Atractylodes Species Research Articles

Complete chloroplast genome sequencing and phylogeny of wild Atractylodes lancea from Yuexi, Anhui province

This study investigated the choroplast genome sequence of wild Atractylodes lancea from Yuexi in Anhui province by high-throughput sequencing, followed by characterization of the genome structure, which laid a foundation for the species identification, analysis of genetic diversity, and resource conservation of A. lancea. To be specific, the total genomic DNA was extracted from the leaves of A. lancea with the improved CTAB method. The chloroplast genome of A. lancea was sequenced by the high-throughput sequencing technology, followed by assembling by metaSPAdes and annotation by CPGAVAS2. Bioiformatics methods were employed for the analysis of simple sequence repeats(SSRs), inverted repeat(IR) border, codon bias, and phylogeny. The results showed that the whole chloroplast genome of A. lancea was 153 178 bp, with an 84 226 bp large single copy(LSC) and a 18 658 bp small single copy(SSC) separated by a pair of IRs(25 147 bp). The genome had the GC content of 37.7% and 124 genes: 87 protein-coding genes, 8 rRNA genes, and 29 tRNA genes. It had 26 287 codons and encoded 20 amino acids. Phylogenetic analysis showed that Atractylodes species clustered into one clade and that A. lancea had close genetic relationship with A. koreana. This study established a method for sequencing the chloroplast genome of A. lancea and enriched the genetic resources of Compositae. The findings are expected to lay a foundation for species identification, analysis of genetic diversity, and resource conservation of A. lancea.

Complete chloroplast genome of Atractylodes japonica native to the Korean Peninsula and Atractylodes species identification challenges

Atractylodes japonica Koidz. ex. Kitam. (Asteraceae) is a perennial plant, of which the dried rhizome has been utilized as a traditional herbal medicine in East Asia. Here, we sequenced and annotated the complete chloroplast genome of A. japonica native to Korea. The circular genome was 153,207 bp in length and was composed of a large single-copy region (84,256 bp), a small single-copy region (18,657 bp), and a pair of inverted repeat regions (25,147 bp each). The annotation and manual curation of genes predicted a total of 114 genes, of which 80 were classified as protein-coding genes, 30 were transfer ribonucleic acid (RNA) genes, and 4 were ribosomal RNA genes. Phylogenetic analysis using 13 genomes from 6 Atractylodes species showed intermingled clustering of 9 genomes from 4 species, A. chinensis, A. japonica, A. koreana, and A. lancea, indicating ambiguity in their taxonomic identification.

Evolution and Phylogeny of Soybean Mosaic Virus Based on 143 Complete Genomes.

Soybean mosaic virus (SMV) of the genus Potyvirus is an important virus in cultivated soybeans. Here, we obtained 7 SMV genomes from soybean germplasms using RNA sequencing and conducted a comprehensive evolutionary and phylogenetic study of 143 SMV genomes derived from 10 plant species and 12 countries. The phylogenetic tree we constructed using coding DNA sequences revealed the existence of nine clades of SMV isolates/strains. Recombination analysis revealed 76 recombinant events and 141 recombinants in total. Clades 1 and 3 contain the most common SMV pathotypes, including G1 through G7, which are distributed worldwide. Clade 2 includes several Chinese SMV pathotypes. The SMV isolates were further divided into two groups. The SMV isolates in the first group, including clades 8 and 9, were identified from Pinellia and Atractylodes species, whereas those in the second group (clades 1 through 7) were mostly found in cultivated soybeans. The SMV polyprotein undergoes positive selection, whereas most mature proteins, except for the P1 protein, undergo negative selection. The P1 protein of SMV isolates in group 1 may be highly correlated with host adaptation. This study provides strong evidence that recombination and plant hosts are powerful forces driving the genetic diversity of the SMV genome.

Phylogenomic analyses based on the plastid genome and concatenated nrDNA sequence data reveal cytonuclear discordance in genus Atractylodes (Asteraceae: Carduoideae).

