Revealing Medicinal Constituents of Bistorta vivipara Based on Non-Targeted Metabolomics and 16S rDNA Gene Sequencing Technology.

Bistorta vivipara is a medicinal plant with a long history, but there are few studies on the effects of its medicinal components and endophytic bacteria on the accumulation of secondary metabolites. Therefore, in this study, non-targeted metabolomics techniques and 16s rDNA techniques were used to study B. vivipara from different regions. A total of 1290 metabolites and 437 differential metabolites were identified from all samples. Among them, flavonoids, isoflavonoids, and benzopyrans are the main medicinal components of B. vivipara; these have potential anticancer, antiviral, and antioxidant properties, as well as potential applications for the treatment of atrial fibrillation. In addition, irigenin, an important medicinal component, was identified for the first time. The endophytic bacterial communities in the root tissues of B. vivipara from different regions were also different in composition and richness. Hierarchical clustering heat map analysis showed that Proteobacteria and Actinobacteriota bacteria significantly affected the accumulation of many medicinal components in the roots of B. vivipara.

Genome assembly and microsatellite marker development using Illumina and PacBio sequencing in Persicaria maackiana (Polygonaceae) from Korea.

BACKGROUND: Persicaria maackiana (Regel) is a potential medicinal plant that exerts anti-diabetic effects. However, the lack of genomic information on P. maackiana hinders research at the molecular level. OBJECTIVE: Herein, we aimed to construct a draft genome assembly and obtain comprehensive genomic information on P. maackiana using high-throughput sequencing tools PacBio Sequel II and Illumina. METHODS: Persicaria maackiana samples from three natural populations in Gaecheon, Gichi, and Uiryeong reservoirs in South Korea were used to generate genomic DNA libraries, perform genome de novo assembly, gene ontology analysis, phylogenetic tree analysis, genotyping, and identify microsatellite markers. RESULTS: The assembled P. maackiana genome yielded 32,179 contigs. Assessment of assembly integrity revealed 1503 (93.12%) complete Benchmarking Universal Single-Copy Orthologs. A total of 64,712 protein-coding genes were predicted and annotated successfully in the protein database. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs, 13,778 genes were annotated into 18 categories. Genes that activated AMPK were identified in the KEGG pathway. A total of 316,992 microsatellite loci were identified, and primers targeting the flanking regions were developed for 292,059 microsatellite loci. Of these, 150 primer sets were randomly selected for amplification, and 30 of these primer sets were identified as polymorphic. These primers amplified 3-9 alleles. The mean observed and expected heterozygosity were 0.189 and 0.593, respectively. Polymorphism information content values of the markers were 0.361-0.754. CONCLUSION: Collectively, our study provides a valuable resource for future comparative genomics, phylogeny, and population studies of P. maackiana.

Plastome comparison and phylogenomics of Fagopyrum (Polygonaceae): insights into sequence differences between Fagopyrum and its related taxa.

BACKGROUND: Fagopyrum (Polygonaceae) is a small plant lineage comprised of more than fifteen economically and medicinally important species. However, the phylogenetic relationships of the genus are not well explored, and the characteristics of Fagopyrum chloroplast genomes (plastomes) remain poorly understood so far. It restricts the comprehension of species diversity in Fagopyrum. Therefore, a comparative plastome analysis and comprehensive phylogenomic analyses are required to reveal the taxonomic relationship among species of Fagopyrum. RESULTS: In the current study, 12 plastomes were sequenced and assembled from eight species and two varieties of Fagopyrum. In the comparative analysis and phylogenetic analysis, eight previously published plastomes of Fagopyrum were also included. A total of 49 plastomes of other genera in Polygonaceae were retrieved from GenBank and used for comparative analysis with Fagopyrum. The variation of the Fagopyrum plastomes is mainly reflected in the size and boundaries of inverted repeat/single copy (IR/SC) regions. Fagopyrum is a relatively basal taxon in the phylogenomic framework of Polygonaceae comprising a relatively smaller plastome size (158,768-159,985 bp) than another genus of Polygonaceae (158,851-170,232 bp). A few genera of Polygonaceae have nested distribution of the IR/SC boundary variations. Although most species of Fagopyrum show the same IRb/SC boundary with species of Polygonaceae, only a few species show different IRa/SC boundaries. The phylogenomic analyses of Fagopyrum supported the cymosum and urophyllum groups and resolved the systematic position of subclades within the urophyllum group. Moreover, the repeat sequence types and numbers were found different between groups of Fagopyrum. The plastome sequence identity showed significant differences between intra-group and inter-group. CONCLUSIONS: The deletions of intergenic regions cause a short length of Fagopyrum plastomes, which may be the main reason for plastome size diversity in Polygonaceae species. The phylogenomic reconstruction combined with the characteristics comparison of plastomes supports grouping within Fagopyrum. The outcome of these genome resources may facilitate the taxonomy, germplasm resources identification as well as plant breeding of Fagopyrum.

