Colour-Scent Associations in a Tropical Orchid

Phytochemistry 72 (2011) 735–742

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Phytochemistry

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Colour-scent associations in a tropical orchid: Three colours but two odours ⇑ Roxane Delle-Vedove a, , Nicolas Juillet a,b,1, Jean-Marie Bessière c, Claude Grison a, Nicolas Barthes a, Thierry Pailler b, Laurent Dormont a, Bertrand Schatz a a Centre d’Ecologie Fonctionnelle et Evolutive (CEFE), UMR CNRS 5175, 1919 route de Mende, 34293 Montpellier cedex 5, France b UMR53, Laboratoire des Peuplements Végétaux et Bioagresseurs en Milieu Tropical, 15 Avenue René Cassin, BP 7151, 97415 St Denis de la Réunion messag cedex 9, France c Ecole Nationale Supérieure de Chimie de Montpellier, 8 rue de l’Ecole Normale, 34296 Montpellier cedex 5, France article info abstract

Article history: Colour and scent are the major pollinator attractants to flowers, and their production may be linked by Received 11 October 2010 shared biosynthetic pathways. Species with polymorphic floral traits are particularly relevant to study Received in revised form 1 February 2011 the joint evolution of floral traits. We used in this study the tropical orchid Calanthe sylvatica from Accepted 7 February 2011 Réunion Island. Three distinct colour varieties are observed, presenting lilac, white or purple flowers, Available online 4 March 2011 and named respectively C. sylvatica var. lilacina (hereafter referred as var. lilacina), C. sylvatica var. alba (var. alba) and C. sylvatica var. purpurea (var. purpurea). We investigated the composition of the floral Keywords: scent produced by these colour varieties using the non-invasive SPME technique in the wild. Scent emis- Flower colour polymorphism sions are dominated by aromatic compounds. Nevertheless, the presence of the terpenoid (E)-4,8-dim- Colour-scent associations Scent variation ethylnona-1,3,7-triène (DMNT) is diagnostic of var. purpurea, with the volatile organic compounds DMNT (VOC) produced by some individuals containing up to 60% of DMNT. We evidence specific colour-scent SPME associations in C. sylvatica, with two distinct scent profiles in the three colour varieties: the lilacina-like Pollinator preferences profile containing no or very little DMNT (<2%) and the purpurea-like profile containing DMNT (>2%). Cal- Island colonisation anthe sylvatica var. alba individuals group with one or the other scent profile independently of their pop- Hawkmoth ulation of origin. We suggest that white-flowered individuals have evolved at least twice, once from var. lilacina and at least once from var. purpurea after the colonisation of la Réunion. White-flowered individuals may have been favoured by the particular pollinator fauna characterising the island. These flowering varieties of C. sylvatica, which display three colours but two scents profiles prove that colour is not always a good indicator of odour and that colour-scent associations may be complex, depending on pollination ecology of the populations concerned. Ó 2011 Elsevier Ltd. All rights reserved.

Introduction ing phenology (Parra-Tabla and Vargas, 2007). These floral traits are the principal detectable and attractive signals for pollinators Polymorphism in floral traits is widespread in animal-polli- and are used by animal-pollinated plants generally in addition to nated plants, and ecologists have been long intrigued by its emer- a reward, which ensures their repeated visits. Investigations in gence and maintenance (Weiss, 1995). Except for the case of plant reproduction have long asserted that non-random combina- specific and obligate plant–pollinator associations, where low tions of flower colour, shape and fragrance have evolved in re- intraspecific variation of floral traits has been found (Dufaÿ et al., sponse to directional selection exerted by specific classes of 2004; Hossaert-McKey et al., 2010; Svensson et al., 2006), high effective animal pollinators (Faegri and van der Pijl, 1979; Fenster polymorphism of pollinator-attractant floral traits has been docu- et al., 2004; Johnson et al., 1997; Raguso et al., 2003). Pollinators mented at individual or population levels in numbers of flowering show preferences for specific odour, colour or shape according to plants. Variation occurs for different floral traits such as colour innate preferences but also influenced by their considerable learn- (Dormont et al., 2010; Gigord et al., 2001; Waser and Price, ing capabilities and sensory flexibility (Chittka et al., 1999; Daly 1981; Weiss, 1995), odour (Azuma et al., 2001; Knudsen et al., and Smith, 2000; Goyret et al., 2008; Kelber, 2010). Such associa- 2006; Majetic et al., 2009; Salzmann et al., 2007; Tollsten and tive learning is based on floral characters or floral rewards of differ- Bergström, 1993), flower size (Dickson and Petit, 2006) and flower- ent kinds (nectar, pollen, oils, etc.) or on associations of these traits (Giurfa et al., 1997; Raine and Chittka, 2007). Learning in insect is complex and depend on discrimination capacities of the insect. ⇑ Corresponding author. Tel.: +33 4 67 61 33 00; fax: +33 4 67 41 06 16. These are first conditioned by the species specific sensory machin- E-mail address: [email protected] (R. Delle-Vedove). 1 Present address: Parcs Nationaux de France, Château La Valette, 1037 rue Jean- ery of insects and, environmental conditions as illumination or the François Breton, 34090 Montpellier, France. presence of contrasting floral species (Dyer and Chittka, 2004;

