Phylogeny of Hanabusaya (Campanulaceae), a Korean endemic,based on ITS sequences of nuclear ribosomal DNA

The phylogenetic position of the genus Hanabusaya was verified using the internal transcribed spacer (ITS) sequences data of 181 individuals of 176 taxa in Campanulaceae, including allied genera such as genus Campanula, Adenophora, Symphyandra, and Zeugandra. The phylogenetic analysis showed thatHanabusaya is more closely allied to Adenophora than to Symphyandra, Zeugandra, and Campanula. Accordingly, the connate anthers of Hanabusaya are considered to have evolved independently of Symphyandra or Zeugandra. However, Hanabusaya and sect. Remotiflorae of Adenophora were unresolved within a clade. Therefore, the relationship between Hanabusaya and its allies should be more accurately determined through a broader study on diverse genetic regions.     

Phytochemical and chemotaxonomic study on the dried rhizome of Menispermum dauricum DC

An alkaloid (1), an amide (2), three lactones (3, 6 and 7), two coumarins (4–5), eight lignans (8–15), four alcohols (16–19) and three phenols (20–22) are obtained from the EtOAc soluble portion of the MeOH extract of the rhizome of Menispermum dauricum DC. The chemical structures of the compounds are determined by using spectroscopic methods and further supported by comparison with previous literatures. This is the first report that compounds 4–5, 12–17, 19 and 20–21 are obtained from the family Menispermaceae. Compounds 1–2, 6–11 and 22 are isolated for the first time from Menispermum genus. The chemotaxonomic significance of the compounds is also discussed.     

Echinocodon Hong,a New Genus of Campanulaceae and Its Systematic Position

Echicocodon Hong (Wahlenbergieae); Gen. nov. Calyx 2-5-saepe 4-fidus; lobis utrinque 2-4-lobulis spiniformibus praeditis. Corolla tubulosa; ad medium parterm (3-) 4 (-5)-lobata. Stamina (3-) 4 (-5); libera; sed inter se eonniventia; filamentis basi parum expansis. Ovarium fere inferum; 3-5-1oculare; stigmata 3-5; demum recurva; linearia; ovula ∞. Capsula globosa; 3-5-locularis; spice loculicide dehiscens; valvis calycis lobis alternantibus. Semina ∞ ; minima; ellipsoidea; obtuse triquetra.——Herbae perennes; lactesentes; radicibus palaribus crassiusculis. Folia alterna; pinnatipartita. Flores solitarii terminalesque vel 2-3 in cymam conferti. Genus monotypicum; in provincia Hubei Sinae cresens. Typus generis: Echinocodon lobophyllus Hong. Echinocodon lobophyllus Hong; sp. nov. plate 1 Herbae diffusae; omnino glabrae; radicibus palaribus ad 5 mm crassis. Caulis ad 40 cm longus; ramosissimus. Folia alterna; petiolis 5-10 mm longis; laminis ellipticis; 5-20 mm longis; 3-15 mm latis; basi anguste cuneatis; apice obtusis; pinnatisectis vel ad medium pinnatipartitis. Flores solitarii terminales vel 2-3 in cymam conferti; pedicellis 1-5 cm longis. Calycis lobi ovato-lanceolati; 2-6 mm longi; lobulis spiniformibus exclusis 1-3 mm lati. Corolla purpureo-caerulea; tubulosa; 3-4.5 mm longa; ad medium (3-)4(-5)-lobata; lobis late ovato-diltoideis; ad 3 mm latis. Stamina inter se conniventia; filamentis liberis; 1.5 mm longis; basi expansis; ciliatis; antheris oblongis; 1 mm longis. Stylus 1 mm longus; stigmata linearia; demum recurva. Capsula globosa; 3-5 mm diam.; sed parte supera conica et 2 mm longa. Semina minima; luteo-fusca; ellipsoidea; obtuse triquetra; 0.3 mm longa. China: Hubei Province; Yunxi County; near the town; 300 m alt.; 1 Jul. 1983. Ma Yuan-jun and Hong De-yuan PB83006 (holotype; PE); the same locality; 11 Jul. 1978; Ma Yuan-jun 3921. The new genus; Echinocodon Hong; shows its systematic position in the tribe Wahlenbergieae by the inferior ovary and capsule which loculicidally dehisces above the calyx and is closely related to the genera Codonopsis; Platycodon and Wahlenbergia; from all of which it differs in having the calyx lobes with spinelike lobules and the pinnatisect or pinnatipartite leaves. From Codonopsis it differs also in having the linear stigma lobes; from Platycodon also in the capsule valves alternate with the calyx lobes and from Wahlenhergia also in having the 4(5)-colpate pollen grains. Meiosis in pollen mother cells was examined and; 8 bivalents in MI and 8 chromosomes in MII were always observed (Plate 2: 1-2). The chromosomt number is thus determined to be n=8. The pollen grains taken from the flowers which were fixed in the fields with Carnoy II fixative (6:3:1 absolute alcohol/chloroform/glacial acetic acid) were examined without further treatment under SEM; and under LM after acetolysis. The result shows that they are oblate; 4(5)-colpate (Plate 2: 3-4); with colpi 10.5μm long and 2.5μm wide and with a smooth colpus membrane (Plate 2: 5). The exine is 2-layered; with a distinct baculate layer (Plate 2:7-8). The sexine surface is covered with spinules almost regularly spaced; 0.6μm high and basally divided. The sexine between spinules consists of short ridges (Plate 2: 6). The new genus is closely related to the genera Codonopsis; Wahlenbergia and Platycodon from gross morphological point of view; but its pollen grains differ markedly from all of them. The pollen grains in Codonopsis are longly 5-colpate; and with the sexine consisting of a low relief reticulum; those in Platycodon longly 5-colporate and those in Wahlenbergia 3-porate. The pollen type of the new genus Echinocodon with short colpi is therefore probably derived from the longly colpate type of pollen like that of Codonopsis or directly from longly colporate type like that of Platycodon; and is likely more primitive than thatin Wahlenbergia.     

