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.

Comparative Analysis of Four Buckwheat Species Based on Morphology and Complete Chloroplast Genome Sequences.

Buckwheat is a nutritional and economically crop belonging to Polygonaceae, Fagopyrum. To better understand the mutation patterns and evolution trend in the chloroplast (cp) genome of buckwheat, and found sufficient number of variable regions to explore the phylogenetic relationships of this genus, two complete cp genomes of buckwheat including Fagopyrum dibotrys (F. dibotrys) and Fagopyrum luojishanense (F. luojishanense) were sequenced, and other two Fagopyrum cp genomes were used for comparative analysis. After morphological analysis, the main difference among these buckwheat were height, leaf shape, seeds and flower type. F. luojishanense was distinguishable from the cultivated species easily. Although the F. dibotrys and two cultivated species has some similarity, they different in habit and component contents. The cp genome of F. dibotrys was 159,320 bp while the F. luojishanense was 159,265 bp. 48 and 61 SSRs were found in F. dibotrys and F. luojishanense respectively. Meanwhile, 10 highly variable regions among these buckwheat species were located precisely. The phylogenetic relationships among four Fagopyrum species based on complete cp genomes was showed. The results suggested that F. dibotrys is more closely related to Fagopyrum tataricum. These data provided valuable genetic information for Fagopyrum species identification, taxonomy, phylogenetic study and molecular breeding.

Development of a Reference Standard Library of Chloroplast Genome Sequences, GenomeTrakrCP.

Precise, species-level identification of plants in foods and dietary supplements is difficult. While the use of DNA barcoding regions (short regions of DNA with diagnostic utility) has been effective for many inquiries, it is not always a robust approach for closely related species, especially in highly processed products. The use of fully sequenced chloroplast genomes, as an alternative to short diagnostic barcoding regions, has demonstrated utility for closely related species. The U. S. Food and Drug Administration (FDA) has also developed species-specific DNA-based assays targeting plant species of interest by utilizing chloroplast genome sequences. Here, we introduce a repository of complete chloroplast genome sequences called GenomeTrakrCP, which will be publicly available at the National Center for Biotechnology Information (NCBI). Target species for inclusion are plants found in foods and dietary supplements, toxin producers, common contaminants and adulterants, and their close relatives. Publicly available data will include annotated assemblies, raw sequencing data, and voucher information with each NCBI accession associated with an authenticated reference herbarium specimen. To date, 40 complete chloroplast genomes have been deposited in GenomeTrakrCP (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA325670/), and this will be expanded in the future.

Unidentifiable by morphology: DNA barcoding of plant material in local markets in Iran.

Local markets provide a rapid insight into the medicinal plants growing in a region as well as local traditional health concerns. Identification of market plant material can be challenging as plants are often sold in dried or processed forms. In this study, three approaches of DNA barcoding-based molecular identification of market samples are evaluated, two objective sequence matching approaches and an integrative approach that coalesces sequence matching with a priori and a posteriori data from other markers, morphology, ethnoclassification and species distribution. Plant samples from markets and herbal shops were identified using morphology, descriptions of local use, and vernacular names with relevant floras and pharmacopoeias. DNA barcoding was used for identification of samples that could not be identified to species level using morphology. Two methods based on BLAST similarity-based identification, were compared with an integrative identification approach. Integrative identification combining the optimized similarity-based approach with a priori and a posteriori information resulted in a 1.67, 1.95 and 2.00 fold increase for ITS, trnL-F spacer, and both combined, respectively. DNA barcoding of traded plant material requires objective strategies to include data from multiple markers, morphology, and traditional knowledge to optimize species level identification success.

Chloroplast genome structures in Gentiana (Gentianaceae), based on three medicinal alpine plants used in Tibetan herbal medicine.

