Rapidly accumulating environmental sequencing data have revealed that eukaryotic microbes are far more diverse and complex than previously thought. However, meta-barcoding and meta-genomics surveys are severely limited by the fact that the majority of environmental sequences do not match a sequence with associated phenotypic/taxonomic information in reference databases. Phytoplankton, a polyphyletic group of single-celled photosynthetic organisms that play key roles in aquatic food webs and global biogeochemical cycles, comprise an important part of this undescribed diversity. PHENOMAP will address this major “phenotype gap” for marine phytoplankton by undertaking targeted phenotypic description of key cryptic lineages. The work plan integrates a suite of state of the art methods that will be applied to two outstanding existing resources: the Roscoff Culture Collection, which is the largest and most diverse service collection of living microalgal strains in the world, and the Tara protist sample collection that contains over 15,000 fixed plankton samples from the worldwide Tara Oceans expeditions (2009-2018). Additional targeted sampling at 3 French marine stations will complement these resources. Phenotypic analyses will include light, fluorescence and electron microscopy for both live and fixed samples, as well as photosynthetic pigment analysis for cultures. For fixed samples, fluorescent in-situ hybridisation will be used to target cells belonging to the most abundant cryptic environmental lineages. Genetic barcoding of morphologically identified single cells isolated from fixed and live samples will complete the experimental strategy. We aim to formally describe at least 100 new taxa (including many high taxonomic rank lineages) and link genotypic to phenotypic information for hundreds of additional existing species, adding significant value to integrated community databases (PR2, UniEuk, Ecotaxa) that are increasingly central to studies on phytoplankton biology, ecology and evolution. Widespread dissemination of scientific outputs and development of innovative educational and outreach resources is integral to the PHENOMAP approach.

Alpha-cyanobacteria are the most abundant and ubiquitous photosynthetic prokaryotes in both marine and freshwater ecosystems. This ecological success as well as the numerous available strains, genomes and metagenomes make them highly pertinent models in microbial ecology, which can be studied at all organization scales from genes to ecosystems. Yet, their long evolutionary history, similar morphologies, high phenotypic plasticity and high degree of functional variation among closely related lineages, have so far made it difficult to classify them in a rigorous and consensual way, while a reliable systematics for this group is critical to unravel the links between phylogenetic diversification, differential functional capacities and colonization of specific environmental niches. Although several standardized classifications based on comparative genomics have recently been proposed for the whole Bacteria domain or more restricted phylogenetic groups, they all have largely ignored the wealth of experimental and in situ data available on alpha-cyanobacteria and therefore appear inappropriate to tackle these questions in this ecologically important group. In this context, the main objectives of the TaxCy project will be to : i) propose an integrative formulation of the species concept for alpha-cyanobacteria by combining the existent and newly generated data on phenotypes, genotypes and habitat, with a particular focus on both marine and freshwater uncultivated taxa that have been largely overlooked so far, ii) determine how the different genotypes differentiate functionally and ecologically from their close (intra-species variability) and more distant relatives (inter-species variability) and iii) describe and model environmental and metabolic niches to decipher the importance of particular reactions and pathways in the survival of the different species in their specific niches. In practice, starting from an initial set of candidate species selected by pre-screening the ca. 500 alpha-cyanobacteria strains of the Roscoff Culture Collection (RCC), the delineation of valid species will be done using an iterative, multi-step approach combining comparative physiology, comparative genomics and metagenomic analyses in order to identify strains that share a number of phenotypic, genotypic, functional and ecological characteristics, which differentiate them from all other species. To fill diversity gaps, the initial set of candidate species will be complemented by targeting specific aquatic environments for isolating new strains that will then be characterized using the same protocol. Each validated species will then be formally described according to the bacteriological code and type strains will be made axenic and deposited in the RCC and other collections. Based on this rigorous and reasoned taxonomy, complemented by the development, update or expansion of large, manually-curated open-access databases for inter-comparison of marker genes (CyanoMarks), genomes (Cyanorak) and traits (CyanoTraits), we will then i) unveil the functional specificities of each species potentially involved in their adaptation to these niches and ii) use the latest developments in genome-scale metabolic modeling to build realistic metabolic and ecological niche models for alpha-cyanobacteria species and higher taxonomic ranks. This will allow us not only to describe, but also predict their realized environmental niches, based on their within-species metabolic capacities as well as to assess the importance of specific metabolic pathways and/or of specific genomic regions on metabolic niches. The TaxCy project will therefore provide novel insights into the relationships between systematics, function and niche partitioning for key members of aquatic ecosystems and should give us a better appraisal of the ecosystemic services potentially offered by alpha-picocyanobacteria.

