Ecological Entomology (2007), 32, 211–220 DOI: 10.1111/j.1365-2311.2006.00860.x

© 2007 The AuthorsJournal compilation © 2007 The Royal Entomological Society 211

Correspondence: Benedicte R. Albrectsen, Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, Sweden. E-mail: benedicte.albrectsen@plantphys.umu.se

Does the differential seedling mortality caused by slugs alter the foliar traits and subsequent susceptibility of hybrid willows to a generalist herbivore?

B E N E D I C T E R . A L B R E C T S E N 1 , L A U R A G U T I E R R E Z 2 , R O B E R T S . F R I T Z 3 , R O B E R T D . F R I T Z 3 and C O L I N M . O R I A N S 2 1 Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, Sweden , 2 Department of Biology,

Tufts University, Medford, Massachusetts, U.S.A. , and 3 Department of Biology, Vassar College, Poughkeepsie,

New York, U.S.A.

Abstract . 1. Many Salicaceae species naturally form hybrid swarms with parental and hybrid taxa that differ in secondary chemical profile and in resistance to herbivores. Theoretically, the differential mortality in the seedling stage can lead to changes in trait expression and alter subsequent interactions between plants and herbivores. This study examines whether herbivory by the generalist slug Arion subfuscus , which causes extensive mortality in young willow seedlings, causes shifts in (a) the foliar chemistry of F2 willow hybrids ( Salix sericea and Salix eriocephala ), and (b) the subsequent susceptibility to Japanese Beetles, Popillia japonica .

2. In 2001, two populations of F2 seedlings were generated: those that survived slug herbivory (80 – 90% of seedlings placed in the field were killed by the slugs) were designated as S-plants, whereas C-plants (controls) experienced no mortality.

3. Common garden experiments with cuttings from these populations, in 2001 and 2002, revealed extensive variation in the phenolic chemistry of F2 hybrids, but revealed no significant difference between S- and C-plants, although the levels of foliar nutrients, proteins and nitrogen tended to be higher in S-plants.

4. Concentrations of salicortin and 2 ′ -cinnamoylsalicortin explained 55 and 38% of the the variation in leaf damage caused by Japanese beetles, and secondary chemistry was highly correlated within replicate clones (salicortin R 2 = 0.85, 2-cinnamoylsalicortin R 2 = 0.77, condensed tannins R 2 = 0.68).

5. Interestingly, Japanese beetle damage and condensed tannins were positively correlated within the S-plants, but not in the C-plants, suggesting that slugs had selected for plants with a positive relationship between tannins and P. japonica damage. This is unlikely to be a consequence of a preference for tannins, but is suggested to be related to the elevated nutrient levels in the S-plants, perhaps in combination with the complex-binding properties of tannins.

6. The damage was highly correlated within replicate clones and a model choice analysis suggested that Japanese beetle damage may be explained by four factors: concentrations of salicortin, condensed tannins, and nitrogen, as well as the specific leaf area (thick leaves were damaged less).

Key words . Arion subfuscus , herbivory , hybridization , Popillia japonica , phenolic glycosides , plant defence , salicaceae , Willow .

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Introduction

Hybridization is extremely common in plants, and differential resistance among hybrids and their pure parents has ecological and evolutionary consequences. The variation in defensive traits among hybrid and parental taxa is known to influence the distri-bution and abundance of herbivores, predators, and other mem-bers of the community ( Fritz et al. , 1994; Whitham et al. , 2003 ). If certain combinations of defensive traits confer a fitness ad-vantage, this could not only influence resistance to other herbiv-ores (the objective of this study) but could ultimately lead to the introgression of defensive traits either between species or to spe-ciation (e.g. Whitham et al. , 1991; Arnold, 1994; Ellstrand et al. , 1996; Arnold, 1997; Rieseberg & Carney, 1998; Orians, 2000 ).

Trait variation is especially pronounced among hybrids. Although F1 hybrids are largely intermediate between the pa-rental taxa ( Orians, 2000 ), recombination in later generations (back-cross, F2, etc.) creates unique trait combinations ( Hochwender et al. , 2000 ). Because some herbivores are impor-tant agents of plant mortality ( Fritz & Simms, 1992 ), differen-tial herbivory may determine the pattern of defensive trait variation in hybrids ( sensu Fritz et al. , 2001 ), and the distribu-tion and abundance of other herbivores as well.

Herbivores that cause extensive mortality at the seedling stage ( Harper, 1977; Stratton, 1992; Crawley, 1997; Fritz et al. , 2001 ) have the potential to favour certain trait combinations and to alter the subsequent susceptibility to herbivores. In temperate habitats, slugs are important agents of seedling mortality. Nystrand and Granström (2000) showed that slugs cause severe damage to 1-week-old seedlings of Pinus sylvestris , with mor-tality levels of up to 37%, and Fritz et al. (2001) reported mor-tality levels up to >80% on young willow seedlings. As important, slugs are highly selective in their choice of host spe-cies ( Cates & Orians, 1975; Dirzo, 1980; Fritz et al. , 2001 ). One therefore would predict slugs to generate non-random hybrid survival, and that this would alter the subsequent susceptibility to other herbivores.

