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50% of the aerial mass) and two levels of light (sun vs. shade) and water (one vs. two waterings). Water had no appre- ... relative importance to plant performance varies as plants progress .... seedlings were situated under the different levels of water and light, so the ... Simulated herbivory significantly decreased all morpho-.
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Herbivory has a greater impact in shade than in sun: response of Quercus pyrenaica seedlings to multifactorial environmental variation Elena Baraza, José M. Gómez, José A. Hódar, and Regino Zamora

Abstract: Many biotic and abiotic factors affect seedling establishment in woody plants. In Mediterranean environments, the major factors affecting tree regeneration are light, water, and herbivory. We investigated the response of some morphological and chemical traits of Quercus pyrenaica Willd. seedlings to simulated herbivory (hand removal of 50% of the aerial mass) and two levels of light (sun vs. shade) and water (one vs. two waterings). Water had no appreciable direct effect on morphological or chemical traits. Shaded seedlings grew less but had greater total leaf area. Simulated herbivory decreased the total leaf area, and root and aerial mass. Among the chemical characteristics, shaded seedlings had higher levels of nitrogen and lower levels of condensed tannins. In colorimetric assays of tannins, clipped seedlings had lower absorbances than did unclipped plants, and this effect was more pronounced in the sun than in the shade. Our experiment shows that light availability and herbivory affect the development and defence of Q. pyrenaica seedlings. Although Q. pyrenaica tolerated shade and simulated herbivory, both factors decreased biomass and chemical defence, which could affect the seedlings’ future performance. Key words: environmental context, light, mammal herbivory, oak seedlings, Quercus pyrenaica, secondary compounds. Résumé : Plusieurs facteurs biotiques et abiotiques affectent l’établissement des plantules chez les plantes ligneuses. Dans les milieux méditerranéens les principaux facteurs affectant la régénération des arbres sont la lumière, l’eau et l’herbivorie. Les auteurs ont étudié la réaction de certains caractères morphologiques et chimiques des plantules du Quercus pyrenaica Willd., à l’herbivorie simulée (élimination manuelle de 50 % de la masse aérienne), à deux niveaux d’illumination (soleil vs ombre) et à l’eau (un vs deux arrosages). L’eau n’a pas d’effet direct appréciable sur les caractères morphologiques et chimiques. Les plantules poussent moins à l’ombre mais développent une plus grande surface foliaire totale. L’herbivorie simulée diminue la surface foliaire totale et le poids des racines et des parties aériennes. Parmi les caractères chimiques, les plants ombragés ont des teneurs en azote plus élevées et plus faibles en tannins condensés. Des essais colorométriques sur les tannins montrent que les plantules coupées ont des absorbances moindres que les plants non coupés, et que cet effet est plus prononcé au soleil qu’à l’ombre. L’expérience montre que la disponibilité de la lumière et l’herbivorie affectent le développement et la défense des plantules du Q. pyrenaica. Bien que le Q. pyrenaica tolère l’ombre et l’herbivorie simulée, les deux facteurs diminuent la biomasse et la défense chimique, ce qui peut affecter leur performance future. Mots clés : contexte environnemental, lumière, herbivorie des mammifères, plantules de chêne, Quercus pyrenaica, composés secondaires. [Traduit par la Rédaction]

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Introduction Forest regeneration is influenced by biotic factors including browsing, competition, and insect pests (Ammer 1996; Buckley et al. 1998; Meiners et al. 2000; Weltzin et al. 1998; Van der Wal et al. 2000; Wada et al. 2000), by abiotic factors including resources such as light, water, and nutrients (Ibañez and Schupp 2001; Riley and Jones 2003), and by Received 14 October 2003. Published on the NRC Research Press Web site at http://canjbot.nrc.ca on 7 April 2004. E. Baraza, J.M. Gómez, J.A. Hódar, and R. Zamora. Departamento de Biología Animal y Ecología, Facultad de Ciencias, Universidad de Granada, Granada, E-18071, Spain. 1

Corresponding author (e-mail: [email protected]).

