Biochemical Systematics and Ecology, VoL 17, No. 7/8, pp. 551-557, 1989 0305-1978/89 $3.00+0.00 Printed in Great Britain. © 1989 Pergamon Press plc.

Intra- and Interplant Leaf Sesquiterpene Variability in Copaifera langsdorfii • Relation to Microlepidopteran Herbivory

CYNTHIA A. MACEDO and JEAN H. LANGENHEIM Department of Biology, University of California, Santa Cruz, CA 95064, U.S.A.

Key Word Index--Copaifera; tropical Leguminosae; Stenoma; sesquiterpenes; intraplant and intrapopulation variation; lepidop- teran herbivory; oecophorid.

Abstract--Oecophorid herbivory in Copaifera langsdorfiileaves along with sesquiterpene composition, concentration of most of the individual sesquiterpenes and total yield did not significantly differ between the lower and upper portions of tree canopies. Although sesquiterpene variation in leaves collected throughout individual tree canopies was less than variation among trees, leaves which were eaten by oecophorid larvae had slightly lower yields than those unattacked. Individual C. langsdorfiitrees within the population were significantly different from one another in sesquiterpene yield, oecophorid herbivory and in the con- centration of seven out of the 11 sesquiterpene compounds. Leaf sesquiterpenes appear to be more important in inhibiting herbi- vory by Stenoma aft. assignata than leaf moisture and nitrogen content and toughness.

Introduction Secondary compound variability is important in restricting herbivores and other plant parasites and predators [1, 2]. For instance, in tree-folio- vore interactions, variability within a canopy or among trees may restrict a foliovore if it en- counters leaves that have greater dosages of a compound or different combinations of a suite of compounds [3-8]. Herbivores may also induce changes while feeding on a plant [e.g. 9- 11] or changes may occur over a longer period of time [12, 13].

Although terpenes (most notably monoterp- enes) have been well studied chemosystematic- ally [14-18], their ecological relationships are less understood, especially in tropical environ- ments. The present investigation is part of a long term chemosystematic and ecological study of the tropical terpenoid resin producing genera Hymenaea and Copaifera. These two genera have been investigated to analyse the hypothesis that in tropical environments biotic pressures by organisms, such as insect herbi- vores and pathogens, may have played a signifi- cant role in the evolution and maintenance of copious resin production and in quantitative terpene variability [19-22]. Hymenaea and Copmfera are prominent leguminous trees (sub-

(Received 28 July 1989)

family Caesalpiniodeae) in African and Neo- tropical lowland ecosystems. The two genera often co-occur and have leaf resins primarily composed of the same suite of sesquiterpenes [23, 24].

The objective of this study was to analyse the relationship between herbivory and leaf sesqui- terpene variability: variability in the concentra- tion of individual compounds and total yield (mg/g dry wt) and in composition (% of total) within and among Copaifera langsdorfii trees. The herbivore is a leaf-tying microlepidopteran of major importance, which is apparently re- stricted to Copaifera and temporarily identified as Stenoma aft. assignata (Oecophoridae) (V. O. Becket, personal communication). A previous investigation [7], showed undamaged C. langs- dorfii to produce higher and/or more variable amounts of certain compounds than plants damaged by S. aft. assignata and three other oecophorid leaf-tiers. In addition to sesqui- terpene variability, spatial variability in leaf toughness, percent moisture and nitrogen content were analysed because these para- meters can influence herbivory [25-27]. The oecophorid Stenoma ferocanella attacks Brazilian Hyrnenaea stigonocarpa leaves when they are less tough and higher in nitrogen and moisture, and have lower sesquiterpene yields than younger leaves [4]. In contrast, oecophorids on

551

552 CYNTHIA A. MACEDO AND JEAN H. LANGENHEIM

C langsdorfii attack mature leaves which are generally tougher, lower in moisture and nitrogen and have higher amounts of sesqui- terpenes than immature leaves [7].

C. langsdorfii is extremely variable morpho- logically and is widespread, occurring as a small tree (1-10 m) in open, savanna-like areas to more closed woodlands. Tall (30-40 m) trees occur in subdeciduous and riparian forests in northeastern, central and southern Brazil. The present research site (SC) was on a reserve, Fazenda Canchim, near the city of S~o Carlos, S~o Paulo state, Brazil and is on a plateau ap- proximately 650 m high. The reserve has essen- tially scrub woodland vegetation which includes a great diversity of trees and shrubs. C. langsdor- fiitree's are abundant at the site although other leguminous trees (Anadenthera falcata, Pterodon pubescens and Hymenaea stigonocarpa) are also common.

