Spatial genetic structure in European marbled newts revealed with target enrichment by sequence capture.

European marbled newts come in two species that have abutting ranges. The northern species, Triturus marmoratus, is found in France and the northern part of the Iberian Peninsula, whereas the southern species, T. pygmaeus, is found in the southwestern corner of the Iberian Peninsula. We study the intraspecific genetic differentiation of the group because morphological data show geographical variation and because the Iberian Peninsula is a recognized center of speciation and intraspecific genetic diversity for all kinds of organisms, amphibians included. We use target enrichment by sequence capture to generate c. 7 k nuclear DNA markers. We observe limited genetic exchange between the species, which confirms their distinctiveness. Both species show substantial genetic structuring that is only in part mirrored by morphological variation. Genetically differentiated groups are found in the south (T. marmoratus) and west (T. pygmaeus) of the species ranges. Our observations highlight the position of the Iberian Peninsula as a hotspot for genetic differentiation.

Assessing fine-scale pondscape connectivity with amphibian eyes: An integrative approach using genomic and capture-mark-recapture data.

In the face of habitat loss, preserving functional connectivity is essential to maintain genetic diversity and the demographic dynamics required for the viability of biotic communities. This requires knowledge of the dispersal behaviour of target species, which can be modelled as kernels, or probability density functions of dispersal distances at increasing geographic distances. We present an integrative approach to investigate the relationships between genetic connectivity and demographic parameters in organisms with low vagility focusing on five syntopic pond-breeding amphibians. We genotyped 1056 individuals of two anuran and three urodele species (1732-3913 SNPs per species) from populations located in a landscape comprising 64 ponds to characterize fine-scale genetic structure in a comparative framework, and combined these genetic data with information obtained in a previous 2-year capture-mark-recapture (CMR) study. Specifically, we contrasted graphs reconstructed from genomic data with connectivity graphs based on dispersal kernels and demographic information obtained from CMR data from previous studies, and assessed the effects of population size, population density, geographical distances, inverse movement probabilities and the presence of habitat patches potentially functioning as stepping stones on genetic differentiation. Our results show a significant effect of local population sizes on patterns of genetic differentiation at small spatial scales. In addition, movement records and cluster-derived kernels provide robust inferences on most likely dispersal paths that are consistent with genomic inferences on genetic connectivity. The integration of genetic and CMR data holds great potential for understanding genetic connectivity at spatial scales relevant to individual organisms, with applications for the implementation of management actions at the landscape level.

Genomic phylogeography illuminates deep cyto-nuclear discordances in midwife toads (Alytes).

The advent of genomic methods allows us to revisit the evolutionary history of organismal groups for which robust phylogenies are still lacking, particularly in species complexes that frequently hybridize. In this study, we conduct RAD-sequencing (RAD-seq) analyses of midwife toads (genus Alytes), an iconic group of western Mediterranean amphibians famous for their parental care behavior, but equally infamous for the difficulties to reconstruct their evolutionary history. Through admixture and phylogenetic analyses of thousands of loci, we provide a comprehensive phylogeographic framework for the A. obstetricans complex, as well as a fully resolved phylogeny for the entire genus. As part of this effort, we carefully explore the influence of different sampling schemes and data filtering thresholds on tree reconstruction, showing that several, slightly different, yet robust topologies may be retrieved with small datasets obtained by stringent SNP calling parameters, especially when admixed individuals are included. In contrast, analyses of incomplete but larger datasets converged on the same phylogeny, irrespective of the reconstruction method used or the proportion of missing data. The Alytes tree features three Miocene-diverged clades corresponding to the proposed subgenera Ammoryctis (A. cisternasii), Baleaphryne (A. maurus, A. dickhilleni and A. muletensis), and Alytes (A. obstetricans complex). The latter consists of six evolutionary lineages, grouped into three clades of Pliocene origin, and currently delimited as two species: (1) A. almogavarii almogavarii and A. a. inigoi; (2) A. obstetricans obstetricans and A. o. pertinax; (3) A. o. boscai and an undescribed taxon (A. o. cf. boscai). These results contradict the mitochondrial tree, due to past mitochondrial captures in A. a. almogavarii (central Pyrenees) and A. o. boscai (central Iberia) by A. obstetricans ancestors during the Pleistocene. Patterns of admixture between subspecies appear far more extensive than previously assumed from microsatellites, causing nomenclatural uncertainties, and even underlying the reticulate evolution of one taxon (A. o. pertinax). All Ammoryctis and Baleaphryne species form shallow clades, so their taxonomy should remain stable. Amid the prevalence of cyto-nuclear discordance among terrestrial vertebrates and the usual lack of resolution of conventional nuclear markers, our study advocates for phylogeography based on next-generation sequencing, but also encourages properly exploring parameter space and sampling schemes when building and analyzing genomic datasets.

