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PubMed1975

Investigations of thermotropic phase changes in peripheral nerve of frog and rat. A spin label study.

The temperature-dependent fluidity of myelin of frog and rat peripheral nerve (Nervus ischiadicus) was studied using the spin label technique. In frog nerve a phase change was detected at 38 degree C. In rat nerve no sharp phase change could be established, and the lipid-depleted frog and rat nerve also showed no transition. From the spectral data, it was concluded that in frog and rat nerve the lipid-protein interactions are different, i.e. species dependent. Ca-2+-depletion of frog nerve caused a loss of transition, while rat nerve remained unaffected. Thus it was indicated that, in frog nerve, Ca-2+ is involved in the phase change. In the total lipid extract of frog nerve a phase change centered at 32 degree C occurred, while the total lipid extract of rat nerve again showed no transition. It is suggested that a connexion exists between our results and investigations on the temperature dependence of an axonal conduction block of nerve.

Animals↗

PubMed1979

The filum terminale of the frog spinal cord, a non transformed preparation: I. Morphology and uptake of gamma-aminobutyric acid.

The filum terminale of the frog spinal cord is a rather pure glial cell preparation, largely devoid of neuronal elements. gamma-Aminobutyric acid (GABA) is taken up by the frog filum terminale (FT) via a Na+-dependent, ouabain-inhibited, saturable high affinity transport system with a Km of 2.7 x 10(5) M. The rate of the FT GABA uptake is significantly greater than the velocities observed in the spinal cord. In fact, the Vmax increases caudally beyond the level of the last root, and is maximal in the FT per se. beta-Alanine is a competitive inhibitor of the FT high affinity transport system for GABA (Ki 11.1 x 10(-5) M). In addition to GABA, the FT also takes up beta-alanine, glycine, glutamate and aspartate at rates significantly higher than those shown by the spinal cord of the frog. Light and electron microscope level radioautography clearly shows that GABA uptake occurs primarily in the glial cells and also in ependymal cells present in the FT. In that the FT contains few ependymal cells and a large number of glia, it is fair to state that most of the GABA accumulated by the FT reflects the glial transport of this amino acid. Unlike the adult frog, the spinal cord of the tadpole does not show any regional differences in the rate of GABA transport during early development. However, during later developmental stages, the rates of GABA transport increase in the caudal portion of the tadpole cord as compared to the more rostral areas. Close to metamorphosis, the terminal portion of the tadpole cord, which is destined to become the filum terminals of the frog, accumulates GABA at rates not greatly different from those observed in the FT of the adult frog. Therefore, the tadpole spinal cord is a useful preparation in which to study the dynamic properties of normal non-transformed glia as influenced by a changing neuronal population, whereas the frog FT is a unique preparation for the study of some properties of normal glia largely in the absence of neurons.

Amino Acids↗

PubMed1996

Characterization of the receptor mediating the effect of calcitonin gene-related peptide in the frog adrenal gland.

We have recently reported the presence of calcitonin gene-related peptide (CGRP)-containing nerve fibers in the frog adrenal gland and we have shown that CGRP is a potent stimulator of corticosterone and aldosterone secretion by adrenocortical cells. The aim of the present study was to characterize the type of receptors mediating the effect of CGRP in the frog adrenal gland. Amylin and adrenomedullin, two members of the CGRP family, induced a weak stimulation of corticosterone and aldosterone secretion from perifused frog adrenal slices. In contrast, salmon and human calcitonin had no effect on corticosteroid secretion. Administration of the type-1 CGRP receptor antagonists human CGRP-(8-37) and human CGRP-(19-37) did not significantly affect the secretory response induced by frog CGRP. Concurrently, the type-2 CGRP receptor agonist [acetamidomethyl-Cys2,7]human CGRP ([Cys(ACM)2,7]human CGRP) provoked a dose-dependent stimulation of corticosterone and aldosterone secretion (EC50 = 1.6 x 10(-7) M). Both frog CGRP and [Cys(ACM)2,7]human CGRP induced a significant increase in cAMP production by frog adrenal tissue. These data indicate that, in the frog adrenal gland, the stimulatory effect of CGRP is mediated through activation of a type-2 CGRP receptor positively coupled to adenylyl cyclase.

