Molecular assessment of fungi in “black spots” that deface murals in the Takamatsuzuka and Kitora Tumuli in Japan: Acremonium sect. Gliomastix including Acremonium tumulicola sp. nov. and Acremonium felinum comb. nov

Abstract

Unidentified black spots (or stains) appeared on the plaster walls of the Takamatsuzuka and Kitora Tumuli in the village of
Asuka, Nara Prefecture, Japan. Public attention was drawn to the biodeterioration of the colorful 1,300-year-old murals. A
total of 46 isolates of Acremonium sect. Gliomastix were obtained from various samples (mainly black spots) of the Takamatsuzuka Tumulus (TT) (sampling period, May 2004–December
2006) and the Kitora Tumulus (KT) (June 2004–May 2007). These isolates were assignable to four known taxa and a new species
in the ‘series Murorum’ sensu W. Gams as inferred from the integrated analysis of phenotypic and genotypic (i.e., ITS and 28S rDNA-D1/D2 sequences)
characters: these were Acremonium masseei, A. murorum, A. felinum comb. nov. with the neotype designation, A. polychromum, and A. tumulicola sp. nov., which have been accommodated in the validated series Murorum in the section Gliomastix. The black spots on the murals of the TT and KT were caused mainly by A. masseei and A. murorum, respectively.

KeywordsBiodeterioration–Black spots on murals–Cultural properties–Dark Acremonium
–Molecular systematics

Full text

FULL PAPER
Molecular assessment of fungi in ‘‘black spots’’ that deface
murals in the Takamatsuzuka and Kitora Tumuli in Japan:
Acremonium sect. Gliomastix including Acremonium tumulicola sp.
nov. and Acremonium felinum comb. nov.
Tomohiko Kiyuna
•
Kwang-Deuk An
•
Rika Kigawa
•
Chie Sano
•
Sadatoshi Miura
•
Junta Sugiyama
Received: 19 March 2010 / Accepted: 21 June 2010 / Published online: 7 August 2010
Ó The Mycological Society of Japan and Springer 2010
Abstract Unidentified black spots (or stains) appeared on
the plaster walls of the Takamatsuzuka and Kitora Tumuli
in the village of Asuka, Nara Prefecture, Japan. Public
attention was drawn to the biodeterioration of the colorful
1,300-year-old murals. A total of 46 isolates of Acremo-
nium sect. Gliomastix were obtained from various samples
(mainly black spots) of the Takamatsuzuka Tumulus (TT)
(sampling period, May 2004–December 2006) and the
Kitora Tumulus (KT) (June 2004–May 2007). These iso-
lates were assignable to four known taxa and a new species
in the ‘series Murorum’ sensu W. Gams as inferred from
the integrated analysis of phenotypic and genotypic (i.e.,
ITS and 28S rDNA-D1/D2 sequences) characters: these
were Acremonium masseei, A. murorum, A. felinum comb.
nov. with the neotype designation, A. polychromum, and
A. tumulicola sp. nov., which have been accommodated in
the validated series Murorum in the section Gliomastix.
The black spots on the murals of the TT and KT were
caused mainly by A. masseei and A. murorum, respectively.
Keywords Biodeterioration  Black spots on murals 
Cultural properties  Dark Acremonium 
Molecular systematics
Introduction
The anamorph-genus Acremonium sect. Gliomastix (here-
after abbreviated as dark Acremonium) is characterized by
having ‘‘chondroid hyphae’’ and short, rarely sympodially
proliferating phialides, which are usually darkly pigmented
ameroconidia in slimy heads or in dry, more or less per-
sistent conidial chains; it is also characterized by a lack of
chlamydospores (Gams 1971; Domsch et al. 2007). These
species appear commonly in soil, associated with plants, or
as airborne fungi (Gams 1971; Domsch et al. 2007).
The anamorph-genus Gliomastix Gue
´
guen (1905)is
typified by G. chartarum, which was recognized by Hughes
(1958) as a synonym of G. murorum (Corda) S. Hughes.
Sixty-three years after Gue
´
guen, the genus was mono-
graphed for the first time in 1968 (Dickinson 1968; Hughes
and Dickinson 1968) and characterized by darkly pig-
mented phialoconidia. Gliomastix, the so-called dark
Acremonium, was moved down by Gams (
1971) to a sec-
tion of Acremonium. The concept of Gliomastix murorum
with two varieties was discussed by Hammill (1981).
Recently, Acremonium sensu Gams (1971) with the soil-
borne species has been taxonomically revised and com-
piled by Gams in Domsch et al. (2007). Morphology-based
taxonomic treatments of dark Acremonium (Gams 1971)
T. Kiyuna and K.-D. An contributed equally to this work.
T. Kiyuna  K.-D. An
NCIMB Group, TechnoSuruga Laboratory Co., Ltd.,
330 Nagasaki, Shimizu-ku, Shizuoka, Shizuoka 424-0065, Japan
R. Kigawa  C. Sano  S. Miura
Independent Administrative Institution,
National Research Institute for Cultural Properties,
Tokyo, 13-43 Ueno Park, Taito-ku, Tokyo 110-8713, Japan
J. Sugiyama (&)
TechnoSuruga Laboratory Co., Ltd.,
Chiba Branch Office & Lab, 2102-10 Dainichi,
Yotsukaido, Chiba 284-0001, Japan
e-mail: jsugiyam@tecsrg.co.jp
Present Address:
K.-D. An
Microbe Division/Japan Collection of Microorganisms,
RIKEN BioResource Center, 2-1 Hirosawa, Wako,
Saitama 351-0198, Japan
123
Mycoscience (2011) 52:1–17
DOI 10.1007/s10267-010-0063-6

and Gliomastix (e.g., Dickinson 1968; Hughes and
Dickinson 1968; Hammill 1981) have not yet been chal-
lenged. Lechat et al. (2010) demonstrated the connection of
G. fusigera with a teleomorph in Hydropisphaera.This
genus, similar to Emericellopsis, the teleomorph of Acre-
monium in the strictest sense, is a member of the Bionec-
triaceae, as shown by molecular evidence. Because there
are too many hyaline-spored Acremonium species at the
borderline of these genera, we do not yet follow this
generic separation. On the other hand, there are a few
molecular phylogenetic studies on selected Acremonium
species (Glenn et al. 1996; Rossman et al. 2001; Seifert
et al. 2003; Zuccaro et al. 2003, 2004; Sigler et al. 2004;
Zare et al. 2007). To date, however, no world monographic
studies based on the integrated analysis of phenotypic and
genotypic characters have been made on the genus. Such
actions are urgently needed because the genus contains
taxa that are agriculturally, economically, and medically
important. In this article, we follow Gams’s concept of the
anamorph-genus Acremonium (Gams 1971) in a very broad
sense, in which Gliomastix was classified as a section
beside the other sections Acremonium and Nectrioidea.
In Europe, fungal biodeterioration is well known to affect
aspects of cultural heritage, such as murals (Dhawan et al.
1993; Guglielminetti et al. 1994; Berner et al. 1997;
Karbowska-Berent 2003). The roles of fungi in the deterio-
ration of murals, as well as their decay mechanisms, have
been reviewed by Garg et al. (1995) and Caneva et al. (2008).
Black stains (or spots) caused by dematiaceous anamorphic
fungi (e.g., Cladosporium) on the mural paintings often
cause problems in cultural heritage conservation (Arai et al.
1991; Ciferri 1999; Caneva et al. 2008). Fungal stains (or
spots) can be caused mainly by the secretion of metabolites
or the pigmentation of fungi, especially melanins (e.g.,
Diakumaku et al. 1995; Saiz-Jime
´
nez 1995; Nieto-Ferna
´
n-
dez et al. 2003
). Even after the fungus is dead, the pigmented
cell walls remain on the surface of the substratum. These
substances are particularly resistant to chemical and enzy-
matic degradation (Nieto-Ferna
´
ndez et al. 2003). Species of
dark Acremonium or Gliomastix have been implicated in the
biodeterioration of wall paintings by several authors; e.g.,
cave wall paintings in the Lascaux cave in France (Orial and
Mertz 2006; Orial et al. 2009), indoor mural paintings in
Europe (Nugari et al. 1993), Ajanta wall paintings in India
(Dhawan et al. 1993), and Ozuka Tumulus paintings in Japan
(Emoto and Emoto 1974).
In a cave with prehistoric (15,000-year-old) paintings in
Lascaux, France, in 2001 white molds identified as the
Fusarium solani (Mart.) Sacc. species complex (FSSC)
initially appeared on the cave wall (Orial and Mertz 2006;
Dupont et al. 2007). The next year, emergence of the black
mold Gliomastix murorum (sic) was reported by Orial and
Mertz (2006) and by Orial et al. (2009). In July 2007, novel
black colonizations were observed resulting from demati-
aceous molds of the anamorph-genera Ulocladium and
Scolecobasidium (Bastian and Alabouvette 2009; Orial
et al. 2009; Bastian et al. 2010).
Serious problems with black spots (or stains) on murals
occurred in the Takamatsuzuka Tumulus (hereafter abbre-
viated to TT) and the Kitora Tumulus (KT), both of which
are Special Historic Sites in Asuka-mura (the village of
Asuka), Nara Prefecture, Japan. Both TT and KT had
1,300-year-old mural (wall) paintings, which were drawn
directly onto thin plaster, in the small stone chamber
interior of each tumulus. After the tumuli were excavated,
molds appeared on the mural paintings at both sites (Arai
1984, 1987; Kigawa et al. 2006, 2009). In previous papers
we have reviewed the history of biological issues of both
tumuli (Kiyuna et al. 2008; Sugiyama et al. 2008, 2009;
Kigawa et al. 2009). In February 2001, renovation work
was done in the space adjacent to the stone chamber of TT.
Falling soil and leakage of rainwater had occurred because
the environmental preservation facility was aging (Kigawa
et al. 2009). In December 2001, after remediation work in
the space adjacent to the stone chamber of TT, a dark
Acremonium was isolated for the first time near a painting
named ‘‘blue dragon’’ (Seiryu) on east wall 2 and above a
painting named ‘‘white tiger’’ (Byakko) on west wall 2
(Kigawa et al. 2006, 2009
). In October 2002, black stains
appeared near the painting of blue dragon, east wall 2, and
the painting of women, east wall 3. It was too difficult to
remove the stains on site. From 2004 onward, viscous gels
(i.e., biofilms, which are mixtures of molds, yeasts, and
bacteria) also appeared on the wall plaster (Kigawa et al.
2009). Because of the continuing deterioration of fragile
supports (plaster walls and cut slabs of tuff stone) in
addition to the serious contamination and blackening of the
mural paintings, the Agency for Cultural Affairs decided to
dismantle the stone chamber in March 2005 to save and
restore the murals. In September 2005, cooling of the TT
mound was started to slow down fungal growth before the
stone chamber was dismantled. In February 2006, in spite
of this interior cooling, black spots appeared on paintings
of a group of four women, called the ‘‘Asuka beauties’’
(Asuka Bijin), on west wall 3. In May 2006, the tempera-
ture of the stone chamber interior was kept stable at about
108C, but the black spots expanded on the plaster walls
(Fig. 1) (Kigawa et al. 2007a, 2009). After the excavation
of the mound, the stone walls with the mural paintings
were moved to a restoration facility in the village of Asuka
by the end of August 2007.
Similar black spots were also seen on the walls of the
stone chamber interior of the KT in June and October 2006
(Fig. 1) (Kigawa et al. 2007b, 2008); thereafter, these spots
continuously developed further on the plaster walls in the
KT chamber (Kigawa et al. 2008; Sano et al. 2008).
2 Mycoscience (2011) 52:1–17
123

