Many closely related species are capable of mating to produce hybrid offspring, which are usually sterile. Nevertheless, altering the gametogenesis of hybrid offspring can rescue hybrids from sterility by enabling asexual reproduction. Hybridogenesis is one of the most complicated asexual reproductive modes, and it includes drastic genome reorganization only in the germline; this is achieved through elimination of one parental genome and duplication of the remaining one to restore diploid chromosomal set and overcome blocks in meiotic progression. We investigated a model of hybridogenesis, namely, water frogs from the Pelophylax esculentus complex, for the emergence of asexual reproduction. Further, we assessed the impact of its asexual reproduction on the maintenance of interspecies hybrids from two populations on the western edge of the P. esculentus range, in which hybrids coexist with either both parental species or with only one parental species. After analysing tadpole karyotypes, we conclude that in both studied populations, the majority of diploid hybrid males produced haploid gametes with the P. ridibundus genome after elimination of the P. lessonae genome. Hybrid females exhibited problems with genome elimination and duplication; they usually produced oocytes with univalents, but there were observations of individual oocytes with 13 bivalents and even 26 bivalents. In some hybrid tadpoles, especially F1 crosses, we observed failed germ cell development, while in tadpoles from backcrosses, germ cells were normally distributed and contained micronuclei. By identifying chromosomes present in micronuclei, we estimated that the majority of tadpoles from all crosses were able to selectively eliminate the P. lessonae chromosomes. According to our results, hybridogenesis in hybrids can appear both from crosses of parental species and crosses between sexual species with hybrid individuals. The ability to eliminate a genome and perform endoreplication to ensure gamete formation differed between male and female hybrids from the studied populations. Some diploid hybrid females can rarely produce not only haploid gametes but also diploid gametes, which is a crucial step in the formation of triploid hybrids.

The edible frog, Pelophylax esculentus, is a hybrid form that reproduces via clonal propagation of only one of the parental genomes through generations of hybrids while the genome of other parental species is eliminated during gametogenesis. Such reproductive ability requires hybrids to coexist with one of the parental species or rarely both parental species causing the formation of so‐called population systems. Population systems and reproductive biology of water frogs from the east of the range remained partially unexplored. In this study, we investigated the distributions, population systems, genetic structure, types of gametes, and morphological variability of water frogs of the genus Pelophylax from the northeastern parts of their ranges (Mari El Republic and adjacent territories, Russia). We examined 1,337 individuals from 68 localities using morphological traits combined with DNA flow cytometry and a multilocus approach (fragments of a nuclear and two mitochondrial genes). We revealed five types of population systems: “pure” populations of the parental P. ridibundus (R) and P. lessonae (L), mixed populations of parental species (R‐L) along and with their hybrids (R‐E‐L), as well as mixed populations of P. lessonae and P. esculentus (L‐E). However, the “pure” hybrid (E) and the mixed P. ridibundus and P. esculentus (R‐E) population systems were not found. All hybrids studied by DNA flow cytometry were diploid. Analysis of gametogenesis showed that the majority of hybrid males, as well as hybrid females from the L‐E system, produced gametes with the P. ridibundus genome. However, in the R‐E‐L system, hybrid females were usually sterile. The reproduction of hybrids in such systems is primarily based on crosses of P. esculentus males with P. lessonae females. Molecular analysis showed the presence of mitochondrial and nuclear DNA introgression of the Anatolian marsh frog (P. cf. bedriagae) into both P. ridibundus and P. esculentus. The observations of alleles and haplotypes of P. cf. bedriagae in P. ridibundus and P. esculentus individuals from the same localities suggest de novo formation of local hybrids. However, the presence of the Balkan marsh frog (P. kurtmuelleri) haplotypes in local hybrids supports the hypothesis regarding the migration of old hemiclonal lineages from glacial refugia. Finally, the diagnostic value of various morphological characteristics was discussed.

Background: Cases of polydactyly in natural populations of amphibians have attracted great interest from biologists. At the end of the 1940s, the French biologist Jean Rostand discovered a polymorphic syndrome in some water frog (Anura:Pelophylax) populations that included polydactyly and some severe morphological anomalies (he called it ‘anomaly P’). The cause of this anomaly remains unknown for 70 years. In a previous study, we obtained anomaly P in the laboratory in tadpoles of water frogs that developed together with molluscsPlanorbarius corneus(Mollusca: Gastropoda) collected in the field. We thus proposed the ‘trematode hypothesis’, according to which the infectious agent responsible for anomaly P is a trematode species.Methods: Metacercariae from tadpoles with anomaly P were identified using ITS2 gene sequencing asStrigea robusta(Trematoda: Strigeidae). To verify teratogenic features of the species, cercariae ofS. robustawere tested for the possibility to cause anomalies. Identification of cercariae species was made using morphological and molecular methods (sequencing of ITS2 and28SrRNA). The tadpoles were exposed to parasites at four doses of cercariae (control, low, medium and high) and divided into two groups: “early” (at 25–27 Gosner stages) and “late” (at 29–34 Gosner stages) exposure.Results: A total of 58 (72.5%) tadpoles survived until metamorphosis under the dose-dependent experiment with the trematodeS. robusta. Differences in the survival rates were observed between the exposed and unexposed tadpoles both in the group of “early” tadpoles and “late” tadpoles. The exposure of tadpoles to the cercariae ofS. robustainduced anomaly P in 82% of surviving tadpoles. The severe forms developed only in “early” stages under all doses of cercariae exposure. Polydactyly predominantly developed in the “late” stages; under a light exposure dose, polydactyly also developed in “early” tadpoles. Laboratory-hatched tadpoles reared together with infected snails had different rates of survival and complexity of deformations associated with the period of coexistence.Conclusions: The experiments with direct cercariae exposure provide compelling evidence thatS. robustaleads to anomaly P in tadpoles of water frogs. The manifestation of anomaly P turned out to be dependent on the stage of development, cercariae dose, and the location of the cysts.

A molecular multiplex PCR method for identification of East European green frog species (Pelophylax ridibundus, P. cf. bedriagae and P. lessonae) and their hybrids was developed. This simple and rapid method can be used for identification of species-specific mitochondrial and nuclear DNA. The method is based on species-specific differences in primary structure of the subunit 1 of the mitochondrial cytochrome C oxidase gene (COI) and the intron-1 of the nuclear serum albumin gene (SAI-1). Based on the method, we analyzed numerous individuals of these species and their hybrids from East European Plain, the Crimea, the Caucasus, the Ural, as well as introduced populations from Western Siberia and the Kamchatka. In all cases, identification of species performed by use of the multiplex PCR method coincided with results of study of primary nucleotide sequences.