Atractylodes species are widely distributed across East Asia and are cultivated as medicinal herbs in China, Japan, and Korea. Their unclear morphological characteristics and low levels of genetic divergence obscure the taxonomic relationships among these species. In this study, 24 plant samples were collected representing five species of Atractylodes located in China; of these, 23 belonged to members of the A. lancea complex. High-throughput sequencing was used to obtain the concatenated nrDNA sequences (18S-ITS1-5.8S-ITS2-28S) and plastid genomes. The concatenated nrDNA sequence lengths for all the Atractylodes species were 5,849 bp, and the GC content was 55%. The lengths of the whole plastid genome sequences ranged from 152,138 bp (A. chinensis) to 153,268 bp (A. lancea), while their insertion/deletion sites were mainly distributed in the intergenic regions. Furthermore, 33, 34, 36, 31, and 32 tandem repeat sequences, as well as 30, 30, 29, 30, and 30 SSR loci, were detected in A. chinensis, A. koreana, A. lancea, A. japonica, and A. macrocephala, respectively. In addition to these findings, a considerable number of heteroplasmic variations were detected in the plastid genomes, implying a complicated phylogenetic history for Atractylodes. The results of the phylogenetic analysis involving concatenated nrDNA sequences showed that A. lancea and A. japonica formed two separate clades, with A. chinensis and A. koreana constituting their sister clade, while A. lancea, A. koreana, A. chinensis, and A. japonica were found based on plastid datasets to represent a mixed clade on the phylogenetic tree. Phylogenetic network analysis suggested that A. lancea may have hybridized with the common ancestor of A. chinensis and A. japonica, while ABBA-BABA tests of SNPs in the plastid genomes showed that A. chinensis was more closely related to A. japonica than to A. lancea. This study reveals the extensive discordance and complexity of the relationships across the members of the A. lancea complex (A. lancea, A. chinensis, A. koreana, and A. japonica) according to cytonuclear genomic data; this may be caused by interspecific hybridization or gene introgression.

An overlooked dispersal route of Cardueae (Asteraceae) from the Mediterranean to East Asia revealed by phylogenomic and biogeographical analyses of Atractylodes.

The East Asian-Tethyan disjunction pattern and its mechanisms of formation have long been of interest to researchers. Here, we studied the biogeographical history of Asteraceae tribe Cardueae, with a particular focus on the temperate East Asian genus Atractylodes DC., to understand the role of tectonic and climatic events in driving the diversification and disjunctions of the genus. A total of 76 samples of Atractylodes from 36 locations were collected for RAD-sequencing. Three single nucleotide polymorphism (SNP) datasets based on different filtering strategies were used for phylogenetic analyses. Molecular dating and ancestral distribution reconstruction were performed using both chloroplast DNA sequences (127 Cardueae samples) and SNP (36 Atractylodes samples) datasets. Six species of Atractylodes were well resolved as individually monophyletic, although some introgression was identified among accessions of A. chinensis, A. lancea and A. koreana. Dispersal of the subtribe Carlininae from the Mediterranean to East Asia occurred after divergence between Atractylodes and Carlina L. + Atractylis L. + Thevenotia DC. at ~31.57 Ma, resulting in an East Asian-Tethyan disjunction. Diversification of Atractylodes in East Asia mainly occurred from the Late Miocene to the Early Pleistocene. Aridification of Asia and the closure of the Turgai Strait in the Late Oligocene promoted the dispersal of Cardueae from the Mediterranean to East China. Subsequent uplift of the Qinghai-Tibet Plateau as well as changes in Asian monsoon systems resulted in an East Asian-Tethyan disjunction between Atractylodes and Carlina + Atractylis + Thevenotia. In addition, Late Miocene to Quaternary climates and sea level fluctuations played major roles in the diversification of Atractylodes. Through this study of different taxonomic levels using genomic data, we have revealed an overlooked dispersal route between the Mediterranean and far East Asia (Japan/Korea) via Central Asia and East China.

Analysis of volatile compounds from three species of Atractylodes by Gas Chromatography-Mass Spectrometry

A total of 99 different volatile compounds were detected through Gas Chromatography-Mass Spectrometry (GC-MS) from three species of Atractylodes, namely Atractylodes lancea, Atractylodes japonica, and Atractylodes chinensis. Thirteen-volatile flavor compounds i.e., acid, alcohol, aldehyde, alkane, alkene, alkyne, ester, ketone, monoterpene, oxygenated monoterpene, sesquiterpene, oxygenated sesquiterpene, and oxygenated triterpenoid detected from different species of Atractylodes. It was observed that all the species contained 38 common compounds, while A. lancea contained 7 unique compounds, A. japonica has 4 unique compounds, and A. chinensis hold 6 compounds not detected in the other extracts. In addition, essential oils from A. lancea and A. japonica possessed 11 compounds in common, and A. lancea and A. chinensis possessed 19 compounds in common. The remaining 14 compounds were detected only in A. japonica and A. chinensis. The total content of all components in the species was comparable, with 82.528%, 81.766%, and 81.799% of volatile components being detected for A. lancea, A. japonica, and A. chinensis, respectively. Curzerene was found to be the most predominant compound in both A. lancea (14.1%) and A. chinensis (16.7%), while murolan-3,9(11)-diene-10-peroxy was found predominantly in A. japonica (16.8%). The present study suggests that the identified volatile compounds may possess important biological properties, and could be suitable for application in both oriental medicines and the pharmaceutical industry.

