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Article

Insight into the Phylogenetic Relationships and Evolutionary History of Indocalamus (Bambusoideae) Through Comparative Analyses of Plastomes

by
Chengkun Wang
1,
Yonglong Li
1,
Ling Cui
1,
Jianqing Wang
1,
Meixia Wang
1,
Chunce Guo
1,
Guangyao Yang
1,
Liqin Gao
1,2,* and
Wengen Zhang
1,*
1
Jiangxi Provincial Key Laboratory of Improved Variety Breeding and Efficient Utilization of Native Tree Species, Forestry College, Jiangxi Agricultural University, Nanchang 330045, China
2
Jiangxi Academy of Forestry, Nanchang 330032, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(9), 1018; https://doi.org/10.3390/horticulturae11091018
Submission received: 9 June 2025 / Revised: 15 August 2025 / Accepted: 27 August 2025 / Published: 29 August 2025
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
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Abstract

Indocalamus Nakai, a genus within the tribe Arundinarieae, has significant horticultural and economic value. However, its classification has long been challenging, and due to limited sampling, both intra- and intergeneric phylogenetic relationships, as well as its evolutionary history, remain unclear. In this study, extensive field surveys and comprehensive sample collection were conducted to address these challenges. A total of 31 complete plastomes of Indocalamus species were assembled. All plastomes exhibit a typical quadripartite structure, ranging in length from 139,555 bp to 139,791 bp, and contain 137 genes, including 90 protein-coding genes, 39 tRNA genes, and 8 rRNA genes. Phylogenetic analyses indicate that Indocalamus is polyphyletic and divided into three distinct clades (IV, V, and X). Based on integrated phylogenomic and morphological evidence, we propose a revised classification of Indocalamus into three major sections. Fossil-calibrated divergence estimates reveal that the major clades of Indocalamus are not monophyletic, highlighting a complex reticulate evolutionary history exemplifying the widespread rapid radiation observed in temperate woody bamboos. The intensification of the East Asian monsoon is likely to have played a key role in driving the rapid radiation of these lineages. Additionally, several clade-specific DNA barcodes (trnT-trnE, petN-trnC, petA-psbJ, and petD-rpoA) were identified, which will enhance the identification of Indocalamus and its closely related genera. This study, through extensive sampling and integration of morphological and molecular phylogenetic evidence, provides a preliminary delimitation of the genus Indocalamus, elucidates its complex evolutionary history, and lays a solid foundation for future systematic research and horticultural applications.

1. Introduction

Woody bamboos of Bambusoideae (Poaceae), ca. 1358 species, including two tribes, Arundinarieae and Bambuseae, are notoriously difficult to identify and classify due to their extremely long and irregular flowering cycles, which can range from 10 to 120 years and, in some cases, even up to 150 years [1,2,3]. Given the rarity of flowering specimens, bamboo species identification has traditionally relied on vegetative characteristics. However, these traits often show high homogeneity and morphological plasticity, presenting significant challenges to taxonomy and systematics [4,5,6,7].
As a member of the tribe Arundinarieae, Indocalamus Nakai (1925), ca. 34 species, has been described under approximately 80 scientific names [8]. Except for one or two species found outside China, the vast majority of Indocalamus species are endemic to southern China, such as Sichuan, Guizhou, Guangxi, Guangdong, Hong Kong, Hunan, Hubei, Jiangxi, Hainan, and Fujian, typically at elevations between 300 and 2400 m in evergreen broad-leaved forests [9,10,11,12,13]. Indocalamus species are mid-size bamboo species, characterized by leptomorph rhizomes, solitary branches, persistent culm sheaths, relatively large leaves with distinct transverse veins, racemose or paniculate inflorescences, pedicellate spikelets, three stamens, and two stigmas [9,10,11,12,14,15]. However, fewer than one-quarter of the species have documented floral characteristics, and species identification is therefore mainly based on vegetative traits. It is also noteworthy that this genus shows significant similarity in rhizome type, branching pattern, and leaf morphology with Ferrocalamus Hsueh & Keng f. (1982), Sasa Makino & Shibata (1909), and Sinosasa L.C. Chia ex N.H. Xia et al. (2021), resulting in ongoing taxonomic ambiguity [16,17,18]. Initially, based on morphological data, Yang et al. (1990) [17] divided the genus into two sections according to whether the leaf blades exhibit wavy folds after drying and their altitudinal geographic distribution: Sect. Rugosi H.R. Zhao & Y.L. Yang, characterized by wavy leaf blades and generally found at higher elevations, and Sect. Indocalamus, comprising species with non-wavy leaf blades that are typically distributed at lower altitudes. However, due to the lack of molecular evidence, some morphologically similar species are often difficult to distinguish. Beyond its taxonomic significance, Indocalamus holds important horticultural and economic value. This genus is distinguished by its large leaves, compact and short stature, evergreen habit, and elegant form, which makes it valuable in horticulture, ecology, and landscaping. Some Indocalamus species exhibit strong cold tolerance and good resistance to pests and diseases [10]. Their large, evergreen leaves and good pruning tolerance make them especially suitable for landscaping in northern regions, where hardy and visually appealing bamboos are rare and highly valued. Additionally, the leaves of Indocalamus are rich in flavonoids, serving as a natural source for extraction, and are traditionally used for wrapping Zongzi, a Chinese festival food, reflecting both cultural and commercial importance. Ecologically, Indocalamus species commonly grow along forest margins and stream banks, playing a vital role in water conservation and soil erosion control, making them effective for slope stabilization.
Although previous phylogenetic studies have established a relatively robust framework for the tribe Arundinarieae [19,20,21,22,23,24,25,26], the relationships within Indocalamus and between Indocalamus and closely related genera remain unresolved due to limited sampling and insufficient molecular data. For example, Peng et al. (2008) analyzed GBSSI and ITS sequences and found that I. latifolius and I. longiauritus clustered near F. strictus and G. stellatus [4]; however, the bootstrap support was relatively low, making these relationships weakly supported. Later, Zhang et al. [26] conducted a more comprehensive phylogenetic analysis of Arundinarieae using GBSSI and seven chloroplast intergenic regions (rpl32-trnL, trnT-trnL, rps16-trnQ, rpoB-trnC, trnD-trnT, atpI-atpH, psaA-ORF170, and trnS-trnG), and found that 10 species of Indocalamus were distributed across four separate clades. Notably, I. wilsonii and I. sinicus each formed a distinct lineage, while other species were scattered among different clades and interspersed with species from closely related genera. These findings suggest that Indocalamus is not monophyletic. More recently, Guo et al. [27] supported this view based on complete plastome sequences from 17 species. However, phylogenies inferred from nuclear ribosomal DNA (nrDNA) data of 16 species and ddRAD sequencing data of 19 species conflicted with the plastome-based results, indicating discordance between nuclear and plastid evolutionary histories. Due to limited sampling, the relationships among all 34 recognized Indocalamus species remain unclear; a comprehensive, genus-wide sampling effort is therefore essential to resolve their phylogenetic placement within Arundinarieae [18,21,22,23,24,25,26,27].
As a relatively conserved organellar genome, the chloroplast genome has played a crucial role in resolving phylogenetic relationships among bamboos [6,21,22]. Although numerous bamboo plastomes have been reported, the genomic structure and diversification history of Indocalamus remain poorly understood. To address these gaps, we conducted extensive field surveys and collected samples representing all known Indocalamus species. Using high-throughput sequencing technologies, we successfully assembled 31 complete plastomes. Our research was driven by three primary objectives: (1) to clarify the phylogenetic relationships of Indocalamus, (2) to examine plastome structure and sequence variation, and (3) to reconstruct its evolutionary history. This study successfully assembled complete plastomes for all known species of Indocalamus, thereby significantly enriching its genomic resources. Through in-depth analyses of these plastomes and the identification of molecular markers, and in combination with morphological characteristics and nuclear gene data, we not only achieved a more precise resolution of the phylogenetic relationships between Indocalamus and its closely related genera, but also conducted a preliminary revision of the genus. These findings provide a solid foundation for future species identification, taxonomic treatment, population genetics, and evolutionary studies, while offering important guidance for the conservation, horticultural utilization, and sustainable management of high-quality Indocalamus germplasm resources.

