3.1 ǀ Population and colony genetic analyses
Nestmate relatedness and inbreeding coefficients were determined from 42 workers from six colonies collected in 2019 and 113 workers from 16 colonies collected in 2020. Mean relatedness per colony ranged from 0.18 to 0.66 in 2019 and 0.13 to 0.69 in 2020. Across all studied colonies, the mean relatedness was 0.49 ± SE 0.08 in 2019 and 0.39 ± 0.04 in 2020, which is in the range of earlier estimates (0.34 ± 0.05, Vidal et al., 2021). The same applies to the fixation coefficient (2019: 0.43 ± 0.10, 2020: 0.28 ± 0.03, 2018: 0.30 ± 0.03).
As colonies are obligatorily monogynous, i.e., have only one fertile queen, the lower relatedness suggests multiple mating, which is also evident from the number of genotypes found among nestmates. The mean effective mating frequency of queens calculated from nestmate relatedness following Tarpy and Nielsen (2002) is 2 and 3.5 in our samples from 2019 and 2020, respectively. The actual mating frequencies of individual queens that had been collected when carried in the field, estimated from the genotypes of offspring reared in the laboratory, revealed a large variation from one to seven mating partners per queen (mean 4.3 ± SD 1.6, n = 6). Though adding unrelated males to female sexuals in the laboratory resulted in additional mating as reflected in worker genotypes, mean mating frequencies did not differ (mean 3.3 ± 2.3, n = 12; t -test, t = 0.950,p = 0.346).
The relatedness between carrier and carried female sexual was 0.39 ± 0.03 in 2019 (n = 91) and 0.23 ± 0.08 in 2020 (n = 8), i.e., in the range of colony-wise estimates for relatedness but, in contrast to our earlier study (Vidal et al., 2021), significantly lower than the estimated mean relatedness in these years (t -test, 2019:t = 2.90, p = 0.005; 2020: t = 2.86, p = 0.009). The relatedness of female sexuals to source colonies was 0.29 ± 0.14 (n = 3) in 2019 and 0.21 ± 0.06 (n = 8) in 2020, the respective values for recipient colonies were 0.03 ± 0.21 and -0.04 ± 0.07. Relatedness to the source colony was not different from nestmate relatedness in 2019 (t = 1.98, p = 0.09) but significantly lower in 2020 (t = 3.81, p = 0.001). Relatedness to the target colony did not differ from zero (single-sample t -test, 2019: t = 0.25, p = 0.83; 2020: t = 1.62, p= 0.15).
The large number of pairs of carriers and female sexuals collected in 2019 allowed us to estimate how pairwise relatedness varied with time. The probability of carrier and female sexual being less closely related than 0.25 (the value for halfsisters) decreased from 0.17 in the first phase of our field collection (Aug 14 – 16, n = 6) to 0.43 in the seventh and last phase (Aug 30, n = 14; Spearman rank correlation, rS = 0.81, p = 0.035).