4 | DISCUSSION

4.1 | Chloroplast genome structure analysis of P. kingianum

In the present study, the chloroplast genome sequences of six medicinal plants of Polygonatum were analyzed. According to the assembly annotation results, the chloroplast genomes of Polygonatum , similar to most angiosperms, exhibit a classical tetrad structure and are closed-circular double-stranded DNA molecules (Daniell et al., 2016). The complete genome length ranged from 154, 578 to 155, 807 bp and the lengths of the LSC, SSC, and IR regions were relatively conserved, with no obvious contraction or expansion. However, the chloroplast genome of P. kingianum is the largest and its measured genome size is usually above 155, 700 bp (Guo et al., 2022; Zhang et al., 2023). P. zanlanscianense chloroplast genome was the next largest and it was statistically found that the genome length of the verticillate leaf types of Polygonatum was typically larger than that of the alternate leaf types (Table S15).Polygonatum chloroplast genome encodes 127–131 genes. P. kingianum encodes 127 genes, comprising 84 protein-coding genes, 37 transfer RNA genes, and 6 ribosomal RNA genes. Compared to other species, P. kingianum lacks rpoC1 , trnV-UAC andrrn4.5 genes, which is consistent with the results of previous studies (Wang et al., 2022). The length and composition of the chloroplast genome of P. kingianum differ from those of other species, which may be related to the limited geographic growth environment of P. kingianum .
SSRs are widely distributed in chloroplast genomes and can be used for species identification and genetic diversity analyses (Jiang et al., 2018). A total of 64–76 SSRs are present in the chloroplast genomes of six Polygonatum species (Zhang et al., 2023; Yan et al., 2023). The number of SSRs in Polygonatum is comparable to that inPeucedanum (Liu et al., 2022), Adonis (Nyamgerel et al., 2023), and Paeoniaceae (Cai et al., 2023). The A/T and AT/TA repeat sequences account for the largest number of mono- and dinucleotide repeat sequences, respectively, influencing the overall GC content of genomes (Gichira et al., 2019).

4.2 | Relatively conserved chloroplast genomes inPolygonatum

In the chloroplast genomes of angiosperms, rRNA genes are generally located in the IR region, which to some extent makes the IR region more conserved than the LSC and SSC regions, making the IR region the most conserved region in the chloroplast genome. However, during the evolution of this species, the IR boundary has undergone contraction and expansion, affecting the length of the chloroplast genome (Li et al., 2013; Zhang et al., 2016). The length of Pelargonium hortorum chloroplast genome is 217, 942 bp owing to the extreme expansion of its IR region (Chumley et al., 2006); the IR region length of Pinus thunbergii is shortened to only 495 bp, and the chloroplast genome length is 119, 707 bp (Wakasugi et al., 1994); whereas the IR ofP. hortorum is generally absent (Liu et al., 2020). The IR boundary region of the chloroplast genomes of the six Polygonatum medicinal plants is relatively conserved and does not undergo significant expansion or contraction (Zhang et al., 2023; Yan et al., 2023). However, the rps19 gene of P. sibiricum overlapped with the LSC/IR boundary and differed from that of the other five species, which was hypothesized to be related to the evolution ofP. sibiricum .
The results of mVISTA and nucleotide diversity analyses indicated that the chloroplast genomes of the six Polygonatum species were highly similar. The highly variable regions of the chloroplast genomes of Polygonatum species were mainly concentrated in the LSC and SSC regions, and 21 highly variable Pi-fragments were screened. Among them, psbI-trnS-GCU , psbJ , rpl32 , trnL-UAG ,ccsA , ndhD , and ycf1 were screened as candidate markers. This provides a reference for subsequent molecular identification of Polygonatum species to identify potential chloroplast DNA barcodes.
The selective pressure analysis revealed that matK , ndhA ,petB , and ycf2 genes were subject to positive selection, of which ndhA and petB were photosynthetic system genes.Polygonatum plants are mainly distributed in the understory, thickets or shady areas of mountain slopes, and adaptation to sunlight stress may be an important genetic basis for the evolution of adaptations at the chloroplast level in Polygonatum (Zhang et al., 2023).

4.3 | Species of Polygonatum form three major groups

The genus Polygonatum is rich in germplasm resources and contains various species that are difficult to identify owing to their similar morphology. The classification of this genus has long been controversial and is a concern for taxonomic treatment. The genus Polygonatum was first established by Miller (Zhao et al., 2014). Baker (Baker et al., 1875) divided Polygonatum into three groups,Alternifolia , Verticillata and Oppositifolia, according to the characteristics of phyllotaxis. However, species ofPolygonatum differ in morphological characteristics, such as perianth and bracts, in addition to differences in leaf type.Flora of China synthesized these characteristics and divided the genus into eight groups. Tamura subdivided the genus into two sections:Polygonatum and Verticillata (Tamura et al., 1993). To date, botanists have not reached a unified view of the classification of Polygonatum species based on their morphological characteristics. With the development of molecular biology, phylogenetic studies of species based on chloroplast genome sequences have provided novel perspectives for solving plant affinities. Previous phylogenetic studies of Polygonatum chloroplast genomes have supported the classification of this genus into three groups: sect. Verticillata , sect. Polygonatum , and sect. Sibirica , where sect. Polygonatum and sect. Sibirica and sect. Verticillata is a sister species of sect. Polygonatum +sect. Sibirica (Floden et al., 2018). Sect. Verticillata contains Polygonatum species with verticillate leaf types,sect. Polygonatum contains alternate leaf types, and sect. Sibirica usually has only one species, P. sibiricum (Qin et al., 2024). Consistent with previous findings, the chloroplast genome phylogenetic tree of Polygonatum used in the present study was divided into three clades: sect. Verticillata , sect. Polygonatum , and sect. Sibirica . P. kingianum andP. zanlanscianense clustered in the verticillate leaf taxonsect. Verticillata . P. cyrtonema , P. filipes andP. odoratum clustered into the alternate-leaf taxon sect. Polygonatum and P. sibiricum clustered as a separate monophyletic group, similar to sect. Sibirica ( Meng et al., 2014).
Studies have shown that geographical factors have a certain effect on the chloroplast genome (Yang et al., 2022). P. cyrtonema, for example, is widely distributed in China; however, it is mainly distributed in the middle and lower reaches of the Yangtze River (Zhang et al., 2024). At the same time, the southern part of Anhui and the eastern part of Zhejiang Province are in the middle and lower reaches of the Yangtze River. The Dabie Mountains and Southern Anhui Mountains in southern Anhui is suitable altitude for the growth of P. cyrtonema (Liu et al., 2023). Beijing and Hunan Province are farther away from this region and have different climatic environments; therefore, it is speculated that those may be geographically isolated from the P. cyrtonema in the Yangtze River Basin. Therefore, the phylogenetic tree showed that the P. cyrtonema from Anhui was more closely related to the P. cyrtonema from Zhejiang. We believe that geographical factors influence the phylogeny of Polygonatum species and this requires further exploration.