A study of the lichens of London under contemporary atmospheric conditions (2002-2003)

Davies, L. (1), Bates, J. W. (2), Bell, J. N. B. (1, 2), James, P. W. (3) & Purvis, O. W. (3)
(1) Dept. of Environmental Science and Technology, Imperial College London, London, SW7 2BP, U.K.; (2) Dept. of Biological Sciences, Imperial College London, Silwood Park, Ascot, Berks, SL5 7PY, U.K.; (3) Department of Botany, The Natural History Museum, Cromwell Road, London, SW7 5BD, U.K.

For the first time in the UK a quantitative epiphytic lichen survey has been completed, recording from a single phorophyte, Fraxinus excelsior, at a background location in every London borough. The position of each relevée was recorded using a geographical positioning device and the associated flora, tree bark pH and girth data mapped using a geographical information system (Arc GIS). Distribution maps for each lichen species and associated measurements were then overlaid with computer generated, fine-scale pollution and climatic maps of London. Data were analysed using canonical correspondence analysis in order to identify relationships between lichen occurrence and the environmental variables.
London remains the most polluted area of the UK, with anthropogenic sources of nitrogen, in its various forms, the main pollutant. The study reveals that London’s epiphytic flora is making a spectacular recovery from the almost total loss of corticolous species that had occurred by the 1970s. Seventy-four lichens were recorded from three hundred and thirty-four trees; thirteen species are new to the Capital. Forty percent of the total species, mainly from the families Physciaceae and Teloschistaceae, are believed to be associated with eutrophication. The main environmental variables affecting distribution were transport pollutants and temperature, bark pH, precipitation, and girth.
A gradient of increasing species numbers with distance from the centre is no longer apparent. Diversity increased dramatically at sites where the annual average NOx was below 150 µg/m-3 and most new species were recorded below 100 µg/m-3. Interestingly, the most common species are those also recorded when SO2 was at a peak, although many were then confined to stone.
Bark pH averaged 5.3 in London; this is far less acidic than the unnaturally low pH of 3.5 recorded on Fraxinus excelsior in the 1960s. The rise appears partly responsible for the disappearance of many of the acidophytes and the colonisation on bark of crustose pioneer species of the Lecanoraceae (L. carpinea, L.confusa, L.symmicta).


Candelariella vitellina on asbestos-rich substrata: field and laboratory bioweathering, potential for bioremediation

Favero-Longo, S.E. (1, 2) & Piervittori R. (2)
(1) Centro Interdipartimentale "G. Scansetti" per lo Studio degli Amianti e di Altri Particolati Nocivi, Via Giuria 7, 10125 Torino, Italia; (2) Dipartimento di Biologia Vegetale, Università degli Studi di Torino, Viale Mattioli 25, 10125 Torino, Italia

Candelariella vitellina (Hoffm.) Müll. Arg. is a widespread species on natural and man-made asbestos-rich substrata, sources of fibres which when airborne are potentially dangerous for human health. However, its occurrence on asbestos has not been investigated, even though weathering by C. vitellina may affect the physico-chemical properties of fibres responsible for pathogenic effects. Candelariella vitellina colonization on asbestos-rich serpentinites from the chrysotile (serpentine asbestos) mine of Balangero (Torino, Italy), until 1990 the most important asbestos mine of Western Europe, was investigated by SEM-EDS. Mycobionts were observed penetrating up to 2.5 mm into fibrous veins, and chrysotile, resulting in a strongly modified chemical composition, particularly an impoverishment in Mg. XRPD and Micro-Raman analysis allowed ex situ and in situ evaluation of the mineral composition at the lichen-rock interface, confirming goethite (Fe-hydroxide) formation increased by lichen colonization. Interactions of C. vitellina with chrysotile fibres in axenic cultures were also examined. Apomycobionts were isolated from spores on BBM, their development monitored and identified through sequencing the ITS region. They were then transferred onto a chrysotile-enriched culture medium, where they re-established the close hyphae-fibre interactions, and produced yellow secondary metabolites. Culturing C. vitellina on chrysotile veins outcropping in the abandoned mining-basin of Balangero, has also been attempted. Thalline-fragments, implanted more than 8 months ago by an innovative method (lichen-pockets), continue to show good vitality. Candelariella vitellina can therefore be considered as a potential resource for bioremediation in abandoned asbestos mines and on asbestos-cement.


