CONSERVATION GENETICS AND POPULATION STATUS OF THE FLAME CHUB, HEMITREMIA FLAMMEA By: Kathlina Frances Alford Approved: Anna L. George Joey Shaw Director of TNACI Professor of Biology (Co-Chair) (Co-Chair) Mark S. Schorr Jeff S. Elwell Professor of Biology Dean of the College of Arts and Sciences (Committee Member) A. Jerald Ainsworth Dean of the Graduate School CONSERVATION GENETICS AND POPULATION STATUS OF THE FLAME CHUB, HEMITREMIA FLAMMEA By: Kathlina Frances Alford A Thesis Submitted to the Faculty of the University of Tennessee at Chattanooga in Partial Fulfillment of the Requirements of the Degree of Master’s of Science of Environmental Science The University of Tennessee at Chattanooga Chattanooga, Tennessee August 2013 ii ABSTRACT The southeastern United States has a rich geologic history that contributed to the evolution of an extremely diverse aquatic fauna throughout the region. The Flame Chub, Hemitremia flammea, is a brightly colored, spring endemic minnow species native to the Cumberland, Tennessee, and Coosa river drainages. In this study, the cytochrome-b gene region was analyzed for 230 individuals from 29 populations across the three drainages. Results from maximum parsimony and Bayesian analyses recovered shallow divergence between the 31 haplotypes. AMOVA analyses indicated that most genetic variation distributed within and between populations, not between drainages. Based on these results, this species may not be restricted to spring habitats as was originally presumed and can move within river systems and likely even between drainages. Further analyses using microsatellites and geospatial modeling would refine these results. Species like H. flammea are indicators of the health of groundwater resources that are under increasing anthropogenic pressure. iii DEDICATION To my parents for instilling in me an inquiring mind and a desire for higher learning and for never letting me settle for less than my very best. To my husband for loving me through my quirks everyday no matter what. iv ACKNOWLEDGEMENTS This thesis would not have been possible without the guidance and the help of so many individuals who in one way or another contributed and extended their valuable assistance in the preparation and completion of this study. First and foremost, my utmost gratitude goes to my husband, Lee, without whose wavering support and encouragement would I have completed this study. Dr. Anna George has been so many things to me throughout the years: a boss, a mentor and a friend. Her passion for Southeastern aquatics is contagious and inspiring. Dr. Joey Shaw graciously allowed me to use his genetics laboratory for this project and also served as a co-chair on my committee. Dr. Mark Schorr also served on my committee and provided ecological insight on this study. Dr. Bernie Kuhajda provided perspective, revisions, and reassurance when I needed it most. Dr. David Neely was amazing with his awe-inspiring field expertise and never- ending collection energy. Evan “Bird Dog” Collins was priceless as a collector, GIS technician and friend. Steve Alford, Stephanie Brandt, Matt Hamilton, Ben Stenger, Dr. Matt Thomas and Josh White all provided field assistance, and Greg Knothe and Bob Jett provided GIS mapping powers. The Tennessee Aquarium not only funded my education but also allowed me the time and flexibility to finish this study while working full-time. AZA, NANFA, and the UTC Graduate Association all helped to fund my research and travel to make presentations at multiple conventions. Last but not the least, my extended family, friends, and fellow students endured this long process with me and provided so much encouragement and prayer. Thanks to you all. v TABLE OF CONTENTS DEDICATION v ACKNOWLEDGEMENTS vi LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS xi CHAPTERS 1. INTRODUCTION 1 2. METHODS AND MATERIALS 15 Field Collections 15 Mitochondrial Analyses 16 3. RESULTS 19 Field Collections 19 Mitochondrial Analyses 20 Gene Analyses 20 Parsimony Analyses 20 Bayesian Analyses 21 Haplotype Analyses 21 Population Analyses 22 4. DISCUSSION 24 REFERENCES 31 APPENDICES A. SEQUENCE DATA FOR ALL 31 HAPLOTYPES RECOVERED FOR HEMITREMIA FLAMMEA 54 vi B. GENBANK ACCESSION NUMBERS AND SEQUENCE DATA FOR ALL OUTGROUPS INCLUDED IN ANALYSES 62 C. PROOF OF PROJECT APPROVAL BY THE UTC INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE 70 VITA 72 vii LIST OF TABLES Table 1 All sites sampled for Hemitremia flammea from November 2009 to April 2012 39 Table 2 Sites where Hemitremia flammea were collected from November 2009 to April 2012 42 Table 3 Primers used in mitochondrial analyses of Hemitremia flammea 43 Table 4 Haplotype and nucleotide diversity for each population of Hemitremia flammea 44 Table 5 Φst values for all