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Community Composition and Chlorophyll Levels in Lichens as Indicators of Atmospheric Pollution in Monteverde Costa Rica Kathryn A. Graziano Department of Natural Resources, Cornell University ABSTRACT Lichens and their photosynthetic pigments are co mmonly used as bioindicators of atmospheric pollutants. This study compared lichen community structure and chlorophyll levels between two sites in the Monteverde region, Costa Rica, to determine the effect of local pollution. The first site, Santa Elena, i s rapidly becoming more populated as a result of an increase in ecotourism, leading to heavy vehicle traffic The other site, Bajo del Tigre, is secondary forest surrounded by a sparsely populated area far removed from automobile exhaust. Lichen samples we re collected from each site. Chlorophyll was extracted with 90 percent acetone and degradation was measured as a ratio of absorbance between 435 and 415 nm, in which a lower ratio implied higher chlorophyll degradation and more pollution. No significant di fference was found in chlorophyll between the two sites, and chlorophyll was degraded in all samples. Species diversity and evenness were higher in Santa Elena, while abundance was higher in Bajo del Tigre, resulting in no significant difference in species richness as measured by the Shannon Weiner Index Factors affecting community composition may be too complex in the Tropics to use lichen species or diversity as bioindicators of pollution. Low chlorophyll levels may indicate that pollution has permeated the entire area as acid rain. RESUMEN Lquenes y sus pigmentos fotosintticos son utilizados con frecuencia como bioindicadores de la contaminacin atmosfrica. Esta investigacin compar la estructura de la comunidad de lquenes y los niveles de cloro fila entre dos sitios en la regin de Monteverde, Costa Rica, para determinar el efecto de contaminacin. En el primer sitio, Santa Elena, la poblacin est creciendo rpidamente como resultado de un aumento del ecoturismo, causando mucho trfico de vehcu los. El otro sitio, Bajo del Tigre, es un bosque secundario rodeado de una zona poca poblada, lejos del escape de los automviles. Muestras de lquenes fueron recolectados de cada sitio. Clorofila fue extrada con 90 por ciento de acetona y la degredacin se midi como una relacin de absorbancia entre 435 y 415 nm, en el que una coeficiente ms bajo implica una mayor degradacin de la clorofila y ms contaminacin. No se econtraron diferencias significativas entre los dos sitos, y clorofila fue degradada e n todas las muestras. La diversidad y equidad de especies fueron mayores en Santa Elena, mientras la abundancia fue mayor en Bajo del Tigre, dando por resultado ningna diferencia significativa en la riqueza de especies, medida por el ndice de Shannon Wei ner. Factores que afectan la composicin de la comunidad pueden ser demasiado complejos en los trpicos para usar especies o la diversidad de lquenes como bioindicadores de contaminacin. Es posible que bajos niveles de clorofila indiquen que la contamina cin haya permeado toda la zona.
INTRODUCTION Lichens are a symbiotic relationship between algae and fungi, where the alga forms nutrients through photosynthesis and the fungus supplies water and minerals. Typically, the alga is in the family Cyanobacte riae or Chlorophyceae while the fungus belongs to Ascomycetes, but in rare cases can be a Basidiomycete or a Phycomycete (Conti and Cecchetti 2000). Since lichens lack roots and often grow in nutrient poor habitats, they rely on nutrients and minerals that are absorbed from the atmosphere over the entire surface of the lichen through pores or cracks designed for gas exchange. Water is also readily absorbed from the atmosphere due to the lack of a waxy cuticle in lichens (Conti and Checchetti 2000, Purvis 20 00). As a result, lichens are extremely sensitive to environmental contaminants in both air and water (Conti and Checchetti 2000). They are particularly sensitive to gaseous air pollutants commonly released as a result of human activity, such as SO 2 NO 2 and ozone, as well as acidic compounds. These environmental compounds adversely affect lichen growth and survival (Ra et al), and cause lichens to exhibit certain physiological responses such as accumulation of compounds or degradation of photosynthetic p igments (Conti and Checchetti 2000) as well as changes in community structure (Purvis 2000). These measurable effects make lichens extremely useful as bioindicators of air quality (Conti and Cecchetti 2000), and their utility has been documented as early a s 1866 levels of pollution, and ability of an organism to indicate either presence or amount of atmospheric pollutants (Garty et al. 2001). Sulfur dioxide, from vehi cle traffic pollution and urban emissions, dissolves in rainwater and is absorbed by lichens. Accumulation of these compounds can affect lichen metabolism causing bleaching of thalli