Unique mitochondrial genome structure in diplonemids, the sister group of kinetoplastids

Kinetoplastid flagellates are characterized by uniquely massed mitochondrial DNAs (mtDNAs), the kinetoplasts. Kinetoplastids of the trypanosomatid group possess two types of mtDNA molecules: maxicircles bearing protein and mitoribosomal genes and minicircles specifying guide RNAs, which mediate uridine insertion/deletion RNA editing. These circles are interlocked with one another to form dense networks. Whether these peculiar mtDNA features are restricted to kinetoplastids or prevail throughout Euglenozoa (euglenids, diplonemids, and kinetoplastids) is unknown. Here, we describe the mitochondrial genome and the mitochondrial ultrastructure of Diplonema papillatum, a member of the diplonemid flagellates, the sister group of kinetoplastids. Fluorescence and electron microscopy show a single mitochondrion per cell with an ultrastructure atypical for Euglenozoa. In addition, DNA is evenly distributed throughout the organelle rather than compacted. Molecular and electron microscopy studies distinguish numerous 6- and 7-kbp-sized mitochondrial chromosomes of monomeric circular topology and relaxed conformation in vivo. Remarkably, the cox1 gene (and probably other mitochondrial genes) is fragmented, with separate gene pieces encoded on different chromosomes. Generation of the contiguous cox1 mRNA requires trans-splicing, the precise mechanism of which remains to be determined. Taken together, the mitochondrial gene/genome structure of Diplonema is not only different from that of kinetoplastids but unique among eukaryotes as a whole.