Gasol, Josep M. ... et.al.-- Special volume: Planet Ocean. Scientia Marina 80(Suppl.1) 2016.-- 15 pages, 8 figures, 4 tables, [EN] We carried out monthly photosynthesis-irradiance (P-E) experiments with the 14C-method for 12 years (2003–2014) to determine the photosynthetic parameters and primary production of surface phytoplankton in the Blanes Bay Microbial Observatory, a coastal sampling station in the NW Mediterranean Sea. Our goal was to obtain seasonal trends and to establish the basis for detecting future changes of primary production in this oligotrophic area. The maximal photosynthetic rate PBmax ranged 30-fold (0.5-15 mg C mg Chl a–1 h–1), averaged 3.7 mg C mg Chl a–1 h–1 (±0.25 SE) and was highest in August and lowest in April and December. We only observed photoinhibition twice. The initial or light-limited slope of the P-E relationship, αB, was low, averaging 0.007 mg C mg Chl a–1 h–1 (μmol photons m–2 s–1)–1 (±0.001 SE, range 0.001-0.045) and showed the lowest values in spring (April-June). The light saturation parameter or saturation irradiance, EK, averaged 711 μmol photons m–2 s–1 (±58.4 SE) and tended to be higher in spring and lower in winter. Phytoplankton assemblages were typically dominated by picoeukaryotes in early winter, diatoms in late autumn and late winter, dinoflagellates in spring and cyanobacteria in summer. Total particulate primary production averaged 1.45 mg C m–3 h–1 (±0.13 SE) with highest values in winter (up to 8.50 mg C m–3 h–1) and lowest values in summer (summer average, 0.30 mg C m–3 h–1), while chlorophyll-specific primary production averaged 2.49 mg C mg Chl a–1 h–1 (±0.19, SE) and peaked in summer (up to 12.0 mg C mg Chl a–1 h–1 in August). 14C-determined phytoplankton growth rates varied between ca. 0.3 d–1 in winter and 0.5 d–1 in summer and were within 60-80% of the maximal rates of growth, based on PBmax. Chlorophyll a was a good predictor of primary production only in the winter and autumn. Seasonality appeared to explain most of the variability in the studied variables, while phytoplankton composition played a minor role. Daily integrated primary production was fairly constant throughout the year: similar to previous oxygen-based estimates in winter but considerably lower than these in summer. The difference between 14C- and oxygen-based estimates of primary production could be explained by community respiration. Annually integrated primary production amounted to a rather modest 48 g C m–2 yr–1 (equivalent to 130 mg C m–2 d–1). Although no interannual patterns were detected, our work soundly establishes the seasonal trends for the coastal NW Mediterranean, therefore setting the basis for future detection of change, [ES] Hemos llevado a cabo mediciones mensuales de la relación entre fotosíntesis e irradiancia (curvas P-E) con el método del 14C durante 12 años (2003-2014), para obtener los parámetros fotosintéticos y la producción primaria del fitoplancton superficial en el Observatorio Microbiano de la Bahía de Blanes, una estación de muestreo costera en el noroeste del Mediterráneo. Nuestro objetivo era determinar las tendencias estacionales y establecer la línea de base para detectar cambios futuros de la producción primaria en esta área oligotrófica. La tasa fotosintética máxima PBmax osciló 30 veces (0.5 a 15 mg C mg Chl a–1 h–1), con un promedio de 3.7 mg C mg Chl a–1 h–1 (±0.25, error estándar), y fue máxima en agosto y mínima en abril y diciembre. Sólo se observó fotoinhibicion dos veces. La pendiente inicial de la curva P-E, αB, fue baja, con un promedio de 0.007 mg C mg Chl a–1 h–1 (µmol fotones m–2 s–1)–1 (error estándar ±0.001, rango de 0.001-0.045) y presentó los valores más bajos en primavera (abril-junio). El parámetro de saturación de irradiancia, EK, presentó un promedio de 711 µmol fotones m–2 s–1 (±58.4, error estándar) y tendió a ser mayor en primavera y menor en invierno. El fitoplancton estuvo típicamente dominado por picoeucariotas a principios de invierno, diatomeas a finales de otoño y en el invierno avanzado, dinoflagelados en primavera, y cianobacterias en verano. La producción primaria particulada total promedió 1.45 m–3 h–1 (±0.13 error estándar) con máximos en invierno (hasta 8.50 mg C m–3 h–1) y mínimos en verano (media en verano, 0.30 mg C m–3 h–1), mientras que la producción primaria por unidad de clorofila promedió 2.49 mg C mg Chl a–1 h–1 (±0.19, error estándar) y alcanzó su punto máximo en verano (hasta 12.0 mg C mg Chl a–1 h–1 en agosto). Las tasas de crecimiento del fitoplancton determinadas por 14C oscilaron entre ca. 0.3 d–1 en invierno y 0.5 d–1 en verano, siendo 60-80% de las tasas máximas de crecimiento basadas en los valores de PBmax. La clorofila a resultó ser un buen predictor de la producción primaria sólo en el invierno y el otoño. La estacionalidad explica la mayor parte de la variabilidad en las variables estudiadas, mientras que la composición del fitoplancton juega un papel menor. La producción primaria integrada diaria fue bastante constante durante todo el año, similar a estimaciones anteriores con mediciones de oxígeno en invierno, pero considerablemente más bajas en verano. La diferencia entre las estimaciones de la producción primaria por 14C y las estimaciones en base al oxígeno podrían explicarse por la respiración de la comunidad. Anualmente la producción primaria integrada ascendió a valores más bien modestos de 48 g C m–2 año–1 (equivalentes a 130 mg C m–2 d–1). Aunque no se detectaron patrones interanuales, nuestro trabajo establece las tendencias estacionales para la producción primaria en el litoral Mediterráneo noroccidental, y supone la línea de base para la detección de cambios futuros, Research at the BBMO has been supported over time by many projects. We emphasize the EU projects Picodiv (EVK3-CT1999-00021), BASICS (EVK3-CT–2002-00078) and Micro-3B (FP7-OCEAN–2011 287589), and the Spanish projects Estramar, PROCAVIR (CTM2004-06842), MICROVIS (CTM2007–62140), Modivus (CTM2005-04795/MAR), Gemma (CTM2007-63753-C02-01/MAR), STORM (CTM2009-09352), SUMMER (CTM2008-03309/MAR) and FLAME (CTM2007-62140). The current work of the authors is supported by the grants Doremi (CTM2012-34294), Pegaso (CTM2012-37615), MEFISTO (CTM2013-43767-P), ANIMA (CTM2015-65720) and Remei (CTM2015-70340-R), yet sampling is contributed to from the PI’s own pocket, Peer Reviewed

Ptacnik, Robert ... et al.-- 10 pages, 4 figures, 1 table, supplementary information https://dx.doi.org/10.1038/srep29286, Mixotrophs combine photosynthesis with phagotrophy to cover their demands in energy and essential nutrients. This gives them a competitive advantage under oligotropihc conditions, where nutrients and bacteria concentrations are low. As the advantage for the mixotroph depends on light, the competition between mixo- and heterotrophic bacterivores should be regulated by light. To test this hypothesis, we incubated natural plankton from the ultra-oligotrophic Eastern Mediterranean in a set of mesocosms maintained at 4 light levels spanning a 10-fold light gradient. Picoplankton (heterotrophic bacteria (HB), pico-sized cyanobacteria, and small-sized flagellates) showed the fastest and most marked response to light, with pronounced predator-prey cycles, in the high-light treatments. Albeit cell specific activity of heterotrophic bacteria was constant across the light gradient, bacterial abundances exhibited an inverse relationship with light. This pattern was explained by light-induced top-down control of HB by bacterivorous phototrophic eukaryotes (PE), which was evidenced by a significant inverse relationship between HB net growth rate and PE abundances. Our results show that light mediates the impact of mixotrophic bacterivores. As mixo- and heterotrophs differ in the way they remineralize nutrients, these results have far-reaching implications for how nutrient cycling is affected by light, The experiment was financed by the European Union Seventh Framework Program MESOAQUA (FP7/2009-2013) coordinated by JCN under grant agreement no. 228224. R.P. further acknowledges financial support by the German Research Council (DFG Pt 5/3-1), JMG by Spanish project REMEI (CTM2015-70340) and AC by project PROTOS (CTM2009-08783) from the Spanish Ministry of Economy and Competitiveness, Peer Reviewed

12 pages, 6 figures, 3 tables, supplementary material http://journal.frontiersin.org/article/10.3389/fmicb.2016.02159/full#supplementary-material, Transparent Exopolymer Particles (TEP) are relevant in particle and carbon fluxes in the ocean, and have economic impact in the desalination industry affecting reverse osmosis membrane fouling. However, general models of their occurrence and dynamics are not yet possible because of the poorly known co-variations with other physical and biological variables. Here, we describe TEP distributions in the NW Mediterranean Sea during late spring 2012, along perpendicular and parallel transects to the Catalan coast. The stations in the parallel transect were sampled at the surface, while the stations in the perpendicular transect were sampled from the surface to the bathypelagic, including the bottom nepheloid layers. We also followed the short-term TEP dynamics along a 2-day cycle in offshore waters. TEP concentrations in the area ranged from 4.9 to 122.8 and averaged 31.4 ± 12.0 μg XG eq L−1. The distribution of TEP measured in transects parallel to the Catalan Coast correlated those of chlorophyll a (Chla) in May but not in June, when higher TEP-values with respect to Chla were observed. TEP horizontal variability in epipelagic waters from the coast to the open sea also correlated to that of Chla, O2 (that we interpret as a proxy of primary production) and bacterial production (BP). In contrast, the TEP vertical distributions in epipelagic waters were uncoupled from those of Chla, as TEP maxima were located above the deep chlorophyll maxima. The vertical distribution of TEP in the epipelagic zone was correlated with O2 and BP, suggesting combined phytoplankton (through primary production) and bacterial (through carbon reprocessing) TEP sources. However, no clear temporal patterns arose during the 2-day cycle. In meso- and bathypelagic waters, where phytoplanktonic sources are minor, TEP concentrations (10.1 ± 4.3 μg XG eq l−1) were half those in the epipelagic, but we observed relative TEP increments coinciding with the presence of nepheloid layers. These TEP increases were not paralleled by increases in particulate organic carbon, indicating that TEP are likely to act as aggregating agents of the mostly inorganic particles present in these bottom nepheloid layers, This work was funded by projects funded by the Spanish Ministry of Science STORM (CTM2009-09352/MAR), SUMMER (CTM2008-03309/MAR), DOREMI (CTM2012-34294), REMEI (CTM2015-70340-R), ANIMA (CTM2015-65720-R), PEGASO (CTM2012-37615), and Grup consolidat de Recerca de la Generalitat de Catalunya (2014SGR/1179), Peer Reviewed

12 pages, 8 figures, supplemental material http://www.nature.com/ismej/journal/v11/n4/suppinfo/ismej2016166s1.html, Depending on their relationship with the pelagic particulate matter, planktonic prokaryotes have traditionally been classified into two types of communities: free-living (FL) or attached (ATT) to particles, and are generally separated using only one pore-size filter in a differential filtration. Nonetheless, particulate matter in the oceans appears in a continuum of sizes. Here we separated this continuum into six discrete size-fractions, from 0.2 to 200 μm, and described the prokaryotes associated to each of them. Each size-fraction presented different bacterial communities, with a range of 23–42% of unique (OTUs) in each size-fraction, supporting the idea that they contained distinct types of particles. An increase in richness was observed from the smallest to the largest size-fractions, suggesting that increasingly larger particles contributed new niches. Our results show that a multiple size-fractionation provides a more exhaustive description of the bacterial diversity and community structure than the use of only one filter. In addition, and based on our results, we propose an alternative to the dichotomy of FL or ATT lifestyles, in which we differentiate the taxonomic groups with preference for the smaller fractions, those that do not show preferences for small or large fractions, and those that preferentially appear in larger fractions, This research was funded by projects STORM (CTM2009-09352), ADEPT (CTM2011-23458), DOREMI (CTM2012-34294), REMEI (CTM2015-70340-R) and ANIMA (CTM2015-65720-R) funded by the former Ministry of Science and Innovation and the Ministry of Economy and Competitiveness. MM was supported by a CSIC JAE-Predoc Grant and by the Ministry of Labor, Employment and Social Security. EB was supported by an FPI predoctoral fellowship from the Spanish Ministery of Economy and Competitiveness, Peer Reviewed

10 pages, 3 figures, 3 tables, supporting information https://dx.doi.org/10.1111/1758-2229.12535, Estimation of growth rates is crucial to understand the ecological role of prokaryotes and their contribution to marine biogeochemical cycling. However, there are only a few estimates for individual taxa. Two top-down (grazing) and bottom-up (phosphorus (P) availability) manipulation experiments were conducted under different light regimes in the NW Mediterranean Sea. Growth rate of different phylogenetic groups, including the Bacteroidetes, Rhodobacteraceae, SAR11, Gammaproteobacteria and its subgroups Alteromonadaceae and the NOR5/OM60 clade, were estimated from changes in cell numbers. Maximal growth rates were achieved in the P-amended treatments but when comparing values between treatments (response ratios), the response to predation removal was in general larger than to P-amendment. The Alteromonadaceae displayed the highest rates in both experiments followed by the Rhodobacteraceae, but all groups largely responded to filtration and P-amendment, even the SAR11 which presented low growth rates. Comparing light and dark treatments, growth rates were on average equal or higher in the dark than in the light for all groups, except for the Rhodobacteraceae and particularly the NOR5 clade, groups that contain photoheterotrophic species. These results are useful to evaluate the potential contributions of different bacterial types to biogeochemical processes under changing environmental conditions, This work was supported by grant REMEI (CTM2015-70340-R) from the Spanish Ministry of Economy, Industry and Competitivity. MK was supported by the Czech GA CR project 13-11281S and MSMT Project Algatech Plus, Peer Reviewed

