Publications

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants in the global ocean, yet the microorganisms involved in their degradation and the environmental drivers controlling their distribution remain poorly understood. Using a global metagenomics approach, we assessed the distribution and diversity of hydrocarbon-degrading bacteria (HDB) across different oceanic regions and depths. Our results reveal a widespread distribution of HDB in the ocean, with distinct community compositions between surface and deep waters. PAH concentrations were positively correlated with the abundance of specific HDB taxa, suggesting an intimate relationship between pollutant levels and microbial community structure. This study provides new insights into the biogeography of HDB and their potential role in PAH cycling in the ocean.

Metazoans represent a minute fraction of oceanic biomass but play a crucial role in ocean food webs and carbon sequestration. We used an environmental DNA (eDNA) approach to assess the patterns in metazoan taxa richness in the global ocean by examining the Malaspina 2010 metagenomic sequences dataset. We detected 14 phyla, with a dominance of arthropods and cnidarians. Metazoan taxa richness in the epipelagic ocean was higher than reported in previous global estimates and strongly related to productivity. Taxa richness and diversity decreased from the epipelagic (surface and 100–200 m depth) to the mesopelagic (300–600 m depth), and bathypelagic (1000–4000 m depth) layers. Richness significantly increased with dissolved silica, suggesting a role of diatom-derived materials as a food source and/or ballast for sinking particles. This study represents one of the first applications of eDNA from metagenomes to investigate patterns in global metazoan diversity in the ocean and suggests that eDNA is a powerful approach to assess patterns in metazoan taxa richness in the global ocean.

The deep ocean is a major reservoir of methylmercury (MeHg), a potent neurotoxin that bioaccumulates through marine food webs. However, the microbial communities involved in MeHg degradation and mercury (Hg) reduction in these environments remain largely unexplored. Here, we combined metagenomic and metatranscriptomic analyses to investigate the distribution, diversity, and activity of microorganisms carrying mer genes (involved in Hg detoxification) in bathypelagic waters of the global ocean. Our results reveal that mer genes are widespread and actively expressed in the deep ocean, with distinct community compositions between different oceanic regions. We identified key bacterial taxa involved in MeHg demethylation and Hg reduction, including members of the Alteromonadaceae and Rhodobacteraceae families. This study provides the first global assessment of microbial Hg cycling in the deep ocean and highlights the importance of microbial processes in controlling MeHg levels in this understudied environment.

A major consequence of sea ice decline in the Arctic Ocean is the expected shift in phytoplankton communities towards smaller picophytoplankton cells. However, the consequences for the bacterioplankton remain largely unexplored. We investigated the changes in bacterioplankton community composition, diversity and the potential for iron acquisition along the early spring to summer transition in Dease Strait (Kitikmeot region, Nunavut) in the Canadian Arctic. Metagenomic data revealed a temporal succession of taxa with an overall increase in diversity at the onset of summer. Furthermore, despite the presence of diverse siderophore biosynthetic gene clusters throughout the ice-covered season, we observed a late-spring drop in siderophore transport genes abundance. This suggests an increase in iron bioavailability during the ice melt season. Indeed, co-abundance network and indicator species analyses showed that the early spring community was dominated by siderophore-producer taxa in comparison to the summer community, where non-producing taxa proliferated. Our analysis suggests that, at the onset of summer, the competition for iron in the free-living bacterioplankton community switched from direct to exploitative competition.

The deep ocean is one of the most unexplored biomes, and microbial processes occurring in these environments remain poorly characterized. This includes the detoxification of toxic methylmercury (MeHg), despite the fact that the deep sea is one of the main reservoirs of MeHg in the ocean. Here we characterize the genomic traits associated with mercury (Hg) resistance in the deep ocean isolate Alteromonas mediterranea ISS312, previously shown to have a high tolerance to MeHg. We also examine its transcriptomic response to MeHg exposure to better understand the mechanisms driving MeHg resistance. Our results show that the presence of a complete mer operon and putative horizontally transferred metal resistance genes could be associated with its high tolerance to MeHg. We also identify a transcriptomic response to MeHg involving the upregulation of putative transporters and stress response mechanisms outside the mer operon, as well as downregulation of membrane modifications and metabolic pathways, highlighting the complexity of the response of this species to MeHg exposure. This study advances the understanding of the Hg detoxification potential of heterotrophic bacteria in the deep ocean.

The bathypelagic realm is by volume the largest biome on Earth and harbors a significant fraction of the ocean's microbial biomass. However, the factors controlling the metabolic activity and composition of its microbial communities remain poorly characterized. Here, we used metatranscriptomics to analyze the active prokaryotic communities throughout the water column of five oceanographic stations in the tropical and subtropical Atlantic and Pacific oceans. We found a clear vertical zonation in the active community composition, with bathypelagic waters harboring distinct communities compared to shallower layers. The composition of the active bathypelagic communities was primarily influenced by water mass age and the quality of the organic carbon sources, with older waters harboring communities enriched in taxa specialized in the degradation of recalcitrant organic matter. Our results suggest that niche partitioning in the bathypelagic ocean is driven by the interplay between water mass age and organic carbon quality, and highlight the importance of considering these factors in future studies of deep ocean microbial ecology.

