Fresh and healthy leaves (free from pest and disease damage) were collected from a single individual of P. sinensis var. brevifolia in Ca Thanh Commune, Cao Bang Province, Vietnam (22.69889°N, 105.81694°E). A voucher specimen (TSLGCB2024) has been deposited and is curated by Son Le (leson@vafs.gov.vn). Sample collection was conducted under authorized permits for scientific research as part of a project approved by the Ministry of Science and Technology. The leaves were dried in silica gel at room temperature and are stored at the Institute of Forest Tree Improvement and Biotechnology, Vietnam Academy of Forest Sciences.

Genomic DNA was extracted from desiccated leaf tissue employing the modified CTAB protocol (Porebski et al., 1997). DNA samples were subsequently purified with the Monarch Spin gDNA Extraction Kit (T3010, New England Biolabs, Ipswich, MA, USA). DNA purity, concentration, and integrity were assessed using a NanoDrop OneC UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), a Qubit 4 fluorometer (Thermo Fisher Scientific), and electrophoresis on a 1% agarose gel, respectively. Library preparation was performed following the NEBNext Ultra II DNA Library Prep Kit protocol (E7103, New England Biolabs). The genomic library was sequenced on the MGI platform (150 bp paired-end) at KTest Science Co Ltd. (https://www.ktest.vn/, Ho Chi Minh City, Vietnam). After quality filtering with fastp v1.0.1 (length > 100 bp, and Q-score > 20), approximately 3Gb of clean data was obtained (Chen, 2023).

The complete cp genome of Pseudotsuga sinensis var. brevifolia was de novo assembled using NOVOPlasty v4.3.1 from purified sequencing data (Dierckxsens et al., 2017). The parameter settings included: type_genome–chloro, reference sequence–Pseudotsuga brevifolia cp genome (GenBank accession no. NC_058308), and a seed sequence–petN gene (GenBank no. GU457496). The resulting circular contig was annotated using GeSeq tool (Tillich et al., 2017). The annotations of transfer RNA genes were manually curated by using tRNAScan-SE v2.0 while protein-coding genes (PCGs) were refined by using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and aligning with homologous protein from cp genome of Pseudotsuga sinensis var. wilsoniana (GenBankaccession no. NC_016064) and Pseudotsuga sinensis var. sinensis (GenBank accession no. NC_058742). To calculate coverage depth, all raw reads were mapped back to the assembled cp genome using SAMtools v1.22, and the results were visualized following the pipeline of Ni et al. (2023). The circular cp genome diagram was sketched by using OGDRAW v.1.3.1 (Greiner et al., 2019)

To determine the phylogenetic position of Pseudotsuga sinensis var. brevifolia within the Pinaceae family, a maximum likelihood (ML) phylogenetic tree was reconstructed using IQ-TREE webserver (Minh et al., 2020) with 1,000 bootstrap replicates under the GTR + GAMMA substitution model. The dataset included 25 complete cp genome sequences from Pinaceae species, of which 24 were available in GenBank (Table 1). Multiple sequence alignment was performed using MAFFT v7 (Katoh et al., 2019). The cp genome of Juniperus przewalskii (GenBank: NC_062328, Cupressaceae family) was designated as the outgroup. The resulting ML tree was edited and visualized using FigTree v.1.3.1 (https://tree.bio.ed.ac.uk/software/figtree/).

The complete cp genome of Pseudotsuga sinensis var. brevifolia was successfully assembled with a total of 214,049 reads, yielding an average coverage depth of 262.22×. The complete genome was 122,441 bp in length, with an overall GC content of 38.7%. Structurally, the cp genome exhibits a typical quadripartite organization consisting of a large single-copy (LSC) region of 66,904 bp (38% GC), a small single-copy (SSC) region of 54,839 bp (39.6% GC), and a pair of inverted repeat (IR) regions each 349 bp in length (36.1% GC) (Fig. 1).

