Extraction and Identification of Volatile Organic Compounds Emitted By

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Article Extraction and Identiﬁcation of Volatile Organic Compounds Emitted by Fragrant Flowers of Three Tillandsia Species by HS-SPME/GC-MS

Mame-Marietou Lo 1, Zohra Benfodda 1 , David Bénimélis 1 , Jean-Xavier Fontaine 2, Roland Molinié 2 and Patrick Meffre 1,*

1 UNIV. NIMES, UPR CHROME, CEDEX 1, F-30021 Nîmes, France; [email protected] (M.-M.L.); [email protected] (Z.B.); [email protected] (D.B.) 2 UMR INRAE 1158 Transfrontaliére BioEcoAgro, BIOlogie des Plantes et Innovation (BIOPI), UPJV, UFR de Pharmacie, 80037 Amiens, France; [email protected] (J.-X.F.); [email protected] (R.M.) * Correspondence: [email protected]

Abstract: Numerous volatile organic compounds (VOCs) with a large chemical diversity are emitted by plant flowers. They play an important role in the ecology of plants, such as pollination, defense, adaptation to their environment, and communication with other organisms. The Tillandsia genus belongs to the Bromeliaceae family, and most of them are epiphytes. The aromatic profile of the Tillandsia genus is scarcely described. In this study, we use the headspace solid phase microextraction (HS-SPME) coupled with gas chromatography combined with mass spectrometry (GC-MS) method developed in our laboratory to explore the chemical diversity of the VOCs of fragrant flowers Citation: Lo, M.-M.; Benfodda, Z.; of three species of the genus Tillandsia. We were able to identify, for the first time, 66 volatile Bénimélis, D.; Fontaine, J.-X.; Molinié, compounds (monoterpenes, sesquiterpenes, phenylpropanoids, and other compounds). We identified R.; Meffre, P. Extraction and 30 compounds in T. xiphioides, 47 compounds in T. crocata, and 43 compounds in T. caliginosa. Only Identification of Volatile Organic seven compounds are present in all the species studied. Comparison of the volatile compounds Compounds Emitted by Fragrant profiles by principal component analysis (PCA) between T. xiphoides, T. crocata, and T. caliginosa Flowers of Three Tillandsia Species by species showed a clear difference in the floral emissions of the studied species. Moreover, floral VOCs HS-SPME/GC-MS. Metabolites 2021, profiles allowed to differentiate two forms of T. xiphioides and of T. crocata. 11, 594. https://doi.org/10.3390/ metabo11090594 Keywords: Tillandsia xiphioides; Tillandsia crocata; Tillandsia caliginosa; headspace solid phase microextraction (HS-SPME); gas chromatography-mass spectrometry (GC-MS); volatile organic Academic Editor: Hirokazu Kawagishi compounds (VOCs); terpenoids; phenylpropanoids; PCA analysis; heatmap

Received: 14 July 2021 Accepted: 30 August 2021 Published: 2 September 2021 1. Introduction Publisher’s Note: MDPI stays neutral A wide variety of Volatile Organic Compounds (VOCs) are produced and emitted with regard to jurisdictional claims in by plants, especially in organs such as flowers, leaves, fruits, and roots. More than published maps and institutional affil- 1700 volatile compounds have been identified and can be classified in the following cat- iations. egories: terpenoids, phenylpropanoids, fatty acid and amino acid derivatives, various compounds containing sulfur, as well as furanocoumarins and their derivatives [1,2]. The volatile compounds emitted by flowers generally have a low molecular weight and differ significantly according to the species studied. They play an important role in attracting Copyright: © 2021 by the authors. pollinators, in defense against herbivores, in adaptation to environmental stress, and in Licensee MDPI, Basel, Switzerland. communication between flowering plants [3,4]. Floral scent therefore not only has a role in This article is an open access article several eco-physiological processes, but also enhances the aesthetic character of ornamental distributed under the terms and plants and is widely used in the perfume and fragrance industry, in the food industry, conditions of the Creative Commons and in cosmetics [5]. For all these reasons, the number of scientific studies carried out on Attribution (CC BY) license (https:// floral scent has increased in recent years, motivated by the discovery of new notes useful creativecommons.org/licenses/by/ in perfumery and by the deepening of knowledge in plant physiology [6]. 4.0/).

Metabolites 2021, 11, 594. https://doi.org/10.3390/metabo11090594 https://www.mdpi.com/journal/metabolites Metabolites 2021, 11, 594 2 of 14

The main analytical method used in the study of VOCs is gas chromatography combined with a mass spectrometer (GC-MS). The analysis of volatile profiles of plants remains a relevant method due to its simplicity and the considerable contribution of data, which allows to differentiate species between them as well as individuals within the same species [5]. Regarding isolation methods for volatile compounds, they can be divided into three cate- gories: steam distillation, solvent extraction, and headspace trapping [7]. Steam distillation and solvent extraction are time-consuming methods requiring the use of large quantities of solvents and can lead to the formation of artifacts either by isolating non-volatile matter from the tissue or by heat-produced re-arrangement [8]. Therefore, soft extraction methods such as headspace trapping are increasingly being used. Headspace solid phase microextraction (HS-SPME) is a widely used method for the extraction of volatile compounds from flowers [9]. It allows the extraction, concentration, and introduction of analytes into the GC inlet in a single step. It is a fast technique, which does not require the use of organic solvents and is characterized by a reduced operating cost due to the reuse of extraction fibers [10]. In our previous study the HS-SPME/GC-MS method was developed to study the VOCs emitted by the flowers of a Tillandsia species. Following this, two methods were selected and allowed the identification of 30 volatile compounds from the flowers of Tillandsia xiphioides Ker Gawler [11]. For the present study, these two methods were used to identify volatile compounds emitted by the flowers of Tillandsia crocata and Tillandsia caliginosa. The Tillandsia species belong to the Bromeliaceae family. Most of them are epiphytic. The genus Tillandsia has a very long history of use as a medicinal plant, especially in traditional medicine [12]. Its main chemical constituents belong to several groups, such as flavonoids, triterpenoids, sterols, and phenylpropanoids, which are known to have various important biological activities [12,13]. However, the volatile composition of Tillandsia has been very little studied, although some species have very fragrant flowers. Among these species, T. crocata Baker has a short stem, distichous leaves quickly forming dense clumps [14] with small yellow or orange flowers. It is known for its very intense floral scent that attracts male Euglossine bees. For this reason, a study was carried out in 1991 to determine the composition of the floral scent of T. crocata, which led to the identification of seven volatile compounds [15]. Concerning T. caliginosa, it is a plant with a single stem, sometimes branched, carrying long, succulent leaves covered with grayish white scales. Its small but very odorous flowers have a dark brown color. To our knowledge, no study has yet been done on the composition of the VOCs of T. caliginosa. The objective of this study is to identify, using the HS-SPME/GC-MS method, the VOCs of fragrant flowers of three species of the genus Tillandsia with different colors and flower shapes. The flowers of these species are presented in Table1, these are flowers of two forms of T. xiphoides noted low pubescent xiphioides (LP-xiphi) and high pubescent xiphioides (HP-xiphi), respectively, due to a low and high presence of trichomes on the leaves of these plants; two forms of T. crocata are yellow crocata and orange crocata according to the color of the flower, and one form of T. caliginosa presents flowers of dark brown color. A principal component analysis (PCA) was performed to profile the variations of compounds of the different species T. xiphioides, T. crocata, and T. caliginosa. Having shown relatively similar profiles, a second PCA as well as a heatmap were performed to better visualize the differences in profiles of T. crocata and T. caliginosa. This study is the first to compare floral volatile profiles between fragrant Tillandsia species and provides additional information on the poorly described floral metabolic network of the genus Tillandsia. Metabolites 2021, 11, 594 3 of 14 MetabolitesMetabolites 2021, 11 2021, x FOR, 11, PEERx FOR REVIEW PEER REVIEW 3 of 14 3 of 14 Metabolites MetabolitesMetabolites 2021 2021, 202111, ,x 11, FOR11, x, xFOR PEERFOR PEER PEER REVIEW REVIEW REVIEW 3 of 314 3of of 14 14

