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The reconstruction of the evolutionary history of sex determination in squamate reptiles (lizards and snakes) is complicated by missing data in many lineages, erroneous reports, and often questionable inferences on state homology. Therefore, despite the large effort, the reconstruction of the ancestral sex determination in squamate reptiles is still controversial. With the hope to shed light on this problem, we aspired to identify the sex chromosome gene content in Dibamus deharvengi, the representative of the family Dibamidae, the putative sister clade to all other squamates. Our analyses revealed XX/XY sex-determination system in D. deharvengi: the X chromosome contains genes with homologues scattered across chicken chromosomes 8, 12, 13, 18, 30, and 33, and the Y chromosome seems to largely degenerate. To the best of our knowledge, this combination has never been reported to form sex chromosomes in any amniote lineage. It suggests that the sex chromosomes can represent an apomorphy of a clade including D. deharvengi. Our findings cover an important gap in the knowledge of sex determination in reptiles and further support multiple independent origins of sex chromosomes in this group.

Geckos (Gekkota), the species-rich clade of reptiles with more than 2200 currently recognized species, demonstrate a remarkable variability in diploid chromosome numbers (2n = 16–48) and mode of sex determination. However, only a small fraction of gekkotan species have been studied with cytogenetic methods. Here, we applied both conventional (karyotype reconstruction and C-banding) and molecular (fluorescence in situ hybridization with probes for rDNA loci and telomeric repeats) cytogenetic analyses in seven species of geckos, namely Blaesodactylusboivini, Chondrodactyluslaevigatus, Gekkobadenii, Gekko cf. lionotum, Hemidactylussahgali, Homopholiswahlbergii (Gekkonidae) and Ptyodactylustogoensis (Phyllodactylidae), in order to provide further insights into the evolution of karyotypes in geckos. Our analysis revealed the presence of interstitial telomeric repeats in four species, but we were not able to conclude if they are remnants of previous chromosome rearrangements or were formed by an accumulation of telomeric-like satellite motifs. Even though sex chromosomes were previously identified in several species from the genera Hemidactylus and Gekko by cytogenetic and/or genomic methods, they were not detected by us in any examined species. Our examined species either have poorly differentiated sex chromosomes or, possibly, environmental sex determination. Future studies should explore the effect of temperature and conduct genome-wide analyses in order to identify the mode of sex determination in these species.

Scincoidea, the reptilian clade that includes girdled lizards, night lizards, plated lizards and skinks, are considered as a lineage with diversity in sex-determining systems. Recently, the hypothesis on the variability in sex determination in skinks and even more the absence of sex chromosomes in some of them has been rivalling. Homologous, evolutionary stable XX/XY sex chromosomes were documented to be widespread across skinks. However, sex determination in the other scincoidean families is highly understudied. ZZ/ZW sex chromosomes have been identified only in night lizards and a single species of plated lizards. It seems that although there are different sex chromosome systems among scincoidean lineages, they share one common trait: they are mostly poorly differentiated and often undetectable by cytogenetic methods. Here, we report one of the exceptions, demonstrating for the first time ZZ/ZW sex chromosomes in the plated lizard Zonosaurus madagascariensis. Its sex chromosomes are morphologically similar, but the W is clearly detectable by comparative genomic hybridization (CGH), suggesting that the Z and W chromosomes highly differ in sequence content. Our findings confirm the presence of female heterogamety in plated lizards and provides novel insights to expand our understanding of sex chromosome evolution in scincoidean lizards.

The recent discovery of two independently evolved XX/XY sex determination systems in the snake genera Python and Boa sparked a new drive to study the evolution of sex chromosomes in poorly studied lineages of snakes, where female heterogamety was previously assumed. Therefore, we examined seven species from the genera Eryx, Cylindrophis, Python, and Tropidophis by conventional and molecular cytogenetic methods. Despite the fact that these species have similar karyotypes in terms of chromosome number and morphology, we detected variability in the distribution of heterochromatin, telomeric repeats, and rDNA loci. Heterochromatic blocks were mainly detected in the centromeric regions in all species, although accumulations were detected in pericentromeric and telomeric regions in a few macrochromosomes in several of the studied species. All species show the expected topology of telomeric repeats at the edge of all chromosomes, with the exception of Eryx muelleri, where additional accumulations were detected in the centromeres of three pairs of macrochromosomes. The rDNA loci accumulate in one pair of microchromosomes in all Eryx species and in Cylindrophis ruffus, in one macrochromosome pair in Tropidophis melanurus and in two pairs of microchromosomes in Python regius. Sex-specific differences were not detected, suggesting that these species likely have homomorphic, poorly differentiated sex chromosomes.

Organisms evolved various mechanisms to cope with the differences in the gene copy numbers between sexes caused by degeneration of Y and W sex chromosomes. Complete dosage compensation or at least expression balance between sexes was reported predominantly in XX/XY, but rarely in ZZ/ZW systems. However, this often-reported pattern is based on comparisons of lineages where sex chromosomes evolved from non-homologous genomic regions, potentially differing in sensitivity to differences in gene copy numbers. Here we document that two reptilian lineages (XX/XY iguanas and ZZ/ZW softshell turtles), which independently co-opted the same ancestral genomic region for the function of sex chromosomes, evolved different gene dose regulatory mechanisms. The independent co-option of the same genomic region for the role of sex chromosome as in the iguanas and the softshell turtles offers a great opportunity for testing evolutionary scenarios on the sex chromosome evolution under the explicit control for the genomic background and for gene identity. We showed that the parallel loss of functional genes from the Y chromosome of the green anole and the W chromosome of the Florida softshell turtle led to different dosage compensation mechanisms. Our approach controlling for genetic background thus does not support that the variability in the regulation of the gene dose differences is a consequence of ancestral autosomal gene content.