The microbial-driven mercury (Hg) detoxification process is mainly based on the reduction of ionic mercury to the less toxic elemental Hg. However, information on this process and, especially, on the physiology of Hg-resistant bacteria (HgRB) is still scarce. Here, we use a combination of cultivation, phenotypic and phylogenomic characterization, and time-series transcriptomics to explore the physiology of HgRB from Hg-polluted marine sediments. We isolate, and characterize 74 HgRB strains. Minimum Inhibitory Concentration (MIC) values range from 2.5 to 80 µM for ionic Hg and from 0.25 to 40 µM for MeHg. Pangenome analyses evidence an accessory genome containing key genes putatively involved in heavy metal-related processes, with mer operons in 64% of the 74 strains. Time-series transcriptomics of selected strains show an overall overexpression of the mer operon and genes for carbohydrate metabolism, lipopolysaccharide modifications, and antioxidant responses related to Hg detoxification that occur after 8 h of Hg exposure. This work reveals that HgRB use multiple strategies to protect themselves from the toxic effects of Hg and provides a valuable resource for the bioremediation of Hg-polluted environments.

Mercury (Hg) accumulates in the biosphere as toxic methylmercury (MeHg) through a process that depends on bacterial activity mediated by hgcAB genes. In this study, we investigate the microbial community composition and the mer (detoxification), as well as hgcAB (methylation) operons present in the sediments of the Bilbao estuary, historically polluted by Hg from human activities. We detected a very high abundance of the mer operon (up to 8.9% of the community) and hgcAB (up to 0.45%), together with a great diversity of sequences phylogenetically related to these genes. We identified a potential multifaceted role for Desulfobacterota since they can harbor hgcAB as well as mer genes that belong to three different putative operons: (i) merAB + hgcAB; (ii) merAB without hgcAB and (iii) merA (lacking merB) together with hgcAB genes. The last two gene combinations have not been yet reported in microorganisms from polluted sediments and highlight the complexity and importance of investigating the Hg cycling. In addition, we discovered that Desulfobacterota may regulate the expression of Hg transporters and transcriptional regulators (arsR) in response to variable Hg concentrations. These findings bring the current consensus on the role of Desulfobacterota in MeHg cycling into question and highlight the need for experimental validation.

The ocean contains a large and highly diverse microbial assemblage whose members are responsible for key steps in global biogeochemical cycles. Therefore, characterization of the taxonomy, functional capabilities, and environmental interactions of these microbial communities is crucial to understand the ocean ecosystem. To help expand our knowledge of marine microbiomes we present a dataset of 94 metagenomes from 11 stations sampled during the Malaspina 2010 Expedition (10 metagenomic stations). Seawater samples were collected at three size fractions (0.2–0.8 µm (picoplankton), 0.8–5 µm (nanoplankton), and 5–20 µm (microplankton)) in surface and four oceanic depths: Deep Chlorophyll Maximum (DCM) epipelagic (Epi), mesopelagic (Meso), and bathypelagic (Bathy). The seawater filtered samples were sequenced, assembled, annotated, and binned to produce metagenome-assembled genomes (MAGs). Following quality control filtering, more than 134.7 billion reads and 377 high-quality MAGs remained available for downstream analyses, which collectively increased our catalog of uncultured marine microorganisms considerably.

Traditional culture techniques usually retrieve a small fraction of the marine microbial diversity, which mainly belong to the so-called rare biosphere. However, this paradigm has not been fully tested at a broad scale, especially in the deep ocean. Here, we examined the fraction of heterotrophic bacterial communities in photic and deep ocean layers that could be recovered by culture-dependent techniques at a large scale. We compared 16S rRNA gene sequences from a collection of 2003 cultured heterotrophic marine bacteria with global 16S rRNA metabarcoding datasets (16S TAGs) covering surface, mesopelagic and bathypelagic ocean samples that included 16 of the 23 samples used for isolation. These global datasets represent 60 322 unique 16S amplicon sequence variants (ASVs). Our results reveal a significantly higher proportion of isolates identical to ASVs in deeper ocean layers reaching up to 28% of the 16S TAGs of the bathypelagic microbial communities, which included the isolation of 3 of the top 10 most abundant 16S ASVs in the global bathypelagic ocean, related to the genera Sulfitobacter, Halomonas and Erythrobacter. These isolates contributed differently to the prokaryotic communities across different plankton size fractions, recruiting between 38% in the free-living fraction (0.2–0.8 µm) and up to 45% in the largest particles (20–200 µm) in the bathypelagic ocean. Our findings support the hypothesis that sinking particles in the bathypelagic act as resource-rich habitats, suitable for the growth of heterotrophic bacteria with a copiotroph lifestyle that can be cultured, and that these cultivable bacteria can also thrive as free-living bacteria.

Studies based on protein-coding genes are essential to describe the diversity within bacterial functional groups. In the case of aerobic anoxygenic phototrophic (AAP) bacteria, the pufM gene has been established as the genetic marker for this particular functional group, although available primers are known to have amplification biases. We review here the existing primers for pufM gene amplification, design new ones, and evaluate their phylogenetic coverage. We then use samples from contrasting marine environments to evaluate their performance. By comparing the taxonomic composition of communities retrieved with metagenomics and with different amplicon approaches, we show that the commonly used PCR primers are biased towards the Gammaproteobacteria phylum and some Alphaproteobacteria clades. The metagenomic approach, as well as the use of other combinations of the existing and newly designed primers, show that these groups are in fact less abundant than previously observed, and that a great proportion of pufM sequences are affiliated to uncultured representatives, particularly in the open ocean. Altogether, the framework developed here becomes a better alternative for future studies based on the pufM gene and, additionally, serves as a reference for primer evaluation of other functional genes.