Plastid and mitochondrial phylogenomics reveal correlated substitution rate variation in Koenigia (Polygonoideae, Polygonaceae) and a reduced plastome for Koenigia delicatula including loss of all ndh genes.

Koenigia, a genus proposed by Linnaeus, has a contentious taxonomic history. In particular, relationships among species and the circumscription of the genus relative to Aconogonon remain uncertain. To explore phylogenetic relationships of Koenigia with other members of tribe Persicarieae and to establish the timing of major evolutionary diversification events, genome skimming of organellar sequences was used to assemble plastomes and mitochondrial genes from 15 individuals representing 13 species. Most Persicarieae plastomes exhibit a conserved structure and content relative to other flowering plants. However, Koenigia delicatula has lost functional copies of all ndh genes and the intron from atpF. In addition, the rpl32 gene was relocated in the K. delicatula plastome, which likely occurred via overlapping inversions or differential expansion and contraction of the inverted repeat. The highly supported but conflicting relationships between plastome and mitochondrial trees and among gene trees complicates the circumscription of Koenigia, which could be caused by rapid diversification within a short period. Moreover, the plastome and mitochondrial trees revealed correlated variation in substitution rates among Persicarieae species, suggesting a shared underlying mechanism promoting evolutionary rate variation in both organellar genomes. The divergence of dwarf K. delicatula from other Koenigia species may be associated with the well-known Eocene Thermal Maximum 2 or Early Eocene Climatic Optimum event, while diversification of the core-Koenigia clade associates with the Mid-Miocene Climatic Optimum and the uplift of Qinghai-Tibetan Plateau and adjacent areas.

The complete chloroplast genome of Persicaria perfoliata and comparative analysis with four medicinal plants of Polygonaceae.

Polygonaceae is a large family of medicinal herbs that includes many species used as traditional Chinese medicine, such as Per sicaria per foliata. Here, we sequenced the complete chloroplast genome of P. per foliata using Illumina sequencing technology with the purpose of providing a method to facilitate accurate identification. After being annotated, the complete chloroplast genome of P. per foliata was compared with those of Fagopyrum tataricum, Per sicaria chinensis, Fagopyrum dibotrys, and Fallopia multiflora. The complete chloroplast genome of P. per foliata is 160 730 bp in length, containing a small single-copy region of 12 927 bp, a large single-copy region of 85 433 bp, and a pair of inverted repeat regions of 62 370 bp. A total of 131 genes were annotated, including 8 rRNA genes, 34 tRNA genes, and 84 protein-coding genes. Forty-two simple sequence repeats and 55 repeat sequences were identified. Mutational hotspot analyses indicated that five genes (matK, ndhF, ccsA, cemA, and rpl20) could be selected as candidates for molecular markers. Moreover, phylogenetic analysis showed that all the Polygonaceae species formed a monophyletic clade, and P. per foliata showed the closest relationship with P. chinense. The study provides valuable molecular information to accurately identify P. per foliata and assist in its development and application.

Know your enemy: Application of ATR-FTIR spectroscopy to invasive species control.

