Phylogeny of anaerobic gut fungi

Neocallimastigomycota is the new phylum of fungi recently established as a result of AFTOL project in 2007, to cover anaerobic fungi. This new phylum resulted from kingdom-wide phylogenetic reconstruction of the Fungi based on rRNA gene (18S and 28S) sequence data. However using of rRNA genes is problematic. While 18S regions are highly conserved, ITS show polymorphisms even within single isolates. There is a need to identify genetic loci with sufficient levels of polymorphism at the subgeneric level to allow typing of isolates and open the reliable approach for ecological studies. Protein-coding genes can better elucidate the relationship inside Fungi.
Here we developed redundant primers able to amplify both coding and non-coding regions of two loci which encode the highly conserved proteins, EFla and RBP1. Our aim was to investigate the use of the less conserved intronic regions of RBP1 as a tool for typing of strains at a subgeneric level and to use the coding regions of the genes to generate a robust phylogenetic reconstruction of the anaerobic fungi. The resulting phylogenetic reconstruction has shown that the polyflagellate genera Orpinomyces and Neocallimastix are closest relative (i.e. the polyflagellate zoospores are unique to the anaerobic fungi), whiles the bulbous morphotypes are also closely related to each other and apparently to Anaeromyces. The basal position of the genus Piromyces and the greater level of genetic diversity in this genus were discovered. Comparison of phylogenetic trees based on DNA and protein sequences revealed that the long branch length of the anaerobic fungi relative to their nearest relatives is to a large extent an artefact of the high AT content of the genomes of these fungi.