Impact of an oligofagus herbivore on a native and an ：在本地和一个oligofagus草食动物的影响

Impact of an oligofagus herbivore on a native and an ：在本地和一个oligofagus草食动物的影响 Zas et al. Tolerance and induced defences in invasion ecology 1 Tolerance and induced resistance in a native and an exotic pine species: 2 relevant traits for invasion ecology 3 4 Running title: Tolerance and induced defences in invasion ecology 5 122* 6 Rafael Zas, Xoaquín Moreira and Luis Sampedro 7 18 Misión Biológica de Galicia, Consejo Superior de Investigaciones Científicas 9 Apdo. 28, Pontevedra, E-36080 Spain 210 Centro de Investigación Forestal de Lourizán. Unidad Asociada a MBG-CSIC. 11 Apdo. 127, Pontevedra, E-36080 Spain 12 *13 Corresponding author: 14 Email: lsampe@uvigo.es 15 Phone Number: +34986805078 16 Fax Number: +34986856420 17 18 1 Zas et al. Tolerance and induced defences in invasion ecology 19 Summary 20 1. Current hypotheses predict contrasting roles for natural enemies in determining the success or 21 failure of plant invasions. Differences in plant induced resistance and tolerance to native 22 herbivores between native and exotic species might contribute to resolve this controversy. 23 2. We examined the differences between the native Pinus pinaster and the exotic P. radiata in 24 constitutive resistance, inducibility of chemical defences, realized resistance and tolerance to 25 the large pine weevil Hylobius abietis in NW Spain. In this region, both pine species closely 26 coexist and are threatened by the weevil, a harmful phloem feeder that causes extensive 27 mortality and growth reduction in young pine stands. 28 3. We performed two in vitro cafeteria bioassays, two induction experiments with direct 29 exposure to the weevil and spraying methyl-jasmonate and an exhaustive field study of the 30 genetic variation in tolerance and resistance in forestry genetic trials. 31 4. The weevil significantly preferred the native to the exotic pine when twigs were offered as 32 cut material in Petri dishes. However, the pattern in the field was the opposite, with greater 33 damage on the exotic. Inducibility of stem oleoresin did not differ between species when 34 elicited by the application of methyl-jasmonate. However, after a 72-h experimental exposure to 35 the weevil, stem resin content in the native pine was double that in the exotic pine, suggesting a 36 lower capability of the exotic pine to respond to the insect damage. In the field, family 37 relationships between early damage and several pine fitness correlates revealed a significantly 38 greater tolerance of the native pine to the insect damage. Furthermore, only the native pine 39 showed genetic variation in tolerance to the damage. 40 5. Synthesis. The preference of the herbivore for the native species was counterbalanced by a 41 lower capability for expressing induced resistance to the weevil and reduced tolerance in the 42 exotic species, resulting in no apparent fitness advantage of the exotic P. radiata over the native 43 P. pinaster. Differences in inducibility by and tolerance to native enemies between exotic and 2 Zas et al. Tolerance and induced defences in invasion ecology 44 native host congeners emerge as key traits for understanding how native enemies might 45 contribute to preventing an introduced species from becoming invasive. 46 47 Key-words: biological invasions, biotic resistance hypothesis, enemy release hypothesis, 48 exotic plants, herbivory, Hylobius abietis, invasion ecology, Pinus pinaster, Pinus radiata, 49 plant–herbivore interactions 50 51 Introduction 52 There are currently several much-debated hypotheses about the differences that should be 53 expected in the impact of specialist and generalist herbivores on native and exotic plant 54 species and how these differences may explain the invasive behaviour of some exotics when 55 planted out of their natural ranges (Catford, Jansson & Nilsson 2009 and references therein). 56 In particular, the enemy release hypothesis (ERH) predicts a lower impact of herbivores on 57 introduced plants because specialist enemies of the introduced plants are absent in the new 58 range, and generalist enemies would be better adapted to consume native than novel exotic 59 species (Keane & Crawley 2002, Maron & Vilá 2001). Alternatively, the biotic resistance 60 hypothesis (BRH) predicts that native herbivores can limit invasions as introduced species 61 may be poorly adapted for defending against them (Elton 1958, Maron & Vilá 2001). 62 Lacking a common evolutionary history with the resident herbivores, exotic plant species 63 may be particularly vulnerable to the new enemies in the introduced range, as they have 64 been not selected to evolve defences against them (Parker, Burkepile & Hay 2006). 65 These hypotheses have been widely tested in different plant–animal systems, and 66 results are controversial (Colautti et al. 2004, Chun, van Kleunen & Dawson 2010, Keane & 67 Crawley 2002, Levine, Adler & Yelenik 2004, Liu & Stiling 2006, Maron & Vilá 2001, 68 Parker, Burkepile & Hay 2006, Parker & Hay 2005). There is increasing evidence that 3 Zas et al. Tolerance and induced defences in invasion ecology 69 richness of herbivorous insects, particularly of specialists, is greater in the native than in the 70 introduced ranges. This apparent release from enemies, however, is only partially translated 71 into a lower incidence of herbivory on the exotics than on the native congeners (Colautti et 72 al. 2004, Liu & Stiling 2006), whereas differences in damage may not necessarily result in 73 greater performance of the introduced species (Chun, van Kleunen & Dawson 2010). 74 Results are especially variable and inconclusive when the incidence of herbivory in exotic 75 and native congeners occurring in the same community is compared (community studies 76 sensu Colautti et al. 2004). Some studies found exotics to be more damaged or more 77 impacted in terms of fitness by herbivores than were native species (e.g. Morrison & Hay 78 2011, Parker & Hay 2005), whereas others found the opposite (e.g. Hill & Kotanen 2009, 79 Siemann & Rogers 2003). These apparently contradictory results have prompted 80 considerable research efforts during recent decades. Surprisingly little attention, however, 81 has been paid to exploring the role played by different plant defensive strategies in 82 explaining the observed patterns. 83 Plant defences against herbivores include two different and complementary 84 mechanisms: those aiming for reducing the damage (resistance) and those aiming for 85 reducing the impact of damage on plant fitness (tolerance). Resistance can be further 86 divided into constitutive resistance, which is permanently expressed irrespective of the 87 incidence of herbivores, and induced resistance, which includes traits that are activated in 88 response to herbivore attack. All these mechanisms are considered adaptive and have 89 evolved in response to the selection pressure imposed by the herbivores with which they 90 have shared an evolutionary history (Núñez-Farfán, Fornoni & Valverde 2007). Although 91 the simultaneous evolution and expression of different mechanisms can be constrained 92 (Koricheva, Nykanen & Gianoli 2004, Leimu & Koricheva 2006), the combination of 93 different defensive strategies appears to be quite common and advantageous, especially in 4 Zas et al. Tolerance and induced defences in invasion ecology 94 trees (Leimu & Koricheva 2006). Their large size and long lifespan make trees very 95 apparent to a wide range of herbivores, and commonly their resistance mechanisms do not 96 completely exclude damage (Haukioja & Koricheva 2000). 97 Interpretation of the results from studies on invasion ecology may substantially 98 differ if different plant defensive strategies are considered (Ashton & Lerdau 2008, Chun, 99 van Kleunen & Dawson 2010). For instance, Lombardero et al. (2008) found that the attack 100 of a bark beetle was significantly greater in a native pine than in a co-occurring exotic pine, 101 but the impact of the insect on pine growth was notably greater in the exotic. Thus, their 102 results support the predictions of the ERH when damage (i.e. resistance) is considered, but 103 they agree with the predictions of the BRH when the impact of herbivores on plant fitness 104 (i.e. tolerance) is taken into account. Native and introduced species can therefore differ in 105 how they tolerate the damage inflicted by native enemies. However, to date, little attention 106 has been paid to exploring how these differences may influence the likelihood of a given 107 introduced species of becoming invasive (Ashton & Lerdau 2008, Chun, van Kleunen & 108 Dawson 2010). 