Brown Frog Species Research Articles

Interspecific variation in the defensive responses of brown frogs to alien predators

AbstractAlien species impact native amphibians through various direct effects, including predation, and pose a significant threat to naïve prey populations. In this study, we exposed tadpoles of three brown frog species (Rana dalmatina, Rana latastei, and Rana temporaria) to the olfactory cues of two alien predators, the pond slider (Trachemys scripta) and red swamp crayfish (Procambarus clarkii), and compared their responses to those induced by the aquatic larvae of a widespread native predator, the Southern hawker (Aeshna cyanea). We recorded two tadpole defensive behaviors, the proportion of time they were active and the number of freezing events. Both agile frog species, R. dalmatina and R. latastei, showed moderate responses to red swamp crayfish kairomones and strong responses to both odonate larvae and pond sliders. In contrast, the common frog (R. temporaria) displayed a less intense response to crayfish with respect to odonate larvae, and a negligible response to pond sliders. Long-lasting coexistence with either European pond turtles or pond sliders may explain the strength of agile frogs’ response toward the alien species; while, the historical range of the white-clawed crayfish (Austropotamobius pallipes) overlapped that of the common frog, enhancing the co-option of alien crayfish cues by this anuran species.

Multiple Transitions between Y Chromosome and Autosome in Tago's Brown Frog Species Complex.

Sex chromosome turnover is the transition between sex chromosomes and autosomes. Although many cases have been reported in poikilothermic vertebrates, their evolutionary causes and genetic mechanisms remain unclear. In this study, we report multiple transitions between the Y chromosome and autosome in the Japanese Tago's brown frog complex. Using chromosome banding and molecular analyses (sex-linked and autosomal single nucleotide polymorphisms, SNPs, from the nuclear genome), we investigated the frogs of geographic populations ranging from northern to southern Japan of two species, Rana tagoi and Rana sakuraii (2n = 26). Particularly, the Chiba populations of East Japan and Akita populations of North Japan in R. tagoi have been, for the first time, investigated here. As a result, we identified three different sex chromosomes, namely chromosomes 3, 7, and 13, in the populations of the two species. Furthermore, we found that the transition between the Y chromosome (chromosome 7) and autosome was repeated through hybridization between two or three different populations belonging to the two species, followed by restricted chromosome introgression. These dynamic sex chromosome turnovers represent the first such findings in vertebrates and imply that speciation associated with inter- or intraspecific hybridization plays an important role in sex chromosome turnover in frogs.

Three brown frog species in Denmark have different abilities to colonise new ponds

For 29 consecutive years, the populations of three species of brown frogs, Rana arvalis, R. dalmatina and R. temporaria, were monitored in an open area in South Zealand, Denmark, with no direct influence of agriculture. Population sizes were recorded by counting egg clumps and showed large variations from year to year. The total population of R. arvalis differed by a factor of 100 between the years with the lowest and the highest numbers. A total of 19 initially unoccupied suitable waterbodies could potentially be colonised by the frogs. Rana dalmatina colonised all of the ponds, mostly in the very first year of existence. Rana arvalis colonised 17 ponds after an average of 10.5 years. Rana temporaria colonised eight ponds after an average of 13.4 years. Colonisation by R. dalmatina was independent of changes in total population size, whereas colonisation by R. arvalis predominantly occurred in years with considerable population increases. The results are discussed in relation to the movement patterns and philopatry of juvenile frogs of the three species. Juvenile R. dalmatina disperse far from the breeding site, but most individuals return to their natal site. This allows the species to be an efficient coloniser of new waterbodies and, at the same time, to have stable occurrence at the original site. The two other species show a more erratic type of dispersal and especially R. temporaria often shifts breeding site from year to year.

Multiplex PCR assay to distinguish among three widespread brown frog species

Multiplex PCR assay to distinguish among three widespread brown frog species

The complete mitochondrial genome of Rana coreana (Anura: Ranidae)

Rana coreana is a brown frog species native to the Korean Peninsula. We characterized the complete mitochondrial genome of the species. The mitochondrial genome sequence of R. coreana is 22,262 bp and comprises 13 protein-coding genes, two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and two control regions (CRs). The CR duplication and gene organization were identical to those observed in Rana kunyuensis and Rana amurensis. A total of 13 protein-coding genes were used to examine the phylogenetic relationships between this species and the genus Rana. R. coreana living on the Korean Peninsula, formed a cluster with R. kunyuensis and R. amurensis, with R. coreana showing the closest phylogenetic affinity for R. kunyuensis.

Assessing Population Trends of Species with Imperfect Detection: Double Count Analyses and Simulations Confirm Reliable Estimates in Brown Frogs.

