Seedling herbivory by slugs in a willow hybrid system: developmental changes in damage, chemical defense, and plant performance

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1 Oecologia (2001) 129:87 97 DOI /s Robert S. Fritz Cris G. Hochwender Debra A. Lewkiewicz Sara Bothwell Colin M. Orians Seedling herbivory by slugs in a willow hybrid system: developmental changes in damage, chemical defense, and plant performance Received: 6 August 2000 / Accepted: 26 January 2001 / Published online: 15 May 2001 Springer-Verlag 2001 Abstract We evaluated feeding preference and damage by the slug, Arion subfuscus, on seedlings of two willow species, Salix sericea and S. eriocephala, and their F 1 interspecific hybrids. Trays of seedlings were placed in the field and excised leaves were presented to slugs in choice tests. Slugs preferred feeding on and caused the most damage to S. eriocephala seedlings. S. sericea seedlings were least preferred and least damaged. F 1 hybrid seedlings were intermediate in preference and damage. Slug preference of and damage to these seedlings decreased over time, suggesting developmental changes in resistance. Seedlings were sampled for phenolic glycoside and tannin chemistry weekly to coincide with the field and laboratory experiments. Concentrations of phenolic glycosides and tannins increased linearly with seedling age, coincident with changes in slug preference and damage, indicating a developmental change in defense. Slug deterrence was not detected at low concentrations of salicortin when painted on leaves or discs, but both salicortin and condensed tannins deterred slug feeding at concentrations between 50 and 100 mg/g, levels found in adult willows. Seedling performance was related to damage inflicted by slugs. Due to lower levels of damage when exposed to slugs in the field, S. sericea plants had significantly greater biomass than S. eriocephala plants. Biomass of F 1 hybrids was equal to S. sericea when damaged. However, undamaged S. eriocephala and F 1 hybrid plants had the greatest biomass. Because F 1 hybrid seedlings performed as well as the R.S. Fritz ( ) C.G. Hochwender D.A. Lewkiewicz S. Bothwell Department of Biology, Vassar College, Poughkeepsie, NY 12604, USA fritz@vassar.edu Fax: C.M. Orians Department of Biology, Tufts University, Medford, MA 02155, USA Present address: D.A. Lewkiewicz, Department of Biology, University of California, Riverside, CA 92521, USA most fit parent in all cases, slugs could be an important selective factor favoring introgression of defensive traits between these willow species. Keywords Arion subfuscus Fitness Hybrid Chemical defense Salix Introduction Detailing the relationships between hybridization, resistance to herbivores, and plant fitness will help to clarify the evolutionary implications of plant hybridization. Interspecific hybridization can alter plant resistance to herbivores, such that hybrids either have lower resistance, higher resistance, or intermediate resistance compared to that of parents (for reviews see Strauss 1994; Fritz 1999; Fritz et al. 1999). Often, hybrids do not show lower resistance than both parents (i.e., hybrid susceptibility), but where hybrid susceptibility does occur, it could lead to decreased hybrid fitness relative to parental taxa (e.g., Drake 1981; Morrow et al. 1994; Cummings et al. 1999), potentially resulting in restricted populations and distributions of hybrids. In those cases, hybridization might have few influential evolutionary implications beyond strengthening species boundaries. However, selection can favor certain genes in hybrids, even when overall hybrid fitness is low, and thus hybridization can still have an important evolutionary effect on lineages (Arnold and Hodges 1995; Arnold et al. 1999). When hybrid resistance is equal to or greater than that of the parental taxa, or when selection favors particular genes in hybrids, hybrid plants may have equivalent or higher fitness compared to parents (Arnold and Hodges 1995; Arnold 1997), leading to larger hybrid populations with more widespread distributions. Hybridization could have at least two important evolutionary consequences the creation of and selection for recombinant hybrid progeny (F 2 or backcrosses) that have novel combinations of defense traits (Rieseberg and Wendel 1993; Fritz et al. 1999; Orians, in press), and introgression of defensive

2 88 traits between species (Stutz and Thomas 1964; Manley and Fowler 1969; Wu et al. 1996). In order to understand the influence of hybridization on fitness via herbivore resistance, particular attention should be paid to early developmental stages of plants. Herbivores can have their greatest impact on plant fitness by consuming seedling or juvenile plants (Huntly 1991; Bryant and Julkunen-Tiitto 1995). Further, seedlings often have the highest mortality of any developmental stage (Harper 1977; Crawley 1983). In addition, the environmental factors that influence seedling survival may not be the same for mature plants, so seedlings must be evaluated to understand which factors are critical during plant establishment (Harper 1977). For example, molluscs have a propensity for feeding on seedlings, thereby making themselves an influential mortality agent of seedlings (Hatto and Harper 1969; Dirzo and Harper 1980; Rai and Tripathi 1985), but not of adult plants. In addition, slugs and snails are often highly selective in their choice of host plant species (Cates 1975; Cates and Orians 1975; Dirzo 1980; Whelan 1982; Rai and Tripathi 1985; Rathke 1985; Hanley et al. 1995; Fenner et al. 1999; Scheidel and Bruelheide 1999), so mollusc herbivory can act as an important selective force on the abundance and distribution of plant species (Bruelheide and Scheidel (1999). Differences in the level of defenses expressed by plant species may underlie such molluscan preferences. Some plant species exhibit high levels of defenses in newly emerged seedlings (e.g., Glen et al. 1990; Thakray et al. 1990; Morse et al. 1991), whereas defenses