Molecular and chemical characterization of the most widespread Ocimum species

DNA fingerprinting (AFLP) and chemical analyses of essential oils were utilized to define the extent of variation existing in the genus Ocimum. Research was carried out on 22 Ocimum accessions representing seven species. Concerning the essential oil composition of all investigated accessions, 115 compounds were identified. UPGMA cluster analysis, based on Euclidian distances of essential oil constituents between all pairs of accessions, showed four well-supported clusters (O. tenuiflorum, O. basilicum/O. africanum, O. basilicum, and O. americanum/O. africanum). Relating to the essential oil composition of all of the investigated accessions, 17 compounds were identified as the main ones, and according to them 13 chemotypes were determined. AFLP relationships were determined by neighbor-joining (NJ) cluster analysis based on Dice??s distance matrix and by maximum parsimony (MP) analysis. O. basilicum, O. americanum/O. africanum, O. tenuiflorum, and O. gratissimum represented four clusters supported with high bootstrap values. A neighbor-net diagram allowed the visualization of apparently conflicting data by revealing relationships between genotypes and chemotypes. Concerning the O. africanum species, two distinct chemotypes, geranial/neral (accession 11) and estragol (accession 10), have been established, while all the studied O. americanum accessions belong to the geranial/neral chemotype. This could be additional evidence that O. americanum is one of the parents of O. africanum. Furthermore, the fact that the O. africanum accession (10) as well as O. basilicum ??Purpurascens?? and O. basilicum ??Erevanskii?? accessions belong to the estragol chemotype supports the theory that O. africanum is one of the parents of these two O. basilicum accessions.     

Impact of chemical polymorphism of Thymus pulegioides on some associated plant species under natural and laboratory conditions

Abstract

Allelopathic potential of Thymus pulegioides L. chemical polymorphism was investigated under natural and laboratory conditions. A field analysis of 127 natural habitats hosting chemotypes of T. pulegioides with different ratios of phenolics, geraniol, and ɑ-terpinyl acetate was conducted. Effects of chemotypes, and their main compounds on seed germination and radicle growth of Trifolium pratense L. and Poa pratensis L. were conducted under laboratory conditions. Field analysis showed that Poa species were more plentiful in comparison with Trifolium species, independent of the chemotypical composition of T. pulegioides habitats. Laboratory tests with plant-acceptors showed a stronger inhibitory effect of essential oils on the germination and radicle growth of P. pratensis but in some instances germination was stimulated. Dissimilar effects were observed for the same allelochemical through air and water on the same plant-acceptor. Significantly, different effects of essential oils on radicle growth occurred in T. pratense and P. pratensis: with sensitivity to the phenolic chemotype via air and the ɑ-terpinyl acetate chemotype through water. This demonstrates that chemical polymorphism can expand communication opportunities of T. pulegioides with associated plant species. Combining investigations in natural habitats with laboratory experiments can help understand the effect of chemical polymorphism on plant-plant ecological interactions.     

Chemical Variation in Essential Oils from the Oleo‐gum Resin of Boswellia carteri: A Preliminary Investigation

Frankincense, the oleo‐gum resin of Boswellia species, has been an important element of traditional medicine for thousands of years. Frankincense is still used for oral hygiene, to treat wounds, and for its calming effects. Different Boswellia species show different chemical profiles, and B. carteri, in particular, has shown wide variation in essential oil composition. In order to provide insight into the chemical variability in authentic B. carteri oleoresin samples, a hierarchical cluster analysis of 42 chemical compositions of B. carteri oleo‐gum resin essential oils has revealed at least three different chemotypes, i) an α‐pinene‐rich chemotype, ii) an α‐thujene‐rich chemotype, and iii) a methoxydecane‐rich chemotype.     

Intra-population terpene polymorphism of Thymus pulegioides L.: Evidence for seven chemotypes in a German limestone grassland

