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Genomes of an entire Plasmodium subgenus reveal paths to virulent human malaria Thomas D. Otto1,†,*, Aude Gilabert2, †, Thomas Crellen1,3, Ulrike Böhme1, Céline Arnathau2, Mandy Sanders1, Samuel Oyola1, Alain Prince Okouga4, Larson Boundenga4, Eric Wuillaume5, Barthélémy Ngoubangoye4, Nancy Diamella Moukodoum4, Christophe Paupy2, Patrick Durand4, Virginie Rougeron2,4, Benjamin Ollomo4, François Renaud2, Chris Newbold1,6, Matthew Berriman1,* & Franck Prugnolle2,4,*

1 Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton CB10 1SA, United Kingdom 2 Laboratoire MIVEGEC, UMR 5290-224 CNRS-IRD-UM, Montpellier, France 3 Department of Infectious Disease Epidemiology, Imperial College London, St Mary’s Campus, Norfolk Place, London W2 1PG, United Kingdom 4 Centre International de Recherches Médicales de Franceville, Franceville, Gabon 5 Sodepal, Parc of la Lékédi, Bakoumba, Gabon 6 Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom *Correspondence to: Thomas D. Otto ([email protected]), Matthew Berriman ([email protected]) or Franck Prugnolle ([email protected]) †These authors contributed equally. Abstract: Plasmodium falciparum, the most virulent agent of human malaria, shares a recent common ancestor with the gorilla parasite P. praefalciparum. Although there are further gorilla and chimpanzee- infecting species in the same (Laverania) subgenus as P. falciparum, none are known to be able to establish repeated infection and transmission in humans. To elucidate underlying mechanisms and the evolutionary history of this subgenus, we have analysed multiple genomes from all known Laverania species. Here we estimate the timings of Laverania speciation events, placing P. falciparum speciation 40,000-60,000 years ago followed by a recent population bottleneck. We show that interspecific gene transfers as well as convergent evolution were important in the evolution of these species. Striking copy number and structural variations were observed within gene families and for the first time, features in P. falciparum are revealed that made it the only member of the Laverania able to infect and spread in humans.

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Introduction The evolutionary history of Plasmodium falciparum, the most common and deadliest human malaria parasite, has been the subject of much uncertainty and debate (1, 2). Recently it has become clear that P. falciparum is derived from a group of parasites infecting African Great Apes and known as the Laverania subgenus (2). Until 2009, the only other species known in this subgenus was a parasite of chimpanzees known as P. reichenowi and for which only one isolate was available (3). It is now clear that there are a total of at least seven species in Great Apes that naturally infect chimpanzees (P. gaboni, P. billcollinsi and P. reichenowi), gorillas (P. praefalciparum, P. blacklocki and P. adleri (4, 5), or humans (P. falciparum only) (Fig. 1A). Within this group, P. falciparum is the only parasite that has successfully adapted to humans after a transfer from gorillas and subsequently spread all over the world (2). Since the discovery of the Laverania a number of studies have provided incremental data on the evolution of this subgenus but the mechanisms underlying host specificity and the reasons why only one productive transfer into humans has taken place are still unclear. Comparisons of the P. falciparum genome first to that of P. reichenowi (6) and more recently to that of P. gaboni (7), have attempted to identify genes that displayed rapid or specific evolution. However, the lack of whole genome information for the whole subgenus (particularly P. praefalciparum, the closest sister species to P. falciparum) limited the power of these comparisons. In addition, the studies involving P. gaboni, lacked the subtelomeric regions that harbour many gene families involved in host-parasite interactions and antigenic variation (8). Only a subset of these multigene families has been studied by PCR-based approaches (9) (e.g. the DBLα domain of var genes), preventing a complete reconstruction of the paths that led to the evolution of P. falciparum. To investigate the evolutionary history of the entire Laverania subgenus and to unravel the genomic evolutionary paths that allowed humans to be colonised, we have sequenced multiple genotypes of all known Laverania species.

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Figure 1: Overview of the Laverania genomes analysed. (A) Maximum likelihood tree of the Laverania based on the “Lav12sp” set of orthologues (see Methods). Bootstrap values are provided at nodes. (B) The number of isolates per species sequenced is indicated using the symbol of their host species, together with the values of polymorphism within species, as estimated using the nucleotide diversity π (“Lav15st” set of orthologues; see Supplementary Materials). (C) The quality of the genome assemblies is indicated through the number of scaffolds and the pairwise BLAST scores within the core chromosome 4 and its subtelomeric regions (shaded boxes).

Genome sequencing from six Laverania species Blood samples were taken during successive routine sanitary controls, from four gorillas and seven chimpanzees living in a sanctuary or quarantine facility prior to release (see Supplementary Material and Methods). A total of 15 blood samples were positive for ape malaria parasites by PCR. Despite low parasitemia in most animals, a combination of host DNA depletion, parasite cell sorting and amplification methods enabled sufficient parasite DNA templates to be obtained for short-read (Illumina) and long read (Pacific Bioscience) sequencing (Fig 1B, Table 1). Mixed infections, which were frequent, were resolved by utilising sequence data from single infections, resulting in 19 genotypes (Table S1), and the dominant genotype in each sample was assembled de novo into a reference genome (see Supplementary Material and Methods). Each reference genome was assembled into 44-97 scaffolds (Table 1), with large contigs containing the subtelomeric regions or internal var gene clusters comprising multigene families (Fig. 1C) that are known in P. falciparum and P. reichenowi to be involved in virulence and host-pathogen interactions. In total, genomes were produced for six malaria parasite species: P. praefalciparum, P. blacklocki, P. adleri, P. billcollinsi, P. gaboni and P. reichenowi. The high quality of the assemblies can be seen in the large number of one-to-one

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orthologues obtained between the different reference genomes (4,324 among the seven species and 4,818 between P. falciparum, P. praefalciparum and P. reichenowi). Two to four additional genomes were obtained for each species except for P. blacklocki (Table 1).

s Mb

Fold Fold b

Species Sample ID Primate species size y Primary co-infection Genes Contigs Scaffold Assembl coverage PprfG01 Chimp 104 26 142 73 6,476 P. adleri P. praefalciparum PprfG02 Gorilla 1269 - - - - none PprfG04 Multiple infection, see PadlG03 P. adleri

P. praefalciparum II PprfG03 Gorilla 668 - - - - none P. reichenowi PrG01 Chimp 106 24.5 66 48 5,941 none PrCDC a Chimp 296 24.1 374 2,465 5,895 none PrG02 Chimp 123 - - - - none PrG03 Chimp 112 - - - - none P. billcollinsi PbilcG01 Multiple inf. PgabG02 23.1 306 89 5,637 P. gaboni

PbilcG03 Multiple infection, see PgabG04 P. gaboni

PbilcG04 Multiple infection, see PgabG05 P. gaboni P. blacklocki PblacG01 Gorilla 221 22 311 97 5,346 none P. gaboni PGAB01 Chimp 367 21.8 121 96 5,421 P. vivax PgabG02 Chimp 341 20.9 76 44 5,249 P. billcollinsi PgabG03 Chimp 669 - - - - P. vivax PgabG04 Chimp 679 - - - - P. billcollinsi PgabG05 Chimp 530 - - - - P. reichenowi P. adleri PadlG01 Gorilla 372 22.2 102 82 5,515 none PadlG02 Gorilla 2262 - - - - none

PadlG03 Gorilla 445 - - - - P. praefalciparum a Data from Otto et al(6)b Based on percentage of reads mapping to the reference for each Laverania species (see Table S1) Table 1: Overview of all Laverania samples used in study. All samples generated for this study, except PrCDC were sequenced using Illumina technology with a range of read lengths from 100–250 bp. For isolates (bold) where assemblies were produced using long-reads from Single-Molecule Real-Time sequencing (Pacific Biosciences), the total assembled size, number of contigs, scaffolds and predicted genes are shown. For those we report the assembly. Fold coverage is reported based on mapping reads to the P. falciparum 3D7 reference genome (v3.1). P. billcollinsi sequences were obtained from P. gaboni co-infections, of which some also harboured P. reichenowi species.

Phylogenetic relationships and the emergence of P. falciparum Conservation of gene content and synteny is striking between these complete genomes, enabling us to reconstruct with confidence the relationships between different Laverania species, to compare their relative genetic diversity (Supplementary Text, Fig. S1) and to estimate the age of the different speciation events that led to the extant species. Because of uncertainty regarding exact generation time and in vivo mutation rates (both of which have a linear influence on time estimates), the values that we quote should be regarded as approximate (Supplementary Materials, Table S2). From our Bayesian whole-genome estimates, the ancestor of all current day parasites of this subgenus existed 0.7–1.2 million years ago, a time at which the subgenus divided into two main clades (A and B): Clade A that

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comprises P. adleri and P. gaboni and Clade B that includes the remaining species (Fig. 2A). Our range of values is far more recent than previous estimates (3, 10). Ancestral state reconstruction did not allow us to solve the nature of the host (gorilla or chimpanzee) in which the ancestor lived. Following the group A/B subdivision, several speciation events occurred leading either to new chimpanzee or gorilla parasites. Interestingly, the divergence between P. adleri and P. gaboni in one lineage and P. reichenowi and the ancestor of P. praefalciparum/P. falciparum in the other lineage occurred at approximately the same time (140–230 thousand years ago; Fig. 2A, Table S2), suggesting that the same phenomena may have favoured these host switches. Based on our coalescence estimates, P. falciparum emerged in humans from P. praefalciparum around 40–60 thousand years ago (Fig. 2A), significantly later than the evolution of the first modern humans and their spread throughout Africa (11). Our analysis also indicates significant gene flow between these two parasite species after speciation (Table S2). It has been suggested that P. falciparum arose from a single transfer of P. praefalciparum into humans (7). It has also been proposed (based on the paucity of neutral SNPs within the genome of P. falciparum) that P. falciparum emerged from a bottleneck of a single parasite around 10,000 thousand years ago, after agriculture was established (Fig. 2B) (7, 12). Clearly neither of these hypotheses are correct in light of our results; we estimate that the P. falciparum population declined around 8,000- 14,000 years ago and reached a minimum about 5,000 years ago (Fig. 2C) with an effective population size (Ne) of at least 8,000 (Supplementary Text; generally the census number of parasites is higher than Ne (13)). Neither are these hypotheses consistent with the observation of several ancient gene dimorphisms that have been observed in P. falciparum. A previous analysis using P. reichenowi and limited P. gaboni sequence data, provided some evidence that different dimorphic loci diverged at different points in the tree (14). Looking at each of these P. falciparum loci across the Laverania, we found different patterns of evolution at the msp1, var1csa, and msp3 loci (Fig. S2A). Most strikingly, a mutually exclusive dimorphism (described as MAD20/K1 (15)) in the central 70% of the msp1 sequence, pre-dates the P. falciparum–P. praefalciparum common ancestor, and dimorphism in var1csa (an unusual var gene of unknown function that is transcribed late in the asexual cycle) occurred before the split with P. reichenowi. The gene eba-175 that encodes a parasite surface ligand contains a dimorphism that arose after the emergence of P. falciparum (Fig. S2B). The time to the most recent common ancestor has been estimated as 130–140 thousand years in an analysis (16) that assumed P. falciparum and P. reichenowi diverged 6 million years ago. However, based on our new estimate for P. falciparum–P. reichenowi divergence, we recalibrate their estimate of the most recent common ancestor of the eba-175 alleles to

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be 4 thousand years ago, which is in good agreement with our divergence time for P. falciparum (Supplementary Text). The recent dimorphism cannot however explain the recent observation of an ancient dimorphism near the human and ape loci for glycophorin (17) – an EBA-175 binding protein. Different balancing selection pressures over time may have shaped the formation and maintenance of all of these dimorphic loci. Identifying the interacting host genes (or epistatic interactions with other parasite genes) will shed more light on the underlying mechanisms.

Figure 2 Overview of the dating of the evolution of the Laverania. (A) Coalescence based estimates of the split of the species using intergenic and genic alignments. Dates are displayed on nodes (average of 402–681 mitotic events per year used; MYA - million years ago). The ratios should be robust but the actual dates depend on estimates of generation time and mutation rate (see Table S2 for details). (B) Estimated time line of events in the Laverania relative to human evolution. (C) Multiple sequentially Markovian coalescent estimates of the bottleneck in the P. falciparum population. Assuming our estimate of the number of mitotic events per year, the bottleneck occurred 4,000-6,000 years ago. y-axis is the natural logarithm (Ln). Bootstrapping was performed by 50 replicates by randomly resampling from the segregating sites used as input.

Evolution through introgression, gene transfer and convergence Frequent mixed infections in apes and mosquitoes (18) provide clear opportunities for interspecific gene flow between these parasites. A recent study (7) reported a gene transfer event between P. adleri and the ancestor of P. falciparum and P. praefalciparum of a region on chromosome 4 including key genes involved in erythrocyte invasion (rh5 and cyrpA). We systematically examined the evidence for introgression or gene transfer events across the complete subgenus by testing the congruence of each gene tree to the species tree. Beyond the region that includes rh5 (Fig. S3A,B), few signals of interspecific gene flow were obtained (n =14) suggesting that these events were rare or usually strongly deleterious (Supplementary Text, Fig. S4).

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The Laverania subgenus evolved to infect chimpanzees and gorillas several times independently but, on a genome-wide scale, the convergent evolution of host-specific traits has not left a signature (Supplementary Text, Fig. S5). We therefore examined each CDS independently and were able to identify genes with differences fixed within specific hosts, falling into three categories: 53 in chimpanzee-infective parasites, 49 in gorilla-infective and 12 with fixed traits in both host species (Fig. 3; Table S3A). For at least 66 genes, these differences were unlikely to have arisen by chance (p <0.05) and GO term enrichment analysis revealed that several of these genes are involved in erythrocyte invasion (Table S3B) including rh5 (which has a signal for convergent evolution even when the introgressed tree topology is taken into consideration; Fig. S3C). Rh5 is the only gene identified in P. falciparum that is essential for erythrocyte recognition during invasion, via binding to Basigin. P. falciparum rh5 cannot bind to gorilla Basigin and binds poorly to the chimpanzee protein (19). We notice that one of the convergent sites is known to be a binding site for the host receptor Basigin (20) (Fig. S3C). The gene eba-165 encodes a member of the erythrocyte binding like (EBL) super family of proteins that are involved in erythrocyte invasion. Although eba-165 is a pseudogene in P. falciparum (21), it is not in the other Laverania species (Fig. 4) and may therefore be involved in erythrocyte invasion, like other EBL members. The protein has three convergent sites in gorillas, one falls inside the F2 region, a Duffy Binding-Like (DBL) domain involved in the interactions with erythrocyte receptors. The role of this protein and of these convergent sites in the invasion of gorilla red cells, remains to be determined. Finally, genes involved in gamete fertility (the 6-cysteine protein P230) or previously considered as potential candidate vaccines (doc2) also displayed signals of convergent evolution. Interestingly, among the 12 coding sequences with fixed differences in both great ape parasite species, P230 was the only one found with a position that was different and fixed within the three host species (gorillas, chimpanzees and humans). P230 is involved in gamete development and trans-specific reproductive barriers (22), possibly through enabling male gametes to bind to erythrocytes prior to exflagellation (23). Host-specific residues observed in P230 might affect the efficiency of the binding to the erythrocyte receptors and result from co-evolution between the parasite molecule and the host receptor.

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Figure 3: Convergent evolution analyses: fixed differences between great ape-infecting species. Analysis was performed using the “Lav7sp” set of orthologues but filled circles are for the differences that are fixed within all the isolates available (“Lav15st” set) and for which we could reject neutral evolution (For the gene list see Table S3)

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Subtelomeric gene families To date, the only in depth data on the subtelomeres of the Laverania have come from P. reichenowi and P. falciparum. We provide for the first time a complete picture of the evolution of these important families (Fig. 4) inside the subgenus. Gene content and copy number is summarized in Fig. 4 and Table S4A. The first important observation is that most gene families were likely present in the ancestor of all the Laverania, suggesting an ancient origin. In addition, most families displayed the same gene composition throughout the subgenus and only a subset of them displayed species-specific contraction or expansion (Fig. 4 and Table S4). For these latter families, two groups of parasites clearly differ in their composition: Clade A on one side (with P. adleri and P. gaboni) and some species of Clade B on the other side (P. billcollinsi, P. reichenowi and P. praefalciparum and P. falciparum). P. blacklocki (Clade B) is intermediate in its composition. Such subdivision concerns for instance the largest gene family that is likely common to all other malaria species: the Plasmodium interspersed repeat family (pir, which includes the rif and stevor families in P. falciparum) (Fig. 4). This family has been proposed to be involved in important functions such as antigenic variation, immune evasion, signalling, trafficking, red cell rigidity and adhesion (24) and yet has expanded only in Clade B, after the P. blacklocki split. For other gene families, like the group of exported proteins hyp4, hyp5, mc-2tm and EPF1,

Figure 4 Gene families in the Laverania. Distribution of genes encoding erythrocyte they seem to have expanded only invasion ligands (RH and EBA genes) and other major multigene families. in P. praefalciparum and P. falciparum (and even more in P. falciparum for hyp4 and hyp5). Since the latter three are components of Maurer’s clefts, an organelle involved in protein export(25), some evolution of function in this organelle may have been an important precursor to human infection.

