Cytogenetics of the Hybridogenetic Frog Pelophylax grafi and Its Parental Species Pelophylax perezi.

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.

Genome elimination from the germline cells in diploid and triploid male water frogs Pelophylax esculentus.

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.

Maintenance of pure hybridogenetic water frog populations: Genotypic variability in progeny of diploid and triploid parents.

An intriguing outcome of hybridisation is the emergence of clonally and hemiclonally reproducing hybrids, that can sustain, reproduce, and lead to the emergence of polyploid forms. However, the maintenance of diploid and polyploid hybrid complexes in natural populations remains unresolved. We selected water frogs from the Pelophylax esculentus complex to study how diploid and triploid hybrids, which reproduce hemiclonally via hybridogenesis, are maintained in natural populations. During gametogenesis in diploid hybrids, one of the parental genomes is eliminated, and the remaining genome is endoreplicated. In triploid hybrids, the single-copy genome is typically eliminated, while genome endoreplication does not occur. To investigate how diploid and triploid hybrid frogs reproduce in populations without parental species, we crossed these hybrid animals from two separate pure hybrid populations located in Poland. Using cytogenetic analysis of tadpoles that emerged from the crosses, we established which gametes were produced by parental hybrids. The majority of hybrid females and hybrid males produced one type of gamete with the P. ridibundus genome. However, in both studied populations, approximately half of the diploid and triploid hybrids simultaneously produced gametes with different genome compositions and ploidy levels, specifically, the P. ridibundus and P. lessonae genomes, as well as diploid gametes with genomes of both parental species. Triploid hybrid males and females mostly produced haploid gametes with the P. lessonae genome; however, gametes with the P. ridibundus genome have also been observed. These results suggest that not all hybrids follow the classical hybridogenetic reproduction program and reveal a significant level of alterations in the gametogenesis pathways. In addition, we found a variable survival rate of particular progeny genotypes when we crossed hybrid females with different males suggesting the important role of postzygotic barriers on the maintenance of pure hybrid systems. We suggest that the observed variability in produced gametes and the different survival rate of the progeny with certain genotypes is crucial for the existence of pure hybrid systems.

Morphometry of two cryptic tree frog species at their hybrid zone reveals neither intermediate nor transgressive morphotypes.

Under incomplete reproductive isolation, secondary contact of diverged allopatric lineages may lead to the formation of hybrid zones that allow to study recombinants over several generations as excellent systems of genomic interactions resulting from the evolutionary forces acting on certain genes and phenotypes. Hybrid phenotypes may either exhibit intermediacy or, alternatively, transgressive traits, which exceed the extremes of their parents due to epistasis and segregation of complementary alleles. While transgressive morphotypes have been examined in fish, reptiles, birds, and mammals, studies in amphibians are rare. Here, we associate microsatellite-based genotypes with morphometrics-based morphotypes of two tree frog species of the Hyla arborea group, sampled across a hybrid zone in Poland, to understand whether the genetically differentiated parental species also differ in morphology between each other and their hybrids and whether secondary contact leads to the evolution of intermediate or transgressive morphotypes. Using univariate approaches, explorative multivariate methods (principal component analyses) as well as techniques with prior grouping (discriminant function analyses), we find that morphotypes of both parental species and hybrids differ from each other. Importantly, hybrid morphotypes are neither intermediate nor transgressive but found to be more similar to H. orientalis than to H. arborea.

Impacts of the synthetic androgen Trenbolone on gonad differentiation and development - comparisons between three deeply diverged anuran families.

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 17ß-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 (10-10 M), 0.27 µg/L (10-9 M), 2.7 µg/L (10-8 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 (10-9 M) and 2.7 µg/L (10-8 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).

Shedding Light on a Secretive Tertiary urodelean Relict: Hynobiid salamanders (Paradactylodon persicus s.l.) from Iran, Illuminated by Phylogeographic, Developmental and Transcriptomic Data.

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, Batrachuperus (later: Paradactylodon) have been described: Paradactylodon persicus from northwestern and P. gorganensis from northeastern Iran. Although P. gorganensis 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 Paradactylodon 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 P. persicus, and GenBank-sequences of eastern P. gorganensis 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 P. persicus s.l. Humankind has a great responsibility to protect this species and the unique biodiversity of the Hyrcanian Forest ecosystems.

The programmed DNA elimination and formation of micronuclei in germ line cells of the natural hybridogenetic water frog Pelophylax esculentus.

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.

Variability of NOR patterns in European water frogs of different genome composition and ploidy level.

