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Marine sediments host heterogeneous protist communities consisting of both living benthic microorganisms and planktonic resting stages. Despite their key functions in marine ecosystem processes and biogeochemical cycles, their structure and dynamics are largely unknown. In the present study, with a spatially intensive sampling design we investigated benthic protist diversity and function of surface sediment samples from three subregions of the Mediterranean Sea, through an environmental DNA metabarcoding approach targeting the 18S V4 region of rRNA gene. Protists were characterized at the taxonomic level and trophic function, both in terms of alpha diversity and community composition, testing for potential differences among marine subregions and bathymetric groups. Overall, Alveolata and Stramenopiles were the two divisions that dominated the communities. These dominant groups exhibited significant differences among the three Mediterranean subregions in the alpha diversity estimates based on the detected ASVs, for all computed indices (ASV richness, Shannon and Simpson indices). Protist communities were also found to be significantly different in terms of composition at the order rank in the three subregions p-value < 0.01). These differences were mainly driven by Anoecales, Peridiniales, Borokales, Paraliales and Gonyaulacales, which together contributed almost 80% of the average dissimilarity. Anoecales was the dominant order in the Ionian – Central Mediterranean and Adriatic Sea, but with considerably different relative abundances (52% and 36%, respectively), while Borokales was the dominant order in the Western Mediterranean Sea (33%). Similarly, significant differences among the three marine subregions were also highlighted when protist assemblages were examined in terms of trophic function, both in terms of alpha diversity (calculated on the ASVs for each trophic group) and community composition p-value < 0.01. In particular, the Adriatic Sea stood out for having the highest relative abundance of autotrophic/ mixotrophic components in the surface sediments analyzed. Conversely, no significant differences in protist assemblages were found among depth groups. This study provided new insights into the taxonomic and trophic composition of benthic protist communities found in Mediterranean surface sediments, revealing geographical differences among regional seas. The results were discussed in relation to the Mediterranean environmental features that could generate the differences among benthic protist communities. [ABSTRACT FROM AUTHOR]

Ancient environmental DNA retrieved from sedimentary records (sedaDNA) can complement fossil-morphological approaches for characterizing Quaternary biodiversity changes. PCR-based DNA metabarcoding is so far the most widely used method in environmental DNA studies, including sedaDNA. However, degradation of ancient DNA and potential contamination, together with the PCR amplification drawbacks, have to be carefully considered. Here we tested this approach on speleothems from an Alpine cave that, according to a previous palynomorphological study, have shown to contain abundant pollen grains. This offers a unique opportunity for comparing the two methods and, indirectly, trying to validate DNA-based results. The plant taxa identified by sedaDNA are fewer than those by pollen analysis, and success rate of PCR replicates is low. Despite extensive work performed following best practice for sedaDNA, our results are suboptimal and accompanied by a non-negligible uncertainty. Our preliminary data seem to indicate that paleoenvironmental DNA may be isolated from speleothems, but the intrinsic weakness of PCR-based metabarcoding poses a challenge to its exploitation. We suggest that newly developed methods such as hybridization capture, being free from PCR drawbacks and offering the opportunity to directly assess aDNA authenticity, may overcome these limitations, allowing a proper exploitation of speleothems as biological archives. [ABSTRACT FROM AUTHOR]