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.     

Nucleotide polymorphism and linkage disequilibrium within and among natural populations of European aspen (Populus tremula L., Salicaceae)

Populus is an important model organism in forest biology, but levels of nucleotide polymorphisms and linkage disequilibrium have never been investigated in natural populations. Here I present a study on levels of nucleotide polymorphism, haplotype structure, and population subdivision in five nuclear genes in the European aspen Populus tremula. Results show substantial levels of genetic variation. Levels of silent site polymorphisms, pi(s), averaged 0.016 across the five genes. Linkage disequilibrium was generally low, extending only a few hundred base pairs, suggesting that rates of recombination are high in this obligate outcrossing species. Significant genetic differentiation was found at all five genes, with an average estimate of F(ST) = 0.116. Levels of polymorphism in P. tremula are 2- to 10-fold higher than those in other woody, long-lived perennial plants, such as Pinus and Cryptomeria. The high levels of nucleotide polymorphism and low linkage disequilibrium suggest that it may be possible to map functional variation to very fine scales in P. tremula using association-mapping approaches.     

An excess of nonsynonymous polymorphism and extensive haplotype structure at the PtABI1B locus in European aspen (Populus tremula): a case of balancing selection in an obligately outcrossing plant?

Here, we describe an unusually pronounced haplotype structure at the PtABI1B locus in the obligately outcrossing tree Populus tremula. Both nucleotide diversity and divergence at PtABI1B was low compared to other P. tremula genes suggesting that the gene is located in a region with a low mutation rate. Despite this, PtABI1B shows a very marked excess of nonsynonymous polymorphisms across the entire coding region and linkage disequilibrium (LD) extending across the entire PtABI1B region of approximately 2.6 kb. Such extensive LD is normally not seen in P. tremula. The extensive LD at PtABI1B is caused by the presence of two distinct haplotypes. The haplotype structure is not caused by a lack of recombination in the region, because evidence of recombination can be detected. In addition, several statistical tests strongly reject neutrality for the PtABI1B region, suggesting that the unusual haplotype structure could be actively maintained by balancing selection.     

Testing models of selection and demography in Drosophila simulans

We analyze patterns of nucleotide variability at 15 X-linked loci and 14 autosomal loci from a North American population of Drosophila simulans. We show that there is significantly more linkage disequilibrium on the X chromosome than on chromosome arm 3R and much more linkage disequilibrium on both chromosomes than expected from estimates of recombination rates, mutation rates, and levels of diversity. To explore what types of evolutionary models might explain this observation, we examine a model of recurrent, nonoverlapping selective sweeps and a model of a recent drastic bottleneck (e.g., founder event) in the demographic history of North American populations of D. simulans. The simple sweep model is not consistent with the observed patterns of linkage disequilibrium nor with the observed frequencies of segregating mutations. Under a restricted range of parameter values, a simple bottleneck model is consistent with multiple facets of the data. While our results do not exclude some influence of selection on X vs. autosome variability levels, they suggest that demography alone may account for patterns of linkage disequilibrium and the frequency spectrum of segregating mutations in this population of D. simulans.     

Evolution at the tip and base of the X chromosome in an African population of Drosophila melanogaster

Hitchhiking effects of advantageous mutations have been invoked to explain reduced polymorphism in regions of low crossing-over in Drosophila. Besides reducing DNA heterozygosity, hitchhiking effects should produce strong linkage disequilibrium and a frequency spectrum skewed toward an excess of rare polymorphisms (compared to the neutral expectation). We measured DNA polymorphism in a Zimbabwe population of D. melanogaster at three loci, yellow, achaete, and suppressor of forked, located in regions of reduced crossing-over. Similar to previously published surveys of these genomic regions in other populations, we observed low levels of nucleotide variability. However, the frequency spectrum was compatible with a neutral model, and there was abundant evidence for recombination in the history of the yellow and ac genes. Thus, some aspects of the data cannot be accounted for by a simple hitchhiking model. An alternative hypothesis, background selection, might be compatible with the observed patterns of linkage disequilibrium and the frequency spectrum. However, this model cannot account for the observed reduction in nucleotide heterozygosity. Thus, there is currently no satisfactory theoretical model for the data from the tip and base of the X chromosome in D. melanogaster.      