We studied water frogs from a complex composed of two species: Pelophylax lessonae (Camerano, 1882) (genome LL, 2n = 26) and P. ridibundus (Pallas, 1771) (RR, 2 = 26), and their natural hybrid P. esculentus (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: AgNO3, chromomycin A3, PI and fluorescent in situ hybridization with a 28S rDNA probe. Results obtained by silver staining corresponded well with those stained with CMA3, 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 AgNO3 and CMA3 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).

Batrachochytrium dendrobatidis is present in Poland and associated with reduced fitness in wild populations of Pelophylax lessonae.

The fungus Batrachochytrium dendrobatidis (Bd) is a pathogen associated with global declines of amphibians. We used qPCR to detect Bd in 255 samples from 10 Polish populations of 8 species. We found Bd infection in 3 species (Bombina variegata, Pelophylax lessonae, P. esculentus). The infection intensity in P. lessonae reached a maximum of 58400 genomic equivalents of zoospores (GE), and the 2 most heavily infected individuals died. Previous observations of the populations that included infected individuals showed reduced body size, failure to reproduce, and mortalities of adults. These data highlight the importance of emerging diseases, and the need to recognize them as an important factor in conservation of the genus Pelophylax in Poland and Central and Eastern Europe.

Prespermatogenesis and early spermatogenesis in frogs.

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.

Impaired gonadal and somatic development corroborate vulnerability differences to the synthetic estrogen ethinylestradiol among deeply diverged anuran lineages.

Amphibians are undergoing a global decline. One poorly investigated reason could be the pollution of aquatic habitats by endocrine disrupting compounds (EDCs). We tested the susceptibility to the synthetically stabilized estrogen 17α-ethinylestradiol (EE2) in three deeply diverged anuran species, differing in sex determination systems, types of gonadogenesis and larval ecologies. To understand whether data from the amphibian model Xenopus laevis (Pipidae) are analogous and applicable to only distantly related non-model amphibians, tadpoles of X. laevis, Hyla arborea (Hylidae) and Bufo viridis (Bufonidae) were simultaneously exposed to 50, 500 and 5000ng/L EE2 from hatching until completion of metamorphosis, using a flow-through-system under identical experimental conditions. Comparing molecularly established genetic with histologically assessed phenotypic sex in all species, we have recently shown that EE2 provoked numerous genetic-male-to-phenotypic-female sex reversals and mixed sex individuals, confirming overall its expected feminizing effect. In the present study, we focus on the influence of EE2 on gonadal and somatic development. Anatomy and histology revealed several species-specific effects. In both non-model species, H. arborea and B. viridis, high numbers of anatomically impaired gonads were observed. In H. arborea, exposed to 5000ng/L EE2, numerous underdeveloped gonads were detected. Whereas EE2 did not alter snout-to-vent length and body weight of X. laevis metamorphs, H. arborea showed a treatment-dependent decrease, while B. viridis exhibited an increase in body weight and snout-to-vent length. Apart from a concentration-dependent occurrence of yellowish skin color in several H. arborea, no organ-specific effects were detected. Since EE2 ubiquitously occurs in many aquatic ecosystems and affects sexual and somatic development, among EDCs, it may indeed contribute to amphibian decline. The inter-species variation in developmental EE2-effects corroborates species-specific vulnerability differences towards EDCs between deeply diverged amphibian groups.

The plasticizer bisphenol A affects somatic and sexual development, but differently in pipid, hylid and bufonid anurans.

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.

Sex reversal assessments reveal different vulnerability to endocrine disruption between deeply diverged anuran lineages.

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.

Empirical evidence for large X-effects in animals with undifferentiated sex chromosomes.

Reproductive isolation is crucial for the process of speciation to progress. Sex chromosomes have been assigned a key role in driving reproductive isolation but empirical evidence from natural population processes has been restricted to organisms with degenerated sex chromosomes such as mammals and birds. Here we report restricted introgression at sex-linked compared to autosomal markers in a hybrid zone between two incipient species of European tree frog, Hyla arborea and H. orientalis, whose homologous X and Y sex chromosomes are undifferentiated. This large X-effect cannot result from the dominance or faster-X aspects of Haldane's rule, which are specific to degenerated sex chromosomes, but rather supports a role for faster-heterogametic-sex or faster-male evolutionary processes. Our data suggest a prominent contribution of undifferentiated sex chromosomes to speciation.

Sex-chromosome differentiation parallels postglacial range expansion in European tree frogs (Hyla arborea).

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).

