Abstracts of the Conference "Fauna of Ukraine at the Turn of the 20th–21st Centuries. New Concepts in Zoological Research" (September 2017)

Shabanov D. A., Biriuk O. V., Vladimirova M. V., Kravchenko M. A., Usova O. E. Paradoxes of reproduction of Pelophylax esculentus in the Siverskyi Donets centre of green frog diversity // "New Concepts in Zoological Research". All-Ukrainian Zoological Conference (12–16 September 2017, Kharkiv): Book of Abstracts. – Kharkiv: V. N. Karazin Kharkiv National University, 2017. – P. 52–53. Drogvalenko N. A., Makaryan R. N. Composition of the hemiclonal population system of green frogs of Lake Podpesochnoye (Kreminna, Luhansk Region) // "Fauna of Ukraine at the Turn of the 20th–21st Centuries. New Concepts in Zoological Research". All-Ukrainian Zoological Conference (12–16 September 2017, Kharkiv): Book of Abstracts. – Kharkiv: V. N. Karazin Kharkiv National University, 2017. – P. 53–54. Pustovalova E. S., Biriuk O. V., Shabanov D. A. Comparison of spermatogenesis stability in diploid Pelophylax esculentus from two different types of hemiclonal population systems // "Fauna of Ukraine at the Turn of the 20th–21st Centuries. New Concepts in Zoological Research". All-Ukrainian Zoological Conference (12–16 September 2017, Kharkiv): Book of Abstracts. – Kharkiv: V. N. Karazin Kharkiv National University, 2017. – P. 54–55. Trokhymchuk R. R. Relationship between age, size and fecundity of female common toads (Bufo bufo (L., 1758)) // "Fauna of Ukraine at the Turn of the 20th–21st Centuries. New Concepts in Zoological Research". All-Ukrainian Zoological Conference (12–16 September 2017, Kharkiv): Book of Abstracts. – Kharkiv: V. N. Karazin Kharkiv National University, 2017. – P. 55–56. Shabanova H. V., Korshunov O. V., Shabanov D. A. Study of population dynamics of the common toad, Bufo bufo (L., 1758), using capture-mark-recapture // "Fauna of Ukraine at the Turn of the 20th–21st Centuries. New Concepts in Zoological Research". All-Ukrainian Zoological Conference (12–16 September 2017, Kharkiv): Book of Abstracts. – Kharkiv: V. N. Karazin Kharkiv National University, 2017. – P. 56–57. Fedorova A. O. Frequency analysis of releasing calls of water frogs from Pelophylax esculentus complex // "Fauna of Ukraine at the Turn of the 20th–21st Centuries. New Concepts in Zoological Research". All-Ukrainian Zoological Conference (12–16 September 2017, Kharkiv): Book of Abstracts. – Kharkiv: V. N. Karazin Kharkiv National University, 2017. – P. 85–86. Katrushenko S. A. Anomalies of amphibians in Kharkiv Region // "Fauna of Ukraine at the Turn of the 20th–21st Centuries. New Concepts in Zoological Research". All-Ukrainian Zoological Conference (12–16 September 2017, Kharkiv): Book of Abstracts. – Kharkiv: V. N. Karazin Kharkiv National University, 2017. – P. 111–112. PARADOXES OF REPRODUCTION OF PELOPHYLAX ESCULENTUS IN THE SIVERSKYI DONETS CENTRE OF GREEN FROG DIVERSITY D. A. Shabanov, O. V. Biriuk, M. V. Vladimirova, M. A. Kravchenko, O. E. Usova V. N. Karazin Kharkiv National University, shabanov@karazin.ua More than half a century ago, the Polish hydrobiologist L. Berger registered unusual crossbreeding results in green frogs. He established that Pelophylax esculentus (Linnaeus, 1758) is a hybrid of Pelophylax lessonae (Camerano, 1882) and Pelophylax ridibundus (Pallas, 1771). The outcomes of crosses between hybrids and representatives of the parental species were inexplicable by the concepts of the time. They were subsequently explained by the hemiclonality of P. esculentus. In Berger's crosses, one of the parental genomes of the hybrids (L — the P. lessonae genome) was eliminated from the germ line, while the other (R, P. ridibundus) underwent endoreplication and was transmitted clonally to gametes. Offspring from crosses between hybrids and P. lessonae consisted entirely of hybrids. The genetic factor responsible for this resides in the R genome. This is why L-E-HPS — hemiclonal population systems (HPS) composed of P. lessonae (“L-”) and P. esculentus (“E-”) — predominate in Western Europe. L-E-R-HPS (with both parental species) and E-Ep-HPS (where diploid hybrids, “E-”, reproduce together with triploids, “Ep-”) are also relatively well studied. R-E-HPS are considerably rarer in Western Europe. 