1 Anthocyanins and Delayed Greening: Inhibition of Leafcutter Ants ( Atta cephalotes ) in Inga punctata (Mimosaceae) and Zygia palmana (Fabaceae) Antoinette Hertel Department of Biology, University of Wisconsin Madison Abstract Leaves that undergo d elayed g reening with anthocyanins are suspected of having anti herbivore defenses in addition to low leaf nutrient content. This study examines two species with delayed greening, one with anthocyanins, Zygia palmana , and one without, Inga punctata , to determine w hether anthocyanins deter herbivores more than low nutritional quality . Leaf samples were taken from 20 trees in Monteverde, Costa Rica. Chlorophyll a, chlorophyll b, carotinoids and anthocyanins were extracted from leaves and tested for concentrations a s well as chemical preference to an herbivore, Atta cephalotes . T here was a significant preference for older, fully green le aves in each species (p=.041), as well as a preference for the young leaves of Z. palmana that have little anthocyanins (p=.0009) . This suggests that , while leafcutters prefer older leaves with higher nutritional content, anthocyanins further help to deter herbivores from attacking young leaves . Resumen Las hojas que presentan clorosis retardada utilizando antocianinas, se cree qu e presentan defensas anti herbivÃ³ricas en adiciÃ³n a un bajo contenido de nutrientes. Este estudio examinÃ³ dos especies con clorosis retardada, una con antocianinas, Zigia palmata y una sin, Inga punctata para determinar si las antocianinas repelen a los herbivoros mas que un bajo contenido nutricional. Las muestras de las hojas fueron tomadas de 20 arboles en Monteverde, Costa Rica. ClorofÃla A, clorofila B, carotenoides y antocianinas fueron extraidas de las hojas, las concentraciones fueron estimadas y la preferencia por los hervÃboros probada ( Atta cephalotes ). Hubo preferencia por hojas viejas totalmente verdes (p=0.041), tambiÃ©n hubo preferencia por hojas jovenes de Z. palmata que tuvieron pocas antocianinas (p=0.0009). Esto sugiere que mientras las z ompopas prefieren hojas mas viejas con mayor contenido nutricional, las antocianinas ayudan a repeler herbivoros.
2 Introduction P lants have developed different strategies to combat herbivory (Soule and Werner 1981, Pastor and Bridgeham 1999). Delayed gre ening occurs when plants invest little energy in young leaves that are particularly susceptible to herbivory ( Kursar and Coley 1992, Coley et al . 2005 ) . For example, chlor oplasts and chlorophyll in young leaves with delayed greening are generally 10% of t hat in old er leaves (Kursar and Cole y 1992 F. Ecology, Whatley 1992 ). B ecause of this newer leaves are less nutritional, so herbivores may not be as inclined to eat them (Kursar and Coley 1992) . As these leaves age they get tougher and less susceptible t o herbivory ( Kursar and Cole y 1992 F. Ecology, Kursar and Coley 1992, Coley et al . 2005 ). At this point, it makes energetic sense to invest in chloroplasts and the leaves become green. Delayed gree n ing is important in tropical understory plants that have little access to light (Rundel and Gibson 1996). Therefore, leaves are particularly expensive, since light is limiting photosynthesis, and any loss to herbivores has a greater impact on fitness (Coley and Barone 1996) . Some delayed greening results in young leaves that are white or very light green; however , certain plants anthocyanins , are red . Anthocyanins are flavenoids found in young leaves that often break down as the leaf ages (Coley and Aide 1989 , Lee and Collins 2001 ) . They may protect leaves from photoinhibition caused by ultraviolet wavelengths. In addition anthocyanins are photosynthetically active, yet demand less energy than chlorop hylls , allowing young leaves to photosynthesize, though at a lower rate (Steyn et al . 2002 , H och et al . 