Investigating dietary patterns and human mobility in bone apatite at the Zhuangqiaofen site (5000-3700 BP), Zhejiang Province, China

Accepted Manuscript Investigating dietary patterns and human mobility in bone apatite at the Zhuangqiaofen site (5000-3700 BP), Zhejiang Province, China Yi Guo, Jie Lou, Chen Xiang, Yang Xia, Xinmin Xu PII:

S1040-6182(18)30083-1

DOI:

10.1016/j.quaint.2018.05.047

Reference:

JQI 7457

To appear in:

Quaternary International

Received Date: 25 January 2018 Revised Date:

1 May 2018

Accepted Date: 31 May 2018

Please cite this article as: Guo, Y., Lou, J., Xiang, C., Xia, Y., Xu, X., Investigating dietary patterns and human mobility in bone apatite at the Zhuangqiaofen site (5000-3700 BP), Zhejiang Province, China, Quaternary International (2018), doi: 10.1016/j.quaint.2018.05.047. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Department of Cultural Heritage and Museology, School of Humanities, Zhejiang University, Hangzhou, 310028, China 2 Department of Archaeology and Anthropology, University of Chinese Academy of Science, Beijing, 100049, China 3 Archaeology Institute of Zhejiang Province, Hangzhou, 310014, China

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†=Co-first author *=Correspondence to: e-mail: [email protected]

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Abstract The Taihu Lake basin is one of the cradles of Chinese civilization with abundant archaeological sites, but the humid and acidic soil conditions of this part of China limit the application of studies using stable isotope ratios of bone collagen. To circumvent this problem, we explore the suitability of using bone hydroxyapatite for stable isotope ratio measurements of carbon (δ13C) and oxygen (δ18O) as a substitute to collagen in order to obtain dietary and mobility information about humans (n=22) and animals (n=7) from the Neolithic site of Zhuangqiaofen. The human δ13C values range from –14.0‰ to –11.5‰ and have an average of –12.8 ± 0.7‰ which indicates that they were mainly consuming a C3 diet, most likely rice (Oryza sativa). The range of δ13C values of the dogs (–13.6‰ to –12.5‰; n=5) and pigs (–13.9‰ and –12.7‰; n=2) are similar to the humans and also suggest that they consumed C3 foods. Humans have a wide range of δ18O values (–12.7‰ to –8.5‰) with an average value of –10.8 ± 1.2‰, which suggests that they were mobile and ingested water from a variety of different sources. This human mobility might be attributed to: the need for labor to construct public works at larger sites in the area, trade networks involving jade and pottery in the Jianghuai region or to the unique funerary customs of the Liangzhu Culture that were based on blood relationships. In contrast, the dog δ18O values have a much smaller range (–12.0‰ to –11.4‰) and have an average value of –11.7 ± 0.2‰. The δ18O values of the two pigs are nearly identical at –12.1‰ and –12.2‰ and similar to the dogs, and these results can be used as an isotopic baseline with which to compare the human δ18O results.

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Yi Guo1†*, Jie Lou1†, Chen Xiang1, Yang Xia2, Xinmin Xu3

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Investigating dietary patterns and human mobility in bone apatite at the Zhuangqiaofen site (5000-3700 BP), Zhejiang Province, China

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Keywords: bone apatite; stable isotopes; diet; mobility

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The Taihu Lake basin is believed to be one of the most important areas for the development of Chinese civilization (Zhang, 1995; Zhang, 2012). Near the center of the basin is Taihu Lake, which is the third largest in China (Figure 1a), and the region is located to the south of the lower Yangtze River and west of the modern city of Shanghai (Xu et al., 1996; Wei, 2013). Archaeological excavations have revealed over 200 Neolithic sites in the Taihu basin and the Neolithic cultural sequence has been divided into the Majiabang Culture (c. 7000-6000 BP), the Songze Culture (c. 6000-5300 BP) and the Liangzhu Culture (c. 5000-3700 BP) (Huang, 2009; Wang, 2014).

