Oecologia (1991) 86:552-560 Oecologia Springer-Verlag 1991

The effects of light on foliar chemistry, growth and susceptibility of seedlings of a canopy tree to an attine ant

Colin M. Nichols-Orians* Department of Entomology, The Pennsylvania State University, University Park, PA 16802, USA Received May 24, 1990 / Accepted in revised form January 3, 1991

Summary. Seedlings oflnga oerstediana Benth. (Mimosa- al. (1984) and Mole and Waterman (1988) suggest that ceae) growing in three different light environments (the sun foliage of these species may be of lower nutritional understory, tree-fall gaps and full sun) were tested for value. Coley (1983b) found no differences in the suscep- differences in chemistry (nutrients and tannins), wound- tibility of a shade-tolerant canopy tree to herbivory when induced increases in tannins, growth, and susceptibility growing in low and high light environments. Since the to leaf-cutter ants, Atta cephalotes (L.) (Formicidae: At- concentrations of nutrients typically increase with in- tini). I hypothesized that seedlings of L oerstediana creasing light (Field and Mooney 1986; Denslow et al. would contain higher concentrations of tannins when 1990), this result suggests that a light-induced increase in growing in high light conditions and, therefore, would be carbon-based chemicals may have counteracted the ben- less susceptible to leaf-cutter ants. efits derived from a light-induced increase in nutrients. Foliar concentrations of condensed tannins were Leaf-cutter ants, common defoliators of in the much higher in plants growing in full sun compared to neotropics, are most common in early successional hab- those growing in the understory. The concentrations of itats where light availability is high (Jonkman 1979; condensed tannins did not increase following damage. Fowler 1983; Blanton and Ewel 1985; Jaffe and Vilela Despite higher concentrations of condensed tannins in 1989; Vasconcelos 1990). This distribution may be a sun foliage, leaf-cutter ants found these leaves more result of abiotic factors which vary as a function of dis- acceptable. The preference for sun leaves was consistent turbance (Fetcher et al. 1985; Jaffe and Vilela 1989). with higher concentrations of foliar nutrients. I suggest Research to date also suggests that leaf material in dis- that the magnitude of the increase in condensed tannins turbed habitats is most acceptable to the ants (Fowler was not great enough to override the benefits of increased 1983; Shepard 1985). There are at least two reasons concentrations of foliar nutrients. Finally, based on these foliage may differ in quality. First, pioneer species, which results and those of others, I suggest that foraging by dominate in early successional disturbed habitats, appear leaf-cutter ants may be an important factor determining more susceptible to leaf-cutter ants than shade-tolerant patterns of succession in early successional habitats. species (Fowler 1983; Shepard 1985). This may be be- cause the former allocate fewer resources to secondary Key words" Leaf-cutter ants - Leaf selection - chemicals (Coley 1983a, b; Coley et al. 1985). Second, Foliar nutrients Condensed tannins leaf-cutter ants may find sun foliage more acceptable than shade foliage because of light-induced increases in the concentrations of nutrients (Barrer and Cherrett 1972; Littledyke and Cherrett 1975; Waring et al. 1985; Canopy trees which are shade-tolerant, but require tree- Berish 1986; Larsson et al. 1986; Denslow et al. 1990). fall gaps to reach the forest canopy, produce high con- No study has evaluated how plants growing in different centrations of carbon-based chemicals (Coley 1983a; light environments differ in susceptibility to leaf-cutter Nichols-Orians and Schultz 1990) and much higher con- ants. I have found that laboratory colonies of leaf-cutter centrations are found in sun leaves than shade leaves (e.g. ants prefer sun leaves over shade leaves of Forsythia, a Chandler and Goosem 1982; Waterman et al. 1984; temperate early successional shrub (unpublished data). Mole et al. 1988). Because of this pattern, Waterman et However, sun leaves of shade-tolerant later successional canopy trees may be less acceptable than shade leaves * Present address and address for offprint requests. Department of because the concentrations of carbon-based chemicals Biology, Vassar College, Poughkeepsie, NY 12601, USA typically increase dramatically with increasing light 553

