University of Texas at Tyler Scholar Works at UT Tyler Biology Theses Biology Summer 8-14-2017 REEVALUATING THE OCCURRENCE AND PHYLOGENY OF THE PIGTOE UNIONID MUSSELS FUSCONAIA ASKEWI, FUSCONAIA LANANENSIS, FUSCONAIA FLAVA, AND PLEUROBEMA RIDDELLI IN TEXAS Charles J. Pratt University of Texas at Tyler Follow this and additional works at: https://scholarworks.uttyler.edu/biology_grad Part of the Biology Commons, and the Genetics Commons Recommended Citation Pratt, Charles J., "REEVALUATING THE OCCURRENCE AND PHYLOGENY OF THE PIGTOE UNIONID MUSSELS FUSCONAIA ASKEWI, FUSCONAIA LANANENSIS, FUSCONAIA FLAVA, AND PLEUROBEMA RIDDELLI IN TEXAS" (2017). Biology Theses. Paper 49. http://hdl.handle.net/10950/589 This Thesis is brought to you for free and open access by the Biology at Scholar Works at UT Tyler. It has been accepted for inclusion in Biology Theses by an authorized administrator of Scholar Works at UT Tyler. For more information, please contact [email protected]. REEVALUATING THE OCCURRENCE AND PHYLOGENY OF THE PIGTOE UNIONID MUSSELS FUSCONAIA ASKEWI, FUSCONAIA LANANENSIS, FUSCONAIA FLAVA, AND PLEUROBEMA RIDDELLI IN TEXAS by CHARLES J PRATT A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Biology Srini Kambhampati, Ph.D., Committee Chair College of Arts and Sciences The University of Texas at Tyler July 2017 Acknowledgements I’d like to thank all of my friends and family for their support, this thesis would have been impossible without them. To my dad, Dan Pratt, and my mom, Kara Bennett, thank you for your excellent advice, your unwavering support, and your faith in me. To my brother, Mike, thanks for your support and the fun times we’ve had. A big thank you as well to the faculty and staff in the UT Tyler Biology department. To my advisor, Dr. Srini Kambhampati, and my thesis committee members Dr. Neil Ford and Dr. Lance Williams, thank you for your help and guidance through my thesis project and research. Drs. Kate Hertweck and Josh Banta, thank you for your help with genetics and data analysis. To Chris Parnell, your helpful suggestions for fixing various issues with lab work solved a lot of headaches, thank you. Nearly every other member of the department also helped me at some point, thank you all. To the other graduate students here at UT Tyler, thanks for your help and your friendship, you helped make my stay in Tyler enjoyable and I’ll miss you all. A special thanks to Jared Dickson and Matt Wolkoff for being good lab buddies and for helping with fieldwork, and to Edith Plants-Paris for help with genetics and for providing some samples used in this project. Thanks as well to Dr. Charles Randklev and the team at the Texas A&M Natural Resources Institute for providing additional samples used in this study. Finally, I would like to thank the Sam A. Lindsey endowment for the funding which made this thesis possible. Table of Contents List of Tables ...................................................................................................................... ii List of Figures .................................................................................................................... iii Abstract .............................................................................................................................. iv Chapter 1 Introduction .........................................................................................................1 Chapter 2 Methods Sample Collection and Identification ...............................................................................8 Sequencing and Data Analysis .......................................................................................16 Chapter 3 Results Sequence Alignment and Divergence Measures ............................................................21 Phylogenetic Trees .........................................................................................................22 Chapter 4 Discussion .........................................................................................................30 Chapter 5 Conclusions .......................................................................................................34 References ..........................................................................................................................37 Appendix A. Nucleotide alignment of the ND1 mitochondrial gene ................................42 Appendix B. Nucleotide alignment of the COI mitochondrial gene ................................66 Appendix C. Nucleotide alignment of the 16S rRNA mitochondrial gene ......................86 i List of Tables Table 1. Morphological and genetic species identities and collection locations ...............10 Table 2. Polymerase chain