An aero-aquatic fungus, Peyronelina glomerulata, is shown to have teleomorphic affinities with cyphelloid basidiomycetes

Abstract

On decayed wood near a stream, tiny cyphelloid, hair-bearing, Flagelloscypha-like basidiomata were found coexisting with conidia of an aero-aquatic fungus, Peyronelina glomerulata. An isolate originating from the basidioma produced conidia of P. glomerulata by soaking the culture in water. Three additional strains originating from conidia of P. glomerulata produced immature basidiomata with basidium-like structures on the agar medium after about 4 months incubation. Fine structure of the hyphal septa of P. glomerulata was found to be of the dolipore type. Phylogenetic analysis based on sequences from the D1/D2 regions of the LSU rDNA showed that the strains from conidia and from a basidioma clustered together in the Flagelloscypha clade and nested within the Nia clade of Hymenomycetes. The culture studies and molecular phylogenetic analysis suggested that P. glomerulata has a Flagelloscypha teleomorph, a cyphelloid basidiomycete. The molecular data also indicate that P. glomerulata is phylogenetically related to the marine basidiomycetes, Nia and Halocyphina. Thus, this study revealed that cyphelloid basidiomycetes have evolved into both marine as well as freshwater habitats by morphological adaptations of the teleomorphs in the former and of the anamorph in the latter case.

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FULL PAPER
Mycoscience (2009) 50:156–164 © The Mycological Society of Japan and Springer 2009
DOI 10.1007/s10267-008-0467-8
K. Yamaguchi (*) · A. Nakagiri
Biological Resource Center (NBRC), Department of Biotechnology,
National Institute of Technology and Evaluation, 2-5-8
Kazusakamatari, Kisarazu, Chiba 292-0818, Japan
Tel. +81-438-20-5763; Fax +81-438-52-2329
e-mail: yamaguchi-kaoru@nite.go.jp
Y. Degawa
Kanagawa Prefectural Museum of Natural History, Kanagawa, Japan
Kaoru Yamaguchi · Yousuke Degawa · Akira Nakagiri
An aero-aquatic fungus,
Peyronelina glomerulata
, is shown to have
teleomorphic affi nities with cyphelloid basidiomycetes
1952; Fisher et al. 1976). This species inhabits submerged
decaying twigs and litter in streams and ponds, although
some records may suggest its fungicolous properties
[Arnaud 1952; the Fungal Records Database of Britain and
Ireland (FRDBI) managed by the British Mycological
Society and hosted by CABI, UK; the online catalogue of
International Collection of Micro-organisms from Plants
(ICMP) culture collection managed by Landcare Research,
New Zealand]. Crown-shaped conidia composed of central
20–30 subglobose cells and surrounding 7–17 arms are pro-
duced on the surface of the substrate in a moist atmosphere
(Figs. 1–3). Ecologically, P. glomerulata belongs to the aero-
aquatic fungi, because its conidia fl oat on the surface of
water by entrapping air inside the arms of the conidium
(Fig. 3) (Fisher et al. 1976; Nakagiri and Ito 1997). The
teleomorph of this species and its phylogenetic position are
unknown and have not been studied so far.
Cyphelloid to disk-shaped basidiomata bearing basidia
were found coexisting with conidia of P. glomerulata on
decaying wood near a stream in Odawara, Kanagawa Pre-
fecture, the mid-part of Japan, in April 2005 (Figs. 4–6). We
tried to confi rm the teleomorph–anamorph relationship of
these fungi by culture studies and also from molecular data.
The number of nuclei per cell and fi ne structures of the
hyphal septa were examined to confi rm the basidiomycete
affi nity of P. glomerulata.
The taxonomic position of P. glomerulata was investi-
gated by phylogenetic analysis of the large subunit (LSU)
rDNA (D1/D2 regions) by examining eight strains isolated
from conidia of P. glomerulata and one strain from a cyphel-
loid basidioma (Table 1).