Kelber, 2010). Discrimination capacities could also be modulated gests that specific flower colour-scent associations could be a by previous experiences and the combined use of visual, gustatory consequence of conserved biochemical pathways (Armbruster, or olfactory inputs (Avarguès-Weber et al., 2010; Giurfa et al., 2002) and may not be dissociated—or formed—by natural selection 1994; Kelber, 2010). These capacities are the basis of ‘‘pollinator (Majetic et al., 2007). constancy’’, in which a pollinator visiting one flowering plant is Species polymorphic for floral traits are ideal model systems to more likely to move to another plant of the same morph than study the joint evolution of floral traits and their impact on plant would be expected based on plant frequency in the population reproduction. In this context, orchids are highly pertinent models (Jones and Reithel, 2001; Waser, 1986). This behaviour is expected for studying plant–pollinator interactions as they present a great to exert stabilizing selection, leading to uniformity of attractive flo- variety of floral traits and trait associations. This is mirrored by ral traits (Raguso et al., 2003). For example, studies of pollination the great diversity of reproductive strategies in orchids. One of syndromes give rise to the hypothesis that specialization of flowers the most intriguing of these strategies is deceptive pollination on pollination by nocturnal hawkmoths leads to the production of (i.e. nectarless flowers), which concerns about one third of orchid white-flowered morphs, which emit more aromatic (i.e. benze- species. Several strategies of pollinator attraction are identified in noids compounds) compounds (especially alcohols and esters: these species (see Jersakova et al. (2006) for a review). Deceptive Majetic et al., 2007; Raguso et al., 2003). Recently, the generality pollination often rely on newly emerged naive insects or may ben- of floral syndromes has been questioned based on the apparent efit from facilitation effect by the surrounding rewarding species commonness of generalized pollination systems, although highly community (Johnson et al., 2003; Juillet et al., 2007). Several spe- specialized pollination systems are also well documented cies of deceptive orchids display floral trait polymorphism sup- (Cozzolino et al., 2005; Johnson and Steiner, 2000). A second con- posed to be advantageous to avoid associative learning cern about pollination syndromes is that evolution of floral traits potentially performed by pollinators (Nilsson, 1992; Jersakova is not solely the result of selection by pollinators (or other agents) et al., 2006). In orchids, as in other insect-pollinated plants, the ex- but may also reflect phylogenetic constraints (Knudsen et al., 2006; tent to which colour and odour polymorphism may influence Raguso et al., 2003). reproductive success remains poorly understood (Dormont et al., Thus, floral trait polymorphism at individual or population lev- 2010). We investigated colour-scent associations in the floral- els has been documented in a large number of flowering plants. polymorphic orchid Calanthe sylvatica (Orchidaceae) in Réunion Is- Current explanations of the maintenance of variable floral traits land presenting three different colour varieties. A first study re- in flowering plants traditionally rely on the key role of insects vealed that the three different colour varieties occur in spatially through pollinator-mediated selection. Polymorphism may be separated populations, flower at different periods of the year and maintained by the existence of several effective pollinator species also present differences in flower morphology, for example in spur with different foraging preferences. For example, hawkmoth and length (Juillet et al., 2010). They also achieve different fruit set, hummingbird pollinators impose divergent selection respectively which is higher in white varieties than in coloured ones. Here we on yellow and red morphs of Mimulus aurantiacus (Scrophularia- analysed the scent production of C. sylvatica and attempted to ceae) (Streisfeld and Kohn, 2007). Pollinators may also exert tem- determine whether there are associations between floral colour porally or spatially fluctuating selection pressures, when their and scent in this species. We address three questions: abundance and assemblage structure are variable (Brown and Clegg, 1984; Eckhart et al., 2006; Waser and Price, 1981). More re- (1) What is the scent composition of C. sylvatica flowers? cently, it has been proposed that floral trait variation may also be (2) Does floral scent vary among individuals within colour vari- maintained by multiple and conflicting selection pressures, both eties of C. sylvatica? by biotic agents such as pollinators and herbivores and by abiotic (3) Are there specific colour-scent associations among colour factors (such as heat or drought). For example in Ipomoea purpurea varieties? (Convolvulaceae), pigment compounds serve to attract pollinators to the flowers and also increase tolerance of water and heat stress 2. Results and discussion in vegetative parts (Warren and Mackenzie, 2001). Colour and scent are generally the main stimuli that attract in- 2.1. Scent characterization of C. sylvatica sects to flowers (Chittka and Raine, 2006; Dobson, 1994) and in some cases, the combined effect of colour and odour allow the poll- The scent of C. sylvatica individuals was composed of 24 identi- inators to distinguish between different flowering plants better fied compounds, all of which were present in individuals of all of than if the two signals are presented separately (Galizia et al., the three colour categories but in different relative proportions 2004; Kunze and Gumbert, 2001). Depending on the species, a (Table 1). These compounds were either terpenoids or aromatic combination of both sensory cues can be used. Giurfa et al. compounds. The scent of C. sylvatica flowers is dominated, in most (1994) demonstrated that honeybees could supplement visual cues individuals, by aromatic compounds that are relatively frequent in by using olfactory inputs to enhance their foraging activity. Alter- floral scents (Knudsen et al., 2006) and often cited as typical attr- natively, male solitary bees used sequentially olfactory signals actants for moth pollinators (Hubert et al., 2005; Majetic et al., from a distance and then visual cues to guide them towards Ophrys 2007; Raguso et al., 2003). Three aromatic compounds accounted heldreichii (Streinzer et al., 2009). Recently, authors have high- for a large fraction (55–75%) of the scent profiles of C. sylvatica lighted the existence of variation in scent emissions of different (Table 1): methyl benzoate, benzyl alcohol and benzyl benzoate. colour morphs of the same flowering species (Li et al., 2006; Maj- Four other compounds, namely (E)-4,8-dimethylnona-1,3,7-triène etic et al., 2007; Odell et al., 1999; Salzmann and Schiestl, 2007). (DMNT), (E)-dendrolasine, benzyl cyanide and methyl salicylate Colour-scent relationships may be complex and often rely on were also relatively important in the odour bouquet (each more shared biosynthetic pathways (Armbruster, 2002; Majetic et al., than 5% in at least one colour variety), and dominated the odour 2007), implying that any mutation in a gene coding for an enzyme emission of some individuals (e.g. up to 62% of DMNT in some or a regulatory element will have an impact both on colour and var. purpurea individuals, Table 1). Fifteen compounds presented scent emitted. Zucker et al. (2002) showed for example that the significant differences in abundance among colour varieties suppression of a single key enzyme led to the formation of (Table 1). Of these, 13 presented heterogeneous values in relative white-flowered mutants with higher amounts of aromatic com- abundance for var. alba individuals. Overall, a principal component pounds (methyl benzoate) than in coloured individuals. This sug- analyse (PCA) performed on the relative proportions of all R. Delle-Vedove et al. / Phytochemistry 72 (2011) 735–742 737

