Article

The Discovery of XY Sex Chromosomes in a Boa and Python

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Abstract

For over 50 years, biologists have accepted that all extant snakes share the same ZW sex chromosomes derived from a common ancestor [1–3], with different species exhibiting sex chromosomes at varying stages of differentiation. Accordingly, snakes have been a well-studied model for sex chromosome evolution in animals [1, 4]. A review of the literature, however, reveals no compelling support that boas and pythons possess ZW sex chromosomes [2, 5]. Furthermore, phylogenetic patterns of facultative parthenogenesis in snakes and a sex-linked color mutation in the ball python (Python regius) are best explained by boas and pythons possessing an XY sex chromosome system [6, 7]. Here we demonstrate that a boa (Boa imperator) and python (Python bivittatus) indeed possess XY sex chromosomes, based on the discovery of male-specific genetic markers in both species. We use these markers, along with transcriptomic and genomic data, to identify distinct sex chromosomes in boas and pythons, demonstrating that XY systems evolved independently in each lineage. This discovery highlights the dynamic evolution of vertebrate sex chromosomes and further enhances the value of snakes as a model for studying sex chromosome evolution.

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... Namely, like other reptiles, turtle sex chromosomes vary in the degree of heteromorphism (Figure 2), and some of them carry the genes of the nucleolar organizing region (NOR) [28]. Just like turtles, both lizards and snakes co-opted various ancestral autosomes as sex chromosomes independently [56][57][58]. Unlike turtles, however, the repeated evolution of sex chromosomes in squamates (lizards and snakes) occurred not only as transitions from TSD but also as transitions between male and female heterogamety, a process that has not been detected in turtles. For instance, the recent discovery of convergent XY sex chromosomes in pythons and boas refuted the long-held notion that they share the ZW system previously thought to be ubiquitous and homologous across snakes [57]. ...
... Unlike turtles, however, the repeated evolution of sex chromosomes in squamates (lizards and snakes) occurred not only as transitions from TSD but also as transitions between male and female heterogamety, a process that has not been detected in turtles. For instance, the recent discovery of convergent XY sex chromosomes in pythons and boas refuted the long-held notion that they share the ZW system previously thought to be ubiquitous and homologous across snakes [57]. Also, no case of multiple sex chromosomes is known in turtles, whereas examples exist in squamates [3,58]. ...
... Additionally, a comparison of turtles, crocodilians, squamates, birds and mammals, detected three genes (Dmrt1, Ctnnb1, Ar) with faster amino acid substitution rates when they are Z-linked (Dmrt1 and Ctnnb1, Z-linked in birds and snakes) but not when they are X-linked (Ar, X-linked in mammals), compared to when they are autosomal [22]. Similar to turtles, faster-Z is observed in chicken and other birds [107] whose sex chromosomes are homologous to Staurotypus X, and faster-X is supported in A. carolinensis [104] whose sex chromosomes are homologous to Apalone's Z. On the contrary, the report of faster-Z in snakes [56] needs revisiting, since it is based on data from Boa whose purported ZW chromosomes are now known to be autosomes [57]. The major roadblock to study molecular evolution of sex chromosomes in turtles and other reptiles should be alleviated with the publication of additional chelonian genomes with mapped sex chromosomes. ...
Article
Full-text available
Sex chromosome evolution remains an evolutionary puzzle despite its importance in understanding sexual development and genome evolution. The seemingly random distribution of sex-determining systems in reptiles offers a unique opportunity to study sex chromosome evolution not afforded by mammals or birds. These reptilian systems derive from multiple transitions in sex determination, some independent, some convergent, that lead to the birth and death of sex chromosomes in various lineages. Here we focus on turtles, an emerging model group with growing genomic resources. We review karyotypic changes that accompanied the evolution of chromosomal systems of genotypic sex determination (GSD) in chelonians from systems under the control of environmental temperature (TSD). These transitions gave rise to 31 GSD species identified thus far (out of 101 turtles with known sex determination), 27 with a characterized sex chromosome system (13 of those karyotypically). These sex chromosomes are varied in terms of the ancestral autosome they co-opted and thus in their homology, as well as in their size (some are macro-, some are micro-chromosomes), heterogamety (some are XX/XY, some ZZ/ZW), dimorphism (some are virtually homomorphic, some heteromorphic with larger-X, larger W, or smaller-Y), age (the oldest system could be ~195 My old and the youngest < 25 My old). Combined, all data indicate that turtles follow some tenets of classic theoretical models of sex chromosome evolution while countering others. Finally, although the study of dosage compensation and molecular divergence of turtle sex chromosomes has lagged behind research on other aspects of their evolution, this gap is rapidly decreasing with the acceleration of ongoing research and growing genomic resources in this group.
... Two henophidian snakes (primitive snakes), a python (Python bivittatus) and boid snake (Boa imperator) are thought to have a XX/XY sex-determination system in accordance with male-specific single nucleotide polymorphisms (SNPs) [1]. In the majority of snakes, the sexdetermination system is the ZZ/ZW type as determined by karyotype studies, identification of sex-specific quantitative trait loci, and analysis of insertions/deletions of DNA fragments [1][2][3][4][5][6][7][8][9][10]. ...
... Two henophidian snakes (primitive snakes), a python (Python bivittatus) and boid snake (Boa imperator) are thought to have a XX/XY sex-determination system in accordance with male-specific single nucleotide polymorphisms (SNPs) [1]. In the majority of snakes, the sexdetermination system is the ZZ/ZW type as determined by karyotype studies, identification of sex-specific quantitative trait loci, and analysis of insertions/deletions of DNA fragments [1][2][3][4][5][6][7][8][9][10]. The Z chromosomes probably share the same genetic constitution across caenophidian snakes (advanced snakes) without large morphological modifications, which indicates that the Z chromosome originated from the same ancestral pair of autosomes [2][3][4][5][6][7][8][9]11,12]. ...
... With these considerations in mind, we propose the following hypotheses: (1) based on their heteromorphism, the Siamese cobra sex chromosomes contain a large number of sex-specific loci or gametologs; (2) these loci can be used to examine evolutionary strata among snake lineages; (3) the Siamese cobra contains sex-specific regions autapomorphic in the snake lineage; and (4) under the hypothesis of an amniote super-sex chromosome, sex-specific loci in the Siamese cobra might show partial homology with sex chromosomes shared with other amniotes. With the rapid development of next-generation sequencing (NGS) technologies, a number of novel approaches have been developed to investigate sex-specific DNA loci [1,[50][51][52][53]. In the present study, we addressed the four hypotheses using Diversity Arrays Technology (DArTseq™), which employs a combination of genome complexity reduction and NGS to allow identification of sex-specific regions using high-throughput multiple loci [51,52]. ...
Article
Elucidation of the process of sex chromosome differentiation is necessary to understand the dynamics of evolutionary mechanisms in organisms. The W sex chromosome of the Siamese cobra (Naja kaouthia) contains a large number of repeats and shares amniote sex chromosomal linkages. Diversity Arrays Technology provides an effective approach to identify sex-specific loci that are epoch-making, to understand the dynamics of molecular transitions between the Z and W sex chromosomes in a snake lineage. From a total of 543 sex-specific loci, 90 showed partial homology with sex chromosomes of several amniotes and 89 loci were homologous to trans-posable elements. Two loci were confirmed as W-specific nucleotides after PCR amplification. These loci might result from a sex chromosome differentiation process and involve putative sex-determination regions in the Siamese cobra. Sex-specific loci shared linkage homologies among amniote sex chromosomes, supporting an ancestral super-sex chromosome.
... The degree of divergence between X and Y or Z and W sex chromosomes is independently observed across amniote lineages with remarkable variation [39]. Synonymous substitution rates of XY (or ZW) gametologous genes, which are homologous genes located in the nonrecombining region of differentiated sex chromosomes, can be used to trace the evolutionary history of sex chromosomes [14,16,32,[40][41][42][43][44][45][46][47][48]. ...
... By contrast, when a new sex-determining gene arises on the existing sex chromosome (termed 'homologous turnover') [61,73], turnover between XY and ZW determination systems on the same chromosome arises in the course of evolution. Caenophidian snakes share the same ancestral ZW chromosomes, with varying degrees of W degeneration; however, pythons have an XY system, leading to the emergence of a new sexdetermining locus, although only a few specimens have been examined [41,47]. The question of how and why these turnovers arise remains unclear but is assumed to result from sexual conflict, genetic drift, and mutation accumulation [73][74][75][76][77][78][79][80]. ...
... Female heterogamety (ZZ/ZW system) occurs in caenophidian snakes [20,[23][24][25]27,28,41,[44][45][46], whereas for noncaenophidian snakes (i) facultative parthenogenesis in pythons and boas leads to exclusively female progeny [191][192][193][194], (ii) inheritance of a color mutation in the ball python (Python regius) indicates a XX/XY sex determination [195], and (iii) a recent study suggests that a transition from ZW to XY may have occurred for Boa imperator and Python bivittatus based on male-specific genetic markers as well as transcriptomic and genomic data [41]. A report of heteromorphic ZZ/ZW sex chromosomes in the Madagascar boa based on conventional cytogenetics was recently confirmed by molecular cytogenetic methods in Acrantophis sp. ...
Article
Full-text available
Heteromorphic sex chromosomes, particularly the ZZ/ZW sex chromosome system of birds and some reptiles, undergo evolutionary dynamics distinct from those of autosomes. The W sex chromosome is a unique karyological member of this heteromorphic pair, which has been extensively studied in snakes to explore the origin, evolution, and genetic diversity of amniote sex chromosomes. The snake W sex chromosome offers a fascinating model system to elucidate ancestral trajectories that have resulted in genetic divergence of amniote sex chromosomes. Although the principal mechanism driving evolution of the amniote sex chromosome remains obscure, an emerging hypothesis, supported by studies of W sex chromosomes of squamate reptiles and snakes, suggests that sex chromosomes share varied genomic blocks across several amniote lineages. This implies the possible split of an ancestral super-sex chromosome via chromosomal rearrangements. We review the major findings pertaining to sex chromosomal profiles in amniotes and discuss the evolution of an ancestral super-sex chromosome by collating recent evidence sourced mainly from the snake W sex chromosome analysis. We highlight the role of repeat-mediated sex chromosome conformation and present a genomic landscape of snake Z and W chromosomes, which reveals the relative abundance of major repeats, and identifies the expansion of certain transposable elements. The latest revolution in chromosomics, i.e., complete telomere-to-telomere assembly, offers mechanistic insights into the evolutionary origin of sex chromosomes.
... Poorly differentiated sex chromosomes homologous with differentiated ZZ/ZW sex chromosomes of the caenophidian snakes were reported in the past in boas and pythons [4,39,40]. Recent investigations showed a more complex history of snake sex chromosomes with XX/XY sex chromosomes evolving independently in Neotropical boas and pythons and ZZ/ZW sex chromosomes in Malagasy boas [41,42]. ...
... Indeed, the same repetitions accumulated independently on clearly non-homologous sex chromosomes several times, as well as on autosomes [16,[77][78][79]. Moreover, differentiated sex chromosomes with degenerated W is a clear apomorphy of caenophidian snakes [52], and the other snakes have generally poorly differentiated sex chromosomes without notable accumulations of repeats [80] and at least in some cases non-homologous to those of caenophidian sex chromosomes [41]. ...
