DNA analysis of archaeological sheep remains from China

Journal of Archaeological Science 34 (2007) 1347e1355 http://www.elsevier.com/locate/jas

DNA analysis of archaeological sheep remains from China Da-Wei Cai a,b, Lu Han b, Xiao-Lei Zhang b, Hui Zhou a,b,*, Hong Zhu a a

Ancient DNA Laboratory, Research Center for Chinese Frontier Archaeology of Jilin University, Changchun 130012, China b College of Life Science, Jilin University, Changchun 130023, China Received 14 June 2006; received in revised form 11 October 2006; accepted 20 October 2006

Abstract Recent research has thrown considerable light on the history of the domestic sheep, but has not extended to ancient sheep specimens. In the present study, ancient DNA analysis was carried out on eight archaeological sheep remains recovered from Erlitou archaeological site in Henan Province (ca. 2100e1800 B.C.) to explore the genetic structure of ancient sheep and the phylogenetic relationship between ancient and modern sheep. We analyzed the control region sequences and coding regions of mitochondrial DNA from the remains by direct sequencing and restriction fragment length polymorphism analysis, respectively. Our results reveal that all ancient sheep belong to lineage A defined by modern sheep sequences. Phylogenetic analysis shows that neither argali (Ovis ammon) nor urial (Ovis vignei) mtDNA is closely related to Erlitou ancient sheep. In addition, our results suggest that ancient DNA analysis can serve as a powerful tool in tracing prehistoric population movement. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Ancient DNA; Domestic sheep; Mitochondrial DNA; Lineage; Haplotype

1. Introduction Plant and animal domestication is the most important developments in the past 13,000 years of human history (Diamond, 2002). It contributed to the rise of civilization, single-handedly transformed global demography, and provides a sizable amount of our food and clothing today (Diamond, 2002). As one of the most early domesticated animals, domestic sheep (Ovis aries) provide a great deal useful products such as meat, milk, and fur to human society and play an important role in agriculture, economy, culture, and even religion dating from very early periods in the Neolithic Age. Evidence from Near East archaeological sites suggested that sheep were probably first domesticated in the Fertile Crescent region of the Near East around 9000e8000 BP (Ryder, 1984). Several wild sheep species such as mouflon (O. musimon or O. orientalis), urial (O. vignei) and argali (O. ammon) have been proposed as * Corresponding author. Ancient DNA Laboratory, Research Center for Chinese Frontier Archaeology of Jilin University, Changchun 130012, China. Tel./fax: þ86 431 849 8031. E-mail address: [email protected] (H. Zhou). 0305-4403/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jas.2006.10.020

ancestors of modern domestic sheep (Ryder, 1984) or are thought to have contributed to specific breeds (Zeuner, 1963). Mitochondrial DNA (mtDNA) has been widely used to explore the origins of domestic sheep. According to previous phylogenetic trees constructed by mtDNA, there were two maternal lineages A and B in domestic sheep (Hiendleder et al., 1998). Lineage A was mainly found in breeds from Asia, while lineage B was observed at the highest frequency in breeds sourced from Europe. Most recently, a novel maternal lineage C, besides lineages A and B, was found in domestic sheep breeds from China and Near East (Guo et al., 2005; Luo et al., 2005; Pedrosa et al., 2005). On the basis of Cyt b gene, divergence time was estimated to be around 170,000e160,000 years ago for lineages A and B, whereas the divergence of lineage C proved to have occurred earlier (ca. 750,000e450,000 BP) (Pedrosa et al., 2005). These times greatly predated domestication (ca. 9000e8000 BP) and suggested that at least three geographically independent sheep domestication events occurred. However, previous studies of sheep were mostly focused on the modern breeds from Central Asia, the Near East and Europe, but did not extend to ancient sheep from East Asia.

