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 OTUs) than has been described previously. The vast majority of the diversity described was represented by rare OTUs (≤ 0.01% of reads) belonging primarily to Cryptomycota, Dikarya 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 OTUs that were detected in intermediate abundance (0.01-1% of reads) and sporadically in dominant taxa. Putative bacterivorous (Ciliophora and Cercozoa) as well as parasitic and saprotrophic taxa (Perkinsozoa and Cryptomycota) 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.

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.

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).

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.

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Planktonic heterotrophic prokaryotes make up the largest living biomass and process most organic matter in the ocean. Determining when and where the biomass and activity of heterotrophic prokaryotes are controlled by resource availability (bottom-up), predation and viral lysis (top-down) or temperature will help in future carbon cycling predictions. We conducted an extensive survey across subtropical and tropical waters of the Atlantic, Indian and Pacific Oceans during the Malaspina 2010Global Circumnavigation Expedition and assessed indices for these three types of controls at 109 stations (mostly from the surface to 4,000 m depth). Temperature control was approached by the apparent activation energy in eV (ranging from 0.46 to 3.41), bottom-up control by the slope of the log-log relationship between biomass and production rate (ranging from À0.12 to 1.09) and top-down control by an index that considers the relative abundances of heterotrophic nanoflagellates and viruses (ranging from 0.82 to 4.83). We conclude that temperature becomes dominant (i.e. activation energy >1.5 eV) within a narrow window of intermediate values of bottom-up (0.3-0.6) and top-down 0.8-1.2) controls. A pervasive latitudinal pattern of decreasing temperature regulation towards the Equator, regardless of the oceanic basin, suggests that the impact of global warming on marine microbes and their biogeochemical function will be more intense at higher latitudes. Our analysis predicts that 1°C ocean warming will result in increased biomass of heterotrophic prokaryoplankton only in waters with <26°C of mean annual surface temperature.bacterioplankton, bottom-up, heterotrophic prokaryotes, latitudinal gradients, microbial oceanography, ocean warming, temperature control, top-down

The seasonal dynamics of the small eukaryotic fraction (cell diameter, 0.2 to 5 m) was investigated in a mesotrophic lake by tyramide signal amplification-fluorescence in situ hybridization targeting seven different phylogenetic groups: Chlorophyceae, Chrysophyceae, Cryptophyceae, Cercozoa, LKM11, Perkinsozoa (two clades), and Fungi. The abundance of small eukaryotes ranged from 1,692 to 10,782 cells ml Recently developed molecular methods based on the amplification and sequencing of rRNA genes have made it possible to investigate picoeukaryote assemblage composition (pigmented or nonpigmented unicellular eukaryotes with cell diameters of Ͻ2 m or Ͻ5 m according to the studies) in various aquatic systems, independently of morphological identification and cultivation (14,23,27,28,29,39). The essential role of picoplankton (both eukaryotic and prokaryotic) as a contributor to plankton biomass and to carbon and nutrient cycling has long been established (9), but the unexpected diversity among the smallest eukaryotes (cell diameters, Ͻ5 m) was only recently revealed. Most of these data were obtained in oceanic systems, but a few recent studies conducted in lakes have also highlighted the broad diversity of 18S rRNA sequences affiliated with numerous phylogenetic groups: Chlorophyceae, Chrysophyceae, Cryptophyceae, Cercozoa, Fungi, Choanoflagellida, Bicosoecida, Ciliophora, Haptophyceae, Perkinsozoa, LKM11, Hyphochytridiomycota, Katablepharidaceae, Dinophyceae, and Eustigmatophyceae (22,23,24,34). Thus, it has been possible to observe clear seasonal changes in smalleukaryote structure in an oligomesotrophic lake (23), and the lake-based studies generally report a dominance of heterotrophic cells within the lacustrine small-eukaryote assemblage. Moreover, the recurrent presence of sequences affiliated with parasitic groups has been highlighted in lakes of various trophic statuses (22,23). Lepère et al. (25) reported the unexpected importance of two groups: first, fungi affiliated with two clades of chytrids known as parasites of various groups of microalgae; and second, members of the phylum Perkinsozoa belonging to two clades closely related to Perkinsus marinus and Parvilucifera infectans, which are parasites of bivalves and dinoflagellates, respectively (30), and whose systematic position has been controversial, since they are phylogenetically related to the Apicomplexa or the Dinoflagellata (6, 13).Although these data brought new insight into the structural diversity of lacustrine small eukaryotes, the relative importance, dynamics, and functional roles of these microorganisms from various phylogenetic groups are still largely unknown. We now need to research specific in situ abundances of previously undetected taxa. In this study, specially developed oligonucleotide probes, designed on the basis of molecular data obtained from sequencing (20,21,22,23,24,25,34), were used for fluorescence in situ hybridization (FISH) coupled with tyramide signal amplification (TSA) to investigate the composition, abundance, and dy...

The diversity of attached and free-living Actinobacteria and Betaproteobacteria, based on 16S rRNA gene sequences, was investigated in a mesotrophic lake during two periods of contrasting phytoplankton dominance. Comparison analyses showed a phylogenetic difference between attached and free-living communities for the two bacterial groups. For Betaproteobacteria, the betaI clade was detected at all sampling dates in free-living and attached bacterial communities and was the dominant clade contributing to 57.8% of the total retrieved operational taxonomic units (OTUs). For Actinobacteria, the acIV cluster was found to be dominant, followed by acI contributing to 45% and 25% of the total retrieved OTUs, respectively. This study allows the determination of eight new putative clades among the Betaproteobacteria termed lbI-lbVIII and a new putative clade named acLBI belonging to the Actinobacteria. The seasonal dynamics of phytoplankton and zooplankton communities have been reflected as changes in distinct bacterial phylotypes for both attached and free-living communities. For attached communities, relationships were observed between Actinobacteria and Chrysophyceae, and between Betaproteobacteria and Dinophyceae and Chlorophyceae biomass. On the other hand, within free-living communities, few actinobacterial clades were found to be dependent on either nutrients or phytoplankton communities, whereas Betaproteobacteria were mainly associated with biological parameters (i.e. phytoplankton and copepod communities).