Hybridisation is a powerful evolutionary force that can lead to diverse reproductive outcomes, including the emergence of asexual lineages such as hybridogenesis. During hybridogenesis, found in water frog hybrids Pelophylax esculentus, one parental genome is eliminated during gametogenesis while the other is clonally propagated to the gametes. Although this reproductive mode allowed hybrids to spread across Europe, the ability to undergo hybridogenesis varies in hybrids throughout their range. To explain this gametogenic variability, we investigated the role of genomic introgression. Using cytogenetic analysis, we examined gametogenesis in diploid and triploid hybrid males from four eastern Ukrainian population systems, known for high hybrid abundance and diverse gametogenic pathways. Most hybrids from the studied localities showed canonical hybridogenetic reproduction, characterised by the expected hybrid genome composition and no detectable intergenomic rearrangements or introgressions. We also identified hybrids with disrupted or absent genome elimination, which were incapable of hybridogenetic reproduction and exhibited reduced fertility and aneuploid gamete formation. These individuals exhibited intergenomic rearrangements, with chromosomal regions showing interchanges of parental chromosomes. Additionally, some chromosomal fragments did not match either parental genome, suggesting introgression from an unidentified lineage. Notably, half of the triploid hybrids had rearranged chromosomes, likely originating from diploid hybrids that failed parental genome elimination and transmitted diploid gametes. Our study reveals that hybridogenesis in P. esculentus is not a conserved mechanism but rather significantly relies on the genomic background. These findings highlight the role of genomic introgression in shaping reproductive diversity and influencing the evolution and persistence of asexual vertebrates.

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 The Czech Academy of Sciences Institute of Animal Physiology and Genetics Liběchov Czech Republic

Laboratory of NonMendelian Evolution The Czech Academy of Sciences Institute of Animal Physiology and Genetics Liběchov Czech Republic

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Abbott, R., D. Albach, S. Ansell, et al. 2013. “Hybridization and Speciation.” Journal of Evolutionary Biology 26: 229–246.

Akın, Ç., C. Can Bilgin, P. Beerli, et al. 2010. “Phylogeographic Patterns of Genetic Diversity in Eastern Mediterranean Water Frogs Were Determined by Geological Processes and Climate Change in the Late Cenozoic.” Journal of Biogeography 37: 2111–2124.

Berger, L. 1969. “Some Characteristics of the Crosses Within Rana esculenta Complex in Postlarval Development.” Annales Zoologici 27: 373–416.

Berger, L. 1977. “Systematics and Hybridization in the Rana esculenta Complex.” In The Reproductive Biology of Amphibians, 367–388. Springer.

Biriuk, O. V., D. A. Shabanov, A. V. Korshunov, et al. 2016. “Gamete Production Patterns and Mating Systems in Water Frogs of the Hybridogenetic Pelophylax esculentus Complex in North‐Eastern Ukraine.” Journal of Zoological Systematics and Evolutionary Research 54: 215–225.

Borkin, L. J., A. V. Korshunov, G. A. Lada, et al. 2004. “Mass Occurrence of Polyploid Green Frogs (Rana esculenta Complex) in Eastern Ukraine.” Russian Journal of Herpetology 11: 203–222.

Chmielewska, M., D. Dedukh, K. Haczkiewicz, et al. 2018. “The Programmed DNA Elimination and Formation of Micronuclei in Germ Line Cells of the Natural Hybridogenetic Water Frog Pelophylax esculentus.” Scientific Reports 8: 7870.

Chmielewska, M., M. Kaźmierczak, B. Rozenblut‐Kościsty, et al. 2022. “Genome Elimination From the Germline Cells in Diploid and Triploid Male Water Frogs Pelophylax esculentus.” Frontiers in Cell and Developmental Biology 10: 1008506.

Choleva, L., M. Dolezalkova‐Kastankova, V. Labajova, et al. 2023. “Formation of Hemiclonal Reproduction and Hybridogenesis in Pelophylax Water Frogs Studied With Species‐Specific Cytogenomic Probes.” bioRxiv (Cold Spring Harbor Laboratory).

Christiansen, D. G. 2009. “Gamete Types, Sex Determination and Stable Equilibria of All‐Hybrid Populations of Diploid and Triploid Edible Frogs (Pelophylax esculentus).” BMC Evolutionary Biology 9: 135.