Extreme weather and globalisation leave our climate vulnerable to invasion by alien species, which have negative impacts on the economy, biodiversity, and ecosystem services. Rapid and accurate identification is key to the control of invasive alien species. However, visually similar species hinder conservation efforts, for example hybrids within the Japanese Knotweed complex.We applied the novel method of ATR-FTIR spectroscopy combined with chemometrics (mathematics applied to chemical data) to historic herbarium samples, taking 1580 spectra in total. Samples included five species from within the interbreeding Japanese Knotweed complex (including three varieties of Japanese Knotweed), six hybrids and five species from the wider Polygonaceae family. Spectral data from herbarium specimens were analysed with several chemometric techniques: support vector machines (SVM) for differentiation between plant types, supported by ploidy levels; principal component analysis loadings and spectral biomarkers to explore differences between the highly invasive Reynoutria japonica var. japonica and its non-invasive counterpart Reynoutria japonica var. compacta; hierarchical cluster analysis (HCA) to investigate the relationship between plants within the Polygonaceae family, of the Fallopia, Reynoutria, Rumex and Fagopyrum genera.ATR-FTIR spectroscopy coupled with SVM successfully differentiated between plant type, leaf surface and geographical location, even in herbarium samples of varying age. Differences between Reynoutria japonica var. japonica and Reynoutria japonica var. compacta included the presence of two polysaccharides, glucomannan and xyloglucan, at higher concentrations in Reynoutria japonica var. japonica than Reynoutria japonica var. compacta. HCA analysis indicated that potential genetic linkages are sometimes masked by environmental factors; an effect that can either be reduced or encouraged by altering the input parameters. Entering the absorbance values for key wavenumbers, previously highlighted by principal component analysis loadings, favours linkages in the resultant HCA dendrogram corresponding to expected genetic relationships, whilst environmental associations are encouraged using the spectral fingerprint region.The ability to distinguish between closely related interbreeding species and hybrids, based on their spectral signature, raises the possibility of using this approach for determining the origin of Japanese knotweed infestations in legal cases where the clonal nature of plants currently makes this difficult and for the targeted control of species and hybrids. These techniques also provide a new method for supporting biogeographical studies.

Screening of an Endophyte Transforming Polydatin to Resveratrol from Reynoutria Japonica Houtt and the Optimization of Its Transformation Parameters.

Resveratrol showed various kinds of bioactivities, such as antioxidant, antimicrobial, anticancer effects and, therefore, has been used widely as an important ingredient in medication, healthy foods and cosmetics. However, in nature, resveratrol usually exists at low content and more often exists as polydatin. Therefore, it becomes important to find the cost-effective and environmental-friendly way to transform polydatin to resveratrol. In this study, endophytes were isolated from the rhizome tissue of Reynoutria japonica and screened for transforming polydatin to resveratrol using reversed-phase high-performance liquid chromatography (RP-HPLC) and confirmed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy. A bacterium identified as Bacillus aryabhattai using 16S rRNA phylogenetic tree analysis showed highest transformation rate. The transforming conditions were optimized including substrate concentration, substrate addition time, culture temperature and inoculation ratio. Our results demonstrated that the bacteria isolated from R. japonica rhizome tissue showed high activity in transforming polydatin into resveratrol. Crude extract of R. japonica root and rhizome (RJE) was also tested as substrate and it was found that the transformation was significantly inhibited at 10.0 mg/mL RJE. Emodin at equivalent concentration of 10.0 mg/mL RJE showed no inhibition activity, and glucose content in RJE was trace and far from enough to exhibit the inhibitory activity. Successive solvent partition followed by an inhibition activity assay revealed that the ethyl acetate fraction showed the main inhibition activity. However, due to the coexistence of polydatin and compounds with inhibitory activity, the concentration of RJE can only be used at limited concentration as substrate.

Complete plastome sequencing resolves taxonomic relationships among species of Calligonum L. (Polygonaceae) in China.

BACKGROUND: Calligonum (Polygonaceae) is distributed from southern Europe through northern Africa to central Asia, and is typically found in arid, desert regions. Previous studies have revealed that standard DNA barcodes fail to discriminate Calligonum species. In this study, the complete plastid genomes (plastome) for 32 accessions of 21 Calligonum species is sequenced to not only generate the first complete plastome sequence for the genus Calligonum but to also 1) Assess the ability of the complete plastome sequence to discern species within the group, and 2) screen the plastome sequence for a cost-effective DNA barcode that can be used in future studies to resolve taxonomic relationships within the group. RESULTS: The whole plastomes of Calligonum species possess a typical quadripartite structure. The size of the Calligonum plastome is approximately 161 kilobase pairs (kbp), and encodes 113 genes, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Based on ML phylogenetic tree analyses, the complete plastome has higher species identification (78%) than combinations of standard DNA barcodes (rbcL + matK + nrITS, 56%). Five newly screened gene regions (ndhF, trnS-G, trnC-petN, ndhF-rpl32, rpl32-trnL) had high species resolution, where ndhF and trnS-G were able to distinguish the highest proportion of Calligonum species (56%). CONCLUSIONS: The entire plastid genome was the most effective barcode for the genus Calligonum, although other gene regions showed great potential as taxon-specific barcodes for species identification in Calligonum.

