Environmentally induced limb malformations in mink frogs (Rana septentrionalis).

In recent years, there has been an increase in the incidence of frog deformities throughout many of the northern states of North America. The most readily noticed malformations involve the hindlimbs of peri-metamorphic animals. We have analyzed skeletal preparations of metamorphosing mink frogs (Rana septentrionalis) collected from a site in Minnesota, in order to develop a better understanding of the possible causes. In this paper we describe the categories of abnormalities found at this site. The spectrum of deformities includes missing limbs, truncated limbs, extra limbs (including extra pelvic girdles), and skin webbings. We also describe a newly recognized malformation of the proximal-distal limb axis, a bony triangle. In this abnormality, the proximal and distal ends of the bone are adjacent to one another forming the base of a triangle. The shaft of the bone is bent double and protrudes laterally, the midpoint of the bone forming the apex of the triangle. In this paper we consider several recently proposed explanations for the recent outbreak of amphibian deformities. Based on our analysis, we conclude that the spectrum of abnormalities seen in these frogs is remarkably similar to the range of abnormalities that has been reported as a result of exposure of developing vertebrates to exogenous retinoids. Given the potential implications of this possibility for the welfare of humans as well as wildlife, further studies are needed to determine whether environmental retinoids are responsible for the frog deformities at the site we have examined.     

Antimicrobial properties of two purified skin peptides from the mink frog (Rana septentrionalis) against bacteria isolated from the natural habitat

Numerous peptides exhibiting antimicrobial properties have been isolated from the skins of many amphibian species. These peptides offer an innate chemical defense system against various microbial agents that exist in the amphibian's environment. Amphibian skin peptides are typically tested for antimicrobial activity against microbial strains that are pathogenic to humans, but not on potential pathogenic or opportunistic bacteria that exist in the organism's habitat. Two peptides, a brevinin-2-related peptide and temporin-1SPb previously isolated from secretions of the mink frog, Rana septentrionalis, were tested for antimicrobial activity on bacterial isolates endemic to the frog's habitat. Ten isolates were identified, using 16S rRNA gene sequencing techniques, in the genera Pseudomonas, Serratia, Bacillus, Aeromonas, Burkholderia, Microbacterium, and Delftia. Bacterial isolates were tested with peptides at concentrations ranging from 0.8 microM to 1000 microM to determine the minimum inhibitory concentration (MIC) to inhibit growth. Growth of four of the isolates was inhibited by temporin-1SPb at the concentrations used, but all of the isolates were inhibited by the brevinin-2-related within the range of peptide concentrations used. This demonstrates the efficacy of both peptides as a component of the frog's innate chemical defense system.     

Purification and characterization of antimicrobial peptides from the skin secretions of the mink frog (Rana septentrionalis)

Skin secretions were obtained from male, female, and juvenile specimens of the mink frog (Rana septentrionalis) by electric stimulation and shown to contain 10 peptides that differentially inhibited the growth of microorganisms. The elution profiles of secretions from the three groups following reverse-phase HPLC were almost identical indicating that there were no major sexual or developmental differences in chemical composition. Four peptides of the brevinin-1 family, with potent antimicrobial activity and strong hemolytic activity, two members of ranatuerin-2 family and three members of the temporin family, were purified and characterized structurally. A 21-amino-acid C-terminally alpha-amidated peptide (GIWDTIKSMGKVFAGKILQNL.NH(2)) with broad-spectrum antimicrobial activity was also isolated from the skin secretions. This peptide shows limited structural similarity with the N-terminal region of brevinin-2 peptides previously isolated from R. temporaria skin but lacks the C-terminal cyclic heptapeptide domain associated with this family. Molecular and morphological data support the placement of R. septentrionalis in the R. catesbeiana species group, but analysis based upon the distribution of the molecular forms of the antimicrobial peptides is indicative of a closer phylogenetic relationship between R. septentrionalis and the frogs of the R. pipiens and R. boylii groups.     

