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1.  Niche Partitioning of Marine Group I Crenarchaeota in the Euphotic and Upper Mesopelagic Zones of the East China Sea▿† 
Applied and Environmental Microbiology  2011;77(21):7469-7478.
Marine group I Crenarchaeota (MGI) represents a ubiquitous and numerically predominant microbial population in marine environments. An understanding of the spatial dynamics of MGI and its controlling mechanisms is essential for an understanding of the role of MGI in energy and element cycling in the ocean. In the present study, we investigated the diversity and abundance of MGI in the East China Sea (ECS) by analysis of crenarchaeal 16S rRNA gene, the ammonia monooxygenase gene amoA, and the biotin carboxylase gene accA. Quantitative PCR analyses revealed that these genes were higher in abundance in the mesopelagic than in the euphotic zone. In addition, the crenarchaeal amoA gene was positively correlated with the copy number of the MGI 16S rRNA gene, suggesting that most of the MGI in the ECS are nitrifiers. Furthermore, the ratios of crenarchaeal accA to amoA or to MGI 16S rRNA genes increased from the euphotic to the mesopelagic zone, suggesting that the role of MGI in carbon cycling may change from the epipelagic to the mesopelagic zones. Denaturing gradient gel electrophoretic profiling of the 16S rRNA genes revealed depth partitioning in MGI community structures. Clone libraries of the crenarchaeal amoA and accA genes showed both “shallow” and “deep” groups, and their relative abundances varied in the water column. Ecotype simulation analysis revealed that MGI in the upper ocean could diverge into special ecotypes associated with depth to adapt to the light gradient across the water column. Overall, our results showed niche partitioning of the MGI population and suggested a shift in their ecological functions between the euphotic and mesopelagic zones of the ECS.
doi:10.1128/AEM.00294-11
PMCID: PMC3209141  PMID: 21873485
2.  Resolution of Prochlorococcus and Synechococcus Ecotypes by Using 16S-23S Ribosomal DNA Internal Transcribed Spacer Sequences 
Cultured isolates of the marine cyanobacteria Prochlorococcus and Synechococcus vary widely in their pigment compositions and growth responses to light and nutrients, yet show greater than 96% identity in their 16S ribosomal DNA (rDNA) sequences. In order to better define the genetic variation that accompanies their physiological diversity, sequences for the 16S-23S rDNA internal transcribed spacer (ITS) region were determined in 32 Prochlorococcus isolates and 25 Synechococcus isolates from around the globe. Each strain examined yielded one ITS sequence that contained two tRNA genes. Dramatic variations in the length and G+C content of the spacer were observed among the strains, particularly among Prochlorococcus strains. Secondary-structure models of the ITS were predicted in order to facilitate alignment of the sequences for phylogenetic analyses. The previously observed division of Prochlorococcus into two ecotypes (called high and low-B/A after their differences in chlorophyll content) were supported, as was the subdivision of the high-B/A ecotype into four genetically distinct clades. ITS-based phylogenies partitioned marine cluster A Synechococcus into six clades, three of which can be associated with a particular phenotype (motility, chromatic adaptation, and lack of phycourobilin). The pattern of sequence divergence within and between clades is suggestive of a mode of evolution driven by adaptive sweeps and implies that each clade represents an ecologically distinct population. Furthermore, many of the clades consist of strains isolated from disparate regions of the world's oceans, implying that they are geographically widely distributed. These results provide further evidence that natural populations of Prochlorococcus and Synechococcus consist of multiple coexisting ecotypes, genetically closely related but physiologically distinct, which may vary in relative abundance with changing environmental conditions.
doi:10.1128/AEM.68.3.1180-1191.2002
PMCID: PMC123739  PMID: 11872466
3.  Culture Isolation and Culture-Independent Clone Libraries Reveal New Marine Synechococcus Ecotypes with Distinctive Light and N Physiologies▿  
Applied and Environmental Microbiology  2006;72(11):7193-7204.
Marine microbial communities often contain multiple closely related phylogenetic clades, but in many cases, it is still unclear what physiological traits differentiate these putative ecotypes. The numerically abundant marine cyanobacterium Synechococcus can be divided into at least 14 clades. In order to better understand ecotype differentiation in this genus, we assessed the diversity of a Synechococcus community from a well-mixed water column in the Sargasso Sea during March 2002, a time of year when this genus typically reaches its annual peak in abundance. Diversity was estimated from water sampled at three depths (approximately 5, 70, and 170 m) using both culture isolation and construction of cyanobacterial 16S-23S rRNA internal transcribed sequence clone libraries. Clonal isolates were obtained by enrichment with ammonium, nitrite, or nitrate as the sole N source, followed by pour plating. Each method sampled the in situ diversity differently. The combined methods revealed a total of seven Synechococcus phylotypes including two new putative ecotypes, labeled XV and XVI. Although most other isolates grow on nitrate, clade XV exhibited a reduced efficiency in nitrate utilization, and both clade XV and XVI are capable of chromatic adaptation, demonstrating that this trait is more widely distributed among Synechococcus strains than previously known. Thus, as in its sister genus Prochlorococcus, light and nitrogen utilization are important factors in ecotype differentiation in the marine Synechococcus lineage.
