Seasonal Dynamics of Algae-Infecting Viruses and Their Inferred Interactions with Protists.

Viruses are a highly abundant, dynamic, and diverse component of planktonic communities that have key roles in marine ecosystems. We aimed to reveal the diversity and dynamics of marine large dsDNA viruses infecting algae in the Northern Skagerrak, South Norway through the year by metabarcoding, targeting the major capsid protein (MCP) and its correlation to protist diversity and dynamics. Metabarcoding results demonstrated a high diversity of algal viruses compared to previous metabarcoding surveys in Norwegian coastal waters. We obtained 313 putative algal virus operational taxonomic units (vOTUs), all classified by phylogenetic analyses to either the Phycodnaviridae or Mimiviridae families, most of them in clades without any cultured or environmental reference sequences. The viral community showed a clear temporal variation, with some vOTUs persisting for several months. The results indicate co-occurrences between abundant viruses and potential hosts during long periods. This study gives new insights into the virus-algal host dynamics and provides a baseline for future studies of algal virus diversity and temporal dynamics.

Protist Diversity and Seasonal Dynamics in Skagerrak Plankton Communities as Revealed by Metabarcoding and Microscopy.

Protist community composition and seasonal dynamics are of major importance for the production of higher trophic levels, such as zooplankton and fish. Our aim was to reveal how the protist community in the Skagerrak changes through the seasons by combining high-throughput sequencing and microscopy of plankton collected monthly over two years. The V4 region of the 18S rRNA gene was amplified by eukaryote universal primers from the total RNA/cDNA. We found a strong seasonal variation in protist composition and proportional abundances, and a difference between two depths within the euphotic zone. Highest protist richness was found in late summer-early autumn, and lowest in winter. Temperature was the abiotic factor explaining most of the variation in diversity. Dinoflagellates was the most abundant and diverse group followed by ciliates and diatoms. We found about 70 new taxa recorded for the first time in the Skagerrak. The seasonal pattern in relative read abundance of major phytoplankton groups was well in accordance with microscopical biovolumes. This is the first metabarcoding study of the protist plankton community of all taxonomic groups and through seasons in the Skagerrak, which may serve as a baseline for future surveys to reveal effects of climate and environmental changes.

Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests.

High animal and plant richness in tropical rainforest communities has long intrigued naturalists. It is unknown if similar hyperdiversity patterns are reflected at the microbial scale with unicellular eukaryotes (protists). Here we show, using environmental metabarcoding of soil samples and a phylogeny-aware cleaning step, that protist communities in Neotropical rainforests are hyperdiverse and dominated by the parasitic Apicomplexa, which infect arthropods and other animals. These host-specific parasites potentially contribute to the high animal diversity in the forests by reducing population growth in a density-dependent manner. By contrast, too few operational taxonomic units (OTUs) of Oomycota were found to broadly drive high tropical tree diversity in a host-specific manner under the Janzen-Connell model. Extremely high OTU diversity and high heterogeneity between samples within the same forests suggest that protists, not arthropods, are the most diverse eukaryotes in tropical rainforests. Our data show that protists play a large role in tropical terrestrial ecosystems long viewed as being dominated by macroorganisms.

Seasonal Dynamics of Haptophytes and dsDNA Algal Viruses Suggest Complex Virus-Host Relationship.

Viruses influence the ecology and diversity of phytoplankton in the ocean. Most studies of phytoplankton host-virus interactions have focused on bloom-forming species like Emiliania huxleyi or Phaeocystis spp. The role of viruses infecting phytoplankton that do not form conspicuous blooms have received less attention. Here we explore the dynamics of phytoplankton and algal viruses over several sequential seasons, with a focus on the ubiquitous and diverse phytoplankton division Haptophyta, and their double-stranded DNA viruses, potentially with the capacity to infect the haptophytes. Viral and phytoplankton abundance and diversity showed recurrent seasonal changes, mainly explained by hydrographic conditions. By 454 tag-sequencing we revealed 93 unique haptophyte operational taxonomic units (OTUs), with seasonal changes in abundance. Sixty-one unique viral OTUs, representing Megaviridae and Phycodnaviridae, showed only distant relationship with currently isolated algal viruses. Haptophyte and virus community composition and diversity varied substantially throughout the year, but in an uncoordinated manner. A minority of the viral OTUs were highly abundant at specific time-points, indicating a boom-bust relationship with their host. Most of the viral OTUs were very persistent, which may represent viruses that coexist with their hosts, or able to exploit several host species.

Haptophyte Diversity and Vertical Distribution Explored by 18S and 28S Ribosomal RNA Gene Metabarcoding and Scanning Electron Microscopy.

