First Record of Comparative Plastid Genome Analysis and Phylogenetic Relationships among Corylopsis Siebold & Zucc. (Hamamelidaceae).

Corylopsis Siebold & Zucc. (Hamamelidaceae) is widely used as a horticultural plant and comprises approximately 25 species in East Asia. Molecular research is essential to distinguish Corylopsis species, which are morphologically similar. Molecular research has been conducted using a small number of genes but not in Corylopsis. Plastid genomes of Corylopsis species (Corylopsis gotoana, Corylopsis pauciflora, and Corylopsis sinensis) were sequenced using next-generation sequencing techniques. Repeats and nucleotide diversity that could be used as DNA markers were also investigated. A phylogenetic investigation was carried out using 79 protein-coding genes to infer the evolutionary relationships within the genus Corylopsis. By including new plastomes, the overall plastid genome structure of Corylopsis was similar. Simple sequence repeats of 73-106 SSRs were identified in the protein-coding genes of the plastid genomes, and 33-40 long repeat sequences were identified in the plastomes. The Pi value of the rpl33_rps18 region, an intergenic spacer, was the highest. Phylogenetic analysis demonstrated that Corylopsis is a monophyletic group and Loropetalum is closely related to Corylopsis. C. pauciflora, C. gotoana, and C. spicata formed a clade distributed in Japan, whereas C. sinensis, C. glandulifera, and C. velutina formed a clade that was distributed in China.

Influence of geographic origin and tissue type on the medicinal chemical compounds of Semiliquidambar cathayensis.

Semiliquidambar cathayensis is widely used in traditional Chinese medicine owing its high concentrations of polyphenol, triterpenoidic acid, and flavonoids. This study aimed to explore the impact of geographical origin and tissue type on the contents of chemical compounds of S. cathayensis, as determined by colorimetric and chromatographic methods. Therefore, we quantitively evaluated chemical compounds found in the tissues of various organs of plants collected in six different regions. Overall, we found that geographical origin affected the content of medicinal compounds in S. cathayensis leaves, with plants from Jingzhou county showing the best therapeutic potential. However, no specific correlation was observed with latitude. It is noteworthy that the amount of paeoniflorin and other compounds can be used as biomarkers of geographical origin and tissue type. Most medicinal compounds accumulated mainly in the leaves, whereas ursolic and oleanolic acids accumulated in the roots. These results show that the comprehensive medicinal value of the leaves of S. cathayensis in Jingzhou county is the highest, but the root should be selected first to collect oleanolic acid and ursolic acid.

Description of a new species of Hamamelistes forming galls on Fothergilla spp. (Hamamelidaceae) and the generic limits of Hormaphidini (Sternorrhyncha: Aphididae: Hormaphidinae).

Hamamelistes and Hormaphis aphids of the tribe Hormaphidini are distributed disjunctly in eastern North America and Eurasia. Host-alternating species have life cycles encompassing generations in a gall on witch-hazel (Hamamelis spp.) and generations on leaves of birch (Betula spp.). In Hamamelistes, generations on witch-hazel induce globular pouch galls on flower or leaf buds. Herbarium specimens of a related Hamamelidaceae genus, Fothergilla, contain large galls in place of the seed head. We obtained a fresh sample of these elongate pouch galls collected from F. milleri in Alabama, USA. The galls were formed in place of fruiting structures and contained numerous aphids. Examination of morphology and the cytochrome oxidase subunit I barcode DNA sequence confirmed that the aphids are an undescribed species of Hamamelistes. Here, we describe the new species, Hamamelistes blackmani Dederich von Dohlen sp. n., from the morphology of foundresses, immatures, and winged forms in the gall. The life cycle is presumed monoecious. In addition, we review the evidence for including other genera in Hormaphidini and recommend that this tribe be restricted to Hamamelistes and Hormaphis.