Initially, the identity of these fungi at species level
remained uncertain (Kigawa et al. 2007a; Sugiyama et al.
2009). To elucidate the cause of black spots of the TT and
KT murals, we surveyed the mycobiota from May 2004 to
December 2006 and obtained 46 isolates of dark Acre-
monium. In the course of an integrated analysis of phe-
notypic and genotypic characters, we attempted to identify
these isolates at species level. We provide here a sys-
tematic and nomenclatural treatment of Acremonium sect.
Gliomastix ‘series Murorum’ (herein validated), and pro-
pose Acremonium tumulicola sp. nov. based on three KT
isolates and Acremonium felinum comb. nov. with
neotypification.
Materials and methods
Sampling, isolating, and culturing
A total of 224 samples, which included moldy spots, vis-
cous gels (biofilms), and mixtures of plaster fragments and
soil, were collected from the stone chamber interior, from
spaces between the stone walls, and from the stone
chamber exterior of TT between May 2004 and August
2007. In addition, a total of 149 samples were collected
from the stone chamber interior and exterior of KT
between June 2004 and September 2007. The isolation
methods used were the smear and moist chamber methods
(Sugiyama et al. 2008, 2009; Kiyuna et al. 2008). The
isolates have been maintained on potato dextrose agar
(PDA; Nihon Pharmaceutical, Tokyo, Japan). Detailed data
on the isolates identified as Acremonium sect. Gliomastix
from both tumuli and accession numbers of DNA
sequences in GenBank are listed in Table 1. Twenty-one
selected living isolates are deposited as vouchers with the
Japan Collection of Microorganisms (JCM), RIKEN Bio-
Resource Center, Wako, Saitama Prefecture, Japan, as JCM
17164–17184 (Table 1). The remaining living isolates
from both tumuli are maintained at the Biology Laboratory
of the National Research Institute of Cultural Properties,
Tokyo, as lyophilized vouchers (Table 1).
Cultural and morphological observations
A total of 46 isolates, comprising 35 and 11 isolates from
TT and KT, respectively, were used in the cultural and
morphological observations. Detailed data on the isolates
are shown in Table 1. All isolates were grown using the
media and growth conditions mainly adopted by Gams
(1971). Isolates were incubated on malt extract agar (MA;
Oxoid, Cambridge, UK) at 208C, oatmeal agar (OA; Bec-
ton-Dickinson, Baltimore, MD, USA) at 208C, potato
dextrose agar (PDA; Nihon Pharmaceutical) at 208C, each
for 20 days in darkness. In the selected isolates, growth
rates were recorded using the average of three colony
diameters; i.e., one conidial suspension per MA plate was
Fig. 1 Black spots on the mural paintings in the Takamatsuzuka
Tumulus (a–f) and Kitora Tumulus (g, h). a A group of women, the
‘‘Asuka beauties,’’ on the west wall plaster [photograph taken on 17
May 2006 (sample no. T6517-1) by the Agency for Cultural Affairs,
Japan]. b–d Conidiophores and conidia in slide preparations made
directly from the part of the mural painting indicated by a red circle in
a. e Part of a group of women on the west wall plaster (photograph
taken on 13 December 2006 by the Agency for Cultural Affairs).
f Right part of the red line on the west wall (photograph taken on 17
May 2006 by the Agency for Cultural Affairs). g Black powder-like
colonies on the north wall [photograph taken on 27 October 2006
(sample no. K61027-1) by the Agency for Cultural Affairs]. h Conidia
in a slide preparation taken directly from the part of the substrata in
Fig. 1g. Bars b 20 lm; c, d, h 5 lm
Mycoscience (2011) 52:1–17 3
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Table 1 Strain data of Takamatsuzuka and Kitora isolates, with the GenBank accession numbers for rDNA sequences determined in this study
Species Isolate no. JCM no. Source
a
Sampling date GenBank accession no.
28S ITS
Acremonium masseei T4519-5-1 17164 White moldy colonies on the floor of the stone chamber of TT 19 May 2004 AB540433 AB540507
T6517-1-1 17165 Black spots on the paintings of the group of women on west wall 3 in the stone chamber of TT 17 May 2006 AB540434 AB540508
T6517-2-1 Black spots on the paintings of the group of women on west wall 3 in the stone chamber of TT 17 May 2006 AB540435 AB540509
T6517-3-1 Black spots above the paintings of the group of women on west wall 3 in the stone chamber of TT 17 May 2006 AB540436 AB540510
T6517-5-1 Black spots between the paintings of the moon and the white tiger on west wall 2 in the stone chamber of TT 17 May 2006 AB540437 AB540511
T6517-6-1 Black spots below the paintings of the moon on west wall 2 in the stone chamber of TT 17 May 2006 AB540438 AB540512
T6517-7-1 17166 Black spots above the painting of the white tiger on west wall 2 in the stone chamber of TT 17 May 2006 AB540439 AB540513
T6517-8-1 17167 White moldy colonies on the paintings of the group of women on east wall 3 in the stone chamber of TT 17 May 2006 AB540440 AB540514
T6517-11-1 Black spots on the paintings of the group of women on west wall 3 in the stone chamber of TT 17 May 2006 AB540441 AB540515
T6713-1-1 Black spots on the paintings of the group of women on west wall 3 in the stone chamber of TT 13 July 2006 AB540442 AB540516
T6713-2-1 Black moldy colonies above the paintings of the group of women on west wall 3 in the stone chamber of TT 13 July 2006 AB540443 AB540517
T6713-4-1 Black moldy colonies below the paintings of the moon on west wall 2 in the stone chamber of TT 13 July 2006 AB540444 AB540518
T6713-8-1 Black moldy colonies above the paintings of the group of women on east wall 3 in the stone chamber of TT 13 July 2006 AB540445 AB540519
T6713-12-1 Black moldy colonies on the northeast area of the ceiling (stone 3) in the stone chamber of TT 13 July 2006 AB540446 AB540520
T6713-14-1 Black moldy colonies above the group of men on west wall 1 in the stone chamber of TT 13 July 2006 AB540447 AB540521
T61017-1-1 Black spots on the paintings of the group of women on west wall 3 in the stone chamber of TT 17 October 2006 AB540448 AB540522
T61017-2-1 Black spots on the paintings of the group of women on west wall 3 in the stone chamber of TT 17 October 2006 AB540449 AB540523
T61017-3-1 Black spots above the paintings of the group of women on west wall 3 in the stone chamber of TT 17 October 2006 AB540450 AB540524
T61017-4-1 Black spots between the paintings of the moon and the white tiger on west wall 2 in the stone chamber of TT 17 October 2006 AB540451 AB540525
T61017-5-1 Black spots below the paintings of the moon on west wall 2 in the stone chamber of TT 17 October 2006 AB540452 AB540526
T61017-9-1 Black spots on northeast area of the ceiling (stone 3) in the stone chamber of TT 17 October 2006 AB540453 AB540527
T61017-10-1 Black spots on northwest area of the ceiling (stone 3) in the stone chamber of TT 17 October 2006 AB540454 AB540528
T61213-1-6 17168 Black moldy colonies and viscous gels on the paintings of the group of women on west wall 3 in the stone chamber of TT 13 December 2006 AB540455 AB540529
T61213-2-1 Blackish viscous gels on the paintings of the group of women on west wall 3 in the stone chamber of TT 13 December 2006 AB540456 AB540530
T61213-3-9 Black spots above the paintings of the group of women on west wall 3 in the stone chamber of TT 13 December 2006 AB540457 AB540531
T61213-4-3 Black moldy colonies between the paintings of the moon and the white tiger on west wall 2 in the stone chamber of TT 13 December 2006 AB540458 AB540532
T61213-5-4 Black moldy colonies below the paintings of the moon on west wall 2 in the stone chamber of TT 13 December 2006 AB540459 AB540533
T61213-6-1 17169 Viscous gels above the painting of the white tiger on east wall 2 in the stone chamber of TT 13 December 2006 AB540460 AB540534
T61213-9-1 Black moldy colonies on northeast area of the ceiling (stone 3) in the stone chamber of TT 13 December 2006 AB540461 AB540535
T61213-10-1 17170 Black moldy colonies on northwest area of the ceiling (stone 3) in the stone chamber of TT 13 December 2006 AB540462 AB540536
T61213-11-1 Black moldy colonies on the paintings of the group of women on west wall 3 in the stone chamber of TT 13 December 2006 AB540463 AB540537
T61213-15-1 Black moldy colonies below the paintings of the group of men on west wall 1 in the stone chamber of TT 13 December 2006 AB540464 AB540538
K61027-2-1 17171 Black powdered molds on the east wall in the stone chamber of KT 27 October 2006 AB540465 AB540539
Acremonium murorum T6713-14-2 17172 Black moldy colonies above the group of men on west wall 1 in the stone chamber of TT 13 July 2006 AB540466 AB540540
K6330-2 17173 Black needle-like molds on the east wall in the stone chamber of KT 30 March 2006 AB540467 AB540541
K6630-3-1 17174 Black moldy colonies on the ceiling in the stone chamber of KT 30 June 2006 AB540468 AB540542
K61027-1-1 17175 Black powdered molds on the north wall in the stone chamber of KT 27 October 2006 AB540469 AB540543
K61027-3-1 17176 Black sooty molds on the south wall in the stone chamber of KT 27 October 2006 AB540470 AB540544
K7511-1 17177 Black sooty molds in the east area of the north wall in the stone chamber of KT 11 May 2007 AB540471 AB540545
4 Mycoscience (2011) 52:1–17
123

