DIATOMS AND DINOFLAGELLATES DIVERSITY INHABITING A COASTAL UPWELLING SYSTEM: A METABARCODING APPROACH

Small eukaryotic plankton has been traditionally characterized using conventional microscopy techniques. Current advances in sequencing technologies allow the cost-effective study the diversity within microbial plankton based on DNA sequences. This technique has been implemented in the last 15 years for investigating prokaryotic diversity, but its application to unravel the diversity and ecology of eukaryotic organisms is still incipient. In this study we analyze the diversity of the diatoms and dinoflagellates in the marine communities using rDNA sequencing techniques, as well as the taxonomic resolution provided by the V4 region of the 18S rRNA gene. Twenty monthly 6L seawater samples for DNA metabarcoding were collected and filtered through 3 μm polycarbonate filter in a station off the Ría de A Coruña (NW Iberian Peninsula). DNA was extracted, the V4 region of the 18S rRNA was PCR amplified and subsequently sequenced using the High Throughput Sequencing (HTS) platform Illumina. Amplicon sequence variants (ASVs) were differentiated using DADA2 implemented in R. Sequences were aligned against PR2 v4.12.0 and SILVA 132 18S rRNA databases as references, as well as studied using BLAST and phylogenetic trees. Sequence-based taxonomic approach found 128 and 416 metabarcodes corresponding to diatoms and dinoflagellates, respectively. Sequences had limited resolution at species or even genus level, and overall the taxonomic resolution of diatoms was substantially higher than those of dinoflagellates

Nano- and microplankton diversity inhabiting a coastal upwelling system: a metabarcoding approach

Small eukaryotic plankton has been traditionally characterized using conventional microscopy techniques. Current advances in sequencing technologies allow the costeffective study the diversity within microbial plankton based on DNA sequences. This technique has been implemented in the last 15 years for investigating prokaryotic diversity, but its application to unravel the diversity and distribution of eukaryotic organisms is still incipient. Alleged advantages of this approach include the ability of detecting the smaller fraction of the community that trend to be overlooked in microscopy studies, as well as improving the resolution of taxonomic identification for groups whose morphological study is challenging due the paucity of morphological characters. In this study we characterize the diversity of the nano- and microplankton marine communities using microscopy and rDNA sequencing techniques. The aim of this work is to compare results of both techniques, assessing whether rDNA data can provide new insights into the study of the diversity of eukaryotic planktonic communities. Twenty monthly samples were collected in a station off the Ría de A Coruña (NW Iberian Peninsula). Planktonic samples for DNA studies were collected by filtering seawater samples through a 3 μm pore size polycarbonate filter. DNA was extracted, the V4 region of the 18S rRNA was PCR amplified and subsequently sequenced using the High Throughput Sequencing (HTS) platform Illumina. Amplicon sequence variants (ASVs) were differentiated using DADA2 implemented in R. Sequences were aligned against PR2 v4.12.0 and SILVA 132 18S rRNA databases as references. Microscopic identification of plankton taxa (phytoplankton and protozoa) was made using the Uthermöhl technique. In total 1182 ASVs were identified based on rDNA data, which contrasts with only 65 species identified by microscopy. Morphological identifications of eukaryotic plankton was restricted mainly to diatoms, dinoflagellates and ciliates, while rDNA data allowed the detection of additional taxonomic groups (i.e. protist). Some of them were very abundant, such as the algae Cryptophyceae and Mamiellophyceae or the heterotrophs Ascomycota or parasitic Stramenopiles. Therefore, our study further evidenced that DNA metabarcoding is a valuable tool to uncover part of the great diversity within microbial plankton, improving the detection of small eukaryotes

High dark CO2 fixation rates by active chemolithoautotrophic microbes along the water column (100-5000m) off Galicia (NW Iberian margin)

Poster communicationOur results provide evidence for the significant contribution to chemolithotrophy by specific archaeal and bacterial groups in the dark ocean

Changes in activity and community composition shape bacterial responses to size-fraccionated marine DOM

