P. vivax remain little characterized.The ideal malaria parasite populations for initial mapping of genomic regions contributing to phenotypes such as drug resistance and virulence, through genome-wide association studies, are those with high genetic diversity, allowing for numerous informative markers, and rare meiotic recombination, allowing for strong linkage disequilibrium (LD) between markers and phenotype-determining loci. However, levels of genetic diversity and LD in field populations of the major human malaria parasite P. vivax in 238 field isolates from areas of low to moderate malaria endemicity in South America and Asia, where LD tends to be more extensive than in holoendemic populations, and in two monkey-adapted strains . We found varying levels of SNP diversity and LD across populations, with the highest diversity and strongest LD in the area of lowest malaria transmission. We found several clusters of contiguous markers with rare meiotic recombination and characterized a relatively conserved haplotype structure among populations, suggesting the existence of recombination hotspots in the genome region analyzed. Both silent and nonsynonymous SNPs revealed substantial between-population differentiation, which accounted for ~40% of the overall genetic diversity observed. Although parasites clustered according to their continental origin, we found evidence for substructure within the Brazilian population of P. vivax. We also explored between-population differentiation patterns revealed by loci putatively affected by natural selection and found marked geographic variation in frequencies of nucleotide substitutions at the pvmdr-1 locus, putatively associated with drug resistance.We examined single-nucleotide polymorphisms (SNPs) and LD patterns across a 100-kb chromosome segment of P. vivax, using relatively low densities of markers, but underscore the risk of false positives caused by population structure at both local and regional levels.These findings support the feasibility of genome-wide association studies in carefully selected populations of http://www.biomedcentral.com/1741-7007/8/90See commentary:  Plasmodium vivax, the most widespread of the four human malaria parasites, causes 132 to 391 million episodes of disease each year [P. vivax is the main cause of malaria morbidity, with enormous public health burden. The recent emergence of drug-resistant strains and severe  disease challenges the traditional view of vivax malaria as a benign infection and calls for new strategies to examine molecular mechanisms underlying drug resistance and increased virulence [P. vivax are currently limited by the lack of practical methods for its long-term propagation and cloning in vitro [P. vivax. However, patterns of genetic diversity and LD remain little characterized in field populations of this parasite and most nucleotide polymorphism data currently available are for antigen-coding genes [P. vivax, which are heavily contaminated with host's DNA, are available for use in next-generation sequencing projects for large-scale characterization of new genetic markers.Linkage analysis of laboratory crosses and population-based genome-wide association studies are powerful approaches to map genetic loci contributing to drug resistance and virulence in malaria parasites . Experiin vitro , which ain vitro . The ideng genes . These dng genes . Due to P. falciparum isolates sampled from four continents. Diversity was highest and LD lowest in populations from holoendemic Africa, while diversity was lowest and LD highest in populations from hypoendemic South America, with intermediate patterns seen in Southeast Asia. Parasite populations clustered according to their continental origins, with most variation found within locations in highly endemic areas. Nevertheless, substantial divergence was seen between subpopulations in South America [P. vivax [P. vivax. In addition, microsatellites mutate at very high rates and other neutral genetic markers with different rate and mode of evolution, such as intergenic or synonymous single-nucleotide polymorphisms (SNPs), could offer a different picture [Microsatellite markers have revealed clear geographic differences in levels of genetic diversity and LD in  America . Putativ America , have reP. vivax -12, suggP. vivax ,11. Howe picture . Signifi picture .P. vivax. We sampled parasites from areas of low to moderate endemicity in South America and Asia, where LD tends to be more extensive than in holoendemic populations. We show varying levels of SNP diversity and LD across populations , with substantial genetic differentiation among populations. Frequencies of nucleotide substitutions at pvmdr-1 gene, putatively associated with drug resistance, varied markedly across locations. Although these findings support the use of genome-wide association approaches to map genes underlying drug resistance and other traits in P. vivax, they underscore the risk of false positives if population structure, at both local and regional levels, is left uncorrected in association studies.To explore the potential for future genome-wide association studies, we examined SNP diversity and LD across a 100-kb chromosome segment of P. vivax from areas of low to intermediate levels of malaria endemicity and two monkey-adapted strains, Belém and Salvador-I. The field isolates originated from three sites in the Amazon Basin of Brazil  and three sites  across South and Southeast Asia  does not match the ranking of malaria endemicity at the time of sample collection (Vietnam > Cambodia > Brazil > Sri Lanka). The trend towards a negative correlation between levels of genetic diversity and malaria transmission contrasts with the pattern observed for settings ,15. Thisr = 0.138, P < 0.001, Mantel correlation test). The correlation remained significant when only silent SNPs were considered , but not when only nonsynonymous sites were considered . The little variation at nonsynonymous SNP loci over time underscores the potential biases introduced by natural selection in studies of malaria parasite diversity. Whether or not similar temporal patterns of genetic divergence occur in other endemic areas remains to be investigated.Parasites were systematically sampled over 30 months (April 2004 through October 2006) in one of the study sites in Brazil, Granada ,16. We hr2 statistic) decayed with increasing physical distance between pairs of segregating sites in the Brazilian population  and to different blocks or no block  in pairwise population comparisons. This analysis was limited to SNPs that were segregating in both populations analyzed (99 to 227 SNP assignments compared). Overall, the vast majority of SNP pairs had a concordant assignment, especially in comparisons involving Brazil and Cambodia (93%), Vietnam and Sri Lanka (91%), and Brazil and Sri Lanka (83%). These data are consistent with a conserved haplotype structure across lciparum . Lower pFST statistics revealed substantial chromosome-level differentiation between Brazilian and Asian samples . Significant divergence was found in all pairwise comparisons within Brazil and Asia  indicates that a considerable proportion (~40%) of the diversity at assayed SNP loci results from differentiation among geographic populations of P. vivax. For all between-population comparisons, except Cambodia versus Sri Lanka, we observed a greater FST for silent SNPs relative to nonsynonymous SNPs   defined P. falciparum populations [P. vivax. We thus analyzed separately this population and found that parasites did not cluster according to their collection site . In fact, most minor clusters in Brazil comprised samples from at least two of these locations  resistance in P. vivax  and Asia (Cambodia versus Vietnam) . The low FST value estimated for the comparison between Cambodian and Vietnamese parasites is particularly noteworthy, given the long time interval between dates of sample collection in these sites.We next explored between-population differentiation patterns revealed by loci putatively affected by natural selection. We scored SNPs at two loci, ) Figure , with lapvmdr-1 thought to be associated with CQ resistance, Y976F and F1076L [pvmdr-1 locus leads to CQ resistance [We focused on two nonsynonymous substitutions in d F1076L -23. Doubsistance . If thispvmdr-1 alleles that are CQ-resistant [P. falciparum [P. vivax appears to be modulated by pvmdr-1 copy number. Gene amplification correlates with increased susceptibility to CQ and decreased susceptibility to amodiaquine, artesunate and mefloquine [pvmdr-1 copy number or select for Y976F alleles, further complicating geographic comparisons of allele frequencies and associated phenotypes.The finding of parasites with wild-type esistant ,24 suggelciparum , in vitrfloquine . DiffereP. vivax populations from areas with low to moderate levels of malaria transmission. The most favorable conditions for association studies with relatively low marker density were observed in Brazil, where parasites were reasonably diverse  and strong LD was observed at relatively short intermarker distances (~40 kb) but gradually declined with increasing physical distance between pairs of markers. In contrast, the similar levels of LD, along the whole 100-kb region analyzed, in the Asian populations may increase the probability of detecting false-positive associations between phenotype-associated loci and genetic markers located at considerable map distances. These findings, although limited to a single chromosome segment that comprises only 0.4% of the whole genome of the parasite, indicate that genome-wide association studies can represent a feasible strategy to map genetic regions associated with drug resistance, virulence and other phenotypes of interest in carefully selected P. vivax populations. Genome-wide studies with higher marker density are required to confirm these findings.Here we describe long-ranging chromosome-level LD and relatively conserved haplotype blocks in P. falciparum [pvmdr-1 locus among populations. We note that, because of the underlying geographic structure, allele frequency differences observed among populations may be unrelated to the genetics of the particular phenotype under study, resulting in false-positive results or reduced power of association studies. Even when studies are restricted to a single continental origin, false positives may still result from major differences in the ancestry of local parasites, such as those found in Brazil. Accounting for the geographic structure is a major practical issue in future population-based studies of genetic determinants of P. vivax traits. Statistical methods have now been developed [We have also observed substantial genetic differentiation among populations, at both local and regional levels. Consistent with previous studies of lciparum , we founeveloped  to addreP. vivax populations, with the most favorable conditions for genome-wide association studies, relatively high diversity and strong LD that declines with increasing intermarker distance, observed in