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The genus Allium is a well-known model system for cytological research, and its cytological characteristics are considered important for species identification. In this study, we counted chromosome numbers of 21 populations from 12 taxa comprising seven sections (Butomissa, Sacculiferum, Schoenoprasum, Oreiprason, Reticulatobulbosa, Rhizirideum, Tenuissima) and five subgenera (Butomissa, Cepa, Polyprason, Reticulatobulbosa, Rhizirideum) from Kyrgyzstan, Mongolia, and South Korea. The chromosome numbers varied with 2n=16 or 2n=32. New ploidy levels were found in A. karelinii (2n=2x=16) and A. burjaticum (2n=2x=16) from Kyrgyzstan and Mongolia, respectively. Photomicrophotographs of metaphase chromosomes and karyotype analyses of all Allium taxa studied are provided.

Thymus quinquecostatus is the economically important plants and herbal resource of the Korean traditional medicine, belonging to the mint family (Lamiaceae, Labiatae). In this study, we determined the complete chloroplast genome sequence of T. quinquecostatus var. quinquecostatus using the Illumina MiSeq platform and reconstructed the phylogenetic tree of the selected Lamiaceae taxa. Results showed that the chloroplast genome of T. quinquecostatus var. quinquecostatus is 151,802 bp in size, with a large single-copy (LSC) region (82,921 bp), a small single-copy (SSC) region (17,669 bp), and two inverted repeat (IR) regions (IRa and IRb; 25,606 bp each). Additionally, the chloroplast genome contains 114 unique genes, including 80 protein-coding, four ribosomal RNA, and 30 transfer RNA genes. Phylogenetic analysis revealed that T. quinquecostatus var. quinquecostatus is most closely related to genera Mentha-Clinopodium within the subfamily.

Scrophularia have traditionally been used as herbal medicines to treat neuritis, sore throats, and laryngitis. In particular, S. takesimensis, a Korean endemic species with restricted distribution on Ulleung Island, holds significant resource and genetic value. However, its pharmacological properties have not been thoroughly evaluated. Thus, we provide detailed morphological characteristics and genomic information for S. takesimensis in this study. Moreover, its pharmacological activity was evaluated in an ovalbumin-induced asthma rat model, using extracts of S. takesimensis roots (100 or 200 mg/kg). The distinguishing features of S. takesimensis from related species include the presence or absence of stem wings, leaf shape, and habitat. The chloroplast (cp) genome of this species is 152,420 bp long and exhibits a conserved quadripartite structure. A total of 114 genes were identified, which included 80 protein-coding genes, 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. The gene order, content, and orientation of the S. takesimensis cp genome was highly conserved and consistent with the general structure observed in S. buergeriana and S. ningpoensis cp genomes. Confirming the anti-inflammatory effects of S. takesimensis extract (STE) using an established mouse model of ovalbumin-induced asthma, we observed reduced asthmatic phenotypes, including inflammatory cell infiltration, mucus production, and suppression of T helper 2 (Th2) cell. Furthermore, STE treatment reduced Th2 cell activation and differentiation. This study underscores the medicinal value of S. takesimensis. The importance of preserving S. takesimensis was revealed and crucial insights were provided for further research on its utilization as a medicinal resource.

The genus Philadelphus (Hydrangeaceae) in South Korea is critically revised based on macro- and micro-morphological characteristics from over 1,000 herbarium specimens, and fresh material from wild populations. Findings show that: (1) The presence/absence and the density of the hairs on the disk, pistil, calyx, leaves, and petiole and the degree of division of the style are insufficient characters for recognizing species in Philadelphus. (2) Philadelphus koreanus, P. robustus, P. scaber, and P. seoulensis were confirmed to be synonyms of P. tenuifolius. (3) Philadelphus lasiogynus is newly synonymized into P. tenuifolius based on the type material. (4) Philadelphus pekinensis has been excluded from the Korean flora because of prior misidentifications of herbarium specimens, whose identity is very sparsely pubescent P. tenuifolius. It is therefore confirmed that only P. tenuifolius has been recorded in South Korea. Detailed morphological descriptions, photographs, illustrations, distribution maps, and information on the herbarium materials of P. tenuifolius in South Korea are provided.

Abstract Osteoporosis affects millions of people worldwide. As such, this study assessed the macrophage-dependent in vitro anti-osteoporosis, phytochemical profile and hepatotoxicity effects in zebrafish larvae of the stem bark extracts of P. africana. Mouse bone marrow macrophages (BMM) cells were plated in 96-well plates and treated with P. africana methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 µg/ml for 24 h. The osteoclast tartrate-resistant acid phosphatase (TRAP) activity and cell viability were measured. Lipopolysaccharides (LPS) induced Nitrite (NO) and interleukin-6 (IL-6) production inhibitory effects of P. africana bark extracts (Methanolic, 150 µg/ml) and β-sitosterol (100 µM) were conducted using RAW 264.7 cells. Additionally, inhibition of IL-1β secretion and TRAP activity were determined for chlorogenic acid, catechin, naringenin and β-sitosterol. For toxicity study, zebrafish larvae were exposed to different concentrations of 25, 50, 100, and 200 µg/ml P. africana methanolic, ethanolic and water bark extracts. Dimethyl sulfoxide (0.05%) was used as a negative control and tamoxifen (5 µM) and dexamethasone (40 µM or 80 µM) were positive controls. The methanolic P. africana extracts significantly inhibited (p