Simple SummaryEstimating the abundance of populations is a key task of zoologists, but animal species often are difficult to detect- As a consequence, obtaining estimates of abundance can be problematic. For many frogs, the count of the number of egg clutches is the standard approach. How many egg clutches are missed during these counts? What are the consequences of missing some egg clutches on the estimates of population decline? First, we used the double-observer approach to estimate the detection probability of the clutches of two frog species (the agile frogs Rana latastei and R. dalmatina) and found that, for both species, clutches are very easy to detect. Subsequently, by using simulations, we showed that if the detection probability is very high, such as in this case, assessing if populations are stable or declining is straightforward. The situation is much trickier for species that are difficult to detect. Information on the detection probability of a species can be used to optimize their monitoring strategies.Most animal species are detected imperfectly and overlooking individuals can result in a biased inference of the abundance patterns and underlying processes. Several techniques can incorporate the imperfect detection process for a more accurate estimation of abundance, but most of them require repeated surveys, i.e., more sampling effort compared to single counts. In this study, we used the dependent double-observer approach to estimate the detection probability of the egg clutches of two brown frog species, Rana dalmatina and R. latastei. We then simulated the data of a declining population at different levels of detection probability in order to assess under which conditions the double counts provided better estimates of population trends compared to naïve egg counts, given the detectability of frog clutches. Both species showed a very high detection probability, with average values of 93% for Rana dalmatina and 97% for R. latastei. Simulations showed that not considering imperfect detection reduces the power of detecting population trends if detection probability is low. However, at high detection probability (>80%), ignoring the imperfect detection does not bias the estimates of population trends. This suggests that, for species laying large and easily identifiable egg clutches, a single count can provide useful estimates if surveys are correctly timed.

The effects of intra- and interspecific competitions on personality and individual plasticity in two sympatric brown frogs

We studied how individuals modify their behavior in response to inter- and intraspecific competitors and how these changes affected the pattern of variation between populations and species. As study models, we used tadpoles of two brown frogs, Rana latastei and R. dalmatina. Since R. latastei is always sympatric to R. dalmatina, whereas R. dalmatina is sympatric to R. latastei only in the periphery of its range, we predicted a stronger response to heterospecifics in R. latastei than in R. dalmatina and, within each species, in syntopic than in allotopic populations. To test these predictions, we raised tadpoles, from either syntopic or allotopic populations, in either syntopy or allotopy and repeatedly tested them in open field trials in the presence of a caged conspecific, a caged heterospecific, or an empty cage. As predicted, we found that, on average, R. latastei tadpoles modified their behavior across treatments more than R. dalmatina tadpoles and individuals from the syntopic population changed more than their conspecifics from the allotopic population. In both species, the pattern of variation at the individual level mirrored that at the population and species levels providing no evidence for an individual-by-environment interaction (Itimes E). Besides these differences, however, individuals of the two species also showed unpredicted and context-independent behavioral differences, suggesting that there might be more to interspecific behavioral variation than the effect of selection by heterospecific competitors.Significance statementDoes the distribution range of a species influence the evolution of plastic behaviors to heterospecific competitors? And how do differences in plasticity affect animal personality? To answer these questions, we raised tadpoles of two brown frog species, Rana dalmatina and R. latastei, and studied how the amount and the type of their swimming varied with the presence of the other species. R. latastei, whose small distribution range fully overlaps with that of R. dalmatina, plastically responds to it, whereas R. dalmatina, which is sympatric to R. latastei only in the periphery of its broader range, does not. These interspecific differences mirrored those among individuals: tadpoles of both species show repeatable behaviors, but only those of R. latastei plastically changed their behavior with the presence of the other species; however, neither R. latastei nor R. dalmatina show among-individual variation in plasticity.

Interspecific size- and sex-related variation in the cranium of European brown frogs (Genus Rana)

The vertebrate skull is considered as phylogenetically conserved, but shows extensive diversification in many clades due to various environmental, climate and habitat influences. We explored the cranial size and shape differences of three closely related brown frog species, Rana temporaria, R. dalmatina and R. graeca to emphases the interspecific variation and intraspecific sexual differences, the allometric patterns in cranial shape changes and the relationship between cranial shape, phylogeny, and ecological similarity. Brown frogs significantly differ in cranial size and shape. Interspecific cranial shape changes are under strong influence of size variation. When size is considered, our results show that R. dalmatina is the most divergent species, while differences between R. dalmatina and R. temporaria cranial shapes are diminished when size is removed and peculiar features of R. graeca cranium arise (the most robust and rounded cranium). In both cases, with size and without size, major shape changes are related to the position of quadratum and width of the cranium at the level of anterior margin of eye. When comparing non-allometric shape with ecological similarity and phylogenetic relatedness we found that ecologically similar but phylogenetically more distant species shared the cranial morphology. Intraspecific patterns of cranial phenotypic variation in brown frogs showed significant cranium sexual size and shape dimorphism and absence of allometric scaling between sexes. More detailed studies on the relation of morphology and ecology in brown frogs are necessary to explain the mechanism behind cranial size and shape variation.

