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Diatom taxonomy has evolved in recent years, with many new species described and new approaches such as molecular genetics showing the existence of cryptic diversity within currently accepted species. This cryptic diversity is not well understood even for common freshwater genera such as Fragilaria and Ulnaria. The purpose of our study was to define taxon-specific ecological profiles and geographical distributions for unique molecular identifiers (amplicon sequence variants, ASVs) linked to curated taxonomy for these genera. Our goal is to contribute to the development of ecological assessment methods, and to the understanding why we often observe so many diatom species co-occurring in a single sample. We filtered a large (770 samples) metabarcoding dataset with linked environmental data covering several countries in Europe for genetic variants (ASVs) assigned to currently accepted species of our target genera. We studied the geographical distribution of the ASVs, and tested for ASV-pair co-occurrence. We modelled ASV-specific preferences for pH, alkalinity, total nitrogen, total phosphorus and conductivity, and analysed their preference for lakes or streams as habitat. Our study confirmed that there seems to be no general geographical barrier for the distribution of freshwater benthic diatom ASVs in Europe, but that dispersal is not rapid enough to hide historical events. The Fragilaria and Ulnaria ASVs in our study showed considerable overlap in geographical distribution, habitat and ecological preferences. We found evidence that only large differences in preferences for the analysed water chemistry variables prevented the co occurrence of ASVs at the same sites. Instead, Fragilaria and Ulnaria ASVs co-occurred frequently in samples. We found subtle differences in ecological preferences for some ASV pairs, which might in part explain the co occurrence by the avoidance of direct competition. However, the great overlap in distribution and ecological preferences suggests that other factors not studied here were also responsible for the observed co-occurrences and high richness of ASVs found at many sites. To our knowledge, we are the first to use ASVs in combina tion with a curated taxonomy to understand co-occurrence, specific ecological profiles and large-scale geographical distribution for unique identifiers not biased by the quality of reference databases, clustering methods, or non-harmonized morphological identification. Thus, our results can now be used in subsequent projects to interpret ASV occurrences, e.g. for development of ecological assessment methods.

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.

The development of DNA metabarcoding and High-Throughput Sequencing for diatoms is nowadays offering an interesting approach to assess their communities in freshwater and marine ecosystems. In the context of the implementation of these genomic methods to environmental monitoring, protocol constraints are moving from scientific to operational applications, requiring operational guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies.The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design included four freshwater and two marine samples from sites of diverse ecological characteristics. The impact of the preservation and storage was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, community composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflects the site origin. Only rare taxa (below 100 reads) differed among methods and durations. Thus, importance of preservation method choice is important for low-density species (rare, invasive, threatened or toxic species). However, for biomonitoring purposes, freshwater ecological index values were not affected whatever the preservation method and duration considered (including ethanol preservation), reflecting the site ecological status.This study proved that diatom metabarcoding is robust enough to replace or complement the current approach based on morphotaxonomy, paving the way to new possibilities for biomonitoring. Thus, accompanied by operational standards, the method will be ready to be confidently deployed and prescribed in future regulatory monitoring.

International audience; Implementation of DNA metabarcoding for diatoms for environmental monitoring is now moving from a research to an operational phase, requiring rigorous guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies and now need to be rationalised. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design used samples from four freshwater and two marine sites of diverse ecological characteristics. The impact of the sample preservation and storage duration was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, diatom assemblage composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflected the site origin. Only rare taxa (< 100 reads) differed among preservation methods and storage durations. For biomonitoring purposes, freshwater ecological index values were not affected by the preservation method and storage duration tested (including ethanol preservation), all treatments returning the same ecological status for a site. This study contributes to consolidating diatom metabarcoding. Thus, accompanied by operational standards, the method will be ready to be confidently deployed and prescribed in future regulatory monitoring.