Abstract The pollen and orbicule morphology of the Korean Piperales (Aristolochia, Asarum, Houttuynia, Piper, and Saururus) were investigated via scanning electron microscopy. Piperales pollen is a monad, its size ranging from very small to large (P = 7.78–51.4 μm, E = 6.68–43.1 μm), and having a mainly circular to sub-circular shape. The aperture type is constant in the genus [inaperturate (Aristolochia), tri to pentaporate (Asarum), and monosulcate (Houttuynia, Piper, and Saururus)]. There are four distinct types of exine ornamentation: Fossulate with perforate, microreticulate with gemmae, microperforate with granula, and microechinate. The orbicules (minute sporopollenin granules) were observed in all studied taxa and thus, may be a possible symplesiomorphic characteristic of Piperales. Further, the observed orbicule surface ornamentation was similar to pollen exine patterns, for example muri, gemmae, or granula. This resemblance between orbicule and pollen exine ornamentation may imply a similar biosynthesis pattern of sporopollenin of pollen exine and orbicules. The phenogram resulting from a cluster analysis using palynological characters was generally consistent with the known molecular phylogeny of Piperales. This initial study will help understand the palynological diversity and provide detailed information of pollen and orbicule characteristics in Piperales.

Davallia mariesii T. Moore ex Baker, a member of the section Trogostolon (Copel.) M. Kato and Tsutsumi (Davalliaceae M.R. Schomb.), is a lithophytic or epiphytic herb that grows on rocks and tree trunks in montane forests. This study analyzed the morphological, anatomical, micromorphological, and palynological characteristics of D. mariesii using a digital slide scanner and a field-emission scanning electron microscope and presented an expanded and updated description. A circumendodermal band was observed in the anatomical structure of the stipe, making D. mariesii the second species in the family Davalliaceae with such a band. The frond anatomical studies revealed that the epidermal cells of the indusium were thicker than those of the epidermis on both sides and that hypostomatic fronds with stomata chambers were present. Diacytic, anisocytic, and tetracytic stomatal complexes were observed on abaxial surfaces. The indusia covered numerous sporangia. Leptosporangium consisted of an apical cap, a basal cap, an annulus, and a stalk. The spore had an ellipsoidal outline, a monolete aperture, and verrucae with colliculate ornamentation. The obtained results provide systematic data for the phylogeny of Davalliaceae and establish a basis for future taxonomic delimitation of other taxa.

Allium section Rhizirideum is reviewed for South Korea and neighboring northeastern China based on critical observation of wild populations and herbarium materials. Species delimitations are re-evaluated on the basis of morphological and somatic chromosome numbers, resulting in the recognition of five species. Allium dumebuchum from Ulleungdo Island, South Korea, is described as a new species. This species is most similar to A. senescens due to its habits, but is clearly distinguished particularly by its rhomboid scapes in cross-secion, light purple perianth color, entire and narrowly triangular inner filaments, and flowering season from late September. One previously recognized species is placed into synonymy: A. pseudosenescens (under A. senescens). Photographs and a key to species of Allium section Rhizirideum in South Korea and northeastern China are provided in addition to information on nomenclatural types, synonymies, chromosome numbers, distribution, and specimens examined.

The medicinal plant, Aspilia africana, has been traditionally used in several African countries to treat many diseases such as tuberculosis, cough, inflammation, malaria, osteoporosis, and diabetes. In this study, we developed a protocol for in vitro propagation of A. africana using indirect shoot organogenesis from leaf and root explants of in vitro-grown seedlings and assessed the tissues at different developmental stages. The highest callus induction (91.9 ± 2.96%) from leaf explants was in the Murashige and Skoog (MS) medium augmented with 1.0 mg/L 6-Benzylaminopurine (BAP) and 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) while from root explants, the highest callus induction (92.6 ± 2.80%) was in the same plant tissue culture medium augmented with 0.5 mg/L BAP and 1.0 mg/L 2,4-D. The best shoot regeneration capacity from leaf-derived calli (i.e., 80.0 ± 6.23% regeneration percentage and 12.0 ± 6.23 shoots per callus) was obtained in medium augmented with 1.0 mg/L BAP and 0.05 mg/L α-Naphthaleneacetic acid (NAA); the best regeneration capacity for root-derived calli (i.e., 86.7 ± 6.24% shoot regeneration percentage and 14.7 ± 1.11 shoots per callus) was obtained in the MS medium augmented with 1.0 mg/L BAP, 0.05 mg/L NAA, and 0.1 mg/L Thidiazuron (TDZ). Regenerated plantlets developed a robust root system in 1/2 MS medium augmented with 0.1 mg/L NAA and had a survival rate of 93.6% at acclimatization. The in vitro regenerated stem tissue was fully differentiated, while the young leaf tissue consisted of largely unorganized and poorly differentiated cells with large intercellular airspaces typical of in vitro leaf tissues. Our study established a protocol for the indirect regeneration of A. africana and offers a basis for its domestication, large-scale multiplication, and germplasm preservation. To the best of our knowledge, this is the first study to develop an indirect regeneration protocol for A. africana and conduct anatomical assessment through the different stages of development from callus to a fully developed plantlet.

A taxonomic revision of Korean endemic Spiraea chartacea is presented compared with its related three species, S. blumei, S. turczaninowii (≡ S. pubescens Turcz.), and S. chinensis, of section Chamaedryon in Korea. Species delimitations are re-evaluated based on critical observations of wild populations and extensive herbarium material. The shape of leaf, the type and distribution of trichome in stem, leaf, peduncle, pedicel, hypanthium, sepal, carpel, and follicle are useful diagnostic traits at the specific level. Almost all morphological and leaf anatomical observations of S. chartacea belong within the range of S. turczaninowii. Therefore, S. turczaninowii is placed into synonymy of S. chartacea by priority. Photographs and a key to three species of Spiraea section Chamaedryon are provided in addition to complete descriptions including information on nomenclatural types, synonymies, chromosome numbers, distributions, and specimens examined. Keywords: Anatomy, Morphology, Rosaceae, Section Chamaedryon, Spiraea L.