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Because Laverania are known to have varying host tropism and highly specific preferences for their host species, we looked at variations in gene number and relatedness between gorilla and chimpanzee specific parasites. Some stevor sequence types are differentially associated with chimpanzee or gorilla infecting parasites (Fig. S6). Interaction with host-factors could be a major role of the stevor family; expression on the infected erythrocyte surface and stevor binding to host glycophorin C in P. falciparum(26) have been reported. The family of acyl-CoA synthetase genes, reported to be expanded and diversified in P. falciparum (27) (Fig. S7A) are in fact expanded across in Laverania and have four fewer copies in P. falciparum. Three other gene families with significant differences within the Laverania, are namely DBLmsp, glycophorin binding protein and CLAG (Fig. S8). Focusing on protein domains within the subtelomeric families, the Plasmodium RESA N-terminal domain has clear host-specific differences in copy number variation in gorilla parasites compared to chimpanzee ones (Fig. S7B, Table S4B).

A pam B - DBLα CIDRα CIDRα pam DBLε CIDRα *! pam DBLζ DBLε/DBLn CIDRβ

C - ATS 0.3 DBLβ CIDRζ

ATS

DBLα *!

*!

P. adleri ! P. gaboni! P. blacklocki! P. billcollinsi! P. reichenowi! P. praefalciparum! P. falciparum! # NTS CIDR DBL DBL DBL D Species! var!NTS! CIDRα! CIDRβ! CIDRδ! CIDRγ! DBLα! DBLβ! DBLδ! DBLε! DBLγ! DBLn! DBLζ! pam! pam! pam1! pam2! pam3! ATS! Padleri! 58! 1! 1! 1! 0! 0! 2! 28! 0! 99! 45! 58! 31! 0! 2! 7! 1! 7! 19! Pgaboni! 61! 1! 1! 0! 0! 0! 2! 6! 0! 75! 20! 45! 17! 0! 16! 4! 4! 19! 17! Pblacklocki! 43! 0! 0! 0! 0! 0! 0! 0! 0! 0! 26! 4! 6! 0! 0! 8! 0! 0! 0! Pbillcollinsi! 35! 20! 31! 25! 0! 5! 27! 9! 32! 4! 0! 2! 1! 0! 0! 0! 0! 0! 21! Preichenowi! 92! 77! 85! 60! 1! 27! 87! 49! 78! 18! 42! 0! 8! 1! 1! 2! 1! 1! 62! 1.0 Ppraefalciparum!112! 65! 85! 47! 7! 29! 92! 79! 70! 96! 81! 1! 29! 5! 5! 6! 5! 5! 94! Pfalciparum! 67! 56! 56! 37! 2! 17! 59! 17! 52! 15! 15! 0! 6! 1! 1! 2! 1! 1! 59! Figure 5 species. A BLAST cut-off of 50% global identity was used. More connected domains are more similar. Maximum likelihood trees were generated for DBLα (B), and the conserved C terminal domain termed Acidic Terminal Sequence (ATS), (C). P. falciparum domains clustering with Clade A are indicated (*). The heatmap (D) shows the amount of var genes longer than 2500bp and the frequency of domains per species

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Evolution of var genes The var genes, crucial mediators of pathogenesis and virulence through cytoadherence and immune evasion, are the most studied and the best known P. falciparum gene family. These genes show striking difference between them in Clades A and B (Fig. 5). This gene family is unique to the Laverania and encodes proteins with variable numbers of highly polymorphic Duffy Binding Like (DBL) domains and cysteine rich interdomain regions (CIDR) involved in the interaction with specific host cell surface proteins, and a more conserved intracellular domain (8). DBL and CIDR domains have a number of subtypes identified in P. falciparum denoted by Greek letters. Our previous analysis of the P. reichenowi genome (6), and other recent studies on partial var genes from a subset of the Laverania (9), established the ancient origin of these sequences, their relationship to the current var repertoire of P. falciparum and the existence of unusual domains in P. gaboni. Here we show that the total domain composition and genomic organisation is conserved in Clade B parasites, with the exception of P. blacklocki (Fig. 5a; Fig. S9A). In contrast, the evolution of these genes in Clade A parasites (P. adleri and P. gaboni) and in P. blacklocki has followed a different course. They almost completely lack CIDR domains, have fewer DBLα domains but contain an excess of other DBL types and untypeable domains named x1 and x2 by Larremore et al. (9). Analysis of full length sequences however puts these latter domains within the DBLε subtype (Fig. S10). A tree of all of the DBLα domains highlights the fact that those of Clade A parasites are unusual (Fig. 5B). Moreover, clustering within the DBLα of Clade A is a DBLα pseudogene on chromosome 4 of both P. falciparum and P. praefalciparum that is isolated in these two species but is part of an additional internal var gene cluster in the other species. Furthermore, its sequence is highly conserved across all species suggesting that the pseudogene may still be functional. A further difference on chromosome 4 between Clade A and B parasites relates to the orientation of the internal var gene clusters (Fig. S9A). In all of the Clade B parasites they are transcribed from the reverse strand whereas in Clade A they are located on the forward strand. The orientations of these clusters could have implications on their relative capacities to recombine with other family members and create further diversity. If we compare sequence relatedness between each individual domain across all species then it is apparent that they have diverged widely. This is particularly evident in the CIDR and intracellular domains, suggesting that polymorphism in host receptors, the use of different receptors or changes in signalling may be driving the divergence (Fig. S10).

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The number of DBL domains currently associated in P. falciparum with var2csa (DBLpam) has also increased in Clade A. This gene is involved in cytoadhesion to the placenta in placental malaria but is also a central intermediary in the process of switching between expressed var genes during antigenic variation (28). The DBLpam domains clearly have a different architecture in Clade A parasites and are absent from P. billcollinsi and amplified in P. praefalciparum implying that historically they may have had a different function (Fig. 5 and Fig. S10). In addition, though the overall similarity between P. falciparum var2csa genes is around 80%, we find in two P. praefalciparum parasites, var2csa genes that shares a 6kb region of 99.61% identity to a set of current P. falciparum field isolates (Fig. S9B) suggesting either introgression or long term functional conservation and strong purifying selection.

P. falciparum-specific evolution To infer P. falciparum specific adaptive changes, we considered the P. falciparum / P. praefalciparum and P. reichenowi genome trios and then applied a branch-site test and calculated McDonald Kreitman (MK) ratios to detect events of positive selection that occurred in the P. falciparum lineage. The two tests identified 171 genes (out of 4,818) with signatures of positive selection in the human parasite species only (Table S5). Of these, 138 genes had a significant dN/dS ratio and 35 genes had an MK ratio significantly higher than 1. Two genes (rop14 and PF3D7_0609900) were significant in both tests. Among those 171 genes, almost half (n=83) encoded proteins of unknown function. Analysis of those with functional annotation indicated that genes involved in pathogenesis and/or the entry into host, in actin movement and organization and in drug response were significantly over-represented. Other genes, expressed in different stages of the P. falciparum life cycle (e.g. sera4 or emp3 involved during the erythrocytic stages, P230 involved in gametocytes, trsp or lisp1 involved in the hepatic stages or plp4, CelTOS or Cap380 involved in the mosquito stages) also showed a significant signal of adaptive evolution (Table S5).

Discussion How did P. falciparum arise? We have shown that the successful infection of humans occurred quite recently, around 40,000-60,000 years ago, and involved numerous parasites rather than a single one as previously proposed. After the establishment in its new host, the parasite population went through a bottleneck around 5,000 years ago during the period of rapid human population expansion due to farming (Fig. 2C). Irrespective of the analytical approach used, across the subgenus, we found

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entry into host, particularly red cells, to be crucial in determining host specificity. A key historic event in the eventual colonisation of humans is likely to have been the horizontal transfer of a region of chromosome 4 from P. adleri to the ancestor of P. falciparum / P. praefalciparum that contained both the rh5 and the cyrpA genes, the latter forming an essential complex with ripr/rh5 in the invasion process. Comparison of P. falciparum lines capable of infecting Aotus monkeys to those that cannot, suggests that rh5 is necessary but not sufficient for host transfer (29). We have identified in this gene sites that are host-specific, species-specific and praefalciparum/falciparum specific which taken together suggest that it is crucial to a host switch. To investigate what other changes must have been involved, we looked for genetic signatures shared by parasite species that infect the same primate host and identified some host specific signals within the core genomes. We have identified genes involved in sexual and mosquito stages together with a number of genes of unknown function expressed across the life cycle, the latter of which clearly require further investigation. Due to the completeness of the genomes, we were able to analyse subtelomeric gene families in detail. Because these families are intimately involved in host-parasite interactions we were surprised to discover that only one of them (stevor) showed evidence of host specific evolution. One feature of malaria parasites is their ability to continuously reinfect the same host throughout its life. This ability is due, at least in part, to the size and polymorphism of the var repertoire(30). It is likely that early in the evolution of this parasite, when host population sizes were relatively small, that this characteristic was essential to continue transmission and overcome herd immunity of a limited host population. We show that the var genes must have been present in the common ancestor of the Laverania but have evolved differently in the two major branches. However, the differences between the repertoires of Clade A and B parasites are not host specific, despite showing clear signs of species-specific evolution. The direct comparison of P. falciparum with P. praefalciparum identified differences in reticulocyte binding proteins (duplication of rh2 and pseudogenization of rh3), in gene dimorphism (msp1, msp3 and eba-175) and in adaptive evolution of genes expressed in the sexual stages. We also observe a reduced number of pir genes, a loss of four acyl-coA synthetases as well as a set of genes showing lineage specific signals of adaptive evolution at all stages of the life cycle. As a result of our analyses we propose the following series of events for the emergence of P. falciparum as a major human pathogen. First, facilitatory mutations are likely to have occurred in rh5 that in the first instance allowed invasion of both gorilla and human red cells. Modern humans emerged more than 200,000 years ago (31) and existed as small isolated populations (11). Our evidence suggests that P. falciparum and P. praefalciparum started to diverge around 40,000-60,000 years ago. In the following 40,000 years with low population densities in humans and gorillas there would have not been

13 bioRxiv preprint doi: https://doi.org/10.1101/095679; this version posted December 20, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

high selection pressure to optimise infectivity in either the hosts or vectors implying parasites would need to move between hosts but inefficiently. We find evidence for gene flow between lineages throughout this period. The expansion of the human population with the advent of farming likely led to strong evolutionary pressure for mosquito species (specifically Anopheles gambiae) to feed primarily on humans (32). Therefore, the existing human infective (P. falciparum) genotypes would be selected for human and appropriate vector success and the fittest would rapidly expand. Subsequent rapid accumulation of mutations that favoured growth in humans are likely to have occurred to increase human specific reproductive success that both produced a specific parasite genotype expansion (that would also appear as an emergence from a bottleneck) and also resulted in the much lower probability of a direct transfer of P. falciparum back to apes. Our estimate of the rapid expansion of P.falciparum within the last 5000 years is also consistent with the proposed timing of the population expansion of Anopheles(33) and with estimates of the age of haemoglobinopathies known to be protective against malaria(34). With experiments on gorillas and chimpanzees not possible it will be difficult directly to prove the precise combination of different alleles that allowed the emergence of P. falciparum. Nevertheless, the data and analyses presented here will be invaluable in future studies of host specificity in Plasmodium. Author Contributions : TDO, BO, FR, CN, MB, FP designed the study. CA, APO, LB, EW, BN, ND, CP, PD, VR, FP collected and assessed samples. CA performed the WGA and cell sorting on one sample. SO performed the WGA on the samples; MS organised the sequencing. TDO did assembly and annotation. UB did manual gene curation; AG, FP performed the evolutionary analyses on core genomes. TDO, CN, MB performed the analyses of gene families and dimorphisms. TC performed the dating analyses. TDO, AG, CN, MB, FP wrote the manuscript. All authors read and approved the paper.

Acknowledgments: This work was funded by ANR ORIGIN JCJC 2012, LMI ZOFAC, CNRS, CIRMF, IRD and the Wellcome Trust (grant WT 098051 to the Sanger Institute, 104792/Z/14/Z CN). TC holds a MRC DTP Studentship. We thank Gavin Rutledge for performing the sWGA and Julian Rayner and Francisco J. Ayala for helpful discussions.

Data Availability All sequences have been submitted to the European Nucleotide Archive. The accession numbers of the raw reads, and assembly data can be found in Table S6. The genomes are being submitted to EBI, project ID PRJEB13584. As the assemblies are currently private, but are available on request.

14 bioRxiv preprint doi: https://doi.org/10.1101/095679; this version posted December 20, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Supplemental Figures & Tables

A ML tree of the Laverania strains Padl2 100 Padl1 g Padl3 100 Pgab5 Pgab1 Pgab4 f 100 Pgab3 Pgab2 Pblac1 Pbilc3 100 b Pbilc1 c d Pbilc4 PrCDC 100 Pr2 Pr1 e 100 Pr3 Pprf3 Pprf4 100 86 Pprf2 h Pprf1 0.01 100 PfIT 100 PfDD2 Human parasites 100 Pf7G8 a Pf P. falciparum PfHB3 Pf3D7

Gorilla-infecting parasites Chimpanzee-infecting parasites 0.000 0.005 0.010 Padl P. adleri Pgab P. gaboni Pblac P. blacklocki Pbilc P. billcollinsi Pprf P. praefalciparum Pr P. reichenowi comparisons comparisons Within host species

Between host species

B Identification of the two P. praefalciparum lineages 1) Extended majority rule tree 2) ML tree consistent with the consensue tree P adl b P ga

P 1 P 100 adl blac

adl

3

P 1 P P bil adl c 4 3 1 2 2 gab gab gab P P P 5 gab P gab

P

41 P bla P bil c 98 18 P c 1 bil 4 c 1 84

100 P r P bilc 3 r um I 100 96 P . pr aefalcipa P 99 pr / 2 / 4 f P prf 1 3 I 100 r um I 98 P f r aefalcipa P . p

3) ML tree showing recombination between the divergent P . pr aefalcipa P r 1 76 r um II P. praefalciparum lineages P adl P r 3 P prf 3 r 2 C P D P gab 75 rC 39 P

P 23 70 blac

Pf 1 H B 3 P bil c 4 f 100

pr

P 1 f 2 P f 85 f Pf 3 pr D P pr 7 7 G P P Pf f 8 DD

I P T . pr aefalcipa 2 r um I

f 100 P 78 P r

f

pr P . pr aefalcipar um I & II P

15 bioRxiv preprint doi: https://doi.org/10.1101/095679; this version posted December 20, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig. S1. (A) Maximum Likelihood tree and nucleotide diversity of Laverania isolates. Tree was obtained using the sequences of 424 genes (“Lav25st” set of orthologues). The results of the test of comparison of the polymorphism between and within host species are given on the right: polymorphism in P. falciparum “a” < polymorphism in the chimpanzee-infecting species “b” < polymorphism in the gorilla-infecting species “c”; within the chimpanzee-infecting species, polymorphism is lower in P. billcollinsi “d” and higher in P. gaboni “f”; within the gorilla-infecting species, polymorphism is lower in P. adleri “g” and higher in P. praefalciparum “h” (see Supplementary Text). (B) Identification of two P. praefalciparum lineages. (1) Extended majority rule tree, built using RAxMLv8.1.20(61) and the “Lav25st” set of 424 orthologues. The consensus tree, reveals two divergent praefalciparum lineages that seems however to recombine. Two examples of the observed single-gene tree topologies are shown. The first (2) is consistent with the consensus tree but the second (3) indicates that recombination has occurred between the two P. praefalciparum lineages. For readability we excluded the G0 in the names of the genotypes.

16

A! msp1 ! var1csa! msp3 !

K1! Pr!

Pprf! Pf! Pr! Pprf! Pf! Pr! MAD20!

0.07 0.07 0.07

P. adleri P. gaboni P. blacklocki P. billcollinsi P. reichenowi P. praefalciparum P.falciparum

B - eba-175 ! Pblc (2)! Pprf! eba175????!

Pf (10)!

Pprf (2)! Pr (2)!

