ASSESSMENT OF PHIALOPHORA SPECIES BASED ON RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACERS AND MORPHOLOGY

Fifty-one isolates of Phialophora americana, P. parasitica, P. richardsiae, and P. verrucosa (deuteromycetes) were initially characterized by restriction enzyme mapping of the ribosomal DNA region containing the conserved 5.8 S gene and the more variable intergenic spacers. Based on this mapping, the 34 isolates in the P. americana/P. verrucosa complex were separated into eight phenotypic groups, the 9 isolates of P. parasitica were split into four groups, and all 6 isolates of P. richardsiae were within a single group. Twenty-four isolates representing 12 of the 13 restriction map groups were chosen for sequence analysis of the same region. The results indicated that isolates selected from the same group shared identical DNA sequences. Comparisons of the sequences revealed a wide range of variation among the species, from a minimum of 13 to a maximum of 196 base pair differences. The gene regions (3' end of the 18S, 5.8S, and 5' end of the 25S genes) were highly conserved, while the internal transcribed spacers were variable among the species. Phenetic similarity clustering produced groupings consistent with morphological species designations for all four species. Phylogenetic bootstrap analysis also supported the separation of isolates into at least four groups corresponding to morphology. Each of the species were placed into separate clades indicating that each was monophyletic. Maximum likelihood analyses supported these conclusions. Phialophora americana and P. verrucosa, considered by some to be synonymous, were closely related but clearly separated into distinct clades. Two isolates, initially designated as P. verrucosa, consistently branched off separately from all other Phialophora isolates and may represent a previously undescribed species. Phialophora parasitica was found to be genetically variable, while P. richardsiae was genetically homogeneous, which is consistent with morphological findings.