Biomonitoring air pollution with the desert lichen Ramalina maciformis

Garty, J. (1, 2), Tamir, O. (1), Levin, T. (1), Weissman, L. (1) & Lehr, H. (1)
(1) Department of Plant Sciences; (2) Institute for Nature Conservation Research, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

The epilithic fruticose lichen Ramalina maciformis was selected to investigate the environmental impact of anthropogenic activity on its elemental content and physiological integrity. In the first year of study, element concentrations (Ca, Cr, Cu, K, Mg, Na, Pb, S, Sr and Zn) in thalli from an unpolluted site (Tellalim), collected in August 1997, transferred to 24 biomonitoring sites and retrieved in April 1998, were compared with those of the material from Tellalim, which served as control. Lichen element concentrations were correlated with the potential quantum yield of photosystem II (PSII) and cell membrane integrity. Transplanted thalli in several sites in the polluted area of Ramat Hovav accumulated large amounts of most elements. Ca, Cu and Sr showed an inverse correlation with the Fv/Fm ratio, expressing the potential quantum yield of PSII. Ca, Cr, Cu, Mg, Na, S and Sr showed a positive correlation with electrical conductivity corresponding with cell-membrane disintegration. In the second year of study, lichen transplants accumulated large amounts of mineral elements in 10 biomonitoring sites in the polluted Ramat Hovav area relative to lichens in the control site over a 7-month period (May-November 1999). Statistical analysis revealed a positive correlation for B, Mn, Ni and Sr with electrical conductivity, a positive correlation for Ni and ethylene production, and a negative correlation for B, Cu, Mn, Na, Ni, Sr and Zn with Fv/Fm ratios. The study highlights the value of R. maciformis as a biomonitor.


Lichen biomonitoring and measuring lichen growth in Greenland

Hansen, E. S.
Botanical Museum, University of Copenhagen, Gothersgade 130, DK-1123 Copenhagen K, Denmark

"Zackenberg Basic" is a long-term monitoring programme, including monitoring climatic changes in North East Greenland. Operated at Zackenberg Station (74°30´N, 20°30´W), it is owned by the Danish Polar Center. Fourteen plots were established in saxicolous and terricolous lichen communities in 1994. Five new plots were established in 2000, together with twenty-five additional stations along the ZERO (= "Zackenberg Ecological Research Operations")-line from Young Sund to the top of Aucellabjerg, 1040 m a.s.l. in 2000. All permanent plots and stations will be monitored every 5th year, next time in 2005. All vegetation and climatic changes including temperature, precipitation and UV-B radiation expected to influence lichens will be recorded.
The Greenland Institute of Natural Resources in Nuuk and associated researchers carried out large-scale vegetation investigations in different reindeer grazing areas in West Greenland during these years. Investigation areas are analysed by satellite photos and lichen cover measurements, using e.g. the ITEX-concept. In a similar study, the degree of reindeer grazing on lichens was estimated near the Isortoq Reindeer Station (60°59´N, 47°32´W) in South West Greenland in 1998. Most investigated plots in the Isortoq area have been moderately grazed by reindeer, while some have been slightly grazed and only one or two plots have been heavily grazed.
A study of the growth of saxicolous lichens was carried out near the Mittivakkat Gletscher (65°41´N, 37°48´W) on Ammassalik Ø in South East Greenland in 2001. Fifty-four permanent plots were established on boulders and rocks in front of the glacier. The maximum diameter of selected lichen thalli was measured and related to the known position of the ice margin in 1933, 1958 and 1970. Lichens will be measured again in 2006.


200 years of lichen surveillance in the city of Turin (N Italy)

Isocrono, D., Matteucci, E., Pensi, E., Ferrarese, A. & Piervittori, R.
Dipartimento di Biologia Vegetale Università di Torino, Torino, Italy

Since the 1970s, decreasing pollution has led to lichen recolonisation in some urban areas. This phenomenon was noted also in Turin, a medium-sized industrial city in Italy, by means of a dataset dating back to 1792. Four locations are permanent observation sites since 1900 and one since 1800.
15 epiphytic species were recorded in the 19th century. During the 1920s, the urban lichen flora indicated reasonable environmental conditions: 8 species were found in the city centre, 15 in the surrounding hills. During the following years, a progressive impoverishment occurred: a single species was found in 1960 and no species at all in 1975, 1980 and 1989. A casual search in 1996 indicated recolonization by 6 species in some urban parks. Two years later, a substantial improvement was noted, when 24 species were found. A lichen desert was, however, still present in the south-western part of the city. Today, lichens are present throughout the urban area. The highest lichen diversity was found on the hill E of Turin, while the lowest biodiversity values and thalli with the smallest size were limited to the northern part of the city, most affected by vehicle traffic, industries and a civic dump. Successful colonizers are mainly foliose, sorediate or isidiate species. More than half of the species, normally bearing apothecia, were, however, sterile. Recolonization appears to be initially correlated mainly with the decrease of SO2 and Pb. However, from 1998 to present, levels of these pollutants were low, and other pollutants, e.g. total suspended particles, may have influenced the composition of lichen assemblages. This hypothesis is supported by the high number of nitrophilous species found.