Complete chloroplast genome sequence determination of Rheum species and comparative chloroplast genomics for the members of Rumiceae.

KEY MESSAGE: Complete plastomes of Rheum species facilitated to clarify the phylogeny of Polygonaceae, and comparative chloroplast genomics contributed to develop genetic markers for the authentication of Rheum species. Rheum (Polygonaceae) is widely distributed throughout the temperate and subtropical areas of Asian interior. Rheum species are usually perennial herbs, and half of them are endemic to China with important medicinal properties. On account of similar morphological characteristics, species delimitation of Rheum still remains unclear. Chloroplast genomes of eight Rheum species, Rumex crispus and Oxyria digyna were characterized. Based on the comparison of genome structure of these species and the two published Rheum species, it is shown that plastome sequences of these species are relatively conserved with the same gene order, and three Sect. Palmata species remarkably showed high sequence similarities. Some hotspots could be used to discriminate the Rheum species, and 17 plastid genes were subject to positive selection. The phylogenetic analyses indicated that all the Polygonaceae species were clustered in the same group and showed that Rheum species, except for Rheum wittrockii, formed a monophyletic group with high maximum parsimony/maximum likelihood bootstrap support values and Bayesian posterior probabilities. The molecular dating based on plastomes indicated that the divergences within Polygonaceae species were dated to the Upper Cretaceous period [73.86-77.99 million years ago (Ma)]. The divergence of Sect. Palmata species was estimated to have occurred around 1.60 Ma, indicating that its diversification was affected by the repeated climatic fluctuation in the Quaternary.

Structural and kinetic studies of a novel nerol dehydrogenase from Persicaria minor, a nerol-specific enzyme for citral biosynthesis.

Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp280, Leu294 and Ala303). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily.

Chasing ghosts: allopolyploid origin of Oxyria sinensis (Polygonaceae) from its only diploid congener and an unknown ancestor.

Reconstructing the origin of a polyploid species is particularly challenging when an ancestor has become extinct. Under such circumstances, the extinct donor of a genome found in the polyploid may be treated as a 'ghost' species in that its prior existence is recognized through the presence of its genome in the polyploid. In this study, we aimed to determine the polyploid origin of Oxyria sinensis (2n = 40) for which only one congeneric species is known, that is diploid O. digyna (2n = 14). Genomic in situ hybridization (GISH), transcriptome, phylogenetic and demographic analyses, and ecological niche modelling were conducted for this purpose. GISH revealed that O. sinensis comprised 14 chromosomes from O. digyna and 26 chromosomes from an unknown ancestor. Transcriptome analysis indicated that following divergence from O. digyna, involving genome duplication around 12 million years ago (Ma), a second genome duplication occurred approximately 6 Ma to give rise to O. sinensis. Oxyria sinensis was shown to contain homologous gene sequences divergent from those present in O. digyna in addition to a set that clustered with those in O. digyna. Coalescent simulations indicated that O. sinensis expanded its distribution approximately 6-7 Ma, possibly following the second polyploidization event, whereas O. digyna expanded its range much later. It was also indicated that the distributions of both species contracted and re-expanded during the Pleistocene climatic oscillations. Ecological niche modelling similarly suggested that both species experienced changes in their distributional ranges in response to Quaternary climatic changes. The extinction of the unknown 'ghost' tetraploid species implicated in the origin of O. sinensis could have resulted from superior adaptation of O. sinensis to repeated climatic changes in the region where it now occurs.

Identification of a drimenol synthase and drimenol oxidase from Persicaria hydropiper, involved in the biosynthesis of insect deterrent drimanes.

