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UV-Blocking Chemicals in Forest Canopy and Understory Lichens Katherine Rose Heal Department of Chemistry, University of Minnesota In stitute of Technology ___________________________________________________ _________________ ABSTRACT Lichens produce over eight hundred secondary metabo lites, many of which are found nowhere else in nature. Many of these chemicals have UV-blocking p roperties, including usnic acid and many depsides. These chemicals could affect colonization success b etween understory and canopy lichens. In Monteverd e, Costa Rica, a total of 244 lichens were extracted f rom six trees, half from canopy branch tips and hal f from the trunk in the understory. Canopy coverage was s ignificantly higher in the understory (t-test, F = 88.78, p < 0.05). In the pooled population, usnic acid was present in the branch tips more often than on the t runks (after Yates correction factor X2 c = 26.23, df = 1, p = 3.03E-07). Conversely, depsi des were present more often on the trunks (after Yates correction factor X2 c = 26.23, df = 1, p = 3.02E-07). The differences i n the chemical constituents, which are associated with di fferences in lichen community composition, may refl ect adaptations that allow niche partitioning along lig ht gradients. ___________________________________________________ ______ RESUMEN Lquenos producen ms que ochocientos metabolites s econdarios, muchos de que son descubiertos solo en lquenos. Muchos de estos qumicos tienen caracter sticos de UV-bloqueadores, incluyendo cido usnic y muchos depsides. Estos qumicos pueden afectar int eracciones en las comunidades de los lquenos el el dosel y sotobosque. En Monteverde, Costa Rica, un total de 244 lquenos fueron quitados de seis rbol es, mitad de las ramas y metad de los troncos. Haba m enos cobertura de ramas en el dosel (t-test, F = 88 .78, p < 0.05). En la poblacin total, cido usnic haba en lquenos de las ramas ms que en los troncos (de spus de correctin de Yates X2 c = 26.23, df = 1, p = 3.03E-07). En la otra mano, h aba ms lquenos con depsides en los troncos que en las ramas (despus d e correctin de Yates X2 c = 26.23, df = 1, p = 3.02E07). Las diferencas entre la composisin qumica puede ser de traslape de nicho entre la communidad. INTRODUCTION Many symbiotic relationships occur in nature, but few so tightly knit as the colonies of lichens. These growths are often mista ken as one organism, but are interactions between mycobionts and photobionts. Th e mycobiont, a fungus, is supplied with carbohydrates from the photobiont, a green alg a or cyanobacterium (Purvis 2000). As a result, lichen colonies of multiple photobiont s and fungal organisms are able to occupy more diverse, often harsher, regions than ei ther the mycobiont or the photobiont could occupy alone (Brodo 2001). Lichens are successful colonizers on a seemingly i nfinite number of substrates, from tree bark to tortoise shells. They are presen t in the coldest, hottest, driest, and wettest parts of the world (Brodo 2001). The cloud forest of Monteverde, Costa Rica is no exception. Lichens can be found on rocks on the forest floor as well as on branch tips
in the canopy. A survey in the Monteverde region revealed over 200 morphospecies of tree-dwelling lichens (Waring 2007). Little resear ch has examined the differences, if any, between these understory and canopy lichens. The chemistry of lichens is important for their su rvival. Lichens are known to produce several secondary chemical compounds for de fense against herbivory, pathogens, and UV irradiation (Huneck 1999). Some o f these secondary metabolites likely aid lichens successful colonization. Lichen s that produce anti-herbivory chemicals like usnic acid, perlatolic acid, and certain depsi des are more successful in areas that are subject to extensive herbivory, such as lichens pre yed upon by caribou in Alaska (Falk et al. 2007). Similarly, lichens producing higher con centration of UV-screening chemicals are more prevalent in more light-exposed areas (Bje rke and Dahl 2002). Usnic acid and atranorin (a depside) act as effective sun block for lichens (Solhoug et al. 2003). They are so effective, in fact, that they have been extr acted from lichens for human use (Rancan et al. 2002, Solhoug et al. 2003). Both us nic acid and depsides are common species-specific chemicals in lichens worldwide, al