Estimation of nucleotide substitution rates in Eurotiomycete fungi.

In the entire fungal kingdom, only DNA substitution rates in the SSU rRNA gene (Berbee and Taylor 1993, 2001) and amino acid substitution rates (Heckman et al. 2001) have been estimated and used to date fungal divergences. However, these molecules are not sufficiently variable to date events at or below the genus level. DNA sequences of protein-coding genes and the internal transcribed spacer (ITS) region are sufficiently variable, but their substitution rates are not known. In this article, we investigated the DNA substitution rates in the protein-coding genes and the ITS region by comparison with the SSU rDNA divergence in a representative fungal lineage, Eurotiomycetes (5plectomycetes) (Eriksson and Winka 1998). The Eurotiomycete lineage is a monophyletic class of Ascomycota (Berbee and Taylor 1992) and includes many economically important fungi, such as Penicillium chrysogenum (antibiotic production) and Aspergillus oryzae (soy sauce production), as well as many human pathogens such as Coccidioides immitis (lung disease), Histoplasma capsulatum (lung disease), and Trichophyton rubrum (athlete’s foot). Estimating DNA substitution rates in quickly evolving molecules such as the ITS and protein-coding genes will not only provide a means to estimate the divergence times between lineages at and below the genus level, but in conjunction with coalescent theory, it may provide information for estimating epidemiological parameters such as effective population size (Watterson 1975) and recombination rates (Hey and Wakeley 1997). With the aid of published Eurotiomycete phylogenies based on SSU rDNA (Ogawa, Yoshimura, and Sugiyama 1997; Sugiyama, Ohara, and Mikawa 1999), we searched for pairs of closely related species which had not only SSU rDNA sequences but also sequences of homologous protein-coding genes. Next, we compared the extent of differentiation at synonymous sites of protein-coding genes between sister taxa. It was found that saturation at synonymous sites of protein-coding loci is attained before divergence of the SSU rDNA sequences reaches 1%–2%. A 1%–2% divergence usually corresponds to divergences seen within a genus or among closely related genera. Therefore, the protein genes should be well suited to dating divergences at and below the genus level.