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Dairying enabled Early Bronze Age Yamnaya steppe expansions


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During the Early Bronze Age, populations of the western Eurasian steppe expanded across an immense area of northern Eurasia. Combined archaeological and genetic evidence supports widespread Early Bronze Age population movements out of the Pontic–Caspian steppe that resulted in gene flow across vast distances, linking populations of Yamnaya pastoralists in Scandinavia with pastoral populations (known as the Afanasievo) far to the east in the Altai Mountains1,2 and Mongolia3. Although some models hold that this expansion was the outcome of a newly mobile pastoral economy characterized by horse traction, bulk wagon transport4–6 and regular dietary dependence on meat and milk5, hard evidence for these economic features has not been found. Here we draw on proteomic analysis of dental calculus from individuals from the western Eurasian steppe to demonstrate a major transition in dairying at the start of the Bronze Age. The rapid onset of ubiquitous dairying at a point in time when steppe populations are known to have begun dispersing offers critical insight into a key catalyst of steppe mobility. The identification of horse milk proteins also indicates horse domestication by the Early Bronze Age, which provides support for its role in steppe dispersals. Our results point to a potential epicentre for horse domestication in the Pontic–Caspian steppe by the third millennium bc, and offer strong support for the notion that the novel exploitation of secondary animal products was a key driver of the expansions of Eurasian steppe pastoralists by the Early Bronze Age. Analysis of ancient proteins suggests that Early Bronze Age dairying and horse domestication catalysed eastern Yamnaya migrations.
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Nature | Vol 598 | 28 October 2021 | 629
Dairying enabled Early Bronze Age Yamnaya
steppe expansions
Shevan Wilkin1,2 ✉, Alicia Ventresca Miller1,3, Ricardo Fernandes1,4,5, Robert Spengler1,
William T.-T. Taylor1,6, Dorcas R. Brown7, David Reich8,9,10,1 1, Douglas J. Kennett12,
Brendan J. Culleton13, Laura Kunz14, Claudia Fortes14, Aleksandra Kitova15, Pavel Kuznetsov16,
Andrey Epimakhov17,18, Victor F. Zaibert19, Alan K. Outram20, Egor Kitov21,22,
Aleksandr Khokhlov16, David Anthony7,11 & Nicole Boivin1,23,24,25 ✉
During the Early Bronze Age, populations of the western Eurasian steppe expanded
across an immense area of northern Eurasia. Combined archaeological and genetic
evidence supports widespread Early Bronze Age population movements out of the
Pontic–Caspian steppe that resulted in gene ow across vast distances, linking
populations of Yamnaya pastoralists in Scandinavia with pastoral populations (known
as the Afanasievo) far to the east in the Altai Mountains1,2 and Mongolia3. Although
some models hold that this expansion was the outcome of a newly mobile pastoral
economy characterized by horse traction, bulk wagon transport4–6 and regular dietary
dependence on meat and milk5, hard evidence for these economic features has not
been found. Here we draw on proteomic analysis of dental calculus from individuals
from the western Eurasian steppe to demonstrate a major transition in dairying at the
start of the Bronze Age. The rapid onset of ubiquitous dairying at a point in time when
steppe populations are known to have begun dispersing oers critical insight into a
key catalyst of steppe mobility. The identication of horse milk proteins also indicates
horse domestication by the Early Bronze Age, which provides support for its role in
steppe dispersals. Our results point to a potential epicentre for horse domestication
in the Pontic–Caspian steppe by the third millennium , and oer strong support for
the notion that the novel exploitation of secondary animal products was a key driver
of the expansions of Eurasian steppe pastoralists by the Early Bronze Age.
The pastoralist populations of the Eurasian steppe have long been a
source of archaeological and historical fascination. Although the later
history of steppe pastoralists—including the rise of the Xiongnu and
Mongol empires in the east—is reasonably well-established, the early
emergence and expansion of pastoralist groups in the steppe occurred
before the historical era and has largely been reconstructed on the
basis of archaeological and linguistic data
. More recently, ancient
DNA evidence has provided insights into early steppe populations,
revealing evidence for a major influx of steppe ancestry into Europe in
the Late Neolithic that effectively transformed the European genetic
. Archaeogenetic data also link these same populations
(referred to as Yamnaya) with pastoral Afanasievo populations far to the
east in the Altai Mountains1,2 and Mongolia3. Combined archaeological
and genetic evidence supports widespread population movements in
the Early Bronze Age (about 3300 to 2500 ) from the Pontic–Caspian
steppe that resulted in gene flow across vast distances, linking Yamnaya
pastoralist populations in Scandinavia with groups that expanded
into Siberia9.
Although the Yamnaya expansions are well-established, the driving
forces behind them remain unclear. A widely cited theory holds that
the early spread of herders across Eurasia was facilitated by a newly
mobile pastoral economy that was made possible by a combination
of horse traction and bulk wagon transport
. Together with regular
dietary dependence on meat and milk
, this opened up the steppe to
exploitation and occupation by pastoralist communities. Yet for all its
persuasiveness, the model remains inadequately supported by direct
Received: 4 April 2021
Accepted: 5 July 2021
Published online: 15 September 2021
Open access
Check for updates
1Department of Archaeology, Max Planck Institute for the Science of Human History, Jena, Germany. 2Institute for Evolutionary Medicine, Faculty of Medicine, University of Zürich, Zürich,
Switzerland. 3Department of Anthropology, University of Michigan, Ann Arbor, MI, USA. 4School of Archaeology, University of Oxford, Oxford, UK. 5Faculty of Arts, Masaryk University,
Brno-střed, Czech Republic. 6Department of Anthropology, University of Colorado, Museum of Natural History, Boulder, CO, USA. 7Department of Anthropology, Hartwick College, Oneonta, NY,
USA. 8Department of Genetics, Harvard Medical School, Boston, MA, USA. 9Broad Institute of Harvard and MIT, Cambridge, MA, USA. 10Howard Hughes Medical Institute, Harvard Medical
School, Boston, MA, USA. 11Department of Human Evolutionary Biology, Harvard University, Cambridge, MA, USA. 12Department of Anthropology, University of California, Santa Barbara, CA,
USA. 13Institutes of Energy and the Environment, The Pennsylvania State University, University Park, PA, USA. 14Functional Genomics Centre Zürich, University of Zürich/ETH, Zürich, Switzerland.
15Center for Egyptological Studies, Russian Academy of Sciences, Moscow, Russian Federation. 16Samara State University of Social Sciences and Education, Samara, Russian Federation. 17South
Ural State University, Chelyabinsk, Russian Federation. 18Institute of History and Archaeology, Ural Branch of the Russian Academy of Sciences, Yekaterinburg, Russian Federation. 19Institute of
Archaeology and Steppe Civilizations, Al-Farabi Kazakh National University, Almaty, Kazakhstan. 20Department of Archaeology, University of Exeter, Exeter, UK. 21Center of Human Ecology,
Institute of Ethnology and Anthropology, Russian Academy of Sciences, Moscow, Russian Federation. 22Faculty of History, Archaeology, and Ethnology, Al-Farabi Kazakh National University,
Almaty, Kazakhstan. 23School of Social Science, The University of Queensland, Brisbane, Queensland, Australia. 24Department of Anthropology and Archaeology, University of Calgary, Calgary,
Alberta, Canada. 25Department of Anthropology, National Museum of Natural History, Smithsonian Institution, Washington, DC, USA. e-mail:;
Content courtesy of Springer Nature, terms of use apply. Rights reserved
630 | Nature | Vol 598 | 28 October 2021
archaeological or biomolecular data. Archaeological evidence for
the use of bulk wagon transport by the Eneolithic Maikop and Early
Bronze Age Yamnaya groups exists in the form of carts and bridling
materials10, but two other critical components of the model—a reliance
on domesticated horses and ruminant dairying—remain archaeologi-
cally unproven.
The domestication status of Eurasian horses has long been
, and recent archaeogenetic findings
have shifted our
understanding of early horses at the Eneolithic site of Botai in north-
ern Kazakhstan by identifying them as Equus przewalskii rather than
the modern-day domestic horse (Equus caballus)
. Although horses
do appear in Early Bronze Age assemblages on the steppe, it remains
unclear whether they were being ridden
, or indeed whether they
were part of pastoral herds or simply hunted. On the eastern Eurasian
steppe, growing evidence suggests that horses were not ridden
or milked19 before about 1200, and horses may have been uncom-
mon in early pastoralist assemblages
. Early ruminant dairying on the
western steppe has also been inadequately demonstrated, as human
stable isotope data from the region suggests—but cannot confirm—
dairy consumption
. Palaeoproteomics, which is the only method
that is able to evince individual dairy consumption (rather than milk
production) and provide taxonomic resolution, has so far been mini-
mally applied to steppe populations. Across Yamnaya and Afanasievo
populations, dairying evidence is available only for a few individuals
from the eastern steppe who have ancestry from western steppe groups;
the earliest individual provides only a taxonomically ambiguous rumi-
nant (Ovis/Bos)peptide result19.