(Chandler and Goosem 1982, Waterman et al. 1984; creases in condensed tannins, c) growth, and Mole et al. 1988; Mole and Waterman 1988). d) susceptibility to A. cephatotes. In this paper I report how leaves of seedlings of a shade-tolerant canopy tree, Inga oerstediana Benth. [for- merly var. minutula Schery (Nichols-Orians Atta cephalotes/Fungus and Schultz 1990)], differ in toughness, water content, nutrient and tannin concentrations, and -binding Colonies of A. cephalotes are abundant within La Selva, capacity when growing in different light environments. and foraging ants often encounter plants growing under I also determined whether the concentrations of tannins different light conditions (pers. obs.). The fungus which increase following damage. Finally, I report how seed- the ants cultivate is the sole food source of the developing lings growing in different light environments differ in larvae (Quintan and Cherrett 1979). To enhance the growth and susceptibility to the leaf-cutter ant, Atta growth of their fungal symbiont the ants deposit cephalotes (L.). Leaf-cutter ants do not find Inga highly previously-ingested fungal enzymes on leaf fragments acceptable as compared to other plant species (Arkcoll before incorporating them into the fungus garden (Boyd 1984; pers. obs.), perhaps because members of the genus and Martin 1975; Martin et al. 1975). These enzymes produce high concentrations of tannins (Koptur 1985), appear critical for vigorous fungal growth (Mudd and especially condensed tannins (Nichols-Orians and Bateman 1979), so any chemicals that inhibit enzyme Schultz 1990). I hypothesized that seedlings of L oer~ activity and subsequent fungal growth should be avoided stediana would be less susceptible to A. cephalotes when by leaf-cutter ants. Condensed tannins are one class of growing in full sun than when growing in the forest chemicals known to inhibit fungi (Zucker 1983). understory because of light-induced increases in the con- Researchers have not always found tannins to be centrations of condensed tannins (sensu Mole and Wa- important deterrents to leaf-cutter ants (Littledyke and terman 1988). Condensed tannins are effective inhibitors Cherrett 1976; Kawanashi and Rauffauf 1986; Howard of fungi and their enzymes (Friend 1979; Zucker 1983; 1987, 1988, 1990; Nichols-Orians and Schultz 1990). Seaman 1984; Cherrett et al. 1989). Since leaf-cutter ants However, most studies have been qualitative (presence/ use the leaves to support the growth of a symbiotic absence) and may have missed the concentration-depen- fungus, quantitative differences in the concentrations of dent role of tannins in this system. Since the attine fungus tannins should deter the ants. However, there is evidence produces tannases and oxidases that can that foraging ants drink sap as they cut. Therefore, an inactivate tannins (Cherrett et al. 1989), tannins may alternative hypothesis is that light-induced increases in only inhibit the ants and the fungus at high concentra- tannins would deter the ants because of lower sap quality tions. One quantitative study (Nichols-Orians and (Quinlan and Cherrett 1979). Schultz 1990) showed that extracts of young leaves of I. oerstediana which had very high concentrations of tannins were more inhibitory of a fungaI pectinase than The system extracts of mature leaves; young leaves were also less acceptable to the ants than mature leaves. Study site Other leaf traits may differ among light environments, and these could influence leaf selection by the ants as This study was conducted at the Organization for Tropi- well. They may select leaves rich in nutrients (Berish cal Studies' La Selva Biological Station (10 ~ 26' N, 1986). Differences in water content may or may not be 83 ~ 59' W) in the Atlantic lowlands of Costa Rica. The important (Bowers and Porter 1981; Blanton and Ewel area is a wet premontane forest (Holdridge et al. 1971), 1985). Finally, differences in leaf toughness may prevent and receives a mean annual rainfall of 4000 mm (Hart- foraging ants from harvesting chemically acceptable leaf shorn 1983), with a short dry season from late January material (Waller 1982, Nichols-Orians and Schultz 1989, to April (La Selva unpublished records). Data were col- 1990). lected between February 1987 and May 1989.

