Narrow Contact Zone Research Articles

Genetic identification, morphology and distribution of Natrixhelvetica subspecies in southern and western Switzerland (Reptilia, Squamata, Serpentes).

Most of Switzerland is inhabited by the nominotypical subspecies of the barred grass snake (Natrixhelveticahelvetica), which is characterized by mitochondrial DNA lineage E. Only in the northeast of the country, the common grass snake (N.natrix) occurs and hybridizes with N.h.helvetica in a narrow contact zone. However, we discovered that in southern and western Switzerland barred grass snakes representing another mtDNA lineage (lineage C) are widely distributed. Lineage C is typical for Alpine populations of the southern subspecies N.h.sicula. Our microsatellite analyses of the Swiss samples revealed differences between the two subspecies and also a substructure with two clusters in each subspecies. Furthermore, we discovered a contact and hybrid zone of N.h.helvetica and N.h.sicula along the northern shore of Lake Geneva and also confirm that interbreeding with alien common grass snakes (N.n.moreotica, mtDNA lineage 7) occurs there. This finding is of concern for nature conservation and measures should be taken to prevent further genetic pollution. Using morphometrics, we found no differences between the two subspecies of N.helvetica, while N.natrix was slightly distinct from N.helvetica.

INFLUENCE OF WELDING PARAMETERS ON THE PERFORMANCE CHARACTERISTICS OF THE THERMOCATODES

Methods of controlling welding parameters, physical and physico-chemical transformations that occur during welding are considered in the work. The physico-chemical mechanism of welding is considered - the deformation of the crystal lattice, the transfer of metal on the contacting surfaces, the mixing of deformation products, diffusion, phase transformations, the appearance of new chemical compounds - intermetallics, oxides, carbides. Studies of the influence of the main parameters of friction welding on the formation of the structure of the welded joints of the investigated alloys have been carried out. The microstructures of welded joints OT4 + VN-2AE and VT6 + VN-2AE are given. The obtained dependence of the relative strength of the OT 4 + VN-2AE connection on the parameters of the welding process. The given microstructures were obtained by SEM from the surface of OT 4 + VN-2AE and VT6 samples, based on which it was determined that the destruction begins with the formation of microcracks in the contact zone on the side of the VN-2AE niobium alloy. It was determined that a layer saturated with oxygen is formed in the diffusion zone due to which the β-modification of Ni2O5 with a significant specific volume is formed, which causes high internal stresses and the formation of microcracks. The reason for the decrease in the mechanical properties of the welded joint OT 4 + ВН-2АЕ was established - the formation of a phase (Ti-Nb) with an orthorhombic lattice, the degree of curvature of which determines the degree of strengthening of the layer and depends on the niobium content. These data are confirmed by X-ray structural analysis. The analysis of the concentration curves of niobium and titanium shows that titanium does not diffuse into the narrow contact zone of the niobium alloy, and niobium forms a wide diffusion zone in the contact zone of the OT 4 alloy. It was established that niobium contributes to the increase in the solubility of aluminum in λ-titanium, which leads to increase in weldability plasticity and corrosion resistance. It was determined that the low mechanical properties of the welded joints of the VN-2AE alloy with the OT-4 and VT-6 titanium alloys are due to the same nature - the oxidation of the niobium alloy at the welding temperature. It was established that the weak link of the welded joint is the surface of the niobium alloy.

Morphological and genetic diversification of Old-World marbled newts, with the description of a new and ‘not-at-all-cryptic’ subspecies from the Iberian Peninsula (Triturus, Salamandridae)

Abstract Morphological and genetic variation of organisms is generally lower in northern than in southern regions of the Palearctic. This ubiquitous geographical pattern has been associated with extinction and recolonization processes in the north versus persistence in the Mediterranean area, as governed by climate change in the Pleistocene. One area of differentiation and complexity is the Iberian Peninsula where two distinct sister-species of marbled newts possess adjoining and geographically sharply delimited ranges over the north (Triturus marmoratus) and the south of the peninsula (T. pygmaeus). Within the latter species two forms are here documented, with different colouration patterns and non-overlapping ranges. The southern form is striped, much as in T. marmoratus, and occurs to the south of the Guadalquivir River. The northern form, here described as a new subspecies, has a reticulated dorsal colouration. It occurs only north of the Guadalquivir and is thus sandwiched in between two striped marbled newt taxa. Both T. pygmaeus subspecies engage inside the Doñana National Park in a ca. 2,500 m narrow contact zone. Although an inferred substantial selection against hybrids goes a long way to support species status, a reanalysis of published genetic data is required to solve this issue, along with the wider investigation of the same type of (microsatelite) data for the southern, nominotypical subspecies.

