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

BACKGROUND AND AIMS: 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. METHODS: 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. KEY RESULTS: 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. CONCLUSIONS: 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.

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.

[Textual research on "Zhu" in Chinese classical prescriptions].

Through consulting the ancient herbs and medical books,combining with modern literature and field investigation,this paper makes textual research on herbal medicine,and makes textual research on the name,origin,position and changes of collection and processing of the medicinal herbs in Ancient Classical Prescriptions. " Zhu" wasn't distinguished before the Northern and Southern Dynasties,but the efficacy of the techniques recorded in the documents at that time was similar to that of today's Atractylodis Rhizoma.Tao Hongjing put forward the theory of " red and white" in the Northern and Southern Dynasties. " Cangzhu" and " Baizhu" gradually differentiated,but there was no clear distinction between them. Since Song and Yuan Dynasties, " Baizhu" has been respected and classified in the literature. In Ming Dynasty, " Baizhu" was cultivated and differentiated into different quality specifications according to its growth pattern and origin. Maoshan is the genuine production area of " Cangzhu". The quality of wild " Baizhu" produced in Yuqian city is the best. In modern times,the origins of " Cangzhu" and " Baizhu" were defined according to plant taxonomy. " Zhu" before Song Dynasty was close to Atractylodes lancea, " Baishu" had been respected since the Song Dynasty. So when Song people revised the previous literature,they unified " Zhu" into " Baizhu" and used it for later generations. Its safety and effectiveness have been confirmed,and the wild resources of modern atractylodes are scarce. Therefore,it is suggested that when classical famous prescriptions are developed,the prescription recorded as " Baizhu" does not need to be changed in its origin,that is,A. macrocephala at present or can choose the appropriate basis according to the principle of local righteousness.Textual research shows that the origin of " Cangzhu" in the classical prescription should be A. lancea.

Textual research on "Zhu" in Chinese classical prescriptions / 中国中药杂志

Through consulting the ancient herbs and medical books,combining with modern literature and field investigation,this paper makes textual research on herbal medicine,and makes textual research on the name,origin,position and changes of collection and processing of the medicinal herbs in Ancient Classical Prescriptions. " Zhu" wasn't distinguished before the Northern and Southern Dynasties,but the efficacy of the techniques recorded in the documents at that time was similar to that of today's Atractylodis Rhizoma.Tao Hongjing put forward the theory of " red and white" in the Northern and Southern Dynasties. " Cangzhu" and " Baizhu" gradually differentiated,but there was no clear distinction between them. Since Song and Yuan Dynasties, " Baizhu" has been respected and classified in the literature. In Ming Dynasty, " Baizhu" was cultivated and differentiated into different quality specifications according to its growth pattern and origin. Maoshan is the genuine production area of " Cangzhu". The quality of wild " Baizhu" produced in Yuqian city is the best. In modern times,the origins of " Cangzhu" and " Baizhu" were defined according to plant taxonomy. " Zhu" before Song Dynasty was close to Atractylodes lancea, " Baishu" had been respected since the Song Dynasty. So when Song people revised the previous literature,they unified " Zhu" into " Baizhu" and used it for later generations. Its safety and effectiveness have been confirmed,and the wild resources of modern atractylodes are scarce. Therefore,it is suggested that when classical famous prescriptions are developed,the prescription recorded as " Baizhu" does not need to be changed in its origin,that is,A. macrocephala at present or can choose the appropriate basis according to the principle of local righteousness.Textual research shows that the origin of " Cangzhu" in the classical prescription should be A. lancea.

[Study on commercial specification of atractylodes based on Delphi method].

This research adopts "Delphi method" to evaluate atractylodes traditional traits and rank correlation. By using methods of mathematical statistics the relationship of the traditional identification indicators and atractylodes goods rank correlation was analyzed, It is found that the main characteristics affectingatractylodes commodity specifications and grades of main characters wereoil points of transaction,color of transaction,color of surface,grain of transaction,texture of transaction andspoilage. The study points out that the original "seventy-six kinds of medicinal materials commodity specification standards of atractylodes differentiate commodity specification" is not in conformity with the actual market situation, we need to formulate corresponding atractylodes medicinal products specifications and grades.This study combined with experimental results "Delphi method" and the market actual situation, proposed the new draft atractylodes commodity specifications and grades, as the new atractylodes commodity specifications and grades standards. It provides a reference and theoretical basis.