Developing a suitable growth substrate for lichen forced cultivation under an artificial environment

Komine, M., Iwasaki, Y., Yamamoto, Y. & Hara, K.
Akita Prefectural University, Akita City, Japan

Lichens have been used as a resource for medicine, dyeing etc since ancient times, and may provide a useful industrial resource today. The most serious problem hindering the industrial use of lichens is obtaining sufficient material because of their slow growth. Tissue culture and forced cultivation methods are available, but few studies about lichen forced cultivation have yet been carried out and there is a need to develop this new methodology.
The main objectives of this study were to develop a forced cultivation technique for lichens, and to investigate the most suitable growth substrate for lichen cultivation.
The lichen species used was Cladonia scabriuscula. Rock wool plate and bryophytes were used as growth substrates. Each substrate was packed on a square tray and lichen thalli planted in the substrate. Cultivation experiments were conducted in 6 growth chambers, with the following environmental conditions: temperature: 25° C, light intensity: 47 µmol m-2 s-1, humidifying: 12h/24h, light period: 8h/16h, 12h/12h and 16h/8h (light/dark). Each chamber contained two trays, creating 12 treatments. Survival and growth rates of lichen thalli were measured after 3 months.
Higher growth and survival rates were obtained for the 16h/8h light period, 12h humidifying, and on the bryophyte substrate. By contrast, almost all lichens on the rock wool substrate decomposed or were contaminated because of high humidity. Humidity conditions over the bryophyte substrate were relatively drier than on the rock wool substrate, and drying condition was supposed to be reinforced by the long light period and short humidifying time. Moderately dry conditions are necessary for lichen growth and survival. Bryophytes may provide a suitable growth substrate.


Analysis of solid particulate matter from the surface of Hypogymnia physodes thalli

Lackovicová, A. (1), Guttová, A. (1) & Kristín, J. (2)
(1) Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 14, 845 23 Bratislava, Slovakia; (2) Central Laboratory of Electrone-optical Methods, Faculty of Natural Sciences, Comenius University, Mlynská dolina, 845 15 Bratislava, Slovakia

The composition of particles attached to the surface of Hypogymnia physodes thalli was analysed by scanning electron microscopy (SEM) coupled with quantitative, non-destructive in situ EDS microanalysis. Chemical composition of particles is expressed as percentage by weight. The results of EDS were compared with total thallus contaminant concentrations determined by AAS. Sampling was performed in industrial hotspot towns (Rudnany, Krompachy, Mochovce) and in relatively clean areas (National Nature Reserves in the Tatry, Poloniny and Slovenské Rudohorie mountains) in Slovakia.
Photodocumentation established a relationship between particle morphology and the type of locality. Particles of secondary aerosols featuring sphaeroidal shapes (diameter 2-27 µm) prevailed on thalli in industrial areas. Rectangular, dust fragments (diameter 1-38 µm) dominated in clean areas.
Total element content was in most cases similar to the composition of surface particulate matter. Discrepancies may be due to different factors, especially the physical properties of the thallus and localisation of particles within it. A smooth thallus surface facilitates ‘wash-off’ of particulate matter poorly attached to the surface. Particles dislodged from the thallus surface may become entrapped within cracks within the thallus leading to their persistence within thalli.


Site condition monitoring with twig epiphytes

Larsen, R.S. (1,2), Wolseley, P.A.(1), Søchting, U.(2) & Chimonides, P.J.(1)
(1) The Natural History Museum, London, United Kingdom; (2) Botanical Institute, University of Copenhagen, Denmark

Twig flora is believed to provide the earliest floristic response to changes in atmospheric conditions. This is due to two factors: 1) Twigs provide new bark every year, unaffected by previous air pollution and bark stem flow chemistry. Current air quality is thus a major factor affecting the twig micro-environment. 2) Lichens and bryophytes living on twigs are mostly pioneer species adapted to efficient dispersal, and lack competition from an established epiphytic flora.
Changes in epiphytic flora on Quercus twigs were monitored at four sites in Tycanol NNR, Wales over an 8 year period (in 1995 and 2003). Increased bark pH on trunks over time was associated with a reduction in frequency of "acidophytes" (Hypogymnia and Usnea spp.) and to a lesser extent with an increase in "nitrophytes" (Physcia and Xanthoria spp.).
Regional differences in twig flora were investigated by comparing Tycanol, Wales with Kaas Forest, Denmark. Total nitrogen content in Hypogymnia physodes together with bark pH from twigs and trunks was assessed at all sites.
The Danish lichen and bryophyte flora is characterised by a high frequency of nitrophytes and near absence of acidophytes. This is associated with the highest %N content in H. physodes, the highest twig bark pH and high trunk bark pH.
Twig epiphytic flora provide a promising early warning system to detect local changes in atmospheric conditions.