Table 1. Characteristics of the ﬂowers of three species of the genus Tillandsia used in this study. TableTableTableTable 1. Table1. Characteristics Characteristics 1. 1. Characteristics 1.Characteristics Characteristics of of the theof of theflowers flowers ofthe theflowers flowers flowers of of three threeof of three ofthree species species three species species speciesof of the theof of thegenus genus ofthe thegenus genus TillandsiagenusTillandsia Tillandsia Tillandsia Tillandsia used used used usedin in used this thisin in this study instudythis this study study. . study. . . Name LP-Xiphi HP-Xiphi Yellow crocata Orange crocata T. caliginosa NameNameNameName Name LPLP-LP-XiphiXiphiLP-LPXiphi-Xiphi- Xiphi HPHPHP--XiphiHPXiphiHP-Xiphi-Xiphi- Xiphi YellowYellowYellowYellowYellow crocata crocata crocata crocata crocata Orange Orange OrangeOrangeOrange crocata crocata crocata crocata crocata T.T. caliginosa caliginosaT.T. caliginosaT. caliginosa caliginosa

HeightHeightHeight * (mm) * (mm)* (mm) 50 5050 50 5050 20 2020 15 1515 15 1515 HeightHeightHeight * (mm) * (mm) 5050 50 50 50 50 20 2020 15 15 15 15 15 15 BreadthBreadthBreadthBreadthBreadth * * (mm) (mm) * *(mm) (mm)* (mm) 2020 2020 20 2020 2020 20 55 5 5 5 55 5 5 5 55 5 5 5 Breadth * OdorOdor Odorlevel level level Scented20ScentedScented Scented 20ScentedScented ScentedScented 5Scented ScentedScentedScented 5 ScentedScentedScented 5 Odor(mm)Odor level level ScentedScented ScentedScented ScentedScented ScentedScented ScentedScented ** = = average *average *= =average* average= average values values values values. values. . . . Odor level Scented Scented Scented Scented Scented AA principal principalAA principal Aprincipal principal component component component component component* = analysis analysis average analysis analysis analysis values. (PCA) (PCA) (PCA) (PCA) (PCA)was was was performedwas performed was performed performed performed to to profile profileto to profile to profile profile the the thevariations variationsthe thevariations variations variations of of com- com-of of com- of com- com- poundspoundspoundspoundspounds of of the theof of the different of differentthe thedifferent different different species species species species species T. T. xiphioides xiphioidesT. T. xiphioides T.xiphioides xiphioides, ,T. T. ,crocata crocataT., T. ,crocata T.crocata ,crocata ,and and, and, T. andT., andcaliginosa caliginosaT. T. caliginosa T. caliginosa caliginosa. .Having Having. Having. Having. Having shown shown shown shown shown rela- rela- rela- rela- rela- tivelytively2.tivelytively Resultssimilar tivelysimilar similar similar similar profiles, profiles, profiles, profiles, profiles, a a second second a asecond seconda second PCA PCA PCA PCA as as PCA well wellas as well as wellas as well a aas heatmap asheatmap a as aheatmap heatmapa heatmap were were were were performed performed were performed performed performed to to better betterto to better to better visualize bettervisualize visualize visualize visualize thethe 2.1.the differencesdifferencesthe thedifferences Identificationdifferences differences inin profilesinprofiles in profilesin profiles profiles ofof T.ofT. of crocata T.crocataof T. crocata T.crocata crocata andand and T.andT. and caliginosaT.caliginosa T. caliginosa T.caliginosa caliginosa. . ThisThis. .This This study.study This study study isstudyis the theis is the firstisfirstthe thefirst first toto first comparetocompare to compareto compare compare floralfloral floral volatile volatile volatile profi profiles profi betweenlesles between between fragrant fragrant fragrant Tillandsia Tillandsia Tillandsia species species species and and provides and provides provides additional additional additional infor- infor- infor- floralfloral volatileCAR/PDMS volatile profi profiles fiberles between between with fragrant optimal fragrant Tillandsia extraction Tillandsia species conditionsspecies and and provides was provides used additional to additional study infor- the infor- volatile mationmationmation on the on on poorlythe the poorly poorly described described described floral floral floralmetabolic metabolic metabolic network network network of the of of genusthe the genus genus Tillandsia. Tillandsia. Tillandsia. mationcompoundsmation on theon thepoorly emittedpoorly described described from floral the floral flowers metabolic metabolic of network three networkTillandsia of the of thegenusspecies. genus Tillandsia. Tillandsia. Through the use of two HS-SPME/GC-MS methods, a total of 66 compounds were identified from the floral 2.2. Results Results2.2. Results 2.Results Results emissions of LP-xiphi, HP-xiphi, yellow crocata, orange crocata, and T. caliginosa (Table2, 2.1. Identification 2.1.Figure2.1. Identification2.1.2.1. Identification Identification 1Identification). Among these compounds, only seven— β-myrcene (6), limonene (8), eucalyptol (9),CAR/CAR/αCAR/-terpineolCAR/PDMSPDMSCAR/PDMSPDMSPDMS fiberfiber (15),fiber fiber with withfiber geranylwith with optimal optimalwith optimal optimal optimal acetate extractionextraction extraction extraction (18),extraction conditionsconditionsβ -farneseneconditions conditions conditions waswas was (23), was usedused was used andused to toused studytostudy αto -farnesene studyto study thestudythe the volatilevolatilethe thevolatile (24)—arevolatile volatile compoundscompoundspresentcompoundscompoundscompoundsin emittedemitted all emitted emitted the emitted fromfrom species from from the thefrom the studied. flowersflowersthe theflowers flowers flowers ofof The threeofthree of threecompoundsof three TillandsiathreeTillandsia Tillandsia Tillandsia Tillandsia species.species. were species. species. species. categorized ThroughThrough Through Through Through thethe into the useusethe the fouruse ofuseof use twooftwo differentof twoof two two HSHS-classes:HS-SPME/GCSPME/GCHS-HSSPME/GC-SPME/GC-SPME/GC monoterpenes,--MSMS-MS -methods, MSmethods,-MS methods, methods, methods, sesquiterpenes, a a total total a atotal total aof of total 66 66of of compounds66 compoundsof 66 phenylpropanoids, compounds66 compounds compounds were were were were identified identifiedwere identified identified and identified other from from from from compoundsthe thefrom thefloral floralthe thefloral floral emis-floralemis- including emis- emis- emis- sionssionsnitrogenoussionssions of ofsions LP LPof of- LP-xiphi, of xiphi,LP- LPxiphi,-xiphi, compounds,- HP xiphi,HP HP--xiphi, HPxiphi, HP-xiphi,-xiphi,- yellow xiphi,yellow esters, yellow yellow yellow crocata crocata andcrocata crocata crocata, ,fattyorange orange, orange, orange acid, orange crocata crocata derivativescrocata crocata crocata, ,and and, and, T. andT., andcaliginosa caliginosaT. (‘others’ T. caliginosa T. caliginosa caliginosa (Table (Table family). (Table (Table (Table2 2, ,Figure Figure2 Qualitative ,2 Figure, 2Figure, Figure 1). Among these compounds, only seven—β-myrcene (6), limonene (8), eucalyptol (9), α- 1). Amongdifferences1).1). Among1). Among Among these these inthese compounds, these the compounds, compounds, profile compounds, of only flower only onlyseven only seven volatilesseven— sevenβ—-myrcene—β—-β ofmyrcene-βmyrcene- thesemyrcene (6), species(6), limonene (6), (6), limonene limonene limonene were (8), observed.