Palearctic Water Frogs Research Articles

Piecing the barcoding puzzle of Palearctic water frogs (Pelophylax) sheds light on amphibian biogeography and global invasions.

Palearctic water frogs (genus Pelophylax) are an outstanding model in ecology and evolution, being widespread, speciose, either threatened or threatening to other species through biological invasions, and capable of siring hybrid offspring that escape the rules of sexual reproduction. Despite half a century of genetic research and hundreds of publications, the diversity, systematics and biogeography of Pelophylax still remain highly confusing, in no small part due to a lack of correspondence between studies. To provide a comprehensive overview, we gathered >13,000 sequences of barcoding genes from >1700 native and introduced localities and built multigene mitochondrial (~17 kb) and nuclear (~10 kb) phylogenies. We mapped all currently recognized taxa and their phylogeographic lineages (>40) to get a grasp on taxonomic issues, cyto-nuclear discordances, the genetic makeup of hybridogenetic hybrids, and the origins of introduced populations. Competing hypotheses for the molecular calibration were evaluated through plausibility tests, implementing a new approach relying on predictions from the anuran speciation continuum. Based on our timetree, we propose a new biogeographic paradigm for the Palearctic since the Paleogene, notably by attributing a prominent role to the dynamics of the Paratethys, a vast paleo-sea that extended over most of Europe. Furthermore, our results show that distinct marsh frog lineages from Eastern Europe, the Balkans, the Near East, and Central Asia (P. ridibundus ssp.) are naturally capable of inducing hybridogenesis with pool frogs (P. lessonae). We identified 14 alien lineages (mostly of P. ridibundus) over ~20 areas of invasions, especially in Western Europe, with genetic signatures disproportionally pointing to the Balkans and Anatolia as the regions of origins, in line with exporting records of the frog leg industry and the stocks of pet sellers. Pelophylax thus emerges as one of the most invasive amphibians worldwide, and deserves much higher conservation concern than currently given by the authorities fighting biological invasions.

New records of the anomaly P syndrome in two water frog species (Pelophylax ridibundus and P. lessonae) in Russia

The “anomaly P” was described in Palearctic water frogs of the genus Pelophylax by Jean Rostand as complex morphological anomalies of water frogs, including polydactyly, brachymely, hind limb oedema, bone outgrowths, spikes, flexions and additional limbs in the inguinal region. In 2016, the anomaly P syndrome was rediscovered in central Russia, confirming the hypothesis concerning its wider distribution. Here, three new records of this syndrome in two species of western Palearctic water frog from Russia are described.

Balancing a Cline by Influx of Migrants: A Genetic Transition in Water Frogs of Eastern Greece

Variation patterns of allozymes and of ND3 haplotypes of mitochondrial DNA reveal a zone of genetic transition among western Palearctic water frogs extending across northeastern Greece and European Turkey. At the western end of the zone, allozymes characteristic of Central European frogs known as Pelophylax ridibundus predominate, whereas at the eastern end, alleles characteristic of western Anatolian water frogs (P. cf. bedriagae) prevail. The ND3 haplotypes reveal 2 major clades, 1 characteristic of Anatolian frogs, the other of European; the European clade itself has distinct eastern and western subclades. Both the 2 major clades and the 2 subclades overlap within the transition zone. Using Bayesian model selection methods, allozyme data suggest considerable immigration into the Nestos River area from eastern and western populations. In contrast, the ND3 data suggest that migration rates are so high among all locations that they form a single panmictic unit; the best model for allozymes is second best for mitochondrial DNA (mtDNA). Nuclear markers (allozymes), which have roughly 4 times as deep a coalescent history as mtDNA data and thus may reflect patterns over a longer time, indicate that eastern and western refugial populations have expanded since deglaciation (in the last 10,000 years) and have met near the Nestos River, whereas the mtDNA with its smaller effective population size has already lost the signal of partitioning into refugia.

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.

