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Taxonomic Re-evaluation of Megacollybia Species in Korea.
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PMID:  24808730     Owner:  NLM     Status:  PubMed-not-MEDLINE    
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The diversity of the genus Megacollybia in Korea was examined based on morphological observation and analysis of molecular data. Currently, the genus is consisted of nine species with a global distribution. However, only M. platyphylla has been reported in the floral survey of Korea. During our re-evaluation of the taxonomic diversity of Megacollybia in Korea, six Megacollybia specimens collected in 2012 were identified based on internal transcribed spacer (ITS) sequences and morphology. Here, we report two Megacollybia species, M. clitocyboidea and M. marginata, as newly recorded species from Korea. The microscopic features of the two species are provided in this study. To our knowledge, this is the first phylogenetic analysis of Korean Megacollybia species.
Authors:
Chang Sun Kim; Jong Won Jo; Young-Nam Kwag; Jae-Gu Han; Bhushan Shrestha; Gi-Ho Sung; Sang-Kuk Han
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Type:  Journal Article     Date:  2014-03-31
Journal Detail:
Title:  Mycobiology     Volume:  42     ISSN:  1229-8093     ISO Abbreviation:  Mycobiology     Publication Date:  2014 Mar 
Date Detail:
Created Date:  2014-05-08     Completed Date:  2014-05-08     Revised Date:  2014-05-12    
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Nlm Unique ID:  100960027     Medline TA:  Mycobiology     Country:  Korea (South)    
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Languages:  eng     Pagination:  22-6     Citation Subset:  -    
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Journal Information
Journal ID (nlm-ta): Mycobiology
Journal ID (iso-abbrev): Mycobiology
Journal ID (publisher-id): MB
ISSN: 1229-8093
ISSN: 2092-9323
Publisher: The Korean Society of Mycology
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© The Korean Society of Mycology
open-access:
Received Day: 31 Month: 10 Year: 2013
Accepted Day: 15 Month: 2 Year: 2014
Print publication date: Month: 3 Year: 2014
Electronic publication date: Day: 31 Month: 3 Year: 2014
Volume: 42 Issue: 1
First Page: 22 Last Page: 26
PubMed Id: 24808730
ID: 4004943
DOI: 10.5941/MYCO.2014.42.1.22

Taxonomic Re-evaluation of Megacollybia Species in Korea
Chang Sun Kim1
Jong Won Jo1
Young-Nam Kwag1
Jae-Gu Han2
Bhushan Shrestha2
Gi-Ho Sung3
Sang-Kuk Han1
1Forest Biodiversity Division, Korea National Arboretum, Pocheon 487-820, Korea.
2Institute of Life Science and Biotechnology, Sungkyunkwan University, Suwon 440-746, Korea.
3Mushroom Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Eumseong 369-873, Korea.
Correspondence: Corresponding author: hansk75@forest.go.kr (S.-K. Han)
Correspondence: Corresponding author: sung97330@gmail.com (G.-H. Sung)

Megacollybia platyphylla (Pers.: Fr.) Kotl. & Pouzar, the type species of Megacollybia Kotl. & Pouzar (Marasmiaceae, Agaricales, Basidiomycota), is macroscopically characterized by its radially fibrillose-streaked, gray-brown pileus, its very broad, distant, white lamellae, and white mycelial strands attached to the stipe base [1]. This genus was considered monotypic until early 2007. However, Hughes et al. [2] confirmed that this genus is consisted of several other species besides M. platyphylla, including: M. clitocyboidea R. H. Petersen, Takehashi & Nagas; M. fusca J. L. Mata, Aime & T. W. Henkel; M. marginata R. H. Petersen, O. V. Morozova & J. L. Mata; M. rodmanii R. H. Petersen, K. W. Hughes & Lickey (with f. murina R. H. Petersen & K. W. Hughes); and M. subfurfuracea R. H. Petersen and M. texensis R. H. Petersen & D. P. Lewis; based on internal transcribed spacer (ITS) region of ribosomal RNA and detailed morphological observations [2, 3]. The distribution of the type species M. platyphylla is restricted to Europe [2, 3]. Recently, Coimbra et al. [4] described another species of Megacollybia, M. rimosa V. R. M. Coimbra & F. Wartchow, from Brazil mainly based on morphological characteristics (characterized by the rimose to cracked pileus, pruinose stipe surface, clavate to lageniform pileocystidia and presence of clamp-connections). In Korea, only M. platyphylla has been reported [5]. In light of the recent revision of the genus Megacollybia, it is necessary to re-evaluate the taxonomic diversity of Megacollybia species in Korea with molecular data coupled with morphological observations [2]. During a survey of the diversity of Korean mushrooms in 2012, we collected six specimens of Megacollybia (KA12-0113, KA12-0146, KA12-0211, KA12-0459, KA12-0596, and KA12-1329). Here, we report two Megacollybia spp. new to Korea, M. clitocyboidea and M. marginata, based on molecular data and morphological observations.