Twenty-two trees with a mean height of 6.1 m and dbh of 11.5 cm were investigated. C. langs- dorfii are spaced on average 7 m apart at the study site. Trees in the eastern portion of the research area, which were included in the present study, are generally larger than those on the western portion that were also studied previ- ously [7, 28, 29]. A more open area in the western portion grades into a more closed woodland in the southeastern portion. The mean annual precipitation is 1476 mm with 80% occurring from October to March. The mean temperature during the rainy season is 21.5°C and 18°C during the dry season.

Herbivore activity at the reserve is greatest during the warm, rainy season. C. langsdorfii is facultatively deciduous and frequently drops its leaves during the dry, cold season and flushes new leaves just prior to the onset of the rainy season. By November, most trees have mature sclerophylous leaves [28]. S. aft. assignata larvae are present near the beginning of the rainy season, after most leaves have matured on C. langsdorfi~ C. langsdorfii leaves consist of from 6-10 leaflets per leaf. Larvae tie and feed on these leaflets. As they develop, the larvae tie more leaflets into their nests. A larva can even- tually tie up to 14 leaflets from more than one leaf into a nest. S. aft. assignata has two genera- tions during the rainy season; the first lasts from about October to December, when they pupate.

Adults from laboratory reared pupae emerged in about 3 weeks. The second generation early instars are present by mid January and pupate in the first half of March.

From November 1984 to March 1985 and again from September 1985 to March 1986 upper and lower canopy leaves were collected at the beginning, middle and end of the rainy season. Although the Copaifera trees have fairly open canopies at the site, fully exposed upper canopy leaves at the top of trees and the most shaded lower canopy leaves were selected for comparison. Mature leaves tied into nests were also collected to compare with the non-nest, mature leaves. Leaves were analysed for tough- ness, percent leaf moisture, nitrogen (for 1984- 1985 only) and sesquiterpenes. Upper and lower canopy comparisons for total sesquiterpene yield were made for both years. Comparisons of upper versus lower canopy leaves for composi- tion and concentration of individual sesquiter- penes were only made for 1985-1986 because leaf sesquiterpenes were analysed from more trees during this period. Mature leaves were col- lected throughout tree canopies to also analyse intraplant sesquiterpene variability during this period.

Every 2 weeks during the rainy season at least one, approximately 1 m long branch, randomly selected from the upper and lower canopies of trees, was searched for leaf-tiers. The number of leaves per branch and larvae per branch were determined along with the number of leaflets per leaf. At the same time a damage score, i.e. estimating the percent of the tree canopy in nests, was recorded for each tree in order to assess the impact of So aft. assignata on C. langs- dorfii leaves.

Results Since no significant differences occurred in ses- quiterpenes, toughness and moisture content between November and February collected leaves (C. A. Macedo, unpublished data), those collected in early February were used for com- parisons of these parameters. This was also the best period for comparison of tier to leaflet ratios in the tree canopies because it corres- ponded to the population peak for S. aft. assig- nata.

In general, the mean leaf sesquiterpene

INTRA- AND INTERPLANT SESOUITERPENE VARIABILITY 553

concentration and composition of individual compounds, and total yield were similar in the upper and lower portions of trees (Tables 1 and 2). The upper canopies of trees had, however, more variable concentrations of cyperene, ~-copaene and caryophyllene oxide; the lower canopies had more variable concentrations of y-muurolene and 6-cadinene. The mean total yield did not differ significantly between the upper and lower canopies of trees during either the 1984-1985 or 1985--1986 seasons.

There was a significant increase ( P < 0.05) in the number of tiers/leaflet from the first to the second year, and a slight increase in the damage score from the first to the second year (Table 3). The upper and lower portions of trees were not significantly different, however, in their mean tier to leaflet ratios for either year. Thus, although larval density differed (increased) from the first to second year, larval distribution within a canopy did not.