doi:10.1128/AEM.00358-06
PMCID: PMC1636174  PMID: 16936060
4.  Diversity and Distribution of Marine Synechococcus: Multiple Gene Phylogenies for Consensus Classification and Development of qPCR Assays for Sensitive Measurement of Clades in the Ocean 
Marine Synechococcus is a globally significant genus of cyanobacteria that is comprised of multiple genetic lineages or clades. These clades are thought to represent ecologically distinct units, or ecotypes. Because multiple clades often co-occur together in the oceans, Synechococcus are ideal microbes to explore how closely related bacterial taxa within the same functional guild of organisms co-exist and partition marine habitats. Here we sequenced multiple gene loci from cultured strains to confirm the congruency of clade classifications between the 16S–23S rDNA internally transcribed spacer (ITS), 16S rDNA, narB, ntcA, and rpoC1 loci commonly used in Synechococcus diversity studies. We designed quantitative PCR (qPCR) assays that target the ITS for 10 Synechococcus clades, including four clades, XV, XVI, CRD1, and CRD2, not covered by previous assays employing other loci. Our new qPCR assays are very sensitive and specific, detecting down to tens of cells per ml. Application of these qPCR assays to field samples from the northwest Atlantic showed clear shifts in Synechococcus community composition across a coastal to open-ocean transect. Consistent with previous studies, clades I and IV dominated cold, coastal Synechococcus communities. Clades II and X were abundant at the two warmer, off-shore stations, and at all stations multiple Synechococcus clades co-occurred. qPCR assays developed here provide valuable tools to further explore the dynamics of microbial community structure and the mechanisms of co-existence.
doi:10.3389/fmicb.2012.00213
PMCID: PMC3377940  PMID: 22723796
microbial ecology; cyanobacteria; Synechococcus; microbial diversity; quantitative PCR; multiple gene locus phylogeny; biogeography; ecotype
5.  Abundance and Distribution of Ostreococcus sp. in the San Pedro Channel, California, as Revealed by Quantitative PCR†  
Ostreococcus is a genus of widely distributed marine phytoplankton which are picoplanktonic in size (<2 μm) and capable of rapid growth. Although Ostreococcus has been detected around the world, little quantitative information exists on its contribution to planktonic communities. We designed and implemented a genus-specific TaqMan-based quantitative PCR (qPCR) assay to investigate the dynamics and ecology of Ostreococcus at the USC Microbial Observatory (eastern North Pacific). Samples were collected from 5 m and the deep chlorophyll maximum (DCM) between September 2000 and August 2002. Ostreococcus abundance at 5 m was generally <5.0 × 103 cells ml−1, with a maximum of 8.2 × 104 cells ml−1. Ostreococcus abundance was typically higher at the DCM, with a maximum of 3.2 × 105 cells ml−1. The vertical distribution of Ostreococcus was examined in March 2005 and compared to the distribution of phototrophic picoeukaryotes (PPE) measured by flow cytometry. The largest contribution to PPE abundance by Ostreococcus was ∼70% and occurred at 30 m, near the DCM. Despite its relatively low abundance, the depth-integrated standing stock of Ostreococcus in March 2005 was ∼30 mg C m−2. Our work provides a new technique for quantifying the abundance of Ostreococcus and demonstrates the seasonal dynamics of this genus and its contribution to picoeukaryote biomass at our coastal sampling station.
doi:10.1128/AEM.72.4.2496-2506.2006
PMCID: PMC1449040  PMID: 16597949
6.  Groups without Cultured Representatives Dominate Eukaryotic Picophytoplankton in the Oligotrophic South East Pacific Ocean 
PLoS ONE  2009;4(10):e7657.
Background
Photosynthetic picoeukaryotes (PPE) with a cell size less than 3 µm play a critical role in oceanic primary production. In recent years, the composition of marine picoeukaryote communities has been intensively investigated by molecular approaches, but their photosynthetic fraction remains poorly characterized. This is largely because the classical approach that relies on constructing 18S rRNA gene clone libraries from filtered seawater samples using universal eukaryotic primers is heavily biased toward heterotrophs, especially alveolates and stramenopiles, despite the fact that autotrophic cells in general outnumber heterotrophic ones in the euphotic zone.
Methodology/Principal Findings
In order to better assess the composition of the eukaryotic picophytoplankton in the South East Pacific Ocean, encompassing the most oligotrophic oceanic regions on earth, we used a novel approach based on flow cytometry sorting followed by construction of 18S rRNA gene clone libraries. This strategy dramatically increased the recovery of sequences from putative autotrophic groups. The composition of the PPE community appeared highly variable both vertically down the water column and horizontally across the South East Pacific Ocean. In the central gyre, uncultivated lineages dominated: a recently discovered clade of Prasinophyceae (IX), clades of marine Chrysophyceae and Haptophyta, the latter division containing a potentially new class besides Prymnesiophyceae and Pavlophyceae. In contrast, on the edge of the gyre and in the coastal Chilean upwelling, groups with cultivated representatives (Prasinophyceae clade VII and Mamiellales) dominated.
Conclusions/Significance
Our data demonstrate that a very large fraction of the eukaryotic picophytoplankton still escapes cultivation. The use of flow cytometry sorting should prove very useful to better characterize specific plankton populations by molecular approaches such as gene cloning or metagenomics, and also to obtain into culture strains representative of these novel groups.
doi:10.1371/journal.pone.0007657
PMCID: PMC2764088  PMID: 19893617
7.  Novel lineages of Prochlorococcus and Synechococcus in the global oceans 
The ISME Journal  2011;6(2):285-297.