Aspilia africana (Pers.) C. D. Adams is an important medicinal plant, that has been used as traditional medicine in many African countries for the treatment of various health problems, including inflammatory conditions, osteoporosis, tuberculosis, cough, measles, diabetes, diarrhea, malaria, and wounds. We developed an efficient and reproducible protocol for in vitro regeneration of A. africana from nodes. We assessed the effects of plant tissue culture media on A. africana growth, cytokinins for in vitro shoot regeneration and proliferation, and auxins for the rooting of regenerated shoots. Furthermore, chlorophyll content, photosynthetic rates, anatomy (leaves, stems, and roots), and Fourier transform near-infrared (FT-NIR) spectra (leaves, stems, and roots) of the in vitro regenerated and maternal A. africana plants were compared. Murashige and Skoog media, containing vitamins fortified with benzylaminopurine (BA, 1.0 mg/l), regenerated the highest number of shoots (13.0 ± 0.424) from A. africana nodal segments. 1-naphthaleneacetic acid (NAA, 0.1 mg/l) produced up to 13.10 ± 0.873 roots, 136.35 ± 4.316 mm length, and was the most efficient for rooting. During acclimatization, the in vitro regenerated A. africana plants had a survival rate of 95.7%, displaying normal morphology and growth features. In vitro regenerated and mother A. africana plants had similar chlorophyll contents, photosynthetic rates, stem and root anatomies, and FT-NIR spectra of the leaf, stem, and roots. The established regeneration protocol could be used for large-scale multiplication of the plant within a short time, thus substantially contributing to its rapid propagation and germplasm preservation, in addition to providing a basis for the domestication of this useful, high-value medicinal plant.

Trichosanthes is a genus in Cucurbitaceae comprising 90–100 species. Trichosanthes species are valuable as herbaceous medicinal ingredients. The fruits, seeds, and roots of species such as T. kirilowii and T. rosthornii are used in Korean traditional herbal medicines. T. rosthornii is only found in China, whereas in South Korea two varieties, T. kirilowii var. kirilowii and T. kirilowii var. japonica, are distributed. T. kirilowii var. kirilowii and T. kirilowii var. japonica have different fruit and leaf shapes but are recognized as belonging to the same species. Furthermore, although its members have herbal medicine applications, genomic information of the genus is still limited. The broad goals of this study were (i) to evaluate the taxonomy of Trichosanthes using plastid phylogenomic data and (ii) provide molecular markers specific for T. kirilowii var. kirilowii and T. kirilowii var. japonica, as these have differences in their pharmacological effectiveness and thus should not be confused and adulterated. Comparison of five Trichosanthes plastid genomes revealed locally divergent regions, mainly within intergenic spacer regions (trnT-UGU–trnL-UAA: marker name Tri, rrn4.5–rrn5: TRr, trnE-UUC–trnT-GGU: TRtt). Using these three markers as DNA-barcodes for important herbal medicine species in Trichosanthes, the identity of Trichosanthes material in commercial medicinal products in South Korea could be successfully determined. Phylogenetic analysis of the five Trichosanthes species revealed that the species are clustered within tribe Sicyoeae. T. kirilowii var. kirilowii and T. rosthornii formed a clade with T. kirilowii var. japonica as their sister group. As T. kirilowii in its current circumscription is paraphyletic and as the two varieties can be readily distinguished morphologically (e.g., in leaf shape), T. kirilowii var. japonica should be treated (again) as an independent species, T. japonica.

The genera Arnebia and Lithospermum (Lithospermeae-Boraginaceae) comprise 25–30 and 50–60 species, respectively. Some of them are economically valuable, as their roots frequently contain a purple-red dye used in the cosmetic industry. Furthermore, dried roots of Arnebia euchroma, A. guttata, and Lithospermum erythrorhizon, which have been designated Lithospermi Radix, are used as traditional Korean herbal medicine. This study is the first report on the floral micromorphology and complete chloroplast (cp) genome sequences of A. guttata (including A. tibetana), A. euchroma, and L. erythrorhizon. We reveal great diversity in floral epidermal cell patterns, gynoecium, and structure of trichomes. The cp genomes were 149,361–150,465 bp in length, with conserved quadripartite structures. In total, 112 genes were identified, including 78 protein-coding regions, 30 tRNA genes, and four rRNA genes. Gene order, content, and orientation were highly conserved and were consistent with the general structure of angiosperm cp genomes. Comparison of the four cp genomes revealed locally divergent regions, mainly within intergenic spacer regions (atpH-atpI, petN-psbM, rbcL-psaI, ycf4-cemA, ndhF-rpl32, and ndhC-trnV-UAC). To facilitate species identification, we developed molecular markers psaA- ycf3 (PSY), trnI-CAU- ycf2 (TCY), and ndhC-trnV-UAC (NCTV) based on divergence hotspots. High-resolution phylogenetic analysis revealed clear clustering and a close relationship of Arnebia to its Lithospermum sister group, which was supported by strong bootstrap values and posterior probabilities. Overall, gynoecium characteristics and genetic distance of cp genomes suggest that A. tibetana, might be recognized as an independent species rather than a synonym of A. guttata. The present morphological and cp genomic results provide useful information for future studies, such as taxonomic, phylogenetic, and evolutionary analysis of Boraginaceae.