[Identification of atractylodis macrocephalae rhizoma and atractylodis rhizoma from their adulterants using DNA barcoding].

Atractylodis Macrocephalae Rhizoma and Atractylodis Rhizoma were widely used in strengthening spleen under different disease conditions, and were easily and often misused each other. Therefore, DNA barcode was used to distinguish Atractylodis Macrocephalae Rhizoma and Atractylodis Rhizoma from their adulterants to ensure the safe use. The sequence lengths of ITS2 of Atractylodes macrocephala, Atractylodis Rhizoma (A. lancea, A. japonica and A. coreana) were both 229 bp. Among the ITS2 sequences of A. macrocephala, only one G/C transversion was detected at site 98, and the average GC content was 69.42%. No variable site was detected in the ITS2 sequences of A. lancea. The maximum K2P intraspecific genetic distances of both A. japonica and A. coreana were 0.013. The maximum K2P intraspecific genetic distances of A. macrocephala, A. lancea, A. japonica and A. coreana were less than the minimum interspecific genetic distance of adulterants. The ITS2 sequences in each of these polytypic species were separated into pairs of divergent clusters in the NJ tree. DNA barcoding could be used as a fast and accurate identification method to distinguish Atractylodis Macrocephalae Rhizoma, Atractylodis Rhizoma, from their adulterants to ensure its safe use.

[Restriction endonuclease digest - melting curve analysis: a new SNP genotyping and its application in traditional Chinese medicine authentication].

Single nucleotide polymorphisms (SNP) is an important molecular marker in traditional Chinese medicine research, and it is widely used in TCM authentication. The present study created a new genotyping method by combining restriction endonuclease digesting with melting curve analysis, which is a stable, rapid and easy doing SNP genotyping method. The new method analyzed SNP genotyping of two chloroplast SNP which was located in or out of the endonuclease recognition site, the results showed that when attaching a 14 bp GC-clamp (cggcgggagggcgg) to 5' end of the primer and selecting suited endonuclease to digest the amplification products, the melting curve of Lonicera japonica and Atractylodes macrocephala were all of double peaks and the adulterants Shan-yin-hua and A. lancea were of single peaks. The results indicated that the method had good stability and reproducibility for identifying authentic medicines from its adulterants. It is a potential SNP genotyping method and named restriction endonuclease digest - melting curve analysis.

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.

[Genetic diversity of Atractylodes macrocephala by ISSR].

OBJECTIVE: To assess the genetic diversity of Atractylodes macrocephala in 12 cultivated populations and 3 wild populations. METHOD: The molecular marker technique ISSR was used to investigate the genetic diversity of 365 samples from 15 populations in Zhejiang, Anhui and Hebei Provinces. Data was analyzed by POPGEN 32, and a cluster diagram was presented by UPGMA. RESULT: One hundred and two amplified fragments were obtained using 13 ISSR primers. 94 polymorphic loci were identified as 92.16%. Nei's genetic diversity index (He) was 0.406 5, Shannon diversity index (I) was 0.590 3, and the coefficient of gene differentiation (Gst) was 0.202 5. The genetic similarity coefficient among the populations ranged from 0.690 7 to 0.960 5 in an average of 0. 825 6. No significant difference was found among the populations based on the clustering analysis. CONCLUSION: There is a low level of genetic differentiation in higher genetic diversity of the A. macrocephala populations. No obvious regional or species diversities were identified. This is consistent with current status of cultivation and gerplasm circulation in A. macrocephala.

[The change rules and correlation between specific leaf weight and dry weight of rhizoma of Atractylodes macrocephala].

OBJECTIVE: To study the change rules of specific leaf weight, dry weight of rhizoma and the drying rate of annual and biennial Atractylodes macrocephala. METHODS: We measured the specific leaf weight and dry weight of rhizoma, and accounted the drying rate and correlation by dugging the annual and biennial plants each 15 days. There were 3 rhizoma types of biennial Atractylodes macrocephala with Drumstick/Frog type, Er-cha type and Wu-zi type. RESULTS: It showed the curve of specific leaf weight of annual plant was "N" style, which of biennial plant was linear. The dry weight and the drying rate of all rhizoma types increased point-blank during the whole growth period. CONCLUSION: The correlation between specific leaf weight and dry weight of rhizoma is obvious in biennial Atractylodes macrocephala but is contrary in annual Atractylodes macrocephala.

