Twisting to freedom: The evolution of copulation termination techniques across 48 species of sepsids (Diptera, Sepsidae)

Studies of insect mating behaviour usually focus on what happens before and during copulation. Few pay close attention to the actions needed to end copulation. However, genital separation after copulation is likely to be an important cause of mechanical stress and injuries because it often involves the withdrawal of heavily armoured male intromittent organs from membranous female reproductive tracts. Difficult and/or slow separations can also reduce male and female fitness by increasing their exposure to predation. We here report the results of a comparative study of separation behaviour in 48 species of Sepsidae (Diptera) and one outgroup. We find a surprising amount of qualitative and quantitative behavioural variability within and between species. We characterize and reconstruct three types of behaviours: 1) The sepsid ancestor likely used `back-off; a gentle separation technique that does not involve any pulling or twisting (https://youtu.be/EbkJvOaubZ0). 2) This potentially gave rise to the most common `pull9 technique where the male turns 180 degrees and pulls in an opposite direction from the female (https://youtu.be/oLf4xGpkk1s). This separation can be quick and straightforward, but in some species the `pull9 is slow and protracted and we routinely find dead males and/or females attached to their living partners in the latter (difficult: https://youtu.be/MbYPbXN6jr0; failure: https://youtu.be/leTiXefFzCc). 3) Finally, several species use `twist9, a new technique where the male rotates >360 degrees from the initial mounting position (https://youtu.be/WMUXbIPyLbk). We document that species capable of using `twist9 have shorter and less variable separation times than those using "pull". However, many species capable of `twist9 also retain the ability to use `pull9 (`back-off9/9pull9= 8%; `pull9 only= 41%; `twist9/ `pull9= 24%; `twist9 only = 27%). Overall, our study suggests that separation behaviour can vary among closely related species and highlights the significance of studying variable behavioural traits in a phylogenetic context.