Population genetic structure and hybrid zone analyses for species delimitation in the Japanese toad (Bufo japonicus).

Hybridization following secondary contact may produce different outcomes depending on the extent to which genetic diversity and reproductive barriers have accumulated during isolation. The Japanese toad, Bufo japonicus, is distributed on the main islands of Japan. In the present study, we applied multiplexed inter-simple sequence repeat genotyping by sequencing to achieve the fine-scale resolution of the genetic cluster in B. j. japonicus and B. j. formosus. We also elucidated hybridization patterns and gene flow degrees across contact zones between the clusters identified. Using SNP data, we found four genetic clusters in B. j. japonicus and B. j. formosus and three contact zones of the cluster pairs among these four clusters. The two oldest diverged lineages, B. j. japonicus and B. j. formosus, formed a narrow contact zone consistent with species distinctiveness. Therefore, we recommend that these two subspecies be elevated to the species level. In contrast, the less diverged pairs of two clusters in B. j. japonicus and B. j. formosus, respectively, admixed over a hundred kilometers, suggesting that they have not yet developed strong reproductive isolation and need to be treated as conspecifics. These results will contribute to resolving taxonomic confusion in Japanese toads.

Genetic data to describe the hybrid zone between Bufo bufo (Linnaeus, 1758) and Bufo verrucosissimus (Pallas, 1814) in northeastern Türkiye

Abstract Hybrid zones are geographic areas where individuals from distinct taxa meet, mate, and hybridize. These zones may have complex histories, but many of them originated relatively recently, during climatic oscillations in the Quaternary, following range shifts of formerly isolated, well-differentiated lineages. The Bufo bufo species group comprises four species distributed over the Western Palearctic. Whereas the contact zone between Bufo bufo and B. spinosus in western Europe has been well characterized, little is known about other species contacts. Here we focused on the contact between B. verrucosissimus and B. bufo in northeastern Türkiye, using mtDNA and microsatellite markers to describe genetic structure and patterns of admixture in the hybrid zone based on Bayesian clustering and cline analyses. Both species meet in a narrow contact zone at Rize province, with restricted introgression suggesting barriers to hybridization consistent with species status. Spatial population genetic analyses of microsatellite data pinpoint a possible enclave population of B. bufo at the Borçka district in Artvin province, isolated within the B. verrucosissimus range. The centers of the microsatellite and mtDNA-based clines are slightly displaced, with B. verrucosissimus mtDNA introgressing about 33 km W of the nuclear contact. Hybrid zone dynamics seem to be associated with range shifts mediated by Pleistocene glacial cycles and/or sex-biased dispersal.

Can demographic histories explain long-term isolation and recent pulses of asymmetric gene flow between highly divergent grey fox lineages?

Secondary contact zones between deeply divergent, yet interfertile, lineages provide windows into the speciation process. North American grey foxes (Urocyon cinereoargenteus) are divided into western and eastern lineages that diverged approximately 1 million years ago. These ancient lineages currently hybridize in a relatively narrow zone of contact in the southern Great Plains, a pattern more commonly observed in smaller-bodied taxa, which suggests relatively recent contact after a long period of allopatry. Based on local ancestry inference with whole-genome sequencing (n = 43), we identified two distinct Holocene pulses of admixture. The older pulse (500-3500 YBP) reflected unidirectional gene flow from east to west, whereas the more recent pulse (70-200 YBP) of admixture was bi-directional. Augmented with genotyping-by-sequencing data from 216 additional foxes, demographic analyses indicated that the eastern lineage declined precipitously after divergence, remaining small throughout most of the late Pleistocene, and expanding only during the Holocene. Genetic diversity in the eastern lineage was highest in the southeast and lowest near the contact zone, consistent with a westward expansion. Concordantly, distribution modelling indicated that during their isolation, the most suitable habitat occurred far east of today's contact zone or west of the Great Plains. Thus, long-term isolation was likely caused by the small, distant location of the eastern refugium, with recent contact reflecting a large increase in suitable habitat and corresponding demographic expansion from the eastern refugium. Ultimately, long-term isolation in grey foxes may reflect their specialized bio-climatic niche. This system presents an opportunity for future investigation of potential pre- and post-zygotic isolating mechanisms.

