Volatile compounds profile of Bromeliaceae flowers

Volatile compounds play a vital role in the life cycle of plants, possessing antimicrobial and anti-herbivore activities, and with a significant importance in the food, cosmetic, chemical, and pharmaceutical industry. This study aimed to identify the volatile compounds emitted by flowers of thirteen species belonging to four genera of Bromeliaceae, using headspace solid-phase micro-extraction and detection by gas chromatography-mass spectrometry. A total of 71 volatile compounds belonging to nine chemical groups were identified. The compounds identified represented more than 97 % of the major components in Aechmea bicolor, Ae. bromeliifolia, Ae. distichantha, Ae. fasciata, and Vriesea friburgensis. In the Ananas varieties, over 99 % of the components were identified, and around 90 % in V. simplex. V. friburgensis presented the largest diversity of volatiles with 31 compounds, while Alcantarea nahoumii presented only 14. All three Ananas varieties presented the same 28 compounds in relatively similar abundance, which has been confirmed by principal component analysis. Current taxonomy and pollination syndrome studies available can adequately explain the variation in volatile compounds among species. Rev. Biol. Trop. 64 (3): 1101-1116. Epub 2016 September 01.

Bromeliaceae Juss family belongs to the Poales order, with 58 genus and 3 352 species (Luther, 2012).Bromeliaceae presents a wide diversity of forms and are found in almost every neotropical ecosystem, from sea level in beaches, mangroves, and shoals to altitudes of 4 000 m above sea level in the Andes (Benzing, 2000).
These compounds may possess antimicrobial and antiherbivore activity, also repelling microorganisms and animals or attracting natural predators, protecting the plant through tritrophic interactions (Hammer, Carson, & Riley, 2003;Arab & Bento, 2006;Lucas-Barbosa, Loon, & Dicke, 2011), which also suggests their involvement in the protection of the reproductive parts of plants during flowering (Kessler, Halitschke, & Poveda, 2011;Parra-Garcés, Caroprese-Araque, Arrieta-Prieto, & Stashenko, 2010).On the other hand, like all inheritable characters, chemical compounds that make up scents may also reflect the taxonomic affinities of a species.The characterization of volatile compounds may contribute to taxonomic and phylogenetic studies considering that some volatile compounds may be specific to certain plant groups (see for example Nogueira, Bittrich, Shepherd, Lopes, & Marsaioli, 2001).
This study aims to identify volatile compounds from flowers of thirteen ornamental species belonging to four genera of Bromeliaceae (using headspace solid-phase micro-extraction with detection by gas chromatography-mass spectrometry), and to bring a new contribution revealing the potential that these plants have in the industry, in the synthesis of natural products, as well as in future studies of ecological processes involving plant-animal interactions, and taxonomy studies from the principal components analysis.

Solid-phase microextraction:
This technique presents a low cost of execution, good repeatability, quickness, and is solvent-free.Collected flowers were immediately placed in 20 mL capped vials and allowed to equilibrate for 20 min at 37 for 20 °C.Volatile compounds were collected from the headspace of each sample by solid-phase microextraction (SPME), using Supelco SPME fibers coated with divinylbenzene-polydimethylsiloxane (DVB/PDMS, 65 μm) during 20 min at 37 °C.The fiber was then withdrawn into the needle and transferred for injection in the GC-MS system with splitless injection mode at 240 °C (Almeida, Gonçalves, Galego, Miguel, & Costa, 2006).
Gas chromatography and mass spectrometry: GC-MS analyses were conducted according to Custódio, Serra, Nogueira, Gonçalves, and Romano (2006) with modifications.The analyses of volatile compounds were performed on a gas chromatograph GC 2 010 (Shimadzu Corp., Kyoto, Japan) coupled to a mass spectrometer QP 2 010 Plus (Shimadzu Corp., Kyoto, Japan).Samples were separated using a capillary column (RTX-5MS 30 m x 0.25 mm x 0.25 µm).The temperature program started at 40 °C for 2 min, increasing at 4 °C per min to 130 °C, remaining at 130 °C for 1 min, increasing at 7 °C per min to 230 °C, remaining at 230 °C for 4 min, totaling 44 min of analysis.Helium was used as the carrier gas at linear velocity of 36.1 cm/s.The interface was maintained at 280 °C and the detector operated in the scanning mode (m/z 45-450).Data integration was performed using the Lab-Solutions-GCMS Ver.2.5 software (Shimadzu Corp., Kyoto, Japan).

Volatile identification and semi-quantification:
The volatile compounds were identified by Wiley 138 and FFNSC libraries.The relative abundance of the compounds was calculated based on the MS results.
The data were subjected to the multivariate principal components analysis using Statistica (Statsoft, 2004).

