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Distribution and anti-herbivoral role of extra extrafloral nectaries and leaflet pair number on Inga sierrae (Fabaceae: ...

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Title:
Distribution and anti-herbivoral role of extra extrafloral nectaries and leaflet pair number on Inga sierrae (Fabaceae: Mimosaceae)
Translated Title:
Distribución y función anti-herbivoral de nectarios extraflorales y número de parejas en las hojuelas en los árboles de Inga sierrae (Fabaceae: Mimosaceae) ( )
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text in English
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Gough, Andraya
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Subjects / Keywords:
Herbivory
Herbivoría
Plants--Insect resistance
Plantas--Resistencia a insectos
Plants--Adaptation
Plantas--Adaptación
Monteverde Biological Station (Costa Rica)
Estación Biológica de Monteverde (Costa Rica)
Tropical Ecology Spring 2003
Ecología Tropical Primavera 2003

Notes

Abstract:
The purpose of this project is to investigate the distribution and anti-herbivore impact of extra-extrafloral nectaries (EEFNs) and leaflet pair number on Inga sierrae (Fabacaeae: Mimosaceae) in Monteverde, Costa Rica. I recorded the number of EEFNs and leaflet pairs per leaf for forty leaves collected from sixty trees from three different locations. EEFNs, though not commonly found on I. sierrae, did significantly reduce the percent herbivory on leaves (1-way ANOVA, P < 0.0001 and Exponential Regression Analysis; R2 = 0.0001). The most commonly encountered leaflet pair number was three. However, when the number of leaflet pairs decreased, percent herbivory increased (Exponential Regression Analysis, R2 = 0.9905, P < 0.0001 and 1-way ANOVA, P < 0.0001). Heavy herbivory pressure could be causing a species change toward increased production of EFNs.
Abstract:
El objetivo de este proyecto es investigar la distribución y el impacto anti-herbívoro de los nectarios extra extraflorales (EEFNs) y números de parejas de hojuelas en los árboles Inga sierrae en Monteverde, Costa Rica. Yo documenté el número de EEFNs en parejas de hojuelas por hoja para cuarenta hojas recogidas de sesenta árboles de tres localidades diferentes. EEFNs, aunque no se encuentran muy a menudo en I. sierrae, reducen significativamente el porcentaje del daño por herbíveros de las hojas. Más frecuentemente el número de parejas de hojuelas encontrado por cada hoja fue tres. Sin embargo, cuando este número disminuyó, el porcentaje de daño por herbíveros aumentó y vice versa. Mucho daño por herbíveros podrían causar un cambio dentro de la especies hacia una producción aumentada de EEFNs.
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Student affiliation: Department of Biology, Occidental College

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Distribution and Anti herbivoral Role of Extra Extrafloral Nectaries and Leaflet Pair Number on Inga sierrae (Fabaceae: Mimosaceae) Andraya Gough Department of Biology, Occidental College _____________________________________________________________________________________ Abstract The purpose of this project is to investigate the distribution and anti herbivore impact of extra extrafloral nectaries (EEFNs) and leaflet pair number on Inga sierrae (Fabacaeae: Mimosaceae) in Monteverde, Costa Rica. I recorded the number of EEFNs and leaflet pairs per leaf for forty leaves collected from sixty trees from three different locations. EEFNs, though not commonly found on I. sierrae did significan tly reduce the percent herbivory on leaves (1 way ANOVA, P < 0.0001 and Exponential Regression Analysis; R 2 = 0.0001). The most commonly encountered leaflet pair number was three. However, when the number of leaflet pairs decreased, percent herbivory incre ased (Exponential Regression Analysis, R 2 = 0.9905, P < 0.0001 and 1 way ANOVA, P < 0.0001). Heavy herbivory pressure could be causing a species change toward increased production of EFNs. Resumen El objecto de este proyecto es investigar la distribucin y el impacto de anti herbivore de los nectararios extraflorales (EEFNs) y nmeros de hojuela de parejas en los rboles de Inga sierrae en Monteverde en Costa Rica. Yo document el nmero de EEFNs y hojuela de pareja por hoja para cuarenta hojas que yo recoj desde seisenta rboles de tres localidades diferentes. EEFNs aunque no son comunes a encontrar en I. sierrae reducen significamente el porcentaje de la herbividad de las hojas. Ms frequentemente el nmer o de las hojuelas de parejas por cada hoja fue tres, aunque, cuando el nmero disminuio, el percentaje de herbividad aument y el opuesto ocurri tambin. Herbividad pesado pueden ser causando un cambio de especies para aumentado producion de EFNs. Introd uction Herbivory can be physiologically costly to plants both in the short term and long term. Herbivores, and in particular folivores, decrease the fitness of plants by destroying their photosynthetic surfaces (Raven et al. 1991). Because plants are stat ionary, they must develop physical, chemical or biotic defenses in response to herbivore damage (Koptur 1991). Biotic defenses, including both dynamic and complex mutualisms, have not been studied as extensively. Inga spp. are tall, leguminous, sub canop y trees commonly found in disturbed areas such as pastures and along forest edges only on the Pacific side of Costa Rica (Bello et al. 2000). The leaves are alternate, parapinnate, and compound with 2 4

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Opposite leaflet pairs, approximately 12 x 18 cm (Be llo et al. 2000). Inga sierrae has a winged rachis and when mature, stiff and thick convex, leathery leaves with rough pubescence (Bello et al. 2000). Between the leaflet pairs on the rachis are conspicuous extrafloral nectaries (Coley & Barone 1996). Nec tary glands found outside the flower, called extrafloral nectaries (EFNs). These EFNs are often found near productive parts of the plants, or are active on young vulnerable leaves and solicit biotic defenses. Third level trophic organisms will often come t o these nectaries and, in exchange for the sugars and amino acids they receive from the plant, will remove herbivores (Koptur 1991). Ants and wasps (Hymenoptera) have been documented visiting several species of trees including Inga spp (Koptur 1991). Herb ivore damage is extensive on Inga spp and as with most plants, usually happens on younger, more tender leaves. Folivores on Inga sp include: katydids and grasshoppers (Othoptera), lepidopteran larvae, beetles (Coleoptera), monkeys (Cebidae in Costa Rica) sloths (Bracypodidae), leaf cutting ants and bees (Hymenoptera) (Koptur 1983). These nectaries are active on younger tender leaves but stop producing nectar (1985) of elevatio nal gradients and Inga spp nectaries indicate increased concentrations of tannins in higher elevation plants. These tannins retard the development of caterpillars, making them vulnerable to predators. She also reported the presence of parasitic wasps (Hym enoptera) and flies (Diptera) on Inga sp (Koptur 2000). Parasitoids complete their life cycle within or on the body of a host, ultimately killing it. Parasitoids provide protection for trees by ovipositing on the larvae that prey on the leaves. Inga sierr ae trees in Monteverde, Costa Rica have been found to have extra extrafloral nectaries (EEFNs) (Masters, Pers. Comm.). These are nectaries usually found on the base of a leaflet pair on the pulvinus, near the expected nectary glands. I investigated the fre quency of EEFNs nectaries on I. sierrae and their effectiveness in reducing herbivory. Additionally, since the number of leaflet pairs vary, and with it the number of EFNs, I suspect that there will be lower herbivory on leaves with more leaflet pairs. I h ypothesize that leaves with greater number of EFNs and leaflet pairs will experience less herbivory. I infer that the tree is producing additional EFNs in response to a decrease in biotic defenses in an attempt to attract the few third trophic level specie s that may be present in the area. Materials and Methods STUDY SITE This study was conducted in a tropical lower montane cloud forest in Monteverde, Costa Rica on the property of the Estacin Biolgica de Monteverde at approximately 1525 1555m (Nadkarni & Wheelwright 2000; Sterlmach 2001). Samples were taken from three sites, no larger than one hectare. Twenty trees were found in a pasture or pasture edge in small stands of two or three trees. The second site for twenty trees was the edge of secondary forest. Most of these trees were moderately distant from co nspecifics. Twenty trees were on the edge of a secondary growth forest. The last

