Seven Unrecorded Indigenous Fungi from Mudeungsan National Park in Korea
Minseo Choa, Sun Lul Kwona, Young Mok Heob, Young Min Leea, Hanbyul Leec, Changmu Kimd,Byoung Jun Ahne and Jae-Jin Kima
aDivision of Environmental Science and Ecological Engineering, College of Life Sciences and Biotechnology, Korea University,Seoul, Republic of Korea; bR&I Center, COSMAX BTI, Seongnam, Republic of Korea; cDivision of Polar Life Sciences, Korea Polar Research Institute, Incheon, Republic of Korea; dDivision of Biological and Genetic Resources Assessment, National Institute of Biological Resources, Incheon, Republic of Korea; eDepartment of Forest Products and Industry, Division of Forest Industrial Materials, National Institute of ForestScience, Seoul, Republic of Korea
ABSTRACT
Fungi act as important decomposers in the forest environment. They recycle essential nutrients, promote plant growth through mycorrhizal relationships, and act as food for small animals. Samples of 265 indigenous fungal species were collected from Mudeungsan National Park in 2020. These species were identified based on morphological, molecular, and phylogenetic analyses using the internal transcribed spacer (ITS), nuclear large subunit rRNA(LSU), and RNA polymerase II second largest subunit (rpb2) regions. Subsequently, seven species were identified as unrecorded species in Korea: Cordyceps cicadae, Dentocorticium bicolor,Hymenochaete nanospora, Physisporinus crataegi, Rigidoporus piceicola, Russula raoultii, and Scutellinia crinita. This study reveals their detailed macro- and microscopic morphological characteristics with phylogenetic trees to report them as unrecorded species in Korea.
ARTICLE HISTORY
Received 21 February 2022
Revised 13 July 2022
Accepted 31 July 2022
KEYWORDS
Indigenous fungi;unrecorded species; ITS;LSU; taxonomy
1. Introduction
Fungi play important roles in forest environments.They act as wood decomposers and food for small animals. They also promote or inhibit growth of plants through mycorrhizal formation or parasitism,respectively. Studies have estimated that 2.2–3.8 million fungal species live on Earth [1]. However, about 10% of the fungal species, between 220,000 and 380,000, were assumed as macrofungal species in worldwide [1,2]. Meanwhile, only about 1900 macrofungal species have been recorded in Korea until 2013[3], and currently, it is estimated that more than 2200 macrofungal species are identified in Korea [4]. This suggests that continuous research is required to discover new and unrecorded indigenous fungal species.
Mudeungsan National Park is located in Gwangju Metropolitan City and the province of Jeollanam-do in the western part of Korea. Mudeungsan was designated as a provincial park in 1972 and redesignated as a national park in 2012 owing to its importance in preserving biodiversity and natural resources in Korea. Flora and fauna in Mudeungsan National Park have been studied extensively to determine their diversity and composition [5–8].However, studies surveying fungal diversity have not yet been conducted.
As a project to discover indigenous fungi in Korea,246 fungal specimens were collected from Mudeungsan National Park in 2020. The surveys were conducted in the east region of Mudeungsan National Park. Among them, 200 specimens had previously been identified using DNA molecular and morphological analyses, whereas seven were identified as unrecorded in Korea: Cordyceps cicadae, Dentocorticium bicolor, Hymenochaete nanospora, Physisporinus crataegi,Rigidoporus piceicola, Russula raoultii, and Scutellinia crinita. Five species are wood-decaying fungi, except C. cicadae, which has parasitic form, and Ru. raoultii, which grows solitary on soil covered with woody debris. This study describes detailed macroand micro-morphological characteristics of the species and provides phylogenetic trees to report them as newly recorded species in Korea.
2. Materials and methods
2.1. Sampling
Mudeungsan National Park (35°03'06ʺ~35°12'59ʺ N, 126°53'41ʺ~127°05'01ʺ E) is located across Buk-gu of Gwangju Metropolitan City, and Damyang-gun and Hwasun-gun of Jeollanam-do Province, Korea.Collection of fungal species in Mudeungsan National Park commenced from May to September 2020. Afterwards, they were dried at 60℃ for 72h and stored in silica gel. The specimens were deposited in the Korea University Collection(KUC) and National Institute of BiologicalResources (NIBR).