Brazil. However, we have also observed substantial genetic differentiation among populations at both local and regional levels, especially in the Brazilian population. Although these results suggest that association studies are feasible in selected P. vivax populations, they highlight the need for correcting for population structure to avoid false-positive associations.We found varying levels of SNP diversity and LD across P. vivax infection from three locations in Brazil and one location each in three Asian countries . The 23 samples from Vietnam were collected in January-December 1995 from patients attending the outpatient clinic of the Lam Dong Provincial Hospital, in the town of Bao Loc, 150 km northwest of Ho Chi Mihn City, Lam Dong Province [P. vivax infection in the rural communities surrounding Bao Loc, on the southern highlands of Vietnam, were estimated to be around 2.5-7.5% at the time of sample collection [vivax malaria were reported from Trincomalee . Over the past few years malaria transmission has declined steadily in Sri Lanka, from 210,039 cases of malaria in 2001 to only 196 cases country-wide in 2007 [We collected venous or finger-prick blood samples from 432 patients with slide-confirmed a Figure . All sitbelow 1% ,30. The below 1% ,16,30, tbelow 1% , and thoProvince . Prevalellection . The 29 llection . Between in 2007 . In contDNA from field samples was extracted using standard protocols referenced in the original publications. Additional DNA samples, from monkey-adapted strains, Belém  and Salvador-I , were provided by the Malaria Research and Reference Reagent Resource Center (MR4), ATCC . To obtain adequate DNA concentrations for SNP assays, parasite DNA was submitted to whole-genome amplification (WGA) prior to typing. WGA was performed on 10 ng of genomic DNA, with high-fidelity multiple displacement technology , using aP. vivax isolates [AY003872 and AY216936-AY216939). DNA sequences were derived from two isolates from Brazil , one frm strain ), one frm strain ), and onm strain ). SNPs we sample . We desiP. vivax  in the pvcrt-o gene [P. vivax [P. falciparum orthologue of pvcrt-o, which encodes the protein chloroquine resistance transporter (PfCRT), confer CQ resistance to this species [pvcrt-o alleles and CQ resistance in P. vivax [multidrug resistance 1 gene of P. vivax (pvmdr-1) [We also examined nucleotide replacements at two loci encoding digestive-vacuole membrane proteins that are potentially involved in chloroquine (CQ) resistance in t-o gene  that werP. vivax ,45. K76T species , but theP. vivax ,45. We apvmdr-1) ,22,24,45pvmdr-1) . Interespvmdr-1) . All SNPpvmdr-1) . Primer pvcrt-o and pvmdr-1 loci was based on complete genotypes; infections with one or more SNPs without allele calls were excluded. The number of isolates considered for further analysis is shown in the additional file Estimation of allele frequencies in malaria parasite populations is complicated by the co-occurrence of multiple clones within infections. Counting all alleles identified within an infection results in overestimation of frequencies of rare alleles and underestimation of common alleles. To minimize bias, we excluded 101 infections in which > 1 allele was observed in any of the SNP loci. For the analysis of chromosome-level SNP diversity and LD patterns, we also excluded 93 infections with allele calls for < 60 markers across chromosome 8. Analysis of the Population-level diversity was measured with the SNP π statistic, defined as the average number of pairwise differences at assayed SNPs between all members of a population . SNP π vr2 [2 tests. Correlation between the physical distance between markers and LD was assessed using the Pearson's coefficient of correlation (r). We defined chromosome 8 haplotype blocks as clusters of adjacent markers over which evidence of meiotic recombination was minimal, using the four-gamete rule [A key factor in the success of association studies is the level of LD observed within and across populations. We examined the evidence for LD within each country and in two subpopulations from Brazil (Granada and Plácido de Castro) with enough samples. The LD statistic r2  was calcr2 . Statistete rule . For eacete rule . To compete rule . These cFST statistic using FSTAT software [K populations characterized by a set of allele frequencies at each locus. We run the program 10 times each for K values between 1 and 6. Each analysis involved 100,000 iterations, with 50,000 burn-in cycles. We used the linkage model to account for the LD among markers across the same chromosome. We computed the posterior probability for each K and show here clustering patterns associated with the strongest statistical support. Again, separate analyses were made for the whole dataset and for Brazilian and Asian samples.We assessed population differentiation using the θ estimator  of FST ssoftware ; 95% consoftware . We carrsoftware . Becausesoftware , we compsoftware  to examiThe parasitized blood samples described in this paper were collected under protocols approved by the relevant ethical review committees in the respective countries. Patients or their parents or legal guardians provided written informed consent before donating samples. Ethical clearance was also obtained from the Institutional Review Board (IRB) of the University of São Paulo, Brazil; the IRB of the NIAID, USA; the National Research Ethics Committee of the Brazilian Ministry of Health; and the Cambodian National Ethics Committee for Health Research.Conceived and designed the experiments: POS, NDK, DFW, MUF. Performed the experiments: POS. Contributed parasite samples and epidemiologic data: MdSN, NDK, NSdS, KKGS, RMG, CA, JMS, DS, RMF, SG, TT, GG, RA, FK, MUF. Analyzed the data: POS, MUF. Drafted the paper: POS, MUF. All authors read and approved the final version of the manuscript.Table S1. Origin of field isolates of P. vivax analyzed in this study.Click here for fileFigure S1. Variation in LD in relation to distance between SNP loci along chromosome 8 of P. vivax in two Brazilian subpopulations. Data are shown for Granada and Plácido de Castro. LD was measured with the r2 statistic ; closed (blue) circles denote significant LD , while open circles denote nonsignificant LD. A significant negative correlation between r2 values and physical distance between SNPs was found for both Granada  and Plácido de Castro .Click here for fileTable S2. Matrix of FST values showing pairwise comparisons for 10 nonsynonymous SNPs  and 75 silent SNPs .Click here for fileFigure S2. Principal component analysis (PCA) of population structure of P. vivax. This analysis considered a subset of 29 SNPs selected to minimize intermarker LD. Plots show the three first principal components. Each circle or square represents a parasite isolate or strain and the color is assigned according to the geographic origin of parasites: green circles, Granada; red circles, Plácido de Castro; open circles, Porto Velho ; pink squares, Cambodia; blue squares, Sri Lanka; light brown squares, Vietnam . The strains Salvador I and Belém are denoted by orange and black circles, respectively. Upper panel, plot of the first two principal components; lower panel, plot of the first and third principal components. Note that the clustering patterns revealed by the subset of 29 SNPs are quite similar to that detected with 57 informative SNPs  of population structure of P. vivax in Brazil. Plots show the three first principal components. Each circle represents a parasite isolate or strain and the color is assigned according to the geographic origin of parasites: green, Granada; red, Plácido de Castro; open, Porto Velho. The strains Salvador I and Belém are denoted by orange and black colors, respectively. Upper panel, plot of the first two principal components; lower panel, plot of the first and third principal components.Click here for fileFigure S4. Principal component analysis (PCA) of population structure of P. vivax in Asia. Plots show the three first principal components. Each square represents a parasite isolate and the color is assigned according to the geographic origin of parasites: pink, Cambodia; blue, Sri Lanka; light brown, Vietnam. Upper panel, plot of the first two principal components; lower panel, plot of the first and third principal components.Click here for fileFigure S5. Predicted structure and location of amino acid replacements in PvMDR-1 and PvCRT-O. PvMDR-1 (upper panel) has two hydrophobic domains, each with six transmembrane α-helices, and a cytosolic domain harboring nucleotide-binding domains 1 (NBD1) and NBD2, each containing an ATP-binding site. PvCRT-O (lower panel) has 10 predicted transmembrane helices, with C- and N-terminal domains located in the parasite cytoplasm. Black dots represent nonsynonymous SNPs assayed in this study; the only open dot in PvMDR-1 represents a synonymous SNP that was also assayed.Click here for fileTable S3. Characterization of the 85 biallelic single-nucleotide polymorphisms (SNPs) along P. vivax chromosome 8 assayed in this study.Click here for fileTable S4. Sequences of primers used to amplify 85 single-nucleotide polymorphisms (SNPs) along chromosome 8 of P. vivax, 5 SNPs in the pvcrt-o gene and 6 SNPs in the pvmdr-1 gene.Click here for file


Zoogoneticus quitzeoensis is widely distributed across Central México, thus making it a good model for phylogeographic analyses in this area.Tectonic, volcanic and climatic events that produce changes in hydrographic systems are the main causes of diversification and speciation of freshwater fishes. Elucidate the evolutionary history of freshwater fishes permits to infer theories on the biotic and geological evolution of a region, which can further be applied to understand processes of population divergence, speciation and for conservation purposes. The freshwater ecosystems in Central Mexico are characterized by their genesis dynamism, destruction, and compartmentalization induced by intense geologic activity and climatic changes since the early Miocene. The endangered goodeid Z. quitzeoensis through a sequential approach, based on both microsatellite and mitochondrial cytochrome b sequences. Most haplotypes were private to particular locations. All the populations analysed showed a remarkable number of haplotypes. The level of gene diversity within populations was d = 0.987 (0.714 – 1.00). However, in general the nucleotide diversity was low, π = 0.0173 (0.0015 – 0.0049). Significant genetic structure was found among populations at the mitochondrial and nuclear level . We distinguished two well-defined mitochondrial lineages that were separated ca. 3.3 million years ago (Mya). The time since expansion was ca. 1.5 × 106 years ago for Lineage I and ca. 860,000 years ago for Lineage II. Also, genetic patterns of differentiation, between and within lineages, are described at different historical timescales.We addressed the phylogeography, evolutionary history and genetic structure of populations of ca. 3.3 Mya) than to the current hydrographic configuration of the basins. In