RESULTS
HS-SPME/GC-MS analysis of the volatile profile emitted by flowers of 13 species belonging to four genera of Bromeliaceae was performed.A total of 80 compounds were extracted and 71 compounds were identified.Nine chemical groups were found in the identified compounds: alcohols, terpenoids, aldehydes, esters, ketones, ethers, furans, oxides, and styrene (Table 1).The compounds identified represented over 97 % of the major components of this fraction.In Ae. bicolor, Ae. bromeliifolia, Ae. distichantha, Ae. fasciata, V. friburgensis and the three varieties of Ananas, over 99.45 % of the compounds were identified, while in V. simplex 90.69 % of the compounds were identified (Table 1, and supplementary material).
V. friburgensis showed the highest diversity of volatile compounds, with 31 chemicals, while Al.nahoumii showed only 14 compounds.The three varieties of Ananas contained the same 28 compounds in relatively similar abundance, as verified by principal component analysis (Fig. 1).
The group of terpenes showed the greatest number of compounds and the highest percentage in the fraction analyzed for most species,     Eleven compounds belonging to the class of alcohols were identified, with a total abundance ranging from 6.12 % to 59.69 % in V. michaelli and Ae.bromeliifolia, respectively.Among them, the most abundant were n-hexanol, (Z)-hex-3-enol, (E)-sabinene hydrate and 1-terpineol (Table 1).n-Hexanol was identified in high abundance in most species studied, particularly Al. nahoumii (31.54 %), Ae. bromelifoliia (29.68 %) and V. paraibica (21.21 %).
The principal component analysis based on the volatile compounds of Bromeliaceae flowers from 13 species showed the first three components retaining 62.81 % of the initial information (Table 2).This total variance is considered high, with high heterogeneity of the samples' chemical composition.
The dispersion diagram of the scores of the first three main components (Fig. 1) showed three groups, and two isolated species, demonstrating the variability of volatile compounds among these bromeliad species.

