Shirako, T. | bioRxiv Science

bioRxiv2021

Development and evaluation of PCR primers for environmental DNA (eDNA) metabarcoding of Amphibia

Biodiversity monitoring is important for the conservation of natural ecosystems in general, but particularly for amphibians, whose populations are pronouncedly declining. However, amphibians ecological traits (e.g., nocturnal or aquatic) often prevent their precise monitoring. Environmental DNA (eDNA) metabarcoding--analysis of extra-organismal DNA released into the environment--allows the easy and effective monitoring of the biodiversity of aquatic organisms. Here, we developed and tested the utility of original PCR primer sets. First, we conducted in vitro PCR amplification tests with universal primer candidates using total DNA extracted from amphibian tissues. Five primer sets successfully amplified the target DNA fragments (partial 16S rRNA gene fragments of 160-311 bp) from all 16 taxa tested (from the three living amphibian orders Anura, Caudata, and Gymnophiona). Next, we investigated the taxonomic resolution retrieved using each primer set. The results revealed that the universal primer set "Amph16S" had the highest resolution among the tested sets. Finally, we applied Amph16S to actual metabarcoding and evaluated its detection capability by comparing the species detected using eDNA and physical survey (capture-based sampling and visual survey) in multiple agricultural ecosystems across Japan (160 sites in 10 areas). The eDNA metabarcoding with Amph16S detected twice as many species as the physical surveys (16 vs. 8 species, respectively), indicating the effectiveness of Amph16S in biodiversity monitoring and ecological research for amphibian communities.

ecology↗

bioRxiv2021

Identification of Lagopus muta japonica plant food resources in the Northern Japan Alps using DNA metabarcoding

DNA metabarcoding was employed to identify plant-derived food resources of the Japanese rock ptarmigan (Lagopus muta japonica), registered as a natural living monument in Japan, in the Northern Japanese Alps in Toyama Prefecture, Japan, in July to October, 2015-2018. By combined use of rbcL and ITS2 local databases of 74 alpine plant species found in the study area, a total of 43 plant taxa were identified and could be assigned to 40 species (93.0%), two genera (4.7%), and one family (2.3%). Rarefaction analysis of each sample collection period showed that this study covered more than 90% of the plant food resources found in the study area. Of the 21 plant families identified using the combined rbcL and ITS2 local databases, the most dominant families were Ericaceae (98.1% of 105 fecal samples), followed by Rosaceae (42.9%), Apiaceae (35.2%), and Poaceae (19.0%). In all fecal samples examined, the most frequently encountered plant species were Vaccinium ovalifolium var. ovalifolium (69.5%), followed by Empetrum nigrum var. japonicum (68.6%), Vaccinium sp. (54.3%), Kalmia procumbens (42.9%), and Tilingia ajanensis (34.3%). Rarefaction analysis of each collection period in the study revealed that this study covered more than 90% (from 91.0% in July to 97.5% in September) of the plant food resources found in the study area, and 98.1% of the plant food taxa were covered throughout the entire study period. Thus, DNA metabarcoding using the rbcL and ITS2 local databases of alpine plants in combination and rarefaction analysis are considered to be well suited for estimating the dominant food plants in the diet of Japanese rock ptarmigans. Further, the local database constructed in this study can be used to survey other areas with similar flora.

ecology↗