Triggers and biological processes controlling male or female gonadal differentiation vary in vertebrates, with sex determination (SD) governed by environmental factors or simple to complex genetic mechanisms that evolved repeatedly and independently in various groups. Here, we review sex evolution across major clades of vertebrates with information on SD, sexual development and reproductive modes. We offer an up-to-date review of divergence times, species diversity, genomic resources, genome size, occurrence and nature of polyploids, SD systems, sex chromosomes, SD genes, dosage compensation and sex-biased gene expression. Advances in sequencing technologies now enable us to study the evolution of SD at broader evolutionary scales, and we now hope to pursue asexomicsintegrative research initiative across vertebrates. The vertebratesexomecomprises interdisciplinary and integrated information on sexual differentiation, development and reproduction at all biological levels, from genomes, transcriptomes and proteomes, to the organs involved in sexual and sex-specific processes, including gonads, secondary sex organs and those with transcriptional sex-bias. Thesexomealso includes ontogenetic and behavioural aspects of sexual differentiation, including malfunction and impairment of SD, sexual differentiation and fertility. Starting from data generated by high-throughput approaches, we encourage others to contribute expertise to building understanding of thesexomesof many key vertebrate species.This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)’.

The lizards of the species-rich clade Scincoidea including cordylids, gerrhosaurids, skinks, and xantusiids, show an almost cosmopolitan geographical distribution and a remarkable ecological and morphological divergence. However, previous studies revealed limited variability in cytogenetic traits. The sex determination mode was revealed only in a handful of gerrhosaurid, skink, and xantusiid species, which demonstrated either ZZ/ZW or XX/XY sex chromosomes. In this study, we explored the karyotypes of six species of skinks, two species of cordylids, and one gerrhosaurid. We applied conventional and molecular cytogenetic methods, including C-banding, fluorescence in situ hybridization with probes specific for telomeric motifs and rDNA loci, and comparative genomic hybridization. The diploid chromosome numbers are rather conserved among these species, but the chromosome morphology, the presence of interstitial telomeric sequences, and the topology of rDNA loci vary significantly. Notably, XX/XY sex chromosomes were identified only in Tiliqua scincoides, where, in contrast to the X chromosome, the Y chromosome lacks accumulations of rDNA loci. We confirm that within the lizards of the scincoidean clade, sex chromosomes remained in a generally poor stage of differentiation.

Sex chromosomes are a great example of a convergent evolution at the genomic level, having evolved dozens of times just within amniotes. An intriguing question is whether this repeated evolution was random, or whether some ancestral syntenic blocks have significantly higher chance to be co-opted for the role of sex chromosomes owing to their gene content related to gonad development. Here, we summarize current knowledge on the evolutionary history of sex determination and sex chromosomes in amniotes and evaluate the hypothesis of non-random emergence of sex chromosomes. The current data on the origin of sex chromosomes in amniotes suggest that their evolution is indeed non-random. However, this non-random pattern is not very strong, and many syntenic blocks representing putatively independently evolved sex chromosomes are unique. Still, repeatedly co-opted chromosomes are an excellent model system, as independent co-option of the same genomic region for the role of sex chromosome offers a great opportunity for testing evolutionary scenarios on the sex chromosome evolution under the explicit control for the genomic background and gene identity. Future studies should use these systems more to explore the convergent/divergent evolution of sex chromosomes. This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)’.

Differentiation of sex chromosomes is thought to have evolved with cessation of recombination and subsequent loss of genes from the degenerated partner (Y and W) of sex chromosomes, which in turn leads to imbalance of gene dosage between sexes. Based on work with traditional model species, theory suggests that unequal gene copy numbers lead to the evolution of mechanisms to counter this imbalance. Dosage compensation, or at least achieving dosage balance in expression of sex-linked genes between sexes, has largely been documented in lineages with male heterogamety (XX/XY sex determination), while ZZ/ZW systems are assumed to be usually associated with the lack of chromosome-wide gene dose regulatory mechanisms. Here, we document that although the pygopodid geckos evolved male heterogamety with a degenerated Y chromosome 32–72 Ma, one species in particular, Burton's legless lizard (Lialis burtonis), does not possess dosage balance in the expression of genes in its X-specific region. We summarize studies on gene dose regulatory mechanisms in animals and conclude that there is in them no significant dichotomy between male and female heterogamety. We speculate that gene dose regulatory mechanisms are likely to be related to the general mechanisms of sex determination instead of type of heterogamety.This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)’.

Amniotes possess variability in sex determination, ranging from environmental sex determination to genotypic sex determination with differentiated sex chromosomes. Differentiated sex chromosomes have emerged independently several times. Their noteworthy convergent characteristic is the evolutionary stability, documented among amniotes in mammals, birds, and some lineages of lizards, snakes and turtles. Combining the analysis of multiple partial transcriptomes with the comparison of copy gene numbers between male and female genomes, we uncovered partial gene content of the highly differentiated ZZ/ZW sex chromosomes in the gecko genus Paroedura. The differentiated ZZ/ZW sex chromosomes of these geckos share genes with the part of the chicken chromosome 4 homologous with the XX/XY sex chromosomes of viviparous mammals and the ZZ/ZW sex chromosomes of lacertid lizards, as well as with the chicken chromosome 15, homologous with the XX/XY sex chromosomes of iguanas and ZZ/ZW sex chromosomes of softshell turtles. Along with other analogous cases, this finding reinforces the observation that particular chromosomes are repeatedly coopted for the function of sex chromosomes in amniotes. Notably, according to the phylogenetic distribution, the subclade of the genus Paroedura represents a rare case of the reversal of the for a considerable evolutionary time highly differentiated ZZ/ZW sex chromosomes back to poorly differentiated state.

