W&M ScholarWorks Dissertations, Theses, and Masters Projects Theses, Dissertations, & Master Projects 2001 Genetic Investigation of Interspecific and Intraspecific Relationships Within the Genus Rapana Arminda L. Gensler College of William and Mary - Virginia Institute of Marine Science Follow this and additional works at: https://scholarworks.wm.edu/etd Part of the Genetics Commons Recommended Citation Gensler, Arminda L., "Genetic Investigation of Interspecific and Intraspecific Relationships Within the Genus Rapana" (2001). Dissertations, Theses, and Masters Projects. Paper 1539617768. https://dx.doi.org/doi:10.25773/v5-v5kh-2t15 This Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Dissertations, Theses, and Masters Projects by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. GENETIC INVESTIGATIONS OF INTERSPECIFIC AND INTRASPECIFIC RELATIONSHIPS WITHIN THE GENUS RAPANA A Thesis Presented to The Faculty of the School of Marine Science The College of William and Mary in Virginia In Partial Fulfillment Of the Requirements for the Degree of Master of Science by Arminda L. Gensler 2001 APPROVAL SHEET This thesis is submitted in partial fulfillment of The requirements for the degree of Master of Science Arminda L. Gensler Approved, October 2001 John E.Xjraves. Ph.D. Co-Advisor Roger Mann, Ph.D. Co-Advisor Kimberlv S. Reece, Ph.D. John M. Brubaker, Ph.D. TABLE OF CONTENTS Page ACKNOWLEDGMENTS................................................................................................ iv LIST OF TABLES............................................................................................................... v LIST OF FIGURES............................................................................................................. vi ABSTRACT......................................................................................................................... vii INTRODUCTION............................................................................................................... 2 Introduction to R.venosa....................................................................................... 2 The genus Rapana................................................................................................... 4 History of the R. venosa invasions .........................................................................8 Black Sea. .................................................................................................. 8 Spread through the Mediterranean and other European waters ..............9 Chesapeake Bay invasion ...........................................................................9 Biological invasions ................................................................................................ 12 Population size, genetic diversity and invasion .................................................... 15 Molecular techniques ............................................................................................. 19 Loci selected for sequencing ................................................................................ 22 OBJECTIVES...................................................................................................................... 25 MATERIALS AND METHODS....................................................................................... 27 Sampling and DNA isolation ..................................................................................27 PCR reactions ........................................................................................................ 40 Cloning .....................................................................................................................44 Sequencing ...............................................................................................................45 Data Analysis - R. venosa population genetics .................................................... 46 Data Analysis - Differentiation of R. venosa and R. bezoar............................... 47 RESULTS............................................................................................................................. 49 Genetic variation in R. venosa............................................................................... 49 Genetic divergence between R. venosa and R. bezoar........................................ 61 DISCUSSION...................................................................................................................... 68 Genetic variation in R. venosa............................................................................... 71 Genetic differentiation between R. venosa and R. bezoar.................................. 77 General discussion ................................................................................................. 79 Further studies ........................................................................................................ 85 CONCLUSIONS................................................................................................................. 89 APPENDIX A ..................................................................................................................... 91 APPENDIX B ..................................................................................................................... 98 APPENDIX C ..................................................................................................................... 101 LITERATURE CITED........................................................................................................ 128 VITA..................................................................................................................................... 134 iii ACKNOWLEDGEMENTS This project could not have been completed without the advice and assistance of many individuals. I am grateful for the help and intellectual and financial support provided by my co-advisors, Drs. John Graves and Roger Mann. I would also like to thank Drs. Kim Reece and John Brubaker for serving on my committee. A number of researchers from around the world provided samples of Rapana venosa and R. bezoar for my use. Korean snails were obtained by Drs. Albert Choi and Jong-Geel Je, and Ms. Rebecca Green. Samples from Trabzon, Turkey, were collected with the assistance of Mehmet Aydin, Hacer Emiral, and Cem Erkebay, all graduate students of Dr. Kadir Seyhan. Warm thanks to Dr. Seyhan who acted as my host during my stay in Turkey, and who greatly facilitated collection of R. venosa as well as information about this non-native snail in the Black Sea. Drs. Jean-Pierre Joly and P. Goulletquer collected R. venosa in France, and Dr. Brian Morton obtained samples of R. bezoar from Hong Kong. The Molluscan Ecology Working Group at VIMS kindly shared tissue samples from their Chesapeake Bay R. venosa. Dr. W. Stickle procured samples of a related gastropod species, Stramonita haemastoma, from Grande Isle, Louisiana. I would like to thank everyone in the VIMS Molluscan Ecology Working Group. Dr. Juli Harding provided graphics assistance and images of R. venosa for my use. Finally, special thanks to everyone in, or associated with, the VIMS Fisheries Genetics Laboratory, especially Jan McDowell and Dr. Cheryl Morrison, for providing support and encouragement through all stages of this project. iv LIST OF TABLES Table 1. Allozyme studies reveal differing amounts of genetic diversity among introduced populations of molluscs ............................................................. 18 Table 2. Individual identification numbers, sample locations, and number of clones per individual sequenced for the cytochrome b analysis of Rapana venosa............................................................................................ 30 Table 3. Individual identification numbers, sample locations, and number of clones per individual sequenced for the ITS analysis of Rapana venosa................................................................................................ 31 Table 4. Individual identification numbers, sample locations, and number of clones per individual sequenced for the ITS analysis of Rapana bezoar... 37 Table 5. Concentrations and components for “cocktails” used for both ITS and cytochrome b PCR reactions .................................................................... 42 Table 6. Aligned haplotypes and polymorphic sites for the 731 bp cytochrome b data set...................................................................................... 50 Table 7. Diversity statistics for the twenty-five Rapana venosa individuals sequenced at the 731 bp cytochrome b locus................................................ 52 Table 8. Matrix of percent sequence divergence between sample locations for cytochrome b ............................................................................. 54 Table 9. Comparison of pairs of samples by pairwise distance 4>st values for Rapana venosa samples ............................................................................ 55 Table 10. Matrix of percent sequence diversity and divergence within and between putative species for the ITS gene region ......................................... 63 Table 11. Range of ITS diversity within individual clones and sample locations for Rapana venosa........................................................................