2.2 ǀ Genetic analyses
We determined the genotypes of workers and female sexuals at seven microsatellite loci as described before (CE2-3A, CE2-4A, CE2-4E, CE2-5D, CE2-12D, Card 8, and Cobs 13; e.g., Lenoir et al. 2007, Vidal et al. 2021). In short, DNA was extracted from whole ants using the CTAB method (modified from Sambrook & Russell, 2001) and diluted in 30 μl of TE buffer. Polymerase Chain Reactions (PCRs) were performed in a 20 μl reaction volume using 1 μl of DNA with 19 μl of master-mix (7 μl H2O, 10 μl GoTaq, and 1 μl of each forward and reverse primers). Samples were amplified following Lenoir et al. (2005) with an initial denaturation step at 94 °C for 3 min, 40 cycles at 94 °C for 45 s, with an annealing temperature according to the primer for 45 s (supplementary table 2.1), followed by a step of 72 °C for 45 s, and a final extension step at 72 °C for 7 min. Allele sizes were determined by GeneScan® 3.1 software (Applied Biosystems) and GeneScan® 500 TAMRA dye as a size standard. Relatedness among nestmates and pairwise relatedness between female sexuals and their carriers were calculated for samples from the two different years separately following Queller and Goodnight (1989) using the related package (Pew et al., 2015) on R software-4.0.3 and Relatedness v4.2 (Queller & Goodnight, 1994). Fixation coefficients (FST) were estimated using GenAlEx v6.51b2 (Peakall & Smouse, 2006). The frequency of sibling mating α was calculated from FST = α/(4-3α) (Pamilo, 1985).