are not immediately expressed in some species (Horrill and Richards 1986). Changes in seedling palatability can affect risk of damage by molluscs. For example, slugs and snails are often highly selective among chemical and morphological phenotypes of plants (Jones 1962; Cates 1975; Westerbergh and Nyberg 1995), and as seedlings age, the likelihood of attack often decreases (Bryant and Julkunen-Tiitto 1995; Hanley et al. 1995; Fenner et al. 1999). Thus, slugs may act as a major selective force favoring greater seedling defense within a lineage, as well as altering interspecific plant abundance. We have been studying herbivory by arthropods on two willow species (Salix eriocephala and S. sericea) and their interspecific hybrids to determine the patterns of relative susceptibility of hybrid and parent plants. S. eriocephala is a relatively fast growing species that utilizes condensed tannins as a foliar defense, whereas S. sericea is a slower growing species that utilizes phenolic glycosides (salicortin and 2 -cinnamoylsalicortin) (Orians et al. 1997). The concentrations of both phenolic glycosides and condensed tannin in hybrids in the field and in F 1 and F 2 hybrids produced from crosses of pure parents are generally intermediate, suggesting additive inheritance of these traits (Fritz et al. 1994; Orians and Fritz 1995; Orians et al. 2001; C. M. Orians, unpublished data). Seedlings of both parental species and F 1 hybrids are susceptible to slug herbivory in the field, but a developmental shift in the palatability of willows to slugs seems to occur before the end of the first year of growth. Given the potentially important impact of slugs on willow seedlings at our site, we evaluated the interaction between slugs and willow seedlings in relation to chemical defense. Specifically, we addressed the following questions: (1) Does foliar feeding by slugs differ among parental and hybrid willow seedlings in choice tests, and do levels of consumption change over time? (2) Do parental and hybrid willow seedlings sustain different levels of damage by slugs in the field, and do those damage levels change over time? (3) Do chemical defenses of parental and hybrid willows change with seedling age (are there developmental changes in defense)? (4) Do putative chemical defenses deter slug herbivory? (5) Do differences in slug damage experienced by plants in the field alter the relative performance among taxa? Materials and methods Study area and system This study was conducted in 1997 and 1998 at the Sosnowski site; a low-lying, willow swamp about 3 km from Milford, Otsego County, N.Y. Two willow species, Salix sericea and S. eriocephala, and their interspecific hybrids occur naturally at the site, along with two other willows, S. discolor and S. bebbiana. For this study we performed intraspecific and interspecific crosses using genetically pure S. sericea and S. eriocephala plants (Fritz et al. 1996) to produce three independent crosses of S. sericea, S. eriocephala, and F 1 seeds. RAPD markers were used to determine parents purity (Hardig et al. 2000). Seeds were germinated in trays of potting soil (Metro Mix 360) during the second week of June 1997 and the second week of May Two- to 3-week-old seedlings were transplanted into potting soil into 72-celled trays. The trays of seedlings were maintained in the greenhouse until used for experiments with regular watering and fertilization with Peters all-purpose 20: 20: 20 fertilizer as needed. The slug Arion subfuscus (Draparnaud) (Mollusca: Arionidae) is the primary herbivore of willow seedlings at our study site (personal observation). A. subfuscus was introduced from Europe (Chichester and Getz 1969, 1973) and is one of several introduced slug species in North America that are dominant molluscan herbivores in many communities. This species is among the most common slugs in many habitats, with the exception of forests (Chichester and Getz 1969). Early records documented A. subfuscus in Massachusetts by the 1840s (Chichester and Getz 1969). Therefore, this species has been established in the northeastern United States for at least 150 years. It is unknown how abundant native slug populations were prior to the introduction of European slug species or how they might have interacted with plant species (D. Strayer, personal communication). A. subfuscus is very common from May through July at our site, and it is an important herbivore of willow seedlings. Slug damage to young willow seedlings is often severe (personal observation). We collected and maintained a population of slugs throughout the summer for use in two experiments. In 1997, slugs were housed together in a large fiber bait box. In 1998, slugs were housed individually in 470-ml plastic containers lined with moist filter paper or paper towels. Slug containers in both years were buried in the ground in a shady spot at our field site to protect slugs from heat stress. Slugs were provided ad libitum with Romaine lettuce, except during periods of food-deprivation before experiments. Feeding choice experiments (laboratory) We conducted feeding preference experiments using excised leaves of parental and hybrid plants to control for confounding

