Comparative and phylogenetic analysis of the complete chloroplast genomes of 10 Artemisia selengensis resources based on high-throughput sequencing.

BACKGROUND: Artemisia selengensis, classified within the genus Artemisia of the Asteraceae family, is a perennial herb recognized for its dual utility in culinary and medicinal domains. There are few studies on the chloroplast genome of A. selengensis, and the phylogeographic classification is vague, which makes phylogenetic analysis and evolutionary studies very difficult. RESULTS: The chloroplast genomes of 10 A. selengensis in this study were highly conserved in terms of gene content, gene order, and gene intron number. The genome lengths ranged from 151,148 to 151,257 bp and were typical of a quadripartite structure with a total GC content of approximately 37.5%. The chloroplast genomes of all species encode 133 genes, including 88 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Due to the contraction and expansion of the inverted repeats (IR), the overlap of ycf1 and ndhF genes occurred at the inverted repeats B (IRB) and short single copy sequence (SSC) boundaries. According to a codon use study, the frequent base in the chloroplast genome of A. selengensis' third codon position was A/T. The number of SSR repeats was 42-44, most of which were single nucleotide A/T repeats. Sequence alignment analysis of the chloroplast genome showed that variable regions were mainly distributed in single copy regions, nucleotide diversity values of 0 to 0.009 were calculated by sliding window analysis, 8 mutation hotspot regions were detected, and coding regions were more conserved than non-coding regions. Analysis of non-synonymous substitution (Ka) and synonymous substitution (Ks) revealed that accD, rps12, petB, and atpF genes were affected by positive selection and no genes were affected by neutral selection. Based on the findings of the phylogenetic analysis, Artemisia selengensis was sister to the genus Artemisia Chrysanthemum and formed a monophyletic group with other Artemisia genera. CONCLUSIONS: In this research, the present study systematically compared the chloroplast genomic features of A. selengensis and provided important information for the study of the chloroplast genome of A. selengensis and the evolutionary relationships among Asteraceae species.

The molecular investigations on the subgenus Artemisia Less. of the genus Artemisia L. (Asteraceae) in Turkey / Investigações moleculares sobre o subgênero Artemisia Less. do gênero Artemisia L. (Asteraceae) na Turquia

The genus Artemisia L. of the family Asteraceae is systematically very complex. The aim of this study was to evaluate taxonomic positions of taxa of the subgenus Artemisia belonging to the genus Artemisia in Turkey using some molecular techniques. In this molecular study, 44 individuals belong to 14 species of the subgenus Artemisia were examined. Analyses were performed on the combined dataset using maximum parsimony, maximum likelihood and Bayesian inference and Molecular parameters obtained from co-evaluations of sequences of the psbA-trnH, ITS and ETS regions of examined individuals were used in the phylogenetic tree drawing. According to the results of this study, two molecular groups have been formed based on the DNA sequence similarity of the species, but there are no obvious morphological characters corresponding to two molecular groups. There is no also agreement between the two molecular groups and the two morphological groups formed according to the hairiness condition of the receptacle of species. Due to the lack of molecular significance of their receptacles with or without hair, dividing of the subgenus Artemisia species into new subgenera or sections was not considered appropriate. Likewise, it has been found that with or without hair on the corolla lobes of the central hermaphrodite disc flowers have no molecular significance. It was found that there were no gene flow and hybridization between the 14 species of the subgenus Artemisia and these 14 species were found completed their speciation. This study is important as it is the first molecular based study relating with belong to subgenus Artemisia species growing naturally in Turkey. In addition, new haplotypes related to the populations of Turkey belonging to the subgenus Artemisia taxa were reported by us for the first time and added to the GenBank database.

