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.

Reptiles display a wide diversity of sex-determining mechanisms ranging from temperature-dependent sex determination (TSD) to genotypic sex determination (GSD) with either male (XY) or female (ZW) heterogamety. Despite this astounding variability, the origin, structure, and evolution of sex chromosomes remain poorly understood. In turtles, TSD is purportedly ancestral while GSD arose multiple times independently. Here we test whether independent (XY or ZW) or morphologically divergent heterogametic sex chromosome systems evolved in tryonichids (Cryptodira) using the GSD spiny softshell turtle, Apalone spinifera, a species with previously unidentified sex chromosomes. A female-specific signal from comparative genomic hybridization (CGH) was detected in a Giemsa/4',6-diamidino-2-phenylindole faint portion of a microchromosome, indicating the presence of a ZZ/ZW system in A. spinifera. In situ hybridization of a fluorescently labeled 18S rRNA probe identified a large nucleolar organizer region block in the female-specific region of the W (co-localizing with the female-specific CGH signal) and a smaller block on the Z. The heteromorphic ZZ/ZW micro-sex chromosome system detected here is identical to that found in another tryonichid, the Chinese softshell turtle Pelodiscus sinensis, from which A. spinifera diverged ∼95 million years ago. These results reveal a striking sex chromosome conservation in tryonichids, compared to the divergent sex chromosome morphology observed among younger XX/XY systems in pleurodiran turtles. Our findings highlight the need to understand the drivers behind sex chromosome lability and conservation in different lineages and contribute to our knowledge of sex chromosome evolution in reptiles and vertebrates.

Most turtle species possess temperature-dependent sex determination (TSD), but genotypic sex determination (GSD) has evolved multiple times independently from the TSD ancestral condition. GSD in animals typically involves sex chromosomes, yet the sex chromosome system of only 9 out of 18 known GSD turtles has been characterized. Here, we combine comparative genome hybridization (CGH) and BAC clone fluorescent in situ hybridization (BAC FISH) to identify a macro-chromosome XX/XY system in the GSD wood turtle Glyptemys insculpta (GIN), the youngest known sex chromosomes in chelonians (8-20 My old). Comparative analyses show that GIN-X/Y is homologous to chromosome 4 of Chrysemys picta (CPI) painted turtles, chromosome 5 of Gallus gallus chicken, and thus to the X/Y sex chromosomes of Siebenrockiella crassicollis black marsh turtles. We tentatively assign the gene content of the mapped BACs from CPI chromosome 4 (CPI-4) to GIN-X/Y. Chromosomal rearrangements were detected in G. insculpta sex chromosome pair that co-localize with the male-specific region of GIN-Y and encompass a gene involved in sexual development (Wt1-a putative master gene in TSD turtles). Such inversions may have mediated the divergence of G. insculpta sex chromosome pair and facilitated GSD evolution in this turtle. Our results illuminate the structure, origin, and evolution of sex chromosomes in G. insculpta and reveal the first case of convergent co-option of an autosomal pair as sex chromosomes within chelonians.

Comparative genomics continues illuminating amniote genome evolution, but for many lineages our understanding remains incomplete. Here, we refine the assembly (CPI 3.0.3 NCBI AHGY00000000.2) and develop a cytogenetic map of the painted turtle (Chrysemys picta—CPI) genome, the first in turtles and in vertebrates with temperature-dependent sex determination. A comparison of turtle genomes with those of chicken, selected nonavian reptiles, and human revealed shared and novel genomic features, such as numerous chromosomal rearrangements. The largest conserved syntenic blocks between birds and turtles exist in four macrochromosomes, whereas rearrangements were evident in these and other chromosomes, disproving that turtles and birds retain fully conserved macrochromosomes for greater than 300 Myr. C-banding revealed large heterochromatic blocks in the centromeric region of only few chromosomes. The nucleolar-organizing region (NOR) mapped to a single CPI microchromosome, whereas in some turtles and lizards the NOR maps to nonhomologous sex-chromosomes, thus revealing independent translocations of the NOR in various reptilian lineages. There was no evidence for recent chromosomal fusions as interstitial telomeric-DNA was absent. Some repeat elements (CR1-like, Gypsy) were enriched in the centromeres of five chromosomes, whereas others were widespread in the CPI genome. Bacterial artificial chromosome (BAC) clones were hybridized to 18 of the 25 CPI chromosomes and anchored to a G-banded ideogram. Several CPI sex-determining genes mapped to five chromosomes, and homology was detected between yet other CPI autosomes and the globally nonhomologous sex chromosomes of chicken, other turtles, and squamates, underscoring the independent evolution of vertebrate sex-determining mechanisms.

