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Hallstatt miners consumed blue cheese and beer during the Iron Age and retained a non-Westernized gut microbiome until the Baroque period

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We subjected human paleofeces dating from the Bronze Age to the Baroque period (18th century AD) to in-depth microscopic, metagenomic, and proteomic analyses. The paleofeces were preserved in the underground salt mines of the UNESCO World Heritage site of Hallstatt in Austria. This allowed us to reconstruct the diet of the former population and gain insights into their ancient gut microbiome composition. Our dietary survey identified bran and glumes of different cereals as some of the most prevalent plant fragments. This highly fibrous, carbohydrate-rich diet was supplemented with proteins from broad beans and occasionally with fruits, nuts, or animal food products. Due to these traditional dietary habits, all ancient miners up to the Baroque period have gut microbiome structures akin to modern non-Westernized individuals whose diets are also mainly composed of unprocessed foods and fresh fruits and vegetables. This may indicate a shift in the gut community composition of modern Westernized populations due to quite recent dietary and lifestyle changes. When we extended our microbial survey to fungi present in the paleofeces, in one of the Iron Age samples, we observed a high abundance of Penicillium roqueforti and Saccharomyces cerevisiae DNA. Genome-wide analysis indicates that both fungi were involved in food fermentation and provides the first molecular evidence for blue cheese and beer consumption in Iron Age Europe.
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Article
Hallstatt miners consumed blue cheese and beer
during the Iron Age and retained a non-Westernized
gut microbiome until the Baroque period
Graphical abstract
Highlights
dGut microbiome and diet of European salt miners determined
using paleofeces
dUntil the Baroque, the microbiome resembled that of modern
non-Westernized people
dFood-fermenting fungi in Iron Age feces indicates blue
cheese and beer consumption
Authors
Frank Maixner, Mohamed S. Sarhan,
Kun D. Huang, ..., Albert Zink,
Hans Reschreiter, Kerstin Kowarik
Correspondence
frank.maixner@eurac.edu (F.M.),
kerstin.kowarik@nhm-wien.ac.at (K.K.)
In brief
Maixner et al. describe the gut
microbiome and diet of European salt
miners using paleofeces dating from the
Bronze Age to the Baroque period. This
analysis provides evidence for recent
changes in the gut microbiome due to
industrialization and for the consumption
of fermented food and beverages in Iron
Age Europe.
Bronze Age
2200 1000 15
Iron Age
Roman
Time
Early Middle
Ages
Late Middle
Ages
Modern
Time
400 1000 1492 Present
BC AD
Paleofeces
Sex and mt
haplogroup
Gut
microbiome
Parasites
Animal diet
Plant diet
Fermented
food
Fermented
beverage
Multidisciplinary analysis
M
ultidisciplinar
y
anal
y
sis
Maixner et al., 2021, Current Biology 31, 1–14
December 6, 2021 ª2021 The Authors. Published by Elsevier Inc.
https://doi.org/10.1016/j.cub.2021.09.031 ll
Article
Hallstatt miners consumed blue cheese and beer
during the Iron Age and retained a non-Westernized
gut microbiome until the Baroque period
Frank Maixner,
1,12,13,16,17,
*Mohamed S. Sarhan,
1,12,16
Kun D. Huang,
2,3,16
Adrian Tett,
2,4
Alexander Schoenafinger,
1,5,12
Stefania Zingale,
1,12
Aitor Blanco-Mı
´guez,
2
Paolo Manghi,
2
Jan Cemper-Kiesslich,
6
Wilfried Rosendahl,
7,8
Ulrike Kusebauch,
9
Seamus R. Morrone,
9
Michael R. Hoopmann,
9
Omar Rota-Stabelli,
10
Thomas Rattei,
4
Robert L. Moritz,
9
Klaus Oeggl,
5
Nicola Segata,
2,14,16
Albert Zink,
1,16
Hans Reschreiter,
11,16
and Kerstin Kowarik
11,15,16,
*
1
Institute for Mummy Studies, EURAC Research, Viale Druso 1, 39100 Bolzano, Italy
2
Department CIBIO, University of Trento, Via Sommarive 9, 38123 Povo (Trento), Italy
3
Department of Sustainable Agro-Ecosystems and Bioresources, Fondazione Edmund Mach, Via Edmund Mach 1, 38010 San Michele
all’Adige (TN), Italy
4
CUBE (Division of Computational Systems Biology), Centre for Microbiology and Environmental Systems Science, University of Vienna,
Althanstraße 14, 1090 Vienna, Austria
5
Institute of Botany, University of Innsbruck, Sternwartestraße 15, 6020 Innsbruck, Austria
6
Interfaculty Department of Legal Medicine & Department of Classics, University of Salzburg, Ignaz-Harrer-Straße 79, 5020 Salzburg, Austria
7
Reiss-Engelhorn-Museen, Zeughaus C5, 68159 Mannheim, Germany
8
Curt-Egelhorn-Zentrum Arch
aomtrie, D6,3, 61859 Mannheim, Germany
9
Institute for Systems Biology, 401 Terry Avenue North, Seattle, WA 98109, USA
10
Center Agriculture Food Environment (C3A), University of Trento, 38010 San Michele all’Adige (TN), Italy
11
Prehistoric Department, Museum of Natural History Vienna, Burgring 7, 1010 Vienna, Austria
12
Twitter: @EuracMummy
13
Twitter: @FrankMaixner
14
Twitter: @cibiocm
15
Twitter: @KowarikKerstin
16
These authors contributed equally
17
Lead contact
*Correspondence: frank.maixner@eurac.edu (F.M.), kerstin.kowarik@nhm-wien.ac.at (K.K.)
https://doi.org/10.1016/j.cub.2021.09.031
SUMMARY
We subjected human paleofeces dating from the Bronze Age to the Baroque period (18
th
century AD) to in-
depth microscopic, metagenomic, and proteomic analyses. The paleofeces were preserved in the under-
ground salt mines of the UNESCO World Heritage site of Hallstatt in Austria. This allowed us to reconstruct
the diet of the former population and gain insights into their ancient gut microbiome composition. Our dietary
survey identified bran and glumes of different cereals as some of the most prevalent plant fragments. This
highly fibrous, carbohydrate-rich diet was supplemented with proteins from broad beans and occasionally
with fruits, nuts, or animal food products. Due to these traditional dietary habits, all ancient miners up to
the Baroque period have gut microbiome structures akin to modern non-Westernized individuals whose diets
are also mainly composed of unprocessed foods and fresh fruits and vegetables. This may indicate a shift in
the gut community composition of modern Westernized populations due to quite recent dietary and lifestyle
changes. When we extended our microbial survey to fungi present in the paleofeces, in one of the Iron Age
samples, we observed a high abundance of Penicillium roqueforti and Saccharomyces cerevisiae DNA.
Genome-wide analysis indicates that both fungi were involved in food fermentation and provides the first mo-
lecular evidence for blue cheese and beer consumption in Iron Age Europe.
INTRODUCTION
Paleofeces are naturally preserved ancient feces found in dry
caves, desert areas, waterlogged environments, and frozen hab-
itats. Specific environmental processes such as desiccation or
freezing prevent their deterioration in mummies, ancient latrines,
bogs, and soils.
1
Previous studies have shown that paleofecal
material still contains plant macro- and microfossils, parasite
eggs, and even ancient biomolecules (DNA, proteins, metabo-
lites).
