Bull. Natl. Mus. Nat. Sci., Ser. B, 36(3), pp. 107–115, August 22, 2010

A New Record of Elaphomyces guangdongensis(Elaphomycetaceae, Eurotiales, Fungi) from Taiwan
Kentaro Hosaka1*, Taiga Kasuya2, Hannah T. Reynolds3 and Gi-Ho Sung1
1

Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, 305–0005 Japan
2
Laboratory of Plant Parasitic Mycology,
Graduate School of Life and Environmental Sciences, University of Tsukuba,
Ten-nodai 1–1–1, Tsukuba, Ibaraki, 305–8572 Japan3
Department of Biology, Duke University, Durham, NC 27708 U.S.A.
*
E-mail: khosaka@kahaku.go.jp(Received 19 May 2010; accepted 23 June 2010)

Abstract During the mycological survey in Central Taiwan in 2009, a few specimens of thegenus Elaphomyces were collected in two localities. Their unusually black peridium and habitat in
Pasania stands are characteristics not known from any other species of Elaphomyces. More detailed morphological observations, however, revealed that they are E. guangdongensis, a species
known only from southeastern China and associated with Castanopsis. This is the first report of E.
guangdongensis outside the type locality, and also the first record of the species from Pasania
stands. Macroscopic and microscopic (both LM and SEM) illustrations as well as detailed descriptions of the specimens from Taiwan are provided.
Key words : Ascomycota, biogeography, Castanopsis, ectomycorrhizae, fungi, Pasania, taxonomy, truffle.

Introduction
The genus Elaphomyces (Elaphomycetaceae,
Eurotiales, Ascomycota) is an ectomycorrhizal
fungus (Theodorou and Reddell, 1991) known
from both Northern and Southern Hemispheres(Castellano et al., 1989). It is known as a “trufflelike” fungus because fruit bodies are usually produced below-ground (hypogeous). The higherlevel phylogeny of the kingdom Fungi revealed
that truffle-like forms have evolved multiple
times in distantly related lineages (Hibbett andThorn, 2001; Hosaka et al., 2006; Landvik et al.,1996). Most truffle-like fungi, however, belong to
one of two major phyla, i.e., Basidiomycota andAscomycota.
Within the Basidiomycota, it is strongly suggested that hypogeous, truffle-like forms have
evolved multiple times from above-ground (epigeous) ancestors (Hibbett and Thorn, 2001;

Peintner et al., 2001). It is hypothesized that thetrend of evolution from epigeous to hypogeous
forms is an adaptation for spore dispersal bysmall animals (Thiers, 1984). Indeed, some truffle-like fungi produce chemical odors to attract
small animals and insects (Pacioni et al., 1991).
Furthermore, many animals both from Northernand Southern Hemispheres are highly dependent
on fruit bodies of such truffle-like fungi for theirdiet (Claridge, 2002; Fogel and Trappe, 1978;
Johnson, 1996; Malajczuk et al., 1987).
Within Ascomycota, to which Elaphomyces
belongs, a vast majority of truffle-like taxa belong to the family Tuberaceae and related taxa in
the Pezizales, including the well-known edible
truffles in the genus Tuber (Hansen and Pfister,2006). The phylogenetic position and taxonomic
treatment of Elaphomyces, however, have been
controversial until relatively recently (Trappe,1979). One of the first phylogenetic studies with

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Kentaro Hosaka et al.