Table 1 Mean relative proportions of compound (±SE) in the three colour varieties of C. sylvatica. RI: retention index; P: probability for the F test; CV: coefficient of variation. For each compound, a different letter after the mean value indicates a significant difference among the three varieties (Tukey–Kramer multiple comparison test). Compounds marked with stars were identified by RI and MS comparison with: known commercial standards (⁄⁄) or compound synthesized by ecological method (⁄).

Compounds RI P Var. alba (n = 18) Var. lilacina (n = 19) Var. purpurea (n = 21) Mean ± SE (Min–Max) CV Mean ± SE (Min–Max) CV Mean ± SE (Min–Max) CV Terpenoids 12.49 ± 3.53 (0.62–42.11) 0.28 2.29 ± 0.33 (0.44–5.63) 0.15 30.88 ± 4.61 (6.09–74.2) 0.15 Myrcene⁄⁄ 991 0.58 0.22 ± 0.05a (0–0.8) 1.06 0.22 ± 0.13a (0.01–2.51) 2.55 0.11 ± 0.06a (0–1.17) 2.32 a-Terpinene 1011 0.01 0.19 ± 0.05ab (0–0.83) 1.14 0.32 ± 0.07b (0–1.01) 0.9 0.08 ± 0.04a (0–0.8) 2.17 p-Cymene⁄⁄ 1026 0.07 0.11 ± 0.04a (0–0.69) 1.44 0.19 ± 0.04a (0.02–0.6) 0.85 0.09 ± 0.02a (0–0.35) 1.02 Limonene⁄⁄ 1030 <0.01 0.34 ± 0.11a (0–1.8) 1.39 0.31 ± 0.05a (0.09–0.86) 0.71 1.75 ± 0.49b (0.05–9.53) 1.29 (E)-Ocimene 1047 0.11 0.03 ± 0.02a (0–0.27) 2.47 0.06 ± 0.03a (0–0.53) 2.38 0.14 ± 0.05a (0–0.62) 1.62 p-Cymenene 1086 <0.01 0.03 ± 0.01a (0–0.18) 1.38 0.28 ± 0.09b (0.02–1.6) 1.41 0.04 ± 0.01a (0–0.21) 1.17 Terpinolene⁄⁄ 1087 0.04 0.13 ± 0.05ab (0–0.77) 1.65 0.18 ± 0.07b (0–1.03) 1.75 0.005 ± 0.003a (0–0.07) 3.34 (Z)-dimethylnonatriene⁄ 1091 <0.01 0.2 ± 0.07a (0.02–1.17) 1.4 0.07 ± 0.02a (0–0.25) 1.06 0.92 ± 0.23b (0.08–4.68) 1.16 (E)-Dimethylnonatriene⁄ 1113 <0.01 8.76 ± 2.82a (0–37.1) 1.37 0.13 ± 0.06a (0.01–1.16) 2.04 20.42 ± 4.1b (2.59–62.03) 0.92 (E)-Nerolidol⁄⁄ 1563 <0.01 0.85 ± 0.3a (0.03–5.03) 1.53 0.22 ± 0.05a (0–0.76) 1.04 2.2 ± 0.5b (0.02–8.85) 1.04 (E)-Dendrolasine 1576 <0.01 1.38 ± 0.81a (0–14.78) 2.48 0.12 ± 0.02a (0.02–0.28) 0.76 4.67 ± 1.07b (0.02–19.85) 1.05 (Z)-Dendrolasine 1557 0.45 0.25 ± 0.08a (0–1.18) 1.37 0.19 ± 0.05a (0–0.66) 1.13 0.46 ± 0.24a (0–4.93) 2.37 Aromatics 87.51 ± 3.53 (57.89–99.38) 0.04 97.71 ± 0.33 (94.37–99.55) 0 69.12 ± 4.61 (25.80–93.92) 0.07 (Z)-Phenylethanal-oxime 1252 0.21 0.68 ± 0.22a (0.01–3.49) 1.39 0.88 ± 0.25a (0.03–4.1) 1.26 0.4 ± 0.1a (0.01–1.74) 1.12 (E)-Phenylethanal-oxime 1280 0.24 0.77 ± 0.18a (0.03–2.68) 1.01 0.89 ± 0.25a (0.1–4.1) 1.21 0.47 ± 0.1a (0.03–1.79) 0.98 Benzyl cyanide 1145 <0.01 7.6 ± 0.66b (3.51–14.9) 0.37 7.76 ± 0.65b (3.25–13.34) 0.36 3.91 ± 0.49a (0.59–7.8) 0.58 2-Phenylethanol 1123 <0.01 0.81 ± 0.2a (0.05–3.45) 1.02 2.65 ± 0.47b (0.27–7.56) 0.78 0.73 ± 0.2a (0.03–3.94) 1.27 2-Phenethyl derivative 1310 <0.01 0.53 ± 0.11a (0.03–2.05) 0.85 1.53 ± 0.16b (0.27–3.03) 0.46 0.4 ± 0.06a (0.05–0.95) 0.67 Methyl benzoate 1099 0.97 28.04 ± 2.9a (11.69–57.07) 0.44 26.84 ± 2.96a (6.7–49.16) 0.48 27.53 ± 3.8a (4.62–56.41) 0.63 Isoamyl benzoate 1444 <0.01 0.26 ± 0.04a (0.12–0.61) 0.6 1.04 ± 0.26b (0.12–5.07) 1.08 0.41 ± 0.05a (0.05–1.25) 0.59 Benzaldehyde⁄⁄ 965 <0.01 1.91 ± 0.22ab (0.61–4.74) 0.48 2.64 ± 0.37b (0.24–5.82) 0.61 1.17 ± 0.2a (0.1–3.37) 0.78 Benzyl alcohol⁄⁄ 1039 0.81 21.99 ± 3.03a (2.92–49.86) 0.59 23.6 ± 3.74a (0.29–51.87) 0.69 20.63 ± 3.11a (3.63–63.02) 0.69 Benzyl benzoate 1770 <0.01 19.98 ± 2.02b (5.09–35.52) 0.43 27.57 ± 2.2c (12.79–42.51) 0.35 6.77 ± 1.49a (0.04–29.32) 1.01 Methyl salicylate⁄⁄ 1199 0.02 4.88 ± 1.13ab (0.22–18.97) 0.98 2.00 ± 0.51a (0.16–8.74) 1.1 5.72 ± 1.02b (0.13–15.24) 0.82 Indole⁄⁄ 1301 0.18 0.06 ± 0.01a (0.03–0.13) 0.42 0.31 ± 0.18a (0.02–2.62) 2.52 0.98 ± 0.55a (0.01–10.16) 2.56

