Complete chloroplast genome sequences and DNA barcode regions (ITS, matK, and rbcL) of T. fruticosum and T. paniculatum were retrieved from the National Center for Biotechnology Information (NCBI) GenBank database on January 2, 2026. These three markers were selected because they are widely applied in plant species identification and standard DNA barcoding studies (Techen et al., 2014; Algarni, 2022). This study is a preliminary in silico assessment; thus, we prioritized sequences with verified voucher information. We cross-referenced species names with the World Flora Online (WFO) database to manage taxonomic synonyms (e.g., treating T. triangulare as a synonym of T. fruticosum) to ensure taxonomic reliability. All available sequences for each marker were initially collected from GenBank. Sequence quality was carefully evaluated based on annotation completeness, sequence length, gene coverage, and consistency after alignment. Preliminary screening was performed to detect sequences showing unusually high intraspecific genetic distances or incomplete gene coverage. Such sequences were considered potential outliers. After filtering, the dataset consisted of n = 12 sequences for ITS, n = 9 for matK, and n = 9 for rbcL per species, which were used for downstream analyses. Two complete chloroplast genome sequences (one per species) were used for comparative plastome analyses. The accession numbers and metadata of all sequences are provided in On-line Supplemental Table S1.

DNA barcode sequences for ITS, matK, and rbcL were aligned separately using multiple sequence alignment implemented in MEGA X software (Stecher et al., 2025) using MUSCLE with default gap opening and extension penalties. Pairwise genetic distances were calculated under the p-distance model. Although the sample size is limited in this preliminary evaluation, the p-distance model remains suitable for assessing these markers at this taxonomic scale (Hadi et al., 2016). Alignments were manually inspected and edited to ensure positional homology. Species identification performance was assessed using BLASTn searches against the NCBI nucleotide database. The top-hit method was applied, and a query was considered correctly identified when the highest bit-score match showed ≥98% sequence identity, ≥90% query coverage, and corresponded to the expected species. We emphasize that the low identification success of certain markers (e.g., matK) in this study likely reflects the current limitations of public sequence databases rather than the markers’ inherent performance.

The plastome structures were analyzed to compare total genome size, overall GC content, and the organization of the large single-copy (LSC), small single-copy (SSC), and IR regions. To ensure consistency and minimize potential annotation bias, both plastomes were re-annotated using GeSeq (Tillich et al., 2017) with identical parameter settings, with tRNA genes further verified using ARAGORN v1.2.38 (Laslett and Canback, 2024). Circular plastome maps were visualized using Chloroplot (Zheng et al., 2020). To evaluate the structural organization and consistency of the chloroplast genomes, we generated circular maps (Fig. 1 ). These maps provide a comprehensive visualization of gene arrangements, GC content, and the quadripartite structure, which clearly illustrate the high level of structural conservation between the two Talinum species. SSRs were detected using MISA (Beier et al., 2017). Intron-containing genes were identified and classified using CPGView (Liu et al., 2023).

Whole-plastome alignment was used to calculate nucleotide diversity (π) in DnaSP v6.0 (Rozas et al., 2017). Sliding window analysis was conducted with a window length of 600 bp and a step size of 200 bp. Windows with π > 0.008 were defined as divergence hotspots. The boundaries between the LSC, SSC, and IR regions were compared using IRscope (Amiryousefi et al., 2018). As this study serves as a foundational framework, we recommend that future research with larger, field-collected population samples validate these findings to support robust phylogenetic conclusions.

Pairwise genetic distance analysis revealed clear differences in the discriminatory performance of the three DNA barcode regions (ITS, matK, and rbcL) for distinguishing T. fruticosum and T. paniculatum (Fig. 2). After excluding potential outlier sequences to reduce the influence of sequencing or annotation errors, all three markers showed a detectable barcode gap; however, the magnitude of separation between intra- and interspecific distances differed markedly among regions. The ITS region exhibited the highest level of sequence divergence and the widest barcode gap. Intraspecific distances within T. fruticosum were zero, whereas T. paniculatum showed low but detectable intraspecific variation, with values ranging from 0 to 3.15% (mean = 1.03%; n = 8). In contrast, interspecific distances between the two species were substantially higher, ranging from 5.64% to 8.18% (mean = 6.73%; n = 1 2). Importantly, the maximum intraspecific distance (3.15%) was clearly lower than the minimum interspecific distance (5.64%), resulting in a wide and non-overlapping barcode gap. This clear separation was also evident in the histogram distribution, where intra- and interspecific distances formed two distinct clusters (Fig. 2A). These results demonstrate that ITS provides strong discriminatory power for species identification in Talinum. This finding is consistent with previous large-scale studies showing that ITS is among the most variable and informative regions for plant DNA barcoding, particularly for closely related species (Hollingsworth et al., 2011; Duan et al., 2019).

