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

Truffology 4 (1): 1–7 (2021)

Original peer-reviewed article （原著論文 ； 査読有）Balsamia oblonga (Helvellaceae), a new species from a subalpine
forest in Japan
日本の亜高山帯において採集された新種ナガミノツチレイシタケ
Balsamia oblonga （ノボリリュウ科）
Kohei Yamamoto1*, Naoki Endo2, Muneyuki Ohmae3, Takamichi Orihara4山本 航平 1*, 遠藤 直樹 2, 大前 宗之 3, 折原 貴道 4
1

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

2

Fungus/Mushroom Resource and Research Center, Faculty of Agriculture, Tottori University, 4-101 Koyama, Tottori
680-8553, Japan
鳥取大学農学部附属菌類きのこ遺伝資源研究センター， 〒 680-8553 鳥取県鳥取市湖山町南 4 丁目 101

3

Hokken Co. Ltd., 7-3 Ekihigashimachi, Mibu-machi, Shimotsuga-gun, Tochigi 321-0222, Japan
株式会社北研，〒 321-0222 栃木県下都賀郡壬生町駅東町 7-3

4

Kanagawa Prefectural Museum of Natural History, 499 Iryuda, Odawara-shi, 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* Corresponding author （主著者）
E-mail: kohei081@yahoo.co.jp

Abstract
Balsamia is a member of the family Helvellaceae, and all but one of the 25 species form subterranean truﬄe-likeascomata. This genus is broadly distributed across the Northern Hemisphere, i.e., Europe, North America, and Asia
(China). However, this genus had not been found in Japan. In September 2013, an unidentified truﬄe-like fungus
was collected in a subalpine forest in Gunma Prefecture, Japan. Morphological observations and molecularphylogenetic analyses revealed that this fungus belongs to the genus Balsamia. Here, we describe the new
species B. oblonga. This species is characterized by large, oblong ascospores (up to 33.5 µm) with a large lengthto-width ratio (up to 3.3). Phylogenetic analyses resolved this species as a distinct species-level branch.

要旨
Balsamia はノボリリュウ科に属する子嚢菌類で、 全 25 種の内 1 種を除くすべてが地中性のトリュフ型の子実体を形成する。
これまでにヨーロッパ、 北米および中国における分布が報じられてきたが、 日本からは未発見であった。 2013 年 9 月に群馬県の亜高山帯において採集された未同定菌について、 形態観察と分子系統解析を行った結果、 本邦初となる Balsamia
属種であることが明らかになった。 本属既知種と比較した結果、 子嚢胞子が大型の長楕円形 （長径最大 33.5 µm） で、縦横比（最大 3.3）が大きい点でいずれにも合致せず、系統学的にも独立種であることが支持された。 以上の結果に基づき、本種を B. oblonga として記載した。 本属の和名は、 褐色で突起に覆われる外皮と白色の基本体から構成される子実体が、植物のレイシの果実 （ライチ） に類似することから、 ツチレイシタケ属とする。 また、 B. oblonga の和名は、 上記の子嚢胞子の特徴に基づきナガミノツチレイシタケとする。
Article Info: Submitted: 15 December 2020
© The Japanese Association for Truffle Science (JATS), 2021

Accepted: 1 March 2021

Published: 31 March 2021

1

Truffology - Volume 4, Issue 1, 2021

Yamamoto et al.: Balsamia oblonga, a new species from Japan

Introduction

observations and molecular phylogenetic analyses, this fungus was

The genus Balsamia (Helvellaceae) is characterized by brown to

identified as a new species of Balsamia. Here, we describe thisblack sequestrate ascomata covered by warts, ovoid, eight-spored

species as Bal. oblonga.

asci, and distinct ellipsoid to cylindrical ascospores (Southworth et al.,2018). This genus is considered ectomycorrhizal (EM) (Tedersoo &Materials and methods