The sesquiterpenoid polygodial, which belongs to the drimane family, has been shown to be an antifeedant for a number of herbivorous insects. It is presumed to be synthesized from farnesyl diphosphate via drimenol, subsequent C-12 hydroxylation and further oxidations at both C-11 and C-12 to form a dialdehyde. Here, we have identified a drimenol synthase (PhDS) and a cytochrome P450 drimenol oxidase (PhDOX1) from Persicaria hydropiper. Expression of PhDS in yeast and plants resulted in production of drimenol alone. Co-expression of PhDS with PhDOX1 in yeast yielded drimendiol, the 12-hydroxylation product of drimenol, as a major product, and cinnamolide. When PhDS and PhDOX1 were transiently expressed by agro-infiltration in Nicotiana benthamiana leaves, drimenol was almost completely converted into cinnamolide and several additional drimenol derivatives were observed. In vitro assays showed that PhDOX1 only catalyses the conversion from drimenol to drimendiol, and not the further oxidation into an aldehyde. In yeast and heterologous plant hosts, the C-12 position of drimendiol is therefore likely to be further oxidized by endogenous enzymes into an aldehyde and subsequently converted to cinnamolide, presumably by spontaneous hemiacetal formation with the C-11 hydroxyl group followed by oxidation. Purified cinnamolide was confirmed by NMR and shown to be deterrent with an effective deterrent dose (ED50 ) of about 200-400 µg g-1 fresh weight against both whiteflies and aphids. The putative additional physiological and biochemical requirements for polygodial biosynthesis and stable storage in plant tissues are discussed.

Arctic plant origins and early formation of circumarctic distributions: a case study of the mountain sorrel, Oxyria digyna.

Many plant species comprising the present-day Arctic flora are thought to have originated in the high mountains of North America and Eurasia, migrated northwards as global temperatures fell during the late Tertiary period, and thereafter attained a circumarctic distribution. However, supporting evidence for this hypothesis that provides a temporal framework for the origin, spread and initial attainment of a circumarctic distribution by an arctic plant is currently lacking. Here we examined the origin and initial formation of a circumarctic distribution of the arctic mountain sorrel (Oxyria digyna) by conducting a phylogeographic analysis of plastid and nuclear gene DNA variation. We provide evidence for an origin of this species in the Qinghai-Tibet Plateau of southwestern China, followed by migration into Russia c. 11 million yr ago (Ma), eastwards into North America by c. 4 Ma, and westwards into Western Europe by c. 1.96 Ma. Thereafter, the species attained a circumarctic distribution by colonizing Greenland from both sides of the Atlantic Ocean. Following the arrival of the species in North America and Europe, population sizes appear to have increased and then stabilized there over the last 1 million yr. However, in Greenland a marked reduction followed by an expansion in population size is indicated to have occurred during the Pleistocene.

Macroevolutionary assembly of ant/plant symbioses: Pseudomyrmex ants and their ant-housing plants in the Neotropics.

Symbioses include some of the clearest cases of coevolution, but their origin, loss or reassembly with different partners can rarely be inferred. Here we use ant/plant symbioses involving three plant clades to investigate the evolution of symbioses. We generated phylogenies for the big-eyed arboreal ants (Pseudomyrmecinae), including 72% of their 286 species, as well as for five of their plant host groups, in each case sampling more than 61% of the species. We show that the ant-housing Vachellia (Mimosoideae) clade and its ants co-diversified for the past 5 Ma, with some species additionally colonized by younger plant-nesting ant species, some parasitic. An apparent co-radiation of ants and Tachigali (Caesalpinioideae) was followed by waves of colonization by the same ant clade, and subsequent occupation by a younger ant group. Wide crown and stem age differences between the ant-housing genus Triplaris (Polygonaceae) and its obligate ant inhabitants, and stochastic trait mapping, indicate that its domatium evolved earlier than the ants now occupying it, suggesting previous symbioses that dissolved. Parasitic ant species evolved from generalists, not from mutualists, and are younger than the mutualistic systems they parasitize. Our study illuminates the macroevolutionary assembly of ant/plant symbioses, which has been highly dynamic, even in very specialized systems.

Environmental and genetic correlates of allocation to sexual reproduction in the circumpolar plant Bistorta vivipara.

