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

Truffology 7 (1): 1–8 (2024)

Original peer-reviewed article （原著論文 ； 査読有）

A new distributional record of Elaphomyces fuscus from Japan: as a newhost of Tolypocladium japonicum
ゴマタマツチダンゴの日本における新たな分布記録 ： タンポタケ
モドキの新宿主として
Taiga Kasuya1*, Ei Kawahara2, Kentaro Hosaka3
糟谷 大河 1*, 河原 栄 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

Department of Pathology, Fukui General Hospital, 58-16-1 Egami-cho, Fukui, Fukui 910-8561, Japan
福井総合病院病理診断科， 〒 910-8561 福井県福井市江上町 58-16-1

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
An Elaphomyces species parasitized by Tolypocladium sp. was observed in Itako, Ibaraki Prefecture, central Japan.
Based on morphological observations and molecular identification using nuclear ribosomal DNA sequences ofboth Elaphomyces and Tolypocladium spp., the present fungus and its parasite were identified as Elaphomyces
fuscus and Tolypocladium japonicum, respectively. We report E. fuscus as a first record outside the type locality andas a new host of T. japonicum.

要旨
茨城県潮来市において、トリポクラディウム属菌が寄生したツチダンゴ属菌の一種が観察された。本菌ならびにトリポクラディ
ウム属菌のそれぞれについて、 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分子同定を行った。 その結果、 本菌はゴマタマツチダンゴ Elaphomyces fuscus、 寄生していたトリポクラディウム属菌はタンポタ
ケモドキ Tolypocladium japonicum とそれぞれ同定された。 ゴマタマツチダンゴを基準標本産地以外から初めて記録するとともに、 タンポタケモドキの新宿主として報告する。
Article Info: Submitted: 18 May 2023

Accepted: 10 January 2024

Published: 31 May 2024

Introduction

a mushroom foray organized by Ibaraki Nature Museum was heldAmong ca. 20 known species of the genus Elaphomyces T. Neesin Suigo Prefectural Forest Park, Itako, Ibaraki Prefecture, central(Eurotiales, Elaphomycetaceae) in Japan, E. fuscus M. Shirakawa

Japan. During the foray, several collections of Tolypocladium sp.

and E. marmoratus M. Shirakawa have been described recently(Hypocreales, Ophiocordycipitaceae) parasitized to fruiting bodies

as new taxa (Hatakeyama & Orihara, 2022; Shirakawa & Tanaka,

of Elaphomyces sp. were obtained by its participants (Fig. 1A, B).

2020). These two species were described based on specimens

Simultaneously ascomata of Elaphomyces sp. without stromata ofcollected in secondary mixed forests of Ome, Tokyo, and known

Tolypocladium sp. were also collected at the same locality (Fig. 1C).

only from the type locality (Shirakawa & Tanaka, 2020). In 2022,

Based on morphological observations and molecular identification© The Japanese Association for Truffle Science (JATS), 2024

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Kasuya et al.: Distributional record of Elaphomyces fuscus

Fig. 1. Fruiting bodies of Tolypocladium japonicum and Elaphomyces fuscus. A: Stromata of T. japonicum in the natural habitat (INM-2-229315). B: Stromata of T.
japonicum arising from fruiting bodies of E. fuscus. (INM-2-229315). C: Immature ascomata of E. fuscus (INM-2-229316).

using nuclear ribosomal DNA sequences of both Elaphomycesparaffin. Deparaffinized thin sections (6 µm thick) were stained withand Tolypocladium spp. specimens, they were identified as E.

hematoxylin and eosin (Feldman & Wolfe, 2014), a routine staining

fuscus and Tolypocladium japonicum (Lloyd) C.A. Quandt, Kepler

method for formalin-fixed paraffin-embedded (FFPE) tissue sections& Spatafora, respectively. Our morphological observations and(Bass et al., 2014; Van den Tweel et al., 2010). Measurements ofmolecular identification of both specimens revealed that it is the first

various lengths were done on photomicrographs of the FFPE fungal

distributional record of E. fuscus outside the type locality along with asections and analyzed with a free image analyzing application, Image

new host fungus of T. japonicum.

