Testis differentiation in anuran amphibians is the result of two opposing processes: degeneration of the distal part, and development of the proximal part, which becomes a functional male gonad. ...Undifferentiated gonad differentiates directly into a testis without a transition phase. We described the morphology of developing testes in Rana temporaria and Hyla arborea, and made careful histology and ultrastructure in Pelophylax lessonae. The developing testis was divided into 10 stages (I-III, undifferentiated gonad, IV-X, testis). The earliest morphological symptoms of testis differentiation were observed in 4- to 5-week-old tadpoles at Gosner stage 27-28. At that time an undifferentiated gonad, composed of 6-9 metameres, differentiates into a testis. The proximal metameres (2-3 in the right gonad and 3-4 in the left one) differentiate into a functional testis, while the distal ones degenerate. The difference between left and right gonad size is maintained until adulthood (stage X). Degeneration of the distal part progresses along the posterior-anterior gradient and starts at stage IV. It affects first the germ cells with accompanying precursor Sertoli cells, and then the mesenchymal cells and fibroblasts. Finally the external epithelium forms a "sleeve" around the almost empty distal part. The total length of the testes stays the same until stage VIII at Gosner stage 41 (age 74-148 days). Active spermatogenesis starts at stage IX (juveniles after their first hibernation), during which the distal part eventually disappears and the proximal part starts growing considerably due to progressing spermatogenesis.
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► Uncovering cryptic biodiversity of threatened European amphibians (Hyla arborea group). ► Divergence times, diversity, distributions for three recently recognized tree frog ...lineages. ► Range borders, secondary contact zones, signatures of a postglacial range expansion. ► Data support conservation by fine-tuning measures to lineage-specific Quaternary refugia. ► Suture zones as natural arenas to generate biodiversity and study speciation.
We characterize divergence times, intraspecific diversity and distributions for recently recognized lineages within the Hyla arborea species group, based on mitochondrial and nuclear sequences from 160 localities spanning its whole distribution. Lineages of H. arborea, H. orientalis, H. molleri have at least Pliocene age, supporting species level divergence. The genetically uniform Iberian H. molleri, although largely isolated by the Pyrenees, is parapatric to H. arborea, with evidence for successful hybridization in a small Aquitanian corridor (southwestern France), where the distribution also overlaps with H. meridionalis. The genetically uniform H. arborea, spread from Crete to Brittany, exhibits molecular signatures of a postglacial range expansion. It meets different mtDNA clades of H. orientalis in NE-Greece, along the Carpathians, and in Poland along the Vistula River (there including hybridization). The East-European H. orientalis is strongly structured genetically. Five geographic mitochondrial clades are recognized, with a molecular signature of postglacial range expansions for the clade that reached the most northern latitudes. Hybridization with H. savignyi is suggested in southwestern Turkey. Thus, cryptic diversity in these Pliocene Hyla lineages covers three extremes: a genetically poor, quasi-Iberian endemic (H. molleri), a more uniform species distributed from the Balkans to Western Europe (H. arborea), and a well-structured Asia Minor-Eastern European species (H. orientalis).
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.
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.
We analysed variation of dorsal yellow patterns in 2077 individuals that represented 23 populations from the northern parts of the Sudetes and the Carpathian Mountains. We distinguished four types of ...yellow patterns: spotted (50.1%), spotted-and-striped (42.8%), zig-zag (5.8%), and striped (1.3%). Spotted form dominated in the east (69.2%, the Carpathians) and its frequency decreased to the west (46.4%, the Sudetes), where spotted-and-striped forms became more common. The rarest in both mountain ranges (0.9% in the Carpathians, 1.3% in the Sudetes) was striped type. The exception was the westernmost population represented by 15.8% of the striped individuals. We did not find evidence of differences between yellow colouration variants and genotypes of 557 individuals defined by 10 microsatellite loci. No differences were found between females and males. We concluded that patterning does not constitute a unique feature at population and mountain ranges levels.Keywords. Amphibians, colouration, dorsal yellow patterns, Poland, Salamandra salamandra.
We studied water frogs from a complex composed of two species:
(Camerano, 1882) (genome LL, 2n = 26) and
(Pallas, 1771) (RR, 2 = 26), and their natural hybrid
(Fitzinger, 1843) of various ploidy and ...genome composition (RL, 2n = 26, and RRL or RLL, 3n = 39). Tetraploids RRLL were found (4n = 52) in juveniles. We applied cytogenetic techniques: AgNO
, chromomycin A
PI and fluorescent
hybridization with a 28S rDNA probe. Results obtained by silver staining corresponded well with those stained with CMA
, PI and FISH. As a rule, NORs are situated on chromosomes 10. The number of Ag-NORs visible on metaphase plates was the same as the number of Ag-nucleoli present in interphase nuclei of the same individual. In all analyzed metaphases, NORs exhibited variations in size after AgNO
and CMA
stainings. Sixty-six individuals (out of 407 analyzed) were polymorphic for the localization and number of NORs. Fifty-one diploids had NORs only on one chromosome of pair 10. Three triploids (LLR and RRL) displayed two NORs, and two other triploid RRL individuals displayed one, instead of expected three NORs. In ten individuals extra NORs were detected on chromosomes other than 10 (chromosomes 2 and 9).
The Hyrcanian Forests present a unique Tertiary relict ecosystem, covering the northern Elburz and Talysh Ranges (Iran, Azerbaijan), a poorly investigated, unique biodiversity hotspot with many ...cryptic species. Since the 1970s, two nominal species of Urodela, Hynobiidae,
(later:
) have been described:
from northwestern and
from northeastern Iran. Although
has been involved in studies on phylogeny and development, there is little data on the phylogeography, systematics, and development of the genus throughout the Hyrcanian Forests; genome-wide resources have been entirely missing. Given the huge genome size of hynobiids, making whole genome sequencing hardly affordable, we aimed to publish the first transcriptomic resources for
from an embryo and a larva (9.17 Gb RNA sequences; assembled to 78,918 unigenes). We also listed 32 genes involved in vertebrate sexual development and sex determination. Photographic documentation of the development from egg sacs across several embryonal and larval stages until metamorphosis enabled, for the first time, comparison of the ontogeny with that of other hynobiids and new histological and transcriptomic insights into early gonads and timing of their differentiation. Transcriptomes from central Elburz, next-generation sequencing (NGS) libraries of archival DNA of topotypic
, and GenBank-sequences of eastern
allowed phylogenetic analysis with three mitochondrial genomes, supplemented by PCR-amplified mtDNA-fragments from 17 museum specimens, documenting <2% uncorrected intraspecific genetic distance. Our data suggest that these rare salamanders belong to a single species
s.l. Humankind has a great responsibility to protect this species and the unique biodiversity of the Hyrcanian Forest ecosystems.
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.
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.
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