Flower color polymorphism in Iris lutescens (Iridaceae): Biochemical analyses in light of plant–insect interactions
Graphical abstract
The different accumulation of delphinidin derivatives is mainly responsible for flower color difference between purple- and yellow-flowered I. lutescens, while the color polymorphism is not associated with significant difference in the scent emitted between the two morphs.
Introduction
Ecologists have long been intrigued by the emergence and maintenance of polymorphism in floral traits (Schaefer et al., 2004, Weiss, 1995). Indeed, as pollinator visitation rate is generally correlated with plant fitness in entomogamous species (i.e. 80% of plant species, Potts et al., 2010), stabilizing selection mediated by associative learning is expected to occur on floral traits, leading to low intra-specific variation (Ashman and Majetic, 2006, Dormont et al., 2010, Salzmann and Schiestl, 2007). However, polymorphism has been documented for numerous floral traits such as color, scent, size and phenology (Delle-Vedove et al., 2011). While flower color is diverse throughout the angiosperms, variation of flower color within species is uncommon; and species that show a true, stable, genetically based polymorphism are comparatively rare (Kay, 1978, Weiss, 1995). Flower color polymorphism has been well described in a limited number of species, such as Ipomoea purpurea (Convolvulaceae, Clegg and Durbin, 2000), Linanthus parryae (Polemoniaceae, Schemske and Bierzychudek, 2001), Mimulus aurantiacus (Scrophulariaceae, Streisfeld and Kohn, 2005), Hesperis matronalis (Brassicaceae, Majetic et al., 2007), and the orchids Dactylorhiza sambucina (Gigord et al., 2001) and Calanthe sylvatica (Juillet et al., 2010).
Flower color is among the most important visual signals in pollinator attraction (Menzel and Shmida, 1993) and generalist pollinators use color and scent to find flowers when first exploring the world (Chittka and Raine, 2006, Giurfa et al., 1995, Lunau and Maier, 1995). Consistently, previous studies suggested that flower color polymorphism could be maintained by contrasting pollinator preferences for particular color morphs. For example, hawkmoth and hummingbird pollinators impose divergent selection, respectively, on yellow and red morphs of Mimulus aurantiacus (Streisfeld and Kohn, 2007). Pollinators may also exert temporally or spatially fluctuating selection pressures, when their abundance and assemblage structure are variable (Eckhart et al., 2006, Salzmann and Schiestl, 2007). In the food-deceptive orchid Dactylorhiza sambucina, negative frequency-dependent selection mediated by the learning abilities of pollinators has been observed (Gigord et al., 2001; but see Pellegrino et al., 2005).
Three groups of pigments are responsible for coloration in plants (Tanaka et al., 2008): the composite group flavonoids (pale yellow to yellow)/anthocyanins (orange to blue), the betalains (yellow to red, found only in the order Caryophyllalles), and the carotenoids (yellow to red). Flavonoids and anthocyanins are the major contributors to flower color (Tanaka et al., 2010). In addition to producing color to attract pollinators, all of these pigments function in protecting plants against damage caused by UV and visible light (Tanaka et al., 2008). Flavonoids and anthocyanins are known to function in the responses of plants to stress (drought, cold) and in resistance to attack by microbes and herbivores (Chalker-Scott, 1999, Harborne and Williams, 2000a). Carotenoids also play essential roles in photosynthesis. These pigments can thus affect plant survival in several ways, and various selection pressures can act indirectly on flower color traits. Selection pressures exerted by a range of abiotic factors such as precipitation, soil or temperature (Dick et al., 2011, Schemske and Bierzychudek, 2001, Schemske and Bierzychudek, 2007, Warren and Mackenzie, 2001) and biotic agents such as herbivores and pathogens (Frey, 2004) could be involved in maintaining polymorphism. Whether pollinators are the main selective agents influencing flower polymorphisms, or whether these polymorphisms are driven mainly by selective forces other than pollinators, remains under debate, and the answer may depend on the studied species (Dormont et al., 2010, Strauss and Whittall, 2006).
