Volatile compounds profile of flowers

Everton Hilo de Souza 1*, Adna P. Massarioli 2, Ivani A. M. Moreno 2, Fernanda V. D. Souza 3, Carlos A. S. Ledo 3, Severino M. Alencar 2 & Adriana P. Martinelli 1* 1. University of São Paulo (CENA), Av. Centenário 303, 13416-903, Piracicaba, SP, ; [email protected], [email protected] 2. University of São Paulo (ESALQ), Av. Pádua Dias 11, 13418-900, Piracicaba, SP, Brazil; [email protected], [email protected], [email protected] 3. Embrapa Cassava and , Rua Embrapa s/n, 44380-000, Cruz das Almas, BA, Brazil; [email protected], [email protected]

Received 16-III-2015. Corrected 07-III-2016. Accepted 01-IV-2016.

Abstract: Volatile compounds play a vital role in the life cycle of , possessing antimicrobial and anti-her- bivore 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 gen- era 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 com- pounds identified represented more than 97 % of the major components in Aechmea bicolor, Ae. bromeliifolia, Ae. distichantha, Ae. fasciata, and Vriesea friburgensis. In the 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.

Key words: Bromeliaceae, gas chromatography, headspace, mass spectrometry, principal component analysis, terpenoids, volatile compounds.

Bromeliaceae Juss family belongs to the pollinate most of the bromeliad species, with order, with 58 genus and 3 352 species flowers presenting colorful and attractive bracts (Luther, 2012). Bromeliaceae presents a wide and abundant nectar (Benzing, 2000; Kessler diversity of forms and are found in almost & Krömer, 2000; Araujo, Fischer, & Sazima, every neotropical ecosystem, from sea level 2004). Bats are also notable pollinator agents in beaches, mangroves, and shoals to alti- in some species that present scented flowers, of tudes of 4 000 m above sea level in the Andes nocturnal anthesis (Sazima, Vogel, & Sazima, (Benzing, 2000). 1989; Knudsen & Tollsten, 1995; Sazima, Volatile compounds are critical for the Buzato, & Sazima, 1995; Aguilar-Rodríguez life cycle, especially for pollination and et al., 2014). In addition to ornithophily and seed dispersion, which assures plant reproduc- chiropterophily, there are also records of pol- tion and their evolutive success (Pichersky & lination of Bromeliaceae by butterflies, bees, Gershenzon, 2002; Knudsen & Gershenzon, and beetles (Benzing, 2000; Kessler & Krömer, 2006; Suinyuy, Donaldson, & Johnson, 2013; 2000; Canela & Sazima, 2005; Siqueira Filho Aguilar-Rodriguez et al., 2014). Hummingbirds & Machado, 2001; Schmid, Schmid, Zillikens,

Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1101-1116, September 2016 1101 & Steiner, 2011). The majority of Bromeliaceae (Dudareva & Pichersky, 2006; Cheng, 2010; has scentless flowers, except in a few cases Darjazi, 2011; Paibon et al., 2011), due to the such as Hohenbergia ridleyi (Baker) Mez. increased preference for natural food addi- (Siqueira Filho, 1998); in Tillandsia crocata tives and other compounds of biological ori- (E. Morren) Baker (Gerlach & Schill, 1991); gin (Huang, Lee, & Chou, 2001). These are Canistrum aurantiacum E. Morren (Siqueira mainly terpenoids, phenylpropanoids, hydro- Filho & Machado, 2001); Puya sp. (Knudsen, carbons, alcohols, aldehydes, ketones, ethers Tollsten, Groth, Bergström, & Raguso, 2004; and esters derived from fatty acids, represent- Bromelia antiacantha Bertol. (Canela & Sazi- ing approximately 1 % of the known secondary ma, 2005), and Tillandsia macropetala Wawra metabolites in plants (Dudareva, Pichersky, (Aguilar-Rodríguez et al., 2014). In these spe- & Gershenzon, 2004). In the Bromeliaceae cies, a delicate and sweet scent is associ- family, volatile compounds were studied in ated with bee pollination. On the other hand, the Ananas genus and mostly the volatile Encholirium glaziovii Mez and some Vriesea compounds. In Ananas over 280 compounds have an unpleasant scent and copious amounts were identified, the most abundant being esters, of nectar, attracting bats (Sazima et al., 1989; terpenes, ketones and aldehydes (Tokitomo, Sazima et al., 1995). Bromelia antiacantha Steinhaus, Suttner, & Schieberle, 2005; Liu, Bertol. flowers have a strong sweet scent which Wei, Sum, & Zang, 2008; Wei et al., 2011). becomes lighter throughout the day (Canela & Aguilar-Rodríguez et al. (2014) identified nine Sazima, 2005). Tillandsia macropetala Wawra volatile compounds (three fatty acid derivatives flowers are pollinated by bats and present and six terpenoids) in T. macropetala, and cor- faintly sweet odor in the early hours of the related their presence to bat-pollination during night, when the nectar volume is the highest a study of floral and reproductive biology. (Aguilar-Rodriguez et al., 2014). This study aims to identify volatile com- These compounds may possess antimi- pounds from flowers of thirteen ornamental spe- crobial and antiherbivore activity, also repel- cies belonging to four genera of Bromeliaceae ling microorganisms and animals or attracting (using headspace solid-phase micro-extraction natural predators, protecting the plant through with detection by gas chromatography-mass tritrophic interactions (Hammer, Carson, & spectrometry), and to bring a new contribution Riley, 2003; Arab & Bento, 2006; Lucas-Bar- revealing the potential that these plants have in bosa, Loon, & Dicke, 2011), which also sug- the industry, in the synthesis of natural prod- gests their involvement in the protection of the ucts, as well as in future studies of ecological reproductive parts of plants during flowering processes involving plant-animal interactions, (Kessler, Halitschke, & Poveda, 2011; Parra- and taxonomy studies from the principal com- Garcés, Caroprese-Araque, Arrieta-Prieto, & ponents analysis. Stashenko, 2010). On the other hand, like all inheritable characters, chemical compounds MATERIALS AND METHODS that make up scents may also reflect the taxo- nomic affinities of a species. The characteriza- Plant material: Entire flowers were tion of volatile compounds may contribute obtained from plants of 13 species belonging to taxonomic and phylogenetic studies con- to four Bromeliaceae genera: Aechmea bicolor sidering that some volatile compounds may L. B. Sm. (ESA 120990), Ae. bromeliifolia be specific to certain plant groups (see for Baker ex Benth. & Hook. f. (ESA 121275), Ae. example Nogueira, Bittrich, Shepherd, Lopes, distichantha Lem. (ESA 121275), Ae. fasciata & Marsaioli, 2001). Baker (ESA 120987), Ae. nudicaulis Griseb. Volatile compounds also have significant (ESA 120991), Ananas macrodontes E. Mor- importance in the food industry, cosmetics, per- ren (ESA 121286), An. comosus (L.) Merr. fumes, chemical and pharmaceutical industries var. bracteatus (Lindl.) Coppens & F. Leal

1102 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1101-1116, September 2016 (ESA 121284), An. comosus (L.) Merr. var. 1 min, increasing at 7 °C per min to 230 °C, erectifolius (L.B.Sm.) Coppens & F. Leal (ESA remaining at 230 °C for 4 min, totaling 44 min 121285), Alcantarea nahoumii (Leme) J.R. of analysis. Helium was used as the carrier gas Grant (ESA 120986), Vriesea friburgensis Mez at linear velocity of 36.1 cm/s. The interface (ESA 121282), V. michaelii W. Weber (ESA was maintained at 280 °C and the detector 121280), V. paraibica Wawra (ESA 121276) operated in the scanning mode (m/z 45-450). and V. simplex Beer (ESA 120989), grown in a Data integration was performed using the Lab- greenhouse, with flowers collected between the Solutions-GCMS Ver. 2.5 software (Shimadzu months of August 2011 and February 2012. All Corp., Kyoto, Japan). species presented anthesis between 6:30 and 8 am. Flowers were collected at anthesis (8 am) Volatile identification and semi-quanti- from the middle part of inflorescences, in three fication: The volatile compounds were identi- replicates, each flower from a different plant. A fied by Wiley 138 and FFNSC libraries. The representative plant of each species was depos- relative abundance of the compounds was ited at the Escola Superior de Agricultura “Luiz calculated based on the MS results. de Queiroz” (ESALQ) University of São Paulo The data were subjected to the multivariate (USP) herbarium. principal components analysis using Statistica (Statsoft, 2004). Solid-phase microextraction: This tech- nique presents a low cost of execution, good RESULTS repeatability, quickness, and is solvent-free. Collected flowers were immediately placed HS-SPME/GC-MS analysis of the vola- in 20 mL capped vials and allowed to equili- tile profile emitted by flowers of 13 species brate for 20 min at 37 for 20 °C. Volatile belonging to four genera of Bromeliaceae compounds were collected from the head- was performed. A total of 80 compounds were space of each sample by solid-phase micro- extracted and 71 compounds were identified. extraction (SPME), using Supelco SPME fibers Nine chemical groups were found in the identi- coated with divinylbenzene-polydimethylsilox- fied compounds: alcohols, terpenoids, alde- ane (DVB/PDMS, 65 μm) during 20 min at hydes, esters, ketones, ethers, furans, oxides, 37 °C. The fiber was then withdrawn into and styrene (Table 1). The compounds iden- the needle and transferred for injection in the tified represented over 97 % of the major GC-MS system with splitless injection mode at components of this fraction. In Ae. bicolor, 240 °C (Almeida, Gonçalves, Galego, Miguel, Ae. bromeliifolia, Ae. distichantha, Ae. fas- & Costa, 2006). ciata, V. friburgensis and the three varieties of Ananas, over 99.45 % of the compounds Gas chromatography and mass spec- were identified, while in V. simplex 90.69 % of trometry: GC-MS analyses were conducted the compounds were identified (Table 1, and according to Custódio, Serra, Nogueira, Gon- supplementary material). çalves, and Romano (2006) with modifica- V. friburgensis showed the highest diver- tions. The analyses of volatile compounds were sity of volatile compounds, with 31 chemi- performed on a gas chromatograph GC 2 010 cals, while Al. nahoumii showed only 14 (Shimadzu Corp., Kyoto, Japan) coupled to a compounds. The three varieties of Ananas mass spectrometer QP 2 010 Plus (Shimadzu contained the same 28 compounds in relatively Corp., Kyoto, Japan). Samples were separated similar abundance, as verified by principal using a capillary column (RTX-5MS 30 m x component analysis (Fig. 1). 0.25 mm x 0.25 µm). The temperature program The group of terpenes showed the greatest started at 40 °C for 2 min, increasing at 4 °C number of compounds and the highest percent- per min to 130 °C, remaining at 130 °C for age in the fraction analyzed for most species,

Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1101-1116, September 2016 1103 , j , f , , j b , , j j i , citrus , nut j b , musty f , musty fruity , herb

j

, j , pine g i h a, j , herb j , oil j , musty b , resin , anise , citrus g g, j , grapefruit a, i f , fruity , camphor , pine j j a, j , turpentine d , green a a, j a , wood , flower , lime b , wood j f j a , lemon , fruity , mint a, j j b g, h a a, i , fruity Aroma descriptor c , resin , flower a , oil e , herb , fruity b , wood a a , citrus , pine , minty j i j b f b , sweet a, f, g, j a , mushroom a a , resin , flower a, f, j a , turpentine , turpentine , turpentine

sweet , resin , pine j

, fruity , flower , herb , green f a , , wood , pine , green , nut , fresh , green e b a j b i j f d a, j a, i g a, b, c a a a j a, f, j a sweet herb - moss lemon sweet camphor vanilla fresh green spice lime lemon grass balsamic cedar sweet - green spicy spice green wood turpentine pine moss grass 0.59 1.57 1.55 0.91 1.58 5.53 4.42 SIM 13.82 0.75 0.72 0.46 9.95 PAR 18.68 21.21 0.54 0.31 2.72 0.89 2.86 MIC FRI 0.33 6.16 1.44 2.07 8.59 3.38 3.00 12.29 6.11 ALC 31.54 0.66 1.69 1.19 7.10 1.78 4.95 2.91 1.35 15.06 MAC Alcohols Terpenes 0.23 2.01 0.98 0.86 1.94 0.58 1.17 2.03 TABLE 1 TABLE ERE 18.09 1.11 0.38 1.83 0.47 5.32 4.57 1.22 BRA 10.29 14.66 Bromeliad species (Peak area %) 1.54 2.68 0.50 0.51 1.31 7.97 2.33 NUD 24.43 15.57 of Bromeliaceae by HS-SPME/GC-MS of Bromeliaceae 9.35 2.89 6.30 2.57 4.33 2.76 6.34 FAS DIS 3.07 1.28 5.97 4.13 1.26 6.80 39.26 5.52 0.72 BRO 24.49 29.68 BIC Percent area of volatile compounds and retention time in a mass spectrum in 13 species in a mass spectrum time compounds and retention of volatile Percent area 1.00 0.93 6.16 9.28 17.81 a (min) 8.22 7.75 7.80