To address the heavily debated question of what drove Yamnaya
expansions across the steppe6,2325, we conducted proteomic analysis
of dental calculus sampled from 56steppe individuals who span the
Eneolithic to Late Bronze Age, and who date from between 4600 and
1700. Our samples from the Eneolithic (about 4600 to 3300) are
from 19individuals from 5sites: Murzikha2 (6 individuals), Khvalynsk1
and Khvalynsk2 (9individuals), Ekaterinovka Mys (1individual), Leb-
yazhinka5 (1individual) and Khlopkov Bugor (2individuals) (Fig.1,
Supplementary Fig.1a). Ancient DNA results from Khvalynsk and other
Eneolithic sites in the Volga and northern Caucasus
support the
existence of an Eneolithic population across this region that was geneti-
cally similar to the Yamnaya population, but who lacked the additional
farmer (Anatolian) ancestry that would arrive later on the steppe
. Pub-
lished stable isotope and archaeological studies applied to Eneolithic
populations from the Pontic region point to an economy based on
fishing, the gathering of local plants and the keeping of domesticated
.Given the importance of the horse in reconstructions of
early pastoralist expansions, we also examined dental calculus from two
individuals from the well-known site of Botai. With faunal assemblages
dominated by horse remains
and early lipid studies of ceramics
indicating horse milking at the site by 3500
, the site is central to
discussions of early horse milking and dairying in the Eurasian steppe.
Our Bronze Age samples come from 35individuals from 20sites in the
Volga–Ural steppes that can be divided into two chronological groups:
the Early Bronze Age (about 3300 to 2500) era of Yamnaya-culture
mobile pastoralism
; and the Middle–Late Bronze Age transition
(about 2500–1700), when chariots, fortified settlements and
new western-derived influence genetic ancestries appeared with the
Sintashta culture
. The cemetery sites and the number of individu-
als (in parentheses) from the Early Bronze Age are: Krasikovskyi1 (2)
Krasnokholm3 (1), Krivyanskiy9 (2), Kutuluk1 (2), Leshchevskoe1 (1),
Lopatino1 (1), Mustayevo5 (2), Nizhnaya Pavlovka (1), Panitskoe (1),
Podlesnoe (1), Pyatiletka (1) and Trudovoy (1); and, from the Middle–
Late Bronze Age transition, Bolshekaraganskyi (1), Kalinovsky1 (2),
Kamennyi Ambar5 (3), KrasikovskyiI (1), Krivyanskiy9 (3), Lopatino1
and Lopatino2 (2), Potapovka1 (1), Shumayevo2 (1) and Utevka6 (5)
(Supplementary Fig.1b, c). Archaeological and stable isotope find
ings6,22 indicate that the diet of Early Bronze Age Yamnaya groups was
focused on herd animals, specifically cattle, sheep and goat. Horse
remains also appear in quantity on a few steppe archaeological sites,
but the status of Early Bronze Age horses—whether domesticated or
hunted—has remained unclear
. The Middle–Late Bronze Age transi-
tion saw a shift to greater horse exploitation and chariot use, within
the context of an ongoing dietary focus on domesticated livestock.
Of the 56ancient human dental calculus samples we tested, 55 were
successfully extracted and produced identifiable protein data. Of these
55, 48 (87%) were determined to have strong signals for preservation
through an assessment of proteins commonly found within the oral
cavity; detailed information on this assessment is provided in Methods,
Supplementary Table3.
The earliest samples in our study (about 4600 to 4000) are from
5Eneolithic sites in southwestern Russia located on or close to the
Volga River and its tributaries. Of the samples from these 19individu-
als, 11 were successfully extracted and well-preserved, and 10of these
did not show any evidence for dairy consumption (Figs.1a, 2a). The
calculus of one individual contained two peptides specific to bovine
(Bos, Bubalis and Bison) α-S1-casein, a milk curd protein. However, as
the only dietary peptides contained in this sample were specific to
casein and evidence for the most commonly recovered dairy protein
β-lactoglobulin (BLG) was lacking, dairy consumption in this individual
could not be confidently confirmed. In general, casein peptides appear
to preserve more poorly than BLG in archaeological calculus, and thus
are most often identified together with other dairy protein peptides
rather than alone
. Additionally, within the two identified casein
peptides, there is only one possible amino acid deamidation site, which
renders any estimation of the antiquity of these peptides exceedingly
challenging. A previously published paper
demonstrates the extreme
variability in deamidation of amino acids in milk proteins, which fur-
ther limits our ability to confirm the authenticity of this dairy finding.
The calculus from the two additional Botai individuals demonstrated
adequate preservation, but also lacked evidence for dairy consumption.
For the Early Bronze Age individuals (dating to the onset of the Yam-
naya cultural horizon), dairy peptides were recovered from 15 of the
16individual calculus samples we analysed (Fig.1b, 2b). All 15indi-
viduals with positive dairy results contained multiple peptide spec-
tral matches to ruminant dairy proteins (including BLG), and some
individuals also contained α-S1 casein, α-S2-casein or both. Although
many of the milk peptides were only specific to higher taxonomic levels
(such as Pecora, an infraorder within Artiodactyla (cow, sheep, goat,
buffalo, yak, reindeer, deer and antelope)), others enabled more spe-
cific taxonomic classifications, including to family, genus or species.
We found Ovis, Capra and Bos attributions, and the calculus of many
individuals contained dairy peptides from several species. Notably,
we identified Equus milk peptides from the protein BLGI in 2 of 17Early
Bronze Age individuals, both from the southwestern site of Krivyan-
skiy9 (3305 to 2633 calibrated years  (Supplementary Table5 pro-
vides individual accelerator mass spectrometry dating information)).
Although the genus Equus includes horse, donkey and kiang, only horse
species (E.caballus, E.przewalskii, Equus hemionus and Equus ferus)
are archaeologically attested in the steppe in the Early Bronze Age,
supporting the Equus identification as horse.
For the Middle–Late Bronze Age transition, calculus samples from 15
of 19 individuals were positive for evidence of ruminant milk consump-
tion (Figs.1c, 2c). Similar to the Early Bronze Age, we identified BLG,
α-S1-casein and α-S2-casein, as well as the whey protein α-lactalbumin.
Taxonomic identifications again ranged from the Pecora infraorder to
genus-level identifications (including Ovis and Bos), but without any
specific identifications for Capra or Equus. Supplementary Table4 pro-
vides a full accounting of all identified dairy proteins for each individual.
Overall, our results point to a clear and marked shift in milk con-
sumption patterns between the Eneolithic and Early Bronze Age in the
Pontic–Caspian Steppe. The majority of Eneolithic individuals (10 out
of 11 (92%)) in our assemblage lack any evidence for milk consumption,
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Nature | Vol 598 | 28 October 2021 | 631
Utevka 6
Kriviyanskiy IX
Kalinovka 2/2
Kammenyi Abmar
Lopatino II 1/1
Kriviyanskiy IX
Lopatino I1/1
Nizhnaya Pavlovka 1/1
Kutuluk I 2/2 Krasikovskyi 1/1
Pyatileka 1/1
Trudovoy1/1Krasnokholm III 1/1
Mustayevo V 1/2
Leshchevskoe 4/5
Kholpkovsky Bugor
Khavalynsk 1 and 2
Murzikha 20/2
Eneolithic sites People with dairy/total per site Early Bronze Age sites Middle/Late Bronze Age sites
Fig. 1 | Map sh owing sites t hat yielded i ndividual s with prese rved ancien t
proteins. ac, Eneolithic (a), Early Bron ze Age (b) and Middle–L ate Bronze
Age (c) sites in the Pont ic–Caspian re gion, showing t he number of indiv iduals
with a posi tive dairy ident ificatio n out of the total nu mber of individ uals with
preser ved ancient prote ins for each site. S trong evidenc e of preservat ion of
equine or rum inant milk protein i dentifie rs are depicte d with black anim al
icons; the sin gle individual w ith equivocal ly identifi ed caseinpept ides is shown
with a grey ic on. For a map of all sites (in cluding those w ithout prese rved
proteins), see Supp lementar y Fig.1. Base maps w ere created usin g QGIS 3.1 2
(, and use Natural Ear th vector map d ata from ht tps: // The horseimage is repro duced from
ref. 33; shee p silhouette, pu blic domain (
Content courtesy of Springer Nature, terms of use apply. Rights reserved
632 | Nature | Vol 598 | 28 October 2021
whereas the overwhelming majority of Early Bronze Age individuals
(15 out of 16 (94%)) contain ample proteomic evidence for dairy con-
sumption in their calculus. Although a single individual at Eneolithic
Khvalynsk with somewhat equivocal evidence for the consumption
of dairy from cattle may indicate small-scale dairy use, the reliability
of this single identification is questionable. Our findings suggest that
regular dairy consumption in the Pontic–Caspian Steppe began only
at the time of the Eneolithic-to-Early Bronze Age transition. Although
neighbouring Eneolithic farming populations in Europe appear to have
been dairying39, those living across the steppe frontier did not adopt
milking practices, which suggests the presence of a cultural frontier.
The proteomic data are in broad agreement with findings from lipid
analyses in the Ukraine (Supplementary Information section 2, Sup-
plementary Table2). They also agree with stable isotope analysis of
individuals from Eneolithic-to-Bronze-Age Samara showing a corre-
sponding shift from a heavy reliance on fish, deer and other riverine
forest (C3) resources to a greater reliance on terrestrial and grassland
(C3 and C4) animal products22,40.
One important advantage of proteomic data is their ability, in some
cases, to provide species-specific protein identifications. Our study
offers evidence for the Bronze Age milking of sheep, goat and cattle,
which fits with evidence for the herding of these animals. The lush
valleys of the Pontic–Caspian Steppe provided ample forage and hydra-
tion for mixed herds of arid-adapted sheep and goat, as well as more
water-reliant cattle
. Although a recent study has shown that lactase
persistence—which results from the presence of an allele that enables
production of lactase into adulthood—was rare in steppe populations
of the Early Bronze Age43, we find that the western steppe community
was regularly consuming dairy that could have included fresh milk and/
or other processed products with reduced lactose, such as yogurts,
cheeses or fermented milk beverages.
Our study of dental calculus from the Eneolithic site of Botai to the
east, where early horse milking has been suggested by lipid analysis
(albeit equivocally
), did not yield milk proteins. Although two samples
are insufficient for drawing broad conclusions, this finding does not sup-
port widespread milk consumption at the site13,45,46. However, two cal-
culus samples from Early Bronze Age individuals of the Pontic–Caspian
region do provide evidence for the consumption of horse milk. Com-
bined with archaeogenetic evidence15 that places the Botai horses
on a different evolutionary trajectory than the domesticated DOM2
E.caballus lineage, this finding—if backed up by further sampling and
analysis—would seem to firmly shift the focus of sustained early horse
domestication on the Eurasian steppe to the Pontic–Caspian region.
So far, the oldest horse specimens that carry the DOM2 lineage date
to between 2074 to 1625 calibrated years , at which time the line-
age is archaeologically attested in present-day Russia, Romania and
. Our identification of—to our knowledge—the earliest horse
milk proteins yet identified on the steppe or anywhere else reveals the
presence of domestic horses in the western steppe by the Early Bronze
Age, which suggests that the region (where the first evidence for horse
chariots later emerged at about 2000 
) may have been the initial
epicentre for domestication of the DOM2 lineage during the late fourth
or third millennium .
Overall, our findings offer strong support to the notion of a second-
ary products revolution
in the Eurasian steppe by the Early Bronze
Age. This change in subsistence economy, indicated by dietary stable
isotopes in human bones as well as by proteomics, was accompanied by
the widespread abandonment of Eneolithic riverine settlement sites,
the appearance of kurgan cemeteries in the previously unexploited arid
plateaus between the river valleys, and the inclusion of wheeled vehi-
cles and occasional horse bones in Yamnaya graves. At the same time,
the steppe Yamnaya population expanded westward into Europe and
eastward to the Altai Mountains (a range of 6,000km)
. Although we
cannot offer direct insight into the question of horse riding or traction
on the basis of our data, evidence for milked horses certainly makes
horse domestication more likely, and may indicate that horses had a role
in the spread of Yamnaya groups. The triad of animal traction, dairying
and horse domestication appears to have had an instrumental role in
transforming Pontic–Caspian economies and opening up the broader
steppe to human habitation by the Early Bronze Age. If some or even all
of these elements were present before the Bronze Age, it is only from
this latter period that we witness their intensive and sustained exploita-
tion amongst numerous groups. Although other factors will no doubt
also have been important, the emergence of more mobile, pastoralist
societies adapted to survival on the cold and arid steppe—where horses
may have opened up snow-covered pasturage for other animals
, and
milk would have provided a sustained source of protein, nutrients
and fluids—was undoubtedly critical to the expansion of Bronze Age
pastoralists such the Yamnaya groups.
Online content
Any methods, additional references, Nature Research reporting sum-
maries, source data, extended data, supplementary information,
KB N-7
KB N-8
KHA1 N-39
KHA1 N-61/62
KHA2 N-1
KHA2 N-2
KHA2 N-4
MUR2 K-1 N-130
MUR2 N-130.1
MUR2 N-94/7
MUR2 N-98
PSM count
PSM count
KRA3 K.1 N-1
KRI9 K.2 N-2
KRI9 K.4 N-21
KRS K.1 N-1
KRS K.2 N-1
KUT1 K.3 N-4
KUT1 K.4 N-1
LES K.1 N-1
LOP1 K.3p N-1
MUS5 K.1 N-1
MUS5 NK.8 N-2
NP K.2 N-3
POD K.1 N-3
PYA K.6 N-2
TRU K.5 N-1
PSM count
BKK K.25 Pit-24
KAL K.1 N-4
KAL1 K.1 N-6
KB5 K.2 A 987
KB5 K.2 MR11 CK-1 A944
KB5 K.4 MR5 CK-2 A 937
KRI9 K.1 N-30
KRI9 K.5 N-6
KRS K.3 N-1
LOP K.3 N-2
LOP2 K.1 N-1 K.2
POT K.5 N-3
SHU2 K.6 N-1
UTE6 K.4 N-1
UTE6 K.4 N-5
UTE6 K.4 N-6
UTE6 K.6 N-2 K-1
UTE6 K.6 N-6
Fig. 2 | Hist ogram of taxon omic speci ficity o f dairy pep tide spec tral matche s per indivi dual. ac, Histogra ms for individual s with evidenc e for consumption
of dairy, from the En eolithic (a), Early Bronze Ag e (b) and Middle and Lat e Bronze Age (c). PSM, pept ide spectra l match.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Nature | Vol 598 | 28 October 2021 | 633
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No statistical methods were used to predetermine sample size. The
experiments were not randomized, and investigators were not blinded
to allocation during experiments and outcome assessment.
Protein extraction and data analysis methods
Sample collection. Dental calculus was collected at the Department of
Archaeology at Samara State University and the Museum at the Institute
of Plant and Animal Ecology at the Ural Branch of the Russian Academy
of Sciences. Calculus was collected in sterile tubes and hand-carried to
the Max Planck Institute for the Science of Human History (MPI-SHH)
in Jena. Calculus from the Botai site was sampled at the site; it was col-
lected in sterile tubes and shipped to MPI-SHH. Each calculus sample
was removed using a clean dental scaler, and implements were cleaned
with alcohol swabs between the sampling of different individuals. Con-
tamination from modern human keratin and environmental collagen
that may have occurred owing to previous sampling for ancient DNA
or stable isotope analysis was reduced through the use of nitrile gloves
during collection, and samples were taken directly from the teeth into
clean, 2-ml Eppendorf tubes, in which they were stored until protein
extraction in the Palaeoproteomics Laboratory at the MPI-SHH.
Protein extraction. For samples with a ‘Z’ designation, proteins were
extracted using a modified low-volume filter-aided sample prepara-
tion that has previously been described
. To decrease contamination,
1 ml of 0.5 M EDTA was then added to each sample tube, samples were
rotated for 5 min followed by centrifugation at 20,000 rcf for 10 min
to remove the any contamination on the outer layer of calculus; the
supernatant was then removed and retained. Then, 1 ml of 0.5 M EDTA
was added to each decontaminated sample, and the sample was al-
lowed to decalcify on a rotator for 5–7 days at room temperature until
completion. After demineralization, samples were centrifuged at top
speed (20,000 rcf) for 5 min. Eight hundred µl of EDTA supernatant
was removed andstored for future analysis. Separately, 50 µl of urea
solution (8 M) was added to a 30-kDa Millipore Microcon filter unit.