15 years ago a region was discovered in Kharkiv Oblast that was subsequently named the Siverskyi Donets Centre of Diversity of the P. esculentus complex. R-E-Ep-HPS and R-E-HPS are typical for it; sexually mature P. lessonae are absent there. One might expect that: 1) in such systems hybrids transmit L genomes that have acquired the hemiclonal transmission factor through recombination with R; 2) some form of hybrid produces a substantial proportion of diploid gametes. These assumptions were not borne out (Biriuk et al., 2016). It has been established that P. esculentus in this centre more commonly transmit clonal R genomes or a mixture of R and L (the phenomenon of hybrid amphispermy, or more precisely, hybrid amphigamety). The proportion of diploid gametes among the total gamete pool is low, and is probably substantially lower than the proportion of triploid hybrids in many HPS. As a result of these studies, the mechanism maintaining HPS composition has become even less comprehensible in terms of current understanding. Population-ecological mechanisms probably operate in this region that substantially alter generational composition in the course of development through selective elimination of a significant proportion of individuals (Biriuk et al., 2016). Registration of the composition of individuals of different ages from natural HPS, determination of the diversity of their ontogenetic strategies (Usova et al., 2015), and broad application of simulation modelling (Shabanov, 2015) are necessary. These studies should constitute the next stage of investigation of the Siverskyi Donets Centre of Diversity of the P. esculentus complex. COMPOSITION OF THE HEMICLONAL POPULATION SYSTEM OF GREEN FROGS OF LAKE PODPESOCHNOYE (KREMINNA, LUHANSK REGION) N. A. Drogvalenko, R. N. Makaryan V. N. Karazin Kharkiv National University, jaegernicholaus@gmail.com Pelophylax esculentus (Linnaeus, 1758) is a natural hybrid of two species of green frogs, Pelophylax ridibundus (Pallas, 1771) and Pelophylax lessonae (Camerano, 1882). P. esculentus reproduces hemiclonally, transmitting clonal genomes of the parental species. They reproduce within HPS (hemiclonal population systems), in the majority of which hybrids breed together with individuals of the parental species. Hybrids exist as diploid (LR) and triploid forms (LLR and LRR); the letters denote the genomes of the parental species. In most European HPS, triploids produce haploid recombinant gametes and reproduce in crosses with diploid hybrids that produce diploid gametes. In some Central European HPS, triploids are represented by males that produce clonal diploid gametes. In Lake Podpesochnoye near the town of Kreminna in Luhansk Region, in 2010 the Tambov batrachologist G. A. Lada registered an HPS composed of P. ridibundus and triploid female P. esculentus (LLR). The data were confirmed by DNA cytometry of 10 individuals (Biriuk et al., 2016). In the Siverskyi Donets basin, the majority of LLR females produce haploid gametes; data were obtained by analysis of lampbrush-type chromosomes (Dedukh et al., 2015). In that case, the mechanism of reproduction of triploid females in Lake Podpesochnoye is unclear, and this HPS itself requires thorough investigation. In this connection, the initial stage of the study aimed to verify the composition of this HPS. For this purpose we collected samples of frogs from Lake Podpesochnoye during the spawning period (04.06.2016) and after it (02.10.2016). Blood samples were taken from the captured frogs to determine ploidy (by erythrocyte length), and tissue samples were taken for karyological analysis. From the sample of 04.06.2016 (30 individuals), 1 individual was morphologically identified as P. esculentus. This was a female with a mean erythrocyte length of 28 μm, corresponding to a triploid. The 29 individuals morphologically identified as P. ridibundus belonged to both sexes. The mean erythrocyte length (18–24 μm) of these individuals is characteristic of diploids. From the sample of 02.10.2016 (12 individuals), 2 individuals were morphologically identified as P. ridibundus. Their erythrocyte lengths (20 and 23 μm) correspond to diploids. Ten individuals were morphologically identified as female P. esculentus. Their erythrocyte lengths (27–30 μm) correspond to triploids. The ploidy of two P. esculentus individuals with the smallest erythrocyte length (27 μm) was confirmed by karyoanalysis of somatic tissue cells. Metaphase plates contained 39 chromosomes; the haploid set comprised 13 chromosomes. Thus, our data confirm the existence in Lake Podpesochnoye of an R-ELLRf-HPS, i.e. a hemiclonal population system of green frogs composed of P. ridibundus and triploid female P. esculentus (with genomic composition LLR). COMPARISON OF SPERMATOGENESIS STABILITY IN DIPLOID PELOPHYLAX ESCULENTUS FROM TWO DIFFERENT TYPES OF HEMICLONAL POPULATION SYSTEMS E. S. Pustovalova, O. V. Biriuk, D. A. Shabanov V. N. Karazin Kharkiv National University, mykhailova.o.v@gmail.com The hybridogenetic complex of green frogs (Pelophylax esculentus complex) comprises two parental species, Pelophylax lessonae (Camerano, 1882) and Pelophylax ridibundus (Pallas, 1771), as well as their diploid and allotriploid hybrids, Pelophylax esculentus (Linnaeus, 