MATERIALS AND METHODS
Specimens and morphological observations

The collected Megacollybia specimens (Table 1) were dried and deposited in the herbarium of Korea National Arboretum (KA) (Fig. 1). Reliable DNA sequence data for other Megacollybia spp. and outgroup species were retrieved from Genbank based on the study of Hughes et al. [2] for the phylogenetic analyses (Table 1, Fig. 2). Macro-morphological characteristics were determined based on the field notes and color photos of basidiomata. Micro-morphological characteristics of dried specimens were examined after sectioning and rehydrating in 3% KOH solution. Basidia, cheliocystidia, caulocystidia, and basidiospores were stained with 1% phloxine and 1% Congo-red solutions. Microscopic observations were made using an Olympus BX53 microscope and Jenoptik ProgRes C14 Plus Camera (Jenoptik Corporation, Jena, Germany). Measurements of microscopic characteristics were made using the ProgRes Capture Pro v.2.8.8. software (Jenoptik Corporation).

PCR amplification and sequencing

DNA was isolated from fresh fruit bodies (approximately 0.1 g) using a modified CTAB procedure of Doyle and Doyle [6]. ITS regions of the ribosomal RNA were amplified using ITS5 and ITS4 primers. PCR mixtures contained 0.5 pmol of each primer, 0.25 mM dNTPs, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM of MgCl2, 2.5 U of Taq DNA polymerase, and 15 ng of template DNA. PCR conditions for ITS were as follows: an initial denaturation step at 94℃ for 4 min, followed by 34 cycles at 94℃ for 40 sec, 52℃ for 40 sec, and 72℃ for 60 sec, and a final elongation step at 72℃ for 8 min. The PCR products were purified and sequenced by Macrogen Inc., Seoul, Korea. Sequences generated from the present study were deposited in Genbank (Table 1).

Phylogenetic analyses

We edited raw sequences using PHYDIT 3.2 [6]. For phylogenetic analyses, DNA sequences were aligned using ClustalX 1.81 [7] with manual adjustment using PHYDIT. We excluded ambiguously aligned regions in the phylogenetic analyses. Maximum parsimony (MP) analysis was performed using the heuristic search option in PAUP* 4.0 [8] with the following settings: all characters were equally weighted, gaps were treated as missing characters, starting trees were obtained by random addition with 1,000 replicates, and tree bisection-reconnection (TBR) branch swapping algorithm was used. Maximum likelihood (ML) phylogenetic tree with bootstrap analysis was constructed using the general time-reversible (GTR) evolutionary model and the gamma distribution of rate heterogeneity. Nodal supports for MP and ML analyses were estimated using nonparametric bootstrapping with 1,000 replicates. The option of each replicate for bootstrapping analyses was chosen as a heuristic search of 100 stepwise random addition replicates and TBR branch swapping for each bootstrap replicate. For Bayesian inference (BI), MrBayes 3.1 [9] was employed to infer the phylogenetic relationships. Posterior probabilities (PP) were approximated using the metropolis-coupled Markov Chain Monte Carlo method. Two parallel runs were conducted with one cold and three heated chains for 5 million generations, respectively, starting with a random tree. The trees were sampled every 100 generations. We interpreted the convergence of two independent runs had converged when the average standard deviation of the split frequencies dropped below 0.01. The trees obtained before the convergence were discarded using the burn-in command, and the remaining trees were used to calculate a 50% majority consensus tree and to estimate PP. PP values below 0.95 were not considered significant. Based on the studies of Moncalvo et al. [10] and Hughes et al. [2], we selected three species, Clitocybula oculus AFTOL-ID 1554, Gerronema strombodes TENN60718 and G. subclavatum DAOM194858 as outgroups.


RESULTS AND DISCUSSION
Identification of Megacollybia species in Korea.

The alignment of the ITS regions of ribosomal RNA included 29 taxa and 879 characters, of which 129 were parsimony-informative. MP tree was 334 steps long, with a consistency index (CI) of 0.8653, a retention index (RI) of 0.8892, and a homoplasy index (HI) of 0.7694. ML analyses resulted in a tree with a log-likelihood (-ln) of 1553.13. The Bayesian analysis was conducted using GTR + I + G model, and the first 45,000 trees were discarded as burn-in (burninfrac = 0.45) because the ITS dataset of four chains reached convergence after approximately 4,500,000 generations. The phylogenetic inference from MP, ML, and BI analyses was consistent in placing the major clades in phylogenetic relationships. MP tree was presented with MP and ML bootstrap support values and BI-PP for each node (MPBS/MLBS/PP) (Fig. 2).

Six specimens of Korean Megacollybia (KA12-0113, KA12-0146, KA12-0211, KA12-0459, KA12-0596, and KA12-1329) were placed in two separate clades in the present phylogenetic study. Out of these six samples, three Megacollybia specimens KA12-12-0211, KA12-0459, and KA12-0596 were placed in the M. clitocyboidea clade with high support values (MPBS/MLBS/PP = 99/96/0.93), whereas the remaining three (KA12-0113, KA12-0146, and KA12-1329) were placed in the M. marginata clade. Two specimens, KA12-0146 and KA12-1329, formed a group with high support values (MPBS/MLBS/PP = 88/88/1.0) within the M. marginata clade that was not strongly supported. Sequence comparisons with M. marginata TENN06752 (type material of M. marginata) showed that the ITS sequences of KA12-0146 and KA12-1329 were identical to that of M. marginata TENN06752, with 99.2% (637/642 bp) and 99.4% (638/642 bp), respectively.

