Genome skimming and NMR chemical fingerprinting provide quality assurance biotechnology to validate Sarsaparilla identity and purity.

Sarsaparilla is a popular natural health product (NHP) that has been reported to be one of the most adulterated botanicals in the marketplace. Several plausible explanations are documented including economically motivated product substitution, unintentional errors due to ambiguous trade name associated with several different taxa, and wild harvesting of incorrect non-commercial plants. Unfortunately, this includes the case of an adulterant species Decalepis hamiltonii, a Red listed medicinal plant species by the International Union for Conservation of Nature (IUCN) and declared as a species with high conservation concern by the National Biodiversity Authority of India (NBA). This study provides validated genomic (genome skimming & DNA probes) and metabolomic (NMR chemical fingerprints) biotechnology solutions to prevent adulteration on both raw materials and finished products. This is also the first use of Oxford Nanopore on herbal products enabling the use of genome skimming as a tool for quality assurance within the supply chain of botanical ingredients. The validation of both genomics and metabolomics approach provided quality assurance perspective for both product identity and purity. This research enables manufactures and retailers to verify their supply chain is authentic and that consumers can enjoy safe, healthy products.

Separation of Seven Polyphenols from the Rhizome of Smilax glabra by Offline Two Dimension Recycling HSCCC with Extrusion Mode.

An offline two-dimensional recycling high-speed countercurrent chromatography (2D R-HSCCC) strategy with extrusion mode was developed for isolating polyphenols from the rhizome of Smilax glabra. Firstly, the ethyl acetate extract was divided into two fractions, Fr.1 and Fr.2, by silica gel column chromatography. Then, HSCCC was applied to separate polyphenols from the two fractions using a solvent system consisting of petroleum ether-ethyl acetate-methanol-water (1:3:0.5:5, v/v). Fifty milligrams of Fr.1 was separated by conventional HSCCC, yielding 5-O-caffeoylshikimic acid (1, 15.8 mg) and taxifolin (2, 4.8 mg). Offline 2D R-HSCCC with extrusion mode was used to separate Fr.2, and astilbin (4, 37.3 mg), neoisoastilbin (5, 8.8 mg), engeletin (7, 7.9 mg), and a mixture of two polyphenols were obtained from 100 mg of Fr.2. The mixture of two polyphenols was further separated by pre-HPLC, yielding neoastilbin (3, 15.2 mg) and isoastilbin (6, 9.9 mg). The purities of these seven compounds were all over 96.0%. Their structures were identified by MS and NMR. The results demonstrated that the strategy based on offline 2D R-HSCCC with extrusion mode was a powerful tool to separate the main compounds from the rhizome of Smilax glabra and valued for the preparative separation compounds with broad K-values and similar structures.

Understanding the formation of Mediterranean-African-Asian disjunctions: evidence for Miocene climate-driven vicariance and recent long-distance dispersal in the Tertiary relict Smilax aspera (Smilacaceae).

Tethyan plant disjunctions, including Mediterranean-African-Asian disjunctions, are thought to be vicariant, but their temporal origin and underlying causes remain largely unknown. To address this issue, we reconstructed the evolutionary history of Smilax aspera, a hypothesized component of the European Tertiary laurel forest flora. Thirty-eight populations and herbarium specimens representing 57 locations across the species range were sequenced at seven plastid regions and the nuclear ribosomal internal transcribed spacer region. Time-calibrated phylogenetic and phylogeographic inferences were used to trace ancestral areas and biogeographical events. The deep intraspecific split between Mediterranean and African-Asian lineages is attributable to range fragmentation of a southern Tethyan ancestor, as colder and more arid climates developed shortly after the mid-Miocene. In the Mediterranean, climate-induced vicariance has shaped regional population structure since the Late Miocene/Early Pliocene. At around the same time, East African and South Asian lineages split by vicariance, with one shared haplotype reflecting long-distance dispersal. Our results support the idea that geographic range formation and divergence of Tertiary relict species are more or less gradual (mostly vicariant) processes over long time spans, rather than point events in history. They also highlight the importance of the Mediterranean Basin as a centre of intraspecific divergence for Tertiary relict plants.

[Identification of Smilax glabra and its related species based on psbA-trnH sequence].

