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 cultureand 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.

Determination of Diversity, Distribution and Host Specificity of Korean Laccaria Using Four Approaches

The genus Laccaria (Hydnangiaceae, Agaricales) plays an important role in forest ecosystems as an ectomycorrhizal fungus, contributing to nutrient cycles through symbiosis with many types of trees. Though understanding Laccaria diversity and distribution patterns, as well as its association with host plants, is fundamental to constructing a balanced plant diversity and conducting effective forest management, previous studies have not been effective in accurately investigating, as they relied heavily on specimen collection alone. To investigate the true diversity and distribution pattern of Laccaria species and determine their host types, we used four different approaches: specimen-based analysis, open database search (ODS), NGS analysis, and species-specific PCR (SSP). As a result, 14 Laccaria species have been confirmed in Korea. Results regarding the species distribution pattern were different between specimen-based analysis and SSP. However, when both were integrated, the exact distribution pattern of each Laccaria species was determined. In addition, the SSP revealed that many Laccaria species have a wide range of host types. This study shows that using these four different approaches is useful in determining the diversity, distribution, and host of ECM fungi. Furthermore, results obtained for Laccaria will serve as a baseline to help understand the role of ECM fungi in forest management in response to climate change.

Guild Patterns of Basidiomycetes Community Associated With Quercus mongolica in Mt. Jeombong, Republic of Korea

Depending on the mode of nutrition exploitation, major fungal guilds are distinguished as ectomycorrhizal and saprotrophic fungi. It is generally known that diverse environmental factors influence fungal communities; however, it is unclear how fungal communities respond differently to environment factors depend on fungal guilds. In this study, we investigated basidiomycetes communities associated with Quercus mongolica using 454 pyrosequencing. We attempted to detect guild pattern (ectomycorrhizal or saprotrophic fungal communities) by comparing the influence of geography and source (root and surrounding soil). A total of 515 mOTUs were detected from root (321) and soil (394) of Q. mongolica at three sites of Mt. Jeombong in Inje County. We found that patterns of diversity and community structure were different depending on the guilds. In terms of alpha diversity, only ectomycorrhizal fungi showed significant differences between sources. In terms of community structure, however, geography significantly influenced the ectomycorrhizal community, while source appeared to have a greater influence on the saprotrophic community. Therefore, a guild-based view will help to elucidates novel features of the relationship between environmental factors and fungal communities.

Four New Species of Amanita in Inje County, Korea

Amanita (Agaricales, Basidiomycota) is one of the most well-known genera composed of poisonous mushrooms. This genus of almost 500 species is distributed worldwide. Approximately 240 macrofungi were collected through an ongoing survey of indigenous fungi of Mt. Jeombong in Inje County, Korea in 2014. Among these specimens, 25 were identified as members of Amanita using macroscopic features. Specimens were identified to the species level by microscopic features and molecular sequence analyses of the internal transcribed spacer and large subunit of nuclear ribosomal RNA. We molecularly identified 13 Amanita species, with seven species matching previously recorded species, four species (A. caesareoides, A. griseoturcosa, A. imazekii, and A. sepiacea) new to Korea, and two unknown species.

Re-evaluation of the Genus Antrodia (Polyporales, Basidiomycota) in Korea

The wood decay fungi Antrodia P. Karst. play important ecological roles and have significant industrial and economic impacts as both wood degraders and sources of pharmaceutical and biotechnological products. Although each Antrodia species has distinct morphological characteristics, the misidentification rate is especially high due to their simple morphological characters. A combination of morphological and internal transcribed spacer region sequence analyses revealed that 27 of 89 specimens previously identified by morphology alone were correct, whereas 35 of these specimens were misidentified as other Antrodia species. We report here that seven Antrodia species exist in Korea (A. albida, A. heteromorpha, A. malicola, A. serialis, A. sinuosa, A. sitchensis, and A. xantha) and based on these specimens, we provide taxonomic descriptions of these species, except for A. serialis, which was only confirmed by isolate.

A Checklist of the Basidiomycetous Macrofungi and a Record of Five New Species from Mt. Oseo in Korea

Basidiomycetous macrofungi play important roles in maintaining forest ecosystems via carbon cycling and the mobilization of nitrogen and phosphorus. To understand the impact of human activity on macrofungi, an ongoing project at the Korea National Arboretum is focused on surveying the macrofungi in unexploited areas. Mt. Oseo was targeted in this survey because the number of visitors to this destination has been steadily increasing, and management and conservation plans for this destination are urgently required. Through 5 field surveys of Mt. Oseo from April to October 2012, 116 specimens of basidiomycetous macrofungi were collected and classified. The specimens were identified to the species level by analyzing their morphological characteristics and their DNA sequence data. A total of 80 species belonging to 57 genera and 25 families were identified. To the best of our knowledge, this is the first study to identify five of these species-Artomyces microsporus, Hymenopellis raphanipes, Pholiota abietis, Phylloporus brunneiceps, and Sirobasidium magnum-in Korea.

Delimitation of Russula Subgenus Amoenula in Korea Using Three Molecular Markers

Distinguishing individual Russula species has been difficult due to extensive phenotypic plasticity and obscure morphological and anatomical discontinuities. Due to highly similar macroscopic features, such as the presence of a red-cap, species identification within the Russula subgenus Amoenula is particularly difficult. Three species of the subgenus Amoneula have been reported in Korea. We used a combination of morphology and three molecular markers, the internal transcribed spacer (ITS), 28S nuclear ribosomal large subunit (LSU), and RNA polymerase II gene (RPB2), for identification and study of the genetic diversity of Russula subgenus Amoenula in Korea. We identified only two species in Korea (R. mariae and R. violeipes); these two species were indistinguishable according to morphology and LSU, but were found to be reciprocally monophyletic species using ITS and RPB2. The markers, ITS, LSU, and RPB2, have been tested in the past for use as DNA barcoding markers, and findings of our study suggest that ITS and RPB2 had the best performance for the Russula subgenus Amoneula.