Phylogenetic Identification of Korean Gymnopus spp. and the First Report of 3 Species: G. iocephalus, G. polygrammus, and G. subnudus.

Gymnopus is a cosmopolitan genus of agaric fungi and consists of ~300 species. In Korea, Gymnopus represents common saprobic mushrooms, and 12 species have been reported in Korea. Several Gymnopus specimens were collected in Korea between 2008 and 2015. To identify them exactly, phylogenetic analysis was performed by means of the internal transcribed spacer region of ribosomal-DNA sequences from the collected Gymnopus specimens. Among them, G. iocephalus, G. polygrammus, and G. subnudus have not been reported in Korea. A phylogenetic tree and images are provided.

Gymnopus (Pers.) Roussel (Omphalotaceae, Agaricales) is a large fungal genus, which consists of ~300 species and is distributed all over the world. It is characterized by a collybioid mushroom; convex to applanate or slightly concave pileus; free, emarginate or adnate and crowded to fairly distant lamellae; central stipe; white spore print; nonamyloid or nondextrinoid hyphae with clamp; ellipsoid to oblong, thin-walled, hyaline, nonamyloid basidiospores; often present cheilocystidia; and usually absent pleurocystidia [1]. The morphological characteristics of Gymnopus are similar to those of other genera: Marasmius (Marasmiaceae, Agaricales) Fr. and Collybia (Fr.) Staude (Tricholomataceae, Agaricales), except for its pileipellis: Gymnopus is distinguished from other collybioid mushrooms by the pileipellis containing a frequently encrusted pigment and a cutis or trichoderm made of projected hyphae [2]. These genera can be easily discriminated by molecular biological assays as well because they are classified into different families.

Gymnopus represents common saprobic fungi on leaf litter and dead wood, and ~12 species have been reported in Korea [34]. Nonetheless, we presume the diversity of Korean Gymnopus to be much greater than the reported species because 12 known species is a small number as compared to the high worldwide diversity. To study the diversity of indigenous Gymnopus spp., the specimens were collected in Korea. Phylogenetic analysis was performed to identify them. Among them, G. iocephalus (= Collybia iocephala), G. polygrammus (= Collybia polygramma), and G. subnudus (= Collybia subnuda) have not been reported in Korea. Thus, morphological examination of the macroand microscopic characteristics was conducted, and we report the 3 Gymnopus species as new in Korea, with detailed descriptions.

MATERIALS AND METHODS

Materials studied

A total of 14 specimens were collected (Table 1). Among them, the specimens that are named Korea University Collection (KUC) were collected and preserved by the Environmental Biotechnology Laboratory (Korea University, Seoul, Korea). Five G. subnudus specimens were obtained from Korea Mushroom Resource Bank (KMRB; Seoul, Korea). Gymnopus sp. NIBRFG0000106907 (KUC20080914-03) was obtained from the National Institute of Biological Resources (NIBR, Incheon, Korea).

Table 1

Specimens analyzed in this study

Molecular analysis

Genomic DNA samples were extracted from dry specimens with the AccuPrep Genomic DNA Extraction Kit (Bioneer, Daejeon, Korea). PCR amplification of the internal transcribed spacer (ITS) region was performed according to the previously described method [567]. To analyze each species, closely related sequences of Gymnopus spp. and reference sequences from other studies were retrieved from GenBank [148]. The determined sequences were proofread and aligned using MAFFT 7.130 [9] and modified manually in the MacClade 4.08 software [10]. Datasets were tested by MrModeltest 2.3 using the Akaike information criterion (AIC) criteria with default options [11]. The GTR + I + G model was chosen for the AIC criteria as a result of the test. Bayesian analysis was performed with MrBayes 3.2.1 [12]. A phylogenetic tree was constructed by a method described elsewhere [7].

Morphological examination

Measurements and drawings were made for the unrecorded species from slide preparations mounted in Melzer's reagent under an Olympus BX51 light microscope (Tokyo, Japan) [13]. More than 30 measurements at different positions were made to ascertain the average dimensions of each part. In case of basidiospores, 5% of the measurements were excluded from each end of the range and are given in parentheses. Munsell colors were used as color standards [14]. Voucher specimens were deposited in the Herbarium of NIBR.

