The complete chloroplast genome of Cynanchum hemsleyanum and its phylogenetic analysis

Abstract

C. hemsleyanum chloroplast genome is 157,356 bp with a quadripartite structure, 37.99% GC, and 132 genes (87 protein-coding, 37 tRNA, 8 rRNA). Phylogenomic analysis places it as sister to C. thesioides with 100% bootstrap support. This resource aids molecular identification, genetic diversity, and evolutionary studies in Apocynaceae.

Introduction

Cynanchum hemsleyanum (Oliv.) Liede & Khanum (2016) is a Chinese endemic vine belonging to Cynanchum (Apocynaceae), mainly distributed in Shaanxi, Sichuan, Yunnan, Guizhou, Guangxi, Hubei and Jiangxi. It is a hygrophilous perennial vine growing in mountain forests, valleys and moist shrubs. As a traditional Chinese medicinal herb, it is slightly bitter and neutral in nature, and its roots, stems and leaves are used to treat kidney-deficiency lumbago, spermatorrhea, postpartum hypogalactia, as well as snake and insect bites (Fang and Liao 2006). Current studies on C. hemsleyanum are limited to morphological taxonomy, chemical constituents and pharmacological activities, with a notable lack of molecular and genomic data, which restricts the research on its genetic diversity, phylogenetic relationships and germplasm utilization (Zhang et al. 2022). The chloroplast genome is a robust molecular marker for plant evolutionary and genetic studies, suitable for phylogenetic analysis and species identification (Jansen and Ruhlman 2012). Therefore, this study sequenced and assembled the complete chloroplast genome of C. hemsleyanum, which fills the genomic gap of this species, supports its germplasm conservation and utilization, and provides a valuable genomic resource for resolving phylogenetic relationships within Apocynaceae.

Material and methods

Sample collection and authentication

Fresh, healthy leaf tissue of C. hemsleyanum were collected from Wuhan City, Hubei Province, China (114°17′17″ E, 30°21′28″ N) (Figure 1). The plant material was taxonomically authenticated based on morphological characteristics, and the voucher specimen (voucher number: whpu-2024-003) was deposited in the Herbarium of the School of Life Science and Technology, Wuhan Polytechnic University (contact person: Chao Xiong; e-mail: xiongchao080190@126.com).

Figure 1.

Morphological characteristics of Cynanchum hemsleyanum. A: Flower; B: Stem and latex secretion; C: Leaf; D: Whole plant. All photographs were taken by Chao Xiong at the sampling site in Wuhan, Hubei Province, China (114°17′17′′ E, 30°21′28′′ N). C. hemsleyanum is a perennial herbaceous twining vine, with white latex exuding from broken stems. Its leaves are opposite and ovate-cordate, flowers are star-shaped, and the plant often forms dense colonies in the wild.

DNA extraction, sequencing, and chloroplast genome assembly

Genomic DNA was extracted from silica gel-dried leaf tissues of C. hemsleyanum using a plant genomic DNA kit (Tiangen Biotech (Beijing) Co., Ltd., China). DNA quality was assessed using Qubit fluorometry, spectrophotometry and agarose gel electrophoresis. A paired-end library was constructed using the Illumina TruSeq DNA PCR-Free Kit and sequenced on the Illumina NovaSeq 6000 platform. Raw reads were quality filtered using Geneious Prime v.2022.1.1 (Biomatters Ltd., Auckland, New Zealand). De novo assembly was performed using GetOrganelle v.1.7.5 (Jin et al. 2020) with the parameters: -k 21,45,65,85,105 and -R 15, with plant chloroplast genome settings, and the circular genome was verified using Bandage (Wick et al. 2015).

Chloroplast genome annotation and physical map construction

The chloroplast genome was annotated using Geneious Prime v.2022.1.1 (Kearse et al. 2012) with C. rostellatum (ON_882042) as reference. Protein-coding, tRNA, and rRNA genes were predicted. Nucleotide composition and genome structure were analyzed, and the physical map of the chloroplast genome was generated using CPGAVAS2 (Shi et al. 2019). The annotated genome was deposited in GenBank under accession number PX927066.

Nucleotide diversity analysis

Nucleotide diversity (π) was estimated using DnaSP v6.12 (Rozas et al. 2017) to assess sequence variation across the chloroplast genome. The complete chloroplast genome sequences of Cynanchum species were aligned using MAFFT v7.490 (Katoh and Standley 2013) with default parameters. A sliding window analysis was performed with a window size of 600 bp and a step size of 200 bp to calculate π values across the genome. Highly variable regions were identified based on π values.