11-Hydroxycorticosteroids↗

PubMed1989

Effect of Des-Gly10-(im-Bzl-D-His6)LHRH-ethylamide on hypophysial gonadotrophs and ovary of the juvenile frog Rana cyanophlyctis (Schn.).

The effects of synthetic luteinizing hormone-releasing hormone (LHRH) (0.01 and 0.1 microgram) agonist on pituitary and ovary were studied in the juvenile frog Rana cyanophlyctis with respect to their body weights. Injections (ip) were given 6 days a week for 30 days and frogs were sacrificed on the 31st day. The pituitary sections were stained with AB-PAS-OG technique. The staining intensity, cytoplasmic granulations, and length of hypophysial gonadotrophs (B2 cells) were increased (P less than 0.05) due to LHRH treatment. In controls, the B2 cells were small and faintly stained. Treatment with LHRH (0.01 or 0.1 microgram) once a day or twice a day (0.01 microgram) to frogs weighing less than 6 g body weight had no effect on gonadosomatic index (GSI) or size frequency distribution of oocytes even though gonadotrophs were hypertrophied and hyperactive. Whereas administration of LHRH (0.01 or 0.1 microgram) once a day to frogs weighing around 8 g caused a significant (p less than 0.05) increase in ovary weight, GSI, and diameter of the largest oocytes. Further, oocytes were recruited to second growth phase (i.e., incorporation of yolk in oocytes) due to LHRH treatment. The above findings suggest that synthetic LHRH agonist has the stimulatory effect on oocyte growth and recruitment of vitellogenic oocytes in juvenile frogs weighing around 8 g. The LHRH induces precocious maturity by incorporating yolk in oocytes. The pituitary responsiveness to LHRH agonist is also evident in juvenile frogs. The ovarian receptivity/responsiveness to endogenous gonadotrophins seems to progressively increase with the increase in the body weight of the juvenile frogs.

Animals↗

PubMed1992

Frog lymph heart synthesizes and stores immunoreactive atrial natriuretic peptide.

The presence of immunoreactive atrial natriuretic peptide (irANP) and ANP gene expression in the frog lymph heart was examined by a radioimmunoassay (RIA) combined with HPLC and by Northern blot hybridization of total RNA. Serial dilution curve of the lymph heart extract was paralleled with the RIA standard curve. The lymph heart contained 153.32 +/- 35.80 pg of irANP/mg of wet tissue. The major form of irANP in the frog lymph heart was high molecular weight on reverse-phase and gel permeation high performance liquid chromatography as in the frog atria and ventricles. The frog lymph heart, as well as frog atria and ventricles, was shown to express mRNA coding for ANP. Dense core secretory granules similar to those observed in the mammalian atria were also found in the frog lymph heart. The presence of irANP and the expression of ANP gene in the frog lymph heart suggest that the lymph heart may participate in the regulation of homeostasis of lymph circulation and blood volume change through the synthesis and release of ANP.

Animals↗

PubMed1983

Heterogeneity of capillary diameters in skeletal muscle of the frog.

Capillary diameters in sartorius muscle of frogs were measured in vivo by means of a new computer video method, based on the passage of red blood cells (RBCs) through the capillary (C. Ellis, R. Sanfranyos, and A. Groom (1983), Microvasc. Res. 26, 139-150). The distribution of capillary diameters from 21 frogs was represented by a histogram with a mean +/- SD of 16.7 +/- 4.4 microns (N = 83). The measured dimensions (mean +/- SD) of frog RBCs, which have a flattened ellipsoidal shape, were: major axis = 24.1 +/- 2.6 microns (N = 149); minor axis = 16.5 +/- 1.5 microns (N = 158); thickness at center = 5.4 +/- 0.8 microns (N = 32). Frog RBCs travel through capillaries with their major axes predominantly parallel to the direction of flow; therefore, RBCs pass through capillaries without deformation provided that the diameter of the capillary is larger than the minor axis of the cell. By standardizing the measured values of capillary diameter in terms of mean minor dimension of the RBCs (ratio of means for frog being 1.0, approx), we were able to compare the diameter distribution in an amphibian with that in a mammal (rat). If RBC size alone mattered, both standardized distributions should superimpose; however, that for frog was shifted to the right of that for rat, indicating that frog RBCs are less deformable than RBCs of rat. This highlights the necessity, in the microcirculation, for matching capillary diameter to both size and deformability of the red cell.

