A new PCR-RFLP-based method for an easier systematic affiliation of European water frogs

We describe a non‐invasive, PCR‐RFLP‐based method that allows reliable determination of the European water frog species Pelophylax lessonae and Pelophylax ridibundus and the hybrid form Pelophylax esculentus. Maximum‐likelihood analysis of ITS2 sequences revealed two robust monophyletic clades corresponding to water frogs of the P. lessonae and P. ridibundus groups. Three restriction enzymes (KpnI, HaeII, and SmaI) were used to digest three conserved ITS2 domains. Taxonomic identification was unambiguous; the three restriction enzymes gave the same results. A French reference sample was identified using allozyme electrophoresis. Our PCR‐RFLP method confirmed circa 83% of identification of the allozyme method. We conclude that the difference between identifications was caused by introgression.     

Taxonomic composition and ploidy level among European water frogs (Anura: Ranidae: Pelophylax) in eastern Hungary

The western Palaearctic water frogs in the genus Pelophylax comprise several distinct species and three hybridogenetic hybrid forms. In this study, we focus on the Pelophylax esculentus complex, which consists of two sexual species, Pelophylax ridibundus and Pelophylax lessonae, and their hybridogenetic hybrid, Pelophylax esculentus. Specifically, we investigated taxonomic composition and ploidy level of water frogs sampled in three different types of wetland habitats in the Hortobágy National Park (HNP), eastern Hungary. Using variation in serum albumin intron 1 (SAI‐1) and 15 microsatellite loci, we detected the presence of all members of the P. esculentus complex in the studied localities. In one locality, all three taxa occurred syntopically, while in others water frog populations consisted of P. ridibundus and P. esculentus exclusively. The genomic composition of the 63 examined hybrid specimens analysed with microsatellites showed the occurrence of diploid genotypes only. We used a population genetic approach (allelic richness, gene diversity, multilocus genotypes and multilocus disequilibrium) to infer the breeding system of water frogs at HNP. Our data indicate that at least in two populations, hybrids form gametes with clonally transmitted P. ridibundus genome and produce a new hybrid generation by mating with P. lessonae.     

Mode of hybridogenesis and habitat preferences influence population composition of water frogs (Pelophylax esculentus complex,Anura: Ranidae) in a region of sympatric occurrence (western Slovakia)

Coexistence of sperm‐dependent asexual hybrids with their sexual progenitors depends on genetic and ecological interactions between sexual and asexual forms. In this study, we investigate genotypic composition, modes of hybridogenetic gametogenesis and habitat preferences of European water frogs (Pelophylax esculentus complex) in a region of sympatric occurrence. Pelophylax esculentus complex comprises parental species P. ridibundus and P. lessonae, whose primary hybridization leads to hybridogenetic lineages of P. esculentus. Hybrids clonally transmit one parental genome and mate with the other parental species, forming a new generation of hybrids. In the region of western Slovakia, we found syntopic occurrence of diploid and triploid hybrids with P. lessonae, syntopic occurrence of all three taxa as well as the existence of pure P. ridibundus populations. All triploid hybrids were exclusively male possessing one ridibundus and two different lessonae genomes (RLL). Sex ratio in diploid hybrids was substantially female‐biased. Irrespective of the population composition, diploid hybrids excluded the lessonae genome from their germ line and produced ridibundus gametes. Contrarily, RLL males unequivocally eliminated the ridibundus genome and produced diploid lessonae sperms. Perpetuation of RLL males in studied populations is most likely achieved by their mating with diploid hybrid females. The composition of water frog populations is also shaped by taxon‐specific habitat preferences. While P. ridibundus preferred larger water bodies (gravelpits, fishery ponds, dead river arms), P. lessonae was most frequently found in marshes and smaller sandpits. Pelophylax esculentus occupied predominately similar habitats as its sexual host P. lessonae.     

