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![Genetic identity of the phased Pelophylax genomes based on 81 individuals and five families. RR denotes P. ridibundus mothers and daughters, RL denotes P. esculentus fathers and sons. R denotes sexual genomes, [R] and [L] denote clonal genomes. (A) An illustration of the laboratory crossing experiments that resulted in R[R] daughters and R[L] sons; photos show real individuals participating in the experiment; * late prophase I of the P. esculentus male´s first meiotic division using comparative genomic in-situ hybridization with lessonae specific probes (red color) and ridibundus specific probes (green color); see Doležálková et al.³⁴. (B1) fineRADstucture heatmap of haplotypes similarity (2219 SNPs): co-ancestry matrix based on a RADseq dataset, above the diagonal are the individual scores, below are the population averages. The color scale legend on the right indicates the relatedness between haploid genomes. A cluster of clonal [L] genomes is apparent in the upper right of the graph (black/dark blue color), while sexual R and clonal [R] on the down left (rose/red color). (B2) STRUCTURE ancestry bar plots of the respective individuals, Q-value scale indicated on the left. Phased haploid genomes of mothers are indicated with ♀ and fathers with ♂, other genomes representing progeny with families´ membership indicated with frames. (C) Principal component analysis (PCA) of phased microsatellite data based on 15 loci. (D) Results of PCA based on phased genotypes of ddRADseq data (2219 SNPs). The clusters determine sexual R/clonal [R] genomes and clonal [L] genomes. Each point represents an individual haploid multilocus genotype; one color and symbol denote one family.](publication/348518215/figure/fig1/AS:980930600636417@1610883488214/Genetic-identity-of-the-phased-Pelophylax-genomes-based-on-81-individuals-and-five.png)
Genetic identity of the phased Pelophylax genomes based on 81 individuals and five families. RR denotes P. ridibundus mothers and daughters, RL denotes P. esculentus fathers and sons. R denotes sexual genomes, [R] and [L] denote clonal genomes. (A) An illustration of the laboratory crossing experiments that resulted in R[R] daughters and R[L] sons; photos show real individuals participating in the experiment; * late prophase I of the P. esculentus male´s first meiotic division using comparative genomic in-situ hybridization with lessonae specific probes (red color) and ridibundus specific probes (green color); see Doležálková et al.³⁴. (B1) fineRADstucture heatmap of haplotypes similarity (2219 SNPs): co-ancestry matrix based on a RADseq dataset, above the diagonal are the individual scores, below are the population averages. The color scale legend on the right indicates the relatedness between haploid genomes. A cluster of clonal [L] genomes is apparent in the upper right of the graph (black/dark blue color), while sexual R and clonal [R] on the down left (rose/red color). (B2) STRUCTURE ancestry bar plots of the respective individuals, Q-value scale indicated on the left. Phased haploid genomes of mothers are indicated with ♀ and fathers with ♂, other genomes representing progeny with families´ membership indicated with frames. (C) Principal component analysis (PCA) of phased microsatellite data based on 15 loci. (D) Results of PCA based on phased genotypes of ddRADseq data (2219 SNPs). The clusters determine sexual R/clonal [R] genomes and clonal [L] genomes. Each point represents an individual haploid multilocus genotype; one color and symbol denote one family.
Source publication
Hybridogenesis is a reproductive tool for sexual parasitism. Hybridogenetic hybrids use gametes from their sexual host for their own reproduction, but sexual species gain no benefit from such matings as their genome is later eliminated. Here, we examine the presence of sexual parasitism in water frogs through crossing experiments and genome-wide da...
Citations
... In the R-E HPS, most hybrids transmit the (L)genome but diploid hybrids here often produce a mix of gametes with either (L)-or (R)genome Doležálková et al., 2016;Pustovalova et al., 2022). This phenomenon was first shown in P. esculentus males and described as amphispermy (Vinogradov et al., 1991;Doležálková-Kaštánková and Mazepa et al., 2021). Because this reproductive mode is not restricted to males, it was proposed to be called amphigameticity (Pustovalova et al., 2022). ...