compounds revealed that the two first axes explained 78.54% of Light, 1998), and are thus potentially biologically relevant in the the total variance (Fig. 1a). Axis 1 was mainly influenced by the rel- pollination of C. sylvatica. Further GC-EAD analysis will allow us ative amount of two compounds: methyl benzoate and DMNT in future studies to discriminate the biological role of scent com- (Fig. 1b) from which DMNT was the sole to present significant dif- pounds in the C. sylvatica system. These results are consistent with ferences among varieties (Table 1). Most individuals were clus- what we know or suppose on C. sylvatica pollinators. The pollinator tered into two groups: one cluster contained all individuals of fauna of Réunion Island is characterized by a poorly diversified var. lilacina and some of the var. alba individuals and the second insect fauna, except in nocturnal Lepidoptera, and it features a par- cluster contained almost all var. purpurea individuals and most of ticularly high diversity of Sphingidae species (Martiré and Rochat, the remaining var. alba individuals. 2008). The diurnal hawkmoth Macroglossum milvus was observed Dendrolasine and benzyl cyanide are not common in flower visiting C. sylvatica var. lilacina (T. Pailler, personnel observation). scents, each having been reported only once in floral scent, the first Complementary field observations are necessary to extend our in Listera ovata (Orchidaceae; Nilsson, 1981) and the second in knowledge of the pollinators of C. sylvatica. Magnolia kobus (Magnoliaceae; Azuma et al., 2001). Both are known to have repellent or attractant effects on different insects (Fatouros 2.2. Colour-scent associations in C. sylvatica and the potential impact et al., 2005; Kite and Leon, 1995). Nevertheless in C. sylvatica, these of biochemical constraints compounds show minor variations, in contrast to DMNT, which dominates scents of some var. purpurea individuals (Table 1) and 2.2.1. Between lilac- and purple-coloured varieties for which variation among individuals allows discriminating be- Despite the large amount of aromatic compounds emitted by all tween colour varieties. DMNT has been identified in floral scents varieties of C. sylvatica, scent differentiation between the lilac and of many angiosperm families, although generally in low quantities purple varieties is mostly accounted for by their terpenoid compo- (Azuma et al., 2001; Knudsen et al., 2006). This compound is com- sition, especially regarding the amount of DMNT: individuals of monly associated with indirect plant defence and is attractive for var. purpurea produce on average large amounts of DMNT hymenopteran natural enemies of phytophagous insects (Boland (mean ± SE = 20.42 ± 4.1%) and var. lilacina individuals always pro- et al., 1992). However, DMNT has also been identified in large duce low (<1.5%) quantities of it (0.13 ± 0.06%). amounts in diverse highly specialized pollination systems involving Purple pigments are frequently anthocyanins (Clegg and Dur- flies (Miyake and Yafuso, 2005), moths (Levin et al., 2001; Svensson bin, 2000), which are present in the genus Calanthe (Arditti, et al., 2005) and bees (Wiemer et al., 2008). For example, the (E) 1992). The result here found seem to be contradictory to the ‘‘bio- isomer of DMNT is the major component (>99.8%) of scents of the chemical pathway’’ hypothesis, as flavonoid pigments, especially bee-pollinated orchid Cyclopogon elatus (Wiemer et al., 2008). To- anthocyanins, are produced by the aromatic pathways (Zucker gether with morphological measurements (flower size and spur et al., 2002) and are thus not directly related to production of ter- length; Juillet et al., 2010), scent analysis suggests that C. sylvatica, penoids. Salzmann et al. (2007) also found an association of the red at least var. alba and lilacina, might display a sphingid pollination Dactylorhyza romana morph with production of larger proportions syndrome. The three major aromatic compounds found in C. sylvat- of the terpenoid linalool. Nevertheless, red coloration in flowers is ica profiles are known to elicit medium to strong response in sometimes due to carotenoid pigments, which are linked to the antennae of some sphinx moths (Raguso et al., 1996; Raguso and terpenoid pathway (Dudareva et al., 2004), if carotenoids are 738 R. Delle-Vedove et al. / Phytochemistry 72 (2011) 735–742

Fig. 1. Principal component analysis of the relative proportions of 24 compounds emitted by each of the three varieties of C. sylvatica (A) and factor loadings in the PCA (B). In (A), solid lines link each individual point to the variety’s barycentre. responsible of coloration in Calanthe, production of greater propor- ‘‘intermediate’’. A first discriminant function analysis (DFA) on tions of terpenoid scent compounds could simply be a byproduct of the relative proportion of scent compounds allowed separation of being red. In the absence of a complete study of pigments involved the three colour varieties (F = 201.78, df = 48, p = 3.76 Â 10À9), in lilac and red colorations, the hypothesis that biochemical con- but not completely, especially for var. alba individuals. In fact, straints lead to associations between colour and scent cannot be without prior knowledge of the colour variety, 5 of the 18 var. alba firmly rejected. individuals were misclassified in coloured varieties. We then tested another hypothesis of assignation. Given that the relative 2.2.2. Between coloured and white varieties proportion of DMNT allows discriminating among colour varieties The PCA reveal that individuals clustered in two groups, the first (Table 1, Fig. 2a), we recoded var. alba individuals according to the composed by all individuals of var. lilacina and some of the var. presence or absence of DMNT. This allowed us to test whether var. alba individuals and the second by almost all var. purpurea individ- alba individuals are well assigned into the lilacina or purpurea-like uals and most of the remaining var. alba individuals. Some var. alba scent profiles. This new classification was well supported by a 8 individuals and one var. purpurea individual were classified as second DFA (F = 296.40, df = 24, p = 4.32 Â 10À ), with all

