Taxonomic reassessment of Middle Eastern water frogs: Morphological variation among populations considered as Rana ridibunda, R. bedriagae or R. levantina

Eight morphometric features of water frogs of 14 localities in Turkey, Syria, Jordan and Israel were compared with those of Rana ridibunda in Kazakhstan, Armenia and Greece (Thrace). These study sites include the type localities of R. ridibunda , R. r. caralitana , R. esculenta var. bedriagae and R. levantina . Multivariate comparisons (principal-component analysis, discriminant analysis) based on the log₁₀-transformed variables demonstrate that the data set includes only two taxa that differ significantly in size and shape. By applying a morphospecies criterion, R. ridibunda is represented exclusively by the three reference populations, whereas all other populations (in Turkey, Syria, Jordan and Israel) represent the same species , R. bedriagae .     

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.     

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 approximately 5Mya, 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.     

Allozyme variation among Rana balcanica,R. levantina,and R. ridibunda (Amphibia: Anura)1

The allozyme variation among water frogs of the species R. balcanica, R. levantina and R. ridibunda, all formerly considered as one species (R. ridibunda Pallas, 1771), was studied using horizontal starch gel electrophoresis. Blood samples (N # 63) of frogs were collected from five populations in Greece and Israel. Samples (N = 9) of the hybrid frog R. esculenta collected from a locality in Germany were used as an outgroup for phylogenetic analyses. Fifteen enzymes controlled by twenty presumptive loci were identified. Thirteen loci were polymorphic within or among the studied populations. Genetic differentiation among the species was considerably greater than among populations of the same species. Even at Nestos River where R. ridibunda and R. balcanica occur in the same habitats, individuals could be assigned to either species due to characteristic differences of the genotypes (GPI1). This indicates reproductive isolation among these species. The reconstruction of phylogenetic relationships among the three species based on the allozyme data corroborated the model presented on the basis of bioacoustic data: R. ridibunda and R. balcanica (Nei's genetic distance D = 0.0820) are sibling species pertaining to an Eurasian lineage, whereas R. levantina (distance to the European species D = 0.1780 - 0.1955) together with R. perezi represent an independent afroasian lineage.     

Variation of BM224 microsatellite in green frogs of the genus Rana

The variable microsatellite repeat BM224 has been discovered in the genomes of eight species of green frogs (Rana ridibunda, R. cf bedriagae, R. cretensis, R. esculenta, R. lessonae, R. shquiperica, R. saharica, R. nigromaculata). Earlier, this repeat had been observed in members of the genus Bufo. In this paper, a possibility of usage of this genetic marker for species identification is discussed.     

Population genetics of a successful invader: the marsh frog Rana ridibunda in Britain

We investigated the genetic outcome of successful invasion by an alien species, the marsh frog Rana ridibunda, in Britain. Twelve adults translocated from Hungary into Kent (Romney) in 1935 resulted rapidly in a large localized population. A further successful translocation in 1973 from Romney to Sussex (Lewes), together with other range extensions, provided an opportunity to test bottleneck effects during colonization events. Romney and Lewes frogs had similar genetic diversities to those in Hungary at 14 random amplified polymorphic DNA marker (RAPD) and five microsatellite loci. The introduced populations were, however, differentiated genetically from each other and from a reference population in Hungary. Fitness assessments (larval growth and survival) revealed no differences between the Lewes and Romney populations. Despite starting with few founders, significant bottleneck effects on R. ridibunda in Britain were therefore undetectable, presumably because population expansions were rapid immediately after the translocations.     

Variations in <Emphasis Type="Italic">BM224</Emphasis> microsatellite in green frogs of genus <Emphasis Type="Italic">Rana</Emphasis>

The variable microsatellite repeat BM224 has been revealed in the genomes of eight species of green frogs (Rana ridibunda, R. cf. bedriagae, R. cretensis, R. esculenta, R. lessonae, R. shquiperica, R. saharica, R. nigromaculata). Previously, this repeat was observed in members of the genus Bufo. In this paper, the possibility of using this genetic marker for species identification is discussed.     

