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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.

Abstract Sexual 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.

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.

Abstract Hybridogenesis is a reproductive tool for sexual parasitism. Hybridogenetic hybrids use gametes from their sexual host for their own reproduction, but sexual species gain no benefit from such matings as their genome is later eliminated. Here, we examine the presence of sexual parasitism in water frogs through crossing experiments and genome-wide data. We specifically focus on the famous Central-European populations where Pelophylax esculentus males (hybrids of P. ridibundus and P. lessonae) live with P. ridibundus. We identified a system where the hybrids commonly produce two types of clonal gametes (hybrid amphispermy). The haploid lessonae genome is clonally inherited from generation to generation and assures the maintenance of hybrids through a process, in which lessonae sperm fertilize P. ridibundus eggs. The haploid ridibundus genome in hybrids received from P. ridibundus a generation ago, is perpetuated as clonal ridibundus sperm and used to fertilize P. ridibundus eggs, yielding female P. ridibundus progeny. These results imply animal reproduction in which hybridogenetic taxa are not only sexual parasites, but also participate in the formation of a sexual taxon in a remarkable way. This occurs through a process by which sexual gametes are being captured, converted to clones, and returned to sexual populations in one generation.

Abstract Reproductive isolation barriers maintain the integrity of species by preventing interspecific gene flow. They involve temporal, habitat or behavioral isolation acting before fertilization, and postzygotic isolation manifested as hybrid mortality or sterility. One of the approaches of how to study reproductive isolation barriers is through the analysis of hybrid zones. In this paper, we describe the structure of a hybrid zone between two crested newt species (Triturus cristatus and T. carnifex) in the southern part of the Czech Republic using morphological, microsatellite, and mitochondrial (mtDNA) markers. Specifically, we tested the hypothesis that the structure of the hybrid zone is maintained by species‐specific habitat preferences. Comparing the genetic structure of populations with geographical and ecological parameters, we found that the hybrid zone was structured primarily geographically, with T. cristatus‐like populations occurring in the northeast and T. carnifex‐like populations in the southwest. Despite T. cristatus tending to occur in deeper ponds and T. carnifex on localities with more shading, the effect of both ecological parameters on the structure of the zone was minimal. Next, we corroborated that T. carnifex individuals and some hybrids possess mtDNA of T. dobrogicus, whose nuclear background was not detected in the studied hybrid zone. Hybridization between T. carnifex and T. dobrogicus (resulting in unidirectional mtDNA introgression) had to predate subsequent formation of the hybrid zone between T. cristatus and T. carnifex. Populations of crested newts in the southern part of the Czech Republic thus represent a genetic mosaic of nuclear and mitochondrial genomes of three species.

Abstract It is the first time that Crimean-Congo haemorrhagic fever virus (CCHFV), causing potentially lethal disease of humans, has been reported from the Middle East region and from the tortoise tick Hyalomma aegyptium from a tortoise host, whose epidemiological significance may have remained almost completely overlooked so far. We used RT-PCR to screen for 245 ticks collected from 38 Testudo graeca tortoise individuals. Results of our genetic screening provide unambiguous evidence of occurrence of CCHFV in this region and host, suggesting a potentially important role of H. aegyptium in CCHF epidemiology.

We have studied the population genetic structure of slightly admixed populations of crested newts (Triturus cristatus and T. carnifex) in a continuously fragmented landscape, located in northern Salzburg (Austria) and neighbouring Bavaria (Germany). Crested newts are listed as Critically Endangered in the provincial Red List of Salzburg and strictly protected by the EU Habitats Directive. We used seven polymorphic microsatellite loci to evaluate genetic diversity and processes that may determine the genetic architecture of populations. Genetic diversity was moderate, pairwise FST-values were comparatively high showing significant genetic differentiation and limited gene flow. Isolation by distance was significant for the whole data set, but not significant when calculated for T. cristatus- and T. carnifex-like populations separately. Bayesian analyses of population structure, using three different programs showed similar results. Spatial statistics reveal that the geographical isolation of populations is very high.

The southwestern Balkans is inhabited by three endemic water frog species, Pelophylax shqipericus, P. epeiroticus and P. kurtmuelleri. Although these species are genetically and bioacoustically distinct from each other, their morphological differentiation is less pronounced as is generally observed in the genus Pelophylax. In this study, we analyzed morphological data of 215 individuals of these Balkan endemics in order to evaluate their morphological variability and facilitate their identification in the field. Moreover, since P. kurtmuelleri is a disputable taxon, we compared it with closely related P. ridibundus from Central Europe. The most pronounced morphological differences expressed by the analysis of variance (ANOVA) and the discriminant analysis of the principal components (DAPC) were found between P. kurtmuelleri and the two other Balkan species. Pelophylax kurtmuelleri and P. ridibundus, as well as P. shqipericus and P. epeiroticus were only slightly differentiated by morphology, which demonstrates that the morphological characters measured do not reflect the genetic divergence of the species and are weak indicators of their taxonomic status. Morphological characters suitable for species identification include a combination of indices L/CINT, T/CINT, L/T, and DP/CINT (L -- snout--vent length, CINT-length of the metatarsal tubercle, T -- length of the tibia, DP -- length of the first toe), and qualitative traits like the pattern and shape of the lateral spots, presence/absence of yellow pigment in the flanks and thighs, foot webbing colouration, size and shape of the metatarsal tubercle, and colouration of the male vocal sacs. [ABSTRACT FROM AUTHOR]

