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 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 applied successfully to related species, expanding the utility of the pipeline, while simultaneously providing the first definitive evidence that the bog turtle Glyptemys muhlenbergii has an XX/XY sex chromosome system. Together these chapters provide data about the proximate mechanisms of SDM evolution in turtles, which are necessary to begin to understand the ultimate explanations for why SDM evolution is so labile across taxa.