The genus Gentiana is the largest in the Gentianaceae family with ca. 400 species. However, with most species growing on the Qinghai-Tibet plateau, the processes of adaptive evolution and speciation within the genus is not clear. Also, the genomic analyses could provide important information. So far, the complete chloroplast (cp) genome data of the genus are still deficient. As the second and third sequenced members within Gentianaceae, we report the construction of complete cp sequences of Gentiana robusta King ex Hook. f. and Gentiana crassicaulis Duthie ex Burk., and describe a comparative study of three Gentiana cp genomes, including the cp genome of Gentiana straminea Maxim. published previously. These cp genomes are highly conserved in gene size, gene content, and gene order and the rps16 pseudogene with exon2 missing was found common. Three repeat types and five SSR types were investigated, and the number and distribution are similar among the three genomes. Sixteen genome divergent hotspot regions were identified across these cp genomes that could provide potential molecular markers for further phylogenetic studies in Gentiana. The IR/SC boundary organizations in Gentianales cp genomes were compared and three different types of boundaries were observed. Six data partitions of cp genomes in Gentianales were used for phylogenetic analyses and different data partitions were largely congruent with each other. The ML phylogenetic tree was constructed based on the fragments in cp genomes commonly available in 33 species from Lamiids, including 12 species in Gentianales, 1 in Boraginaceae, 10 in Solanales, and 10 in Lamiales. The result strongly supports the position of Boraginaceae (Ehretia acuminata) as the sister of Solanales, with the bootstrap values of 97 %. This study provides a platform for further research into the molecular phylogenetics of species in the order Gentianales (family Gentianaceae) notably in respect of speciation and species identification.

The complete chloroplast genome sequence of Cynanchum auriculatum Royle ex Wight (Apocynaceae).

Cynanchum auriculatum is a climbing vine belonging to the Apocynaceae family and shows very similar morphology to Cynanchum wilfordii, a medicinal plant. The complete chloroplast genome of C. auriculatum was generated by de novo assembly using the small amount of whole genome sequencing data. The chloroplast genome of C. auriculatum was 160 840 bp in length and consisted of four distinct regions, such as large single copy region (91 973 bp), small single copy region (19 667 bp), and a pair of inverted repeat regions (24 600 bp). The overall GC contents of the chloroplast genome were 37.8%. A total of 114 genes were predicted and included 80 protein-coding genes, 30 tRNA genes, and four rRNA genes. Phylogenetic analysis with the reported chloroplast genomes revealed that C. auriculatum is most closely related to Cynanchum wilfordii, a medicinal plant.

Rapid identification of a narcotic plant Papaver bracteatum using flow cytometry.

In May 2011, numerous poppy plants closely resembling Papaver bracteatum Lindl., a type of narcotic plant that is illegal in Japan, were distributed directly from several large flower shops or through online shopping throughout Japan, including the Tokyo Metropolitan area. In order to better identify the narcotic plants, the relative nuclear DNA content at the vegetative stage was measured by flow cytometric (FCM) analysis in 3 closely-related species of the genus Papaver section Oxytona, namely P. orientale, P. pseudo-orientale, and P. bracteatum, based on the difference between the chromosome numbers of these species. The results showed that the nuclear DNA content differed between these 3 species, and that most of the commercially distributed plants examined in this study could be identified as P. bracteatum. The remaining plants were P. pseudo-orientale, a non-narcotic plant. In addition, the FCM results for the identification of P. bracteatum completely agreed with the results obtained by the morphological analysis, the inter-genic spacer sequence of rpl16-rpl14 (PS-ID sequence) of chloroplast DNA, and the presence of thebaine. These results clearly indicate the usefulness of FCM analysis for the identification of P. bracteatum plants, including when they are in their vegetative stage.

Nucleotide based validation of Ocimum species by evaluating three candidate barcodes of the chloroplast region.

The genus Ocimum comprises of several medicinally important species which frequently fall prey to adulteration due to misidentification. A proficient method is hence required to solve the problems that exist in differentiating its various morphotypes. In plants, candidate DNA barcodes of the chloroplast and nuclear regions have proved to be a great success in the validation of several plant families. Hence, this study involves the use of the molecular-based DNA barcoding method to identify some of the most common and useful species of the genus Ocimum (Tulsi). Here, DNA amplification of three candidate barcodes of the chloroplast genome viz. matK, rbcL and psbA-trnH was performed, to access their ability to produce high sequence variability. The discrimination among species was performed using the Kimura 2-parameter and maximum composite likelihood methods. On analysing the sequence data, the psbA-trnH region proved to be the most suitable candidate barcode and gave an overall variation of 7.3% at the interspecies level. A clear differentiation was found at the species level, showing a maximum distance of 0.264 between dissimilar species. Also, phylogenetic analysis led to the successful identification of hybrids, while it failed to do so at the variety level. Hence, it can be inferred that DNA barcoding is ideal for species-level identification of the genus Ocimum.