Fig. S2. Dimorphisms in the Laverania. (A) Examples of ancient dimorphisms based on maximum likelihood phylogenetic trees. Dimorphism in msp1 arose in the P. falciparum–P. praefalciparum ancestor, after the divergence of P. reichenowi and dimorphism in var1csa evolved in the P. reichenowi–P. praefalciparum–P. falciparum ancestor after the divergence of P. billcollinsi. There is also evidence of a bi-allelic distribution of msp3 in P. falciparum, P. praefalciparum and P. reichenowi. (B) Dimorphism in eba-175 is more recent. The alignment shown two mutually exclusive indels (arrow) in the P. falciparum sequences, not present in other Laverania species. The colours represent different nucleotides. For the P. falciparum sequences, we used full sequences from the Pf3K dataset (Supplementary Materials).

17 A B Species tree Topology A Topology B P. adleri P. adleri P. adleri D(P.adlerprae/P.adler) D(P.adlerfalci/P.adler) D(r) D(r) P P P. praefalciparum 0.4 1. CyRPA P P P. falciparum 0.3 2. PF3D7_0423900 3. PF3D7_0424000 P P P 0.3 4. RH5 P P P 0.2 P. praefalciparum P. praefalciparum P 0.2

Density 1 P. falciparum P. falciparum P 1 2 0.1 3 2 3 4 1 2 3 0.1 4 1 2 3 Intergenic regions: A A B B1 B1,2 B NA 4 4 5’ 3’ CyRPA PF3D7_0424000 EBA-165 0.0 0.0 Topology: PF3D7_0423600 ETRAMP4 PF3D7_0423900 RH5 RH4 0 10 20 0 10 20 0 10 20 0 10 20 3’ 5’ CDS: A A B B B1 B A A

1 No P. blacklocki sequence data available 2 Short alignment (77 nt) 3 No P. blacklocki , P. billcollinsi & P. adleri sequence data available

C

P ------MNLSKCFSLQNAIKKTKYQENNLTLLPIKSTEEEKYGIKRKDIE--NANDIKNDIDNDKKNVKINNVKDHSTYIKSYLNTNVNDGLKYLF*MPSHDSYIKKYSLFNKKNDSILLNEKNDVNNN----NKVDYKNVNFLQYDFKGLSNYNIADSIDIVQEKEGHLDFIIIPHYSYIEYCKHLSLISVLHRLSTYEKYKDVIAFFNDINKRYDKVKDKCNEIKNDLITTIKKLEHPYDVNNKNEDSYIYDTFEGIDA------MNLSKCLSFENAKKKTKNQENNLTLLPIKSSEEEKSNIKHKDIGNVNKDDINNDIDNDKKDVKINNVKDHSTYIKSYLNKNVNDGLKYLFMPSHNAYIKKYSLFNKMNDDMLLNEKKDVNNNKEDDKNINHKNVNFLQNDFKGLPKNNIADSIDIVQEKEGHLDFIIIPYYSHVEYCKHLSIISVFHRLSTYGEYKDVIAFVKNINEKYDKVKDKCNEIKNDLITTIKKLENPHDVNNKNEDSNIYNTFEDIDDI- P ------MNLSKCLSFENAKKKTKNQENNLTLLPIKSSEEEKSNIKHKDIGNVNKDDINNDIDNDKKDVKINNVKDHSTYIKSYLNKNVNDGLKYLFMPSHNAYIKKYSLFNKMNDDMLLNEKKDVNNNKEDDKNINHKNVNFLQNDFKGLPKNNIADSIDIVQEKEGHLDFIIIPYYSHVEYCKHLSIISVFHRLSTYGEYKDVIAFVKNINEKYDKVKDKCNEIKNDLITTIKKLENPHDVNNKNEDSNIYNTFEDIDDI------MNLSKCLSFENAKKKTKNQENNLTLLPIKSSEEEKSNIKHKDIGNVNKDDINNDIDNDKKDVKINNVKDHSTYIKSYLNKNVNDGLKYLFMPSHNAYIKKYSLFNKMNDDMLLNEKKDVNNNKEDDKNINHKNVNFLQNDFKGLPKNNIADSIDIVQEREGHLDFIIIPYYSHVEYCKHLSIISVFHRLSTYGEYKDVIAFVKNINEKYDKVKDKCNEIKNDLITTIKKLENPHDVNNKNEDSNIYNTFEDIDDI- MIRIKKKIILSILYIHLFILNSLSFENAKKKTKNQENNLTLLPIKSSEEEKSNIKHKDIGNVNKDDINNDIDNDKKDVKISNVKDHSTYIKSYLNKNVNDGLKYLFMPSHNAYIKKYSLFNKMNNDMLLNEKKDVNNNKEDDKNINHKNVNFLQNDFKGLPKNNIADSIDIVQDKEGHLDFIIIPYYSHVEYCKHLSIISVFHRLSTYGEYKDVIAFVKNINEKYDKVKDKCNEIKNDLITTIKKLENPHDVNNKNEDSNIYNTFEDIDDI- MIRIKKKLILTILYIHLFILNRLSFENAIKKTKNQENNLTLLPIKSTEEEKSDIK------NKKDIKKEIDNDKENIKRNNATDHSTYIKSYLNINVNDGLKYLFMPSHNSFIKKYSVLNQINDGMLENKKNDVKNN-EDHNNVDYKNVNFLQYDFKELSNYNIADSIDIIQEKEGHLDFIIIPYYIYIDYHKHISYNSIYHKLSTFWKYKDVDAFIKNINEKYDQVKSKCNDINNDLIATIKKLEHPYDINNKNEDSYRYDISEEIDD-- P MIRIKKKLILTILYIHLFILNRLSFENAIKKTKNQENNLTLLPIKSTEEEKSDIK------NKKDIKKEIDNDKENIKRNNATDHSTYIKSYLNINVNDGLKYLFMPSHNSFIKKYSVLNQINDGMLENKKNDVKNN-EDHNNVDYKNVNFLQYDFKELSNYNIADSIDIIQEKEGHLDFIIIPYYIYIDYHKHISYNSIYHKLSTFWKYKDVDAFIKNINEKYDQVKSKCNDINNDLIATIKKLEHPYDINNKNEDSYRYDISEEIDD-- MIRIKKKLILTILYIHLFILNRLSFENAIKKTKNQENNLTLLPIKSTEEEKSDIK------NKKDIKKEIDNDKENIKRNNATDHSTYIKSYLNINVNDGLKYLFMPSHNSFIKKYSVLNQINDGMLENKKNDVKNN-EDHNNVDYKNVNFLQYDFKELSNYNIADSIDIIQEKEGHLDFIIIPYYIYIDYHKHISYNSIYHKLSTFWKYKDVDAFIKNINEKYDQVKSKCNDINNDLIATIKKLEHPYDINNKNEDSYRYDISEEIDD-- MIRIKKKLILTILYIHLFILNRLSFENAIKKTKNQENNLTLLPIKSTEEEKNDIK------NTKDIKKEIDNDKENIKRNNATDHSTYIKSYLNTNVNDGLKYLFMPSHNSFIKKYSVLNQINDGMLENKKNDVKNN-EDHKNVDYKNVNFLQYDFKELSNYNIADSIDIIQEKEGHLDFIIIPYYIYIDYHKHISYNSIYHKLSTFWKYKDVDAFIKNINEKYDQVKSKCNDINNDLIATIKKLEHPYDINNKNEDSYRYDISEEIDD-- P MRRIKKKIIFTILYIHLFILNSLSFENAVKKTKNQENNLALLPIKSTEEEKYGIKQKDIENSNENDIKKDRDNDKKNVKINNVTDHSTYIKSYLNRNVNDGLKFLFIPSHNAYVKKYSLFNKMNDDILLNEKNDVNNNKEDNKNINNKNVNFLQYDFKGLSKYSTADSIDIVQEKEGHLDFIIIPFYSYVDYCKHISINSVFHRLSTYIKYKDVLAFFKNINERYEKVKDKCNEIKNDLITTINKIEHPYDINNKNEDSYIYETIEDIDD-- P. adleri MIRIKKKLILTIIYIHLFILNRLSFENAIKKTKNQENNLTLLPIKSTEEEKNDIK------NGKDIKKEIDNDKENIKTNNIKDHSTYIKSYLNTNVNDGLKYLFIPSHNSFIKKYSVFNQINDGMLLNEKNDVKNN-EDYKNVDYKNVNFLQYVFKELSNYNIADSIDIFQEKEGHLDFVIIPHYTFLDYYKHLSYNSIYHKYSTHGMYIGVDAFIKKINERYDQVKSKCNDIKNDLIGTIKKLEHPYDINNKNDDSYRYDISEEIDD-- MIRIKKKLILTIIYIHLFILNRLSFENAIKKTKNQENNLTLLPIKSTEEEKNDIK------NGKDIKKEIDNDKENIKTNNIKDHSTYIKSYLNTNVNDGLKYLFIPSHNSFIKKYSVFNQINDGMLLNEKNDVKNN-EDYKNVDYKNVNFLQYVFKELSNYNIADSIDIFQEKEGHLDFVIIPHYTFLDYYKHLSYNSIYHKYSTHGMYIGVDAFIKKINERYDQVKSKCNDIKNDLIGTIKKLEHPYDINNKNDDSYRYDISEEIDD-- P. praefalciparum MIRIKKKLILTIIYIHLFILNRLSFENAIKKTKNQENNLTLLPIKSTEEEKDDIK------NGKDIKKEIDNDKENIKTNNAKDHSTYIKSYLNTNVNDGLKYLFIPSHNSFIKKYSVFNQINDGMLLNEKNDVKNN-EDYKNVDYKNVNFLQYDFKELSNYNIANSIDILQEKEGHLDFVIIPHYTFLDYYKHLSYNSIYHKYSTHGKYIAVDAFIKKINERYDQVKSKCNDIKNDLIATIKKLEHPYDINNKNDDSYRYDISEEIDDKS MIRIKKKLILTIIYIHLFILNRLSFENAIKKTKNQENNLTLLPIKSTEEEKDDIK------NGKDIKKEIDNDKENIKTNNAKDHSTYIKSYLNTNVNDGLKYLFIPSHNSFIKKYSVFNQINDGMLLNEKNDVKNN-EDYKNVDYKNVNFLQYDFKELSNYNIANSIDILQEKEGHLDFVIIPHYTFLDYYKHLSYNSIYHKYSTHGKYIAVDAFIKKINERYDQVKSKCNDIKNDLIATIKKLEHPYDINNKNDDSYRYDISEEIDDKS MIRIKKKLILTIIYIHLFILNRLSFENAIKKTKNQENNLTLLPIKSTEEEKDDIK------NGKDIKKEIDNDKENIKTNNAKDHSTYIKSYLNTNVNDGLKYLFIPSHNSFIKKYSVFNQINDGMLLNEKNDVKNN-EDYKNVDYKNVNFLQYHFKELSNYNIANSIDILQEKEGHLDFVIIPHYTFLDYYKHLSYNSIYHKYSTYGKYIAVDAFIKKINETYDKVKSKCNDIKNDLIATIKKLEHPYDINNKNDDSYRYDISEEIDD-- MIRIKKKLILTIIYIHLFILNRLSFENAIKKTKNQENNLTLLPIKSTEEEKDDIK------NGKDIKKEIDNDKENIKTNNAKDHSTYIKSYLNTNVNDGLKYLFIPSHNSFIKKYSVFNQINDGMLLNEKNDVKNN-EDYKNVDYKNVNFLQYHFKELSNYNIANSIDILQEKEGHLDFVIIPHYTFLDYYKHLSYNSIYHKSSTYGKYIAVDAFIKKINETYDKVKSKCNDIKNDLIATIKKLEHPYDINNKNDDSYRYDISEEIDD-- P. falciparum MIRIKKKLILTIIYIHLFILNRLSFENAIKKTKNQENNLTLLPIKSTEEEKDDIK------NGKDIKKEIDNDKENIKTNNAKDHSTYIKSYLNTNVNDGLKYLFIPSHNSFIKKYSVFNQINDGMLLNEKNDVKNN-EDYKNVDYKNVNFLQYHFKELSNYNIANSIDILQEKEGHLDFVIIPHYTFLDYYKHLSYNSIYHKSSTYGKYIAVDAFIKKINETYDKVKSKCNDIKNDLIATIKKLEHPYDINNKNDDSYRYDISEEIDD-- MIRIKKKLILTIIYIHLFILNRLSFENAIKKTKNQENNLTLLPIKSTEEEKDDIK------NGKDIKKEIDNDKENIKTNNAKDHSTYIKSYLNTNVNDGLKYLFIPSHNSFIKKYSVFNQINDGMLLNEKNDVKNN-EDYKNVDYKNVNFLQYHFKELSNYNIANSIDILQEKEGHLDFVIIPHYTFLDYYKHLSYNSIYHKSSTYGKCIAVDAFIKKINETYDKVKSKCNDIKNDLIATIKKLEHPYDINNKNDDSYRYDISEEIDD-- 1 MIRIKKKLILTIIYIHLFILNRLSFENAIKKTKNQENNLTLLPIKSTEEEKDDIK------NGKDIKKEIDNDKENIKTNNAKDHSTYIKSYLNTNVNDGLKYLFIPSHNSFIKKYSVFNQINDGMLLNEKNDVKNN-EDYKNVDYKNVNFLQYHFKELSNYNIANSIDILQEKEGHLDFVIIPHYTFLDYYKHLSYNSIYHKSSTYGKCIAVDAFIKKINETYDKVKSKCNDIKNDLIATIKKLEHPYDINNKNDDSYRYDISEEIDD--262

