Evolutionary relationships among "jakobid" flagellates as indicated by alpha- and beta-tubulin phylogenies

Jakobids are free-living, heterotrophic flagellates that might represent ea rly-diverging mitochondrial protists. They share ultrastructural similariti es with eukaryotes that occupy basal positions in molecular phylogenies, an d their mitochondrial genome architecture is eubacterial-like, suggesting a close affinity with the ancestral alpha-proteobacterial symbiont that gave rise to mitochondria and hydrogenosomes. To elucidate relationships among jakobids and other early-diverging eukaryotic lineages, we characterized al pha- and beta-tubulin genes from four jakobids: Jakoba libera, Jakoba incar cerata, Reclinomonas americana (the "core jakobids"), and Malawimonas jakob iformis. These are the first reports of nuclear genes from these organisms. Phylogenies based on alpha-, beta-, and combined alpha- plus beta-tubulin protein data sets do not support the monophyly of the jakobids. While beta- tubulin and combined alpha- plus beta-tubulin phylogenies showed a sister g roup relationship between J. libera and R. americana, the two other jakobid s, M. jakobiformis and J. incarcerata, had unclear affinities. In all three analyses, J. libera, R. americana, and M. jakobiformis emerged from within a well-supported large "plant-protist" clade that included plants, green a lgae, cryptophytes, stramenopiles, alveolates, Euglenozoa, Heterolobosea, a nd several other protist groups, but not animals, fungi, microsporidia, par abasalids, or diplomonads. A preferred branching order within the plant-pro tist clade was not identified, but there was a tendency for the J. libera-R . americana lineage to group with a clade made up of the heteroloboseid amo eboflagellates and euglenozoan protists. Jakoba incarcerata branched within the plant-protist clade in the beta- and the combined alpha- plus beta-tub ulin phylogenies. In alpha-tubulin trees, J. incarcerata occupied an unreso lved position, weakly grouping with the animal/fungal/microsporidian group or with amitochondriate parabasalid and diplomonad lineages, depending on t he phylogenetic method employed. Tubulin gene phylogenies were in general a greement with mitochondrial gene phylogenies and ultrastructural data in in dicating that the "jakobids" may be polyphyletic. Relationships with the pu tatively deep-branching amitochondriate diplomonads remain uncertain.