Complete chloroplast genome and phylogenetic analysis of the korean endemic plant Semiaquilegia quelpaertensis D.C. Son & K.H. Lee (Isopyreae, Ranunculaceae)

Abstract

We sequenced the chloroplast genome of Semiaquilegia quelpaertensis and analyzed its phylogenetic relationships. The genome exhibited a typical quadripartite structure of 162,312 bp, comprising a large single-copy region (91,355 bp), a small single-copy region (17,331 bp), and two inverted repeat regions (26,813 bp each). A total of 127 genes were identified, including 81 protein-coding sequences (CDSs), 38 transfer RNA genes, and eight ribosomal RNA genes. Phylogenetic analysis using 77 CDSs from 48 taxa indicated that S. quelpaertensis is sister to S. adoxoides. These findings provide genomic fundamental data for evolutionary and conservation studies of endemic Semiaquilegia species.

Introduction

Semiaquilegia species are distinguished by morphological properties, including underground stem shape, inflorescence length, petal morphology, and the number of stamens and staminodes (Fu and Orbélia 2001; Son et al. 2017). Moreover, they differ markedly in distribution and habitat types. Semiaquilegia adoxoides is widespread across subtropical–temperate regions of China, Korea, and Japan, occupying diverse habitats and substrates. In contrast, the remaining taxa are narrow endemics with restricted distributions and specialized habitats: Semiaquilegia danxiashanensis and S. guangxiensis are restricted to Danxia landforms in Guangdong and limestone areas of Guangxi (Guilin and Liuzhou), China, respectively, whereas S. quelpaertensis is confined to Jeju-do, Republic of Korea, where it inhabits submontane broadleaf forests (Fu and Orbélia 2001; Huang et al. 2017; Son et al. 2017; Zhou et al. 2019). This distributional pattern suggests that habitat adaptation may have contributed to diversification and speciation within Semiaquilegia (Qin et al. 2024).

Chloroplast genomic data are useful for resolving phylogenetic relationships and may provide clues to evolutionary divergence associated with habitat differentiation; moreover, adaptive evolution in chloroplast genomes has been reported in plant taxa occupying different habitats (Gu et al. 2023). However, no complete chloroplast genome sequence is currently available for S. quelpaertensis, and phylogenetic analyses incorporating plastome-scale data are lacking. Therefore, we determined the complete chloroplast genome sequence of S. quelpaertensis and conducted phylogenetic analyses using chloroplast genome from taxa belonging to the tribe Isopyreae.

Our findings are expected to enhance our understanding of speciation and evolutionary processes within Semiaquilegia and provide valuable genomic resources for rare endemic plants with restricted distributions, thereby establishing a foundation for future conservation research.

Materials and methods

Sampling and DNA extraction

Semiaquilegia quelpaertensis D.C. Son & K.H. Lee (2017) was identified and fresh leaf samples were collected from Jeju-si, Jeju, Republic of Korea (33.437722°N, 126.640028°E) by Kang-Hyup Lee, a researcher at the Korea National Arboretum, on April 7, 2021 (Figure 1). The voucher specimen (KHB1635596) was deposited in the National Arboretum Herbarium (KH; http://www.nature.go.kr; contact: Dong Chan Son, sdclym@korea.kr).

Figure 1.

Photographs of S. quelpaertensis. A. This species was first discovered in Hallasan National Park, Jeju-si, Jeju-do, Republic of Korea, and grows in submontane broadleaf forests and moist valleys at 800–850 m elevation. B. Leaves are ternate, and the leaflets are shallowly lobed. C. Flowers are large, with broadly elliptic to obovate sepals and tubular petals reflexed along the lower margin. D. Follicles are 4–5, free, widely divergent, ovoid to oblong, and apically with a small beak formed by the persistent style. E. Seeds are obovoid, blackish brown, and conspicuously rugose-tuberculate on the surface.

Total genomic DNA was extracted from dried leaf tissue using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. High-quality genomic DNA (gDNA) was selected and purified by 2% agarose gel electrophoresis. The gDNA was sequenced on the Illumina MiSeq platform (Illumina, Inc., CA, USA), generating 301 bp paired-end reads, with a total of 10,953,574 reads obtained.

Assembly and annotation of the chloroplast genome

Chloroplast genome assembly was performed in Geneious Prime v.2025.2.2 using the chloroplast genome of S. adoxoides (GenBank accession no. PV546613) as a reference, obtained from the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov). The completeness of the assembled genome was assessed by examining read coverage depth using the Draw_SequencingDepth.py script provided by Ni et al. (2023). Genome annotation was conducted using GeSeq (Tillich et al. 2017), and gene order was verified by comparison with the chloroplast genome of S. adoxoides. The circular map of the chloroplast genome and intron structures of cis- and trans-splicing genes were visualized using CPGView (http://www.1kmpg.cn/cpgview).