This study examined the way in which seedling mortality caused by a slug ( Arion subfuscus ) among F2 hybrids of Salix sericea and Salix eriocephala (willows) affected the consequent damage to the hybrids by a common generalist herbivore (the Japanese beetle, Popillia japonica ) . Defence and nutritional chemistry were analysed in the leaves to determine the traits with importance for Japanese beetle damage. In addition to quantify-ing phenolic glycosides, a critical determinant of resistance in hybrid and pure willow species ( Rowell-Rahier, 1984; Matsuda & Matsuo, 1985; Tahvanainen et al. , 1985; Denno et al. , 1990; Kelly & Curry, 1991; Rank, 1992; Kolehmainen et al. , 1994 Orians et al. , 1997; Fritz et al. , 2001; Hallgren et al. , 2003; Glynn et al. , 2004; Bailey et al. , 2005 ), other traits, including condensed tannins, protein, nitrogen, and specific leaf area, were measured. Because phenolic glycosides deter feeding by slugs ( Fritz et al. , 2001 ), it was predicted that concentrations of phenolic glyco-sides would be higher in plants that survived herbivory.

Several attributes make the present study system ideal for this work. First, there is extensive variation in the resistance to slugs and other herbivores ( Fritz et al. , 1994; Orians et al. , 1997; Fritz et al. , 2003 .). Second, slugs cause high seedling mortality

( Fritz et al. , 2001 ). Third, the secondary chemistry is well- characterised. The levels of condensed tannins are high in S. eriocephala and the levels of phenolic glycosides are high in S. sericea , intermediate levels of tannins and phenolic glyco-sides are found in F1-hybrids, and variable secondary chemistry characterises other hybrids (F2s and back-crosses) ( Orians & Fritz, 1995; Hochwender et al. , 2000 ). Because generalist her-bivores, such as slugs and Japanese beetles, are deterred by phe-nolic glycosides ( Orians et al. , 1997; Fritz et al. , 2001 ), seedlings with high concentrations of phenolic glycoside were predicted to survive slug herbivory or alternatively slug herbivory would select for other traits or trait combinations including nutrient levels that would in turn determine the resistance of hybrids to Japanese beetles. If so, it would indicate that herbivory at the seedling stage has the potential to modify the effects of herbiv-ores that attack later ontogenetic stages.

Materials and methods

Willow hybrids

Hybridization is very common in willows ( Argus, 1986; Fritz et al. , 1994; Hjältén, 1998; Nordman et al. , 2005 ). The two study species, Salix sericea (Marshall 1785) and Salix eriocephala (Michx. 1803) are largely sympatric within North America and hybridise naturally ( Argus, 1986 ). Salix eriocephala produces high concentrations of condensed tannins, approximately ∼ 10% of the dry leaf mass ( Albrectsen et al. , 2004 ). Salix sericea pro-duces high concentrations of salicortin and 2 ′ -cinnamoylsali-cortin, two phenolic glycosides that account for > 10 and > 1.5% of the dry leaf mass, respectively ( Orians & Fritz, 1995 ). The ability to generate crosses of known parentage and use ei-ther seedlings or clones to examine survival, growth and resist-ance (e.g. Fritz et al. , 2001 ) makes it an excellent system to study the consequences of differential seedling mortality to sub-sequent resistance.

Study site

These studies were performed at the Sosnowski willow re-search facilities in Milford (NY, USA). The vegetation is domi-nated by S. eriocephala , S. sericea , and their hybrids ( Hardig et al. , 2000 ), and consequently herbivores of these willows are naturally abundant.

Herbivores

In north-east North America the Dusky slug, Arion subfuscus (Draparnaud 1805) and the scarab Japanese beetle, Popillia japonica (Newman 1841), both exotic species and generalists, are common herbivores on willow. Both herbivores generally avoid S. sericea but consume S. eriocephala and their hybrids ( Orians & Floyd, 1997; Orians et al. , 1997; Fritz et al. , 2001 ). Although both salicortin and condensed tannins deter slugs ( Fritz et al. , 2001 ), higher concentrations of tannins are required to achieve similar levels of deterrence. Moreover, there is a 5 – 8-week window before willow seedlings produce tannins at

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concentrations sufficient to deter slugs ( Albrectsen et al. , 2004 ). Both these factors are likely to explain why seedlings of S. erio-cephala are much more susceptible to slugs than are seedlings of S. sericea .

Adult P. japonica aggregate on willows during the summer months, starting in mid-July, and preferentially attack the tannin-rich S. eriocephala over phenolic glycoside containing hybrids and S. sericea ( Orians et al. , 1997; Orians & Floyd, 1997 ). Mechanistically this is because phenolic glycosides deter the beetles , and not because condensed tannins stimulate them ( Orians et al. , 1997 ). Other factors may also be important: Hamilton et al. (2005) provide evidence that P. japonica feeding is stimulated by nutritional chemistry.

Experimental design

A phenotypic selection experiment was performed using F2 seedlings from 15 crosses between F1 parents in June and July 2000. F1 plants had been produced from genetically pure S. sericea and S. eriocephala that were growing in a breeding garden at the field site. Seeds of all crosses were mixed and were germinated on beds of a commercial potting mixture. Germinated seedlings were transferred to individual cells in 72-cell trays and maintained with water and nutrients in open-ended greenhouses until they reached 5 weeks of age (the 4 – 5-leaf stage).

