The complete chloroplast genome of Kummerowia stipulacea (Maxim.) Makino (Fabaceae)

Abstract

Kummerowia stipulacea (Maxim.) Makino is a traditional medicinal herbal native to East Asia and introduced to the eastern United States. Here, we report its first complete chloroplast genome (146,837 bp; GC 34.94%), exhibiting a typical quadripartite structure with a large single-copy region (87,357 bp), a small single-copy region (18,844 bp), and two inverted repeats (20,318 bp each). It encodes 122 genes: 78 protein-coding, 36 tRNA, and 8 rRNA genes. Phylogenetic analysis of 75 protein-coding genes indicated that K. stipulacea is closely related to K. striata, providing a genomic baseline for Fabaceae research.

1. Introduction

Kummerowia stipulacea (Maxim.) Makino 1914 is an annual herb belonging to the genus Kummerowia in the Fabaceae family. Native to East Asia (China, Japan, Korea, and the Russian Far East), K. stipulacea has also been introduced to the eastern United States. Exhibiting strong natural reproductive capacity, this species typically inhabits roadsides, grasslands, mountain slopes, and fixed or semi-fixed sand dunes at elevations ranging from 100 to 1200 meters. K. stipulacea grows prostrate, spreading, or has erect stems up to 7–15 cm tall (Zhang et al. 2014). As a traditional herbal medicine, K. stipulacea is widely utilized in East Asian folk medicine, including in China and Korea (Lin et al. 2007). Modern pharmacological studies have shown that K. stipulacea is a good source of natural estrogens, which could be used as herbal medicines for the treatment of menopausal dysfunctions in the future (Wang et al. 1998, Yoo et al. 2005). Furthermore, characterized by its soft stems and leaves, distinct aromatic profile, high nutritional value, and substantial energy content, K. stipulacea serves as a high-quality forage grass for livestock and poultry (Lee and Lee 2002).

The chloroplast genome of angiosperms has been shown to be a valuable tool for inferring species trees, analyzing population genetics, studying chloroplast gene function, and understanding chloroplast metabolism (Dobrogojski et al. 2020). Furthermore, it facilitates the investigation of species adaptation to extreme environments (Daniell et al. 2016, Wang et al. 2024). However, to date, the complete chloroplast genome of K. stipulacea has not been reported, which hinders a deeper understanding of its genomic characterization and evolutionary history.

In this study, the chloroplast genome of K. stipulacea was assembled for the first time, and its phylogenetic position in Papilionoideae was confirmed. This newly assembled chloroplast genome provides a fundamental resource for comparative genomic and genetic studies within Kummerowia and the broader Fabaceae.

2. Materials and methods

2.1. Plant materials and sampling

Fresh and mature leaves from an adult K. stipulacea specimen were collected in Chengdu City, Sichuan Province, China (30.53146^oN, 104.30634^oE) (Figure 1), in strict accordance with the Regulations of the People’s Republic of China on the Protection of Wild Plants. The species was identified based on key morphological characters: stems with upward-pointing white hairs, obovate and emarginate leaflets, and flowers distributed at stem apices, which distinguish it from the closely related K. striata. A specimen was deposited at the herbarium of Chengdu University of Traditional Chinese Medicine (contact Huang Zhu, zhuhuang@cdu.edu.cn, voucher number: CDU20251012002).

Figure 1.

Morphology of K. stipulacea. Photograph of a specimen showing its prostrate, multi-branched habit, trifoliolate leaves with obovate to widely obovate leaflets arranged on short petioles, and the axillary flowers with a purplish corolla, which are characteristic of the species. Image credit: Qingjie Li (qingjieli2005@163.com).

2.2. Genome sequencing, assembly, and annotation

For whole-genome sequencing, a modified CTAB method was employed for the extraction of total genomic DNA (Doyle and Doyle 1987). The construction of paired-end libraries, characterized by an insertion size of 350 base pairs, was undertaken in accordance with the manufacturer’s instructions. The subsequent process of sequencing was performed on a DNBSEQ-T7 platform (MGI Tech Co., Ltd., Shenzhen, China), resulting in a total of 7.24 Gb of paired-end reads. Subsequently, the chloroplast genome of K. stipulacea was de novo assembled by GetOrganelle v1.7.7.1 (Jin et al. 2020) using the following parameters: “-F embplant_pt” and “-k 21,45,65,85,105”. The chloroplast genome assembly was annotated using Plann v1.1 (Huang and Cronk 2015) with the K. striata chloroplast genome (GenBank accession number: NC_044114) as reference. The obvious annotation errors were corrected by Geneious (Kearse et al. 2012). The structures of cis-splicing and trans-splicing genes were visualized using CPGview2 (Liu et al. 2023).

