https://orcid.org/0009-0007-9286-4883
Figures
Abstract
The genus Polygonatum (Asparagaceae) comprises perennial herbaceous plants with significant economic and medicinal value. In this study, we analyzed the complete chloroplast (cp) genome of Polygonatum sinopubescens and compared it with closely related species. The primary objective was to elucidate structural variations, species divergence, and phylogenetic relationships among taxa. The cp genome of P. sinopubescens exhibits the typical quadripartite structure, consisting of a large single-copy (LSC) region, a small single-copy (SSC) region, and a pair of inverted repeats (IRs), with a total sequence length of 155,307 bp and a GC content of 37.68%. The present analysis revealed a high degree of consistency in gene order and GC content between P. sinopubescens and other Polygonatum species. A total of 112 genes were annotated, including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. The genome contained 67 simple sequence repeats (SSRs), and codon usage was biased toward codons ending in A/T; among the 30 codons with RSCU > 1, 93.3% ended with A/T. Nucleotide polymorphism analysis identified nine highly variable regions, and selection pressure analysis revealed that only ndhA, ycf2, accD, and rbcL genes were under positive selection (Ka/Ks > 1), which was observed in only a subset of species. Phylogenetic analyses indicated that Polygonatum is a monophyletic group that can be divided into three major clades. P. sinopubescens was placed in sect. Polygonatum and was most closely related to P. filipes. This study provides a comprehensive characterization of the cp genome of P. sinopubescens and clarifies its phylogenetic placement, offering important references for species identification, evolutionary studies, and phylogenetic research within Polygonatum.
Citation: Mo Z-M, Wei C-M, Yu H-Y, Yang C-D (2025) Chloroplast genome analysis and phylogenetic position of Polygonatum sinopubescens and comparison with related species. PLoS One 20(12): e0338103. https://doi.org/10.1371/journal.pone.0338103
Editor: Mojtaba Kordrostami, Nuclear Science and Technology Research Institute, IRAN, ISLAMIC REPUBLIC OF
Received: May 7, 2025; Accepted: November 18, 2025; Published: December 5, 2025
Copyright: © 2025 Mo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its S1 File.
Funding: the National Natural Science Foundation of China (32160287); Basic Research Program Project of Tongren Science and Technology Bureau (Tongshi Scientific Research [2021] No. 73); The Youth Science and Technology Talent Growth Project of Regular Higher Education Institutions in Guizhou Province (Qian Jiao He KY[2022] No. 066); Supported by the Special Project of the Forestry Department of Guizhou Province (Investigation of Endemic Seed Plant Resources in Tongren City). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript, Basic Research Program Project of Tongren Science and Technology Bureau (Tongshi Scientific Research 〔 2025 〕 No. 58), Funds for the Protection and Restoration of Forests and Grasslands in 2024 from the Central Finance, QCZH [2023]82.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
The genus Polygonatum Mill. (Asparagaceae) comprises perennial herbaceous plants, with approximately 39 species distributed in China [1]. “Polygonati Rhizoma”is a medicinal and edible plant known for its pharmacological properties, including anti-aging, anti-tumor, hypoglycemic, and immune-enhancing effects [2–4]. Due to its high medicinal value, over a hundred commercial pharmaceutical and healthcare products are derived from Polygonatum [5]. Based on morphological, palynological, cytological, and molecular biological studies [6–10], recent research has classified Polygonatum into three sections: section Verticillata, section Polygonatum, and section Sibirica [11–13]. The Chinese Pharmacopoeia primarily records the dried rhizomes of Polygonatum odoratum, Polygonatum sibiricum, Polygonatum kingianum, and Polygonatum cyrtonema as medicinal ingredients [14]. However, due to morphological similarities among Polygonatum species, botanical identification is often challenging, leading to frequent adulteration in the market [15]. Common adulterants include Polygonatum zanlanscianense, Polygonatum cirrhifolium, Polygonatum verticillatum, and other species from section Verticillata [16,17]. These adulterants generally exhibit inferior medicinal properties, and some may compromise clinical safety and efficacy [18,19]. Therefore, effective identification methods are needed to ensure the quality of Polygonati Rhizoma medicinal materials.
The chloroplast (cp) is a crucial organelle for photosynthesis and energy conversion in plant cells. In angiosperms, the cp genome is maternally inherited and is characterized by structural stability, conserved coding sequences, and rich genetic information, making it a valuable resource for species identification and genetic variation studies [20]. Most higher plants possess a typical quadripartite chloroplast genome structure, comprising a large single-copy (LSC) region, a small single-copy (SSC) region, and a pair of inverted repeats (IRs) [21]. With the advent of high-throughput sequencing technologies, cp genomes have been widely employed in plant phylogenetics, species identification, genetic diversity analysis, and genetic engineering [22–24]. The complete chloroplast genome, used as a super-barcode, has shown great potential in the identification of medicinal plants. Wu et al. [25] applied this technique to successfully distinguish Fritillaria species recorded in the Chinese Pharmacopoeia from their close relatives and adulterants. This technology has also been widely applied in other medicinal plants and has achieved good results [26]. For example, Cui et al. [27] accurately identified three closely related species of Amomum (A. villosum, A. villosum var. xanthioides, and A. longiligulare). Similarly, Zhu et al. [28] confirmed that the complete chloroplast genome dataset provides the strongest discriminating power for Dendrobium officinale and its related species. Chen et al. [29] demonstrated that the chloroplast genome not only enables precise identification of Thalictrum fargesii, but that its highly variable regions (such as ndhD-psaC and rpl16-rps3) also hold promise for developing specific molecular markers for identifying ethnomedicines and their contaminants. In addition, this technique has been successfully applied to identify Mussaenda pubescens [30], Sophora tonkinensis [31], members of the subfamily Aroideae [32], and to determine their phylogenetic positions. Wang et al. [33] combined chloroplast genome and internal transcribed spacer(ITS) sequences to confirm that C. × ventricosum is most closely related to C. calceolus and supportsd its origin as an interspecific hybrid between C. calceolus and C. macranthos. Similarly, studies on the genus Lasianthus not only clarified phylogenetic relationships using the complete chloroplast genome but also identified an efficient identification marker composed of ITS2 + psaI-ycf4 [34].