Evidence for Integrity of Parental Genomes in the Diploid Hybridogenetic Water Frog Pelophylax esculentus by Genomic in situ Hybridization

The Western Palearctic water frogs Pelophylax ridibundus and P. lessonae were identified as parental (sexual) species and P. esculentus as their interspecific, hybridogenetically reproducing hybrid with hemiclonal heredity. We used genomic in situ hybridization (GISH) to identify parental chromosomes of P.lessonae and P.ridibundus in diploid P. esculentus karyotypes (2n = 26). GISH probes were made by fluorochrome labeling of total genomic DNA extracted from the sexual progenitors. The labeled probe from one species was hybridized to chromosomes of P. esculentus in the presence of excess of unlabeled genomic DNA from the other species. Thus, the P. lessonae probe was blocked by P. ridibundus unlabeled DNA, and vice versa. We successfully discriminated each of the 13 respective parental chromosomes in metaphase complements of the hybrids according to species-specific hybridization signals. GISH enabled us to confirm additional differences between parental chromosomes in size (smaller chromosomes belong to P. lessonae) and in the presence of DAPI-positive centromeric heterochromatin (detected in chromosomes of P. ridibundus, but not in P. lessonae). The fact that no visible intergenomic exchanges were found in metaphase chromosomes of diploid P. esculentus provides important information on the genomic integrity of hemiclonal transmission and supports hybridogenesis as a reproductive mode at the chromosome level for the specimens examined.

Genome Dosage Effect and Hybrid Morphology — The Case of the Hybridogenetic Water Frogs of thePelophylax esculentusComplex

Western Palearctic water frogs offer a unique possibility to study the genome dosage effect (GDE). There are two morphologically distinct species, Pelophylax ridibundus RR and Pelophylax lessonae LL, and their hybridogenetic hybrid Pelophylax esculentus (RL, LLR or RRL). It is supposed that RL have intermediate morphological features, LLR are more similar to P. lessonae, and RRL more similar to P. ridibundus. We tested if the morphology of the water frogs reflects the GDE, and whether it can be used in the field for determination of the genome composition. Mean values of the indices DP/CI, T/CI and F/T followed the order LL-LLR-RL-RRL-RR. After applying discriminant and canonical analyses 89% RR, 95% LL, 91% RL, 84% LLR and 52% RRL were correctly classified. Surprisingly, the L haplotype had bigger influence on morphology than the R haplotype — all hybrid genotypes were morphologically closer to P. lessonae than to P. ridibundus.

Evolution of serum albumin intron-1 is shaped by a 5′ truncated non-long terminal repeat retrotransposon in western Palearctic water frogs (Neobatrachia)

A 5' truncated non-LTR CR1-like retrotransposon, named RanaCR1, was identified in the serum albumin intron-1 (SAI-1) of at least seven species of western Palearctic water frogs (WPWF). Based on sequence similarity of the carboxy-terminal region (CTR) of ORF2 and/or the highly conserved 3' untranslated region (3' UTR), RanaCR1-like elements occur also in the genome of Xenopus tropicalis and Rana temporaria. Unlike other CR1 elements, RanaCR1 contains a CA microsatellite in its 3' UTR. The low nucleotide diversity of the 3' UTR compared to the CTR and to SAI-1 suggests that this region still plays a role in WPWF, either as a structure-stabilizing element, or within a species-specific transcriptional network. Length variation of water frog SAI-1 sequences is caused by deletions that extend in some cases beyond the 5' or 3' ends of RanaCR1, probably a result of selection for structural and functional stability of the primary transcript. The impact of RanaCR1 on SAI-1 evolution is also indicated by the significant negative correlation between the length of both SAI-1 and RanaCR1 and the percentage GC content of RanaCR1. Both SAI-1 and RanaCR1 sequences support the sister group relationship of R. perezi and R. saharica, which are placed in the phylogenetic tree at a basal position, the sister clade to other water frog taxa. It also supports the monophyly of the R. lessonae group; of Anatolian water frogs (R. cf. bedriagae), which are not conspecific with R. bedriagae, and of the European ridibunda group. Within the ridibunda clade, Greek frogs are clearly separated, supporting the hypothesis that Balkan water frogs represent a distinct species. Frogs from Atyrau (Kazakhstan), the type locality of R. ridibunda, were heterozygous for a ridibunda and a cf. bedriagae specific allele.