... Since ACA-Lg F is syntenic with the part of sex chromosomes in Boa spp. [41], we added 'missing genes in chicken' as an additional syntenic block to our analysis for accuracy. The exclusion of this artificial block from the analyses does not change the significance of the results. ...
Article
Sex chromosomes are a great example of a convergent evolution at thegenomic 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 sexchromosome evolution: empirical and theoretical insights with a focus onvertebrates (Part II)’.
... Several species of turtles and lizards have male heterogamety (XY males and XX females). In contrast, some turtles and lizards and all snakes have female heterogamety (ZZ males and ZW females) except boids (Gamble et al. 2017). Some turtles and lizards have no noticeable heteromorphic sex chromosomes. ...
... These chromosomes may contain sex-specific genes or sequences that can reveal their sex determination mode (XY or ZW) (Gamble et al. 2017;Gamble et al. 2015;Hill et al. 2018;Lambert et al. 2016;Nielsen et al. 2020;Ogata et al. 2018). ...
... Restriction site-associated DNA sequencing, such as RADseq and DArTseq, has been proposed as a method for developing sex-linked markers i.e. sex-linked loci in taxa with homomorphic sex chromosomes Lambert et al. 2016). This has successfully been used to develop sex-linked markers and to infer the sexdetermining mode for several amphibian, squamate and fish species (Brelsford et al. 2016;Gamble et al. 2017;Lambert et al. 2016;Nielsen et al. 2020;Palaiokostas et al. 2013;Utsunomia et al. 2017;Wilson et al. 2014). Diversity Arrays Technology, the developer of DArTseq™, has developed a methylation-sensitive DArTseq (DArTseqMet) that uses two different restriction enzyme isoschizomers (one CpG methylation-sensitive and the other not) to identify sex-specific markers. ...
Thesis
Full-text available
Sex in vertebrates can be determined by genes on specific chromosomes (genotypic sex determination, GSD), by environmental factors such as temperature (e.g., temperature-dependent sex determination, TSD) or by the combination of both. Reptiles, especially lizards, exhibit the greatest diversity in sex-determining mechanisms (SDM) among amniotes. This diversity suggests frequent transitions between sex-determining modes. Among the lizards, the Agamidae family (commonly known as agamids) is known to show diverse modes of reproduction as well as modes of sex determination mechanism, possibly even among congenerics. This family contains more than 500 species across Africa, Asia and Australia under six subfamilies and includes species in which both temperature and genes interact to determine sex. The multiple modes of sex determination in agamid lizards have evolved many times, suggesting multiple and independent evolutions of sex-determining modes within the animal kingdom. Most of the studies of sex determination in agamids have focused on the species under one sub-family from one continent, i.e., Australian species of the sub-family Amphibolurinae. Only little is known about the agamids from other subfamilies. As a result, the diversity and evolution of sex determination mechanisms remain unidentified among a significant group of agamid lizards, yet these have the potential to uncover novel sex determination mechanisms, including sex chromosomes. Filling in this knowledge gap would provide insight into the overall understanding of the phylogenetic relationship and evolutionary history of sex determination mechanisms. In my thesis, I examined aspects of evolution and ecology of sex determination across the family Agamidae with a combination of incubation experiments, cytogenetics and genomics. The studies conducted under this thesis have expanded the knowledge of labile sex-determination mechanisms in reptiles, keeping agamid lizards as models. The studies on the sex determination in this group were previously concentrated mainly on the Australian clade of Amphibolurinae (subfamily), while this research went beyond this boundary and initiated an investigation including species from other subfamilies. The study identified the sex determination mode and sex chromosomes in a threatened Australian agamid species, reported variation of sex-determination modes between populations and closely related species and explored chromosomal synteny among the subfamilies of the agamid lizards using P. vitticeps sex-chromosome BACs. The results presented here are still preliminary, and to fully understand the process of sex determination and sex chromosome evolution in the studied species, additional studies using advanced molecular cytogenetic and genomic techniques are needed, with particular priority to gain access to samples where the gonads have been dissected.
... Determining heterogametic sex by identifying sex chromosomes is not easy if the sex chromosomes are cryptic. These chromosomes may contain sex-specific genes or sequences that can reveal their sex-determination mode (XY or ZW) [8][9][10][11][12][13][14]. This may be true even in DNA 2021, 1 50 the case of species with temperature-dependent sex determination (TSD) and, therefore, lacking sex-specific chromosomes or where species show diverse sex ratio patterns complicated by gene-temperature interactions. ...
... In these cases, the development of sex-linked markers can provide valuable insights by enabling genetic sex to be identified and correlated with phenotypic sex. Such markers have been successfully identified in several reptilian taxa [8,11,12,[14][15][16][17], where they have been used to identify sex-determination modes and cases of sex reversal [18,19]. ...
... Restriction site-associated DNA sequencing, such as RADseq and DArTseq, has been proposed as a method for developing sex-linked markers, i.e., sex-linked loci in taxa with homomorphic sex chromosomes [8,10]. This has successfully been used to develop sex-linked markers and infer the sex-determining mode for several amphibian, squamate, and fish species [10,11,14,[28][29][30][31][32][33]. Diversity Arrays Technology (Bruce, Australia), the developer of DArTseq™, has developed a methylation-sensitive DArTseq (DArTseqMet) that uses two different restriction enzyme isoschizomers (one of them CpG methylation-sensitive) to identify sex-specific markers. ...
Article
Full-text available
Sex-determination mechanisms and sex chromosomes are known to vary among reptile species and, in a few celebrated examples, within populations of the same species. The oriental garden lizard, Calotes versicolor, is one of the most intriguing species in this regard, exhibiting evidence of multiple sex-determination modes within a single species. One possible explanation for this unusual distribution is that in C. versicolor, different modes of sex determination are confined to a particular population or a species within a cryptic species complex. Here, we report on a population genetic analysis using SNP data from a methylation-sensitive DArT sequencing analysis and mitochondrial DNA data obtained from samples collected from six locations: three from Bangladesh and three from Thailand. Our aim was to determine whether C. versicolor is best described as a single species with multiple lineages or as multiple species, as well as if its sex-determination mechanisms vary within or between species. We present evidence that the latter possibility is the case and that C. versicolor comprises a complex of cryptic species. We also identify sex-linked markers within these species and use them to identify modes of sex determination. Overall, our results suggest that different sex-determination modes have evolved among closely related species and within populations of Agamid lizards.
... Most of the Serpentes lineages have 2n = 36 chromosomes (16 macro + 20 microchromosomes), which is considered a plesiomorphic feature for the suborder [25][26][27][28]. However, Boidae differs from other families by presenting the greatest chromosomal diversity among the Booidea families, with 2n ranging from 36 to 44 chromosomes, including an undifferentiated XY sex chromosome system [24,25,29]. Robertsonian and non-Robertsonian rearrangements, including fission events [26], have often been proposed to explain this karyotype diversity. ...
... Meanwhile, the ancestral state 2n = 36 is retained along the diversification and split among Eunectes, Epicrates, and Chilabothrus genera in the Paleogene/Neogene (Figure 4). Interestingly, the undifferentiated putative XY sex chromosomes of Boa constrictor [29,46] remained conserved in species with 2n = 36 (putative pair 4 in Eunectes and Epicrates) and with 2n = 40 (pair 5 in Corallus hortulana). On the other hand, in Corallus caninus (2n = 44), the putative XY chromosomes of Boa constrictor underwent fission events, giving rise to the smallest acrocentric chromosomes of the complement, pairs 11 and 12 (Figures 3 and 5). ...
... This chromosomal homology of the putative XY (4th pair), across Boidae lineages, suggests that other genera of the family may also share an undifferentiated XY system of sex chromosomes, even though Corallus caninus presents the putative Boa XY as four small acrocentric chromosomes (pairs 11 and 12) ( Figure 5). Furthermore, consecutive parthenogenetic births in Eunectes, Epicrates and Chilabothrus, producing exclusively females, also support an XY system shared among Boidae species [29,[47][48][49]. In Corallus caninus, the putative Boa XY is homologous to pairs 11 and 12 due to a fission event, however, it is uncertain whether a functional XY sex chromosome system exists in this lineage or if a multiple XY system evolved through this fission event. ...
Article
Full-text available
The Boidae family is an ancient group of snakes widely distributed across the Neotropical region, where several biogeographic events contributed towards shaping their evolution and diversification. Most species of this family have a diploid number composed of 2n = 36; however, among Booidea families, the Boidae stands out by presenting the greatest chromosomal diversity, with 2n ranging between 36 and 44 chromosomes and an undifferentiated XY sex chromosome system. Here, we applied a comparative chromosome analysis using cross-species chromosome paintings in five species representing four Boidae genera, to decipher the evolutionary dynamics of some chromosomes in these Neotropical snakes. Our study included all diploid numbers (2n = 36, 40, and 44) known for this family and our comparative chromosomal mappings point to a strong evolutionary relationship among the genera Boa, Corallus, Eunectes, and Epicrates. The results also allowed us to propose the cytogenomic diversification that had occurred in this family: a process mediated by centric fissions, including fission events of the putative and undifferentiated XY sex chromosome system in the 2n = 44 karyotype, which is critical in solving the puzzle of the karyotype evolution of boid snakes.
... Finally, they concluded that "identifying the molecular underpinnings of the henophidian sex determination system will require further investigation [e.g., identification of sex specific markers via RAD-seq]" [305]. Such RAD-seq experiments were published by another group one year later [306]. In their study these authors looked for the presence of an excess of female or male-specific markers in a boa (Boa imperator), a python (Python bivittatus) and a rattlesnake (Crotalus atrox), used as a caenophidian snake control [306]. ...
... Such RAD-seq experiments were published by another group one year later [306]. In their study these authors looked for the presence of an excess of female or male-specific markers in a boa (Boa imperator), a python (Python bivittatus) and a rattlesnake (Crotalus atrox), used as a caenophidian snake control [306]. As expected for a ZW species, they found an excess of female-specific RAD markers for the rattlesnake. ...
... On the contrary, the primers of the python male-specific RAD markers confirmed by PCR failed to amplify in a sex-specific manner in two related species: the ball python (P. regius) and the carpet python (Morelia spilota) [306]. The extent of this XY system among other python species is therefore unknown. ...
Article
Full-text available
Among tetrapods, the well differentiated heteromorphic sex chromosomes of birds and mammals have been highly investigated and their master sex-determining (MSD) gene, Dmrt1 and SRY, respectively, have been identified. The homomorphic sex chromosomes of reptiles have been the least studied, but the gap with birds and mammals has begun to fill. This review describes our current knowledge of reptilian sex chromosomes at the cytogenetic and molecular level. Most of it arose recently from various studies comparing male to female gene content. This includes restriction site-associated DNA sequencing (RAD-Seq) experiments in several male and female samples, RNA sequencing and identification of Z- or X-linked genes by male/female comparative transcriptome coverage, and male/female transcriptomic or transcriptome/genome substraction approaches allowing the identification of Y- or W-linked transcripts. A few putative master sex-determining (MSD) genes have been proposed, but none has been demonstrated yet. Lastly, future directions in the field of reptilian sex chromosomes and their MSD gene studies are considered.
... Durante muitos anos, biólogos acreditavam que todas as espécies de serpentes compartilhavam o mesmo cromossomo sexual ZW. Entretanto, segundo Vicoso et al. (2013), serpentes da espécie Python não possuem cromossomos sexuais ZW, e de acordo com Gamble et al. (2017), essa espécie possui cromossomos XY. ...