though the chemicals can differ significantly in concentration within one species ( Rundel 1969). There is a correlation between the production of usnic acid and exposure t o irradiation, suggesting the importance of the metabolite in successful coloniza tion (Bjerke et al. 2002, Rundel 1969). A previous study investigating the effect of light exposure on species richness of lichens revealed that, although the diversity of UV -blocking compounds was greater under brighter conditions, the percentage of UV-pro tected lichen morphospecies in the did not change with light intensity (Waring 2007). In fact, the frequency of morphospecies containing UV-blocking chemicals was over 85% along the entire light gradient (Waring 2007), demonstrating the importanc e of UV-blocking chemicals in lichen colonization of sunlit habitats. However, th e frequencies of different UV-blocking chemicals in sunlit and shady habitats have remaine d unstudied. Areas of success along gradients of those morphospecies could reveal colon ization dynamics such as niche partitioning among lichens. Although little is known about canopy lichens, I p redicted that canopy lichens on branch tips would contain UV-blocking secondary com pounds more often than the lichen colonies in the more shaded understory. I also exp ected to see differences in the types of UV-blocking chemicals depending on the site from wh ich the lichen was extracted. MATERIALS AND METHODS Specimen Collection This study was conducted in Lower Montane Rainfores t around the Estacin Biolgica in Monteverde, Costa Rica. I selected lichens from two microhabitats per tree (trunk at breast height and branch tips) on six canopy elemen t trees, and I extracted twenty lichens from each location on each tree. Lichens were stor ed for no more than 48 hours in paper bags until I performed laboratory tests. For each tree, I recorded the canopy coverage in the understory (trunk) and the canopy (tree crown) using a canopy densiometer to test for differences in sunlight exposure.
Experimental Methods To test for the presence of UV-blocking compounds, I performed chemical spot tests of K (10% KOH), C (undiluted household bleach), and KC ( C on filter paper with K previously applied) on each of the lichen colonies. To perform each spot test, I removed a small portion of the lichen (approximately 1 cm x 0.1 cm) and placed it onto a sheet of white filter paper. I then applied a drop (approxi mately 0.5 mL) of the appropriate solution to the lichen. After no more than 10 minu tes, I removed the lichen from the filter paper and recorded the color transferred to the filter paper using a hand lens (10X). Using Brodos Lichens of North America as guide to commonly encountered lichen substances I identified which, if any, secondary ch emical compounds were present in each specimen sample. Statistical Analysis I conducted a t-test to determine whether there wer e differences in the canopy coverage between the canopy and understory canopy coverage. I determined whether or not the six separate trees populations could be pooled by usin g a heterogeneity chi-squared test. A chi-squared one-sample test for goodness of fit was then used to characterize the observed frequencies of UV-blocking chemical produc ing lichens. RESULTS Two hundred and forty four lichens were extracted f rom six trees with at least twenty specimens collected from the two locations (branch tips and trunk) on each tree. Canopy coverage in the understory (91.68 % 1.6 %) and in tree crowns (52.36 % 14.7 %) was significantly different (t-test, F = 88.78, p < 0.0 5), with the trunk being in greater shade. Overall, 122 of the lichens produced usnic acid at high enough concentrations to be recognized, 115 produced a depside compound and 7 h ad no recognizable secondary chemical (Figure 1). The heterogeneity chi-squared test revealed that t he populations could be pooled (Tables 1 and 2). Of the lichens producing usnic a cid, 80 were on branch tips and 42 were on the trunk. The pooled data revealed a signi ficant difference between the frequency of lichens containing usnic acid in the b ranch tips and on the trunk (Figure 2). Similarly, the pooled data showed a significant dif ference between the frequency of lichens containing depsides in the branch tips and on the trunk; lichens with depsides were observed more often on the trunks than on the branch tips (Figure 3).
