The complete chloroplast genome of Lagochilus ilicifolius Bunge ex Bentham, Labiat. Gen. 1834 (Lamiaceae) and its phylogenetic analysis

Abstract

Lagochilus ilicifolius Bunge ex Bentham, Labiat. Gen is a perennial herb with much-branched stems native to Nei Mongol, Ningxia, Gansu, N Shaanxi. It can be used clinically as a hemostatic agent. The chloroplast genome length is 151,466 bp. It contained two inverted repeat regions of 25,660 bp each, a large single-copy region of length 82,504 bp, and a small single-copy region of length 17,642 bp. Also, the GC content is 38.6%. There were 133 genes annotated, including 88 known protein-coding genes, 37 tRNAs, and eight rRNAs. The phylogenetic tree was constructed using Bayesian method for plastome data of 29 species. The entire chloroplast genome of L. ilicifolius within the Lamiaceae is the first to reveal genetic taxonomy at the molecular level, and the new phylogenetic tree data can be used for future evolutionary studies.

Introduction

Lagochilus ilicifolius is a perennial plant in the genus Lamiaceae of the family Lagochilus (Gen 1994). The aqueous extracts of the whole plant of Lagochilus have obvious in vitro anti-inflammatory activity, and can effectively scavenge DPPH (2,2-diphenyl-1-picrylhydrazyl, a stable free radical that can be used to measure the free radical scavenging activity of antioxidants) free radicals, showing certain in vitro antioxidant activity (Jiao et al. 2014). Analysis of the nonpolar constituents of the above-ground parts of L. ilicifolius allowed identification of 52 components (accounting for 92.61% of the total amount of nonpolar components), which include mainly fatty acid esters, long-chain alkanes, monoterpenes, sesquiterpenes, sterols, and aromatic compounds, and so on, mainly including fatty acid esters, long-chain alkanes, monoterpenes, sesquiterpenes, steroids, and aromatic compounds (Chen et al. 2012). Erythro‑1‑[(4‑O‑β‑d‑glucopyranosyl‑3‑methoxyl)‑phenyl]‑2‑[(5′‑methoxyl)‑pinoresinol]‑propane‑1,3‑diol (1), a newly identified lignan from the L. ilicifolius, showed relatively strong cytotoxicity against PC12 cell line which was derived from a rat adrenal medullary chromosomal tumor (Qian et al. 2015).

A study of L. ilicifolius from northern China, Mongolia and Russia using five cpDNAs spacers, confirmed that L. ilicifolius belonged to two lineages, one in northern China and the other in Inner Mongolia-Russia (Gao et al. 2014).

Materials and methods

The fresh leaves were collected in Ordos, Inner Mongolia Autonomous Region, China (108°38′22.54″ E, 39°49′13.62″ N) (Figure 1). Professor Tingguo Kang from the Liaoning University of Traditional Chinese Medicine in Shenyang, China, authenticated the sample vouchers. A specimen was deposited at the herbarium of Liaoning University of Traditional Chinese Medicine (Liang Xu 861364054@qq.com, L. ilicifolius number: 210711191006023LY). All operations were carried out in accordance with Specification on Good Agriculture and Collection Practices for Medicinal Plants (GACP; Number: T/CCCMHPIE 2.1-2018).

Figure 1.

The morphological characteristics of L. ilicifolius. The photographs were taken by Xu Liang in Ordos, Inner Mongolia Autonomous Region, China (108°38′22.54″ E, 39°49′13.61″ N). The most characteristic feature of the specimen: leaves cuneate-rhombic, upwards, short awned spines at the ends of the teeth, glabrous on both sides; flowers whorled cyme, white tomentum on the outside of the corolla.