3 89 factors that could influence slug herbivory of seedlings in the field, such as changes in slug abundance over time and changing environmental conditions. This experiment was conducted weekly to coincide with the weekly field choice experiments (see below). Seedlings used in these feeding choice experiments were obtained from replicate trays of seedlings of the same crosses and ages as seedlings in the field choice experiments. Seedlings were maintained under identical conditions in the greenhouse. We placed single, excised leaves of the same age and of similar sizes from seedlings of the three taxa abaxial side up along three equally spaced radii in a petri dish lined with moist filter paper. We placed a slug that had been deprived of food for at least 24 h into each petri dish and left them overnight to feed. Experiments were established between 2000 and 2100 hours. The next morning between 0800 and 0900 hours, we scored leaves for slug feeding in 10% damage intervals (0=0% feeding damage; 10=100% feeding damage) by visually assessing the proportion of leaf area removed. We analyzed damage scores using Wilcoxon signed-rank tests to determine differences in damage scores among taxa and over time. Slugs used in these trials had been collected from the field near where willows grow; thus some of them may have had prior exposure to willow seedlings, including both parent species and hybrids. Since seedlings in the field had low chemical concentrations at the time slugs were collected, it is less likely that slugs used in the trials had learned aversions to seedling. However, we cannot discount the possibility that slugs may have had aversions or preferences for willow seedlings. Feeding choice experiments (field trays) This experiment was designed to determine slug herbivory patterns among seedlings of S. sericea, S. eriocephala, and their interspecific F 1 hybrids. In 1998, we randomly planted seedlings of the three taxa, 24 S. sericea, 24 S. eriocephala, and 24 hybrid seedling for each tray, in 23 trays (72 cells per tray). Colored plastic toothpicks were used to mark the locations of each taxon of seedling. Trays remained in the greenhouse until they were put in the field. A new set of trays was placed in the field in designated slug-dense areas on a weekly basis. The first set of trays was placed into the field during the first week of July (week 5, measured as weeks since seed germination) and the last set was placed in the field on the second week in August (week 10). After 1 week in the field, we returned trays to the greenhouse and scored each seedling for slug damage. Damage scores, ranging from 0 5, were given for 20% damage increments (0=0% damage, 5=100% damage). We compared damage scores among taxa and weeks using two-way ANOVAs. Initial seedling leaf number was used as a covariate to correct for plant size differences. Analysis of ranked data did not alter results, so we present analyses using non-transformed data. To determine if damage scores for each taxon changed over time, we compared the differences in mean damage score among weeks for each taxon. Taxa were then compared by week. We performed contrasts among taxa within each week to test the fit of observed slug damage to the hypothesized patterns of hybrid resistance (Fritz et al. 1996). We first compared the damage scores of the F 1 hybrids to the average of the two parents (midparent). If F 1 hybrids were significantly different than the mid-parent value, we then compared hybrid damage scores to damage score of the nearest parent (S. eriocephala in all cases). When this difference was significant, the pattern was classified as partial dominance; if not significant it was classified as dominance. If the hybrid versus midparent difference was not significant, we then compared the damage scores for the two parents. When this difference was significant, we then classified hybrids as having an additive pattern. When this difference was not significant, we classified hybrids as having no difference in resistance compared to either parent. To monitor slug density near the locations of field trays at the time trays were placed in the field, we conducted weekly censuses. We marked 1 2 m rectangular plots of similar habitat located within 1 m of each field tray location. We systematically searched for A. subfuscus individuals within our plots at night ( hours) using head lamps. We compared slug numbers among weeks using a Kruskal-Wallis test. Changes in chemical defense in seedlings Adult S. sericea plants have phenolic glycosides and adult S. eriocephala plants have condensed tannins that act as defenses (Orians and Fritz 1995; Orians et al. 1997), but seedling plants have little or no chemical defenses; therefore, developmental changes in the onset of these chemical defenses could affect resistance. To quantify changes in concentrations of defensive chemicals, weekly samples of seedlings of each taxon were collected for chemical analysis from extra trays of seedlings maintained in the greenhouse under identical conditions as those used in the field tray and feeding choice experiments. Seedlings were not collected for chemical samples at the youngest ages used in our experiments, since a large number of seedlings would have to be sampled to provide enough tissue for analysis. Seedlings were cut at the base with scissors, placed in coin envelopes, and returned to the laboratory, where they were vacuum dried. Some seedlings were kept for a time in a freezer before vacuum drying. Care was taken to prevent thawing. Analysis of salicortin, 2 -cinnamoylsalicortin, and tannin concentrations followed protocols in Orians and Fritz (1995). Data were analyzed by performing regressions between the week that samples were taken and concentration of the each foliar chemical. Deterrence of defensive chemicals To estimate the concentrations of salicortin and condensed tannins necessary to deter slug herbivory, we conducted chemical painting experiments. In 1997, we painted leaves from young S. eriocephala seedlings that only produce very low concentrations of tannins (see Results) with various concentrations of salicortin or condensed tannin solution. Salicortin was isolated from bulk samples of S. sericea leaves, and condensed tannins were isolated from bulk samples of S. eriocephala leaves. Solutions were prepared using acetone as a solvent, and concentrations were determined based on leaf dry weight. Choice tests between control leaves with and without acetone suggest that slugs did not discriminate against acetone (unpublished data). We painted leaves that had an estimated average dry weight of mg with 7 µl of various solutions of salicortin or tannin and allowed leaves to dry before introducing slugs. We placed slugs, deprived of food for at least 24 h, into petri dishes lined with moist filter paper with one