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

Truffology 5 (1): 3–13 (2022)

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

Taxonomic re-examination and phylogeny of neglected Japanese
black deer truﬄes, Elaphomyces miyabeanus and E. nopporensis
クロツチダンゴ Elaphomyces miyabeanus およびコクロツチダンゴ
E. nopporensis の分類学的再検討と系統
Takamichi Orihara 1*, Michael A. Castellano 2, Muneyuki Ohmae 3, Yoshinori Kaneko 4,
Kentaro Hosaka5
折原 貴道 1*, Michael A. Castellano2, 大前 宗之 3, 金子 義紀 4, 保坂 健太郎 5
1

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

2USDA, Forest Service, Northern Research Station, Corvallis, Oregon 97331, USA

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

Yamakita-machi, Asigarakami-gun, Kanagawa, Japan
神奈川県足柄上郡山北町

4

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: t_orihara@nh.kanagawa-museum.jp

Abstract
Two Japanese black deer truffles, Elaphomyces miyabeanus and E. nopporensis, were described from Hokkaido
in 1929 by Dr. Sanshi Imai. Since then, there have been no reliable, formal records of either species. Both species
were collected from the same locality on the same day, and are morphologically similar to each other. We
successfully located authentic specimens including syntypes of each species, which had been scattered and lost,
and we critically examined their morphology. We also collected fresh ascomata of E. miyabeanus / E. nopporensis
from the type locality and central Honshu. Based on examination of the rediscovered authentic specimens andthe fresh ascomata along with the original descriptions by Dr. Sanshi Imai, we concluded that E. nopporensis was
small, dried, old ascomata of E. miyabeanus and should be synonymized into the latter species. Phylogenetic
analyses based on the internal transcribed spacer region of the nuclear ribosomal DNA placed E. miyabeanus
within sect. Ceratogaster subsect. Sclerodermei along with E. anthracinus. The resultant tree also showed that E.
miyabeanus was conspecific with Elaphomyces sp. collected in Norway, suggesting that the species is broadly
distributed across northern Eurasia.

要旨
クロツチダンゴ Elaphomyces miyabeanus およびコクロツチダンゴ E. nopporensis は、 今井三子博士により北海道から 1929年に記載された黒色のツチダンゴ類である。 しかし、 新種記載以降、 両種の確実な報告は無く、 その実態は不明である。
両種は同日、 同一産地 （現在の道立自然公園野幌森林公園付近） にて採集されており、 形態的に酷似している。 著者© The Japanese Association for Truffle Science (JATS), 2022

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Orihara et al.: Elaphomyces miyabeanus taxonomy and phylogeny

らは北海道大学総合博物館および日本きのこセンター菌蕈研究所にて、両種のシンタイプを含む複数の標本を再発見した。
さらに、 タイプ産地および本州中部 （山梨県） にて、 両種と形態的特徴の一致する子実体を新たに採集した。 これらの標本および原記載の情報から両種の形態的比較検討を行った結果、 コクロツチダンゴ E. nopporensis は、 乾燥の進んだ、 古い小型のクロツチダンゴ E. miyabeanus 子実体であると結論した。核リボソーム rDNA ITS 領域に基づく分子系統解析の結果、
E. miyabeanus はツチダンゴ属 Ceratogaster 節 Sclerodermei 亜節に含まれ、 E. anthracinus に近縁であること、 ノルウェー産の標本と同一種であることが示された。 この結果から、 本種はユーラシア大陸北部に広く分布していることが示唆された。
Article Info: Submitted: 8 February 2022

Accepted: 21 March 2022

Published: 31 March 2022

Introductionof Elaphomyces. We therefore tried to locate authentic specimens of

The deer truffle genus, Elaphomyces T. Nees (Elaphomycetaceae,

E. miyabeanus and E. nopporensis in several Japanese herbaria andEurotiales, Ascomycota) is one of the largest genera composed ofmade field surveys throughout Japan to locate fresh specimens of

truffle-like fungi. Currently, Elaphomyces comprises approximately

both species. As a result, we successfully located several herbarium

170 infrageneric taxa (according to the MycoBank database,specimens of E. miyabeanus and E. nopporensis collected by S. Imaisearched on 3 January, 2022) both from the Northern and Southern

and taxonomically re-evaluated the two species. Furthermore, weHemispheres. Elaphomyces spp. are ectomycorrhizal, which is

obtained fresh materials of E. miyabeanus from several localities andexceptional in the Eurotiales, and some Elaphomyces spp. are also

examined its phylogenetic position based on the internal transcribed

known as hosts of the fungicolous Tolypocladium spp.

spacer (ITS) region of the nuclear ribosomal DNA (nrDNA).

In Japan, Elaphomyces has attracted attention by mycologists
since very early in the taxonomic history of Japanese truffle-like

Materials and methods

fungi, mainly as hosts of mycoparasitic fungi (e.g., Kawamura, 1914;

Taxon sampling and morphological observation

Kobayasi, 1960; Lloyd, 1916; Umemura, 1923; Yasuda, 1919). It

Authentic herbarium specimens of E. miyabeanus and E.

was Dr. Sanshi Imai (1900–1976) who revealed a potentially highnopporensis collected by S. Imai were investigated in Hokkaido

diversity of Japanese Elaphomyces spp. for the first time, describing

University Museum (SAPA), National Museum of Nature andfive new species as well as the widely distributed E. granulatus Fr.

