Laying Down the Foundations for Qualitative and Quantitative Freshwater Zooplankton Metabarcoding Surveys

Metabarcoding has been proven to be a highly effective tool for surveying biodiversity in aquatic ecosystems. Despite the recent explosion of metabarcoding studies, relatively few efforts have focused on freshwater zooplankton communities, even though they form an essential component of freshwater ecosystems. Here, we evaluate some essential aspects of metabarcoding surveys to provide a solid basis for the development of qualitative and quantitative metabarcoding surveys for freshwater zooplankton communities. We developed and validated taxon‐specific and universally applicable metabarcoding primers for Cladocera , Copepoda and Rotifera . These primers were subsequently used to assess optimal sample collection, preservation and DNA extraction protocols and gain insights into the key biases that may influence the interpretation of freshwater zooplankton metabarcoding results. The presented primers performed well when applied to both bulk community samples and eDNA samples, although the latter seemed to slightly decrease the performance of metabarcoding analyses. We found significant effects of subsample size, sample preservation, DNA extraction and lysis methods for bulk community samples with inadequate protocols likely to underestimate Cladocera diversity. Primer amplification efficiency was found to be the primary driver of biases in the quantitative interpretation of metabarcoding data, regardless of the taxonomic target or sequencing depth. General correction factors based on primer amplification efficiency may thus be sufficient to enhance the quantitative nature of metabarcoding surveys.