Lichen biomonitoring of ammonia emission and nitrogen deposition around a pig stockfarm

Loppi, S. (1), Frati, L. (1), Santoni, S. (1), Nicolardi, V. (1), Gaggi, C. (1), Brunialti, G. (1), Guttova, A. (2) & Gaudino, S. (3)
(1) University of Siena, Italy; (2) Slovak Academy of Sciences, Slovakia; (3) APAT Roma, Italy

The effect of high ammonia emissions and nitrogen deposition on lichens was investigated at 4 sites along a transect (200, 400, 1000, 2500 m) from a pig stockfarm (ca. 7 000 animals) in central Italy. Epiphytic lichen diversity was measured and transplanted thalli of Xanthoria parietina and Flavoparmelia caperata exposed together with passive NH3 samplers (diffusion tubes) at each site.
Ammonia concentrations dramatically decreased from the centre of the stockfarm to the sampled sites, correlated with bark pH. Total lichen diversity did not show any association with either NH3 concentrations or bark pH, but the diversity of strictly nitrophytic species was highly correlated with these parameters. Physconia grisea was the best indicator species for NH3 pollution. Total N accumulated in X. parietina and F. caperata was correlated with NH3 concentrations. F. caperata samples exposed in the centre of the stockfarm showed visible signs of damage, while these symptoms were not evident in X. parietina, confirming the former is a sensitive species, and latter, a resistant species to NH3 pollution.


Data quality in lichen biomonitoring studies

Loppi, S. (1), Giordani, P. (2), Brunialti, G. (2) & Isocrono, D. (3)
(1) University of Siena, Italy; (2) University of Genova, Italy; (3) University of Torino, Italy

The results of quality control (QC) and quality evaluation (QE) tests on the influence of field crews on data quality in lichen biomonitoring surveys were investigated.
Personnel involved in Italian lichen mapping studies underwent QC and QE tests to compare lichen biodiversity (LB) counts measured by different crews from different parts of Italy.
LB was measured as the sum of frequencies of epiphytic lichens in a sampling grid of 30x50 cm divided into 10 units of 10x15 cm. All lichen species within the grid were recorded and their frequency, namely the number of grid units in which the species was found.
Quality Control - Sampling grids were placed by a group of expert lichenologists on the bole of selected trees. Each crew was allowed 30 min to sample trees without any diagnostic keys or chemical tests. One tree was re-sampled to check the data precision. Results of each operator were compared with the scores of the expert group (control).
Quality Evaluation - Accuracy and precision were used to assess the % error of LB counts. Three types of accuracy/precision measures were considered: 1) quantitative, to evaluate the LB counts; 2) qualitative, to evaluate the number of lichen taxa recognized within the grid; 3) taxonomic, to evaluate the correct identification of lichen taxa within the grid.
Results showed that 75% quantitative accuracy and 90% quantitative precision can be regarded as satisfactory measurement quality objectives (MQOs) for lichen biodiversity data. 65% proved to be sufficient MQO for accuracy of taxonomic identification in the field. Results also indicate a need for further taxonomic training.


Dating moraines and rock glaciers on Western Spitsbergen, Svalbard

Martin, J.
Eurouniversity, Mustamäe Rd. 4, Tallinn 10621, Estonia

Svalbard glaciers are set to be the principal contributor to sea level rise in the High Arctic outside Greenland. Lichenometric data collected along the western coast of Spitsbergen demonstrate continuous ice cover regression over the last 3500 years. Three lichen species i.e. Rhizocarpon geographicum, Xanthoria elegans and Pseudephebe minuscula were used as indicators. Growth curves were constructed for these lichen species using objects of a known age. Kongsfjorden, the oldest investigated glacier moraine of Midre Loven formed ca 300 years ago during the "Little Ice Age". Glacier Pedersen, the longest exposed "rock glaciers" was ca 800 years old (Viking period). The other rock ridge had a lichenometric age of ca 2000 years and formed during the neoglacial advancement. Dating of rock surfaces in Isfjorden include rock glaciers at Vardeborgsletta and Linnedahlen. Lichenometric datings demonstrate marine surface terraces were covered by a permanaent ice sheet, which melted between 1000 and 1400 years ago. Rock glaciers beneath the mountain slope and in Linnedahlen were of a similar age. Most terminal moraines in Grønfjorden formed at the beginning of the last century. A few moraines appeared earlier, ca 300 years ago. Glacier Aldegonda had relatively young moraines forming 100-200 years ago. Older formations, 800-900 years old, were discovered on the northern slope. In Hornsund, ridges of rock glaciers were divided into two groups: ridges between Steinvika and Russepunten exposed between 400 and 675 years ago. The other group, including five moraines, was formed ca 1000 years ago. The dates of the first group coincide with the Magdalena-fjord glaciation stage. The other group coincides with glacier advancement 3500 to 2000 years ago, and with the ensuing glacier regression 2000 to 750 years ago. Synchronous moraine formation was demonstrated, in many cases, over the investigated territory.