109 On the other hand, induced responses to herbivory caused by native insects may be 110 elicited more strongly in native than in alien plants (Stout & Bostock 1999). There is 111 evidence that plants have evolved sophisticated mechanisms to detect herbivore-derived 112 molecules that act as signals of herbivore damage and mediate the specificity in the 113 response in plant defence responses (Heil 2009, Hilker & Meiners 2010). Induced responses 114 to the damage of native herbivores may therefore be more efficiently elicited in native plant 115 species, which have coevolved with the herbivore, than in exotics, which may lack 116 mechanisms to recognize the native insect as a harmful enemy. This would imply that native 117 herbivores could limit invasions, as suggested by the BRH, but as far as we know, no 118 previous studies have specifically tested this appealing idea. 5 Zas et al. Tolerance and induced defences in invasion ecology 119 The aim of this paper was to explore the role of different defensive strategies in 120 explaining the diverse impact of a harmful native insect herbivore on the native P. pinaster 121 and the exotic P. radiata. Specifically, we analysed the differences between the two pine 122 species in i) constitutive resistance to this insect, by means of in vitro bioassays, ii) 123 inducibility of chemical defences elicited by chemical elicitors and insect feeding, iii) 124 realized resistance (constitutive plus induced) and iv) short- and medium-term tolerance to 125 the damage, and their genetic variation, under field conditions. Although invasive in many 126 countries of the southern hemisphere, P. radiata is not invasive in Europe (Carrillo-Gavilán 127 & Vilá 2010). It has been suggested that the populations of phytophages and pathogens from 128 closely related host species in the introduced area might contribute to the inability of this 129 species to invade in Europe, but this hypothesis remains untested. We aimed to gain insight 130 into the implications of constitutive and induced resistance and of tolerance and resistance 131 strategies in explaining how native herbivores might contribute to prevent an introduced 132 species from becoming invasive. 133 134 Material and Methods 135 Study system 136 Our study was carried out in the coastal area of Galicia (NW Spain), where two main pine 137 species currently dominate the forested area: the native Pinus pinaster and the introduced P. 138 radiata. The former, Maritime pine, occurs naturally in the western part of the 139 Mediterranean basin, where it is an important forest species with high ecological and 140 economic value. The natural distribution of radiata pine is restricted to a very small area in 141 California (US). Nowadays, however, it is among the most widely planted tree species in the 142 world, especially in the southern hemisphere (Lavery & Mead 1998), where no pine species 143 occurs naturally, and where this pine species is considered a threatening invasive plant 6 Zas et al. Tolerance and induced defences in invasion ecology 144 causing important economic and ecological problems (Richardson & Higgins 1998). In 145 Spain, P. radiata was introduced in the nineteenth century, although it was not widely 146 planted until the mid-twentieth century. Here, however, it rarely spreads from planting sites 147 (Carrillo-Gavilán & Vilá 2010). 148 In Spain, both pine species coexist closely, are planted in similar sites and exhibit 149 similar ecological habits. They are light pioneer species with high growth rates and similar 150 dispersal strategies (Barberó et al. 1998, Lavery & Mead 1998). As in other sun-loving 151 colonizer species, early growth is related to plant fitness, because only the fast growers can 152 take their crowns to the vital sunlight and thus survive and reproduce. Because they are 153 obligate seeders living in fire-prone regions, early reproduction is also crucial for fitness of 154 the species (Barberó et al. 1998, Richardson & Higgins 1998). 155 In Spain, both pines are attacked by diverse insect herbivores that cause important 156 growth reduction and mortality, especially at the seedling stage. The native large pine 157 weevil, Hylobius abietis, in particular, stands out as an herbivore that directly affects pine 158 fitness by causing extensive tree mortality at young ages (Lieuter et al. 2004). This pine 159 weevil is a polyphagous herbivore that feeds on the bark and phloem of conifer seedlings 160 and small branches of adult trees, mainly Pinus sp. and Picea sp. If the damage is intense, 161 the wounds on the stems of young seedlings may cause stem girdling and, consequently, the 162 loss of the leader and even the death of the seedling (Lieuter et al. 2004). This weevil occurs 163 naturally in Europe and northern Asia where it is one of the most harmful pests affecting the 164 regeneration of managed coniferous forests. Clear-felling of conifers provides ideal 165 conditions for weevil breeding, leading to outbreaks that can severely damage the natural or 166 artificial regeneration in the cleared area (Nordlander 1991). After one or two years, adults 167 fly away to find new breeding sites. Although weevils can also feed on small branches of 168 adult stands and in young stands established on areas not previously occupied by conifers, 7 Zas et al. Tolerance and induced defences in invasion ecology 169 the damage inflicted on those stands, and its impact on plant fitness, is minimal (Orlander et 170 al. 2000). Indeed, this insect herbivore is considered a ‘silvicultural pest’ that causes serious 171 damage only after the clear-felling of large areas of conifers (Lieuter et al. 2004). Genetic 172 variation in resistance to this insect has been found in both pine species (Zas et al. 2008, Zas 173 et al. 2006), although the weevil has only coexisted with the native P. pinaster, not with the 174 exotic P. radiata (Lieuter et al. 2004). 175 To address our research questions we used three different and independent 176 experimental approaches. First, we evaluated the realized resistance and tolerance to H. 177 abietis damage in an extensive field genetic trial with both pine species naturally infected by 178 the weevil, where damage, subsequent growth and other fitness correlates were assessed 179 over five years. Additionally, we studied pine growth in a neighbouring identical twin trial 180 not attacked by the weevil. Second, the preference of adult weevils for cut twigs of the two 181 pine species was evaluated in in vitro feeding bioassays. Third, differences in inducibility 182 between the two pines were evaluated by assessing the increase in concentration of chemical 183 defences (stem resin) after exposing the pine seedlings to either chemical induction with 184 methyl jasmonate (MeJa) or biotic induction by direct H. abietis feeding. 185 186 Field trials 187 A family ， fertilization trial of P. pinaster and P. radiata, located in Rianxo (Galicia, NW 188 Spain; 42.36º N; 8.46º W; altitude 90 m a.s.l.) and planted in March 2003, was naturally 189 infected by the large pine weevil H. abietis soon after planting. The site had previously been 190 occupied by a mature stand of P. pinaster, clearcut in 2002. Weevils were attracted to the 191 cut area by the volatiles emanating from the fresh conifer stumps, where females lay their 192 eggs (Nordlander 1991). Both the newly arrived adults and the new generation emerging 193 from the stumps caused an intensive H. abietis attack on the established seedlings that lasted 8 Zas et al. Tolerance and induced defences in invasion ecology 194 until the end of the second season. All the seedlings of the two trials had the appropriate size 195 for being fed on by the weevil, with diameter distributions at the time of damage (99% 196 confidence intervals of 4.8-16.6 mm and 4.4-15 mm for P. radiata and P. pinaster, 197 respectively) within the range of twig diameters that the weevil prefers (Orlander et al. 198 2000). The spatial analysis of the damage severity across all the seedlings in the trial 199 revealed that the infestation was spatially uniform in the experimental area (see variograms 200 in Zas et al., 2006, 2008). 201 Two adjacent independent trials were established, one where the main species was P. 202 radiata and P. pinaster was used as a control, and the other with P. pinaster as the main 203 species and P. radiata as the control. The experimental layout, exactly the same for both 204 species, consisted of a split-plot design superimposed across a randomized block design 205 with 10 blocks, with 9 fertilization treatments randomly assigned to the 9 whole plots in 206 which each block was divided and 32 genetic entries randomly distributed within each 207 whole plot. Fertilization (see details in Zas et al. 2006, 2008) was not a central focus of the 208 present study but was considered in the statistical analyses to account for the corresponding 209 variation. 