BACKGROUND
malaria is a globally important disease which exhibits major differences in local epidemiology and ecology, with great variation within africa where most cases are caused by plasmodium falciparum  <cit> .
local differences in selection by infectious diseases has impacted on human genetic variation  <cit> , and malaria parasites have played a significant role in this  <cit> .
conversely, selection operating on parasites must vary locally due to varying transmission ecology, differences in innate susceptibility of mosquito or human populations, degrees of acquired immunity in humans, or drug pressure.
malaria parasites in highly endemic regions are subject to competition due to superinfection by different genotypes, and are subject to strong acquired immune responses, whereas low endemicity requires more prolonged maintenance of asexual parasite infection as there are only rare opportunities for transmission of sexual stages during seasons when mosquitoes are present  <cit> .
malaria parasites have a compact ~ <dig> megabase haploid genome of  <dig> chromosomes containing more than  <dig>  genes  <cit> , with a high recombination rate  in a brief diploid stage that occurs after parasite mating in the mosquito each transmission cycle  <cit> , and thus should allow relatively efficient mapping of signatures of natural selection  <cit> .
recent genome-wide analyses of plasmodium falciparum revealed significant global population structure  <cit> , consistent with previous microsatellite genotyping surveys that indicated substantial divergence between southeast asia and africa, and slight differentiation between east and west africa  <cit> .
local subdivisions in p. falciparum population structure in southeast asia may be important for understanding the current emergence of artemisinin antimalarial drug resistance .
in contrast, there is little evidence of parasite population subdivision within the large endemic region of west africa  <cit> , although infection endemicity ranges from very high in the south where there is abundant rainfall to low in the north where rainfall is limited  <cit> .
initial analyses to scan for loci under selection within west africa have been performed on populations sampled from senegal  <cit> , the gambia , and guinea  <cit> .
differences between populations are evident in signatures of selection surrounding genes involved with chloroquine  and anti-folate  resistance that reflect differences in historical drug use among the countries  <cit> .
direct comparison between a highly endemic population in guinea and a population with lower endemicity in the gambia indicated another locus with alleles at highly differentiated frequencies, containing the gametocyte development gene gdv <dig> that is essential for parasite transmission  <cit> .
however, more local differences between parasite populations within an endemic country in africa have not yet been investigated by genome-wide surveys with adequate sample sizes to detect differences in selective signatures.
to test for selection that might occur due to varying patterns of transmission seasonality, varying antimalarial drug use, or other causes, p. falciparum population samples were analysed from two different areas of ghana .
clinical isolates from p. falciparum malaria patients at two sites separated by ~350 km were sequenced and the resulting high quality genome sequence data from  <dig> isolates were analysed.
malaria transmission in kintampo, in the central forest-savannah transitional zone, occurs for much of each year  <cit> , while navrongo, in the sudan savannah zone near the northern border with burkina faso, experiences more markedly seasonal transmission  <cit> .
major mosquito vectors in ghana include an.
gambiae sensu stricto , an.
coluzzii , an.
arabiensis, and an.
funestus  <cit> , with survey data suggesting that an.
arabiensis and an.
coluzzii may be more common in the north than in central areas of the country  <cit> .
the overall genome-wide snp patterns in the two p. falciparum populations analysed here were not significantly different, and most of the strong selective signatures were evident in both populations, several of these signatures mapping to loci encoding known targets of antimalarial drugs and immunity.
however, haplotype-based tests indicated a small number of chromosomal regions at which signatures of recent directional selection were stronger in one or other population, and these putatively contain one or more genes under locally varying selection with potential relevance to malaria control.fig.
<dig> location of sites in ghana at which malaria infections were sampled for study of plasmodium falciparum genome sequences.
the epidemiology of malaria has been previously described in kintampo  <cit>  and navrongo  <cit> , which are separated by approximately 350 km.
the figure incorporates graphics from d-maps.com  and the malaria atlas project   <cit>
RESULTS
parasite allele frequency distributions and within-host infection diversity
illumina short read sequence data obtained from  <dig> of the clinical infection isolates  were mapped to the 3d <dig> p. falciparum reference genome sequence, enabling high quality genome-wide snp calling and analysis for  <dig> isolates  passing the quality filtering described in the methods .
this identified  <dig>  biallelic snps in the combined population, with majority read allele calls for all isolates across a total of  <dig>  positions, and <5 % missing isolate data for the remaining  <dig>  snps.
for  <dig>  snps  the minor frequency allele in the total population sample was observed as the majority read allele for only a single isolate, similar to proportions in previously examined west african populations with approximately similar sample sizes  <cit> , leaving  <dig>  non-singleton snps in the overall dataset.
the majority  of all snps were observed within genes, as a more extreme a + t bias within intergenic regions  restricts ability to map reads uniquely  <cit> .
the within-infection diversity in each isolate was assessed using the fws fixation index  <cit> .
the distribution of fws scores was similar across isolates in each local population, ranging from  <dig>  -  <dig>   in kintampo, and from  <dig>  -  <dig>   in navrongo .
the proportions of isolates with fws scores above  <dig> , indicating infections dominated by single genotypes at the time of sampling, were  <dig>  of  <dig> isolates in kintampo and  <dig>  of  <dig> isolates in navrongo.
within each isolate, the majority allele at each snp  was counted towards analyses of population-based allele frequencies for the following tests.fig.