2. Materials and Methods

2.1. Sampling, DNA Extraction, Sequencing, Assembly, and Annotation

To comprehensively investigate the phylogenetic relationships and evolutionary history of Indocalamus, field surveys and sampling were conducted at the type localities of 34 species. Fresh, pest- and disease-free young leaves were carefully collected from voucher specimens at these designated type localities. All the collected samples were then archived in the herbarium of Jiangxi Agricultural University, China (JXAU).
Total genomic DNA was extracted from silica gel-dried foliage leaves by using a modified cetyltrimethylammonium bromide (CTAB) method [28]. Illumina paired-end libraries were constructed and sequenced (Novogene Bioinformatics Technology Co., Ltd., Beijing, China), generating approximately 6 GB of raw data for each sample. To improve the accuracy of genome assembly, FastQC v0.11.9 and Fastp v0.12.4 [29] were applied with default parameters to filter out unpaired and low-quality reads, as these reads may introduce errors and biases during the assembly process.
Following quality control, the filtered reads were used for chloroplast genome assembly by using GetOrganelle v1.7.4 [30] with k-mers set at 45, 65, 85, 105, and 115. The Genome Graph Format (GFA) results generated by GetOrganelle v1.7.4 were then imported into Bandage v0.8.1 [31] to assess the integrity of chloroplast genome scaffold assembly and connections. Subsequently, the clean reads were mapped to the draft genome in Geneious 9.1.4 [32] to verify the concatenation of contigs. The resulting chloroplast genome sequences were annotated with CPGAVAS2 (http://47.96.249.172:16019/analyzer/home, accessed on 26 August 2025) [33], followed by manual curation in Geneious. Finally, the complete chloroplast genomes were visualized using Chloroplot v0.2.4 [34].

2.2. Phylogenetic Analyses

To rebuild the phylogenetic framework of Indocalamus and refine the understanding of its evolutionary history, plastome sequences from 34 species of the genus were analyzed. Three species (I. jingpingensis, I. latifolius, and I. pedulis) were sourced from the dataset of Guo et al. [27]. Three outgroup species were selected to represent two sister clades of the Arundinarieae tribe: Bambusa multiplex, Dendrocalamus latiflorus (Bambuseae tribe), and Olyra latifolia (Olyreae tribe). An additional 43 representative plastomes from 24 genera across 12 major clades of the tribe Arundinarieae were included to ensure comprehensive taxonomic coverage. All sequences were retrieved from the NCBI GenBank database and Guo et al.’s dataset [27] (Supplementary Table S1).
Maximum Likelihood (ML) and Bayesian Inference (BI) approaches were used for phylogenetic reconstruction. Chloroplast genomes were aligned by using MAFFT v7.450 [35] and filtered with trimAl v1.4 [36]. ML analyses were conducted in IQ-TREE v2.2.0 [37] with 5000 ultrafast bootstrap replicates under the GTR + F + I + G4 model, selected by ModelFinder [38]. BI analyses were performed by using MrBayes v3.2.6 [39] with the same model. Markov Chain Monte Carlo (MCMC) simulations were run for 20 million generations with sampling every 1000 generations. Convergence was assessed based on the average standard deviation of split frequencies (ASDF < 0.01), and the first 25% of trees were discarded as burn-in.

2.3. Estimation of Divergence Time

Divergence times within Indocalamus were inferred using BEAST v2.6.3 [40] following a calibration scheme adapted from Wang et al. [41]. Four calibration points were applied: (1) Bambusoideae cf. Chusquea (35–90 Mya; Strömberg, 2011 [42]) for the crown Bambusoideae; (2) Arundinarieae crown (6.88–20.96 Mya, median 12.72 Mya); (3) Clade IV crown (2.16–6.58 Mya, median 4.01 Mya); and (4) Clade V crown (1.24–3.82 Mya, median 2.38 Mya). Calibrations were implemented as uniform distributions. Analyses utilized a Yule process tree prior, an uncorrelated lognormal relaxed clock, and the GTR substitution model. Markov Chain Monte Carlo (MCMC) simulations were run for 250 million generations, sampling every 1000 generations. Convergence was evaluated in Tracer v1.7.2 (ESS > 200). Median node ages were summarized with TreeAnnotator v2.6.6. Diversification patterns were visualized with lineage-through-time (LTT) plots using the R package ape v5.8, based on the maximum clade credibility tree and 1000 posterior samples.

2.4. Plastome Structure Comparison and Sequence Divergence Analyses

To assess structural variation in Indocalamus plastomes, genome size, gene content, and GC content were calculated in Geneious 9.1.4 [32]. Genome sequences were aligned using MAFFT v7.450. Structural rearrangements and inversions were identified using Mauve v20150266. Expansion and contraction at inverted repeat (IR) junctions were visualized with CPJSdraw v0.01 [43]. Nucleotide diversity (pi) was analyzed with DnaSP v5.10.01 [44] using a sliding window approach to detect highly variable regions.

2.5. Codon Usage Analyses

Codon usage patterns were investigated by extracting protein-coding genes using PhyloSuite v1.2.2 [45]. Relative Synonymous Codon Usage (RSCU) values were calculated with CodonW v1.4.4 [46]. Heatmaps were generated to visualize codon usage patterns using TBtools v2.224 [47].

