Unexpected biodiversity of ciliates in marine samples from below the photic zone

Marine microbial eukaryotes play critical roles in planktonic food webs and have been described as most diverse in the photic zone where productivity is high. We used high‐throughput sequencing (HTS) to analyse the spatial distribution of planktonic ciliate diversity from shallow waters (<30 m depth) to beyond the continental shelf (>800 m depth) along a 163 km transect off the coast of New England, USA. We focus on ciliates in the subclasses Oligotrichia and Choreotrichia (class Spirotrichea), as these taxa are major components of marine food webs. We did not observe the decrease of diversity below the photic zone expected based on productivity and previous analyses. Instead, we saw an increase of diversity with depth. We also observed that the ciliate communities assessed by HTS cluster by depth layer and degree of water column stratification, suggesting that community assembly is driven by environmental factors. Across our samples, abundant OTUs tend to match previously characterized morphospecies while rare OTUs are more often undescribed, consistent with the idea that species in the rare biosphere remain to be characterized by microscopy. Finally, samples taken below the photic zone also reveal the prevalence of two uncharacterized (i.e. lacking sequenced morphospecies) clades – clusters X₁ and X₂ – that are enriched within the nano‐sized fraction (2–10 μm) and are defined by deletions within the region of the SSU‐rDNA analysed here. Together, these data reinforce that we still have much to learn about microbial diversity in marine ecosystems, especially in deep‐waters that may be a reservoir for rare species and uncharacterized taxa.     

Distinct protistan assemblages characterize the euphotic zone and deep sea (2500 m) of the western North Atlantic (Sargasso Sea and Gulf Stream)

Protistan diversity was characterized at three locations in the western North Atlantic (Sargasso Sea and Gulf Stream) by sequencing 18S rRNA genes in samples from euphotic (< or = 125 m) and bathypelagic depths (2500 m). A total of 923 partial-length protistan sequences were analysed, revealing 324 distinct operational taxonomic units (OTUs) determined by an automated OTU-calling program set to 95% sequence similarity. Most OTUs were comprised of only one or two sequences suggesting a large but rare pool of protistan diversity. Many OTUs from both depth strata were associated with recently described novel alveolate and stramenopile lineages while many OTUs from the bathypelagic were affiliated with Acantharea, Polycystinea and Euglenozoa and were not observed in euphotic zone libraries. Protistan assemblages from the euphotic zone and the deep sea were largely composed of distinct OTUs; only 28 of the 324 protistan OTUs were detected in both shallow and deep sea clone libraries. The diversity of protistan assemblages in the deep sea was distinctly lower than the diversity of euphotic zone assemblages. Protistan assemblages from the Gulf Stream were the most diverse for either depth strata. Overall, protistan assemblages from different stations but comparable depths were more similar than the assemblages from different depths at the same station. These data suggest that particular groups of protistan OTUs formed distinct 'shallow' and 'deep-sea' assemblages across widely spaced oceanic locales.     

Vertical gradients in species richness and community composition across the twilight zone in the North Pacific Subtropical Gyre

Although metazoan animals in the mesopelagic zone play critical roles in deep pelagic food webs and in the attenuation of carbon in midwaters, the diversity of these assemblages is not fully known. A metabarcoding survey of mesozooplankton diversity across the epipelagic, mesopelagic and upper bathypelagic zones (0–1500 m) in the North Pacific Subtropical Gyre revealed far higher estimates of species richness than expected given prior morphology‐based studies in the region (4,024 OTUs, 10‐fold increase), despite conservative bioinformatic processing. Operational taxonomic unit (OTU) richness of the full assemblage peaked at lower epipelagic–upper mesopelagic depths (100–300 m), with slight shoaling of maximal richness at night due to diel vertical migration, in contrast to expectations of a deep mesopelagic diversity maximum as reported for several plankton groups in early systematic and zoogeographic studies. Four distinct depth‐stratified species assemblages were identified, with faunal transitions occurring at 100 m, 300 m and 500 m. Highest diversity occurred in the smallest zooplankton size fractions (0.2–0.5 mm), which had significantly lower % OTUs classified due to poor representation in reference databases, suggesting a deep reservoir of poorly understood diversity in the smallest metazoan animals. A diverse meroplankton assemblage also was detected (350 OTUs), including larvae of both shallow and deep living benthic species. Our results provide some of the first insights into the hidden diversity present in zooplankton assemblages in midwaters, and a molecular reappraisal of vertical gradients in species richness, depth distributions and community composition for the full zooplankton assemblage across the epipelagic, mesopelagic and upper bathypelagic zones.     

