Chloroplast genome assembly and phylogenetic analysis of Melodinus fusiformis Champ. ex Benth. (Apocynaceae)

Abstract

Melodinus fusiformis (Apocynaceae) is a medicinally important species with limited genomic data. Here, we report the first complete chloroplast genome of M. fusiformis, which is 154,913 bp in length with a GC content of 38.05% and encodes 130 genes, including 85 protein-coding genes, 37 tRNAs, and eight rRNAs. Phylogenetic analysis shows that M. fusiformis and M. tenuicaudatus are sister species forming a monophyletic clade with Periploca, Mandevilla, and Pentalinon species. This study provides essential genomic resources for molecular identification, phylogenetic research, and the development of M. fusiformis medicinal resources.

Introduction

Melodinus fusiformis Champ. ex Benth. 1852 in Hook. Kew Bull. Bot. 4: 332. 1852, a species of woody vine in the genus Melodinus of the Apocynaceae family, is commonly used in Yao medicine. It is widely distributed in Guangdong, Guangxi, Guizhou, and Sichuan, China, commonly found in mountainous sparse forests at altitudes of 300–1400 m, along hillside roads, or near valley streams (Institute of Botany 2025). The entire plant of M. fusiformis can be used medicinally to promote blood circulation, dispel wind-dampness, tonify the lungs, and stimulate lactation (Chang-Jiang et al. 2012). It is particularly effective in treating rheumatic heart disease and traumatic injuries. Modern pharmacological studies indicate that M. fusiformis is rich in various bioactive compounds, including triterpenoids such as alphitolic acid and oleonolide, as well as physiologically active alkaloids. These compounds exhibit antibacterial, anti-inflammatory, analgesic, and antioxidant properties, with several alkaloids demonstrating significant antitumor potential (Xiao et al. 1992; Chang-Jiang et al. 2012; Wang et al. 2012; Fang et al. 2017; Teng et al. 2025).

However, species identification of M. fusiformis primarily relies on morphological methods, which require extensive taxonomic expertise and are susceptible to phenotypic plasticity interference. Although molecular identification can accurately distinguish closely related species through genetic information, the scarcity of genomic data for the genus Melodinus in existing databases limits the depth of this technology. Notably, the chloroplast genome, due to its maternal inheritance, structural conservation, and moderate evolutionary rate, has emerged as an efficient molecular marker for plant phylogeny and species identification (Yue and Bing-Bing 2024). Currently, no complete chloroplast genome of M. fusiformis is publicly available. Therefore, this study assembled and annotated the chloroplast genome of M. fusiformis to provide crucial molecular evidence for its identification and phylogenetic research.

Materials and methods

Fresh leaves from an individual plant were collected in Jinxiu Town, Jinxiu Yao Autonomous County, Laibin City, Guangxi Zhuang Autonomous Region, China (Latitude N24.137803°, Longitude E110.102417°; Altitude: 545 m) and preserved with desiccant. The plant was identified by Jiahua Chen. Voucher specimens were deposited in the Herbarium of the Institute of Medicinal Plant Development (https://www.implad.ac.cn/, the contact person is Jiahua Chen and the email is 825981332@qq.com) under voucher number JXHC019 (Figure 1).

Figure 1.

Photograph of Melodinus fusiformis. (A) Leaves and flowers of M. fusiformis. This species is a stout woody liana with gray-brown bark. Young branches, leaves, petioles, and inflorescences are pubescent, becoming glabrous with age. Leaves are subleathery, elliptic to oblong, measuring 4.5–12 cm long and 1–5.3 cm wide, with an acuminate apex and a cuneate to rounded base. Lateral veins number about 15 pairs, spreading obliquely and forming a reticulate pattern near the margins. Petioles are 4–6 mm long. Terminal cymes bear 6–12 white flowers with oblong or oblanceolate corolla lobes and a scaly corona with 2–3 lobes. (B) The fruit corresponds to M. fusiformis. The green, ellipsoid berries measure 3.5–5.3 cm in length and 2.2–4 cm in width. Both photographs were taken by the author of this article, Xianglan Liang.