J (Abramoff et al., 2004; Schneider et al., 2012). Measurementsof the sizes of ascospores of Elaphomyces specimens, which wereMaterials and methods

much larger than the width of fungal tissue sections, were done on

Sample collecting and morphological observations

photomicrographs of ascospores smeared on a glass slide mounted

Fresh samples were collected in secondary mixed forests dominatedwith 3% KOH. In addition, the surface features of the ascosporesby Quercus serrata Murray and Pinus densiflora Siebold & Zucc. atwere observed by scanning electron microscopy (SEM). For SEM,

Suigo Prefectural Forest Park, Itako, Ibaraki Prefecture in July 2022.

a small portion from spore mass was put onto double-sided adhesiveFresh samples were photographed and observed macroscopicallytape on a specimen holder and coated with platinum-palladium using

and then they were air-dried using a food dehydrator (Snackmastera JFC-1600 Ion Sputter Coater (JEOL, Tokyo, Japan). Specimen was

Express FD-60; Nesco/American Harvest, Milwaukee, WI, USA)examined with a JSM-6480LV SEM (JEOL, Tokyo, Japan) operating

under 46 °C for 46 hours. Dried specimens examined in this study

at 10 kV.

were deposited at the mycological herbaria of Ibaraki Nature Museum(INM) and National Museum of Nature and Science (TNS) in Japan.

DNA preparation, PCR and sequencing

For light microscopy, dried fungal tissues were revived with water

DNA extraction, PCR and DNA sequencing of specimens

and fixed with neutral buffered 10% formalin. The fixed tissues

examined in the present study were carried out according to thewere dehydrated, replaced with xylene, and then embedded withmethods introduced by Kasuya et al. (2012, 2022). DNA was

2© The Japanese Association for Truffle Science (JATS), 2024

Truffology - Volume 7, Issue 1, 2024

Kasuya et al.: Distributional record of Elaphomyces fuscus
extracted from an inner peridium and/or a gleba of Elaphomyces

nos.: OQ991235–OQ991238).

sp., and fertile portions of Tolypocladium specimens. DNA
sequence data were obtained from the internal trascribed spacer

Results

region (ITS) and a part of the large subunit (LSU) of the nuclear

Morphological observations

rDNA. For amplifying the ITS region, the primer combination

One to four stromata of Tolypocladium sp. grown directly fromof ITS5 and ITS4 (White et al., 1990) was used. For amplifying

underground ascomata of Elaphomyces sp. (Fig. 1A, B), they

the LSU, the combination of LR0R and LR5 (Vilgalys & Hester,

were clavate, unbranched, with a slightly compressed apex, 5–20

1990) was used.

mm long, 5–7 mm broad at fertile parts which were tan-coloredto blackish brown, on a cylindrical stipe, surface roughened by

Molecular identification

ostioles, solid in the center but holes possibly eaten by fly larvae

Newly generated ITS and LSU sequences from Japanese

were observed (Figs 1A and 2A, B). Stipes were 25–65 mm long,specimens were used for DNA barcoding for species identification.

2–5 mm broad, cylindrical, sometimes bifurcate, white, cream or

The obtained raw sequences were assembled using ATGC version

pale yellow to pale gray, lacking the rhizomorphous structure on

6.0.1 (GENETYX Corporation, Tokyo, Japan). Assembled sequences

the basal part (Figs. 1B and 2A). Perithecia were wholly or partiallywere analyzed using GenBank (https://www.ncbi.nlm.nih.gov/

immersed, ovoid to ellipsoid, 400–450 ×130–180 μm, with papillate

genbank/) Basic Local Alignment Search Tool (BLAST) search

ostioles. Ascospores were hyaline, long cylindrical, disarticulated(blastn) to confirm their phylogenetic affinities. Default settings

into part-spores within asci; part spores were cylindrical, 10–20 ×of blastn option were used for the analyses. A total of four newly

3.0–3.4 μm (Fig. 2C). In addition to the stipe of Tolypocladium sp.,

generated and assembled sequences from this study were depositedanother two primordia of possible this fungus were visible in theto the International Nucleotide Sequence Databases (INSD; accession

FFPE thin sections (Fig. 2D).