When a clonal genome finds its way back to a sexual species: evidence from ongoing but rare introgression in the hybridogenetic water frog complex

Besides several exceptions, asexual metazoans are usually viewed as ephemeral sinks for genomes, which become ‘frozen’ in clonal lineages after their emergence from ancestral sexual species. Here, we investigated whether and at what rate the asexuals are able to introgress their genomes back into the parental sexual population, thus more or less importantly affecting the gene pools of sexual species. We focused on hybridogenetic hybrids of western Palaearctic water frogs (Pelophylax esculentus), which originate through hybridization between P. ridibundus and P. lessonae, but transmit only clonal ridibundus genome into their gametes. Although usually mating with P. lessonae, P. esculentus may upon mating with P. ridibundus or another hybrid produce sexually reproducing P. ridibundus offspring with the introgressed ex‐clonal genome. We compared the rate of nuclear amplified fragment length polymorphism (AFLP) and mitochondrial introgression in two types of populations, that is, those where P. ridibundus occurs in isolation and those where it lives with the hybridogens. Although significant differentiation (Φpt) between sexual and clonal ridibundus genomes suggested limited gene flow between sexuals and hybridogens, a non‐negligible (~5%) proportion of P. ridibundus bore introgressed mtDNA and AFLP markers. Whereas transfer of mtDNA was exclusively unidirectional, introgression of nuclear markers was bidirectional. The proportion of introgressed P. ridibundus was highest in syntopic populations with P. esculentus, proving an ongoing and site‐specific interspecific genetic transfer mediated by hybridogenetic hybrids. It turns out that asexual hybrids are not just a sink for genes of sexual species, but may significantly influence the genetic architecture of their sexual counterparts.     

Are invasive marsh frogs (Pelophylax ridibundus) replacing the native P. lessonae/P. esculentus hybridogenetic complex in Western Europe? Genetic evidence from a field study

The water-frog L–E system, widespread in Western Europe, comprises the pool frog Pelophylax lessonae and the hybridogenetic edible frog P. esculentus, which originated from hybridization between pool frogs and marsh frogs (P. ridibundus). In P. esculentus, the lessonae (L) genome is eliminated during meiosis and has to be gained anew each generation from a P. lessonae partner, while the ridibundus (R′) genome is transmitted clonally. It therefore accumulates deleterious mutations, so that R′R′ offspring from P. esculentus×P. esculentus crosses are normally unviable. This system is now threatened by invasive P. ridibundus (RR) imported from Eastern Europe and the Balkans. We investigated the genetic interactions between invasive marsh frogs and native water frogs in a Swiss wetland area, and used genetic data collected in the field to validate several components of a recently postulated mechanism of species replacement. We identified neo-ridibundus individuals derived from crosses between invasive ridibundus and native esculentus, as well as newly arisen hybridogenetic esculentus lineages stemming from crosses between invasive ridibundus (RR) and native lessonae (LL). As their ridibundus genomes are likely to carry less deleterious mutations, such lineages are expected to produce viable ridibundus offspring, contributing to species replacement. However, such crosses with invasive ridibundus only occurred at a limited scale; moreover, RR×LL crosses did not induce any introgression from the ridibundus to the lessonae genome. We did not find any ridibundus stemming from crosses between ancient esculentus lineages. Despite several decades of presence on the site, introduced ridibundus individuals only represent 15 % of sampled frogs, and their spatial distribution seems shaped by specific ecological requirements rather than history of colonization. We therefore expect the three taxa to coexist stably in this area.     

Extensive mitochondrial heteroplasmy in hybrid water frog (Pelophylax spp.) populations from Southeast Europe

Water frogs of the genus Pelophylax (previous Rana) species have been much studied in Europe for their outstanding reproductive mechanism in which sympatric hybridization between genetically distinct parental species produces diverse genetic forms of viable hybrid animals. The most common hybrid is P. esculentus that carries the genomes of both parental species, P. ridibundus and P. lessonae, but usually transfers the whole genome of only one parent to its offsprings (hybridogenesis). The evolutionary cost of transfer of the intact genome and hence the hemiclonal reproduction is the depletion of heterozygosity in the hybrid populations. Pelophylax esculentus presents an excellent example of the long‐term sustained hybridization and hemiclonal reproduction in which the effects of the low genetic diversity are balanced through the novel mutations and periodic recombinations. In this study, we analyzed the mitochondrial (mt) and microsatellites DNA variations in hybrid Pelophylax populations from southern parts of the Pannonian Basin and a north–south transect of the Balkan Peninsula, which are home for a variety of Pelophylax genetic lineages. The mtDNA haplotypes found in this study corresponded to P. ridibundus and P. epeiroticus of the Balkan – Anatolian lineage (ridibundus–bedriagae) and to P. lessonae and a divergent lessonae haplotype of the lessonae lineage. The mtDNA genomes showed considerable intraspecific variation and geographic differentiation. The Balkan wide distributed P. ridibundus was found in all studied populations and its nuclear genome, along with either the lessonae or the endemic epeiroticus genome, in all hybrids. An unexpected finding was that the hybrid populations were invariably heteroplasmic, that is, they contained the mtDNA of both parental species. We discussed the possibility that such extensive heteroplasmy is a result of hybridization and it comes from regular leakage of the paternal mtDNA from a sperm of one species that fertilizes eggs of another. In this case, the mechanisms that protect the egg from heterospecific fertilization and further from the presence of sperm mtDNA could become compromised due to their differences and divergence at both, mitochondrial and nuclear DNA. The heteroplasmy once retained in the fertilized egg could be transmitted by hybrid backcrossing to the progeny and maintained in a population over generations. The role of interspecies and heteroplasmic hybrid animals due to their genomic diversity and better fitness compare to the parental species might be of the special importance in adaptations to miscellaneous and isolated environments at the Balkan Peninsula.     

