Lychnophora pinaster in endangered campos rupestres: phenolic compounds and population ecogeography.

Lychnophora pinaster, known as arnica-mineira, is endemic to campos rupestres, at risk of extinction. The present study aimed to characterize the ecogeography and phenolic constituents of 11 L. pinaster populations collected in the mesoregions North, Jequitinhonha, Metropolitan of Belo Horizonte and Campos das Vertentes in the state of Minas Gerais, Brazil. Phenolic constituents were identified and quantified by Ultra-high performance liquid chromatography- mass spectrometry. Lychnophora pinaster occurs in sites at high altitude (700 and 1498 m), annual rainfall of up to 1455 m, soils with low fertility and predominantly loamy texture. Therefore, it can be considered tolerant to acidic soils, with low availability of nutrient. The most abundant substances in all populations were vitexin (18 - 1345 ng/g) and chlorogenic acid (60 - 767 ng/g). The 11 populations formed four groups in relation to the phenolic constituents, with group 1 consisting of the populations of the North Mesoregion (GM, OD) and Jetiquinhonha (DIMa), group 2, the Metropolitan of Belo Horizonte (SRM, NLSC, SM, RPS, CTRA), group 3, the North Mesoregion (ODMa and DI), and group 4, the Campos das Vertentes (CC). Among the populations, only those from the Metropolitan of Belo Horizonte showed correlation of soil properties with phenolic constituents.

High-Resolution Liquid Chromatography-Mass Spectrometry-Based Metabolomics for the Classification of Chuquiraga (Barnadesioideae, Asteraceae): New Phenylpropanoid Derivatives as Chemical Markers for Chuquiraga spinosa.

Despite their relatively poorly investigated phytochemistry, species of the genus Chuquiraga are widely commercialized. The present study reports the use of a high-resolution liquid chromatography-mass spectrometry-based metabolomics approach coupled with exploratory and supervised multivariate statistical analyses for species classification and chemical marker identification of four species of Chuquiraga (C. jussieui, C. weberbaueri, C. spinosa, and Chuquiraga sp.) from Ecuador and Peru. Based on these analyses, a high percentage of correct classifications (87% to 100%) allowed the prediction of the taxonomic identity of Chuquiraga species. Through the metabolite selection process, several key constituents with the potential to be chemical markers were identified. Samples of C. jussieui displayed alkyl glycosides and triterpenoid glycosides as discriminating metabolites, while Chuquiraga sp. displayed high concentrations of p-hydroxyacetophenone, p-hydroxyacetophenone 4-O-glucoside, p-hydroxyacetophenone 4-O-(6-O-apiosyl)-glucoside, and quinic acid ester derivatives as the main metabolites. Caffeic acid was characteristic for C. weberbaueri samples, whereas C. spinosa displayed higher concentrations of the following new phenylpropanoid ester derivatives: 2-O-caffeoyl-4-hydroxypentanedioic acid (24), 2-O-p-coumaroyl-4-hydroxypentanedioic acid (34), 2-O-feruloyl-4-hydroxypentanedioic acid (46), 2,4-O-dicaffeoylpentanedioic acid (71), and 2-O-caffeoyl-4-O-feruloylpentanedioic acid (77).

Evidence of altitudinal gradient modifying genomic and chemical diversity in populations of Lychnophora pinaster Mart.

Lychnophora pinaster Mart. (Asteraceae) is endemic to the Brazilian Cerrado. It is distributed along the altitudinal gradient of the mountainous ranges of the state of Minas Gerais. This study aimed to evaluate the influence of altitude on the genetic diversity of L. pinaster populations and the effects of altitude and climatic factors on essential oil chemical composition. Essential oils from L. pinaster populations from the north (North 01, North 02, and North 03, 700-859 m) and the Metropolitan region of Belo Horizonte (MhBH 01 and MrBH 02, 1366-1498 m) were analyzed. SNP markers from L. pinaster in these regions and Campos das Vertentes (CV 01, CV 02, and CV 03, 1055-1292 m) were also analyzed. The main compounds in essential oils were 14-hydroxy-α-humulene (North 01 and North 03), cedr-8(15)-en-9-α-ol (North 02), 14-acetoxy-α-humulene (MrBH 01), and 4-oxo-15-nor-eudesman-11-ene (MrBH 02). Hierarchical cluster and heatmap analyses showed that the North and MrBH populations included five different groups, indicating the chemical composition of essential oils is distinct in each population. Furthermore, principal component analysis showed that higher altitudes (1366 m and 1498 m) in the MrBH influence the chemical composition of essential oils, and climatic factors determine the chemical composition in North region. The genetic diversity showed that most alleles are in Hardy-Weinberg equilibrium and imply high genetic variation and genetic polymorphisms between populations. Furthermore, the results of Mantel tests (R = 0.3861517; p = 0.04709529; R = 0.9423121; p = 0.02739726) also showed that higher altitude (>1360 m) shapes the genetic diversity at the MrBH. The genetic structure showed that higher altitudes (>1360 m) contribute to the structure of the MrBH populations, but not to North and CV populations. Therefore, the altitudinal ranges of Minas Gerais mountainous ranges determine the higher genetic and chemical diversity of L. pinaster populations.