... As amostras foram encaminhadas para análise no Laboratório de Genética da Conservação -GECON, onde o DNA foi extraído a partir de um protocolo utilizando NaCl (Bardakci e Skibinski, 1994) e quantificado a partir da espectrofotometria pelo Nanodrop 2000c UV-Vis THERMO SCIENCE®, sendo as amostras diluídas em tampão TE (Tris−HCl 10 mM, EDTA 1 mM) para 5 ng/μL. As reações de PCR foram realizadas com a utilização de 1 µM dos primers M3F e M3R, seguindo o protocolo de Gamble et al. (2017). Amplificações via Reação em Cadeia da Polimerase (PCR) de um trecho específico do cromossomo Y foram conduzidas utilizando o par de primers M3F: 5'-GCTGATTATTCCAGCGGCAT-3' e M3-R: 5'-GGATTCCAAGTCCACAACGG-3', descritos por Gamble et al. (2017). ...
... As reações de PCR foram realizadas com a utilização de 1 µM dos primers M3F e M3R, seguindo o protocolo de Gamble et al. (2017). Amplificações via Reação em Cadeia da Polimerase (PCR) de um trecho específico do cromossomo Y foram conduzidas utilizando o par de primers M3F: 5'-GCTGATTATTCCAGCGGCAT-3' e M3-R: 5'-GGATTCCAAGTCCACAACGG-3', descritos por Gamble et al. (2017). As condições de amplificação e as concentrações de reagentes na PCR também foram de acordo com Gamble et al. (2017), seguido de avaliação dos produtos da PCR em eletroforese, utilizando gel de agarose 1% e marcador de peso molecular de 100 pares de bases (PB). ...
... For decades, it was speculated that all snakes might have homologous ZZ/ZW sex chromosomes, which are heteromorphic and highly differentiated in caenophidian snakes but homomorphic and poorly differentiated in henophidian and scolecophidian snakes [20,21,[24][25][26]. However, this view was proved incorrect when two non-homologous XX/XY systems were detected in a python (Python bivittatus; Pythonidae) and two species of boa (Boa constrictor, B. imperator; Boidae) by single-nucleotide polymorphism (SNP) analysis of RAD-seq genomic data [27]. Notably, the sex chromosomes of P. bivittatus are partially homologous to ZZ/ZW sex chromosomes of caenophidian snakes, while the sex chromosomes in the two boas are homologous to an autosome of caenophidian snakes. ...
... T. melanurus is a member of the lineage Amerophidia and sister to all other henophidian and caenophidian snakes [2,5]. The ball python P. regius is closely related to P. bivittatus, a species with an XX/XY sex determination system [27], and C. ruffus is a member of the lineage relatively closely related to pythons [2,5]. The sand boas of the genus Eryx are phylogenetically nested between lineages of boas with documented ZZ/ZW (A. sp. ...
... However, we cannot rule out that environmental sex determination might also be present in some henophidian snakes, although it has not yet been reported in any snake [9]. Poorly differentiated sex chromosomes are more prone to turnovers than highly differentiated sex chromosomes [73], which can-together with differences in lineage ages-explain the emerging pattern of the higher variability in sex chromosome systems in snakes from the scolecophidian and henophidian groups compared with the evolutionary stable ZZ/ZW sex chromosomes of caenophidian snakes [14,16,24,27]. Molecular methods such as RAD-seq or whole-genome coverage analyses have been successful in uncovering sex determination systems not only in snakes but also in other squamate lineages with poorly differentiated sex chromosomes [27,[74][75][76], and might offer a way to resolve sex determination systems in scolecophidian and henophidian snakes in the future. ...
Article
Full-text available
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.
... Species with an abundance of male-specific RAD markers have an XX/XY sex chromosome system, and species with an abundance of female-specific RAD markers have a ZZ/ZW system (Gamble et al., 2015;Pan et al., 2016). This method has been used to identify homomorphic sex chromosomes across a wide range of taxa (Fowler & Buonaccorsi, 2016;Gamble et al., 2015;Jeffries et al., 2018;Nielsen, Daza, Pinto, & Gamble, 2019;Pan et al., 2016) and detect important and unexpected transitions among sex chromosomes (Gamble et al., 2017;Nielsen, Banks, Diaz, Trainor, & Gamble, 2018). Such methods, particularly when combined with modern cytogenetics (Deakin et al., 2019), are building a greater foundation on which to study the evolutionary processes governing sex chromosome origins, degeneration and stability. ...
... The 2n = 30 karyotype of A. expectatus is unique among sphaerodactylid geckos (Table S3). It can be derived from a 2n = 36 karyotype of all acrocentric chromosomes, like that of the sphaerodactylid genera Teratoscincus (Manilo, 1993;Zeng et al., 1998) Chicken chromosome 2/Anolis 6 is homologous to the sex-linked chromosome in Python bivittatus (Gamble et al., 2017) and caenophidian snakes (Matsubara et al., 2006;Vicoso, Emerson, Zektser, Mahajan, & Bachtrog, 2013). Having become sex-linked in three independent squamate lineages, this linkage group appears to be a frequent sex chromosome candidate. ...
Article
Current understanding of sex chromosome evolution is largely dependent on species with highly degenerated, heteromorphic sex chromosomes, but by studying species with recently evolved or morphologically indistinct sex chromosomes we can greatly increase our understanding of sex chromosome origins, degeneration, and turnover. Here, we examine sex chromosome evolution and stability in the gecko genus Aristelliger. We used RADseq to identify sex‐specific markers and show that four Aristelliger species, spanning the phylogenetic breadth of the genus, share a conserved ZZ/ZW system syntenic with avian chromosome two. These conserved sex chromosomes contrast with many other gecko sex chromosome systems by showing a degree of stability among a group known for its dynamic sex determining mechanisms. Cytogenetic data from A. expectatus revealed homomorphic sex chromosomes with an accumulation of repetitive elements on the W chromosome. Taken together, the large number of female‐specific A. praesignis RAD markers and the accumulation of repetitive DNA on the A. expectatus W karyotype suggests that the Z and W chromosomes are highly differentiated despite their overall morphological similarity. We discuss this paradoxical situation and suggest that it may, in fact, be common in many animal species.
... Like many of its reptilian relatives, sex determination in the ancestral snake species is thought to have involved temperature and lacked the influence of sex chromosomes. For example, boa can still undergo occasional parthenogenesis and produce viable WW offspring [49], consistent with having one of the most primitive vertebrate sex chromosome pairs hitherto reported. At least three recombination suppression events occurred between Z and W in the ancestors of advanced snakes, leading to the generally degenerated W chromosome that has been observed in the five-pacer viper [22]. ...
... The proto-sex chromosome subsequently underwent structural changes, such as rearrangements, gene degradation, repeat accumulations, and heterochromatinization. However, certain snakes, such as python and boa, may also exhibit an XX/XY system [49]. Two major transitions, which resulted in sex chromosome turnover between these two systems, may have occurred in the Pythonidae and Boidae families [65,83]. ...
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The distinctive biology and unique evolutionary features of snakes make them fascinating model systems to elucidate how genomes evolve and how variation at the genomic level is interlinked with phenotypic-level evolution. Similar to other eukaryotic genomes, large proportions of snake genomes contain repetitive DNA, including transposable elements (TEs) and satellite repeats. The importance of repetitive DNA and its structural and functional role in the snake genome, remain unclear. This review highlights the major types of repeats and their proportions in snake genomes, reflecting the high diversity and composition of snake repeats. We present snakes as an emerging and important model system for the study of repetitive DNA under the impact of sex and microchromosome evolution. We assemble evidence to show that certain repetitive elements in snakes are transcriptionally active and demonstrate highly dynamic lineage-specific patterns as repeat sequences. We hypothesize that particular TEs can trigger different genomic mechanisms that might contribute to driving adaptive evolution in snakes. Finally, we review emerging approaches that may be used to study the expression of repetitive elements in complex genomes, such as snakes. The specific aspects presented here will stimulate further discussion on the role of genomic repeats in shaping snake evolution.
... RAD-seq has also proven powerful in the identification of single nucleotide polymorphisms (SNPs) and genetic sex markers (Brown et al., 2016;Carmichael et al., 2013;Etter, Preston, Bassham, Cresko, & Johnson, 2011;Fowler & Buonaccorsi, 2016;Gamble et al., 2017;Gamble et al., 2015; ...
... RAD-seq has been applied recently in the discovery of sex markers in various species of fish, reptiles and crustaceans (Brown et al., 2016;Carmichael et al., 2013;Fowler & Buonaccorsi, 2016;Gamble et al., 2017;Gamble et al., 2015;Gamble & Zarkower, 2014;Mathers et al., 2015;Palaiokostas, Bekaert, Davie, et al., 2013;Palaiokostas, Bekaert, Khan, et al., 2013;Palaiokostas et al., 2015) and in some cases, these studies have been able to construct linkage maps based on single nucleotide polymorphisms (SNPs) and map a major sex determining locus to certain linkage groups. In our study, we have been able to identify presence-absence markers pertaining to sex only, and so more extensive sequencing of the roach genome would be required to construct linkage maps and further characterise this marker. ...
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Oestrogenic wastewater treatment works (WwTW) effluents discharged into UK rivers have been shown to affect sexual development, including inducing intersex, in wild roach (Rutilus rutilus). This can result in a reduced breeding capability with potential population level impacts. In the absence of a sex probe for roach it has not been possible to confirm whether intersex fish in the wild arise from genetic males or females, or whether sex reversal occurs in the wild, as this condition can be induced experimentally in controlled exposures to WwTW effluents and a steroidal oestrogen. Using restriction site‐associated DNA sequencing (RAD‐seq), we identified a candidate for a genetic sex marker and validated this marker as a sex probe through PCR analyses of samples from wild roach populations from non‐polluted rivers. We also applied the sex marker to samples from roach exposed experimentally to oestrogen and oestrogenic effluents to confirm suspected phenotypic sex reversal from males to females in some treatments, and also that sex‐reversed males are able to breed as females. We then show, unequivocally, that intersex in wild roach populations results from feminisation of males, but find no strong evidence for complete sex reversal in wild roach at river sites contaminated with oestrogens. The discovered marker has utility for studies in roach on chemical effects, wild stock assessments, and reducing the number of fish used where only one sex is required for experimentation. Furthermore, we show that the marker can be applied non‐destructively using a fin clip or skin swab, with animal welfare benefits.
... When the new locus is dominant to the previous sex determining system there is an instant turnover in which chromosome is acting as the sex chromosome (e.g. Boas and Pythons (Gamble et al. 2017)). ...
... In some cases, the emergence of a new sex determining locus leads to a transition between XY and ZW systems, as has occurred in snakes and amphibians. Although most snakes share the same ancestral ZW chromosomes, with varying degrees of W degeneration, multiple pythons were found to have transitioned to an XY system (Augstenová et al. 2018;Gamble et al. 2017). Although one of the new XY systems shares gene content with the ancestral ZW chromosomes, the other new XY does not, ...
