Phylogenomic insights from the complete chloroplast genome of Phyllostachys lithophila (poaceae: bambusoideae: arundinarieae) in Taiwan

Abstract

Phyllostachys lithophila Hayata 1916 is a unique bamboo species endemic to Taiwan, typically found at elevations ranging from 500 to 1,500 meters. This study provides a detailed analysis of the complete chloroplast genome of P. lithophila for the first time. The genome spans 139,664 base pairs (bp) and consists of a large single-copy (LSC) region of 83,192 bp, a small single-copy (SSC) region of 12,869 bp, and two inverted repeat (IR) regions, each 21,798 bp in length. The plastid genome encodes a total of 129 genes, including 83 protein-coding genes, 38 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Phylogenetic analysis confirms that P. lithophila shares a closer phylogenetic relationship with species of Phyllostachys within the Arundinarieae tribe, while being phylogenetically distinct from the morphologically similar P. makinoi.

Introduction

Phyllostachys is a taxon within the tribe Arundinarieae (subfamily Bambusoideae), comprising numerous species of significant economic and ecological value. Phyllostachys has garnered attention due to the edibility of its bamboo shoots and the architecture of its culms, such as P. edulis. Many studies have been conducted on the chloroplast genomes within Phyllostachys (Zhang et al. 2011; Zhang et al. 2011; Wu and Song 2012; Gao and Gao 2016; Huang et al. 2019; Cao et al. 2020; Hu et al. 2021; Zheng et al. 2021; Zhou et al. 2021; Wang et al. 2023; Kung and Chang 2024; Liu et al. 2024;). Phyllostachys lithophila Hayata 1916 (Hayata 1916) is a temperate bamboo belonging to the Bambusoideae. This species primarily inhabits elevations between 500 and 1,500 meters, with a distribution concentrated in the mountainous regions of central and northern Taiwan (Figure 1). It is particularly abundant in Shizhuo and Fenqihu, located in Chiayi County. P. lithophila is highly similar to P. makinoi in morphology, and they always appear at the same area at the same time. At present, shoot morphology remains the most reliable diagnostic feature for distinguishing between the two species. However, identification becomes difficult during non-shooting seasons. In recent years, molecular techniques have been applied to investigate bamboo systematics and resolve taxonomic ambiguities. In Taiwan, more and more species have been reported such as Ampelocalamus naibuensis (Zhang and Chen 2016), Bambusa oldhamii (Wu et al. 2009), Dendrocalamus latiflorus (Wu et al. 2009), and P. makinoi (Kung and Chang 2024). However, to date, no studies have focused on P. lithophila. This study represents the first report of the complete chloroplast genome sequence of P. lithophila.

Figure 1.

Image of phyllostachys lithophila (species images were taken by Kung-Ning kung at shizhuo chiayi county, Taiwan. A. Habitat; B. Plant; C. Leaf; D. Branches of culm; E. Internode of culm; F. Shoots; G. Shoots).

Materials and methods

Fresh leaf samples of P. lithophila were collected from Xiding Village, Fanlu Township, Chiayi County, Taiwan (23°25′23.0ʺN, 120°39′07.4ʺE) for DNA extraction and sequencing. The samples were silica gel-dried and stored at −20 °C. A voucher specimen was deposited at the Herbarium of the Department of Forestry and Natural Resources, National Chiayi University (Index Herbariorum code: CHIA) under the collection number K. N. Kung 3184, curated by Associate Professor Kun Cheng Chang (kcchang@mail.ncyu.edu.tw).

