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Additional file 4: Figure S25. RBP2a alignment. Complete alignment of RBP2a protein sequences.

Additional file 3: Figure S17. DBP1 alignment. Complete alignment of DBP1 protein sequences. The presence of the DBL domain (Pfam: PF0311) and the trans-membrane domain is indicated.

Additional file 2: Figure S1. Apicoplast and mitochondrial genomes. Circos plots of the assembled apicoplast (top) and mitochondrial (bottom) genomes. The presence of protein-coding genes (grey), ribosomal-RNAs (green), and transfer-RNAs (red) is denoted by bars. For each gene category, outermost circle denotes genes on the forward strand, and the innermost circle denotes genes on the reverse strand. The two circular representations nearest to the center represent GC-skew and GC-content, respectively. Figure S2. Busco assembly assessment. Gene content in Plasmodium simium and other Plasmodium genome assemblies. BUSCO was run using the eukaryota odb9 data set containing 303 BUSCO groups. Figure S3. Protein phylogeny. Maximum likelihood phylogeny based on 3204 concatenated Plasmodium simium protein-coding genes with 1:1 orthologs across a selection of Plasmodium species (see Methods). Tree was constructed using RaxML with the GTRGAMMA model and Plasmodium gallinaceum as outgroup. Branch support from 100 bootstrap replicates. Figure S4. Gene families. A) The number of gene family members among Plasmodium genomes. Diameters of circles are proportional to the log10-transformed number of family members, as shown on the right. B) Bar plot showing the distribution of average read coverage across all annotated P. simium genes. The 5th and 95th percentiles (genes with the highest and lowest read coverage, respectively) are highlighted in dark. Above the plots the proportion of genes belonging to the PIR gene family is shown as pie charts for the two extreme percentiles and the remaining genes ('Rest'). Using a Fisher's exact two-sided test, the proportion of PIR genes in both the 95th and the 5th percentile are significantly higher than in the 'Rest' genes (p = 5.8e-80 and p = 5.1e-67, respectively). Figure S5. Pir/Vir gene clustering. Clustering of PIR protein sequences based on BLASTP similarity. Network threshold is a bit-score of 40 and visualized using the edge-weighted spring embedded layout in cytoscape 3. Figure S6. SNP counts. Top: Number of high-confidence SNPs detected in each sample. Plasmodium simium samples shown in dark red, and Plasmodium vivax samples in orange (see also Additional file 1: Tables S3 & S4). Bottom: For a given SNP loci data is not available from all samples. The bar chart shows how many SNP loci (left y-axis) that have data (coverage) in a given number of samples (x-axis). Only SNPs with data from at least 55 samples were used in this study, as indicated by the blue bars. Purple bars represent discarded SNPs. The cumulative fraction of SNPs is shown as red line (right x-axis). For example, slightly more than half of the SNP loci have data from at least 55 samples, whereas app. 75% of samples have data from at least 25 samples. Figure S7. PCA plot. A) Plot of the first two dimensions from a principal component analysis of 124,968 SNPs. Plasmodium vivax samples are denoted by their geographic origin. B) Magnification of American vivax samples. C) Cumulative contribution (in percent) of each eigenvector. The separate clustering of Plasmodium simium and American P. vivax samples was also observed when plotting 2nd versus 3rd dimensions, and 3rd versus 4th dimensions (not shown). Figure S8. MDS plot. Plot of Multidimensional Scaling of Plasmodium simium and Plasmodium vivax samples. Figure S9. SNP phylogeny. Phylogenetic tree constructed from SNP sites. Identical to Fig. 1, but with sample IDs shown at terminal branches. Figure S10. Phylogenetic network. Network from nucleotide diversity distances produced in SplitsTree using the NeighborNet network. A magnification of the central hubs (red rectangle) is shown at the bottom right. Figure S11. Admixture. Q-estimates from unsupervised ADMIXTURE clustering analysis at K from 2 to 10. Plasmodium vivax samples are ordered by geographic origin. The lowest cross-validation error was observed for K = 3. Figure S12. Gene DXY diversity. A) For different intervals of average DXY values (x-axis), the number of genes with thee values are plotted (y-axis, log10-scaled). B) Box plot showing the distributions of DXY values for members of selected gene families. The right-most, yellow box shows the DXY values for all genes