1758). Reproduction of P. esculentus occurs within hemiclonal population systems (HPS). It involves complex processes in germ-line cells: elimination of the non-clonal genome and endoreduplication (in diploids) of the clonal genome (Dedukh et al., 2015). Gametogenesis in P. esculentus proceeds with disturbances; aneuploid and polyploid cells are recorded in the testes (Biriuk, 2017). In Kharkiv Oblast, regions differing in HPS composition have been described. The Siverskyi Donets floodplain and its associated ponds are inhabited by R-E-Ep-HPS, i.e. HPS composed of one parental species (P. ridibundus, “R-”), diploid (“E-”), and triploid (“Ep-”) P. esculentus. The Mzha and Udy river basins are inhabited by R-E-HPS. As clonal genome transmission continues, the stability of hybrid reproduction increases (Shabanov, 2015). We hypothesised that, owing to recombination of clonal genomes, which is possible in triploids in R-E-Ep-HPS (Christiansen & Reyer, 2009), the stability of gametogenesis in such HPS would be higher. To test this hypothesis, we determined the distribution of the number of univalents in meiotic divisions and chromosomes in mitotic divisions of 10 male P. esculentus from Tymchenky settlement (R-E-HPS) and 7 diploid male P. esculentus from Nyzhniy Dobrytskyi pond (R-E-Ep-HPS). The processing methodology has been described previously (Veherina et al., 2013; Biriuk et al., 2015). For males from R-E-HPS, 102 meiotic plates were analysed, of which 28 (27%) were normal (26 univalents). For males from R-E-Ep-HPS, 35 plates out of 103 (34%) were normal; the difference between males from different HPS types is non-significant. Among mitotic plates, 10 of 40 (25%) were normal (26 chromosomes) for males from R-E-Ep-HPS and 8 of 17 (47%) for males from R-E-Ep-HPS. The difference in the proportion of normal karyotypes among mitotic cells (using a one-tailed criterion) is close to significance (p=0.053). In three males from R-E-HPS, no normal mitoses were recorded; normal mitoses were recorded in all diploids from R-E-Ep-HPS. The results obtained are consistent with the hypothesis of more effective adaptation of clonal genomes to hemiclonal reproduction in R-E-Ep-HPS. RELATIONSHIP BETWEEN AGE, SIZE, AND FECUNDITY OF FEMALE COMMON TOADS (Bufo bufo (L., 1758)) R. R. Trokhymchuk V. N. Karazin Kharkiv National University, rtrokhymchuk@gmail.com Over the past decade, studies of the development of tailless amphibians in Kharkiv Oblast have led to the development of the concept of intra-population ontogenetic strategies (Maro et al., 2008; Shabanov et al., 2010; Usova et al., 2015; Usova, 2016). According to this concept, individuals within a single population differ in their developmental strategy: growth rate, age at first reproduction, seasonal fecundity, and probable lifespan. The skeletochronology method (Smirina, 1983) provides valuable opportunities for studying these strategies. The aim of our study was to determine the parameters of ontogenetic strategies of common toads Bufo bufo (L., 1758) and their variability within a single habitat. Toads were caught during spawning on 2–3 April 2016 at Iskovyi Pond (surroundings of Homilshanski Lisy National Nature Park, Zmiiivskyi district, Kharkiv Oblast). Pairs of toads were placed in water-filled containers. Egg clutches were photographed and returned to the pond. The number of eggs was determined from photographs. Body length was measured, the longest toe of the hind foot was clipped, and the animals were released into the pond. The 3rd phalanx of the longest toe of the hind foot was used for skeletochronology; the methodology developed for green frogs was employed (Usova, 2014). Analysis of bone structure and counting of lines of arrested growth were carried out from digital micrographs of temporary preparations on a PC. When determining the age of toads, a correction for bone resorption (+2 years) was applied. At the time of writing, the age, body length, and fecundity of 15 female common toads have been determined. The females studied belonged to two size groups: small (45–55 mm; 6–8 years) and large (94–106 mm; 7–10 years). Egg clutches were small (2,500–3,300 eggs), medium (3,800–5,200), or large (5,500–6,700). Small females produced small and medium clutches, and large females produced clutches ranging from small to large. Six-year-old females belonged to both size groups and produced small clutches. Large clutches were produced by females aged 7 and 8 years; maximum fecundity was characteristic of eight-year-old toads. The oldest female (10 years) was large (105 mm) and laid a small clutch (approximately 3,270 eggs). The results obtained demonstrate a complex, non-linear relationship between age, size, and fecundity in common toads. The approach used proved