• The relative performance of iterative programs (e. g. MAFFT, MUSCLE, PRRN) improves with an increasing number of input sequences and/or decreasing sequence identity. The non-iterative, progressive programs show the opposite trend. With increasing number of sequences and decreasing sequence identity the progressive alignment approach is more likely to introduce errors, which cannot be corrected at a later alignment stage ("once a gap, always a gap" [37]). These errors are corrected by iterative programs during their refinement stage.