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While many sites have been studied with stable isotope ratio analysis in north China (e.g. Hu et al., 2005; Cui et al., 2006; Wang et al., 2012; Si et al., 2014; Ma et al., 2016a; 2016b; Wang et al., 2017), there has been considerably less research completed on sites in southern China. This is mainly due to the fact that the warm and humid climate and acidic soils of south China are not suitable for the preservation of organic biomolecules such as collagen, which is frequently used in paleodiet reconstruction (e.g. Lee-Thorp, 2008). As a result of these problems, only two sites have been previously isotopically investigated using collagen in the Taihu basin, Songze (Zhang et al., 2003) and Sanxingcun (Hu et al., 2007). This lack of suitable organic material has severely hampered efforts to reconstruct the subsistence strategies and life ways of the Neolithic cultures of the Taihu Lake basin. To circumvent this problem, studies on skeletal remains have turned to the analysis of hydroxyapatite or bioapatite in bone and tooth enamel, as it has the ability to possibly preserve for longer periods than bone collagen (Lee-Thorp et al., 1989; Qu et al., 2014). Here we present a pilot study of bone bioapatite from humans (n=33) and animals (dogs (n=7) and pigs (n=2)) from the Neolithic site of Zhuangqiaofen located in the Taihu Lake basin. The aim of this research is to investigate the dietary patterns and human mobility of Zhuangqiaofen site via the approach of stable isotope analysis of bioapatite samples. First we need to determine if the bone bioapatite at this site is suitable for carbon (δ13C) and oxygen (δ18O) stable isotope ratio analysis and free of diagenetic alteration. Thus, we investigate the bone apatite crystal structure with the methods of X-ray diffraction (XRD) and Fourier Transform Infra-Red Spectroscopy (FTIR) (Sponheimer et al., 1999; Ma et al., 2016c). Further, the isotopic results are examined to learn more information about subsistence strategies and human mobility in the Taihu Lake basin.

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1. Introduction

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2. Isotopic analysis of bioapatite The stable isotope ratio analysis of δ13C and δ18O values in apatite can be used to determine dietary patterns and water sourcing/availability during the period of tissue (bone, teeth) mineralization (Bocherens, 2011; Dotsika, 2011;King, 2013; Sjögren, 2013; Hamre, 2016; Medin, 2017). The details of the technique and the different 2

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3.1 The Zhuangqiaofen site

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3. Materials and methods

The Zhuangqiaofen site is located in Pinghu City, Zhejiang Province, China (30°40′N, 120°35′E), about 5 km north from Hangzhou Bay (Figure 1a). It was excavated in 2003 and 2006 and is linked to the Liangzhu Culture (c. 5000-3700 BP) by artifact analysis. Three man-made earthen platforms and 270 burials have been excavated (Xu and Cheng, 2005; Cheng et al., 2006). The tombs are rectangular earthen pits with most individuals buried in the extended position. Many of the burials contain a rich array of grave goods (pottery, stone, jade, bone, antler, ivory and wooden artifacts) (Figure 1 b,c,d), and the site represents the largest Liangzhu cultural cemetery found in China to date (Xu and Cheng, 2005). Because of the sample, shape and stratigraphy evidence, it is believed that belongs to Liangzhu culture period (5000-3700BP) by archaeologists. In addition, approximately 100 ash-pits, ash-trenches and sacrificial pits have been found. Here, 42 bone samples selected from the Zhuangqiaofen site are investigated, including 33 human samples, 7 dog samples and 2 pig samples (Table 1).

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applications as well as the drawbacks have been well described in numerous publications (e.g. Tykot, 2004; Lee-Thorp, 2008). Briefly, the different photosynthetic pathways of C3 and C4 plants have distinct carbon stable isotope ratios. The δ13C results of C3 plants range between –30‰ to –23‰ and the δ13C values of C4 plants range between, –16‰ to –9‰ (O'Leary, 1981; Hastorf et al., 1989). These isotopic signatures are passed onto to the consumers of these plants, both humans and animals, in the food chain. There is a fractionation factor of ~12‰ between diet and the carbonate ion (CO32-) in bone apatite (DeNiro, 1978). In China, δ13C values can be used to distinguish the consumption of C3 (rice, wheat, etc.) and C4 based foods, mainly millets (Guo et al., 2015; Wang et al. 2017). While bone collagen results provide information about the dietary protein intake, bone apatite reflects the whole diet that was consumed (Lee-Thorp, 2008). Oxygen isotope ratios in apatite are determined by the water, which is ingested either as liquid or in food sources (Bryant et al., 1994; 1996; Fenner et al., 2016). In rainwater, δ18O values are related to the δ18O values of the source water (usually the ocean surface), the number and intensity of rainout events, elevation and air temperature (Clark and Fritz, 1997). Most areas tend to have fairly constant rainwater δ18O values through time so the δ18O results of humans and animals can be linked to the geography in which they lived. Thus, δ18O measurements can be used to reconstruct human and animal water sources, habitat and potential mobility patterns (White et al., 1998; Sjögren et al., 2013; Hamre, 2016).