Science, Japan (TNS) and Tottori Mycological Institute (TMI). The

and E. variegatus Vittad. (Imai, 1929, 1934, 1938, 1939). Of these,

specimens were photographed and were subjected to microscopic

E. miyabeanus S. Imai (Japanese name: Kuro-tsuchidango) and E.

observation.

nopporensis S. Imai (Japanese name: Kokuro-tsuchidango) are theField surveys were conducted in various types of forests infirst two black deer truffles described from Japan (Imai, 1929). Both

Hokkaido and Honshu. Fresh ascomata were collected using a

species were collected several times from “Nopporo Forest” (i.e.,

garden rake and were photographed in the field. After morphological

Nopporo Forest Park as it is known today), Hokkaido Prefecture,observation, the collected ascomata were air-dried for 2 days at 48°C

on the same day. Some of those specimens were parasitized byor freeze-dried and then stored in sterile plastic bags. The specimens

Tolypocladium jezoense (S. Imai) Quandt, Kepler & Spatafora.

are deposited in Kanagawa Prefectural Museum of Natural History,

Morphologically, both species appear similar but E. miyabeanus canJapan (KPM).

be distinguished from E. nopporensis by having larger ascomata, a

For light microscopy, hand-cut sections of dried specimens wereperidium with partial pyramidal warts and slightly larger ascosporespresoaked in 70% ethanol and 3% KOH. After briefly rinsing out those(Imai, 1929). It should also be noted that only mature or old ascomata

sections with distilled water, they were mounted in lacto-glycerol. A

of E. nopporensis were collected by S. Imai.

MT5310L microscope (Meiji Techno Co., Ltd., Saitama, Japan) was

Since the original description, there have been no reliable, formal

used for the observation. Photomicrographs were captured using arecords of either species although similar black truffles have been

WRAYMER NOA-2000 camera (WRAYMER Inc., Osaka, Japan).

sporadically recorded as hosts of T. jezoense from Honshu (Shimizu,

Measurement under the microscope was performed with MicroStudio1997). Also, it was not clear whether the type specimens of either

software (WRAYMER Inc.). Measurement of spores includes outerspecies were extant. To clarify the true diversity of Japanese black

ornamentation and an outermost layer if any. Scanning electron

Elaphomyces spp., we first need to know the taxonomic entity andmicroscopy (SEM) was performed with a HITACHI TM-4000Plus

phylogeny of the species based on the current systematic framework

Tabletop Microscope (Hitachi High-Technologies, Tokyo, Japan).

4© The Japanese Association for Truffle Science (JATS), 2022

Truffology - Volume 5, Issue 1, 2022

Orihara et al.: Elaphomyces miyabeanus taxonomy and phylogeny
Small fragments of a dried gleba and spore mass were set on a sample

algorithm (Lefort et al., 2017). For calculating branch supports in thisstage with a double-sided carbon tape and were observed at 15 kV.

analysis, the SH-like approximate likelihood-ratio test (SH-aLRT;
Anisimova & Gascuel, 2006) was conducted instead of bootstrapping.

DNA extraction, PCR amplification and sequencing

Bayesian analyses were conducted with MrBayes 3.2 (Ronquist

DNA was extracted from peridial tissue of a fresh E.

& Huelsenbeck 2003). The ITS dataset was partitioned in the same

miyabeanus specimen (KPM-NC 29301) and dried, syntypes of E.

pattern as in RAxML analysis, and the resultant two partitions were

miyabeanus deposited in TMI using the protocol of Izumitsu et al.

independently subjected to a substitution-model estimation using(2012). PCR amplification of the ITS of nrDNA followed Orihara

jModelTest2 under AIC. The K80+I+G was selected as the best-

et al. (2012) with the exception of reducing the elongation time

fit model for the partial 18S-5.8S-partial 28S, and GTR+G was

before the final elongation step to 60S. The primer pair used wasfor the ITS1-ITS2 partition. Bayesian posterior probabilities (PPs)ITS1F (Gardes & Bruns, 1993) / ITS4 (White et al., 1990). PCR

were estimated by the Metropolis-coupled Markov chain Monteamplification from the syntypes was not successful. Amplified

Carlo method (Geyer, 1991). The two parallel runs were conducted

PCR product was purified with IllustraTM ExoProStarTM

with one cold and seven heated chains each for 3M generations.

(Cytiva, Tokyo, Japan) based on the manufacturer’s instructions.

Temperature parameter of the seven heated chains in both runs was

Sequencing steps of Orihara et al. (2012) were followed.

set to 0.20 (as a default). Trees were saved to a file every 1000th
generation. We determined that the two runs reached convergence

Phylogenetic analyses

when the average standard deviation of split frequencies (ASDSF)

For the ITS dataset, we retrieved nucleotide sequences thatwas continuously lower than 0.01. The ASDSF was monitored

covered all the taxa of sect. Ceratogaster shown in Paz et al. (2017)

every 5000 generations. Trees obtained before reaching convergencefrom the International Nucleotide Sequence Database (INSD).

were discarded as the burn-in, and the remaining trees were used to

We also included sequences of unidentified Elaphomyces spp. andcalculate a 50% majority consensus topology and to determine PP

environmental sequences from ectomycorrhizal (ECM) roots hit

values for individual branches.

by the BLAST search in the NCBI website (https://blast.ncbi.nlm.
nih.gov/Blast.cgi). ITS sequences used for the phylogenetic analyses

Resultsare listed in Table 1. Species in sect. Ascoscleroderma were selected

Investigation of old Elaphomyces specimens in the mycologicalfor outgroups. The dataset was aligned with the online version of

herbaria

MAFFT version 7 (Katoh & Standley, 2013) under default settings

In November 2017, three of the authors (i.e., T. Orihara, M.A.

