Loss of pair formation predates the evolution of male-less society in termites.

Asexual lineages are rare in social animals with biparental care, where successful reproduction typically requires coordinated behavior between two individuals of opposite sex. Male-less lineages of the termite Glyptotermes nakajimai provide a unique opportunity to unravel how sexual reproduction can be lost in such animals. Here, we show that modification of the mate-pairing process predated the evolution of asexual populations. Termite colonies are typically initiated by a mating pair that searches for a nest site through a tandem courtship behavior. Our comparative analysis of tandem running in Glyptotermes termites revealed that two related species, G. fuscus and G. satsumensis, exhibit both female-leader and male-leader tandem runs. However, tandem running was rare and ephemeral in both sexual and asexual lineages of G. nakajimai. Furthermore, our comparative studies indicated typical monogamous pairing was uniquely lost in G. nakajimai, while pairs initiate nests in G. fuscus and G. satsumensis. Our study evidenced that a clear disruption of termites' classic reproductive behavioral sequence, coupled with an alternative mode of colony foundation, was likely a precondition for the evolution of asexuality in species with biparental care.