Climate and hybridization shape stomatal trait evolution in Populus.

Stomata play a critical role in regulating plant responses to climate. Where sister species differ in stomatal traits, interspecific gene flow can influence the evolutionary trajectory of trait variation, with consequences for adaptation. Leveraging six latitudinally distributed contact zones spanning the natural hybrid zone between Populus trichocarpa × Populus balsamifera, we used whole genome resequencing and replicate common garden experiments to test the role that interspecific gene flow and selection play in stomatal trait evolution. While species-specific differences in the distribution of stomata persist between P. balsamifera and P. trichocarpa, hybrids on average resemble P. trichocarpa. Admixture mapping identified several candidate genes associated with stomatal trait variation in hybrids, including TWIST, a homolog of SPEECHLESS in Arabidopsis, which initiates stomatal development via asymmetric cell divisions. Geographic clines revealed candidate genes deviating from genome-wide average patterns of introgression, suggesting restricted gene flow and the maintenance of adaptive differences. Climate associations, particularly with precipitation, indicated selection shapes local ancestry at candidate genes across contact zones. These results highlight the role of climate in shaping stomatal trait evolution in Populus and demonstrate how interspecific gene flow creates novel genetic combinations that may enhance adaptive potential in changing environments.