Therefore, utilizing the cp genome for species identification within Polygonatum can enhance the safety and efficacy of medicinal applications and promote the sustainable development of Polygonatum resources. Polygonatum sinopubescens is an endemic species discovered in Yinjiang, Guizhou Province [35]. Morphologically, P. sinopubescens is distinguished from Polygonatum filipes by its densely hairy stems (approximately 30 cm tall), petioles with soft hairs, young leaves densely covered with short hairs on the abaxial surface, inflorescences bearing 2 ~ 3 flowers per peduncle, pedicels covered with long soft hairs, filaments measuring 7 ~ 11 mm in length with pubescent upper portions, and obovoid berries, These characteristics classify P. sinopubescens within sect. Polygonatum of Polygonatum [36]. Nutritional composition analysis has further confirmed that P. sinopubescens is a high-quality functional plant resource with both medicinal and edible applications [37]. This study reports the complete chloroplast genome of P. sinopubescens, expanding the genomic resources for Polygonatum and providing a valuable reference for species classification, genetic diversity research, and medicinal applications.
2. Materials and methods
2.1. Plant materials
The plant samples were collected from Yinjiang Tujia Autonomous County, Tongren City, Guizhou Province (27°43’1.98“N,108°28’15.21” E). They were identified as P. sinopubescens of the Polygonatum genus by Professor Yang Chuandong of Tongren University (Fig 1). The fresh leaves collected were stored in dry ice and sent to Qingke Company for DNA extraction and third-generation chloroplast gene sequencing.
2.2. Chloroplast DNA extraction
Fresh young leaves of the P. sinopubescens sample were frozen in liquid nitrogen, and high-quality genomic DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method. The DNA concentration was measured using the Thermo Scientific NanoDrop software, and DNA quality was assessed by 1% agarose gel electrophoresis. An Illumina genomic library was constructed and subjected to 2 × 150 bp sequencing using the NovaSeq X Plus platform (Illumina, San Diego, CA, USA) at Qingke Biotechnology (Beijing, China).
2.3. Chloroplast DNA sequencing, assembly and data processing
For chloroplast genome assembly, clean data were processed using GetOrganelle v1.7.5 [38], with the seed database as a reference. Genome assembly was performed using SPAdes, and the assembly order of chloroplast contigs was verified by alignment against the NT database. Contigs with consistent sequence order were selected as the final genome assembly. The starting position and orientation of the chloroplast genome sequence were determined based on a reference genome, along with the identification of possible partition structures (LSC/IR/SSC), resulting in the finalized chloroplast genome sequence. Gene annotation, including predictions of protein-coding genes, tRNA genes, and rRNA genes, was performed using GeSeq, with manual correction of gene boundaries and exon/intron junctions. The circular genome map was visualized using OGDRAW [39]. The final annotated chloroplast genome was submitted to NCBI, and the registration number PQ858224 was obtained.
2.4. Comparative bioinformatic analysis
The relative synonymous codon usage (RSCU) values were calculated using the Cusp software (EMBOSS v6.6.0.0) to determine codon preference. Microsatellite loci were analyzed using the MISA software (version 1.0), with parameters set to ≥10 repeats for mononucleotides, ≥ 5 repeats for dinucleotides, ≥ 4 repeats for trinucleotides, and ≥3 repeats for tetranucleotides, pentanucleotides, and hexanucleotides [40].The contraction and expansion of the IR regions were visualized using the IRscope online tool(https://irscope.shinyapps.io/irapp/) to investigate changes in the LSC/IRb/SSC/IRa boundary positions [41]. Phylogenetic analysis was performed using PhyloSuite_v1.2.3, with MAFFT alignment of P. sinopubescens and nine closely related species, followed by nucleotide diversity (Pi) analysis using DnaSP software (version 6.0). The window length and step size parameters were set to 600 and 200, respectively [42]. The Ka, Ks, and Ka/Ks ratios for the shared protein-coding genes (PCGs) across 10 Polygonatum species were extracted and calculated using CPStools and KaKs_calculator3 [43]. It is generally accepted that Ka/Ks < 1 indicates negative selection (purifying selection), meaning harmful mutations are eliminated, and the gene function is conserved. A Ka/Ks ratio of 1 suggests that the gene is in a neutral evolutionary state, with mutations not affected by natural selection. A Ka/Ks ratio > 1 suggests positive selection (adaptive evolution), where beneficial mutations are retained, aiding species adaptation to the environment.
