Annals of Microbiology (2019) 69:1001–1021
https://doi.org/10.1007/s13213-019-1446-3

ORIGINAL ARTICLE

A time travel story: metagenomic analyses decipher the unknown
geographical shift and the storage history of possibly smuggled antiquemarble statues
Guadalupe Piñar 1

1

1& Caroline Poyntner & Hakim Tafer & Katja Sterflinger

1

Received: 3 December 2018 / Accepted: 30 January 2019 / Published online: 23 February 2019
# The Author(s) 2019

Abstract
In this study, three possibly smuggled marble statues of an unknown origin, two human torsi (a female and a male) and a smallhead, were subjected to molecular analyses. The aim was to reconstruct the history of the storage of each single statue, to infer the
possible relationship among them, and to elucidate their geographical shift. A genetic strategy, comprising metagenomic analysesof the 16S ribosomal DNA (rDNA) of prokaryotes, 18S rDNA of eukaryotes, as well as internal transcribed spacer regions offungi, was performed by using the Ion Torrent sequencing platform. Results suggest a possible common history of storage of thetwo human torsi; their eukaryotic microbiomes showed similarities comprising many soil-inhabiting organisms, which may
indicate storage or burial in land of agricultural soil. For the male torso, it was possible to infer the geographical origin, due to thepresence of DNA traces of Taiwania, a tree found only in Asia. The small head displayed differences concerning the eukaryotic
community, compared with the other two samples, but showed intriguing similarities with the female torso concerning thebacterial community. Both displayed many halotolerant and halophilic bacteria, which may indicate a longer stay in arid andsemi-arid surroundings as well as marine environments. The microbiomes retrieved from each statue showed to be very specific,
but some individual members showed to be biological markers for the geographical regions through which the pieces traveledand for the conditions under which they were stored.
Keywords Metagenomics . Microbiome . Cultural heritage objects . Geographical shifts . Storing conditions

Introduction
Each piece of art harbors a specific and absolute unique pile ofinformation about its own history. Every step in the history ofan object leaves its own genetic information: the plant fromwhich a canvas or paper was made, the organic binder used toprepare a paint, the animal from which a parchment was made,and the hands by which an object was manufactured andElectronic supplementary material The online version of this article(https://doi.org/10.1007/s13213-019-1446-3) contains supplementarymaterial, which is available to authorized users.
* Guadalupe Piñar
guadalupe.pinar@boku.ac.at
1

Department of Biotechnology, Vienna Institute of Biotechnology(VIBT), University of Natural Resources and Life Sciences,
Muthgasse 11, 1190 Vienna, Austria

through which it was passed during its history (Bower et al.
2010; O’Sullivan et al. 2016). In addition to the information
given by the material, each object carries a specificmicrobiome (Teasdale et al. 2017), mainly consisting of bacteria and fungi. The microflora again can be used as an indicator for the geographical and climatic regions through whicha piece of art traveled, the conditions under which it might
have been stored, or the soil in which it might have beenburied. Microbes—as humans—leave a specific mark on
and in an object: their unique fingerprint represented by
DNA (Burger et al. 2000).
Nowadays, using high-throughput molecular methods, it ispossible to extract and to analyze even smallest traces of DNA
from an object, to amplify these fragments, to visualize, andby the help of bioinformatics and international databases, toassign single molecules of DNA to a certain plant, a bacterium, and a fungus or even to humans (Fu et al. 2013). The
compilation of these data results in an individual Bbiologicalpedigree.^ The biochemical and molecular methods applied

1002

are the same that are in use in forensics—for example to identify an offender—or in medicine, where the microbiome of thehuman skin is currently in the focus of research (Schommer
and Gallo 2013).
To date, molecular analyses applied on cultural heritage
studies have mainly focused on the identification of microbialcommunities associated with specific biodeterioration phenomena displayed by different materials. Such studies haveenabled the implementation of conservative and disinfecting
treatments as well as the monitoring during and after the conservation treatments of valuable objects (Sterflinger and Piñar2013). Recently, the ongoing scientific and technologicalprogress has led to metagenomics, metatranscriptomics,
metaproteomics, and metabolomics, which are giving a more
complete overview of the present microorganisms, their activity, and the expressed proteins and produced metabolites in a
specific environment. These state-of-the-art methodologies
are now developing at a very fast pace, and next-generation
sequencing (NGS) methods are becoming applicable in thefield of cultural heritage (Adamiak et al. 2017, 2018),
allowing to answer the two most basic questions in microbial
ecology: Bwho is out there?^ and Bwhat are they doing?^
Nevertheless, the biological information contained inpieces of art has the potential to answer many other interestingquestions that may arise when dealing with cultural heritage
objects. The state-of-the-art genetic analyses can, for instance,
provide amazing insights into the geographical origins of artifacts (geolocation), the selection of materials at the time of
manufacturing, the storage conditions, the species composition of materials, and the use history of an object (Bower et al.
2010; Teasdale et al. 2017). These answers may help to understand many open questions in a variety of fields, such asarcheology, history, restoration, philology, and last but notleast, criminology. In some cases, curators and custom officers
are confronted with valuable smuggled objects of an unknown
origin. In these cases, analyses that could enable to make agrouping of some of the objects, as well as to infer the conditions of their storage and their geographical shift, could help toreconstruct the history of these objects, a kind of time travel.
In this study, three possibly smuggled marble statues of an
unknown origin were transitorily deposited in the Museumof Art History (KHM) in Vienna, Austria. Curators and conservators of the museum put many efforts to infer the origin
and the possible relationship among the three statues throughpetrological and morphological analyses, without success.
Therefore, they considered the possibility of constituting aninterdisciplinary team, recruiting microbiologists andbioinformaticians, in order to include a genetic strategy,
which could provide novel insights into these questions.
Metagenomic analyses of the 16S ribosomal DNA (rDNA)of prokaryotes, 18S rDNA of eukaryotes, as well as internaltranscribed spacer (ITS) regions of fungi were performed byusing the Ion Torrent sequencing platform, and the generated

Ann Microbiol (2019) 69:1001–1021

data were used to infer the storage history and the geographical shift of the samples, as well as to consider the possible
relationship among them.

Material and methods
Sampling
Three statues of unknown origin, two of them representing
human torsi, one female (S1) and one male (S2), and the third
one representing a small young girl head (S3), were depositedin the KHM, Vienna, Austria (Fig. 1). Samples were takenfrom the surface of the three statues with the help of sterile
scalpels and needles by gently scratching. Approximately 40–
60 mg of material, mainly powdered material, dust, dirt, andeven some textile fibers, was collected independently fromfour different points of each statue without damaging the marble stone. The material collected from each point was immediately placed into sterile Eppendorf tubes. Later, in the laboratory, the material collected from each point of the same
statue was pooled in order to obtain about 160–250 mg ofmaterial for DNA extraction analyses per statue. DNA extraction was performed at the next day of sampling.
Quality assurance (QA), in order to avoid any kind ofcontamination or bias, was performed by using only highquality reagents for DNA extraction, including single-use
manufactured vials to avoid cross-contamination as well as
high-quality reagents for down steps, as PCR amplificationand sequencing. All reagents used for library preparation andsequencing are tested and single packet/reaction, to avoid
contaminations. In addition, the steps of (1) DNA extraction
and amplification, (2) the DNA library preparation, and (3)the sequencing were performed in three separated laboratories dedicated only and exclusively to one of the mentioned
steps. DNA extraction was performed in a laminar flow bankonly used for ancient DNA (aDNA) preparations, and PCR
reactions were performed in a PCR workstation equippedwith UV lamps.

DNA extraction
Total DNA was isolated directly from the powdered material collected and pooled from each statue (160–250 mg/
statue, as mentioned above) using the FastDNA SPIN Kit
for soil (MP Biomedicals, Illkirch, France) as recommended
by the manufacturers. The DNA concentrations wereassessed by using the Qubit 2.0 fluorometer (Thermo
Fisher Scientific, MA, USA), which uses fluorescent dyes
to determine the concentration of nucleic acids, with the
Qubit dsDNA HS Assay Kit.

Ann Microbiol (2019) 69:1001–1021

1003

Fig. 1 The three antique marble
statues. a S1, female torso. b S2,
male torso. c S3, small head

PCR amplification
All PCR reactions were executed in a Bio-Rad C1000 Thermal
Cycler. PCR reactions performed to amplified eukaryotes andfungi were carried out with the 2× PCR Master Mix from
Promega (Vienna, Austria) (50 units/ml of Taq DNA
Polymerase supplied in an appropriate reaction buffer(pH 8.5), 400 μM dNTPs, 3 mM MgCl2) diluted to one time,to which 12.5 pmol/μl of each primer (stock 50 pmol/μl; VBCBiotech, Vienna, Austria) and 400 μg/ml BSA (stock
20 mg/ml; Roche Diagnostics GmbH, Basel, Switzerland) wereadded.
Quality control (QC) was performed in order to exclude thepossibility of cross-contamination, and in each PCR reaction,a negative control was included (no DNA template).
For the amplification of the eukaryotic 18S rDNA, fragments targeting the V4 region were amplified with the primers
5 2 8 F ( G C G G TA AT T C C A G C T C C A A ) a n d 7 0 6 R(AATCCRAGAATTTCACCTCT). The thermocycling program
was as follows: 5-min denaturation at 95 °C, followed by 35 cycles of 1-min denaturation at 95 °C, 1-min annealing at 53 °C,
and 1-min extension at 72 °C. Five-minute denaturation at 72 °C
was performed as a final extension step. Reactions were performed in 2× 50 μl, and a 3.5-μl DNA template was added toeach reaction tube. The duplicate amplicons of each sample (2×50 μl) were pooled, and 7 ml was visualized on 2% (w/v) agarose
gels, stained in an ethidium bromide solution (10 mg/ml), anddocumented using a UVP documentation system (Bio-Rad
Transilluminator; Universal Hood, CA, USA). The remaining
PCR reaction was purified using the QIAquick PCR
Purification Kit (Qiagen, Hilden, Germany).
For the amplification of fungal ITS regions, fragments of
450–600 bp, corresponding to the ITS1 and the ITS2 regions,and the 5.8S ribosomal RNA (rRNA) gene situated between
them were amplified with the primer pairs ITS1 forward and
ITS4 reverse (White et al. 1990). The thermocycling program
was as follows: 5-min denaturation at 95 °C, followed by
35 cycles of 1-min denaturation at 95 °C, 1-min annealing at
55 °C, and 1-min extension at 72 °C. Five-minutedenaturation at 72 °C was performed as a final extension step.
Thereafter, the obtained PCR products were further amplified
in a nested PCR using the forward primer ITS1 and the reverse
primer ITS2. The cycling scheme was the same as before, but
the annealing T was 58 °C, and the reactions were performed
in 2× 50 μl volume containing each 3.5 μl of template DNA.
The duplicate amplicons of each sample (2× 50 μl) werepooled and visualized as mentioned above. The remaining
PCR reaction was purified using the NucleoSpin Gel and
PCR Clean-up kit (Macherey-Nagel, Düren, Germany).
For the amplification of the prokaryotic 16S rDNA, theDNA was amplified using the Ion 16S Metagenomics Kit(Thermo Fisher Scientific, MA, USA). The kit includes two
primer sets that selectively amplify the corresponding hypervariable regions of the 16S region in bacteria (primer set V24-8 and primer set V3-6,7-9). After amplification, the reactions obtained from each sample with the two primer sets werepooled and mixed with Agencourt AMPure XP reagent(Beckman Coulter, CA, USA) for purification, using a magnetic rack as described by the manufacturers.
The concentration of all the purified amplicons (prokaryotes, fungi, and eukaryotes) was assessed with the Qubit 2.0
Fluorometer (Thermo Fisher Scientific, MA, USA).

Library construction and quantification
DNA libraries were constructed using the Ion Plus Library Kit
for the AB Library Builder System (Thermo Fisher Scientific,
MA, USA) following the library preparation protocol for short
amplicons, provided by the manufacturers. The amplicons
were processed as barcoded libraries, by using the P1 adapter
and the barcoded A adapters provided in the Ion Xpress
Barcode Adapter Kit. The adapters were diluted according to
the amount of DNA input, as recommended by the
manufacturers.
The resulting fungal and eukaryotic DNA libraries were
subsequently quantified by qPCR using the Ion Library
Quantitation Kit (Thermo Fisher Scientific, MA, USA) andthe prokaryotic DNA libraries using the Ion Universal Library1004

Quantitation Kit (Thermo Fisher Scientific, MA, USA).
Reactions were performed as described by the manufacturers,
to calculate the dilution factor of each library for a final concentration of 50 pmol. After individual quantitation, thebarcoded libraries of each sample (amplified with prokaryote-,eukaryote-, and fungus-specific primers) were pooled in equimolar amounts to ensure equal representation of eachbarcoded library in the sequencing run.

Ann Microbiol (2019) 69:1001–1021&&

Template preparation and sequencing&
Template preparation was performed by the Ion Chef System(Thermo Fisher Scientific, MA, USA) using the Ion PGM HiQ Chef Kit and the sequencing by using the Ion Personal
Genome Machine (PGM) using the Ion PGM Hi-Q
Sequencing Kit with the Ion 318 Chip v2 following the instructions of the manufacturers (Thermo Fisher Scientific,
MA, USA). All barcoded libraries amplified with the prokaryote primer sets were pooled in one chip, and all barcoded
libraries amplified with eukaryote- and fungus-specificprimers were pooled in a second chip.

Statue 1 (SAMN10477674): fungi1 (SRX5063102),
RUN: FungiTO1.fastq (SRR8245025); bacteria1(SRX5063103) RUN: bac1RfamAllClean.fastq(SRR8245024); eukaryote1 (SRX5063105) RUN:
Euk4TrimmedOnly.fastq (SRR8245022)
Statue 2 (SAMN10477675): fungi2 (SRX5063107),
RUN: FungiTO2.fastq (SRR8245020); bacteria2(SRX5063104) RUN: bac2RfamAllClean.fastq(SRR8245023); eukaryote2 (SRX5063106) RUN:
Euk5TrimmedOnly.fastq (SRR8245021)
Statue 3 (SAMN10477695): fungi3 (SRX5063108) RUN:
FungiTO3.fastq (SRR8245019); bacteria3(SRX5063109) RUN: bac3RfamAllClean.fastq(SRR8245018); eukaryote3 (SRX5063110) RUN:
Euk6TrimmedOnly.fastq (SRR8245017)

Data availability
All data generated or analyzed during this study are includedin this published article.