UNLABELLED: • PREMISE OF THE STUDY: Sexual reproduction often requires more energy and time than clonal reproduction. In marginal arctic conditions, species that can reproduce both sexually and clonally dominate. Plants with this capacity may thrive because they can alter reproduction depending on environmental conditions. Bistorta vivipara is a circumpolar herb that predominately reproduces clonally, but certain environmental conditions promote higher investment in flowers (and possible sexual reproduction). Despite largely reproducing clonally, the herb has high levels of genetic variation, and the processes underlying this paradoxical pattern of variation remain unclear. Here we identified environmental factors associated with sexual investment and examined whether sexual reproduction is associated with higher levels of genetic variation.• METHODS: We sampled 20 populations of B. vivipara across the high Arctic archipelago of Svalbard. In each population, we measured reproductive traits, environmental variables, and collected samples for genetic analyses. These samples permitted hypotheses to be tested regarding sexual investment and ecological and genetic correlates.• KEY RESULTS: Increased soil nitrogen and organic matter content and decreased elevation were positively associated with investment in flowers. Increased investment in flowers significantly correlated with more genotypes per population. Linkage disequilibrium was consistent with predominant clonality, but several populations showed higher genetic variation and lower differentiation than expected. There was no geographical genetic structure.• CONCLUSIONS: In B. vivipara, sexual investment is positively associated with habitat quality. Bistorta vivipara predominantly reproduces clonally, but occasional outcrossing, efficient clonal reproduction, and dispersal by bulbils can explain the considerable genetic variation and weak genetic structure in B. vivipara.

Refugial isolation and range expansions drive the genetic structure of Oxyria sinensis (Polygonaceae) in the Himalaya-Hengduan Mountains.

The formation of the Mekong-Salween Divide and climatic oscillations in Pleistocene were the main drivers for the contemporary diversity and genetic structure of plants in the Himalaya-Hengduan Mountains (HHM). To identify the relative roles of the two historical events in shaping population history of plants in HHM, we investigated the phylogeographic pattern of Oxyria sinensis, a perennial plant endemic to the HHM. Sixteen chloroplast haplotypes were identified and were clustered into three phylogenetic clades. The age of the major clades was estimated to be in the Pleistocene, falling into several Pleistocene glacial stages and postdating the formation of the Mekong-Salween Divide. Range expansions occurred at least twice in the early and middle Pleistocene, but the spatial genetic distribution rarely changed since the Last Glacial Maximum. Our results suggest that temporary mountain glaciers may act as barriers in promoting the lineage divergence in O. sinensis and that subsequential range expansions and secondary contacts might reshape the genetic distribution in geography and blur the boundary of population differentiation created in the earlier glacial stages. This study demonstrates that Pleistocene climatic change and mountain glaciers, rather than the Mekong-Salween Divide, play the primary role in shaping the spatial genetic structure of O. sinensis.

Phylogeography of the arid shrub Atraphaxis frutescens (Polygonaceae) in northwestern China: evidence from cpDNA sequences.

Climatic fluctuations during the Pleistocene are usually considered as a significant factor in shaping intraspecific genetic variation and influencing demographic histories. To well-understand these processes in desert northwest China, we selected arid adapted Atraphaxis frutescens as the study species. Two cpDNA regions (psbK-psbI, psbB-psbH) were sequenced in 272 individuals from 33 natural populations across the range of this shrub, and 10 haplotypes were identified. It was found to contain high levels of total gene diversity (H T = 0.858), and low levels of within-population diversity (H S = 0.092). Analysis of molecular variance (AMOVA) indicates that genetic differentiation primarily occurs among groups of populations. Based on BEAST (Bayesian Evolutionary Analysis Sampling Trees) analysis, we suggest that intraspecific differentiation of the species, resulting from isolated populations, accompanied enhanced desertification during the middle and late Pleistocene. The expansion of the Gurbantunggut and Kumtag deserts in this area appears to have triggered divergence among populations of the western, central, and eastern portions of the region and shaped genetic differentiation among them. Two possible independent glacial refugia were predicted, the Ili Valley and the northern Junggar Basin. Extensive development of arid habitats (desert margin and arid piedmont grassland) coupled with a more equable climate because the early Holocene are factors likely to have generated recent expansion of A. frutescens.

