Lichens and Lichen-Feeding
Moths (Arctiidae: Lithosiinae) as Bioindicators of Air Pollution in the Rocky
Mountain Front Range
Sara SimonsonColorado State UniversityFort Collins, Coloradoа 80523 saras@lamar.colostate.edu Abstract This review summarizes some of the recent methodological developments in determining the responses and sensitivity of lichens to air pollutants, and in chemical analysis used in taxonomic identification and detecting bioaccumulation.а Whenever possible, recent case studies are used as examples ofа practical application.а Also included is a summary of the ecology of Lithosiinae moths.аа Finally, a preliminary evaluation is presented on the potential for using lichens and lichen feeding moths as pollution monitors in the Rocky Mountain Front Range region. Introduction Lichens Lichens are a group of non-vascular plants composed of fungal (mycobiont) and algal (photobiont) species growing in a symbiotic relationship.а Lichens are classified and named according to the fungal partner, with many thousands of species of lichens sharing a much smaller number of photobiont species.а Lichens are considered to be relatively well known in the Rocky Mountain Region, especially in Colorado (McCune and Goward 1995, Weber and Whitman 1990). Lichens as Bioindicators of Air Pollution Lichens were recognized as potential indicators of air pollution as early as the 1860's in Britain and Europe (Hawksworth and Rose 1976).а Since then, lichens have played prominent roles in air pollution studies throughout the world because of their sensitivity to different gaseous pollutants,а particularly sulfur dioxide.а They have also been found to act as accumulators of elements, such as trace metals, sulfur, and radioactive elements (Stolte et al. 1993, Ahmadjian 1993).аа During the period 1973-1988, approximately 1500 papers were published on the effects of air pollution on lichens (Richardson 1988, in Ahmadjian 1993), and many general reviews of lichens and air pollution have been compiled (Ahmadjian 1993).а Lichen Feeding Moths A number of Lepidoptera are lichenivorous.а For example, caterpillars of Lithosiinae feed on epiphiytic algae of trees and rocks or on the algal component of lichens (Habeck 1987, in Scoble 1992, Rawlins 1984) .а At least sixteen species in this subfamily are known from the Rocky Mountain Front Range (Opler 1996).а Adults are easily identified, and certain aspects of the ecology of these moths are well known (Opler, personal communication).а Although lepidopterists have expressed concern about secondary effects on lichen feeders, studies to determine these effects have apparently not been attempted (Hawksworth 1976).аа The Rocky Mountain Front Range Air pollution is of critical importance in the Rocky Mountain Front Range region (generally defined).а Topographic position and weather patterns create an airshed that extends eastward from the mountains from Denver to the Wyoming border.а This regional airshed traps pollution from the urbanization that is rapidly increasing along the eastern slope from areas south of Pueblo to north of Fort Collins.аа Of great concern to Front Range cities such as Fort Collins are pollutants such as carbon monoxide, fine particulates, and a long list of organic compounds.аа Volatile compoundsа and oxides of nitrogen are also ofа concern because the assist in the formation of ozone (Brian Woodruff, Environmental Planner, City of Fort Collins, personal communication).а Radioactive elements are not generally air borne, but when attached to fine particulates they can become part of the "brown cloud" (Rocky Flats Environmental Technology Site, personal communication).а Of great concern to land managing agencies in this region are the effects of airborne pollutants on natural resources.аа Determining the responses and sensitivity of lichens to air pollutants: Responses to Air Pollution Lichens have been used often as receptor-based biomonitors in air quality studies.а Lichen characters measured in air pollution studies include morphological, physiological, and population characteristics.а Historically, lichens have been used in a qualitative way, with observations of population changes and morphological effects serving as indicators of pollutants.аа In the last few decades quantitative measurements of the chemical content of lichens and sensitive physiological processes have increasingly been used to indicate pollutants. Possible responses to air pollution stress include chlorophyll degradation, changes in photosynthesis and respiration, alterations in nitrogen fixation, membrane leakage, accumulation ofа toxic elements, and possible changes in spectral reflectance, lichen cover, morphology, community structure, and reproduction .