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twenty were on the property of Alan and Karen Masters planted on fallow pasture from seeds collected in nearby areas. The distance between nearest neighbor for these trees was usually not more than 1m. FREQUENCY DISTRIBUTION I looked at the distribution of the EEFNs and leaflet pair number with a sample size of sixty trees. I documented forty random leaves per tree and counted the number of EEFNs as well as noting the number of leaflet pairs on each leaf. This made a total sample size of 2, 400 leaves. The leaves were collected by hand and with pole clippers for higher branches and examined with a hand lens. Due to the frequent unavailability of the pole clippers, most of the leaves were collected from branches that were within 2m of the forest floor. Each leaf was labeled for organizational purposes. From this information, I was able to create a frequency distribution of the EEFNs and leaflet pair numbers. I then looked at th e distribution of EEFNs and leaflet pair number for trees that were planted and those found in natural populations. PERCENT HERBIVORY The next phase of the project involved recording the percent herbivory on the leaves and comparing this to the number o f EEFNs on the leaves as well as leaflet pair number. I collected mature, toughened leaves that were fully expanded but had not been exposed to non herbivory related damage. The leaves were collected from both saplings and adults, although none of the tree s were smaller than 2m due to lack of sapling material, nor were any larger than 20m. To measure herbivory, I used a grid on transparent paper. Each convex leaf was carefully flattened (making sure not to tear any of the edges) and the grid placed over it. I extrapolated the missing parts of the leaf from the projected shape or from the paired leaflet across, which is nearly a mirror image. Herbivory was calculated for each leaflet as the number of grid boxes missing divided by the total number of grid boxe s that the leaf would have been. I then added all the missing parts for each leaflet and divided by the total leaflet area for each leaf. In my inventory of herbivory, I included gall formation, leaf rollers and large discolorations on the surface of the l eaf that reduced photosynthetic area. I then looked at the percent herbivory of EEFNs and leaflet pair number for trees that were planted and those found in natural populations. DATA ANALYSIS I created frequency distributions to determine the population wide occurrence of EEFNs and leaflet pair numbers. Utilizing an unpaired t test, I was able to statistically determine if a difference existed between the percent herbivory between trees with EEFNs and those without. I also used a 1 way ANOVA and regressi on analyses to see if there was a difference between trees with varying number of EEFNs numbers of EEFNs and percent herbivory. I employed the same tests for leaflet pair numbers. A Chi Square test was used to determine whether these frequencies deviated f rom what was expected. I also used regression analyses to see if there was a difference between number of galls per leaf and number of EEFNs per leaf. I then divided the sample size between trees in natural populations and trees planted and created frequen cy distributions for leaves from trees that had been planted and those that occur naturally. I applied an

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unpaired t test and 1 way ANOVA to determine if there was a difference in number of EEFNs, leaflet pair number, and percent herbivory between the natu ral populations and those planted. Results FREQUENCY DISTRIBUTION There was no statistical difference between trees with the typical number of EEFNs (1 way ANOVA, P > 0.14). Excluding leaflet pair number, about 75% of the trees had at least one EEFN. H owever, of all the leaves sampled, approximately 85% of the leaves did not; there is a significant difference between the frequency of leaves with EEFNs and those without (X 2 = 1221.22, df = 1, cv = 3.84) (Figure 1). The frequency of leaves with EEFNs decr eased as number of EEFNs increased, except for 6 EEFNs which were nearly as common as finding two EEFNs (Figure 2). There was no significant difference between leaves with two and six EEFNs (X 2 = 0.68, df = 1, cv = 3.84) (Figure 2) There is a significant d ifference between leaves with 1 4 EEFNs and 5 9 EEFNs (X 2 = 82.04, df = 1, cv = 3.84). On any given leaf, there is a an 87% chance of finding three leaflet pairs (Figure 3). There was a 7.0% chance of finding leaves with two leaflet pairs, and a 5.7% cha nce of finding leaves with four leaflet pairs (Figure 3). There was no significant difference between the frequency of leaves with two leaflet pairs and four leaflet pairs (X 2 = 2.96, df = 1, cv = 3.84) (Figure 3). Only 0.125% of the leaves had leaves with a single leaflet pair, which was significantly different from the frequency of leaves with two and four leaflet pairs (X 2 = 148.80, df = 2, cv = 5.99) (Figure 3). Though EEFNs were infrequently encountered in the total population, the frequency distribut ion of number of EEFNs per leaf significantly differed between leaves found on planted trees and those found in natural populations (1 way ANOVA, P = 0.028) (Figure 4). The frequency of EEFNs per leaf were more evenly distributed on planted trees than thos e found in natural populations. There was also a significant difference between the distribution of leaflet pair numbers per leaf found on planted trees versus the ones found on natural population trees (1 way ANOVA, P = 0.0322) (Figure 5). The frequency of two leaflet pairs per leaf is lower on planted population trees, while the frequency of four leaflet pairs is higher in planted populations (2 x 2 Contingency Table, X 2 = 217.20, df = 1, cv = 3.84). The most commonly encountered leaflet pair per leaf wa s three between both populations. PERCENT HERBIVORY There was significantly less herbivory on trees that had EEFNs. (T test, P < 0.0001). The mean number of EEFNs, the mean number of EEFNs, and the percent herbivory on population of trees found on the M significantly different from those found in natural populations (t test, P < 0.0001, P < 0.0001, P < 0.0001) (Appendix A, Table 1). There was less percent herbivory per tree that had EEFNs versus those that did not (Exponential Regress ion Analysis, R 2 = 0.80; P < 0.0001) (Figure 6). For natural populations, there is no significant decrease in percent

