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Accurate reconstruction of ancestral states is a critical evolutionary analysis when studying ancient proteins and comparing biochemical properties between parental or extinct species and their extant relatives. It relies on multiple sequence alignment (MSA) which may introduce biases, and it remains unknown how MSA methodological approaches impact ancestral sequence reconstruction (ASR). Here, we investigate how MSA methodology modulates ASR using a simulation study of various evolutionary scenarios. We evaluate the accuracy of ancestral protein sequence reconstruction for simulated data and compare reconstruction outcomes using different alignment methods. Our results reveal biases introduced not only by aligner algorithms and assumptions, but also tree topology and the rate of insertions and deletions. Under many conditions we find no substantial differences between the MSAs. However …