Phylogenetic analysis

To analyze the phylogenetic relationships of S. quelpaertensis and related taxa, complete chloroplast genome sequences of 43 taxa within the tribe Isopyreae, including the study species, were retrieved from the NCBI. Five taxa of Trollius (subfamily Ranunculoideae) were included as outgroups, resulting in a total of 48 taxa. For each taxon, only one copy of duplicated sequences was retained. A total of 77 shared protein-coding sequences (CDSs) were extracted and aligned using MAFFT implemented in PhyloSuite v.1.2.2 (Zhang et al. 2020). The best-fitting nucleotide substitution model for maximum likelihood (ML) analysis was selected using ModelFinder v.2.2.0 (Kalyaanamoorthy et al. 2017). An ML phylogenetic tree was then constructed using IQ-TREE v.1.2.3 (Nguyen et al. 2015) under the TVM+F + R2 model with 1,000 bootstrap replicates to assess branch support. The resulting phylogenetic tree was visualized using FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree).

Results

Chloroplast genome

The complete chloroplast genome of S. quelpaertensis is 162,312 bp long (Figure 2), with a GC content of 41.7%. The genome was assembled with an average read coverage of 2613.64 × (Figure S1). The plastome exhibited a typical quadripartite structure, in which two inverted repeat (IR) regions separated the large single-copy (LSC) and small single-copy (SSC) regions. The lengths of the LSC, SSC, and IR regions were 91,355 bp, 17,331 bp, and 26,813 bp, respectively. The complete chloroplast genome sequence has been deposited in the NCBI database under accession no. PX987619.

Figure 2.

Chloroplast genome map of S. quelpaertensis. The map comprises six distinct tracks arranged from the center outward. The innermost track shows dispersed repeats, connected by red and green arcs indicating forward and reverse directions, respectively. The next track highlights long tandem repeats as blue bands, followed by a track marking short tandem repeats (microsatellites), represented as green bands. Another track depicts the GC content across the plastome. The outermost track displays genes as colored boxes, with inner boxes indicating clockwise transcription and outer boxes indicating counterclockwise transcription.

The genome contains a total of 127 genes, including 81 CDSs, 38 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. Among these, duplicated genes located in the IR regions comprised four CDSs (ndhB, rpl23, rps7, and ycf2), eight tRNA genes (trnA-UGC, trnG-GCC, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, and trnV-GAC), and four rRNA genes (rrn4.5, rrn5, rrn16, and rrn23).

Among cis-splicing genes, eight (atpF, ndhA, ndhB, petB, petD, rpl2, rpoC1, and rps16) contained a single intron, whereas one (pafI) contained two introns (Figure S2A). One trans-splicing gene, rps12, was identified; its 5′ exon is located in the IR region and 3′ exon in the LSC region, spanning both the regions (Figure S2B). In this plastome, infA was confirmed to be a pseudogene.

Phylogenetic analysis

Phylogenetic analyses based on chloroplast genome CDSs divided the Isopyreae taxa into two major groups (Figure 3). One group comprised Dichocarpum, Leptopyrum, Paraquilegia, and Thalictrum, whereas the other group included Aquilegia, Enemion, Isopyrum, Semiaquilegia, and Urophysa. Within the latter group, Semiaquilegia was closely related to Aquilegia and Urophysa. Furthermore, within Semiaquilegia, S. quelpaertensis formed a clade with S. adoxoides, and this relationship was strongly supported by a bootstrap value of 100.

Figure 3.

