Phylogenetic relationships of the Chinese brown frogs (genus Rana) inferred from partial mitochondrial 12S and 16S rRNA gene sequences

Based on partial sequences of the 12S and 16S ribosomal RNA genes, we estimated phylogenetic relationships among brown frogs of the Rana temporaria group from China. From the phylogenetic trees obtained, we propose to include Rana zhengi in the brown frogs. Monophyly of the brown frogs was not unambiguously supported, but four well-supported clades (A, B, C, and D) always emerged, although relationships among them remained unresolved. Clade A contained brown frogs with 24 chromosomes and was split into two distinct subclades (Subclade A-1: R. chensinensis and R. huanrenensis; Subclade A-2: R. dybowskii). Polytomous relationships among populations of R. chensinensis and R. huanrenensis suggested the necessity of further taxonomic assessment. Rana kunyuensis proved to be the sister group to R. amurensis, and these two species formed Clade B. Clade C was composed of R. omeimontis and R. chaochiaoensis, and Clade D included R. sauteri, which has been placed in other ranid genera. These relationships did not change after adding published data, and monophyly of Subclade A-1, A-2, and other East Asian brown frogs with 24 chromosomes (R. pirica and R. ornativentris) was ascertained, though their relationships were unresolved. Clade C, together with R. japonica and R. longicrus, also formed a monophyletic group. Brown frogs related to Clades A and C were estimated to have dispersed from continental Asia to adjacent regions through multiple events.     

Evolutionary relationships of Eastern Palearctic Brown Frogs, genus Rana: paraphyly of the 24-chromosome species group and the significance of chromosome number change

The Eurasian 'brown frogs' are a morphologically conservative assemblage consisting of the European Rana temporaria and a large number of similar species considered to be related. Although the chromosome number of the great majority of Rana species is 26, there are several species of brown frogs (including R. arvalis, R. chensinensis, R. omativentris and R. dybowskii ) which instead have 24 chromosomes. Yet comparative study of isozyme variation at 25 gene loci from these and seven other European, Caucasian, and east Asian species indicates that the 24-chromosome brown frogs are not a monophyletic group. To the exclusion of R. arvalis , the east Asian 24-chromosome species form a distinct clade related to the 26-chromosome species R. temporaria, R. dalmatina, R. japonica , and R. tagai more closely than to R. arvalis. This result is reinforced by the comparative position of the nucleolar organizer region (NOR) in the karyotypes of these frogs, as shown by NOR-banding of metaphase chromosomes. The NOR is on chromosome No. 2 in R. arvalis but on chromosome No. 10 in the other three 24-chromosome species. The systematic positions of the Causasian species A. camerani and R. macrocnemis relative to the other species are unclear except that these frogs are distinct enough to be considered part of an outgroup clade. However, in general, the systematics of the entire brown frog group remains unresolved due to great genetic divergences between species and the evident homoplasy of the brown frog morphotype. The parallel origins of reduced karyotypes among the brown frogs demonstrate that caution should be exercised when judging systematic relationships based upon gross karyology in the absence of more detailed information.     

Quantitative estimation of area parameters (with representatives of genus rana as a case study)

A quantitative method of species "point" area analysis is considered that provides the interpolation of species distribution to the whole territory on the basis of its relationships with climatic and relief variables. It is shown that application of standart statistical interpolation techniques is incorrect. The proposed approach is based on interpolation onto the whole territory the species-specific relations with environmental variables detected in single "points". The basic method for the task proves to be the factor analysis. Within the scope of the study, we have considered the methods for quantitative representation of species relationships with climatic and relief variables. The analysis efficiency is demonstrated by an example of three species of brown frogs: Rana temporaria, R. arvalis u R. amurensis.     

Effects of pH and aluminium on embryonic and early larval stages of Swedish brown frogs Rana arvalis, R. temporaria and R. dalmatina

The effects of pH and aluminium on embryonic and early larval stages of Swedish brown frogs Rana arvalis, R. temporaria and R. dalmatina were tested in laboratory bioassays. In all three species egg mortality and time needed for embryonic development to hatching increased when pH declined, but no significant effects were found on embryonic development when aluminium level was elevated. In R. arvalis and R. temporaria larval mortality was affected by both pH and aluminium. In both species the frequency of occurrence of larval deformities increased in acid water, and there was a synergistic effect of pH and aluminium. In R. arvalis swimming behaviour was disturbed by high levels of aluminium at pH 5. In all three species the frequency of stressed larvae increased when pH was depressed and aluminium concentration elevated, and there was a synergistic effect when both were combined. The three species differed significantly in egg mortality, time needed for embryonic development, larval mortality, larval deformities and larval stress al low pH and high aluminium levels. R. arvalis showed the highest acid tolerance and R. dalmatina was the most sensitive to low pH.     

