is high compared to smaller populations with similar mutation rates; therefore, selection must be very strong in order to maintain genetic homogeneity. The populations sampled fo r this study were fairly large, making selection an improbable explanation. Furtherm ore, the degenerate nature of the genetic code would still allow mutations at the th ird codon position even in the presence of strong background selection, yet, there was an overall lack of va riation at all codon positions equally, indicating that there was no particular bias for mutation at the third position. Neither background selection or selective sweeps provide satisfactory explanations for the lack of sequence variation in this study

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48 Rapid speciation, or adaptive radiation is a common theme in molluscan evolutionary studies, and is often suppor ted by shifts in morphology recorded by paleontological records (Vermeij 1978). For example, rapid speciation among Lake Turkana molluscs has been postulated based on paleontological and climatic records (Williamson 1981). In some instances, rapid speciation may result in morphological divergence between taxa so quickly that phylogenetic signal does not accumula te in the nucleotide sequences. It is possible that this scenario may account fo r patterns witnessed among members of the corona complex. Recent rapid speciation imp lies that the morphotypes within the corona complex are reproductively isolated from one another and the event occurred quickly and recently; therefore species specific molecula r differences have not accumulated. The long evolutionary history of these morphotype s inferred from the P liocene fossil record (ca. 5 mybp; Petuch 1988; 1994), makes this scenario less likely. Alternatively, a rapid speciation event preceded by a selective sw eep that homogenized mtDNA would account for the lack of variation. Again, such strong selection is unlikely gi ven the large size of most crown conch populations. Furthermore, much of the morphovariation attributed to species differences in this group is read ily found intraspecifica lly within a single population (Tucker 1994; Poland pers. com.; pers. obs.). Finally, I propose that the most likely explana tion for the data produced from the current study is one of an evolutionarily recen t isolation of popula tions within the M. corona group without accompanying speciation. This scenario may adequately explain the patterns of genetic similarity un covered in this study. Drama tic changes in sea-level over the last 2 my altered the co astal topology of the southeas tern United States, which had

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49 significant impacts on the dist ribution and patterns of relatedness among regional intertidal fauna (Avise 1994; Schulze et al. 2000). It is likely that these events played a similar role in shaping phylogeographic patter ns within the corona complex; however, in order to implicate such events it is necessa ry to infer a time frame for mtDNA divergence within this group. Divergence Timing Timing and tempo of divergence within a group of conspecifics or between species is often carried out using the foss il record or a major geologic event that alters gene flow between two groups (i.e. closure of the transi sthmian seaway by formation of Isthmus of Panama or closure of Bearing Strait). Evid ence from the Florida fossil record indicates that regional gastropod fauna have undergone multiple mass extinction events over the last 2 – 5 my, making it difficult to accurately calibrate a molecular clock based on these types of data (Petuch 1995). Similarly, the cl imatic and geologic events that have been responsible for the aforementioned extinction ev ents are difficult to correlate to specific divergences. Additionally, no mtDNA molecu lar clock has been published for taxa within the corona complex; therefore, in an attempt to provide a temporal perspective for the divergence within this group it is necessary to draw infere nces about rates of change from other sources. In a variety of marine invert ebrates, including a geminate species pair in the genus Melongena the rates of mtDNA divergence have b een calculated using both the fossil records and the formation of barriers separa ting sister taxa. The rate of sequence

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50 divergence between the eas tern Pacific species M. patula and the Western Atlantic M. melongena estimated from mtRFLP analysis ranges from 2.1 – 2.5%/million years (MYR) (Collins 1989). This calculation is based upon a final emergence date for the isthmus of Panama of 3 million years befo re present (mybp); however, if an earlier closure date is used of 3.5 mybp the corre sponding rate would be 1.8 2%/MYR. The earlier closure date may be reasonable in li ght of the uncertainty relating to the changes in the region leading up to final closur e (reviewed in Collins 1989; 1996; Cronin & Dowsett 1996). Estimated in the same study, the geminate snail pairs in the genera Purpura and Vasum had higher divergence rates of 2.8 – 4.1%/MYR, again these rates must be considered within the time fra me from the beginning through complete emergence of the isthmus. Furthermore, since all of these estimates are derived from mtRFLP analyses they represent an averag e over the entire mitochondrial genome. As such, it can be expected that any single locus may exhibi t higher or lower rates. Similarly, there is significan t body of evidence from studies of COI and 16S data that the rates for homologous loci in different taxa may vary considerably. Estimates from the shrimp genus Alpheus yield a rate of 1.4%/MYR for COI, while the rate of COI sequence differentiation in bivalve family Arcid ae ranges from 0.7 – 1.2%/MYR (Knowlton & Weigt 1998; Marko 2002). These rates are lo wer than those estimated from hydrobiine gastropods (1.8%/MYR) and the 2.4%/MY R from gastropods in the genera Tegula and Norissia (Hellberg & Vacquier 1999; Wilke 2003). Despite the variability among different rates and the uncertainty regardi ng the consistency of rates even between conspecific, I have attempted to use these data to infer a general time frame for the divergence among populations within the coro na complex. As such, these estimates

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51 should be interpreted with caution; however, th ey still allow the disc rimination of ancient versus recent divergence estimates. Previous studies place the separation of M. patula and M. melongena in a time frame concordant with the closure of the transist hmian seaway (3.1 – 3.5 mybp) (Collins 1989). Using this time range, yields an estimate d substitution rate for 16S of 0.8 – 0.9%/MYR between these species. Applying the 16S rate from these congenerics to estimate divergence within the corona comple x yields a time range of 220,000 – 250,000 ybp (Late Pleistocene). Comparing this time frame to the paleontological and climatic records of Florida provides a plausible frame work to interpret this separation. At the beginning of the Pleistocene seas were mu ch higher than they are presently, and throughout the epoch sea levels oscillated in concert with the a dvancement and recession of glaciers much further north. For example, from beginning of the Pleistocene, sea level decreased until approximately 150,000 ybp, and th en began to rise again until about 115,000 – 130,000 ybp (Muhs et al. 2002). During this last gl acial minimum sea level in the southeastern United States was consider ably higher (5 – 7 m) (Mercer 1972). To put this into perspective consider that the average sl ope of land in Florida ranges from about 1:3000 in some of the southern regions to 1:1000 further north (Davis 1997). A five meter rise in sea level would translate into a 5 – 15 km inland advance of Florida’s coastlines relative to their present positions. Similarly, estimates from the Environmental Protection Agency indicate that a 2 m rise in sea level would inundate approximately 2,200 square miles in south Florida (Titus 1988). Given the dramatic impacts of changing sea level on the coastal topology in Fl orida it is possible that rapidly fluctuating

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52 sea level over the last 500,000 years may ha ve resulted in the fragmentation of populations within the corona complex. Othe r divergences estimated from the 16S rate also correlate to specific climatic or geologi c events. For instance, the estimated time of divergence between co rona group taxa and M. bispinosa falls within the Tortonian stage of the Late Miocene (7.4– 8.4 mybp). The ge ologic details of shorelines during this period were erased by erosion and deposition during sea level fl uctuations in subsequent epochs. Nevertheless, the mass extinction of marine gastropods during the Late Miocene may be indicative of an ecological catast rophe during this period, which may have contributed to the cladogenic event (P etuch 1993; 1995). This scenario appears consistent with the presence of M. bispinosa in the Florida fossil records from the Early Pliocene (Petuch 1994). Finall y, estimates of divergences between the corona complex and the geminate species M. melongena (2.8 – 3.3 mybp) and M. patula (3.3 – 3.8 mybp) place these events in the Midto Late Pliocene. The timing of these divergences correlate well with the time frame given for the closure of the Isthmus of Pana ma. Because I only included a single M. melongena 16S sequence from GenBank it is possible that the molecular rate for 16S within this group may be significantly lower or higher than what I have estimated. Furthermore, insufficient sampling from the other geminate species may account for the possibility of an inaccurate 16S substitution rate. Although this is always a possibility, the evolutionary tempo of 16S calculated here is within the range of other rates (0.1 – 3.1%/MYR) estimated from various invertebrates (Cunningham et al 1992; Sturmbauer et al. 1996; Cunningham & Collins 1998; Romano & Palumbi 1997; Gantenbein & Largiader 2003).

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53 Unfortunately, the COI sequence from M. melongena was not available on GenBank and I was unable to obtain tissue from this species for sequencing; therefore, the substitution rate for this locus could not be calibrated using the geminate pair as with 16S. Instead, an approximate molecular rate for COI was de termined using the coalescent time between corona complex taxa and M. patula calculated from 16S (3.3 – 3.8 mybp). This method yields a COI rate of 2.5 – 2.8%/MYR, which dates the divergence between corona group taxa and M. bispinosa to 5.9 – 6.6 mybp. Both of thes e separations along with the divergence between M. patula and M. bispinosa (4.9 – 5.5 mybp) predate the closure of the transisthmian seaway, and are more in line with records of Pliocene fossils of M. bispinosa in Florida. Additionally, this rate is consistent with published rates for COI from other marine gastropods (Hellberg & Vacquier 1999; Wilke 2003), and for the averages calculated from RFLP analysis of the mtDNA genome from geminate species of Melongena Based on the COI rate, the divergence within the corona complex dates to the Late Pleistocene (285,000 – 320,000 ybp), is w ithin an equitable range of that time frame calculated from 16S. Although these mo lecular rates may not be accurate, they do appear to be reasonable enough approximations to allow me to evaluate the divergence within the corona complex. Even if the rate of evolution for 16S from Melongena spp. was two-fold lower than what I have estimat ed here, the divergences within the corona group would still not predate the Mid-Pleistocene. Both data sets are congruent in the revela tion of a much more recent divergence within the corona complex than previously hypothesized based on fo ssils records and morphological analyses of extant species. Prev iously, authors have speculated that due to

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54 the presence of Melongena spp. as far back as the Mi ocene in Florida that these morphologically variable snails must have speciated as a result of isolation during dramatic sea-level changes during the early Pliocene (Clench & Turner 1956; Petuch 1994; Tucker 1994). The genetic patterns, how ever, are much more consistent with repeated localized extinction and recoloniza tion as a result of changing climates and coastal geology during the Pleistocene. It is possible that the inter tidal regions of the western panhandle of Florida may have acted as a refugia for crown conchs during high seas encountered during the Pliocene. Furthermore, Pleistocene seas did not completely inundate the Florida peninsula, which may have created ref ugia either in the northern central Florida regions, or further sout h, around Lake Okeechobee (Petuch 1994; Jones 1997) Evidence from Pleistocene coast lines and biogeography of regional fauna indicate that temperatures were warmer a nd seas were much higher at the beginning of the Pleistocene (ca. 75 m above current le vels; 1 mybp) (Webb 1990; Petuch 1994; Jones 1997; Scott 1997). Sea-level decreased dr amatically until about 150,000 ybp (ca. 50 m below current level), and as seas dropped sn ails may have expanded southward until seas rose again about 130,000 ybp. At th is point it is possible that crown conchs were isolated in northern or southern refugi a. Sea-level dropped a final time during the last glacial maximum approximately 20,000 ybp, and has risen at various rates to present day levels (Webb 1990; Scott 1997). As sea-levels ro se over the last 12,000 years localized populations of crown conchs were probably extirpated, while others may have been dispersed to newly formed embayments and estuaries. Climatic and coastal changes throughout the last 300,000 years co incide well with genetic patterns within the complex. The greater degree of diverg ences seen in Orange Beach Alabama and the individual

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55 from one of southern most population (BS18) are adequately explained by refugia formed in the north western range of this sp ecies as well as the southern range, followed by range expansion as sea-levels decreased. Other Intertidal Invertebrates Vicariance has often been invoked to explain concordant patterns of genetic structuring in a variety of marine animals (reviewed in Avise 1994, 2000). Similarly, patterns of relatedness among crown conchs may be expl ained by recent vicariant events, yet the relationships revealed here do not parallel a ny of those previously reported for marine invertebrates from this region. Clearly, despite shared historical pr essures, other factors hold significant influence over the evolution of various taxa. One su ch factor that may have played a pivotal role in influencing the contemporar y genetic structure of crown conchs is the occurrence of a non-planktonic larval stage in their life history. To illustrate this point I contrast the patterns revealed from studies of the eastern oyster, Crassostrea virginica with that of the crown conchs. Crassostrea virginica has been shown to exhibit significant genetic struct ure over the same range occupied by crown conchs (Reeb & Avise 1990). There are cl early numerous biological distinctions between these two taxa; however the planktonic larvae pr oduced by oysters may account for the differences in genetic structure between the two. After extirp ation from an area, the higher dispersal potential of oysters may ha ve made it possible for them to recolonize areas much more quickly; therefore, allowi ng more time for isolated populations to differentiate. Of course, the counter argum ent is that this dispersal ability would homogenize populations through gene flow, yet this only holds true if gene flow is

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56 maintained after these dispersal events. Obvi ously, factors such as currents, climate, and instability of near shor e habitats during and after dispersa l events most likely altered gene flow and the patterns of conn ectivity between contemporary populations. In contrast, this is quite different than what populations of crown conchs may have experienced during the same time period. As a result of a much lowered dispersal rate in crown conchs, the rapidly changing climate and restriction to near shore habitats probably prevented the invasion of regions that oyster would have colonized in the same time span. Once again, support for this idea comes from studies of other invertebrates from this region, including the horseshoe crab ( Limulus polyphemus ; Saunders et al. 1986), the Florida stone crab ( Mineppe mercenaria ; Bert 1986), a spionid polychaete ( Streblospio benedicti ; Schulze et al. 2000), the blue crab ( Callinectes sapidus ; McMillen-Jackson et al 1994), and the intertidal gastropod Crepidula spp. (Collin 2001) all of which produce planktonic larvae and share similar patterns of genetic structure. Long distance dispersal ability combined with historical influences have produced parallel patterns of ge netic relatedness among these disparate taxa, while at the same tim e similar vicariant events combined with reduced vagility have resulted in dissim ilar genetic patterns in crown conchs. Taxonomy Melongena spp. examined in this stu dy appear to be a monophylet ic group. Furthermore, results from these data indicat e that populations of crown conchs in the southeastern United States are composed of a single evolu tionary lineage that exhibits minimal genetic variation over a large (> 1900 km) heter ogeneous geographic range. Estimates of divergence point to a recent time (late Plei stocene) for the isol ation of the extant

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57 populations. Although it is inappropriate to assign species designa tions based solely on genetic distances, it is clear that current sp ecific and subspecific definitions within the complex do not correspond to distinct groupi ngs based on mtDNA data. Species within the complex were originally defined on basi s of shell sculpture (spination), size, and shape, as well as geographic location. These definitions are in need of reassessment, and any future taxonomic re-evaluations should cons ist of multiple lines of independent data such as soft part anatomy, reproductive compatibility, and possibly further genetic analysis to include sequences of nuclear loci (although micros atellite data yield similar conclusions – see chapter 3). At this time there is no co mpelling data (morphological or molecular) to support the current taxonomic de signations within this group, as such, it is recommended that the group be treated as a si ngle taxonomic entity until such data are forthcoming. The specific nomen corona has precedent and shoul d be applied to all individuals throughout their range in the southeastern United States. Conclusions All three hypotheses given in the introduction can be rejected based on the data obtained from analyses of mtDNA sequences. The coro na complex is not composed of genetically and geographically differentiable units, a nd the patterns of rela tedness among populations are not concordant with those published for ot her intertidal invertebrates from the SE U.S.. Finally, although larval dispersal potentia l has played a substant ial role in shaping the genetic picture of crown conchs, it is ultimately the interplay between historical events (vicariance) and life-history that have determined the contemporary patterns of relatedness within the corona complex.

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58 References Adamkewicz, S. L. and M. G. Harasewych 1996. Systematics and biogeography of the genus Donax (Bivalvia: Donacidae) in eastern North America. American Malacological Bulletin 13:97-103. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl, P. Wang-Iverson and S. G. Bon itz 1993. Current protocols in molecular biology. Greene Publishing Associates and Wiley-Interscience. New York. Avise, J. C. 1994. Molecular markers, natural history and evolution. Chapman & Hall. New York. Avise, J. C., D. Walker and G. C. John s 1998. Speciation durations and Pleistocene effects on vertebrate phylogeography. Proceedings of the Royal Society of London. Series B 265:1707-1712. Avise, J. C. 2000. Stability, equilibrium and molecular aspects of conservation in marine species. Hydrobiologia 420:xi-xii. Bensasson, D., D.-X. Zhang, D. L. Hart l and G. M. Hewitt 2001. Mitochondrial pseudogenes: evolution's misplaced witne sses. Trends in Ecology and Evolution 16:314-321. Berger, E. M. 1983. Population genetics of marine molluscs. In Russell-Hunter, W. D (Ed), The Mollusca Volume 6 Ecology. Academic Press, Inc. London. 563-596. Bert, T. M. 1986. Speciation in west ern Atlantic stone crabs (genus Menippe ):the role of geological processes and climatic events in the formation and distribution of species. Marine Biology 93:157-170. Bhaud, M. 1998. The spreading potential of polychaete larvae does not predict adult distributions; consequences for conditi ons of recruitment. Hydrobiologia 375/376:35-47. Boore, J. L. 1999. Animal mitochondrial ge nomes. Nucleic Acids Research 27(8):17671780.

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66 OB MH LW SI MIB R SPCA PI BT FTD SJP ECS(3)[1] (1) [2] [2] (5) (1) (1)[2] (2) (2)[1] [2] [2] (2) [2] BS ( 1 ) CCES(1) BL (1) FB (1) LMK ( 1 ) Georgia Alabama Florida Gulf of Mexico Atlantic Ocean FIGURE 2.1: Sample locations in Florida and Alabama used in mtDNA analysis. The numbers of individuals sequenced from each site are indicated in parentheses for COI and brackets for 16s. OB=Orange Beach SJP=Saint Joseph’s Peninsula, FTD & CCES=Tampa Bay, PI=Pine Island, ECS=Cape Sable, BT=Big Torch, MH=Matheson Hammock, LW=Lake Worth, SI=Sebastian Inlet, MIBR= Merritt Island Banana River, SPCA=Spruce Creek Estuary. Map created using Generic Mapping Tools Software (ver. 3.0; Wessel & Smith 1995) available online at http://www.aquarius.geomar.de/omc_intro.html.

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67FIGURE 2.2: Phylogenetic tree for relationships of Melongenid gastropods and several outgroup taxa, based on the combined 524 bp and 1207 bp sequences from the COI mitochondrial gene. Maximum likelihood tree (l og likelihood –5122.28) reconstructed under the best fit model of substitution (TVM+I+G), ca lculated using Modeltest version 3.06. Node values indicate bootstrap support above 50%. Bootstrap values in parentheses indicate node support from analysis of taxa within the genus Melongena carried out using only the 1207 bp fragment of COI under the assumptions of the HKY85+G substitution model. Sequences obtained from GenBank are indicated by accession numbers and those generated from this study are listed with sample numbers. Bold type = individuals represented by 1207 bp sequence of COI. A,B,C,D indicate individuals with identical haplotypes. M .bicolor ( BT-04 ) M .bis p inosa ( YP-01 ) M .bis p inosa ( YP-03 ) M p atula ( MP-07 ) M .bicolor ( BT-03 ) C M .bicolor ( BT-02 ) M .bicolor ( BT-01 ) C M .corona corona ( PI-10 ) M .corona corona ( PI-25 ) M sp rucecreekensis ( SPCA-11 ) M .corona corona ( ECS-16 ) M .bicolor ( BT-05 ) M .corona j ohnstonei ( OB-30 ) D M .corona j ohnstonei ( OB-29 ) D M .corona j ohnstonei ( OB-20 ) D M bicolor ( MIB R -01 ) B M .bicolor ( SI-08 ) M .corona corona ( FTD-10 ) M sp rucecreekensis ( SPCA-25 ) B M .bicolor ( MIB R -03 ) A M .bicolor ( FB-01 ) A M .bis p inosa ( YP-06 ) I schnochiton australis ( AY296815 ) M itra cucumerina ( AY296839 ) Pleuro p loca g i g antea ( AF373885 ) F asciolaria tuli p a ( AF373884 ) Strombus luhuanus ( AY296831 ) Bab y lonia j a p onica ( AF373888 ) Ne p tunea anti q ua ( AF373886 ) Ne p tunea p ol y costata ( U86326 ) Bus y con carica ( U86323 ) Bus y con p erversum ( AF373887 ) Bus y con sinistrum ( Bsin-01 ) Nerita atramento s a ( AY296824 ) M .bicolor ( BS-18 ) M .corona j ohnstonei ( BL-01 ) M .bicolor ( LMK-01 ) A M .corona corona ( CCES-06 ) A Cre p idula convexa ( AF388739 ) Corona Complex 5.0 % sequence divergence 78 55 72 88 (100) 53 (70) 65 76 (100) 99 89 (100)

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68 FIGURE 2.3: Phylogenetic relationships among gastropods in the genus Melongena and outgroup taxa, based on 16S rDNA sequences. Tree derived using maximum likelihood (ML) analysis under the assumptions of the TVM+G substitution model (log likelihood –2419.49). Nodes values are bootstrap estimates exceeding 50%. Sequences obtained from GenBank are indicated by accession numbers and those generated from this study are listed with sample numbers. =Corona complex 16S haplotype 1; †= Corona complex 16S haplotype 2; = M. patula 16S haplotype 1; §= M. patula 16S haplotype 2; = M. bispinosa 16S haplotype. Crepidula complanata (AF545968) Vasum muricatum (AF338156) Crepidula marginalis (AF545970) Vasum caestus (AF338155) Melongena bicolor (MH-03) Melongena bicolor (MH-01) Melongena bicolor (LW-02) Melongena sprucecreekensis (SPCA-11) Busycon sinistrum (Bsin-02) Busycon sinistrum (Bsin-01) Melongena bispinosa (YP-01) Melongena bispinosa (YP-03) Melongena melongena (AF338149) Melongena patula (MP-09) Melongena patula (AF338150) Melongena patula (MP-07) Melongena patula (MP-21) Fasciolaria hunteria (Fas-01) Plicopurpura patula (AF338152) Melongena corona corona (ECS-03) Melongena corona corona (ECS-04) Melongena bicolor (SI-01) Melongena bicolor (SI-02) Melongena bicolor (MIBR-01) Melongena corona johnstonei (OB-20) Melongena patula (MP-15) Melongena bicolor (MIBR-03)† Melongena bicolor (LW-03)† Melongena corona johnstonei (SJP-03) Melongena corona johnstonei (SJP-02) Busycotypus spiratum (AY010327) Corona Complex 100 98 59 99 100 100 100 98 98 57 5% sequence divergence

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69 Primer Name Sequence mcCOI1F 5'AGC AGG G AA TTT AGC TCA CG -3' mcCOI1 R 5'CCT GTT A AC CCC CAA CTG TG -3' mcCOI2F 5'ATC CAC GGT GCC AAA ATC -3' mcCOI2R 5'ATT CCC CGT CAT CGC ATA -3' mcCOI3F 5'AAT CAT AAG GAT ATT GGC AC -3' mcCOI3R 5'AGT CCT AGG AAA TGT TGA GG -3' mcCOI4F 5'AAT ATG ATC GGG GTT GGT TG -3' mcCOI4R 5'TGC TAC ATT AAC CCC AAT AAA CA -3' TABLE 2.1: Oligonucleotide primers designed for PCR amplification of COI fragment in Melongena spp mcCOI4F & R were used in initia l amplification of 1250 bp fragment, and others were used as internal sequence primers during cycle sequencing amplifications. All primers anneal at 50oC during PCR and 55oC for cycle sequencing.