2.2. Molecular approach
Genomic DNA of the dried specimens was extracted using AccuPrep® Genomic DNA Extraction Kit(Bioneer, Daejeon, Korea). The internal transcribed spacer (ITS) region was amplified with ITS5/ITS4 or ITS1F/ITS4 primer sets [9,10]. The nuclear large subunit rRNA (LSU) region was amplified with the primer sets, LR0R/LR5 or LR0R/LR7 [11,12]. For Rigidoporus piceicola, bRPB2-6F/bRPB2-7.1R primer set was used to amplify RNA polymerase II second largest subunit (rpb2) region [13]. The PCR products were purified using AccuPrep® PCR Purification Kit and AccuPrep® Gel Purification Kit (Bioneer) according to the manufacturer’s instructions. DNA sequencing was performed by Cosmogenetech (Seoul, Korea), and each sequencing result was edited using SeqMan Lasergene package version 7.0.0 (DNAStar Inc., Madison, WI). Reference sequences were collected from the NCBI GenBank database (www.ncbi.nlm.nih.gov/genbank/),and the edited sequences were compared with the reference sequences using BLAST. Each sequence was assembled and aligned using MEGA version 7 and MAFFT version 7.130 [14,15]. Phylogenetic analyses were performed using the maximum likelihood (ML) method, using RAxML with GTR+G model, and 1000 bootstrap replicates were used for tree inference[16]. All analyses were performed using CIPRES [17].The obtained trees were edited using FigTree version 1.4.3. [18] and Adobe Illustrator CS6 (Adobe Systems, Inc., San Jose, CA). Bootstrap support values above 70% are shown in the tree. All newly generated sequences of the seven species have been deposited in GenBank (Table 1).
2.3. Morphological observation
For morphological characteristics, microscopic features were observed using Olympus BX51 light microscope (Olympus, Tokyo, Japan), and pictures were taken using DP20 microscope camera(Olympus). The observations were performed using 5% KOH and Congo red solutions. The special color terms follow the Munsell Soil Color Book [19]. The following abbreviations are used: L=mean spore length, W=mean spore width, and Q=L/W ratio.
3. Results and discussion
Seven unrecorded species were identified using phylogenetic analyses of DNA sequences, particularly the ITS or LSU regions (Table 1). Six species were enough to be identified using ITS or LSU regions. However, Ri. piceicola needed multigene analysis (ITS, LSU, and rpb2) [20]. At the molecular level, each of the seven species was compared with its reference sequence and clearly identified with a high percentage of homology (Table 1). The identification results revealed the orders of the species: Polyporales (two spp.), Hymenochaetales (two spp.),Hypocreales (one sp.), Pezizales (one sp.), and Russulales (one sp.) (Table 1).
Seven species were clearly distinguished through ML method using ITS or LSU or rpb2 sequence datasets for phylogenetic analyses. According to the phylogenetic trees (Figures S1–S7, Supporting Information), C. cicadae formed monophyletic groups with high bootstrap values and its sister group was C. tenuipes (Figure S1). D. bicolor was clearly separated from other taxa with high bootstrap values (Figure S2). H. nanospora formed monophyletic groups with high bootstrap values and its sister group was H. cana (Figure S3). P. crataegi formed monophyletic groups with high bootstrap values and its sister group was P. vitreus (Figure S4). Ri. piceicola was not clearly separated from Ri.obducens, Ri. populinus, and Ri. subpopulinus using ITS or LSU regions, so combined tree of ITS, LSU,and rpb2 regions is provided for accurate identification[20]. As a result, Ri. piceicola formed monophyletic groups with high bootstrap values (Figure S5).Ru. raoultii was clearly separated from the other taxa with high bootstrap values (Figure S6). S. crinita was clearly separated from the other taxa, but the clade was divided within the S. crinita group(Figure S7). Therefore, further research on the genus Scutellinia is required to determine why the clade was divided.