2
Ancient paleofeces have therefore recently been used
as a source of information to study prehistoric nutrition pat-
terns
3–5
and health
6,7
and to analyze single representatives
8,9
or the overall composition of the intestinal microbiome of our an-
cestors.
10–12
One of the few archaeological sites where well-preserved
paleofeces can be found is the protohistoric salt mines of the
Current Biology 31, 1–14, December 6, 2021 ª2021 The Authors. Published by Elsevier Inc. 1
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Please cite this article in press as: Maixner et al., Hallstatt miners consumed blue cheese and beer during the Iron Age and retained a non-Westernized
gut microbiome until the Baroque period, Current Biology (2021), https://doi.org/10.1016/j.cub.2021.09.031
Austrian UNESCO World Heritage area Hallstatt-Dachstein/Sal-
zkammergut. Protohistoric salt mines offer ideal preservation
conditions for organic materials. The high salt concentrations
and the constant annual temperature of around 8C inside the
isolated mine workings preserve organic artifacts very well.
The Hallstatt salt mines located in the Eastern Alps (Figure 1)
offer one of the world’s oldest and most continuous record of un-
derground salt mining.
13–15
Large-scale underground mining in
the Hallstatt salt mountains dates back at least to the 14
th
cen-
tury BC (late Bronze Age). Several protohistoric (Bronze, Iron
Age) and historic (14
th
century AD to present) mining phases
are well documented. The site also gave name to the early period
of the Iron Age in Europe, the so-called Hallstatt Period (800 to
400 BC). Dense layers of production waste reaching several
meters of thickness were excavated from the protohistoric
Bronze Age and Iron Age mine workings of Hallstatt, uncovering
thousands of wooden tools and construction elements, imple-
ments made from fur, rawhide, hundreds of woolen textile frag-
ments, grass, bast ropes, and human excrements.
16
These ob-
jects provide insights into the daily life of a Bronze Age and
Iron Age mining community ranging from mining technology, or-
ganization of production, and resource management to human
health, dietary habits, social organization of production pro-
cesses, and social status within a mining system. These aspects
have been studied extensively in Hallstatt based on a combina-
tion of data sources encompassing the protohistoric mine work-
ing, Bronze Age meat-curing facilities, and large Iron Age
cemetery.
14,16,17
AB
C
Early Iron Age
Bronze Age
2610 2604 2611 2612
1000
1492
Late Middle
Ages
Bronze Age
Iron Age
Roman
Time
2200
1000
400
BC AD
15
Present
Modern
Time
Early Middle
Ages
D
Tuschwerk
1301-1121 cal BC
Kernverwässerungswerk
650-545 cal BC
Josefstollen-Querschlag
652-544 cal BC
Edlersbergwerk-oben
1720-1783 cal AD
Baroque period
Figure 1. The Hallstatt salt mine and radiocarbon-dated paleofeces samples used in this study
(A) The salt mines are located in Upper Austria.
(B) Finding sites of the four paleofeces samples in the Bronze Age, Iron Age, and Baro que mining area. The symbol color corresponds to the radiocarbon date of
the paleofeces.
(C) Macroscopic appearance of the four paleofeces samples. The scale bar corresponds to 1 cm of length. The sample description includes the sample ID, the
mine workings name, and the radiocarbon date. The provided radiocarbon date range corresponds to the Cal 2-sigma values with the highest probability.
(D) Temporal assignment of the radiocarbon-dated paleofeces to the major European time periods from the Bronze Age onward.
See also Figure S1 for details of the salt crystals surrounding sample 2612. Data S1A and S1B provide additional information about the samples and the
radiocarbon dating.
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2Current Biology 31, 1–14, December 6, 2021
Please cite this article in press as: Maixner et al., Hallstatt miners consumed blue cheese and beer during the Iron Age and retained a non-Westernized
gut microbiome until the Baroque period, Current Biology (2021), https://doi.org/10.1016/j.cub.2021.09.031
Article
Here we focus on the question of the structure and evolution of
dietary habits as well as the human gut microbiome in one of Eu-
rope’s most important early production communities. We used
microscopic, metagenomic, and proteomic analysis to charac-
terize nutrition patterns of the protohistoric (Bronze Age, early
Iron Age) and historic (Baroque period, 18
th
century AD) miners
and metagenomic analysis to determine the structure and
evolution of the gut microbiome. Our findings will enhance the
understanding of early European dietary habits (especially the
production and consumption of processed foodstuffs) and pro-
vide further evidence of the recent change in gut microbiome
structure as result of industrialization and Westernization
processes.
RESULTS
Paleofeces from Bronze Age to Baroque Period contain
ancient endogenous DNA
In this study, we initially subjected four paleofeces samples,
collected from Bronze Age and Iron Age Hallstatt mine workings,
to radiocarbon dating, then to in-depth microscopic and molec-
ular analysis (Figures 1A–1C; STAR Methods;Data S1A). The
four paleofeces samples can be macroscopically differentiated
into three samples containing a high amount of fibrous plant ma-
terial (2610, 2604, 2611) and one more homogeneous sample
(2612) that does not contain any visible larger plant fragments
(Figures 1 andS1A). Radiocarbon analyses date the roughly
structured samples to the Late Bronze Age (2610) and Iron Age
(2604, 2611), which is in perfect accordance with the proposed
period of usage of the mine workings where the paleofeces
have been found.
15
In contrast, the fine-textured paleofeces
2612 sampled in an Iron Age minedates to the Baroque period
(18
th
century AD) (Figure 1C; Data S1B). For this part of the salt
mine, however, it is historically documented that the mine work-
ings had started to be reused from the beginning of 18
th
century
onward.
18
Independently of the paleofeces’ age, their storage
time since excavation (some samples were recovered in the
year 1983), or the mode of excavation (wet sieving versus direct
sampling) (Figure S1), we could retrieve biomolecules (DNA and
protein) from all samples for the subsequent molecular analysis
(Data S1 and S2;STAR Methods). Proteomics analysis provided
the first evidence for the presence of endogenous biomolecules
in the paleofeces material. The most abundant peptides were as-
signed to human intestinal tract proteins that are involved in food
digestive processes (Data S2B, S2D, S2F, and S2H). The DNA of
the paleofeces material was further subjected to a deep shotgun
sequencing approach resulting in 57,130,584 to 221,314,691
quality-filtered reads (Data S1C). A first taxonomic overview us-
ing DIAMOND against the NCBI NR database revealed that the
majority of reads in the samples are assigned to Bacteria
(93.9% to 78.9% of all assigned reads), with Firmicutes and Bac-
teroidetes being the most abundant phyla of this kingdom (Fig-
ures S2A–S2D). Less than 7.5% of the reads were eukaryotic,
with up to 6.7% fungal reads in sample 2604. The Metazoa
and Viridiplantae reads, important for the molecular reconstruc-
tion of the diet, comprised 0.5% to 0.01% of all assigned reads.
Further analysis of the human DNA in the paleofeces revealed an
endogenous DNA content between 0.26% and 0.06%, sufficient
for molecular sex and mitochondrial haplogroup assignment
(Data S1D). The highly fragmented human reads display a very
low deamination pattern at the 50ends (Figures S2E–S2H). In
the most recent sample (2612), the reads appear even less frag-
mented and display almost no DNA damage. Considering the
age of these samples, the DNA damage is exceptionally low.