On June 29, 60 trays (4320 F2 seedlings) were placed on the ground in the field among naturally occurring willows. Slug densities at this field site often reach 17 per m 2 . Five additional trays with F2 seedlings that were not exposed to slugs were used as controls. Herbivory on seedlings was monitored in each tray daily. Trays were returned to the greenhouse when either eight or fewer seedlings remained in the treatment trays. Overall mor-tality ranged from 80 to 90% in each tray. Surviving seedlings from treatment trays (S-plants) and control trays (C-plants) were grown for 5 weeks before being numbered and trans-planted into 8-L pots containing top soil, vermiculite, and peat moss (4: 1: 1). All plants were overwintered at the field site.

In the experiments described below, cuttings from subsets of S- and C-plants were used. The cuttings were rooted and grown in 7.6-L pots in a mixture of top soil, vermiculite, and peat moss (3: 1: 1), and fertilizer was added (13 g of Osmocote © ; N: P: K, 10: 10: 10 per pot). The plants were held free of pests until they were moved to common gardens to be exposed to Japanese beetles.

To assess the response of P. japonica to C- and S-plants, three common garden experiments were performed (Experiment A, B, and C; Table 1). Experiment A, using a single clone, was designed to correlate damage levels with chemical traits. Experiment B, using two replicate clones of each plant, was designed to deter-mine whether both damage levels and beetle densities were con-sistent between replicates. If the patterns of attack are similar between replicate clones, it provides strong evidence for the role of genetics in determining resistance. In Experiment C the nutri-tional traits measured were changed (the levels of nitrogen were measured instead of protein) because the preliminary evidence indicated that the nitrogen concentration varied among S- and C-plants and might be important. Table 1 provides a description of the traits measured in each experiment. All the experiments are complimentary because they used different subsets of S- and C-plants, and were performed in successive years (2001 and 2002).

Experiment A, Japanese beetle, summer 2001 . On July 19th 2001 60 C- and 60 S-plants were randomly placed in a 10 × 12 pot irrigated grid, with approximately 1 m between neighbour-ing plants, in a common garden. After 3 weeks (on August 14th) the average damage by Japanese beetles was calculated from 25 leaves per plant (five randomly chosen leaves on five randomly chosen branches) that had been classified into one of six leaf-damage intervals, at 20% increments that included 0 as a separate class. The damage was compared with the chemistry from leaves of a different set of identical clones that were har-vested in June prior to the emergence of Japanese beetles. Chemical measurements included concentrations of salicortin, 2 ′ - cinnamoylsalicortin, condensed tannin, and protein.

Experiment B, Japanese beetle, summer 2002 . In March 2002 branches were cut from clones of C- and S-plants that were created in 2001 to obtain replicate clones. The cuttings were rooted with the help of liquid Dip ‘n’ Grow © . A double set of 36 clones ( c . 18 S- and 18 C-plants, a total of 72 plants) were set out in a 6 × 12 pot grid and irrigated daily at the time that beetles started emerging (July 17th). On July 31st the number of beetles on each plant was counted and the damage was assessed as damaged leaves relative to total number of leaves. The total number of leaves per plant ranged from 51 to 343, and the number of damaged leaves ranged from 0 to 69. Five leaves per plant were then harvested for chemical analysis (prior to attack) as described in Experiment A. The chemical mea surements in-cluded determinations of the levels of salicortin, 2 ′ -cinnamoyl-salicortin, condensed tannin, and protein.

Table 1. Overview of experiments. F2 hybrids that had survived slug herbivory (S-plants) and controls (C-plants) were exposed to attack by Popillia japonica in common garden experiments. Measured leaf traits included phenolics (condensed tannins and phenolic glycosides), protein concentration, nitrogen concentration, specifi c leaf area (SLA), and beetle damage (% Damage). N is the number of samples analyzed and roman numerals indicate the month of measurement.

Experiment Year Population N Phenolics Protein N SLA % Damage

A 2001 C 53 VII VII VIII S 57

B 2002 C 35 VII VII VII S 37

C 2002 C 56 V V VII IX S 58

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Experiment C, Japanese beetle, autumn 2002 . As in Experiment B, two cuttings of each F2 genotype were used (60 S- and 60 C-plants with two cuttings each gave a total of 240 plants). These genotypes were different from those used for Experiment B. Cuttings were moved to a common garden on June 6th 2002 into a 15 × 16 pot grid and irrigated daily. The damage was scored on September 19th and evaluated as the percentage damage at the plant level, by assessing the number of damaged leaves relative to the total number of leaves. The correlation between attacks by Japanese beetles between two identical clones was calculated. The mean damage for the two clones was evaluated for correlation with the chemical traits of leaves (harvested as described in Experiment A) measured on these clones in June 2002. The chemical measurements included determinations of the concentrations of salicortin, 2 ′ -cinnamoylsalicortin, condensed tannin, nitrogen, and the specific leaf area (leaf area divided by the dry leaf mass).

Sample preparation and chemical analyses

Sample preparation . Leaves were clipped at the petiole, transported on ice to the laboratory and vacuum dried for ∼ 48 h. Dried leaf samples were ground into powder using a Kleco Pulverizer and stored in the freezer until analysis.

