Biol. Stud. 2018: 12(3–4); 17–26 • DOI:


V. O. Stakh, Iu. M. Strus, I. S. Khamar


The results of the analysis of the genetic structure in population systems of green frogs on the territory of Lviv and Volyn regions are presented. The material was collected in 2011–2012 in water bodies representing three nature regions of Ukraine – Forecarpathians, Roztochia, and Western Polissia. Three taxonomic groups of green frogs were in a focus of the study: Marsh frog – Pelophylax ridibundus (Pallas, 1771), Pool frog – Pelophylax lessonae (Camerano, 1882) and their hybrid – Edible frog – Pelophylax esculentus (Linnaeus, 1758). DNA was extracted from 91 individuals and analyzed using of 10 pairs of primers: Rrid059, Rrid082, Rrid171, Res5, Res14, Res16, Res22, RlCA1b5, RlCA18, RlCA19. A majority of those are highly polymorphic and diagnostic for species identification. During the analysis, we used programs based on principles of Bayesian statistics and Monte-Carlo Markov Chain algorithms: Structure, BAPS, and NewHybrids. Linkage groups were searched using the GenePop software, and hidden null-alleles were detected using Micro-Checker program. For the first time, in the studied area the genetic structure of populations and population systems were described. After the analysis of genetic diversity of frogs sampled from the Pelophylax ridibundus population and from hemiclonal population systems of mixed R-E-L type, we found that the smallest genetic diversity is observed in the population of Marsh frog from the Nyzhankovychi area (Forecarpathians). More diverse are hemiclonal population systems of green frogs sampled in water bodies of “Cholgynskyi” ornithological reserve (Ukrainian Roztochia) and Shatsk National Nature Park (Western Polissia). Also, for the first time, the hybrid composition of studied localities is described. Hybrids of the first generation (F1) and backcrosses were detected in water bodies of Shatsk National Nature Park and ornithological reserve “Cholgynskyi”.

Keywords: green frogs, microsatellite loci, Structure, BAPS, NewHybrids, backcrosses, water bodies of Western Ukraine

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1. Anderson E., Thompson E. A model-based method for identifying species hybrids using multilocus genetic data. Genetics, 2002; 160(3): 1217-1229.

2. Arnaud-Haond S., Alberto F., Teixeira S. et al. Assessing genetic diversity in clonal orga­nisms: low diversity or low resolution? Combining power and cost efficiency in selecting markers. Journal of Heredity, 2005; 96(4): 434-440.

3. Arnaud-Haond S., Belkhir K. GENCLON: a computer program to analyse genotypic data, test for clonality and describe spatial clonal organization. Molecular Ecology Notes, 2007; 7: 15-17.

4. Berger L. Is Rana esculenta lessonae Camerano a distinct species? Annales Zoologici, 1964; 22(13): 245-261.

5. Berger L., Berger A. Persistence of all-hybrid water frog populations (Rana kl. esculenta) in northern German. Genet. Pol, 1994; 35(1-2): 73-80.

6. 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; 54(3): 215-225.

7. Bohling J.H., Adams J.R., Waits L.P. Evaluating the ability of Bayesian clustering methods to detect hybridization and introgression using an empirical red wolf data set. Molecular Eco­logy, 2013; 22: 74-86.

8. Carlsson J. Effects of microsatellite null alleles on assignment testing. Journal of Heredity, 2008; 99(6): 616-623.

9. Charney N.D. Relating hybrid advantage and genome replacement in unisexual salamanders. Evolution, 2011; 66-5: 1387-1397.

10. Corander J., Marttinen P., Sirén J. et al. Enhanced Bayesian modelling in BAPS software for learning genetic structures of populations. BMC Bioinformatics, 2008; 9: 539: 1-14.
PMid:19087322 PMCid:PMC2629778

11. Corander J., Cheng L., Marttinen P. et al. BAPS: Bayesian Analysis of Population Structure. Manual v. 6.0. Department of Mathematics and statistics University of Helsinki, 2013. 28 p.

12. Dawson K., Belkhir K. A Bayesian approach to the identification of panmictic populations and the assignment of individuals. Genetical Research, 2001; 78(1): 59-78.

