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Polyandry in hymenopteran social insects is surprisingly rare, despite its likely colony-level fitness benefits. Ordinarily, a male's fitness will be at odds with that of a colony when the genetic representation within it is diluted by multiple mating by the queen. Consequently, males are expected to be under selection to limit female re-mating, for example via secretions of their accessory glands. I hypothesized that if accessory glands in some way regulate female mating frequency, an evolutionary transition from single to multiple mating would likely be accompanied with a change in the morphology of the accessory glands. The accessory gland morphology was examined in the fungus-gardening ants, which have made a single transition from single to multiple mating. The evolution of polyandry within this clade corresponds to the loss of male accessory glands, lending tentative support to the idea that they may be involved in regulating mating frequency.