<dig> within-infection f
ws fixation indices for each clinical isolate with genome-wide snp data from kintampo  and navrongo , ordered by increasing index value.
the distribution of f
ws values was not significantly different between the populations .
the horizontal line marks f
ws =  <dig> , above which an isolate may be considered to contain a single predominant genotype;  <dig>  of  <dig> isolates in kintampo and  <dig>  of  <dig> isolates in navrongo
the snp allele frequency spectra in each of the two local populations were examined by calculation of tajima’s d value for the  <dig>  genes with  <dig> or more snps in at least one of the two populations .
there was a strong correlation between the two populations  in the distributions of tajima’s d values across all genes .
tajima’s d values of above  <dig>  were observed for  <dig> genes in kintampo and  <dig> genes in navrongo, with  <dig> of these genes having values of above  <dig>  in both populations .
reference genomic map positions and annotations may be viewed on plasmodb   <cit>  using the gene id numbers.fig.
<dig> correlation of tajima’s d indices shows similar overall genome-wide allele frequency distributions of snps within genes in each of the two ghanaian population samples, kintampo  and navrongo .
the scatterplot compares tajima’s d indices in both populations for each of  <dig>  genes containing  <dig> or more snps
testing for differentiation between populations
principal component analysis using the  <dig>  snps with no missing data in the complete dataset of  <dig> isolates did not show any separation between isolates sampled from the kintampo and navrongo populations .
to scan for individual snps which might have allele frequency differentiation between the populations, the fixation index fst was calculated for each of the  <dig>  non-singleton snps genome-wide .
there was minimal differentiation between populations , and only  <dig> snps had fst values above  <dig> , with the highest fst value being  <dig>  .
the observed numbers of snps with fst values above  <dig>  was not significantly different from null expectations due to sampling variance .fig.
<dig> genome-wide f
st scan for differentiation between the kinpampo  and navrongo  population samples, for each of  <dig>  non-singleton snps genome-wide.
the green line indicates the genome-wide mean f
st =  <dig> .
the positions of snps with f
st values above  <dig>  are shown in additional file 1: table s <dig>  but the numbers of these did not differ from null expectations based on sampling from a single pooled population
evidence of directional selection within local populations
to identify loci putatively under recent positive selection, indices of long range haplotypes were analysed.
using the standardised integrated haplotype score  for all snps above allele frequencies of 5 % in the combined population sample from ghana  identified  <dig> chromosomal regions with two or more snps above the top  <dig>  % of the randomly expected distribution  and at least  <dig> snp with |ihs| >  <dig> .
seven of these regions were also identified in both of the local population samples when applying this cut-off for data from kintampo and navrongo separately , with a further  <dig> of the regions observed when a slightly less stringent cut-off was applied  as may be appropriate given the lower sample sizes in the separate populations compared with the combined population.
the largest and most strongly supported region of elevated |ihs| values extended over approximately 303 kb on chromosome  <dig> .
another region of high |ihs| values in both population samples covers approximately 50 kb on chromosome  <dig>  having core snps within the ama <dig> antigen gene.
both of these regions were also detected in analyses of other west african population samples  <cit> .
elevated |ihs| values were observed around the antifolate drug target genes dhfr and dhps, as well as the chloroquine resistance transporter gene crt, although it was only in the case of dhps that a signature covered the gene in both of the local population samples.
there was no detectable signature around the multi-drug resistance mdr <dig> gene .
a small number of regions had elevated |ihs| values detected in only one or other of the two populations , or had an apparently stronger signature in one population than the other, but some of these few apparent differences may be due to random sampling variation.fig.
<dig> scan for evidence of recent directional selection by analysis of the standardised integrated haplotype score |ihs| for individual snps in kintampo  and navrongo .
additional file 1: table s <dig> gives the co-ordinates and genes in  <dig> chromosomal regions containing windows of contiguous extended haplotype homozygosity attributed to two or more snps within the top  <dig>  % of the expected distribution , with at least  <dig> snp having |ihs| > 5 in these regions for the combined ghana population.
most of these chromosomal regions are similar in both populations, as shown schematically in figure 6
fig.
