Mass of genes rather than master genes underlie the genomic architecture of amphibian speciation.

The genetic architecture of speciation, i.e., how intrinsic genomic incompatibilities promote reproductive isolation (RI) between diverging lineages, is one of the best-kept secrets of evolution. To directly assess whether incompatibilities arise in a limited set of large-effect speciation genes, or in a multitude of loci, we examined the geographic and genomic landscapes of introgression across the hybrid zones of 41 pairs of frog and toad lineages in the Western Palearctic region. As the divergence between lineages increases, phylogeographic transitions progressively become narrower, and larger parts of the genome resist introgression. This suggests that anuran speciation proceeds through a gradual accumulation of multiple barrier loci scattered across the genome, which ultimately deplete hybrid fitness by intrinsic postzygotic isolation, with behavioral isolation being achieved only at later stages. Moreover, these loci were disproportionately sex linked in one group (Hyla) but not in others (Rana and Bufotes), implying that large X-effects are not necessarily a rule of speciation with undifferentiated sex chromosomes. The highly polygenic nature of RI and the lack of hemizygous X/Z chromosomes could explain why the speciation clock ticks slower in amphibians compared to other vertebrates. The clock-like dynamics of speciation combined with the analytical focus on hybrid zones offer perspectives for more standardized practices of species delimitation.

Meiosis reveals the early steps in the evolution of a neo-XY sex chromosome pair in the African pygmy mouse Mus minutoides.

Sex chromosomes of eutherian mammals are highly different in size and gene content, and share only a small region of homology (pseudoautosomal region, PAR). They are thought to have evolved through an addition-attrition cycle involving the addition of autosomal segments to sex chromosomes and their subsequent differentiation. The events that drive this process are difficult to investigate because sex chromosomes in almost all mammals are at a very advanced stage of differentiation. Here, we have taken advantage of a recent translocation of an autosome to both sex chromosomes in the African pygmy mouse Mus minutoides, which has restored a large segment of homology (neo-PAR). By studying meiotic sex chromosome behavior and identifying fully sex-linked genetic markers in the neo-PAR, we demonstrate that this region shows unequivocal signs of early sex-differentiation. First, synapsis and resolution of DNA damage intermediates are delayed in the neo-PAR during meiosis. Second, recombination is suppressed or largely reduced in a large portion of the neo-PAR. However, the inactivation process that characterizes sex chromosomes during meiosis does not extend to this region. Finally, the sex chromosomes show a dual mechanism of association at metaphase-I that involves the formation of a chiasma in the neo-PAR and the preservation of an ancestral achiasmate mode of association in the non-homologous segments. We show that the study of meiosis is crucial to apprehend the onset of sex chromosome differentiation, as it introduces structural and functional constrains to sex chromosome evolution. Synapsis and DNA repair dynamics are the first processes affected in the incipient differentiation of X and Y chromosomes, and they may be involved in accelerating their evolution. This provides one of the very first reports of early steps in neo-sex chromosome differentiation in mammals, and for the first time a cellular framework for the addition-attrition model of sex chromosome evolution.

When Sex Chromosomes Recombine Only in the Heterogametic Sex: Heterochiasmy and Heterogamety in Hyla Tree Frogs.

Sex chromosomes are classically predicted to stop recombining in the heterogametic sex, thereby enforcing linkage between sex-determining (SD) and sex-antagonistic (SA) genes. With the same rationale, a pre-existing sex asymmetry in recombination is expected to affect the evolution of heterogamety, for example, a low rate of male recombination might favor transitions to XY systems, by generating immediate linkage between SD and SA genes. Furthermore, the accumulation of deleterious mutations on nonrecombining Y chromosomes should favor XY-to-XY transitions (which discard the decayed Y), but disfavor XY-to-ZW transitions (which fix the decayed Y as an autosome). Like many anuran amphibians, Hyla tree frogs have been shown to display drastic heterochiasmy (males only recombine at chromosome tips) and are typically XY, which seems to fit the above expectations. Instead, here we demonstrate that two species, H. sarda and H. savignyi, share a common ZW system since at least 11 Ma. Surprisingly, the typical pattern of restricted male recombination has been maintained since then, despite female heterogamety. Hence, sex chromosomes recombine freely in ZW females, not in ZZ males. This suggests that heterochiasmy does not constrain heterogamety (and vice versa), and that the role of SA genes in the evolution of sex chromosomes might have been overemphasized.

