1 Herbivory and alkaloid concentration in Cecropia obtusifolia (Cecropiaceae) trees with and without ants Katherine A. Riha University of Wisconsin Madison ________________________________________________________________________ ABSTRACT Herbivore damag evolved various defenses. Cecropia obtusifolia has a facultative mutualism with Azteca ants. Selection could favor plants without ants to compensate in some way, like increasing concen trations of secondary compounds. In this study, percent herbivory and alkaloid concentration were measured in C. obtusifolia trees with and without ants in Monteverde, Costa Rica. Herbivory levels were low in both occupied ( mean sd = 2.702 2.139 ) and unoccupied (3.029 3.662) trees, and no significant difference was found in the concentration of alkaloids ( mean sd with ants = 0.57 0.34, without ants = 0.63 0.45) These results could be a consequence of the dry season. RESUMEN El exito repro ductivo de las plantas se ve altamente afectado por la herbivora debido a esto, las plantas han evolucionado varias defensas. Cecropia obtusifolia tiene un mutualismo facultativo con hormigas del gnero Azteca La seleccin puede favorecer plantas sin hormigas para compensar de alguna manera, como aumentando la concentracin de compuestos secundarios. En este estudio, el porcentaje de herbivora y concentracin de alcaloides se midio para plantas de C. obtusifolia con y sin hormigas en Monteverde, Cost a Rica. Los niveles de herbivora fueron bajos tanto en plantas ocupadas ( promedio ed = 2.702 2.139 ) como desocupadas (3.029 3.662), y no existen diferencias en la concentracin de alcaloides (promedio ed con hormigas = 0.57 0.34, sin hormigas = 0.63 0.45). Estos resultados son una consecuencia de la poca seca. INTRODUCTION Plants employ a variety of defense mechanisms, both physical and chemical, to protect them from herbivore attack. Some defenses include trichomes (Traw and Feeny, 2008 ), spines (Gowda and Palo, 2003), leaf toughness (Loney et al., 2006), alkaloids (Elger et al., 2009), cyanogenic glycosides (Goodger and Woodrow, 2002), and phenolics (Elger et al., 2009). These mechanisms are believed to increase plant fitness in the pr esence of herbivores, but also incur costs. Because of this, they are often found in high levels in seedlings and juveniles, which can experience a large reduction in fitness with herbivore damage (Gowda and Palo, 2003).
2 Another way plants may defend th emselves is by evolving and maintaining specialized ant mutualisms. For example, in mainland Costa Rica, three of four Cecropia species have evolved mutualisms with Azteca ants that nest within their hollow trunks. The ants are therefore provided with ho using and also a proteinaceous food source from the Mllerian bodies produced by the leaves. By housing Azteca ants, Cecropia trees are aggressively defended: the ants attack herbivores that try to feed on leaves (Longino, 1989). In a study by Schupp (19 86), unoccupied Cecropia had 5.4 times as much leaf damage as occupied saplings during the dry season. Vasconcelos and Casimiro (1997) had similar findings: leaf cutting ants attacked 71 100 percent of trees unoccupied by A. alfari colonies, while only 15 45 percent of occupied trees were attacked. Both studies concluded that ants play a role in decreasing herbivory, thereby increasing plant fitness. Cecropia trees unoccupied by ants may compensate by increasing their chemical defenses. In highly mutual istic and obligate associations, such as that between Acacia and Pseudomyrmex ants, plants with defending ants have no obvious chemical defenses (Janzen, 1966). Cecropia polyphlebia also does not house ants and it has been found to not only have low Mll erian body production (Janzen, 1973), but also slightly higher leaf alkaloid concentrations as compared to C. obtusifolia species with ant mutualisms (Gottheiner, 1998). Alkaloids are secondary constituents that are believed to provide herbivore defense d ue to their toxic effects on many animals (Hartmann, 1991). High alkaloid levels are known to reduce nutrient utilization, feed efficiency, animal productivity, and in some cases cause death in grazing animals and leaf feeding insects (Achakzai et. al, 20 09). Selection should favor Cecropia plants without ants to produce greater concentrations of chemicals toxic to herbivores (Smiley, 1986). In this study, I focused on a single Cecropia species, C. obtusifolia which has a facultative mutualism with A zteca ants (Schupp, 1986). Because of this, some individuals house ants, while others do not. In this paper, I compare percent herbivory