Crossing experiments reveal gamete contribution into appearance of di-and triploid hybrid frogs in Pelophylax esculentus population systems

Speciation through hybridization is connected with appearance of interspecies hybrids which can survive and reproduce owing to changes in their gametogenesis. In animals, these changes lead to appearance of clonal animals, which for successful reproduction usually depend on parental species and lack of recombination during gamete formation. Polyploidization can resolve these problems and may lead to emergence of new species. Pelophylax esculentus complex (complex of European water frogs) represents one of the appropriate models for studying interspecies hybridization and processes of polyploidization. Hybrid nature of the P. esculentus (RL genotype, 2n=26) was confirmed after crossings of two parental species P. ridibundus (RR genotype, 2n=26) and P. lessonae (LL genotype, 2n=26). Nevertheless absence of one parental species (P. lessonae) and abundance of triploid hybrid frogs (RRL and LLR genotypes, 3n=39) in population systems at the East of Ukraine challenged us to understand how di- and triploid hybrids can appear and prosper in population systems where hybrids exist only with P. ridibundus (R-E type population system). To answer this question we performed cytogenetic analysis of tadpoles appeared after artificial crossing experiments of diploid and triploid hybrids. Moreover, we identified karyotypes transmitted in growing oocytes of females participated in the crossings. Genome composition of mature frogs and tadpoles was established using FISH revealing interstitial (TTAGGG)n repeat sites that differed in two parental species. After crossings of six triploid hybrid females with RRL genotype and one female with LLR genotype with diploid hybrid males and triploid hybrid males with RRL genotype, tadpoles with karyotypes corresponding to P. ridibundus karyotypeappeared.Lampbrushchromosomesobtainedfrom oocytes of all triploid females participated in the crossings were represented by 13 bivalents corresponding to P. ridibundus chromosomes. Analysis of lampbrush chromosomes from oocytes of additional 11 hybrid females with RRL genome composition also revealed oocytes with 13 bivalents corresponding to P. ridibundus chromosomes. We suppose that such oocytes can overcome meiosis and form haploid gametes withP. ridibundusgenome. After crossings oftwo pairs of diploid hybrids we obtained triploid tadpoles with RRL andLLRkaryotypes.Oocytesfromdiploidhybrid females participated in the crossing and four additional diploid hybrid females contained 26 bivalents corresponding to P. ridibundus and P. lessonae chromosomes.Suchoocytespresumablycanformdiploidgametes after meiotic division. One diploid female after crossing with P. ridibundus male produced both P. ridibundus and diploid P. esculentus tadpoles and had oocytes with 26 bivalents corresponding to P. ridibundus and P. lessonae chromosomes. Other six diploid hybrid females had oocytes with 13 bivalents corresponding to P. ridibundus chromosomes. Crossings of seven diploid males with P. ridibundus females or triploid females with RRL genome composition resulted in appearance of tadpoles with karyotypes corresponding to P. ridibundus karyotype. Thus diploid males most probably produced haploid gametes with P. ridibundus genome. We suggest that triploid hybrid frogs cannot reproduce independently from diploid hybrids. In studied population systems, diploid hybrid females are likely to be responsible for appearance of triploid hybrids as well as new diploid hybrids

The effect of phylogeographic history on species boundaries: a comparative framework in Hyla tree frogs.

Because it is indicative of reproductive isolation, the amount of genetic introgression across secondary contact zones is increasingly considered in species delimitation. However, patterns of admixture at range margins can be skewed by the regional dynamics of hybrid zones. In this context, we posit an important role for phylogeographic history: hybrid zones located within glacial refugia (putatively formed during the Late-Pleistocene) should be better defined than those located in post-glacial or introduced ranges (putatively formed during the Holocene and the Anthropocene). We test this hypothesis in a speciation continuum of tree frogs from the Western Palearctic (Hyla), featuring ten identified contacts between species spanning Plio-Pleistocene to Miocene divergences. We review the rich phylogeographic literature of this group and examine the overlooked transition between H. arborea and H. molleri in Western France using a multilocus dataset. Our comparative analysis supports a trend that contacts zones resulting from post-glacial expansions and human translocations feature more extensive introgression than those established within refugial areas. Integrating the biogeographic history of incipient species, i.e. their age since first contact together with their genetic divergence, thus appears timely to draw sound evolutionary and taxonomic inferences from patterns of introgression across hybrid zones

A case of natural triploidy in European diploid green toad (Bufo viridis), with some distributional records of diploid and tetraploid toads

A triploid female (3n = 33) was found in diploid species Bufo viridis from the Crimea Peninsula, Ukraine. The case is recognized as an occasional autotriploidy. No morphological differences were found between this triploid female and other diploid specimens of the sample. Three categories of triploids in the Bufo viridis group are classified. Based on six green toad samples identified by chromosome number, the distribution of diploid and polyploid toads in Kazakhstan is discussed

Mass occurrence of polyploid green frogs (Rana esculenta complex) in Eastern Ukraine

