Supplementary Material for: Cytogenetic Insights into the Evolution of Chromosomes and Sex Determination Reveal Striking Homology of Turtle Sex Chromosomes to Amphibian Autosomes

Turtle karyotypes are highly conserved compared to other vertebrates; yet, variation in diploid number (2n = 26-68) reflects profound genomic reorganization, which correlates with evolutionary turnovers in sex determination. We evaluate the published literature and newly collected comparative cytogenetic data (G- and C-banding, 18S-NOR, and telomere-FISH mapping) from 13 species spanning 2n = 28-68 to revisit turtle genome evolution and sex determination. Interstitial telomeric sites were detected in multiple lineages that underwent diploid number and sex determination turnovers, suggesting chromosomal rearrangements. C-banding revealed potential interspecific variation in centromere composition and interstitial heterochromatin at secondary constrictions. 18S-NORs were detected in secondary constrictions in a single chromosomal pair per species, refuting previous reports of multiple NORs in turtles. 18S-NORs are linked to ZW chromosomes in <i>Apalone</i> and<i>Pelodiscus </i>and to X (not Y) in <i>Staurotypus</i>. Notably, comparative genomics across amniotes revealed that the sex chromosomes of several turtles, as well as mammals and some lizards, are homologous to components of <i>Xenopus tropicalis </i>XTR1 (carrying <i>Dmrt1</i>). Other turtle sex chromosomes are homologous to XTR4 (carrying<i>Wt1</i>). Interestingly, all known turtle sex chromosomes, except in Trionychidae, evolved via inversions around <i>Dmrt1 </i>or <i>Wt1</i>. Thus, XTR1 appears to represent an amniote proto-sex chromosome (perhaps linked ancestrally to XTR4) that gave rise to turtle and other amniote sex chromosomes