Environmental DNA (eDNA) metabarcoding has revolutionized biomonitoring in both marine and freshwater ecosystems. However, for semi‐aquatic and terrestrial animals, the application of this technique remains relatively untested. We first assess the efficiency of eDNA metabarcoding in detecting semi‐aquatic and terrestrial mammals in natural lotic ecosystems in the UK by comparing sequence data recovered from water and sediment samples to the mammalian communities expected from historical data. Secondly, using occupancy modelling we compared the detection efficiency of eDNA metabarcoding to multiple conventional non‐invasive survey methods (latrine surveys and camera trapping). eDNA metabarcoding detected a large proportion of the expected mammalian community within each area. Common species in the areas were detected at the majority of sites. Several key species of conservation concern in the UK were detected by eDNA sampling in areas where authenticated records do not currently exist, but potential false positives were also identified. Water‐based eDNA metabarcoding provided comparable results to conventional survey methods in per unit of survey effort for three species (water vole, field vole and red deer) using occupancy models. The comparison between survey ‘effort’ to reach a detection probability of ≥.95 revealed that 3–6 water replicates would be equivalent to 3–5 latrine surveys and 5–30 weeks of single camera deployment, depending on the species. Synthesis and applications. eDNA metabarcoding can be used to generate an initial ‘distribution map’ of mammalian diversity at the landscape level. If conducted during times of peak abundance, carefully chosen sampling points along multiple river courses provide a reliable snapshot of the species that are present in a catchment area. In order to fully capture solitary, rare and invasive species, we would currently recommend the use of eDNA metabarcoding alongside other non‐invasive surveying methods (i.e. camera traps) to maximize monitoring efforts.

Our understanding of trophic interactions of small insectivorous mammals has been drastically improved with the advent of DNA metabarcoding. The technique has continued to be optimised over the years, with primer choice repeatedly being a vital factor for dietary inferences. However, the majority of dietary studies examining the effect of primer choice often rely on in silico analyses or comparing between species that occupy an identical niche type. Here, we apply DNA metabarcoding to empirically compare the prey detection capabilities of two widely used primer sets when assessing the diets of a flying (lesser horseshoe bat; Rhinolophus hipposideros) and two ground-dwelling insectivores (greater white-toothed shrew; Crocidura russula and pygmy shrew; Sorex minutus). Although R. hipposideros primarily rely on two prey orders (Lepidoptera and Diptera), the unique taxa detected by each primer shows that a combination of primers may be the best approach to fully describe bat trophic ecology. However, random forest classifier analysis suggests that one highly degenerate primer set detected the majority of both shrews’ diet despite higher levels of host amplification. The wide range of prey consumed by ground-dwelling insectivores can therefore be accurately documented from using a single broad-range primer set, which can decrease cost and labour. The results presented here show that dietary inferences will differ depending on the primer or primer combination used for insectivores occupying different niches (i.e., hunting in the air or ground) and demonstrate the importance of performing empirical pilot studies for novel study systems.

We focus on a case study along an English canal comparing environmental DNA (eDNA) metabarcoding with two types of electrofishing techniques (wade‐and‐reach and boom‐boat). In addition to corroborating data obtained by electrofishing, eDNA provided a wider snapshot of fish assemblages. Given the semi‐lotic nature of canals, we encourage the use of eDNA as a fast and cost‐effective tool to detect and monitor whole fish communities.

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