The matK region showed moderate discriminatory ability. Intraspecific distances were low in both species, ranging from 0 to 0.39% (n = 5) in T. fruticosum and from 0 to 0.13% (n = 4) in T. paniculatum. Interspecific distances were clearly higher, ranging from 2.59% to 2.98% (mean = 2.72%; n = 9). Although the barcode gap in matK was narrower than that observed for ITS, there was still no overlap between intra-and interspecific distances, as the maximum intraspecific distance (0.39%) was much lower than the minimum interspecific distance (2.59%) (Fig. 2B). These results indicate that matK can reliably discriminate the two Talinum species in our dataset. However, it is important to note an interpretive inconsistency: while distance-based analysis confirmed a distinct barcode gap, the BLAST-based identification success rate for matK was relatively low (11%). This discrepancy arises because distance-based analysis compares genetic variation within our curated dataset, whereas BLAST-based identification relies heavily on the comprehensiveness and quality of reference sequences in public databases. The current limited representation of Talinum sequences in GenBank likely hinders BLAST performance for this marker, rather than reflecting an inherent lack of discriminatory power in matK itself. This pattern agrees with previous reports that matK evolves more slowly than ITS and often provides intermediate discriminatory power in plant barcoding studies (CBOL Plant Working Group, 2009).

In contrast, rbcL exhibited the lowest level of sequence variation among the three markers. Intraspecific distances were extremely low, ranging from 0 to 0.19% (n = 3) in T. fruticosum and from 0 to 0.58% (n = 6) in T. paniculatum. Interspecific distances were also relatively low, ranging from 0.96% to 1.35% (n = 9). Although a barcode gap was still present (maximum intra = 0.58%, minimum inter = 0.96%), the separation between intra- and interspecific distances was narrow compared with ITS and matK (Fig. 2C). This limited divergence reflects the conservative nature of rbcL and explains its reduced resolution for closely related species. Similar limitations of rbcL for species-level discrimination have been widely reported in plant barcoding literature (Jamdade et al., 2021).

Overall, the comparative analysis clearly indicates that ITS is the most effective DNA barcode for discriminating T. fruticosum and T. paniculatum, followed by matK with moderate resolution, and rbcL with the weakest resolving power. These results support the use of ITS as the primary barcode for species identification in Talinum, in agreement with recommendations from previous global assessments of plant DNA barcodes. The combination of ITS with plastid markers such as matK and rbcL may further improve robustness, but plastome-scale analyses are expected to provide the highest level of resolution for closely related taxa.

BLAST-based identification showed that ITS achieved 100% correct species assignment, whereas matK and rbcL showed lower success rates (11% and 0%, respectively). As discussed above, the lower success of matK in BLAST searches highlights the necessity of expanding reference sequence databases to fully utilize the diagnostic potential of this marker. Beyond confirming the conserved nature of rbcL and the higher variability of ITS, the present findings have practical implications for real-world species authentication. In routine identification, highly conserved markers such as rbcL may be insufficient for discriminating closely related Talinum species, particularly when morphological traits overlap. In contrast, ITS and selected plastome hotspot regions provide higher resolution and may improve reliability in distinguishing authentic materials from substitutes or adulterants. Taken together, our results suggest that a combined-marker strategy, integrating nuclear ITS as a primary barcode with confirmatory plastid regions, may provide a more reliable framework for authentication than reliance on single-locus barcodes.