RESULTS AND DISCUSSION

Phylogenetic analysis

A total of 61 taxa were used to construct the phylogenetic tree (Fig. 1). Despite the differences among the target taxa, several parts of our phylogenetic tree were found to be similar to trees from other studies [148]. Most of our specimens were clustered with the 6 known species: G. confluens (Pers.) Antonín, Halling & Noordel.; G. dryophilus (Bull.) Murrill; G. iocephalus (Berk. & M. A. Curtis) Halling; G. luxurians (Peck) Murrill; G. polygrammus (Mont.) J. L. Mata; and G. subnudus (Ellis ex Peck) Gilliam. The specimen KUC20140820A-21 was identified as G. confluens, which is the most common Gymnopus species in Korea and a well-known edible and medicinal mushroom [1516]. KUC20140627-37 ended up in a clade of G. dryophilus. It is also a common Korean Gymnopus species, known as an edible and medicinal mushroom [1516]. G. luxurians KUC20080725-28 was previously reported in Korea [8] but is paraphyletic in our phylogenetic tree. In one study on Korean G. luxurians, this strain was found to be monophyletic with other G. luxurians strains [8]. Analysis of additional DNA regions may be needed to identify such specimens exactly. G. iocephalus, G. polygrammus, and G. subnudus have not been reported in Korea. Thus, additional examination of morphological features of these specimens was carried out.

Fig. 1

The phylogenetic tree of Gymnopus spp. based on internal transcribed spacer region sequences. The dataset was created from 61 taxa and 1,032 characters. The specimens examined in this study are boldfaced. GenBank accession numbers are shown in parentheses. The numbers above branches indicate posterior probabilities. The scale bar indicates nucleotide substitutions per position.

Two specimens—KUC20080914-03 and KUC20150819-52—are in their own clade without any reference sequences. They were identified at the genus level: Gymnopus sp. They are located in a clade of G. polyphyllus with long branches and match G. barbipes KJ416266 according to a BLAST search but show low similarity (KUC20150819-52: 552/591, 93%). We presume that they are new species candidates, and further research is needed.

Taxonomy

Gymnopus iocephalus (Berk. & M. A. Curtis) Halling, Mycotaxon 63: 364 (1997) (Fig. 2).

Pileus 2~4 cm; convex with incurved margin when young, becoming planoconvex; sometimes margin uplifted when aged; yellowish brown (10YR5/6) to very pale brown (10YR8/2) when fresh, becoming very pale brown (10YR7/4) when dry, radially streaked; surface moist, but dries rapidly; margin somewhat wrinkled. Lamellae adnate to adnexed; close, Stipe 4~8 cm long, 3~4 mm wide; central, terete, somewhat equal; fibrous, light brownish grey (10YR6/2). Partial veil absent. Odor not distinctive. Cespitose on leaf litter.

Hyphal system monomitic, generative hyphae with clamps, somewhat thick-walled, 2.5~5.0 µm diam. Basidia clavate with 4 sterigmata and basal clamp; 18.5~29 × 5.5~7 µm. Cheilocystidia and pleurocystidia absent. Basidiospores smooth, inamyloid, lacrymoid to elliptical, (6.1~) 6.4~9.1 (~9.7) × (3.2~) 3.7~5.1 (~5.4) µm.

Note: The fruit body of KUC20140804-02 is somewhat larger than the previously described fruit body of G. iocephalus, but microscopic characteristics are a match [17]. Although an odor was reported in a previous description, our specimen has no distinctive odor. The odor of fresh Korean G. iocephalus is uncertain because we examined a dried specimen. To verify these data, additional specimens of Korean G. iocephalus are needed. In the phylogenetic tree (Fig. 1), G. iocephalus KUC20140804-02 is located in the monophyletic group of G. iocephalus with 100% posterior probabilities.

Specimen examined: Korea, Seoul, Bukhansan National Park, Mt. Dobong, 37°41'8'' N, 127°2'10'' E, on the leaf litter, 4 Aug 2014, Seokyoon Jang, KUC20140804-02 (NIBRFG0000138670, GenBank accession No. KX513745).

