In this post we present selected parts of the very interesting paper titled “Ancient Egyptian mummy genomes suggest an increase of Sub-Saharan African ancestry in post-Roman periods“, by Verena J. Schuenemann et al.
Abstract Egypt, located on the isthmus of Africa, is an ideal region to study historical population dynamics due to its geographic location and documented interactions with ancient civilizations in Africa, Asia and Europe. Particularly, in the first millennium BCE Egypt endured foreign domination leading to growing numbers of foreigners living within its borders possibly contributing genetically to the local population. Here we present 90 mitochondrial genomes as well as genome-wide data sets from three individuals obtained from Egyptian mummies. The samples recovered from Middle Egypt span around 1,300 years of ancient Egyptian history from the New Kingdom to the Roman Period. Our analyses reveal that ancient Egyptians shared more ancestry with Near Easterners than present-day Egyptians, who received additional sub-Saharan admixture in more recent times. This analysis establishes ancient Egyptian mummies as a genetic source to study ancient human history and offers the perspective of deciphering Egypt’s past at a genome-wide level.
Introduction Until now the study of Egypt’s population history has been largely based on literary and archaeological sources and inferences drawn from genetic diversity in present-day Egyptians. Both approaches have made crucial contributions to the debate but are not without limitations. On the one hand, the interpretation of literary and archaeological sources is often complicated by selective representation and preservation and the fact that markers of foreign identity, such as, for example, Greek or Latin names and ethnics, quickly became ‘status symbols’ and were adopted by natives and foreigners alike. On the other hand, results obtained by modern genetic studies are based on extrapolations from their modern data sets and make critical assumptions on population structure and time. The analysis of ancient DNA provides a crucial piece in the puzzle of Egypt’s population history and can serve as an important corrective or supplement to inferences drawn from literary, archaeological and modern DNA data.
Despite their potential to address research questions relating to population migrations, genetic studies of ancient Egyptian mummies and skeletal material remain rare, although research on Egyptian mummies helped to pioneer the field of ancient DNA research with the first reported retrieval of ancient human DNA. Since then progress has been challenged by issues surrounding the authentication of the retrieved DNA and potential contaminations inherent to the direct PCR method. Furthermore, the potential DNA preservation in Egyptian mummies was met with general scepticism: The hot Egyptian climate, the high humidity levels in many tombs and some of the chemicals used in mummification techniques, in particular sodium carbonate, all contribute to DNA degradation and are thought to render the long-term survival of DNA in Egyptian mummies improbable. Experimental DNA decay rates in papyri have also been used to question the validity and general reliability of reported ancient Egyptian DNA results. The recent genetic analysis of King Tutankhamun’s family is one of the latest controversial studies that gave rise to this extensive scholarly debate. New data obtained with high-throughput sequencing methods have the potential to overcome the methodological and contamination issues surrounding the PCR method and could help settle the debate surrounding ancient Egyptian DNA preservation.
Here, we provide the first reliable data set obtained from ancient Egyptians using high-throughput DNA sequencing methods and assessing the authenticity of the retrieved ancient DNA via characteristic nucleotide misincorporation patterns and statistical contamination tests to ensure the ancient origin of our obtained data.
Results
Samples and anthropological analysis All 166 samples from 151 mummified individuals used in this study were taken from two anthropological collections at the University of Tübingen and the Felix von Luschan Skull Collection, which is now kept at the Museum of Prehistory of the Staatliche Museen zu Berlin, Stiftung preußischer Kulturbesitz (individuals: S3533, S3536, S3544, S3552, S3578, S3610). According to the radiocarbon dates, the samples can be grouped into three time periods: Pre-Ptolemaic (New Kingdom, Third Intermediate Period and Late Period), Ptolemaic and Roman Period. During their conservation in the Tübingen and Berlin collections the remains underwent different treatments: some were preserved in their original mummified state, while others were macerated for anthropological analysis or due to conservation problems.
The excellent bone preservation and the more or less good soft tissue preservation made a wide-ranging analysis possible.
For the DNA analysis we sampled different tissues (bone, soft tissue, tooth), macerated and non-macerated, to test for human DNA preservation.
Processing and sequencing of the samples We extracted DNA from 151 mummified human remains and prepared double-stranded Illumina libraries with dual barcodes. Then we used DNA capture techniques for human mitochondrial DNA and for 1.24 million genomic single nucleotide polymorphisms (SNPs) in combination with Illumina sequencing, through which we successfully obtained complete human mitochondrial genomes from 90 samples and genome-wide SNP data from three male individuals passing quality control.
Comparison of the DNA preservation in different tissues We tested different tissues for DNA preservation and applied strict criteria for authenticity on the retrieved mitochondrial and nuclear DNA to establish authentic ancient Egyptian DNA.
Importantly, mtDNA haplotypes were identical for all samples from the same individuals. Our results thus suggest that DNA damage in Egyptian mummies correlates with tissue type.