Spatial Diversity of Bacterioplankton Communities in Surface Water of Northern South China Sea

The South China Sea is one of the largest marginal seas, with relatively frequent passage of eddies and featuring distinct spatial variation in the western tropical Pacific Ocean. Here, we report a phylogenetic study of bacterial community structures in surface seawater of the northern South China Sea (nSCS). Samples collected from 31 sites across large environmental gradients were used to construct clone libraries and yielded 2,443 sequences grouped into 170 OTUs. Phylogenetic analysis revealed 23 bacterial classes with major components α-, β- and γ-Proteobacteria, as well as Cyanobacteria. At class and genus taxon levels, community structure of coastal waters was distinctively different from that of deep-sea waters and displayed a higher diversity index. Redundancy analyses revealed that bacterial community structures displayed a significant correlation with the water depth of individual sampling sites. Members of α-Proteobacteria were the principal component contributing to the differences of the clone libraries. Furthermore, the bacterial communities exhibited heterogeneity within zones of upwelling and anticyclonic eddies. Our results suggested that surface bacterial communities in nSCS had two-level patterns of spatial distribution structured by ecological types (coastal VS. oceanic zones) and mesoscale physical processes, and also provided evidence for bacterial phylogenetic phyla shaped by ecological preferences.     

Diversity and abundance of ammonia-oxidizing prokaryotes in sediments from the coastal Pearl River estuary to the South China Sea

In the present study the diversity and abundance of nitrifying microbes including ammonia-oxidizing archaea (AOA) and betaproteobacteria (beta-AOB) were investigated, along with the physicochemical parameters potentially affecting them, in a transect of surface sediments from the coastal margin adjacent to the Pearl River estuary to the slope in the deep South China Sea. Nitrifying microbial diversity was determined by detecting the amoA (ammonia monooxygenase subunit A) gene. An obvious community structure shift for both AOA and beta-AOB from the coastal marginal areas to the slope in the deep-sea was detected, while the OTU numbers of AOA amoA were more stable than those of the beta-AOB. The OTUs of beta-AOB increased with the distance from the coastal margin areas to the slope in the deep-sea. Beta-AOB showed lower diversity with dominant strains in a polluted area but higher diversity without dominant strains in a clean area. Moreover, the diversity of beta-AOB was correlated with pH values, while no noticeable relationships were established between AOA and physicochemical parameters. Beta-AOB was more sensitive to transect environmental variability and might be a potential indicator for environmental changes. Additionally, the surface sediments surveyed in the South China Sea harboured diverse and distinct AOA and beta-AOB phylotypes different from other environments, suggesting the endemicity of some nitrifying prokaryotes in the South China Sea.     

Bacterial Community Composition of South China Sea Sediments through Pyrosequencing-Based Analysis of 16S rRNA Genes

Background

Subseafloor sediments accumulate large amounts of organic and inorganic materials that contain a highly diverse microbial ecosystem. The aim of this study was to survey the bacterial community of subseafloor sediments from the South China Sea.

Methodology/Principal Findings

Pyrosequencing of over 265,000 amplicons of the V3 hypervariable region of the 16S ribosomal RNA gene was performed on 16 sediment samples collected from multiple locations in the northern region of the South China Sea from depths ranging from 35 to 4000 m. A total of 9,726 operational taxonomic units (OTUs; between 695 and 2819 unique OTUs per sample) at 97% sequence similarity level were generated. In total, 40 bacterial phyla including 22 formally described phyla and 18 candidate phyla, with Proteobacteria, Firmicutes, Planctomycetes, Actinobacteria and Chloroflexi being most diverse, were identified. The most abundant phylotype, accounting for 42.6% of all sequences, belonged to Gammaproteobacteria, which possessed absolute predominance in the samples analyzed. Among the 18 candidate phyla, 12 were found for the first time in the South China Sea.