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3.2 Bone apatite preparation The bones were prepared according to the protocol outlined in Lee-Thorp et al. (1989; 1991). Briefly, the bones were sonicated in an ultrasonic bath for 10 min to dislodge sediment. Then, the bone surface was removed using a sander-blaster to get rid of 3

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3.3.1 X-ray diffraction analysis

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The finely ground bone apatite powder was placed on a glass slide and measured with a Rigaku D/MAX-2550/PC Powder X-ray Diffractometer, located at the Department of Chemistry, Zhejiang University. The phase compositions of the bone apatite were identified through the XRD peaks by MDI Jade 8.0 software and the XRD patterns of the samples were produced by Origin 8.6. 3.3.2 Fourier Transform Infra-Red Spectroscopy

The bone apatite powder and potassium bromide (1:100) were mixed, ground to a fine powder with an agate mortar and pestle, and made into a KBr pellet. Absorbance and infrared spectra were obtained between 4000 and 400 cm-1 wave numbers using a VECTOR 22 Fourier Transform Infra-Red Spectroscopy (FTIR) located in the Department of Chemistry, Zhejiang University. Resolution was set at 1 cm-1. The pure KBr background spectrum was subtracted from the sample spectrum and the absorbance spectra were baseline-calibrated and plotted using the software of OMNIC 8.0 and Origin 8.6. The bone apatite crystallinity index, e.g., PCI (phosphate crystallinity index), BPI (B-carbonate on Phosphate Index) and P/C (CO3/PO4), were calculated according to the protocols addressed by previous studies (Michel et al., 1995; Sponheimer and Lee-Thorp, 1999), and the results are listed in Table 1.

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3.3 XRD, FTIR and isotopic analyses

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surface contamination. ~30 mg of bone powder was sampled using a Dremel drill and the powder was transferred to a centrifuge tube and immersed in 2% sodium hypochlorite (NaOCl) solution for 1-2 days at 4°C to eliminate organic matter. The residue was then centrifuged at 3000 rpm for 5 min and rinsed three times with distilled water. In order to remove diagenetic carbonates, 1 M acetic acid was added to each tube and refreshed every 1-2 days at 4°C until the effervescence ceased.(Somerville, 2015) Afterwards, the remains were washed three times with distilled water and the samples were freeze-dried. The prepared bone powder was then analyzed for XRD, FTIR and δ13C and δ18O results.

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3.3.3 Stable isotope analysis The δ13C and δ18O isotopic ratios of carbonate in bone apatite were measured by an Isoprime 100 Isotope Ratio Mass Spectrometry (IRMS) coupled with a multi-flow system at the Stable Isotope Laboratory in Department of Archaeology and Anthropology, University of Chinese Academy of Sciences. The bone apatite powder was packed into sealed glass tubes and flushed with high-purity helium. Then 1 ml of ultrapure phosphoric acid (H3PO4) at 70°C was injected into every tube using a disposable medical syringe. After the reaction was maintained at 80°C for an hour, the carbon dioxide released in the tube was eventually transported by the helium carrier gas 4

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4. Results and Discussion

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4.2 δ13C results of animals and humans