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Ancient DNA offers a direct means to assess past genetic structure and diversity of animals, which can help to reveal the origin and history of domestic animals. For example, ancient and modern mtDNA phylogeography in European cattle indicates a derived origin from the Near East and supports a different history for African cattle (Troy et al., 2001). The use of ancient DNA has also proved crucial evidence for dog domestication, where it seems that the Old World, particularly East Asia, was an important area (Leonard et al., 2002). Considering archaeological remains showing an early presence of domestic sheep in ancient China (ca. 4000 BP) (Chen, 1994; Tu et al., 1989) and the geographical distribution of lineage A and C, ancient DNA data from China will enhance our understanding about the origins of domestic sheep. Erlitou archaeological site is located along the Luo River, about 9 km southwest of Yanshi City in Henan Province of China (Fig. 1), characterized by a continental monsoon climate of the North Temperate Zone, with four distinct seasons and annual average temperature of 13e15  C. Loess soil is one of the most significant geologic features of this area. The site covers an area of 4,000,000 m2, with deposits up to 4 m thick. According to the excavation data, Chinese archaeologists have found there were some manufacturing workshops of bone artefact, pottery making and bronze smelting. Moreover, they have found 400 tombs, bronze and jade sacrificial vessels and the foundation of a large palace. All these have proven Erlitou was the earliest capital city ever found in China. Radiocarbon dates (ca. 2100e 1800 B.C.) put it to be appropriate for the Xia dynasty in Chinese history. Many animal remains of sheep, swine, cattle etc. were unearthed from this site. Presently, most bones are mainly used for identification of taxa using morphological features (Li, 2004). In this study, we have two main objectives: (1) to determine the genetic structure of ancient sheep and (2) to explore the phylogenetic relationship between ancient and modern sheep.

2. Materials and methods 2.1. Sample collection Nine sheep remains were provided by Institute of Archaeology, Chinese Academy of Social Sciences (Table 1). Morphological and morphometric analyses have concluded that all were domestic sheep. These samples were excavated from the Erlitou archaeological site during the period 2000e 2004. Immediately after excavation, the remains were cleaned with a soft brush and washed under running water, and allowed to dry naturally. Subsequently, the remains underwent a morphological study, before being stored safely in sealed plastic bags, within closed boxes, in a storage room. In general, the remains used for ancient DNA analysis demonstrated good macroscopic preservation. The teeth were white, smooth and compact, almost without damaged external surfaces. Five archaeologists have ever had access to the samples during the excavation, washing, and subsequent morphological study. 2.2. Sample decontamination and DNA extraction Teeth were immersed in 1 N hydrochloric acid for 3e 5 min, and washed using ultra-pure water and 100% ethyl alcohol in turn. Subsequently, each side of the teeth was exposed to ultraviolet (UV) light (1.0 J/cm2, 254 nm) for 15 min. Teeth were ground to fine powder under liquid nitrogen in a 6750 Freezer Mill (Metuchen, USA). Ancient DNA was extracted from the tooth powder by using a modified silica-spin column method (Yang et al., 1998). The powdered samples (1.5e2 g) were incubated at 53  C overnight with 3 ml of lysis buffer (0.465 M EDTA pH 8.0, 0.5% SDS and 0.4 mg/ml Proteinase K) in a shaking airbath. After centrifugation, 2 ml of the supernatant was transferred into CentriconÒ YM-10 microconcentrator, reduced to less than 100 ml, and purified with QIAquickÒ spin column (QIAGEN GmbH, Germany) according to its manual. 2.3. PCR amplification and sequencing of mtDNA

Fig. 1. Geographical distribution of Erlitou archaeological site (available at http://encarta.msn.com/map_701511747/China.html).

Two sets overlapping primers (Table 2) were designed from reference sequence (AF010406), to amplify a 271 bp mtDNA fragment which contains the partial tRNApro and control region sequence between nucleotide positions (nps.) 15,391e 15,661 (Reference NC_001941). PCR amplification was conducted in a MastercyclerÒ personal Thermal Cycler (Eppendorf, Hamburg, Germany) in a 50 ml reaction volume containing 50 mM KCl, 10 mM TriseHCl, 2.5 mM MgCl2, 0.2 mM dNTP, 2.0 mg/ml BSA, 0.5 mM of each primer, 2 ml DNA sample and 1 U Taq polymerase (Promega, USA). PCR parameters were: 94  C for 3 min (an initial denaturing), followed by 36 cycles with 94  C for 55 s (denaturing), 52e55  C for 55 s (annealing), 72  C for 55 s (extension) and a final extension of 10 min at 72  C. Five microlitres of PCR product were separated by electrophoresis on 2% agarose gel (Biowest, Germany). PCR