13. Doležálková M., Pruvost N. B. M., Plötner J. et al. All-male hybrids of a tetrapod Pelophylax esculentus share its origin and genetics of maintenance. Biology of Sex Differences, 2018; 9(13): 1-11.
PMid:29609661 PMCid:PMC5880063

14. Dufresnes C., Denoël M., di Santo L. et al. Multiple uprising invasions of Pelophylax water frogs, potentially inducing a new hybridogenetic complex. Scientific reports, 2017; 7: 6506: 1-9.
PMid:28747630 PMCid:PMC5529583

15. Earl D.A., VonHoldt B.M. STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv. Genet. Resour, 2012; 4: 359-361.

16. Falush D., Stephens M., Pritchard J.K. Inference of population structure using multilocus genotype data: dominant markers and null alleles. Molecular Ecology Notes, 2007; 7: 574-578.
PMid:18784791 PMCid:PMC1974779

17. François O., Ancelet S., Guillot G. Bayesian clustering using hidden Markov random fields in spatial population genetics. Genetics, 2006; 174(2): 805-816
PMid:16888334 PMCid:PMC1602073

18. Gao H., Williamson S., Bustamante C. A Markov chain Monte Carlo approach for joint inference of population structure and inbreeding rates from multilocus genotype data. Genetics, 2007; 176(3): 1635-1651.
PMid:17483417 PMCid:PMC1931536

19. Garner T.W.J., Gautschi B., Rothlisberger S. et al. Set of CA repeat microsatellite markers derived from the pool frog, Rana lessonae. Molecular Ecology, 2000; 9: 2155 - 2234.

20. Guillot G., Santos F., Estoup A. Analysing georeferenced population genetics data with Geneland: a new algorithm to deal with null alleles and a friendly graphical user interface. Bioinformatics, 2008; 24(11): 1406-1407.

21. Herczeg D., Vörös J., Christiansen D.G. et al. Taxonomic composition and ploidy level among European water frogs (Anura: Ranidae: Pelophylax) in eastern Hungary. J Zool Syst Evol Res, 2017; 55(2): 129-137.

22. Hoffmann A., Plötner J., Pruvost N. et al. Genetic diversity and distribution patterns of diploid and polyploid water frogs (Pelophylax esculentus) across Europe. Molecular Ecology, 2015; 24(17): 4371-4391.

23. Holm S. A simple sequentially rejective multiple test procedure. Scandinavian Journal of Statistics,1979; 6(2): 65-70.

24. Hotz H., Uzzel T. Biochemically Detected Sympatry of Two Water Frog Species: Two Different Cases in the Adriatic Balkans (Amphibia, Ranidae). Proceedings of the Academy of Natural Sciences of Philadelphia. 1982; 134: 50-79.

25. Hotz H., Uzzel T., Guex G.-D. et al. Microsatellites: a tool for evolutionary genetic studies of western Palearctic water frogs. Mitt. Mus. Nat. kd. Berl., Zool. Reihe, 2001; 77(1): 43-50.

26. Jakobsson M., Rosenberg N.A. CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality analysis of population structure. Bioinformatics, 2007; 23: 1801-1806.

27. Huelsenbeck J., Andolfatto P. Inference of population structure under a Dirichlet process model. Genetics, 2007; 175(4): 1787-1802.
PMid:17237522 PMCid:PMC1855109

28. Kaeuffer R., Reale D., Coltman D.W. et al. Detecting population structure using STRUCTURE software: effect of background linkage disequilibrium. Heredity, 2007; 99: 374-380.

29. Kanginakudru S., Metta M., Jakati R.D. et al. Genetic evidence from Indian red jungle fowl corroborates multiple domestication of modern day chicken. BMC Evolutionary Biology, 2008; 8:174: 1-14.
PMid:18544161 PMCid:PMC2474866

30. Kravchenko M.A., Shabanov D.A. Possible Ways of Transformation of Population Systems of Pelophylax esculentus complex (Ranidae, Anura, Amphibia). Proceeding of the Ukranian Herpetological Society, 2008; 1:15-20. (In Russian)

31. Nekrasova O.D. Interspecific Variability and Colouring Polymorphism of Green Frogs Rana esculenta Complex (Amphibia, Ranidae) in Hybrid Populations. Vestnik zoologii, 2002; 36(4): 47-54. (In Russian)

32. Ogielska M. Reproduction of amphibians. Enflield, NH: Science Publishers, 2009. 422 p.

33. Oosterhout van C., Hutchinson W.F., Wills D.P.M. et al. MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology Notes, 2004; 4: 535-538.

34. Peakall R., Smouse P. GenAlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research-an update. Bioinformatics, 2012; 28: 2537-2539.
PMid:22820204 PMCid:PMC3463245

35. Pella J., Masuda M. The Gibbs and split-merge sampler for population mixture analysis from genetic data with incomplete baselines. Canadian Journal of Fisheries and Aquatic Sciences, 2006; 63(3): 576 - 596.

36. Perez-Enriquez R., Medina-Espinoza J.A, Max-Aguilar A. et al. Genetic tracing of farmed shrimp (Decapoda: Penaeidae) in wild populations from a main aquaculture region in Mexico. Rev. Biol. Trop, 2018; 66(1): 381-393.

