Leaf samples were procured from an individual S. glomeratum tree specimen located in Phuoc Minh district, Tay Ninh province, Vietnam (11°19′55.3″N, 106°18′02.0″E). A taxonomic expert verified the plant identity, and the voucher specimen was labeled UMP_2024.02.05_TramTron and deposited in the herbarium of the Faculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City. Fresh leaves were desiccated using silica gel at room temperature and stored for subsequent experiments. No permits were required to collect S. glomeratum samples.

Total genomic DNA was isolated from dehydrated leaf tissues following the modified cetyltrimethylammonium bromide protocol (Porebski et al., 1997). The extracted DNA was further purified using a commercial Monarch Genomic DNA Purification Kit (#T3010, New England Biolabs, Ipswhich, MA, USA) according to the manufacturer’s instructions. The extracted genomic DNA, with a quality and purity (A₂₈₀/₂₆₀ ratio of approximately 1.8–2.0), was stored at –20°C until it was used to construct the sequencing library.

Library preparation was accomplished using the NEBNext Ultra II DNA Library Prep kit (#E7103, New England Biolabs). High-throughput sequencing (2 × 150 bp) was performed using a MiSeq sequencer (Illumina, San Diego, CA, USA). After quality control using FastQC (Andrews, 2010) and purification using Trimmomatic (Bolger et al., 2014), the clean reads were used to de novo assemble the complete cp genome using GetOrganelle v1.7.7.0 (Jin et al., 2020) and NOVOPlasty v4.3.1 (Dierckxsens et al., 2016) pipelines with the cp genome of Syzygium polyanthum (Wight) Walp. (accession number: NC_072979) as a reference (Nguyen et al., 2023). The assembled S. glomeratum cp genome was annotated using the GeSeq tool (Tillich et al., 2017). All protein-coding genes and tRNA genes were confirmed by BLAST and tRNAscan-SE v2.0 (Chan and Lowe, 2019), respectively. The annotated gene content was manually curated using Geneious Prime v2024.0.2. A circular map of the cp genome was generated using OGDRAW v1.3.1 (Greiner et al., 2019).

A total of 42 Syzygium cp genomes were retrieved from the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/) (Table 1). In addition, the cp genome of Backhousia citriodora F. Muell. (accession number ON422330), a member of the Myrtaceae family was used as an outgroup. To reconstruct the phylogenetic tree, individual protein-coding genes (PCGs) from all cp genomes were extracted and aligned using Geneious Prime v2024.0.2. The TrimAl tool was used to remove gaps and poorly aligned regions from the sequence alignments (Capella-Gutiérrez et al., 2009). Afterward, the aligned PCGs were concatenated in Geneious Prime, resulting in a combined alignment dataset. The optimal nucleotide substitution model was GTR+I+G, identified using jModelTest v2 (Posada, 2008). Bayesian inference (BI) and maximum likelihood (ML) phylogenetic trees were reconstructed using MrBayes v3.2.7a (Huelsenbeck and Ronquist, 2001) and IQTREE v1.6.12 (Nguyen et al., 2015), respectively. For the ML analysis, IQ-TREE was executed with 1,000 bootstrap replicates. The BI analysis was run for 1,000,000 generations, which resulted in a split frequency lower than 0.01. The resulting ML and BI phylogenetic trees were visualized and annotated using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

The complete cp genome of S. glomeratum was successfully assembled and annotated in this study and was deposited in GenBank under accession number PP734015. This circular cp genome map was 158,469 bp in length and had an overall GC content of 37.0% (Fig. 1). This genome revealed a typical quadripartite structure consisting of a large single-copy region (LSC, 87,962 bp in length) and a small single-copy region (SSC, 18,391 bp in length) separated by two inverted repeat regions (IRa and IRb, 26,058 bp). The GC content of LSC, SSC, and IR regions were 34.8%, 31.0%, and 42.7%, respectively. Generally, the cp genome of S. glomeratum was similar to the typical quadripartite cp genome of angiosperms (Jansen and Ruhlman, 2012). Compared with the reported cp genomes of Syzygium species, no special structural variations (i.e., gene loss, IR loss, and large inversion) were found in the cp genome of S. glomeratum.

A total of 130 genes were annotated in the S. glomeratum cp genome, including 85 PCGs, 8 ribosomal RNA genes, and 37 transfer RNA genes (Table 2). The gene content and orientation are highly conserved compared with other cp genomes of Syzygium species (Asif et al., 2013; Zhang et al., 2019; Chen et al., 2022; Nguyen et al., 2023). Among the 130 annotated genes, 18 contained introns, with 15 genes having a single intron and 3 genes (rpl2, rps12, and clpP) containing two introns. Seventeen coding genes were duplicated in IR regions, including six PCGs (rpl2, rpl23, ycf2, ndhB, rps7, rps12), seven tRNA genes (trnI-CAU, trnL-CAA, trnV-GAC, trnI-GAU, trnA-UGC, trnR-ACG, trnN-GUU), and four rRNA genes (rrn16, rrn23, rrn4.5, rrn5). The rps12 was identified as a trans-splicing gene with three exons located in distinct regions of the cp genome. In particular, exon 1 was located in the LSC region, and exons 2 and 3 were in the IR regions.

We used ML and BI methods for reconstructing phylogenetic trees based on the cp genome CDS of 43 species (S. glomeratum, 41 Syzygium species, and B. citriodora used as an outgroup). The resulting ML and BI trees had an identical topology with strongly supported values (bootstrap > 70 and posterior probability > 0.95) in many nodes (Fig. 2). A close relationship among six Syzygium species was previously reported based on morphological markers (Cheong and Ranghoo-Sanmukhiya, 2013). It has been shown that S. glomeratum has a closer relationship with sister groups (including S. venosum, S. coriaceum, and S. petrinense) (Cheong and Ranghoo- Sanmukhiya, 2013). In this study, based on 79 PCGs from the cp genome, S. glomeratum formed a monophyletic group with S. cinereum and S. claviflorum. Furthermore, the tree topology closely aligns with prior phylogenetic studies based on cp genomic (Eguiluz et al., 2017; Nguyen et al., 2023; Huynh et al., 2024; Sun et al., 2024). However, the recent study by Low et al. (2022), which utilized single nucleotide polymorphism data from nuclear loci in the Syzygium genus, shows inconsistency with our findings (Low et al., 2022). Specifically, it indicated that S. glomeratum is closely related to S. buxifolium rather than S. cinereum (Low et al., 2022). This incongruence between nuclear and cp phylogenies could be attributed to factors, such as: convergent evolution, lineage sorting, or reticulate evolution (Nishimoto et al., 2003; Yu et al., 2013). Syzygium is a large genus with more than 1200 species, but only a few cp genome sequences of Syzygium were reported, further studies are needed to understand fully resolved evolutionary and phylogenetic relationships within the genus.