Metabolic fingerprinting for discrimination of DNA-authenticated Atractylodes plants using 1H NMR spectroscopy

Identifying different species of the genus Atractylodes which are commonly used in Chinese and Japanese traditional medicine, using chromatographic approaches can be difficult. 1H NMR metabolic profiling of DNA-authenticated, archived rhizomes of the genus Atractylodes was performed for genetic and chemical evaluation. The ITS region of the nuclear rDNA was sequenced for five species, A. japonica, A. macrocephala, A. lancea, A. chinensis, and A. koreana. Our samples had nucleotide sequences as previously reported, except that part of the A. lancea cultivated in Japan had a type 5, hybrid DNA sequence. Principal component analysis (PCA) using 1H NMR spectra of extracts with two solvent systems (CD3OD, CDCl3) was performed. When CDCl3 extracts were utilized, the chemometric analysis enabled the identification and classification of Atractylodes species according to their composition of major sesquiterpene compounds. The 1H NMR spectra using CD3OD contained confounding sugar peaks. PCA removal of these peaks gave the same result as that obtained using CDCl3 and allowed species distinction. Such chemometric methods with multivariate analysis of NMR spectra will be useful for the discrimination of plant species, without specifying the index components and quantitative analysis on multi-components.Graphic abstract

Chloroplast genome variation and phylogenetic relationships of Atractylodes species

BackgroundAtractylodes DC is the basic original plant of the widely used herbal medicines “Baizhu” and “Cangzhu” and an endemic genus in East Asia. Species within the genus have minor morphological differences, and the universal DNA barcodes cannot clearly distinguish the systemic relationship or identify the species of the genus. In order to solve these question, we sequenced the chloroplast genomes of all species of Atractylodes using high-throughput sequencing.ResultsThe results indicate that the chloroplast genome of Atractylodes has a typical quadripartite structure and ranges from 152,294 bp (A. carlinoides) to 153,261 bp (A. macrocephala) in size. The genome of all species contains 113 genes, including 79 protein-coding genes, 30 transfer RNA genes and four ribosomal RNA genes. Four hotspots, rpl22-rps19-rpl2, psbM-trnD, trnR-trnT(GGU), and trnT(UGU)-trnL, and a total of 42–47 simple sequence repeats (SSR) were identified as the most promising potentially variable makers for species delimitation and population genetic studies. Phylogenetic analyses of the whole chloroplast genomes indicate that Atractylodes is a clade within the tribe Cynareae; Atractylodes species form a monophyly that clearly reflects the relationship within the genus.ConclusionsOur study included investigations of the sequences and structural genomic variations, phylogenetics and mutation dynamics of Atractylodes chloroplast genomes and will facilitate future studies in population genetics, taxonomy and species identification.

Gas purge micro solvent extraction: A rapid and powerful tool for essential oil chromatographic fingerprints

In this work, a new an innovative micro liquid extraction technique named “gas purge micro solvent extraction” (GP-MSE) have been tested, hyphenated with gas chromatography-mass spectrometry (GC–MS), to evaluate its capability to extract volatile fractions from vegetal samples respect to classical steam distillation extraction procedure, by comparing essential oil fingerprints from three different Atractylodes species. GP-MSE showed higher extraction capabilities and sensitivities (in particular for semi-volatile components), obtaining more structured essential oil fingerprints with high reliabilities (relative standard deviation of the peak areas between 0.34% and 5.83%), requiring at the same time smaller sample amounts and reduced extraction times. Essential oil profile data were also submitted to principal component analysis and included cosine angle in order to highlight differences among the Atractylodes species under investigation. GP-MSE can be considered an alternative, simple, fast, sensitive and environmentally friendly extraction method, with high extraction efficiencies and reproducibility toward both volatile and semi-volatile compounds.

