Traditional DSFI analysis and conversion to ecological status classes
Sampling of 53 Danish stream ecosystem sites using the kick-sampling method (Bradey & Ormerod, 2002) and subsequent DSFI analysis was performed by the Laboratory of Fish Ecology (Fiskeøkologisk Laboratorium, Helsingør, Denmark) in accordance with the Danish standardised DSFI protocol (Skriver, 1999). The samples used for the DSFI analysis was stored in 96 % ethanol at 4 °C until further analysis. After DSFI analysis, the scores were converted to ecological quality classes using a previously proposed distribution (Baattrup-Pedersen et al., 2004). An overview of all samples, their DSFI quality category and ecological quality class is shown in Table 1.

Sample homogenisation and DNA extraction

Prior to DNA extraction, samples were decanted using a mesh sieve (0.5 mm) and homogenised with a blender (JB 5160 BK, Braun GmbH, Germany) using 10 second cycles at speed setting 3. Total genomic DNA was extracted from 0.25 g of homogenised material using the QIAamp PowerFecal DNA kit (Qiagen, USA) according to manufacturer’s recommendations. Extracted DNA was eluted to 50 µL elution buffer from the kit. Concentration of the extracted DNA was determined using Quant-IT Broad Range DNA assay kit (Thermo Fisher Scientific, USA) and an M200 Infinite PRO plate reader (TECAN, Switzerland).