Time-Scale Shifting of Volatile Semiochemical Levels in Wild Type Lychnophora ericoides (Brazilian arnica) and Pollinator Records.

Lychnophora ericoides is a Brazilian folk phytomedicine from Cerrado's "campus rupestris". Its volatile organic compounds includes bisabolene-derivatives as major compounds. Herein we provide the chemical profiling of constitutive volatile sesquiterpenes from L. ericoides leaves, timeframe emissions surveys, and pollinators records. In situ samples of L. ericoides were harvested. A headspace-solid phase micro extraction method of pre-concentration was optimized. Identification was done through GC-MS. Isolation and structural elucidation were performed whenever necessary. Pollinators were registered in pictures and video. Short time-series and harmonic regressions determined rhythms of single compounds, and average chromatographic signal area was used to determine mono and sesquiterpene rhythms. Concluding, optimized headspace-solid phase micro extraction method of terpenes level analysis was reached. α-Pinene, ß-pinene, α-terpinene, para-cymene, limonene, γ-terpinene, terpinen-4-ol, dehydro-sesquicineole, and ß-guaiene were identified using GC-MS data. 11-dehydro cadinol and ortho-acetoxy bisabolol were elucidated. Sesquiterpenes concentrations were higher due to temperature rise, lower leaf age, and flowering seasons. Harmonic regressions determined that daylight might control levels of terpenes. Hummingbird, hemiptera insects, and wasps were recorded visiting Compositae capitulum for the first time. We studied nondomestic plants from in situ conditions and concluded that bisabolene-derivative levels were more abundant than monoterpenes during flowering throughout the summer.

Metabolomic analysis applied to chemosystematics and evolution of megadiverse Brazilian Vernonieae (Asteraceae).

Vernonia sensu lato is the largest and most complex genus of the tribe Vernonieae (Asteraceae). The tribe is chemically characterized by the presence of sesquiterpene lactones and flavonoids. Over the years, several taxonomic classifications have been proposed for Vernonia s.l. and for the tribe; however, there has been no consensus among the researches. According to traditional classification, Vernonia s.l. comprises more than 1000 species divided into sections, subsections and series (sensu Bentham). In a more recent classification, these species have been segregated into other genera and some subtribes were proposed, while the genus Vernonia sensu stricto was restricted to 22 species distributed mainly in North America (sensu Robinson). In this study, species from the subtribes Vernoniinae, Lepidaploinae and Rolandrinae were analyzed by UHPLC-UV-HRMS followed by multivariate statistical analysis. Data mining was performed using unsupervised (HCA and PCA) and supervised methods (OPLS-DA). The HCA showed the segregation of the species into four main groups. Comparing the HCA with taxonomical classifications of Vernonieae, we observed that the groups of the dendogram, based on metabolic profiling, were in accordance with the generic classification proposed by Robinson and with previous phylogenetic studies. The species of the genera Stenocephalum, Stilpnopappus, Strophopappus and Rolandra (Group 1) were revealed to be more related to the species of the genus Vernonanthura (Group 2), while the genera Cyrtocymura, Chrysolaena and Echinocoryne (Group 3) were chemically more similar to the genera Lessingianthus and Lepidaploa (Group 4). These findings indicated that the subtribes Vernoniinae and Lepidaploinae are non-chemically homogeneous groups and highlighted the application of untargeted metabolomic tools for taxonomy and as indicators of species evolution. Discriminant compounds for the groups obtained by OPLS-DA were determined. Groups 1 and 2 were characterized by the presence of 3',4'-dimethoxyluteolin, glaucolide A and 8-tigloyloxyglaucolide A. The species of Groups 3 and 4 were characterized by the presence of putative acacetin 7-O-rutinoside and glaucolide B. Therefore, untargeted metabolomic approach combined with multivariate statistical analysis, as proposed herein, allowed the identification of potential chemotaxonomic markers, helping in the taxonomic classifications.