Comparative transcriptome analysis of leaf, stem, and root tissues of Semiliquidambar cathayensis reveals candidate genes involved in terpenoid biosynthesis.

BACKGROUND: Semiliquidambar cathayensis is a traditional medicinal plant and endemic species in China. Its roots, branches, leaves, bark, and nectar are known to have therapeutic effects against rheumatoid arthritis, lumbar muscle strain, and several other diseases. However, limited knowledge regarding the molecular properties of S. cathayensis highlights the need for further research in order to elucidate the underlying pathways governing the synthesis of its active ingredients and regulation of its accumulation processes. METHODS: We conducted transcriptome sequencing of the leaf, stem and root epidermises, and stem and root xylems of S. cathayensis with three biological replicates. Moreover, candidate genes involved in terpenoid biosynthesis, such as IDI, FPPS, DXR, SQS, GPPS, and HMGR were selected for quantitative real-time PCR analysis. RESULTS: We identified 88,582 unigenes. Among which, 36,144 unigenes were annotated to the nr protein database, 21,981 to the Gene Ontology database, 11,565 to the Clusters of Orthologous Groups database, 24,209 to the Pfam database, 21,685 to the SWISS-PROT database, and 12,753 to the Kyoto Encyclopedia of Genes and Genomes (KEGG), with 5072 unigenes common to all six databases. Of those annotated using the KEGG database, 187 unigenes were related to the terpenoid metabolism pathway, and expression analysis of the related genes indicated that the mevalonate and methylerythritol 4-phosphate pathways play different roles in terpenoid biosynthesis in different tissues of S. cathayensis. CONCLUSIONS: These findings greatly expand gene resources of S. cathayensis and provide basic data for the study of the biosynthetic pathways and molecular mechanisms of terpenoids.

Different pruning level effects on flowering period and chlorophyll fluorescence parameters of Loropetalum chinense var. rubrum.

"Pruning" is a simple and efficient way to control the flowering period, but it is rarely used in perennial woody ornamental plants. In this paper, Loropetalum chinense var. rubrum was pruned in different degrees, and the relationship between pruning intensity and flowering number, and flowering time and chlorophyll fluorescence parameters were compared. After statistics, it was found that pruning could advance blossoms of L. chinense var. rubrum; also, light and heavy cutting could both obtain a larger number of flowers. In addition, through correlation analysis, it was found that during the flowering period, the Rfd parameter of the unpruned treatment had a very significant positive correlation with the number of flowers FN, which was 0.81. In other pruning treatment groups, Rfd and FN also presented a certain positive correlation, indicating that the Rfd parameter can be used to predict the number of flowers during the flowering process of L. chinense var. rubrum. The research results provided a new idea for the regulation of the flowering period of L. chinense var. rubrum and other woody ornamental plants and laid the foundation for the diversified application of L. chinense var. rubrum.

In vitro anticancer activity of extracted oil from Parrotiopsis jacquemontiana (Decne) Rehder.

BACKGROUND: Parrotiopsis jacquemontiana, commonly referred to as "Beranj" in the local community, is widely used traditionally and has numerous health benefits. However, no studies have been conducted to investigate its anticancer potential, particularly its extracted oil. PURPOSE: The present study was put forth to appraise the anticancer potential of Parrotiopsis jacquemontiana extracted oil against liver (hcclm3 and hepg2) and breast cancer (mda-mb 231 and mcf-7) cell lines relative to normal cell lines (lo2 and mcf-10a) via MTT assay. METHODS: Flow cytometry indicated the apoptotic effect whereas invasion and migration capabilities of oil against cancer cells were determined by Matrigel invasion chamber and wound-scratch assays. RESULTS: The results of oil revealed a time and dose-dependent increase in cell proliferation inhibition, conferring to least IC50 shown against hcclm3 (144.9 ± 0.75 µg/ml) and mda-mb 231 (145.7 ± 0.32 µg/ml) cell line at 72 h, whereas no cytotoxic effect on normal cells was observed. In addition, the oil significantly (p < 0.001) suppressed the migration and invasion of hcclm3 and mda-mb 231 cells, showing noteworthy anti-metastatic potential. Furthermore, cell death was confirmed by Annexin‒V/PI staining where the maximum apoptotic percentage was calculated for oil (200 µg/ml) alongside mda-mb 231 conferring to 15.36 ± 1.22, 26.7 ± 1.2, and 36.43 ± 1.65 at 24, 48, and 72 h whereas 12.33 ± 1.05, 19.36 ± 1.62, and 29.3 ± 0.79 was recorded alongside hcclm3 at similar time intervals, respectively. CONCLUSION: In conclusion, the extracted oil exhibited strong anti-proliferative, anti-metastatic, and apoptotic effects and therefore may have potential applications in cancer treatment, however, further studies of oil regarding the action mechanisms and compounds involved in anticancer therapy are necessary.