Prunus africana is an endangered medicinal plant and hence new propagation methods are urgently required to increase its populations. Unfortunately, propagation through seeds is challenging due to its long flowering cycle and recalcitrant seeds. We developed a protocol for micropropagation using nodal segment explants. A woody plant medium supplemented with vitamins, 15 g L−1 sucrose, and 1.0 mg L−1 6-benzylaminopurine (BAP) supported the optimum rate (100%) of axillary shoot initiation. Supplementation with 15 g L−1 sucrose and 1.5 mg L−1 indole-3-acetic acid (IAA) provided the optimum rate (75%) of root initiation. Rooted plantlets were successfully planted in sterilized horticultural soil containing perlite (2:1 v/v) and the survival rate was 98% following acclimatization. The photosynthetic rate assessed using FlourPen FP110 series showed that the ratio of variable fluorescence to maximum fluorescence mean value for in vitro regenerated P. africana (0.830 ± 0.0008) was similar to that of the maternal P. africana plant (0.825 ± 0.005), indicating similarity in their photosynthetic performance; a pivotal process for growth and development. The Fourier transform near-IR (FT-NIR) spectrometer analysis of the in vitro regenerated and the maternal P. africana plant samples exhibited homogeneity in the absorbance peaks at 8,273, 6,344, and 4,938–4,500 cm−1 associated with lipids, starch, and proteins. The genetic fidelity of regenerated plants was confirmed using the randomly amplified polymorphic DNA (RAPD) technique. Our protocol is suitable for use in large-scale P. africana to meet the increasing demands for it in the global market.

Floral micromorphology and pollen morphology of two Leonurus (Lamiaceae) species were examined and compared using scanning electron microscopy to evaluate the diagnostic value of these features to facilitate future studies on floral biology and taxonomy. Floral epidermal patterns were similar in both species, with the conical to central-conical epidermal cells on the adaxial side of the lower lip (corolla). Capitate, peltate, scale-like glandular, and non-glandular trichomes were distributed on the surface of the floral organs. Notably, scale-like anther glands and floral stomata were found on the anthers and abaxial side of the calyx, respectively. Pollen grains had bi-reticulate exine with angular primary lumina and rounded secondary lumina. These characteristics provide indirect evidence of a close association between plant-pollinator interactions and effective pollination. In addition, quantitative traits of pollen grains and trichome types on the adaxial side of the lip differed between the two species. These characteristics may have diagnostic and taxonomic value for the genus Leonurus and family Lamiaceae.

Scrophulariae Radix, derived from the dried roots of Scrophularia ningpoensis Hemsl. or S. buergeriana Miq, is a traditional herbal medicine used in Asia to treat rheumatism, arthritis, and pharyngalgia. However, the effects of Scrophularia buergeriana, S. koraeinsis, and S. takesimensis on osteoclast formation and bone resorption remain unclear. In this study, we investigated the morphological characteristics and harpagoside content of S. buergeriana, S. koraiensis, and S. takesimensis, and compared the effects of ethanol extracts of these species using nuclear factor (NF)-κB ligand (RANKL)-mediated osteoclast differentiation. The harpagoside content of the three Scrophularia species was analyzed by high-performance liquid chromatography–mass spectrometry (HPLC/MS). Their therapeutic effects were evaluated by tartrate-resistant acid phosphatase (TRAP)-positive cell formation and bone resorption in bone marrow-derived macrophages (BMMs) harvested from ICR mice. We confirmed the presence of harpagoside in the Scrophularia species. The harpagoside content of S. buergeriana, S. koraiensis, and S. takesimensis was 1.94 ± 0.24 mg/g, 6.47 ± 0.02 mg/g, and 5.50 ± 0.02 mg/g, respectively. Treatment of BMMs with extracts of the three Scrophularia species inhibited TRAP-positive cell formation in a dose-dependent manner. The area of hydroxyapatite-absorbed osteoclasts was markedly decreased after treatment with the three Scrophularia species extracts. Our results indicated that the three species of the genus Scrophularia might exert preventive effects on bone disorders by inhibiting osteoclast differentiation and bone resorption, suggesting that these species may have medicinal and functional value.

To guarantee the safety and efficacy of herbal medicines, accurate identification and quality evaluation are crucial. The ripe dried seeds of Cuscuta australis R.Br. and C. chinensis Lam. are known as Cuscutae Semen (CS) and are widely consumed in Northeast Asia; however, the seeds of other species can be misidentified as CS owing to morphological similarities, leading to misuse. In this report, we propose a multilateral strategy combining microscopic techniques with statistical analysis and DNA barcoding using a genus-specific primer to facilitate the identification and authentication of CS. Morphology-based identification using microscopy revealed that the useful diagnostic characteristics included general shape, embryo exudation, hairiness, and testa ornamentation, which were used to develop an effective identification key. In addition, we conducted DNA barcoding-based identification to ensure accurate authentication. A novel DNA barcode primer was produced from the chloroplast rbcL gene by comparative analysis using Cuscuta chloroplast genome sequences, which allowed four Cuscuta species and adulterants to be discriminated completely. Therefore, this investigation overcame the limitations of universal DNA barcodes for Cuscuta species with high variability. We believe that this integrated approach will enable CS to be differentiated from other species, thereby improving its quality control and product safety in medicinal markets.

Dimocarpus longan, Litchi chinensis, and Nephelium lappaceum are commercially valuable subtropical and tropical fruits of the Sapindaceae family. Arillus and seeds of the three species have very similar morphologies; however, the arillus of D. longan is used as the herbal medicine Longan Arillus and seeds of L. chinensis are used as Litchi Semen in Korean and Chinese pharmacopoeias. The adulteration of herbal medicines with inauthentic species, including the use of Aril and seed fractions acquired from a single species for two herbal medicines (e.g., Longan Arillus and Litchi Semen), is often driven by economic motives. DNA markers are a tool for the detection of adulterants in commercial products. To establish rapid and reliable assays for the genetic identification of authentic Longan Arillus and Litchi Semen, we developed DNA markers with high specificity and sensitivity based on internal transcribed spacer (ITS) sequences. The newly developed DNA markers and multiplex PCR assay may contribute to efforts to protect against adulteration, quality control, and the standardization of herbal medicines.