The genus Cordyceps Fr. is characterized by fusiform to clavate fertile heads of the stroma and filiform ascospores [21]. Additionally, this genus is widely used for medicinal applications, especially in Asia. Although 183 species are accepted as Cordyceps worldwide [22], only 46 species are listed in the NIBR database in Korea. Occurrence of Cordyceps is affected by conditions, such as humidity,temperature, elevation, and life cycle of its host species [21]. The host species are mostly insects, but other Cordyceps species, fungi Elaphomyces, spiders,nematodes, and plant tissues have also been reported as the hosts [23,24]. Many synonyms have been listed because of their sexual and asexual morphs [21]. Therefore, additional studies are required to correct sexual and asexual names and to reclassify hidden Cordyceps species.
The genus Dentocorticium (Parmasto) M.J. Larsen & Gilb was separated from the extinct genus Laeticorticium [25]. Currently, six species are listed in the genus, and five previously listed species have been reclassified as other genera [26–29].Dendrodontia and Fuscocerrena have been identified as synonyms of Dentocorticium based on phylogenetic analysis [30]. These two genera have macroscopic features similar to Dentocorticium [30];therefore, accurate observations of microscopic characteristics and molecular analyses are required to identify them correctly. Dentocorticium is characterized by effused basidiocarps, brown skeletal hyphae,and irregularly branched dendrohyphidia [30].
The genus Hymenochaete Lev is characterized by brown effused basidiocarps, hymenial setae, and small basidiospores [31,32]. More than 120 species have been reported globally [33], but only 12 species were reported in Korea. Phylogenetic analysis revealed that in addition to Hymenochaete, H. tabacina (Sowerby) Lev is closely related to the poroid genera [33,34]. Consequently, the new genus Pseudochaete was separated [33,34]. Therefore,molecular approaches are required because of the similar morphological characteristics of these two genera [33].
The genus Physisporinus P. Karst. has eight species worldwide and is new to Korea. Physisporinus species are characterized by soft basidiocarps, and lack of cystidia [20,35–37]. Phylogenetic analysis showed that Physisporinus and several species of Rigidoporus Murrill were closely related in the Polyporales clade [34,38–40]. However, it is difficult to distinguish between the two genera because of their similar morphological characteristics [20]. Therefore, accurate phylogenetic analysis is required to correctly identify the species in these genera [20].
The genus Rigidoporus Murrill has morphological characteristics similar to genera Leucophellinus Bondartsev & Singer, Oxyporus (Bourdot & Galzin) Donk, and Physisporinus P. Karst [35,37,41].Rigidoporus is distinguished by ochraceous and corky basidiocarps, and hyphoid cystidia [20]. According to phylogenetic analyses, the four genera are closely related [34]. Several species of Oxyporus and Rigidoporus were classified in the order Hymenochaetales, but species of Physisporinus and some species of Rigidoporus were classified in the order Polyporales [34,38,39]. Therefore, additional research is required for accurate classification.
The genus Russula Pers. is the second largest genus in the class Agaricomycetes, with has 1331 recorded species; it is estimated that almost 3000 species exist globally [42,43]. However, only 82 species of this genus were reported in Korea. In some studies, Russula was studied using the LSU region, and it was proved that the LSU region is an effective tool for identifying this species [44,45]. Russula is characterized by a large, bright-colored pileus and spores with echinulate surfaces [46,47].The species in this genus are difficult to distinguish through morphological characteristics because of their large population [48,49]. Since an accurate description of taxonomy is difficult, molecular and phylogenetic analyses are necessary [47].
The genus Scutellinia (Cooke) Lambotte is a cosmopolitan fungi which have been reported worldwide, usually in the Northern Hemisphere [50]. This genus is characterized by orange to red apothecia and blackish brown hairs around the margin of apothecia [51]. Although over 100 species are listed in the genus, only four species were reported in Korea [22]. Among the species, 10 species that occur in soil have globose to subglobose ascospores, and the others that occur on decayed wood have ellipsoid ascospores [50–54]. Phylogenetic analysis showed that the genus Scutellinia is well divided in the family Pyronemataceae [51].
This study identified several unrecorded species in Mudeungsan National Park. These records are important resources for determining the diversity and composition of fungal species in Korea. The survey started in 2020; therefore, it is expected that many new and unrecorded species are yet to be discovered. Thus, additional surveys are required at different locations, altitudes, and seasons.