Biriuk O. Cytogenetic features of spermatogenesis in diploid and triploid hemiclonal interspecific hybrids of Pelophylax esculentus complex

The dissertation contains results of studies about 740 representatives of hybridоgenetic complex of green frogs (Pelophylax esculentus complex). Two different karyological techniques (squashed preparation and dropping of cell suspension) have been adapted for and applied to studying the spermatogenesis in hybrid green frogs. Ag-staining of chromosome preparations has been used to check whether the length of the nucleolus organizer region differed in the parental species. The analysis revealed no significant differences in the relative length of NORs in P. lessonae and P. ridibundus chromosomes. The conclusion was that the length of NOR could not be used as reliable species-specific marker. However, Ag-staining was suggested to be used for ploidy determination of hybrids by determining the maximal number of nucleoli in the nuclei of somatic cells. Karyological studies of germ cells show that spermatogenesis of hemiclonal interspecific hybrids P. esculentus is significantly less stable than spermatogenesis in parental species P. ridibundus. There was a lot of abnormalities within germ line cells in P. esculentus testis. Нaploid, aneuploid and polyploid cells in mitosis and meiosis have been registered. For instance, average part of normal spermatocyte I in mature hybrid males was about 29% comparing to 77% in parental species. Abnormal spermatocytes I appeared to be an effect of disturbance of elimination or reduplication process. Furthermore, DNA flow cytometry analysis of testes' cell suspension have showed particularly low part of haploid cells (on average 37%) in testis of P. esculentus in contrast to that in gonads of parental species (on average 86%). Karyological investigation of spermatogenesis of immature green frogs has revealed a similar trend. Overall, spermatogenesis was significantly less stable in immature frogs than in mature representatives of the same species or hybrid form. The study of the genomic composition of gametes have revealed only a small part of P. esculentus (7% of males and 13% of females among the diploids), yielded gametes with the clonal genome of the parental species P. lessonae, which was absent in the Sіversky Donets center of diversity of Pelophylax esculentus complex. At the same time, approximately one-third of males (31%) produced two types of haploid gametes with genomes of parental species (hybrid amphispermy). Sіversky Donets center of diversity of Pelophylax esculentus complex is the region of mass occurrence of animals with hybrid amphispermy, which has never been reported before. Based on the presence of various types of hybrids and patterns of gamete production we have divided all studied populations into three geographical groups.