Astacus Astacus Research Articles

Does the narrow-clawed crayfish, Pontastacus leptodactylus (Eschscholtz, 1823) (Decapoda: Astacidea: Astacidae), contributes to the decline of the noble crayfish, Astacus astacus Linnaeus, 1758 in the Belarusian Lakeland?

Abstract The noble crayfish, Astacus astacusLinnaeus, 1758, is the rarest crayfish species in Belarus, distributed mainly within the Belarusian Lakeland, a region of numerous lakes of glacial origin in northern Belarus. The territory has been surveyed for the species to recognize its conservation status. From a total of 44 previously known A. astacus habitats, the species now occurs in 23 of them, five of which cohabit with the narrow-clawed crayfish, Pontastacus leptodactylus (Eschscholtz, 1823) which occupies the majority of habitats where A. astacus populations have been lost. Based on the long-term capture data, we tracked the displacement of A. astacus by P. leptodactylus in Karavaino Lake. To our knowledge, this observation represents the first published field evidence showing the progressive and successful outcompeting of A. astacus by P. leptodactylus. Based on our results, as well as by comparing it with previously published information on the crayfish distribution in Belarus, we highlight the unrealised scale of P. leptodactylus expansion in its non-native area of occurrence in Belarus. Due to the invasive potential of P. leptodactylus, we stress the need for research on the influence of P. leptodactylus on overall biodiversity within its introduced range.

Selective Breeding Is an Underexplored Route Towards Conservation of the Endangered Noble Crayfish

Selective Breeding Is an Underexplored Route Towards Conservation of the Endangered Noble Crayfish

Crayfish invasions at a long-term ecological research site formerly occupied by the noble crayfish Astacus astacus

Crayfish invasions at a long-term ecological research site formerly occupied by the noble crayfish Astacus astacus

High variation of virulence in Aphanomyces astaci strains lacks association with pathogenic traits and mtDNA haplogroups

Introduced into Europe from North America 150 years ago alongside its native crayfish hosts, the invasive pathogen Aphanomyces astaci is considered one of the main causes of European crayfish population decline. For the past two centuries, this oomycete pathogen has been extensively studied, with the more recent efforts focused on containing and monitoring its spread across the continent. However, after the recent introduction of new strains, the newly-discovered diversity of A. astaci in North America and several years of coevolution with its European host, a new assessment of the traits linked to the pathogen’s virulence is much needed. To fill this gap, we investigated the presence of phenotypic patterns (i.e., in vitro growth and sporulation rates) possibly associated with the pathogen’s virulence (i.e., induced mortality in crayfish) in a collection of 14 A. astaci strains isolated both in North America and in Europe. The results highlighted a high variability in virulence, growth rate and motile spore production among the different strains, while the total-sporulation rate was more similar across strains. Surprisingly, growth and sporulation rates were not significantly correlated with virulence. Furthermore, none of the analysed parameters, including virulence, was significantly different among the major A. astaci haplogroups. These results indicate that each strain is defined by a characteristic combination of pathogenic features, specifically assembled for the environment and host faced by each strain. Thus, canonical mitochondrial markers, often used to infer the pathogen’s virulence, are not accurate tools to deduce the phenotype of A. astaci strains. As the diversity of A. astaci strains in Europe is bound to increase due to translocations of new carrier crayfish species from North America, there is an urgent need to deepen our understanding of A. astaci’s virulence variability and its ability to adapt to new hosts and environments.

Plastic Debris in the Stomach of the Invasive Signal Crayfish Pacifastacus leniusculus from a Baltic Coastal River

The signal crayfish Pacifastacus leniusculus is one of the most widespread non-indigenous crayfish in Europe and is of interest to aquaculture in many countries. Once they escape into the wild, they disperse and become a potential source of food exploited for consumption by local inhabitants. The ingestion of plastics by the invasive signal crayfish in the Wieprza River, a tributary to the Baltic Sea, was determined by the FTIR identification of plastic compounds found in stomachs. The occurrence of plastic debris in the stomachs of P. leniusculus is reported for the first time. Plastic particles with the size range between 70 and 450 µm were observed only among crayfish inhabiting the lower, urbanized part of the river (7.3% of specimens). The presence of PTFE (polytetrafluoroethylene), cellophane, PP (polypropylene), PE (polyethylene), PMMA (polymethyl methacrylate), and nylon was confirmed with the highest frequency of PTFE in the stomachs of crayfish. Fibres were the predominant type of microplastics in crayfish stomachs. The results indicated the size-selective uptake and ingestion of plastics depending on the traits of the species and environment.