Data analyses
Raw reads were trimmed and filtered with cutadapt (Martin
2011). After trimming, only reads longer than 100 nt andcontaining at least one primer were kept. The prokaryotic
reads were first grouped based on their hypervariable region(HVR) of origin. The HVR was inferred by running cmscan(Nawrocki and Eddy 2013) with the prokaryotic 16S rRNA
model from Rfam (Kalvari et al. 2018) on each read and comparing the start and end position of the match with the corresponding HVR position in the Rfam model. Reads mapping
outside of the HVR were discarded while the other reads were
trimmed and filtered with cutadapt similar to what was done
for the eukaryotic and fungal reads.
Taxonomy assignment was made with DADA based on the
protocol published in Callahan et al. (2016). For the prokaryotic reads, the protocol was applied on each group of HVR
separately as well as on all HVR at the same time.

Geographical assignment
For each genus found in the samples, the geographic spread
was plotted by fetching information from the Global
Biodiversity Information Facility. Geographical assignmentof the species was done with the help of the R rgbif package(Chamberlain and Boettiger 2017).

Nucleotide sequence accession number
The nucleotide sequences were deposited in the GenBank
database under the accession number PRJNA506995:

Results and discussion
In this study, we investigated the microbiomes of three antique marble statues of unknown origin. Two different theories arose, the first arguing that the statues might have beenfound under the rubble of an old building and the second
arguing that they could have a criminal background of being
smuggled. In order to provide novel insights into the originof the three statues, we applied a genetic strategy including
metagenomic analyses, by using the Ion Torrent sequencing
platform, to test the suitability and the potential of the strategy to retrieve the maximal genetic information contained on
the surface of the statues. The resulting information was used
to reconstruct the history of the storage of each single object,
to infer the possible relationship among the three statues, andto elucidate their geographical shift. To this end, DNA libraries, targeting seven variable regions of the 16S rDNA fragments of prokaryotes, as well as a variable region of the 18S
rDNA of eukaryotes and the ITS regions of fungi, were
performed from each statue and further analyzed. To ourknowledge, this is the first metagenomic approach in cultural
heritage studies targeting so many regions in one single sample. The results showed to be very complex, and the challenge in this study, as in other Bomics^ studies, was theinterpretation of the results. Bioinformatic pipelines were
optimized in order to get results from lower taxonomic
levels, as genus and species, what is not always possible
when using universal primers.

Ann Microbiol (2019) 69:1001–1021

DNA extraction, yield, and amplification procedure
The DNAs were directly extracted from the pooled powderedmaterial collected from each statue and yielded a concentration of 11.7 ng/μl, 0.054 ng/μl, and 0.5 ng/μl for S1–S3,
respectively. DNAs were subjected to PCR analyses using
all the three primer sets (prokaryotes, eukaryotes, and fungi)
described in the section BMaterial and methods.^ All PCR
reactions showed positive results when using the eukaryotespecific primer pair, the fungal ITS-specific primer pair, as
well as the Ion 16S Metagenomics Kit.

Sequencing analyses
Two sequencing runs were carried out; the first run was performed with the three pooled barcoded libraries derived fromthe amplicons obtained with the Ion 16S Metagenomics Kit of
all three samples, generating 4,307,464 total reads, with anaverage of 1,305,936 reads per sample (supplementary
Table 1). The second run was performed with the six pooled
barcoded libraries derived from the amplicons obtained witheukaryote- and fungus-specific primers of all three samples,
generating 4,195,209 total reads, with an average of 585,818
reads per sample (supplementary Table 2).
Eukaryotic community
The DNAs obtained from the three statues were processed foreukaryotic and fungal metagenomic analyses, respectively.
The obtained DNA libraries were barcoded and pooled as
mentioned in the section BMaterial and methods^ and weresequenced in a single run, generating a total of 830 Mb.
Results derived from the eukaryotic DNA libraries revealed similarities between the two torsi (S1 and S2), both
displaying a low biodiversity concerning the amount of detected phyla, namely Ascomycota, Basidiomycota, andEmbryophyta (or land plants) (Fig. 2). In contrast, the smallhead (S3) showed a much higher diversity and some otheradditional phyla were detected in lower proportions (Figs. 2
and 3a). Even if the main targeted organisms in all three samples were fungi, it is worth noting that those phyla present inlower relative proportions showed to be important biologicalmarkers for specific environments.
The phylum Chlorophyta (green algae) accounted for 8%of the eukaryotic sequences retrieved from S3, and this phylum was negligible in the other samples (Fig. 4). Members
belonging to three of the four recognized classes within the
Chlorophyta were identified in S3: the freshwater or terrestrial
Chlorophyceae (97%) and Trebouxiophyceae (3%), as well as
the Ulvophyceae (0.5%). The latter group is best known as
marine macroalgae (green seaweeds) in coastal ecosystems(Cocquyt et al. 2010). Within the Chlorophyceae class, thegenera Heterochlamydomonas (82%), Fasciculochloris

1005(3%), Chlorosarcinopsis (3%), Chlorosarcina (4%), andChlorococcum (5%) were identified. All of them have been
described as terrestrial species (McLean and Trainor 1965),
but some of them are also found in freshwater and marine
environments and as aerial isolates. Chlorococcum sp. has
been previously described as one of the dominant organisms
contributing to biodeterioration of stone monuments in Korea(Klochkova et al. 2006). Within the Trebouxiophyceae class,
Trebouxia was the dominant genus. Members of this genus are
the most widespread photobionts in lichens but are only sporadically found in soil or bark outside of a lichen. They all
appear to be desiccation tolerant; i.e., they can survive drying
to water contents of below 10% (Carniel et al. 2015) and, thus,
are a good indicator for an osmophilic environment.
The phylum Cercozoa showed to be present only in S3, with arelative abundance (4% of eukaryotes). This phylum embraces
numerous ancestrally biciliate zooflagellates, euglyphid, and other amoebae (Cavalier-Smith and Chao 2003). The Cercozoa
contribute to a great biomass in soil (Urich et al. 2008) but arealso abundant in every marine habitat. A high percentage of thesesequences could not be identified at lower taxonomic levels (datanot shown). However, 29% of the sequences affiliated withmembers of the family Cercomonadidae, which are found in soil,
salt, brackish, and fresh water (Mylnikov and Karpov 2004); 3%
with members of the order Glissomonadida; and 1% with the
Marimonadida, namely with the genus Pseudopirsonia, which
are marine, naked, ancestrally gliding amoeboflagellates and flagellates (Howe et al. 2011).
Some additional eukaryotic phyla were detected only in S3,
as the phyla Ochrophyta and Chytridiomycota (0.3% and0.8% of total eukaryotic reads, respectively). Even though
they were detected in very low proportions, they represented
important ecological markers in this sample. The phylum
Ochrophyta is a major line of eukaryotes, most of them algae,
which are a primary component of plankton (Patterson 1989).
Several genera were identified, such as Xanthonema,
Spumella, Eustigmatos, and Chlamydomyxa, all reported tobe common in fresh waters. The phylum Chytridiomycota,often called chytrids, are mainly aquatic organisms (Barr
1990; Zhang et al. 2016), found in peats, bogs, rivers, ponds,
springs, and ditches. The presence of these two phyla in S3
supports the suspect that this sample was in contact with wateror even a marine environment.
Other sequences retrieved in very low proportion, but reinforcing the hypothesis that S3 was in contact with water, aresequences related with the Dinophyceae class of the dinoflagellates (0.06% of eukaryotic reads). Dinoflagellates constitute one of the main groups of marine and freshwater protists(Gómez 2012). In terms of a number of species, dinoflagellates are one of the largest groups of marine eukaryotes, andsome species are endosymbionts of marine animals. In addition, we detected some parasites, as members of theIchthyosporea class (0.04% of eukaryotes), namely the genus

1006

Ann Microbiol (2019) 69:1001–1021
100

Fig. 2 Relative abundance of the
eukaryotic community at thephylum level. S1, female torso;
S2, male torso; S3, small head

95

90

Ochrophyta

Phyla (%)

85

Chytridiomycota
Cercozoa

80

Chlorophyta
Embryophita

75

Basidyomicota
Ascomycota

70

65

60

S1

Ichthyophonus, which species are known to be unicellular
eukaryotic parasites of marine and freshwater fishes (Ragan

a

S1

S2

et al. 1996), and members of the Tubulinea of the Amoebozoa(0.03% of eukaryotes), which are free-living amoebae (FLA).

b

S2

S1

73
(2.8%)

69(6.8%)

3204(28.9%)

1096(24.1%)

22(4.1%)

6(35.4%)
253(7.8%)

6(0.3%)

65(7.2%)

2150(25.3%)

201
(22%)

S3

S2

76
(2.6%)

8(13.6%)

5(19.1%)

S3

Non−core:
0 (0%)

S3

Non−core:
0 (0%)

Fig. 3 Venn diagrams showing the OTUS shared by all three samples. S1, female torso; S2, male torso; S3, small head. a OTUs derived from eukaryotic
DNA libraries. b OTUs derived from prokaryotic DNA libraries

Ann Microbiol (2019) 69:1001–1021

a

1007

b

c

Fig. 4 Krona charts displaying the relative abundance of Embryophyta (land plants) at the genus level. a S1, female torso. b S2, male torso. c S3, small head

Within this group, we identified the genus Vermamoeba,
which infects various organisms, as freshwater fishes(Milanez et al. 2017).
The Embryophyta (or land plants) showed to be present inall three samples in relatively low proportions (2%, 1%, and
0.3% of eukaryotes for S1–S3, respectively), but they represented important biological markers. In fact, all samples
showed signs of storage in contact with agricultural soils,
where the most detected land plants were crop plants.
The order Poales was dominant in all three samples (77%,65%, and 52% of Embryophyta in S1–S3, respectively)(Fig. 4), and it was possible to identify plants of the genus
Aegilops in all samples (48%, 61%, and 32% of Embryophytain S1–S3, respectively), which are commonly known as goatgrasses. The statue S1 was also in contact with other crop
plants, as members of the Zea genus (29%). This genus includes large grasses, such as Zea mays, commonly known as
maize or corn, which is one of the most important crops forhuman societies all over the world. In addition, we could
detect in this statue DNA of plants of the Glycine genus (order
Fabales, 23%), of which the best-known species is the soybean. The majority of the Glycine species are found only inAustralia, but the soybean’s native range is in East Asia.
In S2, we could detect DNA from some other crop plants,
such as the genus Triticum, or common wheat (4%), which arewidely cultivated for their cereal grains and are a worldwide
staple food. In addition, this sample revealed traces of DNA
from plants of the genus Capsicum (or peppers) of the family
Solanaceae (4%), which are cultivated worldwide, but withthe highest production in East Asia. Also DNA of plants of thegenus Cucumis was found in this sample (4%), which is the
major group of angiosperms and includes gherkins, melons,and cucumbers, as examples.
S3 differed from the other two samples and showed thepresence of one of the largest subtribes of temperate grasses,
namely plants of the genus Loliinae (20%). In addition, plantsof the genus Brassica (order Brassicales, 31%), which are
informally known as cabbages, or mustard plants, weredetected in this sample. The genus Brassica is known for its
important agricultural and horticultural crops and includes
species and varieties commonly used for food, such as broccoli, cauliflower, cabbage, choy sum, rutabaga, and turnip,
and some seeds used in the production of canola oil and thecondiment mustard. The genus is native to Western Europe,
the Mediterranean, and temperate regions of Asia. Finally,
17% of sequences of Embryophyta were related to the order
Malvales, namely to the genus Malva. This genus consists ofabout 25–30 species of herbaceous annual, biennial, and perennial plants and is widespread throughout the temperate,
subtropical, and tropical regions of Africa, Asia, and Europe.
Nevertheless, the most relevant finding when analyzing the
DNA of land plants was the relative abundance of DNA (24%of total reads of land plants) of the genus Taiwania in S2.
Taiwania, with the single living species Taiwania
cryptomerioides, is a large coniferous tree in the cypress family Cupressaceae. It is native and endemic to Eastern Asia,
growing in the mountains of central Taiwan and locally inSouthwest China, adjoining Myanmar, and northern
Vietnam (according to the Global Biodiversity Information
Facility). The outliers outside Asia are located in botanicalgardens (supplementary Fig. 1). This tree was in very highdemand in the past for timber, particularly for temple building
and making furniture and coffins (BChinese coffin tree^).
Therefore, the species was decreasing and, nowadays, haslegal protection in China and Taiwan (Hayata 1906). The detection of DNA of Taiwania tree on the surface of S2 enabled
the geolocation of this statue, as staying or being transported
through Asia.
Fungal community
As mentioned in the section BEukaryotic community,^ themain targeted organisms in the eukaryotic 18S rDNA libraries
were fungi, accounting for 98% and 99% of the total reads forS1 and S2, respectively, and a lower proportion (86.6%) for
S3. The phylum Ascomycota accounted for 92%, 82%, and1008

Ann Microbiol (2019) 69:1001–1021

86%, while the phylum Basidiomycota represented 6%, 17%,
and 0.6% of all reads in the eukaryotic DNA libraries for S1–
S3, respectively. It is worth mentioning that data derived
from18S rRNA sequence analyses, even if showing a higher
diversity of fungi than ITS analyses, are not variable enoughto infer identification at taxonomic levels lower than genus.
Therefore, analyses of fungal ITS regions were implemented
to identify fungal species.
Ascomycota Within the Ascomycota, it was possible to infersimilarities, but also differences at lower taxonomic levels
among the three samples (Fig. 5). The order Eurotiales(97%, 93%, and 90% of Ascomycota reads for S1–S3, respectively), with the genus Aspergillus, showed to be dominant inall three samples. ITS analyses enabled to identify fungi at the
species level. The species Aspergillus sydowii was present inall three samples. This species is a saprophytic fungus foundmainly in soil, but it has been recently proven to be present indust and indoor environments (Visagie et al. 2014).
Aspergillus proliferans was present in S1 and S2; this species
has been widely found in indoor environments and food(Chen et al. 2017). Aspergillus penicillioides was present onlyin S1. This fungus is typically found on substrates with low
water activity, such as dried food. It is also known as a causal
agent of foxing on paper and books and of brown spots onancient Egyptian paintings (Koestler 2003). Aspergillus ruber
was present only in S2. This fungus has been described asplant pathogen, i.e., in red peppers (Ham et al. 2016), and theirpresence in S2 may correlate with the presence of plants of thegenus Capsicum (or peppers) also detected in this sample. S3
100
98
96

Others94

Order (%)