Picocyanobacteria represented by Prochlorococcus and Synechococcus have an important role in oceanic carbon fixation and nutrient cycling. In this study, we compared the community composition of picocyanobacteria from diverse marine ecosystems ranging from estuary to open oceans, tropical to polar oceans and surface to deep water, based on the sequences of 16S-23S rRNA internal transcribed spacer (ITS). A total of 1339 ITS sequences recovered from 20 samples unveiled diverse and several previously unknown clades of Prochlorococcus and Synechococcus. Six high-light (HL)-adapted Prochlorococcus clades were identified, among which clade HLVI had not been described previously. Prochlorococcus clades HLIII, HLIV and HLV, detected in the Equatorial Pacific samples, could be related to the HNLC clades recently found in the high-nutrient, low-chlorophyll (HNLC), iron-depleted tropical oceans. At least four novel Synechococcus clades (out of six clades in total) in subcluster 5.3 were found in subtropical open oceans and the South China Sea. A niche partitioning with depth was observed in the Synechococcus subcluster 5.3. Members of Synechococcus subcluster 5.2 were dominant in the high-latitude waters (northern Bering Sea and Chukchi Sea), suggesting a possible cold-adaptation of some marine Synechococcus in this subcluster. A distinct shift of the picocyanobacterial community was observed from the Bering Sea to the Chukchi Sea, which reflected the change of water temperature. Our study demonstrates that oceanic systems contain a large pool of diverse picocyanobacteria, and further suggest that new genotypes or ecotypes of picocyanobacteria will continue to emerge, as microbial consortia are explored with advanced sequencing technology.
doi:10.1038/ismej.2011.106
PMCID: PMC3260499  PMID: 21955990
cyanobacteria; Prochlorococcus; Synechococcus; diversity; global ocean; 16S-23S rRNA ITS
8.  Temporal variability and coherence of euphotic zone bacterial communities over a decade in the Southern California Bight 
The ISME Journal  2013;7(12):2259-2273.
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 <4 years apart. The surface exhibited more pronounced seasonality than the DCM. Inter-depth Bray-Curtis similarities repeatedly decreased as the water column stratified each summer. Environmental factors were better predictors of shifts in community composition than months or elapsed time alone; yet, the best predictor was community composition at the other depth (that is, 0–5 m versus DCM).
doi:10.1038/ismej.2013.122
PMCID: PMC3834854  PMID: 23864126
marine bacterioplankton; ARISA; community ecology; microbe–microbe interactions; time series
9.  Relationship between Abundance and Specific Activity of Bacterioplankton in Open Ocean Surface Waters 
Marine microbial communities are complex and dynamic, and their ecology impacts biogeochemical cycles in pelagic ecosystems. Yet, little is known about the relative activities of different microbial populations within genetically diverse communities. We used rRNA as a proxy for activity to quantify the relative specific activities (rRNA/ribosomal DNA [rDNA or rRNA genes]) of the eubacterial populations and to identify locations or clades for which there are uncouplings between specific activity and abundance. After analyzing 1.6 million sequences from 16S rDNA and rRNA (cDNA) libraries from two euphotic depths from a representative site in the Pacific Ocean, we show that although there is an overall positive relationship between the abundances (rDNAs) and activities (rRNAs) among populations of the bacterial community, for some populations these measures are uncoupled. Different ecological strategies are exemplified by the two numerically dominant clades at this site: the cyanobacterium Prochlorococcus is abundant but disproportionately more active, while the heterotrophic SAR11 is abundant but less active. Other rare populations, such as Alteromonas, have high specific activities in spite of their low abundances, suggesting intense population regulation. More detailed analyses using a complementary quantitative PCR (qPCR)-based approach of measuring relative specific activity for Prochlorococcus populations in the Pacific and Atlantic Oceans also show that specific activity, but not abundance, reflects the key drivers of light and nutrients in this system; our results also suggest substantial top-down regulation (e.g., grazing, viruses, or organismal interactions) or transport (e.g., mixing, immigration, or emigration) of these populations. Thus, we show here that abundance and specific activity can be uncoupled in open ocean systems and that describing both is critical to characterizing microbial communities and predicting marine ecosystem functioning and responses to change.
doi:10.1128/AEM.02155-12
PMCID: PMC3536108  PMID: 23087033
10.  High-resolution SAR11 ecotype dynamics at the Bermuda Atlantic Time-series Study site by phylogenetic placement of pyrosequences 
The ISME Journal  2013;7(7):1322-1332.
Advances in next-generation sequencing technologies are providing longer nucleotide sequence reads that contain more information about phylogenetic relationships. We sought to use this information to understand the evolution and ecology of bacterioplankton at our long-term study site in the Western Sargasso Sea. A bioinformatics pipeline called PhyloAssigner was developed to align pyrosequencing reads to a reference multiple sequence alignment of 16S ribosomal RNA (rRNA) genes and assign them phylogenetic positions in a reference tree using a maximum likelihood algorithm. Here, we used this pipeline to investigate the ecologically important SAR11 clade of Alphaproteobacteria. A combined set of 2.7 million pyrosequencing reads from the 16S rRNA V1–V2 regions, representing 9 years at the Bermuda Atlantic Time-series Study (BATS) site, was quality checked and parsed into a comprehensive bacterial tree, yielding 929 036 Alphaproteobacteria reads. Phylogenetic structure within the SAR11 clade was linked to seasonally recurring spatiotemporal patterns. This analysis resolved four new SAR11 ecotypes in addition to five others that had been described previously at BATS. The data support a conclusion reached previously that the SAR11 clade diversified by subdivision of niche space in the ocean water column, but the new data reveal a more complex pattern in which deep branches of the clade diversified repeatedly across depth strata and seasonal regimes. The new data also revealed the presence of an unrecognized clade of Alphaproteobacteria, here named SMA-1 (Sargasso Mesopelagic Alphaproteobacteria, group 1), in the upper mesopelagic zone. The high-resolution phylogenetic analyses performed herein highlight significant, previously unknown, patterns of evolutionary diversification, within perhaps the most widely distributed heterotrophic marine bacterial clade, and strongly links to ecosystem regimes.