Neuroprotective Activities of Constituents from Phyllosticta capitalensis, an Endophyte Fungus of Loropetalum chinense var. rubrum.

One new dioxolanone derivative, guignardianone G (1) and twelve known compounds (2-13) were isolated from the 95 % ethanol extract of the plant endophytic fungus Phyllosticta capitalensis cultured in rice medium. Among these known compounds, isoaltenuene (3), brassicasterol (7), 5,6-epoxyergosterol (8), citreoanthrasteroid A (9), demethylincisterol A (10), and chaxine C (11) were reported from Phyllosticta sp. for the first time. The structure of 1 was elucidated by 1D- and 2D-NMR experiments and HR-ESI-MS data analysis, and its absolute configuration was established through the comprehensive use of the methods of modified Mosher methods, calculations of ECD spectra and optical rotation values. The neuroprotective activity of compounds (1-9, 11-13) were evaluated on PC12 cells damage induced by glutamate, and compounds 9 and 12 showed potential neuroprotective activities with half effective concentration (EC50 ) of 24.2 and 33.9 µM, respectively.

Chloroplast phylogenomic insights into the evolution of Distylium (Hamamelidaceae).

BACKGROUND: Most Distylium species are endangered. Distylium species mostly display homoplasy in their flowers and fruits, and are classified primarily based on leaf morphology. However, leaf size, shape, and serration vary tremendously making it difficult to use those characters to identify most species and a significant challenge to address the taxonomy of Distylium. To infer robust relationships and develop variable markers to identify Distylium species, we sequenced most of the Distylium species chloroplast genomes. RESULTS: The Distylium chloroplast genome size was 159,041-159,127 bp and encoded 80 protein-coding, 30 transfer RNAs, and 4 ribosomal RNA genes. There was a conserved gene order and a typical quadripartite structure. Phylogenomic analysis based on whole chloroplast genome sequences yielded a highly resolved phylogenetic tree and formed a monophyletic group containing four Distylium clades. A dating analysis suggested that Distylium originated in the Oligocene (34.39 Ma) and diversified within approximately 1 Ma. The evidence shows that Distylium is a rapidly radiating group. Four highly variable markers, matK-trnK, ndhC-trnV, ycf1, and trnT-trnL, and 74 polymorphic simple sequence repeats were discovered in the Distylium plastomes. CONCLUSIONS: The plastome sequences had sufficient polymorphic information to resolve phylogenetic relationships and identify Distylium species accurately.

Growth and respiratory metabolic adaptation strategies of riparian plant Distylium chinense to submergence by the field study and controlled experiments.