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Table 1 Samples used in this study, their excavated location, genus identification, element and tooth eruption Sample

Excavated location

Species

Element

Tooth eruption

ELS1 ELS2 ELS3 ELS4 ELS5 ELS6 ELS7 ELS8 ELS9

2003YL V T36H198 2003YL V T37H211 2004YL V T85uA 2003YL V T36uB 2001YL V T6H22 2002YL V T26tA 2004YL V T79t 2003YL V T33tB east 2000YL III T2H13s

O. O. O. O. O. O. O. O. O.

Left mandible Left mandible Left mandible Left mandible Right mandible Left mandible Right mandible Dissociated Right mandible

P4(E) þ M1 þ M2 dp3 þ dp4 þ Ml þ M2 (0.5) dp3 þ dp4 þ Ml dp3 þ dp4 þ M1 dp2 þ dp3 þ dp4 þ M1 þ M2(E) dp4 þ Ml dp2 þ dp3 þ dp4 þ M1 Left I1 P4

aries aries aries aries aries aries aries aries aries

products were purified using QIAEXÒ Gel Extraction Kit (QIAGEN) and were subjected to direct sequencing. The sequencing reaction was carried out on an ABI 310 automated DNA sequencer (Applied Biosystems, USA) using ABI Prism Big Dye Terminator v3.1 Cycle Sequencing kit. The primers used for amplification were also used for sequencing, and each fragment was sequenced in both directions. The obtained electropherograms were assembled to examine any base pair ambiguities using Chromas 2.22 (www.technelysium.com.au). 2.4. Restriction enzyme analysis of mtDNA Hiendleder et al. (1999) reported the polymorphic HinfI recognition site to nucleotide positions 5562e5566 in the COI gene can be used to assay lineage A (5562 HinfI) and B (þ5562 HinfI). Further identification of sheep lineage and authenticity, the primers COIF (50 -GAGATGACCAAATCTAC AACG-30 ) and COIR (50 -GCTCCAATTATCAGAGGAAC-30 ) were designed to amplify a 121 bp (nps. 5547e5597, Reference NC_001941) fragment encompassing this polymorphic HinfI site. PCR conditions follow as above paragraphs, RELP method described by Hiendleder et al. (1999).

blanks were set up with each batch of PCRs to check if PCR reagents had been contaminated. 2.6. Data analysis Ancient mtDNA sequences were compared to 143 sequences from different geographic regions available from the GenBank database (Table 3). DNA sequences were aligned with the program of Clustal X 1.83 (Thompson et al., 1997). Haplotypes were identified using NSA 3.1 software (http://perso. wanadoo.fr/daniel.montagnon/NSAAng.htm). The neighbourjoining (NJ) and unweighted pair group method with arithmetic mean (UPGMA) trees were constructed using the program MEGA 3.1 (http://www.megasoftware.net/) (Kumar et al., 2004), with a Tamura-Nei model and a bootstrap test (number of replications ¼ 1000). The goat (Capra hircus) sequence was used as an outgroup (Parma et al., 2003). The medianjoining network (Bandelt et al., 1999) was drawn using the program NETWORK4.1.1.2 (www.fluxus-engineering.com/ sharenet.htm) to investigate the possible relationships among haplotypes. Erlitou ancient sheep sequences data in this study have been submitted to GenBank with accession numbers DQ401036eDQ401043. 3. Results

2.5. Contamination controls 3.1. mtDNA variation in ancient sheep The analysis of ancient DNA in this study followed strict contamination control protocols for ancient DNA (Yang et al., 2004). All the experiments were carried out in a dedicated ancient DNA laboratory into which no modern sheep DNA had ever been introduced. All pre-PCR and post-PCR procedures were performed respectively in a separate room where PCRs are never carried out. PCRs were carried out in another clean room with positive pressure and air filtration system. The laboratory surfaces needed to be cleaned by using DNA-OFFÔ (Q.BIO gene, Germany) and UV-irradiation before the experiment. All reagents and dedicated equipment were UV irradiated for 45 min before use. DNA-free reagents and one-off articles were used. All workers wore sterilized laboratory coats, coveralls with hood, facemasks and gloves (which were frequently changed). Extraction blanks were prepared in parallel with each ancient DNA extraction to check whether extraction reagents had been contaminated, and water