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herbivory as average EEFN number per tree increases (Simple Regression Analysis, R 2 = 0.02, P > 0.05) (Figure 7). There is a significant d ecrease in percent herbivory as average number of EEFNs per tree increases for planted trees (Exponential Regression Analysis, R 2 = 0.85, P < 0.0001) (Figure 7). The most dramatic decrease, greater than 15% in the percent herbivory per leaf is from zero EE FNs to one EEFN (Figure 8a). The average percent herbivory for leaves with no EEFNs was 27.2%, while the average percent herbivory for leaves with at least on EEFN was 10.6%. The percent herbivory significantly decreased as the number of EEFNs increased (1 way ANOVA, P < 0.0001) and Exponential Regression Analysis; R 2 = 0.94, P < 0.0001) (Figure 8b). The percent herbivory significantly decreased for both planted and natural populations as number of EEFNs increased (Polynomial Regression Analysis R 2 = 0.89 P < 0.0001, Polynomial Regression Analysis, R 2 = 0.89, P < 0.0001) (Figure 9). The percent herbivory decreased as number of leaflet pairs increased (Exponential Regression Analysis, R 2 = 0.99, P < 0.0001 and 1 way ANOVA, P = 2.62 E 35) (Figure 10). The percent herbivory also significantly decreased as number of leaflet pairs increased for both the natural and the planted population (Polynomial Regression Analysis R 2 = 0.91; P < 0.0001 and R 2 = 0.9 998; P < 0.0001). Potential herbivores found on the plant included many of the insects found in previous studies. The following insects, both adult and larval, were found on or near the leaves of I. sierrae : Coleoptera, Lepidoptera, Hymenoptera, Hemiptera and Orthoptera. Several arachnids were also seen, but their impact on herbivory is unknown. There were no mammals seen preying on the leaves. Two species of ants that vigorously defended the leaves were seen visiting the EEFNs. Gall number decreased as n umber of EEFN increased (Simple Regression Analysis, R 2 = 0.9905, P < 0.0001) (Figure 12). DISCUSSION There was no statistical difference in herbivore damage between trees that had normal numbers of EEFNs. This means that the I. sierrae trees are subject to equivalent herbivore pressures, and that other factors such as location or size were not an important factor in this study. This means that the differences in percent herbivory stem from other factors such as increased EEFN production. Though over 75% of the trees had EEFNs, over 85% of leaves surveyed did not have any EEFNs at all. The production of EEFNs is probably costly to the tree. It would maximum benefit of herbivory reduction. The most commonly found leaflet pair number per leaf was three. The production of four leaflet pairs may be an adaptation to increase the number of EFNs The production of four leaflet pairs may be an adaptation to increase the numbe r of EFNs. The production of two leaflet pairs may have to do with a lack of resources to allocate to that particular leaf. There was a significant difference between the percent herbivory on trees with

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EEFNs and those that did not. Having EEFNs on the tr ee seems to have increased fitness for the entire tree. Even though all leaves did not have EEFNs, they may have been enough to attract the needed amount of biotic defenses. Increasing the number of EEFNs may increase the number of visits by any nectar see king insect, making the overall activity at the site higher, discouraging smaller or less aggressive insects. An increased number of EEFNs may have increased herbivore defense by ants. In accordance with the findings of Haff (1995), there were at least two species of ants, possibly three, visiting the EEFNs (Koptur 1991). The number of parasitoid visitors which prey on the larvae of ovipositing herbivores could also have increased attracted to the additional EFNs, reducing the percent herbivory found on I. sierrae leaves. The increase in leaflet pair number per leaf correlating with a decrease in herbivory supports these ideas. As leaflet pair number increases so do the number of EFNs. Between each leaflet pair is an EFN, so if there are four leaflet pairs, there are 4 EFNs. The increase in leaflet pairs could be a strategy not only to increase photosynthetic capacity, but also to increase the number of EFNs. Production of two leaflet pairs, less than the 3 leaflet pair norm, may be due to the lack of resour ces to allocate to a particular leaf. It is interesting to note that there was no significant difference between the frequency of leaves with two leaflet pairs and those with four leaflet pairs. This is in spite of the fact that, in this study, two leaflet pairs sustained much more damage than four leaflet pairs which tended to decrease percent herbivory to the leaf. One possible explanation is that it may be that, though the percent of herbivory is changing, the quantity of herbivory is remaining constant as leaflet pair is increasing. Gall number per leaf decreases as EEFN frequency per leaf increases. If the increased EFN production also increases visitation by and abundance of mutualistic organisms, this may increase defense and decrease desirability of the leaf when a female galling insect tries to oviposit. leaflet pairs per leaf, and reduced percent herbivory. This could be due to the fact that the seeds planted on this pro perty are from a few, unusual individuals. The increased production of EFNs is most likely genetic as is indicated by the localized population of genetically similar individuals. Additionally, if the production of EEFNs was a physiological response to high predation, then the frequency of EEFNs would be more common in the population. However, the reduction in herbivory may not necessarily be because of the increase in EEFNs. Insects tend to be attracted to the food and waste of people. The proximity of thes e trees to their house may increase the number of insects present, especially insects that would be biotic defenses to the I. sierrae Moreover since most of the plants in their yard have been placed intentionally, competition may be reduced allowing I. s ierrae to allocate more resources to leaf toughness, leaflet pair and EEFNs. It was noted that large saplings from the secondary forest edge that had EEFNs may be offspring of the planted trees. The average number of EEFNs per tree may not significantly r educe the percent herbivory on natural population of trees because the frequency is too low to have an impact on the entire tree. As the frequency of average number of EEFNs increased on

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planted trees the average percent herbivory decreased. The high densi ties of EEFNs may be attracting enough visitors to confer fitness for the entire tree. With increasing elevation there is often a decrease in the species richness of insects (Koptur 1994). The cause of increased herbivory may be that as elevation increase s, there is a disproportionate number of mutualistic organisms that decrease. In order to compensate for the increased herbivore pressure, the trees may invest more in costly chemical protection and EEFN production (Koptur 1994). Since this study was condu cted at a high elevation, it would be interesting to find out whether the I. sierrae on increased leaflet pair number. The direct mutualism between ants and EFNs and the indir ect mutualism between parasitoids may have led to an increase in production of EEFNs in the I. sierrae trees in the Monteverde Cloud Forest Preserve. It could be that we are witnessing a change in species morphology and/or it could be that the introduction and propagation of these particular alleles has increased the abundance of I. sierrae trees with EEFNs. Improvements on this project include data from the wet season when insect abundances are higher. For example, I noticed Chrysomelid beetles preying on the young leaves of the planted trees that were present only after I had finished collecting data. Future research would include investing the relationship between fitness and percent herbivory. Another interesting study would be the co st benefit analysis of the production of EEFNs and leaflet pair number. Also, it is imperative to create an inventory of the species visiting the nectaries, ovipositing on the leaves, parasitizing the galls, and causing the herbivory damage on the I. sierr ae trees. Acknowledgements I would like to thank my advisor Alan Masters for his guidance through the course of this project and his family for the use of their Inga sierrae trees, and Mauricio Garca, Rick Smith and Andrew Rodstrom were invaluable resou rces. Additionally, I give thanks to those apart of La Estacin Biolgica Monteverde for the use of their I. sierrae trees, and CIEE for all their support. And of course, thanks to the I. sierrae for all the leaves they donated. A special thanks goes to my roommates: Ruth, Crystal, and Chrissi, for nursing me through this paper. Literature Cited Bello, E., W.A. Haber and W. Zuchowski. 2000. An Introduction to Cloud Forest Trees. Monteverde Costa Rica Mountain Gem Publications. Monteverde, Costa Rica. Pp. 62. Ann. Rev. Ecol. Syst. 27: 305 335.