This high preservation is most likely due to the rapid desiccation
of the samples in the salt mine, which may result in reduced hy-
drolytic damage of the biomolecules. Our analyses show that the
four paleofeces come from male individuals that carry distinct
mitogenomes with low contamination estimates (1% to 2%),
indicating that each sample represents unique unmixed ancient
feces.
Ancient paleofeces display a gut microbiome structure
similar to modern non-Westernized individuals
We compared the microbiome structure of the paleofeces to a
large number of contemporary metagenomes (n = 823) (Data
S1E). Principal coordinate analysis (PCoA) performed on a spe-
cies-level taxonomic composition shows that the paleofeces
from the Bronze Age to the Baroque period cluster with stool
samples from contemporary non-Westernized individuals (Fig-
ure 2A) with diets mainly consisting of unprocessed foods and
fresh fruits and vegetables.
19
This clustering is similarly observed
for encoded metabolic pathways (Figure 2B). All the paleofecal
samples were distinct from the oral and, more importantly,
from the soil samples, suggesting little evidence of soil contam-
ination, which is sometimes observed in ancient metagenomics
studies.
20
The source prediction analysis further supports the
sample preservation (Data S1F).
To further assess the paleofeces samples, we analyzed the
prevalence of the top 15 most abundant species in the paleofe-
ces compared to 8,968 gut microbiomes of healthy Westernized
and non-Westernized adults (Data S1G). As a result, 13 out of the
15 most abundant species were identified to be associated with
human gut environment, of which 11 species were found to be
more prevalent in modern non-Westernized compared to West-
ernized populations. Five of these species, Bifidobacterium
angulatum,Lactobacillus ruminis,Catenibacterium mitsuokai,
Prevotella copri, and Clostridium ventriculi, were over twice as
prevalent in non-Westernized populations (Figure 2C; Data
S1H). One of the two species not associated with the human
gut is the halophilic archaeon Halococcus morrhuae, which sur-
vives on a high concentration of salt.
22
It was observed in low
abundance in the paleofeces sample 2612, the only sample
that was not subjected to wet sieving. Therefore, we assume
that the archaeon was introduced from the environment via the
salt crystals (Figure S1B). In the paleofeces samples 2604 and
2611, we also identified Clostridium perfringens, a known intes-
tinal foodborne pathogen,
23
that also occurs free living in the soil
and other environments.
24
Since an infection with C. perfringens
causes acute diarrhea and the paleofeces does not indicate any
characteristics pointing to that disease, we assume that the
presence of this bacterium is due to an environmental
contaminant rather than a remnant of food spoilage in the
miners’ gut. When all paleofecal microbiome members were
considered in population prevalence analysis, 100 out of
158 species were found in R5% stool samples from modern
healthy adult individuals and 65% of these species are overrep-
resented in non-Westernized populations in comparison with
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Please cite this article in press as: Maixner et al., Hallstatt miners consumed blue cheese and beer during the Iron Age and retained a non-Westernized
gut microbiome until the Baroque period, Current Biology (2021), https://doi.org/10.1016/j.cub.2021.09.031
Article
the Westernized equivalent (Data S1I). A similar result was ob-
tained when we decreased the prevalence threshold to 1%
(Data S1J).
The Prevotella copri complex, which is highly prevalent in non-
Westernized populations and prevalent in previously investi-
gated ancient samples,
9
was identified in all paleofecal samples,
representing, on average, 7.3% (1.6%–14.7%) of the relative
abundance (Figure 2C; Data S1K). Consistent with previous find-
ings,
9
we found multiple clades of the complex to be present in
each of the paleofeces samples (Figure 2D) with the exception
of Clade D, which was barely detectable in sample 2604
and 2612. All other clades were detected with relative abun-
dances > 0.01% in all samples (Figure 2D; Data S1L). Of note,
in contrast to other samples, the sample 2604 displayed higher
abundance of bacterial species such as Lactobacillus brevis,
Bifidobacterium merycicum,Bifidobacterium angulatum, and
Lactobacillus plantarum (Data S1K) that are known to be of pro-
biotic activities or involved in processing of dairy products.
25
Microscopic and molecular reconstruction of the
Hallstatt miners’ diet
Next, we aimed to reconstruct the dietary components in the pa-
leofeces using both a microscopic and a molecular survey. The
above-mentioned structural differences between the paleofeces
became even more evident in the microscopic analyses. The
Baroque period sample 2612 was much finer textured than all
other samples from protohistory (Figure 1C). This was also re-
flected in the macro-remain composition of the paleofeces,
showing that samples 2610, 2604, and 2611 contained a lot of
seeds contrary to 2612, which consisted of frequent tissues of
2604
2611
2612
2610
10
−2
10
−1
10
0
10
1
Relative abundance (%)
100500
Prevalence (%)
Turicibacter sanguinis
Intestinibacter bartlettii
Oscillibacter sp. CAG241
Clostridium ventriculi
Prevotella copri
Collinsella aerofaciens
Bifidobacterium adolescentis
Halococcus morrhuae*
Roseburia faecis
Ruminococcus bromii
Methanobrevibacter smithii
Catenibacterium mitsuokai
Clostridium perfringens
Lactobacillus ruminis
Bifidobacterium angulatum
Non-westernized (n = 725)
Westernized (n = 8243)
AB C
D
2610
2604
2611
2612
Relative abundance (%)
1086420
P. copri complex
Clade A
Clade C
Clade B
Clade D
PC1 (44.97%)
0.1
0.0
-0.1
-0.2
0.2
0.10.0-0.1-0.2-0.3 0.2
PC2 (22.08%)
Human
Soil
PC1 (33.61%)
0.025
-0.025
-0.075
0.075
0.050
0.000
-0.050
-0.100
0.100.050.00-0.05-0.01 0.15
PC2 (17.19%)
Non-westernised
Westernised
PC1 (78.69%)
0.0
-0.1
-0.2
-0.3
-0.4
0.1
0.10.0-0.1-0.2 0.30.2 0.5 0.60.4
PC2 (7.84%)
Human
Soil
PC1 (22.04%)
0.10
0.00
-0.10
0.20
0.15
0.05
-0.05
0.25
-0.15 0.100.05-0.05 0.0-0.10-0.20 0.15
PC2 (7.29%)
Non-westernised
Westernised
PC1 (58.86%)
0.10
-0.05
0.05
-0.15
0.20
0.00
-0.10
0.15
0.10.0-0.1-0.2-0.3-0.4 0.2
PC2 (8.67%)
Stool
Oral cavity
PC1 (45.27%)
0.10
0.15
0.05
-0.05
0.00
-0.10
0.20
0.10.0-0.1-0.2-0.3-0.4
PC2 (18.01%)
Stool
Oral cavity
2610 2604 2611 2612
Ancient samples:
Figure 2. Overview of microbial composition and metabolic pathways of paleofeces samples in comparison to a large collection of contem-
porary metagenomes
(A) Principal coordinate analysis (PCoA) based on microbial abundance profiled using MetaPhlAn 3.0
21
between four paleofeces samples and 823 contemporary
samples characterized by sampling environment, body site, and non-Westernized lifestyle.
(B) Principal coordinate analysis (PCoA) based on metabolic pathway abundance profiled using HUMAnN 3.0
21
between four paleofeces samples and the same
contemporary samples used in (A).
(C) Prevalence of the top 15 most enriched species of four paleofeces samples in non-Westernized and Westernized datasets comprising 8,968 stool samples
from healthy adult individuals. Asterisk indicates species that is likely from external contamination.
