# Basidiomycetes or lecanoromycetes? Revealing secondary metabolite production in lichens with nanospectroscopy

## Project Summary

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:

1. 1.

To confirm the presence of basidiomycete yeast in the Bryoria fremontiiBryoria tortuosa species complex, and determine if the yeasts are involved in the production of secondary matabolites;

2. 2.

To develop a reliable AFM-IR method to image yeasts in lichens; and

3. 3.

To determine if yeasts are similarly involved in other lichen species with the new method.

## Introduction: What is a lichen?

Lichens have been important to humans since pre-history as medicines, foods, dyes, perfumes, and bioindicators, yet remain shrouded in mystery. Figure \ref{fig:lichens} shows the various growth forms of lichens and hints at their unique chemistry. Lichens result from the symbiosis (= ”living together”, a close association between two or more organism) between heterotrophic and autotropic organisms and have been looked to as model systems in studies of symbiosis and evolution for over a century. However, the relatively recent advances in genetic technology and the birth of the microbial ecology discipline have recently raised questions about even our most basic understanding of lichens.

### What We Thought We Knew: The Traditional Model

Historically, lichens have been considered a mutualistic or controlled parasitic relationship between a fungal partner (the mycobiont, a heterotroph that consumes sugars and produces CO$${}_{2}$$) and one or more algal and/or cyanobacterial photosynthetic partners (the photobionts, autotrophs produce sugars). The model holds that the carbon-hungry mycobiont provides protection and shelter for the more delicate photobionts in exchange for fixed carbon. Such associations have proven through time to be specific and repeatable, and given rise to the estimated 20,000 species of lichens thought to exist.11As symbioses, lichens are miniature ecological communities and cannot be classified into species; notwithstanding, lichens have been classified according to the genetic makeup of the fungal partner since 1984. Thus, a lichen species actually refers to a species of lichenized fungi. —(Hawksworth 2000) (Brodo 2001) Most typically lichenized fungi are of the phylum Ascomycota (ascomycetes), which seem to ”infect” algae of the genus Trebouxia to form a lichen. However, recent observations have suggested there may be additional, overlooked organisms participating in the lichen symbiosis.

\label{fig:lichens}The various forms and colors of lichens. (a) Crustose lichens grow as a crust over their substrate; the yellow Rhizocarpon. lecanorinum gets its color from rhizocarpic acid while the grey Rhizocarpon lacks it. (b) Foliose lichens appear leaf-like or foliar, with distinct upper and lower surfaces; Rusavskia elegans