2.5. Phylogenetic analysis
Phylogenetic analysis was conducted using the complete chloroplast genomes, with Maianthemum as the outgroup. Except for P. sinopubescens, all chloroplast genome sequences were retrieved from GenBank. The total sequence matrix was aligned using the MAFFT plugin in PhyloSuite v1.2.3, and the optimal substitution model was selected using ModelFinder based on the Bayesian Information Criterion (BIC). A maximum likelihood (ML) phylogenetic tree was reconstructed under the TVM + F + I + I + R3 model using IQ-TREE with 5000 ultrafast bootstrap replicates. The Bayesian (BI) phylogenetic tree was constructed using MrBayes under the GTR + I + G + F model. The phylogenetic tree was further refined using FigTree v1.4.4.
3. Results
3.1. Structure and characteristics of chloroplast group in P. sinopubescens
The P. sinopubescens chloroplast genome was a double-stranded circular molecule with a typical quadripartite structure, comprising a large single-copy region (LSC), a small single-copy region (SSC), and two inverted repeat regions (IRs). The total genome length was 155,307 bp (Fig 2), with an overall GC content of 37.68%. The LSC region was 84,252 bp in length with a GC content of 35.72%, the SSC region measured 18,455 bp with a GC content of 31.56%, and each IR region spanned 26,300 bp with a GC content of 42.98%. A total of 112 genes were annotated, including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes, These genes are primarily involved in photosynthesis and self-replication. Among them, 11 genes contain introns, with 7 genes containing one intron and 4 genes containing two introns (Table 1).
3.2. simple repeat sequence
Simple sequence repeats (SSRs) in the chloroplast genome of P. sinopubescens were detected using the MISA software (version 1.0) tool. The results showed (Fig 3) that a total of 67 SSR sequences were identified, categorized into 5 types. Among these, the most abundant were mononucleotide SSRs, with 38 sequences, accounting for 56.72% of the total; followed by 15 dinucleotide SSRs, which accounted for 22.39%; trinucleotide, tetranucleotide, and pentanucleotide SSRs numbered 4, 8, and 2, respectively, with proportions ranging from 0% to 11.94%. For mononucleotide repeats, A and T repeats dominated, comprising 97.37% of the total; dinucleotide repeats were predominantly AT/TA (80%). In trinucleotide SSRs, repeats composed of A and T bases (such as AAT and ATT) accounted for 75% of the total trinucleotide SSRs. Additionally, the LSC region contained the most SSR sequences, representing 76.12% of all SSRs. The REPuter-generated results indicated the identification of 59 dispersed repeat sequences, including 7 complementary repeat sequences (C) and 52 palindromic repeat sequences (P).
3.3. Codon usage frequency analysis
Based on the protein-coding genes of the complete chloroplast genome, the codon usage frequency in P. sinopubescens was calculated. A total of 61 codons encoding 20 amino acids. Among these, leucine (Leu) was the most frequently used amino acid, with a total of 2,673 occurrences, followed by isoleucine (Ile) and serine (Ser), with 2,267 and 2,048 occurrences, respectively. Cysteine (Cys) was the least frequently used amino acid, with only 304 occurrences. The most frequently used synonymous codon was ATT, encoding isoleucine (Ile), with 1,082 instances (4.14%), while the least frequently used codon was TGC, encoding cysteine (Cys), with only 68 instances (0.26%). Codon usage bias was analyzed using Cusp (EMBOSS v6.6.0.0) software to calculate the Relative Synonymous Codon Usage (RSCU) values. High codon usage bias was detected for 30 codons with an RSCU > 1, while low codon usage bias was observed for 29 codons with an RSCU < 1. These results indicate that the chloroplast genome of P. sinopubescens exhibits a significant codon usage bias. Additionally, no codon usage bias was detected for methionine and tryptophan (RSCU = 1), and the third position of all highly preferred codons (RSCU > 1) primarily included 28 A/T codons (Fig 4).
3.4. IR contraction and expansion
The contraction and expansion of the IR regions reveal structural variations at the LSC/IR/SSC junctions. Using the IRscope online program, we analyzed the expansion and contraction of the IR regions in the chloroplast genomes of 10 Polygonatum species (Fig 5). The results showed that the length of the IR regions was relatively conserved, ranging from 25,008 bp in P. odoratum to 26,415 bp in P. sibiricum, and the gene content at the IR/SC boundaries was generally consistent. The genes rpl22, rps19, ndhF, ycf1, and psbA were located at the IR boundary regions, and significant differences were observed in the contraction and expansion of the IR regions. In most Polygonatum species, the rps19 gene was entirely located in the IRb region, positioned 13 bp or 17 bp from the IRb boundary. In contrast, rps19 was absent in P. cyrtonema and P. odoratum, which may be attributed to the extended length (1,492 bp) of the rpl2 gene that bridges the LSC and IRb regions, with distances of 754 bp and 663 bp from the IRb boundary, respectively. The ndhF gene spanned the junction between IRb and SSC, with 22–34 bp located within the IRb region. The ycf1 gene formed the junction between SSC and IRa, and its distance from the IRa boundary ranged from 883 bp to 895 bp. In addition, rpl2 and trnH were present in the IRa region, while psbA was located in the LSC region.