Ancient DNA sequences of 271 bp D-loop region were successfully retrieved from eight samples (except for sample ELS9). Four different haplotypes were determined, and some of the ancient specimens shared the same haplotype. Comparing to the reference sequence (GenBank AF010406), a total of Table 2 Primers for PCR amplifications mtDNA site

Primer

Primer sequence

Fragment length (bp)

tRNAPro - HVRI HVRI

L15391a H15534 L15496a H15661

50 -CCACTATCAACACCCAAAG-30 50 -AAGTCCGTGTTGTATGTTTG-30 50 -TTAAACTTGCTAAAACTCCCA-30 50 -AATGTTATGTACTCGCTTAGCA-30

144 166

a The numbers give the 50 -end of the primers according to AF010406. L and H refer to the light and the heavy strands, respectively.

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Table 3 DNA sequences used in this study and the locations where the sample were originally collected Namea

Breedb

GenBank accession numbers

Country/region

Ovis aries

Z35228eZ35268, Z35293

New Zealand

Ovis aries

Merino, Romney, Coopworth, Perendate  Romney Merinolandschaf

Germany

Ovis Ovis Ovis Ovis Ovis Ovis Ovis Ovis Ovis Ovis Ovis Ovis Ovis Ovis Ovis Ovis Ovis Ovis

RasaArgonesa Astrachan Edilbey Gizarr Akkaraman Daglic Kivircik Small tail Han Tibetan Tong Hu Mongolian Kazakh Fat-rumped Texel Polled Dorset n.a. n.a. n.a.

AF039577eAF039578, AF010406eAF010407 L29055 AY091500 AY091499 AY091498 AY091497 AY091496 AY091495 AY829423e30, AY829376 AY829416eAY829422 AY829411eAY829415 AY829389eAY829396 AY829397eAY829405 AY829383eAY829388 AY829406eAY829410 AY829377eAY829382 AJ238304 AJ238298 AJ238302 AJ238297, AJ251327 AJ238292, AJ238293, AJ238299 AF242347 AJ238300, AJ238303, AJ238296, AJ238294, AF242348 AY091492 AY091493, AY091494 AJ238301, AJ238305 AJ238295 AY091490, AY091491 AF039580, AY091489 AY091487, AY091488, AF039579 AF076911eAF076917

aries aries aries aries aries aries aries aries aries aries aries aries aries aries aries ammon sairensis ammon adametzi ammon hodgsoni

Ovis ammon dalai-lamae Ovis ammon ammon Ovis ammon darwini

n.a. n.a. n.a.

Ovis ammon collium Ovis ammon nigrimontana

n.a. n.a.

Ovis Ovis Ovis Ovis Ovis

n.a. n.a. n.a. n.a. n.a.

a b

ammon severtzovi vignei bochariensis vignei arkal aries musimon Canadensis canadensis

Spain Kazakstan/Tschimkent Kazakstan/Alma-Ata Tadjikistan Turkey/central Anatolia Turkey/west Turkey Turkey/Aegean China China China China China China Netherlands Australia China/Tuoli, Xinjiang China/Bybrki, Xinjiang China/Qinghai China/Tibet China/GanSu Mogolia/Altai China/GanSu Kazakstan/Karaganda Kazakstan/Kara Tau Uzbekistan/Taskent Uzbekistan/Taskent Turkmenistan/southeast Kazakstan/Ust-Urt Germany/Hessen Canada/Rocky Mountains

Nomenclature is according to Vorontsov et al. (1972). n.a., not applicable.

11 polymorphic sites (representing 4.8% of the total DNA sequence analyzed) were found; all of these variable positions were single nucleotide substitutions, and none of them were transversions (Table 4). Previous studies showed that each

lineage contain a unique sequence motif (Table 5), which can be used to predict the lineage of domestic sheep (Luo et al., 2005). On the basis of variation positions of each sample, all Erlitou sheep belong to lineage A.