that not member of a gene family. Figure S13. DBP phylogeny. Neighbor-Joining tree of Plasmodium DBP protein sequences. Sequences were aligned using mafft and tree produced with CLUSTALW. Support from 1000 bootstrap replicates. Tree visualized with FigTree, genetic distance shown below tree. Plasmodium simium and Plasmodium vivax sequences derived from this study are highlighted in red and blue, respectively. Remaining sequences are suffixed by their genome of origin (PvivP; P. vivax P01, PvivS; P. vivax SalI, PcynM; P. cynomolgi M, PcynB; P. cynomolgi B, PknoH; P. knowlesi H). Figure S14. RBP phylogeny. Neighbor-Joining tree of Plasmodium RBP protein sequences. Sequences were aligned using mafft and tree produced with CLUSTALW. Support from 1000 bootstrap replicates. Tree visualized with FigTree, genetic distance shown below tree. Plasmodium simium and Plasmodium vivax sequences derived from this study are highlighted in red and blue, respectively. Remaining sequences are suffixed by their genome of origin (PvivP; P. vivax P01, PvivS; P. vivax SalI, PcynM; P. cynomolgi M, PcynB; P. cynomolgi B, PknoH; P. knowlesi H). Figure S15. Coverage across RBP gene loci. Average read coverage across gene loci when mapping human Plasmodium simium (top) and Plasmodium vivax (bottom) reads onto the P. vivax P01 genome. The RBPs genes are shown above plots with the P. vivax P01 gene identifiers below plots. Genes absent in P. simium are highlighted in grey. Note that the P. vivax P01 genome contains two annotated RBP1a and RBP2d genes, respectively. The average read coverage per gene is shown as dots for each individual sample, and the combined distributions are outlined by boxes. A single P. simium sample, AF22, has a typical coverage of around 200X but is omitted from this representation for clarity. The coverage of the P. simium CDC strain is highlighted as red dots. Figure S16. coverage across flanking regions. The average read coverage across Plasmodium vivax DBP and RBP genes is compared to the average read coverage across flanking genomic regions. The log2 ratio between coverage at flanking region and coverage at gene was calculated for four regions: 10 kb-5 kb upstream of gene (UP2), 5 kb-0 kb upstream of gene (UP1), 0 kb-5 kb downstream of gene (DOWN1), 5 kb-10 kb downstream of gene (DOWN2). Boxplot denotes the range of ratios for Plasmodium simium samples (red boxes), American Plasmodium vivax samples (light blue), and remaining Plasmodium vivax samples (blue). Up- and downstream are defined based on genome coordinates irrespective of gene orientation. After Bonferroni correction, only the 'UP1' region at RBP3 for P. simium samples (p = 0.011, denoted by asterisk) had a probability below 0.05 of the mean being above zero assuming a normal distribution. Figure S18. Haplotype network of DBP1 sequences. Haplotype network (minimum spanning network) produced using PopART. Numbers of mutations are indicated by hatch marks on edges. Plasmodium simium samples are shown in red, Plasmodium vivax in green, and P. vivax-like in purple. The previously published P. simium CDC strain sequence (ACB42432) is shown in black and bold. Figure S19. Read support for DBP1 deletion patterns. Among Plasmodium vivax and Plasmodium simium samples multiple deletion patterns in the DBP1 gene are observed (top). Deletions found in P. vivax samples are arbitrarily denoted vivax 1-4. For each sample, the number of reads supporting a given deletion is shown (bottom). Samples with reads supporting multiple deletion forms are indicated by asterisks. Figure S20. Dotplot. Similarity DNA dot plots of DBP1 genes. Top plots show Plasmodium simium and bottom plots show Plasmodium vivax DBP1 genes. Introns are denoted by grey rectangles and the deleted region (or site of deleted region in P. simium) is highlighted in red. Figure S21. Read coverage across the DBP1 deletion in Plasmodium simium samples. Artemis representation of read coverage across the Plasmodium simium DBP1 deletion. Reads from Plasmodium vivax samples AM01 & AM02 (two top panels) and P. simium AF22 & AF36 samples (two bottom panels) were mapped onto the P. simium AF22 assembly. The site of deletion is indicated by vertical red arrows. Note the lack of P. vivax reads spanning the deletion site. Figure S22. read coverage across the DBP1 deletion in Plasmodium vivax samples. Artemis representation of read coverage across the Plasmodium simium DBP1 deletion. Reads from P. simium AF22 & AF36 samples were mapped onto the Plasmodium vivax P01 genome. The site of deletion is indicated by red