Through genomic phylogeography, previously unrecognised biodiversity can be revealed. The alpine newt serves as a case in point: this taxon carries highly distinct mtDNA clades and has a severely fragmented range. We obtain genome-wide data with target enrichment by sequence capture to delineate cryptic species and disentangle their phylogenetic relationships. Furthermore, we explore potential niche divergence and glaciation-driven distribution dynamics. On the basis of the uncovered genetic structure, we distinguish five main groups that we propose should be treated as distinct species. Limited interspecific genetic admixture often occurs away from current contact zones between these species, in line with a scenario of current range reduction, compared to the Last Glacial Maximum. A decline in suitable habitat also explains the fragmented nature of current species ranges. We uncover pronounced mito-nuclear discordance. We show that an ancient mtDNA lineage endemic to the Vlasina Plateau on the border between Serbia and Bulgaria, previously interpreted to be a 'ghost lineage', in fact represents a distinct species. However, it is nested considerably deeper inside the alpine newt species complex than mtDNA suggests. Our study illustrates how genomic phylogeography allows intricate evolutionary histories to be untangled.

Abstract Taxonomic identification of introduced amphibian populations – a crucial first step for conservation management – often relies on mitochondrial DNA (mtDNA) barcoding, which can be intricate with recent histories of introgressive hybridization. The European green toad ( Bufotes viridis ) is characterized by cryptic phylogeographic subspecies and extensive cyto-nuclear discordance, particularly between European B. v. viridis and Asian B. v. sitibundus . Using an extensive reference dataset (2440 individuals from 730 localities) of two mtDNA genes, and haplotype-level analysis, we identified two well-established allochthonous green-toad populations in eastern and southern France. Both are assigned to B. v. viridis , despite distinct mitochondrial lineages: a typical viridis haplotype and a sitibundus haplotype largely associated with B. v. viridis ; their native distributions suggest independent origins. This case shows that mtDNA barcoding can remain informative when native diversity is well characterized and highlights green toads as a pioneer species highly adaptable to human-modified landscapes that warrants attention in bioinvasion monitoring.

Measuring gene flow across hybrid zones can provide a direct evaluation of reproductive isolation (RI) between evolutionary divergent lineages. Geographic-explicit modelling of gene flow across hybrid zones, known as cline analysis, thus offers a gold standard for species delimitation under the biological species criterion. We here relate divergence time to post-zygotic RI as inferred from hybrid zone cline width from studies on amphibians, reptiles, birds, mammals and insects. The data confirm an overall significant negative correlation between cline width and divergence time (within-clade consistently significant only in amphibians). Out of 108 hybrid zones, hybridizing taxa were younger than 10 million years (Ma) in 104 cases, and younger than 7 Ma in 94 cases, implying that older lineage pairs are usually reproductively isolated. In amphibians, the correlation suggested that shallow and steep hybrid zones, characteristic of subspecies and species, are found with divergence times of < 2.6 Ma and > 7 Ma, respectively. In all taxa we revealed a trend of young lineages being increasingly described as new species over time by simulating past sets of taxonomically named species. Overall, the inferred proportion of species with post-Pliocene ages was highest in birds (27.3%) and mammals (21.8%), but lower in amphibians and squamates (< 10%). In amphibians we document a 56% increase from 1980 to 2010 in the number of newly described species that fall below the hybrid zone-derived divergence reference point of 2.6 Ma. While this trend so far impacts only a small proportion of the total amount of amphibian species, we commend that taxonomic hypotheses that delimit Pleistocene-aged lineages as species, or conversely, that consider lineages with Miocene ages or older as conspecific, should be carefully evaluated to avoid taxonomic inflation and deflation.