Plant genomic DNA was extracted from the samples using a precipitation-based method, and the purity, concentration, and integrity were assessed (Koren et al. 2017). DNA concentration was measured using a Qubit fluorometer, purity was evaluated by a NanoDrop spectrophotometer, and integrity was checked by pulsed-field gel electrophoresis. Samples that passed these quality controls were used for subsequent third-generation high-fidelity (HiFi) library construction and sequencing.

The chloroplast genome of M. fusiformis was sequenced using the PacBio Sequel II platform, which generates long HiFi reads suitable for organelle genome assembly. After quality control, the filtered reads were used for de novo assembly with OATK v1.0 (Zhou et al. 2025). The assembled chloroplast genome was annotated using the CPGAVAS2 (Shi et al. 2019). Visualization of the chloroplast genome structure, including gene distribution, repeat elements, cis-splicing and trans-splicing genes, was performed using the CPGview (Liu et al. 2023).

Fourteen chloroplast genomes were selected from NCBI BLAST results as phylogenetic tree construction sequences. Shared CDS sequences were extracted using PhyloSuite v1.2.3 (Zhang et al. 2020; Xiang et al. 2023), multiple sequence alignment was conducted with MAFFT v7.505 (Katoh and Standley 2013), sequences were concatenated using PhyloSuite v1.2.3, and trimming was performed using Gblocks v0.91b (Dereeper et al. 2008). A maximum-likelihood phylogenetic tree was constructed with IQ-TREE v2.3.6, implementing the optimal model TVM + F + I + R3 selected by Bayesian information criterion (BIC). The analysis included 1000 replicates each for SH-aLRT and UFBoot tests (Hoang et al. 2018; Minh et al. 2020).

Results

Raw data of M. fusiformis were obtained. The complete chloroplast genome is 154,913 bp in length with an average sequencing depth of 3134× (Figure S1). The chloroplast genome exhibits a typical quadripartite structure: a large single-copy region (LSC, 85,463 bp), a small single-copy region (SSC, 17,876 bp), and two inverted repeat regions (IR, 25,787 bp each) (Figure 2). The average GC content is 38.05%.

Figure 2.

The circular map of Melodinus fusiformis chloroplast genome, generated by CPGview. From the outside inward, the first ring represents gene distribution, with codon usage bias indicated in parentheses; genes are color-coded by functional category. Genes on the inner circle transcribe clockwise, while those on the outer circle transcribe counterclockwise. The legend for functional categories is annotated in the bottom-left corner. The outer side of the second ring shows positions within the chloroplast genome, with the inner dark gray area representing whole-genome GC content distribution. The third ring displays genomic region divisions, including LSC, SSC, IRA, and IRB. The fourth ring illustrates microsatellites using multicolored bars. The fifth ring represents long tandem repeats, indicated by blue bars. The sixth ring depicts dispersed repeats, with different colors denoting distinct types. For details, refer to the CPGview homepage.

A total of 130 functional genes were annotated, including 85 protein-coding genes (PCGs), 37 tRNAs, and eight rRNAs. The IR regions contain duplicated copies of six PCGs (rpl2, rpl23, ycf2, ndhB, rps7, rps12), seven tRNAs (trnI-CAU, trnL-CAA, trnV-GAC, trnI-GAU, trnA-UGC, trnR-ACG, trnN-GUU), and four rRNAs (rrn16, rrn23, rrn4.5, rrn5S). Eleven PCGs (rps16, atpF, rpoC1, petB, petD, rpl16, rpl2, ndhB, ndhA) contain single introns, while two PCGs (ycf3, clpP) possess double introns (Figure S2). The rps12 gene undergoes trans-splicing, with two of its three exons located in the IR regions (Figure S3).

Phylogenetic analysis revealed two major clades: one comprising the genera Tabernaemontana, Alstonia, and Rauvolfia, and the other consisting of Melodinus species and other related taxa. Within the latter clade, M. fusiformis and M. tenuicaudatus are sister species, forming a monophyletic group (Figure 3).

Figure 3.