Christiansen, D. G., C. Jakob, M. Arioli, S. Roethlisberger, and H.‐U. Reyer. 2010. “Coexistence of Diploid and Triploid Hybrid Water Frogs: Population Differences Persist in the Apparent Absence of Differential Survival.” BMC Ecology 10: 14.

Christiansen, D. G., and H.‐U. Reyer. 2009. “From Clonal to Sexual Hybrids: Genetic Recombination via Triploids in All‐Hybrid Populations of Water Frogs.” Evolution 63: 1754–1768.

Comeault, A. A., and D. R. Matute. 2018. “Genetic Divergence and the Number of Hybridizing Species Affect the Path to Homoploid Hybrid Speciation.” Proceedings of the National Academy of Sciences 115: 9761–9766.

Coyne, J. A., and H. A. Orr. 2004. Speciation. Sinauer Associates, Oxford University Press.

Dawley, R. M., and J. P. Bogart. 1989. Evolution and Ecology of Unisexual Vertebrates. New York State Museum Publications.

Dedukh, D., and A. Krasikova. 2021. “Delete and Survive: Strategies of Programmed Genetic Material Elimination in Eukaryotes.” Biological Reviews 97: 195–216.

Dedukh, D., J. Litvinchuk, A. Svinin, S. Litvinchuk, J. Rosanov, and A. Krasikova. 2019. “Variation in Hybridogenetic Hybrid Emergence Between Populations of Water Frogs From the Pelophylax esculentus Complex.” PLoS One 14: e0224759.

Dedukh, D., S. Litvinchuk, J. Rosanov, et al. 2015. “Optional Endoreplication and Selective Elimination of Parental Genomes During Oogenesis in Diploid and Triploid Hybrid European Water Frogs.” PLoS One 10: e0123304.

Dedukh, D., S. Litvinchuk, J. Rosanov, D. Shabanov, and A. Krasikova. 2017. “Mutual Maintenance of Di‐ and Triploid Pelophylax esculentus Hybrids in R‐E Systems: Results From Artificial Crossings Experiments.” BMC Evolutionary Biology 17: 220.

Dedukh, D., S. Riumin, M. Chmielewska, et al. 2020. “Micronuclei in Germ Cells of Hybrid Frogs From Pelophylax esculentus Complex Contain Gradually Eliminated Chromosomes.” Scientific Reports 10: 8720.

Doležálková, M., A. Sember, F. Marec, P. Ráb, J. Plötner, and L. Choleva. 2016. “Is Premeiotic Genome Elimination an Exclusive Mechanism for Hemiclonal Reproduction in Hybrid Males of the Genus Pelophylax?” BMC Genomic Data 17: 100.

Doležálková‐Kaštánková, M., D. Dedukh, V. Labajová, E. Pustovalova, and L. Choleva. 2024. “Inheritance Patterns of Male Asexuality in Hybrid Males of a Water Frog Pelophylax esculentus.” Scientific Reports 14: 22221.

Doležálková‐Kaštánková, M., G. Mazepa, D. L. Jeffries, et al. 2021. “Capture and Return of Sexual Genomes by Hybridogenetic Frogs Provides Clonal Genome Enrichment in a Sexual Species.” Scientific Reports 11: 1633.

Doležálková‐Kaštánková, M., N. B. M. Pruvost, J. Plötner, H.‐U. Reyer, K. Janko, and L. Choleva. 2018. “All‐Male Hybrids of a Tetrapod Pelophylax esculentus Share Its Origin and Genetics of Maintenance.” Biology of Sex Differences 9: 13.

Doležálková‐Kaštánková, M., P. Pyszko, and L. Choleva. 2022. “Early Development Survival of Pelophylax Water Frog Progeny Is Primarily Affected by Paternal Genomic Input.” Frontiers in Bioscience‐Landmark 27: 233.

Dubey, S., J. Leuenberger, and N. Perrin. 2014. “Multiple Origins of Invasive and “Native” Water Frogs (Pelophylax spp.) in Switzerland.” Biological Journal of the Linnean Society 112: 442–449.

