While some C2 species exhibit elevated C4 biochemistry, none show significant net gain of carbon through this nascent C4 cycle.¹ The role of this modest C4 metabolism is uncertain, though it has been proposed to supply carbon skeletons to the sheath cells for re-assimilation of ammonia that is released during photorespiration.² How the C4 and Calvin cycles become integrated remains unknown.¹ A scarcity of C2-C4 intermediate species makes addressing this question difficult. Comparative genomics of numerous parallel C2 and C4 species pairs is one approach. Another approach is to focus on the few lineages which do have species exhibiting C2-C4 intermediacy. Flaveria (Asteraceae) and Blepharis (Acanthaceae) represent two such lineages in which the answer to these outstanding questions in C4 evolution may be found. We are sequencing numerous species of both genera to enable robust comparative analyses. Other important questions in C4 evolution include whether or not reversion from C4 is possible, how the evolutionary “choice” of C4 metabolic subtype occurs, and the reason for phylogenetic “hotspots” of C4 evolution in the grasses, sedges, and the eudicot order Caryophyllales. By sequencing representative C3 and C4 sister species from all C4 branches of the Angiosperm tree we will be able to address these and many other questions in C4 evolution.