Madagascar-endemic chameleons with large, conspicuous occipital lobes currently comprise 12 species that were traditionally classified in the phenetic Calumma cucullatum group. We here use a combination of DNA sequences from Restriction-Associated DNA sequencing (RADseq), museomics shotgun sequencing from historical name-bearing types, and Sanger-sequenced mitochondrial and nuclear gene fragments, to revisit the phylogeny and species delimitation in this group. We find robust phylogenomic evidence for polyphyly of the group, with C. cucullatum sister to the C. furcifer group, and species of the C. nasutum group intercalated between C. malthe and the remainder of ingroup species. Furthermore, according to our mitochondrial tree, C. tsycorne does not belong to the group. Several species such as C. brevicorne and C. malthe are recovered as paraphyletic in the mitochondrial tree while being unambiguously monophyletic in the phylogenomic tree; this mito-nuclear discordance may reflect ancestral introgressive hybridization. In our phylogenomic estimations, most currently recognized species are inferred as deep monophyletic lineages, and the RADseq clustering analysis recovered these as well-defined units. Exceptions were C. jejy, which is placed as sister taxon to the morphologically distinct C. peltierorum with rather shallow sequence divergence, and the C. brevicorne / C. crypticum complex. Combining molecular and morphological data suggests that populations of C. malthe from northern Madagascar represent a distinct species, here described as C. krystalae sp. nov. Our data reveal that chameleons, despite their elaborate phenotypic differences used in intraspecific signalling and their interspecific genital differences, can be characterized by complex evolutionary patterns probably involving hybridization and introgression.

The Himalaya harbors a unique faunal diversity shaped by major geological events and the resulting paleoenvironmental changes. Retracing the evolution of Himalayan species thus offers a window to understand how mountain formation and climatic shifts influenced their diversification. Using genomic analyses and paleoclimatic species distribution modeling, we reconstructed the evolutionary history of the Himalayan toad (Duttaphrynus himalayanus), a montane amphibian widely distributed across the Himalayan range. Our analyses reveal two major phylogeographic lineages in D. himalayanus-east and west of the Thakkhola basin (upper Kali Gandaki River)-that diverged during the Late Pliocene (similar to 3.2 Mya) and seemingly show traces of recent gene flow. Patterns of divergence in the eastern lineage suggests additional phylogeographic structure between Nepalese and Bhutan ranges in the central Himalaya. The two lineages show differences in climatic space (especially precipitation), but not necessarily ecological divergence, suggesting that topographic barriers are a primary factor of differentiation. Our results suggest that D. himalayanus is a relatively young montane lineage, compared to other Himalayan amphibians, that diversified within the uplifted Himalaya rather than originating from paleo-Tibetan or Indochinese ancestors. The resulting hidden diversity seems primarily shaped by topographic barriers, notably the Kali Gandaki, despite episodic connectivity that was possibly facilitated by paleolake corridors formed during the Late Quaternary. These findings imply that taxonomic revisions in D. himalayanus are required, and call to further similar studies in other Himalayan herpetofauna to identify common drivers of biogeographic diversification across these mountain environments.

Abstract The olm, Proteus anguinus , is one of the most iconic vertebrates of Europe. Since its scientific description in 1768, its extreme adaptations to subterranean life in the enigmatic underground waters of the Dinaric Karst have fascinated scholars and naturalists worldwide for more than two centuries. Yet, the evolution and taxonomic diversity of its populations remained largely unresolved, as complex patterns of phenotypic differentiation and molecular variation collide with an intricate nomenclatural history. In this work, we review recent biogeographic advances, notably based on range-wide phylogeographic analyses implementing genomic data, and combined with distributional data, species distribution modelling, red list assessment, morphological analyses and a critical species delimitation evaluation, recognize nine species in immediate danger of extinction. Based on an extensive nomenclatural search, we determined that six correspond to available names and three are newly described in this work. These species all exhibit highly restricted distributions within the Dinaric karst, collectively representing an exceptional example of subterranean microendemism among European vertebrates. Linking influential historical research with modern zoological methods, this revision provides an up-to-date science-based taxonomic framework for future studies of comparative evolution and ecology of Proteus , while establishing a sound basis for species-level conservation management of one of Europe’s most remarkable vertebrate diversifications.

Abstract Comparative phylogeography provides a powerful framework to identify the historical processes shaping biodiversity hotspots by testing whether co-distributed species exhibit shared patterns of diversification. Southeast Asia harbors exceptional biodiversity, yet the extent to which common paleogeographic and climatic drivers have structured diversification across taxa remains poorly understood. Here, we investigated the evolutionary history of five widespread Microhyla species complexes distributed across the Indochinese Peninsula and adjacent regions using dense mitochondrial sampling (1,388 individuals) combined with genome-scale ddRAD sequencing (280 individuals). Across all complexes, both approaches recovered multiple lineages and remarkably congruent phylogeographic breaks and contact/intergradation zones associated with major Indochinese regions, including Myanmar, the Tenasserim-Malay Peninsula, southern Vietnam, and northern Vietnam-southern China, supporting the hypothesis that Indochina functions as a mosaic of stable biogeographic units. However, lineage divergence varied substantially among complexes, suggesting that common biogeographic drivers interacted with species-specific demographic histories. Phylogenomic analyses and cyto-nuclear discordance further revealed historical introgression in every complex, indicating that diversification involved both long-term allopatric isolation and reticulate evolution. These findings portray the Indochinese biodiversity hotspot as a dynamic evolutionary system where cycles of fragmentation and reconnection have repeatedly reshaped lineage boundaries. Moreover, the complex phylogeographic structure recovered exemplifies the urgent need for taxonomic revisions, for which genome-scale data provide an essential framework to validate mitochondrial hypotheses and assess admixture patterns for species delimitation. Finally, regions such as the southern Annamites and Tenasserim Hills emerged as recurrent hotspots of genetic diversity across independent lineages, highlighting their importance for conserving not only species/lineage richness but also the evolutionary processes and adaptive potential that sustain biodiversity.

