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Eastern boundary upwelling systems (EBUS) contribute a disproportionate fraction of the global fish catch relative to their size and are especially susceptible to global environmental change. Here we present the evolution of communities over 50 d in an in situ mesocosm 6 km offshore of Callao, Peru, and in the nearby unenclosed coastal Pacific Ocean. The communities were monitored using multi-marker environmental DNA (eDNA) metabarcoding and flow cytometry. DNA extracted from weekly water samples were subjected to amplicon sequencing for four genetic loci: (1) the V1–V2 region of the 16S rRNA gene for photosynthetic eukaryotes (via their chloroplasts) and bacteria; (2) the V9 region of the 18S rRNA gene for exploration of eukaryotes but targeting phytoplankton; (3) cytochrome oxidase I (COI) for exploration of eukaryotic taxa but targeting invertebrates; and (4) the 12S rRNA gene, targeting vertebrates. The multi-marker approach showed a divergence of communities (from microbes to fish) between the mesocosm and the unenclosed ocean. Together with the environmental information, the genetic data furthered our mechanistic understanding of the processes that are shaping EBUS communities in a changing ocean. The unenclosed ocean experienced significant variability over the course of the 50 d experiment, with temporal shifts in community composition, but remained dominated by organisms that are characteristic of high-nutrient upwelling conditions (e.g., diatoms, copepods, anchovies). A large directional change was found in the mesocosm community. The mesocosm community that developed was characteristic of upwelling regions when upwelling relaxes and waters stratify (e.g., dinoflagellates, nanoflagellates). The selection of dinoflagellates under the salinity-driven experimentally stratified conditions in the mesocosm, as well as the warm conditions brought about by the coastal El Niño, may be an indication of how EBUS will respond under the global environmental changes (i.e., increases in surface temperature and freshwater input, leading to increased stratification) forecast by the IPCC.

Microbial predators such as choanoflagellates are key players in ocean food webs. Choanoflagellates, which are the closest unicellular relatives of animals, consume bacteria and also exhibit marked biological transitions triggered by bacterial compounds, yet their native microbiomes remain uncharacterized. Here we report the discovery of a ubiquitous, uncultured bacterial lineage we name Candidatus Comchoanobacterales ord. nov., related to the human pathogen Coxiella and physically associated with the uncultured marine choanoflagellate Bicosta minor. We analyse complete ‘Comchoano’ genomes acquired after sorting single Bicosta cells, finding signatures of obligate host-dependence, including reduction of pathways encoding glycolysis, membrane components, amino acids and B-vitamins. Comchoano encode the necessary apparatus to import energy and other compounds from the host, proteins for host-cell associations and a type IV secretion system closest to Coxiella’s that is expressed in Pacific Ocean metatranscriptomes. Interactions between choanoflagellates and their microbiota could reshape the direction of energy and resource flow attributed to microbial predators, adding complexity and nuance to marine food webs.

Characterizing complex microbial communities with single-cell resolution has been a long-standing goal of microbiology. We present Microbe-seq, a high-throughput method that yields the genomes of individual microbes from complex microbial communities. We encapsulate individual microbes in droplets with microfluidics and liberate their DNA, which we then amplify, tag with droplet-specific barcodes, and sequence. We explore the human gut microbiome, sequencing more than 20,000 microbial single-amplified genomes (SAGs) from a single human donor and coassembling genomes of almost 100 bacterial species, including several with multiple subspecies strains. We use these genomes to probe microbial interactions, reconstructing the horizontal gene transfer (HGT) network and observing HGT between 92 species pairs; we also identify a significant in vivo host-phage association between crAssphage and one strain of Bacteroides vulgatus . Microbe-seq contributes high-throughput culture-free capabilities to investigate genomic blueprints of complex microbial communities with single-microbe resolution.

Universal primers for SSU rRNA genes allow profiling of natural communities by simultaneously amplifying templates from Bacteria, Archaea, and Eukaryota in a single PCR reaction. Despite the potential to show relative abundance for all rRNA genes, universal primers are rarely used, due to various concerns including amplicon length variation and its effect on bioinformatic pipelines. We thus developed 16S and 18S rRNA mock communities and a bioinformatic pipeline to validate this approach. Using these mocks, we show that universal primers (515Y/926R) outperformed eukaryote-specific V4 primers in observed versus expected abundance correlations (slope = 0.88 vs. 0.67–0.79), and mock community members with single mismatches to the primer were strongly underestimated (threefold to eightfold). Using field samples, both primers yielded similar 18S beta-diversity patterns (Mantel test, p < 0.001) but differences in relative proportions of many rarer taxa. To test for length biases, we mixed mock communities (16S + 18S) before PCR and found a twofold underestimation of 18S sequences due to sequencing bias. Correcting for the twofold underestimation, we estimate that, in Southern California field samples (1.2–80 μm), there were averages of 35% 18S, 28% chloroplast 16S, and 37% prokaryote 16S rRNA genes. These data demonstrate the potential for universal primers to generate comprehensive microbiome profiles.