Viruses play important roles in the ocean's biogeochemical cycles. Yet, deep ocean viruses are one of the most under-explored fractions of the global biosphere. Little is known about the environmental factors that control the composition and functioning of their communities or how they interact with their free-living or particle-attached microbial hosts.

Research on marine microbial communities is growing, but studies are hard to compare because of variation in seawater sampling protocols. To help researchers in the inter-comparison of studies that use different seawater sampling methodologies, as well as to help them design future sampling campaigns, we developed the EuroMarine Open Science Exploration initiative (EMOSE). Within the EMOSE framework, we sampled thousands of liters of seawater from a single station in the NW Mediterranean Sea (Service d'Observation du Laboratoire Arago [SOLA], Banyuls-sur-Mer), during one single day. The resulting dataset includes multiple seawater processing approaches, encompassing different material-type kinds of filters (cartridge membrane and flat membrane), three different size fractionations (>0.22 µm, 0.22–3 µm, 3–20 µm and >20 µm), and a number of different seawater volumes ranging from 1 L up to 1000 L. We show that the volume of seawater that is filtered does not have a significant effect on prokaryotic and protist diversity, independently of the sequencing strategy. However, there was a clear difference in alpha and beta diversity between size fractions and between these and 'whole water' (with no pre-fractionation). Overall, we recommend care when merging data from datasets that use filters of different pore size, but we consider that the type of filter and volume should not act as confounding variables for the tested sequencing strategies. To the best of our knowledge, this is the first time a publicly available dataset effectively allows for the clarification of the impact of marine microbiome methodological options across a wide range of protocols, including large-scale variations in sampled volume.

Strong purifying selection is considered a major evolutionary force behind small microbial genomes in the resource-poor photic ocean. However, very little is currently known about how the size of prokaryotic genomes evolves in the global ocean and whether patterns reflect shifts in resource availability in the epipelagic and relatively stable deep-sea environmental conditions. Using 364 marine microbial metagenomes, we investigate how the average genome size of uncultured planktonic prokaryotes varies across the tropical and polar oceans to the hadal realm. We find that genome size is highest in the perennially cold polar ocean, reflecting elongation of coding genes and gene dosage effects due to duplications in the interior ocean microbiome. Moreover, the rate of change in genome size due to temperature is 16-fold higher than with depth up to 200 m. Our results demonstrate how environmental factors can influence marine microbial genome size selection and ecological strategies of the microbiome.

Mercury (Hg) methylation genes (hgcAB) mediate the formation of the toxic methylmercury and have been identified from diverse environments, including freshwater and marine ecosystems, Arctic permafrost, forest and paddy soils, coal-ash amended sediments, chlor-alkali plants discharges and geothermal springs. Here we present the first attempt at a standardized protocol for the detection, identification and quantification of hgc genes from metagenomes. Our Hg-cycling microorganisms in aquatic and terrestrial ecosystems (Hg-MATE) database, a catalogue of hgc genes, provides the most accurate information to date on the taxonomic identity and functional/metabolic attributes of microorganisms responsible for Hg methylation in the environment. Furthermore, we introduce 'marky-coco', a ready-to-use bioinformatic pipeline based on de novo single-metagenome assembly, for easy and accurate characterization of hgc genes from environmental samples. We compared the recovery of hgc genes from environmental metagenomes using the marky-coco pipeline with an approach based on coassembly of multiple metagenomes. Our data show similar efficiency in both approaches for most environments except those with high diversity (i.e., paddy soils) for which a coassembly approach was preferred. Finally, we discuss the definition of true hgc genes and methods to normalize hgc gene counts from metagenomes.

Natural microbial communities are an important source of therapeutically relevant small molecules. However, studying the biosynthetic potential of complex microbiomes at the global scale remains challenging. Here we systematically investigated the biosynthetic potential of the ocean microbiome—one of the largest microbiomes on Earth—by reconstructing ~40,000 putative biosynthetic gene clusters (BGCs) from ~10,000 metagenome samples. We found BGCs to be abundant in the ocean, with ~3% of microbial genomes encoding at least one cluster. We discovered that the majority of oceanic BGC diversity remains uncharted, with most clusters representing novel families. Moreover, we show that BGC composition varies in response to environmental parameters, such as temperature and oxygen, implying an adaptation of microbial secondary metabolism to environmental conditions. Finally, we identify candidate clusters for the biosynthesis of known molecules, enabling studies of their ecology and evolution. This work provides a resource of unprecedented scale to explore microbial biosynthetic potential in the ocean and serves as a blueprint for microbiome-scale studies of natural product diversity.