Materials and methods
Fungal materials and strains
Submerged twigs, litter, and wood were collected from
streams and ponds in various parts of Japan (see Table 1)
and incubated in a moist chamber at room temperature
Received: September 8, 2008 / Accepted: November 14, 2008
Abstract On decayed wood near a stream, tiny cyphelloid,
hair-bearing, Flagelloscypha-like basidiomata were found
coexisting with conidia of an aero-aquatic fungus, Peyrone-
lina glomerulata. An isolate originating from the basidioma
produced conidia of P. glomerulata by soaking the culture
in water. Three additional strains originating from conidia
of P. glomerulata produced immature basidiomata with
basidium-like structures on the agar medium after about 4
months incubation. Fine structure of the hyphal septa of P.
glomerulata was found to be of the dolipore type. Phyloge-
netic analysis based on sequences from the D1/D2 regions
of the LSU rDNA showed that the strains from conidia and
from a basidioma clustered together in the Flagelloscypha
clade and nested within the Nia clade of Hymenomycetes.
The culture studies and molecular phylogenetic analysis
suggested that P. glomerulata has a Flagelloscypha teleo-
morph, a cyphelloid basidiomycete. The molecular data
also indicate that P. glomerulata is phylogenetically related
to the marine basidiomycetes, Nia and Halocyphina. Thus,
this study revealed that cyphelloid basidiomycetes have
evolved into both marine as well as freshwater habitats by
morphological adaptations of the teleomorphs in the former
and of the anamorph in the latter case.
Key words Aero-aquatic fungi · Cyphelloid fungi · Flagell-
oscypha · Peyronelina glomerulata · Teleomorph–anamorph
relationship
Introduction
Peyronelina is a monotypic genus containing Peyronelina
glomerulata Arnaud ex Fisher, Webster & Kane (Arnaud

157
(∼25°C) for at least 3 months. The material was examined
under a dissecting microscope regularly once a week for the
fi rst month, then every 4 weeks for the following 2 months.
Conidia and basidiomata that appeared on the substrates
were picked up with a needle and inoculated onto YM agar
(1% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt
extract, 1.5% agar, pH 5.6) containing tetracycline hydro-
chloride (50 mg/l). Nine strains [eight newly isolated strains
(including one Flagelloscypha strain) and one strain (NBRC
32867) maintained in NITE Biological Resource Center
(NBRC) collection] were used for this study (see Table 1).
Strains were maintained by culturing on potato sucrose agar
(PSA: extract from 200 g/l potato, 2% sucrose, 2% agar, pH
5.6) and potato carrot agar (PCA: extract from 20 g/l potato,
extract from 20 g/l carrot, 2% agar, pH 6.0) at room
temperature.
Culture studies to examine teleomorph–anamorph
relationship
For inducing the teleomorph in culture, Peyronelina strains
(NBRC 104517, NBRC 104518, NBRC 104521) were inocu-
lated on sterilized twigs, which were stuck on 50 ml solid
PCA medium in a 500-ml Erlenmeyer fl ask. Sterilized water
was added in the fl ask to keep the twigs wet. Some fl asks
were incubated at room temperature for at least 4 months,
12
3
Figs. 1–3. Conidia of Peyronelina glomerulata. 1, 2 Crown-shaped
conidium composed of central subglobose cells and arms. 3 Conidia
produced on the surface of a twig. Note the released conidia fl oating
on the water. 1, 2 Scanning electron micrographs; 3 dissecting micro-
graph. Bars 1, 2 10 μm; 3 50 μm
5
6
4
Figs. 4−6. Cyphelloid to disk-shaped basidiomata of Flagelloscypha-
like teleomorph on natural substrates. 4 Cyphelloid basidiomata
(arrows) and conidia of Peyronelina glomerulata (arrowhead) on
decaying wood near a stream. 5 Cyphelloid hair-bearing basidioma. 6
Basidia bearing four sterigmata (arrows) produced on basidioma. 4
Dissecting micrograph; 5, 6 light micrographs. Bars 4 0.1 mm; 5 100 μm;
6 10 μm

158
while the others were incubated at 4°C for a month after
preincubation at room temperature for a month, then trans-
ferred into the program incubator (CFH-300; Tomy Seiko,
Tokyo, Japan). The following light–dark cycle and tempe-
rature were employed: 12 h light period at 20°C, 12 h
dark period at 10°C, for simulating early spring natural
conditions.