Phylogeography of the Cow Knob Salamander (Plethodon punctatus Highton): populations on isolated Appalachian mountaintops are disjunct but not divergent

Although many studies have examined how taxa responded to Pleistocene climate fluctuations in the Appalachian Mountains, impacts on high-elevation endemics of Central Appalachia are not yet understood. We use mitochondrial (ND4 & Cytb) and nuclear (GAPD) DNA sequences to investigate the phylogeography of the Cow Knob Salamander (Plethodon punctatus), a woodland species from Central Appalachian highlands thought to have origins in the Pleistocene. Data from 72 tail tips representing 25 sites revealed that the species comprises two geographically cohesive mitochondrial clades with a narrow, putative contact zone on Shenandoah Mountain. Molecular clock estimates indicate the clades diverged in the Middle Pleistocene. The population size of the Southern clade appears to have remained stable for at least 50,000 years. Despite spanning several isolated mountain systems, the Northern clade has exceptionally low genetic diversity, probably due to recent demographic expansion. Palaeodemographic hypothesis testing supported a scenario in which a founder effect characterized the Northern clade as it diverged from the Southern clade. Species distribution models predicted no suitable habitat for the species during the Last Glacial Maximum. Ultimately, Pleistocene glacial climates may have driven the species from the northern half of its current range, with recolonization events by members of the Northern clade as climates warmed. Density dependent processes may now maintain a narrow contact zone between the two clades.

Hybridization in late stages of speciation: Strong but incomplete genome-wide reproductive isolation and 'large Z-effect' in a moving hybrid zone.

In organisms reproducing sexually, speciation occurs when increasing divergence results in pre- or post-zygotic reproductive isolation between lineages. Studies focusing on reproductive isolation origin in early stages of speciation are common and many rely on genomic scans to infer introgression providing limited information on the genomic architecture of reproductive isolation long-term maintenance. This study analyses a natural hybrid zone between two species in a late stage of speciation. We used ddRADseq genotyping in the contact between Podarcis bocagei and P. carbonelli to examine admixture extent, analyse hybrid zone stability and assess genome-wide variation in selection against introgression. We confirmed strong but incomplete reproductive isolation in a bimodal hybrid zone. New findings revealed population genetic structure within P. carbonelli in the contact zone; geographical and genomic clines analysis suggested strong selection against gene flow, but a relatively small proportion of the loci can introgress, mostly within the narrow contact zone. However, geographical clines revealed that a few introgressed loci show signs of potential positive selection, particularly into P. bocagei. Geographical clines also detected a signal of hybrid zone movement towards P. bocagei distribution. Genomic cline analysis revealed heterogeneous patterns of introgression among loci within the syntopy zone, but the majority maintain a strong association with the genomic background of origin. However, incongruences between both cline approaches were found, potentially driven by confounding effects on genomic clines. Last, an important role of the Z chromosome in reproductive isolation is suggested. Importantly, overall patterns of restricted introgression seem to result from numerous strong intrinsic barriers across the genome.

Sympatric co-existence of two ecotypes of Impatiens noli-tangere (Balsaminaceae) with different morphology and flowering phenology.

In angiosperms, intraspecific variation of flowering phenology may affect reproductive isolation and, consequently, speciation. This study focused on Impatiens noli-tangere (Balsaminaceae), which is distributed over broad latitudinal and altitudinal ranges in Japan. We aimed to reveal the phenotypic mixture of two ecotypes of I. noli-tangere with different flowering phenology and morphological traits in a narrow contact zone. Previous studies have shown that I. noli-tangere has early- and late-flowering types. The early-flowering type makes buds in June and is distributed at high-elevation sites. The late-flowering type makes buds in July and is distributed at low-elevation sites. In this study, we analyzed the flowering phenology of individuals at an intermediate elevation site where the early- and late-flowering types grow in sympatry (contact zone). We found no individuals showing intermediate flowering phenology at the contact zone, and early- and late-flowering types were clearly distinguishable. We also found that the differences in many other phenotypic traits between the early- and late-flowering types were maintained, including the number of flowers produced (total number of chasmogamous and cleistogamous flowers), leaf morphology (aspect ratio, number of serrations), seed traits (aspect ratio), and flower bud formation positions on the plant. This study showed that these two flowering ecotypes maintain many different traits in sympatry.

