Phytochemistry 94 (2013) 123–134 Contents lists available at SciVerse ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Flower color polymorphism in Iris lutescens (Iridaceae): Biochemical analyses in light of plant–insect interactions Hui Wang a,1, Lucie Conchou b,1, Jean-Marie Bessière c, Guillaume Cazals d, Bertrand Schatz b,2, ⇑ Eric Imbert a, ,2 a Institut des Sciences de l’Évolution de Montpellier (ISEM), UMR 5554 CNRS-Université Montpellier 2, Bâtiment 22, Université Montpellier 2, place E. Bataillon, 34095 Montpellier Cedex 5, France b Centre d’Ecologie Fonctionnelle et Evolutive (CEFE), UMR 5175 CNRS, 1919 route de Mende, 34293 Montpellier Cedex 5, France c Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), 8, rue de l’Ecole Normale, 34296 Montpellier Cedex 5, France d Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-Université Montpellier 1 et 2, Bâtiment Chimie (17), Université Montpellier 2, place E. Bataillon, 34095 Montpellier Cedex 5, France article info abstract Article history: We describe a flower color polymorphism in Iris lutescens, a species widespread in the Northern part of Received 13 February 2013 the Mediterranean basin. We studied the biochemical basis of the difference between purple and yellow Received in revised form 13 May 2013 flowers, and explored the ecological and evolutionary consequences of such difference, in particular Available online 18 June 2013 visual discrimination by insects, a potential link with scent emitted and the association between color and scent. Anthocyanins were found to be present in much greater concentrations in purple flowers than Keywords: in yellow ones, but the anthocyanin composition did not differ between color morphs. Likewise, no quan- Flower color polymorphism titative difference in anthocyanin content was found between vegetative tissues of the two morphs. Flo- Iris lutescens ral anthocyanins were dominated by delphinidin 3-O-(p-coumaroylrutinoside)-5-O-glucoside (also called Anthocyanins Floral scent delphanin) and its aliphatic derivatives. Small amounts of delphinidin 3-O-(p-caffeoylrutinoside)-5-O- Terpenoids glucoside and its aliphatic derivatives were also characterized. Based on a description of bumblebees’ Plant–insect interactions (one of the main pollinators of I. lutescens) color perception, purple and yellow flowers of I. lutescens could Color-scent association be visually discriminated as blue and blue-green, respectively, and likely by a wide variety of other insects. The overall chemical composition of the scent produced was not significantly different between morphs, being dominated by terpenoids, mainly myrcene, (E)-b-ocimene and limonene. A slight color- scent correlation was nevertheless detected, consistent with the shared biosynthetic origin of both pig- ments and volatile compounds. Therefore in this species, the difference in the amounts of pigments responsible for flower color difference seems to be the major difference between the two morphs. Poll- inators are probably the main selective agent driving the evolution of flower color polymorphism in I. lutescens, which represents a suitable species for investigating how such polymorphism is maintained. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction polymorphism has been documented for numerous floral traits such as color, scent, size and phenology (Delle-Vedove et al., Ecologists have long been intrigued by the emergence and 2011). While flower color is diverse throughout the angiosperms, maintenance of polymorphism in floral traits (Schaefer et al., variation of flower color within species is uncommon; and species 2004; Weiss, 1995). Indeed, as pollinator visitation rate is generally that show a true, stable, genetically based polymorphism are com- correlated with plant fitness in entomogamous species (i.e. 80% of paratively rare (Kay, 1978; Weiss, 1995). Flower color polymor- plant species, Potts et al., 2010), stabilizing selection mediated by phism has been well described in a limited number of species, associative learning is expected to occur on floral traits, leading such as Ipomoea purpurea (Convolvulaceae, Clegg and Durbin, to low intra-specific variation (Ashman and Majetic, 2006; 2000), Linanthus parryae (Polemoniaceae, Schemske and Bierzych- Dormont et al., 2010; Salzmann and Schiestl, 2007). However, udek, 2001), Mimulus aurantiacus (Scrophulariaceae, Streisfeld and Kohn, 2005), Hesperis matronalis (Brassicaceae, Majetic et al., 2007), ⇑ Corresponding author. Tel.: +33 0 467144910. and the orchids Dactylorhiza sambucina (Gigord et al., 2001) and E-mail address: [email protected] (E. Imbert). Calanthe sylvatica (Juillet et al., 2010). 