OBJECTIVE: To identify Smilax glabra and its related species using DNA barcoding technique and psbA-trnH sequence. METHODS: Total genomic DNA was isolated as template and the chloroplast gene psbA-trnH region was amplified by PCR technology and sequenced bidirectionally. The sequences and the related data were analyzed using the software CodonCode Aligner and MEGA 6.0; The intra- and inter-specific Kimura-2-Parameter(K2P) distances were calculated and the phylogenetic tree was constructed using the Neighbor-joining method. RESULTS: The maximum K2P genetic distance of the plants of Smilax glabra was lower than the minimum K2P genetic distance of its related species. In the cluster dendrogram, the plants of Smilax glabra from various sources showed the monophyletic and simultaneously distinguished from the closely relative species. CONCLUSION: psbA-trnH barcoding could be used to distinguish Smilax glabra and its related species effectively, and provide important molecular evidence for identification of original plant of Smilacis Glabrae Rhizoma and clinic safety in medicinal use.

Developmental origins of the conjoined twin mature embryo sacs in Smilax davidiana, with special notes on the formation of their embryos and endosperms.

PREMISE OF THE STUDY: Smilax davidiana, a perennial deciduous vine in Smilacaceae, is widely distributed in East and South China. A recent investigation revealed the existence of twin embryo sacs in this species. This study describes their mature structure, analyzes their developmental origins, and discusses the formation of their embryos and endosperms. METHODS: Conventional paraffin section, light microscopy, and various staining techniques were used to document the course of megasporogenesis and mature embryo sac structure in pistillate flowers. KEY RESULTS: Two chalazal megaspores of the tetrad became functional, giving rise to twin embryo sacs. Two mature embryo sacs of the same size (equal-conjoined twins); a dominant mature embryo sac and a subordinate one (unequal-conjoined twins); or very rarely, fully fused twin mature embryo sacs sharing a common chalazal domain are formed. CONCLUSION: A single embryo occurring in conjoined twins is the outcome of two eggs competing for sperm rather than a result of contention of two pre-embryos (or young embryos) for nutrition. Conjoined twin embryo sacs could provide a new pathway to the formation of pentaploid endosperms.

Karyotype characterization reveals active 45S rDNA sites located on chromosome termini in Smilax rufescens (Smilacaceae).

The genus Smilax (Smilacaceae) includes species of medicinal interest; consequently, their identification is important for the control of raw material used in the manufacture of phytotherapeutic products. We investigated the karyotype of Smilax rufescens in order to look for patterns that would be useful for comparative studies of this genus. To accomplish this, we developed procedures to grow plants and optimize root pretreatment with mitotic fuse inhibitors to obtain metaphase spreads showing clear chromosome morphology. The karyotype, analyzed in Feulgen-stained preparations, was asymmetric, with N = 16 chromosomes gradually decreasing in size; the larger ones were subtelocentric and the smaller chromosomes were submetacentric or metacentric. Nearly terminal secondary constrictions were visualized on the short arm of chromosome pairs 7, 11, and 14, but they were clearly detected only in one of the homologues of each pair. The nucleolus organizer regions (NORs) were mapped by silver staining and fluorescent in situ hybridization of 45S rDNA probes. Silver signals (Ag-NORs) colocalized with rDNA loci were detected at the termini of the short arm of 6 chromosomes. The secondary constriction heteromorphism observed in Feulgen-stained metaphases suggests that differential rRNA gene expression between homologous rDNA loci can occur, resulting in different degrees of chromatin decondensation. In addition, a heteromorphic chromosome pair was identified and was interpreted as being a sex chromosome pair in this dioecious species.

Phylogeography of North American herbaceous Smilax (Smilacaceae): Combined AFLP and cpDNA data support a northern refugium in the Driftless Area.