reaction primers for amplification and sequencing of the ND1, COI, and 16S rRNA mitochondrial genes. ........................................................................17 Table 3. Polymerase chain reaction parameters for amplifying the ND1, COI, and 16S rRNA mitochondrial genes ................................................................................................18 Table 4. Best-fit evolutionary model of nucleotide substitution as determined by Akaike information criterion (AIC) score. .....................................................................................19 Table 5. DNA sequence alignment lengths, variable sites, and parsimony-informative sites ....................................................................................................................................21 Table 6. Mean inter- and intra-specific variation for the ND1, COI, and 16S rRNA mitochondrial genes ...........................................................................................................23 ii List of Figures Figure 1. East Texas mussel sample locations, including both field sites and donated samples.. ...............................................................................................................................9 Figure 2. Shell morphology of Fusconaia askewi, Fusconaia lananensis, Fusconaia flava, and Pleurobema riddellii .........................................................................................15 Figure 3. Maximum Likelihood Gene Tree for ND1 .........................................................24 Figure 4. Bayesian Gene Tree for ND1 .............................................................................25 Figure 5. Maximum Likelihood Gene Tree for COI ..........................................................26 Figure 6. Bayesian Gene Tree for COI ..............................................................................27 Figure 7. Maximum Likelihood Gene Tree for 16S ..........................................................28 Figure 8. Bayesian Gene Tree for 16S ...............................................................................29 iii Abstract REEVALUATING THE OCCURRENCE AND PHYLOGENY OF THE PIGTOE UNIONID MUSSELS FUSCONAIA ASKEWI, FUSCONAIA LANANENSIS, FUSCONAIA FLAVA, AND PLEUROBEMA RIDDELLI IN TEXAS Charles Jacob Pratt Thesis Chair: Srini Kambhampati, Ph.D. The University of Texas at Tyler July 2017 The recent development of DNA-based species classification methods has led to the revision of many morphology-based species designations as genetic data has provided new information for species delineation. Analysis of standardized DNA ‘barcoding’ gene regions has proven particularly effective in resolving the taxonomic status of taxa that display phenotypic and habitat overlap. Freshwater Unionid and Margaritiferid mussels comprise one of the most diverse aquatic taxa in North America, but are also among the most threatened, with over 70% of the approximately 300 native species considered at-risk or in decline. Although mussels face extensive threats from anthropogenic activity, conservation efforts have been hampered by limited knowledge of mussel ecology, their uncertain phylogeny and species status, and difficulty of identification. Within East Texas, six of the 37 native mussel species are listed as threatened and three are under consideration for federal Endangered Species Act listing. In this study, a phylogenetic analysis was performed on three mitochondrial genes to examine the occurrence and phylogeny of four Unionid mussel species, the Texas Pigtoe, Fusconaia askewi (Marsh 1896); Triangle Pigtoe, iv Fusconaia lananensis (Frierson 1901); Wabash Pigtoe, Fusconaia flava (Rafinesque 1820); and Louisiana Pigtoe, Pleurobema riddellii (Lea 1861), using an expanded sample dataset including the majority of these species’ known occurrence in Texas. As all of these mussels except F. flava are listed as threatened by the state of Texas, and both F. lananensis and P. riddellii are under consideration for federal Endangered Species Act listing, revisions to the taxonomy of this group will have important implications for future conservation planning. Phylogenetic analysis found three monophyletic clades consisting of F. flava, P. riddellii, and intermixed F. askewi and F. lananensis. Phylogenetic analysis and comparative divergence measures identified F. askewi and F. lananensis as a single monophyletic clade, with F. flava and P. riddellii supported as separate monophyletic lineages. The high degree of observed morphological and habitat similarity and lack of genetic divergence from the Texas Pigtoe, Fusconaia askewi, suggests that the Triangle Pigtoe, Fusconaia lananensis, is not a valid species and that