FIGURE 7 Phylogenetic tree constructed with the whole sequences of the chloroplast genomes of the above 59 species.

3.6 | Selective pressure analysis

We calculated the Ka/Ks values of Polygonatum protein-coding genes (PCGs) based on KaKs_Calculator2. The genes subject to positive selection are matK , ndhA , petB , and ycf2 , with a maximum Ka/Ks value of 1.41. The genes that were positively selected can be categorized into five groups according to their functions: (1) maturation enzyme gene matK ; (2) photosynthetic system genes ndhA and petB ; and (4) the ycf2 gene with unknown function. The remaining genes, all with Ka/Ks values less than 1, were subjected to purifying selection, with atpI being the lowest at 0.048 (Figure S3).

3.7 | Phylogenetic analysis

Phylogenetic trees were constructed using the maximum likelihood (ML) approach based on 59 complete chloroplast genome sequences ofPolygonatum medicinal plants published in the NCBI database (Dioscorea aspersa and Dioscorea alata ) to illustrate the genetic relationships of these species. The results showed that the two outgroups were distinct from Polygonatum . The nodes of the phylogenetic tree in this study had a high support rate and strong reliability for the phylogenetic analysis (Figure 7, Table S15). In the phylogenetic trees, all alternate leaf types of Polygonatum species were clustered on one large clade and species with verticillate leaves were divided into sect. Sibirica and sect. Verticillata group, confirming that the leaf type affects the classification of the genus. Of these, sect. Polygonatum and sect. Sibirica clustered together and exhibited a sister relationship with sect. Verticillata. Among the sixPolygonatum species, P. sibiricum formed a monophyletic group (sect. Sibirica ) with 100 % support. P. cyrtonema was the earliest clade to differentiate, followed P. odoratum , P. filipes and other related species clustered into a sister clade in sect. Polygonatum . P. kingianum was the earliest species differentiated from sect. Verticillata. P. zanlanscianense and other verticillate leaf type species of Polygonatum clustered into sister clades with P. kingianum. These results are consistent with those of the previous studies.
In addition, we downloaded the chloroplast genomes of six otherPolygonatum species from the database of the National Center for Biotechnology Information (NCBI) to explore the chloroplast genome affinities of Polygonatum in different regions. The phylogenetic results showed that P. cyrtonema was collected in Anhui Province and had the closest relationship with P. cyrtonema (MZ579646, MZ150839) stemming from Zhejiang Province and a relatively distant relationship with Hunan (OL436258), Beijing (MW248135), and Jiangxi (MZ029094). P. zanlanscianense (from Yunnan Province) was closely related to Guizhou Province (MW373522), followed by Hubei Province (OL405020) and Hunan Province (ON534059).