Search

Diatoms are micro-algal indicators of freshwater pollution. Current standardized methodologies are based on microscopic determinations, which is time consuming and prone to identification uncertainties. The use of DNA-barcoding has been proposed as a way to avoid these flaws. Combining barcoding with next-generation sequencing enables collection of a large quantity of barcodes from natural samples. These barcodes are identified as certain diatom taxa by comparing the sequences to a reference barcoding library using algorithms. Proof of concept was recently demonstrated for synthetic and natural communities and underlined the importance of the quality of this reference library. We present an open-access and curated reference barcoding database for diatoms, called R-Syst::diatom, developed in the framework of R-Syst, the network of systematic supported by INRA (French National Institute for Agricultural Research), see http://www.rsyst.inra.fr/en. R-Syst::diatom links DNA-barcodes to their taxonomical identifications, and is dedicated to identify barcodes from natural samples. The data come from two sources, a culture collection of freshwater algae maintained in INRA in which new strains are regularly deposited and barcoded and from the NCBI (National Center for Biotechnology Information) nucleotide database. Two kinds of barcodes were chosen to support the database: 18S (18S ribosomal RNA) and rbcL (Ribulose-1,5-bisphosphate carboxylase/oxygenase), because of their efficiency. Data are curated using innovative (Declic) and classical bioinformatic tools (Blast, classical phylogenies) and up-to-date taxonomy (Catalogues and peer reviewed papers). Every 6 months R-Syst::diatom is updated. The database is available through the R-Syst microalgae website (http://www.rsyst.inra.fr/) and a platform dedicated to next-generation sequencing data analysis, virtual_BiodiversityL@b (https://galaxy-pgtp.pierroton.inra.fr/). We present here the content of the library regarding the number of barcodes and diatom taxa. In addition to these information, morphological features (e.g. biovolumes, chloroplasts…), life-forms (mobility, colony-type) or ecological features (taxa preferenda to pollution) are indicated in R-Syst::diatom.[br/] Database URL: http://www.rsyst.inra.fr/

International audience; Diatoms are main bioindicators used to assess the ecological quality of rivers, but their identification is difficult and time-consuming. Next Generation Sequencing (NGS) can be used to study communities of microorganisms, so we carried out a test of the reliability of 454 pyrosequencing for estimating diatom inventories in environmental samples. We used small subunit ribosomal deoxyribonucleic acid (SSU rDNA), ribulose-1, 5-bisphosphate carboxylase (rbcL), and cytochrome oxidase I (COI) markers and examined reference libraries to define thresholds between the intra- and interspecific and intra- and intergeneric genetic distances. Based on tests of 1 mock community, we used a threshold of 99% identity for SSU rDNA and rbcL sequences to study freshwater diatoms at the species level. We applied 454 pyrosequencing to 4 contrasting environmental samples (with one in duplicate), assigned taxon names to environmental sequences, and compared the qualitative and quantitative molecular inventories to those obtained by microscopy. Species richness detected by microscopy was always higher than that detected by pyrosequencing. Some morphologically detected taxa may have been persistent frustules from dead cells. Some taxa detected by molecular analysis were not detected by morphology and vice versa. The main source of divergence appears to be inadequate taxonomic coverage in DNA reference libraries. Only a small percentage of species (but almost all genera) in morphological inventories were included in DNA reference libraries. DNA reference libraries contained a smaller percentage of species from tropical (27.1–38.1%) than from temperate samples (53.7–77.8%). Agreement between morphological and molecular inventories was better for species with relative abundance >1% than for rare species. The rbcL marker appeared to provide more reproducible results (94.9% species similarity between the 2 duplicates) and was very useful for molecular identification, but procedural standardization is needed. The water-quality ranking assigned to a site via the Pollution Sensitivity diatom index was the same whether calculated with molecular or morphological data. Pyrosequencing is a promising approach for detecting all species, even rare ones, once reference libraries have been developed.