Testing hypothesis about the biogeography of genes using large data resources such as Tara Oceans marine metagenomes and metatranscriptomes requires significant hardware resources and programming skills. The new release of the 'Ocean Gene Atlas' (OGA2) is a freely available intuitive online service to mine large and complex marine environmental genomic databases. OGA2 datasets available have been extended and now include, from the Tara Oceans portfolio: (i) eukaryotic Metagenome-Assembled-Genomes (MAGs) and Single-cell Assembled Genomes (SAGs) (10.2E+6 coding genes), (ii) version 2 of Ocean Microbial Reference Gene Catalogue (46.8E+6 non-redundant genes), (iii) 924 MetaGenomic Transcriptomes (7E+6 unigenes), (iv) 530 MAGs from an Arctic MAG catalogue (1E+6 genes) and (v) 1888 Bacterial and Archaeal Genomes (4.5E+6 genes), and an additional dataset from the Malaspina 2010 global circumnavigation: (vi) 317 Malaspina Deep Metagenome Assembled Genomes (0.9E+6 genes). Novel analyses enabled by OGA2 include phylogenetic tree inference to visualize user queries within their context of sequence homologues from both the marine environmental dataset and the RefSeq database. An Application Programming Interface (API) now allows users to query OGA2 using command-line tools, hence providing local workflow integration. Finally, gene abundance can be interactively filtered directly on map displays using any of the available environmental variables. Ocean Gene Atlas v2.0 is freely-available at: https://tara-oceans.mio.osupytheas.fr/ocean-gene-atlas/.

Genes of unknown function are among the biggest challenges in molecular biology, especially in microbial systems, where 40–60% of the predicted genes are unknown. Despite previous attempts, systematic approaches to include the unknown fraction into analytical workflows are still lacking. Here, we present a conceptual framework, its translation into the computational workflow AGNOSTOS and a demonstration on how we can bridge the known-unknown gap in genomes and metagenomes. By analyzing 415,971,742 genes predicted from 1749 metagenomes and 28,941 bacterial and archaeal genomes, we quantify the extent of the unknown fraction, its diversity, and its relevance across multiple organisms and environments. The unknown sequence space is exceptionally diverse, phylogenetically more conserved than the known fraction and predominantly taxonomically restricted at the species level. From the 71 M genes identified to be of unknown function, we compiled a collection of 283,874 lineage-specific genes of unknown function for Cand. Patescibacteria (also known as Candidate Phyla Radiation, CPR), which provides a significant resource to expand our understanding of their unusual biology. Finally, by identifying a target gene of unknown function for antibiotic resistance, we demonstrate how we can enable the generation of hypotheses that can be used to augment experimental data.

Microbial reduction of inorganic divalent mercury (Hg2+) and methylmercury (MeHg) demethylation is performed by the mer operon, specifically by merA and merB genes, respectively, but little is known about the mercury tolerance capacity of marine microorganisms and its prevalence in the ocean. Here, combining culture-dependent analyses with metagenomic and metatranscriptomic data, we show that marine bacteria that encode mer genes are widespread and active in the global ocean. We explored the distribution of these genes in 290 marine heterotrophic bacteria (Alteromonas and Marinobacter spp.) isolated from different oceanographic regions and depths, and assessed their tolerance to diverse concentrations of Hg2+ and MeHg. In particular, the Alteromonas sp. ISS312 strain presented the highest tolerance capacity and a degradation efficiency for MeHg of 98.2% in 24 h. Fragment recruitment analyses of Alteromonas sp. genomes (ISS312 strain and its associated reconstructed metagenome assembled genome MAG-0289) against microbial bathypelagic metagenomes confirm their prevalence in the deep ocean. Moreover, we retrieved 54 merA and 6 merB genes variants related to the Alteromonas sp. ISS312 strain from global metagenomes and metatranscriptomes from Tara Oceans. Our findings highlight the biological reductive MeHg degradation as a relevant pathway of the ocean Hg biogeochemical cycle.

The Arctic Ocean is a unique marine environment characterized by extreme seasonality, cold temperatures, and ice cover. Despite its importance for global biogeochemical cycles, the microbial communities inhabiting this ocean remain poorly characterized at the genomic level. Here, we present a compendium of 530 metagenome-assembled genomes (MAGs) from 41 Tara Oceans polar samples spanning the epipelagic and mesopelagic zones of the Arctic Ocean. These MAGs represent 526 bacterial and 4 archaeal species, of which 68% could not be assigned to any known species. We found that Arctic MAGs are enriched in genes involved in cold adaptation, photoheterotrophy, and the degradation of algal-derived organic matter. Comparison with global ocean MAG catalogues revealed that 41% of the Arctic MAGs were exclusively found in polar waters, highlighting the unique composition of Arctic microbial communities. This resource provides new insights into the genomic diversity and metabolic potential of Arctic Ocean microbiomes.

The deep sea, the largest ocean's compartment, drives planetary-scale biogeochemical cycling. Yet, the functional exploration of its microbial communities lags far behind other environments. Here we analyze 58 metagenomes from tropical and subtropical deep oceans to generate the Malaspina Gene Database. Free-living or particle-attached lifestyles drive functional differences in bathypelagic prokaryotic communities, regardless of their biogeography. Ammonia and CO oxidation pathways are enriched in the free-living microbial communities and dissimilatory nitrate reduction to ammonium and H2 oxidation pathways in the particle-attached, while the Calvin Benson-Bassham cycle is the most prevalent inorganic carbon fixation pathway in both size fractions. Reconstruction of the Malaspina Deep Metagenome-Assembled Genomes reveals unique non-cyanobacterial diazotrophic bacteria and chemolithoautotrophic prokaryotes. The widespread potential to grow both autotrophically and heterotrophically suggests that mixotrophy is an ecologically relevant trait in the deep ocean. These results expand our understanding of the functional microbial structure and metabolic capabilities of the largest Earth aquatic ecosystem.

