Early-diverging wood-decaying fungi detected using three complementary sampling methods.

Wood-decaying fungi are essential components of degradation systems in forest ecosystems. However, their species diversity and ecological features are largely unknown. Three methods are commonly used to investigate fungal diversity: fruiting body collection, culturing, and environmental DNA analysis. Because no single method fully characterises fungal diversity, complementary approaches using two or more methods are required. However, few studies have compared the different methods and determined the best way to characterise fungal diversity. To this end, we investigated wood-decomposing Dacrymycetes (Agaricomycotina, Basidiomycota) using a complementary approach combining fruiting body collection, culturing, and environmental DNA analysis, thereby offering an effective approach for investigating the diversity of saprotrophic mushrooms. Fruiting body collection, culturing, and environmental DNA analysis detected 11, 10, and 16 operational taxonomic units (OTUs; 25 OTUs in total) and identified three, seven, and seven novel lineages, respectively. The three methods were complementary to each other to detect greater Dacrymycetes diversity. The culturing and environmental DNA analysis identified three early-diverging lineages that were not identified in the fruiting body collection suggesting that diverse lineages lacking observable fruiting bodies remain undiscovered. Such lineages may be important to understand Dacrymycetes evolution. To detect early branches of Dacrymycetes more efficiently, we recommend a combined approach consisting of a primary environmental DNA survey to detect novel lineages and a secondary culture survey to isolate their living mycelia. This approach would be helpful for identifying otherwise-undetectable lineages, and could thus uncover missing links that are important for understanding the evolution of mushroom-forming fungi.