The goal of this study was to describe the spatial segregation and diet of Pond bats (Myotis dasycneme). A wide range of water-bound habitats throughout the Netherlands was sampled, including marshes, lakes, rivers, wetlands and waterways. For all the locations water depth and soil type were determined. Life animals were captured during 471 nights using a mist net. No Pond bats were captured during 134 of those nights. Water depth and soil type was based on top 10 vector maps (www.pdok.nl/geo-services) with information about both variables. Each captured individual was placed in a separate cotton holding bag until it was weighted, sexed, and the reproductive status and age were assessed by observation of external characteristics. Before dissecting, the dry weight of each faecal pellet was measured with an electronic scale. All samples were dissected under a Carl Zeiss Discovery V20 stereomicroscope. All identifiable fragments were photographed and stored for later use. Genetic analysis: The pellets were ground to a fine powder in liquid nitrogen with a mortar and pestle using the protocol of the commercial Qiagen QIAamp DNA Stool Mini Kit in a special Ancient DNA facility dedicated to work with samples with degraded DNA and following established protocols to avoid contamination such as the inclusion of extraction blanks. Subsequently, aliquots of each extraction were further purified using Promega PCR purification columns. Amplifications of the ~313 bp long mitochondrial COI mini-barcoding marker were performed using forward primer ZBJ-ArtF1c 5’-AGATATTGGAACWTTATATTTTATTTTTGG-3’ and reverse primer ZBJ-ArtR2c 5’- WACTAATCAATTWCCAAATCCTCC-3’. The ~157 bp long mitochondrial 16S barcoding marker was amplified using the forward primer P7_FO-16S 5’- RGACGAGAAGACCCTATARA-3’ and P7_R0-16S 5’-ACGCTGTTATCCCTAARGTA-3’. Primers were labelled for DNA metabarcoding with IonExpress labels. The PCR was carried out in 30 microliter reactions containing 0.20 µl Qiagen taq 5u/µl, 3 µl 10x Qiagen buffer, 2 µl 2,5mM dNTP’s, 0,5 µl ...