Animals↗

PubMed1984

Occurrence of skin alkaloids in non-dendrobatid frogs from Brazil (Bufonidae), Australia (Myobatrachidae) and Madagascar (Mantellinae).

Several taxa of small frogs from the southern hemisphere contain alkaloids similar or identical to compounds previously known only from neotropical poison frogs of the family Dendrobatidae. Skin of the Brazilian toad Melanophryniscus moreirae (family Bufonidae) contains a new alkaloid 8-hydroxy-8-methyl-6-(5'-hydroxy-2'-methyl-hexylidene)-1-azabicycl o-[4.3.0] nonane (C16H29NO2), which is designated pumiliotoxin 267C. Such a structure is typical of the pumiliotoxin-A class of dendrobatid alkaloids. Melanophyryniscus moreirae contains smaller quantities of an alkaloid (C19H33NO3) identical in chromatographic and mass spectral properties to the dendrobatid alkaloid allopumiliotoxin 323B. Allopumiliotoxin 323B and an isomer of 267C occur with unidentified alkaloids in skin of the Australian frog Pseudophryne semimarmorata (family Myobatrachidae) and also in the skin of the Madagascan frog Mantella aurantiaca (family Ranidae, subfamily Mantellinae). In addition to new compounds, Mantella aurantiaca and M. madagascariensis also contain other alkaloids (e.g. histrionicotoxin and pumiliotoxin B) that were known previously only in dendrobatid frogs. Such alkaloids have not been detected in a phylogenetically wide array of other anuran amphibians, and the dendrobatid alkaloids thus become an evolutionary enigma. Certain of these compounds may have arisen convergently from new biosynthetic pathways in several families of frogs, or these alkaloids may represent parallel expression of shared-primitive pathways that are unexpressed or lost in related frogs.

Alkaloids↗

PubMed1995

Reinnervation of frog sympathetic ganglia with somatic nerve fibres.

The formation of synapses in the last two ganglia of the sympathetic chain of the frog Rana esculenta was investigated after anastomosing the 6th spinal nerve to the denervated ganglia in order to evaluate the reinnervation of deafferented sympathetic neurons with somatic cholinergic axons. The same ganglia were examined both electrophysiologically and morphologically from 25 to 280 days after the operation. In response to electrical stimulations of the anastomosed spinal nerve, synaptic transmission was analysed with intracellular microelectrodes placed into B or C sympathetic neurons. Synaptic density was quantified using electron microscopy by a synaptic index defined as the ratio of the number of synapses encountered to the number of perykarya examined. After ganglionic deafferentation, post-synaptic membrane differentiations persisted without any pre-synaptic element and an index of the 'vacated' post-synaptic differentiations was calculated. Although somatic axons were growing into all ganglia studied, no sign of neuronal reinnervation was detected in ganglia of 8 of the 31 frogs (26%) taken from 29 to 210 days after the anastomosis. Moreover, in 18 out of 31 frogs (58%) analysed at different times after the operation, the ganglia were reinnervated with regenerating preganglionic axons in spite of care taken to avoid it. However, even after 3 months, certain neurons of these ganglia were not reinnervated and the synaptic index approximated the value of normal ganglia only in the 8th ganglion. In addition, post-synaptic membrane differentiations could still persist and coexist with normal synapses. It was only beyond three months after the anastomosis that the ganglia of 5 of the 31 frogs (16%) were reinnervated with regenerating somatic axons. Reinnervated B and C neurons were polyinnervated. But in 3 out of these 5 frogs the ganglia were also reinnervated with preganglionic axons and several B and C neurons received a double reinnervation. The synaptic indices were far from the value of normal ganglia except for the 8th ganglion of one frog reinnervated by both types of axons and the indices of vacated differentiations were close to that of ganglia with no reinnervated neurons. Contrary to mammals, frog somatic axons are, therefore, relatively ineffective at reinnervating sympathetic neurons, probably because in amphibian ganglia, synapses between the pre- and post-synaptic elements require higher specificity.