Fig. 2. Morphological features of Elaphomyces fuscus with mature stromata of Tolypocladium japonicum (INM-2-229315). A: The ascoma of E. fuscus withstromata of T. japonicum after formalin-fixation. B–I: Whole structures of ascomata of E. fuscus with stromata of T. japonicum showing by the formalin-fixed andparaffin-embedded (FFPE) thin (6 µm thick) sections stained with hematoxylin and eosin. B: Layers of perithecia just beneath the surface of the fertile parts of T.
japonicum (large arrows), ectomycorrhizal root tips (small arrows) and the inner structures of E. fuscus are clearly visible. Asterisks show holes possible eaten by
fly larvae in the stroma of T. japonicum. C: Segmentalized and articulated ascospores of T. japonicum. D: A stipe of mature stroma of T. japonicum growing outof the surface of E. fuscus (asterisk), with another two primordia (arrows) of possible T. japonicum in its vicinity. E: Peridium and surface warts of the ascoma of
E. fuscus. F: The vertical section of ectomycorrhizal roots covering the ascoma of E. fuscus. Formation of the Hartig net beyond the epidermis to enclose several
layers of cortical cells are frequently observed (arrows). G: A large-sized ascospore of E. fuscus. H: A small-sized ascospore of E. fuscus. I: An ascus of E. fuscus
containing five ascospores or more. Spores do not show ornamentation. Bars: A = 1 cm; C, F–I: 10 µm; D, E = 1 mm.
© The Japanese Association for Truffle Science (JATS), 20243

Kasuya et al.: Distributional record of Elaphomyces fuscus

Truffology - Volume 7, Issue 1, 2024

Underground ascomata of Elaphomyces sp. parasitized byTolypocladium sp. were 20–25 × 22–25 mm, globose, subglobose tooval, rigid and covered with soil, earthy crust and ectomycorrhizal
roots (Figs. 1B and 2A, B). Warts of ascomata surfaces were conical,
dark yellow, partly with pale purplish tinge, the peridium was whitein the outer layer and brown in the inner layer, not marbled, and theinside of the peridium was packed with powdery, dark brown to
blackish gleba (Figs. 1B and 2A, B). The warts were dark yellow
under microscopy, 334 ± 81.0 µm high, and were covered with thin
(80–150 µm) layer of hyaline hyphae on the peridium (Fig. 2E).
The peridium was composed of compactly woven hyaline hyphae,outer layer was composed of hyaline hyphae, and inner layer wasof brown hyphae. The membranous septae radiated from the centerand connected to the peridium (Fig. 2B, D). An inner gleba waspacked with numerous dark brown to blackish ascospores andsparsely distributed hyalinous hyphae which presumably originatedfrom parasitized Tolypocladium (Fig. 2B, D and E). Ascospores
were globose to subglobose, and the mean of the quotient was 1.08
± 0.06 µm. The dispersion of spore lengths (mean = 24 µm, median= 26 µm) was high, (15–) 20–28 (–33) µm (Fig. 3, box-whisker
plots of INM-2-229315) with ornamentation, and the distribution
pattern did not show Gaussian but they were biphasic (Fig. 3, scatter

Fig. 3. Scattered plots and box-whisker plots showing the distribution ordispersion of spore lengths of E. fuscus with (INM-2-229315, n = 87) andplots of INM-2-229315) with larger and smaller ones. Surfaces

without (INM-2-229316, n = 107) mature stromata of T. japonicum. Scatterof large-sized ascospores were reticulate, 1.5–3.0 µm high, darkplots show biphasic patterns of the distribution. Box and whisker plots show

reddish brown under the microscope, and apices of each reticulation

the high dispersion and the difference of medians. An asterisk (*) indicates thewere conical, capitulate or obtuse head and irregularly shaped,

significant difference (p = 0.0000039) using Mann-Whitney U test.

broadly base to costate with labyrinthine pattern (Fig. 2G). Surfaces

and ectomycorrhizal roots (Figs. 1C and 4A). Warts of ascomata

of small-sized ascospores were also dark reddish brown undersurfaces were conical, dark yellow, partially with pale purplish

microscopy, and reticulate with conical, capitulate or obtuse tips andtinge; the outer part of the peridium was white, partially with

with polygonal to labyrinthine bases, 1.5–2.5 µm high (Fig. 2H).

purplish tinge, and the inner part of the peridium was brown, not

A few, 12–15 µm in lengths of asci containing smooth ascospores

marbled; the inside of the peridium was packed with gleba whichwere observed (Fig. 2I).