doi:10.1038/ismej.2013.32
PMCID: PMC3695298  PMID: 23466704
BATS; ecotype; pyrosequencing; SAR11
11.  Isolation of Prasinoviruses of the Green Unicellular Algae Ostreococcus spp. on a Worldwide Geographical Scale▿ † 
Ostreococcus spp. are extremely small unicellular eukaryotic green algae found worldwide in marine environments, and they are susceptible to attacks by a diverse group of large DNA viruses. Several biologically distinct species of Ostreococcus are known and differ in the ecological niches that they occupy: while O. tauri (representing clade C strains) is found in marine lagoons and coastal seas, strains belonging to clade A, exemplified by O. lucimarinus, are present in different oceans. We used laboratory cultures of clonal isolates of these two species to assay for the presence of viruses in seawater samples from diverse locations. In keeping with the distributions of their host strains, we found a decline in the abundance of O. tauri viruses from a lagoon in southwest France relative to the Mediterranean Sea, whereas in the ocean, no O. tauri viruses were detected. In contrast, viruses infecting O. lucimarinus were detected from distantly separated oceans. DNA sequencing, phylogenetic analyses using a conserved viral marker gene, and a Mantel test revealed no relationship between geographic and phylogenetic distances in viruses infecting O. lucimarinus.
doi:10.1128/AEM.01799-09
PMCID: PMC2798645  PMID: 19897754
12.  Diversity of picoeukaryotes at an oligotrophic site off the Northeastern Red Sea Coast 
Aquatic Biosystems  2013;9:16.
Background
Picoeukaryotes are protists ≤ 3 μm composed of a wide diversity of taxonomic groups. They are an important constituent of the ocean’s microbiota and perform essential ecological roles in marine nutrient and carbon cycles. Despite their importance, the true extent of their diversity has only recently been uncovered by molecular surveys that resulted in the discovery of a substantial number of previously unknown groups. No study on picoeukaryote diversity has been conducted so far in the main Red Sea basin-a unique marine environment characterized by oligotrophic conditions, high levels of irradiance, high salinity and increased water temperature.
Results
We sampled surface waters off the coast of the northeastern Red Sea and analyzed the picoeukaryotic diversity using Sanger-based clone libraries of the 18S rRNA gene in order to produce high quality, nearly full-length sequences. The community captured by our approach was dominated by three main phyla, the alveolates, stramenopiles and chlorophytes; members of Radiolaria, Cercozoa and Haptophyta were also found, albeit in low abundances. Photosynthetic organisms were especially diverse and abundant in the sample, confirming the importance of picophytoplankton for primary production in the basin as well as indicating the existence of numerous ecological micro-niches for this trophic level in the upper euphotic zone. Heterotrophic organisms were mostly composed of the presumably parasitic Marine Alveolates (MALV) and the presumably bacterivorous Marine Stramenopiles (MAST) groups. A small number of sequences that did not cluster closely with known clades were also found, especially in the MALV-II group, some of which could potentially belong to novel clades.
Conclusions
This study provides the first snapshot of the picoeukaryotic diversity present in surface waters of the Red Sea, hence setting the stage for large-scale surveying and characterization of the eukaryotic diversity in the entire basin. Our results indicate that the picoeukaryotic community in the northern Red Sea, despite its unique physiochemical conditions (i.e. increased temperatures, increased salinity, and high UV irradiance) does not differ vastly from its counterparts in other oligotrophic marine habitats.
doi:10.1186/2046-9063-9-16
PMCID: PMC3766091  PMID: 23962380
Picoeukaryotes; Red sea; Protists; SSU rRNA; Microbial diversity
13.  Seasonal dynamics of active SAR11 ecotypes in the oligotrophic Northwest Mediterranean Sea 
The ISME Journal  2014;9(2):347-360.
A seven-year oceanographic time series in NW Mediterranean surface waters was combined with pyrosequencing of ribosomal RNA (16S rRNA) and ribosomal RNA gene copies (16S rDNA) to examine the environmental controls on SAR11 ecotype dynamics and potential activity. SAR11 diversity exhibited pronounced seasonal cycles remarkably similar to total bacterial diversity. The timing of diversity maxima was similar across narrow and broad phylogenetic clades and strongly associated with deep winter mixing. Diversity minima were associated with periods of stratification that were low in nutrients and phytoplankton biomass and characterised by intense phosphate limitation (turnover time<5 h). We propose a conceptual framework in which physical mixing of the water column periodically resets SAR11 communities to a high diversity state and the seasonal evolution of phosphate limitation competitively excludes deeper-dwelling ecotypes to promote low diversity states dominated (>80%) by SAR11 Ia. A partial least squares (PLS) regression model was developed that could reliably predict sequence abundances of SAR11 ecotypes (Q2=0.70) from measured environmental variables, of which mixed layer depth was quantitatively the most important. Comparison of clade-level SAR11 rRNA:rDNA signals with leucine incorporation enabled us to partially validate the use of these ratios as an in-situ activity measure. However, temporal trends in the activity of SAR11 ecotypes and their relationship to environmental variables were unclear. The strong and predictable temporal patterns observed in SAR11 sequence abundance was not linked to metabolic activity of different ecotypes at the phylogenetic and temporal resolution of our study.
doi:10.1038/ismej.2014.129
PMCID: PMC4303628  PMID: 25238399
14.  Plastid 16S rRNA Gene Diversity among Eukaryotic Picophytoplankton Sorted by Flow Cytometry from the South Pacific Ocean 
PLoS ONE  2011;6(4):e18979.