Sex differences in age structure, growth rate and body size of common frogs Rana temporaria in the subarctic

The thermal environment and length of the activity season are important factors in shaping life-history trait variation in ectotherms. Many ectothermic vertebrates living at high latitudes or altitudes tend to be larger and older than their conspecifics living at lower latitudes or altitudes. However, detailed data on age, body size and growth variation—and how they may differ between males and females—are still scarce, especially from extreme high-latitude environments. We studied growth (body length increment), age and size structure of common frogs (Rana temporaria) in subarctic Finland (69°04′N) by applying skeletochronological methods to individually marked adults (n?=?169) captured and recaptured between 1999 and 2003. We found that breeding males were on average younger (mean?=?8.5?years) than females (11.9?years) and that males started reproducing earlier (≥3–4?years of age) than females (>4–5?years). The oldest encountered individual was an 18-year-old female, which to our knowledge is the oldest wild common frog ever reported. Females were on average larger (mean body length?=?76.6?mm) than males (70.7?mm), and this appeared to be mainly due to their older age as compared to males. While body length increased and growth rate decreased with age in both sexes, growth rate declined significantly faster with age in males than in females. The latter finding provides a proximate explanation for the observation that even after accounting for age differences among sexes (females?>?males), females were longer than males.     

Loss of conspicuous coloration has co-evolved with decreased body size in populations of poison dart frogs

Larger signal size is known to facilitate the learning process of predators to warning signals. Further, smaller objects are generally harder to detect than large, which suggests that smaller sized prey are less likely to benefit from an aposematic strategy compared to crypsis. However, whether body size changes in concert with shifts between crypsis and aposematism in natural populations, remains largely unexplored. I tested whether body size was larger in visually conspicuous population than in cryptic populations among recently diverged populations of the Strawberry Poison frog, Oophaga pumilio. By analysing spectral reflectance and body size data from individuals from 18 discrete populations I found a larger mean body size in conspicuous populations, which was confirmed by an analysis of a subset of 12 populations accounting for phylogenetic history. This shows that the loss of conspicuous colour likely co-evolved repeatedly with a decrease in body size. Thus, selection on body size may influence evolutionary shifts between aposematism and crypsis and vice versa.     

Reproduction and hybrid load in all-hybrid populations of Rana esculenta water frogs in Denmark

All-hybrid populations of the water frog, Rana esculenta, are exceptional in consisting of independently and to some extent sexually reproducing interspecific hybrids. In most of its range R. esculenta reproduces hemiclonally with one of the parental species, R. lessonae or R. ridibunda, but viable populations of diploid and triploid hybrids, in which no individuals of the parental species have been found, exist in the northern part of the range. We test the hypothesis that nonhybrids arise every year in these all-hybrid populations, but die during larval development. Microsatellite markers were used to determine the genotypes of adults and abnormal and healthy offspring in three all-hybrid populations of R. esculenta in Denmark. Of all eggs and larvae, 63% developed abnormally or died, with some being nonhybrid (genomes matching one of the parental species), many being aneuploid (with noninteger chromosome sets), a few being tetraploid, and many eggs possibly being unfertilized. The 37% surviving and apparently healthy froglets were all diploid or triploid hybrids. In all three populations, gametogenesis matched the pattern previously described for all-hybrid R. esculenta populations in which most triploid adults have two R. lessonae genomes. This pattern was surprising for the one population in which triploid adults had two R. ridibunda genomes, because here it leads to a deficiency of gametes with an R. lessonae genome and should compromise the stability of this population. We conclude that faulty gametogenesis and mating between frogs with incompatible gametes induce a significant hybrid load in all-hybrid populations of R. esculenta, and we discuss compensating advantages and potential evolutionary trajectories to reduce this hybrid load.     

Growth, maturation and survival of frogs Rana temporaria L.

A total of 1560 breeding frogs were collected in three consecutive years at sites in the west of Ireland. Alt were weighed and measured and the ages of 702 individuals were determined by skeletochronology. The length-weight relationship differed between gravid females, spent females and males, with the gravid females showing the largest weight gain for any increase in length. The majority of males bred for the first time at two years, but most females delayed maturity until they were a year older. While the two-year olds were of similar size, the females subsequently grew at a faster rate and achieved larger maximal sizes than the males. However, both sexes exhibited a decelerating growth rate with age. There was no apparent difference between the sexes in post-maturation survival, which was estimated to be about 50% annually. The oldest frogs were seven years old.     