control leaf painted with acetone, and one treatment leaf painted with salicortin or tannin solution. In the first trial, we prepared 20 petri dishes using a 75 mg/g salicortin solution and 20 petri dishes using a 50 mg/g tannin solution. In the second trial, 30 petri dishes were prepared using 100 mg/g salicortin and 30 petri dishes were prepared using 100 mg/g tannin solutions. Leaves were scored for damage in 20% increments. Data were analyzed using two-tailed paired t-tests. In 1998, to further examine the effects of salicortin, we used Romaine lettuce instead of S. eriocephala leaves to eliminate the possible synergistic effects of tannins or other defense compounds in the leaves. Lettuce is the standard reference food for slug preference tests. We painted 3.5 cm 2 disks of lettuce (average dry weight: 9.42 mg) with 30 µl of various concentrations of salicortin in acetone to achieve the following concentrations, based on dry weight: 1 mg/g, 3 mg/g, 6 mg/g, 10 mg/g, 20 mg/g, 30 mg/g, 50 mg/g, and 100 mg/g. These concentrations were chosen to span a possible developmental range of concentrations that might occur in seedlings. Control disks were painted with 30 µl of acetone. We placed slugs that had been deprived of food for 15 h into petri dishes lined with moist filter paper with one control and one treatment disk. After 8 h, we assessed slug damage in 20% intervals. Due to constraints in slug numbers during any 1 week, three sepa-

4 90 rate trials involving various combinations of salicortin concentrations were spread out over a period of 3 weeks. For each concentration, we used paired t-tests to compare control and treatment disks. Since conditions between the three trials did not differ, we grouped the results of trials. Because the painting assays lasted 8 h in a humid environment, chemical breakdown is likely. Therefore, these results provide a relative measure of how deterrence changes with increasing chemical concentration, not an absolute measure of deterrence of a particular concentration. Performance of damaged and undamaged seedlings Performance of damaged seedlings was determined by measuring dry biomass at week 12. We collected the surviving seedlings from trays placed in the field în weeks 6, 7 and 8. Trays were returned to the greenhouse after exposure to slugs and permitted to grow under greenhouse conditions (watering and ambient light), but received no further fertilizer supplements. Performance of two trays of undamaged seedlings maintained in the greenhouse throughout the study was also determined by measuring dry biomass at week 12, with leaf number having been measured at week 11. Seedling biomass data were adjusted for the initial number of leaves on each seedling at the time they were placed in the field (or, in the case of the undamaged seedlings, 1 week prior to harvesting) by entering initial leaf number as a covariate in an analysis of variance. This measure of residual biomass (hereafter just biomass) is therefore a measure of seedling performance with initial size taken into account. We compared biomass among the taxa for undamaged plants and at each week for damaged plants using two-way ANOVA. The effect of week that damage occurred was not significant. Therefore, we combined weeks and compared differences among the taxa using t-tests of least square means (corrected by use of sequential Bonferroni adjustments) generated in PROC GLM (SAS 1990). Table 1 Results from pairwise Wilcoxon signed-rank test showing significance values of differences between damage scores of taxa by week for the feeding preference experiments. Only one significant difference would have been expected by chance alone given 18 separate tests Week E vs H E vs S H vs S Results Feeding choice experiments (laboratory) Fig. 1 Plot of percent leaf damage scores (mean ± 1SE) for Salix sericea, S. eriocephala, and F 1 hybrids from plants of different age (weeks) offered to the slug A. subfuscus in tripartite choice tests The results of the feeding choice experiments showed significant differences among taxa and over time in percent leaf consumption by slugs (Fig.1). In 1998, from week 5 to 7, S. eriocephala had significantly higher damage scores than hybrids, and hybrids had significantly higher damage scores than S. sericea (Fig. 1, Table 1). A marked decline in feeding on S. eriocephala and hybrid leaves occurred between weeks 7 through 9 (Fig. 1). Mean percent damage of S. eriocephala dropped from 64% to 2.6% from week 7 to 9. During the same period, percent damage to F 1 hybrids decreased from 46% to 5% and S. sericea dropped from 17% to 5%. In weeks 9 and 10, all three taxa had very low damage scores; the differences among them were fewer and seem biologically less relevant because of the low damage levels (Fig. 1, Table 1). In similar feeding choice experiments conducted in 1997, S. eriocephala seedlings had the highest damage scores of the three taxa, S. sericea seedlings had the lowest scores, and hybrids had intermediate scores (data not shown). Slugs continued to feed on lettuce provided for them in their storage containers (personal observation), supporting the hypothesis that decline in consumption of willow leaves is related to changes in palatability, and not a general decrease in slug s consumption rate at that time. Feeding choice experiments (field trays) The percent damage to willow seedlings placed in trays in the field for 1 week differed among taxa (Table 2, Fig.2). Significant differences in damage among tray locations (blocks) occurred for weeks 5 7 and 10. For weeks 6 and 7, significant interactions between taxon and tray location also existed (Table 2). In addition, initial leaf number had significant effects during weeks 8 and 9. Still, the overriding relationship between damage and taxon was not obscured by any other significant effect; S. sericea had lower percent damage than did S. eriocephala (Fig. 2). For weeks 5 7, slug damage on S. eriocephala was between 63% and 85%, while only between 17% and 24% on S. sericea.

