Strains of 21 fungal species (including 17 species of aquatic hyphomycetes) known to occur in local streams, were used to generate reference sequences. Fungal strains were obtained from various streams in Germany, via isolation and cultivation of single spores. The strains were axenically cultured on malt extract agar (20 g/L malt extract, 20 g/L agar) at 16 C in darkness and subcultures were deposited at the Leibniz Institute DSMZ.</p><p>DNA was extracted from 21 fungal isolates using the Qiagen DNeasy PowerPlant Pro Kit. Following DNA extraction, a ca. 4500 bp fragment of the rRNA operon, including small subunit (SSU), ITS1, 5.8S, ITS2, and partial large subunit (LSU) regions, was amplified using the forward NS1short (CAGTAGTCATATGCTTGTC) and reverse RCA95m primers with a 5' mismatch spacer (ACCTATGTTTTAATTAGACAGTCAG). Following the PacBio manufacturer's guidelines on multiplexing SMRT libraries, symmetric 16 mer barcodes were added to the primers. PCRs were conducted in 25 uL reactions using 0.5 uL high processivity Herculase II Fusion enzyme (Agilent Technologies), 5 uL of 5x PCR buffer, 0.62 uL of each primer (10 uM), 0.25 uL dNTPs (250 mM each), filled up with PCR grade water with 2 min denaturation at 95 C, 20 cycles of 94 C for 30 sec, 55 C for 30 sec and 70 C for 4 min and final elongation at 70 C for 10 min. Samples were then pooled equimolarly and sequenced on a PacBio RSII at Macrogen (Seoul, South Korea).