applicable; the described studies should be continued. STUDY OF POPULATION DYNAMICS OF THE COMMON TOAD, BUFO BUFO (L., 1758), USING CAPTURE-MARK-RECAPTURE H. V. Shabanova, O. V. Korshunov, D. A. Shabanov V. N. Karazin Kharkiv National University, ashabanova1307@gmail.com The common toad, Bufo bufo (L., 1758), is a convenient object for monitoring population dynamics of a breeding assemblage. In the context of climate change and the impoverishment of amphibian fauna, such studies are of great relevance. The study was conducted at Iskovyi Pond — an artificial water body near the village of Haidary (Zmiiivskyi district, Kharkiv Oblast, surroundings of Homilshanski Lisy National Nature Park). The toad population was studied using the capture-mark-recapture method during the breeding season over the period 2000–2017. Animals were captured by hand, circumnavigating the pond along its shallow-water margin and collecting the maximum available number. Body length of each toad was measured; marking was performed by clipping a combination of toes indicating the year of marking (a group mark), after which animals were released into the pond. No additional mark was applied to toads already marked in previous years. Clipped toes were preserved for skeletochronological analysis. Over the entire study period (18 breeding seasons), 13,139 males and 3,309 females were captured; 10,721 males and 2,958 females were marked; and 2,307 males and 236 females were recaptured. In all years, males predominated in the collected samples (sex ratio males:females ranging from 1.5:1 to 12.1:1, mean 3.8:1). Population size estimates were calculated separately for males and females. Population size was estimated using several methods: the positive and negative Jackson methods; Bailey's triple-catch method. For males recaptured within a single breeding period, the Petersen-Lincoln index was applied (a “closed” population assumption, where no recruitment or loss occurs). A simulation model was also developed that, using the Solver module of Microsoft Excel, fits parameters to empirical data. Parameters were selected by minimising the value of Pearson's chi-squared goodness-of-fit criterion between the calculated and recorded numbers of individuals in recapture events. The mean population size over the observation period was approximately 7,000 males and 2,500 females in the breeding assemblage. Population size fluctuated considerably across years; in particular, the number of males varied by almost a factor of three, while variation was somewhat smaller for females. The cause of these fluctuations was presumably substantial variation in mortality and recruitment rates within the breeding assemblage, which we hypothesise to result primarily from the influence of weather conditions. Over the course of the study, progressive degradation of Iskovyi Pond has been observed as a result of its reduction in area, which raises concern about the future of the toad population under study. The authors sincerely thank many colleagues and students of the Biology Faculty of V. N. Karazin Kharkiv National University who participated in the labour-intensive field studies. FREQUENCY ANALYSIS OF RELEASING CALLS OF WATER FROGS FROM PELOPHYLAX ESCULENTUS COMPLEX A. O. Fedorova V. N. Karazin Kharkiv National University, Biology Faculty, 4 Svobody Sq., Kharkiv, Ukraine, 61022, annaph94@gmail.com Interspecies hybrids of water frogs Pelophylax esculentus (Linnaeus, 1758) and representatives of the parental species Pelophylax ridibundus (Pallas, 1771) inhabit the vicinity of the V. N. Karazin KhNU biostation (village of Haidary, Zmiiivskyi district, Kharkiv Oblast). Bioacoustic methods are useful in describing the diversity of tailless amphibians. Among all the calls they produce, the most complex and informative are the male mating calls. Differences in such calls are described and used both for taxonomic purposes and for field identification of frog forms. Releasing calls, which can be heard when attempting to grasp frogs in amplexus, are less well studied. Releasing calls are typical of both sexes, which distinguishes them from mating calls. In this study we compared releasing calls of male and female P. ridibundus and P. esculentus on the basis of a call-specific frequency distribution obtained through Fourier analysis. Releasing calls of 72 water frogs (31 males and 12 females P. ridibundus, 27 males and 2 females P. esculentus) were recorded for analysis. The frogs were held behind the torso to imitate amplexus. Recording was performed using a digital camera. The frequency distribution of 4 cyclic calls was obtained using the Audacity 2.0.5 programme. Processing of results was carried out in the Statistica 8.0 programme. We calculated a matrix of distances