(i.e., the alignment algorithm is automatically selected from FFT-

Castellano and K. Hosaka) intensively searched old, unsorted

NS-1, FFT-NS-2, FFT-NS-i or L-INS-i). Subsequently, the sites withspecimens of truffle-like fungi deposited in SAPA, and we foundobvious alignment errors were manually adjusted in SeaView version

one E. miyabeanus specimen parasitized by Tolypocladium jezoense

4 (Gouy et al., 2010). We referred to the results of the Gblocks option(= Cordyceps jezoensis S. Imai) and one E. nopporensis specimen,(Castresana, 2000) in SeaView to exclude ambiguously aligned sites

both of which were collected and identified by S. Imai (Fig. 1A–C).

for the following analyses.

The former was one of the syntypes of T. jezoense and the earliest

Maximum likelihood (ML) analyses were performed with RAxML

record of both E. miyabeanus and T. jezoensis known today. The

8.2.10 (Stamatakis, 2014) and the online version of PhyML 3.0

latter was one of the syntypes of E. nopporensis. The first author(Guindon et al., 2010). In the RAxML analysis, the best substitutionsubsequently searched old specimens collected by S. Imai in TMI inmodel for the whole dataset was estimated by the Akaike Information

November 2018, and found several additional specimens identified as

Criterion (AIC) under jModelTest2 (Darriba et al., 2012; Guindon &E. miyabeanus and E. nopporensis including syntypes of both species

Gascuel, 2003). The ITS dataset was partitioned into partial 18S-5.8S-(Fig. 1D–I). All of these dried specimens in SAPA and TMI werepartial 28S and ITS1-ITS2 so that different α-shape parameters,preserved in good condition. We could not find any specimens of E.

GTR rates, and empirical base frequencies could be assigned to eachmiyabeanus and E. nopporensis in TNS.

partition. RAxML analysis was conducted under the GTR+G modelwith rapid bootstrap replicates set to 1000. PhyML analysis was

Field sampling of fresh ascomata

implemented under default settings with the GTR+G model, which

We conducted field samplings several times in the type locality of

was automatically estimated as a best-fit model based on the SMS

E. miyabeanus and E. nopporensis (i.e., the Nopporo Forest Park,© The Japanese Association for Truffle Science (JATS), 2022

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Orihara et al.: Elaphomyces miyabeanus taxonomy and phylogeny

Table 1. INSD (GenBank) accession numbers of the ITS sequences used for the phylogenetic analyses (UECM: uncultured ectomycorrhizal roots)
表 1． 分子系統解析に用いた ITS 領域塩基配列の INSD (GenBank) 登録番号
Taxa
E. aculeatus

Locality
SPAIN, Asturias

Voucher no.
IC 27111115 (Herb. pers.
A. Paz)

Reference
Paz et al. (2017)

ITS acc. no.
KX238821

E. aculeatus

SPAIN, Asturias

IC 10041103 (Herb. pers.
A. Paz)Paz et al. (2017)

KX238844

E. anthracinus

NORWAY, Vestland Co., Stord

O-F-22176

Unpubl. data by Larsson, E. andMolia, A.

KR029773

E. anthracinus

NORWAY, Vestland Co., Stord

O-F-22177

Unpubl. data by Larsson, E. andMolia, A.

KR029774

E. anthracinus
E. anthracinus
E. anthracinus
E. anthracinus f. talosporus
E. foetidus
E. leonis

SPAIN, Cantabria
SPAIN, Cantabria
SPAIN, Cantabria
SPAIN, Asturias
SPAIN, Caceres
SPAIN, Cantabria

LIP-0001144 (lectotype)
Herb. pers. A. Paz
Herb. pers. A. Paz
LIP-0001145 (holotype)
LIP-0001138 (epitype)
IC 14111101 (Herb. pers.
A. Paz)Paz et al. (2017)
Paz et al. (2017)
Paz et al. (2017)
Paz et al. (2017)
Paz et al. (2017)
Paz et al. (2017)

KX238803
KX238804
KX238813
KX238805
KX238797
KX238814

E. leucosporus

SPAIN, Cantabria

IC 24051203 (Herb. pers.
A. Paz)Paz et al. (2017)

KX238816

E. leveillei
E. maculatus
E. morettii var. echinatus

SPAIN, Asturias
SPAIN, Cantabria
NORWAY, Vest-Agder

LIP-0001148 (epitype)
LIP-0001149
IC 27091306 (Herb. pers.
A. Paz)Paz et al. (2017)
Paz et al. (2017)
Paz et al. (2017)

