*3.7. Positive Selection Analysis*

In order to detect the protein-coding genes under selection in Lythraceae, the sequences for each gene were aligned separately using the Muscle (codon) implemented in MEGA [68], and the Maximum likelihood phylogenetic tree based on complete cp genome sequences was constructed using IQ-tree [69]. The site-specific model was performed to test for natural selection using the CODEML algorithm [70] implemented in EasyCodeML [71]. Six codon substitution models described as M0, M1a, M2a, M3, M7, and M8 were investigated. This model allowed the ω ratio to vary among sites with a fixed ω ratio in all branches in order to test for site-specific evolution in the gene phylogeny. Two likelihood ratio tests were performed to detect positively selected sites: M1a (neutral) vs. M2a (positive selection), M7 (β) vs. M8 (β and ω), and M0 (one-ratio) vs. M3 (discrete), which were compared using a site-specific model [72].
