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

Abstract

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 Apalone and Pelodiscus and to X (not Y) in Staurotypus. 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 Xenopus tropicalis XTR1 (carrying Dmrt1). Other turtle sex chromosomes are homologous to XTR4 (carrying Wt1). Interestingly, all known turtle sex chromosomes, except in Trionychidae, evolved via inversions around Dmrt1 or Wt1. 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.

Chromosomal Rearrangements during Turtle Evolution Altered the Synteny of Genes Involved in Vertebrate Sex Determination

“…Notably, our mapping data in the context of the phylogenetic relationships of the species examined here ( Fig. 1 A) indicate that the ancestral location of Foxl2 was in a macroautosome (chromosome 9 based on decreasing size) [Montiel et al, 2016], from which it translocated to the micro-sex chromosomes in Apalone [Badenhorst et al, 2013]. Whether the translocation occurred after Apalone 's split from Pelodiscus, with whom it shares a homologous ZZ/ZW sex chromosome B system [Badenhorst et al, 2013], or at the split of the Trionychidae family lineage warrants further investigation, as we were not successful in mapping Foxl2 in Pelodiscus .…”

“…Metaphase chromosome preparations were obtained from cell cultures following standard procedures as described previously [Badenhorst et al, 2015;Montiel et al, 2016Montiel et al, , 2017 for 12 turtle species with either TSD or GSD ranging in diploid number from 2n = 28 to 2n = 66 ( Fig. 1 A): Chrysemys picta (CPI), Trachemys scripta (TSC), Chelydra serpentina (CSE), Glyptemys insculpta (GIN), Staurotypus triporcatus (STR), Sternotherus odoratus (SOD), Apalone spinifera (ASP), Pelodiscus sinensis (PSI), Podocnemis unifilis (PUN), Pelomedusa subrufa (PSU), Chelodina oblonga (COB), and Emydura macquarii (EMA).…”

“…BAC DNA ( ∼ 1 μg) was extracted and labeled by standard nick translation (Abbott Molecular) using either biotin-16-dUTP or digoxigenin-11-dUTP (Roche) and coprecipitated with human Cot-1 DNA and Chrysemys Cot-1 DNA to suppress repetitive DNA sequences. BAC-FISH was carried out using these labeled BAC probes, as described by Montiel et al [2016Montiel et al [ , 2017. Briefly, chromosome slides were incubated at 65 ° C for 2 h, denatured for 1 min 45 s at 70 ° C in 70% formamide/2× SSC, dehydrated through an ethanol series, and air dried.…”

“…The FISH signal and hybridization pattern determined the BAC clones' relative positions within chromosomes, and chromosomes were identified according to their morphology, size, shape, and DAPI-banding ( Fig. 3 ) as previously described [Montiel et al, 2016]. A minimum of 10 complete metaphase spreads were examined and analyzed for each gene per specimen.…”

“…Turtle karyotypes are characterized by 2 chromosomal components, macro-and microchromosomes, as those of other reptiles (except crocodilians) and birds [Olmo, 2008]. Importantly, changes in turtle diploid number involve mostly variation in the number of microchromosomes [Olmo, 2008;Montiel et al, 2016]. In general, microchromosomes are defined as those that are nearly indistinguishable from each other by shape and centromere position due to their small size.…”