from loss of chlorophyll in the algal cells (Purvis, 2000). Phaeophytin i s a product of chlorophyll degradation, and variation in the normal ratio of phaeophytin to chlorophyll indicates suffering in lichen (Conti and Cechetti 2000). This ratio between phaeophytin and chlorophyll can be measured with a spectrophotometer as the light transmittance at 435 nm over transmittance at 415 nm (Ronen and Galun 1984). A ratio of 1.4 indicates that chlorophyll is unchanged, while any reduction in this value indicates chlorophyll degradation from stress. This is considered to be an appropri ate index for measuring the impact of high concentrations of SO 2 and other degrading pollutants in lichens (Conti and Cecchetti 2000). Furthermore, different species respond with different levels of sensitivity to environmental stressors. Certain species are unable to survive in mild or excessive pollution (Purvis 2000). Esseen and Renhorn (1998) found a significantly lower abundance of lichens along the forest edge than deeper within the forest. In a previous study done in the Monteverde region, it was f ound that lichen richness, cover, and abundance increased as distance from the road increased (Toy 2005). In Santa Elena, Costa Rica, rapid expansion of eco tourism has led to an increase in automobile traffic in town. Between 1974 and1994, the Monteverde Cloud Forest Preserve increased visitation from 471 to 49,793 visitors (Aylward et al 1995). In 1975, there less than a hundred visitors to Monteverde per year. In the mid 1990s, there were around 50,000. Now, as many as 250,000 tourists visit Monteverde every year (www.monteverdeinfo.com). Increased tourism is associated with more buses, taxis, cars,
etc, contributing to higher levels of air pollution, which could be a potential adverse effect of ecotourism in Monteverde and other ecotourism sites. Lichen community composition and chlorophyll levels could be used to compare the air quality in the center of town to a forested site to determine if there is enough pollution to cause damage to surrounding organisms. An overall assessment of the effect of poll ution on lichens within Monteverde should include a comparison of both physiological response and community composition between lichens found in the roadside area of Santa Elena and the forest of Bajo del Tigre. Measuring species richness, abundance, and d iversity of each site in addition to comparing the chlorophyll content between sites could reveal definitive assessment of pollution rates and affect of contaminants on lichens. I hypothesize that species richness, diversity, and abundance will be higher i n Bajo del Tigre and that the ratio of chlorophyll to phaeophytin in lichens collected in the forest of Bajo del Tigre will be higher than in Santa Elena. MATERIALS AND METHODS S TUDY S ITES This study compared two sites within Puntarenas, Costa Rica. Th e first site encompassed the city center of Santa Elena, which receives the most vehicle traffic in the area and is a hub for taxis, buses, and other vehicles associated with tourism and commerce. The second site was in Bajo del Tigre, which is part of th in Monteverde, and is characterized as P remontane M oist F orest. Elevation of both sites are between 1,300 and 1,450 m above sea level and receive about 2 to 2.5 m of rainfall per year (Haber et al. 2000). D ATA C OLLECTION F OR S PECIES C OMPOSITION I spent four hours of sampling in each site as a way to compare species richness and diversity with equal sampling effort. First, each morphospecies was determined and given a letter. Then, each clump of lichen larger than 1 cm in diameter was counted as an individual. Any lichen above eye level was not counted to avoid confusion in identification. In Bajo del Tigre, I counted lichen along walking paths to mimic the high levels of sunlight found in the Santa Elena site and to reduce the variables other than pollution between the sites. P IGMENT E XTRACTION Two morphospecies, both in the genus Sticta were determined common enough in both study sites to be collected for chlorophyll extraction (Fig. 1). In Santa Elena, 10 samples of each morphospecies were collected from individual or patches of trees within 25 m of the road. In Bajo del Tigre, I collected 10 samples of each morphospecies from trees on the edges of walking trails within the forest. Methods of pigment extraction wer e adapted from Brown and Hooker (1977) and Ronen and Galun (1984). First, I washed each sample in 5 mL absolute acetone for one
