Unique ASCII characters, except letters and '*', can be used as special symbols for alignment gaps, missing data, and identical sites. Frequently used symbols for identical sites, alignment gaps, and missing data are '.', '-', and '?', respectively. This error message means that you have attempted to use the same symbols for two or more of these types of sites, or a chosen symbol is not appropriate. For example, do not use N (the ambiguous site symbol for DNA/RNA sequences), or X (the ambiguous site symbol for protein sequences) because they are already available as the IUPAC symbols for molecular sequences.