Martínez-Hernández, Francisco J. et al.-- 13 pages, 6 figures, 1 table, data availability, supplementary information https://dx.doi.org/10.1038/ncomms15892, Microbes drive ecosystems under constraints imposed by viruses. However, a lack of virus genome information hinders our ability to answer fundamental, biological questions concerning microbial communities. Here we apply single-virus genomics (SVGs) to assess whether portions of marine viral communities are missed by current techniques. The majority of the here-identified 44 viral single-amplified genomes (vSAGs) are more abundant in global ocean virome data sets than published metagenome-assembled viral genomes or isolates. This indicates that vSAGs likely best represent the dsDNA viral populations dominating the oceans. Species-specific recruitment patterns and virome simulation data suggest that vSAGs are highly microdiverse and that microdiversity hinders the metagenomic assembly, which could explain why their genomes have not been identified before. Altogether, SVGs enable the discovery of some of the likely most abundant and ecologically relevant marine viral species, such as vSAG 37-F6, which were overlooked by other methodologies, This work has been supported by Spanish Ministry of Economy and Competitiveness (refs CGL2013-40564-R and SAF2013-49267-EXP), Generalitat Valenciana (ref. ACOM/2015/133 and ACIF/2015/332), the USA National Science Foundation (OCE#1536989), the USA Department of Energy (DE-SC0010580), and Gordon and Betty Moore Foundation (grants 3305, 3790, and 5334). [...] Work at BBMO was funded by Spanish project CT2015-70340-R. Work at CRG, BIST and UPF was in part funded by the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’ and the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Maria de Maeztu 2016-2019’, Peer Reviewed

3 pages, 2 figures, supplementary information https://dx.doi.org/10.1038/ismej.2017.79, Aerobic anoxygenic phototrophic (AAP) bacteria are microorganisms that can harvest light energy using bacteriochlorophyll a to supplement their predominantly organotrophic metabolism. Growth enhancement by light has repeatedly been demonstrated in laboratory experiments with AAP isolates. However, the ecological advantage of light utilization is unclear, as it has never been proven in the natural environment. Here, we conducted manipulation experiments in the NW Mediterranean and found that AAP bacteria display high growth rates which are controlled to a large extent by intense grazing pressure and phosphorous availability. Foremost, we found that, contrarily to the bulk bacterioplakton, AAP bacteria display higher growth rates when incubated under light-dark cycles than in complete darkness. These results represent the first direct evidence that natural populations of marine AAP bacteria can be stimulated by light, This work was supported by grant REMEI (CTM2015-70340-R) from the Spanish Ministry of Economy, Industry and Competitivity. MK was supported by the GA ČR project 13-11281S and the MŠMT project Algatech Plus, Peer reviewed, Peer Reviewed

14 pages, 5 figures, 2 tables, supporting information https://dx.doi.org/10.1111/mec.14421, Biotic and abiotic particles shape the microspatial architecture that defines the microbial aquatic habitat, being particles highly variable in size and quality along oceanic horizontal and vertical gradients. We analysed the prokaryotic (bacterial and archaeal) diversity and community composition present in six distinct particle size classes ranging from the pico- to the microscale (0.2 to 200 μm). Further, we studied their variations along oceanographic horizontal (from the coast to open oceanic waters) and vertical (from the ocean surface into the meso- and bathypelagic ocean) gradients. In general, prokaryotic community composition was more variable with depth than in the transition from the coast to the open ocean. Comparing the six size-fractions, distinct prokaryotic communities were detected in each size-fraction, and whereas bacteria were more diverse in the larger size-fractions, archaea were more diverse in the smaller size-fractions. Comparison of prokaryotic community composition among particle size-fractions showed that most, but not all, taxonomic groups have a preference for a certain size-fraction sustained with depth. Species sorting, or the presence of diverse ecotypes with distinct size-fraction preferences, may explain why this trend is not conserved in all taxa, The present work was possible thanks to the Generalitat de Catalunya Quality Research Group 2014SGR/1179 and projects STORM (CTM2009-09352), ADEPT (CTM2011-23458), NEMO (CTM2011-13137-E), ANIMA (CTM2015-65720-R) and REMEI (CTM2015-70340-R) funded by the former Ministry of Science and Innovation and the Ministry of Economy and Competitiveness. M. Mestre was supported by a JAE-Predoc grant, a Deutscher Akademischer Austausch Dienst (DAAD) grant, and by the Ministry of Labour, Employment and Social Security. The Leibniz society and the MIBI group at IGB are thanked for funding and technical as well as scientific support, respectively. I. Ferrera was funded by a postdoctoral fellowship from the Leibniz society and the IGB. E. Borrull benefited from a FPI predoctoral fellowship from the Spanish Ministry of Economy and Competitiveness. HPG was funded by DFG (German Science Foundation) projects Aquameth (GR1540-21/1), MicroPrime (GR1540-23/1) and Aggregates (GR1540-28/1), Peer Reviewed

11 pages, 8 figures, supplementary material https://doi.org/10.3389/fmicb.2017.02360, A major challenge in microbial ecology is linking diversity and function to determine which microbes are actively contributing to processes occurring in situ. Bioorthogonal non-canonical amino acid tagging (BONCAT) is a promising technique for detecting and quantifying translationally active bacteria in the environment. This technique consists of incubating a bacterial sample with an analog of methionine and using click-chemistry to identify the cells that have incorporated the substrate. Here, we established an optimized protocol for the visualization of protein-synthesizing cells in oligotrophic waters that can be coupled with taxonomic identification using Catalyzed Reporter Deposition Fluorescent in Situ Hybridization. We also evaluated the use of this technique to track shifts in translational activity by comparing it with leucine incorporation, and used it to monitor temporal changes in both cultures and natural samples. Finally, we determined the optimal concentration and incubation time for substrate incorporation during BONCAT incubations at an oligotrophic site. Our results demonstrate that BONCAT is a fast and powerful semi-quantitative approach to explore the physiological status of marine bacteria, This work was supported by grants EcoRARE (CTM2014-60467-JIN), funded by the Spanish Government and the European Regional Development Fund (ERDF), and REMEI (CTM2015-70340-R) funded by the Spanish Government, Peer Reviewed

Special issue on Marine Microbial Ecology.-- 11 pages, 5 figures, supporting information https://doi.org/10.1111/1462-2920.14002, The bathypelagic ocean is one of the largest ecosystems on Earth and sustains half of the ocean's microbial activity. This microbial activity strongly relies on surface‐derived particles, but there is growing evidence that the carbon released through solubilization of these particles may not be sufficient to meet the energy demands of deep ocean prokaryotes. To explore how bathypelagic prokaryotes respond to the absence of external inputs of carbon, we followed the long‐term (1 year) dynamics of an enclosed community. Despite the lack of external energy supply, we observed a continuous succession of active prokaryotic phylotypes, which was driven by recruitment of taxa from the seed bank (i.e., initially rare operational taxonomic units [OTUs]). A single OTU belonging to Marine Group I of Thaumarchaeota, which was originally rare, dominated the microbial community for ∼ 4 months and played a fundamental role in this succession likely by introducing new organic carbon through chemolithoautotrophy. This carbon presumably produced a priming effect, because after the decline of Thaumarchaeota, the diversity and metabolic potential of the community increased back to the levels present at the start of the experiment. Our study demonstrates the profound versatility of deep microbial communities when facing organic carbon deprivation, This study was partially supported by grants DOREMI (CTM2012–34294), HOTMIX (CTM2011–30010/MAR), REMEI (CTM2015-70340-Rf), SUAVE (CTM2014-54926-R), funded by the Spanish Government, and EcoRARE (CTM2014-60467-JIN), funded by the Spanish Government and the European Regional Development Fund (ERDF), Peer Reviewed

19 pages, 10 figures, 4 tables, supplementary material https://doi.org/10.3354/meps12493, We studied phytoplankton community structure in surface waters of the fixed coastal station of the Blanes Bay Microbial Observatory (NW Mediterranean Sea). A chemotaxonomic approach based on HPLC analysis of phytoplankton pigments, followed by CHEMTAX algorithm implementation, was applied to a set of monthly samples taken during a 14 yr period (2000-2014). Additional samples were taken for nutrient analyses, flow cytometric measurements and during part of the period for phytoplankton cell counts by optical microscopy. Overall, the most abundant groups in terms of chlorophyll a (chl a) were haptophytes, diatoms and prasinophytes. In general, diatoms were the most important components of the total chl a maxima (T_Chl a). We observed a marked seasonality of T_Chl a and several phytoplankton groups (prasinophytes, diatoms, haptophytes, cryptophytes and pelagophytes) with autumn-winter or winter-spring maxima and summer minima, coinciding with similar variation in major nutrient concentrations. Prochlorococcus presented a fall-winter maximum and a spring-summer minimum, while Synechococcus peaked in April and August, and dinoflagellates were relatively important in summer. Superimposed to this general pattern, prasinophytes and diatoms responded positively to episodic fertilization events associated with freshwater runoff caused by rain storms. Most phytoplankton groups presented a decreasing linear interannual trend that could be associated with a reduction in nutrient availability. A possible driver for this oligotrophication is the improvement of wastewater treatment in the region, The present study was supported in part by the project REMEI (CTM2015-70340-R, MINECO) as well as the Grups Consolidats de Recerca 2014 SGR/761 and 2014 SGR/1179 of the Generalitat de Catalunya, Peer reviewed

11 pages, 6 figures, 2 tables, Transparent Exopolymer Particles (TEPs) are a subclass of organic particles with high impact in biogeochemical and ecological processes, such as the biological carbon pump, air-sea interactions, or the microbial loop. However, the complexity in production and consumption makes TEP dynamics hardly predictable, calling for the need of descriptive studies about the in situ dynamics of these particles. We followed monthly TEP dynamics and combined them with a dataset of environmental variables during three years in a coastal site of the oligotrophic North Western Mediterranean (Blanes Bay). TEP concentration, ranging from 11.3 to 289.1 μg XG eq L (average 81.7 ± 11.7 μg XG eq L), showed recurrent peaks in early summer (June–July). TEP were temporally disconnected from chlorophyll a maxima, that occurred in late winter and early spring (maxima 1.21 μg L), but they were significantly related to the abundance of specific phytoplankton groups (diatoms and dinoflagellates) and also coincided with periods of low nutrient concentrations. The fraction of particulate organic carbon in the form of TEP (the TEP:POC and TEP:PM ratios) were also highest in early summer, indicating that TEP-enriched particles of low density accumulate in surface waters during stratified periods. We hypothesize that the accumulation of these particles affects the microbial food web by enhancing the activity of specific prokaryotic extracellular enzymes (esterase, β-glucosidase and alkaline phosphatase) and promoting the abundance of heterotrophic nanoflagellates, This work was funded by projects STORM (CTM2009-09352/MAR), PEGASO (CTM2012-37615), ANIMA (CTM2015-65720-R) and REMEI (CTM2015-70340-R) of the Spanish Ministry of Science or Economy. [...] EOR was supported by a Juan de la Cierva Fellowship, Peer Reviewed

The anthropogenic perturbation of the phosphorus (P) marine biogeochemical cycle due to synthetic organophosphorus compounds remains unexplored. The objective of this work was to investigate the microbial degradation of organophosphate triesters (OPEs), widely used as plasticizers and flame retardants, in seawater and their effects on the physiology and composition of microbial communities. Experiments were performed in July 2014 using surface seawater from the Blanes Bay Microbial Observatory (NW Mediterranean) to which OPEs were added at environmentally relevant concentrations. The concentrations of OPEs in the dissolved-phase generally decreased after 24 hours of incubation at in situ conditions. The fitted first order reaction constants were significantly different than zero for the trihaloalkyl phosphate, tris(2-chloroethyl) phosphate and trialyl phosphate tricresyl phosphate. In general, OPEs triggered an increase of the percentage of actively respiring bacteria, total bacterial activity, and the number of low-nucleic acid bacteria, and a decrease in the percentage of membrane-compromised bacteria. Members of some bacterial groups, in particular Flavobacteria, increased their specific activity, indicating that seawater contains bacteria with the potential to degrade OPEs. In aged seawater that was presumably depleted of labile dissolved organic carbon and inorganic P, alkaline phosphatase activities significantly decreased when OPEs were added, indicating a relief on P stress, consistent with the role of OPEs as potential P sources. © 2019, The Author(s)., This work was supported by Fundación BBVA award to MVC and by the Spanish MEIC through projects ISOMICS (CTM2015-65691-R) and REMEI (CTM2015-70340-R). MS was supported by grant EcoRARE (CTM2014-60467-JIN), funded by the Spanish Government and the European Regional Development Fund (ERDF) and a Viera y Clavijo contract funded by the ACIISI and the ULPGC. The research group of Global Change and Genomic Biogeochemistry is supported by the Catalan Goverment (2017SGR800). We thank J Castro-Jimenez for support on the OPE analyses. MVC is indebted to J Pinhassi for support., Peer reviewed