Relative roles of mutation and recombination in generating allelic polymorphism at an MHC class II locus in Peromyscus maniculatus

The MHC class II loci encoding cell surface antigens exhibit extremely high allelic polymorphism. There is considerable uncertainty in the literature over the relative roles of recombination and de novo mutation in generating this diversity. We studied class II sequence diversity and allelic polymorphism in two populations of Peromyscus maniculatus, which are among the most widespread and abundant mammals of North America. We find that intragenic recombination (or gene conversion) has been the predominant mode for the generation of allelic polymorphism in this species, with the amount of population recombination per base pair exceeding mutation by at least an order of magnitude during the history of the sample. Despite this, patchwork motifs of sites with high linkage disequilibrium are observed. This does not appear to be consistent with the much larger amount of recombination versus mutation in the history of the sample, unless the recombination rate is highly non-uniform over the sequence or selection maintains certain sites in linkage disequilibrium. We conclude that selection is most likely to be responsible for preserving sequence motifs in the presence of abundant recombination.     

The signature of positive selection at randomly chosen loci

In Drosophila and humans, there are accumulating examples of loci with a significant excess of high-frequency-derived alleles or high levels of linkage disequilibrium, relative to a neutral model of a random-mating population of constant size. These are features expected after a recent selective sweep. Their prevalence suggests that positive directional selection may be widespread in both species. However, as I show here, these features do not persist long after the sweep ends: The high-frequency alleles drift to fixation and no longer contribute to polymorphism, while linkage disequilibrium is broken down by recombination. As a result, loci chosen without independent evidence of recent selection are not expected to exhibit either of these features, even if they have been affected by numerous sweeps in their genealogical history. How then can we explain the patterns in the data? One possibility is population structure, with unequal sampling from different subpopulations. Alternatively, positive selection may not operate as is commonly modeled. In particular, the rate of fixation of advantageous mutations may have increased in the recent past.     

Estimates of Linkage Disequilibrium and the Recombination Parameter Determined from Segregating Nucleotide Sites in the Alcohol Dehydrogenase Region of Drosophila Pseudoobscura

The alcohol dehydrogenase (Adh) region of Drosophila pseudoobscura, which includes the two genes Adh and Adh-Dup, was used to examine the pattern and organization of linkage disequilibrium among pairs of segregating nucleotide sites. A collection of 99 strains from the geographic range of D. pseudoobscura were nucleotide-sequenced with polymerase chain reaction-mediated techniques. All pairs of the 359 polymorphic sites in the 3.5-kb Adh region were tested for significant linkage disequilibrium with Fisher's exact test. Of the 74,278 pairwise comparisons of segregating sites, 127 were in significant linkage disequilibrium at the 5% level. The distribution of five linkage disequilibrium estimators D(ij), D(2), r(ij), r(2) and Dij were compared to theoretical distributions. The observed distributions of D(ij), D(2), r(ij) and r(2) were consistent with the theoretical distribution given an infinite sites model. The observed distribution of Dij differed from the theoretical distribution because of an excess of values at -1 and 1. No spatial pattern was observed in the linkage disequilibrium pattern in the Adh region except for two clusters of sites nonrandomly associated in the adult intron and intron 2 of Adh. The magnitude of linkage disequilibrium decreases significantly as nucleotide distance increases, or a distance effect. Adh-Dup had a larger estimate of the recombination parameter, 4Nc, than Adh, where N is the effective population size and c is the recombination rate. A comparison of the mutation and recombination parameters shows that 7-17 recombination events occur for each mutation event. The heterogeneous estimates of the recombination parameter and the inverse relationship between linkage disequilibrium and nucleotide distance are no longer significant when the two clusters of Adh intron sites are excluded from analyses. The most likely explanation for the two clusters of linkage disequilibria is epistatic selection between sites in the cluster to maintain pre-mRNA secondary structure.     