Since total leaf sesquiterpene yield and leaf parameters did not differ significantly between years (Tables 1 and 3), means for both years were taken for leaves not tied or eaten by S, aft. assignata to compare with those tied and eaten (Table 4). The mean sesquiterpene yield was slightly lower in nest compared to non-nest

TABLE 1. COMPARISON OF MEAN SESQUITERPENE CONCENTRATION AND TOTAL YIELD (mg/g DRY WT) IN UPPER AND LOWER CANOPY C. LANGSDORFIILEAVES BY t- AND F--TESTS

Sesquiterpenes Upper Lower tl" F (mg/g dry wt) canopy canopy (1985-1986)

(l-Cubebene 0.04 ± 0.08 0.02 ± 0.04 0.75 4.37** c¢-Copaene 0.18±0.21 0.17±0.21 0.21 1.04 Cyperene 0.33±1.14 0.10±0.19 0.92 37.50** ~-Copeene 0.04±0.15 0.00±0.01 1.18 260.27** Caryophyllene 0.42±0.57 0.42±0.54 0.00 0.02 ~-Humulene 0.04± 0.08 0.04± 0.07 0.02 1.26 ?-Muurolene 0.04±0.06 0.17±0.60 -0.98 91.55"* a + ~-Selinene 2.15±2.44 2.19±2.91 -0.05 1.42 y-Cadinene 0.34± 0.65 0.48 ± 0.66 --0.68 1.04 6-Cadinene 0,02±0.03 0.06±0,14 -1.34 29.79** Caryophyllene oxide 0.26±0.36 0.12+0.15 1.68 5.76** Total yield 4.10±3.87 4.21±4.24 --0.17 1.20 Total yield (1984-1985) 3.19±2.06 4.46±2.29 --1.30 1.20

*Significant at 0.05. **Significant at 0.01. n =21 trees for 1985-1986; n--10 trees for 1984-1985. tan approximation of twas used when an F-test showed the proba-

bility of equal variance was <0.05.

TABLE 2. COMPARISONS OF MEAN SESQUITERPENE COMPOSITION (% OF TOTAL) BETWEEN THE UPPER AND LOWER CANOPY LEAVES OF C. LANGSDORFII DURING 1985-1986

Sesquiterpenes Upper Lower t (% of total) canopy canopy

a-Cubabene 1.0± 2.1 0.5± 1.0 0.99 a-Copaene 4.4± 5.1 4.0± 5.0 0.26 Cyperene 8.1 ± 27.8 2.4± 4,5 0.93 ~-Copaene 1.0± 3.7 0_+ 0.2 1.24 Caryophyllene 10.2±13.9 10.0±12.8 0.02 ~-Humulene 1.0± 1.5 1.0± 1.7 0 y-Muurolene 1.0± 1.5 4.0± 14.3 --0.96 a + ~-Selinene 52.4 ± 59.5 52.0 ± 69.1 0.02 y-Cadinene 8.3 ± 15.9 11.4 ± 15.7 --0.64 6-Cadinene 0.5± 0.7 1.4± 3.3 -1.22 Caryophyllene oxide 6.3±8.8 2.9±3.6 1.64

n=21 trees.

TABLE 3. COMPARISONS OF MEANS FOR LEAF PARAMETERS AND HERBIVORY BE'P/VEEN THE UPPER AND LOWER CANOPY OF C.

LANGSDORFII DURING 1984-1985 AND 1985-1986

Lower canopy Upper canopy

Leaf toughness (g to puncture)

1984-1985 461 ± 153 394± 98 1985-1986 328 ± 44 321 ± 47

% Leaf moisture 1984-1985 46±4 45± 5 1985-1986 44± 3 42±4

Leaf nitrogen content (% dry wt)

1984-1985 2,23% 2.25% No. tiers/leaf

1984-1985 0.02 ± 0.01 0.02 ± 0.02 1985-1986 0,08 ± 0.07 0.09 ± 0.10

Damage score 1984-1985 2.5± 1.0 1985-1986 2.8± 1.0

From each of the lower and upper canopies: n=21 leaves for leaf toughness and moisture for both years; n=10 for damage score for 1984-1985; n=21 for 19e5-1986. n=10 for leaf nitrogen content from each of the lower and upper canopy.

leaves; the means for percent moisture and toughness between the two groups were similar (Table 4). Older, pre-senescing leaves also did not differ significantly in their mean sesquiter- pene yield compared to mature leaves (~=4.85 _+ 2.14).

