Interspecific hybridization can disrupt canonical gametogenic pathways, leading to the emergence of clonal and hemiclonal organisms. Such gametogenic alterations usually include genome endoreplication and/or premeiotic elimination of one of the parental genomes. The hybrid frog Pelophylax esculentus exploits genome endoreplication and genome elimination to produce haploid gametes with chromosomes of only one parental species. To reproduce, hybrids coexist with one of the parental species and form specific population systems. Here, we investigated the mechanism of spermatogenesis in diploid P. esculentus from sympatric populations of P. ridibundus using fluorescent in situ hybridization. We found that the genome composition and ploidy of germ cells, meiotic cells, and spermatids vary among P. esculentus individuals. The spermatogenic patterns observed in various hybrid males suggest the occurrence of at least six diverse germ cell populations, each with a specific premeiotic genome elimination and endoreplication pathway. Besides co-occurring aberrant cells detected during meiosis and gamete aneuploidy, alterations in genome duplication and endoreplication have led to either haploid or diploid sperm production. Diploid P. esculentus males from mixed populations of P. ridibundus rarely follow classical hybridogenesis. Instead, hybrid males simultaneously produce gametes with different genome compositions and ploidy levels. The persistence of the studied mixed populations highly relies on gametes containing a genome of the other parental species, P. lessonae.

Department of Biology and Ecology Faculty of Science University of Ostrava Ostrava Czech Republic

Laboratory of Amphibian Population Ecology Department of Zoology and Animal Ecology School of Biology 5 N Karazin Kharkiv National University Kharkiv Ukraine

Laboratory of Fish Genetics Institute of Animal Physiology and Genetics of the CAS v v i Libechov Czech Republic

Zobrazit více v PubMed

Abbot R, Albach D, Ansell S, Arntzen JW, Baird SJE, Bierne N, Boughman J, Brelsford A, Buerkle CA, Buggs R, Butlin RK, Dieckmann U, Eroukhmanoff F, Grill A, Cahan SH, Hermansen JS, Hewitt G, Hudson AG, Jiggins C, Jones J, Keller B, Marczewski T, Mallet J, Martinez-Rodriguez P, Möst M, Mullen S, Nichols R, Nolte AW, Parisod C, Pfennig K, Rice AM, Ritchie MG, Seifert B, Smadja CM, Stelkens R, Szymura JM, Väinölä R, Wolf JBW, Zinner D. Hybridization and speciation. Journal of Evolutionary Biology. 2013;26:229–246. doi: 10.1111/j.1420-9101.2012.02599.x. PubMed DOI

Arnold ML, Hodges SA. Are natural hybrids fit or unfit relative to their parents? Trends in Ecology & Evolution. 1995;10(2):67–71. doi: 10.1016/S0169-5347(00)88979-X. PubMed DOI

Battaglia E. Sulla terminologia dei processi apomittici. Nuovo Giornale Botanico Italiano. 1947;54:674–696. doi: 10.1080/11263504709440462. DOI

Berger L. Sex ratio in the F1 progeny within forms of Rana esculenta complex. Genetica Polonica. 1970;12:87–101.

Berger L. Viability, sex and morphology of F2 generation within forms of Rana esculenta complex. Zoologica Poloniae. 1971;21(4):345–393.

Berger L. Systematics and hybridization in the Rana esculenta complex. In: Taylor DH, Guttman SI, editors. The reproductive biology of amphibians. Springer; Boston: 1977. pp. 367–388. DOI

Berger L. Systematyka i systemy genetyczne zab zielonych Europy. Przegrad Zoologiczny. 1983;28:47–61.

Bi K, Bogart JP. Probing the meiotic mechanism of intergenomic exchanges by genomic in situ hybridization on lampbrush chromosomes of unisexual Ambystoma (Amphibia: Caudata) Chromosome Research. 2010;18:371–382. doi: 10.1007/s10577-010-9121-3. PubMed DOI

Biriuk OV, Shabanov DA, Korshunov AV, Borkin LJ, Lada GA, Pasynkova RA, Rosanov JM, Litvinchuk SN. Gamete production patterns and mating systems in water frogs of the hybridogenetic Pelophylax esculentus Complex in northeastern Ukraine. Journal of Zoological Systematics and Evolutionary Research. 2016;54(3):215–225. doi: 10.1111/jzs.12132. DOI

Birstein VJ. Localization of NORs in karyotypes of four Rana species. Genetica. 1984;64:149–154. doi: 10.1007/BF00115338. DOI

