Gametogenesis is a fundamental biological process that ensures both genetic recombination and the continuity of successive generations. Interspecific hybrids can reproduce through modified mechanisms, such as hybridogenesis, by transmitting clonal, unrecombined genomes of only one of the parental species via their gametes. Pelophylax grafi (RP) is a natural hybrid frog composed of mixed genomes (subgenomes) of two related species, Pelophylax perezi (P) and Pelophylax ridibundus (R), and coexists in populations with P. perezi. This study tested the involvement of programmed genome elimination in gamete production of P. grafi, providing new insight into reproductive mechanisms of hybrid vertebrates. Using comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH), we examined the genomic constitution of germline cells in tadpoles and adult male and female P. grafi. Controlled crosses between P. perezi and P. grafi produced F1 hybrid tadpoles, whose genotypes confirmed that P. grafi parents transmitted the R subgenome through their gametes. In the early germline cells (gonocytes) of these tadpoles, P chromosomes were selectively eliminated via micronuclei formation during interphase. The occasional presence of the R genome and mixed R/P genome micronuclei suggests variability and imperfect fidelity in the elimination process. In adult hybrids, the majority of diplotene oocytes, spermatogonial stem cells (SSC) and spermatocytes carried R subgenomes. We demonstrated that programmed genome rearrangement in Pelophylax hybrids is an evolutionarily conserved mechanism underlying this unique reproductive strategy.

Amphibian Biology Group Department of Evolutionary Biology and Conservation of Vertebrates University of Wrocław Sienkiewicza 21 50 335 Wrocław Poland

CEFE CNRS University Montpellier EPHE IRD 1919 Route de Mende 34293 Montpellier France

Department of Biology and Ecology Faculty of Science University of Ostrava Chittussiho 10 710 00 Ostrava Czech Republic

Institute of Heart Diseases Faculty of Medicine Wroclaw Medical University Borowska 213 50 556 Wroclaw Poland

Laboratory of Fish Genetics Institute of Animal Physiology and Genetics Czech Academy of Sciences Rumburská 89 277 21 Liběchov Czech Republic

Laboratory of Non Mendelian Evolution Institute of Animal Physiology and Genetics Czech Academy of Sciences Rumburská 89 277 21 Liběchov Czech Republic

Zobrazit více v PubMed

Scali V. Metasexual stick insects: Model pathways to losing sex and bringing it back. In: Schön I., Martens K., Peter D., editors. Lost Sex: The Evolutionary Biology of Parthenogenesis. Springer; Dordrecht, The Netherlands: 2009. pp. 317–345.

Graf J.-D., Polls-Pelaz M. Evolutionary genetics of the Rana esculenta complex. In: Dawley R.M., Bogart J.P., editors. Evolution and Ecology of Unisexual Vertebrates. New York State Museum; Albany, NY, USA: 1989. pp. 289–302.

Lamatsch D.K., Stöck M. Sperm-dependent parthenogenesis and hybridogenesis in teleost fishes. In: Schön I., Martens K., Peter D., editors. Lost Sex: The Evolutionary Biology of Parthenogenesis. Springer; Dordrecht, The Netherlands: 2009. pp. 399–432.

Ogielska M. Development and Reproduction of amphibian Species, Hybrids, and Polyploids. In: Ogielska M., editor. Reproduction of Amphibians. Science Publisher; Enfield, NH, USA: 2009. pp. 343–410.

Schultz R.J. Hybridization, unisexuality, and polyploidy in the teleost Poeciliopsis (Poeciliidae) and other vertebrates. Am. Nat. 1969;103:605–619. doi: 10.1086/282629. DOI

Tunner G. Die klonale Struktur einer Wasserfroschpopulation. J. Zool. Syst. Evol. Res. 1974;12:309–314. doi: 10.1111/j.1439-0469.1974.tb00173.x. DOI

Berger L. Western Palearctic water frogs (Amphibia, Ranidae): Systematics, genetics and population compositions. Experientia. 1983;39:127–130. doi: 10.1007/BF01958859. DOI

Alves M.J., Coelho M.M., Collares-Pereira M.J. Evolution in action through hybridisation and polyploidy in an Iberian freshwater fish: A genetic review. Genetica. 2001;111:375–385. doi: 10.1023/A:1013783029921. PubMed DOI

