en-US Leaf Toughness and Cyanide Defense of Passiflora dioscoreifolia in Varied Light Habitats en-US en-US James Bellush en-US en-US Department of Biology, Moravian College en-US en-US ABSTRACT en-US en-US Coevolution can lead to complex biological relationships between tropical plants and herbivores, as with Passiflora (Passifloraceae) vines and heliconiine butterflies (Nymphalidae: Heliconiiae). Constant herbivory from heliconiine butterflies has led to the development of cyanide-based defensive compounds by Passiflora , in addition to the more general defense of toughening leaves, found in all plants. To observe the effect of light availability on leaf toughness and cyanide content in Passiflora dioscoreifolia, leaves from twenty plants in sun and shade habitats were sampled in the forests of Monteverde, Costa Rica and tested for their leaf toughness and cyanide content. Plants in the sun habitat possessed more expensive defensive compounds, such as lignin and cellulose. Plants with a reduced photosynthetic capacity in the shade habitat possessed less costly compounds, such as cyanide. Leaves are tougher in sun habitat plants, providing protection against all herbivores, including Heliconius . Shade habitat leaves are less tough, thus requiring additional cyanide for protection. Resources, such as light, impact allocation of resources for defense compounds in the dynamic coevolutionary relationship between Passiflora and Heliconius. en-US en-US INTRODUCTION en-US en-US Resource allocation for plants is a constant cost-benefit analysis, taking into account available resources to be budgeted for immediate and future metabolic needs. The theory of resource allocation states that investment of resources from primary production (photosynthesis) should allow for maximized growth and fitness (Bloom et al. 1985). Sufficient resources need to be allocated toward present growth and reproduction as well as protection against agents who can inhibit future growth, such as herbivores. Herbivory is especially prevalent in tropical forests, where, on average,10% of the annual plant production is damaged (Coley et al. 1985). The targets of insect herbivores are younger plant leaves, which are more preferred over older leaves (Thomas 1987). Therefore energy is directed towards the production of various defense compounds. Various selective pressures and resource limitations, such as light, determine the differences in these defense compounds among specific plants (Janzen 1974). en-US The understory habitat of Passiflora dioscoreifolia , is characterized by low light. The average photon flux density (PFD) reaching plants in a neotropical forest is less than 0.5% full sunlight (Rundel & Gibson 1998). Most light comes as fleeting sunflecks, severely inhibiting photosynthetic activity. Therefore, light availability for an understory plant such as P. dioscoreifolia can have significant effects on the photosynthetic capacity. en-US I measured defensive compounds in leaves of P. dioscoreifolia for two different light habitats, one shady and one sunny, in order to determine if there are differences in defensive compounds related to light availability. Plants in the sun will have more photosynthetic output, so that more costly lignins and cellulose can be used to increase leaf toughness. Leaf toughness has been shown to be the most effective defense against
en-US insect herbivory (Sagers & Coley 1995). Shade plants, on the other hand, will likely utilize cheaper compounds, such as cyanide and monoterpenes. en-US In the context of the coevolutionary relationship between Heliconius and Passiflora , certain plants could be left more vulnerable to herbivory due to the allocation of resources towards defense compounds based upon light availability. en-US en-US METHODS en-US en-US Study Organism en-US The study organism that I have chosen to use is Passiflora dioscoreifolia , an understory vine common in many neotropical forests. Passiflora occurs in tree fall gaps and also along forest edges. Like all plants, passion vines use toughness to protect themselves against generalist herbivores but also have developed a cyanide-based defense. Cyanide is concentrated in youngest leaves and is lost as leaves age and toughen (Gleadow & Woodrow 1999). In addition, passion vines show a host of leaf shapes, egg mimic glands, and EFNs that attract ants. These are believed to be coevolutionary responses to heliconiine butterflies, who have broken through the cyanide defense with enzyme inhibitors. en-US en-US Study Site en-US Plants were collected from the forest behind the Monteverde Biological Station Forest, which is classified as a premontane moist/wet transition zone. Light availability was measured by use of a canopy densiometer. A sunny habitat was one that allowed 65% of the light to penetrate the canopy whereas a shady habitat allowed less than 35% of light to penetrate the canopy. From each of these two habitats, 10 plants were sampled. Only healthy plants were sampled; plants that were badly herbivorized or had leaves that were not fully formed were not sampled. Also plants of intermediate canopy cover were not used