Hibernation behavior of Rana lessonae and R. esculenta in their natural habitat

We studied the hibernation behavior of the water frog Rana lessonae and its hybridogenetic associate R. esculenta in their natural habitat during three successive winters. Animals caught in pitfall traps at a fenced pond were individually marked with PIT tags and some (n=36) were additionally equipped with radio transmitters. Of the animals caught, 85% left the fenced pond for hibernation. More R. esculenta remained inside the fenced area compared to R. lessonae. R. lessonae emigrated earlier in autumn and came back later in spring than R. esculenta, but the distance to their hibernation sites did not differ. Both species left the fenced pond earlier in the year when ambient temperatures were lower. All radio-tracked animals hibernated in woodland, 3–7 cm below the surface in soil, under moss, fallen leaves or small branches. Soil temperatures at the actual hibernation sites were significantly higher than at randomly chosen control sites. A surprising finding was that most frogs changed their hibernation sites during winter, and often more than once. Movements were more frequent in the warmer first half of the winter than in the cooler second half, but some animals were active even on days with mean temperatures below 1°C. These results show that both species do not spend the whole winter torpid in one particular hibernation site but move around, especially at higher temperatures. Most of the animals lost weight during the winter, and the weight loss was greater in females than in males and higher in warm than in cold winters. To what extent weight loss and survival is influenced by the chosen hibernation sites and the amount of movement during winter, and whether this contributes to the differences in species and sex ratios found in mixed populations, needs more investigation. Received: 4 August 1999 / Accepted: 15 November 1999     

Regional specificity of anuran larval skin during metamorphosis: dermal specificity in development and histolysis of recombined skin grafts

Summary Skins from back and tail were dissected from tadpoles of Rana japonica prior to resorption of the tail and separated into epidermis and dermis by treatment with neutral protease. Homotypically and heterotypically recombined skins were constructed from the separated epidermis and dermis and transplanted into the tail of the original tadpole. Skin grafts using dermis from tail region degenerated simultaneously with resorption of the tail. However, skin grafts containing dermis from back region survived on the posterior part of the juvenile frog beyond metamorphosis. Furthermore, all epidermis underlaid with dermis from back region formed secretory glands and became flattened epithelia characteristic of adult back skin, regardless of region from which the epidermis came. Even when epidermis isolated from tail skin was cultured inside a back skin graft, the tail epidermis survived forming an epithelial cyst and developed secretory glands. These results suggest that regional specificities of anuran larval skin, i.e., development of back skin and even histolysis of tail skin, are determined by regionally specific dermis. The results also suggest that some of epidermal cells of tail skin are able to differentiate into epithelial cells similar to back skin of the adult under the influence of back dermis.     

Control and interaction of the cardiovascular and respiratory systems in anuran amphibians

In anuran amphibians, respiratory rhythm is generated within the central nervous system (CNS) and is modulated by chemo- and mechanoreceptors located in the vascular system and within the CNS. The site for central respiratory rhythmogenesis and the role of various neurotransmitters and neuromodulators is described. Ventilatory air flow is generated by a positive pressure, buccal force pump driven by efferent motor output from cranial nerves. The vagus (cranial nerve X) also controls heart rate and pulmocutaneous arterial resistance that, in turn, affect cardiac shunts within the undivided anuran ventricle; however, little is known about the control of central vagal motor outflow to the heart and pulmocutaneous artery. Anatomical evidence indicates a close proximity of the centers responsible for respiratory rhythmogenesis and the vagal motoneurons involved in cardiovascular regulation. Furthermore, anurans in which phasic feedback from chemo- and mechanoreceptors is prevented by artificial ventilation exhibit cardiorespiratory interactions that appear similar to those of conscious animals. These observations indicate interactions between respiratory and cardiovascular centers within the CNS. Thus, like mammals and other air-breathing vertebrates, the cardio-respiratory interactions in anurans result from both feedback and feed-forward mechanisms.     

Modality interactions alter the shape of acoustic mate preference functions in gray treefrogs

Sexual selection takes place in complex environments where females evaluating male mating signals are confronted with stimuli from multiple sources and modalities. The pattern of expression of female preferences may be influenced by interactions between modalities, changing the shape of female preference functions, and thus ultimately altering the selective landscape acting on male signal evolution. We tested the hypothesis that the responses of female gray treefrogs, Hyla versicolor, to acoustic male advertisement calls are affected by interactions with visual stimuli. We measured preference functions for several call traits under two experimental conditions: unimodal (only acoustic signals presented), and multimodal (acoustic signals presented along with a video‐animated calling male). We found that females were more responsive to multimodal stimulus presentations and, compared to unimodal playbacks, had weaker preferences for temporal call characteristics. We compared the preference functions obtained in these two treatments to the distribution of male call characteristics to make inferences on the strength and direction of selection expected to act on male calls. Modality interactions have the potential to influence the course of signal evolution and thus are an important consideration in sexual selection studies.     

Cryopreservation of spermatozoa of the terrestrial Puerto Rican frog, Eleutherodactylus coqui

World-wide reports of amphibian population declines have led to increased interest in the reproductive biology of anurans. As a model system, here we present evidence for the effective cryoprotection of sperm from the Puerto Rican frog, Eleutherodactylus coqui, using mixtures of fetal bovine serum (FBS) and dimethylsulfoxide (Me(2)SO), glycerol or sucrose extenders. Using a fluorescent dye exclusion assay, we found that 53.9 and 50.4% of all sperm with intact membranes prior to freezing maintained membrane integrity after rapid freezing and thawing when protected with either a FBS/glycerol or FBS/sucrose solution, respectively. The methods reported here may be useful for similar work with many of the more than 700 other species in this genus.     

