Compound tests for the detection of hitchhiking under positive selection

Many statistical tests have been developed for detecting positive selection. Most of these tests draw conclusions based on significant deviations from the patterns of polymorphism predicted by the neutral model. However, many non-equilibrium forces may cause similar deviations, and thus the tests usually have low statistical specificity to positive selection. The main challenge is hence to construct test statistics that are reasonably powerful in detecting positive selection, but are relatively insensitive to other forces. Recently, Zeng et al. (2006) proposed a new test, DH, which is a compound of Tajima's D and Fay and Wu's H, and showed that DH has reasonably high statistical specificity to positive selection. In this report, we expand the idea of a compound test by combining Fay and Wu's H or DH with the Ewens-Watterson (EW) test. We refer to these 2 new tests as HEW and DHEW, respectively. Compared to the DH test, HEW and DHEW are more robust against the presence of recombination, and are also more powerful in detecting positive selection. Furthermore, the DHEW test, similar to DH, is also relatively insensitive to background selection and demography. The HEW test, on the other hand, tends to be somewhat less conservative than DH and DHEW in some cases.     

Effect of recombination on the accuracy of the likelihood method for detecting positive selection at amino acid sites

Maximum-likelihood methods based on models of codon substitution accounting for heterogeneous selective pressures across sites have proved to be powerful in detecting positive selection in protein-coding DNA sequences. Those methods are phylogeny based and do not account for the effects of recombination. When recombination occurs, such as in population data, no unique tree topology can describe the evolutionary history of the whole sequence. This violation of assumptions raises serious concerns about the likelihood method for detecting positive selection. Here we use computer simulation to evaluate the reliability of the likelihood-ratio test (LRT) for positive selection in the presence of recombination. We examine three tests based on different models of variable selective pressures among sites. Sequences are simulated using a coalescent model with recombination and analyzed using codon-based likelihood models ignoring recombination. We find that the LRT is robust to low levels of recombination (with fewer than three recombination events in the history of a sample of 10 sequences). However, at higher levels of recombination, the type I error rate can be as high as 90%, especially when the null model in the LRT is unrealistic, and the test often mistakes recombination as evidence for positive selection. The test that compares the more realistic models M7 (beta) against M8 (beta and omega) is more robust to recombination, where the null model M7 allows the positive selection pressure to vary between 0 and 1 (and so does not account for positive selection), and the alternative model M8 allows an additional discrete class with omega = d(N)/d(S) that could be estimated to be >1 (and thus accounts for positive selection). Identification of sites under positive selection by the empirical Bayes method appears to be less affected than the LRT by recombination.     

The role of natural selection in genetic differentiation of worldwide populations of Drosophila ananassae

The main evolutionary forces leading to genetic differentiation between populations are generally considered to be natural selection, random genetic drift, and limited migration. However, little empirical evidence exists to help explain the extent, mechanism, and relative role of these forces. In this study, we make use of the differential migration behavior of genes located in regions of low and high recombination to infer the role and demographic distribution of natural selection in Drosophila ananassae. Sequence data were obtained from 13 populations, representing almost the entire range of cosmopolitan D. ananassae. The pattern of variation at a 5.1-kb fragment of the furrowed gene, located in a region of very low recombination, appears strikingly different from that of 10 noncoding DNA fragments (introns) in regions of normal to high recombination. Most interestingly, two main haplotypes are present at furrowed, one being fixed in northern populations and the other being fixed or in high frequency in more southern populations. A cline in the frequency of one of these haplotypes occurs in parallel latitudinal transects. Taken together, significant clinal variation and a test against alternative models of natural selection provide evidence of two independent selective sweeps restricted to specific regions of the species range.     

