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ORIGINAL PAPER
The identity of Penicillium sp. 1, a major contaminantof the stone chambers in the Takamatsuzuka and KitoraTumuli in Japan, is Penicillium paneum
Kwang-Deuk An Æ Tomohiko Kiyuna ÆRika Kigawa Æ Chie Sano Æ Sadatoshi Miura ÆJunta Sugiyama
Received: 16 March 2009 / Accepted: 13 August 2009 / Published online: 26 September 2009
� Springer Science+Business Media B.V. 2009
Abstract Penicillium appeared as the major dweller
in the Takamatsuzuka Tumulus (TT) and Kitora
Tumulus (KT) stone chambers, both located in the
village of Asuka, Nara Prefecture, in relation to the
biodeterioration of the 1,300-year-old mural paintings,
plaster walls and ceilings. Of 662 Penicillium isolates
from 373 samples of the TT (sampling period, May
2004–2007) and the KT (sampling period, June 2004–
Sep 2007), 181 were phenotypically assigned as
Penicillium sp. 1 which shared similar phenotypic
characteristics of sect. Roqueforti in Penicillium subg.
Penicillium. Fifteen representative isolates of Penicil-
lium sp. 1, 13 from TT and 2 from KT, were selected for
molecular phylogenetic analysis. The 28S rDNA
D1/D2, ITS, b-tubulin, and lys2 gene sequence-based
phylogenies clearly demonstrated that the three known
species P. roqueforti, P. carneum and P. paneum in
sect. Roqueforti, and all TT and KT isolates grouped
together. In addition to this, TT and KT isolates formed
a monophyletic group with the ex-holotype strain CBS
101032 of P. paneum Frisvad with very strong
bootstrap supports. So far, P. paneum has been isolated
only from mouldy rye breads, other foods, and baled
grass silage. Therefore, this is the first report of
P. paneum isolation from samples relating to the
biodeteriorated cultural properties such as mural
paintings on plaster walls.
Keywords Biodeterioration �Penicillium sect. Roqueforti � Molecular phylogeny �Murals � Takamatsuzuka and Kitora Tumuli
Introduction
Penicillium, the phialidic, anamorphic, species-rich
genus of the Trichocomaceae in the Eurotiomycetes,
inhabits a variety of substrates and environments.
Kwang-Deuk An and Tomohiko Kiyuna authors contributed
equally to this work.
Electronic supplementary material The online version ofthis article (doi:10.1007/s10482-009-9373-0) containssupplementary material, which is available to authorized users.
K.-D. An � T. Kiyuna
TechnoSuruga Laboratory Co., Ltd, NCIMB Group,
330 Nagasaki, Shimizu-ku, Shizuoka,
Shizuoka-ken 424-0065, Japan
R. Kigawa � C. Sano � S. Miura
Independent Administrative Institution, National Research
Institute for Cultural Properties, 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-ken 284-0001, Japan
e-mail: jsugiyam@tecsrg.co.jp
Present Address:K.-D. An
Japan Collection of Microorganisms, RIKEN
BioResource Center, 2-1 Hirosawa, Wako,
Saitama-ken 351-0198, Japan
123
Antonie van Leeuwenhoek (2009) 96:579–592
DOI 10.1007/s10482-009-9373-0
Members of this genus are important in human life;
they commonly appear as soil, storage, indoor and
airborne fungi or as food contaminants (e.g., Pitt
1979; Domsch et al. 2007; CBS databases [http://
www.cbs.knaw.nl/fungi/BioloMICS.aspx]). Fungal
biodeterioration is well known to affect cultural
properties such as catacombs (Saarela et al. 2004) and
murals (Berner et al. 1997; Dhawan et al. 1993;
Guglielminetti et al. 1994) in Europe. In addition, the
roles of fungi in the deterioration of murals, as well as
their decay mechanism, have been reviewed by Nu-
gari et al. (1993) and Garg et al. (1995); those studies
listed, at the species level and including Penicillium
spp., fungi that have been isolated from deteriorated
murals.
On the other hand, in Japan, microbiological
surveys related to the conservation of stone chamber
interiors and murals were conducted for the Ozuka
and Chibusan Tumuli in Fukuoka Prefecture (Emoto
and Emoto 1974), the Torazuka Tumulus in Ibaraki
Prefecture (Emoto et al. 1983; Arai 1984, 1990a), and
the Takamatsuzuka (Arai 1984, 1987, 1990b; Kigawa
et al. 2004, 2006b) and Kitora (Kigawa et al. 2005,
2006a) Tumuli, both in Nara Prefecture. Anamorphic
fungi such as species of Cladosporium, Doratomyces,
Fusarium, Penicillium, and Trichoderma were
detected from samples taken from these tumuli (cf.
Tables 1 and 3 in Kiyuna et al. 2008). In most cases,
however, identification of Penicillium isolates in
these previous studies was limited to the generic
level.
The Takamatsuzuka Tumulus (TT) and the Kitora
Tumulus (KT), which are thought to have been built
in late 7th century, are well known in Japan as
invaluable cultural heritage sites because of their
beautiful mural paintings, which were drawn directly
onto thin plaster in the small stone chamber interior.
The mural paintings of the TT were discovered in
1972, whereas the KT was discovered in 1983 and
excavated in early 2004. The Japanese government
designated the TT and KT as Special Historic Sites in
1973 and 2000, respectively.
The TT is located in the village of Asuka, Nara
Prefecture, Japan. It is a circular burial mound
measuring approximately 20 m in diameter. The
stone chamber was constructed of cut slabs of
volcanic tuff; the interior area is approximately
2.7 m deep, 1.0 m wide, and 1.1 m tall (for the
schematic diagram, see Fig. 1 in Kiyuna et al. 2008).
The colorful paintings adorn the inner walls and a
ceiling of the stone chamber with various colors,
including red, green, yellow, blue, and gold. The
figures comprise star constellations (Seishuku) on the
ceiling, and the sun and the moon (Nichi-Getsu),
the four heavenly guardian gods (Shishin), and groups
of men and women on the four walls. The group of
women on the west wall is called the ‘‘Asuka
beauties’’ (Asuka bijin) (see Fig. 2 in Kiyuna et al.
2008). All the paintings were designated as National
Treasures by the government in 1974. In accordance
with specialists’ advice, the Japanese Agency for
Cultural Affairs decided in 1973 to preserve the
murals on site and they were never opened to the
public. It was thought that the beauty and stability of
the plaster paintings could be preserved if conditions
inside the stone chamber would be kept as before,
naturally buried state; high humidity (100% RH) and
cool temperature (14–20�C) could be established. In
addition, the stone chamber was kept in darkness
except for regular inspections by the staff involved in
its conservation.
The KT is also located 1.2 km south of the TT in
the same village of Asuka. Its circular tomb is
approximately 14 m diameter; the stone chamber
interior is approximately 2.4 m deep and 1 m wide
and tall; a diagram of the stone chamber and related
facilities is not illustrated here, but the KT adjacent
small room corresponds to the adjacent space of the
TT. Similar colorful murals are drawn on the thin
plaster wall within the stone chamber, and a star chart
on the ceiling is in the same style as in the TT. The
interior environmental conditions are quite similar to
those of the TT, and no conspicuous fungal colonies
were seen for some time after the excavation. In
2004, in the course of the excavation, species of
Acremonium, Fusarium, Penicillium, and Tricho-
derma appeared as the main contaminants (Kigawa
et al. 2005, 2006a, 2007). In the case of the KT, the
paintings on the plaster wall had become partly
detached from their support. Considering such situ-
ation, it was decided to relocate these paintings to a
controlled environment in July 2004. By the end of
2008, all the paintings except for possible ones
hidden by thin wall layer, were relocated.
