Hybridization and polyploidy play an important role in animal speciation. European water frogs of the Pelophylax esculentus complex demonstrate unusual genetic phenomena associated with hybridization, clonality and polyploidy which presumably indicate an initial stage of reticulate speciation. The Seversky Donets River drainage in north-eastern Ukraine is inhabited by both sexes of the diploid and triploid hybrid P. esculentus and only one parental species Pelophylax ridibundus. Based on the presence of various types of hybrids, all populations studied can be divided into three geographical groups: I) P. ridibundus-P. esculentus without triploids; II) P. ridibundus-P. esculentus without diploid hybrids; and III) P. ridibundus-P. esculentus with a mixture of diploids and triploids. A study of gametogenesis revealed that diploid P. esculentus in populations of the first type usually produced haploid gametes of P. ridibundus and a mixture of haploid gametes that carried one or another parental genome (hybrid amphispermy). In populations of the second type, hybrids are derived from crosses of P. ridibundus males with triploid hybrid females producing haploid eggs with a genome of P. lessonae. Therefore, we suggest that clonal genome duplication in these eggs might be the result of suppression of second polar body formation or extra precleavage endoreduplication. In populations of the third type, some diploid females can produce diploid gametes. Fertilization of these eggs with haploid sperm can result in triploid hybrids. Other hybrids here produce haploid gametes with one or another parental genome or their mixture giving rise to new diploid hybrids.

Pelophylax esculentus is an interspecific hybrid which reproduces hemiclonally by crossing with the parental species (P. ridibundus or P. lessonae). The structure of hemiclonal population systems is of great interest. The majority of investigations into populations of water frogs deal with samples of mature animals, while subadults are less studied. We collected a random sample of 73 small water frogs from three localities in the Siverskyi Donets River floodplain. All frogs were measured, injected with colchicines and killed after anesthesia. We determined the species specifity, sex, age and ploidy of every individual. In addition, we measured testis length and studied the germ cells of all males by means of karyological analysis. We calculated the portion of triploids in the largest subsample from the vicinity of the Biological Station of V.N. Karazin Kharkiv National University and carried out a meta-analysis of previous publications dealing with composition of green frog samples from this locality. The proportion of triploids in our sample appeared to be approximately the same as 12 years ago. However, this proportion in our sample differed significantly from that obtained in recent samples of green frogs belonging to other age groups (adults and metamorphs) from the same locality. The share of triploids of P. esculentus is the lowest in the sample of metamorphs. This proportion increases in froglets because of early death of representatives of parental species which originated from hybrid-hybrid crossing. Then the number of triploids among adult P. esculentus individuals declines again probably because of their lower viability compared to diploids. We have revealed that subadult diploids have erythrocytes smaller than 28 microns, while triploids’ erythrocytes exceed 27 microns. Therefore, in borderline cases true ploidy could be determined only by the karyological technique. The average ratio between testis length and body length appeared to be larger in the parental species than in both diploid and triploid hybrids. Karyological analysis has revealed that P. esculentus had significantly lower portions of spermatocytes I with normal karyotype (13 bivalents) in the testis in comparison with P. ridibundus, but this value increases in adult hybrids. We suggest that increasing of spermatogenesis stability in adult frogs as opposed to subadults might be the consequence of both selection of germ cell lines in the testis and more frequent survival of individuals with stable gametogenesis.