Samples were denatured, reduced and alkylated with 30 µl of sodium
dodecyl sulfate (SDS)-lysis buffer (4% w/v SDS, 100 mM Tris/HCL pH 8.2
and 0.1 M DTT), incubated at 95°C for 5 min, and 100 µl of iodoaceta-
mide (IAA) solution (0.5 M IAA and 8 M UA) was added to the filter units
and was mixed at 600 rpm for 1 min in the dark. Following incubation,
the samples were centrifuged at 14,000g for 10 min. Two hundred µl
of UA (8 M) was added to the filter unit, followed by the lysed sample
supernatant and units centrifuged for 20 min at 14,000g. UA was used
twice to remove the IAA and followed by centrifugation for 12–15 min
at top speed. One hundred µl of 0.5 M NaCl was added to each filter
unit, spun for 12 min at 14,000g. This step was repeated and the flow-
through discarded. The filter units were transferred to new units, and
120 µl of trypsin solution (3 µl of 0.4 µg µl−1 trypsin in 117 µl of 0.05 M
triethylammoniumbicarbonate) was added to each unit. Units were
thermomixed at 600 rpm for 1 min and then incubated overnight at
37°C. Following digestion, samples centrifuged for 20 min at 14,000g
and acidified with 5% TFA to a pH of <2.
Stage tips (Thermo Scientific StageTips 200 µl C18 tips) were cleaned
with 100% methanol, followed by 60% acetonitrile (ACN) solution (60%
ACN, 0.1% TFA and 39.9% ddH
O). Then, each was equilibrated with
2 washes of 150 µl of 3% ACN solution (3% ACN, 0.1% TFA and 96.9%
O). Samples were then loaded onto the tips and twice washedwith
3% ACN and 0.1% TFA and the flowthrough discarded. Peptides were col-
lected in new tubes from the stage tip with 150 µl 60% ACN solution and
each was dried in an evaporator and stored at −80°C until liquid chro-
matography with tandem mass spectrometry (LC–MS/MS) analysis.
Samples with a ‘DA’ were extracted using a single pot, solid phase
enhanced sample preparation (SP3) modified for archaeological den-
tal calculus samples. One millilitre of 0.5 M EDTA was added to each
sample, and samples were then placed on a rotator for 5 min and cen-
trifuged at top speed (20,000 rcf) for 10 min. The entire supernatant
was removed and retained, and an additional 500 µl of 0.5 M EDTA for
demineralization was added and samples were placed back onto the
rotator for 5–7 days.
Following demineralization, samples were centrifuged at 20,000 rcf
for 10 min, and 400 µl of the supernatant was removed and retained.
To increase denaturation, reduce and alkylate, 200 µl of 6 M guanidine
hydrochloride and 30 µl of 40 mM CAA, 100 mM TCEP were added
to the pellet and remaining supernatant and mixed through resus-
pension. Samples were then placed on a heating block (Cell Media,
Thermoshaker Pro) and heated to 99°C for 10 min. Upon removing
samples from heat, 20 µl of a20 µg µl−1 50/50 mixture of hydrophilic
and hydrophobic SeraMag SpeedBeads was added to each sample, and
to increase protein–bead adhesion, 350 µl of 100% ethanol was then
added to each tube. Samples were then placed on the ThermoMixer
for 5 min at 1,000 rpm at 24°C. Upon removal from the ThermoMixer,
tubes were placed on a magnetic rack, which moved the beads to the
wall side of the tube. With the proteins now adhering to the beads,
the entire supernatant was removed and retained for possible later
analysis. To remove any non-proteinaceous materials, 3 washes of
200 µl 80% ethanol were carried out. Once the beads were thoroughly
washed, 100 µl of 100 mM ammonium bicarbonate was added to each
tube, as well as 0.2 µg of trypsin. Samples were then placed on the Ther-
moMixer at 37°C at 750 rpm. After 10 min, samples were resuspended
and left on the ThermoMixer overnight (18 h) for protein digestion.
Following digestion, sample tubes were centrifuged at 20,000 rcf for
1 min, and then placed back onto the magnetic rack. The entire super-
natant was removed and transferred to a clean tube. Each sample was
then acidified with 5% TFA to reduce the pH to <2. Acidified sample
tubes were again centrifuged at top speed for five minutes to push
any remaining non-proteinaceous materials into a pellet and improve
stage tip clean up. Stage tips were prepared with 150 µl MeOH, and
centrifuged at 2,000 rcf, followed by 60% ACN, 0.1% TFA and another
round of centrifugation. To equilibrate the stage tips, we added
150 µl 3% ACN, 0.1% TFA, followed by another centrifugation step, and
these steps were then repeated. Samples were added to each stage
tip, and centrifuged for 3 min at 2000 rcf, or until the entire sample
had passed the stage tip. This was followed by an additional 2 rinse
steps with 3% ACN, 0.1% TFA . Samples were not eluted at the MPI-SHH,
but retained on stage tips in the −20°C freezer until shipment to the
Functional Genomics Center Zürich at the University of Zürich. A full
detailed protocol is available at (
High performance LC–MS/MS analysis. The samples were sent on
stage tips to the Functional Genomics Center. There, the peptides were
eluted from the stage tips and dried. After resolubilization in 10 µl of
3% ACN, 0.1% formic acid, the peptide level was normalized using the
DeNovix DS-11 Series Spectrophotometer.
LC–MS/MS analysis. For samples with a laboratory identifier that
starts with Z (Supplementary Table3), mass spectrometry analysis was
performed on a Q Exactive HF mass spectrometer (Thermo Scientific)
equipped with a Digital PicoView source (New Objective) and coupled
to a M-Class UPLC (Waters). Solvent composition at the two channels
was 0.1% formic acid for channel A and 0.1% formic acid, 99.9% ACN for
channel B. Column temperature was 50°C. For each sample, 4 µl of pep-
tides were loaded on a commercial ACQUITY UPLC M-Class Symmetry
C18 Trap column (100 Å, 5 µm, 180 µm × 20 mm, Waters) followed by
ACQUITY UPLC M-Class HSS T3 column (100 Å, 1.8 µm, 75 µm × 250 mm,
Waters). The peptides were eluted at a flow rate of 300 nl min
by a
gradient from 5 to 40% B in 62 min. Column was cleaned after the run by
increasing to 98% B and holding 98% B for 5 min before re-establishing
the loading condition. Samples were acquired in a given order.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
The mass spectrometer was operated in data-dependent mode,
acquiring full-scan mass spectra (350−1,500 m/z) at a resolution of
120,000 at 200 m/z after accumulation to a target value of 3,000,000,
and a maximum injection time of 50 ms, followed by higher-energy col-
lision dissociation (HCD) fragmentation on the six most intense signals
per cycle. HCD spectra were acquired at a resolution of 120,000 using a
normalized collision energy of 28 and a maximum injection time of 247
ms. The automatic gain control was set to 100,000 ions. Charge state
screening was enabled. Singly, unassigned and charge states higher
than six were rejected. Only precursors with intensity above 18,000
were selected for MS/MS. Precursor masses previously selected for MS/
MS measurement were excluded from further selection for 30 s, and
the exclusion window was set at 10 ppm. The samples were acquired
using internal lock mass calibration on m/z 371.1012 and 445.1200.
For samples with laboratory identifiers starting with DA (Supple-
mentary Table3), mass spectrometry analysis was performed on a Q
Exactive mass spectrometer (Thermo Scientific) equipped with a Digital
PicoView source (New Objective) and coupled to a nanoAcquity UPLC
(Waters). Solvent composition at the two channels was 0.1% formic acid
for channel A and 0.1% formic acid, 99.9% ACN for channel B. Column
temperature was 50°C. For each sample, 4 µl of peptides were loaded
on a commercial ACQUITY UPLC M-Class Symmetry C18 Trap column
(100 Å, 5 µm, 180 µm × 20 mm, Waters) followed by ACQUITY UPLC
M-Class HSS T3 column (100 Å, 1.8 µm, 75 µm × 250 mm, Waters). The
peptides were eluted at a flow rate of 300 nl min
by a gradient from
8 to 22% B in 49 min and to 32% B in additional 11 min. Column was
cleaned after the run by increasing to 95% B and holding 95% B for 5 min
before re-establishing the loading condition. Samples were acquired
in a given order.
The mass spectrometer was operated in data-dependent mode,
acquiring a full-scan mass spectra (300−1,700 m/z) at a resolution of
70,000 at 200 m/z after accumulation to a target value of 3,000,000,
and a maximum injection time of 110 ms followed by HCD fragmenta-
tion on the 12 most intense signals per cycle. HCD spectra were acquired
at a resolution of 35,000 using a normalized collision energy of 25 and
a maximum injection time of 110 ms. The automatic gain control was
set to 50,000 ions. Charge state screening was enabled. Singly, unas-
signed and charge states higher than seven were rejected. Only precur-
sors with intensity above 9,100 were selected for MS/MS. Precursor
masses previously selected for MS/MS measurement were excluded
from further selection for 30 s, and the exclusion window was set at 10
ppm. The samples were acquired using internal lock mass calibration
on m/z 371.1012 and 445.1200.