<dig> chromosomal regions with elevated |ihs| values within the combined ghana population , and within each of the local populations .
red shading indicates windows containing two or more snps within the top  <dig>  % of the expected distribution  and at least  <dig> snp with a |ihs| value >  <dig>  blue shading indicates windows containing two or more snps with |ihs| >  <dig>  but none having a value >  <dig>  the regions, and genes within them, are listed in additional file 1: table s <dig>  the location of four drug resistance genes mentioned in the text are shown with green markers: dhfr , mdr <dig> , crt  and dhps .
asterisks show the positions of three elevated |ihs| windows for which the rsb analysis  indicated stronger evidence of selection in one or other of the two populations
to test for evidence of potential selective differences using an alternative approach, the cross population rsb metric  <cit>  was applied to compare the average haplotype length associated with each snp in the two populations.
this identified four chromosomal loci with population-specific evidence of directional selection, one in kintampo and three in navrongo .
the rsb signature for kintampo within chromosome  <dig> spans a region of 22 kb covering  <dig> genes.
the rsb signatures for navrongo within chromosomes  <dig>   <dig> and  <dig> span 12 kb , 38 kb  and 48 kb  respectively.fig.
<dig> scan for evidence of population specific directional selection using the rsb cross-population test for extended haplotypes.
positive values indicate snps associated with longer haplotypes in the kintampo population sample, whereas negative values indicate snps associated with longer haplotypes in the navrongo population sample.
one region contains at least  <dig> snps with rsb values above  <dig> indicating a potentially stronger signature of selection in kintampo on chromosome  <dig> while  <dig> regions contain at least  <dig> snps with rsb values below − <dig> indicating a potentially stronger signature of selection in navrongo on chromosomes  <dig>   <dig> and  <dig> .
the regions, and genes within them, are detailed in fig.
<dig> and additional file 1: table s <dig>  three of these four regions were associated with high |ihs| windows in both populations
three of the four rsb signatures  coincide with regions that had high |ihs| signatures .
the genes within these regions are shown in fig.
<dig> and listed in additional file 1: table s <dig>  further available information on these are accessible using the plasmodb browser   <cit> .
the functions of most of these genes are unknown, but some have been previously characterised and are potential targets of differential selection .
for example, the region on chromosome  <dig> contains the trap gene  encoding the thrombospondin related adhesive protein, also known as sporozoite surface protein  <dig>  which is vital for hepatocyte cell invasion and is a leading vaccine candidate antigen to which some naturally acquired immune responses are allele specific  <cit> .
this chromosome  <dig> region is also immediately adjacent to the rh2a, rh2b, and msp <dig> genes encoding polymorphic merozoite proteins involved in erythrocyte invasion.
the region on chromosome  <dig> contains the crmp <dig> gene  encoding the cysteine repeat modular protein  <dig> which is essential for mosquito transmission  <cit> , adjacent to the ccp <dig> gene  encoding an lccl  domain-containing protein expressed in parasite gametocytes  <cit> .
the region on chromosome  <dig> is in close proximity to the sub-telomere and contains paralogous copies of genes putatively encoding acyl-coa binding proteins  as well as single members of the phist and hyp multigene families.fig.
<dig> locations of genes within three narrow genomic regions of p. falciparum that had significant rsb scores indicating differences in selection between kintampo and navrongo populations, also coinciding with regions of high |ihs| values in both population samples.
graphics are extracted as snapshots from the respective chromosomal regions in the genome browser at www.plasmodb.org.
the chromosomal sequence co-ordinates are shown below each map in kilobase scale.
bars above each map show the extent of the rsb signatures .
particular genes noted in the results text are boxed for illustration
discussion
the overall population genetic structure of parasites at both of the endemic sites sampled in ghana was very similar despite differences in transmission seasonality and local vector species abundances.
the within-isolate fixation index fws, which is inversely related to the level of genomic complexity per infection, had a similar range of values in each population.
the overall levels of within-infection parasite genomic complexity in each of the populations sampled here are similar to those estimated for other highly endemic west african populations in burkina faso, mali, and guinea, and higher than for a population of lower endemicity in the gambia  <cit> .
regarding the individual values, it is worth noting that although an infection with fws value close to  <dig>  is dominated by a single population of identical or closely related parasites  <cit> , diverse parasite genotypes may exist at low levels or sequestered in organ capillaries which may become abundant in the peripheral blood after a few hours or days  <cit> .
similar overall allele frequency distributions observed in both of the sampled populations was not surprising, given their proximity  and a high level of gene flow generally among west african malaria parasite populations  <cit> .
