Lawrence Berkeley National Laboratory Recent Work Title Plankton networks driving carbon export in the oligotrophic ocean Permalink

The biological carbon pump is the process by which CO2 is transformed to organic carbon via photosynthesis, exported through sinking particles, and finally sequestered in the deep ocean. While the intensity of the pump correlates with plankton community composition, the underlying ecosystem structure driving the process remains largely uncharacterised. Here we use environmental and metagenomic data gathered during the Tara Oceans expedition to improve our understanding of carbon export in the oligotrophic ocean. We show that specific plankton communities, from the surface and deep chlorophyll maximum, correlate with carbon export at 150 m and highlight unexpected taxa such as Radiolaria, alveolate parasites, as well as Synechococcus and their phages, as lineages most strongly associated with carbon export in the subtropical, nutrient-depleted, oligotrophic ocean. Additionally, we show that the relative abundance of just a few bacterial and viral genes can predict most of the variability in carbon export in these regions. Guidi et al. Page 2 Nature. Author manuscript; available in PMC 2016 September 22. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts Marine planktonic photosynthetic organisms are responsible for approximately fifty percent of Earth’s primary production and fuel the global ocean biological carbon pump1. The intensity of the pump is correlated to plankton community composition2,3, and controlled by the relative rates of primary production and carbon remineralisation4. About 10% of this newly produced organic carbon in the surface ocean is exported through gravitational sinking of particles. Finally, after multiple transformations, a fraction of the exported material reaches the deep ocean where it is sequestered over thousand-year timescales5. Like most biological systems, marine ecosystems in the sunlit upper layer of the ocean (denoted the euphotic zone) are complex6,7, characterised by a wide range of biotic and abiotic interactions8-10 and in constant balance between carbon production, transfer to higher trophic levels, remineralisation, and export to the deep layers11. The marine ecosystem structure and its taxonomic and functional composition likely evolved to comply with this loss of energy by modifying organism turnover times and by the establishment of complex feedbacks between them6 and the substrates they can exploit for metabolism12. Decades of groundbreaking research have focused on identifying independently the key players involved in the biological carbon pump. Among autotrophs, diatoms are commonly attributed to being important in carbon flux because of their large size and fast sinking rates13-15 while small autotrophic picoplankton may contribute directly through subduction of surface water16 or indirectly by aggregating with larger settling particles or consumption by organisms at higher trophic levels17. Among heterotrophs, zooplankton such as crustaceans impact carbon flux via production of fast-sinking fecal pellets while migrating hundreds of meters in the water-column18,19. These observations, focusing on just a few components of the marine ecosystem, highlight that carbon export results from multiple biotic interactions and that a better understanding of the mechanisms involved in its regulation will require an analysis of the entire planktonic ecosystem. Advanced sequencing technologies offer the opportunity to simultaneously survey whole planktonic communities and associated molecular functions in unprecedented detail. Such a holistic approach may allow the identification of communityor gene-based biomarkers that could be used to monitor and predict ecosystem functions, e.g., related to the biogeochemistry of the ocean20-22. Here, we leverage global-scale ocean genomics datasets from the euphotic zone10,23-25 and associated environmental data to assess the coupling between ecosystem structure, functional repertoire, and carbon export at 150 m. Carbon export and plankton community composition The Tara Oceans global circumnavigation crossed diverse ocean ecosystems and sampled plankton at an unprecedented scale20,26 (see Methods). Hydrographic data were measured in situ or in seawater samples at all stations, as well as nutrients, oxygen and photosynthetic pigments (see Methods). Net Primary Production (NPP) was derived from satellite measurements (see Methods). In addition, particle size distributions (100 μm to a few mm) and concentrations were measured using an Underwater Vision Profiler (UVP) from which carbon export, corresponding to the carbon flux (Fig. 1a) at 150 m, was calculated to range from 0.014 to 18.3 mg.m−2.d−1 using methods previously described (see Methods). One Guidi et al. Page 3 Nature. Author manuscript; available in PMC 2016 September 22. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts should keep in mind that fluxes are calculated from images of particles. These estimates are derived from an approximation of Stokes’ law relating the equivalent spherical diameter of particles to carbon flux (see Methods). This exponential approximation is reasonable assuming similar particle composition across all sizes, as highlighted by the standard deviations of parameters in Eq. 5 (see Methods). Furthermore, because of instrument and method limitations, particles <250 μm were not used, which may underestimate total carbon fluxes. Finally, these fluxes are instantaneous because they do not integrate space and time as sediment traps would. However, the approach allowed us to assemble the largest homogeneous carbon export dataset during a single expedition, corresponding to more than 600 profiles over 150 stations. This dataset is of similar magnitude to the body of historical data available in the literature that includes the 134 deep sediment trap-based carbon flux time-series27 from the JGOFS program and the 419 thorium-derived particulate organic carbon (POC) export measurements28. From 68 globally distributed sites, a total of 7.2 Tb of metagenomics data, representing ~40 million non-redundant genes, around 35,000 Operational Taxonomic Units (OTUs) of prokaryotes (Bacteria and Archaea) and numerous mainly uncharacterized viruses and picoeukaryotes, have been described recently23,25. In addition, a set of 2.3 million eukaryotic 18S rDNA ribotypes was generated from a subset of 47 sampling sites corresponding to approximately 130,000 OTUs24 (Fig. 1a). Finally, 5,476 viral “populations” were identified at 43 sites from viral metagenomic contigs, only 39 (<0.1%) of which had been previously observed25 (see Methods). These genomics data combined across all domains of life and viruses together with carbon export estimates and other environmental parameters were used to explore the relationships between marine biogeochemistry and euphotic plankton communities (see Methods) in the top 150 m of the oligotrophic open ocean. Our study did not include high latitude areas due to the current lack of available molecular data and results should not be extrapolated to deeper depths. Using a method for regression-based modeling of high multidimensional data in biology (specifically a sparse Partial Least Square analysis sPLS29, Extended data Fig. 1), we detected several plankton lineages for which relative sequence abundance correlated with carbon export and other environmental parameters, most notably with NPP, as expected (Fig. 1b and see Supplementary Table 1). These included diatoms, dinoflagellates and metazoa (zooplankton), lineages classically identified as key contributors to carbon export. Plankton community networks associated with carbon export While the analysis presented in Fig. 1b supports previous findings about key organisms involved in carbon export from the euphotic zone14,15,17-19, it is not able to capture how the intrinsic structure of the planktonic community relates to this biogeochemical process. Conversely, although other recent holistic approaches10,30,31 used species co-occurrence networks to reveal potential biotic interactions, they do not provide a robust description of sub-communities driven by abiotic interactions. To overcome these issues, we applied a systems biology approach known as Weighted Gene Correlation Network Analysis (WGCNA32,33) to detect significant associations between the Tara Oceans genomics data and carbon export. This method delineates communities in the euphotic zone that are the Guidi et al. Page 4 Nature. Author manuscript; available in PMC 2016 September 22. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts most associated with carbon export rather than predicting organisms associated with sinking particles. In brief, the WGCNA approach builds a network in which nodes are features (in this case plankton lineages or gene functions) and links are evaluated by the robustness of cooccurrence scores. WGCNA then clusters the network into modules (hereafter denoted subnetworks) that can be examined to find significant subnetwork-trait relationships. We then filtered each subnetwork using a Partial Least Square (PLS) analysis that emphasizes key nodes (based on the Variable Importance in Projection (VIP) scores; see Methods and Extended data Fig. 1). These particular nodes are mandatory to summarize a subnetwork (or community) related to carbon export. In particular, they are of interest for evaluating (i) subnetwork robustness and (ii) predictive power for a given trait (see Methods and Extended data Fig. 1). We applied WGCNA to the relative abundance tables of eukaryotic, prokaryotic and viral lineages23-25 and identified unique subnetworks significantly associated with carbon export within each dataset (see Methods and Supplementary Tables 2, 3, 4). The eukaryotic subnetwork (subnetwork-trait relationship to carbon export, Pearson cor. = 0.81, p = 5e−15) contained 49 lineages (Extended data Fig. 2a and Supplementary Table 2) among which 20% represented