Smith, 2013) based on the observation of EM roots formed between

Morphological observations

Bal. platyspora Berk. and Tilia (Ceruti & Bussetti, 1962) and between

Specimen was collected in Katashina-mura, Gunma PrefectureBal. alba Harkn. and Pinus (Agerer & Rambold, 2004–2020), andin September 2013. Specimen was photographed and oven-dried at

isotopic measurements of the ascoma of Bal. magnata Harkn. (Hobbie

60°C. For light microscopy, hand-cut sections of the dried specimen

et al., 2001).

were reconstituted in water for 24 h and then mounted in water or 5%

Balsamia was described based on a truffle-like species, Bal.

KOH. Melzer’s reagent was used to test the amyloid reaction of thevulgaris Vittad. (Vittadini, 1831), and currently includes 25 species.

asci and ascospores. All measurements were made using PhotoRuler

By the 1900s, Bal. ambigua Petitb., Bal. fusispora Schulzer, Bal.

version 1.1.3 (http://inocybe.info/). Q value was obtained from theplatyspora (= Bal. fragiformis Tul. & C. Tul.), and Bal. polysperma

length-to-width ratio of mature ascospores. The specimen is deposited

Vittad. from Europe and Bal. alba, Bal. filamentosa Harkn., Bal.

in the herbarium of the Kanagawa Prefectural Museum of Natural

magnata, and Bal. nigrans Harkn. from western North America

History (KPM) and National Museum of Nature and Science, Tokyo

had been described (Tulasne & Tulasne, 1843; Berkeley, 1844;(TNS) in Japan.

Schulzer, 1870; Harkness, 1899; Petitberghien, 1966). More recently,
Southworth et al. (2018) described Bal. cascadensis D. Southw.,

DNA sequencing and phylogenetic analyses

J.L. Frank & Castellano, Bal. latispora D. Southw., J.L. Frank &DNA was extracted as described by Izumitsu et al. (2012) with

Castellano, Bal. lazyana Trappe, D. Southw. & Amaranthus, Bal.

minor modifications (Yamamoto et al., 2019). PCR amplification

limuwensis D. Southw., J.L. Frank & Castellano, Bal. pallida D.

of the internal transcribed spacer (ITS) region and the large

Southw., J.L. Frank & Castellano, Bal. quiercicola D. Southw.,

subunit (LSU) of nuclear ribosomal DNA was performed using

M.E. Sm. & J.L. Frank, and Bal. trappei D. Southw., J.L. Frankthe primer pair ITS1F (Gardes & Bruns, 1993) and LR5 (Vilgalys& Castellano from western North America, and Bal. alba, Bal.

& Hester, 1990). PCR was performed in a total volume of 25 μl

ﬁlamentosa, Bal. magnata, Bal. nigrans, and Bal. setchellii (E. Fisch.)containing 5 µL dNTPs (0.4 mM each), 0.75 µL each primer (0.3 µM

Trappe, D. Southw. & Castellano (=Pseudobalsamia setchellii E.

each), 12.5 µL 2× PCR buffer for KOD FX Neo (Toyobo, Osaka,

Fisch.) were emended. Hansen et al. (2019) expanded the definition

Japan), 0.5 µL 1.0 U μL−1 KOD FX Neo (Toyobo), 1.25 µL templateof Balsamia based on morphological and phylogenetic studies,

DNA, and 4.25 µL sterile distilled water. The PCR conditions weretransferring a cup-shaped epigeous fungus, Acetabula aestivalis

94 °C for 2 min and 35 cycles of 98 °C for 10 s, 58 °C for 30 s, and

R. Heim & L. Rémy, to Balsamia and synonymizing the genus

68 °C for 45 s. The PCR products were purified using illustra™

Barssia. They also transferred four Mediterranean Barssia species

ExoProStar™ (GE Healthcare, Buckinghamshire, UK) and sent to(i.e., Bar. gunerii H.H. Doğan, Bozok & Taşkın, Bar. hellenica

Macrogen Japan (Tokyo, Japan) for sequencing using four primers:

Kaounas, Agnello, P. Alvarado & Slavova, and Bar. maroccana G.

