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Anthocyanin pigments in plant reproductive organs have long been known to serve as a visual signal to attract animals, promoting pollination and/or seed dispersal (Gould et al., 2009). In vegetative organs, however, anthocyanins might be deployed to repel animals. For example, to explain the autumnal reddening in the leaves of many deciduous trees, Archetti (2000) and Hamilton & Brown (2001) suggested that anthocyanins function as a visual warning to deter browsing by insect herbivores. Accordingly, red leaves would signal enhanced investment in defensive compounds that impair insect fitness. The signal would, in turn, be used by herbivores to select which plant to colonize. Aphids that switch host in autumn would colonize the least defended plants, and aphid infestation would be reduced in the redder, more strongly defended plants (Archetti, 2009). This so-named ‘co-evolution hypothesis’ has received indirect support in recent years from a variety of studies (Hamilton & Brown, 2001; Hagen et al., 2003, 2004; Archetti & Leather, 2005; Karageorgou et al., 2008).
Although originally proposed to explain the reddening or yellowing of the entire lamina in senescing leaves, Archetti’s (2000) and Hamilton & Brown’s (2001) hypotheses might similarly apply to more localized patterns of anthocyanin accumulation in nonsenescing leaves (Lev-Yadun et al., 2002). In many plants, anthocyanic pigmentation is restricted to the stipules, petiole, major veins, trichomes, marginal teeth and/or leaf apex (Wheldale, 1916; Hatier & Gould, 2009). Of particular interest in relation to animal communication are the anthocyanins in the epidermal and/or subepidermal cells at leaf margins. These can provide a prominent red border that presents a sharp chromatic contrast against the green lamina (Fig. 1a–d). Although there has been no published systematic survey of species bearing leaves with red margins, they are evidently very common. Red margins are often used by taxonomists as a character for species identification (Bayly & Kellow, 2006; Versieux & Wanderley, 2007; Redden, 2008), and as a marker to study genetic linkages in crop species as diverse as maize (Flint-Garcia et al., 2005), banana (Jarret et al., 1993), lettuce (Sabharwal & Doležel, 1993), mustard (Nick et al., 1993) and rice (Hadagal et al., 1981). There are also numerous reports of anthocyanic leaf margins being induced in response to mineral nutrient deficiencies (Walker, 1956; Johanson & Walker, 1963; Nyborg & Hoyt, 1970; Haque & Walmsley, 1973; Balo et al., 1975; Hassouna, 1977; Raese, 2002).
Figure 1. Red- (a, c, f) and green-margined (b, d, g) leaves of Pseudowintera colorata: (a, b) photographs of individual branches; (c, d) photographs of leaf laminae; (e) polygodial chemical structure; (f, g) photomicrographs of transverse sections. I, idioblast.
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The function (if any) of anthocyanins at the leaf margin is unknown, but their extremely restricted histological distribution within the leaf argues against many of the physiological hypotheses for foliar anthocyanins, such as photoprotection and antioxidant activities (Gould, 2004; Hatier & Gould, 2009). However, because many herbivores initiate feeding at the leaf edges, it is at least possible that the marginal anthocyanins function as a visual signal to indicate that the leaves contain unpalatable compounds. Edge-feeding is prominent among Orthopteran, Coleopteran and Lepidopteran larvae (Bernays, 1998), and the ability to detect red hues is well established among members of Lepidoptera and Coleoptera (Briscoe & Chittka, 2001).
During the process of edge-feeding, a plant loses not only those resources contained within the leaf portion eaten, but also the future photosynthetic potential of that portion. A damaged margin enhances evaporative water loss and could place water stress on the adjacent tissues, as well as increase the risk of infection by pathogens. Thus, edge-feeding is costly, and can be more detrimental than other types of herbivory, such as phloem feeding, wherein the leaves are left relatively intact. Relative growth rates of Solidago altissima, for example, have been shown to be reduced significantly by edge-feeding beetles, but are unaffected by sap-feeding aphids (Meyer, 1993). It is not surprising, therefore, that secondary metabolites which are considered to be involved in defence against insect herbivores are often more concentrated at the periphery than in the interior regions of a leaf lamina (Gutterman & Chauser-Volfson, 2000; Kester et al., 2002; Shroff et al., 2008; Hughes et al., 2010). Anthocyanins may provide the means for a plant to advertise defensive compounds in areas in which herbivores prefer to initiate feeding.
