Volatile Constituents from Catasetum (Orchidaceae) Species with Occurrence in the Brazilian Amazon

Background: Catasetum Rich. ex Kunth is a genus of Neotropical orchids distributed in Central and South American regions. In the Brazilian Amazon, there are more than 60 species of Catasetum. The floral aromas of orchids are little known, particularly of Catasetum species. This work aimed to analyze the chemical constituents of the volatile concentrates of eight Catasetum specimens from the Amazon: C. alatum (1), C. albovirens (2), C. barbatum (1), C. ciliatum (2), C. galeritum (1), and C. gnomus (1). Methods: Gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analyzed and identified the constituents of the volatile concentrates, and principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used in the multivariate statistical analysis. Results: The Catasetum main constituents in descending order and above 10% were trans-geranylgeraniol, 1,4-dimethoxybenzene, linalool, 2-phenylethyl acetate, geraniol, 7-epi-1,2-dehydro-sesquicineole, 1,8-cineole, benzyl acetate, limonene, methyl salicylate, (E)-β-farnesene, anisyl butyrate, cis-carvone oxide, cadin-4-en-10-ol, indole, α-pinene, and δ-cadinene. Conclusions: Multivariate statistical analysis of Catasetum species showed that C. barbatum, C. albovirens, and C. gnomus are distinct from the other studied species, while C. alatum, C. ciliatum, and C. galeritum presented the same primary classes of compounds. These results contribute to a better understanding of the genus Catasetum chemotaxonomy.


Introduction
Flowering plants and their volatile compounds attract birds, insects, and other animals, including mammals, as pollinators for their reproduction. More than 1700 flower scent compounds, covering 990 taxa, have already been identified [1][2][3]. The flower perianth is primarily responsible for the scent emission, though all floral organs might contribute to the emission of scents. Floral scents are stored in the oily glands, such as the trichomes before it is released into the air as volatile compounds, and in addition to flowers, volatile compounds emitted by other plant organs have involved in its defense mechanisms. Therefore, floral volatiles play a significant role in the plant's reproductive process by attracting pollinators and acting as repellents and physiological protectors against abiotic stress [2,4]. Floral volatiles are expected to be used in the composition of perfumes, cosmetics, flavors, and therapeutic applications. However, the volatiles emitted by flowers are also the main signals captured by insects to select gratifying flower species associated with the respective flower colors [3,5]. Floral scents are composed of volatile compounds, usually lipophilic and low molecular weight. Based on their origin, function, and biosynthesis, floral scents are grouped into three main classes of compounds: Terpenoids, phenylpropanoids/benzenoids, and fatty acid and derivatives [3,6].
Orchidaceae is the most prominent flowering plant family, with 736 genera and ca 28,000 species, which shows a wide diversity of epiphytic and terrestrial specimens, colonizing almost every earth's habitat and renowned for their abundance of morphological types, with an unending number of beautiful variations and very well represented in the monocotyledons' floral evolution [7,8]. The orchids distributed in the natural environment have small sizes and have a limited production of flowers. These restrictions are circumvented by producing visual and olfactory signals to attract pollinators. Developing highly specialized pollination mechanisms to attract effective pollinators is a common strategy in orchids. Specializing these mechanisms leads to the formation of syndromes, in which the set of floral characteristics, including fragrances, is associated with attracting a particular group of pollinators. Euglossini syndrome is widely known among orchids and is characterized by the absence of food resources, with only the production of specialized floral fragrances collected by male Euglossini bees. Floral fragrances not only act as signaling but also represent a reward for bees because some components of these mixtures will compose their pheromones, which is the essential prerequisite for sexual recognition and selection during mating [9][10][11][12].
Catasetum Rich. ex Kunth is one of the genera of Neotropical orchids pollinated by male Euglossini bees. The genus has about 130 species distributed mainly in Central and South American tropical regions. Floral fragrances play a vital role in the diversification of Catasetum. Hybridizations that result in changes in the composition of fragrances also generate differences in pollinators, restricting gene flow and contributing to the origin of new strains. Inflorescences of Catasetum are usually unisexual with distinct morphology and sexual determination according to each individual's environmental and nutritional characteristics. Catasetum usually shows male or female flowers, but in some situations, may form nonfunctional hermaphroditic flowers and/or flowers of both sexes. Sex expression is controlled by plant size and light intensity. Large plants under strong sunlight usually develop female flowers, whereas younger and smaller plants under moderate light develop male flowers. They are sympatric species that use floral fragrances as a fundamental part of the reproductive isolation mechanism. The composition of these aromas is represented by a mixture of constituents with attractive and repellent action of different proportions, detected in small amounts by male Euglossini bees. Therefore, orchids attract particular species of bees that, in synergy with other floral filters (e.g., morphological characteristics), act as their effective pollinators. [13][14][15][16].
The present work aimed to extract the volatile concentrates and identify the chemical constituents of the flowers of eight specimens from six species of Catasetum: C. alatum (1), C. albovirens (2), C. barbatum (1), C. ciliatum (2), C. galeritum (1), and C. gnomus (1), with occurrence in the Brazilian Amazon. In addition, submit the chemical composition data of these specimens to multivariate analysis, targeting their association with other species/specimens of Catasetum taxonomically close or previously analyzed.   Geographic distribution: Endemic in Brazil. Occurrence for the North region of Brazil, State of Rondônia, in riparian or gallery forest areas [17].
Geographic distribution: Endemic in Brazil, occurring in the states of Amazonas, Pará, Maranhão, Mato Grosso, and Tocantins, in anthropic areas such as rupestrian fields, high and flooded forests, seasonal deciduous forests, and savannas [17].
Two specimens of C. albovirens were analyzed. Specimen Calb-1 was sampled initially in the municipality of Muaná, Ilha do Marajó, and specimen Calb-2 was collected initially in the municipality of Tucuruí, Pará state, Brazil. Table 2 lists the constituents of their volatile concentrates.  Synonimy: Catasetum lamosii Rolfe [17]. Geographic distribution: Endemic in Brazil, occurring in the states of Amazonas, Pará, Maranhão, Mato Grosso, and Tocantins, in anthropic areas such as rupestrian fields, high and flooded forests, seasonal deciduous forests, and savannas [17].