Dothideales
Pleosporales

92

Capnodiales
90

Hypocreales
Microascales

88

Sordariales
Chaetothyriales

86

Onygenales
Eurotiales

84
82
80

S1

S2

S3

Fig. 5 Relative abundance of the Ascomycota community at the orderlevel. S1, female torso; S2, male torso; S3, small headdiffered from the other two samples, and the dominant species
was Aspergillus microcysticus, which was isolated from a
Savannah soil in Somalia (Samson et al. 2011). Members of
the genus Penicillium were detected surprisingly only in S1,with the species Penicillium glandicola and Penicillium
citrinum. These species have been detected growing in association with plants, such as cereals (Vinokurova et al. 2003),
which were also identified as dominant land plants in thissample. Nevertheless, both species have been recently identified as causative agents of the biodeterioration on rock surfaces (Ogórek et al. 2016; Boniek et al. 2017).
The order Onygenales was negligible in S1 and S2 but
accounted for 6% of the Ascomycota in S3 (Fig. 5), in which
fungal members of the family Gymnoascaceae were all identified. This family includes species of some genera (asGymnoascus) that have been found in sediments of salt lakes,
compost, and cornfield soils and described as halophilic fungi(Zhou et al. 2016). Interestingly, only S3 showed the presenceof members of the order Chaetothyriales, even though in avery low proportion (0.03% of Eurotiomycetes class).
Members of the order Sordariales showed to be represented in all three samples (Fig. 5) but dominated in S2 (0.3%, 6%,
and 2% of Ascomycota reads for S1–S3, respectively). ITS
analyses revealed that all members of this order belonged to
the family Chaetomiaceae, with Chaetomium as the dominantgenus in S1 and S3. Chaetomium species are capable of colonizing various substrates and are well known for their ability
to degrade cellulose. The species Chaetomium murorum was
identified in S1; this species has been identified in geographically widely distributed indoor environments (Wang et al.
2016). In contrast, the genus Thielavia with the species
Thielavia basicola dominated in S2. This species is a phytopathogenic fungus, known to attack the roots of at least 100
angiosperm plants (Alam et al. 2012), as members of the family Cucurbitaceae, which correlates with some of the land
plant sequences retrieved from this sample.
Members of the order Microascales could be identified in
S1 and S3 (0.4% and 2% of Ascomycota) but were absent in
S2 (Fig. 5). ITS analyses allowed identifying the species
Microascus verrucosus in S1. The genus Microascus comprises species commonly isolated from soil, decaying plantmaterial, and indoor environments, but few species are also
recognized as opportunistic pathogens.
The order Hypocreales was detected in all three samples,
accounting for 0.1%, 0.5%, and 0.3% of Ascomycota(Fig. 5), with Gibberella and Fusarium as the dominant genera.
Both genera comprise a large variety of plant pathogens (riceand grain) with worldwide distribution. Gibberella intricans
was identified only in S1, which is an opportunistic pathogen
of durians (Sivapalan et al. 1998). The durian is the fruit ofseveral tree species belonging to the genus Durio, abundant
in Southeast Asia. Fusarium sp. and, in less proportion,
Acremonium sp. were identified only in S3. Acremonium

Ann Microbiol (2019) 69:1001–1021

contains many species that are saprophytic, which was isolated
from dead plant material and soil. The presence of members of
the order Hypocreales in all three samples seems to correlatewith the presence of plants and as normal inhabitants of soils.
The order Capnodiales was present in S1 and S2 and madeup 1% and 0.1% of Ascomycota, respectively (Fig. 5). ITS
sequencing allowed to infer lower taxonomic levels andshowed differences between the samples. The genus
Cladosporium, with species of Cladosporium halotoleransand Cladosporium sphaerospermum, was detected only in
S1. Both species have been found in hypersaline aqueous
habitats worldwide and are described as halo- or osmotolerant
as a recurrent feature. Both species have been isolated fromhypersaline water (Zalar et al. 2007). This finding is in linewith the many halotolerant-halophilic bacteria detected in thissample. In contrast, the genus Vermiconia of the Extremaceae
family, with the species Vermiconia calcicola, was identified
in S2. This species is an extremotolerant rock-inhabiting black
fungus, which was isolated and described for the first time inmarble monuments in Italy (Isola et al. 2016). Surprisingly, in
S3, members of the order Capnodiales were detected only
when analyzing ITS regions, and two species, namely
V. calcicola and Extremus antarcticus, were identified. The
latter species was originally isolated from rock samples in
the Antarctica (Quaedvlieg et al. 2014) but also isolated andidentified as a rock-inhabiting black fungus in Italian monumental sites (Isola et al. 2016).
The order Pleosporales accounted for 0.5%, 0.01%, and
0.3% of Ascomycota in S1–S3, respectively (Fig. 5). Further
analyses of ITS regions allowed identifying Alternaria
alternata as the dominant species in S1, in addition to
Alternaria chlamydospora. Alternaria alternata causes leaf
spot and other diseases on over 380 host species of plant.
Interestingly, A. chlamydospora is primarily found in salty
soils (Mouchacca 1973). Some additional species were identified in S1 in lower proportions, such as Didymella urticicola,
Phoma saxea, Stagonosporopsis sp., and Preussia sp. All ofthem are plant pathogenic, saprobic, and endophytic species(Chen et al. 2015). S2 and S3 showed much less diversity
inside this order, being Phoma saxea the only species identified in S2 and as the dominant species in S3.
Deeper analyses of 18S rDNA sequences enabled to detect
the presence of other fungal taxa, even if in very low proportions. In this way, members of the order Dothideales represented 0.01% of the Ascomycota in S2; members of thePezizomycetes class were identified in S1 and S3 (0.3% and0.07% of Ascomycota, respectively). The genus
Pseudombrophila was detected in S1, which is found on deadwood, soil, or rotten plant remains and on substrates often
contaminated with urine by animals. In contrast, in S3, allsequences affiliated with the genus Tuber. The true truffles
belong to this genus, one of the few ectomycorrhizal
Ascomycetes, which are highly valued as delicacies (Mello1009

et al. 2006). In addition, members of the Saccharomycetales
were detected in S2 and S3 (0.2% and 0.03% of Ascomycota,respectively). The genus Cyberlindnera with the species
Cyberlindnera jadinii was detected only in S2. This fungus
has been used since the early 1900s as fodder yeast for livestock and as a dietary supplement for humans.
Basidiomycota Within the Basidiomycota, the majority of thesequences affiliated with members of the Agaricomycetes
class (99% of Basidiomycota for S1 and S2 and 78% for S3,
data not shown), in which it was not possible to infer lower
taxonomic levels in S1 and S2, but in sample S3. The lattersample showed the presence of relatives of the genera
Pleurotus and Pluteus (order Agaricales) commonly calledoyster, abalone, or tree mushrooms. In addition, S3 showedthat 22% of the Basidiomycota sequences were affiliated withthe order Malasseziales (Exobasidiomycetes class), namely
with species of the genus Malassezia. This genus comprises
yeast species that are part of the normal skin microbiota, butsome species can also be involved in skin disorders (Velegraki
et al. 2015). Malassezia spp. have not yet been cultured fromthe environment; nevertheless, metagenomic studies haveidentified Malassezia phylotypes from marine habitats(Amend 2014).
In addition, other basidiomycetes detected in very low proportion allowed inferring additional differences among thethree samples, as members of the Microbotryomycetes class,
only detected in S1 (0.6% of Basidiomycota). ITS analyses
further enabled to assign these sequences to the genus
Rhodotorula, namely to Rhodotorula kratochvilovae.
Rhodotorula is common environmental yeast isolated from different ecosystems and environments, including sites with extreme conditions. In contrast, members of the order
Ustilaginales (0.5% of Basidiomycota) were detected only in
S2. Members of this order are plant pathogens and are known
as the smut fungi, which are the causal agent of corn smut
disease (Stirnberg and Djamei 2016). Their presence in thissample may be associated with the detected corn plants (Zea).
Prokaryotic community
First, it is worth mentioning that in this study, we applied forthe first time in cultural heritage studies a metagenomic strategy implying the amplification of seven of the nine variable
regions of the 16S rDNA of prokaryotes, simultaneously. The
Ion 16S Metagenomics Kit (Thermo Fisher Scientific) wasused. This kit takes advantage of using two primer pools toamplify seven of the nine variable regions of the prokaryotic
16S rDNA (V2, V3, V4, V6, V7, V8, and V9). The results(supplementary Fig. 2) clearly highlighted that some variable
regions overestimate specific phyla, as it was very evident for
the V9 region, which almost uniquely amplified the phylum
Proteobacteria. In contrast, other variable regions

1010

Ann Microbiol (2019) 69:1001–1021

for 1% and 0.7% of total reads for S1 and S3, respectively, andthe phylum Planctomycetes for 0.7% of total reads in S1 andS3, and the latter phyla were negligible in S2.
Results derived from prokaryotes supported those obtained
from eukaryotes, and sequences derived from all samples
showed strong evidence of contact with agricultural soils.
All samples showed high abundance of Actinobacteria(Fig. 6). Members of this phylum are widespread in soils
throughout the world and are successful colonizers of different
extreme environments. However, important differences wereobserved among the three samples concerning the biodiversityof this phylum (supplementary Fig. 3). S2 showed the lowest
biodiversity within the Actinobacteria, and only members of
the order Micromonosporales (76% of Actinobacteria),
Frankiales (17% of Actinobacteria), and Micrococcales (2%of Actinobacteria) were represented with relatively high abundance in this sample. Within the order Micromonosporales,
the genus Micromonospora dominated, accounting for 49% of
all Actinobacteria, followed by the genera Plantactinospora(10%) and Verrucosispora (3%). Bacteria belonging to theMicromonospora genus have been widely isolated from mangrove soils, where they represent high populations (Honget al. 2009). The genus Plantactinospora represents mainlybacteria associated with plant tissues, such as endophytic
Actinomycetes which was isolated from Camptothecaacuminata Decne, a Chinese traditional medicine plant endemic to Southwest China (Zhu et al. 2012). Species of thegenus Verrucosispora have been isolated from peat bog anddeep-sea sediments but also from mangrove sediments in
China (Xi et al. 2012). Within the Frankiales, the only

underestimated or even completely missed specific phyla, as
the V4, which was not targeting the phylum Planctomycetes in
S3 (supplementary Fig. 2c). This fact is of great relevance,
because the V4 region is the one that is standardly used inmost of the metagenomic approaches performed with theIllumina sequencing platform. In summary, these results showthat the targeting of specific variable regions of the 16S rDNA
gene can influence the results and remark the necessity totarget more than one variable region simultaneously, in order
to diminish the bias caused when analyzing single hypervariable regions of the 16S rDNA (Fouhy et al. 2016).
All three samples were processed for 16S rDNA
metagenomic sequencing, generating a total of 774 Mb.
Computational analyses were performed, merging the results
obtained from all targeted variable regions, and revealed 22
bacterial phyla in all three marble samples. Nevertheless, only
eight phyla showed to count for at least 0.5% of the total reads
in at least one sample. At the level of phyla, differences could
be observed among the three samples (Figs. 3b and 6); members of the Actinobacteria phylum showed to be dominant in
S1, representing 43% of the total sequences and 32% and 29%of total sequences for S2 and S3, respectively. In contrary, theFirmicutes phylum dominated in S2 and S3, with 62% and
55% of the total sequences, respectively, representing 35% ofsequences for S1. The Proteobacteria phylum showed to bealso representative in all three samples (15%, 6%, and 12% for
S1–S3, respectively). Interestingly, the phyla Bacteroidetes
and Acidobacteria accounted for 2% and 1% of the total reads
only in S1, and the phylum Tectomicrobia accounted for 1%of total reads only in S3. The phylum Chloroflexi accounted
100%

Fig. 6 Relative abundance of thebacterial community at thephylum level. S1, female torso;
S2, male torso; S3, small head

90%

80%

Phyla (%)

70%

othersPlanctomycetes

60%

Chloroflexi
Tectomicrobia

50%

Acidobacteria
Bacteroidetes

40%

Proteobacteria
Firmicutes

30%

Actinobacteria
20%

10%

0%

S1

S2

S3

Ann Microbiol (2019) 69:1001–1021

detected genus was Blastococcus (16% of Actinobacteria).
Bacteria of this genus are commonly isolated from the surfaceof marble and calcareous stones (Urzì et al. 2004).
In contrast, S1 and S3 showed a much higher biodiversity
concerning the phylum Actinobacteria. At least seven orders
could be detected within the Actinobacteria class in all two
samples with relatively high abundance, at least in one of them.
Some orders were present in only one sample, such as the order
Corynebacteriales, which made up 23% of the phylum
Actinobacteria in S1, which were almost negligible in the othertwo samples (supplementary Fig. 3). The Corynebacterium_1
group showed to be dominant (17% of Actinobacteria).
Members of this group have been described mainly in swine
manure (Song et al. 2017). At the species level, it was possible
to identify the species Corynebacterium maris (5% ofActinobacteria), which are halotolerant bacteria isolated forthe first time from the coral Fungia granulosa in the Gulf ofEilat, Israel (Ben-Dov et al. 2009), and in lower proportion in
Corynebacterium efficiens, which have been isolated from soil
and vegetables (Fudou et al. 2002). Two additional genera weredetected within this order in S1, namely Mycobacterium andDietzia (each representing 2% of Actinobacteria), and bothwere isolated from aquatic and terrestrial habitats, as well asfrom clinical material (Koerner et al. 2009).
Bacteria of the order Micrococcales dominated in S1 (16%of the phylum Actinobacteria) but were also present in S2 andS3 (2% and 5% of Actinobacteria, respectively). The genera
Brachybacterium and Kocuria were the most dominant in S1(each 3% of Actinobacteria), followed by the genera
Citricoccus and Curtobacterium (each 1%), in addition to othergenera with relatively lower proportions as Arthrobacter andPseudarthrobacter (both 0.7% of Actinobacteria) as well as
Agromyces (0.5%). Species of the genus Brachybacterium
have been isolated from poultry deep litter, stool samples, soils,
sediments, and seawater (Collins et al. 1988). Bacteria belonging to all abovementioned genera are typically species found insoils. Kocuria and Citricoccus have been widely isolated fromsoils, and some species are halotolerant and alkalitolerant (Liet al. 2005; Wang et al. 2015). The Curtobacterium genus has a
global distribution with a predominant presence in soil ecosystems and is described mainly in association with plants, especially in the phyllosphere (Chase et al. 2016). Agromyces are
soil actinomycetes with formation of true mycelia (Zgurskaya
et al. 1992). In contrast, the most dominant genus in S3 was
Arthrobacter, with the species Arthrobacter crystallopoietes.
This species has been described as desiccation-resistant bacteria, and this capability may explain their numerical dominancein soil (Boylen 1973).
Bacteria of the order Streptomycetales were present in relatively high abundance in S1 (14% of the phylum
Actinobacteria) and S3 (9%). The genus Streptomyces dominated in both samples (13% and 8%, respectively). Bacteria
belonging to this genus are ubiquitous in soils and marine