12 pages, 5 tables, 6 figures, Transparent exopolymer particles (TEP) play a key role in ocean carbon export and structuring microbial habitats, but information on their distribution across different ocean basins and depths is scarce, particularly in the dark ocean. We measured TEP vertical distribution from the surface to bathypelagic waters in an east-to-west transect across the Mediterranean Sea (MedSea) and the adjacent North East Atlantic Ocean (NEA), and explored their physical and biological drivers. TEP ranged from 0.6 to 81.7 µg XG eq L−1, with the highest values in epipelagic waters above the deep chlorophyll maximum, and in areas near the Gibraltar and Sicily Straits. TEP were significantly related to particulate organic carbon (POC) in all basins and depth layers (epipelagic vs. deep), but the contribution of TEP to POC was higher in the NEA (85%, 79% and 67% in epi-, meso- and bathypelagic waters, respectively) than in the MedSea (from 53% to 62% in epipelagic waters, and from 45% to 48% in meso- and bathypelagic waters), coinciding with higher carbon to nitrogen particulate organic matter ratios in the NEA. The TEP connectivity between epipelagic waters and mesopelagic waters was less straightforward than between mesopelagic waters and bathypelagic waters, with a 23% and 55% of the variance in the relationship between layers explained respectively. Prokaryotes were found to be a likely net source of TEP as inferred by the significant direct relationship observed between prokaryotic heterotrophic abundance and TEP. This assumption was confirmed using experimental incubations, where prokaryotes produced TEP in concentrations ranging from 0.7 (Western Mediterranean, bathypelagic) to 232 (Western Mediterranean, mesopelagic) µg XG eq. L−1 day−1, This work was funded by project HOTMIX (CTM2011-30010/MAR) of the Spanish Ministry of Economy and Innovation, co-financed with FEDER funds. Financial support for the writing of this manuscript was also provided by project FLUXES (CTM2015-69392-C3) to XAAS, MFM and JA, REMEI (CTM2015-70340-R) to JMG, and MNat Scientific Unit of Excellence (UCE.PP2017.03) to IR, Peer reviewed

12 pages, 6 figures, supplemental material https://www.frontiersin.org/articles/10.3389/fmicb.2019.00760/full#supplementary-material, Experiments with bacteria in culture have shown that they often display “feast and famine” strategies that allow them to respond with fast growth upon pulses in resource availability, and enter a growth-arrest state when resources are limiting. Although feast responses have been observed in natural communities upon enrichment, it is unknown whether this blooming ability is maintained after long periods of starvation, particularly in systems that are energy limited like the bathypelagic ocean. Here we combined bulk and single-cell activity measurements with 16S rRNA gene amplicon sequencing to explore the response of a bathypelagic community, that had been starved for 1.6 years, to a sudden organic carbon supply. We observed a dramatic change in activity within 30 h, with leucine incorporation rates increasing over two orders of magnitude and the number of translationally active cells (mostly Gammaproteobacteria) increasing 4-fold. The feast response was driven by a single operational taxonomic unit (OTU) affiliated with the Marinobacter genus, which had remained rare during 7 months of starvation. Our work suggests that bathypelagic communities harbor a seed bank of highly persistent and resourceful “feast and famine” strategists that might disproportionally contribute to carbon fluxes through fast responses to occasional pulses of organic matter, This work was supported by grants DIFUMIC (2017SGR/1568, Generalitat de Catalunya), DOREMI (CTM2012-34294), HOTMIX (CTM2011-30010/MAR), REMEI (CTM2015-70340-R), and ANIMA (CTM2015-65720-R) funded by the Spanish Government, and EcoRARE (CTM2014-60467-JIN), funded by the Spanish Government and the European Regional Development Fund (ERDF). CRG was supported by a Juan de la Cierva contract (IJCI-2015-23505) and MS by a Viera y Clavijo contract funded by the ACIISI and the ULPGC, Peer Reviewed

16 pages, 6 figures, 1 table, supporting information https://dx.doi.org/10.1111/mec.15026, Microbial taxa range from being ubiquitous and abundant across space to extremely rare and endemic, depending on their ecophysiology and on different processes acting locally or regionally. However, little is known about how cosmopolitan or rare taxa combine to constitute communities and whether environmental variations promote changes in their relative abundances. Here we identified the Spatial Abundance Distribution (SpAD) of individual prokaryotic taxa (16S rDNA-defined Operational Taxonomic Units, OTUs) across 108 globally-distributed surface ocean stations. We grouped taxa based on their SpAD shape (“normal-like”- abundant and ubiquitous; “logistic”- globally rare, present in few sites; and “bimodal”- abundant only in certain oceanic regions), and investigated how the abundance of these three categories relates to environmental gradients. Most surface assemblages were numerically dominated by a few cosmopolitan “normal-like” OTUs, yet there was a gradual shift towards assemblages dominated by “logistic” taxa in specific areas with productivity and temperature differing the most from the average conditions in the sampled stations. When we performed the SpAD categorization including additional habitats (deeper layers and particles of varying sizes), the SpAD of many OTUs changed towards fewer “normal-like” shapes, and OTUs categorized as globally rare in the surface ocean became abundant. This suggests that understanding the mechanisms behind microbial rarity and dominance requires expanding the context of study beyond local communities and single habitats. We show that marine bacterial communities comprise taxa displaying a continuum of SpADs, and that variations in their abundances can be linked to habitat transitions or barriers that delimit the distribution of community members, This work was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) through the Consolider‐Ingenio program (Malaspina 2010 Expedition, ref. CSD2008‐00077), with contributions from grant CTM2015‐70340R, CTM2015‐65720‐R, CTM2015‐69936‐P and King Abdullah University of Science and Technology (KAUST). [...]. This is a contribution of Grup Consolidat de Recerca of the Catalan Government 2014SGR/1179. CRG was supported by a Juan de la Cierva (IJCI‐2015‐23505, MINECO, Spain) fellowship and RL by a Ramón y Cajal fellowship (RYC‐2013‐12554, MINECO, Spain). MS was supported by grant EcoRARE (CTM2014‐60467‐JIN), funded by the Spanish Government and the European Regional Development Fund (ERDF), REMEI (CTM2015‐70340‐Rf) funded by the Spanish Government, and a Viera y Clavijo contract funded by the ACIISI and the ULPGC. MM was supported by CONICYT (FONDAP‐IDEAL 15150003). SGA was funded by BIOSENSOMICS through the ‘Convocatoria 2015 de ayudas Fundación BBVA a investigadores y creadores culturales'. RRM is supported by CONICYT FONDECYT 11170748, Peer Reviewed

13 pages, 2 figures, 1 table, supplementary data https://doi.org/10.1093/plankt/fbz008, Growing attention to phytoplankton mixotrophy as a trophic strategy has led to significant revisions of traditional pelagic food web models and ecosystem functioning. Although some empirical estimates of mixotrophy do exist, a much broader set of in situ measurements are required to (i) identify which organisms are acting as mixotrophs in real time and to (ii) assess the contribution of their heterotrophy to biogeochemical cycling. Estimates are needed through time and across space to evaluate which environmental conditions or habitats favour mixotrophy: conditions still largely unknown. We review methodologies currently available to plankton ecologists to undertake estimates of plankton mixotrophy, in particular nanophytoplankton phago-mixotrophy. Methods are based largely on fluorescent or isotopic tracers, but also take advantage of genomics to identify phylotypes and function. We also suggest novel methods on the cusp of use for phago-mixotrophy assessment, including single-cell measurements improving our capacity to estimate mixotrophic activity and rates in wild plankton communities down to the single-cell level. Future methods will benefit from advances in nanotechnology, micromanipulation and microscopy combined with stable isotope and genomic methodologies. Improved estimates of mixotrophy will enable more reliable models to predict changes in food web structure and biogeochemical flows in a rapidly changing world., We acknowledge funding from a Friedrich Wilhelm Bessel Award, Humboldt Foundation (Germany) to BEB, which enabled the writing of this paper. JMG was supported by grant REMEI (CTM2015-70340-R), Peer Reviewed

14 pages, 4 figures, tables, supporting information https://doi.org/10.1111/mec.15068.-- Data Availability Statement:: DNA sequences from clone libraries: Genbank accessions KP887163 –KP887808. High‐throughput sequences: Genbank Sequence Read Archive under Bioproject ID PRJNA495818, Bacteroidetes is one of the dominant phyla of ocean bacterioplankton, yet its diversity and population structure is poorly understood. To advance in the delineation of ecologically meaningful units within this group, we constructed near full-length 16S rRNA gene clone libraries from contrasting marine environments in the NW Mediterranean. Based on phylogeny and the associated ecological variables (depth and season), 24 different Bacteroidetes clades were delineated. By considering their relative abundance (from iTag amplicon sequencing studies), we described the distribution patterns of each of these clades, delimiting them as ecologically significant taxonomic units (ESTUs). Spatially, there was almost no overlap among ESTUs at different depths. In deep waters there was predominance of Owenweeksia, Leeuwenhoekiella, Muricauda-related genera, and some depth-associated ESTUs within the NS5 and NS2b marine clades. Seasonally, multi-annual dynamics of recurring ESTUs were present with dominance of some ESTUs within the NS4, NS5 and NS2b marine clades along most of the year, but with variable relative frequencies between months. A drastic change towards the predominance of Formosa-related ESTUs and one ESTU from the NS5 marine clade was typically present after the spring bloom. Even though there are no isolates available for these ESTUs to determine their physiology, correlation analyses identified the environmental preference of some of them. Overall, our results suggest that there is a high degree of niche specialisation within these closely related clades. This work constitutes a step forward in disentangling the ecology of marine Bacteroidetes, which are essential players in organic matter processing in the oceans, This work was supported by grants MAGGY (CTM2017‐87736‐R) to SGA and REMEI (CTM2015‐70340‐R) to J.M.G. from the Spanish Ministry of Science and Innovation (MICINN). M.S. was supported by a Viera y Clavijo contract funded by the ACIISI and the ULPGC. Financial support was provided by PhD JAE‐Predoc fellowship from CSIC to CDV. P.S. was supported by project Malaspinomics (CTM2011‐15461) from the Spanish Ministry of Economy & Competitivity, Peer Reviewed

13 pages, 6 figures, 1 table, supplementary information https://doi.org/10.1038/s41396-019-0401-4, We studied the long-term temporal dynamics of the aerobic anoxygenic phototrophic (AAP) bacteria, a relevant functional group in the coastal marine microbial food web, using high-throughput sequencing of the pufM gene coupled with multivariate, time series and co-occurrence analyses at the Blanes Bay Microbial Observatory (NW Mediterranean). Additionally, using metagenomics, we tested whether the used primers captured accurately the seasonality of the most relevant AAP groups. Phylogroup K (Gammaproteobacteria) was the greatest contributor to community structure over all seasons, with phylogroups E and G (Alphaproteobacteria) being prevalent in spring. Diversity indices showed a clear seasonal trend, with maximum values in winter, which was inverse to that of AAP abundance. Multivariate analyses revealed sample clustering by season, with a relevant proportion of the variance explained by day length, temperature, salinity, phototrophic nanoflagellate abundance, chlorophyll a, and silicate concentration. Time series analysis showed robust rhythmic patterns of co-occurrence, but distinct seasonal behaviors within the same phylogroup, and even within different amplicon sequence variants (ASVs) conforming the same operational taxonomic unit (OTU). Altogether, our results picture the AAP assemblage as highly seasonal and recurrent but containing ecotypes showing distinctive temporal niche partitioning, rather than being a cohesive functional group, This research was funded by grant REMEI (CTM2015-70340-R) from the Spanish Ministry of Economy, Industry and Competitiveness and Grant 2017SGR/1568 from CIRIT-Generalitat de Catalunya to Consolidated Research Groups, Peer Reviewed

13 pages, 5 figures, 2 tables, supplementary material https://doi.org/10.1016/j.scitotenv.2019.04.361, Organic pollutants are continuously being introduced in seawater with uncharacterized impacts on the engines of the marine biogeochemical cycles, the microorganisms. The effects on marine microbial communities were assessed for perfluoroalkyl substances, organophosphate esters flame retardants and plasticizers, polycyclic aromatic hydrocarbons, and n-alkanes. Dose-response experiments were performed at three stations and at three depths in the NW Mediterranean with contrasted nutrient and pollutant concentrations. In these experiments, the microbial growth rates, the abundances of the main bacterial groups, measured by Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH), and extracellular enzymatic activities, were quantified. Increasing concentrations of organic pollutants (OPs)promoted different responses in the communities that were compound, organism and nutrient availability (trophic status). The largest differences between OP treatments and controls in the growth rates of both heterotrophic and phototrophic microbial groups were observed in seawater from the deep chlorophyll maxima. Furthermore, there was a compound specific stimulation of different extracellular enzymatic activities after the exposure to OPs. Our results revealed that marine microbial communities reacted not only to hydrocarbons, known to be used as a carbon source, but also to low concentrations of organic pollutants of emerging concern in a complex manner, reflecting the variability of various environmental variables. Multiple linear regressions suggested that organic pollutants modulated the bacterial growth and extracellular enzymatic activities, but this modulation was of lower magnitude than the observed pronounced response of the microbial community to nutrient availability, This work has been financed by the Spanish Ministry of Economy and Competitiveness through projects ISOMICS (CTM2015-65691-R), DO-RE-MI (CTM2012-34294), ANIMA (CTM2015-65720-R) and REMEI (CTM2015-70340-R) and by Fundación BBVA award to MVC. ECG acknowledges a FI fellowship from the Generalitat de Catalunya (AGAUR), Peer Reviewed

