Ancient Mitochondrial DNA Analysis of the Roman/Parthian Period Cemetery

at the Site of Tall Šēḫ Ḥamad, Syria

J.G.L. KENNEDY and D.A. MERRIWETHER. Department of Anthropology, State University of New York, Binghamton.

ABSTRACT The site of Tall Šēḫ Ḥamad presented a unique opportunity to conduct genetic analysis of a distinct mortuary population

dating to the Roman/Parthian period (200 BCE - 300 CE). The archaeological and historical evidence indicate a region in

constant political flux. This frontier, existing at the extremities of both the Roman and Parthian empires, created a unique

sphere of potential interactions both on the individual level and broader scale. This created a varied archaeological context

played out in mortuary practices and material artifacts at the site (Novak et al. 2000). In order to better understand these

contexts, but also the long-term processes at the site, a multi-proxy approach was used incorporating molecular techniques

and archaeological mortuary evidence to develop an understanding of the interments from the Roman/Parthian era

occupation at Tall Šēḫ Ḥamad, Syria. Ancient mitochondrial DNA profiles were generated for forty individuals and analyzed

to ascertain how they statistically correlated with mortuary archaeological information. Through this analysis it became

apparent that the individuals of Tall Šēḫ Ḥamad were not buried based upon matrilineal relationships. However, genetic

distance measured between different burial types potentially indicates a socio-economic distinction between classes at Tall

Šēḫ Ḥamad that may have impacted marriage practices.

BACKGROUND The site of Tall Šēḫ Ḥamad is situated approximately 70 km

northeast of the modern Syrian provincial capital of Deir az-Zor on

the left bank of the Khabur River, north of the Euphrates confluence

(Fig. 1). Archaeological excavations conducted by the Freie

Universität Berlin under the direction of Dr. Hartmut Kühne revealed

continuous occupation of this site from the Middle Assyrian I period

(around 1300 BCE, equivalent to the Early Iron Age) to the third

century CE. Of particular interest to this study is the Town II region

(Fig. 2) where a distinct Roman period cemetery is situated over the remains of an earlier Assyrian settlement (Kühne

2005). There are five types of Roman period burial types seen at the site. They consist of: mud brick graves (53.4%),

earthen or pit graves (31.8%), amphora graves (9.1%), sarcophagi (4.9%) and a few cremations (0.8%). Cremations are

not represented in the genetic assemblage.

Using a combination of archaeological information and mtDNA data the following questions have been proposed:

• Is there a significant genetic correlation among individuals with similar types of graves?

• If an association is found between different types of burials, what can then be determined about the burial

practices of this population?

• Is there a significant genetic correlation among individuals who were buried with similar objects?

• Again, if an association is present between individuals who were buried with similar objects, what conclusions can

be drawn about the sociality of death and burial based upon genetic affiliation?

MATERIALS AND METHODS One hundred and forty individuals were chosen from the assemblage at Tall Šēḫ Ḥamad based on observed

preservation, temporal associations, stratigraphy, grave type and form. These samples were stored in Berlin, Germany

under the supervision of Dr. Kühne, the director of the Tall Šēḫ Ḥamad excavations, and were sub-sampled and

transported to the D.A. Merriwether ancient DNA laboratory in August of 2009.

Two samples were taken from each individual. Teeth with complete roots were preferred and collected when available,

but when preservation was poor, ribs and long bone fragments were also chosen. Teeth are the optimal material as they

can be directly associated with a particular individual in situ and are easily transported. Samples were extracted using a

modified version of the Yang et al. (1998) protocol outlined in Lee et al. (2009). Extracts were amplified using four primer

sets targeting the Hypervariable region I (HVI) of the mitochondrial D-loop. The samples were then prepared and

sequenced under standard laboratory protocols in order to obtain the raw data needed for the production of individual

genetic profiles. Out of the 140 individuals collected, 105 were extracted with 40 samples exhibiting successful DNA

amplification for HVI. The successful samples were prepared under standard laboratory protocols described in

Merriwether et al. (1999) and direct cycle sequenced using a ABI 3730XL automated sequencer (Applied Biosystems,

Inc.). Sequences were analyzed and aligned with Sequencer 4.10.1 (Gene Codes, Inc.).

Population FST statistics were calculated in Arlequin 3.5 (Excoffier and Lischer 2010). The FST value is a standardized

measure of genetic variance among populations. It measures the likelihood for gene flow among the user-determined

groups (Excoffier and Lischer 2010). Zero or negative values within the matrix indicate no barriers to gene flow while

positive values indicate that something is impacting the gene flow among individuals. The hypothetical groupings (Table 1)

were created based upon the archaeological data provided in Novák et al. (2000).

Figure 1. Map of region with the site of Tall Šēḫ Ḥamad indicated in

red during the Roman/Parthian Period.

Figure 2. Map of excavation site with the Lower

Town II region circled in red (adapted from Novak

et al. 2000).