KX238856
KX238799
KX238854

E. persoonii

SPAIN, Cantabria

IC 18031201 (Herb. pers.
A. Paz)Paz et al. (2017)

KX238828

E. septatus

SPAIN, Cantabria

IC 10041107 (Herb. pers.
A. Paz)Paz et al. (2017)

KX238807

E. spirosporus
E. virgatosporus

SPAIN, Caceres
SPAIN, Asturias

LIP-0001152 (holotype)
IC 26051213 (Herb. pers.
A. Paz)Paz et al. (2017)
Paz et al. (2017)

KX238796
KX238811

E. iuppitercellus
E. miyabeanus

CAMEROON, East ProvinceJAPAN, Yamanashi Pref.,Narusawa Village

M3 (from an ECM root tip) Castellano et al. (2016)
KPM-NC 29301
This study

KT694140
OM286885

Elaphomyces sp.

MEXICO, Mexico State,
Temascaltepec

GO-2009-040

Unpubl. data by Garibay Orijel, R.
and Pina Paez, C.

KJ594995

Elaphomyces sp.

MEXICO, Mexico State,
San Simon de Guerrero

GO-2009-028

Unpubl. data by Garibay-Orijel, R.
and Pina Paez, C.

KJ594996

Elaphomyces sp.
Elaphomyces sp.
Elaphomyces sp.
Elaphomyces sp.
Elaphomyces sp.
Elaphomyces sp. (sp. 6)

ROMANIA, ECM of Quercus robur
USA
PANAMA
USA, FL
USA, FLNORWAY, Viken, UllensakerLM 2779
FLAS_AC727
AC-2017a
FLAS-F-60353
FLAS-F-61848
O-F-22082

Suz et al. (2014)
Unpubl. data by Corrales, A. et al.
Unpubl. data by Corrales, A. et al.
Unpubl. data by Richter, B. et al.
Unpubl. data by Kaminsky, B. et al.
Unpubl. data by Larsson, E. andMolia, A.

KM576390
MH496798
KY825739
MF074844
MH399885
KR029769

Elaphomyces sp. (sp. 6)

NORWAY, Oslo fylke, OsloO-F-245336

Unpubl. data by Larsson, E. andMolia, A.

KR029770

Elaphomyces sp. (sp. 6)

NORWAY, Viken, Ringerike

O-F-245524

Unpubl. data by Larsson, E. andMolia, A.

KR029771

UECM of Quercus crispula
UECM of Quercus kelloggii

JAPANUSA, CA, Riverside County,
James Reserve

M0400
JR9

Unpubl. data by Ishida T. A.et al.
Unpubl. data by Taniguchi et al.

KC791026
KC791026

Hokkaido Pref.). On 1 October 2018, one of the authors (M. Ohmae)

to E. miyabeanus from a subboreal coniferous forest in Yamanashi

collected a single overmature ascoma that was morphologicallyPrefecture, central Honshu (Fig. 2D–H). ITS sequence was

identical to E. nopporensis (Fig. 2A): the ascoma had a rough, black

successfully obtained from this specimen (KPM-NC 29301; Table 1)

peridium that became papery due to dehydration and over-maturity,and subjected to the following phylogenetic analyses. Additionally,

and a shrunken blackish gleba. Subsequently, additional old ascomata

we collected black Elaphomyces ascomata that appeared very similar

were collected at a different site in the type locality on 22 Sep. 2019.

to E. miyabeanus from Ehime Prefecture, Shikoku (KPM-NC 29304),

Trials of ITS sequencing from both specimens failed.

but this specimen was different from E. miyabeanus in the ascospore

We also collected ascomata that were morphologically identical6morphology (Fig. 2I; see “Notes” below for more details).
© The Japanese Association for Truffle Science (JATS), 2022

Orihara et al.: Elaphomyces miyabeanus taxonomy and phylogeny

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Fig. 1. Authentic specimens of E. miyabeanus and E. nopporensis recorded in Imai (1929) and rediscovered from SAPA and TMI herbaria. A–C: SAPA specimensof E. miyabeanus parasitized by Tolypocladium jezoense (upper of A and B; syntype of T. jezoense [Imai, 1929]) and E. nopporensis (lower of A and C; isosyntypeof E. nopporensis). D–I: TMI specimens Newly designated lectotype of E. miyabeanus (D [middle], E, G; TMI 37400) and one of the syntypes of E. nopporensis (F, H,
I; TMI 37401). Both of the specimens were collected by Dr. S. Imai on 4 July 1926. G: Ascospores of E. miyabeanus. H: Ascospores of E. nopporensis. Note that theouter warty spore walls and the inner cores are separated in most ascospores. I: SEM photomicrograph of partially angular ascospore (right) of E. nopporensis
and remnants of spore walls (arrows). Bars: E–F = 1 cm; G–H = 20 µm; I = 10 µm.
図 1． Imai (1929) における記載に用いられたクロツチダンゴ E. miyabeanus およびコクロツチダンゴ E. nopporensis 標本 （北海道大学総合博物館 [SAPA] お
よび日本きのこセンター菌蕈研究所 [TMI] 所蔵）． A–C: SAPA 所蔵のエゾハナヤスリタケ Tolypocladium jezoense に寄生された E. miyabeanus 標本 （A 上部
および B ； 本標本は Tolypocladium jezoense のシンタイプに相当） および E. nopporensis 標本 （A 下部および C ； 本標本は E. nopporensis のアイソシンタイ
プに相当）． D–I: 新たにレクトタイプ指定された TMI 所蔵の E. miyabeanus 標本 （D ［中央］ , E, G; TMI 37400） および E. nopporensis シンタイプ標本 （F, H, I;
TMI 37401）． いずれも今井三子博士により 1926 年 7 月 4 日採集． G: Elaphomyces miyabeanus 子嚢胞子． H: Elaphomyces nopporensis 子嚢胞子． 多くの胞子において、 短い刺状突起に覆われた胞子外壁と， その内部の胞子中心部が分離している． I: Elaphomyces nopporensis の一部角張った子嚢胞子 （右側） および胞子外壁の残骸 （矢印） の SEM 画像．

