The first complete chloroplast genome of Calamus erectus (Arecaceae): Phylogenetic placement and divergence time estimation

Plant specimen collection and voucher specimen preparation

C. erectus specimens were collected from Savar, Dhaka. The collected specimens were identified based on relevant literature and diagnostic morphological characters (Uddin and Hassan, 2018). A voucher specimen was prepared following standard herbarium procedures (Singh and Subramaniam, 2008) and deposited in the Dhaka University Salar Khan Herbarium (DUSH), Department of Botany, University of Dhaka, Bangladesh, under the accession number DUSH10865.

Retrieval and quality assessment of whole-genome sequencing reads

Whole-genome sequencing data for C. erectus were retrieved from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the accession number SRR15672877. The dataset comprises publicly available paired-end Illumina reads generated on the HiSeq X Ten platform, with a nominal read length of 150 bp. Raw sequencing reads were subjected to quality assessment using FastQC v.0.12.1 (Rahman et al., 2025) to evaluate key quality parameters, such as GC content, sequence length distribution, per-base sequence quality, and the presence of adapter contamination or overrepresented sequences.

Assembly and annotation of the plastome

High-quality paired-end reads were assembled into a complete circularized plastome employing GetOrganelle v.1.7.7.0. The assembly was performed with default settings optimized for embryophyta, using a maximum read extension rounds of 15 with multiple k-mer sizes (21, 45, 65, 85, and 105) (Jin et al., 2020). Subsequently, the assembled plastome was annotated using the CPGAVAS2 web server (Shi et al., 2019), which enables automated gene prediction, functional annotation, and boundary verification of Cp genome features. A circular gene map of the annotated plastome was generated using the OGDraw server (Greiner et al., 2019). The full plastome of C. erectus has been deposited in the NCBI GenBank repository (accession number PX797484.1).

Coverage depth analysis and cis-trans splicing genes

To assess sequencing depth and assembly completeness, raw paired-end reads were aligned to the assembled plastome using the BWA-MEM algorithm in UGENE v.53.0 (Okonechnikov et al., 2012). The resulting alignments were used to evaluate read coverage depth across the Cp genome. Cis- and transsplicing genes were identified based on the annotated plastome employing the CPGView server (Liu et al., 2023).

Repeats and codons evaluation

SSRs within the plastome were identified with the MISA web server (Beier et al., 2017). The analysis was performed to detect and characterize the distribution and frequency of microsatellite motifs across the Cp genome. Relative synonymous codon usage (RSCU) values were calculated to evaluate codon usage bias using the RSCU module implemented in MEGA v.11 (Tamura et al., 2021). To facilitate comparative analysis, RSCU values were visualized as a heatmap generated using a customized Python script (heatmap.py) incorporating the pandas, seaborn, and matplotlib libraries (Ahmed and Rahman, 2025).

Phylogenetic analyses

The complete plastome sequence of C. erectus, together with those of its closely related taxa within the subfamily Calamoideae, were retrieved from the NCBI GenBank database. To properly root the phylogeny, Baxteria australis and Dasypogon bromeliifolius (Dasypogonaceae), a lineage closely related to Arecaceae, were included as outgroup taxa. The sequences were aligned using the MAFFT server (Katoh and Standley, 2013). The resulting alignment was further refined via TrimAl (Capella-Gutiérrez et al., 2009) to eliminate poorly aligned and ambiguously homologous sites, followed by manual inspection to ensure alignment accuracy. The final curated alignment was used to infer phylogenetic relationships under both maximum likelihood (ML) and Bayesian inference (BI) frameworks. ML analysis was conducted in MEGA v.11 (Tamura et al., 2021) using the GTR + Γ + I nucleotide substitution model, with nodal support assessed through 1,000 bootstrap replicates. Bayesian phylogenetic reconstruction was carried out using BEAST v2.7.3. Prior to analysis, the XML input file and model parameters were generated using the BEAUti package, with the GTR + Γ substitution model specified for sequence evolution (Drummond et al., 2012). Convergence and mixing were assessed using Tracer v.1.7.2 (Rambaut et al., 2018), ensuring effective sample size values exceeded 500 following the removal of initial 25% of samples as burn-in. The maximum clade credibility tree was subsequently constructed using TreeAnnotator, and the resulting phylogenetic tree was visualized in FigTree v.1.4.4 (Ahmed et al., 2023).

