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Herbivore-induced defenses in Passiflora biflora

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Title:
Herbivore-induced defenses in Passiflora biflora
Translated Title:
Inducidos por las defensas herbívoras en Passiflora biflora ( )
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English
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Morris, Melissa
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Plants--Insect resistance   ( lcsh )
Cyanides   ( lcsh )
Passiflora   ( lcsh )
Costa Rica--Puntarenas--Monteverde Zone   ( lcsh )
Plantas--Resistencia a insectos
Cyanides
Passiflora
Costa Rica--Puntarenas--Zona de Monteverde
Tropical Ecology Spring 2005
Ecología Tropical Primavera 2005
Genre:
Reports   ( lcsh )
Reports

Notes

Abstract:
Herbivore-induced defenses in plants save energy in the absence of herbivory (Karban et al. 1997). Despite this, Passiflora biflora has not been shown to have short-term induced defenses in past studies (Baptiste 2002). P. biflora might have a slower reaction to chronic herbivory either through greater leaf toughness or greater cyanide concentrations. To test this, leaves from three sites were assessed for herbivory defense. The first two sites were butterfly gardens with high and low populations of butterflies from the genus Heliconius, for whom Passiflora vines are the host plant. Defense levels were compared with nearby plants outside the garden at a third site. Light intensity, percent herbivory, leaf toughness, and cyanide were measured at each site to quantify herbivory defense. Light intensity was significantly higher in the garden with lower Heliconius populations (p=0.0086). Herbivory, measured by number of caterpillars encountered, was significantly higher in the garden with high Heliconius density. Leaf toughness was significantly lower at the roadside site (p=0.0231), but similar for the two butterfly gardens. Cyanide concentrations were significantly lower in the garden with fewer Heliconius, compared to the other two sites (p=0.002). While the cyanide concentration for vines outside the garden was higher, low leaf toughness there indicates younger vines, which are reported to have higher cyanide content (Magee 1995). Both gardens had similar leaf toughness, indicating similar age, yet significant differences in cyanide content. This implies that at higher and chronic levels of herbivory, Passiflora biflora shows induced defenses to herbivory.
Abstract:
Las defensas de las plantas inducidas por la herbivoría ahorran energía en ausencia de herbivoría (Karban et al. 1997). A pesar de esto, no se ha demostrado que Pasiflora biflora tenga ninguna defensa inducida a corto plazo (Baptiste 2002). Puede ser que P. biflora presente una reacción más lenta a la herbivoría crónica, ya sea debido a la mayor dureza de sus hojas o a sus mayores concentraciones de cianuro. Se recolectaron hojas de tres sitios diferentes y se analizaron para medir la defensa en la herbivoría. Los primeros dos sitios fueron jardines de mariposa con poblaciones altas y bajas de mariposas del género Heliconius para las cuales las enredaderas de Passiflora son las plantas anfitrionas. Los niveles de defensa se compararon con plantas en las afueras del jardín. La intensidad de la luz, el porcentaje de herbivoría, la dureza de las hojas, y la concentración de cianuro se midieron en cada sitio para cuantificar la defensa en la herbivoría. La intensidad de la luz fue significativamente más alta en el jardín con poblaciones bajas de Heliconius (p=0.0086). La herbivoría, medida de acuerdo con el número de orugas encontradas, fue significativamente más alta en el jardín con densidad alta de Heliconius. La dureza de las hojas fue significativamente más baja en el sitio al borde de la carretera (p=0.0231), pero este parámetro fue semejante en los dos jardines de mariposa. Las concentraciones del cianuro fueron significativamente más bajas en el jardín con menos Heliconius, en comparación a los otros dos sitios (p=0.002). Mientras que la concentración de cianuro para las enredaderas fuera del jardín fue más alta, una menor dureza de hojas en ese sitio indicó que las enredaderas allí eran más jóvenes y, por lo tanto, contenían una concentración más alta (Magee 1995). Los dos jardines tuvieron durezas de hojas semejantes, lo que indicó que tenían edades semejantes pero, sin embargo, presentaban diferencias significativas en su contenido de cianuro. Esto implica que a niveles más altos y crónicos de herbivoría, Pasiflora biflora muestra defensas inducidas a la herbivoría.
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Herbivore-induced defenses in plants save energy in the absence of herbivory (Karban et al. 1997). Despite this, Passiflora biflora has not been shown to have short-term induced defenses in past studies (Baptiste 2002). P. biflora might have a slower reaction to chronic herbivory either through greater leaf
toughness or greater cyanide concentrations. To test this, leaves from three sites were assessed for herbivory defense. The first two sites were butterfly gardens with high and low populations of butterflies from the genus Heliconius, for whom Passiflora vines are the host plant. Defense levels were compared with nearby plants outside the garden at a third site. Light intensity, percent herbivory, leaf toughness, and
cyanide were measured at each site to quantify herbivory defense. Light intensity was significantly higher in the garden with lower Heliconius populations (p=0.0086). Herbivory, measured by number of caterpillars encountered, was significantly higher in the garden with high Heliconius density. Leaf
toughness was significantly lower at the roadside site (p=0.0231), but similar for the two butterfly gardens. Cyanide concentrations were significantly lower in the garden with fewer Heliconius, compared to the other two sites (p=0.002). While the cyanide concentration for vines outside the garden was higher, low leaf toughness there indicates younger vines, which are reported to have higher cyanide content (Magee 1995). Both gardens had similar leaf toughness, indicating similar age, yet significant differences in cyanide content. This implies that at higher and chronic levels of herbivory, Passiflora biflora shows induced defenses to herbivory.