The Crayfish Plague Pathogen Aphanomyces astaci in Ireland.

Crayfish plague is a devastating disease of European freshwater crayfish and is caused by the oomycete Aphanomyces astaci (Ap. astaci), believed to have been introduced to Europe around 1860. All European species of freshwater crayfish are susceptible to the disease, including the white-clawed crayfish Austropotamobius pallipes. Ap. astaci is primarily spread by North American crayfish species and can also disperse rapidly through contaminated wet gear moved between water bodies. This spread, coupled with competition from non-indigenous crayfish, has drastically reduced and fragmented native crayfish populations across Europe. Remarkably, the island of Ireland remained free from the crayfish plague pathogen for over 100 years, providing a refuge for A. pallipes. However, this changed in 1987 when a mass mortality event was linked to the pathogen, marking its introduction to the region. Fortunately, crayfish plague was not detected again in Ireland until 2015 when a molecular analysis linked a mass mortality event in the Erne catchment to Ap. astaci. Since then, the pathogen has appeared across the island. Between 2015 and 2023, Ap. astaci was detected in 18 water catchments, revealing multiple genotypes. Intriguingly, the pathogen in Ireland is present without its natural host species. The uneven distribution of various genetic lineages strongly suggests the human-mediated transport of zoospores via contaminated water equipment as a primary cause of spread. This review details the timeline of these events, Ap. astaci's introduction into Ireland, and its rapid spread. As well, this review references the genotypes that have been determined, and discusses the issue of non-indigenous crayfish species in Ireland and management efforts.

Genetic diversity of noble crayfish in Finland based on ITS1 microsatellite-like repeat variation: implications to the conservation and management

During the last two centuries, native noble crayfish (Astacus astacus) has been introduced and re-introduced into many waters in Finland, to widen the area of distribution, and to re-establish the collapsed populations. Recent studies have revealed narrowed genetic diversity in A. astacus populations, especially in North Europe, due to crayfish plagues and due to the past translocation policies with numerous introductions also with small number of individuals. However, preliminary data based on the microsatellite like-repeat variation in ITS1 (Internal Transcribed Spacer 1) have indicated also genetic heterogeneity among Swedish and Finnish populations. In this study, the genetic diversity of 38 Finnish A. astacus populations was analysed based on variation in ITS1 region. Stocking histories of populations were obtained from official records and from local fishermen. Eighth out of 38 populations showed divergence in paired comparison with other analysed populations (Population Divergence Test, p < 0.05) indicating either possibility of autochthonous origin or donor. Potential autochthonous populations, as well as refugee areas within original distribution range and within designated protection area, should be further distinguished and monitored to maintain the remaining genetic diversity of the populations.

Diversity and distribution of Aphanomyces astaci in a European hotspot of ornamental crayfish introductions

Ornamental trade has become an important introduction pathway of non-native aquatic species worldwide. Correspondingly, there has been an alarming increase in the number of established crayfish of aquarium origin in Europe over the previous decade. The oomycete Aphanomyces astaci, the pathogen causing crayfish plague responsible for serious declines of European crayfish populations, is dispersed with introduced North American crayfish. The role of ornamental taxa in introducing and spreading different genotypes of this pathogen in open waters remains unclear. We investigated the distribution, prevalence, and diversity of A. astaci in Budapest, Hungary, which became a hotspot of aquarium crayfish introductions. Their establishment in this area was facilitated by locally abundant thermal waters. We screened for A. astaci in six host taxa from 18 sites sampled between 2018 and 2021: five cambarids (Cambarellus patzcuarensis, Faxonius limosus, Procambarus alleni, P. clarkii, P. virginalis) and one native astacid (Pontastacus leptodactylus). The pathogen was confirmed at five sampled sites in four host taxa: P. virginalis, P. clarkii, F. limosus, and for the first time in European open waters also in P. alleni. Genotyping was successful only in individuals from two different brooks where multiple host species coexisted but revealed unexpected patterns. Mitochondrial B-haplogroup of A. astaci, previously usually reported from Pacifastacus leniusculus or infected European species, was detected in P. virginalis at both sites, and in both F. limosus and P. virginalis sampled from a thermally stable tributary of Barát brook in 2018. In contrast, A-haplogroup of A. astaci was detected in coexisting F. limosus, P. virginalis and P. clarkii sampled in the same watercourse just a few hundred meters downstream in 2020. Additional genotyping methods indicated that a previously unknown A. astaci strain was associated with the latter haplogroup. One P. virginalis individual from 2020 was apparently co-infected by strains representing both mitochondrial haplogroups. The results indicated multiple sources of A. astaci in Budapest, likely directly associated with the introduction of ornamental species, interspecific transmission of this pathogen among ornamental hosts, and potential for a quick spatial or temporal turnover of dominant A. astaci strains at a certain locality. This highlights that in regions with high richness of potential A. astaci hosts, host taxon/pathogen genotype combinations become unpredictable, which might prevent reliable genotyping of pathogen sources in local crayfish mass mortalities.