2.6. Repeats and SSR Analyses

Simple sequence repeats (SSRs) were identified using MISA-web (https://webblast.ipk-gatersleben.de/misa/, accessed on 26 August 2025) [48], with the following minimum thresholds: 10 for mononucleotides, 5 for dinucleotides, 4 for trinucleotides, and 3 for tetra- to hexanucleotides. Dispersed repeats (forward, reverse, palindromic, and complementary) were detected using the web-based tool REPuter (https://bibiserv.cebitec.uni-bielefeld.de/reputer, accessed on 26 August 2025) [49] with parameters set to a minimum repeat size of 30 bp, Hamming distance of 3, and a maximum of 80 computed repeats.

3. Results

3.1. Sampling, Sequencing, and Assembly

High-throughput sequencing of 31 Indocalamus samples yielded 20–60 million paired-end reads per sample. After quality control, retained high-quality data enabled de novo assembly of 31 complete chloroplast genomes across all species. As shown in Figure 1, all Indocalamus species examined exhibited a typical quadripartite structure of angiosperm plastomes, consisting of a large single-copy (LSC) region, a small single-copy (SSC) region, and two inverted repeat regions (IRA and IRB). Genome sizes ranged narrowly from 139,555 (I. tessellatus) to 139,791 (I. pseudosinicus) bp, with a consistent GC content of 38.9%. The LSC region spanned 83,074 bp (I. confertus) to 83,635 bp (I. suichuanensis), while the SSC region varied between 12,384 bp (I. sinicus) and 12,912 bp (I. hunanensis). Inverted repeat lengths showed greater variation, from 21,788 bp (I. pseudosinicus) to 21,835 bp (I. inaequilaterus). All plastomes were annotated to contain 137 genes, including 90 protein-coding sequences (CDS), 39 transfer RNAs (tRNA), and 8 ribosomal RNAs (rRNA). Detailed genomic features are provided in Supplementary Tables S1–S3.

3.2. Phylogenetic Reconstruction

Chloroplast genome-based phylogenetic analysis reconstructed the fundamental evolutionary framework of bamboo taxa (Figure 2), with herbaceous Olyra and tropical woody (Bambusa and Dendrocalamus) forming strongly supported sister clades to Arundinarieae (bootstrap support BSML = 100%, posterior probability PPBI = 1.00). Within Arundinarieae, all 12 major clades were resolved with high confidence. Thirty-four species of Indocalamus were distributed across three clades: Clade X contained only one species, i.e., I. sinicus; Clade V included twenty-seven Indocalamus species interspersed with Gelidocalamus, Phyllostachys and Yushania, including twelve species (I. macrophyllus, I. lacunosus, I. chebalingensis, etc.) clustering with G. rutilans, G. latifolius, and G. stellatus, four species (I. hispidus, I. pedulis, I. youxiuensis and I. longiauritus) with G. longiinternodus (BSML = 100%, PPBI = 1.00), and five species (I. emeiensis, I. auriculatus, I. hunanensis, I. confertus and I. wuxiensis) with Bashania fargesii. The remaining Indocalamus species in Clade V exhibited intergeneric relationships with Phyllostachys, Chimonobambusa, or Yushania. Clade IV grouped six Indocalamus species with Gelidocalamus, Ferrocalamus, and Shibataea, including I. inaequilaterus, I. tongchunensis, I. amplexicaulis, I. babartus, and I. pseudosinicus clustering with Gelidocalamus (BSML = 100%, PPBI = 1.00), and I. jingpingensis forming a sister relationship with F. rimosivaginus (being closely related to Shibataea kumasaca; BSML = 100%, PPBI = 1.00). Notably, Indocalamus and Gelidocalamus consistently exhibited close phylogenetic affinity and congruent species distribution patterns across multiple clades.

3.3. Divergence Time Estimation

Divergence time analysis (Figure 3) indicates that the stem lineage of Arundinarieae originated in the Late Eocene (about 36.33 Mya; 95% HPD: 35.00–40.78 Mya), with crown group diversification occurring later in the mid-Miocene (about 13.69 Mya; 95% HPD: 10.00–18.42 Mya). Within this temporal framework, divergence patterns in traditional Indocalamus reveal that the genus is polyphyletic, comprising several independently originated lineages. Clade X originated in the mid to late Miocene (about 9.05 Mya; 95% HPD: 7.34–11.64 Mya), Clade IV may have emerged in the late Miocene to early Pliocene (about 6.29 Mya; 95% HPD: 5.49–6.58 Mya), while Clade V diverged in the late Pliocene to early Pleistocene (about 2.97 Mya; 95% HPD: 2.34–3.77 Mya).
All extant Indocalamus species are distributed across three distinct clades with markedly different divergence times. Clade X contains only I. sinicus, which originated in the late Miocene and has remained a single lineage without further diversification, representing an early-diverging lineage within the genus. Clade IV includes six Indocalamus species. I. jingpingensis split from its common ancestor with Ferrocalamus rimosivaginus in the mid-Pliocene (about 4.43 Mya; 95% HPD: 3.33–5.28 Mya). This was followed by a gradual divergence spanning approximately 3.60 million years, culminating in the separation of I. tongchunensis from the common ancestor of G. dongdingensis around 0.83 Mya (95% HPD: 0.36–1.43 Mya). Clade V, which harbors the largest number of Indocalamus species, began diverging more recently, with I. dayongensis splitting from the Indocalamus-Yushania common ancestor at about 1.37 Mya (95% HPD: 0.82–2.01 Mya), followed by rapid diversification within 1.25 million years, and I. confertus and I. wuxiensis diverging from their common ancestor as recently as 0.12 Mya (95% HPD: 0.02–0.29 Mya).
Overall, members of Clade IV diverged earlier than those of Clade V, whereas both clades split much later than I. sinicus of Clade X. These contrasting divergence times across the three clades highlight the complex, polyphyletic origin of the genus and the distinct evolutionary trajectories of its constituent lineages.
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Figure 2. Phylogenetic tree of Indocalamus using maximum likelihood (ML) and Bayesian inference (BI) methods based on plastomes. ML tree topology is shown with ML bootstrap values, and BI posterior probabilities are indicated on the nodes. Only bootstrap values (BS) ≥ 75% and posterior probabilities (PP) ≥ 0.75 are indicated at each node; otherwise, dashes are used. The asterisk (*) indicates support of 100% BS or 1.00 PP. V–XI indicate different clades of tribe Arundinarieae based on plastome data. The characteristics of the culms, branches, and culm sheaths, along with the details of the oral setae of the culm sheaths, are shown in panels (A–I) for representative species from three clades of Indocalamus. (A) I. auriculatus, (B) I. wuxiensis, (C) I. decorus, (D) I. dayongensis, (E) I. tessellatus, (F) I. sinicus, (G) I. inaequilaterus, (H) I. tongchunensis, (I) I. babartus. Scale bars = 1 cm.
Figure 2. Phylogenetic tree of Indocalamus using maximum likelihood (ML) and Bayesian inference (BI) methods based on plastomes. ML tree topology is shown with ML bootstrap values, and BI posterior probabilities are indicated on the nodes. Only bootstrap values (BS) ≥ 75% and posterior probabilities (PP) ≥ 0.75 are indicated at each node; otherwise, dashes are used. The asterisk (*) indicates support of 100% BS or 1.00 PP. V–XI indicate different clades of tribe Arundinarieae based on plastome data. The characteristics of the culms, branches, and culm sheaths, along with the details of the oral setae of the culm sheaths, are shown in panels (A–I) for representative species from three clades of Indocalamus. (A) I. auriculatus, (B) I. wuxiensis, (C) I. decorus, (D) I. dayongensis, (E) I. tessellatus, (F) I. sinicus, (G) I. inaequilaterus, (H) I. tongchunensis, (I) I. babartus. Scale bars = 1 cm.
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Figure 3. Divergence times of major clades in traditional Indocalamus. The chronogram of Indocalamus and relatives is based on BEAST analysis. (A) Lineage-through-time plot, the red plot represents the maximum clade credibility tree, and the gray plots denote 1000 trees. (B) Four calibration points are marked by black circles. Blue characters and shades of light blue indicate the approximate periods of two intensifications of the East Asian monsoon climate. Node bars represent the 95% highest posterior density intervals for node ages, with mean ages of some important nodes in Arundinarieae indicated (node numbers represent the estimated divergence times in million years).
Figure 3. Divergence times of major clades in traditional Indocalamus. The chronogram of Indocalamus and relatives is based on BEAST analysis. (A) Lineage-through-time plot, the red plot represents the maximum clade credibility tree, and the gray plots denote 1000 trees. (B) Four calibration points are marked by black circles. Blue characters and shades of light blue indicate the approximate periods of two intensifications of the East Asian monsoon climate. Node bars represent the 95% highest posterior density intervals for node ages, with mean ages of some important nodes in Arundinarieae indicated (node numbers represent the estimated divergence times in million years).
Horticulturae 11 01018 g003