Microbial predators such as choanoflagellates are key players in ocean food webs. Choanoflagellates, which are the closest unicellular relatives of animals, consume bacteria and also exhibit marked biological transitions triggered by bacterial compounds, yet their native microbiomes remain uncharacterized. Here we report the discovery of a ubiquitous, uncultured bacterial lineage we name Candidatus Comchoanobacterales ord. nov., related to the human pathogen Coxiella and physically associated with the uncultured marine choanoflagellate Bicosta minor. We analyse complete 'Comchoano' genomes acquired after sorting single Bicosta cells, finding signatures of obligate host-dependence, including reduction of pathways encoding glycolysis, membrane components, amino acids and B-vitamins. Comchoano encode the necessary apparatus to import energy and other compounds from the host, proteins for host-cell associations and a type IV secretion system closest to Coxiella's that is expressed in Pacific Ocean metatranscriptomes. Interactions between choanoflagellates and their microbiota could reshape the direction of energy and resource flow attributed to microbial predators, adding complexity and nuance to marine food webs.

Significance Although viruses are well-characterized regulators of eukaryotic algae, little is known about those infecting unicellular predators in oceans. We report the largest marine virus genome yet discovered, found in a wild predatory choanoflagellate sorted away from other Pacific microbes and pursued using integration of cultivation-independent and laboratory methods. The giant virus encodes nearly 900 proteins, many unlike known proteins, others related to cellular metabolism and organic matter degradation, and 3 type-1 rhodopsins. The viral rhodopsin that is most abundant in ocean metagenomes, and also present in an algal virus, pumps protons when illuminated, akin to cellular rhodopsins that generate a proton-motive force. Giant viruses likely provision multiple host species with photoheterotrophic capacities, including predatory unicellular relatives of animals., Giant viruses are remarkable for their large genomes, often rivaling those of small bacteria, and for having genes thought exclusive to cellular life. Most isolated to date infect nonmarine protists, leaving their strategies and prevalence in marine environments largely unknown. Using eukaryotic single-cell metagenomics in the Pacific, we discovered a Mimiviridae lineage of giant viruses, which infects choanoflagellates, widespread protistan predators related to metazoans. The ChoanoVirus genomes are the largest yet from pelagic ecosystems, with 442 of 862 predicted proteins lacking known homologs. They are enriched in enzymes for modifying organic compounds, including degradation of chitin, an abundant polysaccharide in oceans, and they encode 3 divergent type-1 rhodopsins (VirR) with distinct evolutionary histories from those that capture sunlight in cellular organisms. One (VirRDTS) is similar to the only other putative rhodopsin from a virus (PgV) with a known host (a marine alga). Unlike the algal virus, ChoanoViruses encode the entire pigment biosynthesis pathway and cleavage enzyme for producing the required chromophore, retinal. We demonstrate that the rhodopsin shared by ChoanoViruses and PgV binds retinal and pumps protons. Moreover, our 1.65-Å resolved VirRDTS crystal structure and mutational analyses exposed differences from previously characterized type-1 rhodopsins, all of which come from cellular organisms. Multiple VirR types are present in metagenomes from across surface oceans, where they are correlated with and nearly as abundant as a canonical marker gene from Mimiviridae. Our findings indicate that light-dependent energy transfer systems are likely common components of giant viruses of photosynthetic and phagotrophic unicellular marine eukaryotes.

The marine picoeukaryote Bathycoccus prasinos has been considered a cosmopolitan alga, although recent studies indicate two ecotypes exist, Clade BI (B. prasinos) and Clade BII. Viruses that infect Bathycoccus Clade BI are known (BpVs), but not that infect BII. We isolated three dsDNA prasinoviruses from the Sargasso Sea against Clade BII isolate RCC716. The BII-Vs do not infect BI, and two (BII-V2 and BII-V3) have larger genomes (~210 kb) than BI-Viruses and BII-V1. BII-Vs share ~90% of their proteins, and between 65% to 83% of their proteins with sequenced BpVs. Phylogenomic reconstructions and PolB analyses establish close-relatedness of BII-V2 and BII-V3, yet BII-V2 has 10-fold higher infectivity and induces greater mortality on host isolate RCC716. BII-V1 is more distant, has a shorter latent period, and infects both available BII isolates, RCC716 and RCC715, while BII-V2 and BII-V3 do not exhibit productive infection of the latter in our experiments. Global metagenome analyses show Clade BI and BII algal relative abundances correlate positively with their respective viruses. The distributions delineate BI/BpVs as occupying lower temperature mesotrophic and coastal systems, whereas BII/BII-Vs occupy warmer temperature, higher salinity ecosystems. Accordingly, with molecular diagnostic support, we name Clade BII Bathycoccus calidus sp. nov. and propose that molecular diversity within this new species likely connects to the differentiated host-virus dynamics observed in our time course experiments. Overall, the tightly linked biogeography of Bathycoccus host and virus clades observed herein supports species-level host specificity, with strain-level variations in infection parameters.