Dufresnes, C., M. Denoël, L. Di Santo, and S. Dubey. 2017. “Multiple Uprising Invasions of Pelophylax Water Frogs, Potentially Inducing a New Hybridogenetic Complex.” Scientific Reports 7: 6506.

Dufresnes, C., S. Dubey, B. Wielstra, D. Jablonski, and M. Denoël. 2024. “Panacea or Nemesis? Re‐Assessing the Reliability of Serum‐Albumin Intron 1 for Genotyping Western Palearctic Water Frogs.” Alytes 41: 5–17.

Dufresnes, C., and G. Mazepa. 2020. “Hybridogenesis in Water Frogs.” Els 1: 718–726.

Dufresnes, C., B. Monod‐Broca, A. Bellati, et al. 2024. “Piecing the Barcoding Puzzle of Palearctic Water Frogs (Pelophylax) Sheds Light on Amphibian Biogeography and Global Invasions.” Global Change Biology 30: e17180.

Fedorova, A., E. Pustovalova, M. Drohvalenko, et al. 2025. “Hybridogenetic Reproduction of Pelophylax Water Frogs From Different RE Hemiclonal Population Systems From Eastern Ukraine: Selective Mortality, Clonal and Ploidy Diversity.” Zoological Journal of the Linnean Society 204, no. 3: zlaf066. https://doi.org/10.1093/zoolinnean/zlaf066.

Graf, J. D., and M. Polls‐Pelaz. 1989. “Evolutionary Genetics of the Rana esculenta Complex.” In Evolution and Ecology of Unisexual Vertebrates, 289–302. New York State Museum Publications.

Hauswaldt, J. S., M. Hoer, M. Ogielska, et al. 2012. “A Simplified Molecular Method for Distinguishing Among Species and Ploidy Levels in European Water Frogs (Pelophylax).” Molecular Ecology Resources 12: 797–805.

Herrera, J. C., A. D'Hont, and P. Lashermes. 2007. “Use of Fluorescence In Situ Hybridization as a Tool for Introgression Analysis and Chromosome Identification in Coffee (Coffea arabica L.).” Genome 50: 619–626.

Holsbeek, G., J. Mergeay, H. Hotz, J. Plotner, F. A. M. Volckaert, and L. de Meester. 2008. “A Cryptic Invasion Within an Invasion and Widespread Introgression in the European Water Frog Complex: Consequences of Uncontrolled Commercial Trade and Weak International Legislation.” Molecular Ecology 17: 5023–5035.

Hotz, H., G. Mancino, S. Bucciinnocenti, M. Ragghianti, L. Berger, and T. Uzzell. 1985. “Rana ridibunda Varies Geographically in Inducing Clonal Gametogenesis in Interspecies Hybrids.” Journal of Experimental Zoology 236: 199–210.

Howell, W. M., and D. A. Black. 1980. “Controlled Silver‐Staining of Nucleolus Organizer Regions With a Protective Colloidal Developer: A 1‐Step Method.” Experientia 36: 1014–1015.

Kolenda, K., A. Pietras‐Lebioda, S. Hofman, M. Ogielska, and M. Pabijan. 2017. “Preliminary Genetic Data Suggest the Occurrence of the Balkan Water Frog, Pelophylax kurtmuelleri, in Southwestern Poland.” Amphibia‐Reptilia 38: 187–196.

Litvinchuk, S. N. 2018. “Testicular Anomalies in the Hybridogenetic Frog Pelophylax esculentus (Amphibia: Anura: Ranidae).” KNE Life Sciences 4: 92–96.

Litvinchuk, S. N., D. V. Skorinov, A. Y. Ivanov, and O. A. Ermakov. 2024. “Detection of Glacial Refugia and Post‐Glacial Colonization Routes of Morphologically Cryptic Marsh Frog Species (Anura: Ranidae: Pelophylax) Using Environmental Niche Modeling.” Diversity 16: 94.

Lymberakis, P., N. Poulakakis, G. Manthalou, C. S. Tsigenopoulos, A. Magoulas, and M. Mylonas. 2007. “Mitochondrial Phylogeography of Rana (Pelophylax) Populations in the Eastern Mediterranean Region.” Molecular Phylogenetics and Evolution 44: 115–125.