Marine heterotrophic bacteria are key players in ocean biogeochemical cycles, yet their diversity and distribution remain poorly characterized. Here, we analyzed a large culture collection of marine heterotrophic bacteria isolated from various oceanographic regions and depths to assess their diversity and biogeographic patterns. The collection comprised 2003 isolates from surface to bathypelagic waters across tropical and subtropical oceans. We found that the culturable fraction of marine heterotrophic bacteria is dominated by a few cosmopolitan genera, including Alteromonas, Erythrobacter, and Marinobacter. However, the majority of isolates represented rare taxa with restricted geographic distributions. Our results suggest that while a core set of heterotrophic bacteria is globally distributed, regional environmental conditions shape the composition of local communities. This culture collection provides a valuable resource for future studies on the physiology and ecology of marine heterotrophic bacteria.

Cultivable bacteria represent only a fraction of the diversity in microbial communities. However, the official procedures for classification and characterization of a novel prokaryotic species still rely on isolates. Nevertheless, due to single cell genomics, it is possible to retrieve genomes from environmental samples by sequencing them individually, and to assign specific genes to a specific taxon, regardless of their ability to grow in culture. In this study, a complete description was performed for uncultured Kordia sp. TARA_039_SRF, a proposed novel species within the genus Kordia, using culture-independent techniques. The type material was a high-quality draft genome (94.97% complete, 4.65% gene redundancy) co-assembled using ten nearly identical single amplified genomes (SAGs) from surface seawater in the North Indian Ocean during the Tara Oceans Expedition. The assembly process was optimized to obtain the best possible assembly metrics and a less fragmented genome. The closest relative of the species was Kordia periserrulae, which shared 97.56% similarity of the 16S rRNA gene, 75% orthologs and 89.13% average nucleotide identity. The functional potential of the proposed novel species included proteorhodopsin, the ability to incorporate nitrate, cytochrome oxidases with high affinity for oxygen, and CAZymes that were unique features within the genus. Its abundance at different depths and size fractions was also evaluated together with its functional annotation, revealing that its putative ecological niche could be particles of phytoplanktonic origin. It could putatively attach to these particles and consume them while sinking to the deeper and oxygen depleted layers of the North Indian Ocean.

Surveying microbial diversity and function is accomplished by combining complementary molecular tools. Among them, metagenomics is a PCR free approach that contains all genetic information from microbial assemblages and is today performed at a relatively large scale and reasonable cost, mostly based on very short reads. Here, we investigated the potential of metagenomics to provide taxonomic reports of marine microbial eukaryotes. We prepared a curated database with reference sequences of the V4 region of 18S rDNA clustered at 97% similarity and used this database to extract and classify metagenomic reads. More than half of them were unambiguously affiliated to a unique reference whilst the rest could be assigned to a given taxonomic group. The overall diversity reported by metagenomics was similar to that obtained by amplicon sequencing of the V4 and V9 regions of the 18S rRNA gene, although either one or both of these amplicon surveys performed poorly for groups like Excavata, Amoebozoa, Fungi and Haptophyta. We then studied the diversity of picoeukaryotes and nanoeukaryotes using 91 metagenomes from surface down to bathypelagic layers in different oceans, unveiling a clear taxonomic separation between size fractions and depth layers. Finally, we retrieved long rDNA sequences from assembled metagenomes that improved phylogenetic reconstructions of particular groups. Overall, this study shows metagenomics as an excellent resource for taxonomic exploration of marine microbial eukaryotes.

Ocean microbial communities strongly influence the biogeochemistry, food webs, and climate of our planet. Despite recent advances in understanding their taxonomic and genomic compositions, little is known about how their transcriptomes vary globally. Here, we present a dataset of 187 metatranscriptomes and 370 metagenomes from 126 globally distributed sampling stations and establish a resource of 47 million genes to study community-level transcriptomes across depth layers from pole-to-pole. We examine gene expression changes and community turnover as the underlying mechanisms shaping community transcriptomes along these axes of environmental variation and show how their individual contributions differ for multiple biogeochemically relevant processes. Furthermore, we find the relative contribution of gene expression changes to be significantly lower in polar than in non-polar waters and hypothesize that in polar regions, alterations in community activity in response to ocean warming will be driven more strongly by changes in organismal composition than by gene regulatory mechanisms.

Aerobic anoxygenic phototrophic (AAP) bacteria are widespread in the ocean and contribute to marine carbon cycling through their unique photoheterotrophic metabolism. However, the long-term dynamics of these organisms remain poorly understood. Here, we analyzed the seasonal and interannual variability of AAP bacteria at the Blanes Bay Microbial Observatory (NW Mediterranean) over a 10-year period using amplicon sequencing of the pufM gene, which encodes a key protein in bacterial phototrophy. We found that AAP bacteria exhibited strong seasonal patterns, with highest abundances in summer and lowest in winter. The community composition also varied seasonally, with distinct assemblages dominating in different seasons. Interannual variability was lower than seasonal variability but still significant, suggesting that long-term environmental changes can influence AAP bacterial communities. Our results provide new insights into the temporal dynamics of these important photoheterotrophic bacteria in coastal marine ecosystems.

