In diet metabarcoding analyses, insufficient taxonomic coverage of PCR primer sets generates false negatives that may dramatically distort biodiversity estimates. In this paper, we investigated the taxonomic coverage and complementarity of three cytochrome c oxidase subunit I gene (COI) primer sets based on in silico analyses and we conducted an in vivo evaluation using fecal and spider web samples from different invertivores, environments, and geographic locations. Our results underline the lack of predictability of both the coverage and complementarity of individual primer sets: (a) sharp discrepancies exist observed between in silico and in vivo analyses (to the detriment of in silico analyses); (b) both coverage and complementarity depend greatly on the predator and on the taxonomic level at which preys are considered; (c) primer sets’ complementarity is the greatest at fine taxonomic levels (molecular operational taxonomic units [MOTUs] and variants). We then formalized the “one‐locus‐several‐primer‐sets” (OLSP) strategy, that is, the use of several primer sets that target the same locus (here the first part of the COI gene) and the same group of taxa (here invertebrates). The proximal aim of the OLSP strategy is to minimize false negatives by increasing total coverage through multiple primer sets. We illustrate that the OLSP strategy is especially relevant from this perspective since distinct variants within the same MOTUs were not equally detected across all primer sets. Furthermore, the OLSP strategy produces largely overlapping and comparable sequences, which cannot be achieved when targeting different loci. This facilitates the use of haplotypic diversity information contained within metabarcoding datasets, for example, for phylogeography and finer analyses of prey–predator interactions.

Dietary studies are critical for understanding foraging strategies and have important applications in conservation and habitat management. We applied a robust metabarcoding protocol to characterize the diet of the critically endangered freshwater fish Zingel asper and conducted modelling and simulation analyses to characterize and identify some of the drivers of individual trophic trait variation in this species. We found that intra-specific competition and ontogeny had minor effects on the trophic niche of Z. asper. Instead, our results suggest that the majority of trophic niche variation was driven by seasonal variation in ecological opportunity (in our case, the seasonal variation in the availability of preferred prey types). Overall, our results are in line with the optimal foraging theory and suggest that Z. asper is specialized on a few ephemeropteran prey species (Baetis fuscatus and Ecdyonurus) but adapts its foraging by becoming more opportunistic as its favoured prey seasonally decline. Despite the now widespread usage of metabarcoding, very few studies have attempted to study inter- and intra-populational individual trophic traits variation with metabarcoding data. This study illustrates how metabarcoding data obtained from feces can be combined with modelling and simulation approaches to test hypotheses in the conventional analytic framework of trophic analysis.

Aim: Invasive alien species are a growing problem worldwide due to their ecological, economic and human health impacts. The "killer shrimp" Dikerogammarus villosus is a notorious invasive alien amphipod from the Ponto-Caspian region that has invaded many fresh and brackish waters across Europe. Understandings of large-scale population dynamics of highly impactful invaders such as D. villosus are lacking, inhibiting predictions of impact and efficient timing of management strategies. Hence, our aim was to assess trends and dynamics of D. villosus as well as its impacts in freshwater rivers and streams.Location: Europe. Methods:We analysed 96 European time series between 1994 and 2019 and identified trends in the relative abundance (i.e. dominance %) of D. villosus in invaded time series, as well as a set of site-specific characteristics to identify drivers and determinants of population changes and invasion dynamics using meta-regression modelling.We also looked at the spread over space and time to estimate the invasion speed (km/year) of D. villosus in Europe. We investigated the impact of D. villosus abundance on recipient community metrics (i.e. abundance, taxa richness, temporal turnover, Shannon diversity and Pielou evenness) using generalized linear models.Results: Population trends varied across the time series. Nevertheless, community dominance of D. villosus increased over time across all time series. The frequency of occurrences (used as a proxy for invader spread) was well described by a Pareto distribution, whereby we estimated a lag phase (i.e. the time between introduction and spatial expansion) of approximately 28 years, followed by a gradual increase before new occurrences declined rapidly in the long term. D. villosus population change was associated with decreased taxa richness, community turnover and Shannon diversity. Main Conclusion:Our results show that D. villosus is well-established in European waters and its abundance significantly alters ecological communities. However, the multidecadal lag phase prior to observed spatial expansion suggests that initial introductions by D. villosus are cryptic, thus signalling the need for more effective early detection methods.