inoculated in the center and incubated at 58,108,158,208,
258,308,378, and 408C, each for 7 days in the dark. The
colony colors of the isolates on all media were determined
by using the Kornerup and Wanscher color standard
(1978). Microscopic slides were prepared from portions of
the colonies grown on MA plate cultures and were
mounted in lactophenol mounting medium without dye
(Gams et al. 1987; Bills and Foster 2004). Microscopic
examinations were made using a BX51 microscope
(Olympus, Tokyo, Japan) with Normarski interference
contrast at up to 1,0009. All micrographs were taken with
a Coolpix 5000 digital camera (Nikon, Tokyo, Japan).
Phylogenetic analyses
DNA extraction, PCR amplification, and sequencing
The isolates and reference strains from various culture
collections used for the DNA sequencing are listed in
Tables 1 and 2. Their genomic DNA was extracted using
a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany)
according to the manufacturer’s instructions. The two
gene regions sequenced were the nuclear 28S rDNA D1/D2
region (hereafter abbreviated as 28S or rDNA D1/D2) and
internal transcribed spacer (ITS)–5.8S rDNA. The primers
used included NL1 and NL4 (O’Donnell 1993) for 28S,
ITS5, and ITS4 (White et al. 1990) for ITS. Polymerase
chain reaction (PCR) was performed using puReTaq
Ready-To-Go PCR beads (GE Healthcare, Buckingham-
shire, UK). Thermal cycling was performed using a
GeneAmp PCR System 9700 (Applied Biosystems, Foster
City, CA, USA). An initial denaturation at 958C for 5 min
was followed by 40 cycles of denaturation at 948C for 30 s,
annealing at 558C for 30 s, extension at 728C for 1 min,
and then a final extension at 728C for 10 min. The ampli-
fied DNA fragments were purified with a QIAquick PCR
Purification Kit (Qiagen). Direct sequencing for the PCR
products was performed using the BigDye Terminator
Cycle Sequencing Kit v3.1 (Applied Biosystems), and the
tubes were incubated in a GeneAmp PCR System 9700
(Applied Biosystems). The sequencing primers ITS5, ITS2,
ITS3, and ITS4 (White et al. 1990) were used for the
amplification of ITS, and the primers NL1, NL2, NL3,
and NL4 (O’Donnell 1993) for 28S. The completed
reactions were cleaned using a DyeExTM 2.0 Spin Kit
(Qiagen). Sequences were determined using electropho-
resis in an ABI3130xl DNA sequencer (Applied Bio-
systems). The sequences determined in this study were
deposited in GenBank/EMBL/DDBJ. Their accession
numbers are given in Tables 1 and 2. Other known
sequences downloaded for comparison for the respective
molecular phylogenetic analyses from GenBank are
shown in Table 3.
Table 1 continued
Species Isolate no. JCM no. Source
a
Sampling date GenBank accession no.
28S ITS
Acremonium felinum K4615-9 17178 Soil from the space between the west wall and soil flowing into the stone
chamber in area B of the stone chamber of KT
15 June 2004 AB540472 AB540546
Acremonium polychromum T6713-22-1a 17179 Air in adjacent room B of TT 13 July 2006 AB540473 AB540547
T6713-22-1b 17180 Air in adjacent room B of TT 13 July 2006 AB540474 AB540548
K5225-12-5 17181 Air from the north area in the adjacent small room of KT 25 February 2005 AB540475 AB540549
Acremonium tumulicola K5916-10-3 17182 Viscous substances on the stone wall in the adjacent small room of KT 16 September 2005 AB540476 AB540550
K6303-1-7 17183 Moldy colonies on the floor in the stone chamber of KT 3 March 2006 AB540477 AB540551
K6613-2
b
17184 White salt-like masses on the central part of the paintings of the vermillion bird
(Suzaku) on the south wall in the stone chamber of KT
13 June 2006 AB540478 AB540552
a
TT, Takamatsuzuka Tumulus; KT, Kitora Tumulus
b
Ex-type strain
Mycoscience (2011) 52:1–17 5
123