(8), eucalyptol (8), (8), eucalyptol eucalyptol eucalyptol (9), (9), α (9),- (9),α α- -α- terpineolterpineolterpineolterpineolterpineol (15), (15), (15), (15),geranyl geranyl (15), geranyl geranyl geranyl acetate acetate acetate acetate acetate (18), (18), (18), (18),β β -(18),-farnesene farneseneβ -βfarnesene- βfarnesene-farnesene (23) (23) (23), ,(23)and and (23), and, α andα, -and-farnesene farneseneα α-farnesene -αfarnesene-farnesene (24) (24) (24)— —(24) are(24)are—— arepre —preare arepresent sentpre present sentin insent in in in Table 2. Identificationallall the allthe ofall theall volatilespecies speciesthe thespecies species species compoundsstudied studied studied studied studied. .The The. fromThe. compounds Thecompounds. The compounds floral compounds compounds emissions were were were were categorized categorized ofwere T.categorized categorized xiphioides categorized into into, T.into fourinto crocatafour into four fourdifferent different four, anddifferent different differentT. caliginosaclasses:classes: classes: classes: classes: mon- .mon- mon- mon- mon- oterpenes,oterpenes,oterpenes,oterpenes,oterpenes, sesquiterpenes,sesquiterpenes, sesquiterpenes, sesquiterpenes, sesquiterpenes, phenylpropanoidsphenylpropanoids phenylpropanoids phenylpropanoids phenylpropanoids, , andand, ,and andother,other and other other compoundsothercompounds compounds compounds compounds includingincluding including including including nitroge-nitroge- nitroge- nitroge- nitroge- nousnous compounds,nous compounds, compounds, esters esters ,esters and, and,fatty and fatty acidfatty acid derivativesacid derivatives derivatives (‘otherComposition (‘other (‘others’ family)s’ s’family) (%)family). Qualitat. Qualitat. Qualitative differ-iveive differ- differ- Compoundnousnous compounds, compounds, esters esters, and, and fatty fatty acid acid derivatives derivatives (‘other (‘others’ family)s’ family). Qualitat. Qualitative ivediffer- differ- # Family ences in the profile ofRT flower (min) volatilesYellow of theseOrange species wereT. Caliginosaobserved. encesencesences inences the in in theprofilein the theprofile profile profile of flower of of flower of flower flower volatiles volatiles crocatavolatiles volatiles of these of of these of these crocataspecies these species species species were were were observed. were observed. observed. observed. LP-Xiphi HP-Xiphi 1 M α-thujene b, d 14.15 nd 0.2 nd 0.3 nd TableTableTableTable 2. Table2. Identification Identification 2. 2. Identification 2.Identification Identification of of volatile volatileof of volatile ofvolatile volatile compounds compounds compounds compounds compounds from from from fromfloral floral from floral floral emissions emissionsfloral emissions emissions emissions of of T. T.of ofxiphioides xiphioidesT. ofT. xiphioides T.xiphioides xiphioides, ,T. T. ,crocata crocataT., T. ,crocata T.crocata, crocata,and and, and, T. andT., caliginosa and caliginosaT. T. caliginosa T.caliginosa caliginosa. . . . . 2 M α-pinene c, d 14.46 0.3 0.3 0.3 0.5 nd 3 M Sabinene c, d 16.05 0.2Composition 0.3Composition (%) 0.4 (%) 0.3 nd RT CompositionCompositionComposition (%) (%) (%) 4 M β-phellandrene b, d RT RTRT16.23 RT 0.3 0.5 0.6 0.5 nd ## # #FamilyFamily # FamilyFamilyFamily CompoundCompoundCompoundCompoundCompound YellowYellowYellow Orange OrangeOrange cro- cro- cro- 5 M β c, d (min)16.46 (min)YellowYellow Orange ndOrange cro- cro-0.2T. Caliginosa 0.4 LP-Xiphi 0.3HP-Xiphind -pinene (min)(min)(min) crocata cata T. CaliginosaT.T. CaliginosaT. Caliginosa Caliginosa LP -LP XiphiLP-LPXiphi-Xiphi- XiphiHP HP- XiphiHPHP-Xiphi-Xiphi- Xiphi 6 M β-myrcene c, d 16.78crocatacrocatacrocatacrocata 0.5 cata cata catacata 1.2 1.6 4.7 6.6 b, d 11 17 1 1M M MM MM αα-α-thujenethujene-phellandreneαα-thujene-αthujene-thujene b, d b, b,d d b, d d 14.1514.1514.1514.15 17.51 14.15 ndnd nd nd nd nd 0.20.2 0.2 0.2 0.2 nd ndnd nd nd nd nd 0.30.3 0.3 0.3 0.30.7 ndnd nd nd ndnd c, d c, d 22 28 2 2M M MM MM αα--pinenepineneαLimoneneα-pinene-αpinene-pinene c, d c, dc, d c, d 14.4614.4614.4614.46 18.52 14.46 0.30.3 0.3 0.3 0.3 6.4 0.30.3 0.3 0.3 0.3 11.4 0.30.3 0.3 0.3 0.310.8 0.50.5 0.5 0.5 10.10.5 ndnd nd nd nd 11.5 9 M c, d c, d 18.66 8.1 11.5 12.1 7.2 7.9 33 3 3 3M M MM M SabineneSabineneSabineneEucalyptolSabineneSabinene c, d c, dc, d c, d 16.0516.0516.0516.05 16.05 0.20.2 0.2 0.2 0.2 0.30.3 0.3 0.3 0.3 0.40.4 0.4 0.4 0.4 0.30.3 0.3 0.3 0.3 ndnd nd nd nd 10 M β b,c, d d 18.98 nd nd 0.4 18.8 9.3 4 M β-phellandrene-ocimene b, d b, b,d d b, d 16.23 0.3 0.5 0.6 0.5 nd 4 4 4 4M MM M β-phellandreneβ-βphellandrene-βphellandrene-phellandrenec, d 16.2316.2316.23 16.23 0.3 0.30.3 0.3 0.5 0.50.5 0.5 0.6 0.60.6 0.6 0.5 0.50.5 0.5 nd ndnd nd 11 M γ-terpinene c, d 19.5 nd nd 0.2 0.3 nd 5 5 M M β-pineneβ β-pinene c, d c, dc, d c, d 16.46 16.46nd nd 0.2 0.2 0.4 0.4 0.3 0.3 nd nd 5 125 5 M MM M β-pineneTerpinoleneβ-pinene-pinene b, d 16.4616.4616.46 20.57 nd ndnd nd0.2 0.20.2 nd0.4 0.40.4 nd 0.3 0.30.3 0.3 nd ndnd nd 6 M β-myrcene c, d c, d c, d 16.78 0.5 1.2 1.6 4.7 6.6 6 136 6 6M MM MM β-myrceneββ-βmyrcene--linaloolβmyrcene-myrcene c, d c, d d 16.7816.7816.78 21.0216.78 0.5 0.50.5 0.5 nd 1.2 1.21.2 1.2 nd 1.6 1.61.6 1.6 nd 4.7 4.74.7 4.71.6 6.6 6.66.6 16.16.6 b, d 77 147 7 7M M MM MM αα--phellandrenephellandreneαα-phellandrene-αphellandreneβ-phellandrene-fenchol b,c, d d b, b,d d b, d 17.5117.5117.5117.51 23.817.51 ndnd nd nd nd nd ndnd nd nd nd nd ndnd nd nd nd 0.3 0.70.7 0.7 0.7 0.7nd ndnd nd nd nd nd c, d c, d 88 158 8 8M M MMM M LimoneneLimoneneLimoneneαLimonene-terpineolLimonene c, d c, dc, d c, d 18.5218.5218.5218.52 24.71 18.52 6.46.4 6.4 6.4 6.4 1.2 11.411.411.4 11.4 11.4 1.9 10.810.810.8 10.8 10.8 2.1 10.110.110.1 10.1 10.1 0.8 11.511.511.5 11.5 11.5 0.9 16 M c, dc, d 26.64 nd 0.3 nd 1.2 1.1 9 9 9 M MM EucalyptolEucalyptolGeraniolEucalyptol c, d c, dc, d c, d 18.6618.66 18.66 8.1 8.18.1 11.511.5 11.5 12.112.1 12.1 7.2 7.27.2 7.9 7.97.9 9 9 M M EucalyptolEucalyptol b, e 18.6618.66 8.1 8.1 11.511.5 12.112.1 7.2 7.2 7.9 7.9 17 M Methyl geranate c, d 29.02 nd nd nd nd 1.9 1010 1010 10 MM MM M ββ--ocimeneocimeneβ-βocimene-βocimene-ocimene c, d c, dc, d c, d 18.9818.9818.9818.98 18.98 ndnd nd nd nd ndnd nd nd nd 0.40.4 0.4 0.4 0.4 18.818.818.8 18.8 18.8 9.39.3 9.3 9.3 9.3 c, d 1111 1111 11 MM MM M γγ--terpineneterpineneγγ-terpinene-γterpinene-terpinene c, d c, dc, d c, d 19.519.519.5 19.5 19.5 nd nd nd nd nd ndnd nd nd nd 0.20.2 0.2 0.2 0.2 0.30.3 0.3 0.3 0.3 ndnd nd nd nd