There was more variability in herbivory and sesquiterpene concentrations, and yield among trees, than between the upper and lower canopies within trees (Table 5). There was a

554 CYNTHIA A. MACEDO AND JEAN H. LANGENHEIM

TABLE 4. COMPARISON OF MEANS OF LEAF PARAMETERS AND SES- QUITERPENE TOTAL YIELD BETWEEN LEAVES NOT TIED OR EATEN AND LEAVES TIED AND EATEN BY S. AFF. ASSlGNATA DURING 1984- 1985 AND 1985-1986

Leaves not tied Leaves tied or eaten and eaten t

Sesquiterpene total yield 3.99_+3.12 2.60_+ 2.00 1.35 (mg/g dry wt) % Moisture 45 ± 4 44 ± 9 0.52 Toughness 377±86 381±136 0.13 (g to puncture)

For leaves not tied or eaten: n=42 for toughness and moisture; n= 29 for sesquiterpene total yield. For leaves tied and eaten: n-- 24 for moisture and toughness; n--11 for sesquiterpene total yield.

TABLE 5. COMPARISON OF VARIATION IN MEANS OF LEAF PARA- METERS, LEAF SESQUITERPENE CONCENTRATIONS AND TOTAL YIELD, AND HERBIVORY WITHIN TREES (UPPER VS LOWER CANOPY) AND AMONG TREES FOR 1985-1986

Sesquiterpenes Within trees Among trees F P > F (mg/g dry wt) error (df=15) (df=16)

a-Cubabene 0.004 0.006 1.32 0.297 a-Copaene 0.024 0.079 3,31 0.013 Cyperene 0.006 0,054 9.84 0.0001 ~-Copaene 0.018 0.016 0.87 0.610 Caryophyllene 0.080 0.080 7.71 0.0001 ~-Humulene 0.002 0.012 7.13 0.0002 y-Muurolene 0.257 0.227 0.88 0.597

+ ~-Selinene 1.149 10.536 9.16 0.0001 y-Cadinene 2,061 13.670 6.22 0.0005 ~-Cadinene 0,012 0.014 1.21 0.357 Caryophyllene oxide 0,107 0.079 0.74 0.725 Total yield 2.250 28.737 12.77 0.0001 % Moisture 33.857 41.710 1.23 0,351 Toughness 1602.083 1874.044 1.17 0.443 No. Tier/leaf 0.001 0.014 11.26 0.0001

General linear model analysis for unbalanced data was used. n=18; n=2 leaves per tree (1 upper, 1 lower canopy).

highly significant difference among trees in the tier to leaflet ratio (P< 0.0001). The concentra- tions of cyperene, caryophyllene, ~-humulene, y + 13-selinene, y-cadinene, and total yield were also all highly significantly different (P< 0.0005) among trees.

There was no significant difference for either year in leaf toughness and percent moisture between the upper and lower portions of the canopy. Nitrogen content also did not differ significantly between the two levels during 1984-1985 (Table 3). Furthermore, the variability within and among trees was similar for the per- cent leaf moisture and toughness (Table 5).

An analysis of variance (ANOVA) of terpene total yield for leaves collected throughout the canopies of five trees also showed greater ses- quiterpene variability among trees, which was highly significant (F=10.06, P=0.0001), com- pared to variability within trees.

Discussion The similar oecophorid levels in the upper and lower portions of tree canopies would be expected since leaf sesquiterpene total yield, moisture, toughness and nitrogen were all similar at the different canopy levels. The overall composition was also similar with only a few compounds differing between the two levels (Table 2). These results support past studies that showed little compositional variation within indi- vidual Hymenaea and Copaifera trees, and hence the conclusion that composition is under tight genetic control [7, 30-34]. The high variance in percent composition of some compounds is partially a reflection of differences among trees in their concentration of these compounds and their total yield. The significant within-tree variability in the concentrations of cyperene, (~-copaene, ~,-muurolene, (~-cadinene, and caryo- phyllene oxide could be important in relation to herbivory, Cyperene and ~/-muurolene have been shown to adversely affect the success and feeding behavior of oecophorid leaf-tiers [4, 7, 29], and both caryophyllene and caryophyllene oxide inhibit fungal growth and herbivory by leaf-cutting ants which rear fungi [35-37]. Although there is strong evidence that caryo- phyllene adversely affects lepidopterans, including oecophorids [3, 4, 7], the affect of the oxide on oecophorids has been investigated only recently [38].