3.4. Genome Structural Variations and Sequence Divergences

Due to the polyphyletic composition of the genus Indocalamus, with its species spread across three clades and interwoven with closely related genera forming Clades IV and V, this study conducted a comparative analysis of the chloroplast genomes of Indocalamus and its close relatives within these clades, exploring their structure and evolutionary relationships.
By using the program of Mauve, a whole-genome collinearity analysis of 61 chloroplast genomes was performed, and showed that high synteny was observed within and between the clades, with no rearrangements or inversions detected (Supplementary Figure S1). The analysis of IR boundary expansion and contraction revealed no significant large-scale structural changes among the three clades. At the junction of LSC/IRb, the genes rpl22 and rps19 were located near the boundary; rps15 and ndhF were situated at the junction of IRb/SSC, while ndhH spanned the boundary of SSC/IRa. Notably, in I. latifolius, the insertion of seven bases (CTCCCTC) within the ndhH gene led to a premature stop codon, converting it into a pseudogene. Comparative analysis of boundary dynamics revealed a clade-specific insertion event at the LSC/IRb junction, involving the sequence “GGTTATTCCCCG.” (Figure 4) This insertion likely originated in the common ancestor of Clade V and was later partially lost in a sublineage comprising 15 species of Gelidocalamus and Indocalamus, with the sequence gradually shortened to “GGTTATTCCA” or “GGTTATTCC.” In Phyllostachys edulis, it was entirely lost. Interestingly, this insertion was not detected in Clades I, IV, or other related lineages. (Figure 4, Figures S2 and S3).
Given the distribution of Indocalamus species across Clades IV, V, and X, nucleotide diversity (Pi) was assessed both within and among these clades, based on their phylogenetic relationships (Figure 5). The analysis focused on Clade IV, the combined clades V + X, and the broader group IV + V + X. In Clade IV, Pi values ranged from 0 to 0.0064, with an average of 0.0017. In clades V + X, the range was 0 to 0.0052, with an average of 0.0006. Across all three clades (IV + V + X), Pi values extended from 0 to 0.0074, averaging 0.0014. A total of 53 highly variable sites were identified in Clade IV, compared to 9 in clades V + X and 28 in the combined clades. As shown in Figure 5, the 53 sites in Clade IV were distributed across 22 genes and intergenic regions, with eight sites exhibiting Pi values greater than 0.005. The nine highly variable sites in clades V + X were all located in three intergenic regions. In the IV + V + X group, 28 sites were found spanning nine genes and intergenic regions. Overall, Clade IV exhibited markedly higher nucleotide variability than clades V + X. Notably, all three highly variable regions identified in clades V + X were shared across the three clades.

3.5. Codon Usage Bias

Codon usage bias analysis revealed that Indocalamus species and their closely related genera across the three clades exhibited similar codon usage preferences (Figure 6). Specifically, the number of preferred codons (RSCU > 1) was consistently 29 across all clade members, while codons with RSCU = 1 were typically 2 or 3. Notably, in Drepanostachyum semiorbiculatum, three codons showed no usage bias, with 29 or 30 codons displaying low usage preference (RSCU < 1). Among the 29 preferred codons, only two did not end with A or U. In terms of total codon counts, species in Clade V ranged from 20,531 in I. latifolius to 20,880 in Chimonobambusa angustifolia, with an average of 20,858.4 codons. Clade X included 20,872 codons in I. sinicus. In Clade IV, codon counts ranged from 20,777 in Ferrocalamus rimosivaginus to 20,880 in Shibataea chiangshanensis. Within Indocalamus, I. latifolius had the fewest codons (20,531), while I. pseudosinicus had the most (20,875). Among all amino acids, leucine had the highest usage frequency (2251–2268 codons), whereas cysteine had the lowest (227–233 codons).

