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Diatoms (Bacillariophyta) are ubiquitous microalgae which produce a siliceous exoskeleton and which make a major contribution to the productivity of oceans and freshwaters. They display a huge diversity, which makes them excellent ecological indicators of aquatic ecosystems. Usually, diatoms are identified using characteristics of their exoskeleton morphology. DNA-barcoding is an alternative to this and the use of High-Throughput-Sequencing enables the rapid analysis of many environmental samples at a lower cost than analyses under microscope. However, to identify environmental sequences correctly, an expertly curated reference library is needed. Several curated libraries for protists exists; none, however are dedicated to diatoms. Diat.barcode is an open-access library dedicated to diatoms which has been maintained since 2012. Data come from two sources (1) the NCBI nucleotide database and (2) unpublished sequencing data of culture collections. Since 2017, several experts have collaborated to curate this library for rbc L, a chloroplast marker suitable for species-level identification of diatoms. For the latest version of the database (version 7), 605 of the 3482 taxonomical names originally assigned by the authors of the rbc L sequences were modified after curation. The database is accessible at https://www6.inra.fr/carrtel-collection_eng/Barcoding-database .

Current freshwater biomonitoring with diatoms is based on microscopic examination of the morphology of their silica skeleton. This standardized approach is time consuming and requires a high degree of taxonomic expertise. Metabarcoding combined with high-throughput sequencing (HTS) has great potential for next-generation biomonitoring applications but requires standardization. Molecular inventories are strongly influenced by the DNA extraction method used, but the effect of extraction protocols has not been tested to enable selection of the best DNA extraction method for HTS metabarcoding. We used 5 DNA extraction methods combining various types of cell lysis and DNA purification to extract DNA from 8 pure diatom cultures and 8 samples from streams and lakes with differing water quality. We compared the methods based on: 1) quality and purity of the extracted DNA, 2) community inventories obtained from HTS targeting the ribulose-1, 5-bisphosphate carboxylase (rbcL) barcode, and 3) similarity between molecular and microscopy-based inventories of community composition and the Specific Pollution-sensitivity Index [SPI]. A method based on GenElute™-LPA had higher extraction efficiency than the 4 commercial kits but had the highest polymerase chain reaction inhibition level. All 5 methods were efficient for HTS, and method did not affect operational taxonomic unit richness. We observed variations in the relative abundance of some taxa within Nitzschia, Amphora, Encyonema, Gomphonema, and Navicula between 2 of the 5 methods, but method did not affect global diatom community composition or SPI values. SPI values calculated from microscopy-based inventories and molecular inventories based on all 5 extraction methods were strongly correlated. For convenience purposes (high DNA quantity and low cost), we encourage standardization of HTS diatom biomonitoring based on the SA-Gen method.

Sea turtles are distributed in tropical and subtropical seas worldwide. They play several ecological roles and are considered important indicators of the health of marine ecosystems. Studying epibiotic diatoms living on turtle shells suggestively has great potential in the study of turtle behavior because diatoms are always there. However, diatom identification at the species level is time consuming, requires well-trained specialists, and there is a high probability of finding new taxa growing on turtle shells, which makes identification tricky. An alternative approach based on DNA barcoding and high throughput sequencing (HTS), metabarcoding, has been developed in recent years to identify species at the community level by using a DNA reference library. The suitabilities of morphological and molecular approaches were compared. Diatom assemblages were sampled from seven juvenile green turtles (Chelonia mydas) from Mayotte Island, France. The structures of the epibiotic diatom assemblages differed between both approaches. This resulted in different clustering of the turtles based on their diatom communities. Metabarcoding allowed better discrimination between turtles based on their epibiotic diatom assemblages and put into evidence the presence of a cryptic diatom diversity. Microscopy, for its part, provided more ecological information of sea turtles based on historical bibliographical data and the abundances of ecological guilds of the diatom species present in the samples. This study shows the complementary nature of these two methods for studying turtle behavior.

A high number of species often represents a relevant redundancy in terms of ecological adaptation strategies. Collecting species to groups based on their functional adaptations can handle this redundancy and obtain the \"real\" functional complexity of ecosystems. Functional traits are proxies of adaptation strategies under particular environmental conditions, and a set of functional traits are interpreted as life-strategies. Organisms with life-strategies occupying a similar niche can be collected in ecological groups (functional group/guild). In this study, we review the latest trait-based approaches and existing attempts at functional classifications in phytobenthos studies. Advantages and shortcomings of these classifications are discussed with perspectives of their utility in ecological status assessment.

Classical biomonitoring techniques have focused primarily on measures linked to various biodiversity metrics and indicator species. Next-generation biomonitoring (NGB) describes a suite of tools and approaches that allow the examination of a broader spectrum of organisational levels - from genes to entire ecosystems. Here, we frame ten key questions that we envisage will drive the field of NGB over the next decade. While not exhaustive, this list covers most of the key challenges facing NGB, and provides the basis of the next steps for research and implementation in this field. These questions have been grouped into current- and outlook-related categories, corresponding to the organization of this paper.

