Developed primer sets and samples sequenced for primer validation. Two independent PCR replicates were run and sequenced for each sample. A: Binding sites of the two primer sets (fwhF1+fwhR1 and fwhF2+fwhR2) targeting a 178 and 205 bp fragment internal to the COI Folmer barcoding region (Folmer et al. 1994). The fwhR2 primer was affected by a design error, thus an improved version (fwhR2n) was developed. B: Overview of the sequenced benthic communities and mock samples to test and validate the developed primer sets. Five mock communities (four multi and one single species) from Elbrecht and Leese (2015), as well as three kick samples collected from streams in Romania (Călățele River: L2, Almaşul River: R2, Valea Racilor River: Z2), were collected and tested using the fwh1 and fwh2 primer sets (except for sample DceM that could only be amplified using the fwh1 primer set).