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 .     

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.     

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

Temporal characteristics of advertisement calls of water frogs of 10 localities in Turkey, Syria, Israel and Egypt were compared with those of Rana ridibunda in Kazakhstan, Armenia and Greece (Thrace) as reference populations. These study sites include the type localities of R. ridibunda , R. r. caralitana , R. esculenta var. bedriagae und R. levantina . The temperature-dependent as well as the temperature-independent call parameters clearly revealed two species. R. ridibunda is represented by the three reference populations and, in addition, a population in central Turkey. The other populations (in Turkey, Syria, Israel and Egypt) represent R. bedriagae . R. bedriagae is the oldest available name for water frogs of these regions and was given priority over R. r. caralitana and R. levantina .     

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.     

Mating calls of water frogs (Ranidae) from Lake Skutari,Yugoslavia, and the relationship to water frogs of other regions1

According to the results of a bioacoustic analysis of the mating calls recorded at the northern end of Lake Skutari, Yugoslavia, this region is inhabited by three water frog phenotypes: Rana ridibunda, R. lessonae and hybrids of these two species. The mating call of R. ridibunda consists of an average of 3.55 pulse groups at all water temperatures. Other parameters of the call, however, are greatly influenced by temperature. The conclusion that the second phenotype is the species R. lessonae was based on the characteristics of the mating call. At a water temperature of 20°C, a call consists of 29 pulse groups repeated at a rate of 24 Hz. Analysis revealed almost complete agreement between this mating call and those of Central and Eastern European R. lessonae. The systematic implications of this finding are discussed. The mating calls of the hybrids of R. ridibunda and R. lessonae are intermediate between those of the parents.     

Vocal response of male European water frogs (Rana Esculenta complex) to mating and territorial calls

The responses of male European water frogs (the two species Rana lessonae and Rana ridibunda and their hybrid, Rana esculenta) to playback of their mating and territorial calls were studied during the mating season.In order to select biologically relevant intensities for the presentation of the recorded calls, the sound pressure of the calls produced by the frogs themselves was established prior to the experiment. At a distance of 1 m the most intense calls were those of R. ridibunda, with a sound pressure of 110 dB (peak SPL). The smaller males of R. esculenta gave calls about 5 dB lower in intensity. The calls of R. lessonae, the smallest phenotype, were still less intense, 10 dB lower than those of R. ridibunda.The territorial calls of all three phenotypes elicited territorial calls in all of the males tested, as a rule accompanied by approach to the sound source. The sound pressure required to elicit a vocal response was nearly the same for each of the three different territorial calls. Sometimes encounter calls and warning calls were given in addition to territorial calls.When the mating calls were presented at low intensity, in some cases the males responded with their own mating calls. Mating calls at higher intensity elicited the same behavior that appeared following presentation of territorial calls, but significantly higher sound pressures were required to elicit such a response to mating calls than to territorial calls. The males of R. lessonae and R. esculenta did not respond to the mating calls of R. ridibunda, and each of them had significantly lower thresholds to the mating call of its own phenotype than to that of the other. The males of R. ridibunda responded only to conspecific mating calls.The vocal-response thresholds are compared with those of the electrodermal response reacting to the same stimuli. The significance of the different calls of the European water frogs is discussed.     

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 and ecological relationships of marsh frog populations in Israel