Molecular dating analysis

Molecular divergence time estimation was conducted exclusively using the RelTime-ML approach implemented in MEGA, following the methodological framework described by Mello (2018). The RelTime-ML approach estimates relative divergence times under a maximum-likelihood framework without assuming a strict molecular clock. Calibration constraints were obtained from the TimeTree database (Kumar et al., 2017), which summarizes divergence time estimates from previously published molecular dating studies. Two calibration points were applied in the analysis. The first calibration was assigned to the most recent common ancestor of Calamus erectus and Eugeissona tristis, with a median age of 75.0 million years ago (MYA) and a confidence interval of 8.1–80.2 MYA. This estimate was based on divergence time records summarized in TimeTree (Couvreur et al., 2011; Faye et al., 2016; Antonelli et al., 2017; Li et al., 2019; Ramírez-Barahona et al., 2020). The second calibration was assigned to the most recent common ancestor of Raphia vinifera and Metroxylon warburgii, with a median age of 60.0 MYA and a confidence interval of 7.5–76.7 MYA, based on estimates summarized from earlier studies (Couvreur et al., 2011; Faye et al., 2016; Antonelli et al., 2017; Li et al., 2019). The resulting time-calibrated phylogeny was used to infer divergence patterns and approximate evolutionary timelines among the studied taxa.

Quality evaluation of the raw reads

Quality assessment using FastQC indicated that the Illumina paired-end sequencing data were of high quality (Illumina, San Diego, CA, USA). Per-base Phred quality scores consistently exceeded Q30 across the entire read length for both forward and reverse reads, reflecting high base-calling accuracy. Median quality values remained close to Q41 for most positions, demonstrating uniform sequencing performance along the reads. Although a gradual decrease in quality was observed toward the 3′ ends, particularly in the reverse reads, the scores remained well within acceptable thresholds for downstream analyses. Overall, the FastQC results were found consistent with those reported in previous plastome studies (Ahmed and Rahman, 2024; Rahman et al., 2025) and confirmed that the sequencing data were suitable for accurate plastome assembly and subsequent analyses of C. erectus.

Assembled and annotated plastome

The complete Cp genome of C. erectus was successfully assembled and annotated, yielding a circular plastome of 157,887 bp. The genome exhibited the characteristic circular organization of angiosperm plastome, comprising a large single-copy (LSC) region of 85,756 bp, a small single-copy (SSC) region of 17,593 bp, and two inverted repeat (IR) regions (IRA and IRB) of 27,269 bp each (Fig. 1). The total GC content of the plastome was 37.34%, with distinct regional variation. The IR regions showed the maximum GC content (42.47%), while the LSC and SSC regions demonstrated lower GC contents of 36.54% and 33.18%, respectively. This pattern is consistent with the enrichment of ribosomal RNA (rRNA) genes within the IR regions. The total plastome size, quadripartite structure, and GC content observed in C. erectus are highly comparable to those reported for Calamus tetradactylus (Zhang et al. 2024), indicating a high degree of structural conservation of plastomes within the genus Calamus.

Fig. 1

Graphical representation of the Calamus erectus plastome. The circular map illustrates gene arrangement and the boundaries between the large single-copy (LSC), small single-copy (SSC), and inverted repeat (IR) regions. Genes located on the inner side of the circle are transcribed in the clockwise direction, whereas genes positioned on the outer side are transcribed counterclockwise. Functional categories of genes are indicated by distinct colors. The innermost ring depicts the variation in GC content across the plastome.

A total of 131 functional genes were annotated in the plastome of C. erectus, comprising 86 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight rRNA genes (Table 1). The protein-coding genes represented several gene families, including genes encoding subunits of photosystem I and II, the cytochrome b/f complex, ATP synthase, and NADH dehydrogenase, reflecting the conserved photosynthetic and respiratory functions of the Cp genome. In addition, the plastome contained genes involved in transcription and translation, such as the RNA polymerase subunits (rpoA, rpoB, and rpoC1), ribosomal protein genes, and the translation initiation factor infA. Genes associated with chloroplast biogenesis and maintenance were also identified, including matK, accD, clpP, cemA, ccsA, along with several conserved open reading frames (ycf1, ycf2, ycf3, and ycf4), which are known to play vital roles in plastome stability and functionality.

Table 1

Protein-coding genes in the plastome of Calamus erectus.

Several genes, including ndhB, rpl23, rps12, rps19, rps7, rpl2, and ycf2, were duplicated within the IR regions, contributing to the higher GC content of these regions. Compared with other congeners, C. erectus possessed a slightly smaller gene complement, with 131 genes (Table 2). Despite these minor variations in gene number and overall genome size, both plastome length and GC content were highly conserved across the genus Calamus, indicating strong structural conservation of Cp genomes within the genus.

Table 2

Comparative features of the plastomes of Calamus species used.

