Complete chloroplast genome of a coastal herb, Linaria japonica Miquel 1865 (Plantaginaceae)

Abstract

Linaria japonica Miquel 1865 (Plantaginaceae) is a perennial herb found in coastal areas of north-east China, Japan, Korea, and the Far East Russia. Here, we report the complete chloroplast genome of L. japonica and determine its systematic position in 11 tribes of Plantaginaceae. The size of complete chloroplast genome was 150,660 bp, consisting of a pair of inverted repeats (25,647 bp each) separated by a large single-copy region (81,766 bp) and a small single-copy region (17,600 bp). Our phylogenomic analyses using 78 protein-coding genes show that Linaria belongs to the tribe Antirrhineae. Within Linaria, L. japonica is sister to L. buriatica Turcz.

Introduction

Linaria japonica Miquel 1865 (Plantaginaceae) is a perennial herb found in coastal areas of north-east China, Japan, Korea, and the Far East Russia. This species is not only used as traditional medicine due to its diuretic, purgative, and laxative properties (Kitagawa et al. 1973; Otsuka 1993), but also cultivated as ornamentals with showy and colorful flowers (Korea National Arboretum 2025). Linaria japonica can be distinguished from the related species, L. vulgaris Miller by shape of leaf blade (ovate to oblong vs. linear) and length of corolla spur (3–6 mm vs. 10–15 mm) (Figure 1; Hong et al. 1998; Kim and Flora of Korea Committee 2018). Despite the morphological uniqueness of L. japonica, few molecular phylogenetic analyses to determine its systematic position in the genus have been addressed (Fernández-Mazuecos et al. 2013; Yousefi et al. 2017).

Figure 1.

The morphological feature of linaria japonica (photographed by C kim; apr. 29, 2023; gangreung-si, Gangwon, South Korea; voucher number: HNIBRVP41776 [HIBR]). (a) Perennial herb with stems that are often branched and can be ascending or trailing. Leaves often irregularly whorled or alternative upward, sessile. Corolla bright yellow, spur straight. (b) Leaves ovate, obovate, or oblong, base obtuse to subcuneate, apex obtuse to subacute. (c) Capsules globose or oval often appearing as a continuation of the flower stalk after petal have fallen. (d) Seeds compressed, reniform or obliquely ellipsoid, blackish.

Coastal areas with high biodiversity are ecologically and economically important and have been affected by climate change and human interference. Linaria japonica only grows in coastal sand dunes on the east area of the Korean Peninsula (Shim et al. 2009). Although L. japonica has not yet been designated as an endangered species in Korea, the population size of this species is rapidly decreasing due to its limited distribution and habitat disturbances such as reclamation, shoreline development, and increasing tourism (Shim et al. 2009; Han et al. 2013). According to a recent study that analyzed the future distribution of coastal plants in Korea, the current distribution of L. japonica was expected to decrease (Park and Choi 2020). However, no phylogeographic research or conservation genetics of this species have been evaluated. Chloroplast genomes can provide useful markers for phylogeographic research and conservation measures. Here, we provide the complete chloroplast genome of L. japonica using next generation sequencing (NGS) technology.

Materials and methods

Plant material and DNA extraction

The fresh leaves of L. japonica were collected from Gangreung-si, Gangwon-do, South Korea (37.8631° N, 128.8479° E; Figure 1) and dried using silica gel for DNA extraction. The voucher specimen (HNIBRVP41776) was deposited at the herbarium of Honam National Institute of Biological Resources (HIBR [http://hnibr.re.kr]; contact person Yongsung Kim, orchidpark@hnibr.re.kr). We extracted genomic DNA from the samples using a DNeasy Plant Mini Kit (Qiagen Co., Valencia, CA, USA).