Metabolites 2021, 11, 594 4 of 14

Table 2. Cont.

Composition (%) Compound # Family RT (min) Yellow Orange T. Caliginosa crocata crocata LP-Xiphi HP-Xiphi 18 M Geranyl acetate c, e 30.59 0.3 0.3 0.2 19.6 9.4 19 M Geranyl butyrate b, e 34.75 nd nd nd 1.5 1.3 20 M Geranyl tiglate b, e 37.39 nd nd nd nd 1.4 21 M Geranyl hexanoate b, e 38.27 nd nd nd 0.9 1.2 22 S α-bergamotene b, e 32.16 0.2 nd nd 0.5 0.8 23 S β-farnesene b, e 32.55 5.5 1.2 1.0 3.3 3.5 24 S α-farnesene b, e 33.69 1.2 0.4 0.4 1.2 1.2 25 S β-bisabolene b, e 33.86 0.3 0.2 nd 0.8 1.7 26 S α-bisabolene b, e 34.52 nd nd nd 0.5 0.9 27 S Nerolidol c, e 34.98 0.3 0.4 nd 18.7 14.6 28 S Denderalasin b, e 35.17 nd nd nd 4.7 5.0 29 S α-patchoulene b, e 35.55 0.4 nd nd 0.7 0.8 30 S Farnesol c, e 37.81 9.3 4.3 1.8 nd nd 31 S Farnesal b, e 38.16 0.6 0.3 nd nd nd 32 S Farnesyl acetate b, e 39.67 4.5 0.5 1.3 nd nd 33 P Benzaldehyde c, d 15.8 0.5 0.2 0.2 nd nd 34 P Phenylacetaldehyde c, d 18.92 2.3 0.3 nd nd nd 35 P Methyl benzoate c, d 20.89 0.8 0.3 2.2 nd nd 36 P Benzene ethanol b, d 21.57 0.9 1.3 1.5 nd nd 37 P Benzylacetate c, d 23.43 5.5 nd 1.3 nd nd 38 P Methyl salicylate c, d 24.63 0.6 1.1 2.4 nd nd 39 P Phenylethyl acetate c, d 26.87 14.2 nd 2.5 nd nd 40 P Cinnamaldehyde b, e 27.5 0.5 0.6 0.5 nd nd 41 P Alcool cinnamique c, e 28.61 1.2 1.2 0.8 nd nd 42 P Eugenol c, e 29.96 0.9 14.5 12.3 nd nd 43 P Methyl eugenol c, e 31.18 0.4 1.0 nd nd nd 44 P Cinnamyl acetate b, e 32.37 nd 0.7 nd nd nd 45 P Isoeugenol c, e 32.45 5.1 0.6 0.9 nd nd 46 P Methyl isoeugenol b, e 33.49 2.8 0.8 nd nd nd 47 P Zingerone b, e 36.5 nd 0.5 2.7 nd nd 48 P Benzyl benzoate c, e 38.83 4.5 8.9 1.8 nd nd 49 P Phenethyl benzoate b, e 40.24 0.2 5.1 1.4 nd nd 50 P Benzyl salicylate b, e 40.51 1.7 5.1 nd nd nd 51 P Phenethyl salicylate a, e 42.09 nd 0.2 nd nd nd 52 O Methyl hexanoate b, d 14.24 nd nd 0.3 nd nd 53 O Hexyl acetate c, d 17.58 nd nd 0.2 nd nd 54 O Methyl nicotinate c, d 22.59 13.3 15.2 10.8 nd nd 55 O Indole b, e 28.22 1.2 0.6 nd nd 1.1 56 O Methyl tetradecanoate b, e 37.85 nd nd 1.7 nd nd 57 O p-tolyl octanoate b, e 38.36 1.1 0.2 nd nd nd 58 O 11-hexadecenal b, e 39.24 nd nd 0.5 nd nd 59 O Hexadecanal b, e 39.43 nd nd 1.3 nd nd 60 O Phenethyl octanoate b, e 39.97 0.3 0.4 nd nd nd 61 O 1-hexadecanol b, e 40.42 2.0 1.0 10.2 nd nd 62 O Methyl palmitoleate a, e 40.96 nd nd 1.2 nd nd 63 O Methyl palmitate a, e 41.14 nd 4.3 1.7 nd 1.7 64 O Benzyl decanoate a, e 41.76 0.3 0.2 nd nd nd 65 O 11-hexadecenyl acetate a, e 42.43 nd nd 0.7 nd nd 66 O Hexadecyl ethanoate a, e 42.66 0.5 nd 6.9 nd nd # = compound number, in bold: compounds present in all species, M = Monoterpene, S = Sesquiterpene, P = Phenylpropanoid, O = Other, nd = not detected, a = identification performed by comparing the mass spectrum with that of the NIST library, b = identification performed by comparing the mass spectrum with that of the NIST library and by comparison of RI (retention index) with RI of published literatures and online library (https://webbook.nist.gov/chemistry/cas-ser.html, accessed on 28 August 2021), c = identification performed by comparing the mass spectrum with that of the NIST library, by comparison of RI (retention index) with RI of published literatures and online library and by comparison of retention time and mass spectrum of the authentic standard, d = efficient extraction with the first method, low values of temperature and extraction time (30 ◦C and 20 min), e = efficient extraction with the second method, high values of temperature and extraction time (75 ◦C and 65 min). For the complete table, see Table S1 in the Supplementary Materials. Metabolites 2021, 11, 594 5 of 14 Metabolites 2021, 11, x FOR PEER REVIEW 6 of 14

FigureFigure 1. Chromatographic1. Chromatographic separation separation was was carried carried out on out a DB-5MSon a DB column,-5MS column and the, and extraction the extraction were performedwere performed with CAR/PDMS with CAR/PDMS fiber for 65fiber min for at 65 an min extraction at an extraction temperature temperature of 75 ◦C. ( Aof) 75 TIC °C. of ( orangeA) TIC of crocataorange.(B )crocata TIC of. yellow(B) TICcrocata of yellow.(C) crocata TIC of.T. (C caliginosa) TIC of .(T.D caliginosa) TIC of. LP-xiphi. (D) TIC of (E )LP TIC-xiphi. of HP-xiphi. (E) TIC of For HP- xiphi. For chromatograms obtained using the first extraction method (low values of temperature chromatograms obtained using the first extraction method (low values of temperature and extraction and extraction time), see Figure S1 in the Supplementary Material. The identification numbers cor- time), see Figure S1 in the Supplementary Material. The identification numbers correspond to those respond to those shown in Table 2. shown in Table2. For T. caliginosa, 43 volatile compounds were identified for the first time in floral For T. xiphioides, a total of 30 volatile compounds were identified in the floral emissions ofemissions. both forms The of this volatile species. profile The volatileis quite profile similar is largelyto that dominatedof the species by terpenoids,T. crocata; however which , arethere 28 of are the fewer 30 compounds sesquiterpenes identified., which Indeed, are only the five first in extractionT. caliginosa method, and the allowed compounds the isolationbelonging of more to the monoterpenes, ‘others’ family of whichare more the majornumerous ones in are T. limonene caliginosa (8) than and in eucalyptol T. crocata (9) and forin LP-xiphi T. xiphioides and .β The-linalool first extraction (13) for HP-xiphi. method With allowe thed useus to of isolate higher mostly values oflimonene temperature (8), eu- andcalyptol extraction (9), time,and phenylethyl the second extraction acetate (39) method, while allowed the second the isolation method ofallowed higher molecularus to extract weightmostly compounds. hexadecyl Theethanoate major (66) ones, 1 are-hexadecanol geranyl acetate (61), (18)phenylethyl and nerolidol acetate (27) (39) for, and LP-xiphi methyl andnicotinate indole (55), (54) nerolidol from the (27), flowers and of denderalisin T. caliginosa. (28) for HP-xiphi. Among the compounds identified in both forms of T. xiphioides, only indole (55) and methyl palmitate (63) are present2.2. Chemometrics in the HP-xiphi Analysis form of and Volatile not in Organic the LP-xiphi Compounds form. ForInT. order crocata to, identify 47 volatile volatile compounds compounds were that identified contribute in the to floral differences emissions or similarities of both formsbetween of this the species. species This studied, completes data on the the list 66 of identified seven VOCs, volatiles consisting were analyzed of benzaldehyde using prin- (33)cipal (3%), component benzyl acetate analyses (37) (PCA). (9%), PCA eucalyptol is a multivariate (9) (16%), analysis limonene that (8) allows (3%), methylthe extraction sal- icylateof important (38) (2%), informationE-β-ocimene from (10) a data (48%), table and in which phenylethyl observations acetate are (39) described (8%), identified by several byintercorrelated Gerlach and Schill dependent in 1991 variables [15]. In and contrast represents to T. xiphioides them as ,a whereset of new no compounds orthogonal vari- of theables phenylpropanoid called principal family components. were identified, In Figure for2A,T. it crocatais shown, these that compoundsthe first principal are very com- present.ponent Indeed, (PC1) contributes phenylpropanoids to 32.7% are, and secondary the second metabolites principal derived component from (PC2) phenylalanine contributes byto the 17.29% shikimate of the biosynthetic total variance pathway. in the floral Only volatile compounds peak forming area data. an On aldehyde, this same an figure alcohol,, we orcan an alkane/alkenesee three distinct by groups reduction; indeed at C9, the position, flowers or ofethers the two and forms esters of T. by xiphioides addition are of anclus- alkyltered group together, on hydroxyl the flowers groups of orthe carboxyl two forms group, of T. can crocata be volatile are clustered [16]. Thus, together among, and the the identifiedflowers compounds,of T. caliginosa the form first the extraction third group. method This allowed shows usa high to isolate degree mainly of clustering benzyl of acetateflowers (37), belonging phenylethyl to the acetate same (39), species eucalyptol, thus suggesting (9), and limonene a unique (8) floral for the volatile yellow profcrocataile for andeach limonene species (8) of andthe genus eucalyptol Tillandsia (9) for. PC1 the allowed orange crocatadiscrimination. The second of LP extraction-xiphi and method HP-xiphi allowedflowers us that to isolate are clearly more separated compounds from and yellow the major crocata ones, orange are farnesol crocata, (30),and caliginosa phenylethyl flow- acetateers. This (39), separation isoeugenol is (45), due methylto higher nicotinate intensities (54), of benzylmonoterpenoids acetate (37), andβ-farnesene sesquiterpenoids (23), andin farnesylthe floral acetate emissions (32) of for both yellow formscrocata of T. andxiphioides benzyl (Figure benzoate 2B). (48),PC2 methylshowed nicotinate a difference (54),in eugenolfloral volatiles (42), farnesol of T. caliginosa (30), benzyl compared salicylate to the (50), two and forms methyl of T. eugenol crocata (43). This for was orange due to crocata. Moreover, for the species T. crocata, some VOCs are present only in one of the two