general, mitochondrial and nuclear data supported the same relationships between populations, with the exception of some reduced populations in highly polluted basins (Lower Lerma River), where the effects of genetic drift are suggested by the different analyses at the nuclear and mitochondrial level. Further, our findings are of special interest for the conservation of this endangered species.Our mtDNA data indicates that the evolution of the different genetic groups is more related to ancient geological and climatic events (Middle Pliocene,  Primary freshwater fishes are strictly confined to freshwater basins, limiting their dispersal capacity. The evolution and dispersal of primary freshwater fishes are closely tied to the palaeogeography and history of connections, captures or separation of the water bodies they inhabit . AccordiThe freshwater ecosystems of Central Mexico are characterized by their genesis dynamism, destruction, and compartmentalization induced by intense tectonic and volcanic activity. Its major physiographic feature is the Mesa Central, a large and isolated tropical highland, which includes the geological active Transmexican Volcanic Belt (TMVB), defined as the southern limit of the massive uplifted and as the transition area between the Nearctic and Neotropical provinces . The tecMany studies have discussed the biogeography of the Mesa Central, and have described the vicariant events that have resulted in subsequent differentiation in beetles , salamanRecent molecular studies have demonstrated the genetic signatures these volcanic, tectonic, and climatic events have left in some freshwater fish species of the Mesa Central, such as the Poecilids , CypriniZoogoneticus is represented by two species, Z. tequila  and Z. quitzeoensis . The former is a microendemic species of the upper Ameca River basin, while the latter is widely distributed across the hydrological basins that drain the TMVB. Previous works have demonstrated that the populations of Z. quitzeoensis from Cuitzeo and Zacapu are significantly divergent when compared with populations from Lower Lerma [Zoogoneticus quitzeoensis is considered an endangered species by the Mexican Official Norm of Ecology, and its sister species, Z. tequila, is now reported as extinct in the wild [Z. quitzeoensis provides an interesting case-study for examining various features of the evolutionary and demographic history of the geologically active TMVB and its biota. Also, it can serve as a model to understand the processes and events that rule the biodiversity assemblages of the area and to promote its conservation.Within the endemic freshwater fish fauna of the Mesa Central, the Goodeinae is one of the most diverse groups (around 41 species), characterized by its particular life history, including internal fertilization, matrotrophy and viviparity, and a high degree of genetic divergence ,21,22. Wer Lerma  and Amecer Lerma -26. Becathe wild . Thus, ZIn this sense, phylogeographical approaches have generally served to establish patterns of evolutionary history in distinct geographical populations. However, they have also been successfully used to infer historical demographic processes such as gene flow, effective population sizes or evolutionary trajectories . ElucidaMitochondrial DNA is preferentially and commonly used in most phylogeographic studies , althougZoogoneticus quitzeoensis across its whole distribution range. Based on our results, we then infer the historical biogeographical scenario of its populations and propose future strategies for its conservation.Herein we describe the phylogeography, evolutionary and demographic history of b gene in 80 individual Z. quitzeoensis specimens from 12 populations  and no evidence of positively selected sites was detected with any of the two methods used. Full details of substitution parameters and evolutionary models are given in Additional file By sequencing 1140 bp of the entire mitochondrial cytochrome Weighted parsimony analyses generated 95 equally parsimonious trees . The three methods  produced largely congruent tree topologies, with proportionally similar bootstrap and posterior probabilities supporting major lineage and population divergences  from the 3 sampled sites in the Ameca River Basin , and the other clade  contained individuals from the localities sampled in the Lower Lerma Basin and Lake Chapala. Distances between Ameca and Chapala-Lower Lerma clades were ML = 0.93% ± 0.2 and p = 0.92% ± 0.2.Within Lineage I, we found two main clades. One clade (Ameca Clade) included 18 haplotypes (Hn = 20), and the second one, the San Francisco del Rincón population (Middle Lerma Clade: Hn = 5). The ML between the two clades was 1.17% and p = 1.21   and long distance colonization associated with subsequent fragmentation followed by range expansion for clades 3.5 (Clade La Luz-Orandino) and 4.2  [Allopatric fragmentation could have rendered the geographical pattern of the full cladogram of r Lerma) .In Lineage II, the inference key  suggesteST values obtained for the comparisons of all the populations ranged from 0.006 to 0.940 (Table ST = 0.518 – 0.940), except for comparisons with two populations from Lake Cuitzeo . Significant ΦST values were also obtained for all the comparisons of Los Veneros with the rest of the populations, except for those involving populations within the same basin . The only significant ΦST values between samples within the same basin were found in the Lower Lerma River, except for Orandino and La Platanera . Although the La Luz and La Platanera populations occur in the same basin (Lower Lerma), their FST values were among the highest. Notwithstanding, the other population inhabiting the Lower Lerma Basin (i.e. Orandino) showed significant yet much lower FST values when compared with La Luz and La Platanera (FST = 0.271 and 0.224 respectively).Pairwise in Table . HighestST = 0.836, P < 0.001 and FST = 0.262, P < 0.001 respectively). For the subsequent analyses, populations were grouped in different hierarchical arrangements according to previous information and to uncover groupings obtained in the previous analyses  and by their biogeographical arrangement . When the basins where each lineage was found were considered separately, only Lineage I showed significant structure for mtDNA (Table P < 0.001) of the total variance was explained as differences among groups for mtDNA and 11.18% in the case of microsatellite data (P < 0.01). Division of the populations into the groups suggested by the phylogenetic and NCA analysis maximised among-group variance for the mtDNA data . Thus, the structure previously obtained in the phylogenetic and NCA was statistically supported for the mtDNA, but not for the microsatellite data.The AMOVA performed for the mitochondrial and nuclear data, revealed significant structure among populations . Otherwise, non significant correlation was found considering the genetic distances obtained using the microsatellite data   increased rapidly between K = 1 and K ≤ 6, whereas beyond K > 6 lnPr(X|K) started to oscillate. But when we considered Δ K, we obtained clear peak at K = 5  to their inferred cluster.The Bayesian structure analysis for the microsatellite data clearly revealed a genetic structure among the specimens analysed. According to the maximum likelihood value, we estimated a number of genetic clusters of o et al. . All popK = 6, Orandino represented a separate cluster. Slightly different results, regarding the position of the Lower Lerma populations, were rendered by the two types of marker for population relationships , and the other one was found in 3 individuals from Magdalena and Moloya (MAG-MOL 2) . However, the nucleotide diversity exhibited by most of the tested populations (π = 0.0015 – 0.0049) was low. Mean pairwise nucleotide diversity (k) ranged from 1.71 to 5.69 within populations  were not significant, thus not rejecting the null hypothesis of stationarity. Conversely, Tajima's D-statistic and Fu's statistic (Fs) were significantly negative and supported the expansion model for the different groups tested (Table D) or haplotype distribution (e.g. Fu's F). In a variety of cases and for large samples sizes, Fu's F has been proved to be the most powerful test to detect population growth [6 years before present. Within Lineage I, the estimated time since population expansion for the two clades obtained  were ca. 1.4 × 106 (τ = 5.08) and ca. 1.08 × 106 (τ = 5.647), respectively. In Lineage II, we estimated ca. 860,000 years since population expansion according to the value of τ = 3.09. For the clades within Lineage II, the estimates of time since population expansion were ca. 790,000 years for Cuitzeo-Zacapu (τ = 2.83) and ca. 550,000 years for San Francisco del Rincón (τ = 1.95).All the populations analysed showed a remarkable number of haplotypes Table . The lev0.987 0.74 – 1.00.π = 0.001 – 0.0049n growth . Values b and microsatellites) distinguished two independent lineages, supporting the conclusions of previous studies [Z. quitzeoensis. Lineage I inhabits areas west of the Middle Lerma, including the Lower Lerma, Ameca and the Chapala Lake basins. Lineage II occurs at sites east of the Angulo River and the Middle Lerma Basin, including the Cuitzeo and Zacapu lakes. Our NCA also supported this conclusion, indicating the two main lineages were not nested together; moreover, the 21 mutation steps between the two lineages exceeded the 95% parsimony limits (14 mutation steps). This result suggests allopatric fragmentation between the two clades.The trees obtained by the different methods  using both types of markers .The formation of the two lineages of on years . These tThe dispersion event we refer to, from the Chapala-Lower Lerma to the Middle Lerma-Cuitzeo-Zacapu area, could have been promoted by a period of high precipitation and humidity occurred in Central Mexico in the early Pliocene (5.2 – 3.6 Mya). This could have caused an increase of the water bodies level in this area causing them to get in contact, as was previously proposed for other lakes in Central Mexico ,39. The Skiffia lermae-S. multipunctata, dated around 3.2 Mya [Since recent geological times, some of the regions where these two lineages occur have been connected, and at present constitute one single hydrologic system (Lerma River). However, our findings of ancestral isolation between Lower Lerma-Chapala and Middle Lerma-Zacapu-Cuitzeo are supported by at least one pair of sister species with the same cladogenetic pattern,  3.2 Mya . This in 3.2 Mya ,43. FurtThe results of all our analyses revealed significant genetic structure and differentiation among populations between and within the two lineages, but with certain differences as indicated by the two types of molecular markers.versus restricted dispersal and gene flow. However, the fact that haplotype LUZ-40 emerged as the most probable outgroup in the network, and the significant negative value of the Fu's Fs, and Tajima's D statistics seem to better support the range expansion and colonization scenario. The