Hitchhiking effect of a beneficial mutation spreading in a subdivided population

A central problem in population genetics is to detect and analyze positive natural selection by which beneficial mutations are driven to fixation. The hitchhiking effect of a rapidly spreading beneficial mutation, which results in local removal of standing genetic variation, allows such an analysis using DNA sequence polymorphism. However, the current mathematical theory that predicts the pattern of genetic hitchhiking relies on the assumption that a beneficial mutation increases to a high frequency in a single random-mating population, which is certainly violated in reality. Individuals in natural populations are distributed over a geographic space. The spread of a beneficial allele can be delayed by limited migration of individuals over the space and its hitchhiking effect can also be affected. To study this effect of geographic structure on genetic hitchhiking, we analyze a simple model of directional selection in a subdivided population. In contrast to previous studies on hitchhiking in subdivided populations, we mainly investigate the range of sufficiently high migration rates that would homogenize genetic variation at neutral loci. We provide a heuristic mathematical analysis that describes how the genealogical structure at a neutral locus linked to the locus under selection is expected to change in a population divided into two demes. Our results indicate that the overall strength of genetic hitchhiking--the degree to which expected heterozygosity decreases--is diminished by population subdivision, mainly because opportunity for the breakdown of hitchhiking by recombination increases as the spread of the beneficial mutation across demes is delayed when migration rate is much smaller than the strength of selection. Furthermore, the amount of genetic variation after a selective sweep is expected to be unequal over demes: a greater reduction in expected heterozygosity occurs in the subpopulation from which the beneficial mutation originates than in its neighboring subpopulations. This raises a possibility of detecting a "hidden" geographic structure of population by carefully analyzing the pattern of a selective sweep.     

The Hitchhiking Effect on the Site Frequency Spectrum of DNA Polymorphisms

The level of DNA sequence variation is reduced in regions of the Drosophila melanogaster genome where the rate of crossing over per physical distance is also reduced. This observation has been interpreted as support for the simple model of genetic hitchhiking, in which directional selection on rare variants, e.g., newly arising advantageous mutants, sweeps linked neutral alleles to fixation, thus eliminating polymorphisms near the selected site. However, the frequency spectra of segregating sites of several loci from some populations exhibiting reduced levels of nucleotide diversity and reduced numbers of segregating sites did not appear different from what would be expected under a neutral equilibrium model. Specifically, a skew toward an excess of rare sites was not observed in these samples, as measured by Tajima's D. Because this skew was predicted by a simple hitchhiking model, yet it had never been expressed quantitatively and compared directly to DNA polymorphism data, this paper investigates the hitchhiking effect on the site frequency spectrum, as measured by Tajima's D and several other statistics, using a computer simulation model based on the coalescent process and recurrent hitchhiking events. The results presented here demonstrate that under the simple hitchhiking model (1) the expected value of Tajima's D is large and negative (indicating a skew toward rare variants), (2) that Tajima's test has reasonable power to detect a skew in the frequency spectrum for parameters comparable to those from actual data sets, and (3) that the Tajima's Ds observed in several data sets are very unlikely to have been the result of simple hitchhiking. Consequently, the simple hitchhiking model is not a sufficient explanation for the DNA polymorphism at those loci exhibiting a decreased number of segregating sites yet not exhibiting a skew in the frequency spectrum.     

The mean and variance of the number of segregating sites since the last hitchhiking event

 Tight linkage may cause a reduction of nucleotide diversity in a chromosomal region if an advantageous mutation appears in that region which is driven to fixation by directional selection. This process is usually called genetic hitchhiking. If selection is strong, the entire process takes place during a time period of length 2s ln (2N) that is very short relative to 2N generations [s is the selection coefficient of the advantageous mutation and N the effective diploid population size]. On the time scale of 2N generations, which is characteristic for neutral evolution, we may therefore call this process a hitchhiking event. Using coalescent methods, we analyzed a model in which a hitchhiking event occurred in a chromosomal region of zero-recombination in the past at time x. Such a hitchhiking “catastrophe” wipes out completely genetic variation that existed in a population before that time. Standing variation observed at present must therefore be due to mutations that have arisen since time point x. Assuming that all newly arising mutations are neutral, we derived expressions for the expectation, variance and also for the higher moments of the number of nucleotide sites segregating in a sample of n genes as a function of x. The result for the first moment is then used to estimate the time back to the last hitchhiking event based on DNA polymorphism data from Drosophila. Assuming that directional selection is the sole determinant of the level of genetic variation in the gene regions surveyed, we obtained estimates of x that were typically in the order of 0.1N generations. Received 14 May 1996; received in revised form 26 August 1996     