5 91 Table 2 F-values of 2-way ANOVA comparing proportion damage, and F-values of contrasts between Salix eriocephala (E) vs S. sericea (S), E vs hybrids (H), and S vs H in field tray damage trials generated from the two-way ANOVA. Hybrid patterns of resistance detected included partial dominance of hybrid susceptibility (PD), an additive pattern (A), and no difference (ND) Week Initial leaf Taxon Location Taxon by Hybrid vs Parents Hybrid vs Hypothesis number location midparent near parent supported *** 42.98*** *** 63.61*** PD *** 8.22*** 2.15* 34.77*** 61.53*** PD *** 12.41*** 13.99*** 13.78*** 10.67*** PD *** 42.17*** *** A *** 10.42*** *** A * ND *P<0.05; **P<0.01; ***P<0.001 Fig. 2 Plot of weekly percent damage (mean±1 SE) caused by the slug A. subfuscus for S. sericea, S. eriocephala, and F 1 hybrid willow seedlings. New trays of undamaged seedlings were placed in the field in successive weeks Damage incurred by the three taxa decreased significantly over time (P< for all taxa). Damage on S. sericea declined most noticeably between weeks 7 and 8 from a high of 19% to less than 3%. Damage of S. eriocephala was high in weeks 5 and 6 of the study. However, from week 6 through week 10, damage to S. erio-cephala seedlings declined dramatically from 85% to 11%. By week 10, no significant differences in damage existed between these two taxa (Table 2). The qualitative pattern of damage to F 1 seedlings was similar to S. erio-cephala seedlings; high levels of damage occurred early (53 64%), and a rapid decrease in damage followed, with F 1 seedlings having comparable damage scores to S. sericea by week 10 (Fig. 2). The patterns observed in the field closely resemble the results found in the preference tests (Figs. 1, 2), with correlations being significant for each taxon (S. eriocephala: F 1,4 =77.2, P=0.0009, r 2 =0.938; F 1 hybrids: F 1,4 =77.3, P=0.0009, r 2 =0.939; S. sericea: F 1,4 =8.1, P=0.046, r 2 =0.587). Although F 1 willow seedlings generally suffered intermediate damage due to slug herbivory compared to parental species (Fig. 2), their resistance in relation to parental species changed over time. In weeks 5 7, the hypothesis of partial dominance was supported (i.e., there was a significant dominance deviation in damage from the midparent value toward the more susceptible S. eriocephala) (Table 2). For weeks 8 and 9, damage of hybrids compared to the midparent value fit the additive hypothesis. By week 10, the no difference hypothesis was supported. Damage by slugs among seedlings within a taxon did not depend on seedling leaf number for S. eriocephala or for F 1 hybrids for weeks 5 8 or for S. sericea in any week (data not shown). However, for S. eriocephala in weeks 9 and 10, and for F 1 hybrids during week 9, significant negative regressions between leaf number and percent damage occurred (damage = lf#, F=22.4, P=0.0001; damage = lf#, F=8.2, P=0.006; damage = lf#, F=9.3, P=0.004, respectively, where lf# = leaf number). Slug abundance also declined over the course of the field tray experiments in Slug abundance was highest in weeks 5 7, with average densities ranging from 6.9 to 8.8 slugs/2 m 2. Between weeks 7 and 8, slug abundance decreased from 7.7 to 3.0 slugs/2 m 2. By week 10, slug density was 0.5 slugs/2 m 2. Chemistry of seedlings Phenolic glycoside concentrations were higher in S. sericea than in F 1 hybrid seedlings (Fig. 3A). Over the course of the study, the combined concentrations of salicortin and 2 -cinnamoylsalicortin in S. sericea and F 1 hybrids increased linearly (y= week, F 1,12 =25.4, P=0.0003, r 2 =0.652, y= week, F 1,12 =14.5, P=0.0025, r 2 =0.510, respectively) (Fig. 3A). The negative x-intercepts of these regressions indicate that both taxa probably have little or no phenolic glycosides present in their leaves until about week 5 or 6. Condensed tannin concentrations differed among the willow taxa and changed with seedling age (Fig. 3B). Over the time that tannins were sampled, tannin concentrations were highest in S. eriocephala, intermediate in

6 92 Fig. 4 Mean percent difference (± 1 SE) in damage between acetone controls versus S. eriocephala leaves (1997) or lettuce (1998) painted with salicortin or condensed tannin at the concentrations indicated. Bars with an asterisk differ significantly at P<0.05 Fig. 3 Plot of phenolic glycosides (salicortin and 2 -cinnamoylsalicortin combined) concentrations (mg/g) in leaves of S. sericea and F 1 hybrid (A) and concentrations of condensed tannins of S. eriocephala, F 1 hybrid, and S. sericea (B) seedlings of different ages. Linear regressions show the best fit line through the data points F 1 plants, and lowest in S. sericea (Fig. 3B). Concentrations increased significantly over time for S. eriocephala and F 1 hybrids (y= week, F 1,4 =32.9, P=0.0046, r 2 =0.864, y= week, F 1,3 =11.8, P=0.0415, r 2 =0.729, respectively), but not for S. sericea (y= week, F 1,4 =2.023, P=0.228, r 2 =0.170) (Fig. 3B). Concentrations of tannins were 85 mg/g when tannins were first sampled for S. eriocephala in week 8 (Fig. 3B). Effects of secondary chemicals on preference Both salicortin and condensed tannins deterred feeding by slugs. Slugs ate significantly more of the acetonepainted, control disks than of disks treated with salicortin concentrations of 50 mg/g, 75 mg/g, and 100 mg/g. Lower salicortin concentrations (1 30 mg/g) did not significantly deter slugs from eating the treatment lettuce disks Fig. 5 Plot of the standardized residual biomass (mean ± 1 SE) for S. sericea, S. eriocephala, and F 1 hybrids damaged during weeks 6 8 (combined) or when undamaged, and then allowed to grow until week 12. Different letters within damage treatment indicate significant differences (P<0.05) (Fig.4). Similarly, leaves painted with tannins at concentrations of 50 mg/g and 100 mg/g were eaten significantly less than control leaves, indicating that high concentrations of tannins on leaves are an effective deterrent to slug feeding. Lower concentrations of tannins were not tested, so their effectiveness at deterring slug feeding was not determined.

