Stakh et al. (2016) Features of GPS of Green Frogs of Korjakov Yar

Stakh V. O., Bobrova A. A., Yermakov D. V., Meleshko O. V., Tarasenko K. S., Korshunov O. V., Kravchenko M. O. Features of the hemiclonal population system of green frogs (Pelophylax esculentus complex) in the Koryakov Ravine (NPP “Homilshansky Forests”) / Proceedings of the scientific conference “State and biodiversity of ecosystems of Shatsk National Natural Park and other protected areas” (Shatsk settlement, September 8–11, 2016). – Lviv: SPOLOM, 2016. – pp. 88‑92. Features of the hemiclonal population system of green frogs (Pelophylax esculentus complex) in the Koryakov Ravine (NPP “Homilshansky Forests”) Stakh V. O.¹, Bobrova A. A.², Yermakov D. V.², Meleshko O. V.², Tarasenko K. S.², Korshunov O. V.², Kravchenko M. O.² ¹Ivan Franko National University of Lviv ²V. N. Karazin Kharkiv National University marinakravchenko2106@gmail.com The locality called “Koryakiv Yar”, located near the village of Haidari in Zmiiv district, Kharkiv region, is notable because in 1914 the Siversko‑Donets Biological Station of the Society of Naturalists at Kharkiv University was established there. This site was chosen because earlier investigations by Society members had shown that the raised oak forest near the former Zaytsevo hamlet was “an exceptionally interesting place in a natural‑historical sense” [1]. During the years of the university’s biological station (now Kharkiv National University named after V. N. Karazin) it was moved about 1.5 km to the northeast, to the former estate of photographer O. M. Ivanitsky. Our work is devoted to green frogs inhabiting a small pond located in the Koryakov Ravine (49°36′57″ N; 36°18′44″ E). This pond lies in a raised oak forest above the floodplain of the Siversky Donets within the Homilshansky Forests National Natural Park. As is known, green frogs (Pelophylax esculentus complex) constitute a hybridogenic complex composed of two parental species: the pool frog Pelophylax lessonae (Camerano, 1882) and the marsh frog Pelophylax ridibundus (Pallas, 1771), as well as various forms of their interspecific hybrids differing in ploidy and genome composition. The latter are referred to by a name analogous to the species: the edible frog Pelophylax esculentus (Linnaeus, 1758). A distinctive feature of P. esculentus is its ability for hemiclonal inheritance – transmission of the genome of one parental species through the gametes without recombination, i.e., clonally. Reproduction of P. esculentus in most cases occurs via hybridogenesis with representatives of the parental species. Such assemblages of different green‑frog forms that reproduce together and transmit both recombinant and clonal genomes are called hemiclonal population systems, HPS (HPS) [9]. The presence of P. lessonae, P. ridibundus and P. esculentus in an HPS is denoted by the first letters of their species names [2, 8, 14 with modifications]. Thus, for Eastern Ukraine the characteristic type is R‑E‑HPS, comprising P. ridibundus and P. esculentus. Polyploid P. esculentus are indicated by Ep; if P. esculentus are represented only by females or only by males, this is marked by the letters f or m. For example, in the Siversky Donets near the Homilshansky Forests NPP occurs R‑E‑Ep‑HPS, which includes P. ridibundus and diploid and triploid P. esculentus of both sexes. In contrast, the pond in the village of Zamulyvka (former Zhovtneve) of Vovchansky district, Kharkiv region, hosts R‑E‑Epf‑HPS, in which triploid hybrids are represented only by females [8]. The study of the Koryakov Ravine HPS began in 1995‑1996 by G. A. Lada – a Tambov herpetologist who collaborated with the Zoological Institute of the Russian Academy of Sciences (St. Petersburg). He discovered in the Koryakov Ravine, as well as in the Iškiv pond (the subject of another paper in this volume), population systems consisting solely of diploid hybrids [8]. At the beginning of the 21st century, only a few green frogs were found in the Koryakov Ravine. This pond is a subject of hydrobiological studies during student practice at V. N. Karazin Kharkiv National University, where isolated tadpoles of green frogs were collected. In 2002 university staff together with members of the Zoological Institute of the RAS began systematic investigations of the green‑frog assemblage in Kharkiv region. These studies led to the identification of a region of massive occurrence of triploid hybrids [4, 12], later named the Siversko‑Donets Center of Diversity of the Pelophylax