(D) Relative abundance of P. copri four clades estimated using MetaPhlAn 3.0
21
in each paleofecal sample.
See also Figure S1 for additional microbial profiles in the DNA ‘wash-out’’ experiment. Data S1 contains additional information about the comparative datasets
and the results obtained by the prevalence and abundance analysis.
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gut microbiome until the Baroque period, Current Biology (2021), https://doi.org/10.1016/j.cub.2021.09.031
Article
Papaver somniferum
Panicum miliaceum
Setaria italica
Hordeum vulgare
Triticum dicoccum
Triticum spelta
Agrostemma githago
Aethusa cynapium
Vaccinium myrtillus
Malus sylvestris
AB
C
Triticum aestivum cultivar CS TA3008, KJ614396.1
Triticum turgidum subsp. durum, KJ614398.1
Triticum aestivum subsp. macha, LC005978.1
Triticum turgidum subsp. dicoccoides, KJ614400.1
Triticum aestivum subsp. spelta, KJ614403.1
2604
2610
Aegilops speltoides var. ligustica, KJ614404.1
Triticum timopheevii subsp. timopheevii, KJ614410.1
Triticum monococcum
Triticum urartu
Aegilops spp.
Secale cereale, KC912691.1
Hordeum spp.
0.10
D
0% 20% 40% 60% 80% 100%
2610
2604
A
B
D
Un
Vicia faba
0
1
2
3
4
5
2610
2604
2611
2612*
Log10 (counts)
Juglans regia
Panicum miliaceum
Hordeum vulgare
Triticum
Papaver somniferum
Sus scrofa
Bos taurus
Trichuris trichiura
Ascaris suum
Wallemia ichthyophaga
Aspergillus pseudoglaucus
Penicillium roqueforti
Magnusiomyces capitatus
Pichia kluyveri
Wickerhamomyces anomalu
s
[Candida] glabrata
Saccharomyces cerevisiae
Kluyveromyces marxianus
2610
2604
2611
2612
2610
2604
2611
2612
**
*
Microscopy
Metagenome Proteome
Log10(species)
0123
Log10(genus)
0123
5 1015202530
Number of peptides
Figure 3. Microscopic and molecular dietary analysis of the Hallstatt paleofeces
(A) Plant macro-remains microscopically detected in the four paleofeces samples. The scale bar indicates 1 mm of length. The heatmap shows the log-scale
macro-remain counts normalized to 3.7 g sample. The sample with asterisk was assessed in a semiquantitative manner. For further details, please referto
Data S1.
(B) Most abundant taxa (plants, nematodes, animals, fungi) detected in the four paleofeces metagenomes and proteomes. The circle size and circle color
correspond to log10 ‘‘normalized’’ number of reads per million at genus and species levels, respectively. The asterisks in the proteome heatmap mean the
peptides were assigned only to genus level.
(C) Phylogenetic assignment of two partial Triticum chloroplast genomes in the 2604 and 2610 metagenomes. The comparative dataset included
complete chloroplast genomes of selected members of the Triticeae tribe (NCBI accession numbers are provided in the figure). The tree was
calculated using the maximum-likelihood algorithm (PhyML) based on 136,160 informative positions. Black circles symbolize parsimony and
neighbor joining bootstrap support (>90%) based on 100 and 1,000 iterations, respectively. The scale bar indicates 10% estimated sequence
divergence.
(legend continued on next page)
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Article
fruit husks and seed coats (Figure 3A; Data S1M). Generally, all
samples displayed a predominance of cereal remains.
Microscopic analysis revealed that the Bronze Age sample
(2610) consisted more or less exclusively of cereal remains,
which originated from barley (Hordeum vulgare), spelt (Triticum
spelta), some emmer (Triticum dicoccum), proso millet (Panicum
miliaceum), and a few weeds, e.g., corn cockle (Agrostemma gi-
thago) and poison parsley (Aethusa cynapium). The Iron Age
samples (2604, 2611) were characterized by a predominance
of cereal remains from barley (Hordeum vulgare), spelt (Triticum
spelta), millets (P. miliaceum,Setaria italica), and a little emmer
(T. dicoccum). Furthermore, in sample 2611, testa remains of
broad beans (Vicia faba) and seeds of opium poppies (Papaver
somniferum) were observed. Crab apples (Malus sylvestris) and
bilberries/cranberries (Vaccinium myrtillus/vitis-idea) in sample
2604 document the consumption of gathered wild fruits. Striking
in the Iron Age sample 2604 was the contamination with weeds,
in particular corn cockle (Agrostemma githago). In the sample
2612 from the Baroque time, the microscopic pattern was
notably different from the other samples. The plant material
was finely ground, and entire fruits were missing apart from a di-
gested mericarp of anise (Pimpinella anisum). The plant tissue
belonged to bran (fragments of cereal testa, pericarp, hairs, hi-
lum, endosperm) of wheat (Triticum sp.). The precise species
was unidentifiable, but according to the rare occurrence of
tube cells in the pericarp fragments, a member of the tetra- or
hexaploid wheat group is suggested. Bran of barley
(H. vulgare) was also observed in minor quantities. Furthermore,
the consumption of legumes is documented by testa remains of
garden bean (Phaseolus vulgare) in this sample.
In addition to the microscopic analysis, we subjected paleofe-
ces biomolecules (DNA and proteins) to molecular dietary ana-
lyses. Both metagenomic and proteomic analyses included a
homology search against different databases, followed by
strict filtering steps of the obtained hits and a subsequent in-
depth analysis of selected identified taxa (STAR Methods;Fig-
ure S3;Data S1N, S1O, S2B, S2D, S2F, and S2H). For the plant
diet, we could confirm the presence of the most abundant
domesticated plant macro-remains, including broomcorn millet
(P. milliaceum), barley (H. vulgare), and wheat (Triticum spp.)
(Figure 3B). In addition, we found DNA-based evidence of the
presence of walnut (Juglans regia) in the sample 2604 and pro-
tein-based support for the occurrence of opium poppy seeds
(P. somniferum) in the sample 2611. In addition to the foxtail mil-
let (S. italica), which appeared with high grain number, all the low
abundant wild plants unveiled by the microscopic investigation
were not identified in our molecular survey, which has undergone
strict filtering to minimize the false positives (STAR Methods).
Further phylogenetic analysis assigned the Triticum spp. chloro-
plast genomes of the samples 2604 and 2610 closest to the
chloroplasts of tetraploid (emmer, durum) and hexaploid (spelt
wheat, bread wheat) wheat varieties, respectively (Figures 3C,
S3A, and S3B; Data S1P). Additional comparison with the bread
wheat genome revealed an equal subgenome (A, B, and D) rep-
resentation in the 2604 and 2610 metagenomic reads, which
suggests, in combination with the microscopic identification of
numerous characteristic grains, glumes, and spikelets, the pres-
ence of hexaploid spelt wheat (T. spelta) in these paleofeces
(Figure 3D). Beside the plant diet, we obtained molecular evi-
dence for the consumption of cattle (Bos taurus) and swine
meats (Sus scrofa) throughout all investigated time periods
(Figures 3B and S3D). Interestingly, the most abundant cattle
proteins in sample 2611 (hemoglobin and coagulation proteins)
indicate the plant diet was supplemented by blood-rich animal
tissues (e.g., muscle, liver) (Data S2F). The molecular analyses
revealed in addition, that individuals from both the Iron Age
(2611) and the Baroque (2612) suffered from intestinal infections
of whipworms (Trichuris trichuria) and roundworm (Ascaris spp.)
