Journal of Biology Research 2016 5(1): 12-19 eISSN:2233-0275 pISSN:2233-1980 http://www.inast.org/jpbr.html

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Is the red coloration in the moranensis related with protection against photodamage or against herbivory?

Luz María Ayestarán, Raúl E. Alcalá*

Departamento de Ecología Evolutiva, Centro de Investigación en Biodiversidad y Conservación. Universidad Autónoma del Estado de . Av. Universidad 1001, Cuernavaca, Morelos, México. C. P. 62209.

ABSTRACT Understanding the relationship between phenotypic variation and environmental pressures has been useful to identify the factors that currently maintain functional traits in populations. The red coloration of derived from anthocyanins is frequently related with a protective role against biotic and abiotic factors. Using a carnivorous plant species as a model system, we evaluated two alternative hypotheses: 1) that red color confers protection against photodamage or 2) it confers protection against herbivory. In summer of 2015, 1153 individuals of Pinguicula moranensis were marked in 58 1-m2 plots within a population located in central in which patches of exhibit contrasting light environments (sunny and shaded habitats) due to different orientation (northeast vs. southwest, respectively). The association between color of plants (red and green) with the spatial variation in light environment was tested through a contingency table. Regarding herbivory, in the sunny habitat 35 pairs of individuals were photographed to quantify the percent of damage through image analysis. The mean values of herbivory were compared through a t-test with dependent groups using the arcsin transformation. The proportion of red plants was 2 one order of magnitude higher in the sunny site (1 = 89.53, P < 0.0001). The mean values for the percentage of herbivory show no differences between both groups (t = -1.688, p = 0.10, n = 35). Overall, our study suggests that there is no a dual role explaining the presence of red coloration in Pinguicula. The absence of effects of the red coloration on the percentage of damage suggests that the interaction with herbivores is not involved in the maintenance of red coloration. In contrast, the strong bias in the density of red plants toward the sunny habitat supports the hypothesis about the protective role against photodamage. Consequently, exposition to direct sunlight seems to be the most likely ecological factor explaining the color polymorphism in Pinguicula.

Keywords: Anthocyanins, Color polymorphism, Light environment, Sunscreen, Phenotypic variation.

INTRODUCTION or multiple roles is particularly interesting for the The study of phenotypic variation has been a central balance between fitness gains and the costs of theme in evolutionary ecology mainly because the synthesis and maintenance or phenotypic traits [10- inter-individual differences in phenotypic traits are 12]. Secondary metabolites are universally present related with the ability to cope with environmental in plants and the evidence about their role and its pressures [1-7]. Understanding the relationship evolutionary significance is increasing [13-17]. A between phenotypic variations and environmental scenario for multipurpose functions could be pressures has been useful to characterize for expected for secondary metabolites given that these example, the mode and magnitude of natural compounds can confer protection against several selection [8] and to identify the ecological factors biotic and abiotic factors [18,19]. Multi- those involved with their origin [9]. functionality would be advantageous as several The presence of specific functional traits with dual direct and indirect costs for the synthesis and storing * Corresponding author E-mail: [email protected]