Submergence tolerance is crucial when thinking in promising species for restoration of ecosystems prone to suffer extreme flooding events. In this study, two-year-old seedlings of Distylium chinense were subjected to one field study and five controlled experiments: unsubmerged and watered daily as controls (CK) and completely submerged for 30, 60, 90 and 120 days, respectively followed by a 60-day recovery period to test the submergence tolerance. The results showed that the survival decreased with the increasing flooding duration. Different submergence duration treatments affected dry mass accumulation and carbohydrate content of roots, stems and leaves. Flooding stress affected the activities of pyruvate decarboxylase (PDC), ethanol dehydrogenase (ADH) and lactic dehydrogenase (LDH) enzymes, which indicated the roots and leaves adapt to long-term flooding by reinforcing their anaerobic respiration and activities of ADH were higher than those of LDH for roots and leaves with stronger alcoholic fermentation mainly. After de-submergence, the recovery patterns of carbohydrate were coincided with those of dry mass accumulation of the roots, stems and leaves. A significant regression equation analysis showed root starch content and dry mass accumulation were the major factors affecting the seedling survival. And D. chinense accumulated substantial amounts of carbohydrate before submergence and invested more in roots and stems than in leaves, which enhances long-term survival under submergence. Carbohydrate storage is a key functional trait that can explain high survival under submergence. D. chinense may have adopted a suite of growth and respiratory metabolic adaptation strategies to survive long-term submergence.

Diversity and bioactivities of fungal endophytes from Distylium chinense, a rare waterlogging tolerant plant endemic to the Three Gorges Reservoir.

BACKGROUND: The present study involves diversity and biological activities of the endophytic fungal community from Distylium chinense, a rare waterlogging tolerant plant endemic to the Three Gorges Reservoir. This study has been conducted hypothesizing that the microbial communities in the TGR area would contribute to the host plant tolerating a range of abiotic stress such as summer flooding, infertility, drought, salinity and soil erosion etc., and they may produce new metabolites, which may possess plentiful bioactive property, especially antioxidant activity. Therefore in the current study, the antioxidant, antimicrobial and anticancer activities of 154 endophytes recovered from D. chinense have been investigated. Furthermore, the active metabolites of the most broad-spectrum bioactive strain have also been studied. RESULTS: A total of 154 fungal endophytes were isolated from roots and stems. They were categorized into 30 morphotypes based on cultural characteristics and were affiliated with 27 different taxa. Among these, the most abundant fungal orders included Diaporthales (34.4%) and Botryosphaeriales (30.5%), which were predominantly represented by the species Phomopsis sp. (24.7%) and Neofusicoccum parvum (23.4%). Fermentation extracts were evaluated, screening for antioxidant, antimicrobial and anticancer activities. Among the 154 isolates tested, 99 (64.3%) displayed significant antioxidant activity, 153 (99.4%) exhibited inclusive antimicrobial activity against at least one tested microorganism and 27 (17.5%) showed exclusive anticancer activity against one or more cancer cell lines. Specifically, the crude extract of Irpex lacteus DR10-1 exhibited note-worthy bioactivities. Further chemical investigation on DR10-1 strain resulted in the isolation and identification of two known bioactive metabolites, indole-3-carboxylic acid (1) and indole-3-carboxaldehyde (2), indicating their potential roles in plant growth promotion and human medicinal value. CONCLUSION: These results indicated that diverse endophytic fungal population inhabits D. chinense. One of the fungal isolate DR10-1 (Irpex lacteus) exhibited significant antioxidant, antimicrobial and anticancer potential. Further, its active secondary metabolites 1 and 2 also showed antioxidant, antimicrobial and anticancer potential.

Rapid Analysis of the Chemical Compositions in Semiliquidambar cathayensis Roots by Ultra High-Performance Liquid Chromatography and Quadrupole Time-of-Flight Tandem Mass Spectrometry.