Giant viruses have remarkable genomic repertoires—blurring the line with cellular life—and act as top–down controls of eukaryotic plankton. However, to date only six cultured giant virus genomes are available from the pelagic ocean. We used at-sea flow cytometry with staining and sorting designed to target wild predatory eukaryotes, followed by DNA sequencing and assembly, to recover novel giant viruses from the Pacific Ocean. We retrieved four ‘PacV’ partial genomes that range from 421 to 1605 Kb, with 13 contigs on average, including the largest marine viral genomic assembly reported to date. Phylogenetic analyses indicate that three of the new viruses span a clade with deep-branching members of giant Mimiviridae, incorporating the Cafeteria roenbergensis virus, the uncultivated terrestrial Faunusvirus, one PacV from a choanoflagellate and two PacV with unclear hosts. The fourth virus, oPacV-421, is phylogenetically related to viruses that infect haptophyte algae. About half the predicted proteins in each PacV have no matches in NCBI nr (e-value < 10−5), totalling 1735 previously unknown proteins; the closest affiliations of the other proteins were evenly distributed across eukaryotes, prokaryotes and viruses of eukaryotes. The PacVs encode many translational proteins and two encode eukaryotic-like proteins from the Rh family of the ammonium transporter superfamily, likely influencing the uptake of nitrogen during infection. cPacV-1605 encodes a microbial viral rhodopsin (VirR) and the biosynthesis pathway for the required chromophore, the second finding of a choanoflagellate-associated virus that encodes these genes. In co-collected metatranscriptomes, 85% of cPacV-1605 genes were expressed, with capsids, heat shock proteins and proteases among the most highly expressed. Based on orthologue presence–absence patterns across the PacVs and other eukaryotic viruses, we posit the observed viral groupings are connected to host lifestyles as heterotrophs or phototrophs. This article is part of a discussion meeting issue ‘Single cell ecology’.

Ecosystems are controlled by ‘bottom-up’ (resources) and ‘top-down’ (predation) forces. Viral infection is now recognized as a ubiquitous top-down control of microbial growth across ecosystems but, at the same time, cell death by viral predation influences, and is influenced by, resource availability. In this Review, we discuss recent advances in understanding the biogeochemical impact of viruses, focusing on how metabolic reprogramming of host cells during lytic viral infection alters the flow of energy and nutrients in aquatic ecosystems. Our synthesis revealed several emerging themes. First, viral infection transforms host metabolism, in part through virus-encoded metabolic genes; the functions performed by these genes appear to alleviate energetic and biosynthetic bottlenecks to viral production. Second, viral infection depends on the physiological state of the host cell and on environmental conditions, which are challenging to replicate in the laboratory. Last, metabolic reprogramming of infected cells and viral lysis alter nutrient cycling and carbon export in the oceans, although the net impacts remain uncertain. This Review highlights the need for understanding viral infection dynamics in realistic physiological and environmental contexts to better predict their biogeochemical consequences. In this Review, Coleman and colleagues discuss recent advances in understanding the biogeochemical impact of viruses, focusing on how metabolic reprogramming of host cells during viral infection alters the flow of energy and nutrients in aquatic ecosystems.

While the impact of light on primary productivity in aquatic systems has been studied for decades, the role light plays in the degradation of photosynthetically-produced biomass is less well understood. We investigated the patterns of light-induced particle breakdown and bacterial assimilation of detrital C and N using13C and15N labeled freeze-thawed diatom cells incubated in laboratory microcosms with a marine microbial community freshly-collected from the Pacific Ocean. Particles incubated in the dark resulted in increased bacterial counts and dissolved organic carbon concentrations compared to those incubated in the light. Light also influenced the attached and free-living microbial community structure as detected by 16S rRNA gene amplicon sequencing. For example, bacterial taxa from the Sphingobacteriia were enriched on dark-incubated particles and taxa from the family Flavobacteriaceae and the genus Pseudoalteromonas were numerically enriched on particles in the light. Isotope incorporation analysis by phylogenetic microarray and NanoSIMS (a method called Chip-SIP) identified free-living and attached microbial taxa able to incorporate N and C from the particles. Some taxa, including members of the Flavobacteriaceae and Cryomorphaceae, exhibited increased isotope incorporation in the light, suggesting the use of photoheterotrophic metabolisms. In contrast, some members of Oceanospirillales and Rhodospirillales showed decreased isotope incorporation in the light, suggesting that their heterotrophic metabolism, particularly when occurring on particles, might increase at night or may be inhibited by sunlight. These results show that light influences particle degradation and C and N incorporation by attached bacteria, suggesting that the transfer between particulate and free-living phases are likely affected by external factors that change with the light regime, such as time of day, depth and season.