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environmental conditions including temperature and soil texture (Grubb 1977). Many of these factors covary, and their relative importance to plant performance varies as plants progress through life-history stages (Lentz and Cipollini 1998; Germaine and McPherson 1999; Gómez et al. 2003). The seedling stage is one of the most vulnerable stages for most plant species (Harper 1977), and processes during these stages are thereby critical for understanding plant regeneration. Light and water are major environmental factors that influence the performance of many tree species in Mediterranean-type ecosystems and determine the most appropriate microhabitat for their regeneration (Zamora et al. 2001; Gómez 2004; Gómez et al. 2003). Apart from the direct impact of light and water on the performance of tree seedlings (Meiners and Handel 2000; Welander and Ottosson 1998,

doi: 10.1139/B04-004

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2000), they also modify the plant’s ability to tolerate or defend against herbivory (Maschinski and Whitham 1989; Herms and Matson 1992; Strauss and Agrawal 1999; Oba et al. 2000). Their availability affects the biochemical functions of the plant and triggers variations in the concentration of different elements such as nitrogen and secondary metabolites (Chapin et al. 1987). Previous studies on foliar chemistry of trees have shown a higher phenolic concentration in high-light environments than in low-light environments (Shure and Wilson 1993; Dudt and Shure 1994). Plants growing under contrasting conditions of light and water may differ in physiological and biochemical features, prompting a different response to damage (Maschinski and Whitham 1989; Coleman and Jones 1991). Pisani and Distel (1999) demonstrated in two species of Prosopis that when conditions for growth were relatively favourable (wet year), defoliated branches were able to compensate for the lost foliage and leaf phenol concentration was lower than in intact control branches. Conversely, when conditions for growth were unfavourable (dry year), defoliated branches were unable to compensate for the loss of foliage and leaf phenol concentration was higher than in control branches. The risk and magnitude of damage suffered by the seedlings is influenced by many chemical, nutritional, and structural traits of the plant (Coley et al. 1985; Zamora et al. 1999; Baumont et al. 2000). Some of these traits, such as chemical and mechanical defences, as well as compensation ability, can offset the negative impact of herbivores, improving the probability of plant recruitment and forest regeneration (Weltzin et al. 1998). Moreover, subsequent changes in plants after herbivory may be crucial in determining their future performance (Van Hees et al. 1996) and could even influence their future interactions with herbivores (Provenza and Malechek 1984; Hunter 1991). Quercus pyrenaica Willd. (Fagaceae) is a resprouting, deciduous oak distributed from southwestern France to northern Morocco, inhabiting the Sierra Nevada (southeastern Spain) from 1600 to 1800 m a.s.l. In these mountains, most of these forests have disappeared because of the combined effects of several management practices. In the remnant forests, a diverse assemblage of vertebrate herbivores, notably wild boar (Sus scrofa), Spanish ibex (Capra pyrenaica), and domestic livestock, frequently damage seedlings and juveniles. We have shown experimentally that these mammalian herbivores kill up to 90% of the Q. pyrenaica seedlings in these Mediterranean mountains areas (Gómez et al. 2003). This oak is a late-successional species that might show shade tolerance as well as a strong link between levels of carbohydrates and concentration of phenolic compounds in the leaf (Coley et al. 2002). In the present study, we experimentally determined the effects of three essential factors (light, water, and herbivory) on morphological and chemical characteristics of Q. pyrenaica seedlings. Specifically, we tested the following predictions: (1) shade decreases growth and the production of chemical compounds; (2) the addition of water improves growth and decreases the production of chemical compounds; (3) herbivory affects growth and architectural patterns; and (4) biochemical response to herbivory differs depending on the environment.

