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 The eponymous mitochondrial DNA in kinetoplastid organisms (also called kDNA) is unique in structure and gene content. Kinetoplastea include the trypanosomatid parasites, where kDNA is organised as a massive network of concatenated circular DNA molecules. Trypanosoma brucei brucei (Tbb) kDNA contains 20-50 practically identical copies of ~23-kb maxicircles and 5-10k highly heterogeneous 1-kb minicircles. Homologous to other eukaryotic mitochondrial genomes, maxicircles encode genes essential to oxidative phosphorylation and mitochondrial translation, including 2 rRNA genes and 18 protein coding genes. Minicircles encode guide RNAs (gRNAs) that direct post-transcriptional editing of the pre-mRNA products from 12 encrypted maxicircle genes by insertion and deletion of uridines by virtue of their complementarity to the fully edited version. Although numerous maxicircle genes are essential to the parasites’ survival in the tsetse fly vector, only the A6 gene, encoding a subunit of the F1FO-ATP synthase, along with the maxicircle-encoded subunits of the mitoribosome required to translate its mRNA, are essential in the mammalian bloodstream stage. 

  T. b. equiperdum (Tbeq) and T. b. evansi (Tbev; the subspecies designation adopted here is under debate) evolved from Tbb on multiple occasions by switching from transmission via tsetse flies, with obligate development in the vector, to direct transmission between mammals, either sexually in horses (Tbeq) or mechanically between many mammalian species via biting flies or vampire bats. Thus, Tbeq and Tbev are polyphyletic taxa and, to complicate things further, monophyletic subgroups of Tbev include strains historically designated as Tbeq and vice versa.   As Tbeq and Tbev are generally considered to have evolved kDNA independence and have undergone partial (and sometimes complete) loss of the organellar genome, key molecular differences between them and Tbb concerns kDNA . Previous studies of three distinct monophyletic groups of Tbeq and Tbev isolates have suggested that (i) Tbev isolates generally lack a maxicircle and (ii), where present, kDNA is dominated by one of three minicircle classes, type A, B, or C. Both observations are being used for diagnostic purposes. However, an in-depth comparative analysis of Tbeq/Tbev kDNA is lacking. 

 We have used next-generation sequencing data and a bespoke assembly pipeline to compare kDNA from 56 Tbeq/Tbev samples to each other and to a Tbb reference. Here, we report three main findings: 

 1) For three groups of Tbeq/Tbev, we confirm that the minicircle genomes consist of thousands of copies of a single type A, B, or C minicircle class specific and therefore diagnostic for each group. 

 2) Unexpectedly, one type A Tbev isolate (Vietnam strain, Gillingwater et al. 2007) had a complete maxicircle. 

 3) A fourth group of isolates has a higher minicircle diversity of 43-45classes per network. These minicircles encode gRNA genes sufficient for directing complete editing of the A6 mRNA. However, editing of the RPS12 mRNA, encoding a subunit of the mitoribosome, appears to be incomplete, suggesting that kDNA independence in these isolates evolved relatively recently in these strains. 

 Altogether, the project sheds light on how the complex evolutionary history of non-tsetse transmitted T. brucei strains has shaped their