To induce conidium production, a strain isolated from
the basidioma (NBRC 104516) was incubated as follows:
the colony of NBRC 104516, which had been cultured on
PCA at 20°C, was cut into 5 × 5 mm agar blocks, then the
agar blocks were submerged into sterilized water and incu-
bated for 2–3 weeks at room temperature.
Scanning electron microscopy
Small piece of the natural substrate and PCA agar blocks
with conidia were fi xed with 1% OsO4 at 4°C for 12 h or at
room temperature for 2 h, then dehydrated in ethanol series
and fi nally substituted with isoamyl acetate. After critical-
point drying and coating with platinum-palladium, the spec-
imens were observed with a JSM-6060 (JEOL, Tokyo,
Japan) operated at 15 kV.
DAPI staining
Hyphal cells of the conidia-forming strains and conidial
cells were treated with 4′,6-diamidino-2-phenylindole
(DAPI) in VECTASHIELD Mounting Medium (Vector
Laboratories, Burlingame, CA, USA) by following the
manufacturer’s protocol. The number of nuclei per cell was
observed using an Axioplan2 imaging microscope (Carl
Zeiss MicroImaging, Tokyo, Japan).
Observation of fi ne structure of hyphal septum
Mycelia cultured on PCA plates were submerged in 3.5%
glutaraldehyde/1/15 M potassium phosphate buffer (pH
7.0) for 10–30 min for prefi xation. Agar disks about 2–3 mm
in diameter were cut out from mycelia and transferred to
vessels containing 3.5% glutaraldehyde/1/15 M potassium
phosphate buffer (pH 7.0) for 1 h at room temperature.
After washing fi ve times with 1/15 M potassium phosphate
buffer (pH 7.0), the sample was fi xed with 2% OsO4 solution
for 1 h at room temperature. After washing three times with
distilled water, the sample was dehydrated in ethanol series.
Ethanol was substituted by acetone and then Spurr’s resin
Tabl e 1. Strains used in this study
Taxon Strain Strain data Basidiomata
production
in culture
nuc-LSU rDNA
(D1/D2 regions)
accession no.
Peyronelina glomerulata AN-1505
(= NBRC 32867)
Dec. 11, 1995 Pond; Kamegajyou-ike,
Misaki-machi, Isumi-
gun, Chiba Pref.
Submerged
decaying culm
of Cyperus sp.
AB455963
6KY-12-10
(= NBRC 104517)
Mar. 5, 2003 Pond; Shinsekiya,
Kimitsu-shi, Chiba
Pref.
Submerged
decaying pod
AB455955
7KY-4-8
(= NBRC 104518)
Apr. 17, 2003 Pond; Jyouganji temple,
Nakajima, Kimitsu-
shi, Chiba Pref.
Submerged
decaying twig
䊊AB455956
20KY-7-6
(= NBRC 104520)
Sep. 22, 2005 Stream; Fuchigasawa,
Seiwa forest, Houei,
Kimitsu-shi, Chiba
Pref.
Submerged
decaying twig
䊊AB455957
21KY-6-3
(= NBRC 102381)
Sep. 30, 2005 Waterfall; Sugadaira
Kogen, Ueda-shi,
Nagano Pref.
Submerged
decaying litter
AB455958
29KY-5-10
(= NBRC 104521)
Apr. 22, 2007 Stream; Goshouzawa,
Iryuda, Odawara-shi,
Kanagawa Pref.
Submerged
decaying twig
AB455959
30KY-8-2
(= NBRC 104128)
Apr. 28, 2007 Pond; Sugadaira Kogen,
Ueda-shi, Nagano
Pref.
Submerged
decaying litter
AB455960
36KY-8-3
(= NBRC 104522)
Sep. 26, 2007 Stream; Iriomote Island,
Okinawa Pref.
Submerged
decaying twig
䊊AB455961
Flagelloscypha sp. 35KY-1-1
(= NBRC 104516)
Aug. 12, 2007 Stream; Goshouzawa,
Iryuda, Odawara-shi,
Kanagawa Pref.