Genetic diversity in mitochondrial 12S rDNA of western Palearctic water frogs (Anura, Ranidae) and implications for their systematics

The nucleotide sequence of a part of the mitochondrial 12S rRNA gene of eight western Palearctic water frog species was analysed. The results are consistent with the species status of Rana bedriagae, Rana bergeri, Rana epeirotica, Rana lessonae, Rana perezi, Rana ridibunda, Rana saharica and Rana shqiperica. The obtained DNA data suggest that lake frogs from Greece and Yugoslavia on the one hand and lake frogs from Georgia, Uzbekistan and Turkmenistan on the other hand represent two distinct species. However, it is not yet clear whether lake frogs from Georgia, Uzbekistan and Turkmenistan belong to R. ridibunda or represent a new species. The very high similarity between the analysed 12S rDNA segments of German R. ridibunda and R. lessonae confirm the finding that mtDNA of R. lessonae was transmitted into the mitochondrial gene pool of R. ridibunda probably as a result of backcrosses with the hybridogenetic hybrid R. kl. esculenta. The results of parsimony analyses speak in favour of very close phylogenetic relations between R. perezi and R. saharica; with a high probability these species represent an adelphotaxon. Furthermore, the clades (R. lessonae + R. shqiperica + R. bergeri) and (R. ridibunda + R. bedriagae) are considered to be sister groups. According to the mt 12S rDNA data R. epeirotica seems to be more closely related to the supraspecific taxon (R. ridibunda + R. bedriagae) than to (R. lessonae + R. shqiperica + R. bergeri). Thus, it can be excluded that R. shqiperica and R. epeirotica represent sister species.

Microsatellites: A tool for evolutionary genetic studies of western Palearctic water frogs

Genotypic variation at six of 67 microsatellite loci we developed from Rana ridibunda, containing di- or trinucleotide simplesequence repeats, confirms the value of microsatellites as an evolutionary genetic tool for studying western Palearctic water frogs, a model system characterized by clonal reproduction in natural hybrid lineages. R. ridibunda, Rana lessonae. and their hemiclonal hybrid Rana esculenta, which transmits only its ridibunda genome to gametes, are highly variable in central Poland and northern Switzerland: with one exception (a locus that is part of a coding region), each locus was polymorphic in each taxon when scorable, and the number of alleles was high (in Swiss lessonae-esculenta populations, 1-6 per locus, total 13, for ridibunda genomes; 1-9 per locus, total 23, for lessonae genomes; 3 shared by both genomes). Estimated repeat numbers ranged from 3 to 14 for trinucleotide- and from 6 to 27 for dinucleotide-repeats. Nonamplifying (null) alleles, problematic because they underestimate genotypic variability if undetected, occur in ridibunda genomes of Swiss R. esculenta at one locus: apparent lessonae null alleles at another locus were subsequently resolved for several segregating alleles evidenced by bands of much lower intensity. Two loci had multiple ridibunda alleles in Swiss R. esculenta and thus discriminate between hemiclones. with a resolution superior to that of allozymes. Adding these two loci to our protein electrophoretic data (7 loci) more than doubled the number of hemiclones detected, showed that several allozyme-defined hemiclones are composites of multiple microsatellite hemiclones, and revealed clonal diversity in populations that are uniclonal by allozyme data. The extent to which microsatellite-defined hemiclones result from primary hybridizations or from subsequent mutations in ridibunda genomes of R. esculenta lineages remains to be determined. A sample of newly metamorphosed R. ridibunda from hybrid × hybrid matings collected at a Swiss population in the fall had a significant heterozygote excess at the two loci that varied in ridibunda genomes; this shows that most, probably all, of them originated from inter- rather than intrahemiclonal matings, in accord with the idea that the usual hybrid × hybrid inviability in lessonae-esculenta systems is caused by deleterious recessive alleles on the clonally inherited ridibunda genomes. Three of four microsatellite loci tested amplified in most species of western Palearctic water frogs.