Widespread unidirectional transfer of mitochondrial DNA: a case in western Palaearctic water frogs

Interspecies transfer of mitochondrial (mt) DNA is a common phenomenon in plants, invertebrates and vertebrates, normally linked with hybridization of closely related species in zones of sympatry or parapatry. In central Europe, in an area north of 48 degrees N latitude and between 8 degrees and 22 degrees E longitude, western Palaearctic water frogs show massive unidirectional introgression of mtDNA: 33.7% of 407 Rana ridibunda possessed mtDNA specific for Rana lessonae. By contrast, no R. lessonae with R. ridibunda mtDNA was observed. That R. ridibunda with introgressed mitochondrial genomes were found exclusively within the range of the hybrid Rana esculenta and that most hybrids had lessonae mtDNA (90.4% of 335 individuals investigated) is evidence that R. esculenta serves as a vehicle for transfer of lessonae mtDNA into R. ridibunda. Such introgression has occurred several times independently. The abundance and wide distribution of individuals with introgressed mitochondrial genomes show that R. lessonae mt genomes work successfully in a R. ridibunda chromosomal background despite their high sequence divergence from R. ridibunda mtDNAs (14.2-15.2% in the ND2/ND3 genes). Greater effectiveness of enzymes encoded by R. lessonae mtDNA may be advantageous to individuals of R. ridibunda and probably R. esculenta in the northern parts of their ranges.     

Taxonomic relationships among Turkish water frogs as revealed by phylogenetic analyses using mtDNA gene sequences

We assessed taxonomic relationships among Turkish water frogs through estimation of phylogenetic relationships among 62 adult specimens from 44 distinct populations inhabiting seven main geographical regions of Turkey using 2897 bp sequences of the mitochondrial Cytb, 12S rRNA and 16S rRNA genes with equally-weighted parsimony, likelihood, and Bayesian methods of inference. Monophyletic clade (Clade A) of the northwesternmost (Thrace) samples is identified as Pelophylax ridibundus. The other clade (Clade B) consisted of two monophyletic subclades. One of these contains specimens from southernmost populations that are regarded as an unnamed species. The other subclade consists of two lineages, of which one corresponds to P. caralitanus and another to P. bedriagae. Taxonomic relationships of these two species are discussed and recognition of P. caralitanus as a subspecies of P. bedriagae is proposed.     

Bioacoustic assessment of the taxonomic status of pool frog populations (Rana lessonae) with reference to a topotypical population

Six structural and temporal features of the mating calls of pool frogs recorded at eight localities in Italy, Germany, Yugoslavia and Albania were compared with those of the calls of topotypical Rana lessonae in northern Italy. All populations shared three out of six characters studied: call duration, intercall interval and call period. The pool frogs of three localities in peninsular Italy significantly differed in three characters from those of the other six localities, including the northern Italian one: pulses per pulse group, pulse groups per call, and pulse groups per second. This study demonstrates the existence of Rana bergeri , a pool frog species different from R. lessonae in peninsular Italy, but does not support the species status of the chemotaxonomically established R. shqiperica , which is considered as a synonym of R. lessonae .     

Variations of molar morphology in the Spalax ehrenbergi superspecies: adaptive and phylogenetic significance

This study compares and contrasts variations of enamel fold pattern on the crowns of the molars of subterranean mole rats ( Spalax ) belonging to two superspecies, S. ehrenbergi and S. leucodon , and involving about 20 chromosomal species, distributed parapatrically and ranging on different soil types. The sample studied involved 397 skulls, subdivided as follows: 280 of S. ehrenbergi from Israel, representing the four chromosomal species 2n = 52, 54, 58 and 60; 59 of S. ehrenbergi from Egypt, Lebanon, Syria, Jordan and Turkey; 58 of S. leucodon from Turkey.
In making the comparisons it was necessary to take into account the marked changes of molar pattern that take place as a result of wear.
There is a reasonably good agreement between differences of molar pattern and genetic and immunological distances. The species in Israel and Egypt form a compact group, clearly differentiated from Turkish leucodon; ehrenbergi from Turkey and Syria are intermediate, in accordance with their geographical distribution.
In Israel, differences associated with soil type are at about the same level as differences between species. Local populations show a higher level of diversity, and in a number of cases neighbouring populations differed significantly, sometimes over very short distances. Population differences within species are generally associated with differences of soil and vegetation, indicating the influence of natural selection. There are also differences across species boundaries, implying a phylogenetic (heritage) effect. The nature of the adaptive link between molar pattern and soil is unknown: possible factors are contamination of the food by soil, and toughness of the food which necessitates changes of enamel fold length to improve grinding efficiency.     