nrrnn Type of UV-Blocking Chemical Present Figure 1. Observed frequency of lichens producing usnic acid (a UV-blocking and antiherbivory compound) and depsides (a collection of compounds that play many biological roles), as collected from six trees in M onteverde, Costa Rica. Table 1. Presence and absence of usnic acid, a kno wn UV-blocking chemical, in lichens on trunks and branch tips of six trees. Heterogene ity chi-square tests revealed no significant differences between each of the trees ( X 2 = 2.63, df = 5, p = 0.755), so the frequencies across trees can be pooled. Usnic Acid Trunk Branch Present Not Present Present Not Present Chi-Square DF P Tree 1 9 13 14 6 3.58 1 0.058 Tree 2 6 14 14 6 6.4 1 0.011 Tree 3 3 19 12 8 9.81 1 0.0017 Tree 4 6 14 13 7 4.91 1 0.027 Tree 5 8 12 13 7 2.51 1 0.11 Tree 6 10 10 14 6 1.67 1 0.2 Total 28.88 Pooled 42 82 80 40 26.87 1 3.02E-07 Heterogeneity Chi-square 2.63 5 0.755
82 40 42 80 0 10 20 30 40 50 60 70 80 90 TrunkBranch TipsObserved Frequency No UsnicAcidPresent Usnic AcidPresent ___________________________________________________ _____________________ Figure 2: Observed frequency of lichens containing usnic acid on the branch tips and trunks within a population of lichens. Lichens on branch tips were more likely to produce usnic acid than lichens on trunks (after Ya tes correction factor X2 c = 26.23, df = 1, p = 3.03E-07) Table 2. Presence and absence of depsides, a group of chemicals known to have UVblocking and a variety of defense properties, in li chens on trunks and branch tips of six trees. The result of heterogeneity chi-square test s shows no significant differences between each of the trees ( X 2 = 2.26, df = 5, p = 0.81), so it is reasonable to assume that the six trees support the same population of lichen s and therefore the frequencies may be pooled. Depsides Trunk Branch Present Not Present Present Not Present Chi-Square df P Tree 1 14 6 10 10 1.76 1 0.197 Tree 2 13 8 6 14 4.21 1 0.04 Tree 3 18 4 8 12 7.77 1 0.0053 Tree 4 12 8 7 13 2.51 1 0.11 Tree 5 11 9 8 12 0.9 1 0.34 Tree 6 10 10 6 14 1.67 1 0.2 Total 18.82 Pooled 78 45 45 75 18.73 1 3.02E-07 Heterogeneity Chi-square 2.26 5 0.81
TrunkBranch TipsObserved frequency No DepsidesPresent DepsidesPresent ___________________________________________________ _____________________ Figure 3: Observed frequency of lichens containing at least one depside compound on the branch tips and trunks within a population of l ichens. Lichens on branch tips were less likely to produce depsides than lichens on tru nks (after Yates correction factor X2 c = 26.23, df = 1, p = 3.02E-07) DISCUSSION Lichens produce over eight hundred secondary compou nds, many of which are found in abundance in lichens but nowhere else in nature (Hu neck 1999). The commonly encountered depsides and usnic acid are examples of compounds that have only been extracted from the outer cortex of lichen colonies (Rancan et al. 2002) (Figure 4a, b). Results here revealed that while lichens with usnic acid are more common in the canopy, lichens with depsides are more successful in the un derstory; this may indicate a trade-off between containing these helpful chemicals. While both types of compound can co-occur in an individual lichen, usnic acid is usually a do minant chemical and present at much higher concentrations when light is present (Hager et al. 2008, Bjerke et al. 2002). In my observations, when usnic acid was present, depsides were either absent or at too low of concentrations to be recognized. This trade-off between usnic acid and depsides is i mportant for understanding the evolution of lichens. Although it is believed that fungi obtained the ability to lichenize at multiple times in ancient history, there is a conse nsus among lichenologists that the evolution of lichens largely depended on the fungi s ability to produce certain chemicals: polyketides (Huneck 1999) (Figure 4c). A previous s tudy showed that different chemotypes of closely related lichens selected diff erent habitats, adding to the evidence