Discussion meeting issue ‘Single cell ecology’ organized and edited by Thomas A. Richards, Ramon Massana and Neil Hall, 10-11 December 2018, The Royal Society, London.-- 7 pages, 2 figures, Recent developments in community and single-cell genomic approaches have provided an unprecedented amount of information on the ecology of microbes in the aquatic environment. However, linkages between each specific microbe's identity and their in situ level of activity (be it growth, division or just metabolic activity) are much more scarce. The ultimate goal of marine microbial ecology is to understand how the environment determines the types of different microbes in nature, their function, morphology and cell-to-cell interactions and to do so we should gather three levels of information, the genomic (including identity), the functional (activity or growth), and the morphological, and for as many individual cells as possible. We present a brief overview of methodologies applied to address single-cell activity in marine prokaryotes, together with a discussion of the difficulties in identifying and categorizing activity and growth. We then provide and discuss some examples showing how visualization has been pivotal for challenging established paradigms and for understanding the role of microbes in the environment, unveiling processes and interactions that otherwise would have been overlooked. We conclude by stating that more effort should be directed towards integrating visualization in future approaches if we want to gain a comprehensive insight into how microbes contribute to the functioning of ecosystems, M.S. was also supported by a Viera y Clavijo contract funded by the ACIISI and the ULPGC. The work of the authors has been supported by grants REMEI (grant no. CTM2015-70340-R) and MIAU-S3 (grant no. RTI2018-101025-B-I00) from the Spanish Ministry of Science, Innovation and Universities and the Grup de Qualitat de la Generalitat de Catalunya (grant no. 2017SGR/1568), Peer Reviewed

8 pages, 3 figures, 2 tables, supplementary information https://doi.org/10.1038/s41598-019-54936-y.-- The TARA expedition dataset is available at http://ocean-microbiome.embl.de/companion.html; the Malaspina expedition dataset is available at https://github.com/GuillemSalazar/MolEcol_2015; and the experimental data have been deposited in the NCBI Sequence Read Archive under the accession number SRP051855, Global ocean expeditions have provided minimum estimates of ocean’s prokaryote diversity, supported by apparent asymptotes in the number of prokaryotes with sampling effort, of about 40,000 species, representing <1% of the species cataloged in the Earth Microbiome Project, despite being the largest habitat in the biosphere. Here we demonstrate that the abundance of prokaryote OTUs follows a scaling that can be represented by a power-law distribution, and as a consequence, we demonstrate, mathematically and through simulations, that the asymptote of rarefaction curves is an apparent one, which is only reached with sample sizes approaching the entire ecosystem. We experimentally confirm these findings using exhaustive repeated sampling of a prokaryote community in the Red Sea and the exploration of global assessments of prokaryote diversity in the ocean. Our findings indicate that, far from having achieved a thorough sampling of prokaryote species abundance in the ocean, global expeditions provide just a start for this quest as the richness in the global ocean is much larger than estimated, This research was funded by the Malaspina Circumnavigation Expedition supported by the Spanish Ministry of Science and Innovation through project Consolider-Ingenio Malaspina 2010 (CSD2008-00077) as well as King Abdullah University of Science and Technology (KAUST) through baseline funding to C.M. Duarte and X. Irigoien; by Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) through projects SPASIMM FIS2016-80067-P (AEI/FEDER, UE) and REMEI (CTM2015-70340-R); and by the Spanish State Research Agency through the María de Maeztu Program for Units of Excellence in R&D (MDM-2017-0711 to IFISC). S.S. is supported by the ETH and Helmut Horten Foundation, Peer Reviewed

13 pages, 6 figures, supplementary information https://doi.org/10.1038/s41396-019-0506-9, Microbial eukaryotes are key components of the ocean plankton. Yet, our understanding of their community composition and activity in different water layers of the ocean is limited, particularly for picoeukaryotes (0.2–3 µm cell size). Here, we examined the picoeukaryotic communities inhabiting different vertical zones of the tropical and subtropical global ocean: surface, deep chlorophyll maximum, mesopelagic (including the deep scattering layer and oxygen minimum zones), and bathypelagic. Communities were analysed by high-tthroughput sequencing of the 18S rRNA gene (V4 region) as represented by DNA (community structure) and RNA (metabolism), followed by delineation of Operational Taxonomic Units (OTUs) at 99% similarity. We found a stratification of the picoeukaryotic communities along the water column, with assemblages corresponding to the sunlit and dark ocean. Specific taxonomic groups either increased (e.g., Chrysophyceae or Bicosoecida) or decreased (e.g., Dinoflagellata or MAST-3) in abundance with depth. We used the rRNA:rDNA ratio of each OTU as a proxy of metabolic activity. The highest relative activity was found in the mesopelagic layer for most taxonomic groups, and the lowest in the bathypelagic. Altogether, we characterize the change in community structure and metabolic activity of picoeukaryotes with depth in the global ocean, suggesting a hotspot of activity in the mesopelagic, This project was supported by the Spanish Ministry of Economy and Competitiveness through project Consolider-Ingenio Malaspina-2010 (CSD2008–00077). Further research was funded by Spanish projects FLAME (CGL2010-16304, MICINN), ALLFLAGS (CTM2016-75083-R, MINECO), INTERACTOMICS (CTM2015-69936-P, MINECO/FEDER) and REMEI (CTM2015-70340-R). CRG was supported by a Spanish FPI grant and RL was supported by a Ramón y Cajal fellowship (RYC-2013-12554, MINECO, Spain), With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI), Peer reviewed

pages, 7 figures, 1 table, supporting information.-- https://doi.org/10.1111/mec.15454.-- DNA sequences and associated metadata: European Nucleotide Database (ENA) under accession numbers ERP109198 and ERS2539749–ERS2539903. The environmental metadata, and the complete non-rarefied OTU and taxonomic tables used in this study are provided as Supporting Information (Tables S1, S2, S3 and S4).-- This is a contribution of Grup Consolidat de Recerca of the Catalan Government 2014SGR/1179, Deep ocean microbial communities rely on the organic carbon produced in the sunlit ocean, yet it remains unknown whether surface processes determine the assembly and function of bathypelagic prokaryotes to a larger extent than deep‐sea physico‐chemical conditions. Here, we explored whether variations in surface phytoplankton assemblages across Atlantic, Pacific and Indian ocean stations can explain structural changes in bathypelagic (ca. 4000 m) free‐living and particle‐attached prokaryotic communities (characterized through 16S rRNA gene sequencing), as well as in prokaryotic activity and dissolved organic matter (DOM) quality. We show that the spatial structuring of prokaryotic communities in the bathypelagic strongly followed variations in the abundances of surface dinoflagellates and ciliates, as well as gradients in surface primary productivity, but were less influenced by bathypelagic physico‐chemical conditions. Amino acid‐like DOM components in the bathypelagic reflected variations of those components in surface waters, and seemed to control bathypelagic prokaryotic activity. The imprint of surface conditions was more evident in bathypelagic than in shallower mesopelagic (200‐1000 m) communities, suggesting a direct connectivity through fast‐sinking particles that escapes mesopelagic transformations. Finally, we identified a pool of endemic deep‐sea prokaryotic taxa (including potential chemoautotrophic groups) that appear less connected to surface processes than those bathypelagic taxa with a widespread vertical distribution. Our results suggest that surface planktonic communities shape the spatial structure of the bathypelagic microbiome to a larger extent than the local physico‐chemical environment, likely through determining the nature of the sinking particles and the associated prokaryotes reaching bathypelagic waters, This work was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) through the Consolider-Ingenio program (Malaspina 2010 Expedition, ref. CSD2008-00077), with contributions from grant CTM2015-70340R, CTM2015-65720-R, CTM2015-69936-P, RTI2018-101025-B-I00, Modeling Nature Scientific Unit (UCE.PP2017.03), CTM2015-69392-C3-2-R (co-financed with FEDER funds) and King Abdullah University of Science and Technology (KAUST). [...] CRG was supported by a Juan de la Cierva fellowship and the GRAMMI project (IJCI-2015-23505 and RTI2018717 099740-J-I00, MICINN, Spain). MSE was supported by a Viera y Clavijo contract funded by the ACIISI and the ULPGC. MM was supported by CONICYT (FONDAP IDEAL15150003 and FONDECYT-POSTDOCTORADO 3190369)., With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

16 pages, 5 figures, 3 tables, supplementary information https://doi.org/10.1186/s12866-020-01884-7, Isolation of marine microorganisms is fundamental to gather information about their physiology, ecology and genomic content. To date, most of the bacterial isolation efforts have focused on the photic ocean leaving the deep ocean less explored. We have created a marine culture collection of heterotrophic bacteria (MARINHET) using a standard marine medium comprising a total of 1561 bacterial strains, and covering a variety of oceanographic regions from different seasons and years, from 2009 to 2015. Specifically, our marine collection contains isolates from both photic (817) and aphotic layers (744), including the mesopelagic (362) and the bathypelagic (382), from the North Western Mediterranean Sea, the North and South Atlantic Ocean, the Indian, the Pacific, and the Arctic Oceans. We described the taxonomy, the phylogenetic diversity and the biogeography of a fraction of the marine culturable microorganisms to enhance our knowledge about which heterotrophic marine isolates are recurrently retrieved across oceans and along different depths, Projects Arctic Tipping Points (ATP, contract #226248), in the FP7 program of the European Union, and DOREMI (CTM2012–34294), from the Spanish Ministry of Economy and Competitiveness, allowed the collection of samples from the Arctic and NW Mediterranean Sea, respectively. Research, including laboratory experiments and analyses, was mainly funded by grant BIOSENSOMICS “Convocatoria 2015 de ayudas Fundación BBVA a investigadores y creadores culturales” and, MAGGY, Plan Nacional I + D + I 2017 (CTM2017–87736-R), awarded to S.G.A. Analyses were also possible thank to the King Abdullah University of Science and Technology (KAUST) through baseline funding to C.M.D. and a subaward agreement OSR-2014-CC-1973-02 between KAUST and Universitat Autònoma de Barcelona (UAB). Additional funding for analyses and interpretation of the data was provided by projects REMEI (ref. CTM2015–70340-R) and MIAU-S3 (ref. RTI2018-101025-B-I00) from the Spanish Ministry of Economy and Competitiveness, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

14 pages, 6 figures, 1 table, supplementary material https://www.frontiersin.org/articles/10.3389/fmicb.2020.01590/full#supplementary-material.-- Data Availability Statement. The raw sequence data have been deposited at the National Center for Biotechnology Information (NCBI). The data can be accessed online (https://www.ncbi.nlm) with the code PRJNA345534, Seasonal dynamics of ocean prokaryotic communities in the free-living fraction have been widely described, but less is known about the seasonality of prokaryotes inhabiting marine particles. We describe the seasonality of bacterial communities in the particulate matter continuum by sampling monthly over two years in a temperate oligotrophic coastal ecosystem and using a serial filtration (including six size-fractions spanning from 0.2 to 200 μm). We observed that bacterial communities in the particulate matter continuum had annual changes following harmonic seasonal oscillations, where alpha, beta, and gamma diversity increased during the warm period and decreased during the cold period. Communities in each size-fraction changed gradually over time, being the communities in larger size-fractions the ones with stronger annual changes. Annual community changes were driven mainly by day length and sea surface temperature, and each size-fraction was additionally affected by other variables (e.g., smaller size-fractions by dissolved PO4 and larger size-fractions by turbidity). While some taxonomic groups mantained their preference for a given size fraction during most of the year, others varied their distribution into different size fractions over time, as e.g., SAR11, which increased its presence in particles during the cold period. Our results indicate that the size-fractionation scheme provides novel seasonal patterns that are not possible to unveil by analyzing only free-living bacteria, and that help to better understand the temporal dynamics of prokaryotes, This research was funded by projects STORM (CTM2009-09352), ADEPT (CTM2011-23458), REMEI (CTM2015-70340-R), ANIMA (CTM2015-65720-R), and MIAU (RTI2018-101025-B-I00) funded by the former Spanish Ministries of Science and Innovation, Ministry of Economy and Competitiveness, and now Science, Innovation and Universities. JH was partially supported by the Chilean National Agency for Research and Development (ANID) (FONDECYT-POSTDOCTORADO 3180152). MM was supported by a CSIC JAEPredoc Grant, by the Ministry of Labor, Employment and Social Security of Spain and by the Chilean National Agency for Research and Development (ANID) (FONDAP-IDEAL 15150003 and FONDECYT-POSTDOCTORADO 3190369). The publication fee was supported by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI), Spain, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI), Peer reviewed

10 pages, 3 figures, 2 tables, supplementary material https://www.frontiersin.org/articles/10.3389/fmicb.2020.01645/full#supplementary-material.-- Data Availability Statement. The raw sequence data have been deposited in the Figshare data repository, together with the non-rarefied OTU table, the taxonomy table and the environmental data used in this study, 10.6084/m9.figshare.12333107, Microbes associated with sinking marine particles play key roles in carbon sequestration in the ocean. The sampling of particle-attached microorganisms is often done with sediment traps or by filtration of water collected with oceanographic bottles, both involving a certain time lapse between collection and processing of samples that may result in changes in particle-attached microbial communities. Conversely, in situ water filtration through submersible pumps allows a faster storage of sampled particles, but it has rarely been used to study the associated microbial communities and has never been compared to other particle-sampling methods in terms of the recovery of particle microbial diversity. Here we compared the prokaryotic communities attached to small (1–53 μm) and large (>53 μm) particles collected from the mesopelagic zone (100–300 m) of two Antarctic polynyas using in situ pumps (ISP) and oceanographic bottles (BTL). Each sampling method retrieved largely different particle-attached communities, suggesting that they capture different kinds of particles. These device-driven differences were greater for large particles than for small particles. Overall, the ISP recovered 1.5- to 3-fold more particle-attached bacterial taxa than the BTL, and different taxonomic groups were preferentially recovered by each method. In particular, typical particle-attached groups such as Planctomycetes and Deltaproteobacteria recovered with ISP were nearly absent from BTL samples. Our results suggest that the method used to sample marine particles has a strong influence in our view of their associated microbial communities, VP received funding from Edith Cowan University (G1003456) and from the School of Science at Edith Cowan University (G1003362) to support this work. CR-G and JG were supported by the grants CTM2015-70340-R and RTI2018-101025-B-I00 of the Spanish Ministry of Science, Innovation and Universities and by the Generalitat de Catalunya Consolidated Research Group 2017SGR/1568. PM acknowledges the support of the Generalitat de Catalunya (MERS 2017 SGR – 1588), With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI), Peer reviewed