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of secondary metabolites have been demonstrated they are involved with energy acquisition [43]. [20-22]. Therefore, it can be predicted that area lost by Secondary metabolites such as anthocyanins and the attack of herbivores could generate stronger other pigments are widely present in plants. For costs due to their dual role. If the polymorphism in example, these pigments are the responsible for the color could be somewhat involved with variation in color of several fruits and [23] and are also the level of defense, as has been demonstrated for frequent in vegetative organs including petioles, other plant species, the red coloration in Pinguicula veins, stipules [24] and leaves [25]. Anthocyanins could be doubly advantageous. could be costly as they absorb visible light. The In this study standing levels of leaf herbivory were competition with chlorophylls could consequently compared between green and red colored reduce the photosynthetic efficiency. Thus, several individuals of Pinguicula moranensis Kunth to test hypotheses have been advanced to account for their the hypothesis that anthocyanic plants exhibit less presence in leaves [19,26]. Some studies showed herbivory. In addition, we tested the hypothesis that that the red coloration produced by the if anthocyanins act as sunscreen a higher proportion concentration of anthocyanins can be induced by or red plants is expected in sites exposed to higher biotic and abiotic factors [27-29]. For example, direct sunlight. there is evidence indicating that in foliage, anthocyanins increase the protection against MATERIALS & METHODS photoinhibition and damage caused by UV- Study system radiation [25,30,31]. In addition, other studies This study was conducted in a region composed by suggest that anthocyanins tend to confer protection a series of hillsides primarily covered by - against the attack of herbivores and pathogens [29, forests in the northern portion of Cuernavaca City 32-35]. However, evidence about the simultaneous (Morelos, Mexico). Along a rocky wall of some 500 role of anthocyanins acting as a protection against m there were about 2000 individuals of Pinguicula herbivores and as a sunscreen in the same plant moranensis growing in three main patches ranging species is limited (but see, 18,36]. from 20 to 50 m2. Two patches were selected as they Members of Pinguicula L. are small-size, rosetted showed contrasting light conditions due to different and dimorphic perennial herbs that obtain mineral orientation of the wall in which plants were nutrients through the capture and assimilation of growing. In the northeast oriented patch (hereafter, prey [37]. In the field, the summer of most sunny habitat) plants are exposed to higher plants is pale-green colored. However, some irradiance as they received direct sunlight from individuals show red coloration ranging from a light 0900 h to 1400 h. In contrast, in the southwest- pigmentation on the leaves surface to individuals oriented patch plants are not exposed to direct bearing entire red colored leaves. Experimental sunlight. As a consequence this site shows shadier evidence showed that red coloration in Pinguicula and more humid conditions and the wall was is derived from the increase in anthocyanins covered by (hereafter, shaded habitat). providing protection against UV radiation and photoinhibition [38]. However plants of Pinguicula Pinguicula moranensis presents seasonally colonize sites that can vary in orientation and dimorphic leaves. The resistance rosette is present consequently could be exposed to contrasting light during the drier and cooler months from November regimes [30,40]. If anthocyanins are involved with to May and it is composed by tiny non-glandular protective roles functioning as a sunscreen it can be leaves. The summer rosette (i.e., capture rosette) is predicted that patches of populations naturally present from June to October, and it is composed of exposed to direct light should show a higher larger leaves (6-12 cm) covered by stalked secreting concentration of these pigments in plants [41]. glands and sessile digestive glands. Nearly 80% of Conversely, the effect of red coloration against the prey capture corresponds to small flying insects. damaged induced by herbivores has not been production occurs mostly the during evaluated in Pinguicula. This information would be summer months. The zygomorphic and useful, as independently from the color, leaves of hermaphroditic, spurred flowers are individually the summer rosette are the organs involved in prey inserted in stalks of up to 25 cm [44]. capture and assimilation [42], and at the same time,

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Spatial pattern of anthocyanic plants RESULTS In summer of 2014 (middle of the growing season) In the sunny site 778 plants were found in the 34 two quadrants (17×2 m and 12×2 m; long and plots, overall density corresponded to 25.9 plants height) were traced respectively in the sunny and per square meter. In this site 188 plants (~32%) shaded habitats. Each quadrant was divided in 1-m2 corresponded to the red morph and 590 had green plots to facilitate the identification of all plants in rosettes. In the shaded site only 375 plants were current demographic studies. In summer of 2015, marked in 24 plots resulting in a density of 15.62 we utilized these quadrants to evaluate the spatial plants per square meter. Only 7 individuals (~2%) pattern of red individuals. A red plant was defined were red colored and 368 were green plants. Red as a plant with at least 50% of the total area being plants were present only in 25% of the 1-m2 plots in red. The association between color of plants (red the shady habitat, whereas in the sunny site they and green) with the spatial variation in light were present in73% of the plots. There was a environment (sunny and shaded) was tested through statistical significant association between color of a contingency table [45]. rosettes and variation in light conditions. In the Herbivory measurement sunny site the proportion of red plants was one order Herbivory was measured only in the sunny site and of magnitude higher than in the shaded site 2 using a paired comparison to avoid confounding (1 =89.53, P<0.0001). effects related with differences in light environment. Regarding herbivory, the overall frequency In each one of the 1-m2 plots all red plants were distribution for the percentage of area lost by located and marked. However, in most plots only 1 herbivory was right skewed as most plants in both individual plant was selected for analysis to avoid groups received low levels of damage (i.e., 0 to 2%) sampling of genetically related individuals. Then, whereas only some individuals received relatively the nearest green plant of similar rosette size was higher damage (i.e., 7-15%). Despite of the selected for comparison (mean size did not differed occurrence of some highly damaged individuals in among both groups, statistical analysis not shown). the group of green plants, the shape of the To control for possible differences in herbivory distribution of the percentage of herbivory was not related to the ontogenetic stages, all measurements different between red and green plants according were made on well established rosettes excluding with the Kolmogorov-Smirnoff test (-0.17, 0.0; seedlings. negative and positive maximum difference; p>0.10, The estimation of herbivory was based on digital Fig. 1). In addition, the mean values for the images. Each focal plant was photographed from a percentage of herbivory was not statistically frontal plane (Nikon reflex D5100, 35mm f:1.8). different between both groups (t=-1.688, p=0.10, Gray cardboards were utilized to isolate the plant n=35, Fig. 2). from the backward. Each photograph included a ruler used to scale the rosette area and the area lost DISCUSSION by herbivory. The area corresponding to the rosette Understanding the ecological factors that maintain surface was estimated with the software Image J phenotypic variation has been a central theme in 1.49v [46], then the area corresponding to holes and evolutionary ecology. Particularly interesting are damage along the leaf margin was measured and those traits exhibiting dual or multiple roles as they summed to estimate the percentage of leaf area increases fitness while reducing costs. In this study damaged by herbivory. we evaluated for the first time the role of color With regard statistical analysis, a Kolmogorov- polymorphism in a carnivorous plant species with Smirnoff test was performed to test if the shape of regard two contrasting hypotheses accounting for the distribution of herbivory values differed the widely occurrence of anthocyanins in leaves. between the red and green plants. In addition, the The photodamage hypothesis [see 47 for details] hypothesis about difference in mean percentage of assumes that the energy derived from light and herbivory between red and green plants was tested absorbed by leaves generally exceed photosynthetic through a t-test with dependent groups using the demands. The excess of energy produces the angular transformation to fulfill normality formation of highly reactive species that in turn can assumptions [45].

