Variation in hybridogenetic hybrid emergence between populations of water frogs from the Pelophylax esculentus complex.

Many closely related species are capable of mating to produce hybrid offspring, which are usually sterile. Nevertheless, altering the gametogenesis of hybrid offspring can rescue hybrids from sterility by enabling asexual reproduction. Hybridogenesis is one of the most complicated asexual reproductive modes, and it includes drastic genome reorganization only in the germline; this is achieved through elimination of one parental genome and duplication of the remaining one to restore diploid chromosomal set and overcome blocks in meiotic progression. We investigated a model of hybridogenesis, namely, water frogs from the Pelophylax esculentus complex, for the emergence of asexual reproduction. Further, we assessed the impact of its asexual reproduction on the maintenance of interspecies hybrids from two populations on the western edge of the P. esculentus range, in which hybrids coexist with either both parental species or with only one parental species. After analysing tadpole karyotypes, we conclude that in both studied populations, the majority of diploid hybrid males produced haploid gametes with the P. ridibundus genome after elimination of the P. lessonae genome. Hybrid females exhibited problems with genome elimination and duplication; they usually produced oocytes with univalents, but there were observations of individual oocytes with 13 bivalents and even 26 bivalents. In some hybrid tadpoles, especially F1 crosses, we observed failed germ cell development, while in tadpoles from backcrosses, germ cells were normally distributed and contained micronuclei. By identifying chromosomes present in micronuclei, we estimated that the majority of tadpoles from all crosses were able to selectively eliminate the P. lessonae chromosomes. According to our results, hybridogenesis in hybrids can appear both from crosses of parental species and crosses between sexual species with hybrid individuals. The ability to eliminate a genome and perform endoreplication to ensure gamete formation differed between male and female hybrids from the studied populations. Some diploid hybrid females can rarely produce not only haploid gametes but also diploid gametes, which is a crucial step in the formation of triploid hybrids.

Fifteen shades of green: The evolution of Bufotes toads revisited.

The radiation of Palearctic green toads (Bufotes) holds great potential to evaluate the role of hybridization in phylogeography at multiple stages along the speciation continuum. With fifteen species representing three ploidy levels, this model system is particularly attractive to examine the causes and consequences of allopolyploidization, a prevalent yet enigmatic pathway towards hybrid speciation. Despite substantial efforts, the evolutionary history of this species complex remains largely blurred by the lack of consistency among the corresponding literature. To get a fresh, comprehensive view on Bufotes phylogeography, here we combined genome-wide multilocus analyses (RAD-seq) with an extensive compilation of mitochondrial, genome size, niche modelling, distribution and phenotypic (bioacoustics, morphometrics, toxin composition) datasets, representing hundreds of populations throughout Eurasia. We provide a fully resolved nuclear phylogeny for Bufotes and highlight exceptional cyto-nuclear discordances characteristic of complete mtDNA replacement (in 20% of species), mitochondrial surfing during post-glacial expansions, and the formation of homoploid hybrid populations. Moreover, we traced the origin of several allopolyploids down to species level, showing that all were exclusively fathered by the West Himalayan B. latastii but mothered by several diploid forms inhabiting Central Asian lowlands, an asymmetry consistent with hypotheses on mate choice and Dobzhansky-Muller incompatibilities. Their intermediate call phenotypes potentially allowed for rapid reproductive isolation, while toxin compositions converged towards the ecologically-closest parent. Across the radiation, we pinpoint a stepwise progression of reproductive isolation through time, with a threshold below which hybridizability is irrespective of divergence (<6My), above which species barely admix and eventually evolve different mating calls (6-10My), or can successfully cross-breed through allopolyploidization (>15My). Finally, we clarified the taxonomy of Bufotes (including genetic analyses of type series) and formally described two new species, B. cypriensis sp. nov. (endemic to Cyprus) and B. perrini sp. nov. (endemic to Central Asia). Embracing the genomic age, our framework marks the advent of a new exciting era for evolutionary research in these iconic amphibians.

Mutual maintenance of di- and triploid Pelophylax esculentus hybrids in R-E systems: results from artificial crossings experiments.