Conclusions

This study provided a novel insight into the composition of bacterial communities of the South China Sea subseafloor. Furthermore, abundances and community similarity analysis showed that the compositions of the bacterial communities are very similar at phylum level at different depths from 35-4000 m.     

基于amoA基因扩增子高通量测序的氨氧化古菌多样性分析方法

【背景】对于环境样品中氨氧化古菌(Ammonia-oxidizing archaea,AOA)多样性的研究,利用amoA功能基因作为分子标记会比16SrRNA基因有更强的特异性和更高的分辨率,能更准确地反映环境样品中氨氧化古菌的种群结构和分布特征。然而,目前对amoA基因扩增子高通量测序的分析存在两大限制因素:一是缺乏相应的amoA基因参考数据库;二是AOA amoA基因在种水平上的相似性阈值未知,分析过程中没有明确的划分种水平操作分类单元(Operational taxonomic unit,OTU)的阈值。【目的】构建基于amoA功能基因序列分析氨氧化古菌多样性的方法,为基于高通量测序的功能微生物多样性分析提供参考。【方法】基于目前已通过分离纯化或富集培养获得的34株氨氧化古菌及功能基因数据库中收录的环境样品amoA基因序列,构建氨氧化古菌amoA基因参考数据库。通过菌株间两两比对获得的amoA基因相似度与16SrRNA基因相似度的相关性分析,确定amoA基因在种水平上的相似性阈值。基于MOTHUR软件平台,利用建立的参考数据库和确定的阈值对南海一个垂直水体剖面样品的amoA基因序列进行多样性分析。【结果】构建了含有26 091条序列信息的古菌amoA基因参考数据库,确定了89%作为分析过程中古菌amoA基因划分种水平OTU的阈值,对南海水体样品氨氧化古菌的多样性分析结果很好地显示了南海不同深度水层水体中氨氧化古菌的种群结构和系统发育关系,有效揭示了南海氨氧化古菌的垂直分布差异。【结论】建立了基于amoA基因高通量测序的氨氧化古菌多样性分析方法,此方法可以有效分析环境样品中氨氧化古菌的多样性。     

Phylogenetic diversity of planktonic archaea in the estuarine region of East China Sea

To examine the diversity and structure of archaeal communities in the Yangtze River estuarine region of East China Sea (ECS), the 16S rRNA gene clone libraries of two typical sites were constructed with the archaea-specific primers. In total, 71 clones randomly selected were screened by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and 21 clones with unique RFLP pattern were sequenced. All the sequences are clustered into the two groups of Marine Group I (MGI) and Marine Group II (MGII) which are affiliated with the phyla Crenarchaeota and Euryarchaeota, respectively. MGI clones dominate both libraries with 20 MGI sequences obtained. The majority of sequences are closely related to uncultured marine archaea except for two sequences of which the nearest neighbor is a newly identified isolate of nitrifying marine archaeon Nitrosopumilus maritimus (98% identity). The results indicate that ECS coastal waters are inhabited by archaeal community with low dominance and high diversity corresponding to the complex estuarine environments, suggesting that archaea may perform an important role in the estuarine ecosystem.     

Community Structure of Archaea from Deep-Sea Sediments of the South China Sea

Using the archaeal 16S rRNA gene, we determined the community structures of archaea of subseafloor sediments (～9-11 m below seafloor) from two geographically distant cores (MD05-2896, south, water depth 1,657 m; MD05-2902, north, water depth 3,697 m) in the South China Sea. Euryarchaeota accounted for 61.4% of total archaeal clone libraries at MD05-2896 and 56.2% at MD05-2902. At both locations, the Euryarchaeota-related sequences were dominated by Marine Benthic Group D, Terrestrial Miscellaneous Eryarchaeotal Group, and South African GoldMine Euryarchaeotal Group; the Crenarchaeota-related sequences were dominated by Marine Benthic Group B, Marine Group I, pSL12, and C3. The community structure showed no significant difference with depth at each location, suggesting the lack of stratification of archaeal populations in the deep-sea marine sediments in the South China Sea. On the other hand, the community structure is significantly different between the two sites, which may be related to geographical difference in the South China Sea.     

Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness

Although copious qualitative information describes the members of the diverse microbial communities on Earth, statistical approaches for quantifying and comparing the numbers and compositions of lineages in communities are lacking. We present a method that addresses the challenge of assigning sequences to operational taxonomic units (OTUs) based on the genetic distances between sequences. We developed a computer program, DOTUR, which assigns sequences to OTUs by using either the furthest, average, or nearest neighbor algorithm for each distance level. DOTUR uses the frequency at which each OTU is observed to construct rarefaction and collector's curves for various measures of richness and diversity. We analyzed 16S rRNA gene libraries derived from Scottish and Amazonian soils and the Sargasso Sea with DOTUR, which assigned sequences to OTUs rapidly and reliably based on the genetic distances between sequences and identified previous inconsistencies and errors in assigning sequences to OTUs. An analysis of the two 16S rRNA gene libraries from soil demonstrated that they do not contain enough sequences to support a claim that they contain different numbers of bacterial lineages with statistical confidence (P > 0.05), nor do they contain enough sequences to provide a robust estimate of species richness when an OTU is defined as containing sequences that are no more than 3% different from each other. In contrast, the richness of OTUs at the 3% level in the Sargasso Sea collection began to plateau after the sampling of 690 sequences. We anticipate that an equivalent extent of sampling for soil would require sampling more than 10,000 sequences, almost 100 times the size of typical sequence collections obtained from soil.     

Introducing DOTUR, a Computer Program for Defining Operational Taxonomic Units and Estimating Species Richness

Although copious qualitative information describes the members of the diverse microbial communities on Earth, statistical approaches for quantifying and comparing the numbers and compositions of lineages in communities are lacking. We present a method that addresses the challenge of assigning sequences to operational taxonomic units (OTUs) based on the genetic distances between sequences. We developed a computer program, DOTUR, which assigns sequences to OTUs by using either the furthest, average, or nearest neighbor algorithm for each distance level. DOTUR uses the frequency at which each OTU is observed to construct rarefaction and collector's curves for various measures of richness and diversity. We analyzed 16S rRNA gene libraries derived from Scottish and Amazonian soils and the Sargasso Sea with DOTUR, which assigned sequences to OTUs rapidly and reliably based on the genetic distances between sequences and identified previous inconsistencies and errors in assigning sequences to OTUs. An analysis of the two 16S rRNA gene libraries from soil demonstrated that they do not contain enough sequences to support a claim that they contain different numbers of bacterial lineages with statistical confidence (P > 0.05), nor do they contain enough sequences to provide a robust estimate of species richness when an OTU is defined as containing sequences that are no more than 3% different from each other. In contrast, the richness of OTUs at the 3% level in the Sargasso Sea collection began to plateau after the sampling of 690 sequences. We anticipate that an equivalent extent of sampling for soil would require sampling more than 10,000 sequences, almost 100 times the size of typical sequence collections obtained from soil.     

Temporal and Spatial Diversity of Bacterial Communities in Coastal Waters of the South China Sea

Bacteria are recognized as important drivers of biogeochemical processes in all aquatic ecosystems. Temporal and geographical patterns in ocean bacterial communities have been observed in many studies, but the temporal and spatial patterns in the bacterial communities from the South China Sea remained unexplored. To determine the spatiotemporal patterns, we generated 16S rRNA datasets for 15 samples collected from the five regularly distributed sites of the South China Sea in three seasons (spring, summer, winter). A total of 491 representative sequences were analyzed by MOTHUR, yielding 282 operational taxonomic units (OTUs) grouped at 97% stringency. Significant temporal variations of bacterial diversity were observed. Richness and diversity indices indicated that summer samples were the most diverse. The main bacterial group in spring and summer samples was Alphaproteobacteria, followed by Cyanobacteria and Gammaproteobacteria, whereas Cyanobacteria dominated the winter samples. Spatial patterns in the samples were observed that samples collected from the coastal (D151, D221) waters and offshore (D157, D1512, D224) waters clustered separately, the coastal samples harbored more diverse bacterial communities. However, the temporal pattern of the coastal site D151 was contrary to that of the coastal site D221. The LIBSHUFF statistics revealed noticeable differences among the spring, summer and winter libraries collected at five sites. The UPGMA tree showed there were temporal and spatial heterogeneity of bacterial community composition in coastal waters of the South China Sea. The water salinity (P=0.001) contributed significantly to the bacteria-environment relationship. Our results revealed that bacterial community structures were influenced by environmental factors and community-level changes in 16S-based diversity were better explained by spatial patterns than by temporal patterns.     

