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The epiphytic lichen Evernia prunastri is sensitive to air pollution and reacted by total retreat to the worsening of air quality during the peak of SO2 pollution in Central Europe (1950s–1990). Since 1990, after a significant decrease in air pollution, epiphytic lichens recolonized previously polluted areas, including E. prunastri. We investigated the physiological status of E. prunastri, transplanted for six months in 34 sites in the urban area of Bratislava (Slovakia) under current conditions. The content of chlorophylls, cortical and medullar secondary metabolites and soluble proteins were explored. We then examined the relationship of these parameters with the environmental quality status, reflected by the diversity of epiphytic lichens. The results showed that the physiological status of E. prunastri did not change significantly after exposure. Positive correlations were found between lichen diversity in the sampling sites and physiological parameters (photosynthetic pigments and phaeophytinization quotient) in the transplants. Transplants from sampling sites with a greater proportion of nitrophilous lichens displayed a decrease in photosynthetic pigments. Sites where E. prunastri naturally occurred had a lower proportion of nitrophilous species in comparison to sites where E. prunastri was not present. This suggests that the indicator species E. prunastri may also recolonize sites with low eutrophication in urban environments under decreased air pollution, and the information on its presence can help to assess the pressure caused by nitrogen excess. [ABSTRACT FROM AUTHOR]