When species that are reproductively incompletely isolated meet in parapatry and reproduce, a hybrid zone is established. The strength of reproductive isolation between these species and the dynamics of their ranges can be inferred from the extent and direction of gene flow across the hybrid zone. We determine the position, shape and dynamics of the relatively poorly studied hybrid zone between the Danube ( Triturus dobrogicus ) and Italian ( T. carnifex ) crested newts, using genome-wide nuclear DNA data (24 species-diagnostic SNPs) and an mtDNA marker (ND4). Overall, the hybrid zone is narrow, signifying strong selection against hybrids. In the north of the hybrid zone, asymmetric introgression of T. dobrogicus alleles into T. carnifex indicates postglacial expansion by T. carnifex . We also report a highly distinct haplotype lineage that presumably reflects ‘cryptic pseudogenes’; while the relevant sequences do not bear the hallmarks of a pseudogene, their apparent distinctiveness is not supported by an independent mtDNA marker (ND2).

Hybridization is common among animals and plants, but often leads to sterility due to improper chromosome pairing. Some hybrids overcome sterility through modified gametogenesis, enabling clonal or hemiclonal reproduction. In water frog hybrids from the Pelophylax esculentus complex, hybridogenesis typically ensures hemiclonal reproduction. These hybrids inherit haploid chromosomal sets (n = 13) from both parental species, Pelophylax lessonae and Pelophylax ridibundus. During hybrid gametogenesis, one genome is eliminated while the other is clonally transmitted. We analyzed hybrid males and females from Central Europe, where hybrids coexist with P. lessonae and with both parental species, to assess gametogenic stability and whether the P. lessonae genome is consistently eliminated. Most hybrids followed canonical gametogenesis with premeiotic elimination of the P. lessonae genome and endoreplication of the P. ridibundus genome, yielding haploid P. ridibundus gametes. Nevertheless, gametogenesis was frequently altered in males but not in females. Some hybrid males had spermatocytes with 13 or 26 univalents, 26 bivalents, or tetravalents, indicating variability in genome elimination and endoreplication. Some males produced diploid or haploid spermatids with P. ridibundus and P. lessonae genomes. Comparative genome hybridization revealed that some males with altered gametogenesis carried unusual chromosomes with mixed R and L signals. Furthermore, rare triploid males showed substitutions of P. lessonae chromosomes to P. ridibundus chromosomes. Despite altered gametogenesis, they produced spermatocytes and haploid spermatids with P. lessonae chromosomes and two P. ridibundus chromosomes that escaped elimination. Overall, despite a generally consistent pattern of genome transmission, hybrid males exhibit frequent deviations from canonical hybridogenesis across populations.

While various haemoparasites have been reported from anuran hosts, this study provides the first published record of dactylosomatid parasites (Dactylosomatidae Jakowska & Nigrelli, 1955 emend. Levine, 1971) in frogs from Slovakia. Of the 239 anurans screened for apicomplexans, 67 individuals belonging to three species of water frogs, Pelophylax esculentus (Linnaeus, 1758), P. ridibundus (Pallas, 1771), and P. lessonae (Camerano, 1882) were found to be infected with haemogregarines of the genus Dactylosoma Labbé, 1894. Our results demonstrate that the haemogregarine found in all three species of water frogs, collected from three different localities in western Slovakia, represents a single taxon that morphologically resembles the type species, Dactylosoma ranarum (Kruse, 1890). Based on comprehensive morphometric, morphological, and molecular data from newly collected isolates from Pelophylax frogs, including the type host, we provide an integrative assessment of Dactylosoma cf. ranarum. We also conducted molecular screening of dipterans collected from the study sites, which could serve as potential vectors of the parasite, but no representatives of Dactylosoma were detected in any of the examined specimens.