Park J.-Y., Kim I.-S., Ko M.-H. Characteristics of rare males in the cobitid unisexual complex, Cobitis hankugensis-Iksookimia longicorpa. Folia Zool. 2011;60:290–294. doi: 10.25225/fozo.v60.i4.a4.2011. DOI

Suzuki S., Arai K., Munehara H. Karyological evidence of hybridogenesis in greenlings (Teleostei: Hexagrammidae) PLoS ONE. 2017;12:e0180626. doi: 10.1371/journal.pone.0180626. PubMed DOI PMC

Majtánová Z., Dedukh D., Choleva L., Adams M., Ráb P., Unmack P.J., Ezaz T. Uniparental genome elimination in australian carp gudgeons. Genome Biol. Evol. 2021;13:evab030. doi: 10.1093/gbe/evab030. PubMed DOI PMC

Dudzik A., Dedukh D., Crochet P.-A., Rozenblut-Kościsty B., Rybka H., Doniol-Valcroze P., Choleva L., Ogielska M., Chmielewska M. Cytogenetics of the hybridogenetic frog Pelophylax grafi and its parental species Pelophylax Perezi. Genome Biol. Evol. 2023;15:evad215. doi: 10.1093/gbe/evad215. PubMed DOI PMC

Schmidt D.J., Bond N.R., Adams M., Hughes J.M. Cytonuclear evidence for hybridogenetic reproduction in natural populations of the Australian carp gudgeon (Hypseleotris: Eleotridae) Mol. Ecol. 2011;20:3367–3380. doi: 10.1111/j.1365-294X.2011.05206.x. PubMed DOI

Stöck M., Ustinova J., Betto-Colliard C., Schartl M., Moritz C., Perrin N. Simultaneous Mendelian and clonal genome transmission in a sexually reproducing, all-triploid vertebrate. Proc. R. Soc. B Biol. Sci. 2012;279:1293–1299. PubMed PMC

Arai K., Fujimoto T. Genomic constitution and atypical reproduction in polyploid and unisexual lineages of the Misgurnus loach, a teleost fish. Cytogenet. Genome Res. 2013;140:226–240. doi: 10.1159/000353301. PubMed DOI

Sánchez-Montes G., Recuero E., Gutiérrez-Rodríguez J., Gomez-Mestre I., Martínez-Solanot Í. Species assignment in the Pelophylax ridibundus x P. perezi hybridogenetic complex based on 16 newly characterised microsatellite markers. Herpetol. J. 2016;26:99–108.

Dubey S., Dufresnes C. An extinct vertebrate preserved by its living hybridogenetic descendant. Sci. Rep. 2017;7:12768. doi: 10.1038/s41598-017-12942-y. PubMed DOI PMC

Dufresnes C., Denoël M., Di Santo L., Dubey S. Multiple uprising invasions of Pelophylax water frogs, potentially inducing a new hybridogenetic complex. Sci. Rep. 2017;7:6506. doi: 10.1038/s41598-017-06655-5. PubMed DOI PMC

Dufresnes C., Mazepa G. Hybridogenesis in Water Frogs. eLS. 2020;1:718–726.

Demay J., Ciavatti F., Cuevas A., Doniol-Valcroze P., Eble A., Leblanc E., Mansier Y., Martinossi-Allibert I., Nicolas J., Pineau A., et al. Distribution des grenouilles vertes du système perezi-grafi et des autres espèces du genre Pelophylax (Amphibia: Ranidae) dans leur aire méditerranéenne française à l’ouest du Rhône. Bull. Société Herpétologique Fr. 2023;182:1–11.

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. Sci. Rep. 2018;8:7870. doi: 10.1038/s41598-018-26168-z. PubMed DOI PMC

Ogielska M., Chmielewska M., Rozenblut-Kościsty B. Pregametogenesis: The earliest stages of gonad and germline differentiation in anuran amphibians. Biology. 2024;13:1017. doi: 10.3390/biology13121017. PubMed DOI PMC

Ogielska M. Nucleus-like bodies in gonial cells of Rana esculenta [Amphibia, Anura] tadpoles—A putative way of chromosome elimination. Zool. Pol. 1994;39:461–474.

