The origins of domesticated sheep (Ovis sp.) in China remain unknown. Previous workers have speculated that sheep may have been present in China up to 7000 years ago, however many claims are based on associations with archaeological material rather than independent dates on sheep material. Here we present 7 radiocarbon dates on sheep bone from Inner Mongolia, Ningxia and Shaanxi provinces. DNA analysis on one of the bones confirms it is Ovis sp. The oldest ages are about 4700 to 4400 BCE and are thus the oldest objectively dated Ovis material in eastern Asia. The graphitisised bone collagen had δ13C values indicating some millet was represented in the diet. This probably indicates sheep were in a domestic setting where millet was grown. The younger samples had δ13C values indicating that even more millet was in the diet, and this was likely related to changes in foddering practices. © 2014, Macmillan Publishers Limite

Atahan, P

Banati, RB

Dodson, E

Dodson, JR

Hu, S

Li, X

Middleton, RJ

Nan, S

Zhou, X

Scientific Reports

English

ANSTO Publications Online

Oldest Directly Dated Remains of Sheep
in China
John Dodson1, Eoin Dodson1,5, Richard Banati1, Xiaoqiang Li2, Pia Atahan1, Songmei Hu3,
Ryan J. Middleton1, Xinying Zhou2 & Sun Nan4
1Australian Nuclear Science and Technology Organisation, Sydney, Australia, 2Key Laboratory of Vertebrate Evolution and Human
Origins of Chinese Academy of Sciences, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of
Sciences, Beijing, China, 3Institute of Archaeology, Xi’an, Shaanxi, China, 4Chang An University, Xi’an, China, 5Garvan Institute,
Sydney, Australia.
The origins of domesticated sheep (Ovis sp.) in China remain unknown. Previous workers have speculated
that sheep may have been present in China up to 7000 years ago, however many claims are based on
associations with archaeological material rather than independent dates on sheepmaterial. Here we present
7 radiocarbon dates on sheep bone from Inner Mongolia, Ningxia and Shaanxi provinces. DNA analysis on
one of the bones confirms it is Ovis sp. The oldest ages are about 4700 to 4400 BCE and are thus the oldest
objectively dated Ovis material in eastern Asia. The graphitisised bone collagen had d13C values indicating
some millet was represented in the diet. This probably indicates sheep were in a domestic setting where
millet was grown. The younger samples had d13C values indicating that evenmoremillet was in the diet, and
this was likely related to changes in foddering practices
F
ossil sheep of Pliocene age are known for East Asia1, but domesticated sheep are thought to have arrived
several millennia after they were first domesticated in the Fertile Crescent region of Western Asia
e.g. Refs. 2, 3. The pathway to first domestication remains unknown but is believed to have taken place
about 10,500 to 11,000 years ago and relied on the fact that sheep were docile and herded naturally2–4. They were
valuable sources of meat and probably milk and milk products, and the hides were useful for leather. It is likely
that several thousand years later selective breeding added fibre for wool as a new product. Dated evidence from
Turkey at about 8200 cal BC has revealed new insights into the domestication of sheep based on age and sex ratios
and dung analysis5.
The earliest claimed domesticated sheep in China are based on archaeological associations with ages between
5–7000 years from Banpo village near Xi’an and Baoji, also in Shaanxi Province6,7. Other early ages are associated
with sites at Shizhaocun, in Gansu8 and Hetaozhuang inMinhe, Qinghai Province9. None of these ages have been
determined directly on remains of sheep, and they need revisiting to check their veracity. Presumably, sheep
arrived before these early dates and it is believed sheep were first widespread across Gansu-Qinghai sometime
between 5600 and 4000 BP, and on the central Plains of China by 4500-4000 BP10.
While early remains of sheep have been reported for several sites in China, the usually fragmentary and
incomplete nature of those remains has tended to limit whether domesticated animals can be distinguished from
wild varieties11. Ancient DNA analysis is able to resolve this issue and distinguish domestication with a high
degree of certainty, as well as provide specific information about relationships between ancient andmodern sheep
populations. Previous work using ancient DNA analysis on sheep remains from the middle reaches of the Yellow
River valley dating to ca. 2100-1800 BCE found strong genetic links to modern domestic varieties in eastern
Asia12,11. Thus far those studies have been unable to propose specific geographic origins for ancient sheep, due in
part to a scarcity of genetic information from both eastern Asia and surrounding regions.
Stable carbon isotopes on fossil bone collagen are useful for palaeodiet studies. Collagen d13C values indicate
long-term diet averages. In northern China d13C values #18% are typically regarded as mainly C3 food con-
