Continued Adaptation of C(4) Photosynthesis After an Initial Burst of Changes in the Andropogoneae Grasses

C [Formula: see text] photosynthesis is a complex trait that sustains fast growth and high productivity in tropical and subtropical conditions and evolved repeatedly in flowering plants. One of the major C [Formula: see text] lineages is Andropogoneae, a group of [Formula: see text] 1200 grass species that includes some of the world’s most important crops and species dominating tropical and some temperate grasslands. Previous efforts to understand C [Formula: see text] evolution in the group have compared a few model C [Formula: see text] plants to distantly related C [Formula: see text] species so that changes directly responsible for the transition to C [Formula: see text] could not be distinguished from those that preceded or followed it. In this study, we analyze the genomes of 66 grass species, capturing the earliest diversification within Andropogoneae as well as their C [Formula: see text] relatives. Phylogenomics combined with molecular dating and analyses of protein evolution show that many changes linked to the evolution of C [Formula: see text] photosynthesis in Andropogoneae happened in the Early Miocene, between 21 and 18 Ma, after the split from its C [Formula: see text] sister lineage, and before the diversification of the group. This initial burst of changes was followed by an extended period of modifications to leaf anatomy and biochemistry during the diversification of Andropogoneae, so that a single C [Formula: see text] origin gave birth to a diversity of C [Formula: see text] phenotypes during 18 million years of speciation events and migration across geographic and ecological spaces. Our comprehensive approach and broad sampling of the diversity in the group reveals that one key transition can lead to a plethora of phenotypes following sustained adaptation of the ancestral state. [Adaptive evolution; complex traits; herbarium genomics; Jansenelleae; leaf anatomy; Poaceae; phylogenomics.]