Sex chromosome conservation, DMRT1 phylogeny and gonad morphology in diploid Palearctic green toads (Bufo viridis subgroup).

Due to the prevailing sex chromosome homomorphy and large genome size, the knowledge on sex determination systems, sex chromosomes and sex-determining genes in amphibians remains scarce. Using 3 cross-amplifying sex-linked microsatellite markers, we uncover sex determination systems and sex chromosomes in purebred, diploid Palearctic green toads (Bufo viridis subgroup), which had so far only been characterized in laboratory-bred hybrids. Our data support an XY system in B. balearicus, B. viridis and B. variabilis. While females show recombination, it is strongly suppressed (or not detectable) in males. Markers corroborate the largest chromosome pair 1 (homologous to linkage group 1 of Xenopus tropicalis) to represent the sex chromosomes in diploid species of the B. viridis subgroup (B. siculus, B. shaartusiensis, B. balearicus, B. turanensis, B. variabilis, B. viridis, and probably B. boulengeri). This chromosome harbors DMRT1, a key gene of the sexual pathway in deeply divergent animal taxa. However, our phylogenetic analysis of a 600-bp fragment of that gene in diploid green toad taxa reveals that X and Y alleles cluster by species and not by gametolog. This suggests that XY-sequence similarity stems from occasional XY recombination within DMRT1, and we preliminarily reject its role as the master sex determination gene, pending future extension of this evidence to the entire DMRT1 gene. We further create a chain of evidence, which supports the hypothesis that linkage group 1 of X. tropicalis appears to be maintained as the largest chromosome (1), and thus is homologous in anuran karyotype evolution from pipid to hylid, bufonid and ranid anurans.

Gonadal sex differentiation in frogs: how testes become shorter than ovaries.

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.

A stockpile of ova in the grass frog Rana temporaria is established once for the life span. Do ovaries in amphibians and in mammals follow the same evolutionary strategy?

Most anuran amphibians produce high numbers of eggs during several consecutive breeding seasons. The question is still open whether oocytes are formed anew as a result of oogonial proliferation after each spawning or the definitive pool of oocytes is established during the juvenile period and is sufficient for the whole reproductive life span of a female. Our quantitative studies show that primary oogonia in adult female frogs can proliferate, but they fail to differentiate further and do not enter meiosis, and thereby there is no supplementation of new generations of oocytes after each spawning. Ovaries of one-year-old grass frogs contain (median) 53,447 diplotene oocytes, in two-years-old frogs this number decreased to 33,583 and eventually reached 25,679 in virgin mature females. More than 50% decrease in the total oocyte number was accompanied by massive degeneration (atresia) of oocytes. The final number of oocytes in a female forms a stock for 11-12 breeding seasons and exceeds the number of eggs produced during the potential reproductive life span of this species. The phylogenetic context of oocyte recruitment modes in the major clades of vertebrates is discussed in respect to their ability to replenish the stock (a renewable stock in ovaries named "open" vs. a non-renewable stock in ovaries named "closed").

Cryptic diversity among Western Palearctic tree frogs: postglacial range expansion, range limits, and secondary contacts of three European tree frog lineages (Hyla arborea group).

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).

A simplified molecular method for distinguishing among species and ploidy levels in European water frogs (Pelophylax).

Western Palearctic water frogs in the genus Pelophylax are a set of morphologically similar anuran species that form hybridogenetic complexes. Fully reliable identification of species and especially of hybrid ploidy depends on karyological and molecular methods. In central Europe, native water frog populations consist of the Pelophylax esculentus complex, that is, P. lessonae (LL), P. ridibundus (RR) and the hybrid form P. esculentus that can have different karyotypes (RL, LLR and RRL). We developed existing molecular methods further and propose a simple PCR method based on size-differences in the length of the serum albumin intron-1 and the RanaCR1, a non-LTR retrotransposon of the chicken repeat (CR) family. This PCR yields taxon-specific banding patterns that can easily be screened by standard agarose gel electrophoresis and correctly identify species in all of the 160 samples that had been identified to karyotype with other methods. To distinguish ploidy levels in LR, LLR and RRL specimens, we used the ratio of the peak heights of the larger (ridibundus specific) to the smaller (lessonae specific) bands of fluorescently labelled PCR products resolved on a capillary DNA sequencer and obtained a correct assignment of the karyotype in 93% of cases. Our new method will cut down time and expenses drastically for a reliable identification of water frogs of the P. esculentus complex and potentially for identification of other hybridogenetic complexes and/or taxa, and it even serves as a good indicator of the ploidy status of hybrid individuals.