Phytochemical and chemotaxonomic studies on the stems and leaves of Schisandra chinensis (Turcz.) Baill

A comprehensive phytochemical investigation of the stems and leaves of Schisandra chinensis (Turcz.) Baill. resulted in isolation of seventeen compounds, including five lignans: meso-dihydroguaiaretic acid (1), licarin-A (2), pregomisin (3), gomisin A (4), acutissimanide (5), three phenylpropanoids: 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-propane-1,3-diol (6), 2-methoxy-4-(2-propenyl) phenyl β-D-glucopyranoside (7), erigeside 2 (8), six sesquiterpenoids: 7′-hydroxy-abscisic acid (9), burmannic acid (10), (3S,5R,6R,7E)-3,5,6-trihydroxy-7-megastigmen-9-one (11), 3-Cyclohexene-1,2-diol, 4-(3-hydroxybutyl)- 3, 5, 5-trimethyl- (12), (−)-loliolide (13), (3Z,5R,8R,11R)-Caryophyll-3-ene-5,8,15-triol (14), one monoterpenoid: (6R,3Z)-6,7-dihydroxy-3,7-dimethyl-2-octenoic acid (15) and two other compounds: methyl shikimate (16), 4-Hydroxydodec-2-enedioic acid (17). Their chemical structures were confirmed through NMR, HRESIMS and comparison with the data in the literature. This is the first report of compounds 5, 6, 8–15, 17 from the family Schisandraceae and compounds 2, 16 from the genus Schisandra. Furthermore, we performed a chemotaxonomic study of the separated compounds.     

Variations in sterol and triterpene contents of developing Sorghum bicolor grains

The free, esterified and glycosylated sterols and the pentacyclic triterpene esters of developing Sorghum bicolor grains were analysed by GLC and GC-MS. All the pentacyclic triterpenes were completely esterified but were not detected until 24 days after anthesis. Lupanol, multiflorenol, α-amyrin and isoarborinol were identified in the mature grains as components of the triterpene fraction but no 4,4-dimethylsterols could be found at any stage of development. A sixfold increase in total sterol per grain occurred during development. At 8 days after anthesis, 28-isofucosterol was found to be the second most abundant steryl ester. Campesterol was the major steryl glycoside and obtusifoliol was the major 4-monomethylsterol.     

