Phylogenies from amino acid sequences aligned with gaps: the problem of gap weighting

The common but generally overlooked problem of how best to construct phylogenies from orthologous amino acid sequences, when their alignment requires the placement therein of gaps denoting insertions/deletions in the evolutionary history of their genes since their common ancestor, has been studied. Three diverse methods were examined: 1. each missing residue in a gap is weighted as equivalent to the average number of minimum nucleotide replacements in known conjugate amino acid pairs of those same two sequences, which weight necessarily differs for each pair of sequences; 2. each missing residue in a gap is weighted as equivalent to a fixed number of nucleotide replacements; and 3. each gap, regardless of length, is weighted as equivalent to a fixed number of nucleotide replacements. For the flavodoxins, each method yielded a different best tree and suggests that the choice of method may be crucial. For the plant ferredoxins, all methods give results inconsistent with botanical classification and suggests the sequences may not all be orthologous. For the bacterial ferredoxins, the method was less germane than the actual weight used, five different best trees being obtained depending upon the weight. The best tree for all ferredoxins (prokaryotic plus eukaryotic) combined proved to be greatly dependent upon the gap locations with several reasonable aligments yielding different best trees. They also suggest that functional equivalence may well prove to be a poor guide to which residues have a common ancestral codon. The rubredoxin sequences show that a partial internal gene duplication occurred in the Pseudomonas line, probably very soon after its divergence from the other genera. Together, the results clearly indicate that the phylogenetic answer one gets may greatly depend upon how one treats the gaps but they fail to indicate what treatment may be best.