Novel Molecular Synapomorphies Demarcate Different Main Groups/Subgroups of and Piroplasmida Species Clarifying Their Evolutionary Relationships.

The class Hematozoa encompasses several clinically important genera, including , whose members cause the major life-threating disease malaria. Hence, a good understanding of the interrelationships of organisms from this class and reliable means for distinguishing them are of much importance. This study reports comprehensive phylogenetic and comparative analyses on protein sequences on the genomes of 28 hematozoa species to understand their interrelationships. In addition to phylogenetic trees based on two large datasets of protein sequences, detailed comparative analyses were carried out on the genomes of hematozoa species to identify novel molecular synapomorphies consisting of conserved signature indels (CSIs) in protein sequences. These studies have identified 79 CSIs that are exclusively present in specific groups of Hematozoa species, also supported by phylogenetic analysis, providing reliable means for the identification of these species groups and understanding their interrelationships. Of these CSIs, six CSIs are specifically shared by all hematozoa species, two CSIs serve to distinguish members of the order Piroplasmida, five CSIs are uniquely found in all Piroplasmida species except and two CSIs are specific for the genus . Additionally, we also describe 23 CSIs that are exclusively present in all genome-sequenced species and two, nine, ten and eight CSIs which are specific for members of the subgenera , and (excluding and ), respectively. Additionally, our work has identified several CSIs that support species relationships which are not evident from phylogenetic analysis. Of these CSIs, one CSI supports the ancestral nature of the avian- species in comparison to the mammalian-infecting groups of species, four CSIs strongly support a specific relationship of species between the subgenera and and three CSIs each that reliably group with members of the subgenus and within the subgenus respectively. These results provide a reliable framework for understanding the evolutionary relationships among the Piroplasmida species. Further, in view of the exclusivity of the described molecular markers for the indicated groups of hematozoa species, particularly large numbers of unique characteristics that are specific for all species, they provide important molecular tools for biochemical/genetic studies and for developing novel diagnostics and therapeutics for these organisms.