INTRODUCTION
The Entolomataceae Kotl. & Pouzar is a large and highly variable family with a cosmopolitan distribution, including temperate and tropical climates. This family belongs to the order Agaricales that comprises 3 main agaricoid genera: Rhodocybe Maire, Clitopilus (Fr. Ex Rabenh.) P. Kumm., and Entoloma (Fr.) Kumm. sensu lato, among which Entoloma s.l. is the second largest euagaric genus containing more than 1,500 species distributed in arctic to tropical habitats [1-3]. Based on macroscopic and microscopic features, at least 16 taxa can be defined within Entoloma s.l. [4,5]. This genus was traditionally characterized based on the spores that are angular in all views owing to a network of interconnected ridges [6-8].
Since 2013, more than 100 samples of Entoloma species were collected during mycological expeditions in Korea. Among these, some species that were frequently found in the field were already recorded in Korea, but 3 distinct species were subjected to re-examination for reliable identification. Here, we describe 3 new records of Entoloma species in Korea based on morphology and sequence comparisons using 4 gene regions, comprising rpb2, ITS region. 28S, and mtSSU regions of rDNA.
MATERIALS AND METHODS
Sampling and morphological observations
More than 100 samples of Entoloma species were collected during mycological expeditions in Korea since 2013. Samples of species not previously reported from Korea were subjected to morphological examinations and phylogenetic analyses. Detailed information concerning the specimens used in this study is shown in Table 1. Dried specimens made were deposited to the Herbarium of Korea National Arboretum (HKNA).
Macro-morphological characters were based on the field notes and color photos of the fresh fruit-bodies. Samples were sectioned and rehydrated in 3% KOH in order to examine micro-morphological characters using an Olympus BX53 microscope (Olympus, Tokyo, Japan) and Jenoptik ProgRes C14 Plus Camera (Jenoptik Corporation, Jena, Germany). Microscopic characters were measured using the ProgRes Capture Pro v. 2.8.8. software (Jenoptik Corporation, Jena, Germany). In the taxonomic descriptions, Q refers to the quotient of basidiospore length divided by basidiospore width [5].
DNA extraction, PCR, and sequencing
For molecular analyses, DNA was extracted from fruit bodies of the collections using a DNeasy Plant mini Kit (Qiagen Inc., Valencia, CA, USA). ITS1/ITS4 [9], LR0R/LR5 [10,11], MS1/MS2 [9], and rpb2-i6f/rpb2-i7r [2] primers were used to amplify the internal transcribed spacer (ITS) including 5.8S rDNA, 28S rDNA, mtSSU, and rpb2 regions, respectively. The polymerase chain reaction (PCR) products were purified using ExoSAP-IT PCR Cleanup Reagents (Applied Biosystems, Foster City, CA, USA) following the manufacturers protocols. The resulting products were sequenced by Macrogen Sequencing Service (Macrogen, Seoul, Korea) with the primers used for PCR amplifications.
Phylogenetic analyses of sequence data
All sequences were edited using Chromas v. 2.6.6, BioEdit Sequence Alignment Editor v. 7.0.5.3, and MEGA v. 7.0.26 [12]. The edited sequences were aligned using an online version of MAFFT 7 (http://mafft.cbrc.jp/alignment/server/) [13]. For phylogenetic analyses, available ITS sequences of Entoloma species were retrieved from GenBank. The sequence of Tricholoma vaccinum (FJ845444) was used as outgroup. Maximum likelihood (ML) analyses were performed using RAxML HPC Black Box v. 8.2.10 [14] using the default option with general time reversible(GTR) substitution model implemented in the CIPRES cluster server (https://www.phylo.org/) at the San Diego Supercomputer Center. Phylogenetic trees obtained from RaxML analyses were viewed in MEGA v. 7 [12]. Tree robustness was evaluated with 1,000 bootstrap replications. The bootstrap support values were indicated in the tree nodes (bootstrap values ≥ 70%).