Do parapatric populations of the ticks Amblyomma tonelliae Nava, Beati & Labruna, 2014 and Amblyomma sculptum Berlese, 1888 (Acari: Ixodidae) hybridize?

This work aimed to determine if the tick species, Amblyomma sculptum and Amblyomma tonelliae, hybridize along their contact zones in Argentina. Free-living adults and nymphs of A. sculptum and A. tonelliae were collected in seven sampling locations of northern Argentina. In four of them, the two species occur in parapatry (possible hybrid zone) whereas in the other three sites, only one species is known to occur. A total of 65 A. sculptum and 65 A. tonelliae from both, allopatric and parapatric populations, were analysed. The nuclear (ITS2) and mitochondrial (COI and 12SrDNA) gene sequences of each tick were amplified and analysed to verify whether or not they could reveal the presence of hybrids among the parapatric samples. No morphological and molecular evidence was found to support the hypothesis of ongoing natural hybridization. Intrinsic postzygotic barriers may be the cause of lack of gene flow between the two species in areas of co-ocurrence. The results can be explained by the length of time the two lineages spent in allopatry since the middle of the Miocene and before their respective distribution range expanded again reaching a narrow secondary contact zone.

Strongly exclusive distribution with putative character displacement in two flying stag beetles

Character displacement is considered a key driver of evolutionary divergence and adaptation. Few examples of reproductive character displacement with a narrow contact zone have been identified. We examined the genetic structure, body length variation, and genital morphology in the contact and allopatric areas of Platycerus takakuwai and P. viridicuprus to investigate character displacement and gene flow. In the contact area, the species identifications based on endophallic morphology and nuclear genes were identical, whereas mitochondrial gene did not exhibit a perfect match. This incongruence suggests that interspecific hybridization followed by the mitochondrial introgression has likely occurred during historical secondary contact. The species are essentially parapatric in contact area, co-occurring at only one of 28 adjacent sampling sites despite being flying species, and no hybrids based on morphology have been found, which indicates a strongly exclusive distribution. The results showed that the body length variation was consistent with character displacement after controlling for variation along geographic and environmental gradients. Interspecific body size differentiation may have evolved to reduce incorrect mating between the species. Moreover, selective pressure caused by reproductive interference between the two species may act on body size that have likely resulted in strongly exclusive distribution at the edge of their ranges.

Secondary contact rather than coexistence-Erebia butterflies in the Alps.

Secondary contact zones are ideal systems to study the processes that govern the evolution of reproductive barriers, especially at advanced stages of the speciation process. An increase in reproductive isolation resulting from selection against maladaptive hybrids is thought to contribute to reproductive barrier buildup in secondary contact zones. Although such processes have been invoked for many systems, it remains unclear to which extent they influence contact zone dynamics in nature. Here, we study a very narrow contact zone between the butterfly species Erebia cassioides and Erebia tyndarus in the Swiss Alps. We quantified phenotypic traits related to wing shape and reproduction as well as ecology to compare the degree of intra- and interspecific differentiation. Even though only very few first-generation hybrids occur, we find no strong indications for current reinforcing selection, suggesting that if reinforcement occurred in our system, it likely operated in the past. Additionally, we show that both species differ less in their ecological niche at the contact zone than elsewhere, which could explain why coexistence between these butterflies may currently not be possible.

Genetic analysis of hybridization between white-handed (Hylobates lar) and pileated (Hylobates pileatus) gibbons in a contact zone in Khao Yai National Park, Thailand.

Natural hybridization has played various roles in the evolutionary history of primates. Its consequences range from genetic introgression between taxa, formation of hybrid zones, and formation of new lineages. Hylobates lar, the white-handed gibbon, and Hylobates pileatus, the pileated gibbon, are largely allopatric species in Southeast Asia with a narrow contact zone in Khao Yai National Park, Thailand, which contains both parental types and hybrids. Hybrid individuals in the zone are recognizable by their intermediate pelage and vocal patterns, but have not been analyzed genetically. We analyzed mitochondrial and microsatellite DNA of 52 individuals to estimate the relative genetic contributions of the parental species to each individual, and the amount of introgression into the parental species. We obtained fecal samples from 33 H. lar, 15 H. pileatus and four phenotypically intermediate individuals in the contact zone. Both mitochondrial and microsatellite markers confirmed distinct differences between these taxa. Both H. lar and H. pileatus contributed to the maternal lineages of the hybrids based on mitochondrial analysis; hybrids were viable and present in socially normal reproductive pairs. The microsatellite analysis identified ten admixed individuals, four F1 hybrids, which corresponded to phenotypic hybrids, and six H. lar-like backcrosses. All 15 H. pileatus samples were identified as originating from genetically H. pileatus individuals with no H. lar admixture; hence, backcrossing is biased toward H. lar. A relatively low number of phenotypic hybrids and backcrossed individuals along with a high number of parental types indicates a bimodal hybrid zone, which suggests relatively strong bias in mate selection between the species.

