Cytogenetics studies on the evolution of sex chromosomes and genome organization in turtles

Sex-determining mechanisms (SDMs) set an individual’s sexual fate by its genotype (genotypic sex determination, GSD) or environmental factors like temperature (temperature-dependent sex determination, TSD). While sexual reproduction is one of the most taxonomically conserved traits, SDMs are incredibly diverse and evolve frequently. The vast diversity and complexity of SDMs harbored within vertebrates, including turtles still puzzles researchers, and our understanding of how SDMs evolve remains incomplete. This dissertation examines the cytogenetics underpinning of SDM evolution in turtles, exploring the evolution of sex chromosomes and genome organization via chromosomal rearrangements in turtles. In Chapter 2, I characterized the sex chromosome system of Emydura subglobosa turtles by the identification of sex-specific regions that revealed its type of heterogamety. Combining this new information with published data from other turtles, I examined the evolution of sex chromosomes in chelid turtles in a phylogenetic framework and uncovered a novel evolutionary trajectory from what had been previously proposed, which I found involves a Y-autosome fusion. Furthermore, these data also permitted dating this Y-autosome fusion in the Emydura lineage to at least 45 Mya. In Chapter 3, I examined the patterns of synteny of 10 target genes among 12 TSD and GSD focal turtle species to identify genomic regions whose evolution may be associated with transitions in turtle sex determination. I found no evidence to support the hypothesis that changes in the relative position of these gene regulators of sex determination/differentiation are associated with evolutionary transitions in turtle sex determination. However, I uncovered intrachromosomal rearrangements that altered the relative position of some genes that are involved in sexual development within a single chromosome (intrachromosomal rearrangements), fusion/fission events in other chromosomes (interchromosomal rearrangements), and cases of autosome to sex chromosome translocation of a pair of genes in a GSD turtle that render them potential candidate sex-determining genes. In Chapter 4, I used fluorescent in situ hybridization of bacterial artificial chromosome clones (BAC-FISH) from a painted turtle Chrysemys picta library to enhance the physical mapping of the genome assembly of the painted turtle whose contiguity was improved by BioNano optical mapping. This approach improved the C. picta assembly by ~242% compared to the previous assembly, and physical mapping permitted anchoring ~45% of the genome, including 20 genes related to the sex determination network of turtles and vertebrates. Here, I also detected a few discrepancies between BAC-FISH physical mapping and the new assembly, which highlight the importance of molecular cytogenetics data to identify and correct assembly errors. BAC-FISH data was also used to establish the chromosome homology between painted and slider turtles, and between painted turtle and other sauropsid vertebrates. Results revealed shared homology and highly conserved syntenic blocks, as well as numerous instances of chromosomal rearrangements between these species. Together these chapters provide critical insights into the evolution of sex chromosomes and genome organization, especially of chromosomal rearrangements in turtles, which are important to understand the evolution of the remarkable diversity of SDM in turtles, reptiles, and vertebrates