The deterministic effects of alignment bias in phylogenetic inference

Alignment of nucleotide and/or amino acid sequences is a fundamental component of sequence‐based molecular phylogenetic studies. Here we examined how different alignment methods affect the phylogenetic trees that are inferred from the alignments. We used simulations to determine how alignment errors can lead to systematic biases that affect phylogenetic inference from those sequences. We compared four approaches to sequence alignment: progressive pairwise alignment, simultaneous multiple alignment of sequence fragments, local pairwise alignment and direct optimization. When taking into account branch support, implied alignments produced by direct optimization were found to show the most extreme behaviour (based on the alignment programs for which nearly equivalent alignment parameters could be set) in that they provided the strongest support for the correct tree in the simulations in which it was easy to resolve the correct tree and the strongest support for the incorrect tree in our long‐branch‐attraction simulations. When applied to alignment‐sensitive process partitions with different histories, direct optimization showed the strongest mutual influence between the process partitions when they were aligned and phylogenetically analysed together, which makes detecting recombination more difficult. Simultaneous alignment performed well relative to direct optimization and progressive pairwise alignment across all simulations. Rather than relying upon methods that integrate alignment and tree search into a single step without accounting for alignment uncertainty, as with implied alignments, we suggest that simultaneous alignment using the similarity criterion, within the context of information available on biological processes and function, be applied whenever possible for sequence‐based phylogenetic analyses.