Pharmacokinetic Change of Glycyrrhetinic Acid from the Roots and Rhizomes of Glycyrrhiza uralensis by Coadministration with the Rhizomes of Atractylodes japonica, A. macrocephala, or A. chinensis in an Animal Model

Atractylodes rhizomes (Atractylodes japonica Koidz. ex Kitam., A. macrocephala Koidz., and A. chinensis), Asteraceae, are widely used to treat digestive disorders and these rhizomes are commonly paired with the roots and rhizomes of Glycyrrhiza uralensis Fisch., Fabaceae, in herbal formulae, which may lead to mutual interaction in vivo. To assess the influence of three species of Atractylodes rhizomes on the pharmacokinetic features of G. uralensis, the levels of glycyrrhetinic acid in rat plasma samples were quantified using ultra-performance liquid chromatography/ion-trap mass spectrometry after intragastric coadministration. The results demonstrated that simultaneous administration of the extracts of the three Atractylodes rhizomes reduced the mean residence time and last measured concentration of glycyrrhetinic acid in plasma samples. Among the Atractylodes rhizomes, the combination of A. japonica shortened the elimination half-life and time to the maximum concentration of glycyrrhetinic acid. In conclusion, Atractylodes rhizomes, especially A. japonica, might alter the pharmacokinetic characteristics (mostly the duration in vivo) of glycyrrhetinic acid from G. uralensis when these herbs are coadministered. This study provides an understanding for species-specific influences on pharmacokinetic features in herbal combinations.

Distinguishing the rhizomes of Atractylodes japonica, Atractylodes chinensis, and Atractylodes lancea by comprehensive two-dimensional gas chromatography coupled with mass spectrometry combined with multivariate data analysis

Background: In clinical practice, the species of Atractylodes are difficult to identify based on their morphological and chemical features which often leads to confusion. In addition, the composition of volatile components may influence the clinical efficacy of rhizomes of Atractylodes. Materials and Methods: In this study, a comprehensive two-dimensional gas chromatography with mass spectrometry coupled with multivariate data analysis was employed to investigate the differences in the volatile components of the rhizomes of three species of Atractylodes, namely Atractylodes lancea (Thunb.) DC, Atractylodes japonica Koidz. et Kitam, and Atractylodes chinensis (DC.) Koidz. Results: A total of 119 compounds were tentatively identified and confirmed based on the NIST database. Thirty-three samples were well distinguished and the results of two different analytical methods using principal component analysis and partial least-squares discriminant analysis were in satisfactory agreement with one-way analysis of variance. Atractylodin and β-eudesmol can be used to reveal the chemical differentiation and distinguish different species of Atractylodes. Conclusion: The results may provide a reliable reference to quality control and product grade of rhizomes of Atractylodes.

Identification of Crepenynic Acid in the Seed Oil of Atractylodes lancea and A. macrocephala

AbstractAtractylodes rhizome is widely used in traditional Chinese herbal medicine. Although the chemical composition of the root has been studied in detail, the oil content and fatty acid composition of the seeds of Atractylodes species have not been reported. Fatty acyl composition of seeds from Atractylodes lancea and A. macrocephala was determined by gas chromatography and mass spectrometry of fatty acid methyl esters and 3‐pyridylcarbinol esters. The predominant fatty acid in the seeds of both species was linolenic acid, but the unusual acetylenic fatty acid, crepenynic acid (cis‐9‐octadecen‐12‐ynoic acid), was also observed at levels of 18% in A. lancea and 13–15% in A. macrocephala. Fatty acid content was 24% for the samples of A. lancea and 16–17% for samples from A. macrocephala. sn‐1,3 regioselective lipase digestion of seed lipids revealed that crepenynic acid was absent from the sn‐2 position of the seed triacylglycerol. Crepenynic acid was also found in the seed oil of Jurinea mollis at 24% and was not present in the sn‐2 position of the TAG. A contrasting distribution of crepenynic acid was found in the oil of Crepis rubra, suggesting differences in crepenynic acid synthesis or TAG assembly between these species.

Anti-inflammatory and Antinociceptive Constituents of Atractylodes japonica Koidzumi.