Observations on epiphytic community dynamics in deciduous forests around a chemical factory in central Lithuania

Motiejunaite, J.
Institute of Botany, Vilnius, Lithuania

Epiphytic communities were studied annually over a 5-year period in permanent quadrats in deciduous forests surrounding a chemical factory in central Lithuania. Observations revealed intense community dynamics, expressed by changes in species number and abundance (thallus area) within the quadrats, as well as variable dynamics of individual species.
Community composition varied in the study plots and quadrats from monospecific algae communities (plot closest to the emission source (ES)) to rich communities, consisting of 10-12 species per 40 cm2 in the plots situated at the farthest distance from the ES.
The studied communities were affected not only by the environmental variables influencing all trees in the plot, like humidity and air pollution level. All trees were initially subjected to similar light levels, but thinning of forest around two of the plots and increased light levels affected three quadrats, resulting in rapid changes - dominance of photophilous species in one quadrat, and severe damage of ombrophilous species in the other two. Infestations by the fungus Athelia arachnoidea were noted in 10 out of 23 quadrats during the whole observation period. Mollusc grazing was noted in many quadrats, but conspicuous damage recorded in only 4. Bark scaling also influenced, though not significantly, both species diversity (when solitary small thalli fell off together with bark pieces) and lichen cover. Only 7 quadrats out of 23 survived the 5-year period without any "catastrophic" event such as light intensification, grazing, fungal infection, either alone or in combination resulting in dramatic community changes.
Significant changes in most of the observed communities were caused by several factors unrelated to air quality. Care should therefore be taken to assess all relevant factors when applying small plot (quadrat) methods for long-time community observation, especially in the case of epiphytic communities.


Epiphytic lichens as environmental bioindicators in Rome, Italy

Munzi, S. (1), Ravera, S. (2) & Caneva, G. (1)
(1) Dept. of Biology, University of Roma Tre, Rome, Italy; (2) Dept. of Plant Biology, University "La Sapienza", Rome, Italy

A synthesis of studies carried out in Rome over the last twenty years (1982-2003) is presented. Epiphytic lichen research focused on flora, vegetation, air pollution monitoring and accumulation of trace elements in lichen thalli. Phytosociological relevés were carried out in 1982 to investigate the relationship between epiphytic lichen vegetation and urban environmental conditions in 1982. Atmospheric pollution by trace elements was investigated in 1994 using Evernia prunastri (L.) transplants. A first stage of biomonitoring carried out according to the Italian Environmental Protection Agency guidelines ended in 2003.
Results showed: i) floristic composition changed over the period 1982-2003; ii) the lowest lichen diversity was found in the urban centre and the south-western and north-eastern sectors, in relation to direction of prevailing winds, humidity, topography, land-use and other local factors; iii) the "lichen desert" is decreasing in parallel with decreasing concentrations of CO, NOx and SO2.
The most important factors affecting the urban lichen flora of Rome are the individual characteristic of stands, geographical location, direction and intensity of prevailing winds and distance from the Tyrrhenian coast-line. The peculiarity of Rome's climate is reflected by the occurrence of mainly non-Mediterranean taxa. Although located in the centre of the Mediterranean area, the city is characterized by a climate transitional with the central-European type. Rome's protected areas, parks and "ville", are hotspots of lichen diversity, with species that are indicators of ecological continuity in Italy such as Chaenotheca hispidula (Ach.) Zahlbr., Dimerella tavaresiana Vezda, Lecanographa amylacea (Pers.) Egea & Torrente, Gyalecta liguriensis (Vezda) Vezda and G. truncigena (Ach.) Hepp.


Lichens and climate change in Eastern Mediterranean region: A step towards a standardized method

Pirintsos, S. A. (1), Sipman H. (2), Insarov, G. (3), Insarova, I. (4) & Loppi, S. (5)
(1) Department of Biology, University of Crete, P.O. Box 2208, GR-71409 Heraklion, Greece; (2) Botanischer Garten und Botanisches Museum Berlin-Dahlem, Kön-Luise-Str. 6-8, D-14191 Berlin, Germany; (3) Institute of Global Climate and Ecology, Russian Academy of Sciences, Glebovskaya Str., 20 B, 107258 Moscow, Russia; (4) Department of Biology, Moscow State University, Leninskie Gory, 119899 Moscow, Russia; (5) Department of Environmental Sciences "G. Sarfatti", University of Siena, Via P. A. Mattioli 4, I-53100, Siena, Italy

This study has addressed the issue of possible climate change effects on epilithic lichen communities. Saxicolous lichens growing on flat calcareous rocks in open canopy ecosystems represent a candidate biological material for incorporation in the developing monitoring systems of anticipated climate change in the Eastern Mediterranean Region. These lichen communities seem to be less affected than other organisms from management practices and anthropogenic disturbances in an area with long history of intense human-driven changes in natural ecosystems. Additionally, calcareous rocks are a widespread substrate in the semi-arid, Eastern Mediterranean Region, as in the case of Central Crete Mountains, where this study took place. The basic assumption underlying the proposed monitoring system is that anticipated climate change in time can be to some extent simulated by the actually observed changes in epilithic lichen communities in space, as we move along an altitudinal gradient. In this context, the basic parameters of epilithic lichen communities were quantitatively assessed in different altitudes and correlated to the recorded shifts of temperature and humidity along the gradient.
For data collection, a previous methodology was adapted and modified with the use of digital photography and digital image processing. The sampling process consists of taking high-resolution pictures of 10x10 cm quadrats, placed on the surface of randomly selected rocks of approximately the same size. The proposed methodology is simple, formal, easily reproducible and non site-specific, allowing data collection for a real-time monitoring of the anticipated climate-driven changes. Concerning data analysis, several parameters of saxicolous lichen communities are discussed within the context of developing a uniform monitoring method for the Eastern Mediterranean Region.