in the study. From each plant, I collected one leaf representative of three distinct relative ages; newly formed leaves, mature leaves, and old leaves. Newly formed leaves were the first fully formed leaves to open at the terminal end of the vine. Mature leaves were defined as the first full sized leaves below the newly formed leaves. Older leaves were characterized as being fully mature and are located near the bottom of the vine, farther from the terminal end of the vine than the mature leaves. A total of 20 vines were sampled resulting in the collection of 60 leaves. en-US en-US Leaf Toughness and Cyanide Tests en-US Two tests were run for each of the three leaves that were collected from each of the plants. The first test was to determine leaf toughness and the second test was to determine the cyanide content of the leaf. Leaf toughness was determined using a leaf penetrometer. The leaf was placed in the penetrometer and a 9-gram small container was placed on top of the penetrometer while water was slowly added until the leaf broke under the weight of the container. The combined weight of the water in grams (1 mL=1 g) in addition to the 9 grams of the container was recorded as the leaf toughness. The cyanide test was implemented by first creating a sodium picrate stock solution, which contained 2.5 grams of sodium carbonate and 1 gram of moist 0.5% wt. vol. picric acid. Water was added for a total volume of 100 mL. Picrate paper strips were cut from filter
en-US paper, 9 mm x 1 mm, and were later dipped in the sodium picrate solution prior to testing. 0.1 grams of fresh tissue was cut from each leaf and was initially macerated in a small test tube for 20 seconds. Three drops of toluene were added to the leaf, at which point it was macerated again for 25 seconds. A moist picrate paper was positioned inside the vial so that it was not touching the leaf tissue at the bottom and was hanging by nearly half an inch out of the top. The test tube was then quickly capped, covered with aluminum foil, and incubated for 5 hours in a 35 C oven. In the presence of cyanide, the papers turned from yellow to orange/brown, depending on the content of cyanide. The picrate papers were dipped in cuvets filled to 5 mL with water, 10 times to leach the color into the water. The cuvets were agitated and measured in the spectrophotometer at 510 nm (Bradbury et al. 1999, Egan et al. 1998). The percent transmission was recorded and converted into absorbance by way of equation 1. The total cyanide content was calculated in ppm by way of equation 2 (Bradbury et al. 1999, Egan et al. 1998). en-US 1) A= 2log(T): A=absorbance en-US 2) Total cyanide (ppm)= 396(A)(100/z); where z=weight of fresh tissue used (mg) en-US en-US en-US RESULTS en-US en-US Leaf Toughness en-US The study species P. dioscoreifolia showed mean leaf toughness values that varied significantly between light habitats (Fig. 1). It was proposed that leaves from plants in sun habitats would have greater photosynthetic capacity to produce costlier compounds, such as lignin and cellulose, to increase leaf toughness as a defense against herbivores. The data supported this hypothesis. Leaves from plants sampled in sun habitats had significantly greater leaf toughness than leaves sampled from plants in shady habitats (2way ANOVA, F=20.14, df=59, p<0.001). The sun plants possessed a mean toughness of 89.02 grams ( 19.63, s.d.) whereas shade plants measured a mean toughness of 70.78 grams ( 17.85, s.d.), which is a 20.6% reduction. As expected, new leaves (59.81 17.85) were less tough than mature leaves (82.99 13.63) and the oldest leaves were found to be the toughest (96.90 13.72) in both light habitats (2-way ANOVA, F=54.58, df=59, p<0.001). en-US en-US Cyanide Content en-US The cyanide content of the leaves also varied significantly between light habitats (Fig. 2). As was hypothesized, the leaves from plants in shade habitats possessed higher cyanide content than leaves in sun habitats (2-way ANOVA, F= 41.95, df= 59, p < 0.001). The mean cyanide content for the shade habitat plants was 24.40 ppm ( 16.31) whereas the mean cyanide content for the sun habitat plants was 5.40 ppm ( 4.23), which is a 352% reduction in cyanide content across habitats. This trend indicates that plants in resource limiting habitats, such as lower light, have lower photosynthetic capacities and must invest in low construction cost compounds such as cyanide for defense compounds. It was found that older leaves (5.116 5.66) had less cyanide than mature leaves en-US en-US en-US