Table 2 Strain data and rDNA sequences determined in this study for comparison
Species
a
Section
b
Series
b
Strain no.
c
Source Location GenBank accession no.
28S ITS
Acremonium masseei Gliomastix Murorum CBS 794.69 Dung of rabbit Italy AB540479 AB540553
A. masseei Gliomastix Murorum CBS 557.75 Stem of Urtica dioica
together with
Leptosphaeria doliolum
Germany, Wu
¨
rzburg AB540480 AB540554
A. murorum Gliomastix Murorum JCM 23082 Wine cork – AB540481 AB540555
A. murorum var. murorum Gliomastix Murorum CBS 148.81 Forest soil USA, Georgia AB540482 AB540556
Gliomastix murorum var. murorum Gliomastix Murorum NBRC 31241 Soil West Germany AB540483 AB540557
‘G. murorum var. murorum’
d
Gliomastix Murorum NBRC 31044 Timber block of Fagus
crenata
– AB540484 AB540558
G. murorum var. murorum Gliomastix Murorum NBRC 8269 – – AB540485 AB540559
G. murorum var. murorum Gliomastix Murorum NBRC 9144 – – AB540486 AB540560
Acremonium murorum var. felina Gliomastix Murorum DAOM 22657 Soil Ottawa, Ontario, Canada AB540487 AB540561
A. murorum var. felinum Gliomastix Murorum CBS 147.81 Forest soil USA, Georgia AB540488 AB540562
Gliomastix murorum var. felina
d
Gliomastix Murorum NBRC 4871 – – AB540489 AB540563
‘G. murorum var. felina’
d
Gliomastix Murorum NBRC 8515 Soil UK AB540490 AB540564
‘G. murorum var. felina’
d,e
Gliomastix Murorum NBRC 8530 Salt-marsh soil UK AB540491 AB540565
G. murorum var. polychroma
d
Gliomastix Murorum NBRC 9440 Decaying carpophore of
Coriolus sp.
– AB540492 AB540566
Acremonium polychromum Gliomastix Murorum CBS 181.27 Bark of Hevea brasiliensis
(Euphorbiaceae)
Indonesia, Sumatra AB540493 AB540567
A. polychromum Gliomastix Murorum JCM 23084 Stony soil along the coast Papua New Guinea AB540494 AB540568
A. atrogriseum Gliomastix Murorum JCM 23068
T
Noodles Ukraine, Russia AB540495 AB540569
A. brachypenium Gliomastix – CBS 866.73
T
Dead stem of Cocos
nucifera (Palmae)
Sri Lanka AB540496 AB540570
A. cereale Gliomastix Murorum JCM 23071 Sand dune soil, A1 horizon UK AB540497 AB540571
A. dichromosporum Gliomastix – CBS 638.73
IT
Rhizosphere of Triticum
aestivum (Gramineae)
Western Australia AB540498 AB540572
A. longisporum Gliomastix ‘Luzulae’ JCM 23080 Dead leaf sheath of Musa
sapientum in greenhouse
The Netherlands AB540499 AB540573
A. luzulae Gliomastix ‘Luzulae’ JCM 23081 Decaying Picea wood Germany AB540500 AB540574
A. persicinum Gliomastix ‘Persicinum’ JCM 23083
T
Coastal sand under
Ammophila arenaria
France AB540501 AB540575
A. glaucum Acremonium – JCM 23076
T
Woolen overcoat Solomon Islands AB540503 AB540577
A. hansfordii Acremonium – CBS 390.73 Periconia cookei on
Dendrocalamus sp.
India, Bangalore AB540504 AB540578
A. alcalophilum Plectosphaerellaceae
f
– JCM 7366
T
Sludge from a compost
made of pig feces
Japan AB540505 AB540579
6 Mycoscience (2011) 52:1–17
123

Molecular phylogenetic analyses
The sequences were assembled using ChromasPro 1.42
(Technelysium, Tewantin, QLD, Australia). Three subsets
of the segment were also made into data sets: ITS, 28S, and
ITS plus 28S. Multiple alignments were performed using
CLUSTAL W version 1.83 (Thompson et al. 1994); the
final alignments were manually adjusted. Ambiguous
positions and alignment gaps were excluded from the
analysis. The neighbor-joining (NJ) tree was constructed
using the multiple alignments in MEGA ver3.1 (Kumar
et al. 2004), with 1,000 bootstrap replicates (Felsenstein
1985).
The phylogenetic reconstruction approach using a
Bayesian tree based on ITS plus 28S sequences (Rannala
and Yang 1996) was implemented using MrBayes v3.1.2
(Huelsenbeck and Ronquist 2001). The model of DNA
substitution was calculated using Modeltest2.2 (Nylander
2004). The results were obtained by the SYM ? I ? G
model. Bayesian Metropolis-coupled Markov Chain Monte
Carlo (MCMCMC) analyses (Mau et al. 1999) were per-
formed with MrBayes for phylogenetic estimation inferred
from the respective gene sequences. MrBayes was run for
2,500,000 generations. Searches were conducted with four
chains (three cold, one hot) with trees sampled every 100
generations. The average standard deviation of split fre-
quencies was 0.009 at the end of the run. The confidence
levels of nodes were measured by posterior probabilities
Table 3 List of taxa and the accession numbers of rDNA sequences
retrieved from GenBank
Species
a
GenBank accession no.
28S ITS
Acremonium murorum FJ176880 –
A. murorum var. felina AY283559 –
A. strictum AY138483 –
Ambrosiella xylebori DQ470979 –
Gliomastix murorum – AM921702
G. murorum – EU326188
G. murorum var. murorum – EF029216
G. murorum – EF495243
Heleococcum japonicum U17429 –
Hydropisphaera erubescens AF193230 –
H. erubescens AF193231 –
H. erubescens AF193229 –
H. erubescens AY545726 –
H. peziza AY489730 –
Nalanthamala squamicola AF373281 –
Roumegueriella rufula DQ518776 –
R. rufula EF469082 –
a
Species names are noted as registered in GenBank
Table 2 continued
Species
a
Section
b
Series
b
Strain no.
c
Source Location GenBank accession no.
28S ITS
A. rutilum Nectrioidea – JCM 23088
NT
Moist greenhouse wall Germany AB540506 AB540580
Wallrothiella subiculosa Anam. Pseudogliomastix – JCM 23118 Old leaf of Cocos
nucifera (Palmae)
Sri Lanka AB540502 AB540576
T, ex-type strain; IT, ex-isotype strain; NT, ex-neotype strain
–, Source and location are unknown
a
Species names are noted as registered in each Culture Collection
b
Section and series are taxonomic ranks of the genus Acremoniumadopted by Gams (1971, 1975); series names in single quotation marks are not validly published
c
CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; DAOM, Canadian Collection of Fungal Cultures, Ottawa, Canada; JCM, Japan Collection of Microorganisms, Wako,
Japan; NBRC, NITE-Biological Resource Center, Kisarazu, Japan
d
Strain names are doubtful in identification; details are noted in the article
e
Strain CBS 184.30 (=NBRC 8530) was reidentified by W. Gams as A. polychromum
f
Zare et al. (2007)
Mycoscience (2011) 52:1–17 7
123

obtained from the majority-rule consensus after deletion of
the trees during burn-in.
Results and discussion
Cultural and morphological characterization of dark
Acremonium isolates
A total of 46 representative isolates from the TT and KT
stone chamber interiors and exteriors, assignable to Acre-
monium sect. Gliomastix, were culturally and morpholog-
ically characterized (for details, see Table 1). Using the
integrated phenotypic (mentioned here) and genotypic
(mentioned below) analyses, these isolates were identified
as taxa of the ‘series Murorum’ (validated later in this
article) in sect. Gliomastix: Acremonium masseei (Sacc.)
W. Gams, A. murorum (Corda) W. Gams, A. felinum comb.
nov., A. polychromum (J.F.H. Beyma) W. Gams, and A.
tumulicola sp. nov. The cultural and morphological char-
acteristics of the respective taxa are fully described later in
this article (see Figs. 2, 3, 4, 5, 6).
As shown in Table 1, 33 representative strains of
A. masseei, which were isolated from a variety of sub-
strates such as black moldy spots and viscous gels (bio-
films) on plaster walls collected in different periods, are
thought to be genetically the same species. In this study,
A. masseei was isolated only from the stone chamber
interiors of the TT and KT. However, we could not detect
this species from the exterior of either the TT or KT stone
chamber. So far, A. masseei has been isolated from
soil, dung, and plant substrates (Gams 1971; Matsushima
1975; CBS Fungi Database: http://www.cbs.knaw.nl/fungi/
BioloMICS.aspx). This is the first case of isolation of
A. masseei from a biodeteriorated cultural heritage such as
mural paintings.
The optimum temperature for growth on MA, in the two
isolates (T4519-5-1 and T6517-1-1) identified as A. mas-
seei, was 208–258C after 7 days. However, the growth rate
of isolate T6517-1-1 was somewhat higher than that of
T4519-5-1 at 108C (Fig. 7). Therefore, strain T6517-1-1,
isolated after the stone chamber was cooled, is thought to
be active at the low-temperature conditions of the stone
chamber interior.
According to the official records (see the Agency
homepage concerning the TT, http://www.bunka.go.jp/
takamatsu_kitora/hekiga_hozonkanri.html), in December
2001, after remediation work in the space adjacent to the
stone chamber of TT, dark Acremonium [mentioned as
Acremonium (sect. Gliomastix
) sp. in Kigawa et al. 2006,
2009] was detected on the blue dragon painting on east
wall 2 and on the white tiger on west wall 2. The conidia-
bearing structure appearing in the microscopic photographs
in these records is very similar to that of our isolate T6713-
14-2, identified as A. murorum. However, the isolates
obtained by Kigawa and coworkers were not preserved as
vouchers and are no longer available for study. Therefore,
detailed comparison between the previously isolated strains
and our isolates is impossible. The results suggest, how-
ever, that A. murorum was growing in the stone chamber
interior for some time after 2001.
Molecular phylogenetic analyses of dark Acremonium
isolates
The 28S, ITS, and combined ITS–28S dataset contained 46
isolates of Acremonium sect. Gliomastix, i.e., 35 from the
TT and 11 from the KT (see Table 1), and 28 authentic
Fig. 2 Acremonium murorum (K7511-1). a Colonies on malt agar (MA) at 208C, 20 days. b Colonies on oatmeal agar (OA) at 208C, 20 days.
c Colonies on potato dextrose agar (PDA) at 208C, 20 days. d–g Conidiophores. h–k Conidia. Bars d 20 lm; f 10 lm; e, g–k 5 lm
8 Mycoscience (2011) 52:1–17
123