а The most widely used methods to measure these responses are fumigation and gradient studies (Stolte et al. 1993).а Gradient Studies The gradient analysis method assumes that measurable attributes of affected species vary along causative environmental gradients.а These studies are usually done around existing or projected sources of contaminants, with pollutant loadings expected to vary with distance from a source.а Although appropiate in many cases, the use of lichens in gradient studies has some limitations of which the researcher should be aware.а These include the difficulties with identification of species, determination of the best indicator species, and demonstration that the observed patterns reflect pollution stress and not other biotic and/or abiotic factors.а An important consideration in using this approachа is the possibility that responses observed across gradients are a function of changes in other environmental or disturbance variables (Stolte et al. 1993).а Historically, gradient studies have usually involved observations ofа visible injury, such as bleaching and thallus deformation, and changes in community structure, such as species richness, abundance, or cover (Hawksworth and Rose 1976).а Recently, response variables have shifted to physiological processes such as photosynthesis, nitrogenase activity, element uptake, membrane integrity (electrolyte leakage), pigment quantity, degradation, and flourescence (Stolte et al. 1993).а Response variables are often correlated with pollutant exposure (when available). Inconclusive results of preliminary investigations on electrolyte leakage (H2O conductivity) of lichen along a pollution gradient from the Craig to Hayden powerplants in Colorado (1994) demonstrate the complexity of identifying indicator species in field gradient studies.а At increasing distances from the lowland powerplants, high mountain environments dominate.а For this study, the lichen species Usnia nigrens and Rhizoplaca chrysoleuca were selected as potential indicator species.аа Distributions of the selected species did not span the entire study area, and failed to include some of the lower elevation areas near the powerplant site.а The environmental heterogeneity of sites along the gradient may have obscured the effects of pollution on the lichens (Dr. Robert Musselman, USFS Rocky Mountain Forest and Range Experiment Station, personal communication). Gradient methods are usually designed to monitor naturally occurring species in a region.а When climate and pollution factors create unfavorable conditions for lichens, it may be impossible to identify and use a naturally occurring indicator species.а Transplanting lichens is an alternative method that can be used to determine the effects of pollutants on lichens and their photobionts in polluted regions that lack a natural community of lichens.а Lichens or bark discs with thalli can be attached to supports that are then placed at different distances from a pollution source (Schoenbeck 1969, in Ahmadjian 1993).а In Israel, the common lichen Ramalina duriaei was transplanted along a gradient between urban areas.а The lichens were then studied with respect to ATP concentration, chlorophyll degradation, and accumulation of heavy metals (Kardish 1987, Garty et al 1988, in Ahmadjan 1993). Dr. Larry Jackson and others recently (1996) compiled an extensive draft report entitled "Biogeochemistry of Lichens and mosses in and near Mt. Zirkel Wilderness, Routt National Forest, Colorado: Influences of coal-fired power plant emissions".а аThis draft report is not for citation,а although it seems appropriate to summarizeа the study here.а The researchers collected three species of lichens from areas at different distances and in different directions from a power plant which was the suspected source of pollutants affecting the wilderness area and adjacent public lands.а Major, minor, and trace element concentrations were measured,а and isotopic signatures of sulfur were used to differentiate the anthropogenic and environmental sources of atmospheric pollution.а Elevated concentrations of certain compounds were correlated with results from snowpack studies and other atmospheric deposition data.а The use of chemical signatures promises to be very valuable as pollutants can be associated with a particular source.ааааа Fumigation Studies: Fumigation studies most commonly involve measuring response variables of selected physiological processes.