7 Performance of seedlings after slug herbivory Damage by slugs in the field significantly affected the relative sizes of seedlings at week 12. S. sericea and F 1 hybrid seedlings were significantly larger than S. eriocephala seedlings when exposed to damage by slugs in week 6, 7, or 8 (Fig.5). However, for undamaged plants, biomass was greatest for S. eriocephala and F 1 hybrids, and significantly less for S. sericea (Fig. 5). Discussion Parental and hybrid willow seedlings differed both in slug damage in preference tests and in damage inflicted by slugs in field experiments. Chemical defenses of willow seedlings seemed to explain the differences in slug herbivory. Moreover, changes occurred both for slug feeding preference, as well as for seedling damage, suggesting that developmental changes in palatability and resistance to slugs resulted from developmental changes in plant chemistry. Finally, relative seedling performance among taxa was contingent upon herbivore environment. These results strongly suggest that differences in leaf chemistry between the willow species resulted both in differential susceptibility to damage by slugs and in differential performance of seedlings. Because, interspecific hybridization resulted in F 1 progeny with intermediate levels of damage and high performance when damaged, slugs are implicated as an important selective factor favoring introgression of defensive traits between these willow species. Patterns of palatability and damage As seedlings became older, damage in the field and laboratory experiments declined. At least one of three causes may explain the temporal damage patterns of these willow seedlings by slugs. First, seedling growth could have resulted in taller plants that were less accessible to slugs. For example, Rathke (1985) found that slugs avoided climbing and feeding on plants that were as short as 10 cm tall. If this were the case in our study, then declining damage on plants in the field would be related to plant growth. However, we observed slugs feeding on Joe-Pye Weed (Eupatorium sp.) plants at heights well above 50 cm during our censuses. This observation suggests that height did not make plants inaccessible to slugs. Second, a decline in slug abundance over the season could cause decreased damage for all taxa. Based on weekly censuses conducted at 2200 hours, we observed slug abundance to decline. As the season progressed, daytime and evening temperatures rose and humidity declined, potentially altering the time of slug activity. Specifically, dew-fall would have occurred later in the evenings, possibly delaying slug activity until higher levels of humidity were reached. Thus, slug abundance and foraging activity may have been higher later in the evening, but we would not have detected it. While each of these first two explanations may have contributed to the decrease in damage observed in the field, and we cannot exclude a decrease in slug density as a cause of decreased herbivory in the field, they do not sufficiently explain the slug feeding patterns observed in the laboratory experiments. A final explanation for these patterns is that a causal relationship exists between the changes in chemical defenses and changes in susceptibility. The observed developmental changes in palatability and decreases in damage in the field were paralleled by an increase in chemical defenses. Furthermore, damage in the field and damage on leaves exposed to slugs in the laboratory was very highly correlated across weeks for each taxon. Together, these results strongly suggest that a shift in resistance of willow seedlings to slugs occurred. Other studies have also found that palatability of leaves of seedling and adult plants differs. For example, Fenner et al. (1999) found that for 21 of 30 species, seedlings were more palatable than adults. For the remaining species, adults were highly palatable and seedlings were less palatable. In another study, risk of attack by slugs and mortality decreased with seedling age for two plant species (Hanley et al. 1995). Clearly, developmental shifts in palatability are common for seedlings of many plant species. Our results expand the observations that young willow seedlings are more palatable than older seedlings, demonstrating that a change in palatability can occur rapidly (in as little as 2 weeks) and that it can correspond temporally with increasing concentrations of defensive chemicals. Developmental changes in chemical defense of willow seedlings 93 In contrast to year-old S. sericea plants [which have relatively high phenolic glycosides (Orians and Fritz 1995)], 7-week-old seedlings have very low concentrations of phenolic glycosides. Extrapolation of data from S. sericea and hybrids suggests that seedlings less than 5 6 weeks old have low levels of phenolic glycosides in their leaves, but at approximately week 6, these defensive chemicals begin to increase rapidly. Concentrations of phenolic glycosides began to rise linearly to approximately adult levels by week 14. Similarly, phenolic glycosides in hybrids increase linearly to nearly adult levels for hybrids by week 14. Tannin levels were low for S. sericea, and concentrations did not increase over time. For S. eriocephala and F 1 hybrids, tannins were at fairly high levels when we began measuring them, and concentrations rose linearly through week 13. The regression for S. eriocephala has a steep slope, corresponding to a rapid increase in tannins and a prediction that concentrations of tannins are much lower at earlier dates. Thus, this period from 6 14 weeks represents a period when an increase in the defense of willow seedlings occurs. Plant defense theory predicts that early allocation of energy to defense would be costly for seedlings (e.g.,

