Julia A. Packer

The evolution of genome size and eukaryotic genome organisation has puzzled evolutionary biologists for decades. Are differences in DNA content and protein-coding capacity of eukaryotic genomes a result of natural selection, or neutral processes? If they were shaped by natural selection, which features might be connected with these differences: cell volume, ecological strategy, availability of nutrients, etc.? Kinetoplastids are protists with compact nuclear genomes that almost entirely lack introns, but whose mitochondrial DNA content (kDNA) can rival that of their nuclei. We hypothesised that the reduction in non-protein coding nuclear DNA content during the evolution of kinetoplastids is connected with an increase in kDNA content: species with larger fractions of total cellular DNA that is allocated to kDNA will have concomitantly smaller fractions of non-coding DNA in the nuclear genome. We developed a framework for estimation of DNA content in both the mitochondria and nuclei from quantitative fluorescence microscopy data, and inferred the fraction of non-coding DNA in nuclear genomes based on the combination of transcriptomic and fluorescence imaging data. Across 10 genetically distant isolates from diverse free-living kinetoplastid lineages, there is indeed a trend towards a lower proportion of non-protein-coding nuclear DNA when kDNA proportion is higher relative to the total DNA in the cell. This observation is consistent with the hypothesis that in free-living kinetoplastids, mitochondrial and nuclear genomes might co-evolve in terms of their DNA content. This finding raises the possibility that natural selection acts on genome size evolution in kinetoplastids, and contributes evidence and methodology to address this question more broadly in eukaryotes.

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 statement: This 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.