Sex determination is triggered by factors ranging from genotypic (GSD) to environmental (ESD), or both GSD + EE (GSD susceptible to environmental effects), and its evolution remains enigmatic. The presence/absence of sex chromosomes purportedly separates species at the ESD end of the continuum from the rest (GSD and GSD + EE) because the evolutionary dynamics of sex chromosomes and autosomes differ. However, studies suggest that turtles with temperature-dependent sex determination (TSD) are cryptically GSD and possess sex chromosomes. Here, we test this hypothesis in painted turtles Chrysemys picta (TSD), using comparative-genome-hybridization (CGH), a technique known to detect morphologically indistinguishable sex chromosomes in other turtles and reptiles. Our results show no evidence for the existence of sex chromosomes in painted turtles. While it remains plausible that cryptic sex chromosomes may exist in TSD turtles that are characterized by minor genetic differences that cannot be detected at the resolution of CGH, previous attempts have failed to identify sex-specific markers. Genomic sequencing should prove useful in providing conclusive evidence in this regard. If such efforts uncover sex chromosomes in TSD turtles, it may reveal the existence of a fundamental constraint for the evolution of a full spectrum of sex determination (from pure GSD to pure TSD) that is predicted theoretically. Finding sex chromosomes in ESD organisms would question whether pure ESD mechanisms exist at all in nature, or whether those systems currently considered pure ESD simply await the characterization of an underlying GSD architecture.

Summary1 Sex diagnosis is important in ecology, evolution, conservation biology, medicine, and food production. However, sex diagnosis is difficult in species without conspicuous sexual dimorphism or at life stages before such differences develop. This problem is exacerbated when the diagnostic trait is a continuous (non-discrete) variable to which general analytical methods are not commonly applied. 2 Here we demonstrate the use of copy-number variation between males and females of the nucleolar organizing region (NOR) in the genome of Apalone spinifera softshell turtles, which we quantify by real-time PCR. We analyze these continuous data using mixture models that can be applied either in discriminant analysis when a subset of individuals of known sex is used as a training set, or in clustering procedures when all individuals are of unknown sex. 3 Using individuals of known sex, the discriminant analysis exhibited 100% accurate classification rate for both the training set and the test set. Classification rates were also 100% when using the clustering procedure to identify groups and classify individuals in the absence of sex information. Standard curves using only male DNA provided better discrimination than using mixed-sex DNA during qPCR. NOR copy number is an effective sex diagnostic for A. spinifera turtles. Our sexing approach using qPCR of 18S genes should prove useful for other taxa that also possess dimorphic NORs, as is known in some vertebrates and insects. While the 18S copy numbers in our dataset exhibited a non-overlapping binomial distribution, this may not always be the case in future studies of A. spinifera or for other taxa. 4 Importantly however, our sex-typing approach using mixture models provides an attractive alternative under overlapping distributions of these and of other continuous data such as hormone levels, gene expression levels, shape or behavior. We present an example using overlapping distributions of hormone levels in Chelydra serpentina turtles, to demonstrate the broader utility of mixture models for sex-typing, and obtain a high correct classification of 90%.

Karyotypes of 18 rodent species collected in various localities in Thailand were analysed as part of an epidemiological survey of the region using conventional cytogenetic techniques. The aim was to re-assess the reliability of karyotype-based diagnoses of Thai rodents using an updated taxonomic framework. The species examined include Menetes berdmorei (Sciuridae), Mus caroli, Mus cervicolor and Mus cookii, Hapalomys delacouri, Chiropodomys gliroides, as well as several representatives of most of the lineages of the Rattini tribe, that is Rattus exulans, Rattus losea, Rattus tanezumi, Leopoldamys edwardsi, Leopoldamys neilli, Maxomys surifer, Niviventer fulvescens, Berylmys berdmorei, Berylmys bowersi, Bandicota indica and Bandicota savilei (Muridae). The first descriptions of G-and/or C-banding karyotypes are provided for several of these, that is, B. savilei, L. edwardsi, M. surifer, B. berdmorei, B. bowersi, N. fulvescens and H. delacouri. Although largely in agreement with available data, our findings on chromosome morphology differ slightly from those published for L. edwardsi, M. surifer, B. savilei and the two Berylmys species, B. berdmorei and B. bowersi. In addition, we document the novel finding of B-chromosomes in the genera Berylmys, Bandicota and the emblematic Mus. Importantly, few species-specific chromosomal characteristic could be identified within most of the genera investigated in our study and, in contrast to previous claims, the usefulness of karyotypes for diagnosing these Asian murid species appears to be limited.