Fang Yan’s research while affiliated with Yunnan University and other places

Environmental DNA (eDNA) metabarcoding has emerged as a powerful, non-invasive tool for biodiversity assessments. However, the accuracy and limitations of these assessment techniques are highly dependent on the choice of primer pairs being used. Although several primer sets have been used in eDNA metabarcoding studies of amphibians, there are few comparisons of their reliability and efficiency. Here, we employed lab- and field-tested sets of publicly available and de novo-designed primers in amplifying 83 species of amphibian from all three orders (Anura, Caudata, and Gymnophiona) and 13 families present in China to evaluate the versatility and specificity of these primers sets in amphibian eDNA metabarcoding studies. Three pairs of primers were highly effective, as they could successfully amplify all the major clades of Chinese amphibians in our study. A few non-amphibian taxa were also amplified by these primers, which implies that further optimization of amphibian-specific primers is still needed. The simultaneous use of three primer sets can completely cover all the species obtained by conventional survey methods and has even effectively distinguished quite a number of species (n = 20) in the Wenshan National Nature Reserve. No single primer set could individually detect all of the species from the studied region, indicating that multiple primers might be necessary for a comprehensive survey of Chinese amphibians. Besides, seasonal variations in amphibian species composition were also revealed by eDNA metabarcoding, which was consistent with traditional survey methods. These results indicate that eDNA metabarcoding has the potential to be a powerful tool for studying spatial and temporal community changes in amphibian species richness.

Many frogs undergo direct development, in which the embryo develops within the egg and lacks a tadpole stage, being hatched morphologically similar to the adults. Such reproductive behaviour ensures the evolutionary success of their lineages without the limitation of needing a moist substrate for egg deposition. The species in the genus Raorchestes are characterised as small-sized frogs that undergo terrestrial direct development. However, observations of the reproductive behaviour of many species in Raorchestes are lacking. Here, we report the first examination of bush frogs, Raorchestes longchuanensis, from southern China undergoing direct development. Under laboratory conditions, we found that R. longchuanensis can lay two clutches in a single month (as two were laid in the month of June). Only 10–12 eggs were laid per clutch. The hatching period of R. longchuanensis was 25–30 days.