Semiliquidambar cathayensis Chang was a traditional medicinal plant and used to treat rheumatism arthritis and rheumatic arthritis for centuries in China with no scientific validation, while only 15 components were reported. Thus, a rapid, efficient, and precise method based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS) was applied in both positive- and negative-ion modes to rapidly analysis the main chemical compositions in S. cathayensis for the first time. Finally, a total of 85 chemical compositions, including 35 alkaloids, 12 flavonoids, 7 terpenoids, 5 phenylpropanoids, 9 fatty acids, 7 cyclic peptides, and 10 others were identified or tentatively characterized in the roots of S. cathayensis based on the accurate mass within 5 ppm error. Moreover, alkaloid, flavonoid, phenylpropanoid, and cyclic peptide were reported from S. cathayensis for the first time. This rapid and sensitive method was highly useful to comprehend the chemical compositions and will provide scientific basis for further study on the material basis, mechanism and clinical application of S. cathayensis roots.

[Spatial distribution patterns and association of Loropetalum chinense population in karst hills of Guilin, Southwest China]. / æ¡‚æž—å²©æº¶çŸ³å±±æªµæœ¨ç§ç¾¤ç©ºé—´æ ¼å±€åŠå ¶å ³è”æ€§.

The spatial distribution and association of populations can reflect succession patterns and its adaptation strategies to the change of environmental factors, with important significance for vegetation restoration, reconstruction, and biodiversity conservation. We used the point-pattern analytical method to examine the spatial distribution and its association of the Loropetalum chinense population in karst hills of Guilin, China, based on field surveys. On the basis of Ripley K function, we used the pair-correlation function statistic derived to explore difference in the distribution patterns.We used Ripley L function to examine the spatial associations among the three diameter classes. The results showed that the diameter class structure of the population exhibited an irregular inverted "J" type, with the small diameter class occupying a large proportion, indicating an increased population structure with good regeneration capability. The individuals of the three diameter classes had a clumped distribution at the small scale. With the increases of spatial scales, the aggregation intensity gradually weakened and tended to be randomly distributed. There was an independent spatial association between individuals among the three diameter classes at small scale. As the scale increased, the spatial association between individuals with different diameter classes became positive or negative association. The greater the difference in diameter class of L. chinense population, the weaker their spatial correlation was, which might gradually turn into negative association. Our findings contributed to a clearer understanding of the ecological strategies and scale-dependent cha-racteristics of species coexistence and underlying mechanisms during the growth and development of L. chinense population in karst hills of Guilin, providing reference for the restoration, reconstruction, protection and management of forest in karst hills.

Mining and characterization of novel EST-SSR markers of Parrotia subaequalis (Hamamelidaceae) from the first Illumina-based transcriptome datasets.

Parrotia subaequalis is an endangered Tertiary relict tree from eastern China. Despite its important ecological and horticultural value, no transcriptomic data and limited molecular markers are currently available in this species. In this study, we first performed high-throughput transcriptome sequencing of two individuals representing the northernmost (TX) and southernmost (SJD) population of P. subaequalis on the Illumina HiSeq 2500 platform. We gathered a total of 69,135 unigenes for P. subaequalis (TX) and 84,009 unigenes for P. subaequalis (SJD). From two unigenes datasets, 497 candidate polymorphic novel expressed sequence tag-simple sequence repeats (EST-SSRs) were identified using CandiSSR. Among these repeats, di-nucleotide repeats were the most abundant repeat type (62.78%) followed by tri-, tetra- and hexa-nucleotide repeats. We then randomly selected 54 primer pairs for polymorphism validation, of which 27 (50%) were successfully amplified and showed polymorphisms in 96 individuals from six natural populations of P. subaequalis. The average number of alleles per locus and the polymorphism information content values were 3.70 and 0.343; the average observed and expected heterozygosity were 0.378 and 0.394. A relatively high level of genetic diversity (HT = 0.393) and genetic differentiation level (FST = 0.171) were surveyed, indicating P. subaequalis maintained high levels of species diversity in the long-term evolutionary history. Additionally, a high level of cross-transferability (92.59%) was displayed in five congeneric Hamamelidaceae species. Therefore, these new transcriptomic data and novel polymorphic EST-SSR markers will pinpoint genetic resources and facilitate future studies on population genetics and molecular breeding of P. subaequalis and other Hamamelidaceae species.

