The dissertation is devoted to developing new methods and analyzing the effectiveness of already existing methods for studying water frogs of the genus Pelophylax, and using these methods to determine the composition and mechanisms of reproduction of water frogs in hemiclonal population systems.
Water frogs of Pelophylax esculentus complex consist of hybrid P. esculentus (Linnaeus, 1758) and two parental species, namely P. ridibundus (Pallas, 1771) (genotype RR), P. lessonae (Camerano, 1882) (genotype LL), and their interspecies hybrid P. esculentus (genotypes LR, LLR and LRR). Hybrids reproduce through hybridogenesis and live in sympatry with one or both parental species in so-called hemiclonal population systems (HPS).
To study the complex mechanisms of reproduction and maintenance of the composition of various types of HPS, it is necessary to determine the genotypes of the individuals that are part of the HPS, the types of gametes they transmit, etc. This requires a large number of methods and constant modification and development of new ones.
In total, more than 14 different methods were used to analyze more than 1,200 frogs of the genus Pelophylax. Most of frogs were parental species P. ridibundus (genotype RR), P. lessonae (genotype LL), and their interspecies hybrid P. esculentus (genotypes LR, LLR and LRR).
In this work, we developed and implemented a new protocol for obtaining mitotic chromosomes from tadpoles by using their ability to rapidly regenerate in the early stages of development; and also analyzed the effectiveness of the following methods: bioacoustic analysis of release calls, analysis of developmental anomalies, erythrocyte cytometry, sperm cytometry.
Bioacoustic analysis of release calls of five Pelophylax species and three interspecies hybrids showed that male release calls are divided into “ridibundus” and “lessonae” lineages, while hybrids’ calls have intermediate features. Within the lineages release calls of different species are also distinguished. However, the same pattern was not found for females: most of female release calls are mixed in one group. Also, release calls of triploid hybrids did not differ from diploid. Therefore, release calls cannot be used as a reliable method for species or ploidy identification of water frogs.
As a result of the analysis of developmental anomalies, no difference was found in the frequency of anomalies between P. ridibundus and P. esculentus. Also, no difference was observed between diploid and triploid hybrids. However, a significant difference in the frequency of limb malformations was found between the newly metamorphosed juveniles and the adult individuals.
It has been established that measuring the size of erythrocytes or their nuclei is not a reliable method for determining the ploidy of specific individuals, since triploids with cells and nuclei size in the range typical of diploid cells and nuclei are found in some HPS. However, this method is effective for the general detection of the presence of triploids in HPS.
Analysis of sperm size distribution of diploid and triploid P. esculentus hybrids showed that spermatogenesis of interspecies hemiclonal hybrids has many degrees of freedom and measurement of sperm size cannot be a reliable method for identification of males potentially producing diploid gametes.
We used a combination of morphological, molecular, cytogenetic and hybridological methods to comprehensively analyze the composition and pattern of reproduction in R-E-Ep-HPS and R-Epf-HPS.
Based on the results of using a combination of methods to determine the composition of three different HPS, we compared and evaluated of the effectiveness of each method used.