3.6. SSRs and Long Repeats Analysis

A total of 3208 simple sequence repeats (SSRs) and 4102 long repetitive sequences were identified across 61 species within the three clades of Indocalamus (Figure 7). Regarding SSRs, Clade V had a total of 2175 SSRs, averaging 50.58 SSRs per species, with a range of 45–56 SSRs (from A. melicoideus to G. kunsishii). Clade IV had 971 SSRs, with an average of 57.12 SSRs, ranging from 46 to 65 SSRs (from I. barbatus to G. multifolius). Clade X contained only a single species, I. sinicus, in which 62 SSRs were identified. As shown in Figure 7, four types of SSRs—mono-, di-, tri-, and tetranucleotide repeats were detected across 61 species in the three clades. However, pentanucleotide SSRs were not detected in nine species, such as G. annulatus, I. victorialis, I. sinicus, and I. inaequilaterus. Hexanucleotide SSRs were only detected in eight species, including G. longiinternodus, I. youxiuensis, I. longiauritus, and I. tongchunensis. These SSRs showed high similarity among the three clades. Among 1854 mononucleotide repeats, adenine (A) and thymine (T) nucleotides dominated, collectively accounting for 96.7% of the total, while guanine (G) and cytosine (C) nucleotides represented a mere 2.33%. In the 278 dinucleotide repeats, the most common types were AT/TA/TC, with TA (56.1%) being the most frequent. In the case of trinucleotide repeats, AAT, TAT, and TCT were predominant, each contributing approximately one-third (31.9%, 31.9%, and 31.4%, respectively). Among 816 tetranucleotide repeats of 15 types, AAAT was the most frequent (13.8%). For the seven types of pentanucleotide repeats, TTTTA appeared most frequently (74.6%). As for hexanucleotide repeats, only five were found in five species (G. longiinternodus, I. youxiuensis, I. longiauritus, D. semiorbiculatum, and F. rimosivaginus), namely: AAGGGG (1), ACTTCT (1), and TATTAG (3). These SSRs were also highly consistent in their distribution within the chloroplast genome. Across the LSC/SSC/IR regions, the majority (78.9%) were located in the LSC region, with similar proportions in the SSC and IR regions (approximately 10.7%). In terms of genomic location, 57.4% of SSRs were found in intergenic spacer (IGS) regions, 23.8% in CDS regions, and 18.8% in intron regions. Additionally, a total of 4102 long repeat sequences were identified among the three clades. Clade V contained 2808, averaging 65.30 repeats per species. Clade X had 68 repeats, while Clade IV had 1226, with an average of 72.11 repeats. In terms of their distribution, most long repeats in Clade V were located in the LSC region (71.4%). The majority were found in CDS regions (62.8%), which was also the case for Clades X and IV (Supplementary Figures S4–S8).

4. Discussion

4.1. Re-Evaluating the Circumscription of Indocalamus

In previous studies of temperate woody bamboos (trib. Arundinarieae), phylogenetic frameworks were primarily constructed by using chloroplast DNA fragments, complete plastid genomes, or a limited number of molecular markers [19,20,21,22]. These approaches led to the recognition of 12 lineages within the Arundinarieae [19,20,21,22,23,24,25,26]. However, with the advent of next-generation sequencing technologies (such as RAD-seq), recent studies have brought new insights into the phylogenetic relationships within the group. For instance, based on ddRAD data, Zhang et al. redefined the Arundinarieae into five lineages supported by morphological evidence, and proposed the new genus Ravenochloa to accommodate Indocalamus wilsonii as its basionym and type species within the leptomorph lineage [26,27]. These findings highlight significant discrepancies between nuclear- and plastid-based phylogenies, underscoring that the rational use of diverse lines of evidence can better support revisions at the genus level.
Against this backdrop, Indocalamus, characterized by high morphological diversity, has been underrepresented in previous phylogenetic studies, leaving its evolutionary relationships poorly resolved. In the study, a comprehensive sampling of Indocalamus and its close relatives was conducted, and 77 whole cp genomes (including 34 species of Indocalamus) were analyzed to reconstruct their phylogenetic relationships. Our results strongly support the twelve-lineage framework of the Arundinarieae. Specifically, Indocalamus is highly polyphyletic, with species distributed across Clades IV, V, and X, a pattern that parallels the complex distribution observed in its close relative Gelidocalamus [27]. Notably, Clade X includes only I. sinicus, while most Indocalamus species fall within Clade V alongside Gelidocalamus, Yushania, and Bashania. A few species are also placed within Clade IV, alongside Gelidocalamus and Ferrocalamus. Compared to results based on ddRAD data of Guo et al. [27], I. sinicus in Clade X occupies a basal position relative to all other species of Indocalamus and its closely related genera. Within Clade V, Indocalamus species continue to form an internal clade together with related genera, while those in Clade IV similarly constitute a relatively independent subclade.
Based on the phylogenetic analysis of Guo et al. [27], 19 Indocalamus species were grouped into three clades. The clustering patterns of these clades are congruent with those from plastid genome phylogenies, providing strong support for our taxonomic delimitation. Integrating extensive morphological data, such as the number of leaves per branch, coloration of the abaxial leaf surface, branching pattern, culm sheath color, and altitudinal distribution, together with plastid genome results and with reference to ddRAD-based phylogenetic clustering, we propose a revised circumscription of Indocalamus, provisionally divided into three major clades corresponding to three sections, which includes Indocalamus Sect. I Rugosi H.R. Zhao & Y.L. Yang; Indocalamus Sect. II Indocalamus; and Indocalamus Sect. III Tessellatus L.Q. Gao, W.G. Zhang & G.Y. Yang, Sect. nov. (Supplementary Figures S9 and S10). Notably, based on plastid genome data and the findings of Guo et al. [27] six species clustered in Clade IV (Supplementary Figure S9) are proposed for exclusion from the genus Indocalamus, as this clade appears to represent an independently originated lineage closely related to genera such as Gelidocalamus and Ferrocalamus, although further morphological and nuclear data are needed to confirm their placement. Due to the current lack of morphological data for related external groups, the precise taxonomic placement of these species requires further study. Moreover, intergeneric relationships reveal that several Gelidocalamus species consistently cluster with Indocalamus. For example, G. longiinternodus exhibits key diagnostic traits characteristic of Indocalamus, including single-branch growth on one-year-old culms and spring sprouting. Based on these observations, Li et al. [50] proposed the concept of a “core Gelidocalamus,” suggesting that only a few species should be retained in that genus, while the rest would be better reassigned to Indocalamus [40,50]. For instance, species such as G. longiinternodus, G. kunsishii, G. rutilans, G. solidus, and G. subsolidus cluster together with Indocalamus species based on both plastid genome and ddRAD data, rather than grouping with the core Gelidocalamus clade. Therefore, further sampling and data collection are necessary to clarify whether these species should be formally included within the circumscription of Indocalamus.
Although plastid genome results strongly support previous classification frameworks, significant nuclear-plastid conflicts remain. In particular, within the three proposed sections, species in Clade V, including Indocalamus Sect. Rugosi and Sect. Tessellatus, show intermingled relationships. Similar patterns have been frequently observed in related groups such as Gelidocalamus and Sasa, indicating the limitations of plastid data in resolving complex evolutionary histories influenced by rapid radiation and chloroplast capture. Nuclear gene or genome data may better address these issues. Based on Guo et al.’s study [27], although only about two-thirds of Indocalamus species were included, their results largely support our morphology-based delimitation. Combining plastid genome, ddRAD, and morphological data, our study clarifies the distribution patterns of Indocalamus and the congruence between different datasets and morphology, providing a comprehensive discussion to better resolve its complex taxonomy. Unfortunately, comprehensive nuclear gene data across the entire genus are still lacking, and for some groups, only plastid genomes are available. Therefore, building on previous studies [17] and combining morphological and molecular phylogenetic evidence, we provide a preliminary discussion on the delimitation of genus boundaries.
(1) Indocalamus Sect. I Rugosi H.R. Zhao & Y.L. Yang (Supplementary Figure S11)
Lectotype: Indocalamus hispidus H.R. Zhao & Y.L. Yang.
Description: Distributed at higher altitudes, typically in mountainous regions above 1000 m elevation. Shoot sheaths are purple with yellow patches or green with purple patches, except in I. hunanensis. The abaxial surface of the leaf blade is grayish-white, distinctly different from the adaxial surface. Based on altitude distribution and the wrinkling of dried leaves, Zhao et al. established the Rugosi section, which includes three species: I. wilsonii (now transferred to the genus Ravenochloa as Ravenochloa wilsonii), I. auriculatus, and I. hispidus, with I. wilsonii as the type species. Species in this section share distinctive traits such as leaves exhibiting wavy wrinkles upon drying. They predominantly occur in mountainous regions above 1000 m. However, I. wilsonii has been reassigned to Ravenochloa due to its unique phylogenetic position, multiple lines of evidence—including abaxial leaf epidermal micromorphology, population-level vegetative traits, altitudinal distribution, and molecular phylogenetic data—support recognizing Rugosi as an independent taxonomic section comprising 12 species, with I. hispidus as the neotype species. The incomplete monophyly observed in phylogenetic trees may be due to the high conservation of chloroplast genomes, complex allopolyploid origins in temperate bamboos, and a likely recent radiation history.
(2) Indocalamus Sect. II Indocalamus (Supplementary Figure S12)
Type: Indocalamus sinicus (Hance) Nakai.
Description: Culm internodes glossy and glabrous, with flat nodes. Culm sheaths sparsely covered with white or colorless hairs, deciduous. Leaves 8–11 (occasionally up to 18) per small branch; secondary veins 8–9 pairs. As the neotype species of Indocalamus, it shares solitary branches and large leaves with congeners but is readily distinguished by the unusually high number of leaves per small branch. Morphological stability across populations, together with phylogenetic evidence from complete chloroplast genomes, ddRAD data, and nuclear genes (e.g., GBSSI), consistently indicates that it is phylogenetically distant from other Indocalamus lineages, representing an independent evolutionary clade. It is therefore recognized here as a new section containing only this species. Since this species serves as the lectotype of Indocalamus, under a strict definition, the genus Indocalamus would include only this single species. Considering that complete evidence for the other two groups—such as nuclear genomic data for all species and more detailed characters like leaf epidermal micromorphology—is still lacking, we provisionally distinguish the broadly defined Indocalamus according to these sections.
(3) Indocalamus Sect. III Tessellatus L.Q. Gao, W.G. Zhang & G.Y. Yang, Sect. nov. (Supplementary Figure S13)
Type: Indocalamus tessellatus (Munro) P. C. Keng
Description: Generally distributed at altitudes below 1000 m, these species have fresh shoot sheaths that are purple and lack patches, with textures varying from bristly and puberulous to glabrous. Their leaf blades usually display similar coloration on both adaxial and abaxial surfaces, although some bear fine hairs on the abaxial side or along both sides of the midrib. Fresh culm sheaths consistently lack patches and tend to be bristly, puberulous, or glabrous on the back. Based on stable morphological traits observed across populations, geographic distribution, and molecular phylogenetic analyses, these species predominantly cluster within Clade V according to chloroplast genome data; furthermore, ddRAD data indicate that several species also group within a single clade; therefore, combined with morphological and molecular evidence, we consider these groups to warrant recognition as an independent taxonomic section. Currently, this section includes approximately 15 species.