In Pseudotsuga sinensis var. brevifolia cp genome, a total of 113 genes were annotated, consisting of 73 PCGs, 36 tRNA genes, and four rRNA genes (Table 2). Additionally, six PCGs (atpF, rpl2, rpl16, rpoC1, petB, and petD) each contained a single intron, while pafI contained two introns. The rps12 gene was found as trans-splicing. Especially, three copies of the psbI gene were identified in the genome.

Among the 36 tRNA genes, 32 were single-copy. The trnI-CAU gene has two copies as it is located in the IR regions. Additionally, trnT-GGU and trnS-GCU were identified with two and three copies, respectively, in the cp genome. The six tRNA genes (trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC) each contained an intron.

To determine the phylogenetic relationship of Pseudotsuga sinensis var. brevifolia, a phylogenetic tree was reconstructed using 71 unique PCG genes from 25 Pinaceae species cp genomes, including newly sequenced P. sinensis var. brevifolia cp genome. Juniperus przewalskii (Cupressaceae) was used as the outgroup. Bootstrap values at major nodes are high (greater than 75%), indicating strong statistical support for the inferred relationships (Fig. 2). The phylogenetic tree revealed a clear monophyly of genera within Pinaceae, such as Picea, Pinus, Larix, Abies, Keteleeria, Tsuga, Cedrus, and Pseudotsuga. Furthermore, three of the four recognized subfamilies of Pinaceae were recovered as monophyletic groups, with the exception of Laricoideae. Within Laricoideae, two main lineages were identified: “Pseudotsuga + Larix” clade and Cathaya genus. However, Cathaya genus showed a closer relationship to Picea (subfamily Piceoideae) than to the “Pseudotsuga + Larix” clade.

Within the “Pseudotsuga + Larix” clade, Pseudotsuga species formed a monophyletic group distinct from Larix. Notably, the newly sequenced cp genome of P. sinensis var. brevifolia (indicated in bold in the tree) clustered closely with P. sinensis var. brevifolia (GenBank accession no. NC_058308). Moreover, both accessions of P. sinensis var. brevifolia were placed near P. sinensis var. wilsoniana, supporting their taxonomic placement within the genus. This phylogenetic analysis provided valuable insights into the evolutionary relationships within Pinaceae and further validates the genetic distinctiveness of P. sinensis var. brevifolia based on cp genome sequences.

The cp genome of angiosperms is typically approximately in 150 kb with a quadripartite structure containing two IR regions (IR_A and IR_B, ~20 to 30 kb each), which separate the large and small single copy regions (Palmer, 1985; Wicke et al., 2011; Daniell et al., 2016). However, the cp genome of Pinaceae species only range from 107 to 120 kb due of their highly reduced IRs (Wu et al., 2011; Le et al., 2025). In this study, the overall length cp genomes of Pseudotsuga species ranged from 122,348–122,513 bp (Table 3) which was aligned with characteristics of Pinaceae species (Wu et al., 2011b; Le et al., 2025).

The IR regions play an essential role in maintaining the structural stability of cp genome by suppressing large-scale rearrangements and facilitating homologous recombination (Palmer et al., 1987; Nguyen et al., 2025). Although conserved, the IR regions exhibit remarkable evolutionary variation across plant lineages. For example, complete IR loss has occurred independently in several plant lineages, such as legumes of the subfamily Papilionoideae (Moghaddam et al., 2022) or Cupressaceae family (Le et al., 2025). In Pinaceae family, the IR regions are highly reduced, with an average size of 362 ± 101 bp (Lin et al., 2010). As member of Pinaceae, Pseudotsuga species process the extremely contracted IR regions. Particularly, the IR regions range from 204 to 349 bp in length and retain primarily the trnI-CAU gene and adjacent intergenic spacers, with conserved junctions across the IR/SC boundaries (Table 3). Besides, the sister genus Larix (Laricoideae subfamily) exhibits slightly larger IRs. For example, Larix cajanderi has an IR of 436 bp, which includes trnI-CAU and an 86 bp fragment of the psbA gene (Table 3). These observations indicate that the IR regions of Pseudotsuga are more contracted than those of Larix which represent a characteristic, highly conserved feature of the genus.

