Phylogeography and Evolution of the Florida Crown Conch
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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, microsatellite dna, mitochondrial dna, gastropoda, biogeography © Copyright 2003, Kenneth A. Hayes This thesis is dedicated to my wife Pam. Without her unwavering support, patience, love, and companionship this journey 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 Acknowledgements There are a number of people to whom I owe my sincere gratitude for making the last four years a memorable and worthwhile 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 excellence and a more thorough understanding of the world around me, and providing guidance and support in both academic and personal pursuits. I hope that the experience was as enriching for him as it was for me. Dr. James Garey who in addition to serving on my committee 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 comments concerning my thesis. Dr. Joseph Simon, as a mentor and a friend, inspired 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 Nichols, Mike Roberson, Heather Hamilton, Terry Campbell, David Karlen for providing sequencing facility support and for numerous conversations about science, invertebrates, graduate 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 staff who make everything run smoothly and without them very few graduate students would ever finish. 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 advice about the evolution of Melongenidae and speciation in marine gastropods. Funding for this thesis was provided by American Malacological Society, Conchologist of America, Sigma Xi, American Museum of Natural History, Florida Association of Benthologists, University of South Florida Dept of Biology, Biology graduate student organization, and a National Science Foundation Grant in Systematics to Dr. Stephan Karl. Finally, I owe my overwhelming gratitude to my family. My parents who’s encouragement, support, and belief in me have allowed me to pursue my dreams of being a biologist. My sister Rena, 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 child. His generosity and creativity are an inspiration. My wonderful wife to whom I dedicate this thesis. 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 Perspectives 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 i Phylogenetic Analysis 42 Discussion 43 Divergence Timing 49 Other Intertidal Invertebrates 54 Taxonomy 56 Conclusions 57 References 58 Chapter Three – Microsatellite Analysis of Crown 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 ii List of Tables Table 2.1 COI primers 69 Table 2.2 Sequence divergence Dxy 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 Microsatellite 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 (δµ)2 & geographic distances 129 Table 3.8 AMOVA results 132 iii List of Figures Figure 1.1 Distribution of the genus Melongena 21 Figure 1.2 Shell morphology within 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 maximum likelihood tree 68 Figure 3.1 Map of sample sites with number of individuals 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 (δµ)2 estimates 128 iv Phylogeography and Evolution of the Florida Crown Conch (Melongena corona). Kenneth A. Hayes 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. v Chapter 1: Natural History of Crown Conchs & Thesis Overview Introduction to the Family Melongenidae The family Melongenidae (Gill, 1867) consists of seven genera of intertidal, 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 vernacular 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 within these genera (Clench and Turner 1956; Paine