Vertical Profiles of Bacteria in the Tropical and Subarctic Oceans Revealed by Pyrosequencing

Community composition of Bacteria in the surface and deep water layers were examined at three oceanic sites in the Pacific Ocean separated by great distance, i.e., the South China Sea (SCS) in the western tropical Pacific, the Costa Rica Dome (CRD) in the eastern tropical Pacific and the western subarctic North Pacific (SNP), using high throughput DNA pyrosequencing of the 16S rRNA gene. Bioinformatic analysis rendered a total of 143600 high quality sequences with an average 11967 sequences per sample and mean read length of 449 bp. Phylogenetic analysis showed that Proteobacteria dominated in all shallow and deep waters, with Alphaproteobacteria and Gammaproteobacteria the two most abundant components, and SAR11 the most abundant group at family level in all regions. Cyanobacteria occurred mainly in the surface euphotic layer, and the majority of them in the tropical waters belonged to the GpIIa family including Prochlorococcus and Synechococcus, whilst those associated with Cryptophytes and diatoms were common in the subarctic waters. In general, species richness (Chao1) and diversity (Shannon index H′) were higher for the bacterial communities in the intermediate water layers than for those in surface and deep waters. Both NMDS plot and UPGMA clustering demonstrated that bacterial community composition in the deep waters (500 m ∼2000 m) of the three oceanic regions shared a high similarity and were distinct from those in the upper waters (5 m ∼100 m). Our study indicates that bacterial community composition in the DOC-poor deep water in both tropical and subarctic regions were rather stable, contrasting to those in the surface water layers, which could be strongly affected by the fluctuations of environmental factors.     

SSU‐rRNA Gene Sequencing Survey of Benthic Microbial Eukaryotes from Guaymas Basin Hydrothermal Vent

Microbial eukaryotes have important roles in marine food webs, but their diversity and activities in hydrothermal vent ecosystems are poorly characterized. In this study, we analyzed microbial eukaryotic communities associated with bacterial (Beggiatoa) mats in the 2,000 m deep‐sea Guaymas Basin hydrothermal vent system using 18S rRNA gene high‐throughput sequencing of the V4 region. We detected 6,954 distinct Operational Taxonomic Units (OTUs) across various mat systems. Of the sequences that aligned with known protistan phylotypes, most were affiliated with alveolates (especially dinoflagellates and ciliates) and cercozoans. OTU richness and community structure differed among sediment habitats (e.g. different mat types and cold sediments away from mats). Additionally, full‐length 18S rRNA genes amplified and cloned from single cells revealed the identities of some of the most commonly encountered, active ciliates in this hydrothermal vent ecosystem. Observations and experiments were also conducted to demonstrate that ciliates were trophically active and ingesting fluorescent bacteria or Beggiatoa trichomes. Our work suggests that the active and diverse protistan community at the Guaymas Basin hydrothermal vent ecosystem likely consumes substantial amounts of bacterial biomass, and that the different habitats, often defined by distances of just a few 10s of cm, select for particular assemblages and levels of diversity.     

Archaea Appear to Dominate the Microbiome of Inflatella pellicula Deep Sea Sponges

Microbes associated with marine sponges play significant roles in host physiology. Remarkable levels of microbial diversity have been observed in sponges worldwide through both culture-dependent and culture-independent studies. Most studies have focused on the structure of the bacterial communities in sponges and have involved sponges sampled from shallow waters. Here, we used pyrosequencing of 16S rRNA genes to compare the bacterial and archaeal communities associated with two individuals of the marine sponge Inflatella pellicula from the deep-sea, sampled from a depth of 2,900 m, a depth which far exceeds any previous sequence-based report of sponge-associated microbial communities. Sponge-microbial communities were also compared to the microbial community in the surrounding seawater. Sponge-associated microbial communities were dominated by archaeal sequencing reads with a single archaeal OTU, comprising ∼60% and ∼72% of sequences, being observed from Inflatella pellicula. Archaeal sequencing reads were less abundant in seawater (∼11% of sequences). Sponge-associated microbial communities were less diverse and less even than any other sponge-microbial community investigated to date with just 210 and 273 OTUs (97% sequence identity) identified in sponges, with 4 and 6 dominant OTUs comprising ∼88% and ∼89% of sequences, respectively. Members of the candidate phyla, SAR406, NC10 and ZB3 are reported here from sponges for the first time, increasing the number of bacterial phyla or candidate divisions associated with sponges to 43. A minor cohort from both sponge samples (∼0.2% and ∼0.3% of sequences) were not classified to phylum level. A single OTU, common to both sponge individuals, dominates these unclassified reads and shares sequence homology with a sponge associated clone which itself has no known close relative and may represent a novel taxon.     