haplotype arrangement found within the three populations sampled on the Ameca river basin also supports this scenario. Haplotypes from Los Veneros are placed in a basal position in the phylogenetic trees and appeared in all the 2-step clades found in the NCA (within 3.6 Clade). Such patterns could indicate that Los Veneros may represent the ancestral population of the Ameca Basin. The Los Veneros population is geographically close to the San Marcos-Atotonilco lakes (~19 Km), which formed part of the Chapala Palaeolake [Z. quitzeoensis spread from the Chapala Palaeolake region to the Ameca River region via the Zacoalco-Ameca Paleolake , in such case the dispersion event could have taken place at the beginning of the Pleistocene.The two main clades identified within Lineage I correspond to two different hydrologic systems. One clade appears in the Ameca river basin (Clade 3.6) and the other is distributed across the Lower Lerma-Chapala Lake area (Clade 4.2). When Lineage I was tested independently, the statistical associations in the nested contingency analysis were not able to discriminate between range expansion and colonization laeolake , but also lakes ~9 Km, whiPoeciliopsis [Notropis [Chirostoma [Ictalurus [Yuriria [A former connection between the Chapala and Ameca basins have been previously proposed via the Atotonilco and San Marcos lakes   and the populations of La Luz and Orandino (clade 3.5), within the Lower Lerma region, was larger than the distance between the population of La Alberca (clade 3.4), in the Chapala Lake region, and the populations of Orandino and La Luz  and microsatellite analyses . The first of these groups comprises the San Francisco del Rincón population and the second group includes the populations from Zacapu, La Mintzita, San Cristóbal and Belisario, the last three belonging to the Cuitzeo region. In the NCA, only Clade 4.1, which clustered together all the populations of this lineage, showed a significant geographical association, but insufficient to discriminate between long distance colonization and past fragmentation , could point to the long distance colonization of organisms from Zacapu-Cuitzeo and past fragmentation as the most plausible scenario. This assumption is supported by the neutrality tests of Fu's Fs and Tajimas's D statistics.According to our demographic results, both scenarios provided by the NCA inference key are candidates for the five populations distributed across three different basins  [Notropis [All the populations within this lineage showed evidence of recent gene flow, except the one from San Francisco del Rincón. Our results also indicated non significant genetic differentiation for most of the pairwise comparisons, for mtDNA, among the four populations inhabiting the Zacapu and Cuitzeo basins. Haplotypes were shared by the two basins, a fact that disagrees with the hypothesis that Zacapu and Cuitzeo constitute two well-defined biogeographic entities . Althoug(≈1 Mya) . This coNotropis  and otheNotropis . HoweverMitochondrial and nuclear markers reveal different parts of the evolutionary history of the organisms due to their different inheritance modes and mutation rates. Even though mitochondrial markers coalesce faster than nuclear markers due to the smaller effective size and the lack of recombination, microsatellites have a much faster mutation rate which makes them useful to detect more recent processes ,51.FST values were obtained for most of the comparisons of populations from the Lower Lerma basin  for oneticus . When thThe present study is the first attempt to describe, on a fine scale, the evolutionary history of populations of a fish species in Central Mexico. Our results demonstrate the value of the use of mtDNA combined with nuclear microsatellite loci to detect genetic structure and to elucidate the evolutionary history of fish species. The methodology used integrates independent geological and genetic information to test for interactions between historical and contemporary factors in a highly structured endemic fish in Central Mexico.Our results indicate that the evolutionary history and genetic structure among populations of this fish species is closely tied to geological and climatic events that promoted changes in the ancient drainages, since the Middle-Pliocene, rather than to the current configuration of those drainages. In addition to this, the results obtained and differences between molecular markers are an evidence of the effects of genetic drift over the genetic structure in some highly polluted aquatic environments.The information provided by this type of study is essential for the conservation of highly genetically structured species and its phylogeographic hypotheses prompt comparable analyses of other codistributed fish species to test the scenarios proposed.Zoogoneticus quitzeoensis. Specimens were collected from 12 sites distributed across six regions along the Mesa Central of Mexico  were amplified via polymerase chain reaction (PCR) from 80 specimens distributed in 12 sampling sites. The primers used were those used in Machordom & Doadrio [Two overlapping fragments of the cytochrome  Doadrio . The amp Doadrio . PCR proZoogoneticus obtained in PAUP 4.0b10 [6 cycles and using the substitution model obtained for each codon position selected by BIC criterion, 20,000 trees were generated, 1000 of which were burned and discarded. Posterior clade probabilities were used to assess node support. To identify ancestral and derived haplotypes, the trees were rooted using Zoogoneticus tequila, the sister species of Z. quitzeoensis, as outgroup, and a molecular clock of 0.9% per million years was applied to the pairwise uncorrected p genetic distances [We used phylogenetic tree-building algorithms to infer the phylogenetic relationships among sequence haplotypes. Maximum Parsimony Analysis (MP) was performed by heuristic searching with the tree-bisection-reconnection (TBR) branch swapping algorithm and random stepwise sequence addition using 10 replicates. Two different weights were given to the characters; first all characters were equally weighted, and second, transversions (Tv) were assigned eight times the weight of transitions (Ti) according to the empirically determined Tv/Ti ratio for P 4.0b10 . The robP 4.0b10  using thP 4.0b10 . By simuistances .P value of 0.1. In both cases, ambiguities in the consensus sequence were averaged in the analysis.Evidence of positive selection was sought using a codon-based approach as implemented in Datamonkey . This meWe constructed a 95% statistical parsimonious un-rooted haplotype network using TCS 1.18 . As a coST values were calculated among all geographic populations as an estimate of genetic differentiation. To assess the significance of genetic differentiation at different hierarchical levels, an analysis of molecular variance (AMOVA) was performed as described by Excoffier et al. [Pairwise Φr et al. . Populatr et al. . In subsr et al. ) served r et al. .Hd, [k, [Population genetic statistics, such as the number of polymorphic sites (S), haplotype diversity , nucleoHd, ) and theHd, [k, ) were caHd, [k, .r) [D [Fs [2μt, were μ is the sum of per-nucleotide mutation rates in the DNA region under study .To investigate the demographic history of the groups identified in the phylogenetic analyses and through the AMOVA results, a mismatch distribution analysis (MMD) of pairwise substitution differences among haplotypes was performed for the whole data set and the lineages obtained. Deviations from the constant population size model were further tested using the Harpending's raggedness index (r) . To testr) [D  and Fu's) [D [Fs  tests as) [D [Fs . We used) [D [Fs . This wa.9% PMY; ) and t iZ. quitzeoensis from 10 populations, genotyped for five microsatellite loci , to find the true K following the method of Evanno et al. [FST values were calculated for each K to find out which of the structures inferred explained the highest percentage of genetic variation. To assess the level of admixture and gene flow between the central Mexican Basins, we ran a population assignment test using GeneClass 2.0 [Because of the uncertainty that the geographical assignment of individuals to populations could represent biologically significant entities, a Bayesian clustering method was conducted as implemented in the program STRUCTURE 2.1 . We perfo et al. . Global lass 2.0 .OD–D conceived the study, collected the samples, participated obtaining the molecular data and analyses and drafted the manuscript. FA contributed in the data analyses and helped to draft the manuscript. GP–PdL conceived the study, participated in its design and coordination and helped to draft the manuscript. JLG–G participated obtaining the molecular data and analyses. ID conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.Zoogoneticus quitzeoensis. The table provided summarizes the collection sites of Zoogoneticus quitzeoensis, their geographical coordinates, the number of individuals analysed for cytochrome b and microsatellites and Genebank accession numbers.Sampling sites and individuals analysed of Click here for fileb data. The data provided summarizes the parameters of the evolutionary models obtained for the cytochrome b data after the AIC and BIC. Ti/Tv ratio, Empirical base frequencies, Gamma shape parameter and Proportion of invariant sites are provided.Substitution parameters and evolutionary models obtained for the different criterion and partitions of the cytochrome Click here for fileInferences for all clades showing a significant association in the nested clade analysis results provided in Figure Click here for fileP(X|D) for each of the K populations inferred by STRUCTURE. (Middle) Number of Zoogoneticus populations with the highest posterior probability expressed as the ΔK . (Right) Comparisons between LnP(D) (black circles) and FST values (white squares) obtained for the different K values inferred by STRUCTURE. This figure represents graphically the values of LnP(X|D), ΔK and FST for each of the different genetic arrangements inferred by STRUCTURE.(Left) Mean LnClick here for fileZoogoneticus quitzeoensis. Modified from Domínguez-Domínguez et al. (2007). The table describes values of genetic diversity based on microsatellite data.Summary table of diversity and BOTTLENECK statistics for the microsatellite data of Click here for file