genome-wide fst values between the populations were very low, with the small proportion of snps having fst values above  <dig>  not departing from random expectations accounting for sampling variance.
a previous comparison of two west african parasite populations with different levels of endemicity  also showed very low fst values throughout most of the genome  <cit> .
however, there was exceptional differentiation between the guinea and gambia populations over a 15 kb region of chromosome  <dig> incorporating the gametocyte development  <dig> gene   <cit>  which is responsible for early initiation of gametocyte development and vital for enabling parasite infection of mosquitoes  <cit> .
there was no differentiation at this locus between the two populations in ghana studied here .
in contrast, haplotype-based approaches indicated that selection has probably been operating on multiple other loci.
of the  <dig> genomic windows containing elevated |ihs| scores, marking loci likely to have been under recent directional selection in ghana,  <dig> were observed in both of the local population samples.
the most significant window identified a large region located towards one end of chromosome  <dig>  as observed previously in other population samples from senegal, the gambia, and guinea  <cit> , although the mechanism and target of selection remains unknown.
another strong signal was due to core snps in the ama <dig> gene on chromosome  <dig>  encoding an important merozoite target of acquired immunity with extensive sequence polymorphism.
this is likely to be subject to occasional positive selection of new alleles added to the repertoire of existing alleles maintained by balancing selection  <cit> .
strong |ihs| signatures around three major drug resistance genes  highlight the role of selection by antimalarial treatment across ghana.
strongest selection on these genes will have occurred when chloroquine and pyrimethamine-sulphadoxine were widely used in antimalarial therapy in ghana, until artemisinin combination therapy  replaced them as official first line treatment in  <dig>  and there may have been local differences in the decay of these signatures following the introduction of act.
it is also possible that there is some limited ongoing selection, due to use of sulphadoxine – pyrimethamine for intermittent preventive treatment of malaria in pregnancy  <cit> , continued use of chloroquine in some areas despite its proscription  <cit>  or selection by act partner drugs which have included amodiaquine and lumefantrine in ghana.
three regions, on chromosomes  <dig>   <dig> and  <dig>  with high |ihs| values in both of the local populations here were also marked as having stronger evidence of extended haplotypes in one or other of the populations by the rsb cross-population analysis.
this has yielded windows that each contain candidate genes that may be subject to population-specific differences in selection, including the trap gene which encodes a vaccine-candidate target of immunity, genes involved in parasite development in mosquitoes, and members of variant-expressed multigene families.
haplotype based tests of directional selection on malaria parasites in west africa have previously identified selection occurring over very recent time frames, with strong signatures linked to the use of chloroquine and the antifolate drugs sulphadoxine-pyrimethamine as first line malaria therapies  <cit> .
decay of these signatures following changes in drug use appears to be rapid  <cit> , facilitated by the high recombination rate of p. falciparum.
our results here, using a combination of the |ihs| and the rsb tests, provide evidence of subtle differences in local selection signatures in the two ghanaian populations that are likely to represent ongoing or very recent selection events.
future studies to investigate selection operating over longer time frames, and heterogeneity among more distantly related populations with potentially differing demography, may employ additional tests including coalescent-based approaches .
studies to conduct genome-wide analyses on malaria parasites from a larger number of populations sampled across heterogeneous environments are likely to be able to refine the mapping of loci influenced by local selective processes, and would also indicate the geographical scales of such signatures of selection.
coupling these approaches with epidemiological analyses, including further details of local transmission by different vector species, should facilitate future studies to not only detect signatures of selection but identify the causal processes underlying them.
CONCLUSIONS
this study compares population structure and genomic signatures of selection on malaria parasites in two closely situated but ecologically contrasting endemic areas.
parasite genome sequencing yielded high coverage of more than  <dig>  snps for population genomic analyses of  <dig> clinical infection isolates from an area with malaria transmission for most of each year, and  <dig> isolates from an area with seasonally restricted transmission.
the local populations had similar profiles in most respects, particularly with regard to overall snp allele frequency distributions, and proportions of mixed genotype infections.
eleven different chromosomal regions showed elevated integrated haplotype scores in both populations, but another metric comparing extended haplotype homozygosity  indicated differences in the strength of the signal between the populations for three of these chromosomal regions.
a stronger signal was detected within one population for a narrow region of chromosome  <dig>  whereas a stronger signal was seen in the other population for small regions on chromosomes  <dig> and  <dig>  these include genes that are potential targets of locally varying recent selection, such as those involved in parasite development in mosquitoes, members of variant-expressed multigene families, and a leading vaccine-candidate target of immunity.
even in a situation of closely situated populations with very similar population structure and shared selection signatures, analysis across heterogeneous environments has potential to refine the mapping of important loci under temporally or spatially varying selection, including those of potential relevance to epidemiology and control of infection.