Reduced Levels of DNA Polymorphism and Fixed between-Population Differences in the Centromeric Region of Drosophila Ananassae

We have estimated DNA sequence variation within and between two populations of Drosophila ananassae, using six-cutter restriction site variation at vermilion (v) and furrowed (fw). These two gene regions are located close to the centromere on the left and right X chromosome arms, respectively. In the fw region, no DNA polymorphism was detected within each population. In the v region, average heterozygosity per nucleotide was very low in both populations (pi = 0.0005 in the Burma population, and 0.0009 in the India population). These estimates are significantly lower than those from loci in more distal gene regions. The distribution of DNA polymorphisms between both populations was also striking. At fw, three fixed differences between the Burma and India populations were detected (two restriction site differences and one insertion/deletion of approximately 2 kb). At v, each DNA polymorphism in high frequency in the total sample was nearly fixed in one or the other population, although none of them reached complete fixation. The observed pattern of reduced variation within populations and fixed differences between populations appears to correlate with recombination rate. We conclude that recent hitchhiking associated with directional selection is the best explanation for this pattern. The data indicate that different selective sweeps have occurred in the two populations. The possible role of genetic hitchhiking in rapid population differentiation in gene regions of restricted recombination is discussed.     

Natural selection at linked sites in humans

Theoretical and empirical work indicates that patterns of neutral polymorphism can be affected by linked, selected mutations. Under background selection, deleterious mutations removed from a population by purifying selection cause a reduction in linked neutral diversity. Under genetic hitchhiking, the rise in frequency and fixation of beneficial mutations also reduces the level of linked neutral polymorphism. Here we review the evidence that levels of neutral polymorphism in humans are affected by selection at linked sites. We then discuss four approaches for distinguishing between background selection and genetic hitchhiking based on (i) the relationship between polymorphism level and recombination rate for neutral loci with high mutation rates, (ii) relative levels of variation on the X chromosome and the autosomes, (iii) the frequency distribution of neutral polymorphisms, and (iv) population-specific patterns of genetic variation. Although the evidence for selection at linked sites in humans is clear, current methods and data do not allow us to clearly assess the relative importance of background selection and genetic hitchhiking in humans. These results contrast with those obtained for Drosophila, where the signals of positive selection are stronger.     

The hitchhiking effect on linkage disequilibrium between linked neutral loci

We analyzed a three-locus model of genetic hitchhiking with one locus experiencing positive directional selection and two partially linked neutral loci. Following the original hitchhiking approach by Maynard Smith and Haigh, our analysis is purely deterministic. In the first half of the selected phase after a favored mutation has entered the population, hitchhiking may lead to a strong increase of linkage disequilibrium (LD) between the two neutral sites if both are <0.1 s away from the selected site (where s is the selection coefficient). In the second half of the selected phase, the main effect of hitchhiking is to destroy LD. This occurs very quickly (before the end of the selected phase) when the selected site is between both neutral loci. This pattern cannot be attributed to the well-known variation-reducing effect of hitchhiking but is a consequence of secondary hitchhiking effects on the recombinants created in the selected phase. When the selected site is outside the neutral loci (which are, say, <0.1s apart), however, a fast decay of LD is observed only if the selected site is in the immediate neighborhood of one of the neutral sites (i.e., if the recombination rate r between the selected site and one of the neutral sites satisfies r<0.1 s). If the selected site is far away from the neutral sites (say, r > 0.3 s), the decay rate of LD approaches that of neutrality. Averaging over a uniform distribution of initial gamete frequencies shows that the expected LD at the end of the hitchhiking phase is driven toward zero, while the variance is increased when the selected site is well outside the two neutral sites. When the direction of LD is polarized with respect to the more common allele at each neutral site, hitchhiking creates more positive than negative linkage disequilibrium. Thus, hitchhiking may have a distinctively patterned LD-reducing effect, in particular near the target of selection.     

