Electronic Supplementary Materials

<b>Electronic Supplementary Materials </b>for the article "Comparative performance of bulk and eDNA metabarcoding data in freshwater biomonitoring under the Water Framework Directive".<b>Supplementary Material 1. </b>List of sampling sites and their detailed characteristics processed using DNA metabarcoding in this study. (Supplementary Material 1.xlsx)<b>Supplementary Material 2. </b>List of species (DNA dataset) obtained in this study using the DNA metabarcoding approach after merging replicates in TTT. The dataset includes results for individual sampling sites and sample types, with species presence indicated by read counts and absence marked as 0. (Supplementary Material 2.xlsx)<b>Supplementary Material 3. </b>List of metrics used to calculate the Multimetric Index for ten different stream types. (Supplementary Material 3.xlsx)<b>Supplementary Material 4. </b>Reference values for metrics used in partial EQR (Ecological Quality Ratio) calculations by stream type. (Supplementary Material 4.xlsx)<b>Supplementary Material 5. </b>Graphical representation of the number of detected taxa across different taxonomic levels (species, genus, family, order) for three datasets (OTUs, Linnaean species, and ASTERICS) and sample types (bulk, water, sediment). (Supplementary Material 5.tiff)<b>Supplementary Material 6. </b>Sequencing depth across sample types (bulk, water, and sediment eDNA), expressed as log-transformed read counts per locality. Box boundaries represent the 25th and 75th percentiles, the central line indicates the median, and points represent individual site values (Supplementary Material 6.tiff).<b>Supplementary Material 7. </b>Values of individual metrics from ASTERICS and EQR calculations for ecological status assessment: individual sheets for each stream type include metric values (A.) and calculated partial EQRs (B.); additional sheets summarize all partial EQRs, final multimetric EQRs, and classification into five ecological quality classes (Supplementary Material 7.xlsx)<b>Supplementary Material 8a</b>. List of all detected EPT taxa (Ephemeroptera, Plecoptera, Trichoptera), including their occurrence across individual sample types (bulk, water, and sediment) (Supplementary Material 8a.xlsx).<b>Supplementary Material 8b.</b> List of all families contributing to the BMWP score and Number of Families metrics, including their presence across sample types (bulk, water, and sediment) and corresponding BMWP scores (Supplementary Material 8b.xlsx).<b>Supplementary Material 9.</b> Boxplots showing differences in EQR values between sample types, including results of Tukey HSD post-hoc tests. EQR values were calculated excluding metrics EPT taxa, BMWP score, and Number of Families. Box boundaries represent the 25th and 75th percentiles, the central line indicates the median, and points represent individual EQR values for each site. Coloured background bands indicate ecological quality classes (EQC): red – bad, orange – poor, yellow – moderate, green – good, and blue – very good. The figure also includes a table summarising results of parametric (one-way ANOVA) and non-parametric (Kruskal–Wallis) tests comparing EQR values among sample types for the multimetric index as well as for individual metrics. The ANOVA section includes degrees of freedom (between-group and within-group), F-statistic, and p-values (Supplementary Material 9.tiff).