Hybridogenesis is a hemiclonal reproductive strategy in diploid and triploid hybrids. Our study model is a frog P. esculentus (diploid RL and triploids RLL and RRL), a natural hybrid between P. lessonae (LL) and P. ridibundus (RR). Hybridogenesis relies on elimination of one genome (L or R) from gonocytes (G) in tadpole gonads during prespermatogenesis, but not from spermatogonial stem cells (SSCs) in adults. Here we provide the first comprehensive study of testis morphology combined with chromosome composition in the full spectrum of spermatogenic cells. Using genomic in situ hybridization (GISH) and FISH we determined genomes in metaphase plates and interphase nuclei in Gs and SSCs. We traced genomic composition of SSCs, spermatocytes and spermatozoa in individual adult males that were crossed with females of the parental species and gave progeny. Degenerating gonocytes (24%–39%) and SSCs (18%–20%) led to partial sterility of juvenile and adult gonads. We conclude that elimination and endoreplication not properly completed during prespermatogenesis may be halted when gonocytes become dormant in juveniles. After resumption of mitotic divisions by SSCs in adults, these 20% of cells with successful genome elimination and endoreplication continue spermatogenesis, while in about 80% spermatogenesis is deficient. Majority of abnormal cells are eliminated by cell death, however some of them give rise to aneuploid spermatocytes and spermatozoa which shows that hybridogenesis is a wasteful process.

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.

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 (PR) 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.