Genome sequencing, assembly, and annotation

The NGS was performed at Macrogen, Inc. (Seoul, South Korea) using the HiSeq X Ten system (Illumina, San Diego, CA, USA). A total of 47,514,966 reads were produced from NGS. Using the chloroplast genome of L. vulgaris (GenBank accession no. OL977692) as a reference, de novo assembly was conducted with NOVOPlasty v.4.3 (Dierckxsens et al. 2017). CpGAVAS2 (Shi et al. 2019) was used to identify the protein-coding genes (PCGs) and ribosomal RNAs (rRNAs) of the chloroplast genome, and tRNAscan-SE (Lowe and Eddy 1997) was used to identify the transfer RNA (tRNA) sequences. Gene annotations were manually checked and modified to ensure accuracy. To determine hotspot regions, we examined the nucleotide diversity (Pi) of three complete Linaria chloroplast genomes through a sliding window analysis using DnaSP v.6 (Rozas et al. 2017). The circular genome map and cis/trans-splicing genes were drawn using CPGView (http://www.1kmpg.cn/cpgview/; Liu et al. 2023), and the complete chloroplast genome sequence of L. japonica was submitted to GenBank (accession no. OR823819).

Phylogenetic analyses

To determine the systematic position of L. japonica, we included 26 species from 11 tribes of Plantaginaceae. Based on the previous phylogenetic studies (Albach et al. 2005; Xie et al. 2023), we also included three species (Streptocarpus teitensis Christenh [Gesneriaceae], Lindernia ruellioides Pennell [Linderniaceae], Myoporum bontioides A. Gray [Scrophulariaceae]) from Lamiales as outgroups. The construction of phylogenomic tree was performed with maximum likelihood (ML) and Bayesian inference (BI) methods using the concatenated 78 PCGs. A multiple sequence alignment was performed with MAFFT v.7.490 (Katoh et al. 2019) using default alignment parameters. The ML and BI phylogenetic analyses were conducted using IQ-TREE web server (http://iqtree.cibiv.univie.ac.at/; Trifinopoulos et al. 2016) and MrBayes v.3.2.6 (Ronquist et al. 2012), respectively with the parameters (nst = 6, rates = invgamma, ngen = 1,000,000, samplefreq = 1000, burn-in = 25%). Bootstrap support (BS, 1000 pseudoreplicates) and posterior probability (PP) were calculated to examine the relative level of robustness for each clade.

Results

General feature of chloroplast genome

The mean and minimum read mapping depth of assembled chloroplast genome of L. japonica were 3412× and 2587×, respectively (Figure S1). The entire chloroplast genome sequence of L. japonica was 150,660 bp in length. The GC content of the species was 37.8%. Similar to most angiosperms, the L. japonica chloroplast genome revealed a typical quadripartite structure, consisting of a pair of IRs (25,647 bp) split by the LSC (81,766 bp) and SSC (17,600 bp) regions (Figure 2). The chloroplast genome of L. japonica had 130 predicted functional genes encompassing 85 PCGs, 37 tRNA genes, and eight rRNA genes. In these genes, 113 were unique (counting all duplicated genes only once) and 17 were duplicated in the IR regions including six PCGs, seven tRNA genes, and four rRNA genes (Table 1). Furthermore, three PCGs (clpP, rps12, and ycf3) have two introns, while nine PCGs (petB, petD, atpF, ndhA, ndhB, rpl2, rpl16, rps16, and rpoC1) and six tRNA genes (trnA-UGC, trnI-CAU, trnL-CAA, trnN-GUU, trnR-ACG, and trnV-GAC) have one intron. Among the PCGs, 13 were cis-splicing genes (Figure S2). The rps12 gene was trans-spliced, which had the 5′ exon in the LSC region and the 3′ exon and intron in the IR regions (Figure S3). Within Linaria, the ycf15 gene has been lost from chloroplast genomes of L. japonica and L. vulgaris. Hotspot regions showing high nucleotide diversity in the chloroplast genomes of three Linaria species included matK-rps16, rpoB-petN, ndhC-atpE, rpL32-ccA, ndhG-ndhI, and ycf1 (Pi > 0.008; Figure S4).

Figure 2.

