The complete coding region of the maxicircle as a superior phylogenetic marker for exploring evolutionary relationships between members of the Leishmaniinae.

The mitochondrial DNA (mtDNA) is a potentially valuable phylogenetic marker given its presence across all eukaryotic taxa and its relative conservation in structure and sequence. In trypanosomatids, a homologue of the mtDNA referred to as the maxicircle DNA, is located within a specialised structure in the single mitochondrion of the trypanosomatids called the kinetoplast; a high molecular weight network of DNA composed of thousands of catenated minicircles and a smaller number of larger maxicircles. Unique to the kinetoplastid protists, the maxicircle component of this complex network could represent a desirable target for taxonomic inquiry that may also facilitate exploration of the evolutionary history of this important group of parasites. The aim of this study was to investigate the phylogenetic value of the trypanosomatid maxicircle for these applications. Maxicircle sequences were obtained either by assembling raw sequence data publicly accessible in online databases (i.e., NCBI), or by amplification of novel maxicircle sequences from trypanosomatid DNA using long-range (LR) PCR with subsequent Illumina sequencing. This procedure facilitated the generation of nearly complete maxicircle sequences (i.e., excluding the divergent region) for numerous dixenous and monoxenous trypanosomatid species. Annotation of each maxicircle sequence confirmed that their structure was conserved across all taxa examined. Phylogenetic analyses confirmed that Z. australiensis showed a greater genetic relatedness with the dixenous trypanosomatids of the genera Leishmania and Endotrypanum, as opposed to members of the monoxenous genera Crithidia and Leptomonas. Additionally, molecular clock analysis supported that the dixenous Leishmaniinae appeared approximately 75 million years ago during the breakup of Gondwana. In line with previous studies, our results support the Supercontinents hypothesis regarding the origin of dixenous Leishmaniinae. Ultimately, we demonstrate that the maxicircle represents an excellent phylogenetic marker for studying the evolutionary history of trypanosomatids, resulting in trees with very high bootstrap support values.