The comparative analysis of the two Talinum plastomes revealed a high level of overall structural conservation, consistent with the generally stable architecture of angiosperm chloroplast genomes (Fig. 1). Both plastomes exhibited nearly identical quadripartite organization and GC content, indicating strong evolutionary constraint on core plastome structure and nucleotide composition. The total genome size of T. fruticosum was 156,811 bp, whereas T. paniculatum showed a slightly larger plastome of 156,929 bp. This minor size difference was mainly attributable to small variations in the lengths of the single-copy and IR regions.

Despite this overall similarity, several subtle differences were detected. T. paniculatum possessed a slightly larger plastome and marginally expanded single-copy regions compared with T. fruticosum (Table 1). Notably, T. paniculatum contained a higher number of protein-coding genes and tRNA genes than T. fruticosum. Variation in gene number among plastomes of closely related species is relatively uncommon but has been reported and may result from differential gene duplication, annotation differences, or lineage-specific gene loss or retention (Daniell et al., 2016). The increased gene content in T. paniculatum may reflect subtle differences in plastome gene organization and could contribute to species-level plastome divergence. In contrast, the number of rRNA genes was identical in both species, reflecting strong conservation of the ribosomal RNA operon within the IR regions. This conservation is typical of angiosperm plastomes and highlights the functional constraint on genes involved in plastid ribosome assembly and translation (Wicke et al., 2011).

Under the applied MISA criteria, only mono- and dinucleotide SSRs were detected in both plastomes, whereas tri-, tetra-, penta-, and hexa-nucleotide repeats were not observed. The chloroplast simple sequence repeat density was 0.51 SSRs per kb in T. fruticosum and 0.46 SSRs per kb in T. paniculatum. In both species, mononucleotide repeats were the most dominant SSR class, with a strong bias toward A/T-rich motifs. This pattern is consistent with the overall AT-rich nature of chloroplast genomes and has been widely reported in plastome studies across angiosperms (Amenu et al., 2022). In T. fruticosum, a total of 36 A-type and 33 T-type mononucleotide repeats were detected, whereas T. paniculatum contained 33 A-type and 33 T-type repeats. Only a small number of C and G mononucleotide repeats were observed in both plastomes, reflecting the low frequency of GC-rich SSRs in chloroplast genomes. Dinucleotide SSRs, particularly AT and TA motifs, were also detected in both species but at much lower frequencies compared with mononucleotide repeats. The similarity in SSR composition between the two Talinum species indicates a high degree of conservation in microsatellite distribution. However, minor differences in SSR number and motif composition may contribute to species-specific plastome variation. Such plastid SSRs are considered useful molecular markers for population genetic and phylogeographic studies due to their high mutation rates and maternal inheritance (Rogalski et al., 2015; Bravo, 2026).

Comparative analysis of intron-containing genes based on structural visualization (Fig. 3) showed that the cis-splicing gene organization was highly conserved between T. fruticosum and T. paniculatum. In both plastomes, a typical set of intron-containing genes was identified, including rps16, atpF, rpoC1, ycf3, clpP, petB, petD, rpl16, ndhA, and ndhB. These genes contained one or two introns, consistent with the general organization of angiosperm chloroplast genomes (Wicke et al., 2011; Daniell et al., 2016). Visual comparison of exon-intron structures indicated largely similar exon numbers and arrangements between the two species. Minor variations in exon or intron lengths were observed in the graphical representation; however, overall gene structure and exon-intron organization remained comparable.

The trans-splicing gene rps12 exhibited an identical structural arrangement in both plastomes (Fig. 4). In each species, exon 1 was located in the LSC region, whereas exons 2 and 3 were duplicated within the IR regions (IRa and IRb), forming mature transcripts via trans-splicing. This conserved arrangement is characteristic of angiosperm plastomes and reflects structural conservation of trans-splicing gene organization.

A total of nine major divergence hotspots (π > 0.8) were identified (Fig. 5). These regions include trnS–trnG, rpoB–trnC, trnE–trnT, petA–psbJ, psaJ–rpl33, psbN, petD, rps12–rpl32, and ycf1. Most hotspots were located in non-coding regions of the LSC and SSC regions, whereas the IR regions showed comparatively low nucleotide diversity, indicating a high level of sequence conservation typical for closely related angiosperms (Daniell et al., 2016). To facilitate future validation and species identification, the precise genomic locations, lengths, and potential primer regions for these divergence hotspots are summarized in On-line Supplemental Table S2.