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70 COI\16s Corona Complex (14) M. melongena (1) M. patula (4) M. bispinosa (2) Corona Complex (22) -0.02460.02780.0663 M melon g ena (0) nc-0.02570.0674 M patula (1) 0.0908 ( 0.0920 ) nc-0.0705 M bispinosa (3) 0.1569 ( 0.1478 ) nc0.1463 TABLE 2.2: Sequence divergence ( dxy) between taxa within the Corona Complex, M melongena M. patula M. bispinosa for the COI gene (below diagonal long fragment in parentheses) and 16s (above diagonal). The number of individuals sequenced for each gene are given in parentheses. nc = not calculated

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71 FB-01 BS-18 BT-02BT-04BT-05PI-25BL-01OB-20MP-07 YP-01 YP-03YP-06 FB-01 0.006 0.0030.0040.0020.0030.0020.0030.083 0.143 0.1430.141 BS-18 3 0.0080.0080.0080.0080.0040.0060.094 0.164 0.1600.160 BT-02 4 4 0.0010.0020.0030.0040.0050.083 0.141 0.1410.139 BT-04 5 4 1 0.0020.0040.0040.0060.082 0.140 0.1400.138 BT-05 3 4 3 2 0.0040.0040.0040.084 0.142 0.1420.140 PI-25 4 4 4 5 5 0.0040.0050.085 0.139 0.1390.138 BL-01 1 2 2 2 2 2 0.0020.092 0.160 0.1560.156 OB-20 4 3 6 7 5 6 1 0.086 0.143 0.1430.141 MP-07 100 49 100 99 101 102 48 104 0.138 0.1380.136 YP-01 172 86 170 169 171 168 84 172 166 0.0050.003 YP-03 172 84 170 169 171 168 82 172 166 6 0.002 YP-06 170 84 168 167 169 166 82 170 164 4 2 TABLE 2.3: Pairwise genetic distance matrix showing average molecular sequence divergence (above diagonal) and absolute number nucleotide of differences (below diagonal) calculated using COI seque nces from taxa within the genus Melongena Only pairwise comparisons with non-zero values are shown. Sample numbers correspond to those given in Figure 2.1 and Appendix B. Corona complex 1 Corona complex 2 M. bispinosa M. melongena M. patula 1 M. patula 2 Corona complex 1 0.002 0.067 0.024 0.030 0.028 Corona complex 2 1 0.065 0.026 0.028 0.026 M. bispinosa 31 30 0.067 0.072 0.070 M. melongena 11 12 31 0.028 0.026 M. patula 1 14 13 33 13 0.002 M. patula 2 13 12 32 12 1 TABLE 2.4: Pairwise genetic distance matrix showing average molecular sequence divergence (above diagonal) and absolute number of nucleotide differences (below diagonal) calculated using 16S sequences from individuals within the genus Melongena Sample numbers and haplotype designations correspond to those gi ven in Figure 2.3 and Appendix B.

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72 Chapter 3: Microsatellite Analysis of Crow n Conchs: Population Structure and Evolution in a Direct Developing Snail Introduction An understanding of evolutionary processe s that lead to population subdivision and speciation requires knowledge of the roles played by diverse f actors such as dispersal, localized adaptation and life history. In ge neral, the structure of marine populations results from an interplay between a comp lex series of contemporary processes and historical events. Among these influen ces are behavioral, genetic, demographic, oceanographic, geologic, and climatic pro cesses (Hedgecock 1986; Palumbi 1994; 1995; Grosberg & Cunningham 2001). Primarily, it is the strength, scale, and rate of these processes that determine the es tablishment or destruction of population structure, and the ultimate fate of species. The relative importa nce of the various factors in establishing present-day population structure in many ma rine taxa remains unclear, yet certain paradigms generalizing the magnitude and perm anence of different factors in influencing population differentiation have been estab lished by a number of authors (Mayr 1942; Burton & Feldman 1982; Hansen 1983; Avise 1992; Palumbi 1994; Hoskins 1997; Cunningham & Collins 1998). A number of studies have asserted that there are primarily two overriding factors regulati ng population interconnectivity, gene flow and historical processes (McMillan-Jackson et al .1994; Foighil & Jozef owicz 1999; Riginos & Nachman 2001; Broughton et al. 2002).

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73 Traditionally, populations of any particular spec ies in the sea are believed to be more or less linked by the individual dispersal capabi lities of such species. Often populations of species with low dispersal potential are expected to differentiate faster than those with higher dispersal capabilities, resulting in increased speciation frequency (Scheltema 1971; 1986; Burton & Feldman 1982; Collin 2001). A substantial number of studies have investigated the link between potential and realized gene flow in a variety of species (McMillian et al. 1992; Hellberg 1996; Kyle & Boulding 2000). In ge neral, species with direct developing demersal larvae exhibit highl y restricted gene fl ow over moderate to large geographic scales, and those with long lived planktonic larvae show little to no structure over similar ra nges (Berger 1973; Crisp 1978; Hedgecock 1986; Scheltema 1986; Janson 1987). Contrasting this assump tion, a few studies have been a bit more equivocal with regard to comparisons be tween planktonic larvae producing species and those with direct development. For ex ample, in their examination of four Littorina spp Kyle and Boulding (2000) found that one direct developing species from the Northeastern Pacific exhibited similar levels of mark ed population subdivis ion as a congeneric planktotrophic species, and two other species (one direct developer and one planktotrophic) did not exhi bit any significant level of population subdivision. Other studies have shown that many species with widely dispersing pla nktonic larvae do not actually realize their true dispersal capability and many show unanticipated levels of population subdivision (Reeb & Avise 1990; Ka rl & Avise 1992; Bert 1986; Bhaud 1998; Schulze et al. 2000). The disparity between potential and realized gene flow in a number of these species may often be explained by such factors as suitable ha bitat, direction and velocity of regional ocean currents, or ev en selection (Riginos & Nachman 2001).

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74 Another often invoked interpre tation for present day patt erns of distribution and relatedness among populations within certain species is vicariance (Avise 2000; Riddle et al 2000). The fact that recent history plays a vital role in shaping populations, as well as species, makes it even more difficult to decipher the precise role of va rious influences. In an attempt to disassociate the influence of c ontemporary processes from historical events, numerous authors have focused on a wide vari ety of marine, freshwater, and terrestrial fauna inhabiting the southeastern United Stat es. For example, studies revealing shared phylogeographic patterns across taxa as different as stone crabs (Bert 1986), oysters (Reeb & Avise 1990), polychaetes (Schulze et al. 2000), fish (Avise 1994), tortoises (Schwartz 2003), snakes (Jansen & Karl in prep ), shrimp (Staton and Felder 1995), horseshoe crabs (Saunders et al. 1986), and snails (Wilke & Pfenninger 2002) have provided concordant signs indicating some shar ed vicariant event. Molecular (allozyme, mtDNA, microsatellite) studies of marine fauna distribut ed around the peninsula of Florida indicate biogeographic and genetic breaks between sp ecies found in the Gulf of Mexico and the Atlantic north of Cape Canaveral (Bert 1986 ; Reeb & Avise 1990; Cunningham et al. 1991; Schulze et al. 2000; Collin 2001; Young et al. 2002). Sea-level fluctuation and changes in coastal topogr aphy during the Pliocene and Pleistocene provides the common thread that ties these fam iliar patterns among distinct taxa together. Curiously, the expectation would be for long distance dispersers, with potentially high gene flow among populations, to erase such genetic signatures over a relatively short period of time. Conversely, it may be anticipa ted that historically influenced patterns would be exaggerated and reinforced among taxa with minimal dispersal capability. Yet, despite the capability of long di stance dispersal in many of the species investigated so far,

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75 much of the genetic structure remains. Expl aining this persistence requires a thorough understanding of the interplay between dispersal and vicariant processes. Genetic signatures of patter ns established by vicariant events may be maintained by contemporary forces. For instance, specific habitat preferences by particular taxa may influence the overall picture of relatedness among populations. Species restricted to a limited ecological niche are often subject to local differentiation and small scale geographical isolation, whereas organisms that tolerate a wider range of environments are less likely to show similar patterns. Fo r example, mangrove dependent taxa have substantially different genetic patterns than do generalist estuarine species (Avise 1994; Jansen & Karl in prep ). In trying to make sense of the various pa tterns exhibited by ma rine taxa in the southeastern United States a few studies have paid attention to di rect developing taxa (Berlocher 2000), while most have focuse d on species with high dispersal potential, usually those with planktonic larvae (Ber t 1986; Reeb & Avise 1990; McMillen-Jackson et al 1994; Schulze et al 2000). It is assumed that the genetic barriers among populations of direct developi ng species arise as a result of the typical scenarios discussed previously, although; in reality few have sought to evaluate the validity of such assumptions (Palumbi 1994). Based on stereoty pical direct development and specialized habitat preferences, most authors have made just such assumptions regarding the prosobranch snails in the genus Melongena (Clench & Turner 1956; Tucker 1994)

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76 Snails in the genus Melongena are highly philopatric and in the southeastern United States they inhabit non-conti guous intertidal habitats inte rspersed with uninhabitable areas. Over evolutionary time, this situat ion may be expected to produce large numbers of ecologically similar but genetically dis tinct populations, with some even becoming reproductively isolated. Many authors have ta ken the differences in shell morphologies from various populations of crown conchs as an indicator of underlying genetic differentiation, resulting from the previous s cenario. Nomenclatural designations within the corona complex, based primarily on shell color, size, and spination, along with the assumptions of underlying genetic differe ntiation have prompted a number of malacologists to support the asse rtion that the genus is comp osed of multiple species and subspecies that are structured geographical ly (for taxonomic review see Chapter 1). Recent mitochondrial DNA analyses conflict with these assertions revealing levels of sequence divergence far below those found in homologous sequences from other taxa described as species (Thomaz et al. 1996; Holland & Hadfield 2002; Hasse et al 2003). These results bring into ques tion the validity of the taxonom ic definitions within the group, and at the same time raise further que stions about the impact of dispersal and vicariance on the evolution of the complex. Fo r instance, why in the face of such limited dispersal potential have these snails rema ined relatively genetically homogenous at two mtDNA loci, that in other snail species show a much larger degree of variation (Thomaz et al. 1996)? Additionally, why do other mollu sc species with similar life history attributes and similar geographi c ranges exhibit a greater exte nt of genetic structure than does Melongena corona (i.e. Busycon spp.) (Berlocher 2000)? The questions raised by

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77 the low variability in two mtDNA loci indicat ed the need for a more discriminatory technique appropriate for invest igations of intra-population va riability. For this purpose microsatellite loci where chosen. Microsatellites (MS) are nuclear loci containing short, two – five base pair (bp), tandem repeats [e.g., (TC)n (CAA)n (AC)n] and exhibit fa st mutational rates. These rapid rates of mutation result in high levels of genetic vari ability, even among closely related species and populations (Queller et al. 1993; Moritz and Hillis 1996; Luikart and England 1999). This variability, coupled with the ability to identify c odominant genotypes with exact allele sizes, and the ease of amplification via the polymerase chain reaction (PCR) have made microsatellite analys is a powerful tool for studying population structure, evolutionary history, and taxonomic affinities (Queller et al. 1993; Paszek et al. 1998; Streelman et al. 1998; Luikart and England 1999; Broughton et al. 2002; Schwartz 2003). Recently, a number of statis tical methods have been employed in the analysis of microsatellite data revealing subtle patterns and processes of evolution that may not be discovered using other techniques (for review see Luikart and Engla nd 1999). The latest statistical programs can be utilized to anal yze these data for subtle differences in individuals and populations, which may be in terpreted with an understanding of the mechanism and models of micros atellite evolution (Goldstein et al. 1995b; Goldstein et al 1999; Nielsen 1998; Luikar t and England 1999). There are two mechanisms by which microsatellit es are thought to arise and evolve. The first, unequal crossover in meiosis, is now believed to be only a mi nor component in the formation of MS. The predominate mech anism, and generally well supported, for

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78 microsatellites is slip-strand mispairing (SSM), which involves the gain or loss of one or more repeat units (i.e. GAGA) (Levin son and Gutman 1987; Eisen 1999; Twerdi et al. 1999; Harr et al. 2000). Although the primary mechan ism of mutation is fairly well established, there remains some ambiguity re garding which model of mutation is most likely under the SSM mechanism. Currently, most data indicate th at microsatellites mutate unde r one or a mixture of three different models (Estoup and Cornuet 1999). The two classic models, the stepwise mutation model (SMM) originally proposed by Ohta and Kimura (1973), subsequently adapted for microsatellite loci by Valdes et al. (1993), and the infinite-allele model (IAM) proposed by Kimura and Crow (1964) are the two extremes of theoretical models. At one extreme, under the SMM, alleles mutate by the gain or loss of one repeat unit, which may result in the creation of new alle les or alleles that already exist in the population. At the other extreme, the IAM, mu tations can involve any number of repeats and all mutations result in the creation of a new allele previously not found in the population. In between these two models is the two-phase model (TPM) (DiRienzo et al. 1994). Under TPM, mutations introduce the gain or loss of repeats with different probabilities for each repeat number class. Early studies asserted that mutations in microsatellites generally consist of the a ddition or loss of one repeat, and much less frequently several repeats conforming more closely to the SMM (Estoup and Cornuet 1999); however, recent data indicate that a more realistic model of microsatellite mutation may be the TPM or a simplified va riant of it known as the General Stepwise Model (GSM) (Ellegren 2000; Estoup et al 2002).

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79 The goal for this study was to evaluate fine scale differentiation among populations of Melongena corona using microsatellite loci. Using th ese data, the influences of natural history, climatic events, and current pro cesses on the genetic subdivision of these populations will be delineated. These data will then be compared to other species from the SE U.S. in order to evaluate the degr ee and scale of these fo rces in maintaining population structure. Methods Sample Collection and Library Development Snails were collected as discussed in Ch apter 2 (Figure 3.1 and Appendix B). For the development of the microsatellite enriched subgenomic library, total cell DNA (tDNA) was extracted from foot tissue of a single Melongena corona (CCES-06) using a standard phenol chloroform extraction (Ausubel et al 1993). Library development followed the protocol of Severance (2002). Three to five micrograms of tDNA were s ubjected to restriction digestion at 37 C for 2 hr using 100 U of Sau 3A (Boehringer Manheim, Germany). Bgl II linkers (25mer and 21mer; Annovis, Inc., PA) were ligated to the DNA overnight at 12C. These linkers served as priming sites for amplification using three concentrations of ligated tDNA fragments (undiluted, 1:10, and 1:100) via P CR containing a final concentration of 2.5 U Taq polymerase in storage buffer B (20m M Tris-HCl (pH 8.0), 100mM KCl, 0.1mM EDTA, 1mM DTT, 50% glycerol, 0.5% Noni det-P40 and 0.5% Tween-20; Promega, WI), 6g of Bovine Seru m Albumin, 1.5 – 2.5mM MgCl2, 1X reaction buffer (50mM

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80 KCl, 10mM Tris-HCl pH 9.0, 0.1 % Triton X100; Promega, WI), 0.2mM each dNTP, 0.05 M of each Bgl II primer, and 1.5 l of ligation d ilution as the template. Cycling parameters consisted of: initial denaturati on (95C for 180s); 30 cycles of denaturation (95C for 60s); annealing (60C for 60s), extension (72C for 120s); and a final extension (72C for 600s) on an Omni Gene Hybaid thermocycler. The amplification reaction with the undiluted ligation had the high est yield when visual ized via agarose gel electrophoresis and was used in the subsequent steps. Tw enty microliters of the PCR product were denatured and biotin la beled microsatellite oligos 5’-(AC)15TATAAGATA3’, 5’-(TC)15TATAAGATA-3’, and 5’-(CAA)15TATAAGATA-3’ (Annovis, Inc., PA) were added and allowed to hybridize overnight at 48C. The hybridization mixture was resuspended with Streptavdin Magnesphere Paramagnetic Particles (SA-PMP: Promega, WI), which bind to the biotin labeled oli gos now hybridized to fragments containing microsatellite repeats. Using a magnetic tube holder the SA-PMP bound fragments were retained while the unhybridized fragments we re removed by repeated washings. The hybridization procedure was re peated twice, followed by amplification of three concentrations (undiluted, 1: 10, and 1:100) of the recovere d microsatellite enriched fraction of DNA. Amplification procedures followed previously described methods using the Bgl II oligo primers. Amplifications of all three products were combined, cleaned, and concentrated by ethanol precip itation followed by dige stion with 100 U of Sau 3A. Digested products were further purifie d by ethanol precipitation prior to cloning.

