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.

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.

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.

The genome of Triatoma delpontei Romaña & Abalos 1947 is the largest within Heteroptera, approximately two to three times greater than other evaluated Heteroptera genomes. Here, the repetitive fraction of the genome was determined and compared with its sister species Triatoma infestans Klug 1834, in order to shed light on the karyotypic and genomic evolution of these species. The T. delpontei repeatome analysis showed that the most abundant component in its genome is satellite DNA, which makes up more than half of the genome. The T. delpontei satellitome includes 160 satellite DNA families, most of them also present in T. infestans. In both species, only a few satellite DNA families are overrepresented on the genome. These families are the building blocks of the C-heterochromatic regions. Two of these satellite DNA families that form the heterochromatin are the same in both species. However, there are satellite DNA families highly amplified in the heterochromatin of one species that in the other species are in low abundance and located in the euchromatin. Therefore, the present results depicted the great impact of the satellite DNA sequences in the evolution of Triatominae genomes. Within this scenario, satellitome determination and analysis led to a hypothesis that explains how satDNA sequences have grown on T. delpontei to reach its huge genome size within true bugs.