Most Powerful Permutation Invariant Tests for Relatedness Hypotheses Using Genotypic Data

The problem of inferring kinship structure among a sample of individuals using genetic markers is considered with the objective of developing hypothesis tests for genetic relatedness with nearly optimal properties. The class of tests considered are those that are constrained to be permutation invariant, which in this context defines tests whose properties do not depend on the labeling of the individuals. This is appropriate when all individuals are to be treated identically from a statistical point of view. The approach taken is to derive tests that are probably most powerful for a permutation invariant alternative hypothesis that is, in some sense, close to a null hypothesis of mutual independence. This is analagous to the locally most powerful test commonly used in parametric inference. Although the resulting test statistic is a U-statistic, normal approximation theory is found to be inapplicable because of high skewness. As an alternative it is found that a conditional procedure based on the most powerful test statistic can calculate accurate significance levels without much loss in power. Examples are given in which this type of test proves to be more powerful than a number of alternatives considered in the literature, including Queller and Goodknight's (1989) estimate of genetic relatedness, the average number of shared alleles (Blouin, 1996), and the number of feasible sibling triples (Almudevar and Field, 1999).     

Polymorphism,haplotype composition,and selection in the <Emphasis Type="Italic">Mhc-DRB</Emphasis> of wild baboons

General patterns of organization in the major histocompatibility complex (MHC) have been successfully explained by the model of birth-and-death evolution, but understanding why certain MHC genes are maintained together into specific haplotypes remains challenging. The haplotype configurations of the functionally important class II DR region have been described in few primates and display important interspecific variability with respect to the extent of allelic variation, the number of loci and/or combinations of loci present. Understanding the evolutionary mechanisms driving such variation is conditional upon characterizing haplotypes in new species and identifying the selective pressures acting on haplotypes. This study explores the variability of haplotype configurations in the Mhc-DRB region (exon 2) for the first time in wild non-human primates, chacma baboons (Papio ursinus). Paur-DRB haplotypes were characterized through segregation studies and linkage disequilibrium. 23 Paur-DRB sequences and 15 haplotype configurations were identified in 199 animals. The Paur-DRB exon 2 is shown to be subjected to intense positive selection and frequent recombination. An approach recently developed for human vaccine studies was used to classify Paur-DRB sequences into supertypes, based on the physico-chemical properties of amino acids that are positively selected, thus most probably involved in antigen recognition. Sequences grouped into the same supertype (thus presumably sharing antigen-binding affinities) are non-randomly distributed within haplotypes, leading to an increased individual diversity of supertypes. Our results suggest that selection favoring haplotypes with complementary sets of DRB supertypes shapes functionally tuned haplotypes in this natural baboon population.     

Statistical tests for detecting positive selection by utilizing high-frequency variants

By comparing the low-, intermediate-, and high-frequency parts of the frequency spectrum, we gain information on the evolutionary forces that influence the pattern of polymorphism in population samples. We emphasize the high-frequency variants on which positive selection and negative (background) selection exhibit different effects. We propose a new estimator of θ (the product of effective population size and neutral mutation rate), θ_L, which is sensitive to the changes in high-frequency variants. The new θ_L allows us to revise Fay and Wu's H-test by normalization. To complement the existing statistics (the H-test and Tajima's D-test), we propose a new test, E, which relies on the difference between θ_L and Watterson's θ_W. We show that this test is most powerful in detecting the recovery phase after the loss of genetic diversity, which includes the postselective sweep phase. The sensitivities of these tests to (or robustness against) background selection and demographic changes are also considered. Overall, D and H in combination can be most effective in detecting positive selection while being insensitive to other perturbations. We thus propose a joint test, referred to as the DH test. Simulations indicate that DH is indeed sensitive primarily to directional selection and no other driving forces.     

A More Powerful Simultaneous Test Procedure in Configural Frequency Analysis

In the Configural Frequency Analysis (CFA) of KRAUTH and LIENERT (1973 a, b), overfrequented (or underfrequented) cells in multivariate contingency tables are identified by simultaneous binomial tests. As an alternative, finite and asymptotic tests are proposed, which are derived from the (exact conditional) generalized hypergeometrical distribution of the cell frequencies. These tests allow for considerably more powerful decisions than do the conservative binomial tests.     