The genus Oswaldocruzia represents a taxonomically diverse group of nematodes with global distribution. Although Oswaldocruzia species are widespread and exhibit a remarkably wide host range in some species, their genetic diversity and biogeographic patterns remain poorly understood. This study investigated the genetic variability and distribution of Oswaldocruzia spp. in nine anuran species from the genera Bufo, Bufotes, Pelophylax, and Rana across Central Europe and the Balkans. Two species were identified: Oswaldocruzia filiformis and O. ukrainae, each exhibiting a different range of host associations. Phylogenetic analyses based on mitochondrial COI sequences revealed significant haplotype diversity in the generalist O. filiformis, with low geographic and host-associated genetic structuring. In contrast, O. ukrainae, which is closely associated with Bufotes viridis, exhibited only one genetic variant across all samples, highlighting its restricted genetic diversity. The findings emphasize contrasting genetic diversities among nematode parasites exhibiting different levels of host-specificity and expand the known distribution of O. filiformis into new regions of the Balkans. In addition, they highlight the need for additional studies on the ecological and evolutionary factors that influence the genetic diversity of parasites in amphibians.

The central European complex of water frogs (Pelophylax esculentus complex) is one of the best studied hybridogenetic complexes in vertebrates, but also one of the most complicated due to different modes of hybrid reproduction. Although genetic and ecological research on water frogs has been going on for more than fifty years, many areas of Europe are still underexplored, which is also the case of eastern Slovakia. In this study, we fill this gap and investigate local populations from morphological and genetic perspectives. During field surveillance and using genetic markers, we found 146 individuals of P. ridibundus, 17 of P. lessonae and 116 diploid hybrids of P. esculentus in 21 localities. In addition to pure populations of P. ridibundus, we recorded the syntopic occurrence of hybrids with parental species. The sex ratio in parental species was equal, but females strongly dominated in hybrids. Based on several clonality tests, it can be assumed that hybrids primarily eliminate the lessonae genome during gametogenesis and form clonal gametes with the ridibundus genome. The high clonal diversity observed in the ridibundus genome of hybrids is most likely the result of multiple primary hybridization events in sympatric populations. All individuals of P. lessonae, 86.2% of P. esculentus and 8.3% of P. ridibundus had lessonae mtDNA. Introgressed individuals of P. ridibundus shared the same mitochondrial haplotypes with the two other taxa, suggesting relatively recent unidirectional introgression.

Ticks are important vectors of various microorganisms, including bacteria. In this study, we examined Hyalomma aegyptium ticks collected from 240 spur-thighed tortoises Testudo graeca at 42 localities in the Mediterranean and Middle East and analysed them for the presence of bacteria of the genera Anaplasma, Borrelia, Coxiella, and Rickettsia. Altogether, 576 out of 928 analysed ticks (62.1%) were positive for at least one of the tested bacteria. The highest prevalence in individual ticks was found for Borrelia turcica (43.6%), followed by Rickettsia (12.3%) and Anaplasma (6.1%). No sample was positive for Coxiella burnetii. Among Rickettsia, we detected two species, Rickettsia africae and Rickettsia aeschlimannii, and also other unspecified Rickettsia. Anaplasma (100% identity with A. phagocytophilum) was detected at 15 (35%) out of 42 studied localities, any of Rickettsia at 28 (67%), and B. turcica at 32 (76%) localities. The geographic distribution of the studied microorganisms varied, with none of them detected in Syria, and only Rickettsia spp. detected in Morocco. Sequence analysis revealed substantial genetic variability in all detected agents, with the most variable (36 new haplotypes) being glpQ gene used as a marker for B. turcica. We also analysed the prevalence of various co-infections among studied ticks, with the mean number of co-infected ticks per tortoise increased with the number of ticks per tortoise. However, the frequencies of co-infected ticks do not indicate the presence of antagonistic or synergistic facilitative interactions between the agents. According to our data, we could expect that the eco-epidemiological importance of H. aegyptium does not stem from their tortoise hosts but rather from the low host specificity of its larvae and nymphs, feeding on a wider spectrum of reptilian, avian, and mammalian hosts.