Table 4 Ancient sheep nucleotide variation positions

3.2. HinfI restriction enzyme analysis

Hapoltype

Reference sequence (AF010406) ELS1, ELS2, ELS3 ELS4, ELS5, ELS7 ELS6 ELS8

Variation positions 1 5 4 5 9 C

1 5 4 6 4 T

1 5 4 8 4 G

1 5 5 1 3 C

1 5 5 4 7 G

1 5 5 8 3 C

1 5 5 9 7 T

1 5 6 2 2 T

1 5 6 3 5 A

1 5 6 3 8 C

1 5 6 3 9 A

T T T T

C $ $ $

A A A A

$ $ $ T

A A A A

T T T T

C C C C

$ C $ $

G G G G

T T T T

G G G G

Dot ($) denotes identity with the reference sequence.

Mitochondrial DNA control region sequence differences might be used to discriminate two mtDNA lineages (A and B) of domestic sheep (Hiendleder et al., 1998; Wood and Phua, 1996). However, the high mutation rate of this region, Table 5 Sequence motifs of the mtDNA lineages Lineage

Sequence motif

A Ba C

15583Te15597Ce15547Ae15635G Reference mtDNA sequence 15583Te15597Ce15509Ge15551Ge15607Ce15629C

a

Lineage corresponds to mtDNA reference AF010406.

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which is an order of magnitude higher than in coding regions (Hiendleder, 1998), could interfere with unequivocal typing results. Based on the above, HinfI restriction enzyme analysis of mtDNA was performed. All samples showed the negative results (5562 HinfI), according to Hiendleder et al. (1999), and all ancient sheep were identified as lineage A. 3.3. Phylogenetic tree construction In order to determine the phylogenetic position of ancient sheep, the ancient DNA sequences were compared with 143 sequences representing a variety of breeds from all over the word. To optimize and simplify the comparative analysis of sheep haplotypes, 66 haplotypes were identified among 151 sheep sequence using NSA 3.1 software. The NJ and UPGMA trees were constructed to address the relationships between the ancient specimens studied and the breeds used for comparison. As both trees produced the similar patterns, only the NJ tree was shown (Fig. 2). The NJ tree displayed that the domestic sheep were divided into three distinct lineages A, B and C. All Erlitou ancient sheep clustered in lineage A with New Zealand breeds, Merinolandschaf, Polled Dorset, Texel, Astrachan, Hu, Small tail Han, Mongolian, Tong and Kazakh fatrumped. 3.4. Phylogenetic network construction As expected, three star-like major lineages A, B, and C were observed in domestic sheep (Fig. 3). The lineages A, B and C included 15, 16 and 5 haplotypes comprising 48, 56 and 14 individuals, respectively. Within each lineage, there is one major haplotype in the centre (Here defined as haplotype a, b and c, respectively). From haplotype a, there were 8 and 10 mutation steps to haplotype b and c, respectively. Remarkably, Sample ELS6 shared the founder haplotype of lineage A, and perfectly matched 26 sequences present in the database: 12 from New Zealand breeds, 4 from European breeds imported into China in 1997 (2 Texel, 2 Polled Dorset), 1 from Central Asian breeds (1 Gizarr), and 9 from Chinese breeds (3 Tong, 2 Small tail Han, 2 Mongolian, 1 Hu, 1 Kazakh fat-rumped). ELS4eELS5eELS7 shared their sequence with that of a Kazakh fat-rumped sheep from China. ELS1e ELS2eELS3 and ELS8 are the only Erlitou sequences that matched none of the sequences retained in the database. The network profile also showed that breeds from different geographical regions intermingled. Some haplotypes were shared by individuals from different geographical regions. 4. Discussion 4.1. Authenticity of ancient DNA results The ancient DNA results must be proved authentic before they can be used to address research questions (Cooper and Poinar, 2000; Kaestle and Horsburgh, 2002; Poinar, 2003). Due to the careful research design, the authenticity of the obtained sequences can be generally demonstrated through the