Amniotes possess variability in sex determination, from environmental sex determination (ESD), where no sex chromosomes are present, to genotypic sex determination (GSD) with highly differentiated sex chromosomes. Some evolutionary scenarios postulate high stability of differentiated sex chromosomes and rare transitions from GSD to ESD. However, sex chromosome turnovers and two independent transitions from highly differentiated ZZ/ZW sex chromosomes to ESD were previously reported in the lacertid lizards. Here, we examined the homology of sex chromosomes in the wide phylogenetic spectrum of lacertids and their outgroups by comparing gene copy numbers between sexes in genes previously found to be Z-specific in some lacertids. Our current sampling covers 45 species from 26 genera including lineages supposed to possess a derived sex determining systems. We found that all tested lacertids share homologous differentiated ZZ/ZW sex chromosomes, which were present already in their common ancestor living around 85 million years ago. These differentiated sex chromosomes are not present in amphisbaenians and teiid lizards, the close relatives of lacertids. Our study demonstrates how inaccuracies in data can influence the outcome of phylogenetic reconstructions of evolution of sex determination, in this case they overestimated the number of shifts from GSD to ESD and the rate in turnovers of sex chromosomes.

An experimental approach using monoclonal anti-5-methylcytosine antibodies and indirect immunofluorescence was elaborated for detecting 5-methylcytosine-rich chromosome regions in reptilian chromosomes. This technique was applied to conventionally prepared mitotic metaphases of 2 turtle species and 12 squamate species from 8 families. The hypermethylation patterns were compared with C-banding patterns obtained by conventional banding techniques. The hypermethylated DNA sequences are species-specific and are located in constitutive heterochromatin. They are highly reproducible and often found in centromeric, pericentromeric, and interstitial positions of the chromosomes. Heterochromatic regions in differentiated sex chromosomes are particularly hypermethylated.

Amniotes possess astonishing variability in sex determination ranging from environmental sex determination (ESD) to genotypic sex determination (GSD) with highly differentiated sex chromosomes. Geckos are one of the few amniote groups with substantial variability in sex determination. What makes them special in this respect? We hypothesized that the extraordinary variability of sex determination in geckos can be explained by two alternatives: 1) unusual lability of sex determination, predicting that the current GSD systems were recently formed and are prone to turnovers; and 2) independent transitions from the ancestral ESD to later stable GSD, which assumes that geckos possessed ancestrally ESD, but once sex chromosomes emerged, they remain stable in the long term. Here, based on genomic data, we document that the differentiated ZZ/ZW sex chromosomes evolved within carphodactylid geckos independently from other gekkotan lineages and remained stable in the genera Nephrurus, Underwoodisaurus, and Saltuarius for at least 15 Myr and potentially up to 45 Myr. These results together with evidence for the stability of sex chromosomes in other gekkotan lineages support more our second hypothesis suggesting that geckos do not dramatically differ from the evolutionary transitions in sex determination observed in the majority of the amniote lineages.

The voles of the Microtus thomasi/M. atticus species complex demonstrate a remarkable variability in diploid chromosomal number (2n = 38–44 chromosomes) and sex chromosome morphology. In the current study, we examined by in situ hybridization the topology of four satellite DNA motifs (Msat-160, Mth-Alu900, Mth-Alu2.2, TTAGGG telomeric sequences) and two transposons (LINE, SINE) on the karyotypes of nine chromosome races (i.e., populations with unique cytogenetic traits) of Microtus thomasi, and two chromosomal races of M. atticus. According to the topology of the repetitive DNA motifs, we were able to identify six types of biarmed chromosomes formed from either Robertsonian or/and tandem fusions. In addition, we identified 14 X chromosome variants and 12 Y chromosome variants, and we were able to reconstruct their evolutionary relations, caused mainly by distinct mechanisms of amplification of repetitive DNA elements, including the telomeric sequences. Our study used the model of the Microtus thomasi/M. atticus species complex to explore how repetitive centromeric content can alter from chromosomal rearrangements and can shape the morphology of sex chromosomes, resulting in extensive inter-species cytogenetic variability.

Embryos, juveniles, and even adults of many bird species lack pronounced external sexually dimorphic characteristics. Accurate identification of sex is crucial for research (e.g., developmental, population, and evolutionary studies), management of wildlife species, and captive breeding programmes for both conservation and poultry. An accurate molecular sexing method applicable across the entire bird radiation is theoretically possible thanks to the long-term stability of their ZZ/ZW sex chromosomes, but current methods are not applicable in a wide range of bird lineages. Here, we developed a novel molecular sexing method based on the comparison of gene copy number variation by quantitative real-time PCR (qPCR) in conserved Z-specific genes (CHRNA6, DDX4, LPAR1, TMEM161B, VPS13A), i.e. genes linked to Z but absent from W chromosomes. We tested the method across three paleognath and 70 neognath species covering the avian phylogeny. In addition, we designed primers for four Z-specific genes (DOCK8, FUT10, PIGG and PSD3) for qPCR-based molecular sexing in three paleognath species. We have demonstrated that the genes DOCK8, FUT10, PIGG and PSD3 can identify sex in paleognath birds and the genes CHRNA6, DDX4, TMEM161B, and VPS13A can reveal sex in neognath birds. The gene LPAR1 can be used to accurately identify sex in both paleognath and neognath species. Along with outlining a novel method of practical importance for molecular sexing in birds, our study also documents in detail the conservation of sex chromosomes across the avian phylogeny.

