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.     

On the utility of linkage disequilibrium as a statistic for identifying targets of positive selection in nonequilibrium populations

A critically important challenge in empirical population genetics is distinguishing neutral nonequilibrium processes from selective forces that produce similar patterns of variation. We here examine the extent to which linkage disequilibrium (i.e., nonrandom associations between markers) improves this discrimination. We show that patterns of linkage disequilibrium recently proposed to be unique to hitchhiking models are replicated under nonequilibrium neutral models. We also demonstrate that jointly considering spatial patterns of association among variants alongside the site-frequency spectrum is nonetheless of value. Through a comparison of models of equilibrium neutrality, nonequilibrium neutrality, equilibrium hitchhiking, nonequilibrium hitchhiking, and recurrent hitchhiking, we evaluate a linkage disequilibrium (LD) statistic (omega(max)) that appears to have power to identify regions recently shaped by positive selection. Most notably, for demographic parameters relevant to non-African populations of Drosophila melanogaster, we demonstrate that selected loci are distinguishable from neutral loci using this statistic.     

DNA polymorphism in lycopersicon and crossing-over per physical length.

Surveys in Drosophila have consistently found reduced levels of DNA sequence polymorphism in genomic regions experiencing low crossing-over per physical length, while these same regions exhibit normal amounts of interspecific divergence. Here we show that for 36 loci across the genomes of eight Lycopersicon species, naturally occurring DNA polymorphism (scaled by locus-specific divergence between species) is positively correlated with the density of crossing-over per physical length. Large between-species differences in the amount of DNA sequence polymorphism reflect breeding systems: selfing species show much less within-species polymorphism than outcrossing species. The strongest association of expected heterozygosity with crossing-over is found in species with intermediate levels of average nucleotide diversity. All of these observations appear to be in qualitative agreement with the hitchhiking effects caused by the fixation of advantageous mutations and/or "background selection" against deleterious mutations.     

Evidence of gene conversion associated with a selective sweep in Drosophila melanogaster

Since Drosophila melanogaster colonized Europe from tropical Africa 10 to 15 thousand years ago, it is expected that adaptation has played a major role in this species in recent times. A previously conducted multilocus scan of noncoding DNA sequences on the X chromosome in an ancestral and a derived population of D. melanogaster revealed that some loci have been affected by directional selection in the European population. We investigated if the pattern of DNA sequence polymorphism in a region surrounding one of these loci can be explained by a hitchhiking event. We found strong evidence that the studied region around the gene unc-119 was shaped by a recent selective sweep, including a valley of reduced heterozygosity of 83.4 kb, a skew in the frequency spectrum, and significant linkage disequilibrium on one side of the valley. This region, however, was interrupted by gene conversion events leading to a strong haplotype structure in the center of the valley of reduced variation.     

Regions of lower crossing over harbor more rare variants in African populations of Drosophila melanogaster

A correlation between diversity levels and rates of recombination is predicted both by models of positive selection, such as hitchhiking associated with the rapid fixation of advantageous mutations, and by models of purifying selection against strongly deleterious mutations (commonly referred to as "background selection"). With parameter values appropriate for Drosophila populations, only the first class of models predicts a marked skew in the frequency spectrum of linked neutral variants, relative to a neutral model. Here, we consider 29 loci scattered throughout the Drosophila melanogaster genome. We show that, in African populations, a summary of the frequency spectrum of polymorphic mutations is positively correlated with the meiotic rate of crossing over. This pattern is demonstrated to be unlikely under a model of background selection. Models of weakly deleterious selection are not expected to produce both the observed correlation and the extent to which nucleotide diversity is reduced in regions of low (but nonzero) recombination. Thus, of existing models, hitchhiking due to the recurrent fixation of advantageous variants is the most plausible explanation for the data.     

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.     

DNA sequence polymorphism and divergence at the erect wing and suppressor of sable loci of Drosophila melanogaster and D. simulans

Several evolutionary models of linked selection (e.g., genetic hitchhiking, background selection, and random environment) predict a reduction in polymorphism relative to divergence in genomic regions where the rate of crossing over per physical distance is restricted. We tested this prediction near the telomere of the Drosophila melanogaster and D. simulans X chromosome at two loci, erect wing (ewg) and suppressor of sable [su(s)]. Consistent with this prediction, polymorphism is reduced at both loci, while divergence is normal. The reduction is greater at ewg, the more distal of the two regions. Two models can be discriminated by comparing the observed site frequency spectra with those predicted by the models. The hitchhiking model predicts a skew toward rare variants in a sample, while the spectra under the background-selection model are similar to those of the neutral model of molecular evolution. Statistical tests of the fit to the predictions of these models require many sampled alleles and segregating sites. Thus we used SSCP and stratified DNA sequencing to cover a large number of randomly sampled alleles (approximately 50) from each of three populations. The result is a clear trend toward negative values of Tajima's D, indicating an excess of rare variants at ewg, the more distal of the two loci. One fixed difference among the populations and high FST values indicate strong population subdivision among the three populations at ewg. These results indicate genetic hitchhiking at ewg, in particular, geographically localized hitchhiking events within Africa. The reduction of polymorphism at su(s) combined with the excess of high-frequency variants in D. simulans is inconsistent with the hitchhiking and background-selection models.     