Variation after a selective sweep in a subdivided population

The effect of genetic hitchhiking on neutral variation is analyzed in subdivided populations with differentiated demes. After fixation of a favorable mutation, the consequences on particular subpopulations can be radically different. In the subpopulation where the mutation first appeared by mutation, variation at linked neutral loci is expected to be reduced, as predicted by the classical theory. However, the effect in the other subpopulations, where the mutation is introduced by migration, can be the opposite. This effect depends on the level of genetic differentiation of the subpopulations, the selective advantage of the mutation, the recombination frequency, and the population size, as stated by analytical derivations and computer simulations. The characteristic outcomes of the effect are three. First, the genomic region of reduced variation around the selected locus is smaller than that predicted in a panmictic population. Second, for more distant neutral loci, the amount of variation increases over the level they had before the hitchhiking event. Third, for these loci, the spectrum of gene frequencies is dominated by an excess of alleles at intermediate frequencies when compared with the neutral theory. At these loci, hitchhiking works like a system that takes variation from the between-subpopulation component and introduces it into the subpopulations. The mechanism can also operate in other systems in which the genetic variation is distributed in clusters with limited exchange of variation, such as chromosome arrangements or genomic regions closely linked to targets of balancing selection.     

Evidence for genetic hitchhiking effect associated with insecticide resistance in Aedes aegypti.

Information on genetic variation within and between populations is critical for understanding the evolutionary history of mosquito populations and disease epidemiology. Previous studies with Drosophila suggest that genetic variation of selectively neutral loci in a large fraction of genome may be constrained by fixation of advantageous mutations associated with hitchhiking effect. This study examined restriction fragment length polymorphisms of four natural Aedes aegypti mosquito populations from Trinidad and Tobago, at 16 loci. These populations have been subjected to organophosphate (OP) insecticide treatments for more than two decades, while dichlor-diphenyltrichlor (DDT) was the insecticide of choice prior to this period. We predicted that genes closely linked to the OP target loci would exhibit reduced genetic variation as a result of the hitchhiking effect associated with intensive OP insecticide selection. We also predicted that genetic variability of the genes conferring resistance to DDT and loci near the target site would be similar to other unlinked loci. As predicted, reduced genetic variation was found for loci in the general chromosomal region of a putative OP target site, and these loci generally exhibited larger F(ST) values than other random loci. In contrast, the gene conferring resistance to DDT and its linked loci show polymorphisms and genetic differentiation similar to other random loci. The reduced genetic variability and apparent gene deletion in some regions of chromosome 1 likely reflect the hitchhiking effect associated with OP insecticide selection.     

Analysis of a genetic hitchhiking model, and its application to DNA polymorphism data from Drosophila melanogaster

Begun and Aquadro have demonstrated that levels of nucleotide variation correlate with recombination rate among 20 gene regions from across the genome of Drosophila melanogaster. It has been suggested that this correlation results from genetic hitchhiking associated with the fixation of strongly selected mutants. The hitchhiking process can be described as a series of two-step events. The first step consists of a strongly selected substitution wiping out linked variation in a population; this is followed by a recovery period in which polymorphism can build up via neutral mutations and random genetic drift. Genetic hitchhiking has previously been modeled as a steady-state process driven by recurring selected substitutions. We show here that the characteristic parameter of this steady-state model is alpha v, the product of selection intensity (alpha = 2Ns) and the frequency of beneficial mutations v (where N is population size and s is the selective advantage of the favored allele). We also demonstrate that the steady-state model describes the hitchhiking process adequately, unless the recombination rate is very low. To estimate alpha v, we use the data of DNA sequence variation from 17 D. melanogaster loci from regions of intermediate to high recombination rates. We find that alpha v is likely to be > 1.3 x 10(-8). Additional data are needed to estimate this parameter more precisely. The estimation of alpha v is important, as this parameter determines the shape of the frequency distribution of strongly selected substitutions.      

Joint effects of genetic hitchhiking and background selection on neutral variation

Due to relatively high rates of strongly selected deleterious mutations, directional selection on favorable alleles (causing hitchhiking effects on linked neutral polymorphisms) is expected to occur while a deleterious mutation-selection balance is present in a population. We analyze this interaction of directional selection and background selection and study their combined effects on neutral variation, using a three-locus model in which each locus is subjected to either deleterious, favorable, or neutral mutations. Average heterozygosity is measured by simulations (1) at the stationary state under the assumption of recurrent hitchhiking events and (2) as a transient level after a single hitchhiking event. The simulation results are compared to theoretical predictions. It is shown that known analytical solutions describing the hitchhiking effect without background selection can be modified such that they accurately predict the joint effects of hitchhiking and background on linked, neutral variation. Generalization of these results to a more appropriate multilocus model (such that background selection can occur at multiple sites) suggests that, in regions of very low recombination rates, stationary levels of nucleotide diversity are primarily determined by hitchhiking, whereas in regions of high recombination, background selection is the dominant force. The implications of these results on the identification and estimation of the relevant parameters of the model are discussed.     