Genetic diversity and distribution patterns of diploid and polyploid hybrid water frog populations (Pelophylax esculentus complex) across Europe

Polyploidization is a rare yet sometimes successful way for animals to rapidly create geno‐ and phenotypes that may colonize new habitats and quickly adapt to environmental changes. In this study, we use water frogs of the Pelophylax esculentus complex, comprising two species (Pelophylax lessonae, genotype LL; Pelophylax ridibundus, RR) and various diploid (LR) and triploid (LLR, LRR) hybrid forms, summarized as P. esculentus, as a model for studying recent hybridization and polyploidization in the context of speciation. Specifically, we compared the geographic distribution and genetic diversity of diploid and triploid hybrids across Europe to understand their origin, maintenance and potential role in hybrid speciation. We found that different hybrid and parental genotypes are not evenly distributed across Europe. Rather, their genetic diversity is structured by latitude and longitude and the presence/absence of parental species but not of triploids. Highest genetic diversity was observed in central and eastern Europe, the lowest in the northwestern parts of Europe. This gradient can be explained by the decrease in genetic diversity during postglacial expansion from southeastern glacial refuge areas. Genealogical relationships calculated on the basis of microsatellite data clearly indicate that hybrids are of multiple origin and include a huge variety of parental genomes. Water frogs in mixed‐ploidy populations without any parental species (i.e. all‐hybrid populations) can be viewed as evolutionary units that may be on their way towards hybrid speciation. Maintenance of such all‐hybrid populations requires a continuous exchange of genomes between diploids and triploids, but scenarios for alternative evolutionary trajectories are discussed.     

Gamete production patterns and mating systems in water frogs of the hybridogenetic Pelophylax esculentus complex in north‐eastern 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.     

Genetic diversity in water frog hybrids (Pelophylax esculentus) varies with population structure and geographic location

Pelophylax esculentus is a hybridogenetic frog originating from matings between P. ridibundus (RR) and P. lessonae (LL). Typically, diploid hybrids (LR) live in sympatry with one of their parental species, upon which they depend for successful reproduction. In parts of their range, however, pure hybrid populations can be found. These hybrid populations have achieved reproductive independence from their parental species by using triploid hybrids (LLR, LRR) rather than LL and RR as their sexual hosts. These different breeding systems also entail differences in reproduction (clonal versus sexual) and hence offer the opportunity to study how genetic diversity is affected by reproductive mode, population structure and geographic location. We investigated 33 populations in the Scania region (South Sweden) and 18 additional populations from Northern and Central Europe. Within both genomes (L, R), genetic variability increases with the potential for recombination and declines from the main species distribution area southeast of the Baltic Sea to the fringe populations northwest of the Baltic Sea. Within the main study area in Scania, genetic diversity is low and decreases from a core area to the periphery. Genetic differentiation between Scania populations is small but significant and best explained by ‘isolation by distance’. Despite the low genetic variability within the discrete genomes, all‐hybrid P. esculentus populations in southern Sweden are apparently not suffering from direct negative fitness effects. This is probably because of its somatic hybrid status, which increases diversity through the combination of genomes from two species.     

Gamete types, sex determination and stable equilibria of all-hybrid populations of diploid and triploid edible frogs (Pelophylax esculentus)

Background  

Triploid individuals often play a key role in speciation by hybridization. An understanding of the gamete types (ploidy and genomic content) and stability of hybrid populations with triploid individuals is therefore of importance for exploring the role of hybridization in evolution. The all-hybrid populations of the edible frog, Pelophylax esculentus, are unique in their composition and genetic dynamics: Diploid (genotype LR) and triploid (LLR and LRR) hybrids depend on each other's different gamete contributions for successful reproduction and maintenance of the populations, as the parental genotypes P. lessonae (LL) and P. ridibundus (RR) are absent among adults. This study provides data and interpretations on gamete types and sex determination that are essential for understanding the function, evolutionary potential and threats of this intriguing system.     