Microcystin profiles in European noble crayfish Astacus astacus and water in Lake Steinsfjorden, Norway

Lake Steinsfjorden, an important noble crayfish (Astacus astacus) habitat, is often affected by blooms of Planktothrix spp. that produce microcystins (MCs). A poor correlation between MCs by ELISA in the water and in crayfish tissue in a study in 2015 prompted further investigation by LC–HRMS. LC–HRMS analyses of filters from water samples and on selected crayfish tissue extracts from the 2015 study revealed the presence of known and previously unreported MCs. Crayfish samples from May and June 2015 were dominated by MCs from the Planktothrix bloom, whereas in September novel MCs that appeared to be metabolites of MC-LR were dominant, even though neither these nor MC-LR were detected in the water in 2015. A water sample from October 2016 also showed MCs typical of Planktothrix (i.e., [d-Asp3]- and [d-Asp3,Dhb7]MC-RR and -LR), but low levels of MC-RR and MC-LR were detected in the lake water for the first time. In late summer and autumn, the MC profiles of crayfish were dominated by the homonorvaline (Hnv) variant MC-LHnv, a putative metabolite of MC-LR. Taken together, ELISA, LC–HRMS and previous PCR analyses showed that although Planktothrix was part of the crayfish diet, it was not the sole source of MCs in the crayfish. Possibly, crayfish in Lake Steinsfjorden may be ingesting MCs from benthic cyanobacteria or from contaminated prey. Therefore, information on the cyanobacterial or MC content in the water column cannot safely be used to make predictions about MC concentrations in the crayfish in Lake Steinsfjorden. Interestingly, the results also show that targeted LC–MS analysis of the crayfish would at times have underestimated their MC content by nearly an order of magnitude, even if all previously reported MC variants had been included in the analysis.

Declining populations of Astacus astacus drag European Branchiobdella parasita (Annelida: Clitellata) ectosymbionts to serious biodiversity loss

Branchiobdella parasita is specific ectosymbiont of European freshwater crayfish, including endangered noble crayfish (Astacus astacus). Although numerous information on distribution, biology or ecology were reported, genetic structure of B. parasita in European crayfish populations was not studied truthfully. The available molecular data include only cytochrome oxidase subunit I (COI) barcodes of B. parasita coexisting with noble crayfish in Alpine freshwaters in Austria, as well as stone crayfish (Austropotamobius torrentium) and white-clawed crayfish (Austropotamobius pallipes) in Balkan region. Here we report first genetic diversity and haplotype richness analysis of B. parasita populations on a pan-European scale. Specimens (n = 41) were collected from female noble crayfish from three lakes in northwest Poland (Kołtki Małe, Rosko Duże, Skrzynki Małe) and Elkenrother Weiher Lake in southwest Germany, high-quality COI barcodes were obtained and analysed against existing genetic data. The analysed populations were haplotype rich (h = 23) and had high COI haplotype diversity (Hd = 0.8988, I = 1.4554). Our results indicate that diversification rate of European B. parasita populations highly depends on habitat quality, its connection with surface waters and hosts species and mobility. We revealed that Central European populations inhabiting noble crayfish show significant genetic relationship, presenting common ancestral haplotypes and at the same time high genetic divergence from Balkan populations. Based on our results we suggest that ‘parasita-complex’ of phylogenetically related species with similar body shape and structure of chitin jaws, but with differentiating anatomical structure details are present across the continent.

