Inquiline ants are highly specialised social parasites. They usually do not produce their own worker caste but instead use the worker force of the host ant colony to ensure the rearing of their sexual progeny. Several barriers are expected to severely limit their migration, and the mechanism allowing them to disperse remains largely enigmatic. Here, we tested two hypotheses to account for the low level of infestation of inquiline parasites, in populations of the parasite ant Plagiolepis xene and its host P. pygmaea: (1) the establishment of a new P. xene colony is such a rare event that a single colonisation should be expected per population, and (2) once a P. xene colony is established in one location, it has very little chance to succeed in infecting a neighbour genetically unrelated colony. We sampled nests from both species along four separate transects, and genotyped host and parasite individuals at eight polymorphic microsatellite loci. Our genetic data contradict both hypotheses: multiple colonisation events were recorded in all four transects sampled and, in at least one case, P. xene has successfully migrated from one host colony of P. pygmaea to a spatially close unrelated nest. This shows that the dispersion capacity of the social parasite is sufficiently effective to ensure its long-term survival.

Relatedness is a central parameter in the evolution of sociality, because kin selection theory assumes that individuals involved in altruistic interactions are related. At least three reproductive characteristics are known to profoundly affect colony kin structure in social insects: the number of reproductive queens per colony, the relatedness among breeding queens and queen mating frequency. Both the occurrence of multiple queens (polygyny) and multiple mating (polyandry) decrease within-colony relatedness, while mating among sibs increases relatedness between the workers and the brood they rear. Using DNA microsatellites, we performed a detailed genetic analysis of the colony kin structure and breeding system in three ant species belonging to the genus Plagiolepis: P. schmitzii, P. taurica and P. maura. Our data show that queens of the three species mate multiply: queens of P. maura mate with 1-2 males, queens of P. taurica with 3-11 males and queens of P. schmitzii may have 1-14 different mates. Moreover, colonies are headed by multiple queens: P. taurica and P. maura are facultatively polygynous, while P. schmitzii is obligately polygynous. Despite polyandry and polygyny, relatedness within colonies remains high because all species are characterized by sib-mating, with a fixation index F(it) = 0.25 in P. taurica, 0.24 in P. schmitzii and 0.26 in P. maura, and because the male mates of a queen are on average closely related.

Social Hymenoptera are ideal biological models for the study of the selective forces affecting the evolution of multiple mating (polyandry), because sister species can evolve different lifestyles and mating strategies. Single mating is predicted in workerless social parasites, because the key benefit of multiple mating in social insects, that is, the increase in genetic diversity among worker offspring, does not hold for workerless species. We compared the queen mating frequency between the ant Plagiolepis pygmaea and its derived social parasite P. xene . Previous studies showed that queens of the host P. pygmaea are obligately polyandrous. Here, pedigree analyses of mother–offspring combinations indicate that queens of the parasite P. xene did not revert to single mating; more than 50% of queens mated multiply, with 2–4 males. This result shows that reversal from multiple to single mating may be not selected in polyandrous social insect workerless parasites. We propose that such reversion does not occur when multiple mating is virtually cost free.

Multiple functional queens in a colony (polygyny) and multiple mating by queens (polyandry) in social insects challenge kin selection, because they dilute inclusive fitness benefits from helping. Colonies of the ant Plagiolepis pygmaea brash contain several hundreds of multiply mated queens. Yet, within-colony relatedness remains unexpectedly high. This stems from low male dispersal, extensive mating among relatives and adoption of young queens in the natal colony. We investigated whether inbreeding results from workers expelling foreign males, and/or from preferential mating between related partners. Our data show that workers actively repel unrelated males entering their colony, and that queens preferentially mate with related males. These results are consistent with inclusive fitness being a driving force for inbreeding: by preventing outbreeding, workers reduce erosion of relatedness within colonies due to polygyny and polyandry. That virgin queens mate preferentially with related males could result from a long history of inbreeding, which is expected to reduce depression in species with regular sibmating.

Several genetic and nongenetic benefits have been proposed to explain multiple mating (polyandry) in animals, to compensate for costs associated with obtaining additional mates. The most prominent hypotheses stress the benefits of increased genetic diversity. In social insects, queens of most species mate only once or have effective mating frequencies close to one. Yet, in a few species of ants, bees, and wasps, polyandry is the rule. In these species, colonies are usually headed by a single queen, whereas multiple queening adds diversity in several of the remaining species, especially in ants. Here we investigated mating frequency, inbreeding and relatedness between the queens and their mates in the polygynous ant Plagiolepis pygmaea, and the effect of polyandry on the genetic diversity as a function of the effective population size of individual colonies. Our results show that polyandry occurs frequently in the species. However, queens are frequently inseminated by close relatives, and additional sires add little genetic diversity among offspring of individual queens. In addition, the increase in diversity at the colony level is only marginal. Hence, contrary to established notions, polyandry in P. pygmaea seems not to be driven by substantial benefits of genetic diversity. Nonetheless, very small or as yet unidentified genetic benefits to one party (males, workers, queens) in conjunction with low costs of mating may favor polyandry. Alternatively, nongenetic factors, such as convenience polyandry, may be more important than genetic factors in promoting polyandry in P. pygmaea.