DOI: 10.5061/dryad.v6wwpzh2b
title: Data from: Wildlife fecal microbiota exhibit community stability across a semi-controlled longitudinal non-invasive sampling experiment

description: Wildlife microbiome studies are being used to assess microbial links with
 animal health and habitat. The gold standard of sampling microbiomes
 directly from captured animals is ideal for limiting potential abiotic
 influences on microbiome composition, yet fails to leverage the many
 benefits of non-invasive sampling. Application of microbiome-based
 monitoring for rare, endangered, or elusive species creates a need to
 non-invasively collect scat samples shed into the environment. Since
 controlling sample age is not always possible, the potential influence of
 time-associated abiotic factors was assessed. To accomplish this, we
 analyzed partial 16S rRNA genes of fecal metagenomic DNA sampled
 non-invasively from Rocky Mountain elk (Cervus canadensis) near
 Yellowstone National Park. We sampled pellet piles from four different
 elk, then aged them in a natural forest plot for 1, 3, 7, and 14 days,
 with triplicate samples at each time point (i.e., a blocked, repeat
 measures (longitudinal) study design). We compared microbiomes of each elk
 through time with point estimates of diversity, bootstrapped hierarchical
 clustering of samples, and a version of ANOVA–simultaneous components
 analysis (ASCA) with PCA (LiMM-PCA) to assess the variance contributions
 of time, individual and sample replication. Our results showed community
 stability through days 0, 1, 3 and 7, with a modest but detectable change
 in abundance in only 2 genera (Bacteroides and Sporobacter) at day 14. The
 total variance explained by time in our LiMM-PCA model across the entire
 2-week period was not statistically significant (p&gt;0.195) and the
 overall effect size was small (&lt;10% variance) compared to the
 variance explained by the individual animal (p&lt;0.0005; 21% var.).
 We conclude that non-invasive sampling of elk scat collected within one
 week during winter/early spring provides a reliable approach to
 characterize microbiome composition in a 16S rDNA survey and that sampled
 individuals can be directly compared across unknown time points with
 minimal bias. Further, point estimates of microbiome diversity were not
 mechanistically affected by sample age. Our assessment of samples using
 bootstrap hierarchical clustering produced clustering by animal (branches)
 but not by sample age (nodes). These results support greater use of
 non-invasive microbiome sampling to assess ecological patterns in animal
 systems.
description: 1.1 Sample collection Scat samples from 4
 elk were collected near the northern boundary of Yellowstone National Park
 in Montana in March 2016. Animal sampling was conducted non-invasively
 within 15 minutes of defecation. Elk sex and age could not be accurately
 determined due to these samples being collected after observing the elk
 defecating from a distance using binoculars. Based on our observations,
 they were most likely adult females or young males. Fecal samples from
 each scat pile (i.e., individual) were collected from the ground with
 sterile gloves and forceps and placed in sterile whirl-pak sample bags.
 Sample whirl-paks were placed on wet ice in a cooler in the field for
 transportation to the experimental site. The experimental site was located
 on a sparsely forested plot near Evaro, MT with conditions known to be
 suitable as elk habitat, at approximately 4000 ft elevation.
 Three pellets from each animal were frozen at -20° C after
 arriving at the experimental site approximately 6 hours post-defecation.
