Multiple alignments of published sequences for the primer sites. Primer sequence alignments are depicted with consensus bases blank and mismatch bases noted. The six bases at the 3' end of the primers are highlighted yellow. "Clusters" were created by sorting the sequences in Excel and grouping identical sequences. "Species per cluster" numbers indicate the number of species with that mismatch pattern. Only one sequence per species was included in the alignment (final totals of: 584 species for 18S, 633 species for 5.8S, and 943 species for 28S). The "Cluster number" is a sequential numbering of clusters in order of descending "Species per cluster" with sequences from the EMBL Fungal database numbered before sequences from the EMBL Plant database (the latter are presented in green font). Sequences were collected using FASTA  for a set of 12 taxonomically representative fungal sequences for each region. Most mismatch groups containing fewer than 4 species were removed from the figure to save space. The full alignment is also available [see Additional file 1].