Light microscopy of Pakistani Berberis leaf cuticles and its taxonomic implications.

Taxonomy of the genus Berberis is quite complex, due to overlapping morphological characters, making it very difficult to differentiate the species within the genus. In order to resolve this taxonomic complexity, the foliar anatomy of 10 Berberis L. species was carried out, for the first time from Pakistan, using light microscopy (LM). Significant variation in terms of epidermal cells shape, size, cell wall pattern, and stomata type was observed. B. baluchistanica has the largest epidermal cells, Adaxial: length = 45-(53.9 ± 3.6)-62.5 µm; and width = 22.5-(26.3 ± 1.3)-30 µm; Abaxial: length = 37.5-(43.25 ± 2.5)-50 µm; and width = 20-(22.6 ± 0.8)-25. The highest number of stomata was observed in B. glaucocarpa as 62 on the abaxial surface while the lowest number of stomata was recorded in B. baluchistanica as 8 on the adaxial surface. Of 10 investigated species, 6 possess anomocytic type stomata, while 2 species that is, B. aitchisonii and B. parkeriana have both anomocytic and anisocytic stomata while B. baluchistanica and B. calliobotrys have only paracytic type stomata. The highest number of cells per unit area was present on the adaxial surface of B. calliobotrys ranging from 245-(252.4)-260 followed by B. parkeriana with 209-(227.8)-250 on the abaxial surface. Stomatal index (SI) also varied considerably and was the lowest (2.6) percentage in B. baluchistanica and highest (31.9) percentage in B. kunawurensis. A taxonomic key based on micro-morphological characters is provided for species identification.

Pollen morphological variation of Berberis L. from Pakistan and its systematic importance.

Due to overlapping and diverse morphological characters, Berberis is among the most taxonomically complex genera. Palynology is one of the taxonomic tools for delimitation and identification of complex species. In this study, pollens of 10 Berberis species were analyzed through light and scanning electron microscopy. Qualitative as well as quantitative features (pollen shape, size, presence or absence of colpi, colpi length and width, exine thickness, ornamentation, pollen class, aperture, and polar-equatorial ratio) were measured. Five species were observed to have colpate (pantocolpate) with elongated ends, radially symmetrical, isopolar, monads, and psilate-regulate pollens. In polar view, six pollen were spheroidal, two were ovoid, one spherical, and one oblate. Similarly, variation in pollen length was prominent and the largest pollen on polar view was recorded for B. psodoumbellata 60-65 µm (62.4 ± 0.9), while the smallest one was observed for B. lycium 29-35 µm (32.2 ± 1). The observed variation in both quantitative and qualitative features were important in taxonomic identification. This shows that palynological characters are helpful in identification of Berberis genus at the species level.

[Determination of six compounds in Berberidis Cortex and comparative study of its different species].

In order to clarify the characteristic components of Berberidis Cortex,the preparative liquid chromatography and spectral analysis methods were used to separate and identify the unknown components in the water extract of Berberidis Cortex. Two compounds were isolated and identified as bufotenidine and ferulic acid 4-O-ß-D-glucopyranoside. They were both isolated for the first time from Berberidis Cortex and Berberis. In addition,an HPLC method was successfully established for simultaneously determination of six compounds in Berberidis Cortex,and chemometric methods were used to study the chemical differences among three main species of Berberidis Cortex. The results suggested that jatrorrhizine and bufotenidine are the main difference compounds among the three species.Compared with B. kansuensis and B. diaphana,B. vernae contains significantly more jatrorrhizine(P<0. 01),and the content of bufotenidine in B. vernae was significantly higher than that in B. kansuensis(P<0. 05). Considering these results,further research is necessary to reveal the pharmacological activities of bufotenidine and the pharmacodynamic differences between the three species. The results could provide a reference for quality control,the basic research on effective substances,and development of Berberidis Cortex.

Classification of barberry genotypes by multivariate analysis of biochemical constituents and HPLC profiles.