 This initial subsample represents time-point zero samples (and technical
 replicates) with minimal exposure to ambient conditions typical of a
 direct or capture-based sampling scheme. The remaining pellets from each
 elk were placed in square plastic culture plates (25 cm x 25 cm) with a
 grid backing using sterile gloves and forceps. Each culture plate had a
 larger glass plate suspended above it at a height of 4 cm using a cork
 stopper in each corner to allow air flow and prevent direct contact with
 incidental precipitation (although no precipitation occurred on-site
 during the study), and the group of culture plates was surrounded by
 protective wire fencing. One plate was used for each technical replicate,
 with each replicate plate containing samples from all four individuals
 (for photos of the enclosure and a schematic of the experimental layout
 see Supplemental Figure 1). The samples were exposed to ambient conditions
 from March 27th through April 9th (14 days). Three samples from each elk
 were removed from the replicate plates after 1 day, 3 days, 7 days, and 14
 days and immediately frozen at ‑20° C after removal from ambient
 conditions. A total of 60 elk pellets were experimentally
 collected. Temperature was logged in 10-minute
 increments during the study using Thermocron temperature loggers
 (OnSolution Pty Ltd, Australia) distributed above and below the culture
 plates and shielded from direct sunlight. The temperature data were
 aggregated into hourly oscillations, daily max and minimum, and a smoothed
 average temperature. Additional temperature recordings were obtained from
 a NOAA weather station (Point 6, MT) 3.5 miles and 4000 ft above our site
 as reference. 1.1 Sample preparation, DNA extraction
 and sequencing Frozen elk fecal pellets (stored frozen
 at -20° C) were prepared for DNA extractions by separating a standard
 weight (250 mg) cross-section from each pellet using a sterile petri dish
 (10 cm) and sterile safety razor blade for each sample. This fraction was
 placed into a designated sample tube from the Qiagen PowerSoil DNA
 extraction kit (Qiagen Inc., Germantown, MD) and processed using the
 manufacturer’s recommended protocol. The resulting purified metagenomic
 DNA was eluted with 100 µL PCR-grade water and stored at -20° C prior to
 further analysis.             To assess the bacterial
 community present in the fecal DNA extraction, we used a
 generally-conserved (i.e., “universal”) 16S/18S barcoded primer set (536F
 and 907R) designed to amplify the V4 and V5 variable regions of the rRNA
 gene (Holben et al., 2004) and PCR using 1mL of elk fecal sample
 metagenomic DNA standardized to 25ng/mL as template. Once amplified,
 samples were gel purified using the QIAGEN Gel Purification kit (QIAGEN,
 Germantown, MD) following the manufacturer’s recommended protocol for
 downstream direct sequencing. An Illumina MiSeq platform (San Diego, CA,
 USA) was used to obtain 300 base-pair (bp) paired-end sequencing using the
 Illumina MiSeq Reagent Kit.
description: # Data from: Wildlife fecal microbiota exhibit community stability across
 a longitudinal semi-controlled non-invasive sampling experiment
 **Data overview:**
 Directory contains 120 paired-end gzipped FASTQ files from Illumina MiSEQ
 paired-end 300 bp sequencing prior to any manipulation. Each file responds
 to a sequenced 16S metabarcoded bacterial community from an elk fecal
 pellet. This study consisted of 4 elk, repeatedly sampled across 5 time
 points (Day 0, 1, 3, 7, and 14) in triplicate for a total of 60 sampling
 points. Each sampling point has a forward and reverse read file for a
 total of 120 paired files. Also included is a tab separated mapping file
 (.txt) identifying file names with associated sample information. Study
 results can be found in the Frontiers in Microbiomes publication. A brief
 overview is included below. **Study purpose:** We designed an experiment
 to quantify the potential bias of sample age (i.e. time since defecation)
 on fecal microbiota of a North American ungulate, where sampling takes
 place in late winter/early spring conditions, typically within a 2-4 day
 window. The purpose was to assess potential community compositional
 changes through time that might confound experimental observations and
 interpretation when samples come from an unknown timeline. We sampled
 fecal pellets from four Rocky Mountain elk (*Cervus canadensis*)
 non-invasively, ***but at the time of defecation***, near Yellowstone
 National Park, Montana in March 2016. The longitudinal component of this
 fecal microbiota experiment was conducted in a forested plot near Evaro,
 MT beginning on the day of collection after transporting the fecal pellet
 samples in sterile whirl-pak bags on ice. In our design, sample age is
 controlled by repeatedly subsampling elk fecal pellets originally sampled
 from the same individual across day 0, 1, 3, 7, and 14, in triplicate.
 Thus, our hierarchical block experimental design captures compositional
 fecal microbiota change through time within four elk biological replicates
 (blocks) and estimates the variation within each block using three
 technical replicates for each sample/time combination. **File naming
 scheme:** Files are named in order starting with project "ETS"
 (Elk Time Series) followed by a combined digit block beginning with sample
 age [0-14] (1or 2 digits), elk individual biological replicate [1-4], and
 technical replicate [1-3], with dot separated forward (.1) or reverse (.2)
 reads. Example, "ETS_1442.1.fq.gz" is Day [14], Elk [4], Rep
 [2], forward read (.1) gziped fastq file (.fq.gz). For a summary of all
 FASTQ files see the mapping file included as a tab separated text
 document. ## Code: