Effects of defoliation and site quality on growth and defenses of Pinus pinaster and P. radiata

Forest Ecology and Management 382 (2016) 39–50

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Effects of defoliation and site quality on growth and defenses of Pinus pinaster and P. radiata María J. Lombardero a,⇑, Matthew P. Ayres b, Pierluigi Bonello c, Don Cipollini d, Daniel A. Herms e a

Departamento de Producción Vegetal, Universidad de Santiago, 27002 Lugo, Spain Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA c Department of Plant Pathology, Ohio State University, Columbus, OH 43210, USA d Department of Biological Sciences, Wright State University, Dayton, OH 45435, USA e Department of Entomology, Ohio State University, Ohio Agricultural Research & Development Center, Wooster, OH 44691, USA b

a r t i c l e

i n f o

Article history: Received 6 July 2016 Received in revised form 30 September 2016 Accepted 2 October 2016

Keywords: Thaumetopoea pytiocampa Delayed induced response Site quality Plantations Native and non-native species

a b s t r a c t The susceptibility to pests and pathogens is frequently of only minor consideration by foresters when selecting tree species to plant, but growth reductions from pests and pathogens are commonly large relative to productivity differences among tree species. Because the selection of tree species and sites when establishing plantation forests has potential effects on subsequent damage by pests, there is need for consideration of joint effects of tree species, site quality, and risk of pests. We tested here how site quality influences the response of two pines species that differ in their evolutionary history with a primary pest in the system. Our specific research objectives were to (1) measure the growth response of two pine species (the native Pinus pinaster and the introduced P. radiata) to defoliation by the pine processionary moth, across a gradient of nutrient availability and (2) compare the constitutive and delayed defoliation induced chemical defenses of these two pine species on sites of low and high quality. We analyzed variation in foliar nitrogen, terpenes, tannins, total phenols and four defensive proteins. The impact on tree growth from experimentally applied defoliation by PPM was surprisingly low. Even with complete defoliation, large sample sizes, and baseline measurements that accounted for much of the variation among trees, we observed only modest effects on diameter growth and no effects on height growth; volume growth was initially reduced by only 6–8% initially, with full recovery within 3 years of defoliation. Pinus radiata, the faster growing species, had higher concentrations than P. pinaster of foliar N while P. pinaster had up to 12 times higher concentrations of terpenes, these results match with predictions of the resource availability hypothesis (RAH). Pinus radiata foliage was relatively rich in tannins and total phenolics. The pine species also differed in defensive proteins. Pinuspinaster had higher concentrations of peroxidase (POD), polyphenol oxidase (PPO) and chitinase (CHI), while P. radiata had higher concentrations of trypsin inhibitor (TI). Finally there was no evidence for delayed inducible defenses in either pine species following PPM defoliation; both species seem to use other defensive strategies to counter predictable attacks by the herbivore. In our study region, defoliation by PPM may not be a primary consideration in the selection of pine species for production forests, but this conclusion need not hold in regions where pines grow more slowly or are exposed to multiple years of defoliation by PPM. Ó 2016 Elsevier B.V. All rights reserved.

1. Introduction Plantations presently represent about 7% of the world’s forest and about 76% of plantations have production as a primary function (FAO, 2010). The selection of trees to be planted therefore emphasizes productivity, which commonly involves the propaga-

⇑ Corresponding author at: Departamento de Producción Vegetal, Universidad de Santiago, Campus de Lugo, 27002 Lugo, Spain. E-mail address: [email protected] (M.J. Lombardero). http://dx.doi.org/10.1016/j.foreco.2016.10.003 0378-1127/Ó 2016 Elsevier B.V. All rights reserved.

tion of non-native tree species. Susceptibility to pests and pathogens is frequently of only minor consideration by foresters when selecting tree species to plant, but growth reductions from pests and pathogens are commonly large relative to productivity differences among tree species (Wainhouse, 2005). In modern forest production systems, pest impacts can involve any combination of native or non-native herbivores and native or non-native trees (Leather and Barbour, 1987; Lombardero et al., 2008, 2012; Parker and Hay, 2005).

