Online publication; available at: http://jats-truffles.org/truffology/Truffology 1 (1): 5–8 (2018)

Original peer-reviewed article （原著論文 ； 査読有）First report of a rare sequestrate fungus, Rossbeevera yunnanensis (Boletaceae,Boletales) from Japan
希少シクエストレート菌、 ウンナンツチダマタケ Rossbeevera yunnanensis （イグチ目イグチ科） の日本における初報告
Takamichi Orihara
折原 貴道
Kanagawa Prefectural Museum of Natural History, 499 Iryuda, Odawara, Kanagawa 250-0031, Japan
神奈川県立生命の星 ・ 地球博物館， 〒 250-0031 神奈川県小田原市入生田 499
E-mail: t_orihara@nh.kanagawa-museum.jp

Abstract
A sequestrate, truffle-like bolete species, Rossbeevera yunnanensis was described in 2012 based on a single specimen
collected from Chuxiong Yi Autonomous Prefecture, Yunnan Province, China. I collected a second specimen fromHiroshima Prefecture, Japan. This is the first report of this truffle in Japan. The overall morphology of the Japanese
specimen was identical to the holotype from China, and the internal transcribed spacer region and 28S of the nuclear
ribosomal RNA gene between the Japanese and Chinese specimens showed 99.6% and 99.9% similarity, respectively. I
discuss the possibility of long-distance spore dispersal of the species.

要旨
トリュフ型 （シクエストレート性） イグチ類からなるツチダマタケ属 （Rossbeevera） の一種、 Rossbeevera yunnanensis は、 2012
年に中華人民共和国 （中国） 雲南省産の標本 1 点を基に記載された稀産種である。 この度、 広島県廿日市市の落葉広葉樹林コナラ樹下において、 日本新産となる本種子実体を採集したので報告する。 本種は極めて薄い白色～類白色の外皮と、 紡錘形の大型の担子胞子 （平均径 20.1 × 7.8 µm） が特徴で、 外皮は手で触れると淡青色に変色する。 日本産標本は形態的特徴が中国産基準標本とほぼ一致し、 核リボソーム RNA 遺伝子の ITS 領域および 28S （大サブユニット） の配列についても、両標本はそれぞれ 99.6% および 99.9% の相同性を示した。 このことは、 中国南部と日本 （本州） という離れた地域間で、 比較的短期間に本種の分散が生じたことを示唆している。 なお、 本種の和名は 「ウンナンツチダマタケ」 とする。
Article Info: Submitted: 24 June 2017 Accepted: 30 November 2017

Published: 31 March 2018

Introduction

Species of the genus presumably form ectomycorrhizas withThe genus Rossbeevera T. Lebel & Orihara was proposed

trees of Fagaceae, Casuarinaceae, Myrtaceae and Nothofagaceae.

in 2011 to accommodate sequestrate (truffle-like) species

Orihara et al. (2016) proposed a sister, sequestrate genus

phylogenetically related to epigeous bolete genera Leccinum

Turmalinea Orihara & N. Maek . that accommodates four

Gray and Leccinellum Bresinsky & Manfr. Binder s. l. in thespecies and one subspecies from Japan and China. This genus is

Boletaceae (Lebel et al., 2012). The genus currently includes 10

easily distinguished from Rossbeevera by the basidiospores withspecies from East Asia to Australasia, and is characterized by

6–9 longitudinal ridges and a cushion-shaped, colorless sterile

ellipsoid basidiospores with 3–5 longitudinally ridges and bluishbase.

discoloration of the fruitbody surface (Orihara et al., 2016).
© The Japanese Association for Truffle Science (JATS), 2018

Shortly after the proposal of the genus Rossbeevera by Lebel
5

Truffology - Volume 1, Issue 1, 2018

Orihara: Rossbeevera yunnanensis from Japanet al. (2012), Orihara et al. (2012a) described an additionalwith Sequence Scanner v. 1.0 (Applied Biosystems, Foster City,species, R. yunnanensis Orihara & M. E. Sm. based on a single

California, USA), BioEdit v. 7.0.9 (Hall, 1999) and SeaView v.

specimen collected from Mt. Zixi, Chuxiong Yi Autonomous

4 (Galtier et al., 1996). The ITS and 28S rDNA sequences werePrefecture, Yunnan Province, China. Rossbeevera yunnanensis

deposited in the International Nucleotide Sequence Databases

forms the earliest diverging lineage of Rossbeevera (Orihara(INSD) under the accession numbers MF357925 and MF354015,

et al., 2012a, 2016), and is considered an important species torespectively. Nucleotide sequence similarity was examinedclarify the phylogeography of Rossbeevera and Turmalinea. In

using NCBI BLAST searches (https://blast.ncbi.nlm.nih.gov/

the course of field surveys in Japan, I unexpectedly collected

BlastAlign.cgi).

fruitbodies of R. yunnanensis in Hiroshima Prefecture, Japan.
Here I give a short description and accompanying information

Results

on the specimen.