Specialized mucous glands and their possible adaptive role in the males of some species of Rana (Amphibia,Anura)

A structural and ultrastructural study was carried out on the cutaneous glands in some species of Rana (R. dalmatina, R. iberica, R. italica, R. "esculenta," and R. perezi), giving particular attention to the mucous secretory units. Two different types of mucous glands occur in R. dalmatina, R. iberica, and R. italica. Besides the ordinary mucous units, which are randomly distributed over the body surface in both males and females, a further population of mucous glands was observed on the male dorsal skin. The latter is recognizable by the peculiar morphology of the epithelial cells and some characteristics of the secretory product. Specialized mucous glands are absent in both sexes of R. "esculenta" and R. perezi. The possible adaptive role of the specialized mucous glands is discussed in light of the absence of vocal sacs in males of R. dalmatina, R. iberica, and R. italica. Chemosignals released by sexually dimorphic mucous units may replace vocal communication during the breeding season and so play an important role in female attraction and/or territorial announcement. The morphology and possible function of the specialized mucous glands in the three species of Rana are compared with the breeding glands of other frogs and with the hedonic glands of some urodeles (Salamandridae and Plethodontidae), which are known to produce pheromonal substances during courtship.     

哈士蟆油的形态结构观察和化学成分测定

在不同的文献中对“哈士蟆油”存在着完全不同的解释,主要有三种观点：哈士蟆油是“哈士蟆的脂肪”,“雌性哈士蟆的卵巢和输卵管外的脂肪”,“雌性哈士蟆的输卵管”。本研究通过对哈士蟆油干品形态结构的观察、显微切片的观察和化学成分的分析,否定了上述的前两种解释。形态结构、组织化学和成分分析的结果均表明：哈士蟆油是雌性哈士蟆（中国桂蛙Ｒａｎａ ｔｅｍｐｏｒａｒｉａ ｃｈｅｎｓｉｎｅｎｓｉｓ或黑龙江林蛙Ｒ．ａｍ     

Phylogeny of the New World true frogs (Rana)

Phylogenetic relationships among the species of true frogs (Rana) from North, South, and Central America were investigated based on the sequences of approximately 2 kb from the mitochondrial genome, sampled from most of the described species, as well as eight undescribed species. This analysis, combined with previous studies of the phylogeny of New World Rana, served as the basis for a revised classification of the group. The American species of Rana are not monophyletic; the western North American Amerana is more closely related to the R. temporaria group of Eurasia (together, these frogs form the group Laurasiarana). The remaining species from the Americas form the monophyletic group Novirana, which includes: R. sylvatica; Aquarana (the R. catesbeiana group); Ranula (the R. palmipes group, including the mostly upland Levirana species and the mostly lowland Lithobates species); Torrentirana (the R. tarahumarae group, or Zweifelia, plus R. sierramadrensis), Stertirana (the R. montezumae group, or Lacusirana, plus R. pipiens), Nenirana (the R. areolata group), and Scurrilirana (most of the southern and tropical leopard frogs). The mitochondrial sequences supported many of the previous hypotheses of relationships of New World Rana, although there were some differences involving the placement of the species R. pipiens, R. sierramadrensis, and R. sylvatica. Parametric bootstrap analyses indicated significant support for the relationships inferred from the mtDNA sequences, and rejected the previous hypotheses of relationships for these three species.     

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.     

寒露林蛙的核型和Ag-NORs研究

采用常规骨髓细胞制片法对产于湖南省双牌县的寒露林蛙的核型及NORs位点进行了观察和分析.结果表明寒露林蛙与分布于我国南方的其他林蛙相同:2n=26,NF=52,为5+8核型模式.寒露林蛙核型为14M+10SM+2ST,无次溢痕,NORs位于第11对染色体长臂的中部.通过比较分析我国2n=26的6种林蛙的核型等细胞遗传学信息,本文结果支持形态分类和分子系统发生的研究结论,认为寒露林蛙是一有效种.     

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.     

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 1976bp 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 1425bp 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.     