Summary Allozymic variation in proteins encoded by 28 loci was analyzed electrophoretically in 340 mostly adult specimens representing 11 populations, 8 central and 3 isolated, of aquatic marsh frogs, Rana ridibunda in Israel, along a north-south transect of generally increasing aridity. In addition, geographic variation in 3 morphological variables of 144 frogs and in vertebral stripe color polymorphism of 262 frogs were also studied. The results indicate that. (a) Of the 28 loci examined, 12 (= 43%) are largely monomorphic in all populations; out of the remaining loci, 6 were locally and weakly polymorphic and 10 regionally and strongly polymorphic. (b) No fixation of alternative alleles was found in any of the 28 loci and 11 populations studied. The commonest allele predominated across all populations, central as well as isolates, (c) Clinal patterns associated with increasing aridity southwards and eastwards occurred in polymorphism, P; heterozygosity, H; and in allele frequencies of Esterase-1, Xanthine dehydrogenase, Aldehyde oxidase and Albumin. (d) In the 3 estimates of genie variation, mean number of alleles per locus, A, mean proportion of polymorphic loci per population, P, and heterozygous loci per individual, H, marsh frogs displayed average estimates of genetic variation. The 3 estimates were: A =1.14 (range, 1.18–1.57); P = 0.33 (range, 0.14–0.54): H = 0.069 (range, 0.032–0.094). (e) Central populations harbored distinctly more genic variation than isolated populations. (f) Genic similarity between populations was high. (g) Significant deviations from Hardy-Weinberg equilibrium were found in 8 out of 11 populations involving 8 loci, (h) P, H, and allozymic variation in several gene loci were significantly correlated and predictable by environmental variables, primarily those related to water and temperature. (i) A significant amount of morphological variation was found between localities for body length, foot length, and weight in both sexes. Body weight in females was negatively correlated with temperature; and all three morphological variables in females were predicted significantly by a combination of temperature and humidity. (j) The three vertebral stripe color phenotypes, gray, green and red occurred in the following frequencies: 0.59, 0.24, 0.17, respectively. The red morph increased clinally southwards and was significantly correlated with most temperature and water variables. The geographic variation in both the green and red morphs was predicted significantly by climatic variables, both colors blending with local substrates.The spatial patterns and environmental correlates of genetic and morphological variation in Rana ridibunda in Israel suggest that (i) protein polymorphisms are at least partly adaptive and that part is moulded by natural selection rather than by stochastic processes or neutrality; (ii) the environmental variation model seems to be a good predictor of genetic variation in marsh frogs; (iii) body size varies adaptively, presumably determined primarily through thermoregulation; (iv) the spatial pattern of the color polymorphism seems to be adaptively selected by at least two factors: visual predation and climatic determinants.     

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.     

Variation in Male Calling Behaviour and Relation to Male Mating Success in the Strawberry Poison Frog (Dendrobates pumilio)

Calling behaviour and the characteristics of the males’ call are important for mate attraction, female mate choice and male mating success in anurans. In this study I investigated the variation in calling activity and the variation in one spectral (dominant frequency) and four temporal properties (call rate, call duration, duty cycle and pulse rate) of the advertisement call of strawberry poison frogs, Dendrobates pumilio, in two Costa Rican populations during two study periods. Regarding all call properties, no differences were found between populations or years, but between‐male variation was significantly higher than within‐recording and within‐male variation. Dominant frequency was less variable within and among males than temporal call properties and I classified the former as static and the latter as intermediate properties of the call. No call property seemed to be strongly affected by body length or weight. Moreover, I used behavioural observations to relate male calling behaviour to mating success. Calling activity was more variable between males and accounted for more variability in mating success in multiple regression models than any call property. Some call properties (call rate, dominant frequency and pulse rate) were also related to male mating success in one or two study periods. These call properties are probably important for females during courtship to assess their prospective mate and/or for competitive male–male interactions. Furthermore, I compared call properties and mating success of some males for which data were available for both study periods. Pulse rate was lower in the second than in the first year, suggesting age‐related changes in this call property. Moreover, male D. pumilio increased their relative mating success from the first to the second study year. Together with other studies, the data of this study suggest that, in anurans, endurance rivalry contributes more to variation in male mating success than does female choice based on male characteristics.     

Patterns of protein synthesis in oocytes and early embryos of Rana esculenta complex

Summary We have used isotopic labelling and both one-and two-dimensional electrophoretic procedures to analyse the protien synthesis patterns in oocytes and early embryos of three phenotypes of the European green frogs. The results demonstrated that protein patterns of Rana ridibunda and R. esculenta are identical, but that they differ from those of R. lessonae. Progeny of the lethal cross R. esculenta × R. esculenta showed a distinct delay in the appearance of stage-specific proteins during early embryogenesis. The heat-shock response of R. ridibunda and R. esculenta oocytes was found to be identical, but different from that of Xenopus laevis. The implications of these findings, with respect to hybridogenesis in R. esculenta complex and variations in the regulations of heat shock genes in different amphibian species, are discussed.     

A genetic mechanism of species replacement in European waterfrogs?

