Technical challenges in the implementation of macroinvertebrate DNA metabarcoding in river biomonitoring: A case study from peninsular Spain—Advantages, limitations and roadmap

European Union member states are required to monitor the ecological status of their water bodies. The existing official protocols and indices for routine biomonitoring are usually based on the morphological identification of different groups of indicator organisms (biological quality indicators). However, this approach has several limitations in routine use. DNA metabarcoding is presented today as an alternative, as it is potentially able to overcome those limitations. Recent studies have applied DNA metabarcoding in wadeable river biomonitoring throughout the world, including the northwest of the Iberian Peninsula, using benthic macroinvertebrates as bioindicators. This review reanalysed previous data gathered from rivers of the northwest of the Iberian Peninsula as a case study to (a) compare the detection rates of river macroinvertebrate taxa between morphological identification and DNA metabarcoding techniques, both in bulk and water eDNA samples; (b) identify false positive and false negative detections after DNA metabarcoding, as well as other sources of variation that account for the differences in the results of both methodologies; (c) discuss the impact of these differences on DNA metabarcoding reliability for biomonitoring, and propose possible solutions to enhance it; and (d) propose a roadmap for an effective implementation of DNA metabarcoding in river biomonitoring. Several false‐positives, false‐negatives and other methodological sources of error were identified in relation to DNA metabarcoding, as well as other aspects of the sampling methodology and quality water index developments that hinder the routine use of this technique in river biomonitoring. Practical implication . This study shows that several actions need to be taken in order to ensure the effective application of metabarcoding in riverine macroinvertebrate biomonitoring: (1) the compilation of a complete list of species present in the implementation area, (2) the completion of genetic databases for relevant bioindicator species, (3) the development of metabarcoding primer pairs for those species and finally, (4) the adaptation of water quality indices to take advantage of the new taxonomic information provided by metabarcoding.