Differentiated sex chromosomes are believed to be evolutionarily stable, and their emergence was suggested to lead to a remarkable increase in the diversification rate and in disparity in such groups as birds, mammals and snakes. On the other hand, poorly differentiated sex chromosomes are considered to be prone to turnovers. With around 1.700 currently known species formingc. 15% of reptile species diversity, skinks (family Scincidae) are a very diverse group of squamates known for their large ecological and morphological variability. Skinks generally have poorly differentiated and cytogenetically hardly distinguishable sex chromosomes and their sex determination was suggested to be highly variable. Here, we determined X-linked genes in the common sandfish (Scincus scincus) and demonstrate that skinks have shared the same homologous XX/XY sex chromosomes across their wide phylogenetic spectrum for at least 85 million years, approaching the age of the highly differentiated ZZ/ZW sex chromosomes of birds and advanced snakes. Skinks thus demonstrate that even poorly differentiated sex chromosomes can be evolutionarily stable and that large diversity can emerge even in groups with poorly differentiated sex chromosomes. The conservation of sex chromosomes across skinks allows us to introduce the first molecular sexing method widely applicable in this group.

Geckos demonstrate a remarkable variability in sex determination systems, but our limited knowledge prohibits accurate conclusions on the evolution of sex determination in this group. Eyelid geckos (Eublepharidae) are of particular interest, as they encompass species with both environmental and genotypic sex determination. We identified for the first time the X-specific gene content in the Yucat&aacute, n banded gecko, Coleonyx elegans, possessing X1X1X2X2/X1X2Y multiple sex chromosomes by comparative genome coverage analysis between sexes. The X-specific gene content of Coleonyx elegans was revealed to be partially homologous to genomic regions linked to the chicken autosomes 1, 6 and 11. A qPCR-based test was applied to validate a subset of X-specific genes by comparing the difference in gene copy numbers between sexes, and to explore the homology of sex chromosomes across eleven eublepharid, two phyllodactylid and one sphaerodactylid species. Homologous sex chromosomes are shared between Coleonyx elegans and Coleonyx mitratus, two species diverged approximately 34 million years ago, but not with other tested species. As far as we know, the X-specific gene content of Coleonyx elegans / Coleonyx mitratus was never involved in the sex chromosomes of other gecko lineages, indicating that the sex chromosomes in this clade of eublepharid geckos evolved independently.

Telomeres are nucleoprotein complexes protecting chromosome ends in most eukaryotic organisms. In addition to chromosome ends, telomeric-like motifs can be accumulated in centromeric, pericentromeric and intermediate (i.e., between centromeres and telomeres) positions as so-called interstitial telomeric repeats (ITRs). We mapped the distribution of (TTAGGG)n repeats in the karyotypes of 30 species from nine families of turtles using fluorescence in situ hybridization. All examined species showed the expected terminal topology of telomeric motifs at the edges of chromosomes. We detected ITRs in only five species from three families. Combining our and literature data, we inferred seven independent origins of ITRs among turtles. ITRs occurred in turtles in centromeric positions, often in several chromosomal pairs, in a given species. Their distribution does not correspond directly to interchromosomal rearrangements. Our findings support that centromeres and non-recombining parts of sex chromosomes are very dynamic genomic regions, even in turtles, a group generally thought to be slowly evolving. However, in contrast to squamate reptiles (lizards and snakes), where ITRs were found in more than half of the examined species, and birds, the presence of ITRs is generally rare in turtles, which agrees with the expected low rates of chromosomal rearrangements and rather slow karyotype evolution in this group.

Dragon lizards (Squamata: Agamidae) comprise about 520 species in six subfamilies distributed across Asia, Australasia and Africa. Only five species are known to have sex chromosomes. All of them possess ZZ/ZW sex chromosomes, which are microchromosomes in four species from the subfamily Amphibolurinae, but much larger in Phrynocephalus vlangalii from the subfamily Agaminae. In most previous studies of these sex chromosomes, the focus has been on Australian species from the subfamily Amphibolurinae, but only the sex chromosomes of the Australian central bearded dragon (Pogona vitticeps) are well-characterized cytogenetically. To determine the level of synteny of the sex chromosomes of P. vitticeps across agamid subfamilies, we performed cross-species two-colour FISH using two bacterial artificial chromosome (BAC) clones from the pseudo-autosomal regions of P. vitticeps. We mapped these two BACs across representative species from all six subfamilies as well as two species of chameleons, the sister group to agamids. We found that one of these BAC sequences is conserved in macrochromosomes and the other in microchromosomes across the agamid lineages. However, within the Amphibolurinae, there is evidence of multiple chromosomal rearrangements with one of the BACs mapping to the second-largest chromosome pair and to the microchromosomes in multiple species including the sex chromosomes of P. vitticeps. Intriguingly, no hybridization signal was observed in chameleons for either of these BACs, suggesting a likely agamid origin of these sequences. Our study shows lineage-specific evolution of sequences/syntenic blocks and successive rearrangements and reveals a complex history of sequences leading to their association with important biological processes such as the evolution of sex chromosomes and sex determination.