© The Japanese Association for Truffle Science (JATS), 2022

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Fig. 2. A–C: Elaphomyces nopporensis (= small, old, dried ascoma of E. miyabeanus) newly collected from the type locality (KPM-NC 25100). A: Ascoma. B–
C: Ascospores; in most ascospores the outer exosporium was shrunken and separated from the inner dark brown core (arrows). D–H: Newly collected E.
miyabeanus specimens (KPM-NC 29301). D: Ascomata. E: Peridiopellis. F: Inner peridial tissue. G: Ascospores. H. Ascospores in various developmental stages
under SEM. Immature ascospores are covered with an evanescent, plate-like, warty layer (arrow). I: SEM photomicrograph of an ascospores of E. anthracinus
collected from Japan (KPM-NC 29304). Arrows indicate evanescent, outermost veil-like materials. Bars: A, D = 1 cm; B–C, E–G = 20 µm, H–I = 10 µm.
図 2．A–C: タイプ産地（野幌森林公園）で発生が確認されたコクロツチダンゴ E. nopporensis（E. miyabeanus の乾燥した小型の老菌）標本（KPM-NC 25100）．A:
子嚢果． B–C: 子嚢胞子． ほとんどの子嚢胞子で胞子外壁が収縮し、 内部の暗褐色の胞子中心部と分離していることが確認される （矢印）． D–H: 本研究において新たに採集されたクロツチダンゴ E. miyabeanus 標本 （KPM-NC 29301; 山梨県南都留郡鳴沢村産）． D: 子嚢果． E: 最外殻皮． F: 殻皮実質の菌糸． G: 子嚢胞子． H: 様々な成熟段階の子嚢胞子の SEM 画像． 未熟な胞子はいぼ状突起のある消失性の胞子最外層に覆われる （矢印）． I: 日本産
E. anthracinus 子嚢胞子の SEM 画像 （KPM-NC 29304 ； 愛媛県北宇和郡鬼北町産）． 矢印は消失性の胞子最外膜を示す．

8© The Japanese Association for Truffle Science (JATS), 2022

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Orihara et al.: Elaphomyces miyabeanus taxonomy and phylogeny

Bayesian PPs. The total arithmetic and harmonic mean of estimatedPhylogenetic analyses

marginal lnL for runs were -3456.18 and -3490.43, respectively.

The ITS dataset was composed of 607 aligned nucleotide positions
and contained 32 sequences in total, including newly generated

The resulting overall ML and Bayesian topologies were identicalsequence of E. miyabeanus specimen (KPM-NC 29301; GenBank

although statistic support of some branches varied considerablyaccession no.: OM286885). In the RAxML analysis, the final ML

among the three methods (i.e., MLBS/SH-aLRT/PP). We therefore

optimization of log likelihood (lnL) was -3510.721128. In the PhyML

show only the RAxML tree (Fig. 3). The tree mostly recovered overall

analysis, the final lnL was -3570.619692. In the Bayesian inference,

relationships within Elaphomyces sect. Ceratogaster shown by Paz etthe two parallel runs reached convergence after ca. 1.9M generations.

al. (2017) with some minor exceptions (e.g., the relationships among

Accordingly, we discarded the first 1900 trees in each run, and theE. aculeatus Vittad., E. virgatosporus Hollós and E. anthracinus

remaining 1101 trees in each run were summarized to approximate

Vittad. clades). The E. miyabeanus specimen from Yamanashi Pref.,
KX238805 E. anthracinus f. talosporus SPAIN*

62/0.00/0.91
78/1.00/0.90

KX238804 E. anthracinus SPAIN
KX238803 E. anthracinus SPAIN*
KR029774 E. anthracinus NORWAY
KX238813 E. anthracinus SPAIN
KR029773 E. anthracinus NORWAY
KJ594995 Elaphomyces sp. MEXICO