were compact and white to brown when immature, but becoming

There were numerous ectomycorrhizal roots (Figs. 1B and 2B,

powdery and black-brown at maturity (Figs. 1C and 4A). Ascomata

F) both on the surface of the Elaphomyces ascomata and at thehave short protrusions on the top, which turned out to be the possible

stipe base of Tolypocladium sp., even around the primordia ofprimordia of Tolypocladium stromata by the thin FFPE sections,

possible this fungus (Fig. 2B, D). Ectomycorrhizal root tips were

although we have not sequenced this possible primordia. (Fig.

directly connected to the hyphae both on the Elaphomyces and

4B). Ascospores were globose to subglobose, and the mean of the

Tolypocladium fruiting bodies. The vertical section of a mycorrhizal

quotient was 1.12 ± 0.14 µm. The dispersion of spore lengths (meantip covering the ascoma of E. fuscus (Fig. 2F) clearly shows= 20 µm, median = 18 µm) was (11–) 17–23 (–31) µm (Fig. 3, box-

formation of the Hartig net beyond the epidermis to enclose several

whisker plots of INM-2-229316) with ornamentation, and similar

layers of cortical cells and it suggests the typical ectomycorrhizal

to the case of the infected ascomata, the distribution pattern showed

structure of gymnosperm trees (Smith & Read, 2008).

biphasic (Fig. 3, scatter plots of INM-2-229316 and Fig. 4C). Both

Underground Elaphomyces ascomata without stromata ofthe mean and the median of ascospores of Elaphomyces sp. without

Tolypocladium sp. were 8–23 × 8–18 mm, globose, subglobose

mature Tolypocladium stromata were significantly smaller thanto depressed-globose, rigid and covered with soil, earthy crust

those with mature stromata of Tolypocladium sp. Further, numbers

4© The Japanese Association for Truffle Science (JATS), 2024

Truffology - Volume 7, Issue 1, 2024

Kasuya et al.: Distributional record of Elaphomyces fuscus

Fig. 4. Morphological features of Elaphomyces fuscus without mature stromata of Tolypocladium japonicum (INM-2-229316). A: The ascoma of E. fuscus after
formalin fixation. B–D: Microscopic features of E. fuscus showing by the FFPE thin section stained with hematoxylin and eosin. B: The primordium of possibly T.
japonicum (asterisk) growing out of E. fuscus and breaking through the warts. The structure is also visible macroscopically as a short protrusion on the top of
E. fuscus (asterisk in A). C: Unstained spores directly smeared on the glass slide showing the biphasic pattern of the spore sizes. D: A large-sized ascospore. E: A
small-sized ascospore. F: Scanning electron microscopy image of a small-sized ascospore. Bars: A = 1 cm; B = 500 µm; C–E: 10 µm; F = 2 µm.

of large-sized ascospores were much larger and numbers of small-accession nos. MT386337–MT386340) from China, showing asized spores were much smaller than those in Elaphomyces sp. withsimilarity of 93.18% (Table 1). For LSU, the top two hits of BLAST

mature Tolypocladium stromata. Surfaces of large-sized ascospores

search (99.77 to 100.00%) were also T. japonicum (AB027366 andwere reticulate, dark reddish brown under the microscope, and

DQ518761; Table 2).

apices of each reticulation were conical, capitulate or obtuse and

The BLAST search indicated that the ITS sequence of the present

irregularly shaped, broad base to costate with labyrinthine pattern

Elaphomyces specimen (OQ991238, 680 bp) shares the highest(Fig. 4D). Surfaces of small-sized ascospores were also dark reddish

similarities with E. fuscus (Table 3). For ITS, the top two hits ofbrown under the microscope, and reticulate with conical, capitulate

BLAST search were all E. fuscus (99.63 to 99.84% similarities)

or obtuse tips and with polygonal to labyrinthine bases (Fig. 4E, F).

including the sequence from the holotype (Table 3), and the next hit

Only a few asci were observed with non-reticulate ascospores.

was “Uncultured Elaphomyces YM56” (LC175083) from Iwate,
Japan at 97.40% similarity. No LSU sequences from E. fuscus are

Molecular identification

currently available from INSD. The top six hits of BLAST search (all

The BLAST search indicated that sequences of the presentof 99.78%) of LSU sequence (OQ991236, 911 bp) of the present

Tolypocladium specimen (ITS: OQ991237, 565 bp; LSU:

Elaphomyces specimen were E. asperulus Vittad. (KR029753–

OQ991235, 895 bp) share highest similarities with T. japonicum

KR029755 and KR064762) from Denmark, Italy and Sweden (Molia(Tables 1 and 2). For ITS, the top two hits of BLAST search were

et al., 2020), and E. roseoviolaceus A. Molia & E. Larss. (KR029750

both T. japonicum (98.56 to 99.29% similarities), and next hits wereand KR029751) from Norway (Molia et al., 2020).