Male advertisement call characters as phylogeographical indicators in European water frogs

Anuran vocalizations are valuable in the determination of species identity and have the potential to discriminate intraspecific variation. We developed novel bioacoustic sampling techniques, based on Fast Fourier Transforms, to increase the precision and sensitivity of male advertisement call analysis and applied the method to European water frogs. This approach robustly separated the three types of north European water frogs (Rana ridibunda, R. lessonae and their viable, fertile hybrid R. esculenta) by their call subunit characteristics. The hybrid frog exhibited a high frequency call component absent from both the parental species. Furthermore, call analysis demonstrated significant intraspecific differences among populations of all three frog types. Call characters of R. ridibunda changed systematically as a function of longitude. This trend may reflect either clinal variation in selection pressures across Europe, or the consequences of drift following postglacial colonization from eastern refugia. High resolution vocalization analysis therefore provides a potentially useful method for investigating intraspecific differentiation and the phylogeographical origins of anuran distributions. © 2002 The Linnean Society of London. Biological Journal of the Linnean Society, 2002, 77 , 355?365.     

Hemiclonal reproduction slows down the speed of Muller's ratchet in the hybridogenetic frog Rana esculenta

Rare recombination in otherwise asexually reproducing organisms is known to beneficially influence the fitness in small populations. In most of the investigated organisms, asexual and rare sexual generations with recombination follow each other sequentially. Here we present a case where clonal reproduction and rare recombination occur simultaneously in the same population. The hybridogenetic water frog Rana esculenta (E), a hybrid between R. lessonae (L) and R. ridibunda (R) produces gametes that only contain the unaltered maternal R part of their genome. New generations of R. esculenta usually arise from E x L matings. Intraspecific E x E matings produce mostly inviable offspring, but in rare cases, female R. ridibunda arise from such matings which are capable of recombination. In the absence of conspecific males, these R females have to mate with E males, which results in further R females, or with L males, which produces new E lineages. This indirect mechanism reintroduces recombination into the otherwise clonally transmitted R genomes in R. esculenta populations. In this study, we show through Monte Carlo simulations that, in most cases, it is sufficient that only between 1 % and 10 % of mixed water frog populations consist of R females to prevent or significantly reduce the fixation and accumulation of deleterious mutations.     

The water frogs of Greece: Bioacoustic evidence for a new species

The structure of the mating call of lake frogs (referred to as R. ridibunda) from 16 populations in Greece was analyzed for local variation using multivariate statistics. The populations of Thrace and of the island of Samothraki form a group giving the same type of mating call, whereas the mating call of the other populations differs in the degree of temperature dependence of four parameters, and specifically in the number of pulses/pulse group and pulse groups/call. Discriminant functions distinguish even single call series with a probability of 97%, intermediate mating calls are absent, and there is a significant, but slight differentiation of external morphological characters. These results have strong taxonomic implications. We conclude that the lake frogs of Greece comprise two species. The mating call of the lake frogs from Thrace resembles in all parameters that of the Rana ridibunda in the terra typica restricta (Guryev, CIS). Accordingly, the lake frogs of eastern Greece belong to R. ridibunda. The mating call of these lake frogs consists of 20 pulses/pulse group and of 7 pulse groups/call on the average. Most of Greece is inhabited by the second taxon, Rana balcanica sp. n. Its mating call is characterized by 27 pulses/pulse group and 4 pulse groups/call on the average. The two species in Greece do not differ with respect to coloration and size, but several standardized indices vary significantly: body length/digitus primus length; body length/callus internus length; body length/snout-eye distance; body length/tympanum diameter; tibia length/callus internus length; maximal head width/snout-eye distance.     