Total genomic DNA was extracted using the Plant Genomic DNA Extraction Miniprep System (Viogene, Taiwan) following the manufacturer’s protocol. The extracted DNA was submitted to Tri-I Biotech, Inc. (New Taipei City, Taiwan) for high-throughput sequencing. Sequencing was performed using the Illumina MiSeq platform with a 2 × 150 bp paired-end read configuration. Quality assessment, trimming, and de novo assembly of the chloroplast genome were conducted using CLC Genomic Workbench 21 (CLC Inc., Aarhus, Denmark). The assembly was guided by the reference chloroplast genome of Phyllostachys edulis f. bicolor (accession number: OM084949). The final assembled complete chloroplast genome sequence was annotated using the online tools GeSeq (Tillich et al. 2017) to identify and annotate various functional genes in the chloroplast genomes of each sample. The annotated genome sequence of P. lithophila was submitted to GenBank under the accession number MZ662759 (https://www.ncbi.nlm.nih.gov).

To investigate the phylogenetic position of P. lithophila within the genus Phyllostachys, complete chloroplast genome sequences of 13 additional Phyllostachys species were downloaded from the NCBI database. Additionally, sequences of Pseudosasa cantorii (accession number: MF066255) and Pleioblastus amarus (accession number: MH988736) from the tribe Arundinarieae, as well as Bambusa oldhamii (accession number: FJ970915) and Dendrocalamus latiflorus (accession number: FJ970916) from the tribe Bambuseae were included for comparative analysis. Oryza sativa (accession number: NC001320) was used as the outgroup. All sequences were aligned using MAFFT v7 (Katoh and Standley 2013). A phylogenetic tree was reconstructed using both maximum likelihood (ML) and Bayesian inference (BI) approaches to ensure robust inference. The ML analysis was performed using RAxML v8 (Stamatakis 2014) with 1,000 bootstrap replicates to evaluate branch support. For BI analysis, MrBayes v3.2.7 (Ronquist et al. 2012) was employed, implementing two independent Markov chain Monte Carlo (MCMC) runs, each with four chains run for 1,000,000 generations, sampling every 1,000 generations. The initial 25% of samples were discarded as burn-in, and posterior probabilities were calculated to assess the credibility of each clade. The circular chloroplast genome map and visual representation of cis-splicing and trans-splicing gene structures were generated using CPGview (Liu et al. 2023).

Results

The complete chloroplast genome of Phyllostachys lithophila was successfully assembled, yielding a circular DNA molecule of 139,664 bp in length. It exhibits the typical quadripartite structure, consisting of a large single-copy (LSC) region of 83,196 bp, a small single-copy (SSC) region of 12,872 bp, and a pair of inverted repeats (IRa and IRb), each 21,798 bp in length (Figure 2). The overall GC content of the genome is 38.9%, with the LSC, SSC, and IR regions exhibiting GC contents of 37.0%, 33.2%, and 44.2%, respectively. A total of 129 unique genes were annotated, including 83 protein-coding genes, 38 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes.

Figure 2.

Circular map of the complete chloroplast genome of phyllostachys lithophila. The map is composed of six concentric tracks, organized from the center outward as follows: the innermost track illustrates repetitive sequences, connected by red or green arcs to indicate repeat relationships. The second track displays long tandem repeats as short blue bars. The third track shows short tandem repeats (SSRs), represented by short, colored bars. The fourth track outlines the structural regions of the chloroplast genome, including the large single-copy (LSC) region, small single-copy (SSC) region, and a pair of inverted repeats (IRA and IRB). The fifth track represents GC content variation across the genome. The outermost track annotates genes, color-coded by functional categories. Genes located outside the circle are transcribed in the clockwise direction, while those inside are transcribed counterclockwise.

Discussion and conclusion

To clarify the phylogenetic position of Phyllostachys lithophila within the genus Phyllostachys, we performed a comprehensive phylogenomic analysis based on complete chloroplast genome sequences using both Maximum Likelihood (ML) and Bayesian Inference (BI) approaches. The resulting phylogenetic tree (Figure 3) resolved P. lithophila as sister to P. glauca, with full statistical support (ML bootstrap = 100; BI posterior probability = 1.0). Together with P. reticulata, these three taxa formed a monophyletic subclade within Phyllostachys. However, the internal branches within this subclade received relatively low support (ML/BI = 22/1 between P. glauca and the clade comprising P. lithophila and P. reticulata; ML/BI = 28/1 between P. lithophila and P. reticulata), suggesting unresolved relationships that may benefit from expanded taxon sampling or incorporation of nuclear genomic data.