and alkaloid concentrations in individuals of C. obtusifolia with and without ant mutualisms. I expected plants with out ants to have the capacity to compensate by having higher chemical defenses in their leaf tissue. METHODS SITE DESCRIPTION The study was conducted during the dry season in Monteverde, Costa Rica, between April and May 2010. Cecropia trees were foun d in San Luis (1,200 m), Bajo del Tigre (1,300 m), and Santa Elena (1,400 m), all of which are Premontane Moist Forests according to the characteristics of the Holdridge life zones. The mean annual rainfall is 2000 4000 mm, the mean annual temperature is 17 24 C, and the dry season is 4 5 months in duration (Haber, 2000). I chose C. obtusifolia that were 2 8 m tall. To be certain they were C. obtusifolia I chose only trees that had fruits near 50 cm in length, and had rough leaf surfaces with ten or m ore lobes. Once individuals were found, the tree trunks and/or branches were vigorously shaken to determine if ants were present. The third youngest leaves were clipped from trees with ants and trees without ants. Young plant parts generally contain gre ater levels of alkaloids as compared to old parts (Achakzai et al., 2009). Also, the third youngest leaves have lived adequate time to experience herbivore damage. And because young leaves are approximately half as tough, less fibrous, and significantly more nutritious
3 than mature leaves (Coley, 1983), herbivores may target them. Other than the absence of ants, there were no apparent differences between these groups of plants. HERBIVORY H erbivory was calculated for each leaf by placing the leaf under a transparent, plastic grid. First, the total number of squares occupied by leaf tissue was counted. Then, the total number of squares missing leaf tissue was counted. These squares had fifty percent or more leaf tissue removed. Percent herbivory was equ al to the total number of squares missing divided by the total number of squares times 100. ALKALOID CONCENTRATION Collected leaves were dried for at least 12 hours, or until they were completely dry. A mortar and pestal was then used to break the leave s into small pieces and 4.0 g samples were weighed out. The samples were saturated with methanol, and left soaking for 12 15 hours. In the morning, the methanol was filtered out, and then the beakers containing methanol were heated until all methanol eva porated. Next, 5 mL of 2N H 2 SO 4 was added to the beaker. The solution was then transferred to a test tube and washed 3 times with equal volumes of chloroform. The chloroform layers were discarded each time. NH 4 OH basified the solution to a pH of 10. T he alkaloids were again washed 3 times with equal volumes of chloroform. This time, the chloroform layers were preserved in test tubes. The test tubes were placed in a hot water bath to evaporate the chloroform. Now isolated alkaloids were mixed with 4. 0 mL of 0.1N H 2 SO 4 530 nm measured transmittance (Scalley, 1993). RESULTS Eighteen leaves from trees with ants and twenty one leaves from trees without ants were collected and measu red. HERBIVORY The percent herbivory of trees with ants (mean sd = 2.702 2.139) and the percent herbivory of trees without ants (3.029 3.662) were not significantly different (t test, df = 37, t = 0.334, P = 0.7405, see Figure 1). Plants with an ts had 0.9% to 8.9% leaf tissue removed, and those without had 0.2% to 12.5% removed. Figure 1. Mean percent herbivory ( SD) of C. obtusifolia trees with (n=18, 2.702 2.139 ) and without (n=21 3.029 3.662 ) ants. Percent herbivory is not significantly different between the two groups (t test, df=37, t=0.334, P=0.7405).
4 ALKALOID CONCENTRATION Alkaloid concentrations of leaves were not significantly different between plants with ants (n=5) and plants without ants (n=7) (Mann Whitne y u test, df = 1, chi squared = 0.0595, P = 0.8072, see Figure 2). No relation was found between alkaloid concentration and percent herbivory ( Spearman rank z = 0.53, P = 0.59, see Figure 3). Figure 2. Mean absorbance ( SD) of C. obtusifolia tr ees with (n=5, 0.57 0.34) and without (n=7, 0.63 0.45) ants. Transmittance was measured with a spectrophotometer and the values were converted to absorbance with the equation absorbance = log(transmittance/100). No difference was found between the two plant groups (Mann Whitney U test, df=1, chi squared = 0.0595, P = 0.8072). Figure 3. Absorbance versus percent herbivory in C. obtusifolia trees with and without ants. No relation was found between alkaloid concentration and percent herbivory ( Spearman rank, z = 0.53, P = 0.59).