Identification of ancestral sex chromosomes in the frog Glandirana rugosa bearing XX-XY and ZZ-ZW sex-determining systems.

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.

Are glacial refugia hotspots of speciation and cytonuclear discordances? Answers from the genomic phylogeography of Spanish common frogs.

Subdivided Pleistocene glacial refugia, best known as "refugia within refugia", provided opportunities for diverging populations to evolve into incipient species and/or to hybridize and merge following range shifts tracking the climatic fluctuations, potentially promoting extensive cytonuclear discordances and "ghost" mtDNA lineages. Here, we tested which of these opposing evolutionary outcomes prevails in northern Iberian areas hosting multiple historical refugia of common frogs (Rana cf. temporaria), based on a genomic phylogeography approach (mtDNA barcoding and RAD-sequencing). We found evidence for both incipient speciation events and massive cytonuclear discordances. On the one hand, populations from northwestern Spain (Galicia and Asturias, assigned to the regional endemic R. parvipalmata), are deeply-diverged at mitochondrial and nuclear genomes (~4 My of independent evolution), and barely admix with northeastern populations (assigned to R. temporaria sensu stricto) across a narrow hybrid zone (~25 km) located in the Cantabrian Mountains, suggesting that they represent distinct species. On the other hand, the most divergent mtDNA clade, widespread in Cantabria and the Basque country, shares its nuclear genome with other R. temporaria s. s. lineages. Patterns of population expansions and isolation-by-distance among these populations are consistent with past mitochondrial capture and/or drift in generating and maintaining this ghost mitochondrial lineage. This remarkable case study emphasizes the complex evolutionary history that shaped the present genetic diversity of refugial populations, and stresses the need to revisit their phylogeography by genomic approaches, in order to make informed taxonomic inferences.

No evidence that Y-chromosome differentiation affects male fitness in a Swiss population of common frogs.

The canonical model of sex-chromosome evolution assigns a key role to sexually antagonistic (SA) genes on the arrest of recombination and ensuing degeneration of Y chromosomes. This assumption cannot be tested in organisms with highly differentiated sex chromosomes, such as mammals or birds, owing to the lack of polymorphism. Fixation of SA alleles, furthermore, might be the consequence rather than the cause of recombination arrest. Here we focus on a population of common frogs (Rana temporaria) where XY males with genetically differentiated Y chromosomes (nonrecombinant Y haplotypes) coexist with both XY° males with proto-Y chromosomes (only differentiated from X chromosomes in the immediate vicinity of the candidate sex-determining locus Dmrt1) and XX males with undifferentiated sex chromosomes (genetically identical to XX females). Our study finds no effect of sex-chromosome differentiation on male phenotype, mating success or fathering success. Our conclusions rejoin genomic studies that found no differences in gene expression between XY, XY° and XX males. Sexual dimorphism in common frogs might result more from the differential expression of autosomal genes than from sex-linked SA genes. Among-male variance in sex-chromosome differentiation seems better explained by a polymorphism in the penetrance of alleles at the sex locus, resulting in variable levels of sex reversal (and thus of X-Y recombination in XY females), independent of sex-linked SA genes.

Integrating hybrid zone analyses in species delimitation: lessons from two anuran radiations of the Western Mediterranean.

Molecular ecologists often rely on phylogenetic evidence for assessing the species-level systematics of newly discovered lineages. Alternatively, the extent of introgression at phylogeographic transitions can provide a more direct test to assign candidate taxa into subspecies or species categories. Here, we compared phylogenetic versus hybrid zone approaches of species delimitation in two groups of frogs from the Western Mediterranean region (Discoglossus and Pelodytes), by using genomic data (ddRAD). In both genera, coalescent analyses recovered almost all nominal taxa as "species". However, the least-diverged pairs D. g. galganoi/jeanneae and P. punctatus/hespericus admix over hundreds of kilometers, suggesting that they have not yet developed strong reproductive isolation and should be treated as conspecifics. In contrast, the comparatively older D. scovazzi/pictus and P. atlanticus/ibericus form narrow contact zones, consistent with species distinctiveness. Due to their complementarity, we recommend taxonomists to combine phylogenomics with hybrid zone analyses to scale the gray zone of speciation, i.e., the evolutionary window separating widely admixing lineages versus nascent reproductively isolated species. The radically different transitions documented here conform to the view that genetic incompatibilities accumulating with divergence generate a weak barrier to gene flow for long periods of time, until their effects multiply and the speciation process then advances rapidly. Given the variability of the gray zone among taxonomic groups, at least from our current abilities to measure it, we recommend to customize divergence thresholds within radiations to categorize lineages for which no direct test of speciation is possible.