Plant species Methods

Seeds of ali Inga species appear to germinate immediately I located t. oerstediana seedIings growing in three different light following dispersal independent of light level. I. oer- environments (in the understory, in tree-fall gaps, and in full sun). Seedlings growing in the forest understory of La Selva receive stediana is therefore found under a range of light con- 0.4-2% of full sunlight and plants in gaps receive 9-23% of full ditions (pers. obs.). L oerstediana like other Inga species sunlight (Denslow et al. 1990). All seedlings were growing in nu- is naturally attacked by leaf-cutter ants (Shepard 1985; trient-rich soils (alluvial soils) so differential nutrient availability pers. obs.) should not have influencedmy results (e.g. Price et al. 1989). Water I used leaves from I. oerstediana seedlings which were content, leaf toughness, foliar non-structuralcarbohydrate, protein, either found naturally, or grown in shadehouses and condensed tannin concentrations, and protein binding capacity of the seedlings from the different environments were determined. subsequently transplanted into the field, to determine I also conducted an experiment to determine if the concentrations light-induced differences in a) leaf traits (toughness, nu- of condensed tannins increase followingdamage, and if so, whether trients and condensed tannins), b) damage-induced in- light availabilityalters the response. Finally, I determinedhow these 554

seedlings differed in growth rates and susceptibility to leaf-cutter istry was determined using a One-Way ANOVA (light as the main ants. All data were analyzed using the SYSTAT statistical package effect) and Tukey's HSD, following square root transformation of (SYSTAT, Inc.) (see details of models below). all data.