As all samples in our study were digested with trypsin, peptides
had either an arginine or lysine at the C terminus. This resulted in the
C-terminal fragments remaining charged, and therefore identified at
a higher intensity than b-ions (Extended Data Fig.1). The mass spec-
trometry proteomics data were handled using the local laboratory
information management system
and all relevant data have been
deposited to the ProteomeXchange Consortium via the PRIDE (http:// partner repository.
Data analysis. To account for as much variation of milk-associated
proteins as possible during MS/MS ion searches, a supplementary
database of milk protein sequences that had not been reviewed was
curated from UniProtKB in addition to those from ancient horses, as
previously generated
. As a previous publication
, peak lists were gen-
erated from raw files by selecting the top 100 peaks using MSConvert
from the ProteoWizard software package version 3.0.11781
. Sample
analysis results were searched using Mascot
(version 2.6.0) against
the Swiss-Prot database in combination with a curated milk protein
database19. Results were exported from Mascot as .csv files, and further
processed through an internally created tool, MS-MARGE
, to esti
mate the validity of peptide identifications and summarize the findings.
False-discovery rates at both the peptide spectral match and protein
level were calculated using MS-MARGE by counting the number of de-
coy hits after filtering for e-value and minimum peptide support, then
dividing this value by the number of target hits minus the number of
decoys. The resulting value is multiplied by 100 to provide an estimate
of the false-discovery rate. For each individual sample, we aimed for a
protein false-discovery rate of under 5% and a peptide false-discovery
rate of under 2% (Supplementary Table4). A minimum of two individual
peptide spectral matches were required for each specific protein iden-
tification, and only peptide spectral matches with an evalue of below
0.01 were accepted. After filtering criteria were applied, we observed a
range of variation in the numbers of proteins identified, with samples
ranging from 25 to 196 confidently identified protein families.
Sequence similarities between casein and Jeotgalicoccus. During
necessary BLAST searches to authenticate the taxonomic specificity of
ruminant α-S1 casein peptides, we found identical sequence matches to
theoretical proteins for the numerous bacterial firmicute species from
the genus Jeotgalicoccus (NCBI reference sequence: WP_188349304.1).
Upon further investigation, the full amino acid sequence for these
hypothetical bacterial proteins is almost identical to ruminant casein
sequences, which is probably due to laboratory contamination dur
ing the genomic sequencing. As its listing in the NCBI database is not
associated with a publication, we assume this is probably contamina-
tion. Supplementary Figure2 shows the alignment comparing the α-S1
casein sequence for Bos taurus, Bos grunniens, Bubalus bubalis and
Jeotgalicoccus species.
Proteome preservation assessment with the Oral Signature Screen-
ing Database. To confirm the preservation of thecalculus fromindi-
viduals included in this study, the metaproteome from each sample
was examined for a combination of specific protein types. Following a
previous publication6 we compared the data from each sample against
the Oral Signature Screening Database (OSSD) to determine the number
of common laboratory contaminants, contaminants introduced during
handling and curation, regularly recovered human immune proteins
found in the oral cavity, and bacterial proteins common to the human
oral microbiome. Supplementary Table3 contains the overall count
of OSSD proteins pulled from our filtered results, as well as the result
of the oral microbiome protein identifiers + human immune proteins
divided by the total number of OSSD proteins multiplied by 100 to find
the ‘authenticity’ of oral signature proteins in comparison to the total
proteins recovered. To determine who among the individuals passed our
screening, we applied a different threshold to each time period. For the
Early and Middle Bronze Age, we applied a previously published stand-
, and for the Eneolithic period samples we lowered the standard to
40% to take into account increased protein degradation over time. Indi-
viduals who had calculus that fell below the authenticity threshold were
excluded from the study, but remain listed on the preservation table.
Sample authenticity is further supported by an absence of dietary pro-
teins in all positive (archaeological sheep bone with known proteome)
and negative controls (extraction blank), as well as the fact that none
of the control samples showed any evidence of a typical oral protein
signature. Protein preservation varies greatly between different envi-
ronments and can even differ between individuals at the same site37,55,
and this assessment should be conducted on a project-by-project basis.
Bayesian estimates of dietary contributions from freshwater protein
and radiocarbon calibration adjusted for freshwater dietary radio-
carbon reservoir effects for Eneolithic individuals. Chronologies
based on human radiocarbon dates require estimates of individual
aquatic dietary intakes, as well as estimates of aquatic radiocarbon
reservoir effects of consumed aquatic protein
. For the latter, we con-
sidered a wide potential variability of between 0 and 1,000 years, which
covers previously reported archaeological measurements of coeval
terrestrial and aquatic samples and the majority of measurements made
Content courtesy of Springer Nature, terms of use apply. Rights reserved
on modern freshwater species from our study region
. To estimate
the dietary contributions from aquatic protein we used the Bayesian
mixing model ReSources developed within the Pandora & IsoMemo
initiatives ( ReSources is a R-based model
that follows a similar implementation to the Bayesian mixing model
. We defined a two-end member model (terrestrial versus fresh-
water animal protein) with stable nitrogen reference values for these
=7.1±2‰, δ
=10.6±1‰) calculated following a
literature review of previously reported values for bone collagen ex-
tracted from terrestrial and freshwater animal species within the study
region26,60. As with previous similar models, protein reference values
are corrected for offsets between bone collagen and edible meat, and
the implemented model also included a dietary to consumer isotopic
offset56,57. For each human bone collagen δ15N value, ReSources pro-
vided an estimate (expressed as a mean and s.d.) of the dietary intake
of freshwater protein. This estimate was included within the Bayes-
ian chronological model OxCal v.4.4 to express the degree of mixing
between the terrestrial radiocarbon calibration curve IntCal20 and a
freshwater radiocarbon curve
. The latter was defined from IntCal20
by adding a uniform prior of between 0 and 1,000 years. Calibrated
radiocarbon dates for each individual are expressed as 95% credible
intervals. An example of the OxCal code is given below.
Delta_R(“LocalFRE”, U(0,1000));
Mix_Curves(“Date1”, “IntCal20”,”LocalFRE”, 63,26);
R_Date(“OxA-35976, 5965, 20);
Mix_Curves(“Date2”, “IntCal20”,”LocalFRE”, 36,22);
R_Date(“OxA-37350”, 4390, 20);
Radiocarbon sample preparation methods
Bone sample preparation methods for radiocarbon data followed pre-
viously described methods63. In brief, the outer bone surfaces were
removed manually and all samples were soaked in successive washes
of methanol, acetone and dichloromethane for 30min each at room
temperature to remove adhesives and consolidants, and rinsed in
>18.2MΩ cm
water. Bones were demineralized in 0.5N HCl for 24-36h
at 5°C, and then gelatinized in 0.01N HCl for 12h at 60°C. On the basis
of crude gelatin yield and quality, the gelatin was either ultrafiltered
(30-kDa MWCO), or hydrolysed for XAD purification. Resulting material
was then combusted under vacuum in sealed quartz tubes with CuO
and Ag wire, and the resulting CO2 was converted to graphite using H2
reduction over an iron catalyst. Radiocarbon content was measured
on a 500-kV NEC 1.5SDH-1 compact accelerator, and conventional ages
were calculated by normalizing to OXII oxalic acid standards and cor-
recting for fractionation using the δ13C ratio measure on the AMS64.
Reporting summary
Further information on research design is available in theNature
Research Reporting Summary linked to this paper.
Data availability
All raw, peak and result protein data have been uploaded to ProteomEx-
change ( Files are available under
the project accession: PXD022300, and the project DOI is https://doi.
org/10.6019/PXD022300. S.W. can also be contacted at shevan.wilkin@
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Acknowledgements We thank the Max Planck Society for providing the funding for this
project. D.R. is an Investigator of the Howard Hughes Medical Institute. A.E. was supported by
a grant from the Russian Science Federation, grant number 20-18-00402. We thank A. Dittman
for insights on protein mass spectra.
