Complete chloroplast genome sequence of Rhamnella franguloides (Maxim.) Weberb. (Rhamnaceae)

Abstract

Rhamnella franguloides (Maxim.) Weberb. (Rhamnaceae) is a shrub native to East Asia that supports forest understories, and has potential medicinal and ornamental value. In this study, the complete plastome of R. franguloides was sequenced and assembled. The 161,545 bp circular plastome showed a typical quadripartite structure consisting of a pair of inverted repeats (IRs; 26,723 bp), a large single-copy region (LSC; 88,828 bp), and a small single-copy region (SSC; 19,269 bp). It comprises 132 annotated genes including 87 protein-coding genes (PCGs), 37 tRNA genes, and 8 rRNA genes with an overall GC content of 37.1%. Phylogenetic analysis based on complete chloroplast genomes revealed that R. franguloides forms a well-supported sister relationship within the Rhamnella clade, indicating its intermediate phylogenetic position within the genus. This plastome sequence provides a valuable genomic resource for further taxonomic and evolutionary studies of the Rhamnaceae.

Introduction

The genus Rhamnella is a small group within the family Rhamnaceae, which comprises around 61 genera (Medan and Schirarend 2004; Hauenschild et al. 2016; Basso-Alves et al. 2023). Among them, Rhamnella franguloides (Maxim.) Weberb. represents an important East Asian lineage within the genus Rhamnella. It occurs as a deciduous shrub or small tree in China, Korea, and Japan, where it occupies the understory of temperate forests. By producing fleshy drupes, the species provides a key food source for frugivorous birds, which in turn disperse its seeds and help sustain forest dynamics. Because of its unique geographic distribution and distinct phylogenetic placement within Rhamnaceae, R. franguloides serves as a useful species for examining evolutionary relationships across the family.

Although the genus Rhamnella plays an evident ecological and evolutionary role, genomic resources are very restricted. To date, only three plastomes have been described from this genus (Wang et al. 2024), leaving a notable gap in the genomic record of R. franguloides. Thus, plastome data for this species would supply valuable characters for comparative analyses and improve the resolution of phylogenetic studies within Rhamnaceae. Such information would also provide molecular markers that could be applied to population-level and conservation research, which is increasingly urgent for understory plants exposed to habitat loss and climate change.

Materials and methods

The plant material of R. franguloides were collected from Wanju, Jeollabuk-do, Korea (35° 44′ 11.36″ N, 127° 14′ 08.75″ E) on 15 Aug, 2025 and photographed with a digital camera to record the natural habitat (Figure 1). Each specimen was morphologically identified by Joon Moh Park and deposited in the Jeollabuk-do Forest Environment Research Institute under the voucher JFERI2025-08 (contact person: Joon Moh Park, joonmoh@korea.kr). Total genomic DNA was extracted from fresh leaves using a DNeasy Plant Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions.

Figure 1.

The species reference photographs of R. franguloides. (A) a natural habitat of R. franguloides along a forest stream. (B) Close-up of leaves and fruits. Photographs were captured by Joon Moh Park from Wanju, Jellabuk-do, South Korea (35° 44′ 11.36″ N, 127° 14′ 08.75″ E). Main identifying features include its growth as a shrub or small tree, glabrous leaves with 7–9 pairs of lateral veins, and nearly cylindrical drupes measuring 6–8 mm in length with pedicels of 3–4 mm. Scale bar = 2 cm.

The NGS sequencing library was constructed using TruSeq Nano DNA Kit (Illumina, USA) and sequenced by paired-end sequencing using Illumina HiSeq X platform (Macrogen Inc., Korea), yielding approximately 6.4 Gbp raw reads (42,112,110 reads). After quality control and filtering, 6.0 Gbp of high-quality reads (40,067,852 reads, Q30 = 97.2%) were retained. The plastome assembly was performed using NOVOPlasty v2.7.2 (Dierckxsens et al. 2017), employing the complete chloroplast genome of R. rubrinervis (GenBank accession number: NC_088754) as a reference sequence. Assembly validation was conducted using self-mapping and BLAST analysis. Raw Illumina reads were re-aligned against the assembled sequence using BWA v0.7.17 (Li 2013) under default settings, and the alignments were processed with Samtools v1.13 (Danecek et al. 2021). Examination of the aligned read coverage patterns indicated uniform read support across the plastome, with an average depth of 2574× (Figure S1). In addition, BLASTn comparisons with complete plastid genomes of R. martinii (OQ714592.1), R. rubrinervis (NC_088754), and R. wilsonii (NC_066091) provided further confirmation of sequence completeness and accuracy. Annotation of the plastome was performed through the CPGAVAS2 online platform (Shi et al. 2019), which integrates multiple chloroplast genome annotation tools. A plastome map illustrating the gene organization, including the cis- and trans-splicing structure of rps12, was generated using CPGView (Liu et al. 2023). The annotated complete chloroplast genome sequence of R. franguloides was deposited in GenBank (accession number: PX394660.1).