Maximum-likelihood tree illustrating the relationship between Melodinus fusiformis and other Apocynaceae species, with the sequence of Luculia pinceana NC_063658 as the outgroup at the base, and the sequence of Melodinus fusiformis PV751239 indicated by a red dot representing M. fusiformis. Based on BLAST results, 13 chloroplast genomes with the highest sequence similarity within the Apocynaceae family and one chloroplast genome from a related order were selected to construct the phylogenetic tree. The numbers on the nodes represent bootstrap support values, all of which are above 95%, indicating a high level of confidence in the inferred phylogenetic relationships. The following sequences were used: Pentalinon luteum NC_025658 (Tan et al. 2018), Pentalinon luteum OP133568, Mandevilla sanderi NC_079592, Periploca calophylla NC_086729, Periploca floribunda NC_086730, Amsonia elliptica NC_069298, Amsonia tabernaemontana NC_079612, Melodinus tenuicaudatus NC_079613, Rauvolfia tetraphylla PQ260791, Rauvolfia verticillata NC_046841, Alstonia yunnanensis NC_068670, Tabernaemontana bovina NC_079611, Tabernaemontana divaricata MZ073339 (Zhang et al. 2021), and Luculia pinceana NC_063658 (Fatima et al. 2024).

Discussion and conclusions

Melodinus, a genus within the Apocynaceae family, comprises 53 known species (Institute of Botany 2025). However, until now, only the chloroplast genome of Melodinus tenuicaudatus (NC_079613.1, NCBI Accession No. NC_079613.1) has been available in the NCBI database. In this study, we report the first complete chloroplast genome assembly of the medicinal plant M. fusiformis, with a total length of 154,913 bp, which is similar to that of M. tenuicaudatus (154,916 bp). This new genomic resource fills a critical gap in the chloroplast genomic data for both M. fusiformis and the Melodinus genus as a whole.

Phylogenetic analyses reveal that M. fusiformis and M. tenuicaudatus form a monophyletic clade and are sister species. This represents the first genome-wide evidence confirming their close evolutionary relationship and provides new insights into their phylogenetic placement. It should be noted that chloroplast genome-based phylogenetic studies within the Apocynaceae family are still limited. Although previous studies have analyzed species of Merrillanthus and Biondia, these genera were not included in the phylogenetic tree constructed in this study (Liao et al. 2022). Therefore, the phylogenetic position of the Melodinus genus within Apocynaceae still requires further validation with additional genera and more samples.

The availability of the complete chloroplast genome of M. fusiformis not only addresses the significant shortage of molecular data for this species but also lays a solid foundation for its accurate molecular identification, taxonomic clarification, conservation efforts, and sustainable utilization. However, it is important to note that this study is based on a single sample and does not reflect intraspecific genetic diversity of M. fusiformis. Future studies should include multiple populations from different geographic locations to investigate intraspecific variation and evolutionary patterns. Additionally, the chloroplast genome sequences generated here can be used to develop specific molecular markers for rapid PCR-based identification, facilitating accurate molecular authentication of M. fusiformis in the medicinal plant market, and providing a molecular basis for resource conservation, sustainable utilization, and studies of pharmacologically active compound biosynthesis.

Funding Statement

This work was supported by Guangxi Special Project for Science and Technology Bases and Talents (Project No. 2021AC19423); The 2023 Scientific Research Fund Projects of Guangxi Science & Technology Normal University (No. GXKS2023ZDA004); The Scientific Research and Technology Development Plan of Guangxi Laibin, China (Laibin Sci-Tech Tackling 240102).

Ethical approval

Melodinus fusiformis is not a protected species; therefore, no special permit was required for sample collection. The collection of plant materials complied with international ethical standards and did not cause any harm to the local environment. All experimental procedures and research objectives were conducted in accordance with the guidelines and regulations of our institution. There are no ethical concerns or conflicts of interest associated with this study.

Disclosure statement

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

Data availability statement

The genomic sequence data supporting the findings of this study are available in GenBank under the accession number PV751239, with the BioProject, BioSample, and SRA numbers being PRJNA1272332, SAMN48910257, and SRR34493248, respectively.