Metabolites 2021, 11, 594 6 of 14

forms of crocata, such as α-bergamotene (22) and benzyl acetate (37), which are present only in yellow crocata, as well as α-thujene (1), β-pinene (5), geraniol (16), cinnamyl acetate (44), phenethyl salicylate (51), and methyl palmitate (63) have been identified only in orange crocata. For T. caliginosa, 43 volatile compounds were identified for the first time in floral emissions. The volatile profile is quite similar to that of the species T. crocata; however, there are fewer sesquiterpenes, which are only five in T. caliginosa, and the compounds belonging to the ‘others’ family are more numerous in T. caliginosa than in T. crocata and in T. xiphioides. The first extraction method allowed us to isolate mostly limonene (8), eucalyptol (9), and phenylethyl acetate (39), while the second method allowed us to extract mostly hexadecyl ethanoate (66), 1-hexadecanol (61), phenylethyl acetate (39), and methyl nicotinate (54) from the flowers of T. caliginosa.

2.2. Chemometrics Analysis of Volatile Organic Compounds In order to identify volatile compounds that contribute to differences or similarities between the species studied, data on the 66 identified volatiles were analyzed using principal component analyses (PCA). PCA is a multivariate analysis that allows the extraction of important information from a data table in which observations are described by several intercorrelated dependent variables and represents them as a set of new orthogonal variables called principal components. In Figure2A, it is shown that the first principal component (PC1) contributes to 32.7%, and the second principal component (PC2) contributes to 17.29% of the total variance in the floral volatile peak area data. On this same figure, we can see three distinct groups; indeed, the flowers of the two forms of T. xiphioides are clustered together, the flowers of the two forms of T. crocata are clustered together, and the flowers of T. caliginosa form the third group. This shows a high degree of clustering of flowers belonging to the same species, thus suggesting a unique floral volatile profile for each species of the genus Tillandsia. PC1 allowed discrimination of LP-xiphi and HP-xiphi flowers that are clearly separated from yellow crocata, orange crocata, and caliginosa flowers. Metabolites 2021, 11, x FOR PEER REVIEW 7 of 14 This separation is due to higher intensities of monoterpenoids and sesquiterpenoids in the floral emissions of both forms of T. xiphioides (Figure2B). PC2 showed a difference in floral volatiles of T. caliginosa compared to the two forms of T. crocata. This was due to high phenylpropanoidshigh phenylpropanoid intensitiess intensities in both in forms both of formsT. crocata of T.while crocataT. while caliginosa T. caliginosashowed highershowed intensitieshigher intensities of other compounds.of other compounds.

A 10 B 1.0 ● Monoterpenoids ● Sesquiterpenoids 8 ● Phenylpropanoids ● Others compounds 1452 53 ● ●● ● 65●● 58 59 ● ● 5 ● ● 6 11 56 ● 62 66 ● ●61 ●● 0.5 47 ● 4 ● ● ● % ●●● % 35 38 6 12

2 2 ●19 ● 10 ●4 8 8

. . ● ● ● 28 ●●18 ●7 3 ● 9 2 9 ● ● ● 21 ●● ● 2 ● 42 1 ● 1 ● 1 ● 27 ● 8 ● ●● 26 ● 15 : ● ● : 16 9 2 2 13 20 ● ● t ● t 0 ● ●● ● 0.0 22● ● ● 63 ● n ● n ● 36 e ● ● e 25●●29 ● n n 17 49 o o

p −2 ● p ● ● ●40 m ● m ●24 51●44 41

o ● o ● ●

C C 34 −4 ● 43 33 ●54 ●● 50 −0.5 37● ●●48 ● ●23 ● ●32 −6 ● 39● ● Yellow Crocata 55● 46● ●● 57●45●60 ● Orange Crocata ● 64● ●30 −8 ● Caliginosa ●31 ● LP−Xiphi −10 ● HP−Xiphi −1.0

−10 −8 −6 −4 −2 0 2 4 6 8 10 −1.0 −0.5 0.0 0.5 1.0 Component 1: 31.22 % Component 1: 31.22 %

FigureFigure 2. 2.PCA PCA based based on on VOCs VOCs emitted emitted from from yellow yellow crocata, crocata, orange orange crocata, crocata, caliginosa, caliginosa, HP-xiphi HP- andxiphi and LP-xiphi: (A) Score plot of PC1 versus PC2 scores. (B) Loading plot of PC1 and PC2 contributing LP-xiphi: (A) Score plot of PC1 versus PC2 scores. (B) Loading plot of PC1 and PC2 contributing volatile compounds. volatile compounds.

ToTo better better visualize visualize the differences differences in in volatile volatile profiles profiles between between flowers flowers of the of two the twoforms formsof T. crocata of T. crocata and thoseand of those T. caliginosa of T. caliginosa, a second, a secondPCA as PCAwell as as a well heat asmap a heatmapwere performed were performed(Figures 3 (Figuresand 4). The3 and first4). two The components first two componentsof the PCA explained of the PCA 43.14% explained and 23.74% 43.14% of the variation, explaining approximately 66% of the combined variance (Figure 3A). We can see that the two forms of T. crocata have relatively different floral volatile profiles due to their higher intensities of sesquiterpenoids and phenylpropanoids, such as β-farnesene (23), farnesyl acetate (32), benzylacetate (37), and phenylethyl acetate (39), which have higher intensities in floral emanations of yellow crocata compared to orange crocata (Figure 3B). While compounds such as benzyl benzoate (48), eugenol (42), methyl eugenol (43), benzyl salicylate (50), and phenethyl benzoate (49) are more abundant in orange crocata flowers, T. caliginosa flowers are grouped on the right due to higher intensities of other compounds, such as 1-hexadecanol (61), hexadecyl ethanoate (66), methyl tetradecanoate (56), hexadecanal (59), and some phenylpropanoids, such as methyl benzoate (35) and methyl salicylate (38).

A 8 B 1.0 1 ● 51●44 ●49 ● 16 ●63 ●● ●50 6 48● ●8 ●42 15 9 ● ● 60● ● 4 ● 0.5 ● ● 4 54 ● 3 27 ●6

% % ●

4 4 ● ● ●5 7 2 7 43 40 . . ●36 3 3

2 2 ● ● ● 41 64 18 : : ● 2 2 ● 38 25 t 0 ● t 0.0 n n ● e e 55 47 n ● n 31● ● o ● ● ● o ● 57 ●34 56● 62 10 p ● ● p 30 ● 52 53 ● −2 ● ● ● 33 ●●

m m ●11 ● ● ● ● 58 59 ●

o o 24 ● ● ●46 ● 14 C ● C 23 65 ●35 ● ● ● 29 45 ●66 −4 −0.5 ●● ●32 61 22 39●● 37 −6 ● Monoterpenoids ● Yellow Crocata ● Sesquiterpenoids ● Orange Crocata ● Phenylpropanoids −8 ● Caliginosa −1.0 ● Others compounds

−8 −6 −4 −2 0 2 4 6 8 −1.0 −0.5 0.0 0.5 1.0 Component 1: 43.14 % Component 1: 43.14 % Figure 3. Chemometric analysis of 57 VOCs emitted by the flowers of yellow crocata, orange crocata and T. caliginosa. (A) Score plot of PC1 versus PC2 scores. (B) Loading plot of PC1 and PC2 contributing volatile compounds.