5 DISCUSSION Herbivory percentages were very low for both occupied and unoccupied plants. This study was conducted in April, the end of the dry season, and therefore it is probable that herbivores were less active. Coley (1983) observed that 46% of pioneer species were grazed during the dry season, wh ereas 89% of species were grazed in the wet season. The decrease in abundance of herbivores during the dry season may explain why neither occupied nor unoccupied plants experienced much herbivory in this study. The results of a study done by F veri and Va sconcelos (2004) strongly suggested that when Cecropia suffers low incidence of attack, it is able to survive in the absence of defensive ants. Cecropia trees respond to draught in various ways. For example, host tree Mllerian body production decreases and internode humidity also decreases to damaging levels during the dry season. Branch exit holes are also smaller and fewer (Longino, 1991), perhaps an adaptation for reducing water loss. This suggests that trees allocate resources differently during t he dry season, thus possibly affecting their ability to house ants. A. alfari does not forage away from its host resources (Vasconcelos and Casimiro, 1997). Plants may therefore not house ants during the dry season when resources are limited. Or, plants may induce ant activity only in the presence of herbivores (Agrawal and Rutter, 1998). However, the individuals studied showed much variation in the amount of herbivory. Azteca species were not identified in this study, and they vary greatly in their behaviors towards herbivores (Longino, 1991). The amount of tissue removed in an individual most likely depends on which species of Azteca is occupying the plant. Furthermore, A. alfari the least aggressive of all Cecropia inhabiting Azteca is one of the two most abundant species in Costa Rica. Perhaps occupied individuals in this study were housing A. alfari and were only weakly protected, therefore having similar herbivory rates as unoccupied individuals. Frequ ently chopped roadsides and agricultural land generally inhabit Cecropia tress with A. alfari (Longino, 1991) and collected leaves came from roadsides in Santa Elena and pasture land of San Luis. Regarding alkaloids, they may be an induced defense in th is species, only being resistant to herbivory when necessary and can allocate more resources to growth and reproduction when they are not under attack (Karban et. al, 1997 ). The low levels of alkaloids found in this study can be explained by inducible defenses: the lack of insects resulted in low herbivory, and the lack of herbivores resulted in low alkaloid production. It is also possible that alkaloids are not an indu cible defense in this species. Instead, the low concentrations found may be sufficient in protecting the leaves, resulting in the low herbivory found here. A constant alkaloid concentration may mean that ants are unreliable, and plants instead produce al kaloids for protection, and they are effective even at low levels. AKNOWLEDGEMENTS especially his help with herbivory and alkaloid measurements. Thank you Y. Araya for
6 your statistics mastery; and thank you M. Caldern for assisting me in collecting my leaf samples. LITERATURE CITED Achakzai, A.K.K., P. Achakzai, A. Masood, S.A. Kayani, and R.B. Tareen. 2009. Response of plant parts and age on the distribution of se condary metabolites on plants found in Quetta. Pak. J. Bot. 41(5): 2129 2135. Agrawal, A.A. and M.T. Rutter. 1998. Dynamic anti herbivore defense in ant plants: the role of induced responses. Oikos 83: 227 236. Barto, E.K. and D. Cipollini. 2005. Testi ng the optimal defense theory and the growth differentiation balance hypothesis in Arabidopsis thaliana Oecologia 146: 169 178. Coley, P.D. 1983. Herbivory and defensive characteristics of tree species in a lowland tropical forest. Ecological Monogra phs. Vol. 53 no. 2 pp.209 229. Elger, A., D.G. Lemoine, M. Fenner, and M.E. Hanley. 2009. Plant ontogeny and chemical defence: older seedlings are better defended. Oikos 118: 767 773. Fveri, S.B. and H.L. Vasconcelos. 2004. The Azteca Cecropia associati on: are ants always necessary for their host plants? Biotropica 36(4): 641 646. Goodger J.Q.D. and I.E. Woodrow. 