The rhizomes of many Atractylodes species, including Atractylodes chinensis Koidzumi, Atractylodes macrocephala Koidzumi, and Atractylodes japonica Koidzumi, are collectively termed Atractylodis Rhizoma. We prepared n-hexane extracts of the three species and evaluated their anti-inflammatory effects on lipopolysaccharide (LPS)-induced RAW 264.7 cells. Among all n-hexane extracts, those of A. japonica most strongly inhibited nitric oxide (NO) production in LPS-induced RAW 264.7 cells; five sesquiterpenes, atractylon, atractylenolide I, atractylenolide II, atractylenolide III, and 8-epiasterolid, were isolated from A. japonica. The phytochemical content of A. japonica was similar to those of A. chinensis and A. macrocephala. Moreover, the atractylon concentration was higher in A. japonica than in A. chinensis and A. macrocephala. Atractylon significantly inhibited NO and prostaglandin E2 production as well as inducible NO synthase and cyclooxygenase-2 expression in LPS-induced RAW 264.7 cells. Atractylon (40 mg/kg) also significantly reduced the acetic-acid-induced writhing response, carrageenan-induced paw edema, and hot-plate latent pain response in mice. According to the results, A. japonica has anti-inflammatory and antinociceptive effects and atractylon is the major active component of A. japonica. Therefore, atractylon can be used as a bioactivity marker in A. japonica.

Evaluation of Medicinal Categorization of Atractylodes japonica Koidz. by Using Internal Transcribed Spacer Sequencing Analysis and HPLC Fingerprinting Combined with Statistical Tools.

Atractylodes rhizomes have been used as the herbal medicine “Changchul” or “Baekchul,” according to their clinical purpose, in Korea, China, and Japan. Among the Atractylodes species, the medicinal use of Atractylodes japonica has been controversial, as it is categorized as both Changchul and Baekchul in those countries, and, moreover, parts of the rhizome have been differently used, depending on age of the plant, in Korea. Chromatographic fingerprinting by using HPLC combined with chemometric analyses and internal transcribed spacer (ITS) sequencing analysis were conducted to classify and identify 34 crude drugs derived from Atractylodes rhizomes. The identification of the samples, authenticated by their morphological features as A. japonica Koidz. (Changchul and Baekchul), A. chinensis Koidz., and A. macrocephala Koidz., was confirmed as A. japonica, A. chinensis, and A. macrocephala by ITS sequencing. The results from chemometric analyses showed that the chemical components of the crude drugs from A. japonica were significantly different from those from A. macrocephala but were similar to those from A. chinensis. The analyses also suggested that the categorization by age of A. japonica as Changchul or Baekchul is not recommended. The results indicate that A. japonica should be categorized as “Changchul” and should not be further categorized by age.

Molecular Systematics of Genus Atractylodes (Compositae, Cardueae): Evidence from Internal Transcribed Spacer (ITS) and trnL-F Sequences

To determine the evolutionary relationships among all members of the genus Atractylodes (Compositae, Cardueae), we conducted molecular phylogenetic analyses of one nuclear DNA (nrDNA) region (internal transcribed spacer, ITS) and one chloroplast DNA (cpDNA) region (intergenic spacer region of trnL-F). In ITS and ITS + trnL-F trees, all members of Atractylodes form a monophyletic clade. Atractylodes is a sister group of the Carlina and Atractylis branch. Atractylodes species were distributed among three clades: (1) A. carlinoides (located in the lowest base of the Atractylodes phylogenetic tree), (2) A. macrocephala, and (3) the A. lancea complex, including A. japonica, A. coreana, A. lancea, A. lancea subsp. luotianensis, and A. chinensis. The taxonomic controversy over the classification of species of Atractylodes is mainly concentrated in the A. lancea complex. In base on molecular results, the intraspecific division of Atractylodes lancea is not supported, and A. coreana should be treated as a synonym A. chinensis.

The effect of Hypericum perforatum extract on the properties of erythrocyte membrane

The phylogenetic relationships of five Atractylodes species were examined by random amplification of polymorphic DNA (RAPD) assay. A total of 298 bands were amplified by 24 RAPD primers, of which 244 bands (81.88%) were polymorphic. The results indicated that: 1) Atractylodes chinensis had a close genetic relationship with Atractylodes lancea, and A. chinensis should be treated as A. lancea var. chinensis; 2) Atractylodes coreana was closely related to A. lancea and A. chinensis; 3) Atractylodes macrocephala was distantly related to other species of Atractylodes. One primer which produced an A. lancea-specific band with a length of 1800 bp was obtained from 80 RAPD primers. Subsequent analysis showed that all of the 38 individuals of A. lancea could produce the sequence characterized amplified region (SCAR) bands, while the other four species of Atractylodes could not, indicating that A. lancea can be exactly distinguished from the other four species by the RAPD-SCAR marker. Key words: Atractylodes, A. lancea, endangered herb, random amplification of polymorphic DNA (RAPD), sequence characterized amplified region (SCAR).