Fig. 2. Relative amount of DMNT emitted by each colour variety of C. sylvatica (A), and the PCA showing the grouping of individuals according to presence and absence of DMNT in the odour bouquet. Individuals of var. alba are rearranged according to their scent profile, being either lilacina-like or purpurea-like (see text for details). R. Delle-Vedove et al. / Phytochemistry 72 (2011) 735–742 739 individuals being well classified in one or the other scent-profile. not see any special damages on var. purpurea individuals in the The ANOVA on the individual contributions to first axis was thus field, DMNT could be produced in response of biotic pathogens À5 highly significant (F1,56 = 21.76, p = 1.96 Â 10 ). This detailed pressure. study of var. alba individuals revealed that they fall for one part in a lilacina-like profile, and for the other part in a purpurea-like profile, as shown by the DFA and the PCA (Figs. 1a and 2b) and well 2.3.2. Polymorphism within var. alba exemplified by amounts of the terpenoid DMNT in Fig. 2a. Considering the two scent profiles found in var. alba individuals, If biochemical constraints are important in governing colour- we propose that these groups may be derived from two indepen- scent associations, we would expect to find consistent differences dent mutations, once in var. lilacina and once in var. purpurea indi- between coloured and white-flowered varieties, and particularly viduals, after the colonisation of Réunion Island. Loss-of-function an increased production of aromatic compounds in the latter mutations are often the cause of transitions from coloured to white (Majetic et al., 2008; Zucker et al., 2002). Surprisingly, white flowers, and are more common than the reverse transitions (re- individuals in C. sylvatica did not show such a pattern (Table 1). view in Rausher (2008)). Such mutations in colour-coding genes, Our results clearly diverged from expectations of the biochemi- or regulatory elements affecting them, may have occurred in cal-constraint hypothesis as no differentiation was found between C. sylvatica. However, the exact scenario for the evolution of var. coloured and white-flowered varieties of C. sylvatica. purpurea and var. lilacina remains unknown. A detailed genetic study would enable us to test specifically this hypothesis. 2.2.3. Within the white variety As both scent profiles co-occur within white-flowered popula- Another intriguing result of the study is the scent differentia- tions, this would imply in turn that mutant white phenotypes have tion between two groups of white individuals. We present the converged in adapting to conditions ecologically different than PCA figure with the new grouping in two scent profiles in Fig. 2b. those to which the parental coloured varieties are adapted, occur- Var. alba individuals presenting lilacina-like scent (without DMNT) ring at lower altitude and flowering later (Juillet et al., 2010). This and purpurea-like scent (with DMNT) were equally represented in convergence may have been driven by pollinator preferences and our sample (n = 10 and n = 8, respectively), and were present in all the facilitation effect of a guild of nectar-producing white co- three sampled populations. flowering species, which represent a large part of the Réunion Studies of scent variations between colour morphs have identi- orchid flora (Jacquemyn et al., 2005, 2007). This convergence might fied variations in white flower scent among populations (Dormont also have been facilitated by the probable continuity between var. et al., 2010; Majetic et al., 2007). In C. sylvatica, two scent profiles alba habitat (lower altitude) and habitat of the coloured varieties can be found in a single white-flowered population and they (mid to high altitude) before human colonisation (XVIIth century) matched the scent profiles of one or the other coloured variety. and the ensuing forest fragmentation (Strasberg et al., 2005). Selection by abiotic or biotic factors appears more likely to have Var. alba individuals from the two scent profiles were found in shaped colour-scent associations in C. sylvatica than biochemical all three sampled populations. Some studies, mainly focused on constraints. This situation, in which pollinators encounter a flower colour, have hypothesized that this type of dimorphism white-flowered morph displaying two kinds of odours, is to the should be adaptive for nectarless species, as it reduces the speed best of our knowledge unique in orchids. It allows us to raise with which pollinators learn to avoid rewardless flowers, and gen- new exciting questions. In the following, we discuss the origin erates negative frequency-dependent selection that could maintain and maintenance of these two profiles in white-flowered C. the floral colour dimorphism (Gigord et al., 2001; Smithson and sylvatica. MacNair, 1997). To date, this form of selection has never been demonstrated to maintain different scent variants within popula- 2.3. Evolution of colour-scent associations in C. sylvatica tions. A complete study at the population level, quantifying the reproductive success of var. alba individuals according to their 2.3.1. Differentiation between varieties lilacina and purpurea scent profiles and the frequency of these profiles, would enable How did these colour and scent variations arise in Réunion Is- evaluation of this hypothesis. Alternatively, pollinator observation land? C. sylvatica probably colonized the Island from East Africa should enable us to know if the different scent profile attract dif- or Madagascar. Although data on herbarium specimens from these ferent species of them or elicit different pollinator’s behaviour, countries (East African Herbarium, Nairobi, Kenya; Herbarium of leading to the maintenance of these polymorphisms. the National Museum of Natural History, Paris, France) mention various flower colours, it is unknown whether the three colour varieties colonized the island independently or have evolved lo- 3. Concluding remarks cally. Orchids usually do not set fruit in the absence of animal pollinators, and fruit set is often low, particularly for nectarless tropical We demonstrated that white individuals displayed two kinds of species (Tremblay et al., 2005). C. sylvatica is no exception to this odours, and that the odours emitted by white individuals corre- rule (5.17 ± 9.34% of open flower setting fruit; Juillet et al., 2010), spond to the odours emitted by each of the two