Molecular techniques allow non-invasive dietary studies from faeces, providing an invaluable tool to unveil ecological requirements of endangered or elusive species. They contribute to progress on important issues such as genomics, population genetics, dietary studies or reproductive analyses, essential knowledge for conservation biology. Nevertheless, these techniques require general methods to be tailored to the specific research objectives, as well as to substrate- and species-specific constraints. In this pilot study we test a range of available primers to optimise diet analysis from metabarcoding of faeces of a generalist aquatic insectivore, the endangered Pyrenean desman (Galemys pyrenaicus, É. Geoffroy Saint-Hilaire, 1811, Talpidae), as a step to improve the knowledge of the conservation biology of this species. Twenty-four faeces were collected in the field, DNA was extracted from them, and fragments of the standard barcode region (COI) were PCR amplified by using five primer sets (Brandon-Mong, Gillet, Leray, Meusnier and Zeale). PCR outputs were sequenced on the Illumina MiSeq platform, sequences were processed, clustered into OTUs (Operational Taxonomic Units) using UPARSE algorithm and BLASTed against the NCBI database. Although all primer sets successfully amplified their target fragments, they differed considerably in the amounts of sequence reads, rough OTUs, and taxonomically assigned OTUs. Primer sets consistently identified a few abundant prey taxa, probably representing the staple food of the Pyrenean desman. However, they differed in the less common prey groups. Overall, the combination of Gillet and Zeale primer sets were most cost-effective to identify the widest taxonomic range of prey as well as the desman itself, which could be further improved stepwise by adding sequentially the outputs of Leray, Brandon-Mong and Meusnier primers. These results are relevant for the conservation biology of this endangered species as they allow a better characterization of its food and habitat requirements.

1. The Pyrenean desman (Galemys pyrenaicus) is an endangered stream-dwelling insectivore endemic to the Iberian Peninsula and the north of the Pyrenees. It favours riffles over slow habitats such as runs or pools, yet it is still unclear whether habitat preference is based on prey availability or on other factors, such as mechanical constraints on its hunting habits. 2. Desman diet and prey selection were analysed along the pristine Elama Stream and the slightly modified Leitzaran Stream in the Basque Country, Spain.3. In each stream the prey consumed were identified by metabarcoding 94 desman faeces, and prey availability characterized from 10 Surber samples taken in each habitat type (riffles, runs and pools). Invertebrates were sorted and identified, the biomass of each taxon was calculated, and their DNA was also extracted, amplified using PCR, and sequenced to build a reference database. 4. Prey availability and diet varied between streams. Desmans positively selected shredders and invertebrates that live on the substrate, and selected against taxa that live in fine sediment, taxa with hard shells or those that are highly mobile.The diet of desmans was more selective in the Elama, the stream with better ecological status. Food availability did not differ among habitats, and therefore does not explain the preference of desmans for riffles; rather, mechanical constraints to deal with buoyancy and physical habitat heterogeneity were hypothesized to be the reason behind the habitat preference.5. The results suggest that promoting the formation of riffles in streams can improve the situation of some desman populations living in degraded habitats.

1. The Pyrenean desman (Galemys pyrenaicus) is an endangered, semi-aquatic, insectivore mammal, endemic to the northern Iberian Peninsula and the Pyrenees. Owing to its small populations, evasive behaviour, and nocturnal activity, knowledge of its ecological requirements is still limited. Continuing population decline over most of its distribution rangeeven in regions where water quality has clearly improvedpoints to other factors as the main conservation threat. Nevertheless, at present there is a lack of information on its habitat preferences within its area of occupancy (e.g. stream microhabitat characteristics), a key point for assessing or improving its habitat.2. This study used radio-telemetry data to determine the use of space at microhabitat level by the desman, and how this changed depending upon environmental conditions. 3. Desmans were studied in two contrasting rivers in the Basque Country (northern Iberian Peninsula): Elama, a nearly pristine stream, and the Leitzaran, a clean-water stream affected by hydropower diversions. Fifteen desmans were captured and radio-tracked in Elama and 16 were captured and radio-tracked in the Leitzaran, and nocturnal activity points were assigned to one of three habitat types: riffles, runs, or pools. Habitat use was compared against availability to measure habitat selection in each stream and between streams. 4. Desmans selected riffles positively and pools negatively, with this selection being stronger in the Leitzaran. 5. The results highlight the ecological relevance of riffles as foraging habitats of desmans, and therefore as key features for their conservation. It suggests that channel modifications that reduce the areal cover of riffles impair habitat quality for this species. In addition, water diversion for hydropower is likely to be detrimental for desmans, as it reduces discharge and flow velocity in the bypassed river sections.

In this study, the combined effects of hydrological and chemical stressors on benthic macroinvertebrates were evaluated in order to explore the response of the biological community to multiple stressors. The Adige River, located in the south‐eastern Alps, was selected as a case study because representative of the situation of a large river in which the variety of stressors present in the Alpine region act simultaneously. As expected, streamflow showed a seasonal pattern, with high flows in the spring–summer period; however, locally, the natural hydrological regime was altered by the presence of hydropower systems, which chiefly affected low flows. Multivariate analysis showed seasonal and spatial patterns in both chemical and hydrological parameters with a clear gradient in the concentration of nitrate, personal care, and pharmaceutical products moving from headwaters to the main stem of the river. The macroinvertebrate community composition was significantly different in summer and winter and between up and downstream sites. Streamflow alteration chiefly due to water use by hydropower affected community composition but not richness or diversity. Gammarus sp., Hirudinea, and Psychomyia sp., were positively correlated with flow variability, increasing their densities in the sites with higher streamflow variability because of hydropeaking. The results obtained in this study show that the composition of the macroinvertebrate community responded to seasonality and to changes in the main stressors along the river and highlights the importance of the spatial and temporal variability of stressors in this Alpine river. Taking into account, this variability will help the decision‐making process for improving basin management.