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Dynamics, genomics and functional significance of uncultured marine Stramenopiles

Final Report Summary - DYGEMAST (Dynamics, genomics and functional significance of uncultured marine Stramenopiles)

The major role of microbes and in particular marine picoeukaryotes (cells ≤3 μm) in global biogeochemical cycles and their crucial importance in aquatic ecosystems are now clearly recognized. Despite the enormous importance of the microbial compartment, the knowledge on their diversity, their abundance (notably in terms of spatial and temporal dynamics) and their respective functional role on Earth are still limited, even if the recent molecular revolution over the last fifteen years has partially contributed to fill these gaps. The outgoing project “DYGEMAST” aimed to pursue the acquisition of knowledge about the dynamics of abundance and functional role of one of the most important lineages of the picoeukaryotic community, the Marine Stramenopiles (MASTs). Constituted by 18 discrete groups, MASTs appear to have a widespread distribution and to be, sometimes, in high abundance, suggesting that this may be one of the most abundant component of heterotrophic picoeukaryotes in the oceans. However, the number of quantified samples is still limited and, consequently, we only have a partial view of their current temporal and spatial distribution in worldwide marine systems. Furthermore, most picoeukaryotes have escaped cultivation so far, yielding impossible the access to their genomes, which would get us better insights into the metabolic pathways/functions of these uncultured flagellates and understand their ecological success.

In the present project, two axes of research were developed to deepen our knowledge on marine stramenopiles. A first axis was the study of the quantitative dynamics of several MASTs lineages in marine systems. Prior to this, Dr. Jean-François Mangot (J-FM) has optimized an automatic picoeukaryotic cell counting approach by epifluorescence microscopy. Firstly developed for the counting of prokaryotic cells, this approach has been tested and applied on two MAST lineages differing in their cell size (MAST-4 and MAST-1C) in order to facilitate their application and generalisation to other microeukaryotes. At the end of the project, this approach is now mastered and allows an accurate estimation of densities of contrasting cell-size picoeukaryotes in a wide range of samples. Furthermore, our automatic cell counting approach offers the benefit of processing a larger number of fields of view and therefore provides a better estimate of cell abundance. During the second year of the project, J-FM has further applied this automatic cell enumeration system on a spatial survey with a wide geographical coverage. About 129 stations from the Malaspina circumglobal expedition were analysed and the vertical (surface and Deep Chlorophyll Maximum [DCM] depths) and spatial distribution of the two above-mentioned MAST groups were depicted together with a third one, MAST-7, in relation with environmental factors to have access to their ecology. In general, contrasting abundances among the different targeted MASTs lineages were observed with notably highest densities in surface samples taken near the coasts and in low latitudes (near the equator). Open questions regarding both the patterns that govern their distribution and their possible biogeography arise from this study.

A second axis of research was consecrated to the exploration of the functional role of MASTs lineages in marine ecosystems. One way to access their metabolic potential is the acquisition and analysis of their genomes. In this sense, single cell genomics (SCG), a technique by which the genomes of single isolated cells (i.e. single amplified genomes or SAGs) can be sequenced without any step of cultivation, was a revolution. So far, SCG has been mainly applied to prokaryotic cells and studies in unicellular uncultured eukaryotes are still scarce. Thanks to an ongoing collaboration with scientists involved on the Tara Oceans project, J-FM has got access to SAGs affiliated to MASTs from different global oceanic sites. However, although SCG has proved its efficiency to obtain genomic information from uncultured microbes, this approach is still suffering of bias introduced notably during the whole-genome amplification (WGA) procedure, a step preceding the shotgun sequencing of single cells. Given the huge potential of SCG to improve our understanding of eukaryotic ecology and evolution, it is important to know the genomic recovery of single cells, owing to this WGA bias. Yet, studies trying to optimize protistan genome recovery using SCG are missing. During the project, J-FM has tested an approach to increase genome recovery through the co-assembly of separately sequenced SAGs assumed to be from the same species. This co-assembly strategy was initiated on several individual SAGs from picosized eukaryotes affiliated to MASTs, chrysophytes and prasinophytes lineages, and a special attention was paid on two specific clades of the MAST-4 lineage (clades A and E), for which the highest amount of genomic information was recovered. To ensure the correct mixing of related cells, we established two additional criteria essential to discriminate cells from the same species in addition to identical SSU rDNA: a high overall genomic similarity (>95%) and similar tetranucleotide frequency profiles. Interestlingly, SAGs from the same species retrieve different genome regions, and recovery is hardly improved when increasing the sequencing depth of a given SAG. Thanks to this co-assembly strategy, a significant increase of the genomic information captured in both MAST-4 clades was observed, from around 20% of gene functions in individual SAGs to about 70% in the final co-assemblies. Our approach evidences the great potential of SCG to start opening the black box of marine picoeukaryotes. Overall, this approach has permitted us to start accessing to the structural and functional composition of MAST-4 genomes, paving the road for future studies connecting the metabolisms of these organisms with their ecological role in oceanic ecosystems.

The outgoing project fully achieved the initial objectives with the development of state-of-the-art approaches (high-throughput automatic cell enumeration, SAGs co-assembling) applied on specific picoeukaryotic lineages (MASTs) that can be now adapted to a range of protists. The obtained results constitute a step towards a better comprehension of the role of the microbial biodiversity in oceanic systems through the study of one of its important components and could be useful for future reference in a context of global change and potential biodiversity loss. Lastly, during the EC project, results were widely presented and shared among the scientific community and the general public in both Europe and US.