Comparative study of oxidative stress parameters and acetylcholinesterase activity in the liver of Pelophylax esculentus complex frogs

Comparative activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), the phase II biotransformation enzyme glutathione-S-transferase (GST), the concentrations of total glutathione (GSH), sulfhydryl groups (–SH) and the activity of the neurotoxicity biomarker acetylcholinesterase (AChE) were investigated in the livers of species belonging to the Pelophylax esculentus “complex” (parental species Pelophylax ridibundus, Pelophylax lessonae, and their hybrid Pelophylax kl. esculentus) from the wetland, Obedska bara in Serbia. The condition factor (CF) and hepato somatic index (HSI) were also calculated. All three species were caught at same locality and were exposed to the same environmental conditions. Liver SOD activity was lower in P. ridibundus than in P. kl. esculentus and P. lessonae; higher activities of CAT, GR and GST were observed in P. kl. esculentus frogs as compared to their parental species. The activity of GSH-Px was significantly lower in P. kl. esculentus. The activity of AChE was increased in P. lessonae as compared to P. kl. esculentus and P. ridibundus. Similar concentrations of GSH and SH groups were observed in all investigated species. P. kl. esculentus had a higher CF, while the HSI was lower when compared to the parental species. Our findings suggest that the parental species (P. ridibundus and P. lessonae) possess more similar antioxidative responses to environmental conditions than the hybrid species P. kl. esculentus. The obtained results improve our understanding of the biology and physiology of these three closely related species.     

Potential impact of genome exclusion by alien species in the hybridogenetic water frogs (<Emphasis Type="Italic">Pelophylax esculentus</Emphasis> complex)

Globalization and increasing human impact on natural aquatic systems have facilitated the movement of species and the establishment of nonindigenous species enhancing hybridisation opportunities between naturally allopatric species. In this review, we focus on a special case of natural hybrid speciation and the consequences of recent anthropogenic hybridisation in the water frog complex (Pelophylax esculentus complex), which consists of two parental species, Pelophylax lessonae and Pelophylax ridibundus and a hybrid taxon. The hybrid water frogs reproduce hybridogenetically and eliminate the genome of the syntopic water frog species. Although the actual cause triggering chromosome exclusion remains elusive, it has been proposed that chromosome elimination takes place prior to meiosis and may involve enzymatic degradation of the discarded genome. Translocations of water frogs in Western Europe have become frequent the last decade leading to rapid expansion of the range of the marsh frog P. ridibundus. Subsequent hybridisation of the exotic P. ridibundus may dramatically affect the viability and maintenance of hybrid water frog populations throughout Europe. Interestingly, the impact of this introduced species may differ depending on their geographic origin, which defines the ability to induce genome elimination. This may result in fertile or sterile hybrids, making global conservation guidelines challenging. We predict a severe genetic and ecological impact of nonindigenous P. ridibundus prompting for strict conservation measures to reduce species translocations and for studies on the geographic origin of exotic frog species.     

A microsatellite‐based method for genotyping diploid and triploid water frogs of the Rana esculenta hybrid complex

Rana esculenta is a hybrid between Rana lessonae (LL) and Rana ridibunda (RR), and hybrids may be diploid (LR) or triploid (LLR or LRR). Genotypes can be roughly determined from erythrocyte size and morphometry in adult frogs, but accurate genotyping requires more labourious methods. Here I demonstrate that both the L and R genomes have specific microsatellite alleles, and that genotype and ploidy can be accurately inferred from the quantitative ratio of PCR‐amplified (polymerase chain reaction‐amplified) genome‐specific alleles. This method greatly facilitates genotyping in DNA studies of the R. esculenta complex and allows analysis of badly preserved samples and embryos.     