Herbivore numbers, and the composition and concentration of sesquiterpenes, varied more among than within trees. Because leaf moisture and toughness, however, did not vary signifi- cantly within or among trees, these parameters apparently are not as important in determining oecophorid distribution as leaf sesquiterpenes. Although differences among trees in leaf sesqui- terpenes seem more important in influencing herbivore distribution, total yields within some trees did greatly vary, even when compared to differences between trees. For example, the range of total yields of leaves in one tree (nsl0)

INTRA- AND INTERPLANT SESQUITERPENE VARIABILITY 555

was from 2-13 mg/g; the ranges of total yields in two other trees was 6-10 mg/g (n=10) and 1-5 mg/g (n=8). The higher total yields in some of these leaves reach levels found to negatively affect leaf foliovores [4, 7, 38, 39]. Perhaps some trees are more resistant to herbivory because their leaves have a greater range of total yields and/or overall greater yields. The range of total yields in leaves actually tied into nests and fed upon by the oecophorids was from 1-6 mg/g, which is the lower end of the range found for non-nest leaves in the population and lower than the dosages found to adversely affect herbivores. Thus S. aft. assignata apparently does not survive on individual leaves which have higher total yields. C. langsdorfii seedlings greater than one year old at SC have, on average, three times the mean leaf sesquiter- pene yield found in adults and appear to com- pletely lack leaf-tier herbivory [7, 29, 38].

In the present study oecophorid herbivory and the concentrations of cyperene, caryophyl- lene, the selinenes, and resin total yield were all significantly different among trees. The import- ance of greater concentrations of these compounds and total yield in inhibiting herbi- vory has been well documented. A negative relation between cyperene and oecophorid herbivory has been demonstrated in past studies at SC and a nearby site [7, 29]. Caryo- phyllene, the selinenes and yield have dosage dependent toxic effects on lepidopteran herbi- vores and deterrent properties [3, 4, 7, 39]. Thus the differences between trees in these com- pounds and total yield may be important in restricting oecophorid herbivory.

The lack of differences in total yield of leaf sesquiterpenes, herbivory, leaf moisture, nitro- gen content and toughness between upper exposed and lower shaded canopy leaves was not anticipated. Even though the density of larvae on C. langsdorfii leaves increased from one year to the next, the distribution within the canopy did not. Researchers investigating rain- forest trees have found less herbivory in upper canopy leaves [40, 41] which generally are tougher and less moist than shaded leaves [42, 43]. Although lower canopy leaves collected in our scrub woodland study were shaded, the C. langsdorfiitrees at SC have fairly open canopies, especially compared to rainforest trees. Perhaps

temperature and light intensity do not differ sufficiently to affect these leaf parameters and herbivory. The production of tough, sclero- phylous leaves in savanna and scrub woodland species, such as C. langsdorfii, may be influ- enced by factors other than light intensity such as fire and soil properties [44]. Leaves were collected for analysis of moisture content during the rainy season at which time they may not suffer water deprivation anywhere within a tree's canopy. Furthermore, many Brazilian savanna and scrub woodland plants are phreatophytic. Because these plants have long tap roots which reach water tables they rarely suffer water deprivation, even during the dry season [45].

Responses of sesquiterpenes to irradiance may differ between tree species or populations or with ontogeny. Copaifera rnu~]uga saplings, growing in a cacao plantation near Manaus, Brazil had higher yields in their upper compared to lower canopy leaves [33]. However, these differences were not observed in emergent adult C. rnultljuga growing in nearby rainforest. C. langsdorfii seedlings, which were experi- mentally shaded (near the SC reserve), had significantly lower leaf sesquiterpene yields compared to seedlings growing in full sunlight. On the other hand, shaded C. langsdorfii sap- lings growing naturally at SC had higher yields than those in full sunlight [46]. The authors postulated that these higher yields in the shade may be due to greater oecophorid pressures there, which select out lower yielding seedlings.