Effects of pH on K depolarization and contracture in frog muscles.

Membrane potential changes and mechanical tension provoked by 20 or 80 mM K at pH 8.0 or 4.5 were measured in muscle fiber bundles from Rana temporaria and Rana esculenta. The bathing solution contained Cl as the main anion. Low pH increased the depolarizing efficiency of high K to about the same degree in muscles of both species of frogs. The tension-[K] relationship was shifted toward lower [K] in muscles from R. temporaria and toward higher [K] in muscles from R. esculenta. It was concluded that the depolarization-contraction coupling was insensitive to pH changes in the former but not in the latter frog species.     

Genetic relationships among Korean brown frog species (Anura,Ranidae), with special reference to evolutionary divergences between two allied species Rana dybowskii and R. huanrenensis

Allozyme analysis for 41 populations of brown frog species, Rana dybowskii, R. huanrenensis, and R. amurensis from Korea and three reference species (Chinese R. chensinensis and Japanese R. dybowskii and R. tsushimensis), were performed to clarify taxonomic status of Korean brown frogs. The level of average genetic differentiation (Nei's D) among local populations of each species in Korea was very low (D<0.01 2) and Korean and Japanese R. dybowskii also showed conspecific level of differentiation (D=0.070). Whereas, much larger, discrete genetic differences were detected in the interspecific comparisons (D>0.370). In the genetic relationships among five species examined, the 24 chromosome brown frogs (R. dybowskii, R. huanrenensis, and R. chensinensis) did not form a monophyletic group. Rana dybowskii with the chromosome number of 2n=24 was grouped together with R. amurensis with the chromosome number of 2n=26. The hypothesis of reversal change from 24 to 26 in Korean R. amurensis seems to better explain the phylogenetic relationships of east Asian brown frogs than the assumption of parallel reduction in chromosome number from 2n=26 to 24 in R. dybowskii and in the common ancestor of R. huanrenensis and R. chensinensis. The genetic, morphological, and reproductive divergences between Korean R. dybowskii and R. huanrenensis were compared.     

阿尔泰林蛙的物种有效性及其分类地位——来自系统发育分析的证据

为检验阿尔泰林蛙(Rana altaica)的系统发育地位及其物种有效性,该文运用线粒体细胞色素b基因,应用贝叶斯分析和最大简约方法构建了欧哑人陆分布的部分林蛙的系统发育关系.两种分析方法均支持阿尔泰林蛙在田野林蛙(R.arvalis)这一分支的内部.单倍型网络图显示来自阿尔泰地区和中西伯利亚地区所谓的阿尔泰林蛙与田野林蛙有共享单倍型.通过该文母系遗传发育分析结果显爪阿尔泰林蛙种级地位不成立,是田野林蛙的同物异名.另外,该文实验分析结果提示,对中国分布的林蛙内部种组划分应建立在系统进化关系的基础上重新进行评估.     

Phylogenetic relationships of Oriental torrent frogs in the genus Amolops and its allies (Amphibia, Anura, Ranidae)

We investigated the phylogenetic relationships among 20 species of Oriental torrent frogs in the genus Amolops and its allies from China and Southeast Asia based on 1346-bp sequences of the mitochondrial 12S and 16S rRNA genes. Oriental species of the tribe Ranini form a monophyletic group containing 11 clades (Rana temporaria + Pseudoamolops, R. chalconota, four clades of Amolops, Meristogenys, three clades of Huia species, and Staurois) for which the phylogenetic relationships are unresolved. The genus Amolops consists of southern Chinese, southwestern Chinese, Thai, and Vietnamese-Malaysian lineages, but their relationships are also unresolved. The separation of southern and southwestern lineages within China conforms to previous morphological and karyological results. Species of Huia do not form a monophyletic group, whereas those of Meristogenys are monophyletic. Because P. sauteri is a sister species of R. temporaria, distinct generic status of Pseudoamolops is unwarranted.     

The natural stimulus for spawning in Xenopus laevis (Amphibia)

I have already reported (Savage, 1965), preliminary experiments in which "natural spawning", that is, spawning by frogs kept under as natural conditions as aquaria allow, and by frogs not injected or manipulated in any other way, had been induced by a variety of external stimuli, including unialgal cultures of various kinds.
These experiments were planned and executed using as a guide, at every possible point, the statistical investigations of Rana temporaria obtained entirely from field data (Savage, 1935, 1961). The reasons for using Xenopus laevis and not R. temporaria were purely practical. R. temporaria breeds but once a year, and has other disadvantages, but X. laevis is a good laboratory animal.
Since conclusions drawn from mathematical relations in one species have led without important deviation to the expected experimental results in another, it is unlikely that what I now have to report is peculiar to these two species, selected because of availability in one case and ease of working in another. The conclusions are probably quite general. The earlier conclusions have been extended, active materials have been isolated, and other factors relevant to reproduction have been found.     