that secondary metabolite production is not control led by the environment or substrate, but by specific genes (Culberson and Culberson 2004 ). In an extensive genetic study of lichens by Maio et al. (2001), it was found that the genes in lichens that produce the polyketides s ynthases are linked to genes that are probably involved in modification of pathway interm ediates, including the genes that code for the enzymes important for depside synthesi s. Huneck (1999) proposed that usnic acid evolved from the depsides after changes in the clustered genes, but little research has been done to isolate the genes for usnic acid synth ases (Figure 4). Evidence for divergent evolution between depsides and usnic acid has been shown in a study by Culberson et al. (1988) before evolutionary pathways for depsides ha d been proposed. While hybridization is relatively common among fungi, the different chemotypes tested rarely hybridized, indicating their early divergence and speciation (Culberson et al. 1988). These different evolutionary pathways may have permitted niche partitioning by lichen colonies. Niche partitioning in lichens may be partially dependent on the production of particular secondary metabolites. The UV-blocking effectiveness of usnic acid far exceeds that of the depsides, but both compounds play other beneficial roles (Rancan et al. 2002). Usnic acid is an effective antiviral and antibiotic, as well as an apparent antiherbivory agent (Lawrey 1980, Piovano et al 2002). Depsides generally act as UV-screening chemicals, but at a much lower level of effectiveness. They have a wide range of other biotic implications in addition to UV-screening: antifungal, antiherbivory, antibiotic, antiviral, and alleleopatric defense against bryophytes (Bjerke et al. 2002, Hager et al. 2008, Lawrey 1980, Piovano 2002). Although both usnic acid and depsides have similar functions, their slight variations are important for understanding the difference in the success of colonization in the canopy versus the understory. In forest canopies, the effectiveness of the UV-blocking properties seems a likely factor in the success of lichens with usnic acid over QuickTime and a TIFF (LZW) decompressor are needed to see this picture. QuickTime and a TIFF (LZW) decompressor are needed to see this picture. QuickTime and a TIFF (LZW) decompressor are needed to see this picture. A C B Figure 4. Proposed evolutionary pathway by Huneck et al.1999 of usnic acid (a) from depsides (b) from the original chemicals involved in lichenization: polyketides (c). The depside pictured is diffracatic acid, a common depside in lichens, while the polyketide depicted is alfatoxin, a common secondary metabolite of both lichenized and non-lichenized fungi.
those with depsides, but other important factors li kely play a role. A past study in the Monteverde area demonstrated differences in bryophy te and lichen coverage between different sized trees at a similar elevation to my study site (Barr 2007). This study showed that younger trees have more lichen coverage than older trees, and vice versa for bryophyte coverage. This indicates that lichens col onize first and are competitively replaced, to an extent, by mosses, hornworts or liv erworts (Barr 2007, Nadkarni 2000). Another study in Monteverde stripped mature branche s of epiphytes (including the corticolous lichens) and recorded the order of colo nization: lichens arrived first, followed by bryophytes, and finally (after no less than 10 years) vascular plants (Nadkarni 2000). Usnic acid has not been shown to e xhibit allelopathy against bryophytes, whereas depsides have. Alleleopatric p roperties, then, would be much more helpful on the older growth of trees (the trunk), t han on the newer growth, if bryophyte