Dracocephalum L. (Lamiaceae) is the second-largest genus within the subtribe Nepetinae, comprising more than 80 species worldwide (Chen et al., 2022; Rose et al., 2023). Morphologically, Dracocephalum is most similar to Nepeta L. but can be readily distinguished by its calyces with a thickened sinus-like fold between the bases of the calyx lobes (Harley et al., 2004). Many species of Dracocephalum are widely used in traditional medicine and herbal remedies around the world (Fathiazad and Hamedeyazdan, 2011).

The northern dragonhead, Dracocephalum ruyschiana L., is a perennial species distributed from Europe to Mongolia. In Europe, it is listed on Red Lists in several countries due to its declining population (Kleven et al., 2019). In Mongolia, this species is distributed in the northern forest areas (Baasanmunkh et al., 2022). As a member of the polyphenol-rich genus Dracocephalum, the aerial parts of D. ruyschiana, collected in Mongolia, are known to contain flavone tetra-glycosides and benzyl alcohol glycosides, which have antioxidant, antimicrobial activities, and anti-inflammatory effects (Selenge et al., 2013).

The complete chloroplast genomes (plastomes) of land plants have increasingly been sequenced in recent years. Plastids are essential organelles in plant cells, playing critical roles in growth and development (Howe et al., 2003). The plastomes in plants have a typical tetrad structure, consisting of a large single-copy (LSC), a small single-copy (SSC), and two copies of inverted repeats (IRa and IRb) (Wicke et al., 2011). Additionally, plastome sequences have been widely used in plant phylogenetic studies (e.g., Liu et al., 2023; Nyamgerel et al., 2024; Oyuntsetseg et al., 2024; Wang et al., 2024; Yuan et al., 2024). To date, plastome data from 11 Dracocephalum species have been sequenced and are available in the National Center for Biotechnology Information (NCBI) database (Yao et al., 2020; Zhao et al., 2021; Zhang et al., 2024).

This study presents the complete plastome sequence of D. ruyschiana from Mongolia and investigates its phylogenetic relationships within Dracocephalum, providing valuable resources for future research on this genus.

Fresh leaves of D. ruyschiana were collected from Bugant, Selenge province, Mongolia (49°24′47.5″N, 107°15′59.0″E). The specimens were deposited in the herbarium of the National University of Mongolia (UBU0038422). Detailed illustration photos of species were taken in the field surveys by S. Baasanmunkh and D. Munkhutlga.

Genomic DNA was extracted from silica gel-dried leaf material using the CTAB method (Doyle and Doyle, 1987). The sequencing library was constructed from the extracted DNA using the TruSeq DNA Nano Kit and the NextSeq 500 platform (Illumina, San Diego, CA, USA), following the manufacturer’s protocol. Trimmomatic v.0.36 (Bolger et al., 2014) was used to remove adapter sequences and low-quality reads to reduce bias. A base quality plot generated using FastQC v.0.11.5 (Antil et al., 2023) was used to check the overall quality of the data and show the range of quality values for each cycle. NOVOplasty v.4.1.0 was used to perform de novo assembly using various k-mers (Dierckxsens et al., 2016).

Genome annotation of D. ruyschiana was performed using the GeSeq web server (https://chlorobox.mpimp-golm.mpg.de/geseq.html) (Tillich et al., 2017) to predict gene locations. Protein-coding sequences were acurated using BLAST and tRNA was identified using tRNAscan-SE (Chan and Lowe, 2019). A circular map was visualized using the CPGAVAS2 web server (Liu et al., 2012). Long tandem repeats were identified with the TRF online tool (Benson, 1999), with a minimum alignment score of 50 and a maximum period size of 500; the identity of repeats was set to ≥ 90%. Simple sequence repeats (SSRs) were identified using the Microsatellite Identification Tool (MISA) web server (Beier et al., 2017), with minimum repeat thresholds set to 10, 5, 4, 3, 3, and 3 for mono-, di-, tri-, tetra-, penta-, and hexanucleotides, respectively.