To infer the phylogenetic position of R. franguloides within the family Rhamnaceae based on plastome data, plastome sequences of 14 representative taxa belonging to the tribe Rhamneae, which comprises genera most closely related to Rhamnella, were retrieved from the NCBI RefSeq database. Spyridium parvifolium (ON534256) of the tribe Pomaderreae and Ziziphus jujuba (NC_030299) of the tribe Paliureae were designated as outgroups. Complete plastome sequences were aligned using MAFFT v7 (Katoh and Standley 2013) under the auto setting. The resulting alignments were subsequently trimmed using TrimAl (Capella-Gutiérrez et al. 2009). The best-fit model was determined by ModelFinder (Kalyaanamoorthy et al. 2017). Maximum-likelihood (ML) phylogenies were inferred with IQ-TREE v2.0 (Minh et al. 2020) employing the TVM+F + R3 model, and nodal support was evaluated with 5000 ultrafast bootstrap replicates.

Results

The complete chloroplast genome of R. franguloides, with a total length of 161,545 bp, exhibits a typical quadripartite structure (Figure 2). Genes were categorized into functional groups, including those involved in photosystem I and II, cytochrome b6/f complex, ATP synthase, NADH dehydrogenase, RNA polymerase, ribosomal proteins (small and large subunits), Rubisco, tRNAs, rRNAs, and hypothetical chloroplast reading frames. The per-base coverage depth was evaluated to assess assembly accuracy, with an average depth of 2574, confirming the reliability of the assembled genome (Figure S1). The genome comprises a large single-copy (LSC) region of 88,828 bp and a small single-copy (SSC) region of 19,269 bp, which are separated by a pair of inverted repeat (IR) regions of 26,723 bp each. The IR boundaries are highly conserved, with the rps19 and ycf1 genes spanning the LSC/IR and SSC/IR junctions, respectively. This structural stability is consistent with other members of the family. The overall GC content of the plastome is 37.1%, with 35.0% in the LSC, 31.3% in the SSC, and 42.6% in the IR regions. In addition to the structural characterization of the LSC and SSC regions, a comprehensive analysis of the plastome revealed 49 long repeat sequences (≥20 bp). Among these, palindromic repeats were the most frequent (24), followed by forward (21) and reverse (4) repeats, with lengths ranging from 20 to 46 bp. These repetitive elements provide valuable genomic resources and serve as potential molecular markers for future investigations into the genetic diversity and taxonomic resolution of the Rhamnaceae family.

Figure 2.

Complete plastome map of R. franguloides. The circular representation of the plastome comprises six concentric tracks arranged from the outermost to the innermost ring. The outermost track shows annotated genes as variably sized and colored boxes, corresponding to gene length and functional classification. Genes positioned on the outside and inside of this track are transcribed in the clockwise and counterclockwise directions, respectively. The second track illustrates the GC content distribution across the genome. The third track delineates the overall structural organization, including the large single-copy (LSC), small single-copy (SSC), and two inverted repeat (IR) regions. The fourth track highlights short tandem repeats (microsatellites) with colored bars, while the fifth track depicts long tandem repeats as compact blue bars. The innermost track connects forward and reverse repeats with red and green arcs, respectively, indicating repeat orientation and correspondence.

A total of 132 genes (113 are unique genes) of the R. franguloides plastome were annotated, comprising 87 protein-coding genes (79 unique genes), 37 tRNA genes (30 unique genes), and 8 rRNA genes (4 unique genes). Seven protein-coding (rpl2, rpl23, ndhB, ycf2, rps7, rps19, and ycf1), seven tRNA (trnI-CAU, trnL-CAA, trnV-GAC, trnI-GAU, trnA-UGC, trnR-ACG, and trnN-GUU), and four rRNA (rrn16, rrn23, rrn4.5, and rrn5) genes were duplicated within the IR regions. Eleven protein-coding genes contain a single intron, whereas pafI and clpP1 each possess two introns (Figure S2). The rps12 gene was identified as a trans-splicing gene (Figure S3).

