Online publication; available at: https://jats-truffles.org/truffology/

Truffology 6 (1): 1–6 (2023)

Original peer-reviewed article （原著論文 ； 査読有）First report of a subhypogeous basidiomycete, Scleroderma yunnanense(Boletales) from Japan
半地下生担子菌 Scleroderma yunnanense （シラガニセショウロ，イグチ目） の日本初記録
Taiga Kasuya1*, Kazumasa Imai2, Kentaro Hosaka3
糟谷 大河 1*, 今井 一雅 2, 保坂 健太郎 3
1

Department of Biology, Keio University, 4-1-1 Hiyoshi, Kohoku-ku, Yokohama-shi, Kanagawa 223-8521, Japan慶應義塾大学生物学教室， 〒 223-8521 神奈川県横浜市港北区日吉 4-1-1

2

Shin-ike-cho, Chikusa-ku, Nagoya-shi, Aichi 464-0027, Japan〒 464-0027 愛知県名古屋市千種区新池町

3

Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba-shi, Ibaraki 305-0005, Japan
国立科学博物館植物研究部，〒 305-0005 茨城県つくば市天久保 4-1-1* Corresponding author （主著者）

E-mail: tkasuya@keio.jp

Abstract
A subhypogeous sclerodermataceous fungus was collected in Nagoya, Aichi Prefecture, central Japan. Based onmorphological observations and phylogenetic analyses using nuclear ribosomal DNA sequences, the present
fungus was identified as Scleroderma yunnanense, belonging to Sclerodermataceae (Boletales). Scleroderma
yunnanense is new to Japan and characterized by not stellately dehiscent basidiomata when mature, thick (2–7mm) peridium covered by cream to pale yellowish scales, basidiomata covered with whitish mycelial strands,
clamped hyphae, and globose to subglobose basidiospores densely covered with pyramidal warts.

要旨
愛知県名古屋市において、 ニセショウロ型の半地下生菌の一種が採集された。 本菌について、 形態的特徴の観察および子実体より得られた核リボソーム DNA の塩基配列 を用いた系統解析を行った。 その結果、 本菌はイグチ目ニセショウロ科に属する Scleroderma yunnanense と同定された。 本種は日本新産であり、 子実体が成熟しても星形に裂開しない点、 殻
皮が厚く （2–7 mm）、 クリーム色～淡黄色を帯びる鱗片に覆われる点、 子実体の表面に白色の菌糸束が存在する点、 菌
糸にクランプ結合を有する点、 そして担子胞子が球形～類球形で角錐状のいぼ状突起により密に覆われる点で特徴づけられる。 本菌の和名をシラガニセショウロとする。
Article Info: Submitted: 29 December 2022

© The Japanese Association for Truffle Science (JATS), 2023

Accepted: 17 March 2023

Published: 31 July 2023

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Kasuya et al.: Scleroderma yunnanense new to Japan

Introduction

DNA preparation, PCR and sequencing

Among ca. 40 known species of the genus Scleroderma Pers.

DNA extraction, PCR and DNA sequencing of the specimen(Boletales, Sclerodermataceae), at least five subhypogeous or

examined in the present study were carried out according to the

hypogeous species, i.e., S. hypogaeum Zeller, S. michiganense

methods introduced by Kasuya et al. (2012, 2022). DNA sequence(Guzmán) Guzmán, S. paradoxum G.W. Beaton, S. patagonicum

data were obtained from the nuclear ribosomal internal spacer

Nouhra & Hern. Caff. and S. yunnanense Y. Wang have beenregion (ITS) and a part of the large subunit gene (LSU). For

recognized in the world (Beaton & Weste, 1982; Guzmán, 1970;

amplifying the ITS region, the primer combination of ITS5 andNouhra et al., 2012; Zhang et al., 2013). Although sixteen taxa

ITS4 (White et al., 1990) was used. For amplifying the LSU, theof the genus were hitherto recorded in Japan, only one species, S.

combination of LR0R and LR5 (Vilgalys & Hester, 1990) was

capeverdeanum M.P. Martín, M. Dueñas & Tellería has been known

used. PCR and sequencing were carried out following the sameas a subhypogeous taxon (Kasuya et al., 2022). During our continuous

methods previously described in Kasuya et al. (2012, 2022). A

studies of the species diversity of Scleroderma in Japan (Kasuya &total of two newly generated sequences from this study were

Guzmán, 2007; Kasuya et al., 2002, 2022), several subhypogeous

deposited in GenBank (Table 1).

