Challenging Patterns of Fine-Scale Genetic Structure in Galicia and East-to-West Genetic Divergence Across the Iberian Peninsula-When Technical Batches Rewrite History.

The influential 2019 study by Bycroft et al. reported remarkably pronounced ultrafine-scale genetic structure within a small region of Galicia (northwestern Iberia). Using fineSTRUCTURE clustering of ChromoPainter coancestry profiles, the authors identified multiple internally coherent clusters characterized by high levels of within-group haplotype sharing and reduced sharing with neighboring populations. On a broader national scale, they also reported a marked East-West genetic differentiation and North-South homogeneity across the Iberian Peninsula, patterns which they largely attributed to historical population movements associated with the gradual territorial expansion of Christian kingdoms between the 8th and 15th centuries following the initial Islamic conquest. Several subsequent studies have adopted similar interpretations, but all relied on the same genetic resource, the EPICOLON Phase I cohort. Following a detailed reexamination of Bycroft et al., we identify multiple methodological issues, most notably signals consistent with unaccounted batch effects in EPICOLON, some of which were recognized by the authors in earlier studies. In contrast, we highlight recent analyses based on independent genomic datasets that consistently report high levels of genetic homogeneity within Galicia and across central and southern Iberia, with no evidence supporting a pronounced East-West genetic divide across Iberia. In addition, recent studies have documented North African demographic influences in Iberia, particularly in Galicia, predating the period of Islamic rule and therefore challenging the historical interpretation proposed by Bycroft et al. We also show that while fineSTRUCTURE is a powerful tool for detecting subtle population stratification, it may be highly sensitive to technical confounders, which can magnify or distort inferred clustering patterns. This study demonstrates that technical artifacts can strongly affect fine-scale population structure inferred by fineSTRUCTURE, emphasizing the need for rigorous quality control and cautious interpretation of highly resolved genetic clusters.