1011

sediments and are commonly found in the rhizosphere or inside plant roots (Rey and Dumas 2017). The species
Streptomyces albiaxialis was detected only in S1, which is athermotolerant and halotolerant Streptomyces (Kuznetsov
et al. 1992). In addition, it was also possible to detect in a very
low proportion members of the genus Streptacidiphilus. The
genus Streptacidiphilus is composed of acidophilic actinomycetes and has a major role in the turnover of organic matter inacidic habitats (Kim et al. 2003).
The order Streptosporongiales was better represented in S1and S3 (6% and 7% of the phylum Actinobacteria, respectively), with the genus Nocardiopsis as dominant in S1 andActinomadura in S3. Nocardiopsis is a bacterial genus that
produces some antimicrobial compounds and occurs mostly
in saline and alkaline soils (Bennur et al. 2014). Bacteria of
the genus Actinomadura are commonly isolated from soils(Zhao et al. 2015).
The order Propionibacteriales accounted for 3% and 7% of
the phylum Actinobacteria in S1 and S3, respectively. The
most represented genera in both samples were Nocardioides
and Microlunatus, and both genera are common bacteria insoils (Kämpfer et al. 2010; Wang et al. 2017). Finally, theorder Pseudonocardiales accounted for 4% and 6% of thephylum Actinobacteria in S1 and S3, respectively, in which
Pseudonocardia is the dominant genus. In both samples, it
was possible to detect the species Pseudonocardia zijingensis,
which was isolated from a soil sample collected from Zijing
Mountain, Yunnan Province, China (Huang et al. 2002).
Members of the Thermoleophilia class were detected in S1and S3 (24% and 23% of the phylum Actinobacteria, respectively). Uncultivated bacteria of the Solirubrobacterales Elev-16S1332 group as well as cultivated bacteria of the genera
Solirubrobacter and Gaiella showed to be dominant in both
samples, which are bacteria commonly isolated from soil
(Singleton et al. 2003) and water. In addition, members of theclass Acidimicrobiia were detected in both samples (3% of thephylum Actinobacteria in each sample) where it was possible toinfer only the genus Lamia, which members have been isolated
from marine environments (Kurahashi et al. 2009). Curiously,
members of the Rubrobacteria class were only detected in a
relatively high proportion (23% of the phylum Actinobacteria)in S3, where it was possible to identify only the genus
Rubrobacter. Unfortunately, it was not possible to infer at the
species level, but species of this genus have been widely detected
in terrestrial habitats, as stone monuments usually subjected tosalt weathering (Otlewska et al. 2017) as well as in marine environments, and some of them are known to be halotolerant,
thermotolerant, and radiotolerant (Egas et al. 2014).
Interestingly, members of other phyla found in significant
proportions only in S1 strongly indicated that the soil being incontact with this statue must have been from animal farm. For
example, members of the phylum Bacteroidetes were present
only in this sample, accounting for 2% of bacteria (Fig. 6),

1012

with mainly species of Bacteroides. Members of Bacteroides
make up the most substantial portion of the mammalian gastrointestinal flora and play a fundamental role in the processing of complex molecules to simpler ones in the host intestine(Wexler 2007); the species Bacteroides barnesiae was identified, which has been isolated from the cecum of a healthy
chicken (Lan et al. 2006). In addition, members of the family
Rikenellaceae, with the Rikenellaceae_RC9_gut group andthe genus Alistipes, as well as members of the family
Prevotellaceae were identified. Members of these two families are often found in the gastrointestinal tract of different
animals and humans (Rosenberg 2014). Other bacteria that
could be indicators of a contact with soils of animal farms
belonged to the phylum Acidobacteria, accounting for 1% ofbacteria in S1 (Fig. 6), which are among the most common
bacteria in soils worldwide. Nevertheless, the main
Acidobacteria group detected in S1 was subgroup 6, which
members are related to those detected in pasture soils(Navarrete et al. 2015).
In addition, sequences related to the phylum Firmicutes, retrieved in significant proportions only from S1, confirmed thesuspicion that this statue must have been in contact with animal
farm soil (supplementary Fig. 3). Sequences affiliated to the order
Clostridiales were found in a relatively high proportion (8% ofFirmicutes). Within this order, the most dominant members comprised the Christensenellaceae_R-7 group, which includes uncultured bacteria mainly found in rumen, as well as members of thegenera Romboutsia, Intestinibacter, and Clostridium and of thefamilies Ruminococcaceae and Lachnospiraceae, all of themwere identified in the gut microbiota of human and other animals(Gerritsen et al. 2014; Meehan and Beiko 2014). Sequences related to the order Lactobacillales were also detected in high proportion in this sample (12% of Firmicutes). The most detected
genus was Lactobacillus (3% of Firmicutes), which are a majorpart of the group of lactic acid bacteria (LAB) and in humans andconstitute a significant component of the microbiota at a numberof body sites, such as the digestive, urinary, and genital systems.
At lower taxonomic levels, it was possible to identify the species
Lactobacillus kitasatonis, Lactobacillus reuteri, Lactobacillus
pontis, and Lactobacillus salivarius, among the other unidentified
species. Several genera within the family Carnobacteriaceae
were also detected, such as Atopostipes and Carnobacterium(each 3% of Firmicutes). At the level of species, the only one
known species (Atopostipes suicloacale) was identified in thisstudy. This species has been previously isolated from an underground swine manure storage pit (Cotta et al. 2004). Members of
the genus Carnobacterium are ubiquitous LAB, which are isolated from cold and temperate environments, and predominate ina range of foods, including fish, meat, and some dairy products(Leisner et al. 2007). Other members of the familyEnterococcaceae could be detected even in low proportions(0.2% of Firmicutes), with the species Enterococcus cecorum,
which is known as a commensal in the intestines of mammals

Ann Microbiol (2019) 69:1001–1021

and birds, but it has been described as an emerging pathogen inpoultry industry worldwide (Dolka et al. 2017). Sequences related to the order Erysipelotrichales (Firmicutes), which species are
known to be common in the gut microbiome of animals, werealso detected in this sample (4% of Firmicutes), with the genus
Turicibacter as the sole representative (Auchtung et al. 2016).
In contrast, S2 and S3 showed to have a lower biodiversityof Firmicutes and were dominated by members of the order
Bacillales (supplementary Fig. 3), which represent 97% and
82% of Firmicutes, while this order accounted for 76% ofFirmicutes in S1. Nevertheless, the detected genera showed
to be different among the samples. S2 and S3 showed a strong
dominance of Bacillus (39% and 23% of Firmicutes, respectively). At the level of species, Bacillus timonensis andBacillus subtilis were detected in S2. Bacillus timonensis has
been detected in human fecal flora (Kokcha et al. 2012), andB. subtilis is commonly found in the gastrointestinal tract ofruminants and humans and is widely used as a probiotic feed
supplement for farm animals (Wang et al. 2018). In addition,
this species collectively inhabits the rhizosphere, where it contributes to the promotion of plant growth (Hirooka 2014).
In S3, it was possible to detect a wider range of species ofBacillus. Some of them accounted for relatively high proportions, such as Bacillus niacini and Bacillus longiquaesitum,
both isolated from soils (Nagel and Andreesen 1991), but thelatter one was also commonly isolated from microbial communities associated with truffles (Mello et al. 2013). It is
worth noting that traces of DNA of truffles were identified
in this sample (genus Tuber). Other species were detected inthis sample, such as Bacillus nealsonii, which produces endospores that are resistant to UV, gamma-radiation, salinity, anddesiccation (Venkateswaran et al. 2003). In lower proportions,
Bacillus humi, Bacillus benzoevorans, and Bacillus pumilus
were detected as well, all of them most commonly isolated
from soils (Heyrman et al. 2005) and known to be plant
growth-promoting bacilli (Yadav et al. 2011).
The genus Anoxybacillus was additionally detected in S2 (2%of Firmicutes). Many species of this genus are known to bealkaliphilic and thermophilic and commonly occur in geothermal
springs, manure, and milk processing plants (Yamamoto et al.
2006). Other detected genera in S3 were Fictibacillus (2% ofFirmicutes) and Falsibacillus (1%), both commonly isolated
from marine environments (Dastager et al. 2014).
In contrast, S1 showed a much lower proportion of Bacillus(7% of Firmicutes) and the only identified species was
Bacillus alkalitelluris, an alkaliphilic bacterium isolated fromsoil in Korea (Lee et al. 2008). The genus Oceanobacillus was
also detected in this sample in a relatively high proportion (4%of Firmicutes). Oceanobacillus species have been isolated
from diverse environmental samples, including deep-sea sediment cores and salt fields; fermented shrimp paste samples,soy sauce production equipment; and traditional Koreanfermented food. We could identify some species, such as

Ann Microbiol (2019) 69:1001–1021

Oceanobacillus iheyensis, an extremely halotolerant species
isolated from deep-sea sediment (Lu et al. 2001);
Oceanobacillus profundus, a halotolerant bacterium, isolated
from the deep surface of a sediment core in Korea (Kim et al.
2007a); and Oceanobacillus picturae, isolated for the firsttime from a mural painting (Heyrman et al. 2003). Other genera identified in lower proportions (0.9–0.2% of Firmicutes),such as Virgibacillus, Falsibacillus, Fictibacillus,
Pontibacillus, Terribacillus, and Marinococcus, further supported the hypothesis that statue 1 was in contact with a saline
environment, in which many of the species of all detected
genera were able to grow in moderate concentrations of
NaCl and were isolated from saline environments.
S1 showed actually a higher proportion of members of the
Planococcaceae family (39% of Firmicutes), which belong to
the genus Planomicrobium (32% of Firmicutes). At the level of
species, Planomicrobium okeanokoites showed to be dominant.
This species has been isolated from the marine muds and fromsoils in China (Nakagawa et al. 1996). Other species detected inlower proportions were Planomicrobium glaciei (Zhang et al.
2009), which was isolated from a glacier in Northwest China,
and Planomicrobium chinense (Dai et al. 2005), which was isolated from a coastal sediment of the Eastern China Sea, China.
Both species are able to growth at moderate salinities (0–10%
NaCl). The genera Planococcus and Sporosarcina were also
detected, but in lower proportions (1% of Firmicutes).
Members of the Planococcaceae were less dominant in S2 andS3 (3% and 10% of Firmicutes, respectively), and the main genera detected in these samples differed from those in S1. Bacteriaof the genus Domibacillus were dominant in both samples (1%and 5% of Firmicutes in S2 and S3, respectively), in addition to
Sporosarcina (0.2% and 2% of Firmicutes, respectively). At thelevel of species, Sporosarcina soli was detected in S2 andSporosarcina koreensis in S3. Both species were isolated forthe first time from soils in Korea, and both tolerate moderate(5–7%) NaCl concentrations (Kwon et al. 2007). The genus
Chungangia (1% of Firmicutes) was only present in S2. Other
genera detected in lower proportions in S3 were
Paenisporosarcina, Lysinibacillus, and Bhargavaea.
Members of the Staphylococcaceae were present in relatively high abundance only in S1 (14% of Firmicutes) whichwere negligible in the other two samples. The genus
Salinicoccus showed to be dominant (10% of Firmicutes) inthis sample. It was possible to identify the species
Salinicoccus kekensis, a moderate halophilic bacterium isolated from a salt lake in China (Gao et al. 2010), as well as otherspecies, such as Salinicoccus halodurans, Salinicoccus
kunmingensis, and Salinicoccus roseus, which were isolated
from saline soils and a salt mine in China (Chen et al. 2007;
Wang et al. 2008). The genus Jeotgalicoccus accounted, in
addition, for 3% of Firmicutes in S1. Bacteria of this genus
are halotolerant to halophilic bacteria. The species
Jeotgalicoccus psychrophilus was identified, which was

1013

isolated from the Korean fish sauce jeotgal (Yoon et al.
2003). The genus Staphylococcus was also found in this sample, even if in less proportion, with the species Staphylococcus
saprophyticus. This species is part of the normal human florathat colonizes the perineum, rectum, urethra, cervix, and gastrointestinal tract but has also been found as a common gastrointestinal flora in pigs and cows (Ehlers and Merrill 2018).
Members of the Paenibacillaceae were present in relatively high proportions in all three samples, with the dominance ofthe genus Paenibacillus (2%, 12%, and 5% of Firmicutes,
respectively). Paenibacillus spp. are found in soil, often associated with plant roots. These rhizobacteria promote plant
growth and can be exploited for use in agriculture.
Nevertheless, some differences were observed among thethree samples concerning the detected species. Paenibacillus
alginolyticus and Paenibacillus harenae were detected in S1and S3. The first one was isolated from soils and the second
one from desert sand in China (Jeon et al. 2009). In S2, thedetected species were Paenibacillus lautus, which has been
isolated from soil and human intestinal tract, andPaenibacillus glucanolyticus, which is known to degrade cellulose, hemicellulose, and lignin (Mathews et al. 2014). The
genus Cohnella was also detected in all three samples (0.5%,
0.2%, and 5% of Firmicutes, respectively). Members of thisgenus are highly cellulolytic bacteria, which can be found in avariety of habitats. At the level of species, it was possible todetect Cohnella saccharovorans only in S3. This species was
isolated from ginseng soils (Choi et al. 2016). Additional genera were detected in S1, such as Saccharibacillus, with thespecies Saccharibacillus deserti, which was isolated from desert soil in China (Sun et al. 2016).
Members of the Alicyclobacillaceae, with the genus
Effusibacillus, were detected in relatively high abundance only in S2 (6% of Firmicutes). Unfortunately, it was not possible
to infer at the level of species. The genus Exiguobacterium
was detected only in S1 (2% of Firmicutes), in which it waspossible to identify the species Exiguobacterium acetylicum.
This species has been isolated from soils and expressed multiple plant growth promotion attributes such as phosphate solubilization (Selvakumar et al. 2010). Finally, concerning thephylum Firmicutes, some sequences affiliated with uncultivated bacilli in S2 and S3, as the uncultivated clone TSCOR001H18 (2% of Firmicutes sequences in each sample), which has
been detected on rice paddy soils in Japan (Ishii et al. 2009).
Proteobacteria was the third phylum most represented in all
three samples (15%, 6%, and 12% of total reads for S1–S3,
respectively; see Fig. 6). Nevertheless, some differences wereobserved among the three samples (supplementary Fig. 3). The
Alphaproteobacteria class was dominant on S1 and S3, accounting for 40% and 76% of Proteobacteria, respectively, whereas it
accounted for only 18% of Proteobacteria reads of S2. Some
additional differences were noticed at lower phylogenetic levels.
The order Rhizobiales dominated on S1 (13% of Proteobacteria)