extraction process (Brown and Hooker 1977). 0.2 g of lichen thalli were ground in a mortar in the presence of excess carbonate (0.05 g Na 2 CO 3 per 0.2 g of lichen). The carbonate is necessary to further neutralize acids that would artificially degrade chlorophyll and produce phaeophytin. I conducted the entire procedure in dim lighting to reduce damage of pigments. Pigment was extracted for 3 hours in 90 % acetone in a dark and cold place. Each sample was then centrifuged for two minutes and the spectrophotometer was used to measure transmittance at 4 35 and 415 nm against a 90% acetone blank. A OD435/OD415 ratio of 1.4 would indicate no chlorophyll degradation. Any reduction of the value indicates chlorophyll degradation, and therefore environmental stress (Ronen and Galun 1984). FIGURE 1 Sticta sp p (morphospecies A and B) collected for chlorophyll extraction. 10 samples of both morphospecies were collected from the two study sites, Bajo del Tigre and Santa Elena, Costa Rica. Pigments were later extracted and compared between the two sites. RESU LTS L ICHEN D IVERSITY A total of 22 morphospecies were found in both sites. Twelve morphospecies and 617 individual lichens were observed in Santa Elena. In Bajo del Tigre, 20 morphospecies and 980 individual lichens were observed (Figure 2). Despite the trend towards more species in Bajo del Tigre, species richness was not found to be significantly different between the two sites (Chi square = 2, CV = 3.84, df=1, p<0.05). Significantly 1.899, df to have higher evenness than Bajo del Tigre (E = 0.765 in Santa Elena, E = 0.488 in Bajo del Tigre). However, overall abundance of lichens was significantly hi gher in Bajo del Tigre than in Santa Elena (Chi square = 82.91,df=1, p<0.05). Of the 22 total morphospecies, 10 species were common to both sites. The accounting for relative a bundance of shared species. There were only two morphospecies
(D and K) found exclusively in Santa Elena. There were ten morphospecies found in Bajo del Tigre but not in Santa Elena (M,N,O,P,Q,R,S,T,U and V). Santa Elena Bajo del Tigre FIGURE 2 Relative lichen morphospecies abundance in urban Santa Elena and forested Bajo del Tigre found with 4 hours sampling effort in each location. A total of 12 species and 617 individual lichens were observed in Santa Elena. In Bajo del Tigre, 20 morphospecies and 980 individual lichens were observed. L ICHEN C HLOROPHYLL L EVELS Two way ANOVA showed no statistical difference between the ratio of OD 435/OD 415 between the two sites (P = 0.903, df =2), indicating no difference in chlorophyll degradation (Fig. 3). There was a significant difference between chlorophyll levels between morphospecies A and B (P = 0.0091, df = 2), with a trend towards higher chlorophyll to phaeophytin found in morphospecies B.
FIGURE 3 Numbers above bars show mean values of OD 435/OD 415 calculated from 10 samples of morphospecies A and 10 samples of morphospecies B collected from both the forest (Bajo del Tigre) and the town center (Santa Elena). This value corresponds with the amoun t of chlorophyll relative to phaeophytin. Any value less than 1.4 implies degradation of chlorophyll. Lines represent +/ 1 standard deviation. G ENERAL O BSERVATIONS Lichens in Santa Elena appeared and felt much drier than the lichens in Bajo del Tigre. Morphospecies A was much less bright green in Santa Elena, and morphospecies B was duller brown there. DISCUSSION There was no significant difference in lichen richness between Bajo del Tigre and Santa Elena. Contrary to the original hypothesis, ther e was actually higher diversity and evenness in Santa Elena than within the forest, though not statistically significant. Although not supported by many studies (Toy 2005, Esseen and Renhorn 1998), these results are consistent with another study done in th e Monteverde area. Hosford (2005) found that species richness, diversity, and evenness of lichens were greater in disturbed
areas, such as pastures and edges, than in primary forest. Factors such as sunlight and intraspecific competition were thought to af fect this distribution (Hosford 2005). Other studies found that edge effects, which include increased sunlight and wind exposure, may increase lichen species richness and abundance. Neitlich and McCune (1997) found that forests with gaps had higher richn ess and abundance than those without gaps. Wolseley et al (1991) suggested that higher species richness may be found along roads or edges than within forests because some less sensitive species have a competitive advantage in disturbed areas. In fact, some extremely tolerant species are found only in polluted areas, and many types of lichen actually thrive in mild pollution (Purvis 2000). But in the Tropics, lack of knowledge of taxonomy and ecology of lichens makes them less developed as environmental in dicators. In areas where monitoring techniques using lichens have been successful, the lichen communities and taxa were all well described before the study (Wolseley and Aguirre Hudson 1991). Before richness and diversity of lichens can be used as indicat ors in Monteverde, a more thorough assessment of the lichen community would be essential. It may be more useful to look at presence of specific lichen species than to look at overall species richness and diversity. The overlap between the two sites was on ly 35.67 percent. Since some species are more sensitive