coloured varieties. and there is great potential for pollinator agents to select for spe- The consequence is that what the insect smells does not match cific floral traits combinations attractive for them. For nectarless what it sees. Floral polymorphisms are complex and strongly influ- species, the around rewarding floral diversity could shape pollina- enced not only by the pollinators, but also by several other selec- tor preferences and could also lead to mimicry (Johnson et al., tive agents such as herbivores and abiotic factors or even by 2003). For example, var. lilacina could take advantage of the pollin- pleiotropic effects of selection acting on other traits (Dormont ators attracted by the reward shrub Chassalia coralioides, which of- et al., 2010; Rausher, 2008; Schaefer et al., 2004; Schaefer and ten bloom in the same populations and present flowers of the same Ruxton, 2009). Our study demonstrates an original case of col- colour (Juillet et al., 2010). Alternatively, the colour-scent associa- our-scent association and the importance of taking all floral traits tions documented here could also be independent from each other, into account when studying floral polymorphism (Raguso, 2008). resulting from divergent environmental conditions or from selec- The floral odour-colour combination has been treated in recent tive pressures on each trait exerted by forces unrelated to pollina- reviews as one of the main future issues in pollination ecology tors. Especially, we know that DMNT is often associated with (Raguso, 2008; Rausher, 2008; Schaefer and Ruxton, 2009). The indirect plant defence (Boland et al., 1992) and, even if we did case of C. sylvatica suggests several ways to explore the ecological 740 R. Delle-Vedove et al. / Phytochemistry 72 (2011) 735–742 consequences of discordance between visual and olfactory signals forme d’Analyses Chimiques en Ecologie’’ (Platform for Chemical produced by floral traits. Analyses in Ecology) of ‘‘IFR 119 Montpellier Environnement Bio- diversité’’. Mass spectra were recorded in electronic impact (EI) at 70 eV, and identified by comparison with data of the NIST 98 4. Experimental software library (Varian, Palo Alto, CA, USA). In addition, identification of some compounds has been confirmed by RI and Mass Spec- 4.1. Study site and species trum comparison of the samples with commercial known standards GC–MS runs. For (Z)-DMNT and (E)-DMNT, an original The study was conducted on Réunion Island, a volcanic island ecological synthesis has been performed to confirm identification situated in the western Indian Ocean. Reunion Island is character- and isomerism since no commercial reference exist (see Sec- ized by a steep altitudinal gradient ranging from 0 to 3070 m and a tion 4.4.). Comparison of the chromatographs of the orchid samples tropical climate. Mean annual rainfall ranges from 1500 mm on the with that of the control sample from the same population allowed east coast, to more than 8000 mm in high altitude. The Island pre- removing the peaks corresponding either to adsorbent, to degrada- sents a highly diverse orchid flora, with a total of 135 species of tion of NalophanÒ, or to volatiles present in the air surrounding the which 42% are terrestrial (Jacquemyn et al., 2007). bags. Peaks were quantified using the Star Chromatography Soft- Calanthe sylvatica (Thouars) Lindl. is a terrestrial orchid species wareÒ and the proportion of each compound was expressed as with large and showy inflorescences and a pollination system rely- the peak area relative to the total peak area for all compounds. ing on deception (absence of floral rewards) (Jacquemyn et al., 2007; Juillet et al., 2010). The species is colour-polymorphic (De 4.4. Revisited synthesis of DMNT Cordemoy, 1895) with three known varieties differing in colour, morphology, flowering phenology and altitudinal distribution Despite its recurring interest in chemical ecology, DMNT has re- (Juillet et al., 2010). Var. alba individuals grow at low altitude (0– ceived no recent attention by chemists. Only few DMNT syntheses 1100 m), have white inflorescences and their flowering peaks in have been reported and involved Wittig reaction (Maurer et al., the late rainy season (March); var. lilacina and purpurea grow at 1986). This reaction, although being useful so far in classical organ- higher altitudes (from 1100 m up to 1600 m) and bear respectively ic chemistry, requires rigorous experimental conditions, using of lilac and purple/red flowers. Var. lilacina flowers mainly in Decem- hazardous solvents and reagents, and it does not maximize atom ber and var. purpurea from December to May. The three colour economical. Indeed, it takes place at low temperature (À78 °C), un- varieties seldom co-occur in the same location. der anhydrous conditions because of, among others, strong organic Preliminary observations in natural conditions and knowledge base reactant (LDA) and uses large amounts of solvents (THF and of Réunion Island’s pollinator fauna suggest that this species might petroleum ether), both for the reaction and for the numerous treat- be pollinated by the diurnal hawkmoth M. milvus Boisduval ments and purifications. (Sphingidae). Recent cleaner method like metathesis, involving last genera- tion of Grubbs’ catalysts, is getting more and more interest in the 4.2. Odour sampling field of pheromone synthesis (Mori and Tashiro, 2004) but is not suitable for DMNT preparation as it would engage 1,3-butadiene For each colour variety, a total of three populations were randomly and leads to polymerisation products. selected and VOC were extracted using solid-phase micro-extraction We herein report an innovative access to DMNT incorporating a (SPME) technique, a non-destructive solvent-free sampling green chemistry approach: solvent free and highly concentrated technique. The sampling of odour was carried out in situ on 58 individ- reactions. The synthetic strategy includes the sequence aqueous uals (6–10 individuals per population) from nine populations (three Wittig-Horner-Emmons reaction/saponification (Coutrot et al., populations per colour variety), between 09h00 and 12h00. 1992; Villieras and Rambaud, 1982; Villieras et al., 1985), followed Sampling by SPME was performed using 65 lm poly- by a copper-catalysed decarboxylation (Fig. 3)(Gooßen et al., 2007). dimethylsiloxane/divinylbenzene (PDMS–DVB) fibres (SupelcoÒ, Synthetic sample has been analysed by 1H NMR (Bruker NMR

