Complete chloroplast genome of Lonicera ligustrina Wall. (Caprifoliaceae) and its phylogenetic implications

Abstract Lonicera ligustrina is a folk medicinal herb in China and India with highly potential medicinal value. Here, we reported the complete chloroplast (cp) genome of L. ligustrina (GenBank accession number: ON968694). The cp genome was 155,330 bp long, with a large single-copy region (LSC) of 88,855 bp and a small single-copy region (SSC) of 18,647 bp separated by a pair of inverted repeats (IRs) of 23,914 bp. We also reconstructed the phylogeny of Lonicera using maximum likelihood (ML) method, including our data and previously reported cp genomes of related taxa. The current study indicated that L. ligustrina is sister with the Nintooa clade of subgen. Lonicera.


Introduction
The genus Lonicera L., commonly known as honeysuckles, is the largest genus of Caprifoliaceae (Ren et al. 2008;Jacobs et al. 2009). Historically, it has received extensive taxonomic evaluation and phylogenetic inference, including studies based on complete chloroplast genome (Smith and  CONTACT Jian-Hui Li lijianhui@yeah.net College of Chemistry and Materials Engineering, Quzhou University, Quzhou, China Supplemental data for this article can be accessed online at https://doi.org/10. 1080/23802359.2023.2239386 Donoghue 2010; Fan et al. 2018;Wang et al. 2020), but so far, its phylogenetic relationships among sections, subsections, and species are still obscure (Theis et al. 2008;Nakaji et al. 2015;Wang et al. 2020). Lonicera ligustrina Wall.1824, is an evergreen, semi-evergreen, or deciduous shrub belonging to the section Isika of Caprifoliaceae (Hsu and Wang 1988). It can be classified into 3 varieties: L. ligustrina var. ligustrina, L. ligustrina var. pileata (Oliver) Franchet, L. ligustrina var. yunnanensis Franchet (Yang and Landrein 2011). The species varied greatly in morphology, and more research work is still needed to clarify its infraspecific classification (Zeng et al. 2021). At the same time, L. ligustrina, commonly used in folk herbs in China and India also has certain medicinal value (Yashodha et al. 2019;Murthy et al. 2022). Therefore, we assembled and characterized the complete chloroplast genome of L. ligustrina, which is expected to lay a solid foundation for the medical applications and future phylogenetic investigations of Lonicera.

Plant materials and DNA extraction
Fresh leaves of L. ligustrina ( Figure 1)

Plastome sequencing, assembly, and annotation
The next-generation sequencing was performed with an Illumina NovaSeq platform (Illumina, San Diego, CA). The chloroplast genome was assembled via NOVOPlasty 2.6.3 (Dierckxsens et al. 2017), using the L. japonica (NC026839, He et al. 2017) as the initial reference genome. The assembled cp genome was annotated using the online software GeSeq v.1.59 (Tillich et al. 2017). Geneious R11 (Biomatters Ltd., Auckland, New Zealand) was used for inspecting the cp genome structure. The circular gene map of the chloroplast genome was drawn by d by CPGView software (Liu et al. 2023, http://www.1kmpg.cn/cpgview/).

Phylogenetic analysis
Species phylogeny of Lonicera was evaluated based on protein coding regions (CDS) extracted from 27 complete chloroplast genomes from Caprifoliaceae. Heptacodium miconioides (NC042739, Wang et al. 2019) was used as outgroup. We reconstructed a phylogeny employing the GTR þ G model and 1000 bootstrap replicates under the maximum likelihood (ML) inference in RAxML-HPC v.8.2.10 on the CIPRES cluster (Miller et al. 2010).

Genome organization and compositions
The complete chloroplast genome of L. ligustrina is 155,330 bp in length. It has a typical quadripartite structure with a large single-copy (LSC) region of 88,855 bp, a small single-copy (SSC) region of 18,647 bp, and a pair of inverted repeats (IRs) of 23,914 bp ( Figure 2). The GC contents of the total length, LSC, SSC, and IR regions were 38.5%, 36.9%, 33.4% and 43.3%, respectively.

Phylogenetic analysis
A robust phylogeny of Lonicera was obtained based on the CDS data, most nodes in the ML tree were highly supported, and the genus was resolved as a monophyletic clade consisting two well supported clades which consistent with the previous research which divided Lonicera into two subgenera: subgen. Caprifolium and subgen. Lonicera (Figure 3). The subgen. Lonicera clade consisted of species from 4 sections (sect. Coeloxylosteum, sect. Isika, sect. Isocylosteum, sect. Nintooa), but in our phylogenetic tree, only the species from sect. Nintooa were clustered into a monophyletic clade. According to the phylogenetic tree, the sister relationship between L. ligustrina and L. ligustrina clade was observed.

Discussion
The current study presented the complete chloroplast genome of L. ligustrina and a highly resolved phylogenies of Lonicera based on 27 complete chloroplast genome from Caprifoliaceae. Our result supports the classification of the two subgenera, subgen. Lonicera and subgen. Caprifolium, in Lonicera proposed by Rehder (1903Rehder ( , 1913 and Hsu and Wang (1988) and coincides with previous molecular phylogenetic studies (Theis et al. 2008;Nakaji et al. 2015;Wu et al. 2021). Among the four sections of subgen. Lonicera, sect. Nintooa was supported as monophyly, and L. ligustrina is sister with the Nintooa clade with strong bootstrap support ( Figure 3). We expect that the cp genome of L. ligustrina will be a valuable resource for future studies on molecular identification and the better understanding of phylogeny in Lonicera and Caprifoliaceae.

Ethical approval
The material involved in the article does not involve ethical conflicts. This study was permitted by College of Chemistry and Materials Engineering, Quzhou University, China. All collection and sequencing work was strictly executed under local legislation and related laboratory regulations to protect wild resources.

Authors' contributions
The article was designed and conceived by Jian-Hui Li; Li-Li Liu and Zhong-Shuai Sun assembled and annotated the cp genome; Zhong-Shuai Sun collected and identified the plant material; Li-Li Liu contributed significantly to phylogenetic analysis and manuscript preparation; Jian-Hui Li was involved in interpretation of the data and revised the manuscript critically for intellectual content. All authors approved the final version to be published and agreed to be accountable for all aspects of the work.

Disclosure statement
The authors report no conflicts of interest and are responsible for the content and writing of the paper.

Funding
This work was supported by the Natural Science Foundation of China (No.31400321).

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. ON968694. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA894792, SRR22061799 and SAMN31469260 respectively.