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The intrinsic growth rate of candidate tree species, which is a dominant consideration for plant production systems, may frequently influence pest impacts, but in different possible ways. Fast growing tree species have generally greater capacity for compensatory growth after tissue loss (Henery et al., 2010; Herms and Mattson, 1992; Stone, 2001; Zhou et al., 2008). On the other hand, fast-growing species tend to be less defended and therefore sustain more herbivore damage than their slow growing counterparts (Resource Availability Hypothesis, RAH; Coley et al., 1985; Fine et al., 2004). Under the RAH, plants adapt to rich resource environments by evolving high growth rates to take advantage of available resources, which selects for reduced defenses due to a growth – defense tradeoff (Fine et al., 2006; Herms and Mattson, 1992). In contrast, species adapted to low resource environments are predicted to invest relatively more in defenses because replacement of lost tissue is constrained under limited resources. Slow growing plants also tend to have long-lived leaves, which tend to be better defended (Coley, 1988; Iddles et al., 2003; Mooney and Gulmon, 1982), conserve nutrients (Aerts and Chapin, 2000; Escudero et al., 1992), improve carbon balance (Greenway et al., 1992) and be a general adaptation to stress (Ewers and Schmid, 1981, but see Richardson et al., 2010). Theoretical models of trade-offs among growth and defenses frequently consider defenses in general, but plants can employ different kinds of defense (Agrawal and Fishbein, 2006). It can be useful to distinguish between constitutive defenses, which are expressed in the plant independent of previous damage, and inducible defenses, which are facultatively expressed by the plant after damage caused by insects or pathogens (Villari et al., 2014). Inducible responses can include changes in plant chemistry, morphology, phenology, and growth (Haukioja, 1991; Karban and Baldwin, 1997). Plant responses to damage can occur within hours to days (Rapid Induced Responses) or not until the season after damage (Delayed Induced Responses) (Haukioja, 1991). Delayed induction can involve changes in phytochemistry (Haukioja, 1991; Martemyanov et al., 2012; Tuomi et al., 1990) and also increases in mechanical defenses like trichomes (Valkama et al., 2005). The replacement of plant tissue damaged by pests, as well as plant defenses, require expenditures of plant resources, influence plant allocation patterns, and may influence forest productivity and economic returns to landowners (Cipollini et al., 2014; Franceschi et al., 2005; Waring, 1987). However, the extent to which productivity is affected by insect damage can depend greatly on local climate and site fertility (Hawkes and Sullivan, 2001). Sites that permit rapid growth in the absence of pests might also be the best sites in the presence of pests because plant tolerance and the ability to recover can be higher under good growing conditions (Maschinski and Whitman, 1989, but see Wise and Abrahamson, 2007). An alternative possibility is that good growing conditions promote susceptibility to pests because high availability of soil moisture and mineral nutrients promotes plant growth at the expense of constitutive defenses (Growth Differentiation Balance Hypothesis; Herms and Mattson, 1992; Lombardero et al., 2000; Zas et al., 2006). It could also be that the costs of inducible defenses (Karban and Baldwin, 1997) are easier for plants to support on sites that permit high growth because these plants have larger resource pools on which to draw (Cipollini and Bergelson, 2001; Dietrich et al., 2005; Herms and Mattson, 1992; Lombardero et al., 2000; Sampedro et al., 2011). Tuomi et al. (1984) proposed that delayed induced resistance results from defoliation-induced increases in foliar carbon:nutrient balance, resulting in decreased foliar nutrient concentrations and increased phenolic concentrations that decrease herbivore growth and survival in the year(s) following defoliation. Consistent with this, delayed induced resistance of Alaska paper birch (Betula neoalaskana Sarg. = B. resinifera) was suppressed by fertilization (Bryant

et al., 1993). Conifers may be less likely than deciduous trees to display delayed induced resistance because they store relatively more carbohydrate reserves in foliage, which means that defoliation depletes conifers of the carbohydrates to support biosynthesis of foliar phenolics following defoliation (Bryant et al., 1988). Relatively large reductions from defoliation of carbohydrate reserves may also lead to generally greater growth losses from defoliation in conifers vs. deciduous trees (Krause and Raffa, 1996). Because the selection of tree species and sites when establishing plantation forests has potential effects on subsequent damage by pests, there is need for consideration of joint effects of tree species, site quality, and risk of pests. We tested how site quality influences the response of two different pines species to a primary pest (the pine processionary moth, hereafter PPM; Thaumetopoea pityocampa Denis & Schiff., Notodontidae: Thaumetopoeinae). Since P. pinaster (native to the region) has a long history of evolution with PPM, while P. radiata (non-native) does not, we could expect less impact on growth and more effective inducible defenses in P. pinaster vs. P. radiata. Our specific research objectives were to (1) measure the growth response of two pine species (Pinus pinaster and P. radiata) to defoliation by PPM across a gradient of site quality and (2) compare the constitutive and delayed inducible chemical defenses of these two pine species on sites of low vs. high quality. 2. Material and methods 2.1. Study system Our study system included two pine species that are widely planted in Galicia, Spain and elsewhere in the world (Richardson and Higgins, 1998): the native moderate-growing P. pinaster and the non-native faster-growing P. radiata. Pinus pinaster (maritime pine) occurs naturally throughout the Mediterranean region, typically occupying coastal areas in sandy and unfertile soils where other pine species cannot compete. It is the conifer traditionally propagated by Galician landowners since the XVIII century (Ortuño, 1990; Rodriguez-Soalleiro et al., 1997) due to its good adaptation and rapid growth on good soils. Pinus radiata (Monterey or radiata pine) is native to California, was introduced to Spain around 1840 and is now widely planted from the Basque Country to Galicia (Mead, 2013). Pinus pinaster and P. radiata are both hard pines (subgenus Diploxylon), but P. pinaster is within the subsection Pinus and P. radiata within the subsection Attenuatae (Price et al., 1998). The species differ accordingly in some physiological and ecological traits. Pinus pinaster has relatively moderate requirements for water and nutrients but has a high demand for light (Rodriguez-Soalleiro et al., 1997). Pinus radiata has higher requirements for water and nutrients but is more tolerant to shade when juvenile (Ceballos and Ruiz de La Torre, 1979). Pinus pinaster needles typically live for 5.6 years, while P. radiata needles live for 2.5 years, which indicates a difference in annual turnover of foliage mass of about 18 vs. 40% for P. pinaster vs. P. radiata, respectively (Warren and Adams, 2000; but see Eimil-Fraga et al., 2015). Pinus radiata in our study area are attacked by practically all insects and pathogens that attack P. pinaster (Dans et al., 1999; Lombardero et al., 2012). The pine processionary moth is a common defoliator pest in pine forests throughout the Mediterranean countries, although it can feed also on Cedrus spp. (Demolin, 1970; Sbabdji et al., 2009). 2.2. Study area We selected nine even-aged pine plantations in the province of Lugo (Galicia, Spain) in which both pine species were growing

