The duality of the lichen structure (a fungus and a photosynthesizing partner) has recently come under question. Spribille et al. (2016) suggest basidiomycete yeasts are a third, previously unrecognized partner in the lichen symbiosis, contrary to the centuries-old duality dogma that a lichen is a symbiotic relationship between a single fungi and a photosyntheisizing partner, usually an algae. (de Bary 1879, Oulhen 2016) Lichens produce numerous chemicals as secondary metabolites that have been studied extensively for antimicrobial, medicinal, and industrial uses, and lichen chemistry is often used to classify and identify lichens taxonomically. (Cocchietto 2002, Shukla 2010, Nylander 1866, Culberson 1970, Culberson 1972, Culberson 1976) Although the fungal partner (taxonomically a lecanoromycete) has traditionally been thought to be responsible for the production of these secondary metabolites, the recent evidence of Spribille et al. (2016) suggests otherwise. They note, “the assumption that these substances are exclusively synthesized by the lecanoromycete [i.e. the duality model] must now be considered untested.” Despite this, the molecular and biochemical tools needed to test that hypothesis are not yet in place.
Chemical imaging, here meaning spatially resolved spectra (vibrational or mass) of a sample, has been gaining popularity as a technique for mapping chemical composition in two dimensions on biological samples, especially when paired with chemometric analysis. (Gendrin 2008, Boxer 2009, Cunha 2016) Specifically, spatially resolved mass-spectrometric techniques such as nano-SIMS (secondary ion MS) can give spatial resolutions on the order of nanometers and provide the wealth of chemical information associated with MS. (Jiang 2016) Similarly, atomic force infrared microspectroscopy (AFM-IR) can provide the chemical information of an infrared spectrum but with sub-diffraction limit resolution on the order of nanometers, and when combined with chemometrics can be used to classify microorganisms. (Dazzi 2012, Vitry 2016)
The unifying hypothesis of this work maintains: Previously unrecognized basidiomycete yeasts in the epicortex of lichens are responsible, at least in part, for the production of lichen secondary metabolites. To test this, I will apply the aforementioned chemical tools to study secondary metabolite production in lichens. I will section lichens and (1) image them according to Spribille et al. (2016) to locate yeasts and (2) use nano-SIMS to locate secondary metabolites in the lichen and, using chemometrics, correlate them to yeast location; finally (2) I will use this information to develop a chemometric AFM-IR method to determine the location of yeasts, other fungi (lecanoromycetes), algae, and their secondary metabolites within the lichen in a streamlined fashion. By providing chemical images with unprecedented spatial resolution, I will be able to map chemical production to specific organelles and/or cell locations, improving on previous studies — which mapped only to specific macro structures in the lichen — by orders of magnitude. (Liao 2010) This fine-scale chemical information will allow me to trace the chemicals from production to final location to reveal the organisms involved in their production, lending further information about lichen symbiosis and the potential utilization of their secondary metabolites. To that regard, the specfic objectives of this project are:
To confirm the presence of basidiomycete yeast in the Bryoria fremontii–Bryoria tortuosa species complex, and determine if the yeasts are involved in the production of secondary matabolites;
To develop a reliable AFM-IR method to image yeasts in lichens; and
To determine if yeasts are similarly involved in other lichen species with the new method.