 malaria is a globally important disease which exhibits major differences in local epidemiology and ecology, with great variation within africa where most cases are caused by plasmodium falciparum. local differences in selection by infectious diseases has impacted on human genetic variation, and malaria parasites have played a significant role in this. conversely, selection operating on parasites must vary locally due to varying transmission ecology, differences in innate susceptibility of mosquito or human populations, degrees of acquired immunity in humans, or drug pressure. malaria parasites in highly endemic regions are subject to competition due to superinfection by different genotypes, and are subject to strong acquired immune responses, whereas low endemicity requires more prolonged maintenance of asexual parasite infection as there are only rare opportunities for transmission of sexual stages during seasons when mosquitoes are present. malaria parasites have a compact ~ megabase haploid genome of chromosomes containing more than genes, with a high recombination rate in a brief diploid stage that occurs after parasite mating in the mosquito each transmission cycle, and thus should allow relatively efficient mapping of signatures of natural selection. recent genome-wide analyses of plasmodium falciparum revealed significant global population structure, consistent with previous microsatellite genotyping surveys that indicated substantial divergence between southeast asia and africa, and slight differentiation between east and west africa. local subdivisions in p. falciparum population structure in southeast asia may be important for understanding the current emergence of artemisinin antimalarial drug resistance. in contrast, there is little evidence of parasite population subdivision within the large endemic region of west africa, although infection endemicity ranges from very high in the south where there is abundant rainfall to low in the north where rainfall is limited. initial analyses to scan for loci under selection within west africa have been performed on populations sampled from senegal, the gambia, and guinea. differences between populations are evident in signatures of selection surrounding genes involved with chloroquine and anti-folate resistance that reflect differences in historical drug use among the countries. direct comparison between a highly endemic population in guinea and a population with lower endemicity in the gambia indicated another locus with alleles at highly differentiated frequencies, containing the gametocyte development gene gdv that is essential for parasite transmission. however, more local differences between parasite populations within an endemic country in africa have not yet been investigated by genome-wide surveys with adequate sample sizes to detect differences in selective signatures. to test for selection that might occur due to varying patterns of transmission seasonality, varying antimalarial drug use, or other causes, p. falciparum population samples were analysed from two different areas of ghana. clinical isolates from p. falciparum malaria patients at two sites separated by ~350 km were sequenced and the resulting high quality genome sequence data from isolates were analysed. malaria transmission in kintampo, in the central forest-savannah transitional zone, occurs for much of each year, while navrongo, in the sudan savannah zone near the northern border with burkina faso, experiences more markedly seasonal transmission. major mosquito vectors in ghana include an. gambiae sensu stricto, an. coluzzii, an. arabiensis, and an. funestus, with survey data suggesting that an. arabiensis and an. coluzzii may be more common in the north than in central areas of the country. the overall genome-wide snp patterns in the two p. falciparum populations analysed here were not significantly different, and most of the strong selective signatures were evident in both populations, several of these signatures mapping to loci encoding known targets of antimalarial drugs and immunity. however, haplotype-based tests indicated a small number of chromosomal regions at which signatures of recent directional selection were stronger in one or other population, and these putatively contain one or more genes under locally varying selection with potential relevance to malaria control.fig. location of sites in ghana at which malaria infections were sampled for study of plasmodium falciparum genome sequences. the epidemiology of malaria has been previously described in kintampo and navrongo, which are separated by approximately 350 km. the figure incorporates graphics from d-maps.com and the malaria atlas project  parasite allele frequency distributions and within-host infection diversity illumina short read sequence data obtained from of the clinical infection isolates were mapped to the 3d p. falciparum reference genome sequence, enabling high quality genome-wide snp calling and analysis for isolates passing the quality filtering described in the methods. this identified biallelic snps in the combined population, with majority read allele calls for all isolates across a total of positions, and <5 % missing isolate data for the remaining snps. for snps the minor frequency allele in the total population sample was observed as the majority read allele for only a single isolate, similar to proportions in previously examined west african populations with approximately similar sample sizes, leaving non-singleton snps in the overall dataset. the majority of all snps were observed within genes, as a more extreme a + t bias within intergenic regions restricts ability to map reads uniquely. the within-infection diversity in each isolate was assessed using the fws fixation index. the distribution of fws scores was similar across isolates in each local population, ranging from - in kintampo, and from - in navrongo. the proportions of isolates with fws scores above, indicating infections dominated by single genotypes at the time of sampling, were of isolates in kintampo and of isolates in navrongo. within each isolate, the majority allele at each snp was counted towards analyses of population-based allele frequencies for the following tests.fig. within-infection f ws fixation indices for each clinical isolate with genome-wide snp data from kintampo and navrongo, ordered by increasing index value. the distribution of f ws