Species sensitivity analysis as a tool for interpreting diatom metabarcoding for WFD bioassessment

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