eDNA sampling and environmental parameters
In order to cover the expected spatial heterogeneity within reservoirs, samples were collected at intervals of approximately 1 km along the river channel, hereafter referred to as “localities”. Based on the length of the river basin, five localities were sampled in Klíčava and Žlutice, and eight localities were sampled in Římov. In each reservoir, one more locality was sampled in a side basin (Figure 1). At each locality, samples were collected at both banks in the littoral zone and from the surface layer of the pelagic zone, approximately halfway between the two littoral samples. Additional deep water samples were collected along a vertical transect 5 m, 10 m and 20 m at all pelagic sites in Klíčava and Žlutice, and at localities 1, 4, 6 and the side basin in Římov. The number of pelagic samples taken depended on water depth at each site (Figure 1). A single sample each was collected from the inflows to the main reservoirs and the side basins respectively. Littoral, pelagic (depth separated), and inflow samples are hereafter referred to as “habitats”. Each site was sampled twice, with sampling campaigns in the summer and late autumn of 2018 (Table 2). During the Klíčava and Žlutice summer campaigns, samples from 5 m depth in the bay were not collected. During the late autumn campaign in Římov, locality 8 (closest to the main tributary) was covered with ice preventing water sample collection. The total number of samples collected in summer/late autumn was 29/30 in Klíčava, 38/35 in Římov and 28/29 in Žlutice.
At each locality, 2 L of water was sampled by pooling five 400 mL subsamples taken within 50 m of the locality. A Friedinger sampler (Karel Šramhauser – Plastické hmoty, CZ) was used for water collection. To avoid excessive seston, the water samples were pre-filtered in the field with a 40 µm sterile plankton net into sterile plastic bottles that were placed in an isolated box with ice for storage at approximately 4 °C until further processing. Sampling equipment was sterilized by soaking for 10 min in 10% commercial bleach solution (containing 5% sodium hypochlorite), followed by soaking for 10 min in 10% microsol detergent solution and rinsing with purified water between localities. The sampler was then rinsed again in reservoir water at the next locality. After water sample collection, field blanks (0.5 L purified water per sampling campaign) were passed through the sampler, then passed through a sterile plankton net back into the storage bottle.
Meteorological conditions (i.e. air temperature, wind speed and cloud cover), water inflow and water level were measured during each sampling event by a floating tower (Aquaread, UK) with multiparametric sensors (Fiedler, CZ) in the dam section of each reservoir. At each pelagic locality, vertical profiles of water temperature and concentration of dissolved oxygen (DO) were measured using a multiparameter probe YSI EXO II (YSI, USA). Chlorophyll a (ChlA) concentrations of four phytoplankton groups (i.e. green algae, diatoms, cyanobacteria and cryptomonads) were measured by the bbe Moldaenke FluoroProbe GmbH.
The field sampling and experimental protocols were performed in accordance with the guidelines and permission from the Experimental Animal Welfare Commission under the Ministry of Agriculture of the Czech Republic (Ref. No. CZ 01679) and the experimental project was approved by the Ministry of Environment of the Czech Republic (Ref. No. MZP/2019/630/16). All methods were approved by the Experimental Animal Welfare Commission of Biology Centre of the Czech Academy of Sciences.