Although the above examples show contra- dictory results, adult trees of both C. rnu~juga and C. langsdorfii showed no significant differ- ences in total yield between their upper and lower canopy leaves. However, C. rnu~juga and C. langsdorfii upper canopy leaves may have higher yields at other times of the year. Leaves analysed for sesquiterpene yields for both species were collected during flowering. C. langsdorfii flowers are most abundant in the upper canopy and photosynthate could be allo- cated more for reproduction during this period rather than for sesquiterpene production. Some upper canopy leaves yellowed at this time, possibly due to translocation of nitrogen from leaves to reproductive structures. The sesqui- terpenes in presenescing leaves (which were

556 CYNTHIA A. MACEDO AND JEAN H. LANGENHEIM

produced the previous year and can be attacked by leaf-tiers), however, differed little between those of the recently produced mature leaves.

Mature leaves do differ from immature ones, which have comparatively lower concentrations of individual sesquiterpenes and total yield, are less tough and are higher in leaf moisture and nitrogen content [28]. Although sesquiterpenes appear to restrict oecophorid herbivory, other factors may influence their presence on mature versus immature leaves. Immature leaves have higher levels of astringent phenolic compounds and perhaps other secondary compounds, such as unusual amino acids, than mature leaves [28, 47]. Furthermore, these immature leaves are present for only approximately 3 weeks while the larval life-span of S. aft. assignata is approximately 2 months. The changes in sesqui- terpenes (in composition, concentration and total yield) during leaf development may adversely affect the oecophorids. Also, the longer life span and rigidity of mature leaves perhaps offer some protection against preda- tors, as was found for pyratid leaf-tying larvae [48]. Protection from ants, carib beetles and spiders, which have been observed preying on larvae, may be important during the warm rainy season, which is the peak period for most insect activity.

Thus S. aft. assignata may be somewhat restricted to mature leaves and then to only some mature leaves, i.e., those having concentrations of certain compounds--such as caryophyllene and the selinenes and/or total yields below a threshold, above which larvae are adversely affected. Oecophorid herbivory at the SC site has been lower than levels at a nearby site where C. langsdorfii leaf sesquiterpenes are less variable from tree to tree and where micro- lepidopteran outbreaks have occurred [7, 38]. The sesquiterpene profiles of some trees at SC may restrict herbivore loads on them, thus suppressing herbivore outbreaks. The presence of some intraplant variation in leaf sesquiter- pene patterns (but not restricted to upper and lower portions), and especially differences among trees, appears to suppress foliovore activity.

Experimental Twenty-two C. langsdorfii trees at SC were studied from

November to mid-March in 1984-1985 and from September to February 1985-1986, Each tree was measured for height and dbh. Two mature leaves were collected from the upper fully exposed and lowest shaded portions of each tree canopy at the beginning, middle and end of the rainy season for both years. Leaves from 10 trees were air dried and analysed for sesquiterpenes in 1984-1985 and from 21 trees in 1985-1986. During 1985-1986 an average of eight leaves were collected throughout the canopies of five trees for sesquiterpene analy- sis. Additionally, in September 1985, 10 pre-senescing leaves (leaves remaining from the previous rainy season) were collected for sesquiterpene analysis.

Sesquiterpenes were extracted at the University of Cali- fornia at Santa Cruz (UCSC) in high purity grade pentane by grinding leaves in a mortar and pestle with Standard Ottawa sand. N-Tetradecane was added as a GC internal standard; the extract was filtered with a Whatman no. 1 filter paper, the volume concentrated to approximately 0.25 ml with a mild stream of N and analysed directly by GC (31~m DC-WAX, 0.53 mm×30 m silica capillary column, 130°C, He carrier gas, FID, electronic integrator). This method gives results essentially identical to those used in previous studies of Hymenaea and Copaifera leaf sesquiterpenes.

Upper and lower canopy leaves collected for sesquiter- penes were also analysed for leaf toughness and moisture content for both years and leaf nitrogen content for 1984-1985. Leaf toughness values were obtained using a H516-1000MRP force penetrometer to puncture leaflets. Two leaflets were weighed, oven dried for 3 days at 80°C and weighed again for % leaf moisture content. Leaves for total nitrogen were oven dried for 3 days at 80°C, digested according to a micro- Kjeldahl method [49J, and nitrogen assayed colorimetrically using a Technicon 2 Autoanalyzer.