Chameleons are well-known, highly distinctive lizards characterized by unique morphological and physiological traits, but their karyotypes and sex determination system have remained poorly studied. We studied karyotypes in six species of Madagascan chameleons of the genus Furcifer by classical (conventional stain, C-banding) and molecular (comparative genomic hybridization, in situ hybridization with rDNA, microsatellite, and telomeric sequences) cytogenetic approaches. In contrast to most sauropsid lineages, the chameleons of the genus Furcifer show chromosomal variability even among closely related species, with diploid chromosome numbers varying from 2n = 22 to 2n = 28. We identified female heterogamety with cytogenetically distinct Z and W sex chromosomes in all studied species. Notably, multiple neo-sex chromosomes in the form Z1Z1Z2Z2/Z1Z2W were uncovered in four species of the genus (F. bifidus, F. verrucosus, F. willsii, and previously studied F. pardalis). Phylogenetic distribution and morphology of sex chromosomes suggest that multiple sex chromosomes, which are generally very rare among vertebrates with female heterogamety, possibly evolved several times within the genus Furcifer. Although acrodontan lizards (chameleons and dragon lizards) demonstrate otherwise notable variability in sex determination, it seems that female heterogamety with differentiated sex chromosomes remained stable in the chameleons of the genus Furcifer for about 30 million years.

Once believed to be restricted only to endotherms (mammals and birds), several poikilothermic amniote lineages have recently been documented to possess long-term evolutionary stability in their sex chromosomes. However, many important lineages were not included in these tests. Previously, based on molecular evidence, we documented the homology of well-differentiated sex chromosomes among seven families of iguanas (Pleurodonta), with basilisks (Corytophanidae) being the only exception, as the tested genes linked to X, but missing on the Y chromosome, in other iguanas were autosomal or pseudoautosomal in basilisks. In this study, we test the homology of sex chromosomes in the remaining, previously unstudied iguana families (Hoplocercidae, Leiosauridae, Liolaemidae, Polychrotidae) and in the basilisk genus Corytophanes. Our results show that 12 currently recognized families of iguanas share X-specific gene content conserved from the common ancestor living in the Cretaceous period. However, the results in the genus Corytophanes indicate the loss of the ancestral differentiated sex chromosomes from the ancestor of basilisks. Our new data further confirm the extensive stability of sex chromosomes in iguanas, thus enabling molecular sexing based on the comparison of the number of X-specific genes by quantitative PCR (qPCR) in all but one family of this widely diversified clade.

Homologous and differentiated ZZ/ZW sex chromosomes (or derived multiple neo-sex chromosomes) were often described in caenophidian snakes, but sex chromosomes were unknown until recently in non-caenophidian snakes. Previous studies revealed that two species of boas (Boa imperator, B. constrictor) and one species of python (Python bivittatus) independently evolved XX/XY sex chromosomes. In addition, heteromorphic ZZ/ZW sex chromosomes were recently revealed in the Madagascar boa (Acrantophis sp. cf. dumerili) and putatively also in the blind snake Myriopholis macrorhyncha. Since the evolution of sex chromosomes in non-caenophidian snakes seems to be more complex than previously thought, we examined ten species of pythons and boas representing the families Boidae, Calabariidae, Candoiidae, Charinidae, Pythonidae, and Sanziniidae by conventional and molecular cytogenetic methods, aiming to reveal their sex chromosomes. Our results show that all examined species do not possess sex-specific differences in their genomes detectable by the applied cytogenetic methods, indicating the presence of poorly differentiated sex chromosomes or even the absence of sex chromosomes. Interestingly, fluorescence in situ hybridization with telomeric repeats revealed extensive distribution of interstitial telomeric repeats in eight species, which are likely a consequence of intra-chromosomal rearrangements.

Chameleons are well-known, highly distinctive lizards characterized by unique morphological and physiological traits, but their karyotypes and sex determination system have remained poorly studied. We studied karyotypes in six species of Madagascan chameleons of the genus Furcifer by classical (conventional stain, C-banding) and molecular (comparative genomic hybridization, in situ hybridization with rDNA, microsatellite, and telomeric sequences) cytogenetic approaches. In contrast to most sauropsid lineages, the chameleons of the genus Furcifer show chromosomal variability even among closely related species, with diploid chromosome numbers varying from 2n = 22 to 2n = 28. We identified female heterogamety with cytogenetically distinct Z and W sex chromosomes in all studied species. Notably, multiple neo-sex chromosomes in the form Z1Z1Z2Z2/Z1Z2W were uncovered in four species of the genus (F. bifidus, F. verrucosus, F. willsii, and previously studied F. pardalis). Phylogenetic distribution and morphology of sex chromosomes suggest that multiple sex chromosomes, which are generally very rare among vertebrates with female heterogamety, possibly evolved several times within the genus Furcifer. Although acrodontan lizards (chameleons and dragon lizards) demonstrate otherwise notable variability in sex determination, it seems that female heterogamety with differentiated sex chromosomes remained stable in the chameleons of the genus Furcifer for about 30 million years.

The recent advances in DNA sequencing technology are enabling a rapid increase in the number of genomes being sequenced. However, many fundamental questions in genome biology remain unanswered, because sequence data alone is unable to provide insight into how the genome is organised into chromosomes, the position and interaction of those chromosomes in the cell, and how chromosomes and their interactions with each other change in response to environmental stimuli or over time. The intimate relationship between DNA sequence and chromosome structure and function highlights the need to integrate genomic and cytogenetic data to more comprehensively understand the role genome architecture plays in genome plasticity. We propose adoption of the term ‘chromosomics’ as an approach encompassing genome sequencing, cytogenetics and cell biology, and present examples of where chromosomics has already led to novel discoveries, such as the sex-determining gene in eutherian mammals. More importantly, we look to the future and the questions that could be answered as we enter into the chromosomics revolution, such as the role of chromosome rearrangements in speciation and the role more rapidly evolving regions of the genome, like centromeres, play in genome plasticity. However, for chromosomics to reach its full potential, we need to address several challenges, particularly the training of a new generation of cytogeneticists, and the commitment to a closer union among the research areas of genomics, cytogenetics, cell biology and bioinformatics. Overcoming these challenges will lead to ground-breaking discoveries in understanding genome evolution and function.

