*2.2. Codon Usage Analyses and RNA Editing Sites*

Relative synonymous codon usage (RSCU) is the ratio between the use and expected frequencies for a particular codon and a measure of nonuniform synonymous codon usage in coding sequences [32]. On the basis of the sequences of protein-coding genes, the codon usage frequency was estimated for the chloroplast genome of the three *Macrosolen* species (Figure 3). All the protein-coding genes were found to consist of 21,581, 21,598 and 21,520 codons in the chloroplast genomes of *M. cochinchinensis*, *M. tricolor* and *M. bibracteolatus*, respectively (Table S3). Figure 3 shows that the RSCU value increased with the increase in the quantity of codons which coded for a specific amino acid. Most of the amino acid codons show preferences except for methionine and tryptophan. Potential RNA editing sites were also predicted for 29 genes in the chloroplast genomes of the three species. A total of 39 RNA editing sites were identified (Table S4). The amino acid conversion from serine (S) to leucine (L) occurred most frequently, whereas that from proline (P) to serine (S) and from threonine (T) to methionine (M) occurred the least.

**Figure 3.** Codon content of 20 amino acids and stop codons in all of the protein-coding genes of the chloroplast genomes of three *Macrosolen* species.

#### *2.3. IR Constriction and Expansion*

Figure 4 shows the comparison of the boundaries of the LSC/IR/SSC regions of three *Macrosolen* species. The LSC/IR/SSC boundaries and gene contents in the chloroplast genomes of the three species were found to be highly conserved, featuring the same sequence structure and differences in length. In the three species, the *rpl2* gene, which is a normal functional gene, crossed the LSC/IRa boundary, but the *rpl2* pseudogene with a length of 1268 bp formed in the IRb region. The SSC/IRb boundaries of *M. cochinchinensis*, *M. tricolor* and *M. bibracteolatus* were found to be located in the complete *ycf1* gene, and their *ycf1* pseudogenes with lengths of 2457, 2455 and 2448 bp, respectively, were found to be produced in IRa.

**Figure 4.** Comparison of the borders of the large single-copy (LSC), small single-copy (SSC), and inverted repeats (IR) regions among the chloroplast genomes of three *Macrosolen* species. The number above the gene features means the distance between the ends of genes and the borders sites. These features are not to scale. Ψ: pseudogenes.