Eithea lagopaivae, a new critically endangered species in the previously monotypic genus Eithea Ravenna (Amaryllidaceae).

Eithea lagopaivae Campos-Rocha & Dutilh, sp. nov. is described as the second species of the formerly monotypic genus Eithea. It is characterized by a one flowered inflorescence, completely hollow scape, white or lightly magenta-striated flower that is enclosed by spathe bracts fused for more than the lower fifth of its length. Comments on its range, habitat, phenology, as well as photographs and illustrations are provided. In addition, a distribution map and an identification key for the two species of the genus are presented and anatomical and ecological differences compared. Known by only two small populations exposed to several types of threats and without any guarantee of protection, E. lagopaivae is considered a Critically Endangered (CR) species.

Resumo Eithea lagopaivae Campos-Rocha & Dutilh, sp. nov. é descrita como a segunda espécie do gênero previamente monotípico Eithea. Caracteriza-se por apresentar a inflorescência uniflora, escapo totalmente oco, flor alva ou com leves estrias magenta, protegida por brácteas espatáceas fundidas na base por mais de um quinto do seu comprimento. Comentários sobre a sua área de ocorrência, hábitat, dados de fenologia, fotografias e ilustrações são fornecidos. Adicionalmente, um mapa de distribuição e uma chave de identificação para as duas espécies do gênero são apresentados e suas diferenças anatômicas e ecológicas comparadas. Conhecida de apenas duas pequenas populações sujeitas a diversos tipos de ameaça e sem qualquer garantia de proteção, E. lagopaivae é considerada uma espécie Criticamente em Perigo (CR) de extinção.

A metabolomic protocol for plant systematics by matrix-assisted laser-desorption/ionization time-of flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS) has been widely used for the identification and classification of microorganisms based on their proteomic fingerprints. However, the use of MALDI-TOF MS in plant research has been very limited. In the present study, a first protocol is proposed for metabolic fingerprinting by MALDI-TOF MS using three different MALDI matrices with subsequent multivariate data analysis by in-house algorithms implemented in the R environment for the taxonomic classification of plants from different genera, families and orders. By merging the data acquired with different matrices, different ionization modes and using careful algorithms and parameter selection, we demonstrate that a close taxonomic classification can be achieved based on plant metabolic fingerprints, with 92% similarity to the taxonomic classifications found in literature. The present work therefore highlights the great potential of applying MALDI-TOF MS for the taxonomic classification of plants and, furthermore, provides a preliminary foundation for future research.

Low genetic structure in an epiphytic Orchidaceae (Oncidium hookeri) in the Atlantic rainforest of South-eastern Brazil.

BACKGROUND AND AIMS: Oncidium hookeri is a neotropical species of epiphytic Orchidaceae found in the Brazilian Atlantic rainforest at the top of the Mantiqueira Range of mountains. The genetic variation of O. hookeri was studied to assess the distribution of genetic variability within and among six populations localized in Atlantic rainforest remnants. Gene flow among populations and the occurrence of recent bottlenecks were investigated in order to infer the degree of isolation of these populations. METHODS: Thirteen polymorphic loci were used for allozyme electrophoresis. The data were analysed by means of standard statistical approaches, to estimate gene diversity and the genetic structure of the populations. KEY RESULTS: The mean gene diversity and allelic richness were H(e) = 0.099 and A = 1.75, respectively. F-statistics revealed high heterozygote deficiencies in all populations (F(IS) = 0.43-0.82). Several rare alleles were found in all the populations, and three populations presented private alleles. Low genetic differentiation among O. hookeri populations was detected (F(ST) = 0.029); natural selection may be involved in PGM locus differentiation among populations. The genetic differentiation between paired populations was low, bearing no correlation with geographic distance (Mantel test: r = -0.34, P = 0.72). Only two populations showed signs of recent bottlenecks. CONCLUSIONS: The heterozygote deficiency found seems to be caused by pollinator behaviour; the low frequencies of several alleles of different loci can be maintained due to clonal propagation. Despite the stochastic nature of the wind-dispersal of seeds to long distances, this process may promote an effective gene flow among populations, thus avoiding genetic differentiation.

Low allozymic variation in the Bidens pilosa L. complex (Asteraceae).