values was not significantly different between the populations. the horizontal line marks f ws = , above which an isolate may be considered to contain a single predominant genotype; of isolates in kintampo and of isolates in navrongo the snp allele frequency spectra in each of the two local populations were examined by calculation of tajima’s d value for the genes with or more snps in at least one of the two populations. there was a strong correlation between the two populations in the distributions of tajima’s d values across all genes. tajima’s d values of above were observed for genes in kintampo and genes in navrongo, with of these genes having values of above in both populations. reference genomic map positions and annotations may be viewed on plasmodb using the gene id numbers.fig. correlation of tajima’s d indices shows similar overall genome-wide allele frequency distributions of snps within genes in each of the two ghanaian population samples, kintampo and navrongo. the scatterplot compares tajima’s d indices in both populations for each of genes containing or more snps testing for differentiation between populations principal component analysis using the snps with no missing data in the complete dataset of isolates did not show any separation between isolates sampled from the kintampo and navrongo populations. to scan for individual snps which might have allele frequency differentiation between the populations, the fixation index fst was calculated for each of the non-singleton snps genome-wide. there was minimal differentiation between populations, and only snps had fst values above, with the highest fst value being. the observed numbers of snps with fst values above was not significantly different from null expectations due to sampling variance.fig. genome-wide f st scan for differentiation between the kinpampo and navrongo population samples, for each of non-singleton snps genome-wide. the green line indicates the genome-wide mean f st = . the positions of snps with f st values above are shown in additional file 1: table s but the numbers of these did not differ from null expectations based on sampling from a single pooled population evidence of directional selection within local populations to identify loci putatively under recent positive selection, indices of long range haplotypes were analysed. using the standardised integrated haplotype score for all snps above allele frequencies of 5 % in the combined population sample from ghana identified chromosomal regions with two or more snps above the top  % of the randomly expected distribution and at least snp with |ihs| > . seven of these regions were also identified in both of the local population samples when applying this cut-off for data from kintampo and navrongo separately, with a further of the regions observed when a slightly less stringent cut-off was applied as may be appropriate given the lower sample sizes in the separate populations compared with the combined population. the largest and most strongly supported region of elevated |ihs| values extended over approximately 303 kb on chromosome. another region of high |ihs| values in both population samples covers approximately 50 kb on chromosome having core snps within the ama antigen gene. both of these regions were also detected in analyses of other west african population samples. elevated |ihs| values were observed around the antifolate drug target genes dhfr and dhps, as well as the chloroquine resistance transporter gene crt, although it was only in the case of dhps that a signature covered the gene in both of the local population samples. there was no detectable signature around the multi-drug resistance mdr gene. a small number of regions had elevated |ihs| values detected in only one or other of the two populations, or had an apparently stronger signature in one population than the other, but some of these few apparent differences may be due to random sampling variation.fig. scan for evidence of recent directional selection by analysis of the standardised integrated haplotype score |ihs| for individual snps in kintampo and navrongo. additional file 1: table s gives the co-ordinates and genes in chromosomal regions containing windows of contiguous extended haplotype homozygosity attributed to two or more snps within the top  % of the expected distribution, with at least snp having |ihs| > 5 in these regions for the combined ghana population. most of these chromosomal regions are similar in both populations, as shown schematically in figure 6 fig. chromosomal regions with elevated |ihs| values within the combined ghana population, and within each of the local populations. red shading indicates windows containing two or more snps within the top  % of the expected distribution and at least snp with a |ihs| value >  blue shading indicates windows containing two or more snps with |ihs| >  but none having a value >  the regions, and genes within them, are listed in additional file 1: table s the location of four drug resistance genes mentioned in the text are shown with green markers: dhfr, mdr, crt and dhps. asterisks show the positions of three elevated |ihs| windows for which the rsb analysis indicated stronger evidence of selection in one or other of the two populations to test for evidence of potential selective differences using an alternative approach, the cross population rsb metric was applied to compare the average haplotype length associated with each snp in the two populations. this identified four chromosomal loci with population-specific evidence of directional selection, one in kintampo and three in navrongo. the rsb signature for kintampo within chromosome spans a region of 22 kb covering genes. the rsb signatures for navrongo within chromosomes and span 12 kb, 38 kb and 48 kb respectively.fig. scan for evidence of population specific directional selection using the rsb cross-population test for extended haplotypes. positive values indicate snps associated with longer haplotypes in the kintampo population sample, whereas negative values indicate snps associated with longer haplotypes in the navrongo population sample. one region contains at least snps with rsb values above indicating a potentially stronger signature of selection in kintampo on chromosome while regions contain at least snps with rsb values below − indicating a potentially stronger signature of selection in navrongo on chromosomes