1 These authors contributed equally to this work and are considered as joint first Flower color is among the most important visual signals in pol- authors. linator attraction (Menzel and Shmida, 1993) and generalist pollin- 2 These authors contributed equally to this work and are considered as joint last ators use color and scent to find flowers when first exploring the authors. 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.05.007 124 H. Wang et al. / Phytochemistry 94 (2013) 123–134 world (Chittka and Raine, 2006; Giurfa et al., 1995; Lunau and between composition of pigments and of volatiles in plant species Maier, 1995). Consistently, previous studies suggested that flower with flower polymorphism. Recent reviews consider the flower color polymorphism could be maintained by contrasting pollinator scent-color combination to be an outstanding open question in preferences for particular color morphs. For example, hawkmoth pollination ecology (Raguso, 2008; Rausher, 2008; Schaefer and and hummingbird pollinators impose divergent selection, respec- Ruxton, 2009). tively, on yellow and red morphs of Mimulus aurantiacus (Streisfeld We investigated flower color polymorphism in a rewardless and Kohn, 2007). Pollinators may also exert temporally or spatially species common in the Northern part of the Mediterranean region, fluctuating selection pressures, when their abundance and assem- Iris lutescens, which displays a spectacular purple-yellow flower blage structure are variable (Eckhart et al., 2006; Salzmann and color polymorphism within populations (Fig. 1). Information on Schiestl, 2007). In the food-deceptive orchid Dactylorhiza sambuci- the reproductive biology of this species is presented in the Materi- na, negative frequency-dependent selection mediated by the learn- als and methods section below. We analyzed the biochemical basis ing abilities of pollinators has been observed (Gigord et al., 2001; of color and scent, and explored ecological and evolutionary conse- but see Pellegrino et al., 2005). quences of color differentiation. We aimed to determine whether Three groups of pigments are responsible for coloration in pollinator agents (mainly bumblebees and solitary bees) are poten- plants (Tanaka et al., 2008): the composite group flavonoids (pale tial selective agents acting on the evolution of the flower color yellow to yellow)/anthocyanins (orange to blue), the betalains polymorphism. More specifically, we addressed the following four (yellow to red, found only in the order Caryophyllalles), and the questions: (1) What are the pigmentation differences between the carotenoids (yellow to red). Flavonoids and anthocyanins are the two color morphs? (2) Are the two color morphs discriminated be- major contributors to flower color (Tanaka et al., 2010). In addition tween by insects? (3) What floral scents are emitted by the two to producing color to attract pollinators, all of these pigments func- color morphs? (4) Is there an association between floral scent tion in protecting plants against damage caused by UV and visible and flower color in this species? light (Tanaka et al., 2008). Flavonoids and anthocyanins are known to function in the responses of plants to stress (drought, cold) and 2. Results and discussion in resistance to attack by microbes and herbivores (Chalker-Scott, 1999; Harborne and Williams, 2000a). Carotenoids also play essen- 2.1. What are the pigmentation differences between the two color tial roles in photosynthesis. These pigments can thus affect plant morphs? survival in several ways, and various selection pressures can act indirectly on flower color traits. Selection pressures exerted by a 2.1.1. Quantifying pigment contents range of abiotic factors such as precipitation, soil or temperature The amounts of three classes of pigments, flavonoids (mainly (Dick et al., 2011; Schemske and Bierzychudek, 2001, 2007; War- chalcones, flavones and flavonols), anthocyanins and carotenoids, ren and Mackenzie, 2001) and biotic agents such as herbivores were all significantly different between plants of the two color and pathogens (Frey, 2004) could be involved in maintaining poly- morphs in flowers (petals and sepals, except for flavonoids in se- morphism. Whether pollinators are the main selective agents influ- pals, Fig. 2), while contents of none of them differed significantly encing flower polymorphisms, or whether these polymorphisms in leaves (Fig. 2). The largest difference in flowers was for anthocy- are driven mainly by selective forces other than pollinators, re- anins, which were present in about 18- to 28-fold higher concen- mains under debate, and the answer may depend on the studied trations in petals and sepals, respectively, of the purple morph species (Dormont et al., 2010; Strauss and Whittall, 2006). than