Comparison of the chemical composition of three species of smartweed (genus Persicaria) with a focus on drimane sesquiterpenoids.

The genus Persicaria is known to include species accumulating drimane sesquiterpenoids, but a comparative analysis highlighting the compositional differences has not been done. In this study, the secondary metabolites of both flowers and leaves of Persicariahydropiper, Persicariamaculosa and Persicariaminor, three species which occur in the same habitat, were compared. Using gas chromatography-mass spectrometry (GC-MS) analysis of extracts, overall 21/29 identified compounds in extracts were sesquiterpenoids and 5/29 were drimanes. Polygodial was detected in all species, though not in every sample of P. maculosa. On average, P. hydropiper flowers contained about 6.2 mg g FW(-1) of polygodial, but P. minor flowers had 200-fold, and P. maculosa 100,000 fold lower concentrations. Comparatively, also other sesquiterpenes were much lower in those species, suggesting the fitness benefit to depend on either investing a lot or not at all in terpenoid-based secondary defences. For P. hydropiper, effects of flower and leaf development and headspace volatiles were analysed as well. The flower stage immediately after fertilisation was the one with the highest content of drimane sesquiterpenoids and leaves contained about 10-fold less of these compounds compared to flowers. The headspace of P. hydropiper contained 8 compounds: one monoterpene, one alkyl aldehyde and six sesquiterpenes, but none were drimanes. The potential ecological significance of the presence or absence of drimane sesquiterpenoids and other metabolites for these plant species are discussed.

Transcriptome profiling and digital gene expression analysis of Fallopia multiflora to discover putative genes involved in the biosynthesis of 2,3,5,4'-tetrahydroxy stilbene-2-O-ß-D-glucoside.

The compound 2,3,5,4'-tetrahydroxy stilbene-2-O-ß-d-glucoside (THSG) synthesized by Fallopia multiflora (F. multiflora) exhibits pharmacological potency. However, the mechanistic details of its biosynthesis pathway are still vague. To clear this ambiguity, we performed de novo transcriptome assembly and digital gene expression (DGE) profiling analyses of F. multiflora using the Illumina RNA-seq system. RNA-seq generated approximately 70 million high-quality reads that were assembled into 65,653 unigenes (mean length=750 bp), including 26,670 clusters and 38,983 singletons. A total of 48,173 (73.4%) unigenes were annotated using public protein databases with a cut-off e-value above 10(-5). Furthermore, we investigated the transcriptome difference of four different F. multiflora tissues using DGE profiling. Variations in gene expression were identified based on comparisons of transcriptomes from various parts of a high-level THSG- and a low-level THSG-producing F. multiflora plant. Clusters with similar differential expression patterns and enriched metabolic pathways with regard to the differentially expressed genes putatively involved in THSG biosynthesis were revealed for the first time. Our data provides the most comprehensive sequence resource regarding F. multiflora so far. Taken together, the results of this study considerably extend the knowledge on THSG production.

Tertiary montane origin of the Central Asian flora, evidence inferred from cpDNA sequences of Atraphaxis (Polygonaceae).

Atraphaxis has approximately 25 species and a distribution center in Central Asia. It has been previously used to hypothesize an origin from montane forest. We sampled 18 species covering three sections within the genus and sequenced five cpDNA spacers, atpB-rbcL, psbK-psbI, psbA-trnH, rbcL, and trnL-trnF. BEAST was used to reconstruct phylogenetic relationship and time divergences, and S-DIVA and Lagrange were used, based on distribution area and ecotype data, for reconstruction of ancestral areas and events. Our results appear compatible with designation of three taxonomic sections within the genus. The generic stem and crown ages were Eocene, approximately 47 Ma, and Oligocene 27 Ma, respectively. The origin of Atraphaxis is confirmed as montane, with an ancestral area consisting of the Junggar Basin and uplands of the Pamir-Tianshan-Alatau-Altai mountain chains, and ancestral ecotype of montane forest. Two remarkable paleogeographic events, shrinkage of the inland Paratethys Sea at the boundary of the late Oligocene and early Miocene, and the time intervals of cooling and drying of global climate from 24 (22) Ma onward likely facilitated early diversification of Atraphaxis, while rapid uplift of the Tianshan Mountains during the late Miocene may have promoted later diversification.