International audience; DNA barcoding, being developed for biomonitoring, requires a database of reference sequences and knowledge of how much sequences can deviate before they are assigned to separate species. The molecular hunt for hidden species also raises the question of species definitions. We examined whether there are objective criteria for sequence-based species delimitation in diatoms, using Nitzschia palea, an important monophyletic indicator species already known to contain cryptic diversity. Strains from a wide geographical range were sequenced for 28S rRNA, COI and rbcL. Homogeneity indices and the Chao index failed to objectively select a precise number of species existing in N. palea as well as an evolutionary method based on coalescence theory. COI always gave higher diversity estimations than 28S rRNA or rbcL. Mating data did not provide a precise calibration of molecular species thresholds. Rarefaction curves indicated that further MOTUs would be detected with more isolates than we sampled (81 clones, 42 localities). Although some genotypes had intercontinental distributions, there was a positive relationship between genetic and geographical distance, suggesting even higher richness than we assessed, given that many regions were not sampled. Overall, no objective criteria were found for species separation; instead barcoding will need a consensual approach to molecular species limits.

International audience; The recent emergence of barcoding approaches coupled to those of next-generation sequencing (NGS) has raised new perspectives for studying environmental communities. In this framework, we tested the possibility to derive accurate inventories of diatom communities from pyrosequencing outputs with an available DNA reference library. We used three molecular markers targeting the nuclear, chloroplast and mitochondrial genomes (SSU rDNA, rbcL and cox1) and three samples of a mock community composed of 30 known diatom strains belonging to 21 species. In the goal to detect methodological biases, one sample was constituted directly from pooled cultures, whereas the others consisted of pooled PCR products. The NGS reads obtained by pyrosequencing (Roche 454) were compared first to a DNA reference library including the sequences of all the species used to constitute the mock community, and second to a complete DNA reference library with a larger taxonomic coverage. A stringent taxonomic assignation gave inventories that were compared to the real one. We detected biases due to DNA extraction and PCR amplification that resulted in false-negative detection. Conversely, pyrosequencing errors appeared to generate false positives, especially in case of closely allied species. The taxonomic coverage of DNA reference libraries appears to be the most crucial factor, together with marker polymorphism which is essential to identify taxa at the species level. RbcL offers a high resolving power together with a large DNA reference library. Although needing further optimization, pyrosequencing is suitable for identifying diatom assemblages and may find applications in the field of freshwater biomonitoring.

International audience; The Gomphonema parvulum complex includes species displaying considerable morphological variability and a wide geographical distribution. These characteristics make them difficult to identify by microscopy and raise the question of their taxonomic validity and of the possibility of biogeographical differentiation between them. In this context, we isolated 39 G. parvulum s.l. strains from rivers located in a tropical island (Mayotte) and in mainland Europe. By sequencing three DNA fragments (ITS, rbcL and cox1), four clades (A, B, C and D) were clearly identified, and an additional one (B') was distinguishable only on the rbcL sequence. The main four clades can be separated by their morphological criteria, in particular by the shape of the central area, but some overlaps were found between them. We therefore consider that the G. parvulum complex contains at least four semi-cryptic species corresponding to the four main clades. One of them (A) was found only on Mayotte, while two others (C and D) were found only in Europe. The last clade (B) contained strains from both Europe and Mayotte. Pyrosequencing data confirmed the geographical differences in the distribution of these species, suggesting that the G. parvulum complex displays biogeographic structure. (c) 2013 Elsevier GmbH. All rights reserved.

International audience

absent

Sequence analysis of the 18S rDNA gene from 93 taxa belonging to the pennate diatoms plus one centric, Cyclotella meneghiniana Kutzing, were made using two different alignments (Clustal and secondary structure) and two different types of algorithms (neighbour-joining and maximum likelihood). The monophyly of the bacillariacean taxa depends on the type of alignment used for the 18S gene. A secondary structure alignment does not support its monophyly, whereas a Clustal alignment does, but only in the maximum likelihood analysis. The Eunotiales were basal to all other raphid diatoms if a maximum likelihood analysis was used, regardless of the alignment, whereas a neighbour-joining analysis, regardless of the alignment, pulled the Eunotiales inside the raphid diatom sister to one of the bacillariophycean clades in the secondary structure alignment and sister to a monophyletic bacillariophycean clade in the Clustal alignment. The classification of the Bacillariaceae by Krammer & Lange-Bertalot and Round, Crawf...