Condensed tannin . Condensed tannins were analyzed using standard techniques ( Hunter & Forkner, 1999 ; see Albrectsen et al. , 2004 for details). Briefly, approximately 10 mg of leaf powder was weighed into 2-ml microfuge vials, washed with 500 � l of ether, and then the tannins were extracted four times with 200 � l of a 70: 30 acetone: water mixture with 1 mm ascor-bate. The acetone in the final supernatant was removed by evap-oration using a Savant Speed-Vac, and the final volume was brought to 500 � l using distilled water. Samples were then analyzed using the n -butanol assay for proanthocyanidins ( Hagerman & Butler, 1989 ). Condensed tannin concentration (as a percentage of the dry leaf mass) was then calculated.

Phenolic glycosides . Phenolic glycosides were analyzed us-ing standard methods ( Lindroth & Koss, 1996 ). Briefly, 15 mg of each leaf powder was weighed into a 2-ml microfuge vial. Cold methanol (200 � l) was added to each vial, sonicated in a cold water bath for 12 min, and then centrifuged for 5 min at 3200 r.p.m, 942 g. Duplicate 1- � l aliquots of supernatant were spotted onto 20 × 10 cm HPTLC silica plates using CAMAG Linomat IV. A standard curve for salicortin (0.2 – 4.0 mg ml –1 ) and 2 ′ -cinnamoylsalicortin (0.05 – 1.0 mg ml –1 ) was also spotted onto each plate. The plates were developed in a solution of CH 2 Cl 2 , MeOH, and tetrahydrofuran in a ratio of 6: 1: 1, and then scanned on the CAMAG TLC Scanner 3. Chromatograms were analyzed with Camag TLC software (CATS 3.11). The concentration of salicortin and 2 ′ -cinnamoylsalicortin (as a per-centage of the dry leaf mass) was then calculated.

Protein . Ground dry leaf material (3.0 ± 0.2 mg) was ex-tracted in 1.5 ml of 0.1 m NaOH for 2 h at 100 °C. The protein extracts were combined with the Bio-Rad reagent (Coomassie Brilliant Blue) in 96-well microtiter plates, and their absorb-ances were measured at 595 nm. Bovine serum albumin (BSA)

was used as a standard to calculate the percentage BSA equiva-lents per mg of dry leaf mass.

Nitrogen . Samples (12 – 15 mg) were folded into 8 × 5-mm tin weighing capsules (EMAL Tech Inc.), and were analyzed using a CE Elantech NC 2500 Element Analyzer at Boston University.

Statistics . ancova models with interaction were used to ana-lyze the effect of slug treatment and the concentration of various leaf compounds on the damage. Because damage scores were quantified as a proportion (either the percentage of the leaf area or the percentage of leaves on the plant), damage levels were arcsine transformed to normality ( Zar, 1996 ).

Damage levels by Japanese beetles varied naturally according to the time of experiment, exposure time to beetles, location at the field site, and damage assessment method. To enable a com-parison across experiments for leaf chemistry and other leaf traits, a common damage threshold was found. This was found by testing the balance of data points above and below each of three possible threshold levels (0, 10, and 15% damage) by cal-culating the damage odds: p e /(1 − p e ) between less damaged ( p ) and more damaged plants ( p − 1) for each experiment (e) and threshold level. For a threshold that provided identical odds of high damage among experiments, the odds would be equal {[ p 1 /(1 − p 1 )] = [ p 2 /(1 − p 2 )] = [ p 3 /(1 − p 3 )]} and thus the odds ratio would be equal to one. Consequently, the threshold where the odds were most alike and the odds ratio approached one could be chosen. Using that threshold level plants could be grouped and analysed as categorical data (damaged above and below the threshold level), and plants could be analysed across experiments as belonging to either high or low damage levels, respectively.

In 2002 (Experiment B and C) clones were represented by two replicates. The within loan agreement of leaf chemistry and Japanese beetle damage was examined by correlating respec-tively, chemistry and damage to replicated clones using the Pearson product–moment correlation coefficient ( R ). A high agreement in a trait suggests that the trait is clone specific and under genetic control. Because the order in which clone repli-cates appeared in the analyses slightly alters the outcome (anal-yses not shown), the distribution of r ± SE was calculated by re-sampling the order in which members of all clone pairs ap-peared in the analyses (randomly, binomial distribution, n = 1, and p = 0.5, equivalent to tossing a coin). Re-sampling was performed 10 times for each data set and analysis.

To judge the relative importance of various chemical com-pounds in the leaves and other leaf characteristics for damage by P. japonica , a model choice comparison was performed. For this purpose � AIC (Akaike’s Information Criterion) was calcu-lated, which identifies a relevant model when � AIC is less than 2 ( Anderson et al. , 2000 ).

Results

Chemistry of selected and control plants

Because the willow cuttings used in each experiment were randomly chosen both from the survivors of exposure to slugs and from the group of controls that had not been exposed to slug herbivory, the chemical profiles varied, as expected, slightly among

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experimental subsets ( Table 2). The reduced chemical concen-trations in Experiment C are likely to reflect the fact that these leaves were sampled earlier in the year. Overall, the concentrations measured in this experiment were similar to the concentrations ob-served in naturally occurring ( Orians & Fritz, 1995 ) and experi-mental hybrids ( Hochwender et al. , 2000; Fritz et al. , 2001 ).

