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

Abstract

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.

Complementary molecular methods reveal comprehensive phylogenetic diversity integrating inconspicuous lineages of early-diverged wood-decaying mushrooms

“…In addition to the four clades (clade E, H, I, and K) reported by the previous study 4 , eDNA metabarcoding discovered four new clades (clade N, O, P, and Q), and the OTUs close to D. lacrymalis and Dacrymyces variisporus that were not detected by the previous DNA cloning. Compared to the previous study, the improved detections of this study would be attributed to the longer-term and larger-area survey, reduction of primer mismatches by using the degenerate primers, and/or acquisition of a much larger number of sequence reads by eDNA metabarcoding with HTS.…”

“…Clades I and Q, Unilacryma spp., and Dacryonaema spp., which branched deep in the phylogenetic tree, are considered to be important lineages for studying the early evolution of Dacrymycetes 29,33 . Lineages that diverged early in the phylogeny of Dacrymycetes tend to feature small or thin fruiting bodies 4,33 . The fruiting bodies of Unilacryma spp.…”

“…As the study period was short (one year) and the sample size was small, the number of OTUs detected was not saturated. Moreover, some lineages could not be detected probably owing to primer mismatches in the previous study 4 , and the sequence reads generated through DNA cloning were limited. Therefore, it has remained unclear whether our previous findings are caused by differences in methods or insufficient sampling.…”

“…eDNA-cloning and culture isolation methods detected OTUs belonging to unknown clades in the phylogeny that were not identified among the sequences obtained via the fruiting-body survey. However, certain OTUs were detected only as fruiting bodies 4 . As the study period was short (one year) and the sample size was small, the number of OTUs detected was not saturated.…”

“…The various methods designed for the detection of fruiting bodies, isolated mycelia, and eDNA provide complementary biological information regarding fungal taxonomic and phylogenetic diversity and ecological features. The diversity of mushroom-forming fungi has previously been investigated using methods based on the fruiting-body, culture isolation, and/or eDNA 4,[21][22][23][24][25][26][27] . These studies have revealed the unique characteristics of each method.…”