and. the regions, and genes within them, are detailed in fig.  and additional file 1: table s three of these four regions were associated with high |ihs| windows in both populations three of the four rsb signatures coincide with regions that had high |ihs| signatures. the genes within these regions are shown in fig.  and listed in additional file 1: table s further available information on these are accessible using the plasmodb browser. the functions of most of these genes are unknown, but some have been previously characterised and are potential targets of differential selection. for example, the region on chromosome contains the trap gene encoding the thrombospondin related adhesive protein, also known as sporozoite surface protein which is vital for hepatocyte cell invasion and is a leading vaccine candidate antigen to which some naturally acquired immune responses are allele specific. this chromosome region is also immediately adjacent to the rh2a, rh2b, and msp genes encoding polymorphic merozoite proteins involved in erythrocyte invasion. the region on chromosome contains the crmp gene encoding the cysteine repeat modular protein which is essential for mosquito transmission, adjacent to the ccp gene encoding an lccl domain-containing protein expressed in parasite gametocytes. the region on chromosome is in close proximity to the sub-telomere and contains paralogous copies of genes putatively encoding acyl-coa binding proteins as well as single members of the phist and hyp multigene families.fig. locations of genes within three narrow genomic regions of p. falciparum that had significant rsb scores indicating differences in selection between kintampo and navrongo populations, also coinciding with regions of high |ihs| values in both population samples. graphics are extracted as snapshots from the respective chromosomal regions in the genome browser at www.plasmodb.org. the chromosomal sequence co-ordinates are shown below each map in kilobase scale. bars above each map show the extent of the rsb signatures. particular genes noted in the results text are boxed for illustration discussion the overall population genetic structure of parasites at both of the endemic sites sampled in ghana was very similar despite differences in transmission seasonality and local vector species abundances. the within-isolate fixation index fws, which is inversely related to the level of genomic complexity per infection, had a similar range of values in each population. the overall levels of within-infection parasite genomic complexity in each of the populations sampled here are similar to those estimated for other highly endemic west african populations in burkina faso, mali, and guinea, and higher than for a population of lower endemicity in the gambia. regarding the individual values, it is worth noting that although an infection with fws value close to is dominated by a single population of identical or closely related parasites, diverse parasite genotypes may exist at low levels or sequestered in organ capillaries which may become abundant in the peripheral blood after a few hours or days. similar overall allele frequency distributions observed in both of the sampled populations was not surprising, given their proximity and a high level of gene flow generally among west african malaria parasite populations. genome-wide fst values between the populations were very low, with the small proportion of snps having fst values above not departing from random expectations accounting for sampling variance. a previous comparison of two west african parasite populations with different levels of endemicity also showed very low fst values throughout most of the genome. however, there was exceptional differentiation between the guinea and gambia populations over a 15 kb region of chromosome incorporating the gametocyte development gene which is responsible for early initiation of gametocyte development and vital for enabling parasite infection of mosquitoes. there was no differentiation at this locus between the two populations in ghana studied here. in contrast, haplotype-based approaches indicated that selection has probably been operating on multiple other loci. of the genomic windows containing elevated |ihs| scores, marking loci likely to have been under recent directional selection in ghana, were observed in both of the local population samples. the most significant window identified a large region located towards one end of chromosome as observed previously in other population samples from senegal, the gambia, and guinea, although the mechanism and target of selection remains unknown. another strong signal was due to core snps in the ama gene on chromosome encoding an important merozoite target of acquired immunity with extensive sequence polymorphism. this is likely to be subject to occasional positive selection of new alleles added to the repertoire of existing alleles maintained by balancing selection. strong |ihs| signatures around three major drug resistance genes highlight the role of selection by antimalarial treatment across ghana. strongest selection on these genes will have occurred when chloroquine and pyrimethamine-sulphadoxine were widely used in antimalarial therapy in ghana, until artemisinin combination therapy replaced them as official first line treatment in and there may have been local differences in the decay of these signatures following the introduction of act. it is also possible that there is some limited ongoing selection, due to use of sulphadoxine – pyrimethamine for intermittent preventive treatment of malaria in pregnancy, continued use of chloroquine in some areas despite its proscription or selection by act partner drugs which have included amodiaquine and lumefantrine in ghana. three regions, on chromosomes and with high |ihs| values in both of the local populations here were also marked as having stronger evidence of extended haplotypes in one or other of the populations by the rsb cross-population analysis. this has yielded windows that each contain candidate genes that may be subject to population-specific differences in selection, including the trap gene which encodes a vaccine-candidate target of immunity, genes involved in parasite development in mosquitoes, and members of variant-expressed multigene families. haplotype based tests of directional selection on malaria parasites in west africa have previously identified