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In the protist Diplonema papillatum (Diplonemea, Euglenozoa), mitochondrial genes are systematically fragmented with each nonoverlapping piece (module) encoded individually on a distinct circular chromosome. Gene modules are transcribed separately, and precursor transcripts are assembled to mature mRNA by a trans-splicing process of yet unknown mechanism. Expression of the cox1 gene that consists of nine modules, also involves RNA editing by which six uridines are added between Modules 4 and 5. Here, we investigate whether the unusual features of cox1 are shared by all Diplonemea and what the mechanism of trans-splicing might be. We examine three additional species representing both Diplonemea genera, namely D. papillatum described before, and D. ambulator, Diplonema sp.2, and Rhynchopus euleeides and discover that in all Diplonemea, the cox1 gene is discontinuous and split up into nine modules that each reside on a distinct chromosome. Positions of gene breakpoints vary by up to two nucleotides. Further, all taxa have six nonencoded uridines inserted in cox1 mRNA at exactly the same position as D. papillatum. In silico searches do not detect signatures of introns known to engage in trans-splicing, in particular Group I, Group II, spliceosomal, and transfer RNA introns. Nor did we find statistically significant reverse-complementary motifs between adjacent modules and their flanking regions, or residues conserved within or across species. This provides compelling evidence that trans-splicing in Diplonemea mitochondria does not rely on sequence elements in cis but rather proceeds by a mechanism employing matchmaking trans factors, such as RNAs or proteins.