Phylogenetic analysis

To elucidate the phylogenetic position of C. hemsleyanum within Apocynaceae, the complete chloroplast genome sequences of 21 closely related species from genera including Calotropis, Asclepias, Cynanchum, Vincetoxicum, Periploca, Marsdenia, Leichhardtia and Hoya were retrieved from GenBank, with Calotropis procera and Asclepias syriaca designated as the outgroups for phylogenetic tree rooting (Supplementary Table 1). Species selection was based on available chloroplast genome resources and previous phylogenetic studies of Apocynaceae. Multiple sequence alignment was performed using MAFFT v7.490 (Katoh and Standley 2013) with default parameters, and the aligned sequence data were further refined and filtered using BMGE v1.1 (Criscuolo and Gribaldo 2010) to remove poorly aligned regions and enhance phylogenetic informativeness. After trimming, a total of 107,355 aligned nucleotide positions were retained for phylogenetic analysis. Maximum likelihood (ML) phylogenetic analysis was performed using IQ-TREE v1.6.12 (Nguyen et al. 2015) via the IQ-TREE web server (Trifinopoulos et al. 2016). The best nucleotide substitution model (TVM+F + I + G4) was automatically determined by the ‘-m MFP’ option (ModelFinder) based on the Bayesian Information Criterion (BIC). Branch support was evaluated with 1000 ultrafast bootstrap replicates (‘-bb 1000’), and the analysis was run with automatic thread detection (‘-nt AUTO’).

Results

The assembled chloroplast genome showed a minimum coverage depth of 519× and an average coverage depth of 1132× (Supplementary Figure 1). After quality filtering, 966.46 Mb of clean data were retained, with high quality (Q20 = 97.81%, Q30 = 93.85%, GC content = 44.46%), supporting the reliability of the assembly.

The complete chloroplast genome of C. hemsleyanum was 157,356 bp in length with an overall GC content of 37.99%, exhibiting the typical quadripartite structure of angiosperm chloroplast genomes (Figure 2). It consisted of a LSC region of 89,044 bp (36.26% GC), a SSC region of 18,680 bp (32.25% GC), and a pair of IR regions each spanning 24,816 bp (43.25% GC). A total of 132 genes were annotated from the chloroplast genome, encompassing 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes (Figure 2). The detailed annotation information is provided in the supplementary tables (Supplementary Tables 2 and 3). Among the annotated genes, most split genes contained one intron, whereas ycf3 and clpP contained two introns (Supplementary Figure 2).

Figure 2.

Schematic map of the chloroplast genome of C. hemsleyanum. The circular map includes six concentric rings for genomic features. From the innermost to outermost ring, the displayed features are as follows: spatially distributed repetitive sequences (forward repeats in red, reverse repeats in green), tandem repeats (indicated by blue bars), short tandem repeats (marked with green bars), the classic quadripartite genomic architecture (LSC, SSC, and the paired IRa/IRb regions) with their respective annotated lengths, GC content gradients distribution across the genome, and gene annotations color-coded by functional category. The functional classification of the genes is shown in the bottom left corner.

Comparative analysis was performed among four Cynanchum chloroplast genomes, including C. hemsleyanum, C. thesioides (Kang et al. 2021), C. chinense (Chen and Zhang 2022), and C. acutum subsp. sibiricum (Zhang et al. 2023). The chloroplast genome size ranged from 157,356 bp to 158,615 bp, with highly conserved GC contents (37.82-37.99%) and gene compositions among the examined species. The gene content is also largely conserved: C. hemsleyanum and C. chinense both contained 132 genes, while C. thesioides and C. acutum subsp. sibiricum harbored 131 and 130 genes, respectively, with the differences mainly attributable to slight variations in protein-coding gene counts (85–87). Furthermore, all four genomes shared the same quadripartite structure.Nucleotide diversity (π) analysis revealed several highly variable regions across the Cynanchum chloroplast genomes. Sliding window analysis identified ycf1, ndhI and ycf15 as highly variable regions, with π values exceeding 0.08. SSR analysis identified 87 SSR loci in the C. hemsleyanum chloroplast genome, predominantly composed of A/T-rich mononucleotide repeats (74 of the 87 loci).

To clarify the phylogenetic position of C. hemsleyanum within Apocynaceae, a ML phylogenetic tree was constructed based on the complete chloroplast genome sequences of related species, with Calotropis procera and Asclepias syriaca designated as the outgroups. Phylogenetic analysis revealed that C. hemsleyanum clustered with other Cynanchum species into a well-supported monophyletic clade (100% bootstrap support) and showed a close sister relationship with C. thesioides (Figure 3).

Figure 3.