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81 Cloning Twenty-five microliters of DNA enriched for mi crosatellite sequences were digested with 100 U of Bgl II restriction enzyme and ligated into pBSK+ plasmid vector (Stratagene, CA) that was previously digested with Bam HI (Boehringer Manheim, Germany). The ligation reaction was transformed into Nova Blue Competent Cells (Novagen, Inc., WI) following manufacturers suggested protocol. Twenty microliter s of cells were plated on ampicillin and X-gal treated Lauria Broth agar plates and incubated overnight at 37C. Recominant clones were screened using P CR and M13 primers with the same final concentrations as previous amplifications. Cycling parameters consisted of initial denaturation of 120s at 95C followed by 35 cycl es of 30s at 95C, 30s at 55C, and 30s at 72C, and final extension of 600s at 72C. Presence of insert s and their approximate sizes were determined visually using ag arose gel electrophoresis. Four hundred recombinant clones were transferred to 96well microtiter plates containing Lauria Broth/glycerol solution (30% glycerol, 5% NaCl, 1% tript one, 0.5% yeast extract, 0.1% 1N NaOH) and stored at -80C. Eighty-four recombinant clones were sequenced from the microsatellite enriched library using DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech Inc., NJ). Sequences were analyzed on an ABI 310 sequencer (Applied Biosystems, CA). Forward and reverse sequences were ali gned and edited using Sequencher (GeneCodes Corporation, MI). Sixty-two of the eighty-f our (74%) clones contai ned a repeat region, and from these, 16 primer pairs were developed using Primer 3 (http://wwwgenome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi ) and Oligo 4.06 Primer Analysis

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82 Software (National Biosciences, MN). Each primer pair was tested for amplification consistency and variability unde r a different levels of stringency using tDNA extracted from twenty-five individuals from different populations throughout the range. Eight of the sixteen primer pairs were successfully op timized and a single primer from each of eight variable loci was labele d with one of the fluorescent dye s from Virtual Filter Set C (Integrated DNA Technologies, IA). DNA Isolation Isolation of tDNA for the purposes of amplif ication followed the protocols described in Chapter two Genotyping Amplifications of all eight loci were perf ormed singly in 30l PCR reactions containing 0.625 U EnzyPlus polymerase (EnzyPol Ltd., CO), 6g of Bovine Serum Albumin, 1.5 – 2.5mM MgCl2, 1X reaction buffer (50mM KCl, 10 mM Tris-HCl pH 9.0, 0.1 % Triton X-100; Promega, WI), 0.2mM each dNTP, 10 pmols of each primer, and 0.5 – 1l of tDNA. Cycling was carried out on a Hybaid Omni Gene thermocycler (ThermoHybaid) with the following parameters; 240s at 95C, 90s at 55C, 120s at 72C, followed by 35 cycles of 95C for 30s, 55C for 30s, 72C for 30s, and a final extension of 600s at 72C. Multiplex amplification of pairs of loci was attempted with varying degrees of success, however, due to inconsistencies in product quali ty individuals were amplified separately for each locus. Prior to elec trophoresis, the eight loci were combined into two different mixes so that there were four loci in each (Table 3.1). Depending on amplification

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83 quality, 0.25-2 l of each locus were combined in 12l of deionzied formamide containing 0.5l of GeneScan-500 size marker (Applied Biosystems, CA), and denatured for 3 min at 95C. PCR products were combined in such a manner as to avoid overlap of allele sizes with similar fluorescent labels and to insure that the total volume of PCR product in each mix did not exceed 3 l. Samples were subjected to capillary electrophoresis on an ABI Prism 310 Ge netic Analyser ru nning DATA COLLECTION Software (ver. 1.2.2) and GENESCAN An alysis Software (ver. 3.1.2, Applied Biosystems, CA). Alleles were sized us ing the GENESCAN local Southern method (Ghosh et al 1997), and all genotypes were entered into Microsoft Access 2000 database. Statistical Analyses To evaluate neutrality of the loci and to detect the presence of null alleles (nonamplifying alleles) all possibl e locus/population pairs were te sted for deviations from Hardy-Weinberg equilibrium (HWE) usi ng GENEPOP 3.1b (Raymond & Rousset 1995) using the Markov chain method for unbiased es timate of p-values (Guo and Thompson, 1992). Parameters for all tests were set to 10,000 dememorization steps and 1000 batches with 1000 iterations per batch. Genotypic linkage disequilibrium was also tested in GENEPOP 3.1b ratio test with parameters set to 10,000 de memorization steps and 1000 batches with 10,000 iterations pe r batch (Raymond & Rousset 1995; Slatkin & Excoffier 1996). Number of alleles, allele frequencies, allele size range, observed and expected heterozygosity, and variance in repeat nu mber were calculated with MICROSATELLITE

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84 ANALYSER (MSA) (Dieringer and Schlotterer 2003). Average gene diversity (GD) per locus, across populations was estimated w ith FSTAT version 2.9.3 (Goudet 1995; 2001). Pairwise FST (Wright 1969) and RST (Slatkin 1995) measures were calculated in ARLEQUIN (vers. 2.0; Schneider et al. 2002). Matrices of FST and RST values were used to construct dendrograms of the relations hips among populations using MEGA version 3.0 (Kumar et al. 2001). Further characterizat ion of population structure and connectivity were evaluated by estim ating stepwise genetic distance ( )2 (Goldstein et al. 1995a), designed specifically for microsatel lite data, using MSA. These pairwise distances were then used to construct a dendrogram of relatedness among populations using the neighbor-joining algorithm (Sa itou & Nei 1967). To detect inbreeding, multilocus FIS was estimated for each population, and averaged across all populations, using Goudet’s (2001) FSTAT version 2.9.3, significance of FIS values were estimated using 2560 randomizations under HWE expectations. Tests for the presence of a statistically si gnificant relationship betw een genetic distance and geographic distance were carried out us ing the program IBD (Is olation by Distance: Bohonak 2002). Mantel tests (Mantel 1967) we re performed on matrices of pairwise genetic measures of population differentiation ( FST, RST, & ( )2) and pairwise geographic distances to determ ine the significance of any co rrelation. Each matrix was tested for a correlation between the log of both genetic and geographic distances (Slatkin 1993). Quantification of the strength of is olation by distance was done through a reduced major axis regression analysis, which has been shown to be a better estimator of the slope

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85 in isolation by distance analyses than a standard ordinary least squares method (Hellberg 1994; Bohonak 2002). Partitioning of genetic varian ce was evaluated by computing -statistics from FST and RST estimates using ARLEQUIN version 2.0 (M ichalakis & Excoffier 1996; Schneider et al. 2002). Populations were assigned to groups based on currently ac cepted systematic hypotheses, known genetic breaks found in othe r marine species in the region, and in such a manner as to minimize within group variance and maximize the among group variance (Table 3.2). Evaluation of recent population growth and/or decline was carried out using the program BOTTLENECK (Corneut & Luikart 1996). The test s implemented by this software are based upon the assumption that populations having undergone recent reduction in population size will exhibit a re duced number of alleles ( k ) in conjunction with expected heterozygosity ( H ) at polymorphic loci. BOTTLEN ECK calculates the equilibrium distribution of H for each locus according to the observed k the sampled number of genes, and the mutation model. Three mutation models are evaluated; the IAM, the TPM, and the SMM. To evaluate the probabi lity of a recent bottleneck, the program uses a sign test (Corneut & Luikart 1996), a Wilcoxon sign-rank test (Luikart et al. 1998a), and a mode-shift test that dete cts a characteristic shift in allele frequency distributions when a population has gone through a bottleneck (Luikart et al. 1998b). Tests were carried out on a per-population basi s using 1000 iterations per test.

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86 In all cases of multiple tests, a sequential B onferroni correction was applied to eliminate the possibility of a false assignment of significance by chance alone (Rice 1989). Results Microsatellite Diversity Total numbers of alleles per locus was moderately variable, ranging from 8 ( Mco 5) to 28 ( Mco A4), with a mean per locus of 17.9 (Table 3.3; Appendices C & D). A total of 21 private alleles were found distributed among nine of the 16 populations. Often private alleles can be used to gauge the degr ee of connectivity among populations. The population that had the most unique alleles was SPCA with four, followed by CK, FTD, MIBR with three each, SI, ECS, PI with two apiece, and BL and FB with only one each. Interestingly, only 10 individuals were ge notyped from the SI population, yet the two private alleles were found indi cating that it may be in a fa irly high frequency among the population. Not surprisingly, the populations with the most unique alleles were also the ones with the largest samples sizes (Table 3.4) The private alleles we re distributed across all loci, with each locus possessing at least one unique allele, and Mco 6 & Mco E4 containing four and five respectively. (Table 3.4; Appendices C & D). Mean observed heterozygosity ( HO) was moderate to high in all populations, ranging from 0.477 (MH & OB) to 0.841 in LW, and an average of 0.627 over all populations (Table 3.4). As expected, in c onjunction with low allelism, locus Mco 5 had the lowest HO (0.155) averaged over all populations, and Mco 2 had the highest (0.786). Gene diversity ( GD ) was lowest in MH (0.472) and highes t in CK (0.802). As anticipated with

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87 highly polymorphic loci such as microsatellites overall GD was high (mean = 0.669) (Table 3.4). Disequilibrium Tests Exact tests of 122 locus/population pairs revealed statistically signifi cant deviations from Hardy-Weinberg Equilibrium in only thr ee (Mco6 in PI & BH and Mco12 in BH) comparisons after Bonferroni correction (p 0.05; Table 3.4). Sixteen of 403 pair wise lo cus tests indicated significan t linkage disequilibrium (p 0.05), however, none were significant after sequ ential Bonferroni corr ection. None of the uncorrected significant locus pairs were domin ated by any single locus or locus pair Intra-population Measures Measures of inbreeding, FIS, averaged over all loci in e ach population yielded 13 positive values (heterozygote deficiency) and three ne gative values (heterozygote excess) (Table 3.4). The three negative FIS values associated with ECS, LW SI were small, (mean of 0.014) and not significant before or after Bonferr oni correction ( p 0.05). Nine of the positive values (OB, BL, PA, CK, FTD, BH, SPCA, and FB) exhibited significant deviation from random mating expected under HWE, however, none of these values remained significant after Bonferroni correction. Analyses carried out in BOTTLENECK indicated no significant ex cess or deficiencies of heterozygosity under the IAM, TPM, or the SMM (Bonferroni corrected p 0.05) in MIBR (Table 3.5). Prior to Bonferroni corr ection, ten of the 16 populations exhibit a

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88 heterozygosity deficit under the SMM, and f our exhibit excesses unde r either the TPM or the IAM (Table 3.5). Population Structure Initially, all analyses of populat ion subdivision were carried out treating the three sample sites from Tampa Bay (FTD, CCES, & GBW) as independent populations and the then as single population. There were no significant differences in re sults between analyses that group the three into a singl e population or those assuming independence among them. As such, all references to FTD throughout the results and discussion sections includes the other two sample sites from Tampa Bay. Both measures of population subdivision, FST and RST, exhibit similar patterns of populati on differentiation, although; far more FST (111 vs. 68) values remain significant after seque ntial Bonferroni corre ction (Table 3.6). Significant pairwise FST’s among all populations ranged 0.017, between CK and PA, to 0.430 (OB – MH). Of the nine non-significant FST estimates seven involved the Sebastian Inlet (SI) population with only te n individuals sampled, which may account for the lack of differentiation between SI and all other populations. Generally, significant pairwise RST’s were larger than analogous FST’s, and ranged from 0.067 (SJP FTD) to 0.524 (OB – ECS) (Table 3.6). Unrooted dendrograms constructed using both FST’s and RST’s show only minor consistencies, such as differentiating the panhandle populations west of Apalachicola from the others in Fl orida (Figures 3.2 & 3.3). At the same time, both provide support for a clade from the southe rn tip of the Florida peninsula. Mostly, the two measures display incongruities in the relationships am ong geographic regions exhibiting no clear affinity for any partitioni ng of particular regions with the exception

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89 of the two mentioned above (Figures 3.2 & 3.3). The FST based phenogram shows the clearest geographic groupings with an ex treme western panhandle group, another clade north of Cape Canaveral, a group consisting of the eastern panhandl e and north-central gulf coast, and a final clustering of the peni nsular region from Ca pe Canaveral south on the east coast and south of Tampa Bay on th e west coast. (Figure 3.2). Similarly, RST estimates group the far western panhandle togeth er with the exclusion of the more central panhandle region (SJP), otherwise there are no clear clusterings of geographic regions (Figure 3.3). A neighbor-joining tree c onstructed from the stepwise genetic distance ( )2 between all possible pairs of populations show s some features of both the FST and RST based trees (Figure 3.4). The genetic distance based tr ee still resolves the clustering of the far western panhandle region, as well as that of th e southern peninsular region (Figure 3.4). Mostly, the three trees provide only a few unequi vocal clusterings of geographic regions. Only one test for an isolation by distan ce relationship resulted in a significant ( p 0.05) positive correlation. The Mantel test of the log of both geographic and ( )2 distances yielded a small significant positive correlation (r = 0.2293; p 0.0099). All tests carried out on FST and RST values resulted in nonsignificant results. Fu rther analyses conducted using only populations south of 27 latitude (PI, ECS, BS, MH, LW, and SI) failed to produce a single positive correlation between genetic and geographic distances (data not shown).

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90 Table 3.8 contains the data from five, two ba sed on a priori hypotheses and three that gave the highest among group variance (two signif icant), of the fourteen tests (Table 3.2) evaluating the hierarchical partitioning of variance via -statistics (AMOVA). Analyses revealed that 80 – 86% of the microsatel lite variance was found within populations (Table 3.8). Both FST or RST yielded comparable patterns, but RST-based -statistics tended to produce lower values overall. Most groupings, for example Groups 1 – 3 (Table 3.8), were essentially equivalent with regard to variance components, fixation indices, and percent variati on. Most groupings accounted fo r a small percentage of the variation (0.57% to 9.72), and provided only small, bu t significant values of CT (Table 3.8). Only one of the two a priori hypothese s resulted in a significan t explanation of the variance. Grouping Gulf of Me xico relative to Atlantic a ssemblage north and south of Cape Canaveral (phylogeogra phic hypothesis) resulted in only a minimal significant variance component ( CT = 0.030; p = 0.020), and only accounted for 3.04% of the variation. On the other hand, the systematic hypothesis did not yield a significant among group variance component (CT = 0.147; p = 0.150) (Table 3.8), and comparing the Gulf of Mexico to a single Atlantic assemblage resulted in a non-significant value ( CT = 0.015; p = 0.200), which explained the least amount of variation, 0.57% (dat a not shown). All other groups tested resulted in non-significant values for CT, with the exception of only two that explain a moderate portion of the variation. Groups four and five, based on a posteriori geographic clusterings establ ished in NJ trees, resulted in comparable variance components (CT= 0.128 & 0.129; p = 0.00), and explain 12.75 % to 12.88% of the variation respectively (Table 3.8).

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91 Discussion When studying extant taxa it is often difficult to decouple the influences of existing evolutionary processes from those of the evolutionary past. In the present study, microsatellites have been used to eval uate the intraspecific relationships among populations of crown conchs. Most of the ge netic signature resulting from events that predate the Pliocene have esse ntially been washed away and reshaped by the dramatic geologic and climatic events th at have taken place in the la st 5 my (See Chapter 1 for details). Similar to a number of coastal and estuarine species in the southeastern United States, the distribution and relatedness among crown conch populations has been influenced by geologic and climatic events occurring during the late Pleistocene. Although the genetic structure among populatio ns of crown conchs does not exactly mirror that of many sympatrically occurring spec ies, it is evident that recent glacial episodes have influenced these patterns. Although mtDNA analyses (Chapter 2) establishes that the cr own conchs in Florida are part of a single evolutionary lineage, it does not fully resolve the pa tterns of Pleistocene influence on crown conch relatedness. Microsat ellites, which are assumed to be neutral, reveal subtle patterns of subdivision among popula tions of crown conchs in the SE U.S.. In general, inconsistencies between the various trees constructed using FST’s RST’s and ( )2 distances may be the result of the diffe rent properties and assumptions inherent under each algorithm.

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92 Wright’s (1951) FST is probably the most commonly used measure of population subdivision; however, many have asserted th at it may not make use of much of the information provided by microsatellites. Of ten considered a more suitable measure, Slatkin’s (1995) RST was developed as an estimate of Wright’s FST for evaluating population differentiation, subdivision, effec tive migration rates, and times since population divergence (coalescence). Unlike FST, which is primarily based on the variance in allele frequencies, calculation for RST incorporates the variance in repeat number, the number of mutati ons that have occu rred (under SMM), and the increment of the repeat length in order obt ain a better estimate of popul ation parameters (Slatkin 1995). Commonly, both are used in populations studies invo lving microsatellites, and they each have aspects about them that need to be considered when evaluating results. For example, under a strict SMM RST measures are independe nt of mutation rate; however, FST is more sensitive to mutation rate, especia lly when the migration rate is low. In contrast, RST has an extremely high variance relative to FST, possibly making FST a more reliable predictor of population di fferentiation (Balloux & Lugon-Moulin 2002). Several authors have attemp ted to evaluate both measures using simulations and empirical data, yet only a minor consensus ha s been reached regarding which measure is best for any particular study. In their study of sheep, Forbes et al. 1995 found that RST was a better estimator when dealing w ith interspecific divergence; however, FST was more sensitive when evaluating recent separa tions (intraspecific), which is clearly the scenario in the present study. In a comparison of both methods Gaggiotti et al (1999) found that in studies with large sample sizes (n = 50) and numerous loci (= 20) RST out performs the more common FST, however, due to budgetary and time constraints this

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93 situation is rare. In more realistic situations moderate to small sample sizes (n = 10) and fewer loci (< 20), FST-based estimates always yielded a more accurate picture of genetic structure than did RST. From their simulations they c oncluded that the more conservative approach in most situations would be to use FST estimates. Based on these arguments it would appear that FST estimates are more appropriate for the present study, and they provide a more readily expl ainable picture of the gene tic subdivision present among populations of crown conchs when compared to RST and ( )2 estimates. Based on results from FST, most populations show little connectivity among one another (Table 3.6; Figure 3.2). Privat e alleles, in relatively high frequencies, may offer some indication of gene flow among populations, and half of the populations surveyed in this study possessed private alleles (Table 3.4). Almost all, however, were present at low frequencies ( 5%), with the exception of one private allele from Mco E4 found in the Flagler Beach population. The low frequency of private alleles prev ents any inferences regarding gene flow among populations. Furthermore, populations (MIBR & SPCA) with the largest samples surveyed revealed three and four privat e alleles respectively, indicating that more extensiv e sampling of some of the ot her populations in this study may reveal other unique alleles (Table 3.4) The level of allelism at each lo cus in this study is comparable to levels reported in studies of other gastropods. The seven mi crosatellite loci studied from Littorina saxatilis contained 9 and 27 alleles per locus (Sokolova et al. 2002). In their study of Nucella lapillus Kawai et al (2001) report that the number of alleles per locus from 14 microsatellite loci was much lower than that of Littorina ranging from 4 to 9. By

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94 comparison, the number of alleles per locus in the eight loci fr om this study were more in line with those from Littorina saxatilis ranging from 8 to 28. Additionally, levels of observed heterozygosity were slightly highe r in crown conchs with a mean of 0.627, compared to an average of 0.614 in Littorina saxatilis and 0.369 in Nucella lapillus (Table 3.3). Overall most pairwise FST estimates demonstrate that populat ions of crown conchs in this study are not presently conn ected through substantial ge ne flow, yet the lower FST values among neighboring populations indicat e a recently shared genetic pool. Much of the subdivision among extant populations of Melongena corona can be explained by sealevel during the Pleistocene. Unlike the glac ial induced fluctuations in sea-level during the Pliocene, the Pleistocene seas did not completely inundate the entire Florida Peninsula. Sea-level is belie ved to have been as high as 20 m to 75 m above present day level at various times during th e past 1.2 my. At this hei ght, shallow seas would have surely covered much of southern Florida from just north of Lake Okeechobee southward (Webb 1990; Scott 1997); however, portions of the northern peninsula would have been exposed, effectively separating populations betw een the northern Gulf of Mexico and the Atlantic. Given the presumed shallow marine habitats cr eated by Pleistocene coastal patterns and the warmer climate it is not difficult to imagine large populations of Melongena distributed along southern Florida. In this region, Petuch (1994) uncovered a variety of specimens belonging to the genus da ting back to the Pliocene and Pleistocene. He also uncovered a large number of other warm water intertidal gastropods that verify the suitability of preferred habitat during that period. When temperat ures cooled, as they

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95 did as recent as 20,000 ybp, glaciers in Canada spread southward into the northern US causing a precipitous drop in sea-level. A dram atic drop in sea-level, which in geologic terms would have occurred fairly rapidly, altered the coastal landscape of Florida, redistributing populations of Melongena Populations inhabiting the southern shallow water habitats overlying the peninsula were probably displaced east, west, and south along with the reemerging Florida peninsula. As a result, present day populations found on both coasts of southern Florida still bear a genetic signature of having been descended from populations occurring sympatrically in southern localities uncovered by Petuch (1994). Support for this is provided through th e clades formed in the NJ tree constructed from FST values (Figure 3.2). The clustering of pe ninsular populations to the exclusion of the far western panhandle and those above Ca pe Canaveral is consistent with this hypothesis. While most of the populations studied here were considerably differentiated from one another (mean FST = 0.130; mean RST’s = 0.144), some exhibited subtle patterns of connectivity. Various arrangements discernable from FST estimates suggest some commonality among populations south of 27 latitude on the west coast and south of Cape Canaveral on the east coast of penins ular Florida (Figure 3.2). Pulling these patterns together is the fact that all of these populations occur in a region along the Florida coast adjacent to inland areas where Pleistocen e populations once occurred in large numbers (Petuch 1988; 1994). Pleistocene vicariant hypothe ses are strengthened by the results from tests for a significant isolation-by-distan ce relationship. If the pres ent day populations of crown

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96 conchs had arrived at their current locatio ns through a steady southerly migrations along both coasts, from northern (panhandle) source populations it is expected that populations would exhibit a positive isola tion-by-distance relationship. In stead, only a single test of the log of ( )2 distances and the log of geographic distance demonstrat es a significant, yet minimal (r = 0.2293; p 0.0099), isolation-by-distance correlation. This may indicate that the isolating event responsible for this pattern among populations most likely occurred to all populations at or ar ound the same time. Furthermore, if the populations west of Apalachicola (OB, BL, & SJP) are removed from these analyses all tests for isolation-by-distance yield non-signi ficant results, indicat ing that the greater degree of genetic and geogra phic separation between the fa r western panhandle and the remainder of Florida is responsible for the on ly significant result. Despite the lack of strong isolation-bydistance co rrelation, genetic measures us ed here do indicate a pattern of clustering among neighboring populations, wh ich may be indicative of the low vagility of these snails. Additi onally, calculation of -statistics based on the grouping of populations south of 27 latitude reveals the largest significant variance components, and the most substantial fixation indices (Table 3.8). These results comb ined with geologic evidence reinforces the idea that these sout hern populations share a more recent common ancestor with one another than they do with some of the more northern populations. The least complex explanation for the patter n of relatedness among southern Florida populations is that they are likely desce nded from closely related groups that were isolated during the last glacial maxima (ca. 20,000 bp).