210 The plant material for each species consisted of open-pollinated families of mother 211 trees from natural stands or mature plantations in the coastal area of Galicia. In the P. 212 pinaster section, the 32 genetic entries included 28 half-sib families and 3 commercial 213 seedlots of P. pinaster without family structure. One mixed seedlot of P. radiata was 214 interspersed within the P. pinaster seedlings to complete the 90 main rectangular 215 experimental units (whole plots) of 31+1 plants (8 columns ， 4 rows). Conversely, in the P. 216 radiata section there were 30 half-sibs and 1 commercial seedlot of P. radiata, with a mixed 217 seedlot of the material included in the attached P. pinaster experiment. In total, within each 218 trial, 2790 seedlings of the main pine species were interspersed with 90 seedlings of the 9 Zas et al. Tolerance and induced defences in invasion ecology 219 opposite species, providing a unique experimental design for studying the feeding behaviour 220 of H. abietis on these species. 221 Plant growth (total height and basal stem diameter) was measured yearly from 222 December 2003 to December 2007 (1 to 5 growing seasons after planting) in all plants of 223 the two trials. Reproductive effort of each tree was also evaluated at age five and considered 224 as another early fitness correlate. Reproductive effort was assessed according to Climent et 225 al. (2008) as the total count of cones per tree, including the two female strobili cohorts that 226 grow together in the crown. 227 Hylobius abietis damage was measured after the first growing season (February 228 2004) by evaluating the wounds in the stem caused by the insect. The stem height of each 229 tree was divided in 10 sections; the damage was measured in each section according to a 230 four-level wounding scale (0 = undamaged, 1 = some wounds, 2 = many wounds, and 3 = 231 full of wounds or death due to stem girdling below the given section). The weevil damage 232 on each plant was the sum of the 10 stem sections (0-30 score, Zas et al. 2006). We assumed 233 that the lower the site of the stem girdling, the smaller the amount of living tissue remaining 234 alive, and thus the greater the damage caused to the plant. We accounted for this by 235 assigning the maximum score to all stem sections recorded above the site of girdling. 236 237 Cafeteria experiments 238 Two independent in vitro feeding bioassays, a two-choice and a non-choice test, were 239 performed to test for differences in the preference of the weevil for the two species. 240 Ingestion rate (debarked area) was considered a proxy of constitutive resistance against the 241 pine weevil between the two pine species. 242 Plant material for both experiments was randomly selected from the open-pollinated 243 families included in the field trials. Seeds were sown in May 2006 and seedlings were 10 Zas et al. Tolerance and induced defences in invasion ecology 244 cultivated in 2-L pots filled with 1:1 peat:perlite (v:v) in a greenhouse, following 245 conventional nursery watering and fertilizing protocols. One year after they were sown, 246 seedlings of each pine species were grouped in pairs of similar basal diameter and harvested; 247 needles were gently removed and stems placed on ice in coolers. 248 For the two-choice experiment, three 2.5-cm long twigs (upper, middle and lower 249 thirds of the stem) from each seedling were sampled and paired with twigs belonging to the 250 other pine species according to their relative position within the plant. Each pair of fresh 251 cuttings was placed in an 8-cm Petri dish with a moistened filter paper and offered to one 252 preweighed pine weevil for 48 h at room temperature (c. 22 ºC). The total number of plates 253 was 60, corresponding to 20 pairs of seedlings of similar diameter, with 3 stem twigs per 254 seedling. Sampling several twigs from each seedling allowed us to account for the possible 255 effects of heterogeneous levels of defences along the stems of pine seedlings. 256 For the non-choice feeding test we used 20 seedlings of each species which were 257 grouped according to their diameter into 5 groups, with four plants in each group. We 258 sampled 5 twigs of 2.5 cm from each seedling. Twigs of the four seedlings of each group 259 were grouped according to their position along the stem and offered together in Petri dishes 260 to one preweighed weevil as above. This allowed us to analyse the sum of the debarked 261 areas in the four twigs of each plate, without losing information regarding the possible 262 effects of the seedling size and the part of the stem from which the twigs were sampled. The 263 total number of plates was 50, corresponding to 2 species, 5 diameter groups and 5 twigs per 264 seedling. 265 Pine weevils were collected at a neighbouring clear-felled P. pinaster forest (see 266 Moreira et al. 2008a) two weeks before the experiments and maintained at room 267 temperature in the laboratory in plastic buckets with moistened sawdust and bark and twigs 268 of both pine species. Sex was not considered in the bioassays because previous studies have 11 Zas et al. Tolerance and induced defences in invasion ecology 269 revealed little difference in feeding behaviour between sexes (e.g. Borg-Karlson et al. 2006). 270 The weevils were starved for 24 h before the test period. 271 After 48 h, weevils were removed and the debarked area in each twig was estimated 272 with the aid of a transparent millimetric grid. 273 274 Induction experiments 275 In order to assess weevil-driven induced responses, we covered 40 one-year-old seedlings of 276 each pine species with a plastic net cage (plant material and culture as above). We confined 277 1 preweighed adult weevil within the net in half of the seedlings following a completely 278 factorial design with 20 replicates. After a 72-h feeding period, weevils were removed, 279 debarked area was evaluated according to the same scale as in the field trials, and seedlings 280 were harvested to analyse the concentration of resin acids in the stem. The whole stem was 281 cut in 5-mm sections, extracted twice with hexane in an ultrasonic bath (15 min at 20 ºC) 282 and then for 24 h at room temperature, and the non-volatile resin residue was determined 283 gravimetrically and referred to the dry mass of each stem (Sampedro, Moreira & Zas 2011). 284 Resin concentration in pine stems has been shown to be inversely related to wounding by 285 the weevils (Moreira, Sampedro & Zas 2009), and thus represents an effective resistance 286 trait against this insect. 287 In order to check for differences between species in their inducibility potential, we 288 evaluated the pine response in chemical defences to the exogenous application of MeJa. 289 Methyl jasmonate is a well-known plant phytohormone involved in the signalling and 290 triggering of induced responses after insect damage. Experimental MeJa application to 291 conifer seedlings has been reported to induce chemical defensive responses similar to those 292 caused by insects (Moreira, Sampedro & Zas 2009, Sampedro, Moreira & Zas 2011), and it 12 Zas et al. Tolerance and induced defences in invasion ecology 293 has been shown to be very useful as a reference induction value for comparing induction 294 capabilities to different agents (Hudgins, Christiansen & Franceschi 2004). 295 Twelve seedlings from each pine species were cultivated as described before, and 296 during the second growth period (August 2008) half of the seedlings were sprayed with a 297 suspension of 80 mM MeJa (cat. #39270-7; Sigma-Aldrich, St. Louis, MO, USA) in 298 deionized water with 2.5 % (v/v) ethanol; the remaining half was treated only with the 299 carrier solution. Both treatments were applied with a hand sprayer to runoff at an average 300 dose of about 3 mL of solution per plant. The MeJa was applied in a different space of the 301 greenhouse, and the seedlings remained in that isolated room for 24 h to allow the 302 volatilization of excess MeJa. Then, all seedlings were distributed in the greenhouse 303 according to a factorial randomized block design with 6 blocks. Fifteen days after the 304 application of the induction treatments, i.e. when MeJa effects are assumed to be maximized 305 (Martin et al. 2002), the plants were harvested and the non-volatile resin content in the stem 306 was determined as described before. 307 308 Statistical analyses 309 All analyses were carried out in SAS System, using the GLIMMIX and MIXED procedures 310 for analyses of generalized and general linear mixed models, respectively (Littell et al. 311 2006). 