Microsatellites surrounding functionally important candidate genes or quantitative trait loci have received attention as proxy measures of polymorphism level at the candidate loci themselves. In cattle, selection for economically important traits is a long-term strategy and it has been reported that microsatellites are linked to these important loci.Bos taurus autosome 1) and 16 on BTA20, using bovine populations of typical production types and horn status in northern Eurasia. Genetic variability of these loci and linkage disequilibrium among these loci were compared with those of 28 microsatellites on other bovine chromosomes. Four different tests were applied to detect molecular signatures of selection.We have investigated the variation of seven microsatellites on BTA1  were significantly (P < 0.05) higher than for non-syntenic markers (0.15). The Ewens-Watterson test, the Beaumont and Nichol's modified frequentist test and the Bayesian FST-test indicated elevated or decreased genetic differentiation, at SOD1 and AGLA17 markers respectively, deviating significantly (P < 0.05) from neutral expectations. Furthermore, lnRV, lnRH and lnRθ' statistics were used for the pairwise population comparison tests and were significantly less variable in one population relative to the other, providing additional evidence of selection signatures for two of the 51 loci. Moreover, the three Finnish native populations showed evidence of subpopulation divergence at SOD1 and AGLA17. Our data also indicate significant intergenic linkage disequilibrium around the candidate loci and suggest that hitchhiking selection has played a role in shaping the pattern of observed linkage disequilibrium.No marked difference in locus variability was found between microsatellites on BTA1, BTA20 and the other chromosomes in terms of different diversity indices. Average POLL gene, is a possible explanation for this pattern. The potential impact of selective breeding by man on cattle populations is discussed in the context of selection effects. Our results also suggest that a practical approach to detect loci under selection is to simultaneously apply multiple neutrality tests based on different assumptions and estimations.Hitchhiking due to tight linkage with alleles at candidate genes, e.g. the  Expectation of neutrality regarding the mutation-drift equilibrium for microsatellite variation is not always valid due to demographic changes, including genetic bottlenecks and admixture e.g. ,2), and , and 2])FST and diversity parameters from individual genetic loci to detect the effects of selection, hereafter termed the FST-based approach, such as the FDIST program-based method [FST, such as the ln RV [Different statistical methods have been developed to identify outlier loci under the influence of selection -13 and ad method , Bayesiad method , and popd method  methods.he ln RV , the ln he ln RV , and thehe ln RV -20, all POLL gene, characterized by two alleles: P (polled) dominant over H (horn), is responsible for the polled (i.e. hornless) and horn phenotypes in cattle and has been subjected to both natural and artificial selection. Georges et al. [POLL gene and two microsatellites, GMPOLL-1 and GMPOLL-2. These loci are syntenic to the highly conserved gene for superoxide dismutase 1 (SOD1). In addition, in various breeds the POLL gene has been found to be linked to the microsatellites TGLA49, AGLA17, INRA212 and KAP8, located in the centromeric region of BTA1 close to the SOD1 locus [The recent increased availability of large genomic data sets and the identification of a few genes or loci as the targets of domestication or subsequent genetic improvement in cattle have renewed the investigation of the genomic effects of selection. Candidate genes and QTL have been described on both BTA1 -25 and Bs et al.  have demD1 locus ,23,25. TD1 locus  affectinD1 locus ), udder D1 locus ), udder D1 locus ), milk yD1 locus ), fat peD1 locus ), and esD1 locus -30).Bos taurus, we present microsatellite data using a relatively larger number of loci than previously reported, which mainly included the 30 microsatellite markers recommended by the International Society for Animal Genetics (ISAG)/Food and Agriculture Organization of the United Nations (FAO) working group . Among up (e.g. . We compBos taurus) populations including 366 individuals were analyzed. Finnish populations were represented by Finnish Ayrshire , Finnish Holstein-Friesian , Eastern Finncattle , Western Finncattle , and Northern Finncattle . We were able to inference the heterozygotic status at the POLL locus in 19 phenotypically polled cattle of the three Finnish native populations, on the basis of their offspring/parent phenotypes. In addition, there were 19 animals horned (recessive homozygotic) in the Finnish native populations. Istoben , Yakutian , and Kholmogory  cattle were sampled in Russia. Ukrainian Grey  and Danish Jersey  were sampled in Ukraine and Denmark, respectively. During sample collection, the pedigree information and the herdsman's knowledge were used to ensure the animals were unrelated. Additional information on these populations has been reported in previous publications [Microsatellite data from 10 different cattle  amonterature . The 23 terature  and in mhtml and . In thisHEXP), allelic richness (AR), and Weir and Cockerham's FST [Microsatellite variability, expected heterozygosity . Valuesfound in . The stafound in  using a y tables . A totalsion 4.0 . Fisher'sion 4.0  were appPossible departures from the standard neutral model of molecular evolution - potentially revealing demographic events or the existence of selective effects at certain loci - were examined for each locus using the Ewens-Watterson test ,45 and thttp://www.rubic.rdg.ac.uk/~mab/software.html, a currently distributed version of the original FDIST program as described by [θ, Weir & Cockerham's [θ centered on the empirical estimates. Then, the quantiles of the simulated FST within which the observed FST's fell and the P-values for each locus were determined. Initially an island model of population differentiation was used and the procedure repeated 50,000 times to generate 95% confidence intervals for neutral differentiation and to estimate P-values for departure of the loci from these expectations. Simulation parameters were under an infinite allele mutation model for 100 demes, 10 sample populations, sample sizes of 100, and a weighted FST similar to the trimmed mean FST calculated from the empirical distribution. Computed by removing the 30% highest and lowest FST values observed in the empirical data set, the trimmed mean FST is an estimate of the average "neutral" FST value uninfluenced by outlier loci . This mP-values . The apphttp://www.reading.ac.uk/Statistics/genetics/software.html package, which generates 2,000 Markov chain Monte Carlo (MCMC) simulated loci on the basis of the distribution of FST given the data. The method combines information over loci and populations in order to simultaneously estimate FST at the ith locus and the jth population, FST, for all i loci and j populations. A hierarchical model is implemented for FST asBeaumont & Balding's  hierarchi, βj and γij are locus, population and locus-by-population parameters, respectively [αi). Outliers from our data set were identified on the basis of the distribution following [FST as assumed in the above frequentist method of [FST for each population [where αectively . In thisollowing . Rather ethod of , this BApulation ,12.Neμ, where Ne is the effective population size and μ is the mutation rate. A locus linked to a beneficial mutation will have a smaller effective population size and consequently a reduction in variability below neutral expectations. The relative variance in variability, lnRθ, can be assessed instead by estimating the relative variance in repeat number, lnRV, or heterozygosity, lnRH, for loci between populations. The lnRV was calculated using the equation lnRV = ln (Vpop1/Vpop2) where Vpop1 and Vpop2 are the variance in repeat number for population 1 and population 2, respectively [H for each locus for a pair of populations as followsTo test for additional evidence of selection, we used the combination of statistics lnRH, lnRV and lnRθ' in the population pairwise comparisons. The principle behind these tests is that variability at a neutral microsatellite locus is given by θ = 4 ectively . The lnRH denotes expected heterozygosity (see equation 2 in [where ion 2 in ). In addion 2 in .The tests have been shown to be relatively insensitive to mutation rate, deviation from the stepwise mutation model, demographic history of population and sample size . As suggi, j), and for all loci, we calculated Fi and Fj  μ = 1 × 10-2, 1 × 10-3, and 1 × 10-4; ancestor population size Ne = 500, 5,000, and 50,000; times since an assumed bottleneck event T0 = 50, 500, and 5,000 generations; time since divergence t = 50 and 500; and population size before the split N0 = 50 and 500. In order to detect outlier loci potentially selected for the polled trait within the three Finnish native cattle populations, the DetSel program was run for comparison between the two subpopulations representing the definitely polled (n = 19) and horned (n = 19) animals, respectively.The coalescence simulation approach using the DetSel 1.0 program  was usedails see ,49) and FST = 0.117, 95% CI 0.108 - 0.125). FST values for an individual locus varied from 0.017 (SD = 0.011) at AGLA17 on BTA1 to 0.180 (SD = 0.057) at BMS2461 on BTA20. Mean population differentiations for loci on BTA1 and BTA20 were 0.126  and 0.118 , respectively. Neither of the values indicated significant difference from the average for loci on other chromosomes .A complete list of loci and their variability in the 10 cattle populations are shown in Table AR) and expected heterozygosity (HE), were in similar ranges as for microsatellites on BTA1, BTA20 and other autosomes, with the smallest variations observed at AGLA17 . The highest HE of 0.79 was observed at BM2113 (BTA2) and the highest AR of 11.36 at TGLA122 (BTA21). Most FIS values were positive and for some loci significantly positive. Of the 13 negative FIS values, seven occurred for loci on BTA20, and two for loci on BTA1. Loci on BTA1 and BTA20 did not show a significant reduction or increase in mean FIS compared with the loci on other autosomes . Given the range of observations of FIS at an individual locus, there were no marked difference among the three classes of loci .Levels of variation across populations, including allelic richness  higher than for non-syntenic markers  significant observations. However, a substantially smaller proportion  of significant (P < 0.05) pairs was found between non-syntenic markers. In general, significantly higher levels of LD were observed for syntenic markers on BTA1 and BTA20 than that for non-syntenic markers. There was no evidence of LD blocks on either of the chromosomes.The strength of pairwise linkage disequilibrium (LD) between markers was estimated and the average AGLA17, DIK5019, SOD1, AGLA29, BMS2361, BM2113, ETH10, ETH225, CSSM66, ETH152, TGLA227, HAUT24, and CSRM60) on 10 different chromosomes exhibiting significant probabilities for the Ewens-Watterson test based on both homozygosity (PH) and Fisher's exact test (PE) (see Table AGLA17) exhibited a significant (P < 0.05) deficit of heterozygosity and all the other 12 loci exhibited a significant (P < 0.05) excess in genetic diversity relative to the expected values; these patterns are consistent with directional and balancing selection, respectively. The 12 loci generated average P values significantly  below than the expected median value of 0.5. However, average P values of 0.313 for PH  and 0.232 for PE  