Bidens alba, B. subalternans, and B. pilosa form a complex group based on their morphological similarities. Bidens pilosa L. and B. subalternans DC. are herbs with a wide distribution in agricultural and disturbed areas. Bidens alba (L.) DC. varies in size from herb to subshrub and has a coastal distribution. Enzyme electrophoresis was used to evaluate genetic diversity in 12 populations of Bidens. All but three loci (Lap-1, Est, and Got) were monomorphic. Est-1 and Got were polymorphic only in B. alba. Lap-1 was polymorphic only in B. pilosa and B. subalternans. The estimates of genetic variability were low for all three taxa and all of the populations studied. Genetic diversity varied from 0.01 to 0.03. Mean genetic identities were high among populations of each species (0.99 for B. alba and 1.00 for B. pilosa and B. subalternans) and among the three species (1.00). Bidens pilosa and B. subalternans could be considered a single species if the taxonomy of the group were based solely on isozyme data.

Lip anatomy and its implications for the pollination mechanisms of Bulbophyllum species (Orchidaceae).

BACKGROUND AND AIMS: The lip structure of six Brazilian and one Asiatic species of Bulbophyllum with wind-assisted fly pollination (B. involutum, B. ipanemense and B. weddellii) and non-wind-assisted fly pollination (B. epiphytum, B. glutinosum, B. regnellii and B. rothschildianum) was studied to investigate the presence of secretory tissues related to these pollination mechanisms. METHODS: The lip study was carried out through scanning electron microscopy (lip surface) and light microscopy (anatomical features). KEY RESULTS: In most of the species studied, the osmophores (odour glands) were located in the lobes and in the upper surface of the lip callus. Differences in the lip structure were observed between the two groups (the presence of a nectary and the extent of osmophore surface), depending on the mechanism of pollination. Nectaries were found in the cavity callus in B. ipanemense, B. involutum and B. weddellii, even though their pollinators were presumably attracted by the instinct to oviposit. CONCLUSIONS: These findings corroborate the hypothesis that, because pollination in these species is dependent on an unpredictable external factor (wind), nectar is necessary to keep the insect in the flower for a long period. Despite the occurrence of a liquid-like nectar in the flowers of B. epiphytum, B. glutinosum, B. regnelli and B. rothschildianum, no anatomical evidence for nectaries was found in the lips of these species, although a similar structure may occur in another region of the flowers. This observation agrees with the fact that pollination by lip movement in the latter species requires only gravity, with no additional mechanism being needed to keep the flies in the flower.

Histological study of post-pollination events in Spathodea campanulata beauv. (Bignoniaceae), a species with late-acting self-incompatibility.

The reproductive biology of Spathodea campanulata was investigated by means of hand-pollination experiments, observations of pollen tube growth using fluorescence microscopy, and serial sections of ovules in selfed and crossed pistils. Only cross-pollinated flowers developed fruits, and all selfed flowers abscised within 3-4 d. However, self pollen tubes grew successfully to the ovary, penetrating and fertilizing the majority of ovules by 48 h, indicating that S. campanulata is a species with late-acting self-incompatibility. The incidences of ovule penetration, fertilization and endosperm initiation were all significantly slower in selfed vs. crossed pistils, although no other signs of malfunctioning were detected. The possible role of such slow self pollen tube effectiveness as a recognition event is discussed within the context of the slow but not entirely suppressed self pollen tube growth reported for some species with conventional homomorphic self-incompatibility.

Floral and vegetative morphometrics of five Pleurothallis (Orchidaceae) species: correlation with taxonomy, phylogeny, genetic variability and pollination systems.

Morphometric analyses of vegetative and floral characters were conducted in 21 populations of five Pleurothallis (Orchidaceae) species occurring in Brazilian 'campo rupestre' vegetation. A phylogenetic analysis of this species group was also carried out using nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2). Results of the ordination and cluster analyses agree with species' delimitation revealed by taxonomic and allozyme studies. The groups formed in ordination analysis correspond to the pollinator groups determined in a previous pollination study. Relationships among the species in the cluster analysis using only vegetative characters are similar to those found in a previous allozyme study, but those indicated by cluster analysis using only floral characters differ. These results support the hypothesis that floral similarities are due to convergence driven by similar pollination mechanisms, and therefore floral traits may not be good indicators of phylogenetic relationships in this group. The results of the phylogenetic analysis support this conclusion to some extent. There is no correlation between genetic (allozyme) and morphological variability in the populations nor in the way this variability is distributed among conspecific populations. We describe a new subspecies of Pleurothallis ochreata based on differences in vegetative and chemical characters as well as geographic distribution. Absence of differentiation in floral characters, attraction of the same pollinator species, interfertility and genetic similarity support the argument for subspecific rather than specific status.