en-US en-US (14.00 12.03) and newer leaves possessed the highest cyanide content (25.58 22.62) between both habitats (2-way ANOVA, F=12.77, df=59, p<0.001). en-US en-US DI SCUSSION en-US en-US Effects of Light Habitat on Leaf Toughness and Cyanide en-US The trend of leaf toughness and cyanide content of the leaves from P. dioscoreifolia growing in varied light habitats strongly supports the hypothesis that resource availability determines the amount and type of defense compound used by plants (Coley et al. 1985). The data show that the higher photosynthetic capacity of the sun habitat plants allows them to allocate more energy to higher cost defense compounds. Compounds such as tannins, lignin, and cellulose are responsible for toughness and the fact that leaves were tougher in the sun suggests that plants had more energy to invest in them. The data also show that plants growing in shade possess leaves with high cyanide contents, reflecting their low photosynthetic capacity and necessity to allocate fewer metabolic resources to defense compounds. Cyanide is a cheaper alternative to high molecular weight defense compounds such as lignin and cellulose. Lack of lignin and cellulose in leaves of shade plants correspond to reduced leaf toughness as compared to leaves of sun habitat. _____________________________________________ Figure 1. Mean leaf toughness ( SD) of new, mature, and old leaves of Passiflora dioscoreifolia in varied light habitats in Monteverde Cloud Forest. Sun habitats were characterized as having over 65% of light reaching the understory. Shade habitats were characterized as having 35% of light reaching the understory. Lines over the ba rs indicate that new leaves were different from mature and old leaves (Tukey Kramer HSD Test, q=2.41, p < 0.005). ________________________________________ Figure 2. Mean cyanide content ( SD) of new, mature, and old leaves of Passiflora dioscoreifoli a in varied light habitats (same light parameters as Figure 1). Lines over bars indicate that new leaves were different from mature and old leaves (Tukey Kramer HSD Test, q=2.41, p < 0.05) ________________________________________.
en-US Prior studies also show the cost-benefit analysis trend observed in the study of P. dioscoreifolia. Plants with higher photosynthetic rates and more resources are able to devote more energy to costlier immobile defenses, such as tannins and lignin (Coley 1988). With more resources, the plant has a greater budget to invest more energy into expensive defenses, which provides better protection against herbivory (Coley et al. 1985, Hemming & Lindroth 1999). Limited light availability in shade plants decreases photosynthetic rate, lowering the budget of available energy to invest in costly defense compounds. Therefore, the limited metabolic resources are invested in cheaper mobile defenses, such as cyanide (Coley 1988). en-US en-US Effect of Leaf Age on Leaf Toughness and Cyanide en-US Previous studies have also found an increase in leaf toughness as a function of age (Lowman & Box 1983). Because the older leaves were tougher and more effectively protected against herbivory, there was no need to allocate resources for mobile defenses into older leaves (Sagers & Coley 1995). The data indicating lower cyanide content in older leaves also supports this hypothesis (Fig. 2). The newer leaves of P. dioscoreifolia were shown to contain higher contents of cyanide due to their reduced toughness and consequently increased vulnerability to herbivory. en-US The findings from this study illustrate the allocation of energy towards defense compounds of P. dioscoreifolia based upon light availability . The amount of energy that a plant can invest in defense compounds is directly related to the photosynthetic rate. Higher light availability and a greater subsequent amount of energy leads to investment in costlier immobile defensive compounds. Limited light availability and fewer resources in the metabolic budget of the shade plants resulted in their production of cheaper mobile defense compounds, such as cyanide. en-US en-US Impact of Resource Availabilty and Allocation: Growth and Herbivory en-US The findings from this study implicate the major impact that resource availability exerts on plant defense compounds. The results provided insight into cost-benefit analysis and metabolic tradeoff of Passiflora spp . Understory plants are characterized as slow growers, due to their limited sunlight and low rate of leaf turnover (Bradshaw et al. 1964). For such understory plants with limited light, it is more cost effective to invest in immobile defenses. These plants incur a high initial cost, however they avoid continual cost of reinvestment of resources into defense. Slow growers tend to have leaf life spans three times longer than fast growing gap species, therefore investing in the protection of their leaves is of crucial importance (Coley 1988). Investing in costly defense compounds can decrease growth by up to 50%, resulting in the same relative decrease in growth that herbivore damage can cause (Coley 1988). en-US The cost-benefit analysis scenario of whether to invest in costly plant defense compounds or suffer herbivory by investing in cheaper compounds could have implications as to the allocation of resources by Passiflora . Sun plants were found to have costly defense compounds, which are proven to be more effective against herbivory. Since Passiflora are slow growing understory vines, the decrease in growth by investing in costly defense compounds will not be as significant as it would be on a fast growing light gap species (Coley 1988). Therefore, its inherent slow growth has encouraged high investment in defense compounds when resources are available to support the investment.