P -----KSKDIEDQTEDVDDTMEDIESDNTSPNKKKYHFMNKTYKKMMEEYNKKKNKLVQCIKNNENDFNNICMDMKKYGTNLFEQISCDNNNFCNTNGIK*NNYIYYIREGILSTKSKNLNKDILDMTNILEQSELLLINLNKKMSSHIYIDTIKFIHEEMKYIFKKIQHHTNIIIDKIKIIEDKIKLNTNRTFPKDILLQTILDLPNEYALFITSDLLRKMLYNTFYSKEKHLHSLFHHLIYVLQIKFNDVPLa NKEYFQLYKNNKSLIQ------YEDDVQDQLEYVYDTIEDADSDNTHPNKKKYHLINRTYKKMMDEYNLKKNKLIQCIKNNEPDFNNIYMDIKNYSTNRFENISCDNINFCNTNGIKNHYIYYIEKEMLAIKSKDLNKDIKDMTSILQQSELLLINLNKKMGSYIYIDTIKFIHKEMKYIFKRIEHHTNIIIDKIKIIDDKYKISINKTFPKNILLPKILDLSNEYALFITSSTLRQMLYNTFYTKEKHLHNLFHHLIYVLQIKFNDVPVHMEYFPLYKKKKILTQ- P -----YEDDVQDQLEYVYDTIEDADSDNTHPNKKKYHLINRTYKKMMDEYNLKKNKLIQCIKNNEPDFNNIYMDIKNYSTNRFENISCDNINFCNTNGIKNHYIYYIEKEMLAIKSKDLNKDIKDMTSILQQSELLLINLNKKMGSYIYIDTIKFIHKEMKYIFKRIEHHTNIIIDKIKIIDDKYKISINKTFPKNILLPKILDLSNEYALFITSSTLRQMLYNTFYTKEKHLHNLFHHLIYVLQIKFNDVPVHMEYFPLYKKKKILTQ------YEDDMQDQLEYVYDTIEDADSDNTHPNKKKYHLINRTYKKMMDEYNLKKNKLIQCIKNNEPDFNNIYMDIKNYSTNRFENISCDNINFCNTNGIKNHYIYYIEKEMLAIKSKDLNKDIKDMTNILQQSELLLINLNKKMGSYIYIDTIKFIHKEMKYIFKRIEHHTNIIIDKIKIIDDKYKISINKTFPKNILLPKILDLSNEYALFITSSTLRQMLYNTFYTKEKHLHNLFHHLIYVLQIKFNDVPVHMEYFPLYKKKKILTQ------YEDDMQDQLEYVYDTIEDADSDNTHPNKKKYHLINRTYKKMMDEYNLKKNKLIQCIKNNEPDFNNIYMDIKNYSTNRFENISCDNINFCNTNGIKNHYIYYIEKEMLAIKSKDLNKDIKDMTSILQQSELLLINLNKKMGSYIYIDTIKFIHKEMKYIFKRIEHHTNIIIDKIKIIDDKYKISINKTFPKNILLPKILDLSNEYALFITSSTLRQMLYNTFYTKEKHLHNLFHHLIYVLQIKFNDVPVHMEYFPLYKKKKILTQ------RSEETDEEIEEVEDNIQDTDSSNTPSNNKKNDLMNRTFKKMMDEYNTKKNKLIQCIKNHENDFNKICMDMKNFSTNLFQQISCDNINFCDTNGIKYHYDEYINKRILSIKSINLNKDISDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHILKRIQYHTNIINDKTKIIQDKIKLNTWRTFQKDELLKRILDLSNEYSLFITSDDLRQMLYKTFYSKEKYLHNIFHHLIYVLQMKFNDVPIKMEYFQTYKNKKPLTQ- P -----RSEETDEEIEEVEDNIQDTDSSNTPSNNKKNDLMNRTFKKMMDEYNTKKNKLIQCIKNHENDFNKICMDMKNFSTNLFQQISCDNINFCDTNGIKYHYDEYINKRILSIKSINLNKDISDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHILKRIQYHTNIINDKTKIIQDKIKLNTWRTFQKDELLKRILDLSNEYSLFITSDDLRQMLYKTFYSKEKYLHNIFHHLIYVLQMKFNDVPIKMEYFQTYKNKKPLTQ------RSEETDEEIEEVEDNIQDTDSSNTPSNNKKNDLMNRTFKKMMDEYNTKKNKLIQCIKNHENDFNKICMDMKNFSTNLFQQISCDNINFCDTNGIKYHYDEYINKRILSIKSINLNKDISDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHILKRIQYHTNIINDKTKIIQDKIKLNTWRTFQKDELLKRILDLSNEYSLFITSDDLRQMLYKTFYSKEKYLHNIFHHLIYVLQMKFNDVPIKMEYFQTYKNKKPLTQ------RSEETDEEIEEVEDNIQDTDSSNTPSNNKKNDLMNRTFKKMMDEYNTKKNKLIQCIKNHENDFNKICMDMKNFSTNLFQQISCDNINFCDTNGIKYHYDEYINKRILSIKSINLNKDISDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHILKRIEYHTNIINDKTKIIQDKIKLNTWRTFQKDELLKRILDLSNEYSLFITSDDLRQMLYKTFYSKEKYLHNIFHHLIYVLQMKFNDVPIKMEYFQTYKKKKPLTQ- P -----RSEDMDDEIEDVDDTIEDTDSDNTPPNKKKYHFMNRTYKKMMEEYNTKKNKLIQCIKNNENDFNNIYTDVKKYSTNLFEQISCDNIKFCNSNGIRMHYEDYVHKKIISIKSKNLNKDILEMTNIIQQSELLLINLNKKMCSYIYINTIKFIHNEMKYILTRIQHHTNIIINKTKIIEDKIKLYLNRTFPKDILLQNISYISNEYALFITSDVVRQMLYNTFYSKEKHLHKIFNHLIYVLQIKFDDVPIKMEYFQVNKKNKPLSQ- P. adleri -----KSEETDDETEEVEDSIQDTDSNHTPSNKKKNDLMNRTFKKMMNEYNTKKNKLIKCIKNHENDFNKICMDMKNYGTKLFQQISCYNNNFCNTNGIRYHYDEYILKIILSVKSKNLNKDLSDMTNILQQSELLLTNLNKKMSSHIYIDTIKFIHKEMKHILKRIEYHTKIINDKNKIIQDKIKLNIWRTFQKDELLKRILDMSNEYSLFITSDHLRQMLYNTFYSKEKHLNNIFHHLIYVLQMKFNDVPIKMEYFQTYKKNKPLTQ------KSEETDDETEEVEDSIQDTDSNHTPSNKKKNDLMNRTFKKMMNEYNTKKNKLIKCIKNHENDFNKICMDMKNYGTKLFQQISCYNNNFCNTNGIRYHYDEYILKIILSVKSKNLNKDLSDMTNILQQSELLLTNLNKKMSSHIYIDTIKFIHKEMKHILKRIEYHTKIINDKNKIIQDKIKLNIWRTFQKDELLKRILDMSNEYSLFITSDHLRQMLYNTFYSKEKHLNNIFHHLIYVLQMKFNDVPIKMEYFQTYKKNKPLTQ- P. praefalciparum EEIDDKSEETDDETEEVEDSIQDTDSNHTPSKKKKNDLMNRTFKKMMDEYNTKKKKLIKCIKNHENDFNKICMDMKNYGTNLFEQLSCYNNNFCNTNGIRYHYDEYIHKLILSIKSKNLNKDLSDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHIFNRIEYHTKIINDKTKIIQDKIKLNIWRTFQKDELLKRILDMSNEYSLFITSDHLRQMLYNTFYSKEKHLNNIFHHLIYVLQMKFNDVPIKMEYFQTYKKNKPLTQ- EEIDDKSEETDDETEEVEDSIQDTDSNHTPSKKKKNDLMNRTFKKMMDEYNTKKKKLIKCIKNHENDFNKICMDMKNYGTNLFEQLSCYNNNFCNTNGIRYHYDEYIHKLILSIKSKNLNKDLSDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHIFNRIEYHTKIINDKTKIIQDKIKLNIWRTFQKDELLKRILDMSNEYSLFITSDHLRQMLYNTFYSKEKHLNNIFHHLIYVLQMKFNDVPIKMEYFQTYKKNKPL??? -----KSEETDDETEEVEDSIQDTDSNHTPSNKKKNDLMNRTFKKMMDEYNTKKKKLIKCIKNHENDFNKICMDMKNYGTNLFEQLSCYNNNFCNTNGIRFHYDEYIHKLILSVKSKNLNKDLSDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHIFNRIEYHTKIINDKTKIIQDKIKLNIWRTFQKDELLKRILDMSNEYSLFITSDHLRQMLYNTFYSKEKHLNNIFHHLIYVLQMKFNDVPIKMEYFQTYKKNKPLTQ------KSEETDDETEEVEDSIQDTDSNHTPSNKKKNDLMNRTFKKMMDEYNTKKKKLIKCIKNHENDFNKICMDMKNYGTNLFEQLSCYNNNFCNTNGIRYHYDEYIHKLILSVKSKNLNKDLSDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHIFNRIEYHTKIINDKTKIIQDKIKLNIWRTFQKDELLKRILDMSNEYSLFITSDHLRQMLYNTFYSKEKHLNNIFHHLIYVLQMKFNDVPIKMEYFQTYKKNKPLTQ- P. falciparum -----KSEETDDETEEVEDSIQDTDSNHTPSNKKKNDLMNRTFKKMMDEYNTKKKKLIKCIKNHENDFNKICMDMKNYGTNLFEQLSCYNNNFCNTNGIRYHYDEYIHKLILSVKSKNLNKDLSDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHIFNRIEYHTKIINDKTKIIQDKIKLNIWRTFQKDELLKRILDMSNEYSLFITSDHLRQMLYNTFYSKEKHLNNIFHHLIYVLQMKFNDVPIKMEYFQTYKKNKPLTQ------KSEETDDETEEVEDSIQDTDSNHTPSNKKKNDLMNRTFKKMMDEYNTKKKKLIKCIKNHENDFNKICMDMKNYGTNLFEQLSCYNNNFCNTNGIRYHYDEYIHKLILSVKSKNLNKDLSDMTNILQQSELLLTNLNKKMGSYIYIDTMKFIHKEMKHIFNRIEYHTKIINDKTKIIQDKIKLNIWRTFQKDELLKRILDMSNEYSLFITSDHLRQMLYNTFYSKEKHLNNIFHHLIYVLQMKFNDVPIKMEYFQTYKKNKPLTQ- 263 -----KSEETDDETEEVEDSIQDTDSNHTPSNKKKNDLMNRTFKKMMDEYNTKKKKLIKCIKNHENDFNKICMDMKNYGTNLFEQLSCYNNNFCNTNGIRYHYDEYIHKLILSVKSKNLNKDLSDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHIFNRIEYHTKIINDKTKIIQDKIKLNIWRTFQKDELLKRILDMSNEYSLFITSDHLRQMLYNTFYSKEKHLNNIFHHLIYVLQMKFNDVPIKMEYFQTYKKNKPLTQ- 526

Fig. S3. Interspecific gene transfer and convergent evolution in the 3’ end of the chromosome 4. (A) Support for interspecific gene transfer between the gorilla-infecting species P. adleri and the common ancestor of P. praefalciparum and P. falciparum. The topologies observed in the coding and intergenic regions of the end of chromosome 4 are given. (B) The analysis of relative genetic distances in the rh5 region and in the other regions of the genome also support the interspecies transfer. (C) Convergent evolution in the rh5 gene. Amino acid alignment of the rh5 region that carries the significant fixed difference between parasites infecting the chimpanzees and those infecting gorillas (red stars). The positions in the alignment of the 19 strains with rh5 sequence available is given at the top, while the positions at the bottom of the alignments correspond to the position in the P. falciparum 3D7 sequence. Green circles indicate positions that are known to be involved in the interaction with the human receptor Basigin.

18

Fig.S4. Tree topology tests. The species tree (topology A) and the topologies that differ significantly from the species tree topology – when sufficient phylogenic information was available – are given.

Fig. S5. Relationships between number of divergent and convergent substitutions for each branch pair of the Laverania species tree. Relationships are provided for each chromosome (1- 14). See Material and Methods for details.

20 Color Key Color Key

Stevor_SvoreMax heatmap

0 0.5 1 1.5A! 2 0 0.2 0.6 1 B! Value Value Similarity Matrix! Species!

PBILG01_0001500 PBILG01_0024400PBILG01_1476600 PPRG01_0003100PBILG01_0009300 PBILG01_0902700PRG01_0009600 PPRG01_0006100PBILG01_0900400 PPRG01_0049700PRG01_0401600 PBILG01_0001300PBILG01_1477500 PRG01_0102500PRG01_0043400 PRG01_1201000PBILG01_0202500 PF3D7_0832400PRG01_0301400 PPRG01_0834600PPRG01_0024500 PF3D7_1040200PPRG01_00114700 PBILG01_1401200PRG01_0303900 PRG01_0303400PRG01_1479700 PF3D7_1400700PRG01_0300600 PRG01_0026400PBILG01_0023300 PRG01_0016400PBILG01_0200900 PRG01_0903300PRG01_0328800 PRG01_0021600PRG01_0430100 PRG01_0101800PBILG01_1477300 PRG01_0903800PBILG01_1479800 PPRG01_1375200PRG01_1203800 PPRG01_0012400PRG01_0302100 PRG01_0115100PF3D7_1149900 PPRG01_0033800PF3D7_0617600 PF3D7_0901600PRG01_0905200 PRG01_0029400PBILG01_0800700 PBILG01_0045700PRG01_0906400 PRG01_0043600PRG01_0905800 PPRG01_0425200PPRG01_1235500 PBILG01_0300800PPRG01_0900500 PF3D7_0631900PBILG01_0202300 PPRG01_1301000PPRG01_0089500 PF3D7_1300900PPRG01_0536200 PPRG01_1000100PPRG01_0052700 PF3D7_0200400PPRG01_1000600 PPRG01_0034100PBILG01_0034200 PRG01_1400600PRG01_0801500 PPRG01_0023300PPRG01_0009000 PRG01_0901200PPRG01_0424200 PBILG01_0100500PRG01_0015200 PBILG01_0401200PRG01_0403300 PPRG01_0021000PPRG01_1302600 PBILG01_1479600PPRG01_0938500 PRG01_0430400PBILG01_0632200 PRG01_0600800PBILG01_0901200 PBILG01_0400300PRG01_0631000 PRG01_0029000PRG01_0027200 PPRG01_0039000PBILG01_0836200 PBILG01_1000900PBILG01_0036800 PBILG01_0701800PPRG01_0090100 PRG01_0030400PRG01_0000700 PRG01_0305700PBILG01_1300900 PBILG01_1401600PBILG01_0100200 PF3D7_0832900PPRG01_1101100 PPRG01_0424900PPRG01_0001000 PF3D7_0401500PRG01_1480200 PPRG01_0033500PPRG01_1374900 PPRG01_0014200PPRG01_0009800 PPRG01_0040600PF3D7_1479900 PPRG01_0201400PF3D7_0201300 PPRG01_0534900PPRG01_0835400 PRG01_0800500PPRG01_0020100 PPRG01_0113100PRG01_1481400 PRG01_0008900PF3D7_0115400 PRG01_0100800PRG01_0904100 PF3D7_0900900PRG01_0908200 PPRG01_0424600PPRG01_0501000 PF3D7_0222800PPRG01_0005300 PBILG01_0600800PBILG01_0326300 PBILG01_1400500PRG01_0904800 PF3D7_1254100PPRG01_1256100 PF3D7_0101800PPRG01_1255600 PPRG01_0836100PF3D7_0324600 PF3D7_0400800PPRG01_0800800 PF3D7_1372800PPRG01_0021900 PF3D7_1254600PPRG01_0086100 0.2 PF3D7_0200900PF3D7_1254300 PF3D7_0732000PF3D7_1479500 PF3D7_0300400PPRG01_0011000 PF3D7_0700400PPRG01_0038100 PRG01_0902500PRG01_0300800 PPRG01_0003200PPRG01_0006400 PPRG01_0050300PPRG01_0300100 PRG01_0020300PPRG01_0063200 PRG01_0200600PPRG01_0800500 PPRG01_0100200PBILG01_0901800 PPRG01_0400900PF3D7_0102100 PRG01_0010600PRG01_0328100 PBILG01_0631900PBILG01_0601100 PBILG01_1301600PRG01_1302900 PF3D7_0114600PBILG01_0323800 PPRG01_0025200PPRG01_0059100 PPRG01_1480200PPRG01_0025400 PGOAG01_0005900PGOAG01_0041800 PGOAG01_0711000PGOAG01_0006200 PPRG01_0222100PPRG01_0937200 PF3D7_1000800PPRG01_1002200 PF3D7_0402600PPRG01_0070100 PPRG01_0402100PPRG01_0042900 PPRG01_0425900PPRG01_1200200 PGOAG01_0700300PGOAG01_0037700 PPRG01_1374300PGOAG01_0003700 PF3D7_0425500PF3D7_1372500 PPRG01_0834000PF3D7_0832000 PF3D7_0500600PPRG01_0502300 PPRG01_0221500PF3D7_0221400 PBILG01_0042400PBILG01_0037000 PGOAG01_0112600PGOAG01_0008900 PGOAG01_0036500PPRG01_0834900 ! ! ! ! !