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Materials and methods Seeds and seedlings In November 1997, several thousand ripe acorns were collected from at least 20 trees in a forest situated at 37°05′N, 03°26′W (Parque Nacional de Sierra Nevada, Granada province, southern Spain). The acorns were pooled to avoid problems related to interindividual differences in acorn traits and stored in a cold room (2–4 °C) for several weeks. In December 1997, the acorns were planted individually in cylindrical pots (0.3 L) located in a fenced plot situated approximately 4 km from the forest and at the same altitude (1650 m a.s.l.). Acorns germinated from February to March 1998, and seedlings emerged in late May and June, as under natural conditions. The seedlings were kept all summer in the nursery and weeded periodically. In April 1999, 400 seedlings were randomly selected, root pruned to a root length of 20–25 cm, and potted in 2.5-L plastic pots containing a mixture of peat, vermiculite, and topsoil (1:1:2) without litter from the site where the parent trees were growing. Experimental design The experiment involved the following three factors: light, water, and herbivory. Light Light had two intensities: (1) sun, where plants were illuminated with full sunlight (photosynthetic photon flux density (PPFD): 2033.0 ± 3.2 µmol·m–2·s–1), measured by LI-COR LI-200 sz Pyranometer Sensor connected to a Li-1000 data logger (LI-COR Inc., Lincoln, Nebr., USA); (2) shade, where plants were set under a neutral shading net with only 5% of the sunlight (PPFD: 96.2 ± 8.1 µmol·m–2·s–1). These two levels of light represented the two extremes possible under natural conditions. Full light corresponded to radiation in open areas, while shade corresponded to radiation under the canopy of adult oaks. Water Water had two levels: (1) single, where plants were watered with 1.5 L weekly; (2) double, where plants were watered with 1.5 L twice weekly. We chose the single level to avoid mortality due to summer drought in potted seedlings. Only a few of the seedlings died, 15% in sun and 1.5% in shade, with no differences between single and double watering. During the summer of the year 2000, the soil moisture (sensor ThetaProbe ML2x, Delta-T Devices Ltd., Cambridge, UK; error ±1%) of the experimental seedlings showed a significant effect of light and water treatment, and also an interaction between them (F[1,395] = 36.70–1071.17, P < 0.0001 for all cases, two-way ANOVA), because moisture was higher at the double than at the single water level, but also was higher in shade than in sun. Herbivory Herbivory had two levels: (1) clipped, where 50% (or 50% + 1 in the case of odd numbers) of the stems of each seedling were removed with scissors; main and higher branches were chosen to simulate the damage provoked by © 2004 NRC Canada

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ungulates (Gómez et al. 2003); (2) unclipped, where the plants were left untouched as a control. In April 1999, each of the 400 potted seedlings was randomly assigned to one of the eight treatments, resulting in 50 oaks per treatment. Random assignation resulted in no initial between-group differences in plant trunk basal diameter (light F[1,391] = 0.176, P = 0.675; water F[1,391] = 1.915, P = 0.16; herbivory F[1,391 = 0.507, P = 0.477, three-way ANOVAs; data taken in April 1999). The herbivory treatment was applied in September 1999, 6 months after the seedlings were situated under the different levels of water and light, so the environment they were growing in could affect the response to artificial herbivory. Data collection In September 2000, before leaf senescence (1 year after the application of herbivory), the seedlings were harvested, carefully washed to avoid the loss of fine roots, and placed in plastic bags. In the laboratory, the number of leaves and stems from each seedling was counted and the organs (roots, stems, and leaves) were separated and dried in an oven for 1 week at 45 °C to determine their dry mass. The leaves were pressed and scanned to measure leaf area per seedling with a computer imaging system (Medidor de Objetos 1999– 2000, R. Ordiales Plaza®, Almería, Spain). For each seedling, the following morphological traits were determined: (1) number of leaves, (2) number of stems, (3) total leaf area (in mm2), (4) leaf dry mass per unit area or ratio of leaf dry mass to leaf area (in g/m2), (5) root dry mass (in g), (6) leaves plus stems dry mass (in g), and (7) shoot/root ratio. The following chemical variables were quantified: (1) total nitrogen content, expressed as percentage of dry mass and quantified by sulphuric acid digestion of 0.5 g dry powder, distillation in a semiautomatic Kjeldahl distiller (Büchi), and subsequent titration; (2) total phenols, analyzed by the Folin–Ciocalteu method; and (3) condensed tannins, analyzed by the proanthocyanidin assay. These procedures are considered adequate for relative comparisons of similar plant materials (Waterman and Mole 1994; Reed 1995; Yu and Dahlgren 2000; Covello and Gallardo 2001; Coley et al. 2002). Phenolic compounds were extracted from 0.5 g dry powder with 10 mL of 50% (v/v) methanol in an ultrasonic bath for 15 min and ultracentrifuged at 2500 r/min for 15 min. For the analysis of total phenols, an aliquot was diluted with water and assayed with the Folin–Ciocalteu phenol reagent and 20% sodium carbonate. Absorbance was then measured at 740 nm (Hódar and Palo 1997). For the analysis of condensed tannins, the same extract was assayed with butanol – hydrochloric acid reagent (0.7 g ferrous sulphate heptahydrate in 50 mL concentrated HCl and n-butanol added to make 1 L), and absorbance was measured at 550 nm (Waterman and Mole 1994). The phenolic compound concentrations were statistically analyzed with absorbance direct values, without transformation to standard equivalents (see Waterman and Mole 1994 for a full explanation of this procedure). These three chemical traits were analyzed for both leaves and leaves plus stems because although leaves are the most usual plant tissue in chemical studies, the major herbivores