The differences in total gene number among the examined Calamus plastomes were relatively minor and were mainly associated with variations in protein-coding and tRNA gene annotations, whereas the number of rRNA genes remained constant. In C. erectus, 131 annotated gene copies were identified, comprising 86 protein-coding genes, 37 tRNA genes, and eight rRNA genes. By comparison, the other examined Calamus plastomes contained 132–133 annotated gene copies, comprising 86–87 protein-coding genes, 38 tRNA genes, and eight rRNA genes. Thus, the slightly lower total gene number in C. erectus was primarily associated with the presence of one fewer annotated tRNA gene compared with most congeners, while its number of protein-coding genes was comparable to several other Calamus species. Species containing 133 genes possessed one additional protein-coding gene and one additional tRNA gene relative to C. erectus. These small differences may reflect minor variation in annotation criteria, gene-copy recognition, or IR boundary annotation rather than major structural rearrangement of the plastome. Nevertheless, the overall plastome size, GC content, quadripartite organization, and rRNA gene complement were highly conserved among the compared Calamus species, indicating substantial structural conservation of plastomes within the genus.

Coverage depth analysis and cis-trans splicing genes

Read-mapping analysis revealed a high and uniform sequencing depth across the plastome of C. erectus, with a mean coverage of 180.51×. No region exhibited zero coverage, indicating complete representation of the Cp genome (Fig. 2A). The continuous coverage supported a fully circularized plastome assembly without gaps or fragmentation. Several plastid genes in C. erectus contained introns and exhibited cis-splicing. These included clpP, petB, petD, rpl2, rpl16, ndhA, ndhB, rps16, atpF, rpoC1, and ycf3. Among them, petB, petD, rpl2, rpl16, ndhA, ndhB, rps16, atpF, and rpoC1 each harbored a single intron, whereas clpP and ycf3 comprised two introns (Fig. 2B). In contrast, rps12 was identified as a trans-splicing gene, with its 5′ exon located in the LSC region and the duplicated 3′ exons positioned within the IR regions (Fig. 2C). This trans-splicing arrangement is consistent with the conserved Cp genome organization observed in angiosperms (Nyamgerel et al., 2025).

Fig. 2

Analysis of coverage depth and cis-trans splicing in the plastome of Calamus erectus. A. Read-mapping profile showing sequencing coverage depth across the plastome. B. Cis-splicing profile. C. Trans-splicing profile of the rps12 gene.

Repeats and codons

MISA-based analysis identified SSRs across all examined taxa, with mononucleotide repeats representing the most abundant repeat class. In C. erectus, 70 SSRs were detected, comprising 49 mononucleotide, 19 dinucleotide, one trinucleotide, and one tetranucleotide repeat, indicating a strong dominance of short repeat motifs within the plastome. A comparable SSR composition was observed in C. exilis, which contained 72 SSRs, including 49 mononucleotide, 21 dinucleotide, one trinucleotide, and one tetranucleotide repeat. C. walkeri harbored comparatively fewer SSRs (65), with mononucleotide (44) and dinucleotide (20) repeats predominating and a single tetranucleotide repeat detected. Outside the genus Calamus, Raphia vinifera exhibited the highest SSR abundance (87 SSRs), dominated by mononucleotide repeats (64), followed by dinucleotide (20), trinucleotide (1), tetranucleotide (1), and a single pentanucleotide repeat. Eugeissona tristis contained 71 SSRs, primarily mononucleotide (54) and dinucleotide (15) repeats, along with two tetranucleotide repeats (Fig. 3A).

Fig. 3

Evaluation of repeats and codon usage patterns in the plastome of Calamus erectus and its closely related taxa. A. Simple sequence repeat analysis. B. Relative synonymous codon usage analysis.

Codon usage analysis based on RSCU module revealed a clear bias toward A/U-ending codons in the plastome of C. erectus. Synonymous codons ending in U or A generally exhibited RSCU values > 1, whereas G/C-ending codons were mostly underrepresented (RSCU < 1), reflecting the overall A/T-rich composition of the Cp genome (Fig. 3B). From the codon count perspective, the five most abundant codons in the plastome of C. erectus were UUU (Phe; 2175), AAA (Lys; 2107), AUU (Ile; 1851), AAU (Asn; 1802), and AUA (Ile; 1602), indicating a strong preference for codons encoding hydrophobic and polar amino acids that end in A or U. In terms of RSCU, the most strongly preferred codons were AGA (Arg; RSCU = 1.91), UCU (Ser; 1.48), GGA (Gly; 1.47), CAU (His; 1.45), and GAU (Asp; 1.45), highlighting pronounced codon bias. Comparative analyses with closely related species (C. exilis and C. walkeri) and other palms (Raphia vinifera and Eugeissona tristis) revealed highly conserved codon usage patterns. All examined plastomes showed a strong bias toward A/U-ending codons, similar profiles of over- and underrepresented synonymous codons, and consistent stop codon usage (Fig. 3B). This conservation suggests that codon usage bias within Calamus plastomes is largely shaped by shared mutational pressures and evolutionary constraints, and is typical of angiosperm Cp genomes (Ahmed and Rahman, 2025).

