R-Syst::diatom: an open-access and curated barcode database for diatoms and freshwater monitoring.

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. Database URL: http://www.rsyst.inra.fr/.

When is sampling complete? The effects of geographical range and marker choice on perceived diversity in Nitzschia palea (Bacillariophyta).

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.

First evidence of the existence of semi-cryptic species and of a phylogeographic structure in the Gomphonema parvulum (Kützing) Kützing complex (Bacillariophyta).

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.