Borkin LJ, Korshunov AV, Lada GA, Litvinchuk SN, Rosanov JM, Shabanov DA, Zinenko AI. Mass occurrence of polyploid green frogs (Rana esculenta Complex) in eastern Ukraine. Russian Journal of Herpetology. 2004;11:194–213. doi: 10.30906/1026-2296-2004-11-3-203-222. DOI

Borodin PM, Rogatcheva MB, Zhelezova AI, Oda S. Chromosome pairing in inter-racial hybrids of the house musk sherew (Suncus murinus, Insectivora, Soricidae) Genome. 1988;41:79–90. doi: 10.1139/g97-103. PubMed DOI

Brychta BH, Tunner HG. Flow cytometric analysis of spermatogenesis in triploid Rana esculenta. Zoologica Poloniae. 1994;39:507.

Bucci S, Ragghianti M, Mancino GL, Hotz H, Uzzell T. Lampbrush and mitotic chromosomes of the hemiclonally reproducing hybrid Rana esculenta and its parental species. Journal of Experimental Zoology. 1990;255:37–56. doi: 10.1002/jez.1402550107. PubMed DOI

Chmielewska M, Dedukh D, Haczkiewicz K, Rozenblut-Kościsty B, Kaźmierczak M, Kolenda K, Serwa E, Pietras-Lebioda A, Krasikova A, Ogielska M. The programmed DNA elimination and formation of micronuclei in germ line cells of the natural hybridogenetic water frog Pelophylax esculentus. Scientific Reports. 2018;8(1):1–19. doi: 10.1038/s41598-018-26168-z. PubMed DOI PMC

Christiansen DG. Gamete types, sex determination and stable equilibria of all-hybrid populations of diploid and triploid edible frogs (Pelophylax esculentus) BMC Evolutionary Biology. 2009;9(1):135. doi: 10.1186/1471-2148-9-135. PubMed DOI PMC

Christiansen DG, Fog K, Pedersen BV, Boomsma JJ. Reproduction and hybrid load in all-hybrid populations of Rana esculenta water frogs in Denmark. Evolution. 2005;59:1348–1361. doi: 10.1111/j.0014-3820.2005.tb01784.x. PubMed DOI

Christiansen DG, Reyer HU. From clonal to sexual hybrids: genetic recombination via triploids in all-hybrid populations of water frogs. Evolution. 2009;63(7):1754–1768. doi: 10.1111/j.1558-5646.2009.00673.x. PubMed DOI

Coyne JA, Orr HA. Speciation. Sinauer Associates, Inc; Sunderland: 2004.

Dawley RM, Bogart JP. Evolution and ecology of unisexual vertebrates. New York State Museum Publications; Albany: 1989.

Dedukh D, Krasikova A. Delete and survive: strategies of programmed genetic material elimination in eukaryotes. Biological Reviews. 2021;97:195–216. doi: 10.1111/brv.12796. PubMed DOI PMC

Dedukh D, Litvinchuk J, Svinin A, Litvinchuk S, Rosanov J, Krasikova A. Variation in hybridogenetic hybrid emergence between populations of water frogs from the Pelophylax esculentus complex. PLOS ONE. 2019;14(11):e0224759. doi: 10.1371/journal.pone.0224759. PubMed DOI PMC

Dedukh D, Litvinchuk S, Rosanov J, Mazepa G, Saifitdinova A, Shabanov D, Krasikova A. Optional endoreplication and selective elimination of parental genomes during oogenesis in diploid and triploid hybrid European water frogs. PLOS ONE. 2015;10(4):e0123304. doi: 10.1371/journal.pone.0123304. PubMed DOI PMC

Dedukh D, Litvinchuk S, Rosanov J, Shabanov D, Krasikova A. Mutual maintenance of di- and triploid Pelophylax esculentus hybrids in R-E systems: results from artificial crossings experiments. BMC Evolutionary Biology. 2017;17:220. doi: 10.1186/s12862-017-1063-3. PubMed DOI PMC

Dedukh D, Riumin S, Chmielewska M, Rozenblut-Kościsty B, Kolenda K, Kaźmierczak M, Dudzik A, Ogielska M, Krasikova A. Micronuclei in germ cells of hybrid frogs from Pelophylax esculentus complex contain gradually eliminated chromosomes. Scientific Reports. 2020;10(1):1–13. doi: 10.1038/s41598-020-64977-3. PubMed DOI PMC