Adrenergic Fibers↗

PubMedDate not supplied

Localization and characterization of the N-terminal fragment of atrial natriuretic factor (ANF) precursor in the frog heart.

The localization of the N-terminal fragment of the atrial natriuretic factor (ANF) precursor in the heart of the frog Rana ridibunda was examined by the indirect immunofluorescence and the immunogold techniques using an antiserum directed against synthetic rat ANF (Asp11-Ala37). At the optic level, positive material was found in most atrial myocytes. Staining of consecutive sections of frog heart with antibodies against N-terminal and C-terminal regions of the proANF molecule showed that both peptides are contained in the same cardiocytes. In the rat atrium, antibodies against the N-terminal ANF region induced a more intense labeling than in the frog atrium. Electron microscopic studies indicated that all secretory granules present in frog atrial cardiocytes contain N-terminal ANF-like immunoreactive material. The positive material localized in frog atrium was characterized by gel filtration and radioimmunological detection. Serial dilutions of frog atrial extracts exhibited displacement curves which were parallel to that obtained with synthetic human ANF (Asn1-Asp30). Sephadex G-50 gel chromatography of the immunoreactive material showed that the N-terminal ANF-like immunoreactivity eluted in a single peak corresponding to high molecular weight material. These results indicate that the N-terminal fragment of frog proANF is immunologically and biochemically related to the homologous mammalian peptide.

Animals↗

PubMed1994

Behavior of Japanese tree frogs under microgravity on MIR and in parabolic flight.

Japanese tree frogs (Hyla japonica) were flown to the space station MIR and spent eight days in orbit during December, 1990. Under microgravity, their postures and behaviors were observed and recorded. On the MIR, floating frogs stretched four legs out, bent their bodies backward and expanded their abdomens. Frogs on a surface often bent their neck backward and walked backwards. This behavior was observed on parabolic flights and resembles the retching behavior of sick frogs on land--a possible indicator of motion sickness. Observations on MIR were carried out twice to investigate the frog's adaptation to space. The frequency of failure in landing after a jump decreased in the second observation period. After the frogs returned to earth, readaptation processes were observed. The frogs behaved normally as early as 2.5 hours after landing.

Adaptation, Physiological↗

PubMed1982

Hepatic cytochrome P-450-dependent monooxygenase systems of the trout, frog and snake--III. Induction.

1. 3-Methylcholanthrene administration increased levels of cytochrome P-450, benzo[a]pyrene hydroxylase activity and p-nitrophenetole O-deethylase activity in hepatic microsomes from the brown trout (Salmo trutta), leopard frog (Rana pipiens) and the garter snake (Thamnophis). The level of aminopyrine N-demethylase activity was increased in trout microsomes, but not in those from the frog, snake or rat. 2. The shift in the Soret maximum of the reduced carbon monoxide difference spectrum of cytochrome P-450 from 450 to 448 nm, which is observed when 3-methylcholanthrene is administered to rats, was not seen in microsomes from the trout, frog or snake. 3. Benzo[a]pyrene hydroxylase activity induced by 3-methylcholanthrene in the trout, frog and snake was inhibited by relatively low concentrations of SKF 525-A, but that induced in the rat was not. alpha-Naphthoflavone inhibited 3-methylcholanthrene-induced benzo[a]pyrene hydroxylase activity in the trout, frog and rat, but not in the snake. 4. 3-Methylcholanthrene induced an increase in the 455/430 nm peak height ratio of the reduced ethylisocyanide spectrum of microsomes in the rat and trout, but not in the frog or snake. 5. These observations (items 1--4 of the abstract) show that different species of cytochrome P-450 are induced by 3-methylcholanthrene in the four vertebrates. 6. Phenobarbital did not alter the components or the activities of hepatic monooxygenase systems in the trout, frog or snake, even though it was shown to accumulate in the livers of these vertebrates as readily as it does in the liver of the rat.

Animals↗

PubMed1986

Fast turnover of clear vesicle membrane in frog sympathetic ganglion.