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The human (n=22) δ13C values range from –14.0‰ to –11.5‰ and have an average of –12.8 ± 0.7‰ (Figure 4). These results indicate some dietary variability, but that these individuals were mainly dependent on C3 foods. Considering the local environment of the Zhuangqiaofen site, the main C3 food consumed was most likely rice (Oryza sativa). The Taihu Lake basin is one of the birthplaces of Chinese rice agriculture (Zhu, 2004; Fan, 2011). This was especially true during the period of the Liangzhu Culture, as rice has been unearthed from nearly all of the Liangzhu Culture sites (Zhejiang Bureau of Cultural Relics, 1960; Changzhou Museum, 2001; Luojiajiao Archeological Team, 1981). This was also the case at the Zhuangqiaofen site where large quantities of charred rice were found in the ash pits. In addition, a stone plough with a wooden base was unearthed from pit H18. This plough is 1.06 meters long and the head shows clear signs of use. The excavation of this large-scale farming tool implies that a significant amount of agricultural activity was occurring at the site. Fruit pits and seeds of summer squash (C. Pepo L.), wild jujube (Ziziphus jujuba) and pear (Pyrus spp.) were also found in the ash-pits. A large number of fish bones and clamshells were also recovered at the site suggesting that aquatic resources were an important part of the diet.

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The diagenetic evaluation of the 42 bone apatite samples was previously reported by Guo et al. (2017), and will only be briefly discussed here. The XRD results showed that there are quartz and feldspar diffraction peaks in the XRD spectrum of 13 samples (see Table 1), meaning that these specimens were contaminated. However, the other 29 samples (22 human samples, 5 dog samples and 2 pig samples) were found to be uncontaminated with only diffraction peaks of hydroxyapatite present (Figure 2). In addition, the FTIR results of these 29 specimens show clear absorption peaks of hydroxyapatite that also indicates that they were well preserved (Figure 3). Thus, these 29 samples are suitable for stable isotope ratio analysis (Table 1; Figure 4).

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It is interesting to note that the individual (ZQF33) with the most 13C-enriched result (–11.5‰) was found buried in a flexed position. Flexed burials are common in southern China during the early Neolithic and are found more frequently in the middle reaches of the Yangtze River between 8000 – 5000 BP (Han, 2006). ZQF33 was only found buried with two grave goods, a tripod and small pieces of pottery (much less than most of the 5

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The dog (n=5) δ13C values are similar to the humans and range from –13.6‰ to –12.5‰ and have an average of –13.2 ± 0.4‰, which also suggest that they consumed C3-based foods. Domestic dogs have a close relationship with humans and can serve as companions or guards for their owners. They can be either provisioned by their owners or scavenge human refuse around a habitation site (Rick et al., 2011). At many archaeological sites, dogs and their human owners have similar isotopic signatures (Fischer et al., 2007; Allittet et al., 2008; Tankersley and Koster, 2009; Choy and Richards, 2009), and Cannon et al. (2009) proposed that analyzing the isotopic values of archaeological dog remains can serve as a proxy for reconstructing human diet. At the Zhuangqiaofen site, domestic dogs were found being buried with their owners in 12 tombs. The dogs were buried in the flexed position and located in either the northeast or northwest corner of the tombs. This might indicate that the domestic dogs had a close relationship with their owners. In addition, the δ13C values of dogs are all in the range of the humans, which indicates that they were consuming similar diets.

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The δ13C values of the two pigs are –13.9‰ and –12.7‰, which indicates that they were also consuming C3 foods. In contrast to dogs, pigs are regarded as a primary food source for humans, but are also known to survive on human refuse and waste (Halley and Rosvold, 2014). In the Taihu Lake basin, the domestication of pigs is thought to have occurred sometime during the Neolithic, and the bones of domestic pigs as well as ceramic models of pigs were found at the Luojiajiao site (c. 7000 BP) in Zhejiang Province (Zhang, 1981). During the Liangzhu Cultural period, nearly all of the sites have evidence of domestic pig bones (Ding, 2002), and the pigs from the Zhuangqiaofen site have been identified as being domesticated. 4.3 δ18O results of animals and humans The humans have a wide range of δ18O values (–12.7‰ to –8.5‰) with an average value of –10.8 ± 1.2‰ (Figure 4). This suggests that some of the humans were mobile at the Zhuangqiaofen site and/or were ingesting water from a variety of different sources. In general, domestic animals have a close relationship with humans and a constant range of activities, so their δ18O values are generally considered to be the local baseline values of a site. Compared to the humans, the dog δ18O values have a much smaller range (–12.0‰ to –11.4‰) and have an average value of –11.7 ± 0.2‰. The δ18O values of the two pigs are nearly identical at –12.1‰ and –12.2‰ and similar to the dogs.