We have detected and reported the haplotypes of
Fusarium and Trichoderma, the two predominant
fungal colonizers, using the 28S rDNA D1/D2 region
(28S) and EF-1a (=tef1: protein-coding gene
580 Antonie van Leeuwenhoek (2009) 96:579–592
123
translation-elongation factor 1-alpha) gene sequence-
based analyses (Kiyuna et al. 2008). We have also
proposed two new species of Candida assignable to
the C. membranifaciens clade: C. tumulicola and C.
takamatsuzukensis, isolated mainly from biofilm
samples from the stone chamber interior of the
Takamatsuzuka Tumulus (Nagatsuka et al. 2009). In
the previous papers (Sugiyama et al. 2008, 2009;
Kiyuna et al. 2008), we have mentioned that Peni-
cillium isolates, which comprised one of the predom-
inant fungal groups, were obtained from samples in
the stone chamber interiors and adjacent spaces or
rooms of both tumuli. Of 1,780 fungal isolates, 662
were assignable to Penicillium. And 181 of those
isolates have been phenotypically assigned as Peni-
cillium sp. 1.
In the present study, we reveal phenotypically and
genotypically the identity of Penicillium sp. 1 isolates
as the major Penicillium colonizers in the stone
chamber environments in the Takamatsuzuka and
Kitora Tumuli.
Materials and methods
Sample sources, isolation, and culturing
A total of 224 samples, including mold spots, viscous
gels (biofilms), and mixtures of plaster fragments and
soil, were collected from the stone chamber interior,
spaces between the stone walls, and stone chamber
exterior of the Takamatsuzuka Tumulus (TT)
between May 2004 and May 2007. As well, a total
of 149 samples were collected from the stone
chamber interior and exterior of the Kitora Tumulus
(KT) between June 2004 and September 2007.
Isolation methods have been mentioned in Sugiyama
et al. (2008, 2009) and Kiyuna et al. (2008). The
isolates have been maintained on potato dextrose agar
(PDA; Nihon Pharmaceutical, Tokyo, Japan). For
strain data originally identified as Penicillium sp. 1
from the Takamatsuzuka and Kitora Tumuli, see
Table 1 and supplementary Table S1. The 11 selected
isolates are deposited with the Japan Collection of
Microorganisms (JCM), RIKEN BioResource Center,
Wako, Saitama Prefecture, Japan, as JCM 15985–
15995 (Table 1). The remaining P. paneum isolates
from both tumuli are maintained at the Biology
Laboratory of the National Research Institute of
Cultural Properties, Tokyo as lyophilised cultures
(Table 1, supplementary Table S1).
Cultural and morphological observations
A total of 50 isolates, comprising 31 and 19 isolates
from TT and KT, respectively, were used in the
cultural and morphological observations. These iso-
lates were selected from the standpoint of sampling
sites and dates as well from the standpoint of kinds of
substrates. The detailed data on isolates are shown in
supplementary Table S1. All isolates were grown
using the media and growth conditions adopted by
Pitt (1979, 2000) and Frisvad and Samson (2004).
The isolates were incubated on Czapek yeast autol-
ysate (CYA) agar at 25�C, malt extract agar (MEA) at
25�C, 25% glycerol nitrate (G25N) agar at 25�C,
yeast extract sucrose (YES) agar at 25�C, and
creatine sucrose (CREA) agar at 25�C all for 7 days
in darkness, respectively. Growth rates were mea-
sured on CYA inoculated with conidial suspension
and incubated at 5, 10, 15, 20, 25, 30, 37 and 40�C all
for 7 days in the dark. Furthermore, growth rates
were determined in the moist chamber, about 100%
RH. The colony colors of the isolates on all media
were determined by using Kornerup and Wanscher’s
color standard (1978). Microscopic slides were
prepared from portions of the colonies grown on
MEA plate cultures and were mounted in lactophenol
mounting medium without dye (Bills and Foster
2004). Microscopic examinations were made using a
fluorescence microscope BX51 (Olympus, Tokyo,
Japan) with Normarski interphase contrast at up to
91,000 magnification. All micrographs were taken
with a Coolpix P5000 digital camera (Nikon, Tokyo,
Japan).
Ubiquinone determination, a chemotaxonomic
characteristic
Mycelia grown on DifcoTM Potato Dextrose Broth
(Becton–Dickinson, MD, USA) were used for the
extraction of ubiquinones. The isolates and other
authentic strains were cultured at 25�C for 3 days on
a rotary shaker (Taitec, Saitama, Japan) at 125
revolutions per min-1. Extraction, purification, and
determination of ubiquinones by high-performance
liquid chromatography were carried out as described
previously (Kuraishi et al. 1985, 1991). When one
Antonie van Leeuwenhoek (2009) 96:579–592 581
123
Ta
ble
1S
trai
nd
ata
of
TT
and
KT
iso
late
s,w
ith
maj
or
ub
iqu
ino
ne
syst
emd
ata
and
the
Gen
Ban
kac
cess
ion
nu
mb
ers,
for
28
S,IT
S,b-
tub
uli
n,an
dly
s2g
ene
seq
uen
ces
det
erm
ined
inth
isst
ud
y
Iso
late
aJC
Mn
o.
Iso
lati
on
sou
rce
Sam
pli
ng
dat
e
Gen
Ban
kac
cess
ion
no
.U
biq
uin
on
e
syst
emb
28
SIT
Sb-
tub
uli
nly
s2
T4
51
9-5
-51
59
85
Mo
uld
ysp
ots
on
the
flo
or
of
the
sto
ne
cham
ber
of
Tak
amat
suzu
ka
Tu
mu
lus
19
May
20
04
AB
47
92
90
AB
47
93
22
AB
47
93
37
AB
47
93
66
T4
90
6-1
1-8
15
98
6S
urf
ace
of
anin
sect
bo
dy
(Iso
po
da)
fro
mth
ew
all
inth
e
sto
ne
cham
ber
of
Tak
amat
suzu
ka
Tu
mu
lus
6S
ep2
00
4A
B4
79
29
1A
B4
79
32
3A
B4
79
33
8A
B4
79
36
7
T5
91
6-1
-1V
isco
us
gel
sb
elo
wth
efo
refo
ot
of
the
pai
nti
ng
of
the
wh
ite
tig
er
on
the
wes
tw
all
2in
the
sto
ne
cham
ber
of
Tak
amat
suzu
ka
Tu
mu
lus
16
Sep
20
05
AB
47
92
92
AB
47
93
24
AB
47
93
39
AB
47
93
68
T5
91
6-3
-1V
isco
us
gel
sb
elo
wth
ep
ain
tin
gs
of
the
gro
up
of
wo
men
on
the
wes
tw
all
3in
the
sto
ne
cham
ber
of
Tak
amat
suzu
ka
Tu
mu
lus
16
Sep
20
05
AB
47
92
93
AB
47
93
25
AB
47
93
40
AB
47
93
69
T5
91
6-6
-11
59
87
Vis
cou
sg
els
bel
ow
the
pai
nti
ng
so
fth
eg
rou
po
fw
om
en
on
the
east
wal
l3
inth
est
on
ech
amb
ero
fT
akam
atsu
zuk
aT
um
ulu
s
16
Sep
20
05
AB
47
92
94
AB
47
93
26
AB
47
93
41
AB
47
93
70
Q-9
T6
51
7-1
-21
59
88
Bla
cksp
ots
on
the
pai
nti
ng
so
fth
eg
rou
po
fw
om
en
on
the
wes
tw
all
3in
the
sto
ne
cham
ber
of
Tak
amat
suzu
ka
Tu
mu
lus
17
May
20
06
AB
47
92
95
AB
47
93
27
AB
47