Majtánová, Z., D. Dedukh, L. Choleva, et al. 2021. “Uniparental Genome Elimination in Australian Carp Gudgeons.” Genome Biology and Evolution 13: evab030.

Mallet, J. 2007. “Hybrid Speciation.” Nature 446: 279–283.

Marracci, S., V. Michelotti, G.‐D. Guex, H. Hotz, T. Uzzell, and M. Ragghianti. 2011. “RrS1‐Like Sequences of Water Frogs From Central Europe and Around the Aegean Sea: Chromosomal Organization, Evolution, Possible Function.” Journal of Molecular Evolution 72: 368–382.

Marta, A., T. Tichopád, O. Bartoš, et al. 2023. “Genetic and Karyotype Divergence Between Parents Affect Clonality and Sterility in Hybrids.” eLife 12: RP88366.

McKee, B. D. 2004. “Homologous Pairing and Chromosome Dynamics in Meiosis and Mitosis.” Biochimica et Biophysica Acta (BBA)—Gene Structure and Expression 1677: 165–180.

Mikulíček, P., M. Kautman, B. Demovič, and K. Janko. 2014. “When a Clonal Genome Finds Its Way Back to a Sexual Species: Evidence From Ongoing but Rare Introgression in the Hybridogenetic Water Frog Complex.” Journal of Evolutionary Biology 27: 628–642.

Mikulíček, P., and P. Kotlík. 2001. “Two Water Frog Populations From Western Slovakia Consisting of Diploid Females and Diploid and Triploid Males of the Hybridogenetic Hybrid Rana esculenta (Anura, Ranidae).” Zoosystematics and Evolution 77: 59–64.

Miura, I., V. Vershinin, S. Vershinin, et al. 2021. “Hybridogenesis in the Water Frogs From Western Russian Territory: Intrapopulation Variation in Genome Elimination.” Genes 12: 244–244.

Moritz, C., W. M. Brown, L. Densmore, et al. 1989. “Genetic Diversity and the Dynamics of Hybrid Parthenogenesis in Cnemidophorus (Teiidae) and Heteronotia (Gekkonidae).” Evolution and Ecology of Unisexual Vertebrates 466: 87–112.

Ogielska‐Nowak, M. 1978. “DNA Content in Erythrocyte Nuclei of Diploid and Triploid Green Frog Hybrid of Rana esculenta L. Complex.” Zoologica Poloniae 27: 109–115.

Papežík, P., P. Mikulíček, M. Benovics, et al. 2023. “Comparative Mitochondrial Phylogeography of Water Frogs (Ranidae: Pelophylax spp.) From the Southwestern Balkans.” Vertebrate Zoology 73: 525–544.

Pereira, C. S. A., M. A. Aboim, P. Ráb, and M. J. Collares‐Pereira. 2014. “Introgressive Hybridization as a Promoter of Genome Reshuffling in Natural Homoploid Fish Hybrids (Cyprinidae, Leuciscinae).” Heredity 112: 343–350.

Plötner, J., Ç. Akın Pekşen, F. Baier, T. Uzzell, and C. C. Bilgin. 2015. “Genetic Evidence for Human‐Mediated Introduction of Anatolian Water Frogs (Pelophylax Cf. Bedriagae) to Cyprus (Amphibia: Ranidae).” Zoology in the Middle East 61: 125–132.

Plötner, J., T. Uzzell, P. Beerli, et al. 2008. “Widespread Unidirectional Transfer of Mitochondrial DNA: A Case in Western Palaearctic Water Frogs.” Journal of Evolutionary Biology 21: 668–681.

Pruvost, N., P. Mikulíček, L. Choleva, and H.‐U. Reyer. 2015. “Contrasting Reproductive Strategies of Triploid Hybrid Males in Vertebrate Mating Systems.” Journal of Evolutionary Biology 28: 189–204.

Pruvost, N. B. M., A. Hoffmann, and H.‐U. Reyer. 2013. “Gamete Production Patterns, Ploidy, and Population Genetics Reveal Evolutionary Significant Units in Hybrid Water Frogs (Pelophylax esculentus).” Ecology and Evolution 3: 2933–2946.