To our knowledge, only one previous study has thus far addressed a possible antiherbivory role for red leaf margins. Hughes et al. (2010) compared insect damage and leaf phenolic content across 11 Veronica species which differed in leaf margin colour. Contrary to the authors’ hypothesis, the presence of anthocyanins did not correspond to increased phenolic content at the leaf margins. However, the authors acknowledged limitations to their dataset as all plants had been growing together in a common garden and were not, therefore, subjected to natural herbivory pressure, and the study did not account for interspecific differences in leaf structure and biochemistry. Moreover, measurements of total phenolic content may not be the best estimate of defensive strength; the primary role of many phenolics appears to be to protect leaves from photodamage rather than from herbivory (Close & McArthur, 2002), and the phenolics which are known to be involved in defence constitute only a small fraction of the total phenolic pool (Lawler et al., 1998, 1999). Hughes et al. (2010) therefore recommended that future studies focus on specific defensive compounds, rather than general phenolic pools, to explore a possible defensive role of anthocyanic leaf margins, and to utilize intraspecific systems for better control of variation in leaf structure and chemistry. Thus, the hypothesis that red leaf margins reduce insect herbivory remains a possibility that warrants further investigation.
The New Zealand pepper tree, Pseudowintera colorata (Winteraceae), is, for several reasons, a particularly useful model to test for defensive functions of red leaf margins (Perry & Gould, 2010). First, populations exhibit pronounced variation in leaf margin colour and size among individuals (Fig. 1; P. colorata leaves also develop red margins around areas of mechanical damage (Gould et al., 2002), but this induced coloration was not a factor in the present study). Second, the primary defence compound in P. colorata leaves is known; it is the sesquiterpene dialdehyde polygodial (Fig. 1e), which imparts a pungent taste and has potent insect antifeedant properties (Barnes & Loder, 1962; Asakawa et al., 1988). In vitro studies have shown polygodial to be an effective antifeedant at concentrations of 3 mg g−1 (Gerard et al., 1993), whereas P. colorata leaves contain at least 10 mg g−1 (Wayman et al., 2010). Finally, P. colorata herbivores have been documented (http://plant-synz.landcareresearch.co.nz/; accessed November 2011). Thus, P. colorata presents an opportunity both to collect correlative data on margin colour, herbivory damage and polygodial content, and to conduct feeding trials under controlled laboratory conditions using natural herbivores.
In this study, we tested the fundamental requirement of any visual signal – that red leaf margins in P. colorata provide a reliable indication of the defensive status of a plant. We hypothesized that: polygodial concentrations would be highest around the leaf margin, polygodial concentrations in leaves with red margins would be greater than those in leaves with green margins, and leaves with the larger red margins would incur less herbivory. We also tested whether a natural edge-feeding generalist herbivore of P. colorata, the brownheaded leafroller moth (Ctenopseustis obliquana), responds to the red leaf margins as a visual signal, and avoids the more strongly defended plants. We hypothesized that C. obliquana larvae would prefer to eat, and adults to oviposit, on leaves with green margins, but only under light conditions in which colour discrimination was possible.
