Choice of Primer Pairs Affects the eDNA-Based Detection of Eukaryotic Phytoplankton Communities

Environmental DNA (eDNA) has become a promising tool for phytoplankton surveys. However, the accuracy of eDNA-based detection is related to primer selection across diverse environments, and optimal primer pairs selection on phytoplankton community in human impacted ecosystems is still lacking. The aim of this study is to evaluate how primer selection shapes phytoplankton community profiles by eDNA biomonitoring diverse anthropogenically disturbed aquatic systems (rivers, reservoirs, and seas). Four primer pairs targeting the 18S rRNA (V9-1 and V9-2), chloroplast rbcL, and ITS regions, were explored and our results revealed that primer choice critically governed the accuracy of phytoplankton profiling. Significant variations in annotated phytoplankton eDNA sequences in different groups of primer pairs were observed, where the primers 18SV9-1 and rbcL demonstrated superior specificity, amplifying >90% of phytoplankton OTUs. 18S-targeted primers detected the highest species richness, while the ITS primer showed the lowest. Alpha diversity was highest and most consistent for 18S primers. Beta diversity ordination (nMDS/Bray–Curtis) further highlighted primer-dependent community structuring in which 18S primers effectively clustered reservoir and marine samples separately, whereas primer rbcL discriminated habitat-specific signatures across three ecosystems. The primer ITS failed to distinguish among different habitats. Overall, our data demonstrated the critical role of primer optimization in eDNA-based phytoplankton studies, and could provide methodological guidelines for the design of effective monitoring protocols in rapidly urbanizing aquatic systems.