To study the response of bacteria to different size-fractions of naturally occurring dissolved organic matter (DOM), a natural prokaryotic community from North Atlantic mesopelagic waters (1000 m depth) was isolated and grown in (i) 0.1-µm filtered seawater (CONTROL), (ii) the low-molecular-weight (<1 kDa) DOM fraction (L-DOM), and (iii) the recombination of high- (>1 kDa) and low-molecular-weight DOM fractions (H + L-DOM), to test the potential effect of ultrafiltration on breaking the DOM size continuum. Prokaryotic abundance and leucine incorporation were consistently higher in the H + L-DOM niche than in the L-DOM and CONTROL treatments, suggesting a different interaction with each DOM fraction and the disruption of the structural DOM continuum by ultrafiltration, respectively. Rhodobacterales (Alphaproteobacteria) and Flavobacteriales (Bacteroidetes) were particularly enriched in L-DOM and closely related to the colored DOM (CDOM) fraction, indicating the tight link between these groups and changes in DOM aromaticity. Conversely, some other taxa that were rare or undetectable in the original bacterial community were enriched in the H + L-DOM treatment (e.g., Alteromonadales belonging to Gammaproteobacteria), highlighting the role of the rare biosphere as a seed bank of diversity against ecosystem disturbance. The relationship between the fluorescence of protein-like CDOM and community composition of populations in the H + L-DOM treatment suggested their preference for labile DOM. Conversely, the communities growing on the L-DOM niche were coupled to humic-like CDOM, which may indicate their ability to degrade more reworked DOM and/or the generation of refractory substrates (as by-products of the respiration processes). Most importantly, L- and/or H + L-DOM treatments stimulated the growth of unique bacterial amplicon sequence variants (ASVs), suggesting the potential of environmental selection (i.e., changes in DOM composition and availability), particularly in the light of climate change scenarios. Taken together, our results suggest that different size-fractions of DOM induced niche-specialization and differentiation of mesopelagic bacterial communities.Versión del edito

Seasonal niche of planktonic prokaryotes inhabiting surface waters of the upwelling region off NW Iberia

Prokaryotes play an important role in biogeochemical cycling in marine ecosystems, but little is known about their diversity and composition, and it’s even less understood how they may contribute to the ecological functioning of highly variable coastal areas affected by upwelling. Between May 2016 and May 2018, we carried out 26 one-day samplings in the temperate northwestern Iberian upwelling system to investigate the temporal patterns of variability for prokaryotic abundance, diversity and community composition by combining flow cytometry and 16S RNA high- throughput sequencing. A marked seasonality was found for prokaryotic abundance, peaking during summer upwelling and relaxation season (≈May to September), when extracellular release of organic matter from phytoplanktonic blooms is a significant process, and decreasing in downwelling events (≈October to April). Those downwelling conditions, characterized by deeper mixed layers and a homogeneous water column, favored a higher abundance of the archaeal groups, Marine Group II (Euryarchaeota) and Nitrosopelagicus (Thaumarchaeota), as well as of Marinimicrobia bacterium (SAR406 clade) and the group Bacteria_Others. By contrast, upwelling and relaxation conditions characterized by enhanced vertical stratification and hydrographic variability, included a community generally less diverse with core-phylotypes (occurring > 75% of the samples) proliferating, i.e. Flavobacteriaceae (Bacteroidetes), Chloroplast (Cyanobacteria) and Amylibacter (Proteobacteria). Overall, the environmental conditions explained 60% (R2=0.60, AIC=125.64) of the prokaryotic community composition, being temperature, inorganic nutrients, chlorophyll and particulate organic nitrogen the variables that best explained the variation in the prokaryotic community composition (r=0.40). Additional efforts are currently underway on a fine-tuning composition assessment (oligotypes composition from particular core-phylotypes) to study if this variability along the temporal environmental gradient could be associated with the differentiation of ecotypes (oligotype ́s seasonality within particular phylotypes). Overall, the present study provides new insights into the barely explored seasonal niche partitioning of surface prokaryotic community driven by hydrodynamic forcing in an upwelling system, which may support further investigations on the role of bacterioplankton in the biogeochemistry of these ecosystems

Seasonal niche of planktonic prokaryotes inhabiting surface waters of the upwelling region off NW Iberia

Oral communicationProkaryotes play an important role in biogeochemical cycling in marine ecosystems, but little is known about their diversity and composition, and it’s even less understood how they may contribute to the ecological functioning of highly variable coastal areas affected by upwelling. Between May 2016 and May 2018, we carried out 26 one-day samplings in the temperate northwestern Iberian upwelling system to investigate the temporal patterns of variability for prokaryotic abundance, diversity and community composition by combining flow cytometry and 16S RNA high- throughput sequencing. A marked seasonality was found for prokaryotic abundance, peaking during summer upwelling and relaxation season (≈May to September), when extracellular release of organic matter from phytoplanktonic blooms is a significant process, and decreasing in downwelling events (≈October to April). Those downwelling conditions, characterized by deeper mixed layers and a homogeneous water column, favored a higher abundance of the archaeal groups, Marine Group II (Euryarchaeota) and Nitrosopelagicus (Thaumarchaeota), as well as of Marinimicrobia bacterium (SAR406 clade) and the group Bacteria_Others. By contrast, upwelling and relaxation conditions characterized by enhanced vertical stratification and hydrographic variability, included a community generally less diverse with core-phylotypes (occurring > 75% of the samples) proliferating, i.e. Flavobacteriaceae (Bacteroidetes), Chloroplast (

A device for assesing microbial activity under ambient hydrostatic pressure: The in situ microbial incubator (ISMI)