squares. Note the lack of P. simium coverage across the deleted region. Figure S23. PacBio read coverage across the DBP1 deletion in Plasmodium simium. Schematic depiction of PacBio reads mapping across the deletion in DBP1 gene. X-axis denotes genomic positions. Black line at the centre of plot shows the extent of exons. Reads mapping on the forward strand are shown above exons, reverse strand reads underneath exons. Reads are colored according to the sample from which they are derived. The site of deletion is indicated by arrows and thin vertical orange lines. Figure S24. DBP1 PCR. Top: Schematic overview of DBP1 deletion PCR approach. Gel images shown for human Plasmodium vivax samples (top gel image), human Plasmodium simium samples (middle image), and non-human primate (NHP) P. simium samples (bottom image). Expected band sizes with and without deletion event are indicated by red triangles. Bottom: Primer sequences. Figure S26. Read coverage across the RBP2a deletion in Plasmodium simium samples. Artemis representation of read coverage across the Plasmodium simium RBP2a deletion. Reads from vivax samples AM01 & AM02 (two top panels) and P. simium AF22 & AF36 samples (two bottom panels) were mapped onto the P. simium AF22 assembly. The site of deletion is indicated by vertical red arrows. Note the lack of Plasmodium vivax reads spanning the deletion site. Figure S27. Read coverage across the RBP2a deletion in Plasmodium vivax samples. Artemis representation of read coverage across the Plasmodium simium RBP2a deletion. Reads from P. simium AF22 & AF36 samples were mapped onto the P. vivax P01 genome. The site of deletion is indicated by red squares. Note the lack of P. simium coverage across the deleted region. Figure S28. PacBio read coverage across RBP2a deletion in Plasmodium simium. Schematic depiction of PacBio reads mapping across deletions in the RBP2a gene. X-axis denotes genomic positions. Black line at the centre of plots shows the extent of exons. Reads mapping on the forward strand are shown above exons, reverse strand reads underneath exons. Reads are colored according to the sample from which they are derived. Sites of deletions are indicated by arrows and thin vertical orange lines. Figure S29. RBP2a PCR. Top: Schematic overview of RBP2a deletion PCR approach. Gel images shown for Plasmodium vivax and Plasmodium simium samples. Expected band sizes with and without deletion event are indicated by red triangles. Bottom: Primer sequences. Figure S30. Short indels. A) Pie chart showing the percentage of indels being integers of 1-3 base pairs. B) The percentage of insertions (left bar) and deletions (middle) overlapping low-complexity regions in proteins. The percentage of all proteins consisting of low-complexity sequences is shown on the right. Low-complexity annotation downloaded from PlasmoDB. C) Size distributions of genes with and without indels. Genes with indels are listed in Additional file 1: Table S7. Figure S31. DBP1 protein structures. Plasmodium simium DBP1 is predicted to associate with human DARC. Left: Top-view of modelled DBP1 from Plasmodium vivax strain P01, human-infecting P. simium AF22 (PsDBP1) and monkey-infecting P. simium (CDC PsDBP1) sequences. Models were established based on the crystal structure of the P. vivax DBP1, strain Salvador 1, in complex with human DARC (PDB 4nuv). Individual chains of the DBP1 dimer are shown in light and dark blue. DARC (residues 19-30) is coloured in grey. Residue substitutions between the models and the crystal structure are highlighted in magenta. Right: close-up view of the DARC-binding site, as delimited by a dashed square on the left. Colours as in the left panel. The rearrangement of hydrogen-bonds (black dotted lines) is shown for the Lys-Asn substitution. Figure S32. Mitochondrial haplotype network. Haplotype network (minimum spanning network) produced using PopART (see Methods). Numbers of mutations are shown on edges. The two identical Plasmodium simium samples (AF22 and GenBank accession AY722798) are shown in black. Plasmodium vivax haplogroups coloured according to country of origin. Circle sizes indicate the number of sequences in haplogroups. Figure S33. Protein orthology. Venn diagram showing the number of shared gene orthogroups between Plasmodium simium and three other Plasmodium genomes. Figure S34. SNP allele frequencies & coverage. Top: Allele frequencies for SNPs with available data from at least 37 Plasmodium simium or Plasmodium vivax samples. Bottom: Mean read coverage at SNP sites shown for each samples.