PREMISE OF THE STUDY: The genetic structure of North American herbaceous Smilax, a clade of closely related understory herbs that has a wide and disjunct distribution, was investigated to test the hypothesis of whether a northern refugium in the upper Midwest may have existed for these plants during the last glaciation. METHODS: We analyzed 33 populations sampled from California and throughout the eastern United States using AFLP and chloroplast DNA sequence data. KEY RESULTS: Individuals of S. jamesii from northern California formed a clade sister to the eastern North American species, and they likely diverged from each other during the Pleistocene. Among the eastern species, two lineages were found on opposite sides of the Appalachian Mountains. The populations near the "Driftless Area" contained most of the chlorotypes found in the Midwest, including a unique one endemic to this area, and the AFLP data indicated that one population from this area has the highest frequency-down-weighted-marker value. CONCLUSIONS: This study, and others that have focused on mammals, amphibians, and woody plants, provides further evidence for the debate over whether the upper Midwest's "Driftless Area" may have hosted a biologically diverse northern glacial refugium. Herbaceous species of Smilax from eastern North American exhibit an Appalachian discontinuity. They appear to have persisted in multiple refugia both east and west of the Appalachians, with several populations persisting in situ through the Last Glacial Maximum (LGM). We suggest that they experienced localized expansion after the LGM, possibly followed by subsequent contact between the Midwest and the East Coastal lineages.

Complete cpDNA genome sequence of Smilax china and phylogenetic placement of Liliales--influences of gene partitions and taxon sampling.

The complete nucleotide sequence of the chloroplast genome (cpDNA) of Smilax china L. (Smilacaceae) is reported. It is the first complete cp genome sequence in Liliales. Genomic analyses were conducted to examine the rate and pattern of cpDNA genome evolution in Smilax relative to other major lineages of monocots. The cpDNA genomic sequences were combined with those available for Lilium to evaluate the phylogenetic position of Liliales and to investigate the influence of taxon sampling, gene sampling, gene function, natural selection, and substitution rate on phylogenetic inference in monocots. Phylogenetic analyses using sequence data of gene groups partitioned according to gene function, selection force, and total substitution rate demonstrated evident impacts of these factors on phylogenetic inference of monocots and the placement of Liliales, suggesting potential evolutionary convergence or adaptation of some cpDNA genes in monocots. Our study also demonstrated that reduced taxon sampling reduced the bootstrap support for the placement of Liliales in the cpDNA phylogenomic analysis. Analyses of sequences of 77 protein genes with some missing data and sequences of 81 genes (all protein genes plus the rRNA genes) support a sister relationship of Liliales to the commelinids-Asparagales clade, consistent with the APG III system. Analyses of 63 cpDNA protein genes for 32 taxa with few missing data, however, support a sister relationship of Liliales (represented by Smilax and Lilium) to Dioscoreales-Pandanales. Topology tests indicated that these two alignments do not significantly differ given any of these three cpDNA genomic sequence data sets. Furthermore, we found no saturation effect of the data, suggesting that the cpDNA genomic sequence data used in the study are appropriate for monocot phylogenetic study and long-branch attraction is unlikely to be the cause to explain the result of two well-supported, conflict placements of Liliales. Further analyses using sufficient nuclear data remain necessary to evaluate these two phylogenetic hypotheses regarding the position of Liliales and to address the causes of signal conflict among genes and partitions.

Isolation of compound microsatellite markers for the common Mediterranean shrub Smilax aspera (Smilacaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for the common Mediterranean shrub Smilax aspera to study the population genetics of this species. METHODS AND RESULTS: Using the compound microsatellite marker technique, a total of 14 pairs of primers were developed for S. aspera, of which 11 were polymorphic, and three were monomorphic. Levels of polymorphism in the 11 markers were checked in 43 individuals collected from two populations in Greece and Italy. The number of alleles per locus ranged from 10 to 26, with an average of 15.55 alleles per locus. All of these primers also could be amplified from a second species, S. hispida. CONCLUSIONS: These microsatellite markers can be used for future studies of genetic diversity in S. aspera, as well as in other related taxa, and will help us to improve our understanding of the microevolutionary processes of this species in the Mediterranean region.

Relationships and hybridization among Smilax china and its affinities: evidence from allozyme data.

Smilax china L. is a widespread species in China with different ploidy levels. It is morphologically similar to S. davidiana, S. trinervula, and S. glauco-china. In this study, the chromosome number and the variation in allozyme patterns of eight enzyme systems with 25 alleles in 11 populations of S. china and three affinitive species were investigated. The allozyme data, together with morphological and cytological data, suggest that S. glauco-china is not closely related to the other taxa investigated. The diploid species S. davidiana and S. trinervula are involved as ancestor species and share great introgressions with S. china. In S. china, populations from Guilin and Guiyang are allotetraploid; their diploid progenitors probably are diploid populations of S. china and S. trinervula. The results suggest this species arose from multiple origins.