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Guaba Tree Inga punctata (Fabaceae: Mimosoide UCEAP Tropical Biology Fall, pp. 29 41. Inga herbivores and ants and insect visitors to Ecology and Conservation of a Tropical Cl oud Forest. Oxford University Press. New York, New York. pp. 277 278. -----Inga trees: a comparison of ----ies of herbs and trees: modeling the interaction with ants Ant Plant Interactions Oxford University Press. Walton Street, Oxford. -----Inga In D. H. Janzen (Ed.) Costa Rica n Natural History. University of Chicago Press. Chicago, Illinois. Pp. 259 261. Masters, A. 2003. Personal Communication: Unpublished data regarding EEFNs in Inga sierrae and herbivory damage. Nadkarni, N.M. and Wheelwright N.T. (Ed.) 2000. Monteverde: Ecology and Conservation of a Tropical Cloud Forest Oxford University Press. New York, New York. pp. 9. Raven, P.H., R.F. Evert and A.E. Eichhorn. 1991. Biology of Plants. W.H. Freeman and Company and Worth Publishers. New York, New York. pp. 779 783. Sterlmach, D.K. 2001. Levels of Herbivory on Inga sierrae Along an Elevational Gradient. In Tropical Ecology and Conservation Unpublished: Council for International Educational Exchange, Monteverde, Costa Rica, pp. 103 107.

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APPENDIX A. ______________________________________ _______________________ ___________________ Table 1. The differences between the average number of EEFNs, EFNs, and percentage of herbivory of test was used to examine the differences between the EEFNs, leafl et pairs, and percent herbivory on trees that were planted and those found in nature. Planted Trees Trees in Natural Populations P Value Average # of EEFNs 1.62 0.04 <0.0001 Range 0 9 0 2 Standard Error 0.0076 0.1764 Average # of Leaflet Pairs 3.11 2.94 <0.0001 Range 0 4 0 4 Standard Error 0.355 0.362 Average % of Herbivory 17.12 27.91 <0.0001 Range 0 100 0 100 Standard Error 0.481 0.7278 Total Population _____________________________________________________________________________________ Table 2. The average, maximum number for range and standard error for number of EEFNs per leaf for the entire sample. The minimum number for range for all categories was zero. All numbers are rounded to the nearest tenth. _____________________________________________________________________________________________ 0 1 2 3 4 5 6 7 8 9 Average 2.2 10.6 8.3 6.4 5.1 1.9 1.7 0 1.4 .8 Range 100 56.1 32.3 21.2 15.4 5.6 15.6 0 8.1 2.3 Standard Error 0.4 0.8 0.7 1.1 0.7 0.3 0.2 0 0.7 0.8 _____________________________________________________________________________________________ Table 3. The average, range and standard error for number of leaflet pairs per leaf for the entire sample. All numbers are rounded to the nearest tenth. _____________________________________________________________________________________________ 1 2 3 4 Average 81.2 52.7 22.8 12.4 Range 45.7 100 0 100 0 99 0 38.3 Standard Error 17.7 1.7 0.4 0.49 _____________________________________________________________________________________________ Table 4. The average, maximum number for range and standard error for number of galls per leaflet pair with respect to EEFNs. The minimum number for range for all categories was zero. 0 1 2 3 4 5 6 7 8 9 Average 0.2673 0.0133 0.0087 0.0037 0.0025 0.0008 0.0050 0 0.0004 0 Range 45 4 4 2 1 1 2 0 1 0 Standard Deviation 2.20 0.19 0.17 0.07 0.05 0.03 0.09 0 0.02 0

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Planted Populations _____________________________________________________________________________________________ Table 5. The average, maximum number for range and standard error for number of EEFNs per leaf for planted populations. The minimum number for range for all categ ories was zero. _____________________________________________________________________________________________ 0 1 2 3 4 5 6 7 8 9 Average 25.4 10. 5 8. 2 5.7 5.1 1.9 1. 8 0 1.4 0.78 Range 100 56.1 23.3 2 1.2 15. 7 5.6 15.6 0 8.1 2.3 4 Standard Error 0.008 0.004 0 .034 0.064 0 .067 0 .157 0. 01 2 0 0. 301 0. .0387 _____________________________________________________________________________________________ Table 6. The average, range and standard error for number of leaflet pairs per leaf for planted populations. All numbers are rounded to the nearest tenth. _____________________________________________________________________________________________ 1 2 3 4 Average 57.15 46.24 17.89 0.687 Range 47.5 100 14.3 89 0 89.2 0 29.4 Standard Error 3.098 0.247 0.005 0.030 Natural Populations _____________________________________________________________________________________________ Table 7. The average, maximum number for range and standard error for number of EEFNs per leaf for natural populations. The minimum number for range for all categories was zero. _____________________________________________________________________________________________ 0 1 2 3 4 5 6 7 8 9 Average 28.45 10. 87 9.06 0 0 0 0 0 0 0 0 Range 99 5 4.3 32.32 0 0 0 0 0 0 0 0 Standard Error 0.003 0.083 0.376 0 0 0 0 0 0 0 0 _____________________________________________________________________________________________ Table 8. The average, range and standard error for number of leaflet pairs per leaf for natural populations. _____________________________________________________________________________________________ 1 2 3 4 Average 97.8 53.22 25.84 11.91 Range 97.8 0 100 0 99 0 38.3 Standard Error 0 0.032 0.003 0.055



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Distribution and Anti herbivoral Role of Extra Extrafloral Nectaries and Leaflet Pair Number on Inga sierrae Fabaceae: Mimosaceae Andraya Gough Department of Biology, Occidental College _____________________________________________________________________________________ Abstract The purpose of this project is to investigate the distribution and anti herbivore impact of extra extrafloral nectaries EEFNs and leaflet pair number on Inga sierrae Fabacaeae: Mimosaceae in Monteverde, Costa Rica. I recorded the number of EEFNs and leaflet pairs per leaf for forty leaves collected from sixty trees from three different locations. EEFNs, though not commonly found on I. sierrae did significant ly reduce the percent herbivory on leaves 1 way ANOVA, P < 0.0001 and Exponential Regression Analysis; R 2 = 0.9413, P < 0.0001. The most commonly encountered leaflet pair number was three. However, when the number of leaflet pairs decreased, percent herb ivory increased Exponential Regression Analysis, R 2 = 0.9905, P < 0.0001 and 1 way ANOVA, P < 0.0001. Heavy herbivory pressure could be causing a species change toward increased production of EFNs. Resumen El objecto de este proyecto es investigar la distribuci n y el impacto de anti herbivore de los nectararios extraflorales EEFNs y nmeros de hojuela de parejas en los rboles de Inga sierrae en Monteverde en Costa Rica. Yo document el nmero de EEFNs y hojuela de pareja por hoja para cuarenta hojas que yo recoj desde seisenta rboles de tres localidades diferentes. EEFNs, aunque no son comunes a encontrar en I. sierrae reducen significamente el porcentaje de la herbividad de las hojas. Ms frequentemente el nme ro de las hojuela s de parejas por cada hoja fue tres, aunque, cuando el nmero disminuio, el percentaje de herbividad aument y el opuesto ocurri tambin. Herbividad pesado pueden ser causando un cambio de especies para aumentado produccin de EFNs. Intro duction Herbivory can be physiologically costly to plants both i n the short term and long term. Herbivores, and in particular folivores, decrease the fitness of plants by destroying their photosynthetic surfaces Raven et al. 1991. Because plants are sta tionary, they must develop physical, chemical or biotic defenses in response to herbivore damage Koptur 1991. Biotic defenses, including both dynamic and complex mutualisms, have not been studied as extensively. Inga spp are tall, leguminous, sub canop y trees commonly found in disturbed areas such as pastures and along forest edges only on the Pacific side of Costa Rica Bello et al. 2000. The leaves are alternate, parapinnate, and compound with 2 4 opposite leaflet pairs, approximately 12 x 18 cm Bel lo et al. 2000. Inga sierrae has a

