Interciencia ISSN: 0378-1844 [email protected] Asociación Interciencia Venezuela

Castillo Guevara, Citlalli; Rico Gray, Víctor Is cycasin in eumaeus minyas (: ) a predator deterrent? Interciencia, vol. 27, núm. 9, septiembre, 2002, pp. 465-470 Asociación Interciencia Caracas, Venezuela

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CITLALLI CASTILLO-GUEVARA and VÍCTOR RICO-GRAY

arvae exhibit a wide quences for the biology and ecology of and Clark, 1991; Nash et al., 1992; range of defensive strate- these species. Many “chemically de- DeVries, 1994). However, this has not gies to avoid being eaten, fended” Lepidoptera are aposematic, and been tested and the life cycle of E. minyas e.g., mimetic coloration, shelter construc- store plant compounds that are known remains undescribed. We present the re- tion, unpalatability due to urticating hairs, vertebrate toxins, such as cardenolides sults of laboratory and field experiments spines and defensive glands, regurgitation, (Brower, 1984), (Rothschild et to evaluate the protective function of cyca- chemicals sequestered from host plants, se- al., 1979; Boppre, 1990; Montllor et al., sin in the aposematic E. minyas. cretion of volatiles, and noise production 1990), and cyanogens (Jones et al., In particular, the following questions are (see Brower, 1984; Bowers, 1993). Not 1962). Chemical defense of aposematic addressed: In which stages of its life cycle only do they use their bad taste or unpleas- has also been shown to be effec- does E. minyas contain cycasin? Does odor as a defense, they also announce it tive against invertebrate predators, which pure cycasin repel potential predators of to potential predators by means of a con- can learn to subsequently avoid similar E. minyas? Does cycasin turn eggs, larvae spicuous coloration, and gregarious and prey (Montllor and Bernays, 1993). and adults unpalatable? Is cycasin efficient sedentary behavior (Bowers, 1993). Seques- Cycasin is a secondary in protecting eggs and larvae from their tration of defense compounds from larval metabolite present in cycads (Cycadales), natural predators under natural conditions? host plants may require particular physi- belonging to the azoxyglycosid group This research is part of a wider study on ological by larvae to ingest, ac- (Matsumoto and Strong, 1963; Whiting, the interactions between E. minyas and E. cumulate, and store those compounds (Brat- 1963; Kobayashi and Matsumoto, 1965; debora and their host plants Z. loddigesii tsten, 1986; Bowers, 1992). Defense com- Morgan and Hoffman, 1983; Norstog and and D. edule. pounds are used for various purposes, par- Nicholls, 1997; Jones, 2000). Species of ticularly against predators, (Bowers, 1990; the American genera Zamia, Ceratozamia Materials and Methods Duffey, 1980; Blum, 1983; Brower, 1984). and Dioon (Cycadales: Zamiaceae) are Research on acquisition of chemical de- hosts to aposematic of the ge- Study site fenses by insects, particularly in Lepi- nus Eumaeus (Lepidoptera: Lycaenidae). It doptera, has been done using adult indi- has been demonstrated that E. atala se- Field work was accom- viduals. However, it is usually during the questers cycasin from Z. floridana, which plished in an oak forest near Chavarri- larval stage that chemical defenses, seques- is later used as a defense against both ver- llo, in central Veracruz, México (19º24’N, tered from host plants, are ingested, pro- tebrate (Bowers and Farley, 1990) and in- 96º48’W; 1000m altitude), characterized by cessed and stored (Bowers, 1993). Ex- vertebrate (Rothschild et al., 1986; Bowers a calcareous-derived soil and abundant rock amples of chemical defense of Lepidoptera and Larin, 1989) predators. These results outcrops. The climate is temperate-humid, larvae are well known (Brower, 1984; Bow- were based on laboratory experiments, but mean annual temperature is 24.5C°, total ers, 1990; Witz, 1990). no field research has confirmed them. It annual precipitation is ca. 1110mm, with a In aposematic species has also been suggested that cycasin rainy season between June and September unpalatability is coupled with a warning works in E. minyas as a defense mecha- and an extended six-month dry season coloration which can have many conse- nism, providing chemical protection (Clark (García, 1964; Soto et al., 1996). The veg-

KEYWORDS / Aposematic Butterfly / Cycads / Deterrent Effect of Cycasin / Lycaenidae / / Received: 03/01/2002. Modified: 07/30/2002. Accepted: 08/08/2002

