Mies, G.; Mathieson, I.

During the European Neolithic transition, migrating Anatolian farmers admixed with local hunter-gatherers, coinciding with major shifts in diet, environment, and lifestyle that imposed strong selective pressures. Local ancestry inference is widely used to detect selection following admixture, but most methods were developed and validated on present-day populations. Their performance in ancient DNA, where reference panels are smaller, data sparser, and admixture more ancient, remains unresolved. We benchmark six local ancestry inference methods on 176 imputed Neolithic genomes, comparing ancestry proportions, tract length distributions, and selection signatures. While individual-level ancestry estimates are highly correlated across methods, inferred tract lengths and admixture time estimates vary by over an order of magnitude. Integrating results across methods and replicating across methods and in two independent datasets (n=378 and 1,121) identifies robust ancestry deviations at SLC24A5 and FADS1/2, consistent with adaptation on pigmentation and metabolism, respectively. We also identify PER3 (circadian rhythm) and IRAK4 (innate immunity) as candidate loci, but with less consistent signals across methods. Finally, we replicate previous reports of excess hunter-gatherer ancestry at the HLA, but these results are inconsistent across methods and suggest that they may be affected by bias in local ancestry inference. Our findings demonstrate that while local ancestry inference recovers biologically meaningful signals in ancient genomes, results can be sensitive to the methods used for inference, particularly in complex regions like the HLA. Method choice critically influences inferred ancestry patterns and selection signals, underscoring the importance of multi-method validation.