Mendelian Inheritance Pattern and High Mutation Rates of Microsatellite Alleles in the Diatom Pseudo-nitzchia multistriata

The diatom Pseudo-nitzschia multistriata exhibits a diplontic life cycle composed of an extensive phase of vegetative cell division and a brief phase of sexual reproduction. To explore genotypic stability, we genotyped seven polymorphic microsatellite loci in 26 monoclonal strains over 3–16 months in a culture maintenance regime. Moreover, to assess inheritance patterns of the microsatellite alleles, we genotyped 246 F1 strains resulting from four mating experiments between parental strains of know genotype. Results generally conformed expectations according to Mendelian inheritance patterns, but deviations were detected indicating mutations during sexual reproduction. A total of forty-two mutations were detected in the clonal cultures over time. Microsatellites with more core-repeats accumulated mutations faster. The mutation rate varied significantly across loci and strains. A binomial mass function and a computer simulation showed that the mutation rate was significantly higher during the first months of culture (μ≈3×10-3 per locus per cell division) and decreased to μ≈1×10-3 in the strains kept for 16 months. Our results suggest that genetic mutations acquired in both the vegetative phase and sexual reproduction add to the allelic diversity of microsatellites, and hence to the genotypic variation present in a natural population

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote Small Sub-Unit rRNA sequences with curated taxonomy

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR2, http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientist

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

International audienceAlthough 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

Cryptic Diversity: a Long-lasting Issue for Diatomologists

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Assessment of Species Diversity and Distribution of an Ancient Diatom Lineage Using a DNA Metabarcoding Approach

International audienceBackground: Continuous efforts to estimate actual diversity and to trace the species distribution and ranges in the natural environments have gone in equal pace with advancements of the technologies in the study of microbial species diversity from microscopic observations to DNA-based barcoding. DNA metabarcoding based on Next Generation Sequencing (NGS) constitutes the latest advancement in these efforts. Here we use NGS data from different sites to investigate the geographic range of six species of the diatom family Leptocylindraceae and to identify possible new taxa within the family. Methodology/Principal Findings: We analysed the V4 and V9 regions of the nuclear-encoded SSU rDNA gene region in the NGS database of the European ERA-Biodiversa project BioMarKs, collected in plankton and sediments at six coastal sites in European coastal waters, as well as environmental sequences from the NCBI database. All species known in the family Leptocylindraceae were detected in both datasets, but the much larger Illumina V9 dataset showed a higher species coverage at the various sites than the 454 V4 dataset. Sequences identical or similar to the references of Leptocylindrus aporus, L. convexus, L. danicus/hargravesii and Tenuicylindrus belgicus were found in the Mediterranean Sea, North Atlantic Ocean and Black Sea as well as at locations outside Europe. Instead, sequences identical or close to that of L. minimus were found in the North Atlantic Ocean and the Black Sea but not in the Mediterranean Sea, while sequences belonging to a yet undescribed taxon were encountered only in Oslo Fjord and Baffin Bay. Conclusions/Significance: Identification of Leptocylindraceae species in NGS datasets has expanded our knowledge of the species biogeographic distribution and of the overall diversity of this diatom family. Individual species appear to be widespread, but not all of them are found everywhere. Despite the sequencing depth allowed by NGS and the wide geographic area covered by this study, the diversity of this ancient diatom family appears to be low, at least at the level of the marker used in this study

Global distribution and diversity of Chaetoceros (Bacillariophyta, Mediophyceae): integration of classical and novel strategies

Information on taxa distribution is a prerequisite for many research fields, and biological records are a major source of data contributing to biogeographic studies. The Global Biodiversity Information Facility (GBIF) and the Ocean Biogeographic Information System (OBIS) are important infrastructures facilitating free and open access to classical biological data from several sources in both temporal and spatial scales. Over the last ten years, high throughput sequencing (HTS) metabarcoding data have become available, which constitute a great source of detailed occurrence data. Among the global sampling projects that have contributed to such data are Tara Oceans and the Ocean Sampling Day (OSD). Integration of classical and metabarcoding data may aid a more comprehensive assessment of the geographic range of species, especially of microscopic ones such as protists. Rare, small and cryptic species are often ignored in surveys or mis-assigned with the classical approaches. Here we show how integration of data from various sources can contribute to insight in the biogeography and diversity at the genus- and species-level using Chaetoceros as study system, one of the most diverse and abundant genera among marine planktonic diatoms. Chaetoceros records were extracted from GBIF and OBIS and literature data were collected by means of a Google Scholar search. Chaetoceros references barcodes where mapped against the metabarcode datasets of Tara Oceans (210 sites) and OSD (144 sites). We compared the resolution of different data sources in determining the global distribution of the genus and provided examples, at the species level, of detection of cryptic species, endemism and cosmopolitan or restricted distributions. Our results highlighted at genus level a comparable picture from the different sources but a more complete assessment when data were integrated. Both the importance of the integration but also the challenges related to it were illustrated. Chaetoceros data collected in this study are organised and available in the form of tables and maps, providing a powerful tool and a baseline for further research in e.g., ecology, conservation and evolutionary biology