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Genomic analysis of many non-model species has uncovered an incredible diversity of sex chromosome systems, making it possible to empirically test the rich body of evolutionary theory that describes each stage of sex chromosome evolution. Classic theory predicts that sex chromosomes originate from a pair of homologous autosomes and recombination between them is suppressed via inversions to resolve sexual conflict. The resulting degradation of the Y chromosome gene content creates the need for dosage compensation in the heterogametic sex. Sex chromosome theory also implies a linear process, starting from sex chromosome origin and progressing to heteromorphism. Despite many convergent genomic patterns exhibited by independently evolved sex chromosome systems, and many case studies supporting these theoretical predictions, emerging data provide numerous interesting exceptions to these long-standing theories, and suggest that the remarkable diversity of sex chromosomes is matched by a similar diversity in their evolution. For example, it is clear that sex chromosome pairs are not always derived from homologous autosomes. Also, both the cause and mechanism of recombination suppression between sex chromosome pairs remain unclear, and it may be that the spread of recombination suppression is a more gradual process than previously thought. It is also clear that dosage compensation can be achieved in many ways, and displays a range of efficacy in different systems. Finally, the remarkable turnover of sex chromosomes in many systems, as well as variation in the rate of sex chromosome divergence, suggest that assumptions about the inevitable linearity of sex chromosome evolution are not always empirically supported, and the drivers of the birth-death cycle of sex chromosome evolution remain to be elucidated. Here, we concentrate on how the diversity in sex chromosomes across taxa highlights an equal diversity in each stage of sex chromosome evolution.
... Among amniotes, snakes are a valuable model system for studying sex chromosome evolution, as male and female heterogamety has evolved independently in different lineages ( fig. 1A; Matsubara et al. 2006Matsubara et al. , 2019Gamble et al. 2017;Augstenová et al. 2018), providing useful comparisons to established XY and ZW model systems. Caenophidian snakes sensu Zaher et al. (2019), including the Acrochordoidea and Colubriformes (colubrids, elapids, viperids, and relatives), possess homologous ZW chromosomes (Rovatsos et al. 2018; fig. ...
... The inferred age range of the older strata is coincident with the ancestral split between Caenophidia (including Colubriformes and Acrochordoidea) and Henophidia (including boas and pythons) roughly 91 Ma . This is particularly interesting because henophidian snakes have evolved independent XY and ZW sex chromosomes multiple times (Gamble et al. 2017;Augstenová et al. 2018), while all caenophidian snakes sampled to date have homologous ZW sex chromosomes (Matsubara et al. 2006;Rovatsos et al. 2015Rovatsos et al. , 2018. The age of the older strata and ubiquitous presence of ZW sex chromosomes together indicate that the formation of ZW sex chromosomes was a major transition in early caenophidian evolution. ...
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Sex chromosomes diverge after the establishment of recombination suppression, resulting in differential sex-linkage of genes involved in genetic sex determination and dimorphic traits. This process produces systems of male or female heterogamety wherein the Y and W chromosomes are only present in one sex and are often highly degenerated. Sex-limited chromosomes (e.g., Y and W) contain valuable information about the evolutionary transition from autosomes to sex chromosomes, yet detailed characterizations of the structure, composition, and gene content of sex-limited chromosomes is lacking for many species. In this study, we characterize the female-specific W chromosome of the prairie rattlesnake (Crotalus viridis) and evaluate how recombination suppression and other processes have shaped sex chromosome evolution in ZW snakes. Our analyses indicate that the rattlesnake W chromosome is over 80% repetitive and that an abundance of GC-rich mdg4 elements has driven an overall high degree of GC-richness despite a lack of recombination. The W chromosome is also highly enriched for repeat sequences derived from endogenous retroviruses and likely acts as a ‘refugium’ for these and other retroelements. We annotated 219 putatively functional W-linked genes across at least two evolutionary strata identified based on estimates of sequence divergence between Z and W gametologs. The youngest of these strata is relatively gene-rich, however gene expression across strata suggests retained gene function amidst a greater degree of degeneration following ancient recombination suppression. Functional annotation of W-linked genes indicates a specialization of the W chromosome for reproductive and developmental function since recombination suppression from the Z chromosome.
... According to Singh (1972), because the fifth pair is biarmed in most of the other colubrids, a simple pericentric inversions can be assumed to explain the different morphology of this pair. The ZZ/ZW sex determination system was supposed to be a plesiomorphic state in snakes (Mengden, 1981;Matsubara et al., 2016), however recent evidences suggest that different sex chromosome systems evolved multiple times, independently, in different snake lineages, including species with either female (ZZ/ZW) or male (XX/XY) heterogamety along with a discrete number of species with undifferentiated sex chromosomes (Gamble at al., 2017;Mezzasalma et al., 2019). This makes the suborder Serpentes, and more in general the whole order Squamata, which also includes various taxa with temperature-dependent sex determination (Gamble, 2010;Gamble et al., 2017;Pallotta et al. 2017;Alam et al., 2018), a unique study system to analyze the evolution and diversification of different mechanisms of sex determination. ...
... The ZZ/ZW sex determination system was supposed to be a plesiomorphic state in snakes (Mengden, 1981;Matsubara et al., 2016), however recent evidences suggest that different sex chromosome systems evolved multiple times, independently, in different snake lineages, including species with either female (ZZ/ZW) or male (XX/XY) heterogamety along with a discrete number of species with undifferentiated sex chromosomes (Gamble at al., 2017;Mezzasalma et al., 2019). This makes the suborder Serpentes, and more in general the whole order Squamata, which also includes various taxa with temperature-dependent sex determination (Gamble, 2010;Gamble et al., 2017;Pallotta et al. 2017;Alam et al., 2018), a unique study system to analyze the evolution and diversification of different mechanisms of sex determination. Nevertheless, in the family Colubridae, the fourth macrochromosome pair is usually composed of the ZW elements: the Z is metacentric and conserved in most species, while the W is often heteromorphic compared to the Z and largely heterochromatic (see e.g., Mengden, 1981;Mezzasalma et al., 2015a;Rovatsos et al., 2015;Matsubara et al., 2016). ...
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The smooth snake Coronella austriaca is a widespread Palearctic colubrid species. The species has been the subject of several molecular and phylogeographic studies which highlighted the occurrence of distinct genetic lineages in different areas of the species distribution, but scarce cytogenetic data are currently available on the species. In this paper we present a molecular and karyological study performed with several banding, staining methods and NOR-FISH on samples of C. austriaca from different geographical areas (Italy and Greece) of the species distribution. The molecular and phylogenetic analysis unambiguously placed the studied samples in different clades with a clear geographical pattern. The karyotype of the two female samples studied was composed of 2n = 36 chromosomes with 16 macro-and 20 microchromosomes and a mix of plesiomorphic and derivate chromosome features. All macrochromo-somes were biarmed with the exception of pair 5 that was telocentric. NORs were detected on a microchromosome pair. In both females, the pair 4 was heteromorphic (and completely heterochromatic after C-banding in the Italian female), representing the first report of a ZZ/ZW sex chromosome system with female heterogamety in C. austriaca. In addition, the W chromosome showed a different morphology between the two female studied (submetacentric and subtelocentric), highlighting the occurrence of a chromosomal diversification among distinct geographical areas of the species distribution and further supporting that the species contains different diverging evolutionary clades.
... Compared to other vertebrate lineages, recent studies in snakes have generated novel and field-advancing results that have significantly contributed to our understanding of FP 8,11,26 ; thus, snakes represent an ideal model system with which to address outstanding questions. Following the publication of its genome 27 , the king cobra, www.nature.com/scientificreports/ ...
... Both measures of relatedness fell between approximately 0.99 and 1 (Fig. 2b, Table 1), which is far above the expectation of 0.5 in pairwise comparisons between a parent and sexually produced progeny. The highly reduced level of heterozygosity, inflated values of pairwise relatedness, and offspring sex (i.e., male being the expected sex of a parthenogen in species with ZZ/ZW sex determination; see 8,11 ), strongly support the conclusion that these offspring are the product of FP, and provide the first evidence of this mode of reproduction in the king cobra. ...
Article
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Facultative parthenogenesis (FP) is widespread in the animal kingdom. In vertebrates it was first described in poultry nearly 70 years ago, and since then reports involving other taxa have increased considerably. In the last two decades, numerous reports of FP have emerged in elasmobranch fishes and squamate reptiles (lizards and snakes), including documentation in wild populations of both clades. When considered in concert with recent evidence of reproductive competence, the accumulating data suggest that the significance of FP in vertebrate evolution has been largely underestimated. Several fundamental questions regarding developmental mechanisms, nonetheless, remain unanswered. Specifically, what is the type of automixis that underlies the production of progeny and how does this impact the genomic diversity of the resulting parthenogens? Here, we addressed these questions through the application of next-generation sequencing to investigate a suspected case of parthenogenesis in a king cobra (Ophiophagus hannah). Our results provide the first evidence of FP in this species, and provide novel evidence that rejects gametic duplication and supports terminal fusion as a mechanism underlying parthenogenesis in snakes. Moreover, we precisely estimated heterozygosity in parthenogenetic offspring and found appreciable retained genetic diversity that suggests that FP in vertebrates has underappreciated evolutionary significance.
... Female-specific sequences were produced by GBS/RAD-seq using the sex-limited occurrence in the Chinese giant salamander (Andrias davidianus) [8]. Sex-specific molecular markers have been found using GBS/RAD-seq approaches via a similar sex-limited occurrence principle in lizards (Anolis carolinensis), skinks, more than 12 gecko species, and boa and python snakes [9][10][11][12][13]. However, there are no effective methods to exclude higher rates of false positives in sex-linked markers from GBS/RAD-seq screening. ...
... However, there are no effective methods to exclude higher rates of false positives in sex-linked markers from GBS/RAD-seq screening. Variation between individuals, rather than between GBS/RADseq results, has been validated by PCR tests on additional individuals in limited reptile species [9,11,12] and more rarely in amphibian species [7,8]. ...
Article
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We used genotyping-by-sequencing (GBS) to identify sex-linked markers in 43 wild-collected spiny frog (Quasipaa boulengeri) adults from a single site. We identified a total of 1049 putatively sex-linked GBS-tags, 98% of which indicated an XX/XY system, and finally confirmed 574 XY-type sex-linked loci. The sex specificity of five markers was further validated by PCR amplification using a large number of additional individuals from 26 populations of this species. A total of 27 sex linkage markers matched with the Dmrt1 gene, showing a conserved role in sex determination and differentiation in different organisms from flies and nematodes to mammals. Chromosome 1, which harbors Dmrt1, was considered as the most likely candidate sex chromosome in anurans. Five sex-linked SNP makers indicated sex reversals, which are sparsely present in wild amphibian populations, in three out of the one-hundred and thirty-three explored individuals. The variety of sex-linked markers identified could be used in population genetics analyses requiring information on individual sex or in investigations aimed at drawing inferences about sex determination and sex chromosome evolution.
... The ability to observe heteromorphic sex chromosomes under a light microscope has led to numerous discoveries in sex chromosome evolution, but as most vertebrate species do not possess heteromorphic sex chromosomes, other technologies are needed to identify sex chromosomes in these species . Thanks to recent advances in sequencing and cytogenetic methods, empiricists are now able to identify and characterize homomorphic sex chromosomes in diverse taxa (Gamble et al. 2015(Gamble et al. , 2017Augstenová et al. 2018;Nielsen et al. 2018Nielsen et al. , 2019aNielsen et al. , 2019bNielsen et al. , 2020Pan et al. 2019Pan et al. , 2021aRovatsos et al. 2019;Sidhom et al. 2020;Keating et al. 2020Keating et al. , 2021. The recent ability to characterize homomorphic sex chromosomes has allowed researchers to test existing hypotheses in new ways, transforming our understanding of sex chromosome evolution (Bull 1983;Ogata et al. 2007;Uno et al. 2008;Blaser et al. 2013;Gamble et al. 2015a;Augstenová et al. 2018;Jeffries et al. 2018;Hundt et al. 2019;Kottler et al. 2020). ...