Phylogeny and biogeography of South Chinese brown frogs (Ranidae, Anura).

Few studies have explored the role of Cenozoic tectonic evolution in shaping the patterns and processes of extant animal distributions in and around East Asia. In this study, we selected South Chinese brown frogs as a model to examine the phylogenetic and biogeographical consequences of Miocene tectonic events within South China and its margins. We used mitochondrial and nuclear molecular data to reconstruct phylogenetic interrelationships among Chinese brown frogs using Bayesian and maximum likelihood analyses. The phylogeny results show that there are four main clades of Chinese brown frogs. Excepting the three commonly known Chinese brown frog species groups, R. maoershanensis forms an independent clade nearest to the R. japonica group. Phylogeny and P-distance analyses confirmed R. maoershanensis as a valid species. Among South Chinese brown frogs, there are four subclades associated with four geographical areas: (I) R. maoershanensis; (II) R. japonica; (III) R. chaochiaoensis; and (IV) other species of the R. longicrus species group. Divergence times, estimated using mitochondrial sequences, place the vicariance events among the four subclades in the middle to late Miocene epoch. Our results suggest that (1) South Chinese brown frogs originated due to a vicariance event separating them from the R. chensinensis species group at the time of the Geological movement (~18 million years ago, Ma) in southern Tibet and the Himalayan region; (2) the separation and speciation of R. maoershanensis from the R. japonica group occurred due to the dry climate at approximately 16 Ma; (3) South Chinese brown frogs migrated from South China to Japan at the time (~10.8 Ma) that the global sea-level fell and the East China Sea Shelf Basin was swamp facies, when a land gallery may have formed across the sea to connect the two areas; and (4) R. chaochiaoensis separated from other species of the R. longicrus species group during the uplift of the Tibetan Plateau at approximately 9.5 Ma.

Food Habits of the Endemic Long Legged Wood Frog, Rana Pseudodalmatina (Amphibia, Ranidae), in Northern Iran

Abstract Iranian long legged wood frog, Rana pseudodalmatina Eiselt & Schmidtler, 1971 is a brown frog species endemic to the Hyrcanian forest. The objective of the present study is to collect detailed information on the feeding habits of 44 specimens of this species (24 ♂, 20 ♀) by analyzing the stomach contents of individuals from 10 populations inhabiting range. The food habit of R. pseudodalmatina generally varies by the availability of surrounding prey items, and it is a foraging predator, the food of which consists largely of Coleoptera (mainly Carabidae, Dytiscidae and Haliplidae), Diptera (Muscidae) and Hymenoptera (Formicidae), and no difference was found between females and males in the stomach content.

The complete mitochondrial genome of the Endangered European brown frog Rana pyrenaica through RNAseq

We sequenced the complete mitogenome of the Pyrenean frog Rana pyrenaica, which was determined from an Illumina Hi-seq RNAseq run (Illumina Inc., San Diego, CA). The genome is 17,213 bp in size, including 13 protein-coding genes, 21 transfer RNAs, two ribosomal RNAs and a control region. It shows the typical gene order of previously available frog mitogenomes, although it lacks the tRNAPhe. This is the first complete mitogenome described for a Western Palearctic brown frog species.

First data on genetic variability of the siberian wood frog rana amurensis in western siberia and its differentiation from the moor frog rana arvalis

The article presents data on genetic variability in populations of two brown frog species: the moor frog Rana arvalis and the siberian wood frog R. amurensis, in Western Siberia, Russia. Persentage of polymorphic ISSR-PCR-bands in R. arvalis was 63-93 %, in R. amurensis - 90 %, genetic diversity indices were 0.18-0.20 and 0.31, respectively. The high level of genetic variability in the siberian wood frog is contrary to its low population size, restricted distribution in the study area and the boundary position of the population. Some ISSR-PCR-bands were species-specific, they can be used for fast genotyping and further population genetic studies of the siberian wood and the moor frog in their areas of cohabitation.