93
42
AB
47
93
71
T7
21
4-8
-11
59
89
Bla
cksp
ots
on
wes
tern
area
of
the
adja
cen
tsp
ace
of
Tak
amat
suzu
ka
Tu
mu
lus
(du
rin
gex
cav
atio
n)
14
Feb
20
07
AB
47
92
96
AB
47
93
28
AB
47
93
43
AB
47
93
72
Q-9
T7
42
5-3
-1B
lack
sub
stan
ces
insm
all
pit
so
nth
eto
psu
rfac
eo
fw
est
wal
lst
on
e3
of
Tak
amat
suzu
ka
Tu
mu
lus
(du
rin
gre
loca
tio
no
fth
est
on
ech
amb
er)
25
Ap
r2
00
7A
B4
79
29
7A
B4
79
32
9A
B4
79
34
4A
B4
79
37
3
T7
42
5-4
-11
59
90
Bla
cksu
bst
ance
sfr
om
the
top
surf
ace
of
east
wal
lst
on
e3
of
Tak
amat
suzu
ka
Tu
mu
lus
(du
rin
gre
loca
tio
no
fth
est
on
ech
amb
er)
25
Ap
r2
00
7A
B4
79
29
8A
B4
79
33
0A
B4
79
34
5A
B4
79
37
4Q
-9
T7
51
0-5
-11
59
91
Pie
ces
of
sid
ep
last
erfr
om
the
no
rth
ern
sid
eo
fw
est
wal
lst
on
e2
of
Tak
amat
suzu
ka
Tu
mu
lus
(du
rin
gre
loca
tio
no
fth
est
on
ech
amb
er)
10
May
20
07
AB
47
92
99
AB
47
93
31
AB
47
93
46
AB
47
93
75
T7
52
1-8
A-4
15
99
2P
last
icco
ver
ov
erth
eth
iev
ing
ho
le(s
ton
ech
amb
erin
teri
or
sid
e)
of
Tak
amat
suzu
ka
Tu
mu
lus
21
May
20
07
AB
47
93
00
AB
47
93
32
AB
47
93
47
AB
47
93
76
T7
52
8-2
1-1
15
99
3S
ou
ther
nar
eao
fth
ece
ilin
gp
last
ero
fce
ilin
gst
on
e2
of
Tak
amat
suzu
ka
Tu
mu
lus
(du
rin
gre
loca
tio
n)
28
May
20
07
AB
47
93
01
AB
47
93
33
AB
47
93
48
AB
47
93
77
T7
53
0-4
-1B
lack
vis
cou
sg
els
and
pie
ces
of
sid
ep
last
erb
etw
een
east
wal
lst
on
e1
and
ceil
ing
sto
ne
1o
fT
akam
atsu
zuk
aT
um
ulu
s(d
uri
ng
relo
cati
on
)
30
May
20
07
AB
47
93
02
AB
47
93
34
AB
47
93
49
AB
47
93
78
K5
91
6-7
-11
59
94
Dar
k-g
reen
ish
vis
cou
sg
els
bel
ow
the
pai
nti
ng
so
fth
eto
rto
ise
and
snak
e
on
the
no
rth
wal
lin
the
sto
ne
cham
ber
of
Kit
ora
Tu
mu
lus
16
Sep
20
05
AB
47
93
03
AB
47
93
35
AB
47
93
50
AB
47
93
79
K6
20
3-2
-11
59
95
Bla
ckv
isco
us
sub
stan
cein
ah
ole
on
the
east
wal
lin
the
sto
ne
cham
ber
of
Kit
ora
Tu
mu
lus
3F
eb2
00
6A
B4
79
30
4A
B4
79
33
6A
B4
79
35
1A
B4
79
38
0
aT
and
Kin
dic
ate
the
Tak
amat
suzu
ka
Tu
mu
lus
and
Kit
ora
Tu
mu
lus,
resp
ecti
vel
yb
Th
em
ajo
ru
biq
uin
on
esy
stem
was
det
erm
ined
inth
isst
ud
y
582 Antonie van Leeuwenhoek (2009) 96:579–592
123
type of ubiquinone molecule constituted more than
90% of the types found in a particular strain, it was
determined to be the major ubiquinone type (Kuraishi
et al. 1985, 1991).
DNA extraction, PCR amplification,
and sequencing
The isolates used for the DNA sequencing are listed
in Table 1. Their genomic DNA was extracted using
a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany)
according to the manufacturer’s instructions. The
four gene regions sequenced were the 28S rDNA
D1/D2 region (28S), ITS-5.8S rDNA (ITS), part of
the b-tubulin, and part of the lys2 (aminoadipate
reductase) gene. The primers used included NL1 and
NL4 (O’Donnell 1993) for 28S, ITS5 and ITS4
(White et al. 1990) for ITS, Bt2a and Bt2b (Glass and
Donaldson 1995) for b-tubulin, and lys2F and lys2R
(An et al. 2002) for lys2. Polymerase chain reactions
(PCR) were performed using puReTaq Ready-To-Go
PCR beads (GE Healthcare, Buckinghamshire, UK).
Thermal cycling was performed using a GeneAmp
PCR System 9700 (Applied Biosystems, Foster City,
CA, USA). Initial denaturation at 95�C for 5 min was
followed by 40 cycles of denaturation at 94�C for
30 s, annealing at 55�C for 30 s (28S, ITS)/58�C for
30 s (b-tubulin)/56�C for 1 min (lys2), extension at
72�C for 1 min, and then a final extension at 72�C for
10 min. The amplified DNA fragments were purified
with a QIAquick PCR Purification Kit (Qiagen,
Hilden, Germany). The PCR products of the lys2
gene were cloned using a PCR Cloning Kit (Qiagen,
Hilden, Germany). Two to three white clones of each
the PCR products were sampled. Sequencing of the
PCR products and the cloned plasmids was per-
formed using the BigDye Terminator Cycle Sequenc-
ing Kit v3.1 (Applied Biosystems, CA, USA).
Reactions were incubated in a GeneAmp PCR
System 9700 (Applied Biosystems, CA, USA) and
the products cleaned using a DyeExTM 2.0 Spin Kit
(Qiagen, Hilden, Germany). Sequences were deter-
mined using electrophoresis in an ABI3130xl DNA
sequencer (Applied Biosystems, CA, USA). The
sequences determined in this study were deposited
in GenBank/EMBL/DDBJ, and their accession num-
bers are given in Table 1. Other sequences down-
loaded from GenBank (Table 2) are shown in the
respective molecular phylogenetic trees.
28S and ITS molecular phylogenetic analyses
The sequences were assembled using ChromasPro
1.42 (Technelysium Pty Ltd., Tewantin QLD, Aus-
tralia). Multiple alignments were performed using
CLUSTAL W version 1.83 (Thompson et al. 1994),
and then the final alignments were manually adjusted.
Ambiguous positions and alignment gaps were
excluded from the analysis. The alignments and trees
were deposited in TreeBASE, accession number
SN4551. The neighbor-joining (NJ) tree based on
the respective gene sequences was constructed using
the multiple alignments in MEGA ver3.1 (Kumar
et al. 2004), with 1,000 bootstrap replicates (Felsen-
stein 1985).
Molecular phylogenetic analyses of b-tubulin
and lys2 genes
The b-tubulin and lys2 gene analysis for Penicillium,
the phylogenetic tree by NJ, and the maximum
likelihood (ML) and Bayesian (Bayes) analyses were
performed. The alignments and trees were deposited
in TreeBASE, accession number SN4551. NJ trees
based on the respective gene sequences were con-
structed using the multiple alignments in MEGA
ver3.1 (Kumar et al. 2004), with 1,000 bootstrap
replicates (Felsenstein 1985). ML trees based on the
respective gene sequences were also constructed
using the multiple alignments in PHYLIP version
3.6 (Felsenstein 2002), with 100 bootstrap replicates.
The Bayesian approach to phylogenetic reconstruc-
tion (Rannala and Yang 1996) was implemented
using MrBayes v3.1.1-p1 (Huelsenbeck and Ronquist
2001). The model of DNA substitution was calcu-
lated using Modeltest 3.0 (Posada and Crandall
1998). The results were obtained by the K80 model
and gamma correction (G) rate variation among sites
for b-tubulin, and the general time-reversible (GTR)
model and G rate variation among sites for lys2.
