Protist diversity in suboxic and sulfidic waters of the Black Sea

The oxic-anoxic transition zone of the Black Sea comprises a large suboxic zone as well as anoxic and sulfidic waters. While the prokaryotes and biogeochemical cycles that characterize this zone have been frequently studied, little is known about the diversity or ecology of its microbial eukaryotes. Here, we present the first broad qualitative report of the protist species composition in the Black Sea redoxcline using molecular tools. Fingerprint analysis from the whole redoxcline revealed a complex community structure of metabolically active protists with distinct shifts along the redox gradient. Additionally, 18S rRNA gene clone libraries were used to compare protist species composition of suboxic and sulfidic water layers. Among the ciliates, sequences related to Pleuronema and Strombidium were dominant in both water layers whereas sequences affiliated with anaerobic plagiopylids and Cyclidium were detected only in the sulfidic zone. Among the flagellates, mainly stramenopiles (mostly bicosoecids and chrysophytes) occurred throughout the redoxcline. In the sulfidic zone we found stramenopile sequences but also euglenozoans, jakobids and choanoflagellates that were related to clonal sequences from other anoxic marine habitats, thus indicating the existence of globally distributed groups of anoxic flagellates. Higher species diversity in the sulfidic zone and about twice as many novel sequence types of ciliates and stramenopiles compared with the suboxic layer emphasizes the importance of anoxic, sulfidic waters as habitat for high protist diversity although the function of these organisms is yet unknown.     

Comparative genomics reveals new functional insights in uncultured MAST species

Heterotrophic lineages of stramenopiles exhibit enormous diversity in morphology, lifestyle, and habitat. Among them, the marine stramenopiles (MASTs) represent numerous independent lineages that are only known from environmental sequences retrieved from marine samples. The core energy metabolism characterizing these unicellular eukaryotes is poorly understood. Here, we used single-cell genomics to retrieve, annotate, and compare the genomes of 15 MAST species, obtained by coassembling sequences from 140 individual cells sampled from the marine surface plankton. Functional annotations from their gene repertoires are compatible with all of them being phagocytotic. The unique presence of rhodopsin genes in MAST species, together with their widespread expression in oceanic waters, supports the idea that MASTs may be capable of using sunlight to thrive in the photic ocean. Additional subsets of genes used in phagocytosis, such as proton pumps for vacuole acidification and peptidases for prey digestion, did not reveal particular trends in MAST genomes as compared with nonphagocytotic stramenopiles, except a larger presence and diversity of V-PPase genes. Our analysis reflects the complexity of phagocytosis machinery in microbial eukaryotes, which contrasts with the well-defined set of genes for photosynthesis. These new genomic data provide the essential framework to study ecophysiology of uncultured species and to gain better understanding of the function of rhodopsins and related carotenoids in stramenopiles.Subject terms: Genomics, Microbiology     

Unveiling trophic functions of uncultured protist taxa by incubation experiments in the brackish baltic sea

Background

Our knowledge of the phylogeny and diversity of aquatic protists is rapidly increasing due to molecular surveys and next-generation sequencing approaches. This has led to a considerable discrepancy between the taxa known from cultures and those known from environmental 18S rRNA gene sequences. Hence, it is generally difficult to assign ecological functions to new taxa detected by culture-independent molecular approaches.

Methodology/Principal Findings

A combination of unamended dark incubations and 18S rRNA sequencing was chosen to link molecular diversity data of uncultured protists with heterotrophic, presumably bacterivorous, growth. The incubations, conducted with Baltic Sea brackish water, resulted in a consistent shift from a protistan community dominated by phototrophs to one in which heterotrophs predominated. This was determined on the basis of cell abundance and 18S rRNA sequences derived from fingerprint analysis and clone libraries. The bulk of enriched phylotypes after incubation were related to hitherto uncultured marine taxa within chrysophytes, ochrophytes, choanoflagellates, cercozoans, and picobiliphytes, mostly represented in recently established or here defined environmental clades. Their growth in the dark, together with coinciding results from studies with a similar objective, provides evidence that these uncultured taxa represent heterotrophic or mixotrophic species.

Conclusions/Significance

These findings shed some light into the trophic role of diverse uncultured protists especially within functionally heterogeneous groups (e.g., chrysophytes, ochrophytes) and groups that appear to be puzzling with regard to their nutrition (picobiliphytes). Additionally, our results indicate that the heterotrophic flagellate community in the southwestern Baltic Sea is dominated by species of marine origin. The combination of unamended incubations with molecular diversity analysis is thus confirmed as a promising approach to explore the trophic mode of environmentally relevant protist taxa for which only sequence data are currently available.     