The results of three independent experiments are presented. In the first experiment, the afferent fibers of the right side IX and X sympathetic ganglia of five frogs were stimulated at 100 Hz and control unstimulated ganglia were taken from five other frogs. In the second experiment, the right IX and X ganglia from eight other frogs were stimulated at 10 Hz; the left side ganglia of the same eight frogs served as controls. A third experiment was performed in the presence of extracellular horseradish peroxidase, the sympathetic ganglia of three frogs were stimulated at 10 Hz, those of three other frogs were stimulated at 100 Hz and control ganglia exposed to horseradish peroxidase, but not stimulated were taken from another three frogs. Postganglionic compound action potentials were recorded in each experiment to verify the effectiveness of the stimulation. Following stimulation, the ganglia were fixed and prepared for electron microscopy. Between 10 and 20 synaptic profiles were photographed for each ganglia. The numerical density on area (i.e. number of vesicles per micron 2) was calculated separately for the clear vesicles and for the large dense-core vesicles of each profile. Statistical tests indicated no significant difference in the numerical density on area of the clear vesicles, or of the dense-core vesicles between the control and the ganglia stimulated at either 10 or 100 Hz. However, in the experiment done in presence of horseradish peroxidase, a significant increase of the percentage of vesicles labeled with horseradish peroxidase was obtained following stimulation at either 10 or 100 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

PubMed1986

Is there functional cholinergic innervation in the frog duodenum (Rana catesbeiana)?

To determine if functional cholinergic innervation occurs in the frog duodenum or not, the effects of exogenous acetylcholine and electrical transmural stimulation, the contractile activity of an acid extract from the frog duodenum, and the distribution of acetylcholinesterase (AChE) activity in the wall of the frog duodenum were investigated. Acetylcholine caused non-sustained contraction in a dose-dependent manner (100 nM-1 mM). The ED50 value was 17 +/- 2.4 microM. Atropine (500 nM) shifted the dose-response curve for acetylcholine parallel to the right. Transmural stimulation of the frog duodenum caused frequency-dependent (0.5-50 Hz) contraction which was not decreased by atropine (500 nM) at all. The acid extract from the frog duodenum caused contraction of a longitudinal muscle strip of guinea-pig ileum but atropine (500 nM) had no significant effect on the contraction. Only a little AChE activity was found in Auerbach's plexus of the frog duodenum compared with that of the rat ileum. These results suggest that a cholinergic nerve is present in the frog duodenum but its physiological significance is very small.

Acetylcholine↗

PubMed2005

Atrazine concentrations, gonadal gross morphology and histology in ranid frogs collected in Michigan agricultural areas.

The triazine herbicide atrazine has been suggested to be a potential disruptor of normal sexual development in male frogs. The goals of this study were to collect native ranid frogs from sites in agricultural and non-agricultural areas and determine whether hypothesised atrazine effects on the gonads could be observed at the gross morphological and histological levels. Juvenile and adult green frogs (Rana clamitans), bullfrogs (R. catesbeiana) and leopard frogs (R. pipiens) were collected in the summers of 2002 and 2003. Atrazine concentrations were below the limit of quantification at non-agricultural sites, and concentrations did not exceed 2 microg/L at most agricultural sites. One concentration greater than 200 microg atrazine/L was measured once at one site in 2002. Hermaphroditic individuals with both male and female gonad tissue in either one or both gonads, were found at a low incidence at both non-agricultural and agricultural sites, and in both adults and juveniles. Testicular oocytes (TO) were found in male frogs at most of the sites, with the greatest incidence occurring in juvenile leopard frogs. TO incidence was not significantly different between agricultural and non-agricultural sites with the exception of juveniles collected in 2003. Atrazine concentrations were not significantly correlated with the incidence of hermaphroditism, but maximum atrazine concentrations were correlated with TO incidence in juvenile frogs in 2003. However, given the lack of a consistent relationship between atrazine concentrations and TO incidence, it is more likely the TOs observed in this study result from natural processes in development rather than atrazine exposure.

Agrochemicals↗

PubMed2005

Up-regulation of acidic ribosomal phosphoprotein P0 in response to freezing or anoxia in the freeze tolerant wood frog, Rana sylvatica.