1014

and accounted for 4% and 8% of the Proteobacteria in S2 and S3,
respectively. Within this order, the genera Mesorhizobium (3% ofProteobacteria), Bradyrhizobium, and Neorhizobium (both 0.6%
of Proteobacteria) were present in S1, all of them were nitrogenfixing endosymbionts associated with roots of legumes. It is
worth mentioning that members of the Fabales (leguminous
plants) were detected in this sample. Another dominant genus
in S1 was Devosia (1% of Proteobacteria). Species of Devosia
are commonly isolated from rhizospheric soils (Mohd Nor et al.
2017). In contrast, the dominant genus in S2 and S3 wasMicrovirga. This genus comprises mainly soil bacteria, and somespecies are root nodule symbiotic bacteria that fix nitrogen indifferent plant hosts (Ardley et al. 2012).
The Sphingomonadales were present in S1 and S2 (9% and
2% of Proteobacteria, respectively) and almost negligible in
S3. The most dominant genus in both samples was
Sphingomonas. Members of this genus are pigmented bacteria
found in various environments, including both aqueous (freshwater and seawater) and terrestrial habitats, plant root systems,
clinical specimens, and others. Nevertheless, it was possible toinfer differences at the level of species. Sphingomonas
mucosissima and Sphingomonas jaspsi were identified in
S1, being the first species isolated from biological soil crustcollected from sandy arid soils (Reddy and Garcia-Pichel2007), and S. jaspsi was isolated from freshwater samples
collected in Japan (Asker et al. 2007). In contrast,
Sphingomonas kaistensis and Sphingomonas humi were detected in S2, both previously isolated from soils in South
Korea (Kim et al. 2007b; Yi et al. 2010).
The Rhodobacterales accounted for 6%, 7%, and 3% ofProteobacteria in S1–S3, respectively. Samples S1 and S2
showed certain similarities, as the most dominant genus in both
samples was Paracoccus. Paracoccus siganidrum was identified in both samples, and this species was isolated from the fish
gastrointestinal tract (Liu et al. 2013). In addition, Paracoccus
sanguinis was identified only in S1 and Paracoccus yeei onlyin S2. Both species have been identified as clinical specimens(McGinnis et al. 2015). The genus Rubellimicrobium was also
identified in both samples. Species of this genus are reddish
bacteria commonly isolated from soils (Riedel et al. 2014). In
contrast, in S3, the genus Amaricoccus showed to be dominant.
Species of this genus have been isolated from activated sludge(Maszenan et al. 1997).
Members of the Caulobacterales were detected in S1 andS2, but absent in S3. The most dominant genus in both samples was Brevundimonas, and it was possible to identify
Brevundimonas vesicularis in S1. Brevundimonas spp., andin particular B. vesicularis, are emerging global opportunistic
pathogens (Ryan and Pembroke 2018).
Sample S3 differed significantly from the other two samples,
concerning the Alphaproteobacteria group, and the dominantorder was the Rhodospirillales (65% of Proteobacteria), of whichmost reads match the Rhodospirillales MSB-1E8 group (53% of

Ann Microbiol (2019) 69:1001–1021

Proteobacteria). Members of this group are all uncultivated bacteria, which have been found on the microbiome of seabirds andcorals (Grottoli et al. 2018). The genus Skermanella was also
dominant in this sample (5% of Proteobacteria). It was not possible to infer at the level of species, but species of this genus havebeen identified as airborne bacteria in Asian dust, as well as
isolated from freshwater and soils in extreme environments, as
deserts in China (Zhang et al. 2015). In addition, theCandidatus_Alysiosphaera group and the genus Craurococcus
accounted each for 3% of Proteobacteria in this sample, and thelatter one was isolated from soils (Saitoh et al. 1998). This orderaccounted for 9% and 3% of Proteobacteria in S1 and S2, respectively. The group Rhodospirillales MSB-1E8 was also identified in S1, but in lower proportion.
The Betaproteobacteria class showed to be dominant in S2(51% of Proteobacteria) and accounted for 21% and 6% ofProteobacteria in S1 and S3, respectively (supplementary
Fig. 3). However, some differences were observed at the family and genus levels, being the Oxalobacteraceae, with thegenus Massilia dominant on S1. Species of Massilia, even if
known as pathogens, have been also isolated from soils in
China (Zhang et al. 2006). The family Burkholderiaceae, withthe genus Ralstonia, dominated on S2. Species of this genus
are well-known plant pathogens, and their significant dominance in S2 may correlate with the presence of plants detectedin this sample. Sample S2 showed the highest biodiversity of
Betaproteobacteria, and additional genera, such as
Aquabacterium, Pelomonas, and Achromobacter, were identified. Species of all these genera have been isolated fromwater (fresh and marine) and soils. Sample S3 comprises also
species of Ralstonia, in addition to members of the family
Nitrosomonadaceae. All cultivated representatives of the last
family are lithoautotrophic ammonia oxidizers that exert control over nitrification by oxidizing ammonia to nitrate. They
therefore play major roles in the control of the nitrogen cyclein terrestrial, freshwater, and marine environments.
The Gammaproteobacteria class accounted for 33% and 27%
of Proteobacteria in S1 and S2, respectively, and for significantly
less proportion in S3 (3%). Members of the Pseudomonadales
were present in S1 and S2, with the genus Pseudomonas being
dominant in S1 and the genus Acinetobacter in S2.
Pseudomonas are commonly isolated from soils. The species
Pseudomonas pseudoalcaligenes, detected in S1, has been isolated from plant rhizospheres and is an alkaliphilic cyanidedegrading bacterium (Luque-Almagro et al. 2011). In addition,
the genus Alkanindiges, with the species Alkanindiges
illinoisensis, an alkane-degrading bacterium isolated from
oilfield soils (Bogan et al. 2003), was detected only S1.
The order Enterobacterales showed to be relevant only in S1(12% of Proteobacteria), less represented in S2 (3% ofProteobacteria), and absent in S3. Many members of this order
are a normal part of the gut flora found in the intestines ofhumans and other animals. The significant presence of theseAnn Microbiol (2019) 69:1001–1021

bacteria in S1 supports, once more, the hypotheses that this statue
was in contact with farm soil. The genus Pantoea was dominant
in S1 and S2. The species Pantoea agglomerans and Pantoea
ananatis were identified in S1. P. agglomerans is a ubiquitousbacterium commonly isolated from plant surfaces, seeds, fruit,and animal or human feces. P. ananatis is a bacterium with theability to colonize and interact with hosts in both the plant andanimal kingdoms and was found in a wide range of naturalenvironments, as part of the epiphytic and endophytic flora ofvarious plant hosts, and in the insect gut (De Maayer et al. 2014).
In addition, members of Enterobacter, Escherichia/Shigella,
Cronobacter, and Salmonella were detected only in S1 andButtiauxella only in S2.
The order Xanthomonadales accounted for 5% ofProteobacteria in S1 and was very low in S2 and S3 (0.5%
and 0.8% of Proteobacteria, respectively). The genera
Steroidobacter, with the species Steroidobacter flavus, andAcidibacter were identified in S1 and S3. The detected
S. flavus is a microcystin-degrading strain, isolated from forest
soil in China (Gong et al. 2016). The genus Acidibacter wasrecently described as acidophilic, mesophilic, and capable ofreducing ferric iron (Falagán and Johnson 2014). In addition,
the genera Stenotrophomonas and Lysobacter were identified
in S1 and S2. The genus Lysobacter accommodates nonfruiting, gliding bacteria commonly isolated from soils. At
lower taxonomic levels, the species Lysobacter niastensis
was identified. This species was originally isolated fromgreenhouse soil cultivated with cucumber in Korea (Weonet al. 2007). It is worth noting that plants of the genus
Cucumis were detected in S2. Within the genus
Stenotrophomonas, Stenotrophomonas maltophilia was detected. This species is ubiquitous in aqueous environments,
soil, and plants, but it is also an emerging multidrugresistant global opportunistic pathogen (Brooke 2012).
The Salinisphaerales showed to be present only in S3, withmembers of the Salinisphaeraceae family. Unfortunately, it
was not possible to infer lower taxonomic levels. This family
comprises two genera: Salinisphaera and Halophibacterium.
All members of the family are halophilic or halotolerant andhave been isolated from marine/oceanic and high-salinity environments (Vetriani et al. 2014).
The Deltaproteobacteria accounted for 14% of Proteobacteriain S3, in contrast to the lower proportions detected in S1 and S2(6% and 4%, respectively). Members of the orders
Desulfurellales and Myxococcales accounted each for 6% ofProteobacteria in S3. It is worth mentioning that members ofDesulfurellales were negligible in S1 and S2. This order comprises exclusively the family Desulfurellaceae. Members of thisfamily are all strictly anaerobic and moderately thermophilic
bacteria, found mostly in warm-hot sulfur-containing anoxic environments, such as hydrothermal springs and sediments, and areoften in association with microbial mats (Greene 2014). All thereads matched with the uncultured genus Desulfurellaceae H16,

1015

previously detected in sediments of lakes (Ruuskanen et al.
2018). Members of the order Myxococcales were also represented in S1 and S2 (5% and 3% of Proteobacteria). The genera
Haliangium and Sorangium were present in all three samples,with the first one being dominant in S1 and the second in S2 andS3. Species of Haliangium are known as marine myxobacteria
and are halotolerant (Albataineh and Stevens 2018). The genus
Sorangium, with the detected species Sorangium cellulosum,
plays an important role in soil ecology by its ability to degrade
cellulosic materials. In addition, S1 showed the presence of the
Myxococcales Blrii 41 group, which comprises uncultivated bacteria that have been identified in rice field soils (Hori et al. 2007)
as well as the genera Vulgatibacter, Anaeromyxobacter, andPolyangium, all of them being commonly isolated from soils
and rhizosphere, and specifically from saline-alkaline soils(Zhang et al. 2013b). These genera were also present in S3, butabsent in S2. The order Bdellovibrionales accounted for 1% ofProteobacteria in S3 and was present at lower proportions in S1and S2. In S1 and S3, this order was composed exclusively ofmembers of the OM27_clade, which comprises uncultivatedbacteria involved in nutrient cycles in the ocean (Orsi et al. 2016).
Some additional phyla were detected in S1 and S3 but wereabsent in S2 (Fig. 6). Even though they were detected in low
proportions, they represented important ecological markers inthese two samples. The phylum Chloroflexi (1% and 0.8% of
total reads in S1 and S3, respectively) contains bacteria withvarious metabolic features and is one of the most common anddiverse bacterial phyla in sponges with many sponge-specificlineages (Schmitt et al. 2011). Nevertheless, it is worth notingthat all detected sequences belonging to this phylum affiliatedwith uncultivable microorganisms of the no-rank groups GittGS-136, TK10, S085, JG30-KF-CM45, and KD4-96. These
groups have been detected in bat guano (De Mandal et al.
2015) and in manure- and fertilizer-treated soils (Zhang
et al. 2013a); thus, they seem to be biological markers indicating a contact with agricultural land.
The phylum Planctomycetes (0.7% of total reads in both S1and S3) comprises mainly aquatic bacteria found in samples ofbrackish, marine, and fresh waters. The family
Planctomycetaceae accounted for 83% of Planctomycetes inboth samples; within this family, it was possible to identify
some genera in both samples, such as Gemmata, Zavarzinella,
and Singulisphaera, all of them being isolated from acid bogs,
swamps, and wetlands, and the genus Planctomyces was detected only in S3. The family Tepidisphaeraceae was present
in both samples. Members of this family have been isolated
from microbial mats (Kovaleva et al. 2015).
Finally, the phylum Tectomicrobia was detected only in S3(1% of bacteria; see Fig. 6). This phylum consists of uncultured members comprising at least three discrete phylogenetic
clades. The largest one encompasses all Entotheonella sequences sensu stricto, and in this study, half of the reads
matching this phylum belonged to the candidate genus

1016

Entotheonella, which are widely distributed in marine
sponges and seawater (Wilson et al. 2014).

Giving answers to the questions posed
The complex data obtained in this metagenomic study showthat all three statues own a very specific microbiome, with abiodiversity that differs with respect to all groups studied (bacteria, fungi, plants, protozoa, etc.). Nevertheless, theirmicrobiomes enable to answer some of the posed questions:
Were the statues stored individually or together during theirrecent history? Moreover, could all three objects have been
stored under building dust and rubble?

Ann Microbiol (2019) 69:1001–1021

European countries (Visagie et al. 2014), where it is mainly
found as a saprophytic fungus in soils. In S2, A. ruber, which
is a plant pathogen, was relevant. Therefore, a common storage in a household or in building rubble is not considered themost plausible hypothesis for the statues in the recent past.
The data strongly pointed out that most probably both torsi
were stored or even buried temporarily together in agriculturalsoils or even animal farm soils.
In contrast, the girl’s head differed considerably from theother two samples and clearly showed a primarily longer stayin arid and semi-arid surroundings, but in any case, a contact
or even its submersion in seawater. The fungal and eukaryotic
flora differs considerably from the other two objects.
What is the long-term storage history of the three statues?