O gênero Artemisia L. da família Asteraceae é sistematicamente muito complexo. O objetivo deste estudo foi avaliar as posições taxonômicas de táxons do subgênero Artemisia pertencentes ao gênero Artemisia na Turquia usando algumas técnicas moleculares. Neste estudo molecular, 44 indivíduos pertencentes a 14 espécies do subgênero Artemisia foram examinados. As análises foram realizadas no conjunto de dados combinado usando máxima parcimônia, máxima verossimilhança e inferência bayesiana e parâmetros moleculares obtidos a partir de coavaliações de sequências das regiões psbA-trnH, ITS e ETS de indivíduos examinados foram usados no desenho da árvore filogenética. De acordo com os resultados deste estudo, dois grupos moleculares foram formados com base na similaridade da sequência de DNA das espécies, mas não há caracteres morfológicos óbvios correspondentes a dois grupos moleculares. Também não há concordância entre os dois grupos moleculares e os dois grupos morfológicos formados de acordo com a condição de pilosidade do receptáculo da espécie. Devido à falta de significado molecular de seus receptáculos com ou sem cabelo, a divisão das espécies do subgênero Artemisia em novos subgêneros ou seções não foi considerada apropriada. Da mesma forma, verificou-se que com ou sem cabelo nos lobos da corola das flores do disco hermafrodita central não tem significado molecular. Constatou-se que não houve fluxo gênico e hibridização entre as 14 espécies do subgênero Artemisia e essas 14 espécies concluíram sua especiação. Este estudo é importante porque é o primeiro estudo de base molecular relacionado com espécies pertencentes ao subgênero Artemisia crescendo naturalmente na Turquia. Além disso, novos haplótipos relacionados às populações da Turquia pertencentes ao subgênero Artemisia taxa foram relatados por nós pela primeira vez e adicionados ao banco de dados do GenBank.

An investigation on the chloroplast and nuclear genomes of taxa belong to the subgenus Dracunculus (Bess) Rydb. of Artemisia L (Asteraceae) in Turkey / Uma investigação sobre o cloroplasto e os genomas nucleares de táxons pertencentes ao subgênero Dracunculus (Bess) Rydb. de Artemisia L (Asteraceae) na Turquia

Artemisia is one of the biggest genera in the family Asteraceae, with around 500-600 taxa at specific and sub specific levels and organised in 5 subgenera. Due to the high number of taxa, a lot taxonomists are trying to solve the problem of its classification and phylogeny but its natural classification still hasn't been achieved. In this research, 60 individuals belonging to 4 taxa of the subgenus Dracunculus of Artemisia L. in Turkey were examined. For all the examined individuals from both the same and different populations belonging to the taxa of the subgenus Dracunculus, the sequences of the regions both psbA-trnH of chloroplast DNA and ITS of nuclear DNA were determined. Also, the gene regions obtained were recorded in the NCBI GenBank database and an accession number was taken. It was found that there was no gene flow and hybridization between the four studied taxa of the subgenus Dracunculus, and these 4 taxa also completed their speciation. According to the results of this molecular study, A. campestris var. campestris, A. campestris var. marschalliana and A. campestris var. araratica were proposed to be raised from the variety level to the species level. This research is important as it is the first molecular based study relating with the subgenus Dracunculus growing in Turkey.

Artemisia é um dos maiores gêneros da família Asteraceae, com cerca de 500 a 600 táxons em níveis específicos e subespecíficos e organizados em cinco subgêneros. Em razão do grande número de táxons, muitos taxonomistas estão tentando resolver o problema de sua classificação e filogenia, mas sua classificação natural ainda não foi alcançada. Nesta pesquisa, 60 indivíduos pertencentes a quatro táxons do subgênero Dracunculus de Artemisia L. na Turquia foram examinados. Para todos os indivíduos examinados de populações iguais e diferentes pertencentes aos táxons do subgênero Dracunculus, foram determinadas as sequências das regiões psbA-trnH do DNA do cloroplasto e ITS do DNA nuclear. Além disso, as regiões gênicas obtidas foram registradas no banco de dados do NCBI GenBank e um número de acesso foi obtido. Foi constatado que não houve fluxo gênico nem hibridização entre os quatro táxons estudados do subgênero Dracunculus, os quais também completaram sua especiação. De acordo com os resultados deste estudo molecular, A. campestris var. campestris, A. campestris var. marschalliana e A. campestris var. araratica foram propostos para ser elevados do nível de variedade para o nível de espécie. Esta pesquisa é importante porque é o primeiro estudo de base molecular relacionado com o subgênero Dracunculus em crescimento na Turquia.

Flavonoids from Artemisia rupestris and their synergistic antibacterial effects on drug-resistant Staphylococcus aureus.

