*3.3. Selective Pressure Analysis*

In genetics, the Ka/Ks ratio is useful for inferring the direction and magnitude of natural selection acting on homologous PCGs across diverged species. The ratio is a more powerful test of the neutral model of evolution than many others available in population genetics as it requires fewer assumptions [52]. A Ka/Ks ratio <1 implies purifying or stabilizing selection (acting against change), while a ratio of >1 implies positive or Darwinian selection (driving change) and a ratio of exactly 1 indicates neutral selection. Importantly, the Ka/Ks ratio is unlikely to be significantly above 1 without at least some of the mutations being advantageous.

In this study, the Ka/Ks ratio was determined for all 31 PCGs following comparison of the *P. vulgaris* mitogenome with those of *C. lanatus*, *V. vinifera* and *A. thaliana* (Figure 5). Nearly all of the Ka/Ks ratios were <1.0, suggesting that most of the PCGs were under stabilizing selection during evolution. Combining the information in Figure 5 and Table 1, the Ka/Ks ratios of all Complex I–V genes were <1, indicating that these genes were highly conserved in the evolutionary process of higher plants. The large number of mitochondrial genes under stabilizing selection (Ka/Ks < 1) may play important roles in stabilizing the normal functioning of mitochondria [53,54].

**Figure 4.** Relative synonymous codon usage (RSCU) of *P. vulgaris* mitogenome. Codon families are on the X-axis. RSCU values are the number of times of a particular codon, relative to the number of times that the codon would be observed for a uniform synonymous codon usage.

**Figure 5.** Ka/Ks ratios for 31 protein coding genes of *P. vulgaris*, *C. lanatus*, *V. vinifera*, and *A. thaliana*. The blue, orange, and gray boxes indicate Ka/Ks ratios of *P. vulgaris* vs. *C. lanatus*, *P. vulgaris* vs. *V. vinifera*, and *P. vulgaris* vs. *A. thaliana*.

As shown in Figure 5, the Ka/Ks ratios of *ccmB* were >1 between *P. vulgaris* and all of the three selected species, indicating that *ccmB* may have suffered from positive selection since divergence from their last common ancestor. Particularly, the Ka/Ks ratio of *ccmB* between *P. vulgaris* and *V. vinifera* was significantly >1 (4.01), suggesting that some advantage occurred during evolution. Additionally, the Ka/Ks ratios of *ccmFC*, *rps1*, *rps10*, and *rps14* were also >1, indicating that these genes were under positive selection after divergence of the last common ancestor. Since *CcmB* and *ccmFC* genes encode for some important components of the *c*-type cytochrome maturation pathway in mitochondria, we speculate that the adaptive evolution of *P. vulgaris* is closely related to the roles of *c*-type cytochromes in respiratory and photosynthetic electron transport [55–57]. Additionally, *rps1*, *rps10*, and *rps14* genes encode small mitoribosomal subunit proteins, which have been reported to play crucial roles in various biological processes in eukaryotic organisms, such as embryogenesis, leaf morphogenesis, and the formation of reproductive tissues [58–60]. The high Ka/Ks ratios of *rps* genes observed here may be very important for the evolution of *P. vulgaris*. Ka/Ks ratios >1 have also been reported for some other mitochondrial genes, including *atp8*, *ccmFN*, *matR*, and *mttB* [26,33,61,62], indicating that

mitochondrial genes in different plant species may be subjected to diverse selection pressures during evolution. Most importantly, the Ka/Ks ratio of the orthologous gene-pairs is an average over all sites and, even under positive selection, it can be <1 because some sites might be under positive selection while others are under purifying selection [53,61,63].