Arctic freshwaters support biota adapted to the harsh conditions at these latitudes, but the climate is changing rapidly and so are the underlying environmental filters. Currently, we have limited understanding of broad-scale patterns of Arctic riverine biodiversity and the correlates of alpha- and beta-diversity. Using information from a database set up within the scope of the Arctic Council's Conservation of Arctic Flora and Fauna Circumpolar Biodiversity Monitoring Plan, we analysed patterns and correlates of alpha- and beta-diversity in benthic diatom and macroinvertebrate communities across northern Norway, Sweden, and Finland. We analysed variation in total beta-diversity and its replacement and richness difference components in relation to location of the river reach and its drainage basin (Baltic Sea in the south, the Barents Sea in the east and the north, and the Norwegian Sea in the west), in addition to climate and environmental variables. In both macroinvertebrates and diatoms, the replacement and richness difference components showed wide variation. For macroinvertebrates, the richness difference component was the more important, whereas for diatoms, the replacement component was the more important in contributing to variation in beta-diversity. There was no significant difference in beta-diversity between the three main drainage basins, but species composition differed among the drainage basins. Based on the richness difference component of beta-diversity, climate variables were most strongly associated with community variation in macroinvertebrates. In diatoms, both environmental and climate variables were strongly correlated with community compositional variation. In both groups, there were also significant differences in alpha-diversity among the three main drainage basins, and several taxa were significant indicators of one of these drainage basins. Alpha diversity was greater in areas with a continental climate, while the oceanic areas in the west harboured greatly reduced flora and fauna. The correlates of biodiversity were relatively similar in macroinvertebrates and diatoms. Climate variables, in particular temperature, were the most strongly associated with biodiversity patterns in the Arctic rivers of Fennoscandia. Sedimentary geology may be associated with increased productivity and, to a lesser extent, with sensitivity to acidification. There was considerable variation in community composition across Arctic Fennoscandia, indicating the necessity of protecting several stream reaches or even whole catchments within each region to conserve total riverine biodiversity. Furthermore, it is likely that the predicted changes in temperature in Arctic areas will influence riverine diversity patterns across Fennoscandia.

Efforts to renew marine ecosystem monitoring to include advanced technology and cost-effective methods have been repeatedly called for. The current environmental legislation in European Union (EU) requires also ecosystem monitoring beyond the scope of conventional methods and sampling strategies. Despite several studies showing the benefits of new methods, the progress to adopt the methods in national monitoring programmes under legal requirements has been slow. In this study, we have reviewed the current use of a set of methods in marine monitoring programmes under the EU marine strategy framework directive (MSFD), which calls for a holistic view of the marine environment and thus requires tens of monitoring parameters by different methods. Here we assess how widely the new methods are being adopted in the EU member states implementing the MSFD. Our results show a relatively high adoption rate for certain methods, while others are widely ignored. We compare the results also with the monitoring strategies of the four regional sea conventions. We argue that the adoption of methods in European and regional programmes may positively influence the national use of new methods.

We developed and compared the performance of freshwater benthic diatom indices calculated from (i) traditional morphological species identification, (ii) Amplicon Sequence Variants (ASVs) obtained via DNA metabarcoding, and (iii) morphological traits to indicate eutrophication in rivers and lakes in Fennoscandia. Based on the results, we provided recommendations for the future routine use of diatom bioassessment tools in environmental monitoring and assessment. Our results show that ASVs are the most promising candidates to be used in environmental assessment. Indices based on ASVs correlated better with TotP concentrations than morphological taxa data, whereas the trait indices correlated least. We could see by studying the taxonomic assignments of the ASVs that traditional morphotaxa were divided up into several ASVs with different ecological profiles, which explained part of the better index performance and also encourages further studies on diatom diversity and ecological preferences. In general, ASV- and morphotaxon-specific optima differed slightly between streams and lakes, but were significantly correlated with each other. This means that it should be possible to develop a common index that is applicable in both streams and lakes, but boundary values with respect to TotP might need to be set separately for them. More knowledge on diatom traits is required to enable their use for environmental assessment.