A cryptic invasion within an invasion and widespread introgression in the European water frog complex: consequences of uncontrolled commercial trade and weak international legislation

In Western Europe, many pond owners introduce amphibians for ornamental purposes. Although indigenous amphibians are legally protected in most European countries, retailers are circumventing national and international legislation by selling exotic nonprotected sibling species. We investigated to what extent non‐native species of the European water frog complex (genus Pelophylax) have become established in Belgium, using morphological, mitochondrial and nuclear genetic markers. A survey of 87 sampling sites showed the presence of non‐native water frogs at 47 locations, mostly Marsh frogs (Pelophylax ridibundus). Surprisingly, at least 19% of all these locations also harboured individuals with mitochondrial haplotypes characteristic of Anatolian water frogs (Pelophylax cf. bedriagae). Nuclear genotyping indicated widespread hybridization and introgression between P. ridibundus and P. cf. bedriagae. In addition, water frogs of Turkish origin obtained through a licensed retailer, also contained P. ridibundus and P. cf. bedriagae, with identical haplotypes to the wild Belgian populations. Although P. ridibundus might have invaded Belgium by natural range expansion from neighbouring countries, our results suggest that its invasion was at least partly enhanced by commercial trade, with origins as far as the Middle East. Also the invasion and rapid spread of Anatolian lineages, masked by their high morphological similarity to P. ridibundus, is likely the result of unregulated commercial trade. We expect that Anatolian frogs will further invade the exotic as well as the native range of P. ridibundus and other Pelophylax species elsewhere in Western and Central Europe, with risks of large‐scale hybridization and introgression.     

Regulation of an alternative sigma factor σI by a partner switching mechanism with an anti-sigma factor PrsI and an anti-anti-sigma factor ArsI in Streptomyces coelicolor A3(2)

Natural transfer of mitochondrial DNA has occurred between three western Palaearctic waterfrog taxa: Pelophylax lessonae, Pelophylax ridibundus and their hybridogenetic hybrid, Pelophylax kl. esculentus. The transfer is asymmetric with most P. kl. esculentus and approximately one third of all central European P. ridibundus having mtDNA derived from P. lessonae (L-mtDNA). We obtained complete nucleotide sequences of multiple mitochondrial genomes (15,376-78 bp without control regions) from all 3 taxa, including a P. ridibundus frog with introgressed L-mtDNA. The gene content and organization of the mitogenomes correspond to those typical of neobatrachians. Divergence between the mtDNAs of P. lessonae and P. ridibundus is high with an uncorrected p-distance of 11.9% across the entire mitogenome. However, the rate of nucleotide substitution depends on the degree of functional constraint with up to 30-fold differences in levels of divergence. In general, mitochondrial genes encoding the translational machinery evolve very slowly, whereas genes encoding polypeptides of the electron transport system, especially the ND genes, evolve rapidly. Only 25 of 211-213 observed amino acid replacements could be classified as radical and are therefore more likely to be exposed to selection. A disproportionately high number of amino acid substitutions has occurred in the ND4, ND4L and cytb genes of the P. lessonae lineage (including 36% of all radical changes). In contrast to the interspecific divergence, nucleotide polymorphism within L- and R-mtDNA is very low: L-mtDNA haplotypes differed on average by only 19 nucleotides, while there was no variation within two mtDNAs derived from P. ridibundus. This is an expected finding considering that we have sampled a post-glacial expansion area. Moreover, the introgressed L-mtDNA on a P. ridibundus background differed from other L-mtDNAs by only a few substitutions, indicative of a very recent introgression event. We discuss our findings in the context of natural selection acting on L-mtDNA and its potential significance in cytonuclear epistasis.     

Gamete production patterns,ploidy, and population genetics reveal evolutionary significant units in hybrid water frogs (Pelophylax esculentus)

The European water frog Pelophylax esculentus is a natural hybrid between P. lessonae (genotype LL) and P. ridibundus (RR). It reproduces through hybridogenesis, eliminating one parental genome from its germline and producing gametes containing the genome of the other parental species. According to previous studies, this elimination and transmission pattern is very diverse. In mixed populations, where only diploid hybrids (LR) live in sympatry and mate with one or both parental species, the excluded genome varies among regions, and the remaining genome is transmitted clonally to haploid gametes. In all‐hybrid populations consisting of diploid (LR) and triploid (LLR and/or LRR) frogs, diploid individuals also produce gametes clonally (1n in males, 2n in females), whereas triploids eliminate the genome they have in single copy and produce haploid gametes containing the recombined other genome. However, here, too, regional differences seem to exist, and some triploids have been reported to produce diploid gametes. In order to systematically study such regional and genotype differences in gamete production, their potential origin, and their consequences for the breeding system, we sampled frogs from five populations in three European countries, performed crossing experiments, and investigated the genetic variation through microsatellite analysis. For four populations, one in Poland, two in Germany, and one in Slovakia, our results confirmed the elimination and transmission pattern described above. In one Slovakian population, however, we found a totally different pattern. Here, triploid males (LLR) produce sperm with a clonally transmitted diploid LL genome, rather than a haploid recombined L genome, and LR females clonally produce haploid R eggs, rather than diploid LR eggs. These differences among the populations in gamete production go along with differences in genomotype composition, breeding system (i.e., the way triploids are produced), and genetic variation. These differences are strong evidence for a polyphyletic origin of triploids. Moreover, our findings shed light on the evolutionary potential inherent to the P. esculentus complex, where rare events due to untypical gametogenetic processes can lead to the raise, the perpetuation, and the dispersion of new evolutionary significant lineages which may also deserve special conservation measures.     