RESULTS AND DISCUSSION
Phylogenetic analyses
PCR amplifications and sequencing of 4 gene regions (rpb2, ITS, 28S, and mtSSU) were successfully completed for all collections in this study. Sequences were deposited in the GenBank nucleotide database (www.ncbi.nlm.nih.gov), and the accession numbers are listed in Table 1. A GenBank basic local alignment search tool(BLAST) search of sequences from the 4 Korean collections showed > 99% similarity with existing GenBank sequences: KA16-0020 and KA13-0010 with E. aprile (AB520845 and AB520846), KA15-0373 with E. chytrophilum (KC898430), and KA13-1522 with E. hirtipes (JX454867). In the phylogenetic tree based on the ITS region, the sequences obtained in this study clustered within the Entoloma clade and formed a single lineage with E. aprile, E. chytrophilum, and E. hirtipes with strong support (BS values: 99/100/100, respectively; Fig. 1). Although the ITS region including the 5.8S rDNA region was sufficient to differentiate Entoloma species at the species level, the sequences of rpb2, 28S, and mtSSU regions were additionally used to confirm the identity of the species and to resolve their taxonomic positions. The sequences obtained from the 4 gene regions (ITS, 28S, mtSSU and rpb2) are provided (Table 1).
Taxonomy
Entoloma aprile (Britzelm.) Sacc., Syll. fung. (Abellini) 5: 696, 1887 (Fig. 2)
Bas. Agaricus aprilis Britzelm. 1885
Description: Pileus 20–30 mm, campanulate to plicate, then convex, expanding with age, centrally depressed, with straight, entire margin, striate, hygrophanous, brown to dark brown. Lamellae adnate, crowded, up to 5 mm broad, moderately thick, white then pink, pinkish brown with serrulate edge. Stipe 25–40×5.5–7.0 mm, cylindrical, white to pale grayish, tapering or thickened toward the base. Basidiospores 8–11×7–9.5 μm, Q = 1.1–1.3, Qav = 1.1, irregularly nodulose-angled, with 5–7 blunt angles in outline. Basidia 4-spored, 30–58 × 11.5–15 μm, clamped. Hymenophoral trama regular, made up of inflated elements, 38–190 × 4.5–9.3 μm. Pileipellis a differentiated cutis of cylindrical hyphae, 10–13.8 μm wide. Stipitipellis a cutis of narrow, cylindrical hyphae 5–11.5 μm wide. Caulocystidia absent. Clamp connections abundant.
Habitat: On soil in broadleaf forest, appearing in early spring.
Collections examined: KOREA. Gyeongsangnam-do, Daebong Mountain, May 14, 2013, KA13-0010; Gangwon-do, Jeombong Mountain, May 4, 2016, KA16-0020.
Comments: Morozova et al. [15] showed that E. aprile belongs to Rhodopolioid clade in subgenus Entoloma in phylogenetic tree. The rhodopoloioid species are distinguished from other Entoloma taxa by their large basidiomata, uniform predominantly brown colors due to simultaneously encrusted-intracellular plasmatic pigment, and nitrous smells [16]. Morphological characteristics of the specimens examined are in good agreement with previous descriptions of E. aprile [16,17]. In addition, the Korean collections of E. aprile cluster in a highly supported clade with Japanese collections (Fig 1; BS values: 99%). Therefore, these Korean collections were identified asE . aprile.