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following multiple means of examination: (1) a dedicated ancient DNA facility was used in this study; (2) all extraction and PCR reagents blanks were consistently negative throughout the study; (3) throughout the study, no modern sheep DNA was introduced into laboratory; (4) the PCR results were obtained from multiple extractions, amplifications and double-strand sequencings of the same samples well separated in time (at least three times); and (5) all results (including HinfI restriction fragment length polymorphism; RFLP) were repeated multiple times by two researchers (D.W. Cai and L. Han), respectively. All the results showed that the ancient sheep sequences were authentic and reliable. 4.2. Ancient sheep lineage Three lineages A, B and C observed in modern sheep breeds suggest that at least three independent domestication events have occurred (Guo et al., 2005; Pedrosa et al., 2005). Considering the dominance of linage B and the abundant archaeological evidences throughout the Near East, it implies that lineage B was probably of Near Eastern origin (Chen et al., 2006). Hiendleder et al. (2002) proposed the mouflon populations found in Turkey and West Iran (Ovis orientlis anatolica and Ovis orientalis gmelini) as ancestors of lineage B. However, the origin and wild ancestors of lineages A and C remain uncertain. Among the Chinese breeds, lineage A (65%) was popular, followed by lineage B (22%) and C (13%) (Chen et al., 2006; Guo et al., 2005). Remarkably, the pattern of genetic variation of lineage A within Chinese domestic sheep revealed a new subclade, only present in North China, but not in other regions (Chen et al., 2006). These results suggested that lineage A is a key to reveal the origin of Chinese sheep. Therefore, it is interesting to identify the lineages of ancient Chinese sheep. In this study, sequence motif, RFLP and phylogenetic analysis of ancient sheep mtDNA consistently revealed that Erlitou ancient sheep belong to lineage A. Surprisingly, no other lineages were found. Considering the divergence time of lineage A, we suggest that lineage A was a very ancient and important lineage in Chinese domestic sheep. 4.3. Ancient sheep and Chinese domestic sheep Besides the identification of linage, we also paid attention to determine if the ancient sequences obtained from Erlitou sheep samples could be linked to some particular breeds. China has 15 autochthonous sheep breeds and numerous local sheep populations, which are distributed from the high QinghaiTibet Plateau to the low land of East China (Tu et al., 1989). Based on the origin and the geographical distribution of breeds, Chinese domestic sheep can be classified into three genetic groups: Tibetan sheep, Kazak sheep and Mongolian sheep (Zheng, 1980). The Tibetan sheep group is mainly distributed in Qinghai-Tibetan plateau and its neighbouring areas. The Kazak sheep group is mainly found in the desert and mountainous areas in west Xinjiang. The Mongolian sheep group derived from Mongolian plateau is now widely distributed in North, Northeast and Northwest China. Interestingly,

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Fig. 2. Neighbour-joining tree of the 66 haplotypes of modern domestic sheep, ancient sheep and wild sheep based on 203 bp of the mtDNA control region along with a goat as an outgroup. Bootstrap support is indicated at nodes if found in <50% of 1000 bootstrap replications. The number of sequences include in the haplotype is indicated between brackets. Erlitou sheep are indicated with a dot.

we noted that ELS6 shared the founder haplotype with 26 sequences. Nine of 26 sequences belong to Chinese breeds (3 Tong, 2 small tail Han, 2 Mongolian, 1 Hu, 1 Kazakh fat rumped). According to historical record (Tu et al., 1989) and recent study (Lu et al., 2005), Tong, Small tail Han and Hu

sheep were originated from the Mongolian sheep. Considering the geographical distribution of Erlitou Ruin and the historical record, ELS6 may be related to the Mongolian sheep group. In addition, ELS4eELS5eELS7 shared their sequences with that of a Kazakh fat-rumped sheep distributed in western Xinjiang,

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Fig. 3. Phylogenetic networks of 151 sheep mtDNA sequences. Circle areas are proportional to haplotype frequencies. Links represent mutations at the mtDNA nps. indicated alongside. Letters A, B, J, H and G are abbreviations of GenBank accession numbers (A ¼ AF0, B ¼ AF2, J ¼ AJ2, H ¼ AY0, G ¼ AY8).