Flower color and scent often function synergistically to attract pollinators (Burger et al., 2010, Chittka and Raine, 2006, Dötterl et al., 2011, Milet-Pinheiro et al., 2012, Raguso and Willis, 2002, Raguso and Willis, 2005). Furthermore, relationships between color and scent are partly inherent, because the two kinds of traits rely on shared biosynthetic pathways (Armbruster, 2002, Majetic et al., 2007), implying that any mutation in a gene coding for an enzyme or a regulatory element of these pathways could have an impact on both color and scent emitted. Volatile organic compounds involved in floral scent are dominated by fatty-acid derivatives, (mono- or sesqui-) terpenoids and phenylpropanoids/benzenoids (Knudsen et al., 2006). All the terpenoids (carotenoids, mono- and sesquiterpenoids) are synthesized from the same precursors – isopentenyl pyrophosphate and dimethylallyl pyrophosphate (Wu, 2008). Phenylpropanoid/benzenoid volatile compounds and flavonoid/anthocyanin pigments both originate from the same phenylpropanoid biosynthetic pathway (Dudareva et al., 2004, Zvi et al., 2008). In both terpenoids and phenylpropanoids, volatiles are early products of the pathway while pigments are produced at later steps. The fact that biosynthetic pathways are shared suggests the potential for correlated responses in color and scent to a single change in biochemistry. For instance, in a study manipulating pigmentation in Dianthus caryophyllus petals, Zuker et al. (2002) found that removal of petal pigment by antisense suppression of the flavanone 3-hydroxylase gene in the anthocyanin biosynthetic pathway led to higher emission of an aromatic volatile (methyl benzoate). Although a number of studies have investigated scent composition of different color morphs (reviewed in Dormont et al., 2010), as far as we know, no research has directly examined correlation between composition of pigments and of volatiles in plant species with flower polymorphism. Recent reviews consider the flower scent-color combination to be an outstanding open question in pollination ecology (Raguso, 2008, Rausher, 2008, Schaefer and Ruxton, 2009).
We investigated flower color polymorphism in a rewardless species common in the Northern part of the Mediterranean region, Iris lutescens, which displays a spectacular purple-yellow flower color polymorphism within populations (Fig. 1). Information on the reproductive biology of this species is presented in the Materials and methods section below. We analyzed the biochemical basis of color and scent, and explored ecological and evolutionary consequences of color differentiation. We aimed to determine whether pollinator agents (mainly bumblebees and solitary bees) are potential selective agents acting on the evolution of the flower color polymorphism. More specifically, we addressed the following four questions: (1) What are the pigmentation differences between the two color morphs? (2) Are the two color morphs discriminated between by insects? (3) What floral scents are emitted by the two color morphs? (4) Is there an association between floral scent and flower color in this species?
Section snippets
Quantifying pigment contents
The amounts of three classes of pigments, flavonoids (mainly chalcones, flavones and flavonols), anthocyanins and carotenoids, were all significantly different between plants of the two color morphs in flowers (petals and sepals, except for flavonoids in sepals, Fig. 2), while contents of none of them differed significantly in leaves (Fig. 2). The largest difference in flowers was for anthocyanins, which were present in about 18- to 28-fold higher concentrations in petals and sepals,
Concluding remarks
Three important results emerge from the present study. First, the most significant difference in pigmentation was that the concentration of anthocyanins was higher in purple flowers than in yellow flowers, while anthocyanin composition was the same for both morphs. No pigmentation difference (absolute or relative concentrations) was detected in leaves. Thus abiotic factors, herbivores (excluding florivores) and pathogens are unlikely to have influenced the evolution of flower color polymorphism
Study organism
I. lutescens Lam. (Iridaceae) is a perennial rhizomatous species, with a range that extends from Spain through France to Italy. The species occurs in dry places in the Mediterranean region of these countries. It grows 10–30 cm tall, with erect, sword-shaped leaves and one showy flower at the apex of each shoot. Each flower has three pendant, bearded sepals, alternating with three erect petals (Fig. 1). Flowers are hermaphroditic but self-incompatible, and thus highly pollinator-dependent.
Acknowledgements
The authors would like to thank David Carbonell and the staff of the CEFE’s field experiment station for their help in cultivating the irises. We also thank Bruno Buatois for chemical analyses in the labex CEMEB platform PACE (Platform for Chemical Analyses in Ecology) and Professor Christine Enjalbal for chemical analyses in the IBMM technical platform LMP (Laboratory for Physical Measurements). We are grateful to D. McKey and R. Kassen for their comments on the manuscript. Financial support
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- 1
These authors contributed equally to this work and are considered as joint first authors.
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These authors contributed equally to this work and are considered as joint last authors.