Exploring the uncultured microeukaryote majority in the oceans: reevaluation of ribogroups within stramenopiles

Molecular surveys in planktonic marine systems have unveiled a large novel diversity of small protists. A large part of this diversity belongs to basal heterotrophic stramenopiles and is distributed in a set of polyphyletic ribogroups (described from rDNA sequences) collectively named as MAST (MArine STramenopiles). In the few groups investigated, MAST cells are globally distributed and abundant bacterial grazers, therefore having a putatively large impact on marine ecosystem functioning. The main aim of this study is to reevaluate the MAST ribogroups described so far and to determine whether additional groups can be found. For this purpose, we used traditional and state-of-the-art molecular tools, combining 18S rDNA sequences from publicly available clone libraries, single amplified genomes (SAGs) of planktonic protists, and a pyrosequencing survey from coastal waters and sediments. Our analysis indicated a final set of 18 MAST groups plus 5 new ribogroups within Ochrophyta (named as MOCH). The MAST ribogroups were then analyzed in more detail. Seven were typical of anoxic systems and one of oxic sediments. The rest were clearly members of oxic marine picoplankton. We characterized the genetic diversity within each MAST group and defined subclades for the more diverse (46 subclades in 8 groups). The analyses of sequences within subclades revealed further ecological specializations. Our data provide a renovated framework for phylogenetic classification of the numerous MAST ribogroups and support the notion of a tight link between phylogeny and ecological distribution. These diverse and largely uncultured protists are widespread and ecologically relevant members of marine microbial assemblages.     

Termite Hindguts and the Ecology of Microbial Communities in the Sequencing Age

Advances in high‐throughput nucleic acid sequencing have improved our understanding of microbial communities in a number of ways. Deeper sequence coverage provides the means to assess diversity at the resolution necessary to recover ecological and biogeographic patterns, and at the same time single‐cell genomics provides detailed information about the interactions between members of a microbial community. Given the vastness and complexity of microbial ecosystems, such analyses remain challenging for most environments, so greater insight can also be drawn from analysing less dynamic ecosystems. Here, we outline the advantages of one such environment, the wood‐digesting hindgut communities of termites and cockroaches, and how it is a model to examine and compare both protist and bacterial communities. Beyond the analysis of diversity, our understanding of protist community ecology will depend on using statistically sound sampling regimes at biologically relevant scales, transitioning from discovery‐based to experimental ecology, incorporating single‐cell microbiology and other data sources, and continued development of analytical tools.     

Solenicola setigera is the first characterized member of the abundant and cosmopolitan uncultured marine stramenopile group MAST‐3

Culture‐independent molecular methods based on the amplification, cloning and sequencing of small‐subunit (SSU) rRNA genes are a powerful tool to study the diversity of prokaryotic and eukaryotic microorganisms for which morphological features are not conspicuous. In recent years, molecular data from environmental surveys have revealed several clades of protists lacking cultured and/or described members. Among them are various clades of marine stramenopiles (heterokonts), which are thought to play an essential ecological role as grazers, being abundant and distributed in oceans worldwide. In this work, we show that Solenicola setigera, a distinctive widespread colonial marine protist, is a member of the environmental clade MArine STramenopile 3 (MAST‐3). Solenicola is generally considered as a parasite or an epiphyte of the diatom Leptocylindrus mediterraneus. So far, the ultrastructural, morphological and ecological data available were insufficient to elucidate its phylogenetic position, even at the division or class level. We determined SSU rRNA gene sequences of S. setigera specimens sampled from different locations and seasons in the type locality, the Gulf of Lions, France. They were closely related, though not identical, which, together with morphological differences under electron microscopy, suggest the occurrence of several species. Solenicola sequences were well nested within the MAST‐3 clade in phylogenetic trees. Since Solenicola is the first identified member of this abundant marine clade, we propose the name Solenicolida for the MAST‐3 phylogenetic group.     