Evaluation of the state of the gene pool of natural populations of vertebrate animals inhabiting the fragmented landscape of Moscow and Moscow district (with special reference to brown frogs)

A system of urban ecological genetic monitoring 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. anvalis 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 district has laid a foundation for a new branch of science, gene urbanology.     

Expression of genes encoding antimicrobial and bradykinin-related peptides in skin of the stream brown frog Rana sakuraii

Peptidomic analysis of an extract of the skin of the stream brown frog Rana sakuraii Matsui and Matsui, 1990 led to the isolation of a C-terminally alpha-amidated peptide (VR-23; VIGSILGALASGLPTLISWIKNR x NH2) with broad-spectrum antimicrobial activity that shows structural similarity to the bee venom peptide, melittin together with two peptides belonging to the temporin family (temporin-1SKa; FLPVILPVIGKLLNGIL x NH2 and temporin-1SKb; FLPVILPVIGKLLSGIL x NH2), and peptides whose primary structures identified them as belonging to the brevinin-2 (2 peptides) and ranatuerin-2 (1 peptide) families. Using a forward primer that was designed from a conserved region of the 5'-untranslated regions of Rana temporaria preprotemporins in a 3'-RACE procedure, a cDNA clone encoding preprotemporin-1SKa was prepared from R. sakuraii skin total RNA. Further preprotemporin cDNAs encoding temporin-1SKc (AVDLAKIANIAN KVLSSL F x NH2) and temporin-1SKd (FLPMLAKLLSGFL x NH2) were obtained by RT-PCR. Unexpectedly, the 3'-RACE procedure using the same primer led to amplification of a cDNA encoding a preprobradykinin whose signal peptide region was identical to that of preprotemporin-1SKa except for the substitution Ser18-->Asn. R. sakuraii bradykinin ([Arg0,Leu1,Thr6,Trp8] BK) was 28-fold less potent than mammalian BK in effecting B2 receptor-mediated relaxation of mouse trachea and the des[Arg0] derivative was only a weak partial agonist. The evolutionary history of the Japanese brown frogs is incompletely understood but a comparison of the primary structures of the R. sakuraii dermal peptides with those of Tago's brown frog Rana tagoi provides evidence for a close phylogenetic relationship between these species.     

Influence of introduced fish Percottus glenii (Odontobutidae, Pisces)

Monitoring of 28 waterbodies has been carried out since 1994 in the region of reserve "Lake Glubokoe" (Moscow region, Russia). It was revealed that species diversity as well as abundance of larval amphibians correlate negatively with presence of introduced fish, rotan, Perccottus glenii (Odontobutidae). Newts (Triturus cristatus, T. vulgaris) and frogs (Rana temporaria, R. arvalis, R. lessonae) as a rule are not capable to breed in waterbodies colonised by rotan. In contrast, toads (Bufo bufo) breed successfully in such sites. Larvae of toads are comparatively less edible for rotan and pass their metamorphosis. Persistence of amphibians to predation of rotan decreases in the row: B. bufo (R. temporaria, R. arvalis and R. lessonae), T. vulgaris, T. cristatus. The Crested newt (T. cristatus) is the most endangered species and could extinct in next years.     

The reproductive biology of Common frog (Rana temporaria) populations from different altitudes in northern England

The study investigated the ecophysiological differences between populations of the Common frog( Rana temporaria L.) from different altitudes in northern England. Frog embryos in upland northern ponds experience very low temperatures during the period of embryonic development. These embryos were found to have a lower lethal limit for 50% normal development of2–8 °C, compared with3–8 °C for lowland embryos. These values are below most of the lower limiting temperature estimates previously obtained for R. temporaria . Embryos from upland ponds developed faster at low temperatures than embryos from lowland ponds. This may be an adaptation to the shorter growing season and lower temperatures at higher altitudes. Upland female frogs were found to be shorter and produced fewer eggs than lowland females. The differences found between lowland and upland frogs are discussed in terms of their potential adaptive significance.