Hybridisation impairs fertility in offspring, usually through disrupted gametogenesis. Some hybrids benefit from polyploidisation, while others adopt a clonal strategy. Hemiclonality was described for water frog hybrids from the Pelophylax esculentus complex, including parental P. ridibundus (genotype RR), P. lessonae (LL), and their hybrids, P. esculentus (diploid LR or triploid LLR or LRR), which can transmit one parental genome clonally, eliminating the other. Hybrids coexist with one or both parental species within population systems of various dynamics. Our investigation focuses on clonal diversity and reproductive mechanisms in two unique all-female hybrid population systems in eastern Ukraine. These consist exclusively of triploid hybrid females with the LLR genotype and bisexual P. ridibundus, a combination not observed elsewhere in Europe. Microsatellite analysis showed that L-genomes were less diverse and more clonal, while R-genomes were more diverse, aligning with the presence of parental P. ridibundus but only clonal transmission of L-genomes. Crosses of LLR-females with P. ridibundus males revealed only LR and LLR offspring. Presumably, LLR-females transmit gametes with L-genomes. Selective mortality was observed for LR-hybrids of both sexes and LLR-males during tadpole development, but not LLR-females. Therefore, this selective mortality likely stabilises such population systems and shifts the sex ratio towards LLR-females.

European water frogs from the Pelophylax esculentus complex include two sexual species, Pelophylax ridibundus (genome RR) and Pelophylax lessonae (genome LL), and their hybrids (genome LR), which usually clonally transmit one of the parental species' genomes. This reproductive strategy, known as hybridogenesis, allows hybrids to reproduce with parental species, creating diverse population systems. Unlike most well-studied populations in Europe, in the Siverskyi Donets River basin (eastern Ukraine), P. lessonae is absent, while diploid and polyploid hybrids coexist with P. ridibundus (R-E system). To reveal diverse system compositions, genetic divergence, and tadpole selective mortality, we combined novel data from over a decade of observations with previous research on population systems in the Siverskyi Donets River. Two key population types of the R-E system were identified: those with diploid hybrids in northern localities and those with both diploid and triploid hybrids, extending from the mainstream of the Siverskyi Donets River to its tributaries. Both sexes were found among both P. ridibundus and hybrids. Additionally, we found higher genetic diversity in R-genomes compared to L-genomes and discuss the cause of variation differences between the genomes. We highlighted the importance of continuous monitoring to unravel water frog population dynamics and complexity.

The distribution of the neutral component of genetic diversity is the interplay of historical and ongoing processes resulting in the species-specific genetic structure of populations, which can, however, be disrupted by interspecific hybridisation and introgression. In this study, we focused on two species of water frogs, Pelophylax epeiroticus and P. kurtmuelleri, which live in sympatry in the southwestern Balkans, to investigate the rate of hybridisation and population genetic structure using cytogenetic, mitochondrial (ND2) and nuclear DNA (microsatellite) markers. The overall hybridisation rate was 2.6%, with rates reaching up to 10% at specific sites. The course of gametogenesis and the occurrence of later generations of hybrids (beyond the F1 generation) indicate a sexual mode of hybrid reproduction. The bimodal structure of hybrid populations and the rarity of hybrids suggest substantial reproductive isolation between the two species; however, this isolation is unlikely attributable to differences in ecological niche occupation. In P. epeiroticus, sequence variation in the ND2 gene revealed two divergent lineages with a clear geographic pattern that corresponds to the genetic structure in microsatellite markers. In contrast, P. kurtmuelleri populations were not as geographically structured and showed only weak genetic differentiation in both types of markers. Pelophylax epeiroticus was significantly less variable at microsatellite loci compared to P. kurtmuelleri, which, together with the high differentiation of its populations, suggests a stronger influence of genetic drift. We can hypothesise that the differential strength of genetic drift in the two species may lead to unequal interspecific gene flow.