This study seeks to discover flavonoids from a traditional Chinese herb, Artemisia rupestris L., with synergistic antibacterial effects against multidrug-resistant Staphylococcus aureus. Five flavonoids, artemetin (1), chrysosplenetin (2), pachypodol (3), penduletin (4) and chrysoeriol (5) were obtained by various column chromatographic methods. Their chemical structures were determined on the basis of comprehensive spectroscopic analysis and comparison with literature data. Three of the compounds (2, 4 and 5) exhibited synergistic activity when combined with norfloxacin against SA1199B, an effluxing fluoroquinolone-resistant strain. The fractional inhibitory concentration indices (FICIs) of 2, 4 and 5 in combination with norfloxacin were 0.375, 0.079 and 0.266 respectively, suggesting synergy. Compound 5 also showed synergistic effects against EMRSA-15 and EMRSA-16 when combined with ciprofloxacin and oxacillin exhibiting FICIs of 0.024 and 0.375 respectively. Real time ethidium bromide (EtBr) efflux assay, qRT-PCR and molecular docking were employed to explore the mechanisms of the synergistic effects.

Comparative chloroplast genome analysis of Artemisia (Asteraceae) in East Asia: insights into evolutionary divergence and phylogenomic implications.

BACKGROUND: Artemisia in East Asia includes a number of economically important taxa that are widely used for food, medicinal, and ornamental purposes. The identification of taxa, however, has been hampered by insufficient diagnostic morphological characteristics and frequent natural hybridization. Development of novel DNA markers or barcodes with sufficient resolution to resolve taxonomic issues of Artemisia in East Asia is significant challenge. RESULTS: To establish a molecular basis for taxonomic identification and comparative phylogenomic analysis of Artemisia, we newly determined 19 chloroplast genome (plastome) sequences of 18 Artemisia taxa in East Asia, de novo-assembled and annotated the plastomes of two taxa using publicly available Illumina reads, and compared them with 11 Artemisia plastomes reported previously. The plastomes of Artemisia were 150,858-151,318 base pairs (bp) in length and harbored 87 protein-coding genes, 37 transfer RNAs, and 8 ribosomal RNA genes in conserved order and orientation. Evolutionary analyses of whole plastomes and 80 non-redundant protein-coding genes revealed that the noncoding trnH-psbA spacer was highly variable in size and nucleotide sequence both between and within taxa, whereas the coding sequences of accD and ycf1 were under weak positive selection and relaxed selective constraints, respectively. Phylogenetic analysis of the whole plastomes based on maximum likelihood and Bayesian inference analyses yielded five groups of Artemisia plastomes clustered in the monophyletic subgenus Dracunculus and paraphyletic subgenus Artemisia, suggesting that the whole plastomes can be used as molecular markers to infer the chloroplast haplotypes of Artemisia taxa. Additionally, analysis of accD and ycf1 hotspots enabled the development of novel markers potentially applicable across the family Asteraceae with high discriminatory power. CONCLUSIONS: The complete sequences of the Artemisia plastomes are sufficiently polymorphic to be used as super-barcodes for this genus. It will facilitate the development of new molecular markers and study of the phylogenomic relationships of Artemisia species in the family Asteraceae.

Chloroplast genome sequences of Artemisia maritima and Artemisia absinthium: Comparative analyses, mutational hotspots in genus Artemisia and phylogeny in family Asteraceae.

Artemisia L. is a complex genus of medicinal importance. Publicly available chloroplast genomes of few Artemisia species are insufficient to resolve taxonomic discrepancies at species level. We report chloroplast genome sequences of two further Artemisia species: A. maritima (151,061 bp) and A. absinthium (151,193 bp). Both genomes possess typical quadripartite structure comprising of a large single copy, a small single copy and a pair of long inverted repeats. The two genomes exhibited high similarities in genome sizes, gene synteny, GC content, synonymous and non-synonymous substitutions, codon usage, amino acids frequencies, RNA editing sites, microsatellites, and oligonucleotide repeats. Transition to transversion ratio was <1. Maximum likelihood tree showed Artemisia a monophyletic genus, sister to genus Chrysanthemum. We also identified 20 highly polymorphic regions including rpoC2-rps2, trnR-UCU-trnG-UCC, rps18-rpl20, and trnL-UAG-rpl32 that could be used to develop authentic and cost-effective markers to resolve taxonomic discrepancies and infer phylogenetic relationships among Artemisia species.