EDNA monitoring as a tool for evaluating the reintroduction of Austropotamobius pallipes after a crayfish plague outbreak

The crayfish plague, a severe disease caused by the oomycete Aphanomyces astaci, is responsible for most population declines of susceptible crayfish in Europe. This pathogen has been devastating native populations of Austropotamobius pallipes since the 1970s in the Iberian Peninsula. In this study, we report a massive mortality event in one of the most important Spanish populations of A. pallipes. We aimed to: (i) identify the cause of the mortality, and (ii) evaluate the reintroduction viability of the species. Over the course of six months, we used environmental DNA (eDNA) and traditional trap-based methods to detect the presence of A. astaci or of native or invasive crayfish in order to evaluate the reintroduction viability of A. pallipes to the affected population. We did not capture any live crayfish or detect the presence of A. astaci in the reservoir water during the six months following the mass mortality event. Our analyses indicated that it was feasible to initiate a reintroduction program at the site, which will continue to be monitored for three to five years and will help improve the conservation status of A. pallipes.

Remains of decapod crustaceans from the late Cenomanian and early Turonian of the Czech Republic mistakenly assigned to crayfishes (Astacoidea)

Abstract Two species of fossil crustaceans from the Czech Republic are assigned to AstacusFabricius, 1775, a European crayfish: A. laevissimusFritsch & Kafka, 1887 and A. cenomanensisFritsch & Kafka, 1887. Both come from marine sediments, making doubtful their assignment to crayfishes. Specimens assigned to Astacus laevissimus correspond in fact to propodi of etyoid brachyurans. The type material of A. cenomanensis is heterogeneous, comprising fragmentary ctenochelid (Thalassinidea) propodi and isolated cheliped fingers of etyoid crabs. None of the two species belong to crayfishes. Since the studied cheliped material is too fragmentary and poorly preserved to be attributed confidently on species or genus level, A. laevissimus and A. cenomanensis are considered nomina dubia.

Temporal dynamics of the immune response in Astacus astacus (Linnaeus, 1758) challenged with Aphanomyces astaci Schikora, 1906

One of the main drivers of biodiversity loss in freshwater ecosystems are alien invasive species. In Europe, pathogen Aphanomyces astaci Schikora, 1906, is considered as one of the most problematic invasive species, as its introduction caused the severe decimation of the European freshwater crayfish stocks. The most affected are the populations of noble crayfish, keystone species native to European freshwaters. Unfortunately, even after decades of research, we do not understand the temporal dynamics of the noble crayfish immune response during Ap. astaci infection. Here, we studied the changes in the immune response of the noble crayfish during a time course challenge with a highly virulent strain of Ap. astaci. We recorded gross symptoms of the disease, changes in the total haemocyte count, gene expression profiles of putative immune response regulators and pathogen load. Additionally, we conducted a preliminary histological analysis of the pathogen dissemination in host tissues. Based on the occurrence of symptoms we propose three stages in the crayfish plague disease progression: asymptomatic stage, symptomatic stage, and finally death of infected individual. Furthermore, based on the qPCR analysis we could differentiate three Ap. astaci growth phases: initial lag phase, followed by exponential growth phase and finally sporulation phase. We observed that all measured immune response parameters were significantly correlated to the observed increase in the pathogen load (qPCR). Altogether, our results point to the absence of a successful immune response in the noble crayfish to a challenge with a highly virulent strain of Ap. astaci. The noble crayfish immune system was not able to suppress the growth of the intruding pathogen. In general, our observations have to be considered in the context of the specific combination of crayfish plague pathogen virulence and disease resistance of the challenged crayfish population, which defines the temporal dynamics of their interaction.

Detection of novel RNA viruses in wild noble crayfish (Astacus astacus): A virome analysis in Swiss water bodies

European native crayfish populations are undergoing a strong decline due to environmental factors and the introduction of highly competitive non-native species. Pathogens are an additional threat to native crayfish. However, aside from the crayfish plague, other infectious diseases are still widely unknown. This study aimed to investigate viruses present in seven populations of wild noble crayfish (Astacus astacus) in Switzerland, through high-throughput sequencing. Sequence analysis revealed the presence of 11 novel RNA viruses (one bunya-like, four hepe-like, two dicistro-like, three picorna-like, and one permutotetra-like) in the samples. The discovery of a novel bunya-like virus in noble crayfish without associated mortality or macroscopical alterations is of particular interest since it is closely related to the bunya-like brown spot virus, a virus described in 2019 from diseased native white-clawed crayfish (Austropotamobius pallipes) during a mass mortality event in France. It seems that these two closely related viruses have very different impacts on their respective hosts, raising the need for further investigations on virulence factors and host susceptibility towards these viruses. This study provides a basis for future investigations, permitting to gradually fill the knowledge gap in crayfish viral diseases.