Figure 3.

Phylogenetic relationships among 19 species based on complete chloroplast genome sequences, reconstructed using both maximum likelihood (ML) and Bayesian inference (BI) methods. The phylogenetic tree includes pleioblastus amarus (MH988736) (Zhou et al. 2019), pseudosasa cantorii (MF066255) (Ma et al. 2017), bambusa oldhamii (FJ970915) and dendrocalamus latiflorus (FJ970916) (Wu et al. 2009) as representatives of the subfamily bambusoideae, and Oryza sativa (NC001320) (Hiratsuka et al. 1989) as the outgroup. The following phyllostachys species were included in the analysis: P. angusta (MW027348) (Zheng et al. 2021), P. edulis (HQ337796) (Zhang et al. 2011), P. edulis f. holochrysa (PP453781) (Liu et al. 2024), P. glauca (MT657329) (Cao et al. 2020), P. heteroclada f. solida (MW075109) (Hu et al. 2021). P. incarnata (OL457160) (Wang et al. 2023), P. lithophila (MZ662759)(This study), P. makinoi (MZ662758) (Kung and Chang 2024), P. nidularia f. farcta (LC590826) (Zhou et al. 2021), P. nigra var. henonis (HQ154129) (Zhang et al. 2011), P. praecox cv. prevernalis (OL335943) (unpublished), P. propinqua (JN415113) (Wu and Song, 2012), P. reticulata (MN537808) (Huang et al. 2019), and P. sulphurea (KJ722540) (Gao and Gao 2016). The numbers indicated at each node represent branch support values in the format ML/BI, where ML denotes the bootstrap support value from the maximum likelihood analysis, and BI indicates the posterior probability from Bayesian inference.

Although P. lithophila and P. makinoi share substantial morphological similarity and overlapping distributions in central Taiwan, our phylogenetic analyses clearly separated them into distinct clades. P. makinoi was recovered within a strongly supported lineage together with P. edulis f. holochrysa and P. nigra var. henonis (ML/BI = 100/1), confirming that morphological resemblance does not necessarily imply phylogenetic proximity. These results support the recognition of P. lithophila as a taxonomically distinct species and underscore the value of genome-scale data in resolving species boundaries within morphologically conservative bamboo taxa.

At the tribal level, all Phyllostachys species, including P. lithophila, were placed within a strongly supported clade corresponding to the temperate woody bamboo tribe Arundinarieae, together with Pseudosasa cantorii and Pleioblastus amarus. In contrast, the tropical genera Bambusa oldhamii and Dendrocalamus latiflorus were recovered in a separate clade representing the tribe Bambuseae. The clear phylogenetic separation between Arundinarieae and Bambuseae, each with maximal bootstrap and posterior probability support (ML/BI = 100/1), reflects a deep evolutionary divergence between temperate and tropical bamboo lineages.

Overall, the well-resolved placement of P. lithophila within Arundinarieae provides critical molecular evidence for its systematic distinctiveness and taxonomic recognition. These findings not only refine our understanding of phylogenetic relationships among temperate woody bamboos but also provide a valuable foundation for future taxonomic revisions, conservation strategies, and evolutionary studies of East Asian bamboos.

Funding Statement

This study was supported by the Taiwan Forestry Research Institute of Bamboo Forest Regeneration, Yield Enhancement Strategies, and Industry Diversification [114AS-7.4.3-FI-01]

Ethical approval

This article does not contain any involving human participants or animal’s studies.

Disclosure statement

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

Data availability statement

The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov/under the accession no.MZ662759. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA927338, SRR18091080, and SAMN25949384 respectively.