Testing robustness of relative complexity measure method constructing robust phylogenetic trees for Galanthus L. using the relative complexity measure.

BACKGROUND: Most phylogeny analysis methods based on molecular sequences use multiple alignment where the quality of the alignment, which is dependent on the alignment parameters, determines the accuracy of the resulting trees. Different parameter combinations chosen for the multiple alignment may result in different phylogenies. A new non-alignment based approach, Relative Complexity Measure (RCM), has been introduced to tackle this problem and proven to work in fungi and mitochondrial DNA. RESULT: In this work, we present an application of the RCM method to reconstruct robust phylogenetic trees using sequence data for genus Galanthus obtained from different regions in Turkey. Phylogenies have been analyzed using nuclear and chloroplast DNA sequences. Results showed that, the tree obtained from nuclear ribosomal RNA gene sequences was more robust, while the tree obtained from the chloroplast DNA showed a higher degree of variation. CONCLUSIONS: Phylogenies generated by Relative Complexity Measure were found to be robust and results of RCM were more reliable than the compared techniques. Particularly, to overcome MSA-based problems, RCM seems to be a reasonable way and a good alternative to MSA-based phylogenetic analysis. We believe our method will become a mainstream phylogeny construction method especially for the highly variable sequence families where the accuracy of the MSA heavily depends on the alignment parameters.

Sequencing and annotation of the chloroplast DNAs and identification of polymorphisms distinguishing normal male-fertile and male-sterile cytoplasms of onion.

Male-sterile (S) cytoplasm of onion is an alien cytoplasm introgressed into onion in antiquity and is widely used for hybrid seed production. Owing to the biennial generation time of onion, classical crossing takes at least 4 years to classify cytoplasms as S or normal (N) male-fertile. Molecular markers in the organellar DNAs that distinguish N and S cytoplasms are useful to reduce the time required to classify onion cytoplasms. In this research, we completed next-generation sequencing of the chloroplast DNAs of N- and S-cytoplasmic onions; we assembled and annotated the genomes in addition to identifying polymorphisms that distinguish these cytoplasms. The sizes (153 538 and 153 355 base pairs) and GC contents (36.8%) were very similar for the chloroplast DNAs of N and S cytoplasms, respectively, as expected given their close phylogenetic relationship. The size difference was primarily due to small indels in intergenic regions and a deletion in the accD gene of N-cytoplasmic onion. The structures of the onion chloroplast DNAs were similar to those of most land plants with large and small single copy regions separated by inverted repeats. Twenty-eight single nucleotide polymorphisms, two polymorphic restriction-enzyme sites, and one indel distributed across 20 chloroplast genes in the large and small single copy regions were selected and validated using diverse onion populations previously classified as N or S cytoplasmic using restriction fragment length polymorphisms. Although cytoplasmic male sterility is likely associated with the mitochondrial DNA, maternal transmission of the mitochondrial and chloroplast DNAs allows for polymorphisms in either genome to be useful for classifying onion cytoplasms to aid the development of hybrid onion cultivars.

Comparative genetic structure of two mangrove species in Caribbean and Pacific estuaries of Panama.