Winter mortality and growth of juvenile noble crayfish (Astacus astacus) under realistic environmental conditions – Implications for conservation management

Noble crayfish (Astacus astacus) are endangered in many parts of Europe. Conservation efforts, including stocking measures, are widely conducted to safeguard the species and associated ecosystem functions. Due to limited availabilities of adult crayfish, juvenile noble crayfish are frequently released into lakes and rivers during autumn, usually without information on their fate and growth during the following winter. To investigate size- and density dependence of winter mortality and growth of newly stocked noble crayfish we conducted a mesocosm experiment in a small artificial lake in northern Germany. The experiment was carried out at water temperatures between 0.7 °C and 9.1 °C from mid-December until mid-March and simulated stocking densities of 20, 60, and 120 crayfish per square meter with unlimited food availability. Winter mortality rates were generally low and strongly size dependent. Specifically, a winter mortality rate of 9.2% was observed and crayfish with a total length (TL) above 34 mm all survived. The relative risk of mortality in dependence of TL increased by 41–94% (95% CI) with every decreasing centimetre of TL at the onset of winter. Increasing density had no effect on winter survival, but tended to reduce growth. Our work indicates that stocking measures of noble crayfish for conservation reasons can decrease costs and increase prospects of success if juveniles larger than 30 mm TL are used as stocking material.

A new species ofAustropotamobiusSkorikov, 1907 (Decapoda: Astacidae: Astacidae) from the late Miocene (Messinian) of Slovenia, with remarks on the evolution of European crayfishes

AbstractCrayfishes are rather rare in the fossil record, limiting our understanding of their evolutionary history. We describe and discuss a fossil crayfish from the Miocene of Slovenia. This fossil, comprising only the cephalothorax, first pereiopod, and another cheliped, seems closely allied to AustropotamobiusSkorikov, 1907, but also bears an epistome and postorbital carina reminiscent of AstacusFabricius, 1775. For these reasons, it is tentatively assigned to Austropotamobius. It differs from all other Austropotamobius species by the shapes of its epistome cephalic lobe, and postorbital carina, and resemble Austropotamobius torrentium (von Paula Schrank, 1803) most due to its ornamentation. It is therefore described as a new species, Austropotamobius plenicarisp. nov. The new species is associated with brackish water ostracods, most likely reworked from slightly older sediments, suggesting a late Miocene (Messinian) age. The origin of this species and its age show it lived long after the split between Astacus and Austropotamobius, and also probably after the split between the two Austropotamobius species (Au. pallipes and Au. torrentium), and may have been contemporary to, and lived in the area of the diversification of Au. torrentium haplotypes.

Molecular detection of Aphanomyces astaci – An improved species specific qPCR assay

The parasitic oomycete Aphanomyces astaci is the causative agent of crayfish plague, a devastating disease for European freshwater crayfish. Species specific quantitative real-time PCR (qPCR) can offer rapid detection of the pathogen. However, the well established A. astaci qPCR assay recommended by the World Organization for Animal Health (WOAH) amplifies the recently described Aphanomyces fennicus. Consequently, false-positive results may occur. This calls for the improvement of the established species specific A. astaci qPCR assay in order to avoid amplifying A. fennicus while screening for A. astaci.We developed an improved species specific A. astaci qPCR assay and validated the assay across three laboratories, using established procedures including different qPCR master mixes for each respective laboratory. Genomic DNA from A. astaci, A. fennicus and closely related Aphanomyces spp. was analysed and compared with both the improved and established assay. Additionally, DNA from crayfish tissue and environmental samples were analysed with both assays. The improved assay showed similar sensitivity with the established assay for all sample types, while proving highly specific for A. astaci avoiding amplification of A. fennicus and the other tested Aphanomyces spp.Environmental DNA (eDNA) samples collected at River Lierelva in Norway amplified with the established assay, but not with the improved assay indicating false positive. We were able to sequence a 530 bp fragment of the ITS region from these eDNA samples and the consensus sequence showed 99.9–100 % pairwise identity with A. fennicus and 97.2–98 % pairwise identity with A. astaci, suggesting that the occurrence of A. fennicus is not limited to Finland, where it was first discovered.