Bayesian Metropolis-coupled Markov chain Monte
Carlo (MCMCMC) analyses (Mau et al. 1999) were
performed with MrBayes for phylogenetic estimation
inferred from the respective gene sequences. MrBa-
yes was run for 1,500,000 generations for b-tubulin
and 1,000,000 generations for lys2. Searches were
conducted with four chains (three cold, one hot) with
trees sampled every 100 generations. The average
standard deviations of split frequencies were 0.008
Antonie van Leeuwenhoek (2009) 96:579–592 583
123
Ta
ble
2S
trai
nd
ata
use
dfr
om
mo
lecu
lar
ph
ylo
gen
etic
anal
yse
sw
ith
the
maj
or
ub
iqu
ino
ne
syst
eman
dG
enB
ank
acce
ssio
nn
um
ber
sfo
r2
8S
,IT
S,b-
tub
uli
n,
and
lys2
gen
e
seq
uen
ces
Tax
on
Str
ain
no
.N
om
encl
atu
ral
stat
us
So
urc
e(s
ub
stra
te)
and
ori
gin
Gen
Ban
kac
cess
ion
no
.aU
biq
uin
on
e
syst
em2
8S
ITS
b-t
ub
uli
nly
s2
Pen
icil
liu
ma
lbo
core
miu
mIB
T1
06
82
Ex
-ho
loty
pe
Sal
ami,
Den
mar
k–
AJ0
04
81
9–
––
P.
all
iiD
AR
74
61
3A
nta
rcti
ca,
orn
ith
og
enic
soil
s
inp
eng
uin
colo
ny
area
s,
Win
dm
ill
Isla
nd
s
–A
F2
18
78
7–
––
P.
all
iiIB
T3
05
6F
oo
d,
Gre
atB
rita
in–
AJ0
05
48
4–
––
P.
atr
am
ento
sum
NR
RL
79
5E
x-n
eoty
pe
Fre
nch
Cam
emb
ert
chee
se,
US
AA
F0
33
48
3–
––
Q-9
P.
au
ran
tio
gri
seu
mC
BS
32
4.8
9E
x-n
eoty
pe
AB
47
92
86
AB
47
93
18
AY
67
42
96
AB
47
93
65
–
P.
bre
vico
mp
act
um
CB
S2
57
.29
Ex
-neo
typ
e–
–A
Y6
74
43
7–
Q-9
P.
bre
vico
mp
act
um
JCM
22
84
9E
x-n
eoty
pe
AB
47
92
75
AB
47
93
06
––
Q-9
P.
cam
emb
erti
CB
S2
99
.48
Ex
-neo
typ
eF
ren
chC
amem
ber
tch
eese
,U
SA
AB
47
92
82
AB
47
93
14
AY
67
43
68
AB
47
93
61
Q-9
b
P.
carn
eum
CB
S1
12
29
7E
x-h
olo
typ
eM
ou
ldy
rye
bre
ad,
Den
mar
kA
B4
79
27
9A
B4
79
31
0A
Y6
74
38
6A
B4
79
35
8Q
-9b
P.
chry
sog
enu
mC
BS
30
6.4
8E
x-n
eoty
pe
Ch
eese
,U
SA
––
AY
49
59
81
–Q
-9
P.
chry
sog
enu
mJC
M2
28
26
Ex
-neo
typ
eC
hee
se,
US
AA
B4
79
27
4A
B4
79
30
5–
AB
47
93
53
Q-9
P.
com
mu
ne
CB
S3
11
.48
Ex
-iso
typ
eC
hee
se,
US
AA
Y2
13
61
6–
––
–
P.
con
cen
tric
um
NR
RL
20
34
–D
Q3
39
56
1–
––
P.
cop
rop
hil
um
NR
RL
13
62
7S
org
hu
m,
Afr
ica
AF
03
34
69
––
––
P.
cord
ub
ense
NR
RL
13
07
2A
F5
27
05
5–
––
–
P.
dig
ita
tum
CB
S1
36
.65
Fru
its
of
Cit
rus
med
ica
lim
on
ium
?(R
uta
cea
e),
Net
her
lan
ds
AB
47
92
76
AB
47
93
07
AY
67
44
04
AB
47
93
55
–
P.
ech
inu
latu
mC
BS
31
7.4
8E
x-i
soty
pe
Cu
ltu
reco
nta
min
ant,
Can
ada
––
AY
67
43
41
–Q
-9
P.
ech
inu
latu
mJC
M2
28
36
Ex
-iso
typ
eC
ult
ure
con
tam
inan
t,C
anad
aA
B4
79
28
4A
B4
79
31
6–
AB
47
93
63
Q-9
P.
exp
an
sum
CB
S3
25
.48
Ex
-neo
typ
eF
ruit
so
fM
alu
ssy
lves
tris
(Ro
sace
ae)
,U
SA
AB
47
92
78
AB
47
93
09
AY
67
44
00
AB
47
93
57
–
P.
gla
dio
liN
RR
L9
39
Ex
-neo
typ
eG
lad
iolu
sco
rm,
Afr
ica
AF
03
34
80
––
––
P.
gla
nd
ico
laC
BS
49
8.7
5E
x-e
pit
yp
eM
ou
ldy
win
eco
rk,
Po
rtu
gal
AB
47
92
77
AB
47
93
08
AY
67
44
15
AB
47
93
56
–
P.
gri
seo
fulv
um
NR
RL
23
00
Ex
-neo
typ
eA
F0
33
46
8–
––
Q-9
P.
gri
seo
fulv
um
NR
RL
35
23
–D
Q3
39
54
9–
––
P.
hir
sutu
mJC
M2
28
35
Ex
-neo
typ
eA
ph
id,
gre
enfl
y,
Net
her
lan
ds
AB
47
92
83
AB
47
93
15
–A
B4
79
36
2Q
-9
P.
hir
sutu
mC
BS
13
5.4
1E
x-n
eoty
pe
Ap
hid
,g
reen
fly
,N
eth
erla
nd
s–
–A
Y6
74
32
8–
Q-9
P.
infl
atu
mC
BS
68
2.7
0E
x-h
olo
typ
eR
oo
tsu
rfac
eo
fP
icea
ab
ies,
Den
mar
k–
––
AB
48
08
87
–
P.
ma
dri
tiN
RR
L3
45
2E
x-h
olo
typ
eG
ard
enso
il,
Sp
ain
AF
03
34
82
AF
03
34
82
––
–
584 Antonie van Leeuwenhoek (2009) 96:579–592
123
Ta
ble
2co
nti
nu
ed
Tax
on
Str
ain
no
.N
om
encl
atu
ral
stat
us
So
urc
e(s
ub
stra
te)
and
ori
gin
Gen
Ban
kac
cess
ion
no
.aU
biq
uin
on
e
syst
em2
8S
ITS
b-t
ub
uli
nly
s2
P.
ols
on
iiC
BS
23
2.6
0E
x-n
eoty
pe
Ro
ot
of
Pic
eaa
bie
s,A
ust
ria
––
AY
67
44
45
––
P.
pa
neu
mC
BS
10
10
32
Ex
-ho
loty
pe
Mo
uld
yry
eb
read
,D
enm
ark
AB
47
92
73
AB
47
93
12
AY
67
43
87
AB
47
93
21
Q-9
b
P.
pa
neu
mC
BS
46
5.9
5M
ou
ldy
bak
er’s
yea
st,
Den
mar
kA
B4
79
28
0A
B4
79
31
1A
Y6
74
38
8A
B4
79
35
9Q
-9b
P.
po
lon
icu
mN
RR
L9
95
Ex
-ho
loty
pe
So
il,
Po
lan
dA
F0
33
47
5A
F0
33
47
5–
––
P.
roq
uef
ort
iC
BS
22
1.3
0E
x-n
eoty
pe
Fre
nch
Ro
qu
efo
rtch
eese
,U
SA
––
AY
67
43
83
–Q
-9b
P.
roq
uef
ort
iJC
M2
28
42
Ex
-neo
typ
eF
ren
chR
oq
uef
ort
chee
se,
US
AA
B4
79
28
1A
B4
79
31
3–
AB
47
93
60
Q-9
b
P.
scle
roti
gen
um
NR
RL
34
61
Ex
-neo
typ
eP
lan
ttu
ber
,S
wee
tp
ota
to,
Jap
anA
F0
33
47
0–
––
–
P.