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of Q. pyrenaica at the study site were ungulates, which browse on the entire seedlings (Gómez et al. 2003). Each chemical analysis was made on a random subsample of experimental seedlings ranging from 47 to 54. Statistical analysis Three-way ANOVAs (GLM procedure; SAS/STAT Version 6.12, SAS Institute Inc. 1997) were used, introducing each characteristic of the seedling as a dependent variable, the treatments as fixed factors, and the seedlings as the experimental units. We used type III sum of squares because of the unbalanced nature of the data after removing from the analyses seedlings that died before the end of the experiment. To improve homoscedasticity, an arcsine transformation was performed on proportional data and logarithmic transformation on the remaining variables (Zar 1996). Because we repeated the same model for several plant traits, we used the fixed Bonferroni correction to avoid experiment-wise type I error, adjusting α to 0.05 in the statistical tests, which resulted in different P values.

Results Morphological traits The number of leaves correlated strongly with the number of stems (r = 0.778, P < 0.0001), and therefore only the first trait was included in the main analysis. Seedlings in the sun had more leaves and greater leaf mass per unit area, root dry mass, and leaves plus stems dry mass, but less total leaf area and a lower shoot/root ratio than did shade-grown ones (Table 1, Fig. 1). In addition, a significant light × herbivory interaction appeared for the number of leaves (Table 1), because the effect of herbivory proved to be significant only for shade-grown seedlings (Fig. 1). Simulated herbivory significantly decreased all morphological traits except the number of leaves, which was higher in clipped than in unclipped seedlings (Table 1, Fig. 1). As a single factor, water treatment had no significant effect after fixed Bonferroni correction on any trait (Table 1). The light × water interaction was significant only for number of leaves and for root dry mass (Table 1), because sun-grown oaks produced more leaves and had heavier roots in the double water level, whereas in the shade treatment this extra watering had no effect for these two traits (Fig. 1). Chemical traits The three chemical traits, nitrogen, phenols, and tannins, were significantly affected by light when quantified both in leaves and in leaves plus stems (Table 2). Whereas nitrogen was higher in shade-grown seedlings than in sun-grown seedlings, the opposite was found for phenols and tannins (Fig. 2). The light × water interaction was significant for nitrogen, because double-watered seedlings in shade had more nitrogen than single-watered seedlings, whereas in sun, the highest concentration was found in single-watered plants (Fig. 2). The tannin concentration was significantly affected by herbivory in leaves but not in leaves plus stems (Table 2), the tannin conentration being lower in leaves of clipped © 2004 NRC Canada

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Table 1. ANOVA results (F-values) for comparisons among all the treatment effects on morphological traits of Quercus pyrenaica seedlings.

Light Water Herbivory Light × water Light × herbivory Water × herbivory Light × water × herbivory Residual R2

df

NLa

TLA

LMA

RMa

AM

S/R

1 1 1 1 1 1 1 354

60.289**** 4.701* 7.861*** 8.039*** 11.05*** 0.570 0.130

45.395**** 1.128 20.636**** 5.096* 0.124 2.074 1.093

307.902**** 3.550 11.976*** 2.482 0.719 1.106 2.892

154.944*** 5.808* 9.304**** 11.42*** 2.259 1.793 0.038

14.625*** 1.482 49.198**** 7.025** 1.479 0.421 0.257

170.58**** 1.547 34.861**** 0.004 0.417 0.085 3.827

0.202

0.145

0.485

0.336

0.152

0.365

Note: Asterisks denote the following significance levels: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. After performing a Bonferroni correction, differences were considered significant at P < 0.0008. Significant values are in bold. NL, number of leaves; TLA, total leaf area; LMA, leaf mass per unit area; RM, root dry mass; AM, aerial dry mass, S/R, shoot/root ratio. a The residual is 359.

Fig. 1. Between-treatment difference in the morphological traits of Quercus pyrenaica seedlings. Light: (i) sun, full sunlight, (ii) shade, 5% of the sunlight; water: (i) single, plants were watered weekly, (ii) double, plants were watered twice weekly; and herbivory: (i) clipped, where 50% of the stem was removed, (ii) unclipped, where plants were left untouched. Bars are means + 1 SE. Different letters signify statistical differences (post hoc test, Fisher’s PLSD).