... We can interpret areas with an abundance of mapped sex-specific RADtags as regions within the nonrecombining region of the sex chromosomes. When analyzed alone, RADseq can identify sex chromosome systems but can say nothing of sex chromosome linkage or the size of the nonrecombining region in the focal taxon (Gamble et al. 2015a(Gamble et al. , 2017Fowler and Buonaccorsi 2016;Hundt et al. 2019;Nielsen et al. 2019aNielsen et al. , 2020. However, when analyzed in conjunction with a reference genome, we can both map these sex-specific RADtags to identify the linkage group and analyze the sequences in this region by calling SNPs from the raw data (Gamble 2016;Pan et al. 2019). ...
Article
Sex determination is a critical element of successful vertebrate development, suggesting that sex chromosome systems might be evolutionarily stable across lineages. For example, mammals and birds have maintained conserved sex chromosome systems over long evolutionary time periods. Other vertebrates, in contrast, have undergone frequent sex chromosome transitions, which is even more amazing considering we still know comparatively little across large swaths of their respective phylogenies. One reptile group in particular, the gecko lizards (infraorder Gekkota), show an exceptional lability with regard to sex chromosome transitions and may possess the majority of transitions within squamates (lizards and snakes). However, detailed genomic and cytogenetic information about sex chromosomes is lacking for most gecko species, leaving large gaps in our understanding of the evolutionary processes at play. To address this, we assembled a chromosome-level genome for a gecko (Sphaerodactylidae: Sphaerodactylus) and used this assembly to search for sex chromosomes among six closely related species using a variety of genomic data, including whole-genome re-sequencing, RADseq, and RNAseq. Previous work has identified XY systems in two species of Sphaerodactylus geckos. We expand upon that work to identify between two and four sex chromosome cis-transitions (XY to a new XY) within the genus. Interestingly, we confirmed two different linkage groups as XY sex chromosome systems that were previously unknown to act as sex chromosomes in tetrapods (syntenic with Gallus chromosome 3 and Gallus chromosomes 18/30/33), further highlighting a unique and fascinating trend that most linkage groups have the potential to act as sex chromosomes in squamates.
... In amniotes, sex chromosomes are highly diverse, ranging from cryptic to highly heteromorphic with XY or ZW systems that originated independently from different autosomal pairs (Ezaz et al. 2009a(Ezaz et al. , 2017Valenzuela and Adams 2011;O'Meally et al. 2012;Srikulnath et al. 2014Srikulnath et al. , 2015Gamble et al. 2014;Singchat et al. 2018;Iannucci et al. 2019). For instance, the chicken Z chromosome (Gallus gallus Z chromosome (GGAZ)) is homologous to the short arm of snake chromosome 2, as also found in many other squamate reptiles, whereas the sex chromosomes of advanced snakes show homology with GGA2 and GGA27 (Matsuda et al. 2005;Matsubara et al. 2006Matsubara et al. , 2012Srikulnath et al. 2009aSrikulnath et al. ,b, 2013Srikulnath et al. , 2014Srikulnath et al. , 2015Alföldi et al. 2011;Pokorná et al. 2011;Ezaz et al. 2013Ezaz et al. , 2017Young et al. 2013;Gamble et al. 2017;Augstenová et al. 2018;Singchat et al. 2018). Genome sequence analyses and cross-species chromosome mapping have, however, revealed that unrelated sex chromosomes share linkage homologies across distantly related taxa, and might involve genomic regions orthologous to squamate reptile chromosome 2 (SR2) and snake W sex chromosome (Ezaz et al. 2017;Singchat et al. 2018;Matsubara et al. 2019). ...
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Sex chromosomes in some amniotes share linkage homologies with distantly related taxa in regions orthologous to squamate reptile chromosome 2 (SR2) and the snake W sex chromosome. Thus, the SR2 and W chromosomes may formerly have been part of a larger ancestral amniote super-sex chromosome. Comparison of various sex chromosomal linkage homologies in Toxicofera with those in other amniotes offers an excellent model to assess key cytological differences, to understand the mechanisms of amniote sex chromosome evolution in each lineage and the existence of an ancestral amniote super-sex chromosome. Chromosome maps of four spe-Chromosome Res https://doi.
... In reptiles, independent turnovers and transitions among sex chromosomes systems (XY and ZW) and sexdetermining mechanisms (TSD and GSD) within closely related species are more common than previously thought, being thus, a widespread feature among non-avian reptiles 1,3,4,54,55 . Unlike most snakes, Boa imperator was reported to have XY homomorphic sex chromosomes 26 , and its sister species Boa constrictor, shares ancestry presenting also an XY system. Therefore, it is plausible that transitions between homomorphic ZW and XY have occurred in the Boidae family without much substantial genotypic innovation (e.g. ...
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Most of snakes exhibit a ZZ/ZW sex chromosome system, with different stages of degeneration. However, undifferentiated sex chromosomes and unique Y sex-linked markers, suggest that an XY system has also evolved in ancestral lineages. Comparative cytogenetic mappings revealed that several genes share ancestry among X, Y and Z chromosomes, implying that XY and ZW may have undergone transitions during serpent’s evolution. In this study, we performed a comparative cytogenetic analysis to identify homologies of sex chromosomes across ancestral (Henophidia) and more recent (Caenophidia) snakes. Our analysis suggests that, despite ~ 85 myr of independent evolution, henophidians and caenophidians retained conserved synteny over much of their genomes. However, our findings allowed us to discover that ancestral and recent lineages of snakes do not share the same sex chromosome and followed distinct pathways for sex chromosomes evolution.
... Using comparative gene mapping (i.e., chromosome mapping by means of a cytogenetic technique) and whole-genome sequencing, genomic convergence has been detected in which unrelated sex chromosomes share syntenies across distantly related taxa (Voss et al., 2001;Matsuda et al., 2005;Matsubara et al., 2006Matsubara et al., , 2012Matsubara et al., , 2019Srikulnath et al., 2009aSrikulnath et al., ,b, 2013Srikulnath et al., , 2014Srikulnath et al., , 2015Alföldi et al., 2011;Pokorná et al., 2011;Ezaz et al., 2013Ezaz et al., , 2017Young et al., 2013;Gamble et al., 2017;Augstenová et al., 2018;Singchat et al., 2018Singchat et al., , 2020aIannucci et al., 2019;Lind et al., 2019;Ahmad et al., 2020). The ACAXspecific region contains genomic content orthologous to genes on GGA15 (Alföldi et al., 2011), which is homologous to the Z chromosome of the Chinese softshell turtle (Pelodiscus sinensis) and the spiny softshell turtle (Apalone spinifera) (Kawagoshi et al., 2009(Kawagoshi et al., , 2014. ...
Article
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The majority of lizards classified in the superfamily Iguanoidea have an XX/XY sex-determination system in which sex-chromosomal linkage shows homology with chicken (Gallus gallus) chromosome 15 (GGA15). However, the genomics of sex chromosomes remain largely unexplored owing to the presence of homomorphic sex chromosomes in majority of the species. Recent advances in high-throughput genome complexity reduction sequencing provide an effective approach to the identification of sex-specific loci with both single-nucleotide polymorphisms (SNPs) and restriction fragment presence/absence (PA), and a better understanding of sex chromosome dynamics in Iguanoidea. In this study, we applied Diversity Arrays Technology (DArTseqTM) in 29 phenotypic sex assignments (14 males and 15 females) of green iguana (Iguana iguana). We confirmed a male heterogametic (XX/XY) sex determination mode in this species, identifying 29 perfectly sex-linked SNP/PA loci and 164 moderately sex-linked SNP/PA loci, providing evidence probably indicative of XY recombination. Three loci from among the perfectly sex-linked SNP/PA loci showed partial homology with several amniote sex chromosomal linkages. The results support the hypothesis of an ancestral super-sex chromosome with overlaps of partial sex-chromosomal linkages. However, only one locus among the moderately sex-linked loci showed homology with GGA15, which suggests that the specific region homologous to GGA15 was located outside the non-recombination region but in close proximity to this region of the sex chromosome in green iguana. Therefore, the location of GGA15 might be further from the putative sex-determination locus in green iguana. This is a paradigm shift in understanding linkages on homomorphic X and Y sex chromosomes. The DArTseq platform provides an easy-to-use strategy for future research on the evolution of sex chromosomes in Iguanoidea, particularly for non-model species with homomorphic or highly cryptic sex chromosomes.
... It is true that some syntenic blocks were co-opted for the role of sex chromosomes across amniotes several times, for example the ortholog of GGAZ in birds, in a turtle lineage, and twice independently in geckos, the ortholog of GGA4p which makes up the main part of the sex chromosomes in viviparous mammals, geckos of the genus Paroedura and lacertid lizards [22], and GGA28 in anguimorphan lizards and monotremes [18]. However, other lineages such as the caenophidian and non-caenophidian snakes, the xantusiid lizard Xantusia henshawi, the pygopodid geckos and skinks [63][64][65][66] have evolved sex chromosomes from syntenic blocks not forming sex chromosomes in other amniotes. ...
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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 forming c. 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.
... Facultative parthenogenesis in caenophidian snakes is characterized by the production of a large number of infertile ova, low viability of offspring, and developmental abnormalities (Schuett et al., 1997;Germano and Smith, 2010;Schuett, 2011, 2016;Reynolds et al., 2012). In contrast, boids and pythonids were recently characterized as possessing an XX/XY chromosome sex determination system (Mallery and Carrillo, 2016;Gamble et al., 2017). Contrasting with caenophidian snakes, facultative parthenogenesis in these groups results in the production of viable normal females (XX) (Booth et al., 2011a(Booth et al., , 2011b(Booth et al., , 2014Kinney et al., 2012;Groot et al., 2014;Booth and Schuett, 2016;Shibata et al., 2017). ...
Article
Here we describe a case of facultative parthenogenesis in a diamondback water snake (Nerodia rhombifer). An adult female N. rhombifer kept in captivity produced four unfertilized ova, one stillborn, and one live neonate in July 2016. The neonates had characteristic abnormalities in morphology and were determined as males, suggestive of a parthenogenetic event. Stillborn and live neonates along with the mother and four unrelated N. rhombifer were genetically screened at twelve microsatellite loci. Results confirmed that the reproductive event represented a case of facultative parthenogenesis. In light of previous reports of facultative parthenogenesis in the genera Nerodia and Thamnophis, these results suggest that this reproductive mode may be a widespread phenomenon in the Natricinae as a whole.
... That is, the chicken Z sex chromosome is homologous to parts of chromosome 2p in most snakes and also in other squamate reptiles, whereas the snake Z sex chromosome is homologous to the chicken chromosomes 2p and 27. These conserved linkage homologies are shared across most snake species studied (Matsuda et al., 2005;Matsubara et al., 2006Matsubara et al., , 2012Srikulnath et al., 2009aSrikulnath et al., ,b, 2013Srikulnath et al., , 2014Srikulnath et al., , 2015Ezaz et al., 2017;Laopichienpong et al., 2017a,b;Tawichasri et al., 2017;Singchat et al., 2018Singchat et al., , 2020, whereas two henophidian snakes (primitive snakes), python (Python bivittatus), and boid (Boa imperator), are thought to have XX/XY sex-determination systems (Gamble et al., 2017). Interestingly, sex chromosomal linkage homologies have also been found between chromosomes in different amniote lineages, and unrelated sex chromosomes share linkage homologies across distantly related groups (Ezaz et al., 2017). ...