Rossbeevera yunnanensis Orihara & M.E. Sm., Mycotaxon

Materials and methods
Taxon sampling and morphological observation
Fruitbodies were collected during the fungal foray in

120: 141.
Fig. 1.
Specimen examined: JAPAN, Hiroshima Prefecture, Hatsukaichi
City, Mominoki Forest Park, subhypogeous under Quercus

Hiroshima Prefecture held by Mycological Society of Japan

serrata Murray, 28 Sep. 2013, T. Orihara, KPM-NC 23352.

during 27–28 Sep. 2013. After DNA extraction, the fruitbodiesINSD (GenBank) ID of the nucleotide sequences: ITS region ofwere air-dried for later examination. The specimen is depositedrDNA: MF357925; 28S rDNA: MF354015.

in Kanagawa Prefectural Museum of Natural History (KPM),

Japanese name: Unnan-tsuchidama-take ( “Unnan” = Yunnan;

Japan. For microscopy, hand-cut sections were mounted in water,“tsuchidama-take” = the Japanese name of Rossbeevera).

3% KOH, lacto-glycerol, or 1% phloxine B aqueous solution.
Basidiospore dimensions (e.g., range of spore length × spore

Morphological and molecular comparison with the isotype

width, length of hilar appendages) and their standard deviations

specimen(SD) were determined based on 30 measurements. The 95%

Two fruitbodies were collected in deciduous Quercus

prediction intervals of basidiospore diameter are shown without

forest in a temperate, low mountain area. The specimen

parentheses in taxonomic descriptions. Both endpoints of thewas morphologically identical to the original description of

spore dimensions are shown in parentheses. Two additional

Rossbeevera yunnanensis provided by Orihara et al. (2012a).

spore features are shown; the length to width ratio (Q) andThe fruitbodies were 8 mm and 9.5 mm in diam., and thethe hilar appendage to spore length ratio (HA/S; Orihara et al.,surface quickly turned light blue or blue-green when touched.

2012a). Measurements include the hilar appendage but not spore

The peridium was smooth, and extremely thin or almost absent

ornamentation or the pedicel.

in some parts of the fruitbodies. While the holotype specimen
was only partially mature and the gleba was mostly off-white toDNA extraction, PCR amplification and sequencing

beige, one of the fruitbodies of the Japanese specimen was fully

DNA was extracted using the FTA Classic Card or Indicating

mature, and the gleba was blackish brown. The basidiospores

FTA Cards (Whatman International Ltd, Maidstone, England)

were 17.1– (17.8–) 22.9 (–25) × (7.4–) 7.5– (9.1) –9.2 µm (SD:based on the manufacturer's protocol for plants (www.whatman.

1.41 (length), 0.41 (width)), mean 20 × 8.3 µm in diam., Q =com/References/WGI_1397_PlantPoster_V6.pdf). PCR

2.1–2.9 (mean Q = 2.4), fusoid to fusiform, brown at maturity,

ampliﬁcation of the internal transcribed spacer region (ITS) andwith 3–5 longitudinal ridges up to 1.5 µm high and a basal hilar

28S (large subunit; LSU) of the nuclear rRNA gene (rDNA)

appendage 2–4.8 µm long (HA/S = 0.1–0.2, mean HA/S = 0.15).

followed the protocol in Orihara et al. (2012b). PCR primers

The ITS and 28S rDNA sequences of the Japanese specimen

were ITS1F (Gardes & Bruns, 1993) and ITS4 (White et al.,

(KPM-NC 23352) obtained for the study were 791 bp and 923

1990) for the ITS region, and LR0R and LR5 (Vilgalys & Hester,bp, respectively. The nucleotide similarity of ITS sequence1990) for 28S rDNA. Cycle sequencing of the PCR products

between the Japanese specimen and the holotype was 99.6% (738in forward and reverse directions were completed according tobp /741 bp). The similarity of 28S between the two specimens

Orihara et al. (2012b). Sequences were edited and assembled

was 99.9% (813 bp /814 bp).

6

© The Japanese Association for Truffle Science (JATS), 2018

Truffology - Volume 1, Issue 1, 2018

Orihara: Rossbeevera yunnanensis from Japan

Fig. 1. Rossbeevera yunnanensis collected from Hiroshima Prefecture, Japan (KPM-NC 23352): a. Basidiomata (fruitbodies); b. Trama, hymenia andbasidiospores (stained with 1% phloxine aqueous solution); c. Basidiospores mounted in lactoglycerol; d. Peridium and trama (stained with 1%phloxine aqueous solution). Bars: a = 1 cm; b = 50 µm; c, d = 20 µm.
図 1． ウンナンツチダマタケ Rossbeevera yunnanensis （広島県産標本；KPM-NC 23352）： a. 子実体； b. グレバ基層、子実層および担子胞子 （1%フロキシン水溶液により染色） ； c. 担子胞子 （ラクトグリセロールにより封入） ； d. 外皮およびグレバ基層 （1% フロキシン水溶液により染色）．
スケール ： a = 1 cm; b = 50 µm; c, d = 20 µm.

