Speciation can be initiated by adaptive divergence between populations in ecologically different habitats, but how sexually based reproductive barriers contribute to this process is less well understood. We here test for sexual isolation between ecotypes of threespine stickleback fish residing in adjacent lake and stream habitats in the Lake Constance basin, Central Europe. Mating trials in outdoor mesocosms allowing for natural reproductive behavior reveal that mating occurs preferentially between partners of the same than of the opposed ecotype. Compared to random mating, this sexual barrier reduces gene flow between the ecotypes by some 36%. This relatively modest strength of sexual isolation is surprising because comparing the males between the two ecotypes shows striking differentiation in traits generally considered relevant to reproductive behavior (body size, breeding coloration, nest size). Analyzing size differences among the individuals in the mating trials further indicates that assortative mating is not related to ecotype differences in body size. Overall, we demonstrate that sexually based reproductive isolation promotes divergence in lake-stream stickleback along with other known reproductive barriers, but also caution against inferring strong sexual isolation from the observation of strong population divergence in sexually relevant traits
