Journal of Chemical Ecology, Vol. 27, No. 3, 2001 TRADE-OFFS IN ANTIHERBIVORE DEFENSES IN Piper cenocladum: ANT MUTUALISTS VERSUS PLANT SECONDARY METABOLITES LEE A. DYER,1,2,* CRAIG D. DODSON,2 JON BEIHOFFER,3 and DEBORAH K. LETOURNEAU2,4 1 Department of Ecology and Evolutionary Biology Tulane University New Orleans, Louisiana 70118 2 Western Colorado Center For Tropical Research Mesa State College Grand Junction, Colorado 81501 3 US EPA, National Enforcement Investigation Center Bldg 53, Box 25227, DFC Lakewood, Colorado 80225 4 Department of Environmental Studies University of California Santa Cruz, California 95064 (Received November 16, 1999; accepted November 14, 2000) Abstract—Ant–plant mutualisms may provide indirect evidence for costs of antiherbivore defenses when plants demonstrate trade-offs between allocating resources and energy into ant attractants versus chemical defenses. We tested the hypothesis that ecological trade-offs in defenses are present in Piper cenocladum. This plant possesses two distinct defenses: food bodies that attract predatory ants that destory herbivore eggs and amides that deter herbivores. Previous studies have demonstrated that the food bodies in P. cenocladum are an effective defense because the ants deter herbivory by specialist herbivores. Amides in other Piper species have been shown to have toxic qualities, but we tested the additional hypothesis that these amides have an actual defensive function in P. cenocladum. To test for ecological trade-offs between the two putative defenses, fragments of P. cenocladum were examined for the presence of amides both when the plant was producing food bodies and when it was not producing food bodies. Plants with active ant colonies had redundant defenses, producing food bodies and high levels of amides at the same time, but we detected a trade-off in that they had significantly lower levels of amides than did plants with no ants. To test for the defensive value *To whom correspondence should be addressed. 581 0098-0331/ 00/ 0300-0581$19.50/ 0 2001 Plenum Publishing Corporation 582 DYER, DODSON, BEIHOFFER, AND LETOURNEAU of P. cenocladum amides, we used an ant bioassay and we examined herbivory results from previous experiments with plants that had variable levels of amides. These tests demonstrated that amides are deterrent to omnivorous ants, leaf cutting ants, and orthopterans. In contrast, the resident Pheidole bicornis ants are effective at deterring herbivory by specialist herbivores that oviposit eggs on the plant but not at deterring herbivory by nonresident omnivores. We concluded that although both amides and food body production appear to be costly, redundancy in defenses is necessary to avoid damage by a complex suit of herbivores. Key Words—Piper cenocladum, ant plants, chemical defense, trade-offs, amides, herbivory, predation, Costa Rica, mutualism. INTRODUCTION Defenses against herbivores are assumed to be costly in terms of fitness-enhanc- ing functions (Cates and Orians, 1975; Levin, 1976; Fox, 1981; Gould, 1983, 1988; Gershenzon, 1994; Sagers and Coley, 1995; Elle et al., 1999), but these costs are presumably outweighed by the benefits associated with lower levels of herbivory (Zangerl and Bazzaz, 1992; Simms, 1992). As a result of these costs, ecological and evolutionary defensive trade-offs are expected when resources are limited (Rehr et al., 1973; Simms, 1992). For example, a plant that invests heavily in trichomes as a defense should have low levels of chemical defenses compared to a similar species that has a low density of foliar trichomes or com- pared to an individual of the same species that has decreased its investment in tri- chome production. Such trade-offs can be difficult to detect (Simms, 1992), and some authors have even found that allocation of resources to multiple defenses is common in plants that need to defend against many different types of herbivores (e.g., Lindroth and Hwang, 1996). While the presence of trade-offs is consistent with the hypothesis that defenses are costly, allocation of resources to multiple defenses, or redundancy in defenses (Romeo et al., 1996), does not imply absence of cost nor does it exclude the possibility of ecological trade-offs. Limited resources can still cause reduc- tion of one redundant defense in response to an increase in another. Nevertheless, studies uncovering redundant defenses in ant–plant mutualisms have been used to argue that trade-offs between antiherbivore defenses do not occur and that the defenses are not costly because plants are able to allocate resources and energy into chemical defenses as well as ant attractants (biotic defenses). Steward and Keeler (1988) found that the mean number of different indole alkaloid com- pounds was not lower in species