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Three sets of evidence from our analysis of P. colorata indicate that red leaf margins may function as a visual signal to deter insect herbivores. First, relative to the green leaves, those leaves that bore a wide red margin were significantly richer in polygodial, a potent insect antifeedant (Fig. 3a). The red margins are therefore a reliable signal of increased investment in this defensive compound. Second, the presence of red margins was associated with a reduced propensity for herbivory damage in a natural P. colorata population (Fig. 4b,c). Third, in laboratory trials, the larvae of C. obliquana, a natural herbivore of P. colorata, preferred to feed on green- rather than red-margined leaves, but only when the experiment was conducted under white light which enabled colour vision. The larvae evidently respond to a visual rather than an olfactory signal. Collectively, these data present a compelling case for a new functional role of anthocyanins in leaf margins, and they add empirical support to the co-evolution hypothesis (Archetti, 2000; Hamilton & Brown, 2001) and aposematic coloration in red leaves (Lev-Yadun, 2006, 2009; Lev-Yadun & Gould, 2007, 2009; Lev-Yadun & Holopainen, 2009).
Irrespective of colour, levels of polygodial were consistently greater at the leaf margins than in the leaf interior (Fig. 3a). Previous studies have similarly shown higher concentrations of phenolic compounds (Gutterman & Chauser-Volfson, 2000; Hughes et al., 2010), glucosinolates (Shroff et al., 2008), nicotine (Kester et al., 2002) and trichome density (Rautio et al., 2002), all implicated in herbivory defence, at the leaf margin. By concentrating defensive compounds around the leaf margin, plants may be selectively targeting edge-feeding herbivores. As the interiors of red-margined leaves were also richer in polygodial than those of green leaves (Fig. 3a), anthocyanins may indicate not only that the leaf periphery is especially rich in polygodial, but also the overall differences in investment in defences among individuals.
A conceptual problem with the co-evolution hypothesis is that many insects are incapable of perceiving red hues (Döring & Chittka, 2007; Archetti, 2009). Although the receptors needed to perceive red light have evolved multiple times in Lepidoptera (Briscoe & Chittka, 2001), it is not known whether they are present in C. obliquana larvae. Nonetheless, insects lacking red receptors have been shown to distinguish red from green by using the ratio of green to blue light reflected from a leaf (Doring et al., 2009), and, for P. colorata, the ratio of reflected green to blue light was almost two-fold lower in the green margins (Fig. S1). Aphids lack red receptors, yet apparently discriminate between red and green leaves (Doring et al., 2009). Lepidopteran larvae, including one species within Tortricidae, the family containing C. obliquana, have similarly been shown to discriminate between red and green stimuli (Harris et al., 1995; Singh & Saxena, 2004).
Compared with chemoreception, the importance of visual detection in locating insect host plants has been largely neglected (Reeves, 2011). In our study, C. obliquana larvae did not appear to distinguish between leaves with different margin colours when illuminated under monochromatic green or red light, or in the absence of light. However, under white light, green-margined leaves were clearly preferred (Fig. 5). Under the monochromatic light, the colour contrast between red margin and leaf interior disappears, although an achromatic contrast remains between the part reflecting light (e.g. red margin under red light) and the leaf part not reflecting light (e.g. green interior). Our data indicate that the larvae used chromatic rather than achromatic contrast to distinguish between the two leaf types.
The feeding preference for green leaves under white light was evident only when both leaf types had been sampled. The avoidance of red-margined leaves appears not to be innate but, rather, relies on gustatory or post-ingestive feedbacks. Importantly, C. obliquana larvae did not use olfactory cues or the position of leaves within an enclosure to discriminate between the food sources. Although previous studies have demonstrated an innate preference by some Lepidopteran larvae towards green stimuli (Singh & Saxena, 2004; Hora et al., 2005; Yasui et al., 2006), our study suggests that learning based on colour contrasts may play an important role in distinguishing host quality.
