Sampling of eDNA was conducted at six stations belonging or close to the territory of the city of Hamburg, Germany. Three stations were sampled in the tidal freshwater part of the Elbe estuary downstream (stations A, B) and upstream (station C) of the Hamburg harbor. Three further stations were sampled in two lakes near the estuarine section of the Elbe upstream of the Hamburg harbour: Lake Eichbaum (station D) and Lake Hohendeich (stations E, F; Fig 1; S1 Table). Both lakes are not connected to the Elbe estuary. As all water samples were taken outside of protected areas, no permits were required.

For details on sampling events see S1 Table. The map was created using the Free and Open Source QGIS (Version 3.10 A Coruña; https://www.qgis.org/de/site/). It contains information from OpenStreetMap and OpenStreetMap Foundation, which is made available under the Open Database License. Stations A-C Elbe estuary, Station D Lake Eichbaum, Stations E and F Lake Hohendeich.

At each station, water was collected with a bucket. The bucket was cleaned with commercial bleach-solution at each station and washed twice in the location’s water. Water was filtered via a sterile 50 ml syringe through a sterile Sterivex-VF filter (0.45 μm cartridge filter, PVDF membrane, 10 cm2 filter area; Millipore, Burlington, Massachusetts, USA). At each station, three replicates were taken, filtering 200 ml water for each replicate. For each replicate a fresh water sample was collected. 200 ml was the maximum amount of water that could be pressed through the filter at Elbe estuary stations due to the high load of particles. For consistency, we filtered the same amount of water also at the lake stations, though here filters clogged slower. To assess if higher species diversities would have been recovered if more water had been filtered, we took a fourth replicate at each lake station filtering 1000 ml (this water was taken from the same water as one of the other replicates). The filters were conserved with RNAlater, sealed, placed into a separate plastic zip-lock bag and put on ice immediately. At two stations (Stations B and C), sampling was conducted twice, once at high and once at low tide. These sampling events occurred at most 1.5h from the respective high or low tide event (S1 Table). Two negative controls were taken in the field using distilled water, following the same procedure as for the samples (e.g., taking the distilled water from the cleaned bucket). These were taken after sampling Lake Hohendeich (Station E).

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