Introduction
Macrofungi usually called mushrooms are known to be one of the most important constituents of the forest eco- system with forest trees. Most of them are in temperate regions and constitute a significant part of terrestrial eco- systems. Their edibility, poisonous nature, and medicinal value, have made them economically, biotechnologically, and medically important [1]. The study of fungal biodiv- ersity has been carried out the world over and so far only 6.7% of 1.5 million species of fungi estimated in the world have been described [2], while 4,686 species of fungi are recorded in Korea by 2015 (National Institute of Biological Resources, https://species.nibr.go.kr). To secure, preserve, and manage the genetic biological resources in Korea, a research project entitled survey and discovery of Korean indigenous fungal species has been performed by aid of National Institute of Biological Resources (NIBR) under the Ministry of Environment, Republic of Korea since 2006.
Materials and Methods
In this study, the distribution of macrofungi at Chung- nam, Chungbuk and Jeonnam Provinces, Korea was inv- estigated by analyzing fungal specimens collected during from August 2014 to October 2015. Each specimen was photographed, and details regarding the collection site, habitat, host, substrates, and fruiting bodies of each speci- men were recorded prior to collection. Specimens were then brought to the laboratory and dried over mild heat for several days. Dried specimens were deposited in the NIBR.
Taxonomic classification of species and the associated nomenclature were assigned using the Index Fungorum database (http://www.index-fungorum.org). Measurements and drawings were made from slide preparations mounted in 3% KOH [3] using a BAM-102i light microscope (MRC Lab, Holon, Israel). Twenty randomly selected mature basidiospores and basidia from each specimen were mea- sured. For molecular identification, total DNA was ext- racted from dried specimens using an AccuPrep Genomic DNA Extraction Kit (Bioneer, Daejeon, Korea). The int- ernal transcribed spacer (ITS) and partial nLSU rDNA regions were amplified using primers ITS5 [4] and LR3 [5], as described by Lee and Jung [6]. DNA sequencing was performed at the DNA Synthesis and Sequencing Facility, Macrogen (Seoul, Korea), using the aforemen- tioned primers and an ABI 3730XL DNA Analyzer. The resulting nucleotide sequences were edited using jPHYDIT [7] and deposited in GenBank (accession nos. KX963782 ~KX963793). Specimens were initially identified on the basis of their macro- and microscopic features according to published descriptions (Fig. 1) [8-16].
Fig. 1
Basidiocarps and microscopic features of Otidea bufonia (A), Daldinia childiae (B), Agaricus guizhouensis (C), Lycoperdon scabrum (D), Amanita orientifulva (E), Crepidotus inhonestus (F), Marasmius brunneoaurantiacus (G), Armillaria cepistipes (H), Pluteus hongoi (I), Pluteus variabilicolor (J), Russula grisea (K), and Elmerina cladophora (L). a, asci; b, ascospores; c, paraphyses; d, basidia; e, basidiospores; f, pileipellis; g, generative hyphae; h, skeletal hyphae; i, cheilocystidia; j, pleurocystidia; k, caulocysti- dia (scale bar = 10 μm).
Results and Discussion
Species identities were confirmed by comparison with GenBank reference sequences using BLASTn (Table 1) [17]. A Neighbor-joining (NJ) phylogenetic analysis was implemented in PAUP 4.0b10 [18] with a Jukes-Cantor correction. The robustness of inferred NJ topologies was tested with 1,000 bootstrap replicates (Fig. 2).
Table 1. Closest GenBank matches of 12 undescribed species in this study
|
| ITS, internal transcribed spacer. |
Using a combination of the morphological and phylo- genetic analyses described above, all fungal taxa were enu- merated and classified according to current taxonomies. Among these taxa, twelve species including Agaricus gui- zhouensis, Amanita orientifulva, Armillaria cepistipes, Cre- pidotus inhonestus, Daldinia childiae, Elmerina cladophora, Lycoperdon scabrum, Marasmius brunneoaurantiacus, Oti- dea bufonia, Pluteus hongoi, Pluteus variabilicolor, and Ru- ssula grisea have not been previously reported in Korea.
Taxonomy
Ascomycota Whittaker
Pezizomycetes O.E. Erikss. &Winka
Pezizales J. Schröt.