Analysis of foliar traits Induction experiment

The analyses of foliar traits were done on the leaves of ten seedlings Light conditions may not be the only factor influencing the con- per light treatment when possible. For non-structural carbohy- centrations of tannins. Previous damage may result in an increase drates leaves of only two or three seedlings per treatment were in their concentrations (Rossiter et al. 1988), but a plant's environ- analyzed. Leaves were collected in March and in May of 1987. ment can temper wound-induced responses (Baldwin 1988a). To Previous work with L oerstediana indicates that little seasonal varia- assess whether the concentrations of condensed tannins and protein tion in leaf chemistry exists (Nichols-Orians and Schultz 1990). binding capacity increase following damage, I damaged ten seed- Only mature leaves devoid of epiphylls were collected. The amount lings in each light environment and determined whether the con- of leaf material per seedling was insufficient to allow me to quantify centrations of condensed tannins and protein binding capacity foliar leaf traits and susceptibility to leaf-cutter ants using the same increased after 24 h. Because leaf-cutter ants can defoliate an entire seedling. However I collected leaves for chemical/physical analyses seedling well within a day (unpublished data), I did not extend my from the same habitats as those collected for acceptability bioassays sampling beyond this time period. with leaf-cutter ants. I collected seeds in May 1987 and grew them at 20% of full Leaves collected for water and toughness determination were sunlight in shadehouses until January 1988. In January the seedlings cut at the and placed in floral water picks to prevent wilting were transplanted into one of three habitats, an open clearing, a while I transported them to the laboratory. Those collected for gap, and a forest understory, and allowed to grow until May 1988. chemical determinations were cut at the petiole, frozen within In May, I tore off leaf fragments from each leaflet, produced bet- 15 min, and later lyophilized (Hagerman 1988) before being trans- ween January and May, until half of the leaf area of each leaflet ported to The Pennsylvania State University for chemical analyses. remained. Leaf material from either the right or left side of the vein Those collected during the induction experiments were immediately was removed and preserved for chemical analyses. After 24 h I tore placed on ice and then frozen and lyophilized. off the remainder one half of each leaf, except the mid-vein. Statis- tically significant changes in leaf chemistry were determined using 1. Water content and leaf toughness. I determined the water content a paired t-test for each plant within each light treatment following of leaves of ten seedlings per light treatment by weighing the leaves square root transformation of the data. before and after drying them for 24 h at 70 ~ C. Leaf toughness was also measured for ten seedlings per light treatment using a pen- etrometer (Schultz and Baldwin 1982). Three measurements were Seedling growth taken per leaf and averaged, and the values (g/area) were subse- quently converted to kiloPascals (kPA). Following arcsine square The growth and survival of seedlings growing in the forest under- root transformation, a one-way ANOVA (light as the main effect) story were followed from March 1987 through May 1989. Three and Tukey's HSD was used to assess the significance of differences contiguous 100 m 2 quadrats were located. Within each quadrat ten in water content and leaf toughness. 1 m 2 plots were selected using a random numbers table. A plot was accepted if at least one L oerstediana seedling was present. All 2. Chemical analyses. The concentrations of non-structural carbo- seedlings within these plots were marked and measured. In 1987, the hydrates, proteins and various phenolic traits were measured on height of these seedlings was measured every three weeks until July freeze-dried leaves ground to a powder with a UDY| Cyclone Mill. and then again in January, 1988 and May, 1989. For analysis of non-structural , I extracted 20 mg of I also measured the growth of the seedlings used in the induction leaf powder with 3 ml of 80% methanol three times. The super- experiment described above. Height was measured at the time of natant was diluted to 10 ml and non-structural carbohydrates were defoliation (May 1988) and again one year later (May 1989). Signifi- determined by the phenol-sulfuric acid method (Southgate 1976). cance of differences in growth was determined using a One-Way Protein concentration was determined using the Bio-Rad reagent ANOVA (light as the main effect) and Tukey's HSD, following following an extraction of 20 mg of powder in 10 ml of 0.1 N NaOH square root transformation of the data. for 2 hr in a boiling water bath (Snyder and Desborough 1978; Compton and Jones 1985). I extracted the phenolics from freeze-dried leaf tissue with 70 % Acceptability bioassays acetone. The acetone was then removed under vacuum and the extract diluted to 10 ml with distilled water (see Nichols-Orians and I focused intensively on 2 ant colonies (hereafter 1 & 2) from Schultz 1990). Extracts were analyzed for total phenolics (Folin- February to June 1987 and used 7 others to collect supplemental Denis assay, Swain and Hillis 1959), proanthocyanidin condensed data in May 1988. Leaves collected for bioassays were collected in tannins (butanol/HC1 method, Bate-Smith 1977), leucoanthocyanin the same manner as those for water and toughness determinations. condensed tannins (vanillin method, Broadhurst and Jones 1978; All assays were initiated within an hour of collecting the leaves and Butler et al. 1982), and protein-binding capacity (hemanalysis, were completed within ~3 h. Schultz et al. 1981). Relative concentrations were expressed as % Two different bioassays, the "pickup" and "cutting" assays, tannic acid (Sigma, Lot ~ 11F-0559) equivalents per mg dry weight were used to assess whether plants growing in different light en- (%TAE) for total phenolics and protein binding capacity, and % vironments differ in acceptability to colonies 1 & 2. Because wattle tannin (Acacia sp., from Leon Monnier, Inc., Peabody MA L oerstediana produces compound leaves it was possible to use a USA) equivalents per mg dry weight (%WTE) for condensed tan- leaflet for each assay and perform the two assays with a single leaf. nins. Thus far, surveys indicate that phenolics, specifically con- The "pickup" assay was designed to determine preferences based on densed tannins, are the only secondary compounds abundant in chemical differences, defensive or nutritional, between leaf types Inga spp. (Koptur 1985; Nichols-Orians and Schultz 1990). Because (Howard 1987). This assay has been used repeatedly to identify total phenolics were highly correlated with the two condensed species and leaves which contain chemicals which are toxic to the tannin measures (Pearson correlation coefficients were _> 20.9), I am ants and/or their symbiotic fungus (e.g. Howard and Wiemer 1986; restricting my discussion to the two condensed tannin measures and Howard 1987). While physical features of a leaf (e.g. trichomes) protein-binding capacity. Significance of differences in foliar chem- could influence pickup rates, I did not identify any such physical 555 features in Inga. Furthermore, because the leaf discs were small (all 1988). In this assay both chemical and physical features of the leaf foraging ants were able to pick up these leaf discs), differences in are likely to be very important determinants of leaf utility to leaf- leaf mass could not have influenced the results. cutter ants. One 48 cm 2 rectangle was cut from each leaf type and In the "pickup" assay, two leaf discs, from a seedling of one of placed beside a foraging trail. The number of fragments cut and the three light regimes (understory, small gap or full sun), were removed during the ensuing hour was tallied. Clearly if the results produced with a standard paper punch and placed beside two discs of the "cutting" assay are significantly different from the results of of a highly preferred control, Hamelia patens (Rubiaceae). The use the "pickup" assay it would indicate that a physical feature of the of a control indicated whether ants of these colonies were willing leaf affects harvestability. The "cutting" assay was also repeated ten to pick up leaf discs and standardized for fluctuations in ant activity times per colony using leaves from different plants each time. I also during each assay replication. H. patens was used as the control had measured the leaf area remaining at the end of the hour, but because of its high acceptability to leaf-cutter ants and its abun- the results were identical, so I will present the results for the number dance in the clearing adjacent to the study colonies. Only leaf discs of leaf fragments cut and removed. Again, data were square root from fully expanded leaves, located at nodes two to four from the transformed and analyzed using a Two-Way ANOVA (light and terminus, of five different. H. patens individuals were used as con- colony as the main effects). trols. The different treatments were typically tested on the same day using the same control leaf. In the "pickup" assay, when one disc was removed, it was replaced by a like disc. The chemical acceptability of the two leaf Results types was expressed as the number of leaf discs removed when 20 control discs had been removed. Each trial was replicated three Foliar traits times and the replicates averaged. For each colony, the pickup assay was repeated ten times per leaf type, using leaves from different In general, light level had a strong effect on foliar traits plants each time. Data were square root transformed because of a (Table 1). Only leaf water content and leaf toughness positive correlation between the mean and the standard deviation, and analyzed using a Two-Way ANOVA (leaf type and ant colony showed no differences among light treatments. The con- as the main effects). Although these assays were done over a four centrations of non-structural carbohydrates and proteins month period, the date of the bioassay was not used as a covariate were lowest in understory plants and highest in full sun because its effect was insignificant. plants. Both measures of condensed tannins and protein- To insure that the use of a control did not affect the ranking of binding capacity showed a similar pattern. However, leaves (Richardson and Whittaker 1982), I conducted a "modified there were no differences in the concentrations of leu- pickup" assay on 7 other colonies in May, 1988. In the "modified pickup" assay, I placed two discs of each leaf type (full sun and coanthocyanins and protein-binding capacity between understory) along the foraging trails, and calculated the ranking of plants growing in gaps and those growing in full sun. each leaf type as the number of leaf discs removed during a 20 rain period. Two trials were completed and averaged for each assay. The position of the leaf discs, relative to the nest entrance, was reversed Induction experiment in the second trial to eliminate factors related to order of encounter by the ants. Data were analyzed using a paired t-test first for each colony and then across all colonies. There was no evidence of an increase in tannin produc- The second assay, the "cutting" assay, was used to measure the tion following damage for seedlings growing in any of the harvestability of the leaves (Howard and Wiemer 1986; Howard three light regimes (Table 2). These results indicate that