The pathogenicity of haemogregarines and their effects on the health status of ectothermic hosts remain largely unexplored. In this study, we examined the impact of Hemolivia mauritanica (Sergent et Sergent, 1904) infection on the differential leukocyte count (DLC) as a measurable indicator of health in tortoise hosts. A total of 206 blood smears were analysed, including 181 from spur-thighed tortoises (Testudo graeca Linnaeus) and 25 from marginated tortoises (Testudo marginata Schoepff). Light microscopy was used to identify infected individuals, determine DLC, and quantify parasitaemia levels. Overall, H. mauritanica was detected in 125 of 181 (69%) T. graeca samples and 21 of 25 (84%) T. marginata samples. To assess whether infection influenced DLC, we statistically compared leukocyte profiles between infected and uninfected individuals. Additionally, we evaluated the effects of other factors, including host species, parasitaemia intensity, sex, age, and the month and year of blood collection. Wilcoxon rank-sum tests revealed that parasitaemia and age had a statistically significant effect on DLC in T. graeca. Further analysis using linear models showed a significant association between parasitaemia and DLC, specifically affecting azurophils in T. graeca and basophils in T. marginata. Nine T. graeca tortoises positive for H. mauritanica were co-infected with haemosporidian parasites of the genus Haemocystidium Castellani et Willey, 1904, specifically three with Haemocystidium anatolicum (Orkun et Güven, 2013) and six with Haemocystidium caucasicum (Krasilnikov, 1965). Although co-infection itself was not statistically significant, a separate analysis of Haemocystidium parasitaemia revealed a significant effect on lymphocyte DLC. Furthermore, the frequent presence of mitotic and polychromatophilous erythrocytes in H. mauritanica-infected tortoises suggests a potential increase in erythrocyte regeneration.

Pelophylax esculentus (genotype LR), an interspecific hybrid of P. ridibundus (genotype RR) and P. lessonae (genotype LL), is one of the most studied examples of hemiclonal hybridogenic vertebrates. In most populations, hybrids produce clonal gametes of one parental species and backcross with the other parental species, forming a new generation of hybrids. In this study, we focused on the population system of water frogs in the Siverskyi Donets river basin (eastern Ukraine) with the aim to determine the genetic diversity of parental genomes and clarify how such a system is maintained in nature. Of the 55 frogs analyzed, only one individual was P. ridibundus , 48 belonged to diploid LR and five to triploid LRR hybrids. Using 16 microsatellite loci, we found significantly lower genetic diversity and significantly higher clonality in the L-genome of hybrids compared to the R-genome. This contrasting diversity might be attributed to the absence of P. lessonae in the studied region and the clonal inheritance of the L-genome. Our population-genetic results are partially inconsistent with previous studies focusing on hybrid gametogenesis in the Siverskyi Donets river basin, according to which hybrids form both clonal R and L gametes. We hypothesize that discrepancies between the type of clonally formed gametes and the genotypic composition of the studied population may be due to the selective mortality of some genotypes during larval development and metamorphosis. Selective mortality thus may play an important role in the dynamics of the hybridogenetic systems in eastern Ukraine.

Water frogs of the genus Pelophylax host a variety of parasites, from protozoa to helminths. Among the blood parasites, representatives of Apicomplexa, Trypanosoma and Nematoda show the highest prevalence. In this study, we focused on blood parasites of water frogs living in the Danube Delta, Romania. In total, 74 individuals of P. ridibundus and eight individuals of P. esculentus from six localities were examined. Blood parasites were detected microscopically and using a molecular marker (18S rDNA). 89.77% of frogs from all investigated localities were found to be infected with at least one parasitic group, specifically with haemogregarines (84.09%), nematodes (1.14%), and trypanosomes (63.64%). The parasitemia of haemogregarines and trypanosomes differed significantly among the studied locations. There was no statistically significant difference in parasitemia between male and female hosts. However, adults were found to have a significantly higher parasitemia in comparison with subadults infected with haemogregarines. Correlation between parasitemia and the body length of frogs infected with haemogregarines was also significant (r = 0.226). By comparing the 18S rDNA sequences with the corresponding GenBank sequences, Hepatozoon species identified in water frogs showed a close similarity (98.1-99.8%) to Hepatozoon magna. Trypanosomes showed the highest sequence similarity to Trypanosoma sp. isolate R10 clone L2-3, Trypanosoma ranarum, and Trypanosoma cobitis.