15 pages, 5 figures, 3 tables, supplementary information https://doi.org/10.1038/s41598-020-76590-5, Estimation of prokaryotic growth rates is critical to understand the ecological role and contribution of different microbes to marine biogeochemical cycles. However, there is a general lack of knowledge on what factors control the growth rates of different prokaryotic groups and how these vary between sites and along seasons at a given site. We carried out several manipulation experiments during the four astronomical seasons in the coastal NW Mediterranean in order to evaluate the impact of grazing, viral mortality, resource competition and light on the growth and loss rates of prokaryotes. Gross and net growth rates of different bacterioplankton groups targeted by group-specific CARD-FISH probes and infrared microscopy (for aerobic anoxygenic phototrophs, AAP), were calculated from changes in cell abundances. Maximal group-specific growth rates were achieved when both predation pressure and nutrient limitation were experimentally minimized, while only a minimal effect of viral pressure on growth rates was observed; nevertheless, the response to predation removal was more remarkable in winter, when the bacterial community was not subjected to nutrient limitation. Although all groups showed increases in their growth rates when resource competition as well as grazers and viral pressure were reduced, Alteromonadaceae consistently presented the highest rates in all seasons. The response to light availability was generally weaker than that to the other factors, but it was variable between seasons. In summer and spring, the growth rates of AAP were stimulated by light whereas the growth of the SAR11 clade (likely containing proteorhodopsin) was enhanced by light in all seasons. Overall, our results set thresholds on bacterioplankton group-specific growth and mortality rates and contribute to estimate the seasonally changing contribution of various bacterioplankton groups to the function of microbial communities. Our results also indicate that the least abundant groups display the highest growth rates, contributing to the recycling of organic matter to a much greater extent than what their abundances alone would predict, This study was supported by grants REMEI (CTM2015-70340-R), ANIMA (CTM2015-65720-R) and MIAU (RTI2018-101025-B-I00) from the Spanish Ministry of Research and Innovation, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

19 pages, 6 figures, 2 tables, supporting Information https://doi.org/10.1002/lno.11944, he Mediterranean Sea is a miniature ocean divided by the Sicily Strait into two basins with a marked west to east trophic gradient and separated of the nearby eastern Atlantic Ocean by the Strait of Gibraltar. Here, we test the hypothesis that these physical and environmental barriers favor the development of specific prokaryotic assemblages, leading to changes in community structure both in the vertical and horizontal spatial scales. By analyzing taxonomic and phylogenetic diversity using amplicon sequence variants (ASVs) of the 16S rRNA gene, we show that there is indeed marked vertical segregation of prokaryotic groups, similar to that found in other areas of the ocean, but also a clear horizontal structuring among the two Mediterranean basins and the adjacent Atlantic waters. Prokaryotic diversity increased with depth and toward the Atlantic, whereas the easternmost stations displayed more phylogenetically diverse phylotypes, despite harboring globally less diverse communities. Basin-indicator taxa (ASVs) accounted for a large fraction of the community (between 50% and 80%) in each of the basins at the surface and bathypelagic layers, being associated with different environmental variables. The existence of biogeographic and environmental barriers in the Mediterranean Sea is likely related to the trophic gradient at the surface and the isolation of water bodies in depth due to the Gibraltar and Sicily straits. Our work highlights the importance of studying microbial regional biogeography and provides the basis for future studies on the impact of this regionalization in the function of Mediterranean Sea prokaryotic communities, Sampling was funded by grant HOTMIX (CTM2011-30010/MAR), and processing also by grants REMEI (CTM2015-70340-R) and MIAU (RTI2018-101025-B-I00) from the Spanish Ministry of Science and Innovation, cofunded with FEDER funds. It has also received support from the Spanish government through the “Severo Ochoa Centre of Excellence” accreditation (CEX2019-000928-S) and the Catalan government through excellence group grant 2017SGR/1568. Laura Gómez-Consarnau was funded by grant OCE1924464 from the United States National Science Foundation (NSF), Peer reviewed

23 pages, 7 figures, 4 tables, supplementary data https://doi.org/10.1525/elementa.2020.00165.-- Data accessibility statement: The data are deposited at https://doi.org/10.5281/zenodo.5633941, Transparent exopolymer particles (TEP) and Coomassie stainable particles (CSP) are gel-like particles, ubiquitous in the ocean, that affect important biogeochemical processes including organic carbon cycling by planktonic food webs. Despite much research on both groups of particles (especially TEP) over many years, whether they exist as distinctly stainable fractions of the same particles or as independent particles, each with different driving factors, remains unclear. To address this question, we examined the temporal dynamics of TEP and CSP over 2 complete seasonal cycles at 2 coastal sites in the Northwestern Mediterranean Sea, the Blanes Bay Microbial Observatory (BBMO) and the L’Estartit Oceanographic Station (EOS), as well as their spatial distribution along a coast-to-offshore transect. Biological, chemical, and physical variables were measured in parallel. Surface concentrations (mean ± standard deviation [SD]) of TEP were 36.7 ± 21.5 µg Xanthan Gum (XG) eq L–1 at BBMO and 36.6 ± 28.3 µg XG eq L–1 at EOS; for CSP, they were 11.9 ± 6.1 µg BSA eq L–1 at BBMO and 13.0 ± 5.9 µg BSA eq L–1 at EOS. Seasonal variability was more evident at EOS, where surface TEP and CSP concentrations peaked in summer and spring, respectively, and less predictable at the shore-most station, BBMO. Vertical distributions between surface and 80 m, monitored at EOS, showed highest TEP concentrations within the surface mixed layer during the stratification period, whereas CSP concentrations were highest before the onset of summer stratification. Phytoplankton were the main drivers of TEP and CSP distributions, although nutrient limitation and saturating irradiance also appeared to play important roles. The dynamics and distribution of TEP and CSP were uncoupled both in the coastal sites and along the transect, suggesting that they are different types of particles produced and consumed differently in response to environmental variability, This research was funded by the Spanish Ministry of Economy and Competitiveness through projects PEGASO (CTM2012-37615), BIOGAPS (CTM2016-81008-R), REMEI (CTM2015-70340-R), and DOREMI (CTM12-34294) and the Generalitat de Catalunya, through funding to the Research Group on Marine Biogeochemistry and Global Change (2017 SGR1011) and that of Microbial Diversity and Function in Aquatic Systems (2017SGR/1568) and with the institutional support of the Severo Ochoa Center of Excellence accreditation (CEX2019-000928-S). MZ was supported by a FPU predoctoral fellowship (FPU13/04630) from the Spanish Ministry of Education and Culture. The EOS Monitoring Program is supported by the Institut de Ciències del Mar (ICM-CSIC) and the Parc Natural del Montgrí, les Illes Medes i el Baix Ter (Generalitat de Catalunya), Peer reviewed

12 pages, 5 figures, 1 table, supplementary information https://doi.org/10.1038/s41396-021-01053-2, Bacteria display dynamic abundance fluctuations over time in marine environments, where they play key biogeochemical roles. Here, we characterized the seasonal dynamics of marine bacteria in a coastal oligotrophic time series station, tested how similar the temporal niche of closely related taxa is, and what are the environmental parameters modulating their seasonal abundance patterns. We further explored how conserved the niche is at higher taxonomic levels. The community presented recurrent patterns of seasonality for 297 out of 6825 amplicon sequence variants (ASVs), which constituted almost half of the total relative abundance (47%). For certain genera, niche similarity decreased as nucleotide divergence in the 16S rRNA gene increased, a pattern compatible with the selection of similar taxa through environmental filtering. Additionally, we observed evidence of seasonal differentiation within various genera as seen by the distinct seasonal patterns of closely related taxa. At broader taxonomic levels, coherent seasonal trends did not exist at the class level, while the order and family ranks depended on the patterns that existed at the genus level. This study identifies the coexistence of closely related taxa for some bacterial groups and seasonal differentiation for others in a coastal marine environment subjected to a strong seasonality, This research was funded by grants REMEI (CTM2015-70340-R), MIAU (RTI2018-101025-B-I00), and ECLIPSE (PID2019-110128RB-I00) from the Spanish Ministry of Science and Innovation, by the Grup de Recerca de la Generalitat de Catalunya (2017SGR/1568) and received support from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S). Adrià Auladell was supported by a Spanish FPI grant and IF received the support of the Fundación BBVA through the ‘Becas Leonardo a Investigadores y Creadores Culturales’ 2019 Program, Peer reviewed

42 pages, 5 figures, supplementary information https://www.biorxiv.org/content/10.1101/374298v1.supplementary-material, The smallest members of the sunlit-ocean microbiome (prokaryotes and picoeukaryotes) participate in a plethora of ecosystem functions with planetary-scale effects. Understanding the processes determining the spatial turnover of this assemblage can help us better comprehend the links between microbiome species composition and ecosystem function. Ecological theory predicts that selection, dispersal and drift are main drivers of species distributions, yet, the relative quantitative importance of these ecological processes in structuring the surface-ocean microbiome is barely known. Here we quantified the role of selection, dispersal and drift in structuring surface-ocean prokaryotic and picoeukaryotic assemblages by using community DNA-sequence data collected during the global Malaspina expedition. We found that dispersal limitation was the dominant process structuring picoeukaryotic communities, while a balanced combination of dispersal limitation, selection and drift shaped prokaryotic counterparts. Subsequently, we determined the agents exerting abiotic selection as well as the spatial patterns emerging from the action of different ecological processes. We found that selection exerted via temperature had a strong influence on the structure of prokaryotic communities, particularly on species co-occurrences, a pattern not observed among communities of picoeukaryotes. Other measured abiotic variables had limited selective effects on microbiome structure. Picoeukaryotes presented a higher differentiation between neighbouring communities and a higher distance-decay when compared to prokaryotes, agreeing with their higher dispersal limitation. Finally, drift seemed to have a limited role in structuring the sunlit-ocean microbiome. The different predominance of ecological processes acting on particular subsets of the ocean microbiome suggests uneven responses to environmental change, RL was supported by a Ramón y Cajal fellowship (RYC-2013-12554, MINECO, Spain). IMD was supported by an ITN-SINGEK fellowship (ESR2-EU-H2020-MSCA-ITN-2015, Grant Agreement 675752) and CRG by a Juan de la Cierva (IJCI-2015-23505, MINECO, Spain) fellowship. This work was supported by the projects Malaspina-2010 Expedition (CSD2008-00077, MINECO, Spain), INTERACTOMICS (CTM2015-69936-P, MINECO, Spain), REMEI (CTM2015-70340-P, MINECO, Spain) and MicroEcoSystems (240904, RCN, Norway), Peer reviewed