stevor! PR ! ! Padl PPR PBIL Pf Pprf Pr Pbilc Pblac Pgab Padl PGAB Pblack PF3D7 PF3D7_0221400 PF3D7_0500600 PF3D7_0832000 PF3D7_0425500 PF3D7_1372500 PF3D7_0402600 PF3D7_1000800 PF3D7_0114600 PF3D7_0102100 PF3D7_0700400 PF3D7_0300400 PF3D7_0732000 PF3D7_1479500 PF3D7_0200900 PF3D7_1254300 PF3D7_1254600 PF3D7_1372800 PF3D7_0400800 PF3D7_0324600 PF3D7_0101800 PF3D7_1254100 PF3D7_0222800 PF3D7_0900900 PF3D7_0115400 PF3D7_0201300 PF3D7_1479900 PF3D7_0401500 PF3D7_0832900 PF3D7_0200400 PF3D7_1300900 PF3D7_0631900 PF3D7_0901600 PF3D7_0617600 PF3D7_1149900 PF3D7_1400700 PF3D7_1040200 PF3D7_0832400 PRG01_1302900 PRG01_0010600 PRG01_0328100 PRG01_0200600 PRG01_0020300 PRG01_0902500 PRG01_0300800 PRG01_0904800 PRG01_0908200 PRG01_0100800 PRG01_0904100 PRG01_0008900 PRG01_1481400 PRG01_0800500 PRG01_1480200 PRG01_0305700 PRG01_0030400 PRG01_0000700 PRG01_0029000 PRG01_0027200 PRG01_0631000 PRG01_0600800 PRG01_0430400 PRG01_0403300 PRG01_0015200 PRG01_0901200 PRG01_1400600 PRG01_0801500 PRG01_0043600 PRG01_0905800 PRG01_0906400 PRG01_0029400 PRG01_0905200 PRG01_0115100 PRG01_0302100 PRG01_1203800 PRG01_0903800 PRG01_0101800 PRG01_0021600 PRG01_0430100 PRG01_0903300 PRG01_0328800 PRG01_0016400 PRG01_0026400 PRG01_0300600 PRG01_0303400 PRG01_1479700 PRG01_0303900 PRG01_0301400 PRG01_1201000 PRG01_0102500 PRG01_0043400 PRG01_0401600 PRG01_0009600 P. adleri P. gaboni P. blacklocki P. billcollinsi PPRG01_0834900 PPRG01_0221500 PPRG01_0502300 PPRG01_0834000 PPRG01_1374300 PPRG01_0425900 PPRG01_1200200 PPRG01_0402100 PPRG01_0042900 PPRG01_0070100 PPRG01_1002200 PPRG01_0222100 PPRG01_0937200 PPRG01_1480200 PPRG01_0025400 PPRG01_0025200 PPRG01_0059100 PPRG01_0400900 PPRG01_0100200 PPRG01_0800500 PPRG01_0063200 PPRG01_0050300 PPRG01_0300100 PPRG01_0003200 PPRG01_0006400 PPRG01_0038100 PPRG01_0011000 PPRG01_0086100 PPRG01_0021900 PPRG01_0800800 PPRG01_0836100 PPRG01_1255600 PPRG01_1256100 PPRG01_0005300 PPRG01_0424600 PPRG01_0501000 PPRG01_0113100 PPRG01_0020100 PPRG01_0534900 PPRG01_0835400 PPRG01_0201400 PPRG01_0040600 PPRG01_0014200 PPRG01_0009800 PPRG01_0033500 PPRG01_1374900 PPRG01_0424900 PPRG01_0001000 PPRG01_1101100 PPRG01_0090100 PPRG01_0039000 PPRG01_0938500 PPRG01_0021000 PPRG01_1302600 PPRG01_0424200 PPRG01_0023300 PPRG01_0009000 PPRG01_0034100 PPRG01_1000600 PPRG01_1000100 PPRG01_0052700 PPRG01_0536200 PPRG01_1301000 PPRG01_0089500 PPRG01_0900500 PPRG01_0425200 PPRG01_1235500 PPRG01_0033800 PPRG01_0012400 PPRG01_1375200 PPRG01_0834600 PPRG01_0024500 PPRG01_0049700 PPRG01_0006100 PPRG01_0003100 PBILG01_0042400 PBILG01_0037000 PBILG01_0323800 PBILG01_1301600 PBILG01_0631900 PBILG01_0601100 PBILG01_0901800 PBILG01_1400500 PBILG01_0600800 PBILG01_0326300 PBILG01_1401600 PBILG01_0100200 PBILG01_1300900 PBILG01_0701800 PBILG01_1000900 PBILG01_0036800 PBILG01_0836200 PBILG01_0400300 PBILG01_0901200 PBILG01_0632200 PBILG01_1479600 PBILG01_0401200 PBILG01_0100500 PBILG01_0034200 PBILG01_0202300 PBILG01_0300800 PBILG01_0045700 PBILG01_0800700 PBILG01_1479800 PBILG01_1477300 PBILG01_0200900 PBILG01_0023300 PBILG01_1401200 PBILG01_0202500 PBILG01_0001300 PBILG01_1477500 PBILG01_0900400 PBILG01_0902700 PBILG01_0009300 PBILG01_0024400 PBILG01_1476600 PBILG01_0001500 PPRG01_00114700 %PGOAG01_0036500 PGOAG01_0112600 PGOAG01_0008900 similarityPGOAG01_0003700 PGOAG01_0700300 PGOAG01_0037700 PGOAG01_0711000 PGOAG01_0006200 PGOAG01_0005900 PGOAG01_0041800 ! P. reichenowi! P. praefalciparum! P. falciparum!

Fig. S6. Similarity between stevor genes. (A). Similarity matrix of the stevor genes. The similarity matrix was generated through a BLASTp (expect-val ≤1e-6) and then clustered through the ward2 algorithm in R. Every row and column represents a gene. (B) The dendrogram on the left hand side shows the clustering and similarity between the genes. The binary blue barcode shows in which species each gene copy is present. Different groups can be seen. One cluster has is no present in chimpanzee parasites (black dotted box). Pf - P. falciparum, Pprf P. praefalciparum, Pr - P. reichenowi, Pbilc - P. billcollinsi, Pblac - P. blacklocki, Pgab - P. gaboni and Padl - P. adleri. b. Maximum likelihood tree of the same data.

21

A! P. falciparum 3D7!

Syntenic in all! P. praefalciparum!

Syntenic with four Acyl-CoA! P. reichenowi!

P. gaboni !

P. adleri!

B!

Fig. S7. Acyl-CoA Synthetase expansion on Chromosome 9 and Resa domains. (A) ACT view of five genomes, at the right hand side of chromosome 9. The grey areas indicate co- linearity. P. falciparum has lost this region with four Acyl-coA synthase genes, as this locus is conserved in the other species. (B) Similarity matrix of all genes containing the Resa Pfam domain. The similarity matrix was generated through a BLASTp (expect-val ≤1e-6) and then clustered through the ward2 algorithm in R. Every row and column represents a gene. The dendrogram on the left hand side shows the clustering and similarity between the genes. The

22

binary blue barcode indicates species and gene annotation. Most of the genes are well distributed between the species, the genes annotated as exported proteins (red dotted box), cluster by species and do not occur in Clade A. Some of the PhistA genes seem also to cluster by species (red dotted box). Pf - P. falciparum, Pprf P. praefalciparum, Pr - P. reichenowi, Pbilc - P. billcollinsi, Pblac - P. blacklocki, Pgab - P. gaboni and Padl - P. adleri

A Putative Glycophorin binding! C - clag! ! clag9 ! Chimp! Group B! clag2 ! Pf + Pprf!

clag8 !

all species! Group A!

P. blacklocki!

0.06

clag3.1! B DBLmsp! Group B! clag3.2 ! DBLmsp1!

GroupB! Group A! DBLmsp2!

*! 0.2

P. adleri P. gaboni P. blacklocki P. billcollinsi P. reichenowi P. praefalciparum 0.2 P.falciparum

Fig. S8. Phylogenetic analysis of gene families. Example of three families that show differences within the Laverania. (A) The putative glycophorin binding proteins form four distinct groups. One group contains sequence from all species. The remaining groups are clade, host or species sub-group specific. (B) Differences in the DBLmsp that are expanded in Clade A. The DBLmsp2 is a pseudo gene (*) in Pr. (c) Expansion of clag genes in Clade A.

23

A!

P. adleri! P. gaboni !

P. blacklocki!

P. billcollinsi!

P. reichenowi!

P. praefalciparum!

P. falciparum 3D7!

B!

Fig. S9. Composition, structure and evolution of var genes within Laverania. (A) Schematic of 2nd internal var genes cluster of chromosome 4 in the seven Laverania species. In the Clade A & P. blacklocki, the orientation of the var genes is different compared to that of the other species. Also the high GC ruf (RNA of Unknown function) elements, in circles, do not occur in those genomes. Last it can be also seen that the size of the var genes between the species is different. (B) shows a conserved var2csa between P. falciparum and P. praefalciparum where 6kb is shared between species with over 99% identity. Total length of the alignment is longer than 12kb.

24

A! Domain attribution! Similarity matrix! Species!

DBLε/DBLn!

DBLε! DBLγ! DBL! δ! ! DBLβ!

DBLα!

CIDRα! Color Key Color Key NTS! Color Key CIDRβ! DBLx2 heatmapCIDR ζ! exon2! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! α! β! δ γ ! ! α! β! δ ε γ ζ ! Pf Pprf Pr Pbilc Pblac Pgab Padl 0 0.2 0.6 1 Score! 0 100 ATS 300CDIR CDIR CDIR CDIR CIDRpam DBL DBL DBL DBL DBL DBLn DBLpam1 DBLpam2 DBLpam3 DBL NTS 0 0.2 0.6 1 Value B! Value Value

