Evolutionary history of the Pasque-flowers (Pulsatilla, Ranunculaceae): Molecular phylogenetics, systematics and rDNA evolution.

Pulsatilla (Anemoneae, Ranunculaceae) is sister to Anemone s.s. and contains ca 40 perennial species of considerable horticultural and medical importance. We sequenced 31 of those species, plus nine subspecies, two cultivars and six outgroups, for two nuclear regions (high-copy nrITS and low-copy MLH1) and three plastid regions (rbcL, accD-psaI, trnL intron) in order to generate the first comprehensive species-level phylogeny of the genus. Phylogenetic trees were constructed using both concatenation-based (maximum likelihood and Bayesian inference) and coalescence methods. The better supported among the internal nodes were subjected to molecular clock dating and ancestral area reconstruction, and karyotypic characters identified by us using Fluorescence In Situ Hybridization were mapped across the tree. The preferred species tree from the coalescence analysis formed the basis of a new infrageneric classification based on monophyly plus degree of divergence. The earliest divergent of the three subgenera, Kostyczewianae, is represented by only a single species that is morphologically intermediate between Anemone s.s. and 'core' Pulsatilla. Subgenus Pulsatilla is considerably richer in species than its sister subgenus Preonanthus and contains three monophyletic sections. Species possessing nodding flowers and pectinately dissected leaves are phylogenetically derived compared with groups possessing erect flowers and palmately lobed leaves. Pulsatilla separated from Anemone s.s. at ca 25 Ma. Our results indicate a central Asian mountain origin of the genus and an initial diversification correlated with late Tertiary global cooling plus regional mountain uplift, aridification and consequent expansion of grasslands. The more rapid and extensive diversification within subgenus Pulsatilla began at ca 3 Ma and continued throughout the Quaternary, driven not only by major perturbations in global climate but also by well-documented polyploidy.

Discrimination and Chemical Phylogenetic Study of Four Pulsatilla Herbs Using UPLC-ESI-MS/MS Combined with Hierarchical Cluster Analysis.

A tissue-smashing based ultra-rapid extraction coupled with ultra-performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) method was developed to determine 10 major triterpenoid saponins from Pulsatilla herbs. Compound 4 was characterized as betulinic acid glycoside 3-O-α-arabinopyranosyl-28-O-ß-glucopyranosyl-23-hydroxy with HR-ESI-MS, 1H-NMR and 13C-NMR experiment. The MS spectra result showed that the ionization of compound 4 was more efficient in the positive mode. Meanwhile, the ions at m/z 789.6 and m/z 627.5 were selected as precursor and product ion for the determination, respectively. The chromatographic separation was carried out on a Phenomenex Kinetex C18 column using a gradient mobile phase system composed of 0.1% formic acid both in methanol and water at a flow rate of 0.4 mL/min. The detection was performed by multiple reaction monitoring mode, using electrospray ionization in the positive and negative mode. The total run time was 6 min. The calibration curves possessed good linearity with all coefficients higher than 0.9987. The intra- and interday precisions were no more than 4.9%, and the average recoveries were from 97.6% to 103.4% with RSD <4.7%. Moreover, hierarchical cluster analysis was performed to compare and discriminate the Pulsatilla herbs based on the quantitative data. The hierarchical cluster analysis results demonstrated that Pulsatilla chinensis, Pulsatilla cernua, Pulsatilla dahurica, Pulsatilla turczainovii samples could be easily discriminated from each other based on the contents of triterpenoid saponins and the established method is feasible for quality control of Pulsatilla herbs.

Rapidly discriminate commercial medicinal Pulsatilla chinensis (Bge.) Regel from its adulterants using ITS2 barcoding and specific PCR-RFLP assay.

Pulsatillae radix is a conventional traditional Chinese medicine (TCM) with common name Baitouweng, and has notable effects on inflammation and dysentery. Pulsatilla chinensis (Bge.) Regel is the only source plant of Baitouweng recorded in Chinese Pharmacopoeia, but its adulteration often occurs in the market that possibly affects medicinal efficacy and safety. We have established an internal transcribed spacer 2 (ITS2) barcode library based on 105 plant samples from 12 Pulsatilla species and 10 common adulterants. Results indicate that ITS2 barcoding can accurately distinguish Pulsatilla species from their adulterants. Pulsatilla chinensis can be discriminated from 11 congeneric species by two stable single nucleotide polymorphisms (SNPs) in the ITS2 region. Additionally, a quick specific PCR-RFLP identification assay based on the ITS2 barcode was developed. Using specific primers ITS2/PR1 combined with restriction enzyme Bgl I, Pu. chinensis can rapidly be differentiated from other species via simple and low-cost test procedures. Furthermore, 30 commercial Baitouweng products were tested and only two products were derived from authentic Pu. chinensis. Thus, these two molecular approaches provide practical tools for quick identification of commercial Baitouweng products and can help ensure the safe use of this TCM product.

Genomic Resources of Three Pulsatilla Species Reveal Evolutionary Hotspots, Species-Specific Sites and Variable Plastid Structure in the Family Ranunculaceae.

