Bull. Natl. Mus. Nat. Sci., Ser. B, 45(2), pp. 71–76, May 22, 2019

First Record of Macrolepiota velosa Vellinga & Zhu L. Yang(Agaricaceae) from Myanmar
Kentaro Hosaka1*, Kyung-Ok Nam1, Wah Wah Linn2, Mu Mu Aung2
1

Department of Botany, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan
2
Forest Research Institute, Forest Department, Yezin, Nay Pyi Taw, Myanmar* E-mail: khosaka@kahaku.go.jp(Received 12 March 2019; accepted 27 March 2019)

Abstract The mushroom specimen with large-sized fruit body with a distinct volva at base ofstipe collected in Myanmar was demonstrated to be Macrolepiota velosa, originally describedfrom Yunnan, China. This is the ﬁrst record of the species outside of China and Thailand, and is
the ﬁrst record from Myanmar. This study extended the known distribution of M. velosa to wider
areas, indicating the species may be distributed across tropical to subtropical regions of SoutheastAsia.
Key words : Agaricales, Agaricomycota, Basidiomycota, biogeography, distribution, fungi, inventory, mushrooms, Southeast Asia, volva.

Introduction
The genus Macrolepiota Singer is typicallycharacterized by having medium- to large-sizedbasidiomata with a scaly pileus, free gills, andthick annulus, but lacking a volva at base of stipe(Largent and Baroni, 1988). However, several species are known to possess a distinct volva and a
new genus Volvolepiota Singer has been proposedto accommodate them (Singer, 1959). Recent
molecular phylogenetic studies indicated that Volvolepiota is deeply nested within the rest of Macrolepiota (Ge et al., 2012; Vellinga et al., 2003;
Vizzini et al., 2011), or it consists of a sister clade
to the rest of Macrolepiota (Ge et al., 2010).
Because of their close afﬁnities, Volvolepiota iscurrently treated as a synonym of Macrolepiota.
To clarify fauna and ﬂora (including fungi) ofMyanmar and the surrounding areas, a joint
research team of National Museum of Nature andScience, Japan, and Forest Research Institute,
Myanmar has been conducting biological inventories in Myanmar since 2016. During one of ourﬁeldwork activities in 2016, we have collected apeculiar basidioma with a distinct volva at base ofstipe. Further investigations all indicated that ourmaterial is Macrolepiota velosa originallydescribed from Yunnan, China (Vellinga and Yang,2003). This is the ﬁrst record of the species fromMyanmar and the second record outside of China.
Colored photographs of macroscopic andmicroscopic characters of M. velosa from Myanmar are provided. In addition, DNA sequence
data from the internal transcribed spacer (ITS)
region and the large subunit (LSU) of nuclear
ribosomal DNA were compared with the materials from China and Thailand.
Materials and Methods
Collecting Sites, Collecting Scheme, and Curation of Specimens
One basidiome of a volvate Macrolepiota wascollected by the ﬁrst author from National Kandawgyi Garden, Pyin Oo Lwin, Mandalay Division, Myanmar (N21°59′33.2″, E96°28′16.3″;

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Kentaro Hosaka et al.