T. guangdongense (T.H. Li, Q.Y. Lin & B. Song) V. Papp (INSD© The Japanese Association for Truffle Science (JATS), 2024

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Kasuya et al.: Distributional record of Elaphomyces fuscus

Table 1. Similarities of the ITS sequence (OQ991237, 565 bp) of the present Tolypocladium specimen (INM-2-229315) resulted by BLAST search

INSD accession no.
AB027366
KT873533
MT386340
MT386339
MT386338
MT386337

Taxon names
Tolypocladium japonicum
T. japonicum
T. guangdongense
T. guangdongense
T. guangdongense
T. guangdongense

OriginJapan
Taiwan
China
China
China
China

Sequence similarities
99.29%
98.56%
93.18%
93.18%
93.18%
93.18%

Sequence lengths (bp)
1690
601
597
572
614
597

Table 2. Similarities of the LSU sequence (OQ991235, 895 bp) of the present Tolypocladium specimen (INM-2-229315) resulted by BLAST search

INSD accession no.
AB027366
DQ518761
OP207731
AB027364
OP207735

Taxon names
T. japonicum
T. japonicum
T. capitatum
T. capitatum
T. paradoxum

OriginSequence similarities
Japan
100.00%unknown 99.77%
China
97.43%
Japan
97.43%
Japan
97.43%

Sequence lengths (bp)
1690
886
901
1653
900

Table 3. Similarities of the ITS sequence (OQ991238, 680 bp) of the present Elaphomyces specimen (INM-2-229316) resulted by BLAST search

INSD accession no.
LC500967*
LC500968

Taxon names
Elaphomyces fuscus"uncultured Elaphomyces "(mycorrhizal root tips of E. fuscus )LC523911
E. fuscus"uncultured Elaphomyces YM56"
LC175083
KX238833
E. asperulus
KR029750
E. roseoviolaceus
KR029751
E. roseoviolaceus
KX165346
E. asperulus*Sequence from the holotype of E. fuscus .

OriginJapan
Japan

Sequence similarities Sequence lengths (bp)99.84%
615
99.82%
568

Japan
Japan
Spain
NorwayNorwayNorway99.63%
97.40%
95.90%
95.59%
95.59%
95.26%

546
583
698
2067
2070
602

Discussion

Known distribution: Japan (Imai, 1935; Kobayasi & Shimizu,

Morphological features of Tolypocladium specimens examined in1960), China (Liang et al., 2003), Taiwan (Ke & Ju, 2015) andthe present study such as clavate stromata on a cylindrical stipe, andAustria (Kobayasi & Shimizu, 1960; Mains, 1957).

lacking of the rhizomorphous structure on the basal part of stromata

Japanese name: Tanpotake-modoki (Imai, 1935).

were well agreement in the previous descriptions of T. japonicum(Imai, 1935; Ke & Ju, 2015; Kobayasi & Shimizu, 1960). MicroscopicMorphologically, ascomata of Elaphomyces with mature stromata of T.

features of perithecia and ascospores were also almost identical tojaponicum and without them were very similar. These Elaphomyces

descriptions of T. japonicum (Ke & Ju, 2015; Kobayasi & Shimizu,

specimens shared morphological similarities with E. fuscus in the1960). Moreover, ITS and LSU sequences newly generated in thefollowing features: (1) dark yellow warts of ascomata, (2) whitish

present study showed high similarities with those of T. japonicum

outer layer of the peridium, (3) dark brown to blackish powdery

from Japan and Taiwan (Tables 1 and 2). We therefore identified thegleba, and (4) reticulate, dark reddish brown ascospores under the

Tolypocladium specimens examined as T. japonicum as below.

microscope. Aforementioned characteristics were main diagnostic
features of E. fuscus and the present Elaphomyces specimens wereTolypocladium japonicum (Lloyd) C.A. Quandt, Kepler &morphologically almost identical to the original description of E.

Spatafora, IMA Fungus 5: 126 (2014).

fuscus (Shirakawa & Tanaka, 2020). In addition, the ITS sequence[MycoBank ID: 808705]

newly generated in the present study showed high similarity (99.84%)
Figs. 1 and 2.

