Trophic niche differentiation and phenotypic divergence among cryptic species of Japanese ninespine sticklebacks

Background: Morphologically similar cryptic species often differ in ecological niches. Three cryptic species of ninespine stickleback (genus Pungitius), P. tymensis and the freshwater and brackish types of the P. pungitius-P. sinensis species complex are found in eastern Hokkaido, Japan. All three co-occur in river catchments, inhabiting the upstream (P. tymensis), midstream (freshwater P. pungitius-P. sinensis type), and downstream (brackish P. pungitius-P. sinensis type). Questions: Are differences in trophic ecology among these species persistent? Do the three species differ in geometric body shape? Which ecological and morphological traits best distinguish these three cryptic species? Methods: We used stable isotope analysis to estimate trophic position and different source contributions in order to quantify trophic niche differentiation among three species collected from eastern Hokkaido, Japan. We characterized body shape variation using geometric morphometrics. We conducted exchangeability analysis to test how well different traits could distinguish all three species. Results: Isotopic source and niche overlap estimates clearly indicated a greater contribution of marine prey items to the brackish type than to the other two species and showed little differences between the freshwater type and P. tymensis. Body shape substantially differed between the freshwater type and P. tymensis, which had a deeper, elongated shape, whereas the brackish type was intermediate. Based on morphological and trophic traits, the brackish type and P. tymensis could be clearly distinguished. In contrast, misclassification of P. tymensis as the freshwater type based on stable isotope data and trophic morphology was high, indicating substantial overlap in trophic niche between these species.