Beyond RuBisCO: convergent molecular evolution of multiple chloroplast genes in C₄ plants

The recurrent evolution of the C₄ photosynthetic pathway in angiosperms represents one of the most extraordinary examples of convergent evolution of a complex trait. Comparative genomic analyses have unveiled some of the molecular changes associated with the C₄ pathway. For instance, several key enzymes involved in the transition from C₃ to C₄ photosynthesis have been found to share convergent amino acid replacements along C₄ lineages. However, the extent of convergent replacements potentially associated with the emergence of C₄ plants remains to be fully assessed. Here, we conducted an organelle-wide analysis to determine if convergent evolution occurred in multiple chloroplast proteins beside the well-known case of the large RuBisCO subunit encoded by the chloroplast gene rbcL.

Our study was based on the comparative analysis of 43 C₄ and 21 C₃ grass species belonging to the PACMAD clade, a focal taxonomic group in many investigations of C₄ evolution. We first used protein sequences of 67 orthologous chloroplast genes to build an accurate phylogeny of these species. Then, we inferred amino acid replacements along 13 C₄ lineages and 9 C₃ lineages using reconstructed protein sequences of their reference branches, corresponding to the branches containing the most recent common ancestors of C₄-only clades and C₃-only clades. Pairwise comparisons between reference branches allowed us to identify both convergent and non-convergent amino acid replacements between C₄:C₄, C₃:C₃ and C₃:C₄ lineages.

The reconstructed phylogenetic tree of 64 PACMAD grasses was characterized by strong supports in all nodes used for analyses of convergence. We identified 217 convergent replacements and 201 non-convergent replacements in 45/67 chloroplast proteins in both C₄ and C₃ reference branches. C₄:C₄ branches showed higher levels of convergent replacements than C₃:C₃ and C₃:C₄ branches. Furthermore, we found that more proteins shared unique convergent replacements in C₄ lineages, with both RbcL and RpoC1 (the RNA polymerase beta' subunit 1) showing a significantly higher convergent/non-convergent replacements ratio in C₄ branches. Notably, more C₄:C₄ reference branches showed higher numbers of convergent vs. non-convergent replacements than C₃:C₃ and C₃:C₄ branches. Our results suggest that, in the PACMAD clade, C₄ grasses experienced higher levels of molecular convergence than C₃ species across multiple chloroplast genes. These findings have important implications for our understanding of the evolution of the C₄ photosynthesis pathway.