Egg Size Versus Clutch Size: Variation and Trade-off in Reproductive Output of Rana dalmatina and R. temporaria in a Pond near Bonn (Germany)

The common frog (R. temporaria) is a widespread and abundant species of woodland and agricultural landscapes in Europe and north-west Asia. In contrast, the distribution of the agile frog (R. dalmatina) is rather scattered and restricted mainly to deciduous and mixed forests in western, central and south-eastern Europe. Within their overlapping range, both species often breed in the same ponds. Although ecology of these brown frog species is well known in parts, many aspects of their biology possibly explaining niche differentiation and large and small scale distribution remain uncertain. Interspecific variation in reproductive output may be one key factor explaining local differences in their abundance and dispersal ability. Our study compares the reproductive output of both species in a single pond near Bonn (Germany) using data on egg size (diameter, egg mass), clutch size, body size, age, and body condition.

Quantitative estimation of distribution area parameters: A case study of members of the genus Rana

Quantitative analysis of “point” distribution areas of species is analyzed; this method interpolates species distribution to the whole territory based on its correlations with climatic and landscape variables. It is demonstrated that the application of standard statistical interpolation techniques is inappropriate. The new approach uses the interpolation of the species-specific relations with environmental variables determined at individual points to the whole territory. The basic method for solving this problem is factor analysis. This study analyzed methods for quantifying species relations to particular climate and relief variables. Efficiency of this analysis is demonstrated by a case study of three brown frog species: Rana temporaria, R. arvalis, and R. amurensis.

Genetic differentiation of ezo brown frog (Rana pirica Matsui, 1991) (Anura: Ranidae) on Pacific islands

The mechanisms of colonization of islands by ver� tebrates it a topical problem of zoogeography and the theory of evolution. A promising approach to solving this problem is the genetic analysis of taxonomically close forms inhabiting the mainland and the neighbor� ing islands separated from the latter at different time. This is the first study to reconstruct the pathway of colonization of Pacific islands by brown frogs on the basis of analysis of the sequence of the mitochondrial gene encoding cytochrome c oxidase I of related spe� cies combined with geographical data. It is assumed that gene flow between the geographical populations of Rana pirica inhabiting Kunashir, the Smaller Kuril Chain, and Hokkaido took place after the separation of Sakhalin Island. Until recently, the taxonomic status and the num� ber of species of brown frogs of Sakhalin and Kuril islands was the subject of discussions (for review, see [2]). This especially applies to the brown frogs of the R. chensinensis complex. The study of sequences of the gene for mitochondrial cytochrome b showed that the frogs of this group from the southern Sakhalin are almost identical to R. pirica (Matsui, 1991) from Hok� kaido and significantly differ from R. dybowskii (Guenther, 1876) from Primorie. It is believed that Hokkaido, Sakhalin, and South Kuril islands are inhabited by R. pirica, whereas the mainland part of the Far East of Russia and some offshore islands are inhabited by R. dybowskii, although the genetics of the populations inhabiting the South Kuril islands has not been studied. At the same time, the morphological dif� ferences between the Sakhalin, Hokkaido, and Kuril populations of R. pirica as well as certain morphologi� cal similarity of the Sakhalin populations of this frog with R. dybowskii from Khabarovsk krai leave the issue on the genetic differentiation pattern and subspecies structure of R. pirica to be elucidated.

Intra-specific variability and unusual organization of the repetitive units in a satellite DNA from Rana dalmatina: Molecular evidence of a new mechanism of DNA repair acting on satellite DNA

We have characterized the S1 satellite from eight European populations of Rana dalmatina by Southern blot, cloning and a new method that determines the sequence variability of repetitive units in the genome. This report completes our previous studies on this satellite DNA family, thus providing the first characterization of the overall variability of the structure and genomic organization of a satellite DNA within a species and among related species. The S1 satellite from R. dalmatina has a pericentromeric location on ten chromosome pairs and presents two homologous repeats S1a (494 bp) and S1b (332 bp), mostly organized as composite S1a–S1b repetitive units. In other brown frog species, both repeats have different sequences and locations, and are usually organized as separate arrays, although composite S1a–S1b repeats represent a minor, widely variable component in Rana italica. The average genomic sequences indicate that the species contains an enormous number of variants of each repeat derived from a unique, species-specific common sequence. The repeat variability is restricted to specific base changes in specific sequence positions in all population samples. Our data show that the structure and evolution of S1 satellite family is not due to crossing-over and gene conversion, but to a mechanism that maintains the ability of the satellite DNA to assemble in constitutive heterochromatin by replacing altered satellite segments with new arrays generated by rolling circle amplification. The mode of action of this repair process not only directly explains the intra- and inter-specific variability of the structure and organization of the S1 satellite repeats from European brown frogs, but also accounts for all general features of satellite DNA in eukaryotes, including its discontinuous evolution. This repair mechanism can maintain the satellite structure in a species indefinitely, but also promote a rapid generation of new variants or types of satellite DNA when environmental conditions favor the formation of new species.