ITS1F, ITS4 (White et al., 1990), LR0R (Vilgalys & Hester, 1990),

Moreno, Manjón, Carlavilla & P. Alvarado) and North America (Bar.

and LR5. The resulting bidirectional sequences were edited using

oregonensis Gilkey) to Balsamia.

4Peaks 1.8 (http://nucleobytes.com/4peaks) and assembled using

By contrast, in Asia, Bal. platyspora was reported from China only

MEGA X (Kumar et al., 2018). Newly generated sequences wereonce (Liu & Tao, 1990), and this genus had not been found in Japan.

deposited in the DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp)

In recent years, three new species of Balsamia (Bal. guozigouensis (L.

under accession numbers LC600234 (ITS) and LC600235 (LSU).

Fan & Y.Y. Xu) L. Fan & Y.Y. Xu, Bal. lishanensis L. Fan & Y.Y. Xu,

For phylogenetic analyses, 102 ITS sequences and 34 LSU

and Bal. luyashanensis (L. Fan & Y.Y. Xu) L. Fan & Y.Y. Xu) weresequences from Balsamia spp. were retrieved from NCBI (https://

described from China (Xu et al., 2018, 2020), and the diversity of

www.ncbi.nlm.nih.gov/nucleotide) and included in the dataset.

this genus in East Asia is becoming more clearly known. During ourWynnella subalpina Q. Zhao, Zhu L. Yang & K.D. Hyde and Helvellataxonomic study of sequestrate fungi from subalpine forest in Japan,

dryophila Vellinga & N.H. Nguyen (Helvellaceae) were selected as

an unidentified ascomycete was examined. Based on morphologicaloutgroups. The sequences were aligned following Yamamoto et al.

2© The Japanese Association for Truffle Science (JATS), 2021

Truffology - Volume 4, Issue 1, 2021

Yamamoto et al.: Balsamia oblonga, a new species from Japan(2020). Ambiguously aligned sites were removed using the Gblocks

Results

server (http://molevol.cmima.csic.es/castresana/Gblocks_server.

An ascoma was collected from a subalpine forest dominated byhtml) under the least stringent setting. Topological conflicts between

Abies and Betula which were potential host trees. The macroscopic

the two trees (ITS, 724 bp; LSU, 839 bp) were checked directly bycharacters, i.e., a sequestrate, reddish-brown ascoma without folding

topological comparison of the preliminary maximum likelihood (ML)and apical depression, and opened glebal chambers (Fig. 1), weretrees. Next, the two datasets were combined into a single 1563 bp

similar to those of Bal. ambigua, Bal. lazyana, Bal. lishanensis, Bal.

dataset and deposited in TreeBASE (accession URL: http://purl.org/

platyspora, Bal. polysperma, Bal. trappei, and Bal. vulgaris (Hawker,

phylo/treebase/phylows/study/TB2:S27402). ML analysis of the

1954; Montecchi & Sarasini, 2000; Southworth et al., 2018; Xu et

combined dataset was conducted based on Yamamoto et al. (2020)

al., 2020). However, the ascospores were narrower than those of Bal.

using raxmlGUI 1.31 (Silvestro & Michalak, 2012) under a generalvulgaris and longer than those of the remaining species (Fig. 2).

time reversible model of nucleotide substitution, with a discrete

ITS (1093 bp) and LSU (869 bp) sequences were obtained fromgamma distribution (+G) and invariant sites (+I), selected by MEGA

the specimen. A NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.