Submerged
decaying wood
AB455962
Flagelloscypha japonica NBRC 101830
(= JCM 12855)
– – – – AB455964
Halocyphina villosa NBRC 32086 – – – – AB455965
Halocyphina villosa NBRC 32087 – – – – AB455966
Nia vibrissa NBRC 32089 – – – – AB455967
Nia vibrissa NBRC 32090 – – – – AB455968

159
(Spurr 1969). The sample was embedded in the resin and
solidifi ed by the following resin polymerizing program:
50°C for 5 h, then 70°C for 48–60 h. Thin sections of mycelia
were made using an ultramicrotome (Ultracut UCT; Leica
Microsystems, Wetzlar, Hessen, Germany) equipped with a
diamond knife, then picked up on the formvar-coated slot
grids. Sections were stained with 3% uranyl acetate for 2 h
followed by lead citrate (Reynolds 1963) for 5 min and
examined under a H-7600 transmission electron microscope
(Hitachi, Tokyo, Japan) operated at 100 kV.
DNA isolation and PCR amplifi cation
Mycelia cultured on PSA or PCA plates were harvested
using a spatula and put into 2-ml plastic tubes. DNA was
extracted using the Nucleon PhytoPure Genomic DNA
Extraction Kit (Amersham Biosciences, Piscataway, NJ,
USA). Polymerase chain reaction (PCR) was performed by
using the TaKaRa Ex Taq Hot Start Version Kit (Takara
Bio, Otsu, Shiga, Japan). LSU rDNA (D1/D2 region) frag-
ments were amplifi ed using primers NL1 and NL4
(O’Donnell 1993). A total 50 μl mixture [33.7 μl distilled
water, 5 μl DNA extract (template), 5 μl PCR buffer, 4 μl
dNTP, 0.3 μl Taq DNA polymerase, 1 μl each primer (fi nal
conc., 1 μM)] was employed for PCR. Amplifi cation of the
DNA fragments was performed using the GeneAmp PCR
System 9700 (Applied Biosystems, Foster City, CA, USA)
under the following thermal cycling program: an initial
denaturation at 95°C for 3 min, 30 cycles of denaturation
at 95°C for 30 s, annealing at 55°C for 30 s, extension at
72°C for 1 min, a fi nal extension at 72°C for 5 min, and a
4°C soak. Amplifi ed DNA was purifi ed by using the GFX
PCR DNA and Gel Band Purifi cation Kit (Amersham
Biosciences).
DNA sequencing
Sequencing reactions were performed using the Big Dye
Terminator v3.1 Cycle Sequencing Kit (Applied Biosys-
tems) and the primers NL1 and NL4 on the GeneAmp PCR
System 9700 (Applied Biosystems) under the following
program: an initial denaturation at 96°C for 3 min, 25 cycles
of denaturation at 96°C for 10 s, annealing at 50°C for 5 s,
extension at 60°C for 4 min, and a 4°C soak. Sequencing
reaction products were purifi ed with the Agencourt
CleanSEQ Kit (Agencourt Bioscience, Beverly, MA, USA)
and sequenced with the ABI PRISM 3130 Genetic Ana-
lyzer (Applied Biosystems). Contiguous sequences were
assembled by using the Sequencher ver. 4.7 (Gene Code,
Ann Arbor, MI, USA). Sequences obtained in this study
were deposited at DDBJ/EMBL/GenBank.
Phylogenetic analysis
Forty-eight sequences from 38 taxa of homobasidiomycetes
including cyphelloid fungi (by referring to Bodensteiner
et al. 2004) were downloaded from DDBJ/EMBL/GenBank.
The data set (42 taxa) including our own sequences of Pey-
ronelina, Flagelloscypha, Nia, and Halocyphina strains (see
Table 1) was aligned using Clustal X ver. 1.83 (Thompson
et al. 1997). The resulting alignment was manually refi ned
and gap positions were removed using Se-Al v2.0a11
(Rambaut 2007). The alignment was deposited at Tree-
BASE (http://www.treebase.org/treebase/index.html; study
No. S2217, matrix No. M4214). A phylogenetic tree was
constructed based on the neighbor-joining (NJ) method
(Saitou and Nei 1987) and the Knuc value (Kimura 1980) by
using Clustal X. The topology of the tree was evaluated by
the bootstrap resampling method (Felsenstein 1985) with
1000 replicates. The NJplot program (Perrière and Gouy
1996) was used for plotting the phylogenetic tree.