312 Damage data from the field experiments was analysed with a generalized mixed 313 model appropriate for split-plot designs, in which the two main factors (fertilization and 314 genetic entry) are applied to nested experimental units of different size. The dependent 315 variable, which ranged from 0 to 30, was assumed to be similar to a count-data variable and 316 was thus modelled with a Poisson distribution. Fertilization, genetic entry and fertilization 317 by genetic-entry interaction were considered fixed effects. Blocks and the block × 13 Zas et al. Tolerance and induced defences in invasion ecology 318 fertilization interaction (i.e. the whole plots) were considered random factors in order to 319 analyse each factor with the appropriate error term (Littell et al. 2006). To avoid any 320 possible confounding effects associated with size differences between the two pine species, 321 we also included the diameter at the time of the attack as a covariate in the model. Within 322 each site and species, differences in consumption between the pine species by the pine 323 weevil were analysed with specific contrasts on linear combinations of the family means, 324 using the CONTRAST statement of the GLIMMIX procedure in SAS. Family least-squares 325 means were derived from the generalized mixed models and used to estimate the 326 relationship between the damage by the insect and pine survival at the family level. Survival 327 being a clear component of pine fitness, the slope of this relationship reflects the tolerance 328 to the insect attack (Simms 2000). How pines were able to recover growth after insect 329 damage was also considered as a measure of tolerance to the insect. Thus, for each pine 330 species, we estimated the relationship at the family level between damage by the insect and 331 consecutive pine growth one and five years after the attack. Consecutive pine growth was 332 measured as the relative growth rate in height or diameter after the attack (e.g. (H-H) / H, 500 333 where H and H are the pine heights in year five and in the year of the attack). This 50 334 measure was considered to represent the relative investment of plant resources in recovering 335 after attack. Differences in tolerance between the two pine species were estimated by testing 336 for differences in the slope of these regressions with an analysis of covariance (ANCOVA). 337 To double check that the differences in the slopes between the two species are not due to 338 different ranges of weevil damage, we further confirmed the results of this analysis using 339 only the common range of weevil damage (i.e. restricting the analyses to scores from 12 to 340 20). 341 Intraspecific genetic variation for tolerance to H. abietis damage was evaluated by 342 analysing different pine fitness correlates with a general mixed model equivalent to that 14 Zas et al. Tolerance and induced defences in invasion ecology 343 described before for the analysis of weevil damage, but including the damage and damage ， 344 family interaction as covariates (Pilson 2000). A significant family ， damage interaction 345 indicates that the effect of damage on pine fitness, that is tolerance, varies among families. 346 The two-choice test bioassay was analysed with a doubly repeated-measures analysis 347 where the two dimensions of repeated measures were represented by the pair of pine 348 seedlings (with three parts of the same subjects) and the Petri dish in which they shared the 349 same pine weevil. Analysis was performed with a repeated-measures mixed model 350 including the species as a between-subject factor, and the stem part and their interaction as 351 within-subject factors. The Petri dish and the pine weevil weight were also included in the 352 model as a random effect and a fixed covariate, respectively. The covariance structure of the 353 repeated measures was modelled with the Huynh–Feldt structure, as it gave the lowest 354 Akaike information criteria and was consistent with the sample schedule (Littell et al. 2006). 355 The Kenward–Roger correction was used for the estimation of the degrees of freedom, as 356 recommended by Littell et al. (2006, p. 188). 357 The non-choice experiment was analysed with a repeated-measure mixed model 358 where the five stem parts are repeated measures of the same subjects. The mixed model was 359 fitted for the sum values of the four wounding-area values per plate, and the pine weevil 360 weight was included as a covariate. A first-order autoregressive covariance structure was 361 used to model the covariance of the repeated measures. Degrees of freedom were again 362 estimated by the Kenward–Roger method. 363 The induction of chemical defences by either weevil feeding or MeJa application 364 was analysed with a factorial ANCOVA using the debarked area as covariate and assuming 365 all effects (species, induction treatment and their interaction) as fixed factors. 366 367 15 Zas et al. Tolerance and induced defences in invasion ecology 368 Results 369 Weevil damage on the two pine species 370 Results from the field trials indicate that H. abietis fed significantly more on P. radiata than 371 on P. pinaster seedlings. The P. radiata seedlings growing interspersed within the P. 372 pinaster trial were attacked the most (Fig. 1a; see also Table S1 in Supporting Information), 373 and they were significantly more consumed than 24 of the 31 P. pinaster genetic entries in 374 the trial (data not shown). Consistently, the P. pinaster seedlings growing interspersed 375 within the P. radiata trial were significantly less attacked (Fig. 1b, Table S1), and 26 out of 376 the 31 P. radiata genetic entries were significantly more damaged than the control P. 377 pinaster seedlings (data not shown). 378 However, pine weevils preferred P. pinaster (the native) more than P. radiata (the 379 exotic) twigs when both species were offered as cut material in Petri dishes (Fig. 2, 380 Appendix S2). Weevil feeding was significantly more intense in P. pinaster than in P. 381 radiata, both in the two-choice (Fig. 2a, Table S2A) and in the non-choice (Fig. 2b, Table 382 S2B) experiments. 383 384 Short-term tolerance to pine weevil damage 385 The relationship at the family level between actual damage and subsequent survival in the 386 field was negative and very strong in both species (Fig. 3). Damage by the pine weevil 387 explained more than 50% of the family variation in survival. The slopes significantly 388 differed between the two pine species (ANCOVA F = 9.34, p = 0.003), with a steeper 1,58 389 relation in the case of P. radiata, indicating that the native P. pinaster was more tolerant of 390 the damage than the exotic P. radiata (Fig. 3). Results remained the same when the analysis 391 was restricted to the common range of damage scores for the two species (scores 12-20) 392 (ANCOVA F = 4.53, p = 0.038). The relationships with survival at age 5 followed the 1,51 16 Zas et al. Tolerance and induced defences in invasion ecology 393 same trends, confirming the relevance of the early attack as a determinant of medium-term 394 mortality. 395 396 Medium-term tolerance to the pine weevil damage 397 The family relationship between insect damage and the immediate consecutive pine growth 398 in P. pinaster was significant and negative (Fig. 4a). Pine growth after insect damage was 399 less in those families that were more damaged and greater in those where the damage was 400 less, indicating that the insect attack disrupted regular growth. This relationship, which 401 could be interpreted as early growth costs of tolerance or induced resistance, was not 402 significant in the exotic P. radiata (Fig. 4a), where differences in consecutive family growth 403 appeared to be unaffected by the damage level. Figure 4b summarizes the medium-term 404 vegetative tolerance, that is, the potential of each family for height recovery 5 years after the 405 insect attack, measured as 5- to 1-year relative growth regressed against the early damage. 406 Relative compensatory effort in the 5 years following attack was greater in the more 407 damaged families of P. pinaster, whereas we found no significant response in P. radiata. 408 Comparing the growth patterns in the studied stands with those in two neighbouring twin 409 trials of the two pine species that remained free of weevil damage also suggested that the 410 native pine was better able to recover from weevil damage (see Appendix S3). The negative 411 effect of the weevil damage on pine growth disappeared at age five in P. pinaster, but still 412 remained significant in P. radiata, suggesting a compensatory growth in the native but not 413 in the exotic. 