*2.3. Analysis of Evolutionary Patterns of* PLP\_deC *Genes*

We analyzed the relationships of *D. o*ffi*cinale* and *P. equestris* homologous gene pairs to further analyze the evolutionary patterns of PLP\_deC genes. We obtained six homologous gene pairs (*PeGAD1*–*DoGAD1*, *DoGAD2*–*DoGAD4*, *PeAAD1*–*DoAAD3*, *DoAAD2*–*PeAAD2*, *PeHDC1*–*DoHDC1*, and *PeGAD2*–*PeGAD3*) and calculated their Ka, Ks, and Ka/Ks values. The estimation of the synonymous (Ks) and nonsynonymous (Ka) nucleotide substitution rates is one of the important parameters for molecular evolutionary analyses, which are, respectively, defined as the number of synonymous substitutions per synonymous site and the number of nonsynonymous substitutions per nonsynonymous site per year or per generation. It is generally recognized that Ka/Ks > 1, Ka/Ks = 1, and Ka/Ks < 1 indicate positive, neutral, and purifying selection, respectively [20]. The experimental results showed that the Ka/Ks values of two homologous pairs (*DoAAD2*–*PeAAD2*, *PeHDC1*–*DoHDC1*) were lower than 0.3, the Ka/Ks values of the three homologous pairs (*PeAAD1*–*DoAAD3, DoGAD2*–*DoGAD4,* and *PeGAD1*–*DoGAD1*) were between 0.3 and 1, and the Ka/Ks value of one of the gene pairs (*PeGAD2*–*PeGAD3*) was greater than 1 (Table 3). These data indicated that most homologous *PLP\_deC* gene pairs are subjected to purifying selection. To further understand the influence of selection on the homologous gene pairs, we performed a sliding window analysis. The grey regions shown in Figure 3 represent the conserved domains. The Ka/Ks values for the conserved domains were typically less than 1, indicating that these pairs have purifying selection. These findings suggest that purifying selection may have played a key role in the evolution of this gene family.



**Figure 3.** Sliding window analysis of Ka/Ks for each gene pair. The window size is 150 bp, and the step size is 9 bp. The grey region represents the conserved domain.