This is confirmed by the heatmap (Figure 4); indeed, it is shown that the 57 volatile compounds detected in the floral emissions of T. caliginosa and in the two forms of T. cro-

Metabolites 2021, 11, x FOR PEER REVIEW 7 of 14

high phenylpropanoids intensities in both forms of T. crocata while T. caliginosa showed higher intensities of other compounds.

2 2 ●19 ● 10 ●4 8 8

p −2 ● p ● ● ●40 m ● m ●24 51●44 41

o ● o ● ●

−10 −8 −6 −4 −2 0 2 4 6 8 10 −1.0 −0.5 0.0 0.5 1.0 Component 1: 31.22 % Component 1: 31.22 % Figure 2. PCA based on VOCs emitted from yellow crocata, orange crocata, caliginosa, HP-xiphi and LP-xiphi: (A) Score plot of PC1 versus PC2 scores. (B) Loading plot of PC1 and PC2 contributing Metabolites 2021, 11, 594 volatile compounds. 7 of 14

To better visualize the differences in volatile profiles between flowers of the two forms of T. crocata and those of T. caliginosa, a second PCA as well as a heatmap were performed and(Figure 23.74%s 3 and of 4 the). The variation, first two explaining components approximately of the PCA explained 66% of the43.14% combined and 23.74% variance of the (Figurevariation,3A). explaining We can see approximately that the two 66% forms of the of combinedT. crocata variancehave relatively (Figure different3A). We can floral see volatilethat the profiles two forms due of to T. their crocata higher have intensities relatively of different sesquiterpenoids floral volatile and phenylpropanoids,profiles due to their suchhigher as βintensi-farneseneties of (23), sesquiterpenoids farnesyl acetate and (32), phenylpropanoids benzylacetate (37),, such and as phenylethylβ-farnesene (23) acetate, far- (39),nesyl which acetate have (32), higherbenzylacetate intensities (37), inand floral phenylethyl emanations acetate of yellow(39), whichcrocata havecompared higher inten- to orangesities incrocata floral (emanationsFigure3B ). Whileof yellow compounds crocata compared such as benzylto orange benzoate crocata (48),(Figure eugenol 3B). W (42),hile methylcompounds eugenol such (43), as benzylbenzyl salicylatebenzoate (50),(48), andeugenol phenethyl (42), methyl benzoate eugenol (49) are (43), more benzyl abundant salicy- inlate orange (50), andcrocata phenethylflowers, benzoateT. caliginosa (49) areflowers more abundant are grouped in orange on the crocata right flowers due to, higherT. calig- intensitiesinosa flowers of otherare grouped compounds, on the suchright asdue 1-hexadecanol to higher intensities (61), hexadecyl of other compounds ethanoate, (66),such methylas 1-hexadecanol tetradecanoate (61), (56), hexadecyl hexadecanal ethanoate (59), (66) and, somemethyl phenylpropanoids, tetradecanoate (56), such hexadecanal as methyl benzoate(59), and (35)some and phenylpropanoids methyl salicylate, such (38). as methyl benzoate (35) and methyl salicylate (38).

A 8 B 1.0 1 ● 51●44 ●49 ● 16 ●63 ●● ●50 6 48● ●8 ●42 15 9 ● ● 60● ● 4 ● 0.5 ● ● 4 54 ● 3 27 ●6

% % ●

4 4 ● ● ●5 7 2 7 43 40 . . ●36 3 3

2 2 ● ● ● 41 64 18 : : ● 2 2 ● 38 25 t 0 ● t 0.0 n n ● e e 55 47 n ● n 31● ● o ● ● ● o ● 57 ●34 56● 62 10 p ● ● p 30 ● 52 53 ● −2 ● ● ● 33 ●●

m m ●11 ● ● ● ● 58 59 ●

−8 −6 −4 −2 0 2 4 6 8 −1.0 −0.5 0.0 0.5 1.0 Component 1: 43.14 % Component 1: 43.14 % FigureFigure 3. 3.Chemometric Chemometric analysis analysis of of 57 57 VOCs VOCs emitted emitted by by the the ﬂowers flowers of of yellow yellow crocata, crocata, orange orange crocata crocata andand T.T. caliginosa.caliginosa. ( (AA)) Score Score plot plot of ofPC1 PC1 versus versus PC2 PC2 scores. scores. (B) Loading (B) Loading plot of plot PC1 of and PC1 PC2 and contrib- PC2 contributinguting volatile volatile compounds. compounds.

ThisThis is is confirmed confirmed by by the the heatmap heatmap (Figure (Figure4 );4) indeed,; indeed it, it is is shown shown that that the the 57 57 volatile volatile compoundscompounds detected detected in in the the floral floral emissions emissions of ofT. caliginosaT. caliginosaand and in thein the two two forms forms of T. of crocata T. cro-, are grouped in four large groups in the dendogram of the heatmap. The 14 compounds of group 1, consisting of methyl salicylate, methyl benzoate, methyl tetradecanoate, methyl palmitoleate, β-ocimene, hexyl acetate, methyl hexanoate, 11-hexadecenal, γ-terpinene, β hexadecanal, -fenchol, 11-hexadecenyl acetate, 1-hexadecanol, and hexadecyl ethanoate, are more abundant in the flowers of T. caliginosa. The 10 volatile molecules of Group 2, consisting of β-myrcene, limonene, α-pinene, eucalyptol, sabinene, β-phellandrene, β-pinene, zingerone, α-terpineol, and eugenol, are relatively abundant in the floral emissions of T. caliginosa and highly present in orange crocata flowers compared to yellow crocata. Group 3, consisting of 17 compounds, is formed mostly by sesquiterpenes and phenylpropanoids, which are abundant in yellow crocata flowers, while Group 4 is formed by 16 volatile compounds, which are abundant in orange crocata floral emissions. Metabolites 2021, 11, x FOR PEER REVIEW 8 of 14

cata, are grouped in four large groups in the dendogram of the heatmap. The 14 compounds of group 1, consisting of methyl salicylate, methyl benzoate, methyl tetradecanoate, methyl palmitoleate, β-ocimene, hexyl acetate, methyl hexanoate, 11-hexadecenal, γ- terpinene, hexadecanal, β-fenchol, 11-hexadecenyl acetate, 1-hexadecanol, and hexadecyl ethanoate, are more abundant in the flowers of T. caliginosa. The 10 volatile molecules of Group 2, consisting of β-myrcene, limonene, α-pinene, eucalyptol, sabinene, β-phellandrene, β-pinene, zingerone, α-terpineol, and eugenol, are relatively abundant in the floral emissions of T. caliginosa and highly present in orange crocata flowers compared to yellow Metabolites 2021, 11, 594 crocata. Group 3, consisting of 17 compounds, is formed mostly by sesquiterpenes8 of 14 and phenylpropanoids, which are abundant in yellow crocata flowers, while Group 4 is formed by 16 volatile compounds, which are abundant in orange crocata floral emissions.

4 Methyl salicylate (38) Methyl benzoate (35) Methyl tetradecanoate (56) methyl palmitoleate b - ocimene (10) Hexyl acetate (53) 2 Methyl hexanoate (52) 11 - hexadecenal (58) g - terpinene (11) Hexadecanal (59) b - fenchol (14) 11 - hexadecenyl acetate (65) 0 1 - hexadecanol Hexadecyl ethanoate (66) b - myrcene (6) Limonene (8) a - pinene (2) Eucalyptol (9) −2 Sabinene (3) b - phellandrene (4) b - pinene (5) Zingerone (47) a - terpineol (15) Eugenol (42) −4 Phenylacetaldehyde (34) Benzaldehyde (33) p - tolyl octanoate (57) Benzyl decanoate (64) Methyl isoeugenol (46) Indole (55) b - bisabolene (25) Farnesol (30) Farnesal (31) b - farnesene (23) Isoeugenol (45) Farnesyl acetate (32) a - farnesene (24) a - patchoulene (29) Benzylacetate (37) a - bergamotene (22) Phenylethyl acetate (39) Phenethyl benzoate (49) Methyl palmitate (63) a - thujene (1) Cinnamyl acetate (44) Geraniol (16) Phenethyl salicylate (51) Nerolidol (27) Benzyl benzoate (48) Benzyl salicylate (50) Benzene ethanol (36) Methyl eugenol (43) Cinnamaldehyde (40) Alcool cinnamique (41) Geranyl acetate (17) Methyl nicotinate (54) Phenethyl octanoate (60) C C C C C C C C C O O O O O Y Y Y Y Y Y Y e e e e e e e a a a a a a a a a r r r r r a a a a a l l l l l l l l l l l l l l l l l l l l l l l i i i i i i i i i n n n n n o o o o o o o g g g g g g g g g g g g g g w w w w w w w i i i i i i i i i n n n n n n n n n e e e e e

o o o o o o o o o C C C C C C C

C C C C C s s s s s s s s s r r r r r r r a a a a a a a a a o o o o o o o r r r r r o o o o o

c c c c c c c 4 5 9 7 8 2 3 1 6 c c c c c a a a a a a a a a a a a t t t t t t t a a a a a a a t t t t t a a a a a

3 7 5 6 2 1 4

1 5 3 2 4

Figure 4. Heatmap, the levels of each VOC in the three types of ﬂowers were normalized in the range of −4 to 4. Blue (−4) and yellow (4) represent the lowest and highest levels, respectively.