Turtles demonstrate variability in sex determination and, hence, constitute an excellent model for the evolution of sex chromosomes. Notably, the sex determination of the freshwater turtles from the family Chelidae, a species-rich group with wide geographical distribution in the southern hemisphere, is still poorly explored. Here we documented the presence of an XX/XY sex determination system in seven species of the Australasian chelid genera Chelodina, Emydura, and Elseya by conventional (karyogram reconstruction, C-banding) and molecular cytogenetic methods (comparative genome hybridization, in situ hybridization with probes specific for GATA microsatellite motif, the rDNA loci, and the telomeric repeats). The sex chromosomes are microchromosomes in all examined species of the genus Chelodina. In contrast, the sex chromosomes are the 4th largest pair of macrochromosomes in the genera Emydura and Elseya. Their X chromosomes are submetacentric, while their Y chromosomes are metacentric. The chelid Y chromosomes contain a substantial male-specific genomic region with an accumulation of the GATA microsatellite motif, and occasionally, of the rDNA loci and telomeric repeats. Despite morphological differences between sex chromosomes, we conclude that male heterogamety was likely already present in the common ancestor of Chelodina, Emydura and Elseya in the Mesozoic period.

Sex determination in varanids, Gila monsters, beaded lizards, and other anguimorphan lizards is still poorly understood. Sex chromosomes were reported only in a few species based solely on cytogenetics, which precluded assessment of their homology. We uncovered Z-chromosome-specific genes in varanids from their transcriptomes. Comparison of differences in gene copy numbers between sexes across anguimorphan lizards and outgroups revealed that homologous differentiated ZZ/ZW sex chromosomes are present in Gila monsters, beaded lizards, alligator lizards, and a wide phylogenetic spectrum of varanids. However, these sex chromosomes are not homologous to those known in other amniotes. We conclude that differentiated sex chromosomes were already present in the common ancestor of Anguimorpha living in the early Cretaceous or even in the Jurassic Period, 115-180 Ma, placing anguimorphan sex chromosomes among the oldest known in vertebrates. The analysis of transcriptomes of Komodo dragon (Varanus komodoensis) showed that the expression levels of genes linked to anguimorphan sex chromosomes are not balanced between sexes. Besides expanding our knowledge on vertebrate sex chromosome evolution, our study has important practical relevance for breeding and ecological studies. We introduce the first, widely applicable technique of molecular sexing in varanids, Gila monsters, and beaded lizards, where reliable determination of sex based on external morphology is dubious even in adults.

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.

Among amniotes, squamate reptiles are especially variable in their mechanisms of sex determination; however, based largely on cytogenetic data, some lineages possess highly evolutionary stable sex chromosomes. The still very limited knowledge of the genetic content of squamate sex chromosomes precludes a reliable reconstruction of the evolutionary history of sex determination in this group and consequently in all amniotes. Female heterogamety with a degenerated W chromosome typifies the lizards of the family Lacertidae, the widely distributed Old World clade including several hundreds of species. From the liver transcriptome of the lacertid Takydromus sexlineatus female, we selected candidates for Z-specific genes as the loci lacking single-nucleotide polymorphisms. We validated the candidate genes through the comparison of the copy numbers in the female and male genomes of T. sexlineatus and another lacertid species, Lacerta agilis, by quantitative PCR that also proved to be a reliable technique for the molecular sexing of the studied species. We suggest that this novel approach is effective for the detection of Z-specific and X-specific genes in lineages with degenerated W, respectively Y chromosomes. The analyzed gene content of the Z chromosome revealed that lacertid sex chromosomes are not homologous with those of other reptiles including birds, but instead the genes have orthologs in the X-conserved region shared by viviparous mammals. It is possible that this part of the vertebrate genome was independently co-opted for the function of sex chromosomes in viviparous mammals and lacertids because of its content of genes involved in gonad differentiation.

Geckos in general show extensive variability in sex determining systems, but only male heterogamety has been demonstrated in the members of their legless family Pygopodidae. In the pioneering study published more than 45 years ago, multiple sex chromosomes of the type X1X1X2X2/X1X2Y were described in Burton's legless lizard (Lialisburtonis) based on conventional cytogenetic techniques. We conducted cytogenetic analyses including comparative genomic hybridization and fluorescence in situ hybridization (FISH) with selected cytogenetic markers in this species and the previously cytogenetically unstudied Papua snake lizard (Lialis jicari) to better understand the nature of these sex chromosomes and their differentiation. Both species possess male heterogamety with an X1X1X2X2/X1X2Y sex chromosome system; however, the Y and one of the X chromosomes are not small chromosomes as previously reported in L. burtonis, but the largest macrochromosomal pair in the karyotype. The Y chromosomes in both species have large heterochromatic blocks with extensive accumulations of GATA and AC microsatellite motifs. FISH with telomeric probe revealed an exclusively terminal position of telomeric sequences in L. jicari (2n = 42 chromosomes in females), but extensive interstitial signals, potentially remnants of chromosomal fusions, in L.burtonis (2n = 34 in females). Our study shows that even largely differentiated and heteromorphic sex chromosomes might be misidentified by conventional cytogenetic analyses and that the application of more sensitive cytogenetic techniques for the identification of sex chromosomes is beneficial even in the classical examples of multiple sex chromosomes.

