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Conservation genetics must find the balance between the technical challenges of DNA sampling while promoting animal welfare. In amphibians, buccal swabs offer a least intrusive source of DNA, but most herpetologists still refrain to use them, partly due to doubts regarding their effectiveness to provide enough material now that next-generation sequencing (NGS) analyses have become routine. In this article, we hope to change their minds through an empirical demonstration that buccal swabs offer DNA amounts and success rates for a widely used NGS approach (RAD-sequencing) that are equivalent to those of more intrusive yet commonly used samples in frogs and toads. We thus call for a shift of DNA sampling practices, and stress the fact that beyond their proven reliability, buccal swabs facilitate the issue of collection permits by increasingly restrictive ethical committees, especially when it comes to endangered species. With this purpose in mind, we share our long-term experience with amphibian buccal swabs through visual and textual pointers.

With five currently recognized species that form several secondary contact zones, slow worms (Anguidae: Anguis) offer a valuable model to study the fate of evolutionary lineages in the face of hybridization and genetic introgression. The relationships between the Western Slow Worm Anguis fragilis and the Italian Slow Worm Anguis veronensis are particularly puzzling. Their respective distributions remain poorly known on the edges of their parapatric ranges, as both species lack external differentiation. Contra earlier mitochondrial phylogenies, new phylogenomic inferences have shown that A. fragilis and A. veronensis are sister taxa, thus casting doubts on their specific status. In this study, we analyze the A. fragilis/veronensis transition in southeastern France, based on one mitochondrial (ND2) and two nuclear (PRLR and HA1) genetic markers in 81 specimens from 61 localities. The ranges of A. fragilis and A. veronensis roughly extends northwest and southeast of the Rhône-Durance valleys, respectively, with clear signs of introgressive hybridization in the areas of contact (notably the eastern parts of the lower Rhône valley). Based on the three molecular markers analyzed, gene flow does not seem to reach outside the narrow hybrid zone, which likely indicates (incomplete) intrinsic reproductive isolation. Hence, we provisionally suggest maintaining A. veronensis as a separate species from A. fragilis. More generally, patterns of genetic divergence, external differentiation, and hybridization (both historical and contemporary) in Anguis ssp. supports a speciation continuum spanning from cryptic, genetically compatible alloparapatric lineages to phenotypically distinct, deeply diverged and fully reproductively isolated taxa able to coexist in sympatry.

Restriction site-Associated DNA sequencing (RADseq) has great potential for genome-wide systematics studies of non-model organisms. However, accurately assembling RADseq reads into orthologous loci remains a major challenge in the absence of a reference genome. Traditional assembly pipelines cluster putative orthologous sequences based on a user-defined clustering threshold. Because improper clustering of orthologs is expected to affect results in downstream analyses, it is crucial to design pipelines for empirically optimizing the clustering threshold. While this issue has been largely discussed from a population genomics perspective, it remains understudied in the context of phylogenomics and coalescent species delimitation. To address this issue, we generated RADseq assemblies of representatives of the amphibian generaDiscoglossus, Rana, LissotritonandTriturususing a wide range of clustering thresholds. Particularly, we studied the effects of the intra-sample Clustering Threshold (iCT) and between-sample Clustering Threshold (bCT) separately, as both are expected to differ in multi-species data sets. The obtained assemblies were used for downstream inference of concatenation-based phylogenies, and multi-species coalescent species trees and species delimitation. The results were evaluated in the light of a reference genome-wide phylogeny calculated from newly generated Hybrid-Enrichment markers, as well as extensive background knowledge on the species’ systematics. Overall, our analyses show that the inferred topologies and their resolution are resilient to changes of the iCT and bCT, regardless of the analytical method employed. Except for some extreme clustering thresholds, all assemblies yielded identical, well-supported inter-species relationships that were mostly congruent with those inferred from the reference Hybrid-Enrichment data set. Similarly, coalescent species delimitation was consistent among similarity threshold values. However, we identified a strong effect of the bCT on the branch lengths of concatenation and species trees, with higher bCTs yielding trees with shorter branches, which might be a pitfall for downstream inferences of evolutionary rates. Our results suggest that the choice of assembly parameters for RADseq data in the context of shallow phylogenomics might be less challenging than previously thought. Finally, we propose a pipeline for empirical optimization of the iCT and bCT, implemented in optiRADCT, a series of scripts readily usable for future RADseq studies.