Discussion

substitution in the 28S rDNA. These ﬁndings suggest that theyThis is the first report of R. yunnanensis from Japan andspread across Asia relatively recently, despite the fact that those

only the second known specimen of the species in the world

two collection localities are far apart from each other (i.e.,(Orihara et al., 2012a). The species is characteristic in the genus

Chuxiong, Yunnan Province, China vs. Hiroshima Prefecture,

Rossbeevera in the remarkably thin peridium that turns light blueJapan; ca. 3,150 km in distance). Although it is generally

or blue green, and the large, fusoid to fusiform basidiospores

believed that sequestrate, truffle-like basidiomycetes rely on(average dimension: 20.1 × 7.8 µm; Orihara et al., 2012a). The

mycophagy by insects or mammals for spore dispersal (Claridge

overall morphology of the Japanese specimen is almost identical& May, 1994; Fogel & Peck, 1975; Maser et al., 1978), theto the Chinese holotype specimen. This confirms the accuracy

results suggest that R. yunnanensis may be able to disperse itsof the description by Orihara et al. (2012a) despite the fact

spores over long distances.

that it was based only on a single specimen. The basidiospore
dimension of the Japanese specimen was slightly wider thanAcknowledgementsthat of the holotype. This is probably due to the difference of theI am grateful to Dr. Matthew E. Smith for critically reading

degree of maturity noted above.

the manuscript and for giving me valuable suggestions. I thankNucleotide comparison using BLAST searches showed thatMs. Suyama-Watanabe Mai for supporting the molecular

there were only three nucleotide substitutions in the ITS region

experiments for this study. This study was supported partiallybetween the Chinese and Japanese specimens, and only one

by JSPS KAKENHI Grant-in-Aid for Young Scientists (B)© The Japanese Association for Truffle Science (JATS), 20187

Truffology - Volume 1, Issue 1, 2018

Orihara: Rossbeevera yunnanensis from Japan(no. 17K15184) and the 27th Pro Natura Fund from Pro Naturainterrelationships with emphasis on Oregon coniferous forests.

Foundation Japan (PNFJ).

Ecology 59: 799–809.

References
Claridge A.W., May T.W. (1994) Mycophagy among Australianmammals. Australian Journal of Ecology 19: 251–275.

Orihara T., Smith M.E., Ge Z.-W., Maekawa N. (2012a) Rossbeevera
yunnanensis (Boletaceae, Boletales), a new sequestrate species
from southern China. Mycotaxon 120: 139–147.
Orihara T., Smith M.E., Shimomura N., Iwase K., Maekawa N. (2012b)

Fogel R., Peck S.B. (1975) Ecological studies of hypogeous fungi. I.

Diversity and systematics of the sequestrate genus Octaviania inColeoptera associated with sporocarps. Mycologia 67: 741–747.

Japan: two new subgenera and eleven new species. Persoonia 28:Hall T.A. (1999) BioEdit: a user-friendly biological sequencealignment editor and analysis program for Windows 96/98/NT.
Nucleic Acids Symposium Series 41: 95–98.

85–112.
Orihara T., Lebel T., Ge Z.-W., Smith M.E., Maekawa N. (2016)
Evolutionary history of the sequestrate genus Rossbeevera (Boletaceae)

Galtier N., Gouy M., Gautier C. (1996) SEAVIEW and PHYLO_reveals a new genus Turmalinea and highlights the utility of ITS

WIN: Two graphic tools for sequence alignment and molecularminisatellite-like insertions for molecular identification. Persoonia

phylogeny. Computer Applications in the Biosciences 12: 543–548.

37: 173–198.

Gardes M., Bruns T.D. (1993) ITS primers with enhanced speciﬁcity

Vilgalys R., Hester M. (1990) Rapid genetic identification andfor basidiomycetes: application to the identiﬁcation of mycorrhizae

mapping of enzymatically ampliﬁed ribosomal DNA from severaland rusts. Molecular Ecology 2: 113–118.

Cryptococcus species. Journal of Bacteriology 172: 4238–4246.

Lebel T., Orihara T., Maekawa N. (2012) The sequestrate genus

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

Rosbeeva T.Lebel & Orihara gen. nov. (Boletaceae) from Australasia

sequencing of fungal ribosomal RNA genes for phylogenetics.

and Japan: new species and new combinations. Fungal Diversity 52:

In: Innis M.A., Gelfand D.H., Sninsky J.J., White T.J. (eds.) PCR

49–71+73.

protocols: a guide to methods and applications. Academic Press,

Maser C., Trappe J.M., Nussbaum R.A. (1978) Fungal-small mammal

8USA, pp. 315–322.

© The Japanese Association for Truffle Science (JATS), 2018