FOUNDING FISH: GENE DUPLICATION CONTRIBUTES TO IMMUNOLOGICAL DIVERSITY IN BOTTLENECKED POPULATIONS OF INTRODUCED RAINWATER KILLIFISH A Thesis submitted to the faculty of A 5 San Francisco State University 3(o In partial fulfillment of MS the requirements for the Degree Master of Science In Biology: Marine Biology by Danielle Nicole Desmet San Francisco, California December 2015 Copyright by Danielle Nicole Desmet 2015 CERTIFICATION OF APPROVAL I certify that I have read FOUNDING FISH: GENE DUPLICATION CONTRIBUTES TO IMMUNOLOGICAL DIVERSITY IN BOTTLENECKED POPULATIONS OF * INTRODUCED RAINWATER KILLIFISH by Danielle Nicole Desmet, and that in my opinion this work meets the criteria for approving a thesis submitted in partial fulfillment of the requirement for the degree Master of Science in Biology (Marine Biology) at San Francisco State University. C. Sarah Cohen, Ph.D. Professor, Biology Eric J. Routman, Ph.D. Professor, Biology Vanessa Miller-Sims, Ph.D. Adjunct Faculty FOUNDING FISH: GENE DUPLICATION CONTRIBUTES TO IMMUNOLOGICAL DIVERSITY IN BOTTLENECKED POPULATIONS OF INTRODUCED RAINWATER KILLIFISH Danielle Nicole Desmet San Francisco, California 2015 Population bottlenecks, and subsequent loss of genetic diversity, are a common occurrence for introduced species. The MHC Class IIDB locus must constantly adapt to detect shifting pathogen communities and is used in this study to assess functional diversity in 2 regions with introduced populations of Rainwater killifish, Lucania parva. L. parva was likely introduced to San Francisco Bay from the Pecos River, New Mexico through a series of sport fish stockings to southern then northern California during the 1940’s and 1950’s. Demographic patterns of San Francisco and New Mexico populations were assessed using microsatellite, D-loop and MHCIIDB diversity. A single D-loop allele was found in all populations and three of four microsatellite loci were fixed at shared alleles, suggesting a strong bottleneck and common ancestry between all populations sampled. MHC diversity was lowest in New Mexico and one very similar allele was shared between the regions sampled. Inferred functional diversity was also assessed using physiochemical properties of MHC codons under positive selection and alleles were grouped into supertypes. Supertype usage and allelic diversity was relatively uniform between San Francisco and New Mexico. Despite low allelic diversity, functional diversity was spread across duplicated MHCIIDB loci. Two supertypes (out of four) were found to dominate in all populations. This reduced MHC diversity may be a result of relaxed parasite mediated selection as decreased immune response, assortative mating or new parasite pressures. I c< A1 ' rrect representation of the content of this thesis. C. Sarah Cohen, Chair, Thesis Committee Date PREFACE AND/OR ACKNOWLEDGEMENTS I would like to thank Daniel Chase for his help collecting samples from San Francisco Bay and Evan Carson, Colleen Caldwell, Stephen Davenport and Chris Hoagstrom for their assistance in obtaining samples from New Mexico. I am also grateful to Laura Melroy, Vanessa Miller-Sims, Riley Smith, Kathryn Nuessly and Benson Chow for their assistance in the field, and to Bradley Johnson and Peter Drell for statistical advice. Collection and euthanization methods were approved by the California Department of Fish and Wildlife (SC-12713, permit holder Danielle Desmet) and the San Francisco State University Institutional Review Board and Institutional Animal Care and Use Committee (IACUC protocol #A13-04). v TABLE OF CONTENTS List of Table........................................................................................................................ viii List of Figures....................................................................................................................... ix List of Appendices................................................................................................................. x Introduction.............................................................................................................................1 Methods...................................................................................................................................9 Fish Collection........................................................................................................... 9 Molecular Methods....................................................................................................9 Data Analysis.......................................................................................................................12 Sequence Analysis................................................................................................... 12 MHC loci..................................................................................................................12 MHC class II variability and tests for selection and supertypes............................ 13 Results...................................................................................................................................16 Microsatellites.......................................................................................................... 16 Mitochondrial Control Region Sequences............................................................. 16 MHC IIDB Sequences............................................................................................. 16 MHC loci................................................................................................................. 18 Selection................................................................................................................... 19 Functional Diversity................................................................................................. 19 Discussion............................................................................................................................ 21 Common Ancestry...................................................................................................21 Recovery and Drift...................................................................................................23 Selection...................................................................................................... 25 MHC and Functional Diversity............................................................................... 27 Conclusions.......................................................................................................................... 30 Funding.................................................................................................................................31 Reference..............................................................................................................................32 Appendices ..................................................................................................................... 49 LIST OF TABLES Table Page Populations sampled............................................................................................42 Pairwise Fst values................................................................................................42 Loci summary......................................................................................................42 Diversity measures by population............................................................. 43 Analysis of molecular variance........................................................................... 44 Codons under positive selection.......................................................................... 44 LIST OF FIGURES Figures Page Map of study sites with MHCIIDB exon 2 frequencies..............................................45 Loci prevalence by population.....................................................................................45 Codon level analysis of selection................................................................................ 46 Supertype tree............................................................................................................... 47 Supertype prevalence by population........................................................................... 48 LIST OF APPENDICES Appendix Page Microsatellite heterozygosity................................................................................ 49 Loci Fishers Exact Test..........................................................................................49 Positively selected codon and PBR........................................................................ 50 Supertype Fishers Exact Test.................................................................................50 Codon level analysis of selection........................................................ 51 PCR gel of imaged loci...........................................................................................51 Supertype trees.................................................................................................... 53-54 Alignment of MHC exons......................................................................................55 Alignment of amino acid haplotypes..................................................................... 56 Alignment of MHC introns.................................................................................... 57 x 1 Introduction Eco-immunology is an emerging field that focuses on the relationship between an ecosystem and the immune systems of the resident organisms.