Biodiversity analyses can greatly benefit from coherent species delimitation schemes and up-to-date distribution data. In this article, we have made the daring attempt to delimit and map described and undescribed lineages of anuran amphibians in the Eastern Palaearctic (EP) region in its broad sense. Through a literature review, we have evaluated the species status considering reproductive isolation and genetic divergence, combined with an extensive occurrence dataset (nearly 85k localities). Altogether 274 native species from 46 genera and ten families were retrieved, plus eight additional species introduced from other realms. Independent hotspots of species richness were concentrated in southern Tibet (Medog County), the circum-Sichuan Basin region, Taiwan, the Korean Peninsula and the main Japanese islands. Phylogeographic breaks responsible for recent in situ speciation events were shared around the Sichuan Mountains, across Honshu and between the Ryukyu Island groups, but not across shallow water bodies like the Yellow Sea and the Taiwan Strait. Anuran compositions suggested to restrict the zoogeographical limits of the EP to East Asia. In a rapidly evolving field, our study provides a checkpoint to appreciate patterns of species diversity in the EP under a single, spatially explicit, species delimitation framework that integrates phylogeographic data in taxonomic research.

Old World tree frogs from the family Rhacophoridae, one of the most species-rich groups of amphibians worldwide, are becoming a model in ecological and evolutionary research, notably for their tremendous diversity of breeding systems. In this study, we provide the most comprehensive temporally and spatially explicit phylogeographical framework for the tropical neo-genus Zhangixalus, with a specific focus on the evolution of inter- and intraspecific polymorphism in their egg-laying strategies. We combined ~500 sequences spanning ~4 kb of mitochondrial genes, representative of nearly all known Zhangixalus species from 373 localities. We recovered a robust timetree featuring ~60 phylogeographical lineages assigned to 36 taxa, illustrating a vast under-appreciation of the diversity of the genus. Patterns of diversification were intimately linked to climatic trends since the Mid-Miocene, leading to a remarkable biogeographical scenario for the onset of Taiwanese diversity. Finally, in contrast to the macro-evolution of conserved breeding strategies in rhacophorids, the striking variation of nesting behaviour documented across Zhangixalus species, whereby foamy egg nests are alternatively fossorial, deposited on the edge of the water or suspended on the overhanging vegetation, seems to have evolved rapidly from arboreal ancestors in all major clades, implying micro-evolutionary processes and/or phenotypic plasticity.

Genetic variation is often lower at high latitudes, which may compromise the adaptability and hence survival of organisms. Here we show that genetic variability is negatively correlated with northern latitude in European green toads (Bufotes viridis). The result holds true for both putatively neutral microsatellite variation and supposedly adaptive MHC Class IIB variation. In particular, our findings have bearing on the conservation status of this species in Sweden, on the northern limit of its distribution where local populations are small and fragmented. These genetically impoverished populations are closely related to other populations found around the Baltic Sea basin. The low neutral and adaptive variation in these fringe populations compared to population at central ranges confirms a pattern shared across all other amphibians so far studied. In Sweden, the situation of green toads is of concern as the remaining populations may not have the evolutionary potential to cope with present and future environmental challenges.

