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

Background. The recurrent evolution of the C-4 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 C-4 pathway. For instance, several key enzymes involved in the transition from C-3 to C-4 photosynthesis have been found to share convergent amino acid replacements along C-4 lineages. However, the extent of convergent replacements potentially associated with the emergence of C-4 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. Methods. Our study was based on the comparative analysis of 43 C-4 and 21 C-3 grass species belonging to the PACMAD Glade, a focal taxonomic group in many investigations of C-4 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 C-4 lineages and 9 C-3 lineages using reconstructed protein sequences of their reference branches, corresponding to the branches containing the most recent common ancestors of C-4-only Glades and C-3-only Glades. Pairwise comparisons between reference branches allowed us to identify both convergent and non-convergent amino acid replacements between C-4:C-4, C-3:C-3 and C-3:C-4 lineages. Results. 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 C-4 and C-3 reference branches. C-4:C-4 branches showed higher levels of convergent replacements than C-3:C-3 and C-3:C-4 branches. Furthermore, we found that more proteins shared unique convergent replacements in C-4 lineages, with both RbcL and RpoC1 (the RNA polymerase beta' subunit 1) showing a significantly higher convergent/non-convergent replacements ratio in C-4 branches. Notably, more C-4:C-4 reference branches showed higher numbers of convergent vs. non-convergent replacements than C-3:C-3 and C-3:C-4 branches. Our results suggest that, in the PACMAD Glade, C-4 grasses experienced higher levels of molecular convergence than C-3 species across multiple chloroplast genes. These findings have important implications for our understanding of the evolution of the C-4 photosynthesis pathway.

Automated summary

This study uses comparative analysis of chloroplast proteins in C-4 and C-3 grass species to reveal higher levels of molecular convergence in C-4 grasses. These findings have important implications for our understanding of the evolution of the C-4 photosynthesis pathway.