Metadata standards and practical guidelines for specimen and DNA curation when building barcode reference libraries for aquatic life

DNA barcoding and metabarcoding is increasingly used to effectively and precisely assess and monitor biodiversity in aquatic ecosystems. As these methods rely on data availability and quality of barcode reference libraries, it is important to develop and follow best practices to ensure optimal quality and traceability of the metadata associated with the reference barcodes used for identification. Sufficient metadata, as well as vouchers, corresponding to each reference barcode must be available to ensure reliable barcode library curation and, thereby, provide trustworthy baselines for downstream molecular species identification. This document (1) specifies the data and metadata required to ensure the relevance, the accessibility and traceability of DNA barcodes and (2) specifies the recommendations for DNA harvesting and for the storage of both voucher specimens/samples and barcode data.info:eu-repo/semantics/publishedVersio

Environmental filtering and mass effect are two important processes driving lake benthic diatoms: Results of a DNA metabarcoding study in a large lake

International audienceEnvironmental filtering is often found to dominate assembly rules in diatoms. These microalgae are diverse, especially at subspecies level, and tend to exhibit a niche phylogenetic conservatism. Therefore, other rules, such as competition or mass effects, should be detectable when environmental gradients and dispersal barriers are limited. We used metabarcoding to analyse benthic littoral diatom communities in 153 sites in a large lake (Geneva) exhibiting weak geographical barriers and weak environmental gradients outside river estuaries. We assessed assembly rules using variance partitioning, phylogenetic and source tracking analyses. No phylogenetic over-dispersion of communities, indicative of exclusive competition, was detected. Instead, we found these communities to be phylogenetically over-clustered, indicating environmental filtering, which was even stronger near river estuaries where environmental gradients are stronger. Finally, using a Bayesian method (SourceTracker), we found that rivers flowing into the lake bring communities that settle, especially in sites close to estuaries. Rivers with the highest discharges are primarily responsible for immigration, explaining 27% of lake composition. Therefore, despite favourable conditions to observe other rules, our results support that diatom communities are prominently assembled by environmental filtering and immigration processes, in particular from rivers. However, this does not exclude that other assembly rules may be at play at a finer spatial, temporal and/or phylogenetic scale

Phytool, a ShinyApp to homogenise taxonomy of freshwater microalgae from DNA barcodes and microscopic observations

Methods for biomonitoring of freshwater phytoplankton are evolving rapidly with eDNA-based methods, offering great complementarity with microscopy. Metabarcoding approaches have been more commonly used over the last years, with a continuous increase in the amount of data generated. Depending on the researchers and the way they assigned barcodes to species (bioinformatic pipelines and molecular reference databases), the taxonomic assignment obtained for HTS DNA reads might vary. This is also true for traditional taxonomic studies by microscopy with regular adjustments of the classification and taxonomy. For those reasons (leading to non-homogeneous taxonomies), gap-analyses and comparisons between studies become even more challenging and the curation processes to find potential consensus names are time-consuming. Here, we present a web-based application (Phytool), developed with ShinyApp (Rstudio), that aims to make the harmonisation of taxonomy easier and in a more efficient way, using a complete and up-to-date taxonomy reference database for freshwater microalgae. Phytool allows users to homogenise and update freshwater phytoplankton taxonomical names from sequence files and data tables directly uploaded in the application. It also gathers barcodes from curated references in a user-friendly way in which it is possible to search for specific organisms. All the data provided are downloadable with the possibility to apply filters in order to select only the required taxa and fields (e.g. specific taxonomic ranks). The main goal is to make accessible to a broad range of users the connection between microscopy and molecular biology and taxonomy through different ready-to-use functions. This study estimates that only 25% of species of freshwater phytoplankton in Phytobs are associated with a barcode. We plead for an increased effort to enrich reference databases by coupling taxonomy and molecular methods. Phytool should make this crucial work more efficient. The application is available at https://caninuzzo.shinyapps.io/phytool_v1

Which barcode to decipher freshwater microalgal assemblages? Tests on mock communities

International audienceMicroalgae taxa can be identified with short DNA barcodes. Eight primer pairs for 16S and five for 23S were tested in silico and with mock communities including cyanobacteria and eukaryotic microalgae. We conclude that the 23S primer pair ECLA23S_F1/ECLA23S_R1 was the best candidate to decipher freshwater phytoplankton communities

Assessing the relevance of DNA metabarcoding compared to morphological identification for lake phytoplankton monitoring

International audiencePhytoplankton is a key biological group used to assess the ecological status of lakes. The classical monitoring approach relies on microscopic identification and counting of phytoplankton species, which is time-consuming and requires high taxonomic expertise. High -throughput sequencing, combined with metabarcoding, has recently demonstrated its potential as an alternative approach for plankton surveys. Several studies have confirmed the relevance of the diatom metabarcoding approach to calculate biotic indices based on species ecology. However, phytoplankton communities have not yet benefited from such validation. Here, by comparing the results obtained with the two methods (molecular and microscopic counting), we evaluated the relevance of metabarcoding approach for phytoplankton monitoring by considering different metrics: alpha diversity, taxonomic composition, community structure and a phytoplankton biotic index used to assess the trophic level of lakes. For this purpose, 55 samples were collected in four large alpine lakes (Aiguebelette, Annecy, Bourget, Geneva) during the year 2021. For each sample, a metabarcoding analysis based on two genetic markers (16S and 23S rRNA) was performed, in addition to the microscopic count. Regarding the trophic level of lakes, significant differences were found between index values obtained with the two approaches. The main hypothesis to explain these differences comes from the incompleteness, particularly at the species level, of the barcode reference library for the two genetic markers. It is therefore necessary to complete reference libraries for using such species -based biotic indices with metabarcoding data. Besides this, species richness and diversity were higher in the molecular inventories than in the microscopic ones. Moreover, despite differences in taxonomic composition of the floristic lists obtained by the two approaches, their community structures were similar. These results support the possibility of using metabarcoding for phytoplankton monitoring but in a different way. We suggest exploring alternative approaches to index development, such as a taxonomy -free approach