Table 1. Chemical and physical leaf traits Leaf trait Seedling [mean(SE)] of L oerstediana seedlings from different light environments. Values with Understory Gap Full sun different letters are significantly different at P<0.05 Water content (%) 66.2 a 66.8 a 66.6 a n=10 (0.6) (0.7) (1.3) Toughness (kiloPascals) 222 a 229 a 249 a n = 10 (8.8) (9.7) (11.0) Non-Structural Carbohydrates 5.1 a 6.3 b 9.2 c (% glucose/rag dry wt) (0.4) (0.1) (0.4) n=3 n=2 n=3 Protein (% BSA/mg dry wt) 20.2 a 26.8 b 29.9 c (0.7) (1.1) (1.0) n=9 n=10 n=10 Condensed tannins 1. Leueoanthocyanins 6.6 a 11.8 b 12.0 b (%WTE/mg dry wt) (0.5) (0.8) (1.0) n=9 n=10 n=lO 2. Proanthocyanidins 5.3 a 10.3 b 12.8 c (%WTE/mg dry wt) (0.4) (0.8) (1.2) n=9 n=10 n=10 Protein binding capacity 4.4 a 5.7 b 6.8 b (%YAE/mg dry wt) (0.3) (0.4) (0.5) n-9 n=10 n=10 556