The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX), which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.
doi:10.1371/journal.pone.0018979
PMCID: PMC3084246  PMID: 21552558
15.  A novel clade of Prochlorococcus found in high nutrient low chlorophyll waters in the South and Equatorial Pacific Ocean 
The ISME journal  2010;5(6):933-944.
A novel high-light (HL)-adapted Prochlorococcus clade was discovered in high nutrient and low chlorophyll (HNLC) waters in the South Pacific Ocean by phylogenetic analyses of 16S ribosomal RNA (rRNA) and 16S–23S internal transcribed spacer (ITS) sequences. This clade, named HNLC fell within the HL-adapted Prochlorococcus clade with sequences above 99% similarity to one another, and was divided into two subclades, HNLC1 and HNLC2. The distribution of the whole HNLC clade in a northwest to southeast transect in the South Pacific (HNLC-to-gyre) and two 8°N to 8°S transects in the Equatorial Pacific was determined by quantitative PCR using specific primers targeting ITS regions. HNLC was the dominant HL Prochlorococcus clade (2–9% of bacterial 16S rRNA genes) at the three westernmost stations in the South Pacific but decreased to less than 0.1% at the other stations being replaced by the eMIT9312 ecotype in the hyperoligotrophic gyre. The highest contributions of HNLC Prochlorococcus in both Equatorial Pacific transects along the latitudinal lines of 170°W and 155°W were observed at the southernmost stations, reaching 16 and 6% of bacterial 16S rRNA genes, respectively, whereas eMIT9312 dominated near the Equator. Spearman Rank Order correlation analysis indicated that although both the HNLC clade and eMIT9312 were correlated with temperature, they showed different correlations with regard to nutrients. HNLC only showed significant correlations to ammonium uptake and regeneration rates, whereas eMIT9312 was negatively correlated with inorganic nutrients.
doi:10.1038/ismej.2010.186
PMCID: PMC3131852  PMID: 21124492
16S rRNA; Equatorial Pacific; HNLC; ITS; Prochlorococcus; qPCR
16.  Distribution Patterns and Phylogeny of Marine Stramenopiles in the North Pacific Ocean 
Marine stramenopiles (MASTs) are a diverse suite of eukaryotic microbes found in marine environments. Several MAST lineages are thought to contain heterotrophic nanoflagellates. However, MASTs remain uncultured and data on distributions and trophic modes are limited. We investigated MASTs in provinces on the west and east sides of the North Pacific Subtropical Gyre, specifically the East China Sea (ECS) and the California Current system (CALC). For each province, DNA was sampled from three zones: coastal, mesotrophic transitional, and more oligotrophic euphotic waters. Along with diatoms, chrysophytes, and other stramenopiles, sequences were recovered from nine MAST lineages in the six ECS and four CALC 18S rRNA gene clone libraries. All but one of these libraries were from surface samples. MAST clusters 1, 3, 7, 8, and 11 were identified in both provinces, with MAST cluster 3 (MAST-3) being found the most frequently. Additionally, MAST-2 was detected in the ECS and MAST-4, -9, and -12 were detected in the CALC. Phylogenetic analysis indicated that some subclades within these lineages differ along latitudinal gradients. MAST-1A, -1B, and -1C and MAST-4 size and abundance estimates obtained using fluorescence in situ hybridization on 79 spring and summer ECS samples showed a negative correlation between size of MAST-1B and MAST-4 cells and temperature. MAST-1A was rarely detected, but MAST-1B and -1C and MAST-4 were abundant in summer and MAST-1C and MAST-4 were more so at the coast, with maximum abundances of 543 and 1,896 cells ml−1, respectively. MAST-4 and Synechococcus abundances were correlated, and experimental work showed that MAST-4 ingests Synechococcus. Together with previous studies, this study helps refine hypotheses on distribution and trophic modes of MAST lineages.
doi:10.1128/AEM.06952-11
PMCID: PMC3346489  PMID: 22344659
17.  Diversity and Abundance of Bolidophyceae (Heterokonta) in Two Oceanic Regions 
Applied and Environmental Microbiology  1999;65(10):4528-4536.