Doležálková M, Sember A, Marec F, Ráb P, Plötner J, Choleva L. Is premeiotic genome elimination an exclusive mechanism for hemiclonal reproduction in hybrid males of the genus Pelophylax? BMC Genetics. 2016;17:100. doi: 10.1186/s12863-016-0408-z. PubMed DOI PMC

Doležálková-Kaštánková M, Mazepa G, Jeffries DL, Perrin N, Plötner M, Plötner J, Guex GD, Mikulíček P, Poustka AJ, Grau J, Choleva L. Capture and return of sexual genomes by hybridogenetic frogs provides clonal genome enrichment in a sexual species. Scientific Reports. 2021;11(1):1–10. doi: 10.1038/s41598-021-81240-5. PubMed DOI PMC

Doležálková-Kaštánková M, Pruvost NBM, Plötner J, Reyer HU, Janko K, Choleva L. All-male hybrids of a tetrapod Pelophylax esculentus share its origin and genetics of maintenance. Biology of Sex Differences. 2018;9:1–13. doi: 10.1186/s13293-018-0172-z. PubMed DOI PMC

Drohvalenko M, Pustovalova E, Fedorova A, Shabanov D. First finding of triploid hybrid frogs Pelophylax esculentus (Anura: Ranidae) in Mozh river basin (Kharkiv region, Ukraine) Biodiversity, Ecology and Experimental Biology. 2022;23(2):61–67. doi: 10.34142/2708-5848.2021.23.2.04. DOI

Dubey S, Maddalena T, Bonny L, Jeffries DL, Dufresnes C. Population genomics of an exceptional hybridogenetic system of Pelophylax water frogs. BMC Evolutionaty Biology. 2019;19:164. doi: 10.1186/s12862-019-1482-4. PubMed DOI PMC

Dufresnes C, Mazepa G. Hybridogenesis in water frogs. eLS. 2020;1:718–726. doi: 10.1002/9780470015902.a0029090. DOI

Graf JD, Müller WP. Experimental gynogenesis provides evidence of hybridogenetic reproduction in the Rana esculenta complex. Experientia. 1979;35:1574–1576. doi: 10.1007/BF01953200. PubMed DOI

Graf JD, Polls-Pelaz M. Evolutionary genetics of the Rana esculenta Complex. In: Dawley RM Bogart JP, eds., editor. Evolution and ecology of unisexual vertebrates. New York State Museum Publications; Albany: 1989. pp. 289–302.

Günther R. Zur populationsgenetik der mitteleuropäischen wasserfrösche des Rana esculenta—synkleptons (Anura, Ranidae) Zoologischer Anzeiger. 1983;211(1/2):43–54.

Günther R, Uzzell T, Berger L. Inheritance patterns in triploid Rana esculenta (Amphibia, Salientia) Mitteilungen des Zoologischen Museums Berlin. 1979;55:35–57.

Heppich S, Tunner HG, Greilhuber J. Premeiotic chromosome doubling after genome elemination during spermatogenesis of the species hybrid Rana esculenta. Theoretical and Applied Genetics. 1982;61:101–104. doi: 10.1007/BF00273874. PubMed DOI

Tunner HG, Heppich S. Premeiotic genome exclusion during oogenesis in the common edible frog, Rana esculenta. Die Naturwissenschaften. 1981;68(4):207–208. doi: 10.1007/BF01047207. PubMed DOI

Hoffman A, Plötner J, Pruvost NBM, Christiansen DG, Röthlisberger S, Choleva L, Mikulíček P, Cogălniceanu D, Sas-Kovács I, Shabanov D, Morozov-Leonov S, Reyer HU. Genetic diversity and distribution patterns of diploid and polyploid hybrid water frog populations (Pelophylax esculentus complex) across Europe. Molecular Ecology. 2015;24:4371–4391. doi: 10.1111/mec.13325. PubMed DOI

Hotz H, Semlitsch RD, Gutmann E, Guex GD, Beerli P. Spontaneous heterosis in larval life-history traits of hemiclonal frog hybrids. Proceedings of the National Academy of Sciences of the United States of America. 1999;96(5):2171–2176. doi: 10.1073/pnas.96.5.2171. PubMed DOI PMC

Ishishita S, Tsuboi K, Ohishi N, Tsuchiya K, Matsuda Y. Abnormal pairing of X and Y sex chromosomes during meiosis I in interspecific hybrids of Phodopus campbelli and P. sungorus. Scientific Reports. 2015;5(1):1–9. doi: 10.1038/srep09435. PubMed DOI PMC