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.      

Patterns of sequence variability and divergence at the diminutive gene region of Drosophila melanogaster: complex patterns suggest an ancestral selective sweep

To identify putatively swept regions of the Drosophila melanogaster genome, we performed a microsatellite screen spanning a 260-kb region of the X chromosome in populations from Zimbabwe, Ecuador, the United States, and China. Among the regions identified by this screen as showing a complex pattern of reduced heterozygosity and a skewed frequency spectrum was the gene diminutive (dm). To investigate the microsatellite findings, nucleotide sequence polymorphism data were generated in populations from both China and Zimbabwe spanning a 25-kb region and encompassing dm. Analysis of the sequence data reveals strongly reduced nucleotide variation across the entire gene region in both the non-African and the African populations, an extended haplotype pattern, and structured linkage disequilibrium, as well as a rejection of neutrality in favor of selection using a composite likelihood-ratio test. Additionally, unusual patterns of synonymous site evolution were observed at the second exon of this locus. On the basis of simulation studies as well as recently proposed methods for distinguishing between selection and nonequilibrium demography, we find that this "footprint" is best explained by a selective sweep in the ancestral population, the signal of which has been somewhat blurred via founder effects in the non-African samples.     

Molecular evolution of the Est-6 gene in Drosophila melanogaster: contrasting patterns of DNA variability in adjacent functional regions

We have investigated nucleotide polymorphism at the esterase 6 gene (Est-6) gene, including the complete coding region (1686 bp), as well as the 5'-flanking (1183 bp) and 3'-flanking (193 bp) regions of the gene, in 30 strains of Drosophila melanogaster and in one strain of Drosophila simulans. The level of silent variation is similar in the coding and in the 3'-flanking region, but smaller in the 5'-flanking region. Strong linkage disequilibrium occurs within each region; and also, although less pronounced, between the 5'-flanking region and the rest of the gene, including the 3'-flanking region. We suggest that the pattern of nucleotide polymorphism of Est-6 may be shaped by: (1) directional and balancing selection acting on the promoter and the coding region; and (2) interactions between the two regions that involve variable degrees of hitchhiking. The patterns of linkage disequilibrium, as well as the statistics Z(nS) (Genetics 146 (1997)1197) and B and Q (Genet. Res. 74 (1999) 65), may be interpreted as there being multiple targets of selection within the gene. The previously reported Est-6 allozyme latitudinal clines may be accounted for by the interaction between selective processes in the promoter and coding regions.     

Hitchhiking: A Comparison of Linkage and Partial Selfing

Genetic hitchhiking occurs when alleles at unselected loci are changed in frequency because of an association with alleles at a selected locus. This association may be mediated either by linkage or partial selfing (inbreeding) and can affect the gene frequency and gametic disequilibrium at the neutral loci. Hitchhiking from partial selfing (unlinked loci) occurs more quickly than linkage hitchhiking and generally has a greater effect. In addition, partial-selfing hitchhiking can cause increases or changes in sign in gametic disequilibrium between neutral loci. The effects of the two types of hitchhiking with different levels of dominance, zygotic frequencies and number of selected loci are also examined. The general conditions for linkage and partial-selfing hitchhiking are outlined and the implications of hitchhiking are discussed for marker or electrophoretic loci.     

DNA variability and recombination rates at X-linked loci in humans.

We sequenced 11,365 bp from introns of seven X-linked genes in 10 humans, one chimpanzee, and one orangutan to (i) provide an average estimate of nucleotide diversity (pi) in humans, (ii) investigate whether there is variation in pi among loci, (iii) compare ratios of polymorphism to divergence among loci, and (iv) provide a preliminary test of the hypothesis that heterozygosity is positively correlated with the local rate of recombination. The average value for pi was low 0.063%, SE = 0.036%, about one order of magnitude smaller than for Drosophila melanogaster, the species for which the best data are available. Among loci, pi varied by over one order of magnitude. Statistical tests of neutrality based on ratios of polymorphism to divergence or based on the frequency spectrum of variation within humans failed to reject a neutral, equilibrium model. However, there was a positive correlation between heterozygosity and rate of recombination, suggesting that the joint effects of selection and linkage are important in shaping patterns of nucleotide variation in humans.     

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.     

A genome-wide departure from the standard neutral model in natural populations of Drosophila

We analyze nucleotide polymorphism data for a large number of loci in areas of normal to high recombination in Drosophila melanogaster and D. simulans (24 and 16 loci, respectively). We find a genome-wide, systematic departure from the neutral expectation for a panmictic population at equilibrium in natural populations of both species. The distribution of sequence-based estimates of 2Nc across loci is inconsistent with the assumptions of the standard neutral theory, given the observed levels of nucleotide diversity and accepted values for recombination and mutation rates. Under these assumptions, most estimates of 2Nc are severalfold too low; in other words, both species exhibit greater intralocus linkage disequilibrium than expected. Variation in recombination or mutation rates is not sufficient to account for the excess of linkage disequilibrium. While an equilibrium island model does not seem to account for the data, more complicated forms of population structure may. A proper test of alternative demographic models will require loci to be sampled in a more consistent fashion.     