colonization comes after lichen colonization. Suly ma and Coxson (2001) showed that bryophyte coverage was directly correlated to canop y coverage. Thus, the presence of depsides on the trunks of trees, where canopy cover age is high, could be more important in alleleopatry than UV-screening, although further research is needed to support this assertion. The expense of production is also a determining fac tor for whether or not a chemical is used for defense in plants, animals, an d lichens. Usnic acid, though effective, is extremely short-lived, forcing the lichen to pro duce it constantly, albeit at concentrations related to exposure (Bjerke et al. 2 002, Rancan et al. 2002, Rundel 1969). Depsides, on the other hand, last longer and barely change in concentrations despite changes in abiotic factors (Rancan et al. 2002). T he expensive-to-maintain usnic acid may be used as a blanket defense among those lichen s where UV-irradiation seems a very important factor, whereas the longer-lasting depsid es could be used where they would be less at risk for UV-irradiation. Other research has revealed chemotypes as a more pr edictable indicator of species than phenotypes, demonstrating the secondary metabo lites importance in taxonomic classification (DePriest 1993). Little research ha s been done in the Monteverde area on the dynamics between different phenotypes or chemot ypes of lichens. Further research on selectivity of certain lichens could reveal more extensive community interactions, as well as contribute to classification and taxonomy o f these fascinating colonizers. Recognizing the importance of niche partitioning be tween lichens producing different secondary metabolites is a critical step in further understanding the amazing association between mycobiont and photobiont: on both a communi ty and evolutionary scale. ACKNOWLEDGEMENTS First let me give many thanks to Karen Masters for being excited about lichens with me. A noteworthy thanks needs to go to Pablo Allen for support throu ghout my project and in times of trouble. Anyone w ho helped with the climbing process deserves to be ack nowledged here: including Jessica, Nate, Eric, and Cyrus. Big thanks to Carolyn and Juliana for help on the final paper. Lastly, Bob Law is my savior. ___________________________________________________ _____________________ LITERATURE CITED
BARR, S. 2007. Succession of tropical epiphyte lichens and bryophytes at two elevations in the zone of Monteverde, Costa Rica. CIEE Fall 2 007: 9-17. BJERKE, J.W. AND T. DAHL. 2002. Distribution patterns of usnic acid-produ cing lichens along local radiation gradients in West Greenland. Nova Hedwigia 75:487-506 BJERKE, J.W., K. LERFALL, AND A. ELVEBAKK. 2002. Effects of ultraviolet radiation and PAR on the content of usnic and divaricatic acids i n two arctic-alpine lichens. Photochemical and Photobiological Sciences 9:678-6 85. BRODO, I.M., S.D. SHARNOFF, AND S. SHARNOFF. 2001 Lichens of North America. Yale University Press, New Haven, U.S.A. CULBERSON, C.F., AND W.L. CULBERSON. 2004. Future directions in lichen chemistry. The Bryologist 230-234. CULBERSON, C.F., W.L. CULBERSON, AND A. JOHNSON. 1988. Gene flow in lichens. American Journal of Botany 75:1135-1139 DEPRIEST, P.T. 1993. Variation in the Cladonia chlorophaea complex I: Morphological and chemical variation in southern Appalachian pop ulations. The Bryologist 96:555-563 FALK, A., T.K. GREEN, AND P. BARBOZA. 2008. Quantitative determination of secondary metabolites in Cladina stellaris and other lichens by micellar electrokinetic chromatography. Journal of Chromato graphy 1182: 141-144. HAGER, A., G. BRUNAUER, R. TURK, AND E. STOCKER-WORGOTTER. 2008. Production and bioactivity of common lichen metabolites as exe mplified by Heterodea muelleri (Hamp) Nyl. Journal of Chemical Ecology. 