Comparative identification of phytoplankton taxonomic and functional group approach in karst lakes using classical microscopy and eDNA metabarcoding for ecological status assessment

Phytoplankton is one of the five biological quality elements used to assess the ecological status of lakes within the Water Framework Directive. Classical morphological Utermöhl method and eDNA metabarcoding by Ilumina sequencing the hypervariable V9 region of the eukaryotic SSU rRNA gene were used to analyse the qualitative and quantitative composition of the phytoplankton and compared at the taxonomic and FG level to highlight advantages and disadvantages of eDNA metabarcoding method over classical microscopy. Samples were collected from April to September in seven Croatian natural karst lakes. Cluster analysis based on the Bray–Curtis similarity of taxa biomass (microscopy) and number of sequences (eDNA metabarcoding) clearly separated lakes showing that eDNA metabarcoding is sensitive to species change. Overlap at the species level between methods was found primarily in the taxa of Cryptophyta, Miozoa, and Ochrophyta, while some very common taxa of Bacillariophyta, Charophyta, and Chlorophyta identified by microscopy were not detected by eDNA metabarcoding, possibly due to incompleteness of the reference databases. At a higher organizational level, the results showed poor overlap of taxonomic and functional group composition and poor comparability of relative biomass to relative number of sequences, indicating the need to complete reference databases and standardize quantification to further develop eDNA metabarcoding for ecological status assessment.