Central European water frog
Pelophylax esculentus
is a hybrid that lives sympatrically and forms genetic systems with one of its parental species, in this case L-E (
P. lessonae - P. esculentus
). ...Hybrids are restored in each generation due to hybridogenesis, a unique mode of hemiclonal reproduction. We investigated changes in the species composition, mate choice, and genome transmission in naturally formed pairs in the L-E hybridogenetic system from the Raków fish pond complex (SW Poland). Our observations showed that the genetic system remained the same for over a decade but the share of the taxa and the sex ratios within the species changed. The frequency of
P. lessonae
decreased by about 20% in favor of
P. esculentus
. The proportion of males to females of
P. lessonae
increased twofold and of
P. esculentus
fourfold. We also found that the stability of the L-E population was ensured by a specific female mate choice pattern with the dominance of homotypic
P. lessonae
and heterotypic ♀
P. esculentus
× ♂
P. lessonae
pairs (almost 90% of the pairs collected from nature). The youngest females and males that formed a successful amplexus were 3 years old. We noted a male-male competition manifested by the exclusion of young males (before the third hibernation) from the participation in the mating. An analysis of genome transmission to gametes revealed that females
P. lessonae
transmitted Lx, while
P. esculentus
transmitted the Rx and/or Lx genomes. Males of
P. lessonae
transmitted the Lx or Ly genomes, while
P. esculentus
transmitted the Lx, Ly, and/or Rx genomes. The high proportion of the L genomes transferred to gametes enables both the restoration of the parental species
P. lessonae
and the regular renewal of a new generation of hybrids.
Significance statement
Central European water frog complex includes two species:
Pelophylax ridibundus
(genotype RR) and
P. lessonae
(LL), and their natural hybrid,
P. esculentus
(RL). Hybrid individuals of both sexes reproduce due to a specific mode of reproduction, i.e., hybridogenesis, in which hybrids eliminate one of the parental genomes (R or L) from the germ line prior to meiosis and transmit the other one (not recombined, i.e., clonal) to the gametes. The hybrids live in mixed populations with one of the parental species and are restored in each generation. This, in turn, allows a kind of balance to be maintained in a population due to specific mate choice and genome transmission. Our research showed that the stability of the studied population was ensured by female preference to
P. lessonae
and male-male competition manifested by the exclusion of males under 3 years of age from the participation in the mating.
Occasional XY recombination is a proposed explanation for the sex-chromosome homomorphy in European tree frogs. Numerous laboratory crosses, however, failed to detect any event of male recombination, ...and a detailed survey of NW-European Hyla arborea populations identified male-specific alleles at sex-linked loci, pointing to the absence of XY recombination in their recent history. Here, we address this paradox in a phylogeographic framework by genotyping sex-linked microsatellite markers in populations and sibships from the entire species range. Contrasting with postglacial populations of NW Europe, which display complete absence of XY recombination and strong sex-chromosome differentiation, refugial populations of the southern Balkans and Adriatic coast show limited XY recombination and large overlaps in allele frequencies. Geographically and historically intermediate populations of the Pannonian Basin show intermediate patterns of XY differentiation. Even in populations where X and Y occasionally recombine, the genetic diversity of Y haplotypes is reduced below the levels expected from the fourfold drop in copy numbers. This study is the first in which X and Y haplotypes could be phased over the distribution range in a species with homomorphic sex chromosomes; it shows that XY-recombination patterns may differ strikingly between conspecific populations, and that recombination arrest may evolve rapidly (<5000 generations).
The growing interest in the lability of sex determination in non‐model vertebrates such as amphibians and fishes has revealed high rates of sex chromosome turnovers among closely related species of ...the same clade. Can such lineages hybridize and admix with different sex‐determining systems, or could the changes have precipitated their speciation? We addressed these questions in incipient species of toads (Bufonidae), where we identified a heterogametic transition and characterized their hybrid zone with genome‐wide markers (RADseq). Adult and sibship data confirmed that the common toad B. bufo is female heterogametic (ZW), while its sister species the spined toad B. spinosus is male heterogametic (XY). Analysis of a fine scale transect across their parapatric ranges in southeastern France unveiled a narrow tension zone (∼10 km), with asymmetric mitochondrial and nuclear admixture over hundreds of kilometers southward and northward, respectively. The geographic extent of introgression is consistent with an expansion of B. spinosus across B. bufo’s former ranges in Mediterranean France, as also suggested by species distribution models. However, widespread cyto‐nuclear discordances (B. spinosus backrosses carrying B. bufo mtDNA) run against predictions from the dominance effects of Haldane's rule, perhaps because Y and W heterogametologs are not degenerated. Common and spined toads can thus successfully cross‐breed despite fundamental differences in their sex determination mechanisms, but remain partially separated by reproductive barriers. Whether and how the interactions of their XY and ZW genes contribute to these barriers shall provide novel insights on the debated role of labile sex chromosomes in speciation.