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intermixed, and which had not been subject to previous defoliation by PPM within the life of the trees that we studied. Stands were similar (Table 1) except that they were chosen to span a gradient of site quality for growth (chiefly from differences in soils that influence nutrient and water availability to trees). In each stand, we selected 30 interspersed trees of each species, and then randomly allocated ten trees of each species to one of three experimental treatments: severe defoliation, moderate defoliation and control trees. Plots were monitored for 6 years (2002–2007). Climate data were available from a nearby station at Cervantes, Lugo (42 49 12N, 6 55 54W). Annual precipitation was quite variable among years with 2006 being the driest (mean monthly total ± SD = 82 ± 25 L/m2) with precipitation mainly only in winter months and 2002 the wettest (135 ± 4 L/m2) with precipitation also in spring and fall; 2006 was also the warmest year in our study and 2002 the coolest (average daily temperature ± SD of 9.4 ± 1.6 vs. 8.0 ± 1.2 °C). 2.3. Defoliation treatments In September 2003 we collected 360 similarly sized egg batches of PPM from both P. pinaster and from P. radiata in stands outside our uninfested study stands but within the same region. PPM females lay their eggs in summer as a single batch using one pine needle cluster as support. This permitted us to collect and move individual egg batches intact with their supporting needle cluster. Trees allocated to moderate and severe defoliation treatments received 1 and 3 egg batches respectively, while control trees received no egg batches. Batches were attached to trees individually with a wire and positioned within the upper part of crowns similarly to match natural oviposition. We checked each egg batch every day during hatching time to ensure that they hatched successfully. In our area, PPM larvae feed and grow within collective nests through fall and part of winter. In late February, just before pupation, colonies were removed to prevent stand infestation. As we planned, trees allocated to high defoliation lost nearly 100% of their crowns while trees allocated to moderate defoliation lost 50% of their crowns (representative photograph in Supplemental materials, Fig. 1). 2.4. Tree growth For all 540 experimental trees, we measured stem diameter (at 1.30 m above ground) and total height for two years prior to defoliation and 4 years after defoliation (2002–2007). Bole volume of each tree (m3) was estimated from height and diameter as V = a
D b
H c, with coefficients a, b, and c for each species from Diéguez-Aranda et al. (2009). Measurements were made in later winter of each year, after feeding by PPM, but before the start of