Author contributions S.W. and N.B. designed the study; S.W., A. Khokhlov, E.K., V.F.Z., A.K.O.,
D.R.B., P.K., A. Kitovaand A.E. participated in sample collection; S.W. conducted the protein
extractions and analysed the data; L.K. and C.F. ran the samples on the LC–MS/MS; D.R., R.F.,
B.J.C., D.K., A. Khokhlov and E.K. provided radiocarbon dates; S.W. and N.B. wrote the draft
with the help of A.V.M., W.T.-T.T., R.S., D.A., E.K. and A. Khokhlov, and with input from all other
Funding Open access funding provided by Max Planck Society.
Competing interests The authors declare no competing interests.
Additional information
Supplementary information The online version contains supplementary material available at
Correspondence and requests for materials should be addressed to Shevan Wilkin or Nicole
Peer review information Nature thanks the anonymous, reviewer(s) for their contribution to
the peer review of this work. Peer reviewer reports are available.
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Extende d Data Fig. 1 | MS 2 spectra for d airy protei ns. a, BLG peptide s pecific
to Ovis or Bovinae for DA420. b, BLG I pe ptide specif ic to Equus for DA420.
c, Equus BLG I pe ptide for Z438. d, MS2 spe ctra for a Capra-specif ic BLG peptid e
for Z438. e, α-S1 casein f rom DA430 specif ic to Bovinae. f, Se cond α-S1 casein
peptide sp ecific to B ovidae, also from DA43 0. Horse, goat and cow i mages
are reproduc ed from ref. 37; sheep silho uette, publi c domain
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nature research | reporting summary October 2018
Corresponding author(s):
Shevan Wilkin
Nicole Boivin
Last updated by author(s): Jun 14, 2021
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Data collection Mass spectrometry analysis was performed on a Q Exactive HF mass spectrometer (Thermo Scientific) equipped with a Digital PicoView
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Sample size Sample size was determined by the number of archaeological individuals that had accumulated dental calculus available for collection.
Data exclusions Dental calculus samples that did not pass the preservation threshold were excluded from further analysis, however, these samples are still
listed and their preservation score is provided in Supplementary Table S3.
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Specimen provenance Dental calculus samples were collected from Samara State University Department of Archaeology Collection Saplesand the
scientific collections of the Museum at the Institute of Plant and Animal Ecology (Ural Branch of the Russian Academy of
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Dating methods AMS radiocarbon dating was conducted to dates some individuals. Bone sample preparation methods for radiocarbon data
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... Many of the dairy peptides matched both to a milk protein reference and a Jeotgalicoccus sequence (a facultatively anaerobic, and halotolerant group, first reported in fermented seafood). Wilkin et al. [49] recently noted that the Jeotgalicoccus sequence WP_188349304.1 is likely to have high levels of milk contamination from laboratory analysis; as such, Jeotgalicoccus was not considered a valid result in taxonomic attribution. While deamidation patterns have been used to investigate the preservation quality of ancient dietary proteins [50], recently the high variability of deamidation patterns in dairy proteins has been demonstrated, rendering their application to samples with low peptide recovery challenging [51]. ...
Full-text available
The detection of dairy processing is pivotal to our understanding of ancient subsistence strategies. This culinary process is linked to key arguments surrounding the evolution of lactase persistence in prehistory. Despite extensive evidence indicating the presence of dairy products in ceramics in the European Neolithic, questions remain about the nature and extent of milk (and lactose) processing and consumption. In order to investigate past patterns of dairy processing, here we analyse ancient proteins identified from Late Neolithic Funnel Beaker ceramics, scrutinizing the principle that curd and whey proteins partition during the production of dairy foods from milk. Our results indicate the presence of casein-rich dairy products in these vessels suggesting the creation of curd-enriched products from raw milk. Moreover, this analysis reveals the use of multiple species for their dairy products in the Late Neolithic, adding to a growing body of evidence for the period. Alongside palaeoproteomic analysis, we applied well-established lipid residue analysis. Differential interpretations between these two approaches show that palaeoproteomics is especially useful where the effects from isotope mixing may underestimate the frequency of dairy products in archaeological ceramics, highlighting the potential utility of a multi-stranded approach to understand life histories of vessel use.
... Possible bit wear in premolar teeth of horses from Botai (Kazakhstan) dating to <3500 BCE were extensively debated during the past three decades (4)(5)(6). Information from the Botai site such as horse demography, horse dung finds, potential paddock fences, or horse milk traces in pot shards (7,8), as well as horse milk peptides in the calculus of Yamnaya individuals from Krivyanskiy 9 (Russia;~3000 BCE) (9), suggests that domestication became widely established during the second half of the fourth millennium BCE. However, these do not provide direct evidence for riding. ...
The origins of horseback riding remain elusive. Scientific studies show that horses were kept for their milk ~3500 to 3000 BCE, widely accepted as indicating domestication. However, this does not confirm them to be ridden. Equipment used by early riders is rarely preserved, and the reliability of equine dental and mandibular pathologies remains contested. However, horsemanship has two interacting components: the horse as mount and the human as rider. Alterations associated with riding in human skeletons therefore possibly provide the best source of information. Here, we report five Yamnaya individuals well-dated to 3021 to 2501 calibrated BCE from kurgans in Romania, Bulgaria, and Hungary, displaying changes in bone morphology and distinct pathologies associated with horseback riding. These are the oldest humans identified as riders so far.
... From ~5 ka, steppe pastoralists of the Yamnaya culture expanded east-and westward, linking Asia and Europe (Allentoft et al., 2015;Lazaridis et al., 2022). This created a geographic corridor which, aided by the development of horse-riding and chariotry (Anthony, 2007;Anthony and Brown, 2011;Wilkin et al., 2021), led to the dispersal of crops, herds, and commensal species from one continent to another. Regarding the Iberian Peninsula, an increasing gene inflow from Northern and Central European human populations with steppe ancestry has been detected (Olalde et al., 2019;Villalba-Mouco et al., 2021;Patterson et al., 2022), dating the earliest evidence from ~4.5-4 ka, from individuals who coexisted with locals without steppe ancestry (Olalde et al., 2019). ...
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The Punta Lucero III cave is a natural trap where abundant vertebrate remains were accumulated during the Meghalayan (Late Holocene). To better understand the paleoenvironmental conditions in which this record was accumulated, the micromammal assemblage, comprising a minimum number of 1396 individuals belonging to 19 taxa, was studied using the Mutual Ecogeographic Range and the Habitat Weighting Method. Throughout ~2600 years, the micromammal community's quick turnover reflected a shift from patchy forests and humid meadows to open, shrubbier grasslands. The Late Holocene Thermal Maximum's humid and mild climatic conditions underwent a cooling and aridification phase, coeval with the Iron Age Cold Epoch. These concluded in a slight temperature rising, coeval with the Roman Warm Period. Macromammals experienced a shift from wild populations to domestic herds. Therefore, this work discusses a broader context for this mammalian turnover from a human cultural perspective.
... The highest proportion of steppe ancestry is found in northeast Europe, in populations that speak Uralic languages, while many IE-speaking regions of southern Europe have substantially less steppe ancestry (47,55,70) possibly reflecting dilution of steppe ancestry via subsequent migrations. Furthermore, ancient DNA data from domestic horses indicate that the expansion of steppe ancestry into Europe was not driven by horses (75), although there is evidence of horse milking by the Yamnaya (76). IE languages therefore appear to have a more complex history than can be explained by a simple model; perhaps some IE languages were spread by farmers and others by steppe pastoralists, or perhaps some IE languages spread by demic and others by cultural diffusion. ...
Full-text available
Nearly 20 y ago, Jared Diamond and Peter Bellwood reviewed the evidence for the associated spread of farming and large language families by the demographic expansions of farmers. Since then, advances in obtaining and analyzing genomic data from modern and ancient populations have transformed our knowledge of human dispersals during the Holocene. Here, we provide an overview of Holocene dispersals in the light of genomic evidence and conclude that they have a complex history. Even when there is a demonstrated connection between a demographic expansion of people, the spread of agriculture, and the spread of a particular language family, the outcome in the results of contact between expanding and resident groups is highly variable. Further research is needed to identify the factors and social circumstances that have influenced this variation and complex history.
... Each of these hypotheses has strong arguments for their existence; nevertheless, one of the recent study on the evolutionary origin of the LP genotype concludes that the increase in the percentage of LP in the European population was unlikely related to steppe movements and their genotypes, and began no earlier than after 3000 BP [15]. Other studies showed that the large-scale migrations of steppe nomads were connected precisely with the beginning of milk consumption and domestication of horses: the advantage of a permanent source of protein combined with the potential epicenter of horse domestication allowed steppe people to begin large-scale migrations of their Pontic-Caspian steppe across Eurasia [37]. In addition to genetic variation in humans, high genetic diversity has been observed in cows from such regions where dairy farming is well practiced and humans are lactose persistent [38]. ...