BACKGROUND: Mangroves are ecologically important and highly threatened forest communities. Observational and genetic evidence has confirmed the long distance dispersal capacity of water-dispersed mangrove seeds, but less is known about the relative importance of pollen vs. seed gene flow in connecting populations. We analyzed 980 Avicennia germinans for 11 microsatellite loci and 940 Rhizophora mangle for six microsatellite loci and subsampled two non-coding cpDNA regions in order to understand population structure, and gene flow within and among four major estuaries on the Caribbean and Pacific coasts of Panama. RESULTS: Both species showed similar rates of outcrossing (t= 0.7 in A. germinans and 0.8 in R. mangle) and strong patterns of spatial genetic structure within estuaries, although A. germinans had greater genetic structure in nuclear and cpDNA markers (7 demes > 4 demes and Sp= 0.02 > 0.002), and much greater cpDNA diversity (H(d)= 0.8 > 0.2) than R. mangle. The Central American Isthmus serves as an exceptionally strong barrier to gene flow, with high levels nuclear (F(ST)= 0.3-0.5) and plastid (F(ST)= 0.5-0.8) genetic differentiation observed within each species between coasts and no shared cpDNA haplotypes between species on each coast. Finally, evidence of low ratios of pollen to seed dispersal (r = -0.6 in A. germinans and 7.7 in R. mangle), coupled with the strong observed structure in nuclear and plastid DNA among most estuaries, suggests low levels of gene flow in these mangrove species. CONCLUSIONS: We conclude that gene dispersal in mangroves is usually limited within estuaries and that coastal geomorphology and rare long distance dispersal events could also influence levels of structure.

LC-MS-based chemotaxonomic classification of wild-type Lespedeza sp. and its correlation with genotype.

In this study, 39 specimens belonging to Lespedeza species (Lespedeza cyrtobotrya, L. bicolor, L. maximowiczii, and Lespedeza cuneata) (Leguminosae) were classified phenotypically and genotypically. We constructed a phylogenetic tree based on the combined nrDNA (internal transcribed spacer; ITS) and cpDNA (trnL-trnF) sequences with the aim of classifying the genotypes. Samples were mainly divided into three genotypes. Samples of L. cyrtobotrya and L. bicolor were mixed in a single branch, whereas samples of L. maximowiczii and L. cuneata were clustered within species, respectively. We performed a liquid chromatography-electrospray ionization-mass spectrometry-based metabolite profiling analysis to classify the phenotypes. Multivariate statistical analyses such as principal component analysis (PCA) and hierarchical clustering analysis (HCA) were used for the clustering pattern analysis and distance analysis between species, respectively. According to the PCA and HCA results, leaves were classified into four phenotypes according to species. In both the genetic and chemotaxonomic classification methods, the distance between L. cyrtobotrya and L. bicolor was the closest between species, and L. cuneata was the farthest away from the other three species. Additionally, orthogonal partial least squares-discriminant analysis was employed to identify significantly different phytochemicals between species. We classified L. cyrtobotrya and L. bicolor by identifying significantly different phytochemicals. Interestingly, leaves and stems showed different phenotypic classifications based on the chemotaxonomic classification. Stem samples of the other three species were mixed regardless of species, whereas L. cyrtobotrya stem samples were clustered within species. The phenotypic classification of leaves coincided more with the genotypic classification than that of stems. Key message We classified four wild-type Lespedeza sp. by analyzing the combined nrDNA (ITS) and cpDNA (trnL-trnF) sequences. We also classified leaves and stems of Lespedeza sp. by applying liquid chromatography-mass spectroscopy-based metabolite profiling.

Experimental evidence for the ancestry of allotetraploid Trifolium repens and creation of synthetic forms with value for plant breeding.

BACKGROUND: White clover (Trifolium repens) is a ubiquitous weed of the temperate world that through use of improved cultivars has also become the most important legume of grazed pastures world-wide. It has long been suspected to be allotetraploid, but the diploid ancestral species have remained elusive. Putative diploid ancestors were indicated by DNA sequence phylogeny to be T. pallescens and T. occidentale. Here, we use further DNA evidence as well as a combination of molecular cytogenetics (FISH and GISH) and experimental hybridization to test the hypothesis that white clover originated as a hybrid between T. pallescens and T. occidentale. RESULTS: T. pallescens plants were identified with chloroplast trnL intron DNA sequences identical to those of white clover. Similarly, T. occidentale plants with nuclear ITS sequences identical to white clover were also identified. Reciprocal GISH experiments, alternately using labeled genomic DNA probes from each of the putative ancestral species on the same white clover cells, showed that half of the chromosomes hybridized with each probe. F1 hybrids were generated by embryo rescue and these showed strong interspecific chromosome pairing and produced a significant frequency of unreduced gametes, indicating the likely mode of polyploidization. The F1 hybrids are inter-fertile with white clover and function as synthetic white clovers, a valuable new resource for the re-incorporation of ancestral genomes into modern white clover for future plant breeding. CONCLUSIONS: Evidence from DNA sequence analyses, molecular cytogenetics, interspecific hybridization and breeding experiments supports the hypothesis that a diploid alpine species (T. pallescens) hybridized with a diploid coastal species (T. occidentale) to generate tetraploid T. repens. The coming together of these two narrowly adapted species (one alpine and the other maritime), along with allotetraploidy, has led to a transgressive hybrid with a broad adaptive range.

