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.