Identification and development of microsatellite (SSRs) makers of Exbucklandia (HAMAMELIDACEAE) by high-throughput sequencing.

Hamamelidaceae (Saxifragales, previously Rosales) comprises approximately six subfamily, 30 genera and 140 species, most of which are Tertiary relicts. Exbucklandia is the only genus of the subfamily Exbucklandioideae, Hamelidaceae, containing only 2-4 species. Of them, the species E. longipetala H. T. Chang is endemic to China and listed as endangered in The Biodiversity Red List of China: Higher Plant, yet some taxonomists put forward that E. longipetala should be merged into E. tonkinensis (Lecomte) H. T. Chang. Currently, there was nearly no phylogeographic studies on this genus possibly due to the deficiency of efficient molecular markers. In this study, we sequenced the genome of E. tonkinensis based on high throughput sequencing technology, and obtained approximately 6 G raw data, which was further de novo assembled into 303,481 contigs. Based on them, 15,326 SSRs were identified from 13,596 contigs, and primers were successfully designed for 10,660 SSRs. A total of 139 paired primers were synthesized, 106 of them were successfully amplified in six Exbucklandia individuals with expected PCR product size, and 24 demonstrated to be polymorphic among three Exbucklandia populations. Accordingly, the expected and observed heterozygosity were between 0.097-0.717 and 0.098-0.583. Based on these efforts, future researches on genetic diversity and population structure of Exbucklandia can be performed to understand its phylogenetic origins and phylogeographic pattern.

Wound healing potential of oil extracted from Parrotiopsis jacquemontiana (Decne) Rehder.

ETHNOPHARMACOLOGICAL RELEVANCE: Oil extracted from Parrotiopsis jacquemontiana stem traditionally used for wound healing, body aches and dermatitis. In this study we have evaluated oil for its phytoconstituents, antioxidant, antimicrobial and wound healing activities. METHODS: Phytochemical characterization of oil was determined by standard qualitative procedures, gas chromatography mass spectrometry technique (GC-MS) and Fourier transform infra-red spectroscopy (FT-IR). The in vitro antioxidant aptitude was determined by scavenging of DPPH radical, hydroxyl ion, nitric oxide, inhibition of ß-carotene bleaching assay and iron chelation power assay. The antimicrobial potential of oil was investigated by disc diffusion method against multidrug resistant (MDR) bacterial isolates and fungal strains. Wound healing was performed in vivo with determination of wound contraction rates, histopathology, hemostatic potential and hydroxyproline estimation. RESULTS: GC-MS analysis indicated that oil was constituted mainly of 2, 6-dimethyl-8-oxoocta-2, 6-dienoic acid, methyl ester (18.2%), syringol (17.8%), catechol (12.4%), guaiacol (5.2%), p-cresol (5.4%) and phenol, 2-propyl- (3.7%). FT-IR analysis revealed several important functional groups in its chemical composition especially phenolic O-H compound stretching. Scavenging of DPPH radical, hydroxyl ion, nitric oxide, inhibition of ß-carotene oxidation and iron chelation power assays indicated strong antioxidant activities of oil. Further it efficiently inhibited growth of multidrug resistant isolates of Staphylococcus aureus, S. lugdenesis, Klebsiella pneumoniae, Escherichia coli, Coagulase -ve staphylococci and Pseudomonas aeruginosa. The minimum inhibitory concentrations ranged between (32-256) (µg/mL) of oil. The oil also strongly inhibited the growth of various fungal isolates with low level of minimum inhibitory concentrations (64-256) µg/mL. Remarkable rate for wound closure and epithelization, hemostatic potential and marked increase (p < 0.05) in hydroxyproline content was observed for oil during wound healing in rat. CONCLUSION: The results suggested that oil can be used as a potential source of wound healing therapeutics.