esculentus complex [9]. Between 2002 and 2013, flow cytometric DNA analysis established the genotypes of 45 frogs from the Koryakov Ravine. The authors express sincere gratitude to S. M. Lytvynchuk and Y. M. Rozanov for performing the cytometric analysis and providing its results. According to these data, in the Koryakov Ravine 20 immature (including metamorphs) P. ridibundus, 8 immature and 11 ♂ ♂ P. esculentus (2n), 1 immature and 1 ♀ P. esculentus (3n) with genome composition LLR, as well as 1 immature individual and 2 ♂ ♂ P. esculentus (3n) with genome composition LRR were found (L denotes the P. lessonae genome, R – the P. ridibundus genome). The apparent absence of diploid P. esculentus females is probably random and related to the fact that only one female was found in the pond during that period. In 2015 the situation changed dramatically. In May 2015 a spawning aggregation was observed in the pond, in which, according to O. V. Korshunov’s counts, no fewer than 67 males, all unequivocally identified by vocalization as P. esculentus [10], were present. Surprisingly, after this intense spawning, only a few tadpoles were found in the habitat. Repeated searches yielded only two tadpoles; their ploidy, determined by carianalysis, showed both to be diploids [7]. In 2015 an assessment of the size and composition of the Koryakov Ravine HPS was also started using marking and recapture. The sample collected in June–July 2015 (according to Y. Artemova, T. Beshentsova and O. Meleshko) comprised 5 ♂ ♂ P. ridibundus, 1 ♀ and 72 ♂ ♂ P. esculentus (2n), 4 ♀ ♀ and 1 ♂  P. esculentus (3n). The sample collected in July 2016 consisted of 4 ♀ ♀ and 3 ♂ ♂ P. ridibundus, 1 ♀ and 27 ♂ ♂ P. esculentus (2n), 1 ♀ and 1 ♂ P. esculentus (3n). The difference between the 2015 and 2016 samples was not statistically significant (p = 0.53). Thus, long‑term observations indicate that the Koryakov Ravine hosts an R‑E‑Ep‑HPS with a pronounced male bias among diploid hybrids. The population size estimated by the Petersen method [6] in 2015 was 413 ± 177 individuals. Unfortunately, no 2015 marks were recovered in the 2016 sample. Flow cytometry shows that the proportion of P. ridibundus is higher among immature individuals than among sexually mature ones. We interpret this as supporting hypothesis [2] that the share of diploids among sexually immature frogs is higher because it includes parental‑species individuals generated through hybrid‑induced backcrossing (so‑called hybridolysis). Such crosses are known to produce non‑viable offspring [13]. The number of tadpoles in the pond increased dramatically in 2016: a single excursion in July 2016 yielded 15 individuals. To elucidate possible reasons for the lack of tadpoles in 2015 and their abundance in 2016, we tested male fertility. Each individual received an injection of 1 µg surfacton – a synthetic gonadotropin‑releasing hormone – per gram of body mass [3]. Two hours later, urine was massaged from the male to obtain urinary sperm, and the number of active spermatozoa per µL was counted. Of three examined P. ridibundus males, one had no spermatozoa in the urinary sperm, another showed a reduced count (≈ 5 000 µL⁻¹), and the third had a normal count (> 15 000 µL⁻¹). Among 28 P. esculentus males, spermatozoa were absent in 23; in three individuals (including the sole triploid in the sample) sperm counts were reduced (≈ 3 000 in the triploid, 3 000 and 7 000 in two diploids), and only two males displayed normal sperm numbers (> 15 000 µL⁻¹). It should be noted that several males from the 2016 sample exhibited poorly developed secondary sexual characteristics: underdeveloped nuptial pads and vocal sacs. One such male was dissected for sex determination and was found to have underdeveloped testes. All such feminized males failed to produce active sperm under hormonal stimulation. [IMG_1] Comparison of intra‑group variability of green frogs from two localities. Vertical lines indicate the 0.95 % confidence interval of the mean value for phenetic distances between different individuals within each group. These results suggest that the population explosion of green frogs observed in 2015 resulted from one “successful” introduction or a few introductions in which a single clonal genome (most likely the male P. lessonae genome contributed by a male) was transmitted, leading to male infertility in P. esculentus. To test this hypothesis, we analyzed phenetic diversity of P. esculentus males and representatives of all three forms of the Pelophylax esculentus