Liz 500 internal size standard

We extracted DNA from 116 workers per colony, as well as the mother queen, by placing a single antenna or leg in 100 μl of 10% Chelex solution (Chelex 100, 100–200 mesh, Bio-Rad), then incubating for 20 minutes at 95°C. We refrigerated or froze the supernatant before PCR.
We genotyped each worker at five variable loci (LIST2004, RUFA05, RUFA13, RUFA19, VMA3) using two multiplex PCR reactions [24 (link)–26 ]. Products at each locus were distinguished by employing simple dye-labeled primers (Applied Biosystems) and a 3-primer method [27 (link)], as well as fragment size for those labeled with the same dye. Each 10 μl PCR reaction included 1 μl of extracted DNA, 5 μl Qiagen master mix (Qiagen Type-It Microsatellite Kit, Qiagen Inc.), 0.2 μl of each reverse primer, 0.2 μl (dye-labeled) or 0.1 μl (3-primer labeled) of each forward primer, 0.15 μl FAM-labeled 3-primer tag for each 3-primer-labeled primer pair, and water. PCR reaction conditions were 95°C for 15 minutes, 35 cycles of 95°C for 30 seconds, 50°C for 90 seconds, 72°C for 60 seconds, followed by 60°C for 30 minutes. The fragment analysis was performed on an ABI-3730xl sequencer using 0.5 μl PCR product combined with 15 μl HiDi Formamide and 0.15 μl LIZ 500 internal size standard (Applied Biosystems). Allele sizes were called using GeneMarker (SoftGenetics LLC) and checked twice by eye.

The total DNA was extracted from gill lamellae using E.Z.N.A E-Z 96 Tissue DNA Kit (Omega-Biotek) following the manufacturer’s protocol. Individuals were genotyped at 20 polymorphic microsatellite loci: BFRO-018 [60] (link), BWF1, BWF2 [61] , Cla-Tet01, Cla-Tet03, Cla-Tet06, Cla-Tet09, Cla-Tet10, Cla-Tet13, Cla-Tet15, Cla-Tet17, Cla-Tet18 [62] (link), Cocl-Lav04, Cocl-Lav06, Cocl-Lav10, Cocl-Lav18, Cocl-Lav27, Cocl-Lav49, Cocl-Lav52 [63] , C2-157 [64] (link). The loci were co-amplified in four 2.5 µl PCR multiplex reactions as described earlier [65] using the QIAGEN Multiplex PCR kit following the manufacturer’s protocol. The PCR products were denatured in Hi-Di Formamide, containing LIZ-500 internal size standard (Applied Biosystems) and separated using an ABI-3130×l Genetic Analyzer (Applied Biosystems). The alleles were scored using the automatic binning function, with predefined bins, as implemented in Gene-Mapper 3.7 software (Applied Biosystems). All alleles were subsequently verified visually by two independent persons. In addition, the included replicate and blank samples were manually verified to ensure the validity of the data. Finally, we manually verified all identified private alleles to ensure that they are not an artefact from inconsistent scoring.

We extracted DNA from approximately twenty workers or gynes per colony, as well as the mother queen when present, by placing a single antenna or leg in 100 μL of 10% Chelex solution (Chelex 100, 100–200 mesh, Bio-Rad), then incubating for 20 minutes at 95°C. We then refrigerated or froze the supernatant before PCR. Variable loci were selected based on preliminary screening of published loci [31 -33 (link)]. We used dye-labeled primers (Applied Biosystems) in combination with a 3-primer labeling method [34 (link)] to perform multiplex PCR with 4–6 primers, depending on the species (Additional file 1: Table S1). Each 10 μL PCR reaction included 1ul extracted DNA, 5 μL Qiagen master mix (Qiagen Type-It Microsatellite Kit, Qiagen Inc.), 0.2 μL of each reverse primer, 0.2 μL (dye-labeled) or 0.1 μL (3-primer labeled) of each forward primer, 0.15 μL FAM-labeled 3-primer tag for each 3-primer-labeled primer pair, and water to total 10 μL. PCR reaction conditions were 95°C for 15 minutes, 35 cycles of 95°C for 30 seconds, 50°C for 90 seconds, 72°C for 60 seconds, followed by 60°C for 30 minutes. Fragment analysis was performed on an ABI-3730 × l sequencer using 0.5 μL PCR product combined with 15 μL HiDi Formamide and 0.15 μL LIZ 500 internal size standard (Applied Biosystems). Allele sizes were called using GeneMarker (SoftGenetics LLC) and checked twice by eye.