Extant Sequence Reconstruction: The accuracy of ancestral sequence reconstructions evaluated by extant sequence cross-validation

Ancestral sequence reconstruction (ASR) is a phylogenetic method widely used to analyze the properties of ancient biomolecules and to elucidate mechanisms of molecular evolution. Despite its increasingly widespread application, the accuracy of ASR is currently unknown, as it is generally impossible to compare resurrected proteins to the true ancestors. Which evolutionary models are best for ASR? How accurate are the resulting inferences? Here we answer these questions using a cross-validation method to reconstruct each extant sequence in an alignment with ASR methodology, a method we term "extant sequence reconstruction" (ESR). We thus can evaluate the accuracy of ASR methodology by comparing ESR reconstructions to the corresponding known true sequences. We find that a common measure of the quality of a reconstructed sequence, the average probability, is indeed a good estimate of the fraction of correct amino acids when the evolutionary model is accurate or overparameterized. However, the average probability is a poor measure for comparing reconstructions from different models, because, surprisingly, a more accurate phylogenetic model often results in reconstructions with lower probability. While better (more predictive) models may produce reconstructions with lower sequence identity to the true sequences, better models nevertheless produce reconstructions that are more biophysically similar to true ancestors. In addition, we find that a large fraction of sequences sampled from the reconstruction distribution may have fewer errors than the single most probable (SMP) sequence reconstruction, despite the fact that the SMP has the lowest expected error of all possible sequences. Our results emphasize the importance of model selection for ASR and the usefulness of sampling sequence reconstructions for analyzing ancestral protein properties. ESR is a powerful method for validating the evolutionary models used for ASR and can be applied in practice to any phylogenetic analysis of real biological sequences. Most significantly, ESR uses ASR methodology to provide a general method by which the biophysical properties of resurrected proteins can be compared to the properties of the true protein.