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. Sci. Rep. 2020;10:8720. doi: 10.1038/s41598-020-64977-3. PubMed DOI PMC

Dedukh D., Krasikova A. Delete and survive: Strategies of programmed genetic material elimination in eukaryotes. Biol. Rev. 2022;97:195–216. doi: 10.1111/brv.12796. PubMed DOI PMC

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

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

Heppich S., Tunner H.G., Greilhuber J. Premeiotic chromosome doubling after genome elimination during spermatogenesis of the species hybrid Rana esculenta. Theor. Appl. Genet. 1982;61:101–104. doi: 10.1007/BF00273874. PubMed DOI

Bucci S., Ragghianti M., Mancino G., Berger L., Hotz H., Uzzell T. Lampbrush and mitotic chromosomes of the hemiclonally reproducing hybrid Rana esculenta and its parental species. J. Exp. Zool. 1990;255:37–56. doi: 10.1002/jez.1402550107. PubMed DOI

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

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:e0123304. doi: 10.1371/journal.pone.0123304. PubMed DOI PMC

Zlotina A., Dedukh D., Krasikova A. Amphibian and avian karyotype evolution: Insights from lampbrush chromosome studies. Genes. 2017;8:311. doi: 10.3390/genes8110311. PubMed DOI PMC

Pustovalova E., Choleva L., Shabanov D., Dedukh D. The high diversity of gametogenic pathways in amphispermic water frog hybrids from Eastern Ukraine. PeerJ. 2022;10:e13957. doi: 10.7717/peerj.13957. PubMed DOI PMC

Choleva L., Doležálková-Kaštánková M., Labajová V., Sember A., Altmanová M., Lukšíková K., Chung Voleníková A., Dalíková M., Nguyen P., Pustovalova E., et al. Formation of hemiclonal reproduction and hybridogenesis in Pelophylax water frogs studied with species-specific cytogenomic probes. bioRxiv. 2023;277:12

Marracci S., Michelotti V., Guex G.D., Hotz H., Uzzell T., Ragghianti M. RrS1-like sequences of water frogs from central Europe and around the Aegean Sea: Chromosomal organization, evolution, possible function. J. Mol. Evol. 2011;72:368–382. doi: 10.1007/s00239-011-9436-5. PubMed DOI

Biriuk O.V., Shabanov D.A., Korshunov A.V., Borkin L.J., Lada G.A., Pasynkova R.A., Rosanov J.M., Litvinchuk S.N. Gamete production patterns and mating systems in water frogs of the hybridogenetic Pelophylax esculentus complex in north-eastern Ukraine. J. Zool. Syst. Evol. Res. 2016;54:215–225. doi: 10.1111/jzs.12132. DOI

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 Genet. 2016;17:100. doi: 10.1186/s12863-016-0408-z. PubMed DOI PMC

Skierska K., Lagner A., Rozenblut-Kościsty B., Kosiba P., Kolenda K., Ogielska M. Population structure, mate choice, and genome transmission in naturally formed pairs in a Pelophylax lessonae–Pelophylax esculentus hybridogenetic system. Behav. Ecol. Sociobiol. 2023;77:92. doi: 10.1007/s00265-023-03366-y. DOI

Dedukh D., Riumin S., Kolenda K., Chmielewska M., Rozenblut-Kościsty B., Kaźmierczak M., Ogielska M., Krasikova A. Maintenance of pure hybridogenetic water frog populations: Genotypic variability in progeny of diploid and triploid parents. PLoS ONE. 2022;17:e0268574. doi: 10.1371/journal.pone.0268574. PubMed DOI PMC

Zaleśna A., Choleva L., Ogielska M., Rábová M., Marec F., Ráb P. Evidence for integrity of parental genomes in the diploid hybridogenetic water frog Pelophylax esculentus by genomic in situ hybridization. Cytogenet. Genome Res. 2011;134:206–212. doi: 10.1159/000327716. PubMed DOI

Koref-Santibañez S. The karyotypes of Rana lessonae Camerano, Rana ridibunda Pallas and of the hybrid form Rana ‘esculenta’ Linne (Anura) Mitt. Zool. Mus. Berl. 1979;55:115–124.

