To examine which phenotypic traits are significantly differentiated between species and if the divergence is an adaptive response to divergent habitats, we conducted a common garden experiment for six pairs of populations sampled from different localities (fig. 1). Linear mixed-effect models detected that 14 of 19 traits showed significant differences, and only three grain traits (weight, length, and thickness of grains), awn length, and the flag leaf length were not significantly different between species (fig. 4a andsupplementary table S4, Supplementary Material online). Similarly, PCA showed that the combined samples are organized into two distinct groups (or species), with PC1 and PC2 accounting for 46.1% and 16.9% of the variance, respectively (fig. 5a). A three-way analysis of variance (ANOVA) (Butlin et al. 2014) on first two PCs and all 18 traits further indicate that all traits except for PC2 showed significant differences between species and the differences remain significant after correction for multiple testing, except for three traits (panicle exsertion, number of spikelets per panicle, and attitude of flag leaf). By contrast, no significant differentiation was found between regions within species and between localities within regions for all traits (table 1). In general, relative to the perennial O. rufipogon, the annual O. nivara is shorter and thicker, flowers earlier, has shorter and less exserted panicles with more compactness, and has more spikelets per panicle with shorter anthers and awns (fig. 4a), consistent with previous reports (Grillo et al. 2009; Banaticla-Hilario et al. 2013; Guo et al. 2016).