Entoloma chytrophilum Wölfel, Noordel. & Dähncke, in Wölfel & Noordeloos, Öst. Z. Pilzk. 10: 190, 2001 (Fig. 3)
Description: Pileus 5–18 mm broad, plano-convex to concave with depressed center, not hygrophanous, with straight then deflexed undulating margin, radially fibrillose, dark blue, slightly discoloring to bluish violaceus. Lamellae moderately distant, adnate-emarginate with small decurrent tooth, ventricose, white, becoming pinkish, with entire concolorous edge. Stipe 17–38 × 1–2 mm, cylindrical, fibrillose-striate, slightly squamulose in the upper part, concolorous with pileus or grey-blue, with white basal tomentum. Smell strong, fungoid. Basidiospores 7.8–11 × 5.5–7.0 μm, Q = 1.3–1.7, nodulose, heterodiametrical. Basidia 1–4-spored, 35–47.5 × 9–11.5 μm, clavate, clamped. Lamellae edge fertile. Cheilocystidia absent. Pileipellis slightly inflated hyphae 8–18 μm wide with blue intracellular pigment. Clamp connections present.
Habitat: On rotten wood in mixed forests.
Collection examined: KOREA. Gangwon-do, Gangneung-si, Nochu Mountain, August 18, 2015, KA15-0373.
Comments: Entoloma coelestinum has been recorded in Korea and the specimen we collected is similar to E. coelestinum morphologically. However, basidiospores of E. coelestinum are smaller than E. chytrophilum (6.9–8.3 × 5.2–6.2 μm vs. 7.8–11 × 5.5–7.0 μm) [15]. In the molecular phylogenetic analysis, the collection used in this study formed a distinct single lineage in the E. chytrophilum clade and it formed a sister clade to E. coelestinum (Fig. 3). Therefore, morphological and molecular data support its identity as E. chytrophilum. Morozova et al. [15] described E. lepidissimum var. pauciangulatum is now treated as a synonym of E. chytrophilum. In RAxML tree of the present study, we also confirmed that E. lepidissimum var. pauciangulatum clusters together with E. chytrophilum. In this study, it was not possible to examine our collection to take a photo under the microscope. Therefore, morphological description and molecular examination were provided here.
Entoloma hirtipes (Schumach.) M.M. Moser, in Gams, Kl. Krypt.-Fl., Bd II b/2, ed. 4 (Stuttgart) 2b/2:206, 1978 (Fig. 4)
Bas. Agaricus hirtipes Schumach. 1803
Description: Pileus 20–50 mm, campanulate to plicate, expanding with central umbo, entire margin, hygrophanous, striate, long convoluted, greyish to brown to blackish to brown, glabrous. Lamellae adnate 3–5 mm broad, ventricose, cream-greyish finally pinkish to brownish, with finely wavy edge. Stipe 40–80 × 2–5 mm, cylindrical, brown to greyish, easily broken. Basidiospores 11–14 × 8–9.5 μm, Q = 1.2–1.5, Qav = 1.41, with 5–7 angles. Basidia 4-spored, 30–41 × 10–13 μm, cylindrical claviform. Pileipellis graybrownish, 5–10 μm wide. Pileocystidia not observed. Stipitipellis a cutis of cylindrical hyphae 4–8 μm wide. Caulocystidia 35–50 × 4–7 μm, sub-cylindric, numerous, apex often from subcapitulate to capitulate.
Habitat: Widespread in a range of habitats, on soil in broadleaf forest.
Collection examined: KOREA. Gangwon-do, Odae Mountain, October 22, 2013, KA13-1522.
Comments: Entoloma hirtipes can be readily distinguished by the much larger basidiospores and mainly in montane or subalpine areas [18]. These characters were similar to the collection examined in this study and detailed morphological features were sufficient to identify this collection as E. hirtipes. In RAxML tree (Fig. 1), this collection formed a separate cluster with E. hirtipes. Therefore, identity of the Korean collection was confirmed as E. hirtipes by morphology and molecular sequence analyses.
CONCLUSION
The present study describes three new records of Entoloma species in Korea. These species were identified based on morphological examinations and molecular sequence analyses. To date, only 103 species of Entoloma have been recorded in Korea [19-22], indicating that less than 10% of Entoloma species occur in Korea. Further studies are needed to increase understanding of this genus in Korea.