complex from Lake Pysochne (51°56′94″ N; 23°90′23″ E). Each individual was described by eight characters: dorsal‑medial stripe pattern, skin surface, dorsal spots, ventral coloration, throat, snout, legs, and relative position of the posterior limb articulations (see figure). For each pair of individuals within each group (by taxonomic affiliation, sex, and origin) a phenetic distance was calculated as the proportion of differing characters. Thus, a distance of 1 corresponds to individuals differing in all eight traits, while a distance of 0.125 corresponds to individuals differing in only one trait. According to the figure, males of P. esculentus from the Koryakov Ravine are markedly more homogeneous than individuals from other groups (differences are highly statistically significant). This indicates a high genetic similarity, possibly resulting from the spread of a single clonal genome in the ravine. A paradox arises: for this clonal genome to spread in the pond it would have had to ensure successful reproduction of its carrier, yet at the present stage we observe that it causes sexual development disorders. In any case, further studies of the Pelophylax esculentus complex HPS in the Koryakov Ravine are of considerable scientific interest. 1. Arnoldi V. M. Siversko‑Donets Biological Station of the Society of Naturalists at Kharkiv University // Kharkiv, 1918. 16 p. 2. Biriuk O. V., Usova E. E., Meleshko E. V., Shabanov D. A. Stability of spermatogenesis and selection manifestations at the cellular and individual levels in immature representatives of the Pelophylax esculentus complex // Vysnyk Dnipropetrovsk University. Biology, Ecology. 2016. 24(1). pp. 193–202. 3. Bobrova A. A., Makaryan R. M., Sheiko V. P., Shabanov D. A. Fertility disorders in interspecific hybrids of green frogs from the Siversko‑Donets Center of Diversity of the Pelophylax esculentus complex // Biology and Valiology. 2014. Issue 16. pp. 7–15. 4. Borkin L. Y., Zinenko A. I., Korshunov A. V. et al. Mass polyploidy in the hybridogenic complex Rana esculenta (Ranidae, Anura, Amphibia) in Eastern Ukraine // Materials of the Ukrainian Herpetological Society Conference. K.: Zoological Museum of the NNP M NASU, 2005. pp. 23–26. 5. Lada G. A. On the need to preserve unique “pure” diploid populations of the edible frog (Rana esculenta Linnaeus, 1758) in the Belgorod and Kharkiv regions // Problems of protection and rational use of natural ecosystems and biological resources. Penza, 1998. pp. 333–335. 6. Koli G. Analysis of vertebrate populations. Moscow: Mir, 1979. 362 p. 7. Pustovalova E. S., Batureva E. D., Kharchenko T. S. Ploidy of tadpoles of green frogs from the Iškiv pond and Koryakov Ravine (Zmiiv district, Kharkiv region) // Biology: from molecule to biosphere: Materials of the X International Conference of Young Scientists (December 2–4, 2015, Kharkiv, Ukraine). Kharkiv: FOP Shapovalova T. M., 2015. pp. 183‑184. 8. Shabanov D. A. Evolutionary ecology of population systems of the hybridogenic green‑frog complex (Pelophylax esculentus complex) of the Left‑bank forest‑steppe of Ukraine: author’s abstract of dissertation … Doctor of Biological Sciences, specialization 03.00.16 — Ecology. Dnipro, 2015. 36 p. 9. Shabanov D. A., Korshunov O. V., Kravchenko M. O. What green frogs inhabit Kharkiv region? Terminological and nomenclatural aspects of the problem // Biology and Valiology. 2009. Issue 11. pp. 116–125. 10. Shabanov D. A. Ponds in the “Great Haidars”: Iškiv pond, Koryakov Ravine, Lower Dobritsky pond // /Iškiv‑Koryakov‑Dobritsky [Electronic resource] 11. Biriuk O., Shabanov D., Korshunov O. et al. Gamete production patterns and mating systems in water frogs (hybridogenetic Pelophylax esculentus complex) in North‑Western Ukraine // Journal of Zoological Systematics and Evolutionary Research. 2015. Vol. 54. No. 3. pp. 215–225. 12. Borkin L. J., Korshunov A. V., Lada G. A. et al. Mass occurrence of polyploid green frogs (Rana esculenta complex) in Eastern Ukraine // Russian Journal of Herpetology. 2004. Vol. 11 (3). pp. 194–213. 13. Günther R., Plötner J. Zur Problematik der klonalen Vererbung bei Rana kl. esculenta (Anura) // Beiträge zur Biologie und Bibliographie (1960‑1987) der europäischen Wasserfrösche / R. Günther, R. Klewen. – Duisburg: Verlag für Ökologie und Faunistik, 1988. pp. 23‑46. 14. Uzzell T. M., Berger L. Electrophoretic phenotypes of Rana ridibunda, Rana lessonae and their hybridogenic associate Rana esculenta // Proc. Acad. Nat. Sci. Phila. 1975. Vol. 127. pp. 13‑24.