Evaluation of the state of the gene pool of natural populations dwelling in the fragmented landscape of Moscow and Moscow region (with special reference to brown frogs)

A system of urban ecological genetic monitoring of vertebrates has been developed for the first time in Russia and in the world. As a model, two species of brown frogs, Rana arvalis Nilss. and R. temporaria L. (16 populations, seven isozyme loci) were used. An evaluation of the gene pool state in urban frog populations has shown that the diversity in the Moscow frog populations was lower than that in natural populations (up to 80 and 50% in respectively R. arvalis and R. temporaria). Mean heterozygosities per locus were higher in large natural populations than in small urban isolates: in R. arvalis, these values were 0.16 and 0.06; in R. temporaria, 0.34 and 0.18, respectively. The number of polymorphic loci was also higher in natural populations than in the urban ones: 4 versus 2 in R. arvalis and 5 versus 4 in R. temporaria. Using superoxiddismutase as an example, fixation of different alleles of the same locus in different small isolates was shown. The gene pool condition of all but one urban populations of brown frog was evaluated as unsatisfactory, and that of the R. arvalis populations, as critical. These changes of the gene pool are explained mainly by gene drift accompanied by inbreeding, which was caused by human-induced fragmentation of the range and a decrease in population size of the species. The results of this study was employed in the development of the Moscow governmental program on restoration of the gene pools of vanishing animal species on specially protected natural urban territories. The series of works on long-term monitoring and assessment of the state of natural populations of model species in anthropogenic landscapes of Moscow and Moscow region has laid a foundation for a new branch of science, gene urbanology.

Distinct Specific Status of the Korean Brown Frog, Rana amurensis coreana (Amphibia: Ranidae)

We used morphological and genetic data to assess the taxonomic status of Rana amurensis coreana and R. a. amurensis. Morphological comparisons revealed these two subspecies to be different from each other in size of body, nature of tubercles on dorsal stripe, degree of development in toe webbing, and condition of lateral spots of trunk. They were also different in sequence of mitochondrial 16S rRNA gene, with genetic distance as large as those observed among different species of brown frogs. Therefore, Korean populations previously considered a subspecies of R. amurensis should be regarded as a distinct species (=R. coreana).

The first characterisation of the overall variability of repetitive units in a species reveals unexpected features of satellite DNA

We investigated the overall variability of the S1a satellite DNA repeats in ten European populations of Rana temporaria by a new procedure that determines the average sequence of the repeats in a genome. The average genomic sequences show that only 17% of the S1a repeat sequence (494 bp) is variable. The variable positions contain the same major and minor bases in all or many of the population samples tested, but the percentages of these bases can greatly vary among populations. This indicates the presence in the species of an enormous number of repeats having a different distribution of bases in these variable positions. Individual genomes contain thousands of repeat variants, but these mixtures have very similar characteristics in all populations because they present the same type of restricted and species-specific variability. Southern blots analyses and sequences of cloned S1a repeats fully support this conclusion. The S1 satellite DNA of other European brown frog species also presents properties indicating the same type of variability. This first characterisation of the overall repeat variability of a satellite DNA in a species has revealed features that cannot be determined by gene conversion and crossing over. Our results suggest that a specific directional process based on rolling circle amplification should play a relevant role in the evolution of satellite DNA.

Reproductive isolating mechanisms and molecular phylogenetic relationships among Palearctic and Oriental brown frogs.

Crossing experiments were made among various brown frog species and populations collected from Japan, China, Russia and Taiwan. The main purpose of these experiments was to confirm the existence of reproductive isolating mechanisms among Rana pirica from Japan, R. chensinensis from China and R. chensinensis from Russia, and between these three taxa and the other brown frogs distributed in the Palearctic and Oriental regions. It was found that there was no or a slight gametic isolation among the three taxa. While there was a nearly equal number of male and female offspring in the control groups, the hybrid frogs were all males, and completely sterile upon attaining sexual maturity. Thus, each of the Japanese R. pirica and the Russian R. chensinensis is a valid species, distinct from the Chinese R. chensinensis. The phylogenetic tree based on nucleotide sequence data from the mitochondrial 12S and 16S rRNA genes of the Palearctic and Oriental brown frogs showed that the three taxa are included in a cluster together with the other species with 2n=24 chromosomes. The present crossing experiments and molecular data support the hypothesis that each of them is a separate but closely related species.