(Figure 3B and S3D–S3G). Finally, all samples showed a contin-
uous low background with fungal DNA mainly coming from
different Ascomycota.
Molecular evidence for blue cheese and beer
consumption during the Iron Age
In contrast to all other samples, the Iron Age sample 2604 dis-
played an exceptionally high abundance of Penicillium roqueforti
and Saccharomyces cerevisiae proteins (Data S2D) and DNA
(Data S1N), making up to 7%–22% of total eukaryotic reads.
This was characteristic of this sample as compared with the
other samples that did not show such prevalence—even the
sample 2611, which was taken from a similar context and dated
back to the same time point. To authenticate the data and gain
further insights into their potential ecological significance, we
mapped the high-quality reads of sample 2604 against the refer-
ence genomes of these two fungi (Figure S4A; Data S1O). With
11–133coverage, we were able to reconstruct >92% of both ge-
nomes, displaying even coverage and SNP distribution. To
confirm whether these two fungi are of ancient origin and not
modern contaminants, we initially checked the ancient DNA
damage pattern of the mapped reads. Both fungi displayed
typical ancient DNA damage patterns, with levels comparable
to the human endogenous DNA (Figures S4B and S4C). Hence,
and considering their extraordinarily high abundance and exclu-
sive incidence in this sample, we assumed their endogenous
originality to the coprolite microbial community. Additionally,
both fungi are commonly used nowadays in food processing:
P. roqueforti is used for cheese fermentation, and S. cerevisiae
is used for fermenting bread and alcoholic beverages including
beer, mead, and wine. Therefore, we assume that they could
have been involved in food processing at that time. To test this
assumption, we used the reconstructed genomes for further
comparative phylogeny and population genetic analyses to infer
whether they had been truly involved in food processing or were
just transient environmental microbes.
(D) Wheat subgenome (A, B, and D) representation in the 2604 and 2610 metagenomes (Data S1), aligned to the modern hexaploid bread wheat reference
genome (accession number GCA_900519105). Both the wheat chloroplast and nuclear reads were highly fragmented and display aDNA-specific damage
patterns (Figures S3H–S3K).
See also Figure S3 for details about the comparative analysis, phylogenet ic assignment, and damage pattern of selected plant, animal, and parasite DNA. Data S1
provides further details of the macro-remains, comparative datasets, dietary DNA, and mapping statistics. Data S2 provides additional information about the
comparative datasets and proteomics results.
ll
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Please cite this article in press as: Maixner et al., Hallstatt miners consumed blue cheese and beer during the Iron Age and retained a non-Westernized
gut microbiome until the Baroque period, Current Biology (2021), https://doi.org/10.1016/j.cub.2021.09.031
Article
First, we compared our putative P. roqueforti strain to 33 other
sequenced modern P. roqueforti strains coming from different
functional niches.
26
The comparative dataset included 18
cheese-fermenting and 15 non-cheese-fermenting strains (Data
S1Q), in addition to Penicillium psychrosexualis and Penicillium
carneum as an outgroup. After mapping the raw reads of all
strains to the reference P. roqueforti genome FM164 and data
filtering, we resolved 120,337 SNPs, which were used for inferring
maximum likelihood (ML) phylogenetic relationships among the
tested strains (STAR Methods). Consistent with the original pub-
lication of Dumas and colleagues,
26
the resulting phylogeny re-
vealed four distinct clades: a Roquefort cheese clade, a non-
Roquefort cheese clade containing blue cheeses others than
Roquefort, a silage/food spoilage clade, and a wood/food
spoilage clade (Figure 4A). Initially, the phylogenetic analysis
separated the non-Roquefort cheese clade from the other, then
the Roquefort cheese clade was diverged from the other food
spoilage clades. The ancient P. roqueforti strain showed highest
similarity to the non-Roquefort cheese strains, being clustered
together with their corresponding clade as an earlier divergent.
The reason behind such early divergence might be attributed to
the recent acquisition of some genomic regions—most impor-
tantly, CheesyTer and Wallaby—by the non-Roquefort cheese
strains. This gene acquisition most likely happened via repeated
multiplication of selected spores of the best cheeses on bread
used as a growth medium in the late 19
th
century and early 20
th
century before the advent of microbiological in vitro culturing
techniques.
27
Thereby, modern non-Roquefort strains were
exposed to extensive selection coupled to horizontal gene trans-
fer events from other cheese-producing Penicillium spp. or even
other genera.
26,27–29
Importantly, our Iron Age strain did not
contain any of those recently acquired fragments, which comes
in congruence with the hypothesis that such domestication
events occurred during the last two centuries.
We further used the tool ADMIXTURE to infer the degree of
admixture among the strains. By assuming the presence of 3 an-
cestries (K = 3), we could clearly distinguish the non-Roquefort,
Roquefort, and food spoilage strains (Figure 4B). Our putative
strain displayed 70% cheese-producing ancestry (60% of the
non-Roquefort and 10% of Roquefort cheese) and 30% food-
spoiler ancestry. Both the phylogenetic placement and ADMIX-
TURE profile indicate that the ancient P. roqueforti has already
been under positive selection toward the non-Roquefort cheese
cluster, a selection process that most likely occurred during the
process of cheeseproduction. Some archeological findings exca-
vated from the mines might have been usedfor that purpose (Fig-
ure 4C), as they showed some traces of fatty food products.
Next, we compared the ancient S. cerevisiae genome to 157
recent strains coming from different ecological niches, i.e.,
food, alcoholic beverages (e.g., beer, wine, sake, and spirits),
biofuels, and laboratories, as well as wild strains (Data S1R).
ML phylogenetic analysis, based on 375,629 SNP positions,
distinguished 2 main clades. The first main clade splits into two
subclades, with one containing most of the beer strains (beer 1
clade) that show a successive sub-clustering based on the origin
of the strains (Figure 4D). The other subclade (henceforth referred
to as ‘‘mixed’’ clade) included a mixture of bread, wine, beer, and
spirit strains. The second main clade is composed of two sub-
clades: a wine clade and another beer clade (beer 2). All other
wild, laboratory, and sake strains fall to the base of the whole phy-
logeny. The ancient S. cerevisiae strain clustered basal to the
second main clade, which includes the wine and beer 2 strains.
Further population structure analysis displayed high admixture
in our putative strain, resembling primarily the wine ancestral
population (47%), followed by 29.2% beer ancestries (Fig-
ure S4D) and only 19% wild strain ancestry. Therefore, and
considering the ML phylogenetic assignment, we assume that
the possibility of our strain to be of wild origin is unlikely. The re-
sults rather indicate higher similarity to wine and beer strains.
Principal component analysis (PCA) provided further indication
for the domestication of our strain in alcoholic beverage fermen-
tation. Along the PC1 that explains 25.42% of the variation, our
strain clustered closer to the strains of beer 2 than to the strains
of the wine clade (Figure 4E). This was further supported with pro-
teomic analysis (Data S2D) that unveiled that most of the peptides
assigned to the genus Saccharomyces derived from proteins
involved in alcohol fermentation pathways (e.g., glycolysis). To
further narrow down the possible routes of domestication, we
decided to differentiate the strains based on functional marker
genes (Data S1S). According to recent literature,
31,32,33
the genes
RTM1,BIO1/BIO6, and the chromosomal regions A/B/C can be
used to differentiate yeast strains based on their functional
niches. The gene RTM1 is a strong domestication marker respon-
sible for conferring resistance against the toxicity of molasses
and other rich-sugar substrates and is assumed to be positively
selected in beer yeast strains.