Inferring the Evolutionary Histories of the Adh and Adh-Dup Loci in Drosophila Melanogaster from Patterns of Polymorphism and Divergence

The DNA sequences of 11 Drosophila melanogaster lines are compared across three contiguous regions, the Adh and Adh-dup loci and a noncoding 5' flanking region of Adh. Ninety-eight of approximately 4750 sites are segregating in the sample, 36 in the 5' flanking region, 38 in Adh and 24 in Adh-dup. Several methods are presented to test whether the patterns and levels of polymorphism are consistent with neutral molecular evolution. The analysis of within- and between-species polymorphism indicates that the region is evolving in a nonneutral and complex fashion. A graphical analysis of the data provides support for a hypothesized balanced polymorphism at or near position 1490, site of the amino acid replacement difference between Adhf and Adhs. The Adh-dup locus is less polymorphic than Adh and all 24 of its polymorphisms occur at low frequency--suggestive of a recent selective substitution in the Adh-dup region. Adhs alleles form two distinct evolutionary lineages that differ one from another at a total of nineteen sites in the Adh and Adh-dup loci. The polymorphisms are in complete linkage disequilibrium. A recombination experiment failed to find evidence for recombination suppression between the two allelic classes. Two hypotheses are presented to account for the widespread distribution of the two divergent lineages in natural populations. Natural selection appears to have played an important role in governing the overall patterns of nucleotide variation across the two-gene region.     

Contrasting patterns of nonneutral evolution in proteins encoded in nuclear and mitochondrial genomes

We report that patterns of nonneutral DNA sequence evolution among published nuclear and mitochondrially encoded protein-coding loci differ significantly in animals. Whereas an apparent excess of amino acid polymorphism is seen in most (25/31) mitochondrial genes, this pattern is seen in fewer than half (15/36) of the nuclear data sets. This differentiation is even greater among data sets with significant departures from neutrality (14/15 vs. 1/6). Using forward simulations, we examined patterns of nonneutral evolution using parameters chosen to mimic the differences between mitochondrial and nuclear genetics (we varied recombination rate, population size, mutation rate, selective dominance, and intensity of germ line bottleneck). Patterns of evolution were correlated only with effective population size and strength of selection, and no single genetic factor explains the empirical contrast in patterns. We further report that in Arabidopsis thaliana, a highly self-fertilizing plant with effectively low recombination, five of six published nuclear data sets also exhibit an excess of amino acid polymorphism. We suggest that the contrast between nuclear and mitochondrial nonneutrality in animals stems from differences in rates of recombination in conjunction with a distribution of selective effects. If the majority of mutations segregating in populations are deleterious, high linkage may hinder the spread of the occasional beneficial mutation.     

DNA variation in a conifer,Cryptomeria japonica (Cupressaceae sensu lato)

We investigated the nucleotide variation of a conifer, Cryptomeria japonica, and the divergence between this species and its closest relative, Taxodium distichum, at seven nuclear loci (Acl5, Chi1, Ferr, GapC, HemA, Lcyb, and Pat). Samples of C. japonica were collected from three areas, Kantou-Toukai, Hokuriku, and Iwate. No apparent geographic differentiation was found among these samples. However, the frequency spectrum of the nucleotide polymorphism revealed excesses of intermediate-frequency variants, which suggests that the population was not panmictic and a constant size in the past. The average nucleotide diversity, pi, for silent sites was 0.00383. However, values of pi for silent sites vary among loci. Comparisons of polymorphism to divergence among loci (the HKA test) showed that the polymorphism at the Acl5 locus was significantly lower. We also observed a nearly significant excess of replacement polymorphisms at the Lcyb locus. These results suggested possibilities of natural selection acting at some of the loci. Intragenic recombination was detected only once at the Chi1 locus and was not detected at the other loci. The low level of population recombination rate, 4Nr, seemed to be due to both low level of recombination, r, and small population size, N.     

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.     

Nucleotide variation in Quercus crispula Blume

Quercus is attractive for evolutionary studies, primarily for developing the concepts of the species, speciation and adaptation; however, remarkably little is known about levels of nucleotide polymorphism in the nuclear functional genes of this genus. This article provides the first characterization of levels of nucleotide polymorphism in 11 gene fragments in natural populations of a Quercus species, Quercus crispula Blume. Results show that the level of nucleotide variation in this oak is generally higher than that in conifers, as high as that in a European oak, but lower than that in an aspen. The level of population recombination is relatively high. Within-population inbreeding is negligible and between-population differentiation is modest. The decay of linkage disequilibrium is significantly faster in the species-wide samples and the three northernmost populations than in the other populations. Statistical tests support the hypothesis of a recent bottleneck for several populations in the southern part of Japan. The amounts and patterns of nucleotide variation, recombination and linkage disequilibrium, and genetic differentiation observed among populations of this species are contradictory to our expectations, given the recent colonization history of the northern Japan populations.     