elevation ca. 1,100 m) on August 28, 2016. Specimen was photographed and macroscopic observation was conducted. Specimen was cut into
half and dried with low heat and good air circulation, using a food dehydrator for 24 hours. In
addition to dried materials, small fragments ofclean, internal tissue from freshly collected material were cut using a clean, sterile razor blade.
Contamination of visible soil particles and othermaterials was strictly avoided. The tissue fragments were soaked in DMSO buffer (Seutin etal., 1991) with an addition of 100 mM Tris-HCl(pH 8.0) and 0.1 M sodium sulﬁte (Na2SO3)
under 4°C, following the procedures of Hosaka(2009) and Hosaka and Castellano (2008).
Specimens collected during the ﬁeldwork weredeposited at Forest Research Institute, Myanmar,and the duplicate at the fungal herbarium of theNational Museum of Nature and Science, Tsukuba, Japan (TNS). All tissue samples werestored in freezers (−80°C) at the Center for
Molecular Biodiversity Research, National
Museum of Nature and Science.
Light Microscopy
For light microscopic observations, a small
portion from dried basidioma was mounted in
3% (w/v) KOH or Meltzerʼs reagent on glass
slides. Those samples were examined with
Olympus BX53 microscope under Nomarski
interference contrast. Thirty randomly selectedbasidiospores were measured under a lightmicroscope at 1000× magniﬁcation.
DNA Preparation, PCR, and Sequencing
DNA was extracted from the tissue fragments
stored in DMSO buffer. Tissues were groundunder liquid nitrogen using a mortar and pestle.
DNA extractions used a modiﬁed CTAB extraction followed by glass milk puriﬁcation methods
as summarized by Hosaka (2009) and Hosaka
and Castellano (2008).
DNA sequence data were obtained from theITS and LSU regions of the nuclear ribosomal
DNA. For amplifying the ITS, the primer combination of ITS5 and ITS4 (White et al., 1990) wasused. For amplifying the LSU, the primer combination of LR0R and LR5 (Vilgalys and Hester,1990) was used. PCR reactions were carried outusing 20 μL reaction volumes each containing:
1 μL genomic DNA, 1 μL dNTPs (4 mM), 1 μL ofeach primer (8 μM), 0.5 units of Taq polymerase(TaKaRa, Tokyo, Japan), 2μL MgCl2 (25 mM),
2 μL Bovine Serum Albumin (BSA). PCR products were electrophoresed in 1% agarose gelsstained with ethidium bromide and visualized
under UV light. When ampliﬁcation bands wereconﬁrmed, PCR products were then puriﬁedusing the ExoSap-IT (Millipore, Molsheim,France) and directly sequenced using the Big
Dye Terminator Cycle Sequencing Kit on
ABI3500 (Applied Biosystems Inc., Foster City,CA, USA), following the manufacturerʼs instructions.
Phylogenetic Analyses
The obtained raw sequences were edited using
ATGC version 7.1.0 (GENETYX Corporation,Tokyo, Japan). Edited sequences were analyzed
using the GenBank BLAST search (blastn).
Default settings of blastn option were used.
Because our sequences (both ITS and LSU)
showed a signiﬁcant hit (more than 99% similarities) with Macrolepiota velosa, we have retrieved
all available sequences (ITS and LSU) of M.
velosa for further analyses.
DNA sequences of the ITS and LSU (GenBank accession nos. MK634569 and MK634568,respectively) were aligned manually using thedata editor of BioEdit ver. 7.0.1 (Hall, 1999). The
dataset was then analyzed by maximum parsimony (MP) analysis. MP analyses were conducted under the equally weighted parsimony
criterion using PAUP* version 4.0b10 (Swofford,2002), with heuristic search option (with TBRand Multrees on). Support for the individual
nodes was tested with bootstrap (BS) analysis
under the equally-weighted parsimony criterion.
BS analysis was based on 100 BS replicatesusing the heuristic search option (TBR and Multrees options off), with ten random addition
sequences.

Macrolepiota velosa from Myanmar

73

Fig. 1. Macrolepiota velosa (KH-MYA16-018). A. Basidioma in situ. B. Pileus showing ﬁbrillose surface. C.
Basidioma with distinct volva at base of stipe. D. Lamellae and annulus. E. Volva. F. Dried basidioma. Scalebars＝2 cm.

Kentaro Hosaka et al.

74

Results and Discussion
Macrolepiota velosa Vellinga & Zhu L. Yang,
Mycotaxon 85: 184. 2003.
Basidioma (Fig. 1) large-sized, 14 cm high.
Pileus 10 cm in diam., plano-convex, with a wide
indistinct umbo (Fig. 1B), ﬁbrillose, covered
with dense, purplish brown squamules at center,much scarcer near margin (Fig. 1A, B, F).
Lamellae (Fig. 1D) free, moderately crowded,
white. Stipe (Fig. 1A, C, D) purplish brown,slightly paler above annulus, cylindrical,
13×0.7–0.9 cm, tapering at apex, slightly
enlarged at base. Annulus (Fig. 1D) ascending,white above, purplish brown below, with margin
irregularly waved. Volva (Fig. 1C, E) white, limbate, membranous, with white rhizomorphs atbase (Fig. 1E). Taste and odor indistinct.
Basidiospores (Fig. 2A, B) amygdaloid-ellipsoid in side view, ellipsoid in front view,
8–11×5.5–7 μm (average＝9.2×6.2 μm), Q(length/width ratio) 1.28–1.68 (average Q＝1.49), smooth, thick-walled, dextrinoid, with api-Fig. 2. Light microscopy of Macrolepiota velosa (KH-MYA16-018). A. Basidiospores in Meltzerʼs reagent. B.
Basidiospores (reddish brown) with globose lamellar cells (hyaline). C. Basidia showing 4 sterigmata (arrow).
D. 2-spored basidia. Scale bars＝10 μm.

