Volatile compounds profile of Bromeliaceae flowers

Perfil de compuestos volátiles de las flores en las Bromeliaceae .

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, such as Ae. bicolor (74.92 %), Ae. distichantha (77.54 %) and V. friburgensis (71.27 %). Among the Ananas varieties, terpenes corresponded to 69.01 %, representing 66.21 % (A. comosus var. bracteatus), and 70.97 % (A. comosus var. erectifolius) of the total composition. The compound β-myrcene was observed in all species except Al. nahoumii, with abundance ranging from 0.31 to 39.26 % in V. michaelii, and Ae. distichantha, respectively. Linalool was only observed in Al. nahoumii, with approximately 12.94 % of the total compounds found (Table 1, Fig. 2B, and supplementary material).

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 %).

Five aldehydes were identified among the compounds: n-hexanal, phenylacetaldehyde, n-nonanal, (E)-non-2-enal and decanal, the last three being found in all species of Aechmea analyzed. Phenylacetaldehyde was observed in Ae. bromeliifolia (8.17 %), and V. michaelli (1.32 %).

Gaultheric acid was found in highest abun- was emitted by flowers of the three varieties dance in V. michaelii (63.13 %), V. paraibica of the genus Ananas (Fig. 2C), V. friburgensis (10.02 %), while in the other species the values (Fig. 2D) and V. simplex, with abundance rangwere below 2 % (Table 1). Eucalyptol, an ether, ing from 5.16 to 6.08 % (Table 1).

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 correlation of each component and the variables allowed for the evaluation of the discriminatory power of this analysis. For PC1 (30.78 %), the variables that mostly influenced the separation of accessions based on volatile compounds were the presence of eucalyptol, alloaromadendren, and cadina-1(6),4-diene, with correlation values of 0.99, 0.98 and 0.98, respectively. For PC2 (19.17 %), the variables with highest influence were cadina-3,5-diene, unknown 7 and muurola-4(14),5-diene, with correlation values of 0.96, 0.93 and 0.93, respectively. For PC3 (12.85 %), the most significant variables were α-humulene, (E)-α- bergamotene and β-bisabolene, with correlations of 0.76, 0.75 and 0.75, respectively.

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.

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).

Two other compounds observed in high abundance in most species studied here were: γ-terpinene, which has a citrusy, sweet (Acree & Arn, 2004), gasoline and turpentine scent (Qiao et al., 2008); and α-humulene, which has a woody (Acree & Arn, 2004) and fresh scent (Cuevas-Glory, Ortiz-Vezquez, Sauri-Duch, & Pino, 2013) and has been shown to have antiinflammatory properties. It is also found in the essential oil of Cordia verbenacea DC (Boraginaceae) (Fernandes et al., 2007).

Aldehydes have strong odors that recall citrus fruits, roses and fresh cut grass, being widely used in perfumery (Bauer et al., 2001). Phenylacetaldehyde is an aromatic compound also found in Fagopyrum esculentum Moench (Polygonaceae) (Janes, Kantar, Kreft, & Prosen, 2009) and several species of flowers (Robert & Meagher, 2002). This compound has long been used to attract various species of moths in traps for biological control (Robert, & Meagher, 2002; Smith, Allen, & Nelson, 1943; Cantelo, & Jacobson, 1979), and has a floral/honey odor (Whetstine, Cadwallader, & Drake, 2005).

High abundance of n-hexanol has also been observed in plum fruits, Prunus domestica L. (Rosaceae) (Gomez, Ledbetter, & Hartsell, 1993), Caralluma europaea (Guss.) N.E.Br. (Apocynaceae) flowers (Formisano et al., 2009), Camellia sinensis (L.) Kuntze (Theaceae) (Han, Zhou, Cui, & Fu, 2006) and essential oil of Pimenta guatemalensis (Lundell) Lundell (Chaverri & Cicció, 2015). Jabalpurwala, Smoot and Rouseff (2009), studying volatile compounds in flowers of different species of Citrus, also observed high levels of n-hexanol in Citrus grandis (L.) Osbeck (Rutaceae), and correlated the levels with the pollination by bees, which is crucial for reproduction due to self-incompatibility.

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.

The authors acknowledge Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (2009/18255-0), and Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq (476.131/2008-1), for financial support. APM also acknowledges CNPq for research fellowships (305.785/20087 and 310.612/2011-0).