3. Discussion The identification of VOCs of three species of the genus Tillandsia was achieved through the use of the HS-SPME/GC-MS method. By using the CAR/PDMS fiber and two extraction methods with different values of extraction time and temperature developed in a previous study, the majority of the volatile compounds emitted by the flowers of Tillandsia species were extracted efficiently. Indeed, it was shown that the CAR/PDMS fiber allowed to extract the maximum of volatile compounds from T. xiphioides flowers compared to PDMS/DVB and DVB/CAR/PDMS fibers; moreover, considering the volatility of analytes Metabolites 2021, 11, 594 9 of 14

affecting the optimal conditions of temperature and duration of extraction, it is necessary to use two extraction methods in order to trap the majority of volatiles emitted by Tillandsia flowers [11]. After the GC-MS analysis, the profiles of the volatiles emitted by the flowers of the different species studied were compared qualitatively. This is the first time that a comparison of the VOCs emitted by different fragrant flowers of the genus Tillandsia has been performed. The results showed a difference in volatile profiles between the flowers of the species T. xiphioides, T. crocata, and T. caliginosa. Indeed, the flowers of the two forms of T. xiphioides present a profile of volatile compounds much more enriched in monoterpenes and sesquiterpenes, compared to the flowers of T. crocata and T. caliginosa, which present relatively more similar profiles. This could be confirmed by a morphological difference but also by a difference in pollinator type between T. xiphioides species and those of T. crocata and T. caliginosa. Indeed, it is shown that T. caliginosa and T. crocata are morphologically closer—these species present the same types of foliage and flowers; moreover the old name given to T. caliginosa is T. crocata var. tristis [17]. This difference in the composition of floral volatiles could also be confirmed by the attraction of different pollinators [18] for T. crocata, whose floral odor attracts Euglossine bees [15] and T. xiphioides, which is considered sphingophilous [19]. Indeed, it is shown that plants pollinated by the same ecological guild show similar visual and olfactory signals [20]. Even though it is not the only characteristic that can play a role in the attraction of pollinators, the floral odor constituted by a complex mixture of volatile compounds belonging to different chemical families plays a very important role in the stimulation and attraction of pollinators. These floral volatile compounds can be classified in the family of terpenoids, phenylpropanoids/benzenoids, and fatty acid derivatives, according to their biosynthetic pathways. Found predominantly in the flowers of both forms of T. xiphioides, terpenoids are derived from two precursors with five carbons isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP) and represent the largest class of floral volatile compounds. The latter are synthesized in plants from two independent pathways: the mevalonic acid pathway, where synthesis is carried out in the cytosol, giving rise to precursors of volatile sesquiterpenes (C15); and the methyl-erythritol phosphate pathway, where synthesis is carried out in the plastids mainly responsible of the formation of monoterpenes (C10) and volatile diterpenes (C20)[1]. Regarding the odor of this group of compounds, monoterpenoid hydrocarbons generally have a spicy and/or resinous odor, oxygenated monoterpenes have a sweet or citrus odor, and sesquiterpenes have a green and floral odor; however, diterpenes are rarely present in floral fragrances due to their low volatility [7]. Although close in the composition of floral volatiles, the two forms of T. xiphioides present, nevertheless, a slight difference in the intensities of certain compounds, which can affect the floral odor and lead to a difference in odor for the two forms of this species [11]. Found mainly in the flowers of both forms of T. crocata, phenylpropanoids represent the second class of VOCs emitted by plants. The first molecule of the phenylpropanoid pathway is phenylalanine, which is derived from the shikimate biosynthetic pathway. The deamination of this molecule by phenylalanine ammonia-lyase (PAL) forms the phenylpropanoid skeleton producing trans-cinnamic acid. All phenylpropanoid compounds are derived from trans-cinnamic acid by a succession of hydroxylation, methylation, and reduction steps [21]. Among the phenylpropanoids identified in this study, benzylacetate and phenylethyl acetate were found mainly in the floral emissions of yellow crocata. Benzyl acetate is a compound with a pleasant odor and is commonly found in plants such as jasmine, hyacinth, gardenia, and azalea, and is thought to be formed from the oxidation of cinnamoyl CoA [7,8,22]. Phenylethyl acetate is widely used in the cosmetic, food, and pharmaceutical industries for its pleasant rose smell. It is present in rose essential oils and is the main aromatic volatile ester emitted by rose flowers. Traditionally, this compound is obtained by acetylation of phenylethyl alcohol; moreover, the acetylation of aromatic alco- hols catalyzed by alcohol acetyltransferase leads to volatile acetate esters in plants [23,24]. Other phenylpropanoids, such as eugenol, methyl eugenol, benzyl benzoate, phenethyl Metabolites 2021, 11, 594 10 of 14

benzoate, and benzyl salicylate, were very present in the floral emissions of orange crocata. In the phenylpropanoid biosynthetic pathway, eugenol and isoeugenol are obtained from coniferyl acetate through eugenol synthase and isoeugenol synthase. Subsequently, a methylation reaction of eugenol, catalyzed by an O-methyltransferase, allows us to obtain methyl eugenol [21]. Eugenol and methyl eugenol are major constituents of several essential oils, such as clove, basil, cinnamon, lavender, and jasmine essential oils. Eugenol is widely used in perfumery due to its powerful spicy odor, and methyl eugenol is widely used as a flavoring agent in food products [25–27]. In plants such as Clarkia breweri and Petunia hybrida, benzyl benzoate and phenethyl benzoate are obtained from benzyl alcohol and phenylethanol, respectively, through benzoyl-CoA: benzyl alcohol/phenylethanol benzoyltransferase [28,29]. In addition to its activity in the treatment of scabies and lice, benzyl benzoate is used as a fixative in perfumery [26]. Benzyl salicylate is the benzyl ester of salicylic acid, it is widely used in the cosmetics industry and is used in the composition of many perfumes [30]. Volatile aromatic esters generally contribute to a sweet fruity smell [31]. These results show that there is a slight difference in the floral volatile profiles of the two forms of T. crocata, especially in the intensities of some volatile compounds. This difference could be associated with the difference in floral colors of the two forms of this species. Indeed, it was shown that plants of the same species having a variation of floral color also presented a variation in the composition of floral volatiles. For example, a study on orchids showed that the color morphs of O. simia presented different volatile profiles; the white flowers emitted more benzenoid compounds and lipid products than the purple flowers [32]. However, there are studies that did not find a clear difference in the odor chemistry of color morphs of the same species [33,34]. Thus, even if the slight difference in volatile profiles of yellow and orange crocata flowers could be associated with the difference in flower colors, it could also be due to natural selection by pollinators or mutations in genetic drift or metabolic pathways. In T. caliginosa, floral emissions showed, in addition to phenylpropanoids, higher intensities of compounds of the ‘others’ family than in the ‘others’ species. Indeed, compounds such as 1-hexadecanol, hexadecyl ethanoate, methyl tetradecanoate, and hexadecanal are very present in T. caliginosa. These compounds are derivatives of fatty acids, which constitute another large family of volatile compounds. They are produced following a series of reactions catalyzed by lipoxygenases, hydroperoxide lyases, isomerases, and dehydrogenases from polyunsaturated fatty acids and phospholipids [1]. They generally bring a waxy and oily note to the floral scent of the plants. It should also be noted that the flowers of both forms of T. crocata as well as T. caliginosa have shown a high intensity of methyl nicotinate. The latter is a vasodilator, which is used in topical preparations as a rubefacient in case of muscle pain [35].