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0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Percentageof herbivory Figure 1. Frequency distribution for the percentage of damage by herbivory in red plants (continuous line) and green plants (broken line) of Pinguicula moranensis.

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Figure 2. Percentage of damage by herbivory in red and green plants of Pinguicula moranensis. Mean (filled square), standard error (box) and 95% confidence limit (whiskers) are shown for each group.

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produce oxidative damage [47,48]. The available photodamage, establishing whether photodamage is evidence derived from some plant species indicates the main selective agent is a different issue [47]. We that anthocyanins could provide an antioxidant considered that our system offers the opportunity to activity that quench reactive chemical species. disentangle the causes that maintain color Therefore, the temporal fluctuation and the induction polymorphism. This is due to Pinguicula seems to of anthocyanins in leaves have been interpreted as suffer low damage by herbivory. For example, leaf advantageous against photodamage [49-53] and damage in P. vallisneriifolia Webb ranged from 0.2 against UV-B radiation [54, 55]. to 2% [58]. Experimental evidence with P. In this study we found that the proportion of red moranensis showed that the stalked glands that plants in the site receiving direct sunlight by several secrete droplets of useful to retain prey hours at day was one order of magnitude higher than currently maintain a strong physical defense against in the shaded habitat. In fact in the sunny site, almost herbivory reducing the mean damage to 1.6% [59]. all 1-m2 plots had red plants whereas this morph was Thus, defense in Pinguicula seems not to be nearly absent in the shaded habitat. Our results are in dependent of the red coloration as the defense accordance with the photodamage hypothesis [see derived from the stalked secretive glands is highly 26], from which a positive correlation between the effective. exposition to direct sunlight with the expression of Our study lacks of a direct experimental approach the red coloration in plants can be expected. For performed in controlled conditions to evaluate the example, total anthocyanins in Iris pumila L. was direct effect of leaf color on the rate of consumption higher for plants majorly exposed to direct sunlight of generalist herbivores. However, we utilized a and for all populations during summer months [41]. design including a paired comparison in which plants In our study the concentration of anthocyanins was within the sunny site were separated by no more than not measured. However, the red coloration in the 50 cm. Thus, based on the evidence we gathered the subartic Pinguicula vulgaris L. resulted as a presence of red coloration in Pinguicula is not consequence of the experimental induction of playing a dual role. The absence of effects of the red anthocyanins after excavated and potted plants were coloration on the percentage of damage suggests that exposed to UV-B radiation. Thus, it can be assumed the interaction with herbivores is not involved in the that the red coloration we observed was due to the maintenance of red coloration. In contrast, the strong presence of anthocyanins. The red coloration was bias in the density of red plants toward the sunny interpreted as advantageous in the open and habitat supports the hypothesis about the protective perturbed areas of subartic latitudes where plants role against photodamage. Consequently, exposition could be exposed to higher dangerous radiation [38]. to direct sunlight seems to be the most likely This interpretation about the experimental induction ecological factor explaining the color polymorphism of anthocyanins has major implications considering in Pinguicula. that plants of Pinguicula face a high environmental heterogeneity regarding light environment [39,40]. CONCLUSION Therefore the evidence about differences in the proportion of red plants derived from the natural ACKNOWLEDGENT patchy distribution of P. moranensis supports the We thank Sara G. Sánchez for tracing the quadrants photodamage hypothesis regarding the maintenance that greatly facilitated the sampling of individuals. of red coloration in plants. Lorena Sánchez provided field assistance to mark Alternatively, it has been hypothesized that and photograph plants. This manuscript was anthocyanins are involved in a protective role against enhanced by the comments provided by Marines de herbivores, although the mechanisms explaining the la Peña-Domene made on an early draft. differences in herbivory associated to color polymorphism are contrasting [18, 19, 26, 56,57]. In REFERENCES our study, neither the mean percentage of herbivory nor the frequency distribution of damage differed [1] Darwin, C. 1859. On the origin of species by between red and green plants. It has been stated that means of natural selection. J. Murray, London. although many phenolics including anthocyanins may play a role in protecting plants against

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