[Dynamics of nutrient concentration and microbial community composition during fine root decomposition in subtropical Mytilaria laosensis and Cunninghamia lanceolata plantations.] / äºšçƒ­å¸¦ç±³è€æŽ’å’Œæ‰æœ¨ç»†æ ¹åˆ†è§£è¿‡ç¨‹ä¸­å »åˆ†ä¸Žå¾®ç”Ÿç‰©ç¾¤è½ç»„æˆçš„å˜åŒ–.

We conducted a 12-month fine root decomposition experiment under 19-year-old Mytilaria laosensis and Cunninghamia lanceolate plantations to explore the dynamics of nutrient concentration and microbial community composition. The aim of this study was to provide insights into nutrient cycling under plantations with different tree species. Our results showed that the initial concentrations of phosphorus (P) and potassium (K) were significantly higher in the fine root of M. laosensis than those in C. lanceolata, which significantly decreased with decomposition. Nitrogen (N) concentration in fine roots of both species increased with decay time. The variation of N concentration in fine root of C. lanceolata lagged behind that in M. laosensis. During the decomposition, magnesium (Mg) concentration in fine root of C. lanceolata showed no significant changes, but that of M. laosensis decreased at the initial decay stage and increased thereafter and was significantly lower than that of C. lanceolata at the 8th month. The ratio of fungi to bacteria (F/B) of both species decreased at the initial stage and then increased, with significantly higher F/B in fine root of M. laosensis than that of C. lanceolate after one-year decay. Redundancy analysis (RDA) showed that changes in N and K concentrations and C/N ratio explained 37.2%, 14.5% and 14.8% of the variations in microbial community composition of C. lanceolata fine root respectively. However, during the decay of M. laosensis fine root, concentrations of Mg and K were key factors, accounting for 35.9% and 17.6% of the variations in microbial community composition, respectively. We concluded that other nutrients beyond N, such as Mg, might also be an important factor affecting root decomposition in different tree species.

Genetic diversity and ex situ conservation of Loropetalum subcordatum, an endangered species endemic to China.

BACKGROUND: Loropetalum subcordatum is an endangered species endemic to China that is characterized by narrow distribution, small population size, and delayed fertilization. However, the genetic diversity of the entire extant natural and ex situ populations has not been assessed to date. In this study, we evaluated the genetic diversity and structure of six natural populations and a single ex situ population (the only known ex situ population of L. subcordatum) using sequence-related amplified polymorphism data. RESULTS: In total, 553 reliable DNA bands, of which 359 (63.28%) were polymorphic, were amplified by polymerase chain reaction with combinations of 15 primers. Low average gene diversity within populations and high genetic differentiation were detected in L. subcordatum. A Mantel test demonstrated that there was a positive correlation between genetic and geographic distances, indicating that significant genetic divergence was likely the result of geographic isolation among natural populations. Furthermore, based on genetic structure patterns, populations of L. subcordatum were divided into three clusters. Group 1 was composed of specimens from Libo, Guizhou Province (GZ) and Huanjiang, Guangxi Zhuang Autonomous Region (GX). Group 2 was composed of Mt. Wuguishan, Guangdong Province (GD). Group 3 was composed of three populations in Hong Kong Special Administrative Region. Additionally, clonal reproduction probably existed in GD population. According to the genetic information analysis and field survey, the ex situ population did not match its source population (GD) in terms of genetics, and its habitat was different from the original natural habitat. We observed that a few individual GD seeds were needed to improve ZS ex situ in the future. CONCLUSIONS: Compared to previous SRAP-based studies of endangered plants, L. subcordatum had extremely low average gene diversity within populations and high genetic differentiation among populations. At present, the unique ex situ population has not been successful due to non-representative samples being taken, a smaller population size, and man-made changes in habitat. Potential strategies are suggested to improve the conservation of this species.

Phytochemical investigation and antimicrobial appraisal of Parrotiopsis jacquemontiana (Decne) Rehder.