Hemiclone diversity in the hybridogenetic frog Rana esculenta outside the area of clone formation: the view from protein electrophoresis

European water frog hybrids Rana esculenta reproduce hemiclonally, by hybridogenesis: In the germ line they exclude the genome of the parental species Rana lessonae and produce haploid, unrecombined gametes with a genome of the parental species Rana ridibunda . These hybrids coexist with and depend as sexual parasites on the host parental species R. lessonae (the L-E population system); matings with R. lessonae restore somatic hybridity in each generation of R. esculenta . We investigated 15 L-E system populations in northern Switzerland, which is outside R. ridibunda 's native range. Frequency of hybrids in samples varied from 8% in marsh ponds to 100% in gravel pits and forest ponds. Clonal diversity (variation among R. ridibunda genomes of hybrids), detected by six protein electrophoretic marker loci, revealed a total of eight hemiclones and locally ranged from uniclonal populations in southern parts of the survey region to six coexisting hemiclones in the north. All alleles distinguishing hemiclones occur commonly in the nearest native R. ridibunda populations of east-central Europe; the most probable source of clonal diversity in our samples is multiple clone formation by primary hybridizations in the sympatry area of R. ridibunda and R. lessonae and subsequent dispersal of hemiclonal lineages. A positive correlation between amount of clonal diversity and hybrid frequency, predicted by the Frozen Niche Variation (FNV) model (each hemiclone is characterized by a relatively narrow niche, coexistence is possible through niche partitioning), was not found; this contrasts with hemiclonally reproducing fish hybrids ( Poeciliopsis ). Historical factors, such as availability of different colonizing hemiclones may be strong enough to override the signal from operation of the FNV.     

Additional records of the Ring-necked Parakeet,Psittacula krameri,from Egypt and the Middle East

Abstract

Additional new records and references from the literature are given for the Ring-nek-ked Parakeet, Psittacula krameri, in Egypt, Jordan, Israel and Syria. The species only succeeds in surviving in areas where there is little pressure from hunting, for example the city area of Cairo and several localities in Israel.     

Deleterious alleles and differential viability in progeny of natural hemiclonal frogs

Abstract.-Spontaneous deleterious mutations are expected to accumulate through Muller's ratchet in clonally reproducing organisms and may lead to their extinction. We study deleterious mutations and their effects in a system of European frogs. Rana esculenta (RL), natural hybrids R. ridibunda (RR) X R. lessonae (LL), reproduce hemiclonally; both sexes exclude the L genome in the germ line and produce unrecombined R gametes; hybridity is restored each generation by matings of RL with coexisting LL. Different allozyme-defined hybrid hemiclones (R genome haplotypes) are thought to have originated independently from primary hybridizations RR x LL. Natural matings between two hybrids usually lead to inviable RR tadpoles. This inviability is thought to result from unmasked deleterious alleles on the clonally transmitted R genomes. Most simply it reflects homozygosity for recessive deleterious alleles at particular loci; alternatively (consistent with absence of RR adults in multiclonal populations) it may reflect hemiclone-specific sets of incompletely recessive deleterious mutations that cumulatively cause inviability when two such genomes are combined. If inviability results from the former, progeny of two hybrids of different hemiclones, whether allopatric or coexisting, should be viable, because it is improbable that their R genomes share recessive deleterious alleles at the same set of loci; if inviability results from the latter, progeny of hybrids of different hemiclones should be inviable, especially when hybrid lineages are old. We tested these hypotheses in artificial crosses, using frogs from three regions: hemiclonal hybrids outside R. ridibunda's range from northern Switzerland (two abundant coexisting allozyme-defined hemiclones; estimated lineage age < or = 5,000 generations) and from Sicily, Italy (one hemiclone; estimated age > or = 25,000 generations) and R. ridibunda from Poland. We generated RR progeny, which we reared under benign conditions in the laboratory, by crossing (1) two hybrids from the same region (H x H local); (2) two hybrids from different regions (H X H foreign); (3) hybrids and R. ridibunda (H X R); and (4) two R. ridibunda (R X R). Survival to metamorphosis was similar and high for R x R, H X H foreign, and H X R, whereas all tadpoles of H X H local died before metamorphosis. This supports the hypothesis that homozygosity for recessive deleterious mutations at particular loci causes inviability. Crosses within and between the two coexisting hemiclones from Switzerland were, however, equally inviable. This result may reflect episodic sexual recombination in RR progeny from exceptional successful interclonal hybrid X hybrid matings, followed by matings of such RR with LL. This process would both slow down or halt Muller's ratchet and disrupt genetic independence of coexisting hemiclones, so that the same remaining deleterious R alleles could exist in different allozyme-defined hemiclones. Whereas all data are consistent with the prediction of Muller's ratchet operating on clonally transmitted R genomes of natural hybrid lineages, they are insufficient to demonstrate such operation, because deleterious recessives that mutated after clone formation and those that preexisted in the R. ridibunda source populations that formed the hemiclonal lineages are not distinguished. The possibility of episodic sexual recombination must be carefully taken into account when studying Muller's ratchet in natural populations of this Rana system.     