4.2. Rapid Adaptive Radiation Evolution of Indocalamus and Its Related Genera

Rapid radiation is considered a key factor contributing to the reticulate evolutionary patterns observed in temperate woody bamboos. To investigate the evolutionary history of Indocalamus, we estimated the divergence times of Arundinarieae based on complete chloroplast genome data. Using one reliable fossil calibration point and three secondary calibration points, we inferred the divergence times of Indocalamus and its closely related genera [19,22]. The results revealed that the crown group emerged in the middle Miocene (around 13.96 Mya). The three clades containing Indocalamus crown diversification occurred at around 9.05 Mya, 6.29 Mya, and 2.47 Mya, respectively. The lineage-through-time (LTT) plot shows two turning points in lineage accumulation, occurring around 10 Mya and 6 Mya, which correspond closely with the divergence times of several major clades. In evolutionary studies of Arundinarieae, rapid radiation has been widely recognized, with many genera such as Gelidocalamus, Sasa, Chimonocalamus, and Shibataea reported to exhibit complex polyphyletic origins [18,22,27,41,51]. Indocalamus is no exception; it includes 34 extant species scattered across three main clades, within which species remain intermingled with close relatives. The divergence times of these three clades further support that Indocalamus is a complex polyphyletic group with extensive distribution and pronounced differentiation, representing the most striking case of polyphyly within Arundinarieae. Besides the impact of rapid radiation, erroneous taxonomic assignments may also contribute significantly to the present complex phylogenetic patterns. Based on morphological studies, we have redefined the generic boundaries of Indocalamus. All species of the revised Indocalamus (except those in Indocalamus Sect. Indocalamus) are distributed within Clade V and remain intermingled with closely related genera. This pattern likely results from recent rapid radiation, which has driven the reticulate evolution observed in this group.
Building upon this, Arundinarieae is recognized as a complex group that underwent rapid radiation [22,27,41]. Paleoclimatic fluctuations, especially the intensified East Asian monsoon around 15.5–13 Mya and 8–7 Mya [52,53,54,55,56,57,58], likely promoted rapid diversification and expansion of temperate woody bamboos. Morphological diversification of rhizome types (pachymorph and leptomorph) provided ecological advantages, enabling adaptation to varied habitats and facilitating niche occupation during the rainy season. Although rapid radiation caused swift species divergence with notable morphological differences, genetic divergence remained limited [23,27,59]. Additionally, incomplete lineage sorting, gene flow, and frequent hybridization due to overlapping habitats contributed to reticulate evolution. Similarly, Indocalamus and its relatives likely experienced rapid diversification in a short evolutionary window, resulting in discordance between morphology-based classification and molecular phylogeny [27]. Shared habitat preferences (e.g., understory or riparian zones) further complicated species boundaries. Based on previous studies, this research reestimated divergence times of all known Indocalamus species and related genera using plastome data, offering valuable phylogenetic insights.

