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Fluorescence microscopy as a tool for the visualization of lichen substances within Bryoria thalli

In some species of Bryoria (Parmeliaceae), including Bryoria bicolor (Ehrh.) Brodo & D. Hawksw. and B. fuscescens (Gyeln.) Brodo & D. Hawksw., thallus reactions with spot tests are patchy, and phrases such as ‘‘Pd+ bright red at least in parts’’ have been used in descriptions for many years (e.g. Hawksworth 1972). It has recently been discovered that the extracellular lichen substances (‘extrolites’) in individual thalli of some species in the genus are more varied than hitherto assumed (Hawksworth et al. 2011; Myllys et al. 2011). We hypothesized that this variation in extrolites and the patchiness phenomenon could be due to one or more of several factors, including: 1) chemosyndromic variation; 2) variations in concentration and the sensitivity of detection methods; 3) differences between basal, median and apical regions of the thallus; or 4) the localization of particular compounds in particular anatomical features, such as pseudocyphellae and soralia. As the localization of compounds is known to occur in at least one species of the genus, viz. the yellow pigment vulpinic acid in the soralia of Bryoria fremontii (Brodo & Hawksworth 1977), we decided to explore the last possibility first. Long-wave ultra-violet (UV) fluorescence (at 350 nm) has proved a valuable tool in the separation of thalli of similar crustose and macrolichens, and is also used routinely in the examination of thin-layer chromatographic (TLC) plates; xanthones fluoresce shades of yellow, orange and red, while depsides and depsidones generally fluoresce blue to white or shades of grey, although atranorin gives a yellow hue (Orange 2010). In addition, fluorescence microscopy has been used to explore the location of lichen products in sections of a range of macrolichens (Kauppi & Verseghy-Patay 1990). We therefore decided to explore whether fluorescence microscopy could be used to determine the localization of extrolites in whole lichen thalli, as a supplement to reagent tests, especially as material subjected to Pd reactions has to be discarded. In addition, we wished to determine whether fluorescence would disclose sites: 1) not revealed by reagent tests, that is sites where compounds were present in concentrations too low to yield visible spot test reactions; and 2) which fluoresced in different colours, suggesting the presence of more than one compound.