11.07 12.13 17.29 17.64 18.74 19.36 19.73 20.23 13.16 13.33 15.57 32.42 10.44 10.49 12.52 12.67 12.97 10.29 R t A A A A A A A A A A by chemical class -Menthatriene Volatile compounds Volatile p )-Sabinene hydrate )-3-Hexenol )-Non-3-en-1-ol )-3-Hexenol -Menth-2-en-1-ol E Z E Z Phenethyl alcohol p ( Octanol 1-Terpineol (-)-α-Trepineol β-Pinene α-Phellandrene δ-3-Carene ( Cedrol Camphene o-Cymene β-Myrcene Sabinene n-Hexanol α-Thujene 1,3,8- α-Pinene ( ( N 5 6 7 8 9 10 16 20 21 4 11 15 17 18 19 3 12 13 14 1 2

1104 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1101-1116, September 2016 , d , j , j , herb , j j b, g g , sweet j , fruity j , sweet j green

f , , sweet f , parsley wood

, h, j f a, j, k , lemon , fruity citrus j f a, j , minty , green j , muscat a, f, g j a b, f , wood , sweet , citrus j , lavander j a f, j a, j a a Aroma descriptor a fruity , earthy

, citrus , , anisic a a, j , lemon , tea b a, b , spicy j a b j, k j g , plastic j , fruity , fresh a a j , gasoline , turpentine a , herb , spice a, j , lemon , orange , green a, b, g, j , citrus , balsamic , warm , fresh a , spicy , citrus j b j a, b a a a a a, j b d , wax , wax , anisic a a j green sweet solvent - sweet lemon gasoline herb - wood - musty - wood - - - flower pine flower spicy - - herb - wood terpentine - wood - wood 5.56 3.74 0.14 7.85 1.05 2.53 2.27 1.42 5.46 2.15 3.40 3.74 1.32 1.57 SIM 1.24 0.55 4.43 2.73 PAR 0.17 1.41 1.26 MIC 10.23 FRI 6.75 3.83 0.20 0.77 0.12 2.13 1.85 1.12 4.82 1.87 3.52 2.80 1.01 0.62 0.68 0.90 10.47 ) ALC 12.94 30.69 7.55 5.07 0.58 2.70 1.66 2.23 3.90 6.08 1.68 0.79 0.86 0.73 0.86 12.44 MAC Continued 6.32 0.23 2.05 1.33 2.12 3.99 4.69 1.05 0.49 0.69 0.57 0.66 ERE 11.25 18.43 TABLE 1 ( TABLE 5.73 1.39 8.06 0.92 1.97 1.77 2.69 5.04 2.88 2.34 1.30 0.77 0.78 1.00 BRA Bromeliad species (Peak area %) 1.25 8.05 4.46 1.85 9.63 NUD 5.98 2.46 8.96 2.69 1.41 4.86 FAS DIS 2.07 0.56 1.74 0.60 2.20 8.91 10.19 3.02 1.54 BRO BIC 0.53 0.40 2.69 4.52 18.18 21.19 a (min)

14.11 13.70 13.95 14.03 14.04 15.35 27.18 27.74 27.80 28.06 28.08 28.51 28.63 29.14 29.17 29.23 16.37 16.85 17.40 25.32 25.76 26.59 26.62 28.29 28.39 28.87 28.99 R t B C C C C A C C A C C C C A A A A C C C C C C A A C A by chemical class Volatile compounds Volatile )- β-Ocimene )-Caryophyllene )-α-Bergamotene -Cymene E E E α-Terpinene p Sylvestrene ( Limonene γ-Terpinene β-Elemene β-Maaliene α-Gurjunene ( Caryophyllene α-Maaliene Alloaromadendrene α-Selinene Muurola-4(14),5-diene Neo-allo-ocimene Terpinolene Linalool 4,8-dimethylnona-1,3,7-triene Bicycloelemene α-Cubebene Isoledene α-Copaene γ-Maaliene ( Cadina-3,5-diene α-Humulene N 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1101-1116, September 2016 1105 , , j l e , l , j j , soil , fruity e j , soap a, l geranium