A comparative study of the leaf micromorphology of Korean Piperales, including medicinal materials, was performed through light microscopy and scanning electron microscopy, to evaluate their taxonomic significance. Piperales possessed both amphistomatic and hypostomatic leaves. The epidermal area ranged from 38 to 5077 μm2, and the stomatal area ranged from 201 to 2129 μm2. The stomatal index on the abaxial surface was higher than that on the abaxial surface. Anomocytic stomata occurred most commonly, but actinocytic, anisocytic, tetracytic, and staurocytic stomata were also found in certain taxa. Secretory idioblasts were found on all taxa studied except Aristolochia. Three main types of trichomes were defined—(1) glandular trichome; (2) simple multicellular trichome; and (3) two-armed multicellular Y-shaped trichome. Although the quantitative data on its own had somewhat limited taxonomic value, the various qualitative characteristics (e.g., epidermal surfaces, stomata types and positions, trichome types and density, and secretory idioblast types) had great taxonomic value. These characteristics might be taxonomically relevant and useful for developing an identification key. Additionally, we evaluated and supported the previous taxonomic system of Korean Asarum, using leaf micromorphological characteristics. Finally, through the application for authentication of herbal medicine, we revealed that leaf micromorphological characteristics can be used for accurate authentication.

Disporum sessile roots are used as a medicinal herb. To ensure the accurate identification of D. sessile, we sequenced its chloroplast genome using the Illumina MiSeq platform. Results showed that the chloroplast genome of D. sessile is 159,102 bp in size, with a large single-copy (LSC) region (85,456 bp), a small single-copy (SSC) region (17,108 bp), and two inverted repeat (IR) regions (IRa and IRb; 28,269 bp each). Additionally, the chloroplast genome contains 112 unique genes, including 78 protein-coding, four ribosomal RNA, and 30 transfer RNA genes. Phylogenetic analysis revealed that D. sessile is most closely related to Colchicum autumnale.

Ipomoea L. is the largest genus within the Convolvulaceae and contains 600–700 species. Ipomoea species (morning glories) are economically valuable as horticultural species and scientifically valuable as ecological model plants to investigate mating systems, molecular evolution, and both plant–herbivore and plant–parasite interactions. Furthermore, the dried seeds of I. nil or I. purpurea are used in Korean traditional herbal medicines. In this study, chloroplast (cp) genomes were sequenced from six Ipomoea species, namely, I. nil and I. purpurea and, for the first time, I. triloba, I. lacunosa, I. hederacea, and I. hederacea var. integriuscula. The cp genomes were 161,354–161,750 bp in length and exhibited conserved quadripartite structures. In total, 112 genes were identified, including 78 protein-coding regions, 30 transfer RNA genes, and 4 ribosomal RNA genes. The gene order, content, and orientation of the six Ipomoea cp genomes were highly conserved and were consistent with the general structure of angiosperm cp genomes. Comparison of the six Ipomoea cp genomes revealed locally divergent regions, mainly within intergenic spacer regions (petN-psbM, trnI-CAU-ycf2, ndhH-ndhF, psbC-trnS, and ccsA-ndhD). In addition, the protein-coding genes accD, cemA, and ycf2 exhibited high sequence variability and were under positive selection (Ka/Ks > 1), indicating adaptive evolution to the environment within the Ipomoea genus. Phylogenetic analysis of the six Ipomoea species revealed that these species clustered according to the APG IV system. In particular, I. nil and I. hederacea had monophyletic positions, with I. purpurea as a sister. I. triloba and I. lacunosa in the section Batatas and I. hederacea and I. hederacea var. integriuscula in the section Quamoclit were supported in this study with strong bootstrap values and posterior probabilities. We uncovered high-resolution phylogenetic relationships between Ipomoeeae. Finally, indel markers (IPOTY and IPOYCF) were developed for the discrimination of the important herbal medicine species I. nil and I. purpurea. The cp genomes and analyses in this study provide useful information for taxonomic, phylogenetic, and evolutionary analysis of the Ipomoea genome, and the indel markers will be useful for authentication of herbal medicines.

Achyranthes bidentata Blume is an important herbal medicine. However, little genetic information about Achyranthes is available. To facilitate species identification, we sequenced its complete chloroplast genome using the Illumina MiSeq platform. The data show that the chloroplast genome of A. bidentata is 151,441 bp in size and comprises an 83,890 bp large single-copy region, a 17,261 bp small single-copy region, and two inverted repeat (IR) regions, IRa and IRb (each 25,145 bp). The genome contains 112 unique genes, including 79 protein-coding genes, 4 ribosomal RNAs genes, and 30 transfer RNAs genes. Phylogenetic analysis revealed that A. bidentata is most closely related to Alternanthera philoxeroides.

Actaea (Ranunculaceae; syn. Cimicifuga) is a controversial and complex genus. Dried rhizomes of Actaea species are used as Korean traditional herbal medicine. Although Actaea species are valuable, given their taxonomic classification and medicinal properties, sequence information of Actaea species is limited. In this study, we determined the complete chloroplast (cp) genome sequences of three Actaea species, including A. simplex, A. dahurica, and A. biternata. The cp genomes of these species varied in length from 159,523 to 159,789 bp and contained 112 unique functional genes, including 78 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. Gene order, orientation, and content were well conserved in the three cp genomes. Comparative sequence analysis revealed the presence of hotspots, including ndhC-trnV-UAC, in Actaea cp genomes. High-resolution phylogenetic relationships were established among Actaea species based on cp genome sequences. Actaea species were clustered into each Actaea section, consistent with the Angiosperm Phylogeny Group (APG) IV system of classification. We also developed a novel indel marker, based on copy number variation of tandem repeats, to facilitate the authentication of the herbal medicine Cimicifugae Rhizoma. The availability Actaea cp genomes will provide abundant information for the taxonomic and phylogenetic analyses of Actaea species, and the Actaea (ACT) indel marker will be useful for the authentication of the herbal medicine.