Viruses provide top-down control on microbial communities, yet their direct study in natural environments was hindered by culture limitations. The advance of bioinformatics enables cultivation-independent study of viruses. Many studies assemble new viral genomes and study viral diversity using marker genes from free viruses. Here we use cellular metatranscriptomics to study active community-wide viral infections. Recruitment to viral contigs allows tracking infection dynamics over time and space. Our assemblies represent viral populations, but appear biased towards low diversity viral taxa. Tracking relatives of published T4-like cyanophages and pelagiphages reveals high genomic continuity. We determine potential hosts by matching dynamics of infection with abundance of particular microbial taxa. Finally, we quantify the relative contribution of cyanobacteria and viruses to photosystem-II psbA (reaction center) expression in our study sites. We show sometimes >50% of all cyanobacterial+viral psbA expression is of viral origin, highlighting the contribution of viruses to photosynthesis and oxygen production., Here, Sieradzki et al. use metatranscriptomics to study active community-wide viral infections at three coastal California sites throughout a year, identify potential viral hosts, and show that viruses can contribute a substantial amount to photosystem-II psbA expression.

Short timescale observations are valuable for understanding microbial ecological processes. We assessed dynamics in relative abundance and potential activities by sequencing the small sub-unit ribosomal RNA gene (rRNA gene) and rRNA molecules (rRNA) of Bacteria, Archaea, and Eukaryota once to twice daily between March 2014 and May 2014 from the surface ocean off Catalina Island, California. Typically Ostreococcus, Braarudosphaera, Teleaulax, and Synechococcus dominated phytoplankton sequences (including chloroplasts) while SAR11, Sulfitobacter, and Fluviicola dominated non-phytoplankton Bacteria and Archaea. We observed short-lived increases of diatoms, mostly Pseudo-nitzschia and Chaetoceros, with quickly responding Bacteria and Archaea including Flavobacteriaceae (Polaribacter & Formosa), Roseovarius, and Euryarchaeota (MGII), notably the exact amplicon sequence variants we observed responding similarly to another diatom bloom nearby, 3 years prior. We observed correlations representing known interactions among abundant phytoplankton rRNA sequences, demonstrating the biogeochemical and ecological relevance of such interactions: (1) The kleptochloroplastidic ciliate Mesodinium 18S rRNA gene sequences and a single Teleaulax taxon (via 16S rRNA gene sequences) were correlated (Spearman r = 0.83) yet uncorrelated to a Teleaulax 18S rRNA gene OTU, or any other taxon (consistent with a kleptochloroplastidic or karyokleptic relationship) and (2) the photosynthetic prymnesiophyte Braarudosphaera bigelowii and two strains of diazotrophic cyanobacterium UCYN-A were correlated and each taxon was also correlated to other taxa, including B. bigelowii to a verrucomicrobium and a dictyochophyte phytoplankter (all r > 0.8). We also report strong correlations (r > 0.7) between various ciliates, bacteria, and phytoplankton, suggesting interactions via currently unknown mechanisms. These data reiterate the utility of high-frequency time series to show rapid microbial reactions to stimuli, and provide new information about in situ dynamics of previously recognized and hypothesized interactions.

Despite the routine use of standards and blanks in virtually all chemical or physical assays and most biological studies (a kind of “control”), microbiome analysis has traditionally lacked such standards. Here we show that unexpected problems of unknown origin can occur in such sequencing runs and yield completely incorrect results that would not necessarily be detected without the use of standards. Assuming that the microbiome sequencing analysis works properly every time risks serious errors that can be detected by the use of mock communities., Mock communities have been used in microbiome method development to help estimate biases introduced in PCR amplification and sequencing and to optimize pipeline outputs. Nevertheless, the strong value of routine mock community analysis beyond initial method development is rarely, if ever, considered. Here we report that our routine use of mock communities as internal standards allowed us to discover highly aberrant and strong biases in the relative proportions of multiple taxa in a single Illumina HiSeqPE250 run. In this run, an important archaeal taxon virtually disappeared from all samples, and other mock community taxa showed >2-fold high or low abundance, whereas a rerun of those identical amplicons (from the same reaction tubes) on a different date yielded “normal” results. Although obvious from the strange mock community results, we could have easily missed the problem had we not used the mock communities because of natural variation of microbiomes at our site. The “normal” results were validated over four MiSeqPE300 runs and three HiSeqPE250 runs, and run-to-run variation was usually low. While validating these “normal” results, we also discovered that some mock microbial taxa had relatively modest, but consistent, differences between sequencing platforms. We strongly advise the use of mock communities in every sequencing run to distinguish potentially serious aberrations from natural variations. The mock communities should have more than just a few members and ideally at least partly represent the samples being analyzed to detect problems that show up only in some taxa and also to help validate clustering. IMPORTANCE Despite the routine use of standards and blanks in virtually all chemical or physical assays and most biological studies (a kind of “control”), microbiome analysis has traditionally lacked such standards. Here we show that unexpected problems of unknown origin can occur in such sequencing runs and yield completely incorrect results that would not necessarily be detected without the use of standards. Assuming that the microbiome sequencing analysis works properly every time risks serious errors that can be detected by the use of mock communities.

The 16S protocol detailed here is designed to amplify prokaryotes (bacteria and archaea) using paired-end 16S community sequencing on the Illumina platform. Primers515F-806R target the V4 region of the 16S SSU rRNA. For running these libraries on the MiSeq and HiSeq, please make sure you read the supplementary methods of Caporaso et al. (2012). You will need to make your sample more complex by adding 5-10% PhiX to your run.