Full-text available
Background Lactase persistence — the ability to digest lactose through adulthood — is closely related to evolutionary adaptations and has affected many populations since the beginning of cattle breeding. Nevertheless, the contrast initial phenotype, lactase non-persistence or adult lactase deficiency, is still affecting large numbers of people worldwide. Methods We performed the largest multiethnic genetic study of lactase deficiency on 24439 people in Russia to date. The percent of each population group was estimated according to the local ancestry inference results. Additionally, we calculated frequencies of rs4988235 GG genotype in Russian regions using the information of current location and birthplace data in client’s questionnaire. Results It turned out that among all studied population groups the frequency of GG genotype in rs4988235 was higher than in average in the European populations. In particular, the prevalence of lactase deficiency genotype in the East Slavs group was 42.8% (95% CI: 42.1–43.4%). We also investigated the regional prevalence of lactase deficiency by current place of residence. Conclusions Our study emphasizes the diagnostic significance of genetic testing, i.e. specifically for lactose intolerance parameter, as well as the scale of the problem of lactase deficiency in Russia which needs to be addressed by healthcare and food industry.
Studies of the ancient and modern genomes now allow us to date and track past movements of populations around the world. We have found that in very few areas have populations existed undisturbed since their initial settlement. Most regions have witnessed repeated invasions, followed by population displacement or blending. Populations distinct enough to be considered a deme may expand or contract independently of neighboring peoples.
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The Kura-Araxes (KA) cultural phenomenon (dated to the Early Bronze Age, c. 3500/3350-2500 BCE) is primarily characterised by the emergence of a homogeneous pottery style and a uniform ‘material culture package’ in settlements across the South Caucasus, as well as territories extending to the Ancient Near East and the Levant. It has been argued that KA societies practised pastoralism, despite a lack of direct examination of dietary and culinary practices in this region. Here, we report the first analyses of absorbed lipid residues from KA pottery to both determine the organic products produced and consumed and to reconstruct subsistence practices. Our results provide compelling evidence for a diversified diet across KA settlements in Armenia, comprising a mixed economy of meat and plant processing, aquatic fats and dairying. The preservation of diagnostic plant lipid biomarkers, notably long-chain fatty acids (C 20 to C 28 ) and n -alkanes (C 23 to C 33 ) has enabled the identification of the earliest processing of plants in pottery of the region. These findings suggest that KA settlements were agropastoral exploiting local resources. Results demonstrate the significance of applying biomolecular methods for examining dietary inferences in the South Caucasus region.
The cultural layers of ancient (3rd–2nd millennia BCE) settlements are unique study objects. Top-down, they consist of modern-day soils overlapping the ancient buried soil, strongly altered by anthropogenic pressure. Cultural layers always contain the remains of artifacts and human life in the settlement, such as bones and ceramics. Settlement sites contain cultural layers that are a promising object for studying the ancient anthropogenic mineral formation. Still, such studies should follow the study of the principal physical and chemical properties of soils. Soils of a Bronze Age settlement were studied along with the natural soils of the floodplain terrace of the Volga river, which form a common area with the terrace of the Samara River. Paleourbanozems (soils formed on cultural layers of ancient settlements) with anthropogenic horizons built into the system of natural soil horizons are formed on the settlement site. The Krasnosamarskoe settlement revealed two generations of solonetzic soils located one above another and differing in the thickness of solonetzic jointing (including thin-columnar solonetzic soils). These solonetzic soils were formed during various stages of the Bronze Age, but subsequently, they morphologically merged into a single horizon. The author investigated the stages of soil cover formation of river valleys in connection with the long-term anthropogenic impact with a specific focus on the Bronze Age societies of the Samara Volga region.
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Civilisations including ancient ones, have shaped the global ecosystems in many ways through a co-evolution of landscapes and humans. However, the cultural legacies of ancient and lost civilisations are seldom considered in conservation. Here using a continental-scale dataset containing over 1,000 data records on the localities, land cover, protection status and cultural values related to ancient steppic burial mounds (so-called kurgans), we evaluated how these iconic and widespread landmarks can contribute to grassland conservation in the Eurasian steppes, which is one of the most endangered biomes on Earth. By using Bayesian logistic generalized regressions and proportional odds logistic regressions, we aimed to reveal the potential of mounds in preserving grasslands considering landscapes with different levels of land use transformation. We also compared the conservation potential of mounds situated inside and outside protected areas and assessed whether the presence of cultural, historical or spiritual values support the maintenance of grasslands on them. We revealed that kurgans have enormous importance in preserving grasslands in transformed landscapes outside protected areas, where they can act as habitat islands, and provide an additional pillar for conservation by contributing to habitat conservation and improvement of habitat connectivity. We found that besides their steep slopes hindering ploughing, the existence of cultural, historical or religious values could almost double the chance for grassland occurrence on kurgans due to the related extensive land use and the respect of local communities. As the estimated number of steppic mounds is about 600,000 and also similar historical features exist in all continents, our results can be upscaled to a global level. Our results also suggest that an integrative socio-ecological approach in conservation might support the positive synergistic effects of conservational, landscape and cultural values.
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The origins, prevalence and nature of dairying have been long debated by archaeologists. Within the last decade, new advances in high-resolution mass spectrometry have allowed for the direct detection of milk proteins from archaeological remains, including ceramic residues, dental calculus, and preserved dairy products. Proteins recovered from archaeological remains are susceptible to post-excavation and laboratory contamination, a particular concern for ancient dairying studies as milk proteins such as beta-lactoglobulin (BLG) and caseins are potential laboratory contaminants. Here, we examine how site-specific rates of deamidation (i.e., deamidation occurring in specific positions in the protein chain) can be used to elucidate patterns of peptide degradation, and authenticate ancient milk proteins. First, we characterize site-specific deamidation patterns in modern milk products and experimental samples, confirming that deamidation occurs primarily at low half-time sites. We then compare this to previously published palaeoproteomic data from six studies reporting ancient milk peptides. We confirm that site-specific deamidation rates, on average, are more advanced in BLG recovered from ancient dental calculus and pottery residues. Nevertheless, deamidation rates displayed a high degree of variability, making it challenging to authenticate samples with relatively few milk peptides. We demonstrate that site-specific deamidation is a useful tool for identifying modern contamination but highlight the need for multiple lines of evidence to authenticate ancient protein data.
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The deep population history of East Asia remains poorly understood due to a lack of ancient DNA data and sparse sampling of present-day people1,2. We report genome-wide data from 166 East Asians dating to 6000 BCE – 1000 CE and 46 present-day groups. Hunter-gatherers from Japan, the Amur River Basin, and people of Neolithic and Iron Age Taiwan and the Tibetan plateau are linked by a deeply-splitting lineage likely reflecting a Late Pleistocene coastal migration. We follow Holocene expansions from four regions. First, hunter-gatherers of Mongolia and the Amur River Basin have ancestry shared by Mongolic and Tungusic language speakers but do not carry West Liao River farmer ancestry contradicting theories that their expansion spread these proto-languages. Second, Yellow River Basin farmers at ~3000 BCE likely spread Sino-Tibetan languages as their ancestry dispersed both to Tibet where it forms up ~84% to some groups and to the Central Plain where it contributed ~59-84% to Han Chinese. Third, people from Taiwan ~1300 BCE to 800 CE derived ~75% ancestry from a lineage also common in modern Austronesian, Tai-Kadai and Austroasiatic speakers likely deriving from Yangtze River Valley farmers; ancient Taiwan people also derived ~25% ancestry from a northern lineage related to but different from Yellow River farmers implying an additional north-to-south expansion. Fourth, Yamnaya Steppe pastoralist ancestry arrived in western Mongolia after ~3000 BCE but was displaced by previously established lineages even while it persisted in western China as expected if it spread the ancestor of Tocharian Indo-European languages. Two later gene flows affected western Mongolia: after ~2000 BCE migrants with Yamnaya and European farmer ancestry, and episodic impacts of later groups with ancestry from Turan.
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Consuming the milk of other species is a unique adaptation of Homo sapiens, with implications for health, birth spacing and evolution. Key questions nonetheless remain regarding the origins of dairying and its relationship to the genetically-determined ability to drink milk into adulthood through lactase persistence (LP). As a major centre of LP diversity, Africa is of significant interest to the evolution of dairying. Here we report proteomic evidence for milk consumption in ancient Africa. Using liquid chromatography tandem mass spectrometry (LC-MS/MS) we identify dairy proteins in human dental calculus from northeastern Africa, directly demonstrating milk consumption at least six millennia ago. Our findings indicate that pastoralist groups were drinking milk as soon as herding spread into eastern Africa, at a time when the genetic adaptation for milk digestion was absent or rare. Our study links LP status in specific ancient individuals with direct evidence for their consumption of dairy products. Consuming the milk of other species is a unique adaptation of Homo sapiens. Here, the authors carry out proteomic analysis of dental calculus of 41 ancient individuals from Sudan and Kenya, indicating milk consumption occurred as soon as herding spread into eastern Africa.