A total amount of 0.2 μg DNA per sample was used as input material for the DNA library preparations. Sequencing library was generated using NEB Next^® Ultra™ DNA Library Prep Kit for Illumina (NEB, Ipswich, MA) following manufacturer’s recommendations and index codes were added to each sample. Genomic DNA sample was fragmented by sonication to a size of 350 bp, and ligated with the full-length adapter for Illumina sequencing, followed by further PCR amplification. After PCR products were purified by AMPure XP system (Beckman Coulter, Beverly, MA), DNA concentration was measured by Qubit^®3.0 Flurometer (Invitrogen, Waltham, MA). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using Illumina PE Cluster Kit (Illumina, San Diego, CA) according to the manufacturer’s instructions. High-quality reads were assembled into chloroplast genome using a de novo assembler SPAdes v3.14.1 (Bankevich et al. 2012). Finally, it was annotated by PGA (Qu et al. 2019) with Lagopsis supina (NC058246) as reference genome. The Illumina short sequences were compared to the chloroplast genome sequences using BWA software and finally the coverage was calculated using samtools depth (high coverage of over ×100). The horizontal coordinate is the chloroplast length and the vertical coordinate is the coverage depth (Figure S1) (Li et al. 2009; Li 2013). The maps of the chloroplast genome, cis-splicing genes, and trans-splicing genes of L. ilicifolius were processed by CPGview (Liu et al. 2023).

To analyze the relationship between L. ilicifolius and other species in the family Lamiaceae, the whole chloroplast genomes of 28 species and an outgroup taxon (Akebia quinata) were selected from NCBI. Using mafft-7.037 software (Katoh and Standley 2013), 70 shared protein genes were selected for comparison. After sequence alignment using MAFFT, the sequences were then aligned using Gblocks (Xiao et al. 2021) 0.91b, which is used to extract conserved sites from multiple sequence alignment results for the next evolutionary analysis. The maximum-likelihood evolutionary tree was then constructed using the iqtree-1.6.12 software (Xue et al. 2022) with a self-expansion value of 1000. The selection of the optimal substitution model (TVM + F + I + G4) was performed according to Bayesian information criterion (BIC) method implemented in ModelFinder (Kalyaanamoorthy et al. 2017).

Results

The chloroplast genome of L. ilicifolius is 151,466 bp, including a large single-copy region (LSC) of 82,504 bp, a small single-copy region (SSC) of 17,642 bp, and a pair of inverted repeats of 25,660 bp. The overall GC content of the chloroplast genome is 38.6%. The average sequencing depth was ×1099 (Figure S1) (Li et al. 2009; Li 2013). The 133 genes encoding are 88 protein coding genes, eight rRNA genes, and 37 tRNAs. trnK-UUU, rps16, trnG-UCC, atpF, rpoC1, trnL-UAA, trnV-UAC, petB, petD, rpl16, rpl2, ndhB, trnI-GAU, trnA-UGC, and ndhA genes each contain one intron, clpP, ycf3 genes contain two introns, whereas rps12 appeared as a trans-spliced gene with its 5′ end located in the LSC region and the duplicated 3′ end in both IR regions. The L. ilicifolius chloroplast genome and an annotated map of cis- and trans-splicing genes (Figure 2, Figures S2 and S3, respectively) were processed with CPGview (Liu et al. 2023).

Figure 2.

A circular map of the entire chloroplast genome of L. ilicifolius using CPGview (Liu et al. 2023). The map encompasses six tracks that depict various features of the genome. Starting from the center, the first track displays dispersed repeats, consisting of both direct and palindromic repeats, which are delineated by red and green arcs. The second track highlights long tandem repeats represented by short blue bars. In the third track, short tandem repeats or microsatellite sequences are illustrated as colored bars. Each color corresponds to a specific type of repeat, and accompanying descriptions provide valuable information about the characteristics of each repeat type. The colors and their respective repeat types are as follows: black: c (complex repeat); green: p1 (repeat unit size = 1); yellow: p2 (repeat unit size = 2); purple: p3 (repeat unit size = 3); blue: p4 (repeat unit size = 4); orange: p5 (repeat unit size = 5); red: p6 (repeat unit size = 6). The chloroplast genome contains an LSC region, an SSC region, and two IR regions, and they are shown on the fourth track. The GC content along the genome is shown on the fifth track. Genes within the genome visualization are meticulously color-coded based on their functional classification. The transcription directions of the inner genes are represented in a clockwise manner, while the outer genes are shown in an anticlockwise orientation. To assist with interpretation, the key for gene functional classification is provided in the bottom left corner of the visualization.

