Lichens and Lichen-Feeding Moths (Arctiidae: Lithosiinae) as Bioindicators of Air Pollution in the Rocky Mountain Front Range

Sara Simonson
Colorado State University
Fort Collins, Colorado  80523


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.  Whenever possible, 
recent case studies are used as examples of  practical application.  Also included 
is a summary of the ecology of Lithosiinae moths.   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.  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.  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).  Since then, lichens have 
played prominent roles in air pollution studies throughout the world because of 
their sensitivity to different gaseous pollutants,  particularly sulfur dioxide.  
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).   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).  Lichen Feeding 

A number of Lepidoptera are lichenivorous.  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) .  At least sixteen species in this 
subfamily are known from the Rocky Mountain Front Range (Opler 1996).  Adults are 
easily identified, and certain aspects of the ecology of these moths are well known 
(Opler, personal communication).  Although lepidopterists have expressed concern 
about secondary effects on lichen feeders, studies to determine these effects have 
apparently not been attempted (Hawksworth 1976).   

The Rocky Mountain Front Range

Air pollution is of critical importance in the Rocky Mountain Front Range region 
(generally defined).  Topographic position and weather patterns create an airshed 
that extends eastward from the mountains from Denver to the Wyoming border.  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.   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.   Volatile 
compounds  and oxides of nitrogen are also of  concern because the assist in the 
formation of ozone (Brian Woodruff, Environmental Planner, City of Fort Collins, 
personal communication).  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).  Of great concern to land 
managing agencies in this region are the effects of airborne pollutants on natural 
resources.   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.  
Lichen characters measured in air pollution studies include morphological, 
physiological, and population characteristics.  Historically, lichens have been used 
in a qualitative way, with observations of population changes and morphological 
effects serving as indicators of pollutants.   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 of  toxic elements, and possible changes in spectral 
reflectance, lichen cover, morphology, community structure, and reproduction .  The 
most widely used methods to measure these responses are fumigation and gradient 
studies (Stolte et al. 1993).  Gradient Studies 

The gradient analysis method assumes that measurable attributes of affected species 
vary along causative environmental gradients.  These studies are usually done around 
existing or projected sources of contaminants, with pollutant loadings expected to 
vary with distance from a source.  Although appropiate in many cases, the use of 
lichens in gradient studies has some limitations of which the researcher should be 
aware.  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.  An important 
consideration in using this approach  is the possibility that responses observed 
across gradients are a function of changes in other environmental or disturbance 
variables (Stolte et al. 1993).  

Historically, gradient studies have usually involved observations of  visible 
injury, such as bleaching and thallus deformation, and changes in community 
structure, such as species richness, abundance, or cover (Hawksworth and Rose 1976).  
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).  Response variables are often correlated with pollutant exposure (when 

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.  At increasing distances from the lowland 
powerplants, high mountain environments dominate.  For this study, the lichen 
species Usnia nigrens and Rhizoplaca chrysoleuca were selected as potential 
indicator species.   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.  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.  When climate and pollution factors create unfavorable conditions for 
lichens, it may be impossible to identify and use a naturally occurring indicator 
species.  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.  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).  

In Israel, the common lichen Ramalina duriaei was transplanted along a gradient 
between urban areas.  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".   
This draft report is not for citation,  although it seems appropriate to summarize  
the study here.  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.  Major, minor, and trace element concentrations were measured,  and isotopic 
signatures of sulfur were used to differentiate the anthropogenic and environmental 
sources of atmospheric pollution.  Elevated concentrations of certain compounds were 
correlated with results from snowpack studies and other atmospheric deposition data.  
The use of chemical signatures promises to be very valuable as pollutants can be 
associated with a particular source.      Fumigation Studies:

Fumigation studies most commonly involve measuring response variables of selected 
physiological processes.  Sensitive processes  include activity, K+  efflux/total, 
electrolyte leakage, photosynthesis, and respiration pigment status.  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).    

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.  the continuously 
stirred tank reactors (CSTR)).  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.  

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).  
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).   

Field fumigation studies are conducted using techniques such as Zonal Air Pollutant 
Study (ZAPS) and simulated acidic rain exposures.   The ZAPS system refers to means 
of delivering air pollutants in the field for relatively long-term testing with no 
control over environmental conditions.  This system has been utilized in Alaska to 
fumigate caribou forage lichens with SO2  (Moser 1982, in Stolte et al. 1993).  Rain 
simulators have been used in combination with ZAPS field situations to reproduce the 
combined effects of gaseous pollutants.  The more sophisticated systems are capable 
of reproducing many of the physical and chemical characteristics of precipitation.   
Exposure chambers used for plants that may lend  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).  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.  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.  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).  The methods of TLC to identify specific lichen metabolite 
products should be considered necessary to obtain a species determination.  High-
performance liquid chromatography (HPLC) may be used to support or better define TLC 
results (Stolte et al. 1993).  The use of  chemical techniques may avoid the 
difficulties of identification of species naturally occurring in an area.    
Chemical Analysis