Citlalli Castillo-Guevara. Ph.D. in Ecology and Management of Natural Resources. Address: Departamento de Ecología Vegetal, Instituto de Ecología, A.C., Apdo. 63, Xalapa, Veracruz 91070, México. e-mail: [email protected] Víctor Rico-Gray. Ph.D., Tulane University. Senior Research Scientist, Departamento de Ecología Vegetal, Instituto de Ecología, A.C. e-mail: [email protected]

SEP 2002, VOL. 27 Nº 9 0378-1844/02/09/465-06 $ 3.00/0 465 etation is characterized (Flores, 1995) by a dae, Fabricius 1804) as predators. This 0.075, p= 0.7877) were found in the num- mixture of oak forest [Quercus oleoides, Q. ant inhabits the study site and was ob- ber of visits between solutions (control, laurina, Q. peduncularis (Fagaceae), Nec- served on Z. loddigesii. Before the ex- X= 84.375 +5.123, N= 675; experimental, tandra sanguinea (Lauraceae), Bursera si- periments, were collected from a X= 86.875 +6.465, N= 695). maruba (Burseraceae)] and palm groves of colony in Francisco Javier Clavijero Bo- To test the defensive Brahea dulcis (Arecaceae). Epiphytes are tanical Garden (Xalapa, Veracruz, Méxi- function of cycasin under natural condi- present in the Araceae, Bromeliaceae, Or- co; 19º30’N, 96º57’W; 1280m altitude). tions, a predator exclusion experiment was chidaceae and Cactaceae (Castillo, 1985). The sample included workers, soldiers conducted at the study site. Egg clusters of The main shurbs and herbs are (Flores, and larvae in order to ensure that ant E. minyas were located on individuals of Z. 1995) Acacia cornigera, A. pennatula (Mi- behaviour was the least affected by sam- loddigesii and three predator exclusion mosaceae), Zamia loddigesii, and Dioon pling, (Jorge Valenzuela-González, per- treatments were applied: 1) fronds covered edule (Zamiaceae). sonal communication). Ants were placed with mesh, 2) fronds with a band of in 25 x 13cm plastic containers covered Tanglefoot® at the base, and 3) fronds with Species with mesh, and were deprived of food Tanglefoot® and mesh; the control were 48h before the experiments. The contain- fronds with egg clusters under natural con- Zamia loddigesii (Zami- ers were then uncovered and placed ditions. Different treatments were used to aceae, Miquel 1843) (voucher, L. M. White- within a square (0.26m2) of Tanglefoot® determine differential effects of the differ- lock 11/26/1963, XAL) is a small plant (up (Tanglefoot Co., Grand Rapids, MI, ent groups of predators (mainly for to 1m tall) with 1 to 6 fronds, inhabiting USA), where different food solutions and ants, but other flying and crawling in- tropical dry and deciduous forests and sec- were offered to the ants (see below). sects as well). Eighty-three eggs (total for ondary vegetation (Vovides et al., 1983). It To test if pure cycasin the study site at the time) grouped in egg is distributed along the coast of the Gulf of deterred S. geminata individuals, ants clusters located on 15 Z. loddigesii indi- Mexico up to Guatemala, from 0 to 1000m were offered two solutions, 1) control viduals were used, 32 on control plants and in elevation (Vovides et al., 1983; Jones, (0.75g sucrose + 0.30ml water), and 2) 51 on treatment plants (16 with mesh, 19 2000). Z. loddigesii is protected under the experimental (0.75 g sucrose + 0.30ml of with Tanglefoot®, 16 with Tanglefoot® and status of threatened species both interna- 1mg/ml cycasin in water). Cycasin was mesh). Over 15 days (the time for egg tionally (CITES Appendix II) and nation- the same as that used for chemical analy- hatching), the plants were visited every day ally (Annonymous, 1994). sis. The number of ant visits to each so- and the number of eggs preyed upon, Eumaeus minyas (Lycae- lution was recorded during 90 min. When hatched and unhatched was registered. The nidae, Hübner 1809) is distributed from an ant lowered its head and touched a so- exclusion experiment was continued apply- México to Costa Rica, where the larvae lution, it was considered a visit. The ex- ing the same treatments to the recently have been reported to consume fronds and periment was replicated eight times with hatched larvae, plus 15 more first-instar lar- female reproductive cones from Z. fur- different ants in the same conditions vae collected elsewhere in the study site. A furacea, Z. skinnery and Z. loddigesii (De- mentioned above. total of 70 larvae, 26 on control plants and Vries, 1976; 1983a; Clark and Clark, To test whether cycasin 44 on treatment plants (19 with mesh, 15 1991). E. minyas is considered aposematic, in eggs, larvae and adults rendered them with Tanglefoot®, 10 with Tanglefoot® and exhibiting a flashy warning coloration unpalatable and deterred S. geminata in- mesh) were observed. The number of lar- (DeVries, 1977; Clark and Clark, 1991; dividuals, individuals