414 415 Genetic variation in tolerance 416 Growth and survival during the period studied were strongly determined by the damage 417 produced by the pine weevil at age 1, as evidenced by the significant covariation of the 17 Zas et al. Tolerance and induced defences in invasion ecology 418 initial damage on the studied variables in the ANCOVA analyses (Table 1). We found 419 evidence of genetic variation for tolerance to H. abietis in P. pinaster, but not in P. radiata. 420 In the case of P. pinaster, the damage ， family interaction was marginally significant for 421 pine survival after the insect damage, representing the proximal effects of the attack, and 422 significant for pine height at age 5, suggestive of vegetative compensatory effort and 423 opportunity fitness. However, the covariation of weevil damage on the early reproductive 424 effort did not vary among the P. pinaster families (Table 1). We did not detect genetic 425 variation in tolerance in P. radiata in any case, nor was there covariation of weevil damage 426 with early reproductive effort in this species. Radiata pine families survived and grew 427 similarly irrespective of the level of damage suffered, whereas early cone production was 428 not affected by insect damage. 429 430 Induced response of pine to weevil damage 431 We did not detect significant differences between species in the constitutive resin content in 432 control plants (Fig. 5a). After exposure to insect feeding for 72 h, both species reacted to the 433 weevil damage by increasing the resin concentration in the whole stem. However, the 434 quantitative response in resin differed between the two species (significant species ， 435 induction interaction). The response of the native P. pinaster was 2-fold that of the exotic 436 pine, even though no significant differences were observed in the wounding intensity 437 = 1.2, p = 0.274). between the species (F1,27 438 The application of MeJa also significantly increased the resin content in the two pine 439 species. The response to MeJa application was, however, similar in the two pines (no 440 significant species ， MeJa interaction, Fig. 5b). Thus, the resin content in the stem of both 441 constitutive and MeJa-induced plants did not differ between species, suggesting similar 442 physiological capabilities to express MeJa elicited induction. 18 Zas et al. Tolerance and induced defences in invasion ecology 443 Discussion 444 Induced resistance and biological invasions 445 In contrast to the expectations of the BRH, the large pine weevil consumed more on the 446 native P. pinaster than on the exotic P. radiata when both species were offered as cut 447 material in in vitro bioassays. As cut stem twigs are unable to respond to the damage caused 448 by the weevils, results of the in vitro bioassays likely reflect the variation in preference due 449 to constitutive resistance between the two species, which appeared to be higher in the exotic 450 than in the indigenous pine species. However, under field conditions, damage by the pine 451 weevil was clearly greater on the exotic than on the native species, suggesting lower 452 effective resistance of the former. Both species were planted simultaneously, and the pine 453 weevil attack started early after planting and lasted several months until weevil damage was 454 assessed. During this time, weevils were continuously feeding on the pine seedlings, which 455 should have had enough time to respond by activating induced resistance mechanisms. As 456 an evidence of this, the resin canal density in the xylem of the P. pinaster seedlings in the 457 attacked stand was nearly twice as high as that found in the nearby unattacked stand; the 458 differences were attributed to the defensive response induced by the weevil damage 459 (Moreira et al. 2008b). 460 The results of our induction experiment revealed large differences between the 461 species in the induction of resin in the stem after wounding. Although the damage inflicted 462 by the weevil during the induction experiment did not differ between pine species, the 463 increment in the resin content in the indigenous species just 72 hours after insect damage 464 was more than 2-fold that in the non-indigenous species. Moreover, differences between 465 species in this main resistant trait were only significant after exposure to the insect. 466 Conversely, the response to the application of MeJa did not differ between the species, 467 suggesting similar MeJa inducible physiological capabilities (i.e. inducibility potential) 19 Zas et al. Tolerance and induced defences in invasion ecology 468 between the two species. On the basis of the differences between the response to the pine 469 weevil and to the MeJa application, we infer, as predicted by the BRH, that the native pine 470 species is better adapted to this local insect, being able to elicit a stronger induced resistance 471 mechanism that likely will deter further damage. These results suggest that, because of the 472 greater induced resistance in the native species, the initial preference of the insect for the 473 native pine could switch with time towards greater damage on the exotic, as observed in the 474 field. The lack of preference between the two pine species shown by the weevil in the 475 induction experiment does not conflict with this idea, as the period of time that the weevils 476 were feeding on the seedlings (just 72 h) was probably insufficient for the induced defences 477 to become effective against the weevil. 478 Induced resistance is a key strategy for defending Pinus sp. against bark beetles and 479 phloem feeders (Franceschi et al. 2005, Lieuter et al. 2004) and varies greatly among 480 species of the Pinaceae (Hudgins, Christiansen & Franceschi 2004). Plants are known to be 481 able to recognize specific cues of their enemies and elicit the appropriate defensive 482 mechanisms (Heil 2009, Hilker & Meiners 2010). Thus, plant species can respond 483 specifically to different enemies (Lieutier 2002), with larger responses of the trees to the 484 enemies that normally attack them (Raffa 1991). Results presented here are in concordance 485 with this cue specificity of the induced responses, which were more intensely elicited in the 486 pine species with a common evolutionary history with the enemy. 487 Empirical evidences of the ability of plants to recognize different herbivorous 488 species and elicit differential responses against them are, however, equivocal (Heil 2009). 489 For example, the specificity of the tree response to the bark beetle–fungus association may 490 be more related to protein–protein interactions with the fungus species than to plant 491 recognition of the insect species (Karban & Kuc 1999). Hylobius abietis could also vector 20 Zas et al. Tolerance and induced defences in invasion ecology 492 different native pathogenic fungus species to which P. radiata may be not adapted, but 493 information on the transmission of diseases by weevils is very limited (Lieuter et al. 2004). 494 Although other factors (e.g. the environmental and ontogenetic differences between 495 the field trials and the bioassays with greenhouse potted plants) may have differentially 496 affected the results, our findings indicate that the intensity and/or rapidity of the induced 497 responses to a native insect can differ between native and non-native host species. If 498 induced defences are more specific mechanisms than constitutive resistance (Franceschi et 499 al. 2005), the differences in inducibility between native and exotic species may be relevant 500 for understanding the role of local enemies in invasion ecology. The lower inducibility of 501 the exotic pine species supports the predictions of the BRH (Elton 1958), as it could imply 502 greater damage in the field, which could contribute towards preventing the exotic pine from 503 becoming invasive in the area. As pointed out by Orians & Ward (2010), however, little 504 attention has been paid to the implications of induced resistance in invasion ecology. To 505 date, only two studies have examined induced defences in exotic plants, comparing patterns 506 of inducible defences in native and introduced ranges (Cipollini et al. 2005, Eigenbrode et 507 al. 2008). These studies explore how induced defences have evolved after the relaxation of 508 herbivore pressure in the exotic range, using either chemical elicitors or mechanical 509 wounding to assess inducibility. However, responses to specific herbivore species can 510 potentially differ from those to artificial induction treatments (as observed in the present 511 study), and these differences could be highly relevant in the context of invasion ecology. To 512 our knowledge, results presented here are the first reporting how failure to elicit effective 513 induced responses against local herbivores might be a factor in impeding an introduced 514 species from becoming invasive. 