INTRODUCTION: Recently, there has been a growing interest in the use of edible barberry and their extracts as a source of natural antioxidants in food and pharmaceutical industries. OBJECTIVE: The aim of this study was to evaluate the biochemical constituents of 18 samples of barberry fruits and classification of barberry genotypes by multivariate analysis. METHODS: Total phenolic, total flavonoid, total anthocyanin, total tannin, total carbohydrate contents and antioxidant activity were determined using Folin-Ciocalteu, aluminum chloride, colorimetric, vanillin, anthron and DPPH (2,2'-diphenyl-1-picrylhydrazyl) assays, respectively. High-performance liquid chromatography (HPLC) system is used for quantitative determination of phytochemical constituents. The multivariate data analysis (principal component analysis and hierarchical cluster analysis) and heat map data visualisation techniques were performed to classify barberry genotypes using Minitab and GraphPad Prism software, respectively. RESULTS: The highest amounts of total phenolics and flavonoids were obtained in fruit extracts of G3 (Berberis vulgaris). The highest total anthocyanin content and antioxidant activity were observed in G8 (B. vulgaris) and G16 (B. vulgaris), respectively. HPLC analysis of phytochemicals (gallic acid, caffeic acid, chlorogenic acid, p-coumaric acid, cinnamic acid, rutin, apigenin, and quercetin) revealed that gallic acid and p-coumaric acid were found as the most abundant phytochemical compounds. Based on multivariate analysis and heat map visualisation techniques, Berberis genotypes were classified into three main clusters. CONCLUSIONS: These results showed that barberry species (especially B. vulgaris and B. carataegina) are promising sources of natural antioxidants and biochemical compounds beneficial to be used in the food industry and that the multivariate analysis was a suitable approach to classify the barberry samples.

Colonization in North American Arid Lands: The Journey of Agarito (Berberis trifoliolata) Revealed by Multilocus Molecular Data and Packrat Midden Fossil Remains.

Here we conduct research to understand the evolutionary history of a shrubby species known as Agarito (Berberis trifoliolata), an endemic species to the Chihuahuan Desert. We identify genetic signatures based on plastid DNA and AFLP markers and perform niche modelling and spatial connectivity analyses as well as niche modelling based on records in packrats to elucidate whether orogenic events such as mountain range uplift in the Miocene or the contraction/expansion dynamics of vegetation in response to climate oscillations in the Pliocene/Pleistocene had an effect on evolutionary processes in Agarito. Our results of current niche modelling and palaeomodelling showed that the area currently occupied by Berberis trifoliolata is substantially larger than it was during the Last Interglacial period and the Last Glacial Maximum. Agarito was probably confined to small areas in the Northeastern and gradually expanded its distribution just after the Last Glacial Maximum when the weather in the Chihuahuan Desert and adjacent regions became progressively warmer and drier. The most contracted range was predicted for the Interglacial period. Populations remained in stable areas during the Last Glacial Maximum and expanded at the beginning of the Holocene. Most genetic variation occured in populations from the Sierra Madre Oriental. Two groups of haplotypes were identified: the Mexican Plateau populations and certain Northeastern populations. Haplogroups were spatially connected during the Last Glacial Maximum and separated during interglacial periods. The most important prediction of packrat middens palaeomodelling lies in the Mexican Plateau, a finding congruent with current and past niche modelling predictions for agarito and genetic results. Our results corroborate that these climate changes in the Pliocene/Pleistocene affected the evolutionary history of agarito. The journey of agarito in the Chihuahuan Desert has been dynamic, expanding and contracting its distribution range and currently occupying the largest area in its history.

Restriction site-associated DNA sequencing, genotyping error estimation and de novo assembly optimization for population genetic inference.

Restriction site-associated DNA sequencing (RADseq) provides researchers with the ability to record genetic polymorphism across thousands of loci for nonmodel organisms, potentially revolutionizing the field of molecular ecology. However, as with other genotyping methods, RADseq is prone to a number of sources of error that may have consequential effects for population genetic inferences, and these have received only limited attention in terms of the estimation and reporting of genotyping error rates. Here we use individual sample replicates, under the expectation of identical genotypes, to quantify genotyping error in the absence of a reference genome. We then use sample replicates to (i) optimize de novo assembly parameters within the program Stacks, by minimizing error and maximizing the retrieval of informative loci; and (ii) quantify error rates for loci, alleles and single-nucleotide polymorphisms. As an empirical example, we use a double-digest RAD data set of a nonmodel plant species, Berberis alpina, collected from high-altitude mountains in Mexico.

[A new herbs traceability method based on DNA barcoding-origin-morphology analysis--an example from an adulterant of 'Heiguogouqi'].

The fruit of Lycium ruthenicum is a common folk medicine in China. Now it is popular for its antioxidative effect and other medical functions. The adulterants of the herb confuse consumers. In order to identify a new adulterant of L. ruthenicum, a research was performed based on NCBI Nucleotide Database ITS Sequence, combined analysis of the origin and morphology of the adulterant to traceable varieties. Total genomic DNA was isolated from the materials, and nuclear DNA ITS sequences were amplified and sequenced; DNA fragments were collated and matched by using ContingExpress. Similarity identification of BLAST analysis was performed. Besides, the distribution of plant origin and morphology were considered to further identification and verification. Families and genera were identified by molecular identification method. The adulterant was identified as plant belonging to Berberis. Origin analysis narrowed the range of sample identification. Seven different kinds of plants in Berberis were potential sources of the sample. Adulterants variety was traced by morphological analysis. The united molecular identification-origin-morphology research proves to be a preceding way to medical herbs traceability with time-saving and economic advantages and the results showed the new adulterant of L. ruthenicum was B. kaschgarica. The main differences between B. kaschgarica and L. ruthenicum are as follows: in terms of the traits, the surface of B. kaschgarica is smooth and crispy, and that of L. ruthenicum is shrinkage, solid and hard. In microscopic characteristics, epicarp cells of B. aschgarica thickening like a string of beads, stone cells as the rectangle, and the stone cell walls of L. ruthenicum is wavy, obvious grain layer. In molecular sequences, the length of ITS sequence of B. kaschgarica is 606 bp, L. ruthenicum is 654 bp, the similarity of the two sequences is 53.32%.

[Species and distribution of the medicinal plants peculiar to Guizhou].

OBJECTIVE: To review the species and distribution of the medicinal plants peculiar to Guizhou and provide evidence for application, protection and collection. METHOD: Open-air investigation, data collection and specimen identification. RESULT: More than eighty kinds of the medical plants peculiar to Guizhou have been identified. CONCLUSION: Guizhou has a diversity of medicinal plants. The area of distribution of most species is restricted and the population is small. Some of the species have higher medicinal and scientific research values.

Taxonomic and phytogeographic implications from ITS phylogeny in Berberis (Berberidaceae).

A phylogeny based on the internal transcribed spacer (ITS) sequences from 79 taxa representing much of the diversity of Berberis L. (four major groups and 22 sections) was constructed for the first time. The phylogeny was basically congruent with the previous classification schemes at higher taxonomic levels, such as groups and subgroups. A notable exception is the non-monophyly of the group Occidentales of compound-leaved Berberis (previously separated as Mahonia). At lower levels, however, most of previous sections and subsections were not evident especially in simple-leaved Berberis. Possible relationship between section Horridae (group Occidentales) and the simple-leaved Berberis clade implies paraphyly of the compound-leaved Berberis. A well-known South America-Old World (mainly Asia) disjunctive distribution pattern of the simple-leaved Berberis is explained by a vicariance event occurring in the Cretaceous period. The ITS phylogeny also suggests that a possible connection between the Asian and South American groups through the North American species ( Berberis canadensis or B. fendleri) is highly unlikely.

[Distribution of four alkaloids in plants of Berberis].

In order to assess a simple and effective method for sensitive detection of alkaloids in Berberis plants, HPLC with gradiation elution is described. This method was developed for determination of four kinds of alkaloids including berbamine, jatrorrhizine, berberine and palmatine in methanolic extracts from bark and core of root and stem of Berberis. The linear correlations of berberine, jatrorrhizine, berbamine and palmatine were in the range of 0.028 - 4.74 microg (r = 0.9998), 0.012 - 2.0 microg (r = 0.9996), 0.026 - 0.52 microg (r = 0.9999), 0.015 - 2.56 microg (r = 0.9998), respectively. The content of alkaloids had obvious difference in Berberis plants from different habitats and different parts.

[Determination of four alkaloids in Berberis plants by HPLC].

OBJECTIVE: In order to assess Berberis, a simple and effective high-performance liquid chromatographic (HPLC) method was established. METHOD: By applying HPLC with gradiation elution, this method was developed for determination of four kinds of alkaloids including berbamine, Jatrorrhizine, berberine and palmatine in methanolic extracts from root bark, root, stem bark and stem of Berberis. RESULT: The alkaloids berbamine, jatrorrhizine, berberine and palmatine shown good linear correlations in the range of 0.028-4.74 microg (r = 0.9998); 0.012-2.0 microg (r = 0.9996); 0.026-0.52 microg (r = 0.9999); 0.015-2.56 microg (r = 0.9998), respectively. The correlation coefficients of the calibration curve for the analytes exceeded 0.9998. CONCLUSION: The optimized HPLC method was applied to analyze various samples, and the results shown that the content of alkaloids were obvious difference in Berberis from different area, different species and different parts.