Use of atp6 in fungal phylogenetics: An example from the Boletales

Complete nucleotide sequences have been determined for atp6 from Suillus lu teus and cox3 from Suillus sinuspaulianus (Boletales, Hymenomycetes, Basidi omycota), which code for ATPase subunit 6 and cytochrome oxidase subunit 3, respectively. These sequences were used to design PCR primers for the ampl ification of partial atp6 and cox3 sequences from other members of the Bole tales and outgroup taxa. In atp6 and coxs from Russula rosacea, one of the outgroup taxa, we observed a number of in-frame TGA(trp) codons, which impl y a Neurospora crassa-type mitochondrial code in R. rosacea and possibly in basidiomycetes in general. Interestingly, however, most basidiomycetes oth er than R. rosacea appear to strongly prefer the TGG(trp) codon, which is u nusual, given the strong A + T bias in fungal mitochondrial genomes. Pairwi se comparisons were performed between atp6 sequences from increasingly dive rgent fungal lineages, and results show that all three codon positions beco me saturated in substitutions after an estimated divergence time of approx 300 Ma. This means that atp6 is likely to provide phylogenetic resolution w ithin fungal classes but not at higher taxonomic levels. Also, because of t he strong A + T bias in fungal mitochondrial genomes, APT transversions wer e found to be more common than any other type of substitution, resulting in transversions being about two to three times more common in most pairwise sequence comparisons. Finally, atp6 sequences were used to infer phylogenet ic relationships between 27 taxa from the Boletales and 4 outgroup taxa. An alyses were performed (i) on nucleotide sequence data using parsimony (succ essive approximation) as well as maximum likelihood methods and (ii) on ded uced amino acid sequences using distance methods based on empirical substit ution probabilities. Results from the various analyses are largely concorda nt with each other as well as with prior analyses of partial mitochondrial large-subunit rDNA (mtLSU rDNA). Analysis of the combined atp6 and mtLSU rD NA sequences results in increased bootstrap support for several key branche s. Relationships that have been resolved for the first time in the current analysis are discussed. (C) 1999 Academic Press.