Majtánová Z., Choleva L., Symonová R., Ráb P., Kotusz J., Pekárik L., Janko K. Asexual reproduction does not apparently increase the rate of chromosomal evolution: Karyotype stability in diploid and triploid clonal hybrid fish (Cobitis, Cypriniformes, Teleostei) PLoS ONE. 2016;11:e0146872. doi: 10.1371/journal.pone.0146872. PubMed DOI PMC

Dedukh D., Mazepa G., Shabanov D., Rosanov J., Litvinchuk S., Borkin L., Saifitdinova A., Krasikova A. Cytological maps of lampbrush chromosomes of European water frogs (Pelophylax esculentus complex) from Eastern Ukraine. BMC Genet. 2013;14:26. doi: 10.1186/1471-2156-14-26. PubMed DOI PMC

Berger L., Rybacki M., Hotz H. Artificial fertilization of water frogs. Amphib. Reptil. 1994;15:408–413. doi: 10.1163/156853894X00452. DOI

Chmielewska M., Kaźmierczak M., Rozenblut-Kościsty B., Kolenda K., Dudzik A., Dedukh D., Ogielska M. Genome elimination from the germline cells in diploid and triploid male water frogs Pelophylax esculentus. Front. Cell Dev. Biol. 2022;10:1008506. doi: 10.3389/fcell.2022.1008506. PubMed DOI PMC

Cuevas A., Patrelle C., Ciavatti F., Gendre T., Sourrouille P., Geniez P., Doniol-Valcroze P., Crochet P.A. A new PCR-RFLP method for the identification of parental and hybridogenetic western European Water Frogs, including the Pelophylax perezi-grafi system. Salamandra. 2022;58:218–230.

Gall J.G., Murphy C., Callan H.G., Wu Z.A. Lampbrush chromosomes. Methods Cell Biol. 1991;36:149–166. PubMed

Callan H.G. Lampbrush Chromosomes. Springer; Berlin/Heidelberg, Germany: 1986. DOI

Wagner E., Ogielska M. Oogenesis and ovary development in the natural hybridogenetic water frog, Rana esculenta L. II. After metamorphosis until adults. Zool. Jb Physiol. (Jena) 1993;97:369–382.

Ogielska M., Wagner E. Oogenesis and ovary development in the natural hybridogenetic water frog, Rana esculenta L. I Tadpole stages until metamorphosis. Zool. Jb Physiol. (Jena) 1993;97:349–368.

Rozenblut-Kościsty B., Chmielewska M., Dudzik A., Crochet P.-A., Ogielska M. Gonadal development and micronuclei formation in hybrid frogs of the Pelophylax perezi × P. grafi complex. 2025. Amphibian Biology Group, Department of Evolutionary Biology and Conservation of Vertebrates, University of Wrocław, Sienkiewicza 21, Wrocław, Poland. in preparation .

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:e0224759. doi: 10.1371/journal.pone.0224759. PubMed DOI PMC

Schmeller D.S., Seitz A., Crivelli A., Veith M. Crossing species’ range borders: Interspecies gene exchange mediated by hybridogenesis. Proc. Biol. Sci. 2005;272:1625–1631. doi: 10.1098/rspb.2005.3129. PubMed DOI PMC

Plötner J. Beiheft Zeitschrift Für Feldherpetologie. Laurenti Verlag; Bielefeld, Germany: 2005. Die Westpaläarktischen Wasserfrösche; pp. 1–160.

Szydłowski P., Chmielewska M., Rozenblut-Kościsty B., Ogielska M. The frequency of degenerating germ cells in the ovaries of water frogs (Pelophylax esculentus complex) Zoomorphology. 2017;136:75–83. doi: 10.1007/s00435-016-0337-4. DOI

Dedukh D., Majtánová Z., Ráb P., Ezaz T., Unmack P.J. Gradual chromosomal lagging drive programmed genome elimination in hemiclonal fishes from the genus Hypseleotris. Sci. Rep. 2024;14:26866. doi: 10.1038/s41598-024-78278-6. PubMed DOI PMC

Gernand D., Rutten T., Varshney A., Rubtsova M., Prodanovic S., Brüß C., Kumlehn J., Matzk F., Houben A. Uniparental chromosome elimination at mitosis and interphase in wheat and pearl millet crosses involves micronucleus formation, progressive heterochromatinization, and DNA fragmentation. Plant Cell. 2005;17:2431–2438. doi: 10.1105/tpc.105.034249. PubMed DOI PMC

Gernand D., Rutten T., Pickering R., Houben A. Elimination of chromosomes in Hordeum vulgare × H. bulbosum crosses at mitosis and interphase involves micronucleus formation and progressive heterochromatinization. Cytogenet. Genome Res. 2006;114:169–174. doi: 10.1159/000093334. PubMed DOI