tric
olo
rA
TC
C1
04
13
–A
Y3
73
93
5–
––
P.
ven
etu
mIB
T5
46
4F
low
erb
ulb
,D
enm
ark
–A
J00
54
85
––
–
P.
verr
uco
sum
CB
S6
03
.74
Ex
-neo
typ
eA
B4
79
28
5A
B4
79
31
7A
Y6
74
32
3A
B4
79
36
4Q
-9
P.
viri
dic
atu
mN
RR
L9
61
AF
03
34
78
AF
03
34
78
––
Q-9
Eu
pen
icil
liu
mb
aa
rnen
seN
RR
L2
08
6E
x-n
eoty
pe
So
il,
Net
her
lan
ds
AF
03
34
81
––
––
E.
cate
na
tum
CB
S4
31
.69
So
il,
US
A–
–A
Y6
74
43
3–
–
E.
cru
sta
ceu
mC
BS
58
1.6
7S
oil
,P
akis
tan
AB
47
92
89
AB
47
93
21
––
–
E.
cru
sta
ceu
mC
BS
24
4.3
2E
x-n
eoty
pe
So
il,
Eg
yp
tA
B4
79
28
7A
B4
79
31
9–
––
E.
mo
lle
CB
S4
56
.72
Ex
-ho
loty
pe
So
il,
Pak
ista
nA
B4
79
28
8A
B4
79
32
0–
–Q
-9
E.
tula
ren
seN
RR
L5
27
3E
x-h
olo
typ
eS
oil
un
der
Pin
us
po
nd
ero
sa&
Qu
ercu
ssp
.,U
SA
AF
03
34
87
AF
03
34
87
––
Q-9
IBT
Dep
artm
ent
of
Bio
tech
no
log
y,T
ech
nic
alU
niv
ersi
tyo
fD
enm
ark
,L
yn
gb
y,D
enm
ark
,A
TC
CA
mer
ican
Ty
pe
Cu
ltu
reC
oll
ecti
on
,M
anas
sas,
Vir
gin
ia,U
SA
,N
RR
LA
gri
cult
ura
l
Res
earc
hS
erv
ice
(AR
S)
Cu
ltu
reC
oll
ecti
on
,N
ort
her
nU
tili
zati
on
Reg
ion
alR
esea
rch
Cen
ter,
Peo
ria,
Illi
no
is,
US
A,
NB
RC
NIT
EB
iolo
gic
alR
eso
urc
eC
ente
r,K
isar
azu
,C
hib
a,
Jap
an,
CB
SC
entr
aalb
ure
auv
oo
rS
chim
mel
cult
ure
s,U
trec
ht,
Th
eN
eth
erla
nd
s,JC
MJa
pan
Co
llec
tio
no
fM
icro
org
anis
ms,
RIK
EN
Bio
Res
ou
rce
Cen
ter,
Wak
o,
Sai
tam
a,Ja
pan
,
DA
RD
epar
tmen
to
fA
gri
cult
ure
,B
iolo
gic
alan
dC
hem
ical
Res
earc
hIn
stit
ute
,R
yd
alm
ere,
NS
W,
Au
stra
lia
aB
old
acce
ssio
nn
um
ber
sin
dic
ate
seq
uen
ces
det
erm
ined
inth
isst
ud
y,
wh
erea
so
ther
seq
uen
ces
wer
eo
bta
ined
fro
mG
enB
ank
bT
he
maj
or
ub
iqu
ino
ne
syst
emw
ere
det
erm
ined
inth
isst
ud
y,
wh
erea
sth
ere
mai
nin
gu
biq
uin
on
ed
ata
wer
eci
ted
fro
mK
ura
ish
iet
al.
(19
91
)
–N
od
ata
Antonie van Leeuwenhoek (2009) 96:579–592 585
123
and 0.006, respectively, at the end of the run. The
confidence levels of nodes were measured by poster-
ior probabilities obtained from the majority rule
consensus after deletion of the trees during burn-in.
Results and discussion
Cultural and morphological characterization
of Penicillium sp. 1 isolates
Cultural and morphological characterization of Pen-
icillium sp. 1 was based on 50 representative isolates
from the TT and KT stone chamber interiors and
exteriors (for details, see Table 1 and supplementary
Table S1).
Colonies on CYA at 25�C for 7 days grew rapidly
and were 30–40 mm in diameter, radially sulcate,
velutinous, predominantly greyish turquoise (25CD-
6) or greyish green (28B2-3), and had a white (25A1-
2) margin; the reverse was pale yellow. No growth
was observed at 37�C for 7 days. Colonies on MEA
at 25�C for 7 days grew rapidly, and were 43–50 mm
in diameter, velutinous, and predominantly pale
green (26A-3) to greenish white (26A-2), with a
greenish white (25A-2) margin; the reverse was pale
yellow. Colonies on YES at 25�C for 7 days grew
rapidly, and were 40–43 mm in diameter, radially
sulcate, velutinous, and predominantly greyish white
(25A-2) to pale green (25A-3); the reverse was pale
yellow. Colonies on G25N at 25�C for 7 days were
26–30 mm in diameter, velutinous, and predomi-
nantly greyish green (25B-5), with a white (25A-1)
margin; the reverse was pale yellow. Colonies on
CREA at 25�C for 7 days attained a diameter of 18–
20 mm and were velutinous, predominantly pale
green (25A-3). The optimum temperature for growth
on CYA, in the four selected Penicillium sp. 1
isolates (T4519-5-5, T5916-6-1, T6517-1-2 and
K5916-6-1), was 20–25�C; the colonies attained ca.
50–60 mm diameter after 7 days. The growth rates
were similar to the moist chamber, about 100% RH.
These results suggested that TT and KT isolates were
mesophilic.
Conidiophores arose from the subsurface and sub-
merged hypha, and stripes reached lengths of 100–200
(–250) lm, characteristically with very rough and
tuberculate walls. The penicillus was typically terver-
ticillate, occasionally quaterverticillate, with appressed
elements; the rami were 20–25 (–30) lm long with
tuberculate walls; the metulae were 12–15 lm long; and
the phialides were ampulliform, 7.5–8.5 (–10) lm long,
with short collula. Conidia were spherical, (3–) 3.5–4
(–4.5) lm in diameter, with smooth or finely rough
walls, pale green to dark green, borne in long chains, and
closely packed. No sclerotium-like structures were
observed.
The above-mentioned cultural and morphological
characterization of Penicillium sp. 1 isolates differed
from those of P. roqueforti and P. carneum, both in
Penicillium subg. Penicillium sect. Roqueforti by a
pale yellow colony reverse on CYA and YES. The
phenotypic characteristics of Penicillium sp. 1 iso-
lates agreed well with those of the ex-holotype (CBS
101032) of P. paneum Frisvad, the third species in the
same section and its previous descriptions (Boysen
et al. 1996; Frisvad and Samson 2004), except for
producing finely rough conidia. As a whole, the
results of our morphological observations were in
concordance with those of O’Brien et al. (2008).
Further studies are needed to clarify whether or not
the finely rough conidial formation is a common
characteristic of P. paneum.
Chemotaxonomic characterization
of Penicillium sp. 1 isolates
Three selected Penicillium sp. 1 isolates (T5916-6-1,
T7214-8-1, and T7425-4-1) and the ex-holotype
strain CBS 101032 and CBS 465.95 of P. paneum,
which were determined in this study, had Q-9 as the
major ubiquinone system, a chemotaxonomic char-
acteristic (Table 1). It agreed with that of the
remaining two species within the same section
Roqueforti, P. roqueforti and P. carneum. As a
result, three known species comprising P. roqueforti,
P. carneum and P. paneum in the same section
Roqueforti, have been characterized by the same
ubiquinone system Q-9 (Table 2; cf. Kuraishi et al.
1991).
Molecular phylogenetic position
of Penicillium sp. 1 isolates
Here we show evidence from our molecular phyloge-
netic analyses of four different gene sequences (28S,
ITS, b-tubulin, and lys2; Figs 1, 2, supplementary
586 Antonie van Leeuwenhoek (2009) 96:579–592
123
Figs. S1 and S2) that the selected 15 isolates of
Penicillium sp. 1, i.e., 13 from the TT and 2 from the
KT (Table 1), are assignable to Penicillium paneum in
sect. Roqueforti subg. Penicillium.