Allopatric divergence and secondary contact of two weak fish species (Macrodon ancylodon and Macrodon atricauda) from the South Atlantic

The king weakfish Macrodon ancylodon (Scianidae) has been regarded as a widespread representative of Macrodon in the South Atlantic, managed as a single stock in Brazilian fisheries. Nonetheless, molecular and morphological data showed that populations from cold and subtropical waters in southeastern and southern Brazil actually corresponded to another taxon (Macrodon atricauda or Southern king weakfish). Apparently, the environmental and geomorphological heterogeneity in the South Atlantic would account for their allopatric differentiation with a putative but largely overlooked barrier in a narrow area between the northeastern and southeastern coast of Brazil. Therefore, we performed phylogenetic and phylogeographic analyses based on mitochondrial and nuclear markers in populations of M. ancylodon and M. atricauda with increased sampling efforts within and nearby their alleged range boundaries (Southern Bahia to Rio de Janeiro coast) to infer their biogeographic history and verify putative contact zones and/or introgression. Mitochondrial markers recovered both taxa as reciprocally monophyletic species that have diverged in Pleistocene (~1.5 million years ago), probably related to isolation of ancestor populations by the exposure of Abrolhos shelf (southern Bahia) and the Vitoria-Trindade chain during sea level retreats in glaciation periods. This vicariant effect combined with distinct ecological features between northeastern (warm waters) and southeastern coast (cold waters) should have favored their allopatric speciation. On the other hand, syntopic populations of M. atricauda and M. ancylodon were clearly recognized within a narrow contact zone (~60 km) between the estuaries of the Mucuri (northeastern Brazil) and Conceição da Barra (southeastern Brazil) without evidence of introgression, as a result of secondary contact. Thus, estuarine areas along this region represent a hotspot for conservation of fisheries resources, since it encompasses two differentiated stocks of king weakfish.

Elevation of Divergent Color Polymorphic and Monomorphic Lizard Lineages (Squamata: Agamidae) to Species Level

The Australian Tawny Dragon lizard (Ctenophorus decresii), as currently recognized, comprises two genetically divergent lineages, northern and southern, that differ notably in male coloration. A narrow contact zone exists between the lineages with asymmetric and limited hybridization, indicating incompatibility and highlighting the need for further taxonomic assessment. Here, we evaluate morphological variation in C. decresii and elevate the lineages to separate species. The southern lineage retains the name C. decresii (Duméril and Bibron, 1837), and we formally reinstate C. modestus for the northern lineage, which was previously a synonym of C. decresii (Amphibolurus modestus,Ahl, 1926). We redescribe C. modestus and C. decresii and highlight important considerations for reevaluation of their conservation statuses. Recognition of C. modestus represents another species that may have differentiated in relatively mesic mountainous refugia during Pleistocene glacial-interglacial cycles, and underscores the importance of divergence in male coloration as a driver of speciation in the rock dragon species group.

Asymmetric acoustic signal recognition led to asymmetric gene flow between two parapatric frogs

AbstractCorrect discrimination between courtship signals could help to maintain genetic integrity between closely related species. However, asymmetric usage of signals might cause asymmetric gene flow across the contact zone. Buergeria choui and B. otai are sibling-species with a parapatric distribution pattern in Taiwan, having two narrow contact zones on the east and west sides of the island. Combining behavioural experiments with genome-wide RAD-seq analyses, we test whether the ability of signal recognition influences genetic introgression across their species boundary. The playback experiments show that all B. choui populations respond strongest to their own ‘cricket’ trills, while the western population of B. otai have evolved a strong level of reproductive character displacement by showing the inclusive usage of the unique ‘chicken’ signals. In contrast, the eastern B. otai population uses both ‘chicken’ and ‘cricket’ trills, and has a stronger preference for the latter. The weak reproductive character displacement in the eastern population has led to asymmetry genetic introgression from B. choui toward B. otai. Our results support the prediction that a more specialized signal-user, compared to its sibling, generalized signal-user, might have a higher probability of maintaining their genetic integrity in the secondary contact region.