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97 The close association of far western pa nhandle populations (OB & BL) is also explainable under the conditions of Pleisto cene sea-level varia tion. Much of the panhandle has a substantially higher land eleva tion relative to south Florida, so while southern Florida was mostly submerged during the last glacial maximum, only the lowest portions of the panhandle were inundated. Supp ort for this is provided in the form of fossil vegetation and the locati on of marine fossils from the area (Richards 1938; Webb 1990; Jones 1997; Scott 1997). Flooding of low lying land around Little Lagoon, Alabama and Pensacola, FL created a shallow water habitat connecting the two protected embayments where current day populations of OB and BL exist. As sea-level dropped, land barriers again severed any direct conne ction between the two regions the snails currently inhabit. The geneti c grouping of these two populations is consistent with this scenario. The larger and more significant FST estimates between these far western panhandle populations and the rema ining localities can be attri buted to the persistence of these snails in the panhandle area for a l onger period of time. Although crown conchs have inhabited the southeastern United Stat es for at least the last 35 my, peninsular populations would have expanded into Florida only after its emergence some 25 my ago. Furthermore, populations that inhabited penins ular Florida during th e late Miocene (ca. 10 mybp) would have been extirpated and forced northward as the sea completely inundated the land. This took plac e several times prior to the Pleistocene, with the last complete submergence of the peninsula o ccurring just over 1 mybp (Deevey 1950; Webb 1990; Allmon 1996; Scott 1997). Estimates of FST from pairwise comparisons of populations from Panacea south to Tampa Bay st rengthen this scenario, with the highest significant FST among populations in this region be ing 0.019 (FTD – CK). Furthermore,

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98 FST values among populations on the northeast coas t of Florida, north of 28 latitude, and parallel regions on the west coast are genera lly lower than estimates from comparisons between northeast coast populations and those directly to the south (Table 3.6). This pattern is consistent with e xpansion of snails into regions on the northwest and northeast coasts of Florida from common source popul ations. During higher sea levels, when much of peninsular Florida was covered w ith shallow water, these source populations may have inhabited coastal regions from Panacea east to just north of present day Flagler Beach. A drop in sea level, exposing the peni nsular land mass, would have isolated and forced the dispersal of many of these populations to their curr ent locations on either coast of Florida.. Prior to analysis it was conjectured that the northern most Atlantic site (FB) may have been founded by migrants from southern populations via the In tracoastal waterway warming periods (NOAA 2003). Based on FST estimates it would a ppear that the close relationship between populations in this region (FB & SPCA) may be attributed to migration; yet, measures of inbreeding ( FIS) and tests for recent bottleneck fail to support this hypothesis. Lack of support from th ese values does not rule out the recent establishment of the northern most population by founders from those to the south of Flagler Beach. If the founding group was sufficiently large an d grew rapidly any genetic signature of the event may no longer exist. At present, it is not possible to definitively determine if the close genetic association between the two populat ions north of Cape Canaveral is the result of a recent founding event, or due to Pleistocene sea-level fluctuations.

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99 Habitats occupied by crown conchs in Florid a are often heavily imp acted by a variety of anthropogenic activities, especially in areas heavily visited by tourists and those under development. The Florida Keys are visited by over 2.5 million visitors annually and have undergone extensive development in the last two decades (Leeworthy & Vanasse 1999). Much of this development has resulted in ne ar shore habitat loss and degradation, and it was anticipated that the two populations (BH & BS) sampled from the Keys may exhibit signs of genetic bottleneck. This was not th e case, either because these populations are not being impacted by the devel opment, or the effect is too weak to detect by the methods used in this study. Natural events such as hurricanes and below normal temperatures can also impact crown conch populations. In 1998 hurricane Geor ges made land fall near Orange Beach Alabama, and populations of Melongena spp. in this area, including the Big Lagoon population, suffered mass mortalities as a re sult (Walker 1998; Walker pers. com). Interestingly, only six (15.8%) of the 38 i ndividuals collected from OB in 2000 were male, whereas all other populations from this study exhibited a typical 1:1 sex ratio (Appendix B). No deviation from 1:1 se x ratio was detected among the Big Lagoon population; however the first trip to the region to collect snai ls yielded only three snails. Another 28 snails were collect ed on a second trip in 2001, an d there appeared to be a larger number of snails in that region when compared to the previous visit. Despite indications of drastic population reducti ons in this region in 1998, neither population exhibit a significant FIS or a positive test for recent bot tleneck (after Bonferroni) (Table 3.4 & 3.5). Again, this may be attributed to the fact that the eff ect from the reduction

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100 may be too weak because of the large prereduction population sizes, or that the methods used here are unable to detect these bo ttlenecks. The inbreeding coefficient ( FIS) may not register the event simply because it may be a poor indicator of inbreeding when using highly variable markers, particularly micros atellites. Microsatellites, even under close inbreeding, exhibit high levels of hetero zygosity because most individuals are heterozygous. Before Bonferroni correction, both popul ations indicate a deficiency in heterozygosity, but so do eight others (T able 3.5). Gene diversity and observed heterozygosity, also are lower for both popul ations than the average across all populations, yet the values are still well within range of heterozygosity values seen in other intertidal gastropods (Kawai et al 2001; Samadi et al. 2001; Sokolov et al 2002). The average FIS value across loci in BL was the second highest at 0.141, which may be considered a moderate inbreeding coeffi cient. Combining the results from BOTTLENECK with those from some of the ot her tests, both OB and BL app ear to exhibit some signs of typical genetic signatures that follow a recent bottleneck (reduced dive rsity; loss of rare alleles (OB); higher FIS values (BL)). Given these e quivocal measures, it is unclear whether applying the particularly conservative Bonferroni correcti on across all data may in fact overlook valuable bi ological information. To insure important processe s occurring in populations of crown conchs are not missed, it is worth while to take a second look at th e eight other populations that demonstrate significant heterozygosity deficits (prior to Bonferroni) (Table 3.5). It is possible that these populations have undergone recent reductions as a result of natural pressures. The impact of hurricanes has alre ady been discussed, however, all organisms that occupy the

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101 intertidal environment may be s ubjected to extremes in salinit y, temperature, and light. It is possible that snails may be sensitive to thes e natural pressures. It has been reported by several authors that Melongena suffers high mortalities during colder than normal temperatures (Clench & Turner 1956; Ca ldwell 1959; Albertson 1980; Loftin 1987; Walker 1998; pers. obs.). Although mean annual temperature has remained fairly constant over the last few decades, temperatur es in Florida alternate between years of somewhat warmer winter months (Dec. – Fe b.) and years with far below normal winter temperatures (NOAA 2003). During these cold snaps populations may suffer substantial losses, especially those in the more northerly locations. Again, the data are equivocal on this point. If repeated population losses were occurring every 3 – 5 generations, it would be expected to lower overall he terozygosity and purge some of the rare alleles, effectively lowering the average number of alleles per locu s. Again, this does not appear to be the case for the populations that exhi bited significant values (prior to Bonferroni correction) from tests for a recent bottlen eck. For reference, the aver age observed heterozygosity values calculated across loci from microsatel lites from other intertidal gastropods were 0.352 in Cerithium lividulum (Samadi et al. 2001), 0.614 in Littorina saxatilis (Sokolov et al 2002), and 0.365 in Nucella lapillus (Kawai et al 2001). Additionally, the average number of alleles per locu s for each of these species was 10.5, 19.2, and 5.93. Average values across loci for the eight populations of Melongena spp. considered here range from 0.477 (OB & MH) to 0.698 (MIBR), and the average number of alleles per locus range from 4.37 (OB) to 12.25 (TB) (Table 3.4). Again, the equivocal nature of these data may indicate weaknesses in the ability of the methods used here, but more likely reflects the highly variable natu re of microsatellites. As mentioned previously, the high

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102 mutation rate and typically high levels of he terozygosity associated with microsatellites may translate into maintenance of higher values even under elevated levels of inbreeding. Furthermore, the high fecundity of crown c onchs may result in rapid population increases following reductions, which may effectively wa sh out any reduction in heterozygosity (Nei et al 1975). In the end the only conclusion that can be drawn is that although some of the populations in this study may suffer reoccurring reductions in population size and others have surely recently undergone a re duction in population size (OB & BL), rapid population growth and the high in trinsic heterozygosity associ ated with microsatellites probably hide any genetic signal a bottleneck may produce. Although the Pleistocene vicari ance hypothesis may explain some of the patterns of relatedness among M. corona populations in the SE United States, it is necessary to consider other possible explanations. The swimming behavior of newly emerged juveniles and data from other direct deve loping invertebrates s uggests that occasional rafting by juveniles may explai n the various patterns of mo rphological variance exhibited by M. corona (Highsmith 1985; Helmuth et al. 1994). Studies of ra fting in populations of the intertidal gastropods Bedeva hanleyi, and Barleeia spp indicate that rafting dispersal can rival the potential seen in many planktonic developers (Martel & Chia 1991; Hoskin 2000). If M. corona juveniles were rafting or floating among populations it is expected that relationshi ps among populations would be somewhat correlated to ocean currents in the region during peri ods of juvenile emergence from capsules (for review of currents see Bumpus 1973; Mooers & Maul 1998; He & Weisberg 2002) The correlation would, of course, be dependent upon the number of migrants and the

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103 frequency of such events. Examining the patter ns of near shore currents does not provide any support for this hypothesis. Although, th ere is some clustering of localized populations, there is no consiste nt pattern that would indi cate rafting among populations. Various historical events have been invoked to explain a variety of patterns in different taxa studied from the southeastern United Stat es. In the case of oysters, Reeb and Avise (1990) determined that despite the high poten tial for dispersal, that populations still exhibited a significant phylogenetic break in mtDNA north and south of Cape Canaveral on Florida’s east coast. The break was explaine d by the role of histor ical factors shaping peninsular Florida. Substantiating their co nclusions similar genetic breaks have been reported in a number of other co astal species (Bert 1986; Saunders et al. 1986; Schulze et al 2000). Unlike Melongena spp. many of these other taxa have the potential for long distance dispersal via planktonic larvae, wh ich may help explain why the patterns exhibited by crown conchs ar e not completely concordant with a number of other taxa studied. Although populations of crown conc hs do not show a clear phylogeographic pattern they do exhibit fairly large pairwise FST values indicating an absence of panmixia, yet at the same time some of the lowest pairwise FST values occur between adjacent populations. This pattern is consistent with the dispersal abilities of crown conchs. Finally, it is interesting to note that Jansen and Karl ( in prep. ), in their study of marsh snakes, found mtDNA control region hapl otype patterns sim ilar to those of Melongena Although, these are very different taxa with hi ghly varied natural hi story attributes, and the divergence in the marsh snakes appears mu ch older, it is possi ble that some common vicariant event has shaped their shared evolutionary patterns. Both taxa exhibit structure

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104 that can be explained by isola tion in south Florida shallow brackish/marine refugia during historically high sea-level. In the case of marsh snakes th e refugia probabl y consisted of mangroves swamps growing in the same shallo w water areas that provided ideal habitat for crown conchs. The congruence in these tren ds further strengthens this explanation for patterns found among populations of Melongena spp. Conclusions In the present study, microsatellite data have provided further indication of the complex patterns that emerge from vari ous influences on the evolution of marine taxa. At the same time they have offered insight into the forces that have shaped crown conch evolution during the past few million years. In general, an absence of cohesion among populations, combined with the lack of any s ubstantial isolation-by -distance relationship, indicates that much of the biogeographic structuring may be related to historical dynamics rather than present day migration and dispersal processes. From these data it is clear the Pleistocene vicariant events, in c onjunction with behavior al and developmental patterns, have played a prevailing role in altering the relationshi ps among populations of crown conchs in the southeastern United States.

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105 References Albertson, H. D. 1980. Long term effects of high temperatures and low salinities on specimens of Melongena corona and Nassarius vibex Ph.D. Dissertation Coral Gables, University of Miami, pp:222. Allmon, W. D., S. D. Emslie, D. S. J ones and G. S. Morgan 1996. Late neogene oceanographic change along Florida's west coast: Evidence and mechanisms. Journal of Geology 104(2): 143-162. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl, P. Wang-Iverson and S. G. Bon itz 1993. Current protocols in molecular biology. New York, Greene Publishing A ssociates and Wiley-Interscience. Avise, J. C. 1992. Molecular population stru cture and the biogeographic history of regional fuana: A case history with lessons for conservation bi ology. Oikos 63: 62-76. Avise, J. C. 1994. Molecular markers, natural history and evolution. New York, Chapman & Hall. Avise, J. C. 2000. Phylogeography: The hist ory and formation of species. Cambridge, MA., Harvard University Press. Balloux, F. and N. Lugon-Moulin 2002. The esti mation of population differentiation with microsatellite markers. Molecular Ecology 11: 155-165. Berger, E. M. 1973. Gene-enzyme varia tion in three sympatric species of Littorina Biological Bulletin 145: 83-90. Berlocher, S. H. 2000. Allozyme variation in Busycon whelks (Gastropoda : Melongenidae). Biochemical Genetics 38(9-10): 285-295. Bert, T. M. 1986. Speciation in west ern Atlantic stone crabs (genus Menippe ): the role of geological processes and climatic events in the formation and distribution of species. Marine Biology 93: 157-170. Bhaud, M. 1998. The spreading potential of pol ychaete larvae does not predict adult distributions; consequences for conditions of recruitment. Hydrobiologia 375/376:3547.

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114 Twerdi, C. D., J. C. Boyer and R. A. Farber 1999. Relative rate of insertion and deletion mutations in a microsatellite sequence in cu ltured cells. Proceedings of the National Academy of Science, USA 96: 2875-2879. Valdes, A. M., M. Slatkin and N. B. Freime r 1993. Allele frequencie s at microsatellite loci: the stepwise mutation mode l revisited. Genetics 133: 737-749. Walker, J. M. 1998. A population study of Melongena corona Gmelin on Perdido Key, FL. Marine Science, University of Alabama: 72. Webb, S. D. 1990. Historical biogeography. In Myers, R.L. and J.J. Ewel (Eds), Ecosystems of Florida. University of Central Florida Press, Orlando, FL 70-102. Wessel, P. and W. H. F. Smith 1995. New vers ion of the generic mapping tools released. Eos, Transactions, American Geophysic al Union, Electronic Supplement 76: 329. Wilke, T. and M. Pfenninger 2002. Separating hist oric events from r ecurrent processes in cryptic species: phylogeo graphy of mud snails ( Hydrobia spp.). Molecular Ecology 11: 1439-1451. Wright, S. 1951. Wright, S. 1951. The genetical structure of populations. Annals of Eugenics. 15:323-354. Annals of Eugenics 15: 323-354. Wright, S. 1969. Evolution and the genetics of populations Volume 2 The theory of gene frequencies. Chicago, Univer sity of Chicago Press. Young, A. M., C. Torres, J. E. Mack and C. W. Cunningham 2002. Morphological and genetic evidence for vicariance and refugi um in Atlantic and Gulf of Mexico populations of the hermit crab Pagurus longicarpus Marine Biology 140: 1059-1066.

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115 OB BL BS MH LW SI MIB R SPCA CK PI BH FTD PA SJP ECS FB(38) (10) (46) (40) (40) (48) (25) (40) (30) (29) (30) (55) (23) (10) (45) (31) FIGURE 3.1: Map indicating sample locations in Florida and Alabama used in microsatellite analysis with the number of individuals genotyped from each indicated in parentheses. OB=Orange Beach, BL=Big Lagoon, SJP=Saint Joseph’s Peninsula, PA=Panacea, CK=Cedar Key, FTD=Tampa Ba y, PI=Pine Island, ECS=Cape Sable, BH=Bahia Honda, BS=Barnes Sound, MH=Matheson Hammock, LW=Lake Worth, SI=Sebastian Inlet, MIBR=Merritt Island Ba nana River, SPCA=Spruce Creek Estuary, FB=Flagler’s Beach. Map created using Generic Mapping Tools Software (ver. 3.0; Wessel & Smith 1995) available online at http://www.aquarius.geoma r.de/omc_intro.html.

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116 FIGURE 3.2: Neighbor-joining dendrogram constructed using pairwise FST estimates of eight microsatellite loci from Melongena corona populations. Abbreviations correspond to population designations in Table 3.3 and Figure 3.1. South Florida & Cape Canaveral West Central & East Panhandle Florida North of Cape Canaveral West ern Florida Panhandle & Alabama MH MIBR Pi LW ECS BS SI BH Pa FTD CK SPCA FB SJP OB BL 0.05

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117 FIGURE 3.3: Neighbor-joining dendrogram constructed from pairwise RST estimates of eight microsatellite loci from Melongena corona populations. Abbreviations correspond to population designations in Table 3.3 and Figure 3.1. South Florida & Cape Canaveral West Central & East Panhandle Florida North of Cape Canaveral West ern Florida Panhandle & Alabama BH FTD Pa SI LW MH BS ECS CK SJP Pi MIBR FB SPCA BL OB 0.05

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118 FIGURE 3.4: Neighbor-joining dendrogram c onstructed using pairwise ( )2 distances estimated from microsatellite data from Melongena corona populations. Abbreviations correspond to population designations in Table 3.3 and Figure 3.1. South Florida & Cape Canaveral West Central & East Panhandle Florida North of Cape Canaveral West ern Florida Panhandle & Alabama BH Pa FTD LW MH BS ECS CK Pi SI SJP FB MIBR SPCA BL OB 10

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119 Locus & Flour. Size Range (bp) Mix 1 Mco A4–TET250 –333Mco 6 –FAM162 -210Mco 10 –FAM276 -336Mco 12 –TET200 -220Mix 2 Mco 2 -TET390 –444Mco 3–FAM160 –238Mco E4–TET234 –318Mco 5–HEX180 –216 TABLE 3.1 : Two mixes of the eight loci run on the ABI 310.

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120 TABLE: 3.2 : Fourteen assemblages of crown c onch populations used in hierarchical partitioning of variance analysis (AMOVA). Groups 1 and 2 were a priori hypotheses based on taxonomic designations and phyl ogeographic patterns of intertidal invertebrates with overlapping ranges. All others were a posteriori hypothesis aimed at obtaining a population stru cture with the highest sign ificant among group variance component. Population abbreviations follow those given in Figure 3.1. Group # Description of Population Groupings 1 Current systematic hypotheses – Panhandle ( M.c. johnstonei), Gulf Coast (M. c. corona), Atlantic ( M. bicolor ), Spruce Creek ( M. sprucecreekensis ) 2 Phylogeographic hypothesis – Gulf of Mexico (OB south to ECS) vs. the Atlantic grouped north (SPCA, FB) and south of Cape Canaveral (BH, BS north to MIBR) 3 Orange Beach Alabama (OB) vs. all other populations 4 Central Peninsula Populations (CK south to PI, and SI), Western Panhandle Florida (BL, SJP), North of Cape Canaveral (FB, SPCA), S outhern Florida populations (ECS, BS), and all others. 5 Central Florida Populations (PA south to PI, SI and LW) vs. Western Panhandle Florida (BL, SJP) vs. North of Cape Canaveral (F B, SPCA) vs. Southern Florida populations (ECS, BS) vs. all others. 6 Gulf of Mexico populations (OB south to PI) vs. the Atlantic & Keys (ECS, BH, and BS north to FB) 7 Gulf of Mexico & Keys (OB south to BS and BH) vs. Atlantic (MH north to FB) 8 Panhandle populations (OB east to PA) vs. Gulf Coast & Keys (CK south to BS & BH) vs. Atlantic populations (MH north to FB) 9 Panhandle populations west of Apalachicol a (OB east to SJP) vs. PA and all 10 Western Panhandle (OB, BL) vs. all other populations 11 Western Panhandle (OB, BL) vs. PA south to FTD, BH and LW vs. MH vs. BS & ECS vs. FB south to SI and SJP vs. PI 12 Panhandle populations (OB to PA ) vs. Peninsular populations 13 Western Panhandle (OB, BL) vs. PA south to FTD, BH and LW vs. PI and MH north to FB vs. BS and ECS. 14 OB east to PA south to FTD, SPCA, FB, and BH vs. ECS & BS north to MIBR

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121TABLE 3.3: Primer sequences and characteriza tion of microsatellite loci in Melongena spp from 20 populations in the Southeastern United States. 1Sequence from clone; 2Basepairs; F= forward primer; R=reverse primer; A=number of alleles N=number of individuals genotyped; Ho=Mean Observed Heterozygosity; He=Mean Expected Heterozygosity; SD=Standard Deviation Locus & Accession # Repeat Sequence1 Primer Sequence (5’ – 3’) Size Range (bp)2 A N Ho SD He SD TA(oC) / MgCl2(mM) Mco2 F: (TET)-CGA CAG GTG GCG TTA GGT T AY239792 (GAA)38 R: GTT GGA TTT ATT TGT CTG GTT CG 390 – 444 17 458 0.786 0.02 0.810 0.11 55 / 2.5 Mco3 F: (FAM)-TCT GAA AGA ATT TTC GCT TCT TA AY233793 (GTTT)19 R: CCT GGT CAA TAA TCT TCA CAA AA 160 – 238 20 502 0.753 0.01 0.761 0.15 55 / 1.5 Mco -A4 (GAAAA)4 F: (TET)-TGC TTA GAT TGG AGG TGT TGG AY233798 (GAA)16 R: CGT CGG GAC AGA TTG TGA TAC 250 – 333 28 505 0.756 0.04 0.808 0.16 55 / 1.5 Mco -E4 F: (TET)-TTT TAG TGG AAA GAC ACA CAT GC AY233799 (CA)26 R: GAG ACC CAA ACG AAA ATG GA 234 – 318 26 500 0.705 0.07 0.805 0.14 55 / 2.5 Mco -5 (GT)7GC(GT)4 F: (HEX)-TGC CGC CAC AGA TTA GTC C AY233796 (GTTT)4 R: CGG CCA AGT TTC CCA ATA A 180 – 216 8 515 0.155 0.00 0.158 0.16 55 / 2.5 Mco -6 F: (FAM)-TTG CAC TGA ATG GGA GCT ATT AY233794 (GA)20 R: AGC GTG TGT GTC CTG CAT TA 162 210 17 508 0.687 0.10 0.825 0.07 55 / 2.5 Mco -10 F: CGT GCA TGT TAC TTC CCA CA AY233797 (CAAA)16 R: (FAM)-GAT TCC GTT GCA ACT TTT CGT 276 336 16 514 0.697 0.01 0.688 0.20 55 / 1.5 Mco -12 F: (TET)-AGG ATT AAT GGG AAA TCA TTG CT AY233795 (GC)5(AC)13 R: GAG CTT GAA GTA CAC GCT TGA 200 220 11 518 0.473 0.05 0.543 0.20 55 / 2.5 Average Across Loci 17.9 0.627 0.21 0.675 0.23