2.3. Phylogenetic analysis

We performed phylogenetic analysis of K. stipulacea and nine other species from Papilionoideae, including Campylotropis macrocarpa (MG867566), Desmodium heterocarpon (MG867567), Hylodesmum podocarpum (MG867568), K. striata (NC_044114), Lespedeza maritima (MG867570), Mucuna macrocarpa (MG867571), and Ohwia caudata (MG867572) using Millettia pinnata (NC_016708) and Indigofera tinctoria (NC_026680) as outgroups. The coding sequences of protein-coding genes (PCGs) present in all ten chloroplast genomes of Papilionoideae species were aligned by MAFFT-LINSI v7.313 (Katoh and Standley 2013). The resulting alignments were concatenated to construct a supermatrix. A maximum likelihood phylogenetic tree was generated using RAxML v8.2.11 (Stamatakis 2006) under the GTRGAMMA model with 1000 bootstrap replicates. In addition, nucleotide diversity (pi) among the ten chloroplast genomes was calculated using DnaSP v5.10 (Librado and Rozas 2009), with window length and step size set to 800 bp and 200 bp, respectively.

3. Results

The chloroplast genome of K. stipulacea (GenBank accession: PX648295) is a circular structure spanning 146,837 bp. This structure contains two inverted repeat (IR) regions of 20,318 bp each, separated by a large single-copy (LSC) region of 87,357 bp and a small single-copy (SSC) region of 18,844 bp (Figure 2). The mean coverage depth of the assembled genome was determined to be 2,878× (Supplementary Figure 1).

Figure 2.

Physical map of K. stipulacea chloroplast genome. Genes from different functional groups are color-coded. The boundaries of the LSC, SSC, and IR regions are delineated within the inner circle, with dark gray bars signifying GC content.

The chloroplast genome contains 122 genes, including 78 PCGs, 36 tRNA genes, and 8 rRNA genes (Supplementary Table 1). Of these, six PCGs and six tRNA genes contain a single intron, and two PCGs (ycf3 and clpP) contain two introns. The structures of eight cis-spliced genes are shown in Supplementary Figure 2, and the structure of the trans-spliced gene rps12 was verified and illustrated in Supplementary Figure 3. The overall GC content is 34.94%, while the GC content for the first, second, and third codons of PCGs is 44.23%, 36.90%, and 26.04%, respectively (Supplementary Table 1). The 78 PCGs contain a total of 21,605 codons, with leucine and cysteine being the most and least frequently used amino acids, respectively, at frequencies of 10.46% and 1.14%, respectively.

Phylogenetic analysis of the 75 shared PCGs across all ten chloroplast genomes elucidated their evolutionary relationships (Figure 3), revealing that K. stipulacea is closely related to K. striata with 100% bootstrap support.

Figure 3.

 A maximum likelihood tree showing the phylogenetic relationships between K. stipulacea and nine other papilionoideae species. Bootstrap values are shown at each internal node. The following sequences were used: MG867566 (Jin et al. 2019), MG867567 (Jin et al. 2019), MG867568 (Jin et al. 2019), NC_044114 (Jin et al. 2019), MG867570 (Jin et al. 2019), MG867571 (Jin et al. 2019), MG867572 (Jin et al. 2019), NC_016708 (Kazakoff et al. 2012), and NC_026680 (unpublished).

Comparative analysis of the ten chloroplast genomes identified several highly variable regions with high nucleotide diversity (pi > 0.16), including the genic regions of two PCGs (ycf4 and psaI), one tRNA gene (trnD-GUC), and four intergenic regions (trnD-GUC—trnY-GUA, trnY-GUA—trnE-UUC, trnE-UUC—psbM, and trnQ-UUG—psaI) (Supplementary Figure 4).

4. Discussion and conclusion

The chloroplast genome of K. stipulacea exhibits features consistent with previously reported Desmodieae plastomes, which maintain a conserved quadripartite structure and similar gene content (Jin et al. 2019). The hypervariable regions identified here, such as ycf4 and psaI, align with previous Fabaceae studies and represent promising molecular markers for phylogenetic analyses (Liu et al. 2018, Qin et al. 2025).

Phylogenetic analysis confirmed that K. stipulacea is closely related to K. striata, supported by both molecular data and shared morphological traits, including prostrate habit, trifoliolate leaves, and small oval pods. In contrast, related genera such as Lespedeza and Campylotropis differ in growth form and inflorescence type, highlighting the distinctiveness of the Kummerowia clade.

Notably, the ycf2 gene spans the LSC/IRb boundary, with the IRa copy being truncated and likely pseudogenized. This type of gene loss and pseudogenization at IR/SC junctions has been documented as a common structural feature in angiosperm plastome evolution (Cauz-Santos 2025).

Overall, this work provides the first plastome resource for K. stipulacea, serving as a foundation for future taxonomic, evolutionary, and ecological studies in Kummerowia and related Fabaceae lineages.

Funding Statement

This work was supported by the National Natural Science Foundation of China (No. 52401305) and the Sichuan Science and Technology Program (Nos. 2024NSFSC1659, 2025ZNSFSC1420).

Ethical approval

This study complied with all applicable national regulations for plant material collection and did not involve human or animal subjects.

Disclosure statement

The authors alone are responsible for the content and composition of the paper. Additionally, fresh and mature leaves from an adult plant of K. stipulacea were taken with the permission of the community management department. No potential conflict of interest was reported by the authors.

Data availability statement

The chloroplast genome assembly and raw sequencing data generated in this study are available in NCBI (https://www.ncbi.nlm.nih.gov/) under the accession numbers PX648295 and SRR36601806 (BioSample: SAMN54308308; BioProject: PRJNA1393613).