Elucidating polyploidization of bermudagrasses as assessed by organelle and nuclear DNA markers.

Clarification of relationships among ploidy series of Cynodon accessions could be beneficial to bermudagrass breeding programs, and would enhance our understanding of the evolutionary biology of this warm season grass species. This study was initiated to elucidate polyploidization among Cynodon accessions with different ploidy series collected from Turkey based on chloroplast and nuclear DNA. Forty Cynodon accessions including 7 diploids, 3 triploids, 10 tetraploids, 11 pentaploids, and 9 hexaploids were analyzed using chloroplast DNA restriction fragment-length polymorphism (cpDNA RFLP), chloroplast DNA simple sequence repeat (cpDNA SSR), and nuclear DNA markers based on neighbor-joining (NJ) and principle component analyses (PCA). All three-marker systems with two statistical algorithms clustered the diploids apart from the other ploidy levels. Assuming autopolyploidy, spontaneous polyploidization followed by rapid diversification among the higher ploidy levels than the diploids is likely in Cynodon's evolution. Few tetraploid and hexaploid accessions were clustered with or closely to the group of diploids, supporting the hypothesis above. Eleven haplotypes as estimated by cpDNA RFLP and SSR markers were detected. This study indicated that the diploids had different organelle genome from the rest of the ploidy series and provided valuable insight into relationships among ploidy series of Cynodon accessions based on cp and nuclear DNAs.

Classification and differentiation of the genus Peganum indigenous to China based on chloroplast trnL-F and psbA-trnH sequences and seed coat morphology.

The genus Peganum (Zygophyllaceae) consists of six species and one subspecies; three of which are distributed in China, P. harmala Linn, P. nigellastrum Bunge and P. multisectum (Maxim.) Bobr. A probable new or intermediate species, Peganum sp., has been suggested in the wild in northwest China. Traditional classification in genus Peganum has focused on hairs on the plant surface. In this study, seed coat characteristics of Peganum species were investigated using light and scanning electron microscopy, demonstrating clear differences in morphology between species. In addition, DNA sequence data from two sequences (chloroplast: trnL-F, psbA-trnH) were used to differentiate Peganum sp. and study polygenetic relationships. Diversity in DNA sequences among Peganum species was found, with inter-specific sequence divergence ranging from 0.6% to 5.6% in psbA-trnH, and 0.0% to 1.8% in trnL-F. The variations within species were low: from 0.0% to 0.4% in psbA-trnH and 0.0% to 0.4% in trnL-F. Therefore, Peganum species can now be easily identified as separate entities based on variations in DNA sequences. Phylogenetic trees were constructed from the combined data set for the two gene fragments, and the results indicate that Peganum sp. is monophyletic and sister to P. harmala and P. nigellastrum, while P. multisectum is also monophyletic. DNA data further confirmed that P. multisectum is an independent species and that a new species, Peganum sp., exists within the genus Peganum growing wild in China.

Identification and characterization of plastid trnF(GAA) pseudogenes in four species of Solanum (Solanaceae).

Non-functional trnF pseudogenes that rarely occur in embryophytes have been found in Solanaceae. We have sequenced the trnL-F intergenic spacer of four species of Solanum, and found duplicated regions of the original trnF gene. These repeats were 94-260 bp long causing large length variation in the trnL-F intergenic spacer resulting from differences in pseudogene copy number (2-4). The duplicated trnF regions are comprised of several highly structured motifs, which were partial residues, or entire parts of the Anticodon, T- and D-domains of the original gene, but all lacked the acceptor stems at the 5'- or 3'-end. Pseudogenes included several transitions and transversions in their sequences compared to the original trnF gene. Among pseudogene copies, T-domains were more frequent and fragmented than D-domain elements. Our results demonstrate that although chloroplast evolution is uniform such structural duplications in the sequences used for phylogenetic reconstructions should be treated with great caution.