BACKGROUND: The European continent is presently colonized by nine species of the genus Pulsatilla, five of which are encountered only in mountainous regions of southwest and south-central Europe. The remaining four species inhabit lowlands in the north-central and eastern parts of the continent. Most plants of the genus Pulsatilla are rare and endangered, which is why most research efforts focused on their biology, ecology and hybridization. The objective of this study was to develop genomic resources, including complete plastid genomes and nuclear rRNA clusters, for three sympatric Pulsatilla species that are most commonly found in Central Europe. The results will supply valuable information about genetic variation, which can be used in the process of designing primers for population studies and conservation genetics research. The complete plastid genomes together with the nuclear rRNA cluster can serve as a useful tool in hybridization studies. METHODOLOGY/PRINCIPAL FINDINGS: Six complete plastid genomes and nuclear rRNA clusters were sequenced from three species of Pulsatilla using the Illumina sequencing technology. Four junctions between single copy regions and inverted repeats and junctions between the identified locally-collinear blocks (LCB) were confirmed by Sanger sequencing. Pulsatilla genomes of 120 unique genes had a total length of approximately 161-162 kb, and 21 were duplicated in the inverted repeats (IR) region. Comparative plastid genomes of newly-sequenced Pulsatilla and the previously-identified plastomes of Aconitum and Ranunculus species belonging to the family Ranunculaceae revealed several variations in the structure of the genome, but the gene content remained constant. The nuclear rRNA cluster (18S-ITS1-5.8S-ITS2-26S) of studied Pulsatilla species is 5795 bp long. Among five analyzed regions of the rRNA cluster, only Internal Transcribed Spacer 2 (ITS2) enabled the molecular delimitation of closely-related Pulsatilla patens and Pulsatilla vernalis. CONCLUSIONS/SIGNIFICANCE: The determination of complete plastid genome and nuclear rRNA cluster sequences in three species of the genus Pulsatilla is an important contribution to our knowledge of the evolution and phylogeography of those endangered taxa. The resulting data can be used to identify regions that are particularly useful for barcoding, phylogenetic and phylogeographic studies. The investigated taxa can be identified at each stage of development based on their species-specific SNPs. The nuclear and plastid genomic resources enable advanced studies on hybridization, including identification of parent species, including their roles in that process. The identified nonsynonymous mutations could play an important role in adaptations to changing environments. The results of the study will also provide valuable information about the evolution of the plastome structure in the family Ranunculaceae.

[Study on HPLC fingerprint characteristics and chemotaxonomy of Pulsatilla medicinal plants].

OBJECTIVE: To establish HPLC characteristic fingerprints of the saponins in Pulsatilla medicinal plants, and provide the basis for authentication and classification of Pulsatilla species. METHOD: The HPLC profiles were determined at 35 degrees C on a Symmetry C18 column (4.6 mm x 250 mm,5 microm) eluted with water (A) and acetonitrile (B) as mobile phases in a linear gradient elution with the flowrate of 0.5 mL x min(-1). The elution program was as follows: 0-8 min, 90% A to 77% A, 8-25 min, changed to 71% A, 25-40 min, to 60% A, 40-50 min, to 50% A, 50-75 min, to 10% A, 75-80 min, to 0% A. The detection wavelength was set at 210 nm. RESULT: The different species of Pulsatilla showed different HPLC fingerprints, but with 10 common peaks. A cluster analysis of 14 accessions indicated that they were divided into four groups: all accessions from P. koreana were classified into group I, P. ambigua in group II, P. dahurica and P. turczaninovii in group III, and P. chinensis in group IV, respectively. The significant differences between P. koreana and P. dahurica, and between P. turczaninovii and P. ambigua were observed. CONCLUSION: The results obtained were in agreement with the traditional taxonomic study. The method was rapid and precise, not only can be used to classify and authenticate Pulsatilla species, but also provides important references for HPLC fingerprints and quality control of Pulsatilla medicinal plants.

The use of AFLP markers in conservation genetics--a case study on Pulsatilla vernalis in the Polish lowlands.

Pulsatilla vernalis is a rare species in the Polish lowlands, strongly threatened by anthropogenic disturbance of its habitats. A grave decrease in its populations has been observed during the past 60-80 years (analogous populations in Eastern Austria and the Czech Republic are almost or completely extinct). An analysis of the genetic diversity of populations in the Polish lowlands was performed to estimate its level and distribution. The AFLP method was used for the study of seven populations. An analysis using five pairs of selective primers revealed 446 scorable fragments; 62.1% of them were polymorphic. The average gene diversity indices was 0.17 (the mean value for all the populations), ranging from 0.139 to 0.204. A weak relationship between diversity and population size was revealed. Most of the genetic diversity was contributed to by the within-population level (AMOVA) and only a weak geographical structure was shown by UPGMA clustering. Four populations formed population-specific clusters while three others (from one region) were intermixed. These preliminary results show a moderate genetic diversity of the studied populations, which was still rather high when compared with their size. This result, together with the low between-population differentiation in the region, suggests that these populations are the remnants of larger populations that, only a few decades ago, were much less isolated.