PGABG01_0616300.DBLn.7078 PGABG01_0032500.DBLn.5896 PGABG01_0008700.DBLn.4426 PGABG01_0401000.DBLn.5884 PGABG01_0014500.DBLn.4375 PGABG01_1239000.DBLn.4360 PGABG01_0021200.DBLn.4504 PADL01_0301200.DBLn.4315 PGABG01_0022200.DBLn.1561 PGABG01_0600100.DBLn.1552 PADL01_0531800.DBLn.1564 PGABG01_1239300.DBLn.1558 PADL01_0012900.DBLn.1564 PADL01_1100200.DBLn.1570 PGABG01_0323300.DBLe.1663 PGABG01_0630000.DBLn.2680 PADL01_0322800.DBLn.2674 PGABG01_0630000.DBLn.1558 PADL01_0402400.DBLn.1591 PADL01_0020100.DBLn.1609 PADL01_0011800.DBLn.1540 PGABG01_1239300.DBLn.4441 PADL01_0402400.DBLn.3301 PGABG01_0022200.DBLn.2815 PGABG01_0600100.DBLn.2815 PADL01_0531800.DBLn.2842 PF3D7_0600400.DBLpam1.289 PPRFG01_0400100.DBLpam1.7510 PRG01_0904400.DBLpam1.250 PADL01_0322800.DBLn.1519 PADL01_0700200.DBLn.1477 PADL01_0808000.DBLe.1546 PGABG01_0411100.DBLn.1489 PGABG01_0029400.DBLn.1510 PGABG01_0022800.DBLn.1249 PGABG01_0418600.DBLn.1510 PGABG01_0401000.DBLn.4558 PGABG01_0032500.DBLn.4576 PADL01_0025800.DBLn.5704 PGABG01_0023300.DBLn.3004 PADL01_0011500.DBLn.4201 PGABG01_0014500.DBLn.2992 PGABG01_1239000.DBLn.3061 PGABG01_0418500.DBLn.4582 PBLACG01_0027400.DBLn.1534 PBLACG01_0616600.DBLn.2560 PGABG01_0031300.DBLn.3718 PBILCG01_1481200.DBLn.7540 PBILCG01_0700100.DBLn.6064 PBLACG01_1240800.DBLn.1441 PBLACG01_0035500.DBLn.619 PADL01_1200900.DBLpam1.1678 PADL01_0301200.DBLpam1.307 PGABG01_0710500.DBLpam1.307 PADL01_0042600.DBLn.1792 PADL01_0042500.DBLpam1.1798 PADL01_1241100.DBLpam1.322 PGABG01_0301500.DBLpam1.226 PADL01_0711200.DBLpam1.1732 PBLACG01_0728700.DBLpam1.166 PBLACG01_0011400.DBLpam1.184 PBLACG01_1240800.DBLpam1.181 PBLACG01_0616600.DBLpam1.163 PBLACG01_0800100.DBLpam1.532 PBLACG01_0400300.DBLpam1.196 PBLACG01_1211600.DBLpam1.169 PBLACG01_1211300.DBLpam1.205 PGABG01_0401000.DBLpam1.3217 PGABG01_0021200.DBLpam1.1804 PADL01_0003000.DBLpam1.3535 PADL01_0001200.DBLpam1.4396 PADL01_0412300.DBLe.7117 PADL01_0025800.DBLn.1789 PPRFG01_0001600.DBLn.2464 PPRFG01_0423800.DBLpam1.157 PPRFG01_1254000.DBLpam1.157 PPRFG01_0731000.DBLpam1.157 PRG01_0003400.DBLpam1.154 PPRFG01_0076000.DBLpam1.157 PPRFG01_0801200.DBLpam1.154 PF3D7_1200600.DBLpam1.154 PADL01_0005800.DBLn.4069 PADL01_0420300.DBLn.4147 PADL01_0419900.DBLn.4186 PADL01_0420000.DBLn.4111 PADL01_0012400.DBLn.4111 PADL01_0711300.DBLn.4006 PADL01_0007800.DBLn.3886 PGABG01_0711000.DBLn.2719 PGABG01_0029700.DBLn.1705 PADL01_0412300.DBLn.4948 PADL01_0411900.DBLn.4954 PADL01_0412500.DBLn.4927 PADL01_1200800.DBLn.2110 PADL01_1200900.DBLn.3481 PGABG01_0400600.DBLn.4978 PADL01_0710700.DBLn.4747 PADL01_0711200.DBLn.4789 PPRFG01_0086700.DBLe.7462 PPRFG01_1100200.DBLe.5515 PPRFG01_0836400.DBLe.7039 PRG01_0026700.DBLe.7057 PPRFG01_0034400.DBLe.7186 PPRFG01_0001600.DBLe.7213 PPRFG01_0500200.DBLe.7027 PPRFG01_0005800.DBLe.6904 PBILCG01_0020700.DBLe.4618 PPRFG01_0018600.DBLe.4816 PPRFG01_0058700.DBLe.4810 PPRFG01_0062900.DBLe.7258 PF3D7_0632500.DBLe.7258 PRG01_0041000.DBLe.5980 PPRFG01_0027900.DBLe.6928 PADL01_0710900.DBLe.9373 PADL01_0005800.DBLe.9394 PADL01_1100200.DBLe.11278 PADL01_0012900.DBLe.11296 PPRFG01_1254000.DBLe.11992 PPRFG01_1151300.DBLe.4642 PGABG01_0418600.DBLe.8554 PGABG01_0027900.DBLe.4042 PADL01_0041900.DBLe.11479 PADL01_0011800.DBLe.3685 PPRFG01_0423800.DBLe.13864 PRG01_0043100.DBLe.3373 PADL01_0020100.DBLn.2779 PADL01_0301200.DBLn.5446 PADL01_0402000.DBLe.4891 PADL01_0011800.DBLe.2605 PADL01_0402400.DBLe.4348 PGABG01_1239300.DBLe.5476 PADL01_0711200.DBLn.5833 PADL01_0710700.DBLn.5794 PGABG01_0400600.DBLn.5977 PADL01_1200800.DBLn.3109 PADL01_1200900.DBLn.4426 PADL01_0412500.DBLn.5896 PADL01_0412300.DBLn.5971 PADL01_0411900.DBLn.6001 PGABG01_0411100.DBLn.5314 PGABG01_0410900.DBLn.5371 PGABG01_0410500.DBLn.5410 PADL01_0012900.DBLn.6463 PADL01_0322800.DBLn.5446 PADL01_0700200.DBLn.5440 PADL01_0041900.DBLe.5332 PADL01_0020100.DBLe.7588 PGABG01_0029600.DBLn.394 PGABG01_0022800.DBLe.4945 PADL01_0700200.DBLn.4312 PADL01_0322800.DBLx.4291 PADL01_0012900.DBLx.5284 PADL01_1100200.DBLx.5269 PADL01_0020100.DBLe.6454 PGABG01_0411100.DBLx.4096 PGABG01_0410900.DBLx.4234 PGABG01_0410500.DBLx.4189 PADL01_0402000.DBLx.3871 PGABG01_0418600.DBLn.4249 PGABG01_0022800.DBLn.3988 PADL01_0012400.DBLn.5236 PGABG01_1218300.DBLe.5140 PADL01_0008000.DBLe.5077 PGABG01_0630000.DBLe.5905 PADL01_0005800.DBLe.5023 PADL01_0710900.DBLn.5125 PADL01_0711300.DBLn.5149 PADL01_1219800.DBLn.5326 PADL01_1220100.DBLn.5392 PGABG01_1239100.DBLe.6019 PADL01_0808000.DBLn.5983 PGABG01_0021600.DBLn.2392 PGABG01_0015400.DBLn.5050 PGABG01_0031300.DBLn.1234 PGABG01_0710700.DBLn.5260 PADL01_0420000.DBLn.5098 PADL01_0042500.DBLn.5392 PADL01_1241000.DBLe.5164 PADL01_0007800.DBLe.4837 PADL01_0042600.DBLn.5254 PADL01_0420300.DBLn.5221 PGABG01_0711000.DBLn.3736 PGABG01_0029700.DBLn.2722 PADL01_0037900.DBLe.3520 PADL01_0028700.DBLn.2917 PGABG01_0323300.DBLe.7651 PGABG01_0010100.DBLe.3574 PGABG01_0021400.DBLe.4885 PGABG01_0600100.DBLe.7363 PGABG01_0022200.DBLe.7369 PADL01_0531800.DBLe.7444 PGABG01_0025200.DBLe.388 PGABG01_1211200.DBLe.3376 PGABG01_0808900.DBLe.3622 PGABG01_0013100.DBLe.4840 PGABG01_0400700.DBLe.4795 PGABG01_0700100.DBLe.4735 PGABG01_0711200.DBLe.4831 PGABG01_1218500.DBLn.4714 PGABG01_0009400.DBLn.4672 PRG01_0003400.DBLe.4885 PPRFG01_0731000.DBLe.4747 PPRFG01_0076000.DBLe.4789 PF3D7_1200600.DBLe.4702 PPRFG01_0801200.DBLe.4861 PPRFG01_0423800.DBLe.4774 PPRFG01_1254000.DBLe.4843 PADL01_0531900.DBLe.8062 PADL01_0700200.DBLe.7552 PADL01_0001200.DBLe.8077 PADL01_0005100.DBLe.3115 PADL01_0419900.DBLe.6262 PGABG01_1239300.DBLe.2581 PADL01_0400100.DBLe.493 PGABG01_1239100.DBLe.8254 PGABG01_1211200.DBLe.5785 PADL01_0003000.DBLe.7246 PADL01_0042500.DBLe.6514 PADL01_0025700.DBLe.577 PADL01_0301200.DBLe.6490 PADL01_1241000.DBLe.6283 PADL01_0420000.DBLe.6139 PADL01_0007800.DBLe.5995 PADL01_0011500.DBLe.6538 PADL01_0033200.DBLe.7981 PADL01_0020100.DBLe.4714 PADL01_1241100.DBLe.6688 PADL01_0023800.DBLe.7993 PADL01_0420300.DBLe.6268 PADL01_0042600.DBLe.6310 PADL01_1100200.DBLe.8551 PPRFG01_0733300.DBLe.4774 PPRFG01_00100700.DBLe.4768 PPRFG01_0534500.DBLe.4846 PADL01_1200800.DBLe.5188 PADL01_1200900.DBLe.6559 PADL01_0808000.DBLe.8062 PADL01_0012900.DBLe.8623 PGABG01_0022200.DBLe.9883 PADL01_0113100.DBLe.8110 PGABG01_0711000.DBLe.4933 PGABG01_0029700.DBLe.3919 PGABG01_0027900.DBLe.2983 PGABG01_0022800.DBLe.7249 PGABG01_0418600.DBLe.7495 PGABG01_0323300.DBLe.10189 PGABG01_0410500.DBLe.7759 PGABG01_0410900.DBLe.7765 PGABG01_0411100.DBLe.7585 PADL01_0531800.DBLe.9919 PGABG01_0600100.DBLe.9796 PPRFG01_0400100.DBLe.4801 PPRFG01_0049200.DBLe.4810 PF3D7_0533100.DBLe.4852 PPRFG01_0083200.DBLe.2497 PGABG01_0418500.DBLe.7771 PADL01_0322800.DBLe.6505 PADL01_0419900.DBLe.8800 PRG01_0711500.DBLe.6730 PRG01_0414700.DBLe.6643 PPRFG01_0011400.DBLe.6208 PPRFG01_0058800.DBLe.5749 PADL01_0420300.DBLe.9013 PADL01_1100200.DBLn.3736 PADL01_1100200.DBLe.2731 PADL01_0012900.DBLe.2719 PGABG01_1239100.DBLe.7057 PADL01_1100200.DBLe.7435 PADL01_0808000.DBLn.7072 PGABG01_0022200.DBLe.8677 PADL01_0531800.DBLe.8752 PGABG01_0600100.DBLe.8716 PADL01_1200800.DBLe.4075 PADL01_1200900.DBLe.5437 PGABG01_0027900.DBLe.1708 PGABG01_0022800.DBLe.5974 PGABG01_0418600.DBLe.6226 PADL01_0710900.DBLe.10264 PADL01_0005800.DBLe.10309 PADL01_0301200.DBLe.9274 PADL01_0300500.DBLe.523 PADL01_1100200.DBLe.12163 PADL01_0012900.DBLe.12196 PADL01_0030600.DBLe.415 PGABG01_0400600.DBLe.10078 PGABG01_1211200.DBLe.8578 PPRFG01_1254000.DBLe.14386 PADL01_0020100.DBLe.9931 PF3D7_0100300.DBLe.1441 PF3D7_0600400.DBLe.1450 PF3D7_0937600.DBLe.1420 PADL01_1220100.DBLe.9820 PADL01_1219800.DBLe.10603 PGABG01_0630000.DBLe.9979 PADL01_0700200.DBLe.11230 PADL01_0322800.DBLe.12616 PGABG01_0700100.DBLe.7795 PADL01_0042500.DBLe.10204 PADL01_0033200.DBLe.10549 PGABG01_0022200.DBLe.12298 PGABG01_0418600.DBLe.9655 PGABG01_0022800.DBLe.9292 PGABG01_0027900.DBLe.5143 PADL01_0041900.DBLe.12571 PGABG01_0600100.DBLe.10894 PADL01_0531800.DBLe.10891 PBILCG01_0001700.DBLe.5791 PRG01_1222900.DBLe.7819 PPRFG01_1151300.DBLe.5746 PPRFG01_0113800.DBLe.6097 PPRFG01_0536800.DBLe.6268 PGABG01_0301500.DBLe.6529 PGABG01_0301700.DBLe.1144 PPRFG01_0423800.DBLe.14974 PADL01_0420300.DBLe.9919 PADL01_0011800.DBLe.4771 PPRFG01_0836500.DBLe.7558 PPRFG01_0029600.DBLe.5935 PPRFG01_1375600.DBLe.6094 PPRFG01_1375500.DBLe.841 PF3D7_0600200.DBLe.6250 PPRFG01_0083200.DBLe.6181 PPRFG01_0039800.DBLe.4276 PPRFG01_0534500.DBLe.8713 PF3D7_0533100.DBLe.8674 PF3D7_0632500.DBLe.8323 PPRFG01_0001600.DBLe.8281 PPRFG01_0018600.DBLe.5860 PPRFG01_0034400.DBLe.8239 PPRFG01_0062900.DBLe.8326 PBILCG01_0020700.DBLe.5692 PRG01_0041000.DBLe.7036 PPRFG01_0500200.DBLe.8098 PPRFG01_0423800.DBLe.11899 PGABG01_0323300.DBLe.11212 PPRFG01_0423800.DBLe.6034 PPRFG01_1254000.DBLe.6103 PPRFG01_0731000.DBLe.6022 PPRFG01_0801200.DBLe.6169 PRG01_0003400.DBLe.6085 PPRFG01_0076000.DBLe.6070 PF3D7_1200600.DBLe.5944 PPRFG01_0423800.DBLe.8080 PPRFG01_1254000.DBLe.8143 PRG01_0003400.DBLe.8122 PPRFG01_0731000.DBLe.8062 PPRFG01_0027800.DBLe.4639 PPRFG01_0076000.DBLe.8104 PPRFG01_0801200.DBLe.8230 PADL01_1212600.DBLe.6832 PADL01_0028700.DBLe.6046 PADL01_0037900.DBLe.6577 PGABG01_0710500.DBLe.4381 PGABG01_0021500.DBLe.562 PGABG01_1239100.DBLe.10888 PADL01_0001200.DBLe.9598 PRG01_0402600.DBLe.6538 PGABG01_1218300.DBLe.10948 PADL01_1200900.DBLe.9700 PADL01_0411900.DBLe.8842 PADL01_0412300.DBLe.8860 PADL01_0412500.DBLe.8893 PPRFG01_0836400.DBLe.8110 PPRFG01_0086700.DBLe.8527 PPRFG01_0027900.DBLe.7987 PGABG01_0418500.DBLe.10036 PADL01_0012400.DBLe.10408 PADL01_0402100.DBLe.5923 PPRFG01_1256400.DBLe.6130 PGABG01_0808900.DBLe.6832 PADL01_0023800.DBLe.10699 PPRFG01_0041100.DBLe.4801 PPRFG01_0062800.DBLe.6211 PF3D7_1200400.DBLe.6205 PGABG01_0001000.DBLe.1834 PGABG01_0410500.DBLe.8785 PADL01_0025800.DBLe.9391 PGABG01_0410900.DBLe.8752 PGABG01_0411100.DBLe.8569 PGABG01_1218500.DBLe.6379 PGABG01_0009400.DBLe.6364 PADL01_0003000.DBLe.8767 PADL01_0808000.DBLe.10726 PADL01_1241100.DBLe.8215 PADL01_0007800.DBLe.7405 PPRFG01_0402300.DBLe.1606 PADL01_1200800.DBLe.8320 PGABG01_0711000.DBLe.6457 PGABG01_0029700.DBLe.5443 PGABG01_0013100.DBLe.6628 PPRFG01_0018500.DBLe.8086 PRG01_0710600.DBLe.6547 PPRFG01_0063000.DBLe.10393 PPRFG01_0021400.DBLe.8179 PRG01_1480400.DBLe.6793 PPRFG01_0939000.DBLe.6028 PPRFG01_0031300.DBLe.6034 PPRFG01_0800100.DBLe.6091 PPRFG01_0005600.DBLe.466 PRG01_0043100.DBLe.6025 PPRFG01_0058700.DBLe.7186 PADL01_0531900.DBLe.9493 PADL01_0402000.DBLe.8815 PPRFG01_00100700.DBLe.8536 PPRFG01_1100100.DBLe.6055 PPRFG01_0005800.DBLe.9280 PPRFG01_1100200.DBLe.7924 PRG01_0026700.DBLe.9412 PGABG01_0711200.DBLe.6547 PGABG01_0023300.DBLe.4828 PADL01_0011500.DBLe.7810 PF3D7_0733000.DBLe.5134 PADL01_0419900.DBLe.11155 PF3D7_0800200.DBLe.6226 PPRFG01_0034900.DBLe.6070 PADL01_0005100.DBLe.4591 PADL01_0711200.DBLe.8572 PGABG01_0021600.DBLe.7456 PGABG01_0020100.DBLe.4321 PGABG01_0031300.DBLe.5998 PRG01_0003400.DBLe.7108 PPRFG01_0801200.DBLe.7207 PPRFG01_0731000.DBLe.7051 PPRFG01_0076000.DBLe.7096 PPRFG01_0423800.DBLe.7063 PPRFG01_1254000.DBLe.7126 PPRFG01_0423800.DBLe.9232 PPRFG01_1254000.DBLe.9283 PPRFG01_0731000.DBLe.9208 PPRFG01_0076000.DBLe.9208 PPRFG01_0801200.DBLe.9331 PPRFG01_0086700.DBLe.9499 PPRFG01_0027900.DBLe.8950 PBILCG01_0020700.DBLe.6679 PPRFG01_0500200.DBLe.9055 PPRFG01_0836400.DBLe.9091 PPRFG01_0423800.DBLe.12877 PF3D7_1200600.DBLe.6976 PF3D7_0632500.DBLe.9310 PPRFG01_0001600.DBLe.9265 PRG01_0041000.DBLe.8020 PPRFG01_0018600.DBLe.6847 PPRFG01_0027800.DBLe.5749 PRG01_0003400.DBLe.9268 PPRFG01_0062900.DBLe.9310 ! PPRFG01_0034400.DBLe.9226 ! ! ! PADL01_0030600.DBLe.415 PADL01_0300500.DBLe.523 PADL01_0025700.DBLe.577 PADL01_0400100.DBLe.493 PF3D7_0632500.DBLe.9310 PF3D7_1200600.DBLe.6976 PF3D7_0800200.DBLe.6226 PF3D7_0733000.DBLe.5134 PF3D7_1200400.DBLe.6205 PF3D7_1200600.DBLe.5944 PF3D7_0632500.DBLe.8323 PF3D7_0533100.DBLe.8674 PF3D7_0600200.DBLe.6250 PF3D7_0937600.DBLe.1420 PF3D7_0600400.DBLe.1450 PF3D7_0100300.DBLe.1441 PF3D7_0533100.DBLe.4852 PF3D7_1200600.DBLe.4702 PF3D7_0632500.DBLe.7258 PRG01_0003400.DBLe.9268 PRG01_0041000.DBLe.8020 PRG01_0003400.DBLe.7108 PRG01_0026700.DBLe.9412 PRG01_0043100.DBLe.6025 PRG01_1480400.DBLe.6793 PRG01_0710600.DBLe.6547 PRG01_0402600.DBLe.6538 PRG01_0003400.DBLe.8122 PRG01_0003400.DBLe.6085 PRG01_0041000.DBLe.7036 PRG01_1222900.DBLe.7819 PRG01_0711500.DBLe.6730 PRG01_0414700.DBLe.6643 PRG01_0003400.DBLe.4885 PRG01_0043100.DBLe.3373 PRG01_0041000.DBLe.5980 PRG01_0026700.DBLe.7057 PADL01_0402000.DBLx.3871 PADL01_1100200.DBLx.5269 PADL01_0012900.DBLx.5284 PADL01_0322800.DBLx.4291 PADL01_0711200.DBLe.8572 PADL01_0005100.DBLe.4591 PADL01_0011500.DBLe.7810 PADL01_0531900.DBLe.9493 PADL01_0402000.DBLe.8815 PADL01_1200800.DBLe.8320 PADL01_0007800.DBLe.7405 PADL01_1241100.DBLe.8215 PADL01_0003000.DBLe.8767 PADL01_0025800.DBLe.9391 PADL01_0402100.DBLe.5923 PADL01_0412500.DBLe.8893 PADL01_0411900.DBLe.8842 PADL01_0412300.DBLe.8860 PADL01_1200900.DBLe.9700 PADL01_0001200.DBLe.9598 PADL01_0037900.DBLe.6577 PADL01_0028700.DBLe.6046 PADL01_1212600.DBLe.6832 PADL01_0011800.DBLe.4771 PADL01_0420300.DBLe.9919 PADL01_1220100.DBLe.9820 PADL01_0020100.DBLe.9931 PADL01_0301200.DBLe.9274 PADL01_1200800.DBLe.4075 PADL01_1200900.DBLe.5437 PADL01_0531800.DBLe.8752 PADL01_1100200.DBLe.7435 PADL01_0012900.DBLe.2719 PADL01_1100200.DBLe.2731 PADL01_0420300.DBLe.9013 PADL01_0419900.DBLe.8800 PADL01_0322800.DBLe.6505 PADL01_0531800.DBLe.9919 PADL01_0113100.DBLe.8110 PADL01_0012900.DBLe.8623 PADL01_0808000.DBLe.8062 PADL01_1200900.DBLe.6559 PADL01_1200800.DBLe.5188 PADL01_1100200.DBLe.8551 PADL01_0042600.DBLe.6310 PADL01_0420300.DBLe.6268 PADL01_0023800.DBLe.7993 PADL01_1241100.DBLe.6688 PADL01_0020100.DBLe.4714 PADL01_0011500.DBLe.6538 PADL01_0033200.DBLe.7981 PADL01_0007800.DBLe.5995 PADL01_0420000.DBLe.6139 PADL01_1241000.DBLe.6283 PADL01_0301200.DBLe.6490 PADL01_0042500.DBLe.6514 PADL01_0003000.DBLe.7246 PADL01_0419900.DBLe.6262 PADL01_0005100.DBLe.3115 PADL01_0001200.DBLe.8077 PADL01_0531900.DBLe.8062 PADL01_0700200.DBLe.7552 PADL01_0531800.DBLe.7444 PADL01_0037900.DBLe.3520 PADL01_1241000.DBLe.5164 PADL01_0007800.DBLe.4837 PADL01_0005800.DBLe.5023 PADL01_0008000.DBLe.5077 PADL01_0020100.DBLe.6454 PADL01_0020100.DBLe.7588 PADL01_0041900.DBLe.5332 PADL01_0402400.DBLe.4348 PADL01_0011800.DBLe.2605 PADL01_0402000.DBLe.4891 PADL01_0011800.DBLe.3685 PADL01_0005800.DBLe.9394 PADL01_0710900.DBLe.9373 PADL01_0412300.DBLe.7117 PADL01_0808000.DBLe.1546 PADL01_0808000.DBLn.7072 PADL01_1100200.DBLn.3736 PADL01_0028700.DBLn.2917 PADL01_0420300.DBLn.5221 PADL01_0042600.DBLn.5254 PADL01_0042500.DBLn.5392 PADL01_0420000.DBLn.5098 PADL01_0808000.DBLn.5983 PADL01_1220100.DBLn.5392 PADL01_1219800.DBLn.5326 PADL01_0711300.DBLn.5149 PADL01_0710900.DBLn.5125 PADL01_0012400.DBLn.5236 PADL01_0700200.DBLn.4312 PADL01_0700200.DBLn.5440 PADL01_0322800.DBLn.5446 PADL01_0012900.DBLn.6463 PADL01_0411900.DBLn.6001 PADL01_0412300.DBLn.5971 PADL01_0412500.DBLn.5896 PADL01_1200800.DBLn.3109 PADL01_1200900.DBLn.4426 PADL01_0710700.DBLn.5794 PADL01_0711200.DBLn.5833 PADL01_0301200.DBLn.5446 PADL01_0020100.DBLn.2779 PADL01_0007800.DBLn.3886 PADL01_0711300.DBLn.4006 PADL01_0012400.DBLn.4111 PADL01_0420000.DBLn.4111 PADL01_0419900.DBLn.4186 PADL01_0420300.DBLn.4147 PADL01_0005800.DBLn.4069 PADL01_0711200.DBLn.4789 PADL01_0710700.DBLn.4747 PADL01_1200900.DBLn.3481 PADL01_1200800.DBLn.2110 PADL01_0412500.DBLn.4927 PADL01_0412300.DBLn.4948 PADL01_0411900.DBLn.4954 PADL01_0025800.DBLn.1789 PADL01_0042600.DBLn.1792 PADL01_0011500.DBLn.4201 PADL01_0025800.DBLn.5704 PADL01_0322800.DBLn.1519 PADL01_0700200.DBLn.1477 PADL01_0531800.DBLn.2842 PADL01_0402400.DBLn.3301 PADL01_0011800.DBLn.1540 PADL01_0402400.DBLn.1591 PADL01_0020100.DBLn.1609 PADL01_0322800.DBLn.2674 PADL01_1100200.DBLn.1570 PADL01_0012900.DBLn.1564 PADL01_0531800.DBLn.1564 PADL01_0301200.DBLn.4315 PPRFG01_0005600.DBLe.466 PPRFG01_1375500.DBLe.841 ε PGABG01_0021500.DBLe.562 PGABG01_0025200.DBLe.388 PGABG01_0029600.DBLn.394 PADL01_0419900.DBLe.11155 PADL01_0808000.DBLe.10726 PADL01_0023800.DBLe.10699 PADL01_0012400.DBLe.10408 PADL01_0531800.DBLe.10891 PADL01_0041900.DBLe.12571 PADL01_0033200.DBLe.10549 PADL01_0042500.DBLe.10204 PADL01_0322800.DBLe.12616 PADL01_0700200.DBLe.11230 PADL01_1219800.DBLe.10603 PADL01_0012900.DBLe.12196 PADL01_1100200.DBLe.12163 PADL01_0710900.DBLe.10264 PADL01_0005800.DBLe.10309 PADL01_0041900.DBLe.11479 PADL01_0012900.DBLe.11296 PADL01_1100200.DBLe.11278 PF3D7_1200600.DBLpam1.154 PF3D7_0600400.DBLpam1.289 PPRFG01_0034400.DBLe.9226 PPRFG01_0062900.DBLe.9310 PPRFG01_0018600.DBLe.6847 PPRFG01_0027800.DBLe.5749 PPRFG01_0001600.DBLe.9265 PPRFG01_0836400.DBLe.9091 PPRFG01_0500200.DBLe.9055 PPRFG01_0027900.DBLe.8950 PPRFG01_0086700.DBLe.9499 PPRFG01_0801200.DBLe.9331 PPRFG01_0076000.DBLe.9208 PPRFG01_0731000.DBLe.9208 PPRFG01_1254000.DBLe.9283 PPRFG01_0423800.DBLe.9232 PPRFG01_1254000.DBLe.7126 PPRFG01_0423800.DBLe.7063 PPRFG01_0076000.DBLe.7096 PPRFG01_0731000.DBLe.7051 PPRFG01_0801200.DBLe.7207 PPRFG01_0034900.DBLe.6070 PPRFG01_1100200.DBLe.7924 PPRFG01_0005800.DBLe.9280 PPRFG01_1100100.DBLe.6055 PPRFG01_0058700.DBLe.7186 PPRFG01_0800100.DBLe.6091 PPRFG01_0939000.DBLe.6028 PPRFG01_0031300.DBLe.6034 PPRFG01_0021400.DBLe.8179 PPRFG01_0018500.DBLe.8086 PPRFG01_0402300.DBLe.1606 PPRFG01_0062800.DBLe.6211 PPRFG01_0041100.DBLe.4801 PPRFG01_1256400.DBLe.6130 PPRFG01_0027900.DBLe.7987 PPRFG01_0086700.DBLe.8527 PPRFG01_0836400.DBLe.8110 PPRFG01_0801200.DBLe.8230 PPRFG01_0076000.DBLe.8104 PPRFG01_0027800.DBLe.4639 PPRFG01_0731000.DBLe.8062 PPRFG01_1254000.DBLe.8143 PPRFG01_0423800.DBLe.8080 PPRFG01_0076000.DBLe.6070 PPRFG01_0423800.DBLe.6034 PPRFG01_1254000.DBLe.6103 PPRFG01_0731000.DBLe.6022 PPRFG01_0801200.DBLe.6169 PPRFG01_0500200.DBLe.8098 PPRFG01_0062900.DBLe.8326 PPRFG01_0034400.DBLe.8239 PPRFG01_0018600.DBLe.5860 PPRFG01_0001600.DBLe.8281 PPRFG01_0534500.DBLe.8713 PPRFG01_0039800.DBLe.4276 PPRFG01_0083200.DBLe.6181 PPRFG01_1375600.DBLe.6094 PPRFG01_0836500.DBLe.7558 PPRFG01_0029600.DBLe.5935 PPRFG01_0113800.DBLe.6097 PPRFG01_0536800.DBLe.6268 PPRFG01_1151300.DBLe.5746 PPRFG01_0011400.DBLe.6208 PPRFG01_0058800.DBLe.5749 PPRFG01_0083200.DBLe.2497 PPRFG01_0049200.DBLe.4810 PPRFG01_0400100.DBLe.4801 PPRFG01_0733300.DBLe.4774 PPRFG01_0534500.DBLe.4846 PPRFG01_1254000.DBLe.4843 PPRFG01_0423800.DBLe.4774 PPRFG01_0801200.DBLe.4861 PPRFG01_0731000.DBLe.4747 PPRFG01_0076000.DBLe.4789 PPRFG01_1151300.DBLe.4642 PPRFG01_0027900.DBLe.6928 PPRFG01_0062900.DBLe.7258 PPRFG01_0058700.DBLe.4810 PPRFG01_0018600.DBLe.4816 PPRFG01_0005800.DBLe.6904 PPRFG01_0500200.DBLe.7027 PPRFG01_0001600.DBLe.7213 PPRFG01_0034400.DBLe.7186 PPRFG01_0836400.DBLe.7039 PPRFG01_1100200.DBLe.5515 PPRFG01_0086700.DBLe.7462 PPRFG01_0001600.DBLn.2464 PBLACG01_0035500.DBLn.619 PGABG01_0410900.DBLx.4234 PGABG01_0410500.DBLx.4189 PGABG01_0411100.DBLx.4096 PGABG01_0031300.DBLe.5998 PGABG01_0020100.DBLe.4321 PGABG01_0021600.DBLe.7456 PGABG01_0711200.DBLe.6547 PGABG01_0023300.DBLe.4828 PGABG01_0013100.DBLe.6628 PGABG01_0711000.DBLe.6457 PGABG01_0029700.DBLe.5443 PGABG01_0009400.DBLe.6364 PGABG01_1218500.DBLe.6379 PGABG01_0411100.DBLe.8569 PGABG01_0410900.DBLe.8752 PGABG01_0001000.DBLe.1834 PGABG01_0410500.DBLe.8785 PGABG01_0808900.DBLe.6832 PGABG01_0710500.DBLe.4381 PGABG01_0301500.DBLe.6529 PGABG01_0301700.DBLe.1144 PGABG01_0027900.DBLe.5143 PGABG01_0022800.DBLe.9292 PGABG01_0418600.DBLe.9655 PGABG01_0700100.DBLe.7795 PGABG01_0630000.DBLe.9979 PGABG01_1211200.DBLe.8578 PGABG01_0418600.DBLe.6226 PGABG01_0022800.DBLe.5974 PGABG01_0027900.DBLe.1708 PGABG01_0600100.DBLe.8716 PGABG01_0022200.DBLe.8677 PGABG01_1239100.DBLe.7057 PGABG01_0418500.DBLe.7771 PGABG01_0600100.DBLe.9796 PGABG01_0411100.DBLe.7585 PGABG01_0410900.DBLe.7765 PGABG01_0410500.DBLe.7759 PGABG01_0418600.DBLe.7495 PGABG01_0022800.DBLe.7249 PGABG01_0027900.DBLe.2983 PGABG01_0029700.DBLe.3919 PGABG01_0711000.DBLe.4933 PGABG01_0022200.DBLe.9883 PGABG01_1239100.DBLe.8254 PGABG01_1211200.DBLe.5785 PGABG01_1239300.DBLe.2581 PGABG01_0700100.DBLe.4735 PGABG01_0711200.DBLe.4831 PGABG01_0400700.DBLe.4795 PGABG01_0013100.DBLe.4840 PGABG01_0808900.DBLe.3622 PGABG01_1211200.DBLe.3376 PGABG01_0600100.DBLe.7363 PGABG01_0022200.DBLe.7369 PGABG01_0010100.DBLe.3574 PGABG01_0021400.DBLe.4885 PGABG01_0323300.DBLe.7651 PGABG01_1239100.DBLe.6019 PGABG01_0630000.DBLe.5905 PGABG01_1218300.DBLe.5140 PGABG01_0022800.DBLe.4945 PGABG01_1239300.DBLe.5476 PGABG01_0027900.DBLe.4042 PGABG01_0418600.DBLe.8554 PGABG01_0323300.DBLe.1663 PGABG01_1218500.DBLn.4714 PGABG01_0009400.DBLn.4672 PGABG01_0029700.DBLn.2722 PGABG01_0711000.DBLn.3736 PGABG01_0710700.DBLn.5260 PGABG01_0031300.DBLn.1234 PGABG01_0015400.DBLn.5050 PGABG01_0021600.DBLn.2392 PGABG01_0418600.DBLn.4249 PGABG01_0022800.DBLn.3988 PGABG01_0410500.DBLn.5410 PGABG01_0410900.DBLn.5371 PGABG01_0411100.DBLn.5314 PGABG01_0400600.DBLn.5977 PGABG01_0029700.DBLn.1705 PGABG01_0711000.DBLn.2719 PGABG01_0400600.DBLn.4978 PRG01_0003400.DBLpam1.154 PGABG01_0031300.DBLn.3718 PGABG01_0418500.DBLn.4582 PGABG01_1239000.DBLn.3061 PGABG01_0014500.DBLn.2992 PGABG01_0023300.DBLn.3004 PGABG01_0401000.DBLn.4558 PGABG01_0032500.DBLn.4576 PGABG01_0418600.DBLn.1510 PGABG01_0022800.DBLn.1249 PGABG01_0029400.DBLn.1510 PGABG01_0411100.DBLn.1489 PRG01_0904400.DBLpam1.250 PGABG01_0600100.DBLn.2815 PGABG01_0022200.DBLn.2815 PGABG01_1239300.DBLn.4441 PGABG01_0630000.DBLn.1558 PGABG01_0630000.DBLn.2680 PGABG01_1239300.DBLn.1558 PGABG01_0022200.DBLn.1561 PGABG01_0600100.DBLn.1552 PGABG01_0021200.DBLn.4504 PGABG01_1239000.DBLn.4360 PGABG01_0401000.DBLn.5884 PGABG01_0014500.DBLn.4375 PGABG01_0008700.DBLn.4426 PGABG01_0032500.DBLn.5896 PGABG01_0616300.DBLn.7078 PBILCG01_0020700.DBLe.6679 PBILCG01_0020700.DBLe.5692 PBILCG01_0001700.DBLe.5791 PBILCG01_0020700.DBLe.4618 PBILCG01_0700100.DBLn.6064 PBILCG01_1481200.DBLn.7540 PADL01_1241100.DBLpam1.322 PADL01_0301200.DBLpam1.307 PPRFG01_0423800.DBLe.12877 PPRFG01_00100700.DBLe.8536 PPRFG01_0063000.DBLe.10393 PPRFG01_0423800.DBLe.11899 PPRFG01_0423800.DBLe.14974 PPRFG01_1254000.DBLe.14386 PPRFG01_00100700.DBLe.4768 PPRFG01_0423800.DBLe.13864 PPRFG01_1254000.DBLe.11992 PBLACG01_1240800.DBLn.1441 PBLACG01_0616600.DBLn.2560 PBLACG01_0027400.DBLn.1534 PGABG01_0418500.DBLe.10036 PGABG01_1218300.DBLe.10948 PGABG01_1239100.DBLe.10888 PGABG01_0323300.DBLe.11212 PGABG01_0600100.DBLe.10894 PGABG01_0022200.DBLe.12298 PGABG01_0400600.DBLe.10078 PGABG01_0323300.DBLe.10189 PADL01_0001200.DBLpam1.4396 PADL01_0003000.DBLpam1.3535 PADL01_0711200.DBLpam1.1732 PADL01_0042500.DBLpam1.1798 PADL01_1200900.DBLpam1.1678 PPRFG01_0801200.DBLpam1.154 PPRFG01_0076000.DBLpam1.157 PPRFG01_0731000.DBLpam1.157 PPRFG01_1254000.DBLpam1.157 PPRFG01_0423800.DBLpam1.157 PGABG01_0301500.DBLpam1.226 PGABG01_0710500.DBLpam1.307 PPRFG01_0400100.DBLpam1.7510 PBLACG01_1211300.DBLpam1.205 PBLACG01_1211600.DBLpam1.169 PBLACG01_0400300.DBLpam1.196 PBLACG01_0800100.DBLpam1.532 PBLACG01_0616600.DBLpam1.163 PBLACG01_1240800.DBLpam1.181 PBLACG01_0728700.DBLpam1.166 PBLACG01_0011400.DBLpam1.184 PGABG01_0021200.DBLpam1.1804 PGABG01_0401000.DBLpam1.3217 ! ! ! !