2003 , Pomar and BarcelÃ³ 2007 ) . Anthocyanins may also provide a visual cue for herbivores, indicating low leaf quality (Karageorgou and Manetas 2006 , Lee and Lowry 1980, Close and Beadle 2003). Anthocyanins may have antifungal properties that protect leaves f rom pathogens ( Chalker Scott 1999, Coley and Aide 1989 ) . If so they may also protect leaves from leafcutter ants that grow fungal gardens to feed their brood (Close and Beadle 2003). The leaf cutter ant, Atta cephalotes , is an important herbivore in the understory that avoids plants with antifungal agents while targeting tender leaves with high nutrients (Rockwood 1976, Chalker Scott 1999, Rockwood 1977 , Hubbel l et al . 1983 ) . Previous studies have shown that leafcutters selectively chose leaves without a nthocyanins. One study showed that A. cephalotes was repelled 20 times more often when anti fungal agents were added to the leaf (Hubbell et al . 1983). In addition is has been found that anthocyanins are harmful to the fungus gardens of Atta ants and whe n offered leaf discs of 20 different anthocyanin containing species, leafcutters selected discs with low anthocyanin concentrations (Coley and Aide 1989). However, all of these studies simply examined anthocyanin concentrations and did not factor in nutrie nt differences with age. Therefore, a lthough they avoid plants with anthocyanins and delayed greening, it is unclear if their avoidance is due to a lack of nutritional content in the young leaves or the presence of anthocyanins. This experiment will ev aluate the importance of low nutritional content and possible anti herbivore properties of anthocyanins in young leaves of plants with delayed greening. In order
3 to explore this , I will compare chemical components of two closely related species , Inga punc tata ( Mimosaceae) and Zygia palmata (Fabaceae). I. punctata has delayed greening and no visible anthocyanins , while Z. palmata has delayed greening with anthocyanins. The plants will be tested for herb ivor e preferences in relation to the age of leaves an d in relation to a nthocyanin presence . It has been shown that leaves with low nutrient content have one forth the photosynthetic activity (Longstreth and Nobel 1980). Therefore n utritional variation will be based on the concentrations of photosynthetic c hemical compounds that are found in high concentrations when plant nutrients are high (Krause and Weis 1991 , Longstreth and Nobel 1980 ) . These photosynthetic compounds include c hlorophyll a, chlorophyll b and carotinoids. H igher concentrations will show greater nutrient levels in leaves . It is expected that ants will show preference for older leaves with more nutrient content as well as show a preference for leaves without anthocyanins. Methods Study site and organisms This study was conducted in Mont everde, Costa Rica from April to May 2010 . Two species with delayed greening were chosen: Zygia palmana and Inga punctata. Z. palmana has red young leaves, which is a sign of having high concentrations of anthocyanins . I. punctata , on the other hand, ha s young leaves that are almost white with no sign of red. In addition, belong to the same tribe, Ingeae . T o test the effects of delayed greening , leaf samples were harvested, tested for concentration of photosynthetic pigments, including anthocyanin and then presen ted to Atta cepha lotes , leafcutter ants . Leaf collection Samples of young, adolescent, and mature leaves were collected from 10 different plants in each species I. punctata and Z. palmana . Leaf age was determined based on color and texture o f the leaves. O ld er leaves were greener and tougher than young leaves in both species. After collection leaves were immediately taken to the lab for extractions. Chemical concentrations Chemical extractions were used to quantify concentrations of photos ynthetic pigments . I tested for concentrations of ch lorophyll a, chlorophyll b, carotinoi ds, and anthocyanins. Two grams of leaf material were measured, cut into small pieces, and placed into a beaker. Seven milliliters of 85% acetone was added to the b eaker , along with 2ml of 6.5 phosphate buffer (used to mimic chloroplast conditions) . The mixture was the n allowed to sit for 15 minutes with shaking every 5 minutes fo r 30 seconds . After 15 minutes , I poured the mixture into a test tube and centrifuged it at 4000 rpm for 2 minutes. The s olution was the n decanted; 2ml of the solution was added to 8ml of 85% acetone and transferred into a cuvet. I measured concentration levels using a spectro photo meter . Percent t ransmittance r eadings were recorded at 66 3nm, 646nm, 470nm, 532nm, and 653nm. I converted the readings to absorbance with the equation: Abs= log(%T/100)