Las defensas de las plantas inducidas por la herbivora ahorran energa en ausencia de herbivora (Karban et al. 1997). A pesar de esto, no se ha demostrado que Pasiflora biflora tenga ninguna defensa inducida a corto plazo (Baptiste 2002). Puede ser que P. biflora presente una reaccin ms lenta a la herbivora crnica, ya sea debido a la mayor dureza de sus hojas o a sus mayores concentraciones de cianuro. Se recolectaron hojas de tres sitios diferentes y se analizaron para medir la defensa en la herbivora. Los primeros dos sitios fueron jardines de mariposa con poblaciones altas y bajas de mariposas del gnero Heliconius para las cuales las enredaderas de Passiflora son las plantas anfitrionas. Los niveles de defensa se compararon con plantas en las afueras del jardn. La intensidad de la luz, el porcentaje de herbivora, la dureza de las hojas, y la concentracin de cianuro se midieron en cada sitio para cuantificar la defensa en la herbivora. La intensidad de la luz fue significativamente ms alta en el jardn con poblaciones bajas de Heliconius (p=0.0086). La herbivora, medida de acuerdo con el nmero de orugas encontradas, fue significativamente ms alta en el jardn con densidad alta de Heliconius. La dureza de las hojas fue significativamente ms baja en el sitio al borde de la carretera (p=0.0231), pero este parmetro fue semejante en los dos jardines de mariposa. Las concentraciones del cianuro fueron significativamente ms bajas en el jardn con menos Heliconius, en comparacin a los otros dos sitios (p=0.002). Mientras que la concentracin de cianuro para las enredaderas fuera del jardn fue ms alta, una menor dureza de hojas en ese sitio indic que las enredaderas all eran ms jvenes y, por lo tanto, contenan una concentracin ms alta (Magee 1995). Los dos jardines tuvieron durezas de hojas semejantes, lo que indic que tenan edades semejantes pero, sin embargo, presentaban diferencias significativas en su contenido de cianuro. Esto implica que a niveles ms altos y crnicos de herbivora, Pasiflora biflora muestra defensas inducidas a la herbivora.
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Cyanides
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Costa Rica--Puntarenas--Monteverde Zone
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Cyanides
Passiflora
Costa Rica--Puntarenas--Zona de Monteverde
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Ecologa Tropical Primavera 2005
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Herbivore induced defenses in Passiflora biflora Melissa Morris Department of Biological and Environmental Sciences, University of Tennessee Chattanooga Abstract Herbivore induced defenses in plants save energy in the absence of herbivory (Karban et al. 1997). Despite this, Passiflora biflora has not been shown to have short term induced defenses in past studies (Baptiste 2002). P. biflora might have a slower reaction to chronic herbivory either through greater leaf toughness or greater cyanide conc entrations. To test this, leaves from three sites were assessed for herbivory defense. The first two sites were butterfly gardens with high and low populations of butterflies from the genus Heliconius for whom Passiflora vines are the host plant. Defen se levels were compared with nearby plants outside the garden at a third site. Light intensity, percent herbivory, leaf toughness, and cyanide were measured at each site to quantify herbivory defense. Light intensity was significantly higher in the gard en with lower Heliconius populations (p=0.0086). Herbivory, measured by number of caterpillars encountered, was significantly higher in the garden with high Heliconius density. Leaf toughness was significantly lower at the roadside site (p=0.0231), but s imilar for the two butterfly gardens. Cyanide concentrations were significantly lower in the garden with fewer Heliconius compared to the other two sites (p=0.002). While the cyanide concentration for vines outside the garden was higher, low leaf toughn ess there indicates younger vines, which are reported to have higher cyanide content (Magee 1995). Both gardens had similar leaf toughness, indicating similar age, yet significant differences in cyanide content. This implies that at higher and chronic le vels of herbivory, Passiflora biflora shows induced defenses to herbivory. Resumen Las defensas de las plantas inducidas por la herbivora ahorran energa en ausencia de herbivora (Karban et al. 1997). A pesar de esto, no se ha demostrado que Pasiflora biflora tenga ninguna defensa inducida a corto plazo (Baptiste 2002). Puede ser que P biflora presente una reaccin ms lenta a la herbivora crnica, ya sea debido a la mayor dureza de sus hojas o a sus mayores concentraciones de cianuro. Se recolect aron hojas de tres sitios diferentes y se analizaron para medir la defensa a la herbivora. Los primeros dos sitios fueron jardines de mariposa con poblaciones altas y bajas de mariposas del gnero Heliconius para las cuales las enredaderas de Passiflora son las plantas anfitrionas. Los niveles de defensa se compararon con plantas en las afueras del jardn. La intensidad de la luz, el porcentaje de herbivora, la dureza de las hojas, y la concentracin de cianuro se midieron en cada sitio para cuantifica r la defensa a la herbivora. La intensidad de la luz fue significativamente ms alta en el jardn con poblaciones bajas de Heliconius (p=0.0086). La herbivora, medida de acuerdo con el nmero de orugas encontradas, fue significativamente ms alta en e l jardn con densidad alta de Heliconius La dureza de las hojas fue significativamente ms baja en el sitio al borde de la carretera (p=0.0231), pero este parmetro fue semejante en los dos jardines de mariposa. Las concentraciones del cianuro fueron si gnificativamente ms bajas en el jardn con menos Heliconius en comparacin a los otros dos sitios (p=0.002). Mientras que la concentracin de cianuro para las enredaderas fuera del jardn fue ms alta, una menor dureza de hojas en ese sitio indic que l as enredaderas all eran ms jvenes y, por lo tanto, contenan una concentracin ms alta (Magee 1995). Los dos jardines tuvieron durezas de hojas semejantes, lo que indic que tenan edades semejantes pero, sin embargo, presentaban diferencias significa tivas en su contenido de cianuro. Esto implica que a niveles ms altos y crnicos de herbivora, Pasiflora biflora muestra defensas inducidas a la herbivora.