Maximum likelihood phylogenetic tree of Cynanchum hemsleyanum and 21 related species in Apocynaceae based on complete chloroplast genome sequences. Bootstrap support values based on 1000 replicates are indicated at each branch node. Calotropis procera (NC_041440), (Ma et al. 2024) and Asclepias syriaca (NC_022432), (Straub et al. 2013), were used as outgroup taxa. The following chloroplast genome sequences were included in the analysis: Cynanchum thesioides (MW864598), (Kang et al. 2021), C. hemsleyanum (PX927066; this study), C. sibiricum (OQ390041), (Zhang et al. 2023), C. chinense (MW415427), (Chen and Zhang 2022), C. wilfordii (KX352467), (Park et al. 2016), C. bungei (OK271106), (Pei et al. 2022), C. auriculatum (NC_029460), (Jang et al. 2016), Vincetoxicum hainanense (NC_051946), (Ma et al. 2024), V. versicolor (NC_052877), (Yu et al. 2021), V. bungei (PV747498), (Ma et al. 2024), Periploca chrysantha (NC_086731), (Tian et al. 2025), P. calophylla (NC_086729), (Tian et al. 2025), P. forrestii (ON321894), (Tian et al. 2025), P. floribunda (NC_086730), (Tian et al. 2025), marsdenia tinctoria (OP133574), (Wang et al. 2023), Leichhardtia flavescens (MW719052), (Rodda and Niissalo 2021), Hoya lockii (NC_085235), (Nguyen et al. 2026), H. pottsii (NC_042246), (Tan et al. 2018), H. liangii (NC_042245), (Tan et al. 2018), and H. carnosa (NC_045868), (Ma et al. 2024). Complete information on all taxa, GenBank accession numbers, and corresponding references is provided in Supplementary Table S1.

Discussion and conclusion

The quadripartite structure, GC content distribution, and gene composition of the C. hemsleyanum chloroplast genome are highly conserved among Cynanchum species, consistent with the general evolutionary stability of angiosperm chloroplast genomes (Kang et al. 2021). This conservation likely reflects the strong functional constraints imposed on chloroplast genes involved in photosynthesis and essential cellular processes.

Comparative analysis among four Cynanchum plastomes revealed minor variations in genome size and IR boundaries, whereas gene content and genome organization remained highly conserved. The slightly longer IR region in C. hemsleyanum compared with other Cynanchum species may indicate lineage-specific IR boundary shifts, which have been frequently reported in angiosperm plastomes and are often associated with boundary-adjacent genes such as rps19 and ycf1 (Wang et al. 2023a), although the functional significance of these boundary shifts remains unclear. However, this variation falls within the range reported in Apocynaceae (Wang et al. 2023b), suggesting a minor structural divergence rather than substantial evolutionary differentiation. In addition, the identified chloroplast SSR loci, predominantly composed of A/T-rich repeats, represent valuable molecular resources for future population genetic studies, germplasm identification, and conservation of C. hemsleyanum (Provan et al. 2001), facilitating genetic diversity assessment and monitoring of wild populations.

The high genomic similarity among the sampled Cynanchum species provides useful chloroplast genomic evidence for phylogenetic inference. The ML tree recovered C. hemsleyanum as sister to C. thesioides with strong support, consistent with previous studies based on broader taxon sampling (Ma et al. 2024; Zhang et al. 2024). However, additional nuclear genomic data and broader taxon sampling will be necessary to further resolve evolutionary relationships within Cynanchum, as plastid genomes represent only a single maternal lineage and may not fully capture species-level evolutionary histories. Overall, this study provides the first complete chloroplast genome of C. hemsleyanum, expands the genomic resources available for Cynanchum, and offers molecular evidence for future phylogenetic, taxonomic, and conservation studies of this medicinal species.

Glossary

Abbreviations

LSC

Large single copy

SSC

Small single copy

IR

Inverted repeat

ML

Maximum likelihood

rRNA

Ribosomal RNA

tRNA

Transfer RNA

SSR

simple sequence repeat.

Funding Statement

This work was supported by the Research and Innovation Initiatives of WHPU under Grant [no. 2025Y24].

Ethical approval

This article does not contain any studies with human participants or animals performed by any authors. The species described in this paper is not endangered, protected, or personally owned. The plant material was collected in accordance with guidelines provided by the authors’ institution (School of Life Science and Technology, Wuhan Polytechnic University) and national regulations.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at (https://www.ncbi.nlm.nih.gov) under the accession no. PX927066. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA1437846, SRR37634593, and SAMN56523218, respectively.

Additional information

Supplementary Figure 1. Read mapping depth of the plastome sequence. Clean read mapping depth is presented with blue bars. X and Y axis present nucleotide position of plastome and read mapping depth, respectively. The min (519×), max (1,728×), and average (1,132×) depths.

Supplementary Figure 2. Schematic map of the cis-splicing genes (A) and trans-splicing gene (B, rps12 gene) in the chloroplast genome of C. hemsleyanum.