Gametogenesis produces gametes as a piece of genetic information transmitted to the offspring. While during sexual reproduction, progeny inherits a mix of genetic material from both parents, asexually reproducing organisms transfer a copy of maternal or paternal DNA to the progeny clonally. Parthenogenetic, gynogenetic and hybridogenetic animals have developed various mechanisms of gametogenesis, however, their inheritance is not fully understood. Here, we focused on the inheritance of asexual gametogenesis in hybrid Pelophylax esculentus (RL), emerging after crosses of P. lessonae (LL) and P. ridibundus (RR). To understand the mechanisms of gametogenesis in hybrids, we performed three-generation experiments of sexual P. ridibundus females and hybrids from all-male hybrid populations. Using fluorescent in situ hybridization, micronuclei analysis, flow cytometry and genotyping, we found that most adult hybrid males simultaneously produced two types of clonal sperm. Also, most male tadpole progeny in two successive backcrossed generations simultaneously eliminated L and R parental genomes, while some progeny produced only one type of sperm. We hypothesize that the reproductive variability of males producing two kinds of sperm is an adaptive mechanism to reproduce in mixed populations with P. ridibundus and may explain the extensive distribution of the all-male lineage across the European River Basin.

• Klíčová slova
• Amphispermy, Crossing experiments, FISH., Male hybridogenesis, Micronuclei, Monospermic, Spermatogenesis,
• MeSH
• genotyp MeSH
• hybridizace genetická * MeSH
• hybridizace in situ fluorescenční MeSH
• nepohlavní rozmnožování * genetika   MeSH
• Rana esculenta genetika   MeSH
• Ranidae genetika   fyziologie   MeSH
• spermie fyziologie   MeSH
• typy dědičnosti genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Hybrid taxa from the genus Pelophylax can propagate themselves in a modified way of sexual reproduction called hybridogenesis ensuring the formation of clonal gametes containing the genome of only one parental (host) species. Pelophylax grafi from South-Western Europe is a hybrid composed of P. ridibundus and P. perezi genomes and it lives with a host species P. perezi (P-G system). Yet it is unknown, whether non-Mendelian inheritance is fully maintained in such populations. In this study, we characterize P. perezi and P. grafi somatic karyotypes by using comparative genomic hybridization, genomic in situ hybridization, fluorescent in situ hybridization, and actinomycin D-DAPI. Here, we show the homeology of P. perezi and P. grafi somatic karyotypes to other Pelophylax taxa with 2n = 26 and equal contribution of ridibundus and perezi chromosomes in P. grafi which supports F1 hybrid genome constitution as well as a hemiclonal genome inheritance. We show that ridibundus chromosomes have larger regions of interstitial (TTAGGG)n repeats flanking the nucleolus organizing region on chromosome no. 10 and a high quantity of AT pairs in the centromeric regions. In P. perezi, we found species-specific sequences in metaphase chromosomes and marker structures in lampbrush chromosomes. Pericentromeric RrS1 repeat sequence was present in perezi and ridibundus chromosomes, but the blocks were stronger in ridibundus. Various cytogenetic techniques applied to the P-G system provide genome discrimination between ridibundus and perezi chromosomal sets. They could be used in studies of germ-line cells to explain patterns of clonal gametogenesis in P. grafi and broaden the knowledge about reproductive strategies in hybrid animals.

• Klíčová slova
• Pelophylax grafi, Pelophylax perezi, comparative genomic hybridization, fluorescent in situ hybridization, hybridogenesis, karyotype,
• MeSH
• centromera * genetika   MeSH
• hybridizace in situ fluorescenční MeSH
• karyotypizace MeSH
• Ranidae * genetika   MeSH
• srovnávací genomová hybridizace MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• Klíčová slova
• Pelophylax esculentus, aneuploidy, endoreplication, genome elimination, hybridogenesis, in situ hybridization, polyploidy, spermatogenesis,
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• Amphispermy, Bivalents, FISH, Gametogenesis, Hybridogenesis, Meiosis, Pelophylax, Spermatid,
• MeSH
• gametogeneze * MeSH
• hybridizace in situ fluorescenční MeSH
• lidé MeSH
• sperma MeSH
• žáby * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Ukrajina MeSH