sumers: those with values d13C between 218% and 212% are interpreted to have mixed C3 and C4 food
consumption; and those with d13C values $212% are interpreted to be mainly C4 consumers13–15.
In summary, these data suggest domesticated sheep came to China in the Neolithic and became widespread
during the Bronze Age when societies became sedentary, but the ages of these events remain tentative in the
absence of reliable dates. Here we identified sheep bone palaeontologically from sites in northern Shaanxi
Province, and confirmed the identification ofOviswith DNA analysis on one sample. The AMS ages determined
OPEN
SUBJECT AREAS:
ENVIRONMENTAL
SOCIAL SCIENCES
AGROECOLOGY
Received
29 July 2014
Accepted
4 November 2014
Published
24 November 2014
Correspondence and
requests for materials
should be addressed to
J.D. (john.dodson@
ansto.gov.au)
SCIENTIFIC REPORTS | 4 : 7170 | DOI: 10.1038/srep07170 1
on the bone samples is independent of any archaeological asso-
ciation. In addition the stable isotopic composition was measured
to provide insight into the diet of the sheep.
Study Specimens. The present study is part of a broader analysis on
the impact of Prehistoric to Bronze Age cultures on environments of
northern and western China16–22. In the course of this, several bone
samples were collected from sediment at Shihushan, Dongxiafeng
and Youyao in northern Shaanxi, and Haba Lake in Ningxia
(Figure 1). The bones were removed from clayey sediment and
transported to Xi’an for identification. In each case the bones were
in a close archaeological context with pottery sherds, charcoal and
sometimes associations of slag associated with metal working. The
cultural setting is equivalent to the Yangshao. Six of the bone samples
were identified as sheep (Ovis), and an additional sample from
Huoshiliang (Shaanxi Province) was provided by the Shaanxi
Provincial Museum. There exists a full list of taxa identified at
Huoshiliang site23. About 10 gm of each Ovis bone was transported
to the laboratories of the Australian Nuclear Science and Technology
Organisation (ANSTO) in Sydney for further analysis.
Methods
In the course of this, several bone samples were collected from sediment at Shihushan
(probably belonging to the Yangyao Culture), and Youyao (Zhukaigou Culture: 2200-
1500 BC) in northern Shaanxi, the Dongxiafeng site (belonging to the Erlitou Culture:
1900-1500 BC) in northern Shaanxi, Haba Lake in Ningxia (Xia Dynasty: 2199-1600
BC), and Huoshiliang (Longshan to Xia Culture: 2150-1900 BC) (Figure 1). The
bones were removed from clayey sediment and transported to Xi’an for identification.
In each case the bones were in a close archaeological context with pottery sherds,
charcoal and sometimes associations of slag associated with metal working.
Collagen was extracted from each sample and tested to determine whether pre-
servation was sufficient and contaminants had been effectively removed to achieve
reliable radiocarbon results. For this the total collagen yield (% by weight), nitrogen
and carbon percentages and the C/N atomic ratio were measured, following estab-
lished protocols23–26. Once the collagen had been extracted and determined to meet
standards for reliable dating, they were processed to graphite27. The 14C content was
measured on the STAR Accelerator at ANSTO and AMS ages were calculated after
estimating fractionation effects from the d13C values determined on the samples.
DNA analysis was carried out on five of the bone samples to provide independent
corroboration that the bones were indeed from sheep. DNA extraction was conducted
on subsamples of the same bone fragments that were prepared for radiocarbon
analysis.
In preparation, the metal utensils used during sample processing were wiped with
70% ethanol and UV irradiated, mortars and pestles were soaked in 11–14% sodium
hypochlorite solution (w/v) for 30 min, rinsed withMilliQwater, and then dried in an
oven set at 60uC before being used to grind bone samples. Separate mortars and
pestles were used for each sample (Table 1). Approximately 500 mg of cleaned bone
was used for DNA analysis.
Bone fragments were carefully crushed, as opposed to grinding, in order to prevent
localised high temperatures and further DNA damage. A negative control was used
from this point onwards, in order to detect introduced contaminating DNA, from the
other samples or elsewhere (Table 1).
DNA extraction was performed according to a published protocol that relies on
DNA absorption to silica particles28.
Figure 1 | Map showing the location of key sites where sheep bone was analysed for this paper. Figure 1 was designed, composed and prepared by XZ
using Correl Draw software.
Table 1 | Samples and bone fragments used for DNA analysis, including original samples sizes and DNA Nanodrop results
Site Sample ID Bone type Bone Fragment mass (mg) Bone Powder mass (mg) DNA conc. (ng/uL)
Shihushan OZP087/SI3001 Compact 554 429 52.2
Shihushan OZP086/SI2998 Spongiose 556 385.4 9.6
