Clonemate cotransmission supports a role for kin selection in a puppeteer parasite.

Host manipulation by parasites is a fascinating evolutionary outcome, but adaptive scenarios that often accompany even iconic examples in this popular field of study are speculative. Kin selection has been invoked as a means of explaining the evolution of an altruistic-based, host-manipulating behavior caused by larvae of the lancet fluke Dicrocoelium dendriticum in ants. Specifically, cotransmission of larval clonemates from a snail first host to an ant second host is presumed to lead to a puppeteer parasite in the ant's brain that has clonemates in the ant abdomen. Clonal relatedness between the actor (brain fluke) and recipients (abdomen flukes) enables kin selection of the parasite's host-manipulating trait, which facilitates transmission of the recipients to the final host. However, the hypothesis that asexual reproduction in the snail leads to a high abundance of clonemates in the same ant is untested. Clonal relationships between the manipulator in the brain and the nonmanipulators in the abdomen are also untested. We provide empirical data on the lancet fluke's clonal diversity within its ant host. In stark contrast to other trematodes, which do not exhibit the same host-manipulating behavioral trait, the lancet fluke has a high abundance of clonemates. Moreover, our data support existing theory that indicates that the altruistic behavior can evolve even in the presence of multiple clones within the same ant host. Importantly, our analyses conclusively show clonemate cotransmission into ants, and, as such, we find support for kin selection to drive the evolution and maintenance of this iconic host manipulation.