Michail Rovatsos

Cretaceous park of sex determination: sex chromosomes are conserved across iguanas.

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 species-rich 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.

CONSERVED SEX CHROMOSOMES ACROSS ADAPTIVELY RADIATED ANOLIS LIZARDS

Vertebrates possess diverse sex‐determining systems, which differ in evolutionary stability among particular groups. It has been suggested that poikilotherms possess more frequent turnovers of sex chromosomes than homoiotherms, whose effective thermoregulation can prevent the emergence of the sex reversals induced by environmental temperature. Squamate reptiles used to be regarded as a group with an extensive variability in sex determination; however, we document how the rather old radiation of lizards from the genus Anolis, known for exceptional ecomorphological variability, was connected with stability in sex chromosomes. We found that 18 tested species, representing most of the phylogenetic diversity of the genus, share the gene content of their X chromosomes. Furthermore, we discovered homologous sex chromosomes in species of two genera (Sceloporus and Petrosaurus) from the family Phrynosomatidae, serving here as an outgroup to Anolis. We can conclude that the origin of sex chromosomes within iguanas largely predates the Anolis radiation and that the sex chromosomes of iguanas remained conserved for a significant part of their evolutionary history. Next to therian mammals and birds, Anolis lizards therefore represent another adaptively radiated amniote clade with conserved sex chromosomes. We argue that the evolutionary stability of sex‐determining systems may reflect an advanced stage of differentiation of sex chromosomes rather than thermoregulation strategy.

A ZZ/ZW Sex Chromosome System in the Thick-Tailed Gecko ( Underwoodisaurus milii ; Squamata: Gekkota: Carphodactylidae), a Member of the Ancient Gecko Lineage

Geckos (Gekkota) are a highly diversified group of lizards with an exceptional diversity in sex-determining systems. Despite this intriguing documented variability, data on sex determination in many lineages is still scarce. Here, we document the previously overlooked heteromorphic ZZ/ZW sex chromosomes in the thick-tailed gecko, Underwoodisaurus milii, a member of the ancient lineage of pygopodoid geckos. The finding of female heterogamety within pygopodoid geckos was unexpected, as until now only male heterogamety and environmental sex determination have been reported in this group, and female heterogamety was known only in distantly related gecko families separated from the pygopodoid geckos around 150 million years ago. The W chromosome in U. milii is highly heterochromatic and contains a large number of telomeric-like repeats comparable to around 50% of all telomeric-like sequences present in male genomes. The accumulation of these repeats might have been responsible for the considerable size expansion of the W chromosome in comparison to the Z chromosome. The heteromorphic ZZ/ZW sex chromosomes with accumulated telomeric-like repeats in the thick-tailed geckos further illustrate the exceptional diversity of sex-determining systems in geckos and add important information to our understanding of the evolution and phylogeny of sex-determining systems in reptiles.

Sex determination in Madagascar geckos of the genus Paroedura (Squamata: Gekkonidae): are differentiated sex chromosomes indeed so evolutionary stable?

Among amniote vertebrates, geckos represent a clade with exceptional variability in sex determination; however, only a minority of species of this highly diverse group has been studied in this respect. Here, we describe for the first time a female heterogamety in the genus Paroedura, the group radiated in Madagascar and adjacent islands. We identified homomorphic ZZ/ZW sex chromosomes with a highly heterochromatic W chromosome in Paroedura masobe, Paroedura oviceps, Paroedura karstophila, Paroedura stumpffi, and Paroedura lohatsara. Comparative genomic hybridization (CGH) revealed that female-specific sequences are greatly amplified in the W chromosome of P. lohatsara and that P. gracilis seems to possess a derived system of multiple sex chromosomes. Contrastingly, neither CGH nor heterochromatin visualization revealed differentiated sex chromosomes in the members of the Paroedura picta—Paroedura bastardi—Paroedura ibityensis clade, which is phylogenetically nested within lineages with a heterochromatic W chromosome. As a sex ratio consistent with genotypic sex determination has been reported in P. picta, it appears that the members of the P. picta—P. bastardi—P. ibityensis clade possess homomorphic, poorly differentiated sex chromosomes and may represent a rare example of evolutionary loss of highly differentiated sex chromosomes. Fluorescent in situ hybridization (FISH) with a telomeric probe revealed a telomere-typical pattern in all species and an accumulation of telomeric sequences in the centromeric region of autosomes in P. stumpffi and P. bastardi. Our study adds important information for the greater understanding of the variability and evolution of sex determination in geckos and demonstrates how the geckos of the genus Paroedura provide an interesting model for studying the evolution of the sex chromosomes.

Evolutionary stability of sex chromosomes in snakes.

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.

Conservation of sex chromosomes in lacertid lizards.

Sex chromosomes are believed to be stable in endotherms, but young and evolutionary unstable in most ectothermic vertebrates. Within lacertids, the widely radiated lizard group, sex chromosomes have been reported to vary in morphology and heterochromatinization, which may suggest turnovers during the evolution of the group. We compared the partial gene content of the Z‐specific part of sex chromosomes across major lineages of lacertids and discovered a strong evolutionary stability of sex chromosomes. We can conclude that the common ancestor of lacertids, living around 70 million years ago (Mya), already had the same highly differentiated sex chromosomes. Molecular data demonstrating an evolutionary conservation of sex chromosomes have also been documented for iguanas and caenophidian snakes. It seems that differences in the evolutionary conservation of sex chromosomes in vertebrates do not reflect the distinction between endotherms and ectotherms, but rather between amniotes and anamniotes, or generally, the differences in the life history of particular lineages.

