Competition Between The Tadpoles of Toads Versus Frogs: A Study of Four Japanese Anuran Taxa

Competition within and among species can play a key role in structuring the assemblages of anuran tadpoles. Previous studies have reported that tadpoles of the invasive cane toad (Rhinella marina) are more strongly disadvantaged by the presence of native frog tadpoles than by the same number of conspecific toad tadpoles. That effect might arise from a lack of coevolution of the invasive toad with its competitors; and/or from a generalized superiority of frog tadpoles over toad tadpoles. To clarify those possibilities, we conducted experimental trials using the larvae of a native rather than invasive toad (Bufo japonicus formosus in Japan) exposed to larvae of native anurans (the sympatric frogs Rana japonica and Rana ornativentris and the parapatric toad Bufo japonicus japonicus). In intraspecific competition trials, higher densities of B. j. formosus prolonged the larval period and reduced size at metamorphosis, but did not affect survival. In interspecific competition trials, the effects of the other anuran species on B. j. formosus were similar to the effects of the same number of conspecific larvae. This similarity in impact of interspecific versus intraspecific competition argues against any overall competitive superiority of frog larvae over toad larvae. Instead, the vulnerability of larval cane toads to frog tadpoles may result from a lack of coevolutionary history.

A new species of the Rana japonica group (Anura, Ranidae, Rana) from China, with a taxonomic proposal for the R. johnsi group

Rana jiulingensissp. nov., a new species from Hunan and Jiangxi, southeastern China, is described. The new species is assigned to the R. japonica group. The clade comprising R. jiulingensissp. nov. and R. dabieshanensis from Anhui is the sister taxon of R. omeimontis from Sichuan. Rana jiulingensissp. nov. can be distinguished by the significant divergences in the 16S and COI genes, and the combination of following morphological characters: body size medium, SVL 48.3–57.8 mm in adult males and 48.2–57.5 mm in adult females; dorsolateral fold straight; digits without circummarginal grooves; dorsal skin smooth; tibio-tarsal articulation reaching forward beyond the tip of snout; heels overlapping; webbing formula of toes: I 1⅓ – 2 II 1⅓ – 2⅓ III 1½ – 2⅔ IV 3 – 1⅓ V; absence of vocal sacs in males; and presence of creamy white nuptial pad with tiny hoar spines on the finger I and reddish tubercles on loreal and temporal regions in breeding males. Furthermore, based on our results and the previous literature, R. zhengi is synonymized with R. sangzhiensis, and a new species group, the Rana johnsi group, is proposed for the R. johnsi and R. sangzhiensis. Currently, the Rana contains 41 recognized species, and the phylogenetic placements of several species remain unresolved.

An application of PCR-RFLP species identification assay for environmental DNA detection

Recent advancement of environmental DNA (eDNA) methods for surveying species in aquatic ecosystems has been used for various organisms and contributed to monitoring and conservation of species and environments. Amphibians are one of the promising taxa which could be monitored efficiently by applying quantitative PCR (qPCR) or next generation sequencing to eDNA. However, the cost of eDNA detection using these approaches can be quite high and requires instruments that are not usually installed in ecology laboratories. For aiding researchers in starting eDNA studies of amphibians, especially those not specialized in molecular biology, we developed a cost efficient protocol using PCR-RFLP method. We attempted to detect eDNA of three Japanese Rana species (Rana japonica, Rana ornativentris, and Rana tagoi tagoi) in various spatial scales including an area close to the Fukushima nuclear power plant where the environment is recovering after the disaster in 2011. Our PCR-RFLP protocol was successful in detecting Rana species in static water in both laboratory and field; however, it could not detect Rana species in non-static water samples from the field. Even a more sensitive detection method (standard qPCR) was unable to detect frogs in all non-static water samples. We speculate that our new protocol is effective for frogs living in lentic habitats, but not for lotic habitats which may still require the gold standard of field observation for detection approach.

Selective constraint acting on TLR2 and TLR4 genes of Japanese Rana frogs

Toll-like receptors (TLRs) are an important component of innate immunity, the first line of pathogen defence. One of the major roles of TLRs includes recognition of pathogen-associated molecular patterns. Amphibians are currently facing population declines and even extinction due to chytridiomycosis caused by the Batrachochytrium dendrobatidis (Bd) fungus. Evidence from other vertebrates shows that TLR2 and TLR4 are involved in innate immunity against various fungi. Such genes therefore may play a functional role in amphibian-chytridiomycosis dynamics. Frogs from East Asia appear to be tolerant to Bd, so we examined the genetic diversity that underlies TLR2 and TLR4 from three Japanese Ranidae frog species, Rana japonica, R. ornativentris and R. tagoi tagoi (n = 5 per species). We isolated 27 TLR2 and 20 TLR4 alleles and found that these genes are evolutionarily conserved, with overall evidence supporting purifying selection. In contrast, site-by-site analysis of selection identified several specific codon sites under positive selection, some of which were located in the variable leucine rich repeat domains. In addition, preliminary expression levels of TLR2 and TLR4 from transcriptome data showed overall low expression. Although it remains unclear whether infectious pathogens are a selective force acting on TLRs of Japanese frogs, our results support that certain sites in TLRs of these species may have experienced pathogen-mediated selection.

Novel Vasotocin-Regulated Aquaporins Expressed in the Ventral Skin of Semiaquatic Anuran Amphibians: Evolution of Cutaneous Water-Absorbing Mechanisms

Until now, it was believed that only one form of arginine vasotocin (AVT)-regulated aquaporin (AQP) existed to control water absorption from the ventral skin of semiaquatic anuran amphibians, eg, AQP-rj3(a) in Rana japonica. In the present study, we have identified a novel form of ventral skin-type AQP, AQP-rj3b, in R. japonica by cDNA cloning. The oocyte swelling assay confirmed that AQP-rj3b can facilitate water permeability. Both AQP-rj3a and AQP-rj3b were expressed abundantly in the ventral hindlimb skin and weakly in the ventral pelvic skin. For the hindlimb skin, water permeability was increased in response to AVT, although the hydroosmotic response was not statistically significant in the pelvic skin. Isoproterenol augmented water permeability of the hindlimb skin, and the response was inhibited by propranolol. These events were well correlated with the intracellular trafficking of the AQPs. Immunohistochemistry showed that both AQP-rj3 proteins were translocated from the cytoplasmic pool to the apical membrane of principal cells in the first-reacting cell layer of the hindlimb skin after stimulation with AVT and/or isoproterenol. The type-b AQP was also found in R. (Lithobates) catesbeiana and R. (Pelophylax) nigromaculata. Molecular phylogenetic analysis indicated that the type-a is closely related to ventral skin-type AQPs from aquatic Xenopus, whereas the type-b is closer to the AQPs from terrestrial Bufo and Hyla, suggesting that the AQPs from terrestrial species are not the orthologue of the AQPs from aquatic species. Based on these results, we propose a model for the evolution of cutaneous water-absorbing mechanisms in association with AQPs.