Gymnopus polygrammus (Mont.) J. L. Mata Mycotaxon 86: 313 (2003) (Fig. 3).

Pileus 1.5~2 cm; convex with incurved margin when young, becoming planoconvex; smooth, sometimes margin uplifted when aged; dark yellowish brown (10YR4/6) to very pale brown (10YR8/2) when fresh, becoming dark brown (10YR3/3) when dry, radially streaked; surface moist, but dries rapidly. Lamellae adnexed to free; subdistant, Stipe 3~4 cm long, 2~3 mm wide; central, terete, somewhat equal; fibrous, yellowish brown (10YR5/6). Partial veil absent. Odor not distinctive. Scattered to subcespitose on leaf litter.

Hyphal system monomitic, generative hyphae with clamps, thin or somewhat thick-walled, 2.5~4.5 µm diam. Basidia clavate with 4 sterigmata and basal clamp; 16.5~27.5 (~29) × 4~7 µm. Cheilocystidia and pleurocystidia absent. Basidiospores smooth, inamyloid, lacrymoid to elliptical, (5~) 5.2~7.4 × 2.6~3.5 µm.

Note: The fruit body of KUC20140804-01 is smaller than in the previous description of G. polygrammus, but microscopic characteristics are in good agreement except for the width of spores [18]. The small size of the fruit body was attributed to individual variation. Additional G. polygrammus specimens are needed to confirm this characteristic. In the phylogenetic tree (Fig. 1), G. polygrammus KUC20140804-01 is located in the monophyletic group of G. polygrammus with 100% posterior probabilities.

Specimen examined: Korea, Seoul, Bukhansan National Park, Mt. Dobong, 37°41'8'' N, 127°2'10'' E, on the leaf litter, 4 Aug 2014, Seokyoon Jang, KUC20140804-01 (NIBRFG0000138669, GenBank accession No. KX513746).

Gymnopus subnudus (Ellis ex Peck) Gilliam, Mycotaxon 4: 136 (1976) (Fig. 4).

Synonym: Collybia subnuda (Ellis ex Peck) Gilliam.

Pileus 2~4 cm; convex with incurved margin when young, becoming broadly convex; dry; very pale brown (10YR7/4) when dry; radially streaked; margin somewhat wrinkled and broadly lined; involute when dry. Lamellae adnate; distant to subdistant. Stipe 3~7 cm long, 3~5 mm wide; central, terete or compressed; somewhat equal; fibrous, very pale brown (10YR7/4) when dry. Partial veil absent. Odor not distinctive.

Hyphal system monomitic; generative hyphae with clamps, somewhat thick-walled, 2.0~5.0 µm diam. Basidia clavate with 4 sterigmata and basal clamp; 27~35 × 4.5~8 µm. Cheilocystidia variously shaped (often fusiform) with basal clamp, thin-walled, somewhat projecting, sometimes lobed, (25.5~) 27~34.5 (~37) × 4~5.5 µm. Pleurocystidia absent. Basidiospores smooth, lacrymoid to elliptical in front view, somewhat reniform in side view, (7.7~) 8.7~10.9 (~11.3) × 3.1~4.1 (~4.3) µm.

Specimen examined: Korea, Gyeonggi-do, Mt. Yongmun, 37°32'55'' N, 127°34'21'' E, on the leaf litter, 14 August 2015, Seokyoon Jang, KUC20150814-11 (NIBRFG0000141859, GenBank accession No. KX513747); Korea, Gyeonggi-do, Gwacheon-si, Seoul Grand Park. 37°25'18'' N, 127°1'29'' E, on leaf litter, 11 Sep 2015, Seokyoon Jang, KUC20150911-19 (NIBRFG0000141933, GenBank accession No. KX513748).

Note: The characteristics of our specimens are consistent with the previous description of Gymnopus subnudus except the shape of basidiospores [17]. G. subnudus is characterized by its somewhat longer basidiospores than other Korean Gymnopus spp. In the phylogenetic tree (Fig. 1), the sequences of our specimens were placed in the monophyletic group of G. subnudus with 97% of posterior probabilities. Its sistergroup species was G. peronatus, and it is corresponded with the previous study [4].