Using P. brevicompactum JCM 22849 (Penicillium
subg. Penicillium sect. Coronata) as an outgroup taxon,
the NJ tree (supplementary Fig. S1) was inferred from
594 bp of 28S sequences of 44 isolates of Penicillium
and relatives. In the 28S phylogeny, all the TT and KT
isolates were placed in a cluster comprised only of
P. paneum, including the ex-holotype strain CBS
101032 (isolated from mouldy rye bread in Denmark)
of P. paneum. However, the bootstrap value showed
that the reliability of the phylogeny was not sufficient to
endorse a taxonomic position at a species level because
of a comparatively low confidence level (80%).
Fig. 1 Cultural and morphological characteristics of Penicillium sp. 1 (isolate T5916-6-1). Colonies on CYA (a), MEA (b), G25N
(c), CREA (d), and YES (e) at 25�C, 7 days. Conidiophores and penicilli (f, g), and conidia (h). Bars 10 lm
Antonie van Leeuwenhoek (2009) 96:579–592 587
123
Using the same outgroup taxon as the 28S phylog-
eny, the NJ tree (Fig. 2) was inferred from 519 bp of
ITS sequences of 43 isolates of Penicillium and
relatives. In the ITS phylogeny, all the TT and KT
isolates were placed in the cluster containing only P.
paneum, including its ex-holotype strain CBS 101032.
The bootstrap support showed a comparatively high
value, 94%. Incidentally, ITS is now expected to be the
primary target gene among possible barcodes for fungi
(e.g., All Fungi Barcoding [http://www.allfungi.com/
index.php]). An assessment of the DNA barcode for
Penicillium subg. Penicillium was done by Seifert
et al. (2007) using ITS, CO1 (mitochondrial gene
cytochrome oxidase I; also known as Cox1), and
b-tubulin gene sequences. CO1 provided barcodes for
Penicillium subgen. Penicillium, superior to the reso-
lution of ITS, but inferior to that of the b-tubulin gene.
Our case study of the barcode for TT and KT fungal
isolates is now in progress. The results will appear
elsewhere in the near future.
Using Eupenicillium catenatum CBS 431.69 as an
outgroup, the NJ, ML, and Bayesian trees (Fig. 3)
were inferred from 254 bp of b-tubulin sequences of
31 isolates of Penicillium and relatives. In the
b-tubulin phylogeny, all the TT and KT isolates
were placed in a cluster containing only P. paneum,
ITS
519bpNJ
T5916-6-1T7425-3-1T4519-5-5K5916-7-1T7510 5 1T7510-5-1Penicillium paneum CBS 101032HT
T7528-21-1Penicillium paneum CBS 465.95
T7425-4-1T5916-3-1K6203 2 1
94
Sect. RoquefortiK6203-2-1T7214-8-1T5916-1-1T6517-1-2
T7530-4-1
T7521-8A-453
63
T7521-8A-4T4906-11-8
Penicillium roqueforti JCM 22842NT
Penicillium carneum CBS 112297HT
Penicillium hirsutum JCM 22835NT
Penicillium allii DAR 74613Penicillium allii IBT 3056
99
53
Penicillium venetum IBT 5464Penicillium allii IBT 3056
Penicillium albocoremium IBT 10682HT
Penicillium verrucosum CBS 603.74NT
Penicillium viridicatum NRRL 961Penicillium polonicum NRRL 995HT
Penicillium echinulatum JCM 22836 IT
Sect. Viridicata
Penicillium echinulatum JCM 22836Penicillium camemberti CBS 299.48NT
Penicillium tricolor ATCC 10413Penicillium aurantiogriseum CBS 324.89NT
Penicillium griseofulvum NRRL 3523Penicillium concentricum NRRL 2034
Penicillium expansum CBS 325 48NT
Sect. Penicillium
Penicillium expansum CBS 325.48Penicillium digitatum CBS 136.65
Eupenicillium molle CBS 456.72HT
Eupenicillium crustaceum CBS 244.32NT
Penicillium madriti NRRL 3452HT
Penicillium chrysogenum JCM 22826NT
Penicillium glandicola CBS 498 75ET
54
Sect. Chrysogena
Sect. Digitata
S t P i illi
Sect. Penicillium
Penicillium glandicola CBS 498.75Eupenicillium crustaceum CBS 581.67
Eupenicillium tularense NRRL 5273HT
Penicillium brevicompactum JCM 22849 NT
0.005 substitutions/siteOutgroup
Sect. Penicillium
Fig. 2 Phylogenetic
relationships among 15 TT
and TK isolates and 28
known Penicillium isolates
for which the sequences
were obtained from
GenBank, based on NJ
analysis of ITS sequence
data of 519 aligned
nucleotide sites 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. T of the
respective isolate numbersindicates isolates from TT,
whereas K indicates those
from KT. Right verticalbars indicate the section(s)
and clade(s) defined by
Samson et al. (2004).
Strains with a superscriptindicate the strains derived
from; HT, Holotype; NT,
Neotype; IT, Isotype; and
ET, Epitype
588 Antonie van Leeuwenhoek (2009) 96:579–592
123
including its ex-holotype strain CBS 101032, with
strong statistical supports. The bootstrap values were
99 and 100%, respectively, and the posterior proba-
bility was 1.00. The respective three species of sect.
Roqueforti were well-supported by the bootstrapping,
as was the topology of the species phylogeny
demonstrated by Boysen et al. (1996). Very recently,
O’Brien et al. (2008) adopted b-tubulin and acetyl
CoA synthetase sequences to identify their isolates in
this section. The b-tubulin sequence-based phylogeny
of TT and KT Penicillium sp. 1 isolates were
identical with that of P. paneum isolated from baled
grass silage by O’Brien et al. (2008).
In fungi, aminoadipate reductase converts 2-ami-
noadipate to 2-aminoadipate 6-semialdehyde. An
et al. (2002) have already shown that the lys2 gene
(encoding of aminoadipate reductase) fragment is
useful for the phylogenetic analyses of ascomycetes.
However, the prokaryotes that biosynthesize lysine
through the aminoadipate pathway have no lys2 gene;
they synthesize lysine from the aminoadipate using a
different pathway (An et al. 2002, 2003; Nishida and
Nishiyama 2000). The lys2 gene is fungal-specific
(An et al. 2002).