Pelophylax esculentus (genotype LR), an interspecific hybrid of P. ridibundus (genotype RR) and P. lessonae (genotype LL), is one of the most studied examples of hemiclonal hybridogenic vertebrates. In most populations, hybrids produce clonal gametes of one parental species and backcross with the other parental species, forming a new generation of hybrids. In this study, we focused on the population system of water frogs in the Siverskyi Donets river basin (eastern Ukraine) with the aim to determine the genetic diversity of parental genomes and clarify how such a system is maintained in nature. Of the 55 frogs analyzed, only one individual was P. ridibundus, 48 belonged to diploid LR and five to triploid LRR hybrids. Using 16 microsatellite loci, we found significantly lower genetic diversity and significantly higher clonality in the L-genome of hybrids compared to the R-genome. This contrasting diversity might be attributed to the absence of P. lessonae in the studied region and the clonal inheritance of the L-genome. Our population-genetic results are partially inconsistent with previous studies focusing on hybrid gametogenesis in the Siverskyi Donets river basin, according to which hybrids form both clonal R and L gametes. We hypothesize that discrepancies between the type of clonally formed gametes and the genotypic composition of the studied population may be due to the selective mortality of some genotypes during larval development and metamorphosis. Selective mortality thus may play an important role in the dynamics of the hybridogenetic systems in eastern Ukraine.
... The first type represents classic hybridogenesis with pre-meiotic elimination of the R genome and production of clonal L gametes (these males are referred to as monospermic males) 15,36,43,45 . The second type of hybrid males can simultaneously produce both L and R clonal gametes [34][35][36][37]41,43,45,46 . This phenomenon has been termed hybrid amphispermy 35 , and males exhibiting this trait are referred to as amphispermic males. ...
... B1 males from the family 12-AB (12-AB3 and 12-AB5) showed signals of L sperm while P-male M3 had doubled signals of L and R sperm indicating amphispermy (see Fig. 4). To verify the doubled signal of the amphispermic male M3, we compared its profile with an earlier confirmed amphispermic male that produced two types of sperm leading to RR and RL progeny (male M11 in a publication 46 ). ...
... Secondly, we detected simultaneous formation of L and R spermatids that was confirmed by flow cytometry of mature sperms. Such hybrid amphispermy was observed in hybrid males from Czech, Ukrainian and German R-E systems [35][36][37]43,45,46,65 . Cytogenetic and molecular methods applied to individuals of different ontogenetic stages allowed us to track amphispermic inheritance through three generations. ...
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.
... Bimaternal reproduction means an egg develops into a new individual through DNA replication without fertilization. Compared with bipaternal reproduction, bimaternal reproduction is more common in nature, such as in lizards, frogs, and fish [46][47][48]. In contrast, bipaternal reproduction is only found in very few lower animals [49]. ...
Haploid cells are a kind of cells with only one set of chromosomes. Compared with traditional diploid cells, haploid cells have unique advantages in gene screening and drug-targeted therapy, due to their phenotype being equal to the genotype. Embryonic stem cells are a kind of cells with strong differentiation potential that can differentiate into various types of cells under specific conditions in vitro. Therefore, haploid embryonic stem cells have the characteristics of both haploid cells and embryonic stem cells, which makes them have significant advantages in many aspects, such as reproductive developmental mechanism research, genetic screening, and drug-targeted therapy. Consequently, establishing haploid embryonic stem cell lines is of great significance. This paper reviews the progress of haploid embryonic stem cell research and briefly discusses the applications of haploid embryonic stem cells.
... Therefore, hybrids typically have to coexist with one of the parental species (Graf and Polls-Pelaz 1989;Plötner 2005). Studies on populations that included P. esculentus revealed a high diversity of gametogenic pathways in this hybrid (Dedukh et al. 2015(Dedukh et al. , 2019Doležálková-Kaštánková et al. 2021;Chmielewska et al. 2022;Pustovalova et al. 2022), whereas P. grafi hybrids are currently thought to form only one population type with unknown pathways of gamete formation. ...
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.