Lichen monitoring in the central region of the Czech Karst

Svoboda, D.
Department of Botany, Charles University in Prague, Benátská 2, 128 01 Prague 2, Czech Republic

The lichen flora in CHKO (Czech Karst protected landscape area) was investigated. CHKO is located in central Bohemia, SW of Prague, in a temperate, xerothermic zone with low annual precipitation of 500 mm/y. The area is partially forested, with characteristic limestone and basaltic ecosystems on hills, rocky slopes and ravines. The Czech Karst region is quite polluted, presently the average winter SO2 concentration is ca 15 µg/m3.
The main aim of this preliminary study was to test the applicability of the Lichen Diversity Value (LDV) method in the Czech Republic to assess pollution levels. The zone method of Hawksworth and Rose was additionally applied.
The study area was divided into 43 1x1 km squares and in each square, measurements were taken ideally on 4 trees, or a minimum of 3 trees. The results of both methods were presented in maps. According to the Hawksworth and Rose method, zones 4-7 were found, but it was not possible to evaluate the results in terms of solely SO2 pollution because of the mixture of pollutants present. A modified scale was proposed for the region.
Measurements obtained by the LDV method yield similar results: LDVs varied between 18-65 and served as a basis for a preliminary scale applicable for the region. High LDVs were found in the central forested part, up-wind from Beroun town.
The LDV method is often limited by the lack of suitable trees. It is often difficult to find a solitary tree in a central European forest and trees may be shaded by shrubs.


Chemical constituents from lichens for pharmaceutical and industrial uses

Takahashi, K. (1), Kinoshita, K. (1), Koyama, K. (1), Yamamoto, Y. (2) & Yoshimura, I. (3)
Meiji Pharmaceutical University, Tokyo 204-8588, Japan (1); Akita Prefectural University, Akita 010-0146, Japan (2); Hattori Botanical Laboratory, Kochi 781-2120, Japan (3)

Many lichens produce characteristic secondary metabolites known as lichen substances. During our search for new bioactive compounds, we discovered compounds of pharmaceutical and industrial use in the lichens Protousnea spp., Lecidea confluens (G. Web.) Ach., and mycobiont cultures from Arthonia cinnabarina (DC.) Wallr. and Amygdalaria panaeola (Ach.) Hertel & Brodo.
Pharmaceuticals uses – (1) Lichen mixtures of Protousnea spp. have been used by natives on the island of Tierra del Fuego as a skin conditioner. Scince tyrosinase is an enzyme producing melanin pigments, the anti-tyrosinase activity might be effective in whitening facial skin. Two compounds, 3,5-dihydroxy-1-propylbenzene and bis(2,4-dihydroxy-6-propylphenyl)methane obtained from Protousnea spp., showed anti-tyrosinase activity which might be beneficial for skin care. (2) As already reported, monoamine oxidase B (MAO-B) inhibitors can be effectively used, in combination with L-DOPA (L-dihydroxyphenylalanine), to treat Parkinson’s disease. Confluentic acid, isolated from Lecidea confluens, showed a selective inhibitory effect of MAO-B. (3) Some lichen mycobionts cultured without the algal partner can often synthesize novel and extraordinary constituents. We successfully cultured the mycobiont of Arthonia cinnabarina derived from its spores. New compounds, cinnabarinol, arthoniafurone A and B, were obtained from the culture which showed cytotoxicity agaist U937 human leukemia cells.
Industrials uses – Amygdalaria panaeola was collected in Finland in 1990 and its mycobiont isolated from thallus fragments and spores. The mycobiont was cultured on malt-yeast liquid medium. The medium showed a fluorescent yellowish green color. Novel fluorescent substances, panaefluoroline A to F, were isolated using chromatographic techniques, and show clear fluorescence, stable both in water and organic solvents for long periods. These compounds are very interesting, each chemical structure differing according to amino acid origin. Fluorescent characters remained in all compounds which have potential application in dyes.