а Sensitive processesа include activity, K+а efflux/total, electrolyte leakage, photosynthesis, and respiration pigment status.а Problems with fumigation studies exist because sensitivity also varies with factors such as concentration and duration of exposure, environmental conditions, and status of the thallus (Stolte et al. 1993).ааа Laboratory fumigation studies are designed to show measurable responses to air pollutants under controlled conditions in more or less enclosed exposure systems that can range from plastic bags to elaborate chamber systems with automatic control of environmental parameters and pollutant concentrations (e. g.а the continuously stirred tank reactors (CSTR)).а These studies have been shown to provide some valuable insights on how pollutants affect lichen metabolism and they often provide a means of confirming field studies.а However, many problems exist in using laboratory observations to predict sensitivity under field conditions that may include long-term exposure to multiple pollutants and dynamic environmental conditions (Richardson 1988, in Ahmadjian 1993).а Fumigation field studies of lichens from areas around pollution sources have proven to be more useful in predicting the potential impact of a pollution source on various species (Ahmadjian 1993), because they allow environmentally realistic observations of how specific physiological or morphological changes correlate with specific pollutants (Stolte et al. 1993).аа Field fumigation studies are conducted using techniques such as Zonal Air Pollutant Study (ZAPS) and simulated acidic rain exposures.аа The ZAPS system refers to means of delivering air pollutants in the field for relatively long-term testing with no control over environmental conditions.а This system has been utilized in Alaska to fumigate caribou forage lichens with SO2а (Moser 1982, in Stolte et al. 1993).а Rain simulators have been used in combination with ZAPS field situations to reproduce the combined effects of gaseous pollutants.а The more sophisticated systems are capable of reproducing many of the physical and chemical characteristics of precipitation.аа Exposure chambers used for plants that may lendа themselves to field studies of lichens include open-top field chambers, and the mini-cuvette, which encloses a branch or a portion of a branch in a mature tree canopy, and allows measurement of physiological variables in situ (Stolte et al. 1993).а In the U. S., Corina Gries is investigating the sensitivity of lichens to atmospheric levels of SO2, NO2, and O3 using improved fumigation techniques in testing species used in NPS and USFS air quality studies (Geiser 1992). Physiological analytical methods are useful for both gradient and fumigant studies.а Recently developed/improved techniques to detect physiological responses include the membrane permeability test (measuring efflux and conductivity), photoacoustic spectroscopy (measuring photosynthetic parameters), chlorophyll degradation (measuring phaeophytin), oxygen-electrode method (measuring oxygen exchange), and others (Ahmadjian 1993). Chemical analysis in lichen studies Taxonomic Identification Lichen taxonomy depends heavily on determination of lichen chemistry because lichens produce an abundance of unusual and distinctive secondary metabolites.а Some of these metabolites may be determined by relatively simple spot tests or microcrustal techniques, but increasingly, lichen chemistry is determined by thin-layer chromatography (TLC).а The methods of TLC to identify specific lichen metabolite products should be considered necessary to obtain a species determination.а High-performance liquid chromatography (HPLC) may be used to support or better define TLC results (Stolte et al. 1993).а The use ofа chemical techniques may avoid the difficulties of identification of species naturally occurring in an area.ааа Chemical Analysis Numerous instrumental methods exist for the determination of most metals in lichens, each with a multitude of advantages and disadvantages.а The choice of methods for other pollution elements and compounds is more restricted.а аAlthough chemical analysis is often regarded as more quantitative than traditional observational studies, the demonstration of causal relationships requires careful development of the study objectives, sampling design and sample analysis.а In addition, the various aspects of sample preparation that must be considered prior to chemical analysis include cleaning, washing, and drying of the raw sample, particle size reduction, homogenous subsampling, and destruction of organic matter.