32,34
The genes BIO1 and BIO6,
which are involved in de novo biosynthesis of biotin, are highly
selected in sake fermenting yeasts, due to lack of biotin in the
fermentation substrates, such as rice.
35
The regions A, B, and
C are horizontally transferred genomic regions from other yeast
genera, e.g., Kluyveromyces,Pichia, and Zygosaccharomyces.
36
These regions contain 39 genes distributed over 3 different chro-
mosomes and are assumed to play a role in wine fermentation.
Therefore, we searched the presence of these marker genes in
our comparative dataset, including our ancient strain. In accor-
dance with the literature, almost all beer strains—either of clade
1 or clade 2—were positive for RTM1, while the wine clade was
mainly positive for the genomic regions A/B/C. The mixed clade
contained both RTM1 and the regions A/B/C. The sake clade
exclusively contained the BIO1/BIO6 genes and partially the
RTM1 gene. The wild strains, isolated from cacao in Africa, clus-
tered in the basal clade and did not contain any of these marker
genes (Figure 4D; Data S1T), contrary to our strain that contained
the RTM1 and lackedthe BIO1/BIO6 genes andthe regions A/B/C.
Based on the previous findings—i.e., (1) the ancient DNA dam-
age profile, (2) the high prevalence of S. cerevisiae reads, (3) the
presence of fermentable cereal substrates such as wheat and
barley, (4) the phylogenetic assignment of the ancient yeast
strain, (5) the yeast admixture profile, and (6) the distribution of
marker genes—we assume that this yeast is of ancient origin
and has been involved in beer fermentation, although the
mode of fermentation is unknown (i.e., bottom, top, or sponta-
neous fermentation).
DISCUSSION
Our interdisciplinary analyses of the samples have given detailed
insight into the microbiome evolution and dietary habits and food
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gut microbiome until the Baroque period, Current Biology (2021), https://doi.org/10.1016/j.cub.2021.09.031
Article
Marker genes
RTM1
BIO1/BIO6
Regions A/B/C
Beer1
Mixed
Wine
Beer2
West Africa (WA)
Asia
Origin/Clade
A
D
B
Non-Roquefort
cheese
Roquefort cheese
Non-cheese
2604
Proq_LCP06129
Proq_LCP06138
Proq_LCP06173
Proq_LCP06133
Proq_FM215
Proq_LCP00146
Proq_LCP06132
Proq_LCP06134
Proq_LCP06135
Proq_LCP06137
Proq_LCP06136
Proq_LCP06128
Proq_LCP06130
Proq_LCP04157T
Proq_LCP00148
Proq_LCP02939
Proq_FM317
Proq_LCP06131
Proq_LCP06039
Proq_LCP05419
Proq_LCP06064
Proq_LCP06060
Proq_LCP04111
Proq_LCP06037
Proq_LCP06040
Proq_LCP06036
Proq_LCP03969
Proq_LCP04180
Proq_LCP03914
Proq_FM259
Proq_LCP06043
Proq_LCP05885
Proq_LCP06059
−0.10
−0.05
0.00
0.05
0.10
−0.1 0.0 0.1 0.2
PC1 (25.42%)
PC2 (20.08%)
2604
Beer1
Mixed
Wine
Beer2
Asian
West African
Not specified
E
Britain
US
Belgium/Germany
beer019
wine009
beer033
beer042
bio003
beer094
wine008
beer081
wine019
beer029
beer078
wine012
beer055
beer057
beer035
wild007
beer050
spirits010
sake003
beer012
spirits005
beer016
beer062
wine001
bio005
beer024
beer039
spirits011
beer089
beer020
beer058
wine004
wine013
beer067
beer005
wine014
beer008
beer047
wine018
sake004
bread001
wine010
spirits001
beer034
beer069
beer014
beer007
wild002
wine015
beer026
beer082
beer040
beer054
beer045
wine007
beer025
beer075
wine017
beer006
beer028
spirits003
beer085
beer017
beer065
beer072
beer092
beer088
spirits007
beer071
beer010
bio002
spirits009
beer074
wine006
beer021
0
80
ree
b
beer093
wild001
beer043
spirits002
beer011
beer023
Spar
beer090
bread002
beer064
beer003
sake006
wine005
spirits008
bread004
sake002
spirits004
beer097
beer053
beer018
beer041
beer056
beer087
beer036
sake005
beer015
beer060
beer048
2604
lab001
beer076
wild004
sake007
beer066
bio004
beer079
beer022
beer059
beer073
beer096
bread003
beer004
beer070
beer037
wine002
beer032
beer102
beer049
beer046
spirits006
sake001
wine003
beer095
wild003
beer083
beer001
beer068
beer100
beer027
beer002
beer098
beer061
wild005
7
7
0
r
eeb
beer031
bio001
beer052
beer009
beer084
lab002
beer030
beer044
beer086
beer091
beer099
beer038
wild006
wine016
wine011
beer013
beer051
beer063
beer101
Tree scale: 0.1
Beer2
Asia
WA
Wine
Mixed
Beer1
Outgroup
Lumber/food spoiler
Non-Roquefort
Roquefort
Silage/food spoiler
Proq LCP02939
Proq LCP06173
Proq LCP06130
Pcar LCP05629
Proq LCP06128
2604
Proq FM317
Proq LCP06039
Proq LCP06135
Proq LCP00146
Proq LCP06131
Proq FM215
Proq LCP03914
Ppsy CBS128137
Proq LCP05885
Proq LCP06040
Proq LCP04180
Proq LCP06059
Proq LCP06134
Proq LCP06060
Proq LCP06064
Proq LCP06136
Proq LCP06133
Proq LCP00148
Proq FM259
Proq LCP06037
Proq LCP06138
Proq LCP04111
Proq LCP04157T
Proq LCP06129
Proq LCP06036
Proq LCP06132
Proq LCP03969
Proq LCP05419
Proq LCP06043
Proq LCP06137
Tree scale: 0.1
C
Figure 4. Genome-wide SNP analysis of ancient fungal ‘‘strains’’ versus modern industrial and wild/environmental strains
(A) Maximum likelihood (ML) phylogenetic analysis of the Penicillium roqueforti genome assembled from the sample 2604 in addition to other previously published
P. roqueforti genomes.
26
A total number of 120,359 SNP positions were used for the analysi s. P. roqueforti FM164 was used as a reference, while P. carneum and
P. psychrosexualis were used as outgroups. The scale bar depicts 0.1 substitutions per residue. Colored strips indicate the P. roqueforti population as previously
inferred.
26
For further information on the comparison dataset, please refer to Data S1Q.
(B) Population structure analysis of P. roqueforti 2604 with the same previous dataset, considerin g 3 ancestries (K = 3 with lowest cross-validation error), based on
120,337 SNPs. The order of labels corresponds to the clustering in panel (A).
(C) Wooden containers that have been found among other archeological findings in the mines and assumed to be used as cheese strainers
(D) ML phylogenetic analysis of Saccharomyces cerevisiae genome assembled from the sample 2604 compared with other published S. cerevisiae genomes.
30
The dataset for the analysis included 375,629 SNPs. The Saccharomyces paradoxus CBS432 was used as an outgroup. The scale bar depicts 0.1 substitutions
per residue. The colored strips indicate the clade/origin as reported previously.