DNA elimination is a radical form of gene silencing and occurs both in somatic and germ cells. The programmed DNA elimination occurs during gametogenesis in interspecies hybrids that reproduce by ...hybridogenesis (stick insects, fishes, and amphibians) and concerns removal of whole genomes of one of the parental species and production of clonal gametes propagating the genome of the other species. The cellular mechanisms differ considerably in hybridogenetic insects and fishes but remains unknown in edible frogs Pelophylax esculentus, natural hybrids between Pelophylax lessonae and Pelophylax ridibundus. Here we report DNA elimination mechanism in early developing gonads of diploid and triploid hybrid frogs, studied by TEM, immunofluorescence, and cytochemistry. In gonocytes of both sexes (primary oogonia and prespermatogonia), micronuclei emerge as detached nuclear buds formed during interphase. We found depletion of nuclear pore complexes in micronuclear membrane and chromatin inactivation via heterochromatinization followed by degradation of micronuclei by autophagy. Micronuclei formation does not lead to apoptotic cell death showing that genome elimination is a physiological process. Chromatin elimination via micronuclei in P. esculentus is unique among hybridogenetic animals and contributes to broadening the knowledge about reproductive modes in animals.
Multiple anthropogenic stressors cause worldwide amphibian declines. Among several poorly investigated causes is global pollution of aquatic ecosystems with endocrine disrupting compounds (EDCs). ...These substances interfere with the endocrine system and can affect the sexual development of vertebrates including amphibians. We test the susceptibility to an environmentally relevant contraceptive, the artificial estrogen 17α-ethinylestradiol (EE2), simultaneously in three deeply divergent systematic anuran families, a model-species, Xenopus laevis (Pipidae), and two non-models, Hyla arborea (Hylidae) and Bufo viridis (Bufonidae). Our new approach combines synchronized tadpole exposure to three EE2-concentrations (50, 500, 5,000 ng/L) in a flow-through-system and pioneers genetic and histological sexing of metamorphs in non-model anurans for EDC-studies. This novel methodology reveals striking quantitative differences in genetic-male-to-phenotypic-female sex reversal in non-model vs. model species. Our findings qualify molecular sexing in EDC-analyses as requirement to identify sex reversals and state-of-the-art approaches as mandatory to detect species-specific vulnerabilities to EDCs in amphibians.
•Prespermatogenesis in amphibians is restricted to the larval and early juvenile period.•Primary spermatogonia are represented by gonocytes and spermatogonial stem cells.•Gonocytes differ from ...spermatogonial stem cells in size and some morphological features.•One spermatogonial stem cell in frogs gives rise to 8–256 secondary spermatogonia.
Spermatogenesis in frogs was for the first time divided into two phases: prespermatogenesis, when gonocytes proliferate in developing tadpole testes, and active spermatogenesis when spermatogonial stem cells (i.e. descendants of gonocytes), either self-renew or enter into meiotic cycles within cysts formed by Sertoli cells. We argue that amphibian larval gonocytes are homologues to mammalian gonocytes, whereas spermatogonial stem cells (SSCs) in adult frogs are homologous to mammalian single spermatogonia (As). Gonocytes constitute sex cords, i.e. the precursors of seminiferous tubules; they are bigger than SSCs and differ in morphology and ultrastructure. The nuclear envelope in gonocytes formed deep finger-like invaginations absent in SSCs. All stages of male germ cells contained lipid droplets, which were surrounded by glycogen in SSCs, but not in gonocytes. Mitochondria in gonocytes had enlarged edges of cristae, and in SSCs also lamellar mitochondria appeared. Minimal duration of prespermatogenesis was 46days after gonadal sex differentiation, but usually it lasted longer. SSCs give rise to secondary spermatogonia (equal to mammalian A, In, and B). Their lowest number inside a cyst was eight and this indicated the minimal number of cell cycles (three) of secondary spermatogonia necessary to enter meiosis. We sorted them according to the number of cell cycles (from 8 to 256 cells). This number is similar to that recorded for mammals as the result of a single As proliferation. The number of secondary spermatogonia correlates with the volume of a cyst. The general conclusion is that spermatogenesis in amphibians and mammals follows basically the same scheme.