[Genetic relationships of Atractylodes plants].

The phylogenetic relationships of the genus Atractylodes DC. was analyzed using the nuclear ribosomal ITS and three chloroplast fragments, including atpB-rbcL, psbB-psbF and trnL-trnF intergenic spacer (IGS) sequences. Phylogenetic analyses revealed that A. lancea subsp. luotianensis and A. lancea, A. chinensis var. liaotungensis and A. coreana form monophyletic terminal clade, separately. The trees, within each the pairwise genetic distances, did not support Hu's classification. Authors suggested that A. lancea Subsp. luotianensis should be included in A. lancea rather than be treated as a separate subspecies. A. carlinoides was placed in the basal position of Atractylodes, which had a distant relationship with the others of the genus. The results lead us to suggest that A. chinensis var. liaotungensis be put into A. coreana, A. chinensis as a subspecies of A. lancea.

[Establishment and application of AFLP fingerprinting system in Atractylodes macrocephala germplasm].

OBJECTIVE: To establish the AFLP Fingerprinting system in the germplasm of Atractylodes macrocephala Koidz. METHODS: 10 wild or cultivated Atractylodes macrocephala were used for AFLP fingerprinting analysis by EcoRI and MseI restriction enzymes with silver staining. RESULTS: Using 2X CTAB buffer extraction method can obtain the best genomic DNA samples. According to the AFLP polymorphism, sixteen out of forty primer pairs were selected to be suitable for AFLP analysis. Total 3003 polymorphic bands were obtained from the 16 sets of primer. Based on the AFLP results, the 10 samples of Atractylodes macrocephala germplasm were classified into four types. CONCLUSION: The establishment of the AFLP fingerprinting system in Atractylodes macrocephala will be used in the identification of germplasms and breeding of the species.

[Studies on genetic diversity in cultivated populations of Atractylodes macrocephala].

OBJECTIVE: To investigate the genetic diversity of main germplasm of Atractylodes macrocephala in China and the genetic differentiation of the germplasm of A. macrocephala. METHOD: A molecular marker ISSR was used to analyze the genetic diversity of 7 populations of A. macrocephala and a population of A. lancea. RESULT: Twelve primers were used in the PCR amplification of 86 samples of A. macrocephala and 5 samples of A. lancea. Sixty-three bands with sizes ranged from 100 to 2500 bp were generated from 12 primers. Of all the 63 bands, 55 bands were polymorphic among 86 individuals of A. macrocephala, the percentage of polymorphic bands were 87.30% at the species level. The percentage of polymorphic bands (PPL) for a single population ranged from 58.73% to 71.43% (mean, 64.85%). Among the 7 populations, a population from Panan, GM exhibited highest variability (PPL =71.43%; HE = 0.2835; I = 0.4267). A dendrogram constructed by an unweighted pair group method of cluster analysis showed that populations from Panan constructed one branch and separated from other populations. In the AMOVA analysis, low level of genetic differentiation among populations was detected, 90.52% of the variability existed in population. CONCLUSION: The genetic diversity of cultivated A. macrocephala in China is high, which is good for the production of high quality herb medicine.

[Construction and application of special analysis database of geoherbs based on 3S technology].

In this paper,the structures, data sources, data codes of "the spacial analysis database of geoherbs" based 3S technology are introduced, and the essential functions of the database, such as data management, remote sensing, spacial interpolation, spacial statistics, spacial analysis and developing are described. At last, two examples for database usage are given, the one is classification and calculating of NDVI index of remote sensing image in geoherbal area of Atractylodes lancea, the other one is adaptation analysis of A. lancea. These indicate that "the spacial analysis database of geoherbs" has bright prospect in spacial analysis of geoherbs.

[Studies on population biology of transitional types of genus Atractylodes in Anhui Province].

OBJECTIVE: To explore the differentiation mechanism of population biology between "Cangzhu" and "Baizhu", commonly used traditional Chinese drugs. METHOD: The wild populations of Atractylodes in Anhui Province were investigated successively, in which specimens were studied, and the cultivated population was observed. RESULT: The wild A. macrocephala and A. lancea, A. lancea subsp. luotianensis and A. lancea presented a series of successive transitions, which was caused by the change of ecological environment. A. macrocephala, A. lancea and A. lancea subsp. luotianensis were extreme types of the series of transitions, which were the germplasm of "Baizhu", "Cangzhu" and "Hancangzhu". The transitional types between A. macrocephala and A. lancea were similar of A. japonica, whose effect was the intermediate of "Baizhu" and "Cangzhu". CONCLUSION: The development and differentatiation of commodites, the source of germplasm and the selection of geo-herbs of "Baizhu", "Cangzhu" and "Hancangzhu" are corresponding with the development of population biology of genus Atractylodes.