Reabsorption of water and electrolytes in the urinary bladder of intact frogs (genus Rana).

1. Water and electrolyte reabsorption of the urinary bladder epithelia has been studied in intact, fully hydrated frogs (Rana temporaria, R. lessonae, R. ridibunda). 2. The rates of water reabsorption were lower in frogs on wet soil than in those on dry soil and related to the degree of terrestrialism: R. temporaria greater than R. lessonae greater than R. ridibunda. 3. Samples of urine stored up to 24 hr within the urinary bladder were analysed for osmolality and the concentration of urea, ammonia, sodium, potassium, magnesium and calcium. 4. Selective reabsorption of sodium was detected in all species, that of calcium only in R. ridibunda. The efficiency of electrolyte reabsorption was also related to the degree of terrestrialism. 5. In conclusion, in fully hydrated frogs reabsorption by the bladder epithelia contributes significantly to the water and electrolyte conservation.     

Frog alien species: a way for genetic invasion?

European water frogs are characterized by anthropic introductions and Rana ridibunda may be considered as an invasive species. As such translocations may result in introgression of exotic genes in native populations, i.e. genetic pollution, we studied genetic characteristics (on 11 allozymic loci) of natural versus introduced water frogs. Our study contributed to (1) disclose 3 genetic markers allowing the identification of exotic frogs; (2) quantify the proportion of exotic frogs found in natural populations; and (3) suggest how genetic pollution may arise in these frogs.     

Phylogeographic patterns of genetic diversity in eastern Mediterranean water frogs have been determined by geological processes and climate change in the Late Cenozoic

AIM: Our aims were to assess the phylogeographic patterns of genetic diversity in eastern Mediterranean water frogs and to estimate divergence times using different geological scenarios. We related divergence times to past geological events and discuss the relevance of our data for the systematics of eastern Mediterranean water frogs. LOCATION: The eastern Mediterranean region. METHODS: Genetic diversity and divergence were calculated using sequences of two protein-coding mitochondrial (mt) genes: ND2 (1038 bp, 119 sequences) and ND3 (340 bp, 612 sequences). Divergence times were estimated in a Bayesian framework under four geological scenarios representing alternative possible geological histories for the eastern Mediterranean. We then compared the different scenarios using Bayes factors and additional geological data. RESULTS: Extensive genetic diversity in mtDNA divides eastern Mediterranean water frogs into six main haplogroups (MHG). Three MHGs were identified on the Anatolian mainland; the most widespread MHG with the highest diversity is distributed from western Anatolia to the northern shore of the Caspian Sea, including the type locality of Pelophylax ridibundus. The other two Anatolian MHGs are restricted to south-eastern Turkey, occupying localities west and east of the Amanos mountain range. One of the remaining three MHGs is restricted to Cyprus; a second to the Levant; the third was found in the distribution area of European lake frogs (P. ridibundus group), including the Balkans. MAIN CONCLUSIONS: Based on geological evidence and estimates of genetic divergence we hypothesize that the water frogs of Cyprus have been isolated from the Anatolian mainland populations since the end of the Messinian salinity crisis (MSC), i.e. since c. 5.5-5.3 Ma, while our divergence time estimates indicate that the isolation of Crete from the mainland populations (Peloponnese, Anatolia) most likely pre-dates the MSC. The observed rates of divergence imply a time window of c. 1.6-1.1 million years for diversification of the largest Anatolian MHG; divergence between the two other Anatolian MHGs may have begun about 3.0 Ma, apparently as a result of uplift of the Amanos Mountains. Our mtDNA data suggest that the Anatolian water frogs and frogs from Cyprus represent several undescribed species.