For Thymus pulegioides L. (Lamiaceae), occurring in almost entire Europe, about 20 different essential oil chemotypes are described in approximately 25 studies. However, only few studies mention chemotype diversity on population level, describing up to five chemotypes growing together. The aim of the study was to investigate the chemotype diversity within one T. pulegioides population of a limestone grassland in Germany. Essential oil compounds from samples of 93 thyme cushions were extracted with solid phase extraction (SPE) and analysed by GC-MS. Cluster analysis and nonmetric multi-dimensional scaling (NMDS) of the samples’ essential oil composition revealed the existence of seven different chemotypes: linalyl acetate-chemotype (56 individuals), geraniol-citral-linalyl acetate-chemotype (5), geraniol-citral-chemotype (7), thymol-chemotype (8), carvacrol-chemotype (2), linalool-chemotype (1), and β-caryophyllene-germacrene D-β-bisabolene-chemotype (14). The pattern of major monoterpenes from all chemotypes could be explained by the inhibition of specific steps in monoterpene biosynthesis. The sesquiterpene-dominant chemotype might be caused by a block in a very early step of monoterpene production or a regulatory/channeling mechanism. While the geraniol-citral-linalyl acetate-chemotype has been found for the first time, the other chemotypes have been found in other regions and the β-caryophyllene-germacrene D-β-bisabolene-type is similar to other sesquiterpene chemotypes rarely found in other areas. The large intra-population chemical polymorphism in this study raises questions about the chemotype diversity of thyme in other regions of Germany and potential correlations between essential oil composition and abiotic factors or biotic interactions.     

Differentiating Thymus vulgaris chemotypes with ISSR molecular markers

Common thyme (Thymus vulgaris L.) is a Mediterranean evergreen shrub of the Lamiaceae that shows chemical polymorphism in its natural populations. In Mediterranean habitats six basic chemotypes have been described according to the main component in its essential oil: thymol, carvacrol, linalool, geraniol, α-terpineol, thuyanol-4, of which the thymol chemotype is widely used in cultivation and as a spice. In this study ten selected clones were analysed in terms of chemical composition of the volatile oil and genetic fingerprint. The study question was whether individual chemotypes can be distinguished by molecular markers. Chemical composition was analysed by GC-MS, and for assessing the genetic polymorphism ISSR marker system was chosen as it is reliable and easy to use even in case of less studied species. The studied T. vulgaris clones represented five different chemotypes and could be distinguished using 12 ISSR primers. Clones belonging to the thymol chemotype showed the most distinct separation. The study shows the potential of using molecular markers in breeding and selection of T. vulgaris, being able to differentiate different chemotypes.     

GENE FLOW IN LICHENS

Experimental studies of the evolutionary biology of lichen fungi have been hampered by massive difficulties of in vitro culture and artificial crosses are still not possible. Gene flow in these organisms is demonstrated here for the first time by the analysis of secondary products in the progeny of individuals from natural populations of mixed chemotypes of the Cladonia chlorophaea complex. All of the chemotypes in this study have been interpreted as distinct sibling species. In the Appalachian Mountains of North Carolina, however, the grayi and merochlorophaea chemotypes are found to belong to a single interbreeding populations that is reproductively isolated from the cryptochlorophaea chemotype. In the Coastal Plain, the cryptochlorophaea chemotype hybridizes with the local endemic perlomera chemotype. This study has major impact for species concept in lichens because consideration of neither morphologial tendencies nor biogenetic relationships of the secondary products could have predicted its result.     

Chemical variation in the lichen genus <Emphasis Type="Italic">Letrouitia</Emphasis> (Ascomycota,Letrouitiaceae)

Secondary metabolites from 193 specimens belonging to 15 species of Letrouitia were analyzed by HPLC. Significant quantities of the anthraquinones parietin and fragilin were found in most species and occasionally minor quantities of emodin, 5-chloroemodin, 7-chloroemodin, 7-chloroteloschistin, 7-chlorofallacinal and 7-chloroparietinic acid were present. Eight previously unknown lichen substances were identified. A chemotype containing seven new dibenzofurans (8-chlorodioxocondidymic acid, 8-chlorodioxodidymic acid, 8-chloroxodidymic acid, dioxocondidymic acid, dioxodidymic acid, letrouitic acid and oxodidymic acid) was found in 3 species. In addition, a chemotype containing four unknowns (possibly chlorodepsidones) occurred in L. subvulpina. Eight different chemotypes were identified in the genus. The secondary chemistry was important for the precise identification of some species in Letrouitia. The similarity in secondary chemistry between Letrouitiaceae and Teloschistaceae is not particularly strong, as the shared compounds are also known to occur in several other lichen families.     