Sex-Chromosome Recombination in Common Frogs Brings Water to the Fountain-of-Youth.

According to the canonical model of sex-chromosome evolution, the degeneration of Y or W chromosomes (as observed in mammals and birds, respectively) results from an arrest of recombination in the heterogametic sex, driven by the fixation of sexually antagonistic mutations. However, sex chromosomes have remained homomorphic in many lineages of fishes, amphibians, and nonavian reptiles. According to the "fountain-of-youth" model, this homomorphy results from occasional events of sex reversal. If recombination arrest in males is controlled by maleness per se (and not by genotype), then Y chromosomes are expected to recombine in XY females, preventing their long-term degeneration. Here, we provide field support for the fountain-of-youth, by showing that sex-chromosome recombination in Rana temporaria only depends on phenotypic sex: naturally occurring XX males show the same restriction of recombination as XY males (average map length ∼2 cM), while XY females recombine as much as XX females (average map length ∼150 cM). Our results challenge several common assumptions regarding the evolution of sex chromosomes, including the role of sexually antagonistic genes as drivers of recombination arrest, and that of chromosomal inversions as underlying mechanisms.

A reciprocal translocation radically reshapes sex-linked inheritance in the common frog.

X and Y chromosomes can diverge when rearrangements block recombination between them. Here we present the first genomic view of a reciprocal translocation that causes two physically unconnected pairs of chromosomes to be coinherited as sex chromosomes. In a population of the common frog (Rana temporaria), both pairs of X and Y chromosomes show extensive sequence differentiation, but not degeneration of the Y chromosomes. A new method based on gene trees shows both chromosomes are sex-linked. Furthermore, the gene trees from the two Y chromosomes have identical topologies, showing they have been coinherited since the reciprocal translocation occurred. Reciprocal translocations can thus reshape sex linkage on a much greater scale compared with inversions, the type of rearrangement that is much better known in sex chromosome evolution, and they can greatly amplify the power of sexually antagonistic selection to drive genomic rearrangement. Two more populations show evidence of other rearrangements, suggesting that this species has unprecedented structural polymorphism in its sex chromosomes.

Phylogeography of a cryptic speciation continuum in Eurasian spadefoot toads (Pelobates).

Cryptic phylogeographic diversifications provide unique models to examine the role of phylogenetic divergence on the evolution of reproductive isolation, without extrinsic factors such as ecological and behavioural differentiation. Yet, to date very few comparative studies have been attempted within such radiations. Here, we characterize a new speciation continuum in a group of widespread Eurasian amphibians, the Pelobates spadefoot toads, by conducting multilocus (restriction site associated DNA sequencing and mitochondrial DNA) phylogenetic, phylogeographic and hybrid zone analyses. Within the P. syriacus complex, we discovered species-level cryptic divergences (>5 million years ago [My]) between populations distributed in the Near-East (hereafter P. syriacus sensu stricto [s.s.]) and southeastern Europe (hereafter P. balcanicus), each featuring deep intraspecific lineages. Altogether, we could scale hybridizability to divergence time along six different stages, spanning from sympatry without gene flow (P. fuscus and P. balcanicus, >10 My), parapatry with highly restricted hybridization (P. balcanicus and P. syriacus s.s., >5 My), narrow hybrid zones (~15 km) consistent with partial reproductive isolation (P. fuscus and P. vespertinus, ~3 My), to extensive admixture between Pleistocene and refugial lineages (≤2 My). This full spectrum empirically supports a gradual build up of reproductive barriers through time, reversible up until a threshold that we estimate at ~3 My. Hence, cryptic phylogeographic lineages may fade away or become reproductively isolated species simply depending on the time they persist in allopatry, and without definite ecomorphological divergence.

Diversification and speciation in tree frogs from the Maghreb (Hyla meridionalis sensu lato), with description of a new African endemic.

