BackgroundWe describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing.ResultsOur phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented.ConclusionsOur comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.

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.

Explaining the diversity of vertebrate sex-determining mechanisms ranging from genotypic (GSD) to temperature-dependent (TSD) remains a developmental and evolutionary conundrum. Using a phylogenetic framework, we explore the transcriptional evolution during gonadogenesis of several genes involved in sexual development, combining novel data from Chrysemys picta turtles (TSD) and published data from other TSD and GSD vertebrates. Our novel C. picta dataset underscores Sf1 and Wt1 as potential activators of the thermosensitive period and uncovered the first evidence of Dax1 involvement in male development in a TSD vertebrate. Contrasting transcriptional profiles revealed male-biased Wt1 expression in fish while monomorphic expression is found in tetrapods but absent in turtles. Sf1 expression appears highly labile with transitions among testicular, ovarian, and non-sex-specific gonadal formation patterns among and within lineages. Dax1's dual role in ovarian and testicular formation is found in fish and mammals but is dosage-sensitive exclusively in eutherian mammals due to its X-linkage in this group. Contrastingly, Sox9 male-biased and Aromatase female-biased expression appear ancestral and virtually conserved throughout vertebrates despite significant heterochronic changes in expression as other elements likely replaced their function in early gonadogenesis. Finally, research avenues are highlighted to further study the evolution of the regulatory network of sexual development. Developmental Dynamics 242:307-319,

Vertebrate sexual fate is decided primarily by the individual’s genotype (GSD), by the environmental temperature during development (TSD), or both. Turtles exhibit TSD and GSD, making them ideal to study the evolution of sex determination. Here we analyze temperature-specific gonadal transcriptomes (RNA-sequencing validated by qPCR) of painted turtles (Chrysemys picta TSD) before and during the thermosensitive period, and at equivalent stages in soft-shell turtles (Apalone spinifera—GSD), to test whether TSD’s and GSD’s transcriptional circuitry is identical but deployed differently between mechanisms. Our data show that most elements of the mammalian urogenital network are active during turtle gonadogenesis, but their transcription is generally more thermoresponsive in TSD than GSD, and concordant with their sex-specific function in mammals [e.g., upregulation of Amh, Ar, Esr1, Fog2, Gata4, Igf1r, Insr, and Lhx9 at male-producing temperature, and of β-catenin, Foxl2, Aromatase (Cyp19a1), Fst, Nf-kb, Crabp2 at female-producing temperature in Chrysemys]. Notably, antagonistic elements in gonadogenesis (e.g., β-catenin and Insr) were thermosensitive only in TSD early-embryos. Cirbp showed warm-temperature upregulation in both turtles disputing its purported key TSD role. Genes that may convert thermal inputs into sex-specific development (e.g., signaling and hormonal pathways, RNA-binding and heat-shock) were differentially regulated. Jak-Stat, Nf-κB, retinoic-acid, Wnt, and Mapk-signaling (not Akt and Ras-signaling) potentially mediate TSD thermosensitivity. Numerous species-specific ncRNAs (including Xist) were differentially-expressed, mostly upregulated at colder temperatures, as were unannotated loci that constitute novel TSD candidates. Cirbp showed warm-temperature upregulation in both turtles. Consistent transcription between turtles and alligator revealed putatively-critical reptilian TSD elements for male (Sf1, Amh, Amhr2) and female (Crabp2 and Hspb1) gonadogenesis. In conclusion, while preliminary, our data helps illuminate the regulation and evolution of vertebrate sex determination, and contribute genomic resources to guide further research into this fundamental biological process.

Global climate is warming rapidly, threatening vertebrates with temperature-dependent sex determination (TSD) by disrupting sex ratios and other traits. Less understood are the effects of increased thermal fluctuations predicted to accompany climate change. Greater fluctuations could accelerate feminization of species that produce females under warmer conditions (further endangering TSD animals), or counter it (reducing extinction risk). Here we use novel experiments exposing eggs of Painted Turtles (Chrysemys picta) to replicated profiles recorded in field nests plus mathematically-modified profiles of similar shape but wider oscillations, and develop a new mathematical model for analysis. We show that broadening fluctuations around naturally male-producing (cooler) profiles feminizes developing embryos, whereas embryos from warmer profiles remain female or die. This occurs presumably because wider oscillations around cooler profiles expose embryos to very low temperatures that inhibit development, and to feminizing temperatures where most embryogenesis accrues. Likewise, embryos incubated under broader fluctuations around warmer profiles experience mostly feminizing temperatures, some dangerously high (which increase mortality), and fewer colder values that are insufficient to induce male development. Therefore, as thermal fluctuations escalate with global warming, the feminization of TSD turtle populations could accelerate, facilitating extinction by demographic collapse. Aggressive global CO2 mitigation scenarios (RCP2.6) could prevent these risks, while intermediate actions (RCP4.5 and RCP6.0 scenarios) yield moderate feminization, highlighting the peril that insufficient reductions of greenhouse gas emissions pose for TSD taxa. If our findings are generalizable, TSD squamates, tuatara, and crocodilians that produce males at warmer temperatures could suffer accelerated masculinization, underscoring the broad taxonomic threats of climate change.