4 The level of a bsorbance was then used to calculate the concentrations of the desired plant chemicals (Lichtenthaler and Welbur 1983) . Chlorophy ll a (mg/g)= [12.21Abs (663) 2.81Abs (646) ] x Purified volume(ml ) 200(leaf mass(g)) Chlorophyll b (mg/g)= [20.13Abs (663) 5.03Abs (646) ]x Purified volume(ml ) 200(leaf mass(g)) Carotinoids (mg/g)= [ 1000Abs (470) 3.27(chl a) 1049(chl b)]x Purifie d Volume(ml) 45400(leaf mass (g)) Anthocyanins (mg/g)= Abs (532) 0.25Abs (653) 2.45x10^4(leaf mass (g) ) Leafcutter ant preferences To test herbivore preference to leaf chemical compositions young, adolescent, and mature leaves were collected from the same twenty plants. E xtractions were per formed by measuring 3.75g of leaves and cutting them i nto small pieces . Leaf cuttings were placed into a bowl and ground for 1 minute . I added 20ml of 80% methanol and 1% HCl in a 1:1 ratio and continued g rind ing the leaves for another 7 minutes. The mixture was then filtered into a vial and stored in the dark until needed . E xtractions were tested on A. cephalotes while they foraged on trails . The extractions were taken to an ant nest and tests were preform ed 7m away from a nest entrance . Oat flakes w ere dipped into the extractions , allowed to dry and the liquid coated oats were placed on the , in a line , spaced approximately 3cm apart . Six oats were observed for 15 minutes per test . Each test contained a young, adolescent, and old leaf for two different plants. Selection of an oat was determined by observation of any form of oat movement toward the nest caused by an ant (i.e. pushing or picking up). Any time an oat was se lected the oat was removed from the line and a new oat with the same coating was used to replace it. Results Chemical Concentrations Overall , a nthocyanin concentrations (Fig. 1.a) showed a significant decline with age (Anova, df=2, F=6.238, p=0.0037) . Ho wever when plant species were considered separately, this difference was largely due to Z. palmana. Zygia palmana showed very high concentrations of anthocyanins in young leaves (meanÂ± sd=7.71E 6 Â± 7.82E 6 mg/g ) w hile medium (meanÂ± sd=1.05E 6 Â± 3.05E 6 mg/g ) and old (meanÂ± sd= 6.01E 7 Â± 3.42E 6 mg/g ) showed significantly less (Anova, df=2, F=7.07, p=.0034). H owever concentrations of anthocyanins in I. punctata did not differ with age (Anova, df=2, F=.191, p=0.8272 ) due to a very small difference in concentr at ion levels between young(meanÂ± sd=1.7E 6 Â± 1.27E 6 mg/g ) ,
5 medium (mean Â± sd=2.08E 6 Â± 1.99E 6 mg/g ), and old (meanÂ± sd= 1.62E 6 Â± 2.00E 6 mg/g ) leaves. Chlorophyll a showed an increasing concentration with age (Fig 1.b). Levels of c hlorophyll a in Z. palma na increased from young (mean Â± sd=0.14 Â± 0.06 mg/g ) to medium (mean Â± sd=0.21 Â± 0.11 mg/g ) and finally showed the highest concentrations in old leaves (mean Â± sd = 0.34 Â± 0.22 mg/g ). Likewise, Inga punctata showed an increase in concentrations from young (mean Â± sd= 0.07 Â± 0.02 mg/g ), medium (mean +/ sd= 0.14+0.08 mg/g ), to old (meanÂ± sd= 0.19 Â± 0.03 mg/g ). This increase with age was significant (An ova, df=2, F=10.32, p=0.0002). Chlorophyll b also increased w ith age (Fig 1.c.) . In I. punctata chlorophyll b increas ed from young (mean Â± sd= 0.066 Â± 0.052 ) to medium (mean Â± sd= 0.13 Â± 0.12) to old (mean Â± sd= 0.14 Â± .064) . This pattern was different in Z. palmana in that medium aged leaves had the highest concentration (young : mean Â± sd= 0.092 Â± 0.063 , medium : mean Â± sd= 0.13 Â± 0.067 , old: mean Â± sd= 0.095 Â± 0.13 ). Yet the overall trend between the two species showed a significant increase in concentration (Anova, df=2, F=3.28, p=0.045). Carotinoids (Fig 1.d.) showed a significant increase in both species (Anova, df=2, F= 12.57, p <0.00 01). Both plants increased from young ( I. punctata : meanÂ± sd=0.064Â±0.026; Z. palmana : meanÂ± sd=0.085Â±0.036) , to medium ( I. punctata : meanÂ± sd= 0.11Â±0.064 ; Z. palmana : meanÂ± sd= 0.10Â±0.037 ), to old ( I. punctata : meanÂ± sd= 0.13Â±0.035 ; Z. palmana : meanÂ± sd=0.20 Â±0.11). (a)