Overall the slug treatment did not result in a significant in-crease of salicortin as predicted, although S-plants tended to be slightly richer in salicortin. This pattern was also true for 2 ′ -cin-namoylsalicortin and significantly so in the subset used for Experiment C. The tannin levels varied less consistently among experiments. They were insignificantly higher in the S-plants in Experiment A and C, but significantly lower in Experiment B. In Experiment B the protein concentration was higher in S-plants ( P = 0.002), whereas in Experiment C the levels of nitrogen exhibited a non-significant increase in S-plants ( P = 0.19).

Susceptibility to damage by Japanese beetles

Damage levels by P. japonica were higher in 2001 (Experiment A) compared with 2002, and, as expected, the cumulative dam-age was higher in the autumn (Experiment C) than in the summer of 2002 (Experiment B; Fig. 1). There was no simple effect of slug treatment on damage levels, and there was no interaction between slug treatment and experiment (A, B, or C) (anova model summary: r 2 = 0.30, f 5,285 = 24.11, P < 0.0001. Effect tests: experiment, f 2,286 = 59.74, P < 0.0001; treatment, f 1,285 = 0.0074, P = 0.93; experiment*treatment, f 2,285 = 0.18, P = 0.83).

The damage on F2 hybrids differed among clones but not within clone pairs. Initially 36 (Experiment B) and 120 (Experiment C) clone pairs could be correlated with respect to damage. After wilted individuals (the result of local clogging of the irrigation system) were removed from the data sets, 35 (B) and 102 (C) pairs were left for analyses. The degree of damage was remarkably correlated within clone pairs: r mean = 0.68 ± 0.004, P < 0.0001 (Experiment B); r mean = 0.83 ± 0.002, P < 0.0001 (Experiment C). In contrast, there was no correlation be-tween the number of beetles found on pairs of clones at a single survey event (Experiment B) ( r mean = −0.01 ± 0.009, P > 0.90). The likelihood that a damaged plant was visited by adult beetles was 50 and 90% when a plant was damaged by more than 22.6

and 52.3%, respectively (an inverse prediction from logistic regression, testing the presence of adult beetles according to damage level, R 2 = 0.16, n = 72, χ 2 1 = 8.41, P = 0.003).

Clone specifi city for chemistry

The secondary chemistry and protein concentration was ana-lyzed for all plants in Experiment B, and the within-clone correla-tion of chemistry could therefore be assessed for this data set. The agreement was extremely high for the secondary metabolites (salicortin, 2 ′ -cinnamoylsalicortin, and condensed tannin), whereas there was a striking lack of agreement with respect to protein concentration. The same re-sampling method was used as previously described for data on damage (10 re-sampling events;

Table 2. Mean concentration (± SE) of chemical traits in the leaves of plants that had survived slug herbivory (S-plants) and control plants (C-plants) in each experiment (A, B, and C). Protein is measured as the percentage Bovine Serum Albumin equivalents (BSAE) mg −1 dry leaf mass; N is measured as the percentage of weight of dry leaf mass. Differences between S- and C-plants are shown as probabilities resulting from a one-way anova comparison.

Experiment

Salicortin (% dry leaf mass)

2 ′ - cinnamoylsalicortin (% dry leaf mass)

Condensed tannin (% dry leaf mass)

Nutritional compounds

C S C S C S C S

A 7.36 ± 0.46 7.08 ± 0.47 0.64 ± 0.10 0.63 ± 0.09 24.80 ± 1.10 26.31 ± 1.22 Protein 19.47 ± 0.65 18.65 ± 0.62 P = 0.57 P = 0.91 P = 0.27 P = 0.37

B 5.99 ± 0.65 7.21 ± 0.63 0.24 ± 0.06 0.34 ± 0.07 20.64 ± 1.73 15.58 ± 1.14 13.86 ± 0.27 15.04 ± 0.27 P = 0.18 P = 0.28 P = 0.02 P = 0.002

C 4.64 ± 0.46 5.77 ± 0.49 0.32 ± 0.07 0.60 ± 0.10 15.13 ± 1.67 17.45 ± 1.56 N 2.60 ± 0.06 2.71 ± 0.06 P = 0.13 P = 0.03 P = 0.67 P = 0.19

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Fig. 1. The percentage of damage to willow hybrids (F2) by Japanese beetles in three common gardens (A = summer 2001, B = summer 2002, and C = autumn 2002). The white bars indicate damage to clones of plants that had survived slug selection during the seedling stage in 2000 (S-plants) and the grey bars show the susceptibility of clones from even-aged control plants (C-plants).

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n = 35) and obtained the following Pearson product – moment correlation coefficient averages: salicortin r mean = 0.849 ± 0.0019, P < 0.0001; 2 ′ -cinnamoylsalicortin r mean = 0.765 ± 0.0024, P < 0.0001; condensed tannin r mean = 0.678 ± 0.0031, P < 0.0001; protein r mean = 0.137 ± 0.0027, P = 0.43.

Damage – relationship to secondary chemistry

The concentration of salicortin and 2 ′ -cinnamoylsalicortin in willow leaves was negatively related to damage by P. japonica ( Fig. 2a,b; Table 3) for both treatments in all experiments. The relationship with salicortin ( Table 3a ) and 2 ′ - cinnamoylsalicortin ( Table 3b ), was tested using a nested ancova model with treat-ment nested within experiment as a covariate. The effects of experiment and phenolic glycoside concentration were highly significant ( Table 2 ). The concentration of salicortin explained more of the variation ( R 2 = 0.55) than did the concentration of 2 ′ -cinnamoylsalicortin ( R 2 = 0.38). Moreover the damage curves decrease steeply with increasing phenolic glycoside con-centration, indicating a nonlinear relationship with a threshold concentration above which P. japonica will not feed ( Fig. 2a,b ).