Integrating dispersal, breeding and abundance data with graph theory for the characterization and management of functional connectivity in amphibian pondscapes.

Context: Robust assessment of functional connectivity in amphibian population networks is essential to address their global decline. The potential of graph theory to characterize connectivity among amphibian populations has already been confirmed, but the movement data on which modelled graphs rely are often scarce and inaccurate. While probabilistic methods that account for intraspecific variability in dispersal better reflect the biological reality of functional connectivity, they must be informed by systematically recorded individual movement data, which are difficult to obtain for secretive taxa like amphibians. Objectives: Our aim is to assess the applied potential of probabilistic graph theory to characterize overall connectivity across amphibian pondscapes using fine-scale capture-recapture data, and to inform conservation management based on the role of ponds on functional connectivity. Methods: We monitored an amphibian community in a pondscape located in a Spanish "dehesa" for 2 years. Photoidentification was used to build capture histories for individuals of six species, from which dispersal kernels and population sizes were estimated to model probabilistic graphs. Results: We obtained kernels of variable robustness for six species. Node importance for connectivity varied between species, but with common patterns such as shared road crossing areas and the presence of coincident interconnected pond clusters. Conclusions: The combination of photoidentification, capture-recapture data and graph theory allowed us to characterize functional connectivity across the pondscape of study accounting for dispersal variability and identify areas where conservation actions could be most efficient. Our results highlight the need to account for interspecific differences in the study and management of amphibian pondscapes. Supplementary Information: The online version contains supplementary material available at 10.1007/s10980-022-01520-x.

Diverse aging rates in ectothermic tetrapods provide insights for the evolution of aging and longevity.

Comparative studies of mortality in the wild are necessary to understand the evolution of aging; yet, ectothermic tetrapods are underrepresented in this comparative landscape, despite their suitability for testing evolutionary hypotheses. We present a study of aging rates and longevity across wild tetrapod ectotherms, using data from 107 populations (77 species) of nonavian reptiles and amphibians. We test hypotheses of how thermoregulatory mode, environmental temperature, protective phenotypes, and pace of life history contribute to demographic aging. Controlling for phylogeny and body size, ectotherms display a higher diversity of aging rates compared with endotherms and include phylogenetically widespread evidence of negligible aging. Protective phenotypes and life-history strategies further explain macroevolutionary patterns of aging. Analyzing ectothermic tetrapods in a comparative context enhances our understanding of the evolution of aging.

Sex-related differences in aging rate are associated with sex chromosome system in amphibians.