of E. minyas were vae that had disappeared, died or still sur- Nash et al., 1992). collected in the study site: 33 eggs vived was counted daily. To establish the (0.02g), 15 larvae of the four instars larval instar, larval length every third day Chemical analyses (0.70g), and 2 adults (0.20g). Extracts until pupation (ca. 16 days) was measured. were prepared with EtOH 70%, sepa- Observations were extended up to 32 days Chromatography on sili- rately for each stage. These were used to because different-aged cohorts were ob- ca-gel plates (Whatman PE SIL G/UV) prepare three experimental solutions served. The number of resulting pupae and was used to determine presence or ab- (0.75g sucrose + 0.30ml of egg, larval or of larvae that reached adulthood was also sence of cycasin in all stages of E. adult extract). Ants collected previously counted. minyas. From individuals collected in No- were offered four solutions, a control one vember 1999, we used 36 eggs (0.0293g), (0.75g sucrose + 0.30ml water), and three Statistical analyses 16 larvae of the four instars (2.045g), 1 experimental ones (with egg, larva or pupa (0.1039g), and 5 adults (0.5351g). adult extract). This experiment was also The total number of vis- The fresh samples were processed using replicated eight times, with different ants its to a particular solution was analyzed the technique described by Yagi et al. in the same conditions and the number of with a heterogeneity x2-test (Zar, 1999) to (1980) and Bowers and Larin (1989). For ant visits to each solution was recorded determine whether pure cycasin deterred comparison we used standard pure cycasin during 90 min trial periods. potential predators. The number of visits (Biochemical 66950), and computed the To test whether the to a particular solution were transformed reference factor (Rf) for each sample. presence of 70% ethanol used in the ex- (square root of X + 0.5) and then ana- Vouchers are deposited in a personal collec- traction procedure affected predator be- lyzed with a one-way ANOVA (Abacus tion at the Departamento de Ecología Vege- havior, or if it masked the presence or Concepts, Inc. 1996; Zar, 1999) to deter- tal, Instituto de Ecología, A.C. cancelled out the effect of cycasin, we mine whether the presence of cycasin in counted visits of individuals of S. butterflies offered protection. Field experiments geminata to a control (0.75g sucrose + To test for the efficiency 0.30ml water) and to an experimental of cycasin as a chemical defense of eggs Palatability experiments, solution (0.75g sucrose + 0.30ml 70% under natural conditions, the percentage modified from Bowers and Larin (1989) EtOH). The number of visits were trans- of surviving eggs was computed. To test were conducted using individuals of Sole- formed using the square root of X + 0.5, cycasin as a chemical defense of larvae nopsis geminata (: Formici- no significant differences (ANOVA, F1,14= in natural conditions, we used a ‘survival’

466 SEP 2002, VOL. 27 Nº 9 Figure 1. Mean (+SE) number of visits by experimental ant preda- Figure 2. Survival curves for E. minyas larvae with and without exclu- tor, Solenopsis geminata, to control (0.75g sucrose + 0.30ml water) sion of predators. Treatments of exclusion: mesh, tanglefoot, and experimental (0.75 g sucrose + 0.30ml of egg, larva and adult tanglefoot and mesh. Without exclusion: control. Time is period of lar- extracts of E. minyas) solutions. val state (16 days). Predation events with exclusion, Predation events without exclusion. or ‘failure time’ analysis (Muenchow, Adult butterflies of E. adult monarch butterflies by comparing 1986; Pyke and Thompson, 1986). The minyas oviposit on new fronds, of which the cromatographic separation of extracts analysis calculates the probability of the larvae may consume up to 90% during from insects and from food plants occurrence of predation events during one their development. Larvae may also for- (Harborne, 1988). observation period. The start of the obser- age on the raquis and, while uncommon, Visits of S. geminata vations was designated as time zero, and butterflies may also oviposit on female were significantly higher to the water/su- the predation events, disappearance of reproductive cones, which are also crose solution than to the water/pure cyca- larvae, were subsequently counted over harmed by larvae. sin solution (x2= 80.7249, p<0.001). Simi- 16 days. If a given larva was preyed Eggs are deposited in larly, significant differences (ANOVA, upon, it was considered an uncensored small clusters (mean of egg by cluster= F3,28= 11.591, p<0.0001) were found (Fig- data point, and, if predation never oc- 5.238 +0.547, N= 42), they are pale pink, ure 1) among ant visits to the different so- curred, i.e., the larvae survived, it was turning white after one day, and hatch af- lutions prepared with E. minyas extracts considered a censored data point. To ter approximately 11 days. Larvae are and the control solution: sucrose/water (X= compute the functions among variables bright red with seven transversal yellow 132.37 +19.77, N= 1059), egg extract (X= the product-limit Kaplan-Meier nonpara- stripes and undergo four instar changes in 43.50 +4.89, N= 348), larva extract (X= methric method (Abacus Concepts, Inc. 16 days. Pupae are orange with black 45.25 +14.57, N= 362), and adult extract 1996) was used, and the log-rank statistic marks, pupation lasting approximately 19 (X= 38 +8.79, N= 304). There were signifi- (Mantel-Cox) was used to test for differ- days. Larvae and pupae are gregarious cantly more visits to the water/sucrose solu- ences between the control and a given (DeVries, 1976; 1977; 1983b). The dis- tion (Fisher’s, p<0.001), and there were no treatment (treatments were grouped). An tinctive characteristics of adults are an or- significant differences among the other among-treatments comparison showed ange-red abdomen, and black wings with treatments (Fisher’s, p>0.05). that >20% of the events were censored metallic marks and white margins. It and, as suggested by Pyke and Thompson takes approximately 46 days from ovipo- In situ predator exclusion experiment (1986), instead of the survival analysis sition to adulthood. we used a 3x2 contingency table (Zar, No predation was found 1999). All results are shown as mean + Cycasin detection and palatability in the exclusions, as all of the 51 eggs in standard error. experiments exclusion survived. However, out of 32 eggs in control plants 22 (68.75%) sur- Results Cromatographic compari- vived and 10 (31.25%) were predated. son indicated that cycasin was detected in Three eggs fell from the frond due to Life history all stages of the life cycle of E. minyas: heavy rain, 15 did not hatch, and 55 standard Rf= 71.6; eggs= 71.0; larvae= hatched (i.e., 66% passed from egg to 1st The only herbivore ob- 63.1; pupa= 53.7; and adults= 63.3. instar larvae). Hatching time was 11 days served foraging on the fronds of Z. loddi- Variation in larvae and pupae were attrib- (X= 11.28 +0.39, N= 18), and it took gesii was E. minyas. During the rainy uted to differences in the molecular struc- from one to four days for all eggs in a season Z. loddigesii is frequently visited ture of cycasin due to metabolic changes cluster to hatch (X= 1.85 +0.27, N= 13). by E. minyas. Similar to other cycads within a stage. There are some differences Fifty six of the initial 70 (Norstog and Nicholls, 1997), Z. loddige- in pattern due to the metabolic conjugation larvae were killed. In 26 larvae of control sii grows slowly and irregularly (only a in the of some of the plant compo- plants, 25 (96.15%) were predated, and in few centimeters per year) and produces nents (Harborne, 1988). These variations 44 larvae of exclusion plants, 31 (70.45%) one to two fronds per year during the in Rf were also observed in evaluations in were predated. The number of surviving rainy season. the variations of cardenolide content of larvae (Figure 2) was significantly higher

SEP 2002, VOL. 27 Nº 9 467 2 (x 1= 4.72, p= 0.029) when predators and larvae were subject to attack by (Polis, 1981; Wagner and Wise, 1996). were excluded (N= 13) than when they fungi like Beauveria bassiana (Bals.) Cannibalism in groups of E. minyas was were not (N= 1). We found no significant Vuill., Deuteromycota. Egg mortality can also observed in Costa Rica and Panama; 2 differences (x 2= 1.31, p>0.05) in preda- also be attributed to factors intrinsic to those slowest to molt were eaten by the tion events among exclusion treatments the adult. For example, feeding quality fast-molting members (Phil DeVries, per- (mesh= 14, Tanglefoot®= 9, Tanglefoot®+ and quantity while larvae or adult can sonal communication). We suggest that mesh= 8). Only 14 (20%) of the original significantly influence egg production and cannibalism among E. minyas larvae is a 70 larvae pupated, and all but one pupa maturation (Scott, 1986; Braby and Jones, direct, easier and less energy-costly way reached adulthood, a process lasting 19 1995). The egg stage is critical for the to sequester cycasin. days (X= 19.44 +0.24, N= 16). future survival of an E. minyas indi- As toxin levels vary Cannibalism was an im- vidual, the more eggs that hatch into lar- considerably