Haptophyta encompasses more than 300 species of mostly marine pico- and nanoplanktonic flagellates. Our aims were to investigate the Oslofjorden haptophyte diversity and vertical distribution by metabarcoding, and to improve the approach to study haptophyte community composition, richness and proportional abundance by comparing two rRNA markers and scanning electron microscopy (SEM). Samples were collected in August 2013 at the Outer Oslofjorden, Norway. Total RNA/cDNA was amplified by haptophyte-specific primers targeting the V4 region of the 18S, and the D1-D2 region of the 28S rRNA. Taxonomy was assigned using curated haptophyte reference databases and phylogenetic analyses. Both marker genes showed Chrysochromulinaceae and Prymnesiaceae to be the families with highest number of Operational Taxonomic Units (OTUs), as well as proportional abundance. The 18S rRNA data set also contained OTUs assigned to eight supported and defined clades consisting of environmental sequences only, possibly representing novel lineages from family to class. We also recorded new species for the area. Comparing coccolithophores by SEM with metabarcoding shows a good correspondence with the 18S rRNA gene proportional abundances. Our results contribute to link morphological and molecular data and 28S to 18S rRNA gene sequences of haptophytes without cultured representatives, and to improve metabarcoding methodology.

Benthic protists: the under-charted majority.

Marine protist diversity inventories have largely focused on planktonic environments, while benthic protists have received relatively little attention. We therefore hypothesize that current diversity surveys have only skimmed the surface of protist diversity in marine sediments, which may harbor greater diversity than planktonic environments. We tested this by analyzing sequences of the hypervariable V4 18S rRNA from benthic and planktonic protist communities sampled in European coastal regions. Despite a similar number of OTUs in both realms, richness estimations indicated that we recovered at least 70% of the diversity in planktonic protist communities, but only 33% in benthic communities. There was also little overlap of OTUs between planktonic and benthic communities, as well as between separate benthic communities. We argue that these patterns reflect the heterogeneity and diversity of benthic habitats. A comparison of all OTUs against the Protist Ribosomal Reference database showed that a higher proportion of benthic than planktonic protist diversity is missing from public databases; similar results were obtained by comparing all OTUs against environmental references from NCBI's Short Read Archive. We suggest that the benthic realm may therefore be the world's largest reservoir of marine protist diversity, with most taxa at present undescribed.

Seasonal diversity and dynamics of haptophytes in the Skagerrak, Norway, explored by high-throughput sequencing.

Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high-throughput sequencing. From outer Oslofjorden, S Norway, nano- and picoplanktonic samples were collected monthly for 2 years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta-specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA-sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September-October (autumn) and lowest in April-May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP-3-5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in-depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters.

Deep-branching novel lineages and high diversity of haptophytes in the Skagerrak (Norway) uncovered by 454 pyrosequencing.

Microalgae in the division Haptophyta may be difficult to identify to species by microscopy because they are small and fragile. Here, we used high-throughput sequencing to explore the diversity of haptophytes in outer Oslofjorden, Skagerrak, and supplemented this with electron microscopy. Nano- and picoplanktonic subsurface samples were collected monthly for 2 yr, and the haptophytes were targeted by amplification of RNA/cDNA with Haptophyta-specific 18S ribosomal DNA V4 primers. Pyrosequencing revealed higher species richness of haptophytes than previously observed in the Skagerrak by microscopy. From ca. 400,000 reads we obtained 156 haptophyte operational taxonomic units (OTUs) after rigorous filtering and 99.5% clustering. The majority (84%) of the OTUs matched environmental sequences not linked to a morphological species, most of which were affiliated with the order Prymnesiales. Phylogenetic analyses including Oslofjorden OTUs and available cultured and environmental haptophyte sequences showed that several of the OTUs matched sequences forming deep-branching lineages, potentially representing novel haptophyte classes. Pyrosequencing also retrieved cultured species not previously reported by microscopy in the Skagerrak. Electron microscopy revealed species not yet genetically characterised and some potentially novel taxa. This study contributes to linking genotype to phenotype within this ubiquitous and ecologically important protist group, and reveals great, unknown diversity.

Characterisation of three novel giant viruses reveals huge diversity among viruses infecting Prymnesiales (Haptophyta).

We have isolated three novel lytic dsDNA-viruses from Raunefjorden (Norway) that are putative members of the Mimiviridae family, namely Haptolina ericina virus RF02 (HeV RF02), Prymnesium kappa virus RF01 (PkV RF01), and Prymnesium kappa virus RF02 (PkV RF02). Each of the novel haptophyte viruses challenges the common conceptions of algal viruses with respect to host range, phylogenetic affiliation and size. PkV RF01 has a capsid of ~310 nm and is the largest algal virus particle ever reported while PkV RF01 and HeV RF02 were able to infect different species, even belonging to different genera. Moreover, PkV RF01 and HeV RF02 infected the same hosts, but phylogenetic analysis placed them in different groups. Our results reveal large variation among viruses infecting closely related microalgae, and challenge the common conception that algal viruses have narrow host range, and phylogeny reflecting their host affiliation.

454 pyrosequencing to describe microbial eukaryotic community composition, diversity and relative abundance: a test for marine haptophytes.