Results
Teleomorph–anamorph relationship
Cyphelloid hair-bearing basidiomata about 0.1 mm in diam-
eter, which resembled apothecia of discomycetes, were
found coexisting with conidia of Peyronelina glomerulata on
decaying wood near a stream in Odawara, Kanagawa Pre-
fecture in April 2005 (Fig. 4). Basidia bearing four sterig-
mata of the tapering spore-ejecting type were observed at
the upper part of the basidioma, but basidiospores were not
found (Fig. 6). The cyphelloid basidiomata were again
found on submerged decaying wood at a stream in Kana-
gawa Prefecture in August 2007 (Fig. 7). The strain (NBRC
104516) was obtained by isolating a culture from a single
basidioma. The culture of NBRC 104516 was found to
produce conidia of P. glomerulata after submerging PCA
agar blocks containing mycelia in water (Figs. 8, 9).
Isolates from conidia (NBRC 104518, NBRC 104520,
NBRC 104522) of P. glomerulata produced cup- to disk-
shaped immature basidiomata on PCA after incubation for
about 4 months at room temperature (Figs. 10–14). The
basidiomata show morphology of the genus Flagelloscypha,
Lachnella, or Pseudolasiobolus (Agerer 1983). The charac-
teristics of the surface hairs, i.e., tapering to the apex and
encrusted with fi nely or coarsely acicular crystals or rhombic
crystals (Figs. 10–14), indicate assignment of the fungus to
Flagelloscypha (Agerer 1975, 1979). Although the basidi-
omata on the agar media were further incubated for an
additional 2–3 months, basidia and basidiospores were not
found on them, whereas deformed basidia-like structures
and ejected basidiospores on the media around the basidi-
oma were observed (Figs. 15, 16). Attempts to induce sexual
reproduction by culturing on twigs with PCA medium in the
program incubator were unsuccessful as well, even after the
vernalization treatment (keeping the culture at 4°C for a
month).
The number of nuclei per cell was examined by DAPI
staining in the strains of P. glomerulata-producing conidia.
One or two (mainly two, rarely three or four) nuclei were
observed in each hyphal cell, and one to four nuclei were
observed in the subglobose cells and the arm cells of the
conidia (Figs. 17, 18).

160
79
8
Figs. 7−9. Cyphelloid to disk-shaped basidiomata of Flagelloscypha sp.
(NBRC 104516) and conidia of Peyronelina glomerulata produced by
the strain. 7 Basidiomata on decaying wood submerged in a stream.
8, 9 Conidia produced on submerged potato carrot agar (PCA) blocks
containing mycelia. 7 Dissecting micrograph; 8, 9 scanning electron
micrographs. Bars 7 0.2 mm; 8 50 μm; 9 10 μm
14 15
11
13
12
10
16
Figs. 10−16. Basidiomata and basidium-like structures produced by
Peyronelina glomerulata strains. 10 Basidiomata (arrows) produced on
potato carrot agar (PCA) after incubating for about 4 months. Note
coexisting conidia of Peyronelina glomerulata (arrowheads). 11, 14
Cup- to disk-shaped basidiomata with hairs. 12 Tapering hairs around
basidioma. 13 Surface of hairs. 15 Immature or deformed basidium-like
structures with sterigma-like projections (arrows) on the upper surface
of basidioma. 16 Discharged basidiospores scattered around basidioma
on agar. 10−12 NBRC 104518; 13−16 NBRC 104522. 10 Dissecting
micrograph; 11, 12 light micrographs; 13−16 scanning electron micro-
graphs. Bars 11 200 μm; 12 20 μm; 13 2 μm; 14 100 μm; 15 10 μm; 16
5 μm

161
18a
18b
17a
17b
17c
Figs. 17−18. Nuclear staining with 4′,6-diamidino-2-phenylindole
(DAPI) of Peyronelina glomerulata strain (NBRC 104518). 17a−c Veg -
etative hypha with hyphal septations (arrows). 18a,b Globose cells and
arm cells of a conidium. 17a, 18a Nomarski optics; 17b,c, 18b DAPI
staining. Bars 17 5 μm; 18 20 μm
19
CW
SS
PC
*
*
Fig. 19. Transmission electron micrograph of dolipore/parenthesome
septum of Peyronelina glomerulata strain (NBRC 104517). Pore cap or
parenthesome (PC), septal swelling (SS), electron-transparent zone
(asterisks), and cross wall (CW). Bar 100 nm
The ultrastructure of hyphal septa of P. glomerulata
strain (NBRC 104517) was observed to be a dolipore septum
surrounded by a pair of the perforated pore cap or paren-
thesome (Fig. 19), which is the typical type of septa of
agaricalean basidiomycetes (Kahn and Kimbrough 1982;
Markham 1994).