between the frequency distributions specific to each individual. Pearson's correlation coefficient served as the measure of difference between acoustic frequencies (the angle between vectors drawn from the coordinate origin to points whose coordinates are described by the frequency distribution of the comparison). Differences between individuals from the same group or from different groups were compared by ANOVA. The difference between males of different species is 1.5 times greater than the difference between males of the same species. The difference between females of two species, however, is almost identical to the difference between females of the same species. There is also a noticeable difference between male and female P. esculentus compared to the differences between males and females within the same species (1.5 and 2.7 times, respectively). Differences between male and female P. ridibundus are less pronounced. The results of multidimensional scaling (MDS) show that males of the two species form two slightly overlapping groups, while interspecies differences in females, as well as sex-related differences, are weaker within each species. It can be concluded that the analysed frequency characteristics of releasing calls are species-specific for males. This study needs to be continued with a possible search for specific frequencies that are species- and sex-specific. Comparison of frequency analysis with analysis of temporal characteristics also needs to be undertaken. The author thanks scientific supervisor Professor Shabanov D. A. for guidance and assistance with this study, and also thanks Viktoriia Paramonova for invaluable help with material collection and call recording. ANOMALIES OF AMPHIBIANS IN KHARKIV REGION S. A. Katrushenko V. N. Karazin Kharkiv National University, katrushenkos@mail.ua The aim of the study was to investigate the diversity of external morphological anomalies in amphibians of Kharkiv Oblast. The material comprised amphibian samples from the stock collections of the Museum of Nature of V. N. Karazin Kharkiv National University (Pelophylax esculentus complex, Rana arvalis, Lissotriton vulgaris, Bufotes viridis, Pelobates vespertinus, Bombina bombina, Hyla orientalis, Triturus cristatus; samples collected mainly between 2003 and 2007) and samples of live amphibians (B. bufo, B. viridis, P. esculentus complex; samples collected in 2016–2017). A total of 1,907 amphibians collected on the territory of Kharkiv Oblast were processed. Anomalies were determined visually, according to the classifications of V. L. Vershinin (2015) and O. D. Nekrasova (2008; 2014). Data processing was performed in STATISTICA 8.0 (using Pearson's chi-squared criterion and Spearman's rank correlation coefficient rs). For each species, the occurrence of individuals with anomalies (Pas) and the partial occurrence of anomalies (Ap) were determined (Borkin et al., 2012). Data on amphibian anomalies were compared with the characteristics of the collection localities, assessed using data from the ecological-geographical atlas of Ukraine (Baranovskyi, 2006). Fifteen variants of morphological anomalies were detected: brachydactyly, oligodactyly, ectromelia, polyphalangy, toe thickening, phalanx rotation, syndactyly, schizodactyly, ectrodactyly, cutaneous outgrowth on a toe and on the foot, taumelia, hemimelia, disturbances of skin pigmentation, and anophthalmia. It was shown (using the chi-squared criterion) that when an individual bears one anomaly, it is more likely to bear additional anomalies, which may be associated with increased developmental instability. The studied species differ significantly (p≤0.00001) in anomaly frequency. A high proportion of anomalous individuals is observed in B. viridis (8.3%) and L. vulgaris (45.6%). R. arvalis, P. vespertinus, H. orientalis, and T. cristatus showed almost no deviations. No association was found between the number of anomalies and the type of water body from which the amphibians were collected, whereas an association with the type of terrestrial habitat was found (p≤0.00001). The greatest number of anomalies occurs in amphibians from upland oak forests, steppes, and agricultural landscapes; the fewest in amphibians collected from floodplains, pine forests, and low-rise and high-rise urban developments. In B. viridis, the association between age and number of anomalies is significant (p≤0.00001), which supports the interpretation of low survival of aberrant individuals. The number of amphibian anomalies is associated (p<0.05) with the degree of pollution and ecological conditions of the habitats, but in an unexpected manner: the highest proportion of anomalous individuals (16.5%) occurs in relatively clean areas, while the lowest (5.8%) is found in polluted areas.