6 (b) (c)
7 (d) Figure 1. Concentration levels of a) anthocyanins, b)chlorophyll a, c)chlorophyll b, and d) carotinoids in young, medium, and old leaves of I. punctata and Z. palmana . Ten of each species were found in the Monteverde area and tested . Preference of extractions Leafcutter ants (Fig 2) preferred older leaves of I. punctata (Friedman R ank T est, df=2, Chi square=6.41, p=0.041). Oats with young leaf extracts were taken least often ( 1.7 flakes/15 minutes Â±1.5) followed by medium ( 2.8 flakes/15 minutesÂ±1.4) and old ( 2.8 flakes/15 minutes Â±1.5). Multiple comparison tests showed preference for older leaf extracts (Y M: M ultiple C omparison T est, df=1, q=3.06; Y O: M ultiple C omparison T est, df=1, q=2.69; M O: M ultiple C omparison T est, df=1, q= 0.32 ) . There was also a significant difference in preference for older leaves Z. palmana (Friedman Rank Test, df=2, Chi square=14, p=0.0009) . Young ( 0.40 flakes/15 minutes Â±0.52) were taken considerably less in comparison to medium (2.6 flake s/15 minutes Â±0.84) and old (2.8 flakes/15 minutes Â±2.0). Additionally , there were significant relationships in preference between young and medium (Multiple Comparison Test, df=1, q= 4.74) and young and old (Multiple Comparison Test, df=1, q=3.79).
8 Figur e 2. Number of selections of young, medium and old leaf extractions of I. punctata and Z. palmana by A. cephalotes , tested at two different nests in the Montverde area on ant foraging trails. Discussion C oncentrations of pigments varied with age in both species . Chlorophyll a, chlorophyll b, and carotinoids sho wed an increase from young to old leaves in both species . This incr ease mirrored the delayed greening in both species , as the older leaves had more of these photo synthetic compounds . This might a lso reflect the higher nutritional content of older leaves increase in photosynthetic properties not only gave evidence of more photosyn thetic abilities of older leaves (Krause and Weis 1991 , Longstreth and Nobel 1980 , Droop et al . 1982) . The loss of chlo rophyll b in old est leaves of Z. palmana is representative of leaf age. It is likely that the leaves chosen to represent the oldest category were already beginning to break down their chlorophyll and die due to age (Bachmann et al . 1994) . The results a lso show a clear decline in anthocyanins in Z. palmana which was expected . This shows that as leaves toughen and age , they lose their need for anthocyanins in their leaves. In regard to I. punctata , the low but consistent amount of anthocyanins w as surpr ising. One explanation for their presence is that they are present in leaves to provide photo protection but are not high enough in concentration to turn the leaves red (Steyn et al . 2002, Hoch et al . 2003) . Previous studies show that leaf cutter ants avoid leaves with low nutrients as well as those with anthocyanins ( Rockwood 1976 , Rockwood 1977, Hubbell et al . 1983, Rockwood and Hubbell 1987). However it is not known whether anthocyanins are avoided due to their own
9 chemical properties or because the y are in young leaves with low nutrients. Others have suggested that they do not directly function as deterrents but are a visual indicator of other phenols in leafs with anthocyanins (Lee and Lowry 1980, Close and Beadle 2003 ). However, t his study resul ted in a strong trend showing that anthocyanins were deterring the ant herbivores in addition to showing the benefits of delayed greening . Additionally this study removed any visual component which suggests that visual cues are not important . Although I. punctata also showed a signif icant difference in ant intere st between young and old , the difference in young and old leaves of Z. palmana was