basidiomata of sclerodermataceous fungus were collected from
Nagoya, Aichi Prefecture. Based on morphological observations andPhylogenetic analyses

phylogenetic analyses using nuclear ribosomal DNA sequences, the

Newly generated ITS and LSU sequences from the Japanesepresent fungus was identified as S. yunnanense. This species was

specimen were used for the phylogenetic analyses. Additionally, 30

originally described from China and it is known as a subhypogeous

ITS and eight LSU sequences of Scleroderma were retrieved from

species (Zhang et al., 2013). Our morphological observations andthe NCBI GenBank databases (https://www.ncbi.nlm.nih.gov/) andphylogenetic analyses of the Japanese specimen revealed that it is theincluded in the analyses (Table 1). DNA sequences were initiallyfirst distributional record of S. yunnanense in Japan.

aligned using Muscle v.3.6 (Edgar, 2004), followed by manual

Materials and methods
Sample collecting and morphological observationsalignment in the data editor of BioEdit ver. 7.0.1 (Hall, 1999). A
total of 66 ITS and 98 LSU nucleotide positions were respectively
excluded from the analyses because of the presence of ambiguously

Fresh subhypogeous basidiomata were collected from fallenaligned regions. Phylogenetic analyses were performed for theleaves of broad-leaved trees and rich soil in Heiwagaoka Park,

combined dataset of ITS and LSU sequences under maximum

Meito-ku, Nagoya, Aichi Prefecture in July 2021. Basidiomata wereparsimony (MP) and maximum likelihood (ML) criteria. MP

photographed and observed macroscopically. Fresh basidiomata

analysis was conducted by PAUP version 4.0b10 (Swofford, 2002)

were air-dried using a food dehydrator (Snackmaster Express FD-60;

following the same method reported by Kasuya et al. (2022). ML

Nesco/American Harvest, Milwaukee, WI, USA) under 46°C for 46

analysis of the combined dataset of ITS and LSU was performed

hours. For light microscopy, hand-cut sections of dried basidiomata

using MEGA X (Kumar et al. 2018) after testing the best models

were mounted in 3% (w/v) KOH or 70% (v/v) ethanol reagent.

according to the methods previously introduced by Kasuya et al.

Dimensions of basidiospores were measured from KOH-mounted(2022). Tamura 3-parameter (Tamura, 1992) with gamma-distributed

sections. Fifty randomly selected basidiospores were measured underrate heterogenetic and a proportion of invariant sites (T92+G+I) wasa light microscope at 1000× magnification. All measurements werechosen as the optimal substitution model for the ML analysis of theperformed with Photoruler 1.1.3 (http://inocybe.info/_userdata/ruler/

combined ITS and LSU dataset. Three sequences of S. areolatum

PhotoRuler.html). In addition, the surface features of basidiospores

Ehrenb. (Table 1) were selected for outgroups, which were strongly

were observed by scanning electron microscopy (SEM). For SEM, asupported as the sister of the major clade containing S. yunnanense

small portion from glebal tissue was put onto double-sided adhesive(Ortiz-Rivero et al., 2021).

tape on a specimen holder and coated with platinum-palladium using
a JFC-1600 Ion Sputter Coater (JEOL, Tokyo, Japan). Specimen was
examined with a JSM-6480LV SEM (JEOL, Tokyo, Japan) operating

Results
Morphological observations

at 10 kV. Specimen examined in this study was deposited at the

The specimen examined was morphologically identical to S.

mycological herbarium of National Museum of Nature and Science

yunnanense (Zhang et al., 2013) in its following features: (1)(TNS) in Japan.

irregularly dehiscent basidiomata when mature, (2) thick (2–7 mm)

2© The Japanese Association for Truffle Science (JATS), 2023

Truffology - Volume 6, Issue 1, 2023

Kasuya et al.: Scleroderma yunnanense new to Japan

Table 1. Specimen identification, voucher specimen and/or isolate numbers, origin and GenBank accession numbers for ITS and LSU sequences used forthe present phylogenetic analyses