BACKGROUND: Parrotiopsis jacquemontiana (Decne) Rehder. is locally used for skin infections and in wound healing. In this study we have evaluated methanol extract of its leaves and derived fractions against the clinical multi-drug resistant bacterial strains. METHODS: P. jacquemontiana leaves powder extracted with 95% methanol (PJM) and fractionated in escalating polarity of solvents; n-hexane (PJH), chloroform (PJC), ethyl acetate (PJE), n-butanol (PJB) and the remaining as aqueous fraction (PJA). Clinical as well as environmental 19 bacterial strains and 8 fungal strains were screened for minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC). Preliminary phytochemical investigation for various phytochemical classes was also carried out. RESULTS: PJM contained the coumarins, phenols, flavonoids, tannins, alkaloids, glycosides, saponins, sterols, phlobatannins, steroids, phytosterols, triterpenoids, acids, quinones, proteins, vitamin C, betacyanins, oils and resins while anthraquinones, phytosteroids, carbohydrates and anthocyanins were not detected. Disc diffusion assay (1 mg/disc) indicated the sensitivity of all the MDR strains of bacteria with PJM, PJE and PJB, while no inhibition was recorded with PJA. PJH and PJC inhibited the growth of all the strains of Staphylococcus aureus, Pseudomonas aeruginosa and Coagulase negative staphylococci used in this study. Maximum zone of inhibition (35.5 ± 1.32 mm) was obtained with PJM against Staphylococcus lugdenesis MDR (6197). Comparatively lower MIC (8-64 µg/ml) and MBC (32-256 µg/ml) values were recorded for PJM and PJE. In case of fungal strains only PJM, PJE and PJB markedly inhibited the growth and lower MIC (8-128 µg/ml) and MFC (32-512 µg/ml) values were determined for PJM and PJE. CONCLUSION: The remarkable inhibition of various bacterial and fungal strains at low doses of the extract/fractions suggested the strong antibacterial, antifungal and anti-candidal potential of P. jacquemontiana leaves.

Pollination by fungus gnats and associated floral characteristics in five families of the Japanese flora.

Background and aims: Pollination by fungus gnats (Mycetophilidae and Sciaridae) is uncommon, but is nevertheless known to occur in 20 genera among eight angiosperm families. Because many fungus gnat-pollinated plants possess a dark red floral display, we hypothesized that fungus gnat pollination is more widespread among plants with similar floral display than currently known. We thus studied the pollination biology of flowers with dark red pigmentation in five families, focusing particularly on plants having small, flat, actinomorphic flowers with exposed nectaries and short stamens, because these floral characteristics mirror those of a known fungus gnat-pollinated genus (Mitella). Methods: We observed daytime and night-time floral visitors for a total of 194.5 h in Aucuba japonica (Garryaceae), Euonymus spp. (Celastraceae), Disanthus cercidifolius (Hamamelidaceae), Micranthes fusca (Saxifragaceae) and Streptopus streptopoides (Liliaceae). Visitors were categorized into functional groups, and a pollination importance index (PII) was calculated for each functional group based on visitation frequency, pollen load and behaviour on flowers. Key results: Fungus gnats were dominant among the 1762 insects observed (36-92 % depending on the plant species) and were the most important pollinators among all plants studied (PII: 0.529-1). Fungus gnat visits occurred during the daytime and, more frequently, at dusk. Most often, pollen grains became clumped on the ventral side of the head and/or thorax as the short-proboscid fungus gnats foraged on nectar and came into contact with anthers located close to the flower base. Conclusions: Pollination by fungus gnats is probably more common than previously thought, especially in habitats similar to those of the plants studied (moist forest understorey, streamside or subalpine meadow) where fungus gnats are abundant year-round. Our results further suggest that there may be a previously unnoticed association between fungus gnat pollination and dark red coloration, and a shared overall floral architecture among the plants studied.