... In Austria and Hungary, for example, L-E populations were found in which over 94% of hybrid individuals were of female sex (Tunner 1974, 1976, 1978, 2001, Tunner und Dobrowsky 1976, Gubanyi und Creemers 1994. In contrast, R-E populations from the Oder river contain only diploid LR males that inherit the L and/or the R genome (Uzzell et al. 1977, Berger and Günther 1991, Günther and Plötner 1988, Doležálková-Kaštánková et al. 2021. Hybrid males capable to produce both types of gametes (L and R), are called amphispermic (Vinogradov et al. 1991). ...
... According to Vinogradov et al. (1991), amphispermic males may account for 14-17% of LR males in an R-E population, but their frequency can be even higher. Doležálková-Kaštánková et al. (2021) confirmed amphispermy in 25% of diploid hybrid males from sample of two populations. Because there are no esculentus females in such R-E populations, LR males can only reproduce by mating with RR females (Uzzell et al. 1977). ...
... Because there are no esculentus females in such R-E populations, LR males can only reproduce by mating with RR females (Uzzell et al. 1977). If LR males form sperm with an R genome, LR x RR crosses can also result in RR females (Uzzell et al. 1977, Günther and Plötner 1988, Berger and Günther 1991, Doležálková et al. 2016, Doležálková-Kaštánková et al. 2018, 2021, since in analogy to the XX/XY system of sex determination in mammals, the clonally inherited R genome usually carries one or more female-determining X factor(s) (e.g. (Günther and Plötner 1988). ...
Der mitteleuropäische Wasserfroschkomplex umfasst Pelophylax lessonae (Genotyp LL), Pelophylax ridibundus (RR) und deren Hybriden Pelophylax esculentus (LR), der sich hemiklonal durch Rückkreuzen mit LL in lessonae-esculentus (L-E)-Populationen oder RR in ridibundus-esculentus (R-E)-Populationen reproduziert. Außerdem können Hybriden in reine Hybridpopulationen (E) bilden, in denen triploide Individuen die Elternarten funktionell ersetzen. Bislang ist wenig über die molekularen Mechanismen bekannt, die der klonalen Gametenbildung in der Keimbahn der Hybridform zugrunde liegen. In dieser Studie wurden erstmalig 160 Gene der Elternarten untersucht, die an der Gametenbildung beteiligt sind. Zusätzlich wurden 131 SNPs von 52 dieser Gene von 652 Wasserfröschen aus 26 Populationen analysiert. Im Ergebnis wurden 14 SNPs von 10 Genen entdeckt, deren Frequenzen mit dem Populationssystem assoziiert waren. In Übereinstimmung mit ihren Funktionen könnten diese Gene im Zusammenheng mit den system-spezifischen hybridogenetischen Reproduktionsmodi stehen. Sowohl transkriptomische als auch SNP-Daten lieferten Hinweise auf genetische Introgression, d. h. einen Transfer lessonae-spezifischer Allelen in den ridibundus-Genpool oder umgekehrt. Außerdem wurde bei P. lessonae eine kryptische genetische Diversität beobachtet. SNP-Analysen ergaben auch, dass LR-Individuen aus E-Populationen eine höhere genetische Ähnlichkeit mit LR-Individuen aus R-E als aus L-E-Populationen aufweisen. Diese Ergebnisse werfen die Frage nach dem Ursprung der E-Populationen auf, von denen bisher angenommen wurde, dass sie aus L-E-Populationen hervorgegangen sind. Die neuen molekularen Daten stehen mit den hemiklonalen Fortpflanzungsmodi von P. esculentus im Einklang und unterstreichen, dass diese wahrscheinlich auf komplexen Wechselwirkungen zwischen verschiedenen Genen und Faktoren basieren.
... For such a phenomenon as the production of several types of sperm by one male, Vinogradov et al. (1991) proposed the term "hybrid amphispermy". A similar phenomenon was observed by Pruvost et al. (2013), Biriuk et al. (2016), Doležálková-Kaštánková and Mazepa (2021), Svinin et al. (2021), and most recently by Pustovalova et al. (2022). ...
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.