Polysaccharide-containing fraction from Artemisia argyi inhibits tumor cell-induced platelet aggregation by blocking interaction of podoplanin with C-type lectin-like receptor 2.

Tumor cell-induced platelet aggregation (TCIPA) is a mechanism that involves the protection of tumor cells in the circulation and the promotion of tumor cell invasion and metastases. The C-type lectin-like receptor 2 (CLEC-2) that binds podoplanin (PDPN) is on the platelet surface and facilitates the TCIPA. Selective blockage of the PDPN-mediated platelet-tumor cell interaction is thereby a plausible strategy for inhibiting metastases. In a search for antagonists of PDPN- and tumor cell-induced platelet aggregation, traditional Chinese medicines were screened and it was found that the water extract of Artemisia argyi leaves selectively inhibited the PDPN-induced platelet aggregation. Bioactivity-guided fractionation analysis was performed for defining a polysaccharide-containing fraction (AAWAP) characterized by inhibition of PDPN activity and tumor cell-induced platelet aggregation. The pharmacological effects of AAWAP on PDPN-activated CLEC-2 signaling were determined by using Western blot and alpha screening analyses. AAWAP was non-toxic to the cells and platelets and it suppressed PDPN- and tumor cell-induced platelet aggregation by irreversibly blocking the interaction between PDPN and CLEC-2 in a dose-dependent manner. These findings indicate that AAWAP is an antagonist of the PDPN-CLEC-2 interaction. This action by AAWAP may result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients.

Comparison and Discrimination of Artemisia argyi and Artemisia lavandulifolia by Gas Chromatography-Mass Spectrometry-Based Metabolomic Approach.

Background: Artemisia argyi and A. lavandulifolia are two morphologically similar herbal medicines derived from the Artemisia genus. Although the two Artemisia herbs have been used as herbal medicines for a long time, studies on their phytochemicals and bioactive compositions are still limited, and no research has been devoted to compare the volatile compounds in A. argyi and A. lavandulifolia. Objective: To compare the volatile constituents in A. argyi and A. lavandulifolia and to explore chemical markers for discrimination and quality evaluation of the two Artemisia herbal medicines. Methods: A GC-MS-based metabolomic approach was employed to compare and discriminate A. argyi and A. lavandulifolia from the aspect of volatile compounds. Multivariate statistical methods, including principal component analysis and orthogonal partial least-squares discriminate analysis, were applied to explore chemical markers for discrimination of the two Artemisia herbal medicines. Results: Thirty volatile compounds were identified, and the chemical profiles of volatile compounds in A. argyi and A. lavandulifolia were quite similar. Principal component analysis and orthogonal partial least-squares discrimination analysis results indicated that the two Artemisia herbal medicines could be distinguished effectively from each other. Ten volatile compounds were selected as potential chemical markers for discrimination of the two Artemisia herbal medicines. Conclusions: The GC-MS-based metabolomics could be an acceptable strategy for comparison and discrimination of A. argyi and A. lavandulifolia as well as authentication of herbal medicines derived from other closely related species. Highlights: GC-MS based metabolomic approach was firstly applied to compare and discriminate Artemisia argyi and Artemisia lavandulifolia.

Detecting useful genetic markers and reconstructing the phylogeny of an important medicinal resource plant, Artemisia selengensis, based on chloroplast genomics.