Schematic map of the chloroplast genome of linaria japonica. The map represented by six circles that illustrate the connection among distributed repeats (red line: forward direction, green line: reverse direction), tandem repeats (blue bars), short tandem repeats (green bars), the sizes and locations of quadripartite structure (LSC, SSC, and IRA and IRB), GC contents, and the genes with colored boxes, from the center outward. The number in parentheses after the genes indicates the codon usage bias.

Table 1.

Gene composition within chloroplast genome of Linaria japonica.

Groups of genes		Names of genes	No.
RNA genes	Ribosomal RNAs	rrn4.5 x2, rrn5 x2, rrn16 x2, rrn23 x2	8
	Transfer RNAs	trnA-UGC x2,a, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-GCC, trnG-UCCa, trnH-GUG, trnI-CAU x2, trnI-GAU x2,a, trnK-UUUa, trnL-CAA x2, trnL-UAAa, trnL-UAG, trnM-CAU, trnN-GUU x2, trnP-UGG, trnQ-UUG, trnR-ACGX2, trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC x2, trnV-UACa, trnW-CCA, trnY-GUA	37
Protein genes	Photosystem I	psaA, psaB, psaC, psaI, psaJ	5
	Photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ	15
	Cytochrome	petA, petBa, petDa, petG, petL, petN	6
	ATP synthases	atpA, atpB, atpE, atpFa, atpH, atpI	6
	Large unit of Rubisco	rbcL	1
	NADH dehydrogenase	ndhAa, ndhBX2,a, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK	12
	ATP-dependent protease subunit P	clpPb	1
	Envelope membrane protein	cemA	1
Ribosomal proteins	Large units of ribosome	rpl2X2,a, rpl14, rpl16a, rpl20, rpl22, rpl23X2, rpl32, rpl33, rpl36	11
	Small units of ribosome	rps2, rps3, rps4, rps7 x2, rps8, rps11, rps12X2,b, rps14, rps15, rps16a, rps18, rps19	14
Transcription/translation	RNA polymerase	rpoA, rpoB, rpoC1a, rpoC2	4
	Initiation factor	infA	1
	Miscellaneous protein	accD, ccsA, matK	3
	Hypothetical proteins and conserved reading frames	ycf1, ycf2X2, ycf3b, ycf4	5
Total			130

^X2, duplicated gene; ^agene with one intron; ^bgene with two introns.

Phylogenetic relationship

On the basis of the concatenated 78 PCGs, the results of the ML and BI analyses were congruent (Figure 3; BI tree not shown). Within Plantaginaceae, the monophyly of all tribes was well supported. Three species of Linaria were included in the tribe Antirrhineae, forming a monophyletic group. Within Linaria, L. japonica was sister to L. buriatica Turcz (Figure 3).

Figure 3.

Maximum likelihood tree of 26 plantaginaceeae species and three outgroups inferred from 78 protein-coding genes. The target species, L. japonica is marked in green. Tribes of plantaginaceae classification follows Xie et al. (2023). The GenBank accession number is given in parentheses after the species name. Bootstrap value (BS) and bayesian posterior probability (PP) are indicated before each node; an asterisk represents that a node BS = 100% for ML analysis and PP = 1.00 for bayesian’s inference analysis. Red cross on the top right corner of the species indicates putative loss of ycf15 gene. The following sequences were used: Angelonia angustifolia OM362910 (Yue et al. 2022); antirrhinum majus MW877560 (Mower et al. 2021); aragoa abietina MW877561 (Mower et al. 2021); bacopa monnieri MN736955 (unpublished); callitriche stagnalis ON571658 (unpublished); deinostema violacea OQ129604 (Xie et al. 2023); digitalis lanata KY085895 (Zhang et al. 2017); digitalis purpurea NC068046 (Zhao et al. 2023); ellisiophyllum pinnatum OQ129606 (Xie et al. 2023); hemiphragma heterophyllum MN383191 (Wu and Zhang 2019); hippuris vulgaris OQ058820 (You et al. 2021); lagotis brevituba MW182582 (Fan et al. 2021); limnophila sessiliflora ON000200 (Dong et al. 2023); linaria buriatica OQ129607 (Xie et al. 2023); linaria japonica OR823819 (this study); linaria vulgaris OL977692 (Zhao et al. 2023); lindernia ruellioides OQ784231 (unpublished); littorella uniflora MW877563 (Mower et al. 2021); myoporum bontioides MN044642 (Fowler et al. 2020); neopicrorhiza scrophulariiflora NC057075 (Zhang et al. 2019); penstemon cyaneus MK391143 (Stettler et al. 2021); penstemon fruticosus MK947104 (Stettler et al. 2021); plantago nubicola MW877564 (Mower et al. 2021); plantago ovata MW877583 (Mower et al. 2021); russelia equisetiformis OQ129608 (Xie et al. 2023); scoparia dulcis MZ242235 (Li et al. 2022); streptocarpus teitensis MF596485 (Kyalo et al. 2018); veronica persica KT724052 (Choi et al. 2016); veronicastrum sibiricum KT724053 (Choi et al. 2016).