515 516 21 Zas et al. Tolerance and induced defences in invasion ecology 517 Tolerance in the context of biological invasions 518 Differences in tolerance to herbivore damage can also be important in interpreting the 519 invasion ecology hypotheses (Ashton & Lerdau 2008, Chun, van Kleunen & Dawson 2010). 520 Besides the release from specialist enemies, the ERH also assumes a lower impact of 521 generalist enemies on the introduced species, because native consumers will be better 522 adapted to consume native, rather than exotic hosts (Keane & Crawley 2002). According to 523 the BRH, however, the exotic hosts may be either more heavily damaged by the resident 524 herbivores than native plants or just poorly adapted to tolerate the damage caused by novel 525 enemies with which they lack an evolutionary history (Parker & Hay 2005). This idea is 526 well accepted among foresters, who commonly consider that trees can be more susceptible 527 to pests and pathogens when they are planted off-site, i.e. outside the soil, climate system 528 and biotic interactions to which they are adapted (Lombardero, Vázquez-Majuto & Ayres 529 2008). 530 In support of the BRH, we found that the native pine was more tolerant of the 531 coevolved herbivore than the exotic pine, suggesting no fitness advantage of the exotic pine 532 species in its introduced area. For the same level of insect damage, survival of P. pinaster 533 families was higher than that of P. radiata, indicating a lower impact of the herbivore on the 534 former. Fitness evaluation is especially complicated in forest trees with long lifespans and 535 reproduction. However, early survival is a clear component of pine fitness, and thus the 536 relationship between survival and damage can be utilized to assess tolerance to the attack. 537 Early growth in light-demanding species such as the pines studied is another component of 538 plant fitness, which evaluates the opportunity of a tree to arrive to the dominant stratus, and 539 thus reproduce and contribute to the next generation. Slow-growing pines are expected to be 540 suppressed by dominant trees and die. Considering early growth, the native P. pinaster 541 appeared to be, again, more tolerant than the exotic P. radiata. Although the pine weevil 22 Zas et al. Tolerance and induced defences in invasion ecology 542 reduced the expected regular size of P. pinaster during the immediate following years after 543 the attack, the positive relationship between initial weevil damage and relative medium term 544 growth reveals that this pine species was able to likely compensate after just 5 years. In 545 contrast, in P. radiata, neither immediate consecutive growth nor medium-term relative 546 growth was related to initial damage, suggesting no compensatory growth capability in the 547 exotic pine. The differences in the growth trends in the attacked and unattacked stands were 548 in concordance with this finding, although the results from this comparison should be 549 interpreted with care as the two sites may not have been equally suitable for the two pine 550 species (see Appendix S3). 551 The detection of intraspecific genetic variation in tolerance of P. pinaster to the 552 insect supports the possibility of evolution of tolerance traits as an adaptation to this (and 553 other) local insects. The cost–benefits balance, together with the temporal and spatial 554 heterogeneity in the selective pressure imposed by these (and other) insects, would explain 555 why these apparent adaptive traits are not fixed within the native population and genetic 556 variation for tolerance is maintained. 557 The lower tolerance of the exotic pine species suggest poor adaptation to its new 558 range, which is in consonance with the predictions of the BRH. In a recent study, 559 Lombardero et al. (2008), studying the impact of the local bark beetle Tomicus piniperda on 560 the same pine species in the same region, also found the native P. pinaster to be more 561 tolerant than the exotic P. radiata. Although the insect damage was more than twice as great 562 in the native P. pinaster as in the exotic P. radiata, the productivity of the exotic was more 563 adversely affected, suggesting higher tolerance of the native species. The results were 564 consistent across a wide range of tree ages and beetle incidence levels. Additionally, 565 productivity of the non-native P. radiata was further reduced by the pathogen Sphaeropsis 23 Zas et al. Tolerance and induced defences in invasion ecology 566 sapinea, a fungus that seems to be associated with T. piniperda and was only present in the 567 exotic pine. 568 Few studies have considered the importance of tolerance in invasion ecology (Chun, 569 van Kleunen & Dawson 2010). After the formulation of the Evolution of Increased 570 Competitive Ability (EICA) hypothesis (Blossey & Notzold 1995), however, it has been 571 postulated that the invasiveness of exotic species might be related to increased tolerance of 572 the introduced populations as a result of a relaxation in herbivore pressure in the new range 573 (Stastny, Schaffner & Elle 2005, Zou, Rogers & Siemann 2008). However, P. radiata was 574 introduced in Spain very few generations ago, so few opportunities to evolve tolerance are 575 expected in this species. Besides, the lack of genetic variation in tolerance found in the 576 present study would further constrain the possibility to evolve towards higher tolerance. As 577 stated by Lombardero et al. (2008), the most obvious implications of EICA in forestry 578 would be for tree breeding programs, since natural selection is constrained in trees by their 579 long generation times. 580 In summary, we found that P. pinaster and P. radiata coexisting in NW Spain 581 differed markedly in their responses to a native insect herbivore. Although the insect 582 intrinsically preferred the native pine species with which it shared an evolutionary history, 583 the native P. pinaster responded more strongly to the attack and was more tolerant of it than 584 the exotic P. radiata. Thus, in this area, the exotic P. radiata shows no advantages 585 compared with the native pine against this enemy. These findings are in agreement with the 586 fact that P. radiata is not invasive in this area (Carrillo-Gavilán & Vilá 2010). Other authors 587 have already suggested that the failure of invasions by alien conifers in Central Europe 588 might be, among other reasons, due to the presence of pathogens and phytophages in the 589 introduced range because of the presence of native conifer congeners (Adamowski 2004, 590 Carrillo-Gavilán & Vilá 2010). Indeed, exotic conifers in Europe are known to be colonized 24 Zas et al. Tolerance and induced defences in invasion ecology 591 by as much herbivore insect species as native conifers (Bertheau et al. 2009), and the 592 majority of native insects species that colonize alien trees are generalist herbivores 593 (Bertheau et al. 2010). Additionally, there is some evidence that herbivory by generalist 594 vertebrates might limit the invasiveness of P. radiata in some areas of the Southern 595 Hemisphere (Becerra & Bustamante 2008). However, that P. radiata might shows signs of 596 invasiveness in Europe in the absence of herbivory and that this pine has become invasive in 597 other parts of the world because of escape from herbivory, remains untested. Based on the 598 results presented here, the weak ability to elicit induced defences and the low tolerance of 599 the exotic species against native herbivores appeared as key concepts for understanding how 600 native enemies may contribute to limit the invasiveness potential of exotic plants, as stated 601 by the BRH. Further studies are needed to confirm this by testing whether P. radiata may 602 show signs of invasiveness in the absence of herbivory in Europe. 603 604 Acknowledgements 605 Research was supported by the grants RTA2007-100 and AGL2010-18724. L.S. and X.M. 606 received financial support from DOC-INIA and PREDOC-INIA grant programs, 607 respectively. We thank field technicians from the Lourizán Center (Xunta de Galicia) for 608 their help in field trial establishment and maintenance. We thank Rodrigo Costas and Ana 609 Soliño for assistance with the bioassays, and Patricia Martíns, Diana Blanco, Beatriz 610 Rodriguez-Morales, Sara Varela and Benito Santos for their valuable help in field 611 assessments. Comments and suggestions by Dr. María Josefa Lombardero, Prof. Matthew 612 Ayres and two anonymous referees helped to improve the manuscript. We are also very 613 grateful for the exhaustive language edition by Prof. Alison Moldenke and David Brown. 614 615 25 Zas et al. Tolerance and induced defences in invasion ecology 616 References 617 Adamowski, W. (2004) Why don't alien conifers invade the Bialowieza Forest? Weed 618 Technology, 18, 1453-1456. 