Artemisia selengenesis is not only a health food, but also a well-known traditional Chinese medicine. Only a fraction of the chloroplast (cp) genome data of Artemisia has been reported and chloroplast genomic materials have been widely used in genomic evolution studies, molecular marker development, and phylogenetic analysis of the genus Artemisia, which makes evolutionary studies, genetic improvement, and phylogenetic identification very difficult. In this study, the complete chloroplast genome of A. selengensis was compared with that of other species within Artemisia and phylogenetic analyses was conducted with other genera in the Asteraceae family. The results showed that A. selengensis is an AT-rich species and has a typical quadripartite structure that is 151,215 bp in length. Comparative genome analyses demonstrated that the available chloroplast genomes of species of Artemisia were well conserved in terms of genomic length, GC contents, and gene organization and order. However, some differences, which may indicate evolutionary events, were found, such as a re-inversion event within the Artemisia genus, an unequal duplicate phenomenon of the ycf1 gene because of the expansion and contraction of the IR region, and the fast-evolving regions. Repeated sequences analysis showed that Artemisia chloroplast genomes presented a highly similar pattern of SSR or LDR distribution. A total of 257 SSRs and 42 LDRs were identified in the A. selengensis chloroplast genome. The phylogenetic analysis showed that A. selengensis was sister to A. gmelinii. The findings of this study will be valuable in further studies to understand the genetic diversity and evolutionary history of Asteraceae.

Synergistic antinociceptive activity of combined aqueous extracts of Artemisia campestris and Artemisia herba-alba in several acute pain models.

In this study, total phenolic and flavonoid contents, acute toxicity and the antinociceptive activity of Artemisia campestris and Artemisia herba-alba, individually and in combination, were investigated using multiple forms of pain in animals. Our results have been shown that plants are relatively safe without clinical signs of toxicity in animals. Thus, extracts were presented high levels in phenolic and flavonoid contents. Artemisia decoctions with 100, 200, 400 mg/kg b-w studied dose, clearly attenuate chemical and thermal noxious stimuli in writhing, formalin and hot-plate tests, and significantly reduced paw oedema in formalin test. Additionally, binary combination forms exhibited a great improvement in intensity and amplitude of antinociceptive activity in comparison with both plants used individually by a relative interference with opioid system. Our findings suggested the central and peripheral analgesic properties and confirmed the folkloric medicinal use of these plants in pain symptom treatment.

Comparison of Artemisiae argyi Folium and Artemisiae lavandulaefoliae Folium by simultaneous determination of multi-components with single reference standard method and chemometric analysis.

INTRODUCTION: Artemisiae argyi Folium and Artemisiae lavandulaefoliae Folium, two morphologically similar herbal medicines derived from Artemisia genus. Although the two Artemisia herbs have been used as medicines for a long time in China, the study of their phytochemical and bioactive composition is limited. OBJECTIVE: To comprehensively compare and evaluate the composition of Artemisiae argyi Folium and Artemisiae lavandulaefoliae Folium, and find the chemical makers for quality evaluation of the two Artemisia herbal medicines. METHODOLOGY: Eight compounds including six phenolic acids and two flavonoids were assayed by a single reference standard for simultaneous determination of multi-components method using 3-caffeoylquinic acid as the reference standard. The quantitative data were further analysed by chemometric approaches to compare and distinguish the two herbal medicines. RESULTS: The credibility and feasibility of the single reference standard for simultaneous determination of the multi-components method were carefully validated. The validated method was applied to analyse 16 batches of Artemisiae argyi Folium and 10 batches of Artemisiae lavandulaefoliae Folium samples. The quantitative results showed that 3,5-di-O-caffeoylquinic acid was the most abundant constituent, and the contents of flavonoids were notably different between the two herbal medicines. The chemometric analysis results indicated the two flavonoids, jaceosidin and eupatilin could be used as chemical markers for quality evaluation of the two herbal medicines. CONCLUSION: The single reference standard for simultaneous determination of the multi-components method coupled with chemometrics analysis could be a well-acceptable strategy to compare and evaluate the quality of Artemisiae argyi Folium and Artemisiae lavandulaefoliae Folium.

Effect of Artemisia annua and Artemisia afra tea infusions on schistosomiasis in a large clinical trial.