Introduced Rana ridibunda currentlyreplace the native waterfrogs R. lessonaeand R. esculenta in several areas ofcentral Europe. The unusual reproductive systemin waterfrogs of the Rana esculentacomplex suggests that this replacement may bedriven by a genetic mechanism: Ranaesculenta, a hybrid between R. ridibundaand R. lessonae, eliminates the lessonae genome from the germline and clonallytransmits the ridibunda genome(hybridogenesis). Hybrids form mixedpopulations with R. lessonae (L-E-system)in which they persist by backcrossing with theparental species. Matings between hybrids areunsuccessful, because their ridibundagenomes contain fixed recessive deleteriousmutations. When introduced into a L-E-system,R. ridibunda can mate with both nativetaxa, producing R. ridibunda offspringwith R. esculenta, and R. esculentaoffspring with R. lessonae (primaryhybridizations). If primary hybrids arehybridogenetic, they produce viable R.ridibunda offspring in matings with otherhybrids, because their clonal genomes areunlikely to share the deleterious allelespresent in the ancient clones. Thus, R.ridibunda will increase in the population atthe expense of both native taxa, eventuallyleaving a pure R. ridibunda population.We provide three lines of evidence for thisprocess from a currently invaded population inSwitzerland: (1) Primary hybridizations takeplace, as roughly 10% of hybrids in thepopulation possess ridibunda genomesderived from the introduced frogs. (2)Hybridogenesis occurs in primary hybrids,although at a low frequency. (3) Many hybrid ×hybrid matings in the population indeed produceviable offspring. Hence, the proposed geneticmechanism appears to contribute to the speciesreplacement, although its importance may belimited.     

Distribution and habitat use of water frog hybrid complexes in France

Hybrid zones are either distributed along clines or in a mosaic of patches. This distribution may depend upon variation in taxon habitat use. Habitat use and distribution of diverse taxa of water frogs (Rana ridibunda, R. lessonae, R. perezi, R. kl. grafi and R. kl. esculenta) in France are analysed to determine whether water frog complexes conform to the mosaic or clinal model. Biogeographical scenarios may be invoked in order to explain the distribution of water frogs. However, the distribution of R. perezi and R. kl. grafi, being restricted to regions characterized by Mediterranean or Oceanic climatic conditions, suggests that these frogs do not endure cold winters. R. ridibunda is widespread in Southern France and its distribution suggests multiple introductions. It is concluded that water frogs conform to the mosaic zone model rather than to the tension zone model because: (i) taxa exhibited differences in habitat use, (ii) pure parental species were documented and (iii) hybrids are not unfit relative to parental species.     

Glycolytic adjustments in tissues of frog Rana ridibunda and land snail Helix lucorum during seasonal hibernation

The present work aimed to contribute to the understanding of the adaptation of the glycolytic pathway in tissues of frog Rana ridibunda and land snail species Helix lucorum during seasonal hibernation. Moreover responses of glycolytic enzymes from cold acclimated R. ridibunda and H. lucorum were studied as well. The drop in Po₂ in the blood of hibernated frogs and land snails indicated lower oxygen consumption and a decrease in their metabolic rate. The activities of glycolytic enzymes indicated that hibernation had a differential effect on the glycolyis in the two species studied and also in the tissues of the same species. The activity of l-LDH decreased significantly in the skeletal muscle and heart of hibernated R. ridibunda indicating a low glycolytic potential. Similar biochemical responses were observed in the same tissues during cold acclimation. The continuous increase in the activities of glycolytic enzymes studied, except for HK, might indicate a compensation for the impacts of low temperature on the enzymatic activities. In contrast to R. ridibunda, the activities of the enzymes increased and remained at higher levels than those of the prehibernation controls indicating maintenance of glycolytic potential in the tissues of hibernating land snails.     

Are Tropical Highland Frog Calls Cold–adapted? The Case of the Andean Frog Hyla labialis1

Across a wide range of temperatures established in the laboratory, we tape–recorded the advertisement calls of 76 freshly caught Hyla labialis males from three elevationally separated populations in the Eastern Andes of Colombia. Each male was tested once at a single temperature and returned to his capture site after measurement of his snout–vent length. We measured and averaged three characteristics of five to ten successive calls for each individual: number of pulses per call, pulse repetition rate, and call duration. We found that calling activity occurred within temperature ranges that overlapped among frogs from different elevations, but widened and shifted downward with increasing altitude of origin. Males from all sites called at temperatures higher, but not lower, than those naturally occurring during their nightly activity period. No decline in vocal performance was apparent when frogs extended their calling activity into the range of high temperatures selected for basking. Both snout–vent length and temperature affected pulse repetition rate and call duration, while the number of pulses per call was temperature–independent. Compared to the smaller males from lower elevations, the larger, high–mountain males had calls with significandy more pulses, a lower pulse repetition rate, and longer duration. Within each population, rising temperatures caused pulse repetition rate to increase and call duration to decrease significantly, whereas the number of pulses per call remained unchanged. Pulse repetition rate of highland males was the factor least affected by temperature, and it was less sensitive to night temperatures than to day temperatures. This, together with their capacity to call at low temperatures, suggests that highland frog calls are cold adapted.     

