Nubia, situated along the Nile River between Aswan and Khartoum, boasts a rich history of human occupation and interaction between diverse populations from Africa and West Eurasia. Archaeological expeditions throughout the 20th century explored the relationships between Nubian groups and those from north of the Sahara and sub-Saharan Africa, contributing to ongoing debates about the region's history. A particularly significant and enduring relationship existed between Nubia and Egypt, intensifying over millennia. The introduction of Christianity in 542 CE and the Arab conquests starting in the 7th century CE significantly impacted Nubia, with Egypt often acting as an intermediary for West Eurasian influence. Ancient DNA analysis, when integrated with archaeology, offers valuable insights into the genetic ancestry of ancient populations. This study presents genome-wide data from 66 individuals from Kulubnarti, a site located between the Nile's Second and Third Cataracts, during the early Christian Period (~650–1000 CE). Kulubnarti provides an ideal setting to investigate Nubian genetic ancestry in the mid- to late-1st millennium CE and offers a unique opportunity to address fine-scale questions raised by archaeological and bioarchaeological research. The site's location in the Batn el Hajar region, separating Lower and Upper Nubia, and its two distinct cemeteries, which exhibit differences in morbidity and mortality suggestive of social stratification, are central to the research questions. Previous studies based on cranial and dental traits suggested similarities between Kulubnarti Nubians and those from Wadi Halfa, but genome-wide data offer higher resolution to understand biological relationships. Existing genetic studies of modern Nubians show a mix of sub-Saharan African and West Eurasian ancestry, largely attributed to the Arab conquest. Therefore, analyzing paleogenomic data from Kulubnarti enables a direct investigation of the ancestry and relationships of a Nubian group predating the Islamic migrations, mitigating the obscuring effects of more recent admixture events.

Prior research on Nubia's population history has relied on various methodologies, yielding sometimes contradictory conclusions. Craniometric and dental analyses have been employed to assess biological relationships and continuity across different periods, but the resolution of these methods is limited compared to genomic data. Studies of modern Nubian populations have utilized genetic markers to investigate ancestry and population structure, revealing a mixture of sub-Saharan African and West Eurasian ancestry. However, interpreting these modern datasets requires careful consideration of the influence of post-Christian Period migrations, especially the Arab conquest, which introduced substantial West Eurasian ancestry. The archaeological record of Kulubnarti, with its two distinct cemeteries exhibiting differences in grave goods and bioarchaeological indicators of socioeconomic disparity, has prompted questions about the genetic relationships between the individuals interred in these locations. This study aims to address these unresolved questions using ancient DNA analysis, providing a higher resolution perspective on the complex population history of Nubia.

This study analyzed ancient DNA from 111 individuals from Kulubnarti, extracting DNA from petrous bones in dedicated ancient DNA cleanroom facilities. The DNA was extracted using published protocols and sequenced using Illumina NextSeq500 or HiSeqX10 instruments. Genome-wide SNP capture data was generated using in-solution target hybridization, enriching for sequences overlapping the mitochondrial genome and 1,233,013 genome-wide SNPs. Quality control measures were implemented to ensure data authenticity, including checks for contamination and DNA damage patterns characteristic of ancient DNA. A total of 66 individuals with sufficient data quality were included in the analysis. Radiocarbon dating was performed on 29 individuals to establish the temporal context of the samples. A Bayesian chronological model was used to estimate the start and end dates of the Christian-style burials in each cemetery. Genetic relatedness between individuals was assessed using a previously described method, identifying relatives up to the third/fourth degree. Runs of homozygosity (ROH) were analyzed to investigate patterns of consanguinity and population-level relatedness within the Kulubnarti population. Principal component analysis (PCA) was performed using smartpca to visualize the genetic relationships between the Kulubnarti individuals and both ancient and modern populations. Several statistical methods were employed to infer ancestry proportions and admixture history. *f*-statistics were used to identify admixture between Nilotic and West Eurasian ancestry components. qpAdm was used to estimate the proportions of these ancestries and identify the most likely sources of the West Eurasian component. A model competition approach was adopted to determine the most likely distal source of West Eurasian-related ancestry. The X chromosome was analyzed separately from autosomes to detect potential sex-biased admixture. mtDNA and Y chromosome haplogroups were also identified. qpWave was used to assess the cladality of the two Kulubnarti cemeteries and to compare ancient Kulubnarti Nubians with modern Nubian groups. Finally, the DATES software was used to estimate the timing of admixture events within the Kulubnarti population.

