Rerooting the evolutionary tree of malaria parasites

Abstract

Malaria parasites (Plasmodium spp.) have plagued humans for millennia. Less well known are related parasites (Haemosporida), with diverse life cycles and dipteran vectors that infect other vertebrates. Understanding the evolution of parasite life histories, including switches between hosts and vectors, depends on knowledge of evolutionary relationships among parasite lineages. In particular, inferences concerning time of origin and trait evolution require correct placement of the root of the evolutionary tree. Phylogenetic reconstructions of the diversification of malaria parasites from DNA sequences have suffered from uncertainty concerning outgroup taxa, limited taxon sampling, and selection on genes used to assess relationships. As a result, inferred relationships among the Haemosporida have been unstable, and questions concerning evolutionary diversification and host switching remain unanswered. A recent phylogeny placed mammalian malaria parasites, as well as avian/reptilian Plasmodium, in a derived position relative to the avian parasite genera Leucocytozoon and Haemoproteus, implying that the ancestral forms lacked merogony in the blood and that their vectors were non-mosquito dipterans. Bayesian, outgroup-free phylogenetic reconstruction using relaxed molecular clocks with uncorrelated rates instead suggested that mammalian and avian/reptilian Plasmodium parasites, spread by mosquito vectors, are ancestral sister taxa, from which a variety of specialized parasite lineages with modified life histories have evolved.

Fig. 1.

Series of topologies for the Haemosporida in the style of Martinsen et al. (19). (A) The classic topology based on these traits places Leucocytozoon, which lacks these traits, as a sister to all other Haemosporida. (B) The topology of Perkins and Schall (13) based on cytochrome b sequences rooted with Theileria. (C) The topology of Martinsen et al. (19), based on sequences from two mitochondrial genes, one apicoplast gene, and one nuclear gene, with Leucocytozoon assigned as the outgroup taxon. (D) The data of Martinsen et al. reanalyzed under a Bayesian relaxed molecular clock model, in which Plasmodium becomes paraphyletic with respect to the other genera and Leucocytozoon is sister to the avian Haemoproteus and Parahaemoproteus clade. (E) An enlarged cytochrome b dataset analyzed under a relaxed clock model again places the root between the clades of mammalian and avian/reptilian parasites. This topology is also found under maximum likelihood optimization (Fig. 4). A minimum set of inferred character changes for hemozoin pigment (red circles) and merogony (green squares) is indicated. LEU, Leucocytozoon; PLA, Plasmodium; HEP, Hepatocystis; HAE, Haemoproteus; PAR, Parahaemoproteus; POL, Polychromophilus. Mammalian lineages are shown in black; avian/reptilian lineages, in blue.

Fig. 2.

Series of cladograms based on data analyzed by Martinsen et al. (19). (A–D) Our outgroup-free rooting (A) and three alternative topologies: midpoint rooting (B), UPGMA, in which Hepatocystis spp. form their own clade (orange) and one lizard Plasmodium lineage falls outside the others (C); and outgroup rooting (D). The size of a clade reflects relative taxon sampling. Purple indicates Leucocytozoon; blue, Haemoproteus (dove); red, bird and lizard Plasmodium; yellow, bird Parahaemoproteus; green, mammal Plasmodium. (E and F) Marginal densities of tree likelihoods of all data from Martinsen et al. (19) (E) and cytochrome b data (F).

Fig. 3.

Phylogenetic tree produced using a relaxed clock with an HKY + gamma model of nucleotide substitution and uncorrelated, lognormally distributed rates. Relative rates of evolution on branches increase from blue through black to red, and also increase as shown by fills from white to red. High rates of nucleotide substitution particularly characterize mammalian Hepatocystis and avian Leucocytozoon; also see Fig. 4.

Fig. 4.

Maximum likelihood phylogram based on cytochrome b data using a GTR + gamma model of nucleotide substitution. The tree was rooted between mammal Plasmodium/Hepatocystis and all other parasite lineages. Colored lines indicate >95% bootstrap values. Breadths of cartooned clades are proportional to species sampling in this analysis. (Scale bar: 5% nucleotide substitution.)