Lenormand T, Engelstadter J, Johnston SE, Wijnker E, Haag CR. Evolutionary mysteries in meiosis. Philosophical Transactions of the Royal Society B: Biological Sciences. 2016;371(1706):20160001. doi: 10.1098/rstb.2016.0001. PubMed DOI PMC

Mallet J. Hybrid speciation. Nature. 2007;446:279–283. doi: 10.1038/nature05706. PubMed DOI

Mazepa G, Doležálková M, Choleva L, Plötner J, Biriuk O, Drohvalenko M, Korshunov O, Shabanov D, Wolf J, Perrin N. Sex uncovered: the evolutionary biology of reproductive systems. Roscoff: Inserm; 2018. Distinct fate of the asexual genomes in two convergently evolved Pelophylax hybridogenetic systems; p. 57.

McKee BD. Homologous pairing and chromosome dynamics in meiosis and mitosis. Biochimica et Biophysica Acta. 2004;1677:165–180. doi: 10.1016/j.bbaexp.2003.11.017. PubMed DOI

Mikulícek P, Kotlík P. Two water frog populations from western Slovakia consisting of diploid females and diploid and triploid males of the hybridogenetic hybrid Rana esculenta (Anura, Ranidae) Mitteilungen aus dem Museum fuer Naturkunde in Berlin Zoologische Reihe. 2001;77:59–64. doi: 10.1002/mmnz.20010770110. DOI

Neaves WB, Baumann P. Unisexual reproduction among vertebrates. Trends in Genetics. 2011;27(3):81–88. doi: 10.1016/j.tig.2010.12.002. PubMed DOI

Ogielska M. Nucleus-like bodies in gonial cells of Rana esculenta [Amphibia, Anura] tadpoles-a putative way of chromosome elimination. Zoologica Poloniae. 1994;39:3–4.

Ogielska M, Bartmańska J. Development of testes and differentiation of germ cells in water frogs of the Rana esculenta-complex (Amphibia, Anura) Amphibia-Reptilia. 1999;20:251–263. doi: 10.1163/156853899X00286. DOI

Plötner J. Die westpaläarktischen Wasserfrösche: von Märtyrern der Wissenschaft zur biologischen Sensation. Laurenti; Bielefeld: 2005.

Pruvost NBM, Hoffmann A, Reyer HU. 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. doi: 10.1002/ece3.687. PubMed DOI PMC

Pruvost NBM, Mikulíček P, Choleva L, Reyer HU. Contrasting reproductive strategies of triploid hybrid males in vertebrate mating systems. Journal of Evolutionary Biology. 2015;28(1):189–204. doi: 10.1111/jeb.12556. PubMed DOI

Ragghianti M, Bucci S, Marracci S, Casola C, Mancino G, Hotz H, Guex GD, Plötner J, Uzzell T. Gametogenesis of intergroup hybrids of hemiclonal frogs. Genetics Research. 2007;89:39–45. doi: 10.1017/S0016672307008610. PubMed DOI

Ragghianti M, Guerrini F, Bucci S, Mancino G, Hotz H, Uzzell T, Guex GD. Molecular characterization of a centromeric satellite DNA in the hemiclonal hybrid frog Rana esculenta and parental species. Chromosome Research. 1995;3(8):497–506. doi: 10.1007/BF00713965. PubMed DOI

Renner O. Zur Terminologie des pflanzlichen Generationswechsels. Biologisches Zentralblatt. 1916;36:337–374.

Reyer HU, Arioli-Jakob C, Arioli M. Post-zygotic selection against parental genotypes during larval development maintains all-hybrid populations of the frog Pelophylax esculentus. BMC Evolutionary Biology. 2015;15(1):1–16. doi: 10.1186/s12862-015-0404-3. PubMed DOI PMC

Rieger R, Michaelis A, Green MM. Glossary of genetics classical and molecular. 5th edn Springer; Berlin Heidelberg New York: 1991.