3.5. Nucleotide diversity analysis and selective pressure
Nucleotide diversity (Pi) of ten chloroplast genes was analyzed using DnaSP software (version 6.0) to identify mutational hotspot regions in the chloroplast genomes of Polygonatum species. The results showed that nine regions had Pi values greater than 0.010, namely trnK-UUU, rps16-trnQ-UUG, trnS-GCU-trnG-UCC, trnC-GCA, petA-psbJ, ndhF, rpl32, ccsA-ndhD, and ycf1 (Fig 6). These high-Pi regions represent potential divergence loci within the chloroplast genomes of the ten Polygonatumspecies analyzed. Among them, five mutational hotspots (trnK-UUU, rps16-trnQ-UUG, trnS-GCU-trnG-UCC, trnC-GCA, petA-psbJ) were located in the LSC region, while four (ndhF, rpl32, ccsA-ndhD, ycf1) were located in the SSC region. The rpl32gene fragment, located in the SSC region, exhibited the highest level of variation, with a coefficient of 0.01826. Notably, no highly variable sites were detected in the IR regions, further supporting the high conservation of the IR regions in the chloroplast genomes of Polygonatum species. These nine high-Pi sequences can serve as potential DNA markers for elucidating genetic differentiation among different taxa within the genus Polygonatum.
To investigate the molecular evolutionary processes of chloroplast protein-coding genes in Polygonatum, we estimated the ratio of nonsynonymous (Ka) to synonymous (Ks) substitutions using 78 shared protein-coding genes (CDS) for selection pressure analysis (Fig 7). The results showed that the Ka/Ks values of most genes were lower than 1, indicating that these protein-coding genes have been subjected to strong purifying selection. Only a very small number of genes exhibited Ka/Ks > 1 (including ndhA, ycf2, accD, and rbcL), and this pattern was observed only in a few species. Among them, the ycf2 gene showed the highest Ka/Ks value in P. verticillatum (2.31676), followed by P. sibiricum(2.00985) and P. kingianum (1.08535). The ndhA gene exhibited a Ka/Ks value of 1.34745 in P. verticillatum, the accD gene had a Ka/Ks value of 1.00734 in P. kingianum, and the rbcL gene showed Ka/Ks values of 1.8394 in P. franchetii and 1.1006 in P. sibiricum. These genes exhibited relatively high substitution rates and evolutionary rates in specific species, suggesting evidence of positive selection. Functionally, the positively selected genes can be classified into photosynthesis-related genes (e.g., ndhA, rbcL) and other functional categories (e.g., ycf2, accD), indicating that most of the genes under positive selection are closely associated with the photosynthetic system.
3.6. Phylogenetic analysis
Chloroplast genomes are widely employed in phylogenetic analyses across diverse plant taxa. To clarify the phylogenetic position of Polygonatum species, Maianthemum henryi (Baker) LaFrankie and Maianthemum fuscum (Wall.) LaFrankie were selected as outgroups. A total of 44 complete chloroplast genome sequences were used to construct phylogenetic trees using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. The ML and BI trees exhibited congruent topologies (Fig 8), with most nodes receiving strong statistical support, thereby confirming the monophyly of Polygonatum, Maianthemum, and Disporopsis. Polygonatum was resolved as a sister clade to Heteropolygonatum (BS = 100; PP = 1). Within Polygonatum, three major clades were identified: sect. Verticillata, sect. Polygonatum, and sect. Sibirica (BS = 100; PP = 1), with sect. Sibirica comprising only a single species, P. sibiricum. P. sinopubescens and P. filipes formed a distinct and strongly supported clade (BS = 100; PP = 1), indicating a close evolutionary relationship. Furthermore, the pharmacopoeial species P. odoratum, P. cyrtonema, P. sibiricum, and P. kingianum, which are listed in the Chinese Pharmacopoeia, were clearly distinguishable from other Polygonatum species with high support values, underscoring their distinct genetic identities.
Only ML tree was shown, because of the highly identified topologies of ML tree and BI tree. The value of ML supports and Bayesian posterior probabilities were shown above the branches. The cp genomes newly sequenced in this study are highlighted with red font marks.
4. Discussion
4.1. Characteristics analysis of the chloroplast whole genome
In this study, the complete chloroplast genome of P. sinopubescens was analyzed. The genome exhibits a typical quadripartite structure, consisting of a large single-copy (LSC) region, a small single-copy (SSC) region, and two inverted repeat (IR) regions forming a circular double-stranded molecule. The total genome length is 155,307 bp, which is comparable to previously reported chloroplast genomes of other Polygonatum species [44]. The chloroplast genome displays a higher AT content than GC content, with the GC content in the IR regions being higher than that in the LSC and SSC regions. Our results indicate that the total length, GC content, and gene composition of the P. sinopubescens chloroplast genome are nearly identical to those of other Polygonatum species [42]. In total, the chloroplast genome of P. sinopubescens encodes 112 genes, including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Among them, 11 genes contain introns, and 22 genes are located within the IR regions.
Simple sequence repeats (SSRs) are an important class of codominant DNA molecular markers that have been widely used in species identification, phylogeography, and population genetics due to their high abundance, random distribution in genomes, and rich polymorphism information [45–47]. In this study, a total of 67 SSRs were detected, among which mononucleotide SSRs were the most frequent in all genomes and were predominantly composed of A/T motifs, accounting for 97.37% of the total. Dinucleotide repeats ranked second in abundance, with AT/TA motifs being the most common, representing 80% of this category. These results indicate that SSRs in the chloroplast genomes of Polygonatum species are strongly biased toward A and T bases, consistent with findings from other Polygonatum taxa [42,44,48] and similar to the SSR composition observed in the chloroplast genomes of most angiosperms [49–52]. SSRs rich in A/T have higher mutation rates and are more likely to generate polymorphic loci, making them suitable as high-resolution genetic markers. The cpSSRs identified in this study hold promise as valuable molecular marker resources for Polygonatum species identification, genetic diversity assessment, and phylogenetic studies.