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Table 2. ANOVA results (F-values) for comparisons among all the treatment effects on chemical traits of Quercus pyrenaica seedlings. Leaves plus stems Light Water Herbivory Light × water Light × herbivory Water × herbivory Light × water × herbivory Residual R2

Leaves only

df

Nitrogen

Phenols

Tannins

1 1 1 1 1 1 1 48

26.117**** 0.024 1.230 4.432* 0.107 0.024 2.597

6.393* 1.954 0.064 2.453 1.110 2.260 0.012

137.423*** 0.127 2.185 2.857 6.386* 0.854 0.682

0.652

0.682

0.722

df 1 1 1 1 1 1 1 54

Nitrogena

Phenols

Tannins

21.421**** 0.345 0.327 9.052** 0.883 2.633 0.468

30.958**** 0.291 5.116* 0.034 0.645 3.002 0.468

27.925**** 0.841 13.543*** 4.198* 5.496* 0.155 0.497

0.349

0.361

0.448

Note: Asterisks denote the following significance levels: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. After performing a Bonferroni correction, differences were considered significant at P < 0.016 for leaves plus stems and P < 0.025 for leaves only. Significant values are in bold. a The residual is 47.

Fig. 2. Differences between treatments in the chemical traits of Quercus pyrenaica seedlings under the same treatments as in Fig. 1. Phenols are estimated as absorbance at 740 nm, and tannins as absorbance at 550 nm. Bars are means + 1 SE. Different letters signify statistical differences (post hoc test, Fisher’s protected least significant difference).

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seedlings. The light × herbivory interaction was significant for this trait (Table 2) because for leaves, the effect of herbivory was significant only for sun-grown seedlings (Fig. 2). For leaves plus stems in the shade treatment, tannins were more concentrated in clipped seedlings (nonsignificant differences), whereas in the sun treatment values were greater in unclipped ones (Fig. 2). The concentration of chemical compounds differed in leaves with respect to leaves plus stems (nitrogen, F[1,109] = 120.977, P < 0.0001; tannins, F[1,118] = 22.999, P < 0.0001; phenols, F[1,118] = 9.717, P < 0.023; one-way ANOVAs). Whereas nitrogen and phenols were more concentrated in leaves, tannins were more concentrated in leaves plus stems (Fig. 2).

Discussion The experiment has shown that light intensity severely affected morphological traits of Q. pyrenaica seedlings. Shade seedlings had fewer and larger leaves than did sun seedlings, resulting in a greater total leaf area but a lower leaf mass per unit area. In addition, shade seedlings showed a higher shoot/root ratio because they developed a smaller root system than did sun seedlings. Such changes in morphology and in biomass allocation after shading have been reported for other species of oaks (Ziegenhagen and Kausch 1995; Welander and Ottosson 1998; Ke and Weger 1999; Balaguer et al. 2001; Van Hees and Clerkx 2003), and it is the usual response of plants to increase the efficiency of light conversion to biomass in shady environments (Niinemets 1999; Valladares 2001). Indeed, in terms of absolute size, shade seedlings were significantly smaller than sun seedlings, suggesting that when water is not limiting, light can be a limiting resource when supplied at low levels, even during early life stages of a shade-tolerant species such as Q. pyrenaica. The water treatment did not affect plant performance as a single factor, but it did when interacting with light for the number of leaves and the root mass, because only in the sun did seedlings with double watering produce more leaves and root mass. Oaks growing in the shade could not use a water surplus for growth because of the lack of light. In contrast, for sun-grown seedlings, more water meant greater growth possibilities (Kullberg and Welander 2000). According to our experimental results, Q. pyrenaica seedlings can tolerate some degree of shoot removal. This is a consequence of the ability of Q. pyrenaica to resprout (Calvo et al. 2003). In our case, seedlings did not die because of clipping at any light or water level. Furthermore, all clipped seedlings resprouted, partially compensating (50% on average) for the aerial biomass lost to herbivory, presumably by reallocating the nutrients stored in the main roots. Nevertheless, simulated herbivory did affect several morphological traits of the seedlings. Clipped seedlings produced more but smaller leaves, resulting in a decreased total leaf area and leaf dry mass per unit area. Simulated herbivory also reduced the root mass of the oaks seedlings, as observed in many other plant species (Houle and Simard 1996; Strauss and Agrawal 1999). These results indicate that the growth of Q. pyrenaica seedlings and juveniles is directly limited by browsing (see also Andersson and Frost 1996; Frost and Rydin 1997; Van Hees et al. 1996). In addition,

