Long-Term integrated monitoring (microscopy & metabarcoding) of phytoplankton communities at an eLTER coastal station in the northern Adriatic Sea (2018–2024)

Phytoplankton represents a key component of marine ecosystems. Traditionally, phytoplankton analysis has been carried out using light microscopy (LM), but this method often does not allow a precise taxonomic characterization, particularly for nano-sized taxa. In this study, we analysed the phytoplankton biodiversity at the eLTER coastal station of the Senigallia-Susak transect (northern Adriatic Sea) during 2018₋2024, combining LM and metabarcoding (MB). For MB, V4 and V9 regions of the 18S rRNA gene were targeted and the PR2 and SILVA reference databases were used for taxonomic assignment. Comparison between LM and MB showed a consistent seasonal pattern for diatoms, although marked differences were found for the heterogeneous phytoflagellate group, with changes depending on the marker and reference database used. Phytoplankton showed a marked seasonal variability, with the most intense blooms in winter, largely attributable to diatoms (Skeletonema marinoi). Phytoflagellates were the dominating group during the other seasons. In this group, MB highlighted a hidden biodiversity, revealing a number of taxa (e.g. Picochlorum eukaryotum, Bathycoccus prasinos) not identified by microscopy. Regarding diatoms, MB provided a higher taxonomical resolution only for some cryptic genera (e.g. Pseudo-nitzschia), while dinoflagellate identification was markedly resolved by MB for a high number of species (genera Alexandrium, Gymnodinium, Gyrodinium). Comparing LM and MB approaches, a limited overlap in terms of detected species was observed (~ 12%), confirming their complementarity. This study highlighted the importance of a multi-method approach for a more complete characterization of phytoplankton biodiversity and provide a long-term time series that may support the study of population dynamics.