Memoria de tesis doctoral presentada por Adrià Auladell Martín para obtener el título de Doctor en Microbiologia por la Universitat Autònoma de Barcelona (UAB), realizada bajo la dirección del Dr. Josep M Gasol del Institut de Ciències del Mar (ICM-CSIC) y de la Dra. Isabel Ferrera Ceada del Centro Oceanográfico de Málaga, Instituto Español de Oceanografía (IEO-CSIC).-- 265 pages, figures, tables.-- The code to produce all the analysis and visualizations for each chapter are available here:
Chapter I. https://github.com/adriaaulaICM/bbmo_niche_sea
Chapter II. https://gitlab.com/aauladell/AAP_time_series
Chapter III. https://figshare.com/s/51e6d1fc462950b1f582
Chapter IV. https://figshare.com/s/65d241e03c6393446e43
The data for Chapter I and II are found in the same repositories, with the location indicated in the repository manual.
The data for Chapter III and IV are still unpublished. Both chapters present the summary information (abundance tables, taxonomy, basic environmental data…) but the raw data is still unavailable, [EN] The oceans are ecosystems dominated by microbes, in which bacteria and archaea play key roles in biogeochemical cycling. In temperate oceans, seasonal changes in environmental conditions deeply influence the marine microbiome. In this thesis I analyse the seasonality of the marine microbiome of a coastal ocean site, using the long-term time series of the Blanes Bay Microbial Observatory (BBMO) to understand the seasonal changes through several molecular approaches. Using amplicons of the 16S rRNA gene, I evaluate the dynamics of the main bacterial groups in this coastal oligotrophic station during 11 years and test how similar the temporal niches of closely related taxa are, and what are the environmental parameters modulating their patterns of seasonality. I further explore how conserved the niche is at higher taxonomic levels. The community presented recurrent seasonality for 297 out of 6825 amplicon sequence variants (ASVs), which constituted almost half of the total relative abundance (47%). For certain genera, niche similarity decreased as nucleotide divergence in the 16S rRNA gene increased, a pattern compatible with the selection of similar taxa through environmental filtering. Additionally, I observed evidence of seasonal differentiation within various genera as seen by the distinct seasonal patterns of closely related taxa. I then switch the focus to the seasonal patterns of a specific functional group. Using the pufM gene as a marker gene for the aerobic anoxygenic phototrophic bacteria (AAPs) −a relevant photohete-rotrophic functional group in the marine microbial food web− I evaluated their long-term temporal dynamics through multivariate and co-occurrence analyses. Phylogroup K (Gammaproteobacteria) was the greatest contributor to community structure over all seasons, with phylogroups E and G (Alphaproteobacteria) being prevalent in spring. Diversity indices showed a clear seasonal trend, with maximum values in winter, which was inverse to that of AAP abundance. I afterwards extend these analyses to 21 biogeochemical relevant functions through 7 years of metagenomic data from the BBMO. Most genes presented a seasonal abundance trend: photoheterotrophic processes were enriched during spring, phosphorous-related genes were dominant during summer coinciding with phosphate limitation conditions, and assimilatory nitrate reductases correlated negatively with nitrate availability. Additionally, I identified the main taxa driving each function in each season and showed that, for some groups, the seasonality of bacterial families is different than that of their gene repertoire, so that different taxa within the same group present different functional specialization. Finally, I complement this descriptive view of the temporal changes with manipulation experiments to test how bottom-up and top-down processes exert selection on specific bacterial genomic species over the seasons. I experimentally modified the presence of predators, viruses, nutrient limitation (by diluting the samples with filtered seawater) and light availability in seawater from the BBMO in different seasons and assessed the growth of different organisms defined by metagenome assembled genomes (MAGs) under the manipulated conditions. Overall, I recovered 262 MAGs mainly from the Rhodobacterales, Flavobacteriales and Alteromonadales classes. Season and treatment greatly influenced community composition, with 26% of the MAGs indicative of the control treatments, 24% of both the control and predator-reduced treatments, 12.8% indicators of both the virus-reduced and the diluted treatments, and 7.3% of the predator-reduced treatment only. Flavobacteriaceae MAGs developed mostly in the predator-reduced treatment with distinct species in each season, whereas Alteromonadaceae and Sphingomonadaceae taxa developed preferably in the virus-reduced and diluted treatments indistinctively of season. Overall, this dissertation provides new insights into the seasonal patterns of key taxonomic and functional groups in the coastal surface ocean through the integration of information obtained using several molecular techniques and experimental approaches applied to a long-term time series, [CAT] Els oceans són ecosistemes dominats per microbis i els bacteris i els arqueus hi juguen papers clau en els cicles biogeoquímics. En oceans temperats, els canvis estacionals determinen la composició del microbioma a través de les adaptacions al nínxol de les diferents espècies. En aquesta tesi analitzo l’estacionalitat del microbioma marí usant una sèrie temporal de llarga durada obtinguda a l’Observatori Microbià de la Badia de Blanes, per entendre els canvis estacionals mitjançant diverses aproximacions moleculars. A partir de seqüències d’amplicons del gen de l’RNA ribosòmic (16S) avaluo la dinàmica estacional dels principals grups bacterians durant onze anys, examinant com de similars són els nínxols temporals de taxons estretament relacionats, i quins són els paràmetres que modulen els seus patrons d’estacionalitat. També he explorat com de conservat és aquest nínxol en els nivells taxonòmics més alts. La comunitat presenta patrons estacionals de recurrència en 297 de les 6725 variants d’amplicons que hi apareixen, la qual cosa suposa gairebé la meitat de l’abundància relativa total (47%) de seqüències. Per a determinats gèneres, la similitud de nínxol disminueix amb l’increment de divergència en nucleòtids del gen del RNAr 16S, un patró compatible amb selecció de taxons similars per mitjà del filtratge ambiental. També he observat diferents patrons estacionals entre taxons del mateix gènere. A continuació vaig centrar l’anàlisi en els patrons estacionals d’un grup funcional concret. Utilitzant el gen pufM com a marcador dels bacteris aeròbics anoxigènics fotoheterotròfics −un grup funcional rellevant a la xarxa tròfica marina− avaluo la seva dinàmica temporal a través d’anàlisis multivariants i de co-ocurrència. El filogrup K (Gammaproteobacteria) és el grup dominant a l’estructura de la comunitat durant totes les estacions de l’any, amb els filogrups E i G (Alphaproteobacteria) dominants durant la primavera. Els índexs de diversitat presenten un patró estacional clar, amb els valors màxims durant l’hivern i presentant una relació inversa amb l’abundància. Després vaig ampliar aquest anàlisi a 21 funcions biogeoquímiques fent ús de set anys de dades metagenòmiques de l’observatori de Blanes. La majoria dels gens presenten un patró estacional d’abundància: els processos fotoheterotròfics enriquits durant la primavera, els gens relacionat amb l’adquisició de fòsfor dominants durant l’estiu coincidint amb una major limitació de fòsfor, i els enzims de reducció assimilatòria de nitrat correlacionant negativament amb la disponibilitat de nitrat. També he identificat els taxons principals que contenen cada gen funcional i he demostrat que, per alguns grups, l’estacionalitat a nivell de família és diferent de la del seu repertori gènic, indicant que els taxons dins del mateix grup presenten especialització funcional. Finalment, complemento la visió descriptiva dels canvis temporals amb experiments de manipulació per avaluar com els processos bottom-up i top-down influencien la selecció d’organismes durant les diferents estacions. He modificat experimentalment la presència de depredadors, de virus, la limitació per nutrients (diluint les mostres amb aigua sense microorganismes) i la llum en mostres de la Badia de Blanes en diferents estacions i he avaluat el creixement de diferents organismes definits a partir de genomes construïts a partir de metagenomes (MAGs, de les sigles en anglès). He recuperat 262 MAGs, principalment de les classes Rhodobacterales, Flavobacteriales i Alteromonadales. L’estació de l’any i el tractament influeixen la composició de la comunitat, amb el 26% dels MAGs identificats com a indicadors dels tractaments control, el 24% indicant tant el tractament control com el de reducció de depredadors, el 12.8% indicant tant el tractament de reducció de virus com el tractament diluït, i el 7.3% indicant el tractament de reducció de depredadors. Els MAGs afiliats a Flavobacteriaceae creixien majoritàriament al tractament amb reducció de depredadors, amb diferents espècies a cada estació, mentre que les especies afiliades a Alteromonadaceae i Sphingomonadaceae creixien preferentment als tractaments de reducció vírica i al tractament diluït indistintament de l’estació. En termes generals, aquesta tesi presenta resultats nous sobre els patrons estacionals de grups taxonòmics i funcionals rellevants a l’oceà costaner superficial per mitjà de la integració d’informació obtinguda usant diverses tècniques moleculars i diverses aproximacions experimentals aplicades a una sèrie temporal de llarga durada, During the realization of this thesis, Adrià Auladell Martín held a PhD Fellowship FPI (BES-2016-077820), funded by the Spanish Ministry of Science and Innovation and was a student of UAB’s PhD program in Microbiology. This thesis was also supported by the REMEI (CTM2015-70340-R), ECLIPSE (PID2019-110128RB-I00), and MIAU (RTI2018-101025-B-I00) projects, Peer reviewed

13 pages, 7 figures, 1 table, supplementary material https://www.frontiersin.org/articles/10.3389/fmicb.2023.1078469/full#supplementary-material.-- Data availability statement: The raw sequence data have been deposited in the Figshare data repository, together with the non-rarefied OTU table, the taxonomy table and the environmental data used in this study, doi: 10.6084/m9.figshare.21385215.v1, Antarctic polynyas are highly productive open water areas surrounded by ice where extensive phytoplankton blooms occur, but little is known about how these surface blooms influence carbon fluxes and prokaryotic communities from deeper waters. By sequencing the 16S rRNA gene, we explored the vertical connectivity of the prokaryotic assemblages associated with particles of three different sizes in two polynyas with different surface productivity, and we linked it to the magnitude of the particle export fluxes measured using thorium-234 (234Th) as particle tracer. Between the sunlit and the mesopelagic layers (700 m depth), we observed compositional changes in the prokaryotic communities associated with the three size-fractions, which were mostly dominated by Flavobacteriia, Alphaproteobacteria, and Gammaproteobacteria. Interestingly, the vertical differences between bacterial communities attached to the largest particles decreased with increasing 234Th export fluxes, indicating a more intense downward transport of surface prokaryotes in the most productive polynya. This was accompanied by a higher proportion of surface prokaryotic taxa detected in deep particle-attached microbial communities in the station with the highest 234Th export flux. Our results support recent studies evidencing links between surface productivity and deep prokaryotic communities and provide the first evidence of sinking particles acting as vectors of microbial diversity to depth in Antarctic polynyas, highlighting the direct influence of particle export in shaping the prokaryotic communities of mesopelagic waters, The project that gave rise to these results received the support of a fellowship to VP from “la Caixa” Foundation (ID 100010434) and from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no 847648 (fellowship code LCF/BQ/PI21/11830020). VP also received funding from Edith Cowan University (G1003456) and from the School of Science at Edith Cowan University (G1003362) to support this work. CR-G was supported by the grants RTI2018-101025-B-I00 and a Ramon y Cajal contract (RYC2019-026758-I) and JG by grants CTM2015-70340-R and PID2021-125469NB-C31 of the Spanish Ministry of Science and Innovation and by the Generalitat de Catalunya Consolidated Research Group 2017SGR/1568. [...] This work acknowledges the “Severo Ochoa Centre of Excellence” accreditation (CEX2019-000928-S), With funding from the Spanish government through the "Severo Ochoa Centre of Excellence" accreditation (CEX2019-000928-S)., Peer reviewed

19 pages, 6 figures, 1 table, supporting information https://doi.org/10.1111/1462-2920.16367.-- Data Availability Statement: All the code used for the analyses can be found in the following repository: https://github.com/adriaaulaICM/key_biogem_genes. The data to reproduce the analyses can be found in the same repository. The reader can find in the data folder a FASTA file for all the gene variants, the abundance count table, the taxonomic assignation (both with GTDB and Uniref databases), the functional annotation for each variant, and the seasonality test, Microbes drive the biogeochemical cycles of marine ecosystems through their vast metabolic diversity. While we have a fairly good understanding of the spatial distribution of these metabolic processes in various ecosystems, less is known about their seasonal dynamics. We investigated the annual patterns of 21 biogeochemical relevant functions in an oligotrophic coastal ocean site by analysing the presence of key genes, analysing high-rank gene taxonomy and the dynamics of nucleotide variants. Most genes presented seasonality: photoheterotrophic processes were enriched during spring, phosphorous-related genes were dominant during summer, coinciding with potential phosphate limitation, and assimilatory nitrate reductases appeared mostly during summer and autumn, correlating negatively with nitrate availability. Additionally, we identified the main taxa driving each function at each season and described the role of underrecognized taxa such as Litoricolaceae in carbon fixation (rbcL), urea degradation (ureC), and CO oxidation (coxL). Finally, the seasonality of single variants of some families presented a decoupling between the taxonomic abundance patterns and the functional gene patterns, implying functional specialization of the different genera. Our study unveils the seasonality of key biogeochemical functions and the main taxonomic groups that harbour these relevant functions in a coastal ocean ecosystem, This research was funded by grants REMEI (CTM2015-70340-R), MIAU (RTI2018-101025-B-I00), and ECLIPSE (PID2019-110128RB-100) from the Spanish Ministry of Science and Innovation, and received support from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000924-S). Adrià Auladell was supported by a Spanish FPI grant from the Ministry of Science and Innovation. Isabel Ferrera had the support of a ‘2019 Leonardo Grant for Researchers and Cultural Creators’ from the BBVA Foundation, Peer reviewed

21 pages, 6 figures, 1 table.-- Data Availability: All supplemental figures and tables can be found on GitHub (https://github.com/x-rv/Manuscript-2023). The 16S rRNA gene sequences of the isolates obtained in this study were deposited in GenBank under accession numbers OP342842 to OP344484. Amplicon sequencing data of the V4-V5 region of the 16S rRNA gene used in this study are publicly available in the European Nucleotide Archive under BioProject PRJEB60085, Isolation of microorganisms is a useful approach to gathering knowledge about their genomic properties, physiology, and ecology, in addition to allowing the characterization of novel taxa. We performed an extensive isolation effort on samples from seawater manipulation experiments that were carried out during the four astronomical seasons in a coastal site of the northwest Mediterranean to evaluate the impact of grazing, viral mortality, resource competition reduction, and light presence/absence on bacterioplankton growth. Isolates were retrieved using two growth media, and their full 16S rRNA gene was sequenced to assess their identity and calculate their culturability across seasons and experimental conditions. A total of 1,643 isolates were obtained, mainly affiliated to the classes Gammaproteobacteria (44%), Alphaproteobacteria (26%), and Bacteroidia (17%). Isolates pertaining to class Gammaproteobacteria were the most abundant in all experiments, while Bacteroidia were preferentially enriched in the treatments with reduced grazing. Sixty-one isolates had a similarity below 97% to cultured taxa and are thus putatively novel. Comparison of isolate sequences with 16S rRNA gene amplicon sequences from the same samples showed that the percentage of reads corresponding to isolates was 21.4% within the whole data set, with dramatic increases in the summer virus-reduced (71%) and diluted (47%) treatments. In fact, we were able to isolate the top 10 abundant taxa in several experiments and from the whole data set. We also show that top-down and bottom-up controls differentially affect taxa in terms of culturability. Our results indicate that culturing marine bacteria using agar plates can be successful in certain ecological situations, This research was supported by grant RTI2018-101974-B-C22 funded by the Spanish Ministry of Economy and Competitiveness and grants TM2015-70340-R, RTI2018-101025-B-100 and PID2021-125469NB-C32 funded by the Spanish Ministry of Science and Innovation. This work acknowledges the Severo Ochoa Centre of Excellence accreditation (CEX2019-000928-S). Xavier Rey-Velasco was supported by Spanish FI-SDUR and FPU grants, Peer reviewed