The study reveals several key findings regarding the genetic history of Kulubnarti Nubians during the Christian Period: 1. **Admixed Ancestry:** All Kulubnarti individuals exhibited a mixture of Nilotic (~43%) and West Eurasian (~57%) ancestry. The West Eurasian ancestry showed significant similarity to Bronze and Iron Age populations from the Levant, suggesting an ultimate origin in that region, though it likely entered Nubia via Egypt. 2. **No Genetic Differentiation between Cemeteries:** Despite archeological evidence of social stratification between the two cemeteries (R and S), no significant genetic differentiation was found between the individuals buried in them. Seven pairs of close relatives were even found across the cemeteries, suggesting substantial gene flow between the groups. 3. **Female-Biased West Eurasian Ancestry:** The West Eurasian ancestry was disproportionately associated with female ancestors (64.4% on the X chromosome versus 57.5% on the autosomes), suggesting increased female mobility or exogamy. 4. **Continuous Admixture over a Millennium:** Estimates using DATES indicate that the admixture occurred across a broad timeframe, not just as a single pulse, potentially over the past millennium. Individual estimates ranged widely, suggesting continuous or recurrent gene flow. 5. **No Direct Descent to Modern Nubians:** Modern Nubian populations (Mahas, Danagla, and Halfawieen) did not descend directly from the Kulubnarti population, indicating substantial additional genetic input after the Christian Period, likely through post-Christian Period migrations. 6. **Limited Population-Level Relatedness:** Despite the archaeological evidence suggesting a low population density at Kulubnarti, the analysis of ROH suggests a relatively large mating pool, likely due to connections with a broader meta-population. This finding is consistent with the absence of genetic differentiation between the two cemeteries.

The findings of this study address long-standing questions about the genetic history and social structure of Nubia during the Christian Period. The identification of admixed ancestry in the Kulubnarti population confirms the long-standing interaction between Nubian and West Eurasian populations, with the ultimate origin of the West Eurasian component being linked to the Levant. The absence of genetic differentiation between the socially stratified cemeteries refutes hypotheses suggesting distinct origins or populations for the two groups. This finding highlights the complexity of social organization, where social divisions do not always translate into genetic distinctions. The female-biased West Eurasian ancestry points to patterns of migration and social interaction, possibly suggesting a patrilocal society with greater female exogamy. The continuous admixture over a millennium emphasizes the dynamic nature of genetic exchange in the region. The lack of direct genetic continuity between the ancient Kulubnarti population and modern Nubians underscores the significant impact of post-Christian Period migrations on the genetic landscape of present-day Nubia.

This study provides novel insights into the genetic history of Nubia during the Christian Period. The findings demonstrate the complex interplay of social structure and genetic patterns, showing how social stratification may not always correlate with genetic differentiation. The discovery of female-biased West Eurasian ancestry and continuous admixture highlights the importance of considering sex-specific migration patterns and the dynamic nature of population interactions. Future research should focus on expanding ancient DNA analyses to earlier periods in Nubia and other regions of the Nile Valley to better understand the long-term processes shaping this fascinating region's genetic and cultural landscapes.

One limitation of this study is the relatively small sample size from Kulubnarti, which could affect the statistical power to detect subtle genetic differences. Also, while the study uses a large number of SNPs, ancient DNA analysis always presents challenges with regards to potential biases from DNA degradation and incomplete coverage. Furthermore, reliance on modern populations as proxies for ancient ancestral groups has inherent limitations because genetic drift and admixture affect the accuracy of ancestry estimates. The interpretation of sex-biased admixture relies on population-level modeling and may be influenced by the quality and size of the dataset. The generation time assumed for the DATES analysis can also influence the interpretation of admixture timing.