Catasetum barbatum Lindl.
Botanical description: Epiphytic, occasionally terrestrial. Fusiform pseudobulbs, about 15.0 to 5.0 × 3.0 to 5.0 cm long. Narrow, plicate leaves with a midrib and two lateral ones. Inflorescence suberect or arched with up to 20 flowers. Flowers ( Figure 5) male resupinate, greenish with brown spots. Sepals are lanceolate to dorsal erect, the lateral ones reflexed on the pedicel. Petals lanceolate and erect, margins finely serrated, somewhat revolute. The lips include hairs that may be white or greenish; the basal callus may be simple or bifurcated, usually surrounded by pilosity. Column light green or brownish. Cream anther. Yellow pollinia are hard and compressed on a white laminar stipe and a white viscid disc. Flowering in April and May [17]. Geographic distribution: Not endemic in Brazil, but found in upland and floodplain forests, broadleaf forests, mangrove and palm groves, and vegetation on rocky outcrops in the states of Amazonas, Pará, Roraima, Tocantins, Alagoas, Bahia, Ceará, Maranhão, Paraíba, Pernambuco, Piauí, Distrito Federal, Goiás, Mato Grosso do Sul, Mato Grosso, and Minas Gerais [17].
The specimen of C. barbatum in this study was sampled initially in the municipality of Ourilândia do Norte, Pará, Brazil. Table 3 lists the constituents of its volatile concentrate.  Hook. [17].
The complexity of floral scents in the species of Catasetum investigated so far varies considerably. In the present work, more than 93% of the scent profile has been characterized, while the number of identified constituents varied from 1 in C. micranthum to 74 in C. uncatum [23,24]. This variation in scent floral complexity across Catasetum species certainly reflects an inherent characteristic for each species. As expected in angiosperms, floral scents of Catasetum are species-specific, although dominated by some significant constituents usually shared by several species [25]. Most Catasetum species have 2 or 3 main constituents that account for more than 70% of the fragrances. These constituents are potent attractants to many Euglossa and Eulaema bees, but the attractiveness to individuals and species is reduced as more components compose the mixtures so that specific scents attract only a few pollinator species [11,19].
The pivotal role of floral fragrances in pollinator shifts and as a reproductive isolating mechanism in Catasetum was previously highlighted [16]. However, floral scents may not be enough to assure the effective reproductive isolation in Catasetum. Sympatric species usually produce similar fragrances, thereby attracting the same pollinator species. In these cases, different reproductive isolating mechanisms (e.g., geographical, morphological/mechanical, temporal/seasonal), acting alone or together, will be necessary to contribute to or prevent the hybridization [16,19]. Presently, considering the well-defined separation of the pollinating genera of Catasetum and the higher sensorial similarity between the closely related bee species, have been speculated that the olfactory adaptations have shaped the evolution of floral fragrances of Catasetum due to the partitioning with pollinator's bees, particularly from the genera Euglossa and Eulaema [16,19].
Therefore, pollinator affinity with phylogeny is correlated with differences found in floral aromas. More generally, the question is why flowers produce different odors or why mixtures of odors tend to be species-specific. The answer to this question demands more complex functional analyses, attributing phylogenetic, physiological, and ecological influences to the chemical variation of floral scents [25].