en-US It is more cost effective for these sun plants to invest in costly compounds to prevent herbivory, at the cost of a minor decrease in growth. en-US Shade plants do not have the ability to produce costly immobile defense compounds. With a bare minimum of light to sustain basic metabolic activities, these plants are not capable of any additional investments of energy. Coley (1988) found that fast growing gap specialists utilized cheaper mobile defense compounds, in order to devote more energy into growth. However, with a meager metabolic budget, the shade plants were forced to invest in the cheapest possible defense. The less costly cyanide is ab le to deter general herbivores, however it is ineffective against its current predator, heliconiine butterflies. en-US Investing in costly defense compounds could have as great an effect on growth as herbivory. Costly defense compound investment however does not have as significant an effect on growth in slow growing, understory species. Overall, the effect of defense compounds on growth is significantly less than effects of resource availability (Grime 1977). Aside from limited resources, the main threat to growth of such slow growing, understory species, such as Passiflora , is herbivory. Plants in high light habitats can allocate more energy towards costly defense compounds that better protect their leaves against herbivory. Limited light plants can only afford to invest in cheaper defense compounds, such as cyanide. This system of resource allocation towards defense compounds could impact future plant-herbivore interactions. If such plants are vulnerable to herbivory with these cheaper defense compounds by limited resource habitat plants, such as Passiflora , than selective pressures will drive the adaptation of more effective defense mechanisms. en-US en-US en-US ACKNOWLEDGMENTS en-US en-US I would like to thank my project advisor, Alan Masters, for all of his guidance and assistance during my study. I thank Jose Carlos Calderon and Pablo Allen for all their help and I would also like to thank Danny Whonsetler and Lizzie Schiller for assisting in the taking of pictures in the field. en-US en-US LITERATURE CITED en-US en-US Bloom, A.J., F.S. Chapin, and H.A. Mooney. 1985. Resource limitation in plants: an economic analogy. Annual Review of Ecology and Systematics 16: 363-392. en-US Bradbury, M.G., S.V. Egan, and J.H. Bradbury. 1999. Picrate paper kits for determination of total cyanogenesis in cassava roots and all forms of cyanogens in cassava products. Journal of the en-US Science of Food and Agriculture 79: 593-601. en-US Bradshaw, A.D., M.J. Chadwick, D. Jowett, and R.W. Snydon. 1964. Experimental investigations into the mineral nutrition of several grass species. Journal of Ecology 52: 665-676. en-US Coley, P.D., J.P. Bryant, and F.S. Chapin. 1985. Resource availability and plant anti-herbivore defense. en-US Science 230 (4728): 895-899. en-US Coley, P.D. 1988. Effects of plant growth rate and leaf lifetime on the amount and type of anti-herbivore defense. Oecologia 74: 531-536. en-US Egan, S.V., H.H. Yeoh, and J.H. Bradbury. 1998. Simple picrate paper kit for determination of cyanogenic potential of cassava flour. Journal of the Science of Food and Agriculture 76: 39-48. en-US Gleadow, R.W. and I.E. Woodrow. 1999. Temporal and spatial variation in cyanogenic glycosides in Eucalyptus cladocalyx . Tree Physiology 20: 591-598.
