Three new records of mushroom species from Hangyeong-Andeok Gotjawal, South Korea

Taxon sampling

The specimens used in this study were collected from Hawsun Gotjawal, Seogwipo, Jeju, South Korea, in 2020. A D700 camera (Nikon, Tokyo, Japan) was used to capture images of fruit bodies. The size of the fruit bodies was measured indoors after recording the location information and field substrate. The specimens were deposited in the herbarium of the Biodiversity Research Institute, Jeju Technopark, South Korea, where a constant temperature (25℃) and humidity (50%) were maintained after drying at 60℃ for 24 hr.

Macroscopic and microscopic characteristics

Macroscopic characteristics, such as the size, shape, and color of the pileus, lamellae, and stipe, were observed and described based on captured images and dried specimens. The color was analyzed using the Korea Standard Color Analysis (KSCA) and was indicated with the KS symbol [10]. Microscopic features, including the size, shape, and thickness of basidia, basidiospores, cheilocystidia, pleurocystidia, as well as the number of sterigmata of basidia, were observed and described using an Eclipse 80i microscope (Nikon, Tokyo, Japan). The samples were stained with 1% Congo red and 1% phloxine and then washed with 3% potassium hydroxide.

DNA extraction and sequencing

Genomic DNA was extracted from dried specimens weighing approximately 100 mg using a 5-min Mushroom DNA Extraction Kit (Catalogue number D-1016, Biofactories, Los Angeles, CA, USA), following the manufacturer’s protocol. The universal primer sets ITS1 and ITS4 [11] were used to amplify the internal transcribed spacer (ITS) region. Subsequently, polymerase chain reaction and sequencing of the amplified products were performed by Bionics Inc. (Seoul, South Korea). The resulting sequences were compared with the ITS sequences in GenBank using the Basic Local Alignment Search Tool provided by the National Center for Biotechnology Information.

Molecular phylogenetic analyses

The ITS sequences from GenBank were used to analyze phylogenetic relationships, referencing Kiyashko et al. [12] and Chew et al. [13]. Sequences were aligned using the MEGA X software [14]. A maximum likelihood (ML) tree was constructed using the general time-reversible (GTR) model combined with the gamma distribution (G) and invariant sites (I) model. The GTR+G+I model was selected as the best evolutionary model for ML by jmodeltest2 [15]. Bootstrap testing was performed with 1,000 replicates. The accession numbers of the GenBank sequences, along with their respective scientific names, are provided in Figs 1, 2, and 3.

Fig. 1

Maximum likelihood (ML) tree of internal transcribed spacer (ITS) sequences of Gymnopilus. The bootstrap values above 50% are shown next to the branches (1,000 replicates). JBRI-M20-057 is the accession number of our specimen.

Fig. 2

Maximum likelihood (ML) tree of internal transcribed spacer (ITS) sequences of Marasmius. The bootstrap values above 50% are shown next to the branches (1,000 replicates). JBRI-M20-022 is the accession number of our specimen.

Fig. 3

Maximum likelihood (ML) tree of internal transcribed spacer (ITS) sequences of Mycena. The bootstrap values above 50% are shown next to the branches (1,000 replicates). The type specimen is indicated as “T.” JBRI-M20-136 is the accession number of our specimen. 1

Taxonomy

Gymnopilus dilepis (Berk. & Broome) Singer, Lilloa 22:560, 1949 [16] (Fig. 4)

Pileus (Fig. 4a) 12.10–24.90 mm in diameter, convex shape, surface squamulose, orange (KS 1.25YR 7/10) to dark red (KS 8.75R 4/10) toward the center; Lamellae (Fig. 4b) lamellulae in between, pale orange (KS 5YR 8/4); Stipe (Fig. 4) 33.68–38.71 mm, central, cylindrical, pinstripe, pale orange (KS 5YR 8/4) to pale red (KS 10R 7/6); Basidia (Fig. 5a–5j) 6.81–9.07 × 19.85–33.99 µm, clavate with thin walls, hyaline, four sterigmata with spores at the tip; Basidiospores (Fig. 5k and 5l) 5.36–6.94 × 3.78–4.70 µm, elliptical with thick walls, verrucose; Cheilocystidia (Fig. 6a, 6c–6h) 6.88–12.27 × 19.39–28.83 µm, clavate with a thin handle, thin-walled; Pleurocystidia (Fig. 6b, 6i–6n) 4.99–9.98 × 20.08–29.29 µm, clavate with a thin handle, thin-walled.

Habitat: Occurred on dead wood. Hwasun Gotjawal, the collection site of D. dilepis was covered by both evergreen and deciduous broad-leaved trees.

Specimen examined: South Korea, Jeju, Seogwipo, Hwasun Gotjawal (33°15'55.8792" N, 126° 19'51.9564" E), June 19, 2020, S. H. Lee (JBRI-M20-057; GenBank accession number OR177789).

Remarks: Contrary to the pleurocystidia observed in this study, they were described as absent by Suwannarach et al. [17]. Additionally, Thomas et al. [18] reported that pleurocystidia are absent or extremely rare.

