DOI: 10.5061/dryad.000000022
title: Thermal stratification and fish thermal preference explain vertical eDNA distributions in lakes

description: Significant advances have been made towards surveying animal and plant
 communities using DNA isolated from environmental samples. Despite rapid
 progress, we lack a comprehensive understanding of the “ecology” of
 environmental DNA (eDNA), particularly its temporal and spatial
 distribution and how this is shaped by abiotic and biotic processes. Here,
 we tested how seasonal variation in thermal stratification and animal
 habitat preferences influence the distribution of eDNA in lakes. We
 sampled eDNA depth profiles of five dimictic lakes during both summer
 stratification and autumn turnover, each containing warm- and cool-water
 fishes as well as the cold-water stenotherm, lake trout (Salvelinus
 namaycush). Habitat use by S. namaycush was validated by acoustic
 telemetry and was significantly related to eDNA distribution during
 stratification. Fish eDNA became “stratified” into layers during summer
 months, reflecting lake stratification and the thermal niches of the
 species. During summer months, S. namaycush, which rarely ventured into
 shallow waters, could only be detected at the deepest layers of the lakes,
 whereas the eDNA of warm-water fishes was much more abundant above the
 thermocline. By contrast, during autumn lake turnover, the fish species
 assemblage as detected by eDNA was homogenous throughout the water column.
 These findings contribute to our overall understanding of the “ecology” of
 eDNA within lake ecosystems, illustrating how the strong interaction
 between seasonal thermal structure in lakes and thermal niches of species
 on very localised spatial scales influences our ability to detect species.
description: <b>eDNA
 metabarcoding</b> Sampling was conducted at the
 IISD Experimental Lakes Area (IISD-ELA), a remote research and monitoring
 facility in north-western Ontario, Canada. We sampled two lakes in summer
 and autumn of 2017 and repeated the summer and autumn sampling in five
 lakes in 2018. There are 14 species of fish across all the study lakes
 (mean 8, range 6-10 species per lake). All lakes have overlapping
 community compositions, including Salvelinus namaycush, a cold-water top
 predator, in every lake. Water samples were taken at six depths, dispersed
 vertically throughout the water column at the deepest centre point of each
 lake. The sampling points were distributed at six evenly spaced intervals,
 but because the lakes were different depths, absolute measurements differ
 between the lakes. We refer to the shallowest depth as sampling point one
 and the deepest depth as point six. Four 500 ml replicate water samples
 were taken per depth (for a total of 24 samples per lake per season) using
 an electrical pump and Jayflex PVC tubing (Winnipeg Johnston Plastics, MB,
 Canada) secured to a weight. In total, 336 samples were taken throughout
 the entire study (24 samples x 2 lake states x seven lake replicates).
 Water was filtered onto 47 mm 0.7 μm pore GF/F filters using an electric
 vacuum pump and filtering manifold (Pall Corporation, ON, Canada). The
 filters were immediately stored in screw-cap tubes at -20 ⁰C and then
 shipped on dry ice to McGill University, Montréal for molecular analysis.
 DNA was extracted from filters using the Qiagen Blood and Tissue kit. All
 samples were treated with the OneStep PCR Inhibitor Removal Kit (Zymo
 Research, Irvine, California). DNA was amplified in triplicate 12.5 µl
 reactions using 12S MiFish-U primers selected to target fish assemblages
 tagged with Illumina adapters. PCR replicates from each sample were
 combined and cleaned with a 1 : 0.875 ratio of AMPure beads. Samples were
 dual-indexed with v2 Nextera DNA indexes (Illumina). The samples were
 cleaned again with AMPure beads, quantified and equimolarised to 3 ng/μl
 for sequencing. Sequencing was conducted using 2 x 250 bp Illumina MiSeq
 at Génome Québec, Montréal. We used custom scripts to remove adapters,
 merge paired sequences, check quality and generate amplicon sequencing
 variants (ASVs). Samples were received as demultiplexed fastq files from
 Génome Québec. Non-biological nucleotides were removed (primers, indices
 and adapters) using cutadapt. Paired reads were merged using PEAR. Quality
 scores for sequences were analysed with FASTQC. Amplicon sequencing
 variants (ASVs) were generated using the UNOISE3 package, which uses a
 denoising pipeline to remove sequencing error and to cluster sequences
 into single variants (100% similarity). The full pipeline is available
 from https://github.com/CristescuLab/YAAP. After ASVs were generated, we
 assigned taxonomy using BLAST+ and BASTA, a last common ancestor
 algorithm. We used a custom reference database which contained only fish
 known to exist in the Lake of the Woods region (Ontario, CA), downloaded
 from the NCBI database on 12 August 2018. <b>Acoustic
 telemetry</b> Salvelinus namaycush were
 captured by angling and surgically implanted with coded, acoustic,
 pressure-sensing telemetry tags (model V13P-1L; Vemco, Innovasea, Bedford,
 NS). Between five and ten tagged adults were monitored in each lake during
 the study period. The pressure sensor on each tag was calibrated in the
 lake it was deployed in prior to implantation to ensure accurate depth
 readings (resolution: 0.08-0.15 m). The tags randomly emitted signals
 every 120-300 s (lakes 373, 626 and 239) or every 110-250 s (lakes 223 and
 224). A number of data logging receivers (VR2W, 69 kHz; Vemco, Innovasea,
 Bedford, NS) were deployed under water at specific locations in the lake
 such that the “listening radius” of each receiver (spherical volume ~350 m
 diameter) overlapped slightly with the other receivers, resulting in
 maximum coverage of the lake. Each receiver was attached to a floating
 buoy and suspended ~2 m below the water’s surface or ~2-4 m above the
 bottom of the lake (dependent on mooring apparatus design). The receivers
 logged acoustic signals emitted by the tags through an omnidirectional
 hydrophone. Data (fish ID, date, time, pressure sensor reading) were
 continuously collected except when receivers were removed from the lake
 and downloaded (~8 h duration per lake, semi-annually). The pressure
 sensor data were converted to depth information using Vemco VUE software
 for each detection for the duration of the study (yielding ~200-700 depth
 detections for each fish in a typical 24-hr period). After downloading,
 duplicate detections (single tag signals detected by more than one
 receiver) were removed. In order to assess whether different time periods
 of cumulative eDNA persistence in the lakes affected the relationship
 between eDNA counts and telemetry data, we grouped telemetry data for each
 fish at different temporal scales, ranging from the day of eDNA sample
 collection, as well as one week, and one month prior to sample collection.
 The total number of detections of all fish were grouped into depth
 intervals reflecting the vertical distribution of the eDNA sampling (6
 intervals per lake). We adjusted for varying depth interval size and
 variation in the total amount of telemetry detections for each lake over
 the relevant time period.
description: All instructions for use are found in the README file.