Merozoite surface protein-3 alpha as a genetic marker for epidemiologic studies in Plasmodium vivax: a cautionary note

To generate a global sample of the genetic diversity of MSP-3α [PlasmoDB [36] ID: PVX_097720], all publically available sequences were retrieved. In addition, in order to increase the probability of sampling the most divergent msp-3α alleles, sequences were newly obtained from ten geographically and temporally diverse laboratory isolates from across the parasite’s broad distribution (Figure 1 and Additional file 1). These strains (with their year of isolation, if available) were: North Korean (1953), Indonesia I (1990), Thai III, Vietnam-Palo Alto (before 1978), India VII (2001), Salvador I (1970), Panama I (1969), Brazil I (1994), Mauritania I (1998), and Chesson from New Guinea (1944). To analyse msp-3α diversity at a finer, local scale, sequences of 10 clinical isolates collected in Tumeremo, Venezuela (Bolívar State) in 2003-2004 were also generated. Sequences available in the National Center for Biotechnology Information nucleotide database included 17 sequences of isolates collected from three provinces of Thailand in 2001-2002 by Mascorro et al [16] that allowed assessment of local diversity in another region (accession codes for the P. vivax sequences used are shown in Additional file 1). Combined, a sample of 48 complete P. vivax msp-3α coding sequences was analysed.

To further understand the evolution of the msp-3α gene and its standing diversity, its ortholog in the macaque parasite Plasmodium cynomolgi, the most closely related species to P. vivax known, was amplified, cloned and sequenced from nine strains. The P. cynomolgi samples spanned the species’ South East Asian distribution and included isolates from Peninsular Malaysia (Berok, B Strain, PT1, PT2, Gombak, and Mulligan), Cambodia (Cambodian), Sri Lanka (P. c. ceylonensis), and Burma (RO). Generating population polymorphism data for the gene’s ortholog in a closely related species allows more sophisticated tests of natural selection and the ability to observe the pattern of nucleotide variation in a lineage of the gene unbiased by P. vivax specific demographic events.

Due to the previously known variance in gene length among members of the MSP-3 family of P. vivax (ranging from 1,100 to 3,700 bp) and among MSP-3α alleles, amplification by PCR was performed using two different enzymes in order to increase the probability of amplifying msp-3α alleles that potentially differed in length: AmpliTaq Gold (Applied Biosytems, Roche, USA) and the TaKaRa LA Taq (TaKaRa Mirus Bio Inc, Shiga, Japan) known to better amplify longer products. There were two or more independent reactions per sample. 50 μL PCR reactions included approximately 20 ng of total genomic DNA, 1X PCR buffer, 0.05-0.1 mM of each dNTP, and 0.2-0.4 μM of each primer. Other reagent concentrations and the primers used depended on the polymerase used: TaKaRa reactions included 2.5 U/μM polymerase and primer sequences of 5’ AAG AAA ATT TAC TYS AAA GGS AGT TAA CCG 3’ and 5’ TGT TCT CAA YCG ACA TGC RAA TTR GCT AG 3’; for AmpliTaq Gold reactions, 2.5 mM MgCl₂ and 0.03 U/μM of polymerase was used with the primers 5’ ATG AAA CAC ACC CGC AGC GTC 3’ and 5’ GCT CAA AAA TAG GTG ATT CAT ATC GG 3’. The TaKaRa thermocycle protocol was: an initial denaturation step at 94°C for 1 minute; 35 cycles of denaturation at 94°C for 30 seconds followed by annealing and elongation at 65°C for 3 minutes; and a final elongation step of 10 minutes at 72°C. The AmpliTaq Gold protocol was: an initial denaturation step at 94°C for 4 minutes; 35 cycles of denaturation (94°C for 1 minute), annealing (54-60°C for 1 minute), and elongation (72°C for 2 minutes); and a final elongation step of 10 minutes at 72°C. PCR products were excised from 0.9% agarose gels and purified using the QIAquick DNA gel extraction kit (QIAgen™, Hilden, Germany), then cloned using the pGEM®-T Easy Vector System (Promega, WI, USA). Both strands for multiple clones were sequenced using an Applied Biosystems 3730 capillary sequencer. Sequences generated in this study have the NCBI accession codes: [GenBank: KC935422-KC935447].