Statistical power analysis of neutrality tests under demographic expansions, contractions and bottlenecks with recombination

Several tests have been proposed to detect departures of nucleotide variability patterns from neutral expectations. However, very different kinds of evolutionary processes, such as selective events or demographic changes, can produce similar deviations from these tests, thus making interpretation difficult when a significant departure of neutrality is detected. Here we study the effects of demography and recombination upon neutrality tests by analyzing their power under sudden population expansions, sudden contractions, and bottlenecks. We evaluate tests based on the frequency spectrum of mutations and the distribution of haplotypes and explore the consequences of using incorrect estimates of the rates of recombination when testing for neutrality. We show that tests that rely on haplotype frequencies-especially Fs and ZnS, which are based, respectively, on the number of different haplotypes and on the r2 values between all pairs of polymorphic sites-are the most powerful for detecting expansions on nonrecombining genomic regions. Nevertheless, they are strongly affected by misestimations of recombination, so they should not be used when recombination levels are unknown. Instead, class I tests, particularly Tajima's D or R2, are recommended.     

A Bayesian method for jointly estimating allele age and selection intensity.

The problem of jointly estimating the intensity of past selection affecting an allele and the allele's age is formulated in a Bayesian framework. The prior distribution of allele age given its frequency is obtained from existing population genetics theory. The prior distribution of selection intensity is assumed to reflect the fact that positive selection on a new mutant is more likely to be weak than strong. The general approach is illustrated by the development of an importance sampling method applicable to low-frequency alleles. This method can be used either when the haplotypes of closely linked marker loci are known or when the lengths of linked ancestral chromosomal segments can be inferred. The method is illustrated with an application to the A-allele of G6PD in Africa. Because changes in allele frequency and recombination are both intrinsically stochastic, there are limits to the accuracy achievable with any method.     

DNA tests of neutral theory: applications in marine genetics

The principal methods of using DNA sequence information to test the neutral theory of evolution and polymorphism are described. These include the use of synonymous and nonsynonymous substitutions for detecting purifying and positive selection, the analysis of nucleotide diversity, mismatch analysis and the HKA, McDonald-Kreitman, Tajima and Ewens-Watterson tests. Analysis of the covariation of different kinds of molecular markers and the relationship between genetic variation and fitness is also considered. Examples of the use of these approaches in a wide variety of marine organisms are described. It is emphasised that tests of neutral theory, in addition to providing important fundamental knowledge about the action of evolutionary forces, provide valuable information about the influence of environmental and demographic factors.     

Unconditional versions of several tests commonly used in the analysis of contingency tables

By focusing on a confidence interval for a nuisance parameter, Berger and Boos (1994, Journal of the American Statistical Association 89, 1012-1016) proposed new unconditional tests. In particular, they showed that, for a 2 x 2 table, this procedure generally was more powerful than Fisher's exact test. This paper utilizes and extends their approach to obtain unconditional tests for combining several 2 x 2 tables and testing for trend and homogeneity in a 2 x K table. The unconditional procedures are compared to the conditional ones by reanalyzing some published biomedical data.     

Nucleotide variation at the dopa decarboxylase (<Emphasis Type="Italic">Ddc</Emphasis>) gene in natural populations of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We studied nucleotide sequence variation at the gene coding for dopa decarboxylase (Ddc) in seven populations of Drosophila melanogaster. Strength and pattern of linkage disequilibrium are somewhat distinct in the extensively sampled Spanish and Raleigh populations. In the Spanish population, a few sites are in strong positive association, whereas a large number of sites in the Raleigh population are associated nonrandomly but the association is not strong. Linkage disequilibrium analysis shows presence of two groups of haplotypes in the populations, each of which is fairly diverged, suggesting epistasis or inversion polymorphism. There is evidence of two forms of natural selection acting on Ddc. The McDonald-Kreitman test indicates a deficit of fixed amino acid differences between D. melanogaster and D. simulans, which may be due to negative selection. An excess of derived alleles at high frequency, significant according to the H-test, is consistent with the effect of hitchhiking. The hitchhiking may have been caused by directional selection downstream of the locus studied, as suggested by a gradual decrease of the polymorphism-to-divergence ratio. Altogether, the Ddc locus exhibits a complicated pattern of variation apparently due to several evolutionary forces. Such a complex pattern may be a result of an unusually high density of functionally important genes.     