Some remarks on current research on western Palearctic water frogs

Zoosystematics and EvolutionVolume 77, Issue 1 p. 3-4 Article Some remarks on current research on western Palearctic water frogs Jörg Plötner, Jörg Plötner Berlin/Marburg(Germany)Search for more papers by this authorDirk Scmeller, Dirk Scmeller Berlin/Marburg(Germany)Search for more papers by this author Jörg Plötner, Jörg Plötner Berlin/Marburg(Germany)Search for more papers by this authorDirk Scmeller, Dirk Scmeller Berlin/Marburg(Germany)Search for more papers by this author First published: 22 April 2008 https://doi.org/10.1002/mmnz.20010770102AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume77, Issue12001Pages 3-4 RelatedInformation

The effect of toe-clipping on survival in metamorphs of western Palearctic water frogs (Anura, Ranidae)

Amputation of one to eight toe was used to mark 1963 metamorphs of western Palearctic water frogs. The metamorphs developed in outdoor rearing areas and their survival was checked after their first hibernation. Survival varied from 70 to 87% (mean 81.2) for particular classes of cut toes, but showed no correlation with the number of clipped toes. No infection or inflammation was observed in cut toes. Metamorphs were also classified by type of toe-clipping, ploidy of progenies, types of crosses, and origin of metamorphs. Furthermore, no correlation between survival and number of toes clipped in any of these subgroupings was found.

Mitochondrial phylogeography of Rana ( Pelophylax) populations in the Eastern Mediterranean region

Phylogenetic relationships in the western fraction of Rana ( Pelophylax) have not been resolved till now, even though several works have been devoted to the subject. Here, we infer phylogenetic relationships among the species distributed in the area of the Eastern Mediterranean, comparing partial mitochondrial DNA sequences for the cytochrome b and 16S rRNA genes. The obtained molecular data clearly indicate that Western Palearctic water frogs underwent a basal radiation into at least 3 major lineages (the perezi, the lessonae, and the rindibunda/bedriagae lineages) advocating an upper Miocene speciation. Moreover, we consider that within the rindibunda/bedriagae lineage, R. ( P.) ridibunda, R. ( P.) epeirotica, R. ( P.) cretensis, R. ( P.) bedriagae, R. ( P.) cerigensis and R. ( P.) kurtmuelleri were differentiated from a common ancestor through a series of vicariant and dispersal events, during the last ∼5 Mya, even though the specific rank of some taxa may be questionable, such as R. cerigensis in respect with R. bedriagae and R. kurtmuelleri in respect with R. ridibunda.

Molecular investigations in western palearctic water frogs

In the green frog hybrids there exists an exclusive cytogenetic reproductive mechanism, referred to as hybridogenesis, whose evolutionary value is still under debate. As a contribution to the knowledge of the possible link between reproductive strategies and phylogenetic relationships, the analysis of repetitive DNA elements in the genomes of the Balkan green frogs, Rana epeirotica and R. shqiperica was carried out to compare their molecular DNA organization with those already known for R. ridibunda and R. lessonae. The Balkan species were chosen, because they give rise to spontaneous, although infrequent and not hybridogenetic hybrids with R. ridibunda. The elements of the RrSl family, a centromeric satellite DNA previously isolated in R. ridibunda, are clustered at centromeric regions also in both Balkan species, although the molecular organization of the RrSl sequences proved to be most similar in R. ridibunda and R. epeirotica. Furthermore, a repetitive DNA family (R1BamHI) is interspersed throughout the genome and is highly conserved in all species examined. Notably, the RIBamHI sequences, correlated to members of the hAT family, represent the first transposable elements found in the genus Rana. In the light of the results obtained so far, further studies are in progress with the aim of isolating and characterizing new families of repeated DNA, as additional results are required to compare the genomes of the different frog species and infer phylogenetic deductions on the genome evolution accompanying the speciation process and occurring inside the Palearctic green frog system.

Palaeoclimatic changes explain Anatolian mountain frog evolution: a test for alternating vicariance and dispersal events.