In order to calculate genetic diversity and infer phylogenetic relationships, three alignments were constructed: P. vivax and P. cynomolgi species-specific alignments and an interspecies alignment of both P. vivax and P. cynomolgi. Alignment was performed using the MUSCLE algorithm [37] in SeaView4 [38] on translated sequences, followed by manual editing. Phylogenetic relationships were estimated using Bayesian methods as implemented in MrBayes v3.1.2 [39] and using the neighbor joining (NJ) method [40] implemented in MEGA5 v5.05 [41]. The distance based NJ tree was constructed using the Kimura 2-parameter model of nucleotide substitution to calculate a pairwise nucleotide distance matrix, with invariant rates among sites and 1000 bootstrap replicates to assess the confidence of clades. The pairwise distance matrix was also used separately to identify pairs of more similar sequences.

For the Bayesian methods, a general time reversible + gamma + evolutionarily invariable (GTR + G + I) model was used. This model was the one with the fewest parameters to best fit the data as estimated by MEGA5. Bayesian support for nodes was inferred in MrBayes using 42 x 10⁶ Markov Chain Monte Carlo (MCMC) steps, with sampling every 100 generations. 50% of the samples were discarded as burn-in. Convergence is reached after the average standard deviation of the posterior probability is below 0.01 and the value of the potential scale reduction factor is between 1.00 and 1.02 [39].

Genetic diversity among isolates was estimated using the average number of pairwise nucleotide differences per site (overall mean distance, d) in MEGA5. The Jukes-Cantor model of nucleotide substitution was used to correct for multiple mutations when calculating distance, and 1000 bootstrap replicates determined the standard error. To investigate possible signatures of selection, the diversity at synonymous (dS) and nonsynonymous (dN) sites was calculated separately using the Nei-Gojobori method [42] with the Jukes-Cantor correction. To determine whether the estimated difference between dS and dN deviated significantly from the null expectation, the standard error of dS and dN was calculated with 1000 bootstrap replicates and tested using a two-tailed Z test [43], a commonly used evolutionary genetic test (e.g., [44‐48]). A null hypothesis of neutrality was assumed, with the expectation that synonymous and nonsynonymous positions would have similar levels of diversity (dS = dN). Genetic differentiation between subpopulations was estimated using the F_ST statistic [49] as calculated in DnaSP v5 [50]. Two further tests of neutrality were performed in DnaSP, Tajima’s test based on the frequency of variants as summarized by the D statistic [51], and the McDonald and Kreitman test [52] comparing polymorphism and divergence in the P. vivax and P. cynomolgi alignment.

To identify putative recombination events, the RDP algorithm [53] within the RDP3 program [54] was used to perform an automated screen of the P. vivax alignment. Default parameters for the detection of recombination break points and donor-recipient pairs were used with a significance cut-off of 0.05. Only the events where the donor and recipient sequences could be unambiguously identified were recorded.

The PCR-RFLP analysis of Bruce et al [10] that has been frequently used to genotype P. vivax clinical isolates for MSP-3α was simulated in order to compare PCR-RFLP haplotype diversity to the pattern of nucleotide diversity inferred from the set of full length sequences. The protocol is described in Bruce et al [10]. Briefly, msp-3α genotypes are distinguished by variation in gene length due to insertion-deletion (further referred to as indels) mutations and by sequence variability at restriction enzyme cut sites. For the first step, alleles are separated using variation in the size of the product from amplifying the indel rich region of the gene by nested PCR. Sequences of similar length are then further differentiated on the basis of the banding patterns resulting from digestion of the nested PCR product separately with two restriction enzymes (Alu I and Hha I).

