Summary: The short‐term variation in the community structure of freshwater small eukaryotes (0.2–5 μm) was investigated in a mesotrophic lake every 2–3 days over one summer by coupling three molecular methods: 454 amplicon pyrosequencing, qPCR and TSA ‐ FISH . The pyrosequencing approach unveiled a much more extensive small‐eukaryotic diversity (991 OTU s) than has been described previously. The vast majority of the diversity described was represented by rare OTU s (≤ 0.01% of reads) belonging primarily to C ryptomycota , D ikarya and photosynthetic organisms, which were never detected as abundant in any of the samples. The small eukaryote community was characterized by a continual and important reassembly. These rearrangements involved the 20 ‘core taxa’ (≥ 1% of reads), and, were essentially due to a handful of OTU s that were detected in intermediate abundance (0.01–1% of reads) and sporadically in dominant taxa. Putative bacterivorous ( C iliophora and C ercozoa ) as well as parasitic and saprotrophic taxa ( P erkinsozoa and C ryptomycota ) were involved in these changes of diversity. A putative infection of microalgae by a lacustrine perkinsozoan was also reported for the first time in this study. Open questions regarding both the patterns that govern the rapid small eukaryote reassemblies and the possible biogeography of these organisms arise from this study.

Recent advances in next-generation sequencing (NGS) technologies spur progress in determining the microbial diversity in various ecosystems by highlighting, for example, the rare biosphere. Currently, high-throughput pyrotag sequencing of PCR-amplified SSU rRNA gene regions is mainly used to characterize bacterial and archaeal communities, and rarely to characterize protist communities. In addition, although taxonomic assessment through phylogeny is considered as the most robust approach, similarity and probabilistic approaches remain the most commonly used for taxonomic affiliation. In a first part of this work, a tree-based method was compared with different approaches of taxonomic affiliation (BLAST and RDP) of 18S rRNA gene sequences and was shown to be the most accurate for near full-length sequences and for 400 bp amplicons, with the exception of amplicons covering the V5-V6 region. Secondly, the applicability of this method was tested by running a full scale test using an original pyrosequencing dataset of 18S rRNA genes of small lacustrine protists (0.2–5 µm) from eight freshwater ecosystems. Our results revealed that i) fewer than 5% of the operational taxonomic units (OTUs) identified through clustering and phylogenetic affiliation had been previously detected in lakes, based on comparison to sequence in public databases; ii) the sequencing depth provided by the NGS coupled with a phylogenetic approach allowed to shed light on clades of freshwater protists rarely or never detected with classical molecular ecology approaches; and iii) phylogenetic methods are more robust in describing the structuring of under-studied or highly divergent populations. More precisely, new putative clades belonging to Mamiellophyceae, Foraminifera, Dictyochophyceae and Euglenida were detected. Beyond the study of protists, these results illustrate that the tree-based approach for NGS based diversity characterization allows an in-depth description of microbial communities including taxonomic profiling, community structuring and the description of clades of any microorganisms (protists, Bacteria and Archaea).

Single-celled eukaryotes (protists) are critical players in global biogeochemical cycling of nutrients and energy in the oceans. While their roles as primary producers and grazers are well appreciated, other aspects of their life histories remain obscure due to challenges in culturing and sequencing their natural diversity. Here, we exploit single-cell genomics and metagenomics data from the circumglobal Tara Oceans expedition to analyze the genome content and apparent oceanic distribution of seven prevalent lineages of uncultured heterotrophic stramenopiles. Based on the available data, each sequenced genome or genotype appears to have a specific oceanic distribution, principally correlated with water temperature and depth. The genome content provides hypotheses for specialization in terms of cell motility, food spectra, and trophic stages, including the potential impact on their lifestyles of horizontal gene transfer from prokaryotes. Our results support the idea that prominent heterotrophic marine protists perform diverse functions in ocean ecology.

Pico-sized eukaryotes play key roles in the functioning of marine ecosystems, but we still have a limited knowledge on their ecology and evolution. The MAST-4 lineage is of particular interest, since it is widespread in surface oceans, presents ecotypic differentiation and has defied culturing efforts so far. Single cell genomics (SCG) are promising tools to retrieve genomic information from these uncultured organisms. However, SCG are based on whole genome amplification, which normally introduces amplification biases that limit the amount of genomic data retrieved from a single cell. Here, we increase the recovery of genomic information from two MAST-4 lineages by co-assembling short reads from multiple Single Amplified Genomes (SAGs) belonging to evolutionary closely related cells. We found that complementary genomic information is retrieved from different SAGs, generating co-assembly that features >74% of genome recovery, against about 20% when assembled individually. Even though this approach is not aimed at generating high-quality draft genomes, it allows accessing to the genomic information of microbes that would otherwise remain unreachable. Since most of the picoeukaryotes still remain uncultured, our work serves as a proof-of-concept that can be applied to other taxa in order to extract genomic data and address new ecological and evolutionary questions.