2.1 Sampling, filtration and extraction
Sampling, filtration and extraction methods are described in detail in Leese, Sander et al. 2021. In brief, we sampled in total 102 water samples from the 86 km long Kinzig river every two weeks within a Long-Term Ecological Research (LTER; Mirtl et al., 2018) site at the Rhine-Main-Observatory [RMO,https://deims.org/9f9ba137-342d-4813-ae58-a60911c3abc1] from 24.05.2017 to 29.08.2018. We took three 1-L samples per sampling day at the surface in the middle of the stream, 10 cm above the stream bed in the middle of the stream, with varying stream depth between 0.48 and 2.56 m, and at the riverbank (Fig. 1). After sampling, water samples were put on ice in the field and later stored in a freezer at -20 °C until further processing. Water samples were filtered using a vacuum pump and DNA captured on 0.45 µm Cellulose Nitrate membrane filters (Nalgene, diameter 47 mm). For each day and person who filtered, a negative filter control was included, where only the surrounding air was filtered to control for air and filter contamination. Filters were stored in 1.5 mL Eppendorf tubes filled with 96 % denatured ethanol. DNA extraction was performed following the salt-EtOH-precipitation protocol reported in Weiss & Leese (2016). Subsequent steps included RNA digestion using 1.5 µl RNase A (10 mg/ml, Thermo Fisher Scientific) per sample and clean-up of the samples using a Qiagen MinElute Kit to obtain high-quality DNA.