Abstract The boundary between the Palearctic and Oriental biogeographic realms is one of the oldest unresolved problems in Eurasian biogeography. In Central and South Asia, this transition is remarkable and has often been linked to the Indus River Valley, yet the role of adjacent mountain systems and arid landscapes has remained largely untested because critical regions, especially Afghanistan, have been chronically under-sampled due to difficult, long-term socio-political situation. Here we use a unique amphibian material from Afghanistan and adjacent northwestern Pakistan to test whether the Hindu Kush marks the effective boundary between these two realms. Combining molecular barcoding across all seven amphibian genera inhabiting the region with updated distribution records, we find a strikingly consistent lineage turnover centered on the eastern Hindu Kush and adjacent arid systems. Palearctic taxa reach their southeastern limits along the massif, whereas Oriental taxa extend only to its southern foothills. By contrast, Oriental lineages cross the Indus River without detectable phylogeographic break, indicating that the river valley does not function as a major biogeographic boundary for the taxa examined. The Hindu Kush also harbors endemic relict taxa of both Palearctic and Oriental affinities, revealing a dual role as both refugium and dispersal barrier. Thus, our results identify the Hindu Kush as the de facto boundary between the Palearctic and Oriental realms in this part of Eurasia and show that under-sampled arid-montane regions can disproportionately improve global biogeographic frameworks.

Advances in molecular techniques have improved our ability to quantify genetic introgression, but they also blur distinctions between hybrids and parental taxa in conservation and taxonomy. Here, we highlight challenges of identifying and defining hybrids within zoological nomenclature. Using high-throughput sequencing data, we show that the holotype of a recently described midwife toad subspecies, Alytes almogavarii inigoi, originates from a wide hybrid zone with its sister taxon, Alytes almogavarii almogavarii. Although the International Code of Zoological Nomenclature permits names based on admixed hybrid individuals (except F1 hybrids), this case raises questions about the applicability of the name to the intended population. To address this, we introduce a quantitative method for distinguishing admixed from parental specimens, using thresholds of foreign ancestry corresponding to known levels of backcrossing. Our findings reveal that the holotype carries 15% foreign ancestry, which is more than a second-generation backcross and thus too much to represent a distinct, non-admixed population. We therefore provide a replacement name by redescribing the Central Pyrenean lineage of Alytes almogavarii from a genetically pure population. This case illustrates the ambiguity surrounding admixed type specimens under current nomenclatural rules and emphasizes the growing need for clearer guidelines as taxonomy enters the genomic era.

The biodiversity of the Himalaya remains significantly understudied and, at the same time, is increasingly threatened by habitat loss due to rising anthropogenic pressures and climate change. Lazy Toads of the genus Scutiger are endemic to the Himalaya-Tibet orogen and form a diverse and characteristic component of the Himalayan montane zone. In our study, we re-assess material of Scutiger sikimmensis s. lat. using both molecular and morphological data. Our findings support the description of a new species, Scutiger khumbu sp. nov., with the nominotypical subspecies distributed in the Nepalese Khumbu Himal, and the subspecies Scutiger khumbu makalu subsp. nov. from the adjacent Makalu range