active growth in the coming year; thus, our defoliation treatment occurred late in the 2003–04 growing season. Results show data only until 2006 because 2007 did not show any further patterns. 2.5. Phytochemistry We collected current year needles from tips of branches of the study trees in November, within the time when PPM does most of their feeding. We analyzed samples of current year foliage collected from each tree in November of the year preceding experimental defoliation treatments (2002) and the year after (2004). We were able to perform chemical analyses of 5 trees of each species  2 treatments (control and severe defoliation)  2 sites (the lowest quality site, Villardíaz, and one high quality site, O Picato). Chemical analyses of foliage before and after defoliation included total N, tannins, Folin-Denis phenolics, soluble phenolics, terpenes, total soluble proteins and four defensive proteins: peroxidase activity (POD), poliphenoloxidase activity (PPO), chitinase activity (CHI), trypsin inhibitor activity (TI). In all cases, freshly collected foliage was transported on ice to a freezer. Subsets of foliage from each tree were lyophilized and ground to pass through a 40 mesh screen before analysis of % N (on a Carlo-Erba carbon– nitrogen analyzer), total Folin-Denis phenolics (using tannic acid standard), and total condensed tannins (using quebracho tannin standard) (Nitao et al., 2001). Terpenoids were analyzed from fresh tissue by gas chromatography–flame ionization detection following methods of Wallis et al. (2008). Soluble phenolics were measured by HPLC following the methods of Bonello and Blodgett (2003). Defensive proteins were analyzed from fresh tissue following the methods of Cipollini et al. (2011). Ours is the first study that we know of measuring defensive proteins in either P. radiata or P. pinaster, but see Bordeaux et al. (2011) and Kovalchuk et al. (2015) for studies with other pines. 2.6. Statistical analyses Tree growth measurements (diameter, height, and volume) were each analyzed with a general linear model that included pine species, defoliation, and site as main effects, and growth during the year previous to treatment (2002–03) as a covariate. Defoliation was treated as a continuous effect (0, 50, or 100% for control, low defoliation, and high defoliation, respectively) to test for a linear trend in effects of defoliation (following Mize and Schultz, 1985). Pretreatment growth (the covariate) was adjusted to a mean of 0 for each site so that it did not absorb the effects of site. Site was treated as a fixed rather than a random effect for the primary analyses of defoliation effects because sites were deliberately selected to include low and high quality sites and because both species and each defoliation treatment were replicated at each site. However,

Table 1 Characteristics of the study plots in 2002 prior to experimental defoliation treatments in 2003. N = 30 trees by species and site. Pine stand

Villardíaz Rocas Cortafuegos Teixeiro2 Costantini O Picato Embalse Biduedo Teixeiro1 a

Coordinates

43 43 43 43 42 42 43 42 43

13 10 10 03 54 52 09 56 04

00N, 24N, 07N, 50N, 01N, 51N, 49N, 19N, 01N,

7 6 6 7 7 7 6 7 7

06 52 53 29 13 16 52 14 29

Elevation (m)

Age

530 487 518 526 740 699 381 661 521

8 7 7 6 9, 7 6 6 6 6

30W 39W 45W 12W 38W 59W 14W 10W 20W

Height/age in 2002 (average of 2 species  30 trees).

Diameter (cm) ± SE

Height (m) ± SE

P. pinaster

P. radiata

P. pinaster

P. radiata

4.3 ± 1.1 4.6 ± 1.4 4.5 ± 0.9 4.8 ± 1.0 7.8 ± 2.2 5.4 ± 1.6 4.6 ± 0.8 5.9 ± 1.4 5.4 ± 1.2

4.5 ± 0.7 4.5 ± 0.9 4.3 ± 0.5 5.1 ± 1.0 6.3 ± 1.2 5.3 ± 1.3 4.5 ± 0.8 5.3 ± 0.9 6.2 ± 1.4

3.5 ± 0.5 3.6 ± 0.4 3.5 ± 0.4 3.3 ± 0.4 4.6 ± 0.6 3.4 ± 0.3 3.7 ± 0.5 4.1 ± 0.6 3.7 ± 0.4

3.5 ± 0.5 3.5 ± 0.5 3.8 ± 0.4 3.4 ± 0.4 4.5 ± 0.6 3.9 ± 0.5 3.9 ± 0.5 3.8 ± 0.5 4.5 ± 0.6

Site qualitya

0.43 0.51 0.52 0.56 0.58 0.60 0.64 0.66 0.68

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we also ran the equivalent model with site as a random effect to obtain estimates of variance among sites (with each species separately to allow for the theoretical possibility that species would not respond the same to the range of site qualities). Diameter and height measurements were approximately normally distributed. Volume growth was well normalized with a square-root transformation. We tested for hypothesized effects of site on growth reductions from defoliation by examination of F-tests in the model described above for site  defoliation and site  species  defoliation. We also evaluated the strength and form of relations between site quality and growth reductions from defoliation by estimating the slope and SE of the defoliation effect for each species at each site (with a model that included defoliation and the covariate). From this model, the slope for each species at each site represented the growth reduction from 100% defoliation. This was converted to % reduction in growth by multiplying by mean growth  100

and plotted as a function of site quality, where site quality was quantified as height growth per year of young trees prior to the start of our study (Table 1). Measurements of phytochemistry were analyzed with a general linear model that included site, year, defoliation  year, site  year, defoliation  site  year, and tree (as a random effect) nested within site and defoliation. We used an expected mean squares model in which site was tested over tree (site, defoliation) and all other terms were tested over the mean square error. We analyzed all individual compounds that were generally detectable (>50% of the samples). Tannins and Folin-Denis phenols were so highly correlated (r2 = 0.90 and 0.84 for P. pinaster and P. radiata, respectively) that they were essentially redundant; so we only report measurements of total (Folin-Denis) phenols. POD was only occasionally detected in either species of pine and therefore was not included in statistical analyses. We evaluated the correlation structure among different measures of phytochemistry and

Fig. 1. Volume growth of Pinus pinaster (left) and P. radiata (right) following experimental defoliation treatments in the autumn of 2003. See supplementary materials Table 1 for corresponding statistical analyses.