Summary Microbial community analysis via high-throughput sequencing of amplified 16S rRNA genes is an essential microbiology tool. We found the popular primer pair 515F (515F-C) and 806R greatly underestimated (e.g. SAR11) or overestimated (e.g. Gammaproteobacteria) common marine taxa. We evaluated marine samples and mock communities (containing 11 or 27 marine 16S clones), showing alternative primers 515F-Y (5′-GTGYCAGCMGCCGCGGTAA) and 926R (5′-CCGYCAATTYMTTTRAGTTT) yield more accurate estimates of mock community abundances, produce longer amplicons that can differentiate taxa unresolvable with 515F-C/806R, and amplify eukaryotic 18S rRNA. Mock communities amplified with 515F-Y/926R yielded closer observed community composition versus expected (r2 = 0.95) compared with 515F-Y/806R (r2 ∼ 0.5). Unexpectedly, biases with 515F-Y/806R against SAR11 in field samples (∼4–10-fold) were stronger than in mock communities (∼2-fold). Correcting a mismatch to Thaumarchaea in the 515F-C increased their apparent abundance in field samples, but not as much as using 926R rather than 806R. With plankton samples rich in eukaryotic DNA (> 1 μm size fraction), 18S sequences averaged ∼17% of all sequences. A single mismatch can strongly bias amplification, but even perfectly matched primers can exhibit preferential amplification. We show that beyond in silico predictions, testing with mock communities and field samples is important in primer selection.

Recent advances in studying the dynamics of marine microbial communities have shown that the composition of these communities follows predictable patterns and involves complex network interactions, which shed light on the underlying processes regulating these globally important organisms. Such 'holistic' (or organism- and system-based) studies of these communities complement popular reductionist, often culture-based, approaches for understanding organism function one gene or protein at a time. In this Review, we summarize our current understanding of marine microbial community dynamics at various scales, from hours to decades. We also explain how the data illustrate community resilience and seasonality, and reveal interactions among microorganisms.

Short time-scale observations are valuable for understanding microbial ecological processes. We assessed dynamics in relative abundance and potential activities by sequencing the small sub-unit ribosomal RNA gene (rRNA gene) and rRNA molecules (rRNA) ofBacteria,Archaea, andEukaryotaonce to twice-daily between March 2014 and May 2014 from the surface ocean off Catalina Island, California. TypicallyOstreococcus, Braarudosphaera, Teleaulax, and Synechococcusdominated phytoplankton sequences (including chloroplasts) while SAR11,Sulfitobacter, andFluviicoladominated non-phytoplanktonBacteriaandArchaea. We observed short-lived increases of diatoms, mostlyPseudo-nitzschiaandChaetoceros, with quickly respondingBacteriaandArchaeaincludingFlavobacteriaceae(Polaribacter&Formosa),Roseovarius, andEuryarchaeota(MGII), notably the exact amplicon sequence variants we observed responding similarly to another diatom bloom nearby, three years prior. We observed correlations representing known interactions among abundant phytoplankton rRNA sequences, demonstrating the biogeochemical and ecological relevance of such interactions: 1) The kleptochloroplastidic ciliateMesodinium18S rRNA gene sequences and a singleTeleaulaxtaxon (via 16S rRNA gene sequences) were correlated (Spearmanr=0.83) yet uncorrelated to aTeleaulax18S rRNA gene OTU, or any other taxon (consistent with a kleptochloroplastidic or karyoklepty relationship) and 2) the photosynthetic prymnesiophyteBraarudosphaera bigelowiiand two strains of diazotrophic cyanobacterium UCYN-A were correlated and each taxon was also correlated to other taxa, includingB. bigelowiito a verrucomicrobium and a dictyochophyte phytoplankter (allr> 0.8). We also report strong correlations (r> 0.7) between various ciliates, bacteria, and phytoplankton, suggesting interactions via currently unknown mechanisms. These data reiterate the utility of high-frequency time-series to show rapid microbial reactions to stimuli, and provide new information aboutin-situdynamics of previously recognized and hypothesized interactions.

Mock communities have been used in microbiome method development to help estimate biases introduced in PCR amplification, sequencing, and to optimize pipeline outputs. Nevertheless, the necessity of routine mock community analysis beyond initial method development is rarely, if ever, considered. Here we report that our routine use of mock communities as internal standards allowed us to discover highly aberrant and strong biases in the relative proportions of multiple taxa in a single Illumina HiSeqPE250 run. In this run, an important archaeal taxon virtually disappeared from all samples, and other mock community taxa showed >2-fold high or low abundance, whereas a rerun of those identical amplicons (from the same reaction tubes) on a different date yielded “normal” results. Although obvious from the strange mock community results, due to natural variation of microbiomes at our site, we easily could have missed the problem had we not used the mock communities. The “normal” results were validated over 4 MiSeqPE300 runs and 3 HiSeqPE250 runs, and run-to-run variation was usually low (Bray-Curtis distance was 0.12±0.04). While validating these “normal” results, we also discovered some mock microbial taxa had relatively modest, but consistent, differences between sequencing platforms. We suggest that using mock communities in every sequencing run is essential to distinguish potentially serious aberrations from natural variations. Such mock communities should have more than just a few members and ideally at least partly represent the samples being analyzed, to detect problems that show up only in some taxa, as we observed.ImportanceDespite the routine use of standards and blanks in virtually all chemical or physical assays and most biological studies (a kind of “control”), microbiome analysis has traditionally lacked such standards. Here we show that unexpected problems of unknown origin can occur in such sequencing runs, and yield completely incorrect results that would not necessarily be detected without the use of standards. Assuming that the microbiome sequencing analysis works properly every time risks serious errors that can be avoided by the use of suitable mock communities.