Plant Material
The orchids Catasetum alatum, C. albovirens, C. barbatum, C. ciliatum, C. galeritum, and C. gnomus (Figures 1-6), which provided the flowers for this work, are live plants cultivated in pots containing charcoal and wood shavings, existing in the private nursery of Mr. Luiz Otávio Adão Teixeira, located in the Amazon Garden Condominium, BR-316, km 6, 67015-795 Ananindeua, PA, Brazil (coordinates: 1 • 22 20.96" S/48 • 23 34.14 W). These orchid specimens were previously sampled in various localities and cities of the Brazilian Amazon, as already described for each one in the Results. Each plant exemplar (exsiccate) was deposited in the Herbarium of Emílio Goeldi Museum, Belém, Para state, Brazil. The orchid flowers were collected during the flowering period, at 6 am, to extract their volatile constituents.

Obtaining and Analyzing Volatile Concentrates
The orchid flowers were subjected to micro distillation-extraction in a Likens & Nickerson-type apparatus (3 flowers each, 15 g in total, 2 h, duplicate) to obtain their volatile concentrates, using n-pentane (99% HPLC grade, 3 mL) as the solvent [26].
The volatile concentrates of orchids were submitted to GC and GC-MS analysis. It was performed on a GCMS-QP2010 Ultra system (Shimadzu Corporation, Tokyo, Japan), equipped with an AOC-20i auto-injector and the GCMS-Solution software containing the Adams (2007), Mondello (2011), and Nist (2011) libraries [27][28][29]. A Rxi-5ms (30 m × 0.25 mm; 0.25 µm film thickness) silica capillary column (Restek Corporation, Bellefonte, PA, USA) was used. The conditions of analysis were as follows. Injector temperature: 250 • C; Oven temperature programming: 60-240 • C (3 • C min −1 ); Helium as the carrier gas, adjusted to a linear velocity of 36.5 cm s −1 (1.0 mL min −1 ); split mode injection (split ratio 1:20) of 1.0-2.0 µL of the n-pentane solution; electron ionization at 70 eV; ionization source and transfer line temperatures of 200 and 250 • C, respectively. The mass spectra were obtained by automatically scanning every 0.3 s, with mass fragments in the 35-400 m/z. The retention index was calculated for all volatile components using a homologous series of C8-C40 n-alkanes (Sigma-Aldrich, Milwaukee, WI, USA) according to the linear equation of van den Dool and Kratz (1963) [30]. Individual components were identified by comparing their retention indices and mass spectra (molecular mass and fragmentation pattern) with those existing in the GCMS-Solution system libraries [27][28][29]. The quantitative data regarding the volatile constituents were obtained using a GC2010 Series gas chromatograph, operated under similar conditions to those of the GC-MS system. The relative amounts of individual components were calculated by peak-area normalization using a flame ionization detector (GC-FID). Chromatographic analyses were performed in duplicate.

Multivariate Statistical Analysis
Principal Component Analysis (PCA) was applied to verify the interrelationship of the samples of volatile concentrates analyzed with the classes of identified compounds, monoterpene hydrocarbons (MH), oxygenated monoterpenes (OM), sesquiterpene hydrocarbons (SH), oxygenated sesquiterpenes (OS), oxygenated diterpenes (OD), benzenoids/phenylpropanoids, and fatty acids and derivatives. The data matrix was standardized for multivariate analysis by subtracting the mean and dividing it by the standard deviation. Hierarchical Cluster Analysis (HCA), considering the Euclidean distance and complete linkage, was used to verify the similarity of the samples based on the distribution of the constituents selected in the PCA analysis (Software Minitab, free version 390, Minitab Inc., State College, PA, USA) [31].

Conclusions
In conclusion, the present study showed that previous reports were not found in the literature concerning the chemotaxonomy of the Catasetum species. Thus, considering their classes of compounds, Catasetum albovirens, C. gnomus, and C. barbatum can be distinguished from the other studied species, while C. alatum, C. galeritum, and C. ciliatum showed the same primary compound classes. Also, there are two chemotypes of C. ciliatum, the first one rich in oxygenated monoterpene (67.5%) and the second rich in benzenoids/phenylpropanoids (53.5%). Thus, we think these findings could contribute to a better understanding of the chemical profiles of Catasetum species.