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employed principle component analyses to account for correlations among individual terpenes in P. pinaster and among defensive proteins in P. radiata. All phytochemical measures were in units of compound mass/leaf mass except that defensive proteins were per total soluble protein and soluble phenolics were expressed as total (HPLC) peak area per sample. Analyses were performed with the statistical package JMP (SAS Institute Inc.).

3. Results 3.1. Tree growth Growth differed between pine species as expected, in that P. radiata grew faster than P. pinaster by 12–21% for volume and 36–59% for height (Figs. 1 and 3; F1, 475 > 35, P < 0.001, Supplementary materials, Table 1). Also as expected, there were strong and

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significant effects of site on growth: for volume and height growth respectively, the estimated SD among sites was 21–26% of mean and 17–24% of the mean (Supplementary materials, Table 1). Diameter growth was quite similar between species (Fig. 2; except was 11% higher for P. radiata in 2005–06), but was responsive to site conditions (SD among sites = 17–29% of mean). For both species, the sites that afforded the highest average height growth were the same as those sites with the highest average diameter growth and volume growth: r (Pearson correlation coefficient) = 0.51 to 0.86. Furthermore, the best sites for P. pinaster tended to be the best sites for P. radiata (r = 0.90 for average volume growth). Across the nine sites, relative site quality tended to be about the same across years for each species with respect to diameter and volume growth: r = 0.69–0.93. However, relative site quality was more variable among years for height growth (e.g., site-specific height growth in 2004–05 was a poor predictor of the next year; r = 0.09 and 0.39 for P. pinaster and P. radiata, respectively).

Fig. 2. Diameter growth of Pinus pinaster (left) and P. radiata (right) following experimental defoliation treatments in the autumn of 2003. See supplementary materials Table 1 for corresponding statistical analyses.

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Defoliation by PPM had significant but modest effects on tree growth in both pine species (Figs. 1–3). Overall, there were average reductions in volume growth of only 8 and 6% in the first and second years of growth after defoliation (main effect of defoliation: F1, 475 = 12.17, P = 0.0005 for 2004–05 and F1, 475 = 7.67, P = 0.0058 for 2005–06; Supplementary materials, Table 1). Reductions in volume growth due to defoliation were due to reduced diameter growth, but only in the second year (Fig. 2, Supplementary materials, Table 1). Height growth was not significantly affected by defoliation in either species or in any year (Fig. 3, Supplementary materials, Table 1). There were only modest differences among sites in the effects of defoliation on tree growth, and there was no evidence in either species of systematic patterns related to site quality (Fig. 4). In 2004–05, there were three sites where volume growth of P. pinaster was reduced from defoliation by 20–50%, but there was no evidence of greater reductions in lower or higher quality

sites (Fig. 4, upper left). For P. radiata, growth reductions during 2004–05 were less and restricted to only 2 or 3 sites (Fig. 4, lower left). However, effects on P. pinaster had largely disappeared by 2005–06, while modest effects remained evident in some sites for P. radiata (Fig. 4, upper and lower right). To the extent that defoliated trees showed reduced growth during 1–2 years following treatment, they tended to compensate with increased growth in subsequent years 3–4. Thus, there were no detectable effects of defoliation in 2003 on overall tree growth (diameter, height, or volume) for either species during the interval from 2003 to 2007 (F1, 231 < 1.52, P > 0.20; data not shown). 3.2. Phytochemistry The phytochemistry of P. pinaster and P. radiata differed in almost every aspect that we measured (Supplementary materials, Tables 2 and 3). Concentrations of foliar N were lower and more

Fig. 3. Height growth of Pinus pinaster (left) and P. radiata (right) following experimental defoliation treatments in the autumn of 2003. See supplementary materials Table 1 for corresponding statistical analyses.