As the link between nucleic acids, proteins, and genetic material, codons play a crucial role in the transmission of genetic information and provide reliable insights into gene function and species evolution [53,54]. In this study, a total of 61 codons were identified, among which leucine (Leu) was the most frequently encoded amino acid, followed by isoleucine (Ile) and serine (Ser). The most frequently used codons for these amino acids are TTA, ATT, and TCT. Previous studies have demonstrated that GC content is closely associated with mutational pressure or natural selection, whereas interspecific differences in codon usage frequency may be related to evolutionary status, ecological environment, and nucleotide composition [55]. Elucidating the characteristics of codon bias and its variation is of great significance for advancing our understanding of molecular evolution and the biodiversity of heterologous gene expression across species [56,57]. Based on the relative synonymous codon usage (RSCU) analysis, most high-frequency and highly expressed codons ended with A or U, further supporting the A/U bias at the third codon position in medicinal Polygonatum species. Moreover, SSR analysis revealed a pronounced preference for A and T nucleotides. Given that A/T base pairs, which form two hydrogen bonds, are more easily disrupted than G/C base pairs, the preference for A and T nucleotides in the chloroplast genomes of Polygonatum species may contribute to their strong adaptive capacity and pronounced structural variation in response to environmental changes. However, the underlying mechanisms behind this phenomenon require further investigation.
4.2. Comparative analysis of chloroplast genomes
The contraction or expansion of IR/SC boundaries is a major driver of chloroplast genome size variation. In Polygonatum, the boundary genes are primarily rpl22, rps19, ndhF, ycf1, and psbA, which is consistent with previous studies on this genus [58], suggesting that boundary characteristics are relatively conserved among closely related species [59]. Studies of chloroplast genomes in monocotyledonous plants have shown that the rps19 gene is located in the IR region [60]. In Polygonatum, many rps19 genes are entirely located within the IR region [46,61]; however, in this study, we found that the rps19 gene of P. sibiricum was partially located in the LSC region, which may be attributed to IR contraction. In P. cyrtonema and P. odoratum, the rps19 gene was missing, which may have resulted from the elongation of the rpl2 gene, thereby bridging the LSC and IRb regions. The stability of the IR/SC boundary suggests that Polygonatum species may have experienced relatively low selective pressure during evolution, consistent with their broad ecological adaptability and strong species differentiation ability [62].
Evaluation of Ka/Ks values for protein-coding genes containing RNA editing sites can provide insights into functional diversity, structural variation, and evolutionary processes. The Ka/Ks ratio is commonly used to determine whether protein-coding genes are subject to selective pressure and has been widely recognized as a key metric for assessing adaptive evolutionary rates and positive selection. Our selective pressure analysis indicated that most genes have undergone purifying selection, consistent with a pattern of conservative evolution. Notably, ndhA, ycf2, accD, and rbcL exhibited signatures of positive selection. Among these, ndhA and rbcL are photosynthesis-related and systemic genes, respectively. Given that Polygonatum species predominantly grow on shaded forest slopes, in thickets, or under canopies, their adaptation to light stress may represent an important genetic basis for chloroplast genome evolution in this genus [58].
Nucleotide diversity analysis revealed that the highly variable regions of the Polygonatum chloroplast genome were mainly located in the LSC and SSC regions. Nine hypervariable Pi fragments were identified: trnK-UUU, rps16-trnQ-UUG, trnS-GCU-trnG-UCC, trnC-GCA, petA-psbJ, ndhF, rpl32, ccsA-ndhD, and ycf1. These mutational hotspots provide potential chloroplast DNA barcode references for the molecular identification of Polygonatum species in future studies.
4.3. Phylogenetic analysis
The phylogeny and classification of the genus Polygonatum have long been controversial. In this study, a phylogenetic tree was reconstructed based on complete chloroplast genome sequences. The results provided strong support for the monophyly of Polygonatum (Fig. 8), with Polygonatum and Heteropolygonatum resolved as sister clades. Within Polygonatum, three well-supported clades were identified: sect. Polygonatum, sect. Sibirica, and sect. Verticillata, with sect. Verticillata representing a relatively ancestral lineage within the genus. This finding is consistent with the results of Shi Naixing [53].The phylogenetic tree confirmed the systematic position of P. sinopubescens within Polygonatum, showing that P. sinopubescens and P. filipes form a sister group within sect. Polygonatum. This relationship is supported by previous morphological, cytological, and molecular evidence [35,63,64], further corroborating the close phylogenetic relationship between the two species. Additionally, strong support was found for the monophyly of P. sibiricum, consistent with findings from other studies [65–67].In summary, our study enriches the genomic resources of Polygonatum and provides valuable insights into the phylogenetic relationships within the genus. The findings on P. sinopubescens also have important implications for the exploration and conservation of Polygonatum genetic resources.
Supporting information
S1 File. The new dataset generated by this study has been included in the supplementary materials, while the other datasets are all from the following public domain resources: https://www.ncbi.nlm.nih.gov/#!/edu/home/principal/inicio.
https://doi.org/10.1371/journal.pone.0338103.s001
(ZIP)
References
- 1.