Linnaean and Wallacean shortfalls (Uncertainties on species taxonomy and distribution, respectively) are major factors hampering efficient conservation planning in the current context of biodiversity erosion. These shortfalls concern even widespread and abundant species in relatively well-studied regions such as the Mediterranean biodiversity hotspot which still hosts a large fraction of unrecognised biodiversity, notably in small vertebrates. Species delimitations have long been based on phylogenetic analyses of a small number of standard markers, but accurate lineage identification in this context can be obscured by incomplete lineage sorting, introgression or isolation by distance. Recently, integrative approaches coupling various sets of characters or analyses of contact zones aiming at estimating reproductive isolation (RI) have been advocated instead. Analyses of introgression patterns in contact zone with genomic data represent a powerful way to confirm the existence of independent lineages and estimate the strength of their RI at the same time. The Spiny-footed Lizard Acanthodactylus erythrurus (Schinz, 1833) is widespread in the Iberian Peninsula and the Maghreb and exhibits a large amount of genetic diversity, although the precise number and distribution of its genetic lineages remain poorly understood. We applied a RADseq approach to obtain a genome wide SNPs dataset on a contact zone in central Morocco between the previously described Rif and Middle-Atlas lineages. We show that these two lineages exhibit strong RI across this contact zone, as shown by the limited amount and restricted spatial extant of gene flow. We interpret these results as evidence for species-level divergence of these two lineages. Our study confirms the usefulness of RADseq approaches applied on contact zones for cryptic diversity studies and therefore to resolve Linnaean and Wallacean shortfalls.

As reflected by the two rules of speciation (Haldane's rule and the large X-/Z-effect), sex chromosomes are expected to behave like supergenes of speciation: they recombine only in one sex (XX females or ZZ males), supposedly recruit sexually antagonistic genes and evolve faster than autosomes, which can all contribute to pre-zygotic and post-zygotic isolation. While this has been mainly studied in organisms with conserved sex-determining systems and highly differentiated (heteromorphic) sex chromosomes like mammals, birds and some insects, these expectations are less clear in organismal groups where sex chromosomes repeatedly change and remain mostly homomorphic, like amphibians. In this article, we review the proposed roles of sex-linked genes in isolating nascent lineages throughout the speciation continuum and discuss their support in amphibians given current knowledge of sex chromosome evolution and speciation modes. Given their frequent recombination and lack of differentiation, we argue that amphibian sex chromosomes are not expected to become supergenes of speciation, which is reflected by the rarity of empirical studies consistent with a ‘large sex chromosome effect’ in frogs and toads. The diversity of sex chromosome systems in amphibians has a high potential to disentangle the evolutionary mechanisms responsible for the emergence of sex-linked speciation genes in other organisms. This article is part of the theme issue ‘Genomic architecture of supergenes: causes and evolutionary consequences’.

Crocodile newts (Echinotriton and Tylototriton) are emblematic of the biodiversity of South-East Asia. In this study, we provide an account of their evolution, diversity and phylogeography based on mitochondrial (16.2 kb) and nuclear sequences (3.2 kb), combining barcoding data from nearly 1200 specimens collected over ~180 localities, representative of all lineages known to date. While phylogenies of nuclear data lacked resolution, the mitochondrial tree showed dynamic allopatric speciation that followed climate cooling during the last 10 million years, a pattern shared with many other tropical and subtropical amphibians. In crocodile newts, this implies weak dispersal and local adaptation as contributing factors of isolation, and was accompanied by macro- and micro-evolutionary changes in reproductive behaviours. Specifically, our resolved phylogeny of Tylototriton allowed the tracing of a major shift from conserved habits of terrestrial clutch-spawning towards more plastic strategies involving both aquatic and land spawning of single eggs. Using this framework, we then revised the taxonomy of crocodile newts by describing a new subgenus and two new species. Our study provides a basis to guide future phylogeographic, speciation and conservation research on these iconic amphibians.