To explore this protocol, the nested PCR amplification was simulated in silico for the 48 P. vivax sequences (see Figure 2). The size of the product expected from amplification with the nested primers was used to organize P. vivax sequences into ‘size classes’. Eight of the 48 samples had a single base pair substitution from the nested primers used by Bruce et al [10]. The substitutions’ effects on primer binding are unknown and successful amplification was assumed, allowing all 48 sequences to be assigned to size classes.

In silico digestion was then performed on the sequences using a Perl script (modified from [55]). The positions of enzyme cute sites were recorded for both enzymes for each sequence and the resultant restriction digestion bands were arranged by size (Figure 2). To be conservative and to identify the most PCR-RFLP haplotypes possible, it was assumed that only bands differing by less than ±30 bp would be indistinguishable during electrophoresis, a very fine scale detection limit for agarose gels with a standard DNA ladder (Life Technologies™, Carlsbad, CA manufacturer’s protocol). Unique combinations of Alu I and Hha I digestion bands for samples within a size class were used to create PCR-RFLP haplotypes. Each sequence within a size class was assigned to the corresponding haplotype. As per the Bruce et al. protocol, bands less than 100 bp in length were disregarded when determining haplotypes.

Sequencing and analysis of an augmented sample of P. vivax isolates resulted in identifying substantial indel and coding sequence variation between isolates for the gene encoding MSP-3α (see Table 1). Due to indels ranging from 9 to over 800 bp in length, total gene length varied from 1779 (Thai isolate A12) to 2601 bp (strain Vietnam-Palo Alto). As a consequence, the number of informative, alignable sites for determining the genetic distance and diversity among isolates was much less than the gene length for most isolates (Table 1). As observed before (e.g., [10, 18]), there were numerous sequences with large (over 300 bp) deletion mutations relative to the Salvador-1 reference strain in the N-terminal half of the gene. However, rather than being due to a single, shared deletion event, such shorter alleles were the result of multiple combinations of indels. Indeed, 20 of the 48 sequences had putative deletions of greater than 300 bp, but among these 20 isolates there were 10 different combinations of indel events. Numerous indels of smaller size were segregating among the sequences, resulting in a total of 28 different indel haplotypes, with an average length of 67.3 bp per indel event, in the total P. vivax sample (Table 1).

Sequence polymorphism between isolates varied, but on average there were a high number of segregating (polymorphic) nucleotide sites between isolates in pairwise comparisons (Table 1). Indeed, all P. vivax isolates sampled here could be distinguished by their full length gene sequence. The number of sites differing between isolates ranged from a single missense mutation resulting in an isoleucine to phenylalanine change at residue 74 for two Venezuelan isolates, to over 100 segregating sites for multiple pairs of isolates. The average number of nucleotide differences between sequences in the sample was 38.0, for an average genetic distance of 0.027 ± 0.003 differences per site (Table 1). Diversity was not distributed uniformly in the gene, however, with polymorphism at synonymous sites (dS = 0.044 ± 0.007) significantly higher than at nonsynonymous sites (dN = 0.023 ± 0.003, p = 0.004). This indicates that these aligned sites could be evolving under functional constraints as evidenced by the significant excess of synonymous changes in almost all comparisons from the two-tailed Z test (Table 1).

Diversity among the Thai (d = 0.033 ± 0.002) and Venezuelan (d = 0.031 ± 0.002) clinical isolates was slightly higher but approximated that seen among the 21 geographically and temporally diverse ‘worldwide’ sample of isolates (d = 0.027 ± 0.003). Combining the 27 clinical isolates from these two distant subpopulations with the other 21 diverse isolates did not result in a change in the amount of genetic diversity captured, as average pairwise divergence for the total sample of 48 complete P. vivax msp-3α sequences remained 0.027 ± 0.003 (Table 1).

