# Is the Reindeer Lichen Cladonia arbuscula Really Producing Isousnic Acid? A Chemotaxonomy Query

## TL;DR

This study investigates whether the lichen Cladonia arbuscula produces isousnic acid, using DNA barcoding and advanced chromatography to confirm its presence in trace amounts.

## Contribution

The study confirms the presence of isousnic acid in Cladonia arbuscula using rigorous chromatographic methods and DNA barcoding.

## Key findings

- Isousnic acid was detected in trace quantities in Cladonia arbuscula using advanced chromatographic purification.
- Routine metabolite profiling failed to detect isousnic acid due to its low concentration.
- A new UHPLC method was developed to distinguish isousnic acid from usnic acid.

## Abstract

Isousnic acid (isoUA) has been detected in a few usnic acid (UA)-producing lichens with chemotaxonomic values. IsoUA was first isolated from a specimen belonging to Cladonia arbuscula s.l. (referred to as C. mitis in the publication). However, the isolation and detection of isoUA in this Cladonia species have not been reproduced and confirmed with clear evidence. This study focused on C. arbuscula s.l. collected in Iceland and aimed to (1) identify the lichen specimen using DNA barcoding and (2) investigate whether isoUA is produced using a series of chromatographic methods. The fungal nuclear ribosomal internal transcribed spacer (nrITS) barcode was sequenced, and the specimen was identified as C. arbuscula, following recent circumscription recommendations. Routine metabolite profiling did not detect isoUA, and it could only be identified after vigorous chromatographic purification and concentration steps using flash chromatography and preparative high-performance liquid chromatography. IsoUA was found in trace quantities (~24 µg/g dry weight), which likely explains its absence in routine metabolite profiling. A rapid ultra-high-performance liquid chromatography (UHPLC) method using a pentafluorophenyl column was developed to separate UA and isoUA. Our study highlights the importance of an integrative approach combining DNA barcoding and detailed chromatographic analyses for lichen chemistry research.