The distribution of native plants of Cheondeungsan Mountain (807 m, N 37°05'00“–37°05'30”, E 128°00'0“–128°02'0”) in Chungcheongbuk-do was determined and the major flora were identified. During field investigations carried out from May 2011 to October 2011, 87 families, 254 genera, and 369 taxonomic groups (327 species, 4 subspecies, 33 varieties, and 5 forms) were confirmed, and the distribution of 219 taxonomic groups was discovered for the first time. The distribution of four endemic plants of Korea, including Ajuga spectabilis Nakai and Salvia chanryoenica Nakai, and that of Penthorum chinense Pursh, a Grade V specific plant species, was found. There were 20 taxa of naturalized plants at Cheondeungsan; the growth and development of plants that are harmful to the ecosystem, such as Ambrosia artemisiifolia L., Ambrosia trifida L., Eupatorium rugosum Houtt., and Aster pilosus Willd., was observed around the forest paths and lowlands.

Cnidium officinale (Ligusticum officinale) is an important herbal medicine. To facilitate species identification, we determined the complete chloroplast genome of C. officinale using the Illumina MiSeq platform. The genome was 148,518 bp in length, comprising a large single copy (LSC) region of 93,977 bp, a small single copy (SSC) region of 17,607 bp, and two inverted repeat regions (IRa and IRb) of 18,467 bp each. The genome contains 113 unique genes, including 79 protein-coding genes, four ribosomal RNAs (rRNAs), and 30 transfer RNAs (tRNAs). Phylogenetic analysis revealed that C. officinale is most closely related to L. tenuissium, with high bootstrap values.

Cuscuta pentagona is a parasitic plant whose seeds are often mixed with the seeds of medicinal Cuscuta species. To facilitate the identification of C. pentagona seeds, we generated the complete chloroplast genome sequence of C. pentagona Engelm. using the Illumina MiSeq platform. The complete chloroplast genome is 86,380 bp long, with a 50,958 bp LSC region, a 7022 bp SSC region, and two inverted repeat (IRa and IRb) regions comprising 14,200 bp. The chloroplast genome consists of 85 unique genes, 57 protein-coding genes, four ribosomal RNA (rRNA) genes, and 24 transfer RNA (tRNA) genes. Two gene families, NADH oxidoreductases and RNA polymerase-related genes, are absent in this genome. Phylogenetic analysis revealed that C. pentagona is closely related to C. reflexa and C. exaltata, with strong support values.

Sanguisorba × tenuifolia Fisch. ex Link is an important herbal medicine. To facilitate species identification, we sequenced its complete chloroplast genome using the Illumina MiSeq platform. The data show that the chloroplast genome of S. × tenuifolia is 155,403 bp in size, comprising an 85,525 bp large single-copy (LSC) region, a 18,726 bp small single-copy (SSC) region, and two inverted repeats (IR) regions, IRa and IRb (each 25,576 bp). The genome contains 112 unique genes, including 79 protein-coding genes, four ribosomal RNAs genes, and 30 transfer RNAs genes. Phylogenetic analysis revealed that S. × tenuifolia is most closely related to Hagenia abyssinica.

Dried rhizomes of Actaea heracleifolia, used as a traditional Korean herbal medicine, are frequently adulterated with other plant species. For accurate species identification, we sequenced the complete chloroplast genome of A. heracleifolia using Illumina MiSeq. A. heracleifolia harbours a 159,578 bp chloroplast genome comprising a large single-copy region (88,770 bp), small single-copy region (18,070 bp) and two inverted repeat (IR) regions (IRa and IRb; each 26,519 bp). The chloroplast genome contains 112 unique genes, including 78 protein-coding genes, 4 ribosomal RNA genes, and 30 transfer RNA genes. Phylogenetic analysis revealed that A. heracleifolia was closely related to Gymnaconitum gymnandrum.

Previous studies have suggested that exuviae can be used for the identification of cicada species, but the precise characteristics that differ among species have not been determined. Thus, we performed the first comparative analyses of the leg morphology, ultrastructure, and mitochondrial DNA sequences of exuviae of four dominant cicada species in Korea, Hyalessa maculaticollis (Motschulsky, 1866), Meimuna opalifera (Walker, 1850), Platypleura kaempferi (Fabricius, 1794) and Cryptotympana atrata (Fabricius, 1775), the source of Cicadidae Periostracum, a well-known traditional medicine. A morphological analysis revealed that the profemur length, femoral tooth angle, and distance between the intermediate and last tooth of the femoral comb are useful characteristics for identification. We also evaluated the usefulness of the size, degree of reflex, and number of spines on the mid-legs and hind legs as diagnostic features. An ultrastructural study showed that Meimuna opalifera has a unique surface pattern on the legs. The sequences obtained using exuviae were identical to previously obtained sequences for adult tissues. Moreover, in a phylogenetic analysis using CO1 sequences, each species formed a monophyletic cluster with high bootstrap support. Accordingly, multiple methodological approaches using exuviae might provide highly reliable identification tools. The integrative data provide useful characteristics for the exuviae-based identification of closely related species and for further taxonomic and systematic studies of Cicadinae.