were observed in the remaining 38 loci which were not under selection. The observation provided further evidence that selection affected genetic diversity at the microsatellites under selection.The Ewens-Watterson test enables detection of deviations from a neutral-equilibrium model as either a deficit or an excess of genetic diversity relative to the number of alleles at a locus (see ). When aFST values, identified four loci  as outliers showing footprints of selection in the analyses, including all 10 populations, at the 5% significance level. Of the four significant loci, three  with higher FST values indicated a sign of directional selection and one locus (AGLA17) appearing in the lower tail of the FST distribution suggested a signature potentially affected by balancing selection  were detected as being directionally selected and two (AGLA17 and TGLA227) as under balancing selection. Overall, across all the populations, two loci, AGLA17 and SOD1, exhibited the strongest evidence of selection with all three statistical approaches, which provided good support to their status as outliers due to selection. Two loci (DIK5019 and TGLA227) exhibited significant departure from the neutral expectations in two out of the three selection tests. Furthermore, 12 loci  can be regarded as candidates affected by selection, but were revealed only in one of the three tests. Interestingly, according to ENSEMBL cow genome http://www.ensembl.org/Bos_taurus/Info/Index the significant locus AGLA17 under balancing selection was about 1.78 cM upstream from the candidate locus for POLL, whereas locus SOD1 under directing selection was located about 3.87 cM downstream from the candidate locus. It should be noted that the FST-based tests of selection are prone to false positives because of sensitivity to demographic history [FST-based tests to be enriched for the true positives in further tests.The results of the analyses with the FDIST2 program are presented in Table n Figure . In the t Figure , which w history . NeverthBos taurus, thus the region under selection is likely to be quite wide. Despite the possible presence of a few false positives, the full set of seven loci  was used for further analyses. The lnRθ methods  use heterozygosity or variance difference, rather than population divergence, to test for selection. Significant results for the lnRθ tests for selective sweeps involve the two loci (AGLA17 and SOD1) detected by the Ewens-Watterson test and the FST-based tests for pairwise combinations (n = 12) of three native Finnish cattle populations and four old native populations from Russia and Ukraine  or very significant (P < 0.01) results were observed at AGLA17 and SOD1, respectively, in the three tests. Both loci (AGLA17 and SOD1) appeared in all three different measures of lnRθ for eight or more comparisons  vs. the native populations from Russia and Ukraine , which suggested that selective sweeps had taken place in the Finnish native populations.Significant results for selective sweeps at loci n = 19) and horned (n = 19) cattle, loci AGLA17 and SOD1 were significantly outside the 99% confidence interval . The locus SOD1 presented a higher differentiation  than expected, suggesting that it could have been affected by the action of diversifying selection among homogeneous gene pools and populations. In contrast, the locus AGLA17 presented a lower genetic differentiation than expected, which could represent signatures of homogenizing selection among populations and/or balancing selection within populations. All three methods identified loci SOD1 and AGLA17 as good candidates for selection on the polled trait. However, several significant loci were detected only by one or two of the tests and thus could not be accepted as reliable outliers with the remaining tests. The results obtained by the three methods are not totally consistent, probably because of the difference in statistical power using multiple measures of variability, each of which measures different parameters and relies on different assumptions, e.g. heterozygosity and variance in allele size [Because revealing outlier loci in genome scans currently depends on statistical tests, one of the main concerns is to highlight truly significant loci while minimizing the detection of false positives . Using aele size , as detaele size -55.P < 0.05) results at SOD1 and AGLG17 according to the three estimators of lnRθ  tests yielded a high number of significant (Rθ Table . This fiFST) or reduced variability (lnRθ). Our proposed tests consider lnRθ of several linked loci for the inference of selection. While the single-locus lnRθ-test is largely independent of the demographical past, the additional power of linked loci is balanced by the cost of an increasing dependence of the demographic past due to the fact that LD is extremely sensitive to the demographic history. Thus, pairwise analyses between sub-populations may decrease the demographic effects in accounting for the selection. As indicated in Figure SOD1 and AGLA17) that were probably subject to selection in the three Finnish native populations. Thus, we concluded that the distribution of variability at these loci could have been shaped by forces other than demographic effects e.g. genetic drift. Although the locus DIK4591 was located on the edge or fell just outside the high probability region of the expected conditional distribution in the Finnish native populations, we must be cautious about the locus because the estimation of Fi parameters is discontinuous as a result of the discrete nature of the data, i.e. the allele counts . Thus, we conducted the three different neutrality tests , setting a 95% ca.1100 kb and ca.10300 kb in this study  on the same chromosome, this indicates a high probability that one or several good candidate genes (or QTLs) is/are tightly linked to some of the microsatellites. In many of the cases examined to date, selective sweeps have affected only a very small region, potentially containing only one or a few genes, except in the case of extremely strong selection (see ). EmpiriAGLA17 and SOD1) probably linked to the candidate gene for the polled trait in the populations investigated. The polled trait is an autosomal dominant trait in cattle and to date the genes controlling this trait have not been specifically identified. However, the gene causing the absence of horns is known to be at the centromeric end of BTA1. Several factors have potentially driven evolution of the functionally important candidate locus including artificial selection and mating system. In Finnish native cattle populations, polled animals were particularly favored during selective breeding. However, we did not detect any locus under selection on BTA20 despite that the fact that several microsatellites including GHRJA surround the growth hormone receptor gene. Growth hormone receptor belongs to the large superfamily of class 1 cytokine receptors. It has various roles in growth, lactation and reproduction in cattle and has been identified as a candidate gene affecting a few key quantitative traits. Therefore, it is not specific to dairy traits but to traits related with growth, lactation and reproduction. Among the cattle populations investigated here, no contrasting differences in growth, lactation or reproduction was observed. In addition, a recent study on the evolution of the cytoplasmic domains of the growth hormone receptor gene in Artiodactyla (see [We detected two microsatellite loci (yla (see ) has sughttp://www.ncbi.nlm.nih.gov/projects/genome/guide/cow/)in bovine makes it possible to develop more precise approaches with other much more frequent markers such as SNP. Genotyping an additional set of high density SNP between AGLA17 and SOD1 markers in the populations investigated will definitely give more precise information on selection and LD in the region.Unfortunately, due to the lack of information on the mutation and recombination rates, as well as the effective population size for these data, estimation of the selection coefficient is not possible here (see ). Given Because the populations studied here are not experimental, they differ for many characteristics other than the polled and horned traits. Thus, some of the genetic differentiation could have been due to other selective forces, e.g. pathogens. In addition, since our data violate at least partly the model assumptions of equal population size and migration rates between populations for the FDIST2 test, the outliers from the test alone should be considered with caution although the multiple neutrality tests based on different assumptions and parameter estimation can minimize the possibility of false positives. Moreover, selection is not the only possibility for changes in the distribution of variation to occur at particular loci, reduced variation or increased differentiation can result from chance alone, e.g. genetic drift, bottlenecks or founder events . To obtaSOD1 and AGLA17) that are "selection candidate" targets associated with the horned/polled trait in cattle. This result could be further confirmed by using a more densely spaced set of markers. It would also be of great interest to see if similar patterns of selection around the POLL gene are observed in commercial beef breeds such as Australian Brangus, Angus and Hereford breeds, where dehorning and breeding practices for polled cattle have been an accepted part of cattle management for generations. Another future challenge is to verify the signal of artificial selection on the POLL gene, possibly using the next generation sequencing technology to detect the nucleotide variation of the gene between polled and horned cattle. In addition, the approach we have taken in this paper can be easily extended to other cases and marker types. For example, diversity among cattle has been directed by man towards different goals  during many generations of selection. Each of these selection events has potentially left a signature of selection on the genes and their neighboring loci that could be detected by using tests such as we have applied here. As a marker technology, SNP would offer the advantage of higher throughput when scanning the genome for evidence of hitchhiking selection.Our microsatellite data from northern Eurasian cattle populations empirically indicate a practical approach for identifying the best candidate loci under hitchhiking selection by simultaneously applying multiple neutrality tests based on different assumptions and parameter estimations. By analyzing microsatellite markers adjacent to functional genes, we identified two loci (The authors declare that they have no competing interests.MHL designed the study, performed the data analysis and wrote the manuscript. TI-T did the laboratory work and contributed to the manuscript writing and data analysis. HL did the laboratory work, contributed to the manuscript writing and data analysis. JK planned and coordinated the whole study, and contributed to the manuscript writing. All the authors read and approved the final manuscript.