Fig. 3 Acremonium felinum (K4615-9). a Colonies on MA at 208C, 20 days. b Colonies on OA at 208C, 20 days. c Colonies on PDA at 208C,
20 days. d–h Conidiophores. i–k Conidia. Bars d 20 lm; f 10 lm; e, g–k 5 lm
Fig. 4 Acremonium tumulicola (K6613-2). a Colonies on MA at 208C, 20 days. b Colonies on OA at 208C, 20 days. c Colonies on PDA at 208C,
20 days. d–h Conidiophores. i, j Conidia. Bars d, f 10 lm; e, g–j 5 lm
Fig. 5 Acremonium masseei (T6517-1-1). a Colonies on MA at 208C, 20 days. b Colonies on OA at 208C, 20 days. c Colonies on PDA at 208C,
1 month. d–h Conidiophores. i–k Conidia. Bars d 20 lm; f 10 lm; e, g–k 5 lm
Mycoscience (2011) 52:1–17 9
123

strains obtained from the public culture collections/biore-
source centers (Table 2).
Using Acremonium alcalophilum JCM 7366
T
(which
belongs to the Plectosphaerellaceae according to Zare et al.
2007) as an outgroup taxon, the NJ tree was inferred from
1,018 bp of the ITS ? 28S sequences dataset. In the
ITS ? 28S phylogeny, all the TT and KT isolates appeared
in five clades: Masseei clade, Murorum clade, Felinum
clade, Polychromum clade, and Tumulicola clade (Figs. 8, 9).
Each clade was supported by a high bootstrap value
(99%) and high Bayesian posterior probability (1.00).
Most of the isolates showed good correlation between
the molecular phylogenetic placement and phenotypic
characteristics.
In the Masseei clade, our 33 isolates along with
A. masseei CBS 557.75 (ex stem of Urtica dioica,
Germany) and 794.69 (ex rabbit dung, Italy) formed a
monophyletic cluster with high bootstrap supports. In
Gams’s description (1971) of A. masseei, CBS 794.69 was
cited and illustrated as fig. 46, which nicely depicts the
morphological characteristics. There was no variation in
the ITS–28S gene sequences from 33 isolates of A. masseei
in the Masseei clade; 32 from TT and 1 from KT. On the
other hand, there is a three-nucleotide difference between
these isolates and A. masseei CBS 557.75 and 794.69. The
genetic diversity of this Masseei clade is not great. Our
molecular phylogeny (Figs. 8, 9) only suggests the exis-
tence of two haplotypes (i.e., a group of TT and KT iso-
lates, and one of the two CBS strains) within the Masseei
clade.
Three KT isolates (K5916-10-3, K6303-1-7, and K6613-2)
formed an independent branch named a novel clade 1
with a high bootstrap value (99%) and high Bayesian
posterior probability (1.00) as mentioned earlier, for
which we introduce the new species name, Acremonium
tumulicola.
Three TT and KT isolates (T6713-22-1a, T6713-221b,
and K5225-12-5) grouped together with JCM 23084 (ex
stony soil along the coast, Papua New Guinea) and CBS
181.27 of A. polychromum and NBRC 8530 (=CBS 184.30,
reidentified by W. Gams as A. polychromum; ex salt-marsh
soil, UK) of ‘G. murorum var. felina’. Among these strains,
CBS 181.27 [=Herb. IMI 62332 (ex bark of Hevea bra-
siliensis, Sumatra)] is the ex-type strain of Oospora poly-
chroma, which was cited and illustrated in Gams’s
description of A. polychromum. No base changes for the
28S and ITS sequences were detected among these six
strains. This assemblage is here called the Polychromum
clade.
One KT isolate (K4615-9) was accommodated within
the Felinum clade, which included DAOM 22657 (ex soil,
Ottawa, Ontario, Canada) and CBS 147.81 (ex forest soil,
Georgia, USA) of ‘A. murorum var. felinum’ (or as Glio-
mastix felina), and NBRC 31044 (ex timber block of Fagus
crenata, locality unknown) of ‘G. murorum var. murorum’.
DAOM 22657 was used for the molecular tree based on
partial large subunit (LSU) rDNA sequences (Seifert et al.
2003), whereas CBS 147.81 was examined by Hammill
(1981), who discussed the differences between
G. murorum
and G. felina. NBRC 31044 is listed in the NBRC online
catalogue (http://www.nbrc.nite.go.jp/e/index.html).
In the Murorum clade (Fig. 9), our six isolates, CBS
148.81 and JCM 23081 of A. murorum var. murorum, three
NBRC strains [NBRC 9144 = CBS 119.67, ex Camaro-
phyllus niveus (now Hygrocybe virginea), Netherlands;
NBRC 31241 = ATCC 16277, ex soil, Germany; NBRC
Fig. 6 Acremonium polychromum (K5225-12-5). a Colonies on MA at 208C, 20 days. b Colonies on OA at 208C, 20 days. c Colonies on PDA at
208C, 20 days. d, e Conidiophores. f Conidia. Bars d–f 10 lm
Colony diameter (mm)
Temperature (ºC)
0
5
10
15
20
25
30
35
40
5 10152025303740
T4519-5-1
T6517-1-1
CBS 557.75
Fig. 7 Effect of temperature on colony growth in Acremonium
masseei, Takamatsuzuka isolates (T4519-5-1 and T6517-1-1), and the
reference strain (CBS 557.75)
10 Mycoscience (2011) 52:1–17
123