populations of Hemitremia flammea 45 Table 6 Haplotypes recovered for Hemitremia flammea , as represented at each site 46 viii LIST OF FIGURES Figure 1 Map of the Cumberland, Tennessee, and Coosa river drainages 47 Figure 2 Map of all sites sampled for Hemitremia flammea 48 Figure 3 Map of sites where Hemitremia flammea were collected 49 Figure 4 Map of sites included in population comparison analyses for Hemitremia flammea 50 Figure 5 Maximum parsimony concensus tree for Hemitremia flammea 51 Figure 6 Bayesian consensus tree using GTR+I+G model for Hemitremia flammea 52 Figure7 Haplotype network for Hemitremia flammea 53 ix LIST OF ABBREVIATIONS µL microliter / 0.001 liter A adenine AMOVA analysis of molecular variance C cytosine CI consistency index CPUE catch per unit effort cyt-b cytochrome-b gene region DNA deoxyribonucleic acid et al. and others G guanine h haplotype diversity HI homoplasy index ICUN International Union for the Conservation of Nature m meter mM millimolar / a concentration of 0.001 mole per liter MP maximum parsimony mya million years ago oC degrees Celsius p probability statistic x PCR polymerase chain reaction r Pearson product-moment correlation coefficient RCI rescaled consistency index sp. species sp. cf. species confer / undescribed species T thymine Taq Thermus aquaticus polymerase TVA Tennessee Valley Authority UAIC University of Alabama Ichthyological Collection UMMZ University of Michigan Museum of Zoology UTEIC University of Tennessee Etnier Ichthyological Collection ya years ago Z Mantel’s test statistic π nucleotide diversity ΦCT phi-ct / genetic variation among metapopulations relative to taxon Φsc phi-sc / genetic variation within populations relative to metapopulations ΦST phi-st / genetic variation within populations relative to the taxon xi CHAPTER 1 INTRODUCTION Simply defined, biogeography is the geography of life. The field of biogeography provides the framework by which scientists study species distributional patterns, speciation events, and ancestor relationships within and between species through space and geologic time (Lomolino et al. 2006). By identifying the processes that impact species distributions we are better able to protect imperiled species and habitats as well as develop strategies to control invasive species. Biogeographical research answers questions about spatial patterns of organisms through habitat assessments, regional species lists, and even molecular studies. The science can be subdivided into more narrow concentrations such as: ecological, historic, terrestrial, aquatic, floral, faunal, and by specific taxonomic groups. When researching within each subdivision, unique limitations must be considered. For example, freshwater organisms are restricted by the water pathways in which they live for dispersal opportunities. Freshwater fishes in particular have a unique set of biogeographic constraints in comparison to terrestrial organisms in the same regions. The ability of freshwater fishes to move in response to climate change or geological events is limited to the patterns of connectivity of the water bodies where they exist. Opportunities for range expansion between isolated drainages are limited to rare events such as stream captures, geologic events (e.g., tectonic plate shifts) and major flooding events. Extinction is often more likely than migration due to their limited dispersal abilities. Climatic changes, disease, interspecific competition and 1 habitat destruction are all threats that freshwater fishes are often unable to escape (Swift et al. 1986, Warren et al. 2000). The southeastern United States is a hotspot for biogeographical research on freshwater organisms. This region has the highest aquatic biodiversity of any temperate region in the world and the highest aquatic biodiversity in the country. This biodiverse region contains many endemic species that don’t exist anywhere else in the world. In part because of the many endemic and rare species, there are a high number of imperiled species in this region. Warren et al. (2000) stated that at least 28% of freshwater species are imperiled in the Southeast, including 6% endangered, 7% threatened and 15% vulnerable. These numbers represent a 125% increase in imperilment since 1980 (Warren et al. 2000), indicating an urgent need for aquatic conservation in this region. In general, decline of native fish populations can be attributed to habitat degradation and fragmentation, both in the Southeast and globally (Burkhead et al. 1997, Warren et al. 2000). Allan & Flecker (1993) described six major causes of species loss: habitat loss, invasive species, overharvesting, pollution, climate change and secondary extinctions that occur when a keystone species is lost. The source of the rich biological diversity