The generic name Hemolivia has been used for haemogregarines characterized by morphological and biological features. The few molecular studies, focused on other haemogregarine genera but involving Hemolivia samples, indicated its close relationship to the genus Hepatozoon . Here we analyze molecular data for Hemolivia from a broad geographic area and host spectrum and provide detailed morphological documentation of the included samples. Based on molecular analyses in context of other haemogregarines, we demonstrate that several sequences deposited in GenBank from isolates described as Hepatozoon belong to the Hemolivia cluster. This illustrates the overall difficulty with recognizing Hemolivia and Hepatozoon without sufficient morphological and molecular information. The close proximity of both genera is also reflected in uncertainty about their precise phylogeny when using 18S rDNA. They cluster with almost identical likelihood either as two sister taxa or as monophyletic Hemolivia within paraphyletic Hepatozoon . However, regardless of these difficulties, the results presented here provide a reliable background for the unequivocal placement of new samples into the Hemolivia / Hepatozoon complex. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sexual 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., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

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 cox 1 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 cox 1 haplogroups in the south-western Balkans, which might be associated with the historical diversification of endemic water frogs in the regional glacial microrefugia.

Reproductive isolation barriers maintain the integrity of species by preventing interspecific gene flow. They involve temporal, habitat or behavioral isolation acting before fertilization, and postzygotic isolation manifested as hybrid mortality or sterility. One of the approaches of how to study reproductive isolation barriers is through the analysis of hybrid zones. In this paper, we describe the structure of a hybrid zone between two crested newt species ( Triturus cristatus and T. carnifex ) in the southern part of the Czech Republic using morphological, microsatellite, and mitochondrial (mtDNA) markers. Specifically, we tested the hypothesis that the structure of the hybrid zone is maintained by species-specific habitat preferences. Comparing the genetic structure of populations with geographical and ecological parameters, we found that the hybrid zone was structured primarily geographically, with T. cristatus -like populations occurring in the northeast and T. carnifex -like populations in the southwest. Despite T. cristatus tending to occur in deeper ponds and T. carnifex on localities with more shading, the effect of both ecological parameters on the structure of the zone was minimal. Next, we corroborated that T. carnifex individuals and some hybrids possess mtDNA of T. dobrogicus , whose nuclear background was not detected in the studied hybrid zone. Hybridization between T. carnifex and T. dobrogicus (resulting in unidirectional mtDNA introgression) had to predate subsequent formation of the hybrid zone between T. cristatus and T. carnifex . Populations of crested newts in the southern part of the Czech Republic thus represent a genetic mosaic of nuclear and mitochondrial genomes of three species., Competing Interests: The authors declare that they have no competing interests., (© 2019 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Background: Fascioloides magna (Trematoda: Fasciolidae) is an important liver parasite of a wide range of free-living and domestic ruminants; it represents a remarkable species due to its large spatial distribution, invasive character, and potential to colonize new territories. The present study provides patterns of population genetic structure and admixture in F. magna across all enzootic regions in North America and natural foci in Europe, and infers migratory routes of the parasite on both continents., Methods: In total, 432 individuals from five North American enzootic regions and three European foci were analysed by 11 microsatellite loci. Genetic data were evaluated by several statistical approaches: (i) the population genetic structure of F. magna was inferred using program STRUCTURE; (ii) the genetic interrelationships between populations were analysed by PRINCIPAL COORDINATES ANALYSIS; and (iii) historical dispersal routes in North America and recent invasion routes in Europe were explored using MIGRATE., Results: The analysis of dispersal routes of the parasite in North America revealed west-east and south-north lineages that partially overlapped in the central part of the continent, where different host populations historically met. The exact origin of European populations of F. magna and their potential translocation routes were determined. Flukes from the first European focus, Italy, were related to F. magna from northern Pacific coast, while parasites from the Czech focus originated from south-eastern USA, particularly South Carolina. The Danube floodplain forests (third and still expanding focus) did not display relationship with any North American population; instead the Czech origin of the Danube population was indicated. A serial dilution of genetic diversity along the dispersion route across central and eastern Europe was observed. The results of microsatellite analyses were compared to previously acquired outputs from mitochondrial haplotype data and correlated with past human-directed translocations and natural migration of the final cervid hosts of F. magna., Conclusions: The present study revealed a complex picture of the population genetic structure and interrelationships of North American and European populations, global distribution and migratory routes of F. magna and an origin of European foci.