Rinaldi V.D., Bolcun-Filas E., Kogo H., Kurahashi H., Schimenti J.C. The DNA damage checkpoint eliminates mouse oocytes with chromosome synapsis failure. Mol. Cell. 2017;67:1026–1036.e2. doi: 10.1016/j.molcel.2017.07.027. 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 Evol. Biol. 2017;17:220. doi: 10.1186/s12862-017-1063-3. PubMed DOI PMC

Rybacki M., Berger L. Types of water frog populations. Zoosyst. Evol. 2001;77:51–57. doi: 10.1002/mmnz.20010770109. DOI

Graf J.-D., Karch F., Moreillon M.-C. Biochemical variation in the Rana esculenta complex: A new hybrid form related to Rana perezi and Rana ridibunda. Experientia. 1977;33:1582–1584. doi: 10.1007/BF01934010. PubMed DOI

Vinogradov A.E., Borkin L.J., Günther R., Rosanov J.M. Two germ cell lineages with genomes of different species in one and the same animal. Hereditas. 1991;114:245–251. doi: 10.1111/j.1601-5223.1991.tb00331.x. PubMed DOI

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

Pustovalova E., Choleva L., Shabanov D., Dedukh D. Genomic introgressions may affect hybridogenetic reproduction in water frog hybrids. Authorea. 2024 doi: 10.22541/au.172489658.85596156/v1. PubMed DOI

Thakur J., Packiaraj J., Henikoff S. Sequence, chromatin and evolution of satellite DNA. Int. J. Mol. Sci. 2021;22:4309. doi: 10.3390/ijms22094309. PubMed DOI PMC

Hajkova P. Epigenetic reprogramming in the germline: Towards the ground state of the epigenome. Phil Trans. R. Soc. B. 2011;366:2266–2273. doi: 10.1098/rstb.2011.0042. PubMed DOI PMC

Mills W.K., Lee Y.C.G., Kochendoerfer A.M., Dunleavy E.M., Karpen G.H. RNA from a simple-tandem repeat is required for sperm maturation and male fertility in Drosophila melanogaster. eLife. 2019;8:e48940. doi: 10.7554/eLife.48940. PubMed DOI PMC

Wei X., Eickbush D.G., Speece I., Larracuente A.M. Heterochromatin-dependent transcription of satellite DNAs in the Drosophila melanogaster female germline. eLife. 2021;10:e62375. doi: 10.7554/elife.62375. PubMed DOI PMC

Hotz H., Uzzel T., Berger L. Hemiclonal hybrid water frogs associated with the sexual host species Rana perezi. Zool. Pol. 1994;39:243–266.

Tunner H.G., 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

Christiansen D.G. A microsatellite-based method for genotyping diploid and triploid water frogs of the Rana esculenta hybrid complex. Mol. Ecol. Notes. 2005;5:190–193. doi: 10.1111/j.1471-8286.2004.00869.x. DOI

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

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

Bove P., Milazzo P., Barbuti R. The role of deleterious mutations in the stability of hybridogenetic water frog complexes. BMC Evol. Biol. 2014;14:107. doi: 10.1186/1471-2148-14-107. PubMed DOI PMC

Burriel-Carranza B., Molina-Duran C., Tamar K., Pérez-Sorribes L., López-Caro J., Cirac M., Fernandez-Guiberteau D., Carranza S. Distribution and evolution of the western European water frogs (genus Pelophylax) from Catalonia, northeastern Spain. PeerJ. 2025;13:e19895. doi: 10.7717/peerj.19895. PubMed DOI PMC

Pekşen Ç., Bilgin M., Bilgin C. Discordance between ventral colour and mtDNA haplotype in the water frog Rana (ridibunda) caralitana, 1988 Arıkan. Amphib.-Reptil. 2010;31:9–20.

Holsbeek G., Jooris R. Potential impact of genome exclusion by alien species in the hybridogenetic water frogs (Pelophylax esculentus complex) Biol. Invasions. 2010;12:1–13. doi: 10.1007/s10530-009-9427-2. DOI

Doležálková-Kaštánková M., Dedukh D., Labajová V., Pustovalova E., Choleva L. Inheritance patterns of male asexuality in hybrid males of a water frog Pelophylax esculentus. Sci. Rep. 2024;14:22221. doi: 10.1038/s41598-024-73043-1. PubMed DOI PMC