Next, to investigate the relationship between indel and sequence polymorphism, the genetic diversity among the seven indel haplotypes that were shared by more than one sequence was calculated (see Table 2). This comparison is solely phenetic, since homology cannot be assumed in indels patterns. Evidence that these indels are indeed convergences can be found in the fact that sequences sharing the same indels often had distant geographic origins and could have a higher level of nucleotide polymorphism among themselves (d = 0.007 ± 0.001 to 0.060 ± 0.005) than the average amount of divergence between any two sequences in the global sample (d = 0.027 ± 0.003). This could be partly explained by higher polymorphism at sites within the indel event regions of the gene, as diversity was reduced in the global sample where only the 1413 sites shared by all P. vivax isolates were considered (Table 2). However, it is noteworthy that alleles sharing the same indel haplotype could be more similar to sequences with different indel patterns than they were to each other within the same indel haplotype category at the 1413 comparable sites. An example is strain Thai III (indel haplotype 1), which had only two point mutations relative to Venezuelan isolate 168 (indel haplotype 7), but had five relative to the Chesson strain also with indel haplotype 1. This would indicate either that similar indel or point mutations are repeatedly recurring on different backgrounds or that pre-existing variation is being re-shuffled among and between indel haplotypes.

Another observation was that seven of the 10 Venezuelan clinical isolates constituted indel haplotype 7 and had a substantially reduced level of nucleotide diversity (d = 0.007 ± 0.001) (Table 2) relative to the entire P. vivax sample. This explains the reduced indel haplotype diversity and reduced average indel size seen in the Venezuelan population (Table 1). It is worth noting, however, that despite this cluster of seven similar sequences, the other three Venezuelan sequences were highly divergent. Their inclusion raised the average pairwise divergence within Venezuela to a value approximately equivalent to that of the diverse global sample (Table 1).

With this evidence that local genetic diversity in both Thailand and Venezuela recapitulated the level of global MSP-3α diversity, a standard measure of population differentiation, the F_ST statistic, was used to investigate whether variation at this locus was indeed distributed randomly across populations. The F_ST value, which compares the amount of diversity within populations to the amount of divergence between, for the Venezuelan and Thai sample populations was moderately high likely due to the geographic distance (F_ST = 0.118).

Plasmodium cynomolgi is the closest known relative of P. vivax[56‐58] and it parasitizes simian hosts, mostly of the genus Macaca, in Southeast Asia [56]. Sequencing of its msp-3α ortholog from nine strains revealed extensive nucleotide polymorphism between strains (d = 0.064 ± 0.004), similar to that seen for P. vivax (Table 1). Although the average number of segregating sites among the nine P. cynomolgi sequences was higher than that observed for P. vivax, there was much less indel diversity among the P. cynomolgi strains sampled. The difference between the smallest and largest alleles was only 9 bp as a result, as opposed to a difference of more than 800 bp between some P. vivax isolates. Maximum gene length for P. cynomolgi (1,968 bp) was substantially shorter than for P. vivax (2,601 bp). This observation suggests that P. vivax may have inherited a shorter gene from its common ancestor with P. cynomolgi. In such a case, rather than deletions being responsible for the shorter alleles seen in P. vivax, large insertions in a shorter ancestral sequence would explain the range in allele length. The direction of indel mutations cannot be inferred from alignment, however, and the larger P. vivax alleles have been found repeatedly to be the predominant type (e.g., [13, 26, 30, 31, 33], as was the case in this sample here. Further, diversity among indel haplotypes for sequences with lengths over 2500 bp (indel haplotypes 3-6) varied, but was within the range seen for all haplotype groups in P. vivax (Table 2). Diversity would be expected to be reduced among more recently arisen allele types, but this failed to be the case for either the longer or shorter alleles. Finally, due to the prevalence of indel mutations of varying lengths, gene length varied almost continuously and thus it was not possible to easily identify distinct allele ‘types’ based solely on size in most cases.