Domains ! ! ! PR DBLx DBLn DBLe DBLpam1 DBLn DBL DBLx Pf Pprf Pr Pbilc Pblac Pgab Padl PPRF PGAB PBILC PF3D7

Score! PBLAC DBLpam1 PADL01

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Fig. S10. Diversity of var genes domains. Annotated similarity matrix between all the domains of the Laverania of Fig. 5, including their species attribution and their cluster attribution. The similarity matrix shows the score of the BLASTp between the domains. It was clustered with the ward2 algorithm in R. The dendrogram is on the left hand side. Each row (column) presents therefore one domain and shows its score of similarity to the other 2,547 domains and itself. To each row is associated on the left the domain type and on the right the species it comes from. (A) Similarity matrix based on the 2548 domains. This figures shows that the ATS (acidic terminal sequence) and NTS (N-terminal sequence) are very distinct. The DBL domains on the other side have more similarity between themselves. Importantly, the genes on the top are similar to each other, but they are annotated as different domains. The DBLn domain is a domain where the duffy binding like Pfam domain had a higher score than the other domains from the vardom server (http://www.cbs.dtu.dk/services/VarDom/). (B) To understand the diversity of DBLε and DBLn and to compare it to the newly described DBLx, a similarity matrix of all domains annotated as DBLε, DBLn, DBLpam1 and DBLx labeled sequences, see Supplemental Information C. It can be seen that have domains are similar to each other and that the DBLx labeled sequences as defined by Larremore et al(9) cluster within the DBLε domains. Pf - P. falciparum, Pprf P. praefalciparum, Pr - P. reichenowi, Pbilc - P. billcollinsi, Pblac - P. blacklocki, Pgab - P. gaboni and Padl - P. adleri.

26

Table S1. Multiple infections in Laverania samples Further information on the primates. Samples were analysed for the presence of multiple-species infections by mapping reads to reference datasets, assembled de novo from Pacific Biosciences reads. Host contamination was identified by mapping against the human genome and removed prior to analysis.