BACKGROUND: Interspecies animal hybrids can employ clonal or hemiclonal reproduction modes where one or all parental genomes are transmitted to the progeny without recombination. Nevertheless, some interspecies hybrids retain strong connection with the parental species needed for successful reproduction. Appearance of polyploid hybrid animals may play an important role in the substitution of parental species and in the speciation process. RESULTS: To establish the mechanisms that enable parental species, diploid and polyploid hybrids coexist we have performed artificial crossing experiments of water frogs of Pelophylax esculentus complex. We identified tadpole karyotypes and oocyte genome composition in all females involved in the crossings. The majority of diploid and triploid hybrid frogs produced oocytes with 13 bivalents leading to haploid gametes with the same genome as parental species hybrids usually coexist with. After fertilization of such gametes only diploid animals appeared. Oocytes with 26 bivalents produced by some diploid hybrid frogs lead to diploid gametes, which give rise to triploid hybrids after fertilization. In gonads of all diploid and triploid hybrid tadpoles we found DAPI-positive micronuclei (nucleus-like bodies) involved in selective genome elimination. Hybrid male and female individuals produced tadpoles with variable karyotype and ploidy even in one crossing owing to gametes with various genome composition. CONCLUSIONS: We propose a model of diploid and triploid hybrid frog reproduction in R-E population systems. Triploid Pelophylax esculentus hybrids can transmit genome of parental species they coexist with by producing haploid gametes with the same genome composition. Triploid hybrids cannot produce triploid individuals after crossings with each other and depend on diploid hybrid females producing diploid eggs. In contrast to other population systems, the majority of diploid and triploid hybrid females unexpectedly produced gametes with the same genome as parental species hybrids coexist with.

Optional Endoreplication and Selective Elimination of Parental Genomes during Oogenesis in Diploid and Triploid Hybrid European Water Frogs.

Incompatibilities between parental genomes decrease viability of interspecific hybrids; however, deviations from canonical gametogenesis such as genome endoreplication and elimination can rescue hybrid organisms. To evaluate frequency and regularity of genome elimination and endoreplication during gametogenesis in hybrid animals with different ploidy, we examined genome composition in oocytes of di- and triploid hybrid frogs of the Pelophylax esculentus complex. Obtained results allowed us to suggest that during oogenesis the endoreplication involves all genomes occurring before the selective genome elimination. We accepted the hypothesis that only elimination of one copied genome occurs premeiotically in most of triploid hybrid females. At the same time, we rejected the hypothesis stating that the genome of parental species hybrid frogs co-exist with is always eliminated during oogenesis in diploid hybrids. Diploid hybrid frogs demonstrate an enlarged frequency of deviations in oogenesis comparatively to triploid hybrids. Typical for hybrid frogs deviations in gametogenesis increase variability of produced gametes and provide a mechanism for appearance of different forms of hybrids.

Cytological maps of lampbrush chromosomes of European water frogs (Pelophylax esculentus complex) from the Eastern Ukraine.

BACKGROUND: Hybridogenesis (hemiclonal inheritance) is a kind of clonal reproduction in which hybrids between parental species are reproduced by crossing with one of the parental species. European water frogs (Pelophylax esculentus complex) represent an appropriate model for studying interspecies hybridization, processes of hemiclonal inheritance and polyploidization. P. esculentus complex consists of two parental species, P. ridibundus (the lake frog) and P. lessonae (the pool frog), and their hybridogenetic hybrid - P. esculentus (the edible frog). Parental and hybrid frogs can reproduce syntopically and form hemiclonal population systems. For studying mechanisms underlying the maintenance of water frog population systems it is required to characterize the karyotypes transmitted in gametes of parental and different hybrid animals of both sexes. RESULTS: In order to obtain an instrument for characterization of oocyte karyotypes in hybrid female frogs, we constructed cytological maps of lampbrush chromosomes from oocytes of both parental species originating in Eastern Ukraine. We further identified certain molecular components of chromosomal marker structures and mapped coilin-rich spheres and granules, chromosome associated nucleoli and special loops accumulating splicing factors. We recorded the dissimilarities between P. ridibundus and P. lessonae lampbrush chromosomes in the length of orthologous chromosomes, number and location of marker structures and interstitial (TTAGGG)n-repeat sites as well as activity of nucleolus organizer. Satellite repeat RrS1 was mapped in centromere regions of lampbrush chromosomes of the both species. Additionally, we discovered transcripts of RrS1 repeat in oocytes of P. ridibundus and P. lessonae. Moreover, G-rich transcripts of telomere repeat were revealed in association with terminal regions of P. ridibundus and P. lessonae lampbrush chromosomes. CONCLUSIONS: The constructed cytological maps of lampbrush chromosomes of P. ridibundus and P. lessonae provide basis to define the type of genome transmitted within individual oocytes of P. esculentus females with different ploidy and from various population systems.