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variable across years and treatments in P. pinaster than in P. radiata: averages of 0.56 to 1.26% (grand mean = 0.89%) vs. 1.13 to 1.29% (grand mean = 1.21%), respectively. Total Folin-Denis phenolics (and condensed tannins) were lower in P. pinaster than in P. radiata. However, the total peak area of soluble phenolics as measured by HPLC did not differ between species (F1, 27 = 0.15, P = 0.85). Total terpenes were high in P. pinaster and barely detectable in P. radiata: grand means = 8.65 vs. 11.86% and 1.58 vs. 0.14%, respectively. Of the proteins, total soluble proteins and trypsin inhibitor (TI) were higher in P. radiata, while guaiacol peroxidase (POD), polyphenol oxidase (PPO), and chitinase (CHI) were higher in P. pinaster. There was correlation structure to the phytochemistry of both species. Within P. pinaster, there were generally high positive correlations among terpenes (least so for limonene and d-3-carene) and total soluble proteins were positively correlated with phenolics and negatively correlated with total N (Supplementary materials, Table 4). In P. radiata, there were also generally positive correlations among terpenes, even though terpenes were at such low concentrations as to be frequently undetectable and again total phenolics and total soluble proteins were positively correlated (Supplementary materials, Table 5). For P. radiata, but not P. pinaster, total N was positively correlated with CHI, and TI was negatively correlated with total soluble proteins. For subsequent analyses, we omitted compounds that were undetectable in the majority of samples and used principal components analysis (PCA) to reduce the dimensionality of terpenes in P. pinaster and defensive proteins in P. radiata (Supplementary materials, Tables 4 and 5). For P. pinaster, the first two PC axes explained 70% of the total variation in 8 individual terpenes. The first axis, which accounted for 51% of the total variation, had positive loadings from each terpene; thus positive scores for PC-1

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indicated high terpenes in general. The second axis had a high positive loading for delta-3-carene, lower positive loadings for betapinene and myrcene, and negative loadings for all three sesquiterpenes. For P. radiata, the first two PC axes explained 57% of the total variation in five protein measurements. The first axis, which accounted for 34% of the total variation, had positive loadings from total soluble protein and CHI, and negative loadings from POD, PPO, and TI. The second axis had a high positive loading for POD, a lower positive loading PPO, and negative loadings for TI. For P. pinaster, there were effects of site and year, but not defoliation on phytochemistry (Figs. 5 and 6; Supplementary Materials, Table 6). Foliar N dropped from 1.2% in 2002 to 0.8% in 2004, without effects of either site or defoliation (Fig. 4, upper). At the same time, total phenolics increased by 15–40% from 2002 to 2004, more on the low growth site (site  year interaction; Fig. 5); there was a tendency for lower Folin-Denis phenolics following defoliation but it was not significant. There was no effect of defoliation or site on the total peak area of soluble phenolics as measured by HPLC. There were no significant effects in P. pinaster of site, year, or defoliation on total terpenes or the first axis of the terpene PCA (Fig. 5; Supplementary materials, Table 5). However, trees on the low growth site tended to have more delta-3carene, beta-pinene and myrcene, and lower concentrations of sesquiterpenes (PC axis 2; Fig. 6, lower). PPO was about twice as high in 2002 as in 2004, but there were no other effects of site, year, or defoliation on the three defensive proteins that were at detectable levels in P. pinaster (Supplementary Materials, Tables 2 and 5). By comparison with P. pinaster, the phytochemistry of P. radiata was quite stable with respect to site, year, and defoliation (Supplementary Materials, Tables 3 and 6). The only effects were on two defensive proteins: PPO was decreased by defoliation,

Fig. 4. Defoliation effects on the growth of Pinus pinaster and P. radiata relative to site quality. Each point represents measurements of 30 trees of that species at that site. The effect is expressed as % growth change relative to controls (±SE) in 2004–05 and 2005–06, following 100% defoliation in the autumn of 2003. Dashed horizontal lines indicate no effect of defoliation on growth.

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Fig. 5. Concentrations of N and Folin-Denis phenolics in foliage of P. pinaster before and after experimental defoliation in autumn of 2003. Corresponding statistical analyses in supplementary materials Table 6. Arrows indicate the temporal progression from before defoliation (all trees in 2002) to after defoliation (control vs. defoliated trees in 2004).

while CHI was higher on the high growth site, where it was decreased by defoliation (Fig. 7). We note that tests for effects of site quality on phytochemistry were constrained because we were only able to do chemical analyses for trees from two sites, but it was helpful that we were able to compare the lowest quality site (Villardíaz) with one of the high quality sites (O Picato; see site quality metrics in Table 1). 4. Discussion 4.1. Effects of defoliation on tree growth The impact on tree growth from PPM was surprisingly low. Even with complete defoliation, large sample sizes, and baseline measurements that accounted for much of the variation among trees, we only observed modest effects on diameter growth and no effects on height growth. Thus, effects on volume were only 6–8% initially, with full recovery within 3 years of defoliation. It might have been that P. pinaster suffered less from defoliation by virtue of its evolutionary history with PPM, or it might have been that P. radiata suffered less, especially on good sites, because of its capacity for high growth. However, both pine species displayed high tolerance to defoliation by PPM and strong capacity for compensatory growth to replace damaged leaves and shoots (McNaughton, 1983; Stowe et al., 2000), which is not generally

