DOI: 10.5061/dryad.hx3ffbgh7
title: Aardvark microsatellite data for southern and east Africa

description: Aim: As climate change accelerates, assessing how climate shapes
 gene flow and neutral and adaptive genetic differentiation on landscapes
 is increasingly important. Aardvarks (Orycteropus afer) are ecologically
 important in sub-Saharan Africa but are sensitive to human pressures and
 increasing aridity. We used individual, population, and landscape genetic
 approaches to infer the influence of landscape, climate, and potential
 adaptive differences on gene flow. Location: We surveyed 8 protected and 4
 privately owned areas in South Africa, 2 protected areas in Eswatini, and
 one location in Kenya during 2016–2018. Methods: We developed
 microsatellite markers and methods for DNA extraction from feces,
 collected and genotyped fecal samples from focal areas, and estimated
 genetic structure. We inferred space use from individual redetections,
 tested for close relatives, and estimated genetic neighborhood distance.
 We applied individual-based landscape genetic analyses at multiple scales
 across South Africa to test hypotheses about genetic differentiation by
 landscape resistance and potential adaptive differences. Results: We
 developed 19 variable microsatellite loci and collected 253 fecal samples
 from 13 focal areas. We genotyped 104 samples successfully at ≥8 loci as
 needed for individual identification. Genetic structure suggested 3
 regional divisions in South Africa. We detected individuals at locations
 ≤7.3 km distant and closely related individuals at ≤44 km; genetic
 neighborhood distance was &lt;55 km. Lower precipitation increased
 landscape resistance and strongly predicted genetic differentiation at
 most spatial scales. Temperature differences at sampling sites also
 influenced structure, suggesting climate-associated adaptive differences.
 Main Conclusions: Genetic structure of aardvarks in South Africa and
 Eswatini is strongly shaped by climate, with arid areas limiting gene
 flow, and reflects apparent isolation by adaptation associated with
 temperature. Dispersal distances likely are &lt;45 km. The markers we
 developed will facilitate studies of space use, dispersal, population
 density, or survival. Aridification will increase fragmentation and we
 recommend monitoring aardvark presence as an indicator of ecosystem change
 associated with aridification.
description: This dataset was generated by designing new microsatellite
 primers, collecting fecal samples from aardvarks in southern Africa and
 one location in East Africa, extracting aardvark DNA from those fecal
 samples, and genotyping those DNA extracts at up to 19 loci. Duplicate
 individuals were removed and samples successfully genotyped at fewer than
 8 loci were not included. Laboratory details are provided here:
 We scraped the exterior surface of the dried fecal
 samples using a razor blade to obtain 0.03–0.045g of feces, attempting to
 avoid sand and dietary matter (i.e., ants or termites). We extracted DNA
 from fecal material using a version of the Aquagenomic and Aquaprecipi
 fecal extraction protocol (Multitarget Pharmaceuticals, Colorado Springs,
 CO) modified as follows. We increased the amount of Aquagenomic solution
 to 450 µL per sample, performed a 15-minute bead-beating step with 1.0 mm
 silica/zirconium beads (BioSpec Products Inc., Bartlesville, OK) to
 facilitate cell lysis, and added 12 mAU proteinase K (Qiagen Inc.,
 Valencia, CA). Lastly, we added 150 µL of Aquaprecipi solution to cell
 lysate to counteract PCR inhibitors present in fecal samples and
 rehydrated the final DNA pellet in 100 µL 1x TE buffer. 
 We attempted to amplify 19 microsatellite loci in four multiplex
 PCR panels, after screening samples for DNA quality by initially running
 all samples in three separate PCR reactions for Panel A (Table 1). We
 conducted PCRs in 10 μL reactions consisting of 5x Qiagen Multiplex PCR
 Master Mix, 10 μg of bovine serum albumin, 0.2 μM of each panel-specific
 primer and 1 μL of genomic DNA. Reactions were brought to volume with
 nuclease-free water. For each locus, one primer was fluorescently tagged
 on the 5’ end with NED, PET, VIC (Applied Biosystems, Carlsbad, CA) or
 6-FAM (Sigma-Aldrich, St. Louis, MO). We included negative and positive
 controls in each PCR to monitor for reagent contamination and align calls
 across runs. Thermalcycling conditions for the multiplexed loci were as
 follows: initial denaturation of 15 minutes at 95 °C, followed by 35
 cycles of [95 °C for 30 seconds, 60 °C for 90 seconds, 72 °C for 60
 seconds], and a final elongation of 30 minutes at 60 °C.  We ran PCRs on
 BioRad C1000, T100, and MyCycler thermalcycler machines (Bio-Rad
 Laboratories Inc., Hercules, CA). We verified
 amplifications by visualizing 4 µL of PCR product from one replicate of
 each sample on a 2% agarose gel prestained with GelRed (Biotium, Fremont,
 CA); products were then diluted accordingly, ethanol-precipitated to
 remove salts, and submitted for genotyping on the ABI 3730 DNA analyzer
 (Applied Biosystems) at the Oregon State University Center for
 Quantitative Life Sciences (Corvallis, OR). We used GeneScan500 LIZ dye
 size standard and called allele sizes in GeneMapper v.4.1 (Applied
 Biosystems). Samples that produced data at fewer than
 50% of the loci in the first three replicates for Panel A were not
 analyzed further. Samples that produced partial genotypes at ≥ 50% of
 microsatellite loci were rerun 3–6 more times depending on the
 completeness of initial replicates, while samples that produced complete
 and consistent genotypes in the first three replicates were considered
 finalized and a consensus genotype was accepted. For a genotype to be
 accepted for a particular locus, each allele in a heterozygote genotype
 had to be observed twice, while the single allele in a homozygote genotype
 had to be observed three times. We ran Panels B-D for samples that had
 generated sufficient data at Panel A. Any sample that consistently showed
 more than two alleles at a single locus was considered contaminated and
 removed.
description: R, or Microsoft Excel or similar spreadsheet programs that can
 read a text (.txt) file.
description: # Aardvark microsatellite data for southern and east Africa
 [https://doi.org/10.5061/dryad.hx3ffbgh7](https://doi.org/10.5061/dryad.hx3ffbgh7) Data for 19 microsatellite loci for aardvarks (Orycteropus afer), genotyped from feces. Missing data are scored as ?, otherwise, fragment sizes are reported. Each locus is reported in two columns, with those columns named according to the convention "LocusName\_1" for the first allele and "LocusName\_2" for the second allele. Sampling location (descriptive, as well as GPS coordinates in WGS84, Decimal Degrees) is included for each sample. Samples included were genotyped at a minimum of 8 loci according to the genotyping criteria; duplicate samples determined to be derived from the same individuals have been removed.