Study of specific random amplification of polymorphic DNA- sequence characterized amplified region (RAPD- SCAR) marker for the endangered Chinese endemic herb Atractylodes lancea

The phylogenetic relationships of five Atractylodes species were examined by random amplification of polymorphic DNA (RAPD) assay. A total of 298 bands were amplified by 24 RAPD primers, of which 244 bands (81.88%) were polymorphic. The results indicated that: 1) Atractylodes chinensis had a close genetic relationship with Atractylodes lancea, and A. chinensis should be treated as A. lancea var. chinensis; 2) Atractylodes coreana was closely related to A. lancea and A. chinensis; 3) Atractylodes macrocephala was distantly related to other species of Atractylodes. One primer which produced an A. lancea-specific band with a length of 1800 bp was obtained from 80 RAPD primers. Subsequent analysis showed that all of the 38 individuals of A. lancea could produce the sequence characterized amplified region (SCAR) bands, while the other four species of Atractylodes could not, indicating that A. lancea can be exactly distinguished from the other four species by the RAPD-SCAR marker. Key words: Atractylodes, A. lancea, endangered herb, random amplification of polymorphic DNA (RAPD), sequence characterized amplified region (SCAR).

DNA authentication of So-jutsu (Atractylodes lancea rhizome) and Byaku-jutsu (Atractylodes rhizome) obtained in the market based on the nucleotide sequence of the 18S–5.8S rDNA internal transcribed spacer region

The internal transcribed spacer 1 (ITS1) region between 18S and 5.8S rDNA was amplified from 25 crude drug specimens of So-jutsu (Atractylodes lancea rhizome) and 11 specimens of Byaku-jutsu (Atractylodes rhizome) obtained in the Chinese market. Based on the comparison of the nucleotide sequences of the PCR products with those determined in five Atractylodes species, we were able to identify the plant origins of these crude drugs. Of the 25 specimens of So-jutsu analyzed, 20 were identified to be derived from either A. chinensis, A. lancea or the interspecific hybrid between these two species, whereas five specimens were identified to be derived from A. japonica, i.e. Byaku-jutsu. In contrast, all of the specimens of Byaku-jutsu analyzed were derived from either A. ovata or A. japonica. A PCR-based method was established for the rapid identification and/or discrimination of So-jutsu and Byaku-jutsu without sequencing.

Identification of Medicinal Atractylodes Based on ITS Sequences of nrDNA

Dried rhizomes of five species of Atractylodes (A. japonica, A. macrocephala, A. lancea, A. chinensis, and A. koreana), Compositae, have been used as crude drugs mainly for the treatment of stomach disorders and for their diuretic properties in Chinese and Japanese traditional medicines. The identification of the botanical origins of these crude drugs is generally difficult from their morphological and chemical features only. In this study, for identification with more reliable, nuclear ribosomal DNA (nrDNA), internal transcribed spacer (ITS) regions of five species of medicinal Atractylodes were sequenced. As a result, specific ITS genotypes were recognized by each species. The four species (A. japonica, A. macrocephala, A. lancea, and A. chinensis) prescribed in Chinese and Japanese Pharmacopoeias as botanical origins of crude Atractylodes drugs could be distinguished by their ITS sequences because they had difference genotypes on the ITS sequences. However, the genotype of A. koreana was the same as that of A. chinensis. Additionally, hybrids between A. lancea and A. chinensis were also recognized as nucleotide additives on their ITS sequences. In this study, several morphological characteristics were researched by their genotype, too. As this result, the hybrids recognized from the genetic analysis had intermediate morphological characteristics between A. lancea and A. chinensis. It was also recognized that A. lancea and A. chinensis except for their hybrids were significant differences. It is therefore suggested that ITS sequences of nrDNA would be useful for the identification of the crude drugs derived from Atractylodes species and their interspecific hybridizations.

Phylogenetic analysis of Atractylodes plants based on chloroplast trnK sequence.

The phylogenetic relationship of Atractylodes lancea, A. chinensis, A. koreana, A. ovata and A. japonica were analyzed by comparing the 2.6 kb sequence in a chloroplast gene trnK encoding tRNALys (UUU). The dried rhizomes of the former three species have been used as the crude drug "So-jutsu" and those of the latter two as "Byaku-jutsu" in Chinese and Japanese traditional medicine ("Kampo-medicine"). The trnK phylogenetic tree revealed that A. ovata is an outgroup of the five Atractylodes species examined and that A. japonica and A. lancea are most closely related. PCR amplification of trnK with HinfI digestion provided us with a simple method to distinguish A. ovata from other Atractylodes species at the molecular level.