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Introduction Plants in the Tropics are subject to greater herbivory than those in temperate reg ions. This is because of an increased abundance of herbivores, and these herbivores are active yearlong. Plants have, therefore, evolved increased chemical, mechanical, and pheneological defenses against herbivory (Coley and Barone 1996; Coley and Aide 1 991). Defenses include delayed greening, increased leaf toughness, low nutritional levels, alkaloids, or secondary compounds, and extra floral nectaries. Induced defenses are beneficial to plant physiology because they do not waste energy when they are n ot needed. If no herbivory is present, the plant may redirect this energy from defense to growth and reproduction (Karban et al. 1997). For example, Asclepias spp (Asclepiadaceae) have higher concentrations of alkaloids if fed upon by Danaus plexippus mon arch larvae (Karban and Baldwin 1997). The passion vines in the genus Passiflora (Passifloraceae) are host plants for butterflies in the genus Heliconius (Gilbert 1983). Heliconius butterflies lay their eggs on Passiflora vines and growing larvae feed on the leaves. Passiflora have evolved ways to shape, and extra floral nectarines (Anderson 2002, Gilbert 1983). Vines have also adapted other defenses such as leaf toughness and cyanide content (Spencer 1988). The butterfly larvae, in turn, have evolved ways overcome the defenses and feed on the leaves. Induced cyanide production in Passiflora biflora has not been shown. A previous study measured the cyanide concen tration two days after artificially induced leaf damage (Baptiste 2002). It was thought that P. biflora might have a slower response to herbivore damage and increase defenses only if there is a larger or more chronic damage from herbivores. Here, I repor t that Passiflora biflora vines in an area with more Heliconius have higher concentrations of cyanide but equal greater leaf toughness, than do Passiflora biflora vines in an area with fewer Heliconius Methods Three sites were used for data collection i n this study. Two were enclosed gardens within the Monteverde Butterfly Garden in Monteverde, Costa Rica. Site 1, mimicking highland habitats, was a garden with low numbers of Heliconius spp. butterflies. Site 2, mimicking lowland habitats, had a much l arger population of Heliconius butterflies. While exact numbers of individuals of each species is unknown, observed Heliconius densities at Site 2 was obviously higher than at Site 1. Density of caterpillars at Site 2 was also much higher than at Site 1. Site 3 was located in a shaded open area next to a road across the street from the Monteverde Butterfly Garden, on the property of Bosque Eterno de los Nios. Passiflora biflora occurred in all three sites. Heliconius spp. butterflies occurred in both garden sites, with Heliconius dysonymus at Site 1 and Heliconius hewitsoni at Site 2. Data collection took place between 21 April and 09 May 2005. Light intensity was read at each site with a light meter, as it has been shown that increased light availab ility may increase cyanide production (Magee 1995).