A period of common storage in the recent past can be acceptedcautiously only for the two torsi (S1 [female] and S2 [male]), but
not for the girl’s head (S3), which differs considerably from theother two objects mainly in terms of eukaryotes. Nevertheless, interms of bacteria, S1 and S3 harbor intriguing similarities, suchas the presence of many halotolerant and halophilic bacteria,
which could indicate a common origin in the past.
There is no definitive indication that all three pieces would
have been stored together for a longer period of time underhouse dust or building rubble, as previously supposed by custom officers. Typical indicators for this environment would bespecies of Aspergillus and Penicillium (Pitt and Hocking2009). Very common Aspergillus species described in indoor
include Aspergillus calidoustus, Aspergillus flavus,
Aspergillus fumigatus, Aspergillus penicillioides, Aspergillus
restrictus, Aspergillus versicolor, Aspergillus westerdijkiae,
and more recently, A. sydowii (Visagie et al. 2014). Among
the Penicillium species, commonly reported species from indoor surveys include Penicillium expansum, Penicillium
digitatum, Penicillium italicum, Penicillium brevicompactum,
Penicillium chrysogenum, Penicillium commune, Penicillium
glabrum, Penicillium olsonii, Penicillium oxalicum, andPenicillium rubens.
In our study, Penicillium spp. were only detected in S1, but
completely absent in S2 and S3. Furthermore, the identified
species P. glandicola and P. citrinum are commonly growing
in association with plants, such as cereals. Thus, their presence
is more in line with the hypothesis that statue S1 was stored inan agricultural soil. In addition, some species of Aspergillus
could be markers of house dust, such as the identified
A. proliferans in S1 and S2, which has been widely found inindoor environments, and A. penicillioides only in S1, which
is typically found in indoor air and house dust and on substrates with low water activity. Nevertheless, these species
were not the dominant ones among the identified Aspergilli.
The most dominant species in samples S1 and S2 was
A. sydowii, which has been identified in indoor environments
and dust in countries of Asia and South America, but not in

To answer this question, we have focused on the following
biological markers that strongly point out some specific environments where the statues could have been stored:i. Biological markers indicating a prolonged contact with
agricultural soils, such as land plants, plant-associatedand phytopathogenic fungi, as well as typical soil andplant-associated bacteria (found in all three samples; see
supplementary Table 3).
ii. Biological markers indicating a storage in courtyard, stables, or animal farm, such as those bacteria which are
typical inhabitants of the gastrointestinal tract of animals,
rumen, or manure (mainly in S1, but also in low
proportion in S2; see supplementary Table 4).
iii. Biological markers indicating a contact with saline orarid environments, such as halotolerant and halophilic
fungi and bacteria isolated from desert soils, sandy arid
soils, saline soils, or even salt mines (in S1 and S3; see
supplementary Table 5)iv. Biological markers indicating a contact with aquatic and/or marine environments, including members of the
Chlorophyta (green algae), Ochrophyta (brown seaweeds), amoeboflagellates, and flagellates of the phylum
Cercozoa, Dinophyceae (Dinoflagellates), and some fungi of the phylum Chytridiomycota, indicating an aquatic
environment in S3. In addition, some bacteria indicatedcontact with aquatic environments for S1 and S3 (seesupplementary Table 6).

Is it possible to infer a geographical origin or a geographicalshift?
The eukaryotic community of objects S1 and S2 is similar andcan allow the conclusion that both objects have been stored
and/or transported together in the recent past. Both objects
have references to a stay in China or contact with materials

Ann Microbiol (2019) 69:1001–1021

from China (e.g., wood of packaging/containers/packaging
crates). In S2, the detection of Taiwania sequences allowed areal geolocation of this sample, because this tree is native andendemic to Eastern Asia. The additional geolocation of an
object due to the detection of bacteria uniquely or frequently
isolated from one country may be used carefully, due to theubiquity of bacteria in different geographical locations withsimilar environmental conditions. Nevertheless, it is importantto point out that many of the identified bacteria in this study,
mainly in S1, were described in environmental samples in
Asia and mainly in China.

1017
Acknowledgements The authors wish to thank Dr. Georg Plattner fromthe KHM (Vienna) for the sampling facilities and for the support.
Funding Open access funding provided by University of Natural
Resources and Life Sciences Vienna (BOKU). This study was fundedby the Federal Criminal Police Office and the Federal Monuments
Office, Vienna, Austria.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict ofinterest.
Open Access This article is distributed under the terms of the Creative

Conclusion
Our data showed the potential of the applied genetic strategy toanswer interesting questions that may arise when dealing with
cultural heritage objects and that may not be related with the stateof conservation, but more with the history of the object. It is
worth mentioning that the challenge was to retrieve, among theimmense amounts of data, those referring to unique groups oforganisms that could be considered as specific of one sample oras biological markers of a specific environment.
All three statues showed each a very specific microbiome.
Nevertheless, each of the kingdoms analyzed gave hints forthe following environments, however with some differences
between the three statues: (a) a contact with arid or saline
environment, (b) a period spent in agricultural soil and/or animal farm, and (c) a period spent in fresh, brackish, or sea
water. A specialty was the clear affiliation of the male torso
with a tree endemic to Taiwan.
Summarizing and based on the genetic fingerprints of thethree objects, conclusions can be drawn about the storage ofthe objects: The female and male torsi both indicate storage in
agricultural soils, with the female torso being the most probably stored even in an animal farm. The eukaryotic communities of both objects are similar and can allow the conclusionthat both objects have been stored or transported together inthe recent past. Both objects have references to a stay in China
or contact with materials from China (e.g., packaging/containers/packaging crates). The girl’s head differs considerablyfrom the two other samples concerning the eukaryotic community but shows intriguing similarities with the female torso
concerning the bacterial community, displaying many
halotolerant and halophilic bacteria. This fact may indicate alonger stay in arid and semi-arid surroundings as well as inmarine environments in the past.
In summary, these strategies offer an immense potential
and reveal more information than we could have imagined
before. Nevertheless, it is important to note that these techniques pose a challenge concerning the interpretation of theimmense amount of data generated.

Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a linkto the Creative Commons license, and indicate if changes were made.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References
Adamiak J, Bonifay V, Otlewska A, Sunner JA, Beech IB, Stryszewska T,Kańka S, Oracz J, Zyźelewicz D, Gutarowska B (2017) Untargeted
metabolomics approach in halophiles: understanding the biodeterioration process of building materials. Front Microbiol 8:2448
Adamiak J, Otlewska A, Tafer H, Lopandic K, Gutarowska B, Sterflinger
K, Piñar G (2018) First evaluation of the microbiome of built cultural heritage by using the Ion Torrent next generation sequencing
platform. Int Biodeterior Biodegrad 131:11–18
Alam M, Dharni S, Abdul-Khaliq, Srivastava SK, Samad A, Gupta MK(2012) A promising strain of Streptomyces sp. with agricultural
traits for growth promotion and disease management. Indian J Exp
Biol 50:559–568
Albataineh H, Stevens DC (2018) Marine myxobacteria: a few good
halophiles. Mar Drugs 16:209
Amend A (2014) From dandruff to deep-sea vents: Malassezia-like fungi
areecologically hyper-diverse. PLoS Pathog 10:e1004277
Ardley JK, Parker MA, De Meyer SE, Trengove RD, O’Hara GW, Reeve
WG, Yates RJ, Dilworth MJ, Willems A, Howieson JG (2012)
Microvirga lupini sp. nov., Microvirga lotononidis sp. nov. andMicrovirga zambiensis sp. nov. are alphaproteobacterial rootnodule bacteria that specifically nodulate and fix nitrogen with geographically and taxonomically separate legume hosts. Int J Syst Evol
Microbiol 62:2579–2588
Asker D, Beppu T, Ueda K (2007) Sphingomonas jaspsi sp. nov., a novel
carotenoid-producing bacterium isolated from Misasa, Tottori,
Japan. Int J Syst Evol Microbiol 57:1435–1441
Auchtung TA, Holder ME, Gesell JR, Ajami NJ, Duarte RT, Itoh K, Caspi
RR, Petrosino JF, Horai R, Zárate-Bladés CR (2016) Complete genome sequence of Turicibacter sp. strain H121, isolated from thefeces of a contaminated germ-free mouse. Genome Announc 4:e00114–e00116
Barr DJS (1990) Phylum Chytridiomycota. In: Handbook of Protoctista.
Margulis, Corliss, Melkonian, & Chapman (eds). Jones & Barlett,Boston, pp 454–466
Ben-Dov E, Ben Yosef DZ, Pavlov V, Kushmaro A (2009) Corynebacterium
maris sp. nov., a marine bacterium isolated from the mucus of the coral
Fungia granulosa. Int J Syst Evol Microbiol 59:2458–2463

1018
Bennur T, Kumar AR, Zinjarde S, Javdekar V (2014) Nocardiopsis species as potential sources of diverse and novel extracellular enzymes.
Appl Microbiol Biotechnol 98:9173–9185
Bogan BW, Sullivan WR, Kayser KJ, Derr KD, Aldrich HC, Paterek JR(2003) Alkanindiges illinoisensis gen. nov., sp. nov., an obligatelyhydrocarbonoclastic, aerobic squalane-degrading bacterium isolated
from oilfield soils. Int J Syst Evol Microbiol 53:1389–1395
Boniek D, de Castro Mendes I, Paiva CAO, de Paula Lana UG, Dos
Santos AFB, de Resende Stoianoff MA (2017) Ecology and identification of environmental fungi and metabolic processes involved in
the biodeterioration of Brazilian soapstone historical monuments.
Lett Appl Microbiol 65:431–438
Bower MA, Campana MG, Checkley-Scott C, Knight B, Howe CJ (2010)
The potential for extraction and exploitation of DNA from parchment: areview of the opportunities and hurdles. J Inst Conserv 33:1–11
Boylen CW (1973) Survival of Arthrobacter crystallopoietes during
prolonged periods of extreme desiccation. J Bacteriol 113:33–37
Brooke JS (2012) Stenotrophomonas maltophilia: an emerging global
opportunistic pathogen. Clin Microbiol Rev 25:2–41
Burger J, Hummel S, Herrmann B (2000) Palaeogenetics and cultural
heritage. Species determination and STR-genotyping from ancient
DNA in art and artefacts. Thermochim Acta 365:141–146
Callahan BJ, Sankaran K, Fukuyama JA, McMurdie P, Holems SP (2016)
Bioconductor workflow for microbiome data analysis: from raw
reads to community analyses [version 2; referees: 3 approved].
F1000Research 5:1492
Carniel FC, Zanelli D, Bertuzzi S, Tretiach M (2015) Desiccation tolerance and lichenization: a case study with the aeroterrestrial
microalga Trebouxia sp. (Chlorophyta). Planta 242:493–505
Cavalier-Smith T, Chao EY (2003) Phylogeny and classification of phylum Cercozoa (Protozoa). Protist 154:341–358
Chamberlain SA, Boettiger C (2017) R Python, and Ruby clients for
GBIF species occurrence data. PeerJ Preprints 5:e3304v1. https://doi.org/10.7287/peerj.preprints.3304v1
Chase AB, Arevalo P, Polz MF, Berlemont R, Martiny JBH (2016)
Evidence for ecological flexibility in the cosmopolitan genus
Curtobacterium. Front Microbiol 7:1874
Chen YG, Cui XL, Pukall R, Li HM, Yang YL, Xu LH, Wen ML, Peng
Q, Jiang CL (2007) Salinicoccus kunmingensis sp. nov., a moderately halophilic bacterium isolated from a salt mine in Yunnan,
South-West China. Int J Syst Evol Microbiol 57:2327–2332
Chen Q, Jiang JR, Zhang GZ, Cai L, Crous PW (2015) Resolving the
Phoma enigma. Stud Mycol 82:137–217
Chen AJ, Hubka V, Frisvad JC, Visagie CM, Houbraken J, Meijer M,
Varga J, Demirel R, Jurjević Ž, Kubátová A, Sklenář F, Zhou YG,
Samson RA (2017) Polyphasic taxonomy of Aspergillus section
Aspergillus (formerly Eurotium), and its occurrence in indoor environments and food. Stud Mycol 88:37–135
Choi JH, Seok JH, Jang HJ, Cha JH, Cha CJ (2016) Cohnella
saccharovorans sp. nov., isolated from ginseng soil. Int J Syst
Evol Microbiol 66:1713–1717
Cocquyt E, Verbruggen H, Leliaert F, De Clerck O (2010) Evolution andcytological diversification of the green seaweeds (Ulvophyceae).
Mol Biol Evol 27:2052–2061
Collins MD, Brown J, Jones D (1988) Brachybacterium faecium gen.
nov. sp. nov., a coryneform bacterium from poultry deep litter. Int
J Syst Evol Microbiol 38:45–48
Cotta MA, Whitehead TR, Collins MD, Lawson PA (2004) Atopostipes
suicloacale gen. nov., sp. nov., isolated from an underground swine
manure storage pit. Anaerobe 10:191–195
Dai X, Wang YN, Wang BJ, Liu SJ, Zhou YG (2005) Planomicrobiumchinense sp. nov., isolated from coastal sediment, and transfer ofPlanococcus psychrophilus and Planococcus alkanoclasticus to
Planomicrobium as Planomicrobium psychrophilum comb. nov.
and Planomicrobium alkanoclasticum comb. nov. Int J Syst Evol
Microbiol 55:699–702

Ann Microbiol (2019) 69:1001–1021
Dastager SG, Mawlankar R, Srinivasan K, Tang SK, Lee JC, Ramana
VV, Shouche YS (2014) Fictibacillus enclensis sp. nov., isolated
from marine sediment. Antonie Van Leeuwenhoek 105:461–469
De Maayer P, Chan WY, Rubagotti E, Venter SN, Toth IK, Birch PR,
Coutinho TA (2014) Analysis of the Pantoea ananatis pan-genome
reveals factors underlying its ability to colonize and interact withplant, insect and vertebrate hosts. BMC Genomics 15:404. https://doi.org/10.1186/1471-2164-15-404
De Mandal S, Zothansanga, Panda AK, Bisht SS, Kumar NS (2015) First
report of bacterial community from a bat guano using Illumina nextgeneration sequencing. Genom Data 4:99–101
Dolka B, Chrobak-Chmiel D, Czopowicz M, Szeleszczuk P (2017)
Characterization of pathogenic Enterococcus cecorum from different poultry groups: broiler chickens, layers, turkeys, and waterfowl.
PLoS One 12:e0185199
Egas C, Barroso C, Froufe HJ, Pacheco J, Albuquerque L, da Costa MS(2014) Complete genome sequence of the radiation-resistant bacterium
Rubrobacter radiotolerans RSPS-4. Stand Genomic Sci 9:1062–1075
Ehlers S, Merrill SA (2018) Staphylococcus saprophyticus. In: StatPearls[Internet]. Treasure Island: StatPearls; https://www.ncbi.nlm.nih.
gov/books/NBK482367/
Falagán C, Johnson DB (2014) Acidibacter ferrireducens gen. nov., sp.
nov.: an acidophilic ferric iron-reducing gammaproteobacterium.
Extremophiles 18:1067–1073
Fouhy F, Clooney AG, Stanton C, Claesson MJ, Cotter PD (2016) 16S rRNA
gene sequencing of mock microbial populations—impact of DNA extraction method, primer choice and sequencing platform. BMC
Microbiol 16:123 http://www.biomedcentral.com/1471-2164/15/404
Fu Q, Meyer M, Gao X, Stenzel U, Burbano HA, Kelso J, Pääbo S (2013)
DNA analysis of an early modern human fromTianyuan Cave,
China. Proc Natl Acad Sci U S A 110:2223–2227
Fudou R, Jojima Y, Seto A, Yamada K, Kimura E, Nakamatsu T, Hiraishi
A, Yamanaka S (2002) Corynebacterium efficiens sp. nov., aglutamic-acid-producing species from soil and vegetables. Int J
Syst Evol Microbiol 52:1127–1131
Gao M, Wang L, Chen SF, Zhou YG, Liu HC (2010) Salinicoccus
kekensis sp. nov., a novel alkaliphile and moderate halophile isolated from Keke Salt Lake in Qinghai, China. Antonie Van
Leeuwenhoek 98:351–357
Gerritsen J, Fuentes S, Grievink W, van Niftrik L, Tindall BJ,
Timmerman HM, Rijkers GT, Smidt H (2014) Characterization of
Romboutsia ilealis gen. nov., sp. nov., isolated from the gastrointestinal tract of a rat, and proposal for the reclassification of fiveclosely related members of the genus Clostridium into the genera
Romboutsia gen. nov., Intestinibacter gen. nov., Terrisporobacter
gen. nov. and Asaccharospora gen. nov. Int J Syst Evol Microbiol
64:1600–1616
Gómez F (2012) A checklist and classification of living dinoflagellates(dinoflagellata, alveolata). Oceánides 27:65–140
Gong ZL, Zhang CF, Jin R, Zhang YQ (2016) Steroidobacter flavus sp.
nov., a microcystin-degrading Gammaproteobacterium isolated
from soil. Antonie Van Leeuwenhoek 109:1073–1079
Greene AC (2014) The family Desulfurellaceae. In: Rosenberg E,
DeLong EF, Lory S, Stackebrandt E, Thompson F (eds) The prokaryotes. Springer, Berlin ISBN 978-3-642-39043-2
Grottoli AG, Dalcin Martins P, Wilkins MJ, Johnston MD, Warner ME, Cai
W-J et al (2018) Coral physiology and microbiome dynamics under
combined warming and ocean acidification. PLoS One 13:e0191156
Ham H, Kim S, Kim MH, Lee S, Hong SK, Ryu JG, Lee T (2016)
Mycobiota of ground red pepper and their aflatoxigenic potential.
J Microbiol 54:832–837
Hayata B (1906) On Taiwania, a new genus of Coniferae from the islandof Formosa. J Linn Soc Bot 37:330–331
Heyrman J, Logan NA, Busse HJ, Balcaen A, Lebbe L, Rodriguez-Diaz
M et al (2003) Virgibacillus carmonensis sp. nov., Virgibacillus
necropolis sp. nov. and Virgibacillus picturae sp. nov., three novel