of Ipomoea that produce extrafloral nectaries compared to nonnectary plants; these results have been used as weak evidence that neither alkaloids nor nectaries are costly (Simms, 1992). Letourneau and Barbosa (1999) found that a mechanical defense (trichomes) in Endospermum TRADE-OFFS IN Piper DEFENSES 583 was induced both in the presence and absence of a biotic defense (ant defend- ers); this result implies that the mechanical defense is costly because it is induced (Baldwin, 1998), but that there is no trade-off between the mechanical and biotic defense. Janzen (1973) found that individuals of five Cecropia species found in areas without mutualistic Azteca ants did not produce mullerian bodies for these ants; he concluded that this biotic defense was metabolically expensive, but he did not examine the system for trade-offs with production of chemical defenses. Only one study with ant–plant mutualisms has indicated that a trade-off may exist between biotic and chemical defenses, suggesting that both types of defenses are costly. Rehr et al. (1973) found that Acacia species without mutu- alistic Pseudomyrmex ants had leaves with cyanogenic glycosides, whereas a species with resident ants did not have cyanogenic glycosides. Furthermore, diets made from the nonant species adversely affected the feeding efficiency of Pro- denia eridania (Noctuidae) larvae. These results have been used as evidence to support the hypothesis that there are costs associated with biotic defenses as well as chemical defenses (Simms, 1992). However, HCN did not affect the larvae in this study (Rehr et al., 1973), thus their conclusions rely on the assumption that the nonant Acacias contained an unidentified defensive compound that affects herbivores; it is equally plausible that nutritional factors other than plant defenses affected larval feeding efficiency. We tested the general hypothesis of trade-offs in energy and resource allo- cation to competing defenses using an ant plant that facultatively produces food bodies in the presence of a specific ant species and that varies in levels of defen- sive secondary compounds. Piper cenocladum (Piperaceae) is an understory shrub that produces lipid- and amino acid-rich food bodies for a resident ant, Pheidole bicornis (Formicidae: Myrmicinae), which protects the plants from spe- cialist herbivores (Letourneau, 1983). Plants in the Piperaceae commonly have high levels of secondary compounds that have antifeedant activities (reviewed by Parmar et al., 1997), and P. cenocladum has high levels of three amides that may be deterrent to herbivores (Dodson et al., 2000; Dyer and Letourneau, 1999a). We conducted a study to test the hypothesis that production of food bodies and secondary compounds is metabolically expensive so that high investment in one defense will lead to absence or lower levels of the other defense. Regardless of the presence of trade-offs, the fact remains that this plant species has redundancy in its antiherbivore defenses; thus we also tested the hypothesis that the multiple defenses are necessary because amides in P. cenocladum deter those consumers that are not deterred by ants: generalist herbivores and omnivores. METHODS AND MATERIALS Study System. Piper cenocladum is an understory shrub (usually less than 4 m tall) common in lowland wet forests in Costa Rica (Burger, 1971). The species 584 DYER, DODSON, BEIHOFFER, AND LETOURNEAU reproduces both vegetatively through layering (fallen shrubs root adventitiously) or fragmentation (petioles and twigs break off and root) and through seed (Gart- ner, 1989; Greig, 1993). The plant is commonly found as a shrub as well as a fragment (Gartner, 1989), and the biology of the two architectures can be very different (Dyer and Letourneau, 1999b). The leaves are large (most leaves fall within the range of 86–430 cm2 when fully expanded) and long-lived (approxi- mately 2 years), and opalescent food bodies are produced on the adaxial surface of sheathing leaf bases (petiolar cavities) when occupied by Pheidole bicornis ants. The herbivores most commonly found feeding on Piper ant plants at our study site are specialist lepidopterans and coleopterans. Feeding damage from generalist herbivores, including leafcutter ants (Hymenoptera: Formici- dae: Atta cephalotes) and orthopterans (Orthoptera: Tetigoniidae, Acrididae: Microtylopteryx hebardi, and Eumasticidae: Homeomastax robertsi) is occasion- ally found. The main herbivores that eat leaf tissue are geometrid moth larvae (Lepidoptera: Geometridae: Cambogia sp. and Eois sp.), skippers (Lepidoptera: Hesperiidae: Quadrus cerealis), saddleback caterpillars (Lepidoptera: Limacodi- dae), weevils (Coleoptera: Curculionidae: Ambates spp.), and at least 10 species of flea beetles (Coleoptera: Chrysomelidae: