Pollen morphology of some selected plant species from Horton Plains,Sri Lanka

The pollen morphology of four species: Gaultheria leschenaultii (Ericaceae), Psychotria zeylanica (Rubiaceae), Rubus leucocarpus (Rosaceae) and Symplocos bractealis (Symplocaceae) from Horton Plains, Sri Lanka was investigated using LM and SEM. Psychotria zeylanica is endemic to Sri Lanka and the other three taxa are common in Horton Plains. Gaultheria leschenaultii has tetrads with 3-colporate, coarsely granular to granulo-rugulate pollen grains; in the polar region and at the colpus margin the exine is smooth with micro-rugulate structure. Psychotria zeylanica has 3- (or 4-) colporate, reticulate-spinulose to granular pollen grains. Rubus leucocarpus has 3-colporate, striate-perforate pollen grains and Symplocos bractealis has 3- (or 4-) colporate pollen grains with a verrucate-perforate exine. Pollen grains of these four species are very indicative of human impact and palaeoclimate.     

Gaultheria steyermarkii (Ericaceae): A new endemic to Cerro Turimiquire,Venezuela

Gaultheria steyermarkii Luteyn, from the isolated Cerro Turimiquire, Venezuela, is described, illustrated, and its relationships toG. buxifolia andG. anastomosans are discussed.     

A chemotaxonomic survey of flavonoids and simple phenols in the leaves of Gaultheria L. and related genera (Ericaceae)

MIDDLETON, D. J., 1992. A chemotaxonomic survey of flavonoids and simple phenols in the leaves of Gaultheria L. and related genera (Ericaceae). Species of Gaultheria and Leucothoe have been screened for a number of flavonoids and simple phenols. Many of these, particularly the American species, are surveyed for the first time. Extensive sampling has also been carried out within some of the species of Gaultheria. Together with previously published data the results are discussed in relation to the position of Gaultheria in the Ericaceae and the infrageneric classification of Gaultheria. The infraspecific variation is commented on.     

Gaultheria gonggashanensis sp. nov. (Ericaceae) from Sichuan,China

Gaultheria gonggashanensis P. W. Fritsch & Lu Lu, a new species from western Sichuan Province, China, is described and illustrated. This species belongs to Gaultheria ser. Trichophyllae Airy Shaw and is most similar to G. sinensis J. Anthony, but differs by its longer stem setae, glabrous petioles and leaf blade midveins adaxially, thick‐chartaceous to subcoriaceous leaf blades, larger calyx lobes, and smaller anther thecae. The species is known with certainty only from the Hailuo Valley on the east slope of Mount Gongga in the Daxue Range.     

Estimating contribution of anthocyanin pigments to osmotic adjustment during winter leaf reddening

The association between plant water stress and synthesis of red, anthocyanin pigments in leaves has led some plant biologists to propose an osmotic function of leaf reddening. According to this hypothesis, anthocyanins function as a solute in osmotic adjustment (OA), contributing to depression of osmotic potential (Ψ_π) and maintenance of turgor pressure during drought-stressed conditions. Here we calculate the percent contribution of anthocyanin to leaf Ψ_π during OA in two angiosperm evergreen species, Galax urceolata and Gaultheria procumbens. Both species exhibit dramatic leaf reddening under high light during winter, concomitant with declines in leaf water potential and accumulation of solutes. Data previously published by the authors on osmotic potential at full turgor (Ψ_π,100) of G. urceolata and G. procumbens leaves before and after leaf reddening were used to estimate OA. In vivo molar concentrations of anthocyanin, glucose, fructose, and sucrose measured from the same individuals were converted to pressure equivalents using the Ideal Gas Law, and percent contribution to OA was estimated. Estimated mean OA during winter was −0.7 MPa for G. urceolata and −0.8 MPa for G. procumbens. In vivo concentrations of anthocyanin (3–10 mM) were estimated to account for ∼2% of OA during winter, and comprised <0.7% of Ψ_π,100 in both species. Glucose, fructose, and sucrose combined accounted for roughly 50 and 80% of OA for G. urceolata and G. procumbens, respectively, and comprised ∼20% of Ψ_π,100. We observed that a co-occurring, acyanic species (Vinca minor) achieved similar OA without synthesizing anthocyanin. We conclude that anthocyanins represent a measurable, albeit meager, component of OA in red-leafed evergreen species during winter. However, due to their low concentrations, metabolic costliness relative to other osmolytes, and striking red color (unnecessary for an osmotic function), it is unlikely that they are synthesized solely for an osmoprotectant role.     

