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

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Sep

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0

aT

and

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dic

ate

the

Tak

amat

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ka

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ora

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ined

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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

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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),

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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

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-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,

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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

––

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pa

neu

mC

BS

10

10

32

Ex

-ho

loty

pe

Mo

uld

yry

eb

read

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enm

ark

AB

47

92

73

AB

47

93

12

AY

67

43

87

AB

47

93

21

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b

P.

pa

neu

mC

BS

46

5.9

5M

ou

ldy

bak

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st,

Den

mar

kA

B4

79

28

0A

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79

31

1A

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74

38

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79

35

9Q

-9b

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po

lon

icu

mN

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95

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pe

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lan

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47

5A

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roq

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pe

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nch

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qu

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67

43

83

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-9b

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roq

uef

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iJC

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b

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scle

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gen

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tric

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04

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verr

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47

92

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x-n

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pe

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der

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of

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tech

no

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ion

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ter,

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o,

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tam

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pan

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tmen

to

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gri

cult

ure

,B

iolo

gic

alan

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hem

ical

Res

earc

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stit

ute

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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

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y,

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erea

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ed

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wer

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ted

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iet

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(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