Complete plastid genome sequence of the Korean endemic species Zabelia densipila M.P. Hong, Y.C. Kim & B.Y. Lee (Caprifoliaceae) and its phylogenetic position

Abstract

Zabelia densipila M.P. Hong, Y.C. Kim & B.Y. Lee is a shrub endemic to South Korea. Its complete plastome is 156,361 bp long, comprising a large single-copy region (87,094 bp), a small single-copy region (17,845 bp), and two inverted repeats (25,711 bp each). It has a GC content of 37.8% and contains 130 genes (85 protein-coding, 37 tRNA, and 8 rRNA). Phylogenetic analysis of 79 protein-coding genes supported the monophyly of Zabelia, with Z. densipila closely related to Z. biflora and Z. dielsii, and recovered Morina as sister to Zabelia. This plastome provides a genomic resource for phylogenetic and evolutionary studies of Zabelia.

Introduction

The genus Zabelia (Caprifoliaceae) has received taxonomic attention because of its complex circumscription and historical relationship with Abelia and related genera (Decaisne 1846; Rehder 1911; Erdtman 1945; Makino 1948; Fukuoka 1969, 1972; Hara 1983; Jacobs et al. 2010). Molecular phylogenetic studies based on nuclear and plastid markers have shown that Zabelia represents a distinct monophyletic lineage, whereas Abelia is paraphyletic or possibly polyphyletic (Jacobs et al. 2010; 2011).

Zabelia densipila M.P. Hong, Y.C. Kim & B.Y. Lee is a deciduous shrub endemic to Korea and was first described from Gangwon-do in 2012 (Hong et al. 2012). It was distinguished from Z. biflora by flowering time, dense hairs on vegetative and reproductive structures, and sequence variation in nuclear ribosomal DNA and chloroplast coding regions. Z. densipila was reported from limestone forest slopes at elevations of 200–600 m and was known only from its type locality.

Plastid genomes (plastomes) are generally conserved in structure and gene content and are widely used for plant phylogenetic and comparative genomic studies (Provan et al. 2001; Ravi et al. 2008; Dobrogojski et al. 2020). With advances in next-generation sequencing technologies, complete plastomes have become increasingly accessible, helping resolve complex evolutionary relationships at various taxonomic levels (Hu et al. 2021). Complete plastomes have been reported for seven Zabelia species: Z. triflora, Z. biflora, Z. corymbosa, Z. integrifolia, Z. buddleioides, Z. tyaihyoni, and Z. dielsii (Cao and Xie 2019; Lee et al. 2019; 2020); however, the plastome of Z. densipila has not been reported.

In this study, we assembled and annotated the complete plastome of Z. densipila from high-throughput sequencing data. In addition, we performed phylogenetic analysis based on complete plastome sequences to investigate its evolutionary position within Zabelia. The plastome of Z. densipila provides valuable insights into the phylogeny, species boundaries, and genome evolution of this species within the genus Zabelia.

Materials and methods

Fresh leaves of Z. densipila were collected from Jeongseon-gun, Gangwon-do, Republic of Korea (37°21′53.8ʺN 128°43′26.9ʺE) on 28 April 2020 by Hee-Young Gil, Sa Bum Jang, and Ye-Rim Choi (Figure 1). The material was collected from a non-protected area, and no specific collection permit was required. A voucher specimen was deposited in the herbarium of Korea National Arboretum (KH) under accession number KHB1635154 (http://www.nature.go.kr, contact: Dong Chan Son, sdclym@korea.kr).

Figure 1.

Photographs of Z. densipila taken by Sa Bum Jang in jeongseon-gun, Gangwon-do (A-D). Permission to use the photographs was obtained from Sa Bum Jang. (A) Habitat of Z. densipila growing on limestone forest slopes. (B, C) Paired flowers showing dense hairs on pedicels, ovaries, and adjacent structures; inflorescences terminal; corolla infundibuliform and externally pilose. (D) Opposite leaves with ovate to elliptical leaf blades and dense hairs on the abaxial surface and petioles.

