Workers in many species of social insects are capable of laying unfertilized eggs, which can develop into haploid males. This causes a conflict about male parentage between queens and workers. In a few species, this may result in matricide, i.e., workers kill the colony’s queen. Queen killing has so far been observed mainly in multi-queen colonies or in annual species, when the queen’s fecundity declines at the end of the reproductive period. Here, we report queen expulsion and matricide in a monogynous, monandrous ant with perennial societies. Workers were seen to aggressively expel both related and unrelated queens from their nest shortly after the end of hibernation. Queen expulsion and matricide led to a significant decrease in the number of workers and brood, but eventually increased the direct fitness of workers through significant male production. Long-term observations revealed a short lifespan of queens, while workers in orphaned colonies survived and produced male offspring over several years.

Collection of colonies and immediate census; information on the maternity of males and the relatedness between queens and workers was obtained by visually comparing the genotypes at up to six microsatellite loci.

Missing data are indicated by blank space.

The supplementary material is available in an excel-file with different tables:

Table 1 summarizes information on the composition of colonies (queens, workers, brood, sex ratio) of the ant Temnothorax crassisispinus when collected in spring 2016 and the following years. It is also indicated, which colonies had expelled or killed the queen in 2016 or 2017.

Table 2 indicates the origin of males produced in the colonies in 2016 and 2017, i.e., whether they were offspring of the queens, the workers, or unknown females (more details in notes to table 2)

Table 3 contains the microsatellite genotypes of queens, workers, and males from 2016 at six loci (L18, L5, GT218, 2MS17, ant3993, GT1) with an interpretation of whether males could be assigned to the queen, to workers, or unknown mothers. Explanations are given in notes to table 3.

Table 4 contains the microsatellite genotypes of queens, workers, and males from 2017 at six loci (L18, L5, GT218, 2MS17, ant3993, GT1) with an interpretation of whether males could be assigned to the queen, to workers, or unknown mothers. Explanations are given in notes to table 4.