Frequent independent origins of environmental sex determination (ESD) are assumed within amniotes. However, the phylogenetic distribution of sex-determining modes suggests that ESD is likely very ancient and may be homologous across ESD groups. Sex chromosomes are demonstrated to be old and stable in endothermic (mammals and birds) and many ectothermic (non-avian reptiles) lineages, but they are mostly non-homologous between individual amniote lineages. The phylogenetic pattern may be explained by ancestral ESD with multiple transitions to later evolutionary stable genotypic sex determination. It is pointed out here that amniote ESD shares several key aspects with sequential hermaphroditism of fishes such as a lack of sex differences in genomes, biased population sex ratios, and potentially also molecular mechanism related to general stress responses. Here, it is speculated that ESD evolves via a heterochronic shift of the sensitive period of sex change from the adult to the embryonic stage in a hermaphroditic amniote ancestor. Also see the video abstract here https://youtu.be/q2mjtlCefu4.

Despite their long history with the basal split dating back to the Eocene, all species of monitor lizards (family Varanidae) studied so far share the same chromosome number of 2n = 40. However, there are differences in the morphology of the macrochromosome pairs 5–8. Further, sex determination, which revealed ZZ/ZW sex microchromosomes, was studied only in a few varanid species and only with techniques that did not test their homology. The aim of this study was to (i) test if cryptic interchromosomal rearrangements of larger chromosomal blocks occurred during the karyotype evolution of this group, (ii) contribute to the reconstruction of the varanid ancestral karyotype, and (iii) test homology of sex chromosomes among varanids. We investigated these issues by hybridizing flow sorted chromosome paints from Varanus komodoensis to metaphases of nine species of monitor lizards. The results show that differences in the morphology of the chromosome pairs 5–8 can be attributed to intrachromosomal rearrangements, which led to transitions between acrocentric and metacentric chromosomes in both directions. We also documented the first case of spontaneous triploidy among varanids in Varanus albigularis. The triploid individual was fully grown, which demonstrates that polyploidization is compatible with life in this lineage. We found that the W chromosome differs between species in size and heterochromatin content. The varanid Z chromosome is clearly conserved in all the analyzed species. Varanids, in addition to iguanas, caenophidian snakes, and lacertid lizards, are another squamate group with highly conserved sex chromosomes over a long evolutionary time.

The reconstruction of the evolutionary dynamics of karyotypes and sex determining systems in squamate reptiles is precluded by the lack of data in many groups including most chameleons (Squamata: Acrodonta: Chamaeleonidae). We performed cytogenetic analysis in 16 species of chameleons from 8 genera covering the phylogenetic diversity of the family and also phylogenetic reconstruction of karyotype evolution in this group. In comparison to other squamates, chameleons demonstrate rather variable karyotypes, differing in chromosome number, morphology and presence of interstitial telomeric signal (ITS). On the other hand, the location of rDNA is quite conserved among chameleon species. Phylogenetic analysis combining our new results and previously published data tentatively suggests that the ancestral chromosome number for chameleons is 2n = 36, which is the same as assumed for other lineages of the clade Iguania, i.e., agamids and iguanas. In general, we observed a tendency for the reduction of chromosome number during the evolution of chameleons, however, in Rieppeleon brevicaudatus, we uncovered a chromosome number of 2n = 62, very unusual among squamates, originating from a number of chromosome splits. Despite the presence of the highly differentiated ZZ/ZW sex chromosomes in the genus Furcifer, we did not detect any unequivocal sexual differences in the karyotypes of any other studied species of chameleons tested using differential staining and comparative genomic hybridization, suggesting that sex chromosomes in most chameleons are only poorly differentiated.

The caenophidian (assigned also as “advanced”) snakes are traditionally viewed as a group of reptiles with a limited karyotypic variation and stable ZZ/ZW sex chromosomes. The W chromosomes of the caenophidian snakes are heterochromatic, and pioneering studies demonstrated that they are rich in repetitive elements. However, a comparative study of the evolutionary dynamics of the repetitive content of the W chromosome across the whole lineage is missing. Using molecular-cytogenetic techniques, we explored the distribution of four repetitive motifs (microsatellites GATA, GACA, AG and telomeric-like sequences), which are frequently accumulated in differentiated sex chromosomes in vertebrates, in the genomes of 13 species of the caenophidian snakes covering a wide phylogenetic spectrum of the lineage. The results demonstrate a striking variability in the morphology and the repetitive content of the W chromosomes even between closely-related species, which is in contrast to the homology and long-term stability of the gene content of the caenophidian Z chromosome. We uncovered that the tested microsatellite motifs are accumulated on the degenerated, heterochromatic W chromosomes in all tested species of the caenophidian snakes with the exception of the Javan file snake representing a basal clade. On the other hand, the presence of the accumulation of the telomeric-like sequences on the caenophidian W chromosome is evolutionary much less stable. Moreover, we demonstrated that large accumulations of telomeric-like motifs on the W chromosome contribute to sexual differences in the number of copies of the telomeric and telomeric-like repeats estimated by quantitative PCR, which might be confusing and incorrectly interpreted as sexual differences in telomere length.

