A phylogenetic tree for the evolution of five representative species from four genera of kinetoplastid protozoa was constructed from comparison of the mitochondrial 9S and 12S rRNA gene sequences and application of both parsmony and evolutionary parsimony algorithms. In the rooted version of the tree, the monogenetic species Crithidia fasciciata is the most deeply rooted, followed by another monogenetic species, Leptomonas sp. The three digenetic speces Trypanosoma cruzi, Trypanosoma brucei, and Leishmania tareDtolae branch from the Leptomonas line. The substitution rates for the T. brucei and T. cruzi sequences were 3-4 times greater than that of the L. tarentolae sequences. This phyloeuettic tree is consistent with our dadistic analysis of the bical evidence including life cycles for these five species. A tentative time scale can be assigned to the nodes of this tree by assnmlng that the common ancestor of the digenetic parasites predated the separation of South America and Africa and postdated the first fossil appearance of its host (inferred by parsimony analysis). This time scale predicts that the deepest node occurred at 264 ± 51 million years ago, at a time commensurate with the fossil origins of the Hemiptera insect host. This implies that the ancestral kinetoplastid and its insect host appeared at approximately the same time. The molecular data suggest that these eukhrybtic parasites have an evolutionary history thkt extends back to the origin of their insect host.The kinetoplastids comprise a widely distributed group of parasitic protozoa that are distinguished by the possession of a single mitochondrion containing a network of catenated mitochondrial DNA minicircles and maxicircles (kinetoplast DNA) in the mitochondrial matrix adjacent to the basal body of the flagellum (1, 2). Two forms of host-protozoan relationships are known: (i) mtonogenetic kinetoplastids that are parasitic in a single invertebrate host and (ii) digenetic kinetoplastids that are parasitic in two hosts, usually an invertebrate and a vertebrate. These lower eukaryotic cells are the subject of both biological and medical interest. The 123 monogenetic species that have been described so far belong to the genera Crithidia, Herpetomonas, Blastocrithidia, and Leptomonas (3). They are parasitic in arthropods (mainly in Insecta, Diptera, and Hemiptera and also in Hymenoptera, Lepidoptera, Orthoptera, and Siphonaptera). The monogenetic species are known as the "lower trypanosomatids" because the digenetic genera Leishmania and Trypanosoma are thought to have arisen from a monogenetic ancestor (4). The morphology of the insect-inhabiting stages of the pathogenic digenetic species resembles that of the monogenetic species.In the absence of a fossil record, a phylogenetic tree must be constructed entirely from biological or, better yet, from molecular sequence data. The mitochondrial 9S and 12S ribosomal RNAs (5-9) represent one such group of conserved sequences that lends itself to analysis. The sequences of both large and small ...