4.1 Influence of sampling time on species richness and community composition 
Our results indicated that differences in species richness were caused by seasonality but not sample replicates, which matched our expectations. High similarity among sampling positions likely occurred due to the complete mixing of eDNA in the water body, with smaller compositional differences occurring by replication, the three water samples taken within the sampling site, and not by systematic differences between positions (Macher et al., 2021). The stream sampled in our study had a water depth <1 m and sampling positions were less then 10 m apart with a mostly turbulent flow, all of which can reduce community heterogeneity (see Shogren et al., 2017; Fremier et al., 2019 for further discussions).
Temporal beta-diversity shifted in a seasonally cyclical and gradual pattern, which was primarily driven by turnover, with turnover and nestedness both returning to the same level after one year. The potential of eDNA in detecting seasonal shifts in community composition has been already shown for marine environments (Jensen et al., 2022; Liu et al., 2022) but not for freshwater.