Table 2. The concentrations of condensed tannins, leucoantho- 5- cyanin and proanthocyanidin, and protein-binding capacity of L oerstediana leaves [mean (SE)] before (day 1) and after (day 2) 4.- damage to plants growing in three different light levels. Values with different letters are significantly different at P< 0.05 E ~~ 3 Light level: Seedlings Reps : P 2 Understory Gap Full sun 0 (n = 10) (n = 10) (n = 7) t- Leucoanthocyanins Ot day 1 : 13.8 a 19.3 b 26.6 c I O- (1.1) (2.2) (2.9) day 2: 13.8 a 20.4 b 27.6 c -1 § t Undemtory Cop Full Sun (1.3) (2.2) (2.8) Difference: 0.0 1.1 1.0 P-value: 0.97 0.23 0.26 Seedling Light Environment Proanthocyanidins Fig. 1. The average (SE) increase in height in one year by I. oer- stediana seedlings growing in the forest understory (n = 9), a tree-fall day 1 : 3.8 a 6.4 b 9.6 c gap (n = 10), and in full sun (n = 5) (0.2) (0.6) (0.5) day 2: 4.0 a 7.0 b 10.6 c (0.2) (0.4) (0.6) . On average, there was no net growth by these Difference: 0.2 0.6 1.0 individuals (Fig. 1). P-value: 0.78 0.22 0.09 The growth of seedlings in higher light levels was Protein-binding capacity rapid. On average, after one year the seedlings trans- day 1: 5.3 a 7.5 b 11.9 c planted into full sun grew 3.6 meters and those in the gap (0.4) (1.1) (1.0) grew 2.1 meters (Fig. 1). day 2: 5.9 a 8.1 b 11.3 c (0.4) (0.7) (1.1) Difference: 0.6 0.6 - 0.6 Acceptability bioassays P-value : 0.34 0.52 0.74 The "pickup" and "modified pickup" assays, used to assess differences in chemical acceptability, demon- strated that ants from all study colonies of A. cephalotes wound-induced responses are unlikely to have any effect found leaves from full sun plants more acceptable than on leaf-cutter ants. those from understory plants (Tables 3 and 4). Plants in Chemical analyses of the foliage collected at Day 1 gaps were intermediate in acceptability (Table 3). During revealed that both measures of condensed tannins and "modified pickup" assays sample sizes were often too protein binding capacity increased with increasing light small to obtain significant differences (at P= 0.05) for (Table 2). The concentrations of condensed tannins in each colony, but statistical analysis across colonies this foliage were higher than observed in the leaves of field seedlings (above). Although I do not know the cause Table 3. Chemical and physical acceptability of leaves of L oer- of this difference, it may be due to a leaf age effect. I have stediana seedlings from different light environments, as measured by found that as leaves age the concentrations of tannins the "pickup" [number removed (SE)] and "cutting" assays [number change (unpublished data). cut and removed (SE)], to leaf-cutter ant foragers. Values with different letters are significantly different at P<0.05

Plant growth Leaf trait Seedlinos Understory Gap Full sun I. oerstediana grew very slowly in the understory. Over A. Chemical acceptability 2 years, the average increase in height of seedlings which survived (n=41) was only 4.5 cm. Mortality was ap- ("pickup" assay) colony #1 (n= 10) 1.4 a 5.3 b 14.6 c proximately 50% for older seedlings and over 90% for (0.1) (0.3) (0.5) newly germinated seedlings. I was unable to get an exact colony #2 (n = 10) 1.2 d 4.1 e 10.6 f mortality percentage because some tags were removed by (0.2) (0.2) (O.4) unidentified mammals. Although the cause of mortality B. Chemical/physical acceptability is unknown for each seedling, leaf and branch falls and herbivory were important (pets. obs.). Similar results ("cutting" assay) colony//1 (n = 10) 0.8 a 4.2 a 25.0 c were obtained when I transplanted ten seedlings into the (0.1) (0.5) (1.0) understory. After one year (May 1988 May 1989), one colony//2 (n= 10) 0.2 a 1.3 a 6.8 b of the seedlings had died while five others had been (O.O4) (0.2) (O.6) knocked over by falling branches and/or browsed by 557