Red leaf margins would be a more effective herbivore deterrent if the signal were perceived by gravid females as well as their larvae. Lepidopteran adults are generally more mobile than larvae (Hagstrum & Subramanyam, 2010), and C. obliquana females deposit multiple eggs during oviposition. Contrary to our hypothesis, the frequency of oviposition by gravid C. obliquana was similar on green- and red-margined leaves, under both white light and darkness (Table 1). Qiu et al. (1998) found that polygodial inhibited oviposition by the diamondback moth, although this involved an assay in which polygodial solution had been applied to the surface of test filter paper discs. Our result may be attributable to the life history of the animals used for oviposition trials, for which the adults had been raised on a general purpose diet (Singh, 1983) and had no previous experience of P. colorata. The oviposition preference of some Lepidoptera has been shown to be influenced by their diet during larval stages (Anderson et al., 1995; Akhtar & Isman, 2003; Chow et al., 2005; Hora et al., 2005; Olsson et al., 2006). As the feeding preference of C. obliquana larvae appears to be a learned response, it is reasonable to hypothesize that adult oviposition preference, too, requires previous experience with P. colorata.
For convenience, leaf margins were categorized as red or green. In reality, however, red margin size represented a continuum (Fig. 4); clusters of anthocyanic cells were present in all P. colorata leaves examined, including those which, to the human eye, appeared green (Fig. 1g). As leaves with the larger margins incurred proportionately less damage by herbivores (Fig. 4b,c) it is of interest to ask whether there is a critical size beneath which a red leaf margin ceases to be effective as a visual cue. We observed a sharp decline in the variance of herbivory damage when the red margin was c. > 2% of the lamina width. Thus, only an extremely small proportion of the lamina is required to be red for it to act as an effective deterrent. Pseudowintera colorata leaves can have red margins that extend 25% or more across the leaf lamina (Fig. 4), but the potential additional antiherbivory benefit of these large margins appears to be slight.
Herbivory damage correlated more strongly with the relative rather than absolute widths of leaf margins. Insect preference is evidently determined by the ratio of red to green leaf areas, rather than by the margin size per se. Relative size may be easier for an insect to evaluate against a concept of the maximally acceptable margin size. Alternatively, given that margin polygodial concentrations exceeded the interior for all leaves, the size of red margins may represent the proportion of lamina that is highly defended, that is, the amount of highly defended lamina relative to less defended lamina.
Unlike older leaves, the margin width at node 1 did not correlate with the extent of herbivory (Fig. 4a). Leaves at this position possessed the broadest margins (Fig. 2a) and, by virtue of their location at the periphery of the canopy, were the most prominent to any approaching herbivore. However, the levels of herbivory on leaves at node 1 were significantly lower than those at lower nodes (Fig. 2b), most probably because these younger leaves had been exposed to herbivores for a shorter duration. The benefits of red margins would be increasingly apparent as the leaves aged.
Although we have referred to red margins as visual ‘signals’, the term has an inherent implication which is yet to be confirmed. ‘Signal’ implies that red margins evolved in response to herbivore pressure (Otte, 1974). The alternative, ‘cue’, implies that the association between red margins and polygodial evolved for another purpose, and insects have subsequently altered their behaviour to make use of it. Because neither scenario can, as yet, be confirmed, we maintain the use of ‘signal’ whilst acknowledging its implications. Despite this limitation, one criterion for a signalling relationship was satisfied. The effect of a red leaf margin appears to translate to an overall benefit for an individual plant. When averaging all three nodes for a single plant, those with the largest mean rank in margin width incurred the least overall herbivory. Evidently, the benefits received by a single leaf from the deterrence of herbivores can be scaled up to benefit an entire plant, and is consistent with red leaf margins in P. colorata having evolved in response to edge-feeding herbivores.
Red leaf margins in P. colorata signal increased polygodial concentrations and correlate with reduced herbivory by edge-feeding insects. In laboratory feeding trials, a preference for green-margined leaves by C. obliquana was dependent on ambient light quality. When the conditions necessary to perceive margin colour were removed, no difference was found in the consumption of red- vs green-margined leaves. Although there has been increasing theoretical support for aposematic coloration in red leaves (Lev-Yadun, 2009; Schaefer & Ruxton, 2011), direct experimental evidence has thus far been lacking. Our study is the first to demonstrate a possible adaptive function for this common pattern in leaf coloration.