Molecular detection,community structure and phylogeny of ericoid mycorrhizal fungi

Through traditional culturing and molecular characterization, we have determined that five putative species and 2 polyphyletic assemblages of fungi produce ericoid mycorrhizae in Gaultheria shallon, other Ericaceae and Epacridaceae. Using phylogenetic analysis of ITS2 sequences in GenBank, we have confirmed that most of these fungi occur in North America, Europe, and Australia. The low recovery rate of culturable ericoid mycorrhizal fungi from Gaultheria shallon may partly be explained by the fact that most mycorrhizal root segments contain an unculturable basidiomycete, revealed by direct amplification, cloning, and sequencing of LSU fungal DNA from root. Molecular characterization and phylogenetic analysis are powerful tools in revealing the geographic distribution and identity of ericoid mycorrhizal fungi.     

Fingerprinting of Cyanobacteria Based on PCR with Primers Derived from Short and Long Tandemly Repeated Repetitive Sequences

The presence of repeated DNA (short tandemly repeated repetitive [STRR] and long tandemly repeated repetitive [LTRR]) sequences in the genome of cyanobacteria was used to generate a fingerprint method for symbiotic and free-living isolates. Primers corresponding to the STRR and LTRR sequences were used in the PCR, resulting in a method which generate specific fingerprints for individual isolates. The method was useful both with purified DNA and with intact cyanobacterial filaments or cells as templates for the PCR. Twenty-three Nostoc isolates from a total of 35 were symbiotic isolates from the angiosperm Gunnera species, including isolates from the same Gunnera species as well as from different species. The results show a genetic similarity among isolates from different Gunnera species as well as a genetic heterogeneity among isolates from the same Gunnera species. Isolates which have been postulated to be closely related or identical revealed similar results by the PCR method, indicating that the technique is useful for clustering of even closely related strains. The method was applied to nonheterocystus cyanobacteria from which a fingerprint pattern was obtained.     

DNA barcoding of Gaultheria L.in China (Ericaceae: Vaccinioideae)

Four DNA barcoding loci,chloroplast loci rbcL,matK,trnH-psbA,and nuclear locus internal transcribed spacer (ITS),were tested for the accurate discrimination of the Chinese species of Gaultheria by using intraspecific and interspecific pairwise P-distance,Wilcoxon signed rank test,and tree-based analyses.This study included 186 individuals from 89 populations representing 30 species.For all individuals,single locus markers showed high levels of sequencing universality but were ineffective for species resolvability.Polymerase chain reaction amplification and sequencing were successful for all four loci.Both ITS and matK showed significantly higher levels of interspecific species delimitation than rbcL and trnH-psbA.A combination ofmatK and ITS was the most efficient DNA barcode among all studied regions,however,they do not represent an appropriate candidate barcode for Chinese Gaultheria,by which only 11 out of 30 species can be separated.Loci rbcL,matK,and trnH-psbA,which were recently proposed as universal plant barcodes,have a very poor capacity for species separation for Chinese Gaultheria.DNA barcodes may be reliable tools to identify the evolutionary units of this group,so further studies are needed to develop more efficient DNA barcodes for Gaultheria and other genera with complicated evolutionary histories.     

Gaultheria marronina sp. nov. (Ericaceae) from Sichuan,China

Gaultheria marronina, a new species from the Hengduan Mountain chain of western Sichuan Province, China, is described and illustrated. This species is similar to G. bryoides P. W. Fritsch & L. H. Zhou from the Gaoligong Mountains in its white fleshy calyx with a maroon capsule but differs in its stem setae 0.34–0.50 mm long, petioles 0.4–0.6 mm long, leaf blades 5.1–6.2 × 2.1–3.1 mm, overwintering flower bud pedicels 0.6–0.8 mm long, calyx lobes 2.2–3.0 mm long, and usually slightly open fruiting calyx, among other characters. The species is known only from two widely separated unprotected populations and is categorized as endangered.     