than others to specific types of pollution and disturbance (Purvis 2000), a study of the species that are not common to both sites could be useful. It would be expected that highly sensitive species w ould be found only in forest and not in disturbed or polluted areas. There were 10 morphospecies found exclusively in Bajo del Tigre (Fig. 2). Identification of these species could be extremely useful to starting a list of species that may be sensitive to pollution or disturbance. If consistent, their presence could be used as an indicator of good air quality or low disturbance. But as mentioned earlier, until a more complete knowledge of the ecology of individual species is made, this method would not be a ble to distinguish between responses to pollution or other affects such as disturbance. Similarly, I found two morphospecies in Santa Elena that were not found in Bajo del Tigre. Presence of these species could be used as an indicator of high pollution or environmental disturbance. Observation of the appearance or increase in cover of a tolerant species is an accepted mode of ecosystem monitoring (Wolseley et al. 1991). Investigation into the identification of these tolerant species in Santa Elena would b e necessary for further use as a bioindicator. There was no significant difference between chlorophyll levels in Bajo del Tigre and Santa Elena, indicating that factors other than pollution are likely responsible for the differences in community composit ion. The OD 435/OD 415 ratio was less than 1.4 for both morphospecies in both sites, meaning that chlorophyll has been degraded and phaeophytin has been formed. One explanation for this could be that many lichen species contain acidic organic substances t hat cause degradation of chlorophyll if the substances are not removed prior to pigment extraction. Although the rinse in pure acetone and addition of Na 2 CO 3 were implemented to reduce the effects of acidification, as suggested by Brown and Hooker (1977), it is possible that acidification from these substances still had an affect on the levels of chlorophyll. So it is possible that, despite the increase in vehicular traffic from tourism, air pollution does not have a strong effect on the photosynthetic pigm ents of lichens.
But it is also likely that both sites were affected by pollution. In a study done in North America, it was found that trees at the windward edge of a forest received 3 to15 times greater deposition of ions from cloud water than trees in t he interior of the forest (Esseen and Renhorn 1998). The study site at Bajo del Tigre could have been affected by edge effects, which are thought to extend from 25 to 50 meters into the forest (Esseen and Renhorn 1998). So, although the lichens in Bajo del Tigre were not directly exposed to pollution from the road, like those in Santa Elena, they may have still been exposed to equal amounts of other forms of pollution, such as acid rain. This would explain the low ratio of chlorophyll to phaeophytin in both sites. Lichens can be extremely useful bioindicators, but factors such as sun and wind exposure make it difficult to isolate pollution as a factor in community composition. So, to use lichens as bioindicators in the Monteverde area, it would be useful to identify specific lichen species found exclusively in high or no pollution for use as a bioindicator. But, this still would not implicate pollution as the only variable affecting the presence or absence of certain species. But chemical analysis of chlor ophyll levels can determine effects of pollution without the influence of other variables. In this case, chlorophyll was degraded in both urban and forested sites. This could imply that pollutants are affecting the lichens through acidified precipitation, in which case the effects of automobile pollution in the town center may be having further reaching effects than anticipated. ACKNOWLEDGEMENTS chemicals and helping me to stay calm and adapt my project when nothing seemed to be working out. Thanks to Bajo del Tigre for allowing me to observe and collect lichens from their property. And finally, many thanks to my friends in the Caitas Crew and all the ho st families, especially Socorro, for being the perfect thing to return home to after a long day. LITERATURE CITED Aylward, Bruce, Katie Allen, Jaime Echeverria and Joseph Tosi. 1995. Sustainable ecotourism in Costa Rica: the Monteverde Cloud Forest P reserve. Biodiversity and Conservation 5: 315 343. Brown, D.H. and T.N. Hooker. The Significance of Acidic Lichen Substances in the Estimation of Chlorophyll and Phaeophytin in Lichens. 1977. New Phytologist 78(3): 617 624. Conti, M.E. and G. Cecchett i. Biological monitoring: lichens as bioindicators of air pollution assessment a review. 2001. Environmental Pollution 114: 471 492. Esseen, Per Anders and Karl Erik Renhorn. 1998. Edge Effects on an Epiphytic Lichen in Fragmented Forests. Conservation Biology 12(6):1307 1317. Garty, J., O. Tamir, I. Hassid, A. Eshel, Y. Cohen, A. Karnieli, and L. Orlovsky. 2001. Photosynthesis, Chlorophyll Integrity, and Spectral Reflectance in Lichens Exposed to Air Pollution. J. Environ. Qual 30:884 893. Haber, William A., Willow Zuchowski, and Erick Bello. An Introduction to Cloud Forest Trees; Monteverde, Costa Rica. Second ed. 2000. Mountain Gem Publications, Montedverde de Puntarenas, Costa Rica. Hosford, Sarah. Community composition a nd diversity of lichens along a disturbance