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Two cm 2 sections of each of twenty leaves were collected from five vines in each of the three sites. Of 20 leaves, 10 were young (between the 1 st and 5 th leaves from the tip) and 10 were m ature (further than 10 leaves from the tip). To ensure consistency, the 1 st 4 th 10 th and 13 th leaves were always chosen if possible (plus or minus one leaf, if available leaf was absent or too small for cyanide detection). All leaves were placed in sep arate plastic bags. Percent herbivory was calculated using an herbivory grid. A transparent herbivory grid was placed on top of each leaf and an outline was drawn. Squares that were absent were counted and the squares of the leaf after herbivory were div ided from number of squares of entire original amount. In addition, I counted the number of caterpillars present on each of 20 vines from each site. Toughness was measured using a leaf penetrometer. Each leaf was placed in the leaf penetrometer and a vi le was placed on top. Water was added to the vile until the leaf penetrometer was able to penetrate the leaf. The water was then weighed and recorded. Cyanide was detected using a Sodium Picrate Test (Siegler 1991). This test involves crushing up the le af sample, placing it into a vile, and dropping three drops of Toluene on the crushed leaf. A 1 x 6 cm strip of Watman No. 1 filter paper that had been soaked in a sodium picrate solution was then placed in the vile above the leaf and held in place by the vile lid. The samples were then placed in a warm box where they were kept at about 33 C for 2 hours. Afterwards the strips, which change from yellow to purple in the presence of cyanide, were soaked for 30 seconds in distilled water. The color absorba nce, which indicates the amount of cyanide, was then read on a spectrophotometer set at 540nm. A low percent transmittance indicated a high concentration of cyanide. Cyanide concentration in g per 25 mg of leaf was calculated using a standard curve of c yanide concentration of percent transmittance (Figure 1). Results I then took the values and ran an Analysis of Variance (ANOVA) to see if the values between sites were significantly different. I ran the same test on the percent herbivory values, the le af toughness values, and light amount values. I then ran a Tukey Kramer HSD means of comparison for all variables, to see which sites were significant from each other. I ran a Chi square test on the caterpillar data to see the amount of caterpillars at t he garden with a large population of Heliconius butterflies was higher than expected when compared to the other sites. I found that the light intensity between sites was significantly different (F=6.63, df=2, P=0.0086). Site 1 had significantly higher lig ht intensity than Sites 2 and 3 (Tukey Kramer HSD, p<0.03?). Site 1 had an average light intensity of 12,894.67 Lux, while Site 2 had an average light intensity of 902.67 Lux, and Site 3 had an average light intensity of 3087.50 Lux (Figure 2). No signif icant difference was found between herbivory of the sites measured with the herbivory grid (F=0.0698, df=2, P=0.9327) (Figure 3). Because many Heliconius larvae eat entire leaves, I felt that the results did not accurately portray the actual herbivory. S ite 2 had many more fully eaten leaves that I was not able to sample. A Chi square test showed that the numbers of caterpillars were different between the sites (X 2 =140.92, df=2). Site 1 had 1 caterpillar on 299 leaves