, daisy , j , honey , nut f j j , fat a , grape , vegetables , green j , flower j e j , balsamic f , cucumber , sweet , fruity j , orange j i a, b a, e , chocolate , fruity j c a a a Aroma descriptor , melon j i , green , green , b , oil , vegetable a , cherry , grassy j j a , tallow a, e c a e , green a a , herb , lemon j b , wood , bean , wood a , green , orange blossom , peanut m a i , apricot a a j , camphor c , citrus j i a a f , citrus a sweet - - fresh clorin soap butter metallic root incense peach fat balsamic - thyme gerb - apple apple hyacinth - herb wood - - green 5.26 4.05 1.02 1.83 6.36 SIM 0.51 0.93 0.82 0.93 PAR 10.02 10.77 0.4 0.51 0.35 0.77 0.93 1.32 MIC 63.13 FRI 1.52 5.58 3.08 0.88 1.83 4.98 ) 2.09 0.54 0.15 1.28 4.22 ALC 5.16 2.33 0.76 1.93 5.52 MAC Continued Furan Esters Ketone 5.37 2.79 0.81 1.45 5.35 ERE TABLE 1 ( TABLE 6.08 4.37 1.62 2.39 8.50 BRA Bromeliad species (Peak area %) 0.52 1.03 0.56 0.84 1.18 1.49 0.78 1.50 NUD 0.83 1.96 1.19 2.48 5.01 2.55 5.00 FAS DIS 0.23 2.77 0.42 0.70 0.59 0.98 1.01 0.20 0.92 0.98 0.61 3.41 8.17 BRO BIC 0.55 0.53 1.62 0.70 1.68 1.00 2.38 0.75 2.30 3.17 a (min) 6.09

32.11 20.61 36.85 20.29 14.02 12.66 18.94 14.66 16.91 29.48 29.79 30.01 30.23 30.30 30.71 30.91 31.79 30.04 30.06 30.19 R t C C C C C C C C C C D by chemical class Volatile compounds Volatile )-Non-2-enal -Nonanal -Hexanal E Lyrantion Diisobutyl phthalate Gaultheric acid Eucalyptol Decanal 2-Pentyl-furan n ( β-Bisabolene β-Curcumene δ-Cadinene α-Cubenene Squalene n Phenylacetaldehyde Cadina-1(6),4-diene β-Selinene δ-Elemene Selina-3,7(11)-diene Viridiflorene γ-Elemene N 65 66 67 Ether 68 64 69 62 63 55 56 57 58 59 Aldehydes 60 61 49 50 51 52 53 54

1106 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1101-1116, September 2016 var. var. comosus

k A = monoterpene; A Ananas ------, penetrating d V. simplex; V. ; BRA = ; BRA a, k Aroma descriptor , citrus d ; SIM = , gasoline nahoumii

b, k , resin a V. paraibica V. flower balsamic Alcantarea SIM ; PAR = ; PAR ; ALC = 2.20 1.84 2.89 0.62 0.37 PAR 0.22 2.05 2.35 0.61 MIC V. michaelii V. nudicaulis . FRI Ae ; MIC = ) 0.99 ALC ; NUD = 0.23 1.16 1.12 0.27 MAC Continued Oxide fasciata Styrene . Unknown ERE Ae Vriesea friburgensis Vriesea TABLE 1 ( TABLE BRA ; FAS = ; FAS ; FRI = Bromeliad species (Peak area %) 1.43 0.30 NUD 4.45 4.07 1.42 1.26 5.66 FAS distichantha . DIS 0.42 5.00 0.44 0.12 Ae An. macrodontes 1.32 1.32 1.24 0.85 0.38 BRO 13.24 ; DIS = ; MAC = BIC 2.00 0.17 0.23 a (min) 8.94 6.82

16.42 12.92 12.95 15.58 18.83 21.94 24.76 28.92 29.87 R erectifolius t Ae bromeliifolia var. var. ; BRO = comosus . An by chemical class Volatile compounds Volatile ; ERE = )-Linalool oxide Aechmea bicolor Z ( Styrene Unknown 1 Unknown 2 Unknown 3 Unknown 4 Unknown 5 Unknown 6 Unknown 7 Unknown 8 Unknown 9 N 70 71 72 73 74 75 76 77 78 79 80 BIC = bracteatus B = homoterpene; C = sesquiterpene; D = triterpene. C = sesquiterpene; B = homoterpene; (2004); Lee & Leem, Kim, Kim, Zheng, = e (2006); Sawamura & Choi, Nishiyama, d = Phi, (2006); Quian Fan, & = c (2005); al. et Mahattanatawee b = Arn (2004); Acree, & = a Yoon & Adhikari, Chambers, al. (2009); k = Lee, (2007); j = Formisano et (2013); i = Brechbill et al. al. (2013); h = Bordiga f = Qiao et al. (2008); g Cuevas-Glory et al. (2013). Adams, & Lin (2012); m = Culleré Hossain, Perry, Wang, (2013); l =

Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1101-1116, September 2016 1107 8