The topology of the phylogenetic tree revealed the close relationship between L. ilicifolius and Phlomoides rotata, Lagopsis supina and Leonurus japonicus, while the phylogenetic relationships between other representatives of the Lamiaceae family were consistent with previous studies. The presented results will help to carry out further studies on Lamiaceae. The complete chloroplast genome sequence of L. ilicifolius could provide new directions for the evolutionary history of Lamiaceae and other species (Figure 3).

Figure 3.

Maximum-likelihood (ML) phylogenetic tree with evolutionary distances for L. ilicifolius and 28 other species. The numbers above the branches show the bootstrap support values. The following sequences were used: Teucrium ornatum (MN814864), Ajuga forrestii (NC048512), Ajuga bracteosa (NC068635), Amethystea caerulea (NC068628), Lamium amplexicaule (MT473770) (Zhao et al. 2021), Eriophyton wallichii (NC053828), Leonurus japonicus (NC038062), Lavandula angustifolia (NC029370), Phlomoides rotata (NC065733), Lagochilus ilicifolius (OP251043) (this study), Colquhounia coccinea (NC058329) (Zhao et al. 2021), Scutellaria baicalensis (NC027262), Scutellaria amoena (NC057255) (Chen and Zhang 2019), Scutellaria barbata (NC059814), Salvia miltiorrhiza (NC020431) (Qian et al. 2013), Salvia splendens (NC050901) (Zhao et al. 2020), Rosmarinus officinalis (NC027259), Salvia japonica (NC035233), Mentha spicata (NC037247) (Wang et al. 2017), Mentha canadensis (NC044082), Clinopodium chinense (NC050943) (Zhang et al. 2020), Thymus mongolicus (NC046520), Agastache rugosa (NC053706), Hyssopus cuspidatus (NC079586), Prunella vulgaris (NC039654) (Han and Zheng 2018), Ocimum basilicum (NC035143), Nepeta cataria (NC051544) (Zhou et al. 2020), and Akebia quinata (NC033913) (Li et al. 2016).

Discussion and conclusions

The topology of the phylogenetic tree is in agreement with the results of the previous studies and provides new genetic information for the analyses reconstructing the position of L. ilicifolius within the family Lamiaceae (Pema et al. 2021). The relationship between L. ilicifolius and other species in the genus Lagochilus is currently unclear. A fully resolved phylogenetic tree was obtained in this study, enriching our understanding of the genetic composition, genome evolution, and genetic diversity of species in the genus Lagochilus. This finding facilitates future comparative and evolutionary analyses of chloroplast genome sequences within the genus Lagochilus.

Funding Statement

This research was funded by Liaoning BaiQianWan Talents Program [No. 2021A039], 2019 Liaoning Provincial Department of Education Scientific Research Project [No. L201942], Major Special Fund for Science and Technology of Inner Mongolia Autonomous Region [No. 2019ZD004], Natural Science Fund Project of Liaoning Province [No. 2020-MS-224], National Natural Science Foundation of China [No. 82373999], Key Project at Central Government Level: The Ability Establishment of Sustainable Use for Valuable Chinese Medicine Resources [No. 2060302], and 2023 Liaoning University of Traditional Chinese Medicine Seedling Breeding Project.

Ethical approval

Lagochilus ilicifolius does not belong to the national key protected wild plants category, and collecting it does not violate the Regulations of the People’s Republic of China on Wild Plants Protection. According to Article 5 of the regulation, the state encourages and supports scientific research on wild plants and on-site and ex-situ protection of wild plants. No ethical approval or specific permission was needed in this research.

Author contributions

T. G. K. revised critically important intellectual content and was involved in validation and supervision; W. J. H. and W. X. M. were responsible for conceptualizing, writing, and revising; Y. Y. Y. and L. X. were responsible for identification and collection, preservation of plant specimens, and conceiving the work; Y. P. X. and Y. Y. S. were responsible for acquisition and interpretation of the data; all authors discussed the results and revised the manuscript.

Disclosure statement

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

Data availability statement

The chloroplast genome sequence data that support the findings of this study are openly available in GenBank of NCBI (https://www.ncbi.nlm.nih.gov/) under the accession no. OP251043. The associated BioProject, SRA, and BioSample numbers are PRJNA870254, SRX17131821, and SAMN30355605, respectively.