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

Abstract

BackgroundThe 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 introduced a robust empirical approach to test molecular convergence along a phylogeny including multiple C3 and C4 taxa. By analyzing proteins encoded by chloroplast genes, we tested if convergent replacements occurred more frequently than expected in C4 lineages compared to C3 lineages. Furthermore, we sought 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. MethodsOur 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 ancestral branches, corresponding to the most recent common ancestor of each lineage. Pairwise comparisons between ancestral branches allowed us to identify both convergent and divergent amino acid replacements between C4-C4, C3-C3 and C3-C4 lineages. ResultsThe 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 divergent replacements in 45/67 chloroplast proteins in both C4 and C3 ancestral branches. Pairs of C4-C4 ancestral branches showed higher levels of convergent replacements than C3-C3 and C3-C4 pairs. 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/divergent replacements ratio in C4 branches. Notably, significantly more C4-C4 pairs of ancestral branches showed higher numbers of convergent vs. divergent replacements than C3-C3 and C3-C4 pairs. Our results demonstrated 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 both our understanding of the evolution of photosynthesis and the goal of engineering improved crop varieties that integrates components of the C4 pathway.