An analysis of geothermal and carbonic springs in the western United States sustained by deep fluid inputs

Hydrothermal springs harbor unique microbial communities that have provided insight into the early evolution of life, expanded known microbial diversity, and documented a deep Earth biosphere. Mesothermal (cool but above ambient temperature) continental springs, however, have largely been ignored although they may also harbor unique populations of micro‐organisms influenced by deep subsurface fluid mixing with near surface fluids. We investigated the microbial communities of 28 mesothermal springs in diverse geologic provinces of the western United States that demonstrate differential mixing of deeply and shallowly circulated water. Culture‐independent analysis of the communities yielded 1966 bacterial and 283 archaeal 16S rRNA gene sequences. The springs harbored diverse taxa and shared few operational taxonomic units (OTUs) across sites. The Proteobacteria phylum accounted for most of the dataset (81.2% of all 16S rRNA genes), with 31 other phyla/candidate divisions comprising the remainder. A small percentage (~6%) of bacterial 16S rRNA genes could not be classified at the phylum level, but were mostly distributed in those springs with greatest inputs of deeply sourced fluids. Archaeal diversity was limited to only four springs and was primarily composed of well‐characterized Thaumarchaeota. Geochemistry across the dataset was varied, but statistical analyses suggested that greater input of deeply sourced fluids was correlated with community structure. Those with lesser input contained genera typical of surficial waters, while some of the springs with greater input may contain putatively chemolithotrophic communities. The results reported here expand our understanding of microbial diversity of continental geothermal systems and suggest that these communities are influenced by the geochemical and hydrologic characteristics arising from deeply sourced (mantle‐derived) fluid mixing. The springs and communities we report here provide evidence for opportunities to understand new dimensions of continental geobiological processes where warm, highly reduced fluids are mixing with more oxidized surficial waters.     

Seawater recirculation through subducting sediments sustains a deeply buried population of sulfate‐reducing bacteria

Subseafloor sulfate concentrations typically decrease with depth as this electron acceptor is consumed by respiring microorganisms. However, studies show that seawater can flow through hydraulically conductive basalt to deliver sulfate upwards into deeply buried overlying sediments. Our previous work on IODP Site C0012A (Nankai Trough, Japan) revealed that recirculation of sulfate through the subducting Philippine Sea Plate stimulated microbial activity near the sediment–basement interface (SBI). Here, we describe the microbial ecology, phylogeny, and energetic requirements of population of aero‐tolerant sulfate‐reducing bacteria in the deep subseafloor. We identified dissimilatory sulfite reductase gene (dsr) sequences 93% related to oxygen‐tolerant Desulfovibrionales species across all reaction zones while no SRB were detected in drilling fluid control samples. Pore fluid chemistry revealed low concentrations of methane (<0.25 mM), while hydrogen levels were consistent with active bacterial sulfate reduction (0.51–1.52 nM). Solid phase total organic carbon (TOC) was also considerably low in these subseafloor sediments. Our results reveal the phylogenetic diversity, potential function, and physiological tolerance of a community of sulfate‐reducing bacteria living at ~480 m below subducting seafloor.     

