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.

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Ciliates are unicellular heterotrophic organisms that play a key role in aquatic planktonic and benthic food webs. Advances in sedimentary DNA (sed-DNA) analysis offer the possibility to integrate these bioindicators in paleoenvironmental reconstructions. In this study, we used the top–bottom paleolimnological approach and metabarcoding techniques applied to sed-DNA to compare the recent and past (i.e. prior to major anthropogenic impacts) ciliate communities of 48 lakes located along an elevation gradient. Our results show an overall decline in the β-diversity in recent time, especially in lowland lakes, which are more strongly exposed to local human pressures. Analyses of the functional groups indicate important restructuration of the food web, including the recent increase in mixotrophs. Moreover, changes in the benthic ciliates were consistent with the widespread increase in deep water anoxia. Our results provided evidence that sed-DNA can uncover information about past ciliate communities on a wide variety of lakes. Overall, our study demonstrates the potential of using ciliates as new paleoindicators, integrating information from the pelagic to the benthic zones, and providing valuable insights into ecosystem functioning through a trait-based functional community approach. As paleoindicator, they thus offer a more holistic view on the long-term changes of aquatic ecosystems.

Recent metabarcoding work in European rivers, including our own studies of Mediterranean rivers in Catalonia (Pérez-Burillo et al. 2020), has shown promising agreement between morphology- (LM) and DNA-based (HTS) assessments of diatom periphyton for the Water Framework Directive. However, in 10 out of the 164 Catalan sites we analysed, the ecological status class was downgraded from “Good”/ “High” with LM to “Moderate”/ “Poor”/ “Bad”by HTS. We call these the “critical” sites and they are especially important because the WFD requires remedial action to be taken by water managers for any river stretch with Moderate or lower status. In order to discover the reasons for downgrading we investigated the contribution of each species to the Indice de Polluosensibilité Spécifique [Specific Pollution-sensitivity Index, IPS] using a “leave-one-out” sensitivity analysis, paying special attention to the critical sites. Discrepancies in IPS between LM and HTS were mainly due to the misidentification and overlooking in LM of a few species, which were better recovered by HTS. This bias was particularly important in the case of Fistulifera saprophila, whose clear underrepresentation in LM was important for explaining 8 out of the 10 critical sites and probably reflected destruction of its very weakly-silicified frustules during sample preparation for LM. Another important bias was brought about by differences between species in the rbcL copy number per cell, which for example affected the relative abundance obtained by HTS for Nitzschia inconspicua and Ulnaria ulna; these species were also identified by the sensitivity analysis as important for the WFD. However, blanket application of a correction factor based on cell volume did not lead to a clear improvement in the agreement between HTS and LM assessments. Only minor IPS discrepancies could be attributed to the incompleteness of the reference library, as most of the abundant and influential species (to the IPS) were well represented there. Finally, we propose that leave-one-out analysis is a valuable method for identifying priority species for isolation and barcoding that are currently not, or only poorly, represented in the DNA reference database.