Protists are major actors of soil communities and play key roles in shaping food webs, community assembly, and ecosystem processes, yet their functional diversity is understudied. High-throughput sequencing data have revealed their ubiquity and diversity, but lack of standardized traits has hampered the integration of functional information, limiting our understanding of soil ecosystems. Here we propose a framework for soil protists, identify a set of common traits to characterize their functional diversity, and apply the framework on a broad-scale, real-world dataset. We reviewed studies on soil protists to identify the traits used in the literature, and define a framework based on 10 key traits that satisfy two criteria: availability of information, and applicability to most taxa. The framework was tested on a dataset of environmental DNA metabarcoding data from 1123 soil samples collected in 48 glacier forelands worldwide. Traits were assigned to all the 570 Molecular Operational Taxonomic Units (MOTUs) detected in our dataset, leading to the production of a global trait-based dataset from glacier forelands. We estimated the functional space of protist communities and evaluated if the selected traits were effective in describing protist diversity. The functional space of protist communities showed that the MOTUs are clustered in three regions, mainly reflecting different nutritional and habitat preferences. The proposed framework is appropriate for multiple applications, including estimation of functional diversity and food web analyses, and provides a basis for ecological studies on soil protists, enabling the functional characterization of this essential but often neglected component of soil biodiversity.

An exhaustive assessment of biodiversity is a major challenge of ecological research, and molecular approaches such as the metabarcoding of environmental DNA are boosting our ability to perform biodiversity inventories. Are we actually able to assess the whole community, to unravel the intricate interactions between organisms and the impacts of global changes on the different trophic levels? The majority of metabarcoding papers published in the last years used just one or two markers and analyzed a limited number of taxonomic groups. Nevertheless, approaches are emerging that might allow “all-taxa biological inventories”. Exhaustive biodiversity assessments can be attempted by combining a large number of specific primers, by exploiting the power of universal primers, or by combining specific and universal primers to obtain good information on key taxa while limiting the overlooked biodiversity. Multiplexes of primers and shotgun sequencing may provide a better coverage of biodiversity compared to standard metabarcoding, but still require major methodological advances. We identify the strengths and limitations of different approaches, and suggest new development lines that might improve broad scale biodiversity analyses in the near future.

A document by Aurelie Bonin. Click on the document to view its contents.

Ice-free areas are increasing worldwide due to the dramatic glacier shrinkage and are undergoing rapid colonization by multiple lifeforms, thus representing key environments to study ecosystem development. Soils have a complex vertical structure. However, we know little about how microbial and animal communities differ across soil depths and development stages during the colonization of deglaciated terrains, how these differences evolve through time, and whether patterns are consistent among different taxonomic groups. Here, we used environmental DNA metabarcoding to describe how community diversity and composition of six groups (Eukaryota, Bacteria, Mycota, Collembola, Insecta, Oligochaeta) differ between surface (0-5 cm) and relatively deep (7.5-20 cm) soils at different stages of development across five Alpine glaciers. Taxonomic diversity increased with time since glacier retreat and with soil evolution; the pattern was consistent across different groups and soil depths. For Eukaryota, and particularly Mycota, alpha-diversity was generally the highest in soils close to the surface. Time since glacier retreat was a more important driver of community composition compared to soil depth; for nearly all the taxa, differences in community composition between surface and deep soils decreased with time since glacier retreat, suggesting that the development of soil and/or of vegetation tends to homogenize the first 20 cm of soil through time. Within both Bacteria and Mycota, several molecular operational taxonomic units were significant indicators of specific depths and/or soil development stages, confirming the strong functional variation of microbial communities through time and depth. The complexity of community patterns highlights the importance of integrating information from multiple taxonomic groups to unravel community variation in response to ongoing global changes.

Environmental DNA metabarcoding is becoming a key tool for biodiversity monitoring over large geographical or taxonomic scales and for elusive taxa like soil organisms. Increasing sample sizes and interest in remote or extreme areas often require the preservation of soil samples and thus deviations from optimal standardized protocols. However, we still ignore the impact of different methods of soil sample preservation on the results of metabarcoding studies and there is no guidelines for best practices so far. Here, we assessed the impact of four methods of soil sample preservation commonly used in metabarcoding studies (preservation at room temperature for 6h, preservation at 4°C for three days, desiccation immediately after sampling and preservation for 21 days, and desiccation after 6h at room temperature and preservation for 21 days). For each preservation method, we benchmarked resulting estimates of taxon diversity and community composition of three different taxonomic groups (bacteria, fungi and eukaryotes) in three different habitats (forest, river bank and grassland) against results obtained under optimal conditions (i.e. extraction of eDNA right after sampling). Overall, the different preservation methods only marginally impaired results and only under certain conditions. When rare taxa were considered, we detected small but significant changes in MOTU richness of bacteria, fungi and eukaryotes across treatments, while the exclusion of rare taxa led to robust results across preservation methods. The differences in community structure among habitats were evident for all treatments, and the communities retrieved using the different preservation conditions were extremely similar. We propose guidelines on the selection of the optimal soil sample preservation conditions for metabarcoding studies, depending on the practical constraints, costs and ultimate research goals.

Environmental DNA and metabarcoding have great potential for the biomonitoring of freshwater environments. However, successful application of metabarcoding to biodiversity monitoring requires universal primers with high taxonomic coverage that amplify highly-variable, short metabarcodes with high taxonomic resolution. Moreover, reliable and extensive reference databases are essential to match the outcome of metabarcoding analyses with the available taxonomy and biomonitoring indices. Benthic invertebrates, particularly insects, are key taxa for freshwater biomonitoring. Nevertheless, so far, no formal comparison has assessed primers for metabarcoding of freshwater macrobenthos. Here we combined in vitro and in silico analyses to test the performance of metabarcoding primers amplifying regions in the 18S rDNA (Euka02 metabarcode), 16S rDNA (Inse01), and COI (BF1_BR2-COI) genes, and developed an extensive database of benthic invertebrates of France and Europe, with a special focus on three key insect orders (Ephemeroptera, Plecoptera and Trichoptera). In vitro analyses on 1514 individuals, belonging to 578 different taxonomic units showed very different amplification rates across primer combinations. The Euka02 marker showed the highest universality, while the Inse01 marker showed excellent performance for the amplification of insects. The BF1_BR2-COI metabarcode showed the highest resolution, while the resolution of Euka02 was often limited. By combining in vitro data with GenBank information, we developed a curated database including sequences representing 822 genera. The heterogeneous performance of the different metabarcodes highlights the complexity of the identification of the best markers, and advocates for the integration of multiple metabarcodes for a more comprehensive and accurate understanding of ecological impacts on freshwater biodiversity.