[Study on the volatile oil by SFE-CO, from crude and processed Rhizoma Atractylodis by GC-MS].

OBJECTIVE: To analyze the chemical constituents of the volatile oil by SFE-CO2 from Rhizoma Atractylodis and its roasted products. METHODS: The chemical constituents of the volatile oil extracted by SFE-CO2 were separated and identified by GC-MS. RESULTS: 33 kinds of constituents were identified from Atractylodes lancea (Thunb.) DC. and its roasted products. The relative contents of 12 kinds of constituents from the processing products were descended and 7 kinds were ascended; 30 kinds of constituents were identified from Atractylodes chinensis (DC.) Koidz. and its roasted products. The relative contents of 10 kinds of constituents from the roasted products were descended and 11 kinds were ascended. CONCLUSION: In the processed products, the relative contents of some low boiling point constituents are descended but some high are ascended.

[Analysis of amino acid contents between wild plant QiBaiZhu and YunnanBaiZhu].

Amino acid contents between in the root of wild plant QiBaiZhu (Atractylodes macracephala Koidz) and YunnanBaiZhu were analyzed. The results showed that the content of essential amino acid in QiBaiZhu was 3.5 times as much as that of YunnanBaiZhu, especially the content of Arg was very rich (1.61%) and was 53.6 times as much as that of YunnanBaiZhu. Wild plant QiBaiZhu has very good development and utilization value in nutrition and medicinal.

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.

[Studies on chemical constituents in essential oil from wild Atractylodes lancea in dabie mountains].

The chemical constituents in essential oil of wild Atractylodes lancea (Thumb.) DC. from Dabie Mountains were studied. The essential oil was extracted by simultaneous distillation and extraction equipment and analyzed by gas chromatography-mass spectrometry method. 49 compounds were identified and they represent 92.49% of the total peak aeras. The extraction method and solvent selection of the essential oil were studied. The oil yields and major constituents from Atractylodes lancea (Thumb.) DC. from Dabie Mountains were contrasted to that had been reported. The results showed that Atractylodes lancea (Thumb.) DC. from Dabie Mountains had more essential oil (10.14%) and there were more constituents in it. The main constituents in the essential oil were hinesol, beta-eudesmol, 1H-cyclopropa (a) naphthalene, 1a,2,3,5,6,7,7a,7b-octaphdro-1,1,7,7a-tetramethyl-, [1aR-(1a. alpha, 7. alpha, 7a. alpha,7b. alpha-)] -and gamma-eudesmol. Both the major constituents and contents were different from that had been reported.

[The naphtha composing characteristics of geoherbs of Atractylodes lancea].

OBJECTIVE: To find the chemical diversity and characteristics of A. lancea on two levels--individuals and populations, and to discover the chemical essentials for forming geoherbs. METHOD: 47 rhizomes of A. lancea were collected in 7 populations, and 6 naphtha components (1. elemol, 2. hinesol, 3. beta-eudesmol, 4. atractylone, 5. atractylodin, 6. atractylenolid I) in the rhizomes were determined by GC-MS combination. Principal Component Analysis and Cluster Analysis were carried out by SPSS. RESULT: Cluster Analysis of the 6 main components indicated that the chemical components of geoherbs were different from those of the non-geonerbs of A. lancea. Other analysis showed as follows: 1. The general oil of geoberbs were lower than that of non-geoherbs(P < 0.01), but components yielding more than 1% (% of the total oil) were more than non-geoherbs(P < 0.01); 2. Hinesol mixing beta-eudesmol was more in non-geoherbs, which atractylodin mixing atractylone was more in geoherbs(P < 0.001); 3. Principal Component Analysis implied that atractylone was the most important component to discriminate geoherbs and non-geoherbs of A. Lancea. CONCLUSION: The naphtha composing characteristics of geoherbs was the special proportionment sale, viz. atractylone: hinesol: beta-eudesmol: atractylodin being(0.70~2.00):(0.04~0.35):(0.09~0.40):1.