Linkage disequilibrium and linkage analysis of the glucose-6-phosphatase gene

Recent studies have indicated that the four most common mutations account for 78% of mutant alleles in the glucose-6-phosphatase (G6Pase) gene. A significant fraction of mutant alleles remain unidentified. Thus, informative polymorphic markers are necessary for linkage analysis in carrier testing and prenatal diagnosis in families where mutations can not be identified. The common mutations appear to be ethnic-specific, suggesting that the individual mutations may have a common founder. With the recent discovery of the nucleotide 1176 polymorphism, we have studied whether these mutations are in linkage disequilibrium with the polymorphism. The results of polymerase chain reaction/allele-specific oligonucleotide analysis show that nucleotide 1176 C is in linkage disequilibrium with mutations R83 C and R83H, and with the splicing mutation 727G→T. The 1176 T polymorphism is in linkage disequilibrium with 459insTA. A GT repeat polymorphism has also been found. However, its heterozygosity is low. The 1176 nucleotide polymorphic marker can be used in carrier and prenatal diagnosis of GSD1a families that have unidentified mutations and are informative for this marker. Received: 27 January 1998 / Accepted: 17 April 1998     

Recombination, dominance and selection on amino acid polymorphism in the Drosophila genome: contrasting patterns on the X and fourth chromosomes

Surveys of nucleotide polymorphism and divergence indicate that the average selection coefficient on Drosophila proteins is weakly positive. Similar surveys in mitochondrial genomes and in the selfing plant Arabidopsis show that weak negative selection has operated. These differences have been attributed to the low recombination environment of mtDNA and Arabidopsis that has hindered adaptive evolution through the interference effects of linkage. We test this hypothesis with new sequence surveys of proteins lying in low recombination regions of the Drosophila genome. We surveyed >3800 bp across four proteins at the tip of the X chromosome and >3600 bp across four proteins on the fourth chromosome in 24 strains of D. melanogaster and 5 strains of D. simulans. This design seeks to study the interaction of selection and linkage by comparing silent and replacement variation in semihaploid (X chromosome) and diploid (fourth chromosome) environments lying in regions of low recombination. While the data do indicate very low rates of exchange, all four gametic phases were observed both at the tip of the X and across the fourth chromosome. Silent variation is very low at the tip of the X (thetaS = 0.0015) and on the fourth chromosome (thetaS = 0.0002), but the tip of the X shows a greater proportional loss of variation than the fourth shows relative to normal-recombination regions. In contrast, replacement polymorphism at the tip of the X is not reduced (thetaR = 0.00065, very close to the X chromosome average). MK and HKA tests both indicate a significant excess of amino acid polymorphism at the tip of the X relative to the fourth. Selection is significantly negative at the tip of the X (Nes = -1.53) and nonsignificantly positive on the fourth (Nes approximately 2.9), analogous to the difference between mtDNA (or Arabidopsis) and the Drosophila genome average. Our distal X data are distinct from regions of normal recombination where the X shows a deficiency of amino acid polymorphism relative to the autosomes, suggesting more efficient selection against recessive deleterious replacement mutations. We suggest that the excess amino acid polymorphism on the distal X relative to the fourth chromosome is due to (1) differences in the mutation rate for selected mutations on the distal X or (2) a greater relaxation of selection from stronger linkage-related interference effects on the distal X. This relaxation of selection is presumed to be greater in magnitude than the difference in efficiency of selection between X-linked vs. autosomal selection.     

A method for estimating the mutation,gene conversion and recombination parameters in small multigene families

A simple two-locus gene conversion model is considered to investigate the amounts of DNA variation and linkage disequilibrium in small multigene families. The exact solutions for the expectations and variances of the amounts of variation within and between two loci are obtained. It is shown that gene conversion increases the amount of variation within each locus and decreases the amount of variation between two loci. The expectation and variance of the amount of linkage disequilibrium are also obtained. Gene conversion generates positive linkage disequilibrium and the degree of linkage disequilibrium decreases as the recombination rate is increased. Using the theoretical results, a method for estimating the mutation, gene conversion, and recombination parameters is developed and applied to the data of the Amy multigene family in Drosophila melanogaster. The gene conversion rate is estimated to be approximately 60-165 times higher than the mutation rate for synonymous sites.     