Using P. brevicompactum JCM 22849 as an
outgroup taxon, the NJ, ML, and Bayesian trees
(not shown herein; see supplementary Fig. S2) were
inferred from 384 bp of the lys2 sequences of 30
isolates of Penicillium and relatives. In the lys2
phylogeny, all the TT and KT isolates were placed in
a cluster comprised only of P. paneum, including its
ex-holotype strain CBS 101032. The bootstrap supports
-tubulin254bpNJ/ML/B
T5916-6-1
T7425-3-1
T4519-5-5NJ/ML/Bayes K5916-7-1
T7510-5-1
T7528-21-1
T7425-4-1
T5916-3-1T5916-3-1
K6203-2-1
T7214-8-1
T5916-1-1Penicillium paneum CBS 101032HT
99 / 100 / 1.00
Sect. Roqueforti
T6517-1-2
T7530-4-1Penicillium paneum CBS 465.95
T7521-8A-4
T4906-11-899 / 92 / 1.00
95 / 92 / 1.00
Penicillium carneum CBS 112297HT
Penicillium roqueforti CBS 221.30NT
Penicillium chrysogenum CBS 306.48NT
Penicillium digitatum CBS 136.65
Penicillium glandicola CBS 498.75ET
94 / 94 / 1.00
Sect. Chrysogena
Sect PenicilliumPenicillium expansum CBS 325.48NT
Penicillium verrucosum CBS 603.74NT
Penicillium aurantiogriseum CBS 324.89NT
Penicillium hirsutum CBS 135.41NT
Penicillium camemberti CBS 299.48NT
Sect. Penicillium
Sect. Viridicata
Penicillium echinulatum CBS 317.48IT
Penicillium olsonii CBS 232.60NT
Penicillium brevicompactum CBS 257.29NT
Eupenicillium catenatum CBS 431.69
0.02 b tit ti / it
Sect. Coronata
Outgroup
0.02 substitutions/site
Sect. Digitata
Fig. 3 Phylogenetic
relationships among 15 TT
and TK isolates and 16
known Penicillium isolates
for which the sequences
were obtained from
GenBank based on NJ, ML,
and Bayesian analyses of b-
tubulin gene sequence data
of 254 aligned nucleotide
sites, using MEGA ver3.1.,
PHYLIP version 3.6., and
MrBayes. Numbers on
branches indicate Bayesian
posterior probability and
bootstrap support values
(%) based on 1,000
replications; bootstrap
values greater than 50% are
indicated. For other
abbreviations, see Fig. 2
Antonie van Leeuwenhoek (2009) 96:579–592 589
123
were 99 and 96%, respectively, whereas the posterior
probability was 0.70. No sequence variation was
observed in the 28S, ITS, and b-tubulin gene
sequences from the Penicillium sp. 1 isolates. In the
lys2 gene, however, T6517-1-2 differs isolates
T7425-4-1, T7510-5-1, T7528-21-1, T4906-11-8
and K6203-2-1 by one or two nucleotides. Conse-
quently, the lys2 gene may suggest a potential to
distinguish populations of P. paneum in genealogical
studies. Further analyses are needed to test the
availability for fungi.
Our molecular phylogenies inferred from the 28S,
ITS, b-tubulin, and lys2 gene sequences clearly
indicate that the 15 selected Penicillium sp.1 isolates
of TT and KT, as well as the ex-holotype and another
strain of P. paneum, formed a monophyletic cluster
with high bootstrap supports and posterior probability.
In our previous paper, we detected several haplo-
types of Fusarium and Trichoderma isolates from the
TT and KT stone chamber interiors and exteriors
(Kiyuna et al. 2008). However, our molecular phy-
logenies suggest that there are no haplotypes within
Penicillium sp. 1 (i.e., P. paneum). As shown in
Table 1 and supplementary Table S1, 50 representa-
tive strains of Penicillium sp. 1, which were isolated
from a variety of substrates such as mouldy spots and
viscous gels (biofilms) on plaster walls, soil, air, and
Isopoda’s body surface collected in different periods,
are thought to be the same species, P. paneum.
On the other hand, the most abundant phylotype
P. namyslowskii (=Geosmithia namyslowskii), a
common soil colonizer, has been reported from the
Chamber of Felines in the prehistoric painted cave of
Lascaux in France (Bastian and Alabouvette 2009).
P. paneum (this paper) and P. namyslowskii (Peterson
2000) shows a difference of 46 nucleotides in the ITS
region. In addition to this, the former is characterized
by the Q-9 system, whereas the latter has Q-8
(23%) ? 9 (77%) as the major ubiquinone system
(Ogawa et al. 1997). Therefore, there is a big
difference between the two.
So far P. paneum has been isolated from mouldy
rye breads, other foods (Frisvad and Samson 2004),
and baled grass silage (O’Brien et al. 2006, 2008).
The present report is thus the first account of
P. paneum isolated from samples relating to the
biodeterioration of cultural properties such as mural
paintings. This novel finding will help elucidate the
cause of biodeterioration of the TT and KT murals.
Very recently an important role of arthropods in the
dispersion of fungal spores and development has been
expressed by Bastian et al. (2009) in the prehistoric
painted cave of Lascaux. In the near future, we will
discuss on roles of P. paneum in the biodeterioration
of mural paintings and plaster walls, and also on the
invasion route to the TT and KT stone chamber
interiors.
In conclusion, the phenotypic and genotypic
characteristics of Penicillium sp. 1 isolates from TT
and KT samples, agreed well with those of the
authentic strains (including the ex-holotype) and
related references of P. paneum Frisvad (Boysen
et al. 1996; Frisvad and Samson 2004; O’Brien et al.
2008).
Acknowledgments We are grateful to the curators of
Centraalbureau voor Schimmelcultures (CBS) in Utrecht and
Japan Collection of Microorganisms (JCM) in Wako for
providing authentic strains (including ex-type) of Penicilliumspp. We also thank Dr. Heide-Marie Daniel (Editor) and an
anonymous reviewer for their critical comments and
suggestions in the revision process of this manuscript. 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. S., 2007-) from the Ministry of Education, Culture, Sports,
Science, and Technology, Japan.
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1
Supplementary Fig. S1 Phylogenetic relationships among 15 TT and TK isolates and
29 known Penicillium isolates for which the sequences were obtained from GenBank,
based on NJ analysis of 28S rDNA-D1/D2 region sequence data of 594 aligned
nucleotide sites using MEGA ver3.1. Numbers on the branch nodes represent bootstrap
support values (%) based on 1000 replications; bootstrap values greater than 50% are
indicated. T indicates isolates from TT, K indicates those from KT. Strains with a
superscript indicate the strains derived from; HT, Holotype; NT, Neotype; IT, Isotype;
and ET, Epitype.
Supplementary Fig. S2 Phylogenetic relationships among 15 TT and TK isolates and
15 known Penicillium isolates for which the sequences were obtained from GenBank
based on NJ, ML, and Bayesian analyses of lys2 gene sequence data of 384 aligned
nucleotide sites, using MEGA ver3.1., PHYLIP ver3.6., and MrBayes. Numbers on
branches indicate Bayesian posterior probability and bootstrap support values (%) based
on 1000 replications; bootstrap values greater than 50% are indicated. For other
abbreviations, see Fig. S1
T7214-8-1
T7425-4-1 T4519-5-5
T5916-1-1
T5916-3-1
Penicillium paneum CBS 101032HT
K5916-7-1
T7530-4-1
K6203-2-1
T7425-3-1
T7510-5-1 Penicillium paneum CBS 465.95
T7521-8A-4
T4906-11-8
T7528-21-1 T6517-1-2
T5916-6-1
Penicillium roqueforti JCM 22842NT
Penicillium carneum CBS 112297HT
Penicillium digitatum CBS 136.65
Eupenicillium crustaceum CBS 244.32NT
Eupenicillium molle CBS 456.72HT
Penicillium chrysogenum JCM 22826NT
Eupenicillium crustaceum CBS 581.67 Penicillium griseofulvum NRRL 2300NT
Penicillium coprophilum NRRL 13627
Penicillium hirsutum JCM 22835NT
Penicillium gladioli NRRL 939NT Penicillium echinulatum JCM 22836IT
Penicillium expansum CBS 325.48NT
Penicillium glandicola CBS 498.75ET
Penicillium commune CBS 311.48IT
Penicillium camemberti CBS 299.48NT
Penicillium madriti NRRL 3452HT
Penicillium aurantiogriseum CBS 324.89NT