Coccolithophores and planktonic foraminifera produce more than 90% of pelagic carbonates, thus being major actors in the global carbon cycle. Calcification of these unicellular organisms is known to be influenced by carbonate ion concentration and calcite saturation state of seawater as well as by other physico-chemical parameters. Carbon dioxide produced by human activities since the industrial revolution has already induced a decrease of ocean pH by about 0.1 units. The objective of CALHIS is to undertake the first very high-resolution evaluation of the impact of this pH drop on pelagic carbonate production. To this end, we have selected 3 categories of oceanic zones covering a wide range of carbonate ion concentration, calcification types and trophic levels: (1) the Mediterranean and Caribbean seas with waters with high carbonate ion concentration, oligotrophy, and organisms with well calcified shells; (2) the north Papuan coast and Patagonian shelf with lower carbonate concentration, higher nutrient levels and lightly calcified shells; (3) the Eastern Pacific margin (Peru and Mexico) characterized by upwelled waters with high nutrient levels and such low carbonate ion concentration and pH that coccolithophores would be predicted to no longer calcify, although some in fact exhibit highly calcified shells. In each of these zones, we have privileged access to research vessels for sampling. We will lead or participate in mini-cruises in each of these coastal areas in order to retrieve surface sediment cores that record the history of sedimentation of the last centuries and to collect water samples from the photic zone to establish present-day relationships between calcification and carbonate chemistry, through genetic, morphological and physico-chemical analyses. A range of coccolithophore genotypes and morphotypes will be isolated into laboratory culture in order to quantify eco-physiological tolerances and calibrate specific biomarkers. From well-dated (14C/ 210Pb) sediments, we will establish records of the state of calcification of foraminifera and coccolithophores, temperature, d13C, concentrations of specific biomarkers, and relative abundance of coccolithophore morphotypes and, when possible, genotypes (i.e. in anoxic sediments). These data will enable accurate quantification of changes in pelagic carbonate production over the last 300 years and determination of whether changes are related to recent global ocean acidification. This project is the logical continuation of a study recently published in Nature by most of the proponents of the proposal.

Oceanic plankton are sensitive to physical and biochemical changes in the surface ocean, and are key modulators of the ocean carbon cycle via photosynthesis and calcification. Fossil plankton remains exported to the seafloor and preserved in sediments thus hold key clues on both past climate changes as well as associated biological responses and feedbacks. The western equatorial Indian Ocean (WEIO) is a major player in tropical ocean-atmosphere climate dynamics, with far-reaching impacts on global climate patterns. In 2021, the SCRATCH cruise onboard the research vessel Marion Dufresne II collected high-quality, complete sediment cores spanning the Pleistocene (last ~2 million years) from the WEIO, with the aim of resolving past climate variability and volcanic activity in this important region. Here, we propose to study the co-evolution of plankton, coral reefs, and climate over the Pleistocene. We will perform paired paleogenetic, biomarker, and microfossil analyses, as well as reconstruct key paleoceanographic variables to constrain background climate change, in four sediment cores recovered during SCRATCH. Using state-of-the-art methodologies across three partner institutes, we will combine expertise on paleoceanography, organic geochemistry, genetics, and micropaleontology to address the hypothesis that cyclical changes in the morphology of plankton fossils represent genetic evolution, forced by external climate cycles. Anticipated results will (1) provide a comprehensive picture of paleoceanographic evolution of the WEIO, including Indian Ocean Dipole mean state, (2) demonstrate for the first time the relationship between genetic and morphological plankton evolution in multiple groups, and (3) shed light on external (climatic, volcanic) drivers of evolution, plankton community dynamics, and pelagic versus neritic marine productivity.

The MIDAS project focuses on the discovery and development of novel sustainable sources of bioactive compounds for specific pharmaceutical applications. The overarching objective of the project will be to bring a targeted set of aquatic extremophile prokaryotic and eukaryotic microorganisms into long-term ex situ laboratory culture and to screen them for specific bioactivities via an integrated biodiscovery pipeline. The intention is to focus bioprospection on novel drug discovery in the anti-cancer field. The project is founded on the hypothesis that organisms living in these extreme environments have evolved a plethora of innovate metabolic strategies and are hence an extremely promising source of unusual metabolites that have applications in the biomedical domain. The project will involve the following interlinked and interdisciplinary activities: (1) Bioinformatic "virtual screening" to identify organisms/lineages possessing metabolic pathways for molecules of interest and determine recurrent species associations and the range of environmental conditions delimiting the distribution of species of interest; (2) This information will be used to downstream inform sampling campaigns with a focus on cold (Arctic) and extremely low nutrient (oligotrophic) marine environments, but also in conditions of extreme pH and/or salinity; (3) A variety of techniques will be used for isolating mono-specific or stable microbiome communities into ex situ culture, including state-of-the-art flow cytometric sorting and microfluidics methods which will be developed/refined during the project; (4) Existing medium- to high-throughput screening pipelines (in part developed in previous EU projects) will be used to screen bulk extracts of novel culture strains for target bioactivities; (5) For a selection of strains exhibiting promising bioactivities, the cultures will be characterized by sequencing the full genome and conducting laboratory experiments to optimize the growth of the strain and/or production of the metabolite of interest. Optimized culture conditions will be employed (at the laboratory scale) to produce sufficient biomass for initial product development steps; (6) Bulk extracts from biomass provided to the commercial partner(s) will be fractionated and tested in various well-characterized cell lines. R&D will then focus on preliminary structural characterization of the molecules of interest and of their mechanism of action (TRL3-5). The originality of the MIDAS project resides in the combination of world-leading expertise in culturing and characterization of aquatic microorganisms, informed by cutting-edge bioinformatics approaches, feeding into established bioprospecting pipelines to promote the efficient discovery of novel bioactive molecules from extremophile organisms that are an extremely promising, but as yet underexploited natural resource. The project will build upon strategic advances from previous EU projects such as PharmaSea (2012-2016), EMBRIC (2015-2019) and Nomorfilm (2015-2019) and involve significant methodological innovation, notably as concerns the sampling, isolation into ex situ culture, and metabolic optimization of extremophile microorganisms. In addition to contributing to novel drug discovery, the project will generate fundamental knowledge to advance our understanding of the ecology and evolution of microbial diversity in these extreme environments, which like all other ecosystem compartments are subject to perturbation by ongoing climate change. All resources generated during the project which are not of immediate commercial interest will be made available to the scientific community via the public culture collections and databases hosted by project partners.