The dynamic biogeography of glacial refugia may cause complex patterns of genetic admixture between parapatric taxa, which in turn can mislead their systematics, diversity, and distributions. We investigated this issue for green toads (Bufotes) inhabiting the circum-Aegean region, a biodiversity hotspot of the Eastern Mediterranean. A previous phylogeographic study based on mitochondrial and microsatellite loci identified the hybrid zone between the European (viridis) and Anatolian (sitibundus) lineages of B. viridis all over the Balkan Peninsula, but subsequent range-wide genomic analyses (>1000 SNPs) located this transition in Turkey, a thousand kilometers eastwards. In order to clarify the diversity and taxonomy of the circum-Aegean populations, we reconciled these conflicting findings by integrating previous data with pure sitibundus individuals. Our results confirmed that the viridis/sitibundus hybrid zone extends in Western Anatolia, but that southeastern European populations feature cytonuclear discordances and a high and structured microsatellite diversity. This remarkable situation may stem from a massive geographic displacement of the hybrid zone during the last glacial fluctuations, an underappreciated yet seemingly common feature among the herpetofauna of the region. Our study thus contributes to the rising view that mitochondrial DNA can be a poor predictor of current phylogeographic structure, hence the need for genomic data, especially for narrowly distributed taxa. Finally, the analyses unambiguously support the distinction of a micro-endemic clade of green toads unique to some Cyclades islands, for which we provide a formal taxonomic description.

An illustration of the human footprint on biodiversity are the faunal movements that have accompanied commercial and cultural exchanges between civilizations throughout history. In this article, we provide an integrative review of biogeographical and archaeological knowledge to understand these processes for the Mediterranean tree frog (Hyla meridionalis), an African species that has reached several regions of south-western Europe and associated islands. By re-analysing molecular (multilocus mitochondrial and genome-wide markers) and bioclimatic (ecological niche modelling) data, we give a comprehensive picture of the genetic diversity, structure and environmental suitability of H. meridionalis across its natural and exotic ranges, which in turn offer specific clues to the putative routes of colonization and associated events. Long-term monitoring efforts suggest northwards shifts of local range margins, potentially due to global warming, and we further demonstrate that this species is the most frequent amphibian to travel via the food supply chain in Western Europe. High dispersal ability, ecological tolerance and proximity to human settlements have made H. meridionalis a recurrent witness to the complexity and diversity of the civilizations that ruled the Western Mediterranean.

While estimates of genetic divergence are increasingly used in molecular taxonomy, hybrid zone analyses can provide decisive evidence for evaluating candidate species. Applying a population genomic approach (RAD-sequencing) to a fine-scale transect sampling, we analyzed the transition between two Iberian subspecies of the common midwife toad (Alytes obstetricans almogavarii and A. o. pertinax) in Catalonia (northeastern Spain), which putatively diverged since the Plio-Pleistocene. Their hybrid zone was remarkably narrow, with extensive admixture restricted to a single locality (close to Tarragona), and congruent allele frequency clines for the mitochondrial (13 km wide) and the average nuclear genomes (16 km wide). We also fitted clines independently for 89 taxon-diagnostic SNPs: Most of them behave like the nuclear background, but a subset (13%) is completely impermeable to gene flow and might be linked to barrier loci involved in hybrid incompatibilities. Assuming that midwife toads are able to disperse in the area of contact, we conclude that these taxa experience partial reproductive isolation and represent incipient species, i.e. Alytes almogavarii and Alytes obstetricans. Interestingly, their evolutionary age and mitochondrial divergence fall below the thresholds proposed in molecular systematics studies, emphasizing the difficulty of predicting the outcome of secondary contacts between young lineages entering the grey zone of speciation., This study was funded by the Swiss National Science Foundation (fellowship P2LAP3_171818 to CD, and grant 31003A_166323 to N. Perrin).

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., Our study was funded by the Swiss National Science Foundation, under grant 31003A_166323 to NP andfellowship P2LAP3_171818 to CD. BA was supported by a fellowshipfrom the Fyssen Foundation, and MV by the Deutsche Forschungsgemeinschaft (grant VE247/16-1—HO 3492/6-1) in the framework of the “TaxonOmics” priority program.