en-US Grime, J.P. 1977. Evidence for existence of three primary strategies in plants and its relevance to ecological and evolutionary theory. American Naturalist 111: 1169-1194. en-US Hemming, J.D.C. and Lindroth, R.L. 1999. Effects of light and nutrient availability on Aspen: growth, phytochemistry, and insect performance. Journal of Chemical Ecology 25: 1687-1714. en-US Janzen, D.H. 1974. Tropical blackwater rivers, animals, and mast fruiting by the Dipterocarpaceae. Biotropica 6: 69-103. en-US Lowman, M.D. and J.D. Box. 1983. Variation in leaf toughness and phenolic content among five species of Australian rain forest tress. Australian Journal of Ecology 8: 17-25. en-US Rundel, P.W., M.R. Sharifi, A.C. Gibson, and K.J. Esler. 1998. Structural and physiological adaptation to en-US light environments in neotropical Heliconia (Heliconiaceae). Journal of Tropical Ecology 14: en-US 789-801. en-US Sagers, C.L. and Coley, P.D. 1995. Benefits and costs of defense in a neotropical shrub. Ecology 76: en-US 1835-1843. en-US Thomas, C.D. 1987. Behavioral determination of diet breadth in insect herbivores: the effect of leaf age on choice of host species by beetles feeding on Passiflora vines. Oikos 48: 211-216.
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Hoja de resistencia y defensa del cianuro de Passiflora dioscoreifolia en hbitats variados de luz
Leaf Toughness and Cyanide Defense of Passiflora dioscoreifolia in Varied Light Habitats
Coevolution can lead to complex biological relationships between tropical plants and herbivores, as with Passiflora (Passifloraceae) vines and heliconiine butterflies (Nymphalidae: Heliconiiae). Constant herbivory from heliconiine butterflies has led to the development of cyanide-based defensive compounds by Passiflora, in addition to the more general defense of toughening leaves, found in all plants. To observe the effect of light availability on leaf toughness and cyanide content in Passiflora dioscoreifolia, leaves from twenty plants in sun and shade habitats were sampled in the forests of Monteverde, Costa Rica and tested for their leaf toughness and cyanide content. Plants in the sun habitat possessed more expensive defensive compounds, such as lignin and cellulose. Plants with a reduced photosynthetic capacity in the shade habitat possessed less costly compounds, such as cyanide. Leaves are tougher in sun habitat plants, providing protection against all herbivores, including Heliconius. Shade habitat leaves are less tough, thus requiring additional cyanide for protection. Resources, such as light, impact allocation of resources for defense compounds in the dynamic coevolutionary relationship between Passiflora and Heliconius.
La coevolucin puede conducir a las complejas relaciones biolgicas entre las plantas tropicales y los herbvoros, como las enredaderas de Passiflora (Passifloraceae) y las mariposas heliconiine (Nymphalidae: Heliconiiae). La herbivora constante de las mariposas heliconiine ha llevado al desarrollo de compuestos defensivos a base de cianuro por Passiflora, adems de la defensa general de las hojas de endurecimiento, que se encuentra en todas las plantas. Para observar el efecto de la disponibilidad de luz sobre la dureza foliar y el contenido de cianuro en Passiflora dioscoreifolia, se tomaron muestras de veinte hojas de los hbitats de sol y sombra en los bosques de Monteverde, Costa Rica y la prueba de su tenacidad a la hoja y contenido de cianuro. Las plantas en el hbitat donde posee compuestos defensivos ms costosos, como la lignina y la celulosa. Las plantas con una capacidad reducida de fotosntesis en el hbitat de sombra poseen compuestos menos costosos, tales como el cianuro. Las hojas son ms duras en las plantas del hbitat de sol, proveyendo proteccin contra todos los herbvoros, como Heliconius. Las hojas del hbitat de sombra son menos resistentes, por lo que requiere proteccin adicional de cianuro. Los recursos, como la asignacin de efectos de luz, de los recursos para los compuestos de defensa en la relacin dinmica entre la co-evolucin Passiflora y Heliconius.
Text in English.
Costa Rica--Puntarenas--Monteverde Zone
Costa Rica--Puntarenas--Zona de Monteverde
Tropical Ecology Summer 2010
Ecologa Tropical Verano 2010
t Monteverde Institute : Tropical Ecology