Background: Genetic architecture of a species is a result of historical changes in population size and extent of distribution related to climatic and environmental factors and contemporary processes of dispersal and gene flow. Population-size and range contractions, expansions and shifts have a substantial effect on genetic diversity and intraspecific divergence, which is further shaped by gene-flow limiting barriers. The Balkans, as one of the most important sources of European biodiversity, is a region where many temperate species persisted during the Pleistocene glaciations and where high topographic heterogeneity offers suitable conditions for local adaptations of populations. In this study, we investigated the phylogeographical patterns and demographic histories of four species of semifossorial slow-worm lizards (genus Anguis) present in the Balkan Peninsula, and tested the relationship between genetic diversity and topographic heterogeneity of the inhabited ranges., Results: We inferred phylogenetic relationships, compared genetic structure and historical demography of slow worms using nucleotide sequence variation of mitochondrial DNA. Four Anguis species with mostly parapatric distributions occur in the Balkan Peninsula. They show different levels of genetic diversity. A signature of population growth was detected in all four species but with various courses in particular populations. We found a strong correlation between genetic diversity of slow-worm populations and topographic ruggedness of the ranges (mountain systems) they inhabit. Areas with more rugged terrain harbour higher genetic diversity., Conclusions: Phylogeographical pattern of the genus Anguis in the Balkans is concordant with the refugia-within-refugia model previously proposed for both several other taxa in the region and other main European Peninsulas. While slow-worm populations from the southern refugia mostly have restricted distributions and have not dispersed much from their refugial areas, populations from the extra-Mediterranean refugia in northern parts of the Balkans have colonized vast areas of eastern, central, and western Europe. Besides climatic historical events, the heterogeneous topography of the Balkans has also played an important role in shaping genetic diversity of slow worms.

Complete mitochondrial genome of the Peloponnese endemic lizard species Anguis cephallonica is presented in this study. The complete sequence is 17 208 bp long and consists of 13 protein-coding genes, 22 tRNA genes, two rRNA genes and one control region. The gene order is same as in the relative species Anguis fragilis . Length of the 22 tRNA genes varies from 64 bp to 73 bp. The Anguis cephallonica mitogenome base composition is: A (30.5%), T (24.2%), C (30.5%), G (14.8%), with an A + T bias (54.7%). Six protein coding genes have incomplete stop codons. This is the first complete mitogenome described in this species as well as in any endemic Peloponnese lizard. Presented complete mitochondrial genome will form a basis for future comparative analysis within the genus Anguis ., Competing Interests: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper., (© 2016 The Author(s). Published by Taylor & Francis.)

Polyploidization is a rare yet sometimes successful way for animals to rapidly create geno- and phenotypes that may colonize new habitats and quickly adapt to environmental changes. In this study, we use water frogs of the Pelophylax esculentus complex, comprising two species (Pelophylax lessonae, genotype LL; Pelophylax ridibundus, RR) and various diploid (LR) and triploid (LLR, LRR) hybrid forms, summarized as P. esculentus, as a model for studying recent hybridization and polyploidization in the context of speciation. Specifically, we compared the geographic distribution and genetic diversity of diploid and triploid hybrids across Europe to understand their origin, maintenance and potential role in hybrid speciation. We found that different hybrid and parental genotypes are not evenly distributed across Europe. Rather, their genetic diversity is structured by latitude and longitude and the presence/absence of parental species but not of triploids. Highest genetic diversity was observed in central and eastern Europe, the lowest in the northwestern parts of Europe. This gradient can be explained by the decrease in genetic diversity during postglacial expansion from southeastern glacial refuge areas. Genealogical relationships calculated on the basis of microsatellite data clearly indicate that hybrids are of multiple origin and include a huge variety of parental genomes. Water frogs in mixed-ploidy populations without any parental species (i.e. all-hybrid populations) can be viewed as evolutionary units that may be on their way towards hybrid speciation. Maintenance of such all-hybrid populations requires a continuous exchange of genomes between diploids and triploids, but scenarios for alternative evolutionary trajectories are discussed., (© 2015 John Wiley & Sons Ltd.)

A complex wide-range study on the haemoproteid parasites of chelonians was carried out for the first time. Altogether, 811 samples from four tortoise species from an extensive area between western Morocco and eastern Afghanistan and between Romania and southern Syria were studied by a combination of microscopic and molecular-genetic methods. Altogether 160 Haemoproteus-positive samples were gathered in the area between central Anatolia and eastern Afghanistan. According to variability in the cytochrome b gene, two monophyletic evolutionary lineages were distinguished; by means of microscopic analysis it was revealed that they corresponded to two previously described species-Haemoproteus anatolicum and Haemoproteus caucasica. Their distribution areas overlap only in a narrow strip along the Zagros Mts. range in Iran. This fact suggests the involvement of two different vector species with separated distribution. Nevertheless, no vectors were confirmed. According to phylogenetic analyses, H. caucasica represented a sister group to H. anatolicum, and both of them were most closely related to H. pacayae and H. peltocephali, described from South American river turtles. Four unique haplotypes were revealed in the population of H. caucasica, compared with seven haplotypes in H. anatolicum. Furthermore, H. caucasica was detected in two tortoise species, Testudo graeca and Testudo horsfieldii, providing evidence that Haemoproteus is not strictly host-specific to the tortoise host species., (© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.)