Advertisement calls of two anuran amphibians,Rana tigrina andTomopterna breviceps

Rana tigrina andTomopterna breviceps occur as sympatric species at Dharwad, India. Sexually mature males produce advertisement calls. The advertisement call of both the species consist of a number of calls produced in series forming a call group. Each call group ofRana tigrina comprises 10–40 calls, whereas that ofTomopterna breviceps consists 13–141 calls. Each call consists of a pulse group with variable number of pulses which lack pulse interval. Calls of both the species exhibit similarities in (i) call consisting of series of calls with a pulse group in each call, (ii) absence of pulse interval within the pulse group, (iii) the amplitude of the first pulse being always small, and (iv) the frequency spectrum beginning from 200 Hz. Based on the similarities in the spectral features of the calls, it is suggested that the two species may be closely related to each other.     

Allele frequencies of the human platelet antigen-1 in the Egyptian population

Background

We report the expression pattern of 5S rDNA in the eggs of water frogs Rana lessonae, Rana ridibunda and Rana esculenta using the quantitative real-time PCR. This kind of research had never been performed before.

Results

5S rDNA relative expression of the Rana ridibunda oocytes is approximately six times higher in comparison to the Rana lessonae oocytes. The oocytes of the investigated Rana esculenta frogs, in respect of 5S rDNA relative expression ratio, were very similar to the Rana ridibunda oocytes.

Conclusion

We suggest the possibility of using 5S rDNA as the internal control gene, in the studies of relative mRNA quantitative assays in water frog oocytes, because of its characteristic specific expression pattern in the Rana lessonae, Rana ridibunda and Rana esculenta oocytes.     

Karyological and serological studies in Rana lessonae,R. ridibunda and in their hybrid R. ‘esculenta’ (Amphibia,Anura)

The karyotypes of the three water frog forms: Rana lessonae, R. ridibunda and R. esculenta were analysed from bone marrow cell preparations of animals captured in several localities of the GDR. In the three forms chromosome morphology was similar (5 large and 8 small pairs), although differences in the relative length of most elements were found; R. esculenta chromosomes were always intermediate.One of the small pairs (Chr. 12) was found to be metacentric in R. lessonae and submetacentric in R. ridibunda. Most R. esculenta individuals examined had one meta-and one submetacentric 12th element, indicating the hybrid nature of this form. However 16.6% esculenta proved to be homozygous for either the metacentric or the submetacentric chromosome 12, while 13% lessonae individuals and 7.7% ridibunda were heterozygous for this element.By starch gel electrophoresis an analysis was undertaken of serum proteins from water frogs coming from regions in which the forms occur together (sympatric populations) and from zones in which only one of them lives (allopatric populations).In Rana lessonae, where only one allele had been previously described, two different alleles were found in animals coming from the GDR.If genetic polymorphism is excluded, between 6.1% and 9.1% individuals from sympatric lessonae and ridibunda populations showed introgression of an albumin allele. No gene introgression was found in allopatric lessonae populations from the Leningrad region or in ridibunda from Alma Ata and southern Bulgaria.     

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.     

Neuroanatomical distribution of MCH in the brain and pituitary of submammalian vertebrates

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide that has been initially characterized from a salmon pituitary extract and subsequently identified in various species from all classes of vertebrates. The present review summarizes the current knowledge regarding the neuroanatomical distribution of MCH-immunoreactive neurons in submammalian vertebrates. In all species examined, MCH-immunoreactive perikarya are confined to the hypothalamus, with the exception of the cyclostome Lampetra fluvialis and the lungfish Protopterus annectens, in which additional populations of MCH-immunoreactive cell bodies occur in the telencephalon, and the frogs Rana ridibunda and Rana esculenta which exhibit MCH-positive perikarya in thalamic nuclei. In teleosts, in the frog R. ridibunda and in the L. fluvialis, MCH is present in the classical hypothalamic-neurohypophysial system indicating that the peptide may play the role of a neurohormone. In other groups, MCH-immunoreactive nerve fibers are widely distributed in various brain regions suggesting that, in these species, MCH in the central nervous system may act as a neurotransmitter or/and a neuromodulator rather than a neurohormone.