High-throughput sequencing (HTS) of the 16S rRNA gene has been used successfully to describe the structure and dynamics of microbial communities. Picocyanobacteria are important members of bacterioplankton communities, and, so far, they have predominantly been targeted using universal bacterial primers, providing a limited resolution of the picocyanobacterial community structure and dynamics. To increase such resolution, the study of a particular target group is best approached with the use of specific primers. Here, we aimed to design and evaluate specific primers for aquatic picocyanobacterial genera to be used with high-throughput sequencing. Since the various regions of the 16S rRNA gene have different degrees of conservation in different bacterial groups, we therefore first determined which hypervariable region of the 16S rRNA gene provides the highest taxonomic and phylogenetic resolution for the genera Synechococcus, Prochlorococcus, and Cyanobium. An in silico analysis showed that the V5, V6, and V7 hypervariable regions appear to be the most informative for this group. We then designed primers flanking these hypervariable regions and tested them in natural marine and freshwater communities. We successfully detected that most (97%) of the obtained reads could be assigned to picocyanobacterial genera. We defined operational taxonomic units as exact sequence variants (zero-radius operational taxonomic units [zOTUs]), which allowed us to detect higher genetic diversity and infer ecologically relevant information about picocyanobacterial community composition and dynamics in different aquatic systems. Our results open the door to future studies investigating picocyanobacterial diversity in aquatic systems.

Bacteroidetes is one of the dominant phyla in marine environments, especially in coastal areas. However, little is known about the drivers of their diversity at fine phylogenetic scales. In the present study, we identified two Bacteroidetes taxa with contrasting ecological features by examining the population dynamics of 16S rRNA gene sequences throughout a 2‐year monthly time series data set from surface seawater at a single coastal station in the NW Mediterranean Sea (Blanes Bay Microbial Observatory). While Polaribacter was most abundant during late winter‐spring, the NS5 marine group was most abundant during summer. We then investigated the distribution of Bacteroidetes internal transcribed spacer (ITS) sequences in samples representing different environmental conditions, including coastal and open ocean, surface and deep chlorophyll maximum (DCM), as well as free‐living and particle‐attached fractions. Clear differences in the diversity and composition of the Bacteroidetes community were observed between all sample types. Differences were largest between the free‐living and particle‐attached fractions: the particle‐attached community had fewer ecotypes with more phylogenetic diversity. At the finer phylogenetic scale, most ecotypes (clusters of >97% similar ITS sequences) showed preferences for a particular environment. Interestingly, a predominant group related to the OM43 clade showed a remarkably low differentiation and occupied multiple niches, suggesting a wider ecological potential. We identified ecologically differentiated units of Bacteroidetes and suggest these should be delineated as species.

Aim: Understanding how historical and contemporary processes shaped and maintain spatial patterns of genetic diversity is a major goal for conservation biologists. Here, we characterized the pattern of neutral genetic diversity and we inferred underlying processes in the habitat‐forming octocoral Paramuricea clavata in the Adriatic Sea, a peculiar phylogeographic region of the Mediterranean Sea. Location: Eastern coast of the Adriatic Sea. Methods: We genotyped seven microsatellites in 454 individuals of P. clavata from 13 populations recolonized after the Last Glacial Maximum (LGM). We estimated the levels of contemporary connectivity and genetic drift and we reconstructed the demographic history of these populations. Results: The pattern of spatial genetic structure resulted from the combination of hierarchical genetic clusters and isolation by distance (IBD). A significant decrease in genetic diversity and an increase of the frequencies of individual alleles likely due to allele surfing from the south to the north of the area were observed as expected after a postglacial sequential recolonization. Based on maximum likelihood analyses, the foundation of these populations was not linked to dramatic change in population size. Main conclusion: Oceanographic barriers to gene flow combined to the restricted dispersal of P. clavata likely maintain the hierarchical structure and the IBD pattern. We suggest that the latitudinal genetic gradient results from a northward “serial founder events” recolonization. By integrating patterns and processes, we bridge the gap between the evolutionary and the conservation biology of P. clavata, providing management guidelines, which will benefit the associated coralligenous biodiversity.

Cyanobacteria are important contributors to primary production in the open oceans. Over the past decade, various photosynthesis-related genes have been found in viruses that infect cyanobacteria (cyanophages). Although photosystem II (PSII) genes are common in both cultured cyanophages and environmental samples, viral photosystem I (vPSI) genes have so far only been detected in environmental samples. Here, we have used a targeted strategy to isolate a cyanophage from the tropical Pacific Ocean that carries a PSI gene cassette with seven distinct PSI genes (psaJF, C, A, B, K, E, D) as well as two PSII genes (psbA, D). This cyanophage, P-TIM68, belongs to the T4-like myoviruses, has a prolate capsid, a long contractile tail and infects Prochlorococcus sp. strain MIT9515. Phage photosynthesis genes from both photosystems are expressed during infection, and the resultant proteins are incorporated into membranes of the infected host. Moreover, photosynthetic capacity in the cell is maintained throughout the infection cycle with enhancement of cyclic electron flow around PSI. Analysis of metagenomic data from the Tara Oceans expedition shows that phages carrying PSI gene cassettes are abundant in the tropical Pacific Ocean, composing up to 28% of T4-like cyanomyophages. They are also present in the tropical Indian and Atlantic Oceans. P-TIM68 populations, specifically, compose on average 22% of the PSI-gene-cassette carrying phages. Our results suggest that cyanophages carrying PSI and PSII genes are likely to maintain and even manipulate photosynthesis during infection of their Prochlorococcus hosts in the tropical oceans.

