Testing the correlation between norstictic acid content and species evolution in the Cetraria aculeata group in Europe

Most lichen-forming fungi are characterized by the production of secondary metabolites. Differences in metabolite patterns have frequently served to distinguish lichen taxa with subsequent controversies about the rank of chemical variants (chemotype, variety, subspecies or species). Using a model system, we investigate whether production of norstictic acid within a group of lichenized ascomycetes is correlated with phylogenetic patterns, population differentiation or single and multilocus haplotypes. Our study is based on DNA sequences of three gene loci (ITS, GPD, mtLSU) together with HPLC (311) and TLC (594) data from a total of 594 samples of three closely related fruticose lichens: Cetraria aculeata and C. muricata without norstictic acid, and C. steppae with norstictic acid. In nature, C. aculeata and C. steppae often occur together and the status of C. steppae as a separate species has been questioned. Our results show geographical but no phylogenetic structure of norstictic acid production and few significant associations between genetic clusters and the occurrence of norstictic acid. All frequently distributed haplotypes display differences in norstictic acid content. The few associations at the population level are most likely a by-product of spatial genetic structure, because norstictic acid was expressed only in individuals from the Mediterranean-Central Asian part of the study area. We conclude that the production of norstictic acid in the C. aculeata group is most likely triggered by the environment (climate, edaphic factors, associated symbionts). Cetraria steppae might be a different evolutionary lineage restricted to warm temperate regions but it is not uniquely characterized by the presence of norstictic acid.