Abstract
Environmental DNA (eDNA) metabarcoding has been used increasingly to assess biodiversity of aquatic vertebrates. However, there still remains to be developed a sampling design of eDNA metabarcoding that can ensure high detection rates of species with minimum total survey effort, especially for large-scale surveys of aquatic organisms. We here tested whether pooling of eDNA samples can be used to evaluate biodiversity of freshwater fishes 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 samples. In the individual samples, 31, 22, 33, and 31 fish lineages (proxies for species) were observed at the respective sites, within which moderate spatial autocorrelation existed. In the pooled samples, 30, 20, 29, and 27, lineages were detected, respectively, even after 15 PCR replicates. Lineages accounting for < 0.05% of the total read count of each site’s individual samples were mostly undetectable in the pooled samples. Moreover, fish communities detected were similar among PCR replicates in the pooled samples. Because of the decreased detection rates, the pooling strategy is unsuitable for estimating fish species richness. However, this procedure is useful potentially for among-site comparison of representative fish communities.
Highlights
Knowledge of species distribution is essential for understanding community dynamics and biodiversity patterns, and for planning management and conservation of threatened and endangered species[1,2,3]
The results indicated that use of a pooling strategy leads to saving labor and to missed detection of fish lineages that were detected in the individual samples, resulting in a slight decrease in detection rates in environmental DNA (eDNA) metabarcoding (Table 2; Figs 2–4)
Negative effects are less pronounced for studies of freshwater fish compared to microbes. These findings suggested that the pooling strategy in eDNA metabarcoding is potentially useful for among-site comparison of representative communities of freshwater fishes and other aquatic vertebrates
Summary
Knowledge of species distribution is essential for understanding community dynamics and biodiversity patterns, and for planning management and conservation of threatened and endangered species[1,2,3]. The analysis of eDNA using gel electrophoresis or quantitative PCR (qPCR) has proven highly successful for targeted detection of one or a few species inhabiting various aquatic environments[10,11,13,14,15,16,17] These PCR-based approaches are powerful tools for monitoring target species, they cannot be used for assessing community composition of organisms. Use of small sample sizes may lead to underestimation of biodiversity because recent eDNA metabarcoding studies indicated that eDNA in aquatic systems appears to be spatially autocorrelated at large spatial scales[31,33,36] (and see[38]) Another labor-saving method is pooling of samples collected from multiple locations before DNA extraction. Communities of aquatic macroorganisms are obviously much less complex than microbial communities in soil and, we hypothesized that pooling of eDNA samples potentially might be effective to assess biodiversity of freshwater fishes
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