Comparative molecular studies emphasized a new biogeographic paradigm for the terrestrial fauna of North Africa, one of the last uncharted ecoregions of the Western Palearctic: two independent east-west divisions across the Maghreb. Through a comprehensive phylogeography, we assessed how this model suits the genetic diversification documented for the tree frog Hyla meridionalis sensu lato. Analyses of mtDNA variation and thousands of nuclear loci confirmed the old split (low-Pliocene) between Tunisian and Algerian populations. These lineages meet but barely admix in the eastern Maghreb (Algerian-Tunisian border), a sign of putatively advanced reproductive isolation. In the western Maghreb, we report a Pleistocene divergence between Moroccan and Algerian populations. Tree frogs thus follow both predictions: a double east-west break that gave rise to two suture zones characteristic of North-African phylogeography. Moreover, some intraspecific mtDNA variation is not mirrored by the nuclear data, emphasizing that evolutionary units should always be designated by multilocus approaches. Last but not least, we describe the Tunisian lineage as a new species endemic to Africa.

Impact of deleterious mutations, sexually antagonistic selection, and mode of recombination suppression on transitions between male and female heterogamety.

Deleterious mutations accumulating on non-recombining Y chromosomes can drive XY to XY turnovers, as they allow to replace the old mutation-loaded Y by a new mutation-free one. The same process is thought to prevent XY to ZW turnovers, because the latter requires fixation of the ancestral Y, assuming dominance of the emergent feminizing mutation. Using individual-based simulations, we explored whether and how an epistatically dominant W allele can spread in a young XY system that gradually accumulates deleterious mutations. We also investigated how sexually antagonistic (SA) polymorphism on the ancestral sex chromosomes and the mechanism controlling X-Y recombination suppression affect these transitions. In contrast with XY to XY turnovers, XY to ZW turnovers cannot be favored by Y chromosome mutation load. If the arrest of X-Y recombination depends on genotypic sex, transitions are strongly hindered by deleterious mutations, and totally suppressed by very small SA cost, because deleterious mutations and female-detrimental SA alleles would have to fix with the Y. If, however, the arrest of X-Y recombination depends on phenotypic sex, X and Y recombine in XY ZW females, allowing for the purge of Y-linked deleterious mutations and loss of the SA polymorphism, causing XY to ZW turnovers to occur at the same rate as in the absence of deleterious and sex-antagonistic mutations. We generalize our results to other types of turnovers (e.g., triggered by non-dominant sex-determining mutations) and discuss their empirical relevance.

Phylogeography of Aegean green toads (Bufo viridis subgroup): continental hybrid swarm vs. insular diversification with discovery of a new island endemic.

BACKGROUND: Debated aspects in speciation research concern the amount of gene flow between incipient species under secondary contact and the modes by which post-zygotic isolation accumulates. Secondary contact zones of allopatric lineages, involving varying levels of divergence, provide natural settings for comparative studies, for which the Aegean (Eastern Mediterranean) geography offers unique scenarios. In Palearctic green toads (Bufo viridis subgroup or Bufotes), Plio-Pleistocene (~ 2.6 Mya) diverged species show a sharp transition without contemporary gene flow, while younger lineages, diverged in the Lower-Pleistocene (~ 1.9 Mya), admix over tens of kilometers. Here, we conducted a fine-scale multilocus phylogeographic analysis of continental and insular green toads from the Aegean, where a third pair of taxa, involving Mid-Pleistocene diverged (~ 1.5 Mya) mitochondrial lineages, earlier tentatively named viridis and variabilis, (co-)occurs. RESULTS: We discovered a new lineage, endemic to Naxos (Central Cyclades), while coastal islands and Crete feature weak genetic differentiation from the continent. In continental Greece, both lineages, viridis and variabilis, form a hybrid swarm, involving massive mitochondrial and nuclear admixture over hundreds of kilometers, without obvious selection against hybrids. CONCLUSIONS: The genetic signatures of insular Aegean toads appear governed by bathymetry and Quaternary sea level changes, resulting in long-term isolation (Central Cyclades: Naxos) and recent land-bridges (coastal islands). Conversely, Crete has been isolated since the end of the Messinian salinity crisis (5.3 My) and Cretan populations thus likely result from human-mediated colonization, at least since Antiquity, from Peloponnese and Anatolia. Comparisons of green toad hybrid zones support the idea that post-zygotic hybrid incompatibilities accumulate gradually over the genome. In this radiation, only one million years of divergence separate a scenario of complete reproductive isolation, from a secondary contact resulting in near panmixia.

Profound genetic divergence and asymmetric parental genome contributions as hallmarks of hybrid speciation in polyploid toads.