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The sites of the Liangzhu Cultural period are believed to have been organized as chiefdoms or were in a pre-state stage with the settlements arranged according to hierarchy where the lower status settlements guarded the larger more important sites (Xie 1995; Zhang, 1995; Dai, 1997). According to the study of Guo (Guo, 2009), the 6

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Zhuangqiaofen site belongs to the second level of the settlement hierarchy, while several first level settlements, like the Fuquanshan site (5000-3700 BP) is located in close proximity (Figure 1b). The Fuquanshan site consists of a large platform built with approximately 16000 cubic meters of soil and has a large altar in the middle of the platform. The construction of these public facilities likely required a significant amount of labor so individuals from the lower status settlements may have been conscripted. A study of the physical anthropology of the skeletons from the late Liangzhu Culture site of Guangfulin (50 km northeast of the Zhuangqiaofen site), found that the humans were engaged in strenuous work (Wang, 2008), and in addition to normal daily activities, individuals may have been involved in the construction of these public work projects. As a lower-level settlement, the inhabitants of Zhuangqiaofen might have traveled to the other settlements to make some projects, then came back to bury, and this might a reason to account for the large variation in the δ18O values.

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There was also a large trade network involving jade and pottery during Liangzhu Cultural period, and these goods were widely circulated throughout the Taihu Lake basin and the wider Jianghuai region (the area that is between the Yangtze River and the Huai River) (Xu, 2013). For example, large quantities of jade and pottery from the Liangzhu Culture were found at the sites of Qingdun (Ji, 1983) and Luzhuang (Luan, 2000) in eastern Jianghuai and the Huating site (Gao, 2000) in northern Jianghuai. Thus, in addition to the flow of these goods, it is likely that individuals were traveling to these different locations, and this would account for the wide variation of δ18O bone apatite values.

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In addition, the unique funerary customs of the Liangzhu Culture that were based on blood relationships may have contributed to the diverse δ18O results of the Zhuangqiaofen individuals. In particular, one family with the same blood relationship is always found buried together in a certain place or village (Qin, 2000). For example, family burials are found at the Yaoshan cemetery (12 cemeteries belonged to the same family, see Wu, 2009) and the Wenjiashan cemetery (each phase of the cemetery was distributed between two lines and the internments maintained the same patterns and burial customs through time) (Archaeology Institute of Zhejiang Province, 2011). The Zhuangqiaofen site was made up of four burial districts and each district represented one family (Xu, 2005). According to the data, the pattern of diets can’t explain the family using the same liquid in the food. At the same time, different family Thus, as a result of strong family ties many individuals may have returned home from other locations to be buried in these family plots, and this could also account for the wide range of δ18O values seen here.

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5. Conclusions

Here we show that bone hydroxyapatite results from humans and animals at the Neolithic site of Zhuangqiaofen can be used to extract dietary information from skeletal material that is devoid of collagen. This is an important finding and will help spur more isotopic research on archaeological sites in southern China. In particular, the human δ13C results indicate that the diet was based on C3 resources, which agree with the archaeological finds of rice, fruits/seeds and fish/clams at the site. The range of δ13C values of the dogs and pigs were similar to the humans and also suggest that they consumed C3 foods. The δ18O values of the dogs and the pigs are tightly clustered and reflect the local environment of the site. In contrast, the wide range of human δ18O 7

ACCEPTED MANUSCRIPT values indicates that there was likely substantial human mobility at the Zhuangqiaofen site. This might be attributed to following: the need for labor to construct public works at larger sites in the area, trade networks involving jade and pottery in the Jianghuai region or to the unique funerary customs of the Liangzhu Culture that were based on blood relationships. Additional research using strontium, carbon and oxygen isotope ratios from enamels to figure out the varies in people’s childhood is planned to better understand these human migration patterns in terms of the geography of the Taihu Lake basin.

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Acknowledgements This work was supported by the Fundamental Research Funds for the Central Universities and the Philosophy and Social Science Project of Zhejiang Province (16NDJC171YB).