Generation of nuclear data In order to analyse the nuclear DNA we selected 40 samples with high mtDNA coverage and low mtDNA contamination. Overall, the nuclear DNA showed poor preservation compared to the mtDNA as depicted by a high mitochondrial/nuclear DNA ratio of on average around 18,000. In many samples, nuclear DNA damage was relatively low, indicating modern contamination. We applied strict criteria for further analysis. Three out of 40 samples fulfilling these criteria had acceptable nuclear contamination rates: Two samples from the Pre-Ptolemaic Periods (New Kingdom to Late Period), and one sample from the Ptolemaic Period. To rule out any impact of potential contamination on our results, we analysed the three samples separately or replicated results using only the least contaminated sample.
Analysis of mitochondrial genomes The 90 mitochondrial genomes fulfilling our criteria were grouped into three temporal categories based on their radiocarbon dates, corresponding to Pre-Ptolemaic Periods (n=44), the Ptolemaic Period (n=27) and the Roman Period (n=19). To test for genetic differentiation and homogeneity we compared haplogroup composition, calculated FST-statistics and applied a test for population continuity on mitochondrial genome data from the three ancient and two modern-day populations from Egypt and Ethiopia, published by Pagani and colleagues, including 100 modern Egyptian and 125 modern Ethiopian samples.
We observe highly similar haplogroup profiles between the three ancient groups. Modern Egyptians share this profile but in addition show a marked increase of African mtDNA lineages L0–L4 up to 20%. Genetic continuity between ancient and modern Egyptians cannot be ruled out by our formal test despite this sub-Saharan African influx, while continuity with modern Ethiopians is not supported.
To further test genetic affinities and shared ancestry with modern-day African and West Eurasian populations we performed a principal component analysis (PCA) based on haplogroup frequencies and Multidimensional Scaling of pairwise genetic distances. We find that all three ancient Egyptian groups cluster together, supporting genetic continuity across our 1,300-year transect. Both analyses reveal higher affinities with modern populations from the Near East and the Levant compared to modern Egyptians. The affinity to the Middle East finds further support by the Y-chromosome haplogroups of the three individuals for which genome-wide data was obtained, two of which could be assigned to the Middle-Eastern haplogroup J, and one to haplogroup E1b1b1 common in North Africa.
Population genetic analysis of nuclear DNA On the nuclear level we merged the SNP data of our three ancient individuals with 2,367 modern individuals and 294 ancient genomes and performed PCA on the joined data set. We found the ancient Egyptian samples falling distinct from modern Egyptians, and closer towards Near Eastern and European samples. In contrast, modern Egyptians are shifted towards sub-Saharan African populations.
The three ancient Egyptians differ from modern Egyptians by a relatively larger Near Eastern genetic component, in particular a component found in Neolithic Levantine ancient individuals.
In contrast, a substantially larger sub-Saharan African component, found primarily in West-African Yoruba, is seen in modern Egyptians compared to the ancient samples.
We did not find significant differences between the three ancient samples, despite two of them having nuclear contamination estimates over 5%, which indicates no larger impact of modern DNA contamination.
We find that ancient Egyptians are most closely related to Neolithic and Bronze Age samples in the Levant, as well as to Neolithic Anatolian and European populations.
When comparing this pattern with modern Egyptians, we find that the ancient Egyptians are more closely related to all modern and ancient European populations that we tested, likely due to the additional African component in the modern population observed above.
The substantially lower African component in our ∼2,000-year-old ancient samples suggests that African gene flow in modern Egyptians occurred indeed predominantly within the last 2,000 years.
Discussion This study demonstrates that the challenges of ancient DNA work on Egyptian mummies can be overcome with enrichment strategies followed by high-throughput DNA sequencing. The use of ancient DNA can greatly contribute towards a more accurate and refined understanding of Egypt’s population history. More specifically, it can supplement and serve as a corrective to archaeological and literary data that are often unevenly distributed across time, space and important constituents of social difference (such as gender and class) as well as modern genetic data from contemporary populations that may not be fully representative of past populations.
The archaeological site Abusir el-Meleq was inhabited from at least 3250BCE until about 700CE and was of great religious significance because of its active cult to Osiris, the god of the dead, which made it an attractive burial site for centuries.
Abusir el-Meleq is arguably one of the few sites in Egypt, for which such a vast number of individuals with such an extensive chronological spread are available for ancient DNA analysis.
By comparing ancient individuals from Abusir el-Meleq with modern Egyptian reference populations, we found an influx of sub-Saharan African ancestry after the Roman Period.