8269, ex Herbarium IFO H-10916, K. Tubaki 103-21] of
G. murorum var. murorum, and NBRC 8515 (=IMI 52460,
ex soil, UK) of ‘G. murorum var. felina’ grouped together
with comparatively high bootstrap value (94%) and high
Bayesian posterior probability (1.00). CBS, JCM, and
NBRC strains named A. murorum var. murorum or
G. murorum var. murorum are authentic in having pheno-
typic characteristics that fit Gams’s (1971, in Domsch et al.
2007) and Hammill’s (1981) descriptions and illustrations
for A. murorum/G. murorum. However, there is a slight
difference between the 11 strains placed in the Murorum
clade in their ITS–28S gene sequences, as shown in Fig. 9.
A first molecular phylogeny of the genus Acremonium,
including sect. Gliomastix, by Glenn et al. (1996), revealed
that the genus is polyphyletic. In that paper, only one
species, A. murorum, was sampled from sect. Gliomastix.
In our molecular phylogeny (see Fig. 9), taxa of sect.
Gliomastix series Murorum (A. masseei, A. murorum, and
A. polychromum) were accommodated in a cluster with
high bootstrap support (99%) and high Bayesian posterior
probability (1.00). However, the remaining branches were
longer and comparatively independent and contained spe-
cies of sects. Acremonium (A. glaucum and A. hansfordii)
and Nectrioidea (A. rutilum). The identities of several
strains with the names G. murorum var. polychroma NBRC
9940 (ex decaying carpophore of Coriolus sp.), ‘G.
murorum var. felina’ NBRC 8530, and ‘G. murorum var.
murorum’ NBRC 31044 and 8515, all of which have been
used in this study, should be carefully reconsidered in
future monographic research.
Tumulicola
Clade
28S
533bp
NJ
Acremonium masseei CBS 557.75
Acremonium masseei CBS 794.69
T6517-8-1
T61213-9-1
T6713-1-1
T61213-4-3
T61213-5-4
T6517-2-1
T6517-11-1
T6713-8-1
T61213-3-9
T61213-1-6
T61017-3-1
T6517-1-1
T6517-5-1
K61027-2-1
T61017-9-1
T61213-15-1
T6517-6-1
T61213-6-1
T6517-3-1
T61017-5-1
T6517-7-1
T61213-10-1
T61017-4-1
T6713-14-1
T61213-2-1
T61017-10-1
T61213-11-1
T61017-1-1
T6713-4-1
T4519-5-1
T6713-2-1
T61017-2-1
T6713-12-1
Gliomastix murorum var. murorum NBRC 9144
K6330-2
Acremonium murorum JCM 23082
Gliomastix murorum var. murorum NBRC 8269
T6713-14-2
‘Gliomastix murorum var. felina’NBRC 8515
Gliomastix murorum var. murorum NBRC 31241
K7511-1
Acremonium murorum var. murorum CBS 148.81
K61027-3-1
K61027-1-1
Acremonium murorum FJ176880
K6630-3-1
K5916-10-3
K6613-2
T
K6303-1-7
Gliomastix murorum var. polychroma NBRC 9440
‘Gliomastix murorum var. murorum’ NBRC 31044
Acremonium murorum var. felina DAOM 22657
K4615-9
Acremonium murorum var. felinum CBS 147.81
NT
Acremonium murorum var. felina AY283559
Acremonium polychromum JCM 23084
K5225-12-5
T6713-22-1b
‘Gliomastix murorum var. felina’ NBRC 8530
Acremonium polychromumCBS 181.27
T6713-22-1a
Acremonium luzulae JCM 23081
Hydropisphaera erubescens AF193230
Acremonium cereale JCM 23071
Acremonium persicinum JCM 23083
T
Nalanthamala squamicola AF373281
Hydropisphaera erubescens AF193231
Hydropisphaera erubescens AF193229
Hydropisphaera erubescens AY545726
Hydropisphaera peziza AY489730
Heleococcum japonicum U17429
Roumegueriella rufula DQ518776
Roumegueriella rufula EF469082
Acremonium rutilum JCM 23088
NT
Acremonium longisporumJCM 23080
Acremonium glaucum JCM 23076
T
Acremonium strictum AY138483
Acremonium hansfordii CBS 390.73
Acremonium dichromosporumCBS 638.73
IT
Acremonium brachypenium CBS 866.73
T
Acremonium atrogriseum JCM 23068
T
Acremonium alcalophilumJCM 7366
T
Gliomastix murorum var. felina NBRC 4871
Wallrothiella subiculosa JCM 23118
Ambrosiella xylebori DQ470979
99
99
99
73
93
96
79
98
99
73
78
52
58
54
60
78
74
99
67
90
92
62
59
63
73
52
62
88
65
99
0.02
Masseei
Clade
Murorum
Clade
Felinum
Clade
Polychromum
Clade
T6713-8-1
T61213-4-3
T61017-5-1
T6713-4-1
T6517-2-1
T6713-12-1
T6713-14-1
T61017-9-1
T61017-3-1
T61017-10-1
T61017-2-1
T4519-5-1
T61213-10-1
T6517-6-1
T61213-15-1
T6517-5-1
T6713-1-1
T61213-5-4
T6517-11-1
T6713-2-1
T6517-3-1
T61017-4-1
T61213-1-6
T61213-6-1
T61213-11-1
T6517-8-1
T61213-2-1
T6517-7-1
T6517-1-1
T61213-9-1
T61017-1-1
T61213-3-9
K61027-2-1
Acremonium masseei CBS 557.75
Acremonium masseei CBS 794.69
Gliomastix murorum var. polychroma NBRC 9440
‘Gliomastix murorum var. felina’ NBRC 8530
T6713-22-1b
K5225-12-5
T6713-22-1a
Acremonium polychromumCBS 181.27
Acremonium polychromum JCM 23084
K5916-10-3
K6613-2
T
K6303-1-7
Acremonium murorum var. felina DAOM 22657
Gliomastix murorum EU326188
‘Gliomastix murorum var. murorum’ NBRC 31044
Acremonium murorum var. felinum CBS 147.81
NT
Gliomastix murorum AM921702
K4615-9
K61027-1-1
K61027-3-1
K6630-3-1
T6713-14-2
Gliomastix murorum var. murorum EF029216
Acremonium murorum JCM 23082
K6330-2
K7511-1
‘Gliomastix murorum var. felina’ NBRC 8515
Gliomastix murorum var. murorum NBRC 31241
Gliomastix murorum EF495243
Gliomastix murorum var. murorum NBRC 8269
Gliomastix murorum var. murorum NBRC 9144
Acremonium murorum var. murorum CBS 148.81
Acremonium luzulae JCM 23081
Acremonium longisporumJCM 23080
Acremonium cereale JCM 23071
Acremonium persicinum JCM 23083
T
Acremonium atrogriseum JCM 23068
T
Acremonium rutilum JCM 23088
NT
Acremonium glaucum JCM 23076
T
Acremonium hansfordii CBS 390.73
Acremonium dichromosporumCBS 638.73
IT
Acremonium brachypenium CBS 866.73
T
Gliomastix murorum var. felina NBRC 4871
Wallrothiella subiculosa JCM 23118
Acremonium alcalophilum JCM 7366
T
99
74
72
76
55
96
99
82
91
53
64
84
82
57
90
85
96
84
99
94
61
0.02
Masseei
Clade
Polychromu
m
Clade
Felinum
Clade
Murorum
Clade
Tumulicola
Clade
ITS
394bp
NJ
Fig. 8 Phylogenetic relationships among 46 Takamatsuzuka Tumu-
lus (TT) and Kitora Tumulus (TK) isolates and 28 known Acremo-
nium (sensu lato) species and with the accession numbers downloaded
from GenBank based on neighbor-joining (NJ) analysis of 28S rDNA-
D1/D2 and internal transcribed spacer (ITS)–5.8S region sequence
data of 533 and 394 aligned nucleotide sites, respectively, using
MEGA ver3.1. Numbers on the branch nodes represent bootstrap
support values (%) based on 1,000 replications; bootstrap values
greater than 50% are indicated. Branches significantly supported by
bootstrap values above 95% are shown with thick lines. T and
K indicate isolates from the TT and KT, respectively. Filled squares
indicate the isolates from the respective stone chamber interiors, and
filled triangle from the adjacent space or small room of both tumuli.
Right vertical bars indicate the phylogenetic group in this study.
T
,
IT
,
and
NT
indicate ex-type, ex-isotype, and ex-neotype strains, respec-
tively. Single quotes (‘’) indicate that species names are doubtful in
identification
Mycoscience (2011) 52:1–17 11
123

A discussion on the roles of dark Acremonium spp. in
the biodeterioration of mural paintings and plaster walls,
and also on the invasion route to the TT and KT stone
chamber interiors, will be published elsewhere.
Systematics
A total of 46 TT and KT isolates herein identified have
been accommodated in the ‘series Murorum’ proposed by
Gams (1971). However, his proposal lacked a Latin diag-
nosis/description and typification. Therefore, we validate
this taxon here:
Acremonium sect. Gliomastix series Murorum W. Gams
ex Kiyuna, An & Sugiy., ser. nov.
Acremonium sect. Gliomastix series Murorum W. Gams,
in Cephalosporium-artige Schimmelpilze (Hyphomycetes),
p. 81. 1971, nom. inval. Art. 36, 37.1.
Series in sectione Gliomasticis. Conidia pigmentata,
parva, minus quam 4.5 lm lata, sursum rotundata, laevia
vel incrustata, non costata.
Species typica: Acremonium murorum (Corda) W. Gams
The brief or full descriptions of five species of Acre-
monium sect. Gliomatsix series Murorum are provided
below.
ITS-28S
1018bp
NJ / bayes
T6713-2-1
T61017-4-1
T6517-11-1
T61213-6-1
T61017-10-1
T61213-1-6
T6713-12-1
T61213-10-1
T61213-2-1
T6713-8-1
T61017-1-1
T61017-9-1
T6517-3-1
T6517-8-1
T6517-1-1
T6713-4-1
K61027-2-1
T6517-5-1
T61017-2-1
T61213-5-4
T6713-1-1
T4519-5-1
T61213-11-1
T6517-2-1
T61213-15-1
T61213-4-3
T61213-9-1
T6517-7-1
T6517-6-1
T61017-3-1
T61017-5-1
T61213-3-9
T6713-14-1
Acremonium masseei CBS 557.75
Acremonium masseei CBS 794.69
K5916-10-3
K6303-1-7
K6613-2
T
Gliomastix murorum var. polychroma NBRC 9440
Acremonium polychromum JCM 23084
T6713-22-1a
K5225-12-5
T6713-22-1b
Acremonium polychromum CBS 181.27
‘Gliomastix murorum var. felina’NBRC 8530
Acremonium murorum var. felina DAOM 22657
K4615-9
Acremonium murorum var. felinum CBS 147.81
NT
‘Gliomastix murorum var. murorum’ NBRC 31044
K6630-3-1
K7511-1
K61027-3-1
Acremonium murorum var. murorum CBS 148.81
K61027-1-1
T6713-14-2
K6330-2
Gliomastix murorum var. murorum NBRC 9144
Gliomastix murorum var. murorum NBRC 31241
Gliomastix murorum var. murorum NBRC 8269
‘Gliomastix murorum var. felina’ NBRC 8515
Acremonium murorum JCM 23082
Acremonium luzulae JCM 23081
Acremonium cereale JCM 23071
Acremonium persicinum JCM 23083
T
Acremonium longisporum JCM 23080
Acremonium rutilum JCM 23088
NT
Acremonium glaucum JCM 23076
T
Acremonium hansfordii CBS 390.73
Acremonium dichromosporum CBS 638.73
IT
Acremonium brachypenium CBS 866.73
T
Acremonium atrogriseum JCM 23068
T
Gliomastix murorum var. felina NBRC4871
Wallrothiella subiculosa JCM 23118
Acremonium alcalophilum JCM 7366
T
99 / 1.00
74 / -
99 / 1.00
89 / -
92 / 0.96
64 / 0.59
96 / 0.97
92 / 1.00
69 / 0.70
86 / 0.98
59 / 0.99
99 / 1.00
68 / 0.83
96 / 0.92
63 /
63 / 0.82
94 / 1.00
97 / 1.00
95 / 0.98
99 / 1.00
96 / 0.81
0.02
Masseei
Clade
Polychromum
Clade
Felinum
Clade
Murorum
Clade
99 / 1.00
99 / 1.00
Sect. Nectrioidea
Sect. Acremonium
Sect. Gliomastix
Series Murorum
‘Series Persicinum’
‘Series Luzulae’
‘Series Persicinum’
Series Murorum
Series Murorum
Series Murorum
Tumulicola
Clade
‘Series Luzulae’
Fig. 9 Phylogenetic
relationships among 46 TT and
TK isolates and 28 known
Acremonium (sensu lato)
species and with the accession
numbers downloaded from
GenBank based on NJ and
Bayesian analyses of combined
ITS–5.8S and 28S rDNA-D1/D2
region sequence data of 1,018
aligned nucleotide sites using
MEGA ver3.1. Numbers on the
branch nodes represent
Bayesian posterior probability
and bootstrap support values
(%) based on 1,000 replications;
bootstrap values greater than
50% are indicated. Branches
significantly supported by
bootstrap value and Bayesian
posterior probability above 99%
and 1.00, respectively, are
shown by thick lines. Further
details as in Fig. 8. Section and
series names according to Gams
(1971, 1975)
12 Mycoscience (2011) 52:1–17
123