Essential oil variability of Thymus hyemalis growing wild in Southeastern Spain

Essential oils obtained by steam-distillation of individual samples of Thymus hyemalis were examined for variability in their volatile components by means of gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The thymol chemotype is widespread and is found in most of the vegetal formations where Thymus hyemalis is predominant and does not interact with other species. The carvacrol chemotype is found in isolated individual plants among those containing thymol. Although the chemotypes are usually mutually exclusive, the two phenols occasionally found with similar quantities in the same plant. The linalool chemotype has not previously been described for Thymus hyemalis. Other nonphenolic compounds (1,8-cineole, borneol, α-pinene) are present in low to medium percentages that reflect localized interactions with other species of thyme (Thymus vulgaris, Thymus baeticus) that sometimes produce hybridized or introgressive, morphologically distinguishable individuals.This chemical variability is analyzed by Principal Component Analysis (PCA) and Cluster Analysis (CA), a closely-related phenolic group of samples being revealed separated from less well represented non-phenolic chemotypes or mixed chemotypes. Multidimensional Scaling Analysis (MDS) based on percentage concentration was used to show the relationships between the most important components of the essential oil, the opposite orientation of vectors that represent phenolic compounds (and their precursors) and linalool, being of note. The data set presents positive correlation between camphor and altitude.     

Characterization of two chemotypes of <Emphasis Type="Italic">Pinus pinaster</Emphasis> by their terpene and acid patterns in needles

The existence of two chemotypes of Pinus pinaster, on the basis of the chemical composition of the resin acids in their needles, is known. An investigation was performed on 54 samples of needles of Spanish Pinus pinaster to study the differences between these chemotypes on the basis of monoterpene, sesquiterpene, neutral diterpene, fatty acid, and resin acid composition. One-hundred and twelve compounds were identified by GC–FID and GC–MS. Statistical analysis of the results established the existence of two groups or chemotypes, in the ratio of 5:1. In one chemotype, total acid compounds were more abundant than neutral compounds, whereas in the other the concentrations of both neutral and acid compounds were similar. Distinction of the chemotypes was based on the presence/absence of a sesquiterpene (germacrene d-4-ol acetate), neutral diterpenes (8(14),13(15)-abietadiene, anticopalol, an isomer of anticopalol, and pimarol), fatty acids (10-octadecenoic, 14-hydroxy-10-octadecenoic, and 13-hydroxy-9-octadenoic acids and an unidentified fatty acid), and resin acids (levopimaric + palustric, eperuic, and anticopalic acids, and three isomers of anticopalic acid); and on the different relative percentages of other compounds of these types. This study gives a wide view of the composition of the needles of Pinus pinaster, improving the differentiation of chemotypes on the basis of terpene and acid composition.     

Variability in Foliar Essential Oils among Different Morphotypes of Lantana Species Complexes,and Its Taxonomic and Ecological Significance

The genus Lantana has many species complexes, and L. camara is one of the aggressive alien weedy species complexes; species delimitation in these complexes is a nightmare for taxonomists. We examined the diversity in the chemical composition of foliar essential oils among morphotypes of Lantana species complexes inhabiting the same ecological gradient, and its taxonomic and ecological significance. The yields of essential oils varied from 0.1 to 0.79% in foliar hydrodistillates of eleven morphotypes, and a total of 39 chemical constituents were detected by GC/MS. The quantitative and qualitative variability in the composition of essential oils among morphotypes was very high, and hence they represent chemotypes. The diversity observed in the composition of essential oils appears to be of genetic origin and thus of taxonomic value. The formation of distinct clusters and sub‐clusters at high distance cluster combine values also substantiates that the patterns of distribution of chemical constituents among morphotypes can be used in delimiting species and infraspecific taxa within the species complexes. The presence of β‐caryophyllene and other such compounds, which are known to prevent herbivory, in morphotypes of Lantana species complexes suggest that these compounds may provide selective advantage to Lantana over native species in the invasion of new and disturbed habitats.     

Geographic variation in the essential oils and morphology of natural populations of Melaleuca alternifolia (Myrtaceae)

In recent decades, Melaleuca alternifolia has been grown in plantations for the commercial production of tea tree oil extracted from harvested leaf and stem material by steam distillation. Plantations are grown from seedlings raised from seeds collected from wild populations of this endemic Australian species. Considerable variation in morphology and leaf oil composition and yield has been observed and studies have demonstrated genetic and phenotypic heterozygosity between populations.Here we examine the variation in leaf oil chemical composition (chemotypes) between geographically defined locations of wild populations of M. alternifolia and investigate the relationships between tree size, chemotype and geographic location.Forty separate populations of M. alternifolia distributed amongst three river catchments (two in a warm moist coastal region and one in cool drier highlands) were studied. Total variation in tree size was significantly greater within individual sites than between them. However, the highland catchment populations exhibited significantly smaller mean tree size and a significantly different chemotype profile than the lowland populations. Contrary to the observation of lower genetic diversity, the highland catchment populations had greater chemotypic diversity. Furthermore, highly significant differences in population chemotypes were demonstrated between catchments.The possibilities that these differences could be ascribed to either genetic divergence or to environmental differences are discussed.     