The evolutionary causes and consequences of allopolyploidization, an exceptional pathway to instant hybrid speciation, are poorly investigated in animals. In particular, when and why hybrid polyploids versus diploids are produced, and constraints on sources of paternal and maternal ancestors, remain underexplored. Using the Palearctic green toad radiation (including bisexually reproducing species of three ploidy levels) as model, we generate a range-wide multi-locus phylogeny of 15 taxa and present four new insights: (i) at least five (up to seven) distinct allotriploid and allotetraploid taxa have evolved in the Pleistocene; (ii) all maternal and paternal ancestors of hybrid polyploids stem from two deeply diverged nuclear clades (6 Mya, 3.1-9.6 Mya), with distinctly greater divergence than the parental species of diploid hybrids found at secondary contact zones; (iii) allotriploid taxa possess two conspecific genomes and a deeply diverged allospecific one, suggesting that genomic imbalance and divergence are causal for their partly clonal reproductive mode; (iv) maternal versus paternal genome contributions exhibit asymmetry, with the maternal nuclear (and mitochondrial) genome of polyploids always coming from the same clade, and the paternal genome from the other. We compare our findings with similar patterns in diploid/polyploid vertebrates, and suggest deep ancestral divergence as a precondition for successful allopolyploidization.

Sex chromosome turnovers and genetic drift: a simulation study.

The recent advances of new genomic technologies have enabled the identification and characterization of sex chromosomes in an increasing number of nonmodel species, revealing that many plants and animals undergo frequent sex chromosome turnovers. What evolutionary forces drive these turnovers remains poorly understood, but it was recently proposed that drift might play a more important role than generally assumed. We analysed the dynamics of different types of turnovers using individual-based simulations and show that when mediated by genetic drift, turnovers are usually easier to achieve than substitutions at neutral markers, but that their dynamics and relative likelihoods vary with the type of the resident and emergent sex chromosome system (XY and/or ZW) and the dominance relationships among the sex-determining factors. Focusing on turnovers driven by epistatically dominant mutations, we find that drift-mediated turnovers that preserve the heterogamety pattern are 2-4× more likely than those along which the heterogametic sex changes. This ratio nevertheless decreases along with effective population size and can even reverse in case of extreme polygyny. This can be attributed to a 'drift-induced' selective force, known to influence transitions between male and female heterogamety, but which according to our study does not affect turnovers that preserve the heterogametic sex.

Random sex determination: When developmental noise tips the sex balance.

Sex-determining factors are usually assumed to be either genetic or environmental. The present paper aims at drawing attention to the potential contribution of developmental noise, an important but often-neglected component of phenotypic variance. Mutual inhibitions between male and female pathways make sex a bistable equilibrium, such that random fluctuations in the expression of genes at the top of the cascade are sufficient to drive individual development toward one or the other stable state. Evolutionary modeling shows that stochastic sex determinants should resist elimination by genetic or environmental sex determinants under ecologically meaningful settings. On the empirical side, many sex-determination systems traditionally considered as environmental or polygenic actually provide evidence for large components of stochasticity. In reviewing the field, I argue that sex-determination systems should be considered within a three-ends continuum, rather than the classical two-ends continuum.

Dmrt1 polymorphism and sex-chromosome differentiation in Rana temporaria.

Sex-determination mechanisms vary both within and among populations of common frogs, opening opportunities to investigate the molecular pathways and ultimate causes shaping their evolution. We investigated the association between sex-chromosome differentiation (as assayed from microsatellites) and polymorphism at the candidate sex-determining gene Dmrt1 in two Alpine populations. Both populations harboured a diversity of X-linked and Y-linked Dmrt1 haplotypes. Some males had fixed male-specific alleles at all markers ("differentiated" Y chromosomes), others only at Dmrt1 ("proto-" Y chromosomes), while still others were genetically indistinguishable from females (undifferentiated X chromosomes). Besides these XX males, we also found rare XY females. The several Dmrt1 Y haplotypes differed in the probability of association with a differentiated Y chromosome, which we interpret as a result of differences in the masculinizing effects of alleles at the sex-determining locus. From our results, the polymorphism in sex-chromosome differentiation and its association with Dmrt1, previously inferred from Swedish populations, are not just idiosyncratic features of peripheral populations, but also characterize highly diverged populations in the central range. This implies that an apparently unstable pattern has been maintained over long evolutionary times.

Sex determination: why so many ways of doing it?