Numerous instrumental methods exist for the determination of most metals in lichens, 
each with a multitude of advantages and disadvantages.  The choice of methods for 
other pollution elements and compounds is more restricted.   Although 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.  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.  Atomic Spectroscopy

Atomic absorption spectroscopy (AAS) has commonly been used in inorganic analysis of 
plants and lichens, especially for the determination of metals.  Historically, arc, 
spark, or flame emission sources have been used in photographic or direct-reading 
spectrographs for the determination of metals.    Recently, many AAS methods have 
been replaced with inductively coupled atomic emission spectroscopy (ICP-AES).   In 
this technique, an argon plasma is used as the atomic emission source and many 
elements may be determined simultaneously from a small sample.  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.   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.   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.  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.  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).  Sulfur 
isotopes and other chemical signatures have recently been used in Colorado to 
identify anthropogenic sources of pollutants in the Mount Zirkel Wilderness area.  
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 used  
in the determination of chlorinated hydrocarbons and other organic pollutants.  
Determination of compounds by these methods is similar to the analysis of pesticides 
in plants.  Ion chromatography has also been used to determine cations and anions in 
plant tissue and may be appropriate for lichen analysis.  

Various other chemical analytical methods are being developed and tested.  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.  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.  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,  
subfamily Lithosiinae, all of whose known larvae feed on lichens, algae, liverworts, 
or mosses.  Lithosiines often occur in semi arid or xeric habitats, feeding on 
lichens and ephemeral patches of algae on rocks or bark.  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.  There is no evidence of an 
obligate relationship to the fungal portion of lichens (Rawlins 1984).  The number 
of algal species known to occur in lichens is extremely small compared with the 
fungal species involved.  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).  As previously 
mentioned, lichen species are named based on the fungal component.  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 several  Lithosiines including Crambidia, Cisthene, 
Lycomorpha and Hypoprepia.  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).  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).  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.  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.  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.  Some 
Hypoprepia species have been reported on mosses (Rawlins 1984).  Chemical Ecology

Lithosiinae moths are aposematically colored, and are known to be toxic to 
predators.  It is not known whether adult toxicity results from sequestration of 
substances in the larval hosts, from their synthesis by adults, or both (Rawlins 

A recent survey for the presence of sequestered lichen compounds  in several 
Lithosiine species may help to unravel this mystery (Hesbacher et al. 1995).  The 
results demonstrate that sequestration of lichen compounds is variable, but 
widespread in wild-caught imagines in Austria and Germany.  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.  Identification of 
lichen compounds was by comparison of HPLC retention times and by comparison of the 
online-recorded UV absorption spectra with those of  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 

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 arida   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).  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.  Some experts 
consider the lichens to be relatively well-known in the Rocky Mountains (McCune 
1995), and reliably identified collections exist at regional herbaria.  However, 
practical field identification guides and local distribution and abundance 
information are not available for general air pollution researchers in this region.  
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.  Development of regional references for 
identification and ecology of lichens will increase the feasibility and success of  
pollution studies involving lichens.  

Categorization of  lichens into tolerance/sensitivity categories has not been 
conducted for most species in the Rocky Mountain Front Range.  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).  For example, in 
Europe, Parmelia sulcata is regarded as pollution tolerant, and Parmelia saxatilis 
is reported to be sensitive.   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).  A considerable amount of research on anthropogenic air pollution using 
lichens and other cryptogams has been conducted in northwestern Colorado.  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.   Air pollution 
concerns of the City of Fort Collins, for example, are typically evaluated on the 
scale of days or weeks.  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).   Some of these problems 
might be resolved using transplants,  age estimates of naturally occurring lichens 
from  Pb isotope ratios (Stolte et al 1993), and/or by investigating  rates of 
absorption and extraction of pollutants over short periods of time during wet and 
dry spells in different species.  

Moths of the family Arctiidae have previously been identified as potential 
indicators of  environmental condition and habitat classification at small scales 
(Scoble 1992).  Although it is possible to monitor trends in populations and 
communities of  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.  However, the recent evidence that 
they sequester lichen compounds suggests that sequestration of other chemical 
compounds is possible (Hesbacher et al. 1995).  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.  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.  Laboratory rearing experiments involving fumigation of 
lichens may prove to be very valuable in determining sensitivity of these moths to 
pollutants.  There is also the potential for  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.  Continued federal and local 
agency support may eventually provide some of the necessary groundwork for future 

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