The reconstruction of the evolutionary dynamics of karyotypes and sex determining systems in squamate reptiles is precluded by the lack of data in many groups including most chameleons (Squamata: Acrodonta: Chamaeleonidae). We performed cytogenetic analysis in 16 species of chameleons from 8 genera covering the phylogenetic diversity of the family and also phylogenetic reconstruction of karyotype evolution in this group. In comparison to other squamates, chameleons demonstrate rather variable karyotypes, differing in chromosome number, morphology and presence of interstitial telomeric signal (ITS). On the other hand, the location of rDNA is quite conserved among chameleon species. Phylogenetic analysis combining our new results and previously published data tentatively suggests that the ancestral chromosome number for chameleons is 2n = 36, which is the same as assumed for other lineages of the clade Iguania, i.e., agamids and iguanas. In general, we observed a tendency for the reduction of chromosome number during the evolution of chameleons, however, in Rieppeleon brevicaudatus, we uncovered a chromosome number of 2n = 62, very unusual among squamates, originating from a number of chromosome splits. Despite the presence of the highly differentiated ZZ/ZW sex chromosomes in the genus Furcifer, we did not detect any unequivocal sexual differences in the karyotypes of any other studied species of chameleons tested using differential staining and comparative genomic hybridization, suggesting that sex chromosomes in most chameleons are only poorly differentiated.

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.

Most pleurodont lizard families (anoles, iguanas and their relatives), with the exception of the basilisks and casquehead lizards (family Corytophanidae), share homologous XX/XY sex chromosomes, syntenic with chicken chromosome 15. Here, we used a suite of methods (i.e. RADseq, RNAseq and qPCR) to identify corytophanid sex chromosomes for the first time. We reveal that all examined corytophanid species have partially degenerated XX/XY sex chromosomes, syntenic with chicken chromosome 17. Transcriptomic analyses showed that the expression of X-linked genes in the corytophanid, Basiliscus vittatus, is not balanced between the sexes, which is rather exceptional under male heterogamety, and unlike the dosage-balanced sex chromosomes in other well-studied XX/XY systems, including the green anole, Anolis carolinensis . Corytophanid sex chromosomes may represent a rare example of a turnover away from stable, differentiated sex chromosomes. However, because of poor phylogenetic resolution among pleurodont families, we cannot reject the alternative hypothesis that corytophanid sex chromosomes evolved independently from an unknown ancestral system.

For a long time, turtles of the family Geoemydidae have been considered exceptional because representatives of this family were thought to possess a wide variety of sex determination systems. In the present study, we cytogenetically studiedGeoemyda spengleriandG. japonicaand re-examined the putative presence of sex chromosomes inPangshura smithii. Karyotypes were examined by assessing the occurrence of constitutive heterochromatin, by comparative genome hybridization andin situhybridization with repetitive motifs, which are often accumulated on differentiated sex chromosomes in reptiles. We found similar karyotypes, similar distributions of constitutive heterochromatin and a similar topology of tested repetitive motifs for all three species. We did not detect differentiated sex chromosomes in any of the species. ForP. smithii, a ZZ/ZW sex determination system, with differentiated sex chromosomes, was described more than 40 years ago, but this finding has never been re-examined and was cited in all reviews of sex determination in reptiles. Here, we show that the identification of sex chromosomes in the original report was based on the erroneous pairing of chromosomes in the karyogram, causing over decades an error cascade regarding the inferences derived from the putative existence of female heterogamety in geoemydid turtles.

The Komodo dragon (Varanus komodoensis) is the largest lizard in the world. Surprisingly, it has not yet been cytogenetically examined. Here, we present the very first description of its karyotype and sex chromosomes. The karyotype consists of 2n = 40 chromosomes, 16 macrochromosomes and 24 microchromosomes. Although the chromosome number is constant for all species of monitor lizards (family Varanidae) with the currently reported karyotype, variability in the morphology of the macrochromosomes has been previously documented within the group. We uncovered highly differentiated ZZ/ZW sex microchromosomes with a heterochromatic W chromosome in the Komodo dragon. Sex chromosomes have so far only been described in a few species of varanids including V. varius, the sister species to Komodo dragon, whose W chromosome is notably larger than that of the Komodo dragon. Accumulations of several microsatellite sequences in the W chromosome have recently been detected in 3 species of monitor lizards; however, these accumulations are absent from the W chromosome of the Komodo dragon. In conclusion, although varanids are rather conservative in karyotypes, their W chromosomes exhibit substantial variability at the sequence level, adding further evidence that degenerated sex chromosomes may represent the most dynamic genome part.

Highly differentiated heteromorphic ZZ/ZW sex chromosomes with a heterochromatic W are a basic principle among advanced snakes of the lineage Colubroidea, while other snake lineages generally lack these characteristics. For the first time, we cytogenetically examined the dragonsnake, Xenodermus javanicus, a member of the family Xenodermatidae, which is phylogenetically nested between snake lineages with and without differentiated sex chromosomes. Although most snakes have a karyotype with a stable chromosomal number of 2n = 36, the dragonsnake has an unusual, derived karyotype with 2n = 32 chromosomes. We found that heteromorphic ZZ/ZW sex chromosomes with a heterochromatic W are present in the dragonsnake, which suggests that the emergence of a highly differentiated W sex chromosome within snakes predates the split of Xenodermatidae and the clade including families Pareatidae, Viperidae, Homalopsidae, Lamprophiidae, Elapidae, and Colubridae. Although accumulations of interstitial telomeric sequences have not been previously reported in snakes, by using FISH with a telomeric probe we discovered them in 6 pairs of autosomes as well as in the W sex chromosome of the dragonsnake. Similarly to advanced snakes, the sex chromosomes of the dragonsnake have a significant accumulation of repeats containing a (GATA)n sequence. The results facilitate the dating of the differentiation of sex chromosomes within snakes back to the split between Xenodermatidae and other advanced snakes, i.e. around 40-75 mya.

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.

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.

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.

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.

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.

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.