Table 3.   Molecular Phylogenetic Methods Method Parsimony Possible trees are compared and each is given a score that is a reflection of the minimum number of character state changes (e.g., amino acid substitutions) that would be required over evolutionary time to fit the sequences into that tree. The optimal tree is considered to be the one requiring the fewest changes (the most parsimonious tree). Distance The optimal tree is generated by first calculating the estimated evolutionary distance between all pairs of sequences. Then these distances are used to generate a tree in which the branch patterns and lengths best represent the distance matrix. Maximum likelihood Maximum likelihood is similar to parsimony methods in that possible trees are compared and given a score. The score is based on how likely the given sequences are to have evolved in a particular tree given a model of amino acid or nucleotide substitution probabilities. The optimal tree is considered to be the one that has the highest probability. Bootstrapping Alignment positions within the original multiple sequence alignment are resampled and new data sets are made. Each bootstrapped data set is used to generate a separate phylogenetic tree and the trees are compared. Each node of the tree can be given a bootstrap percentage indicating how frequently those species joined by that node group together in different trees. Bootstrap percentage does not correspond directly to a confidence limit.