Abstract The Balkan Peninsula hosts a great proportion of Europe’s biodiversity, and this is well illustrated by amphibian richness and endemism. Among them, the yellow-bellied toad Bombina variegata has been a model in ecology and evolution, but several aspects of its phylogeography and taxonomy remain surprisingly poorly understood. In this study, we combine cytochrome b DNA barcoding data (1238 individuals from 355 localities), mitogenome phylogenetics (17.2 kb), gene-based nuclear phylogenetics (3.7 kb from four gene fragments) and multilocus phylogenomics (4759 loci / ~554 kb obtained by double digest Restriction Associated DNA sequencing; ddRAD-seq) to re-assess the diversification of B. variegata, and revisit its nomenclatural history to assign scientific names to phylogeographic lineages. The analyses support four major lineages, one assigned to B. v. variegata (Carpathians and northwestern ranges), one assigned to B. v. pachypus (Apennine Peninsula), and two assigned to B. v. scabra (Dinarides, Hellenides and Balkanides vs. the Rhodope mountains). Spatiotemporal patterns of diversification suggest a role for a Late Miocene marine incursion in the Pannonian Plain (Paratethys) as the initial trigger of divergence, followed by a vicariance event in the Apennines and a “sky island” process of Pleistocene differentiation in the Balkan Peninsula. As it reached the Dinarides during the Late Pleistocene, B. v. variegata potentially hybridized with B. v. scabra and captured its mitochondrial DNA, which resulted in a massive cyto-nuclear discordance across all northwestern European populations. Finally, we show that the two lineages of B. v. scabra significantly differ in morphology and ventral coloration patterns, and describe the Rhodope lineage as a new subspecies.

Speciation, i.e., the formation of new species, implies that diverging populations evolve genetic, phenotypic or ecological factors that promote reproductive isolation (RI), but the relative contributions of these factors remain elusive. Here we test which of genomic, bioacoustic, morphological, and environmental differences best predicts RI across a continuum of divergence in the midwife toads (genus Alytes), a group of Western Mediterranean amphibians, using a total evidence approach. We found that, without strong geographic barriers to dispersal, the extent of introgression across hybrid zones between phylogeographic lineages, which should reflect the strength of RI, predominantly covaries with genomic divergence. Overall phenotypic differentiation becomes substantial only between well established, fully isolated species. These results suggest that speciation in midwife toads initially involve cryptic lineages, which probably evolve RI through intrinsic (genetic) hybrid incompatibilities. As they continue to diverge, these nascent species eventually differentiate externally, which potentially enforces pre-mating barriers and facilitates sympatry. This speciation scenario has practical implications for species delimitation, notably when using hybrid zones and divergence thresholds as proxies for reproductive isolation.

Assessing how genetic diversity is spatially structured underlies many research questions in evolutionary ecology and contributes to understanding the factors implicated in population declines and extirpations, facilitating identification of conservation priorities and decision-making. In this study, we surveyed genomic diversity using genotyping by sequencing in the six subspecies of the midwife toad Alytes obstetricans/almogavarii complex, a group of amphibians from southwestern Europe threatened by habitat loss, climate change and chytridiomycosis. We first illustrate how the structure evident in mitochondrial DNA (mtDNA) and nuclear DNA microsatellites is discordant with the respective distributions of subspecies and patterns of admixture between them. We further document a deeply-divergent mtDNA haplogroup unique to Central Spain that is not reflected by the nuclear diversity, likely corresponding to a ghost mtDNA lineage. Patterns of genetic diversity and structure differ among and within subspecies. The Pyrenean endemics A. a. almogavarii and A. a. inigoi form homogenous genetic groups with high levels of heterozygosity, while the more widespread A. o. pertinax, A. o. boscai and A. o. lusitanicus are geographically structured across the Iberian Peninsula, comprising both genetically diverse and impoverished populations. Finally, A. o. obstetricans probably persisted in a composite glacial refugium north of the Pyrenees, from which it recently expanded across Western Europe, losing much of its genetic variation. Our results should be considered in future red list assessments, management unit delimitation, and ex-situ conservation efforts, and are also relevant to study chytrid epidemiology, for which A. obstetricans has been a model organism for nearly three decades.

