Molecular Phylogenetics of Nav1.4a and Kir2.5 in the Neotropical Electric Knifefish family Hypopomidae (Osteichthys: Gymnotiformes) by Thanara Rajakulendran A thesis submitted in conformity with the requirements for the degree of Masters of Science Graduate Department of Ecology and Evolutionary Biology University of Toronto © Copyright by Thanara Rajakulendran, 2014 Molecular Phylogenetics of Nav1.4a and Kir2.5 in the Neotropical Electric Knifefish family Hypopomidae (Osteichthys: Gymnotiformes) Thanara Rajakulendran Masters of Science Graduate Department of Ecology and Evolutionary Biology University of Toronto 2014 Abstract Electric fishes possess electric organs that they use to electrolocate and communicate. The rate and frequency of electrical discharges are affected by ion channels. In this study, I sequence two genes that likely play key roles in the generation of electric organ discharges (Nav1.4a and Kir2.5) and test their utility as phylogenetic markers in the family Hypopomidae. In addition, I concatenate these two genes with cyt b and RAG2 to assess species relationships within Hypopomidae using parsimony and Bayesian analyses. Both Nav1.4a and Kir2.5 exhibit sufficient interspecific variation to assess species relationships, and the four genes produce concordant phylogenetic patterns. Ion channel genes are therefore useful markers for electric fish phylogenetics. ii Acknowledgments I would like to start by first thanking my supervisor Dr. Nathan Lovejoy for the opportunity to take on this thesis project and for his continued guidance and encouragement along this journey. Nathan’s passion for science and hands-off teaching style has helped me develop valuable skills and become more independent as a researcher. I would like to also thank my supervisory committee members, Dr. Belinda Chang and Dr. Mark Fitzpatrick for their insightful feedback and guidance throughout my research. I would like to thank the National Science and Engineering Research Council of Canada (NSERC) and the National Science Foundation (NSF), as well as Nate for funding my research. I am also thankful for the members of the Lovejoy lab for their invaluable encouragement and friendships. Specifically, I would like to thank Dr. Devin Bloom for patiently teaching me the lab techniques and skills I would require for running and troubleshooting PCRs successfully and Dr. Dominik Halas for taking the time to walk me through complex programming algorithms and helping me jump-start my analyses. In addition, I would like to thank Matthew Kolmann and Charmaine Condy for all the support, encouragement and advice they’ve given me along the way. Lastly, I would like to thank all members of the Lovejoy lab who have made my graduate experience enjoyable and rewarding. Finally, I would like thank my family for all their support, encouragement and especially love during this journey. It was my parents who first introduced me to the world of nature and my older brother who helped nurture an early passion for science. iii Table of Contents Contents Acknowledgments.......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii Abbreviations ............................................................................................................................... viii List of Appendices ......................................................................................................................... ix Chapter 1 Introduction .....................................................................................................................1 1 Gymnotiform biology .................................................................................................................1 2 Hypopomidae ..............................................................................................................................2 2.1 Hypopomid phylogeny.........................................................................................................2 3 Electrogenic and electrosensory system......................................................................................4 3.1 Electric organ (EO) ..............................................................................................................4 3.2 Electric organ discharge (EOD) ...........................................................................................4 4 Ion channels ................................................................................................................................5 4.1 The molecular physiology of electrogenesis........................................................................5 4.2 Voltage gated sodium channels ...........................................................................................6 4.3 Inwardly rectifying potassium channel ................................................................................7 5 Objectives ....................................................................................................................................8 Chapter 2 Methods ...........................................................................................................................9 1 Taxon sampling ...........................................................................................................................9 1.1 Field Collection ....................................................................................................................9 1.2 Taxon sampling and DNA isolation ....................................................................................9 iv 2 Primer design for Kir2.5 .............................................................................................................9 3 Molecular Phylogeny ................................................................................................................10 3.1 PCR and sequencing ..........................................................................................................10 3.2 Alignment ..........................................................................................................................11 3.3 Maximum parsimony (MP) analysis ..................................................................................12 4 Bayesian analysis (BI)...............................................................................................................12 Chapter 3 Results ...........................................................................................................................13 1 Molecular Dataset .....................................................................................................................13 2 Phylogenetic Relationships .......................................................................................................13 2.1 Complete Dataset Analyses ...............................................................................................13 2.2 Individual Gene Analyses ..................................................................................................14 Chapter 4 Discussion .....................................................................................................................16 1 Overall findings .........................................................................................................................16 2 Rhamphichthyoidea phylogeny .................................................................................................16 3 Brachyhypopomus phylogeny ...................................................................................................17 4 Phylogenetic signal across genes ..............................................................................................18 5 Future Directions .......................................................................................................................18 6 Conclusion ................................................................................................................................20 References ......................................................................................................................................21 Appendices .....................................................................................................................................43 v List of Tables Table 1: List of specimens used in this study Table 2: List of all primers used for amplification and sequencing of Nav1.4a, Kir2.5, cyt b, RAG2 genes Table 3: Characteristics of gene matrices used in analyses Table 4: Tree indices for parsimony analyses of total evidence matrix and separate genes vi List of Figures Figure 1: Simplified reconstructions of previously hypothesized Rhamphichthyoidea relationships Figure 2: Reconstruction of previously proposed Brachyhypopomus spp. relationships from Sullivan (1997) Figure 3: Total evidence maximum parsimony consensus phylogram for Hypopomidae Figure 4: Total evidence maximum parsimony consensus phylogram for Hypopomidae with duplicate species pruned Figure 5: Bayesian analysis for Hypopomidae