Insertion-deletion variation at the yellow-achaete-scute region in two natural populations of Drosophila melanogaster

We have surveyed the region of the X chromosome of Drosophila melanogaster which encodes the yellow, achaete and scute genes for restriction map variation. Two natural populations, one from North Carolina, U.S.A. and the other from southern Spain were screened for variation at about 70 restriction sites and for variation due to DNA insertion or deletion events in 120 kilobases of DNA. Mean heterozygosity per nucleotide was estimated to be 0.0024 and 15 large insertions were found in the 49 chromosomes screened. Extensive disequilibrium between polymorphic sites were found across much of the region in the North Carolina population. The frequency of large insertions, which usually correspond to transposable genetic elements, is significantly lower than has been observed in autosomal regions of the genome. This is predicted for X-linked loci by certain models of transposable element evolution, where copy number is restricted by virtue of the recessive deleterious effects of the insertions. Our results appear to support such models. The deficiency of insertions may in this case be enhanced by hitch-hiking effects arising from the high level of disequilibrium.     

Nucleotide variation of the duplicated amylase genes in Drosophila kikkawai

We examined levels and patterns of the nucleotide polymorphism of the Amylase genes with a head-to-head duplication in Drosophila kikkawai. The levels of variation in D. kikkawai were comparable to those in Drosophila melanogaster. Tajima's test, Fu and Li's test, HKA test, and MK test did not show significant departure from neutrality. We found an excess of replacement changes in the within-locus class, representing polymorphism in one of the duplicated genes, compared with the between-locus class, representing polymorphism shared between the duplicated genes. Most replacement changes in the within-locus class were singletons. These results suggest that most replacement changes are deleterious. A contrasting evolutionary pattern, involving concerted evolution in the coding regions but differential evolution in the 5'-flanking regions, was observed. However, unlike the duplicated Amy genes of D. melanogaster, the coding regions of the duplicated genes in D. kikkawai tended to diverge. Using Ohta's model of the small multigene family, we found that recombination (interchromosomal equal crossing-over) rate was one order higher than gene conversion (unequal crossing-over) rate, resulting in a considerable but incomplete homogenization of the duplicated coding regions. Linkage disequilibria were found in the intron as well as within and around the regulatory cis-element sequences of one of the duplicated genes (Amy1). The possible causes of these linkage disequilibria were discussed.     

Reduced variation in the yellow-achaete-scute region in natural populations of Drosophila melanogaster

Restriction map variation in 64 X chromosome lines extracted from three different populations of Drosophila melanogaster was investigated with seven six-nucleotide-recognizing restriction enzymes for a 106-kb region encompassing the yellow gene and the achaete-scute complex that is located in the region of reduced crossing over close to the telomere. Nine restriction site polymorphisms (out of 176 sites scored) and 19 length polymorphisms (15 insertions and 4 deletions) were detected. The estimated level of heterozygosity per nucleotide, H = 0.0003, is much lower than that reported for autosomal and sex-linked loci located in regions with normal levels of crossing over. The overall frequency of polymorphic restriction sites is reduced. Six out of nine restriction site polymorphisms are unique and the other three have frequencies less than 0.17. Some large insertions have reached relatively high frequencies, 0.08 to 0.17. Consistent with the theoretically predicted negative relationship between crossing over and the magnitude of linkage disequilibrium, an increase in the relative number of nonrandom associations was observed in the y-ac-sc region.     

Haplotype structure and population genetic inferences from nucleotide-sequence variation in human lipoprotein lipase.

Allelic variation in 9.7 kb of genomic DNA sequence from the human lipoprotein lipase gene (LPL) was scored in 71 healthy individuals (142 chromosomes) from three populations: African Americans (24) from Jackson, MS; Finns (24) from North Karelia, Finland; and non-Hispanic Whites (23) from Rochester, MN. The sequences had a total of 88 variable sites, with a nucleotide diversity (site-specific heterozygosity) of .002+/-.001 across this 9.7-kb region. The frequency spectrum of nucleotide variation exhibited a slight excess of heterozygosity, but, in general, the data fit expectations of the infinite-sites model of mutation and genetic drift. Allele-specific PCR helped resolve linkage phases, and a total of 88 distinct haplotypes were identified. For 1,410 (64%) of the 2,211 site pairs, all four possible gametes were present in these haplotypes, reflecting a rich history of past recombination. Despite the strong evidence for recombination, extensive linkage disequilibrium was observed. The number of haplotypes generally is much greater than the number expected under the infinite-sites model, but there was sufficient multisite linkage disequilibrium to reveal two major clades, which appear to be very old. Variation in this region of LPL may depart from the variation expected under a simple, neutral model, owing to complex historical patterns of population founding, drift, selection, and recombination. These data suggest that the design and interpretation of disease-association studies may not be as straightforward as often is assumed.