Statistical Tests of Neutrality of Mutations against Population Growth, Hitchhiking and Background Selection

The main purpose of this article is to present several new statistical tests of neutrality of mutations against a class of alternative models, under which DNA polymorphisms tend to exhibit excesses of rare alleles or young mutations. Another purpose is to study the powers of existing and newly developed tests and to examine the detailed pattern of polymorphisms under population growth, genetic hitchhiking and background selection. It is found that the polymorphic patterns in a DNA sample under logistic population growth and genetic hitchhiking are very similar and that one of the newly developed tests, F(s), is considerably more powerful than existing tests for rejecting the hypothesis of neutrality of mutations. Background selection gives rise to quite different polymorphic patterns than does logistic population growth or genetic hitchhiking, although all of them show excesses of rare alleles or young mutations. We show that Fu and Li''s tests are among the most powerful tests against background selection. Implications of these results are discussed.     

Demography or selection on linked cultural traits or genes? Investigating the driver of low mtDNA diversity in the sperm whale using complementary mitochondrial and nuclear genome analyses

Mitochondrial DNA has been heavily utilized in phylogeography studies for several decades. However, underlying patterns of demography and phylogeography may be misrepresented due to coalescence stochasticity, selection, variation in mutation rates and cultural hitchhiking (linkage of genetic variation to culturally‐transmitted traits affecting fitness). Cultural hitchhiking has been suggested as an explanation for low genetic diversity in species with strong social structures, counteracting even high mobility, abundance and limited barriers to dispersal. One such species is the sperm whale, which shows very limited phylogeographic structure and low mtDNA diversity despite a worldwide distribution and large population. Here, we use analyses of 175 globally distributed mitogenomes and three nuclear genomes to evaluate hypotheses of a population bottleneck/expansion vs. a selective sweep due to cultural hitchhiking or selection on mtDNA as the mechanism contributing to low worldwide mitochondrial diversity in sperm whales. In contrast to mtDNA control region (CR) data, mitogenome haplotypes are largely ocean‐specific, with only one of 80 shared between the Atlantic and Pacific. Demographic analyses of nuclear genomes suggest low mtDNA diversity is consistent with a global reduction in population size that ended approximately 125,000 years ago, correlated with the Eemian interglacial. Phylogeographic analysis suggests that extant sperm whales descend from maternal lineages endemic to the Pacific during the period of reduced abundance and have subsequently colonized the Atlantic several times. Results highlight the apparent impact of past climate change, and suggest selection and hitchhiking are not the sole processes responsible for low mtDNA diversity in this highly social species.     

Hitchhiking mapping reveals a candidate genomic region for natural selection in three-spined stickleback chromosome VIII

Identification of genes and genomic regions under directional natural selection has become one of the major goals in evolutionary genetics, but relatively little work to this end has been done by applying hitchhiking mapping to wild populations. Hitchhiking mapping starts from a genome scan using a randomly spaced set of molecular markers followed by a fine-scale analysis in the flanking regions of the candidate regions under selection. We used the hitchhiking mapping approach to narrow down a selective sweep in the genomic region flanking a candidate locus (Stn90) in chromosome VIII in the three-spined stickleback (Gasterosteus aculeatus). Twenty-four microsatellite markers were screened in an approximately 800-kb region around the candidate locus in three marine and four freshwater populations. The patterns of genetic diversity and differentiation in the candidate region were compared to those of a putatively neutral set of markers. The Bayesian FST-test indicated an elevated genetic differentiation, deviating significantly from neutral expectations, at a continuous region of approximately 20 kb upstream from the candidate locus. Furthermore, a method developed for an array of microsatellite markers rejected neutrality in a region of approximately 90 kb flanking the candidate locus supporting the selective sweep hypothesis. Likewise, the genomewide pattern of genetic diversity differed from the candidate region in a bottleneck analysis suggesting that selection, rather than demography, explains the reduced genetic diversity at the candidate interval. The neutrality tests suggest that the selective sweep had occurred mainly in the Lake Pulmanki population, but the results from bottleneck analyses indicate that selection might have operated in other populations as well. These results suggest that the narrow interval around locus Stn90 has likely been under directional selection, but the region contains several predicted genes, each of which can be the actual targets of selection. Understanding of the functional significance of this genomic region in an ecological context will require a more detailed sequence analysis.     