... Detail studies of the latter in Central Europe showed that P. esculentus occurs in the male sex only, being even amphispermic. In this process, a single P. esculentus form two sperm cell types, a clonal haploid ridibundus (R sperm) and lessonae (L sperm) genomes [15][16][17]. ...
... PCR protocol was based on a study by Christiansen and Reyer [36]. Individual genotypes were based on species-specifity of amplified alleles described in Doležálková-Kaštánková et al. [16]. Fragment-length analyses were performed on the ABI 3730 Avant capillary sequencer (Applied Biosystems, Foster City, California, USA) with an internal size standard (GeneScan-500 LIZ, Thermo Fisher Scientific, Waltham, MA, USA); the alleles were scored with GeneMapper v. 3. 7 (Applied Biosystems, Zug, Switzerland). ...
... Variation in survival rates in progeny fathered by males from the R-E system may have been caused by variations in sperm production. The occurrence of different germ cells in the testes may lead to the formation of only R sperm, only L sperm, or both sperm types at once, resulting in variable progeny [16,17,52,57]. ...
Background:
Hybrid taxa exist in nature, but their fitness can vary greatly. Hybrids are usually thought to have lower viability and survival rate than parental species due to the occurrence of two different genomes and divergent evolution in each species. On the other hand, the hybrid vigour of the F1 generation may give hybrids an advantage in mixed populations where they have to live and compete with parental taxa. Post-zygotic selection with endogenous genetic mechanisms may be a significant evolutionary force in hybrid formation. Here we tested principles of post-zygotic reproductive dynamics in mixed populations of Pelophylax water frogs that would help us understand the origin and maintenance of such systems.
Methods:
Within experimental crosses, we combined various diploid Pelophylax genotypes resulting in 211 families. Statistical analysis of progeny was used to measure fertilization success, the rate of embryonic/tadpole mortality and the overall survival of the progeny till the time of metamorphosis. Using Generalized Estimating Equations models and variables defined by a mother/father included in mate pairs, we tested which factor best explains the successful embryonal development.
Results:
The development of Pelophylax offspring significantly varied in survival rate and morphological malformations. These post-zygotic reproductive dynamics were driven by parental combinations of species pairs. The best values in the proportion of developing eggs, embryos, tadpoles and overall survival showed progeny of homospecific P. lessonae crosses. Total survival rates were relatively similar between L-E and R-E population systems but much lower than homospecific crosses in parental taxa. However, once the early stages passed this period, tadpoles mostly of hybrid hemiclonal origin performed even better than pure P. ridibundus progeny. Hybrid × hybrid crosses showed the highest mortality values. Statistical testing revealed that high mortality affected paternal genetic input.
Conclusions:
Combined three water frog taxa and both sexes provided patterns of post-zygotic reproduction dynamics of early development in the widespread population systems in Central Europe. The results further showed high survival rates of hybrid F1s created de novo from parental species despite significant divergence between P. ridibundus and P. lessonae DNA. Potential conservation measures of sexual-asexual systems in natural populations are discussed.
... Available data indicate that when hybrid females are fertile and asexual (often employing PMER), their hybrid brothers are usually sterile [11,21,24]. However, some exceptions exist, e.g., in hybridogenetic taxa like Pelophylax [25] or Squalius [26]. Crossing experiments within several asexual complexes of fishes and reptiles further demonstrate that asymmetry between female hybrid asexuality and male sterility is directly linked to the merging of parental genomes and already occurs in the F1 hybrid generation [9,11,[27][28][29]. ...
Interspecific hybridization may trigger the transition from sexual reproduction to asexuality, but mechanistic reasons for such a change in a hybrid’s reproduction are poorly understood. Gametogenesis of many asexual hybrids involves a stage of premeiotic endoreduplication (PMER), when gonial cells duplicate chromosomes and subsequent meiotic divisions involve bivalents between identical copies, leading to production of clonal gametes. Here, we investigated the triggers of PMER and whether its induction is linked to intrinsic stimuli within a hybrid’s gonial cells or whether it is regulated by the surrounding gonadal tissue.