Helminth communities of two green frogs (Rana perezi and Rana saharica) from both shores of the Alboran Sea

The helminth communities of two populations of green frogs from both shores of the Alborón Sea (Western Mediterranean) were studied. Of the 79 frogs examined for helminths, 39 individuals of the species Rano saharica were collected from Bob-Taza (Morocco), and 40 of the species Rona perezi were collected from the Natural Park of the Sierra de Grazalema (Spain). Although the species richness of helminths was identical in the two sampled areas, the differences observed in the structure of the helminth infracommunities were quite important. Statistically, significant differences were found between the species richness and the diversity of the infracommunities of R. perezi female population and the other three studied statistical populations. The helminth component communities of these two green frogs can be considered as depauperate, although their infracommunities present interactive features.     

Patterns of orientation behavior in marsh frogs (Rana ridibunda Pall.) of southern populations

The behavior of frogs from southern areas with an arid climate released during the reproductive period between their “home” pond and a less distant river is shown to be independent of weather conditions. The experiments were performed on frogs living near the village of Dosang, Astrakhan oblast. The frogs were released 60 to 150 m from their “home” breeding waterbody and 60 to 80 m from an “alien” waterbody. Four experiments were performed on 27 individuals. The movements of the frogs were traced by the method of “tracking by a thread.” The results revealed no preference in the frogs to return to their own breeding ponds, rather than to the nearby river. This behavior is peculiar to frogs of southern populations.     

Small body size increases the regional differentiation of populations of tropical mantellid frogs (Anura: Mantellidae)

The processes affecting species diversification may also exert an influence on patterns of genetic variability within species. We evaluated the contributions of five variables potentially influencing clade diversification (body size, reproductive mode, range size, microhabitat and skin texture) on mtDNA divergence and polymorphism among populations of 40 species of frogs (Mantellidae) from two rainforest communities in Madagascar. We report an inverse association between body size and nucleotide divergence between populations but find no influence of other variables on genetic variation. Body size explained ca. 11% of the variation in nucleotide divergence between populations and was coupled with high F_ST levels and an absence of haplotype sharing in small‐bodied and medium‐sized frogs. Low dispersal ability is likely the proximate mechanism producing higher population differentiation in small mantellids. The lack of genetic cohesion among populations establishes regional genetic fragmentation which in turn has the potential to accelerate rates of allopatric speciation in small frogs relative to large species. However, there is little evidence of increased speciation rates in these or other small‐bodied organisms. We reconcile these contradictory observations by suggesting that lower dispersal ability also curbs colonization of new areas, decelerating diversification in weak dispersers. Our results imply that the intermediate dispersal model also applies to amphibians and may explain inconsistent previous results on the correlation of body size and speciation rate.     

Cerebellar afferents in the frogs,Rana esculenta and Rana temporaria

Summary Afferents to the cerebellum in frogs (Rana esculenta, Rana temporaria) were studied by use of retrograde transport of horseradish peroxidase. Following injections restricted to the molecular layer of the cerebellum cell labelling was found in the contralateral inferior olive and the ventral portion of the caudal medullary raphe. Injections involving the granular layer resulted in labelling in the ventral horn of the cervical spinal cord, the caudal spinal trigeminal nucleus, the nucleus caudalis and the medial portion of the nucleus ventralis of the vestibular nerve, the inferior reticular nucleus and the nucleus of the fasciculus longitudinalis medialis. Following larger injections, which may have spread significantly into the cerebellar, secondary gustatory, trigeminal or vestibular nuclei, labelled cell bodies were also found in the nucleus ruber, nucleus solitarius, the rostral spinal trigeminal nucleus and the rostral rhombencephalic reticular formation. It is unclear whether the fibers from these latter areas innervate the cerebellum of the frog, as they do in mammals, or only reach the underlying areas. This situation emphasizes a limitation of the HRP technique when applied to small structures as is often the case in lower vertebrates.Supported by Grant Gr 276 to U. G.-C. from the Deutsche Forschungsgemeinschaft.     