Holarctic biodiversity has been influenced by climatic fluctuations since the Pliocene. Asia Minor was one of the major corridors for postglacial invasions in the Palearctic. Today this area is characterized by an extraordinarily rich fauna with close affiliation to European, Asian and Indo-African biota. However, exact scenarios of range expansion and contraction are lacking. Using a phylogeographical approach we (i). identify monophyletic lineages among Anatolian mountain frogs and (ii). derive a spatio-temporal hypothesis for the invasion process in Anatolia. We sequenced 540 bp of the mitochondrial 16S rRNA gene from 40 populations of mountain frogs from Anatolia, the Elburz Mountains and the Caucasus. Our samples comprise all known species and subspecies: Rana macrocnemis macrocnemis, R. m. tavasensis, R. m. pseudodalmatina, R. camerani and R. holtzi. They include the type localities of four of these taxa. We used a nested clade analysis (NCA) to infer historical and recurrent events that account for the observed geographical distribution of haplotypes. None of the extant species is monophyletic. Based on a molecular clock calibration using homologous sequences of Western Palearctic water frogs of the same genus, we estimated that a basic radiation into three lineages c. 2 Mya was followed by several dispersal and fragmentation events. The geographical distribution of resident and widespread haplotypes allows us to infer and date scenarios of range expansion and fragmentation that are aligned with dramatic climatic oscillations that have occurred during the last 600000 years. Consequently, Pliocene and Pleistocene climatic oscillations triggered the evolution of Anatolian mountain frogs through an interplay of vicariance and dispersal events.

Climatic oscillations triggered post-Messinian speciation of Western Palearctic brown frogs (Amphibia, Ranidae)

Oscillating glacial cycles over the past 2.4 million years are proposed to have had a major impact on the diversity of contemporary species communities. We used mitochondrial and nuclear DNA sequence data to infer phylogenetic relationships within Western Palearctic brown frogs and to test the influence of Pliocene and Pleistocene climatic changes on their evolution. We sequenced 1976 bp of the mitochondrial genes 16S rRNA and cytochrome b and of the nuclear rhodopsin gene for all current species and subspecies. Based on an established allozyme clock for Western Palearctic water frogs and substitution rate constancy among water frogs and brown frogs, we calibrated a molecular clock for 1425 bp of the 16S and rhodopsin genes. We applied this clock to date speciation events among brown frogs. Western Palearctic brown frogs underwent a basal post-Messinian radiation about 4 million years ago (mya) into five major clades: three monotypic lineages ( Rana dalmatina, Rana latastei, Rana graeca), an Anatolian lineage, and a lineage comprising Rana italica, Rana arvalis, and all Iberian taxa. Polytypic lineages radiated further in concordance with the onset of climatic oscillations ca. 3.2, 2.0, and 1.0–0.6 mya, respectively. The dated fossil record corroborates our paleobiogeographic scenario. We conclude that drastic climatic changes followed by successive temperature oscillations “trapped” most brown frog species in their southern European glacial refugia with enough time to speciate. Substantial dispersal was only possible during extensive interglacial periods of a constant subtropical climate.

Some remarks on current research on western Palearctic water frogs

Some remarks on current research on western Palearctic water frogs

Microsatellites: A tool for evolutionary genetic studies of western Palearctic water frogs

Microsatellites: A tool for evolutionary genetic studies of western Palearctic water frogs

The effect of toe-clipping on survival in metamorphs of western Palearctic water frogs (Anura, Ranidae)

The effect of toe-clipping on survival in metamorphs of western Palearctic water frogs (Anura, Ranidae)

Rana/Pol III: a family of SINE-like sequences in the genomes of western Palearctic water frogs.

The highly repetitive Rana/Pol III family consists of short, tandemly arrayed sequences, scattered throughout the genomes of Palearctic green water frogs. The repeat unit is about 250 bp in length and is a composite element: it contains a SINE-like retroposon with a tRNA structure, flanked by two short direct repeats, and the occurrence of two internal repeats gives evidence that an additional transposition event may have inserted a segment within the already transposed element. Rana/Pol III family is present in the genomes of Rana lessonae, R. ridibunda, and their hybrid form R. esculenta, as well as in R. shqiperica. R. epeirotica, R. cretensis, and the Italian taxon. These sequences are also present in the Iberian R. perezi, although less abundant, but appear to be lacking in the north African species R. saharica. The distribution of Rana/Pol III in the genomes of Palearctic green frogs is in agreement with the phyletic history based on genetic data. The evolutionary pattern proposed for the genus Rana enables us to suppose that the hybridogenetic mechanism is one of the factors accounting for the possible horizontal transfer of Rana/Pol III elements from the central-north Europe species to R. perezi.

Rana/Pol III: A family of SINE-like sequences in the genomes of western Palearctic water frogs

Rana/Pol III: A family of SINE-like sequences in the genomes of western Palearctic water frogs