Specimens examined: JAPAN, Ibaraki Prefecture, Itako, Shimasu,

with those of from the holotype of E. fuscus (Table 3). We therefore
identified those specimens as E. fuscus as below.

Suigo Prefectural Forest Park, (approx. 35°58’52”48N, 140°32’22”32E,
alt. approx. 24 m asl.), July 3, 2022, coll. Y. Otaki, INM-2-229315

Elaphomyces fuscus M. Shirakawa, Mycoscience 61: 319 (2020).

[INSD accession no.: OQ991237 (ITS), OQ991235 (LSU)]; same

[MycoBank ID: 833108]

locality, July 3, 2022, coll. T. Kasuya, TNS-F-83036.
Habitat: On ascomata of Elaphomyces spp.
6

Figs. 1, 2 and 4.
Specimens examined: JAPAN, Ibaraki Prefecture, Itako,© The Japanese Association for Truffle Science (JATS), 2024

Truffology - Volume 7, Issue 1, 2024

Kasuya et al.: Distributional record of Elaphomyces fuscus
Shimasu, Suigo Prefectural Forest Park, (approx. 35°58’52”48N,

Acknowledgements

140°32’22”32E, alt. approx. 24 m asl.), July 3, 2022, coll. T.

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

Kasuya, INM-2-229316 [INSD accession no.: OQ991238 (ITS),

Otsuka of the National Museum of Nature and Science for help with

OQ991236 (LSU)]; same locality, July 3, 2022, coll. Y. Otaki,

assisting molecular experiments. We also deeply thank Ms. Yua Otaki,

TNS-F-83037.

Ms. Mihoko Uzawa, Mr. Yoshinori Inaba and volunteer stuffs ofHabitat: Hypogeous, growing in clusters 3–10 cm underground

Ibaraki Nature Museum for facilitating the fieldwork. We are gratefulunder Quercus serrata and Pinus densiflora trees.

to Dr. Tamao Hatta of Chiba Institute of Science for his skillful

Known distribution: Japan (Tokyo: Shirakawa & Tanaka, 2020;

technical assistance and for helpful suggestions to SEM observation.

Ibaraki: new record).

This work was supported in part by JSPS KAKENHI Grant Numbers

Japanese name: Gomatama-tsuchidango (Shirakawa & Tanaka,

JP20K06805 and JP23K05895.

2020).
Notes: Reticulate ascospores of E. fuscus are main distinguishable
features from E. granulatus Fr., and also the present species differsfrom E. japonicus Lloyd in its reticulate ascospores and yellowish
warts of ascomata (Shirakawa & Tanaka, 2020). Similarities of
ITS and LSU sequences between E. fuscus and E. asperulus arehigh (ITS: 95.26 to 95.90%; LSU: 99.78%), and also, both species

References
Abramoff, M.D., Magelhaes, P.J., Ram, S.J. (2004) Image Processing
with ImageJ. Biophotonics International 11: 36–42.
Bass, B.P., Engel, K.B., Greytak, S.R., Moore, H.M. (2014) A review ofpreanalytical factors affecting molecular, protein, and morphologicalare morphologically similar, but the color of the peridium of

analysis of formalin-fixed, paraffin-embedded (FFPE) tissue: how

the former is not homogeneously purplish hue such as the latter

well do you know your FFPE specimen? Archives of Pathology andspecies (Paz et al., 2017; Shirakawa & Tanaka, 2020). Ascospores

Laboratory Medicine 138: 1520–1530.

of E. asperulus show confluent warty ornamentation while those

Feldman, A.T., Wolfe, D. (2014) Tissue processing and hematoxylin andof E. fuscus are labyrinthine, irregular meshes (Paz et al., 2017;

eosin staining. Histopathology: Methods in Molecular Biology 1180:

Shirakawa & Tanaka, 2020). Although ITS and LSU sequences of

31–43.