Fig. 6. Terpene content of Pinus pinaster foliage before and after experimental defoliation in autumn of 2003. Corresponding statistical analyses in supplementary materials Table 6. See test and supplementary materials Table 5 for interpretations of principle components axes 1 (middle figure) and 2 (lower figure). Arrows indicate the temporal progression from before defoliation (all trees in 2002) to after defoliation (control vs. defoliated trees in 2004).

considered to be the case for evergreen conifers (Krause and Raffa, 1996; Kulman, 1971). As we intended with the study design, there was high variation in growth among sites, presumably due to differences in soils that affect availability of nutrients and water to trees (Sanchez-Rodriguez et al., 2002). However, there was no evidence of increased effects of defoliation on the sites that afforded lower growth for pine trees. Palacio et al. (2012) suggest that regrowth after a short term defoliation by PPM was primarily supplied by current year assimilation; consequently, high resource availability may influence our results. Our study area is among

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as nitrogen and fiber content (Battisti, 1988; Devkota and Schmidt, 1990), silica and phenolics (Schopf and Avtzis, 1987), and terpenes (Niccoli et al., 2008; Paiva et al., 2011; Petrakis et al., 2005; Tiberi et al., 1999). Ovipositing PPM are also reported to favor pines within stands that have relatively rapid height growth and relatively slow volume growth (Peres-Contreras et al., 2008). Visual cues apparently play a role in tree selection by PPM females (Demolin, 1969; Jactel et al., 2006). Nonrandom host selection weakens inferences from studies comparing trees that were or were not naturally attacked (Neuvonen and Haukioja, 1985). Our study does not permit inferences regarding effects on tree growth from repeated defoliation, which one would expect to be greater. Repeated attacks of PPM have been reported to reduce growth of P. nigra (Bouchon and Toth, 1971; Palacio et al., 2012). 4.2. Phytochemistry of P. pinaster vs. P. radiata

Fig. 7. Concentrations of two defensive proteins (polyphenol oxidase and chitinase) in foliage of Pinus radiata before and after experimental defoliation in autumn of 2003. Corresponding statistical analyses in supplementary materials Table 7. Arrows indicate the temporal progression from before defoliation (all trees in 2002) to after defoliation (control vs. defoliated trees in 2004).

the most productive areas of Spain for growth of pine trees and even our low quality sites permitted high productivity compared to other regions where PPM occurs (Gandullo and Serrada, 1977). Other studies of PPM defoliation have tended to report greater losses: e.g., up to 33% reduction in volume growth of P. radiata (Cadahia and Insua, 1970) and 40–64% in shoot length of P. pinaster (Markalas, 1998). Reductions in diameter after PPM defoliation have also been reported for P. brutia (Babur, 2002; Carus, 2004; Kanat et al., 2005), P. sylvestris (Hódar et al., 2003) and P. nigra (Durkaya et al., 2009). Jacquet et al. (2012) concluded that processionary moth defoliation has a significant impact on pine growth from a meta-analysis of 45 case study. However Barrento et al. (2008) showed positive effects of PPM defoliation on P. pinaster growth and Palacio et al. (2012) found no effects on growth of P. nigra after short term defoliation by PPM. A positive feature of our study was that we were able to experimentally apply defoliation treatments to randomly selected trees. As far as we know, ours is the first study to test effects of PPM defoliation on pine growth by experimental manipulation of egg batches on interspersed trees. Previous studies have generally compared stands or trees that were naturally attacked or not, which makes it difficult to separate effects of defoliation from those due to nonrandom selection of stands, sites, and trees by PPM. In fact, PPM females tend to select trees for oviposition based on foliage characteristics such

