Characteristics and phylogenetic analysis of the complete chloroplast genome of Gardneria multiflora Makino 1901 from the family Loganiaceae

Abstract

Gardneria multiflora Makino is an important medicinal plant endemic to China, with its roots and leaves traditionally used for therapeutic purposes. In this study, we report the complete chloroplast genome of G. multiflora. The 144,920 bp genome (GC 37.82%) has a typical circular quadripartite structure: 98,036 bp LSC, 18,382 bp SSC, and two 14,251 bp IRs. It has 125 unique genes, comprising 82 protein-coding, 35 tRNA, and 8 rRNA genes. Phylogenetic analysis revealed that G. multiflora is closely related to G. ovata. The data presented in this study can guide future research on the evolutionary history of this species.

Introduction

Gardneria multiflora Makino (1901, Figure 1), a valuable medicinal plant from the Loganiaceae family, is widely distributed in China, specifically south of the Qinling Mountains-Huaihe River Line and north of the Nanling Mountains in China (Zhang et al. 2021). The genus Gardneria, belonging to the Loganiaceae family, is endemic to Asia and occurs throughout East Asia, from the eastern Himalayas to Japan. It comprises six recognized species: G. multiflora Makino, G. anguatifolia Wall., G. linifolia C.Y. Wu & S.Y. Pao, G. lanceolata Rehd. & Wilson, G. ovata Wall., and G. distincta P.T. Li (Jiang et al. 2012).

Figure 1.

Gardneria multiflora reference image. Photographed by Tao Xu. Lianas or scandent shrubs. Leaf elliptic, papery. Cymes 3-branched. Flowers 5-merous. Corolla yellow, tube short; lobes narrowly elliptic. Stamens inserted near base of corolla tube; filaments short, anthers oblong. Ovary with 1 ovule per locule. Berries 6-8mm in diam. Seeds black.

Multiflora is a species of lianas or scandent shrubs that thrives along forest edges at altitudes ranging from 300 to 2100 m (Editorial Committee of Flora of China 1996). Both its roots and leaves exhibit anti-inflammatory, analgesic, and hemostatic properties, making them widely used in traditional medicine to treat conditions such as arthrophlogosis and sciatica by dispersing wind and promoting blood circulation (He and Peng 2010). Numerous studies have shown that the primary bioactive compounds in G. multiflora are monoterpenoid alkaloids and lignans (Yang et al. 2018). However, research on the molecular aspects of G. multiflora remains limited. To enhance understanding of the phylogenetic position of G. multiflora within the Loganiaceae family, this study aimed to assemble and characterize its complete chloroplast genome (cpDNA). The results will contribute to evolutionary research on G. multiflora and support the inference of phylogenetic relationships with related species.

Materials and methods

Sampling, DNA extraction and sequencing

Fresh leaves of G. multiflora were collected from the medicinal botanical garden of West Anhui University, located in Anhui Province, China (latitude: 31°77′ N, longitude: 115°93′ E). A voucher specimen (No. PLG-20230318-1), has been deposited at West Anhui Health Vocational College (contact: Guangyan Li, email: lgyhjj8812345@126.com). Genomic DNA was extracted from the leaves using a genomic DNA extraction kit sourced from Tiangen Biotech (Beijing, China). Whole genome sequencing was conducted using the BGISEQ-500 platform provided by Hefei Biodata Biotechnologies Inc.

Assembly and annotation

The chloroplast genome of G. multiflora was filtered using the program fastp (Chen et al. 2018) and subsequently assembled with SPAdes assembler version 3.10.0 (Bankevich et al. 2012). Genome annotation was performed using utilizing GeSeq (Tillich et al. 2017) and BLASTx (Gish and States 1993). The finalized completed chloroplast genome sequence of G. multiflora has been submitted to GenBank under the accession number OQ756143.

Phylogenetic analysis

A phylogenetic analysis was conducted using the complete the chloroplast of G. multiflora, along with those of nine other closely related species, with Hemidesmus indicus designated as the outgroup. The cpDNA sequences of the other related species were retrieved from the NCBI GenBank database. Sequence alignment was performed using MAFFT v7.307 (Katoh and Standley 2013). A maximum likelihood (ML) phylogenetic tree was then constructed using FastTree version 2.1.10 with 100 resamples, employing the Generalized Time-Reversible (GTR) model and the Shimodaira-Hasegawa (SH) test to assess branch support. Support values based on the SH test were displayed at the corresponding branches of the resulting phylogenetic tree (Price et al. 2010).

Results

Characteristics of G. multiflora CpDNA

The average coverage depth of the chloroplast genome of G. multiflora is 2036.7×. The read coverage map presented a relatively uniform distribution (Figure S1), indicating the robustness and reliability of the sequencing data used in this study. Analysis with the CPGview tool (Liu et al. 2023) revealed that the chloroplast genome has a typical circular, quadripartite structure with a total length of 144,920 bp. It consists of two inverted repeats (IR) regions of 14,251 bp each, separating a large single-copy (LSC) region of 98,036 bp and a small single-copy (SSC) region of 18,382 bp (Figure 2). The overall GC content of the genome is 37.82%, with the LSC, SSC, and IR regions having GC contents of 36.30%, 31.99%, and 46.81%, respectively.

