A large-scale comparative evaluation of DNA metabarcoding primers for profiling freshwater eukaryotic microalgal...

Algae are one of the primary bioindicators used to monitor environmental quality, and environmental DNA (eDNA) metabarcoding has greatly aided the identification of algal biodiversity. Primer performance is critically important for determining the effectiveness of biomonitoring via eDNA, yet comprehensive evaluations of metabarcoding primers for profiling eukaryotic microalgal biodiversity are lacking. We systematically evaluated a large panel of 93 primer sets targeting the 18S rRNA gene for amplifying eukaryotic algae using simulation-based in silico PCR, followed by in vitro PCR and metabarcoding analysis using 35 of the primer sets with actual eDNA sampled from diverse freshwater habitats. While many primers performed similarly in in silico PCR in terms of the number of amplified species and phylum-level distributions, their performance varied markedly in in vitro PCR, with each set detecting 0-221 algal taxa. The taxonomic specificity for algae, the coverage of different lineages, and taxonomic resolution all varied considerably among primers. The rankings of the primers in terms of their performance for detecting algal diversity differed substantially between in silico and in vitro PCR analyses. Notably, the performance of some commonly used primers for characterizing algal biodiversity was relatively poor. Our results identify candidate primer sets (e.g., V8f/1510R, TAReuk454FWD1/V4r, A-528F/B-706R, 960F/NSR1438, and A-528F/V4RB) with superior capacity for profiling eukaryotic microalgal communities, whereas no consistent pattern in primer performance was detected among the targeted gene regions (V1-V9). Furthermore, we found that the curated PR² database outperformed SILVA and GenBank for identifying algal taxa. Our results have important implications for the development of molecular tools to assess freshwater ecosystem health.