Species names

Herbarium voucher; isolate

OriginScleroderma anomalosporum
INPA 271001; n/a*
BrazilS. areolatum
K (M) 125392; n/a
UK: England
S. areolatum
TNS F-82295; n/aJapan: Chiba, Choshi
S. areolatum
INM 2-213463; n/aJapan: Ibaraki, Kamisu
S. australe
MEL 269386; n/aAustralia
S. bermudense
BZ3961; n/aBelizeS. camassuense
INPA 271114; n/aBrazilS. dictyosporumn/a; IR215
Burkina Faso
S. dictyosporum
SD-4901; n/a
Burkina Faso
S. duckei
INPA 272127; n/aBrazilS. duckei
UFRN Fungos-2795; n/aBrazilS. geaster
MA-Fungi 34025; n/aSpain
S. geaster
MA-Fungi 68558; n/aSpain
S. geaster
E 157436; n/a
GreeceS. guzmanii
IBUG J.A. Garcia Valle 24; n/a
MexicoS. guzmanii
OSC 41254; n/aUSA
S. polyrhizum
MA-Fungi 32214; n/aSpain
S. polyrhizum
ILLS 56824; n/aUSA
S. polyrhizum
MA-Fungi 39352; n/a
Spain
S. sinnamariensen/a; n/a
India: Southwestern Ghats
S. texense
AWW216; n/aUSA
S. texense
F C0296202F; n/aUSA
S. texense
VPI F-0004156; n/aUSA
S. xanthochroum
AWW311; n/a
Malaysia
S. yunnanense
KUN-HKAS 79633A; Ji001A
China: Yunnan
S. yunnanense
KUN-HKAS 79633B; Ji001B
China: Yunnan
S. yunnanense
KUN-HKAS 79633C; Ji001C
China: Yunnan
S. yunnanense
KUN-HKAS 79633D; Ji001D
China: Yunnan
S. yunnanense
KUN-HKAS 80386; n/aChina
S. yunnanense
TNS F-82294; n/aJapan: Aichi, Nagoya
Scleroderma sp.
G509; n/a
Guyana*"n/a" means information not available.
**Identical accession number for ITS and LSU indicates a single DNA sequence containing both regions.
***Sequences newly generated in the present study.

GenBank accession numbers
ITS
LSU
KX792084
n/a*
EU784407
n/a
OQ025272
OQ025269
OQ025273
OQ025270
MT270609
n/aEU718118
DQ644137
KX792085
n/a
FJ840443
n/a
FJ840449
n/a
KX792086
n/a
KX792087
n/aMT270638
n/aMT270642
n/aMT270644
n/aMT270645
n/aMT270648
n/aMT270660
n/aMT270661
n/aMT270662
n/aAB908177**
AB908177**
EU718123
EU718153
MT270649
n/aMT270650
n/a
EU718126
EU718154
JQ639040
n/a
JQ639041
n/a
JQ639402
n/a
JQ639403
n/aMW493647
MW493703
OQ025271*** OQ025268***
KJ786660
KJ786558

peridium covered by cream to pale yellowish scales (Fig. 1A–

analyses resulted in trees that were almost identical in topology.

B), (3) well-developed whitish mycelial strands on the surface

Hence, only the MP tree topology of the combined ITS and LSUof basidiomata (Fig. 1C), (4) clamped hyphae (Fig. 1D–E), anddataset is shown in Fig. 2.

(5) globose to subglobose basidiospores densely covered with

The combined ITS and LSU sequences generated from a Japanesepyramidal warts (Fig. 1F–G). A detailed description and illustrations

specimen and Chinese samples of S. yunnanense were placed withinof the salient features of Japanese specimens are given below.

a strongly supported clade in both MP and ML phylogenies [MP BS(%) /ML BS (%) =100/100; Fig. 2], and were distinct from thosePhylogenetic analyses
The The combined dataset of ITS and LSU consisted of 28
ingroups and three outgroups. It had an aligned in length of 1,808
characters inclusive of gaps, of which 164 characters were constant,
1,552 variable and phylogenetically uninformative, and 256
phylogenetically informative. The MP analysis of the combined ITSof the other Scleroderma species. Present phylogenetic analyses
support the identity of Japanese and Chinese S. yunnanense basedon morphology.

Taxonomy
Scleroderma yunnanense Y. Wang, Mycotaxon 125: 195 (2013).

and LSU dataset yielded 10,000 most parsimonious trees, of which

Fig. 1.

