Resolving the question of trypanosome monophyly: a comparative genomics approach using whole genome data sets with low taxon sampling

Since the first attempts to classify the evolutionary history of trypanosomes, there have been conflicting reports regarding their true phylogenetic relationships and, in particular, their relationships with other vertebrate trypanosomatids, e.g. Leishmania sp., as well as with the many insect parasitising trypanosomatids. Perhaps the issue that has provided most debate is that concerning the monophyly (or otherwise) of genus Trypanosoma and, even with the advent of molecular methods, the findings of numerous studies have varied significantly depending on the gene sequences analysed, number of taxa included, choice of outgroup and phylogenetic methodology. While of arguably limited applied importance, resolution of the question as to whether or not trypanosomes are monophyletic is critical to accurate evaluation of competing, mutually exclusive evolutionary scenarios for these parasites, namely the 'vertebrate-first' or 'insect-first' hypotheses. Therefore, a new approach, which could overcome previous limitations was needed. At its most simple, the problem can be defined within the framework of a trifurcated tree with three hypothetical positions at which the root can be placed. Using BLASTp and whole-genome gene-by-gene phylogenetic analyses of Trypanosoma brucei, Trypanosoma cruzi, Leishmania major and Naegleria gruberi, we have identified 599 gene markers--putative homologues--that were shared between the genomes of these four taxa. Of these, 75 homologous gene families that demonstrate monophyly of the kinetoplastids were identified. We then used these data sets in combination with an additional outgroup, Euglena gracilis, coupled with large-scale gene concatenation and diverse phylogenetic techniques to investigate the relative branching order of T. brucei, T. cruzi and L. major. Our findings confirm the monophyly of genus Trypanosoma and demonstrate that <1% of the analysed gene markers shared between the genomes of T. brucei, T. cruzi and L. major reject the hypothesis that the trypanosomes form a monophyletic group.