Genomic tools like RAD-sequencing can provide the resolution to understand the complex genetic patterns underlying cryptic biological invasions. Here we use this method to clarify the genetic consequences of a rampant invasion by the Italian pool frog (Pelophylax bergeri) north of the Alps, threatening the native P. lessonae and its hybridogenetic hybrid P. esculentus. Patterns of genetic structure were consistent with massive genetic introgression, except for an isolated mountain valley in Switzerland (Joux). Moreover, alien alleles also reached the hybridogens, which should be considered non-native as well. Potentially initiated during antic times, this hybrid swarm likely extends all over W-Europe and rises among the largest amphibian invasions documented on the old continent. Invasion genomics thus hold great promises to reliably inform conservationists, especially in animal groups for which conventional genetic markers may not always be diagnostic between closely-related, morphologically similar species.

Phylogeographical breaks can be viewed as regional hotspots of diversity where the genetic integrity of incipient species is put to the test. We focus on an understudied species transition from the Middle East, namely the Dead Sea Rift in the Levant region, which presumably divided the tree frogs Hyla savignyi and H. felixarabica. Combining multilocus genetic analyses (mitochondrial DNA and RAD-sequencing) with ecological niche modelling, we test whether the rift effectively acts as a biogeographical barrier preventing this pair from admixing and merging. The answer is yes: despite weak signs of introgression, all parapatric populations were assigned to either species without cyto-nuclear discordance. Yet, the projected distributions under present and glacial conditions largely overlapped in the area, meaning their current parapatric ranges do not represent an ecological transition. Instead, we hypothesize that H. savignyi and H. felixarabica are maintained apart by limited opportunities for dispersal across the barren Jordan Valley, combined with advanced reproductive isolation. Therefore, the Dead Sea Rift may represent a rare phylogeographical break, and we encourage international efforts to assess its contribution to the rich biodiversity of the Middle East.

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.

The sociopolitical acceptance necessary for the conservation of controversial species requires scientific knowledge that disentangles empirical facts from myth and misinformation. An epitome of such, the gray wolf (Canis lupus) had been eradicated from most of Western Europe by the early twentieth century. However, a few mysteriously re-appeared in the Alps throughout the twentieth century, leading to systematic hunts encouraged by popular folklore and massive waves of panic. These historical events are reminiscent of the hostile context now surrounding the recolonization of the wolf across former ranges. Through historical DNA sequencing of five rare museum specimens shot post-WWII, we tell the true story of these mystery beasts. The oldest ones (1947–1954) were just the very last survivors of an endemic, extremely resilient wolf population, thought to be extinct decades earlier, while recent ones (1978–1990) most likely originated from captivity. This parable reminds that today more than ever, scientific evidence is necessary to conduct an objective societal debate over the management and conservation of controversial species.

Abstract Biogeographic processes have led to different evolutionary taxa occurring in the northern and southern edges of the Alpine Mountains in Western Europe. The integrity of this diversity is being challenged by frequent human-mediated trans-alpine translocations, sometimes leading to biological invasions. Several alien terrestrial vertebrates of south Alpine origins (Italy, Swiss Ticino) are causing damages to native north Alpine fauna. In this paper, we used molecular tools to characterize the understudied case of the Mediterranean smooth newt (Lissotriton vulgaris meridionalis) expanding in the outskirts of Geneva since its introduction before 1975. Mitochondrial and nuclear DNA sequencing suggest that these exotic populations are a mixture between two diverged L. v. meridionalis lineages from central Italy, and traces of potential hybridization with the native L. v. vulgaris was detected. This situation echoes many other trans-alpine alien introductions. We review all comparable cases of southern to northern Alps introductions in vertebrates, including seven reptiles and four amphibians. The majority of south alpine alien lineages were presumably imported voluntarily by enthusiasts and appear to perform better in the disturbed habitats found in the anthropogenic landscapes of Western Europe compared to their native north Alpine counterparts. Most pose serious threats to related species of similar ecology, through direct competition, predation and introgressive hybridization. Difficulties to detect alien species on time lead to significant conservation costs. Better education together with more appropriate and reactive management plans will be necessary to limit the impact of future alien introductions.