Phylogenetic analysis based on complete chloroplast genome sequences (Figure 3) revealed that the Rhamnella species form a well-supported monophyletic clade. Within this clade, R. martinii and R. wilsonii are closely related (bootstrap = 100) and together form a sister group to R. franguloides (bootstrap = 100). R. rubrinervis is positioned as the most basal lineage within the genus, suggesting that it represents the earliest-diverging member of Rhamnella.

Figure 3.

ML phylogeny of Rhamnella franguloides and its close relatives. The bootstrap values based on 5000 replicates are shown on each node in the phylogenetic tree. The complete plastome sequences of 17 Rhamnaceae species, encompassing three major tribes, were downloaded from the GenBank database: Berchemiella berchemiifolia (NC_037477) (Cheon et al. 2018), B. polyphylla (NC_072275), B. wilsonii (NC_043912) (Li et al. 2019), Frangula alnus (NC_068506) (Shi et al. 2023), Rhamnella franguloides (PX394660) (this study), R. martinii (NC_082535), R. rubrinervis (NC_088754), R. wilsonii (NC_066091) (Wang et al. 2024), Rhamnus cathartica (NC_068507) (Shi et al. 2023), R. globosa (NC_057506) (Xie et al. 2020), R. heterophylla (NC_057481) (Li et al. 2020), R. taquetii (NC_045855) (Jin et al. 2020), R. erythroxylum (NC_088768), R. leptacantha (NC_082339) (Huang et al. 2024), Sageretia thea (NC_082057) (Zhan et al. 2024), Spyridium parvifolium (outgroup, ON534256) (Clowes et al. 2023) and Ziziphus jujube (outgroup, NC_030299) (Ma et al. 2017).

Discussion and conclusion

R. franguloides has long been regarded as a taxonomically challenging species within the genus Rhamnella due to its complex morphological and phylogenetic characteristics (Lu and Sun 2020; Kellermann 2021). Morphologically, the species exhibits substantial overlap with those of related taxa like R. martinii and R. wilsonii, making species delimitation difficult. Frequent morphological convergence and incomplete lineage sorting within Rhamnella further complicate its classification.

In this study, we present the first complete characterization of the chloroplast genome of R. franguloides. The 161,545 bp circular genome displays a typical quadripartite structure comprising 132 annotated genes. Plastome-based phylogenetic analysis showed that R. franguloides forms a strongly supported sister relationship with the R. martinii–R. wilsonii clade. This result is consistent with previous studies using the plastid marker DNA (Richardson et al. 2000; Lu and Sun 2020; Wanichthanarak et al. 2023) and agrees with established taxonomic frameworks recognizing Rhamnella as a monophyletic group closely related to Rhamnus. Multiple sequence alignment (MSA) and identity matrix calculations revealed that the R. franguloides plastome shares high sequence identity (95.91%–97.29%) with other Rhamnella species. Interestingly, it also showed high similarity with the genus Berchemiella (over 96%), suggesting a close evolutionary relationship within the tribe Rhamneae. Therefore, the present molecular evidence further supports the traditional classification of R. franguloides within the genus Rhamnella.

The current phylogenetic understanding of Rhamnaceae remains limited due to insufficient chloroplast genome data. Our data might contribute to a more comprehensive understanding of diversification and speciation processes in Rhamnaceae, particularly within the East Asian floristic region, where the family exhibits remarkable ecological and morphological diversity.

Funding Statement

This paper was supported by research funds of Jeonbuk National University in 2025.

Disclosure statement

No potential conflict of interest was reported by the authors.

Ethical approval

The R. franguloides specimen was not an endangered species. It required no specific permissions or licenses. Research on this species, including the collection of plant material, was conducted following the guidelines provided by Jeonbuk National University in this study.

Data availability statement

The complete chloroplast genome sequence of R. franguloides in this study has been submitted to the NCBI database (https://www.ncbi.nlm.nih.gov/nuccore/PX394660) under the accession number PX394660. The associated BioProject, BioSample, and SRA numbers are PRJNA835661, SAMN51880588, and SRR35627059.