Condensed tannin in leaves had a more complex effect on the damage by Japanese beetles ( Fig. 2c ). The damage was either un-correlated or positively correlated with condensed tannin concen-tration. A positive correlation was found for S-plants only, indicating that the treatment could have led to a difference in the response to condensed tannin by P. japonica . The effect of tannins was further tested using the nested ancova model previously de-scribed, but with leaf tannin concentration as a covariate, and in-deed, the treatment interaction term with tannins was significant ( f 3,3 = 5.81, P = 0.0008; Table 3 ), supporting the hypothesis that slugs may select for plants with a positive relationship between tannins and P. japonica damage ( Fig. 2c; Table 3c ).

Overall, the results are robust across experiments and years. Moreover, the three types of damage assessment all proved suc-cessful as indicators of P. japonica consumption and damage. Thus, fast methods like either counting the number of damaged leaves and the total number of leaves (as in experiment B) or by estimating a whole-plant score as in experiment C are reliable methods for estimating resistance. That damage by Japanese bee-tles is highly visibly and not easily confused with other kinds of damage may be one possible reason for this agreement among scoring methods.

Damage – relationship to protein concentration

Protein concentration is often thought to affect the attractive-ness of a plant to herbivores. There was no within-clone correla-tion for foliar protein concentration ( R = 0.13, P = 0.44), and no relationship between foliar protein and herbivory (linear re-gression: R 2 = 0.005, t 1,69 = 0.54, P = 0.54). To remove the dominant effect of phenolic glycosides, the regression analysis was also performed after removing plants without any damage ( n = 19 plants remained). Again, no relationship was found (linear regression: R 2 = 0.005, t 1,17 = −0.14, P = 0.88). The

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Ac, R2=0.02 F=0.36As, R2=0.36 F=11.57***Bc, R2=0.02 F=0.43Bs, R2=0.21 F=4.33*Cc, R2=0.17 F=4.68*Cs, R2=0.13 F=3.19*

Ac

CcBs

Bc

As

Cs

2 4 6 8

Fig. 2. The effect of secondary compounds (a, salicortin; b, 2 ′ - cinnamoylsalicortin; c, condensed tannins) in the leaves of F2 willow cuttings on the damage caused by Popillia japonica . This assessment was performed in common garden experiments in the summer 2001 (Experiment A), the summer 2002 (Experiment B), and in the autumn 2002 (Experiment C) for plants that as seedlings survived slug selection in 2000 (S-plants) and for controls (C-plants). Nonlinear fi ts of the type y = a + bx + c ( x – k ) 2 are fi tted to the data points and are marked with the experiment letter and plant group (unbroken lines for S-plants and broken lines for C-plants).

Resistance of willows to Japanese beetles 217

© 2007 The AuthorsJournal compilation © 2007 The Royal Entomological Society, Ecological Entomology, 32, 211–220

statistical power, however, was too low, indicating that greater sample sizes would be required to make robust conclusions.

Damage – relationship to nitrogen levels and the specifi c leaf area

Nitrogen levels were measured early in the season on replicate clones grown in the greenhouse, and nitrogen concentration was compared with identical clones damaged by Japanese beetles late in the season (Experiment C). The specific leaf area (area in cm 2 , dry weight in g) was measured on the same leaves that were used for the analysis of primary and secondary leaf chemistry. A damage level of 10% was chosen to divide clones into high and low damage categories by comparing the odds of high damage at three thresholds (0, 10 and 15% damage). The resulting odds were as follows: Experiment A, 8, 2.55, and 0.67; Experiment B, 0.36, 0.14, and 0.09; Experiment C, 0.40, 0.21 and 0.21. Accordingly, in this case, a damage level of 10% most evenly divided the data into high versus low damage groups that could be used for comparisons among experiments. Clones with less damage had more phenolics, lower nitrogen levels, larger leaf specific areas and did not differ in their protein concentra-tion ( Fig. 3). To assess the relative importance of effects that might determine preference by Japanese beetles we per-formed AIC model choice analysis including: salicortin, 2 ′ - cinnamoylsalicortin, condensed tannin, treatment (S- and C-plants), nitrogen, and specific leaf area. Independent model choices suggested that four factors may be important for Japanese beetle damage: salicortin, condensed tannin, nitrogen levels, and the specific leaf area ( Table 4a,b). The effects of slug treatment and 2 ′ -cinnamoylsalicortin were not significant and were left out as independent variables in the model, whereas several interac-tion terms that included these effects were significant suggesting

a generally complex response by the Japanese beetles to these traits ( Table 4a,b ). The model summary for the most appropriate model selection (model 2) was highly significant ( F 7,98 = 15.88, P < 0.0001). Consequently, high levels of secondary compounds were associated with reduced damage by Japanese beetles, as were thick leaves and lower concentrations of nitrogen.