Evolutionary history of the northern leopard frog: reconstruction of phylogeny, phylogeography, and historical changes in population demography from mitochondrial DNA

This study uses a combined methodological approach including phylogenetic, phylogeographic, and demographic analyses to understand the evolutionary history of the northern leopard frog, Rana pipiens. We tested hypotheses concerning how (or if) known geological events and key features of the species biology influenced the contemporary geographic and genetic distribution of R. pipiens. We assayed mitochondrial DNA variation from 389 individuals within 35 populations located throughout the species range. Our a priori expectations for patterns and processes influencing the current genetic structure of R. pipiens were supported by the data. However, our analyses revealed specific aspects of R. pipiens evolutionary history that were unexpected. The phylogenetic analysis indicated that R. pipiens is split into populations containing discrete eastern or western haplotypes, with the Mississippi River and Great Lakes region dividing the geographic ranges. Nested clade analysis indicated that the biological process most often invoked to explain the pattern of haplotype position is restricted gene flow with isolation by distance. Demographic analyses showed evidence of both historical bottlenecks and population expansions. Surprisingly, the genetic evidence indicated that the western haplotypes had significantly reduced levels of genetic diversity relative to the eastern haplotypes and that major range expansions occurred in both regions well before the most recent glacial retreat. This study provides a detailed history of how a widespread terrestrial vertebrate responded to episodic Pleistocene glacial events in North America. Moreover, this study illustrates how complementary methods of data analysis can be used to disentangle recent and ancient effects on the genetic structure of a species.     

Leaf litter input mediates tadpole performance across forest canopy treatments

Understanding the mechanisms limiting the distributions of organisms is necessary for predicting changes in community composition along habitat gradients. In many areas of the USA, land originally cleared for agriculture has been undergoing a process of reforestation, creating a gradient of canopy cover. For small temporary wetlands, this gradient can alter abiotic conditions and influence the resource base of wetland food webs by affecting litter inputs. As distributions of amphibians and many other temporary wetland taxa correlate with canopy cover, we experimentally manipulated shade levels and litter types in pond mesocosms to explore mechanisms limiting species performance in wetlands with canopy cover. Most differences between ponds were mediated by litter type rather than direct effects of shading. Although all three amphibian species tested are open-canopy specialists, spring peepers were the only species to show decreased survival in shaded ponds. Pond litter type generally had strong effects on growth and development rates, with tadpoles of two species in grass litter ponds growing to twice the size of, and metamorphosing 7 days earlier than, those in leaf litter ponds. Contrary to our initial hypothesis, shade level and litter type showed very few significant interactions. Our results indicate that the effects of shading cannot be considered in isolation of vegetation changes in pond basins when evaluating the effects of forest succession on temporary pond communities.     

Microsatellite markers in the endangered Australian northern corroboree frog, Pseudophryne pengilleyi (Anura: Myobatrachidae) and amplification in other Pseudophryne species

Seven microsatellite primer pairs were isolated and characterized in the endangered Australian northern corroboree frog (Pseudophryne pengilleyi). All seven were polymorphic (2–14 alleles) and displayed high heterozygosity (0.036–0.964) in 28 sampled individuals. We also tested the microsatellites on two closely related species. Four were polymorphic in the southern corroboree frog (P. corroboree) and Bibron’s toadlet (P. bibronii). These primers will be useful in studies of conservation genetics and mating systems in Pseudophryne species.     

Genes induced during the early developmental stages of the Cane Toad, Bufo (Chaunus) marinus

Metamorphosis, a critical stage in the development of toads and frogs, involves rapid levels of morphological change. In the current study, we have used microarray analysis to identify shifts in gene expression between tadpole and toadlet stages of the cane toad, Bufo (Chaunus) marinus. Here, we report on nine genes that show the greatest induction during metamorphosis; the gut-associated gastrokine and trefoil factor, blood components haemoglobins alpha/beta, apolipoprotein and serum albumin, a nasal gene olfactomedin, a lens gene gamma-crystallin, and a novel gene with low homology to frog harderin. We present both temporal and spatial expression patterns of these genes identified in developing and adult cane toads. This study extends our knowledge of the molecular basis of toad metamorphosis, and not only offers insights to the genes induced during the general remodelling that occurs but also reveals possible targets for control and manipulation of amphibian pest species, for example, the cane toad in Australia.     

Sperm competition selects for increased testes mass in Australian frogs

Game theory predicts that investment in spermatogenesis will increase with the risk and intensity of sperm competition. Widespread support for this prediction has come from comparative studies of internal fertilizing species reporting positive associations between testes mass and the probability that females mate with more than one male. Data for external fertilizers have generated conflicting results. We investigated how risk of sperm competition affects testes size in two families of Australian frogs: the Myobatrachidae and the Hylidae. We also examined effects of clutch size, egg size and oviposition location as alternative factors that might influence sperm production. Species were ranked according to probability of group spawning, and hence risk of sperm competition. Controlling for body size and phylogenetic relationships, we demonstrated that within the Myobatrachidae, the risk of sperm competition explained a significant amount of variation in testes mass. Oviposition location had a weak influence, with species ovipositing into foam having smaller testes. No significant effects of clutch size or egg size were detected. In hylids, the relationship between testes mass and risk of sperm competition was positive but not significant, again with no predictable effects related to egg size or number. These data provide an important test of sperm competition theory for externally fertilizing taxa.