As predicted by the resource availability hypothesis (RAH), P. radiata, the faster growing species, had higher concentrations than P. pinaster of foliar N (a determinant of photosynthetic capacity; Garnier et al., 1995; Poorter et al., 1990) while P. pinaster had up to 12 times higher concentrations of terpenes. Terpenes have broad spectrum toxicity for insects, but can also be attractants for oviposition by PPM adults (Niccoli et al., 2008; Paiva et al., 2011; Peres-Contreras et al., 2008; Tiberi et al., 1999) and feeding by PPM larvae (Petrakis et al., 2005). The secondary chemistry of P. radiata foliage, while very low in terpenes, was relatively rich in tannins and total phenolics. The antiherbivore properties of tannins include digestibility reduction in ruminants (Robbins et al., 1987) and oxidation activity in caterpillars (Barbehenn and Constabel, 2011). There is also evidence that many tannins are relatively benign to many insect herbivores (Ayres et al., 1997). Low-molecular-weight phenolics can sometimes act as toxins but may also be metabolic intermediates with no primary function (Buchanan et al., 2000). The importance of phenolics in conifer defenses against folivores is ambiguous; there are reports of reduced larval performance in some Lepidoptera (Barre et al., 2003; Pasquier-Barre et al., 2000) including PPM (Schopf and Avtzis, 1987), while others seem unaffected by host-plant phenolics (Barre et al., 2003; Hódar et al., 2015). The pine species also differed in defensive proteins. Pinus pinaster had higher concentrations of POD, PPO and CHI, while P. radiata had higher concentrations of TI. As they are N-based and presumably have high turnover times, RAH predicted more investment in defensive proteins by the faster growing species (P. radiata), but this was not the case. Eimil-Fraga et al. (2015) show that needle longevity of some Atlantic provenances of P. pinaster was positively affected by site index and foliar nutrient concentrations, suggesting that maritime pine adapt to low soil fertility by decreasing needle lifespan and maintaining a few young very efficient leaves, similar to P. radiata. There is clear evidence of the importance of the defensive proteins as antiherbivore defenses against insects and pathogens of herbaceous plants (Swapan and Muthukrishnan, 1999), but studies in pines are limited (e.g., Barto et al., 2008). It will require further study to know if proteins have significance in the defenses of pines compared to the more conspicuous role of terpenes and phenolics. 4.3. Inducible responses to defoliation by pine processionary moths P. pinaster has a long evolutionary history with PPM, and PPM defoliation in one year is a good predictor of risks from PPM the following year, because high abundance of PPM in one year tends to yield high abundance the next year (Sangüesa-Barreda et al., 2014). Therefore, it seems that delayed inducible defenses would

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be adaptive for this pine species. However, there was no evidence for delayed inducible defenses in P. pinaster following PPM defoliation. Similar results were reported by Hódar et al. (2015). One general possibility for the absence of inducible plant defenses is that the herbivore has evolved means to suppress the response, for example by interfering with the transcription of genes that upregulate defensive molecules (Bede et al., 2006; Lawrence et al., 2008; Musser et al., 2002; Sarmento et al., 2011). We are able to discount this possibility for PPM and P. pinaster because P. radiata similarly showed no evidence for inducible defenses, even though it lacks an evolutionary history with PPM. The absence of delayed inducible responses has been similarly reported in some other studies of conifers (Bryant et al., 1988; Haukioja, 1991; Niemela and Tuomi, 1993; but see Roitto et al., 2009). One simple explanation is that delayed inducible defenses of foliage are not in the physiological repertoire of pines in general (Bryant et al., 1988). One factor that could limit inducible responses in our study system is that PPM larvae feed on pine needles from late summer to late winter, when the following year’s buds have been already formed and source:sink relations are generally weak (Haukioja and Honkanen, 1996; Nykanen and Koricheva, 2004). Bryant et al. (1988) proposed that delayed induced defenses are generally constrained in evergreen conifers relative to deciduous trees because defoliation has a more limited effect on the carbon:nutrient ratio and thus host quality of the former. We were unable to conduct bioassays to see how feeding on trees defoliated the previous year affects growth and survival of PPM, but Hódar et al. (2015) did not find effects on larval survival after feeding on previously defoliated trees. Likewise, Lombardero et al. (2013) found no effects of needle pre-treated with methyl jasmonate on PPM feeding. 5. Conclusions The selection of tree species and sites are important decisions for foresters that can have important consequences for production. Good sites for pine growth can reduce damage caused by defoliators and permit rapid recovery from effects of a defoliation. Pinus radiata displays generally higher growth than P. pinaster under good conditions in our study region (even when subjected to a defoliation by PPM). On the other hand, the native P. pinaster, compared to the introduced P. radiata, shows higher levels of constitutive defenses and high plasticity in its use of resources, which could be exploited to improve resistance. Defoliation by PPM may not be a primary consideration for foresters in selecting pine species to plant in highly productive stands, such as in our study area. However our results do not permit inferences regarding effects on tree growth from repeated defoliation or in regions with lower site quality than in our study area. Especially with consideration of the full community of potentially damaging pests and pathogens, it remains true that prudent selection of tree species for production forests is informed by consideration of defenses as well as growth potential. Acknowledgements Funding was provided by Spanish grant AGL2001-3871-C02-01 and state and federal funds appropriated to the Ohio Agricultural Research and Development Center and The Ohio State University, and Wright State University. We thank Dr. Chris Wallis for conducting analyses of phenolics and terpenoids, and Stephanie Enright for assistance with defensive protein analysis. We thank Bruce Birr and William J. Mattson (US Forest Northern Research Station, Rhinelander, WI) for analyses of total nitrogen, condensed tannins, and Folin-Denis phenolics. The manuscript was greatly improved by thoughtful comments from anonymous reviewers.

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