Ecotypic response to ultramafic soils by some plant species of northwestern United States

Soils high in magnesium derived from ultramafic rocks (serpentine, peridotite, and dunite) in northwestern United States support endemic as well as wide-ranging but edaphically indifferent(bodenvag) species. The latter occur widely on diverse rock formations of the region. Severalbodenvag species are shown to respond ecotypically to ultramafic soils. Of 18 species tested, all but three are differentiated into strains either tolerant or intolerant of ultramafic soils. Tests for edaphic preferences were conducted with seedlings and mature transplants on ultramafic soils. Growth performances were determined in greenhouse pot tests, outdoor soil bins, and by transplants in the wild. Herbaceous perennials (e.g.,Achillea millefolium, Fragaria virginiana, Prunella vulgaris, Rumex acetosella) gave the clearest ecotypic differences. Woody species either showed only slight ecotypic response(Spiraea douglasii var.menziesii andGaultheria shallon) or delayed the expression of their genotypic adaptability(Pinus contorta). Where ultramafic abut non-ultramafic soils, those populations ofbodenvag species that grow in non-ultramafic habitats can have a significant proportion of individuals tolerant to ferromagnesian soils (e.g.,Achillea millefolium). This suggests gene flow between populations of contrasting edaphic sites and possibly preadaptedness for the ultramafic habitat. Strains of two introduced weeds(Prunella vulgaris andRumex acetosella) have become ecotypically tolerant to ultramafic soils, probably within the last 75 years.     

LSU rDNA based RFLP assays for the routine identification of Gambierdiscus species

The Gambierdiscus genus is a group of benthic dinoflagellates commonly associated with ciguatera fish poisoning (CFP), which is generally found in tropical or sub-tropical regions around the world. Morphologically similar species within the genus can vary in toxicity; however, species identifications are difficult or sometimes impossible using light microscopy. DNA sequencing of ribosomal RNA genes (rDNA) is thus often used to identify and describe Gambierdiscus species and ribotypes, but the expense and time can be prohibitive for routine culture screening and/or large-scale monitoring programs. This study describes a restriction fragment length polymorphism (RFLP) typing method based on analysis of the large subunit rDNA that can successfully identify at least nine of the described Gambierdiscus species and two Fukuyoa species. The software programs DNAMAN 6.0 and Restriction Enzyme Picker were used to identify a set of restriction enzymes (SpeI, HpyCH4IV, and TaqαI) capable of distinguishing most of the known Gambierdiscus species for which DNA sequences were available. This assay was tested using in silico analysis and cultured isolates, and species identifications of isolates assigned by RFLP typing were confirmed by DNA sequencing. To verify the assay and assess intra-specific heterogeneity in RFLP patterns, identifications of 63 Gambierdiscus isolates comprising ten Gambierdiscus species, one ribotype, and two Fukuyoa species were confirmed using RFLP typing, and this method was subsequently employed in the routine identification of isolates collected from the Caribbean Sea. The RFLP assay presented here reduces the time and cost associated with morphological identification via scanning electron microscopy and/or DNA sequencing, and provides a phylogenetically sensitive method for routine Gambierdiscus species assignment.     

Genetic diversity estimation and core collection construction of Sinojackia huangmeiensis based on novel microsatellite markers

Sinojackia huangmeiensis is a critically endangered tree species in the Styracaceae endemic to China. To create tools to better evaluate the genetic diversity of this species, we used a modified genomic library enrichment method, the PETUER (Probe Extension and TEACl Universal EnRichment) method, to develop genetic markers. The resulting 18 microsatellite loci had high polymorphism with 3–12 alleles per locus and showed negligible stutter. Observed (H_O) heterozygosities were 0.057–0.724, and polymorphism information content (PIC) was 0.109–0.794. No significant linkage disequilibrium between pairs of loci was found. All 123 individuals found in the National Natural Reserve of Longgan Lake was genotyped at the 18 loci and used to estimate a UPGMA dendrogram. Using an iterative clustering method, a set of 18 individuals was established as a core collection to represent genetic diversity and would be useful to facilitate the conservation for S. huangmeiensis. In addition, we tested the 18 loci for cross-species amplification in three other Sinojackia species and the closely related Changiostyrax dolichocarpa. These polymorphic loci will be valuable for future population genetic and phylogenetic studies of S. huangmeiensis and congeneric species, as well as in closely related Styracaceae.     