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winged rachis and when mature, stiff and thick convex, leathery leaves with rough pubescence Bello et al. 2000. Between the leaflet pairs on the rachis are conspicuous extrafloral nectaries Coley & Barone 1996. Nectary glands found outside the flower, called extrafloral nectaries EFNs. These EFNs are often found near reproductive parts of the plants, or are active on young vulnerable leaves and solicit biotic defenses. Third level trophic organisms will often c ome to these nectaries and, in exchange for the sugars and amino acids they receive from the plant, will remove herbivores Koptur 1991. Ants and wasps Hymenoptera have been documented visiting several species of trees including Inga spp Koptur 1991. Herbivore damage is extensive on Inga spp and as with most plants, usually happens on younger, more tender leaves. Folivores on Inga sp include: katydids and grasshoppers Othoptera, lepidopteran larvae, beetles Coleoptera, monkeys Cebidae in Costa Rica, sloths Brachypodidae, leaf cutting ants and bees Hymenoptera Koptur 1983. These nectaries are active on young tender leaves but stop producing nectar when the leaves are mature and toughened Koptur 1991. Suzanne Koptur€s study 1985 of elev ational gradients and Inga spp. nectaries indicate increased concentrations of tannins in higher elevation plants. These tannins retard the development of caterpillars, making them vulnerable to predators. She also reported the presence of parasitic wasps Hymenoptera and flies Diptera on Inga sp Koptur 2000. Parasitoids complete their life cycle within or on the body of a host, ultimately killing it. Parasitoids provide protection for trees by ovipositing on the larvae that prey on the leaves. I. sier rae trees in Monteverde, Costa Rica have been found to have extra extrafloral nectaries EEFNs Masters, Pers. Comm.. These are nectaries usually found on the base of a leaflet pair on the pulvinus, near the expected nectary glands. I investigated the fr equency of EEFNs nectaries on I. sierrae and their effectiveness in reducing herbivory. Additionally, since the number of leaflet pairs vary, and with it the number of EFNs, I suspect that there will be lower herbivory on leaves with more leaflet pairs. I hypothesize that leaves with greater number of EFNs and leaflet pairs will experience less herbivory. I infer that the tree is producing additional EFNs in response to a decrease in biotic defenses in an attempt to attract the few third trophic level speci es that may be present in the area. Materials and Methods STUDY SITE This study was conducted in a tropical lower montane cloud forest in Monteverde, Costa Rica on the property of the Estacin Biolgica de Monteverde at approximately 1525  1555m Nadkarni & Wheelwright 2000; Sterlmach 2001. Samples were taken from three sites, no larger than one hectare. Twenty trees were found in a pasture or pasture edge in small stands of two or three trees. The second site for twenty trees was the edge of secondary forest. Most of these trees were moderately distant from conspecifics Twenty trees were on the edge of a secondary growth forest. The last twenty were on the property of Alan and Karen Masters planted on fallow pasture from seeds collected in nearby areas. The distance between nearest neighbor for these trees was usually n ot more than 1m.

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FREQUENCY DISTRIBUTION  I looked at the distribution of the EEFNs and leaflet pair number with a sample size of sixty trees. I documented forty random leaves per tree and counted the number of EEFNs as well as noting the number of leafle t pairs on each leaf. This made a total sample size of 2, 400 leaves. The leaves were collected by hand and with pole clippers for higher branches and examined with a hand lens. Due to the frequent unavailability of the pole clippers, most of the leaves we re collected from branches that were within 2m of the forest floor. Each leaf was labeled for organizational purposes. From this information, I was able to create a frequency distribution of the EEFNs and leaflet pair numbers. I then looked at the distribu tion of EEFNs and leaflet pair number for trees that were planted and those found in natural populations. PERCENT HERBIVORY  The next phase of the project involved recording the percent herbivory on the leaves and comparing this to the number of EEFNs on the leaves as well as leaflet pair number. I collected mature, toughened leaves that were fully expanded but had not been exposed to non herbivory related damage. The leaves were collected from both saplings and adults, although none of the trees were sma ller than 2m due to lack of sampling material, nor were any larger than 20m. To measure herbivory, I used a grid on transparent paper. Each convex leaf was carefully flattened making sure not to tear any of the edges and the grid placed over it. I extrap olated the missing parts of the leaf from the projected shape or from the paired leaflet across, which is nearly a mirror image. Herbivory was calculated for each leaflet as the number of grid boxes missing divided by the total number of grid boxes that th e leaf would have been. I then added all the missing parts for each leaflet and divided by the total leaflet area for each leaf. In my inventory of herbivory, I included gall formation, leaf rollers and large discolorations on the surface of the leaf that reduced photosynthetic area. I then looked at the percent herbivory of EEFNs and leaflet pair number for trees that were planted and those found in natural populations. DATA ANALYSIS  I created frequency distributions to determine the population wide occurrence of EEFNs and leaflet pair numbers. Utilizing an unpaired t test, I was able to statistically determine if a difference existed between the percent herbivory between trees with EEFNs and t hose without. I also used a 1 way ANOVA and regression analyses to see if there was a difference between trees with varying numbers of EEFNs numbers of EEFNs and percent herbivory. I employed the same tests for leaflet pair numbers. A Chi Square test was u sed to determine whether these frequencies deviated from what was expected. I also used regression analyses to see if there was a difference between number of galls per leaf and number of EEFNs per leaf. I then divided the sample size between trees in natu ral populations and trees planted and created frequency distributions for leaves from trees that had been planted and those that occur naturally. I applied an unpaired t test and 1 way ANOVA to determine if there was a difference in number of EEFNs, leafle t pair number, and percent herbivory between the natural populations and those planted.