Plasmodium sp., including the most serious agent Plasmodium falciparum, depends on the local epidemiological and demographic situations, such as the incidence of infected people, the vector transmission intensity and migration of inhabitants (i.e. exchange between sites). Analysing the structure of P. falciparum populations at a large scale, such as continents, or with markers that are subject to non-neutral selection, can lead to a masking and misunderstanding of the effective process of transmission. Thus, knowledge of the genetic structure and organization of P. falciparum populations in a particular area with neutral genetic markers is needed to understand which epidemiological factors should be targeted for disease control. Limited reports are available on the population genetic diversity and structure of P. falciparum in Thailand, and this is of particular concern at the Thai-Myanmar and Thai-Cambodian borders, where there is a reported high resistance to anti-malarial drugs, for example mefloquine, with little understanding of its potential gene flow.The population structure of the causative agents of human malaria, P. falciparum populations were analysed using 12 polymorphic apparently neutral microsatellite loci distributed on eight of the 14 different chromosomes. Samples were collected from seven provinces in the western, eastern and southern parts of Thailand.The diversity and genetic differentiation of P. falciparum transmission areas (average HS = 0.65 ± 0.17), where the lowest is observed in South America and the highest in Africa. However, uniquely the Yala province, had only a single multilocus genotype present in all samples, leading to a strong geographic differentiation when compared to the other Thai populations during this study. Comparison of the genetic structure of P. falciparum populations in Thailand with those in the French Guyana, Congo and Cameroon revealed a significant genetic differentiation between all of them, except the two African countries, whilst the genetic variability of P. falciparum amongst countries showed overlapping distributions.A strong difference in the nuclear genetic structure was observed between most of the assayed populations. The genetic diversity was comparable to the intermediate level observed in low Plasmodium falciparum shows genetically structured populations across local areas of Thailand. Although Thailand is considered to be a low transmission area, a relatively high level of genetic diversity and no linkage disequilibrium was found in five of the studied areas, the exception being the Yala province (Southern peninsular Thailand), where a clonal population structure was revealed and in Kanchanaburi province (Western Thailand). This finding is particularly relevant in the context of malaria control, because it could help in understanding the special dynamics of parasite populations in areas with different histories of, and exposure to, drug regimens. Plasmodium falciparum, the agent of the most malignant form of malaria that accounts globally for some 300–600 million cases of clinical malaria and 1.5 – 2.7 million deaths each year. Although P. falciparum can cause the most severe of the four human forms of malaria [P. falciparum drug resistance [P. falciparum strains have become more resistant, in terms of both resistance level and frequency in the population, to mefloquine, in addition to the widespread resistance to chloroquine, and sulphadoxine/pyrimethamine observed in many endemic areas, especially within those provinces located along the Thai-Myanmar border , or the Thai-Cambodian border . As a result of declining mefloquine efficacy, a combination of mefloquine and artesunate is now administered in those areas [Plasmodium falciparum gene flow between transmission areas and the existent level of P. falciparum population genetic diversity are directly implicated in the spread of drug resistance. Understanding the genetic complexity and organization (structure) of P. falciparum populations is a crucial aspect for the control of this disease, since the genetic diversity and population structure of P. falciparum in each location will have profound impacts on clonal diversity [Malaria is still one of the most important infectious diseases, endemic in the tropical and sub-tropical parts of about 102 countries, especially in the African continent. In Thailand, although the total number of malaria cases has been decreasing annually [se areas . The Plaiversity , dynamiciversity , persistiversity  and malaiversity .P. falciparum is known to exhibit a diverse and patchy array of population genetic characteristics, which are apparently correlated with local levels of endemicity and transmission intensity [Globally, ntensity . Howeverntensity . It thusP. falciparum transmission rate [P. falciparum in different locations have been reported [P. falciparum populations in Kenya was also observed where this form of malaria is also transmitted at a relatively high frequency [The ion rate ,15, and ion rate , which dion rate -18. At areported ,20, whilreported . Moreoverequency . Geograprequency ,23. Howerequency ,24.P. falciparum, as well as other malaria parasites such as Plasmodium vivax, is essential for predicting how fast given phenotypes, such as drug or host resistance or novel antigenic variants, will originate and spread within and between populations [P. falciparum populations, and to lead to an efficient management of this disease agent in particular areas. Indeed, the genetic composition and evolutionary change of each P. falciparum population is of great importance to ascertain the ecophysiology of the parasite and host-parasite interactions, as well as the evolution of resistance in the pathogen. Studies using the extensive polymorphism in antigen coding loci provide little valid information on the population structure of P. falciparum since, being under strong but varying selection, they reflect the combined effects of population history and natural selection [Understanding of the genetic structure of ulations . Studieselection .P. falciparum population genetic studies have focused on the boundary with Myanmar in the western part using different genetic markers [P. falciparum genetic structure relies on the epidemiological and demographical situations encountered in different areas and may change within a relatively short period of time. In this report, the population genetic structure of P. falciparum, is evaluated in seven provinces of Thailand, using 12 polymorphic and apparently neutral nuclear microsatellite loci as genetic markers. These were also compared with samples from other endemic countries, while using the same loci described published literature for other populations. Such studies could later be extended to other endemic areas displaying different treatment policies and different uses of prophylactic anti-malarial drugs.In Thailand, most  markers ,27,28, bP. falciparum based malaria incidences in Thailand [The parasitized blood samples were collected from patients who attended for malarial diagnosis at clinics in seven provinces of Thailand from 2002 to 2007 Figure . All patThailand . In 2006Thailand . Four ofThailand . The samP. falciparum-infected human blood samples from two sources in Thailand were used in the study: 1) samples from in vitro culture, which had been preserved in liquid nitrogen at the Malaria Research Programme, College of Public Health Sciences, Chulalongkorn University, and 2) blood samples from P. falciparum malaria patients preserved on filter papers. Blood samples collected before 2004 were in vitro cultivated after blood collection from the malaria patient, and the cultivated samples were then cryopreserved in liquid nitrogen until being used in this study. The DNA was extracted from cultured parasites by standard phenol/chloroform extraction of whole blood. Thereafter, between 2004 and 2007, parasitized blood samples were collected from thick film-confirmed falciparum malaria patients, before drug administration. These patients were informed of the objectives of this study, agreed to participate in it, and signed a standard consent form. Blood samples were taken from finger-pricks of P. falciparum-infected  patients, and were absorbed onto 3 MM Whatman filter paper and air-dried. DNA from the dried blood sample was subsequently extracted using a DNeasy Tissue kit (Qiagen) according to the manufacturer's instructions. The blood collection protocol was approved by the ethical committee of the Institute of Health Research, Chulalongkorn University. Socio-demographic information of patients was collected by interview, especially the most likely place of infection, to help identify the parasite's origin. The provincial origins and year of collection of the infected blood samples, and the likely country of infection source where not Thailand, are summarized in Table The P. falciparum from each infected blood sample, the DNA was extracted and used as template for the PCR based amplification of 12 established polymorphic and apparently neutral microsatellite loci distributed on eight of the 14 different chromosomes [et al [For genotyping omosomes , as listomosomes , followis [et al . For comP. falciparum clone infection were analysed, because of the difficulty in defining multi-locus genotypes in samples with a polyclonal infection. To this end, given that these 12 loci are all single genome copy, not in linkage, and that P. falciparum from the blood (schizonts and early trophozoites) has a haploid genome, then isolates with more than one allele at more than one locus were deemed to be from multiple infections. Genetic diversity was measured by the allelic richness per locus and sample (RS) and Nei's unbiased expected heterozygosity (HS), after adaption to haploid data, using FSTAT, v2.9.4 [Only isolates that successfully amplified at all 12 loci within the Thai samples or at the selected seven loci for international samples were included in the respective analyses. Moreover, for population genetics, only those samples that showed a single , v2.9.4 . Differe® software [Linkage disequilibrium analyses between pairs of loci were performed with FSTAT v2.9.4. Genetic variation and differentiation were analyzed using FSTAT. Canonical correspondence analysis (CCA) was carried out to illustrate the measures of population structure, using CANOCOsoftware ,33. Onlysoftware , which aP. falciparum, i.e. schizonts and early trophozoites, present in human blood samples were analysed, the presence of multiple infections was assessed in a given isolate by the presence of more than one allele at any of the microsatellite locus surveyed. The minimal multiplicity of infection for each sample  was analysed locus by locus, and was estimated from the locus that exhibited the highest number of alleles in a given isolate. Therefore, a single P. falciparum isolate infection is deemed to correspond to an isolate exhibiting only one allele at each of the 12 loci investigated. Of course, multiple infections that are genetically distinct overall but identical at the 12 microsatellite loci, for example recombinants or new mutations at other sites, or whose differences lead to null alleles and so would not be detected, would not be distinguished. Although the former would still be likely to be closely related isolates all the same, the later maybe quite different and thus the data represents the minimal rather than the actual level of multiple infections. Accordingly, the minimal proportion of multiple infections in each sample group was assessed for each locus and between loci in a sample group , leaving 346 single isolate infected (72%) samples suitable for genotyping and population genetic analysis. The multiple infected samples were distributed across six of the seven provinces assayed, with frequencies of multiple infections as a ratio of infected individuals to the total number of samples ranging from 44% in Maehongson to 22% in the Ubonratchathani population  in each population was 0.68 ± 0.23, 0.63 ± 0.25, 0.60 ± 0.18, 0.70 ± 0.21, 0.56 ± 0.25, 0.62 ± 0.20 and 0 in the Maehongson, Tak, Kanchanaburi, Ubonratchathani, Trat, Ranong and Yala populations, respectively.For the population genetic analysis, based on 12 polymorphic nuclear microsatellite loci, only the 346 single Linkage disequilibrium (LD) was tested at three levels, (i) for the whole population (ii) for the three regional areas; and (iii) amongst sites.p-value = 0.00076) associations of loci were found.