Elaphomyces was done by Landvik et al. (1996).
Their study clearly demonstrated that Elaphomyces is another independent lineage of the truffle-like form, and the genus is closely related toubiquitous molds such as Penicillium and Aspergillus. More recently, an additional genus
with epigeous fruit bodies and an ectomycorrhizal habitat, i.e., Pseudotulostoma, has been
described from Guyana (Miller et al., 2001;
Henkel et al., 2006) and Japan (Asai et al., 2004)
and demonstrated to be closely related to Elaphomyces (Masuya and Asai, 2004; Geiser et al.,2006). Currently, Elaphomyces and Pseudotulostoma are placed in the family Elaphomycetaceae in the order Eurotiales (Kirk et al.,2008).
It is noteworthy that species of the genus
Elaphocordyceps G.H. Sung & Spatafora are
known to be specific parasites of Elaphomyces(Sung et al., 2007; Nikoh and Fukatsu, 2000).
More than 18 species of Elaphocordyceps are
known to be parasites of Elaphomyces (Sung etal., 2007), and the monophyly of Elaphocordyceps indicates some level of co-evolutionary history between Elaphomyces and Elaphocordyceps. Because the fruit bodies of Elaphomyces
are produced below-ground and not easily obtained during the fieldwork, finding the fruit bodies of Elaphocordyceps is possibly a good indicator of the presence of Elaphomyces. Indeed, somespecies of Elaphomyces, such as E. nopporensis
S. Imai and E. japonica Lloyd, have been reported exclusively as a host of Elaphocordyceps(Imai, 1929).
During the fieldwork in Central Taiwan in
2009, a few specimens of Elaphomyces with peculiar morphology were collected from Pasania
stands and mixed stands of Pasania, Castanopsis,and Quercus spp. Morphological examinations
strongly indicated that the specimens belong toE. guangdongensis B.C. Zhang (Zhang, 1991), a
species hitherto known only from the type locality, southeastern China. To our knowledge, this is
the first report of E. guangdongensis in Taiwan,
and also from Pasania stands. The collections
from Taiwan are described and illustrated with a

discussion on the significance of this finding in
the biogeography of Elaphomyces.
Materials and Methods
Collecting sites, collecting scheme, and curationof specimens
Fieldwork was conducted by KH in the year of
2009 (November 7–14). The main collecting
sites were located in central Taiwan, including
Nantou County and Taichung County. A generalcollection scheme (Castellano et al., 1989) for
hypogeous, truffle-like fungi was followed. At
each collecting site, fruit bodies were collectedjust under the leaf litter by using a garden rake.
At the same time, potential ectomycorrhizal hosttrees near fruit bodies, mostly Pinaceae andFagaceae, were identified to species. If alternativeectomycorrhizal hosts were present near fruit
bodies, those were also recorded.
Each specimen was photographed and macroscopic observation was conducted. All specimens
were cut into half and dried with low heat andgood air circulation. In addition to dried materials, small fragments of glebal tissue from freshlycollected samples were soaked in DMSO buffer(Seutin et al., 1991) with an addition of 100 mM
Tris-HCl (pH 8.0) and 0.1 M sodium sulfite(Na2SO3) under 4°C, following the procedures of
Hosaka (2009) and Hosaka and Castellano(2008).
More detailed identification and description
were conducted after returning to the mycology
lab at the National Museum of Nature and Science, Japan. Specimens collected during thefieldwork were deposited at the fungal herbariumof the National Museum of Nature and Science,
Tsukuba, Japan (TNS).
Light and stereo microscopy
For light microscopic observations, a small
portion from the gleba was mounted in water, 3%
or 5% (w/v) KOH and 30% ethanol solution on
glass slides. Those samples were examined witha Leica DM LB microscope (Leica Microsystems
CMS GmbH, Wetzlar, Germany) under Nomars-