Sexual reproduction is an ancient feature of life on earth, and the familiar X and Y chromosomes in humans and other model species have led to the impression that sex determination mechanisms are old and conserved. In fact, males and females are determined by diverse mechanisms that evolve rapidly in many taxa. Yet this diversity in primary sex-determining signals is coupled with conserved molecular pathways that trigger male or female development. Conflicting selection on different parts of the genome and on the two sexes may drive many of these transitions, but few systems with rapid turnover of sex determination mechanisms have been rigorously studied. Here we survey our current understanding of how and why sex determination evolves in animals and plants and identify important gaps in our knowledge that present exciting research opportunities to characterize the evolutionary forces and molecular pathways underlying the evolution of sex determination.

Phylogeography reveals an ancient cryptic radiation in East-Asian tree frogs (Hyla japonica group) and complex relationships between continental and island lineages.

BACKGROUND: In contrast to the Western Palearctic and Nearctic biogeographic regions, the phylogeography of Eastern-Palearctic terrestrial vertebrates has received relatively little attention. In East Asia, tectonic events, along with Pleistocene climatic conditions, likely affected species distribution and diversity, especially through their impact on sea levels and the consequent opening and closing of land-bridges between Eurasia and the Japanese Archipelago. To better understand these effects, we sequenced mitochondrial and nuclear markers to determine phylogeographic patterns in East-Asian tree frogs, with a particular focus on the widespread H. japonica. RESULTS: We document several cryptic lineages within the currently recognized H. japonica populations, including two main clades of Late Miocene divergence (~5 Mya). One occurs on the northeastern Japanese Archipelago (Honshu and Hokkaido) and the Russian Far-East islands (Kunashir and Sakhalin), and the second one inhabits the remaining range, comprising southwestern Japan, the Korean Peninsula, Transiberian China, Russia and Mongolia. Each clade further features strong allopatric Plio-Pleistocene subdivisions (~2-3 Mya), especially among continental and southwestern Japanese tree frog populations. Combined with paleo-climate-based distribution models, the molecular data allowed the identification of Pleistocene glacial refugia and continental routes of postglacial recolonization. Phylogenetic reconstructions further supported genetic homogeneity between the Korean H. suweonensis and Chinese H. immaculata, suggesting the former to be a relic population of the latter that arose when the Yellow Sea formed, at the end of the last glaciation. CONCLUSIONS: Patterns of divergence and diversity were likely triggered by Miocene tectonic activities and Quaternary climatic fluctuations (including glaciations), causing the formation and disappearance of land-bridges between the Japanese islands and the continent. Overall, this resulted in a ring-like diversification of H. japonica around the Sea of Japan. Our findings urge for important taxonomic revisions in East-Asian tree frogs. First, they support the synonymy of H. suweonensis (Kuramoto, 1980) and H. immaculata (Boettger, 1888). Second, the nominal H. japonica (Günther, 1859) represents at least two species: an eastern (new taxon A) on the northern Japanese and Russian Far East islands, and a southwestern species (n. t. B) on southern Japanese islands and possibly also forming continental populations. Third, these continental tree frogs may also represent an additional entity, previously described as H. stepheni Boulenger, 1888 (senior synonym of H. ussuriensis Nikolskii, 1918). A complete revision of this group requires further taxonomic and nomenclatural analyses, especially since it remains unclear to which taxon the species-epitheton japonica corresponds to.

Sex-Chromosome Homomorphy in Palearctic Tree Frogs Results from Both Turnovers and X-Y Recombination.

Contrasting with birds and mammals, poikilothermic vertebrates often have homomorphic sex chromosomes, possibly resulting from high rates of sex-chromosome turnovers and/or occasional X-Y recombination. Strong support for the latter mechanism was provided by four species of European tree frogs, which inherited from a common ancestor (∼ 5 Ma) the same pair of homomorphic sex chromosomes (linkage group 1, LG1), harboring the candidate sex-determining gene Dmrt1. Here, we test sex linkage of LG1 across six additional species of the Eurasian Hyla radiation with divergence times ranging from 6 to 40 Ma. LG1 turns out to be sex linked in six of nine resolved cases. Mapping the patterns of sex linkage to the Hyla phylogeny reveals several transitions in sex-determination systems within the last 10 My, including one switch in heterogamety. Phylogenetic trees of DNA sequences along LG1 are consistent with occasional X-Y recombination in all species where LG1 is sex linked. These patterns argue against one of the main potential causes for turnovers, namely the accumulation of deleterious mutations on nonrecombining chromosomes. Sibship analyses show that LG1 recombination is strongly reduced in males from most species investigated, including some in which it is autosomal. Intrinsically low male recombination might facilitate the evolution of male heterogamety, and the presence of important genes from the sex-determination cascade might predispose LG1 to become a sex chromosome.