6

4

2

0 Factor 3: 12.84% 3: Factor -2

-4

6 4 2 0 Factor 2: 19.17% -2 -4 -6 -8 Factor 1: 30.78% -10 -12 6 4 2 0 -2 -4 -6 -8 -14

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. such as Ae. bicolor (74.92 %), Ae. distichantha abundance ranging from 6.12 % to 59.69 % in (77.54 %) and V. friburgensis (71.27 %). Among V. michaelli and Ae. bromeliifolia, respectively. the Ananas varieties, terpenes corresponded to Among them, the most abundant were n-hexa- 69.01 %, representing 66.21 % (A. comosus nol, (Z)-hex-3-enol, (E)-sabinene hydrate and var. bracteatus), and 70.97 % (A. comosus var. 1-terpineol (Table 1). n-Hexanol was identified erectifolius) of the total composition. The com- in high abundance in most species studied, par- pound β-myrcene was observed in all species ticularly Al. nahoumii (31.54 %), Ae. bromeli- except Al. nahoumii, with abundance ranging foliia (29.68 %) and V. paraibica (21.21 %). from 0.31 to 39.26 % in V. michaelii, and Ae. Five aldehydes were identified among distichantha, respectively. Linalool was only the compounds: n-hexanal, phenylacetalde- observed in Al. nahoumii, with approximately hyde, n-nonanal, (E)-non-2-enal and decanal, 12.94 % of the total compounds found (Table 1, the last three being found in all species of Fig. 2B, and supplementary material). Aechmea analyzed. Phenylacetaldehyde was Eleven compounds belonging to the observed in Ae. bromeliifolia (8.17 %), and class of alcohols were identified, with a total V. michaelli (1.32 %).

1108 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1101-1116, September 2016 Fig. 2. Chromatographic profile obtained by HS-SPME/GC-MS of volatile compounds in flowers of Bromeliaceae, indicating the major compounds identified: A) Aechmea bicolor, B) Alcantarea nahoumii, C) Ananas comosus var. erectifolius, D) Vriesea friburgensis.

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 rang- were below 2 % (Table 1). Eucalyptol, an ether, ing from 5.16 to 6.08 % (Table 1).

Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1101-1116, September 2016 1109 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

Total Accumulated Accumulated Principal components Eigenvalue variance % eigenvalue percentage PC 1: eucalyptol; alloaromadendrene; cadina-1(6),4-diene 25.2458 30.7876 25.2458 30.7876 PC 2: cadina-3,5-diene; unknown 7; muurola-4(14),5-diene 15.7234 19.1749 40.9693 49.9625 PC 3: α-humulene; (E)-α- bergamotene; β-bisabolene 10.5336 12.8459 51.5029 62.8084 PC 4 6.3267 7.7155 57.8296 70.5239 PC 5 5.1625 6.2958 62.9922 76.8198

The principal component analysis based alcohols, terpenoids, aldehydes, esters, ketones, on the volatile compounds of Bromeliaceae ethers, furans, oxides, and styrene. flowers from 13 species showed the first three The presence of terpenoids in plants is components retaining 62.81 % of the initial associated with defense against herbivores, information (Table 2). This total variance is pathogenicity, and allelopathy to attract polli- considered high, with high heterogeneity of the nators (Langenheim, 1994). Terpenes are asso- samples’ chemical composition. ciated with various fragrances, making them The correlation of each component and widely used to make perfumes and flavorings the variables allowed for the evaluation of the (Bauer, Garbe, & Surburg, 2001). discriminatory power of this analysis. For PC1 The compound β-myrcene has been (30.78 %), the variables that mostly influenced described in more than 200 plant species as the separation of accessions based on volatile being responsible for green, herb, pine, lemon, compounds were the presence of eucalyptol, grapefruit, musty and spicy scents (Mahattana- alloaromadendren, and cadina-1(6),4-diene, tawee, Rouseff, Filomena, & Naim, 2005; Qiao with correlation values of 0.99, 0.98 and 0.98, et al., 2008). It is widely used in the cosmetic respectively. For PC2 (19.17 %), the variables and pharmaceutical industries, as described by with highest influence were cadina-3,5-diene, Behr and Johnen (2009). Linalool has been unknown 7 and muurola-4(14),5-diene, with observed in tangerines and is responsible for their taste and aroma (Sawamura, Minhtu, Oni- correlation values of 0.96, 0.93 and 0.93, shi, Ogawa, & Choi, 2004). respectively. For PC3 (12.85 %), the most sig- Two other compounds observed in high nificant variables were α-humulene, (E)-α- ber- abundance in most species studied here were: gamotene and β-bisabolene, with correlations γ-terpinene, which has a citrusy, sweet (Acree of 0.76, 0.75 and 0.75, respectively. & Arn, 2004), gasoline and turpentine scent The dispersion diagram of the scores of the (Qiao et al., 2008); and α-humulene, which has first three main components (Fig. 1) showed a woody (Acree & Arn, 2004) and fresh scent three groups, and two isolated species, demon- (Cuevas-Glory, Ortiz-Vezquez, Sauri-Duch, & strating the variability of volatile compounds Pino, 2013) and has been shown to have anti- among these bromeliad species. inflammatory properties. It is also found in the essential oil of Cordia verbenacea DC (Boragi- DISCUSSION naceae) (Fernandes et al., 2007). Aldehydes have strong odors that recall This study demonstrated great variabil- citrus fruits, roses and fresh cut grass, being ity of volatile compounds among the brome- widely used in perfumery (Bauer et al., 2001). liad species, with 71 compounds identified Phenylacetaldehyde is an aromatic compound belonging to eight chemical groups, including also found in Fagopyrum esculentum Moench