Acremonium murorum (Corda) W. Gams, Cephalospo-
rium-artige Schimmelpilze (Hyphomycetes), p. 84, 1971.
Fig. 2
:Torula murorum Corda, Icon. Fung. 2:9, 1838
(basionym).
:Gliomastix murorum (Corda) S. Hughes var. murorum,
Can. J. Bot. 36:769, 1958.
=Gliomastix chartarum Gue
´
guen, Bull. Soc. Mycol. Fr.
21:240, 1905.
Colonies on MA reaching 70–80 mm diameter in 10 days
at 208C in darkness, white (25A1), floccose, reverse con-
colorous. Colonies on OA reaching 70–80 mm diameter in
10 days at 208C in darkness, blackish brown (5F8), velvety
to floccose, reverse concolorous. Colonies on PDA reach-
ing 65–70 mm diameter in 10 days at 208C in darkness,
blackish brown (5F8), floccose, reverse concolorous.
Hyphae hyaline, septate, 1.5–2 lm wide. Conidiation
plectonematogenous. Phialides mostly borne singly or 2–3
on a short subtending cell, smooth walled, sometimes
forming a collarette, 20–40 (–50) lm long, tapering from
2–2.5 lm near the base to 1–1.5 lm. Conidia mostly
adhering in heads, rarely in chains, blackish brown (5F8),
warted, ellipsoidal to subglobose, with truncate base,
4–5 9 2–3 (–4) lm. Chlamydospores absent.
Gene sequence data: The GenBank accession numbers
for nuclear 28S rDNAD1/D2 and ITS regions are listed in
Table 1. Isolates examined: one isolate from surfaces of
murals of the stone chamber interior of the TT; T6713-14-2
(July 2006) and five isolates from the murals and walls of
the stone chamber interior of the KT, from March 2006 to
May 2007; K6330-2, K6630-3-1, K61027-1-1, K61027-3-1,
and K7511-1. The detailed data on these isolates are shown
in Table 1.
The phenotypic characteristics of the six isolates agreed
well with the previous descriptions (Dickinson 1968; Gams
1971; Matsushima 1975; Hammill 1981).
Acremonium felinum (Marchal) Kiyuna, An, Kigawa &
Sugiy., comb. nov. Fig. 3
MycoBank no.: MB 518430
:Periconia felina Marchal, Bull. Soc. R. Bot. Belg.
34:141, 1895 (basionym).
:Gliomastix murorum var. felina (Marchal) S. Hughes,
Can. J. Bot. 36:769, 1958.
:Gliomastix felina (Marchal) Hammill, Mycologia
73:231, 1981.
Neotype designated here: A dried culture of CBS 147.81
in herb. TNS F-37403: USA, Clarke County, Georgia, in
forest soil, 1968, isol. et det. T. M., Hammill, No. 33 (date
of accession in CBS: February 1981, received as Glio-
mastix felina).
Colonies on MA reaching 60–70 mm diameter in
10 days at 208C in darkness, white (25A1), floccose,
reverse concolorous. Colonies on OA reaching 30–40 mm
diameter in 10 days at 208C in darkness, blackish brown
(5F8), velvety to floccose, zonate, reverse concolorous.
Colonies on PDA reaching 30–40 mm diameter in 10 days
at 208C in darkness, blackish brown (5F8), floccose,
reverse concolorous.
Hyphae hyaline, septate, 1.5–2 lm wide. Conidiation
plectonematogenous. Phialides mostly single, sometimes
on a short subtending cell, smooth walled, with walls
darkly encrusted in the upper part, sometimes forming a
collarette, 20–40 (–50) lm long, tapering from 2–2.5 lm
near the base to 1–1.5 lm. Conidia mostly adhering in
heads, blackish brown (5F8), some smooth walled and
some rough walled, ellipsoidal, with truncate base,
4–6 9 2–2.5 lm. Chlamydospores absent.
Gene sequence data: The GenBank accession numbers
for nuclear 28S rDNA D1/D2 and ITS regions are listed in
Table 2. Isolates examined: one isolate (K4615-9) from
soil from the space between the west wall and soil flowing
into the stone chamber in area B of the stone chamber of
the KT, 15 June 2004.
The phenotypic characteristics of this isolate (K4615-9)
agreed with descriptions of Gliomastix murorum var. felina
(Dickinson 1968) and G. felina (Hammill 1981). Because
of the occurrence of intermediate strains, Gams (1971)
included G. murorum var. felina in the synonymy
A. murorum. Although certain isolates showed quite pro-
nounced differences, the taxonomic situation around
A. murorum var. murorum and A. murorum var. felinum has
remained unsettled. Acremonium murorum var. murorum
was distinguished from the variety felinum by the for-
mation of more subglobose conidia in chains (Domsch
et al. 2007;Crousetal.2009). Our observations support
the specific distinction of A. felinum from A. murorum as
suggested by Hammill (1981). During a review process of
the manuscript, W. Gams (personal communication)
suggested to propose the designation of neotype because
thetypematerialofPericonia felina Marchal is missing.
We designate a collection CBS 147.81 as neotype of
A. felinum agreeing with the morphology-based brief
descriptions by Hammill (1970
, 1981). It has been char-
acterizedbyDNAsequencedata(rDNAD1/D2andITS)
in this study.
Acremonium tumulicola Kiyuna, An, Kigawa & Sugiy.,
sp. nov. Fig. 4
MycoBank no.: MB 518348
Ab Acremonio felino differt conidiis ellipsoideis basi
rotundata, laevibus et sequentiis regionum rDNA D1/D2 et
ITS.
Mycoscience (2011) 52:1–17 13
123