Pyronemataceae Corda
1. Otidea bufonia (Pers.) Boud., Histoire et Classification des Discomycètes d'Europe: 52 (1907) [19]
The cups are 3~4 cm tall and split on one side, with an irregularly wavy, dark brown inner surface and outer sur- face that is slightly whitish to ochre and smooth to fur- furaceous. The flesh is thin and yellowish to light brown.
Asci are 85~100 × 9~11 μm and eight-spored. Ascos- pores are 11~13 × 7~8 μm, elliptic-fusiform, smooth, and contain two oil drops. Paraphyses are slender and strongly curved at the tip.
Specimen examined: The specimens was collected on Mt. Taehak, Cheonan-si, Chungnam Province, Korea, from the ground of a broad-leaved forest, 4th September 2015, JS150904-08 (GenBank accession no. KX963782).
Sordariomycetes O.E. Erikss. & Winka
Xylariales Nannf.
Xylariaceae Tul . & C. Tul .
2. Daldinia childiae J.D. Rogers & Y.M. Ju, Mycotaxon 72: 512 (1999) [20].
Stromata are irregular and spherical, depressed-spherical to turbinate, 0.9 × 1.1 cm in size, sessile, solitary, and smooth, with either inconspicuous or conspicuous peri- thecial mounds. The surface is brown or vinaceous, and later become black. If the stroma is cut vertically, it shows conspicuous concentric blackish and grayish growth zones.
Asci are at least 70 × 7~8 μm. Ascospores are 6~7 × 5~6 μm, dark brown to black, unicellular, and ellipsoid- inequilateral, with narrowly rounded ends and a straight germ slit that is spore-length on the convex side.
Specimen examined: The specimen was collected on Mt. Weolbong, Asan-si, Chungnam Province, Korea, from the branch of a dead deciduous tree, 18th July 2015, JS 150718-02 (GenBank accession no. KX963783).
Basidiomycota R.T. Moore
Agaricomycetes Doweld
Agaricales Underw.
Agaricaceae Chevall
3. Agaricus guizhouensis Y. Gui, Zuo Y. Liu, K.D. Hyde,Fungal Biology 119: 83 (2015) [21]
The pileus is 5.2~12 cm in diameter, 0.5~0.8 cm thick, convex to hemispherical, and applanate. The surface is smooth and white or covered with pastel yellow to green- ish-grey appressed patches that are irregularly shaped. Lamellae are up to 0.4~0.6 cm wide, free, crowded, and brown to brownish-black. The stipe is 9~15 × 1~1.6 cm, hollow in the center, and cylindrical, with many rhizom- orphs. The surface is white and smooth or fibrillose. The context is white, turning yellowish-white or yellow, then brownish-red or pastel yellow, finally yellowish-green.
Basidia are 30~35 × 7~10 μm, clavate. Basidiospores 8~10 × 4~5 μm and elongate-ellipsoid, with a prominent oblique apiculus, attenuated at the apex, smooth, thick- walled, and brown. Pileipellis are 60~70 × 4~5 μm, cylin-drical and hyaline.
Specimen examined: The specimen was collected on Mt. Songni, Boeun-gun, Chungbuk Province, Korea, from leaf litter in a mixed forest, 3rd September 2014, JS140903- 30-1 (GenBank accession no. KX963784).
4. Lycoperdon scabrum Wi l ld. , Florae Berolinensis Pro-dromus: 409 (1787) [22]
The fruiting body is pyriform, with a prominent pale cream base that is 26 × 3~6 cm and glabrous, with dark sepia spines on the upper surface that are 1~3 mm long and that can be rubbed off. The stipe is white to pale cream, 15 × 20 mm, and more or less cylindrical, with rooting rhizomorphs at the base. The endoperidium is umber, smooth, and somewhat shiny. The gleba is a cottony or fibrous mass containing spores that olivaceous brown when mature.
Basidospores are 4~6 × 4~5 μm and globose to subglo- bose, with a long pedicel.
Specimen examined: The specimen was collected on Mt. Bueong, Asan-si, Chungnam Province, Korea, from soil in a mixed forest, 15th July 2015, JS150715-20 (Gen- Bank accession no. KX963785).
Amanitaceae R. Heim ex Pouzar
5. Amanita orientifulva Zhu L. Yang, M. Weiss & Oberw., Mycologia 96(3): 643 (2004) [23]
The pileus is 4~9 cm in diameter, at first hemispherical, hen convex to plano-convex and yellowish-brown to och- eous. The margin is tuberculate-striate, dirty white to ream-colored. Lamellae free, crowded, white to cream- olored. The stipe is cylindrical, 7~8 cm in length, taper- ng upward, and is dirty white to brownish, with squam- ules. The context is white and hollow in the center. The nnulus is absent.