Total genomic DNA was extracted from silica-dried leaves using a DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA) and sequenced on the Illumina MiSeq platform (Illumina Inc., San Diego, CA) to generate 2 × 150 bp paired-end reads. A total of 3,899,951 read pairs were quality-trimmed using Geneious Prime v.2019.2.3 with an error probability limit of 0.05 (Kearse et al. 2012). The plastome was assembled de novo using NOVOPlasty v.4.3.5 (Dierckxsens et al. 2017), with the matK gene of Z. biflora (GenBank accession no. MK136244) as the seed sequence and default parameters. Sequencing depth was assessed using Draw_SequencingDepth.py (Ni et al. 2023). The assembled plastome was annotated using GeSeq (Tillich et al. 2017) and the annotation was manually inspected in Geneious Prime v.2019.2.3 (Kearse et al. 2012). The circular genome map and cis- and trans-splicing gene maps were generated using CPGview (Liu et al. 2023).

For phylogenetic analysis, the Z. densipila plastome was analyzed with 14 complete plastomes from Linnaeoideae and Morinoideae and three complete Caprifolioideae outgroups downloaded from GenBank. A total of 79 protein-coding genes were aligned using MAFFT v7 (Katoh and Standley 2013). A maximum-likelihood (ML) tree was reconstructed using IQ-TREE (Nguyen et al. 2015) with 5000 bootstrap replicates under the best-fit substitution model (GTR+F+R5) selected according to the Bayesian Information Criterion (Kalyaanamoorthy et al. 2017).

Results

The plastome of Z. densipila is 156,361 bp in length and exhibits a typical quadripartite structure, consisting of a large single-copy region of 87,094 bp, a small single-copy region of 17,845 bp, and a pair of inverted repeats (IRs) of 25,711 bp. The overall GC content is 37.8% (59,093 bp) (Figure 2). Read-mapping analysis corroborated the plastome assembly. The average sequencing depth across the plastome was 704.61×, ranging from 281× to 12,423× (Figure S1). Continuous coverage was observed throughout the plastome, including all four LSC/IR and SSC/IR junctions, with no coverage gaps.

Figure 2.

Circular map of the complete plastid genome of Z. densipila. The map contains six concentric tracks arranged from the center outward. The innermost track shows dispersed repeats, with red and green arcs indicating forward and reverse repeats, respectively. The second track represents long tandem repeats as blue bands, and the third track shows short tandem repeats, or microsatellites, as green bands. The fourth track indicates the large single-copy (LSC), small single-copy (SSC), and inverted repeats (IRs). The fifth track displays GC content variation across the plastome. The outermost track shows annotated genes as colored boxes according to functional category, with genes transcribed clockwise and counterclockwise positioned on opposite sides of the circle.

The plastome contains 130 genes, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. 11 unique cis-splicing genes (rps16, atpF, rpoC1, pafI, clpP1, petB, petD, rpl16, rpl2, ndhB, and ndhA) (Figure S2) were identified, with 13 copies because ndhB and rpl2 are duplicated in the IRs. The rps12 gene was trans-spliced (Figure S3).

ML phylogenetic analysis strongly supported the monophyly of Zabelia (Figure 3). Z. densipila formed a clade with Z. biflora and Z. dielsii. Morina was recovered as sister to Zabelia, whereas members of Linnaeoideae, including Abelia, Diabelia, and Vesalea, formed a sister group to the Zabelia–Morina clade.

Figure 3.

