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