The genus Angelica (Apiaceae) comprises valuable herbal medicines. In this study, we determined the complete chloroplast (CP) genome sequence of A. polymorpha and compared it with that of Ligusticum officinale (GenBank accession no. NC039760). The CP genomes of A. polymorpha and L. officinale were 148,430 and 147,127 bp in length, respectively, with 37.6% GC content. Both CP genomes harbored 113 unique functional genes, including 79 protein-coding, four rRNA, and 30 tRNA genes. Comparative analysis of the two CP genomes revealed conserved genome structure, gene content, and gene order. However, highly variable regions, sufficient to distinguish between A. polymorpha and L. officinale, were identified in hypothetical chloroplast open reading frame1 (ycf1) and ycf2 genic regions. Nucleotide diversity (Pi) analysis indicated that ycf4–chloroplast envelope membrane protein (cemA) intergenic region was highly variable between the two species. Phylogenetic analysis revealed that A. polymorpha and L. officinale were well clustered at family Apiaceae. The ycf4-cemA intergenic region in A. polymorpha carried a 418 bp deletion compared with L. officinale. This region was used for the development of a novel indel marker, LYCE, which successfully discriminated between A. polymorpha and L. officinale accessions. Our results provide important taxonomic and phylogenetic information on herbal medicines and facilitate their authentication using the indel marker.

Aconitum species (belonging to the Ranunculaceae) are well known herbaceous medicinal ingredients and have great economic value in Asian countries. However, there are still limited genomic resources available for Aconitum species. In this study, we sequenced the chloroplast (cp) genomes of two Aconitum species, A. coreanum and A. carmichaelii, using the MiSeq platform. The two Aconitum chloroplast genomes were 155,880 and 157,040 bp in length, respectively, and exhibited LSC and SSC regions separated by a pair of inverted repeat regions. Both cp genomes had 38% GC content and contained 131 unique functional genes including 86 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The gene order, content, and orientation of the two Aconitum cp genomes exhibited the general structure of angiosperms, and were similar to those of other Aconitum species. Comparison of the cp genome structure and gene order with that of other Aconitum species revealed general contraction and expansion of the inverted repeat regions and single copy boundary regions. Divergent regions were also identified. In phylogenetic analysis, Aconitum species positon among the Ranunculaceae was determined with other family cp genomes in the Ranunculales. We obtained a barcoding target sequence in a divergent region, ndhC-trnV, and successfully developed a SCAR (sequence characterized amplified region) marker for discrimination of A. coreanum. Our results provide useful genetic information and a specific barcode for discrimination of Aconitum species.

Lepidii seu Descurainiae Semen, the dried ripe seeds of Descurainia sophia (L.) Webb ex Prantl or Lepidium apetalum Willd., is used widely as a traditional herbal medicine in Northeast Asia. However, seeds of a number of other species have been misidentified as Lepidii seu Descurainiae Semen, and are therefore misused because of morphological similarities among the seeds of these species. To accurately identify Lepidii seu Descurainiae Semen, we investigated the morphology, mucilage reaction, and micromorphology of Lepidii seu Descurainiae Semen and the seeds of other plant species. We used a stereomicroscope and a scanning electron microscope to describe and compare various morphological and micromorphological characteristics of seeds, and a light microscope to determine the presence or absence of mucilage. We evaluated the potential usefulness of mucilage as a morphological marker for seed identification. The shape, outline, and size of seeds were useful characteristics for identification. The mucilage reaction and the three types of seed ornamentation that were identified in this study were particularly valuable for seed authentication. Based on these results, we created an identification key based on morphology and micromorphology of Lepidii seu Descurainiae Semen and adulterant seeds. The method of seed identification using microscopic examination, as described here, is easy and economical, making it a potential key tool for accurate identification of Lepidii seu Descurainiae Semen and seeds of other medicinal plants.

The accurate identification of plant species is of great concern for the quality control of herbal medicines. The Korean Pharmacopoeia and the Pharmacopoeia of the People’s Republic of China define Angelicae Dahuricae Radix (Baek-Ji in Korean and Bai-zhi in Chinese) as the dried roots of Angelica dahurica or A. dahurica var. formosana belonging to the family Apiaceae. Discrimination among Angelica species on the basis of morphological characteristics is difficult due to their extremely polymorphic traits and controversial taxonomic history. Furthermore, dried roots processed for medicinal applications are indistinguishable using conventional methods. DNA barcoding is a useful and reliable method for the identification of species. In this study, we sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes in A. dahurica, A. dahurica var. formosana, and the related species A. anomala and A. japonica. Using these sequences, we designed species-specific primers, and developed and optimized a multiplex sequence-characterized amplified region (SCAR) assay that can simply and rapidly identify respective species, and verify the contamination of adulterant depending on the polymerase chain reaction (PCR) amplification without sequencing analysis in a single PCR reaction. This assay successfully identified commercial samples of Angelicae Dahuricae Radix collected from Korean and Chinese herbal markets, and distinguished them from adulterants. This multiplex SCAR assay shows a great potential in reducing the time and cost involved in the identification of genuine Angelicae Dahuricae Radix and adulterant contamination.

Dried roots of Dipsacus asper (Caprifoliaceae) are used as important traditional herbal medicines in Korea. However, the roots are often used as a mixture or contaminated with Dipsacus japonicus in Korean herbal markets. Furthermore, the dried roots of Phlomoides umbrosa (Lamiaceae) are used indiscriminately with those of D. asper, with the confusing Korean names of Sok-Dan and Han-Sok-Dan for D. asper and P. umbrosa, respectively. Although D. asper and P. umbrosa are important herbal medicines, the molecular marker and genomic information available for these species are limited. In this study, we analysed DNA barcodes to distinguish among D. asper, D. japonicus, and P. umbrosa and sequenced the chloroplast (CP) genomes of D. asper and D. japonicus. The CP genomes of D. asper and D. japonicus were 160,530 and 160,371 bp in length, respectively, and were highly divergent from those of the other Caprifoliaceae species. Phylogenetic analysis revealed a monophyletic group within Caprifoliaceae. We also developed a novel sequence characterised amplified region (SCAR) markers to distinguish among D. asper, D. japonicus, and P. umbrosa. Our results provide important taxonomic, phylogenetic, and evolutionary information on the Dipsacus species. The SCAR markers developed here will be useful for the authentication of herbal medicines.