X. A maximum parsimony (MP) analysis was conducted based on

cgi?PAGE_TYPE=BlastSearch) search showed that ITS sequence

Yamamoto et al. (2020).

from the Japanese specimen was most similar to that of Balsamia
spp. and had the highest homology (88% [640 / 726]) with that ofFig. 1. Balsamia oblonga (KPM-NC 28613) collected from Gunma, Japan. (A) Ascoma showing surface (left) and gleba (right). (B) Surface view of the peridium.
(C) Peridium (upper) and gleba (lower) mounted in 5% KOH. (D) Peridium mounted in 5% KOH. (E–F) Asci mounted in water. (G) Ascospores mounted inwater. Bars: A = 1 cm; B = 500 μm; C = 100 μm; D = 50 μm; E–G = 30 μm.
図 1． 群馬県産 Balsamia oblonga（KPM-NC 28613）． A：子実体． 右は断面． B：外皮表面． C：外皮（上部）および子実層（下部）の断面（5% KOH 封入）．
D ： 外皮 （5% KOH 封入）． E–F ： 子嚢 （水封）． G ： 子嚢胞子 （水封）． スケール ： A = 1 cm; B = 500 μm; C = 100 μm; D = 50 μm; E–G = 30 μm．
© The Japanese Association for Truffle Science (JATS), 2021

3

Truffology - Volume 4, Issue 1, 2021

Yamamoto et al.: Balsamia oblonga, a new species from Japan
Bal. lishanensis (MT232904). Fig. 3 shows the ML phylogeny ofDiagnosis: This species is distinguishable from other Balsamia spp.

the combined ITS and LSU dataset (ln L = −10449.792713). The

by its long, slender ascospores.

MP statistics were as follows: tree length = 1723, consistency index

Etymology: From the Latin oblongus = oblong, referring to the= 0.564, retention index = 0.896, and composite index = 0.552. The

oblong ascospore of this species.

genus Balsamia formed a well-supported clade, and monophyly

Type: JAPAN, Gunma Prefecture, Katashina-mura, a mountain trailof each species in the genus was well supported, except for Bal.

on Mt. Nikko-Shirane, semi-hypogeous under a subalpine forest

platyspora and Bal. vulgaris. The sequence from the Japanese

dominated by Abies veitchii Lindl., A. mariesii Mast., and Betulaspecimen belonged to the clade composed of the six abovementioned

ermanii Cham., 27 Sep. 2013, N. Endo (holotype, KPM-NC 28613;

species (except for Bal. ambigua) and environmental sequences fromisotype, TNS-F-82005).

uncultured EM (Bootstrap support: 96 / - ), and the specimen was

Description: Ascoma ptychothecium, closed, spheroid, 18 × 17 mm;formed a sister lineage to Bal. lishanensis.

surface brick-red, warty. Gleba chambered, white; chambers open,
irregular, up to 0.8 mm wide. Odor not recorded. Peridium brown

Taxonomy

to pale brown in 5% KOH, 100–240 μm thick; outer layer strongly

Balsamia oblonga Koh. Yamam., N. Endo, Ohmae & Orihara,

pigmented, textura globulosa, 50–165 μm thick, composed of 4 tosp. nov.

5 layers of cells 10–42 μm wide, wall up to 8 μm thick; inner layer[MycoBank ID: MB 838245]

textura angularis, 62–88 μm thick, cells 9–21 μm wide, wall up to・

Fig. 1.

25

25

20

20

Z

Width

Width (μm)

G

15

2 μm thick. Glebal tissue of textura intricata; composed of hyphae

P

N

I
H

C

15

T

R

M

U

J

W

D

10

10

5

5

L O

Y

Q

B

V

E
S
X

A

K

F

2.5
17.5

0

20

25

30

35

37.5

Length (μm)

152. Measurements of20
25 species in Balsamia
30 and Barssia yezo-montana.
35
40B =
Fig.
the ascospores of sequestrate
A = Bal. oblonga;