Bayesian multiple testing for two-sample multivariate endpoints

In clinical studies involving multiple variables, simultaneous tests are often considered where both the outcomes and hypotheses are correlated. This article proposes a multivariate mixture prior on treatment effects, that allows positive probability of zero effect for each hypothesis, correlations among effect sizes, correlations among binary outcomes of zero versus nonzero effect, and correlations among the observed test statistics (conditional on the effects). We develop a Bayesian multiple testing procedure, for the multivariate two-sample situation with unknown covariance structure, and obtain the posterior probabilities of no difference between treatment regimens for specific variables. Prior selection methods and robustness issues are discussed in the context of a clinical example.     

A Comparison of Tests for Marginal Homogeneity in Square Contingency Tables

Several asymptotic tests were proposed for testing the null hypothesis of marginal homogeneity in square contingency tables with r categories. A simulation study was performed for comparing the power of four finite conservative conditional test procedures and of two asymptotic tests for twelve different contingency schemes for small sample sizes. While an asymptotic test proposed by STUART (1955) showed a rather satisfactory behaviour for moderate sample sizes, an asymptotic test proposed by BHAPKAR (1966) was quite anticonservative. With no a priori information the performance of (r - 1) simultaneous conditional binomial tests with a Bonferroni adjustment proved to be a quite efficient procedure. With assumptions about where to expect the deviations from the null hypothesis, other procedures favouring the larger or smaller conditional sample sizes, respectively, can have a great efficiency. The procedures are illustrated by means of a numerical example from clinical psychology.     

Recombination, balancing selection and adaptive evolution in the aflatoxin gene cluster of Aspergillus parasiticus

Aflatoxins are toxic and carcinogenic polyketides produced by several Aspergillus species that are known to contaminate agricultural commodities, posing a serious threat to animal and human health. Aflatoxin (AF) biosynthesis is almost fully characterized and involves the coordinated expression of approximately 25 genes clustered in a 70-kb DNA region. Aspergillus parasiticus is an economically important and common agent of AF contamination. Naturally occurring nonaflatoxigenic strains of A. parasiticus are rarely found and generally produce O-methylsterigmatocystin (OMST), the immediate precursor of AF. To elucidate the evolutionary forces acting to retain AF and OMST pathway extrolites (chemotypes), we sequenced 21 intergenic regions spanning the entire cluster in 24 A. parasiticus isolates chosen to represent the genetic diversity within a single Georgia field population. Linkage disequilibrium analyses revealed five distinct recombination blocks in the A. parasiticus cluster. Phylogenetic network analyses showed a history of recombination between chemotype-specific haplotypes, as well as evidence of contemporary recombination. We performed coalescent simulations of variation in recombination blocks and found an approximately twofold deeper coalescence for cluster genealogies compared to noncluster genealogies, our internal standard of neutral evolution. Significantly deeper cluster genealogies are indicative of balancing selection in the AF cluster of A. parasiticus and are further corroborated by the existence of trans-species polymorphisms and common haplotypes in the cluster for several closely related species. Estimates of Ka/Ks for representative cluster genes provide evidence of selection for OMST and AF chemotypes, and indicate a possible role of chemotypes in ecological adaptation and speciation.     

Detecting the signature of selection on immune genes in highly structured populations of wild sheep (Ovis dalli)

The confounding effects of population structure complicate efforts to identify regions of the genome under the influence of selection in natural populations. Here we test for evidence of selection in three genes involved in vertebrate immune function - the major histocompatibility complex (MHC), interferon gamma (IFNG) and natural resistance associated macrophage polymorphism (NRAMP) - in highly structured populations of wild thinhorn sheep (Ovis dalli). We examined patterns of variation at microsatellite loci linked to these gene regions and at the DNA sequence level. Simple Watterson's tests indicated balancing selection at all three gene regions. However, evidence for selection was confounded by population structure, as the Watterson's test statistics from linked markers were not outside of the range of values from unlinked and presumably neutral microsatellites. The translated coding sequences of thinhorn IFNG and NRAMP are fixed and identical to those of domestic sheep (Ovis aries). In contrast, the thinhorn MHC DRB locus shows significant evidence of overdominance through both an excess of nonsynonymous substitution and trans-species polymorphism. The failure to detect balancing selection at microsatellite loci linked to the MHC is likely the result of recombination between the markers and expressed gene regions.     