over short distances and portant factor in the survival of E. min- vae the higher the probability of surviv- through time, palatability to predators can yas. In one predator exclusion treatment ing the larval stage, which, no doubt, is also vary spatiotemporally (Harborne, (Tanglefoot® + mesh), 80% mortality was the most vulnerable and critical stage for 1999). Even though predators have devel- attributed to cannibalism. the survival of an E. minyas individual oped behaviors and physiological re- (96.15% predation in the control). sponses to recognize unpalatable prey and Discussion The mortality rate of thus decrease the effect of defenses (Cal- adults is important as every additional vert et al., 1979; Fink and Brower, 1981), The results show for the day of survival increases reproductive po- a decrease in availability of palatable first time that all stages of E. minyas tential (Brower, 1984). Predation was sig- prey may induce predators to forage on contain cycasin that is sequestered from nificant on 3rd-and 4th-instar larvae (be- less palatable prey (Alcock, 1970; Boy- its host plant Z. loddigesii. Moreover, it tween 8 and 16 days of development). den, 1976). Moreover, young predator in- is shown that pure cycasin and cycasin in Under natural conditions, predation was dividuals may sporadically forage on un- E. minyas (eggs, larvae and adult) can significantly higher on non-protected lar- palatable prey, or predators may forget deter a potential predator (S. geminata) vae. However, predation in 1st– and 2nd- a learned behavior (González et al., under laboratory conditions. This is simi- instar larvae did not differ between pro- 1967). lar to findings by Bowers and Larin tected and non-protected conditions, and It is not clear how cyca- (1989) using E. atala and the ant Campo- death of these larvae should be attributed sin works protecting lepidopterans against notus abdominalis floridanus (Hymenop- to factors other than predation. Younger predators. The responses in predators in- tera: Formicidae) which, however, were larvae are smaller and probably less con- clude those caused by cardiac glycosides not obtained under natural conditions. spicuous than older, larger, larvae (e.g., (vomiting) or pyrrolizidine alkaloids (bit- Our results show that predation in natural length of 3rd instar, X= 15.89mm +0.64, ter taste), which mask the real taste of conditions had a negative effect on the N= 42; 4th instar, X=21.97mm +0.64, N= cycasin (Nash et al., 1992). In addition, survival of E. minyas, suggesting that 32; C. Castillo-Guevara, unpublished). No the presence of pyrazines (in E. atala) some predators can avoid the protective significant differences were obtained can enhance the protection offered by cy- chemical barrier provided by cycasin. among exclusion treatments that would casin (Rothschild, 1984; Rothschild et al., Our results suggest a enable us to suggest the types of preda- 1986). Certain insects (e.g., Seiractia pattern. When the Rf of a sample (egg, tors involved, although a number of ant echo, Lepidoptera: Arctiidae) can circum- larva, pupa or adult) approaches that of species (Pheidole sp., Camponotus seri- vent the protective action of the cycasin the standard, the defense attributed to cy- ceiventris, Solenopsis geminata, and Ec- produced by Zamia floridana (Teas, casin increased, whereas when the Rf is tatomma tuberculatum, Hymenoptera: 1967), supporting the idea that no chemi- different the defense decreased. In the Formicidae), were collected in the study cal defense against predators is absolute predation experiment under natural condi- site. S. geminata and E. tuberculatum are (Steward and Keeler, 1988; Agrawal, tions it was evident that the egg stage is carnivorous and potential predators 1998). However, in the Eumaeus-Zamia the most protected and its Rf was similar (Valenzuela-González et al., 1995). interaction the level of cycasin is higher to the standard, whereas the Rf of the Each stage in the life in Eumaeus than in the host plant larval stage was lower than the standard, cycle of lepidopterans has a specific (Rothschild et al., 1986). Production of and predation of larvae was higher. Simi- guild of predators, and the mechanisms secondary metabolites is not related only lar experiments using pupae and adult used to avoid them seem to be mostly re- to defense against vertebrate or inverte- butterflies, are needed in order to evalu- lated to visually-hunting vertebrate preda- brate herbivores, the system is even more ate predation on these stages. tors (Rausher, 1980; 1981). However, the complex and evidence for these