Capturing diversity of marine heterotrophic protists: one cell at a time

Recent applications of culture-independent, molecular methods have revealed unexpectedly high diversity in a variety of functional and phylogenetic groups of microorganisms in the ocean. However, none of the existing research tools are free from significant limitations, such as PCR and cloning biases, low phylogenetic resolution and others. Here, we employed novel, single-cell sequencing techniques to assess the composition of small (<10 μm diameter), heterotrophic protists from the Gulf of Maine. Single cells were isolated by flow cytometry, their genomes amplified, and 18S rRNA marker genes were amplified and sequenced. We compared the results to traditional environmental PCR cloning of sorted cells. The diversity of heterotrophic protists was significantly higher in the library of single amplified genomes (SAGs) than in environmental PCR clone libraries of the 18S rRNA gene, obtained from the same coastal sample. Libraries of SAGs, but not clones contained several recently discovered, uncultured groups, including picobiliphytes and novel marine stramenopiles. Clone, but not SAG, libraries contained several large clusters of identical and nearly identical sequences of Dinophyceae, Cercozoa and Stramenopiles. Similar results were obtained using two alternative primer sets, suggesting that PCR biases may not be the only explanation for the observed patterns. Instead, differences in the number of 18S rRNA gene copies among the various protist taxa probably had a significant role in determining the PCR clone composition. These results show that single-cell sequencing has the potential to more accurately assess protistan community composition than previously established methods. In addition, the creation of SAG libraries opens opportunities for the analysis of multiple genes or entire genomes of the uncultured protist groups.     

Unveiling in situ interactions between marine protists and bacteria through single cell sequencing

Heterotrophic protists are a highly diverse and biogeochemically significant component of marine ecosystems, yet little is known about their species-specific prey preferences and symbiotic interactions in situ. Here we demonstrate how these previously unresolved questions can be addressed by sequencing the eukaryote and bacterial SSU rRNA genes from individual, uncultured protist cells collected from their natural marine environment and sorted by flow cytometry. We detected Pelagibacter ubique in association with a MAST-4 protist, an actinobacterium in association with a chrysophyte and three bacteroidetes in association with diverse protist groups. The presence of identical phylotypes among the putative prey and the free bacterioplankton in the same sample provides evidence for predator–prey interactions. Our results also suggest a discovery of novel symbionts, distantly related to Rickettsiales and the candidate divisions ZB3 and TG2, associated with Cercozoa and Chrysophyta cells. This study demonstrates the power of single cell sequencing to untangle ecological interactions between uncultured protists and prokaryotes.     

A Preliminary Study of Viral Metagenomics of French Bat Species in Contact with Humans: Identification of New Mammalian Viruses

The prediction of viral zoonosis epidemics has become a major public health issue. A profound understanding of the viral population in key animal species acting as reservoirs represents an important step towards this goal. Bats harbor diverse viruses, some of which are of particular interest because they cause severe human diseases. However, little is known about the diversity of the global population of viruses found in bats (virome). We determined the viral diversity of five different French insectivorous bat species (nine specimens in total) in close contact with humans. Sequence-independent amplification, high-throughput sequencing with Illumina technology and a dedicated bioinformatics analysis pipeline were used on pooled tissues (brain, liver and lungs). Comparisons of the sequences of contigs and unassembled reads provided a global taxonomic distribution of virus-related sequences for each sample, highlighting differences both within and between bat species. Many viral families were present in these viromes, including viruses known to infect bacteria, plants/fungi, insects or vertebrates, the most relevant being those infecting mammals (Retroviridae, Herpesviridae, Bunyaviridae, Poxviridae, Flaviviridae, Reoviridae, Bornaviridae, Picobirnaviridae). In particular, we detected several new mammalian viruses, including rotaviruses, gammaretroviruses, bornaviruses and bunyaviruses with the identification of the first bat nairovirus. These observations demonstrate that bats naturally harbor viruses from many different families, most of which infect mammals. They may therefore constitute a major reservoir of viral diversity that should be analyzed carefully, to determine the role played by bats in the spread of zoonotic viral infections.     