Sex-related differences in mortality are widespread in the animal kingdom. Although studies have shown that sex determination systems might drive lifespan evolution, sex chromosome influence on aging rates have not been investigated so far, likely due to an apparent lack of demographic data from clades including both XY (with heterogametic males) and ZW (heterogametic females) systems. Taking advantage of a unique collection of capture-recapture datasets in amphibians, a vertebrate group where XY and ZW systems have repeatedly evolved over the past 200 million years, we examined whether sex heterogamy can predict sex differences in aging rates and lifespans. We showed that the strength and direction of sex differences in aging rates (and not lifespan) differ between XY and ZW systems. Sex-specific variation in aging rates was moderate within each system, but aging rates tended to be consistently higher in the heterogametic sex. This led to small but detectable effects of sex chromosome system on sex differences in aging rates in our models. Although preliminary, our results suggest that exposed recessive deleterious mutations on the X/Z chromosome (the "unguarded X/Z effect") or repeat-rich Y/W chromosome (the "toxic Y/W effect") could accelerate aging in the heterogametic sex in some vertebrate clades.

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.

Coevolution between MHC Class I and Antigen-Processing Genes in Salamanders.

Proteins encoded by antigen-processing genes (APGs) provide major histocompatibility complex (MHC) class I (MHC-I) with antigenic peptides. In mammals, polymorphic multigenic MHC-I family is served by monomorphic APGs, whereas in certain nonmammalian species both MHC-I and APGs are polymorphic and coevolve within stable haplotypes. Coevolution was suggested as an ancestral gnathostome feature, presumably enabling only a single highly expressed classical MHC-I gene. In this view coevolution, while optimizing some aspects of adaptive immunity, would also limit its flexibility by preventing the expansion of classical MHC-I into a multigene family. However, some nonmammalian taxa, such as salamanders, have multiple highly expressed MHC-I genes, suggesting either that coevolution is relaxed or that it does not prevent the establishment of multigene MHC-I. To distinguish between these two alternatives, we use salamanders (30 species from 16 genera representing six families) to test, within a comparative framework, a major prediction of the coevolution hypothesis: the positive correlation between MHC-I and APG diversity. We found that MHC-I diversity explained both within-individual and species-wide diversity of two APGs, TAP1 and TAP2, supporting their coevolution with MHC-I, whereas no consistent effect was detected for the other three APGs (PSMB8, PSMB9, and TAPBP). Our results imply that although coevolution occurs in salamanders, it does not preclude the expansion of the MHC-I gene family. Contrary to the previous suggestions, nonmammalian vertebrates thus may be able to accommodate diverse selection pressures with flexibility granted by rapid expansion or contraction of the MHC-I family, while retaining the benefits of coevolution between MHC-I and TAPs.

Reconstructing hotspots of genetic diversity from glacial refugia and subsequent dispersal in Italian common toads (Bufo bufo).

Genetic diversity feeds the evolutionary process and allows populations to adapt to environmental changes. However, we still lack a thorough understanding of why hotspots of genetic diversity are so 'hot'. Here, we analysed the relative contribution of bioclimatic stability and genetic admixture between divergent lineages in shaping spatial patterns of genetic diversity in the common toad Bufo bufo along the Italian peninsula. We combined population genetic, phylogeographic and species distribution modelling (SDM) approaches to map ancestral areas, glacial refugia, and secondary contact zones. We consistently identified three phylogeographic lineages, distributed in northern, central and southern Italy. These lineages expanded from their ancestral areas and established secondary contact zones, before the last interglacial. SDM identified widespread glacial refugia in peninsular Italy, sometimes located under the present-day sea-level. Generalized linear models indicated genetic admixture as the only significant predictor of the levels of population genetic diversity. Our results show that glacial refugia contributed to preserving both levels and patterns of genetic diversity across glacial-interglacial cycles, but not to their formation, and highlight a general principle emerging in Mediterranean species: higher levels of genetic diversity mark populations with substantial contributions from multiple genetic lineages, irrespective of the location of glacial refugia.

Phylogenomic inference of species and subspecies diversity in the Palearctic salamander genus Salamandra.