30
The colored dots at the tree edges refer to the presence/absence of functional
marker genes.
31
Blue dots in (A) and (C) indicate bootstrap support >80% based on 1,000 bootstrap replicates.
(E) Principal component analysis based on 136,712 SNP, of the S. cerevisiae strains.
For additional information on the coverage and SNP density, DNA damage, and ADMIXTURE, please refer to Figure S4.Data S1 provides further details about the
comparative datasets, mapping results, and functional marker analysis.
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gut microbiome until the Baroque period, Current Biology (2021), https://doi.org/10.1016/j.cub.2021.09.031
Article
processing techniques of the Hallstatt miners over the past three
millennia. Molecular and microscopic investigations revealed
that the miner’s diet was mainly composed of cereals, such as
domesticated wheats (emmer and spelt), barley, common mil-
lets, and foxtail millets. This carbohydrate-rich diet was supple-
mented with proteins from broad beans and occasionally with
fruits, nuts, or animal products. The food remains in the protohis-
toric sample with abundant entire fruits and seeds were less pro-
cessed than those of the Baroque sample, which consisted of
finely ground wheat. This suggests that the protohistoric miners
consumed the cereals and legumes in a sort of gruel or
porridge,
37
whereas miners in the 18th century AD ate their ce-
reals in a more processed form, e.g., as a bread or biscuit.
In general, such carbohydrate-rich fibrous dietary components
as observedin the Bronze Age and Iron Age samplesare typical for
traditional communities and are considered to be the main drivers
of the non-Westernized microbiome structure.
38,39
Consistent
with this observation, our analysis showed that the Hallstatt pale-
ofecal samples contain microbial features similar to gut micro-
biomes of modern non-Westernized populations (Figures 2Aand
2B). Species identified in the samples, such as Lactobacillus rumi-
nis,Catenibacterium mitsuokai,andPrevotella copri,werealso
found to be highly prevalent in present-day individuals with a
more traditional lifestyle (Figure 2C). Furthermore, our paleofecal
samples were rich in the P. copri complex (Figure 2D), including
the four clades that are nearly ubiquitous and co-present in non-
Westernized populations.
9
Of particular interest, P. copri
members have been shown to be associated with the digestion
of complex carbohydrates,
9,40–42
which are the major component
of an unprocessed fibrous plant diet. Finding paleofeces highly
resembling that of non-Westernized individuals, in terms of micro-
biome structure, supports previous observations.
10,12
It also adds
weight to the hypothesis that the modern industrialized human gut
microbiome hasdiverged from an ancestral state, probably due to
modern lifestyle, diet, or medical advances. Interestingly,this non-
Westernized microbiome structure has been observed in all four
paleofeces dating from the Bronze Age to the Baroque period,
which would indicate quite a recent change in the gut community.
However, to spot the critical time points when this shift in the hu-
man gut microbiomebegan requires more ancient samples span-
ning a wider time range; of particular interest would be samples
from the past two or three centuries, when major dietary and med-
ical changes occurred. Overall, our results support the theory that
the shift from traditional to an industrial Westernized lifestyle might
be the driving force for changing the human gut microbiome from
its ancestral state.
9,43–45
In one of the Iron Age samples (2604), the molecular analyses
indicated consumption of fermented food and beverages. The
fungal analysis revealed a high prevalence of Penicillium roque-
forti and Saccharomyces cerevisiae, which are nowadays
involved in fermenting blue cheeses and alcoholic beverages,
respectively, with clear signs of domestication. Following rapidly
in the wake of ruminant animal domestication (mainly cattle,
sheep, and goat),cheese production represents one of the oldest
and widespread food preservation techniques developed by hu-
mans.
46
The oldest evidence for milk use dates back to the
Neolithic in the Fertile Crescent yet provides only indirect evi-
dence for fermentation.
47
The oldest reported chemical evidence
for processing of milk into fermented products (i.e., kefir dairy) is
dated back to the Early Bronze Age in Western China.
48
Other in-
dications, including actual preserved pieces of cheese, whey
strainers, and recipesfor cheese production, were found in North-
ern Europe, the Middle and Near East, and the Mediterranean ba-
sin.
49–52
Here, we report evidence for the domestication of the
fungus Penicillium roqueforti in the course of food processing in
the 1
st
millennium BC that would likely produce a cheese resem-
bling a blue cheese (non-Roquefort cheese clade, in Figure 4A).
To our knowledge, this represents the earliest known evidence
for directed cheese ripening and affinage in Europe, adding a
crucial aspect to an emerging picture of highly sophisticated culi-
nary traditions in European protohistory.
37
Importantly, the pro-
duction of blue cheese today involves a surface application of
dry salt; therefore, it is characterized by a high salt content of
up to 7.5% (w/w).
53
The cheese curd could have been collected,
desiccated, and inoculated with the fungi in wooden cheese con-
tainers like the ones excavated in the Hallstatt mines (Figure 4C).
The presence of P. roqueforti indicates a major step in ruminant
milk processing from fresh to ripened cheese, which could have
offered, in addition to new flavors, several advantages to the Hall-
statt miners including longer storage (i.e., months) and less
lactose content in the fermented dairy product.
54
The reduced
lactose content may have helped the ancient minors to better
digest milk products, living in a time when lactose persistence fre-
quencies only started to rise in Europe.
55
The presence of salt as
well as the constant temperature (8C) and humidity inside the
Hallstatt mine workings represent ideal conditions for blue cheese
production, following the current cheese production standards.
56
It is noteworthy that the early discovery of the Roquefort cheese
was linked to Roquefort-sur-Soulzon caves in France, which
maintain a temperature of 10Cand90% humidity over the
year. With such conditions protecting the cheese from desicca-
tion, these caves have been used exclusively for centuries for
ripening and aging of the ‘‘Roquefort’ cheese.
57,58
Indications for the production of fermented alcoholic bever-
ages in protohistory are abundant, albeit frequently ambig-
uous,
59
and can be found in the Near East, Middle East, Far
East, and Europe.
60–67
Evidence for the production of grape
wine in Europe and viniculture in the Near East dates back to
the 6
th
and 7
th
millennium BC.
68
Such evidence was mainly
based on chemical residue analysis or archaeobotanical analysis
or indicated in ancient inscriptions. Recently scientists claimed
that they were able to revive an ancient yeast strain from Egyp-
tian potteries and used it to ferment beer.
69
Here we were able
to reconstruct >90% of the S. cerevisiae genome from an Iron
Age-dated paleofecal sample. We used different molecular
analysis at the genome level to infer the possible routes of
domestication for this yeast. Our results suggested it was used
in beer fermentation. Together with the results of the dietary
analysis that showed presence of different fermentable cereals,
e.g., wheat, barley, and millets, we can envisage how the
fermentation was carried out.
It might be assumed that the fermentation was carried out in a
spontaneous manner—i.e., by adding water to wort and allowing
the fermentation process to take place by the wild air-borne
yeasts or the constitutional microbiota of the used cereals.
70
We do not see, however, indications for other yeasts species,
such as Brettanomyces bruxellensis, that co-occur in spontane-
ously fermented beers.
71
In addition, we see clear indications of
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Please cite this article in press as: Maixner et al., Hallstatt miners consumed blue cheese and beer during the Iron Age and retained a non-Westernized
gut microbiome until the Baroque period, Current Biology (2021), https://doi.org/10.1016/j.cub.2021.09.031
Article
domestication and continuous supply of new admixture compo-
nents to this yeast, which might suggest that fermentation ves-
sels were repeatedly used for this purpose or the inoculation of
the fermentation batches has been done by back-slopping
(i.e., inoculation of new fermentation batches with portions of
previous batches).
