*2.2. Codon Usage*

One of the factors shaping the evolution of the chloroplast genome is codon usage, which occurs as a result of bias in mutation [51]. The codon usage bias in the plastome was computed using the protein-coding genes and tRNA gene nucleotide sequences— 104,575 bp, 106,488 bp, 105,750 bp and 99,100 bp for *C. farinosa, C. glandulosa, M. crassifolia* and *M. oblongifolia*, respectively. Supplementary Tables S1–S4 present the relative synonymous codon usage (RSCU) of each codon in the genome; these results suggested that all the genes are encoded by 33,686 codons in *C. farinosa,* 34,303 codons in *C. glandulosa*, 34,064 codons in *M. crassifolia* and 31,920 codons in *M. oblongifolia*. The most frequently occurring codons are for the amino acids leucine 3290 (9.76%), 3599 (10.49%), 3452 (10.13%) and 2951 (9.24%), respectively (Figure 2), which have been found in other flowering plant genomes [52], whereas methionine has the fewest codons in the genome at 620 (1.84%) in *C. farinosa* and 606 (1.89%) in *M. oblongifolia*, and for tryptophan it is 674 (1.96%) in *C. glandulosa* and 695 (2.04%) in *M. crassifolia*. A- and T- endings were discovered to be less frequent than their counterparts G- and C-. The codon usage bias is low in the cp genomes of Capparaceae species (Tables S1–S4). Additionally, the results show that the RSCU values of 27 codons were >1, all with A/T- endings, whereas the other 28 codons were <1, and all ended with G/C. The RSCU values of tryptophan and methionine amino acids are 1, so they are the only amino acids with no codon bias.

**Figure 2.** Amino acid frequencies in the four Capparaceae chloroplast genomes' protein-coding sequences.