Length F = Bal. fusispora; G = Bal. gunerii; H = Bal. guozigouensis;
Bal. alba; C = Bal. ambigua; D = Bal. cascadensis; E = Bal. filamentosa;
I = Bal. hellenica; J = Bal. latispora; K = Bal. lazyana; L = Bal. limuwensis; M = Bal. lishanensis; N = Bal. luyashanensis; O = Bal.
magnata; P = Bal. maroccana; Q = Bal. nigrans; R = Bal. oregonensis; S = Bal. pallida; T = Bal. platyspora; U = Bal. polysperma;

V = Bal. quercicola; W = Bal. setchellii; X = Bal. trappei; Y = Bal. vulgaris; Z = Bar. yezo-montana. Sources of ascospore size:
Southworth et al. (2018) for B, D, E, J–L, O, Q, S, V–X; Petitberghien (1966) for C; Schulzer (1870) for F; Doğan et al. (2018) for G;
Xu et al. (2018) for H and N; Kaounas et al. (2015) for I; Xu et al. (2020) for M; Crous et al. (2014) for P; Gilkey (1939) for R; Pegler
et al. (1993) for T and Y; Montecchi & Sarasini (2000) for U; Kobayasi (1937) for Z. Circles, mean values; squares, median values.
図 2． Balsamia に属す地下生菌およびツチクレタケ （Barssia yezo-montana） の子嚢胞子サイズの比較． 縦軸と横軸はそれぞれ幅と長さを示す． 円および四角の位置はそれぞれ，幅と長さの平均値および中間値を示す． 各アルファベットに対応する種名およびサイズの出典は英文キャプションを参照．

4© The Japanese Association for Truffle Science (JATS), 2021

Truffology - Volume 4, Issue 1, 2021

Yamamoto et al.: Balsamia oblonga, a new species from JapanUSA	
  (KF983485/-­‐)	

70/66	 USA	  (KY706379/-­‐)	
93/91	 USA	  (KU170035/-­‐)	
99/96	 USA	  (KU170034/-­‐)	USA	
  (KU170032/-­‐)	

100/99	 USA	
  (KY706378/-­‐)	USA	
  (KY706377/-­‐)	USA	
  (KY706380/-­‐)	

75/-	
 USA	
  (KF983487/KP859283)	
  Holotype	
87/-	
 USA	
  (MK991863/-­‐)	
59/-	

B.	
  la<spora	USA	  (KF983486/-­‐)	USA	
  (KU170037/-­‐)	USA	
  (KU170036/-­‐)	

51/-	
 USA	
  (KU170033/-­‐)	USA	
  (MF098665/-­‐)	

63/87	USA	
  (DQ974730/JQ925658)	
  Epitype	
100/100	
 USA	
  (KU170028/-­‐)	B.	
  setchellii	USA	
  (KU170030/-­‐)	USA	
  (KP859277/KT968654)	
USA	
  (KU170027/-­‐)	
  Holotype	B.	
  limuwensis	
USA	
  (KU170029/-­‐)	USA	
  (KY706383/-­‐)	
100/100	
USA	
  (KY706386/-­‐)	B.	
  ﬁlamentosa	USA	
  (KM115880/-­‐)	USA	
  (KY706388/-­‐)	  Epitype	
54/56	USA	
  (KF983483/-­‐)	
  Holotype	B.	
  cascadensis	
USA	
  (JN022501/-­‐)	
91/98	USA	  (KY706382/-­‐)	  Epitype	
97/92	USA	  (KY706381/-­‐)	
77/74	USA	  (AY558742/-­‐)	USA	
  (AY558741/-­‐)	
88/83	USA	  (KY706376/-­‐)	
86/85	B.	
  alba	USA	
  (KY706387/-­‐)	
60/82	USA	  (KY706385/-­‐)	
USA	
  