Every 2 weeks during the rainy season for both 1984-1985 and 1985-1986, one ca 1 m long, randomly selected branch from both the upper and lower canopy for each tree was assayed for oecophorid leaf-tiers. The number of leaves/ branch were first counted, followed by the number of larvae/ branch. For each tree, the number of leaflets/leaf was also determined. During both years, 24 fresh leaves tied and eaten and 42 not tied nor eaten by S. aft. assignata were collected for moisture and toughness analysis. Eleven of those tied and eaten, and 29 not tied nor eaten were analysed for sesquiter- pene total yield. To express the impact of oecophorids on trees, damage scores were recorded for each tree. These scores give estimates for % of the tree foliage in nests. Scoring was as follows: 1~0% (of tree foliage in nests); 1.5=1-5%; 2--6-12%; 3--13-25%; 4--26-50%; 5-- > 50%.

Data were analysed using SAS statistical package on a Magnuson M80 computer at UCSC. Voucher specimens were deposited in the herbaria at UCSC, and in Brazil at Instituto Brasileiro de Geografico e Estatistica in Brasilia and the Universidade de Estadual de Campinas (UNICAMP).

Ad(nowledgements--We would like to thank Dr George Shephard (UNICAMP) for being the CNPq Brazilian representa- tive, Drs Jo&o Juares Soars and P. V. Moraes for their assist- ance and use of their laboratory at the Universidade de S&o Carlos, S&o Paulo and Dr V. O. Becker (EMBRAPA) for identifi- cation of and information on the microlepidopterans. Also, special thanks are due to Erik B. Feibert for analysing leaves for leaf nitrogen content and considerable help with field

INTRA- AND INTERPLANT SESQUITERPENE VARIABILITY 557

work. We also appreciate valuable discussion and critical comments on the manuscript given by Francisco Espinosa and Jeanette Rollinger. Support for the study was provided by the National Science Foundation grant DEB 78-10645 to J.H.L.

References 1. Denno, R. F. and McClure, M. S., eds (1983) Variable Plants

and Herbivores in Nature/ and Managed Systems. Aca- demic Press, New York.

2. Hedin, P. A., ed. (1983) Plant Resistance to Insects, ACS Symposium Series 208. American Chemical Society, Washington DC.

3. Langenheim, J. H., Foster, C. E. and McGinley, R. B. (1980) Biochem. Syst. Ecol. 8, 385.

4. Langenheim, J. H. and Hall, G. D. (1983) Biochem. Syst Ecol. 11, 29.

5. Cates, R. G., Redak, R. A. and Henderson, C. B. (1983) in Plant Resistance to Insects (Hedin, P. A., ed.), p. 3. ACS Symposium Series 208, American Chemical Society, Washington DC.

6. Schultz, J. C. (1983) in Plant Resistance to Insects (Hedin, P.A., ed.), p. 37. ACS Symposium Series 208, American Chemical Society, Washington DC.

7. Langenheim, J. H., Convis, C. L., Macedo, C. A. and Stubblebine, W. H. (1986) Biochem. Syst Ecol. 14, 41.

8. Cates, R. G. and Redak, R. A. (1988) in Chemical MediaEon of Coevoludon (Spencer, K. C., ed.), p. 317. Academic Press, New York.

9. Green, T. R. and Ryan, C. A. (1972) Science 176, 776. 10. Haukioja, E. and Neimel~, P. (1979) Oecologia 39, 151. 11. Rhoades, D. F. (1979) in Herbivores: Their Interecdons with

Plant Secondary Metabolites (Rosenthal, G. A. and Janzen, D. H., eds), p. 3. Academic Press, New York.

12. Bear, G. (1974) Z. Angew. Entomol. 76, 196. 13. Benz, G. (1977) Eucarpia/IOBC Work. Group Breed. Resist-

ance Insects Mites. Bull. SROR 197711978, 155. 14. Adams, R. P. (1970) Phytochemistry9, 397. 15. Zavarin, E., Lawrence, L. and Thomas, M. C. (1971) Phyto-

chemistry 10, 379. 16. Smith, R. H. (1972) USDA, Forest Service Technical Report

TSW1. Pacific Southwest Forest and Experiment Station. Berkeley, California.