Chen XQ, Tamura MN. Flora of China. Science Press. Beijing: Beijing and Missouri Botanical Garden Press, St. Louis; 2000.
- 2. Jiao J, Huang W, Bai Z, Liu F, Ma C, Liang Z. DNA barcoding for the efficient and accurate identification of medicinal polygonati rhizoma in China. PLoS One. 2018;13(7):e0201015. pmid:30021015
- 3. Zhang J, Wang Y-Z, Yang W-Z, Yang M-Q, Zhang J-Y. Research progress in chemical constituents in plants of Polygonatum and their pharmacological effects. Zhongguo Zhong Yao Za Zhi. 2019;44(10):1989–2008. pmid:31355552
- 4. Luo L, Qiu Y, Gong L, Wang W, Wen R. A review of Polygonatum Mill. genus: its taxonomy, chemical constituents, and pharmacological effect due to processing changes. Molecules. 2022;27(15):4821. pmid:35956772
- 5. Su W-T, Liu Y-J, Jiang Y-F, Xie J-Q, Pan X-H, Liu J-J, et al. Status of Polygonati Rhizome industry and suggestion for its sustainable development. Zhongguo Zhong Yao Za Zhi. 2018;43(13):2831–5. pmid:30111038
- 6. Baker JG. Revision of the Genera and Species of Asparagaceae. (Continued.). J Linn Soc Bot. 1875;14(80):547–632.
- 7. Tamura MN. Biosystematic studies on the genus Polygonatum (Liliaceae) Ⅲ. Morphology of staminal filament and karyology of eleven Eurasian species. Bot Jahrb Syst. 1993;115(1):1–26.
- 8. Wang JJ, Nie ZL, Meng Y. Cytological advances on tribe Polygonateae (Asparagaceae). Acta Botan Boreali-Occident Sin. 2016;36(04):834–45.
- 9. Shi NX, Wen GS, Zhao MF. Application of DNA molecular identification technology in Polygonatum Mill. J Plant Genet Resourc. 2021;22(05):1209–18.
- 10. Xia M, Liu Y, Liu J, Chen D, Shi Y, Chen Z, et al. Out of the Himalaya-Hengduan Mountains: Phylogenomics, biogeography and diversification of Polygonatum Mill. (Asparagaceae) in the Northern Hemisphere. Mol Phylogenet Evol. 2022;169:107431. pmid:35131418
- 11. Floden A, Schilling EE. Using phylogenomics to reconstruct phylogenetic relationships within tribe Polygonateae (Asparagaceae), with a special focus on Polygonatum. Mol Phylogenet Evol. 2018;129:202–13. pmid:30195040
- 12. Meng Y, Nie Z-L, Deng T, Wen J, Yang Y-P. Phylogenetics and evolution of phyllotaxy in the Solomon’s seal genus Polygonatum (Asparagaceae: Polygonateae). Bot J Linn Soc. 2014;176(4):435–51.
- 13. Zhao L, Zhou S, He X. A phylogenetic study of Chinese Polygonatum (Polygonateae, Asparagaceae). Nord J Bot. 2019;37(2).
- 14.
National Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China: Volume One. Beijing: China Medical Science and Technology Press; 2020.
- 15.
Pang KQ. Identification and evaluation of Polygonatum plants from different origins. Guizhou University; 2022.
- 16.
Liu X. Research on the biological characteristics of Polygonatum, a whorled leafy herb. Anhui University of Chinese Medicine. 2018.
- 17. Zhang MY, Li YM, Cheng WP. Molecular identification of medicinal plants of the genus Polygonatum based on universal DNA barcoding sequences. Chinese Herb Med. 2023;54(01):235–44.
- 18. Wang ZW, Du FQ, Zhang L. Research progress on classification and identification of traditional Chinese medicine Polygonatum odoratum and its easily mixed plants. Northern Horticulture. 2019;2019(24):130–6.