Natural freezing survival by the wood frog, Rana sylvatica, involves multiple organ-specific, freeze-responsive changes in gene expression. The present study provides the first report of freeze-responsive genes in brain. Differential screening of a cDNA library made from brain of frozen wood frogs revealed a freeze-responsive clone encoding a protein of 315 amino acids that was identified as the acidic ribosomal phosphoprotein, P0 (GenBank Accession No. AF176302). The amino acid sequence showed 91-92% identity with the protein from other vertebrates. Thirteen unique amino acid substitutions occurred as compared with mammalian or avian P0 sequences; these may represent structural differences that support protein function at low body temperature. Transcripts of P0 rose by 8-fold in brain of frogs frozen for 24 h at -2.5 degrees C, compared with controls at 5 degrees C, and reached 12-fold higher in 24 h thawed frogs. Immunoblotting showed that P0 protein increased by approximately 3-fold in brain during freezing and remained high after thawing. Freeze up-regulation of P0 was largely brain-specific; transcript levels were unaffected in skeletal muscle and skin and, although transcripts rose approximately 2-fold in liver of frozen frogs, liver P0 protein was unchanged (although P0 protein was much higher overall in liver than in brain). P0 transcripts in wood frog brain were also elevated during anoxia exposure (by approximately 4-fold), but did not change under dehydration stress. The gene was similarly up-regulated under anoxia in brain of the freeze intolerant leopard frog, Rana pipiens. This suggests that P0 expression responds to anoxia stress during freezing. Changes in P0 content in ribosomes may contribute to altered patterns of protein synthesis under anoxia or ischemia.

Amino Acid Sequence↗

PubMed1988

Identification of new atrial natriuretic peptides in frog heart.

It has been observed that mammalian atrial natriuretic peptide (ANP)-like immunoreactivity is found in frog heart, but to date the natriuretic factors have not yet been identified. Isolation from bull-frog heart extract was performed mainly by immunoaffinity chromatography on a column linked with anti-hANP IgG. From the low molecular weight fraction, 24- and 21-amino acid peptides were purified to homogeneity. Both peptides were found to elicit diuretic-natriuretic as well as vasorelaxant activity, and were named "frog ANP-24" and "frog ANP-21", respectively. Complete amino acid sequences of the peptides were established by microsequencing and confirmed by syntheses. Frog ANP-21 was identified as an N-terminally three amino acid deleted form of frog ANP-24. Remarkable sequence homology was observed between frog ANP and mammalian ANP, especially in the regions flanked by two half-cystine residues.

Animals↗

PubMed2003

Inhibition of gonadotropin-induced oviposition and ovarian steroidogenesis in the African clawed frog (Xenopus laevis) by the pesticide methoxychlor.

Concern over the role of environmental toxicants in amphibian population declines has highlighted the need to develop more comprehensive ecotoxicological test methods for this at-risk group. With continued interest in environmental endocrine disrupters (EDs), and the paucity of data pertaining to endocrine disrupting effects in amphibia, such tests should incorporate reproductive and endocrine endpoints. We investigated the effects of in vivo exposure to the pesticide methoxychlor (MXC) on reproductive and endocrine function in adult female African clawed frogs, (Xenopus laevis). Frogs were exposed to MXC (0.5-500 microg/l) in tank water throughout a cycle of oogenesis stimulated by exogenous gonadotropins. Gonadotropin-induced oviposition was delayed, and reduced numbers of unfertilizable eggs of increased size were oviposited by frogs exposed to 500 microg/l MXC. Reduced egg output was mirrored by increased gonado-somatic index in MXC-treated frogs. Post-oviposition, plasma sex steroid profiles were altered in MXC-exposed frogs as estradiol/progesterone and estradiol/testosterone ratios were elevated. Ex vivo synthesis of progesterone by ovarian explants was significantly reduced for frogs exposed to MXC> or = 0.5 microg/l. Additionally, plasma vitellogenin concentrations were significantly depressed in frogs exposed to 500 microg/l MXC. These data indicate that reproductive and endocrine dysfunction can occur in adult amphibia exposed to high concentrations of an environmental toxin with endocrine disrupting activity. Such effects may be indicative of the potential for adverse effects on amphibian wildlife exposed to environmental EDs.

Animals↗

PubMed2003

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.

Animals↗