Lichen substances as natural dyeing compounds

Vesselova, S. & Randlane, T.
Institute of Botany & Ecology, University of Tartu, Lai St. 38, 51005 Tartu, Estonia

The project aims (1) to assess the practical use of lichens as sources of dyeing agents; (2) to identify which specific lichen substances act as dyeing substances; (3) to propose additional lichen species which might be suitable for dyeing.
Eight abundant, widely distributed macrolichen species in Estonia were used in the experiment: Cetraria islandica, Evernia prunastri, Hypogymnia physodes, Parmelia sulcata, Pseudevernia furfuracea, Ramalina farinacea, Usnea filipendula and Xanthoria parietina. Appropriate methods were determined and dyeing experiments using pure wool performed. Lichen substances produced by these 8 species were also identified by TLC.
Various colours (mainly yellow, greenish and brownish) were obtained using different lichens and mordants. Brightest colours were achieved with Cetraria islandica (fumarprotocetraric acid as the dyeing compound) and Parmelia sulcata (salazinic acid as the dyeing compound). Evernia prunastri, Hypogymnia physodes and Pseudevernia furfuracea (containing atranorin and chloroatranorin) gave greenish to pale yellow colours, the intensity evidently depending on the concentration of lichen substances. Depsides (e.g. atranorin) and depsidones (e.g. fumarprotocetraric and salazinic acids) having an aldehyde group were found to act as dyeing compounds in the 8 species examined.
Copper and ferric sulphate can be used as mordants to obtain greenish and brownish colours, while soda bicarbonate darkens and acetic acid turns it yellowish. Most dyed wools turned paler by bleaching.
The abundant, easily collectable macrolichens, Cladina rangiferina, Platismatia glauca, Umbilicaria deusta and Xanthoparmelia conspersa, containing depsides and depsidones with an aldehyde group, are potential sources of natural dyeing compounds in Estonia


Development of new inclusion complexes using usnic acid and natural or synthetic carrier materials

Xavier-Filho, L. (1), Araújo, A. A. S. (1), Susmaga, T. L. B. (1), Watthier, J. (1), Cides, L. C. S. (2) & Matos, J. R. (2)
(1) Instituto de Tecnologia e Pesquisa da Universidade Tiradentes, UNIT, Brazil;
(2) Instituto de Química da Universidade de São Paulo, USP, Brazil

The potential use of natural (quitosan, cyclodextrins) and synthethic (FDU-10, SBA-15) compounds has been extensively studied to improve certain drug properties such as solubility, stability, and/or bioavailability. Drug activity enhancement and selective transfer or side effect reduction can be achieved by molecular encapsulation. The aim of this work was to explore new potential properties of quitosan and cyclodextrin (natural compounds) and FDU-10 and SBA-15 (ordered silicas with cage-like mesoporous structures) and their potential to act as a convenient reservoir for controlled drug delivery systems. For this purpose, usnic acid, an anti-microbial, anti-inflammatory and anti-carcinogenic drug extracted from Cladonia substellata Vainio (originally on sand resting from northeast Brazil) was used. To charge the materials with usnic acid, the drug was dissolved in water and inclusion compounds added in the ratio 1:2 (w/w). This mixture was stirred for 36 h taking care to avoid water evaporation. Dry materials were characterized by thermogravimetry/derivative thermogravimetry (TG/DTG), differential scanning calorimetry (DSC), X-ray diffraction (XRD) and infrared espectroscopy (FTIR). DSC curves in the temperature range 25-500°C for the free usnic acid present a single sharp endothermic peak at melting point at the temperature of 201°C, corresponding to the apparent heat of DH=112 J.g-1. The TG/DTG curves indicated that the thermal decompostion process of usnic acid occurs in two stages in the following temperature range and weight loss: 205-370°C (Dm=47%) and 370-900°C (Dm=53%). This profile was compared with the obtained inclusion complexes. The absence of the melting peak of usnic acid at 201°C showed that no free usnic acid was present in the system with FDU-10, SBA-15 and b-cyclodextrin. On the contrary, the DSC curve of usnic acid/quitosan showed evidence of the presence of free usnic acid and quitosan, indicating the need to optimize the inclusion process. These results were corroborated by XRD and FTIR techniques.


Synthesis of methyl-3-orsellinate by organic synthesis or by altered biosynthetic pathways using lichen immobilisates

Xavier-Filho, L. (1), Pereira, E. C. (2), Vicente, C. (3) & Legaz, M.-E. (3)
(1) Universidade Tiradentes, Avila Nabuco 300, Farôlandia, Sergipe, Brazil; (2) Departamento de Ciencias Geograficas, Universidade Federal do Pernambuco, 5000 Recife, Brazil; (3) Departamento de Fisiología Vegetal, Facultad de Biología, Universidad Complutense, 28040 Madrid, Spain

