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      Annotated 18S and 28S rDNA reference sequences of taxa in the planktonic diatom family Chaetocerotaceae

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          Abstract

          The species-rich diatom family Chaetocerotaceae is common in the coastal marine phytoplankton worldwide where it is responsible for a substantial part of the primary production. Despite its relevance for the global cycling of carbon and silica, many species are still described only morphologically, and numerous specimens do not fit any described taxa. Nowadays, studies to assess plankton biodiversity deploy high throughput sequencing metabarcoding of the 18S rDNA V4 region, but to translate the gathered metabarcodes into biologically meaningful taxa, there is a need for reference barcodes. However, 18S reference barcodes for this important family are still relatively scarce. We provide 18S rDNA and partial 28S rDNA reference sequences of 443 morphologically characterized chaetocerotacean strains. We gathered 164 of the 216 18S sequences and 244 of the 413 28S sequences of strains from the Gulf of Naples, Atlantic France, and Chile. Inferred phylogenies showed 84 terminal taxa in seven principal clades. Two of these clades included terminal taxa whose rDNA sequences contained spliceosomal and Group IC1 introns. Regarding the commonly used metabarcode markers in planktonic diversity studies, all terminal taxa can be discriminated with the 18S V4 hypervariable region; its primers fit their targets in all but two species, and the V4-tree topology is similar to that of the 18S. Hence V4-metabarcodes of unknown Chaetocerotaceae are assignable to the family. Regarding the V9 hypervariable region, most terminal taxa can be discriminated, but several contain introns in their primer targets. Moreover, poor phylogenetic resolution of the V9 region affects placement of metabarcodes of putative but unknown chaetocerotacean taxa, and hence, uncertainty in taxonomic assignment, even of higher taxa.

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          A Method for Studying Protistan Diversity Using Massively Parallel Sequencing of V9 Hypervariable Regions of Small-Subunit Ribosomal RNA Genes

          Background Massively parallel pyrosequencing of amplicons from the V6 hypervariable regions of small-subunit (SSU) ribosomal RNA (rRNA) genes is commonly used to assess diversity and richness in bacterial and archaeal populations. Recent advances in pyrosequencing technology provide read lengths of up to 240 nucleotides. Amplicon pyrosequencing can now be applied to longer variable regions of the SSU rRNA gene including the V9 region in eukaryotes. Methodology/Principal Findings We present a protocol for the amplicon pyrosequencing of V9 regions for eukaryotic environmental samples for biodiversity inventories and species richness estimation. The International Census of Marine Microbes (ICoMM) and the Microbial Inventory Research Across Diverse Aquatic Long Term Ecological Research Sites (MIRADA-LTERs) projects are already employing this protocol for tag sequencing of eukaryotic samples in a wide diversity of both marine and freshwater environments. Conclusions/Significance Massively parallel pyrosequencing of eukaryotic V9 hypervariable regions of SSU rRNA genes provides a means of estimating species richness from deeply-sampled populations and for discovering novel species from the environment.
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            A rapid DNA isolation procedure for samll quantities of fresh leaf tissue

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              Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing.

              Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: ResourcesRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: InvestigationRole: Visualization
                Role: ConceptualizationRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SupervisionRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SupervisionRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                26 December 2018
                2018
                : 13
                : 12
                : e0208929
                Affiliations
                [1 ] Integrative Marine Ecology Department, Stazione Zoologica Anton Dohrn, Napoli, Italy
                [2 ] Royal Botanic Garden Edinburgh, Edinburgh, Scotland, United Kingdom, and Institut de Recerca i Tecnologia Agroalimentaries, Sant Carles de La Ràpita, Catalonia, Spain
                National Cheng Kung University, TAIWAN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                [¤]

                Current address: Faculty of Agriculture and Marine Science, Kochi University, Monobe, Nankoku, Kochi, Japan

                Author information
                http://orcid.org/0000-0001-9697-5301
                http://orcid.org/0000-0002-8641-9739
                Article
                PONE-D-18-20644
                10.1371/journal.pone.0208929
                6306197
                30586452
                db8ed0ba-f065-42a8-8fc9-981720372738
                © 2018 Gaonkar et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 11 July 2018
                : 26 November 2018
                Page count
                Figures: 2, Tables: 1, Pages: 20
                Funding
                Funded by: Italian Ministry of Education, University and Research
                Award ID: RBAP10A2T4
                Award Recipient :
                Funded by: FP7 Research infrastructures (ASSEMBLE)
                Award ID: 227799
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100010661, Horizon 2020 Framework Programme;
                Award ID: 654008
                Award Recipient :
                CCG has been supported by a PhD fellowship from Stazione Zoologica Anton Dohrn (SZN). RB was supported by the project FIRB Biodiversitalia (RBAP10A2T4) funded by the Italian Ministry of Education, University and Research. WK was supported by the EU funded project ASSEMBLE (grant agreement 227799) and Horizon 2020 Framework Programme grant 654008. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
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                Biology and Life Sciences
                Evolutionary Biology
                Evolutionary Systematics
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                Phylogenetic Analysis
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                Taxonomy
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                Research and Analysis Methods
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                Sequence Alignment
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                Evolutionary Processes
                Speciation
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                Custom metadata
                DNA sequences for the different strains have been deposited in GenBank (see S1_Table.xlsx). The 18S and 28S alignments have been placed in fasta format on FigShare, https://figshare.com/articles/Chaetoceros_LSU_and_SSU_reference_sequences_and_metadata_table/7275923. The curated sequences will be included in the PR2 and SILVA databases.

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