## 3 Discussion
This agreement across different datasets and methods provided robust validation of the findings, especially in confirming the clear evolutionary distinction between _P. americana_ and _P. acinosa_. Phylogenetic trees based on divergence time estimates provide a valuable method for examining evolutionary relationships and temporal splits among species [74; 75]. By integrating genetic data and molecular evolution models, divergence time trees can be used to estimate when specific species diverged from their common ancestors. These trees are particularly useful for understanding the rate of genetic change over time and for associating evolutionary splits with historical environmental or ecological changes [76]. In this study, a divergence time tree was constructed using 76 CDSs from the cp genomes of various _Phylolaca_ species. This phylogenetic tree, based on both the ML and BI methods, closely aligns with previous results. Interestingly, it showed strong congruence with the divergence tree reported by [PERSON] et al. (2022), who used a broader cp genome dataset that included both coding and rRNA genes [1]. Although the datasets were not identical, the consistency of the resulting phylogenies further validated the depicted evolutionary relationships, highlighting the reliability of the findings across different analytical approaches. The divergence time estimates provided additional insights into the selective pressures acting on the NADH oxidoreductase gene family within the _Phylolaca_ species. The recent divergence of _P. acinosa_ and _P. insularis_ (approximately 0.0215 mya) aligned with the dN/dS analysis, showing low dN/dS values (ranging from 0.0248 to 0.0683) across most genes, including _ndhH_, suggesting strong purifying selection. This supports the close evolutionary relationship between these species, which are likely to be subjected to fewer adaptive pressures, leading to genetic conservation. In contrast, _P. americana_, which diverged from its closest relatives approximately 10.58 mya, showed evidence of positive selection in _ndhH_ (dN/dS = 1.0871), potentially linked to its broader geographic distribution and the resulting need to adapt to more varied environmental conditions. The earlier divergence of _P. americana_ from species such as _P. thyrsiflora_ and _P. icosata_ may correspond to its exposure to greater ecological challenges or geographic expansion, driving adaptive changes in _ndhH_. This suggests that selective pressures may have shaped _P. americana_ over a significant period, as it adapted to different ecological niches. Integrating divergence time estimates with dN/dS ratios revealed different evolutionary trajectories within the _Phylotaloca_ genus. Recent divergence and low dN/dS values in species such as _P. acinosa_ and _P. insularis_ suggest a conserved evolutionary path, whereas _P. americana_ displays signs of adaptive evolution, likely related to its broader ecological range. Although both _P. americana_ and _P. acinosa_ are traditionally used as medicinal herbs, their distinct evolutionary histories indicate potential differences in their chemical compositions and biological activities, which may influence their medicinal properties. While further research is required to fully understand these implications, the findings underscore the importance of accurate species identification for medicinal use to ensure that species-specific differences are considered. By combining phylogenetic and molecular evolutionary analyses, this study enhanced our understanding of genetic distinctions relevant to the safe and effective use of _Phylotaloca_ species in traditional medicine.