2002. Cyanogenic polymorphism as an indicator of genetic diversity in the rare species Eucalyptus yarraensis (Myrtaceae). Functional Plant Biology 29: 1225 1452. Gottheiner, D.M. 1998. An analysis of the relationship between alkaloids and herbivory in Cecropia obtusifolia and Cecropia polyphlebia EAP Tropical Biology Program Fall 1998. Gowda, J.H. and P.R. Thomas. 2003. Age related chang es in defensive traits of Acacia tortilis Hayne. African Journal of Ecology 41: 218 223. Haber, W.A. 2000. Monteverde Ecology and Conservation of a Tropical Cloud Forest. Oxford University Press Inc. New York, NY. Hartmann, T. 1991. Alkaloids. Herbivor es their interactions with secondary plant metabolites. Volume 1: 79 121. Academic Press Inc, San Diego. Janzen, D.H. 1973. Dissolution of mutualism between Cecropia and its Azteca ants. Biotropica 5: 15 28. Karban, R., A.A. Agrawal, and M. Mangel. 19 97. The benefits of induced defenses against herbivores. Ecology 78: 1351 1355. Loney, P.E., C. McArthur, B.M. Potts, and G.J. Jordan. 2006. How does ontogeny in Eucalyptus species affect patterns of herbivory by Brushtail Possums? Functional Ecology 2 0:982 988. Longino, J.T. 1989. Geographic variation and community structure in an ant plant mutualism: Azteca and Cecropia in Costa Rica. Biotropica 21(2): 126 132. 1991. Taxonomy of the Cecropia inhabiting Azteca ants. Journal of Natural History 25: 1571 1602. Scalley, M.L. 1993. Effectiveness of defense tradeoffs in two species of Cecropia EAP Tropical Biology Program Fall 1993. Schupp, E.W. 1986. Azteca protection of Cecropia : ant occupation benefits juvenile trees. Onecologia 70(3): 379 385.
7 Smiley, J. 1986. Ant constancy at Passiflora extrafloral nectaries: effects on caterpillar survival. Ecology 67(2): 516 521. Traw M.B. and P. Feeny. 2008. Glucosinolates and trichomes track tissue value in two sympatric mustards. Evology 89:763 772. Vasconcelos, H.L. 1991. Mutualism between Maieta guianensis Aubl., a myrmecophytic melastome, and one of its ant inhabitants: ant protection against insect herbivores. Oecologia 87: 295 298. Vasconcelos, H.L. and A.B. Casimiro. 1997. Influence of Azt eca alfari ants on the exploitation of Cecropia trees by a leaf cutting ant. Biotropica 29: 84 92. Zuchowski, W. 2005. A guide to tropical plants of Costa Rica. Zona Tropical, S.J.O.
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Riha, Katherine, A.
La herbivora y la concentracin del alcaloide en los rboles de Cecropia obtusifolia (Cecropiaceae) con y sin hormigas
Herbivory and alkaloid concentration in Cecropia obtusifolia (Cecropiaceae) trees with and without ants
Herbivore damage greatly impacts a plants fitness, and because of this, plants have
evolved various defenses. Cecropia obtusifolia has a facultative mutualism with Azteca ants. Selection could favor plants without ants to compensate in some way, like
increasing concentrations of secondary compounds. In this study, percent herbivory and alkaloid concentration were measured in C. obtusifolia trees with and without ants in
Monteverde, Costa Rica. Herbivory levels were low in both occupied (mean sd = 2.702
2.139) and unoccupied (3.029 3.662) trees, and no significant difference was found in the concentration of alkaloids (mean sd with ants = 0.57 0.34, without ants = 0.63 0.45). These results could be a consequence of the dry season.
El xito reproductivo de las plantas se ve altamente afectado por la herbivora debido a esto, las plantas han evolucionado varias defensas. Cecropia obtusifolia tiene un mutualismo facultativo con las hormigas del gnero Azteca. La seleccin puede favorecer a las plantas sin hormigas para compensar de alguna manera, como aumentando la concentracin de compuestos secundarios. En este estudio, el porcentaje de herbivora y la concentracin de alcaloides se midi para las plantas de C. obtusifolia con y sin hormigas en Monteverde, Costa Rica. Los niveles de herbivora fueron bajos tanto en las plantas ocupadas (promedio ed = 2.702 2.139) como desocupadas (3.029 3.662), y no existen diferencias en la concentracin de alcaloides (promedio ed con hormigas = 0.57 0.34, sin hormigas = 0.63 0.45). Estos resultados son una consecuencia de la poca seca.
Text in English.
Costa Rica--Puntarenas--Monteverde Zone
Costa Rica--Puntarenas--Zona de Monteverde
Tropical Ecology Spring 2010
Ecologa Tropical Primavera 2010
t Monteverde Institute : Tropical Ecology