Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2017 Studies on the molecular underpinnings of sex determination mechanism evolution and molecular sexing tools in turtles Robert Alan Literman Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Developmental Biology Commons, Evolution Commons, and the Molecular Biology Commons Recommended Citation Literman, Robert Alan, "Studies on the molecular underpinnings of sex determination mechanism evolution and molecular sexing tools in turtles" (2017). Graduate Theses and Dissertations. 15350. https://lib.dr.iastate.edu/etd/15350 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Studies on the molecular underpinnings of sex determination mechanism evolution and molecular sexing tools in turtles by Robert Literman A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Ecology and Evolutionary Biology Program of Study Committee: Maria N Valenzuela-Castro, Major Professor Jeff Essner John Nason Michael Shogren-Knaak Jonathan Wendel Iowa State University Ames, Iowa 2017 Copyright © Robert Literman, 2017. All rights reserved. ii DEDICATION This work is dedicated to my family: To my parents who supported me in all endeavors leading here, and to my wife Krystal and our son Rubin, who kept me going through it all. iii TABLE OF CONTENTS Page CHAPTER 1 INTRODUCTION .......................................................................... 1 CHAPTER 2 PUTATIVE INDEPENDENT EVOLUTIONARY REVERSALS FROM GENOTYPIC TO TEMPERATURE DEPENDENT SEX DETERMINATION ARE ASSOCIATED WITH ACCELERATED EVOLUTION OF SEX-DETERMINING GENES IN TURTLES ........................... 6 2.1 Abstract ......................................................................................................... 6 2.2 Introduction .................................................................................................... 7 2.3 Methods ......................................................................................................... 11 2.4 Results ......................................................................................................... 15 2.5 Discussion ...................................................................................................... 22 2.6 Conclusions .................................................................................................... 31 2.7 Acknowledgements ........................................................................................ 32 2.8 Tables and Figures ......................................................................................... 33 CHAPTER 3 QPCR BASED MOLECULAR SEXING BY COPY NUMBER VARIATION IN rRNA GENES AND ITS UTILITY FOR SEX IDENTIFICATION IN SOFT-SHELL TURTLES .................................................. 40 3.1 Abstract ......................................................................................................... 40 3.2 Introduction .................................................................................................... 41 3.3 Materials and Methods ................................................................................... 45 3.4 Results ......................................................................................................... 51 3.5 Discussion ...................................................................................................... 54 3.6 Conclusions .................................................................................................... 56 3.7 Acknowledgements ........................................................................................ 58 3.8 Tables and Figures ......................................................................................... 58 CHAPTER 4 DEVELOPMENT OF SEXING PRIMERS IN GLYPTEMYS INSCULPTA AND APALONE SPINIFERA TURTLES UNCOVERS AN XX/XY SEX-DETERMINING MECHANISM IN THE CRITICALLY-ENDANGERED BOG TURTLE GLYPTEMYS MUHLENBERGII 63 4.1 Abstract ......................................................................................................... 63 4.2 Introduction .................................................................................................... 64 4.3 Methods ......................................................................................................... 66 4.4 Results ......................................................................................................... 70 4.5 Discussion ...................................................................................................... 72 4.6 Acknowledgements ........................................................................................ 76 4.7 Tables and Figures ......................................................................................... 77 iv CHAPTER 5 SUMMARY AND CONCLUSIONS ............................................. 79 5.1 Summary ........................................................................................................ 79 5.2 SDM Evolution 101 ....................................................................................... 80 5.3 Conclusions .................................................................................................... 81 REFERENCES .......................................................................................................... 91 APPENDIX A SUPPLEMENTAL TABLES FOR CHAPTER 2 .......................... 103 APPENDIX B SUPPLEMENTAL MATERIAL FOR CHAPTER 3 ..................... 122 B1 Copy Number Quantification by Real-Time qPCR ....................................... 122 B2 Analytical Flow Chart .................................................................................... 125 B3 R Code ......................................................................................................... 126 B4 Sample Dataset of Apalone spinifera 18S Copy Number Data ..................... 128 B5 Application of Pipeline for Sex Diagnosis of the TSD Chelydra serpentina 130 APPENDIX C SUPPLEMENTAL MATERIAL FOR CHAPTER 4 ..................... 133 C1 Detailed Bioinformatics Methods .................................................................. 133 C2 Supplemental Table ....................................................................................... 136 v NOMENCLATURE ESD Environmental Sex Determination gDNA Genomic DNA GSD Genotypic Sex Determination NGS Next-Generation Sequencing rDNA Ribosomal DNA SDM Sex Determination Mechanism TSD Temperature-Dependent Sex Determination TSP Thermosensitive Period vi ABSTRACT Sex determination mechanisms (SDMs) direct the development of individuals towards a male or female fate, and in vertebrates they are typically controlled by an individual’s genotypic content (genotypic sex determination, GSD) or through an environmental cue experienced during development, mainly temperature (temperature- dependent sex determination, TSD). Among vertebrates, SDMs are surprisingly labile, transitioning between different forms of TSD and GSD in some lineages more than others. Turtles represent a model clade to study SDM evolution, as multiple independent transitions between TSD and GSD have occurred throughout their evolution and a growing number of genomic datasets have become available. This dissertation examines the molecular underpinnings of SDM evolution in turtles while also providing tools that enable studies of sex determination across taxa and of sex- specific traits. In Chapter 2, I examine the molecular evolution of a suite of vertebrate sex determining genes in turtles, contrasting their evolutionary rates to those of other major vertebrate clades and also among turtle lineages. Furthermore, I compare the evolutionary rates of turtle lineages which have undergone SDM transitions versus those that have not. I then compare the relative evolutionary rates of turtles that have transitioned from TSD-to- GSD against lineages which have possibly transitioned from GSD-to-TSD. Finally, I discuss amino acid substitutions which occur in the functional domains of key sex determining genes along transitional branches, providing targets for future research. In Chapter 3, I present an analytical pipeline which can diagnose sex with 100% accuracy in the ZZ/ZW spiny softshell turtle Apalone spinifera by leveraging a previously described sex-biased copy number vii variation of rDNA clusters between the sexes. The pipeline is also applied to a previously published dataset of circulating hormonal concentrations in the snapping turtle Chelydra serpentina , where it shows greater than 85% accuracy in sex diagnosis. In Chapter 4, I lay out a bioinformatics pipeline which details the sample collection, sequencing, and analysis of sex-specific DNA libraries, which can be used to identify sex-linked DNA sequences in any taxon with sufficient genetic differentiation between the sexes. This information is leveraged to create sex-diagnostic PCR primers which are 100% accurate at diagnosing sex in the focal taxa, the ZZ/ZW A. spinifera and the XX/XY wood turtle Glyptemys insculpta . Furthermore, primers designed against the focal taxa data are also