Ann Microbiol (2019) 69:1001–1021
species isolated from deteriorated mural paintings, transfer of thespecies of the genus Salibacillus to Virgibacillus, as Virgibacillus
marismortui comb. nov. and Virgibacillus salexigens comb. nov.,and emended description of the genus Virgibacillus. Int J Syst
Evol Microbiol 53:501–511
Heyrman J, Rodríguez-Díaz M, Devos J, Felske A, Logan NA, De Vos P(2005) Bacillus arenosi sp. nov., Bacillus arvi sp. nov. and Bacillus
humi sp. nov., isolated from soil. Int J Syst Evol Microbiol 55:111–117
Hirooka K (2014) Transcriptional response machineries of Bacillus
subtilis conducive to plant growth promotion. Biosci Biotechnol
Biochem 78:1471–1484
Hong K, Gao AH, Xie QY, Gao H, Zhuang L, Lin HP, Yu HP, Li J, Yao
XS, Goodfellow M, Ruan JS (2009) Actinomycetes for marine drug
discovery isolated from mangrove soils and plants in China. Mar
Drugs 7:24–44
Hori T, Noll M, Igarashi Y, Friedrich MW, Conrad R (2007) Identificationof acetate-assimilating microorganisms under methanogenic conditions in anoxic rice field soil by comparative stable isotope probingof RNA. Appl Environ Microbiol 73:101–109
Howe AT, Bass D, Scoble JM, Lewis R, Vickerman K, Arndt H, CavalierSmith T (2011) Novel cultured protists identify deep-branching environmental DNA clades of Cercozoa: new genera Tremula,
Micrometopion, Minimassisteria, Nudifila, Peregrinia. Protist 162:
332–372
Huang Y, Wang L, Lu Z, Hong L, Liu Z, Tan GYA, Goodfellow M (2002)
Proposal to combine the genera Actinobispora and Pseudonocardia
in an emended genus Pseudonocardia, and description of
Pseudonocardia zijingensis sp. nov. Int J Syst Evol Microbiol 52:
977–982
Ishii S, Yamamoto M, Kikuchi M, Oshima K, Hattori M, Otsuka S, Senoo
K (2009) Microbial populations responsive to denitrificationinducing conditions in rice paddy soil, as revealed by comparative
16S rRNA gene analysis. Appl Environ Microbiol 75:7070–7078
Isola D, Zucconi L, Onofri S, Caneva G, de Hoog GS, Selbmann L (2016)
Extremotolerant rock inhabiting black fungi from Italian monumental sites. Fungal Divers 76:75–96
Jeon CO, Lim JM, Lee SS, Chung BS, Park DJ, Xu LH, Jiang CL, Kim
CJ (2009) Paenibacillus harenae sp. nov., isolated from desert sandin China. Int J Syst Evol Microbiol 59:13–17
Kalvari I, Argasinska J, Quinones-Olvera N, Nawrocki EP, Rivas E, Eddy
SR, Bateman A, Finn RD, Petrov AI (2018) Rfam 13.0: shifting to a
genome-centric resource for non-coding RNA families. Nucleic Acids
Res 46(D1):D335–D342. https://doi.org/10.1093/nar/gkx1038
Kämpfer P, Young CC, Busse HJ, Chu JN, Schumann P, Arun AB, Shen
FT, Rekha PD (2010) Microlunatus soli sp. nov., isolated from soil.
Int J Syst Evol Microbiol 60:824–827
Kim SB, Lonsdale J, Seong CN, Goodfellow M (2003) Streptacidiphilus
gen. nov., acidophilic actinomycetes with wall chemotype I and
emendation of the family Streptomycetaceae (Waksman andHenrici (1943) AL) emend. Rainey et al. 1997. Antonie Van
Leeuwenhoek 83:107–116
Kim YG, Choi DH, Hyun S, Cho BC (2007a) Oceanobacillus profundus
sp. nov., isolated from a deep-sea sediment core. Int J Syst Evol
Microbiol 57:409–413
Kim MK, Schubert K, Im WT, Kim KH, Lee ST, Overmann J (2007b)
Sphingomonas kaistensis sp. nov., a novel alphaproteobacterium
containing pufLM genes. Int J Syst Evol Microbiol 57:1527–1534
Klochkova TA, Kang SH, Cho GY, Pueschel CM, West JA, Kim GH(2006) Biology of a terrestrial green alga, Chlorococcum sp.
(Chlorococcales, Chlorophyta), collected from the Miruksazi stupa
in Korea. Phycologia 45:349–358
Koerner RJ, Goodfellow M, Jones AL (2009) The genus Dietzia: a newhome for some known and emerging opportunist pathogens. FEMS
Immunol Med Microbiol 55:296–305

1019
Koestler RJ (2003). Art, biology, and conservation: biodeterioration ofworks of art. New York: Metropolitan Museum of Art. p. 285. ISBN
978-1-58839-107-0
Kokcha S, Mishra AK, Lagier JC, Million M, Leroy Q, Raoult D,
Fournier PE (2012) Non contiguous-finished genome sequenceand description of Bacillus timonensis sp. nov. Stand Genomic Sci
6:346–355
Kovaleva OL, Merkel AY, Novikov AA, Baslerov RV, Toshchakov SV,
Bonch-Osmolovskaya EA (2015) Tepidisphaera mucosa gen. nov.,
sp. nov., a moderately thermophilic member of the class
Phycisphaerae in the phylum Planctomycetes, and proposal of a
new family, Tepidisphaeraceae fam. nov., and a new order,
Tepidisphaerales ord. nov. Int J Syst Evol Microbiol 65:549–555
Kurahashi M, Fukunaga Y, Sakiyama Y, Harayama S, Yokota A (2009)
Iamia majanohamensis gen. nov., sp. nov., an actinobacterium isolated from sea cucumber Holothuria edulis, and proposal of
Iamiaceae fam. nov. Int J Syst Evol Microbiol 59:869–873
Kuznetsov VD, Zaitseva TA, Vakulenko LV, Filippova SN (1992)
Streptomyces albiaxialis sp. nov.—a new petroleum hydrocarbondegrading species of thermo- and halotolerant Streptomyces.
Microbiology 61:62–67
Kwon SW, Kim BY, Song J, Weon HY, Schumann P, Tindall BJ,
Stackebrandt E, Fritze D (2007) Sporosarcina koreensis sp. nov.
and Sporosarcina soli sp. nov., isolated from soil in Korea. Int J
Syst Evol Microbiol 57:1694–1698
Lan PT, Sakamoto M, Sakata S, Benno Y (2006) Bacteroides barnesiae
sp. nov., Bacteroides salanitronis sp. nov. and Bacteroides
gallinarum sp. nov., isolated from chicken caecum. Int J Syst Evol
Microbiol 56:2853–2859
Lee JC, Lee GS, Park DJ, Kim CJ (2008) Bacillus alkalitelluris sp. nov.,
an alkaliphilic bacterium isolated from sandy soil. Int J Syst Evol
Microbiol 58:2629–2634
Leisner JJ, Laursen BG, Prévost H, Drider D, Dalgaard P (2007)
Carnobacterium: positive and negative effects in the environment
and in foods. FEMS Microbiol Rev 31:592–613
Li WJ, Chen HH, Zhang YQ, Kim CJ, Park DJ, Lee JC, Xu LH, Jiang CL(2005) Citricoccus alkalitolerans sp. nov., a novel actinobacterium isolated from a desert soil in Egypt. Int J Syst Evol Microbiol 55:87–90
Liu Y, Xie QY, Hong K, Li L, Zhao YM, Tang YL, An JY, Zhu PP, Xu
CH (2013) Paracoccus siganidrum sp. nov., isolated from fish gastrointestinal tract. Antonie Van Leeuwenhoek 103:1133–1139
Lu J, Nogi Y, Takami H (2001) Oceanobacillus iheyensis gen. nov., sp.
nov., a deep-sea extremely halotolerant and alkaliphilic species isolated from a depth of 1050 m on the Iheya Ridge. FEMS Microbiol
Lett 205:291–297
Luque-Almagro VM, Blasco R, Martínez-Luque M, Moreno-Vivián C,
Castillo F, Roldán MD (2011) Bacterial cyanide degradation is under review: Pseudomonas pseudoalcaligenes CECT5344, a case ofan alkaliphilic cyanotroph. Biochem Soc Trans 39:269–274
Martin M (2011) Cutadapt removes adapter sequences from highthroughput sequencing reads. EMBnet J 17:10–12
Maszenan AM, Seviour RJ, Patel BK, Rees GN, McDougall BM (1997)
Amaricoccus gen. nov., a gram-negative coccus occurring in regular
packages or tetrads, isolated from activated sludge biomass, anddescriptions of Amaricoccus veronensis sp. nov., Amaricoccus
tamworthensis sp. nov., Amaricoccus macauensis sp. nov., andAmaricoccus kaplicensis sp. nov. Int J Syst Bacteriol 47:727–734
Mathews SL, Pawlak JJ, Grunden AM (2014) Isolation of Paenibacillus
glucanolyticus from pulp mill sources with potential to deconstructpulping waste. Bioresour Technol 164:100–105
McGinnis JM, Cole JA, Dickinson MC, Mingle LA, Lapierre P, Musser
KA, Wolfgang WJ (2015) Paracoccus sanguinis sp. nov., isolated
from clinical specimens of New York State patients. Int J Syst Evol
Microbiol 65:1877–1882
McLean RJ, Trainor FR (1965) Fasciculochloris, a new chlorosphaeraceaenalga from a Connecticut soil. Phycologia 4:145–148

1020
Meehan CJ, Beiko RG (2014) A phylogenomic view of ecological specialization in the Lachnospiraceae, a family of digestive tractassociated bacteria. Genome Biol Evol 6:703–713
Mello A, Murat C, Bonfante P (2006) Truffles: much more than a prized
and local fungal delicacy. FEMS Microbiol Lett 260:1–8
Mello A, Ding G-C, Piceno YM, Napoli C, Tom LM, DeSantis TZ,
Andersen GL, Smalla K, Bonfante P (2013) Truffle Brûlés havean impact on the diversity of soil bacterial communities. PLoS
One 8:e61945
Milanez GD, Masangkay FR, Thomas RC, Ordona MOGO, Bernales
GQ, Corpuz VCM, Fortes HSV, Garcia CMS, Nicolas LC,
Nissapatorn V (2017) Molecular identification of Vermamoeba
vermiformis from freshwater fish in lake Taal, Philippines. Exp
Parasitol 183:201–206
Mohd Nor MN, Sabaratnam V, Tan GYA (2017) Devosia elaeis sp. nov.,
isolated from oil palm rhizospheric soil. Int J Syst Evol Microbiol
67:851–855
Mouchacca J (1973) Deux Alternatria des sols arides d’Égypte:
A. Chlamydosporum sp. nov. et A. Phragmospora van Emden.
Mycopathol Mycol Appl 50:217–225
Mylnikov AP, Karpov SA (2004) Review of the diversity and taxonomy
of cercomonads. Protistology 3:201–217
Nagel M, Andreesen JR (1991) Bacillus niacini sp. nov., a nicotinatemetabolizing mesophile isolated from soil. Int J Syst Bacteriol 41:
134–139
Nakagawa Y, Sakane T, Yokota A (1996) Emendation of the genus
Planococcus and transfer of Flavobacterium okeanokoites Zobell
and Upham 1944 to the genus Planococcus as Planococcus
okeanokoites comb. nov. Int J Syst Bacteriol 46:866–870
Navarrete AA, Venturini AM, Meyer KM, Klein AM, Tiedje JM,
Bohannan BJM, Nüsslein K, Tsai SM, Rodrigues JLM (2015)
Differential response of Acidobacteria subgroups to forest-topasture conversion and their biogeographic patterns in the Western
Brazilian Amazon. Front Microbiol 6:1443. https://doi.org/10.3389/
fmicb.2015.01443
Nawrocki EP, Eddy SR (2013) Infernal 1.1: 100-fold faster RNA homology searches. Bioinformatics 29(22):2933–2935
O’Sullivan NJ, Teasdale MD, Mattiangeli V, Maixner F, Pinhasi R, Bradley
DG, Zink A (2016) A whole mitochondria analysis of the Tyrolean
Iceman’s leather provides insights into the animal sources of Copper
Age clothing. Sci Rep 6:31279. https://doi.org/10.1038/srep31279
Ogórek R, Dyląg M, Kozak B (2016) Dark stains on rock surfaces inDriny Cave (Little Carpathian Mountains, Slovakia). Extremophiles
20:641–652
Orsi WD, Smith JM, Liu S, Liu Z, Sakamoto CM, Wilken S, Poirier C,
Richards TA, Keeling PJ, Worden AZ, Santoro AE (2016) Diverse,
uncultivated bacteria and archaea underlying the cycling of dissolved protein in the ocean. ISME J 10:2158–2173
Otlewska A, Adamiak J, Stryszewska T, Kańka S, Gutarowska B (2017)
Factors determining the biodiversity of halophilic microorganisms
on historic masonry buildings. Microbes Environ 32:164–173
Patterson DJ (1989) Stramenopiles: chromophytes from a protistan perspective. In: Green JC, Leadbeater BSC, Diver WL (eds) The
chromophyte algae: problems and perspectives. Clarendon. ISBN0198577133
Pitt J. and Hocking A (2009) Fungi and food spoilage. Blackie Academic andProfessional, London. https://doi.org/10.1007/978-0-387-92207-2
Quaedvlieg W, Binder M, Groenewald JZ, Summerell BA, Carnegie AJ,
Burgess TI, Crous PW (2014) Introducing the consolidated species concept to resolve species in the Teratosphaeriaceae. Persoonia 33:1–40
Ragan MA, Goggin CL, Cawthorn RJ, Cerenius L, Jamieson AV, Plourde
SM, Rand TG, Söderhäll K, Gutell RR (1996) A novel clade of
protistan parasites near the animal-fungal divergence. PNAS 93:
11907–11912