а Atomic Spectroscopy Atomic absorption spectroscopy (AAS) has commonly been used in inorganic analysis of plants and lichens, especially for the determination of metals.а Historically, arc, spark, or flame emission sources have been used in photographic or direct-reading spectrographs for the determination of metals.ааа Recently, many AAS methods have been replaced with inductively coupled atomic emission spectroscopy (ICP-AES).аа In this technique, an argon plasma is used as the atomic emission source and many elements may be determined simultaneously from a small sample.а ICP has also been combined with mass-spectroscopy (MS) to provide a technique with the advantages of ICP as an atomization source and MS as a sensitive detector.аа X-ray flourescence spectroscopy (XRF) with a wave-length or energy dispersive detector is also a technique used for the determination of metals and some non-metals.аа XRF and AAS methods have been used to compare concentrations of the elements Ni, Cr, Cu, Zn, and Pb in lichens (Stolte et al. 1993). Mass Spectroscopy Mass spectrometry is generally confined to the determination of organic pollutants after a chromatographic separation , or to coupling with ICP for the determination of inorganic elements (Stolte et al. 1993). The in situ analysis of organic compounds in lichens is now possible using laser microprobe mass spectrometry.а The accumulation of air-borne toxics, such as aromatic and chlorinated hydrocarbons, can be detected using these methods (Geiser 1992). In addition, stable isotope ratios may be used to help identify sources of elements such as Pb or S.а Measurement of the stable isotope ratio for S34/S32 has the potential for uniquely distinguishing the contribution of sulfur to an ecosystem from various natural and anthropogenic sources (Jackson and Gough 1989, Krouse 1989, in Stolte et al. 1993).а Sulfur isotopes and other chemical signatures have recently been used in Colorado to identify anthropogenic sources of pollutants in the Mount Zirkel Wilderness area.а Chromatographic Methods Chromatographic methods such as gas chromatography with an electron capture detector (GC-ECD) and gas chromatography with a mass spectrometer (GC-MS) are commonly usedа in the determination of chlorinated hydrocarbons and other organic pollutants.а Determination of compounds by these methods is similar to the analysis of pesticides in plants.а Ion chromatography has also been used to determine cations and anions in plant tissue and may be appropriate for lichen analysis.а Various other chemical analytical methods are being developed and tested.а For example, Sulfur has been determined in plant material by a wide variety of techniques incorporating acid digestion or combustion methods followed by turbidimetric, colorimetric, and titrimetric quantitation.а Lichen Moth Ecology Evolutionary Relationships The large concentrations of mostly phenolic secondary compounds that are accumulated in copious amounts by many lichens have long been suspected to protect these symbiotic organisms from generalist herbivores.а In spite of the deterrent or even toxic effects of many lichen products toward generalist herbivores, there are examples of specialized lichen feeders, including oribatid mites, terrestrial gastropods, and Lepidoptera (Hesbacher et al. 1995, and references herein). Perhaps the largest radiation of Lepidopteran lichenivores occurs in the Arctiidae,а subfamily Lithosiinae, all of whose known larvae feed on lichens, algae, liverworts, or mosses.а Lithosiines often occur in semi arid or xeric habitats, feeding on lichens and ephemeral patches of algae on rocks or bark.а The general lack of ecological information concerning these species may be related to nocturnal feeding habits of the larvae and inactivity during dry spells (Rawlins 1984). Feeding Relationships Although the details of lichenivory in Lepidoptera are largely unknown, it has been suggested that lichenivores essentially feed on algae.а There is no evidence of an obligate relationship to the fungal portion of lichens (Rawlins 1984).а The number of algal species known to occur in lichens is extremely small compared with the fungal species involved.а While fungal partners will only form lichens with one, rarely two, particular algal genera, they are less specific as to the species or strains of algae within those genera (Hawksworth and Rose 1976).а As previously mentioned, lichen species are named based on the fungal component.