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the architectural changes imposed by clipping can lessen the seedling capacity of capturing both light and water, indirectly constraining both the future growth rate and tolerance to herbivory (Andersson and Frost 1996). Although the herbivory × light interaction was significant only for the number of leaves, the effect of herbivory on several seedling morphological traits depended on the light conditions under which the seedlings were growing. Thus, herbivory greatly reduced the leaf area to shade-grown seedlings, where light was limited, and caused a greater reduction in root mass in sun, where there was less soil-water availability. That is, losses of aerial parts of Q. pyrenaica seedlings trigger several phenotypic changes (less photosynthetic area and fewer roots) in the same way but with a different intensity depending on the light environment. Light intensity also affected the chemical traits of the seedlings. Shade-grown seedlings contained more nitrogen but had lower levels of chemical defences than did sungrown seedlings (see also Covello and Gallardo 2001; Rieske 2002), presumably making them more attractive to herbivores (Larsson et al. 1985; Wang and Provenza 1997; Nabeshima et al. 2001; Tipler et al. 2002). Chemical traits were also affected by simulated herbivory, decreasing the tannin concentration, and although not statistically significant after Bonferroni correction, mildly increasing the phenol concentration (Fig. 2). Contrary to our results, Hunter and Forkner (1999) found a significant increase in foliar tannins when studying the biochemical response of Quercus rubra to defoliation damage due to hurricanes. However, Faeth (1992) found an increase of hydrolysable tannins but not of condensed tannins in Quercus emoryi after simulated defoliation, which is consistent with our findings of a decrease in condensed tannins but slight increase of total phenols (including hydrolysable tannins). There was also a significant interaction between light and herbivory for tannin concentration (Table 2). In fact, herbivory decreased tannins much more when seedlings were growing in the sun than in the shade (Fig. 2). Furthermore, herbivory slightly induced secondary compounds only in shade, where clipped seedlings had more phenols (Fig. 2). Other studies have reported significant effect of water availability on chemical compounds in several oaks (Shure et al. 1998 and references therein) depending on the water stress amount. In our experiment, water stress did not seem to provoke an increase of chemical defences. In conclusion, mammal herbivory can severely harm Q. pyrenaica seedlings, lower their growth rate, and alter the production of defensive chemical compounds. More importantly, this experiment also reveals that the light conditions under which seedlings grow can mediate their interaction with herbivores, suggesting that seedlings located in shade are potentially less defended and have less tolerance for damage than do seedlings located in full sunlight (see also McGraw et al. 1990; Canham et al. 1994; Lentz and Cipollini 1998 and Harmer 1999 for similar results). This result is consistent with Hawkes and Sullivan’s (2001) idea that the detrimental effects of leaf herbivory are greater in carbonlimited plants because the loss of leaf tissue primarily increases carbon limitation. This can have important consequences for oak forest regeneration. Other studies with different oak species have found that shrubs improve survival © 2004 NRC Canada

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of oak seedlings, by reducing heat or water stress or providing protection from herbivores (Fuchs et al. 2000; Li and Ma 2003). Under natural conditions, acorn predators and seedlings die massively because of summer drought, limiting Q. pyrenaica regeneration in open microhabitats (Gómez et al. 2003). However, our experiment demonstrates that seedlings growing under canopy shade conditions are less able to tolerate severe damage by ungulates. This could be an additional reason for the low recruitment probability of this shade-tolerant oak species in currently overgrazed Mediterranean environments.

Acknowledgments We thank the Consejería de Medio Ambiente, Junta de Andalucía, and the Directorate of the National Park, for permission and facilities to work in Sierra Nevada. Angel Navarro and Joaquín Sánchez provided invaluable technical support, while Maria Jesús Molina Luzón and Carolina Puerta Piñero helped us with the chemical analyses. Ramon Ordiales Plaza allowed us the use of computer image software. David Nesbitt looked over the English version of the manuscript. This study was supported by projects AGF99– 0618 (to R.Z.), REW02–04041 (HETEROMED) (to R.Z.), and REN2002-04475 (to J.M.G.), and by a predoctoral grant from the Spanish Ministry of Science and Technology to E.B.

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