Microsatellite genotyping and genetic diversity
We extracted genomic DNA from 64 individual T. septentrionalisworkers using a QIAamp DNA Micro Kit (QIAGEN). We implemented the M13-tail polymerase chain reaction (PCR) method (Schuelke, 2000), which involves three primers in the PCR reaction: forward primers (Matthews et al., 2020) with an M13-tail at the 5’ end, an unlabeled reverse primer, and a universal M13 primer labeled with 6-FAM (6-carboxy-fluorescine) fluorescent dye. These were used to amplify nine microsatellite loci (Ts21, Ts25, Ts32, Ts33, Ts39, Ts41, Ts43, Ts46, Ts5) (Matthews et al., 2020).
PCRs were performed in a 10 μL mix containing 1 μL of 10X PCR buffer (1.0X; Applied Biosystems), 1 μL Bioline® dNTP mix (1 mM each; 0.1 mM as a proportion of the total), 1.5 μL of 25 mM MgCl2 (3.75 mM; Applied Biosystems), 0.5 μL of 20 μM BSA (1 μM; New England Biolabs), 0.3 μL of 2 μM tag labeled primer (0.06 μM; forward primer with M13 tag), 0.6 μL of a 2 μM universal dye-labeled primer (0.12 μM; FAM label with M13 tag), 1 μL of 2 μM unlabeled primer (0.2 μM; reverse primer), 0.1 μL of Taq polymerase (0.5 U; Applied Biosystems), and 1 μL of DNA template. Nuclease-free water was used to make up the remaining volume. The following thermocycling profile was used on an Eppendorf Mastercycler: initial denaturation of 4 min at 95°C, followed by 25 (or 30) cycles of 30 s at 95°C, 45 s at the primer-specific annealing temperature found by a temperature gradient program, 45 s at 72°C, then 8 cycles of 30 s at 95°C, 45s at 53°C, and 45 s at 72°C, followed by a final extension of 5 min at 72°C. Diluted PCR products were run on an Applied Biosystems 3730 Genetic Analyzer and fragments were sized with LIZ600 size standard at the University of Texas at Austin DNA Sequencing Facility in Austin, Texas, USA. We scored alleles using Geneious v10.2.3 (Kearse et al., 2012).
For each locus, we used GenAlEx v6.5 (Peakall & Smouse, 2006; Peakall & Smouse, 2012) to estimate the number of alleles (K ), observed and expected heterozygosity (Ho and He), and the probability of identity (PI; the probability of two independent samples having the same genotype). We assessed deviations from Hardy-Weinberg equilibrium (HWE) expectations using GENEPOP v4.2 (Rousset, 2008) across each locus for the overall dataset, as well as across each locus within each population. GENEPOP was also used to test for linkage disequilibrium (LD) across all pairs of loci.