72
Albeit varied evidence for beer production
in protohistoric Europe exists,
59,65–67
these beers could not be
preserved for longer time periods and would have had to be
consumed rapidly after production,
66
which also presupposes
that the beer would have had to be produced either in Hallstatt
itself or in the very near surroundings.
Considering the constant temperature of 8C inside the Hall-
statt mines, it might be expected that this yeast was used for pro-
duction of lager-like beer, when fermentation is carried out at low
temperatures (also known as bottom-fermentation) and results in
a beer that can be stored for longer time periods.
73
Historically,
however, the bottom-fermentation was most likely developed af-
ter the year 1553, when the Duke of Bavaria Albrecht V forbade
brewing during summer months.
74
Additionally, Gonc¸ alves and
colleagues demonstrated that Saccharomyces pastorianus
strains, which are hybrids of S. cerevisiae and another Saccharo-
myces species and are used for production of lager beers,
belong to the main beer clade (Figure 4D).
31
Therefore, we postu-
late that the beer produced at that time is similar to what would
nowadays be known as pale beer, produced mainly by top-fer-
menting S. cerevisiae strains.
Paleofeces material displays an archaeological information
source that provides insights into the diet and gut microbiome
composition of ancestors. Here, we had access to four paleofe-
ces samples from the Hallstatt salt mines dating from the Bronze
Age to the Baroque period. The constant low annual temperature
and high salt concentrations inside the mine preserved both
plant macro-remains and biomolecules (DNA and protein) in
the paleofeces. We demonstrate the indispensable complemen-
tarity of using microscopic and molecular approaches in
resolving the paleofecal dietary residual components and to
reconstruct the ancient gut microbiome. Furthermore, we
extended our paleofeces microbiome analysis to focusing on
key microbes that are involved in food processing, which opens
new avenues in understanding fermentation history. In the future,
additional samples from different time points will provide a more
fine-scaled diachronic picture, which may help us to understand
the role of dietary changes in shaping our gut microbiome and
how much this was further influenced by modern lifestyles or
medical advances recently introduced through industrialization
and Westernization.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
dKEY RESOURCES TABLE
dRESOURCE AVAILABILITY
BLead contact
BMaterials availability
BData and code availability
dEXPERIMENTAL MODEL AND SUBJECT DETAILS
dMETHOD DETAILS
BPaleofeces samples, radiocarbon dating
BMicroscopic analysis of the paleofeces
BMolecular analysis of the paleofeces
BProteomic analysis
dQUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.
cub.2021.09.031.
ACKNOWLEDGMENTS
We acknowledge the following funding sources: Programma Ricerca Budget
prestazioni Eurac 2017 of the Province of Bolzano, Italy, and the South Tyro-
lean grant legge 14 (F.M., M.S.S., S.Z., and A.Z.). Additional support was pro-
vided by the European Regional Development Fund 2014-2020_CALL-FESR
2017 Research and Innovation_Autonomous Province of Bolzano South Ty-
rol_Project: FESR1078-MummyLabs. The authors thank the Department of
Innovation, Research and University of the Autonomous Province of Bozen/
Bolzano for covering the Open Access publication costs. We thank Dr. John
Wilson (ProtiFi, USA) for helpful discussions regarding proteomics sample
preparation. This work was in addition supported by the European Research
Council grant ERC-STG Project MetaPG (N.S.); the US National Institutes of
Health, National Institute for General Medical Sciences under grant no.
GM087221 and the Office of the Director 1S10OD026936; and the US National
Science Foundation award 1920268 (R.L.M.). We would like to thank Eva-Ma-
ria Geigl and the two anonymous reviewers for their insightful comments that
helped to improve the manuscript.
AUTHOR CONTRIBUTIONS
F.M., K.O., N.S., A.Z., H.R., and K.K. conceived the investigation. F.M., A.S.,
R.L.M., K.O., N.S., A.Z., H.R., and K.K. designed experiments. K.O., A.S.,
F.M., A.Z., H.R., and K.K. were involved in the sampling campaign. F.M.,
A.S., U.K., M.R.H., and K.O. conducted the experiments. F.M., M.S.S.,
K.D.H., A.T., A.S., S.Z., A.B.-M., P.M., J.C.-K., W.R., U.K., S.R.M., M.R.H.,
O.R.-S., T.R., and K.O. performed analyses. F.M and M.S.S. wrote the manu-
script with contributions from K.D.H., A.T., A.B.-M., J.C.-K., M.R.H., O.R.-S.,
T.R., R.L.M., K.O., N.S., A.Z., H.R., and K.K.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: May 27, 2021
Revised: August 16, 2021
Accepted: September 14, 2021
Published: October 13, 2021
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Biological samples
Paleofeces sample from the Bronze Age This study 2610
Paleofeces sample from the Iron Age This study 2604
Paleofeces sample from the Iron Age This study 2611
Paleofeces sample from the Baroque time This study 2612
Chemicals, peptides, and recombinant proteins
Sodium phosphate Sigma-Aldrich Cat #342483
Trypsin gold Promega Cat # V528A
Critical commercial assays
S-Trap ProtiFi Cat #K02-mini-10
Deposited data
Paleofeces metagenomic shotgun datasets This study ENA: PRJEB44507
NCBI-nr database
75
https://www.ncbi.nlm.nih.gov/protein/
NCBI-nt database
75
https://www.ncbi.nlm.nih.gov/nucleotide/
PlantiTS database
76
https://github.com/apallavicini/PLANiTS
Chloroplast genome database N/A https://www.ncbi.nlm.nih.gov/genome/organelle
Fungal ITS database
77
https://unite.ut.ee/
BOLD system databases
78
https://v3.boldsystems.org/
Software and algorithms
MetaPhlAn
21
https://github.com/biobakery/
MetaPhlAn/wiki/MetaPhlAn-3.0
DIAMOND
79
https://github.com/bbuchfink/diamond
MEGAN6
80
https://www.wsi.uni-tuebingen.de/lehrstuehle/
algorithms-in-bioinformatics/software/megan6
Krona tool
81
https://github.com/marbl/Krona/wiki
BWA
82
http://bio-bwa.sourceforge.net/
DeDup tool N/A https://github.com/apeltzer/DeDup
DamageProfiler
83
https://damageprofiler.readthedocs.
io/en/latest/index.html
Schmutzi
84
https://github.com/grenaud/schmutzi
Molecular sex determination
85
https://github.com/pontussk/ry_compute
SAMtools
86
http://samtools.github.io/
HaploGrep
87
https://haplogrep.i-med.ac.at/
HUMAnN
21
https://github.com/biobakery/humann
python package scikit-bio N/A http://scikit-bio.org/
bowtie2
88
http://bowtie-bio.sourceforge.net/bowtie2/
FastQ Screen N/A https://github.com/StevenWingett/FastQ-Screen
ANGSD tool
89
https://github.com/ANGSD/angsd
MAFFT
90
https://mafft.cbrc.jp/alignment/software/
ARB software package
91
http://www.arb-home.de/
PhyML
92
https://github.com/stephaneguindon/phyml
BLASTn
93
N/A
Picard tools N/A https://broadinstitute.github.io/picard/
GATK4 N/A https://gatk.broadinstitute.org/hc
(Continued on next page)
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