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Results FREQUENCY DISTRIBUTION  There was no statistical difference between trees with the typical number of EFNs 1 way ANOVA, p > 0.14. Excluding leaflet pair number, about 75% of the trees had at least one EEFN. However, of all the leaves sampled, approximately 85% of the leaves did not; there is a significant difference between the frequency of leaves with EEFNs and those without x 2 = 1221.22, df = 1, cv = 3.84 Figure 1. The frequency of leaves with EEFNs decreased as number of EEFNs increased, except for 6 EEFNs which were nearly as common as finding two EEFNs Figure 2. There was no significant difference between leaves with two and six EEFNs x 2 = 0.68, df = 1, cv = 3.84 Figure 2. There is a significant difference between leaves with 1 4 EEFNs and 5 9 EEFNs x 2 = 82.04, df = 1, cv = 3.84. On any given leaf, there is an 87% chance of finding three leaflet pairs Figure 3. There was a 7.0% chance of finding leaves with two leaflet pairs, and a 5.7% chance of finding leaves with four leaflet pairs Figure 3. There was no significant difference between the frequency of leaves with two leaflet pairs and four leaflet pairs x 2 = 2.96, df = 1, cv = 3.84 Figure 3. Only 0.125% of the leaves had leaves with a single leaflet pair, which was significantly different from the frequency of leaves wi th two and four leaflet pairs x 2 = 148.80, df = 2, cv = 5.99 Figure 3. Though EEFNs were infrequently encountered in the total population, the frequency distribution of number of EEFNs per leaf significantly differed between leaves found on planted trees and those found in nat ural populations 1 way ANOVA, P = 0.028 Figure 4. The frequencies of EEFNs per leaf were more evenly distributed on planted trees than those found in natural populations. There was also a significant difference between the distribution of leaflet pair numbers per leaf found on planted trees versus the ones foun d on natural population trees 1 way ANOVA, P = 0.0322 Figure 5. The frequency of two leaflet pairs per leaf is lower on planted population trees, while the frequency of four leaflet pairs is higher in planted populat ions 2 x 2 Contingency Table, x 2 = 217.20, df = 1, cv = 3.84. The most commonly encountered leaflet pair per leaf was three between both populations. PERCENT HERBIVORY  There was significantly less herbivory on trees that had EEFNs. T test, P < 0.0001. The mean number of EEFNs, the mea n number of EFNs, and the percent herbivory on population of trees found on the Master€s property was significantly different from those found in natural populations t test, p < 0.0001, P < 0.0001, P < 0.0001 Appendix A, Table 1. There was less percent herbivory per tree that had EEFNs versus those that did not Exponential Regression Analysis, R 2 = 0.80; P < 0.0001 Figure 6. For natural populations, there is no significant decrease in percent herbivory as average EEFN number per tree increases Simp le Regression Analysis, R 2 = 0.02, P > 0.05 Figure 7. There is a significant decrease in percent herbivory as average number of EEFNs per tree increases for planted trees Exponential Regression Analysis, R 2 = 0.85, P < 0.0001 Figure 7. The most dram atic decrease, greater than 15%, in the percent herbivory per leaf is from zero EEFNs to one EEFN Figure 8a. The average percent herbivory for leaves with no EEFNs was 27.2%, while the average percent

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herbivory for leaves with at least one EEFN was 10.6% The percent herbivory significantly decreased as the number of EEFNs increased 1 way ANOVA, P < 0.0001 and Exponential Regression Analysis; R 2 = 0.94, P < 0.0001 Figure 8b. The percent herbivory significantly decreased for both planted and natural po pulations as number of EEFNs increased Polynomial Regression Analysis, R 2 = 0.89, P < 0.0001, Polynomial Regression Analysis, R 2 = 0.89, P < 0.0001 Figure 9. The percent herbivory decreased as number of leaflet pairs increased Exponential Regression Analysis, R 2 = 0.99, P < 0.0001 and 1 way ANOVA, P = 2.62E 35 Figure 10. The percent herbivory also significantly decreased as number of leaflet pairs increased for both the natural and the planted population Polynomial Regression Analysis, R 2 = 0.91; P < 0.0001 and R 2 = 0.9998; P < 0.0001. Potential herbivores found on the plant included many of the insects found in previous studies. The following insects, both adult and larval, were found on or near the leaves of I. sierrae : Coleoptera, Lepidoptera, Hymenoptera, Hemiptera, and Orthoptera. Several arachnids were also seen, but their impact on herbivory is unknown. There were no mammals seen preying on the leaves. Two species of ants that vigorously defended the leaves were seen visiting the EFNs. Gall number decreased as number of EEFN increased Simple Regression Analysis, R 2 = 0.9905, P < 0.0001 Figure 12. Discussion There was no statistical difference in herbivore damage between trees that had normal numbers of EFNs. This means that the I. sier rae trees are subject to equivalent herbivore pressures, and that other factors such as location or size were not an important factor in this study. This means that the differences in percent herbivory stem from other factors such as increased EFN producti on. Though over 75% of the trees had EEFNs, over 85% of leaves surveyed did not have any EEFNs at all. The production of EEFNs is probably costly to the tree. It would be in the tree€s best interest to produce the fewest number of EEFNs while receiving th e maximum benefit of herbivory reduction. The most commonly found leaflet pair number per leaf was three. The production of four leaflet pairs may be an adaptation to increase the number of EFNs. The production of two leaflet pairs may have to do with a l ack of resources to allocate to that particular leaf. There was a significant difference between the percent herbivory on trees with EEFNs and those that did not. Having EEFNs on the tree seems to have increased fitness for the entire tree. Even though al l leaves did not have EEFNs, they may have been enough to attract the needed amount of biotic defenses. Increasing the number of EEFNs may increase the number of visits by any nectar seeking insect, making the overall activity at the site higher, discourag ing smaller or less aggressive insects. An increased number of EEFNs may have increased herbivore defense by ants. In accordance with the findings of Haff 1995, there were at least two species of ants, possibly three, visiting the EEFNs Koptur 1991. Th e number of parasitoid visitors which prey on the larvae of

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ovipositing herbivores could also have increased attracted to the additional EFNs, reducing the percent herbivory found on I. sierrae leaves. The increase in leaflet pair number per leaf correlat ing with a decrease in herbivory supports these ideas. As leaflet pair number increases so do the number of EFNs. Between each leaflet pair is an EFN, so if there are four leaflet pairs, there are 4 EFNs. The increase in leaflet pairs could be a strategy n ot only to increase photosynthetic capacity, but also to increase the number of EFNs. Production of two leaflet pairs, less than the 3 leaflet pair norm, may be due to the lack of resources to allocate to a particular leaf. It is interesting to note that t here was no significant difference between the frequency of leaves with two leaflet pairs and those with four leaflet pairs. This is in spite of the fact that, in this study, two leaflet pairs sustained much more damage than four leaflet pairs which tended to decrease percent herbivory to the leaf. One possible explanation is that it may be that, though the percent of herbivory is changing, the quantity of herbivory is remaining constant as leaflet pair is increasing. Gall number per leaf decreases as EEFN frequency per leaf increases. If the increased EFN production also increases visitation by and abundance of mutualistic organisms, this may increase defense and decrease desirability of the leaf when a female galling insect tries to oviposit. The trees p lanted on the Masters€ property have, on average, more EEFNs, more leaflet pairs per leaf, and reduced percent herbivory. This could be due to the fact that the seeds planted on this property are from a few, unusual individuals. The increased production of EFNs is most likely genetic as is indicated by the localized population of genetically similar individuals. Additionally, if the production of EEFNs was a physiological response to high predation, then the frequency of EEFNs would be more common in the po pulation. However, the reduction in herbivory may not necessarily be because of the increase in EEFNs. Insects tend to be attracted to the food and waste of people. The proximity of these trees to their house may increase the number of insects present, esp ecially insects that would be biotic defenses to the I. sierrae Moreover, since most of the plants in their yard have been placed intentionally, competition may be reduced allowing I. sierrae to allocate more resources to leaf toughness, leaflet pair and EEFNs. It was noted that large saplings from the secondary forest edge that had EEFNs may be offspring of the planted trees. The average number of EEFNs per tree may not significantly reduce the percent herbivory on natural population of trees because the frequency is to low to have an impact on the entire tree. As the frequency of average number of EEFNs increased on planted trees the average percent herbivory decreased. The high densities of EEFNs may be attracting enough visitors to confer fitness for the entire tree. With increasing elevation there is often a decrease in the species richness of insects Koptur 1994. The cause of increased herbivory may be that as elevation increases, ther e is a disproportionate number of mutualistic organisms that decrease. In order to compensate for the increased herbivore pressure, the trees may invest more in costly chemical protection and EEFN production Koptur 1994. Since this study was conducted at a high elevation, it would be interesting to find out whether the I. sierrae on