Sigma–Aldrich, Bellefonte, PA, USA). For each sampled individual, Advance 300 MHz – CDCl3) d (ppm): 1.70 (s, 3H, Me), 1.78 (s, 3H, we enclosed the whole inflorescence in a polyethylene terephtalate Me), 1.85 (s, 1.89H Me, E enantiomer 63%), 1.87 (s, 1.11H Me, Z Ò bag (Nalophan , Kalle Nalo GmbH, Wursthüllen, Germany). An enantiomer 37%), 2.13-2.30 (m, 4H, CH2), 5.01–5.22 (m, 3H, CH2 SPME fibre was then introduced with a manual holder into the vinylogous and CH), 5.95 (broad d, 1H, JH-H = 12 Hz, CH), 6.65 (td, bag and was adjusted in close proximity to flowers (1–2 cm) and 1H, JH–H = 18 Hz and JH–H = 12 Hz, CH). left for VOC collection for 2 h. This duration was found in prelimin- DMNT has a Rf of 0.8 in pure hexane (0.3 for citral) on TLC ary tests to be the best collection time. A sample of surrounding air (Merck – 5535 – Kieselgel 60-F254), detection being carried out was collected in the same way as a control. After sampling, fibres by UV or by iodine. were brought back to the laboratory in a cool box, and stored at The diastereomer ratio was easily determined from the integra- À20 °C until analysis. tion of the protons signals (of the methyl carried by C-4) in the 1H