Coalescence, years (x 105) Effective Population size, Ne (x 105) Probably of Parasite Migration Migration Population Status Intergenic, Intergenic, Intergenic, Genic Intergenic Genic Intergenic target Genic Intergenic no UTR no UTR no UTR 1.9 - 3.2 0.4 - 0.7 0.4 - 0.7 P. falciparum Present NA NA NA NA 0 0 0 (1.8 - 3.4) (0 - 2.1) (0 - 2.1) P. praefalciparum Present NA NA NA 8.8 - 14.8 12.2 - 20.7 10 - 17 Pf 0.02 0.14 0.16 (8.1 - 16) (11.6 - 21.8) (9.2 - 18.4) (0.0 - 0.03) (0.09- 0.19) (0.08- 0.27) NA NA NA 11.3 - 19.2 15.9 - 26.9 12.3 - 20.7 NA 0 0 0 P. reichenowi Present (3.7 - 19.9) (15.4 - 27.7) (11.7 - 21.8) P. billcollinsi Present NA NA NA 9.3 - 15.8 - - NA 0 - - (8.9 - 16.5) P. gaboni Present NA NA NA 18.1 - 30.6 - - NA 0 - - (17.6 - 31.6) P. adleri Present NA NA NA 17.7 - 29.9 - - NA 0 - - (16.9 - 31.2) 0.2 - 0.3 0.5 - 0.8 0.4 - 0.6 6.5 - 11.1 16.5 - 27.9 12.4 - 21 NA 0 0 0 Pf - Pprf Ancestral (0.1 - 0.3) (0.4 - 0.8) (0.3 - 0.7) (6 - 11.9) (15.4 - 29.7) (11.1 - 23.2) 0.9 - 1.5 1.6 - 2.7 1.4 - 2.3 29.7 - 50.3 68.6 - 116.2 42.3 - 71.5 NA 0 0 0 Pf - Pprf - Pr Ancestral (0.8 - 1.5) (1.5 - 2.7) (1.3 - 2.4) (27.7 - 53.7) (65.4 - 121.6) (39.1 - 77.1) Pf - Pprf - Pr - 2.3 - 4 4.2 - 7.1* 3.7 - 6.2* 65.8 - 111.3 - - Pgab - Padl 0.61 - - Ancestral Pbilc (2.3 - 4) (62 - 118.5) (0.45 - 0.77) 0.9 - 1.4 1.5 - 2.6* 1.3 - 2.2* 28.3 - 47.9 - - NA 0 - - Pgab - Padl Ancestral (0.8 - 1.5) (26.7 - 50.7) Pf - Pprf - Pr - 4.5 - 7.6 8.1 - 13.6* 7 - 11.8* 98.9 - 167.4 - - NA 0 - - Ancestral Pbilc - Pgab - Padl (4.2 - 8) (93.2 - 178.4)

Table S2. Estimated dates of speciation (time to coalescence), effective population size (Ne) and migration rates for present and ancestral Laverania populations. Values inferred using GPhoCS coalescence model. The results are presented from sequence alignments of coding (genic) regions as well as intergenic regions with and without UTR sequences. The coalescence model parameters have been scaled based on an assumption of 402–681 mitotic events per year. The number in the brackets is the range based on the prediction (95% confidence interval). Values with (*) are linear obtained estimates, as we were not able

28

to obtain good alignments between those species due to the low GC content. The migration rate refers to the probability of a parasite from the source lineage migrating into the target over the time period. The data from these estimates are presented

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References:

1. F. Prugnolle et al., African great apes are natural hosts of multiple related malaria species, including Plasmodium falciparum. Proceedings of the National Academy of Sciences of the United States of America 107, 1458 (Jan 26, 2010). 2. W. Liu et al., Origin of the human malaria parasite Plasmodium falciparum in gorillas. Nature 467, 420 (Sep 23, 2010). 3. B. Ollomo et al., A New Malaria Agent in African Hominids. PLoS Pathog 5, e1000446 (2009). 4. W. Liu et al., Multigenomic Delineation of Plasmodium Species of the Laverania Subgenus Infecting Wild- Living Chimpanzees and Gorillas. Genome biology and evolution 8, 1929 (2016). 5. L. Boundenga et al., Diversity of malaria parasites in great apes in Gabon. Malar J 14, 111 (2015). 6. T. D. Otto et al., Genome sequencing of chimpanzee malaria parasites reveals possible pathways of adaptation to human hosts. Nature communications 5, 4754 (2014). 7. S. A. Sundararaman et al., Genomes of cryptic chimpanzee Plasmodium species reveal key evolutionary events leading to human malaria. Nature communications 7, 11078 (2016). 8. M. J. Gardner et al., Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419, 498 (2002). 9. D. B. Larremore et al., Ape parasite origins of human malaria virulence genes. Nature communications 6, 8368 (2015). 10. M. A. Pacheco et al., Timing the origin of human malarias: the lemur puzzle. BMC evolutionary biology 11, 299 (2011). 11. D. M. Behar et al., The dawn of human matrilineal diversity. American journal of human genetics 82, 1130 (May, 2008). 12. S. K. Volkman et al., Recent origin of Plasmodium falciparum from a single progenitor. Science 293, 482 (Jul 20, 2001). 13. F. P. Palstra, D. J. Fraser, Effective/census population size ratio estimation: a compendium and appraisal. Ecology and evolution 2, 2357 (Sep, 2012). 14. S. W. Roy, The Plasmodium gaboni genome illuminates allelic dimorphism of immunologically important surface antigens in P. falciparum. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 36, 441 (Dec, 2015). 15. K. Tanabe, M. Mackay, M. Goman, J. G. Scaife, Allelic dimorphism in a surface antigen gene of the malaria parasite Plasmodium falciparum. Journal of molecular biology 195, 273 (May 20, 1987). 16. Y. Yasukochi, I. Naka, J. Patarapotikul, H. Hananantachai, J. Ohashi, Genetic evidence for contribution of human dispersal to the genetic diversity of EBA-175 in Plasmodium falciparum. Malar J 14, 293 (Aug 01, 2015). 17. N. Malaria Genomic Epidemiology, G. Band, K. A. Rockett, C. C. Spencer, D. P. Kwiatkowski, A novel locus of resistance to severe malaria in a region of ancient balancing selection. Nature 526, 253 (Oct 8, 2015). 18. B. Makanga et al., Ape malaria transmission and potential for ape-to-human transfers in Africa. Proceedings of the National Academy of Sciences of the United States of America 113, 5329 (May 10, 2016). 19. M. Wanaguru, W. Liu, B. H. Hahn, J. C. Rayner, G. J. Wright, RH5-Basigin interaction plays a major role in the host tropism of Plasmodium falciparum. Proceedings of the National Academy of Sciences of the United States of America 110, 20735 (Dec 17, 2013). 20. K. E. Wright et al., Structure of malaria invasion protein RH5 with erythrocyte basigin and blocking antibodies. Nature 515, 427 (Nov 20, 2014). 21. T. Triglia, J. K. Thompson, A. F. Cowman, An EBA175 homologue which is transcribed but not translated in erythrocytic stages of Plasmodium falciparum. Mol Biochem Parasitol 116, 55 (Aug, 2001). 22. R. S. Ramiro et al., Hybridization and pre-zygotic reproductive barriers in Plasmodium. Proceedings. Biological sciences / The Royal Society 282, 20143027 (May 7, 2015). 23. S. Eksi et al., Malaria transmission-blocking antigen, Pfs230, mediates human red blood cell binding to exflagellating male parasites and oocyst production. Mol Microbiol 61, 991 (Aug, 2006).

24. D. Cunningham, J. Lawton, W. Jarra, P. Preiser, J. Langhorne, The pir multigene family of Plasmodium: antigenic variation and beyond. Mol Biochem Parasitol 170, 65 (Apr, 2010). 25. E. Mundwiler-Pachlatko, H. P. Beck, Maurer's clefts, the enigma of Plasmodium falciparum. Proceedings of the National Academy of Sciences of the United States of America 110, 19987 (Dec 10, 2013). 26. M. Niang et al., STEVOR is a Plasmodium falciparum erythrocyte binding protein that mediates merozoite invasion and rosetting. Cell host & microbe 16, 81 (Jul 9, 2014). 27. L. L. Bethke et al., Duplication, gene conversion, and genetic diversity in the species-specific acyl-CoA synthetase gene family of Plasmodium falciparum. Mol Biochem Parasitol 150, 10 (Nov, 2006). 28. M. Frank, R. Dzikowski, B. Amulic, K. Deitsch, Variable switching rates of malaria virulence genes are associated with chromosomal position. Mol Microbiol 64, 1486 (Jun, 2007). 29. K. Hayton et al., Erythrocyte binding protein PfRH5 polymorphisms determine species-specific pathways of Plasmodium falciparum invasion. Cell Host Microbe 4, 40 (Jul 17, 2008). 30. P. C. Bull et al., Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria. Nat Med 4, 358 (Mar, 1998). 31. A. Scally, R. Durbin, Revising the human mutation rate: implications for understanding human evolution. Nature reviews. Genetics 13, 745 (Oct, 2012). 32. R. Carter, K. N. Mendis, Evolutionary and historical aspects of the burden of malaria. Clinical microbiology reviews 15, 564 (Oct, 2002). 33. M. Coluzzi, A. Sabatini, A. della Torre, M. A. Di Deco, V. Petrarca, A polytene chromosome analysis of the Anopheles gambiae species complex. Science 298, 1415 (Nov 15, 2002). 34. J. Flint, R. M. Harding, A. J. Boyce, J. B. Clegg, The population genetics of the haemoglobinopathies. Bailliere's clinical haematology 6, 215 (Mar, 1993). 35. S. Auburn et al., An effective method to purify plasmodium falciparum dna directly from clinical blood samples for whole genome high-throughput sequencing. PLoS ONE 6, (2011). 36. S. O. Oyola et al., Optimized whole-genome amplification strategy for extremely AT-biased template. DNA research : an international journal for rapid publication of reports on genes and genomes 21, 661 (Dec, 2014). 37. S. O. Oyola et al., Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification. bioRxiv, (2016). 38. A. Boissiere et al., Isolation of Plasmodium falciparum by flow-cytometry: implications for single- trophozoite genotyping and parasite DNA purification for whole-genome high-throughput sequencing of archival samples. Malaria Journal 11, 163 (2012). 39. M. A. Quail et al., in Nat Methods. (United States, 2012), vol. 9, pp. 10-1. 40. H. Manske, D. Kwiatkowski, SNP-o-matic. Bioinformatics 25, 2434 (2009). 41. C. S. Chin et al., Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nature methods 10, 563 (Jun, 2013). 42. S. Assefa, T. M. Keane, T. D. Otto, C. Newbold, M. Berriman, ABACAS: algorithm-based automatic contiguation of assembled sequences. Bioinformatics Vol. 25 No. 15, 1968 (2009). 43. T. Carver et al., Artemis and ACT: viewing, annotation and comparing sequences stored in relational database. Bioinformatics 24, 2672 (2008). 44. T. D. Otto, M. Sanders, M. Berriman, C. Newbold, Iterative Correction of Reference Nucleotides (iCORN) using second generation sequencing technology. Bioinformatics 26(14), 1704 (2010). 45. A. C. English et al., Mind the gap: upgrading genomes with Pacific Biosciences RS long-read sequencing technology. PLoS One 7, e47768 (2012). 46. T. D. Otto, From sequence mapping to genome assemblies. Methods Mol Biol 1201, 19 (2015). 47. T. D. Otto, G. P. Dillon, W. S. Degrave, M. Berriman, RATT: Rapid Annotation Transfer Tool. Nucleic Acids Research, 1 (2011). 48. M. Stanke et al., AUGUSTUS: ab initio prediction of alternative transcripts. Nucleic Acids Research 34, W435 (Jul 1, 2006). 49. H. Li, R. Durbin, Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754 (Jul 15, 2009). 50. A. McKenna et al., The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20, 1297 (Sep, 2010). 51. P. Danecek et al., The variant call format and VCFtools. Bioinformatics 27, 2156 (Aug 1, 2011). 52. L. Li, C. J. Stoeckert, Jr., D. S. Roos, OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res 13, 2178 (Sep, 2003).

31

53. G. Jordan, N. Goldman, The Effects of Alignment Error and Alignment Filtering on the Sitewise Detection of Positive Selection. Molecular biology and evolution 29, 1125 (Apr, 2012). 54. A. Loytynoja, N. Goldman, An algorithm for progressive multiple alignment of sequences with insertions. Proceedings of the National Academy of Sciences of the United States of America 102, 10557 (Jul 26, 2005). 55. A. Loytynoja, N. Goldman, Phylogeny-aware gap placement prevents errors in sequence alignment and evolutionary analysis. Science 320, 1632 (Jun 20, 2008). 56. W. Fletcher, Z. Yang, The Effect of Insertions, Deletions, and Alignment Errors on the Branch-Site Test of Positive Selection. Molecular biology and evolution 27, 2257 (October 1, 2010, 2010). 57. P. Markova-Raina, D. Petrov, High sensitivity to aligner and high rate of false positives in the estimates of positive selection in the 12 Drosophila genomes. Genome Research 21, 863 (Jun, 2011). 58. A. Morgulis, E. M. Gertz, A. A. Schäffer, R. Agarwala, A Fast and Symmetric DUST Implementation to Mask Low-Complexity DNA Sequences. Journal of Computational Biology 13, 1028 (2006/06/01, 2006). 59. J. C. Wootton, S. Federhen, Statistics of local complexity in amino acid sequences and sequence databases. Computers & Chemistry 17, 149 (1993/06/01, 1993). 60. J. Castresana, Selection of Conserved Blocks from Multiple Alignments for Their Use in Phylogenetic Analysis. Molecular biology and evolution 17, 540 (April 1, 2000, 2000). 61. A. Stamatakis, RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312 (May 1, 2014, 2014). 62. H. Shimodaira, M. Hasegawa, Multiple Comparisons of Log-Likelihoods with Applications to Phylogenetic Inference. Molecular biology and evolution 16, 1114 (August 1, 1999, 1999). 63. T. A. Castoe et al., Evidence for an ancient adaptive episode of convergent molecular evolution. Proceedings of the National Academy of Sciences of the United States of America 106, 8986 (Jun, 2009). 64. G. W. C. Thomas, M. W. Hahn, Determining the Null Model for Detecting Adaptive Convergence from Genomic Data: A Case Study using Echolocating Mammals. Molecular biology and evolution 32, 1232 (May, 2015). 65. Z. Yang, PAML 4: Phylogenetic Analysis by Maximum Likelihood. Molecular biology and evolution 24, 1586 (August 1, 2007, 2007). 66. J. Zhang, R. Nielsen, Z. Yang, Evaluation of an Improved Branch-Site Likelihood Method for Detecting Positive Selection at the Molecular Level. Molecular biology and evolution 22, 2472 (December 1, 2005, 2005). 67. J. H. McDonald, M. Kreitman, Adaptive protein evolution at the Adh locus in Drosophila. Nature 351, 652 (1991). 68. A. A. a. J. Rahnenfuhrer, topGO: Enrichment analysis for Gene Ontology. R package version 2.8.0, (2010). 69. S. Guindon et al., New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59, 307 (May, 2010). 70. R. C. Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32, 1792 (2004). 71. M. Gouy, S. Guindon, O. Gascuel, SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Molecular biology and evolution 27, 221 (Feb, 2010). 72. T. Carver et al., BamView: visualizing and interpretation of next-generation sequencing read. Briefings in bioinformatics, (Jan 24, 2013). 73. T. S. Rask, D. A. Hansen, T. G. Theander, A. Gorm Pedersen, T. Lavstsen, Plasmodium falciparum erythrocyte membrane protein 1 diversity in seven genomes--divide and conquer. PLoS Comput Biol 6, (2010). 74. C. UniProt, UniProt: a hub for protein information. Nucleic Acids Res 43, D204 (Jan, 2015). 75. A. M. Waterhouse, J. B. Procter, D. M. Martin, M. Clamp, G. J. Barton, Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189 (May 1, 2009). 76. A. Claessens et al., Generation of antigenic diversity in Plasmodium falciparum by structured rearrangement of Var genes during mitosis. PLoS Genet 10, e1004812 (Dec, 2014). 77. I. Gronau, M. J. Hubisz, B. Gulko, C. G. Danko, A. Siepel, Bayesian inference of ancient human demography from individual genome sequences. Nat Genet 43, 1031 (Oct, 2011). 78. S. Schiffels, R. Durbin, Inferring human population size and separation history from multiple genome sequences. Nat Genet 46, 919 (Aug, 2014).

32

79. S. C. Nkhoma et al., Population genetic correlates of declining transmission in a human pathogen. Molecular ecology 22, 273 (Jan, 2013). 80. D. A. Joy et al., Early origin and recent expansion of Plasmodium falciparum. Science 300, 318 (Apr 11, 2003). 81. T. Ponnudurai et al., Sporozoite load of mosquitoes infected with Plasmodium falciparum. Transactions of the Royal Society of Tropical Medicine and Hygiene 83, 67 (Jan-Feb, 1989). 82. D. Mazier et al., Complete development of hepatic stages of Plasmodium falciparum in vitro. Science 227, 440 (Jan 25, 1985). 83. N. Gerald, B. Mahajan, S. Kumar, Mitosis in the human malaria parasite Plasmodium falciparum. Eukaryotic cell 10, 474 (Apr, 2011). 84. A. H. Freedman et al., Genome sequencing highlights the dynamic early history of dogs. PLoS Genet 10, e1004016 (Jan, 2014). 85. H. H. Chang et al., Malaria life cycle intensifies both natural selection and random genetic drift. Proceedings of the National Academy of Sciences of the United States of America 110, 20129 (Dec 10, 2013). 86. E. K. Karlsson, D. P. Kwiatkowski, P. C. Sabeti, Natural selection and infectious disease in human populations. Nature reviews. Genetics 15, 379 (Jun, 2014). 87. G. Talavera, J. Castresana, Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst Biol 56, 564 (Aug, 2007). 88. S. Guindon, F. Delsuc, J. F. Dufayard, O. Gascuel, Estimating maximum likelihood phylogenies with PhyML. Methods Mol Biol 537, 113 (2009). 89. A. Rambaut. (2014). 90. R. D. C. Team, R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing (2008). 91. D. H. Haft, J. D. Selengut, O. White, The TIGRFAMs database of protein families. Nucleic Acids Res 31, 371 (Jan 1, 2003). 92. L. S. Johnson, S. R. Eddy, E. Portugaly, Hidden Markov model speed heuristic and iterative HMM search procedure. BMC Bioinformatics 11, 431 (2010).

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