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 (Ranaand 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., This study was funded by the Swiss NSF (Grant No. 31003A_166323 to N.P. and Fellowship No. P2LAP3_171818 to C.D.) and the Russian Foundation for Basic Research (Grant No. 20-04-00918 to S.N.L.). C.D. and M.V. were further supported by the Taxon-Omics priority program (SPP1991) of the Deutsche Forschungsgemeinschaft (Grant No. VE247/19-1)

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.

The growing interest in the lability of sex determination in non-model vertebrates such as amphibians and fishes has revealed high rates of sex chromosome turnovers among closely related species of the same clade. Can such lineages hybridize and admix with different sex-determining systems, or could the changes have precipitated their speciation? We addressed these questions in incipient species of toads (Bufonidae), where we identified a heterogametic transition and characterized their hybrid zone with genome-wide markers (RADseq). Adult and sibship data confirmed that the common toad B. bufo is female heterogametic (ZW), while its sister species the spined toad B. spinosus is male heterogametic (XY). Analysis of a fine scale transect across their parapatric ranges in southeastern France unveiled a narrow tension zone (∼10 km), with asymmetric mitochondrial and nuclear admixture over hundreds of kilometers southward and northward, respectively. The geographic extent of introgression is consistent with an expansion of B. spinosus across B. bufo’s former ranges in Mediterranean France, as also suggested by species distribution models. However, widespread cyto-nuclear discordances (B. spinosus backrosses carrying B. bufo mtDNA) run against predictions from the dominance effects of Haldane's rule, perhaps because Y and W heterogametologs are not degenerated. Common and spined toads can thus successfully cross-breed despite fundamental differences in their sex determination mechanisms, but remain partially separated by reproductive barriers. Whether and how the interactions of their XY and ZW genes contribute to these barriers shall provide novel insights on the debated role of labile sex chromosomes in speciation.

The last species list of the European herpetofauna was published by Speybroeck, Beukema and Crochet (2010). In the meantime, ongoing research led to numerous taxonomic changes, including the discovery of new species-level lineages as well as reclassifications at genus level, requiring significant changes to this list. As of 2019, a new Taxonomic Committee was established as an official entity within the European Herpetological Society, Societas Europaea Herpetologica (SEH). Twelve members from nine European countries reviewed, discussed and voted on recent taxonomic research on a case-by-case basis. Accepted changes led to critical compilation of a new species list, which is hereby presented and discussed. According to our list, 301 species (95 amphibians, 15 chelonians, including six species of sea turtles, and 191 squamates) occur within our expanded geographical definition of Europe. The list includes 14 non-native species (three amphibians, one chelonian, and ten squamates).

The original diversity of Pelophylax water frogs has been compromised by multiple biological invasions all over Western Europe. For the European pool frog (P. lessonae), the Joux Valley—a 30 km highland depression in northwestern Switzerland—stands as the last stronghold spared by exotic lineages. In order to manage P. lessonae in the valley, we combined traditional field surveys with environmental DNA metabarcoding and mapped the regional distribution of amphibian species. Both approaches concurred that P. lessonae persists at a single isolated site (Pontet). Continuous monitoring of this population throughout the spring and summer 2019 informed on their wintering quarters (most likely the forest litter immediately surrounding the breeding pond), as well as the timing of migration (end of April), breeding (June), and larval development (June–August). In parallel, we experimented the first use of drone technology for amphibian surveillance: 30 adults were individually counted during an aerial survey at the peak of the breeding season, confirming the small size of the population. Finally, we compared some biotic and abiotic properties among water bodies throughout the valley and flagged a few sites that were ecologically similar to Pontet. In a landscape dominated by pastoral activities where Pelophylax dispersal is virtually impossible, these could be candidates for future translocation efforts. Our study illustrates the application of next-generation monitoring techniques for the urgent management of threatened species and stresses the need for reevaluating the conservation status of P. lessonae in Western Europe, where it appears to subsist in low numbers at one last locality.