55/1.00/0.78

100/1.00/1.00

KJ594996 Elaphomyces sp. MEXICO

Subsect. Sclerodermei

KC791026 UECM of Quercus kelloggii USA, CA

100/1.00/1.00

KR029771 Elaphomyces sp. NORWAY
KR029769 Elaphomyces sp. NORWAY

71/1.00/0.88

KR029770 Elaphomyces sp. NORWAY

92/1.00/0.60
95/1.00/1.00

Elaphomyces
miyabeanus

OM286885 E. miyabeanus JAPANKX238811 E. virgatosporus SPAIN
100/1.00/1.00

KX238821 E. aculeatus SPAIN
KX238844 E. aculeatus SPAIN

97/1.00/1.00
62/1.00/0.93 98/1.00/1.00
59/1.00/0.98

KM576390 Elaphomyces sp. ECM of Quercus robur ROMANIA

AB218094 UECM of Quercus crispula M0400 JAPANKX238816 E. leucosporus SPAIN

KX238799 E. maculatus SPAIN

83/1.00/1.00

KX238807 E. septatus SPAIN

92/1.00/1.00

KX238796 E. spirosporus SPAIN*

61/1.00/0.94- /1.00/0.52

KX238814 E. leonis SPAIN
KX238854 E. morettii var. echinatus NORWAY

KX238856 E. leveillei SPAIN*

82/1.00/0.99

MH496798 Elaphomyces sp. USA
100/1.00/1.00

100/1.00/1.00

KT694140 E. iuppitercellus CAMEROON
78/0.98/0.84

MH399885 Elaphomyces sp. USA, FL

69/1.00/0.91

MF074844 Elaphomyces sp. USA, FL

86/1.00/1.00

KX238828 E. persoonii SPAIN
KY825739 Elaphomyces sp. PANAMA

0.1 substitutions / site

Sect.
Ascoscleroderma

KX238797 E. foetidus SPAIN*

Fig. 3. RAxML Maximum likelihood (ML) tree of Elaphomyces sect. Ceratogaster based on the nuclear rDNA ITS dataset.
Sequence of E. miyabeanus specimen (OM286885) was generated for this study. Values of RAxML rapid bootstrapping(MLBS), the SH-aLRT test by PhyML and Bayesian PPs are designated above or below branches or at nodes (MLBS/
SH-aLRT/PP). The GenBank accession numbers are shown followed by taxon names as registered in GenBank.
Sequences from holotype, lectotype or epitype specimens are indicated as asterisks (*). Species of Elaphomyces sect.
Ascoscleroderma are selected as outgroups. UECM: uncultured ectomycorrhizal root.
図 3． 核 rDNA ITS 領域のデータセットに基づく， RAxML によるツチダンゴ属 Ceratogaster 節の最尤系統樹． クロツチ
ダンゴ E. miyabeanus 標本から新たに得られた配列 （OM286885） を含む． RAxML 高速ブートストラップ法 （MLBS），
PhyML による SH 補正近似尤度比検定 （SH-aLRT test）， およびベイズ事後確率 （PP） の値を各分岐に示す （MLBS/
SH-aLRT/PP）． 本節の姉妹群に相当する， ツチダンゴ属 Ascoscleroderma 節に含まれる種を外群に用いた． UECM:
uncultured ectomycorrhizal root.
© The Japanese Association for Truffle Science (JATS), 2022

9

Truffology - Volume 5, Issue 1, 2022

Orihara et al.: Elaphomyces miyabeanus taxonomy and phylogeny
Japan was placed within sect. Ceratogaster subsect. Sclerodermei

Based on these observations, we conclude that E. nopporensis is(Vittad.) Bellanger & P.-A. Moreau and was shown to be closelya small, dried, old, overmature ascoma of E. miyabeanus and should

related to European E. anthracinus, Elaphomyces sp. from Mexico

be synonymized into the latter species. The characteristic large warts

and an environmental ECM sample from the USA. The ITS1-5.8S-

on the surface of E. miyabeanus ascomata apparently fall off on

ITS2 nucleotide identities between E. miyabeanus and E. anthracinus

old ascomata (= E. nopporensis) or are absent on smaller ascomata.

were 95.46% (589 bp / 617 bp; query coverage: 100%). Elaphomyces

We herein designate lectotype of E. miyabeanus from the syntype

miyabeanus formed a species-level clade with unidentified

specimens preserved in TMI. The lectotype specimen (TMI 37400),

Elaphomyces sp. sequences from Norway (i.e., “Elaphomyces sp.

which was collected on 4 July 1926 by S. Imai, consists of many6” according to the taxon-name label by Larsson E. & Molia A.)

ascomata of various developmental stages and is morphologicallyalthough the support by Bayesian PP was unexpectedly low (i.e.,

one of the most well-preserved syntype specimens. Although we

PP = 0.60). The ITS1-5.8S-ITS2 nucleotide identities between E.

successfully obtained an ITS sequence from the specimen collectedmiyabeanus and one of “Elaphomyces sp. 6” were 98.54% (608 bp /from central Honshu (KPM-NC 29301), we will not designate

617 bp; query coverage: 100%).

epitype of E. miyabeanus until fresh ascomata in good condition with
its nucleotide sequences including the barcoding region are obtained

Taxonomynear the type locality.

Taxonomic relationship between E. miyabeanus and E. nopporensis
Imai (1929) noted that he repeatedly collected E. miyabeanus andElaphomyces miyabeanus S. Imai, Transactions of the Sapporo

E. nopporensis on the same days in the same locality (“Nopporo

Natural History Society 11: 35 (1930).