Article
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Squamate reptile chromosome 2 (SR2) is thought to be an important remnant of an ancestral amniote super-sex chromosome, but a recent study showed that the Siamese cobra W sex chromosome is also a part of this larger ancestral chromosome. To confirm the existence of an ancestral amniote super-sex chromosome and understand the mechanisms of amniote sex chromosome evolution, chromosome maps of two snake species [Russell's viper: Daboia russelii (DRU) and the common tiger snake: Notechis scutatus (NSC)] were constructed using bacterial artificial chromosomes (BACs) derived from chicken and zebra finch libraries containing amniote sex chromosomal linkages. Sixteen BACs were mapped on the W sex chromosome of DRU and/or NSC, suggesting that these BACs contained a common genomic region shared with the W sex chromosome of these snakes. Two of the sixteen BACs were co-localized to DRU2 and NSC2, corresponding to SR2. Prediction of genomic content from all BACs mapped on snake W sex chromosomes revealed a large proportion of long interspersed nuclear element (LINE) and short interspersed nuclear element (SINE) retrotransposons. These results led us to predict that amplification of LINE and SINE may have occurred on snake W chromosomes during evolution. Genome compartmentalization, such as transposon amplification, might be the key factor influencing chromosome structure and differentiation. Multiple sequence alignments of all BACs mapped on snake W sex chromosomes did not reveal common sequences. Our findings indicate that the SR2 and snake W sex chromosomes may have been part of a larger ancestral amniote super-sex chromosome, and support the view of sex chromosome evolution as a colorful myriad of situations and trajectories in which many diverse processes are in action.
... Caenophidian snakes share a common ZW system that evolved after their divergence from boas and pythons (Gamble et al. 2017), but before the divergence of wart and file snakes from other caenophidians (Rovatsos et al. 2015;Matsubara et al. 2016), between 91 and 76 million years (Myr) ago (Kumar et al. 2017). Comparisons of male and female whole-genome shotgun sequence data from five-pacer viper (Deinagkistrodon acutus) (Yin et al. 2016), pygmy rattlesnake (Sistrurus miliarius), and mountain garter snake (Thamnophis elegans) (Vicoso et al. 2013a) yielded dozens of W-linked gene predictions from each species. ...
Article
Different ancestral autosomes independently evolved into sex chromosomes in snakes, birds, and mammals. In snakes and birds, females are ZW and males are ZZ; in mammals, females are XX and males are XY. Although X and Z Chromosomes retain nearly all ancestral genes, sex-specific W and Y Chromosomes suffered extensive genetic decay. In both birds and mammals, the genes that survived on sex-specific chromosomes are enriched for broadly expressed, dosage-sensitive regulators of gene expression, subject to strong purifying selection. To gain deeper insight into the processes that govern survival on sex-specific chromosomes, we carried out a meta-analysis of survival across 41 species-three snakes, 24 birds, and 14 mammals-doubling the number of ancestral genes under investigation and increasing our power to detect enrichments among survivors relative to nonsurvivors. Of 2564 ancestral genes, representing an eighth of the ancestral amniote genome, only 324 survive on present-day sex-specific chromosomes. Survivors are enriched for dosage-sensitive developmental processes, particularly development of neural crest-derived structures, such as the face. However, there was no enrichment for expression in sex-specific tissues, involvement in sex determination or gonadogenesis pathways, or conserved sex-biased expression. Broad expression and dosage sensitivity contributed independently to gene survival, suggesting that pleiotropy imposes additional constraints on the evolution of dosage compensation. We propose that maintaining the viability of the heterogametic sex drove gene survival on amniote sex-specific chromosomes, and that subtle modulation of the expression of survivor genes and their autosomal orthologs has disproportionately large effects on development and disease.
... Rattlesnakes in particular have considerable recombination rate variation within and among chromosomes , thus substitution rate variation across the genome as a consequence of male-biased mutation rates and recombination is reasonable. Indeed, considering the substantial variation in heteromorphism between Z and W chromosomes and associated variation in the size of recombining pseudoautosomal regions among colubroid snake species (Matsubara et al. 2006;Vicoso et al. 2013;Gamble et al. 2017), colubroids would provide a valuable model for studying how recombination rate variation, male mutation bias, and sex linkage influence rates of evolution. ...
Article
Male-biased mutation rates occur in a diverse array of organisms. The ratio of male-to-female mutation rate may have major ramifications for evolution across the genome, and for sex-linked genes in particular. In ZW species, the Z chromosome is carried by males two-thirds of the time, leading to the prediction that male-biased mutation rates will have a disproportionate effect on the evolution of Z-linked genes relative to autosomes and the W chromosome. Colubroid snakes (including colubrids, elapids, and viperids) have ZW sex determination, yet male-biased mutation rates have not been well studied in this group. Here we analyze a population genomic dataset from rattlesnakes to quantify genetic variation within and genetic divergence between species. We use a new method for unbiased estimation of population genetic summary statistics to compare variation between the Z chromosome and autosomes and to calculate net nucleotide differentiation between species. We find evidence for a 2.03-fold greater mutation rate in male rattlesnakes relative to females, corresponding to an average μZ/μA ratio of 1.1. Our results from snakes are quantitatively similar to birds, suggesting that male-biased mutation rates may be a common feature across vertebrate lineages with ZW sex determination.
... RAD-Seq generates short sequences for a small but consistent fraction of the genome and thus allows the sequencing and comparison of multiple individuals from several populations at relatively low cost (Davey et al., 2011) and without requiring additional genomic resources. RAD-Seq has been successfully used to identify sex-specific sequences in nonmodel species from diverse taxa, including fish (Drinan et al., 2018), amphibians (Bewick et al., 2013), nonavian reptiles (Gamble, 2016;Gamble et al., 2015Gamble et al., , 2017Gamble et al., , 2018Gamble & Zarkower, 2014;Nielsen et al., 2018Nielsen et al., , 2019, invertebrates (Carmichael et al., 2013;Mathers et al., 2015;Pratlong et al., 2017) and plants (Kafkas et al., 2015), and to identify the sex chromosomes and the sex locus in some of these species (Wilson et al., 2014). Although dedicated pipelines have been developed to analyse RAD-Seq data specifically for sex determination (Gamble & Zarkower, 2014), most of the above-mentioned studies have used stacks (Catchen et al., , 2013Hohenlohe et al., 2012;Rochette & Catchen, 2017) to cluster RAD-Seq reads into polymorphic markers subsequently filtered with custom in-house scripts. ...
Article
The study of sex determination and sex chromosome organisation in non-model species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, Restriction Site-Associated DNA Sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analyzed a published dataset of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyze new RAD-Seq datasets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in non-model species thanks to its speed of analyses, low resource usage, ease of application, and visualization options. Furthermore, our analysis of new datasets from 15 species provides new insights on sex determination in fish.
... Genome/transcriptome/proteome research has advanced and extended the evolutionary analysis of squamate, including snakes and their toxin development [29][30][31][32] . Achievements referred to genome structure, adaption evolution [33][34][35] , axial patterning and limb development 36,37 , the originate and expansion of toxins 19,36 , toxin variation between species 12,38 , sex chromosome evolution [39][40][41] ,tissue-specific expression 42,43 , gene expression regulation 19,40,44 . However, barring a few exceptions 12, 38,45,46 49 . ...
Article
The many-banded krait, Bungarus multicinctus, has been recorded as the animal resource of JinQianBaiHuaShe in the Chinese Pharmacopoeia. Characterization of its venoms classified chief phyla of modern animal neurotoxins. However, the evolutionary origin and diversification of its neurotoxins as well as biosynthesis of its active compounds remain largely unknown due to the lack of its high-quality genome. Here, we present the 1.58 Gbp genome of B. multicinctus assembled into 18 chromosomes with contig/scaffold N50 of 7.53 Mbp/149.8 Mbp. Major bungarotoxin-coding genes were clustered within genome by family and found to be associated with ancient local duplications. The truncation of glycosylphosphatidylinositol anchor in the 3ʹ-terminal of a LY6E paralog released modern three-finger toxins (3FTxs) from membrane tethering before the Colubroidea divergence. Subsequent expansion and mutations diversified and recruited these 3FTxs. After the cobra/krait divergence, the modern unit-B of β-bungarotoxin emerged with an extra cysteine residue. A subsequent point substitution in unit-A enabled the β-bungarotoxin covalent linkage. The B. multicinctus gene expression, chromatin topological organization, and histone modification characteristics were featured by transcriptome, proteome, chromatin conformation capture sequencing, and ChIP-seq. The results highlighted that venom production was under a sophisticated regulation. Our findings provide new insights into snake neurotoxin research, meanwhilewill facilitate antivenom development, toxin-driven drug discovery and the quality control of JinQianBaiHuaShe.
... The possibility exists of within-species sex-chromosome turnover, which could be further tested using other methods such as genome assembly. However, the influence of noise from highthroughput sequencing technologies and/or random biological variation/association cannot be ruled out, especially with small sample sizes that might show sex-linked loci outside the sex-determination regions or even in autosomes (Gamble et al., 2017). ...
Article
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Bighead catfish (Clarias macrocephalus, Günther, 1864) is an important aquacultural species that plays a crucial role in the economy of Southeast Asia. Crossbreeding between female bighead catfish and male African catfish (C. gariepinus, Burchell, 1822) is used to produce hybrids with vigorous phenotypes. However, sterility of the hybrid is a major obstacle to their mass production. There is an emerging hypothesis that the complexity of the sex-determination system between two parental species might affect sterility. Previous studies investigated the co-existence of XX/XY and ZZ/ZW sex-determination systems in the African catfish population in Thailand, but in bighead catfish the sex-determination system remains poorly understood. In this study, the sex-determination system of the bighead catfish was examined using Diversity Arrays Technology to identify the genomic variants associated with sex-linked regions. The results support the hypothesis of the previous study that the bighead catfish might exhibit a male heterogametic XX/XY sex-determination system with multiple male-linked loci. One of the male-linked loci showed homology with the GTSF1L gene, which is shows a testis-enriched expression pattern. Two of the male-linked loci were partially homologous to transposable element. Male-linked loci on the putative Y sex chromosome were identified as an extremely small proportion of the genome. A PCR-based DNA marker was developed to validate the male-linked loci in the bighead catfish. Our findings provide novel insights into sex-determination mechanisms in clariid catfish and will contribute to genetic improvements in breeding programs.
... High-throughput sequencing methods have assisted molecular characterization of sex chromosomes and, consequently, the studies of sex chromosomes evolution (examples in Hall et al., 2013;Traut et al., 2013;Vicoso et al., 2013;Gamble et al., 2015;Gamble et al., 2017). Although our sex chromosome assemblies are highly fragmented, they represent a substantial advance in the characterization of differentiated sex chromosomes in the anuran lineage, for which data are generally lacking and provide new insights into the evolution of Ps. tocantins sex chromosomes. ...