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counted, Site 2 had 31 caterpillars on 133 leaves counted, and Site 3 had 0 caterpillars on 334 leaves counted (Figure 4). There were significant differences between the sites in average leaf toughness (F=4.0307, df=2, P=0.0231). A Tukey Kramer HSD showed that Site 1 had a significantly tougher leaves than Site 3. Site 2 however was not significantly different from Sites 1 or 3. Average leaf toughness at Site 3 was the lowest at 68.04 g. The average leaf toughness at Sites 1 and 2 were 100.95 g and 110.76 g respectively (Figure 5). C yanide concentrations were significantly different between sites (F=6.9572, df=2, P=0.002). A Tukey Kramer showed that Site 1 was significantly lower than Sites 2 and 3. The average concentration for Site 1 was the lowest 1.26 g/25mg of leaf. The avera ge for Site 2 was 5.78 g/25mg, and the average for Site 3 was nearly equal at 6.51 g/25mg (Figure 6). Discussion My results confirm that cyanide concentration is higher where plants are exposed to a larger number of herbivores. However, leaf toughness was not greater with larger populations of herbivores. While the roadside area had the greatest average cyanide concentration, it also had lowest average leaf toughness. These two aspects indicate that the vines on the roadside were younger. Young leav es of Passiflora normally have high cyanide content and low leaf toughness (Magee 1995; Tajiboy 1885). Vines at Site 3 were also shorter than the vines in the enclosed gardens, suggesting that they were younger. It is important to note that Site 1 had th e lowest cyanide concentrations even though light intensity was higher there. Site 2 had significantly more cyanide even with lower light. Similar leaf toughness, combined with significant differences in light intensity, herbivory, and cyanide concentrati on between Sites 1 and 2, confirm that the amount of herbivory does, in fact, impact the amount of cyanogenic compounds present in leaves. This study clearly demonstrates herbivore induced defenses in Passiflora biflora to differences in Heliconius herbi vory. While other plants might show a more immediate response to herbivory (Karban and Baldwin 1997), Passiflora biflora does show induced defenses to chronic herbivore attack. It would be beneficial for future studies to duplicate my study using the sam e species of Heliconius butterflies in all sites. This would control for any variations between interspecies behaviors. This could also indicate if Passiflora vines react differently to different species of Heliconius Other studies could include differ ent species of Passiflora vines to see if induced herbivory is species specific or shared among all species in the genus Passiflora A study of the time line of the induced defenses would also be helpful. It is known that defenses are not apparent in Pas siflora biflora within two days of leaf damage. However, studies measuring defenses at certain points within a time scale of two weeks, one month, or longer, might shed more light on the induced defenses of Passiflora biflora

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Acknowledgments First off I would like to thank the wonderful people at the Monteverde Butterfly Garden, especially Jim Wolfe, for letting me steal precious butterfly host leaves. Muchsimas gracias a Julio, en el Jardn de Mariposa, para ayudarme en la identificacin de vides de Passiflora Thanks also to Bosque Eterno de los Nios for letting me collect leaves from their property. Thanks also to the wonderful staff at C.I.E.E., especially Alan Masters for being patient with me. Also, thanks to Matt Gasner, Ollie Hymen, and Ja vier Mndez for not getting too annoyed with my constant questions about statistics and other random shit. And finally I would like to thank Jenn Toy for taking pictures of my worksites. Literature Cited Anderson, S.L. 2002. the evolutionary arms race: Coevolution of Heliconius and Passiflora Colorado State University Online. www.colostate.edu/Depts/Entomology/courses/en570/papers_2002/anderson.htm. Baptiste, K.K. 2002. Does herbivory induce cyanide production in Passifl ora biflora ? UCEAP IMV, Tropical Biology and Conservation: The sixteenth annual spring EAP symposium. Coley, P.D., and J. A. Barone. 1996. Herbivory and plant defenses in tropical forests. Annual Review of Ecological Systems. 27: 305 35. Coley, P.D., and T.M. Aide. 1991. Comparison of herbivory and plant defenses in temperate and tropical broad leaved forests. In Price, P.W., T.M. Levisohn, G.W. Fernandes, and W.W. Benson (Eds.). Plant Animal Interactions: Evolutionary Ecology in Tropical and Tempe rate Regions, pp. 25 49. John Wiley & Sons, Inc., New York, New York. Gilbert, L.E. 1983. Coevolution and Mimicry. In Futuyma, D.J., and M. Slatkin (Eds.). Coevolution, pp. 263 281. Sinauer Associates, Inc., Sunderland, Massachusetts. Karban, R., A. A. Agrawal, and M. Mangel. 1997. The benefits of induced defenses against herbivores. Ecology. 78(5): 1351 1355. Karban, R., and I.T. Baldwin. 1997. How a plant perceives damage and signals other ramets, and the specificity of these processes. Indu ced Responses to Herbivory. University of Chicago Press, Chicago, Illinois. Magee, A. 1995. The effects of microhabitat and leaf age on qualitative defenses in Passiflroa UCEAP IMV, Tropical Biology and Conservation: The sixteenth annual spring EAP sy mposium. Martin, M.S. 2001. Effect of altitude on cyanide concentration, leaf toughness, and herbivory in Passiflora biflora ( Passifloraceae ). CIEE Tropical Ecology and Conservation, pp. 13 19. Siegler, D.S. 1991. Cyanide and cyanogenic glycosides. In Rosenthal, G.A., and M.R. Berenbaum (Eds.). Herbivores: Their interactions with secondary plant metabolites, 2 nd Edtion, Volume I: The Chemical Participants, pp. 35 70. Academic Press, New York, New York. Spencer, K. C. 1988. Chemical mediation and c oevolution in Passiflora Heliconius interation. In Tajiboy, R. 1995. Comparison of qualitative and quantitative