Phylogenetic analysis
Phylogenetic analysis of LSU rDNA (D1/D2 regions)
sequence data showed that eight strains from conidia
and one strain from a basidioma are all located within
the homobasidiomycetes and nested within the Nia clade
containing marine species (Nia, Halocyphina, and Calathella
mangrovei) and terrestrial species (Flagelloscypha,
Lachnella, Cyphellopsis, etc.) (Fig. 20). The Peyronelina
strains clustered together with Flagelloscypha minutissima
(Burt) Donk and F. japonica T. Handa & Y. Harada. These
topologies were supported by high (91%) and moderate
(74%) bootstrap values (Fig. 20; bootstrap values with
boldface).

162
Cyphellopsis –
Merismodes clade
Lachnella clade
Flagelloscypha
clade
Nia core clade
Nia clade
euagarics clade
/schizophylloid clade
Henningsomyces –
Rectipilus clade B
Henningsomyces –
Rectipilus clade A
/ marasmioid clade
/resupinatus clade
Coniophora olivacea AF098376
Paxillus involutus AF098385
Favolaschia peziziformis AY572008
Favolaschia calocera AY572007
Favolaschia calocera AY572006100
Entoloma eulividum AF261295
Entoloma undatum AF261314
Calathella columbiana AY570993
67
Phaeosolenia densa AY571019
Phaeosolenia densa AY571018100
Crinipellis stipitaria AY570997
Chaetocalathus liliputianus AY570996
Amyloflagellula inflata AY57099095
Rectipilus idahoensis AY571020
Henningsomyces puber AY571009
Henningsomyces sp. AY571011
Henningsomyces candidus AY571008
56
Rectipilus natalensis AY571021
Henningsomyces sp. AY571010
Limnoperdon incarnatum AF42 6958 F
Resupinatus applicatus AY571022
Resupinatus poriiformis AY57102598
100
50
Resupinatus conspersus AY571024
Calyptella capula AY570995
Calyptella capula AY57099499
64
Simocybe centunculus AF205707
Crepidotus mollis AF205677
Pellidiscus pallidus AY57101774
69
78
100
82
Schizophyllum radiatum AY571023
Auriculariopsis ampla AY570992
Auriculariopsis ampla AY570991100
Fistulina pallida AY571005
Fistulina hepatica AY571004
Fistulina antarctica AY571002
Fistulina antarctica AY57100385
86
100
100
Woldmaria crocea AY571026
Calathella mangrovei AF426 954 M
Halocyphina villosa NBRC 32087 M
Halocyphina villosa NBRC 32086 M
Nia vibrissa NBRC 32089 M
Nia vibrissa NBRC 32090 M
Nia vibrissa AF334750 M
99
100
100
76
Lachnella alboviolascens AY571012
Lachnella villosa AY571014
Lachnella villosa AY571013
99
Flagelloscypha japon ica NBRC 101830
Flagelloscypha minutissima AY571006
Peyronelina glomerulata NBRC 104128 F
Peyronelina glomerulata NBRC 102381 F
Peyronelina glomerulata NBRC 104522 F
Flagelloscypha sp. NBRC 104516 F
Peyronelina glomerulata NBRC 104521 F
100
Peyronelina glomerulata NBRC 104518 F
Peyronelina glomerulata NBRC 32867
Peyronelina glomerulata NBRC 104520 F
Peyronelina glomerulata NBRC 104517 F
90
79
95
100
100
68
65
91
99
74
66
Calathella gayana AY572005
Merismodes fasciculata AY571015
Cyphellopsis anomala AY571000
Merismodes fasciculata AY571016
Cyphellopsis anomala AY570999
Cyphellopsis anomala AY570998
67
61
100
98
88
100
0.01
Knuc
Coniophora olivacea AF098376
Paxillus involutus AF098385
Favolaschia peziziformis AY572008
Favolaschia calocera AY572007
Favolaschia calocera AY572006
Entoloma eulividum AF261295
Entoloma undatum AF261314
Calathella columbiana AY570993
Phaeosolenia densa AY571019
Phaeosolenia densa AY571018
Crinipellis stipitaria AY570997
Chaetocalathus liliputianus AY570996
Amyloflagellula inflata AY570990
Rectipilus idahoensis AY571020
Henningsomyces puber AY571009
Henningsomyces sp. AY571011
Henningsomyces candidus AY571008
Rectipilus natalensis AY571021
Henningsomyces sp. AY571010
Limnoperdon incarnatum AF42 6958 F
Resupinatus applicatus AY571022
Resupinatus poriiformis AY571025
Resupinatus conspersus AY571024
Calyptella capula AY570995
Calyptella capula AY570994
Simocybe centunculus AF205707
Crepidotus mollis AF205677
Pellidiscus pallidus AY571017
Schizophyllum radiatum AY571023
Auriculariopsis ampla AY570992
Auriculariopsis ampla AY570991
Fistulina pallida AY571005
Fistulina hepatica AY571004
Fistulina antarctica AY571002
Fistulina antarctica AY571003
Woldmaria crocea AY571026
Calathella mangrovei AF426 954 M
Halocyphina villosa NBRC 32087 M
Halocyphina villosa NBRC 32086 M
Nia vibrissa NBRC 32089 M
Nia vibrissa NBRC 32090 M
Nia vibrissa AF334750 M
Lachnella alboviolascens AY571012
Lachnella villosa AY571014
Lachnella villosa AY571013
Flagelloscypha japon ica NBRC 101830
Flagelloscypha minutissima AY571006
Peyronelina glomerulata NBRC 104128 F
Peyronelina glomerulata NBRC 102381 F
Peyronelina glomerulata NBRC 104522 F
Flagelloscypha sp. NBRC 104516 F
Peyronelina glomerulata NBRC 104521 F
Peyronelina glomerulata NBRC 104518 F
Peyronelina glomerulata NBRC 32867 F
Peyronelina glomerulata NBRC 104520 F
Peyronelina glomerulata NBRC 104517 F
Calathella gayana AY572005
Merismodes fasciculata AY571015
Cyphellopsis anomala AY571000
Merismodes fasciculata AY571016
Cyphellopsis anomala AY570999
Cyphellopsis anomala AY570998
96
Knuc
74
Fig. 20. Neighbor-joining phylogenetic tree inferred from large subunit
(LSU) rDNA (D1/D2 regions) sequence data (513 bp). Names of
aquatic taxa are in boldface. M, marine; F, freshwater. Bootstrap values
above 50% from 1000 replicates are indicated for the corresponding
branches. The names of clades refer to Bodensteiner et al. (2004).
Samples of which new sequences had been generated in this study are
given with strain numbers. Sequences downloaded from GenBank are
given with accession numbers
Discussion
The culture studies and phylogenetic analysis of the
LSU rDNA (D1/D2 regions) sequence data revealed
that Peyronelina glomerulata has a Flagelloscypha-like
teleomorph.
The morphology of basidiomata on decayed wood and
of those induced from P. glomerulata strains in culture cor-
responds with that of the genus Flagelloscypha (see Figs.
4–7, 10–14). However, basidiomata produced by P. glo-
merulata strains did not mature in culture. Our attempts to
induce maturation of basidiomata were unsuccessful. For
species-level identifi cation of the teleomorph, therefore,

163
further research on mature specimens formed on the natural
substrates or in culture is required.