Macrolepiota velosa from Myanmar

cal central germ pore. Basidia (Fig. 2C, D) clavate, without clamp connections at base, mostly
4-spored (Fig. 2C) but sometimes 2-spored (Fig.
2D), 27–35 (not including sterigma)×9–11 μm,sterigma 2–4 μm long. Cheilocystidia cylindrical, without clamp connections at base, thinwalled, 52–63×6–8.5 μm. Squamules on pileus
composed of slightly thick-walled brown hyphaewith cylindrical to swollen apex, up to
95×24 μm.
Habitat and known distribution in Myanmar: Terrestrial, solitary on the ground in predominantly evergreen, broad-leaved forests
including Dipterocarpaceae, mixed with bamboos and Pinus spp. So far only found in one
locality (National Kandawgyi Garden) in Myanmar.
Materials examined: Myanmar: Mandalay
Division, Pyin Oo Lwin, National Kandawgyi
Garden, alt. 1,100 m, 28 Aug. 2016, K. Hosaka(KH-MYA16-018, deposited as TNS and Forest
Research Institute, Myanmar).
Comments: Macrolepicola velosa was ﬁrst
described from Yunnan Province, China (Vellinga and Yang, 2003). It was then recorded from
Hainan Province, China (Ge et al., 2010). The
available information also indicates that the species is also distributed in Thailand (Chiang Mai),retrieved as a GenBank accession nos. KJ524569
and also mentioned by Ge et al. (2010). However, we could not ﬁnd any other publishedrecords (e.g., morphological observation) of the75

specimen from Thailand.
Our morphological observation was consistent
with previous descriptions of M. velosa (Vellingaand Yang, 2003; Ge et al., 2010). However, thematerial from Myanmar possesses slightly longerbasidia (27–35×9–11 μm) than Chinese materials (25–30×9.5–11.5 μm). Furthermore, we haveinfrequently found 2-spored basidia (Fig. 2D),
which were not observed by previous studies(Vellinga and Yang, 2003; Ge et al., 2010). These
differences are likely intraspeciﬁc variations, but
further studies are warranted.
The BLAST search indicated that the sequences(ITS and LSU) of Myanmar material shareshighest similarities with M. velosa from China
and Thailand. The top 5 hits of ITS BLAST
search were all M. velosa (99.03% or higher similarities), and the next hit was “Macrolepiota sp.
3” (GenBank accession no. KY927722) from
Brazil at 93.83% similarity. The top hit of LSU
BLAST search (99.89%) was also M. velosa(GenBank accession no. JN940273) from
Hainan, China. There was only one CT transition
observed between Chinese and Myanmar samples across the whole range of LSU region.
Phylogenetic analyses of the ITS region
resulted in 6 equally parsimonious trees (Fig. 3).
The alignment consisted of a total of 680 characters, but 675 characters were parsimony uninformative and therefore excluded from the analyses.
The ITS phylogeny (Fig. 3) was midpoint rooted,
and arguably indicated that the materials fromFig. 3. One of 6 most parsimonious trees of Macrolepiota velosa based on the ITS sequences. The tree wasrooted at midpoint. Taxon names are shown as GenBank accession numbers followed by the countries (andmore detailed locality, if available) of origin. The number of branch indicates parsimony bootstrap value. The
asterisk indicates the node collapsed in the strict consensus.

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76

Hainan was more diverged from the rest of samples from Myanmar, Thailand and Yunnan,China. All materials were, however, separated
only by a few substitutions. We therefore conclude that they all represent a single species, M.
velosa.
Our study revealed a new distribution of M.
velosa outside of China and Thailand. The species may be distributed in wider areas in tropicalto subtropical regions of Southeast Asia. Whether
intraspeciﬁc variations observed from morphological and molecular analyses actually represent
local populations or distinct species warrant further investigation.
Acknowledgments
The authors thank Kazuo Nishibori, Megumi
Johnston, and Jun Yamazaki for assisting ourcuratorial work. We also thank the park rangers
and ofﬁcers in Mandalay Division, Myanmar, forassisting our ﬁeldwork. This study was ﬁnanced
in part by a research project of the National
Museum of Nature and Science entitled as “Biological inventory with special attention to Myanmar: investigations of the origin of southern elements of Japanese ﬂora and fauna” and JSPS
KAKENHI Grant Number 18KK0210.
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