Chloroplast genome sequence data is very useful in studying/addressing the phylogeny of plants at various taxonomic ranks. However, there are no empirical observations on the patterns, directions, and mutation rates, which are the key topics in chloroplast genome evolution. In this study, we used Calycanthaceae as a model to investigate the evolutionary patterns, directions and rates of both nucleotide substitutions and structural mutations at different taxonomic ranks.

Our direct counts of chloroplast genome evolution events provide raw data for correctly modeling the evolution of sequence data for phylogenetic inferences.

There were 2861 polymorphic nucleotide sites on the five chloroplast genomes, and 98% of polymorphic sites were biallelic. There was a single-nucleotide substitution bias in chloroplast genomes. A → T or T → A (2.84%) and G → C or C → G (3.65%) were found to occur significantly less frequently than the other four transversion mutation types. Synonymous mutations kept balanced pace with nonsynonymous mutations, whereas biased directions appeared between transition and transversion mutations and among transversion mutations. Of the structural mutations, indels and repeats had obvious directions, but microsatellites and inversions were non-directional. Structural mutations increased the single nucleotide mutations rates. The mutation rates per site per year were estimated to be 0.14-0.34 × 10- 9 for nucleotide substitution at different taxonomic ranks, 0.64 × 10- 11 for indels and 1.0 × 10- 11 for repeats. Conclusions: Our direct counts of chloroplast genome evolution events provide raw data for correctly modeling the evolution of sequence data for phylogenetic inferences.