On the conservation of white-clawed crayfish in the Iberian Peninsula: Unraveling its genetic diversity and structure, and origin.

European crayfish species are a clear example of the drastic decline that freshwater species are experiencing. In particular, the native species of the Iberian Peninsula, the white clawed-crayfish (WCC) Austropotamobius pallipes, is listed as "endangered" by the IUCN and included in Annex II of the EU Habitat Directive and requires especially attention. Currently, implemented conservation management strategies require a better understanding of the genetic diversity and phylogeographic patterns, as well as of its evolutionary history. For this purpose, we have generated the largest datasets of two informative ribosomal mitochondrial DNA regions, i.e., cytochrome oxidase subunit I and 16S, from selected populations of the WCC covering its geographical distribution. These datasets allowed us to analyze in detail the (i) genetic diversity and structure of WCC populations, and (ii) divergence times for Iberian populations by testing three evolutionary scenarios with different mtDNA substitution rates (low, intermediate, and high rates). The results indicate high levels of haplotype diversity and a complex geographical structure for WCC in the Iberian Peninsula. The diversity found includes new unique haplotypes from the Iberian Peninsula and reveals that most of the WCC genetic variability is concentrated in the northern and central-eastern regions. Despite the fact that molecular dating analyses provided divergence times that were not statistically supported, the proposed scenarios were congruent with previous studies, which related the origin of these populations with paleogeographic events during the Pleistocene, which suggests an Iberian origin for these WCC. All results generated in this study, indicate that the alternative hypothesis of an introduced origin of the Iberian WCC is highly improbable. The result of this study, therefore, has allowed us to better understand of the genetic diversity, structure patterns, and evolutionary history of the WCC in the Iberian Peninsula, which is crucial for the management and conservation needs of this endangered species.

A Clinical Study to Appraise the Efficacy of Astacus Fluviatilis in the Management of Urticaria by Using Chronic Urticaria -Quality of Life (CU-QoL)

People often experience urticaria, sometimes referred to as hives, which is characterised by erythematous, edematous, itchy, and transitory plaques that affect the skin and mucous membranes. Acute spontaneous urticaria, chronic spontaneous urticaria, chronic inducible urticaria, and episodic chronic urticaria are the different classifications. Many causes are blamed for the aetiology, but sometimes they are idiopathic. These include infections, medications, diet, psychogenic factors, and respiratory allergies. Clinically, there are irritable, swollen, and red plaques. Homoeopathy effectively cures acute, chronic, and recurrent urticaria by addressing the underlying immune system imbalance and takes on the illness head-on. Numerous effective homoeopathic therapies exist to treat urticaria. One of the significant remedies is Astacus fluviatilis that is very helpful in cases of urticaria. This study was done on 20 patients, where 10 patients were given Astacus fluviatilis and 10 patients were given a placebo. The statistical results showed that Astacus fluviatilis gave better results compared to the control group. Keywords: Urticaria, Treatment, Astacus fluviatilis, Homoeopathy.

Phylogeographic patterns of genetic diversity in Pontastacus leptodactylus (Decapoda: Astacidae): is the hypothesis of the taxonomically rich genus Pontastacus true?

Abstract The narrow-clawed crayfish Pontastacus leptodactylus is a large native European astacid crayfish species distributed in the drainage area of the Azov, Black and Caspian Seas. The species shows immense morphological diversity across its range. Various species and subspecies have been described and, recently, eight species and two subspecies were confirmed in the updated classification of freshwater crayfishes. However, genetic diversity studies of the species are scarce, mostly from limited geographical areas. Therefore, we aimed to analyse the genetic diversity of this species using mtDNA and nuDNA from across their distributional range to verify the presence of described species. Analyses of individuals from 65 populations and 14 countries confirmed the existence of three divergent mtDNA phylogroups corresponding to the geographical area of Central and Southern Europe, Eastern Europe and Asia, and Turkey. Analysis of ancestral ranges revealed the Black Sea basin as the most likely area of origin. The nuDNA analysis showed low diversity partially corresponding to the mtDNA pattern. All analyses showed mitonuclear discordance indicating the existence of a single species. Therefore, we point out the possibility of taxonomic inaccuracy with the current number of valid species within the P. leptodactylus species complex.