Multiple queen-mating occurs in many social insects, but high degrees of mu
ltiple paternity have only been found in honeybees and some yellowjacket wa
sps. Here we report the first case of an ant species where multiple mating
reduces relatedness among female offspring to values significantly lower th
an 0.5. Genetic analysis of a Panamanian population of the leaf-cutter ant
Acromyrmex octospinosus showed that queens mate with at least 4-10 males. T
he detected (minimum) genetically effective paternity of nestmate females w
as 3.9 and estimates of mean relatedness among nestmate females were ca. 0.
33. This implies that multiple queen-mating in Acromyrmex octospinosus redu
ces relatedness to 44% of the value in full-sib colonies (0.75), realizing
84% of the maximum reduction (to 0.25) that would be obtained with an infin
ite number of matings. Queens of Panamanian Acromyrmex octospinosus mate wi
th more males than sympatric queens of Atta colombica, which is contrary to
the positive relationship between queen-mating frequency and colony size f
ound across more distantly related ant species. Possible selective forces t
hat maintain high queen-mating frequencies in leaf-cutter ants are discusse
d.