## 3 Discussion
Overall, the observed patterns of variation and nucleotide diversity in the cp genomes of _Phylolaca_ species contribute to a more comprehensive understanding of genetic relationships within the genus. The results of the dN/dS analysis provide significant insights into the evolutionary pressures acting on the NADH oxidoreductase gene family among _Phylolaca_ species. Most of the genes in this family are subject to purifying selection, as evidenced by dN/dS values below 0.25, indicating strong selective constraints that maintain the critical functions of these genes in cellular respiration by limiting non-synonymous substitutions [66; 67]. However, a notable exception is the _ndhH_ gene in _P. americana_, where a dN/dS value of 1.0871 suggests potential positive selection. This positive selection implies that _P. americana_ may have undergone adaptive changes in response to diverse environmental conditions. Unlike other _Phylolaca_ species, _P. americana_ has a vast native range across North America and parts of Mexico, and has been widely introduced to numerous regions worldwide, including Europe, Africa, and Asia [68; 69]. This broad geographical distribution exposes _P. americana_ to a wide variety of ecological niches, potentially driving adaptive evolution as the species responds to different climatic conditions, soil types, and environmental stressors [70; 71; 72]. Positive selection observed for the _ndhH_ gene further supports the hypothesis of adaptation to these diverse environments. In contrast, the _ndhB_ gene, with a consistent dN/dS value of 0.6533 across all six species, indicates relaxed purifying selection or possibly mild adaptive evolution, allowing for moderate variability while maintaining functional constraints. This may reflect selective pressures that permit some flexibility in function, possibly due to the varied ecological backgrounds of the species, particularly because some species (_P. acinosa_, _P. polyandra_, _P. japonica_) have also been introduced into new regions, such as Europe, which may impose additional environmental challenges [69]. Finally, although positive selection in the _P. american ndhH_ gene was suggested by dN/dS analysis, further research is needed to determine whether specific amino acid substitutions in this gene are located in key functional domains of the protein and how these changes might influence the function of the gene. Understanding the roles of these mutations in adaptive evolution will provide deeper insights into the functional significance of these evolutionary changes. Phylogenetic analysis based on 76 CDSs across 14 species (13 _Phylolaca_ species and 1 _Agdestis_ outgroup) clarified distinct evolutionary relationships within the _Phylolaca_ genus. Notably, _P. acinosa_ and _P. insularis_ formed a close sister group relationship, clustering with _P. polyandra_, _P. japonica_, and _P. latbenia_. These species demonstrated high genetic similarity, which likely reflects a recent divergence event. In contrast, _P. americana_, which is traditionally used as Phytolaceae Radix, formed a distinct cluster, underscoring its evolutionary distinction from _P. acinosa_. This suggests that _P. americana_ has followed a separate evolutionary path that was potentially shaped by its broader geographic range and ecological pressures. Previous studies using the ITS1-5.88-ITS2 region phylogenetic analysis identified _P. dioica_ as the earliest-diverging lineage, with _P. americana_ and _P. acinosa_ forming separate clusters [73]. These results are consistent with the findings of the present study, which showed a significant evolutionary divergence between these species. Additionally, the ML and BI phylogenies constructed in this study closely match two separate phylogenetic trees presented in [PERSON] et al. [1]. In their study, one tree was inferred using the whole cp genome, whereas the other was constructed using 79 CDSs and 4 rRNA genes. Both trees provided similar insights into the phylogenetic relationships among _Phylolaca_ species.