619 Ashton, I.-W. & Lerdau, T. (2008) Tolerance to herbivory, and not resistance, may explain 620 differential success of invasive, naturalized, and native North America temperate vines. 621 Diversity and Distributions, 14, 169-178. 622 Barberó, M., Loisel, R., Quézel, P., Richardson, D. M. & Romane, F. (1998) Pines of the 623 Mediterranean Basin. Ecology and biogeography of Pinus (ed D. M. Richardson), pp. 624 153-170. Cambridge University Press, Cambridge, UK. 625 Becerra, P. I. & Bustamante, R. O. (2008) The effect of herbivory on seedling survival of 626 the invasive exotic species Pinus radiata and Eucalyptus globulus in a Mediterranean 627 ecosystem of Central Chile. Forest Ecology and Management, 256, 1573-1578. 628 Bertheau, C., Brockerhoff, E. G., Roux-Morabito, G., Lieutier, F. & Jactel, H. (2010) Novel 629 insect-tree associations resulting from accidental and intentional biological 'invasions': a 630 meta-analysis of effects on insect fitness. Ecology Letters, 13, 506-515. 631 Bertheau, C., Salle, A., Rossi, J. P., Branhead.dronnet, S., Pineau, X., Roux-morabito, G. & 632 Lieutier, F. (2009) Colonisation of native and exotic conifers by indigenous bark beetles 633 (Coleoptera: Scolytinae) in France. Forest Ecology and Management, 258, 1619-1628. 634 Blossey, B. & Notzold, R. (1995) Evolution of Increased Competitive Ability in Invasive 635 Nonindigenous Plants - a Hypothesis. Journal of Ecology, 83, 887-889. 636 Borg-Karlson, A.-K., Nordlander, G., Mudalige, A., Nordenhem, H. & Unelius, C. R. (2006) 637 Antifeedants in the Feces of the Pine Weevil Hylobius abietis: Identification and 638 Biological Activity. Journal of Chemical Ecology, 32, 943-957. 639 Carrillo-Gavilán, M. A. & Vilá, M. (2010) Little evidence of invasion by alien conifers in 640 Europe Diversity and Distributions, 16, 203-213. 26 Zas et al. Tolerance and induced defences in invasion ecology 641 Catford, J. A., Jansson, R. & Nilsson, C. (2009) Reducing redundancy in invasion ecology 642 by integrating hypotheses into a single theoretical framework. Diversity and 643 Distributions, 15, 22-40. 644 Cipollini, D., Mbagwu, J., Barto, K., Hillstrom, C. & Enright, S. (2005) Expression of 645 constitutive and inducible defenses in native and invasive populations of Alliaria 646 petiolata. Chemical Ecology, 31, 1255-1267. 647 Climent, J., Aránzazu-Prada, M., Calama, R., Chambel, R., Sánchez-de-Ron, D. & Alía, R. 648 (2008) To grow or to seed: Ecotypic variation in reproductive allocation and cone 649 production by young female Aleppo pine (Pinus halepensis, Pinaceae). American 650 Journal of Botany, 95, 833-842. 651 Colautti, R. I., Ricciardi, A., Grigorovich, I. A. & MacIsaac, H. J. (2004) Is invasion success 652 explained by the enemy release hypothesis? Ecology Letters, 7, 721-733. 653 Chun, Y. J., van Kleunen, M. & Dawson, W. (2010) The role of enemy release, tolerance 654 and resistance in plant invasions: linking damage to performance. Ecology Letters, 13, 655 937-946. 656 Eigenbrode, S. D., Andreas, J. E., Cripps, M. G., Ding, H., Biggam, R. C. & Schwarzländer, 657 M. (2008) Induced chemical defenses in invasive plants: a case study with Cynoglossum 658 officinale L. Biological Invasions, 10, 1373-1379. 659 Elton, C. S. (1958) The Ecology of invasions by animals and plants. University of Chicago 660 Press, Chicago. 661 Franceschi, V. R., Krokene, P., Christiansen, E. & Krekling, T. (2005) Anatomical and 662 chemical defenses of conifer bark against bark beetles and other pests. New Phytologist, 663 167, 353-375. 664 Haukioja, E. & Koricheva, J. (2000) Tolerance to herbivory in woody vs. herbaceous plants. 665 Evolutionary Ecology, 14, 551-562. 27 Zas et al. Tolerance and induced defences in invasion ecology 666 Heil, M. (2009) Damage-self recognition in plant herbivore defence. Trends in Plant 667 Science, 14, 356-363. 668 Hilker, M. & Meiners, T. (2010) How do plants "notice" attack by herbivorous arthropods. 669 Biological Reviews, 85, 267-280. 670 Hill, S. B. & Kotanen, P. M. (2009) Evidence that phylogenetically novel non-indigenous 671 plants experience less herbivory. Oecologia, 161, 581-590. 672 Hudgins, J. W., Christiansen, E. & Franceschi, V. R. (2004) Induction of anatomically 673 based defense responses in stems of diverse conifers by methyl jasmonate: a 674 phylogenetic perspective. Tree Physiology, 24, 251-264. 675 Karban, R. & Kuc, J. (1999) Induced resistance against pathogens and herbivores: An 676 overview. Induced plant defenses against pathogens and herbivores (eds A. A. Agrawal, 677 S. Tuzun & E. Bent). The American Phytopathological Society, St. Paul, Minnesota. 678 Keane, R. M. & Crawley, M. J. (2002) Exotic plant invasions and the enemy release 679 hypothesis. Trends in Ecology & Evolution, 17, 164-170. 680 Koricheva, J., Nykanen, H. & Gianoli, E. (2004) Meta-analysis of trade-offs among plant 681 antiherbivore defenses: Are plants jacks-of-all-trades, masters of all? American 682 Naturalist, 163, E64-E75. 683 Lavery, P. B. & Mead, D. J. (1998) Pinus radiata: a narrow endemic from North America 684 takes the world. Ecology and biogeography of Pinus (ed D. M. Richardson), pp. 432-449. 685 Cambridge University Press, Cambridge, UK. 686 Leimu, R. & Koricheva, J. (2006) A meta-analysis of tradeoffs between plant tolerance and 687 resistance to herbivores: combining the evidence from ecological and agricultural studies. 688 Oikos, 112, 1-9. 689 Levine, J. M., Adler, P. B. & Yelenik, S. G. (2004) A meta-anlaysis of biotic resistance to 690 exotic plant invasions. Ecology Letters, 7, 975-989. 28 Zas et al. Tolerance and induced defences in invasion ecology 691 Lieuter, F., Day, K. R., Battisti, A. & Gregoire, J. C. (2004) Bark and wood boring insects 692 in living trees in Europe, a synthesis. Kluwer Academic Publishers, The Netherlands. 693 Lieutier, F. (2002) Mechanisms of resistance in conifers and bark beetle attack strategies. 694 Mechanisms and deployment of resistance in trees to insects (eds M. R. Wagner, K. M. 695 Clancy, F. Lieutier & T. D. Paine), pp. 31-77. Kluwer Academic Press, Dordnecht, The 696 Netherlands. 697 Littell, R. C., Milliken, G. A., Stroup, W. W., Wolfinger, R. D. & Schabenberger, O. (2006) 698 SAS System for mixed models, second edition. SAS Institute, Cary, NC. 699 Liu, H. & Stiling, P. (2006) Testing the enemy release hypothesis: a review and meta-700 analysis. Biological Invasions, 8, 1535-1545. 701 Lombardero, M. J., Vázquez-Majuto, P. & Ayres, M. P. (2008) Role of plant enemies in the 702 forestry of indigenous versus nonindigenous pines. Ecological Applications, 18, 1171- 703 1181. 704 Maron, J. L. & Vilá, M. (2001) When do herbivores affect plant invasion? Evidence for the 705 natural enemies and biotic resistance hypothesis. Oikos, 95, 361-373. 706 Martin, D., Tholl, D., Gershenzon, J. & Bohlmann, J. (2002) Methyl jasmonate induces 707 traumatic resin ducts, terpenoid resin biosynthesis, and terpenoid accumulation in 708 developing xylem of Norway spruce stems. Plant Physiology, 129, 1003-1018. 709 Moreira, X., Costas, R., Sampedro, L. & Zas, R. (2008a) A simple method for trapping 710 Hylobius abietis (L.) alive in northern Spain. Investigación Agraria. Sistemas y Recursos 711 Forestales, 17, 188-192. 712 Moreira, X., Sampedro, L., Solla, A. & Zas, R. (2008b) Alterations of the resin canal system 713 of Pinus pinaster seedlings after fertilization of a healthy and of a Hylobius abietis 714 attacked stand. Trees Structure and Function, 22, 771-777. 29 Zas et al. Tolerance and induced defences in invasion ecology 715 Moreira, X., Sampedro, L. & Zas, R. (2009) Defensive responses of Pinus pinaster 716 seedlings to exogenous application of methyl-jasmonate: concentration effect and 717 systemic response. Environmental and Experimental Botany, 67, 94-100. 718 Morrison, W. E. & Hay, M. E. (2011) Herbivore preference for native vs. exotic plants: 719 generalist herbivore from multiple continents prefer exotic plants that are evolutionarily 720 naïve. PLoS One 6, e17227. 721 Nordlander, G. (1991) Host finding in the pine weevil Hylobius abietis: effects of conifer 722 volatiles and added limonene. Entomologia Experimentalis et Applicata, 59, 229-237. 723 Núñez-Farfán, J., Fornoni, J. & Valverde, P. L. (2007) The evolution of resistance and 724 tolerance to herbivores. Annual Review of Ecology, Evolution, and Systematics, 38, 725 541:566. 726 Orians, C. M. & Ward, D. (2010) Evolution of plant defenses in nonindigenous 727 environments. Annual Review of Entomology, 55, 439-459. 728 Orlander, G., Nordlander, G., Wallertz, K. & Nordenhem, H. (2000) Feeding in the crowns 729 of Scots pine trees by the pine weevil Hylobius abietis Scandinavian Journal of Forest 730 Research, 15, 194-201. 