The diversity and abundance of the Bolidophyceae (Heterokonta), a newly described picoplanktonic algal class which is a sister group to the diatoms, was assessed in the equatorial Pacific Ocean and in the Mediterranean Sea by culture isolation, molecular biology techniques, and pigment analyses. Eight strains of Bolidophyceae were isolated in culture from different mesotrophic and oligotrophic areas. The corresponding small subunit (SSU) rRNA gene sequences allowed us to design two probes specific for the Bolidophyceae. These probes have been used in natural samples (i) to selectively amplify and detect Bolidophyceae sequences and (ii) to quantify the relative abundance of Bolidophyceae within the picoeukaryote community. Sequences available to date indicate that the class Bolidophyceae comprises at least three different clades, two corresponding to the previously described species Bolidomonas pacifica and Bolidomonas mediterranea and the third one corresponding to a subspecies of B. pacifica. Amplification of the SSU rRNA gene from natural samples with universal primers and hybridization using a Bolidomonas-specific probe followed by a eukaryote-specific probe allowed us to estimate the contribution of the Bolidophyceae to the eukaryotic DNA in both Pacific and Mediterranean waters to be lower than 1%. Similarly, high-performance liquid chromatography analyses of fucoxanthin, the major carotenoid present in Bolidophyceae, indicated that less than 4% of the total chlorophyll a in the picoplanktonic fraction in the equatorial Pacific was due to Bolidophyceae. Consequently, although strains of Bolidophyceae have been isolated from samples collected at several stations, this new class seems to have been a minor component of the natural picoeukaryotic populations in the ecosystems investigated, at least during the periods sampled.
PMCID: PMC91603  PMID: 10508085
18.  Long Term Seasonal Dynamics of Synechococcus Population Structure in the Gulf of Aqaba, Northern Red Sea 
Spatial patterns of marine Synechococcus diversity across ocean domains have been reported on extensively. However, much less is known of seasonal and multiannual patterns of change in Synechococcus community composition. Here we report on the genotypic diversity of Synechococcus populations in the Gulf of Aqaba, Northern Red Sea, over seven annual cycles of deep mixing and stabile stratification, using ntcA as a phylogenetic marker. Synechococcus clone libraries were dominated by clade II and XII genotypes and a total of eight different clades were identified. Inclusion of ntcA sequences from the Global Ocean Sampling database in our analyses identified members of clade XII from beyond the Gulf of Aqaba, extending its known distribution. Most of the Synechococcus diversity was attributed to members of clade II during the spring bloom, while clade III contributed significantly to diversity during summer stratification. Clade XII diversity was most prevalent in fall and winter. Clade abundances were estimated from pyrosequencing of the V6 hypervariable region of 16S rRNA. Members of clade II dominated Synechococcus communities throughout the year, whereas the less frequent genotypes showed a pattern of seasonal succession. Based on the prevailing nutritional conditions we observed that clade I members thrive at higher nutrient concentrations during winter mixing. Clades V, VI and X became apparent during the transition periods between mixing and stratification. Clade III became prominent during sumeer stratification. We propose that members of clades V, VI, and X, and clade III are Synechococcus ecotypes that are adapted to intermediate and low nutrient levels respectively. This is the first time that molecular analyses have correlated population dynamics of Synechococcus genotypes with temporal fluctuations in nutrient regimes. Since these Synechococcus genotypes are routinely observed in the Gulf of Aqaba we suggest that seasonal fluctuations in nutrient levels create temporal niches that sustain their coexistence.
doi:10.3389/fmicb.2011.00131
PMCID: PMC3122069  PMID: 21734910
marine cyanobacteria; Synechococcus; succession; diversity; ecotype
19.  Streamlining and Core Genome Conservation among Highly Divergent Members of the SAR11 Clade 
mBio  2012;3(5):e00252-12.
ABSTRACT
SAR11 is an ancient and diverse clade of heterotrophic bacteria that are abundant throughout the world’s oceans, where they play a major role in the ocean carbon cycle. Correlations between the phylogenetic branching order and spatiotemporal patterns in cell distributions from planktonic ocean environments indicate that SAR11 has evolved into perhaps a dozen or more specialized ecotypes that span evolutionary distances equivalent to a bacterial order. We isolated and sequenced genomes from diverse SAR11 cultures that represent three major lineages and encompass the full breadth of the clade. The new data expand observations about genome evolution and gene content that previously had been restricted to the SAR11 Ia subclade, providing a much broader perspective on the clade’s origins, evolution, and ecology. We found small genomes throughout the clade and a very high proportion of core genome genes (48 to 56%), indicating that small genome size is probably an ancestral characteristic. In their level of core genome conservation, the members of SAR11 are outliers, the most conserved free-living bacteria known. Shared features of the clade include low GC content, high gene synteny, a large hypervariable region bounded by rRNA genes, and low numbers of paralogs. Variation among the genomes included genes for phosphorus metabolism, glycolysis, and C1 metabolism, suggesting that adaptive specialization in nutrient resource utilization is important to niche partitioning and ecotype divergence within the clade. These data provide support for the conclusion that streamlining selection for efficient cell replication in the planktonic habitat has occurred throughout the evolution and diversification of this clade.
IMPORTANCE
The SAR11 clade is the most abundant group of marine microorganisms worldwide, making them key players in the global carbon cycle. Growing knowledge about their biochemistry and metabolism is leading to a more mechanistic understanding of organic carbon oxidation and sequestration in the oceans. The discovery of small genomes in SAR11 provided crucial support for the theory that streamlining selection can drive genome reduction in low-nutrient environments. Study of isolates in culture revealed atypical organic nutrient requirements that can be attributed to genome reduction, such as conditional auxotrophy for glycine and its precursors, a requirement for reduced sulfur compounds, and evidence for widespread cycling of C1 compounds in marine environments. However, understanding the genetic variation and distribution of such pathways and characteristics like streamlining throughout the group has required the isolation and genome sequencing of diverse SAR11 representatives, an analysis of which we provide here.
doi:10.1128/mBio.00252-12
PMCID: PMC3448164  PMID: 22991429
20.  Genome Sequence of Ostreococcus tauri Virus OtV-2 Throws Light on the Role of Picoeukaryote Niche Separation in the Ocean▿ 
Journal of Virology  2011;85(9):4520-4529.