Marine Bacteroidetes constitute a very abundant bacterioplankton group in the oceans that plays a key role in recycling particulate organic matter and includes several photoheterotrophic members containing proteorhodopsin. Relatively few marine Bacteroidetes species have been described and, moreover, they correspond to cultured isolates, which in most cases do not represent the actual abundant or ecologically relevant microorganisms in the natural environment. In this study, we explored the microdiversity of 98 Single Amplified Genomes (SAGs) retrieved from the surface waters of the underexplored North Indian Ocean, whose most closely related isolate is Kordia algicida OT-1. Using Multi Locus Sequencing Analysis (MLSA) we found no microdiversity in the tested conserved phylogenetic markers (16S rRNA and 23S rRNA genes), the fast-evolving Internal Transcribed Spacer and the functional markers proteorhodopsin and the beta-subunit of RNA polymerase. Furthermore, we carried out a Fragment Recruitment Analysis (FRA) with marine metagenomes to learn about the distribution and dynamics of this microorganism in different locations, depths and size fractions. This analysis indicated that this taxon belongs to the rare biosphere, showing its highest abundance after upwelling-induced phytoplankton blooms and sinking to the deep ocean with large organic matter particles. This uncultured Kordia lineage likely represents a novel Kordia species (Kordia sp. CFSAG39SUR) that contains the proteorhodopsin gene and has a widespread spatial and vertical distribution. The combination of SAGs and MLSA makes a valuable approach to infer putative ecological roles of uncultured abundant microorganisms.

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.

Upon phosphorus (P) deficiency, marine phytoplankton reduce their requirements for P by replacing membrane phospholipids with alternative non-phosphorus lipids. It was very recently demonstrated that a SAR11 isolate also shares this capability when phosphate starved in culture. Yet, the extent to which this process occurs in other marine heterotrophic bacteria and in the natural environment is unknown. Here, we demonstrate that the substitution of membrane phospholipids for a variety of non-phosphorus lipids is a conserved response to P deficiency among phylogenetically diverse marine heterotrophic bacteria, including members of the Alphaproteobacteria and Flavobacteria. By deletion mutagenesis and complementation in the model marine bacterium Phaeobacter sp. MED193 and heterologous expression in recombinant Escherichia coli, we confirm the roles of a phospholipase C (PlcP) and a glycosyltransferase in lipid remodelling. Analyses of the Global Ocean Sampling and Tara Oceans metagenome data sets demonstrate that PlcP is particularly abundant in areas characterized by low phosphate concentrations. Furthermore, we show that lipid remodelling occurs seasonally and responds to changing nutrient conditions in natural microbial communities from the Mediterranean Sea. Together, our results point to the key role of lipid substitution as an adaptive strategy enabling heterotrophic bacteria to thrive in the vast P-depleted areas of the ocean.

Marine photosynthesis is largely driven by cyanobacteria, namely Synechococcus and Prochlorococcus. Genes encoding for photosystem (PS) I and II reaction centre proteins are found in cyanophages and are believed to increase their fitness. Two viral PSI gene arrangements are known, psaJF→C→A→B→K→E→D and psaD→C→A→B. The shared genes between these gene cassettes and their encoded proteins are distinguished by %G+C and protein sequence respectively. The data on the psaD→C→A→B gene organization were reported from only two partial gene cassettes coming from Global Ocean Sampling stations in the Pacific and Indian oceans. Now we have extended our search to 370 marine stations from six metagenomic projects. Genes corresponding to both PSI gene arrangements were detected in the Pacific, Indian and Atlantic oceans, confined to a strip along the equator (30°N and 30°S). In addition, we found that the predicted structure of the viral PsaA protein from the psaD→C→A→B organization contains a lumenal loop conserved in PsaA proteins from Synechococcus, but is completely absent in viral PsaA proteins from the psaJF→C→A→B→K→E→D gene organization and most Prochlorococcus strains. This may indicate a co-evolutionary scenario where cyanophages containing either of these gene organizations infect cyanobacterial ecotypes biogeographically restricted to the 30°N and 30°S equatorial strip.

Species interaction networks are shaped by abiotic and biotic factors. Here, as part of the Tara Oceans project, we studied the photic-zone interactome using environmental factors, taxonomy, and organismal interaction data based on 313 size-fractionated plankton communities sampled across the globe. We found that weights of organismal interactions are on par with those of environmental factors in structuring community composition. The strongest and most conserved associations connect organisms that are closely related or belong to the same trophic level. This study provides a comprehensive view of the planktonic interactome and demonstrates that organismal interaction data are a critical step toward understanding community structure and its response to environmental change.