Hybrid maize breeding with doubled haploids: III. Efficiency of early testing prior to doubled haploid production in two-stage selection for testcross performance

Early testing prior to doubled haploid (DH) production is a promising approach in hybrid maize breeding. We (1) determined the optimum allocation of the number of S₁ families, DH lines, and test locations for two different breeding schemes, (2) compared the maximum selection gain achievable under both breeding schemes, and (3) investigated limitations in the current method of DH production. Selection gain was calculated by numerical integration in two-stage breeding schemes with evaluation of testcross progenies of (1) DH lines in both stages (DHTC), or (2) S₁ families in the first and DH lines within S₁ families in the second stage (S₁TC-DHTC). Different assumptions were made regarding the budget, variance components, and time of DH production within S₁ families. Maximum selection gain in S₁TC-DHTC was about 10% larger than in DHTC, indicating the large potential of early testing prior to DH production. The optimum allocation of test resources in S₁TC-DHTC involved similar numbers of test locations and test candidates in both stages resulting in a large optimum number of S₁ families in the first stage and DH lines within the best two S₁ families in the second stage. The longer cycle length of S₁TC-DHTC can be compensated by haploid induction of individual S₁ plants instead of S₁ families. However, this reduces selection gain largely due to the current limitations in the DH technique. Substantial increases in haploid induction and chromosome doubling rates as well as reduction in costs of DH production would allow early testing of S₁ lines and subsequent production and testing of DH lines in a breeding scheme that combines high selection gain with a short cycle length. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An investigation of the statistical power of neutrality tests based on comparative and population genetic data

In this report, we investigate the statistical power of several tests of selective neutrality based on patterns of genetic diversity within and between species. The goal is to compare tests based solely on population genetic data with tests using comparative data or a combination of comparative and population genetic data. We show that in the presence of repeated selective sweeps on relatively neutral background, tests based on the d(N)/d(S) ratios in comparative data almost always have more power to detect selection than tests based on population genetic data, even if the overall level of divergence is low. Tests based solely on the distribution of allele frequencies or the site frequency spectrum, such as the Ewens-Watterson test or Tajima's D, have less power in detecting both positive and negative selection because of the transient nature of positive selection and the weak signal left by negative selection. The Hudson-Kreitman-Aguadé test is the most powerful test for detecting positive selection among the population genetic tests investigated, whereas McDonald-Kreitman test typically has more power to detect negative selection. We discuss our findings in the light of the discordant results obtained in several recently published genomic scans.     

Genetic linkage map of Coffea canephora: effect of segregation distortion and analysis of recombination rate in male and female meioses.

Two complementary segregating plant populations of Coffea canephora were produced from the same clone. One population (DH) comprised 92 doubled haploids derived from female gametes, while the other population (TC) was a test cross consisting of 44 individuals derived from male gametes. Based on the DH population, a genetic linkage map comprising 160 loci was constructed. Eleven linkage groups that putatively correspond to the 11 gametic chromosomes of C. canephora were identified. The mapped loci included more than 40 specific sequence-tagged site markers, either single-copy RFLP probes or microsatellites, that could serve as standard landmarks in coffee-genome analyses. Furthermore, comparisons for segregation distortion and recombination frequency between the two populations were performed. Although segregation distortions were observed in both populations, the frequency of loci exhibiting a very pronounced degree of distortion was especially high in the DH population. This observation is consistent with the hypothesis of strong zygotic selection among the DH population. The recombination frequencies in both populations were found to be almost indistinguishable. These results offer evidence in favour of the lack of significant sex differences in recombination in C. canephora.