- 19. Ali M, Liu Y-J, Xia Q-P, Bahadur S, Hussain A, Shao J-W, et al. Pollen micromorphology of eastern Chinese Polygonatum and its role in taxonomy by using scanning electron microscopy. Microsc Res Tech. 2021;84(7):1451–61. pmid:33580981
- 20. Xiao-Ming Z, Junrui W, Li F, Sha L, Hongbo P, Lan Q, et al. Inferring the evolutionary mechanism of the chloroplast genome size by comparing whole-chloroplast genome sequences in seed plants. Sci Rep. 2017;7(1):1555. pmid:28484234
- 21. Liu X-F, Zhu G-F, Li D-M, Wang X-J. Complete chloroplast genome sequence and phylogenetic analysis of Spathiphyllum “Parrish”. PLoS One. 2019;14(10):e0224038. pmid:31644545
- 22. Zhao P, Zhou H-J, Potter D, Hu Y-H, Feng X-J, Dang M, et al. Population genetics, phylogenomics and hybrid speciation of Juglans in China determined from whole chloroplast genomes, transcriptomes, and genotyping-by-sequencing (GBS). Mol Phylogenet Evol. 2018;126:250–65. pmid:29679714
- 23. Li L, Hu Y, He M, Zhang B, Wu W, Cai P, et al. Comparative chloroplast genomes: insights into the evolution of the chloroplast genome of Camellia sinensis and the phylogeny of Camellia. BMC Genomics. 2021;22(1):138. pmid:33637038
- 24. Fang H, Dai G, Liao B, Zhou P, Liu Y. Application of chloroplast genome in the identification of Phyllanthus urinaria and its common adulterants. Front Plant Sci. 2023;13:1099856. pmid:36684764
- 25. Wu L, Wu M, Cui N, Xiang L, Li Y, Li X, et al. Plant super-barcode: a case study on genome-based identification for closely related species of Fritillaria. Chin Med. 2021;16(1):52. pmid:34225754
- 26. Jiang Y, Zhu C, Wang S, Wang F, Sun Z. Identification of three cultivated varieties of Scutellaria baicalensis using the complete chloroplast genome as a super-barcode. Sci Rep. 2023;13(1):5602. pmid:37019975
- 27. Cui Y, Chen X, Nie L, Sun W, Hu H, Lin Y, et al. Comparison and phylogenetic analysis of chloroplast genomes of three medicinal and edible Amomum species. Int J Mol Sci. 2019;20(16):4040. pmid:31430862
- 28. Zhu S, Niu Z, Xue Q, Wang H, Xie X, Ding X. Accurate authentication of Dendrobium officinale and its closely related species by comparative analysis of complete plastomes. Acta Pharm Sin B. 2018;8(6):969–80. pmid:30505665
- 29. Chen S, Safiul Azam FM, Akter ML, Ao L, Zou Y, Qian Y. The first complete chloroplast genome of Thalictrum fargesii: insights into phylogeny and species identification. Front Plant Sci. 2024;15:1356912. pmid:38745930
- 30. Zhou C, Tao F, Long R, Yang X, Wu X, Xiang L, et al. The complete chloroplast genome of Mussaenda pubescens and phylogenetic analysis. Sci Rep. 2024;14(1):9131. pmid:38644374
- 31. Wei F, Tang D, Wei K, Qin F, Li L, Lin Y, et al. The complete chloroplast genome sequence of the medicinal plant Sophora tonkinensis. Sci Rep. 2020;10(1):12473. pmid:32719421
- 32. Jia X, Wei J, Chen Y, Zeng C, Deng C, Zeng P, et al. Codon usage patterns and genomic variation analysis of chloroplast genomes provides new insights into the evolution of Aroideae. Sci Rep. 2025;15(1):4333. pmid:39910236
- 33. Wang Q, An J, Wang Y, Zheng B. The complete chloroplast genome sequences of three Cypripedium species and their phylogenetic analysis. Sci Rep. 2025;15(1):13461. pmid:40251259
- 34. Zhang Y, Song M, Tang D, Li X, Xu N, Li H, et al. Comprehensive comparative analysis and development of molecular markers for Lasianthus species based on complete chloroplast genome sequences. BMC Plant Biol. 2024;24(1):867. pmid:39285331
- 35. An M-T, Lin Y, Wang J-G, Wu J-H, Meng M. A new species of <i>Polygonatum</i> Mill. (Asparagaceae) from Guizhou, China. Bangladesh J Plant Taxon. 2016;23(1):7–11.
- 36. Xiang KW, Chu ZF, Meng R. The discovery and karyotype of Polygonatum sinopubescens, a new record plant in Hunan Province. J Jishou Univ Nat Sci Ed. 2018;39(6):4.
- 37. Yang TY, Yang CD, Lu ZH. Analysis of nutritional components of Polygonatum sinopubescens. Grains and Oils. 2022;35(03):155–8.
- 38. Jin J-J, Yu W-B, Yang J-B, Song Y, dePamphilis CW, Yi T-S, et al. GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biol. 2020;21(1):241. pmid:32912315
- 39. Lohse M, Drechsel O, Bock R. OrganellarGenomeDRAW (OGDRAW): a tool for the easy generation of high-quality custom graphical maps of plastid and mitochondrial genomes. Curr Genet. 2007;52(5–6):267–74. pmid:17957369
- 40. Ren YL, Li XE, Liang JL, et al. Genomic characteristics and phylogenetic analysis of chloroplasts in Berberis japonica. Acta Botanica Northwest. 2024;44(10):1616–162.
- 41. Zhang MJ, Yang XY, Yin T. Genomic characteristics and phylogenetic analysis of chloroplasts in Syzygium grijsii. Plants of Guangxi. 2023;43(10):1849–60.
- 42. Wang J, Qian J, Jiang Y, Chen X, Zheng B, Chen S, et al. Comparative analysis of chloroplast genome and new insights into phylogenetic relationships of Polygonatum and tribe Polygonateae. Front Plant Sci. 2022;13:882189. pmid:35812916
- 43. Huang L, Yu H, Wang Z, Xu W. CPStools: A package for analyzing chloroplast genome sequences. iMetaOmics. 2024;1(2).
- 44. Yan M, Dong S, Gong Q, Xu Q, Ge Y. Comparative chloroplast genome analysis of four Polygonatum species insights into DNA barcoding, evolution, and phylogeny. Sci Rep. 2023;13(1):16495. pmid:37779129
- 45. Hirano R, Ishii H, Htun Oo T, Gilani SA, Kikuchi A, Watanabe KN. Propagation management methods have altered the genetic variability of two traditional mango varieties in Myanmar, as revealed by SSR. Plant Genet Res. 2011;9(3):404–10.