Next generation sequencing of ribosomal DNA is increasingly used to assess the diversity and structure of microbial communities. Here we test the ability of 454 pyrosequencing to detect the number of species present, and assess the relative abundance in terms of cell numbers and biomass of protists in the phylum Haptophyta. We used a mock community consisting of equal number of cells of 11 haptophyte species and compared targeting DNA and RNA/cDNA, and two different V4 SSU rDNA haptophyte-biased primer pairs. Further, we tested four different bioinformatic filtering methods to reduce errors in the resulting sequence dataset. With sequencing depth of 11000-20000 reads and targeting cDNA with Haptophyta specific primers Hap454 we detected all 11 species. A rarefaction analysis of expected number of species recovered as a function of sampling depth suggested that minimum 1400 reads were required here to recover all species in the mock community. Relative read abundance did not correlate to relative cell numbers. Although the species represented with the largest biomass was also proportionally most abundant among the reads, there was generally a weak correlation between proportional read abundance and proportional biomass of the different species, both with DNA and cDNA as template. The 454 sequencing generated considerable spurious diversity, and more with cDNA than DNA as template. With initial filtering based only on match with barcode and primer we observed 100-fold more operational taxonomic units (OTUs) at 99% similarity than the number of species present in the mock community. Filtering based on quality scores, or denoising with PyroNoise resulted in ten times more OTU99% than the number of species. Denoising with AmpliconNoise reduced the number of OTU99% to match the number of species present in the mock community. Based on our analyses, we propose a strategy to more accurately depict haptophyte diversity using 454 pyrosequencing.

Seasonal dynamics of harmful algae in outer Oslofjorden monitored by microarray, qPCR, and microscopy.

Monitoring of marine microalgae is important to predict and manage harmful algal blooms. Microarray Detection of Toxic ALgae (MIDTAL) is an FP7-funded EU project aiming to establish a multi-species microarray as a tool to aid monitoring agencies. We tested the suitability of different prototype versions of the MIDTAL microarray for the monthly monitoring of a sampling station in outer Oslofjorden during a 1-year period. Microarray data from two different versions of the MIDTAL chip were compared to results from cell counts (several species) and quantitative real-time PCR (qPCR; only Pseudochattonella spp.). While results from generation 2.5 microarrays exhibited a high number of false positive signals, generation 3.3 microarray data generally correlated with microscopy and qPCR data, with three important limitations: (1) Pseudo-nitzschia cells were not reliably detected, possibly because cells were not sufficiently retained during filtration or lysed during the extraction, and because of low sensitivity of the probes; (2) in the case of samples with high concentrations of non-target species, the sensitivity of the arrays was decreased; (3) one occurrence of Alexandrium pseudogonyaulax was not detected due to a 1-bp mismatch with the genus probe represented on the microarray. In spite of these shortcomings our data demonstrate the overall progress made and the potential of the MIDTAL array. The case of Pseudochattonella - where two morphologically similar species impossible to separate by light microscopy were distinguished - in particular, underlines the added value of molecular methods such as microarrays in routine phytoplankton monitoring.

Diversity patterns of uncultured Haptophytes unravelled by pyrosequencing in Naples Bay.

Haptophytes are a key phylum of marine protists, including ~300 described morphospecies and 80 morphogenera. We used 454 pyrosequencing on large subunit ribosomal DNA (LSU rDNA) fragments to assess the diversity from size-fractioned plankton samples collected in the Bay of Naples. One group-specific primer set targeting the LSU rDNA D1/D2 region was designed to amplify Haptophyte sequences from nucleic acid extracts (total DNA or RNA) of two size fractions (0.8-3 or 3-20 µm) and two sampling depths [subsurface, at 1 m, or deep chlorophyll maximum (DCM) at 23 m]. 454 reads were identified using a database covering the entire Haptophyta diversity currently sequenced. Our data set revealed several hundreds of Haptophyte clusters. However, most of these clusters could not be linked to taxonomically known sequences: considering OTUs(97%) (clusters build at a sequence identity level of 97%) on our global data set, less than 1% of the reads clustered with sequences from cultures, and less than 12% clustered with reference sequences obtained previously from cloning and Sanger sequencing of environmental samples. Thus, we highlighted a large uncharacterized environmental genetic diversity, which clearly shows that currently cultivated species poorly reflect the actual diversity present in the natural environment. Haptophyte community appeared to be significantly structured according to the depth. The highest diversity and evenness were obtained in samples from the DCM, and samples from the large size fraction (3-20 µm) taken at the DCM shared a lower proportion of common OTUs(97%) with the other samples. Reads from the species Chrysoculter romboideus were notably found at the DCM, while they could be detected at the subsurface. The highest proportion of totally unknown OTUs(97%) was collected at the DCM in the smallest size fraction (0.8-3 µm). Overall, this study emphasized several technical and theoretical barriers inherent to the exploration of the large and largely unknown diversity of unicellular eukaryotes.