Female heterogamety in Madagascar chameleons (Squamata: Chamaeleonidae: Furcifer): differentiation of sex and neo-sex chromosomes.

Amniotes possess variability in sex determining mechanisms, however, this diversity is still only partially known throughout the clade and sex determining systems still remain unknown even in such a popular and distinctive lineage as chameleons (Squamata: Acrodonta: Chamaeleonidae). Here, we present evidence for female heterogamety in this group. The Malagasy giant chameleon (Furcifer oustaleti) (chromosome number 2n = 22) possesses heteromorphic Z and W sex chromosomes with heterochromatic W. The panther chameleon (Furcifer pardalis) (2n = 22 in males, 21 in females), the second most popular chameleon species in the world pet trade, exhibits a rather rare Z1Z1Z2Z2/Z1Z2W system of multiple sex chromosomes, which most likely evolved from W-autosome fusion. Notably, its neo-W chromosome is partially heterochromatic and its female-specific genetic content has expanded into the previously autosomal region. Showing clear evidence for genotypic sex determination in the panther chameleon, we resolve the long-standing question of whether or not environmental sex determination exists in this species. Together with recent findings in other reptile lineages, our work demonstrates that female heterogamety is widespread among amniotes, adding another important piece to the mosaic of knowledge on sex determination in amniotes needed to understand the evolution of this important trait.

Sex chromosomes and karyotype of the (nearly) mythical creature, the Gila monster, Heloderma suspectum (Squamata: Helodermatidae).

A wide variety of sex determination systems exist among squamate reptiles. They can therefore serve as an important model for studies of evolutionary transitions among particular sex determination systems. However, we still have only a limited knowledge of sex determination in certain important lineages of squamates. In this respect, one of the most understudied groups is the family Helodermatidae (Anguimorpha) encompassing the only two venomous species of lizards which are potentially lethal to human beings. We uncovered homomorphic ZZ/ZW sex chromosomes in the Gila monster (Heloderma suspectum) with a highly heterochromatic W chromosome. The sex chromosomes are morphologically similar to the ZZ/ZW sex chromosomes of monitor lizards (Varanidae). If the sex chromosomes of helodermatids and varanids are homologous, female heterogamety may be ancestral for the whole Anguimorpha group. Moreover, we found that the karyotype of the Gila monster consists of 2n = 36 chromosomes (14 larger metacentric chromosomes and 22 acrocentric microchromosomes). 2n = 36 is the widely distributed chromosomal number among squamates. In his pioneering works representing the only previous cytogenetic examination of the family Helodermatidae, Matthey reported the karyotype as 2n = 38 and suggested a different chromosomal morphology for this species. We believe that this was probably erroneously. We also discovered a strong accumulation of telomeric sequences on several pairs of microchromosomes in the Gila monster, which is a trait documented relatively rarely in vertebrates. These new data fill an important gap in our understanding of the sex determination and karyotype evolution of squamates.

Interstitial Telomeric Motifs in Squamate Reptiles: When the Exceptions Outnumber the Rule.

Telomeres are nucleoprotein complexes protecting the physical ends of linear eukaryotic chromosomes and therefore helping to ensure their stability and integrity. Additionally, telomeric sequences can be localized in non-terminal regions of chromosomes, forming so-called interstitial telomeric sequences (ITSs). ITSs are traditionally considered to be relics of chromosomal rearrangements and thus very informative in the reconstruction of the evolutionary history of karyotype formation. We examined the distribution of the telomeric motifs (TTAGGG)n using fluorescence in situ hybridization (FISH) in 30 species, representing 17 families of squamate reptiles, and compared them with the collected data from another 38 species from literature. Out of the 68 squamate species analyzed, 35 possess ITSs in pericentromeric regions, centromeric regions and/or within chromosome arms. We conclude that the occurrence of ITSs is rather common in squamates, despite their generally conserved karyotypes, suggesting frequent and independent cryptic chromosomal rearrangements in this vertebrate group.

Novel X-Linked Genes Revealed by Quantitative Polymerase Chain Reaction in the Green Anole, Anolis carolinensis

The green anole, Anolis carolinensis (ACA), is the model reptile for a vast array of biological disciplines. It was the first nonavian reptile to have its genome fully sequenced. During the genome project, the XX/XY system of sex chromosomes homologous to chicken chromosome 15 (GGA15) was revealed, and 106 X-linked genes were identified. We selected 38 genes located on eight scaffolds in ACA and having orthologs located on GGA15, then tested their linkage to ACA X chromosome by using comparative quantitative fluorescent real-time polymerase chain reaction applied to male and female genomic DNA. All tested genes appeared to be X-specific and not present on the Y chromosome. Assuming that all genes located on these scaffolds should be localized to the ACA X chromosome, we more than doubled the number of known X-linked genes in ACA, from 106 to 250. While demonstrating that the gene content of chromosome X in ACA and GGA15 is largely conserved, we nevertheless showed that numerous interchromosomal rearrangements had occurred since the splitting of the chicken and anole evolutionary lineages. The presence of many ACA X-specific genes localized to distinct contigs indicates that the ACA Y chromosome should be highly degenerated, having lost a large amount of its original gene content during evolution. The identification of novel genes linked to the X chromosome and absent on the Y chromosome in the model lizard species contributes to ongoing research as to the evolution of sex determination in reptiles and provides important information for future comparative and functional genomics.