Mediterranean tree frogs, Hyla gr. meridionalis Boettger, 1874 (Anura: Hylidae) are widespread around the Western Mediterranean Basin, where they naturally occur across the Maghreb (Morocco, Algeria, Tunisia). Individuals of diverse Moroccan origins have been introduced and have expanded throughout the Iberian Peninsula, southern France and northern Italy (Liguria), but also on the Canary and Balearic archipelagos (Recuero et al. 2007; Dufresnes et al. 2019). Early molecular studies uncovered several mitochondrial lineages and suggested a major cryptic diversification within this taxon, with Tunisian and eastern Algerian (Numidia) populations carrying deeply divergent haplotypes compared with the rest of the range (Recuero et al. 2007; Stöck et al. 2008; Stöck et al. 2012). While intron markers showed little differentiation (Stöck et al. 2008; Stöck et al. 2012), genome-wide data obtained from RAD-sequencing have supported the deep split suspected from mtDNA (Dufresnes et al. 2018).

Because it is indicative of reproductive isolation, the amount of genetic introgression across secondary contact zones is increasingly considered in species delimitation. However, patterns of admixture at range margins can be skewed by the regional dynamics of hybrid zones. In this context, we posit an important role for phylogeographic history: hybrid zones located within glacial refugia (putatively formed during the Late-Pleistocene) should be better defined than those located in post-glacial or introduced ranges (putatively formed during the Holocene and the Anthropocene). We test this hypothesis in a speciation continuum of tree frogs from the Western Palearctic (Hyla), featuring ten identified contacts between species spanning Plio-Pleistocene to Miocene divergences. We review the rich phylogeographic literature of this group and examine the overlooked transition between H. arborea and H. molleri in Western France using a multilocus dataset. Our comparative analysis supports a trend that contacts zones resulting from post-glacial expansions and human translocations feature more extensive introgression than those established within refugial areas. Integrating the biogeographic history of incipient species, i.e. their age since first contact together with their genetic divergence, thus appears timely to draw sound evolutionary and taxonomic inferences from patterns of introgression across hybrid zones.

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., We thank C. Cabido, I. Garin, A. Gosá, F. Martínez, J. Rubines, X. Rubio, and G. Sánchez‐Montes for help in sample collection. This study was funded by the Swiss National Science Foundation (fellowship P2LAP3_171818 to CD, and grant 31003A_166323 to NP). MV was supported by the Deutsche Forschungsgemeinschaft (grant VE247/16‐1–HO 3492/6‐1) in the framework of the ‘TaxonOmics' priority program, SNL by the RFBR (grant 20‐04‐00918), and AGN by the Ministerio de Ciencia, Innovación y Universidades, Gobierno de España (MICINN grants CGL2012‐40246 and CGL2017‐86924‐P).

The radiation of Palearctic green toads (Bufotes) holds great potential to evaluate the role of hybridization in phylogeography at multiple stages along the speciation continuum. With fifteen species representing three ploidy levels, this model system is particularly attractive to examine the causes and consequences of allopolyploidization, a prevalent yet enigmatic pathway towards hybrid speciation. Despite substantial efforts, the evolutionary history of this species complex remains largely blurred by the lack of consistency among the corresponding literature. To get a fresh, comprehensive view on Bufotes phylogeography, here we combined genome-wide multilocus analyses (RAD-seq) with an extensive compilation of mitochondrial, genome size, niche modelling, distribution and phenotypic (bioacoustics, morphometrics, toxin composition) datasets, representing hundreds of populations throughout Eurasia. We provide a fully resolved nuclear phylogeny for Bufotes and highlight exceptional cyto-nuclear discordances characteristic of complete mtDNA replacement (in 20% of species), mitochondrial surfing during post-glacial expansions, and the formation of homoploid hybrid populations. Moreover, we traced the origin of several allopolyploids down to species level, showing that all were exclusively fathered by the West Himalayan B. latastii but mothered by several diploid forms inhabiting Central Asian lowlands, an asymmetry consistent with hypotheses on mate choice and Dobzhansky-Muller incompatibilities. Their intermediate call phenotypes potentially allowed for rapid reproductive isolation, while toxin compositions converged towards the ecologically-closest parent. Across the radiation, we pinpoint a stepwise progression of reproductive isolation through time, with a threshold below which hybridizability is irrespective of divergence (15My). Finally, we clarified the taxonomy of Bufotes (including genetic analyses of type series) and formally described two new species, B. cypriensis sp. nov. (endemic to Cyprus) and B. perrini sp. nov. (endemic to Central Asia). Embracing the genomic age, our framework marks the advent of a new exciting era for evolutionary research in these iconic amphibians. ispartof: MOLECULAR PHYLOGENETICS AND EVOLUTION vol:141 ispartof: location:United States status: published