Populations of plants and animals at the edge of a species’ range may exhibit lower genetic diversity due to a combination of historical (e.g., postglacial dispersal) and contemporary (e.g., low effective population size, strong genetic drift or bottlenecks) processes. In this study, we analysed the genetic structure of populations of the European pond turtle (Emys orbicularis) in the northern part of the Pannonian Basin (Slovakia, Hungary, Austria), which represents the edge of the species’ range. Using mitochondrial DNA (mtDNA) and microsatellite markers, we aimed to assign individuals and populations into phylogeographic lineages and to determine genetic variation and population structure. Our study revealed that most individuals from natural populations belong to mtDNA lineage II (widely distributed haplotype IIa, and likely endemic haplotypes IIl and IIo), which is typical for the whole Pannonian region. However, non-native haplotypes were detected in one Slovak population (haplotype Ib) and in the Austrian Donau Auen National Park (haplotype IVa). Microsatellite markers revealed high variability comparable to that in other parts of the range, suggesting that marginal populations may not necessarily have reduced genetic diversity. A genetically mixed population was found in the Donau Auen National Park, consisting of turtles belonging to mtDNA lineage II (taxonomically assessed as subspecies E. orbicularis orbicularis) and lineage IV (taxonomically assessed as E. orbicularis hellenica), which is native to the circum-Adriatic region. The admixture suggests intense hybridization and introgression between the two lineages (subspecies).

One of the most frequently translocated species outside their native range in Europe are water frogs of the genus Pelophylax . Recently, water frogs belonging to the same genus have also been recorded on the island of Gozo in Malta. To trace their origin, we genetically examined 17 individuals from three Gozitan localities where water frogs have been recorded recently. We analysed one mitochondrial (NADH dehydrogenase 2, ND2 ) and one nuclear (serum albumin intron 1, SAI-1 ) fragment to identify the geographic origin of the frogs and a set of microsatellite markers to determine their population-genetic structure and the predicted number of source populations. Based on the ND2 and SAI-1 markers, the water frogs on the island of Gozo originate from southern Anatolia, Turkey. According to sequence variation in ND2 , they were assigned to a caralitanus mtDNA clade, which is endemic to southern Anatolia and taxonomically represents either an evolutionary lineage within P. cf. bedriagae or a separate species P. caralitanus . All Gozo water frogs had only one haplotype in the ND2 and one allele in the SAI-1 fragment, indicating a recent and single introduction event. These results are supported by microsatellite analysis, which revealed low genetic variability and the absence of any population-genetic structure, suggesting that Gozo water frogs originate from only one source population.

AbstractSexual reproduction is the primary mode of reproduction in eukaryotes, but some organisms have evolved deviations from classical sex and switched to asexuality. These asexual lineages have sometimes been viewed as evolutionary dead ends, but recent research has revealed their importance in many areas of general biology. Our review explores the understudied, yet important mechanisms by which sperm‐dependent asexuals that produce non‐recombined gametes but rely on their fertilization, can have a significant impact on the evolution of coexisting sexual species and ecosystems. These impacts are concentrated around three major fields. Firstly, sperm‐dependent asexuals can potentially impact the gene pool of coexisting sexual species by either restricting their population sizes or by providing bridges for interspecific gene flow whose type and consequences substantially differ from gene flow mechanisms expected under sexual reproduction. Secondly, they may impact on sexuals' diversification rates either directly, by serving as stepping‐stones in speciation, or indirectly, by promoting the formation of pre‐ and postzygotic reproduction barriers among nascent species. Thirdly, they can potentially impact on spatial distribution of species, via direct or indirect (apparent) types of competition and Allee effects. For each such mechanism, we provide empirical examples of how natural sperm‐dependent asexuals impact the evolution of their sexual counterparts. In particular, we highlight that these broad effects may last beyond the tenure of the individual asexual lineages causing them, which challenges the traditional perception that asexual lineages are short‐lived evolutionary dead ends and minor sideshows. Our review also proposes new research directions to incorporate the aforementioned impacts of sperm‐dependent asexuals. These research directions will ultimately enhance our understanding of the evolution of genomes and biological interactions in general.

Ophidiomyces ophidiicola (Oo) is a snake fungal pathogen that causes ophidiomycosis. The disease manifests as dermatomycosis and/or systemic mycosis, and can be fatal. It occurs in free-ranging snakes in Asia, Europe and the USA and has also been demonstrated in captive snakes. We tested for the presence of Oo in free-ranging snake populations in the Czech and Slovak Republics (n = 420) between 2019 and 2022, focusing mainly on grass snakes (Natrix natrix) and dice snakes (Natrix tessellata), as well as various captive exotic species (n = 207). After collecting skin swabs, we tested for Oo using the qPCR method. We confirmed fragmented occurrence of Oo in the Czech Republic (total prevalence 15%) and recorded Oo in the Slovak Republic for the first time (total prevalence 33.9%). The highest prevalence was observed in N. tessellata (20.2%), which appears to be the most susceptible species. The pathogen was not detected in captive snakes.