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the Masters€ property have higher level of phenols in addition to the EEFNs and increased leaflet pair number. The direct mutualism between ants and EFNs and the indirect mut ualism between parasitoids, may have led to an increase in production of EEFNs in the I. sierrae trees in the Monteverde Cloud Forest Preserve. It could be that we are witnessing a change in species morphology and/or it could be that the introduction and p ropagation of these particular alleles has increased the abundance of I. sierrae trees with EEFNs. Improvements on this project include data from the wet season when insect abundances are higher. For example, I noticed Chrysomelid beetles preying on the y oung leaves of the planted trees that were present only after I had finished collecting data. Future research would include investing the relationship between fitness and percent herbivory. Another interesting study would be the cost benefit analysis of th e production of EEFNs and leaflet pair number. Also, it is imperative to create an inventory of the species visiting the nectaries, ovipositing on the leaves, parasitizing the galls, and causing the herbivory damage on the I. sierrae trees. Acknowledgemen ts I would like to thank my advisor Alan Masters for his guidance through the course of this project and his family for the use of their Inga sierrae trees, and Mauricio Garcia, Rick Smith and Andrew Rodstrom were invaluable resources. Additionally, I give thanks to those apart of La Estacin Biolgica Monteverde for the use of their I. sierrae trees, and CIEE for all their support. And of course, thanks to the I. sierrae for all the leaves they donated. A spe cial thanks goes to my roommates: Ruth, Crystal, and Chrissi, for nursing me through this paper. Literature Cited Bello E., W.A. Haber and W. Zuchowski. 2000. An Introduction to Cloud Forest Trees Monteverde Costa Rica Mountain Gem Publications. Monteverde, Costa Rica pp. 62. Coley, P.D. and Barone, J.A. 1996. ‚Herbivory and Plant defenses in Tropical Forestsƒ. Ann. Rev. Ecol. Syst. 27: 305 335. Haff, T. 1995. ‚Effects of Elevation, Ants and Parasitoid Wasps on Herbivory in the Guaba Tree Inga p unctata Fabaceae: Mimosoideaeƒ. In UCEAP Tropical Biology Fall, pp. 29 41. Koptur, S. 2000. ‚Interactions among Inga herbivores and ants and insect visitors to foliar nectariesƒ. In Nadkarni, N.M. and N.T. Wheelwright. Eds. Monteverde: Ecology and C onservation of a Tropical Cloud Forest Oxford University Press. New York, New York. pp. 277 278. ----, S. 1994. ‚Floral and extrafloral nectars of Costa Rican Inga trees: a comparison of their constituents and compositionƒ. Biotropica 26: 276. ----, S. 1991. ‚Extrafloral nectaries of herbs and trees: modeling the interaction with ants and parasitoidsƒ. In Huxley, C.R. and D.F. Cutler Eds. Ant  Plant Interactions Oxford University Press. Walton Street, Oxford.

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----, S. 1983. ‚ Inga Guaba, Guajiniq uil, Caite, Paternoƒ. In D.H. Janzen Ed. Costa Rican Natural History University of Chicago Press. Chicago, Illinois. pp. 259 261. Masters, A. 2003. Personal Communication: Unpublished data regarding EEFNs in Inga sierrae and herbivory damage. Nadkarni, N.M. and Wheelwright N.T. Ed. 2000. Monteverde: Ecology and Conservation of a Tropical Cloud Forest Oxford University Press. New York, New York. pp. 9. Raven, P.H., R.F. Evert and A.E. Eichhorn. 1991. Biology of Plants W.H. Freeman and Compa ny and Worth Publishers. New York, New York. pp. 779 783. Sterlmach, D.K. 2001. Levels of Herbivory on Inga sierrae Along an Elevational Gradient. In Tropical Ecology and Conservation Unpublished: Council for International Educational Exchange, Monteverd e, Costa Rica, pp. 103 107.

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______________________________________________________________________________ Figure 1. The frequency of I. sierra e leaves that have extra extrafloral nectaries EEFNs compared to leaves that do not have any EEFNs. Over 85% of the 2, 400 leaves sampled did not have any EEFNs at all. There is a significant difference between the frequency of leaves with EEFNs and those without x 2 = 1221.22, df = 1, cv = 3.84. ______________________________________________________________________________ ______________________________________________________________________________ Figure 2. The frequency I. sierra e leaves that have extra extraflora nectaries EEFNs. Frequency decreases as number of EEFNs increases expect for leaves with 6 EEFNs. There is no significant difference between l eaves with two and six EEFNs. x 2 = 0.6849, df = 1, cv = 3.84. There is a significant difference between leaves with 1 4 EEFNs and 5 9 EEFNs x 2 = 82.04, df = 1, cv = 3.84. ______________________________________________________________________________

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______________________________________________________________________________ Figure 3. Frequency of leaflet pair number on leaves of I. sierrae The median number of leaflets per leaf is three. One leaflet per leaf was rare, only three leaves out of 2,40 0 sampled were found. There was a significant difference between the one pair of leaflets a nd two and four leaflet pairs x 2 = 148.8, df = 2, cv = 5.99. However, there was no significant difference between the frequency of leaves with two and four leaflet s x 2 = 2.96, df = 1, cv = 3.84. ______________________________________________________________________________ ______________________________________________________________________________ Figure 4. The frequency of I. sierrae leaves that have extra extrafloral nectaries EEFNs compared to leaves that do not have any EEFNs in a. planted and b. planted populations. There is a significant difference between the frequency of leaves with EEFNs and those without for na tural and planted populations x 2 = 2 229. 89, df = 1, cv = 3.84; x 2 = 10, 710.26, df = 1, cv = 3.84. ______________________________________________________________________________

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______________________________________________________________________________ Figure 5. Frequency of leaflet pair number on leaves of I. sierra e for a. planted and b. natural populations. The median number of leaflets per leaf is three. The frequency of two leaflet pairs per leaf is lower on planted population trees, while the frequency of fo ur leaflet pairs is higher in planted populat ions 2 x 2 Contingency Table, x 2 = 217.2, df = 1, cv = 3.84. ______________________________________________________________________________ _________________________________________________________________ _____________ Figure 6. Percent herbivory on I. sierrae trees with increasing extra extrafloral nectaries EEFNs. As the average number of EEFNs per tree increases the percent herbivory decreases Exponential Regression Analysis, R 2 = 0.7981, P < 0.0001. The horizontal line indicates the average percentage of herbivory 26.53 on trees without EEFNs. The standard deviation from that average is 3.77. ______________________________________________________________________________