Phytochemical investigation on the fruits of Camptotheca acuminata and their chemotaxonomic significance

A phytochemical investigation on the fruits of Camptotheca acuminata led to the isolation of 13 compounds, including five coumarins (1–5), two alkaloids (6 and 7), two triterpenoids (8 and 9), three lactones (10–12) and a sesquiterpene (13). The structures of these compounds were elucidated by extensive spectroscopic methods and comparion with literature data. All compounds were isolated from the Nyssaceae family for the first time. Additionally, it should be noted that this is the first report of coumarins in the C. acuminata. The chemotaxonomic significance of the isolated compounds was summarised.     

Phytochemical and chemotaxonomic study on the leaves of Rhododendron amesiae

The phytochemical study of the leaves of Rhododendron amesiae (Ericaceae) led to the isolation and identification of 19 compounds, including six diterpenoids (1–6), six triterpenoids (7–12) and seven flavonoids (13–19). The chemical structures of these compounds were identified by spectroscopic data, as well as by comparison with previously reported data in literature. This is the first systematic study on the chemical constituents of Rhododendron amesiae. All the compounds were isolated from this plant for the first time. Compounds 12, 14 and 15 were first isolated and reported from the genus Rhododendron and the family Ericaceae. Furthermore, the chemotaxonomic significance of these compounds was discussed.     

Phytochemical and chemotaxonomic study on the leaves of Rhododendron dauricum L

The phytochemical study of the leaves of Rhododendron dauricum L. (Ericaceae) led to the isolation and identification of 44 compounds, including fourteen triterpenoids (1–14), thirteen flavonoids (15–27), eight phenols (28–35), four grifolin derivatives (36–39), two diterpenoids (40–41), two megastigmanes (42–43) and one sesquiterpenoid (44). The chemical structures of these compounds were identified by spectroscopic data and compared with those previously reported. This is the first report on compounds 25, 31, 34, 40, 43, and 44 from the family Ericaceae, compound 42 from the genus Rhododendron, and compounds 24, 28, 30, 32, 33 and 35 from R. dauricum. The chemotaxonomic significance of these compounds was discussed.     

The Restoration of the Genus Cyclocodon ( Campanulaceae) and Its Evidence from Pollen and Seed-coat

The genus Cyclocodon Griff. was reduced by C. B. Clarke in 1881 into a section of the genus Campanumoea. Our LM and SEM observations on pollen morphology show that the pollen of all the three species in the former is 3-colporate with the exine sparsely high-spinulose, whereas that of the two species in the latter is 5～8-colpate with the exine relatively densely short-spinulose. SEM observations on seed-coat indicate that the primary ornamentation of the seed-coat of the two species in Campanumoea (s. str. ) is characterized by regular and polygonal areoles which are much larger than the radial walls in diameter and by the bead-like secondary ornamentation on the radial walls, while that of the three species in Cyclocodon is characterized by irregular-shaped areoles which are nearly equal to the radial walls in diameter and by the rope-like secondary ornamentation on the radial walls. Thus, the pollen morphology is closely correlated with the seed-coat morphology. Taking the characteristics of pollen, seed-coat and gross morphology into consideration, the genus Cyclocodon is restored, separate from Campanumoea (s. str. ). One new combination, Cyclocodon celebicus (B1.)Hong, is made in the present paper. The genus Cyclocodon is con-sidered closely related to Platycodon rather than to Campanumoa.     

Phytochemical investigation on the roots of Juglans mandshurica and their chemotaxonomic significance

Phytochemical research of the roots of Juglans mandshurica Maxim. (Juglandaceae) verified 37 secondary metabolites, including thirteen diarylheptanoids (1–13), five naphthoquinones (14–18), five tetralones (19–23), three lignans (24–26), three phenols (27–29), two anthraquinones (30–31), two triterpenoids (32–33), two secoiridoids (34–35), one naphthalenyl glycoside (36), and one flavonoid (37). The chemical structures of these constituents were elucidated by NMR spectroscopy and compared with data from the literature. This is the first confirmation of the presence of ten compounds (6, 12, 16, 18, 24–26, 30, 32–33) isolated from the family Juglandaceae, two compounds (1 and 8) from the genus Juglans, and four compounds (27, 31, 34–35) from J. mandshurica. The isolation and chemotaxonomic significance of the metabolites from the roots of J. mandshurica were described in this study.     