The marsh frog (Pelophylax ridibundus complex) has been dubbed the number one amphibian invader in Western Europe. In The Netherlands, marsh frogs are native to the north, centre and west of the country, whereas pool frogs (P. lessonae) are mainly found in the south and east; their hybridogenetic hybrid, the edible frog (P. klepton esculentus), is distributed throughout The Netherlands. Over the past decades, the southeastern province of Limburg has been overrun by marsh frogs. To expose the invasion history of marsh frogs in The Netherlands, we mtDNA barcoded hundreds of water frogs from across the country. Throughout The Netherlands, we primarily detect pool frog mtDNA: the only mtDNA type that is expected to occur here in any native water frog, regardless of taxon, due to asymmetric introgression. By contrast, we identify four distinct marsh frog mtDNA haplotypes in Limburg, originating from four regions of the native marsh frog range: (1) Central, Eastern and Southeastern Europe, (2) western Balkan Peninsula, (3) western and northern Turkey, Iran and eastern Ukraine into Russia, up to the Ural Mountains and (4) central southern Turkey. Additionally, two presumably introduced Central, Eastern and Southeastern European marsh frog haplotypes are detected locally elsewhere in The Netherlands, within the native marsh frog range. Our results align with the broader pattern that invasive marsh frogs constitute a 'cocktail' of genetically diverse lineages. As the invasion front in Limburg approaches native water frog populations, we urge that conservation measures be taken promptly.

Proper taxonomic and phylogeographic frameworks are a prerequisite in evolutionary, ecological, and conservation research, but many species still lack adequate assessments. Earlier studies on the northeast Asian treefrog, Dryophytes japonicus, identified a deep Mio-Pliocene diversification; however, phenotypic assessments were lacking, and the taxonomic identity of the identified clades is under debate. In this study, we assessed the genetic, genomic, morphological, and acoustic diversity and differentiation within the D. japonicus complex to propose taxonomic arrangements and assess taxon-specific threats for each defined clade. Analyses of four mtDNA genes and 42.8 kb nuclear loci obtained by RAD-sequencing (RAD-seq) confirmed two distinct species-level clades that diverged ~6 Mya, one provisionally assigned to D. japonicus in southern Japan and mainland Asia and the other unnamed (D. cf. japonicus) in northern Japan and adjacent Russian islands. According to the mitochondrial data, each species is further divided into two subclades of Pleistocene age that display differences in morphological and call properties that may represent candidate subspecies: D. j. japonicus in southwestern Japan and D. j. stepheni on the Asian mainland and two unnamed lineages in Central and Northern Japan for D. cf. japonicus. Phenotypic differentiation between populations was only partly linked to their phylogenetic relationships. Finally, despite the relatively narrow ranges of many lineages, none crosses the threshold to be currently listed as threatened. The conservation of the diversity of the D. japonicus complex will necessarily require proper population monitoring and additional investigations to evaluate whether the unnamed delimited lineages merit taxonomic descriptions.

The frog leg industry relies on a global, largely underregulated market with potentially important ecological impact such as the uncontrolled harvest of declining wild populations and the introduction of invasive species. Here, we inferred the taxonomic nature and geographic origins of frog legs imported to Switzerland by DNA barcoding. Out of 34 samples, we retrieved eight distinct lineages attributed to five species from four genera, namely Hoplobatrachus rugulosus from Vietnam, Fejervarya cancrivora from Indonesia (invasive on several Pacific islands), two phylogeographic lineages of Limnonectes macrodon from Western and Central Java, L. kadarsani from eastern Indonesia, and three phylogeographic lineages of Pelophylax ridibundus from northern and central southern Turkey (invasive in Western Europe). Only the first two species were correctly declared, which is particularly problematic to track down harvests of the declining and geographically restricted Limnonectes taxa. In this respect, we show that the three Asian genera can be reliably distinguished by basic measurements of the frog legs, which could be used in future forensic controls. Our study calls for more stringent international regulations of the frog trade, including shipment monitoring to document the relative abundance of harvested species and ensure the sustainability of their wild populations.

[This corrects the article DOI: 10.1371/journal.pone.0314477.].

Characterizing the diversity and lability of the amphibian sex chromosomes holds key to understand what drives sex chromosome turnovers and assess the role of sex-linked genes in reproductive isolation and speciation. Here, we show that the heterogametic transition previously reported between the hybridizing toads Bufo bufo (ZW) and Bufo spinosus (XY) is nonhomologous, potentially implicates key genes of the vertebrate sex determination cascade (SOX9, DMRT1, and AMH), and is characterized by a much shorter ZW than XY segment. Integrating this information with published hybrid zone data suggests that both sex chromosomes resist interspecific introgression more than autosomes. These observations substantiate that sex chromosome turnovers preferentially involve chromosomes that host conserved sex-determining genes, imply heterochiasmy as a key factor of sex chromosome differentiation, and are consistent with a large sex chromosome effect, an empirical rule of speciation that is not expected with homomorphic sex chromosomes.