In silico selection of microsatellite loci
We screened 269 human microsatellite loci widely used in catarrhine population genetic studies. We extracted the human (GRCh38/hg38) sequence of each locus with 500 bp flanking regions from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and performed BLAT searches against the 16 available (status: 5 December 2018) non-human Catarrhini reference genomes (Table S1) using the UCSC (genome.ucsc.edu) or Ensembl (ensembl.org) genome browsers with standard settings. In addition, we checked the human sequence for repetitive elements (SINEs, LINES, etc.) in flanking regions using the RepeatMasker Web Server (http://www.repeatmasker.org/) with standard settings. We generated alignments for each locus containing the 16 non-human catarrhine species, the human and the human repeat-masked sequences with Muscle 3.8.31 (Edgar, 2004) in SeaView 4 (Gouy, Guindon, & Gascuel, 2010) and added published primer sequences to the alignments.
Loci were selected for further analysis if they fulfilled the following criteria: (1) primer binding sites are not in repetitive elements thus increasing locus-specific amplifiability and reducing the risk of off-target PCR products particularly in multiplex PCR reactions; (2) primer binding sites are conserved among catarrhines so that loci can be universally amplified in this taxonomic group with >180 species (Mittermeier, Wilson, & Rylands, 2013); (3) the microsatellite motif is relatively short (max. 150 bp) to allow small amplicon size and secure locus amplification from degraded DNA samples, such as faecal samples; and (4) loci are evenly (1-3 loci per chromosome) distributed throughout the genome (using the human genome as a reference) to avoid potential linkage problems.
For loci which passed the selection criteria, we designed new primers using Primer-Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). To allow for multiplexing, primers were designed to have similar annealing temperatures. Locus specificity of primers was checked by BLAT search against the 17 available catarrhine genomes. As primer-binding sites were not always fully conserved among the 17 catarrhines, primers of 21 loci were designed with wobble positions. To simplify library preparation for GBS, we added adapter nucleotide sequences to the 5’ end of the locus-specific primers (5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’ to forward primers, 5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’ to reverse primers; locus-specific primers are provided in Table S2).