Table 1. Defined AMOVA groups for FST statistics.

Figure 3. Examples of finds associated with burials. A. adapted from Fig. 911, B. Fig. 909, C. Fig. 919, and D. Fig. 910;

Novak et al. 2000).

RESULTS While all of the examined FST values are not significant to p < .05 due to the small number of samples analyzed within each

group, they do give an indication of potential genetic distance between groups. In essence, any positive values indicate at

least some level of genetic difference between archaeologically defined groups, small though it may be. Further sampling

would easily resolve these issues and provide more statistically viable results.

• Out of the examined criteria only the burial type category showed positive values for the FST measurement. The

genetic distance between every group and that of individuals buried in amphora (Fig. 4) was above zero. This

indicated a "genetic barrier" between those buried in amphorae and every other type of burial.

The values indicated that there was the greatest genetic distance (0.0753) between those buried in amphorae and

sarcophagus graves (Fig. 5) with the least amount of genetic distance (0.0273) between amphorae and earthen

graves (Fig. 6).

• The next greatest distance (0.0340) was between mud brick graves (Fig. 7) and earthen graves again this value

was minimal but it showed that there was a measureable amount of genetic difference between these individuals.

• Mud brick graves and those buried in sarcophagi appeared to have the least amount of genetic distance with the

only negative FST value (-0.0168). Both of these burial types would have required time, resources and skill to create.

Whether or not an individual was buried with jewelry appears to be of little significance for genetic affiliation with FST values

below zero. Similarly whether individuals were buried with bells shows no genetic distance between any of the groups and

to those individuals buried without bells.

Figure 4. Example of amphora burial. Burial 92/070

(Adapted from Fig. 664 & 665; Novak et al. 2000).

Figure 6. Example of earthen grave. Burial 87/051

(Adapted from Fig. 625; Novak et al. 2000).

A B

C D

Figure 5. Example of sarcophagus burial. Burial

93/085 (Adapted from Fig. 730; Novak et al. 2000).

Figure 7. Example of mud brick tomb. Burial 95/027

(Adapted from Fig. 875 and 876; Novak et al. 2000).

CONCLUSIONS Amphorae, would have been an expensive commodity created by specialized trades people or groups. These individuals

could have been members of this particular occupational group. The amphorae all exhibited use-wear consistent with

having been used for oil and grain storage before use as a burial container. Pathological data indicated that those buried in

earthen graves had higher incidences of enamel hypoplasia, indicative of more nutritional stress and potentially a lower

socio-economic status. Since those buried in amphorae were genetically closest to those buried in earthen graves this

would suggest these individuals may have experienced a similar status. Secondary usage of amphorae may have been an

economic solution for burial.

Those buried in mud brick burials may be potentially within a different class, based on the time and resources needed in

order to properly manufacture the graves. Those buried in mud brick graves appeared pathologically to be in general better

health, having less degenerative joint disease and increased dental caries which suggest a diet high in carbohydrates

(grains).

It is difficult to ascertain whether these groups practiced exclusive endogamy within particular classes (such as an elite

class consisting of those buried in mud brick and sarcophagi graves and a lower class, those buried in earthen and

amphorae graves) since only maternal lineage information is present.

It does not appear that families, based upon matrilineal lines, chose these categories of objects to be placed with the

interred.

ACKNOWLEDGEMENTS We would like to thank the Lum Laboratory at Binghamton University for their laboratory assistance. Special thanks to Alondra Díaz-Lameiro,

Reinhard Bernbeck, Hartmut Kühne, and the Freie Universität Berlin.

REFERENCES Excoffier L, Lischer HEL. 2010. Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Molecular Ecology Resources. 10: 564-567. Kühne H. 2005. Magdalu/Magdala; Tall Šēḫ Ḥamad von der postassyrischen Zeit bis zur römischen Kaiserzeit. Berlin: Dietrich Reimer Verlag. Lee EJ, Anderson LM, Dale V, Merriwether DA. 2009. MtDNA origins of an enslaved labor force from the 18th century Schuyler Flatts Burial Ground in colonial Albany, NY: Africans, Native Americans, and Malagasy? Journal of Archaeological Science. 36(12):2805-2810. Merriwether DA. 1999. Freezer anthropology: new uses for old blood. Philosophical Transactions of the Research Society of London. B 354: 121-129. Novák M, Oettel A, Witzel C. 2000. Der parthisch-römische Friedhof von Tall Šēḫ Ḥamad /Magdala. In Series: Berichte der Ausgrabung Tall Šēḫ Ḥamad/ Dūr-Katlimmu (BATSH); Bd. 5. Berlin: Dietrich Reimer. Yang DY, Eng B, Waye JS, Dudar JC, Saunders SR. 1998. Improved DNA Extraction from Ancient Bones Using Silica-Based Spin Columns. American Journal of Physical Anthropology. 105:539-543.