The salamander genus Salamandra is widespread across Europe, North Africa, and the Near East and is renowned for its conspicuous and polymorphic colouration and diversity of reproductive modes. The phylogenetic relationships within the genus, and especially in the highly polymorphic species S. salamandra, have been very challenging to elucidate, leaving its real evolutionary history and classification at species and subspecies levels a topic of debate and contention. However, the distribution of diversity and species delimitation within the genus are critically important for identifying evolutionarily significant units for conservation and management, especially in light of threats posed by the pathogenic chytrid fungus Batrachochytrium salamandrivorans that is causing massive declines of S. salamandra populations in central Europe. Here, we conducted a phylogenomic analysis from across the taxonomic and geographic breadth of the genus Salamandra in its entire range. Bayesian, maximum likelihood and network-based phylogenetic analyses of up to 4905 ddRADseq-loci (294,300 nucleotides of sequence) supported the distinctiveness of all currently recognised species (Salamandra algira, S. atra, S. corsica, S. infraimmaculata, S. lanzai, and S. salamandra), and all five species for which we have multiple exemplars were confirmed as monophyletic. Within S. salamandra, two main clades can be distinguished: one clade with the Apenninic subspecies S. s. gigliolii nested within the Iberian S. s. bernardezi/fastuosa; and a second clade comprising all other Iberian, Central and East European subspecies. Our analyses revealed that some of the currently recognized subspecies of S. salamandra are paraphyletic and may require taxonomic revision, with the Central- and Eastern-European subspecies all being poorly differentiated at the analysed genomic markers. Salamandra s. longirostris - sometimes considered a separate species - was nested within S. salamandra, consistent with its subspecies status. The relationships identified within and between Salamandra species provide valuable context for future systematic and biogeographic studies, and help elucidate critical evolutionary units for conservation and taxonomy.

Evaluating surrogates of genetic diversity for conservation planning.

Protected-area systems should conserve intraspecific genetic diversity. Because genetic data require resources to obtain, several approaches have been proposed for generating plans for protected-area systems (prioritizations) when genetic data are not available. Yet such surrogate-based approaches remain poorly tested. We evaluated the effectiveness of potential surrogate-based approaches based on microsatellite genetic data collected across the Iberian Peninsula for 7 amphibian and 3 reptilian species. Long-term environmental suitability did not effectively represent sites containing high genetic diversity (allelic richness). Prioritizations based on long-term environmental suitability had similar performance to random prioritizations. Geographic distances and resistance distances based on contemporary environmental suitability were not always effective surrogates for identification of combinations of sites that contain individuals with different genetic compositions. Our results demonstrate that population genetic data based on commonly used neutral markers can inform prioritizations, and we could not find an adequate substitute. Conservation planners need to weigh the potential benefits of genetic data against their acquisition costs.

Evaluación de los Sustitutos de la Diversidad Genética para la Planeación de la Conservación Resumen Los sistemas de áreas protegidas deberían conservar la diversidad genética intraespecífica. Ya que para obtener datos genéticos se requieren recursos, se han propuesto distintas estrategias para generar los planes para los sistemas de áreas protegidas (priorizaciones) cuando los datos genéticos no están disponibles. A pesar de lo anterior, dichas estrategias basadas en sustitutos han sido poco evaluadas. Evaluamos la efectividad del potencial de las estrategias basadas en sustitutos cuya base son los datos genéticos de microsatélites obtenidos en toda la Península Ibérica y correspondientes a siete especies de anfibios y a tres de reptiles. La idoneidad ambiental a largo plazo no representó efectivamente los sitios que contienen una diversidad genética alta (riqueza de alelos). Las priorizaciones basadas en la idoneidad ambiental a largo plazo tuvieron un desempeño similar a las priorizaciones aleatorias. Las distancias geográficas y las distancias de resistencia basadas en la idoneidad ambiental contemporánea no siempre fueron sustitutos efectivos para la identificación de las combinaciones de sitios que contienen individuos con composiciones genéticas diferentes. Nuestros resultados demuestran que los datos genéticos de una población basados en marcadores neutrales de uso común pueden informar a las priorizaciones y que no pudimos encontrar un sustituto adecuado. Los planificadores de la conservación necesitan sopesar los beneficios potenciales de los datos genéticos contra sus costos de adquisición.