14 pages, 7 figures, 1 table. © The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited., Marine prokaryotes play crucial roles in ocean biogeochemical cycles, being their contribution strongly influenced by their growth rates. Hence, elucidating the variability and phylogenetic imprint of marine prokaryotes' growth rates are crucial for better determining the role of individual taxa in biogeochemical cycles. Here, we estimated prokaryotic growth rates at high phylogenetic resolution in manipulation experiments using water from the northwestern Mediterranean Sea. Experiments were run in the four seasons with different treatments that reduced growth limiting factors: predators, nutrient availability, viruses, and light. Single-amplicon sequence variants (ASVs)-based growth rates were calculated from changes in estimated absolute abundances using total prokaryotic abundance and the proportion of each individual ASV. The trends obtained for growth rates in the different experiments were consistent with other estimates based on total cell-counts, CARD-FISH subcommunity cell-counts or metagenomic-OTUs. Our calculations unveil a broad range of growth rates [0.3-10 d-1] with significant variability even within closely related ASVs. Likewise, the impact of growth limiting factors changed over the year for individual ASVs. High numbers of responsive ASVs were shared between winter and spring seasons, as well as throughout the year in the treatments with reduced nutrient limitation and viral pressure. The most responsive ASVs were rare in the in situ communities, comprising a large pool of taxa with the potential to rapidly respond to environmental changes. Essentially, our results highlight the lack of phylogenetic coherence in the range of growth rates observed, and differential responses to the various limiting factors, even for closely related taxa, This research was supported by grants REMEI (CTM2015-70340-R) and MIAU (RTI2018-101025-B-100) funded by the Spanish Ministry of Science and Innovation. We acknowledge the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S). Adrià Auladell and Ona Deulofeu-Capo were supported by Spanish FPI grants, With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)., Peer reviewed

Special issue Diversity and Evolution of Viruses in Ecosystem.-- 12 pages, 5 figures, supplementary materials https://www.mdpi.com/article/10.3390/v16111669/s.-- Data Availability Statement: The datasets generated and analyzed during the current study are available in the Zenodo repository under the following link: https://zenodo.org/records/10886232, Viruses play a pivotal role in ecosystems by influencing biochemical cycles and impacting the structure and evolution of their host cells. The widespread pelagiphages infect Pelagibacter spp., the most abundant marine microbe on Earth, and thus play a significant role in carbon transformation through the viral shunt. Among these viruses, the uncultured lytic pelagiphage vSAG 37-F6, uncovered by single-virus genomics, is likely the most numerous virus in the ocean. While previous research has delved into the diversity and spatial distribution of vSAG 37-F6, there is still a gap in understanding its temporal dynamics, hindering our insight into its ecological impact. We explored the temporal dynamics of vSAG 37-F6, assessing periodic fluctuations in abundance and evolutionary patterns using long- and short-term data series. In the long-term series (7 years), metagenomics showed negative selection acting on all viral genes, with a highly conserved overall diversity over time composed of a pool of yearly emergent, highly similar novel strains that exhibited a seasonal abundance pattern with two peaks during winter and fall and a decrease in months with higher UV radiation. Most non-synonymous polymorphisms occurred in structural viral proteins located in regions with low conformational restrictions, suggesting that many of the viral genes of this population are highly purified over its evolution. At the fine-scale resolution (24 h time series), combining digital PCR and metagenomics, we identified two peaks of cellular infection for the targeted vSAG 37-F6 viral strain (up to approximately 103 copies/ng of prokaryotic DNA), one before sunrise and the second shortly after midday. Considering the high number of co-occurring strains of this microdiverse virus, the abundance values at the species or genus level could be orders of magnitudes higher. These findings represent a significant advancement in understanding the dynamics of the potentially most abundant oceanic virus, providing valuable insights into ecologically relevant marine viruses, This work was supported by the Generalitat Valenciana ACIF2020 grant and by the research grants funded by the Spanish Ministry of Science and Innovation and Agencia Estatal de Investigación (PID2021-125175OB-I00). Blanes Bay metagenomes were obtained thanks to projects CTM2015-70340-R and RTI2018-101025-B-I00, With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), Peer reviewed

Memoria de tesis doctoral presentada por Ona Deulofeu Capo para obtener el título de Doctora en Microbiologia por la Universitat Autònoma de Barcelona (UAB), realizada bajo la dirección del Dr. Josep M Gasol y la Dra. Marta Sebastián Caumel del Institut de Ciències del Mar (ICM-CSIC) y la Dra. Olga Sánchez Martínez de la Universitat Autònoma de Barcelona (UAB).-- 363 pages, [EN] Marine prokaryotes are fundamental to all biological and ecological processes in the ocean and are key drivers of global biogeochemical cycles. Prokaryotic communities comprise thousands of diverse taxa that undergo continuous successions across temporal and spatial gradients shaped by environmental variability. Occasionally, a single taxon may dominate the community —a phenomenon referred to as a "bloom"— potentially playing a disproportionate role in maintaining essential ecosystem functions. Despite their ecological significance, many aspects of the ecology of blooming taxa remain poorly understood, including their recurrence, specific triggering factors, and taxonomic diversity.
This thesis aims to enhance our understanding of blooming bacterial taxa. To this end, we first used insights derived from the Blanes Bay Microbial Observatory (BBMO), a coastal long-term monthly metabarcoding time series in the NW Mediterranean Sea. Our analysis identified a limited number of taxa capable of exhibiting bloom-like behavior (ca. 1% of the total diversity), which belonged predominantly to the Flavobacteriales and Pseudomonadales, known copiotrophic orders. Despite the monthly sampling interval, we observed blooms at an average frequency of 4.6±1.9 times per year, considering both the 0.2–3 μm and 3–20 μm size fractions. No consistent association was found between environmental changes and the occurrence of blooms, but a marked increase in blooming events was observed during the period when the harbor near the BBMO site was restored—particularly within the 3–20 μm bacterioplankton size fraction (Chapter I). The absence of a strong link between environmental variability and blooming events may be due to the relatively sparse monthly sampling resolution. To overcome this limitation, we conducted a short twice-daily frequency sampling at the BBMO, which revealed that bloom events are frequent, transient and typically lasting only 24–48 hours (Chapter II). These findings challenge conventional assumptions, given that marine prokaryotes are generally thought to exhibit low in situ growth rates of around 1 d-1., Furthermore, these ephemeral blooms were sometimes associated with marked increases in heterotrophic activity, indicating that they can significantly alter community function. The rapid emergence of bloomers within such short time frames, and the unclear link with environmental parameters, drove us to test experimentally some of the insights observed in the first two chapters, in the following ones. We assessed the growth potential (i.e. the maximal growth rates in the absence of limiting factors) of individual taxa using manipulation experiments in which known growth-limiting factors—such as predation, nutrient limitation, and viral lysis— were reduced. These experiments revealed a wide spectrum of growth rates, ranging from 0.1 to 10 d-1, indicating that certain taxa can indeed transition from rare to dominant in just 24–48 hours (Chapter III). Furthermore, using another set of manipulation experiments (Chapter IV), we tested whether different carbon sources selected for different bacteria with the potential to bloom, and we found that the same taxa (e.g., Alteromonas and Rhodobacteraceae) responded consistently to the different additions, yet their success in dominating the community depended on the carbon source. Additionally, priority effects—where taxa that are relatively abundant at the beginning could have a competitive advantage over rarer ones—also influenced the response.
Overall, this thesis contributes to the understanding of marine bacterial bloomers by elucidating their taxonomic composition, temporal trends, growth dynamics, and potential functional roles. Our findings suggest that these taxa may contribute significantly to ecosystem resilience by rapidly responding to both anthropogenic and natural environmental perturbations, [ES] Los procariotas marinos son esenciales para la mayoría de los procesos biológicos y ecológicos del océano, desempeñando un papel clave en los ciclos biogeoquímicos globales. Las comunidades de procariotas están compuestas por miles de taxones que experimentan una sucesión continua tanto a escalas temporales como a lo largo de gradientes espaciales definidos por la variación en las condiciones ambientales. En ocasiones, un único taxón puede dominar la comunidad, fenómeno al que denominamos proliferación o bloom. Se cree que durante estos crecimientos explosivos, estos taxones pueden tener un papel relevante en el mantenimiento de las funciones ecosistémicas esenciales. A pesar de su importancia ecológica, diversos aspectos como su recurrencia, los factores desencadenantes o su diversidad taxonómica siguen siendo poco comprendidos.
En esta tesis profundizamos en el conocimiento sobre las bacterias de crecimiento explosivo. Para ello, se utilizaron datos de la serie temporal del Observatorio Microbiano de la Bahía de Blanes (BBMO), una serie temporal mensual de larga duración situada en la costa del mar Mediterráneo noroccidental. Nuestros análisis permitieron identificar un número limitado de taxones que proliferaron (alrededor del 1% de la diversidad total), per tenecientes principalmente a los órdenes Flavobacteriales y Pseudomonadales, conocidos por incluir organismos copiótrofos. A pesar de la resolución temporal mensual, detectamos estas proliferaciones con una frecuencia media de 4,6 ± 1,9 veces al año (considerando ambas fracciones: 0,2–3 μm y 3–20 μm). Aunque no se encontraron asociaciones significativas entre las variables ambientales y la ocurrencia de estos eventos, sí se detectó un aumento de éstos durante las obras del puerto de Blanes, cercano al punto de muestreo, principalmente en la fracción 3–20 μm (Capítulo I)., La ausencia de relación entre la variabilidad ambiental y las proliferaciones bacterianas podría deberse a la baja frecuencia de muestreo. Por ello, se llevó a cabo un muestreo intensivo de corta duración (dos veces al día) en el mismo punto de muestreo, lo que permitió observar que estas proliferaciones son frecuentes, transitorias y de corta duración (24–48 horas; Capítulo II). Estos hallazgos cuestionan suposiciones previas como que las bacterias marinas crecen típicamente a tasas de 1 d-1. Además, estas proliferaciones efímeras se asociaron a aumentos de actividad heterotrófica, indicando alteraciones significativas en las funciones ecosistémicas de la comunidad. La rápida aparición de estas bacterias, junto con su escasa asociación con los parámetros ambientales medidos, motivó la realización de experimentos para testar las observaciones en comunidades naturales.
En el siguiente capítulo se estimaron las tasas de crecimiento de taxones individuales mediante experimentos de manipulación reduciendo distintos factores limitantes de crecimiento: la depredación, la limitación por nutrientes y la lisis viral. Estos experimentos revelaron un amplio rango de tasas de crecimiento, entre 0,1 y 10 d-1, lo que indica que la transición de estos taxones desde raros hasta dominantes en tan solo 24–48 horas es posible (Capítulo III). Además, mediante una serie adicional de experimentos se evaluó si diferentes fuentes de carbono seleccionaban distintos taxones. Los mismos grupos (p. ej., Alteromonas y Rhodobacteraceae) respondían a diversas adiciones, aunque su éxito a la hora de dominar la comunidad dependía del tipo de fuente de carbono. Asimismo, los efectos de prioridad (quien empieza a crecer antes o está bien establecido acaba dominando) también influyeron en la respuesta (Capítulo IV).
En conjunto, esta tesis contribuye a una mejor comprensión de las bacterias marinas con capacidad de crecer de manera explosiva, mediante el análisis de su taxonomía, dinámicas temporales y de crecimiento, o sus potenciales roles funcionales. Nuestros resultados sugieren que estos taxones pueden desempeñar un papel importante en la resiliencia del ecosistema, respondiendo rápidamente a perturbaciones tanto de origen antropogénico como natural, [CAT] Els procariotes marins són essencials per a la majoria dels processos biològics i ecològics de l’oceà i tenen un paper clau en els cicles biogeoquímics globals. Les comunitats procariòtiques estan formades per milers de taxons diversos en successió contínua al llarg de gradients tant temporals com espacials, modelats per la variabilitat ambiental. En aquest context, de vegades un tàxon concret pot arribar a dominar la comunitat —un fenomen conegut com a bloom o proliferació—, que potencialment juga un paper desproporcionat en el manteniment de funcions ecosistèmiques essencials durant aquestes proliferacions. Malgrat la seva importància ecològica, molts aspectes de l’ecologia dels taxons protagonistes d’aquestes proliferacions continuen sent poc coneguts, com ara la seva recurrència, factors desencadenants específics i la diversitat taxonòmica.
En aquesta tesi contribuïm al coneixement sobre els bacteris que protagonitzen aquestes proliferacions duent a terme creixements explosius. Amb aquesta finalitat hem utilitzat dades de l’Observatori Microbià de la Badia de Blanes (BBMO), una sèrie temporal mensual i de llarga durada, situada a la costa del mar Mediterrani nord-occidental. Les nostres anàlisis han permès identificar un nombre limitat de taxons amb capacitat per créixer de manera explosiva (aproximadament l’1% de la diversitat total), majoritàriament dels ordres Flavobacteriales i Pseudomonadales, grups coneguts per incloure organismes copiòtrofs. Tot i la resolució mensual, vam detectar aquestes proliferacions amb una freqüència mitjana de 4,6 ± 1,9 vegades a l’any (considerant les fraccions 0,2–3 μm i 3–20 μm). Malgrat que no vam trobar associacions significatives amb les variables ambientals, sí vam trobar un augment de proliferacions bacterianes específiques durant les obres del port de Blanes, proper al punt de mostreig, especialment en la comunitat de la fracció 3–20 μm (Capítol I)., L’absència d’un vincle fort entre variabilitat ambiental i aquestes proliferacions podria deure’s a la baixa freqüència de mostreig. Per això vam realitzar un mostreig intensiu (dues vegades al dia) al mateix punt (BBMO), que va revelar que aquestes proliferacions són freqüents, efímeres (24–48 h) i presents sobretot a la fracció 3–20 μm (Capítol II). Aquests resultats qüestionen assumpcions prèvies com ara que els bacteris marins creixen a una taxa d’1 d-1. A més, aquestes proliferacions efímeres van anar acompanyades d’un augment marcat de l’activitat heterotròfica, indicant una alteració funcional de la comunitat. La seva ràpida aparició i la poca relació amb els paràmetres ambientals van motivar la realització d’experiments per testar aquestes observacions.
En el següent capítol vam estimar les taxes de creixement de taxons individuals mitjançant experiments on es van reduir factors limitants com la depredació, la manca de nutrients i la lisi viral. Les taxes obtingudes van ser molt variables, entre 0,1 i 10 d-1, confirmant que la transició d’ésser rar a dominant en 24–48 h és factible (Capítol III). A més, mitjançant altres experiments (Capítol IV) vam avaluar si fonts de carboni diferents seleccionaven diferents bacteris, i vam trobar que els mateixos taxons (p. ex. Alteromonas, Rhodobacteraceae) responien a addicions diferents, però el seu èxit a l’hora de dominar la comunitat depenia de la font de carboni. També els “efectes de prioritat” (qui comença a créixer abans o ja està ben establert, acaba dominant) han tingut un efecte en la resposta.
En conjunt, aquesta tesi contribueix a una millor comprensió dels bacteris marins amb la capacitat de dur a terme creixements explosius, identificant la seva composició, les dinàmiques temporals i de creixement així com els seus possibles rols funcionals. Els nostres resultats suggereixen que aquests taxons poden contribuir significativament a la resiliència de l’ecosistema, responent ràpidament a pertorbacions tant naturals com antropogèniques, During the realization of this thesis, Ona Deulofeu Capo held a PhD fellowship FPI (PRE2019-090554) associated to the MIAU (RTI2018-101025-B-100) project, funded by the Spanish Ministry of Science and Innovation. and was a student of the Microbiology PhD program from the Universitat Autònoma de Barcelona. The thesis was also supported by projects REMEI (CTM2015-70340-R) and MICOLOR (PID2021-125469NB-C31, PID2021-125469NBC32) and the “Severo Ochoa Centre of Excellence” accreditation (CEX2019-000928-S), Peer reviewed

