The complete chloroplast genome of Ardisia brevicaulis Diels 1900, one traditional medicinal plant in southern China

Abstract

The complete chloroplast (cp) genome of Ardisia brevicaulis Diels 1900, one traditionally medicinal plant usually used in southern China, was first assembled and reported in this study. The genome size is 156,742 bp (37.1% GC content), containing a large single-copy (LSC) region of 86,329 bp, a small single-copy (SSC) region of 18,417 bp, and a pair of inverted repeat regions (IRs) of 25,998 bp. 134 genes (89 protein-coding, 37 tRNA, and 8 rRNA genes) are annotated in the whole cp genome, including 115 unique genes (81 protein-coding, 30 tRNA, and 4 rRNA genes). Phylogenetic analysis showed that A. brevicaulis is closely related to A. primulifolia and A. villosa, indicating their close phylogenetic relationship. The cp genome of A. brevicaulis could provide valuable genomic information for the phylogeny, molecular identification and discovery of new medicinal plant resources in Ardisia Swartz 1788.

Keywords:

• Ardisia
• chloroplast genome
• medicinal plant
• phylogenetic analysis

Ardisia brevicaulis Diels 1900, one species of Ardisia Swartz 1788 from the family Primulaceae Batsch ex Borkh.1797 (Chen and Pipoly 1996; APG IV 2016), is an important medicinal plant species widely distributed in southern China (González de Mejía and Ramírez-Mares 2011). The root of A. brevicaulis is usually used as a traditional chinese medicine for menstrual disorders, injuries and rheumatic pains (González de Mejía and Ramírez-Mares 2011; Li et al. 2010). Moreover, chemical and pharmacological studies showed that abundant natural products isolated from the root of A. brevicaulis have the potential biological activity of anti-tumor effect (Chen et al. 2011; Zhu et al. 2012). In particular, Ardisiphenol D, a major active resorcinol derivative among all the products, was shown to have potent inhibitory activity against cancer cells both in vitro and in vivo (Zhao et al. 2014). Due to the large number of species (65 species in China and about 400 to 500 species worldwide) in Ardisia and the similar morphology of Ardisia species (Chen and Pipoly 1996), the morphological classification and identification within the genus are difficult (Julius et al. 2021). Molecular phylogeny based on nrITS better revealed that Ardisia is not monophyletic as currently circumscribed, but left the phylogenetic relationship among many species of Ardisia unresolved, including the relationship between A. brevicaulis and its related species (Julius et al. 2021). In molecular phylogenetics, the chloroplast genomes can provide valuable sources of phylogenetic information because of their relatively stable genome structure and high evolutionary rates (Ravi et al. 2008). In this study, we first reported the complete chloroplast (cp) genome of A. brevicaulis and constructed the phylogenetic relationship with other Ardisia species. The results could be helpful for the phylogeny, molecular identification and discovery of new medicinal plant resources in Ardisia.

Materials

The sample of A. brevicaulis was collected from a wild population (108°35′12.12″E, 25°34′52.32 N; 910 m) in Congjiang County, Guizhou Province, China. It is a subshrub (10–15 cm) usually growing in dark and humid places under mixed forests (Figure 1; Chen and Pipoly 1996). It is characterized by its narrowly ovate to elliptic or suboblong leaf blade (7–18 × 2.5–6 cm) with an entire margin and indistinct marginal glandular dots, terminal umbellate inflorescences, globose punctate fruits (c. 6 mm in diameter) with usually purplish red persistent calyx and fruiting stalk (Figure 1; Chen and Pipoly 1996). The specimen of A. brevicaulis in this study was deposited at the herbarium of Guizhou University of Traditional Chinese Medicine (Chenggang Hu, Email: [email protected]) under voucher number WZW20210304.

Figure 1. Images of the living plants of Ardisia brevicaulis and its growing environment. A.growing environment, B. living plants, C. fruits. Photos of species reference image were taken by the authors.

Methods

Genomic DNA was extracted from fresh leaves of A. brevicaulis using Tiangen DNA Extraction Kit (DP305, Tiangen, Beijing, China) following the instructions of the kit. Purified genomic DNA was sheared into fragments (c. 350 bp) to construct a paired-end (PE) library. PE reads (c. 150 bp) were generated based on Illumina NovaSeq 6000 (Beijing Novogene Technology Co. Ltd). In total, 1.14 Gb clean data was retained after removing low quality reads and adaptor sequences. Based on the clean data, we assembled the cp genome of A. brevicaulis using GetOrganelle v1.7.5 (Jin et al. 2020), and annotated it via PGA (Qu et al. 2019), with further manual check and adjustment in Geneious v10.2.2 (Kearse et al. 2012). Annotated cp genome of A. brevicaulis was deposited to NCBI under the accession number ON208988.

The phylogenetic relationship between A. brevicaulis and other Ardisia species was inferred based on the whole cp genome sequences, with Tapeinosperma multiflorum and T. netor as outgroups (Hou et al. 2019; Shi and Liu 2019; Yan et al. 2019). Sequences in this study were aligned using MAFFT v7.397 (Katoh and Standley 2013). A maximum-likelihood (ML) tree was reconstructed in IQ-TREE v2.2.0 (Minh et al. 2020) using the best-fit substitution model (K3Pu + F+R2) chosen by ModelFinder (Kalyaanamoorthy et al. 2017). Branch supports were tested using SH-like approximate likelihood ratio (SH-aLRT) and Ultrafast bootstrap (UFBoot) with 10,000 bootstrap replicates.