As with P. vivax, a significant majority of the diversity seen in P. cynomolgi was constrained to synonymous sites (p = 0.020, Table 1). It is noteworthy, though, that despite it being significantly less than at synonymous sites, diversity at non-synonymous sites in both P. vivax (dN = 0.023 ± 0.003) and P. cynomolgi (dN = 0.058 ± 0.005) was substantial, with amino acid changes accordingly frequent. Intraspecies diversity for MSP-3α was comparable to that seen for other merozoite surface proteins, such as MSP-5 [59], and the 200 L fragment of MSP-1 [60], but was substantially higher than for those thought to be evolving under purifying selection such as MSP-7 [61], MSP-8, and MSP-10 [44].

To further test for evidence of a distribution of genetic variation that deviated from expectations of neutrality and to take advantage of the newly generated set of P. cynomolgi sequences, the test of McDonald and Kreitman was performed using the P. vivax and P. cynomolgi alignment. Although there were substantially more non-synonymous than synonymous differences fixed between species, the ratio of fixed to polymorphic sites did not differ significantly (Fisher’s exact test, p = 0.80), indicating that the observed patterns of divergence were still consistent with neutrality. Tajima’s tests of neutrality for both P. vivax and P. cynomolgi (D = -0.987 and D = 0.392, respectively) were insignificant as well.

In silico simulation of the initial nested PCR step in the PCR-RFLP protocol of Bruce et al [10] resulted in the identification of five different size alleles in the sample of 48 P. vivax sequences (Figure 2). The groups of sequences sharing similarly sized amplicons were termed size classes, and the number of sequences within each varied from 27 in class 1 to just a single isolate (Indonesia-INDO94) in class 5 (Table 3). The expected length of the internal region amplified by nested PCR ranged from 1137 to 1950 bp, consistent with what has been seen previously in locations such as Sri Lanka [32] and Brazil [30]. Alleles with an even larger deletion in the central region of the gene, and thus a smaller amplicon, have been observed in India [26] and Pakistan [24], but none were recovered here. All ten Venezuelan isolates had a predicted nested PCR band size of 1590-1644 bp (gene length: 2232-2295) and were thus in the same class, class 2 (Table 3). However, the geographic origin of the isolates within size classes 1, 3, and 4 (amplicon lengths of 1884-1950, 1449-1461, and 1137-1149 bp respectively) was varied.

In silico digestion of the predicted nested PCR products with Hha I resulted in msp-3α sequences being cut into three to seven bands (Figure 2). Variance among sequences in the size and combinations of these bands, due to sequence diversity at and between cut sites, allowed distinguishing 13 Hha I haplotypes among the 27 sequences in size class 1. Three, two and two different Hha I haplotypes were identified among sequences in size classes 2, 3, and 4, respectively. Sequences also had from 4 to 13 digestion sites for Alu I that resulted in banding patterns that allowed the further discrimination of 10 more msp-3α genotypes, for a total of 31 distinct PCR-RFLP haplotypes from the sample of 48 sequences. As a result, 17 of the 48 sequences were within one of the eight PCR-RFLP haplotypes that was shared by more than one sequence after in silico digestion with both enzymes. This therefore allowed the genetic diversity within these eight haplotypes to be calculated (Table 3). PCR-RFLP analysis, as simulated here, was unable to resolve all samples to distinct genotypes, and in six of the eight PCR-RFLP haplotypes shared by more than one sequence, the sequences within that group originated from distant geographic locations (Table 3).

