Usefulness and limitations of sample pooling for environmental DNA metabarcoding of freshwater fish communities

Environmental DNA (eDNA) metabarcoding has been increasingly used for assessing biodiversity of aquatic vertebrates. However, the effective and efficient sampling design remains to be developed. Pooling of eDNA samples is one of the efficient approaches for eDNA metabarcoding, but its effectiveness has not been shown. We here tested for whether a pooling strategy can be used for evaluating biodiversity of freshwater fishes. Multiple water samples were collected in four satellite lakes of Lake Biwa, Japan. Fish communities detected by eDNA metabarcoding of the mitochondrial 12S region were compared between the individual and pooled datasets. In the individual datasets, 31, 22, 33 and 31 fish lineages (proxies for species) were observed in the respective sites, within which moderate spatial autocorrelation existed. Lineages detected in the pooled datasets were 30, 20, 29 and 27, respectively, even after 15 PCR replicates. Lineages accounting for <0.05% of the total read count of each individual dataset were mostly undetectable in the pooled dataset. Moreover, fish communities detected were similar among PCR replicates in the pooled dataset. Because of the decreased detection rates, the pooling strategy is unsuitable for estimating fish species richness. However, this procedure is potentially useful for among-site comparison of representative fish communities.