Ostreococcus tauri, a unicellular marine green alga, is the smallest known free-living eukaryote and is ubiquitous in the surface oceans. The ecological success of this organism has been attributed to distinct low- and high-light-adapted ecotypes existing in different niches at a range of depths in the ocean. Viruses have already been characterized that infect the high-light-adapted strains. Ostreococcus tauri virus (OtV) isolate OtV-2 is a large double-stranded DNA algal virus that infects a low-light-adapted strain of O. tauri and was assigned to the algal virus family Phycodnaviridae, genus Prasinovirus. Our working hypothesis for this study was that different viruses infecting high- versus low-light-adapted O. tauri strains would provide clues to propagation strategies that would give them selective advantages within their particular light niche. Sequence analysis of the 184,409-bp linear OtV-2 genome revealed a range of core functional genes exclusive to this low-light genotype and included a variety of unexpected genes, such as those encoding an RNA polymerase sigma factor, at least four DNA methyltransferases, a cytochrome b5, and a high-affinity phosphate transporter. It is clear that OtV-2 has acquired a range of potentially functional genes from its host, other eukaryotes, and even bacteria over evolutionary time. Such piecemeal accretion of genes is a trademark of large double-stranded DNA viruses that has allowed them to adapt their propagation strategies to keep up with host niche separation in the sunlit layers of the oceanic environment.
doi:10.1128/JVI.02131-10
PMCID: PMC3126241  PMID: 21289127
21.  Differential Distributions of Synechococcus Subgroups Across the California Current System 
Synechococcus is an abundant marine cyanobacterial genus composed of different populations that vary physiologically. Synechococcus narB gene sequences (encoding for nitrate reductase in cyanobacteria) obtained previously from isolates and the environment (e.g., North Pacific Gyre Station ALOHA, Hawaii or Monterey Bay, CA, USA) were used to develop quantitative PCR (qPCR) assays. These qPCR assays were used to quantify populations from specific narB phylogenetic clades across the California Current System (CCS), a region composed of dynamic zones between a coastal-upwelling zone and the oligotrophic Pacific Ocean. Targeted populations (narB subgroups) had different biogeographic patterns across the CCS, which appear to be driven by environmental conditions. Subgroups C_C1, D_C1, and D_C2 were abundant in coastal-upwelling to coastal-transition zone waters with relatively high to intermediate ammonium, nitrate, and chl. a concentrations. Subgroups A_C1 and F_C1 were most abundant in coastal-transition zone waters with intermediate nutrient concentrations. E_O1 and G_O1 were most abundant at different depths of oligotrophic open-ocean waters (either in the upper mixed layer or just below). E_O1, A_C1, and F_C1 distributions differed from other narB subgroups and likely possess unique ecologies enabling them to be most abundant in waters between coastal and open-ocean waters. Different CCS zones possessed distinct Synechococcus communities. Core California current water possessed low numbers of narB subgroups relative to counted Synechococcus cells, and coastal-transition waters contained high abundances of Synechococcus cells and total number of narB subgroups. The presented biogeographic data provides insight on the distributions and ecologies of Synechococcus present in an eastern boundary current system.
doi:10.3389/fmicb.2011.00059
PMCID: PMC3153035  PMID: 21833315
Synechococcus; picocyanobacteria; biogeography; CCS; eastern-Pacific; qPCR; narB
22.  Spatial Distribution and Transcriptional Activity of an Uncultured Clade of Planktonic Diazotrophic γ-Proteobacteria in the Arabian Sea 
The spatial distribution of an uncultured clade of marine diazotrophic γ-proteobacteria in the Arabian Sea was investigated by the development of a specific primer pair to amplify an internal fragment of nifH by PCR. These organisms were most readily detected in highly oligotrophic surface waters but could also be found in deeper waters below the nutricline. nifH transcripts originating from this clade were detected in oligotrophic surface waters and, in addition, in the deeper and the more productive near-coastal waters. The nifH sequences most closely related to the unidentified marine bacterial group are from environmental clones amplified from the Atlantic and Pacific Oceans. These findings suggest that these γ-proteobacteria are widespread and likely to be an important component of the heterotrophic diazotrophic microbial community of the tropical and subtropical oceans.
doi:10.1128/AEM.71.4.2079-2085.2005
PMCID: PMC1082540  PMID: 15812041
23.  Ocean time-series reveals recurring seasonal patterns of virioplankton dynamics in the northwestern Sargasso Sea 
The ISME Journal  2011;6(2):273-284.
There are an estimated 1030 virioplankton in the world oceans, the majority of which are phages (viruses that infect bacteria). Marine phages encompass enormous genetic diversity, affect biogeochemical cycling of elements, and partially control aspects of prokaryotic production and diversity. Despite their importance, there is a paucity of data describing virioplankton distributions over time and depth in oceanic systems. A decade of high-resolution time-series data collected from the upper 300 m in the northwestern Sargasso Sea revealed recurring temporal and vertical patterns of virioplankton abundance in unprecedented detail. An annual virioplankton maximum developed between 60 and 100 m during periods of summer stratification and eroded during winter convective mixing. The timing and vertical positioning of this seasonal pattern was related to variability in water column stability and the dynamics of specific picophytoplankton and heterotrophic bacterioplankton lineages. Between 60 and 100 m, virioplankton abundance was negatively correlated to the dominant heterotrophic bacterioplankton lineage SAR11, as well as the less abundant picophytoplankton, Synechococcus. In contrast, virioplankton abundance was positively correlated to the dominant picophytoplankton lineage Prochlorococcus, and the less abundant alpha-proteobacteria, Rhodobacteraceae. Seasonally, virioplankton abundances were highly synchronous with Prochlorococcus distributions and the virioplankton to Prochlorococcus ratio remained remarkably constant during periods of water column stratification. The data suggest that a significant fraction of viruses in the mid-euphotic zone of the subtropical gyres may be cyanophages and patterns in their abundance are largely determined by Prochlorococcus dynamics in response to water column stability. This high-resolution, decadal survey of virioplankton abundance provides insight into the possible controls of virioplankton dynamics in the open ocean.