Virus diversity in metagenomes of a lichen symbiosis (Umbilicaria phaea): complete viral genomes,putative hosts and elevational distributions

Viruses can play critical roles in symbioses by initiating horizontal gene transfer, affecting host phenotypes, or expanding their host's ecological niche. However, knowledge of viral diversity and distribution in symbiotic organisms remains elusive. Here we use deep-sequenced metagenomic DNA (PacBio Sequel II; two individuals), paired with a population genomics approach (Pool-seq; 11 populations, 550 individuals) to understand viral distributions in the lichen Umbilicaria phaea. We assess (i) viral diversity in lichen thalli, (ii) putative viral hosts (fungi, algae, bacteria) and (iii) viral distributions along two replicated elevation gradients. We identified five novel viruses, showing 28%–40% amino acid identity to known viruses. They tentatively belong to the families Caulimoviridae, Myoviridae, Podoviridae and Siphoviridae. Our analysis suggests that the Caulimovirus is associated with green algal photobionts (Trebouxia) of the lichen, and the remaining viruses with bacterial hosts. We did not detect viral sequences in the mycobiont. Caulimovirus abundance decreased with increasing elevation, a pattern reflected by a specific algal lineage hosting this virus. Bacteriophages showed population-specific patterns. Our work provides the first comprehensive insights into viruses associated with a lichen holobiont and suggests an interplay of viral hosts and environment in structuring viral distributions.     

Broad Distribution of TPI-GAPDH Fusion Proteins among Eukaryotes: Evidence for Glycolytic Reactions in the Mitochondrion?

Glycolysis is a central metabolic pathway in eukaryotic and prokaryotic cells. In eukaryotes, the textbook view is that glycolysis occurs in the cytosol. However, fusion proteins comprised of two glycolytic enzymes, triosephosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were found in members of the stramenopiles (diatoms and oomycetes) and shown to possess amino-terminal mitochondrial targeting signals. Here we show that mitochondrial TPI-GAPDH fusion protein genes are widely spread across the known diversity of stramenopiles, including non-photosynthetic species (Bicosoeca sp. and Blastocystis hominis). We also show that TPI-GAPDH fusion genes exist in three cercozoan taxa (Paulinella chromatophora, Thaumatomastix sp. and Mataza hastifera) and an apusozoan protist, Thecamonas trahens. Interestingly, subcellular localization predictions for other glycolytic enzymes in stramenopiles and a cercozoan show that a significant fraction of the glycolytic enzymes in these species have mitochondrial-targeted isoforms. These results suggest that part of the glycolytic pathway occurs inside mitochondria in these organisms, broadening our knowledge of the diversity of mitochondrial metabolism of protists.     

Amplification of uncultured single-stranded DNA viruses from rice paddy soil

Viruses are known to be the most numerous biological entities in soil; however, little is known about their diversity in this environment. In order to explore the genetic diversity of soil viruses, we isolated viruses by centrifugation and sequential filtration before performing a metagenomic investigation. We adopted multiple-displacement amplification (MDA), an isothermal whole-genome amplification method with phi29 polymerase and random hexamers, to amplify viral DNA and construct clone libraries for metagenome sequencing. By the MDA method, the diversity of both single-stranded DNA (ssDNA) viruses and double-stranded DNA viruses could be investigated at the same time. On the contrary, by eliminating the denaturing step in the MDA reaction, only ssDNA viral diversity could be explored selectively. Irrespective of the denaturing step, more than 60% of the soil metagenome sequences did not show significant hits (E-value criterion, 0.001) with previously reported viral sequences. Those hits that were considered to be significant were also distantly related to known ssDNA viruses (average amino acid similarity, approximately 34%). Phylogenetic analysis showed that replication-related proteins (which were the most frequently detected proteins) related to those of ssDNA viruses obtained from the metagenomic sequences were diverse and novel. Putative circular genome components of ssDNA viruses that are unrelated to known viruses were assembled from the metagenomic sequences. In conclusion, ssDNA viral diversity in soil is more complex than previously thought. Soil is therefore a rich pool of previously unknown ssDNA viruses.     

Shotgun metagenomics and microscopy indicate diverse cyanophytes,other bacteria,and microeukaryotes in the epimicrobiota of a northern Chilean wetland Nostoc (Cyanobacteria)

