The polyphyly of Plasmodium: comprehensive phylogenetic analyses of the malaria parasites (order Haemosporida) reveal widespread taxonomic conflict

Abstract

The evolutionary relationships among the apicomplexan blood pathogens known as the malaria parasites (order Haemosporida), some of which infect nearly 200 million humans each year, has remained a vexing phylogenetic problem due to limitations in taxon sampling, character sampling and the extreme nucleotide base composition biases that are characteristic of this clade. Previous phylogenetic work on the malaria parasites has often lacked sufficient representation of the broad taxonomic diversity within the Haemosporida or the multi-locus sequence data needed to resolve deep evolutionary relationships, rendering our understanding of haemosporidian life-history evolution and the origin of the human malaria parasites incomplete. Here we present the most comprehensive phylogenetic analysis of the malaria parasites conducted to date, using samples from a broad diversity of vertebrate hosts that includes numerous enigmatic and poorly known haemosporidian lineages in addition to genome-wide multi-locus sequence data. We find that if base composition differences were corrected for during phylogenetic analysis, we recovered a well-supported topology indicating that the evolutionary history of the malaria parasites was characterized by a complex series of transitions in life-history strategies and host usage. Notably we find that Plasmodium, the malaria parasite genus that includes the species of human medical concern, is polyphyletic with the life-history traits characteristic of this genus having evolved in a dynamic manner across the phylogeny. We find support for multiple instances of gain and loss of asexual proliferation in host blood cells and production of haemozoin pigment, two traits that have been used for taxonomic classification as well as considered to be important factors for parasite virulence and used as drug targets. Lastly, our analysis illustrates the need for a widespread reassessment of malaria parasite taxonomy.

Keywords: Plasmodium; base composition bias; malaria; phylogeny; polyphyly.

Figure 1.

The order Haemosporida is characterized by extremely heterogeneous base composition and codon usage. (a) The evolution of GC content is shown optimized across the haemosporidian phylogeny using the contMap function in phytools. Note that the macaque Plasmodium clade has evolved a GC content that is significantly higher than the rest of the ingroup. Branches where significant shifts in GC content occurred as determined by the OUshifts analysis are denoted by asterisks. (b) Principle components analysis of RSCU across the Haemosporida. The macaque Plasmodium clade groups with the outgroup Theileria to the exclusion of the other malaria parasites, indicating similar usage of synonymous codons within degenerate amino acids.

Figure 2.

The favoured haemosporidian phylogeny. The haemosporidian parasite phylogeny recovered from BEAST using the fully partitioned amino acid dataset and lognormal relaxed molecular clock. The outgroup Theileria is not shown for ease of viewing the ingroup topology. Shown as silhouettes are representatives of the vertebrate host group for each haemosporidian lineage. Clades denoted with the letters A–F are referred to in the Discussion.

Figure 3.

The ‘mammal-first' topology is likely driven by base composition convergence between the macaque Plasmodium clade and the outgroup Theileria. Two analyses recovered the ‘mammal-first' topology: (a) the ASTRAL-II species tree analysis using gene trees generated from nucleotide codon alignments, and (b) RAxML supermatrix analysis of the unpartitioned codon alignment. This topology contrasts dramatically with the topologies recovered from analyses that correct for the base composition convergence effect, suggesting the ‘mammal-first' topology is an artefact.

Figure 4.

The phylogenetic hypothesis presented here alters our understanding of malaria parasite host-switching and life-history evolution. (a) All topologies that we recovered from across phylogenetic analyses that were corrected for base composition bias suggest a single switch to mammalian hosts from sauropsid-infecting ancestors, followed by one additional switch back to sauropsid hosts in the Plasmodium lineage. (b) This topology suggests two alternative scenarios for the evolution of blood schizogony (asexual reproduction in the host bloodstream) and vector use. One scenario (left) posits a single gain of blood schizogony and a switch to mosquito (Culicidae) vectors in the lineage leading to all mammalian malaria parasites, followed by loss of blood schizogony and a vector switch in three lineages: Polychromophilus, Hepatocystis and Nycteria. Alternatively (right), the four lineages of Plasmodium identified in our analysis could have evolved blood schizogony and switched to mosquito vectors independently.