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122TABLE 3.4: Population estimates for each locus and av eraged over all loci. Population abbr eviations and sample sizes are given below the population name. HE & HO = Expected and observed Heterozygosity respectively, GD = Gene diversity, A = Number of alleles (number unique to the popula tion), SD = Standard deviation, FIS = measure of inbreeding fo r each locus/population, all loci/population, all populations per locus, and all populations & loci *Indicates populations out of HWE after sequential Bonferroni correction. M=monomorphic locus Population & Sample Size Mco 2 Mco 3 Mco A4 Mco E4 Mco 5 Mco 6 Mco 10 Mco 12 Mean SD Total Alleles FIS All HE 0.608 0.436 0.699 0.650 0.144 0.708 0.553 0.493 0.536 0.185 HO 0.667 0.417 0.571 0.500 0.121 0.486 0.556 0.500 0.477 0.161 GD 0.580 0.415 0.701 0.624 0.116 0.711 0.529 0.492 0.521 0.192 A 5 4 5 5 2 7 4 3 35 Orange Beach, Alabama OB (n=38) FIS -0.150 -0.004 0.185 0.199 -0.049 0.317 -0.051 -0.015 0.084 HE 0.879 0.590 0.723 0.374 0.102 0.762 0.614 0.552 0.574 0.244 HO 0.667 0.500 0.815 0.276 0.069 0.481 0.593 0.467 0.483 0.230 GD 0.883 0.563 0.721 0.348 0.068 0.755 0.615 0.551 0.563 0.256 A 6 5 9 4 3 (1) 6 5 3 41 (1) Big Lagoon State Park BL (n=30) FIS 0.245 0.111 -0.130 0.207 -0.009 0.362 0.036 0.152 0.141 HE 0.862 0.784 0.673 0.795 0.044 0.856 0.671 0.679 0.671 0.265 HO 0.841 0.864 0.619 0.698 0.022 0.721 0.682 0.756 0.650 0.266 GD 0.860 0.784 0.671 0.796 0.022 0.855 0.658 0.678 0.666 0.272 A 11 11 14 11 2 10 9 7 75 St. Joseph Peninsula State Park SJP (n=46) FIS 0.023 -0.102 0.077 0.123 0.000 0.157 -0.036 -0.114 0.023 Continued on the next page

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123TABLE 3.4 (CONTINUED): Population estimates for each locu s and averaged over all loci. Population & Sample Size Mco 2 Mco 3 Mco A4 Mco E4 Mco 5 Mco 6 Mco 10 Mco 12 Mean SD Total Alleles FIS All HE 0.849 0.905 0.927 0.860 0.212 0.899 0.819 0.755 0.778 0.235 HO 0.793 0.964 0.775 0.775 0.154 0.868 0.850 0.692 0.734 0.248 GD 0.850 0.903 0.923 0.861 0.187 0.900 0.819 0.755 0.775 0.243 A 11 13 18 9 2 11 10 8 82 Panacea PA (n=40) FIS 0.067 -0.067 0.161 0.100 0.177 0.035 -0.038 0.084 0.053 HE 0.908 0.919 0.931 0.911 0.192 0.909 0.841 0.834 0.806 0.250 HO 0.900 0.838 0.897 0.800 0.077 0.865 0.861 0.725 0.745 0.276 GD 0.908 0.920 0.927 0.909 0.170 0.908 0.841 0.836 0.802 0.258 A 13 16 22 (2) 17 4 14 (1) 11 9 106 (3) Cedar Key CK (n=40) FIS 0.009 0.090 0.032 0.120 0.549 0.048 -0.024 0.132 0.071 HE 0.873 0.907 0.937 0.865 0.215 0.883 0.861 0.596 0.767 0.246 HO 0.810 0.854 0.829 0.838 0.205 0.870 0.739 0.410 0.694 0.248 GD 0.873 0.906 0.938 0.866 0.192 0.883 0.863 0.599 0.765 0.254 A 15 15 20 13 5 (2) 12 12 (1) 6 98 (3) Tampa Bay FTD, CCES, GBW (n=48) FIS 0.073 0.058 0.115 0.032 -0.067 0.015 0.143 0.315 0.092 HE 0.819 0.827 0.912 0.887 0.210 0.892 0.782 0.627 0.744 0.234 HO 0.778 0.833 0.857 0.720 0.233 0.607* 0.767 0.567 0.670 0.204 GD 0.820 0.827 0.908 0.878 0.209 0.889 0.782 0.610 0.740 0.234 A 7 12 17 10 2 10 7 (1) 6 (1) 71 (2) Pine Island PI (n=30) FIS 0.051 -0.008 0.056 0.179 -0.115 0.317 0.02 0.071 0.095 Continued on the next page

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124TABLE 3.4 (CONTINUED): Population estimates for each locu s and averaged over all loci. Population & Sample Size Mco 2 Mco 3 Mco A4 Mco E4 Mco 5 Mco 6 Mco 10 Mco 12 Mean SD Total Alleles FIS All HE 0.865 0.779 0.854 0.821 M 0.859 0.572 0.121 0.696 0.274 HO 0.872 0.700 1.000 0.775 M 0.675 0.763 0.050 0.691 0.303 GD 0.861 0.780 0.852 0.816 0.000 0.853 0.570 0.098 0.604 0.356 A 9 10 12 9 1 10 (1) 5 (1) 3 59 (2) East Cape Sable ECS (n=40) FIS -0.013 0.103 -0.173 0.050 M 0.209 -0.340 0.489 -0.001 HE 0.505 0.802 0.797 0.888 M 0.822 0.740 0.494 0.721 0.158 HO 0.500 0.667 0.704 0.714 M 0.483* 0.690 0.069* 0.547 0.232 GD 0.504 0.805 0.798 0.878 0.000 0.824 0.741 0.467 0.627 0.295 A 7 7 8 11 1 8 4 2 48 Bahia Honda Key BH (n=29) FIS 0.008 0.172 0.119 0.186 M 0.414 0.069 0.852 0.237 HE 0.864 0.775 0.849 0.806 0.079 0.821 0.509 0.117 0.602 0.331 HO 0.800 0.833 0.880 0.760 0.040 0.720 0.600 0.080 0.589 0.337 GD 0.857 0.774 0.844 0.805 0.040 0.823 0.507 0.078 0.591 0.347 A 11 7 9 10 2 7 4 2 52 Barnes Sound BS (n=25) FIS 0.066 -0.077 -0.042 0.056 0.000 0.126 -0.184 -0.021 0.003 HE 0.804 0.474 0.314 0.772 0.372 0.722 0.094 0.404 0.495 0.251 HO 0.929 0.348 0.304 0.750 0.478 0.545 0.048 0.174 0.447 0.292 GD 0.769 0.445 0.314 0.750 0.370 0.711 0.048 0.368 0.472 0.253 A 7 5 5 8 2 5 2 3 37 Matheson Hammock County Park MH (n=23) FIS -0.207 0.218 0.031 0.000 -0.294 0.233 0.000 0.527 0.052 Continued on the next page

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125TABLE 3.4 (CONTINUED): Population estimates for each locu s and averaged over all loci. Population & Sample Size Mco 2 Mco 3 Mco A4 Mco E4 Mco 5 Mco 6 Mco 10 Mco 12 Mean SD Total Alleles FIS All HE 0.858 0.879 0.837 0.895 M 0.847 0.797 0.737 0.836 0.054 HO 0.800 1.000 0.800 0.600 M 1.000 0.889 0.800 0.841 0.139 GD 0.822 0.872 0.839 0.906 0.000 0.839 0.792 0.733 0.725 0.298 A 8 8 8 8 1 6 6 5 50 Lake Worth Cove LW (n=10) FIS 0.027 -0.146 0.046 0.337 M -0.192 -0.123 -0.091 -0.015 HE 0.867 0.909 0.911 0.956 0.489 0.822 0.784 0.505 0.780 0.183 HO 1.000 0.889 0.600 1.000 0.400 0.800 0.800 0.800 0.786 0.202 GD 0.850 0.910 0.850 0.950 0.494 0.800 0.783 0.489 0.766 0.178 A 5 9 (1) 5 8 (1) 3 4 6 2 42 (2) Sebastian Inlet SI (n=10) FIS -0.176 0.023 0.294 -0.053 0.191 0.000 -0.021 -0.636 -0.027 HE 0.809 0.784 0.795 0.849 0.529 0.704 0.763 0.612 0.730 0.109 HO 0.700 0.805 0.825 0.762 0.548 0.512 0.762 0.674 0.698 0.116 GD 0.806 0.784 0.784 0.850 0.529 0.703 0.762 0.597 0.727 0.111 A 13 (1) 14 17 13 (2) 5 11 11 6 90 (3) Merritt Island Banana River MIBR (n=45) FIS 0.131 -0.027 -0.053 0.104 -0.036 0.271 0.000 -0.129 0.039 HE 0.825 0.726 0.864 0.765 0.091 0.848 0.749 0.521 0.674 0.259 HO 0.745 0.811 0.843 0.615 0.075 0.686 0.720 0.292 0.598 0.272 GD 0.826 0.719 0.864 0.766 0.073 0.838 0.750 0.506 0.668 0.265 A 8 10 14 13 (1) 2 12 (2) 7 5 (1) 71 (4) Spruce Creek Estuary SPCA & SPCB (n=55) FIS 0.099 -0.128 0.024 0.197 -0.030 0.181 0.040 0.424 0.104 Continued on the next page

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126TABLE 3.4 (CONTINUED): Population estimates for each locu s and averaged over all loci. Population & Sample Size Mco 2 Mco 3 Mco A4 Mco E4 Mco 5 Mco 6 Mco 10 Mco 12 Mean SD Total Alleles FIS All HE 0.764 0.679 0.901 0.783 0.095 0.846 0.851 0.641 0.695 0.258 HO 0.774 0.724 0.774 0.700 0.065 0.677 0.839 0.516 0.634 0.249 GD 0.764 0.679 0.895 0.771 0.063 0.832 0.849 0.644 0.687 0.266 A 10 7 12 9 (1) 2 10 9 6 65 (1) Flagler Beach FB (n=31) FIS -0.013 -0.067 0.135 0.092 -0.017 0.186 0.012 0.198 0.078 HE 0.810 0.761 0.808 0.805 0.158 0.825 0.688 0.543 0.675 0.229 SD 0.107 0.149 0.156 0.136 0.157 0.067 0.193 0.199 HO 0.786 0.753 0.756 0.705 0.155 0.687 0.697 0.473 0.627 0.213 SD 0.118 0.187 0.166 0.159 0.176 0.162 0.199 0.268 GD 0.802 0.755 0.802 0.798 0.158 0.820 0.682 0.531 0.669 0.228 SD 0.109 0.158 0.153 0.143 0.170 0.067 0.204 0.210 Mean A 9.13 9.56 12.19 9.88 2.44 8.94 7.00 4.75 Per Locus Over all Populations FIS 0.030 0.003 0.046 0.120 0.012 0.178 -0.013 0.142 0.070 Total A 17 (1) 20 (1) 28 (2) 26 (5) 8 (3) 17 (4) 16 (3) 11 (2) 143 (21)

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127 TABLE 3.5: Results from three different tests for ex cesses or deficiency in heterozygosity estimated in BOTTLENECK (Cornuet & Luikart 1996). Significant ( p 0.05 prior to Bonferroni correction) excesses or deficien cies under the assumptions of the three different mutation models for each population. N = not significant; Y = indicates mode shift from normal distribution Mutation Model IAM TPM SMM Population Mode Shift Sign Test Wilcoxon Test Sign Test Wilcoxon Test Sign Test Wilcoxon Test Orange Beach N N excess, 0.03711 N N N deficit, 0.00977 Big Lagoon N N N N N N deficit, 0.02734 St. Joseph Peninsula N N N N N deficit, 0.01450 deficit, 0.00586 Panacea N N excess, 0.00391 N excess, 0.00977 N N Cedar Key N N N N N N N Tampa Bay N N N N N N deficit, 0.02734 Pine Island N N excess, 0.00977 N N N N East Cape Sable N N N N N N deficit, 0.02734 Bahia Honda N N N N N N N Barnes Sound N N N N N N deficit, 0.01367 Matheson Hammock N N N N N N deficit, 0.00977 Lake Worth Y N excess, 0.01172 N N N N Sebastian Inlet Y N N N excess, 0.00391 N excess, 0.01953 Merritt Island N N N deficit, 0.00073 deficit, 0.00195 deficit, 0.00084 deficit, 0.00195 Spruce Creek Estuary N N N N N deficit, 0.01388 deficit, 0.00586 Flagler's Beach N N N N N N deficit, 0.00977

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128TABLE 3.6 : Population pairwise RST (below diagonal) and FST (above diagonal) estimates. Bold t ype indicates values significantly different from zero at the 5% level af ter sequential Bonferroni correction. OB BL SJP PA CK FTD PI ECSBH BS MH LW SI MIBRSPCAFB OB 0.132 0.167 0.204 0.174 0.198 0.234 0.268 0.272 0.283 0.430 0.266 0.204 0.278 0.158 0.176 BL 0.063 0.034 0.154 0.079 0.120 0.164 0.205 0.194 0.219 0.415 0.206 0.184 0.212 0.056 0.057 SJP 0.251 0.108 0.088 0.048 0.080 0.118 0.162 0.151 0.186 0.302 0.135 0.041 0.166 0.098 0.105 PA 0.269 0.104 0.058 0.017 0.019 0.045 0.108 0.080 0.135 0.238 0.048 -0.012 0.107 0.094 0.075 CK 0.198 0.083 0.009 0.039 0.003 0.036 0.116 0.082 0.126 0.211 0.043 -0.050 0.093 0.069 0.065 FTD 0.345 0.140 0.067 -0.026 0.020 0.023 0.111 0.076 0.117 0.224 0.035 -0.032 0.075 0.071 0.058 PI 0.309 0.213 0.034 0.116 0.045 0.157 0.114 0.076 0.117 0.175 0.035 -0.016 0.070 0.104 0.075 ECS 0.524 0.401 0.208 0.134 0.170 0.169 0.149 0.162 0.023 0.310 0.122 0.043 0.199 0.153 0.147 BH 0.445 0.258 0.197 0.053 0.159 0.031 0.235 0.136 0.175 0.287 0.101 0.053 0.146 0.145 0.134 BS 0.489 0.407 0.173 0.165 0.133 0.211 0.087 0.057 0.250 0.322 0.129 0.051 0.202 0.157 0.151 MH 0.449 0.339 0.071 0.100 0.079 0.120 0.037 0.100 0.194 0.065 0.240 0.176 0.086 0.310 0.269 LW 0.485 0.303 0.166 0.041 0.076 0.056 0.222 0.160 0.103 0.172 0.169 -0.020 0.092 0.134 0.099 SI 0.367 0.311 0.014 -0.021 -0.116 0.077 -0.008 0.173 0.134 0.088 0.110 0.068 -0.005 0.037 0.003 MIBR 0.159 0.076 0.155 0.118 0.133 -0.001 0.122 0.263 0.130 0.258 0.144 0.124 -0.112 0.171 0.152 SPCA 0.141 0.051 0.147 0.173 0.144 0.111 0.123 0.299 0.220 0.286 0.181 0.221 -0.078 0.030 0.010 FB 0.189 -0.002 -0.002 0.082 0.046 0.054 0.011 0.179 0.163 0.170 0.045 0.171 -0.113 0.106 0.074

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129TABLE 3.7: Population pairwise distances ( )2 estimates below diagonal and geographi c distances between sites above (km) OB BL SJP PA CK FTDPI ECS BH BS MH LW SI MIBRSPCAFB OB 19.31267.09 411.90469.83569.59674.17867.25889.78912.301020.111140.781301.681473.841386.961639.57 BL 17.00247.79 392.60440.87545.45653.25846.33872.08891.39999.191118.261280.761452.931366.041618.65SJP 47.2120.17144.81239.74353.98469.83669.34699.92706.35822.20941.271103.771275.941189.051441.66PA 81.8938.6424.43 159.29307.32427.99629.12664.52658.08781.97901.041063.551235.711148.831401.44CK 70.7341.7610.62 20.23262.27429.60693.48693.48772.32846.33965.401127.911300.071213.191354.78FTD 77.2330.5118.31 5.2719.64167.34431.21431.21510.05584.07703.13867.251148.831061.941203.53PI 64.7143.7514.82 32.9325.0140.03263.88263.88341.11416.73535.80698.31870.47783.58925.18ECS 138.6389.6752.43 28.4654.1639.4529.6554.7178.84152.86271.92434.43606.59519.71772.32BH 125.8062.4457.04 15.3563.4016.7466.0830.01188.25262.27381.33543.84716.01629.12770.71BS 131.2099.6848.92 40.5541.1054.5820.3711.2666.1174.01193.08355.59527.75440.87582.46MH 98.0356.6019.95 22.7127.9927.837.7711.2639.7014.04119.07281.58453.74366.85508.44LW 147.2495.5454.35 29.2529.0527.5472.6849.7349.1250.3151.89162.51334.67247.79389.38SI 65.2852.9513.59 50.3816.2148.9812.3959.48100.6334.7025.7167.44172.1685.28226.87MIBR 89.3039.7547.69 29.3941.7329.3768.8377.4344.5094.4762.5755.7086.0186.8954.71SPCA 55.6624.4832.86 57.9849.1755.0643.5894.5586.5099.9556.13113.0958.7629.05141.59FB 55.4922.238.25 32.3727.0930.1012.4950.9358.0354.4017.8583.0725.6845.7816.89-

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130 TABLE 3.8: Results from AMOVA of hierarch ical partitioning of variance ( FST) from five of the 14 tested assemblages of crow n conchs. The first two groups followed a priori hypotheses based on taxonomic designatio ns (1) and phylogeographic patterns of intertidal invertebrate s with overlapping ranges (2). A ll other groups were determined a posteriori in an attempt to obtain a patte rn with the highest significant among group variance component. Refer to Table 3.2 for asse mblage descriptions. Data from the two groupings testing a priori hypotheses are show n along with three gr oups that exhibited the highest among group variance (t wo that were significant). Comparison Level Variance Component Fixation Index ( ) Percentage of Variation P Group 1 Among groups 0.04007 0.01478 1.48 0.15 Among populations within groups 0.32909 0.12323 12.14 0.00 Within groups 2.34144 0.13619 86.38 0.00 Group 2 Among groups 0.08307 0.03039 3.04 0.02 Among populations within groups 0.30911 0.11662 11.31 0.00 Within groups 2.34144 0.14346 85.65 0.00 Group 3 Among groups 0.28662 0.09720 9.72 0.06 Among populations within groups 0.32057 0.12043 10.87 0.00 Within groups 2.34144 0.20592 79.41 0.00 Group 4 Among groups 0.34781 0.12754 12.75 0.00 Among populations within groups 0.03785 0.01591 1.39 0.00 Within groups 2.34144 0.14142 85.86 0.00 Group 5 Among groups 0.35355 0.12878 12.88 0.00 Among populations within groups 0.05047 0.02110 1.84 0.00 Within groups 2.34144 0.14716 85.28 0.00

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131 APPENDICES

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132APPENDIX A.1 VARIABLE SITES FROM COI HAPLOTYPES OF MELONGENA SPP. Nucleotide Position Individual or Haplotype 5 2 4 2 7 3 0 3 9 4 0 4 2 5 1 5 4 5 7 6 9 7 6 7 8 8 4 9 0 1 0 2 1 1 4 1 1 7 1 2 0 1 3 9 1 4 1 1 4 4 1 4 7 1 5 0 1 5 3 1 6 2 1 6 5 1 6 8 1 7 1 1 7 4 1 7 5 1 7 7 1 7 8 1 8 0 1 8 6 1 8 8 1 8 9 1 9 3 1 9 8 2 0 7 A A T C C C TATAGGCACATATGTGTG A G AATACTGTGTTGTTT SPCA_11 . . .................. . ............... B . . .................. . ............... SI_08 . . .................. . ............... ECS_16 . . .................. . ............... BS_18 ? ? ? ? .........T....C... . ............... C . . .................. . ............... BT_02 . . .......G.......... . ............... BT_04 . . .......G.......... . ............... BT_05 . . .......G.......... . ............... PI_10 . . .................. . ............... PI_25 . . .................. . .........C..... FTD_10 . . .................. . ............... BL_01 ? ? ? ? ..............C... . ............... D . . ..C...........C... . ............... MP_07 T . . CG....T.TG......C. T A GGC....C..CA..C YP_01 T A A T T .G.GAAT...GTAA.ACA A .TCGTCA.AC.ACAC YP_03 T A A T T .G.GAAT...GT.A.ACA A .TCGT.A.AC.ACAC YP_06 T A A T T .G.GAAT...GT.A.ACA A .TCGT.A.AC.ACAC

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133APPENDIX A.1 (CONTINUED) Nucleotide Position Individual or Haplotype 2 1 0 2 1 3 2 1 6 2 1 9 2 2 2 2 3 1 2 3 4 2 3 7 2 4 0 2 4 3 2 4 4 2 4 6 2 5 2 2 5 5 2 5 8 2 7 0 2 7 6 2 8 2 2 9 6 2 9 7 3 0 0 3 0 3 3 1 2 3 1 8 3 2 1 3 2 4 3 2 7 3 3 0 3 3 6 3 4 5 3 4 8 3 5 1 3 5 3 3 5 4 3 6 0 3 6 6 3 7 2 3 7 8 3 8 1 3 8 2 A A T C G C T AACACTATAGTTGGGAA A T TTAGTCCGTACGTTC SPCA_11 . . . . . . . . . . . . . . . . . . . . B . . . . . . . . . . . . . . . . . . . . SI_08 . . . . . . . . . . . . . . . . . . . . ECS_16 . . . . . . . . . . . . . . . . . . . . BS_18 . . . . . . . . . . . . . . . . C. . . . C . . . . . . . . . . . . . . . . . . . . BT_02 . . . . . . . . . . . . . . . . . . . . BT_04 . . . . . . . . . . . . . . . . . . . . BT_05 . . . . . . . . . . . . . . . . . . . . PI_10 . . . . . . . . . . . . . . . . . . . . PI_25 . . . . . . . . . . . . . . . . . . . . FTD_10 . . . . . . . . . . . . . . . . . . . . BL_01 . . . . . . . . . . . . . . . . . . . . D . . . . . . . . . . . . . . . . . . . . MP_07 . T A . GG. G. G. A. AA. G. C . GA. T. A. A. T YP_01 G C T A T C GT. TGGATACG. AA. G G CA. AGTT. ATTA. CT YP_03 G C T A T C GT. TGGATACG. AA. G G CA. AGTT. ATTACCT YP_06 G C T A T C GT. TGGATACG. AA. G G CA. AGTT. ATTACCT