Article
Pseudis tocantins is the only frog species of the hylid genus Pseudis that possesses highly heteromorphic sex chromosomes. Z and W chromosomes of Ps. tocantins differ in size, morphology, position of the nucleolar organizer region (NOR) and the amount and distribution of heterochromatin. A chromosomal inversion and heterochromatin amplification on the W chromosome were previously inferred to be involved in the evolution of this sex chromosome pair. Despite these findings, knowledge related to the molecular composition of the large heterochromatic band of this W chromosome is restricted to the PcP190 satellite DNA, and no data are available regarding the gene content of either the W or the Z chromosome of Ps. tocantins. Here, we sequenced microdissected Z and W chromosomes of this species to further resolve their molecular composition. Comparative genomic analysis suggests that Ps. tocantins sex chromosomes are likely homologous to chromosomes 4 and 10 of Xenopus tropicalis. Analyses of the repetitive DNA landscape in the Z and W assemblies allowed for the identification of several transposable elements and putative satellite DNA sequences. Finally, some transposable elements from the W assembly were found to be highly diverse and divergent from elements found elsewhere in the genome, suggesting a rapid amplification of these elements on the W chromosome.
... Novel sex chromosomes may arise when an autosomal gene acquires a sex determining function or through autosome sex chromosome fusion (Lee et al., 2019a;Pennell et al., 2015). Sex chromosome turnovers have occurred many times during reptile evolution (Bista et al., 2021;Gamble et al., 2015Gamble et al., , 2017, possibly by a novel sex determining gene usurping the established gene (Herpin & Schartl, 2015) (e.g., sdY in rainbow trout (Yano et al., 2012), or a change to environmental sex determination and the subsequent evolution of novel genetic systems (Holleley et al., 2015). It would not, therefore, be unexpected to find different candidate sex determining genes within the genomic regions that we have identified in the four reptiles (Figures 1, 4). ...
Article
Reptile sex determination is attracting much attention because the great diversity of sex-determination and dosage compensation mechanisms permits us to approach fundamental questions about mechanisms of sex chromosome turnover. Recent studies have made significant progress in better understanding diversity and conservation of reptile sex chromosomes, with however no reptile master sex determination genes identified. Here we describe an integrated genomics and cytogenetics pipeline, combining probes generated from the microdissected sex chromosomes with transcriptome and genome sequencing to explore the sex chromosome diversity in non-model Australian reptiles. We tested our pipeline on a turtle, two species of geckos, and a monitor lizard. Genes identified on sex chromosomes were compared to the chicken genome to identify homologous regions among the four species. We identified candidate sex determining genes within these regions, including conserved vertebrate sex-determining genes pdgfa, pdgfra amh and wt1, and demonstrated their testis or ovary-specific expression. All four species showed gene-by-gene rather than chromosome-wide dosage compensation. Our results imply that reptile sex chromosomes originated by independent acquisition of sex-determining genes on different autosomes, as well as translocations between different ancestral macro- and microchromosomes. We discuss the evolutionary drivers of the slow differentiation and turnover of reptile sex chromosomes.
... However, many of the reported GSD to ESD transitions suggested within lacertids, skinks, varanids, and chameleons appeared to be based on an erroneous assignment of GSD species as ESD (Rovatsos, Johnson Pokorn a, et al. 2015;Hill et al. 2018;Nielsen et al. 2018;Iannucci et al. 2019;Rovatsos, Vuki c, et al. 2019;Cornejo-P aramo et al. 2020;Kostmann et al. 2021) and it seems that many major clades of amniotes fixed their GSD quite a long time ago (reviewed in Kratochv ıl et al. 2021). The variability in sex determination found in some of these lineages, such as snakes (Rovatsos, Vuki c, et al. 2015;Gamble et al. 2017;Augstenov a et al. 2018), chameleons (Rovatsos, Johnson Pokorn a, et al. 2015;Nielsen et al. 2018;Rovatsos, Vuki c, et al. 2019), or iguanas (Acosta et al. 2019Nielsen, Guzm an-M endez, et al. 2019) concerns turnovers of sex chromosomes, that is, transitions within GSD. Lineages with the well-supported cooccurrence of both GSD and ESD are of particular interest to explore the mechanisms driving the evolution of sex determination systems; however, it seems that they are only a few of them among amniotes: the turtles (Valenzuela and Adams 2011;Bista and Valenzuela 2020), the dragon lizards (Agamidae) (Ezaz et al. 2009;Pokorn a and Kratochv ıl 2009), and the geckos (Pokorn a and Kratochv ıl 2009; Gamble 2010; Gamble et al. 2015). ...
Article
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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; 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 million of years (MY) and potentially up to 45 MY. 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.
... A remarkable diversity of mechanisms exists to initially steer the bipotential gonad toward the ovarian versus testicular fate, in a process known as primary sex determination. Reptiles and amphibians uniquely exemplify this diversity with every major sex-determining mechanism (SDM) represented in these groups, including female heterogametic (ZW) sex chromosomes (e.g., majority of snakes [Matsubara et al., 2006], African clawed frog [Yoshimoto et al., 2010]), male heterogametic (XY) sex chromosomes (e.g., some lizards [Gamble et al., 2014[Gamble et al., , 2015, boas and pythons [Gamble et al., 2017]), polygenic sex determination (e.g., some amphibians [Nakamura, 2009;Miura, 2017;Ruiz-Garciá et al., 2021]), and environmental sex determination (e.g., some squamates [Charnier, 1966;Holleley et al., 2015], many turtles [Bull, 1980], all crocodilians [Lang and Andrews, 1994], and tuatara [Mitchell et al., 2006]) [reviewed extensively elsewhere; for example, see Valenzuela and Lance, 2004;Bachtrog et al., 2014;Capel, 2017]. The distribution of different SDMs across the phylogeny of reptiles and amphibians suggests that evolutionary transitions between SDMs occur frequently and sometimes rapidly [Janzen and Phillips, 2006;Pokorná and Kratochvíl, 2009;Bachtrog et al., 2014;Jeffries et al., 2018]. ...
Article
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Background: Reptiles and amphibians provide untapped potential for discovering how a diversity of genetic pathways and environmental conditions are incorporated into developmental processes that can lead to similar functional outcomes. These groups display a multitude of reproductive strategies, and whereas many attributes are conserved within groups and even across vertebrates, several aspects of sexual development show considerable variation. Summary: In this review, we focus our attention on the development of the reptilian and amphibian ovary. First, we review and describe the events leading to ovarian development, including sex determination and ovarian maturation, through a comparative lens. We then describe how these events are influenced by environmental factors, focusing on temperature and exposure to anthropogenic chemicals. Lastly, we identify critical knowledge gaps and future research directions that will be crucial to moving forward in our understanding of ovarian development and the influences of the environment in reptiles and amphibians. Key messages: Reptiles and amphibians provide excellent models for understanding the diversity of sex determination strategies and reproductive development. However, a greater understanding of the basic biology of these systems is necessary for deciphering the adaptive and potentially disruptive implications of embryo-by-environment interactions in a rapidly changing world.
... Facultative parthenogenesis yielding genetically variable offspring of both sexes was discovered in a xantusiid lizard [211]. Five squamate clades (iguanas, lacertid lizards, varanids, skinks and caenophidian snakes) covering approximately 60% of extant squamates show evolutionary conserved sex chromosomes [206,[212][213][214][215][216], while other lineages, particularly Acrodonta (agamid lizards and chameleons), boas and pythons, and geckos exhibit more variable SD [18,205,[217][218][219]. In two snake families and the Komodo dragon (Varanus komodoensis) with female heterogamety, substantial W-chromosome degeneration and the absence of global Z-chromosome dosage compensation has been shown, dosage balance is largely lacking in Z-specific genes in these species [215,220,221]. ...
Article
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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 a sexomics integrative research initiative across vertebrates. The vertebrate sexome comprises 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. The sexome also includes ontogenetic and behavioural aspects of sexual differentiation, including malfunction and impairment of SD, sexual differentiation and fertility. Starting from data generated by high-through- put approaches, we encourage others to contribute expertise to building understanding of the sexomes of 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)’.
... As proposed by Ohno (1967) using outstanding representatives in snakes, although the species of Boidae was found not to be the case 50 years later (Gamble et al., 2017), sex chromosomes often evolve from a homomorphic to a heteromorphic state, with eventual heterochromatinization and degeneration of the W or Y chromosomes (Charlesworth et al., 2005). The typical examples can be observed in mammals and birds, whose sex chromosomes have been conserved for hundreds of millions of years (Bachtrog et al., 2014;O'Meally et al., 2012). ...
Article
Sex chromosomes constantly exist in a dynamic state of evolution: rapid turnover and change of heterogametic sex during homomorphic state, and often stepping out to a heteromorphic state followed by chromosomal decaying. However, the forces driving these different trajectories of sex chromosome evolution are still unclear. The Japanese frog Glandirana rugosa is one taxon well suited to the study on these driving forces. The species has two different heteromorphic sex chromosome systems, XX‐XY and ZZ‐ZW, which are separated in different geographic populations. Both XX‐XY and ZZ‐ZW sex chromosomes are represented by chromosome 7 (2n = 26). Phylogenetically, these two systems arose via hybridization between two ancestral lineages of West Japan and East Japan populations, of which sex chromosomes are homomorphic in both sexes and to date have not yet been identified. Identification of the sex chromosomes will give us important insight into the mechanisms of sex chromosome evolution in this species. Here, we used a high‐throughput genomic approach to identify the homomorphic XX‐XY sex chromosomes in both ancestral populations. Sex‐linked DNA markers of West Japan were aligned to chromosome 1, whereas those of East Japan were aligned to chromosome 3. These results reveal that at least two turnovers across three different sex chromosomes 1, 3 and 7 occurred during evolution of this species. This finding raises the possibility that cohabitation of the two different sex chromosomes from ancestral lineages induced turnover to another new one in their hybrids, involving transition of heterogametic sex and evolution from homomorphy to heteromorphy.
... Several of these chromosomes have been recruited into a sex-determining role in other amniotes. For example, chicken chromosome 2 is sex linked in Python bivittatus (Gamble et al., 2017), caenophidian snakes (Matsubara et al., 2006; and the sphaerodactylid gecko genus Aristelliger (Keating et al., 2020). Chicken chromosome 4q is part of the sex chromosome system in geckos in the family Pygopodidae . ...
Article
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Sex‐determination systems are highly variable amongst vertebrate groups, and the prevalence of genomic data has greatly expanded our knowledge of how diverse some groups truly are. Gecko lizards are known to possess a variety of sex‐determination systems, and each new study increases our knowledge of this diversity. Here, we used RADseq to identify male‐specific markers in the banded gecko Coleonyx brevis, indicating this species has a XX/XY sex‐determination system. Furthermore, we show that these sex‐linked regions are not homologous to the XX/XY sex chromosomes of two related Coleonyx species, C. elegans and C. mitratus, suggesting that a cis‐sex chromosome turnover—a change in sex chromosomes without a concomitant change in heterogamety—has occurred within the genus. These findings demonstrate the utility of genome‐scale data to uncover novel sex chromosomes and further highlight the diversity of gecko sex chromosomes. Male‐specific genetics markers in the banded gecko, Coleonyx brevis, indicate an XX/XY sex chromosome system. These results support a sex chromosome turnover within the genus Coleonyx.