The genus Pelophylax (water frogs) includes relatively common, widely distributed, and even invasive species, but also endemic taxa with small ranges and limited knowledge concerning their ecology and evolution. Among poorly studied species belong endemics of the southwestern Balkans, namely Pelophylax shqipericus, P. epeiroticus and P. kurtmuelleri. In this study, we focused on the genetic variability of these species aiming to reveal their phylogeographic patterns and Quaternary history. We used 1,088 published and newly obtained sequences of the mitochondrial ND2 gene and a variety of analyses, including molecular phylogenetics and dating, historical demography, and species distribution modeling (SDM). We revelated the existence of two mitochondrial lineages within P. epeiroticus and P. shqipericus that diverged at ~ 0.9 Mya and ~ 0.8 Mya, respectively. Contrarily, no deeply diverged lineages were found in P. kurtmuelleri. Pelophylax kurtmuelleri also shows a close phylogenetic relationship with widely distributed P. ridibundus, suggesting that both represent one evolutionary clade called here P. ridibundus/kurtmuelleri. The estimated split between both lineages in the clade P. ridibundus/kurtmuelleri date back to ~ 0.6 Mya. The divergence between the ridibundus and kurtmuelleri lineages on the ND2 gene is thus lower than the divergence between the two lineages found in P. epeiroticus and P. shqipericus. According to haplotype networks, demographic analyses, and SDM, endemic water frogs survived the last glacial maximum (LGM) in Balkan microrefugia, and their distribution has not changed significantly or even retracted since the LGM. Haplotypes of the kurtmuelleri lineage were also found in northern parts of Europe, where haplotype diversity is however much lower than in the Balkans, suggesting the possible hypothesis of their postglacial expansion to the north.

Parasites might represent a helpful tool in understanding the historical dispersion and phylogeography of their hosts. In order to reveal whether the migration routes and diversification of hosts can be traceable in the genetic structures of their parasites, we investigated the diversity of paramphistomoid trematodes of Pelophylax frogs in 2 geographically distant European regions. Water frogs belonging to the genus Pelophylax represent a striking example of a species with a high variety of ecological adaptations and a rich evolutionary history. The parasites were collected from 2 Balkan endemic species, P. epeiroticus and P. kurtmuelleri, and 2 species in Slovakia, P. esculentus and P. ridibundus. While in Slovakia, Pelophylax frogs harboured 2 species, the diplodiscid Diplodiscus subclavatus and the cladorchiid Opisthodiscus diplodiscoides, only the former was recorded in the south-western Balkans. Remarkably high genetic diversity (16 unique mitochondrial cox1 haplotypes, recognized among 60 novel sequences) was observed in D. subclavatus, and subsequent phylogenetic analyses revealed a strong population-genetic structure associated with geographical distribution. We also evidenced the existence of 2 divergent D. subclavatus cox1 haplogroups in the south-western Balkans, which might be associated with the historical diversification of endemic water frogs in the regional glacial microrefugia.

The Levant represents one of the most important reptile diversity hotspots and centers of endemism in the Western Palearctic. The region harbored numerous taxa in glacial refugia during the Pleistocene climatic oscillations. Due to the hostile arid conditions in the warmer periods they were not always able to spread or come into contact with populations from more distant regions. One large and conspicuous member of the Levantine herpetofauna is the legless anguid lizard Pseudopus apodus. This species is distributed from the Balkans to Central Asia with a portion of its range running along the eastern Mediterranean coast. Mitochondrial and nuclear DNA sequences, microsatellite genotypes, and morphology show that populations in this region differ from the two named subspecies and presumably had a long independent evolutionary history during the Quaternary. Here we describe the Levantine population as a new subspecies and present biogeographic scenarios for its origin and diversification. The new subspecies is genetically highly diverse, and it forms a sister lineage to Pseudopus from the remaining parts of the range according to mtDNA. It is the largest-bodied of the three subspecies, but occupies the smallest range.