Further investigation would be needed to link this influx to particular historic processes. Possible causal factors include increased mobility down the Nile and increased long-distance commerce between sub-Saharan Africa and Egypt. Trans-Saharan slave trade may have been particularly important as it moved between 6 and 7 million sub-Saharan slaves to Northern Africa over a span of some 1,250 years, reaching its high point in the nineteenth century. However, we note that all our genetic data were obtained from a single site in Middle Egypt and may not be representative for all of ancient Egypt. It is possible that populations in the south of Egypt were more closely related to those of Nubia and had a higher sub-Saharan genetic component, in which case the argument for an influx of sub-Saharan ancestries after the Roman Period might only be partially valid and have to be nuanced. Throughout Pharaonic history there was intense interaction between Egypt and Nubia, ranging from trade to conquest and colonialism, and there is compelling evidence for ethnic complexity within households with Egyptian men marrying Nubian women and vice versa. Clearly, more genetic studies on ancient human remains from southern Egypt and Sudan are needed before apodictic statements can be made.
The ancient DNA data revealed a high level of affinity between the ancient inhabitants of Abusir el-Meleq and modern populations from the Near East and the Levant. This finding is pertinent in the light of the hypotheses advanced by Pagani and colleagues, who estimated that the average proportion of non-African ancestry in Egyptians was 80% and dated the midpoint of this admixture event to around 750 years ago. Our data seem to indicate close admixture and affinity at a much earlier date, which is unsurprising given the long and complex connections between Egypt and the Middle East. These connections date back to Prehistory and occurred at a variety of scales, including overland and maritime commerce, diplomacy, immigration, invasion and deportation. Especially from the second millennium BCE onwards, there were intense, historically- and archaeologically documented contacts, including the large-scale immigration of Canaanite populations, known as the Hyksos, into Lower Egypt, whose origins lie in the Middle Bronze Age Levant.
Our genetic time transect suggests genetic continuity between the Pre-Ptolemaic, Ptolemaic and Roman populations of Abusir el-Meleq, indicating that foreign rule impacted the town’s population only to a very limited degree at the genetic level. It is possible that the genetic impact of Greek and Roman immigration was more pronounced in the north-western Delta and the Fayum, where most Greek and Roman settlement concentrated, or among the higher classes of Egyptian society. Under Ptolemaic and Roman rule, ethnic descent was crucial to belonging to an elite group and afforded a privileged position in society. Especially in the Roman Period there may have been significant legal and social incentives to marry within one’s ethnic group, as individuals with Roman citizenship had to marry other Roman citizens to pass on their citizenship. Such policies are likely to have affected the intermarriage of Romans and non-Romans to a degree. Additional genetic studies on ancient human remains from Egypt are needed with extensive geographical, social and chronological spread in order to expand our current picture in variety, accuracy and detail.
NovoScriptorium: We remain sceptical about the accuracy of ancient DNA evaluation for various reasons. Nevertheless, our core belief is to ‘stick with Science’ and hope that Time will reveal the actual Truth of things. Now, about the above results, our opinion is that it wouldn’t be in the wrong direction to claim that they partially confirm the ancient Tradition & History.
For instance, ancient Greek Mythology suggests that Egypt had been a Greek kingdom at some point – long before Alexander the Great. To be accurate, a kingdom where the population was a combination of indigenous people and Greeks. Greeks were also described as the ruling class. Danaus (Δαναός) was the brother of Aegyptus (Αίγυπτος), both children of Belus (Βήλος), who all had Epaphus (Έπαφος) as their ancestor. There is a reference in Apollodorus’ ‘Library’ that a family of Scientists from Rhodes, the Heliades (Ηλιάδαι) have forseen the coming Cataclysm (probably the last big one) and migrated to Egypt in order to save their lives, and there they built Heliopolis (Ηλιούπολις) (after their family name). Reasonably, this suggests very good relationship with Egypt and its leaders at the time. They don’t just let you build a whole new city in a foreign land! Interestingly, Apollodorus also writes about the very close relationships of ancient Egypt (of this distant and unknown era) with the Levantine. Actually, he writes about blood relationships between the two. We can also combine information from ‘The Suppliants‘ of Aeschylus, where the ‘dark-skinned’ women from Egypt, the Danaids (Δαναΐδαι), arriving at Argos (Άργος) are, eventually, recognised as being of Greek genous (whatever ‘Greek‘ meant back then – from a modern scientific point of view, most likely we are talking about a cultural group comprising of indigenous, Western Eurasian and Anatolian ancestries), despite their different external appearance. We can also extract information from Manetho the Egyptian, who in his work “Aegyptiaca” clearly refers to Greeks governing Egypt in very ancient times (‘gods and demi-gods’ era).
Then, we know historically that the Hyksos came to Egypt from the Levantine. The Bible also informs us about people of Semitic origin living there.
Therefore, taking in account the ancient Tradition, the results of this research come as no real surprise; the ancient Egyptians must have been a population comprising of Anatolian, Near Eastern, Northern African and Western Eurasian ancestries.
Research-Selection for NovoScriptorium: Maximus E. Niles
Comments for NovoScriptorium: Isidoros Aggelos
Novo Scriptorium 
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