4. Materials and Methods 4.1. Plant Material and Chemicals All the flowers of the Tillandsia species used for this study come from the Tillandsia PROD plant nursery located in Le Cailar (Occitanie, France) and were harvested from February to May 2020. We used two forms of T. xiphioides, named LP-xiphi and HP-xiphi due to low or high presence of trichomes on the leaves of the plants; two forms of T. crocata, named yellow crocata and orange crocata with yellow and orange colored flowers, respectively; and one form of T. caliginosa species. For T. xiphioides, 10 plants of each form were used, providing 2–3 flowers each. For T. crocata, 7 plants (one flower per plant) of the yellow crocata form and 5 plants of the orange crocata form providing 2 flowers each were used. For T. caliginosa, 8 plants were used and 5 of them provided 2 flowers. The chemicals used for the identification of the compounds were ordered from Sigma- Aldrich (St. Louis, MO, USA): α-pinene (98.5% purity), sabinene (75%), β-pinene (98.5%), β- myrcene (90%), limonene (99%), eucalyptol (99%), β-ocimene (95.4%), γ-terpinene (98.5%), β-fenchol (99%), β-linalool (97%), α-terpineol (90%), geraniol (99%), geranyl acetate (99%), nerolidol (98.5%), farnesol (95%), benzaldehyde (99.5%), phenylacetaldehyde (95%), methyl Metabolites 2021, 11, 594 11 of 14

benzoate (99.5%), benzylacetate (99.7%), methyl salicylate (99%), phenylethyl acetate (97%), alcool cinnamique (97%), eugenol (99.6%), methyl eugenol (98%), isoeugenol (99%), benzyl benzoate (98%), hexyl acetate (99.7%), and methyl nicotinate (98%).

4.2. HS-SPME Conditions In our previous study, the Carboxen/Polydimethylsiloxane (CAR/PDMS) 75 µm fiber was determined to be the most suitable for extracting the maximum amount of volatile compounds emitted by T. xiphioides flowers, so this fiber was used for the present study. The CAR/PDMS and the SPME device were ordered from Supelco (Bellefonte, PA, USA). Prior to use, the fiber was conditioned according to the manufacturer’s recommendations. Extractions were performed by placing a flower in a 20 mL amber vial sealed with a polytetrafluoroethylene (PTFE) septum-lined cap. For each Tillandsia flower, two successive extractions were performed: a first extraction method of 20 min at a temperature of 30 ◦C and a second extraction method of 65 min at 75 ◦C. These extraction methods provide a global view of the volatile compounds present in Tillandsia flowers and were retained after the optimization of the SPME method performed during our previous study. For both extraction methods, the equilibrium and desorption times are, respectively, 7.5 and 4 min.

4.3. Instrumentation and GC-MS Conditions The analysis was performed on an Agilent 7890 B gas chromatograph coupled with an Agilent 5977 A mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with an MPS autosampler, a thermal desorption unit (TDU), and a cooled injection system (CIS) (Gerstel, Mülheim, Germany). MSD Chemstation version F.01.00 data acquisition software (Agilent Technologies, Santa Clara, CA, USA) was used to program the GC-MS. After extraction, desorption was performed thermally in the TDU at the recommended temperature of 300 ◦C for CAR/PDMS. After desorption, the injection was performed in split mode; the analytes were focused on the CIS at −10 ◦C for 2 min, then brought to 250 ◦C at a heating rate of 12 ◦C per second and maintained for 2.5 min. A DB-5MS capillary column (5% diphenyl cross-linked 95% dimethylpolysiloxane, 30 m × 0.25 mm × 0.25 µm) (Agilent Technologies, Santa Clara, CA, USA) was used and the separation conditions were as follows: initial column temperature of 40 ◦C for 2 min, then increased by 4 ◦C/min to 130 ◦C for 1 min, then increased by 7 ◦C/min to 230 ◦C, where it was maintained for 4 min. High purity Helium (99.999%) was used as the carrier gas at a ﬂow rate of 1.2 mL per minute. The temperature of the transfer line was set at 250 ◦C, and the temperature of the ion source at 230 ◦C. The ions were generated by a 70 eV electron beam. The mass range was scanned from m/z 33 to 500 Da.

4.4. Identification MassHunter Qualitative Analysis version B.06.00 (Agilent Technologies, Santa Clara, CA, USA) was used to integrate the majority peaks of the chromatograms, representing 95% of the peaks of the Total Ion Chromatogram (TIC) and being above the analytical noise. First, the volatile compounds were identified by comparing their mass spectra to those of compounds in the commercial databases, National Institut of Standard and Technologies (NIST) and Wiley7 (R > 800). Then, the retention indices of the compounds were calculated relative to the n-alkanes (C8–C20) and compared to those of compounds in the NIST online database (https://webbook.nist.gov/chemistry/cas-ser.html, accessed on 27 August 2021). Finally, the identifications were confirmed by injecting the available standards (mentioned above) into the GC-MS based on the comparison of mass spectra and retention times. For the standard solution, 0.2 µL of each standard was added in 2 mL of hexane, and the solvent was subsequently evaporated with nitrogen. The analysis was performed under the same conditions as for the flowers. Metabolites 2021, 11, 594 12 of 14

4.5. Statistical Analysis For statistical analysis, peak area values of the total ion chromatograms were measured with MassHunter and transferred to Excel (Microsoft Excel 2013, version 15.0.5363.1000). Principal component analysis (PCA) was performed using SIMCA-P software (version 15.15, Umetrics, Umea, Sweden) and a heatmap with the R software (version 4.0.3, company Foundation for Statistical Computing, Vienna, Austria) based on an univariate scaling method. For this purpose, for each plant providing two ﬂowers (the plants of orange crocata and the plants of T. caliginosa), an average of the areas of the peaks of the compounds was calculated in Excel. Odor characteristics were obtained from the “The Good Scents” company network database (www.thegoodscentscompany.com, accessed on 27 August 2021). Heatmap data was clustered (Ward’s method was used to form hierarchical clustering) and visualized (using the pheatmap-package, version 1.0.12).

5. Conclusions This study allowed the identification of 66 volatile compounds in three Tillandsia species with different colors and flower shapes. This identification was performed using the HS-SPME/GC-MS technique, where the use of two extraction methods, developed in a previous study, were necessary in order to efficiently extract the majority of floral volatiles. A comparison of the volatile compound profiles by PCA between T. xiphioides, T. crocata, and T. caliginosa species showed a difference in the floral emissions of the studied species. Indeed, the floral emissions of T. xiphioides were richer in terpenoids, those of T. crocata were enriched in phenylpropanoids, and those of T. caliginosa presented more compounds of the ‘others’ family, including fatty acid derivatives and nitrogenous compounds. In addition, a slight intraspecific difference was also observed in T. crocata, particularly variations in the intensities of some VOCs of yellow and orange crocata. These variations in floral volatile profiles between different Tillandsia species and between different forms of the same species could be associated with several phenomena, such as natural selection by pollinators, morphological and/or color differences, mutations in metabolic pathways, or genetic drift. The differences and variations observed in the VOCs profiles can explain the differences in the floral odor of these species and forms. This study provides additional information on the composition of floral VOCs of Tillandsia species and helps to better understand the diversity within the genus Tillandsia.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/metabo11090594/s1, Figure S1: Chromatograms obtained using the first extraction method (low values of temperature and extraction time), Table S1: Complete table of the identification of volatile compounds from floral emissions of T. xiphioides, T. crocata and T. caliginosa. Author Contributions: Conceptualization, P.M.; methodology, M.-M.L. and R.M.; software, M.-M.L., J.-X.F. and R.M.; validation, M.-M.L. and R.M.; formal analysis, M.-M.L. and R.M.; investigation, M.-M.L.; resources, D.B.; writing—original draft preparation, M.-M.L.; writing—review and editing, R.M. and P.M.; visualization, M.-M.L., J.-X.F. and R.M.; supervision, Z.B., R.M. and P.M.; project administration, P.M.; funding acquisition, P.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by La Region Occitanie and Univ. Nimes. Tillandsia PROD plant nursery (28 chemin du Cailar F-30740 Le Cailar, France) provided all the plants that were necessary for the study (in-kind contribution). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available within the article and Supplementary Materials. Any other data are available upon request from the corresponding author. Metabolites 2021, 11, 594 13 of 14

Acknowledgments: We gratefully acknowledge Tillandsia PROD (28 chemin du Cailar 30740 Le Cailar, France) plant nursery (Pierre Kerrand, Daniel Thomin, [email protected]) for providing us the Tillandsia plants used in this study, for helpful discussions and for their interest in the project. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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