gradient in San Luis, Costa Rica. CIEE Tropical Ecology and Conservation, Fall 2005. Monteverde Info. 2010. http://www.monteverdeinfo.com Neitlich, P.N. and B. McCune. 1997. Hotspots of epiphytic lichen diversity in two young managed forests. Conservation Biology. 11(1):172 182. Nylander, W. 1866. Les lichens du Jardin du Luxembourg. Bull. Soc. Bot. Fr. 13:364 372. Purvis, W. 2000. Li chens. Washington DC: Smithsonian Institution Press Ra, H.S.Y., L.H. Geiser, and R.F.E. Crang. 2005. Effects of season and low level air pollution on physiology and element content of lichens from the U.S. Pacific Northwest. Science of the Total Environme nt 343: 155 167. Ronen, R and Margalith Galun. 1984. Pigment Extraction from Lichens with Dimethyl Sulfoxide (DMSO) and Estimation of Chlorophyll Degradation. Environmental and Experimental Botany 24(3): 239 245. Toy, Jennifer M. Lichen Richness Al ong an Air Pollution Gradient in Monteverde, Costa Rica. CIEE Tropical Ecology and Conservation, 2005. Wolseley, P.A., C. Moncrieff, B. Aguirre Hudson. 1991. Lichens as indicators of environmental change in the tropical forests of Thailand. Global Ecol ogy and Biogeography Letters. 1(6): 170 175.
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Graziano, Kathryn, A.
Composicin de la comunidad y los niveles de clorofila en los lquenes como indicadores de contaminacin atmosfrica en Monteverde, Costa Rica
Community composition and chlorophyll levels in lichens as indicators of atmospheric pollution in Monteverde, Costa Rica
Lichens and their photosynthetic pigments are commonly used as bioindicators of atmospheric pollutants. This study compared lichen community structure and chlorophyll levels between two sites in the Monteverde region, Costa Rica, to determine the effect of local pollution. The first site, Santa Elena, is
rapidly becoming more populated as a result of an increase in ecotourism, leading to heavy vehicle traffic. The other site, Bajo del Tigre, is secondary forest surrounded by a sparsely populated area far removed from automobile exhaust. Lichen samples were collected from each site. Chlorophyll was extracted with 90 percent acetone and degradation was measured as a ratio of absorbance between 435 and 415 nm, in which
a lower ratio implied higher chlorophyll degradation and more pollution. No significant difference was found in chlorophyll between the two sites, and chlorophyll was degraded in all samples. Species diversity and evenness were higher in Santa Elena, while abundance was higher in Bajo del Tigre, resulting in no significant difference in species richness as measured by the Shannon Weiner Index. Factors affecting
community composition may be too complex in the Tropics to use lichen species or diversity as bioindicators of pollution. Low chlorophyll levels may indicate that pollution has permeated the entire area as acid rain.
Los lquenes y sus pigmentos fotosintticos son utilizados con frecuencia como bioindicadores de la contaminacin atmosfrica. Esta investigacin compar la estructura de la comunidad de lquenes y los niveles de clorofila entre dos sitios en la regin de Monteverde, Costa Rica, para determinar el efecto de contaminacin. En el primer sitio, Santa Elena, la poblacin est creciendo rpidamente como resultado de un aumento del ecoturismo, causando mucho trfico de vehculos. El otro sitio, Bajo del Tigre, es un bosque secundario rodeado de una zona poco poblada, lejos del escape de los automviles. Muestras de lquenes fueron recolectados de cada sitio. La Clorofila fue extrada con 90 por ciento de acetona y la degradacin se midi como una relacin de absorbancia entre 435 y 415 nm, en el que un coeficiente ms bajo implica una mayor degradacin de la clorofila y ms contaminacin. No se encontraron diferencias significativas entre los dos sitos, y la clorofila fue degradada en todas las muestras. La diversidad y equidad de especies fueron mayores en Santa Elena, mientras la abundancia fue mayor en Bajo del Tigre, dando por resultado ninguna diferencia significativa en la riqueza de especies, medida por el ndice de Shannon Weiner. Los factores que afectan la composicin de la comunidad pueden ser demasiado complejos en los trpicos para usar las especies o la diversidad de lquenes como bioindicadores de contaminacin. Es posible que los niveles bajos de clorofila indiquen que la contaminacin haya permeado toda la zona como lluvia acida.
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