Table 4. The average number (SE) of leaf Colony: 3 4 5 6 7 8 9 Total discs of L oerstediana seedlings growing in Reps: n=5 n=2 n=2 n=2 n=2 n=2 n=2 (n= 17) the understory or in full sun removed by leaf-cutter ant colonies during 20 min Leaf type "modified pickup" assays Understory 4.5 3.0 0 0.5 1.2 1.2 2.0 1.7 (SE) (1.0) (0.4) - (0.2) (0.2) (0.9) (0.7) (0.6) Full sun 17.3 16.5 5.0 20.2 20.0 22.2 9.5 15.7 (SE) (1.8) (4.2) (1.1) (3.0) (5.3) (1.2) (2.1) (2.3) P-values 0.01 0.29 0.19 0.14 0.24 0.09 0.17 <0.001

showed that leaf discs from full sun plants were removed nms (Nichols-Orians and Schultz 1990). I hypothesized more readily than those from understory plants that seedlings of L oerstediana would be less susceptible (P< 0.001) (Table 4). to A. cephalotes when growing in full sun than in the Leaves of plants growing in full sun also were harvest- forest understory, especially since previous work has ed more during the "cutting" assay than were the leaves demonstrated that high concentrations of condensed tan- of plants growing in gaps or in the forest understory nins deter the ants and inhibit the fungus (Cherrett et al. (Table 3). Although on average gap leaves were cut more 1989; Nichols-Orians 1990; Nichols-Orians and Schultz than understory leaves the difference was not statistically 1990). As predicted, the concentrations of condensed significant (Table 3). Overall, the number of leaf discs tannins and protein binding capacity increased with in- removed during the "pickup" assay was a strong indica- creasing light (Tables 1 and 2). Sun leaves contained tor of the number of leaf fragments cut in the "cutting" 35%-60% higher levels than understory leaves. assay (colony 1, N=23, r=0.71, P<0.01; colony 2, Despite light-induced increases in the concentrations N=23, r=0.73, P<0.01). Thus, physical features did of condensed tannins, A. cephalotes preferred leaves from not affect the selection of leaves from different environ- plants in full sun to those from understory plants (Tables ments by A. cephatotes. 3 and 4). Field observations also indicate that seedlings growing in full sun are more susceptible to leaf-cutter ants than those growing in the forest understory (pers. Discussion obs.). Thus, leaf selection by A. cephalotes appeared to correspond to light-induced increases in foliar nutrients Light availability in tropical forests is highly variable (Table 1). The concentrations of protein and non-struc- (Denslow et al. 1990), and can influence the susceptibility tural increased by 32 % and 45 % respective- of plants to herbivores and pathogens (e.g. Coley 1983a; ly. Augspurger 1984; Harrison 1987; Ernest 1989). Dif- Some have suggested that the ratio of nutrients to ferences in susceptibility to natural enemies may be due tannins dictates the susceptibility of leaves to herbivores to light-induced differences in leaf traits such as nutrients (Wint 1983; Mole and Waterman 1988). The results and secondary chemicals (Mole et al. 1988). An increase presented here do not support this assertion. Leaf-cutter in available light can result in an increase in nutrients and ants preferred sun leaves even though they had a lower carbon-based secondary chemicals (Langenheim et al. nutrient to tannin ratio. This descrepancy may be due to 1981 ; Chandler and Goosem 1982; Feibert and Langen- the fact that the fungus the ants cultivate produces en- heim 1988; Mole et al. 1988; Denslow et al. 1990). How- zymes which degrade phenolic substances (Cherrett et al ever, the production of carbon-based chemicals (such as 1989). Perhaps only high concentrations of tannins in- tannins and other phenolics) varies among species (Coley hibit the ant/fungus system so that at low concentrations et at. 1985; Coley 1988). Only shade-tolerant tree species nutrients dictate selection. appear to contain high concentrations of phenolics In a previous study, Nichols-Orians and Schultz (Chandler and Goosem 1982; Denslow et al. 1987), espe- (1990) found that young leaves had higher concentra- cially when growing under high light conditions (Chand- tions of nutrients and condensed tannins than mature ler and Goosem 1982, Waterman et al. 1984; Mole et al. leaves, yet young leaves were less chemically acceptable 1988; Mole and Waterman 1988). These species-specific to A. cephalotes. This suggests that condensed tannins differences may explain why some species are more sus- deterred the ants. The concentrations of condensed tan- ceptible to herbivores when growing in high light en- nins in the young leaves of that study were nearly three vironments (Harrison 1987; Ernest 1989). times higher than those measured in the mature sun In multi-species studies, researchers have failed to leaves of this study. In another study (Nichols-Orians show that herbivory in the tropics is negatively correlated 1990), I found that the leaves of seedlings growing at high with foliar concentrations of tannins (Coley 1983a; How- light in nutrient-poor soils had extremely high concentra- ard 1987). Since only some species are capable of produc- tions of tannins and were also rejected by foraging ants. ing high concentrations of tannins, multispecies studies Taken together, these studies suggest that condensed could mask the role of tannins. I. oerstediana is a large tannins may deter the ants only when above a concentra- seeded shade-tolerant canopy tree species which is capa- tion threshold. Below that threshold, nutrients dictate ble of producing high concentrations of condensed tan- acceptability. 558