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defenses in Passiflora biflora and Passiflora sexflora UCEAP IMV, Tropical Biology and Conservation: The sixteenth annual spring EAP symposiu m. Tajiboy, R. 1995. Comparison of qualitative and quantitative defenses in Passiflora biflora and Passiflora sexflora UCEAP IMV, Tropical Biology and Conservation: The sixteenth annual spring EAP symposium.

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Figure 1. Standard Curve of Cyanide Concentration to Percent Transmittance. Calculations of cyanide concentrations in g/25mg of plant material based on percent transmittance through a spectrophotometer. Equation for curve: y=101.88+( 20.706*LOG(x )) R 2 = 0.879. Percent transmittance ranged from 30.5% transmittance to 99.6% transmittance in 60 sampled leaves from P. biflora ___________________________________________ ___________________________ __ Figure 2. Measure of average light intensity among P. biflora at three study sites. Site 1 has a significantly higher average light intensity than Sites 2 and 3 (P = 0.0086). Site 1 has a large population of Heliconius butterfl ies, Site 2 has a low population, and Site 3 has an unknown population. Site 1 has an average Lux of 12, 894.67 Site 2 has an average Lux of 902.67 and Site 3 has an average Lux of 3087.50. _________________________________________________________________ _____________________

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Site 1 Site 2 Site 3 Number of Leaves Counted 299 133 334 Number of Caterpillars 1 31 0 Figure 3. Measure of average percent herbivory among P. biflora at three study sites. There is no s ignificant difference between averages at each site. Site 1 has a large population of Heliconius butterflies, Site 2 has a low population, and Site 3 has an unknown population. Site 1 has an average herbivory of 5.03%, Site 2 has an average herbivory of 4.03%, and Site 3 has an average herbivory of 3.88%. Number of Caterpillars Count ed Figure 4. Amount of caterpillars counted at each Site. Site 1 has a large Heliconius population, Site 2 has a low population, and Site 3 has an unknown population A Chi square test reveals that there is difference between the sites (X 2 = 140.92, df=2).

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Figure 5. Measure of average leaf toughness among P. biflora at three study sites. Site 3 has significantly lower leaf toughness average than the averages at Sites 1 and 2 (P=0.0231). Site 1 has a lar ge population of Heliconius butterflies, Site 2 has a low population, and Site 3 has an unknown population. Site 1 has an average leaf toughness (g) of 100.95g, Site 2 has average of 110.76g, and Site 3 has average of 68.04. Figure 6. Measure of average cyanide concentration (g/25mg) among P. biflora at three study sites. Site 1 has a signific antly lower cyanide concentration average than the averages at Sites 2 and 3 (P=0.002). Site 1 has a large population of Heliconius butterflies, Site 2 has a low population, and Site 3 has an unknown population. Site 1 has an average cyanide concentratio n of 1.26g/25mg, Site 2 has average of 5.78g/25mg, and Site 3 has average of 6.51/25mg.