BACKGROUND AND OBJECTIVE: Schistosomiasis (bilharzia), a serious neglected tropical disease affecting millions, has few cost-effective treatments, so two Artemisia wormwood species, A. annua and A. afra, were compared with the current standard praziquantel (PZQ) treatment in an 800 patient clinical trial, August-November of 2015. METHODS: The double blind, randomized, superiority clinical trial had three treatment arms: 400 for PZQ, 200 for A. annua, and 200 for A. afra. PZQ-treated patients followed manufacturer posology. Artemisia-treated patients received 1 l/d of dry leaf/twig tea infusions divided into 3 aliquots daily, for 7 days with 28-day follow-up. RESULTS: Of 800 enrolled patients having an average of >700 Schistosoma mansoni eggs per fecal sample, 780 completed the trial. Within 14 days of treatment, all Artemisia-treated patients had no detectable eggs in fecal smears, a result sustained 28 days post treatment. Eggs in fecal smears of PZQ-treated patients were undetectable after D21. More males than females who entered the trial had melena, but both genders responded equally well to treatment; by D28 melena disappeared in all patients. In all arms, eosinophil levels declined by about 27% from D0 to D28. From D0 to D28 hemoglobin increases were greater in PZQ and A. afra-treated patients than in A. annua-treated patients. Hematocrit increases were greater from D0 to D28 for patients treated with either PZQ or A. annua compared to those treated with A. afra. Gender comparison showed that A. afra-treated males had significantly greater hemoglobin and hematocrit increases by D28 than either PZQ or A. annua-treated males. In contrast, PZQ and A. afra-treated females had greater hemoglobin and hematocrit increases than A. annua-treated females. Both adults and pediatric patients treated with A. annua responded better compared to PZQ treatment. CONCLUSION: Both A. annua and A. afra provided faster effective treatment of schistosomiasis and should be considered for implementation on a global scale.

Towards a better understanding of Artemisia vulgaris: Botany, phytochemistry, pharmacological and biotechnological potential.

Artemisia vulgaris is one of the important medicinal plant species of the genus Artemisia, which is usually known for its volatile oils. The genus Artemisia has become the subject of great interest due to its chemical and biological diversity as well as the discovery and isolation of promising anti-malarial drug artemisinin. A. vulgaris has a long history in treatment of human ailments by medicinal plants in various parts of the world. This medicinal plant possesses a broad spectrum of therapeutic properties including: anti-malarial, anti-inflammatory, anti-hypertensive, anti-oxidant, anti-tumoral, immunomodulatory, hepatoprotective, anti-spasmodic and anti-septic. These activities are mainly attributed to the presence of various classes of secondary metabolites, including flavonoids, sesquiterpene lactones, coumarins, acetylenes, phenolic acids, organic acids, mono- and sesquiterpenes. Studies related to A. vulgaris morphology, anatomy and phytochemistry has gained a significant interest for better understanding of production and accumulation of therapeutic compounds in this species. Recently, phytochemical and pharmacological investigations have corroborated the therapeutic potential of bioactive compounds of A. vulgaris. These findings provided further evidence for gaining deeper insight into the identification and isolation of novel compounds, which act as alternative sources of anti-malarial drugs in a cost-effective manner. Considering the rising demand and various medical applications of A. vulgaris, this review highlights the recent reports on the chemistry, biological activities and biotechnological interventions for controlled and continuous production of bioactive compounds from this plant species.

Quantification of santonin in eight species of Artemisia from Kazakhstan by means of HPLC-UV: Method development and validation.

Santonin, a powerful anthelmintic drug that was formely used to treat worms, is Artemisia cina's main constituent. However, due to its toxicity to humans, it is no longer in use. Kazakhstan is looking to introduce this plant as an anthelmintic drug for veterinary purposes, despite the known toxic properties of the santonin. The objective of this study was to develop a fast and specific method for the identification of santonin and its precise quantitation using HPLC-UV in order to avoid unnecessary intoxication, which is paramount for the development of veterinary medicines. The results obtained showed that santonin appears at around 5.7 minutes in this very reliable HPLC method. The validation of the method was performed by the investigation of parameters such as precision, accuracy, reproducibility and recovery. The method was used to identify and quantify santonin in leaves of A. scoparia, A. foetida, A. gmelinni, A. schrenkiana, A. frigida, A. sublesingiana, A terra-albae, and A. absinthium from Kazakhstan as well as in three different extracts of leaves of A. cina. This study has provided a faster and simpler method for the identification and quantification of this compound in other species of Artemisia of economic importance.

Evaluation of DNA barcode candidates for the discrimination of Artemisia L.

