New grass phylogeny resolves deep evolutionary relationships and discovers C ₄ origins

• Grasses rank among the world's most ecologically and economically important plants. Repeated evolution of the C ₄ syndrome has made photosynthesis highly efficient in many grasses, inspiring intensive efforts to engineer the pathway into C ₃ crops. However, comparative biology has been of limited use to this endeavor because of uncertainty in the number and phylogenetic placement of C ₄ origins. • We built the most comprehensive and robust molecular phylogeny for grasses to date, expanding sampling efforts of a previous working group from 62 to 531 taxa, emphasizing the C ₄-rich PACMAD (Panicoideae, Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae and Danthonioideae) clade. Our final matrix comprises c. 5700bp and is>93% complete. • For the first time, we present strong support for relationships among all the major grass lineages. Several new C ₄ lineages are identified, and previously inferred origins confirmed. C ₃/C ₄ evolutionary transitions have been highly asymmetrical, with 22-24 inferred origins of the C ₄ pathway and only one potential reversal. • Our backbone tree clarifies major outstanding systematic questions and highlights C ₃ and C ₄ sister taxa for comparative studies. Two lineages have emerged as hotbeds of C ₄ evolution. Future work in these lineages will be instrumental in understanding the evolution of this complex trait.