(
KF983482/-­‐)	
93/89	
51/94	
 86/80	USA	
  (KY706390/-­‐)	USA	
  (KY706384/-­‐)	USA	
  (KU170039/-­‐)	
84/95	USA	
  (EU669383/EU669425)	
  Epitype	USA	
  (MT156518/-­‐)	B.	
  nigrans	
100/90	USA	
  (KP859264/KP859282)	
98/95	 USA	  (FJ789590/-­‐)	USA	
  (KF983481/-­‐)	USA	
  (KF983480/-­‐)	USA	
  (KU170038/-­‐)	USA	
  (KF983479/-­‐)	B.	
  magnata	USA	
  (KY706389/-­‐)	
100/100	
USA	
  (KF983477/-­‐)	USA	
  (KF983478/-­‐)	  Epitype	
100/100	
 USA	
  (KF983491/JQ925659)	
  Holotype	USA	
  (KF983490/-­‐)	B.	
  quercicola	
65/66	 USA	  (DQ453695/-­‐)	
62/-	
USA	
  (KU170040/-­‐)	B.	
  pallida	
China	  (MT232904/MT232909)	
98/99	 China	  (MT232905/MT232914)	
100/100	
China	  (MT232907/MT232902)	
  Holotype	
56/-	
 China	  (MT232899/MT232911)	
100/100	
 China	  (MT232901/MT232913)	
B.	
  lishanensis	
China	  (MT232900/MT232910)	
95/99	
76/92	 China	  (MT232721/MT232903)	
China	  (MT232908/MT232912)	
Japan	  (LC600234/LC600235)	
  Holotype	B.	
  oblonga	
Italy	
  (KM243650/KM243656)	
B.	
  polysperma	
UEM	
  of	
  Tilia	
  cordata	
  Russia	
  (MT554399/-­‐)	94/-	
 Norway	
  (KP149494/-­‐)	
Norway	
  (KP149498/-­‐)	
54/93	
Norway	
  (KP149497/-­‐)	
62/-	
UEM	
  Italy	
  (JX474857/-­‐)	
52/-	
B.	
  platyspora	
  lineage	
  1	
UEM	
  Italy	
  (JX474850/-­‐)	UEM	
  Italy	
  (JX474829/-­‐)	
70/-	
UEM	
  
I
taly	
  
(
JX474845/-­‐)	
69/89	
91/88	UEM	
  Italy	
  (JX474847/-­‐)	
77/97	
China	  (MT232906/MT229143)	
98/99	
B.	
  platyspora	
  Norway	
  (KP149493/-­‐)	lineage	
  2	
97/97	 UEM	  of	
  Alnus	
  sp.	
  France	
  (JX989935/-­‐)	96/-	
100/100	
Italy	
  (KM243646/-­‐)	
Italy	
  (KM243647/KM243653)	
 B.	
  vulgaris	
  lineage	  1	
100/98	
 Italy	  (KM243645/KM243651)	
Cyprus	
  (KM115881/-­‐)	B.	
  vulgaris	
  lineage	
  2	
50/-	
UEM	
  of	
  Dryas	
  integrifolia	
  Canada	  (JX630958/-­‐)	USA	
  (KM115879/-­‐)	
  Holotype	B.	
  lazyana	
100/99	 Canada	
  (KU170041/-­‐)	B.	
  trappei	USA	
  (KU170042/-­‐)	
  Holotype	
100/100	
USA	
  (AY558743/-­‐)	USA	
  (MK991852/-­‐)	B.	
  oregonensis	
USA	
  (KF983489/-­‐)	
China	  (MH910334/MH910325)	
97/100	
 China	  (MH910335/MH910326)	
B.	
  luyashanensis	
56/53	
China	  (MH910333/MH910324)	
  Holotype	
China	  (MH910340/MH910331)	
80/99	
 69/87	
 China	  (MH910336/MH910327)	
China	  (MH910337/MH910328)	
  Holotype	
100/100	
China	  (MH910339/MH910330)	
B.	
  guozigouensis	
China	  (MH910338/MH910329)	
China	  (MH910341/MH910332)	
50/-	
99/100	
 Greece	  (KT350941/KT350940)	
  Holotype	B.	
  hellenica	
Greece	  (KT350942/KT350939)	
Morocco	
  (KM243649/KM243655)	
  Holotype	
91/99	
Morocco	
  (JN048885/JN048873)	
B.	
  maroccana	
100/100	
Morocco	
  (KM243648/KM243654)	
100/100	
 Turkey	  (MF619955/MF619953)	
  Holotype	B.	
  gunerii	
Turkey	  (MF619952/MF619957)	
Norway	  (AF046225/-­‐)	B.	
  aes<valis	
Wynnella	
  subalpina	
  China	
  (KX034101/KT581118)	
91/99	-/85	