34:113-1 20. HUNECK, S. 1999. The significance of lichens and their metabo lites. Naturwissenschaften 86:559-570. LAWREY, J.D. 1980. Correlations between lichen secondary chemis try and grazing activity by Pallifera varia. The Bryologist 83: 328-334. MIAO, V., M-F. COEFFET-LEGAL, D. BROWN, S. SINNEMANN, G. DONALDSON, AND J. DAVIES. 2001. Genetic approaches to harvesting lichen pr oducts. Symbiosis 26:143-150 NADKARNI, N. 2000. Colonization of stripped branch surfaces by epiphytes in a lower montane cloud forest, Monteverde, Costa Rica. Biotr opica 32: 358-363. PIOVANO, M., J.A. GARBARINO, F.A. GIANNINI, E.R. CORRECHE, G. GERESIN, A. TAPIA, S. ZACCHINO, AND R. D. ENRIZ. 2002. Evaluation of antifungal and antibacterial activities of aromatic metabolites from lichens. J ournal of the Sociedad Quimico de Chile. 47:235-240 PURVIS, W. 2000. Lichens. Smithsonian Institution Press, Washington, D.C. RANCAN, F., S. ROSAN, K. BOEHM, E. FERNNDEZ, M. E. HIDALGO, W. QUIHOT, C. RUBIO, F. BOEHM, H. PIAZENA, AND U. OLTMANNS. 2002. Protection against UVB radiation by natural filters extracted from lic hens. Journal of Photochemistry and Photobiology 68:133-139. RUNDEL, P. W. 1969. Clinical variation in the production of usnic acid in Cladonia subtenuis along light Gradients. The Bryologist 72:40-44. SOLHAUG, K.A., Y. GAUSLAA, L. NYBAKKEN, AND W. BILGER. 2003. UV-induction of sun-screening pigments in lichens. New Phytologist 158:91-100 SULYMA, R., AND D.S. COXSON. 2001. Microsite displacement of terrestrial lichen s by feather moss mats in late seral pine-lichen woodlan ds of North-Central British
Columbia. The Bryologist 104: 505-516. WARING, B. 2007. Light exposure affects secondary compound di versity in lichen communities in Monteverde, Costa Rica. CIEE Spring 2007: 1-8.
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Heal, Katherine Rose
Productos qumicos para bloquear los rayos ultravioleta en el dosel del bosque y en los lquenes del sotobosque
UV-blocking chemicals in forest canopy and understory lichens
Lichens produce over eight hundred secondary metabolites, many of which are found nowhere else in nature. Many of these chemicals have UV-blocking properties, including usnic acid and many depsides. These chemicals could affect colonization success between understory and canopy lichens. In Monteverde, Costa Rica, a total of 244 lichens were extracted from six trees, half from canopy branch tips and half from the trunk in the understory. Canopy coverage was significantly higher in the understory (t-test, F = 88.78, p < 0.05). In the pooled population, usnic acid was present in the branch tips more often than on the trunks (after Yates correction factor X2 c = 26.23, df = 1, p = 3.03E-07). Conversely, depsides were present more often on the trunks (after Yates correction factor X2 c = 26.23, df = 1, p = 3.02E-07). The differences in the chemical constituents, which are associated with differences in lichen community composition, may reflect adaptations that allow niche partitioning along light gradients.
Los lquenes producen ms de ochocientas metabolitos secundarios, muchos de los cuales se encuentran en ningn otro lugar en la naturaleza. Muchas de estas sustancias tienen propiedades bloqueadoras de los rayos UV, incluyendo el cido snico y depsides muchos. Estos productos qumicos pueden afectar la colonizacin de xito entre los lquenes sotobosque y dosel. En Monteverde, Costa Rica, un total de 244 lquenes se extrajeron de seis rboles, la mitad de puntas de las ramas del dosel y la mitad del tronco en el sotobosque.
Text in English.
Lichens--Ecology--Costa Rica--Puntarenas--Monteverde Zone
Lichen communities--Costa Rica--Puntarenas--Monteverde Zone
Cloud forest ecology--Costa Rica
Lquenes--Ecologa-- Costa Rica--Puntarenas--Zona de Monteverde
Comunidades de lquenes--Costa Rica--Puntarenas--Zona de Monteverde
Ecologa del bosque nuboso--Costa Rica
Tropical Ecology 2008
Ecologa Tropical 2008
t Monteverde Institute : Tropical Ecology