Discussion

F2 hybrids of S. sericea and S. eriocephala showed the expected extensive variation in phenolic chemistry ( Hochwender et al. , 2000 ). Although the traits may vary seasonally, the cutting treat-ment does not affect the character and relative concentrations of the focal defence chemicals ( Orians & Fritz, 1995 ). As replicate clones had similar concentrations of phenolics, our results fur-ther suggest that there is a genetic basis for phenolic production in willow (see also Orians et al. , 2000 ). Therefore, herbivory of slugs could have increased the survival of certain hybrid chemo-types with superior resistance characteristics. This is a predic-tion of the potential positive fitness effects of gene introgression of resistance genes in hybrid swarms (e.g. Whitham et al. , 1991; Arnold, 1994; Ellstrand et al. , 1996; Arnold, 1997; Rieseberg & Carney, 1998; Fritz et al. , 2001 ). There was, however, no sys-tematic significant difference in the phenolic chemistry of S- and C-plant population used in the three experiments in this study although levels of phenolics tended to be elevated in S-plants. There was a tendency of S-plants to have higher protein (Experiment B only) and nitrogen (Experiment C) levels. The protein levels were, however, not similar for replicate clones, which may reflect the effects of either microclimate (Nichols-Orians, 1991) or leaf age ( Wait et al. , 1998, 2002 ) (although in this study the first fully expanded leaf on the shoots was always selected). Tannin levels were lower in Experiment B, but not in Experiments A and C.

Table 3. Summary of nested ancova analyses determining the effect of treatment within each experimental set up and with the concentration of secondary chemicals as covariates on the damage by Japanese beetles to potted F2 willows in common garden experiments. Analyses are performed on arcsine-transformed damage rates. DF, degrees of freedom; SS, sum of squares.

Source R 2 DF SS F ratio P > F

(a) Salicortin Model 0.55 11/280 26.09 30.58 <0.0001 Effect Experiment 2/2 4.72 55.75 <0.0001 Salicortin (Exp.) 3/3 6.11 48.31 <0.0001 Treatment (Exp.) 3/3 0.11 0.85 0.47 Sa.*Tr. (Exp.) 3/3 0.13 0.98 0.40

(b) 2 ′ -cinnamoylsalicortin Model 0.38 11/280 16.05 15.90 <0.0001 Effect Experiment 2/2 7.09 61.83 <0.0001 2 ′ -cinn (Exp.) 3/3 2.06 11.95 <0.0001

Treatment (Exp.) 3/3 0.13 0.76 0.52 2*Tr. (Exp.) 3/3 0.03 0.17 0.91 (c) Tannin Model 0.41 11/245 12.73 15.89 <0.0001 Effect Experiment 2/2 6.92 66.55 <0.0001 Tannin (Exp.) 3/3 1.45 9.28 <0.0001 Treatment (Exp.) 3/3 0.29 1.83 0.14 Ta.*Tr. (Exp.) 3/3 0.91 5.81 0.0008

218 Benedicte R. Albrectsen et al.

© 2007 The AuthorsJournal compilation © 2007 The Royal Entomological Society, Ecological Entomology, 32, 211–220

It was hypothesized that herbivory by slugs would cause an increase in the resistance of S-plants to P. japonica . Contrary to our hypothesis, there was no difference between S- and C-plant populations in their susceptibility to P. japonica . Thus, differential mortality by slugs did not result in the correlational selection of resistance to Japanese beetles. Rather, attack by P. japonica in all three experiments was negatively correlated with the presence of salicortin and 2 ′ -cinnamoylsalicortin. This was even true in Experiment C, where leaf samples collected from one replicate clone in the greenhouse in late May were used to predict the susceptibility of a second replicate clone to

Japanese beetles in September The strong deterrent effects of phenolic glycosides is consistent with our previous work dem-onstrating that purified phenolic glycosides inhibit feeding by Japanese beetles ( Orians et al. , 1997 ). Phenolic glycosides are phenolic monomers that are toxic to generalist herbivore in-sects, but may attract specialists ( Matsuda & Matsuo, 1985; Tahvanainen et al. , 1985; Lindroth, 1991; Rank, 1992; Kolehmainen et al. , 1994; Orians et al. , 1997 ). Willows may roughly be divided into belonging to two groups of chemotypes that are either rich in phenolic glycosides or tannins ( Julkunen-Tiitto, 1986, 1989; Kelly & Curry, 1991; Orians & Fritz, 1995 ).

Table 4a. A comparison of multiregression models evaluating the ef-fect of slug treatment (E), salicortin (S), 2 -cinnamoylsalicortin (2), condensed tannin (T), nitrogen concentration (N), and specifi c leaf area, SLA (A), on the damage caused by Japanese beetles herbivory in the fi eld (arcsine-transformed, Experiment C) as a single effect and with interactions up to the fourth degree. K indicates the number of parame-ters in the model, R 2 indicates the explanatory value and � AIC denotes the difference in information value when compared with the preferred model with the lowest Akaike’s Information Criterion (AIC) value. Models are not signifi cantly inferior if � AIC is less than 2.