Secondary contact zones of closely-related Erebia butterflies overlap with narrow phenotypic and parasitic clines.

Zones of secondary contact between closely related taxa are a common legacy of the Quaternary ice ages. Despite their abundance, the factors that keep species apart and prevent hybridization are often unknown. Here, we study a very narrow contact zone between three closely related butterfly species of the Erebia tyndarus species complex. Using genomic data, we first determined whether gene flow occurs and then assessed whether it might be hampered by differences in chromosome number between some species. We found interspecific gene flow between sibling species that differ in karyotype by one chromosome. Conversely, only F1 hybrids occurred between two species that have the same karyotype, forming a steep genomic cline. In a second step, we fitted clines to phenotypic, ecological and parasitic data to identify the factors associated with the genetic cline. We found clines for phenotypic data and the prevalence of the endosymbiont parasite Wolbachia to overlap with the genetic cline, suggesting that they might be drivers for separating the two species. Overall, our results highlight that some gene flow is possible between closely related species despite different chromosome numbers, but that other barriers restrict such gene flow.

Genetic, morphological and ecological variation across a sharp hybrid zone between two alpine butterfly species.

Identifying the mechanisms involved in the formation and maintenance of species is a central question in evolutionary biology, and distinguishing the selective drivers of populations’ divergence from demographic processes is of particular interest to better understand the speciation process. Hybrid zones are recognized to provide ideal places to investigate the genetic architecture of speciation and to identify the mechanisms allowing diverging species to maintain their integrity in the face of gene flow. Here, we studied two alpine butterfly species, Coenonympha macromma and C. gardetta, which can be found flying together and hybridizing in narrow contact zones in the southern French Alps. We characterized the genomic composition of individuals, their morphology and their local habitat requirements, within and around a hybrid zone. Genetic diversity analysis at 794 SNPs revealed that all individuals within the hybrid zone were highly admixed, which was not the case outside the hybrid zone. Cline analysis showed that, despite ongoing hybridization, 56 out of 122 loci differentially fixed or nearly so between the two species were impermeable to introgression across the sharp hybrid zone (9 km wide). We also found concordance in cline position and width among genetic, morphological and environmental variation, suggesting a coupling of different reproductive barriers. Habitat characteristics such as the presence of trees and shrubs and the start of the growing season were strongly associated with the genetic variation, and we found evidence of divergence at genetic markers associated with morphology and physiology, putatively involved in visual or environmental reproductive isolation. We discuss the various behavioural and ecological factors that might interplay to maintain current levels of divergence and gene flow between this species pair.

From refugia to contact: Pine processionary moth hybrid zone in a complex biogeographic setting.

Contact zones occur at the crossroad between specific dispersal routes and are facilitated by biogeographic discontinuities. Here, we focused on two Lepidoptera sister species that come in contact near the Turkish Straits System (TSS). We aimed to infer their phylogeographic histories in the Eastern Mediterranean and finely analyze their co‐occurrence and hybridization patterns in this biogeographic context.We used molecular mitochondrial and nuclear markers to study 224 individuals from 42 localities. We used discordances between markers and complementary assignment methods to identify and map hybrids and parental individuals.We confirmed the parapatric distribution of Thaumetopoea pityocampa (Lepidoptera: Notodontidae) in the west and Thaumetopoea wilkinsoni in the east and identified a narrow contact zone. We identified several glacial refugia of T. wilkinsoni in southern Turkey with a strong east–west differentiation in this species. Unexpectedly, T. pityocampa crossed the TSS and occur in northern Aegean Turkey and some eastern Greek islands. We found robust evidence of introgression between the two species in a restricted zone in northwestern Turkey, but we did not identify any F1 individuals. The identified hybrid zone was mostly bimodal.The distributions and genetic patterns of the studied species were strongly influenced both by the Quaternary climatic oscillations and the complex geological history of the Aegean region. T. pityocampa and T. wilkinsoni survived the last glacial maximum in disjoint refugia and met in western Turkey at the edge of the recolonization routes. Expanding population of T. wilkinsoni constrained T. pityocampa to the western Turkish shore. Additionally, we found evidence of recurrent introgression by T. wilkinsoni males in several T. pityocampa populations. Our results suggest that some prezygotic isolation mechanisms, such as differences in timing of the adult emergences, might be a driver of the isolation between the sister species.

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.