Multiple origins of invasive and ‘native’ water frogs (Pelophylax spp.) in Switzerland

The marsh frog (Pelophylax ridibundus) has been introduced in many areas in Central and Western Europe as a result of commercial trade with Eastern Europe, and is rapidly replacing the native pool frog (P. lessonae). A large number of Pelophylax species are distributed in Eastern Europe and the strong phenotypic similarity between these species is rendering their identification hazardous. Consequently, alien populations of Pelophylax might not strictly be composed of P. ridibundus as previously suspected. In the present study, we analysed the cytochrome‐b and NADH dehydrogenase subunit 3 genes of introduced and native Pelophylax species from Switzerland (299 individuals) in order to properly identify the source populations of the invaders and the genetic status of the native species. Our study highlighted the occurrence of several genetic lineages of invasive frogs in western Switzerland. Unexpectedly, we also showed that several populations of the native pool frog (P. lessonae) cluster with the Italian pool frog P. bergeri from central Italy (considered by some authors as a subspecies of P. lessonae). Hence, these populations are probably also the result of introductions, meaning that the number of native P. lessonae populations is fewer than expected in Switzerland. These findings have important implications concerning the conservation of the endemic pool frog populations, as the presence of multiple alien species could strongly affect their long‐term subsistence. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 442–449.     

The ploidy and genetic structure of hybrid populations of water frogs Pelophylax esculentus complex (Amphibia, Ranidae) of Ukraine fauna

The complex study, including allozyme variability and cytometry of hybrid populations of green frogs Pelophylax esculentus (L., 1758) complex has confirmed that the only region of Ukraine where allodiploid are encountered frequently is the Severski Donets basin (9% of all hybrids). In other areas, only two polyploidy hybrids (0.9%) and one probably autopolyploid individual of each parental species have been registered. According to allozyme specters, all three polyploidy hybrids from the Severski Donets basin were males and belonged to biotype P. esculentus (=lessonae) — 2 ridibundus, and their population in this region has halved during the past decade.     

Premeiotic chromosome doubling after genome elimination during spermatogenesis of the species hybrid Rana esculenta

Summary Gamete production in the hybridogenetic species hybrid Rana esculenta (Rana ridibunda X Rana lessonae) is preceded by a premeiotic elimination of the R. lessonae genome and subsequent duplication of the remaining R. ridibunda genome, so that only ridibunda chromosomes enter a quasi normal meiosis, and only ridibunda gametes are formed. This is demonstrated by differences in genome specific centromere fluorescence and electrophoretic patterns between somatic and gonadal tissue.     

Genetic detection of multiple exotic water frog species in Belgium illustrates the need for monitoring and immediate action

The recent genetic screening of water frogs (genus Pelophylax) in Belgium has shown that the invasion of two water frog species from Eastern Europe and the Mediterranean region, P. ridibundus and P. cf. bedriagae is widespread. Possibly other exotic water frogs are invading and establishing themselves through commercial trade. We used a genetic identification approach to rapidly detect and identify morphologically cryptic exotic water frog species in a large number of populations throughout the northern part of Belgium. Among a total of 944 individuals, we found 506 non-indigenous specimens, seven of which belonged to species not recorded before with certainty in Belgium or neighbouring regions in the wild. One of them was identified genetically as the Iberian green frog (P. perezi), but was most likely a P. perezi × P. esculentus hybrid. Six individuals of the Levantine frog (P. bedriagae) were found in a pond in the vicinity of a pet shop where the species is sold. All other exotic frogs belonged to four different haplotypes of P. ridibundus, established in Belgium since c. 1970, and two haplotypes of P. cf. bedriagae, a poorly-known eastern Mediterranean sister species of the latter. Overall, our study underscores the extent of exotic water frog invasions associated with the pet trade. Although two of the exotic species were recorded in small numbers, their early detection is essential with regards to adequate control and eradication of invasive species.