Elaphomyces in Taiwan

ki interference contrast. More than forty randomly selected ascospores were measured under alight microscope at 1000 magnification. Surface features of the peridium were also observed
by a stereo microscope at 6 magnification.
Scanning electron microscopy
The surface features of the ascospores wereobserved by scanning electron microscopy(SEM). For SEM, a small portion from the gleba
was dusted onto double-sided adhesive tape on a
specimen holder and coated with platinum-palladium using an E-1030 Ion Sputter Coater(Hitachi, Tokyo, Japan). They were examined witha S-4200 SEM (Hitachi, Tokyo, Japan) operating
at 20 kV.
DNA Preparation, PCR and sequencing
DNA from the specimens collected by KH wasextracted from the tissue fragments stored in
DMSO buffer. Tissues were ground under liquid
nitrogen using a mortar and pestle. DNA extractions used either the Qiagen Plant kit (Qiagen,
Mississauga, ON, Canada) following the manufacture’s protocols or modified CTAB extraction
followed by glass milk purification methods assummarized by Hosaka (2009) and Hosaka andCastellano (2008). DNA from the holotype specimen was extracted differently because no immature glebal tissues were available and only the
powdery spore mass was present. The spore mass
was subjected to bead-beating following the protocol of Hosaka and Castellano (2008), followedby modified CTAB extraction and glass milk
purification as above.
DNA sequence data were obtained from the internal transcribed spacer regions (ITS) and large
subunit (LSU) of the nuclear ribosomal DNA.
For amplifying the ITS region, the primer combination of ITS5 and ITS4 (White et al., 1990) wasused. For amplifying the LSU, the combinationof LR0R and LR5 (Vilgalys and Hester, 1990)
was used. PCR reactions were carried out using
20 m l reaction volumes each containing: 1 m l
Genomic DNA, 1 m l dNTP, (4 mM), 1 m l of eachprimer (8 m M), 0.5 units of Taq polymerase

109(TaKaRa, Tokyo, Japan), 2 m l MgCl2 (25 mM),
2 m l Bovine Serum Albumin (BSA). Cycling
parameters were 1 cycle of 94°C for 3 min, 30
cycles of 94°C for 1 min, 51°C for 30 s and 72°C
for 1 min, with a final extension at 72°C for 15
min. PCR products were electrophoresed in 1%
agarose gels stained with ethidium bromide andvisualized under UV light. When amplification
bands were confirmed, PCR products were thenpurified using the ExoSap-IT (Millipore, Molsheim, France) and directly sequenced using the
Big Dye Terminator Cycle Sequencing Kit(Applied Biosystems Inc., Norwalk, CT, USA),
following the manufacturer’s instructions.
Results and Discussion
DNA Preparation, PCR and sequencing
The ITS sequences were successfully obtained
from two specimens (KH-TW09-030 & 031,
with GenBank accession numbers HM357249
and HM357250, respectively). In addition, LSU
sequence was successfully obtained from one
specimen (KH-TW09-031 with GenBank accession number HM357248). No amplifications,however, were successful from the holotype specimen of E. guangdongensis (HKAS 60250) despite several attempts of DNA extraction andPCR (e.g., using different extraction methods,
concentrating extracted DNA using SpeedVac,
and amplifying shorter fragments). Although the
holotype specimen is less than 30 years old,heavy application of fumigation chemicals inmany herbaria in China (Fuqiang Yu, personalcommunication) might have influenced the quality of DNA. This needs to be solved by using internal primers to amplify even shorter fragments,
and/or by designing taxon specific primers for
Elaphomyces.
Two ITS sequences were identical. A comparison to an unpublished dataset of 207 Elaphomycetaceae ITS sequences, including 43 black
Elaphomyces species (Hannah Reynolds, Duke
University) showed no match for the Taiwanese
specimens. No sequences from E. guangdongensis, E. carbonaceus, or similar taxa are currently

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Kentaro Hosaka et al.

available from the public database. Althoughsequence data are lacking, almost identical morphological characters, geographical proximity(southeastern China and Taiwan), and similarity
in possible mycorrhizal hosts (Fagaceae) indicatethat the holotype specimen from southeastern
China and three specimens from Taiwan are conspecific. Future study is necessary to collectfresh materials of E. guangdongensis from thetype locality to confirm their taxonomic identity.
Taxonomy
Elaphomyces guangdongensis B.C. Zhang,
Mycol. Res. 95: 975. 1991. [Fig. 1–4]
Ascoma globose, subglobose, often irregularly
flattened, dark brown to black, 0.8–2.2 cm diam.,
surface with small black conical warts (Fig. 1A,
C, D, Fig. 3A, B) having pale yellowish toorangish mycelial strands (Fig. 3A, B) and often