Identification of volatile markers for indoor fungal growth and chemotaxonomic classification of Aspergillus species

Microbial volatile organic compounds (MVOCs) were collected in water-damaged buildings to evaluate their use as possible indicators of indoor fungal growth. Fungal species isolated from contaminated buildings were screened for MVOC production on malt extract agar by means of headspace solid-phase microextraction followed by gas chromatography-mass spectrometry (GC-MS) analysis. Some sesquiterpenes, specifically derived from fungal growth, were detected in the sampled environments and the corresponding fungal producers were identified. Statistical analysis of the detected MVOC profiles allowed the identification of species-specific MVOCs or MVOC patterns for Aspergillus versicolor group, Aspergillus ustus, and Eurotium amstelodami. In addition, Chaetomium spp. and Epicoccum spp. were clearly differentiated by their volatile production from a group of 76 fungal strains belonging to different genera. These results are useful in the chemotaxonomic discrimination of fungal species, in aid to the classical morphological and molecular identification techniques.     

Floral plasticity in an iteroparous plant: the interactive effects of genotype, environment, and ontogeny in Campanula rapunculoides (Campanulaceae)

Phenotypic variation in 11 floral and reproductive traits was studied in cloned plants of Campanula rapunculoides replicated in three discrete environments. Using an ANOVA approach, we determined the relative influence of genotype (G), environment (E), G × E interaction, and ontogeny (position on the raceme) on the 11 traits. Two traits, duration of flowering and pollen size, showed no significant variation. All nine remaining traits had significant genotypic variation, accounting for 21-38% of the total phenotypic variation. Correlations among variant traits in seven genotypes were predominantly positive, but several significant correlations in one environment changed sign or were nonsignificant in another environment. Ovule number was negatively correlated with most male function traits: the negative correlation between ovule and pollen number was particularly strong and consistent across environments. Six traits varied significantly across environments, including number of flowers, number of ovules per flower, and duration of the male phase, but pollen traits did not show a significant environmental main effect. The G × E interaction was significant for flower number, corolla size, nectar quality, duration of the male phase, pollen viability, and ovule number. The contribution of interaction variance to the total phenotypic variation (5-14%) was comparable to that of the environment alone (7-21%). Ovule number decreased in flowers on the upper part of the raceme by nearly 25%, but other traits did not vary significantly by floral position. These results suggest that (1) pollen traits are buffered against environmental change more than ovule number or other floral characters, (2) a male-female trade-off exists and is complicated by ontogenic factors, (3) G × E interactions are common but may have small effects, and (4) specific correlation patterns among floral traits can be dependent upon the environment under which they develop.     

The effect of methyl jasmonate on triterpene and sterol metabolisms of Centella asiatica, Ruscus aculeatus and Galphimia glauca cultured plants

Considering that exogenously applied methyl jasmonate can enhance secondary metabolite production in a variety of plant species and that 2,3-oxidosqualene is a common precursor of triterpenes and sterols in plants, we have studied Centella asiatica and Galphimia glauca (both synthesizing triterpenoid secondary compounds) and Ruscus aculeatus (which synthesizes steroidal secondary compounds) for their growth rate and content of free sterols and respective secondary compounds, after culturing with or without 100 microM methyl jasmonate. Our results show that elicited plantlets of G. glauca and to a higher degree C. asiatica (up to 152-times more) increased their content of triterpenoids directly synthesized from 2,3-oxidosqualene (ursane saponins and nor-seco-friedelane galphimines, respectively) at the same time as growth decreased. In contrast, the free sterol content of C. asiatica decreased notably, and remained practically unaltered in G. glauca. However, in the case of R. aculeatus, which synthesizes steroidal saponins (mainly spirostane type) indirectly from 2,3-oxidosqualene after the latter is converted to the plant phytosterol-precursor cycloartenol, while the growth rate and free sterol content clearly decreased, the spirostane saponine content was virtually unchanged (aerial part) or somewhat lower (roots) in presence of the same elicitor concentration. Our results suggest that while methyl jasmonate may be used as an inducer of enzymes involved in the triterpenoid synthesis downstream from 2,3-oxidosqualene in both C. asiatica and G. glauca plantlets, in those of C. asiatica and R. aculeatus it inhibited the enzymes involved in sterol synthesis downstream from cycloartenol.     