We examined the short-term variability, by daily to weekly sampling, of protist assemblages from March to July in surface water of the San Pedro Ocean Time-series station (eastern North Pacific), by V4 Illumina sequencing of the 18S rRNA gene. The sampling period encompassed a spring bloom followed by progression to summer conditions. Several protistan taxa displayed sharp increases and declines, with whole community Bray–Curtis dissimilarities of adjacent days being 66% in March and 40% in May. High initial abundance of parasitic Cercozoa Cryothecomonas longipes and Protaspis grandis coincided with a precipitous decline of blooming Pseudo-nitzschia diatoms, possibly suggesting their massive infection by these parasites; these cercozoans were hardly detectable afterwards. Canonical correspondence analysis indicated a limited predictability of community variability from environmental factors. This indicates that other factors are relevant in explaining changes in protist community composition at short temporal scales, such as interspecific relationships, stochastic processes, mixing with adjacent water, or advection of patches with different protist communities. Association network analysis revealed that interactions between the many parasitic OTUs and other taxa were overwhelmingly positive and suggest that although sometimes parasites may cause a crash of host populations, they may often follow their hosts and do not regularly cause enough mortality to potentially create negative correlations at the daily to weekly time scales we studied.

Analysis of seasonal patterns of marine bacterial community structure along horizontal and vertical spatial scales can help to predict long-term responses to climate change. Several recent studies have shown predictable seasonal reoccurrence of bacterial assemblages. However, only a few have assessed temporal variability over both horizontal and vertical spatial scales. Here, we simultaneously studied the bacterial community structure at two different locations and depths in shelf waters of a coastal upwelling system during an annual cycle. The most noticeable biogeographic patterns observed were seasonality, horizontal homogeneity, and spatial synchrony in bacterial diversity and community structure related with regional upwelling–downwelling dynamics. Water column mixing eventually disrupted bacterial community structure vertical heterogeneity. Our results are consistent with previous temporal studies of marine bacterioplankton in other temperate regions and also suggest a marked influence of regional factors on the bacterial communities inhabiting this coastal upwelling system. Bacterial-mediated carbon fluxes in this productive region appear to be mainly controlled by community structure dynamics in surface waters, and local environmental factors at the base of the euphotic zone.

Many reef-building corals photoacclimate by increasing levels of chlorophyll per microalgal cell with depth, but mechanisms of photoacclimation in coral reef sea anemones remain poorly understood. We determined variation in ambient irradiance and patterns of abundance, microhabitat use, chlorophyll and microalgal cell concentrations in clownfish sea anemones Entacmaea quadricolor from 0 to 43 m depth on a coral reef at Aqaba, Jordan, northern Red Sea. In shallow areas, anemones occupied shaded reef microhabitats exposed to significantly lower irradiance than in the adjacent open water, but on the deep reef slope they occupied unshaded habitats. Anemone abundances were the highest observed thus far in the Red Sea, and peaked at mid depth on the reef slope. Microalgal abundance in anemone tentacles increased four-fold from the shallow to deep reef, while chlorophyll-a concentrations per algal cell did not vary significantly with depth. We conclude that E. quadricolor photoacclimates using two major mechanisms: (1) occurrence in shaded microhabitats when shallow, thus reducing exposure to high irradiance, and (2) increasing microalgal abundance with depth, thereby enhancing photosynthetic efficiency at low irradiance.

Long-term non-linear ecosystem-scale changes in water quality and biotic communities in coastal lagoons have been associated with intensification of anthropogenic pressures. In light of incipient changes in Johnson Bay (an embayment of Chincoteague Bay, Maryland-Virginia, USA), examination of nitrogen sources was conducted through synoptic water quality monitoring, stable nitrogen isotope signatures (δ15N) of in situ bioindicators, and denitrification estimates. These data were placed in the context of long-term and broader spatial analyses. Despite various watershed protection efforts, multiyear summer time studies (2004–2007) suggested that high levels of terrestrially derived nutrients still enter Johnson Bay. Total nitrogen concentrations in Johnson Bay were 132% the concentrations in the broader Chincoteague Bay during the late 1970s (mean 2004–2007 was 40.0 – 73.2 μM). Comparing total nitrogen concentrations in Johnson Bay to St. Martin River (consistently the most eutrophic region of these coastal bays), Johnson Bay has increased from 62.5% to 82.5% of the concentrations in St. Martin River during the late 1970s. Though specific sources of nitrogen inputs have not yet been definitively identified, the long-term increase in total nitrogen concentrations occurred despite increased and continued conservation and protection measures. We suggest that investigating nutrient sources can reveal potentially ineffective nutrient policies and that this knowledge can be applied towards other coastal lagoons.