Comparative assessment of range-wide patterns of genetic diversity and structure with SNPs and microsatellites: A case study with Iberian amphibians.

Reduced representation genome sequencing has popularized the application of single nucleotide polymorphisms (SNPs) to address evolutionary and conservation questions in nonmodel organisms. Patterns of genetic structure and diversity based on SNPs often diverge from those obtained with microsatellites to different degrees, but few studies have explicitly compared their performance under similar sampling regimes in a shared analytical framework. We compared range-wide patterns of genetic structure and diversity in two amphibians endemic to the Iberian Peninsula: Hyla molleri and Pelobates cultripes, based on microsatellite (18 and 14 loci) and SNP (15,412 and 33,140 loci) datasets of comparable sample size and spatial extent. Model-based clustering analyses with STRUCTURE revealed minor differences in genetic structure between marker types, but inconsistent values of the optimal number of populations (K) inferred. SNPs yielded more repeatable and less admixed ancestries with increasing K compared to microsatellites. Genetic diversity was weakly correlated between marker types, with SNPs providing a better representation of southern refugia and of gradients of genetic diversity congruent with the demographic history of both species. Our results suggest that the larger number of loci in a SNP dataset can provide more reliable inferences of patterns of genetic structure and diversity than a typical microsatellite dataset, at least at the spatial and temporal scales investigated.

Telomere attrition with age in a wild amphibian population.

Telomere shortening with age has been documented in many organisms, but few studies have reported telomere length measurements in amphibians, and no information is available for growth after metamorphosis, nor in wild populations. We provide both cross-sectional and longitudinal evidence of net telomere attrition with age in a wild amphibian population of natterjack toads (Epidalea calamita). Based on age-estimation by skeletochronology and qPCR telomere length measurements in the framework of an individual-based monitoring programme, we confirmed telomere attrition in recaptured males. Our results support that toads experience telomere attrition throughout their ontogeny, and that most attrition occurs during the first 1-2 years. We did not find associations between telomere length and inbreeding or body condition. Our results on telomere length dynamics under natural conditions confirm telomere shortening with age in amphibians and provide quantification of wide telomere length variation within and among age-classes in a wild breeding population.

Integrative demographic study of the Iberian painted frog (Discoglossus galganoi): inter-annual variation in the effective to census population size ratio, with insights on mating system and breeding success.

In the face of worldwide amphibian declines, integrative studies combining individual-based information and genetic data represent a powerful approach to produce robust, reliable, and comparable assessments of demographic dynamics. The Iberian painted frog (Discoglossus galganoi) is endemic to Spain and Portugal and shows decreasing population trends across its range, but few studies have attempted to estimate census sizes or assess genetic diversity in wild populations, and little is known about their reproductive biology. We applied an integrative approach based on the combination of capture-mark-recapture data and multilocus genotypes to monitor a breeding population of D. galganoi in central Spain during two consecutive breeding seasons, focusing on the estimation of demographic parameters and their temporal variation. Specifically, we estimated the number of adults (Na ), the effective population size (Ne ), and the effective number of breeders (Nb ), as well as survival and migration rates. We documented a >50% decrease in the estimated number of adults of both sexes between the breeding seasons of 2018 and 2019, probably associated with reduced rainfall in the latter. Estimates of Nb and the Nb /Na ratio were low in both seasons, with a 20-30% decrease in Nb and a 47% increase in the Nb /Na ratio in 2019. Based on the reconstruction of pedigrees from larval and adult genotypes, we provide the first genetic evidence of polygamy in males and females of D. galganoi and the first estimates of breeding success in the species.

Contrasting demographic trends and asymmetric migration rates in a spatially structured amphibian population.