杜鹃花科白珠树属分子系统学和生物地理学研究进展

白珠树属(Gaultheria) 在杜鹃花科(Ericaceae)系统演化中占有十分重要的地位, 其系统位置和演化关系一直备受争议。最近的分子系统学研究认为, 白珠树属已经不再属于传统上的越橘亚科(Vaccinioideae)的綟木族(Andromedeae), 而是与一些相关属组成了白珠树族(Gaultherieae)。对白珠树属产于美洲的类群和相关类群的分子系统学的初步研究则表明, 该属与Diplycosia、Tepuia和Pernettya等属(均为“常绿类群”)关系密切, 可能应将这几个属并入到白珠树属中, 但其属下分类系统关系还需要对产于亚洲的类群进行深入的研究后才能确定。白珠树属与其近缘属的进化历史和生物地理学关系较为复杂, 与杜鹃花科其他大多数属不同, 白珠树属为典型的环太平洋分布。关于白珠树属的起源问题存在两种不同的推测: 一种观点认为该属起源于南半球的冈瓦纳古陆; 另一种观点则认为其起源于北半球的劳亚古大陆。本文概述了近年来白珠树属的分子系统学和生物地理学研究进展, 并对该属尚存的一些问题进行了讨论。     

Variation in Spore Polypeptides from Four Species of Vairimorpha

Spore polypeptides from four species of Vairimorpha were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis for identification and classification. The spore profile for each Vairimorpha species is unique and reproducible. The unweighed pair-group method with arithmethic means is used to construct a phenogram to summarize the relationship of the Vairimorphe species.     

A novel method for screening species-specific gDNA probes for species identification

We report a method called SSH array which combines the suppression subtraction hybridization (SSH) and DNA array techniques to find species-specific DNA probes from genomic DNA (gDNA) for species identification. The method first obtains the differential gDNA fragments between two species by SSH and then hybridizes the differential gDNA fragments with arrays made of multiple whole genomes from several species to screen the unique gDNA fragments for one species. The screened unique gDNA fragments can be used as species-specific probes to differentiate the species they represent from all other species. We used five species of the genus Dendrobrium, D.aurantiacum Kerr, D.officinale Kimura et Migo, D.nobile Lindl., D.chrysotoxum Lindl. and D.fimbriatum Hook., as experimental materials to study the feasibility of the method. The results showed that the method could efficiently obtain different species-specific probes for each of the five species.     

Bar-HRM for Authentication of Plant-Based Medicines: Evaluation of Three Medicinal Products Derived from Acanthaceae Species

Medicinal plants are used as a popular alternative to synthetic drugs, both in developed and developing countries. The economic importance of the herbal and natural supplement industry is increasing every year. As the herbal industry grows, consumer safety is one issue that cannot be overlooked. Herbal products in Thai local markets are commonly sold without packaging or labels. Plant powders are stored in large bags or boxes, and therefore buying local herbal products poses a high risk of acquiring counterfeited, substituted and/or adulterated products. Due to these issues, a reliable method to authenticate products is needed. Here DNA barcoding was used in combination with High Resolution Melting analysis (Bar-HRM) to authenticate three medicinal Acanthaceae species (Acanthus ebracteatus, Andrographis paniculata and Rhinacanthus nasutus) commonly used in Thailand. The rbcL barcode was selected for use in primers design for HRM analysis to produce standard melting profiles of the selected species. Melting data from the HRM assay using the designed rbcL primers showed that the three chosen species could be distinguished from each other. HRM curves of all fifteen test samples indicated that three of tested products did not contain the indicated species. Two closely related species (A. paniculata and R. nasutus), which have a high level of morphological similarity, were interchanged with one another in three tested products. Incorrect information on packaging and labels of the tested herbal products was the cause of the results shown here. Morphological similarity among the species of interest also hindered the collection process. The Bar-HRM method developed here proved useful in aiding in the identification and authentication of herbal species in processed samples. In the future, species authentication through Bar-HRM could be used to promote consumer trust, as well as raising the quality of herbal products.     