4.3. Evolution of the Plastomes and DNA Barcode Identification in Indocalamus

Chloroplasts are essential for photosynthesis and plant growth. Like mitochondria, they have their genomes, which are maternally inherited and highly conserved. Most plastomes have a quadripartite structure, consisting of two inverted repeats (IRs), a large single-copy (LSC) region, and a small single-copy (SSC) region. Due to the stable inheritance, moderate size, and high copy number, the chloroplast genome is a valuable molecular marker for phylogenetic analysis, species identification, and population genetics [60,61,62,63]. Numerous studies have characterized the plastome structure and evolutionary patterns in bamboos. However, little is known about the chloroplast genome evolution of Indocalamus. In this study, we assembled and compared the complete plastomes of all known Indocalamus species, conducting comparative analyses of representative taxa from three major lineages. All plastomes exhibited typical quadripartite structures, with high consistency in genome size, gene content, GC content, and synteny within and between genera. The three lineages were nested among closely related genera and showed strong similarity in repeat sequences. No large-scale rearrangements or inversions were detected. Lineage-specific indels were identified at the IR boundaries, including two insertions/deletions and one species-specific insertion. Notably, a 13-bp insertion (“GGTTATTCCCCG”) at the junction of LSC/IRb showed varying evolutionary fates, such as partial or complete loss in different taxa. Similarly, a 7-bp insertion (“CTCCCTC”) in the ndhH gene of I. latifolius introduced a premature stop codon, rendering the gene a pseudogene.
Repeats and SSRs (simple sequence repeats) were more abundant in Clade IV than in Clade V, suggesting higher variability among the younger lineages. Codon usage bias analysis revealed 29 preferred codons, most of which ended in A/U, consistent with other temperate woody bamboos [64,65]. These findings reflect strong conservation of chloroplast codon usage and support the hypothesis of lineage-specific evolutionary patterns in Indocalamus.
DNA barcoding, based on specific DNA fragments, is valuable in bamboo identification due to its standardized sequences, high variability, suitable fragment length, broad applicability, and ease of use [65,66]. However, commonly used plant barcodes, such as rbcL, matK, and ITS, have shown limited effectiveness in identifying bamboo species [66,67,68,69,70,71,72]. For example, Cai et al. (2012) [66] analyzed 27 representative species of the Arundinarieae tribe and found that the discrimination rates for chloroplast fragments were less than 12% at the genus and species levels. The combined use of multiple fragments resulted in a maximum identification rate of only 15% [66]. Although ITS had higher identification rates, its poor universality makes it insufficient for accurate bamboo species identification. To improve the identification efficiency, some researchers have attempted to screen variable fragments from the chloroplast genomes of Bambusoideae as candidate barcodes. For instance, Zhang et al. [67]. found that trnG-trnT had the most variable sites and recommended it as a preferred barcode. Additionally, Lv et al. [72] compared species identification rates, finding that the chloroplast genome (28.6%) outperformed standard barcodes (5.7%), and nrDNA (65.4%) had better discrimination than ITS (47.2%). This suggests that the nuclear genome offers better discrimination than the plastid genome. However, due to factors like rapid radiation, frequent hybridization, and slow evolutionary rates in bamboo, accurate identification of all species remains challenging.
Several highly variable regions were identified from the chloroplast genomes of three Indocalamus lineages, including trnG-trnT, trnL-trnF, rbcL-accD, petA-psbJ, and ndhF-rpl32. Notably, trnG-trnT, rbcL-accD, and ndhF showed high variability, indicating their suitability across multiple lineages. In contrast, conventional markers such as psbA-trnH, matK, and rps16 exhibited lower discriminatory ability. Lineage-specific highly variable regions, such as trnT-trnE, petN-trnC, petA-psbJ, and petD-rpoA, were identified in lineage IV. These regions may serve as effective markers for species identification within this lineage, though their utility across all lineages may be limited. Overall, higher variability was observed in lineage IV compared to the V + X lineage (Figure 5), suggesting greater potential for marker development. Although trnG-trnT, trnL-trnF, and rbcL-related regions were found to be more suitable for species identification across the three lineages, the resolution provided by individual fragments remains insufficient. To achieve more efficient and accurate species discrimination in bamboo, a multilocus approach combining chloroplast markers with nrDNA or nuclear single-copy gene data is required.

5. Conclusions

This study comprehensively sampled all 31 species of Indocalamus and successfully obtained their chloroplast genomes via high-throughput sequencing, including 17 newly sequenced genomes. Comparative analyses demonstrated that the chloroplast genomes of Indocalamus are highly conserved in terms of genome size, gene content, and gene order, with only minor variations detected at SNP and SSR loci, and no major rearrangements or inversions identified. Phylogenetic reconstruction indicated that Indocalamus is a polyphyletic genus, with species distributed among three distinct clades and interspersed with closely related genera. Based on morphological data combined with chloroplast genome and nuclear gene analyses, this study redefined the generic boundaries of Indocalamus, proposing its division into three principal sections: Indocalamus Sect. I Rugosi H.R. Zhao & Y.L. Yang; Indocalamus Sect. II Indocalamus H.R. Zhao & Y.L. Yang; and Indocalamus Sect. III Tessellatus L.Q. Gao, W.G. Zhang & G.Y. Yang, Sect. nov. Nevertheless, owing to the current lack of comprehensive nuclear gene data covering the entire genus, further revisions incorporating additional morphological characters and nuclear single-copy gene data will be conducted in subsequent studies. DNA barcoding analyses identified several hypervariable regions with clade-specific utility, facilitating rapid species identification within clades. Collectively, the chloroplast genome data elucidate the phylogenetic relationships and evolutionary history of Indocalamus, thereby providing a robust molecular basis for species identification, conservation efforts, and future horticultural development.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11091018/s1, Supplementary Table S1: Taxa, accession number, and source information used in the study.; Supplementary Table S2: Basic information of chloroplast genomes.; Supplementary Table S3: Functional annotation of chloroplast genes.; Supplementary Table S4: List of estimated divergence times in major nodes.; Supplementary Figure S1: The collinearity among the chloroplast genomes of the three clades.; Supplementary Figure S2: The insertion sequence “GGTTATTCCCCG” located at the junction of LSC/IRb.; Supplementary Figure S3: The insertion sequence “CTCCCTC” located at the junction of LSC/IRb.; Supplementary Figure S4: Number of SSRs in LSC, SSC, and IR regions.; Supplementary Figure S5: Number of SSRs in the coding regions (CDS), intergenic region (IGS), and introns.; Supplementary Figure S6: Number of forward, palindromic, and reverse repeats.; Supplementary Figure S7: Number of long repeats in LSC, SSC, and IR regions.; Supplementary Figure S8: Number of long repeats in coding regions (CDS), intergenic regions (IGS), and introns.; Supplementary Figure S9: Phylogenetic tree constructed based on chloroplast genomes and the distribution of species after the grouping of Indocalamus on the phylogenetic tree.; Supplementary Figure S10: Phylogenetic tree of Indocalamus and related taxa constructed based on ddRAD data, modified from Guo et al. (2021).; Supplementary Figure S11: Indocalamus hispidus H. R. Zhao et Y. L. Yang. (A) Population and habitat; (B) rhizome and individual plant; (C) shoot; (D) leaf adaxial surface, leaf sheath, leaf tongue, and leaf base; (E) leaf abaxial surface; (F) below culm node, culm, and mature culm sheath. Scale bars: 1 m (A); 1 cm (B–F).; Supplementary Figure S12: Indocalamus sinicus (Hance) Nakai. (A) Population and habitat; (B,C) leaf tongue, leaf sheath, sheath opening, and leaf base; (D) culm sheath and prophyll ring; (E) rhizome; (F) below the node; (E,F) branching. Scale bars: 1 m (A); 1 cm (B–H).; Supplementary Figure S13: Indocalamus tessellatus (Munro) P. C. Keng. (A) Population and habitat; (B) leaf sheath, sheath opening, and leaf base; (C) culm sheath blade; (D) culm sheath and prophyll ring; (E) culm sheath blade and culm sheath. Scale bars: 1 m (A,B); 1 cm (C–E).