Because of the very similar morphologies and wide diversity of Artemisia L. varieties, they are difficult to identify, and there have been many arguments about the systematic classification Artemisia L., especially concerning the division of species. DNA barcode technology is used to rapidly identify species based on standard short DNA sequences. To evaluate seven candidate DNA barcodes (ITS, ITS2, psbA-trnH, rbcL, matK, rpoB, and rpoC1) regarding their ability to identify closely related species of the Artemisia genus in Xinjiang. The corresponding PCR amplification efficiency, detectable genetic divergence, identification efficiency and phylogenetic tree were assessed. We found that the internal transcribed spacer (ITS) region exhibited the highest interspecific divergence, which was significantly higher than the observed intraspecific variation and showed the highest identification efficiency, followed by ITS2, psbA-trnH and, finally, rpoB. matK, rbcL, and rpoC1 performed poorly in this evaluation. ITS, ITS2, and psbA-trnH were able to perfectly identify the tested species Artemisia annua, A. absinthium, A. rupestris, A. tonurnefortiana, A. austriaca, A. dracunculus, A. vulgaris, and A. macrocephala. Therefore, we propose the ITS, ITS2, and psbA-trnH regions as promising DNA barcodes for the closely related species of Artemisia L. in Xinjiang.

DNA Barcode for Identifying Folium Artemisiae Argyi from Counterfeits.

Folium Artemisiae Argyi is an important herb in traditional Chinese medicine. It is commonly used in moxibustion, medicine, etc. However, identifying Artemisia argyi is difficult because this herb exhibits similar morphological characteristics to closely related species and counterfeits. To verify the applicability of DNA barcoding, ITS2 and psbA-trnH were used to identify A. argyi from 15 closely related species and counterfeits. Results indicated that total DNA was easily extracted from all the samples and that both ITS2 and psbA-trnH fragments can be easily amplified. ITS2 was a more ideal barcode than psbA-trnH and ITS2+psbA-trnH to identify A. argyi from closely related species and counterfeits on the basis of sequence character, genetic distance, and tree methods. The sequence length was 225 bp for the 56 ITS2 sequences of A. argyi, and no variable site was detected. For the ITS2 sequences, A. capillaris, A. anomala, A. annua, A. igniaria, A. maximowicziana, A. princeps, Dendranthema vestitum, and D. indicum had single nucleotide polymorphisms (SNPs). The intraspecific Kimura 2-Parameter distance was zero, which is lower than the minimum interspecific distance (0.005). A. argyi, the closely related species, and counterfeits, except for Artemisia maximowicziana and Artemisia sieversiana, were separated into pairs of divergent clusters by using the neighbor joining, maximum parsimony, and maximum likelihood tree methods. Thus, the ITS2 sequence was an ideal barcode to identify A. argyi from closely related species and counterfeits to ensure the safe use of this plant.

Exploration and classification of chromatographic fingerprints as additional tool for identification and quality control of several Artemisia species.

The World Health Organization accepts chromatographic fingerprints as a tool for identification and quality control of herbal medicines. This is the first study in which the distinction, identification and quality control of four different Artemisia species, i.e. Artemisia vulgaris, A. absinthium, A. annua and A. capillaris samples, is performed based on the evaluation of entire chromatographic fingerprint profiles developed with identical experimental conditions. High-Performance Liquid Chromatography (HPLC) with Diode Array Detection (DAD) was used to develop the fingerprints. Application of factorial designs leads to methanol/water (80:20 (v/v)) as the best extraction solvent for the pulverised plant material and to a shaking bath for 30 min as extraction method. Further, so-called screening, optimisation and fine-tuning phases were performed during fingerprint development. Most information about the different Artemisia species, i.e. the highest number of separated peaks in the fingerprint, was acquired on four coupled Chromolith columns (100 mm × 4.6 mm I.D.). Trifluoroacetic acid 0.05% (v/v) was used as mobile-phase additive in a stepwise linear methanol/water gradient, i.e. 5, 34, 41, 72 and 95% (v/v) methanol at 0, 9, 30, 44 and 51 min, where the last mobile phase composition was kept isocratic till 60 min. One detection wavelength was selected to perform data analysis. The lowest similarity between the fingerprints of the four species was present at 214 nm. The HPLC/DAD method was applied on 199 herbal samples of the four Artemisia species, resulting in 357 fingerprints. The within- and between-day variation of the entire method, as well as the quality control fingerprints obtained during routine analysis, were found acceptable. The distinction of these Artemisia species was evaluated based on the entire chromatographic profiles, developed by a shared method, and visualised in score plots by means of the Principal Component Analysis (PCA) exploratory data-analysis technique. Samples of different quality could be indicated on the score plots. No multi-component analysis was required to reach the goal. Furthermore, differences related to the origin of some of the not-certified samples were shown. The importance of the specific herbal part used for its identification was also presented. In addition, no differences were observed among fingerprints of lyophilised or conditioned-air dried samples. Finally, a classification technique, Soft Independent Modelling by Class Analogy (SIMCA), was successfully evaluated as identification technique for unknown samples. Six additional Artemisia species (29 herbal samples) were identified as not belonging to any of the four modelled classes. The developed chromatographic fingerprints and the evaluation of the entire profiles provide an added value to the distinction, identification and quality control of the simultaneously investigated Artemisia species.