а As would therefore be expected, the host range of most lichenivores is broad, although a definite preference is shown for the hosts on either bark or stone substrates (Rawlins 1984). Rearing has been recorded in severalа Lithosiines including Crambidia, Cisthene, Lycomorpha and Hypoprepia.а Species of these genera preferred to feed on cryptograms with high chlorophyll content, including free algal blooms, mosses, and the algal symbionts of lichens (Rawlins 1984).а As previously observed by Dr. Rawlins, when reared on lichens in the laboratory, Hypoprepia miniata and Hypoprepia fucosa prefer the cortex of the lichen, and avoid the medular portion which is free of algae (Ruth Boada, personal communication).а Lithosiines have been reared on artificial diets of isolated alga denatured by heat, suggesting a lack of the dependance on fungal enzymes that occurs in other mycophagous species.а Although several species have been raised in captivity, it is still unclear what foods would be chosen in natural settings (Rawlins 1984). Although species specific feeding relationships are unknown, some ecological information exists for several Lithosiines that occur in our region.а Lycomorpha and Eudesmia species show preferences for lichens growing on rocks, walls, or cliffs, while Crambidia and Cisthene are found among foliose lichens in trees.а Some Hypoprepia species have been reported on mosses (Rawlins 1984).а Chemical Ecology Lithosiinae moths are aposematically colored, and are known to be toxic to predators.а It is not known whether adult toxicity results from sequestration of substances in the larval hosts, from their synthesis by adults, or both (Rawlins 1984).а A recent survey for the presence of sequestered lichen compoundsа in several Lithosiine species may help to unravel this mystery (Hesbacher et al. 1995).а The results demonstrate that sequestration of lichen compounds is variable, but widespread in wild-caught imagines in Austria and Germany.а Although ecological roles of the sequestered compounds for the herbivores remains unknown, it is thought that they may be utilized for chemical defense against predators or pathogens. For this investigation, both wild caught and laboratory reared specimens were ground, extracted in acetone, and subjected to HPLC analysis.а Identification of lichen compounds was by comparison of HPLC retention times and by comparison of the online-recorded UV absorption spectra with those ofа known lichen substances (Hesbacher et al. 1995). Lichen compounds sequestered by the moths included the anthraquinone parietin, divaricatic acid, atranorin, usnic acid, vulpinic acid, and fumarprotocetraric acid, and several unknown lichen compounds (possibly metabolites).а Lithosiinae of the Rocky Mountain Front Range (Opler 1996): Eilema bicolor Crambidia impura Crambidia casta Crambidia casta Crambidia pura Inopsis pura Cisthene barnesii Lycomorpha grotei Lycomorpha pholus Lycomorpha splendens Hypoprepia miniata Hypoprepia cadaverosa Hypoprepia incluta Hypoprepia fucosa Brucea pulverina Brucea hubbardi Eudesmia aridaаа Discussion Lichens and Lithosiinae of the Rocky Mountain Front Range as Bioindicators More than 600 lichens are reported from Colorado alone (Weber and Wittmann 1992).а A large number of these species are expected to occur along the Rocky Mountain Front Range due to the heterogeneity of habitats in this transition zone.а Some experts consider the lichens to be relatively well-known in the Rocky Mountains (McCune 1995), and reliably identified collections exist at regional herbaria.а However, practical field identification guides and local distribution and abundance information are not available for general air pollution researchers in this region.а This is particularly limiting for studies in community and population monitoring, but may also limit the identification of appropriate indicator species in physiological and bioaccumulation.а Development of regional references for identification and ecology of lichens will increase the feasibility and success ofа pollution studies involving lichens.а Categorization ofа lichens into tolerance/sensitivity categories has not been conducted for most species in the Rocky Mountain Front Range.а A few species have been categorized in studies elsewhere that are widespread in North America (Stolte et al. 1993) and also occur in Colorado (Weber and Wittmann 1992).а For example, in Europe, Parmelia sulcata is regarded as pollution tolerant, and Parmelia saxatilis is reported to be sensitive.