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______________________________________________________________________________ Figure 7. Percent herbivory on I. sierrae trees with increasing extra extrafloral nectaries EEFNs for a. planted population trees and b. natural population trees. For natural populations, there is no significant decrease in percent herbivory as average EEFN number per tree increases Simple Regression Analysis, R 2 = 0.0239, P > 0.05. For planted populations, there is a significant decrease in percent herbivory as average number of EEFN per tree increases Exponential Regression Analysis, R 2 = 0.8518, P < 0.0001. _________________________________________________________________ _____________

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______________________________________________________________________________ Figure 8. a. Percent herbivory for all leaves on I. sierrae with respect to number of extra extrafloral nectaries EEFNs per leaf. b. Percent herbivory de creases as number of EEFN increases with standard error Appendix A, Table 2 Exponential Regression Analysis, R 2 = 0.9413, P < 0.0001. ______________________________________________________________________________ ___________________________________ ___________________________________________ Figure 9 a. Percent herbivory for leaves on I. sierrae found in the planted population with respect to number of extra extrafloral nectaries EEFNs per leaf. b. Percent herbivory for leaves found in natural popu lations. Percent herbivory decreases as number of EEFN increases with standard error Appendix A, Table 5 and 7 Polynomial Regression Analysis, R 2 = 0.8932, P < 0.0001, Polynomial Regression Analysis, R 2 = 0.885, P < 0.0001. ______________________________________________________________________________

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______________________________________________________________________________ Figure 10. Percent herbivory for all leaves of I. sierrae with respect to number of leaflet pa irs per leaf with standard error Appendix A, Table 3. As the number of leaflet pairs increases, the percent herbivory decreases Logarithmic Regress ion Analysis, R 2 = 0.9905; P < 0.0001 and 1 way ANOVA, P = 2.62E 35. ______________________________________________________________________________ ______________________________________________________________________________ Figure 11. Percent herbivory for a. planted and b. natural population leaves of I. sierrae with respect to number of leaflet pairs per leaf with standard error Appendix A, Tabl e 6 and 8. As the number of leaflet pairs increases, the percent herbivory decreases Polynomial Regression Analysis, R 2 = 0.9128; P < 0.0001 and R 2 = 0.9998; P < 0.0001. ___________________________________________________________ ___________________

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______________________________________________________________________________ Figure 12. Percentage of leaves with galls on I. sierrae with respect to number of extra extrafloral nectaries EEFNs per leaf with standard deviation Ap pendix A, Table 4. As number of EEFNs increases the percentage of leaves with galls decreases Simple Regression Analysis. R 2 = 0.9865, P < 0.0001. ______________________________________________________________________________

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APPENDIX A. ______________________________________________________________________________ Table 1. The differences between the average number of EEFNs, EFNs, and percentage of herbivory of the trees in the pasture and secondary forest edge a nd those planted by the Masters € An unpaired T test was used to examine the differences between the EEFNs, leaflet pairs, and percent herbivory on trees that were planted and those found in nature. ______________________________________________________________________________ Planted Trees Trees in Natural Populations P value Average # of EEFNs 1.62 0.04 < 0.0001 Range 0 9 0 2 Standard Error 0.0076 0.1764 Average # of Leaflet Pairs 3.11 2.94 < 0.0001 Range 0 4 0 4 Standard Error 0.355 0.362 Average % of Herbivory 17.12 27.91 < 0.0001 Range 0 100 0 100 Standard Error 0.481 0.7278 Top Population ______________________________________________________________________________ Table 2. The average, maximum number for range and standard error for number of EEFNs per leaf for the entire sample. The minimum number for range for all categories was zero. All numbers are rounded to the nearest tenth. ______________________________________________________________________________ 0 1 2 3 4 5 6 7 8 9 Average 2.2 10.6 8.3 6.4 5.1 1.9 1.7 0 1.4 .8 Range 100 56.1 32.3 21.2 15.4 5.6 15.6 0 8.1 2.3 Standard Error 0.4 0.8 0.7 1.1 0.7 0.3 0.2 0 0.7 0.8 ______________________________________________________________________________ Table 3. The average, range and standard error fo r number of leaflet pairs per leaf for the entire sample. All numbers are rounded to the nearest tenth. ______________________________________________________________________________ 1 2 3 4 Average 81.2 52.7 22.8 12.4 Range 45.7 100 0 100 0 99 0 38.3 Standard Error 17.7 1.7 0.4 0.49

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_______________________________________________________________________ _______ Table 4. The average, maximum number for range and standard error for number of galls per leaflet pair with respect to EEFNs. The minimum number for range for all categories was zero. ______________________________________________________________ ________________ 0 1 2 3 4 5 6 7 8 9 Average 0.2673 0.0133 0.0087 0.0037 0.0025 0.0008 0.0050 0 0.0004 0 Range 45 4 4 2 1 1 2 0 1 0 Standard Deviation 2.20 0.19 0.17 0.07 0.05 0.03 0.09 0 0.02 0 Planted Populations __________________________________________________ ____________________________ Table 5. The average, maximum number for range and standard error for number of EEFNs per leaf for planted populations. The minimum numbe r for range for all categories was zero. ______________________________________________________________________________ 0 1 2 3 4 5 6 7 8 9 Average 25.4 10. 5 8. 2 5.7 5.1 1.9 1. 8 0 1.4 0.78 Range 100 56.1 23.3 2 1.2 15. 7 5.6 15.6 0 8.1 2.3 4 Standard Error 0.008 0.004 0 .034 0.064 0 .067 0 .157 0. 01 2 0 0. 301 0. .0387 ______________________________________________________________________________ Table 6. The average, range and standard error for num ber of leaflet pairs per leaf for planted populations. All numbers are rounded to the nearest tenth. ______________________________________________________________________________ 1 2 3 4 Average 57.15 46.24 17.89 0.687 Range 47.5 100 14.3 89 0 89.2 0 29.4 Standard Error 3.098 0.247 0.005 0.030 Natural Populations _____________________________________________________ _________________________ Table 7. The average, maximum number for range and standard error for number of EEFNs per leaf for natural populations. The minimum number for range for all categories was zero. ____________________________________________________ __________________________ 0 1 2 3 4 5 6 7 8 9 Average 28.45 10. 87 9.06 0 0 0 0 0 0 0 0 Range 99 5 4.3 32.32 0 0 0 0 0 0 0 0 Standard Error 0.003 0.083 0.376 0 0 0 0 0 0 0 0

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______________________________________________________________________________ Table 8. The average, range and standard error for number of leaflet pairs per leaf for natural populations. _______________________________________ _______________________________________ 1 2 3 4 Average 97.8 53.22 25.84 11.91 Range 97.8 0 100 0 99 0 38.3 Standard Error 0 0.032 0.003 0.055