8 94 Herms and Mattson 1992). In support of this argument, Bryant and Julkunen-Tiitto (1995) found a shift in the production of chemical defenses in birch seedlings, with younger seedlings investing fewer resources in defense than older seedlings. The developmental changes in defense in our willow seedlings may have been due to seedlings reaching a large enough size that photosynthesis could support the carbon demand for both defense and continued growth. However, seedlings are still very small at this stage (about 20 cm tall), and would be likely to experience intense competition for light from other plants. Instead, seedling growth rate may have slowed as a consequence of additional allocation to defense. Because we did not measure whether plant growth rate changed over time, we cannot elaborate on whether a tradeoff between growth and defense is altered as a consequence of the increased defense as seedlings grow. Still, the lack of sufficient chemical defense during a period of high vulnerability to slug feeding suggests either that allocation of energy to chemical protection very early on is costly or that chemical defenses are developmentally constrained due to autotoxicity (Zangerl and Bazzaz 1992). Plant chemistry and slug resistance The results of our painting experiments confirm that both salicortin and condensed tannins are effective at deterring slug feeding (Fig. 4). Concentrations of salicortin of mg/g painted onto lettuce were highly effective at deterring slug feeding, but concentrations of 1 30 mg/g did not significantly deter slugs. Several other studies have also demonstrated that chemical compounds can deter mollusc feeding. For example, bitter tasting compounds, such as sesquiterpenes and pyrrolizidine alkaloids have been shown to deter to the land snail, Arianta arbustorum (Speiser et al. 1992; Hägele et al. 1998). Similarly, cyanogenic glycosides (Jones 1962; Horrill and Richards 1985) and glucosinolates (Glen et al. 1990) can act as effective deterrents to slug and snail feeding. While several compounds are well recognized for their ability to deter slug herbivory, the relationship between temporal changes in chemical concentrations in the leaf and slug deterrence has only been studied in one other case to our knowledge. Horrill and Richards (1985) found that cyanogenic seedlings of Trifolium repens had increasing concentrations of cyanide in stems and young leaves over time, and that lethal damage by Arion hortensis decreased with seedling age. Concentrations of phenolic glycosides and tannins for 7 to 10-week-old willow seedlings were much lower than for 1-year-old plants (Fig. 3). During weeks 7 10, however, the concentration of total phenolic glycosides rose from 5 to 50 mg/g in S. sericea seedlings. Based on the painting assay, the concentrations of phenolic glycosides at week 10 are sufficient to deter slugs, but the concentrations present in the earlier weeks would not be. There are three reasons lower concentrations might not have deterred the slugs in the painting assay: 1. Lower concentrations might not affect slug selection. 2. In addition to salicortin, S. sericea seedlings contain small concentrations of 2 -cinnamoylsalicortin and other phenolics. Perhaps the combined effects of salicortin and these other phenolics more effectively deterred slug feeding. Combinations of chemicals in other systems have been demonstrated to synergistically deter herbivores (e.g., Berenbaum 1985; Berenbaum and Neal 1985). 3. As mentioned in Materials and methods, degradation of the salicortin during the 8 h of the experiments may have increased the suitability of salicortin painted leaves. We feel that reason 1 is unlikely given the concomitant decrease in acceptability and increase in chemistry found in the seedlings. We are currently planning further experiments to assess the combined effects of chemicals and the immediate responses of slugs to painted leaves. The change in damage and palatability of S. eriocephala is, in part, explained by the increasing concentrations of tannins in their leaves. During weeks 7 9 [i.e., when palatability and damage declined dramatically (Figs. 1, 2)], tannin concentrations were increasing to levels that would deter slugs ( mg/g; Fig. 3). Because we used individual plants as the sample unit, we were unable to sample younger seedlings for chemical content since smaller plants did not provide enough tissue for tannin analyses. Future sampling of these seedlings will determine whether a sharp threshold in tannin production occurs at younger seedling ages or whether concentrations increase linearly and it is slug preference that has a threshold response. Compared to S. eriocephala, F 1 hybrids have similar patterns of palatability and damage, but they have lower levels of damage. Salicortin concentration seems to explain the lower level of damage on hybrids compared to S. eriocephala (F 1 hybrids have small concentrations of salicortin and S. eriocephala does not), but tannins may explain the pattern of declining damage over time that resembles the changes in damage on S. eriocephala. Performance differences among willow taxa and implications for resistance evolution Growth of willow seedlings was dependent upon the occurrence and timing of damage by slugs. S. sericea and F 1 hybrids had superior growth compared to S. eriocephala in the presence of damage by slugs, but S. eriocephala and F 1 hybrids had superior growth compared to S. sericea in the absence of damage. These differences suggest that a substantial cost of herbivory for S. eriocephala seedlings occurred during the period of vulnerability to slug damage. Conversely, a sizable benefit of defense existed for S. sericea seedlings, with earlier deployment of defenses providing decreased herbivory and