- 46. Hu J, Gui S, Zhu Z, Wang X, Ke W, Ding Y. Genome-Wide Identification of SSR and SNP Markers Based on Whole-Genome Re-Sequencing of a Thailand Wild Sacred Lotus (Nelumbo nucifera). PLoS One. 2015;10(11):e0143765. pmid:26606530
- 47. Zhao Y, Yin J, Guo H, Zhang Y, Xiao W, Sun C, et al. The complete chloroplast genome provides insight into the evolution and polymorphism of Panax ginseng. Front Plant Sci. 2015;5:696. pmid:25642231
- 48. Guisinger MM, Kuehl JV, Boore JL, Jansen RK. Extreme reconfiguration of plastid genomes in the angiosperm family Geraniaceae: rearrangements, repeats, and codon usage. Mol Biol Evol. 2011;28(1):583–600. pmid:20805190
- 49. Zhang S-D, Yan K, Ling L-Z. Characterization and phylogenetic analyses of ten complete plastomes of Spiraea species. BMC Genomics. 2023;24(1):137. pmid:36944915
- 50. Zong D, Qiao Z, Zhou J, Li P, Gan P, Ren M, et al. Chloroplast genome sequence of triploid Toxicodendron vernicifluum and comparative analyses with other lacquer chloroplast genomes. BMC Genomics. 2023;24(1):56. pmid:36721120
- 51. Zhang Z, Zhang D-S, Zou L, Yao C-Y. Comparison of chloroplast genomes and phylogenomics in the Ficus sarmentosa complex (Moraceae). PLoS One. 2022;17(12):e0279849. pmid:36584179
- 52. Wu X-M, Wu S-F, Ren D-M, Zhu Y-P, He F-C. The analysis method and progress in the study of codon bias. Yi Chuan. 2007;29(4):420–6. pmid:17548303
- 53.
Shi NX. Comparative Analysis of Chloroplast Genomics of Polygonatum odoratum and Its Phylogenetic Study. Yunnan Agricultural University; 2022.
- 54. Liu X, Bai Y, Wang Y, Chen Y, Dong W, Zhang Z. Complete chloroplast genome of Hypericum perforatum and dynamic evolution in Hypericum (Hypericaceae). Int J Mol Sci. 2023;24(22):16130. pmid:38003320
- 55. Wang Z, Cai Q, Wang Y, Li M, Wang C, Wang Z, et al. Comparative analysis of codon bias in the chloroplast genomes of Theaceae species. Frontiers in Genetics. 2022;13:824610. pmid:35360853
- 56. Chen J, Ma W, Hu X, Zhou K. Synonymous Codon Usage Bias in the Chloroplast Genomes of 13 Oil-Tea Camellia Samples from South China. Forests. 2023;14(4):794.
- 57. Feng T, Jia Q, Meng X, Chen X, Wang F, Chai W, et al. Evaluation of genetic diversity and construction of DNA fingerprinting in Polygonatum Mill. based on EST-SSR and SRAP molecular markers. 3 Biotech. 2020;10(7):322. pmid:32656055
- 58. Zhang D, Ren J, Jiang H, Wanga VO, Dong X, Hu G. Comparative and phylogenetic analysis of the complete chloroplast genomes of six Polygonatum species (Asparagaceae). Sci Rep. 2023;13(1):7237. pmid:37142659
- 59. Liu L, Wang Y, He P, Li P, Lee J, Soltis DE, et al. Chloroplast genome analyses and genomic resource development for epilithic sister genera Oresitrophe and Mukdenia (Saxifragaceae), using genome skimming data. BMC Genomics. 2018;19(1):235. pmid:29618324
- 60. Henriquez CL, Abdullah, Ahmed I, Carlsen MM, Zuluaga A, Croat TB, et al. Evolutionary dynamics of chloroplast genomes in subfamily Aroideae (Araceae). Genomics. 2020;112(3):2349–60. pmid:31945463
- 61. Yao J, Zheng Z, Xu T, Wang D, Pu J, Zhang Y, et al. Chloroplast Genome Sequencing and Comparative Analysis of Six Medicinal Plants of Polygonatum. Ecol Evol. 2025;15(1):e70831. pmid:39803186
- 62. Wicke S, Naumann J. Molecular Evolution of Plastid Genomes in Parasitic Flowering Plants. Adv Botan Res. 2018;85:315–47.
- 63. Qin Y-Q, Zhang M-H, Yang C-Y, Nie Z-L, Wen J, Meng Y. Phylogenomics and divergence pattern of Polygonatum (Asparagaceae: Polygonateae) in the north temperate region. Mol Phylogenet Evol. 2024;190:107962. pmid:37926394
- 64. Xiang K, et al. Distribution of the newly recorded plant Polygonatum sinopubescens in Hunan, China and its karyotype. J Jishou Univ (Natl Sci Edition). 2018;39(6):50.
- 65. Meng Y, Nie Z-L, Deng T, Wen J, Yang Y-P. Phylogenetics and evolution of phyllotaxy in the Solomon’s seal genusPolygonatum(Asparagaceae: Polygonateae). Bot J Linn Soc. 2014;176(4):435–51.
- 66. Floden A.Molecular phylogenetic studies of the genera of tribe Polygonateae (Asparagaceae: Nolinoideae): Disporopsis, Heteropolygonatum, and Polygonatum. 2017. Available from:https://trace.tennessee.edu/utk_graddiss/4398/
- 67.
Qin YQ. Research on phylogenetic genomics of Polygonatum odoratum in the Asparagaceae family. Jishou University; 2023.