Squamate reptiles possess two general modes of sex determination: (1) genotypic sex determination (GSD), where the sex of an individual is determined by sex chromosomes, i.e. by sex-specific differences in genotype; and (2) temperature-dependent sex determination (TSD), where sex chromosomes are absent and sex is determined by nongenetic factors. After gathering information about sex-determining mechanisms for more than 400 species, we employed comparative phylogenetic analyses to reconstruct the evolution of sex determination in Squamata. Our results suggest relative uniformity in sex-determining mechanisms in the majority of the squamate lineages. Well-documented variability is found only in dragon lizards (Agamidae) and geckos (Gekkota). Polarity of the sex-determining mechanisms in outgroups identified TSD as the ancestral mode for Squamata. After extensive review of the literature, we concluded that to date there is no known well-documented transition from GSD to TSD in reptiles, although transitions in the opposite direction are plentiful and well corroborated by cytogenetic evidence. We postulate that the evolution of sex-determining mechanisms in Squamata was probably restricted to the transitions from ancestral TSD to GSD. In other words, transitions were from the absence of sex chromosomes to the emergence of sex chromosomes, which have never disappeared and constitute an evolutionary trap. This evolutionary trap hypothesis could change the understanding of phylogenetic conservatism of sex-determining systems in many large clades such as butterflies, snakes, birds, and mammals.

Amniote vertebrates possess various mechanisms of sex determination, but their variability is not equally distributed. The large evolutionary stability of sex chromosomes in viviparous mammals and birds was believed to be connected with their endothermy. However, some ectotherm lineages seem to be comparably conserved in sex determination, but previously there was a lack of molecular evidence to confirm this. Here, we document a stability of sex chromosomes in advanced snakes based on the testing of Z-specificity of genes using quantitative PCR (qPCR) across 37 snake species (our qPCR technique is suitable for molecular sexing in potentially all advanced snakes). We discovered that at least part of sex chromosomes is homologous across all families of caenophidian snakes (Acrochordidae, Xenodermatidae, Pareatidae, Viperidae, Homalopsidae, Colubridae, Elapidae and Lamprophiidae). The emergence of differentiated sex chromosomes can be dated back to about 60 Ma and preceded the extensive diversification of advanced snakes, the group with more than 3000 species. The Z-specific genes of caenophidian snakes are (pseudo)autosomal in the members of the snake families Pythonidae, Xenopeltidae, Boidae, Erycidae and Sanziniidae, as well as in outgroups with differentiated sex chromosomes such as monitor lizards, iguanas and chameleons. Along with iguanas, advanced snakes are therefore another example of ectothermic amniotes with a long-term stability of sex chromosomes comparable with endotherms.

Many poikilothermic vertebrate lineages, especially among amphibians and fishes, possess a rapid turnover of sex chromosomes, while in endotherms there is a notable stability of sex chromosomes. Reptiles in general exhibit variability in sex-determining systems; as typical poikilotherms, they might be expected to have a rapid turnover of sex chromosomes. However, molecular data which would enable the testing of the stability of sex chromosomes are lacking in most lineages. Here, we provide molecular evidence that sex chromosomes are highly conserved across iguanas, one of the most speciesrich clade of reptiles. We demonstrate that members of the New World families Iguanidae, Tropiduridae, Leiocephalidae, Phrynosomatidae, Dactyloidae and Crotaphytidae, as well as of the family Opluridae which is restricted to Madagascar, all share homologous sex chromosomes. As our sampling represents the majority of the phylogenetic diversity of iguanas, the origin of iguana sex chromosomes can be traced back in history to the basal splitting of this group which occurred during the Cretaceous period. Iguanas thus show a stability of sex chromosomes comparable to mammals and birds and represent the group with the oldest sex chromosomes currently known among amniotic poikilothermic vertebrates.

BackgroundThe accumulation of repetitive sequences such as microsatellites during the differentiation of sex chromosomes has not been studied in most squamate reptiles (lizards, amphisbaenians and snakes), a group which has a large diversity of sex determining systems. It is known that the Bkm repeats containing tandem arrays of GATA tetranucleotides are highly accumulated on the degenerated W chromosomes in advanced snakes. Similar, potentially homologous, repetitive sequences were found on sex chromosomes in other vertebrates. Using FISH with probes containing all possible mono-, di-, and tri-nucleotide sequences and GATA, we studied the genome distribution of microsatellite repeats on sex chromosomes in two lizard species (the gecko Coleonyx elegans and the lacertid Eremias velox) with independently evolved sex chromosomes. The gecko possesses heteromorphic euchromatic sex chromosomes, while sex chromosomes in the lacertid are homomorphic and the W chromosome is highly heterochromatic. Our aim was to test whether microsatellite distribution on sex chromosomes corresponds to the stage of their heteromorphism or heterochromatinization. Moreover, because the lizards lie phylogenetically between snakes and other vertebrates with the Bkm-related repeats on sex chromosomes, the knowledge of their repetitive sequence is informative for the determination of conserved versus convergently evolved repetitive sequences across vertebrate lineages.ResultsHeteromorphic sex chromosomes of C. elegans do not show any sign of microsatellite accumulation. On the other hand, in E. velox, certain microsatellite sequences are extensively accumulated over the whole length or parts of the W chromosome, while others, including GATA, are absent on this heterochromatinized sex chromosome.ConclusionThe accumulation of microsatellite repeats corresponds to the stage of heterochromatinization of sex chromosomes rather than to their heteromorphism. The lack of GATA repeats on the sex chromosomes of both lizards suggests that the Bkm-related repeats on sex chromosomes in snakes and other vertebrates evolved convergently. The comparison of microsatellite sequences accumulated on sex chromosomes in E. velox and in other eukaryotic organisms suggests that historical contingency, not characteristics of particular sequences, plays a major role in the determination of which microsatellite sequence is accumulated on the sex chromosomes in a particular lineage.

The bedbug, Cimex lectularius, is a well-known human ectoparasite that is reemerging after a long absence of several decades in developed countries of North America and Western Europe. Bedbugs' original hosts were likely bats, and the bedbugs are still common in their roosts. Using morphometry and sequences of mitochondrial cytochrome oxidase subunit I and 16S genes, we showed that the populations on bats and humans are largely isolated and differ in morphology. The character of the morphological difference suggests it to be due to adaptation to different hosts, namely adaptations to different sensory, feeding, and dispersal needs. Using the molecular data, we estimated the time of splitting into bat- and human-parasitizing groups using the isolation-with-migration model. The estimate is surprisingly long ago and seems to predate the expansion of modern human from Africa. The gene flow between bat- and human-parasitizing bedbugs is limited and asymmetric with prevailing direction from human-parasitizing populations to bat-parasitizing populations. The differentiation of the populations fits the concept of host races and supports the idea of sympatric speciation. Furthermore, our findings contradict recently formulated hypotheses suggesting bat roosts as a source of bedbug's resurgence as a human pest. Also, we extend the known host range of the bedbug by two bat species.