Aconitum pseudolaeve Nakai and Aconitum longecassidatum Nakai, which belong to the Aconitum subgenus Lycoctonum, are distributed in East Asia and Korea. Aconitum species are used in herbal medicine and contain highly toxic components, including aconitine. A. pseudolaeve, an endemic species of Korea, is a commercially valuable material that has been used in the manufacture of cosmetics and perfumes. Although Aconitum species are important plant resources, they have not been extensively studied, and genomic information is limited. Within the subgenus Lycoctonum, which includes A. pseudolaeve and A. longecassidatum, a complete chloroplast (CP) genome is available for only one species, Aconitum barbatum Patrin ex Pers. Therefore, we sequenced the complete CP genomes of two Aconitum species, A. pseudolaeve and A. longecassidatum, which are 155,628 and 155,524 bp in length, respectively. Both genomes have a quadripartite structure consisting of a pair of inverted repeated regions (51,854 and 52,108 bp, respectively) separated by large single-copy (86,683 and 86,466 bp) and small single-copy (17,091 and 16,950 bp) regions similar to those in other Aconitum CP genomes. Both CP genomes consist of 112 unique genes, 78 protein-coding genes, 4 ribosomal RNA (rRNA) genes, and 30 transfer RNA (tRNA) genes. We identified 268 and 277 simple sequence repeats (SSRs) in A. pseudolaeve and A. longecassidatum, respectively. We also identified potential 36 species-specific SSRs, 53 indels, and 62 single-nucleotide polymorphisms (SNPs) between the two CP genomes. Furthermore, a comparison of the three Aconitum CP genomes from the subgenus Lycoctonum revealed highly divergent regions, including trnK-trnQ, ycf1-ndhF, and ycf4-cemA. Based on this finding, we developed indel markers using indel sequences in trnK-trnQ and ycf1-ndhF. A. pseudolaeve, A. longecassidatum, and A. barbatum could be clearly distinguished using the novel indel markers AcoTT (Aconitum trnK-trnQ) and AcoYN (Aconitum ycf1-ndhF). These two new complete CP genomes provide useful genomic information for species identification and evolutionary studies of the Aconitum subgenus Lycoctonum.

Accurate taxonomic identification of plant materials in herbal medicines is important for product quality control. The genus Paeonia (Saxifragales) is the source of the herbal preparations Paeoniae Radix (Paeoniae Radix Alba and Paeoniae Radix Rubra) and Moutan Radicis Cotex. However, confusion has arisen regarding their contents due to linguistic and taxonomic ambiguities, similar morphologies and different definitions of Paeoniae Radix in the Korean and Chinese national pharmacopoeias, leading to the distribution of adulterated products. To develop a method for identifying the four Paeonia species used in these medicines, three fluorescently-labeled peptide nucleic acid (PNA) probes were designed against ITS2 sequences containing single nucleotide polymorphisms (SNPs) and used in a real-time PCR melting curve assay. Each of the four Paeonia species was accurately identified using this analysis. The accuracy and analytical stability of the PNA melting curve assay was confirmed using commercially available samples of the four Paeonia species. This assay is a reliable genetic tool to distinguish between different Paeonia-derived herbal medicines and identify the botanical origins of Paeoniae Radix and Moutan Radicis Cortex. This technique may also contribute to quality control and standardization of herbal medicines by providing a reliable authentication tool and preventing the distribution of inauthentic adulterants.

Determining the precise botanical origin of a traditional herbal medicine is important for basic quality control. In both the Chinese and Korean herbal pharmacopoeia, authentic Adenophorae Radix is defined as the roots of Adenophora stricta and Adenophora triphylla. However, the roots of Codonopsis lanceolata, Codonopsis pilosula, and Glehnia littoralis are frequently distributed as Adenophorae Radix in Korean herbal markets. Unfortunately, correctly identifying dried roots is difficult using conventional methods because the roots of those species are morphologically similar. Therefore, we developed DNA-based markers for the identification of authentic Adenophorae Radix and its common adulterants in commercially-processed samples. To develop a reliable method to discriminate between Adenophorae Radix and its adulterants, we sequenced the nuclear ribosomal DNA internal transcribed spacers (nrDNA-ITS) and designed sequence-characterized amplified region (SCAR) primers specific to the authentic and adulterant species. Using these primers, we developed SCAR markers for each species and established a multiplex-PCR method that can authenticate the four herbal medicines in a single PCR reaction. Furthermore, we confirmed that commercially-processed herbal medicines, which often have degraded DNA, could be assessed with our method. Therefore, our method is a reliable genetic tool to protect against adulteration and to standardize the quality of Adenophorae Radix.

Accurate identification of the plant species that are present in herbal medicines is important for quality control. Although the dried roots of Aralia continentalis (Araliae Continentalis Radix) and Angelica biserrata (Angelicae Pubescentis Radix) are used in the same traditional medicine, namely Dok-Hwal in Korean and Du-Huo in Chinese, the medicines are described differently in the national pharmacopeia. Further confusion arises from the distribution of dried Levisticum officinale and Heracleum moellendorffii roots as the same medicine. Medicinal ingredients from all four plants are morphologically similar, and discrimination is difficult using conventional methods. Molecular identification methods offer rapidity and accuracy. The internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal RNA gene (rDNA) was sequenced in all four plant species, and the sequences were used to design species-specific primers. Primers for each species were then combined to allow sample analysis in a single PCR reaction. Commercial herbal medicine samples were obtained from Korea and China and analyzed using the multiplex assay. The assay successfully identified authentic medicines and also identified inauthentic or adulterated samples. The multiplex assay will be a useful tool for identification of authentic Araliae Continentalis Radix and/or Angelicae Pubescentis Radix preparations in Korea and China.