Helminth infracommunities of the frogs Rana catesbeiana and Rana clamitans from Turkey Marsh, Michigan

One hundred twenty-seven bullfrogs, Rana catesbeiana, and 120 green frogs, Rana clamitans, collected in July and August 1987 from Turkey Marsh, Michigan, were examined for helminths. Of the 16 helminth species found, Falcaustra catesbeianae had the highest prevalence and mean intensity in bullfrogs. In green frogs, Megalodiscus temperatus had the highest prevalence and Haematoloechus parviplexus had the highest mean intensity. Taxonomically, 1,030 (30.5%) trematodes and 2,348 (69.5%) nematodes occurred in bullfrogs; 2,874 (96.4%) trematodes, 2 (0.1%) cestodes, and 105 (3.5%) nematodes were found in green frogs. The larger and more aquatic of the 2 hosts, R. catesbeiana, had the highest mean number of helminth species, helminth intensity, diversity, and evenness. Adult frogs of both species had a significantly higher mean number of helminth species than did juveniles. Significant correlations existed between the number of helminth species and frog length. Although the helminth communities of Turkey Marsh frogs are isolationist in character, they are more diverse than other helminth communities studied in amphibians.     

The interspecific range size–body size relationship in Australian frogs

Aim There is substantial residual scatter about the positive range size–body size relationship in Australian frogs. We test whether species’ life history and abundance can account for this residual scatter. Location Australia. Methods Multiple regressions were performed using both cross‐species and independent contrasts analyses to determine whether clutch size, egg size and species abundance account for variation in range size over and above the effects of body size. Results In both cross‐species and independents contrasts models with body size, clutch size and egg size as predictors, partial r² values revealed that only egg size was significantly and uniquely related to range size. Contrary to expectation, neither body size nor clutch size could account for significant variation in range size. Incorporating species abundance as a predictor in further multiple regression analysis demonstrated that while abundance accounted for a significant proportion of range size variation, the contribution of egg size was reduced but still significant. Notably, non‐significant relationships persisted between range size and both body size and clutch size. Conclusions The weak positive correlation between body size and range size in Australian frogs disappears after accounting for species abundance and egg size. Our findings demonstrate that species with both high local abundance and small eggs occupy comparatively wider geographical ranges than species with low abundance and large eggs.     

Choosy females and indiscriminate males: mate choice in mixed populations of sexual and hybridogenetic water frogs (Rana lessonae, Rana esculenta)

For several decades, behavioral ecologists have studied theeffects of the environment on the behavior of individuals;but only fairly recently they have started to ask the reversequestion: how do the behavioral strategies of individuals affectthe composition and dynamics of populations and communities?Although intuitively obvious, this feedback from individualto higher levels is difficult to demonstrate, except in systemswith exceptionally fast and marked responses of the populationsto the behavior of its members. Such a system exists in sperm-dependentspecies. In European water frogs, for instance, successfulreproduction of a hybrid species (R. esculenta, genotype LR)requires mating with one of its parental species (R. lessonae,genotype LL), except in the rare cases where hybrids are triploid.The sexual host LL, however, should avoid matings with the sexual parasite LR, because the resulting LR offspring willeliminate the L genome from their germ line. In this studywe investigate how this conflict is solved. Since water froghybrids come in both sexes, rather than as females only likein other sperm-dependent systems, we performed the tests withboth females and males. One individual was given a choice betweentwo individuals of the opposite sex, one an LL and the otheran LR. In both species, females showed the predicted preferencefor LL males, whereas males did not discriminate between LLand LR females. On the individual level, we interpret the sexdifference in choosiness by the lower costs from mating withthe wrong species (LR) and the higher benefits from matingwith large individuals in males than in females. In "normal"species, male preference for large (i.e. more fecund) femalesis advantageous, but in this system such a choice can resultin mating with the larger LR females. With respect to the structureand dynamics of mixed populations, we discuss that the observed female preference is consistent with the higher mating successof LL males found in nature. Hence, mate female choice is astrong candidate for a mechanism promoting coexistence of thesperm-dependent hybrid and its sexual host. This confirms predictionsfrom previous theoretical models.     