Prokaryotic Nostoc, one of the world's most conspicuous and widespread algal genera (similar to eukaryotic algae, plants, and animals) is known to support a microbiome that influences host ecological roles. Past taxonomic characterizations of surface microbiota (epimicrobiota) of free‐living Nostoc sampled from freshwater systems employed 16S rRNA genes, typically amplicons. We compared taxa identified from 16S, 18S, 23S, and 28S rRNA gene sequences filtered from shotgun metagenomic sequence and used microscopy to illuminate epimicrobiota diversity for Nostoc sampled from a wetland in the northern Chilean Altiplano. Phylogenetic analysis and rRNA gene sequence abundance estimates indicated that the host was related to Nostoc punctiforme PCC 73102. Epimicrobiota were inferred to include 18 epicyanobacterial genera or uncultured taxa, six epieukaryotic algal genera, and 66 anoxygenic bacterial genera, all having average genomic coverage ≥90X. The epicyanobacteria Geitlerinemia, Oscillatoria, Phormidium, and an uncultured taxon were detected only by 16S rRNA gene; Gloeobacter and Pseudanabaena were detected using 16S and 23S; and Phormididesmis, Neosynechococcus, Symphothece, Aphanizomenon, Nodularia, Spirulina, Nodosilinea, Synechococcus, Cyanobium, and Anabaena (the latter corroborated by microscopy), plus two uncultured cyanobacterial taxa (JSC12, O77) were detected only by 23S rRNA gene sequences. Three chlamydomonad and two heterotrophic stramenopiles genera were inferred from 18S; the streptophyte green alga Chaetosphaeridium globosum was detected by microscopy and 28S rRNA genes, but not 18S rRNA genes. Overall, >60% of epimicrobial taxa were detected by markers other than 16S rRNA genes. Some algal taxa observed microscopically were not detected from sequence data. Results indicate that multiple taxonomic markers derived from metagenomic sequence data and microscopy increase epimicrobiota detection.     

Genome-resolved viral ecology in a marine oxygen minimum zone

Oxygen minimum zones (OMZs) are critical to marine nitrogen cycling and global climate change. While OMZ microbial communities are relatively well-studied, little is known about their viruses. Here, we assess the viral community ecology of 22 deeply sequenced viral metagenomes along a gradient of oxygenated to anoxic waters (<0.02 μmol/l O₂) in the Eastern Tropical South Pacific (ETSP) OMZ. We identified 46 127 viral populations (≥5 kb), which augments the known viruses from ETSP by 10-fold. Viral communities clustered into six groups that correspond to oceanographic features. Oxygen concentration was the predominant environmental feature driving viral community structure. Alpha and beta diversity of viral communities in the anoxic zone were lower than in surface waters, which parallels the low microbial diversity seen in other studies. ETSP viruses were largely endemic, with the majority of shared viruses (87%) also present in other OMZ samples. We detected 543 putative viral-encoded auxiliary metabolic genes (AMGs), of which some have a distribution that reflects physico-chemical characteristics across depth. Together these findings provide an ecological baseline for viral community structure, drivers and population variability in OMZs that will help future studies assess the role of viruses in these climate-critical environments.     

Reference Tree and Environmental Sequence Diversity of Labyrinthulomycetes

Labyrinthulomycetes are heterotrophic stramenopiles that are ubiquitous in a wide range of both marine and freshwater habitats and play important roles in decomposition of organic matter. The diversity and taxonomy of Labyrinthulomycetes has been studied for many years, but we nevertheless lack both a comprehensive reference database and up‐to‐date phylogeny including all known diversity, which hinders many global insights into their ecological distribution and the relative importance of various subgroups in different environments. Here, we present a curated reference database and a phylogenetic tree of Labyrinthulomycetes small subunit ribosomal RNA (SSU or 18S rRNA) data. Based on this created reference database, we analyzed high‐throughput environmental sequencing data, revealing many previously unknown environmental clades and exploring the ecological distribution of various subgroups. Particularly, a number of newly identified environmental clades that are widespread in the open ocean. Comparing the manually curated reference database to existing tools for identification of environmental sequences (e.g. PR2 or SILVA databases) suggests that the curated database provides a higher degree of specificity and a lower frequency of misidentification. The phylogenetic framework and database will be a useful tool for future ecological and evolutionary studies.     

Culturing Heterotrophic Protists from the Baltic Sea: Mostly the “Usual Suspects” but a Few Novelties as Well

The study of cultured strains has a long tradition in protistological research and has greatly contributed to establishing the morphology, taxonomy, and ecology of many protist species. However, cultivation‐independent techniques, based on 18S rRNA gene sequences, have demonstrated that natural protistan assemblages mainly consist of hitherto uncultured protist lineages. This mismatch impedes the linkage of environmental diversity data with the biological features of cultured strains. Thus, novel taxa need to be obtained in culture to close this knowledge gap. In this study, traditional cultivation techniques were applied to samples from coastal surface waters and from deep oxygen‐depleted waters of the Baltic Sea. Based on 18S rRNA gene sequencing, 126 monoclonal cultures of heterotrophic protists were identified. The majority of the isolated strains were affiliated with already cultured and described taxa, mainly chrysophytes and bodonids. This was likely due to “culturing bias” but also to the eutrophic nature of the Baltic Sea. Nonetheless, ~ 12% of the isolates in our culture collection showed highly divergent 18S rRNA gene sequences compared to those of known organisms and thus may represent novel taxa, either at the species level or at the genus level. Moreover, we also obtained evidence that some of the isolated taxa are ecologically relevant, under certain conditions, in the Baltic Sea.     