Determination of five active compounds in Artemisia princeps and A. capillaris based on UPLC-DAD and discrimination of two species with multivariate analysis.

Five active compounds, chlorogenic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, jaceosidin, and eupatilin, in Artemisia princeps (Compositae) were simultaneously determined by ultra-performance liquid chromatography connected to diode array detector. The morphological resemblance between A. princeps and A. capillaris makes it difficult to properly identify species properly. It occasionally leads to misuse or misapplication in Korean traditional medicine. In the study, the discrimination between A. princeps and A. capillaris was optimally performed by the developed validation method, which resulted in definitely a difference between two species. Also, it was developed the most reliable markers contributing to the discrimination of two species by the multivariate analysis methods, such as a principal component analysis and a partial least squares discrimination analysis.

Phylogeography of mugwort (Artemisia indica), a native pioneer herb in Japan.

Many phylogeographic studies of various tree species have been conducted to elucidate the locations of refugia and the colonization patterns during the Pleistocene. However, only a few large-scale phylogeographic studies have been conducted on herbaceous plants, especially scarce on herbs that are adapted to disturbance. Artemisia indica is a fast-growing perennial herb found in open habitats. To examine the basic information on the genetic structure of this species, we investigated the chloroplast DNA variation within and among populations across Japan. We detected 26 haplotypes in 604 individuals from 28 Japanese populations. The haplotype A1 had wide geographic distribution, and its close relatives were locally present. Some putative ancestral lineages were found mainly in the Kyushu region. This may be because several lineages migrated from the Eurasian continent to the northern coast in Kyushu via the Korean peninsula during the Pleistocene, and the A1 haplotype expanded northward, whereas others remained in southern areas. Phylogenetic distant haplotypes were present mainly in the Kanto region. Because the geographic distribution pattern of these haplotypes in this region is believed to be unnatural, these haplotypes may be derived from commercial sources for re-vegetation during the last few decades.

Phytochemistry predicts habitat selection by an avian herbivore at multiple spatial scales.

Animal habitat selection is a process that functions at multiple, hierarchically. structured spatial scales. Thus multi-scale analyses should be the basis for inferences about factors driving the habitat selection process. Vertebrate herbivores forage selectively on the basis of phytochemistry, but few studies have investigated the influence of selective foraging (i.e., fine-scale habitat selection) on habitat selection at larger scales. We tested the hypothesis that phytochemistry is integral to the habitat selection process for vertebrate herbivores. We predicted that habitats selected at three spatial scales would be characterized by higher nutrient concentrations and lower concentrations of plant secondary metabolites (PSMs) than unused habitats. We used the Greater Sage-Grouse (Centrocercus urophasianus), an avian herbivore with a seasonally specialized diet of sagebrush, to test our hypothesis. Sage-Grouse selected a habitat type (black sagebrush, Artemisia nova) with lower PSM concentrations than the alternative (Wyoming big sagebrush, A. tridentata wyomingensis). Within black sagebrush habitat, Sage-Grouse selected patches and individual plants within those patches that were higher in nutrient concentrations and lower in PSM concentrations than those not used. Our results provide the first evidence for multi-scale habitat selection by an avian herbivore on the basis of phytochemistry, and they suggest that phytochemistry may be a fundamental driver of habitat selection for vertebrate herbivores.