DISCUSSION
This study demonstrated great variability of volatile compounds among the bromeliad species, with 71 compounds identified belonging to eight chemical groups, including alcohols, terpenoids, aldehydes, esters, ketones, ethers, furans, oxides, and styrene.
The presence of terpenoids in plants is associated with defense against herbivores, pathogenicity, and allelopathy to attract pollinators (Langenheim, 1994).Terpenes are associated with various fragrances, making them widely used to make perfumes and flavorings (Bauer, Garbe, & Surburg, 2001).
The compound β-myrcene has been described in more than 200 plant species as being responsible for green, herb, pine, lemon, grapefruit, musty and spicy scents (Mahattanatawee, Rouseff, Filomena, & Naim, 2005;Qiao et al., 2008).It is widely used in the cosmetic and pharmaceutical industries, as described by Behr and Johnen (2009).Linalool has been observed in tangerines and is responsible for their taste and aroma (Sawamura, Minhtu, Onishi, Ogawa, & Choi, 2004).
Gaultheric acid belongs to the class of ketones and can be found in wines and plant species such as Gaultheria itoana Hayata (Ericaceae) (Chen et al., 2009).It is also abundant in the root bark of Securidaca longepedunculata Fresen (Polygalaceae), exerting a biocide effect against insects that feed on stored grains (Lognay, Marlier, Seck, & Haubruge, 2000).The emission of volatile compounds with biocide effect can be related to pollination, exerting a repellent effect on some insect species.
Eucalyptol was also identified among the volatile compounds in African cycad (Encephalartos) (Zamiaceae) flowers (Suinyuy et al., 2013).Furans and oxides were present in a few species studied, but in low amounts.
Special patterns of scent in flowers can function as the same visual patterns, so differences in intensity and types of volatile compounds emitted, besides serving as guides for insects, help in the search for food rewards.A combination of chemical analyses of floral scents with field observations of the behavior of flower visitors is an effective way to demonstrate the effect of volatiles in the attraction of pollinators (Dobson, 1994).
The dispersion diagram of the first three main components scores showed V. michaelli, V. paraibica and Al.nahoumii forming the first group.These species are phylogenetically close, belonging to the subfamily Tillandsioideae (Barfuss, Samuel, Till, & Stuessy, 2005;Givnish et al., 2011;Versieux et al., 2012), and Alcantarea traditionally being either considered as a subgenus of Vriesea, or a genus itself, both belonging to the tribe Vrieseeae (Grant, 1995).Floral morphology is one of the factors that influence the pollination syndrome (Aguilar-Rodríguez et al., 2014), these species present large flowers with yellow petals, and a tubular corolla.Similarities in composition of the flower volatiles produced among these species can be another factor to consider in establishing the relationship of these species.
Ae. bicolor, Ae. distichantha, and Ae.nudicaulis belong to the Bromelioideae subfamily, with similar flower morphological characteristics.They formed a second group according to their volatile compound composition.Faria, Wend and Brown (2004), studying the cladistic relationships of this genus, showed that Ae. distichantha and Ae.nudicaulis are very close in clade distribution.In addition, there are reports of common pollinators for these two species (Schmid, Schmid, Zillikens, Harter-Marques, & Steiner, 2010;Scrok & Varassin, 2011).There are very limited studies of Ae. bicolor, however morphological similarity is observed between its inflorescence and flower morphological characteristics and those of Ae. nudicaulis.
The dispersion diagram showed the two other Aechmea species studied, Ae. fasciata and Ae.bromeliifolia, isolated in the principal component analysis of the volatile compounds.This demonstrates the considerable variability of flower volatile compounds among this genus.
The third group included An. macrodontes, An. comosus var.erectifolius and An.comosus var.bracteatus and two species of Vriesea (V.simplex and V. friburgensis).For Ananas, this grouping supports the morphological and taxonomic closeness within the genus, considering that the same 28 compounds were observed in Ananas flowers at similar values.
Considering that the flower volatile compound spectrum can be a plant strategy to attract pollinators, the two species of Vriesea may present pollination syndrome similarities with the genus Ananas, thus being grouped together in the principal component analysis.Hummingbirds are common pollinators between these two genus, which can explain the proximity of these species in the PCA results.Regarding Ananas ananassoides, Stah, Nepi, Galetto, Guimarães, and Machado (2012) observed the presence of two Trochilidae species, Hylocharis chrysura and Thalarania glaucopis, the latest also observed by Schmid et al. (2011) in V. friburgensis.
One of the reports of volatile compounds in bromeliads refers to T. macropetala, in which Aguilar-Rodríguez et al. ( 2014) identified nine volatile compounds, two of them similar to those found in this study, namely nonanal in the species Aechmea, Al. nahoumii and V. michaelii, and limonene in Ae. nudicaulis.Those authors pointed out that the pollination syndrome is not necessarily related to a single compound, such as dimethyl disulphide, which although absent in T. macropetala, did not prevent the visit of pollinating bats.In this case, of the nine compounds identified by the researchers, six are also present in other species pollinated by bats.In Werauhia gladiolifora, also pollinated by bats, Bestmann, Winkle, and Helversen (1997) observed 12 volatile compounds, five of them common to those observed in the species of the present work (α-Pinene, β-Pinene, 4,8-dimethyl-1,3,7nonatriene, β-Myrcene, Limonene, α-Copaene) and two common to T. macropetala (β-Pinene e Limonene) a species pollinated by bats (Aguilar-Rodríguez et al., 2014).
Finally, it is important to highlight that the aroma composition varies throughout the day and this issue is important in attracting pollinators (Balao, Herrera, Talavera, & Dötterl, 2011;Aguilar-Rodríguez et al., 2014).Dötterl, Jahreb, Jhumur, and Jürgens (2012) evaluated the volatile compound dynamics throughout the 24 h in which the flowers were open, observing a variation of these compounds, thus enabling a greater diversity of pollinators, and thus ensuring the reproductive success of the species.In our study, the flowers were collected at anthesis, which occurred between 6:30 and 8 a.m. for all species.Further experiments on the volatile compounds dynamics throughout the day may be interesting for pollination attraction studies in these species.
We identified 71 different volatile compounds, some of them having significant importance in the food, cosmetic, perfume, chemical and pharmaceutical industries.The variation in the odor profile observed of Bromeliaceae in this study shows complex variability.Current taxonomy and pollination syndrome studies can adequately explain the variation in volatile compounds among species.Characterization of these compounds in Bromeliaceae may clarify some problems in taxonomy.Further studies using more species from different genera and detailed morphological information and volatile composition associated with their pollinators can clarify the attraction of pollinators by specific odor compounds.

Fig. 1 .
Fig. 1.Dispersion diagram of scores associated with the first three principal components obtained from the analysis of volatile compounds from flowers of 13 species of Bromeliaceae by HS-SPME/GC-MS.

TABLE 1
Percent area of volatile compounds and retention time in a mass spectrum in 13 species of Bromeliaceae by HS-SPME/GC-MS R (min) a Bromeliad species (Peak area %) Aroma descriptor BIC BRO DIS FAS NUD BRA ERE MAC ALC FRI MIC PAR SIM

TABLE 2
Parameters obtained from the principal components analysis detailing the first five principal components obtained by HS-SPME/GC-MS from volatile compounds emitted by flowers of 13 species of Bromeliaceae