covered by thick soil crusts (Fig. 1B, C, Fig. 2),
which are not easily separable from ascoma. Hyphae in the mycelial strands brownish under thecompound microscope, 2–4 m m diam., with walls
thin and smooth, septate (Fig. 3D). Peridium
inner layer 0.8–2 mm thick, fleshy and pliable,
white when young, brown to dark brown whenmature, eventually disintegrating and leaving
only outer layer (Fig. 2); outer layer 350–600 m m
thick including warts, carbonaceous, fragile,
composed of hyphae about 4.5–5 m m diam.,
blackish brown to dark brown, with walls 1 m m
thick; warts about 350 m m high, 150–500 m m
broad at the base, covered with parallel hyphae,surface often covered by exposed spores and appearing grainy under stereo microscope (Fig. 3A,
B). Gleba brown with cottony texture, somewhat
radially arranged when young, but soon becoming purplish black, a powdery spore mass (Fig.

Fig. 1. Ascomata of Elaphomyces guangdongensis. A. Subepigeous habit in Lianhuachih Research Center, Taiwan (KH-TW09-030). B. Fresh ascomata just excavated from the ground (KH-TW09-031). Note thick soil
crusts enclosing each fruit body. C. Close-up of a fresh fruit body, with partially exposed peridium (KHTW09-031). D. Close-up of a fresh fruit body, with almost completely exposed peridium. Bars1 cm.

Elaphomyces in Taiwan

2). Asci could not be observed. Ascospores globose, dark brown to blackish brown, 16.5–19(–22.5) m m diam. including ornamentation; ornamentation composed of parallel to spiral ridges
when viewed under light microscope (Fig. 3C),
under SEM parallel deep ridges formed by rowsof joined spines buried in gelatinous material(Fig. 4A–D).
Habitat: Hypogeous to subepigeous under leaf
litter but with little understory, in pure stands ofPasania cornea and in mixed woods of Pasania
cornea, Castanopsis uraiana, Quercus spp.,
Calocedrus sp. and Cryptomeria japonica.
Presumable ectomycorrhizal hosts: Pasania
cornea, Castanopsis uraiana, and Quercus spp.
Fruiting season: November.
Distribution: Known only from southeastern
China (Guangdong) and central Taiwan (Nantou
County).
Materials examined: Holotype: China,
Guangdong, Boluo, Mt. Luofoushan Natural
Reserve, 22 November 1988, B.-C. Zhang 568

111(HMAS 60250). Other collections: Taiwan, Nantou Co., Yuchi Township, Lianhuachih ResearchCenter [23°5518.7N, 120°5314.1E], alt.
678 m, 9 November 2009, K. Hosaka (KHTW09-030, KH-TW09-031; TNS); Nantou Co.,Sun Moon Lake, Hanbi trail [23°5146.3N,
120°5435.8E], alt. 775 m, 10 November 2009,
K. Hosaka (KH-TW09-038; TNS).
Remarks: This is the first record of E. guangdongensis from Taiwan. As far as we know, thisis the second locality officially reported, besides
the type locality in Guangdong Province, China,
for this species. This is a significant finding
because two specimens (KH-TW09-030 & 031)
were collected from pure stands of Pasania
cornea, which is most likely to be an ectomycorrhizal host of this species. The type specimen
was recorded from Castanopsis stands, and it
indicates that the species can form ectomycorrhizae with at least two distinct tree genera
belonging to Fagaceae. Mycorrhizal hosts of
KH-TW09-038 could not be identified because

Fig. 2. Cross sections of fresh fruit bodies of Elaphomyces guangdongensis (KH-TW09-030). Glebal color
changes from brown when immature (right and top left) to a purplish black, powdery mass when matured.
Note thick soil crusts covering each fruit body. Bar 1 cm.

