Reconstructed protein sequence evolution consistent with the evolution of C 4 photosynthesis via a C 2 ancestor in the Paniceae

The grass tribe Paniceae includes a monophyletic subclade of species, the MPC clade, which specialize in each of the three primary C sub-pathways NADP-ME, NAD-ME and PCK. The evolutionary history of C photosynthesis in this subclade remains ambiguous. Leveraging newly sequenced grass genomes and syntenic orthology data, we estimated rates of protein sequence evolution on ancestral branches for both core enzymes shared across different C sub-pathways and enzymes specific to C sub-pathways. While core enzymes show elevated rates of protein sequence evolution in ancestral branches consistent with a transition from C to C photosynthesis in the ancestor for this clade, no subtype specific enzymes showed similar patterns. At least one protein involved in photorespiration also showed elevated rates of protein sequence evolution in the ancestral branch. The set of core C enzymes examined here combined with the photorespiratory pathway are necessary for the C photosynthetic cycle, a previously proposed intermediate between C and C photosynthesis. The patterns reported here are consistent with, but not conclusive proof that, C photosynthesis in the MPC clade of the Paniceae evolved via a C intermediate.