Integrated morphometric and DNA metabarcoding assessment of ciliate biodiversity in the Adriatic Sea

Ciliates are ecologically important microeukaryotes that transfer energy, remineralise nutrients, and regulate microbial communities. Their sensitivity to environmental changes makes them reliable bioindicators of the state of marine ecosystems. Although their taxonomy and ecology are well documented, conventional monitoring methods often overlook cryptic and rare species. Integrative approaches combining morphological and molecular techniques are therefore essential to capture the full spectrum of ciliate diversity. In this study, a dual taxonomic approach was used to assess the composition of the ciliate community at a coastal site in the central Adriatic Sea (BioPlan project). Water samples were collected using a Niskin bottle and a 53 µm plankton net. Bulk DNA was extracted by high-throughput sequencing of the nuclear 18S rRNA gene (V4 and V9 regions), in parallel with morphometric identification under a light microscope. The comparative analysis revealed a much better taxonomic resolution of tintinnids by microscopy, suggesting that morphometric identification remains a crucial tool for the detection of ecologically important species in the marine environment. Tintinnid genera such as Rhabdonella, Xystonella, Dadayiella, Favella, and Epiplocylis were confirmed exclusively by microscopic observation. Conversely, metabarcoding proved to be more effective in detecting non-tintinnid ciliates, such as Spirotontonia, Pseudotontonia, Bergeriella ovata (V4), Metaurostylopsis, and Mesodinium rubrum (V9), which were not present in morphological analyses. These discrepancies between approaches likely reflect the limitations of the reference databases, low genetic divergence in the target regions, and potential DNA degradation, particularly in net samples. Our results highlight the limitations of metabarcoding when applied independently and support the use of integrated morpho-molecular protocols in protist biodiversity studies. They also emphasise the importance of expanding curated sequence databases. Understanding the fine taxonomic structure and ecological dynamics of ciliate communities is critical for monitoring ecosystem health and predicting ecological responses to environmental change in marine ecosystems.