Discussion and conclusion

In this study, we completed and characterized the chloroplast genome of L. japonica used for medicinal and ornamental purposes. The GC content (37.8%) and number of unique genes (113) of chloroplast genome of L. japonica are quite consistent with other Plantaginaceae members (GC content, 37.4%–38.6%; unique gene number, 113–114), except for Littorella uniflora (L.) Asch. with 39% GC content and 103 unique genes (Xie et al. 2023). When compared with two congeneric species, L. vulgaris and L. buriatica (Xie et al. 2023; Zhao et al. 2023), the absence of ycf15 gene was found in the chloroplast genomes of L. japonica and L. vulgaris, which does not reflect a phylogenetic relationship within Linaria (Figure 3). Indeed, the loss of ycf15 has been observed in a variety of Plantaginaceae lineages, which may have occurred independently throughout the evolution of Plantaginaceae (Xie et al. 2023).

According to Xie et al. (2023), the chloroplast genome size of Plantaginaceae species ranges from 130,833 bp in Littorella uniflora to 165,045 bp in Plantago asiatica L. and the size of IR region ranges from 21,404 bp to 38,724 bp. The chloroplast genome size of L. japonica (150,660 bp) was intermediate among Plantaginaceae, whereas its IR region (25,647 bp) appeared to have a relatively small size. Although the conservation of the IR region is crucial for the stability of chloroplast genome structure, its contraction and expansion are very common in the process of evolution and has been proved to be an important source of chloroplast genome size variation (Downie and Jansen 2015).

Our phylogenetic analyses revealed that Linaria is included in the Antirrhineae and is monophyletic. These results are in line with the previous studies of the phylogenetic relationships of Plantaginaceae (Fernández-Mazuecos et al. 2013; Xie et al. 2023). Linaria japonica has been considered closely related to L. vulgaris because they share the morphological trait of alternate or whorled leaf arrangement (Hong et al. 1998). However, L. japonica was found to be sister to L. buriatica instead of L. vulgaris. Therefore, we suggest that this characteristic is homoplasious within Linaria.

Our complete chloroplast genome data of L. japonica may serve as useful information for studying the morphological character evolution and systematics of Linaria. In addition, based on the results of the nucleotide diversity analysis in three Linaria species, six hotspot regions (matK-rps16, rpoB-petN, ndhC-atpE, rpL32-ccA, ndhG-ndhI, and ycf1) could be applied to the molecular identification and phylogeographic history of L. japonica, thereby providing a guideline for conservation.

Funding Statement

This work was supported by a grant from the Honam National Institute of Biological Resources (HNIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (Grant No. HNIBR202101107).

Ethics statement

The collection of plant material was carried out in accordance with the guidelines of Honam National Institute of Biological Resources, South Korea. No special permission is required because the samples used in this study are not endangered and were not collected from protected areas.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The genome data that supported the findings of this study are openly available in GenBank of NCBI at [https://www.ncbi.nlm.nih.gov] under the accession no. OR823819.1. The associated BioProject, SRA, and Bio-sample numbers are PRJNA1184393, SRR31304091, and SAMN44670255, respectively.