Full-text available
The Eastern Eurasian Steppe was home to historic empires of nomadic pastoralists, including the Xiongnu and the Mongols. However, little is known about the region’s population history. Here, we reveal its dynamic genetic history by analyzing new genome-wide data for 214 ancient individuals spanning 6,000 years. We identify a pastoralist expansion into Mongolia ca. 3000 BCE, and by the Late Bronze Age, Mongolian populations were biogeographically structured into three distinct groups, all practicing dairy pastoralism regardless of ancestry. The Xiongnu emerged from the mixing of these populations and those from surrounding regions. By comparison, the Mongols exhibit much higher eastern Eurasian ancestry, resembling present-day Mongolic-speaking populations. Our results illuminate the complex interplay between genetic, sociopolitical, and cultural changes on the Eastern Steppe.
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The flanks of the Caucasus Mountains and the steppe landscape to their north offered highly productive grasslands for Bronze Age herders and their flocks of sheep, goat, and cattle. While the archaeological evidence points to a largely pastoral lifestyle, knowledge regarding the general composition of human diets and their variation across landscapes and during the different phases of the Bronze Age is still restricted. Human and animal skeletal remains from the burial mounds that dominate the archaeological landscape and their stable isotope compositions are major sources of dietary information. Here, we present stable carbon and nitrogen isotope data of bone collagen of 105 human and 50 animal individuals from the 5th millennium BC to the Sarmatian period, with a strong focus on the Bronze Age and its cultural units including Maykop, Yamnaya, Novotitorovskaya, North Caucasian, Catacomb, post-Catacomb and late Bronze Age groups. The samples comprise all inhumations with sufficient bone preservation from five burial mound sites and a flat grave cemetery as well as subsamples from three further sites. They represent the Caucasus Mountains in the south, the piedmont zone and Kuban steppe with humid steppe and forest vegetation to its north, and more arid regions in the Caspian steppe. The stable isotope compositions of the bone collagen of humans and animals varied across the study area and reflect regional diversity in environmental conditions and diets. The data agree with meat, milk, and/or dairy products from domesticated herbivores, especially from sheep and goats having contributed substantially to human diets, as it is common for a largely pastoral economy. This observation is also in correspondence with the faunal remains observed in the graves and offerings of animals in the mound shells. In addition, foodstuffs with elevated carbon and nitrogen isotope values, such as meat of unweaned animals, fish, or plants, also contributed to human diets, especially among communities living in the more arid landscapes. The regional distinction of the animal and human data with few outliers points to mobility radii that were largely concentrated within the environmental zones in which the respective sites are located. In general, dietary variation among the cultural entities as well as regarding age, sex and archaeologically indicated social status is only weakly reflected. There is, however, some indication for a dietary shift during the Early Bronze Age Maykop period.
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Radiocarbon (C) ages cannot provide absolutely dated chronologies for archaeological or paleoenvironmental studies directly but must be converted to calendar age equivalents using a calibration curve compensating for fluctuations in atmospheric C concentration. Although calibration curves are constructed from independently dated archives, they invariably require revision as new data become available and our understanding of the Earth system improves. In this volume the international C calibration curves for both the Northern and Southern Hemispheres, as well as for the ocean surface layer, have been updated to include a wealth of new data and extended to 55,000 cal BP. Based on tree rings, IntCal20 now extends as a fully atmospheric record to ca. 13,900 cal BP. For the older part of the timescale, IntCal20 comprises statistically integrated evidence from floating tree-ring chronologies, lacustrine and marine sediments, speleothems, and corals. We utilized improved evaluation of the timescales and location variable C offsets from the atmosphere (reservoir age, dead carbon fraction) for each dataset. New statistical methods have refined the structure of the calibration curves while maintaining a robust treatment of uncertainties in the C ages, the calendar ages and other corrections. The inclusion of modeled marine reservoir ages derived from a three-dimensional ocean circulation model has allowed us to apply more appropriate reservoir corrections to the marine C data rather than the previous use of constant regional offsets from the atmosphere. Here we provide an overview of the new and revised datasets and the associated methods used for the construction of the IntCal20 curve and explore potential regional offsets for tree-ring data. We discuss the main differences with respect to the previous calibration curve, IntCal13, and some of the implications for archaeology and geosciences ranging from the recent past to the time of the extinction of the Neanderthals.
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Dairy pastoralism is integral to contemporary and past lifeways on the eastern Eurasian steppe, facilitating survival in agriculturally challenging environments. While previous research has indicated that ruminant dairy pastoralism was practiced in the region by circa 1300 bc, the origin, extent and diversity of this custom remain poorly understood. Here, we analyse ancient proteins from human dental calculus recovered from geographically diverse locations across Mongolia and spanning 5,000 years. We present the earliest evidence for dairy consumption on the eastern Eurasian steppe by circa 3000 bc and the later emergence of horse milking at circa 1200 bc, concurrent with the first evidence for horse riding. We argue that ruminant dairying contributed to the demographic success of Bronze Age Mongolian populations and that the origins of traditional horse dairy products in eastern Eurasia are closely tied to the regional emergence of mounted herding societies during the late second millennium bc.
The genetically attested migrations of the third millennium BC have made the origins and nature of the Yamnaya culture a question of broad relevance across northern Eurasia. But none of the key archaeological sites most important for understanding the evolution of Yamnaya culture is published in western languages. These key sites include the fifth-millennium BC Khvalynsk cemetery in the middle Volga steppes. When the first part of the Eneolithic cemetery (Khvalynsk I) was discovered in 1977–1979, the graves displayed many material and ritual traits that were quickly recognized as similar and probably ancestral to Yamnaya customs, but without the Yamnaya kurgans. With the discovery of a second burial plot (Khvalynsk II) 120 m to the south in 1987–1988, Khvalynsk became the largest excavated Eneolithic cemetery in the Don-Volga-Ural steppes (201 recorded graves), dated about 4500–4300 BCE. It has the largest copper assemblage of the fifth millennium BC in the steppes (373 objects) and the largest assemblage of sacrificed domesticated animals (at least 106 sheep-goat, 29 cattle, and 16 horses); and it produced four polished stone maces from well-documented grave contexts. The human skeletons have been sampled extensively for ancient DNA, the basis for an analysis of family relationships. This report compiles information from the relevant Russian-language publications and from the archaeologists who excavated the site, two of whom are co-authors, about the history of excavations, radiocarbon dates, copper finds, domesticated animal sacrifices, polished stone maces, genetic and skeletal studies, and relationships with other steppe cultures as well as agricultural cultures of the North Caucasus (Svobodnoe-Meshoko) and southeastern Europe (Varna and Cucuteni-Tripol’ye B1). Khvalynsk is described as a coalescent culture, integrating and combining northern and southern elements, a hybrid that can be recognized genetically, in cranio-facial types, in exchanged artifacts, and in social segments within the cemetery. Stone maces symbolized the unification and integration of socially defined segments at Khvalynsk.
Lactase persistence (LP), the continued expression of lactase into adulthood, is the most strongly selected single gene trait over the last 10,000 years in multiple human populations. It has been posited that the primary allele causing LP among Eurasians, rs4988235-A [1], only rose to appreciable frequencies during the Bronze and Iron Ages [2, 3], long after humans started consuming milk from domesticated animals. This rapid rise has been attributed to an influx of people from the Pontic-Caspian steppe that began around 5,000 years ago [4, 5]. We investigate the spatiotemporal spread of LP through an analysis of 14 warriors from the Tollense Bronze Age battlefield in northern Germany (∼3,200 before present, BP), the oldest large-scale conflict site north of the Alps. Genetic data indicate that these individuals represent a single unstructured Central/Northern European population. We complemented these data with genotypes of 18 individuals from the Bronze Age site Mokrin in Serbia (∼4,100 to ∼3,700 BP) and 37 individuals from Eastern Europe and the Pontic-Caspian Steppe region, predating both Bronze Age sites (∼5,980 to ∼3,980 BP). We infer low LP in all three regions, i.e., in northern Germany and South-eastern and Eastern Europe, suggesting that the surge of rs4988235 in Central and Northern Europe was unlikely caused by Steppe expansions. We estimate a selection coefficient of 0.06 and conclude that the selection was ongoing in various parts of Europe over the last 3,000 years.