Author Contributions

W.Z., G.Y., M.W., C.G. and L.G. conceived and designed the study. L.G., Y.L., C.W., L.C. and J.W. did fieldwork. C.W. and L.G. conducted experiments and data analysis. C.W., L.G. and W.Z. wrote the draft. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Guangyao Yang, 31960335), the National Natural Science Foundation of China (Chunce Guo, 31960051), and the Basic Research and Talent Research Project of Jiangxi Academy of Forestry, China (Liqin Gao, 2023522802, 2024522801).

Data Availability Statement

The sequence data have been submitted to the GenBank databases under accession numbers in Supplementary Table S1.

Acknowledgments

We thank the Jiangxi Provincial Key Laboratory of Improved Variety Breeding and Efficient Utilization of Native Tree Species (2024SSY04093) for providing the experimental platform. We also appreciate the support of Peng Zhou (Nanchang University) for assistance with data analysis and visualization. Special thanks go to Lin Yang (Sichuan Agricultural University), Jingbo Ni and Zhuoyu Cai (South China Botanical Garden, Chinese Academy of Sciences), Dayong Huang (Guangxi Academy of Forestry Sciences), Jianghua Huang (Forestry Bureau of Wangmo County), Long Tong (Chongqing Academy of Forestry), Yuan Wu and Hengjun Li (Jiashan National Forest Park, Hunan), Jiansheng Wu (Guangxi Dayaoshan National Nature Reserve), Ruibing Huang (Dasha Forest Farm, Jiangmen City), Daye Liu (Jianfengling National Nature Reserve, Hainan), Hongzhuan Cai (Chishui Bamboo Sea National Forest Park, Guizhou), and Deyu Lai (Baying Forest Farm, Ruijin City) for their valuable help during field investigations. We are also grateful to the local people from Hunan, Sichuan, Guizhou, Chongqing, and other regions for their kind assistance in the field.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Horticulturae 11 01018 g001
Figure 1. Chloroplast genome maps of Indocalamus. The total genome length ranges from 139,555 to 139,791 bp. Colored bars represent various functional gene categories. The genome is partitioned into a large single-copy (LSC) region, a small single-copy (SSC) region, and a pair of inverted repeats (IRa and IRb), highlighted by sector shading. The innermost dark gray circle illustrates the GC content across the genome.
Figure 1. Chloroplast genome maps of Indocalamus. The total genome length ranges from 139,555 to 139,791 bp. Colored bars represent various functional gene categories. The genome is partitioned into a large single-copy (LSC) region, a small single-copy (SSC) region, and a pair of inverted repeats (IRa and IRb), highlighted by sector shading. The innermost dark gray circle illustrates the GC content across the genome.
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Figure 4. (A) Visualization of the expansion and contraction of inverted repeat (IR) boundaries in chloroplast genomes. The topology is based on the ML tree constructed using complete plastomes. (B) Insertion and deletion events in the three clades.
Figure 4. (A) Visualization of the expansion and contraction of inverted repeat (IR) boundaries in chloroplast genomes. The topology is based on the ML tree constructed using complete plastomes. (B) Insertion and deletion events in the three clades.
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Figure 5. Comparative analysis of nucleotide variability (Pi values) among the three clades using a sliding window approach (window length: 600 bp; step size: 200 bp). Panels (A–C) show the Pi plots for individual clades or combined clades, with red dots indicating highly variable regions.
Figure 5. Comparative analysis of nucleotide variability (Pi values) among the three clades using a sliding window approach (window length: 600 bp; step size: 200 bp). Panels (A–C) show the Pi plots for individual clades or combined clades, with red dots indicating highly variable regions.
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Figure 6. The relative synonymous codon usage (RSCU) values of merged CDS for the three clades. The red values indicate higher RSCU values, and the blue values indicate lower RSCU values. V, X, and IV indicate different clades of extant Indocalamus species in the phylogeny of the tribe Arundinarieae.
Figure 6. The relative synonymous codon usage (RSCU) values of merged CDS for the three clades. The red values indicate higher RSCU values, and the blue values indicate lower RSCU values. V, X, and IV indicate different clades of extant Indocalamus species in the phylogeny of the tribe Arundinarieae.
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Figure 7. Plot of SSR repeat pattern numbers in three clades. The number following each species name indicates the total number of SSRs in each plastome. V, X, and IV indicate different clades of extant Indocalamus species in the phylogeny of the tribe Arundinarieae.
Figure 7. Plot of SSR repeat pattern numbers in three clades. The number following each species name indicates the total number of SSRs in each plastome. V, X, and IV indicate different clades of extant Indocalamus species in the phylogeny of the tribe Arundinarieae.
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Wang, C.; Li, Y.; Cui, L.; Wang, J.; Wang, M.; Guo, C.; Yang, G.; Gao, L.; Zhang, W. Insight into the Phylogenetic Relationships and Evolutionary History of Indocalamus (Bambusoideae) Through Comparative Analyses of Plastomes. Horticulturae 2025, 11, 1018. https://doi.org/10.3390/horticulturae11091018

AMA Style

Wang C, Li Y, Cui L, Wang J, Wang M, Guo C, Yang G, Gao L, Zhang W. Insight into the Phylogenetic Relationships and Evolutionary History of Indocalamus (Bambusoideae) Through Comparative Analyses of Plastomes. Horticulturae. 2025; 11(9):1018. https://doi.org/10.3390/horticulturae11091018

Chicago/Turabian Style

Wang, Chengkun, Yonglong Li, Ling Cui, Jianqing Wang, Meixia Wang, Chunce Guo, Guangyao Yang, Liqin Gao, and Wengen Zhang. 2025. "Insight into the Phylogenetic Relationships and Evolutionary History of Indocalamus (Bambusoideae) Through Comparative Analyses of Plastomes" Horticulturae 11, no. 9: 1018. https://doi.org/10.3390/horticulturae11091018

APA Style

Wang, C., Li, Y., Cui, L., Wang, J., Wang, M., Guo, C., Yang, G., Gao, L., & Zhang, W. (2025). Insight into the Phylogenetic Relationships and Evolutionary History of Indocalamus (Bambusoideae) Through Comparative Analyses of Plastomes. Horticulturae, 11(9), 1018. https://doi.org/10.3390/horticulturae11091018

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