Forest”). However, the number of E. nopporensis specimens were

[MycoBank ID: 271439]

relatively small, and no young ascomata of the species were found= Elaphomyces nopporensis S. Imai, Transactions of the Sapporo(Imai, 1929). The peridium of the species is papery, which is a typical

Natural History Society 11: 36 (1930).

character of dried, old ascomata of black deer truffles, and the surface[MycoBank ID: 272439].

of both species are more or less rough although in E. miyabeanus

Figs. 1, 2A–H.

large pyramidal warts partially remain on the apical or rarely on the

Lectotype (designated here!): JAPAN, Hokkaido Pref., Ishikari

basal part (Imai, 1929). According to Imai (1929), the ascospores of

Prov., Nopporo Forest (the present Nopporo Forest Park), 4 Jul. 1926,

E. nopporensis are smaller than those of E. miyabeanus but their sizes

S. Imai, TMI 37400 [MycoBank Typification ID: 10006470].

overlap within the range of those of E. miyabeanus (i.e., 15–17.5 µm

Description: Ascomata solitary or gregarious, 3–30 mm in

in E. nopporensis vs. 15–22 µm in E. miyabeanus).

diam., rigid, hard, globose to subglobose, often with a slight

Our observation of the rediscovered syntypes of both the species

apical projection and irregular depression, surface black, almost

confirmed that the descriptions by Imai (1929) were mostly accurate,smooth to the naked eye but more or less rough under loupe or

except for the ascospore size of E. nopporensis. In some ascomata

stereomicroscope, often with pyramidal-obtuse warts around thethe ascospores were shrunken and the exosporium and outermost

apical projection and basal depression. Mycelia on the surface ofornamentation were often collapsed or separated from the inner core.

ascomata inconspicuous. Outer peridium thin, up to 0.4 mm thick,

As a result, many of the remaining ascospores were smaller in size

black, rigid, fragile, separable from inner peridium. Inner peridium

than those of the E. miyabeanus specimens, but in well-preserved

up to 2 mm thick, off-white to pale brown with a reddish tinge,

ascomata of E. nopporensis the ascospore size was larger than the

more or less firm, rubbery, evanescent when dry and overmature.

original description and close to that of E. miyabeanus (i.e., 15.2–21.9

Gleba filled with a black spore mass with white sterile veins runningµm; n = 30).

more or less radially. The outermost part of gleba appears as a pale

As noted above, the newly collected ascomata from the type
locality (KPM-NC 25100, 27903) represented typical macroscopic

reddish brown hyphal layer. Spore mass more or less firm whenimmature, becoming powdery at maturity. Odor not distinct.

features of E. nopporensis. Similar to the E. nopporensis syntypes, inOuter peridium and warts of thick-walled (0.7–3 µm), filamentous

most ascospores the outermost spore wall was shrunken and separated

or palisade-like, dark brown hyphae 2.5–5.5 µm broad and firmly

from the inner spore core (Fig. 2B–C). In other cases, ascospores

adherent to one another, running in several directions. Inner peridium

looked heavily depressed or somewhat angular, or only the outermostof whitish to pale brown, filamentous hyphae 2.5–10 µm broad,layer remained (Figs. 1H–I, 2B).

running perpendicularly in two directions, walls 0.6–1.5 µm thick.

10© The Japanese Association for Truffle Science (JATS), 2022

Truffology - Volume 5, Issue 1, 2022

Orihara et al.: Elaphomyces miyabeanus taxonomy and phylogeny
Glebal hyphae of densely interwoven, evanescent, partially inflated,revealed that the ascospores had a nearly complete reticulum fully or

dark brown, thin-walled (<1.1 µm) filamentous hyphae 3–10 µm

partially covered with an evanescent vail-like material (Fig. 2I). Those

broad, with inflated, clavulate, terminal cells. Asci not seen, but Imaicharacters match well with the description of the E. anthracinus(1929) described them as 8-spored. Ascospores 15.2–25.5 µm in

by Castellano et al. (2017). We therefore tentatively identify thediam. including ornamentation (average size in lectotype: 21.8 µm;

specimen KPM-NC 29304 as E. anthracinus.

standard deviation: 1.57 [n = 50]), globose, composed of multiple
wall layers: outermost walls 0.8–2 µm thick, dark brown, surface

Discussion

finely reticulate under SEM, densely covered with blunt to acute spines

Taxonomy of black Elaphomyces species, which mostly belong

0.6–2.5(–3) µm long partially adhered to each other to form a minute,to sect. Ascoscleroderma (Clémencet) Bellanger & P.-A. Moreau,

incomplete reticulum, often collapsed or separated from the inner

Ceratogaster and Malacodermei (Vittad.) Tul. & C. Tul., have longwall in old, overly dried specimens; in young ascospores the spiny

been neglected in Japan since Kobayasi (1960) despite the recent

ornamentation is completely or partially covered with evanescent,remarkable progress on global biodiversity of Elaphomyces (e.g.,plate-like, warty layer; inner walls 4–5 µm thick, apparently

Castellano et al., 2011, 2012a, b, c, 2016, 2018, 2021; Paz et al., 2017).

composed of 4–5 layers, but sometimes not distinct especially in old,

However, Hatakeyama & Orihara (2020) reported E. asahimontanus

overmature specimens, dark to medium brown.