The chloroplast genomes of 11 Dracocephalum species and two outgroups were retrieved from the NCBI database for phylogenetic reconstruction. The genomes were aligned using MAFFT v.7.490 (Katoh et al., 2002) as implemented in Geneious Prime 2024.0.5 (http://www.geneious.com). Phylogenetic relationships were determined through a maximum likelihood analysis performed in RaxML v.8.2.11 (Stamatakis, 2014) with 1,000 bootstrap replicates. The best-fitting model for nucleotide substitutions was determined using the Akaike information criterion in jModelTest v.2.1.1073 (Darriba et al., 2012), where the GTR+G+I model was selected. The resulting phylogenetic trees were visualized using FigTree v.1.4.2 (Rambaut, 2012).

The complete chloroplast genome of D. ruyschiana from Mongolia (Fig. 1) was sequenced for the first time. A total of 9.6 Gb of paired-end (150 bp) bases, comprising 63,838,854 reads, was obtained. After trimming, 8.5 Gb of high-quality bases and 56,528,706 reads were retained for assembly. The assembled chloroplast genome was 158,469 bp long and displayed a typical quadripartite structure (Fig. 2). It comprises an LSC region of 82,420bp, an SSC region of 17,830 bp, and two IRs of 25,373bp each (Fig. 2). The overall GC content is 37.7%, distributed as 43.1% in the IRs, 35.8% in the LSC, and 31.6% in the SSC regions. The genome sequence data was submitted to GenBank (NCBI) under the accession number PQ963003.

The chloroplast genome of D. ruyschiana encodes a total of 131 genes, including 8 rRNA, 37 tRNA, and 86 protein-coding genes. Among these, 4 rRNA, 7 tRNA, and 6 protein-coding genes are duplicated in the IR regions (Table 1). The predicted gene count in Dracocephalum species ranges from 125 (D. palmatum) to 133 (D. heterophyllum) (Zhao et al., 2021; Fu et al., 2022; Zhang et al., 2024). The plastome of D. ruyschiana contains 10 cis-splicing genes with introns, two of which have two introns (Fig. 3) and one trans-splicing gene with three exons (Fig. 4). The junction analysis revealed that the rps19 and ycf1 genes span LSC/IRa and IRa/SSC junctions, respectively.

Chloroplast microsatellites can serve as valuable markers for ecological and evolutionary studies due to the non-recombinant, uniparentally inherited nature of organelle genomes (Provan et al., 2001). A total of 34 SSRs were identified, primarily consisting of mononucleotide motifs (18), followed by di-nucleotide (6), tetra-nucleotide (5), tri-nucleotide (2), penta-nucleotide (2), and hexa-nucleotide (1) motif repeats, mostly found in the intergenic spacer region (Fig. 2). Additionally, we identified 24 tandem repeats that generally ranged from 9 to 31 bp in length. The number of SSRs in D. ruyschiana is lower than that in other Dracocephalum species, with the highest count (91 SSRs) found in D. moldavica (Fu et al., 2022). These SSR markers can be utilized to assess genetic variation in population genetic studies, which are essential for the conservation of endangered plants.

The genome alignment included available Dracocephalum species from the NCBI database, encompassing 158,397 bp sites, of which 6,653 were variable. Phylogenetic analysis revealed that Dracocephalum species form a monophyletic group, with D. ruyschiana closely related to D. arguments, supported by strong bootstrap values (Fig. 5). A previous phylogenetic study using nuclear ITS and external transcribed spacer regions, plastid rpl32-trnL, trnL-trnF, ycf1, and ycf1-rps15, and two low-copy nuclear markers (Chen et al., 2022) also clustered Dracocephalum ruyschiana and D. arguments together, suggesting a recent common ancestry for these two species. Additionally, the morphological diagnosis of D. ruyschiana is quite similar to that of D. argunense but differs in its stem being sparsely minute hairy toward vs. stem subglabrous, calyx minutely hairy toward base vs. calyx minutely hairy throughout, and the size of the corolla (Li and Hedge, 1994). Further studies will compare the morphological, physiological, and genetic characteristics of these two species to better understand their evolutionary history.

This study provides valuable genomic resources to enhance research on the Dracocephalum genus, particularly in understanding phylogenetic relationships and chloroplast genome evolution. To gain deeper insights into the evolutionary history of this genus, future studies should include comparative genomic analyses with a broader sampling of Dracocephalum species and closely related genera.