Successful mating and hybridisation in two closely related flatworm species despite significant differences in reproductive morphology and behaviour

Speciation is usually a gradual process, in which reproductive barriers between two species accumulate over time. Reproductive traits, like genital morphology and mating behaviour, are some of the fastest diverging characters and can serve as reproductive barriers. The free-living flatworm Macrostomum lignano, an established model for studying sex in hermaphrodites, and its congener M. janickei are closely related, but differ substantially in their male intromittent organ (stylet) morphology. Here, we examine whether these morphological differences are accompanied by differences in behavioural traits, and whether these could represent barriers to successful mating and hybridization between the two species. Our data shows that the two species differ in many aspects of their mating behaviour, with M. janickei having a five-fold longer copulation duration, copulating less frequently, and having a longer and more delayed suck behaviour (a postcopulatory behaviour likely involved in sexual conflict). Interestingly, and despite these significant morphological and behavioural differences, the two species mate readily with each other in heterospecific pairings, often showing behaviours of intermediate duration. Although both species have similar fecundity in conspecific pairings, the heterospecific pairings revealed clear postmating barriers, as only few heterospecific pairings produced F1 hybrids. These hybrids had a stylet morphology that was intermediate between that of the parental species, and they could successfully backcross to both parental species. Finally, in a mate choice experiment we tested if the worms preferentially mated with conspecifics over heterospecifics, since such a preference could represent a premating barrier. Interestingly, the experiment showed that the nearly two-fold higher mating rate of M. lignano caused it to mate more with conspecifics, leading to assortative mating, while M. janickei ended up mating more with heterospecifics. Thus, while the two species can hybridize, the mating rate differences could possibly lead to higher fitness costs for M. janickei compared to M. lignano.