An initial observation made when comparing these PCR-RFLP alleles to the pattern of nucleotide diversity was that similarly sized amplicons, the basis of distinguishing PCR-RFLP size classes, were often produced by different indel haplotypes. From 27 sequences with gene lengths of 2526-2601 bp (resulting in an 1884-1950 bp amplicon for digestion using the Bruce et al. method) 17 different combinations of indels were observed from sequencing, with indel events averaging 27 bp in length. With numerous indels that averaged a size close to the assumed detection limit of 30 bp segregating into many combinations for class 1, raw amplicon length in many cases simply reflected a convergent sum over these mutation events (indels) and failed to reflect the high levels of MSP-3α polymorphism even within this class. Further, as shown in Table 3, nucleotide diversity within PCR-RFLP size classes (d = 0.024 ± 0.002 to 0.052 ± 0.003) varied, but approximated the amount of diversity seen in the total sample population of globally diverse isolates (d = 0.027 ± 0.003, see Table 1).

All ten Venezuelan MSP-3α isolates fell within the same size class, and included a cluster of 5 isolates (haplotype 2.1) that had markedly less nucleotide diversity (d = 0.002 ± 0.001) than the other PCR-RFLP alleles found in 2 or more samples (d = 0.016 ± 0.002 and 0.058 ± 0.005). However, this reduced PCR-RFLP allelic diversity in Venezuela contradicts that the total nucleotide diversity retained within the Venezuelan sample population was similar to that of Thailand, and the entire sample combined (Table 1), despite that those two groups had much greater allelic diversity. More evidence that MSP-3α genetic variation was not structured by PCR-RFLP haplotypes was that isolates with identical banding patterns, such as PCR-RFLP haplotype 1.1 (Table 3), could again exhibit more average pairwise divergence (d = 0.043 ± 0.003) than was seen on average between sequences in the entire sample (d = 0.027 ± 0.003, Table 1).

Additionally, bands of similar size, especially in the simulated Alu I digestion, were not always homologous. As an example, an isolate from Borneo ([GenBank: AY118174]) [12] and the Panama I strain ([GenBank: KC935447]) had an identical PCR-RFLP haplotype (haplotype 1.8), but converged to an indistinguishable banding pattern through similar sized, but non-homologous bands (Figure 3). From Alu I digestion, the isolate from Borneo had two bands of 258 bp, each coming from differing regions of the msp-3α gene, while the sequence of Panama I had a single band at that size (Figure 3). In turn, the Panama I isolate had six different bands between 146 and 186 bp in length. Such bands would migrate similarly during electrophoresis and thus the two highly divergent sequences could not be distinguished. Indeed, the amount of pairwise difference between these two sequences with the same PCR-RFLP haplotype (d = 0.058 ± 0.005) was almost twice that of the average amount of difference between two sequences in the sample. As a result, PCR-RFLP analysis would have failed to reflect the high level of genetic divergence between the isolates, a subsequent example of identity by state, but not by descent.

The Alu I digestion for 47 of the 48 samples resulted in sequences having two or more different digestion fragments from different regions of the gene that differed by less than the ±30 bp discrimination limit. In three additional cases to the one above, identical Alu I haplotypes would have been inferred erroneously after electrophoresis due to coincident size in non-homologous bands for divergent sequences. Multiple digestion bands coinciding to have a similar size resulted from the Hha I digestion much less frequently, only occurring for six of the 48 isolates; however, similar Hha I banding patterns also often failed to correlate to similarity at the nucleotide sequence level. For example, the average pairwise diversity for three Hha I RFLP haplotypes (haplotypes H1.1, H1.4, and H2.2) was 0.046 ± 0.003, 0.056 ± 0.005, and 0.064 ± 0.005, respectively, all values much greater than was seen on average between sequences chosen randomly (d = 0.027 ± 0.003).

Next, to attempt to resolve relationships among PCR-RFLP haplotypes and geographically dispersed isolates, a pairwise distance matrix of the 48 complete sequences was constructed so as to identify pairs of more similar sequences. Figure 4A shows the tree constructed from the pairwise distance matrix using the NJ method, rooted with P. cynomolgi, with isolates labeled by their geographic origin and PCR-RFLP haplotype. Few nodes had greater than 50% bootstrap support, and the topology differed in the tree constructed using Bayesian phylogenetic inference (Figure 4B). The lack of resolution in the Bayesian analysis was especially apparent, as the standard deviation of the posterior probability did not drop below 0.25 even after allowing 42 million MCMC generations. This signifies statistically robust convergence was not reached for the topology displayed [39].