Dobzhansky-Muller (DM) incompatibilities involving sex chromosomes have been proposed to account for Haldane's rule (lowered fitness among hybrid offspring of the heterogametic sex) as well as Darwin's corollary (asymmetric fitness costs with respect to the direction of the cross). We performed simulation studies of a hybrid zone to investigate the effects of different types of DM incompatibilities on cline widths and positions of sex-linked markers. From our simulations, X-Y incompatibilities generate steep clines for both X-linked and Y-linked markers; random effects may produce strong noise in cline center positions when migration is high relative to fitness costs, but X- and Y-centers always coincide strictly. X-autosome and Y-autosome incompatibilities also generate steep clines, but systematic shifts in cline centers occur when migration is high relative to selection, as a result of a dominance drive linked to Darwin's corollary. Interestingly, sex-linked genes always show farther introgression than the associated autosomal genes. We discuss ways of disentangling the potentially confounding effects of sex biases in migration, we compare our results to those of a few documented contact zones, and we stress the need to study independent replicates of the same contact zone.

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.

The marsh frog (Pelophylax ridibundus sensu lato) is the number one amphibian invader in Western Europe. In Switzerland, marsh frogs were introduced in the 1950-1960s and progressively colonized most of the northern parts of the country. We investigated this invasion using molecular tools. We mapped the cryptic presence of three monophyletic mitochondrial lineages (P. ridibundus, P. kurtmuelleri and P. c. f. bedriagae from southeastern Europe) consistent with registered importations by a local frog-leg industry. High nuclear diversity supports that invasive frogs probably originated from genetically-rich import batches and patterns of population differentiation confirm that multiple independent introduction sites were involved. Moreover, several lines of evidence suggest occasional hybridization with local hybridogenetic water frogs. This invasion emphasizes the issues of frequent amphibian releases and translocations at the international and regional scale for commercial and recreational purposes, and stresses for more adequate legislation, control and information to the general public. Given the parallel invasion by alien pool frogs (i. e., the Italian P. bergeri has replaced the local P. lessonae), the situation of water frogs in Switzerland is critical. The water frog complex makes an alarming symbol of the anthropogenic mark left on wildlife diversity and distributions.

Hybridogenesis is a special mode of hybrid reproduction where one parental genome is eliminated and the other is transmitted clonally. We propose that this mechanism can perpetuate the genome of extinct species, based on new genetic data from Pelophylax water frogs. We characterized the genetic makeup of Italian hybridogenetic hybrids (P. kl. hispanicus and esculentus) and identified a new endemic lineage of Eastern-Mediterranean origin as one parental ancestor of P. kl. hispanicus. This taxon is nowadays extinct in the wild but its germline subsists through its hybridogenetic descendant, which can thus be considered as a “semi living fossil”. Such rare situation calls for realistic efforts of de-extinction through selective breeding without genetic engineering, and fuels the topical controversy of reviving long extinct species. “Ghost” species hidden by taxa of hybrid origin may be more frequent than suspected in vertebrate groups that experienced a strong history of hybridization and semi-sexual reproduction.