Spatial-temporal survey of Microcystis oligopeptide chemotypes in reservoirs with dissimilar waterbody features and their relation to genetic variation

The ability of cyanobacteria to produce toxins and other secondary metabolites is patchily distributed in natural populations, enabling the use of cellular oligopeptide compositions as markers to classify strains into ecologically-relevant chemotypical subpopulations. The composition and spatiotemporal distribution of Microcystis chemotypes within and among waterbodies was studied at different time scales by analyzing (i) Microcystis strains isolated between 1998 and 2007 from different Spanish reservoirs and (ii) individual Microcystis aeruginosa colonies collected from pelagic and littoral habitats in Valmayor reservoir (Spain) during a bloom. No agreement between chemotypes and both morphotypes and genotypes (based on cpcBA-IGS, 16S–23S rRNA ITS and mcyB genes) was found, suggesting that oligopeptide profiles in individual strains evolve independently across morphospecies and phylogenetic genotypes, and that the diversity of microcystin variants produced cannot be explained by mcyB gene variations alone. The presence of identical chemotypes in spatially-distant reservoirs with dissimilar trophic state, lithology or depth indicate that waterbody characteristics and geographical boundaries weakly affect chemotype composition and distribution. At smaller spatiotemporal scales (i.e. during bloom), M. aeruginosa populations showed high number of chemotypes, as well as marked differences in chemotype composition and relative abundance among the littoral and pelagic habitats. This indicates that the factors influencing chemotype composition, relative abundance and dynamics operate at short spatial and temporal scales, and supports emerging hypotheses about interactions with antagonistic microorganisms as possible drivers for widespread chemical polymorphisms in cyanobacteria.     

Phenotypic Variations in the Foliar Chemical Profile of Persea americana Mill. cv. Hass

The Hass avocado tree Persea americana cv. Hass was derived from a single hybrid tree of P. americana var. drymifolia and P. americana var. guatemalensis, and it is propagated clonally by grafting. This cultivar is the most widely planted in the world but its profile of secondary metabolites has been studied rarely despite of its importance in plant protection. We illustrate the variability of the volatilome of mature leaves by describing the average chemical composition and the phenotypic variability found in 70 trees. Contrary to the uniformity expected in the Hass cultivar, high variability coefficients were found for most of the 36 detected foliar volatile compounds; furthermore we found six chemotypes grouping the foliar phenotypes of the sampled trees using hierarchical cluster analysis. About 48% of trees were grouped in one chemotype; five chemotypes grouped the remaining trees. The compounds that determined these chemotypes were: estragole, α‐farnesene, β‐caryophyllene, germacrene D, α‐cubebene and eugenol. This striking variation in a cultivar propagated clonally is discussed in terms of somatic mutation.     

Chemical Diversity in Himalayan Elsholtzia Species

The terpenoid composition of essential oils from the leaves of five Elsholtzia species, viz., E. eriostachya Benth ., E. cristata Willd ., E. polystachya Benth ., E. flava Benth ., and E. pilosa Benth ., collected from the Himalayan region (India), was examined by GC, GC/MS, and NMR analyses. Comparison of the results with previous reports revealed new chemotypes. Cluster analysis was carried out in order to discern the similarities and differences within the essential‐oil compositions at their subspecies/chemotype level. Based on the major constituents of the essential oils, six chemical groups were obtained.     

First record of Coleura (Chiroptera: Emballonuridae) on Madagascar and identification and diagnosis of members of the genus

Abstract

The genus Coleura contains two species: C. seychellensis restricted to the Seychelles Archipelago and C. afra broadly distributed on the African continent and the Arabian Peninsula. We recently captured and collected specimens of Coleura from Madagascar, an island from which it was previously unknown. Using museum specimens and the new material from Madagascar, we review morphological variation in members of this genus and present characters for species identification. On the basis of these comparisons, specimens from Madagascar are referable to C. afra. We present information on the natural history of the recently discovered Malagasy population.     

