Endolichenic Fungal Community Analysis by Pure Culture Isolation and Metabarcoding: A Case Study of Parmotrema tinctorum

Lichen is a symbiotic mutualism of mycobiont and photobiont that harbors diverse organisms including endolichenic fungi (ELF). Despite the taxonomic and ecological significance of ELF, no comparative investigation of an ELF community involving isolation of a pure culture and high-throughput sequencing has been conducted. Thus, we analyzed the ELF community in Parmotrema tinctorum by culture and metabarcoding. Alpha diversity of the ELF community was notably greater in metabarcoding than in culture-based analysis. Taxonomic proportions of the ELF community estimated by metabarcoding and by culture analyses showed remarkable differences: Sordariomycetes was the most dominant fungal class in culture-based analysis, while Dothideomycetes was the most abundant in metabarcoding analysis. Thirty-seven operational taxonomic units (OTUs) were commonly observed by culture- and metabarcoding-based analyses but relative abundances differed: most of common OTUs were underrepresented in metabarcoding. The ELF community differed in lichen segments and thalli in metabarcoding analysis. Dissimilarity of ELF community intra lichen thallus increased with thallus segment distance; inter-thallus ELF community dissimilarity was significantly greater than intra-thallus ELF community dissimilarity. Finally, we tested how many fungal sequence reads would be needed to ELF diversity with relationship assays between numbers of lichen segments and saturation patterns of OTU richness and sample coverage. At least 6000 sequence reads per lichen thallus were sufficient for prediction of overall ELF community diversity and 50,000 reads per thallus were enough to observe rare taxa of ELF.