Heritability of adventitious rooting of grapevine dormant canes

One of the most important viticultural characteristics of a grapevine rootstock is the ability to form roots on dormant lignified canes (rootstrike). North American species of Vitis are the primary source of germplasm for grapevine rootstocks and vary widely in their rate of rootstrike. Breeders have hybridized grape species in order to introgress traits to produce commercial rootstocks. A combination of 26 parents consisting of improved and wild accessions of Vitis spp. was used to generate 27 families. The percentage of rootstrike of dormant canes was observed over several years for 552 individuals. A logistic generalized linear mixed model (GLMM) method was used to estimate the narrow sense heritability (h ²) of rootstrike. Heritability was found to be moderate (h ²?=?0.307?±?0.050). The model also estimated breeding values of all parents and progeny. A GLMM method can be used to estimate breeding values of germplasm to identify individuals with commercially acceptable rates of rootstrike with a defined probability of transmitting this trait to progeny. This is useful for the introgression of traits into potentially new commercial rootstocks. The pattern of normal distribution of rooting indicates that it is possible to identify individuals with good rootstrike from Vitis species that are generally considered to have low rootstrike. Selection of individuals with a higher breeding value will increase the efficiency of rootstock breeding.     

Differential Sensitivity of Lactobacillus spp. to Inhibition by Candidate Topical Microbicides

Preclinical evaluation of vaginal microbicides includes screening against lactobacilli. However, there is no consensus regarding the species to be tested. This study was carried out to determine if results with one species would apply to other species, and to evaluate the utility of turbidometry as a screening tool. One current (PPCM; previously designated sulfuric acid-modified mandelic acid, SAMMA) and two former (cellulose sulfate, CS; and polystyrene sulfonate, PSS) candidate microbicides were evaluated. Bacterial growth was measured turbidometrically and by direct cell count. No microbicide affected Lact. gasseri, measured by either method. Apparent inhibition of Lact. jensenii by CS, PSS, and PPCM, and of Lact. crispatus by CS, occurred with turbidometric measurement. This was not substantiated with direct cell count. PSS and PPCM inhibited Lact. crispatus and Lact. acidophilus with both methods. These findings agree with results from vaginal isolates, which included Lact. gasseri, jensenii, acidophillus, crispatus, rhamnosis, casei, and paracasei. We conclude that sensitivities of similar lactobacilli to at least three microbicides are different. A single species is inadequate for screening vaginal products. Turbidometric evaluation is a sensitive, but not specific, screening method. We recommend that this method be used to screen candidate microbicides against several species of prevalent Lactobacillus species as an initial measure of microbicide safety evaluation.     

The Use of DNA Barcoding in Identification and Conservation of Rosewood (Dalbergia spp.)

The genus Dalbergia contains many valuable timber species threatened by illegal logging and deforestation, but knowledge on distributions and threats is often limited and accurate species identification difficult. The aim of this study was to apply DNA barcoding methods to support conservation efforts of Dalbergia species in Indochina. We used the recommended rbcL, matK and ITS barcoding markers on 95 samples covering 31 species of Dalbergia, and tested their discrimination ability with both traditional distance-based as well as different model-based machine learning methods. We specifically tested whether the markers could be used to solve taxonomic confusion concerning the timber species Dalbergia oliveri, and to identify the CITES-listed Dalbergia cochinchinensis. We also applied the barcoding markers to 14 samples of unknown identity. In general, we found that the barcoding markers discriminated among Dalbergia species with high accuracy. We found that ITS yielded the single highest discrimination rate (100%), but due to difficulties in obtaining high-quality sequences from degraded material, the better overall choice for Dalbergia seems to be the standard rbcL+matK barcode, as this yielded discrimination rates close to 90% and amplified well. The distance-based method TaxonDNA showed the highest identification rates overall, although a more complete specimen sampling is needed to conclude on the best analytic method. We found strong support for a monophyletic Dalbergia oliveri and encourage that this name is used consistently in Indochina. The CITES-listed Dalbergia cochinchinensis was successfully identified, and a species-specific assay can be developed from the data generated in this study for the identification of illegally traded timber. We suggest that the use of DNA barcoding is integrated into the work flow during floristic studies and at national herbaria in the region, as this could significantly increase the number of identified specimens and improve knowledge about species distributions.