considerably greater , suggesting that the presence of anthocyanins , in addition to low nutrie n ts , was effective i n discouraging ants from taking food . These results suggest that in addition to delayed greening , anthocyanins can provide plants an anti herbivore defense . Acknowledgements I would like to thank my professors Alan Masters, Anjali Kumar, and Pablo All en for their advice throughout this project. I would also like to thank the teaching assistants Moncho Calder Ã³ n and Yimen Araya for there assistance with all the littl e details . Also , thank you to the EstaciÃ³n BiolÃ³gica de Monteverde for use of their chemis try lab. Finally, thank you to the Arguedas family, Frank Joyce and Alan and Karen Masters for the use of their land during my study. Literature Cited Bachmann, Andre, Fernandez Lopez, Jose, Ginsburg, Samuel, Thomas, Howard, Bouwkamp, John C., Solomos, Theophanes, and Matile, Phillippe. 1994. Stay green genotypes of Phaseolus vulgaris L.: chloroplast proteins and chlorophyll catabolites during foliar senescence. Hew Phytologist. 126:593 600. Belovsky, Gary E. and Schmitz, Oswald J. 1994. Plant defenses a nd optimal foraging by mammalian herbivores. Journal of Mammalogy. 75: 816 832. Chalker Scott, Linda. 1999. Environmental significance of anthocyanins in plant stress responses. Photochemistry and Photobiology. 70:1 Close, Dugald C. and Beadle, Christopher L. 2003. The ecophysiology of foliar anthocyanin. Botanical Review. 69:149 161. Coley, Phyllis D. and Aide, T. Mitchell. 1989. Red coloration of tropical young leaves: a possible antifungal defense ? Journal of Tropical Ecology. 5:293 300. Coley, P.D. and Barone, J.A. 1996. Herbivory and plant defenses in tropical forests. Annual Review of Ecology and Systematics. 27:305 335. Coley, Phyllis D., Lokvam, John, Rudolph, Kathleen, Bromberg, Keryn, Sackett, Tara E., Wright, Leslie, Brenes Arguedas, Tania, Dvore tt, Dan, Ring, Seth, Clark, Alex, Baptiste, Caroline, Pennington, R. Toby and Kursar, Thomas A. 2005. Divergent defensive strategies of young leaves in two species of Inga. Ecology. 86: 2633 2643. Droop, M.R., Mickelson, M.J., Scott, J.M. and Turner M.F. 1982. Light and nutrient status of algal cells. Journal of the Marine Biological Association of the United Kingdom. 62: 403 434. Hoch, William A., Singsaas, Eric L. and McCown, Brent H. 2003. Resorption protection: Anthocyanins facilitate nutrient recovery in autumn by shielding leaves from potentially damaging light levels. Plant Physiology. 133:1296 1305. Howe, Henry F. and Westley, Lynn C. Ecological Relationships of Plants and Animals. Oxford University Press, Oxford and new York. 1988. Hubbell, Stephen P., Wiemer, David F., and Adejare, Adeboye. 1983. An antifungal terpenoid defends a neotropical tree (Hymenaea) against attack by fungus growing ants. Oecologia: 60:321 327.
10 Karageorgou, Panagiota and Manetas, Yiannis. 2006. The importance of being red when young: anthocyanins and the protection of young leaves of Quercus coccifera from insect herbivory and excess light. Tree Physiology. 26:613 621. Krause, G.H. and Weis, E. 1991. Chlorophyll fluorescence and photosynthesis: the basics. Annual Review of Plant Physiology and Plant Molecular Biology. 42:313 349. Kursar, T.A. and Coley, P.D. 1992. Delayed development of the photosynthetic apparatus in tropical rain forest species. Functional Ecology. 6: 411 422. Kursar, Thomas A. and Coley, Phyllis D. 199 2. Delayed greening in tropical leaves: an antiherbivore defense? Biotropica. 24: 256 262. Lee, David W. and Collins, Timothy M. 2001. Phylogenetic and ontogentic influences on the distribution of anthocyanins and betacyanins in leaves of tropical plants. International Journal of Plant Sciences. 162: 1141 1153. Lee, D.W. and Lowry J.B. 1980. Young leaf anthocyanin and solar ultraviolet. Biotropica. 12:75 76. Lichtenthaler, H. and Wellburn, A. 1983. Determinations of total carotinoids and chlorophylls a and b of leaf extracts in different solvents. Biochemistry Society Transactions. 11:591 592. Longstreth, David J. and Nobel, Park S. 1980. Nutrient influences on leaf photosynthesis. Plant Physiology. 