China. Nevertheless, these results should be taken with great caution as ELS6 also matched with sheep from other breeds (12 New Zealand sheep, 1 Gizarr, 2 Texel and 2 Polled Dorset). 4.4. Ancient sheep and wild sheep Two argali subspecies from Tibet and Mongolia (O. a. hodgsoni and O. a. darwini), were proposed as ancestors of the Tibetan and Mongolian sheep groups, respectively, and the contribution from urial to the Mongolian sheep group also was discussed (Tu et al., 1989). However, analysis based on cytogene (Nadler et al., 1973) and mtDNA (Hiendleder et al., 1998, 2002) have indicated that there were no contributions from urial or argali species to modern domestic sheep, whereas the mouflon has a close relationship with lineage B, and both are derived from a common ancestor. Also, Wu et al. (2003) sampled most of currently recognized subspecies of argali and obtained a clear separation from O. aries sequences. To determine if ancient sheep or the Tibet and Mongolia sheep group could be related to wild sheep, several subspecies of O. ammon (ammon, darwini, adametzi, dalai-lame, hodgsoni, sairensis, collium, nigrimotana and severtzovi) and O. vignei (akal and bochariensis) were used to construct the phylogenetic tree and network. In addition, the bighorn (O. canadensis canadensis) and European mouflon (O. musimon) were also used for the

phylogenetic analysis. Phylogenetic tree and network profile show a clear separation between domestic and wild sheep, which reject the previous hypothesis that the Tibetan and Mongolian sheep group may be derived from argali and urial. Moreover, neither argali nor urial mtDNA is closely related to Erlitou ancient sheep. These results suggested that lineage A has additional wild ancestor, which might become extinct long time ago. Notably, despite different chromosome number, domestic sheep (2n ¼ 54) can interbreed with wild sheep such as mouflon (2n ¼ 54), argali (2n ¼ 56) and urial (2n ¼ 58), and the offspring have normal fertility (Nadler et al., 1973). Furthermore, Vila` et al. (2005) revealed that the current genetic diversity presented in modern domestic mammals does not derive solely from the time of domestication, but could have been increased through extended gene flow from the wild species to the domesticates in the form of backcrosses. These results suggest that the origin of modern sheep breeds was more complex than previously thought. To further search the wild ancestry, the contributions from wild male sheep to Y-chromosome should also be taken into account. 4.5. Implications from this study Besides revealing the origin and history of domestic animals, DNA extracted from faunal remains excavated from

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archaeological sites has also proven to be a valuable resource in tracing prehistoric population movement (Matisoo-Smith and Robins, 2004). Human populations have frequently manipulated animal species, by domestication, and by moving them beyond their endemic range. Meadows et al. (2005) observed high levels of gene flow between breeds of domestic sheep from Asia and Europe, and suggesting that introgression has played a major part during breed development and subsequent selection. The fundamental factor of high levels of gene flow could be attributed to human migratory movements and commercial trade in history (Luikart et al., 2001). These results implied that prehistoric population movement could be traced by detecting the distribution of lineage presented in archaeological sheep remains. For example, assuming the Near Eastern origin of lineage B is correct, lineage B observed in Chinese breeds may be derived from the Near East. It is possible to trace the spread of lineage B through ancient DNA analysis of sheep remains from the archaeological sites located in the possible migratory routes. In this study, it is worthy of note that all samples belonged to lineage A; no other lineages were found. Nevertheless, due to small sample size (only eight samples) we cannot jump to the conclusion on the spread of lineage B. However, our results provide a hint that the spread of lineage B could be revealed after adding more early samples from other archaeological sites in China, which will provide new insights into the origin of Chinese domestic sheep and human migratory movements. 5. Conclusion Ancient DNA analysis was successfully applied to eight archaeological sheep remains recovered from Erlitou archaeological site to explore the genetic structure of ancient sheep and the phylogenetic relationship between ancient and modern sheep. In summary: 1. Ancient DNA was successfully extracted from ancient sheep remains using strict contamination control and decontamination measures. Amplifications of both mtDNA control region and coding region were accomplished. To minimize PCRs errors, double strand sequencings of the same sample were performed. The replicated results strong support the authenticity of ancient DNA samples in this study. 2. Sequence motif, RFLP and Phylogenetic analysis of ancient sheep mtDNA consistently revealed that Erlitou ancient sheep belonged to lineage A. Neither argali nor urial mtDNA is closely related to Erlitou ancient sheep. Moreover, our results reject the previous hypothesis that Chinese Tibet and Mongolian sheep groups may be derived from argali and urial. To further investigate the wild ancestor, the factors of male backcrosses should be of consideration. 3. Our results provide a hint that the spread of lineage induced by human migratory movements and commercial trade in history may be traced through detecting the