Figure 2.

Schematic illustration of the chloroplast genome of G. multiflora. This graphical map, generated using the CPGview online tool, visualizes key features identified in the complete chloroplast genome. It consists of seven concentric layers. The outermost layer shows dispersed repeats, with red arcs indicating forward repeats and green arcs indicating reverse repeats. The second layer displays long tandem repeats, marked by short blue bars. The third layer represents short tandem repeats (microsatellites), illustrated by colored bars of varying shades. The fourth layer outlines the boundaries and lengths of the small single-copy (SSC) and large single-copy (LSC) regions. The fifth layer highlights the inverted repeats (IRA and IRB). The sixth layer depicts the GC content distribution across the plastome. Lastly, the innermost layer presents gene annotation, with genes grouped by functional category and color-coded for clarity.

The chloroplast genome of G. multiflora contains a total of 125 genes, comprising 82 protein-coding genes, 35 tRNA genes, and 8 rRNA genes. Of these, 10 genes are duplicated within the IR regions, including one protein-coding gene, five tRNA genes, and four rRNA genes. The LSC region harbors 88 genes in total, including 64 protein-coding genes and 24 tRNA genes. In contrast, the SSC region contains 12 genes – 11 protein-coding genes and one being a tRNA gene. Additionally, 16 genes contain two exons, while four genes – pafI, clpP1, and two copies of rps12 – contain three exons. Several genes, including rps16, atpF, rpoC1, pafI, clpP1, petB, petD, rpl16, rpl2, ndhB, and ndhA, undergo cis-splicing (Figure S2). Notably, rps12 was identified as a trans-splicing gene (Figure S3).

Phylogenetic analysis

To determine the taxonomic placement of G. multiflora, we aligned its complete chloroplast genome with those of ten related species using MAFFT v7.307 (Katoh and Standley 2013). The resulting maximum likelihood phylogenetic analysis confirmed a close relationship between G. multiflora and G. ovata (Figure 3). These two species formed a strongly supported monophyletic clade, with a bootstrap value of 100%. Furthermore, they were most closely related to the four Strychnos species included in the analysis. The phylogenetic tree constructed in this study provides a valuable framework for future evolutionary and taxonomic research within the Loganiaceae family.

Figure 3.

Maximum-likelihood (ML) phylogenetic tree showing evolutionary relationships between G. multiflora and ten other species with Hemidesmus indicus used as the outgroup. The tree was constructed using 1000 bootstrap replicates and support values are shown at the corresponding branches. The following ten chloroplast genome sequences were included in the analysis: Strychnos nitida (OP581044), Strychnos wallichiana (ON881741) (Jin et al. 2024), Strychnos cathayensis (ON881581) (Jin et al. 2024), Strychnos nux-vomica (MZ440354), Strychnos nux-vomica (MZ440353), Gardneria ovata (MZ440349), Mitreola yangchunensis (MT471262) (Du et al. 2020), Mitrasacme pygmaea (MT330399) (Du et al. 2020), Coffea arabica (PV021911) (Zhang et al. 2020), and Galium aparine (KY562587) (Zhang et al. 2020). The numbers above the tree nodes represent SH-like local support values.

Discussion and conclusion

In this study, we successfully sequenced and characterized the complete chloroplast genome of G. multiflora. To ensure the accuracy of the genome assembly, we conducted thorough validation using multiple software tools. Specifically, the genome was assembled using both SPAdes version 3.10.0 and NOVOPlasty version 4.3 (Dierckxsens et al. 2017). Notably, both approaches yielded identical assembly results, thereby confirming the reliability and precision of the sequence data.

The phylogenetic analysis confirms a close relationship between G. multiflora and G. ovata. Despite their genetic similarity, the two species exhibit distinct morphological differences. Specifically, G. multiflora possesses five calyx lobes, five corolla lobes, and five stamens, whereas G. ovata has four of each. These findings support traditional morphological classifications and provide valuable insights into the evolutionary dynamics within the Loganiaceae family. Notably, this study presents the first complete assembly of the chloroplast genome of G. multiflora. This genomic information represents a valuable genetic resource for the Gardneria genus and holds significant potential for advancing our understanding of G. multiflora’s evolutionary history. The research provides a solid foundation for species identification, detailed evolutionary studies, and the genetic improvement of this important medicinal plant. Furthermore, the chloroplast genes identified in G. multiflora may have promising applications in genetic engineering, potentially leading to the discovery or enhancement of medicinal properties.

Funding Statement

This project is supported by Provincial Quality Engineering Project of Colleges and Universities in Anhui Province (2022jpkc184, 2023sdxx202).

Ethical approval

The sample collection of G. multiflora is legal, and it is not an endangered or a protected species in China. Only a few leaves of G. multiflora were sampled for the assay. Methodological documentation includes deposition of voucher specimens at West Anhui Health Vocational College with corresponding accession code PLG-20230318-1. Specimen identification was conducted by Guangyan Li, following ICNafp guidelines for botanical verification.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The genome sequence data of Gardneria multiflora 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. OQ756143. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA1204316, SRR31853245, and SAMN46017364, respectively.