Phytoplankton play a vital role as primary producers in freshwater ecosystems, contributing to the nutrient cycle, energy flow, and ecological stability. To accurately assess phytoplankton diversity and community composition, this study compared traditional microscopy and environmental DNA (eDNA) metabarcoding in six small lakes located in the Han, Geum, and Nakdong River basins in Korea. eDNA analysis identified 268 species from 161 genera, approximately 2.4 times higher than microscopy, which detected 113 species from 68 genera. The eDNA data were dominated by picocyanobacteria such as Synechococcus and Cyanobium, while microscopy primarily revealed larger taxa, including Stephanodiscus and Scenedesmus. Nonmetric multidimensional scaling (NMDS) based on Bray-Curtis similarity showed clear separation between the two methods, with average similarity values of 0.0326 (1st survey) and 0.0221 (2nd survey) at the species level. Only 6.8% of the 429 total species were commonly detected by both methods, while overlap at the genus level was 18.8%. Spatial heterogeneity in phytoplankton communities based on eDNA was also evident depending on the sampling location, with the centre of the surface showing the highest species richness and overlap, suggesting its suitability for biodiversity monitoring. These findings demonstrate the high resolution and sensitivity of eDNA metabarcoding in capturing phytoplankton diversity and highlight its complementary role in existing biomonitoring programmes. Further improvements in the quantitative reliability of eDNA-based assessments will require efforts such as copy number normalisation, methodological standardisation, and refinement of reference databases.

This study analyzed the epilithic diatom community and ecological health of freshwater streams using environmental DNA (eDNA)-based metabarcoding technology. eDNA metabarcoding is a method that analyzes biological communities by performing PCR amplification followed by next-generation sequencing (NGS), offering higher sensitivity and faster results compared to traditional microscopic analyses. The study compared the eDNA metabarcoding results of ribulose bisphosphate carboxylase large chain gene (rbcL) targeting epilithic diatoms according to Taq polymerases (SuperFi II, GainBlue, EzPCR, and AccuPower). SuperFi II and GainBlue yielded the highest number of reads and zOTUs, with GainBlue showing particularly uniform read distribution, allowing for more accurate analysis for community diversity of epilithic diatoms. On the other hand, EzPCR and AccuPower exhibited lower number of reads and zOTUs, making them less suitable for the community diversity. In terms of community similarity analysis, SuperFi II and GainBlue produced highly similar results, while EzPCR and AccuPower showed significant differences. This study demonstrates that PCR Taq polymerases significantly influence community diversity and similarity analyses of epilithic diatoms, with GainBlue providing the most stable and accurate results. Our findings serve as a valuable foundation for improving the accuracy of eDNA-based metabarcoding analyses of diatoms.