Natural populations often persist at the landscape scale as metapopulations, with breeding units (subpopulations) experiencing temporal extinction and recolonization events. Important parameters to forecast population viability in these systems include the ratio of the effective number of breeders (Nb ) to the total number of adults (Na ) and migration rates among subpopulations. Here, we present the results of a 10-year integrative monitoring program of a metapopulation of the Iberian green frog (Pelophylax perezi) in central Spain. We characterized population dynamics at two main breeding ponds (Gravera and Laguna) using capture-mark-recapture data to estimate Na in each breeding season, and multilocus genotypes to estimate the effective population size (Ne ), Nb , individual breeding success, and migration rates. Both ponds experienced population decline after a dry season, with Gravera subsequently recovering and Laguna suffering a bottleneck associated with genetic impoverishment. In this subpopulation, average allelic richness and private alleles decreased from 2010 (10.87 and 1.67, respectively) to 2018 (8.0 and 0.20). The Nb /Na ratio in Laguna in 2018 was twice as high (0.95) than in Gravera (0.41) or in pre-bottleneck Laguna (0.50), suggesting plasticity or genetic compensation through increased individual breeding success. Migration rates were asymmetric between ponds, with a stronger contribution from Gravera to Laguna (29.9% vs. 16.2% in the opposite direction) that may result in a rescue effect. This study emphasizes the importance of integrative demographic approaches for the monitoring of natural populations based on a better understanding of their spatio-temporal dynamics, which provides valuable information for conservation actions.

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.

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.

Discordant patterns of introgression across a narrow hybrid zone between two cryptic lineages of an Iberian endemic newt.

The study of natural hybrid zones can illuminate aspects of lineage divergence and speciation in morphologically cryptic taxa. We studied a hybrid zone between two highly divergent but morphologically similar lineages (south-western and south-eastern) of the Iberian endemic Bosca's newt (Lissotriton boscai) in SW Iberia with a multilocus dataset (microsatellites, nuclear and mitochondrial genes). STRUCTURE and NEWHYBRIDS analyses retrieved few admixed individuals, which classified as backcrosses involving parental individuals of the south-western lineage. Our results show asymmetric introgression of mtDNA beyond the contact from this lineage into the south-eastern lineage. Analysis of nongeographic introgression patterns revealed asymmetries in the direction of introgression, but except for mtDNA, we did not find evidence for nonconcordant introgression patterns across nuclear loci. Analysis of a 150-km transect across the hybrid zone showed broadly coincident cline widths (ca. 3.2-27.9 km), and concordant cline centres across all markers, except for mtDNA that is displaced ca. 60 km northward. Results from ecological niche modelling show that the hybrid zone is in a climatically homogenous area with suitable habitat for the species, suggesting that contact between the two lineages is unlikely to occur further south as their distributions are currently separated by an extensive area of unfavourable habitat. Taken together, our findings suggest the genetic structure of this hybrid zone results from the interplay of historical (biogeographic) and population-level processes. The narrowness and coincidence of genetic clines can be explained by weak selection against hybrids and reflect a degree of reproductive isolation that is consistent with cryptic speciation.

A rapid rate of sex-chromosome turnover and non-random transitions in true frogs.

The canonical model of sex-chromosome evolution predicts that, as recombination is suppressed along sex chromosomes, gametologs will progressively differentiate, eventually becoming heteromorphic. However, there are numerous examples of homomorphic sex chromosomes across the tree of life. This homomorphy has been suggested to result from frequent sex-chromosome turnovers, yet we know little about which forces drive them. Here, we describe an extremely fast rate of turnover among 28 species of Ranidae. Transitions are not random, but converge on several chromosomes, potentially due to genes they harbour. Transitions also preserve the ancestral pattern of male heterogamety, in line with the 'hot-potato' model of sex-chromosome transitions, suggesting a key role for mutation-load accumulation in non-recombining genomic regions. The importance of mutation-load selection in frogs might result from the extreme heterochiasmy they exhibit, making frog sex chromosomes differentiate immediately from emergence and across their entire length.