Essential oil of two chemotypes of Mentha suaveolens during ontogenesis

The composition of the main constituents of the essential oil of two different chemotypes of Mentha suaveolens has been investigated during development. In the first chemotype (6) a definite increase of 1,2-epoxymen-thylacetate was observed, but piperitone oxide was always the main constituent. In the second chemotype (12) an increase of neo- and dihydrocarveol and their corresponding acetates was observed and dihydrocarvone was the main constituent. The results have been used to formulate a new biosynthetic scheme for the cyclic oxygenated monoterpenes found in essential oils of the genus Mentha.     

Essential‐Oil Polymorphism in the ‘Resurrection Plant’ Myrothamnus moschatus and Associated Ethnobotanical Knowledge

Gas chromatography/mass spectroscopy analysis (GC/MS) of essential oils obtained from populations of the resurrection plant Myrothamnus moschatus, growing in different areas of Madagascar, allowed identification of three main chemotypes in the species. The first one was provided by plants with a high content of trans‐pinocarveol and pinocarvone; the second one involved plants with high percentages of limonene, cis‐ and trans‐p‐mentha‐1(7),8‐dien‐2‐ol, and β‐selinene; and the third chemotype was characterized by plants with high levels of oxygenated sesquiterpenes such as caryophyllene oxide and α‐ and β‐isomers of caryophylla‐4(12),8(13)‐dien‐5‐ol. Chemical data were supported by chemometric technique as the principal component analysis. Furthermore, the relationship between the dioecy and phytochemistry within one population was also considered. Finally, correlations between chemical variations and ethnobotanical data were assessed.     

唐古特大黄(Rheum tanguticum Maxim. ex Balf.)功效组分地理变异及气候响应特征

通过研究道地药材唐古特大黄（Rheum tanguticum Maxim.ex Balf.）四种功效组分的地理变异,以及与多层次气候因子间的响应关系,揭示唐古特大黄不同化学型形成的生态学机制。运用聚类方法研究唐古特大黄成分地理变异的空间规律,并利用相关系数法分析气候因子与唐古特大黄成分的响应关系。结果表明：唐古特大黄功效组分存在明显的地理变异。青海和甘肃地区的唐古特大黄属于结合蒽醌化学生态型;而四川所产唐古特大黄为游离蒽醌化学生态型。不同时间尺度气候因子与唐古特大黄组分间的响应特征为：结合蒽醌类物质与年均温呈强负相关,多酚类物质与年均温和年降水为负相关,而与年均日照时数为正相关;结合蒽醌类物质与最冷季节温度是强负相关,多酚类物质与最冷季节温度和最湿季降雨量呈强负相关;月均温和月均日照对结合蒽醌类物质和多酚类物质影响大,其中1月至6月平均气温和9月至12月平均气温与蒽醌类物质呈强负相关,多酚类物质与5月日照量、6月日照量和7月日照量表现出强正相关。地区间的温度和日照量差异是唐古特大黄不同化学型形成的气候原因。最冷季的低温和最湿季的日照量是影响唐古特大黄品质的关键气候因子和主要时间窗口。温度浮动大,日照量高且降水量少的低温区域有助于结合蒽醌类和多酚类物质的形成和累积。     

Essential oils from New Zealand manuka: triketone and other chemotypes of Leptospermum scoparium

The triketone chemotype of manuka, Leptospermum scoparium (Myrtaceae), is commercially important because of its antimicrobial activity. Oils from 36 individual plants on the East Cape of New Zealand all showed similar high triketone contents (>20% total triketones) with little seasonal variation. Analyses of oils from 261 individual manuka plants collected from 87 sites throughout New Zealand showed that the high triketone chemotype was localised on the East Cape, although oils with triketone levels up to 20% were found in the Marlborough Sounds area of the South Island. Cluster analysis revealed other chemotypes localised on other areas. Ten further chemotypes are described: alpha-pinene; sesquiterpene-rich with high myrcene; sesquiterpene-rich with elevated caryophyllene and humulene; sesquiterpene-rich with an unidentified sesquiterpene hydrocarbon; high geranyl acetate; sesquiterpene-rich with high gamma-ylangene + alpha-copaene and elevated triketones; sesquiterpene-rich with no distinctive components; sesquiterpene-rich with high trans-methyl cinnamate; high linalol; and sesquiterpene-rich with elevated elemene and selinene. Some of the chemotypes contained aroma compounds at relatively high levels, with a geranyl acetate-rich oil being most notable. Possible origins for this complex array of chemotypes are proposed.