Linkage disequilibrium and recombination rate estimates in the self-incompatibility region of Arabidopsis lyrata

Genetic diversity is unusually high at loci in the S-locus region of the self-incompatible species of the flowering plant, Arabidopsis lyrata, not just in the S loci themselves, but also at two nearby loci. In a previous study of a single natural population from Iceland, we attributed this elevated polymorphism to linkage disequilibrium (LD) between variants at loci close to the S locus and the S alleles, which are maintained in the population by balancing selection. With the four S-flanking loci whose diversity we previously studied, we could not determine the extent of the region linked to the S loci in which neutral sites are affected. We also could not exclude the possibility of a population bottleneck, or of admixture, as causes of the LD. We have now studied four more distant loci flanking the S-locus region, and more populations, and we analyze the results using a theoretical model of the effect of balancing selection on diversity at linked neutral sites within and between different functional S-allelic classes. In the model, diversity is a function of the number of selectively maintained alleles and the recombination distances from the selectively maintained sites. We use the model to estimate the number of different functional S alleles, their turnover rate, and recombination rates between the S-locus region and other loci. Our estimates suggest that there is a small region of very low recombination surrounding the S-locus region.     

The relative contributions of recombination and point mutation to the diversification of bacterial clones

Low levels of recombination in bacterial species have often been inferred from the presence of linkage disequilibrium between the alleles at different loci in the population. However, significant linkage disequilibrium is inevitable in organisms that divide by binary fission, and recombinational replacements must be very frequent, compared to point mutation, to dissipate disequilibrium. Recent studies using data from multilocus sequence typing indicate that, in many species, recombinational replacements contribute more greatly to clonal diversification than do point mutations and, in some species, recombination has been sufficient to eliminate any phylogenetic signal from gene trees. Recent efforts to improve understanding of the extent and impact of homologous recombination in the diversification of bacterial clones are discussed.     

A weak effect of background selection on trinucleotide microsatellites in maize

Artificial selection during the domestication of maize is thought to have been predominantly positive and to have had little effect on the surrounding neutral diversity because linkage disequilibrium breaks down rapidly when physical distance increases. However, the degree to which indirect selection has shaped neutral diversity in the maize genome during domestication remains unclear. In this study, we investigate the relationship between local recombination rate and neutral polymorphism in maize and in teosinte using both sequence and microsatellite data. To quantify diversity, we estimate 3 parameters expected to differentially reflect the effects of indirect selection and mutation. We find no general correlation between diversity and recombination, indicating that indirect selection has had no genome-wide impact on maize diversity. However, we detect a weak correlation between heterozygosity and recombination for trinucleotide microsatellites deviating from the stepwise mutation model and located within genes (rho = 0.32, P < 0.03). This result can be explained by a background selection hypothesis. The fact that the same correlation is not confirmed for nucleotide diversity suggests that the strength of purifying selection at or near this class of microsatellites is higher than for nucleotide mutations.     

Molecular evolution of two linked genes, Est-6 and Sod, in Drosophila melanogaster.

We have obtained 15 sequences of Est-6 from a natural population of Drosophila melanogaster to test whether linkage disequilibrium exists between Est-6 and the closely linked Sod, and whether natural selection may be involved. An early experiment with allozymes had shown linkage disequilibrium between these two loci, while none was detected between other gene pairs. The Sod sequences for the same 15 haplotypes were obtained previously. The two genes exhibit similar levels of nucleotide polymorphism, but the patterns are different. In Est-6, there are nine amino acid replacement polymorphisms, one of which accounts for the S-F allozyme polymorphism. In Sod, there is only one replacement polymorphism, which corresponds to the S-F allozyme polymorphism. The transversion/transition ratio is more than five times larger in Sod than in Est-6. At the nucleotide level, the S and F alleles of Est-6 make up two allele families that are quite different from each other, while there is relatively little variation within each of them. There are also two families of alleles in Sod, one consisting of a subset of F alleles, and the other consisting of another subset of F alleles, designed F(A), plus all the S alleles. The Sod F(A) and S alleles are completely or nearly identical in nucleotide sequence, except for the replacement mutation that accounts for the allozyme difference. The two allele families have independent evolutionary histories in the two genes. There are traces of statistically significant linkage disequilibrium between the two genes that, we suggest, may have arisen as a consequence of selection favoring one particular sequence at each locus.