Reproductive traits are some of the fastest diverging characters and can serve as reproductive barriers. The free-living flatworm Macrostomum lignano , 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. Despite these differences, the species mate readily with each other in heterospecific pairings. Although both species have similar fecundity in conspecific pairings, the heterospecific pairings revealed clear postmating barriers, as few heterospecific pairings produced F1 hybrids. These hybrids had a stylet morphology that was intermediate between that of the parental species, and they were fertile. Finally, using a mate choice experiment, we show 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 .

We would like to thank Tim Janicke and Georgina Rivera Ingraham for help with collecting 22 specimens of the study species M. janickei, Steven A. Ramm for providing access to the 23 images of the M. lignano stylets from their study, Jeremias Brand for help with imaging the 24 hybrid morphology, Nikolas Vellnow and Axel Wiberg for helpful discussions, and Gudrun 25 Viktorin, Lukas Zimmerman, Jürgen Hottinger, Urs Stiefel and Daniel Lüscher for technical 26 support. Abstract 33Speciation is usually a gradual process, in which reproductive barriers between two species 34 accumulate over time. Reproductive traits, like genital morphology and mating behaviour, are 35 some of the fastest diverging characters and can serve as reproductive barriers. The free-living 36 flatworm Macrostomum lignano, an established model for studying sex in hermaphrodites, 37 and its congener M. janickei are closely related, but differ substantially in their male 38 intromittent organ (stylet) morphology. Here, we examine whether these morphological 39 differences are accompanied by differences in behavioural traits, and whether these could 40 represent barriers to successful mating and hybridization between the two species. Our data 41shows that the two species differ in many aspects of their mating behaviour, with M. janickei 42 having a five-fold longer copulation duration, copulating less frequently, and having a longer 43 and more delayed suck behaviour (a postcopulatory behaviour likely involved in sexual 44 conflict). Interestingly, and despite these significant morphological and behavioural 45 differences, the two species mate readily with each other in heterospecific pairings, often 46 showing behaviours of intermediate duration. Although both species have similar fecundity in 47 conspecific pairings, the heterospecific pairings revealed clear postmating barriers, as only 48 few heterospecific pairings produced F1 hybrids. These hybrids had a stylet morphology that 49 was intermediate between that of the parental species, and they could successfully backcross 50 to both parental species. Finally, in a mate choice experiment we tested if the worms 51 preferentially mated with conspecifics over heterospecifics, since such a preference could 52 represent a premating barrier. Interestingly, the experiment showed that the nearly two-fold 53 higher mating rate of M. lignano caused it to mate more with conspecifics, leading to 54 assortative mating, while M. janickei ended up mating more with heterospecifics. Thus, while 55 the two species can hybridize, the mating rate differences could possibly lead to higher fitness 56 costs for M. janickei compared to M. lignano. 57 4 Keywords 58 mating behaviour, simultaneous hermaphrodite, mate preference, reproductive isolation, 59 hybridization, genitalia, reproductive barriers, premating barriers, postmating barriers, free-60 living flatworms 61 62 63The biological species concept defines species as groups of individuals that interbreed in 64 nature to produce viable and fertile offspring (Mayr 1...