E. roseoviolaceus also shows high similarity (ITS: 95.59%; LSU:
99.78%) to those of E. fuscus, morphological features of both
species are quite different. Elaphomyces roseoviolaceus has spiny toverrucose ascospores and dark pink to violaceous peridium (Molia
et al., 2020).
Elaphomyces fuscus was previously known only from the type
locality. Thus, this study is the first distributional record of E. fuscus

Hatakeyama, S., Orihara, T. (2022) Taxonomic history of the genus
Elaphomyces, with emphasis on E. granulatus and E. muricatus.
Truffology 5: 69–75 (in Japanese with English summary).
Imai, S. (1935) Studies on the Hypocreaceae of Japan II. Transactions of
the Sapporo Natural History Society 14: 101–106.
Kasuya T., Hosaka K., Uno K., Kakishima M. (2012) Phylogenetic
placement of Geastrum melanocephalum and polyphyly of Geastrum
triplex. Mycoscience 53: 411–426.

outside the type locality. As above mentioned, the vertical section of

Kasuya T., Uzawa M., Hosaka K. (2022) Scleroderma capeverdeanum,a mycorrhizal tip covering the ascoma of E. fuscus (Fig. 2F) showsa subhypogeous fungus new to Japan, collected from the Eucalyptus

the typical ectomycorrhizal structure of gymnosperms. Because

plantation in Ibaraki Prefecture. Truffology 5: 23–32.

the present Elaphomyces specimens were collected underneathQ. serrata and P. densiflora trees, we treat this is a portion of P.
densiflora roots. Previously, ectomycorrhizal association of Q.
serrata with E. fuscus has only been reported (Shirakawa & Tanaka,
2020), therefore we here add P. densiflora as a novel host plant of
E. fuscus. Additionally, we report E. fuscus as a new host fungus ofT. japonicum. Although ascomata of E. fuscus with mature stromata
of T. japonicum and without them are morphologically very similar

Ke, Y.-H., Ju, Y.-M. (2015) Two rare ophiocordycipitaceous fungi newly
recorded in Taiwan. Botanical Studies 56: 30.
Kobayasi, Y., Shimizu, D. (1960) Monographic studies of Cordyceps
1. Group parasitic on Elaphomyces. Bulletin of the National Science
Museum, Tokyo 5: 69–85.
Liang, Z.-Q., Liu, A.-Y., Liu, M.-H. (2003) Two new records of
mycogenous Cordyces in China. Mycosystema 22: 159–160.
Mains, E.B. (1957) Species of Cordyceps parasitic on Elaphomyces.
Bulletin of the Torrey Botanical Club 84: 243–251.

each other, sizes of ascospores are different (Fig. 3). Further studies

Molia, A., Larsson, E., Jeppon, M., Læssøe, T., Larsson, K.H. (2020)are required to clarify the mechanism of formation of different sizes

Elaphomyces section Elaphomyces (Eurotiales, Ascomycota) —of ascospores.

taxonomy and phylogeny of North European taxa, with the introductionof three new species. Fungal Systematics and Evolution 5: 283–300.

© The Japanese Association for Truffle Science (JATS), 2024

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Truffology - Volume 7, Issue 1, 2024

Paz, A., Bellanger, J.M., Lavoise, C., Molia, A., Ławrynowicz, M., Larsson,

Van den Tweel, J.G., Taylor, C.R. (2010) A brief history of pathology:

E., Ibarguren, I.O., Jeppson, M., Læssøe, T., Sauve, M., Rizhard, F.,

preface to a forthcoming series that highlights milestones in the

Moreau P.-A. (2017) The genus Elaphomyces (Ascomycota, Eurotiales):evolution of pathology as a discipline. Virchows Archiv 457: 3–10.

A ribosomal DNAbased phylogeny and revised systematics of European

Vilgalys R., Hester M. (1990) Rapid genetic identification and mapping“deer truffles”. Persoonia 38: 197–239.
Schneider, C.A., Rasband, W.S., Eliceiri, K.W. (2012) NIH Image toof enzymatically amplified DNA from several Cryptococcus species.
Journal of Bacteriology 172: 4238–4246.

ImageJ: 25 years of image analysis. Nature Methods 9: 671–675.

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

Shirakawa, M., Tanaka, M. (2020) Two new deer truffle species,

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

Elaphomyces marmoratus and Elaphomyces fuscus spp. nov., from a

M.A., Gelfand D.H., Sninsky J.J., White T.J. (eds.) PCR protocols.

secondary forest in Japan. Mycoscience 61: 315–322.

Academic Press, New York, pp. 315–322.

Smith, S.E., Read, D.J. (2008) Mycorrhizal symbiosis, 3rd Edition.
Academic Press, London.

8© The Japanese Association for Truffle Science (JATS), 2024