doi:10.1038/ismej.2011.101
PMCID: PMC3260494  PMID: 21833038
phage; BATS; FISH; Prochlorococcus; SAR11; Sargasso
24.  Transcriptome sequencing of two wild barley (Hordeum spontaneum L.) ecotypes differentially adapted to drought stress reveals ecotype-specific transcripts 
BMC Genomics  2014;15(1):995.
Background
Wild barley is adapted to highly diverse environments throughout its geographical distribution range. Transcriptome sequencing of differentially adapted wild barley ecotypes from contrasting environments contributes to the identification of genes and genetic variation involved in abiotic stress tolerance and adaptation.
Results
Two differentially adapted wild barley ecotypes from desert (B1K2) and Mediterranean (B1K30) environments were analyzed for drought stress response under controlled conditions. The desert ecotype lost more water under both irrigation and drought, but exhibited higher relative water content (RWC) and better water use efficiency (WUE) than the coastal ecotype. We sequenced normalized cDNA libraries from drought-stressed leaves of both ecotypes with the 454 platform to identify drought-related transcripts. Over half million reads per ecotype were de novo assembled into 20,439 putative unique transcripts (PUTs) for B1K2, 21,494 for B1K30 and 28,720 for the joint assembly. Over 50% of PUTs of each ecotype were not shared with the other ecotype. Furthermore, 16% (3,245) of B1K2 and 17% (3,674) of B1K30 transcripts did not show orthologous sequence hits in the other wild barley ecotype and cultivated barley, and are candidates of ecotype-specific transcripts. Over 800 unique transcripts from each ecotype homologous to over 30 different stress-related genes were identified. We extracted 1,017 high quality SNPs that differentiated the two ecotypes. The genetic distance between the desert ecotype and cultivated barley was 1.9-fold higher than between the Mediterranean ecotype and cultivated barley. Moreover, the desert ecotype harbored a larger proportion of non-synonymous SNPs than the Mediterranean ecotype suggesting different demographic histories of these ecotypes.
Conclusions
The results indicate a strong physiological and genomic differentiation between the desert and Mediterranean wild barley ecotypes and a closer relationship of the Mediterranean to cultivated barley. A significant number of novel transcripts specific to wild barley were identified. The higher SNP density and larger proportion of SNPs with functional effects in the desert ecotype suggest different demographic histories and effects of natural selection in Mediterranean and desert wild barley. The data are a valuable genomic resource for an improved genome annotation, transcriptome studies of drought adaptation and a source of new genetic markers for future barley improvement.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-995) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-995
PMCID: PMC4251939  PMID: 25408241
Hordeum spontaneum; Transcriptome; Drought tolerance; Genetic diversity
25.  Dissolved Phosphorus Pools and Alkaline Phosphatase Activity in the Euphotic Zone of the Western North Pacific Ocean 
We measured pools of dissolved phosphorus (P), including dissolved inorganic P (DIP), dissolved organic P (DOP) and alkaline phosphatase (AP)-hydrolyzable labile DOP (L-DOP), and kinetic parameters of AP activity (APA) in the euphotic zone in the western North Pacific Ocean. Samples were collected from one coastal station in Sagami Bay, Japan, and three offshore stations between the North Pacific subtropical gyre (NPSG) and the Kuroshio region. Although DIP concentrations in the euphotic zone at all stations were equally low, around the nominal method detection limit of 20 nmol L-1, chlorophyll a (Chl a) concentrations were one order of magnitude greater at the coastal station. DOP was the dominant P pool, comprising 62–92% of total dissolved P at and above the Chl a maximum layer (CML). L-DOP represented 22–39% of the total DOP at the offshore stations, whereas it accounted for a much higher proportion (about 85%) in the coastal surface layers. Significant correlations between maximum potential AP hydrolysis rates and DIP concentrations or bacterial cell abundance in the offshore euphotic zone suggest that major APA in the oligotrophic surface ocean is from bacterial activity and regulated largely by DIP availability. Although the range of maximum potential APA was comparable among the environmental conditions, the in situ hydrolysis rate of L-DOP in the coastal station was 10 times those in the offshore stations. L-DOP turnover time at the CML ranged from 4.5 days at the coastal station to 84.4 days in the NPSG. The ratio of the APA half-saturation constant to the ambient L-DOP concentration decreased markedly from the NPSG to the coastal station. There were substantial differences in the rate and efficiency of DOP remineralization and its contribution as the potential P source between the low-phosphate/high-biomass coastal ecosystem and the low-phosphate/low biomass oligotrophic ocean.
doi:10.3389/fmicb.2012.00099
PMCID: PMC3307022  PMID: 22457661
phosphorus; alkaline phosphatase activity; dissolved organic phosphorus; dissolved inorganic phosphorus; north Pacific Ocean; euphotic zone

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