Phylogenetic analyses

Phylogenetic reconstruction inferred using both ML and BI yielded highly congruent tree topologies, confirming the stable placement of C. erectus within the tribe Calameae of the subfamily Calamoideae. The major tribal clades included in the analysis were each recovered as monophyletic. Lepidocaryeae and Eugeissoneae received strong nodal support, with ML bootstrap/posterior probability values of 100/1 for both clades. Calameae was also resolved as monophyletic, although with comparatively moderate ML bootstrap support and strong Bayesian posterior probability support (69/1). Within Calameae, C. erectus showed a close relationship with C. exilis by maximum bootstrap and posterior probability support. In Lepidocaryeae, Mauritia flexuosa exhibited a close relationship with Eremospatha macrocarpa. The tribe Calameae, represented by 13 taxa in the present analysis, was resolved as more closely related to Lepidocaryeae than to Eugeissoneae (Fig. 4). This topology is consistent with the plastid phylogenomic framework of Yao et al. (2023), in which Calameae and Lepidocaryeae formed a clade, with Eugeissoneae placed outside this relationship. A similar relationship was also recovered by Barrett et al. (2016), who analyzed palm plastomes and protein-coding plastid matrices using different character-sampling and partitioning strategies. However, this relationship differs from that reported by Chen et al. (2022), in which Calameae and Eugeissoneae were recovered as a clade, with Lepidocaryeae placed as sister to them. Such topological incongruence may reflect differences in taxon sampling, plastome dataset construction, alignment filtering, character inclusion or exclusion, partitioning strategy, and phylogenetic inference methods among studies. For example, Chen et al. (2022) analyzed multiple plastome-derived datasets, including complete plastome sequences, coding sequences, non-coding regions, LSC, SSC, and plastomes with one IR removed, whereas Yao et al. (2023) used broader plastid-region sampling across palms. Barrett et al. (2016) also demonstrated that plastome-scale phylogenetic inference in palms can be influenced by matrix composition, character filtering, and partitioning strategy. Therefore, although the present topology supports a closer relationship between Calameae and Lepidocaryeae, the intertribal relationships within Calamoideae may require further investigation with expanded taxon sampling and comparative plastomic datasets.

Fig. 4

Phylogenetic relationships of Calamus erectus within the subfamily Calamoideae. The tree was generated by maximum likelihood (ML) and Bayesian inference (BI) approaches. Nodal support values are indicated as bootstrap values and posterior probabilities (ML/BI).

Molecular dating analysis

Molecular dating analysis based on whole-plastome data estimated that the tribe Calameae diverged approximately 23.90 MYA, corresponding to the Aquitanian stage of the Early Miocene within the Neogene Period (Fig. 5). The crown age of the subfamily Calamoideae was estimated at approximately 46.20 MYA, placing its origin in the Lutetian stage of the Eocene epoch within the Cenozoic Era. Previous molecular dating studies have reported variable estimates for the timing of diversification within Calamoideae. For example, Chen et al. (2022) estimated the crown divergence of Calamoideae at 53.39 MYA, with a broad 95% highest posterior density interval of 16.99–87.13 MYA. Although the present estimate is broadly comparable to previously reported values, it should be interpreted cautiously because divergence time estimates may vary substantially depending on taxon sampling, molecular datasets, analytical methods, and calibration strategy. The broad uncertainty intervals reported in previous studies further indicate that precise timing of diversification within Calamoideae remains difficult to resolve. At the species level, C. erectus was inferred to have diverged approximately 7.59 MYA during the Tortonian stage of the Late Miocene, providing a preliminary temporal framework for understanding its evolutionary history within Calamoideae.

Fig. 5

RelTime-ML chronogram showing the estimated divergence time of Calamus erectus within Calamoideae. Divergence time estimation was conducted in MEGA v.11 using the RelTime-ML method. Calibration nodes are indicated by colored circles, and node-height error bars represent uncertainty estimates.

The current study describes the first complete Cp genome of the vulnerable medicinal palm Calamus erectus, providing an important genomic resource for this species. Analyses of SSRs and codon usage revealed conserved features typical of Calamoideae plastomes. Plastome-based phylogenetic reconstruction confirmed the close relationship between C. erectus and C. exilis, while molecular divergence dating indicated that C. erectus originated during the Tortonian stage of the Late Miocene. These findings may provide a solid framework for future phylogenomic, taxonomic, and conservation-oriented studies within Arecaceae.