Summary• Red-pigmented leaf margins are common, but their functional significance is unknown. We hypothesized that red leaf margins reduce leaf herbivory by signalling to herbivorous insects the presence of increased chemical defences.• Leaves were collected from a natural population of Pseudowintera colorata. Margin size, herbivory damage, anthocyanin content and concentrations of polygodial, a sesquiterpene dialdehyde with antifeedant properties, were quantified. Feeding trials involving larvae of Ctenopseustis obliquana, a generalist herbivore, were conducted on red-and green-margined P. colorata leaves in darkness, or under white, green or red light.• Leaves with wider red margins contained higher concentrations of polygodial and anthocyanins, and incurred less natural herbivory. In trials under white light, C. obliquana consumed disproportionately more green-than red-margined leaf laminae. Larvae exhibited no feeding preference when light was manipulated such that leaf colour discrimination was impaired.• Red leaf margins provide a reliable and effective visual signal of chemical defence in P. colorata. Ctenopseustis obliquana larvae perceive and respond to the colour of the leaf margins, rather than to olfactory signals. Our study provides direct experimental evidence for aposematic coloration in red leaves.

Summary 1.Whether plants use leaf colour to deter herbivores remains controversial. The warning signal hypothesis predicts that red pigmentation is adaptive by reducing herbivory; plants with predominantly red foliage should have higher fitness than those with green leaves. Despite many discussions, this prediction has rarely been tested, and alternative, non-exclusive hypotheses cannot be ruled out. 2.We have exploited leaf colour polymorphism in Pseudowintera colorata to test the warning signal hypothesis and to address possible alternative explanations.3. Consistent with warning signals, redder foliage contained higher concentrations of polygodial, a sesquiterpene dialdehyde with strong antifeedant properties, and incurred less herbivory than green leaves. Redder plants hosted 22% fewer lepidopteran leafroller larvae than neighbouring green plants. 4.However, contrary to the predictions of the hypothesis, there were no differences in fitness parameters between red and green plants. Overall leaf canopy colour was not a significant predictor of the number of seeds per fruit or of mean seed weight. This may be explained by differences in photosynthesis: green P. colorata leaves had 47% higher maximum CO 2 assimilation rates than matched red leaves from neighbouring plants. 5.These results indicate that the benefits of deterring insect herbivores by signalling may be balanced by the higher photosynthetic rate of non-signalling plants. A balance between signalling and photosynthesis is a novel mechanism for the maintenance of leaf colour polymorphisms in nature.6. Synthesis. Anthocyanin pigments may simultaneously serve multiple functions within leaves, and individuals of the same plant species may use different strategies to cope with insect herbivores. Therefore, investigations into the role of these pigments in plant-insect interactions need to consider plant physiology and the diversity of plant defence mechanisms.

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<p>Anthocyanin pigments are synthesised in the leaves of many plants, however the adaptive significance of these pigments is not entirely understood. It has been postulated that their red colours may function as visual signals through coevolution between herbivorous insects and their host tree species, though the hypothesis lacks solid empirical evidence. I investigated the leaf signalling hypothesis using Pseudowintera colorata, focusing on five areas:  1) I exploited the natural polymorphism in leaf colour of P. colorata to test the predictions that (i) bright leaf colour is a reliable signal of a plant’s defensive commitment; (ii) insects in the field avoid trees that are brightly coloured; and (iii) the trees with the brightest leaves will have higher fitness. Relative to green leaves, redder foliage contained higher concentrations of polygodial, a sesquiterpene dialdehyde known to have strong antifeedant properties, and incurred less insect feeding damage. Redder trees hosted fewer Ctenopseustis spp. leafroller larvae than neighbouring matched green trees. Contrary to the predictions of the leaf signalling hypothesis, there was no difference in any of the measured fitness parameters between red and green trees, indicating that the leaf colour polymorphism in P. colorata is stable.  2) Many insects are sensitive to volatile organic compounds (VOCs), however the role of VOCs in plant-herbivore signalling has not been investigated. I analysed VOCs released from undamaged, herbivore- and mechanically-damaged red and green leaves of P. colorata, and the olfactory preferences of brownheaded leafroller (C. obliquana) larvae. While the VOC profiles of browsed and unbrowsed leaves were statistically distinguishable, the VOC profiles released from intact, herbivore-, and mechanically-damaged P. colorata leaves did not reliably identify leaf colour. Moreover, naïve and experienced C. obliquana larvae displayed no preference for the volatiles from mechanically-damaged red or green leaves. Therefore, I concluded that VOC compounds are not likely to play a large role in mediating insect herbivore-plant interactions in P. colorata.  3) Studies of leaf signalling rarely consider the influence of the light-absorbing properties of non-green pigments upon photosynthesis. I compared the photosynthetic and photoinhibitory responses of red and green leaves from matched, neighbouring pairs of P. colorata of contrasting colour. Redder P. colorata leaves in the field had a lower maximum photosynthetic assimilation rate than matched green leaves from neighbouring trees. However, I was unable to detect any measurable advantage in terms of photoprotection in the red P. colorata leaves as indicated by chlorophyll fluorescence profiles. My results indicate that the presence of anthocyanin pigments within non-senescing leaves may impose a slight photosynthetic cost to the plant.  4) I used literature searches, field surveys and laboratory bioassays to identify which invertebrate herbivores are most likely to participate in leaf-signalling interactions with P. colorata. Feeding preference bioassays showed that brownheaded leafrollers (C. obliquana and C. herana) and Auckland tree weta (Hemideina thoracica) preferentially consumed leaf material from green than red P. colorata leaves. Results from these bioassays, combined with my field surveys suggest that Ctenopseustis spp. leafroller larvae are the most likely coevolution partners for P. colorata.  5) There is a well-established link between nitrogen deficiency and leaf reddening. Additionally, leaf nutrients can influence foraging behaviour and performance of insect herbivores. I measured N and C contents of leaves from neighbouring matched pairs of red and green P. colorata. There were no significant differences in the amounts of, or ratio between, N and C between matched red and green leaves. This result indicates that differences in colour and herbivory among P. colorata leaves are not attributable to differences in leaf nutrients. Taken together, my results suggest that foliar anthocyanins in P. colorata do function as visual signals, however their effect on herbivory is small. Additionally, interindividual variation in non-senescing leaf colour in P. colorata may be stable due to a trade off between signalling and photosynthesis. Discussions of leaf signalling need to follow the examples of other fields studying the interactions between plants and insects and move from overly simple models to those that incorporate more of the complexity that is observed in the natural world.</p>