Quaternary range dynamics of ecologically divergent species (Edraianthus serpyllifolius and E. tenuifolius,Campanulaceae) within the Balkan refugium

Aim We investigated Quaternary range dynamics of two closely related but ecologically divergent species (cold‐tolerant Edraianthus serpyllifolius and thermophilic Edraianthus tenuifolius) with overlapping distribution ranges endemic to the western Balkan Peninsula, an important yet understudied Pleistocene refugium. Our aims were: to test predictions of the ‘refugia‐within‐refugia’ model of strong genetic subdivisions due to population isolation in separate refugia; to explore whether two ecologically divergent species reacted differently to Pleistocene climatic fluctuations; and to test predictions of the displacement refugia model of stronger differentiation among populations in the thermophilic E. tenuifolius compared with the cold‐tolerant E. serpyllifolius. Location The western Balkan Peninsula. Methods We gathered amplified fragment‐length polymorphism (AFLP) data and plastid DNA sequences from two to five individuals from 10 populations of E. serpyllifolius and 22 populations of E. tenuifolius, spanning their entire respective distribution areas. AFLP data were analysed using a Bayesian clustering approach and a distance‐based network approach. Plastid sequences were used to depict relationships among haplotypes in a statistical parsimony network, and to obtain age estimates in a Bayesian framework. Results In E. serpyllifolius, both AFLP and plastid sequence data showed clear geographic structure. Western populations showed high AFLP diversity and a high number of rare fragments. In E. tenuifolius, both markers congruently identified a major phylogeographic split along the lower Neretva valley in central Dalmatia. The most distinct and earliest diverging chloroplast DNA (cpDNA) haplotypes were found further south in the south‐easternmost populations. North‐western populations, identified as a separate cluster by Bayesian clustering, were characterized by low genetic diversity and a low number of rare AFLP markers. Main conclusions Clear evidence for multiple Pleistocene refugia is found not only in the high‐elevation E. serpyllifolius, but also in the lowland E. tenuifolius, despite the lack of obvious dispersal barriers, in line with the refugia‐within‐refugia model. Genealogical relationships and genetic diversity patterns support the hypothesis that cold‐adapted E. serpyllifolius responded to climatic oscillations mostly by elevational range shifts, whereas thermophilic E. tenuifolius did so mainly by latitudinal range shifts, with different phases (and probably extents) of range expansion. In contrast to the displacement refugia hypothesis, the two elevationally differentiated species do not differ in their genetic diversity.     

Pollen morphology of Campanula (Campanulaceae) and allied genera in Iran with special focus on its systematic implication

Pollen grains of 47 taxa of Campanulaceae including 35 taxa of Campanula that represent its five subgenera and nine sections are investigated. Moreover, five species and three subspecies representing three sections in Asyneuma and one species of each genera Legousia, Michauxia, Zeugandra and Theodorovia were studied by light and scanning electron microscopy. The basic shape of the pollen grains was spheroidal. The apertures vary from tri- to hexa-porate. The sculpturing pattern of exine was rugulate-echinate, rugulate-microechinate or in few species rugulate-microreticulate and microechinate. The most valuable characters for subgeneric classification were the length and density of echini. The length of echini were significantly long (> 2 μm) in C. sclerotricha, Legousia falcata and Michauxia laevigata. Pollen grains show low variation in different species of subgen. Rapunculus, but were variable among different species of some groups, such as sect. Rupestres, probably indicating their non-monophyly despite homogeneity with respect to other morphological characters. Pollen morphology does not support recognition of Asyneuma, Legousia, Michauxia, Symphyandra, Theodorovia, and Zeugandra as separated from Campanula, since none of them exhibit any unique feature.     