NMR data (dH–E = 1.85 ppm and dH–Z = 1.87 ppm). (E)-DMNT has 4.3. Gas chromatography and mass spectrometry of VOCs been identified as major compound (62%). The obtained values clearly demonstrated the configurational integrity of the C@C dou- Gas chromatography and mass spectrometry (GC–MS) analyses ble bond in the successive reactions used to obtain DMNT. of the SPME extracts were performed using electronic impact ion- This sample analysed by GC/MS (see Section 4.3. for analysing ization mode on a Varian Saturn 2000 ion trap spectrometer, inter- conditions) showed retention indexes of 1110 for (E)-DMNT faced with a Varian CP-3800 apparatus. The Varian CP-3800 was (63%) and 1089 for (Z)-DMNT (37%) which permits the chromato- equipped with a 1079 split-splitless injector (260 °C) and a graphic assignments in our study. 30 m Â 0.25 mm Â 0.25 lm film thickness ID WCOT CPSil-8CB fused silica capillary column (ChrompackÒ, Bergen op Zoom, The 4.5. Data analysis Netherlands), with helium as carrier gas (1 ml/min), and pro- grammed 2 min isothermal at 50 °C, then increasing from 50 °C The scent composition of each C. sylvatica colour variety was to 220 °Cat4°C/min. These analysis were performed at the ‘‘Plate- compared using F tests followed by Tukey–Kramer multiple com- R. Delle-Vedove et al. / Phytochemistry 72 (2011) 735–742 741

Fig. 3. Alternative synthetic strategies for DMNT: A comparative study.

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