3,384 trees were found in the first step of the heuristic search. The

Description: Basidiomata (Fig. 1A–B) subhypogeous, globose,

consistency index, retention index, and rescaled consistency index

depressed globose to subglobose, 3–8 cm broad, dirty white, cream toof the most parsimonious trees are 0.6349, 0.8268, and 0.5249,

pale yellowish brown, sessile or rarely with a short pseudostipe up to 1

respectively. The highest log-likelihood of the resulting ML tree of

cm long, often covered with well-developed whitish mycelial strands

the combined ITS and LSU dataset is -5316.67. The MP and ML(Fig. 1C), arising from whitish mycelial tuft attached to soil. Mycelial© The Japanese Association for Truffle Science (JATS), 2023

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Kasuya et al.: Scleroderma yunnanense new to Japan

Truffology - Volume 6, Issue 1, 2023

Fig. 1. Morphological features of Scleroderma yunnanense (TNS-F-82294). A: Surface of basidiomata. B: Vertical sections of mature basidiomata showing
peridium and gleba. C: Whitish mycelial strands on the surface of basidiomata. D: Hypha with a clamp-connection (black arrow) of mycelial strands on thesurface of a basidioma. E: Mycelial strands on the surface of a basidioma composed of clamped (black arrow) hyphae sparsely covered with amorphous, hyaline
crystalline materials (white arrows). F: Basidiospores. G: Scanning electron microscopy image of a basidiospore. Bars: A = 1 cm; C: 1 mm; D, E = 5 µm; F = 10 µm;
G = 1 µm.

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© The Japanese Association for Truffle Science (JATS), 2023

Truffology - Volume 6, Issue 1, 2023

Kasuya et al.: Scleroderma yunnanense new to Japan

Fig. 2. A phylogenetic tree generated from maximum parsimony (MP) analysis based on the combined dataset of the nuclear rDNA ITS regionand LSU sequences of selected members of Sclerodermataceae. Taxon names indicated by bold are newly generated sequences from thepresent study. Bootstrap support values (BS) of MP and maximum likelihood (ML) greater than 60% are shown for each node (MP/ML), and BS
less than 60% are indicated by an asterisk (*).

strands on surface of basidiomata (Fig. 1D–E) interwoven, septate,in 3% KOH (Fig. 1F), dark reddish brown in 70% ethanol reagent;thin-walled, 2–5 µm broad, hyaline in 3% KOH and 70% ethanol

surface densely echinulate composed of pyramidal warts up to 1.5 µm

reagent, with numerous clamp-connetctions; surface sometimes

long (Fig. 1G).

sparsely covered with amorphous, hyaline crystalline materials (Fig.

Habitat: Subhypogeous, small groups or gregarious among fallen

1E). Peridium (Fig. 1A–B) thick, ca. 2–7 mm thick, two-layered.

leaves, mosses and rich soil under Quercus glauca Thunb. trees.

Exoperidium thin (up to ca. 0.5 mm thick), pale yellowish brown to

Fruiting in the Japanese locality occurs in summer (July).

pale brown, covered with cream to pale yellow scales. Endoperidium

Specimens examined: JAPAN, Aichi Prefecture, Nagoya, Meito-

thick, firm, pale yellowish brown to cream near exoperidium, whiteku, Heiwagaoka (approx. 35°10'39''82N, 136°58'54''20E, alt.

near gleba. Apical part of peridium irregularly dehiscent but not stellate

approx. 64 m asl.), July 16, 2021, coll. K. Imai, TNS-F-82294,

when mature. Gleba (Fig. 1B) firm, white to cream when young, then

GenBank accession no.: OQ025271 (ITS), OQ025268 (LSU).

becoming purplish gray, grayish brown to blackish brown with whitish

Known distribution: China (Zhang et al., 2013) and Japan (newcapillitial threads, finally powdery when mature. Rudimentary hyphaerecord).

of gleba interwoven, septate, thin-walled, 1–2.5 µm broad, hyaline

Japanese name: Shiraga-nise-shoro (newly proposed here;

in 3% KOH and 70% ethanol reagent; surface sparsely covered“Shiraga” = white hair in Japanese, referring to the whitish mycelial

with amorphous, hyaline gelatinous materials. Basidia not observed.

strands on the surface of basidiomata; “nise-shoro” = the Japanese

Basidiospores (Fig. 1F–G) globose to subglobose, 6.5–8 µm in diam.

name of Scleroderma).