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134APPENDIX A.1 (CONTINUED) Nucleotide Position Individual or Haplotype 3 8 4 3 9 3 3 9 6 3 9 8 3 9 9 4 0 5 4 0 9 4 1 1 4 2 3 4 3 2 4 5 6 4 6 2 4 6 5 4 7 1 4 8 0 4 8 1 4 8 3 4 8 6 4 8 9 5 0 4 5 2 2 5 2 3 5 2 8 5 2 9 5 3 1 5 3 4 5 3 7 5 3 8 5 4 3 5 4 6 5 5 5 5 5 8 5 5 9 5 6 2 5 6 4 5 7 0 5 7 6 5 8 2 6 0 0 6 0 3 A A C T T A T CTCCGAGGTCCTCCATA C G GTTGGTATCACCTTC SPCA_11 . . . . . . . . . . . . . . . . . . . . B . . . . . . . . . . . . . . . . . . . . SI_08 . . . . . . . . . . . . . . . . . . . . ECS_16 . . . . . . . . . . . . . . . . . . . . BS_18 . . . . . . . . . . . . . . . ????????? C . . . . . . . . . . . . . . . . . . . . BT_02 . . . . . . . . . . . . . . . . . . . . BT_04 . . . . . . . . . . . . . . . . . . . . BT_05 . . . . . . . . . . . . . . . . . . . . PI_10 . . . . . . . . . . . . . . . . . . . . PI_25 . . . . . . . . . . . . . . . . . . . . FTD_10 . . . . . . . . . . . . . . . . . . . . BL_01 . . . . . . . . . . . . . . . ????????? D . . . . . . . . . . . . . . . . T. . . MP_07 . C G C TA. . G. A. TG. T. C. T A. . AC. . GT. . YP_01 G T C . TATTAGAACTG. TTG. T T A TCCA. AGCT. TCAA YP_03 G T . . TATTAGAACTG. TTG. T T A TCCA. AGCT. TCAA YP_06 G T . . TATTAGAACTG. TTG. T T A TCCA. AGCT. TCAA

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135APPENDIX A.1 (CONTINUED) Nucleotide Position Individual or Haplotype 6 1 8 6 2 1 6 2 2 6 2 7 6 3 0 6 3 6 6 4 8 6 5 1 6 5 7 6 6 0 6 7 0 6 7 6 6 7 7 6 7 8 6 8 1 6 8 4 6 8 7 6 9 0 6 9 9 7 0 2 7 1 4 7 1 7 7 2 3 7 2 6 7 3 8 7 4 5 7 4 7 7 5 0 7 5 3 7 5 9 7 6 2 7 6 5 7 6 7 7 6 8 7 8 0 7 8 1 7 8 6 7 9 4 7 9 5 7 9 8 A C T T C A C TTCGTTTAAATGCTCTT G T CGTACTGTATTGTGA SPCA_11 . . . . . . . . . . . . . . . . . . . . B . . . . . . . . . . . . . . . . . . . . SI_08 . . . . . . . . . . . . . . . . . . . . ECS_16 . . . . A. . . . . . . . . . . . . . . BS_18 ? ? ? ? ? ? ????????????????? ? ? ??????????????? C . . . . . . . . . . . . . . . . . . . . BT_02 . . . . . . . . . . . . . . . . . . . . BT_04 . . . . . . . . . . . . . . . . . . . . BT_05 . . . . . . . . . . . . . . . . . . . . PI_10 . . . . . . . . . . . . . . . . . . . . PI_25 . . . . . . . . . . . . . . . . . . . . FTD_10 . . . . . . . . . . . . . . . . . . . . BL_01 ? ? ? ? ? ? ????????????????? ? ? ??????????????? D . . . . . . . . . . . . . . . . . . . . MP_07 T C C T T CCT. C. . . . CT. C C T. CG. . G. . C. G YP_01 T . T G T . T. CC. TTCCAT. TC. A C TA. TCA. GACA. A. YP_03 T . T G T . T. CCCTTCCAT. TC. A C TA. TCACGACA. A. YP_06 T . T G T . T. CC. TTCCAT. TC. A C TA. TCA. GACA. A.

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136APPENDIX A.1 (CONTINUED) Nucleotide Position Individual or Haplotype 8 0 7 8 1 0 8 1 6 8 2 5 8 3 1 8 3 4 8 4 0 8 4 3 8 5 2 8 5 8 8 6 7 8 7 3 8 9 7 9 0 7 9 1 5 9 2 1 9 2 4 9 2 7 9 3 0 9 3 3 9 3 9 9 4 5 9 4 9 9 5 8 9 7 2 9 7 8 9 8 7 9 9 0 9 9 6 9 9 9 1 0 0 6 1 0 0 8 1 0 2 1 1 0 2 6 1 0 3 8 1 0 4 1 1 0 4 4 1 0 4 7 1 0 5 0 1 0 5 9 A C G G G A C AGACCCTGCTCACGATC T C CGTAACACTTTTTCT SPCA_11 . . . . T. . . . . . . . . . . . . . T. B . . . . . . . . . . . . . . . . . . . T. SI_08 . . . . . . . . . A. . . . . . . . . T. ECS_16 . . . . . . A. . . . . . . . . . . . T. BS_18 ? ? ? ? ? ? ????????????????? ? ? ??????????????? C . . . . T. . . . . . . . . . . . . . T. BT_02 . . . . T. . . . . . . . . . . . . . T. BT_04 . . . . T. . T. . . . . . . . . . . . T. BT_05 . . . . . . . T. . . . . . . . . . . . T. PI_10 . . . . T. . . . . . . . . . . . . . T. PI_25 . . . . T. . . . . . T . . . . . . . T. FTD_10 . . . . . . . . . . . . . . . . . . . T. BL_01 ? ? ? ? ? ? ????????????????? ? ? ??????????????? D . . . . . . . . . . . . . . . . . . . T. MP_07 T A A T GAGTTT. T. G. . C. . TA. GG. G. CCCC. . YP_01 T A A A G T . TT. C. TAT. T. G. T C T TAGG. T. T. CC. CTC YP_03 T A A A G T . TT. C. TAT. T. G. T C T TAGG. T. T. CC. CTC YP_06 T A A A G T . TT. C. TAT. T. G. T C T TAGG. T. T. CC. CTC

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137APPENDIX A.1 (CONTINUED) A,B,C,D are haplotypes shared by more than one individual as i ndicated in Figure 2.2 Individual abbreviations follow thos e given in Figure 2.2 and Appendix B Nucleotide Position Individual or Haplotype 1 0 6 2 1 0 6 5 1 0 7 1 1 0 7 4 1 0 7 7 1 0 8 3 1 0 9 2 1 1 0 7 1 1 1 0 1 1 1 3 1 1 1 9 1 1 2 9 1 1 3 1 1 1 3 4 1 1 3 7 1 1 4 6 1 1 4 9 1 1 5 2 1 1 5 3 1 1 5 5 1 1 5 8 1 1 7 0 1 1 7 6 1 1 7 9 1 2 0 3 1 2 0 4 A T TCTTAGAACCCTATA T CATTTTTAG SPCA_11 . . . . . . . . . . . . . B . . . . . . . . . . . . . SI_08 . . . . . . . . . . . . . ECS_16 . . . . . . . . . . . . . BS_18 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? C . . . . . . . . . . . . A BT_02 . . . . . . . . . . . . A BT_04 . . . . . . . . . . . . A BT_05 . . . . . . . . . . . . . PI_10 . . . . . . . . . . . . . PI_25 . . . . . . . . . . . . . FTD_10 . . . . . . . . . . . . . BL_01 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? D . . . . . . . . . . . . . MP_07 . CAG. . . . AT T. CC. . A YP_01 C CTA. TGGTTTAGAG C TGC. CCCTA YP_03 C CTA. TGGTTTAGAG C TGC. CCCTA YP_06 C CTA. TGGTTTAGAG C TGC. CCCTA

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138 APPENDIX A.2 VARIABLE SITES FROM 475 BP FRAGMENT OF 16S Nucleotide Position Haplotype 1 7 2 3 2 4 1 3 1 1 3 2 1 3 6 1 3 7 1 3 8 1 4 4 1 5 4 1 8 0 2 0 5 2 0 7 2 1 5 2 1 8 2 2 1 2 2 6 2 2 7 2 3 0 2 3 1 2 3 3 2 3 5 2 3 7 2 3 8 2 4 1 2 4 6 2 4 7 2 5 6 2 5 9 2 9 4 3 1 0 3 1 3 3 1 5 3 1 9 3 2 1 3 2 5 3 2 9 3 4 3 4 0 2 4 1 7 4 6 0 Corona complex A A ACGTCTTATGAGGGATAC G T GAAAAGCACGAAAAAACGT Corona complex . ................... A.................. M. melongena G ...CTCC......A..... .GG......A....G.-.C M. patula – 1 G G ..A.T..G.....AGC..A AG......TA....G.... M. patula – 2 G ..A.T..G.....AGC..A AG......TA....G.... M. bispinosa . GT.CA.A.ATGAAA-.GG A C A..TTATGT.TGGTGGTTC Individuals sharing haplotype s are indicated in Figure 2.4.

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139 APPENDIX B LIST OF ALL SAMPLES COLLECTED Comprehensive list of all samples collected during this study (note: not all samples collected were us ed in this study). Coord inates were obtained using Garmin GPS III unit at most sites, or in a few cases afterwards using the online topographic mapping softwa re TopoZone (available at http://topozone.com/ ). Taxonomic classification for species of Melongena follows that of Tucker (1994). Abbreviations given correspond to those used throughout the text. M = Male; F = Female. Sample # Collection Date Coordinates Collector(s) Genus Species Subspecies Sex OB = Orange Beach, Baldwin Co., Alabama at Lee Calloway Bridge Public Beach Access (Little Lagoon) OB-01 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei ? OB-02 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-03 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-04 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-05 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei M OB-06 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-07 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-08 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-09 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-10 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei M OB-11 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-12 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-13 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei M OB-14 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-15 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei M OB-16 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F

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140APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex OB-17 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-18 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-19 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-20 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-21 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-22 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-23 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-24 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-25 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-26 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-27 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei M OB-28 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei M OB-29 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-30 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-31 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-32 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-33 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-34 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-35 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-36 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-37 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F OB-38 7/29/2000 30.2411N, -87.7378W K. Hayes Melongena corona johnstonei F BL = Big Lagoon State Park, Escambia Co, FL in channel connecting Grand Lagoon

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141APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex BL-01 7/29/2000 30.3093N, -87.4014W K. Hayes Melongena corona johnstonei BL-02 7/29/2000 30.3093N, -87.4014W K. Hayes Melongena corona johnstonei BL-03 7/29/2000 30.3093N, -87.4014W K. Hayes Melongena corona johnstonei BL-04 5/25/2001 30.3093N, -87.4014W K. Hayes Melongena corona johnstonei BL-05 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei BL-06 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei BL-07 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei BL-08 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei BL-09 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei BL-10 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei BL-11 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei BL-12 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei M BL-13 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei F BL-14 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei F BL-15 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei F BL-16 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei F BL-17 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei M BL-18 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei F BL-19 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei M BL-20 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei M BL-21 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei M BL-22 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei F BL-23 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei M

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142APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex BL-24 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei M BL-25 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei F BL-26 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei M BL-27 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei F BL-28 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei F BL-29 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei F BL-30 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei M BL-31 5/25/2001 30.3093N, -87.4014W K. Hayes/P. Nichols Melongena corona johnstonei SJP = St. Joseph Peninsula State Park in Eagle Harbor, Gulf Co, FL SJP-01 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei SJP-02 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei SJP-03 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei SJP-04 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-05 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei SJP-06 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei SJP-07 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei M SJP-08 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-09 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei M SJP-10 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei M SJP-11 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei M SJP-12 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-13 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-14 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F

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143APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex SJP-15 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei JF SJP-16 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-17 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-18 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei M SJP-19 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-20 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-21 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei M SJP-22 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-23 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei M SJP-24 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei M SJP-25 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei M SJP-26 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-27 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei M SJP-28 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei M SJP-29 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei M SJP-30 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-31 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-32 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-33 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-34 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-35 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-36 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F SJP-37 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei F

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144APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex SJP-38 11/28/1999 29.7748N, -85.4015W S. Karl Melongena corona johnstonei M SJP-39 5/24/2001 29.7748N, -85.4015W K. Hayes/P. Nichols Melongena corona johnstonei ? SJP-40 5/24/2001 29.7748N, -85.4015W K. Hayes/P. Nichols Melongena corona johnstonei ? SJP-41 5/24/2001 29.7748N, -85.4015W K. Hayes/P. Nichols Melongena corona johnstonei ? SJP-42 5/24/2001 29.7748N, -85.4015W K. Hayes/P. Nichols Melongena corona johnstonei ? SJP-43 5/24/2001 29.7748N, -85.4015W K. Hayes/P. Nichols Melongena corona johnstonei ? SJP-44 5/24/2001 29.7748N, -85.4015W K. Hayes/P. Nichols Melongena corona johnstonei ? SJP-45 5/24/2001 29.7748N, -85.4015W K. Hayes/P. Nichols Melongena corona johnstonei ? SJP-46 5/24/2001 29.7748N, -85.4015W K. Hayes/P. Nichols Melongena corona johnstonei ? SJP-47 5/24/2001 29.7748N, -85.4015W K. Hayes/P. Nichols Melongena corona johnstonei ? SJP-48 5/24/2001 29.7748N, -85.4015W K. Hayes/P. Nichols Melongena corona johnstonei ? EP = East Point, Franklin Co, FL along US 98 across from Hammock Shores near broken seawall EP-01 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M EP-02 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei F EP-03 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M EP-04 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M EP-05 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M EP-06 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei F EP-07 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei F EP-08 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M EP-09 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei F EP-10 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M EP-11 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M

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145APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex EP-12 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M EP-13 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei F EP-14 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M EP-15 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M EP-16 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M EP-17 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei F EP-18 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M EP-19 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei F EP-20 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei F EP-21 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M EP-22 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M EP-23 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M EP-24 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei M EP-25 5/24/2001 29.7418N, -84.8664W K. Hayes/P. Nichols Melongena corona johnstonei PA = Panacea, Wakulla Co, FL mile from Hwy 98 on the south side of Marsh Sound Rd PA-01 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-02 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-03 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-04 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-05 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-06 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-07 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-08 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F

PAGE 155

146APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex PA-09 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-10 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-11 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-12 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-13 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-14 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-15 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-16 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-17 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-18 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-19 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-20 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-21 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-22 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-23 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-24 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-25 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-26 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-27 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-28 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-29 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-30 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-31 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F

PAGE 156

147APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex PA-32 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-33 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-34 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-35 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-36 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-37 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-38 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F PA-39 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei M PA-40 5/24/2001 29.9764N, -84.3778W K. Hayes/P. Nichols Melongena corona johnstonei F DB = Dekle Beach, Taylor Co, FL in channel next to SR 321 headed toward Jug Island DB-01 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei M DB-02 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-03 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei M DB-04 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-05 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-06 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-07 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei M DB-08 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-09 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-10 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei M DB-11 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-12 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-13 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F

PAGE 157

148APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex DB-14 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei M DB-15 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-16 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei M DB-17 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei M DB-18 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-19 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei M DB-20 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei M DB-21 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-22 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-23 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-24 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-25 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-26 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-27 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-28 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-29 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-30 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-31 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-32 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-33 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-34 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei M DB-35 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-36 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F

PAGE 158

149APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex DB-37 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei M DB-38 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei M DB-39 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F DB-40 5/26/2001 29.8409N, -83.6161W K. Hayes/P. Nichols Melongena corona johnstonei F HB = HorseShoe Beach, Dixie Co, FL end of CR 351 intertidal oyster patches bordered by juncus marsh HB-01 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? M HB-02 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? F HB-03 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? M HB-04 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? M HB-05 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? M HB-06 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? F HB-07 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? F HB-08 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? F HB-09 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? M HB-10 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? M HB-11 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? M HB-12 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? F HB-13 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? F HB-14 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? F HB-15 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? HB-16 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? HB-17 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? HB-18 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ????

PAGE 159

150APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex HB-19 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? HB-20 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? HB-21 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? HB-22 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? HB-23 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? HB-24 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? HB-25 5/27/2001 29.4377N, -83.2935W K. Hayes/P. Nichols Melongena corona ???? CK = Cedar Key, Levy Co, FL left side Hwy 24 over 1st bridge heading to Cedar Key across from SW 153rd Ct & FWC offices CK-01 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-02 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-03 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-04 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-05 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-06 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-07 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-08 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-09 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-10 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-11 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-12 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-13 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-14 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-15 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M

PAGE 160

151APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex CK-16 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-17 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-18 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-19 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-20 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-21 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-22 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-23 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-24 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-25 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-26 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-27 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona ? CK-28 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-29 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-30 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-31 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-32 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-33 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-34 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-35 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-36 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-37 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CK-38 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F

PAGE 161

152APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex CK-39 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona M CK-40 5/27/2001 29.1643N, -83.0269W K. Hayes/P. Nichols Melongena corona corona F CR = Crystal River, Citrus County, FL end of CR 44 at public boat ramps CR-01 7/22/2000 28.8967N, -82.5970W K. Hayes Melongena corona corona CR-02 7/22/2000 28.8967N, -82.5970W K. Hayes Melongena corona corona CR-03 7/22/2000 28.8967N, -82.5970W K. Hayes Melongena corona corona CR-04 7/22/2000 28.8967N, -82.5970W K. Hayes Melongena corona corona TS = Tarpon Springs, Pinellas Co, FL east of Howard Park in embayment alongside of the road TS-01 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-02 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-03 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-04 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-05 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-06 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-07 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-08 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-09 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-10 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-11 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-12 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-13 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-14 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-15 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona

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153APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex TS-16 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-17 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-18 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-19 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona TS-20 6/16/2000 28.1514N, -82.7716W K. Hayes/P. Nichols Melongena corona corona BB = Bailey’s Bluff, Pasco Co, FL – west edge of Bailey’s Bluff with intertidal oysters BB-01 12/25/2000 28 12N, -82 47W P. Poland Melongena corona corona BB-02 12/25/2000 29 12N, -82 47W P. Poland Melongena corona corona BB-03 12/25/2000 30 12N, -82 47W P. Poland Melongena corona corona BB-04 12/25/2000 31 12N, -82 47W P. Poland Melongena corona corona BB-05 12/25/2000 32 12N, -82 47W P. Poland Melongena corona corona BB-06 12/25/2000 33 12N, -82 47W P. Poland Melongena corona corona BB-07 12/25/2000 34 12N, -82 47W P. Poland Melongena corona corona BB-08 12/25/2000 35 12N, -82 47W P. Poland Melongena corona corona BB-09 12/25/2000 36 12N, -82 47W P. Poland Melongena corona corona BB-10 12/25/2000 37 12N, -82 47W P. Poland Melongena corona corona BB-11 12/25/2000 38 12N, -82 47W P. Poland Melongena corona corona BB-12 12/25/2000 39 12N, -82 47W P. Poland Melongena corona corona CCES = Courtney Campbell Causeway East End, South Side, Hillsbor ough Co., FL Near Rock Pt with oysters at edge of mangroves CCES-01 5/31/1999 27.9655N, -82.5546W K. Hayes Melongena corona corona CCES-02 5/31/1999 27.9655N, -82.5546W K. Hayes Melongena corona corona CCES-03 5/31/1999 27.9655N, -82.5546W K. Hayes Melongena corona corona M CCES-04 5/31/1999 27.9655N, -82.5546W K. Hayes Melongena corona corona