Pustovalova, E., L. Choleva, D. Shabanov, and D. Dedukh. 2022. “The High Diversity of Gametogenic Pathways in Amphispermic Water Frog Hybrids From Eastern Ukraine.” PeerJ 10: e13957.

Ragghianti, M., S. Bucci, S. Marracci, et al. 2007. “Gametogenesis of Intergroup Hybrids of Hemiclonal Frogs.” Genetics Research 89: 39–45.

Ragghianti, M., F. Guerrini, S. Bucci, et al. 1995. “Molecular Characterization of a Centromeric Satellite DNA in the Hemiclonal Hybrid Frog Rana esculenta and Its Parental Species.” Chromosome Research 3: 497–506.

Reyer, H.‐U., C. Arioli‐Jakob, and M. Arioli. 2015. “Post‐Zygotic Selection Against Parental Genotypes During Larval Development Maintains All‐Hybrid Populations of the Frog Pelophylax esculentus.” BMC Evolutionary Biology 15: 131.

Scali, V., M. Passamonti, O. Marescalchi, and B. Mantovani. 2003. “Linkage Between Sexual and Asexual Lineages: Genome Evolution in Bacillus Stick Insects.” Biological Journal of the Linnean Society 79: 137–150.

Schön, I., K. Martens, and P. van Dijk. 2009. Lost Sex: The Evolutionary Biology of Parthenogenesis. Springer Netherlands.

Shabanov, D., M. Vladymyrova, A. Leonov, et al. 2020. “Simulation as a Method for Asymptotic System Behavior Identification (e.g. Water Frog Hemiclonal Population Systems).” In Information and Communication Technologies in Education, Research, and Industrial Applications, 1175, 392–414. Springer.

Stöck, M., D. Dedukh, R. Reifová, D. K. Lamatsch, Z. Starostová, and K. Janko. 2021. “Sex Chromosomes in Meiotic, Hemiclonal, Clonal and Polyploid Hybrid Vertebrates: Along the “Extended Speciation Continuum”.” Philosophical Transactions of the Royal Society, B: Biological Sciences 376: 20200103.

Suzuki, S., K. Arai, and H. Munehara. 2017. “Karyological Evidence of Hybridogenesis in Greenlings (Teleostei: Hexagrammidae).” PLoS One 12: e0180626.

Svinin, A. O., D. V. Dedukh, L. J. Borkin, et al. 2021. “Genetic Structure, Morphological Variation, and Gametogenic Peculiarities in Water Frogs (Pelophylax) From Northeastern European Russia.” Journal of Zoological Systematics and Evolutionary Research 59: 646–662.

Tamura, K., G. Stecher, and S. Kumar. 2021. “MEGA11: Molecular Evolutionary Genetics Analysis Version 11.” Molecular Biology and Evolution 38: 3022–3027.

The ARRIVE Guidelines 2.0. 2020. https://arriveguidelines.org/arrive‐guidelines.

Tunner, H. G. 1973. “Demonstration of the Hybrid Origin of the Common Green Frog Rana esculenta L.” Science of Nature 60: 481–482.

Tunner, H. G., and S. Heppich‐Tunner. 1991. “Genome Exclusion and Two Strategies of Chromosome Duplication in Oogenesis of a Hybrid Frog.” Naturwissenschaften 78: 32–34.

Uzzell, T., R. Günther, and L. Berger. 1977. “Rana ridibunda and Rana esculenta: A Leaky Hybridogenetic System (Amphibia Salientia).” Proceedings of the Academy of Natural Sciences of Philadelphia 128: 147–171.

Vinogradov, A. E., L. J. Borkin, R. Günther, and J. M. Rosanov. 1991. “Two Germ Cell Lineages With Genomes of Different Species in One and the Same Animal.” Hereditas 114: 245–251.

Zaleśna, A., L. Choleva, M. Ogielska, M. Rábová, F. Marec, and P. Ráb. 2011. “Evidence for Integrity of Parental Genomes in the Diploid Hybridogenetic Water Frog Pelophylax esculentus by Genomic In Situ Hybridization.” Cytogenetic and Genome Research 134: 206–212.

Programmed Genome Elimination Is Evolutionarily Conserved Across Pelophylax Hybrids-As Evidenced by P. grafi Hybridogenetic Reproduction