distribution of lineage presented in ancient sheep remains. More early ancient sheep samples from different archaeological sites in China could provide sufficient genetic data to understand the origin of Chinese domestic sheep and human migratory movements. Acknowledgements The work was supported by the Ministry of Science and Technology of the People’s Republic of China (grant no. 2004BA812B03), project name: Study on the development of economy and technology in the Central Plain area from 2500e1500 B.C. and its relationship with occurrence of China civilization. The work was also supported by the National Science Fund for Fostering Talents in Basic Research (grant no. J0530184). We are grateful to Dr Jing Yuan who supplied ancient sheep samples. We also thank Dr Zhuo-Wei Tang, Dr Yao-Fong Chen, Dr Xu-Long Lai and Dr Suratissa for correcting grammar and helpful comments on the manuscript. References Bandelt, H.J., Forster, P., Rohl, A., 1999. Median joining networks for inferring intraspecific phylogenies. Molecular Biology and Evolution 16, 37e48. Chen, W.H., 1994. Agricultural Archaeology Catalogue of China. Jiangxi Scientific & Technical Publishers, Jiangxi (in Chinese). Chen, S.Y., Duan, Z.Y., Sha, T., Xiangyu, J., Wu, S.F., Zhang, Y.P., 2006. Origin, genetic diversity, and population structure of Chinese domestic sheep. Gene 376, 216e223. Cooper, A., Poinar, H.N., 2000. Ancient DNA: do it right or not at all. Science 289, 1139. Diamond, J., 2002. Evolution, consequences and future of plant and animal domestication. Nature 418, 700e707. Guo, J., Du, L.X., Ma, Y.H., Guan, W.J., Li, H.B., Zhao, Q.J., Li, X., Rao, S.Q., 2005. A novel maternal lineage revealed in sheep (Ovis aries). Animal Genetics 36, 331e336. Hiendleder, S., Mainz, K., Plante, Y., Lewalski, H., 1998. Analysis of mitochondrial DNA indicates that domestic sheep are derived from two different ancestral maternal sources: no evidence for contributions from urial and argali sheep. Journal of Heredity 89, 113e120. Hiendleder, S., 1998. A low rate of replacement substitutions in two major Ovis aries mitochondrial genomes. Animal Genetics 29, 116e122. Hiendleder, S., Phua, S.H., Hecht, W., 1999. A diagnostic assay discriminating between two major Ovis aries mitochondrial DNA haplogroups. Animal Genetics 30, 211e213. Hiendleder, S., Kaupe, B., Wassmuth, R., Janke, A., 2002. Molecular analysis of wild and domestic sheep questions current nomenclature and provides evidence for domestication from two different subspecies. Proceedings of the Royal Society B 269, 893e904. Kaestle, F.A., Horsburgh, K.A., 2002. Ancient DNA in anthropology: method, applications, and ethics. Yearbook of Physical Anthropology 45, 92e130. Kumar, Tamura, Nei, 2004. MEGA3: Integrated Software for Molecular Evolutionary Genetics Analysis and Sequence Alignment. Briefings in Bioinformatics 5, 150e163. Leonard, J.A., Wayne, R.K., Wheeler, J., Valadez, R., Guille´n, S., Vila`, C., 2002. Ancient DNA evidence for Old World origin of New World dogs. Science 298, 1613e1616. Li, W.M., 2004. The development and utilization of animal resources in Erlitou culture. Huaxa Archaeology 2, 40e45 (in Chinese). Lu, S.X., Chang, H., Du, Tsunodak, L., Ren, Z.J., Yang, Z.P., Sun, W., Chang, G.B., Wang, Q.H., Xu, M., Guo, X.Y., Ren, X.L., 2005. Study

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