AbstractBackgroundThe genusMacrostomumconsists of small free-living flatworms and containsMacrostomum lignano, which has been used in investigations of ageing, stem cell biology, bioadhesion, karyology, and sexual selection in hermaphrodites. Two types of mating behaviour occur within this genus. Some species, includingM. lignano, mate via reciprocal copulation, where, in a single mating, both partners insert their male copulatory organ into the female storage organ and simultaneously donate and receive sperm. Other species mate via hypodermic insemination, where worms use a needle-like copulatory organ to inject sperm into the tissue of the partner. These contrasting mating behaviours are associated with striking differences in sperm and copulatory organ morphology. Here we expand the genomic resources within the genus to representatives of both behaviour types and investigate whether genes vary in their rate of evolution depending on their putative function.ResultsWe present de novo assembled transcriptomes of threeMacrostomumspecies, namelyM. hystrix, a close relative ofM. lignanothat mates via hypodermic insemination,M. spirale, a more distantly related species that mates via reciprocal copulation, and finallyM. pusillum, which represents a clade that is only distantly related to the other three species and also mates via hypodermic insemination. We infer 23,764 sets of homologous genes and annotate them using experimental evidence fromM. lignano. Across the genus, we identify 521 gene families with conserved patterns of differential expression between juvenile vs. adult worms and 185 gene families with a putative expression in the testes that are restricted to the two reciprocally mating species. Further, we show that homologs of putative reproduction-related genes have a higher protein divergence across the four species than genes lacking such annotations and that they are more difficult to identify across the four species, indicating that these genes evolve more rapidly, while genes involved in neoblast function are more conserved.ConclusionsThis study improves the genusMacrostomumas a model system, by providing resources for the targeted investigation of gene function in a broad range of species. And we, for the first time, show that reproduction-related genes evolve at an accelerated rate in flatworms.