(i) For within the whole population , out of the 66 possible pairwise linkages, 34 significant , respectively.(ii) For within the three regions, we found that in the Eastern region , Western  and Southern area, , there were 0, 3 and 46 significant combinations of loci (out of 66) that were potentially in linkage  linkage disequilibrium was found in any combined pair of loci except for two combinations in Kanchanaburi (Polyα × TA81 and Ara2 × TA81), and, of course, a total linkage disequilibrium existed in the Yala population, since all samples have the same genotype.(iii) For within all the sites, no significant (FST) was estimated using the FSTAT software. A permutation test was applied  permuting alleles amongst all samples for all loci to test whether FST significantly differed from zero. A significant differentiation  of P. falciparum populations between sites was observed, with a very high differentiation between the Yala population and all the other populations  between the western, eastern and southern parts of Thailand with FST values equal to (i) 0.0478 for the western versus the eastern regions, (ii) 0.1321 for the western versus the southern regions and (iii) 0.1199 for the eastern versus the southern regions.Genetic differentiation  they can pass through several generations, and (ii) these genotypes could carry important traits, such as drug-resistance genes.P. falciparum populations with the already published reports for other populations, the observed patterns at seven common microsatellite loci , were compared with those observed from blood samples of two African countries, Congo (n = 15) and the Cameroon (n = 35), and one South American country, French Guyana (n = 137) (unpublished data). The gene diversity in the Thai populations (HS = 0.65 ± 0.17) was lower than those found in the two African countries , but higher than that of French Guyana (HS = 0.54 ± 0.08). Significant LD was observed in the two low transmission countries . The high LD value observed in Thailand is due to the Yala sample. The canonical correspondence analysis (CCA), searches for multivariate relationships between two data sets  [P. falciparum populations. A graphic representation of the results showed overlapping distributions . This suggests some degree of geographic differentiation. Indeed, all populations in different countries displayed significant genetic differentiation except between the two African countries, Congo and Cameroon  , was alsP. falciparum populations in different continents revealed that African countries with high transmission intensities had the highest population genetic diversity. The genetic diversity decreased with low transmission intensities, as in Thailand (Asia) and French Guyana (South America). The average genetic diversity of P. falciparum populations in Thailand (HS = 0.65 ± 0.17) was, however, close to that of the high transmission intensity level of Papua New Guinea [At the level of continents, the genetic structure of  HS = 0.6 ± 0.17 ww Guinea  and Brazw Guinea , also baP. falciparum in Thailand showed a deviation from random mating, in congruence with the French Guyana population, whilst, in contrast, the two African populations showed likely panmixia. The proportion of multiple isolate infections, and the number of clones in an individual host, which are related to the transmission intensity of malaria parasites in any endemic area, are important parameters that affect P. falciparum mating, because fertilization may occur between gametes of the same or different genotypes in mosquitoes. Therefore, an inverse correlation between the proportion of multiple clone infections and the degree of linkage disequilibrium is expected. Even though the genetic diversity of Thai populations was observed at an intermediate level, and multiple isolate infections occurred frequently, a significant linkage disequilibrium was found which could be caused by, (i) a higher chance of inbreeding (selfing) in this parasite population, or (ii) structure formation within populations into sub-populations of each foci in Thailand (i.e. the Wahlund effect). The latter reason would be more likely to explain this finding because samples from all the foci from across the country (the seven different provinces) were pooled in the analysis.The deduced mating patterns of P. falciparum analysis with 12 polymorphic nuclear microsatellite loci in isolates from multiple populations in seven provinces of Thailand.In Thailand, the malaria incidence rate shows a high spatial heterogeneity across the country, with high incidence regions found nearby the borders with Myanmar and Cambodia . This stP. falciparum populations in the areas bordering with Myanmar revealed a similar level of genetic diversity (0.60 <HS <0.68), whilst the populations bordering with Laos and Cambodia had slightly higher or lower levels of genetic diversity at 0.70 and 0.56 in Ubonratchathani and Trat, respectively. Although the multiple isolate infection rates in those areas bordering with Myanmar  were higher than that of Ubonratchanthani (22%) and comparable to that in Trat (36%), this could be explained by the notion that (i) the parasite mating in those areas is preferentially inbreeding, or (ii) the relatively small amount of sampled individuals in Trat and Ubonratchathani leading to a stochastic masking of the true population structure. The Yala population, however, showed an extreme difference in the gene diversity compared the other samples since it showed only a unique haplotype in all sampled individuals, but again may be a transient clonal expansion of an immigrant infection.The P. falciparum gene pool in each endemic area and affect the epidemiology of the P. falciparum caused malaria in these areas. Besides this migration rate of humans in each foci, there is additionally the factor of the geographic barrier between Tak and Kanchanaburi provinces which may be another cause of genetic differentiation by partial isolation by distance and breaking down of panmixis. Thus, the genetic differentiation found between populations within each province of Thailand may reflect both a variation of the transmission rate in each location and the migration rate of people from one location to another. Certainly seasonal migration has been suspected as a leading cause of malaria transmission in these areas [Anopheles vectors in local areas may also have an effect on the parasite structure. In Thailand, Anopheles aconitus sensu lato, Anopheles baimaii, Anopheles dirus, Anopheles maculatus, Anopheles minimus and Anopheles pseudowillmori have been incriminated as important vectors of P. falciparum in humans, and they are differentially distributed throughout the country [P. falciparum. Thus, although differences in the population structure of P. falciparum between two vectors (Anopheles gambiae and Anopheles funestus) have been preliminarily investigated [In contrast to the Yala population, a low but significant population structure was observed amongst the provinces that border with Myanmar, Laos and Cambodia. In the case of the Myanmar bounding provinces, the significant genetic structure detected between populations  was likely to be due to the fact that the pattern of human movement from neighbouring countries into each of these four provinces was different. In the Tak and Ranong provinces, the numerous movements in to Thailand from Myanmar from immigrant laborers, and from patients resident in Myanmar who cross the border to seek free malaria treatment, account for the high proportion (approximately 70%) of sampled individuals being likely to have been infected in Myanmar. Indeed, the same genotype was found in seven samples collected in 2007 in Kanchanaburi, from patients infected in Thailand and Myanmar. These, imported cases may play an important role on the se areas . However country -38, and stigated , no suchP. falciparum infected blood samples collected one year apart (2005 – 2006 and 2006 – 2007) was observed only in Ranong, where one case was found in a patient infected from Myanmar and the following year was found from a patient infected in Thailand. If a generation time is assumed to be two months, this parasite genotype has been transmitted through six generations without detectable change across the 12 loci (eight/14 chromosomes) due to recombination. This may imply that if this P. falciparum strain has an advantage (fitness) in that particular environment, it will propagate and expand in this population.Importantly, the same multilocus genotypes of P. falciparum haplotypes collected in the same year and over two years were detected. A unique spatial haplotype, distinct from the other populations, was found in all patients in the Yala population, which were obtained from individual residents in four different sub-districts in Bannang Sata and other three nearby districts during May – July 2007. Three possible explanations are available to explain this trend. Firstly, that a P. falciparum isolate with this genotype had a high infectivity in the mosquito's midgut or carried some genetic characteristics, such as drug resistance, which gave it a significant benefit over other haplotypes. Secondly, that there is no inhabitant movement into this area due to the political violence, and so as a result no new haplotype parasite was introduced into this area (no gene flow). Thirdly, there maybe a rare new gene influx by immigration but which was subsequently lost due to selection or stochastic failure to progress to a stable infection threshold. Finally, is the possibility that the decrease in sanitary cover in this area allowed this particular genotype to spread into the population rapidly. Thus, a longitudinal study in the population dynamics in this area is required. Nevertheless, this is the first study to reveal the potential clonal expansion of a single haplotype in a malaria endemic area.In this study, both a spatial and a temporal distribution of Plasmodium falciparum populations are highly structured in Thailand suggesting local and differential routes of genetic evolution for this parasite. Although Thailand is considered as a low transmission area, a high level of genetic diversity and weak linkage disequilibrium were found in most sites, except the transient infections in the Yala province in one year where only a single genotype was found. Moreover, (i) identical genotypes were encountered in the same site but from patients coming from different localities (i.e. Myanmar and Thailand), and (ii) two identical genotypes were observed from two successive years at the same site (i.e. Ranong); these findings are particularly relevant in the context of malaria control. They would help to understand the specific dynamics of malaria and parasite populations in areas displaying a different history of drug applications.The authors declare that they have no competing interests.TP carried out field work, the molecular genetic work, analysis, interpretation of data, and drafted the manuscript. CA carried out the molecular genetic work and data analysis. NK contributed to field work, DNA extraction of cultivated parasite. AS and NS were responsible for parasite cultivation. PD and FR carried out the statistic analysis, guidance for interpretation of data, participated in the manuscript preparation and revision. CS participated in the design of the study, the manuscript preparation and revision. PH participated in the design of the study, the manuscript preparation and revision and coordination. All authors read and approved the final manuscript.Allele frequencies. Allele frequencies at 12 microsatellite loci in P. falciparum from seven Thai populations.Click here for file