Reference Population Design and the Illusion of Genetic Intermediacy in Mediterranean Population Models

Genome wide analyses of human populations rely on reference panels to infer ancestry and historical relationships, yet the sensitivity of downstream inference to reference population composition is not always evaluated directly. In this study, I test how asymmetric reference population design can produce the appearance of genetic intermediacy in Mediterranean population models, using Ashkenazi Jewish populations as a focused case. I compare results across multiple analytical frameworks, including qpAdm admixture modeling, pairwise autosomal FST distances, principal component analysis, identity by descent sharing, Global25 based affinity modeling, and reassessment of published uniparental marker studies. Across methods, I vary European and eastern Mediterranean reference sets to evaluate the stability of inferred ancestry patterns under alternative and historically grounded model configurations. This sensitivity to reference choice is consequential because Northern Italian, Tuscan, and Sardinian proxies do not correspond to the primary historical geography of Jewish settlement in southern Italy and the central Mediterranean, which provides the most relevant European context for evaluating Ashkenazi origins. When Southern European populations, particularly Southern Italian, Sicilian, Maltese, and Aegean groups, are included as explicit comparators, Ashkenazi Jews consistently align within a Southern European and central Mediterranean genetic continuum rather than occupying an intermediate position between Europe and the Levant. In contrast, models that approximate Europe using Northern Italian, Tuscan, or genetically drifted Sardinian proxies shift Ashkenazi Jews toward an apparent Europe Levant intermediate placement. Autosomal FST distances identify the closest affinities with Southern Italians, Cretans, Sicilians, and mainland Greeks, with larger divergence from Levantine populations. qpAdm results likewise support a dominant Southern Italian or closely related central Mediterranean autosomal component, while models relying on Northern Italian proxies are less stable and can fail feasibility criteria. Identity by descent analyses show substantially greater sharing with European populations than with Levantine groups. Reassessment of Y chromosomal evidence indicates that several lineages often treated as Levantine fall within broader Southern European and Mediterranean variation when evaluated at higher resolution with appropriate comparators. These results support the conclusion that apparent genetic intermediacy in Mediterranean population models can arise from reference population exclusion. More broadly, they underscore the need for explicit sensitivity testing of reference population design before interpreting intermediate genetic placement as evidence of population history.