1110 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1101-1116, September 2016 (Polygonaceae) (Janes, Kantar, Kreft, & Prosen, demonstrate the effect of volatiles in the attrac- 2009) and several species of flowers (Robert & tion of pollinators (Dobson, 1994). Meagher, 2002). This compound has long been The dispersion diagram of the first three used to attract various species of moths in traps main components scores showed V. michaelli, for biological control (Robert, & Meagher, V. paraibica and Al. nahoumii forming the 2002; Smith, Allen, & Nelson, 1943; Cantelo, first group. These species are phylogenetically & Jacobson, 1979), and has a floral/honey odor close, belonging to the subfamily Tillandsioi- (Whetstine, Cadwallader, & Drake, 2005). deae (Barfuss, Samuel, Till, & Stuessy, 2005; High abundance of n-hexanol has also Givnish et al., 2011; Versieux et al., 2012), been observed in plum fruits, Prunus domes- and Alcantarea traditionally being either con- tica L. (Rosaceae) (Gomez, Ledbetter, & sidered as a subgenus of Vriesea, or a genus Hartsell, 1993), Caralluma europaea (Guss.) itself, both belonging to the tribe Vrieseeae N.E.Br. (Apocynaceae) flowers (Formisano (Grant, 1995). Floral morphology is one of the et al., 2009), Camellia sinensis (L.) Kuntze factors that influence the pollination syndrome (Theaceae) (Han, Zhou, Cui, & Fu, 2006) and (Aguilar-Rodríguez et al., 2014), these species essential oil of Pimenta guatemalensis (Lun- present large flowers with yellow petals, and dell) Lundell (Chaverri & Cicció, 2015). Jabal- a tubular corolla. Similarities in composition purwala, Smoot and Rouseff (2009), studying of the flower volatiles produced among these volatile compounds in flowers of different species can be another factor to consider in species of Citrus, also observed high levels establishing the relationship of these species. of n-hexanol in Citrus grandis (L.) Osbeck Ae. bicolor, Ae. distichantha, and Ae. nudi- (Rutaceae), and correlated the levels with the caulis belong to the Bromelioideae subfamily, pollination by bees, which is crucial for repro- with similar flower morphological character- duction due to self-incompatibility. istics. They formed a second group accord- Gaultheric acid belongs to the class of ing to their volatile compound composition. ketones and can be found in wines and plant Faria, Wend and Brown (2004), studying the species such as Gaultheria itoana Hayata (Eri- cladistic relationships of this genus, showed caceae) (Chen et al., 2009). It is also abundant that Ae. distichantha and Ae. nudicaulis are in the root bark of Securidaca longepeduncu- very close in clade distribution. In addition, lata Fresen (Polygalaceae), exerting a biocide there are reports of common pollinators for effect against insects that feed on stored grains these two species (Schmid, Schmid, Zillikens, (Lognay, Marlier, Seck, & Haubruge, 2000). Harter-Marques, & Steiner, 2010; Scrok & The emission of volatile compounds with bio- Varassin, 2011). There are very limited studies cide effect can be related to pollination, exert- of Ae. bicolor, however morphological similar- ing a repellent effect on some insect species. ity is observed between its inflorescence and Eucalyptol was also identified among the flower morphological characteristics and those volatile compounds in African cycad (Enceph- of Ae. nudicaulis. alartos) (Zamiaceae) flowers (Suinyuy et al., The dispersion diagram showed the two 2013). Furans and oxides were present in a few other Aechmea species studied, Ae. fasciata and species studied, but in low amounts. Ae. bromeliifolia, isolated in the principal com- Special patterns of scent in flowers can ponent analysis of the volatile compounds. This function as the same visual patterns, so dif- demonstrates the considerable variability of ferences in intensity and types of volatile flower volatile compounds among this genus. compounds emitted, besides serving as guides The third group included An. macrodontes, for insects, help in the search for food rewards. An. comosus var. erectifolius and An. comosus A combination of chemical analyses of floral var. bracteatus and two species of Vriesea scents with field observations of the behav- (V. simplex and V. friburgensis). For Ananas, ior of flower visitors is an effective way to this grouping supports the morphological and

Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1101-1116, September 2016 1111 taxonomic closeness within the genus, con- the volatile compound dynamics throughout the sidering that the same 28 compounds were 24 h in which the flowers were open, observing observed in Ananas flowers at similar values. a variation of these compounds, thus enabling a Considering that the flower volatile com- greater diversity of pollinators, and thus ensur- pound spectrum can be a plant strategy to ing the reproductive success of the species. In attract pollinators, the two species of Vriesea our study, the flowers were collected at anthe- may present pollination syndrome similarities sis, which occurred between 6:30 and 8 a.m. for with the genus Ananas, thus being grouped all species. Further experiments on the volatile together in the principal component analy- compounds dynamics throughout the day may sis. Hummingbirds are common pollinators be interesting for pollination attraction studies between these two genus, which can explain in these species. the proximity of these species in the PCA We identified 71 different volatile com- results. Regarding Ananas ananassoides, Stah, pounds, some of them having significant impor- Nepi, Galetto, Guimarães, and Machado (2012) tance in the food, cosmetic, perfume, chemical observed the presence of two Trochilidae spe- and pharmaceutical industries. The variation in cies, Hylocharis chrysura and Thalarania the odor profile observed of Bromeliaceae in glaucopis, the latest also observed by Schmid this study shows complex variability. Current et al. (2011) in V. friburgensis. taxonomy and pollination syndrome studies One of the reports of volatile compounds can adequately explain the variation in volatile in bromeliads refers to T. macropetala, in compounds among species. Characterization of which Aguilar-Rodríguez et al. (2014) identi- these compounds in Bromeliaceae may clarify fied nine volatile compounds, two of them some problems in taxonomy. Further studies similar to those found in this study, namely using more species from different genera and nonanal in the species Aechmea, Al. nahoumii detailed morphological information and vola- and V. michaelii, and limonene in Ae. nudicau- tile composition associated with their pollina- lis. Those authors pointed out that the pollina- tors can clarify the attraction of pollinators by tion syndrome is not necessarily related to a specific odor compounds. single compound, such as dimethyl disulphide, which although absent in T. macropetala, did ACKNOWLEDGMENTS not prevent the visit of pollinating bats. In this case, of the nine compounds identified The authors acknowledge Fundação by the researchers, six are also present in de Amparo à Pesquisa do Estado de São other species pollinated by bats. In Werauhia Paulo, FAPESP (2009/18255-0), and Con- gladiolifora, also pollinated by bats, Bestmann, selho Nacional de Desenvolvimento Científico Winkle, and Helversen (1997) observed 12 e Tecnológico, CNPq (476.131/2008-1), for volatile compounds, five of them common to financial support. APM also acknowledges those observed in the species of the present CNPq for research fellowships (305.785/2008- work (α-Pinene, β-Pinene, 4,8-dimethyl-1,3,7- 7 and 310.612/2011-0). nonatriene, β-Myrcene, Limonene, α-Copaene) and two common to T. macropetala (β-Pinene e Limonene) a species pollinated by bats (Agu- RESUMEN ilar-Rodríguez et al., 2014). Perfil de compuestos volátiles de las flores en las Finally, it is important to highlight that the Bromeliaceae. Los compuestos volátiles tienen un papel aroma composition varies throughout the day vital en el ciclo de vida de las plantas. Poseen actividad and this issue is important in attracting pol- antimicrobiana y anti-herbivoría biológica y una gran importancia en la industria de alimentos, cosméticos, per- linators (Balao, Herrera, Talavera, & Dötterl, fumes, productos químicos y farmacéuticos. Este estudio 2011; Aguilar-Rodríguez et al., 2014). Dötterl, tuvo como objetivo identificar los compuestos volátiles de Jahreb, Jhumur, and Jürgens (2012) evaluated trece flores de especies, pertenecientes a cuatro géneros

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