Radiocarbon dating of two individuals from the Kuline necropolis of Timacum Minus places them in the 10th century CE (Table ST1), well after the first Slavic-related settlements in the Balkans during the 7th century. Two individuals from Kuline were identical twins (Supplementary section 7) and present a different ancestry profile compared to the other individuals from the same necropolis (Figure 1C), likely deriving from Southwestern Europe (Figure 2; Supplementary section 12.9), although they were buried in the same manner as the others from the necropolis 29. The remaining five individuals clustered in the West-Eurasian PCA (Figure 1C) on top of the “present-day Balkan genetic cline”, close to present-day Serbian-speaking individuals that we newly genotyped for this study, but this apparent similarity is a projection artifact as their ancestry could not be fitted using the same qpAdm models (Supplementary section 12.8). To understand this, we performed a PCA using present-day Germanic- and Slavic-speaking populations (Supplementary section 9; Figure S9) that we expected would be sensitive to more recent drift separating Central, Northern and Eastern European populations. The Kuline individuals are more shifted towards present-day Slavic-speaking populations as compared to individuals in the Central/Northern European cluster, agreeing with the presence of Y-chromosome lineage I2-L621 [I-Y3120] in Kuline, which is common in present-day Slavic-speaking groups and absent in earlier periods. In light of these results, we modeled the ancestry of the Kuline individuals as a mixture of 56% deriving from the local Balkan Iron Age substratum and 44% deriving from Northeastern European Iron Age groups, and obtained a good statistical fit (Figure 2; Supplementary section 12.8). Our results point to a strong demographic impact of Eastern European groups in the Balkans during the Medieval period, likely associated to the arrival of Slavic-speaking populations. Yet, our results rule out a complete demographic replacement, as we observe a significant portion of local Iron Age Balkan ancestry in Kuline individuals. Interestingly, we found sex bias when modeling the X chromosome of the individuals of this necropolis (Supplementary section 12.8). Perhaps the immigrant groups were constituted by a higher number of women, who therefore impacted more greatly in the demographics of the post-Roman Balkans. However, these findings have only been observed in the Kuline individuals with North-European related ancestry (n=5), we suggest more data will be needed to obtain more evidence Slavic sex bias in the Balkans.
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