Rieseberg LH. Chromosomal rearrangements and speciation. Trends in Ecology & Evolution. 2001;16(7):351–358. doi: 10.1016/S0169-5347(01)02187-5. PubMed DOI

Rybacki M, Berger L. Types of water frog populations (Rana esculenta complex) in Poland. Mitteilungen aus dem Museum für Naturkunde in Berlin. Zoologische Reihe. 2001;77:51–57. doi: 10.1002/mmnz.20010770109. DOI

Salas N, Valetti J, Grenat P, Otero M, Martino A. Meiotic behavior of two polyploid species of genus Pleurodema (Anura: Leiuperidae) from central Argentina. Acta Herpetologica. 2014;9(1):109–113. doi: 10.1002/mmnz.20010770109. DOI

Schön I, Martens K, Van Dijk P. The evolutionary biology of parthenogenesis. Springer; Heidelberg: 2009. Lost sex.

Shabanov D, Vladymyrova M, Leonov A, Biriuk O, Kravchenko M, Mair Q, Meleshko O, Newman J, Usova O, Zholtkevych G. Information and communication technologies in education, research, and industrial applications. ICTERI 2019. Communications in Computer and Information Science. Vol. 1175. Springer; Cham: 2020. Simulation as a Method for Asymptotic System Behavior Identification (e.g., Water Frog Hemiclonal Population Systems) DOI

Stöck M, Dedukh D, Reifová R, Lamatsch DK, Starostová Z, Janko K. Sex chromosomes in meiotic, hemiclonal, clonal and polyploid hybrid vertebrates: along the ‘extended speciation continuum’. Philosophical Transactions of the Royal Society B. 2021;376(1833):20200103. doi: 10.1098/rstb.2020.0103. PubMed DOI PMC

Svinin AO, Dedukh DV, Borkin LJ, Ermakov OA, Ivanov AY, Litvinchuk JS, Zamaletdinov RI, Mikhaylova RI, Trubyanov AB, Skorinov DV, Rosanov YM, Litvinchuk SN. Genetic structure, morphological variation, and gametogenic peculiarities in water frogs (Pelophylax) from northeastern European Russia. Journal of Zoological Systematics and Evolutionary Research. 2021;59(3):646–662. doi: 10.1111/jzs.12447. DOI

Svinin AO, Litvinchuck SN, Borkin LJ, Rosanov JM. Distribution and population system types of green frogs (Pelophylax Fitzinger, 1843) in Mari El Republic. Current Study of Herpetology. 2013;13(3/4):137–147.

Torgasheva AA, Borodin PM. Cytological basis of sterility in male and female hybrids between sibling species of grey voles Microtus arvalis and M. levis. Scientific Reports. 2016;6:36564. doi: 10.1038/srep36564. PubMed DOI PMC

Tunner HG. Demonstration of the hybrid origin of the common green frog Rana esculenta. Naturwissenschafte. 1973;60:481–482. doi: 10.1007/BF00592872. PubMed DOI

Tunner H. Die klonale Struktur einer Wasserfröschpopulation. Journal of Zoological Systematics and Evolutionary Research. 1974;12:309–314.

Tunner H, Heppich-Tunner S. Genome exclusion and two strategies of chromosome duplication in oogenesis of a hybrid frog. Naturwissenschaften. 1991;78:32–34. doi: 10.1007/BF01134041. DOI

Tunner H, Heppich-Tunner S. A new population system of water frogs discovered in Hungary. Proceedings of the Sixth Ordinary General Meeting of the Societas Europaea Herpetologica. 1992;19-23:453–460.

Uzzell T, Günther R, Berger L. Rana ridibunda and Rana esculenta: a leaky hybridogenetic system (Amphibia, Salientia) Proceedings of the Academy of Natural Sciences of Philadelphia. 1977;128:147–171.

Vinogradov AE, Borkin LJ, Günther R, Rosanov JM. Two germ cell lineages with genomes of different species in one and the same animal. Hereditas. 1991;114(3):245–251. doi: 10.1111/j.1601-5223.1991.tb00331.x. PubMed DOI

Vorburger C. Non-hybrid offspring from matings between hemiclonal hybrid waterfrogs suggest occasional recombination between clonal genomes. Ecology Letters. 2001;4:628–636. doi: 10.1046/j.1461-0248.2001.00272.x. DOI

Zong E, Fan G. The variety of sterility and gradual progression to fertility in hybrids of the horse and donkey. Heredity. 1989;62(3):393–406. doi: 10.1038/hdy.1989.54. PubMed DOI

Inheritance patterns of male asexuality in hybrid males of a water frog Pelophylax esculentus

Cytogenetics of the Hybridogenetic Frog Pelophylax grafi and Its Parental Species Pelophylax perezi

Genome elimination from the germline cells in diploid and triploid male water frogs Pelophylax esculentus