Phylogenomics of free-living neobodonids reveals they are a paraphyletic group from which all other metakinetoplastids are descended

Kinetoplastea is a major taxon of microbial eukaryotes that includes the well-known trypanosomatid parasites, species of which cause diseases in humans, animals and plants. Free-living kinetoplastids are greatly understudied compared to their parasitic relatives, but are ecologically important microbivores, and collectively comprise the great majority of kinetoplastid diversity. For two decades kinetoplastids have been divided into prokinetoplastids and metakinetoplastids, with the latter further split into four orders, the most diverse of which is Neobodonida. However, the position of the root of the metakinetoplastids and whether neobodonids are a clade has remained unclear due to very limited multi-gene data from free-living kinetoplastids, particularly neobodonids. Here, we present transcriptomic data for eleven newly or recently cultivated free-living neobodonid kinetoplastids. Phylogenomic analyses of a de novo generated data set of 444 inferred orthologs and 49 taxa robustly resolve the kinetoplastid tree, including the position of the root of metakinetoplastids. This divides metakinetoplastids such that Trypanosomatida, Eubodonida, Parabodonida, Allobodonidae (formerly neobodonid clade 1E) and neobodonid clade 1D fall on one side, while neobodonid clades Nd6, 1B (Rhynchomonadidae) and 1C fall on the other. Neobodonids are thus inferred to be a paraphyletic group from which all other metakinetoplastids descend. This analysis is the most thorough examination of metakinetoplastid phylogeny thus far, and forms a new basis for tracing the evolutionary history of the entire kinetoplastid group. Significance statementThis study provides transcriptomic data for 11 novel free-living kinetoplastid species, allowing the most accurate phylogeny of metakinetoplastids to date, revealing that neobodonids are paraphyletic and that eubodonids, parabodonids and trypanosomatids emerged within neobodonids. This new framework is essential for understanding the evolution of this major group of parasites and ecologically important free-living heterotrophs.