Shihushan OZP085/SI2997 Spongiose 496 502.8 42.2
Youyao OZO960/SI2950 Compact 440 464.4 65.4
Dongxiafeng OZO949/SI2921 Compact 491 500 51.2
Negative Control - - - 0 13.5
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SCIENTIFIC REPORTS | 4 : 7170 | DOI: 10.1038/srep07170 2
Results
Seven radiocarbon ages on sheep collagen have been obtained and
have a calibrated age range (with errors) from 4700 to 1570 BCE
(Table 2). Three radiocarbon ages of about 5700 BP (4700 to 4400
BCE) are statistically identical, and they overlap the Neolithic
(Yangshao) and Bronze Age boundary. These are the oldest directly
dated sheep material from eastern Asia. The d13C values of the older
bones indicate the sheep had a diet based largely on C3 plants but the
d13C values are close to the boundary of diets based on C3 and C4
plants (Table 1).
Out of the five bone samples only the Youyao bone produced a
specific amplification product. The 78 nucleotide sequence shares
100% identity with Ovis aries mitochondrial DNA (mtDNA) and
in addition the sequence also shares 100% identity with mtDNA
from O. orientalis anatolica, O. vignei and O. ammon which are
Turkish and Central Asian species, that are possible ancestors of
domestic sheep.
Discussion
The identification of the bones on physical characteristics and DNA
fragment confirms the bone samples are from the genusOvis (sheep).
The radiocarbon age of 5700 BP (4700 to 4400 BCE) for the three
bones from Shihushan is the oldest independent age for sheep in
eastern Asia. Since the bones were found in association with other
domestic species and in an archaeological setting of the Yangshao
Culture it is a reasonable conclusion that the sheep were domesti-
cated. This is further supported by the stable isotope analyses on
graphite prepared from bone collagen that had values ranging from
219.2 to214.3%. In all likelihood somemillet was consumed by the
sheep (the Asian millets are the C4 plants Panicum miliaceum and
Setaria italica), but the bulk of the diet was comprised of C3 plants.
The younger samples from Youyao and Huoshiliang had less nega-
tive d13C values, suggesting that millet was a larger proportion of the
diet in these cases, and this possibly related to changes in foddering
practices occurring over time. This is quite unlike the diet of pigs, rats
and dogs from similar settings which had even less negative values
and suggest they had diets strongly influenced by millet, including
meat from possibly domesticated animals that were fed on millet19.
Deer, cattle and sheep d13C values indicate that in general wild ani-
mals and herds grazed beyond the agricultural zone but sheep had
some C4 plants, possibly millet, in their diet19. The younger Ovis
samples suggest that the amount of millet in the diet increased and
possibly indicates a change in foddering practice. It is unfortunate
that a longer DNA sequence could not be recovered from the col-
lagen, particularly from the older samples, as this could shed light on
the early ancestors of the domesticated sheep.
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Site ANSTO Code C% N% C/N (atomic) d13C (%) (graphite) Conventional 14C age (BP 6 1s) Calibrated age (2s) BCE
Shihushan OZP087/SI3001 43.3 16.0 3.1 219.2 6 0.1 5740 6 40 4695–4490
Shihushan OZP086/SI2998 45.2 15.6 3.4 218.9 6 0.1 5695 6 35 4667–4454
Shihushan OZP085/SI2997 44.5 15.4 3.4 219.0 6 0.1 5665 6 40 4601–4372
Youyao OZO960/SI2950 45.3 15.0 3.4 214.3 6 0.1 3750 6 45 2292–2029
Dongxiafeng OZO949/SI2921 45.0 15.4 3.4 217.0 6 0.1 3360 6 45 1713–1570
Huoshiliang OZO951/SI2891 45.0 15.6 3.4 215.5 6 0.1 3750 6 50 2334–1983
Haba Lake OZN204/SI1592 44.0 15.0 3.3 217.0 6 0.1 4090 6 30 2859–2499
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SCIENTIFIC REPORTS | 4 : 7170 | DOI: 10.1038/srep07170 3
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Acknowledgments
We are grateful for support from the Australian Nuclear Science and Technology
Organisation and the support of National Natural Science Foundation of China (Grant No.
41172161, 40572108).
Author contribution
J.D., X.L., P.A., X.Z. and S.N. participated in the fieldwork, and collected the samples. S.H.
identified the bones and provided the museum specimen for analysis, E.D., R.B. and R.M.
carried out the DNA analysis and interpretation, J.D. and P.A. assisted in the radiocarbon
analysis and all contributed to the writing of the manuscript.
Additional information
Competing financial interests: The authors declare no competing financial interests.
How to cite this article:Dodson, J. et al. Oldest Directly Dated Remains of Sheep in China.
Sci. Rep. 4, 7170; DOI:10.1038/srep07170 (2014).
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SCIENTIFIC REPORTS | 4 : 7170 | DOI: 10.1038/srep07170 4


Oldest directly dated remains of sheep in China

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Oldest directly dated remains of sheep in China

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