Although light-induced increases in condensed tan- My data and those of others suggest that leaf-cutter nins did not deter the ants it is conceivable that damage- ants prefer pioneer species over later successional species induced increases could elevate the concentrations of (Fowler 1983; Shepard 1985) and sun leaves over shade tannins to a level that is deterrent. However, previous leaves (Tables 3 and 4). These preferences may explain damage did not result in an increase in the concentrations why leaf-cutter ant nests are usually located in or near of tannins over a 24 h period (Table 2). Although recent disturbed habitats (Jonkman 1979; Jaffe and Vilela 1989; evidence suggests that manual damage does not perfectly Vasconcelos 1990). Differences in the susceptibility of mimic damage, manual damage is nonetheless species to leaf-cutter ants may be an important factor often associated with some change, more or less, in chem- influencing patterns of succession in early successional istry (Baldwin 1988b; Hartley and Firn 1989). Short- habitats in the neotropics, yet, to my knowledge, this has term induction by L oerstediana seedlings is unlikely to not been evaluated. be an important defense mechanism against leaf-cutter ants. In another study, Howard (1990) found no evidence Acknowledgements. I am grateful to J. Nichols-Orians for assistance for differential susceptibility of previously damaged with field work, E. Hollis and K. Kleiner for help with chemical plants to leaf-cutter ants. However, leaf-cutter ants may analyses, M. Slater for doing laboratory assays with Forsythia, and the staff of the Organization for Tropical Studies (OTS) for making return to previously defoliated plants (pers. obs.), so the this research possible. A special thanks to David and Deborah system would be ideal for testing whether regrowth fo- Clark and Victor Chavarria of OTS for making it possible to live liage differs in chemistry and acceptability. in Puerto Viejo. Early versions of this paper benefited from the Nichols-Orians and Schultz (1990) also found that comments of J. Schultz, H. Appel, T. Givnish, J. Howard, M. leaf toughness was the major factor influencing the sus- Hunter, K. Kleiner and one anonymous reviewer. T. Brodzina ceptibility of L oerstediana juveniles to A. cephalotes. In prepared the manuscript. This research was supported by a Jesse Smith Noyes Fellowship Grant awarded by OTS, a USAID this study leaf toughness did not vary among treatments strengthening grant awarded by the College of Agriculture at The (Table 1) and all leaves of this study were nearly two Pennsylvania State University, and National Science Foundation times less tough than the old leaves of the previous study grants BSR-8605106 (to J. Schultz) and BSR-8815648 (Disserta- (371.7 • 3.8 kPa). Results of the "cutting" assay confirm tion Improvement Grant to C. Nichols-Orians). that physical features did not prevent A. cephalotes from harvesting the most chemically acceptable leaf material (leaves from plants growing in full sun). References

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