The type 1 repeat (T1R) and type 3 repeat (T3R) are remnant repeat regions that were main cause of large rearrangement in LSC region of the cp genomes of Pseudotsuga and other Pinaceae species (Wu et al., 2011b; Sudianto et al., 2016). Their size is much smaller than typical IRs (20–25 kb in angiosperm), but these repeats retain partial functions associated with also increase the diversity of cp genome forms. T1R and T3R repeats were reported to be “hotspots” for rearrangements in the cp genome of Pseudotsuga (Hipkins et al., 1995), Tsuga, and Abies (Tsumura et al., 2000). In our cp genome, two copies of T1R were detected (1,271 bp and 1,233 bp), together with T3Rs of 480 bp. The repeat configuration is shared among three Pseudotsuga plastomes (PV879952, NC_058308, and NC_016064), showing only minor length variation, which indicates that this repeat architecture is conserved within the genus. Likewise, P. sinensis var. sinensis harbors two T1R units (831 bp and 841 bp) and a T3R of 480 bp, further supporting that the co-occurrence of T1R and T3R is a conserved feature across Pseudotsuga, although T1R length may vary among lineages. In contrast, L. cajanderi possesses much shorter T1Rs (481 bp) and lacks T3R, highlighting lineage-specific differences in repeat organization within the subfamily Laricoideae. The maintenance of relatively conserved long T1Rs and T3Rs in Pseudotsuga may reflect unique evolutionary constraints or structural requirements of its plastome.

Multiple studies have provided substantial evidence for the monophyletic evolution of Pseudotsuga, a small genus within the Pinaceae (POWO, 2025). In our study, based on the currently available species, we confirmed the monophyly of the genus, with four species being validated. Our findings are consistent with previous research (Ran et al., 2018; Li et al., 2023). For example, the recent complete cp genome analysis of P. sinensis from Guizhou reported that P. sinensis is sister to P. sinensis var. wilsoniana (Li et al., 2023). Furthermore, when compared with results from transcriptomic data, our study is in agreement with the phylogenomic framework of Pinaceae proposed by Ran et al. (2018), which robustly places Pseudotsuga within the “pinoid” clade alongside Pinus, Larix, Cathaya, and Picea, while separating it from the “abietoid” clade (Abies, Cedrus, Keteleeria, Tsuga, Nothotsuga, and Pseudolarix) (Ran et al., 2018). In brief, these findings indicate that our results corroborate earlier phylogenetic, phylogeographic, and taxonomic conclusions.

Earlier work by Wei et al. (2010), using multiple chloroplast, mitochondrial, and nuclear genes, found strong support for the monophyly of East Asian and North American Pseudotsuga, suggesting an origin in North America followed by migration into Asia via the Bering land bridge (Wei et al., 2010). Our study, employing chloroplast protein-coding sequences, provides even supported values (bootstrap support = 99) and reveals clearer internal branching within the Asian clade. However, the lack of chloroplast genomic data from North American species (P. macrocarpa and P. menziesii) remains a limitation. Therefore, additional cp genome sequences from these related species are needed to rigorously test this hypothesis using chloroplast data.

In conclusion, we reaffirm that the several features of cp genome of Pseudotsuga: quadripartite structure with short IR regions, presence of a T1R and T3R, and loss of all plastid ndh genes, and contraction of IR regions signify the cp genome of all Pseudotsuga species. In addition, our plastid phylogenetic supports that the Pseudotsuga are monophyletic clade and a sister genus with Larix, which formed into subfamily Laricoideae. The complete cp genome sequence of Pseudotsuga sinensis var. brevifolia will be aided for further investigations of this endemic relict woody plant and for in-depth understanding of the evolutionary history of the coniferous cp genomes, especially for identifying species that serve in conservation in danger species.