Kobayasi, currently endemic to Japan, for the first time since

Habitat, distribution and season: mostly hypogeous but occasionally

Kobayasi (1960), and gave detailed morphology and ecology of the

sub-epigeous under various ectomycorrhizal tree species such as

species. In this study, we focused on two historically forgotten black

Quercus spp. and Abies veitchii; Japan and Norway, but potentiallydeer truffles, E. miyabeanus and E. nopporensis, and concluded thatbroadly distributed across northern Eurasia; summer to winter.

they were identical species. It should be noted that both species had

Japanese name: Kuro-tsuchidango (“kuro” = black, “tsuchidango”

not been collected for more than 80 years. Most known Japanese= deer truffle in Japanese).

Elaphomyces spp. were described more than a half century ago, andOther specimens examined: JAPAN, Hokkaido Pref., Ishikari

require critical reassessment and modern descriptions to incorporate

Prov., Nopporo Forest (the present Nopporo Forest Park), parasitizedthem into the current framework of Elaphomyces systematics.

by T. jezoense, 8 Oct. 1923, S. Imai, deposited in SAPA; ibid, 15 Nov.

This study is an important step to understand the diversity of black

1925, S. Imai, TMI 37402; ibid, 4 Jul. 1926, S. Imai, a syntype of E.

Elaphomyces species in East Asia.

nopporensis, TMI 37401 (duplicate in SAPA); ibid, parasitized by

Both E. miyabeanus and E. nopporensis were originally describedT. jezoense, 7 Oct. 1927, S. Imai, a syntype of E. nopporensis, TMI

by Imai (1929). They were, therefore, published on the same date

37403; ibid, 21 Oct. 1928, S. Imai, TMI 37404; ibid, parasitized byand have equal taxonomic priority (cf. Article 11.5 of the current

T. jezoense, 17 Oct. 1929, S. Imai, TMI 37405; ibid, 30 Oct. 1938, S.

version of the International Code of Nomenclature for algae, fungi,

Imai, TMI 37406; Ebetsu-shi, Nishi-nopporo, Nopporo Forest Park,and plants [Shenzhen Code, 2018]; https://www.iapt-taxon.org/

under Quercus crispula, 1 Oct. 2018, M. Ohmae, KPM-NC 25100;

nomen/pages/main/art_11.html). We adopt the name E. miyabeanus

Sapporo-shi Atsubetsu-ku, Atsubetu-cho-konopporo, Nopporo Forest

for the combined species because E. nopporensis is only represented

Park, near Hokkaido Museum and the Centennial Memorial Tower,by an overly dried, old ascomata and does not represent its typicalunder Q. crispula, 22 Sep. 2019, S. Hatakeyama, KPM-NC 27903;

morphology. As a result, E. miyabeanus will be treated as having

Yamanashi Pref., Narusawa Village, northeastern slope of Mt. Fuji,

a priority over E. nopporensis according to Article 11.5 of theunder Abies veitchii, alt. ca. 2100 m, 19 Sep. 2021, Y. Kaneko, KPM-

Shenzhen Code.

NC 29301.

Elaphomyces miyaeanus shares many morphological characteristics

Notes: We also collected several black ascomata that appeared quitewith E. anthracinus such as an almost smooth black peridial surface,similar to E. miyabeanus from Ehime Prefecture (Kitauwa-gun

blackish spore mass, medium-sized ascospores (20–24 µm) with low

Kihoku-cho, Narukawa Gorge, under Castanopsis cuspidata andornamentation (Castellano et al., 2018). However, E. miyabeanus

Quercus spp., 13 Dec. 2021, T. Teramoto, T. Orihara & K. Yamamoto,differs from the latter species in the peridium being partially covered

KPM-NC 29304). The ascomata were 10–22 mm in diam., and thewith large, pyramidal warts and the ascospore ornamentation forming

surface was almost smooth to the naked eye and lacked large warts.

an incomplete reticulation or more or less isolated spines. In addition,

Ascospores were almost the same in size as E. miyabeanus ascosporesthe young ascospores of E. miyabeanus are covered with a plate-(i.e., 18.5–23.1 µm; average 20.5 µm [n = 25]), but SEM observation

like, warty layer (Fig. 2H). Our ITS phylogeny also supported that

© The Japanese Association for Truffle Science (JATS), 2022

11

Truffology - Volume 5, Issue 1, 2022

Orihara et al.: Elaphomyces miyabeanus taxonomy and phylogeny
these two species were closely related but genetically distinct (Fig. 3).

kindly helped our search for herbarium specimens at SAPA andKobayasi (1960) and Japanese Society of Cordyceps Research (2014)

TMI, respectively. This study was financially supported by JSPS

recorded E. anthracinus from Japan, but they did not provide a detailed

KAKENHI Grant-in-Aid for Young Scientists (B) (nos. 17K15184),

morphological description. We collected ascomata morphologically

JSPS KAKENHI Grants 24680085, 20K06805 and the Grant-in-Aid

identical to E. anthracinus from Ehime Prefecture, Japan, andfrom Institute for Fermentation, Osaka (IFO). Additional fieldwork

confirmed that E. anthracinus and E. miyabeanus are morphologicallyand other research activities were financially supported by JSPS

distinguishable (Fig. 2I).

Invitation Fellowship for Research in Japan (Short-term) to MAC.

Paz et al. (2017) proposed E. anthracinus f. talosporus A. Paz& Lavoise for the E. anthracinus specimens that have globosepolyhedral ascospores, but no particular genetic divergence wasReferences

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