In Korea, species of Megacollybia had been identified as M. platyphylla because it was considered as a monotypic genus. However, Hughes et al. [2] reported that the distribution of M. platyphylla may be restricted to Europe and western and central Russia. In our results, none of our six specimens was identified as M. platyphylla, which is consistent with the findings of Hughes et al. [2]. In the present study, three of our six specimens were identified as M. clitocyboidea and the rest of them as M. marginata, both of which were previously reported as temperate Asian species [2]. These two species could be morphologically distinguished by the color of pileus disc, cheilocystidia and the characteristics of marginate lamellae [2]. Megacollybia clitocyboidea is characterized by the color of pileus disc that is mummy brown to clove brown, later discoloring to grayish brown to yellowish brown. The disc surface is minutely scabrous or furfuraceous and the lamellar edge is not marginate. The stipe is buffy brown to deep olive buff. Its cheilocystidia is not or rarely protruded beyond the basidia and it is conspicuously clamped [2]. On the other hand, M. marginata is characterized by the pileus disc usually without scabers or squamules and the lamellar edge is dark brown with distinct margin [2]. Hughes et al. [2] reported that a collection of one particular Megacollybia species from the Czech Republic (TENN58493, they regarded it as M. platyphylla) may represent an intermediate between M. marginata and M. platyphylla. Although this specimen (TENN58493) lacks marginate gills, it should be considered as M. marginata because TENN58493 was placed in the M. marginata clade (Fig. 2). To our knowledge, this is the first phylogenetic analysis for the genus Megacollybia in Korea. Megacollybia clitocyboidea and M. marginata are newly recorded species of the genus Megacollybia in Korea, and further floral survey is required to comprehensively discuss the distribution of M. platyphylla in Korea.

Taxonomic description of two newly recorded Megacollybia species in Korea
Megacollybia clitocyboidea

R. H. Petersen, Takehashi & Nagas., Rep. Tottori Mycol. Inst. 45: 17 (2007; [2]) (Figs. 1A~1C, 1G, 3).

Description

Pileus 30~100 mm diam., surface minutely scabrous or furfuraceous, mummy brown- to clove-colored disc. Lamellae edge not marginate. Stipe 45~110 × 5~10 mm. Pileipellis slender and radial hyphae; conspicuously clamped; pileipellis terminal cells 30~130 × 9~25 µm, digitate, clavate to broadly clavate. Basidia clavate, 25~55 × 10~15 µm, conspicuously clamped, 4-spored. Basidiospores broadly ellipsoid to broadly ovate, 6.5~10.0 × 4.7~7.5 µm. Cheilocystidia not or rarely protruding, digitate, narrowly or broadly clavate, conspicuously clamped, 24~70 × 8~20 µm. Caulocystidia narrowly rounded to subcaptitate ends, 28~150 × 10~17 µm.

Distribution

Eastern Asia.

Specimens examined

Korea, Gyeonggi, Gwangneung Forest, coll. Han et al., 12 Jun 2012 (KA12-0211), 25 Jun 2012 (KA12-0459), 13 Jul 2012 (KA12-0596).

Megacollybia marginata

R. H. Petersen, O. V. Morozova & J. L. Mata, Rep. Tottori Mycol. Inst. 45: 21 (2007; [2]) (Figs. 1D~1F, 1H, 1I, 4).

Description

Pileus 35~90 mm diam., usually without scabers or squamules, dark fuscous brown color disc. Lamellae edge marginate. Stipe 60~110 × 4~8mm. Pileipellis terminal cells 32~105 × 6~25 µm, clavate to slightly erect; conspicuously clamped. Basidia clavate, thin-walled, 36~45 × 7~11 µm, basal clamped, 4-spored. Basidiospores broadly ellipsoid, elliptical to rarely subglobose, 6.2~9.6 × 4~7.2 µm. Cheilocystidia abundant, protruding beyond basidia, clavate to broadly clavate, conspicuously clamped, 30~70 × 8~20 µm. Caulocystidia narrowly clavate to subsphaeropedunculate, 35~85 × 5~15 µm.

Distribution

Czech Republic, far eastern Russia, Korea.

Specimens examined

Korea, Gyeonggi, Gwangneung Forest, coll. Han et al., 31 May 2012 (KA12-0113); Gangwon, Daegwanryeong Natural Recreation Forest, coll. Han et al., 9 Jun 2012 (KA12-0146); Ulleung Island, Nari Basin, coll. Han et al., 5 Sep 2012 (KA12-1329)


ACKNOWLEDGEMENTS

This work was supported by the Korea National Arboretum (Project No. KNA 1-1-10) and the research program of agricultural science and technology development (PJ010279) of the National Institute of Horticultural and Herbal Science.


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Article Categories:
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Keywords: Megacollybia clitocyboidea, Megacollybia marginata, Megacollybia platyphylla, Morphology, Phylogeny, Unrecorded species.

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