Selection at linked sites in the partial selfer Caenorhabditis elegans

Natural selection can produce a correlation between local recombination rates and levels of neutral DNA polymorphism as a consequence of genetic hitchhiking and background selection. Theory suggests that selection at linked sites should affect patterns of neutral variation in partially selfing populations more dramatically than in outcrossing populations. However, empirical investigations of selection at linked sites have focused primarily on outcrossing species. To assess the potential role of selection as a determinant of neutral polymorphism in the context of partial self-fertilization, we conducted a multivariate analysis of single-nucleotide polymorphism (SNP) density throughout the genome of the nematode Caenorhabditis elegans. We based the analysis on a published SNP data set and partitioned the genome into windows to calculate SNP densities, recombination rates, and gene densities across all six chromosomes. Our analyses identify a strong, positive correlation between recombination rate and neutral polymorphism (as estimated by noncoding SNP density) across the genome of C. elegans. Furthermore, we find that levels of neutral polymorphism are lower in gene-dense regions than in gene-poor regions in some analyses. Analyses incorporating local estimates of divergence between C. elegans and C. briggsae indicate that a mutational explanation alone is unlikely to explain the observed patterns. Consequently, we interpret these findings as evidence that natural selection shapes genome-wide patterns of neutral polymorphism in C. elegans. Our study provides the first demonstration of such an effect in a partially selfing animal. Explicit models of genetic hitchhiking and background selection can each adequately describe the relationship between recombination rate and SNP density, but only when they incorporate selfing rate. Clarification of the relative roles of genetic hitchhiking and background selection in C. elegans awaits the development of specific theoretical predictions that account for partial self-fertilization and biased sex ratios.     

Genomic effects of nucleotide substitutions in Drosophila simulans

Selective fixation of beneficial mutations reduces levels of linked, neutral variation. The magnitude of this "hitchhiking effect" is determined by the strength of selection and the recombination rate between selected and neutral sites. Thus, depending on the values of these parameters and the frequency with which directional selection occurs, the genomic scale over which directional selection reduces levels of linked variation may vary widely. Here we present a permutation-based analysis of nucleotide polymorphisms and fixations in Drosophila simulans. We show evidence of pervasive small-scale hitchhiking effects in this lineage. Furthermore, our results reveal that different types of fixations are associated with different levels of linked variation.     

Linkage disequilibria and the site frequency spectra in the su(s) and su(w(a)) regions of the Drosophila melanogaster X chromosome

Over the last decade, surveys of DNA sequence variation in natural populations of several Drosophila species and other taxa have established that polymorphism is reduced in genomic regions characterized by low rates of crossing over per physical length. Parallel studies have also established that divergence between species is not reduced in these same genomic regions, thus eliminating explanations that rely on a correlation between the rates of mutation and crossing over. Several theoretical models (directional hitchhiking, background selection, and random environment) have been proposed as population genetic explanations. In this study samples from an African population (n = 50) and a European population (n = 51) were surveyed at the su(s) (1955 bp) and su(w(a)) (3213 bp) loci for DNA sequence polymorphism, utilizing a stratified SSCP/DNA sequencing protocol. These loci are located near the telomere of the X chromosome, in a region of reduced crossing over per physical length, and exhibit a significant reduction in DNA sequence polymorphism. Unlike most previously surveyed, these loci reveal substantial skews toward rare site frequencies, consistent with the predictions of directional hitchhiking and random environment models and inconsistent with the general predictions of the background selection model (or neutral theory). No evidence for excess geographic differentiation at these loci is observed. Although linkage disequilibrium is observed between closely linked sites within these loci, many recombination events in the genealogy of the sampled alleles can be inferred and the genomic scale of linkage disequilibrium, measured in base pairs between sites, is the same as that observed for loci in regions of normal crossing over. We conclude that gene conversion must be high in these regions of low crossing over.     