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not only Pasania, but also Castanopsis uraiana
and Quercus spp. were present at the site. We
cannot eliminate the possibility that the species
can also form ectomycorrhizae with Quercus
spp. It is possible that E. guangdongensis can
associate with a broad range of host trees inFagaceae.
As discussed by Zhang (1991), E. guangdongensis is most similar to E. carbonaceus Corner& Hawker reported from Singapore (Corner and
Hawker, 1953). However, the sizes of ascosporesand peridial warts can clearly distinguish these
two species. Unfortunately, no DNA sequence
data are currently available from either E. guangdongensis or E. carbonaceus. Comparison of
DNA data from additional materials is critical for
future studies.

Biogeographical implication
Because of the geographical proximity ofsoutheastern China and Taiwan, it is not surprising to find that they share species of ectomycorrhizal fungi. It is, however, uncertain whether E.
guangdongensis expanded its distribution via
land bridges (i.e., vicariance) or by transoceanic
dispersal. Although there is only limited evidence, recent studies suggest that even belowground organisms, like truffles, can cross theoceans (Hosaka et al., 2008; Wedén et al., 2004)
or other geographical barriers such as mountains
ranges and valley systems (Murat et al., 2004;
Grubisha et al., 2007) to expand their distributions.
Castanopsis, one of the recognized ectomycorrhizal hosts for E. guangdongensis, is distributed
throughout the warm temperate regions of Japan,

Fig. 3. Peridial surface, ascospores, and hypha of Elaphomyces guangdongensis. A, B. Peridial surface understereo microscope. A. Specimen from Taiwan (KH-TW09-030). Arrows indicate pale yellowish to orangish
mycelial strands. B. Holotype specimen from China (HMAS 60250). Arrows indicate yellowish to orangish
mycelial strands. Bars500 m m. C. Ascospores under light microscope (KH-TW09-031). Note spore surface
ornamented with parallel ridges. Bar20 m m. D. Hypha with septa in peridium under light microscope.
Bar5 m m.

Elaphomyces in Taiwan

113

Fig. 4. Scanning electron micrographs of ascospores of Elaphomyces guangdongensis. A. Specimen from Taiwan (KH-TW09-030). Note spore ornamentation with parallel or spiral ridges. Bar5 m m. B. Specimen from
Taiwan (KH-TW09-031). Note distinct parallel ridges. Bar5 m m. C. Close-up of spore ornamentation (KHTW09-031). Note parallel deep ridges formed by rows of joined spines. Bar1 m m. D. Holotype specimen
from China (HMAS 60250). Bar5 m m.

where 14 species of Elaphomyces have been reported (Kobayasi, 1960). More intensive surveys,
especially from the southern parts of Japan(Ryukyu Islands, etc), may reveal a wider distribution of E. guangdongensis. In addition, Castanopsis (and other fagaceous trees) is widelydistributed in tropical and subtropical areas ofSoutheast Asia to South Pacific (i.e., New
Guinea). Surveys in these areas will be critical inclarifying the distribution of E. guangdongensis.
Acknowledgements
The authors thank Dr. Sheng-Hua Wu, National Museum of Natural Science (Taichung, Taiwan), Dr. Pi-Han Wang, Tunghai University, Mr.
Ming-Hsiu Kao, Ms. Ying-Hsuan Chen and otherstudents from Wang’s lab for facilitating thefieldwork by KH in Taiwan. Curators of the

HMAS herbarium kindly arranged and provided
the holotype specimen of Elaphomyces guangdongensis for morphological and molecular studies. Dr. Makoto Kakishima, University of Tsukuba, has facilitated the use of the SEM Lab.
Thanks also go to Yumiko Hirayama, Takako
Hosaka, Tsukasa Maejima, Yoshiaki Muramatsu,
Kazuo Nishibori, Aya Nomura, Megumi Ohtsuka
and Haruo Sakamoto for assisting our molecularand curatorial work. This research was financed
in part by the National Museum of Nature andScience, “Biodiversity inventory in the Western
Pacific region” project, a research grant(2009–2011) of the Institute of Fermentation,
Osaka, Japan, and JSPS grants-in-aid for young
scientists (B) to KH, and by the NSF-JSPS East
Asia Pacific Studies Institute to HR.

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