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Table 1. Sample information, FTIR peak data and isotope data. Description δ13C（‰） δ18O（‰）

Sample ID

Location

PCI

BPI

C/P

ZQF1

M167

human

-13.6

-10.4

3.26

0.57

0.19

ZQF2

M30

human

-14.0

-11.3

3.34

0.55

0.17

ZQF3

T302 H5

human

-13.0

-11.9

3.47

0.48

0.15

ZQF4

T603 (2)

human

-12.8

-11.7

3.47

0.49

0.17

ZQF5

T604 (9)

human

-12.1

-11.7

3.57

0.47

0.15

13

ACCEPTED MANUSCRIPT M269

human

-11.7

-12.0

3.78

0.45

0.14

ZQF7

T703 M269

human

-

-

-

-

-

ZQF8

M206

human

-13.1

-10.0

3.40

0.42

0.17

ZQF9

M230

human

-13.3

-12.1

3.59

0.48

0.14

ZQF10

T103 M131

human

-12.9

-10.9

3.85

0.41

0.12

ZQF11

M175

human

-

-

-

-

-

ZQF12

T102 M114

human

-

-

-

-

-

ZQF13

T303 (11)

human

-12.7

-11.3

3.43

0.52

0.17

ZQF14

M74

human

-

-

-

-

-

ZQF15

T703 M270

human

-11.9

-8.7

3.62

0.47

0.16

ZQF16

M205

human

-

-

-

-

-

ZQF17

M205

human

-

-

-

-

-

ZQF18

M27

human

-12.4

-8.9

3.95

0.42

0.12

ZQF19

T302 M144

human

-13.2

-11.1

3.39

0.47

0.15

ZQF20

M46

human

-13.1

-8.5

3.88

0.44

0.11

ZQF21

M204

human

ZQF22

M83

human

ZQF23

M20

human

ZQF24

M33

human

ZQF25

M115

human

ZQF26

M234

human

ZQF27

M207

human

ZQF28

M85

ZQF29

M164

ZQF30

M75

ZQF31

M19

ZQF32

M AN U

SC

RI PT

ZQF6

-12.7

3.35

0.5

0.16

-12.5

-10.9

3.72

0.51

0.15

-

-

-

-

-

-

-

-

-

-

-12.6

-9.7

3.65

0.43

0.14

-12.2

-10.2

3.56

0.43

0.13

-

-

-

-

-

TE D

-13.6

-13.4

-9.4

3.54

0.47

0.14

human

-

-

-

-

-

human

-11.8

-10.8

4.16

0.44

0.13

human

-

-

-

-

-

M83

human

-13.1

-12.3

4.09

0.46

0.14

ZQF33

T203 M128

human

-11.5

-11.8

3.79

0.47

0.16

ZQF34

M100

dog

-13.3

-11.7

3.53

0.48

0.15

AC C

EP

human

ZQF35

M271

dog

-12.5

-11.8

3.23

0.55

0.21

ZQF36

？

dog

-13.6

-11.4

3.67

0.47

0.13

ZQF37

M76

dog

-13.3

-11.8

3.15

0.56

0.19

ZQF38

T103 M85

dog

-

-

-

-

-

ZQF39

M66

dog

-

-

-

-

-

ZQF40

M47

dog

-13.2

-12.0

3.6

0.46

0.14

ZQF41

T603 (9)

pig

-13.9

-12.1

3.19

0.59

0.22

ZQF42

T303 H31

pig

-12.7

-12.2

2.98

0.6

0.27

518 519 520 521 14

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M AN U

SC

RI PT

522 523 524 525

526

EP

TE D

Figure 1. (a) Location of the Zhuangqiaofen site, Zhejiang, China (▲solid triangle); (b) excavation of M128 and the human (ZQF33) with flexed burial; (c) excavation of M47 with many grave goods, including stone-Yue, clay jars, clay pots and jade cones; (d) excavation of H18 with pig skeletons.

AC C

527 528 529 530 531 532 533 534 535 536

15

Figure 2. XRD spectrum of some uncontaminated bone samples

AC C

EP

TE D

537 538 539 540

M AN U

SC

RI PT

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541 542 543 544 545

Figure 3. FTIR spectrum of sample ZQF18

16

EP

TE D

Figure 4. Scatter plot of δ13C and δ18O values of humans and animals from the Zhuangqiaofen site.

AC C

546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567

M AN U

SC

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