Holotype: Japan, Nara Prefecture, Asuka-mura, on the
white salt-like masses on the central part of the painting
named the vermillion bird (Suzaku) on the south wall in
the Kitora Tumulus stone chamber interior, 13 June
2006. Herb. TNS F-37402 (dried culture); the ex-type
strain has been deposited in Japan Collection of Micro-
organisms, RIKEN BioResource Center as JCM 17184
(originally as isolate K6613-2); it is also maintained in
Centraalbureu voor Schimmelcultures (CBS), Utrecht as
CBS 127532.
Etymology: The specific epithet ‘‘tumulicola’’ refers to
the habitat (tumulus) where the sample was collected.
Colonies on MA reaching 30–40 mm diameter in
10 days at 208C in darkness, white (25A1), floccose to
funiculose, reverse concolorous. Colonies on OA reaching
30–40 mm diameter in 10 days at 208C in darkness,
blackish brown (5F8), funiculose to floccose, zonate,
reverse concolorous. Colonies on PDA reaching 30–40 mm
diameter in 10 days at 208C in darkness, blackish brown
(5F8), floccose, reverse concolorous.
Hyphae hyaline, septate, 1.5–2 lm wide. Conidiation
mainly synnematogenous. Phialides mostly arising singly,
sometimes on a short subtending cell, smooth walled, with
walls darkly encrusted in the upper part, sometimes
forming a collarette, 20–40 (–50) lm long, tapering from
2–2.5 lm near the base to 1–1.5 lm. Conidia adhering in
heads, blackish brown (5F8), smooth walled, ellipsoidal,
4–5 9 2–3 lm. Chlamydospores absent.
Teleomorph: Unknown.
Gene sequence data: The GenBank accession numbers
for nuclear 28S rDNA D1/D2 and ITS regions are listed in
Table 1. Habitat: Viscous gels on the stone wall, moldy
colonies on the floor, and white salt-like masses on the
murals.
Isolates examined: Three strains [K5916-10-3 (September
2005), K6303-1-7 (March 2006), and K6613-2 (ex-type)
(June 2006)] isolated from the stone chamber interior and
adjacent small room of the KT; the full strain data are pro-
vided in Table 1.
Cultural characteristics of these three KT isolates
resemble those of A. murorum in funiculose surface texture
on the respective media (MA, OA, and PDA). In other
respects, the morphological characteristics resembled
A. felinum rather than A. murorum. Conidia of A. murorum
adhere in heads or in irregular chains, whereas conidia of
A. felinum and A tumulicola adhere in heads. Acremonium
felinum differs from A. tumulicola by more or less rough-
ened conidia with a truncate base whereas in A tumulicola
they are rounded and smooth. Our molecular phylogenetic
analyses of ITS–28S rDNA sequences show that these
isolates should be regarded as a separate species (see
Fig. 9).
Acremonium masseei (Sacc.) W. Gams, Cephalospo-
rium-artige Schimmelpilze (Hyphomycetes), p. 83, 1971.
Fig. 5
:Trichosporum masseei Sacc., Syll. Fung. (Abellini)
22:1356, 1913 (basionym).
Based on Trichosporium aterrimum Masssee 1899
(nom. illegit. Art. 53).
:Gliomastix masseei
(Sacc.) Matsush., Icon. Microfung.
Matsush. Lect. (Kobe), p. 76, 1975.
Colonies on MA reaching 30–40 mm diameter in 10 days
at 208C in darkness, white (25A1), floccose, reverse con-
colorous. Colonies on OA or PDA reaching 30–40 mm
diameter in 10 days at 208C in darkness, white (on OA
25A1), velvety to floccose, zonate, reverse concolorous; on
PDA white (25CD6), floccose; soluble pigment produced,
reddish brown (2B3); reverse yellowish white (2A2).
Hyphae hyaline to pale brown, septate, 1.5–2 lm wide.
Conidiation mainly synnematogenous. Phialides mostly
borne singly on a short subtending cell, smooth walled,
with walls darkly encrusted in the upper part, forming a
collarette, 20–40 (–50) lm long, tapering from 2.5–3.0 lm
near the base to 1.5–2.0 lm. Conidia phialidic, mostly
adhering in chains, rarely in heads, blackish brown (5F8),
smooth walled, ellipsoidal, with truncate base, intensely
black on both ends, 5–8 (–10) 9 2.5–3 (–4) lm. Chlamy-
dospores absent.
Gene sequence data: The GenBank accession numbers
for nuclear 28S rDNA D1/D2 and ITS regions are listed in
Table 1. Isolates examined: 32 isolates from the murals and
walls of the TT stone chamber interior, from May 2004 to
December 2006, and 1 isolate from the wall of the KT
stone chamber interior in October 2006. The detailed data
on these isolates are shown in Table 1.
Cultural and morphological characteristics of the same
isolates agreed well with those of the two reference strains
(CBS 557.75 and CBS 794.69) and with their published
descriptions (Gams 1971; Matsushima 1975).
Acremonium polychromum (J.F.H. Beyma) W. Gams,
Cephalosporium-artige Schimmelpilze (Hyphomycetes),
p. 81, 1971. Fig. 6
:Oospora polychroma J.F.H. Beyma, Verh. K. Ned. Akad.
Wet., Afd. Natuurk, Sect. 2, 26 (2): 5, 1928 (basionym).
:Gliomastix murorum var. polychroma (J.F.H. Beyma)
C.H. Dickinson, Mycol. Pap. 115:11, 1968.
:Gliomastix polychroma (J.F.H. Beyma) Matsush.,
Icon. Microfung. Matsush. Lect. (Kobe), p. 77, 1975.
Colonies on MA reaching 60–70 mm diameter in 10 days
at 208C in darkness, white (25A1), floccose, reverse con-
colorous. Colonies on OA reaching 50–65 mm diameter in
14 Mycoscience (2011) 52:1–17
123

10 days at 208C in darkness, blackish brown (5F8), velvety
to floccose, zonate, reverse concolorous. Colonies on
PDA reaching 40–50 mm diameter in 10 days at 208Cin
darkness, grayish white (25CD6), floccose, reverse
concolorous.
Hyphae hyaline, septate, 1.5–2 lm wide. Conidiation
phalacrogenous to synnematogenous. Phialides mostly
arising singly, sometimes on a short subtending cell, smooth
walled, with walls darkly encrusted in the upper part, lacking
a visible collarette, 20–30 (–40) lm long, tapering from
2–2.5 lm near the base to 1–1.5 (–2) lm. Conidia adhering
in very long, dry chains, blackish brown (5F8), smooth
walled, ellipsoidal, with truncate base, intensely black on
both ends, 3–5 9 2–2.5 (–3) lm. Chlamydospores absent.
Gene sequence data: The GenBank accession numbers
for nuclear 28S rDNA D1/D2 and ITS regions are listed in
Table 1. Isolates examined: two isolates from the air in
adjacent room B of the TT in July 2006; T6713-22-1a,
T6713-22-1b, and one isolate from the air from the north
area in the adjacent small room of the KT in February
2005; K5225-12-5. The detailed data on isolates are shown
in Table 1.
Phenotypic characteristics of three isolates identified as
A. polychromum agreed well with previous descriptions
(Dickinson 1968; Gams 1971; Matsushima 1975). In our
studies, A. polychromum was isolated only from air in
adjacent areas of the TT and KT. We could not detect this
species from the stone chamber interior or surrounding soil
samples from the TT and KT.
Key to the taxa of Acremonium sect. Gliomastix series
Murorum included in the phylogenetic analyses
1a. Conidia dry, in regular chains 2
1b. Conidia coated in black, sticky mucilage, in heads or in
irregular chains 3
2a. Conidia with truncate base, almost black, mostly
3–4 lm wide A. masseei
2b. Conidia with almost pointed base, olivaceous-brown,
mostly 2–3 lm wide A. polychromum
3a. Conidia almost smooth or some rough-walled, in slimy
heads 4
3b. Conidia almost smooth to coarsely roughened, globose
to ellipsoidal, 3–6 9 2–4.5 lm, adhering in slimy
heads or in chains A. murorum
4a. Conidia ellipsoidal, smooth walled to some rough
walled, with truncate base, 4–6 (–7) 9 2–3 (–4) lm
A. felinum
4b. Conidia almost ellipsoidal with rounded base, smooth
walled, 4–5 9 2–3 lm A. tumulicola
Acknowledgments Our studies and the reproduction of the portions
of the photos shown in Fig. 1 were permitted by the Agency for Cultural
Affairs, Japan. We sincerely thank Dr. Walter Gams, one of the
reviewers, for his critical comments and helpful suggestions, without
which this paper could not have been improved. The comments and
suggestions of Dr. Gen Okada as the Editor also helped improving the
manuscript. We are grateful to Ms. Carolyn Babcock, Curator of
Canadian Collection of Fungus Cultures (CCFC/DAOM) in Ottawa;
and the curators of Centraalbureau voor Schimmelcultures (CBS) in
Utrecht, RIKEN-Japan Collection of Microorganisms (JCM) in Wako,
and NITE-Biological Resource Center (NBRC) in Kazusa for providing
the strains (including ex-type) of Acremonium and Gliomastix spp. This
study was financially supported in part by a Research Grant from the
Institute for Fermentation, Osaka (IFO), to K.-D. An (2008–) and in part
by Grants-in-Aid for Scientific Research (A) (No. 17206060 to S.M.,
2005–2007; No. 19200057 to C. Sano, 2007–) from the Ministry of
Education, Culture, Sports, Science, and Technology, Japan.
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