37. Pidancier N., Miquel C., Miaud C. Buccal swabs as a non-destructive tissue sampling method for DNA analysis in amphibians. Herpetological Journal, 2003; 13(4): 175-178.

38. Plötner J. Die westpaläarktichen Wasserfrösche. Bielefeld: Laurenti-Verlag, 2005. 161 p.

39. Pompanon F., Bonin A., Bellemain E. et al. Genotyping errors: causes, consequences and solutions. Nature reviews: Genetics, 2005; 6: 847-859.

40. Pritchard J.K., Stephens M., Donnelly P. Inference of population structure using multilocus genotype data. Genetics, 2000; 155 (2): 945-959.

41. Pruvost N.B.M., Hoffmann A., Reyer H.-U. Gamete production patterns, ploidy, and population genetics reveal evolutionary significant units in hybrid water frogs (Pelophylax esculentus). Ecology and Evolution, 2013; 3(9): 2933-2946.
PMid:24101984 PMCid:PMC3790541

42. Pruvost N.B.M., Mikulicek P., Choleva L., Reyer H.-U. Contrasting reproductive strategies of triploid hybrid males in vertebrate mating systems. Journal of Evolutionary Biology, 2015; 28: 189-204.

43. Quilodran C.S., Montoya-Burgos J.I., Currat M. Modelling interspecific hybridization with genome exclusion to identify conservation actions: the case of native and invasive Pelophylax waterfrogs. Evolutionary Applications, 2015; 8(2): 199-210.
PMid:25685194 PMCid:PMC4319866

44. Raymond M., François R. GENEPOP Version 1.2: Population Genetics Software for Exact Tests and Ecumenicism. J Heredity, 1995; 86(3): 248-249.

45. Rousset F., Raphaël L. Likelihood and approximate likelihood analyses of genetic structure in a linear habitat: performance and robustness to model mis-specification. Mol. Biol. Evol, 2007; 24(12): 2730-2745.

46. Rousset F., Raphaël L. Likelihood-Based Inferences Under Isolation by Distance: Two-Dimensional Habitats and Confidence Intervals. Mol. Biol. Evol, 2012; 29(3): 957-973.

47. Shabanov D.A., Korshunov O.V., Kravchenko M.O. Which of the water frogs inhabit Kharkiv oblast? Perspectives on terminology and nomenclature. Proceedings of G.S. Skovoroda National Pedagogic University of Kharkiv. Biology and Valeology, 2009; 11: 116- 125. (In Ukrainian)

48. Shabanov D.A., Litvinchuk S.N. Green frogs: life without rules or a special way of evolution? Priroda, 2010; 3: 29-36. (In Russian)

49. Smouse P.E., Banks S.C., Peakall R. Converting quadratic entropy to diversity: Both animals and alleles are diverse, but some are more diverse than others. PLOS ONE, 2017; 12(10): 1-19.
PMid:29088229 PMCid:PMC5663342

50. Stakh V., Belokon M., Khamar I. аt al. Morphological and genetic polymorphism of green frogs (Pelophylax) in water bodies of Western Ukraine. Visnyk of the Lviv University. Series Biology, 2014; 64: 241-249. (In Ukrainian)

51. Stakh V., Reshetylo O., Khamar I. Inter-population morphometric variability of Pelophylax ridibundus (Anura, Amphibia) in the water bodies of Lviv province. Visnyk of the Lviv University. Series BIology, 2016; 72: 180-186.

52. Stakh V.O., Khamar I.S., Reshetylo O.S., Zabytivskyi Yu.М. Phenes of water frogs (Pelophylax) as the indicators of water bodies' contamination in Pre-Carpathians, Roztochia, Lesser and Western Polissia. Studia Biologica, 2017; 11(1): 161-168.

53. Stakh V. Population structure of green frogs in Western Ukraine according to the results of analysis in Structure, BAPS and NewHybrids. Mendeley Data, v1, 2018.

54. Stakh V. The genotypes of green frogs from water bodies of Western Ukraine. Mendeley Data, v1, 2018.

55. Wagner A.P., Creel S., Kalinowski S.T. Estimating relatedness and relationships using microsatellite loci with null alleles. Heredity, 2006; 97: 336-345.

56. Wilson G., Rannala B. Bayesian inference of recent migration rates using multilocus genotypes. Genetics, 2003; 163(3): 1177-1191.

57. Zeisset I., Rowe G., Beebee T.J.C. Polymerase chain reaction primers for microsatellite loci in the north European water frogs Rana ridibunda and R. lessonae. Molecular Ecology, 2000; 9(8): 1173-1174.

58. Zimovin А.І. Bayes factor vs. p-value: evaluation of statistical hypotheses likelihood in psychology. Technologies of intellect development, 2016; 14: 1-18. (In Russian)



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