Marasmius tenuissimus (Sacc.) Singer, Fl. Neotrop. Monogr. 17: 258, 1976 [19] (Fig. 7)

Pileus (Fig. 7a) up to 7.56 × 4.57 mm, convex to plano-convex, sulcate, dull, dry, glabrous, pale yellow (KS 10Y 8/4) to brown (KS 1.25Y 6/8); Lamellae (Fig. 7b) adnate, distant, intervenose, white yellowish green (KS 5GY 9/2); Stipe (Fig. 7b) 0.69 × 1.71 mm, lateral, cylindrical, velutinous, dark brown (KS

Fig. 4

Fruit body images of the G. dilepis specimen in the field, Hwasun Gotjawal. a, b: JBRI-M20-057 (Scale bars = 10 mm).

Fig. 5

Microscopic images of basidia and basidiospores of G. dilepis. a–j: Basidia with sterigmata. k, l: Basidiospores (Scale bar = 10 µm).

Fig. 6

Microscopic images of cheilocystidia and pleurocystidia of G. dilepis. a, c–h: Cheilocystidia. b, i–n: Pleurocystidia (Scale bar: a, b = 50 µm; c–n = 10 µm).

5Y 2/2); Basidia (Fig. 8c–8g) 7.74–9.48 × 32.40–43.07 µm, clavate with thin walls, 4 sterigmata with spores at the tip; Basidiospores (Fig. 8b) 11.42–13.28 × 6.06–6.77 µm, elliptical with thin walls, smooth; Cheilocystidia (Fig. 9) 7.18–21.72 × 19.17–44.06 µm, Siccus-type broom cells, cylindrical to clavate, hyaline, thin-walled; Pleurocystidia absent.

Habitat: Scattered or gregarious on branches. Jeju Gotjawal Provincial Park, the collection site for Ma. tenuissimus is covered by both evergreen and deciduous broad-leaved trees.

Specimen examined: South Korea, Jeju, Seogwipo, Jeju Gotjawal Provincial Park (33°17'3.3108" N, 126°16'19.7616" E), May 19, 2020, S. H. Lee (JBRI-M20-022; GenBank accession number OR177790).

Remarks: The specimen in this study was not sufficiently mature; therefore, the size of the pileus was smaller than that reported in other studies [19-21], and it was difficult to observe basidia with sterigmata.

Mycena seminau A.L.C. Chew & Desjardin Mycologia 106(5): 985, 2014 [13] (Figure 10)

Pileus (Fig. 10a and 10c) 11.13–25.42 mm in diameter, convex to flat shape, pressed apex, smooth and crumpled surface without gloss, light brown (KS 5YR 8/6) to dark brown (KS 8.75YR 2/2), sometimes darker toward the edge. Lamellae (Fig. 10b) lamellulae in between, grayish brown (KS 5YR 5/2). Stipe (Fig. 10c) 36.81–62.74 mm, central, cylindrical, smooth and pruinose, pinstriped, basal tomentum, yellowish-brown (KS 8.75YR 7/4) to dark brown (KS 1.25YR 3/6), and darker toward the top. Basidia (Fig. 11d–11m) 6.59–8.75 × 29.63–42.97 µm, clavate with thin walls, 2 or 4 sterigmata with spores at the tip. Basidiospores (Fig. 11c) 3.92–5.52 × 7.36–10.61 µm, elliptical with thin walls, smooth, small apiculus. Cheilocystidia (Fig. 12a and 12d–12h) 5.17–12.61 × 19.76–51.02 µm, multiform, elliptical to clavate or lageniform, sometimes mucronate, thin-walled. Pleurocystidia (Fig. 12b and 12c) 9.05–9.47 × 19.11–23.62 µm, very rare or absent, only elliptical and lageniform observed, thin-walled.

Habitat: Scattered or gregarious on humus. Hwasun Gotjawal, the collection site for My. seminau was covered by both evergreen and deciduous broad-leaved trees.

Specimen examined: South Korea, Jeju, Seogwipo, Hwasun Gotjawal (33°15'59.3568" N, 126° 19'56.0748" E), September 22, 2020, S. H. Lee (JBRI-M20-136; GenBank accession number OM109694).

Remarks: “Seminau” means shine in Malay. It refers to the bioluminescence of this species [13].

Fig. 7

Fruit body images of the Ma. tenuissimus specimen in the field, Jeju Gotjawal Provincial Park. a, b: JBRI-M20-022 (Scale bars = 10 mm).

Fig. 8

Microscopic images of basidia and basidiospores of Ma. tenuissimus. a: Clustered basidia. b: Basidiospore. c–g: Basidia with sterigmata (Scale bar = 10 µm).

Fig. 9

Microscopic images of cheilocystidia of Ma. tenuissimus. Pleurocystidia are absent (Scale bar = 10 µm).

Fig. 10

Fruit body images of the My. seminau specimen in the field, Hwasun Gotjawal. a–c: JBRI-M20-136 (Scale bars = 10 mm).10 µm.

Fig. 11

Microscopic images of basidia and basidiospores of My. seminau. a, b: Clustered basidia. c: Basidiospores. d–m: Basidia with sterigmata (Scale bar = 10 µm).

Fig. 12

Microscopic images of cheilocystidia and pleurocystidia of My. seminau. a, d–h: Cheilocystidia. b, c: Pleurocystidia (Scale bar: a = 100 µm; b = 50 µm; c–h = 10 µm).