Ann Microbiol (2019) 69:1001–1021
Reddy GS, Garcia-Pichel F (2007) Sphingomonas mucosissima sp. nov. and
Sphingomonas desiccabilis sp. nov., from biological soil crusts in the
Colorado Plateau, USA. Int J Syst Evol Microbiol 57:1028–1034
Rey T, Dumas B (2017) Plenty is no plague: Streptomyces symbiosis withcrops. Trends Plant Sci 22:30–37
Riedel T, Spring S, Fiebig A, Petersen J, Göker M, Klenk HP (2014)
Genome sequence of the pink to light reddish-pigmented
Rubellimicrobium mesophilum type strain (DSM 19309(T)), a representative of the Roseobacter group isolated from soil, andemended description of the species. Stand Genomic Sci 9:902–913
Rosenberg E (2014) The family Prevotellaceae. In: Rosenberg E,
DeLong EF, Lory S, Stackebrandt E, Thompson F (eds) The prokaryotes. Springer, Berlin
Ruuskanen MO, St. Pierre KA, St. Louis VL, Aris-Brosou S, Poulain AJ(2018) Physicochemical drivers of microbial community structure in
sediments of Lake Hazen, Nunavut, Canada. Front Microbiol 9:1138. https://doi.org/10.3389/fmicb.2018.01138
Ryan MP, Pembroke JT (2018) Brevundimonas spp: emerging global
opportunistic pathogens. Virulence 9:480–493
Saitoh S, Suzuki T, Nishimura Y (1998) Proposal of Craurococcus roseus
gen. nov., sp. nov. and Paracraurococcus ruber gen. nov., sp. nov.,
novel aerobic bacteriochlorophyll a-containing bacteria from soil.
Int J Syst Bacteriol 48:1043–1047
Samson RA, Peterson SW, Frisvad JC, Varga J (2011) New species inAspergillus section Terrei. Stud Mycol 69:39–55
Schmitt S, Deines P, Behnam F, Wagner M, Taylor MW (2011) Chloroflexi
bacteria are more diverse, abundant, and similar in high than in low
microbial abundance sponge. FEMS Microbiol Ecol 78:497–510
Schommer NN, Gallo RL (2013) Structure and function of the humanskin microbiome. Trends Microbiol 21:660–668
Selvakumar G, Kundu S, Joshi P, Nazim S, Gupta AD, Gupta HS (2010)
Growth promotion of wheat seedlings by Exiguobacterium
acetylicum 1P (MTCC 8707) a cold tolerant bacterial strain fromthe Uttarakhand Himalayas. Indian J Microbiol 50:50–56
Singleton DR, Furlong MA, Peacock AD, White DC, Coleman DC,
Whitman WB (2003) Solirubrobacter pauli gen. nov., sp. nov., amesophilic bacterium within the Rubrobacteridae related to common soil clones. Int J Syst Evol Microbiol 53:485–490
Sivapalan A, Metussin R, Harndan F, Zain RM (1998) Fungi associatedwith postharvest fruit rots of Durio graveolens and D. kutejensis in
Brunei Darussalam. Australas Plant Pathol 27:274–277
Song W, Wang X, Gu J, Zhang S, Yin Y, Li Y, Qian X, Sun W (2017)
Effects of different swine manure to wheat straw ratios on antibiotic
resistance genes and the microbial community structure during anaerobic digestion. Bioresour Technol 231:1–8
Sterflinger K, Piñar G (2013) Microbial deterioration of cultural heritage
and works of art—tilting at windmills? Appl Microbiol Biotechnol
97:9637–9646
Stirnberg A, Djamei A (2016) Characterization of ApB73, a virulence
factor important for colonization of Zea mays by the smut Ustilago
maydis. Mol Plant Pathol 17:1467–1479
Sun JQ, Wang XY, Wang LJ, Xu L, Liu M, Wu XL (2016)
Saccharibacillus deserti sp. nov., isolated from desert soil. Int J
Syst Evol Microbiol 66:623–627
Teasdale MD, Fiddyment S, Vnouček J, Mattiangeli V, Speller C, Binois
A, Carver M, Dand C, Newfield TP, Webb CC, Bradley DG, Collins
MJ (2017) The York Gospels: a 1000-year biological palimpsest. R
Soc Open Sci 4:170988
Urich T, Lanzén A, Qi J, Huson DH, Schleper C, Schuster SC (2008)
Simultaneous assessment of soil microbial community structureand function through analysis of the metatranscriptome. PLoS
One 3(6):e2527
Urzì C, Salamone P, Schumann P, Rohde M, Stackebrandt E(2004) Blastococcus saxobsidens sp. nov., and emended descriptions of the genus Blastococcus Ahrens and Moll 1970

Ann Microbiol (2019) 69:1001–1021
and Blastococcus aggregatus Ahrens and Moll 1970. Int J
Syst Evol Microbiol 54:253–259
Velegraki A, Cafarchia C, Gaitanis G, Iatta R, Boekhout T (2015)
Malassezia infections in humans and animals: pathophysiology, detection, and treatment. PLoS Pathog 11(1):e1004523
Venkateswaran K, Kempf M, Chen F, Satomi M, Nicholson W, Kern R(2003) Bacillus nealsonii sp. nov., isolated from a spacecraftassembly facility, whose spores are gamma-radiation resistant. Int
J Syst Evol Microbiol 53:165–172
Vetriani C, Crespo-Medina M, Antunes A (2014) The family
Salinisphaeraceae. In: Rosenberg E, DeLong EF, Lory S,
Stackebrandt E, Thompson F (eds) The prokaryotes. Springer,
Berlin, Heidelberg ISBN: 978-3-642-38921-4
Vinokurova NG, Boĭchenko LV, Arinbasarov MU (2003) Formation ofalkaloids from Penicillium species fungi during growth on wheat
kernels. Prikl Biokhim Mikrobiol 39:457–460
Visagie CM, Hirooka Y, Tanney JB, Whitfield E, Mwange K, Meijer M,
Amend AS, Seifert KA, Samson RA (2014) Aspergillus,
Penicillium and Talaromyces isolated from house dust samples collected around the world. Stud Mycol 78:63–139
Wang X, Xue Y, Yuan S, Zhou C, Ma Y (2008) Salinicoccus halodurans
sp. nov., a moderate halophile from saline soil in China. Int J Syst
Evol Microbiol 58:1537–1541
Wang K, Zhang L, Liu Y, Pan Y, Meng L, Xu T, Zhang C, Liu H, Hong S,
Huang H, Jiang J (2015) Kocuria dechangensis sp. nov., an
actinobacterium isolated from saline and alkaline soils. Int J Syst
Evol Microbiol 65:3024–3030
Wang XW, Houbraken J, Groenewald JZ, Meijer M, Andersen B, Nielsen
KF, Crous PW, Samson RA (2016) Diversity and taxonomy of
Chaetomium and chaetomium-like fungi from indoor environments.
Stud Mycol 84:145–224
Wang X, Jiang WK, Cui MD, Yang ZG, Yu X, Hu G, Zhang H, Hong Q(2017) Nocardioides agrisoli sp. nov., isolated from farmland soil.
Int J Syst Evol Microbiol 67:3722–3727
Wang WC, Yan FF, Hu JY, Amen OA, Cheng HW (2018)
Supplementation of Bacillus subtilis-based probiotic reduces heatstress-related behaviors and inflammatory response in broiler
chickens. J Anim Sci 96:1654–1666
Weon HY, Kim BY, Kim MK, Yoo SH, Kwon SW, Go SJ, Stackebrandt E(2007) Lysobacter niabensis sp. nov. and Lysobacter niastensis sp.
nov., isolated from greenhouse soils in Korea. Int J Syst Evol
Microbiol 57:548–551
Wexler HM (2007) Bacteroides: the good, the bad, and the nitty-gritty.
Clin Microbiol Rev 20:593–621
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In:
PCR protocols: a guide to methods and applications. Academic,
New York, pp 315–322
Wilson MC, Mori T, Rückert C, Uria AR, Helf MJ, Takada K, Gernert C,
Steffens UA, Heycke N, Schmitt S, Rinke C, Helfrich EJ,
Brachmann AO, Gurgui C, Wakimoto T, Kracht M, Crüsemann
M, Hentschel U, Abe I, Matsunaga S, Kalinowski J, Takeyama H,
Piel J (2014) An environmental bacterial taxon with a large anddistinct metabolic repertoire. Nature 506:58–62
Xi L, Zhang L, Ruan J, Huang Y (2012) Description of Verrucosispora
qiuiae sp. nov., isolated from mangrove swamp sediment, andemended description of the genus Verrucosispora. Int J Syst Evol
Microbiol 62:1564–1569

1021
Yadav S, Kaushik R, Saxena AK, Arora DK (2011) Diversity and phylogeny of plant growth-promoting bacilli from moderately acidic
soil. J Basic Microbiol 51:98–106
Yamamoto M, Ishii A, Nogi Y, Inoue A, Ito M (2006) Isolation andcharacterization of novel denitrifying alkalithermophiles, AT-1 andAT-2. Extremophiles 10:421–426
Yi TH, Han CK, Srinivasan S, Lee KJ, Kim MK (2010) Sphingomonas
humi sp. nov., isolated from soil. J Microbiol 48:165–169
Yoon JH, Lee KC, Weiss N, Kang KH, Park YH (2003) Jeotgalicoccus
halotolerans gen. nov., sp. nov. and Jeotgalicoccus psychrophilus
sp. nov., isolated from the traditional Korean fermented seafood
jeotgal. Int J Syst Evol Microbiol 53:595–602
Zalar P, de Hoog GS, Schroers HJ, Crous PW, Groenewald JZ, GundeCimerman N (2007) Phylogeny and ecology of the ubiquitous saprobe
Cladosporium sphaerospermum, with descriptions of seven new species from hypersaline environments. Stud Mycol 58:157–183
Zgurskaya HI, Evtushenko LI, Akimov VN, Voyevoda HV,
Dobrovolskaya TG, Lysak LV, Kalakoutskii LV (1992) Emended
description of the genus Agromyces and description of Agromyces
cerinus subsp. cerinus sp. nov., subsp. nov., Agromyces cerinus
subsp. nitratus sp. nov., subsp. nov., Agromyces fucosus subsp.
fucosus sp. nov., subsp. nov., and Agromyces fucosus subsp.
hippuratus sp. nov., subsp. nov. Int J Syst Bacteriol 42:635–641
Zhang YQ, Li WJ, Zhang KY, Tian XP, Jiang Y, Xu LH, Jiang CL, Lai R(2006) Massilia dura sp. nov., Massilia albidiflava sp. nov., Massilia
plicata sp. nov. and Massilia lutea sp. nov., isolated from soils in
China. Int J Syst Evol Microbiol 56:459–463
Zhang DC, Liu HC, Xin YH, Yu Y, Zhou PJ, Zhou YG (2009)
Planomicrobium glaciei sp. nov., a psychrotolerant bacterium isolated from a glacier. Int J Syst Evol Microbiol 59:1387–1390
Zhang Q, Sun J, Liu S, Wei Q (2013a) Manure refinement affects apple
rhizosphere bacterial community structure: a study in sandy soil.
PLoS One 8(10):e76937
Zhang X, Yao Q, Cai Z, Xie X, Zhu H (2013b) Isolation and identificationof myxobacteria from saline-alkaline soils in Xinjiang, China. PLoS
One 8(8):e70466
Zhang ZY, Gao XH, Zhang YJ, Jia M, Lu XJ, Ma YC, Tian F, Xie Q, Tang
SK (2015) Skermanella rubra sp. nov., a bacterium isolated from thedesert of Xinjiang, China. Antonie Van Leeuwenhoek 108:627–632
Zhang T, Wang NF, Zhang YQ, Liu HY, Yu LY (2016) Diversity anddistribution of aquatic fungal communities in the Ny-Ålesund region, Svalbard (High Arctic): aquatic fungi in the Arctic. Microb
Ecol 71:543–554
Zhao J, Guo L, Sun P, Han C, Bai L, Liu C, Li Y, Xiang W, Wang X(2015) Actinomadura jiaoheensis sp. nov. and Actinomadurasporangiiformans sp. nov., two novel actinomycetes isolated frommuddy soil and emended description of the genus Actinomadura.
Antonie Van Leeuwenhoek 108:1331–1339
Zhou N, Zhang Y, Liu F, Cai L (2016) Halophilic and thermotolerant
Gymnoascus species from several special environments, China.
Mycologia 108:179–191
Zhu WY, Zhao LX, Zhao GZ, Duan XW, Qin S, Li J, Xu LH, Li WJ(2012) Plantactinospora endophytica sp. nov., an actinomycete isolated from Camptotheca acuminata Decne., reclassification of
Actinaurispora siamensis as Plantactinospora siamensis comb.
nov. and emended descriptions of the genus Plantactinospora andPlantactinospora mayteni. Int J Syst Evol Microbiol 62:2435–2442