аа Many of the lichens known to be sensitive to pollutants and valuable as indicators in other areas have a fruticose or foliose thallus, and others occur on soil or moss, especially in alpine areas (Stolte et al. 1993).а A considerable amount of research on anthropogenic air pollution using lichens and other cryptogams has been conducted in northwestern Colorado.а Some limited a priori categorization of species in our region may be possible using information from these and other studies, providing valuable insights on the determination of indicator species. Depending on research objectives, some lichen species may be problematic as indicators of short term changes because of their slow growth, and because compounds from past pollution exposure may persist in cells over time.аа Air pollution concerns of the City of Fort Collins, for example, are typically evaluated on the scale of days or weeks.а After investigating the potential for using lichens as bioindicators, the city did not identify indicator species that would reflect pollution changes at the temporal scale of interest (Brian Woodruff, Environmental Planner, City of Fort Collins, personal communication).аа Some of these problems might be resolved using transplants,а age estimates of naturally occurring lichens fromа Pb isotope ratios (Stolte et al 1993), and/or by investigatingа rates of absorption and extraction of pollutants over short periods of time during wet and dry spells in different species.а Moths of the family Arctiidae have previously been identified as potential indicators ofа environmental condition and habitat classification at small scales (Scoble 1992).а Although it is possible to monitor trends in populations and communities ofа these species using light traps, identifying causal relationships for observed changes is even more problematic than in lichens due to increased mobility and unknown life histories. Whether Lithosiine moths are likely to sequester pollutants from their hosts is unknown.а However, the recent evidence that they sequester lichen compounds suggests that sequestration of other chemical compounds is possible (Hesbacher et al. 1995).а Based on the widespread but variable patterns in sequestration of lichen compounds observed in wild-caught moths, it seems likely that sequestration of pollutants would also be variable.а Furthermore, based on suspected general feeding habits on algae and algal components, it does not seem likely that Lithosiines discriminate between sensitive and tolerant lichen species in this region.а Laboratory rearing experiments involving fumigation of lichens may prove to be very valuable in determining sensitivity of these moths to pollutants.а There is also the potential forа rearing of moths in semi-controlled field fumigation experiments. Despite potential problems, lichens and lichen feeding moths may prove to be useful as biological monitors in this region, especially in correlation with traditional atmospheric measurements.а Continued federal and local agency support may eventually provide some of the necessary groundwork for future studies. Literature Cited Ahmadjian V (1993)а The Lichen Symbiosis.а John Wiley and Sons, New York, NY. 250pp.а Geiser L (1992)а Feature Article: International Lichenology Meeting.а Forest Service Air Resource Management Newsletter.а USFS Rocky Mountain Region.а Goldsmith FB (1991) (ed.)а Monitoring for Conservationа and Ecology.а Chapman and Hall, UK. 275 pp.а Gough LP, Erdman JA (1977) Influence of a coal-fired powerplant on the element content of Parmelia Chlorochroa.а The Bryologist 80: 492-501.ааа Hawksworth DL , Rose F (1976)а Lichens as Pollution Monitors.а Edward Arnold Ltd.а London, UK. 59pp.а Hesbacher S et al. (1995)а Sequestration of lichen compounds by lichen-feeding members of the Arctiidae (Lepidoptera).а Journal of Chemical Ecology.а Vol. 21, No. 12.а McCune B, Goward T (1995)а Macrolichens of the Northern Rocky Mountains.а Mad River Press, Eureka, CA.а 208pp.а Opler PAа (1996)а Distribution of Arctiidae.а Moths of Western North America .а Unpublished, draft form.а Rawlins JE (1984) Mycophagy in Lepidoptera.а In Fungus-Insect Relationships.а Wheeler, Q, Blackwell M. eds.а Columbia University Press.а New York. pp. 382-423.аа Scoble MJа (1992)а The Lepidoptera.а Form, Function and Diversity.а Oxford University Press, New York.а 404pp.а Stolte K et al. (1993)а Lichens as Bioindicators ofа Air Quality.а General Technical Report RM-224.а Rocky Mountain Forest and Range Experiment Station, Fort Collins, CO.а Weber WA , Whitman RC (1990)а Catalog of the Colorado Flora: A Biodiversity Baseline.а University Press ofа Colorado.