Due to their terrestrial habitats and aquatic reproduction, many amphibians are both very vulnerable and highly suitable bioindicators. The plasticizer bisphenol A (BPA) is one of the most produced ...chemical substances worldwide, and knowledge on its impacts on humans and animals is mounting. BPA is used for the industrial production of polycarbonate plastics and epoxy resins and found in a multitude of consumer products. Studies on BPA have involved mammals, fish and the fully aquatic anuran model Xenopus laevis. However, our knowledge about the sexual development of non-model, often semi-terrestrial anuran amphibians remains poor. Using a recently developed experimental design, we simultaneously applied BPA to two non-model species (Hyla arborea, Hylidae; Bufo viridis, Bufonidae) and the model X. laevis (Pipidae), compared their genetic and phenotypic sex for detection of sex reversals, and studied sexual development, focusing on anatomical and histological features of gonads. We compared three concentrations of BPA (0.023, 2.28 and 228 μg/L) to control groups in a high-standard flow-through-system, and tested whether conclusions, drawn from the model species, can be extrapolated to non-model anurans. In contrast to previous studies on fish and Xenopus, often involving dosages much higher than most environmental pollution data, we show that BPA causes neither the development of mixed sex nor of sex-reversed individuals (few, seemingly BPA-independent sex reversals) in all focal species. However, environmentally relevant concentrations, as low as 0.023 μg/L, were sufficient to provoke species-specific anatomically and histologically detectable impairments of gonads, and affected morphological traits of metamorphs. As the intensity of these effects differed between the three species, our data imply that BPA diversely affects amphibians with different evolutionary history, sex determination systems and larval ecologies. These results highlight the role of amphibians as a sensitive group that is responsive to environmental pollution.
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•EDC-vulnerability of amphibians requires testing pollution-relevant concentrations.•Simultaneous Bisphenol A (BPA)-exposure of clawed frogs, tree frogs and green toads.•Flow-through-system, genetic sexing, histopathology as high standard approaches.•Species-specific anatomical and histological impairments, no sex reversals.•Model and non-model species differ in their somatic development due to BPA.
Genetic sexing and histology reveal pollution-relevant BPA-concentrations to cause species-specific gonadal and somatic impairments in amphibians from three systematic families.
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.
Abstract
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.
Using a recently developed approach for testing endocrine disruptive chemicals (EDCs) in amphibians, comprising synchronized tadpole exposure plus genetic and histological sexing of metamorphs in a ...flow-through-system, we tested the effects of 17β-Trenbolone (Tb), a widely used growth promoter in cattle farming, in three deeply diverged anuran families: the amphibian model species Xenopus laevis (Pipidae) and the non-models Bufo(tes) viridis (Bufonidae) and Hyla arborea (Hylidae). Trenbolone was applied in three environmentally and/or physiologically relevant concentrations (0.027 µg/L (10
M), 0.27 µg/L (10
M), 2.7 µg/L (10
M)). In none of the species, Tb caused sex reversals or masculinization of gonads but had negative species-specific impacts on gonad morphology and differentiation after the completion of metamorphosis, independently of genetic sex. In H. arborea and B. viridis, mounting Tb-concentration correlated positively with anatomical abnormalities at 27 µg/L (10
M) and 2.7 µg/L (10
M), occurring in X. laevis only at the highest Tb concentration. Despite anatomical aberrations, histologically all gonadal tissues differentiated seemingly normally when examined at the histological level but at various rates. Tb-concentration caused various species-specific mortalities (low in Xenopus, uncertain in Bufo). Our data suggest that deep phylogenetic divergence modifies EDC-vulnerability, as previously demonstrated for Bisphenol A (BPA) and Ethinylestradiol (EE2).
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