We investigated gametogenesis in the Cobitis taenia hybrid complex, which involves sexually reproducing species (Cobitis elongatoides and C. taenia) as well as their hybrids, where females reproduce clonally via PMER while males are sterile. We transplanted spermatogonial stem cells (SSCs) from C. elongatoides and triploid hybrid males into embryos of sexual species and of asexual hybrid females, respectively, and observed their development in an allospecific gonadal environment. Sexual SSCs underwent regular meiosis and produced normally reduced gametes when transplanted into clonal females. On the other hand, the hybrid’s SSCs lead to sterility when transplanted into sexual males, but maintained their ability to undergo asexual development (PMER) and production of clonal eggs, when transplanted into sexual females.
This suggests that asexual gametogenesis is under complex control when somatic gonadal tissue indirectly affects the execution of asexual development by determining the sexual differentiation of stem cells and once such cells develop to female phenotypes, hybrid germ cells trigger the PMER from their intrinsic signals.
The molecular processes underlying genome exclusion and clonal gamete formation in the germline of hybridogenetic water frog hybrids are poorly understood. For the first time, we have characterized the coding sequences of 160 gametogenic genes from the European water frog species Pelophylax lessonae (LL) and Pelophylax ridibundus (RR). In addition, single nucleotide polymorphisms (SNPs) in 52 of these genes were analyzed in both species (60 LL, 252 RR) and their hybridogenetic hybrid, Pelophylax esculentus (340 LR), from population systems that differ in genotypic composition, sex ratio, and modes of inheritance. Ten gametogenic genes may be linked to the population system and, thus, indirectly to the reproductive mode, i.e., the premeiotic exclusion of the ridibundus (R) genome in the lessonae - esculentus system and/or the lessonae (L) genome in the ridibundus - esculentus system. These genes are involved in various processes, including the control of spindle formation and chromosome movement during mitosis and meiosis, epigenetic silencing, cell cycle regulation, double-strand break repair and homologous recombination, including transposable element silencing. In addition, both the transcriptome and SNP data provide evidence for gene flow between the L and R genomes. The results are consistent with the clonal inheritance patterns described for P. esculentus and suggest that genome exclusion is based on complex genomic interactions involving multiple genes and molecular factors. Furthermore, our data suggest that interspecific gene flow has played an important role in the evolution of different inheritance modes in P. esculentus , leading to the emergence of distinct population systems.
Palearctic water frogs (genus Pelophylax) are an outstanding model in ecology and evolution, being widespread, speciose, either threatened or threatening to other species through biological invasions, and capable of siring hybrid offspring that escape the rules of sexual reproduction. Despite half a century of genetic research and hundreds of publications, the diversity, systematics and biogeography of Pelophylax still remain highly confusing, in no small part due to a lack of correspondence between studies. To provide a comprehensive overview, we gathered >13,000 sequences of barcoding genes from >1700 native and introduced localities and built multigene mitochondrial (~17 kb) and nuclear (~10 kb) phylogenies. We mapped all currently recognized taxa and their phylogeographic lineages (>40) to get a grasp on taxonomic issues, cyto-nuclear discordances, the genetic makeup of hybridogenetic hybrids, and the origins of introduced populations. Competing hypotheses for the molecular calibration were evaluated through plausibility tests, implementing a new approach relying on predictions from the anuran speciation continuum. Based on our timetree, we propose a new biogeographic paradigm for the Palearctic since the Paleogene, notably by attributing a prominent role to the dynamics of the Paratethys, a vast paleo-sea that extended over most of Europe. Furthermore, our results show that distinct marsh frog lineages from Eastern Europe, the Balkans, the Near East, and Central Asia (P. ridibundus ssp.) are naturally capable of inducing hybridogenesis with pool frogs (P. lessonae). We identified 14 alien lineages (mostly of P. ridibundus) over ~20 areas of invasions, especially in Western Europe, with genetic signatures disproportionally pointing to the Balkans and Anatolia as the regions of origins, in line with exporting records of the frog leg industry and the stocks of pet sellers. Pelophylax thus emerges as one of the most invasive amphibians worldwide, and deserves much higher conservation concern than currently given by the authorities fighting biological invasions.