Generative hyphae are 6~7 μm in width, thin-walled, and hyaline. Basidia are 40~45 × 13~15 μm, clavate, with a 4-spored sterigmata and basal septa without clamps. Basidiospores are 10~12 × 9~11 μm, globose to subglob- ose, colorless, hyaline, thin-walled, and smooth, with a small apiculus.
Specimen examined: The specimen was collected on Mt. Taehak, Cheonan-si, Chungnam Province, Korea, from soil in a mixed forest, 4th September 2015, JS140806-20 GenBank accession no. KX963786).
Inocybaceae J. Schröt.
6. Crepidotus inhonestus P. Ka r s t . , Me d d n S o c . Fau n a Flora fenn. 13: 160 (1886) [24]
The pileus consists of multiple cap-like structures aris- ing from one or that are gregarious, and is 1~2 μm and laterally or centrally attached to the substrate without a stipe. The surface is smooth, dull, and white to cream- yellow. The margin is acute and even. The fruit body is fleshy, becoming partially hollow at the core, and pale or brownish. The lower surface is smooth and shallowly wrinkled or has broad and poorly developed false gills.
The basidia are cylindrical and 20~25 × 7~8 μm, with 4-spored sterigmata. Cystidia are not seen. Basidiospores are 6~7 × 4~5 μm, smooth, and more or less elliptical.
Specimen examined: The specimen was collected on Mt. Weolbong, Asan-si, Chungnam Province, Korea, from the branch of a dead deciduous tree, 15th July 2015, JS 150715-11 (GenBank accession no. KX963787).
Marasmiaceae Roze ex Kühner
7. Marasmius brunneoaurantiacus Antonín & Buyck,Fungal Diversity 23: 24 (2006) [25].
The pileus is 6~7 mm in diameter, at first hemispheri- cal-campanulate with an incurved margin, then broadly convex. The surface is smooth to slightly wrinkled, dull, and finely velutinous. The stipe is 1~10 × 0.1~0.2 cm, cylindrical, almost equal, smooth, and shiny, with an apex that is whitish, and red brown below. The lamellae are adnexed, broad, subdistant, and pale, nearly concolorous with the pileus.
The basidia are 25~30 × 7~9 μm and clavate. Basidios- pores are 7~8 × 4~5 μm, ellipsoid to oblong-ellipsoid, and thin-walled, without a germ pore. The cheilocystidia are 20~25 × 9~11 μm, and pleurocystidia are 50~95 × 5~13.5 μm and fusoid-ventricose.
Specimen examined: The specimen was collected on Mt. Weolbong, Asan-si, Chungnam Province, Korea, from leaf litter in a mixed conifer-hardwood forest, 4th Sep- tember 2015, JS150904-16-1 (GenBank accession no. KX 963788).
Physalacriaceae Bresinsky
8. Armillaria cepistipes Velen. , Ceske Houby 2: 283 (1920) [26].
The pileus is 1~3 cm in diameter, broadly convex to plano-convex, with a margin that is decurved and even to uplifted and wavy, with a reddish brown context that is very thin and concolorous with the surface. The lamel- lae are ascending, adnate to subdecurrent, and close to crowded. The stipe is 20~30 × 4~10 mm, central, and uniform below or sometimes gradually narrowing.
Basidia are 18~22 × 6~7 μm and rather narrowly clavae and clamped. Cheilocystidia are 15~25 × 4~6 μm and cla- vate. Caulocystidia are 40~60 × 5~7 μm, irregularly cylin- drical to sinuous. Basidiospores are 4~6 × 3~4 μm, broadly ellipsoid to subglobose, smooth, and hyaline. Clamp con- nections are abundant in all tissues.
Specimen examined: The specimen was collected on Mt. Songni, Boeun-gun, Chungbuk Province, Korea, from buried wood remnants of a coniferous tree, 21st Septem- ber 2014, JS140921-54 (GenBank accession no. KX963789).
Pluteaceae Kotl. & Pouzar
9. Pluteus hongoi Singer, Fieldiana Botany 21: 95 (1989)[27].
The pileus is 9 cm in diameter, hemispherical or cam- panulate when young, and expands to convex or plano- convex. The surface is smooth to innately radially fibril- lose. The margin is smooth or slightly translucently striate. Lamellae are crowded, free, and ventricose. The stipe is 110 × 7 mm and cylindrical, with a slightly broad base. The surface is white, sometimes with a yellowish tint near the base, and is usually smooth.