DinoREF: a curated dinoflagellate (Dinophyceae) reference database for the 18S rRNA gene

<p>Mordret, S., Piredda, R., Vaulot, D., Montresor, M., Kooistra, H. C. F. & Sarno, D. (under revision 2018). DinoREF: a curated dinoflagellate (Dinophyceae) reference database for the 18S rRNA gene. <i>Molecular Ecology Resources.</i><br></p><p><b><br></b></p><p><b>Supplementary Material</b><b> 1 </b></p><p> </p><p>List of 18S sequences included in DinoREF including annotations extracted from GenBank and literature sources (see “read-me” for details on metadata).</p><p><b>Supplementary Material 2</b></p><p>Fasta file (.fast) of sequences used to build DinoREF including the GenBank Accession number of each sequence.</p><p><b>Supplementary Material 3</b></p><p>DinoREF taxonomy file 1 (.taxonomy) with the taxonomy provided by GenBank for each sequence included in the database. </p><p><b>Supplementary Material 4</b></p><p>DinoREF taxonomy file 2 (.taxonomy) with the 8-level taxonomy for each sequence included in the database. </p><p><b>Supplementary Material 5</b></p><p>DinoREF taxonomy file 3 (.taxonomy) with the classification by Superclades for each sequence included in the database.</p><p><b>Supplementary Material 6</b></p><p>List of V4 unique sequences. When multiple 18S sequences share the same V4 region, the sequences IDs (GenBank # and species name) are reported, one after the other, in the same row.</p><p><b>Supplementary Material 7</b></p><p>List of OTUs_98%. When multiple V4 sequences cluster in the same OTU at 98%, the sequences IDs (GenBank # and species name) are reported, one after the other, in the same row.</p><p><b>Supplementary Material 8</b></p><p> Fasta file (.fasta) of sequences of early branching dinoflagellates.<br></p

Morphology and molecular phylogeny of Nitzschia bizertensis sp. nov. - A new domoic acid-producer

International audienceA new toxin-producing marine diatom, Nitzschia bizertensis sp. nov., isolated from the Bizerte Lagoon(Tunisia, Southwest Mediterranean Sea) is, based on studies on eight different strains, characterizedmorphologically by light microscopy, transmission and scanning electron microscopy, and phylogeneti-cally using the nuclear rDNA regions: SSU, ITS1, 5.8S, ITS2 and D1–D3 of the LSU. The species belongs tothe sections Lanceolatae or Lineares as defined by Cleve and Grunow (1880). These sections arecharacterized by species having linear-lanceolate valves with an eccentric raphe where the fibulae doesnot extend into the valve, and are otherwise famous for the lack of characters useful for delineation ofspecies. Nitzschia bizertensis differs from most other species in these sections by having a high density ofinterstriae. The morphological and phylogenetic studies and comparisons with previously describedNitzschia species showed Nitzschia bizertensis sp. nov. to be a new species. Batch culture experimentswere conducted for estimations of maximum growth rate and production of domoic acid (DA).Maximum cellular DA content of the examined strains ranged from 2 x 10-4 to 3.6 x 10-2 pg cells-1. Thetotal DA concentration (pg mL-1) was high already in exponential growth phase maybe due toreinoculation of ‘‘old’’ stationary phase cells, and increased into stationary growth phase where itreached a stationary level varying among the strains from ca. 4500 to 9500 pg mL-1. Nitzschia bizertensisrepresents a new domoic acid-producing diatom and is the second toxin producing Nitzschia species. Theresolution of Nitzschia bizertensis and Nitzschia navis-varingica in different parts of the LSU phylogenetictree, and the recovery of the Pseudo-nitzschia species phylogenetically distant from those two speciessuggests that the ability to produce DA either evolved multiple times independently or was lost multipletimes