731 Parker, I. M., Burkepile, D. E. & Hay, M. E. (2006) Opposing effects of native and exotic 732 herbivores on plant invasions. Science, 311, 1459-1461. 733 Parker, J. D. & Hay, M. E. (2005) Biotic resistance to plant invasions? Native herbivores 734 prefer non-native plants. Ecology Letters, 8, 959-967. 735 Pilson, D. (2000) The evolution of plant response to herbivory: siultaneous consiederinf of 736 resistance and tolerance in Brassica rapa. Evolutionary Ecology, 14. 737 Raffa, K. F. (1991) Induced defensive reactions in conifer-bark beetle systems. 738 Phytochemical induction by herbivores (eds D. W. Tallamy & M. J. Raupp). John Wiley, 739 New York. 30 Zas et al. Tolerance and induced defences in invasion ecology 740 Richardson, D. M. & Higgins, S. I. (1998) Pines as invaders in the southern hemisphere. 741 Ecology and biogeography of Pinus (ed D. M. Richardson), pp. 450-474. Cambridge 742 University Press, Cambridge, UK. 743 Sampedro, L., Moreira, X. & Zas, R. (2011) Costs of constitutive and herbivore‐induced 744 chemical defences in pine trees emerge only under low nutrient availability. Journal of 745 Ecology, 99, 818-827. 746 Siemann, E. & Rogers, W. E. (2003) Herbivory, disease, recruitment limitation, and success 747 of alien and native tree species. Ecology, 84, 1489-1505. 748 Simms, E. L. (2000) Defining tolerance as a norm of reaction. Evolutionary Ecology, 14, 749 563-570. 750 Stastny, M., Schaffner, U. & Elle, E. (2005) Do vigour of introduced populations and escape 751 from specialist herbivores contribute to invasiveness?. Journal of Ecology, 93, 27-37. 752 Stout, M. J. & Bostock, R. M. (1999) Specificity of induced responses to arthropods and 753 pathogens. Induced plant defenses against pathogens and herbivores (eds A. A. Agrawal, 754 S. Tuzun & E. Bent), pp. 183-210. APS Press, St. Paul, Minnesota. 755 Zas, R., Sampedro, L., Moreira, X. & Martíns, P. (2008) Effect of fertilization and genetic 756 variation on susceptibility of Pinus radiata seedlings to Hylobius abietis damage. 757 Canadian Journal of Forest Research 38, 63–72. 758 Zas, R., Sampedro, L., Prada, E., Lombardero, M. J. & Fernández-López, J. (2006) 759 Fertilization increases Hylobius abietis L. damage in Pinus pinaster Ait. seedlings 760 Forest Ecology and Management, 222, 137-144. 761 Zou, J., Rogers, W. E. & Siemann, E. (2008) Increased competitive ability and herbivory 762 tolerance in the invasive plant Sapium sebiferum. Biological Invasions, 10, 291-302. 763 764 31 Zas et al. Tolerance and induced defences in invasion ecology 765 SUPPORTING INFORMATION 766 Additional supporting information may be found in the online version of this article: 767 768 Appendix S1 – Table S1 Results of the generalized linear mixed model for the analysis of 769 the damage by H. abietis in the P. pinaster and P. radiata field trials. 770 771 Appendix S2 – Table S2A Results of the mixed model for the analysis of the debarked area 772 in the two-choice feeding experiment. 773 774 Appendix S2 – Table S2B Results of the mixed model for the analysis of the debarked area 775 in the non-choice feeding experiment. 776 777 Appendix S3 Medium-term growth trends of both pine species in the attacked and the 778 healthy stands. 779 780 As a service to our authors and readers, this journal provides supporting information 781 supplied by the authors. Such materials may be re-organized for online delivery, but are not 782 copy-edited or typeset. Technical support issues arising from supporting information (other 783 than missing files) should be addressed to the authors. 784 32 Zas et al. Tolerance and induced defences in invasion ecology 785 Table 1. Tolerance and genetic variation in tolerance to Hylobius abietis damage in Pinus 786 pinaster and P. radiata. The table shows the F-values and corresponding P values of the 787 mixed model for the covariation of the initial weevil damage (wounds) and damage ， family 788 interaction with several components of pine fitness. A significant damage ， family 789 interaction indicates that the effect of damage on the subsequent fitness, that is, tolerance, 790 varied between families 791 Pinus pinaster Pinus radiata Wounds at age 1 Wounds x family Wounds at age 1 Wounds × family Fitness component Fp>F Fp>F Fp>F Fp>F 1,1876* 30,1876* 1,1244* 30,1244* Survival after attack340.5 1.5 583.5 0.8 0.737 <0.001 0.051 <0.001 Height at year 5 152.3 1.5 17.5 1.3 0.133 <0.001 0.031 <0.001 Cone production year 5 16.5 0.8 0.801 0.1 0.819 0.9 0.587 <0.001 792 793 * Denominator degrees of freedom in the case of survival were 2394 for P. pinaster and 794 2262 for P. radiata. 795 33 Zas et al. Tolerance and induced defences in invasion ecology 796 Figure legends 797 Figure 1. Realized resistance of the native Pinus pinaster (black bars) and the exotic P. 798 radiata (white bars) to the pine weevil Hylobius abietis in the field conditions. The pine 799 weevil consumed more on the exotic P. radiata than on the native P. pinaster, both when P. 800 pinaster was planted surrounded by P. radiata (a), and when P. radiata was planted 801 surrounded by P. pinaster (b). The F ratio and associated p-values are presented for the 802 specific contrast between species within the generalized mixed model (see also Table S1); 803 sample size is indicated within each bar. Diameter did not differ between species. 804 805 Figure 2. Preference of the native large pine weevil Hylobius abietis for the native Pinus 806 pinaster (black bars) and the exotic P. radiata (white bars) evaluated in two independent in 807 vitro cafeteria experiments: a two-choice (a) and a non-choice (b) feeding tests (see 808 complete results of the corresponding mixed models in Table S2A and Table S2B). N = 60 809 and N = 25, respectively. Bars represent the debarked area consumed by adult individuals in 810 2.5 cm long twigs of both tree species. Twig diameter was used as covariate. Least-squares 811 means , SE. 812 813 Figure 3. Short term tolerance to Hylobius abietis damage as revealed by the family 814 relationship between H. abietis damage and pine survival one year after the insect attack for 815 Pinus pinaster (filled dots) and P. radiata (open dots). Seedlings were planted in the same 816 field trial, naturally and homogeneously infected by the pine weevil. Both relationships 817 were significant at p < 0.001. Each dot is an open-pollinated family least-squares mean (N = 818 90). 819 820 34 Zas et al. Tolerance and induced defences in invasion ecology 821 822 823 Figure 4. Family relationship between Hylobius abietis damage and subsequent pine growth 824 in Pinus pinaster (black dots) and P. radiata (open dots). (a) Early relative pine growth is 825 -D)/D), where D and the relative diameter growth rate one year after the insect attack ((D1000 826 D are the stem pine diameter in the year of the attack and one year after weevil damage. (b) 1 827 Medium-term compensatory growth, measured as the relative growth rate 5 years after the 828 attack ((H-H)/H), where HH are pine height at age 5 and at the year of insect attack, 5005 and 0 829 respectively. Seedlings were planted in the same field trial naturally and homogeneously 830 infected by the pine weevil. Each dot is an open-pollinated family least-squares mean (N = 831 90). 832 833 Figure 5. Inducibility of pine chemical resistance elicited by the pine weevil (a) and by the 834 exogenous application of methyl jasmonate (MeJa) (b) in Pinus pinaster (black bars) and P. 835 radiata (white bars) seedlings. P values in the associated tables resume the results from the 836 mixed models. Both species responded significantly to both chemical and biotic induction. 837 Although response to MeJa was similar, response to weevil feeding markedly differed 838 between the two species. Within each induction treatment (Control, Induced) differences 839 between species are indicated by the associated significance levels (n.s.= no significant; *** 840 = p<0.001). Least-squares means , SE of two independent experiments are shown: n = 20 in 841 (a) and n = 6 in (b). 842 843 844 35 Zas et al. Tolerance and induced defences in invasion ecology 1 2 3 4 5 6 Figure 1 7 36 Zas et al. Tolerance and induced defences in invasion ecology 1 F = 4.551,37 p = 0.040 F = 5.411,7.98 p = 0.0482 3 4 5 6 7 8 9 Figure 2 10 11 37 Zas et al. Tolerance and induced defences in invasion ecology 1 2 3 4 5 6 7 8 Figure 3 9 10 38 Zas et al. Tolerance and induced defences in invasion ecology 1 2 a) 1602 R= 0.59 p < 0.001 140 120 2 100 R= 0.11 p = 0.070 Early relative pine growth (%) 80 2000b) 2 p < 0.001R = 0.42 1600 1200 800 2R p = 0.156 = 0.07400 0 Medium term relative pine growth (%)155102025 Weevil damage (wounds, 0-30 score) 3 4 Figure 4 5 39 Zas et al. Tolerance and induced defences in invasion ecology 1 2 3 4 5 6 7 8 Figure 5 40