The presence of the green and Kemp's ridley turtles is rare at Atlantic and Mediterranean Spanish waters, but the records have increased during the last decades. We reported a new set of records and reviewed all the historical observations of these species. The analysis of a mitochondrial DNA fragment of the newest records provided insights about the origin of the individuals. The Kemp's ridley turtles arrived from the western Atlantic nesting beaches, although the discovering of a new haplotype suggested the existence of an unknown or low sampled nesting area of origin. Furthermore, the genetic analysis was crucial for the species identification in one specimen, hence recommending the use of genetic markers to confirm the presence of a rare species. All green turtles presented haplotypes exclusive from Atlantic nesting beaches and concentrated in the African populations. Thus, the closest eastern Mediterranean nesting areas were discarded as source populations and a new migration route for this species was described.

To assess how initial size dispersion affects Senegalese sole (Solea senegalensis, Kaup) growth, 128 fish were stocked either under homogeneous (HOM) or heterogeneous (HET) initial size dispersion (standard length, CV 11.8% and 29.7% respectively), and also under low (LD) and high (HD) stocking density (60% and 180% of bottom occupation). After 105 days, growth parameters, evaluated at an individual level using linear mixed-effects models, were not significantly affected by initial size dispersion, LD fish growing significantly faster than HD fish. Variance of individual growth rate distributions of weight and standard length were lowest for LD-HOM fish, indicating the most regular growth, and highest for LD-HET. The diminution of the coefficient of variation along time in HET groups (between 13% and 15.4% for weight, and between 4.3% and 5.8% for standard length), and its slight decrease in the HOM groups (between 1% and 3% for both parameters), is consistent with the absence of strong competition between individuals.

Comparisons of the levels of genetic variation within and between a hatchery F1 (FAR, n=116) of Senegalese sole, Solea senegalensis, and its wild donor population (ATL, n=26), both native to the SW Atlantic coast of the Iberian peninsula, as well as between the wild donor population and a wild western Mediterranean sample (MED, n=18), were carried out by characterizing 412 base pairs of the nucleotide sequence of the mitochondrial DNA control region I, and six polymorphic microsatellite loci. FAR showed a substantial loss of genetic variability (haplotypic diversity, h=0.49±0.066; nucleotide diversity, π=0.006±0.004; private allelic richness, pAg=0.28) to its donor population ATL (h=0.69±0.114; π=0.009±0.006; pAg=1.21). Pairwise FST values of microsatellite data were highly significant (P<0.0001) with mtDNA CR-I (0.228) and with microsatellite data (0.095), respectively. While loss of genetic variability in FAR could be associated with the sampling error when the broodstock was established, the results of parental and sibship inference suggest that most of these losses can be attributed to a high variance in reproductive success among members of the broodstock, particularly among females.

Growth is usually inversely correlated with stocking density of fish in culture. Senegalese sole aquaculture is affected by a high size variability and thus, this work tried to investigate the relationship of growth with density of two populations of 96 individually tagged Senegalese sole (318.7± 7.9 g; mean± standard error of the mean). Fish were reared at low (LD) and high (HD) density (60% and 180% of bottom coverage respectively) for 195 days. After 134 days (period 1), density conditions were exchanged between groups. Mean weight, standard length, maximum width and centroid size were calculated for each of the 11 census days of the experiment. White muscle biopsies were taken in 7 of the census days in order to assess the RNA/DNA ratio, as a biochemical indicator of growth. Stocking density had an important effect on growth, as fish reared under HD showed poor or no growth during a 'lag phase' on the first 61 days of the experiment, leading to a significantly lower specific growth rate (0.23 ± 0.014) for period 1 compared with LD fish (0.34 ± 0.016). Fitting of linear mixed-effects (LME) models for the first 134 days of experiment showed a significant effect of density and sex on all the assessed biometric parameters. These results could be attributed mainly to the first 61 days of the experiment, as no differences were observed between days 61 and 134 in all the measurements, except for standard length, that showed to be lower for HD fish throughout the whole period. Fish reared under high density tended to grow slower than fish held at low density, while females showed faster growth than males, particularly in HD. Nevertheless, due to high size variability, no significant differences could be found in the mean values of weight or standard length after 134 days (467.2± 21.6 g and 28.7± 4.2 cm; 502.6 ± 22.5 g and 29.7± 4.5 cm for HD and LD fish respectively). Size variability could be an indicator of the onset of hierarchies, being stronger and with more females as dominant individuals than males in HD. After exchanging densities, and up to day 195, a similar lagging effect could be observed in LD fish exposed to high density, suggesting that a sudden change in density, more than density itself, could be the responsible for a detrimental effect on growth. RNA/DNA ratios, were significantly lower for HD fish between days 20 and 61.

Morphological changes in Senegalese sole adults (Solea senegalensis Kaup, 1858) reared at two stocking density conditions (low stocking density, 60% of bottom occupation and high stocking density, 180% of bottom occupation) were investigated using geometric morphometrics. Canonical variate analysis on weight matrix scores, including the uniform component (W′), at the end of the experiment revealed differences in body shape between experimental groups. The low density group presented a similar change pattern as that of the high density group but was less intense. Differences were discussed in terms of the effect of a high stocking density on the shape of soles which were very close to commercialization. Results did not indicate a significant size-related shape, likely due to fish age. Our results provide the first promising look at environment-related shape changes in reared sole.