Changes in northern Gulf of Mexico sediment bacterial and archaeal communities exposed to hypoxia

Biogeochemical changes in marine sediments during coastal water hypoxia are well described, but less is known about underlying changes in microbial communities. Bacterial and archaeal communities in Louisiana continental shelf (LCS) hypoxic zone sediments were characterized by pyrosequencing 16S rRNA V4‐region gene fragments obtained by PCR amplification of community genomic DNA with bacterial‐ or archaeal‐specific primers. Duplicate LCS sediment cores collected during hypoxia had higher concentrations of Fe(II), and dissolved inorganic carbon, phosphate, and ammonium than cores collected when overlying water oxygen concentrations were normal. Pyrosequencing yielded 158 686 bacterial and 225 591 archaeal sequences from 20 sediment samples, representing five 2‐cm depth intervals in the duplicate cores. Bacterial communities grouped by sampling date and sediment depth in a neighbor‐joining analysis using Chao–Jaccard shared species values. Redundancy analysis indicated that variance in bacterial communities was mainly associated with differences in sediment chemistry between oxic and hypoxic water column conditions. Gammaproteobacteria (26.5%) were most prominent among bacterial sequences, followed by Firmicutes (9.6%), and Alphaproteobacteria (5.6%). Crenarchaeotal, thaumarchaeotal, and euryarchaeotal lineages accounted for 57%, 27%, and 16% of archaeal sequences, respectively. In Thaumarchaeota Marine Group I, sequences were 96–99% identical to the Nitrosopumilus maritimus SCM1 sequence, were highest in surficial sediments, and accounted for 31% of archaeal sequences when waters were normoxic vs. 13% of archaeal sequences when waters were hypoxic. Redundancy analysis showed Nitrosopumilus‐related sequence abundance was correlated with high solid‐phase Fe(III) concentrations, whereas most of the remaining archaeal clusters were not. In contrast, crenarchaeotal sequences were from phylogenetically diverse lineages, differed little in relative abundance between sampling times, and increased to high relative abundance with sediment depth. These results provide further evidence that marine sediment microbial community composition can be structured according to sediment chemistry and suggest the expansion of hypoxia in coastal waters may alter sediment microbial communities involved in carbon and nitrogen cycling.     

A multiple PCR-primer approach to access the microeukaryotic diversity in environmental samples

The Cariaco Basin off the Venezuelan coast in the Caribbean Sea is the world's largest truly marine body of anoxic water. The first rRNA survey of microbial eukaryotes in this environment revealed a number of novel lineages, but sampled only a fraction of the entire diversity. The goal of this study was to significantly improve recovery of protistan rRNA from the Basin. This was achieved by a systematic application of multiple PCR primer sets and substantially larger sequencing efforts. We focused on the most diverse habitat in the basin, anoxic waters approximately 100m below the oxic-anoxic interface, and detected novel lineages that escaped the single PCR primer approach. All clones obtained proved unique. A 99% sequence similarity cut-off value combined these clones into operational taxonomic units (OTUs), over 75% of which proved novel. Some of these OTUs form deep branches within established protistan groups. Others signify discovery of novel protistan lineages that appear unrelated to any known microeukaryote. Surprisingly, even this large-scale multi-primer rRNA approach still missed a substantial part of the samples' rRNA diversity. The overlap between the species lists obtained with different primers is low, with only 4% of OTUs shared by all three libraries, and the number of species detected only once is large (55%). This strongly indicates that, at least in anoxic environments, protistan diversity may be much larger than is commonly thought. A single sample appears to contain thousands of largely novel protistan species. Multiple PCR primer combinations may be needed to capture these species.     

Pan-oceanic distribution of new highly diverse clades of deep-sea diplonemids

Molecular rRNA gene surveys reveal a considerable diversity of microbial eukaryotes in different environments. Even within a single clade, the number of distinct phylotypes retrieved often goes beyond previous expectations. Here, we have used specific 18S rRNA PCR primers to investigate the diversity of diplonemids, a poorly known group of flagellates with only a few described species. We analysed surface and deep-sea plankton samples from different oceanic regions, including the water-column in the Marmara Sea. We retrieved a large diversity of diplonemid phylotypes, most of which formed two novel distinct clades without cultured representatives. Although most marine diplonemid phylotypes appeared to be cosmopolitan, they showed a marked stratified distribution through the water column, being very scarce or absent in surface waters. The small and specific diplonemid diversity found in surface samples and the fact that most sequences of uncultured diplonemids found in other studies came from deep-sea environments suggest that the two major uncultured diplonemid clades group species preferentially inhabit the deep ocean.