Sampling was conducted over a period of 14 yr (2000 to 2014) at the Blanes Bay Microbial Observatory (BBMO) station, located in the Bay of Blanes, in the Northwestern Mediterranean Coast (Fig. 1), approximately 60 km north of Barcelona. The BBMO station is a shallow site (~20 m depth) approximately 800 m offshore from the town of Blanes (41° 40' N, 2° 48' E). A total of 162 surface water samples were considered for analysis. The first one was taken in September 2000; from 2001 to 2014, samples were in general collected monthly, with some exceptions. A volume of water varying from 0.7 l to 1 l (depending on the sampling season) was filtered onto Whatman GF/F (nominal pore size 0.7 μm; 25 mm diameter) glass fiber filters with low vacuum (0.4 bars) to prevent cells from breaking. Subsequently, the filters were folded, dried, wrapped in aluminum foil and stored frozen at −80°C until analysis. For pigment extraction, the filters were placed in tubes with 2.5 ml of 90% acetone submerged in ice, and the pigments were extracted by sonication for 30 s. Subsequently, the filters were stored at −20°C. After 24 h, the samples were vortexed and filtered through Whatman GF/F glass fiber filters. Prior to 2008, the samples were analyzed at the Institut de Ciències del Mar (CSIC) in Barcelona according to the method of Zapata et al. (2000), using a ThermoQuest chromatograph (hereafter System 1), which included a P2000 solvent module, an A/S 3000 autosampler, a UV-3000 absorbance detector (440 nm), a FL2000 fluorescence detector (excitation = 430 nm, emission = 662 nm), and an SN 4000 controller. After 2008, analyses were carried out at the Centro Oceanográfico de Gijón/Xixón (IEO-CSIC) following the procedure (hereafter System 2) of Latasa (2014), which increased the sensitivity and lowered the detection limit, using an Agilent series 1200 chromatographic system consisting of a G1311A quaternary pump, a G1367C autosampler with a 100 μl capillary loop, a G1316B column thermostat, and a G1315C diode array detector. A significant change added to the System 2 analyses was the use of trans-β-apo-8'-carotenal, as an internal standard, dissolved in the acetone used for sample extraction. In total, 28 pigments were detected at 440 and 665 nm (System 1) or 474 and 664 nm (System 2) and identified by retention time and online diode array detector, - Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR), grant/award no. SGR/1179
- Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR), grant/award no. SGR/761
- Comissió Interdepartamental de Recerca i Innovació Tecnològica, Generalitat de Catalunya, grant/award no. 2005SGR/00872
- Comissió Interdepartamental de Recerca i Innovació Tecnològica, Generalitat de Catalunya, grant/award no. 2009SGR/1177
- Comissió Interdepartamental de Recerca i Innovació Tecnològica, Generalitat de Catalunya, grant/award no. 2014SGR/1179
- Comissió Interdepartamental de Recerca i Innovació Tecnològica, Generalitat de Catalunya, grant/award no. 2017SGR/1568
- Spanish Ministry of Science, Innovation and Universities (MICINN), grant/award no. CTM2015-70340-R, This work contributes to the Institut de Ciències del Mar "Severo Ochoa Centre of Excellence" accreditation CEX2024-001494-S funded by AEI 10.13039/501100011033 of the Spanish Ministry of Science and Innovation, Peer reviewed

Background: Isolation of marine microorganisms is fundamental to gather information about their physiology, ecology and genomic content. To date, most of the bacterial isolation efforts have focused on the photic ocean leaving the deep ocean less explored. We have created a marine culture collection of heterotrophic bacteria (MARINHET) using a standard marine medium comprising a total of 1561 bacterial strains, and covering a variety of oceanographic regions from different seasons and years, from 2009 to 2015. Specifically, our marine collection contains isolates from both photic (817) and aphotic layers (744), including the mesopelagic (362) and the bathypelagic (382), from the North Western Mediterranean Sea, the North and South Atlantic Ocean, the Indian, the Pacific, and the Arctic Oceans. We described the taxonomy, the phylogenetic diversity and the biogeography of a fraction of the marine culturable microorganisms to enhance our knowledge about which heterotrophic marine isolates are recurrently retrieved across oceans and along different depths. Results: The partial sequencing of the 16S rRNA gene of all isolates revealed that they mainly affiliate with the classes Alphaproteobacteria (35.9%), Gammaproteobacteria (38.6%), and phylum Bacteroidetes (16.5%). In addition, Alteromonas and Erythrobacter genera were found the most common heterotrophic bacteria in the ocean growing in solid agar medium. When comparing all photic, mesopelagic, and bathypelagic isolates sequences retrieved from different stations, 37% of them were 100% identical. This percentage increased up to 59% when mesopelagic and bathypelagic strains were grouped as the aphotic dataset and compared to the photic dataset of isolates, indicating the ubiquity of some bacterial isolates along different ocean depths. Finally, we isolated three strains that represent a new species, and the genome comparison and phenotypic characterization of two of these strains (ISS653 and ISS1889) concluded that they belong to a new species within the genus Mesonia. Conclusions: Overall, this study highlights the relevance of culture-dependent studies, with focus on marine isolated bacteria from different oceanographic regions and depths, to provide a more comprehensive view of the culturable marine bacteria as part of the total marine microbial diversity.

Microbes associated with sinking marine particles play key roles in carbon sequestration in the ocean. The sampling of particle-attached microorganisms is often done with sediment traps or by filtration of water collected with oceanographic bottles, both involving a certain time lapse between collection and processing of samples that may result in changes in particle-attached microbial communities. Conversely, in situ water filtration through submersible pumps allows a faster storage of sampled particles, but it has rarely been used to study the associated microbial communities and has never been compared to other particle-sampling methods in terms of the recovery of particle microbial diversity. Here we compared the prokaryotic communities attached to small (1-53 μm) and large (.

Estimation of prokaryotic growth rates is critical to understand the ecological role and contribution of different microbes to marine biogeochemical cycles. However, there is a general lack of knowledge on what factors control the growth rates of different prokaryotic groups and how these vary between sites and along seasons at a given site. We carried out several manipulation experiments during the four astronomical seasons in the coastal NW Mediterranean in order to evaluate the impact of grazing, viral mortality, resource competition and light on the growth and loss rates of prokaryotes. Gross and net growth rates of different bacterioplankton groups targeted by group-specific CARD-FISH probes and infrared microscopy (for aerobic anoxygenic phototrophs, AAP), were calculated from changes in cell abundances. Maximal group-specific growth rates were achieved when both predation pressure and nutrient limitation were experimentally minimized, while only a minimal effect of viral pressure on growth rates was observed; nevertheless, the response to predation removal was more remarkable in winter, when the bacterial community was not subjected to nutrient limitation. Although all groups showed increases in their growth rates when resource competition as well as grazers and viral pressure were reduced, Alteromonadaceae consistently presented the highest rates in all seasons. The response to light availability was generally weaker than that to the other factors, but it was variable between seasons. In summer and spring, the growth rates of AAP were stimulated by light whereas the growth of the SAR11 clade (likely containing proteorhodopsin) was enhanced by light in all seasons. Overall, our results set thresholds on bacterioplankton group-specific growth and mortality rates and contribute to estimate the seasonally changing contribution of various bacterioplankton groups to the function of microbial communities. Our results also indicate that the least abundant groups display the highest growth rates, contributing to the recycling of organic matter to a much greater extent than what their abundances alone would predict.

We studied the long-term temporal dynamics of the aerobic anoxygenic phototrophic (AAP) bacteria, a relevant functional group in the coastal marine microbial food web, using high-throughput sequencing of the pufM gene coupled with multivariate, time series and co-occurrence analyses at the Blanes Bay Microbial Observatory (NW Mediterranean). Additionally, using metagenomics, we tested whether the used primers captured accurately the seasonality of the most relevant AAP groups. Phylogroup K (Gammaproteobacteria) was the greatest contributor to community structure over all seasons, with phylogroups E and G (Alphaproteobacteria) being prevalent in spring. Diversity indices showed a clear seasonal trend, with maximum values in winter, which was inverse to that of AAP abundance. Multivariate analyses revealed sample clustering by season, with a relevant proportion of the variance explained by day length, temperature, salinity, phototrophic nanoflagellate abundance, chlorophyll a, and silicate concentration. Time series analysis showed robust rhythmic patterns of co-occurrence, but distinct seasonal behaviors within the same phylogroup, and even within different amplicon sequence variants (ASVs) conforming the same operational taxonomic unit (OTU). Altogether, our results picture the AAP assemblage as highly seasonal and recurrent but containing ecotypes showing distinctive temporal niche partitioning, rather than being a cohesive functional group.

Bottom-up and top-down controls greatly influence marine microbial community composition and dynamics, which in turn have effects on their culturability. We isolated a high amount of heterotrophic bacterial strains from experiments where seawater environmental conditions had been manipulated and found that decreasing grazing and viral pressure as well as rising nutrient availability are key factors increasing the success in culturing marine bacteria. Our data hint at factors influencing culturability and underpin bacterial cultures as a powerful way to discover new taxa.

Marine prokaryotes play crucial roles in ocean biogeochemical cycles, being their contribution strongly influenced by their growth rates. Hence, elucidating the variability and phylogenetic imprint of marine prokaryotes' growth rates are crucial for better determining the role of individual taxa in biogeochemical cycles. Here, we estimated prokaryotic growth rates at high phylogenetic resolution in manipulation experiments using water from the northwestern Mediterranean Sea. Experiments were run in the four seasons with different treatments that reduced growth limiting factors: predators, nutrient availability, viruses, and light. Single-amplicon sequence variants (ASVs)-based growth rates were calculated from changes in estimated absolute abundances using total prokaryotic abundance and the proportion of each individual ASV. The trends obtained for growth rates in the different experiments were consistent with other estimates based on total cell-counts, catalyzed reporter deposition fluorescence in situ hybridization subcommunity cell-counts or metagenomic-operational taxonomic units (OTUs). Our calculations unveil a broad range of growth rates (0.3-10 d -1) with significant variability even within closely related ASVs. Likewise, the impact of growth limiting factors changed over the year for individual ASVs. High numbers of responsive ASVs were shared between winter and spring seasons, as well as throughout the year in the treatments with reduced nutrient limitation and viral pressure. The most responsive ASVs were rare in the in situ communities, comprising a large pool of taxa with the potential to rapidly respond to environmental changes. Essentially, our results highlight the lack of phylogenetic coherence in the range of growth rates observed, and differential responses to the various limiting factors, even for closely related taxa.

Viruses play a pivotal role in ecosystems by influencing biochemical cycles and impacting the structure and evolution of their host cells. The widespread pelagiphages infect Pelagibacter spp., the most abundant marine microbe on Earth, and thus play a significant role in carbon transformation through the viral shunt. Among these viruses, the uncultured lytic pelagiphage vSAG 37-F6, uncovered by single-virus genomics, is likely the most numerous virus in the ocean. While previous research has delved into the diversity and spatial distribution of vSAG 37-F6, there is still a gap in understanding its temporal dynamics, hindering our insight into its ecological impact. We explored the temporal dynamics of vSAG 37-F6, assessing periodic fluctuations in abundance and evolutionary patterns using long- and short-term data series. In the long-term series (7 years), metagenomics showed negative selection acting on all viral genes, with a highly conserved overall diversity over time composed of a pool of yearly emergent, highly similar novel strains that exhibited a seasonal abundance pattern with two peaks during winter and fall and a decrease in months with higher UV radiation. Most non-synonymous polymorphisms occurred in structural viral proteins located in regions with low conformational restrictions, suggesting that many of the viral genes of this population are highly purified over its evolution. At the fine-scale resolution (24 h time series), combining digital PCR and metagenomics, we identified two peaks of cellular infection for the targeted vSAG 37-F6 viral strain (up to approximately 103 copies/ng of prokaryotic DNA), one before sunrise and the second shortly after midday. Considering the high number of co-occurring strains of this microdiverse virus, the abundance values at the species or genus level could be orders of magnitudes higher. These findings represent a significant advancement in understanding the dynamics of the potentially most abundant oceanic virus, providing valuable insights into ecologically relevant marine viruses.