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154APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex CCES-05 5/31/1999 27.9655N, -82.5546W K. Hayes Melongena corona corona CCES-06 5/31/1999 27.9655N, -82.5546W K. Hayes Melongena corona corona CCES-07 5/31/1999 27.9655N, -82.5546W K. Hayes Melongena corona corona F CCES-08 5/31/1999 27.9655N, -82.5546W K. Hayes Melongena corona corona F CCES-09 5/31/1999 27.9655N, -82.5546W K. Hayes Melongena corona corona M GBW = Gandy Bridge West End, Pinellas Co., FL – St. Peters burg Side near radio towers and old bridge material GBW-01 7/20/1999 27.8723N, -82.6106W K. Hayes Melongena corona corona GBW-02 7/20/1999 27.8723N, -82.6106W K. Hayes Melongena corona corona F GBW-03 7/20/1999 27.8723N, -82.6106W K. Hayes Melongena corona corona F GBW-04 7/20/1999 27.8723N, -82.6106W K. Hayes Melongena corona corona F GBW-05 7/20/1999 27.8723N, -82.6106W K. Hayes Melongena corona corona M GBW-06 7/20/1999 27.8723N, -82.6106W K. Hayes Melongena corona corona F GBW-07 7/20/1999 27.8723N, -82.6106W K. Hayes Melongena corona corona M GBW-08 7/20/1999 27.8723N, -82.6106W K. Hayes Melongena corona corona M GBW-09 7/20/1999 27.8723N, -82.6106W K. Hayes Melongena corona corona M GBW-10 7/20/1999 27.8723N, -82.6106W K. Hayes Melongena corona corona M GBW-11 7/20/1999 27.8723N, -82.6106W K. Hayes Melongena corona corona F GBW-12 7/20/1999 27.8723N, -82.6106W K. Hayes Melongena corona corona M FTD = Fort DeSoto Park Area, Pinellas Co., FL – 1st rest area heading over Skyway Bridge FTD-01 6/1/1999 27.6503N, -82.6772W K. Jensen Melongena corona corona FTD-02 6/1/1999 27.6503N, -82.6772W K. Jensen Melongena corona corona F FTD-03 6/1/1999 27.6503N, -82.6772W K. Jensen Melongena corona corona M FTD-04 6/1/1999 27.6503N, -82.6772W K. Jensen Melongena corona corona M

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155APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex FTD-05 6/1/1999 27.6503N, -82.6772W K. Jensen Melongena corona corona F FTD-06 6/1/1999 27.6503N, -82.6772W K. Jensen Melongena corona corona F FTD-07 6/1/1999 27.6503N, -82.6772W K. Jensen Melongena corona corona M FTD-08 6/1/1999 27.6503N, -82.6772W K. Jensen Melongena corona corona F FTD-09 6/1/1999 27.6503N, -82.6772W K. Jensen Melongena corona corona M FTD-10 6/1/1999 27.6503N, -82.6772W K. Jensen Melongena corona corona M FTD-11 6/1/1999 27.6503N, -82.6772W K. Jensen Melongena corona corona F FTD-12 6/1/1999 27.6503N, -82.6772W K. Jensen Melongena corona corona F FTD-13 6/1/1999 27.6503N, -82.6772W K. Jensen Melongena corona corona FTD-14 6/1/1999 27.6503N, -82.6772W K. Jensen Melongena corona corona FTD-15 6/1/1999 27.6503N, -82.6772W K. Jensen Melongena corona corona FTD = Ft. DeSoto Area, Pinellas Co., FL east end intertidally around mangrove islands FTD-16 6/1/1999 27.6503N, -82.6772W K. Hayes Melongena corona corona F FTD-17 6/1/1999 27.6503N, -82.6772W K. Hayes Melongena corona corona F FTD-18 6/1/1999 27.6503N, -82.6772W K. Hayes Melongena corona corona F FTD-19 6/1/1999 27.6503N, -82.6772W K. Hayes Melongena corona corona F FTD-20 6/1/1999 27.6503N, -82.6772W K. Hayes Melongena corona corona M FTD-21 6/1/1999 27.6503N, -82.6772W K. Hayes Melongena corona corona F FTD-22 6/1/1999 27.6503N, -82.6772W K. Hayes Melongena corona corona F FTD-23 6/1/1999 27.6503N, -82.6772W K. Hayes Melongena corona corona F FTD-24 6/1/1999 27.6503N, -82.6772W K. Hayes Melongena corona corona F FTD-25 6/1/1999 27.6503N, -82.6772W K. Hayes Melongena corona corona F FTD-26 6/1/1999 27.6503N, -82.6772W K. Hayes Melongena corona corona F

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156APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex FTD-27 6/1/1999 27.6503N, -82.6772W K. Hayes Melongena corona corona F PI = Pine Island (Pine Land), Lee Co., FL – Gulf side of Pi neland Rd. past Joyce James Memorial Plaque before marina PI-01 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona M PI-02 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona M PI-03 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona M PI-04 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-05 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona M PI-06 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona M PI-07 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-08 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-09 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona M PI-10 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona M PI-11 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona JF PI-12 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-13 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-14 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-15 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-16 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-17 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-18 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona M PI-19 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona M PI-20 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-21 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona M

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157APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex PI-22 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-23 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-24 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona M PI-25 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-26 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-27 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-28 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-29 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-30 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona F PI-31 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona PI-32 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona PI-33 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona PI-34 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona PI-35 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona PI-36 9/10/2000 26.6565N, -82.1494W K. Hayes/P. Hayes Melongena corona corona CB = Chokoloskee Bay, Collier Co., FL – Along Hwy 29 west of Everglad es City mile past 10,000 Isl. State Park near Picnic Are a CB-01 11/12/2000 25.8401N, -81.3823W P. Poland Melongena corona corona M CB-02 11/12/2000 25.8401N, -81.3823W P. Poland Melongena corona corona F CB-03 11/12/2000 25.8401N, -81.3823W P. Poland Melongena corona corona F CB-04 11/12/2000 25.8401N, -81.3823W P. Poland Melongena corona corona F CB-05 11/12/2000 25.8401N, -81.3823W P. Poland Melongena corona corona F CB-06 11/12/2000 25.8401N, -81.3823W P. Poland Melongena corona corona M CB-07 11/12/2000 25.8401N, -81.3823W P. Poland Melongena corona corona

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158APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex CB-08 11/12/2000 25.8401N, -81.3823W P. Poland Melongena corona corona CB-09 11/12/2000 25.8401N, -81.3823W P. Poland Melongena corona corona CB-10 11/12/2000 25.8401N, -81.3823W P. Poland Melongena corona corona CB-11 11/12/2000 25.8401N, -81.3823W P. Poland Melongena corona corona CB-12 11/12/2000 25.8401N, -81.3823W P. Poland Melongena corona corona CB-13 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-14 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-15 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-16 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-17 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-18 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-19 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-20 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-21 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-22 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-23 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-24 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-25 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-26 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-27 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-28 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-29 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona CB-30 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona

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159APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex CB-31 4/6/2001 25.8401N, -81.3823W R. Hayes Melongena corona corona ECS = East Cape Sable, Monroe Co., FL – Open mud-fl at area at low tide heading east toward Lake Ingram. ECS-01 11/24/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona M ECS-02 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-03 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona M ECS-04 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona M ECS-05 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-06 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-07 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-08 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona M ECS-09 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-10 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-11 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona M ECS-12 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-13 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-14 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-15 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-16 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona M ECS-17 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona M ECS-18 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-19 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-20 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-21 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F

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160APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex ECS-22 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona M ECS-23 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-24 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona M ECS-25 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-26 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-27 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona M ECS-28 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona M ECS-29 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-30 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona M ECS-31 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona M ECS-32 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-33 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-34 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-35 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-36 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-37 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-38 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona F ECS-39 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona M ECS-40 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona M ECS-41 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-42 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-43 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-44 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona

PAGE 170

161APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex ECS-45 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-46 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-47 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-48 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-49 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-50 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-51 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-52 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-53 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-54 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-55 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-56 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-57 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-58 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-59 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-60 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-61 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-62 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-63 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-64 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-65 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-66 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-67 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona

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162APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex ECS-68 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-69 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-70 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-71 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-72 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-73 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-74 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona ECS-75 11/25/2001 25.1250 N, -81.0793 W. K. Hayes/R. Hayes Melongena corona corona CF = Cape Flamingo, Monroe Co., FL – Behind Cottag es on Flaming camping area with silty sand and rubble CF-01 12/12/2000 25 08N -80 57W P. Poland Melongena bicolor F CF-02 12/12/2000 26 08N -80 57W P. Poland Melongena bicolor M CF-03 12/12/2000 27 08N -80 57W P. Poland Melongena bicolor F CF-04 12/12/2000 28 08N -80 57W P. Poland Melongena bicolor M CF-05 12/12/2000 29 08N -80 57W P. Poland Melongena bicolor F CF-06 12/12/2000 30 08N -80 57W P. Poland Melongena bicolor F CF-07 12/12/2000 31 08N -80 57W P. Poland Melongena bicolor M CF-08 12/12/2000 32 08N -80 57W P. Poland Melongena bicolor M CF-09 12/12/2000 33 08N -80 57W P. Poland Melongena bicolor F CF-10 12/12/2000 34 08N -80 57W P. Poland Melongena bicolor F CF-11 12/12/2000 35 08N -80 57W P. Poland Melongena bicolor CF-12 12/12/2000 36 08N -80 57W P. Poland Melongena bicolor CF-13 12/12/2000 37 08N -80 57W P. Poland Melongena bicolor CF-14 12/12/2000 38 08N -80 57W P. Poland Melongena bicolor

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163APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex CF-15 12/12/2000 39 08N -80 57W P. Poland Melongena bicolor CF-16 12/12/2000 40 08N -80 57W P. Poland Melongena bicolor CF-17 12/12/2000 41 08N -80 57W P. Poland Melongena bicolor CF-18 12/12/2000 42 08N -80 57W P. Poland Melongena bicolor CF-19 12/12/2000 43 08N -80 57W P. Poland Melongena bicolor CF-20 12/12/2000 44 08N -80 57W P. Poland Melongena bicolor CF-21 12/12/2000 45 08N -80 57W P. Poland Melongena bicolor CF-22 12/12/2000 46 08N -80 57W P. Poland Melongena bicolor CF-23 12/12/2000 47 08N -80 57W P. Poland Melongena bicolor CF-24 12/12/2000 48 08N -80 57W P. Poland Melongena bicolor CF-25 12/12/2000 49 08N -80 57W P. Poland Melongena bicolor CF-26 12/12/2000 50 08N -80 57W P. Poland Melongena bicolor CF-27 12/12/2000 51 08N -80 57W P. Poland Melongena bicolor CF-28 12/12/2000 52 08N -80 57W P. Poland Melongena bicolor CF-29 12/12/2000 53 08N -80 57W P. Poland Melongena bicolor CF-30 12/12/2000 54 08N -80 57W P. Poland Melongena bicolor CF-31 12/12/2000 55 08N -80 57W P. Poland Melongena bicolor CF-32 12/12/2000 56 08N -80 57W P. Poland Melongena bicolor CF-33 12/12/2000 57 08N -80 57W P. Poland Melongena bicolor CF-34 12/12/2000 58 08N -80 57W P. Poland Melongena bicolor CF-35 12/12/2000 59 08N -80 57W P. Poland Melongena bicolor CF-36 12/12/2000 60 08N -80 57W P. Poland Melongena bicolor CF-37 12/12/2000 61 08N -80 57W P. Poland Melongena bicolor

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164APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex CF-38 12/12/2000 62 08N -80 57W P. Poland Melongena bicolor CF-39 12/12/2000 63 08N -80 57W P. Poland Melongena bicolor CF-40 12/12/2000 64 08N -80 57W P. Poland Melongena bicolor CF-41 12/12/2000 65 08N -80 57W P. Poland Melongena bicolor CF-42 12/12/2000 66 08N -80 57W P. Poland Melongena bicolor CF-43 12/12/2000 67 08N -80 57W P. Poland Melongena bicolor CF-44 12/12/2000 68 08N -80 57W P. Poland Melongena bicolor CF-45 12/12/2000 69 08N -80 57W P. Poland Melongena bicolor CF-46 12/12/2000 70 08N -80 57W P. Poland Melongena bicolor CF-47 12/12/2000 71 08N -80 57W P. Poland Melongena bicolor CF-48 12/12/2000 72 08N -80 57W P. Poland Melongena bicolor CF-49 12/12/2000 73 08N -80 57W P. Poland Melongena bicolor CF-50 12/12/2000 74 08N -80 57W P. Poland Melongena bicolor CF-51 12/12/2000 75 08N -80 57W P. Poland Melongena bicolor CF-52 12/12/2000 76 08N -80 57W P. Poland Melongena bicolor CF-53 12/12/2000 77 08N -80 57W P. Poland Melongena bicolor CF-54 12/12/2000 78 08N -80 57W P. Poland Melongena bicolor CF-55 12/12/2000 79 08N -80 57W P. Poland Melongena bicolor BT = Big Torch Key, Monroe Co., FL – Off of Dorn Rd. BT-01 3/23/2001 24.7208N, -81.4523W J. Walker Melongena bicolor BT-02 3/23/2001 24.7208N, -81.4523W J. Walker Melongena bicolor BT-03 3/23/2001 24.7208N, -81.4523W J. Walker Melongena bicolor BT-04 3/23/2001 24.7208N, -81.4523W J. Walker Melongena bicolor BT-05 3/23/2001 24.7208N, -81.4523W J. Walker Melongena bicolor

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165APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex BT-06 3/23/2001 24.7208N, -81.4523W J. Walker Melongena bicolor BT-07 3/23/2001 24.7208N, -81.4523W J. Walker Melongena bicolor BH = Bahia Honda Key, Monroe, Co., FL – Gulf side of US 1 just before Bahia Honda State Park entrance BH-01 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor F BH-02 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor M BH-03 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor F BH-04 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor F BH-05 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor JM BH-06 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor M BH-07 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor M BH-08 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor F BH-09 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor F BH-10 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor M BH-11 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor JM BH-12 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor M BH-13 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor M BH-14 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor F BH-15 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor F BH-16 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor JF BH-17 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor F BH-18 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor F BH-19 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor F BH-20 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor F BH-21 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor M

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166APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex BH-22 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor F BH-23 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor F BH-24 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor M BH-25 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor JM BH-26 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor JF BH-27 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor M BH-28 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor F BH-29 9/23/2000 24.6638N, -81.2651W K. Hayes/R. Hayes Melongena bicolor M LMK = Lower Matecumbe Key, Monroe Co., FL – Anne’s Beach on the Atlantic side of US 1 LMK-01 3/23/2001 24.8475N, -80.7360W J. Walker Melongena bicolor LMK-02 3/23/2001 24.8475N, -80.7360W J. Walker Melongena bicolor LMK-03 3/23/2001 24.8475N, -80.7360W J. Walker Melongena bicolor LMK-04 3/23/2001 24.8475N, -80.7360W J. Walker Melongena bicolor LMK-05 3/23/2001 24.8475N, -80.7360W J. Walker Melongena bicolor LMK-06 3/23/2001 24.8475N, -80.7360W J. Walker Melongena bicolor LMK-07 3/23/2001 24.8475N, -80.7360W J. Walker Melongena bicolor BS = Barnes Sound, Monroe Co., FL – US 1 MM 110 Atlantic side at edge of mangroves BS-01 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor F? BS-02 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor ? BS-03 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor M BS-04 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor F BS-05 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor F BS-06 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor M BS-07 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor F

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167APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex BS-08 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor F? BS-09 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor M BS-10 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor F BS-11 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor F BS-12 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor F BS-13 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor M BS-14 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor F BS-15 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor M BS-16 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor F BS-17 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor M BS-18 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor F BS-19 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor F? BS-20 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor F BS-21 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor F? BS-22 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor BS-23 1/29/2000 25.1971N, -80.4164W K. Hayes Melongena bicolor BS-24 9/24/2000 25.1971N, -80.4164W K. Hayes/R. Hayes Melongena bicolor BS-25 9/24/2000 25.1971N, -80.4164W K. Hayes/R. Hayes Melongena bicolor MH = Matheson Hammock, Miami-Dade Co., FL – In MH county park sandy intertidal MH-01 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor M MH-02 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor F MH-03 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor M MH-04 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor M MH-05 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor F

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168APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex MH-06 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor M MH-07 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor M MH-08 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor M MH-09 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor F MH-10 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor M MH-11 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor M MH-12 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor F MH-13 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor M MH-14 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor F MH-15 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor M MH-16 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor M MH-17 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor M MH-18 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor M MH-19 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor F MH-20 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor M MH-21 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor M MH-22 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor F MH-23 4/7/2001 25.6793N, -80.2621W P. Poland Melongena bicolor F LW = Lake Worth Cove, Palm Beach Co., FL – John D. MacArt hur State Rec. Area with oysters at west edge of cove LW-01 4/6/2001 26.8340N, -80.0500W P. Poland Melongena bicolor F LW-02 4/6/2001 26.8340N, -80.0500W P. Poland Melongena bicolor F LW-03 4/6/2001 26.8340N, -80.0500W P. Poland Melongena bicolor F LW-04 4/6/2001 26.8340N, -80.0500W P. Poland Melongena bicolor M LW-05 4/6/2001 26.8340N, -80.0500W P. Poland Melongena bicolor F

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169APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex LW-06 4/6/2001 26.8340N, -80.0500W P. Poland Melongena bicolor M LW-07 4/6/2001 26.8340N, -80.0500W P. Poland Melongena bicolor M LW-08 4/6/2001 26.8340N, -80.0500W P. Poland Melongena bicolor F LW-09 4/6/2001 26.8340N, -80.0500W P. Poland Melongena bicolor M LW-10 4/6/2001 26.8340N, -80.0500W P. Poland Melongena bicolor M SI = Sebastian Inlet, Indian River Lagoon – Brevard Co., FL – just inside and north of the inlet with oysters near low water SI-01 1/14/2001 27.8645N, -80.4558W P. Poland Melongena bicolor F SI-02 1/14/2001 27.8645N, -80.4558W P. Poland Melongena bicolor F SI-03 1/14/2001 27.8645N, -80.4558W P. Poland Melongena bicolor M SI-04 1/14/2001 27.8645N, -80.4558W P. Poland Melongena bicolor M SI-05 1/14/2001 27.8645N, -80.4558W P. Poland Melongena bicolor F SI-06 1/14/2001 27.8645N, -80.4558W P. Poland Melongena bicolor F SI-07 1/14/2001 27.8645N, -80.4558W P. Poland Melongena bicolor F SI-08 1/14/2001 27.8645N, -80.4558W P. Poland Melongena bicolor M SI-09 1/14/2001 27.8645N, -80.4558W P. Poland Melongena bicolor F SI-10 1/14/2001 27.8645N, -80.4558W P. Poland Melongena bicolor M MIBR = Merritt Island in Banana River, Brevard Co., FL – Ways ide Park on cement pilings under bridge with small mussels MIBR-01 12/5/1999 K. Hayes Melongena bicolor MIBR-02 12/5/1999 K. Hayes Melongena bicolor MIBR-03 12/5/1999 K. Hayes Melongena bicolor MIBR-04 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-05 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-06 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-07 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M

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170APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex MIBR-08 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-09 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-10 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-11 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-12 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-13 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-14 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-15 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-16 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-17 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-18 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-19 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-20 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-21 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-22 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-23 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-24 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-25 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-26 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-27 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-28 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-29 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-30 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-31 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F

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171APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex MIBR-32 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-33 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-34 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-35 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-36 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-37 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-38 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-39 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-40 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-41 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-42 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-43 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-44 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-45 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-46 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor F MIBR-47 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR-48 7/16/2000 28.3569N, -80.6496W K. Hayes Melongena bicolor M MIBR = Merritt Island in Banana River, Brevard Co., FL – South edge of causeway, just SW of Hospital MIBR-49 1/14/2001 28.3577N, -80.6202W P. Poland Melongena bicolor M MIBR-50 1/14/2001 28.3577N, -80.6202W P. Poland Melongena bicolor F MIBR-51 1/14/2001 28.3577N, -80.6202W P. Poland Melongena bicolor F MIBR-52 1/14/2001 28.3577N, -80.6202W P. Poland Melongena bicolor F MIBR-53 1/14/2001 28.3577N, -80.6202W P. Poland Melongena bicolor F MIBR-54 1/14/2001 28.3577N, -80.6202W P. Poland Melongena bicolor M

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172APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex MIBR-55 1/14/2001 28.3577N, -80.6202W P. Poland Melongena bicolor M MIBR-56 1/14/2001 28.3577N, -80.6202W P. Poland Melongena bicolor M MIBR-57 1/14/2001 28.3577N, -80.6202W P. Poland Melongena bicolor M MIBR-58 1/14/2001 28.3577N, -80.6202W P. Poland Melongena bicolor M MIBR-59 1/14/2001 28.3577N, -80.6202W P. Poland Melongena bicolor M MIBR-60 1/14/2001 28.3577N, -80.6202W P. Poland Melongena bicolor M MIBR-61 1/14/2001 28.3577N, -80.6202W P. Poland Melongena bicolor SPCA = Spruce Creek Estuary Site 1, Volusia Co., FL – Rose Bay on both sides of US 1 just before Harbor Rd. SPCA-01 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-02 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-03 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-04 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-05 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-06 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-07 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-08 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-09 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-10 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-11 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-12 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-13 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-14 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-15 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F

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173APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex SPCA-16 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-17 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-18 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-19 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-20 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-21 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-22 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-23 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-24 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-25 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-26 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-27 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-28 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-29 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-30 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-31 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-32 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-33 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-34 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-35 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-36 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-37 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-38 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-39 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F