sub- The results of the preda- first instars of many lepidopterans are stances under natural conditions is cur- tion experiment suggest that the impact predated by a wide range of inverte- rently lacking and difficult to obtain. Fur- of predation was different for the differ- brates, mostly night feeders (Dempster, thermore, even though not tested, there ent stages. While predation on eggs (con- 1984), and thus and warning seems to be a close association between trol) amounted to 31.25%, predation on coloration may not be a good defense warning coloration and unpalatability in larvae was 96.15%. Furthermore, 21% of mechanism. E. minyas. First instar larvae are pale or- the eggs did not hatch, which is compara- Cannibalism was impor- ange, and after three days of feeding on tively low considering the reported 53% tant to the survival of E. minyas larvae, Z. loddigesii fronds and sequestering cy- non-hatched eggs for E. minyas in Costa especially when they were fully confined casin their color changes to bright red; Rica (Clark and Clark, 1991). Further- (Tanglefoot® + mesh treatment). Ingestion consequently, they are more conspicuous. more, eggs are subject to predation but of conspecifics can result in a direct and Specialization on the also to environmental factors. Some eggs crucial metabolic gain when lipid accu- Zamiaceae and the warning attributes in slid off from the fronds due to heavy mulation is not enough to support stress Eumaeus larvae may suggest a long asso- rain, eggs may dry up during days of ex- due to food shortage, but also in an indi- ciation through evolutionary time. How- treme high temperature (>30ºC), and eggs rect gain, the reduction of competitors ever, protection by cycasin in eggs and

468 SEP 2002, VOL. 27 Nº 9 larvae could have evolved associated to Bowers MD (1993) Aposematic caterpillars: life- Ecology and Natural History of a Neo- predators that are currently extinct and styles of the warnigly colored and unpalat- tropical Rain Forest. University Chicago able. In Stamp N, Casey TM (Eds.) Cater- Press. Chicago. pp. 187-194. thus extant predators are not so easily pillars. Ecological and Evolutionary Con- warned. Finally, we suggest that cycasin straints on Foraging. Chapman and Hall. Duffey SS (1980) Sequestration of plant natural in the aposematic butterfly E. minyas has London. pp. 331-371. products by insects. Ann. Rev. Entomol. 22: a defensive function against predators, 447-477. Bowers MD, Larin Z (1989) Acquired chemical however, its populations may be regulated defense in the lycaenid butterfly, Eumaeus Fink LS, Brower LP (1981) Birds can overcome by the negative effect of its predators, atala. J. Chem. Ecol. 15: 1133-1146. the cardenolide defense of monarch butter- which apparently avoid the chemical de- flies in Mexico. Nature 291: 67-70. Bowers MD, Farley S (1990) The behaviour of fensive barrier. grey jays, Perisoreus canadensis, towards Flores PA (1995) Biología reproductiva de palatable and unpalatable Lepidoptera. Anim. Ryncholaelia glauca (Lindl.) Schltr. (Orchi- ACKNOWLEDGEMENTS Behav. 39: 699-705. daceae: Laeliinae) en un encinar del centro de Veracruz. Thesis. Universidad Veracruza- Boyden TC (1976) Butterfly palatability and na-Xalapa. México. 50 pp. The authors thank J. : experiments with Ameiva lizards. López-Portillo, J.G. García-Franco, V. Evolution 30: 73-81. García E (1964) Modificaciones al Sistema de Parra-Tabla and J.F. Ornelas for their Clasificación Climática de Koeppen (para Braby MF, Jones RE (1995) Reproductive pat- adaptarlo a las condiciones de la República suggestions to earlier drafts of the terns and resource allocation in tropical but- Mexicana). Universidad Nacional Autónoma manuscript; S. Cervantes and V. Vázquez terflies influence of adult diet and seasonal de México, México. 246 pp. for their help with field and laboratory phenotype on fecundity, longevity and egg size. Oikos 72: 189-204. González RC, Behrend ER, Bitterman ME work, G. Carrión for fungi identification; (1967) Reversal learning and forgetting in and J. Valenzuela-González for ant iden- Brattsten LB (1986) Fate of ingested plant birds and fish. Science 158: 519-521. tification and suggestions for experi- allelochemicals in herbivorous insects. In Brattsten LB, Ahmad S (Eds.) Molecular as- Harborne JB (1988) Introduction to ecological ments. We specially thank C. Lara for biochemistry. Academic Press. San Diego. his help in the field and for his sugges- pects of insects plant associations. Plenum. New York. pp. 211-255. pp. 356. tions for statistical analyses. 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