Environmental DNA metabarcoding applied to marine ecosystem: insights from an ecological and methodological perspective

Protists inhabiting the marine environment are extremely diversified and abundant playing a pivotal role in the ecosystem functioning. The benthic protist community composition and its diversity is still little known, despite increasing human pressure exerted on the seabed. For these reasons, it is of primary importance the knowledge and the monitoring of these communities to understand the impacts on them. The advent of metagenomics and high-throughput sequencing (HTS) technology has improved our knowledge on the composition of marine protist communities. This technique is particularly useful for studying communities that are difficult to access, such as marine benthic communities. In fact, the application of this technique to environmental DNA (eDNA) samples permits to retrieve information on the community. Debates about the best way to analyse this kind of data (e.g., qualitative, semi-quantitative, quantitative) are still ongoing and methodological studies are useful to shed light on these hot-topics. During these three years as Ph.D. student, I had the opportunity to apply molecular approaches to ecology with two main objectives: (i) to study the genetic diversity of the benthic protist communities in the Mediterranean Sea using eDNA metabarcoding and (ii) to investigate the technical aspects of this approach using laboratory-based communities with a focus on its application to some ecological patterns. These two aims have been carried forward in parallel over these three years, which has made it difficult to integrate the results of the methodological work with the ecological study, but has provided insights into two possible ways of processing the data generated by the metabarcoding approach. In the first chapter of the thesis, I faced the first main objective investigating the composition of the benthic protist communities found in the surface sediments of three Mediterranean basins (Adriatic, Ionian – Central Mediterranean Sea and Western Mediterranean Sea). The focus of this work was (i) the characterization of the benthic protist assemblages at the taxonomic and functional levels, (ii) the investigation of Mediterranean sub-regions and bathymetric depths to check for differences, and (iii) the inspection of the diversity of the assemblages in terms of alpha diversity and community composition. The results showed slight differences between the three sub-regions studied at the taxonomic order rank. Functional diversity, as revealed by trophic habits, also showed slight differences for the Adriatic Sea compared to the other sub-regions. This study provided a first insight into the benthic protist communities inhabiting the surface sediments of the Mediterranean Sea. This chapter will be soon submitted to the Journal Frontiers in Marine Science. In the second chapter of the thesis, the technical aspect of metabarcoding was analysed focusing also on its correctness in detecting some ecological patterns. In particular, the quantitative aspects of the ecological indices (e.g., alpha and beta diversity) were examined, revealing weaknesses in the metabarcoding-derived data. This study aimed to: (i) evaluate the accuracy of the tested metabarcoding protocol in detecting the selected diatom and dinoflagellate taxa; (ii) assess the potential occurrence of false negatives (i.e., the failure to detect a taxon in a mock community in which it was included); and (iii) test the reliability of metabarcoding-derived alpha and beta diversity estimates computed from both qualitative and quantitative data. Biases were observed and studied through morphological and genetic parameters, which showed a potential, although not exhaustive, role in explaining the observed biases.