Maximum-likelihood phylogenetic tree of Zabelia densipila and 17 related taxa based on the concatenated sequences of 79 plastid protein-coding genes. Bootstrap values are indicated above the nodes. The scale bar at the bottom represents the number of substitutions per site. Accession numbers of the plastomes used in phylogenetic analysis are provided below the tree: Zabelia biflora NC_045063 (Wang et al. 2020a), Z. dielsii MW788540, Z. corymbosa MN524630 (Wang et al. 2020a), Z. integrifolia MN524632 (Wang et al. 2020a), Z. buddleioides MN524628 (Wang et al. 2020a), Z. tyaihyoni MN524634 (Wang et al. 2020a), Z. triflora NC_045053 (Wang et al. 2020a), morina Alba MN524639 (Wang et al. 2020a), M. nepalensis NC_080497, abelia chinensis MW752101, A. uniflora MN787781, diabelia serrata MK033538 (Wang et al. 2020b), D. spathulata MK033549 (Wang et al. 2020b), vesalea coriacea MN524620 (Wang et al. 2020a), triosteum himalayanum NC_045219, T. sinuatum NC_068129 (Liu et al. 2022), lonicera pampaninii OR567254 (Liu et al. 2023).

Discussion and conclusion

The plastome of Z. densipila exhibited a conserved quadripartite structure and a gene composition comparable to those of other Zabelia species. Overall genome organization, GC content, and gene arrangement were consistent with the typical plastome structure observed in angiosperms, indicating a relatively conserved evolutionary pattern within the genus.

Phylogenetic relationships were consistent with previous molecular and plastid phylogenomic studies of Caprifoliaceae (Jacobs et al. 2010; 2011; Wang et al. 2020a). Earlier phylogenetic analyses based on nuclear and plastid markers demonstrated that Zabelia is phylogenetically distinct from the traditional Linnaea clade and may be more closely associated with Morinoideae (Jacobs et al. 2010). Previous plastome analyses supported the monophyly of Zabelia and recovered a sister relationship between Zabelia and Morinoideae (Wang et al. 2020a). Similarly, our phylogenetic analysis strongly supports the monophyly of Zabelia, with Z. densipila forming a clade with Z. biflora and Z. dielsii. Although Z. densipila is morphologically similar to Z. biflora and is distinguished from that species based on floral and indumentum characteristics (Hong et al. 2012), it was recovered as a distinct branch within the genus. This result provides additional evidence that Z. densipila represents an independent taxon endemic to Korea. Z. dielsii is distributed across China and was recovered as a close relative of Z. densipila in this phylogenetic study, suggesting a close evolutionary relationship between the sampled Korean species and continental East Asian species of Zabelia. In addition, Morina was recovered as the sister group to Zabelia, whereas members of Linnaeoideae, including Abelia, Diabelia, and Vesalea, formed a sister group to the Zabelia–Morina clade. These results provide additional evidence supporting the phylogenetic placement of Zabelia outside the Linnaeoideae clade.

Despite recent advances in phylogenomics, phylogenetic relationships within Zabelia remain incompletely understood due to limited genomic resources and taxon sampling. Additional comparative analyses incorporating broader species sampling and independently inherited genomic data are necessary to better understand evolutionary relationships within the genus.

As a species endemic to Korea with a limited distribution, Z. densipila represents an important taxon for biodiversity and evolutionary studies. The complete plastome reported here provides a valuable genomic resource for future phylogenetic and comparative genomic studies of the genus. Further population-level sampling and analyses using nuclear markers will be necessary to support studies of species delimitation, population genetics, and conservation.

Funding Statement

This work was supported by the Korea National Arboretum under Grant KNA1-1-34-26-2.

Ethical approval

The collection of plant material complied with institutional, national, and international guidelines and legislation. No specific ethical approval was required for this study.

Disclosure statement

The authors report no conflict of interest.

Data availability statement

Plastome sequence data for Z. densipila are available in GenBank (https://www.ncbi.nlm.nih.gov/nuccore/PX645513) under accession number PX645513. The associated BioProject, Bio-Sample, and SRA numbers are PRJNA1462469, SAMN58835325, and SRR38396686, respectively.