100/97	Helvella	
  dryophila	
  USA	  (KC122811/KC122772)	
0.2 substitutions/site	

Fig. 3. Maximum likelihood (ML) phylogenetic tree of the combined dataset of internal transcribed spacer (ITS) and large subunit (LSU) sequencesof Balsamia. Wynnella subalpina and Helvella dryophila were used as outgroups. GenBank accession numbers for ITS (left) and LSU (right) are
shown in parentheses. Bootstrap (BS) values (1,000 replicates) > 50% from ML (left) and maximum parsimony (MP) (right) analyses are shown near
the nodes. Branches supported by BS values ≥ 70 % from both ML and MP analyses are depicted as thick lines. UEM = uncultured ectomycorrhiza.
図 3． Balsamia oblonga および Balsamia に属すその他の種の系統関係を示した， ITS および LSU の結合データセットに基づく最尤法 （ML）
系統樹． 外群には Wynnella subalpina ならびに Helvella dryophila を用いた． ITS （左） および LSU （右） の GenBank アクセッション番号を括弧内に示した． ノード付近に， 最尤法 （左） ならびに最節約法 （MP） （右） のブートストラップ値 （BS） を “MLBS/MPBS” として表示した．
MLBS ≥ 70 % かつ MPBS ≥ 70% で支持された分岐を太線で示した． UEM = 外生菌根由来の環境配列．
© The Japanese Association for Truffle Science (JATS), 2021

5

Truffology - Volume 4, Issue 1, 2021

Yamamoto et al.: Balsamia oblonga, a new species from Japanhyaline, septate, 3.5–10.5 μm broad, thin-walled. Paraphyses obscure

Ceruti A., Bussetti L. (1962) Sulla simbiosi micorrizica tra tigli e Boletus

possibly due to maturation. Asci irregularly distributed, with 8

subtomentosus, Russula grisea, Balsamia platyspora e Hysterangium

spores, hyaline, ellipsoid to citriform, 64–90.5 × 32.5–45 μm(excluding stipe) in water, thin-walled; stipe arising from crozier,up to 23.5 μm long, 6.5 μm broad; not reacting in Melzer’s reagent
with pretreatment in 5% KOH. Ascospores oblong, hyaline, smooth,
24.5–33.5 × 9.5–13 μm (mean, 29.4 × 11.3 μm; n = 81), Q = 2.3–3.3(mean, 2.6; n = 81) in water, thin-walled; containing 1–3 (mostly
single) large central oil droplets; not reacting in Melzer’s reagent
with pretreatment in 5% KOH.

Discussion

clathroides. Allionia 8: 55–66.
Crous P.W., Wingfield M.J., Schumacher R.K., Summerell B.A., GiraldoA., Gené J., Guarro J., Wanasinghe D.N., Hyde K.D., Camporesi E.,
Gareth Jones E.B., Thambugala K.M., Malysheva E.F., Malysheva
V.F., Acharya K., Álvarez J., Alvarado P., Assefa A., Barnes C.W.,
Bartlett J.S., Blanchette R.A., Burgess T.I., Carlavilla J.R., Coetzee
M.P., Damm U., Decock C.A., den Breeÿen A., de Vries B., Dutta
A.K., Holdom D.G., Rooney-Latham S., Manjón J.L., Marincowitz
S., Mirabolfathy M., Moreno G., Nakashima C., Papizadeh M.,
Shahzadeh Fazeli S. A., Amoozegar M.A., Romberg M.K., Shivas R.G.,
Stalpers J.A., Stielow B., Stukely M.J., Swart W.J., Tan Y.P., van der