... Namely, thanks to the application of genomic techniques such as RADseq in recent years, several counterexamples from poorly differentiated sex chromosomes have emerged. For instance, in squamate reptiles, it was found that both snake lineages with female heterogamety possess heteromorphic sex chromosomes, while the two snake lineages with male heterogamety have homomorphic, cytogenetically indistinguishable sex chromosomes [159,160], although there is no evidence that the female heterogametic systems should be considerably older. A similar pattern can be found in lacertoidean lizards, where tegus, whiptails and spectacles lizards (families Teiidae and Gymnophthalmidae), presumably with male heterogamety, have only poorly differentiated sex chromosomes, while closely related true lizards (Lacertidae) from their sister clade exhibit female heterogamety with highly differentiated sex chromosomes. ...
Article
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Until recently, the field of sex chromosome evolution has been dominated by the canonical unidirectional scenario, first developed by Muller in 1918. This model postulates that sex chromosomes emerge from autosomes by acquiring a sex-determining locus. Recombination reduction then expands outwards from this locus, to maintain its linkage with sexually antagonistic/advantageous alleles, resulting in Y or W degeneration and potentially culminating in their disappearance. Based mostly on empirical vertebrate research, we challenge and expand each conceptual step of this canonical model and present observations by numerous experts in two parts of a theme issue of Phil. Trans. R. Soc. B. We suggest that greater theoretical and empirical insights into the events at the origins of sex-determining genes (rewiring of the gonadal differentiation networks), and a better understanding of the evolutionary forces responsible for recombination suppression are required. Among others, crucial questions are: Why do sex chromosome differentiation rates and the evolution of gene dose regulatory mechanisms between male versus female heterogametic systems not follow earlier theory? Why do several lineages not have sex chromosomes? And: What are the consequences of the presence of (differentiated) sex chromosomes for individual fitness, evolvability, hybridization and diversification? We conclude that the classical scenario appears too reductionistic. Instead of being unidirectional, we show that sex chromosome evolution is more complex than previously anticipated and principally forms networks, interconnected to potentially endless outcomes with restarts, deletions and additions of new genomic material. 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)’.
... Genomic data are, of course, also indispensable in understanding the origin, evolution, and development of complex traits, in conjunction with detailed morphological and physiological observations. Myriad examples are present in the recent literature, such as the origins of visual and olfactory adaptations for prey capture (Perry et al., 2018), venom evolution (Schield et al., 2019), metabolic adaptations (Lind et al., 2019), sex chromosomes (Gamble et al. 2017), and viviparity (Gao et al. 2019). Genomic assemblies of squamate species are becoming increasingly common for snakes (Kerkkamp et al., 2016) and annotated drafts available for some lizards species within iguanians (Alföldi et al., 2011), anguimorphans (Song et al., 2015), and geckos (Xiong et al., 2016). ...
Article
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Squamates (lizards, snakes, and their kin such as amphisbaenians, or worm lizards) represent the world's most diverse clade of terrestrial vertebrates with 11,000 described extant species, representing key components in many of the world's most diverse ecosystems. With an evolutionary history dating back at least to the Middle Triassic at 242 Ma, the squamate Tree of Life also features numerous diverse but extinct branches, with hundreds of fossil species found all over the world. Despite their biological relevance both today and in the geological past, there remains a centuries-old controversy on how the major lineages of squamates are related to each other, with a direct impact on studies in ecology, evolution, paleontology, toxinology, and other fields. Here, we provide a historical overview of this long research tradition, from 19th century naturalists to 21st century phylogenomics, with special emphasis on several recent advances over the last two decades. These insights have had a dramatic effect on our understanding of the squamate Tree of Life and clarify several possible future research agendas. We provide an integrative perspective derived from genomics, morphology, and the fossil record and propose several points of synthesis in our current knowledge of broadscale squamate evolution and systematics. Key topics of interest include dating the origin and early evolution of lizards, the phylogenetic origin of snakes, the evolution of venom, recent agreements between morphological and molecular squamate evolutionary trees, genomic patterns of evolution, and the integration of morphological and molecular data sets. We conclude by providing perspectives on possible advancements in the field, directing researchers to promising future lines of investigation that are necessary to further expand our synthetic knowledge of squamate evolution.
... To test these hypotheses, it is necessary to identify potential variations in the sex linkage across these loci in different jade perch populations. However, the influence of noise from high-throughput sequencing technologies and/or random biological variation/association can be an issue, especially with small sample sizes that might show sex-linked loci outside the sex-determination regions or even in autosomes (Gamble et al., 2017). ...
Article
Jade perch (Scortum barcoo) is a new teleost in the developing aquaculture freshwater finfish grow-out sector in Australia and China. However, key information on the breeding sex determination system (SDS) remains poorly understood, hampering sex control programs and genetic improvement. In this study, the jade perch SDS was examined by investigating genome-wide single-nucleotide polymorphisms (SNPs) using diversity arrays technology and cytogenetics analysis to identify the genomic variants associated with sex-linked regions. Although the cytogenetic results showed no variation in the chromosomal patterns between males and females, one male-specific locus and 13 male-linked loci were observed, suggesting that jade perch exhibits male heterogametic XX/XY SDS. Male-specific loci on the putative Y sex chromosome were also identified as an extremely small proportion of the genome. A homology search of the SNP loci revealed the male-specific loci were homologous to the Gypsy transposable element. This might be a remnant of an initial accumulation of repeats on the Y chromosome at the early stage of sex chromosome differentiation. The results provide a base for sex control breeding biotechnologies and genetic improvements to promote sexual size dimorphism and other new approaches to improve the commercial value of jade perch.
... However, no massive accumulations of microsatellite repeat motifs were found in HPL or HFR. This might result from the influence of karyotypic reorganization in the lineage of the gecko lizards and the state of homomorphic chromosomes between males and females of both species, similar to pythons [104]. The question remains whether any genomic elements are shared among unrelated sex chromosomal linkages in amniotes, which drive sex chromosome differentiation after the chromosomal rearrangement in each lineage. ...
Article
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Comparative chromosome maps investigating sex chromosomal linkage groups in amniotes and microsatellite repeat motifs of a male house gecko lizard (Hemidactylus frenatus, HFR) and a flat-tailed house gecko lizard (H. platyurus, HPL) of unknown sex were examined using 75 bacterial artificial chromosomes (BACs) from chicken and zebra finch genomes. No massive accumulations of microsatellite repeat motifs were found in either of the gecko lizards, but 10 out of 13 BACs mapped on HPL chromosomes were associated with other amniote sex chromosomes. Hybridization of the same BACs onto multiple different chromosome pairs suggested transitions to sex chromosomes across amniotes. No BAC hybridization signals were found on HFR chromosomes. However, HFR diverged from HPL about 30 million years ago, possibly due to intrachromosomal rearrangements occurring in the HFR lineage. By contrast, heterochromatin likely reshuffled patterns between HPL and HFR, as observed from C-positive heterochromatin distribution. Six out of ten BACs showed partial homology with squamate reptile chromosome 2 (SR2) and snake Z and/or W sex chromosomes. The gecko lizard showed shared unrelated sex chromosomal linkages-the remnants of a super-sex chromosome. A large ancestral super-sex chromosome showed a correlation between SR2 and snake W sex chromosomes.
... In mammals, for instance, humans have two PARs in each end of the sex chromosomes [61] (see Fig. 3), and while PAR1 is common to most eutherian mammals, it was not yet thoroughly investigated if a second PAR exists in other eutherian species [56,57]. In some cases, the degeneration process does not occur or is insignificant, and the resulting sex chromosomes are homomorphic or almost morphologically identical, such as in boas and pythons [62] or in emus [1,63,64]. In other cases, degeneration can be extensive, resulting in highly heteromorphic chromosomes, or even in the loss of one of the sex chromosomes in some extreme cases, as happened in very few mammalian speciesfor example, mole voles from genus Ellobius [1,[65][66][67]. ...
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Sex chromosomes have evolved many times in animals and studying these replicate evolutionary "experiments" can help broaden our understanding of the general forces driving the origin and evolution of sex chromosomes. However this plan of study has been hindered by the inability to identify the sex chromosome systems in the large number of species with cryptic, homomorphic sex chromosomes. Restriction site-associated DNA sequencing (RAD-seq) is a critical enabling technology that can identify the sex chromosome systems in many species where traditional cytogenetic methods have failed. Using newly generated RAD-seq data from twelve gecko species, along with data from the literature, we reinterpret the evolution of sex-determining systems in lizards and snakes and test the hypothesis that sex chromosomes can routinely act as evolutionary traps. We uncovered between 17 and 25 transitions among gecko sex-determining systems. This is approximately ½ to ⅔ of the total number of transitions observed among all lizards and snakes. We find support for the hypothesis that sex chromosome systems can readily become trap-like and show that adding even a small number of species from understudied clades can greatly enhance hypothesis testing in a model-based phylogenetic framework. RAD-seq will undoubtedly prove useful in evaluating other species for male or female heterogamety, particularly the majority of fish, amphibian, and reptile species that lack visibly heteromorphic sex chromosomes, and will significantly accelerate the pace of biological discovery. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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The ability to efficiently and accurately determine genotypes is a keystone technology in modern genetics, crucial to studies ranging from clinical diagnostics, to genotype-phenotype association, to reconstruction of ancestry and the detection of selection. To date, high capacity, low cost genotyping has been largely achieved via "SNP chip" microarray-based platforms which require substantial prior knowledge of both genome sequence and variability, and once designed are suitable only for those targeted variable nucleotide sites. This method introduces substantial ascertainment bias and inherently precludes detection of rare or population-specific variants, a major source of information for both population history and genotype-phenotype association. Recent developments in reduced-representation genome sequencing experiments on massively parallel sequencers (commonly referred to as RAD-tag or RADseq) have brought direct sequencing to the problem of population genotyping, but increased cost and procedural and analytical complexity have limited their widespread adoption. Here, we describe a complete laboratory protocol, including a custom combinatorial indexing method, and accompanying software tools to facilitate genotyping across large numbers (hundreds or more) of individuals for a range of markers (hundreds to hundreds of thousands). Our method requires no prior genomic knowledge and achieves per-site and per-individual costs below that of current SNP chip technology, while requiring similar hands-on time investment, comparable amounts of input DNA, and downstream analysis times on the order of hours. Finally, we provide empirical results from the application of this method to both genotyping in a laboratory cross and in wild populations. Because of its flexibility, this modified RADseq approach promises to be applicable to a diversity of biological questions in a wide range of organisms.
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Summary: The two main functions of bioinformatics are the organization and analysis of biological data using computational resources. Geneious Basic has been designed to be an easy-to-use and flexible desktop software application framework for the organization and analysis of biological data, with a focus on molecular sequences and related data types. It integrates numerous industry-standard discovery analysis tools, with interactive visualizations to generate publication-ready images. One key contribution to researchers in the life sciences is the Geneious public application programming interface (API) that affords the ability to leverage the existing framework of the Geneious Basic software platform for virtually unlimited extension and customization. The result is an increase in the speed and quality of development of computation tools for the life sciences, due to the functionality and graphical user interface available to the developer through the public API. Geneious Basic represents an ideal platform for the bioinformatics community to leverage existing components and to integrate their own specific requirements for the discovery, analysis and visualization of biological data.Availability and implementation: Binaries and public API freely available for download at http://www.geneious.com/basic, implemented in Java and supported on Linux, Apple OSX and MS Windows. The software is also available from the Bio-Linux package repository at http://nebc.nerc.ac.uk/news/geneiousonbl.Contact: peter@biomatters.com