Research articleMicrobes in the dark ocean are exposed to hydrostatic pressure increasing with depth. Activity rate measurements and biomass production of dark ocean microbes are, however, almost exclusively performed under atmospheric pressure conditions due to technical constraints of sampling equipment maintaining in situ pressure conditions. To evaluate the microbial activity under in situ hydrostatic pressure, we designed and thoroughly tested an in situ microbial incubator (ISMI). The ISMI allows autonomously collecting and incubating seawater at depth, injection of substrate and fixation of the samples after a preprogramed incubation time. The performance of the ISMI was tested in a high-pressure tank and in several field campaigns under ambient hydrostatic pressure by measuring prokaryotic bulk 3H-leucine incorporation rates. Overall, prokaryotic leucine incorporation rates were lower at in situ pressure conditions than under to depressurized conditions reaching only about 50% of the heterotrophic microbial activity measured under depressurized conditions in bathypelagic waters in the North Atlantic Ocean off the northwestern Iberian Peninsula. Our results show that the ISMI is a valuable tool to reliably determine the metabolic activity of deep-sea microbes at in situ hydrostatic pressure conditions. Hence, we advocate that deep-sea biogeochemical and microbial rate measurements should be performed under in situ pressure conditions to obtain a more realistic view on deep-sea biotic processes.IEO-CSIC, FWF, KAKENHI, ERC and GAI

Changes in bacterial activity and community composition in response to water mass mixing

PosterMixing zones and boundaries between different water masses are "hot spots" of marine biodiversity and activity. We aimed to investigate the effects of water mass mixing in the dark-ocean microbial communities by collecting and incubating natural bacterial communities from the Mediterranean Water (MW; at 1000 m depth), the Subpolar Modal Water (SPMW, 500m) and the Labrador Sea Water (LSW, 1800 m), and comparing them with artificially mixed communities. Mixing experiment 1 consisted of incubating at in sity conditions the original LSW and MW communities, plus a mixture of both (MIX1, dilution 1:1), whereas the Mixing experiment 2 included the original prokaryotic communities from SPMW and MW and a mixture of both (MIX2, dilution 1:1). Bacterial abundance and activity was monitored every 24 h over 8 days, while bacterial community composition and DOM characterization were assessed at the beginning (day 0), middle (day 4) and at the end of the experiment (day 8). Live prokaryotic cell abundance was higher in the MIX1 and MIX2 treatments as compared to the original communities. Moreover, MIX bacteria showed slightly higher leucine incorporation rates than MW or LSW. These metabolic responses were accompanied by changes in the optical properties of DOM, suggesting a change in the dynamics of the organic matter. Taken together, our results indicate differences in the bio-reactvity of the organic matter after mixing as compared to the original water masses that could influence the composition and activity of the bacterial community

FILO Ciliophora

Capítulo de libroEl conocimiento de la diversidad de los ciliados planctónicos en aguas de Galicia hasta la fecha es muy limitado. La mayor parte de los estudios publicados solo incluyen grandes grupos o algunas especies dominantes, que clásicamente se identificaban por microscopía óptica adaptada y hoy en día se se usan técnicas moleculares basadas en la amplificación, clonación y secuenciación de regiones del gen codificador del ARNr 18S, que permite la identificación filogenética de las "especies" o OTUs que componen las comunidades de ciliados planctónicos, por ejemplo. La lista añadida incluye 25 familias y 88 géneros y especies, recogidos en el marco de proyectos de observación sistemática del IEO... La clasificación usada se basa en Lynn&Small (2002) y la nomenclatura empleada en WORMS (Warren, 2011). Diferenciación por ambientes pelágicos.En prens

Climate and Local Hydrography Underlie Recent Regime Shifts in Plankton Communities off Galicia (NW Spain)

A 29-year-long time series (1990–2018) of phyto- and zooplankton abundance and composition is analyzed to uncover regime shifts related to climate and local oceanography variability. At least two major shifts were identified: one between 1997 and 1998, affecting zooplankton group abundance, phytoplankton species assemblages and climatic series, and a second one between 2001 and 2002, affecting microzooplankton group abundance, mesozooplankton species assemblages and local hydrographic series. Upwelling variability was relatively less important than other climatic or local oceanographic variables for the definition of the regimes. Climate-related regimes were influenced by the dominance of cold and dry (1990–1997) vs. warm and wet (1998–2018) periods, and characterized by shifts from low to high life trait diversity in phytoplankton assemblages, and from low to high meroplankton dominance for mesozooplankton. Regimes related to local oceanography were defined by the shift from relatively low (1990–2001) to high (2002–2018) concentrations of nutrients provided by remineralization (or continental inputs) and biological production, and shifts from a low to high abundance of microzooplankton, and from a low to high trait diversity of mesozooplankton species assemblages. These results align with similar shifts described around the same time for most regions of the NE Atlantic. This study points out the different effects of large-scale vs. local environmental variations in shaping plankton assemblages at multiannual time scales.En prens