<p>Anthocyanin pigments are synthesised in the leaves of many plants, however the adaptive significance of these pigments is not entirely understood. It has been postulated that their red colours may function as visual signals through coevolution between herbivorous insects and their host tree species, though the hypothesis lacks solid empirical evidence. I investigated the leaf signalling hypothesis using Pseudowintera colorata, focusing on five areas:  1) I exploited the natural polymorphism in leaf colour of P. colorata to test the predictions that (i) bright leaf colour is a reliable signal of a plant’s defensive commitment; (ii) insects in the field avoid trees that are brightly coloured; and (iii) the trees with the brightest leaves will have higher fitness. Relative to green leaves, redder foliage contained higher concentrations of polygodial, a sesquiterpene dialdehyde known to have strong antifeedant properties, and incurred less insect feeding damage. Redder trees hosted fewer Ctenopseustis spp. leafroller larvae than neighbouring matched green trees. Contrary to the predictions of the leaf signalling hypothesis, there was no difference in any of the measured fitness parameters between red and green trees, indicating that the leaf colour polymorphism in P. colorata is stable.  2) Many insects are sensitive to volatile organic compounds (VOCs), however the role of VOCs in plant-herbivore signalling has not been investigated. I analysed VOCs released from undamaged, herbivore- and mechanically-damaged red and green leaves of P. colorata, and the olfactory preferences of brownheaded leafroller (C. obliquana) larvae. While the VOC profiles of browsed and unbrowsed leaves were statistically distinguishable, the VOC profiles released from intact, herbivore-, and mechanically-damaged P. colorata leaves did not reliably identify leaf colour. Moreover, naïve and experienced C. obliquana larvae displayed no preference for the volatiles from mechanically-damaged red or green leaves. Therefore, I concluded that VOC compounds are not likely to play a large role in mediating insect herbivore-plant interactions in P. colorata.  3) Studies of leaf signalling rarely consider the influence of the light-absorbing properties of non-green pigments upon photosynthesis. I compared the photosynthetic and photoinhibitory responses of red and green leaves from matched, neighbouring pairs of P. colorata of contrasting colour. Redder P. colorata leaves in the field had a lower maximum photosynthetic assimilation rate than matched green leaves from neighbouring trees. However, I was unable to detect any measurable advantage in terms of photoprotection in the red P. colorata leaves as indicated by chlorophyll fluorescence profiles. My results indicate that the presence of anthocyanin pigments within non-senescing leaves may impose a slight photosynthetic cost to the plant.  4) I used literature searches, field surveys and laboratory bioassays to identify which invertebrate herbivores are most likely to participate in leaf-signalling interactions with P. colorata. Feeding preference bioassays showed that brownheaded leafrollers (C. obliquana and C. herana) and Auckland tree weta (Hemideina thoracica) preferentially consumed leaf material from green than red P. colorata leaves. Results from these bioassays, combined with my field surveys suggest that Ctenopseustis spp. leafroller larvae are the most likely coevolution partners for P. colorata.  5) There is a well-established link between nitrogen deficiency and leaf reddening. Additionally, leaf nutrients can influence foraging behaviour and performance of insect herbivores. I measured N and C contents of leaves from neighbouring matched pairs of red and green P. colorata. There were no significant differences in the amounts of, or ratio between, N and C between matched red and green leaves. This result indicates that differences in colour and herbivory among P. colorata leaves are not attributable to differences in leaf nutrients. Taken together, my results suggest that foliar anthocyanins in P. colorata do function as visual signals, however their effect on herbivory is small. Additionally, interindividual variation in non-senescing leaf colour in P. colorata may be stable due to a trade off between signalling and photosynthesis. Discussions of leaf signalling need to follow the examples of other fields studying the interactions between plants and insects and move from overly simple models to those that incorporate more of the complexity that is observed in the natural world.</p>