Strong ecological but weak evolutionary effects of elevated CO2 on a recombinant inbred population of Arabidopsis thaliana

Increases in atmospheric CO2 concentration have an impact on plant communities by influencing plant growth and morphology, species interactions, and ecosystem processes. These ecological effects may be accompanied by evolutionary change if elevated CO2 (eCO2) alters patterns of natural selection or expression of genetic variation. Here, a statistically powerful quantitative genetic experiment and manipulations of CO2 concentrations in a field setting were used to investigate how eCO2 impacts patterns of selection on ecologically important traits in Arabidopsis thaliana; heritabilities, which influence the rate of response to selection; and genetic covariances between traits, which may constrain responses to selection. CO2 had strong phenotypic effects; plants grown in eCO2 were taller and produced more biomass and fruits. Also, significant directional selection was observed on many traits and significant genetic variation was observed for all traits. However, no evolutionary effect of eCO2 was detected; patterns of selection, heritabilities and genetic correlations corresponded closely in ambient and elevated CO2 environments. The data suggest that patterns of natural selection and the quantitative genetic parameters of this A. thaliana population are robust to increases in CO2 concentration and that responses to eCO2 will be primarily ecological.     

The Effect of Adaptive Mutagenesis on Genetic Variation at a Linked, Neutral Locus

Based on recent studies in single-celled organisms, it has been argued that a fitness benefit associated with a mutation will increase the probability of that mutation occurring. This increase is independent of mutation rates at other loci and is called adaptive mutagenesis. We modeled the effect of adaptive mutagenesis on populations of haploid organisms with adaptive mutation rates ranging from 0 to 1 X 10(-5). Allele frequencies at the selected locus and a neutral linked locus were tracked. We also observed the amount of linkage disequilibrium during the selective sweep and the final heterozygosity after the sweep. The presence of adaptive mutagenesis increases the number of genetic backgrounds carrying the new fitter allele, making the outcomes more representative of the population before the selection. Therefore, more neutral genetic variation is preserved in simulations with adaptive mutagenesis than in those without it due to hitchhiking. Since adaptive mutagenesis is time-dependent, it can generate mutants when other mechanisms of mutation cannot. In addition, adaptive mutagenesis has the potential to confound both phylogeny construction and the detection of natural selection from patterns of nucleotide variation.     

Challenges of detecting directional selection after a bottleneck: lessons from Sorghum bicolor

Multilocus surveys of sequence variation can be used to identify targets of directional selection, which are expected to have reduced levels of variation. Following a population bottleneck, the signal of directional selection may be hard to detect because many loci may have low variation by chance and the frequency spectrum of variation may be perturbed in ways that resemble the effects of selection. Cultivated Sorghum bicolor contains a subset of the genetic diversity found in its wild ancestor(s) due to the combined effects of a domestication bottleneck and human selection on traits associated with agriculture. As a framework for distinguishing between the effects of demography and selection, we sequenced 204 loci in a diverse panel of 17 cultivated S. bicolor accessions. Genomewide patterns of diversity depart strongly from equilibrium expectations with regard to the variance of the number of segregating sites, the site frequency spectrum, and haplotype configuration. Furthermore, gene genealogies of most loci with an excess of low frequency variants and/or an excess of segregating sites do not show the characteristic signatures of directional and diversifying selection, respectively. A simple bottleneck model provides an improved but inadequate fit to the data, suggesting the action of other population-level factors, such as population structure and migration. Despite a known history of recent selection, we find little evidence for directional selection, likely due to low statistical power and lack of an appropriate null model.     

Genetic variation versus recombination rate in a structured population of mice

The correlation between genetic variation and recombination rate was investigated in a structured mouse population. Nucleotide sequence data from 19 autosomal DNA loci from eight inbred strains of mouse (Mus musculus) sampled from three major subspecies were analyzed. The recombination rate was estimated from the comparison of genetic and physical map distances between markers flanking a 10-cM region of each locus. The strains were categorized into four groups (subpopulations) based on geography. By partitioning the genetic diversity into within-group and among-group variation, we detected a positive correlation between the recombination rate and nucleotide diversity within groups. The level of nucleotide differentiation among groups (G(ST)) showed a negative correlation with the rate of recombination. There was no significant correlation between recombination rate and nucleotide diversity when data from different subpopulations were pooled. No correlation was detected between recombination rate and nucleotide divergence of M. musculus and M. spicilegus. These patterns deviate from the strict neutral expectation under the constant nucleotide substitution rate, and they are likely to have been formed either by a hitchhiking effect of positively selected mutants or by background selection of deleterious mutants occurring in a subdivided population. Our series of comparisons show that because a real population always has some structure, incorporation of its information is important in detecting non-neutral evolution.