A study on hearing in frogs (Rana pipiens and Rana clamitans)

Zusammenfassung Die relative Reizschwellkurve für die Schallempfindung bei den Fröschen Rana clamitans und R. pipiens wurde in dem Frequenzbereich von 30–15000 Hz bestimmt. Die Kurve ist dieselbe für beide Geschlechter. Versuche zeigten, daß Schälle entweder inhibierend oder stimulierend auf die Atmungsbewegungen wirken können. Diese Wirkung wird nicht von der Frequenz oder der Intensität der Schälle bestimmt, sondern von dem physiologischen Zustand des Tieres.Die Latenzzeit zwischen Reiz und Effekt ist unabhängig von der Tonfrequenz, wird aber von den Intervallen zwischen aufeinanderfolgenden Reizen beeinflußt; sie wird kürzer, wenn die Intervalle verlängert werden.Dressurversuche mit 18 Tieren, unter Anwendung von Strafung durch elektrische Schläge und einem Dressurton von 300 Hz, ergaben positive Erfolge bei zwei Individuen von R. clamitans.

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Contribution No. 67 of the Department of Biology, Hamilton College, McMaster University, Hamilton, Ontario.     

Correlated evolution of conspicuous coloration and body size in poison frogs (Dendrobatidae)

Conspicuous coloration is often used in combination with chemical defenses to deter predators from attacking. Experimental studies have shown that the avoidance inducing effect of conspicuous prey coloration increases with increasing size of pattern elements and with increasing body size. Here we use a comparative approach to test the prediction from these findings, namely that conspicuous coloration will evolve in tandem with body size. In our analysis, we use a previously published mitochondrial DNA-based phylogeny and comparative analysis of independent contrasts to examine if evolutionary shifts in color pattern have been associated with evolutionary changes in body size in aposematic poison frogs (Anura: Dendrobatidae). Information on body size (snout to vent length) and coloration were obtained from the literature. Two different measures of conspicuousness were used, one based on rankings by human observers and the other based on computer analysis of digitized photographs. The results from comparative analyses using either measure of coloration indicated that avoidance inducing coloration and body size have evolved in concert in poison frogs. Results from reconstruction of character change further indicate that the correlated evolution of size and coloration has involved changes in both directions within each of the different clades of the phylogenetic tree. This finding is consistent with the hypothesis that selection imposed by visually guided predators has promoted the evolution of larger body size in species with conspicuous coloration, or enhanced evolution of conspicuous coloration in larger species.     

Elevational patterns of species richness, range and body size for spiny frogs

Quantifying spatial patterns of species richness is a core problem in biodiversity theory. Spiny frogs of the subfamily Painae (Anura: Dicroglossidae) are widespread, but endemic to Asia. Using spiny frog distribution and body size data, and a digital elevation model data set we explored altitudinal patterns of spiny frog richness and quantified the effect of area on the richness pattern over a large altitudinal gradient from 0-5000 m a.s.l. We also tested two hypotheses: (i) the Rapoport's altitudinal effect is valid for the Painae, and (ii) Bergmann's clines are present in spiny frogs. The species richness of Painae across four different altitudinal band widths (100 m, 200 m, 300 m and 400 m) all showed hump-shaped patterns along altitudinal gradient. The altitudinal changes in species richness of the Paini and Quasipaini tribes further confirmed this finding, while the peak of Quasipaini species richness occurred at lower elevations than the maxima of Paini. The area did not explain a significant amount of variation in total, nor Paini species richness, but it did explain variation in Quasipaini. Five distinct groups across altitudinal gradient were found. Species altitudinal ranges did not expand with an increase in the midpoints of altitudinal ranges. A significant negative correlation between body size and elevation was exhibited. Our findings demonstrate that Rapoport's altitudinal rule is not a compulsory attribute of spiny frogs and also suggest that Bergmann's rule is not generally applicable to amphibians. The study highlights a need to explore the underlying mechanisms of species richness patterns, particularly for amphibians in macroecology.