(mean = 7.6 µm, n = 50) including surface ornamentation, dark brown© The Japanese Association for Truffle Science (JATS), 2023

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Kasuya et al.: Scleroderma yunnanense new to Japan

Discussion
As outlined above, the Japanese specimen was morphologicallyalmost identical to the original description of S. yunnanense (Zhang
et al., 2013). Moreover, our phylogenetic analyses (Fig. 2) show the

Guzmán G. (1970) Monografia del género Scleroderma Pers. emend. Fr.
(Fungi-Basidiomycetes). Darwiniana 16: 233–407.
Hall T.A. (1999) BioEdit: a user-friendly biological sequence alignment
editor and analysis program for Windows 95/98/NT. Nucleic Acids
Symposium Series 41: 95–98.

monophyly of the present species, as indicated by Zhang et al. (2013)

Kasuya T., Guzmán G. (2007) Veligaster nitidum, a pantropicaland Ortiz-Rivero et al. (2021). The present species is morphologicallysclerodermataceous fungus new to Japan and Thailand. Mycosciencesimilar to S. cepa Pers. in its echinulate basidiospores and thick

48: 259–262.

peridium (Guzmán, 1970; Coccia et al., 1990), but is clearly

Kasuya T., Guzmán G., Ramírez-Guillén F., Kato T. (2002) Scleroderma

distinguishable by its clampless hyphae. The present phylogenetic

laeve (Gasteromycetes, Sclerodermatales), new to Japan. Mycoscience

analyses demonstrate that S. polyrhizum (J.F. Gmel.) Pers. andS. texense Berk. are closely related to S. yunnanense (Fig. 2).
Morphologically, these two species differ from S. yunnanense bysubreticulate to reticulate basidiospores (Ortiz-Rivero et al., 2021).
Zhang et al. (2013) reported that specimens of S. yunnanense werecollected under Pinus kesiya var. langbianensis (A. Chev.) Gaussen

43: 475–476.
Kasuya T., Hosaka K., Uno K., Kakishima M. (2012) Phylogenetic
placement of Geastrum melanocephalum and polyphyly of Geastrum
triplex. Mycoscience 53: 411–426.
Kasuya T., Uzawa M., Hosaka K. (2022) Scleroderma capeverdeanum,a subhypogeous fungus new to Japan, collected from the Eucalyptus
plantation in Ibaraki Prefecture. Truffology 5: 23–32.

ex Bui and Betula alnoides Buch.-Ham. ex D.Don in China. The

Kumar S., Stecher G., Li M., Knyaz C., Tamura K. (2018) MEGA X:

Japanese specimen was collected under Quercus glauca trees. While

Molecular Evolutionary Genetics Analysis across computing platforms.

it is unclear whether the mycorrhizal hosts of the present species

Molecular Biology and Evolution 35: 1547–1549.

are varied among geographically distant regions, S. yunnanense

Nouhra E.R., Caffot M.L.H., Pastor N., Crespo E.M. (2012) The species

presumably forms ectomycorrhizae with pinaceous, betulaceous, orof Scleroderma from Argentina, including a new species from thefagaceous trees.

Nothofagus forest. Mycologia 104: 488–495.

Scleroderma yunnanense is known as an edible fungus and is
widely consumed by the local people in the Yunnan area of China(Zhang et al., 2013). Further investigations are required to clarify
whether the Japanese population of the present species is also edibleor not.

Acknowledgements

Ortiz-Rivero J., Watling R., Guzmán-Dávalos L., Martín M.P. (2021)
The many-rooted earthball—Scleroderma geaster and S. polyrhizum
revisited, with the description of a new species. Phytotaxa 510: 1–17.
Swofford D.L. (2002) PAUP*. Phylogenetic analysis using parsimony(*and other methods). MA Sinauer Associates, Sunderland.
Tamura K. (1992) Estimation of the number of nucleotide substitutions
when there are strong transition-transversion and G+C-content biases.
Molecular Biology and Evolution 9: 678–687.

We are very much obliged to Ms. Kyung-Ok Nam and Ms. Megumi

Vilgalys R., Hester M. (1990) Rapid genetic identification and mapping

Otsuka of the National Museum of Nature and Science for help withof enzymatically amplified DNA from several Cryptococcus species.

assisting molecular experiments. We also thank Ms. Nako Hirata for

Journal of Bacteriology 172: 4238–4246.

facilitating the fieldwork. We are grateful to Dr. Tamao Hatta of Chiba

White T.J., Bruns T., Lee S., Taylor J.W. (1990) Amplification and direct

Institute of Science for his skillful technical assistance and for helpful

sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis

suggestions to SEM observation. This work was supported in part by
JSPS KAKENHI Grant Number JP20K06805.

M.A., Gelfand D.H., Sninsky J.J., White T.J. (eds.) PCR protocols.
Academic Press, New York, pp. 315–322.
Zhang C., Xu X.-E., Liu J., He M., Wang W., Wang Y., Ji K. (2013)
Scleroderma yunnanense, a new species from South China. Mycotaxon

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6© The Japanese Association for Truffle Science (JATS), 2023