Review of the neotropical endemics Burmeistera, Centropogon, and Siphocampylus (Campanulaceae: Lobelioideae), with description of 18 new species and a new section

Burmeistera, Centropogon, and Siphocampylus together comprise more than 500 species endemic to the Neotropics, Current knowledge of these genera is unsatisfactory, with much diversity yet to be described. Over the past seven years, one out of every 28 specimens received for identification has represented a new species, many characterized by unique or unusual character states. Siphocampylus smilax (Bolivia) is unique in the genus in its parallelodromous leaf venation and extra-floral nectaries. Centropogon dianae (Peru) cannot be accommodated in any recognized infrageneric taxon, and a new section, Centropogon sect. Niveopsis, is proposed for it. The flowers of C. steinii (Ecuador) are the smallest known in the genus, while C. connatilobatus (Venezuela) is the first species to combine arbusculiform pubescence and connate calyx lobes. Burmeistera venezuelensis is only the second species of the genus known to occur in Venezuela and the first to be endemic; similarly, Centropogon wilburii is only the third species of Centropogon known from Mexico and the first to be endemic to that country. Additional novelties are also described.     

The effect of growth phase on the lipid class,fatty acid and sterol composition in the marine dinoflagellate,Gymnodinium sp. in batch culture

We have studied the effects of growth phase on the lipid composition in batch cultures of Gymnodinium sp. CS-380/3 over 43 days of culturing. The lipid content increased two fold, from late logarithmic (day 6) to linear growth phase (day 22) then decreased at stationary phase (day 43) while the lipid yield (mg l(-1)) increased 30-fold from day 6 to 30 mg l(-1) at day 43. Changes in fatty acid content mirrored those observed for the total lipid, while the sterol content continued to increase with culture age through to stationary phase. The largest changes occurred in the lipid classes, especially the polar lipids and triacylglycerols (oil). The proportion of triacylglycerols increased from 8% (of total lipids) at day 6 to 30% at day 43, with a concomitant decrease in the polar lipid fraction. The proportions of 16:0 and DHA [22:6(n-3)] increased while those of 18:5(n-3) and EPA [20:5(n-3)] decreased with increasing culture age. The proportion of the major sterol, dinosterol, decreased from 41% (day 6) to 29% (day 43), while the major dinostanol epimer (23R,24R) increased from 33% (day 6) to 38% (day 22). Despite small changes in the proportion of the main sterols, the same sterols were present at all stages of growth, indicating their value as a chemotaxonomic tool for distinguishing between strains within the same genus. Growth phase could be a useful variable for optimising the oil and DHA content with potential for aquaculture feeds and a source of DHA-rich oils for nutraceuticals.     

Sesquiterpene lactone-based classification of three Asteraceae tribes: a study based on self-organizing neural networks applied to chemosystematics

This work describes an application of artificial neural networks on a small data set of sesquiterpene lactones (STLs) of three tribes of the family Asteraceae. Structurally different types of representative STLs from seven subtribes of the tribes Eupatorieae, Heliantheae and Vernonieae were selected as input data for self-organizing neural networks. Encoding the 3D molecular structures of STLs and their projection onto Kohonen maps allowed the classification of Asteraceae into tribes and subtribes. This approach allowed the evaluation of structural similarities among different sets of 3D structures of sesquiterpene lactones and their correlation with the current taxonomic classification of the family. Predictions of the occurrence of STLs from a plant species according to the taxa they belong to were also performed by the networks. The methodology used in this work can be applied to chemosystematic or chemotaxonomic studies of Asteraceae.     

Comparative analysis of Laurencia tristicha and Laurencia okamurai based on qualitative and quantitative HPLC determination

A simple and reliable HPLC-DAD method for qualitative and quantitative determination of sesquiterpenes in Laurencia tristicha and L. okamurai was developed, and then applied to compare the sesquiterpenes in the two alga species. Except for the difference in content, L. tristicha and L. okamurai are very similar in chemical compositions. The content of laurinterol, the most abundant component in both species, is much higher in L. okamurai than in L. tristicha, while debromolaurinterol and aplysinol are the second abundant components of L. tristicha and L. okamurai, respectively. The current results confirmed the great chemotaxonomic importance of laurane-type sesquiterpenes, especially the main constituents of laurinterol, debromolaurinterol, and aplysinol, for L. tristicha and L. okamurai.