selection occurring over very recent time frames, with strong signatures linked to the use of chloroquine and the antifolate drugs sulphadoxine-pyrimethamine as first line malaria therapies. decay of these signatures following changes in drug use appears to be rapid, facilitated by the high recombination rate of p. falciparum. our results here, using a combination of the |ihs| and the rsb tests, provide evidence of subtle differences in local selection signatures in the two ghanaian populations that are likely to represent ongoing or very recent selection events. future studies to investigate selection operating over longer time frames, and heterogeneity among more distantly related populations with potentially differing demography, may employ additional tests including coalescent-based approaches. studies to conduct genome-wide analyses on malaria parasites from a larger number of populations sampled across heterogeneous environments are likely to be able to refine the mapping of loci influenced by local selective processes, and would also indicate the geographical scales of such signatures of selection. coupling these approaches with epidemiological analyses, including further details of local transmission by different vector species, should facilitate future studies to not only detect signatures of selection but identify the causal processes underlying them.  this study compares population structure and genomic signatures of selection on malaria parasites in two closely situated but ecologically contrasting endemic areas. parasite genome sequencing yielded high coverage of more than snps for population genomic analyses of clinical infection isolates from an area with malaria transmission for most of each year, and isolates from an area with seasonally restricted transmission. the local populations had similar profiles in most respects, particularly with regard to overall snp allele frequency distributions, and proportions of mixed genotype infections. eleven different chromosomal regions showed elevated integrated haplotype scores in both populations, but another metric comparing extended haplotype homozygosity indicated differences in the strength of the signal between the populations for three of these chromosomal regions. a stronger signal was detected within one population for a narrow region of chromosome whereas a stronger signal was seen in the other population for small regions on chromosomes and these include genes that are potential targets of locally varying recent selection, such as those involved in parasite development in mosquitoes, members of variant-expressed multigene families, and a leading vaccine-candidate target of immunity. even in a situation of closely situated populations with very similar population structure and shared selection signatures, analysis across heterogeneous environments has potential to refine the mapping of important loci under temporally or spatially varying selection, including those of potential relevance to epidemiology and control of infection. 
against a background of very similar population structure and selection signatures in the p. falciparum populations of ghana, three narrow genomic regions showed evidence indicating local differences in historical timing or intensity of selection. the candidates include genes involved in parasite development in mosquitoes, members of variant-expressed multigene families, and a leading vaccine-candidate target of immunity. sampling of closely situated populations across heterogeneous environments has potential to refine the mapping of important loci under temporally or spatially varying selection. genome wide sequence analyses of malaria parasites from widely separated areas of the world have identified contrasting population structures and signatures of selection. the local populations had similar proportions of mixed genotype infections, similar snp allele frequency distributions, and eleven chromosomal regions had elevated integrated haplotype scores in both. to compare relatively closely situated but ecologically contrasting regions within an endemic african country, population samples of plasmodium falciparum clinical isolates were collected in ghana from kintampo in the central forest-savannah area, and navrongo in a drier savannah area ~350 km to the north with more seasonally-restricted transmission. a between-population rsb metric comparing extended haplotype homozygosity indicated a stronger signal within kintampo for one of these regions and in navrongo for two of these regions.
 genome wide sequence analyses of malaria parasites from widely separated areas of the world have identified contrasting population structures and signatures of selection. to compare relatively closely situated but ecologically contrasting regions within an endemic african country, population samples of plasmodium falciparum clinical isolates were collected in ghana from kintampo in the central forest-savannah area, and navrongo in a drier savannah area ~350 km to the north with more seasonally-restricted transmission. parasite dna was sequenced and paired-end reads mapped to the p. falciparum reference genome.  high coverage genome wide sequence data for different clinical isolates enabled analysis of single nucleotide polymorphisms. the local populations had similar proportions of mixed genotype infections, similar snp allele frequency distributions, and eleven chromosomal regions had elevated integrated haplotype scores in both. a between-population rsb metric comparing extended haplotype homozygosity indicated a stronger signal within kintampo for one of these regions and in navrongo for two of these regions. at least one gene in each of these identified regions is a potential target of locally varying selection. the candidates include genes involved in parasite development in mosquitoes, members of variant-expressed multigene families, and a leading vaccine-candidate target of immunity.  against a background of very similar population structure and selection signatures in the p. falciparum populations of ghana, three narrow genomic regions showed evidence indicating local differences in historical timing or intensity of selection. sampling of closely situated populations across heterogeneous environments has potential to refine the mapping of important loci under temporally or spatially varying selection. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 