Maximum likelihood phylogenetic tree inferred from 77 concatenated chloroplast CDSs extracted from 48 complete chloroplast genomes, including 43 taxa of Thalictroideae (Tribe Isopyreae) and five outgroup taxa of Ranunculoideae. Chloroplast genome references used in the analysis (from top to bottom in the figure): Aquilegia alpine PV564728 (Yu et al. 2025); A. atrata PV564732 (Yu et al. 2025); A. japonica PV564767 (Yu et al. 2025); A. oxysepala PV564784 (Yu et al. 2025); A. colchica PV564749 (Yu et al. 2025); A. kubanica PV564772 (Yu et al. 2025); A. lactiflora PV564773 (Yu et al. 2025); A. ecalcarata MT875607 (Xue et al. 2021); A. kansuensis MT875845 (Xue et al. 2021); A. incurvata PV564765 (Yu et al. 2025); A. yabeana MT875673 (Xue et al. 2021); A. rockii MT875778 (Xue et al. 2021); A. yangii MT875691 (Xue et al. 2021); A. chrysantha PV564746 (Yu et al. 2025); A. brevistyla PV564740 (Yu et al. 2025); Semiaquilegia quelpaertensis PX987619 (this study); S. adoxoides PV546613 (Yu et al. 2025); S. danxiashanensis PP541616 (Qin et al. 2024); S. guangxiensis NC_057495 (Qin et al. 2020); Urophysa rockii PV546610 (Yu et al. 2025); Enemion raddeanum MK569486 (Zhai et al. 2019); Isopyrum manshuricum MK569496 (Zhai et al. 2019); Thalictrum aquilegiifolium MZ442608 (unpublished); T. minus PV546612 (Yu et al. 2025); T. baicalense MZ962406 (unpublished); T. simplex OM501080 (unpublished); T. fargesii ON868919 (Chen et al. 2024); T. tenue MK253448 (He et al. 2019); T. petaloideum MK253449 (He et al. 2019); T. elegans OR915995 (Cheng et al. 2024); T. foliolosum MZ196217 (Pu et al. 2022); T. baicalense MZ962406 (unpublished); T. cirrhosum NC_061927 (Zhe et al. 2023); T. foeniculaceum MT922012 (Lin et al. 2021); T. alpinum PP234509 (unpublished); T. coreanum KM206568 (Park et al. 2015); T. thalictroides NC_039433 (Morales‐Briones et al. 2019); Paraquilegia anemonoides MK569490 (Zhai et al. 2019); P. microphylla OP177949 (unpublished); Leptopyrum fumarioides MK569497 (Zhai et al. 2019); Dichocarpum sutchuenense MK253458 (He et al. 2019); D. dalzielii PV546614 (Yu et al. 2025); D. fargesii MK (Zhai et al. 2019); Trollius lilacinus OR449287 (Li et al. 2023); Tr. farreri MK843818 (Yu et al. 2019); Tr. japonicus OR449285 (Li et al. 2023); Tr. chinensis NC_059918 (Li et al. 2023); Tr. macropetalus NC_059918 (Li et al. 2023).

Discussion and conclusion

The chloroplast genome of S. quelpaertensis falls within the size range reported for congeneric taxa (160,548–164,047 bp) and exhibits the typical quadripartite structure of most angiosperms (Qin et al. 2020, 2024; Yu et al. 2025). Phylogenetic analyses based on chloroplast CDSs further indicated that Semiaquilegia is closely related to Aquilegia and Urophysa, with S. quelpaertensis forming a sister relationship with S. adoxoides.

Morphologically, S. quelpaertensis is similar to S. adoxoides in having narrowly elliptic sepals, short pedicels, and subtruncate petals (Son et al. 2017), but differs in having more shallowly lobed basal leaves, larger flowers, more stamens and staminodes, and larger rugose seeds.

There are no reports on the pharmacological or physicochemical properties of S. quelpaertensis. In contrast, previous studies on S. adoxoides have demonstrated various biological activities, including antioxidant, immunomodulatory, anti-inflammatory, and anticancer activities (Liang et al. 2016; Yang et al. 2020). These activities have been associated with polysaccharides and solvent extracts such as ethanol and methanol, and various compounds, including alkaloids, steroids, and glycosides, have been identified. As a congeneric species, S. quelpaertensis may possess similar biological activities; therefore, further studies are needed to investigate its pharmacological and physicochemical properties.

This study provides fundamental information on the chloroplast genome of S. quelpaertensis. These data not only contribute to understanding speciation and evolutionary processes within the genus Semiaquilegia, but also provide a basis for future conservation research through the preservation of genetic resources of endemic plants. Additionally, this study is expected to lay a foundation for future studies on the biological functions and potential applications of this species.

Funding Statement

This work was supported by the Korea National Arboretum under Scientific Research grants KNA1-1-30-25-1

Ethical approval

The materials used in this study are not listed in the IUCN Red List, and the collection area is not designated as a protected site. This study complied with the Act on the Creation and Furtherance of Arboretums and Gardens.

Disclosure statement

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

Data availability statement

The complete chloroplast genome sequence data for Semiaquilegia quelpaertensis is available in GenBank of NCBI (https://www.ncbi.nlm.nih.gov) under accession number PX987619. The associated BioProject, Bio-Sample, and SRA numbers are PRJNA1422392, SAMN55305746, and SRR37198644, respectively.