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174APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex SPCA-40 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis M SPCA-41 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-42 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-43 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCA-44 7/16/2000 29.1028N, -80.9721W K. Hayes Melongena sprucecreekensis F SPCB = Spruce Creek Estuary Site 2, Volusia Co., FL – East side of US 1 after 1st bridge after Spruce Creek Estuary Park SPCB-01 7/16/2000 29.0837N, -80.9666W K. Hayes Melongena sprucecreekensis M SPCB-02 7/16/2000 29.0837N, -80.9666W K. Hayes Melongena sprucecreekensis F SPCB-03 7/16/2000 29.0837N, -80.9666W K. Hayes Melongena sprucecreekensis M SPCB-04 7/16/2000 29.0837N, -80.9666W K. Hayes Melongena sprucecreekensis F SPCB-05 7/16/2000 29.0837N, -80.9666W K. Hayes Melongena sprucecreekensis F SPCB-06 7/16/2000 29.0837N, -80.9666W K. Hayes Melongena sprucecreekensis M SPCB-07 7/16/2000 29.0837N, -80.9666W K. Hayes Melongena sprucecreekensis F SPCB-08 7/16/2000 29.0837N, -80.9666W K. Hayes Melongena sprucecreekensis F SPCB-09 7/16/2000 29.0837N, -80.9666W K. Hayes Melongena sprucecreekensis M SPCB-10 7/16/2000 29.0837N, -80.9666W K. Hayes Melongena sprucecreekensis F SPCB-11 7/16/2000 29.0837N, -80.9666W K. Hayes Melongena sprucecreekensis F FB = Flagler Beach, Flagler Co., FL – Flagler Harbor Preserve at th e end of the boardwalk and the edge of the intercoastal wate rway FB-01 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F FB-02 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F FB-03 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F FB-04 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F FB-05 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor M FB-06 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F

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175APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex FB-07 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F FB-08 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor M FB-09 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F FB-10 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F FB-11 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor M FB-12 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor M FB-13 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor M FB-14 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F FB-15 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor M FB-16 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor M FB-17 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F FB-18 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F FB-19 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F FB-20 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor M FB-21 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor M FB-22 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F FB-23 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F FB-24 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F FB-25 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F FB-26 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor M FB-27 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor M FB-28 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor M FB-29 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor FB-30 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F

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176APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex FB-31 6/8/2001 29.4778N, -81.1363W K. Hayes/P. Hayes Melongena bicolor F YP = Yucatan Peninsula, 10km west of Progreso Mexico. YP-01 11/3/2000 E. Vallejo Melongena bispinosa F YP-02 11/3/2000 E. Vallejo Melongena bispinosa F YP-03 11/3/2000 E. Vallejo Melongena bispinosa F YP-04 11/3/2000 E. Vallejo Melongena bispinosa F YP-05 11/3/2000 E. Vallejo Melongena bispinosa M YP-06 11/3/2000 E. Vallejo Melongena bispinosa F YP-07 11/3/2000 E. Vallejo Melongena bispinosa F YP-08 11/3/2000 E. Vallejo Melongena bispinosa F YP-09 11/3/2000 E. Vallejo Melongena bispinosa F YP-10 11/3/2000 E. Vallejo Melongena bispinosa M YP-11 11/3/2000 E. Vallejo Melongena bispinosa F YP-12 11/3/2000 E. Vallejo Melongena bispinosa F HT = Hemifusus ternatanus & HC = Hemifusus colosseus collected in Taiwan HT-01 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-02 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-03 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-04 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-05 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-06 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-07 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus

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177APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex HT-08 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-09 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-10 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-11 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-12 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-13 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-14 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-15 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-16 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-17 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-18 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-19 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HT-20 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus ternatanus HC-01 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus colosseus HC-02 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus colosseus HC-03 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus colosseus HC-04 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus colosseus HC-05 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus colosseus HC-06 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus colosseus HC-07 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus colosseus HC-08 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus colosseus HC-09 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus colosseus HC-10 05/01/1999 Yuh-Wen Chiu (Chet) Hemifusus colosseus

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178APPENDIX B (CONTINUED) Sample # Collection Date Coordinates Collector(s) Genus Species SubspeciesSex MP-07 06/1992 H. Lessios/T. Collins Melongena patula MP-09 06/1992 H. Lessios/T. Collins Melongena patula MP-15 06/1992 H. Lessios/T. Collins Melongena patula MP-21 06/1992 H. Lessios/T. Collins Melongena patula Bsin-01 10/18/2001 27.6503N, -82.6772W K. Hayes Busycon sinistrum Bsin-02 10/18/2001 27.6503N, -82.6772W K. Hayes Busycon sinistrum Bsin-05 10/18/2001 27.6503N, -82.6772W K. Hayes Busycon sinistrum Fas-01 10/18/2001 27.6503N, -82.6772W K. Hayes Fasciolaria hunteria Fas-02 10/18/2001 27.6503N, -82.6772W K. Hayes Fasciolaria hunteria

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179 APPENDIX C ALLELIC FREQUENCIES FOR EACH LOCUS ACROSS ALL POPULATIONS Locus Mco 2 0.000 0.020 0.040 0.060 0.080 0.100 0.120 0.140 0.160 390393396399402405408411414417420423426429432435438441444 Allele Size (bp)Frequency The bar for Allele size 429 is o ff the graph, and has a value of 0.195.

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180APPENDIX C (CONTINUED) Locus Mco 3 0.000 0.020 0.040 0.060 0.080 0.100 0.120 0.140160 164 168 172 176 180 184 188 192 196 200 204 208 212 216 220 224 228 232 236Allele Size (bp)Frequency The bars for Allele sizes 176, 184, & 188 are off the graph, and have values of 0.165, 0.210, & 0.197 respectively.

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181APPENDIX C (CONTINUED) Locus Mco A4 0.000 0.020 0.040 0.060 0.080 0.100 0.120252 255 258 261 264 267 270 273 276 279 282 285 288 291 294 297 300 303 306 309 312 315 318 321 324 327 330 333Allele Size (bp)Frequency The bar for Allele size 285 is o ff the graph, and has a value of 0.127.

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182APPENDIX C (CONTINUED) Locus Mco E4 0.000 0.010 0.020 0.030 0.040 0.050 0.060 0.070 0.080 0.090 0.100180 184 188 192 196 200 204 208 212 216 220 224 228 232 236 240 244 248 252 256 260 264 268 272 276 280 284 288 292 296 300 304 308 312 316Allele Size (bp)Frequency The bars for Allele sizes 276 & 280 are off the graph, and have values of 0.181 & 0.125 respectively.

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183APPENDIX C (CONTINUED) Locus Mco 5 0.000 0.010 0.020 0.030 0.040 0.050 0.060 180184188192196200204208212216 Allele Size (bp)Frequency The bar for Allele size 206 is o ff the graph, and has a value of 0.907.

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184APPENDIX C CONTINUED Locus Mco 6 0.000 0.010 0.020 0.030 0.040 0.050 0.060 0.070 0.080162 164 166 168 170 172 174 176 178 180 182 184 186 188 190 192 194 196 198 200 202 204 206 208 210Allele Size (bp)Frequency The bars for Allele sizes 194 – 204 are off the graph, and have values of 0.87, 0.135, 0.140, 0.149, 0.166, and 0.141 respectiv ely.

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185APPENDIX C (CONTINUED) Locus Mco 10 0.000 0.020 0.040 0.060 0.080 0.100 0.120 0.140 0.160 276280284288292296300304308312316320324328332336 Allele Size (bp)Frequency The bars for Allele sizes 284, 304, & 312 are off the graph, and have values of 0.165, 0.174, & 0.166 respectively.

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186APPENDIX C (CONTINUED) Locus Mco 12 0.000 0.010 0.020 0.030 0.040 0.050 0.060 0.070 0.080 0.090 0.100 200202204206208210212214216218220 Allele Size (bp)Frequency The bars for Allele sizes 204 – 208 are off the gr aph, and have values of 0.502, 0.286, & 0.102 respectively.

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187APPENDIX D ALLELE FREQUENCIES BY LOCUS FOR EACH POPULATION. SAMPLES SIZES ARE INDICATED IN PARENTHESES. Locus BH (29) BL (30) BS (46) CK (40) ECS (40) FB (31) FTD (48) LW (10)MH (23) MIBR (45) OB (38)PA (40)PI (30)SI (10) SJP (46) SPCA (55) Mco 2 390 1.25 399 1.19 5.00 402 2.50 1.25 12.07 405 1.61 2.38 10.00 2.50 408 33.33 2.00 1.61 3.57 15. 00 2.50 53.03 6.82 411 1.92 3.75 2.38 3.45 414 16.00 6.25 7.69 4.76 5.00 1.72 1.85 1.14 417 8.00 12.50 2.56 5.95 3. 57 1.25 12.07 20.00 3.41 1.06 420 3.85 4.00 12.50 14.10 6.45 4.76 6.25 6.90 5.56 20.00 6.82 13.83 423 69.23 16.67 24.00 12.50 12.82 1.61 21.43 7.14 18.75 1.52 6.90 27.78 9.09 7.45 426 1.92 6.00 16.25 24.36 1.61 13.10 10. 00 7.14 11.25 37.88 31.03 20.37 19.32 429 7.69 16.67 24.00 10.00 7.69 37.10 23.81 40.00 42.86 37.50 3. 03 15.52 22.22 10.00 9.09 23.40 432 13.46 8.33 2.00 7.50 8.97 6.45 5.95 5. 00 14.29 7.50 4.55 6.90 30.00 5.68 10.64 435 8.00 6.25 17.95 1.61 3.57 10.00 17.86 7.50 11.11 20.00 27.27 1.06 438 16.67 4.00 6.25 3.85 29.03 4.76 7.14 11.11 7.95 24.47 441 1.92 8.33 2.00 2.50 1.19 1.25 1.72 3.41 444 1.25 12.90 1.19 1.25 1.72 18.09 Mco 3 160 5.56 164 2.08 1.25 1.22 168 7.32 4.88 12.50 3.33 1.14 0.94 172 1.67 1.35 1.22 5.00 176 31.48 28.33 4.05 2.50 20.69 1.22 2.44 75.00 3.57 5.00 20.45 33.02 180 8.33 5.41 3.75 3.45 7.32 6.10 1.39 7.14 1.67 22.22 3.41

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188APPENDIX D (CONTINUED) Locus BH (29) BL (30) BS (46) CK (40) ECS (40) FB (31) FTD (48) LW (10) MH (23) MIBR (45) OB (38) PA (40) PI (30)SI (10) SJP (46) SPCA (55) 184 9.26 60.00 16.221.25 51.7215.85 9.76 12.507.14 28.33 38.6439.62 188 14.81 1.67 41.67 5.41 38.75 3.45 13.4120.0073.9143.90 11. 1112.5025.0016.6712.502.83 192 25.93 14.58 12.1618.7512.0710.9815. 006.52 6.10 21.4318.3316.67 1.89 196 5.56 6.25 10.812.50 14.63 1.22 5.36 5.00 2.27 13.21 200 12.1615.00 3.66 10.0010. 871.22 8.93 3.33 5.56 2.83 204 1.85 12.50 4.05 13.75 9.76 5.00 6.52 7.32 3.57 3.33 3.41 1.89 208 11.11 4.05 2.50 7.32 3.66 7.14 3.33 5.56 212 10.42 6.76 6.90 1.22 1.22 1.79 1.67 11.11 216 8.11 15.00 6.10 1.79 11.1111.361.89 220 12.50 5.41 3.66 2.17 3.66 7.14 1.67 5.56 3.41 1.89 224 1.22 25.00 2.44 228 1.35 1.72 5.00 232 1.35 1.14 236 1.35 2.27 Mco A4 252 9.26 6.41 1.39 12.903.66 1.25 20.00 10.003.57 18.63 255 27.78 1.85 8.00 3.85 8.33 8.54 5.00 7.50 10.001.79 0.98 258 33.33 9.26 12.00 11.5423.618.06 13.41 35.00 11.25 10.007.14 2.38 2.94 261 6.41 2.78 3.23 7.32 5.00 7.50 7.50 1.79 264 1.28 1.39 9.68 10.98 5.00 5.00 1.79 20.001.19 10.78 267 2.56 10.98 1.79 2.38 270 1.79 1.19 273 1.61 1.79 276 1.28 1.22 20.00 1.25 16.07 1.96 279 3.70 1.39 1.22 5.00 282 5.56 3.70 2.00 4.17 1.22 82.6143.75 1.43 3.57 0.98 285 48.15 19.23 1.22 4. 35 3.75 38.576.25 55.950.98

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189APPENDIX D (CONTINUED) Locus BH (29) BL (30) BS (46) CK (40) ECS (40) FB (31) FTD (48) LW (10) MH (23) MIBR (45) OB (38) PA (40) PI (30)SI (10) SJP (46) SPCA (55) 288 6.00 12.501.61 6.10 2.50 14.29 3.57 1.96 291 18.52 30.002.56 25.001.61 4.88 2.17 6.25 4.29 6.25 1.79 4.76 0.98 294 1.85 14.001.28 4.17 8.06 1.22 1.25 22.86 297 2.56 3.66 2.50 1.19 300 18.0010.268.33 14.524.88 10. 00 2.50 2.50 10.001.19 14.71 303 11.11 1.28 6.10 2.50 1.25 7.14 306 2.44 15.00 1.25 3.75 12.50 0.98 309 3.70 3.70 6.41 16.134.88 32.865.00 3.57 3.57 16.67 312 11.11 6.00 5.13 4.84 2.44 8.70 5.00 17.8620.00 6.86 315 5.56 1.85 4.00 3.85 6.94 17.743.66 5. 00 2.17 1.25 6.25 3.57 40.0010.7120.59 318 3.85 1.25 3.75 3.57 1.19 321 1.28 1.25 5.36 324 3.85 1.25 327 1.28 1.25 1.25 330 1.28 333 2.56 Mco E4 180 2.38 182 0.96 234 1.25 28.57 18.27 246 1.16 268 4.76 270 4.41 272 1.25 2.50 7.35 2.33 274 16.07 5.00 1.35 1.19 15.00 276 17.86 79.31 12.50 35.004.05 39.713.75 10.0024.4237.50 278 10.71 2.00 1.25 5.00 13.331.35 15.0040.00 15.00 10.00 0.96 280 3.57 1.72 20.003.75 30.0027.0325. 007.50 10.71 12.5022.0010.00 25.00

PAGE 199

190APPENDIX D (CONTINUED) Locus BH (29) BL (30) BS (46) CK (40) ECS (40) FB (31) FTD (48) LW (10) MH (23) MIBR (45) OB (38) PA (40) PI (30)SI (10) SJP (46) SPCA (55) 282 1.79 20.00 8.75 12.503.33 4.05 10.00 3.57 1.47 20.00 2.33 284 1.79 4.00 8.75 3.75 3.33 12.1610.00 8.33 47.063.75 10.0020.002.33 0.96 286 2.00 2.50 14.86 1.19 2.00 10.002.33 0.96 288 1.79 1.72 2.00 13.75 14.86 1.19 22.504.00 31.40 290 5.36 36.00 5.00 31.251.67 6.76 15. 002.50 2.38 14.00 2.33 5.77 292 12.50 17.24 6.25 25.00 6.76 5. 00 7.14 16.254.00 22.091.92 294 21.43 10.00 3.75 7.50 1.67 2.70 15. 007.50 7.14 10.0010.00 5.81 0.96 296 16.00 2.50 8.75 1.35 5.00 30.0021.43 1.25 10.0020.00 3.85 298 6.00 2.50 2.50 2.70 4.00 10.00 1.92 300 7.14 3.75 304 1.25 3.49 306 1.67 2.50 0.96 308 10.00 314 10.00 318 2.00 7.50 Mco 5 180 1.72 200 1.28 6.41 2.38 6.06 15.00 202 1.28 3.23 1.28 23.9136.90 11.6715.00 3.77 204 1.19 10.26 206 100.00 96.55 98.00 91.03100.00 96.7789.74100.0076.0958.33 93. 9489.7488.3370.0098.8996.23 208 1.72 2.00 6.41 1.19 1.11 212 1.28 216 1.28 Mco 6 162 0.98

PAGE 200

191APPENDIX D (CONTINUED) Locus BH (29) BL (30) BS (46) CK (40) ECS (40) FB (31) FTD (48) LW (10) MH (23) MIBR (45) OB (38) PA (40) PI (30)SI (10) SJP (46) SPCA (55) 176 0.98 178 5.41 3.23 1.09 182 1.35 186 1.35 1.61 5.43 1.22 3.95 1.79 188 5.41 2.17 1.22 9.21 1.16 0.98 190 2.70 1.25 1.22 6.58 5.81 1.96 192 6.76 4.84 5.43 1.22 11.843.57 3.49 8.82 194 12.07 3.70 4.05 3.75 32.268.70 3.66 5.71 10.537.14 3.49 22.55 196 32.76 20.37 2.00 17.578.75 8.06 13.04 13.41 14.2918.4216.07 20.936.86 198 3.45 7.41 14.0013.5110.009.68 11.9620.00 34.0950.00 2.86 7.89 16.0740.009.30 4.90 200 10.34 42.59 24.0010.8110.00 3.23 17.3915.0040.9118.29 1. 43 6.58 16.0720.0013.9510.78 202 15.52 14.81 22.0012.166.25 19.3519.5710.0013.642.44 40. 0010.5314.2920.0012.7929.41 204 18.97 11.11 10.008.11 18.75 14.5210.8715.004.55 6.10 34. 2913.1612.5020.0024.426.86 206 3.45 24.009.46 27.503.23 3.26 30. 00 1.43 1.32 10.71 4.65 4.90 208 3.45 4.00 1.35 12.50 1.09 10.006.82 1.22 1.79 210 1.25 Mco 10 276 2.63 280 1.67 284 31.03 1.85 64.0011.1152.6314.528.70 27. 782.38 1.19 13.7530.0010.001.14 15.00 288 22.41 7.41 30.00 39.4720.976.52 5. 56 1.19 20.003.33 5.00 5.68 18.00 292 2.00 12.503.95 14.527.61 5.56 97.6240.48 1.25 30.0040.007.95 5.00 296 9.72 6.45 14.13 4.76 25.008.75 11.67 5.68 1.00 300 11.11 6.94 3.23 1.09 2.38 2.78 31.25 2.27 304 32.76 22.22 33.33 4.84 22.8322.22 23.81 6.25 5.00 25.0054.5515.00 308 13.79 4.00 9.72 1.32 4.84 21.7433. 33 13.10 15.0018.3310.00 4.00 312 57.41 4.17 25.819.78 7.14 63.891.25 19.3242.00 316 6.94 2.17 8.33 2.27

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192APPENDIX D (CONTINUED) Locus BH (29) BL (30) BS (46) CK (40) ECS (40) FB (31) FTD (48) LW (10) MH (23) MIBR (45) OB (38) PA (40) PI (30)SI (10) SJP (46) SPCA (55) 320 1.39 2.17 2.38 1.25 1.14 324 1.39 1.09 2.38 1.25 328 5.56 1.19 332 2.17 336 2.78 4.84 10.00 Mco 12 200 1.04 202 12.50 1.28 204 34.48 60.00 96.0028.7595.00 54.8450.0025.0017.3920.93 61. 1132.0533.3360.0052.2267.71 206 65.52 10.00 21.253.75 22.5839.7445.00 78.2659.30 1.39 35.9053.3340.008.89 14.58 208 30.00 4.00 7.50 1.25 11.292.56 5. 00 3.49 37.5010.265.00 15.5614.58 210 2.50 1.28 15.00 9.30 2.56 3.33 212 11.25 4.84 5.13 1.28 1.67 2.22 214 11.25 3.23 10.004. 35 5.81 8.97 5.00 14.442.08 216 2.50 3.23 1.16 5.13 218 2.50 3.85 3.33 220 1.67 Population abbreviations follow those given in Figures 2.1 & 3.1, and Appendix B. (Number sampled from each population)


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Hayes, Kenneth A.,
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Phylogeography and evolution of the Florida crown conch (Melongena corona)
h [electronic resource] /
by Kenneth A. Hayes.
260
[Tampa, Fla.] :
University of South Florida,
2003.
502
Thesis (M.S.)--University of South Florida, 2003.
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Includes bibliographical references.
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Text (Electronic thesis) in PDF format.
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System requirements: World Wide Web browser and PDF reader.
Mode of access: World Wide Web.
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Title from PDF of title page.
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ABSTRACT: Melongena corona and closely related congeners are a conspicuous part of the marine intertidal benthic communities of Florida and southeastern Alabama. Significant genetic differentiation among adjacent populations has been conjectured based on variation in shell morphology, habitat discontinuity, low levels of adult motility, and the presence of an aplanic lecithotrophic larval stage. Furthermore, studies of the highly variable shell morphology often have resulted in confusing specific and subspecific definitions of these gastropods, which are often referred to as the "corona complex". Variation in shell morphology may indicate local adaptation or environmentally induced phenotypic plasticity. In this study I utilized mitochondrial DNA sequences in order to reconstruct the phylogenetic relationships of crown conchs, and nuclear microsatellite loci to investigate the patterns of relatedness within and among populations inhabiting the southeastern United States. Approximately 500 individuals from 20 populations throughout the known range of the Crown Conch were genotyped at eight microsatellite loci. Additionally, a 1200bp portion of the cytochrome oxidase subunit I gene was sequenced along with a 490bp fragment of the 16s ribosomal gene from individuals representing all known species and subspecies of the genus Melongena. Phylogenetic analyses completed with these data provide no support for current taxonomic designations within this group and these genetic data indicate that the corona complex is composed of a single polymorphic species. Furthermore, microsatellite data reveal population structure consistent with restricted gene flow between extant populations and phylogeography heavily influenced by historical sea-level fluctuations during the Late Pleistocene.
590
Adviser: Karl, Stephen A.
653
biogeography.
population genetics.
gastropoda.
microsatellite dna.
mitochondrial dna.
690
Dissertations, Academic
z USF
x Biology
Masters.
773
t USF Electronic Theses and Dissertations.
4 856
u http://digital.lib.usf.edu/?e14.184