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Background: Microbial interactions are fundamental for Earth’s ecosystem functioning and biogeochemical cycling. Nevertheless, they are challenging to identify and remain barely known. Omics-based censuses are helpful in predicting microbial interactions through the statistical inference of single (static) association networks. Yet, microbial interactions are dynamic and we have limited knowledge of how they change over time. Here, we investigate the dynamics of microbial associations in a 10-year marine time series in the Mediterranean Sea using an approach inferring a time-resolved (temporal) network from a single static network. Results: A single static network including microbial eukaryotes and bacteria was built using metabarcoding data derived from 120 monthly samples. For the decade, we aimed to identify persistent, seasonal, and temporary microbial associations by determining a temporal network that captures the interactome of each individual sample. We found that the temporal network appears to follow an annual cycle, collapsing, and reassembling when transiting between colder and warmer waters. We observed higher association repeatability in colder than in warmer months. Only 16 associations could be validated using observations reported in literature, underlining our knowledge gap in marine microbial ecological interactions. Conclusions: Our results indicate that marine microbial associations follow recurrent temporal dynamics in temperate zones, which need to be accounted for to better understand the functioning of the ocean microbiome. The constructed marine temporal network may serve as a resource for testing season-specific microbial interaction hypotheses. The applied approach can be transferred to microbiome studies in other ecosystems

[EN] There is a myriad of microorganisms on Earth contributing to global biogeochemical cycles. In the surface ocean, the smallest microbes (picoplankton) are responsible for an important fraction of the total atmospheric carbon and nitrogen fixation. The ocean picoplankton encompasses both prokaryotes (bacteria and archaea) and tiny unicellular eukaryotes (protists). Despite their overall importance for the functioning of the biosphere, many questions remain unanswered on their biogeography, population dynamics, interactions, and evolution. Answering these questions is essential in the context of global change, as alterations of the ocean microbiome could impact the function of multiple ecosystems. In this thesis, we aim at reducing the knowledge gap on the above topics through the application of High-Throughput Sequencing (HTS) and genomic approaches, using data collected during the circumglobal Tara Oceans and Malaspina-2010 expeditions, as well as at the Gulf of Maine (North Atlantic), and two Northwestern Mediterranean coastal microbial observatories (BBMO and SOLA stations). [...], [ES] Existen un sinfín de microorganismos que contribuyen a los ciclos biogeoquímicos globales. En la superficie oceánica, los microbios más pequeños (picoplancton) son responsables de fijar una gran parte del total de carbono y nitrógeno terrestre. El picoplancton agrupa tanto a procariotas (bacterias y arqueas) como a pequeños eucariotas unicelulares (protistas). A pesar de su importancia en el funcionamiento de la biosfera, existen aún muchas preguntas sin respuesta relacionadas con su biogeografía, dinámica poblacional, interacciones y evolución. Responder dichas preguntas es esencial en el contexto del cambio climático, ya que alteraciones en la microbiota marina podría impactar en el funcionamiento de múltiples ecosistemas. El principal objetivo de la tesis es llenar el vacío existente sobre los temas mencionados a través de la aplicación de técnicas de secuenciación de alto rendimiento (HTS) y de métodos genómicos, usando datos recolectados durante las expediciones globales de Tara Oceans y Malaspina-2010, el Golfo de Maine (Océano Atlántico Norte), y dos observatorios situados en la costa noroeste del mar Mediterráneo (BBMO y SOLA). [...]

Unicellular eukaryotic predators play a crucial role in the functioning of the ocean ecosystem by recycling nutrients and energy that are channeled to upper trophic levels. Traditionally, these evolutionarily diverse organisms have been combined into a single functional group (heterotrophic flagellates), overlooking their organismal differences. Here, we investigated four evolutionarily related species belonging to one cosmopolitan group of uncultured marine picoeukaryotic predators: marine stramenopiles (MAST)-4 (species A, B, C, and E). Co-occurrence and distribution analyses in the global surface ocean indicated contrasting patterns in MAST-4A and C, suggesting adaptation to different temperatures. We then investigated whether these spatial distribution patterns were mirrored by MAST-4 genomic content using single-cell genomics. Analyses of 69 single cells recovered 66 to 83% of the MAST-4A/B/C/E genomes, which displayed substantial interspecies divergence. MAST-4 genomes were similar in terms of broad gene functional categories, but they differed in enzymes of ecological relevance, such as glycoside hydrolases (GHs), which are part of the food degradation machinery in MAST-4. Interestingly, MAST-4 species featuring a similar GH composition (A and C) coexcluded each other in the surface global ocean, while species with a different set of GHs (B and C) appeared to be able to coexist, suggesting further niche diversification associated with prey digestion. We propose that differential niche adaptation to temperature and prey type has promoted adaptive evolutionary diversification in MAST-4. We show that minute ocean predators from the same phylogenetic group may have different biogeography and genomic content, which needs to be accounted for to better comprehend marine food webs

Heterotrophic lineages of stramenopiles exhibit enormous diversity in morphology, lifestyle, and habitat. Among them, the marine stramenopiles (MASTs) represent numerous independent lineages that are only known from environmental sequences retrieved from marine samples. The core energy metabolism characterizing these unicellular eukaryotes is poorly understood. Here, we used single-cell genomics to retrieve, annotate, and compare the genomes of 15 MAST species, obtained by coassembling sequences from 140 individual cells sampled from the marine surface plankton. Functional annotations from their gene repertoires are compatible with all of them being phagocytotic. The unique presence of rhodopsin genes in MAST species, together with their widespread expression in oceanic waters, supports the idea that MASTs may be capable of using sunlight to thrive in the photic ocean. Additional subsets of genes used in phagocytosis, such as proton pumps for vacuole acidification and peptidases for prey digestion, did not reveal particular trends in MAST genomes as compared with nonphagocytotic stramenopiles, except a larger presence and diversity of V-PPase genes. Our analysis reflects the complexity of phagocytosis machinery in microbial eukaryotes, which contrasts with the well-defined set of genes for photosynthesis. These new genomic data provide the essential framework to study ecophysiology of uncultured species and to gain better understanding of the function of rhodopsins and related carotenoids in stramenopiles

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

Motivation: Previous Co-assemblies of marine microeukaryotic flagellates MAST-4 (clades A and E) based on single-cell MDA data may present DNA contamination from other eukaryotes or prokaryotes which has proven to be difficult to detect. Decontaminated assemblies are needed in order to study the role of these relatively abundant and ubiquitous organisms in the ocean, as well as to determine their genomic composition. Comparative genomics can help linking gene composition and ecological success in MAST-4 as well as to determine the degree of genomic differentiation between clades A and E. Results: We have been able to detect and remove traces of contaminant DNA. Afterwards, our comparative genomic studies showed how both protists have similar functional metabolic routes that link them to their environment. Yet, at the same time, our analyses showed a significant degree of divergence at the protein and whole genome level, indicating that they are evolutionary more distant than expected