Penicillium polonicum NRRL 995HT Penicillium sclerotigenum NRRL 3461HT
Penicillium cordubense NRRL 13072
Penicillium viridicatum NRRL 961
Penicillium verrucosum CBS 603.74NT
Eupenicillium tularense NRRL 5273HT
Penicillium atramentosum NRRL 795NT
Eupenicillium baarnense NRRL 2086NT Penicillium brevicompactum JCM 22849NT
56
51
80
0.002
28S 594bp NJ
substitutions/site
Sect. Roqueforti
T4519-5-5
T7528-21-1
Penicillium paneum CBS 101032HT
T7214-8-1
T7510-5-1
T7425-3-1
K6203-2-1
T5916-1-1
T7425-4-1 K5916-7-1
T7521-8A-4
T591661
T5916-3-1
T7530-4-1
Penicillium paneum CBS 465.95
T4906-11-8
T6517-1-2
Penicillium carneum CBS 112297HT
Penicillium roqueforti JCM 22842NT
Penicillium digitatum CBS 136.65
Penicillium expansum CBS 325.48NT
Penicillium glandicola CBS 498.75ET
Penicillium aurantiogriseum CBS 324.89NT
Penicillium hirsutum JCM 22835NT Penicillium echinulatum JCM 22836IT
Penicillium camemberti CBS 299.48NT
Penicillium verrucosum CBS 603.74NT
Penicillium chrysogenum JCM 22826NT
Penicillium inflatum CBS 68270
Penicillium brevicompactum JCM 22849
74 / - / -
59 / 80 / 0.93
99 / - / -
0.02
Sect. Roqueforti
63 / - / -
99 / 77 / 1.00
lys2
384bp
NJ/ML/Bayes
substitutions/site
63 / - / -
92 / 81 / 0.99
Supplementary Table S1 - List of strain data used in the cultural and morphological observations in this study
Isolate a Isolation source Sampling date
T4519-5-5b Moldy spots on the floor of the stone chamber of Takamatsuzuka Tumulus 19 May 2004
T4906-11-8b Surface of an insect body (Isopoda) from the wall in the stone chamber of Takamatsuzuka Tumulus 6 Sep 2004
T5916-1-1b Viscous gels below the forefoot of the painting of the white tiger on the west wall 2 in the stone chamber of Takamatsuzuka Tumulus 16 Sep 2005
T5916-3-1b Viscous gels below the paintings of the group of women on west wall 3 in the stone chamber of Takamatsuzuka Tumulus 16 Sep 2005
T5916-6-1b Viscous gels below the paintings of the group of women on east wall 3 in the stone chamber of Takamatsuzuka Tumulus 16 Sep 2005
T6517-1-2b Black spots on the paintings of the group of women on west wall 3 in the stone chamber of Takamatsuzuka Tumulus 17 May 2006
T7214-8-1b Black spots on western area of the adjacent space of Takamatsuzuka Tumulus (during excavation) 14 Feb 2007
T7425-3-1b Black substances in small pits on the top surface of west wall stone 3 of Takamatsuzuka Tumulus (during relocation of the stone chamber) 25 Apr 2007
T7425-4-1b Black substances from the top surface of east wall stone 3 of Takamatsuzuka Tumulus (during relocation of the stone chamber) 25 Apr 2007
T7510-5-1b Pieces of side plaster from the northern side of west wall stone 2 of Takamatsuzuka Tumulus (during relocation of the stone chamber) 10 May 2007
T7521-8A-4b Plastic cover over the thieving hole (stone chamber interior side) of Takamatsuzuka Tumulus 21 May 2007
T7528-21-1b Southern area of the ceiling plaster (stone chamber side) of ceiling stone 2 of Takamatsuzuka Tumulus (during relocation of the stone chamber)
28 May 2007
T7530-4-1b Black visclous gels and pieces of side plaster between the top of east wall stone 1 and ceiling stone 1 of Takamatsuzuka Tumulus (during relocation of the stone chamber)
30 May 2007
T4921-1-1 Near the painting of the group of men on west wall 1 in the stone chamber of Takamatsuzuka Tumulus 21 Sep 2004
T6517-14-2 Black spots on the eastern area of the adjacent space of Takamatsuzuka Tumulus 17 May 2006
T7417-11-1 Black spots on the north wall of Takamatsuzuka Tumulus (during relocation of the stone chamber) 17 Apr 2007
T7511-5-1 Bottom of west wall stone 3 of Takamatsuzuka Tumulus (during relocation of the stone chamber) 11 May 2007
T6220-1-1 Moldy spots near the paintings of the group of women on west wall 3 in the stone chamber of Takamatsuzuka Tumulus 20 Feb 2006
T6220-6-1 Moldy spots near the hind legs of the painting of the blue dragon on east wall 2 in the stone chamber of Takamatsuzuka Tumulus 20 Feb 2006
T6517-9-1 Viscous gels below the paintings of the group of women on east wall 3 in the stone chamber of Takamatsuzuka Tumulus 17 May 2006
T6713-1-2 Black spots on the paintings of the group of women on west wall 3 in the stone chamber of Takamatsuzuka Tumulus 13 July 2006
T6713-10-1 Purple viscous gels on upper part of the north wall in the stone chamber of Takamatsuzuka Tumulus 13 July 2006
T6713-12-2 Black moldy spots on the ceiling in the stone chamber of Takamatsuzuka Tumulus 13 July 2006
T6713-15-1 Wall of the adjacent space of Takamatsuzuka Tumulus 13 July 2006
T61017-1-2 Black spots on the paintings of the group of women on west wall 3 in the stone chamber of Takamatsuzuka Tumulus 17 Oct 2006
T61017-7-1 White moldy spots above the paintings of the group of women on east wall 3 in the stone chamber of Takamatsuzuka Tumulus 17 Oct 2006
T61114-2-1 Gray moldy spots on a ceiling stone of conservation facility of Takamatsuzuka Tumulus 14 Nov 2006
T61213-1-1 Black spots on the paintings of the group of women on west wall 3 in the stone chamber of Takamatsuzuka Tumulus 13 Dec 2006
T61213-8-1 Viscous gels below the paintings of the group of women on east wall 3 in the stone chamber of Takamatsuzuka Tumulus 13 Dec 2006
T61213-9-2 Black moldy spots on ceiling stone 1 in the stone chamber of Takamatsuzuka Tumulus 13 Dec 2006
T61213-12-1 Viscous gels on the north wall in the stone chamber of Takamatsuzuka Tumulus 13 Dec 2006
K5916-7-1b Dark-greenish viscous gels below the paintings of the tortoise and snake on the north wall in the stone chamber of Kitora Tumulus 16 Sep 2005
K6203-2-1b Black viscous substance in a hole on the the east wall in the stone chamber of Kitora Tumulus 3 Feb 2006
K5225-13-2 Colonies of airborne fungi in the stone chamber of Kitora Tumulus 25 Feb 2005
K5225-19-4 A piece of the fallen plaster from the western area in the stone chamber of Kitora Tumulus 25 Feb 2005
K5902-1-1 Dark-greenish viscous gels on the north wall in the stone chamber of Kitora Tumulus 2 Sep 2005
K5916-1-1 Viscous gels on the paintings of the vermilion bird (Suzaku) on the south wall in the stone chamber of Kitora Tumulus 16 Sep 2005
K6117-1-1 Black substance in hole on the plaster of ceiling in the stone chamber of Kitora Tumulus 17 Jan 2006
K6120-2-1 Soil from a space between ceiling and east wall in the stone chamber of Kitora Tumulus 20 Jan 2006
K6203-10-1 Viscous gels on the lower part of the west wall in the stone chamber of Kitora Tumulus 3 Feb 2006
K6303-1-1 Moldy spots on the floor in the stone chamber of Kitora Tumulus 3 Mar 2006
K6303-10-1 Colonies of airborne fungi in the adjacent small room of Kitora Tumulus 3 Mar 2006
K6330-1 Black needle-like molds on the east wall in the stone chamber of Kitora Tumulus 30 Mar 2006
K6613-1 White salt-like masses on the paintings of the vermillion bird (Suzaku) on the south wall in the stone chamber of Kitora Tumulus 13 June 2006
K6630-1-1 White viscous gels above the painting of the vermilion bird (Suzaku) on the south wall in the stone chamber of Kitora Tumulus 30 June 2006
K6630-5-1 Mushroom-like viscous gels on the north wall in the stone chamber of Kitora Tumulus 30 June 2006
K61208-2-3 Moldy spots on the floor in the stone chamber of Kitora Tumulus 8 Dec 2006
K7323-1-1 Yeast-like colonies on the north wall in the stone chamber of Kitora Tumulus 23 Mar 2007
K7706-1-3 Moldy spots on the east wall in the stone chamber of Kitora Tumulus 6 July 2007
K7905-2-1 Green mold on the floor in the stone chamber of Kitora Tumulus 5 Sep 2007a T and K indicate the Takamatsuzuka Tumulus and Kitora Tumulus, respectivelyb Strains used in molecular phylogenetic analyses