Ants exhibit a striking variety of lifestyles, including highly specialist or mutualist species. The minute blind workers of the African genus Melissotarsus chew tunnels in live trees to accommodate their obligate partner scale insects. Their modified legs are adapted for tunneling, but are unsuited for walking outside, confining these ants to their initial host tree. Here, we investigated whether this unique lifestyle results in complex patterns of genetic diversity at different scales, from

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... e same tree to different populations. Using 19 microsatellite markers, we assessed their mating strategy and colony structure among and across populations in South Africa. We showed that only one queen reproduces within a colony, mated with up to three males. Yet, several inseminated dealate queens are present in colonies; one probably replaces the older queen as colonies age. The reproduction of a single queen per colony at a given time results in genetic differentiation between colonies, even those located on the same tree. Overall, we discussed how the slow process of colony digging under the bark and the lack of worker patrolling above the bark might result in reduced competition between colonies and allow several secluded colonies to cohabit in a cramped space on a tree.