9 consequently higher growth. For undamaged seedlings, slower growth of S. sericea could be very costly in terms of interspecific competition with the faster growing S. eriocephala seedlings. Thus, in the absence of herbivory, a substantial cost of defense appears to exist for S. sericea. Furthermore, as damage declines for S. eriocephala and F 1 hybrids in later weeks, their superior growth rates (compared to S. sericea) may give them a fitness advantage. Still, we did not evaluate the time period that would demonstrate this switch in relative growth of the three taxa. F 1 hybrids had high relative biomass across both damage environments, suggesting that they might have a combination of traits that would result in high fitness either when damaged or undamaged. Reduced herbivory on F 1 hybrids compared to S. eriocephala, probably due to the presence of phenolic glycosides in their leaves, seems to confer the opportunity for greater growth when slugs are present. Moreover, in the absence of herbivory, F 1 hybrids grew as well as S. eriocephala, suggesting that the cost of defense for F 1 hybrids was no greater than the parental species. Based on growth measurements, then, the fitness of F 1 hybrids should be relatively high compared to either parental species. However, the conditions of the experiment were not fully realistic. First, conditions following herbivory would be less hospitable than those in the greenhouse would (i.e., competition for light and nutrients would be more severe). Thus, S. eriocephala and hybrids seedlings, which sustain high damage in the field, should be at a competitive disadvantage compared to S. sericea. More importantly, though, seedlings in the field would be subject to more than a single week of herbivory, and herbivory would potentially begin at seed germination. Continued exposure of susceptible seedlings by slugs can be expected to result in greater seedling mortality. For example, in a study of newly established seedlings planted in common garden plots in 1996, we found 37.3% (215 of 576 seedlings) mortality for S. eriocephala seedlings by slugs, 20.5% (118 of 576 seedlings) mortality for F 1 hybrids, and just 5.9% (34 of 576 seedlings) mortality for S. sericea plants within a 2-week period (Fritz, unpublished data). Furthermore, slug damage can be more severe than what we observed in In 1997, mortality of S. eriocephala and F 1 hybrids in trays subjected to herbivory for just week-long periods were 79.6% and 40%, respectively, whereas mortality of S. sericea was 5.6% (Fritz, unpublished data). In all cases, survival and performance of F 1 hybrids is at least intermediate compared to the parental species. Because chemical defense, realized resistance, and performance of F 1 hybrids were always greater than that of the poorer performing parental species, and possibly as great as the better performing parent, natural selection can potentially act to favor introgression of phenolic glycosides and the early expression of this defense in S. eriocephala. To highlight this point, consider the range of defenses potentially expressed by F 2 hybrid plants. We have found that chemical variation of F 2 95 plants is greater than for F 1 plants, with variation spanning the range between parental species (Hochwender et al. 2001). Herbivory by slugs on these recombinant hybrids could cause selection favoring plants with S. sericea-like defensive traits. In a similar process, but one involving advanced generation hybrids/backcrosses, slugs could favor introgression of defensive traits between these willow species. In support of this prediction, we have found S. eriocephala-like plants introgressed with S. sericea genes that express phenolic glycosides in their leaves (Hardig et al. 2000). Apparent introgression of defensive characteristics has been identified in a few systems, and in each case, resistance to herbivores is a demonstrated advantage of the introgressed traits (Stutz and Thomas 1964; Manley and Fowler 1969; Wu et al. 1996). Thus, these data, taken together, support the alternative view suggested by Arnold and Hodges (1995) and Arnold et al. (1999) that hybrid plants can contribute to the evolutionary diversity of plant species. Whether A. subfuscus can be a strong enough selective force to cause introgression of genes for salicortin production from S. sericea to S. eriocephala will depend, in part, upon the heterogeneity of slug selection. Even during drought years, slugs are present when seedlings are small and most vulnerable to them, but drought years may negatively affect slug populations in following years by reducing their abundance. Thus, there may be temporal variation selection. Selection by slugs may also vary spatially. Some swampy locations are so wet that they are inaccessible to slugs (personal observations). The selection that these herbivorous slugs exert will depend on the abundance of such safe sites. Molluscs have been implicated as important selective agents in the evolution of plant defenses. Westerbergh and Nyberg (1995) showed that populations of Silene dioica in areas subjected to snail herbivory have densely hairy leaves that protect them from slug feeding compared to hairless (glabrous) forms that are highly susceptible to slugs. These populations harbor the recessive, glabrous allele, but no plants with hairless leaves are seen in these populations. Similarly, Jones (1962) and Cates (1975) showed than cyanogenic phenotypes of two plant species had less damage and higher fitness than acyanogenic forms. These studies give additional credence to the argument that A. subfuscus may be an influential selective force favoring introgression of phenolic glycosides. Acknowledgements This project would not have been possible without the generous access that Len and Ellie Sosnowski have provided to their land as a research site since The research was supported by the National Science Foundation (DEB ), the Vassar College Environmental Science Research fund, and Tufts University. W. Marussich, B. Crabb, D. Willies, S. Good, and B. Roche provided additional field assistance. D. Strayer kindly identified the slug.

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