However, an observation from the pairwise distance matrix and one consistent across both phylogenetic methods was that there was no discernible pattern of more similar sequences having a closer geographic origin or more similar PCR-RFLP haplotype. Although five of the Venezuelan clinical isolates that shared the same PCR-RFLP haplotype (haplotype 2.1) grouped together, other Venezuelan sequences obtained from Bolívar State were distributed throughout the tree in both the Bayesian and NJ analyses (Figure 4). Furthermore, the sequences of these clinical isolates collected in Venezuela in 2003-2004 were often most similar to isolates collected distantly in time and space. Examples include Venezuelan isolate 1060, which was most similar to an isolate collected in Brazil before 1988 and an Indonesian isolate from 1990. Another example, Venezuelan isolate 166, grouped with a Vietnamese isolate from before 1978 (Figure 4). The relationship of Venezuelan isolate 1081 to the other P. vivax sequences was difficult to infer, with it having the least genetic distance to two Thai clinical isolates from 2000-2001 in the NJ analysis, but situating within a large, unresolved polytomy in the Bayesian tree (Figure 4). Further, the inability to resolve the cluster of five Venezuelan clinical isolates (isolates 109, 120, 127, 164, 167, and 168) from distant strains (e.g., Panama I, Chesson from New Guinea, Thai III) in either tree (Figure 4) makes it questionable whether MSP3-alpha could be used to resolve relationships at a finer scale. For epidemiologic efforts applied at a local scale, this may preclude using phylogenetics to accurately determine the origins of isolates or migration events with MSP-3α.

Frequent recombination at the MSP-3α locus between diverse ancestral alleles whose variation predates the migration events that led to the sample population’s current geographic distribution would be consistent with the lack of geographic patterning seen (Figure 4). To investigate this, the RDP algorithm was used to screen the alignment of P. vivax msp-3α sequences for recombination events. Recombination events were noticeable by eye in the alignment, where isolates that shared the same variant peptide sequence at a region with divergence in the population in one position in the alignment were often more similar to different sequences at other polymorphic regions in the alignment (Figure 5). An illustrative example is strain Mauritania I (isolated in 1998), which shared the divergent KEGSN peptide beginning at residue 65 with 5 other isolates (Figure 5). None of the Venezuelan isolates had the KEGSN motif, instead they had a sequence of NESSD. However, downstream at residue 213, two Venezuelan isolates (166 and 1060) shared the T(N/S)AEK peptide with Mauritania I and 19 other isolates, not the KKAKS sequence seen for other samples (Figure 5). Then, at residue 609, Mauritania I and in this case Venezuelan isolates 105 and 1060 had the KE motif shared by 18 isolates, while Venezuelan isolate 166 had the TA peptide shared by the other 30 of the 48 isolates (Figure 5).

This is the pattern expected by recombination shuffling the variation at these genic regions into multiple combinations, and numerous events were detected by the RDP algorithm as well. Recombination was commonly detected between isolates from different geographic regions, between isolates in different PCR-RFLP size classes, and throughout the phylogeny (Figure 4). Frequent recombination would also explain the lack of phylogenetic resolution observed and the failure for Bayesian analysis to reach convergence on an optimal topology. Indeed, Mascorro et al [16] showed previously that isolates segregated differently into phylogenies made from four different regions of msp-3α, indicating different segments of haplotypes have different evolutionary histories and thus yield a conflicting phylogenetic signal for the entire gene. As a result of recombination acting to shuffle genetic variation at this locus, circulating msp-3α haplotypes are then complex composites of multiple segments that have each likely been differentially shaped by their respective population of origin’s demography or selective environment. This seems a probable explanation for the inability to resolve relationships between distant P. vivax isolates, despite recovering a high number of segregating sites.