Structural and content diversity of mitochondrial genome in beet: a comparative genomic analysis.

Despite their monophyletic origin, mitochondrial (mt) genomes of plants and animals have developed contrasted evolutionary paths over time. Animal mt genomes are generally small, compact, and exhibit high mutation rates, whereas plant mt genomes exhibit low mutation rates, little compactness, larger sizes, and highly rearranged structures. We present the (nearly) whole sequences of five new mt genomes in the Beta genus: four from Beta vulgaris and one from B. macrocarpa, a sister species belonging to the same Beta section. We pooled our results with two previously sequenced genomes of B. vulgaris and studied genome diversity at the species level with an emphasis on cytoplasmic male-sterilizing (CMS) genomes. We showed that, contrary to what was previously assumed, all three CMS genomes belong to a single sterile lineage. In addition, the CMSs seem to have undergone an acceleration of the rates of substitution and rearrangement. This study suggests that male sterility emergence might have been favored by faster rates of evolution, unless CMS itself caused faster evolution.

Molecular phylogenetics and character evolution of the "sacaca" clade: novel relationships of Croton section Cleodora (Euphorbiaceae).

Phylogenetic relationships of Croton section Cleodora (Klotzsch) Baill. were evaluated using the nuclear ribosomal ITS and the chloroplast trnL-F and trnH-psbA regions. Our results show a strongly supported clade containing most previously recognized section Cleodora species, plus some other species morphologically similar to them. Two morphological synapomorphies that support section Cleodora as a clade include pistillate flowers in which the sepals overlap to some degree, and styles that are connate at the base to varying degrees. The evolution of vegetative and floral characters that have previously been relied on for taxonomic decisions within this group are evaluated in light of the phylogenetic hypotheses. Within section Cleodora there are two well-supported clades, which are proposed here as subsections (subsection Sphaerogyni and subsection Spruceani). The resulting phylogenetic hypothesis identifies the closest relatives of the medicinally important and essential oil-rich Croton cajucara Benth. as candidates for future screening in phytochemical and pharmacological studies.

Detection of Valeriana jatamansi as an adulterant of medicinal Paris by length variation of chloroplast psbA-trnH region.

Two varieties of Paris polyphylla Smith (Melanthiaceae), P. polyphylla Smith var. chinensis (Franch.) Hara, and P. polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz., are used as medicinal Paris in China. Their dried rhizomes are the major source of raw material for some medicines. In recent years, medicinal PARIS has been found to be adulterated with Valeriana jatamansi Jones (Valerianaceae) due to its high market demand and natural resource deficiency. After the chloroplast PSBA-TRNH regions of medicinal Paris and V. jatamansi were sequenced and analyzed, it was found that their characteristic sizes were > 1000 and around 250 bp, respectively. Based on length variation, medicinal Paris and the mixed adulterant were detected and distinguished from each other by amplification and electrophoresis. The amount of V. jatamansi that can be identified as an adulterant of medicinal PARIS was also investigated. A trace amount (1 : 1000) of the adulterant was detected in the sensitivity tests. The established method has been proven to be sensitive and reliable.

Evolution and origins of the Mazatec hallucinogenic sage, Salvia divinorum (Lamiaceae): a molecular phylogenetic approach.

Salvia divinorum Epl. & Játiva-M. (Lamiaceae) is a potent hallucinogenic plant that is classified within Salvia subgenus Calosphace, section Dusenostachys, and hypothesized to be an interspecific hybrid. It is of ethnobotanical significance due to its employment in traditional healing ceremonies by the Mazatecs of Oaxaca, Mexico, and due to its unique pharmacology-a highly selective, non-nitrogenous, κ-opioid receptor agonist. In order to test its phylogenetic position and putative hybridity, we sequenced multiple DNA regions (ITS, trnL-trnF, and psbA-trnH) of 52 species-representing the major lineages of subgenus Calosphace-and six accessions of S. divinorum. Our molecular phylogenetic results suggest that S. divinorum should not be classified within Dusenostachys and that it is not a hybrid. Additionally, we determine that the closest known relative of this psychoactive Mexican sage is S. venulosa, a rare endemic of Colombia.