DOI: 10.5281/zenodo.7382262
title: Pond bat capture and diet data from the Netherlands

description: The goal of this study was to describe the spatial segregation and diet of Pond bats (<em>Myotis dasycneme</em>). 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 10 µM forward primer, 0,5 µl 10 µM reverse primer, 1,5 µl 25mM MgCl<sub>2, </sub>0,5 µl 10 mg/ml BSA, 19.80 µl MiliQ and 2 µl template. Amplifications were performed using the following PCR programme: 5 min denaturation at 95°C followed by 40 cycles of 20 seconds denaturation at 95°C, 20 seconds annealing at 50°C and 1-minute elongation at 72°C. Final elongation was conducted at 72°C for 7 minutes on a C1000 Biorad PCR machine. Primer dimer and other contaminants were removed by using 0.9x Ampure XP beads (Agencourt) to which the PCR products were bound. The beads were washed with 150 microliter 70% EtOH twice and resuspended in 20 microliter TE buffer. Cleaned PCR products were quantified using an Agilent 2100 Bioanalyzer DNA High sensitivity chip. An equimolar pool was prepared of the amplicon libraries at the highest possible concentration. This equimolar pool was diluted according to the calculated template dilution factor to target 10-30% of all positive Ion Sphere Particles. Template preparation and enrichment was carried out with the Ion One Touch 200 Template kit with use of the Ion One Touch System, according to the manufacturer's protocol. The quality control of the Ion One Touch 200 Ion Sphere Particles was done with the Ion Sphere Quality Control kit using a Life Qubit 2.0. The enriched Ion Spheres were prepared for sequencing on a Personal Genome Machine (PGM) with the Ion PGM 200 Sequencing kit as described in the protocol and deposited on an Ion-314 chip (520 cycles per run) in three consecutive sequencing runs. Reads obtained from Ion Torrent sequencing were automatically sorted into separate sequence files based on the MID labels by the Ion Torrent software. The reads were further processed with PRINSEQ (version 0.20.3) with the following settings: a minimum read length of 100 bp, trimming to 140 bp, minimum mean quality of Q24 per read, additional trimming of '3 end bases with a Q lower than 24 and removal of full duplicate sequences. Filtered reads were clustered into Operational Taxonomic Units (OTUs) defined by a sequence similarity of at least 97% using CD-HIT-EST. Singletons were omitted. For each cluster the representative sequences were BLASTed with the NCBI-blast+ software package (version 2.2.28+) against either the NCBI GenBank nucleotide database or a custom database containing all Arthropod sequences located on the Barcode of Life Database. BLAST hits were filtered according to the following criteria: minimum hit length of a 100 bp, minimum hit similarity of 97% and a maximum e-value of 0.05. Reference databases of Dutch species such as http://www.nederlandsesoorten.nl/ were used to check if a species had been recorded for the Netherlands. All species not (yet) known for The Netherlands were reduced to genus level or to the family level if the genus is also unknown to occur. Description of the data files: <strong>pelletsMicroscopy.csv</strong> Details of individual faecal pellets with prey remains analysed using microscopic analysis. ID: pellet ID sex: sex of the caught Pond bat individual age: age class of the bat date: capture night province: province in which the bat was captured x and y: spatial coordinates within the Netherlands waterDepth: water depth in meters at the capture location pelletWeight: weight of the pellet in grams year: capture year dayOfYear: day of the year, since January 1 period: I: end of hibernation till May 20, II May 21- June 29, III: June 30- July 30 and IV: July 31 till hibernation meanPreyWeight: average weight of prey in mg peat: whether the capture site was located in peatland or not propPupae: the proportion of pupae of chironomids: the number of pupae divided by the total number of organisms of all species in a pellet evenness: Pielou’s evenness of the abundance of prey items in a pellet shannon: Shannon index of the diversity of prey items in a pellet saFemale and saMale: sexual activity status of females and males at the time of capture temp: mean temperature (in 0.1 degrees Celsius) during the first two hours after sunset during the capture night wind: mean wind speed (in 0.1 m/s) during the first two hours after sunset during the capture night nPupae: number of Chironomidae pupae found in a pellet nPrey: total number of prey items found in a pellet <strong>pelletsMetabarcoding.csv</strong> Details of individual faecal pellets analysed using metabarcoding. ID: pellet ID sex: sex of the caught Pond bat individual reproductiveState: reproductive status of the bat age: age class of the bat mature: sexually mature (1) or not (0) date: capture night peat: whether the capture site was located in peatland or not province: province in which the bat was captured x and y: spatial coordinates within the Netherlands waterDepth: water depth in meters at the capture location pelletWeight: weight of the pellet in grams <strong>preyWeight.csv</strong> Mean weight (milligram) of taxonomic and developmental prey groups, including information on length (mm) and width (mm) of each group. <strong>orderPellet.csv</strong> Number of pellets in which at least 1 prey of a certain taxon is found, separately per sex of the bat and per analysis method (microscopy or metabarcoding). Total number of analyzed pellets was 365 for females – microscopy, 170 for males – microscopy, 95 for females – metabarcoding, and 65 for males – metabarcoding. <strong>taxaCountsMicroscopy.csv</strong> Number of prey individuals per taxonomic group found by using morphological analyses (microscopy) of faecal pellets of Pond bats. For each group the number of observations are given separately for male and female bats. <strong>taxaCountsMetabarcoding.csv</strong> Number of prey individuals per taxonomic group found by using metabarcoding of faecal pellets of Pond bats. For each group the number of observations are given for both male and female bats.