Identification of ultra-conserved PIR orthologs. (A) PIR protein sequences from five Plasmodium species, including P. yoelii (826 yir genes), P. berghei (135 bir genes), P. chabaudi (196 cir genes), P. vivax (345 vir genes), and P. knowlesi (68 kir genes), were globally aligned in an all-vs.-all pairwise manner using ClustalW. Box plots show the resulting distribution of global percent identity values for each pair of species. For each species pair, one outlier (red circles) representing a pair of proteins with increased cross-species conservation levels is identified, including PY06119 (P. yoelii), PBANKA_010050 (P. berghei), PCHAS_010120 (P. chabaudi), PVX_113230 (P. vivax), and PKH_114850 (P. knowlesi). The inset shows the gene models of these five proteins with the predicted Plasmodium_vir domain (PF05795) indicated in blue and predicted transmembrane domains (TM) indicated in black. The second exon of PY06119 was found to be truncated and an improved version of this gene model (PY06119*) was therefore used in the analysis. (B) Multiple sequence alignment of the five conserved PIR orthologs. Note the exceptional high degree of sequence conservation (indicated by black and gray columns), suggesting an important molecular function for this particular PIR protein in each Plasmodium species.