International audience

International audience; Although diatom taxa have been observed and described for many years using light and electron microscopy, several taxa have called for some clarifications and taxonomic reassessments. This is the case for the order Cymbellales D.G. Mann, which is widely represented in freshwater. The phylogenetic relationships among taxa belonging to this order are not always clear because their taxonomic status has been repeatedly revised. In this study, diatom cells were isolated from rivers in Italy, Luxembourg, Portugal and Spain. In total, 21 18S rDNA gene sequences, representing six genera of Cymbellales (Cymbella C. Agardh, Didymosphenia M. Schmidt, Encyonema Kutzing, Gomphoneis Cleve, Gomphonema Ehrenberg and Reimeria Kociolek & Stoermer) were determined. These sequences were analyzed along with other known GenBank diatom 18S rDNA gene sequences. The results indicate that the Cymbellaceae Greville and Gomphonemataceae Kutzing, especially the genus Gomphonema, are paraphyletic, and that the significance of some of the morphological characteristics traditionally used for classification purposes requires a reassessment. These results also demonstrate the importance of a polyphasic approach combining both morphological and molecular data in attempting to improve the taxonomy and classification system of diatoms.

National audience; Pour utiliser les diatomées en tant qu'indicateur écologique de la qualité des eaux courantes, il est nécessaire de mieux connaître les différentes espèces qui peuplent les rivières. De nombreuses espèces appartenant au genre Gomphonema sont abondamment représentées dans les échantillons benthiques d’eau douce sur le continent européen et dans les îles tropicales. Toutefois, plusieurs taxons sont difficiles à identifier en microscopie optique. C'est le cas du complexe d'espèces Gomphonema parvulum. Cette espèce est connue pour sa forte variabilité morphologique et sa large gamme de tolérance écologique. Les taxonomistes ont souvent des difficultés à distinguer morphologiquement les taxons appartenant à ce complexe d'espèces. La distinction de ces formes est pourtant importante, car elles ont souvent des valeurs écologiques différentes dans les indices de diatomées. Les techniques de biologie moléculaire sont des alternatives intéressantes aux observations microscopiques pour l'étude des diatomées. Plusieurs exemples illustrent la façon dont les séquences d'ADN peuvent être utilisées pour stabiliser la taxonomie (Jahn et al. 2008, Evans et Mann 2009). Les séquences d’ADNr 18S, 28S, ITS, et des gènes rbcL et cox1 de plusieurs souches de Gomphonema parvulum des rivières d’îles tropicales françaises (Mayotte, La Réunion), de France métropolitaine, d’Italie, du Luxembourg, du Portugal et d’Espagne ont été déterminées et comparées. Des analyses morphométriques ont également été réalisées afin d’obtenir des critères morphologiques permettant de séparer les taxa. Quatre groupes de G. parvulum peuvent être distingués. La répartition géographique n’est pas le seul facteur expliquant la séparation de ces quatre clades. Les résultats indiquent donc que le complexe d'espèces G. parvulum pourrait être séparé en quatre taxa distincts.

National audience

Community ecology faces a new challenge as the next-generation sequencing approaches can yield data from hundreds of microbial community samples. This way, combined with accurate and reliable taxonomic assessment, yields hundreds of new data that will contribute to a better understanding of community assemblies formed under various environmental and historical conditions. Algorithms classifying sequences by comparison to a reference library are the most widely used tools for assessing community composition of environmental samples. However, as they are computationally intensive, almost all these algorithms (most standard being BLAST and similar offsprings) use heuristics designed to speed up the database exploration phase, at the cost of being less strict with the quality of the match between a query and a reference. This problem is naturally distributable, as all comparisons (query, reference) are independent. Here, we present a tool enabling comparisons between queries ( say, one million reads) and reference sequences (say, several thousands), and its implementation on two infrastructures: a cluster in MCIA (Mésocentre de Calcul Intensif en Aquitaine) and a production grid EGI. We show how tracking the large number of jobs generated was nearly impossible with gLite, and how this problem could be solved using Dirac. We compare time and quality between a run on Avakas and on the grid EGI. As a perspective, we will develop a user friendly interface enabling this tool to be used routinely on the grid as a diagnostic for a user not acquainted with computing subtleties of the grid.