Single virus genomics: a new tool for virus discovery

Whole genome amplification and sequencing of single microbial cells has significantly influenced genomics and microbial ecology by facilitating direct recovery of reference genome data. However, viral genomics continues to suffer due to difficulties related to the isolation and characterization of uncultivated viruses. We report here on a new approach called 'Single Virus Genomics', which enabled the isolation and complete genome sequencing of the first single virus particle. A mixed assemblage comprised of two known viruses; E. coli bacteriophages lambda and T4, were sorted using flow cytometric methods and subsequently immobilized in an agarose matrix. Genome amplification was then achieved in situ via multiple displacement amplification (MDA). The complete lambda phage genome was recovered with an average depth of coverage of approximately 437X. The isolation and genome sequencing of uncultivated viruses using Single Virus Genomics approaches will enable researchers to address questions about viral diversity, evolution, adaptation and ecology that were previously unattainable.     

Phylogenetic and Ecological Analysis of Novel Marine Stramenopiles

Culture-independent molecular analyses of open-sea microorganisms have revealed the existence and apparent abundance of novel eukaryotic lineages, opening new avenues for phylogenetic, evolutionary, and ecological research. Novel marine stramenopiles, identified by 18S ribosomal DNA sequences within the basal part of the stramenopile radiation but unrelated to any previously known group, constituted one of the most important novel lineages in these open-sea samples. Here we carry out a comparative analysis of novel stramenopiles, including new sequences from coastal genetic libraries presented here and sequences from recent reports from the open ocean and marine anoxic sites. Novel stramenopiles were found in all major habitats, generally accounting for a significant proportion of clones in genetic libraries. Phylogenetic analyses indicated the existence of 12 independent clusters. Some of these were restricted to anoxic or deep-sea environments, but the majority were typical components of coastal and open-sea waters. We specifically identified four clusters that were well represented in most marine surface waters (together they accounted for 74% of the novel stramenopile clones) and are the obvious targets for future research. Many sequences were retrieved from geographically distant regions, indicating that some organisms were cosmopolitan. Our study expands our knowledge on the phylogenetic diversity and distribution of novel marine stramenopiles and confirms that they are fundamental members of the marine eukaryotic picoplankton.     

Phylogenetic and ecological analysis of novel marine stramenopiles

Culture-independent molecular analyses of open-sea microorganisms have revealed the existence and apparent abundance of novel eukaryotic lineages, opening new avenues for phylogenetic, evolutionary, and ecological research. Novel marine stramenopiles, identified by 18S ribosomal DNA sequences within the basal part of the stramenopile radiation but unrelated to any previously known group, constituted one of the most important novel lineages in these open-sea samples. Here we carry out a comparative analysis of novel stramenopiles, including new sequences from coastal genetic libraries presented here and sequences from recent reports from the open ocean and marine anoxic sites. Novel stramenopiles were found in all major habitats, generally accounting for a significant proportion of clones in genetic libraries. Phylogenetic analyses indicated the existence of 12 independent clusters. Some of these were restricted to anoxic or deep-sea environments, but the majority were typical components of coastal and open-sea waters. We specifically identified four clusters that were well represented in most marine surface waters (together they accounted for 74% of the novel stramenopile clones) and are the obvious targets for future research. Many sequences were retrieved from geographically distant regions, indicating that some organisms were cosmopolitan. Our study expands our knowledge on the phylogenetic diversity and distribution of novel marine stramenopiles and confirms that they are fundamental members of the marine eukaryotic picoplankton.     

Viral metagenomics

Viruses, most of which infect microorganisms, are the most abundant biological entities on the planet. Identifying and measuring the community dynamics of viruses in the environment is complicated because less than one percent of microbial hosts have been cultivated. Also, there is no single gene that is common to all viral genomes, so total uncultured viral diversity cannot be monitored using approaches analogous to ribosomal DNA profiling. Metagenomic analyses of uncultured viral communities circumvent these limitations and can provide insights into the composition and structure of environmental viral communities.