Numerous ecological processes, such as bacteriophage infection and phytoplankton–bacterial interactions, often occur via strain-specific mechanisms. Therefore, studying the causes of microbial dynamics should benefit from highly resolving taxonomic characterizations. We sampled daily to weekly over 5 months following a phytoplankton bloom off Southern California and examined the extent of microdiversity, that is, significant variation within 99% sequence similarity clusters, operational taxonomic units (OTUs), of bacteria, archaea, phytoplankton chloroplasts (all via 16S or intergenic spacer (ITS) sequences) and T4-like-myoviruses (via g23 major capsid protein gene sequence). The extent of microdiversity varied between genes (ITS most, g23 least) and only temporally common taxa were highly microdiverse. Overall, 60% of taxa exhibited microdiversity; 59% of these had subtypes that changed significantly as a proportion of the parent taxon, indicating ecologically distinct taxa. Pairwise correlations between prokaryotes and myoviruses or phytoplankton (for example, highly microdiverse Chrysochromulina sp.) improved when using single-base variants. Correlations between myoviruses and SAR11 increased in number (172 vs 9, Spearman>0.65) and became stronger (0.61 vs 0.58, t-test: P1 μm (attached/large) prokaryotes are a major myovirus source. Consideration of microdiversity improved observation of apparent host and virus networks, and provided insights into the ecological and evolutionary factors influencing the success of lineages, with important implications to ecosystem resilience and microbial function.

Marine phytoplankton perform approximately half of global carbon fixation, with their blooms contributing disproportionately to carbon sequestration1, and most phytoplankton production is ultimately consumed by heterotrophic prokaryotes2. Therefore, phytoplankton and heterotrophic community dynamics are important in modelling carbon cycling and the impacts of global change3. In a typical bloom, diatoms dominate initially, transitioning over several weeks to smaller and motile phytoplankton4. Here, we show unexpected, rapid community variation from daily rRNA analysis of phytoplankton and prokaryotic community members following a bloom off southern California. Analysis of phytoplankton chloroplast 16S rRNA demonstrated ten different dominant phytoplankton over 18 days alone, including four taxa with animal toxin-producing strains. The dominant diatoms, flagellates and picophytoplankton varied dramatically in carbon export potential. Dominant prokaryotes also varied rapidly. Euryarchaea briefly became the most abundant organism, peaking over a few days to account for about 40% of prokaryotes. Phytoplankton and prokaryotic communities correlated better with each other than with environmental parameters. Extending beyond the traditional view of blooms being controlled primarily by physics and inorganic nutrients, these dynamics imply highly heterogeneous, continually changing conditions over time and/or space and suggest that interactions among microorganisms are critical in controlling plankton diversity, dynamics and fates. Rapid variation in the phytoplankton and bacterioplankton communities of a spring bloom provides new insights into the biological and physical parameters affecting plankton succession.

Microbial community analysis via high-throughput sequencing of amplified 16S rRNA genes is an essential microbiology tool. We found the popular primer pair 515F (515F-C) and 806R greatly underestimated (e.g. SAR11) or overestimated (e.g. Gammaproteobacteria) common marine taxa. We evaluated marine samples and mock communities (containing 11 or 27 marine 16S clones), showing alternative primers 515F-Y (5'-GTGYCAGCMGCCGCGGTAA) and 926R (5'-CCGYCAATTYMTTTRAGTTT) yield more accurate estimates of mock community abundances, produce longer amplicons that can differentiate taxa unresolvable with 515F-C/806R, and amplify eukaryotic 18S rRNA. Mock communities amplified with 515F-Y/926R yielded closer observed community composition versus expected (r(2) = 0.95) compared with 515F-Y/806R (r(2) ∼ 0.5). Unexpectedly, biases with 515F-Y/806R against SAR11 in field samples (∼4-10-fold) were stronger than in mock communities (∼2-fold). Correcting a mismatch to Thaumarchaea in the 515F-C increased their apparent abundance in field samples, but not as much as using 926R rather than 806R. With plankton samples rich in eukaryotic DNA ( 1 μm size fraction), 18S sequences averaged ∼17% of all sequences. A single mismatch can strongly bias amplification, but even perfectly matched primers can exhibit preferential amplification. We show that beyond in silico predictions, testing with mock communities and field samples is important in primer selection.