Basidia are 30~35 × 6~8 μm and clavate. Basidiospores are 5.5~6.5 × 5~6 μm and ellipsoid or broadly ellipsoid. Pleurocystidia are metuloid, 18~20 × 8~12 μm, and fusi- form. The pileipellis is 40~45 × 7~10 μm. It is solitary.
Specimen examined: The specimen was collected on Mt. Weolbong, Asan-si, Chungnam Province, Korea, from the humus layer of soil in a mixed forest, 14th July 2015, JS150714-14 (GenBank accession no. KX963790).
10. Pluteus variabilicolor Babos, Annales Historico- Natureles Musei Nationalis Hungarici 70: 93 (1978) [28]. The pileus is 5~7 cm wide, smooth, and yellowish- orange, with a darker central umbo, clearly striate or not striate at the margin, with well-defined squamules. Lam- ellae are free, quite crowded, ventricose, and yellowish- orange. The stipe is 40~70 × 412 mm, cylindrical, slightly enlarged at the base, streaked-fibrillose over the entire length, and yellow, with reddish tinges at the base of mat- ure specimens. The context is white-yellowish or yellowish- orange under the pileus surface.
Basidia are 20~30 × 5~7 μm and clavate. Pleurocystidia are lageniform and 60~70 × 30~40 μm. The pileipellis is a hymeniderm consisting of clavate, rounded terminal ele- ments and cylindrical, elongated cells that are 70~90 × 7~ 10 μm. Caulocystidia are present over the whole length of the stipe, 45~50 × 8~10 μm, cylindrical to claviform, and fusiform. Basidiospores are 5.5~7.0 × 4.5~6.0 μm, broadly ellipsoid to subglobose, and thin-walled.
Specimen examined: The specimen was collected on Mt. Wolchul, Yeonggwang-eup, Jungnam Province, Korea, from a deciduous tree, 27th August 2015, JS150827-19 (GenBank accession no. KX963791).
Russulales Kreisel ex P.M. Kirk, P.F. Cannon & J.C. David
Russulaceae Lotsy
11. Russula grisea Fr., Epicrisis Systematis Mycologici: 361 (1838) [29].
The pileus is 4~11 cm wide and globose. The margin is fragile and whole to eroded, with warted striations. The surface is smooth and yellow-brown to light brown. Lam- ellae are adnate, closed, brittle, and white, frequently dev- eloping brown stains. The stipe is 3~5 cm tall, 1~2 cm thick, brittle, evenly white with brownish stains, especially at the base, and solid when young, but becoming nearly hollow at maturity. The veil is absent.
Basidia are 40~45 × 6~7 μm, narrowly clavate. Basidio- spores are 7~8 × 5~6 μm, subglobose to elliptical, and warted. Pleurocystidia are 65~70 × 8~10 μm, cylindrical to fusoid,
Specimen examined: The specimen was collected on Mt. Mangyeong, Asan-si, Chungnam Province, Korea, rom soil in a mixed conifer-hardwood forest, 6th August 2014, JS140806-24 (GenBank accession no. KX963792).
Tremellomycetes Doweld Tremel lales Fr.
Aporpiaceae Bondartsev & Bondartseva
12. Elmerina cladophora (Berk.) Bres., Annales Mycolo- gici 10(5): 507 (1912) [30]
The basidiocarps are 2 × 3 cm, annual, and resupinate. Hymenophores are poroid, corky, cream, pinkish-buff, and curry yellow when dry. Pores are 4~5 cm and circular, angular, or hexagonal. Dissepiments are whole and can be thin or thick.
The hyphal system is dimitic. Generative hyphae are 2~ 3 μm in width, thin-walled, and hyaline. Skeletal hyphae are 3~5 μm, are thick-walled and hyaline. Basidia are 20 ~25 × 10~12 μm, thin-walled, hyaline, vertically septate, and mostly clavate. Basidiospores are 9~12 × 5~6 μm, thin-walled, hyaline, allantoid to ellipsoid.
Specimen examined: The specimen was collected on Mt. Songni, Boeun-gun, Chungbuk Province, Korea, from the trunk of a dead Quercus variabilis, 3rd September 2014, JS140903-22-1 (GenBank accession no. KX963793).