17. Von Rudloff, E. (1975) Biochem. Syst Ecol. 2, 131. 18. Hall, G. D. and Langenheim, J. H. (1987) Biochem. Syst

EcoL 15, 31. 19. Langenheim, J. H. (1969) Science 163, 1157. 20. Langenheim, J. H. (1975) Proceedings of the 7th Inter-

national Botanical Congress, Leningrad, p. 116. 21. Langenheim, J. H. (1981) in Advances in Legume System-

atics (Polhill, R. M. and Raven, P. H., eds), p. 627. Proceed- ings of the International Botanical Congress, Royal Botanical Garden, Kew, U.K.

22. Langenheim, J. H. (1984) in PhysiologicalEcology of Plants of the Wet Tropics (Medina, E., Mooney, H. A. and Vasquez- Yanes, C., eds), p. 189. Junk Publishers, The Hague.

23. Martin, S. S., Langenheim, J. H. and Zavarin, E. (1972) Phytochemistry 11, 3049.

24. Arrhenius, S. P., Foster, C. E., Edmonds, C. G. and Langen- helm, J. H. (1983) Phytochemistry22, 471.

25. Feeny, P. (1970) Ecology51, 565. 26. Mattson, W. J., Jr. (1980) Ann. Re~. Ecol. Syst 11, 119. 27. Scriber, J. M. and Stansky, F., Jr (1981) Ann Rev. Ecol.

Syst 26, 183. 28. Langenheim, J. H., Macedo, C. A., Ross, M. K. and

Stubblebine, W. H. (1986) Biochem. Syst Ecol. 14, 51. 29. Ross, M. K. (1986) M.S. Thesis, University of California,

Santa Cruz. 30. Martin, S. S., Langenheim, J. H. and Zavarin, E. (1974)

Biochem. Syst. Ecol. 2, 75. 31. Kane, T. E. (1977) Senior Biology Thesis, University of Cali-

fornia, Santa Cruz. 32. Langenheim, J. H., Stubblebine, W. H., Lincoln, D. E. and

Foster, C. E. (1978) Biochem. Syst Ecol. 6, 299. 33. Langenheim, J. H., Arrhenius, S. P. and Nascirnento, J. C.

(1981) Biochem. Syst. Ecol. 9, 27. 34. Xena de Enrech, N., Arroyo, M. T. K. and Langenheim, J.

H. (1983) Acta Botanica Venezuelica 14, 239. 35. Arrhenius, S. P. and Langenheim, J. H. (1983) Biochem.

Syst_ Ecol. 11, 361. 36. Hubbel, S. P., Weimer, D. F. and Adjare, A. (1983) Oeco-

Iogia 60, 321. 37. Howard, J. J., Cazin, J., Jr. and Weimer, D. F. (1988) J.

Chem. Ecol. 14, 59. 38. Macedo, C. A. and Langenheim, J. H. (1989) Biochem. Syst.

Ecol. 17, 207. 39. Stubblebine, W. H. and Langenheim, J. H. (1977) J. Chem.

Ecol. 3, 633. 40. Nascimento, J. C. (1980) Ph.D. dissertation, University of

California, Santa Cruz. 41. Lowman, M. D. and Box, J. D. (1982) Ph.D. dissertation,

University of Sydney, Australia. 42. Lowman, M. D. and Box, J. D. (1983) Aust J. Ecol. 8, 17. 43. Oberbauer, S. F. and Strain, B. R. (1986) Am. J. Bot 73,

409. 44. Ferri, M. G. (1961) Inst. Interam. Ci~nc. Agric. (Zona Sul) e

Depto. Prod. Anim. Secr. Agric. S&o Paulo, p. 177. 45. Eiten, G. (1972) Bot Rev. 38, 301. 46. Feibert, E. B. and Langenheim, J. H. (1988) Phytochemistry

27, 2527. 47. Figliulo, R., Naylor, S., Wang, J. and Langenheim, J. H.

(1987) Phytochemisb'y26, 3255. 48. Daaman, H. (1987) Ecology68, 88. 49. Isaac, R. A. and Johnson, W. C. (1976) J. Assoc. Offic.

Analyt Chem. 59, 98.