Interactions among microbes and stratification across depths are both believed to be important drivers of microbial communities, though little is known about how microbial associations differ between and across depths. We have monitored the free-living microbial community at the San Pedro Ocean Time-series station, monthly, for a decade, at five different depths: 5 m, the deep chlorophyll maximum layer, 150 m, 500 m and 890 m (just above the sea floor). Here, we introduce microbial association networks that combine data from multiple ocean depths to investigate both within- and between-depth relationships, sometimes time-lagged, among microbes and environmental parameters. The euphotic zone, deep chlorophyll maximum and 890 m depth each contain two negatively correlated ‘modules' (groups of many inter-correlated bacteria and environmental conditions) suggesting regular transitions between two contrasting environmental states. Two-thirds of pairwise correlations of bacterial taxa between depths lagged such that changes in the abundance of deeper organisms followed changes in shallower organisms. Taken in conjunction with previous observations of seasonality at 890 m, these trends suggest that planktonic microbial communities throughout the water column are linked to environmental conditions and/or microbial communities in overlying waters. Poorly understood groups including Marine Group A, Nitrospina and AEGEAN-169 clades contained taxa that showed diverse association patterns, suggesting these groups contain multiple ecological species, each shaped by different factors, which we have started to delineate. These observations build upon previous work at this location, lending further credence to the hypothesis that sinking particles and vertically migrating animals transport materials that significantly shape the time-varying patterns of microbial community composition.

Observation of short-term temporal variation in bacterial and viral communities is important for understanding patterns of aquatic microbial diversity. We collected surface seawater once daily for 38 consecutive days with seven more samples interspersed over 40 more days at one location ∼2 km from Santa Catalina Island, California. Bacterial communities were analyzed by automated ribosomal intergenic spacer analysis (ARISA) and viral communities were analyzed by terminal restriction fragment length polymorphism (TRFLP) of the conserved T4-like myoviral gene encoding the major capsid protein (g23). Common bacterial and viral taxa were consistently dominant, and relatively few displayed dramatic increases/decreases or 'boom/bust' patterns that might be expected from dynamic predator-prey interactions. Association network analysis showed most significant covariations (associations) occurred among bacterial taxa or among viral taxa and there were several modular (highly-interconnected) associations (P≤0.005). Associations observed between bacteria and viruses (P≤0.005) occurred with a median time lag of 2 days. Regression of all pairwise Bray-Curtis similarities between samples indicated a rate of bacterial community change that slows from 2.1%-0.18% per day over a week to 2 months; the rate stays around 0.4% per day for viruses. Our interpretation is that, over the scale of days, individual bacterial and viral OTUs can be dynamic and patterned; resulting in statistical associations regarded as potential ecological interactions. However, over the scale of weeks, average bacterial community variation is slower, suggesting that there is strong community-level ecological resilience, that is, a tendency to converge towards a 'mean' microbial community set by longer-term controlling factors.

Time-series are critical to understanding long-term natural variability in the oceans. Bacterial communities in the euphotic zone were investigated for over a decade at the San Pedro Ocean Time-series station (SPOT) off southern California. Community composition was assessed by Automated Ribosomal Intergenic Spacer Analysis (ARISA) and coupled with measurements of oceanographic parameters for the surface ocean (0–5 m) and deep chlorophyll maximum (DCM, average depth ∼30 m). SAR11 and cyanobacterial ecotypes comprised typically more than one-third of the measured community; diversity within both was temporally variable, although a few operational taxonomic units (OTUs) were consistently more abundant. Persistent OTUs, mostly Alphaproteobacteria (SAR11 clade), Actinobacteria and Flavobacteria, tended to be abundant, in contrast to many rarer yet intermittent and ephemeral OTUs. Association networks revealed potential niches for key OTUs from SAR11, cyanobacteria, SAR86 and other common clades on the basis of robust correlations. Resilience was evident by the average communities drifting only slightly as years passed. Average Bray-Curtis similarity between any pair of dates was ∼40%, with a slight decrease over the decade and obvious near-surface seasonality; communities 8–10 years apart were slightly more different than those 1–4 years apart with the highest rate of change at 0–5 m between communities

Little is known about the mechanisms through which intensivist physician staffing influences patient outcomes. We aimed to assess the effect of closed-model intensive care on evidence-based ventilatory practice in patients with acute lung injury (ALI).We conducted a secondary analysis of a prospective population-based cohort of 759 patients with ALI who were alive and ventilated on day three of ALI, and were cared for in 23 intensive care units (ICUs) in King County, Washington.We compared day three tidal volume (VT) in open versus closed ICUs adjusting for potential patient and ICU confounders. In 13 closed model ICUs, 429 (63%) patients were cared for. Adjusted mean VT (mL/Kg predicted body weight (PBW) or measured body weight if height not recorded) for patients in closed ICUs was 1.40 mL/Kg PBW (95% confidence interval (CI) = 0.57 to 2.24 mL/Kg PBW) lower than patients in open model ICUs. Patients in closed ICUs were more likely (odds ratio (OR) = 2.23, 95% CI = 1.09 to 4.56) to receive lower VT (or= 6.5 mL/Kg PBW) and were less likely (OR = 0.30, 95% CI = 0.17 to 0.55) to receive a potentially injurious VT (or= 12 mL/Kg PBW) compared with patients cared for in open ICUs, independent of other covariates. The effect of closed ICUs on hospital mortality was not changed after accounting for delivered VT.Patients with ALI cared for in closed model ICUs are more likely to receive lower VT and less likely to receive higher VT, but there were no other differences in measured processes of care. Moreover, the difference in delivered VT did not completely account for the improved mortality observed in closed model ICUs.