The genus Balsamia, excluding the earliest diverging apothecial

Bank M., Wood A.R., Zhang Y., Groenewald J.Z. (2014) Fungal Planet

species Bal. aestivaris K. Hansen, Skrede & T. Schumach.and species

description sheets: 281–319. Persoonia 33: 212–289.

formerly classified as Barssia, is largely divided into two majorDoğan H.H., Bozok F., Taşkın H. (2018) A new species of Barssia

clades (Fig. 3). One comprises species distributed in Eurasia (Bal.

(Ascomycota, Helvellaceae) from Turkey. Turkish Journal of Botanylishanensis, Bal. oblonga, Bal. platyspora, Bal. polysperma, andBal. vulgaris) and North America (Bal. lazyana and Bal. trappei).
The species in this clade are characterized by reddish ascomata andopened glebal chambers. The other clade is composed of 10 North
American species, which implies that significant speciation of
Balsamia occurred within western North America.
An enigmatic Japanese species, Barssia yezo-montana (Kobayasi)

42: 636–643.
Gardes M., Bruns T.D. (1993) ITS primers with enhanced specificity forbasidiomycetes: application to the identification of mycorrhizae andrusts. Molecular Ecology 2: 113–118.
Gilkey H.M. (1939) Tuberales of North America. Oregon State
Monographs: Studies in Botany 1: 1–63.
Gilkey H.M. (1961) New species and revisions in the order Tuberales.
Mycologia 53: 215–220.

Trappe (= Phymatomyces yezo-montanus) is known only from the

Hansen K., Schumacher T., Skrede I., Huhtinen S., Wang X.-H. (2019)

type specimen collected in Hokkaido, Japan (Kobayasi, 1937).

Pindara revisited – evolution and generic limits in Helvellaceae.

Furthermore, the holotype of this species seemed to have been lost

Persoonia 42: 186–204.

(Gilkey, 1961). Recently, Hansen et al. (2019) synonymized Barssia

Harkness H.W. (1899) Californian hypogæous fungi. Proceedings of thewith Balsamia, but they retained the classification of Bar. yezo-

California Academy of Science, Third series, Botany 1: 241–286.

montana as Barssia due to a lack of enough specimens to evaluate. In
August 2017, the first and last authors attempted a field survey in thetype locality of Bar. yezo-montana, which was unsuccessful. Barssia
yezo-montana forms cylindrical asci and globose ascospores (Fig. 2),
and thus, this species is suggested to belong to another genus.

Acknowledgements
We thank Mr. Teruhisa Masaki for supporting DNA sequencing. This
study was financially supported by JSPS KAKENHI Grant-in-Aid forYoung Scientists (B) (nos. 17K15184) for the last author.

Hawker L.E. (1954) British hypogeous fungi. Philosophical Transactionsof the Royal Society B 237: 429–546.
Hobbie E.A., Weber N.S., Trappe J.M. (2001) Mycorrhizal vssaprotrophic status of fungi: the isotopic evidence. New Phytologist
150: 601–610.
Izumitsu K., Hatoh K., Sumita T., Kitade Y., Morita A., Gafur A., Ohta
A., Kawai M., Yamanaka T., Neda H., Ota Y., Tanaka C. (2012) Rapidand simple preparation of mushroom DNA directly from colonies andfruiting bodies for PCR. Mycoscience 53: 396–401.
Kaounas V., Agnello C., Alvarado P., Slavova M. (2015) Barssia hellenica
sp. nov. (Ascomycota, Pezizales), a new hypogeous species from Greece.
Ascomycete.org 7: 213–219.

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