# Model K R 2 D AIC

1 S, N, A, T, ST, SN, SA, TN, NA, STN 12 0.58 0

2 S, N, A, T, SA, ST, NA 8 0.55 −0.60 3 S, N, A, T, E 6 0.40 −25.12 4 S, N, A,T, E, 2 7 0.40 −26.74 5 S, N, A, T, E, 2 (full model to

4th°interaction) 64 0.81 −29.58

6 Intercept 1 0 −65.62

Table 4b. Parameter estimates and signifi cances for model 2 ( Table 4a ). The signs of single-source effects and signifi cances agree for the top three models, indicating a negative effect of salicortin ( P < 0.0001) and positive effects of condensed tannin (n.s.), Nitrogen concentration (0.001), and leaf specifi c area, SLA (0.05). Asterisks indicate signifi -cance at the * 0.05 , ** 0.001 and *** 0.0001 levels. Effects tested with t statistics ( t ).

Source Estimate SE t – ratio P

Intercept −0.66979 0.30915 −2.17 * Salicortin −0.03163 0.00694 −4.56 *** Condensed tannin 0.00044 0.00028 1.60 0.11 Nitrogen 0.14303 0.04212 3.40 ** SLA 2.50795 1.10910 2.26 * (Salicortin-7.29)* (Tannins-256)

−0.00025 0.00006 −4.29 ***

(Salicortin-7.29)* (SLA-0.19)

−0.63206 0.22349 −2.83 **

(N-2.66)*(SLA-0.19) 5.00904 2.17783 2.30 *

Fig. 3. The concentration of phenolics and other leaf traits in clones with either high damage levels (>10%; black bars) or low damage levels (<10%; grey bars). Signifi -cance levels after a two-tail Student’s t -test for every trait and experiment are presented in italics above each comparison.

Salicortin0.0003 <0.0001 <0.0001

0A B C

5

10

% S

alic

ort

in

2-cinnamoylsalicortin 0.0001 <0.12 <0.0004

A B C0

1

% 2

-cin

nam

oyl

salic

ort

in

Tannins 0.11 0.01 0.004

A B C0

10

20

30

% T

ann

ins

Protein 0.98 0.21

A B0

10

20

% P

rote

in

Leaf Specific Area

0.03

0

10

20

C

cm2 /

g

Nitrogen 0.009

0

2.5

5

C

%-

Nit

rog

en

Resistance of willows to Japanese beetles 219

© 2007 The AuthorsJournal compilation © 2007 The Royal Entomological Society, Ecological Entomology, 32, 211–220

Species that represent these chemotypes often hybridise natu-rally, and the secondary defence properties generally follow an additive inheritance pattern ( Orians & Fritz, 1995; Orians et al. , 2000; Hallgren et al. , 2003 ), a pattern that is paralleled with average leaf beetle damage ( Hjältén, 1998; Fritz et al. , 1994; Orians et al. , 1997 ). The chemistry profile was related directly to damage at the individual plant level in a population of F2 hybrids, where recombination is assumed to partially separate the effect of secondary compounds from other feeding stimu-lating factors ( Hochwender et al. , 2000 ). Both salicortin and 2 ′ -cinnamoylsalicortin were negatively correlated with Japanese beetles feeding at the individual plant level when ana-lysed separately, but because of a correlation between these two phenolics only salicortin accounted significantly for variation in damage when both phenolics were entered into the same model. Salicortin, which often exceeds 10% of the dry leaf weight, was most strongly negatively correlated with beetle abundance.

The concentration of condensed tannins was uncorrelated with P. japonica attack when comparing the average slug treat-ment effect, however, and when comparing the relationship between tannin and damage levels for the two treatments in an ancova, the group of willows that had survived slug attack (S-plants) showed a positive relationship between damage and tannin concentration, and this relationship was repeated in all three experiments. The reason for this is unknown. The posi-tive response by P. japonica to tannins in the selected group is unlikely to result from a preference for tannins as there was no positive effect of tannins in the control group of plants. Rather the positive correlation is likely to be a response to an unknown correlated trait that changed, perhaps in combination with compensatory feeding by the beetles, in response to the tannins that complexly bind proteins and make them less easy to digest ( Schultz, 1988; Clausen et al. , 1992; Reed, 1995 ). This study supports resent findings that P. japonica feeding is stimulated by nutritional chemistry ( Hamilton et al. , 2005 ), because nitro-gen was significantly enhanced in plants with high damage levels.

The nitrogen concentration and specific leaf area were better predictors of Japanese beetle damage than the protein concen-tration. Plants with high levels of nitrogen were more damaged when the effects of phenolic glycosides were removed. Similarly the specific leaf area was positively related to dam-age, which means that thick leaves got less damaged than thin leaves.

In summary we found that phenolic glycosides were the most important determinant of susceptibility to Japanese beetles in hybrid willows, and this was independent of previous mortality by slugs. Changes in growth, foliar nitrogen levels and resist-ance that had previously been found as a response to slug mor-tality (Fritz, R.S., Hochwender, C.G., Orians, C.M., Albrectsen, B.R., and Czesak, M.E., unpublished data) were not sufficient to influence the subsequent Japanese beetle damage, although a change that was correlated with tannin concentration suggested that slug selection did influence beetle feeding responses. This provides evidence that differential slug mortality at the seedling stage may modify, albeit slightly, the attack by a generalist herbivore at later ontogenetic stages.

Acknowledgements

We thank Len and Ellie Sosnowski, who have permitted us to conduct research on their property. This work has been supported by the National Science Foundation (DEB 9981568 to CMO and DEB 99 – 81406 to RSF).

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Accepted 30 August 2006 First published online 30 January 2007