The influence of databases enrichment using local macroinvertebrate genetic references for metabarcoding based biodiversity studies in river monitoring

•Incomplete genetic databases impact metabarcoding-based river biomonitoring.•About 21% of macroinvertebrate species in the Iberian Peninsula are not barcoded.•Sequencing local macroinvertebrates improved assignments and biodiversity estimates.•A complete checklist of Iberian river macroinvertebrate species is required. Water resources play a vital role in sustaining ecosystems, human life, food security, and biodiversity. However, these resources currently face serious threats caused primarily by human activities. Because of that, European Union have mandated that all its member states monitor the ecological status of their water bodies. One of the most commonly used bioindicator groups for routine river biomonitoring is benthic macroinvertebrates, which rely mainly on morphological identification at the family level and the use of diverse indices to estimate the ecological status of the river ecosystem. The morphological approach has certain limitations, and metabarcoding is now considered an alternative technique for biodiversity studies and biomonitoring of surface waters. However, the efficacy of this approach depends on the comparison of sequencing data with genetic databases, which are often incomplete and geographically biased. In this study, we collected 92 macroinvertebrate samples from rivers in the northern Iberian Peninsula to identify all possible species and generate new DNA sequences for the COI-5P region. The objectives were to perform region-specific enrichment of reference databases for the geographical area of northern Spain and to assess the impact, before and after this strategy, on the taxonomic assignments used for ecological status inference in metabarcoding samples. The results showed that 21% of morphospecies lacked reference sequences in the BOLD or GenBank databases, indicating that a significant number of macroinvertebrate species in the northwestern Iberian Peninsula lack reference sequences in major genetic databases. Enriching the BOLD databases with new sequences led to the detection of an average of 17.25 more BINs (species-assimilable gene cluster) and 1.54 more IBMWP taxa (river ecological status indicator macroinvertebrate taxa) per sample, causing changes in the ecological status determination for 16% of the samples, which clearly shows that improving the databases with local species sequences could enhance the detection and identification of new IBMWP taxa, potentially impacting ecological status inference from molec