Beyond RuBisCO: convergent molecular evolution of multiple chloroplast genes in C ₄ plants

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 clade, 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 clades and C ₃ -only clades. 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 clade, 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.