Lichens produce depsides, phenolics composed of two or three monocyclic units linked by an ester bond and derived from orsellinic acid. Orsellinic acid is synthesized by an aromatic synthase, which produces an 8C lineal polyketide intermediate before cyclasation. However, labelling experiments have indicated that an additional C-1 unit must be added to the polyketidechain prior to cyclasation to produce ß-methyl orsellinate, the basic unit of depsides of the ß -orcinol series. This additional C1 unit can be derivatized or oxidized to alcohol or aldehyde functions, such as occurs in atranorin. Depsides from the orcinol and ß-orcinol series, such as lecanoric or evernic acids, and atranorin, respectively, are produced by esterification of two orsellinic acid, or orsellinate derivatives, catalyzed by several orsellinate depside hydrolases thus acting as esterases.
Methyl-3-orsellinate can be generated from methyl-3-orcinol through the nitration of aryl function to be then reduced to an amine group. This last derivative could be the substrate of a diazotonization for the corresponding diazonium salt. After this, a cyanide group could be introduced by nucleophilic substitution using potassium cyanide and then, the carboxyl function could be produced after hydrolysis. Alternatively, methyl-3-orsellinate could be produced by alginate-immobilized, atranorin-producing lichen cells loaded with acetate and treated with sodium azide. Acetate is used by acetyl-CoA carboxylase and aromatic synthase (methyl transfer) to produce methyl-3-orsellinate and its oxidation to haematommoyl alcohol is inhibited by sodium azide.


Extraction, purification and identification of usnic acid from Cladonia substellata samples by gas chromatography/mass spectrometry

Xavier-Filho, L., Watthier, J. & Araújo, A. A. S.
Laboratório de Produtos Naturais e Biotecnologia, Instituto de Tecnologia e Pesquisa ITP/ UNIT, Aracaju-SE, Brazil

Lichen metabolites have long been used against many kinds of ailments, also in drugs in some European countries. Studies on antimicrobial activities of lichen species present in NE Brazil (semiarid region) showed that antimicrobial properties are determined by usnic acid. Usnic acid has hydrophobic characteristics, being insoluble in water and glycerol, scarcely soluble in ethanol and readily soluble in hot ether, acetone, benzene and chloroform. After purification, usnic acid crystallises in different shapes according to the solvent used in the crystallization process. In this study a soxhlet device was used to extract usnic acid from the lichen Cladonia substellata Vainio. Samples collected in the Sergipe state of Brazil were submitted for extraction (for 12 hours) with the solvents: chloroform, acetone, ethylic ether and methanol/acetone 1:1 (v/v). Subsequently, the extracted material was submitted to a rotavapor at 60°C until partial evaporation of the solvent, allowing the rest to naturally evaporate. An impure yellow powder was obtained, later purified and crystallized by using cold chloroform and ethanol 1:3 (v/v). After 48 hours (the time necessary for complete precipitation) vacuum filtration was performed. Finally, the filtrate was recrystallized with chloroform, and crystals of usnic acid obtained, as confirmed by gas chromatography and melting point. Pure compounds were identified by gas chromatography coupled with mass spectrometry. Usnic acid obtained with the different solvents was 42% with acetone, 28% with ethylic ether and 38% with methanol/acetone. Comparison of the spectra of the obtained crystals with the database library (NIST 98) confirmed formation of usnic acid with the four solvents.


Screening for antibacterial activities and isolation of antibiotics from mycobiont cultures

Yamamoto, Y. (1), Takeda, M. (1), Hara, K. (1), Komine, M. (1), Inamoto, T. (1), Kawakatsu, M. (2) & Miyagawa, H. (2)
(1) Akita Prefectural University, Akita, Japan; (2) Kyoto University, Kyoto, Japan

As many lichens produce characteristic secondary metabolites, they have been used as folk medicines, dyes and cosmetics since ancient times. However, mass harvesting of lichens as an industrial resource may lead to species extinction. If lichens are therefore to be used in industrial applications, they must be cultured in vitro. We succeeded in isolating, culturing and maintaining mycobionts and photobionts of about 450 lichen species by using both spore and thallus fragment culture methods. This study aimed at investigating the antibacterial property of cultured lichen mycobionts and isolating their antibiotics.
22 mycobionts derived from spores or thallus fragments of lichens were used. Cultured mycobiont cell-aggregates were freeze-dried and submerged in acetone. Acetone solution was filtered and evaporated to yield extracts. Each extract was dissolved in acetone and absorbed on a paper disk. We used 16 kinds of bacteria for the experiment: Actinomyces pyogenes, Bacillus subtilis, Bifidobacterium pseudolongum, Clostridium perfringens, Erysipelothrix rhusiopathiae, Escherichia coli, Lactobacillus acidophilus, Micrococcus luteus, Propionibacterium acnes, Pasteurella multocida, Staphylococcus aureus (2 types), S. epidermidis, Streptococcus faecalis, S. mutans and S. pyogenes. Each disk was set on an agar-plate where each bacterium was cultured. After one or two days, growth inhibition of each bacterium was judged by the diameter of its inhibition circle.
The extract of Haematomma puniceum mycobiont cultured on MY agar-plates showed the highest activity of growth inhibition of many bacteria. The ethyl acetate extract of culture broth of H. puniceum mycobiont yielded two pigments identified as 5-deoxy-7-methylanhydrofuzarubin lactol and 5-deoxy-7-methylbostrycoidin. Both acted as antibiotics in the H. puniceum mycobiont extract.

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