Phylogenetic signal is the tendency for closely related species to display similar trait values as a consequence of their phylogenetic proximity. Ecologists and evolutionary biologists are becoming increasingly interested in studying the phylogenetic signal and the processes which drive patterns of trait values in the phylogeny. Here, we present a new R package, phylosignal which provides a collection of tools to explore the phylogenetic signal for continuous biological traits. These tools are mainly based on the concept of autocorrelation and have been first developed in the field of spatial statistics. To illustrate the use of the package, we analyze the phylogenetic signal in pollution sensitivity for 17 species of diatoms. We present a new R package to measure, test, and explore the phylogenetic signal in biological traits. The package implements functions to plot data, indices to measure the signal, and original methods imported from spatial statistics.

Recent advances in molecular biomonitoring open new horizons for aquatic ecosystem assessment. Rapid and cost-effective methods based on organismal DNA or environmental DNA (eDNA) now offer the opportunity to produce inventories of indicator taxa that can subsequently be used to assess biodiversity and ecological quality. However, the integration of these new DNA-based methods into current monitoring practices is not straightforward, and will require coordinated actions in the coming years at national and international levels. To plan and stimulate such an integration, the European network DNAqua-Net (COST Action CA15219) brought together international experts from academia, as well as key environmental biomonitoring stakeholders from different European countries. Together, this transdisciplinary consortium developed a roadmap for implementing DNA-based methods with a focus on inland waters assessed by the EU Water Framework Directive (2000/60/EC). This was done through a series of online workshops held in April 2020, which included fifty participants, followed by extensive synthesis work. The roadmap is organised around six objectives: 1) to highlight the effectiveness and benefits of DNA-based methods, 2) develop an adaptive approach for the implementation of new methods, 3) provide guidelines and standards for best practice, 4) engage stakeholders and ensure effective knowledge transfer, 5) support the environmental biomonitoring sector to achieve the required changes, 6) steer the process and harmonise efforts at the European level. This paper provides an overview of the forum discussions and the common European views that have emerged from them, while reflecting the diversity of situations in different countries. It highlights important actions required for a successful implementation of DNA-based biomonitoring of aquatic ecosystems by 2030.

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.

Diatoms are used as indicators of freshwater ecosystems integrity. Developing diatom-based tools to assess impact of herbicide pollution is expected by water managers. But, defining sensitivities of all species to multiple herbicides would be unattainable. The existence of a phylogenetic signal of herbicide sensitivity was shown among diatoms and should enable prediction of new species sensitivity. However, diatoms present a cryptic diversity that may lead to variation in their sensitivity to herbicides that would need to be taken into account. Using bioassays, the sensitivity to four herbicides (Atrazine, Terbutryn, Diuron, Isoproturon) was evaluated for 11 freshwater diatom taxa and intraspecific variability was assessed for two of them (Nitzschia palea and Achnanthidium spp.). Intraspecific variability of herbicide sensitivity was always smaller than interspecific variability, but intraspecific variability was more important in N. palea than in Achnanthidium spp. Indeed, one species showed no intraspecific phylogenetic signal (N. palea) whereas the other did (Achnanthidium spp.). On one hand, species boundaries are not set properly for Achnanthidium spp. which encompass several taxa. On the other hand, there is a higher phenotypic plasticity for N. palea. Finally, a phylogenetic signal of herbicide sensitivity was measured at the interspecific level, opening up prospects for setting up reliable biomonitoring tools based on sensitivity prediction, insofar as species boundaries are correctly defined.

The effectiveness of environmental protection measures is based on the early identification and diagnosis of anthropogenic pressures. Similarly, restoration actions require precise monitoring of changes in the ecological quality of ecosystems, in order to highlight their effectiveness. Monitoring the ecological quality relies on bioindicators, which are organisms revealing the pressures exerted on the environment through the composition of their communities. Their implementation, based on the morphological identification of species, is expensive because it requires time and experts in taxonomy. Recent genomic tools should provide access to reliable and high-throughput environmental monitoring by directly inferring the composition of bioindicators’ communities from their DNA (metabarcoding). The French-Swiss program SYNAQUA (INTERREG France-Switzerland 2017–2019) proposes to use and validate the tools of environmental genomic for biomonitoring and aims ultimately at their implementation in the regulatory bio-surveillance. SYNAQUA will test the metabarcoding approach focusing on two bioindicators, diatoms, and aquatic oligochaetes, which are used in freshwater biomonitoring in France and Switzerland. To go towards the renewal of current biomonitoring practices, SYNAQUA will (1) bring together different actors: scientists, environmental managers, consulting firms, and biotechnological companies, (2) apply this approach on a large scale to demonstrate its relevance, (3) propose robust and reliable tools, and (4) raise public awareness and train the various actors likely to use these new tools. Biomonitoring approaches based on such environmental genomic tools should address the European need for reliable, higher-throughput monitoring to improve the protection of aquatic environments under multiple pressures, guide their restoration, and follow their evolution.