AbstractThe chloroplast (plastid) arose via the endosymbiosis of a photosynthetic cyanobacterium by a nonphotosynthetic eukaryotic cell ∼1.5 billion years ago. Although the plastid underwent rapid evolution by genome reduction, its rate of molecular evolution is low and its genome organization is highly conserved. Here, we investigate the factors that have constrained the rate of molecular evolution of protein-coding genes in the plastid genome. Through phylogenomic analysis of 773 angiosperm plastid genomes, we show that there is substantial variation in the rate of molecular evolution between genes. We demonstrate that the distance of a plastid gene from the likely origin of replication influences the rate at which it has evolved, consistent with time and distance-dependent nucleotide mutation gradients. In addition, we show that the amino acid composition of a gene product constraints its substitution tolerance, limiting its mutation landscape and its corresponding rate of molecular evolution. Finally, we demonstrate that the mRNA abundance of a gene is a key factor in determining its rate of molecular evolution, suggesting an interaction between transcription and DNA repair in the plastid. Collectively, we show that the location, the composition, and the expression of a plastid gene can account for >50% of the variation in its rate of molecular evolution. Thus, these three factors have exerted a substantial limitation on the capacity for adaptive evolution in plastid-encoded genes and ultimately constrained the evolvability of the chloroplast.