65:541 543. Murrell, James T. and Wolf, Frederick T. 196 9. The anthocyanin of the autumn leaves of Japanese Barberry. Bulletin of the Torrey Botanical Club. 96: 594 596. Pastor, John and Bridgeham, Scott D. 1999. Nutrient efficiency along nutrient availability gradients. Oecologia. 118: 50 58. Pomar, F. and BarcelÃ³, A. Ros. 2007. Are red leaves photosynthetically active. Biologia Plantarum. 51:799 800. Rockwood, Larry L. 1976. Plant selection and foraging patterns of leaf cutting ants (Atta). Ecology. 57:48 61. Rockwood, Larry L. 1977. Foraging patterns and plant selection in Costa Rican leaf cutting ants. Journal of the New York Entomological Society. 85: 222 233. Rockwood, L.L. and Hubbell, S.P. 1987. Host plant selection, diet diversity, and optimal foraging in a tropical leafcutting ant. Oecologia. 74:55 61. Rundel, Philip W. and Gibson, Arthur C. 1996. Adaptive strategies of growth form and physiological ecology in neotropical lowland rainforest plants. Neotropical Biodiversity and Conservation. 33 71. Seigler, David and Price, Peter W. 1976. Secondary compounds in plants: Primary functions. The American Naturalist. 110:101 105. Soule, J.D. and Werner, P.A. 1981. Patterns of resource allocation in plants, with special reference to Potentilla recta. Bulletin of the Torrey Botanical Club. 108: 311 319. S tamp, Nancy. 2003. Out of the quagmire of plant defense hypothesis. The Quarterly Review of Biology. 78: 23 55. Steyn, W.J., Wand, S.J.E., Holcroft, D.M. and Jacobs, G. 2002. Anthocyanins in vegetative tissues: A proposed unified function in photoprotectio n. New Phytologist. 155: 349 361. Whatley, Jean M. 1992. Plastid development in distinctively coloured juvenile leaves. New phytologist. 120: 417 426.
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Antocianinas y clorosis retardada: Inhibicin de las hormigas zompopas (Atta cephalotes) en Inga punctata (Mimosaceae) y Zygia palmana (Fabaceae)
Anthocyanins and delayed greening: Inhibition of leafcutter ants (Atta cephalotes) in Inga punctata (Mimosaceae) and Zygia palmana (Fabaceae)
Leaves that undergo delayed greening with anthocyanins are suspected of having antiherbivore defenses in addition to low leaf nutrient content. This study examines two species with delayed greening, one with anthocyanins, Zygia palmana, and one without, Inga punctata, to determine whether anthocyanins deter herbivores more than low nutritional quality. Leaf samples were taken from 20 trees in Monteverde, Costa Rica. Chlorophyll a, chlorophyll b, carotinoids and anthocyanins were extracted from leaves and tested for concentrations as well as chemical preference to an herbivore, Atta cephalotes. There was a significant preference for older, fully green leaves in each species (p=.041), as well as a preference for the young leaves of Z. palmana that have little anthocyanins (p=.0009). This suggests that, while leafcutters prefer older leaves with higher nutritional content, anthocyanins further help to deter herbivores from attacking young leaves.
Las hojas verdes que presentan clorosis retardada con antocianinas, se sospecha que presentan defensas anti-herbvoras, adems de un bajo contenido de nutrientes. Este estudio examin dos especies con clorosis retardada, una con antocianinas, Zygia palmana y otra sin, Inga punctata para determinar si las antocianinas disuaden a los herbvoros ms que el bajo contenido nutricional. Las muestras de las hojas fueron tomadas de 20 rboles en Monteverde, Costa Rica. Clorofla a, clorofila b, carotenoides y antocianinas fueron extradas de las hojas, se examinaron las concentraciones, as como la preferencia de los productos qumicos en un herbvoro (Atta cephalotes). Hubo preferencia por las hojas viejas totalmente verdes (p=0.041), tambin hubo preferencia por las hojas jvenes de Z. palmata que tuvieron pocas antocianinas (p=0.0009). Esto sugiere que mientras las zompopas prefieren las hojas ms viejas con mayor contenido nutricional, las antocianinas ayudan a disuadir a los herbvoros.
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