One to four (mainly two) nuclei were observed in each
cell of P. glomerulata strains by DAPI staining (Figs. 17,
18). This observation indicates that conidia-forming hyphae
are probably dikaryotic (secondary hyphae). Further studies
on nuclear behavior are required to clarify whether mating
or self-duplication of nuclei is involved in the develop-
ment of dikaryons. Peyronelina glomerulata strain (NBRC
104517) was found to have hyphae with dolipore septa sur-
rounded by a perforated parenthesome, which has been
typically observed in homobasidiomycetes (Fig. 19) (Kahn
and Kimbrough 1982; Markham 1994). This dolipore struc-
ture is characteristic in having an electron-transparent zone
around a septal swelling, which was also observed in the
marine basidiomycetes Nia vibrissa and Digitatispora
marina (Brooks 1975). These fi ndings of dikaryotic cells and
the dolipore structure of the hyphal septa are clear proof
that P. glomerulata is a basidiomycetous anamorphic
fungus.
Most aero-aquatic fungi have been known as ascomyce-
tous anamorphs (Webster and Weber 2007). Only two aero-
aquatic fungi have been known to have basidiomycetous
teleomorphs, i.e., Aegerita candida Persoon and Aegeritina
tortuosa (Bourdot & Galzin) Jülich, the teleomorphs of
which are Bulbillomyces farinosus (Bresàdola) Jülich (Ken-
drick and Watling 1979) and Subulicystidium longisporum
(Patouillard) Parmasto (Kendrick and Watling 1979),
respectively. The Peyronelina–Flagelloscypha connection is
the fi rst case in which the teleomorph is a cyphelloid basidi-
oma-forming basidiomycete, as the teleomorphs of Aegerita
and Aegeritina produce corticioid basidiomata (Kendrick
and Watling 1979).
Molecular phylogenetic analysis showed that P. glomer-
ulata was nested within the cyphelloid homobasidiomycetes
and clustered with Flagelloscypha, terrestrial cyphelloid
basidioma-forming fungi (Fig. 20). Peyronelina glomerulata
strains were separately clustered into several clades (Fig.
20). This separation partly corresponds to the geographic
origins of the strains. Because we could not fi nd any differ-
ences among the strains in morphology and other pheno-
typic characters, we consider that all these strains belong to
a single species, P. glomerulata.
This study also showed that P. glomerulata is phyloge-
netically related to the marine basidiomycetes Nia vibrissa
R.T. Moore & Meyers and Halocyphina villosa Kohlmeyer
& E. Kohlmeyer (Fig. 20). Nia and Halocyphina are con-
sidered to have evolved from terrestrial cyphelloid fungi
and adapted to marine habitats by forming gasteroid basidi-
omata and appendaged or nondischarged basidiospores
(Nakagiri and Ito 1991; Jones and Jones 1993; Hibbett and
Binder 2001; Bodensteiner et al. 2004). Thus, these cyphel-
loid basidiomycetes succeeded in evolving into marine habi-
tats by modifying their teleomorph structures. No anamorph
is known from these marine basidiomycetes. On the other
hand, Peyronelina–Flagelloscypha showed apparent adap-
tation to freshwater habitats by its aero-aquatic anamorph
instead of its Flagelloscypha teleomorph, which has the ter-
restrial type of discharging basidia and basidiospores. These
phenomena may indicate that the terrestrial cyphelloid
basidiomycetes have adapted to marine and freshwater
environments by different strategies, i.e., by modifying tele-
omorphs and anamorphs, respectively.
Acknowledgments We gratefully thank Dr. Gen Okada, curator of
Japan Collection of Microorganisms (JCM), RIKEN Bioresource
center, for providing a Flagelloscypha japonica strain, and Dr. Aiko
Hirata, Department of Integrated Biosciences, Graduate School of
Frontier Sciences, The University of Tokyo, for her help in uranyl
acetate staining. We sincerely thank Dr. Kenji Tanaka, NBRC, for his
instruction in TEM observation and Ms. Kyoko Toyama, NBRC, for
her technical support on the sequence analysis. This work was sup-
ported in part by research grants (2006–2008 and 2007–2009) of the
Institute for Fermentation, Osaka, Japan.
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