Results

The total length of the cp genome is 156,742 bp, with a total GC content of 37.1%. A typical quadripartite structure was shown in the cp genome, including a large single-copy (LSC) region (86,329 bp), a small single-copy (SSC) region (18,417 bp), and two inverted repeat regions (IRs; 25,998 bp). 134 genes (89 protein-coding, 37 tRNA, and 8 rRNA genes) were annotated in the whole cp genome, containing 115 unique genes (81 protein-coding, 30 tRNA, and 4 rRNA genes). Among them, 15 genes (atpF, ndhA, ndhB, petB, petD, rpl2, rpl16, rpoC1, rps16, trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA, trnV-UAC) contained one intron, while the clpP and ycf3 genes contained two introns. In addition, rps12 gene underwent trans-splicing (Figure 2). The phylogenetic tree in this study revealed that A. brevicaulis is nested in one well-supported clade (Clade1; 100/100%) with other 10 Ardisia species, in which A. brevicaulis is closely related to A. primulifolia and A. villosa (99.7/78%; Figure 3).

Figure 2. Circular map of the chloroplast genome of Ardisia brevicaulis. Genes drawn inside the circle are transcribed clockwise, drawn outside counter clockwise.

Figure 3. The maximum likelihood (ML) tree based on the complete chloroplast genome sequences of Ardisia brevicaulis and related species. Numbers in parentheses are SH-aLRT/UFBoot supports (%). The accession number of GenBank for each species is listed after species name.

Discussion and conclusion

Ardisia is the largest genus from the family Primulaceae and one of the largest tropical genera with an estimate of about 400 to 500 species mainly distributed in the tropical Asia, Americas, Australia, and the Pacific Islands (APG IV 2016; Chen and Pipoly 1996; Frodin 2004). Its classification has received considerable attention from botanists in decades, but mainly focused on morphological classification, and presented great uncertainties (Julius et al. 2021). Recently, a molecular phylogeny of Ardisia based on nrITS and three cpDNA regions suggested the monophyly of subgenera Akosmos, Bladhia and Crispardisia, and the non-monophyly of subgenera Tinus and Icacorea, but cannot distinguish many species within Ardisia well (Ku and Hu 2014). Julius et al. (2021) explored a comprehensive phylogenetic relationships among Ardisia and related genera from 177 samples based on nrITS. It well indicate the non-monophyly of Ardisia, but clearly suggested the need for further study of the phylogenetic relationship within Ardisia because of low supporting rates among many Ardisia species. In this study, the phylogenetic relationship among surveyed Ardisia species was well resolved, showing six well-supported clades (Figure 3). Among them, A. brevicaulis and other 10 Ardisia species were nested in one clade with high supporting rates (100/100%), in which A. brevicaulis is closely related to A. primulifolia and A. villosa (Figure 3). These results probably implied that the chloroplast genomes can better resolve the interspecific relationship within Ardisia. Moreover, since medicinal plants within the same phylogenetic groups may have the same or similar therapeutic compounds/effects (Hao and Xiao 2020; Gong et al. 2022;), it is likely that the phylogenetic results in this study can also provide useful information for the exploration of new medicinal resources in Ardisia. However, it is worth noting that our phylogenetic tree was constructed only based on 30 Ardisia species (including one variety) because of few reports and releases of the chloroplast genomes of Ardisia in NCBI at present. Given the numerous species and wide distribution of Ardisia, in future studies, more chloroplast genomes and more species of Ardisia from all over the world are badly needed to be sequenced and studied to better explore the evolution and phylogenetic relationship within the genus.

Ethical approval

Ardisia brevicaulis Diels 1900 is not protected nor endangered in China. Research and collection of plant material comply with appendices I, II and III of the Convention on the Trade in Endangered Species of Wild Fauna and Flora, which has been valid from 22 June 2022 (https://cites.org/eng/app/appendices.php).

Author contributions

Zhiwei Wang analyzed the data and wrote the paper. Youqiong Hu performed the experiments. Shenghua Wei conceived and designed the study. All authors have read and approved the manuscript for publication, and all authors agreed to be accountable for all aspects of the work.

Acknowledgments

The authors are very grateful to Anling Huang of Guizhou University of Traditional Chinese Medicine for her help in collecting the experimental samples.

Disclosure statement

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

Data availability statement

The data in this study is openly available in GenBank of NCBI at [https://www.ncbi.nlm.nih.gov] under the accession number ON208988. The associated BioProject, SRA, and Bio-Sample numbers involved in this study are PRJNA824992, SRR18700737, and SAMN27502973 respectively.

Additional information

Funding

This research was supported by the National Key R&D Program of China under grant numbers [2018YFC1708100, 2018YFC1708101] and Graduate Research Fund Project of Guizhou under grant number [YJSKYJJ[2021]162].