We examined how alignment of internal transcribed spacers (ITS) of rDNA changes with increasing genetic distance between sequences, using four fungal and four plant datasets. Increasing genetic distance can negatively affect the tree topology and branch-support values in two ways. First, it may cause errors in the alignment and therefore the homology of the characters that are based on the misaligned positions. Second, it may cause errors in the homology assessments of positions that underwent multiple hits as genetic distance increased, therefore causing errors in homology assessments of the states for those characters. These two causes of error in phylogenetic inference were distinguished from one another in our analysis. The errors in alignment caused by increasing genetic distance were primarily due to inserting too few gaps and inserting them at the wrong positions. We suggest that terminals on branches that represent genetic distances of over 0.118 are likely to have negative effects on tree construction caused by misaligned positions and multiple hits. Significant errors in tree resolution, topology, and/or branch-support values were more often caused by multiple hits (and/or absence of interaction of the excluded terminals with the included terminals) than by misaligned positions. This suggests that increasing genetic distance negatively affects our primary homology assessments of character states (due to multiple hits) more severely than our primary homology assessments of characters (due to misaligned positions). We suggest that increasing taxon sampling with the aim of subdividing long branches is the preferred strategy for obtaining reliable alignments; manual adjustment of alignments and/or eliminating ambiguously aligned positions from the analysis are of lesser benefit.

Key words: alignment, genetic distance, internal transcribed spacer, multiple hits, rDNA, tree construction