The uneven distribution of nucleotide diversity along the plastome reflects heterogeneous evolutionary rates among different chloroplast regions. Coding regions and IR regions generally showed lower π values, consistent with their functional constraints and the homogenizing effect of gene conversion between IR copies. In contrast, several single-copy region spacers, particularly within the LSC and SSC regions, exhibited higher nucleotide diversity. This pattern is widely observed in plastome comparative studies and reflects relaxed selective pressure in non-coding regions and previously reported (Wang et al., 2021). Importantly, the diversity hotspots detected by sliding window analysis provide candidate regions for the development of supplementary molecular markers for Talinum. These variable regions may be useful for improving species discrimination and population-level studies, especially in cases where standard plastid barcodes such as rbcL and matK show limited resolution. Similar approaches have been successfully applied in many plant groups to identify highly informative plastome regions for phylogenetic and barcoding purposes.

Comparative analysis of the four IR/SC junctions (JLB, JSB, JSA, and JLA) revealed a highly conserved boundary organization between T. fruticosum and T. paniculatum, with only minor positional shifts (Fig. 6, Table 2). At the LSC/IRb boundary (JLB), rps19 was consistently located adjacent to the junction in both species, showing a slight positional difference of 11 bp. The IRb/SSC boundary (JSB) involved ndhF, which exhibited a boundary shift of 181 bp between the two plastomes, representing the largest observed variation. At the SSC/IRa junction (JSA), ycf1 spanned the boundary in both species, with a positional difference of 109 bp. The IRa/LSC boundary (JLA) was associated with trnH and showed only a negligible 1 bp difference. Overall, the magnitude of IR boundary variation was limited (<200 bp), indicating minor IR expansion/contraction events without structural rearrangement. Such stability is common among closely related species and reflects the evolutionary constraints acting on IR regions, which are known to evolve more slowly than single-copy regions due to copy correction via gene conversion (Wicke et al., 2011; Daniell et al., 2016).

Minor differences in the positioning of genes near IR junctions, as visualized by IRscope, likely represent small-scale boundary shifts rather than major structural rearrangements. Similar subtle IR boundary variation has been reported in many angiosperm lineages and is often sufficient to explain small differences in total plastome size among closely related taxa (Zhu et al., 2016). Importantly, the absence of large IR expansions, contractions, or gene losses indicates that plastome evolution in Talinum is largely conservative at the structural level. This high level of structural conservation further supports the interpretation that species discrimination between T. fruticosum and T. paniculatum is driven primarily by sequence divergence in variable regions rather than by major plastome rearrangements.

As shown in Table 2, the comparison of IR/SC junctions between T. fruticosum and T. paniculatum revealed highly conserved boundary structures with only minor positional shifts. In both plastomes, the LSC/IRb junction is associated with rps19, while the IRb/SSC and SSC/IRa junctions involve ndhF and ycf1, respectively. The IRa/LSC boundary is consistently flanked by trnH in both species. The observed positional differences are minimal (≤181 bp), suggesting slight IR expansion or contraction events that did not substantially alter gene organization. Overall, the high similarity of IR boundary structure supports the conserved nature of plastome architecture within Talinum, consistent with the general stability of IR regions in angiosperm chloroplast genomes.

This study establishes a reproducible comparative plastome framework for T. fruticosum and T. paniculatum and systematically evaluates both standard DNA barcodes and plastome-derived markers for species discrimination. Although the two plastomes exhibit a highly conserved overall structure, several divergence hotspots were detected that provide promising candidates for enhanced molecular identification. ITS demonstrated the highest discriminatory power among the tested loci, supporting its use as a primary barcode, while plastome-derived markers may serve as complementary tools. Nevertheless, broader geographic sampling and experimental validation remain necessary to assess marker stability under real-world conditions. Collectively, these findings provide valuable genomic resources and a methodological foundation for future authentication, taxonomic refinement, and molecular marker development in Talinum.