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Environmental DNA (eDNA) extracted from water is routinely used in river biodiversity research, and via metabarcoding eDNA can provide comprehensive taxa lists with little effort and cost. However, eDNA-based species detection in streams and rivers may be influenced by sampling season, location, and other key factors such as water temperature and discharge. Research linking these factors and also informing on the potential of eDNA metabarcoding to detect shifts in ecological signatures, such as species phenology and functional feeding groups across seasons, is missing. To address this gap, we collected 102 water samples every two weeks for 15 months at a long-term ecological research (LTER) site and at three different positions in the river’s cross section, specifically the water surface, riverbed, and riverbank. We analyzed macroinvertebrate species and molecular Operational Taxonomic Unit (OTU) richness and temporal community turnover across seasons and sampling positions based on COI metabarcoding data. Using Generalized Additive Models, we found a significant influence of sampling season but not sampling position on community composition. Community turnover followed a cyclic pattern, reflecting the continuous change of the macroinvertebrate community throughout the year (‘seasonal clock’). Although water temperature had no influence on the inferred community composition, higher discharge reduced the number of Annelida and Ephemeroptera species detectable with eDNA. Most macroinvertebrate taxa showed the highest detection rates in spring, in particular merolimnic species with univoltine life cycles. Further, we detected an increase in proportion of shredders in winter and of parasites in summer. Our results show the usefulness of highly resolved eDNA metabarcoding time series data for ecological research and biodiversity monitoring in streams and rivers.

Environmental DNA (eDNA) metabarcoding has become a powerful tool for examining fish communities. The demand for methodological standardization and the implementation of eDNA-based assessments into the regulatory monitoring (e.g., Water Framework Directive) are imminent. To ensure methodical accuracy and to meet regulatory standards, various sampling, laboratory and bioinformatic workflows have been established. However, a crucial prerequisite for a comprehensive fish monitoring is the choice of suitable primer pairs to accurately depict the present fish fauna. Various fish-specific primer pairs targeting different genetic marker regions were published over the past decade. However, a dedicated study to evaluate performance of frequently applied fish primer pairs to assess Central European fish species has not yet been conducted. Therefore, we created an artificial community composed of DNA from 45 Central European fish species and examined the discriminatory power and reproducibility of five fish primer pairs. Our study highlights the effect of the primer choice and bioinformatic filtering on the outcome of eDNA metabarcoding results. From the five primer pairs evaluated in our study the tele02 (12S gene) primer pair proved to be best choice for eDNA metabarcoding of Central European freshwater fish. Here, the MiFish-U (12S) and SeaDNA-mid (COI) primer pairs also displayed good discriminatory power and reproducibility. However, more general primer pairs (i.e., targeting vertebrates) were found to be less reliable and generated high numbers of false-positive and false-negative detections. Our study illustrates how careful selection of primer pairs and bioinformatic pipelines can make eDNA metabarcoding a more reliable tool for fish monitoring.

We examined the spread and distribution in the Danube River of a new alien gastropod species, Clathrocaspia knipowitschii (Makarov, 1938) (Gastropoda: Hydrobiidae: Caspiinae). First findings of this species for Hungary, Slovakia, Romania, Bulgaria and Serbia are presented. Clathrocaspia knipowitschii was initially found in 2013 in the Iron Gate stretch of the Danube River at the border between Romania and Serbia. In 2019 and 2020, the species was found at several sites in the lower Danube in Romania, Serbia and Bulgaria, and also upstream in the middle Hungarian Danube in high population densities. The species appears to have spread along more than 800 km in six years. This finding together with the available abundance data indicates that C. knipowitschii is potentially an invasive species, but further observations are needed.