Первое сообщение об обнаружении Северско-Донецкого центра разнообразия зеленых лягуше

Gamete production patterns and mating systems in water frogs of the hybridogenetic Pelophylax esculentus complex in northeastern Ukraine

Hybridization and polyploidy play an important role in animal speciation. European water frogs of the Pelophylax esculentus complex demonstrate unusual genetic phenomena associated with hybridization, clonality and polyploidy which presumably indicate an initial stage of reticulate speciation. The Seversky Donets River drainage in north-eastern Ukraine is inhabited by both sexes of the diploid and triploid hybrid P. esculentus and only one parental species Pelophylax ridibundus. Based on the presence of various types of hybrids, all populations studied can be divided into three geographical groups: I) P. ridibundus—P. esculentus without triploids; II) P. ridibundus—P. esculentus without diploid hybrids; and III) P. ridibundus—P. esculentus with a mixture of diploids and triploids. A study of gametogenesis revealed that diploid P. esculentus in populations of the first type usually produced haploid gametes of P. ridibundus and a mixture of haploid gametes that carried one or another parental genome (hybrid amphispermy). In populations of the second type, hybrids are derived from crosses of P. ridibundus males with triploid hybrid females producing haploid eggs with a genome of P. lessonae. Therefore, we suggest that clonal genome duplication in these eggs might be the result of suppression of second polar body formation or extra precleavage endoreduplication. In populations of the third type, some diploid females can produce diploid gametes. Fertilization of these eggs with haploid sperm can result in triploid hybrids. Other hybrids here produce haploid gametes with one or another parental genome or their mixture giving rise to new diploid hybrids

Cracking cryptic species: external characters to distinguish two recently recognized banded newt species (Ommatotriton ophryticus and O. nesterovi)

Animal science

The Near East as a cradle of biodiversity: a phylogeography of banded newts (genus Ommatotriton) reveals extensive inter- and intraspecific genetic differentiation

The banded newt (genus Ommatotriton) is widely distributed in the Near East (Anatolia, Caucasus and the Levant) - an understudied region from the perspective of phylogeography. The genus is polytypic, but the number of species included and the phylogenetic relationships between them are not settled. We sequenced two mitochondrial and two nuclear DNA markers throughout the range of Ommatotriton. For mtDNA we constructed phylogenetic trees, estimated divergence times using fossil calibration, and investigated changes in effective population size with Bayesian skyline plots and mismatch analyses. For nuDNA we constructed phylogenetic trees and haplotype networks. Species trees were constructed for all markers and nuDNA only. Species distribution models were projected on current and Last Glacial Maximum climate layers. We confirm the presence of three Ommatotriton species: O. nesterovi, O. ophryticus and O. vittatus. These species are genetically distinct and their most recent common ancestor was dated at ∼25Ma (Oligocene). No evidence of recent gene flow between species was found. The species show deep intraspecific genetic divergence, represented by geographically structured clades, with crown nodes of species dated ∼8-13Ma (Miocene to Early Quaternary); evidence of long-term in situ evolution and survival in multiple glacial refugia. While a species tree based on nuDNA suggested a sister species relationship between O. vittatus and O. ophryticus, when mtDNA was included, phylogenetic relationships were unresolved, and we refrain from accepting a particular phylogenetic hypothesis at this stage. While species distribution models suggest reduced and fragmented ranges during the Last Glacial Maximum, we found no evidence for strong population bottlenecks. We discuss our results in the light of other phylogeographic studies from the Near East. Our study underlines the important role of the Near East in generating and sustaining biodiversity

Mass of genes rather than master genes underlie the genomic architecture of amphibian speciation.

The genetic architecture of speciation, i.e., how intrinsic genomic incompatibilities promote reproductive isolation (RI) between diverging lineages, is one of the best-kept secrets of evolution. To directly assess whether incompatibilities arise in a limited set of large-effect speciation genes, or in a multitude of loci, we examined the geographic and genomic landscapes of introgression across the hybrid zones of 41 pairs of frog and toad lineages in the Western Palearctic region. As the divergence between lineages increases, phylogeographic transitions progressively become narrower, and larger parts of the genome resist introgression. This suggests that anuran speciation proceeds through a gradual accumulation of multiple barrier loci scattered across the genome, which ultimately deplete hybrid fitness by intrinsic postzygotic isolation, with behavioral isolation being achieved only at later stages. Moreover, these loci were disproportionately sex linked in one group (Hyla) but not in others (Rana and Bufotes), implying that large X-effects are not necessarily a rule of speciation with undifferentiated sex chromosomes. The highly polygenic nature of RI and the lack of hemizygous X/Z chromosomes could explain why the speciation clock ticks slower in amphibians compared to other vertebrates. The clock-like dynamics of speciation combined with the analytical focus on hybrid zones offer perspectives for more standardized practices of species delimitation

Next-generation phylogeography of the banded newts (Ommatotriton): a phylogenetic hypothesis for three ancient species with geographically restricted interspecific gene flow and deep intraspecific genetic structure

NaturalisAnimal science