Large-Scale Adaptive Hitchhiking Upon High Recombination in Drosophila simulans

Corresponding author: Humberto Quesada, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain., humberto{at}porthos.bio.ub.es (E-mail)

Communicating editor: O. SAVOLAINEN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION APPENDIX LITERATURE CITED

Natural selection is expected to leave a characteristic footprint on neighboring nucleotide variation through the effects of genetic linkage. The size of the region affected is proportional to the strength of selection and greatly reduced with the recombinational distance from the selected site. Thus, the genomic footprint of selection is generally believed to be restricted to a small DNA stretch in normal and highly recombining regions. Here, we study the effect of selection on linked polymorphism (hitchhiking effect) by surveying nucleotide variation across a highly recombining 88-kb genomic fragment in an African population of Drosophila simulans. We find a core region of up to 38 kb with a major haplotype at intermediate frequency. The extended haplotype structure that gradually vanishes until disappearing is unusual for a highly recombining region. Both the presence in the structured genomic domain of a single major haplotype depleted of variability and the detected spatial pattern of variation along the 88-kb fragment are incompatible with neutral predictions in a panmictic population. A major role of demographic effects could also be discarded. The observed pattern of variation clearly provides evidence that directional selection has acted recently on this region, sweeping out variation around a strongly adaptive mutation. Our findings suggest a major role of positive selection in shaping DNA variability even in highly recombining regions.

THE extent to which DNA variation is driven by selective and stochastic factors is one of the greatest standing controversies in molecular evolution (KIMURA 1983 ; GILLESPIE 1991 ). The neutral theory holds that the bulk of mutations that spread through a population are governed by genetic drift (KIMURA 1983 ). Yet, positive selection can play an important role by sweeping out nucleotide variation around selected sites (MAYNARD SMITH and HAIGH 1974 ). Attempts to use this genetic hitchhiking effect as evidence for positive selection in regions of low recombination have been inconclusive, since in many cases the results are compatible with both adaptive and nonadaptive explanations (CHARLESWORTH et al. 1993 ; HUDSON and KAPLAN 1995 ). Multilocus surveys of variation in regions with normal and high levels of recombination may also be compromised given that the footprint of positive selection in these regions is generally short-lived and restricted to small stretches and thus difficult to detect (KAPLAN et al. 1989 ; PRZEWORSKI 2002 ). Considerable debate has recently focused on whether the observation of a genome-wide excess of linkage disequilibrium may reflect demographic rather than selective effects (ANDOLFATTO and PRZEWORSKI 2000 ; PRITCHARD and PRZEWORSKI 2001 ; GILAD et al. 2002 ). Distinguishing between these effects remains a crucial task to determine the role of positive selection in shaping genome variability (OLSON 2002 ; SABETI et al. 2002 ).

Detecting the effect of selection on neighboring variation may be facilitated by surveying variation in genomic regions containing loci under putatively recent selection (NURMINSKI et al. 2001 ; PRITCHARD and PRZEWORSKI 2001 ; SABETI et al. 2002 ). Indeed, a distinctive spatial footprint would result from the action of positive selection, whose effect would decay with distance to the target of selection and eventually disappear (KIM and STEPHAN 2002 ; SABETI et al. 2002 ). In contrast, demographic events occurring in the evolutionary history of the population would affect the level and pattern of variation across the entire surveyed region, as well as across the whole genome (ANDOLFATTO 2001 ).

Positive selection has been proposed to explain the unusual haplotype structure detected in Drosophila simulans for a 1.3-kb region encompassing the rp49 gene (ROZAS et al. 2001 ) and for a 2.5-kb region containing three closely linked paralogous genes (janA, janB, and ocn) that lies 7 kb from rp49 (PARSCH et al. 2001 ). These genes are located in a highly recombining region of chromosome 3R (band 99D) that is monomorphic at the chromosomal level (PARSCH et al. 2001 ). Despite high recombination (0.92 x 10^-8 recombination events per base pair; ROZAS et al. 2001 ), these genes display one haplotype at intermediate frequency in both a European and an African population (rp49), as well as in a worldwide sample (janA and janB). This pattern of variation was shown to be highly incompatible with the neutral equilibrium model and rather difficult to reconcile with recent population admixture (PARSCH et al. 2001 ; ROZAS et al. 2001 ). However, definitive inferences on the role of natural selection can be drawn only from multilocus analyses since, for a single locus, demographic effects could mimic the footprint of natural selection. Here, we have surveyed variation in regions at increasing distances from the rp49 gene that extend over 88 kb. This is among the longest stretches of a highly recombining region of Drosophila whose haplotype structure in a single population has been directly determined by DNA sequencing. We were particularly interested in detecting the spatial pattern of variation associated with positive selection. The survey of variation in such a large region has allowed us not only to unambiguously establish the role of positive selection but also to assess the size of the region affected and to estimate the strength of selection.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION APPENDIX LITERATURE CITED

Fly samples and regions studied:
Fourteen D. simulans lines from Maputo (Mozambique) were used in this study. These were highly inbred strains obtained by 10 generations of sib-mating. The DNAs from the same individuals previously sequenced for the rp49 region (ROZAS et al. 2001 ) were used whenever possible; otherwise, genomic DNA was extracted from single individuals from the same strain using a modification of protocol 48 from ASHBURNER 1989 . The individuals used were homozygous for the previously described rp49 allele, as well as for the other regions surveyed.

On the basis of the annotated sequence of D. melanogaster (GenBank accession no. AE003772; ADAMS et al. 2000 ), 10 regions at increasing distances from the rp49 gene and covering an 88-kb genomic fragment were selected (Fig 1). They consisted primarily of noncoding DNA and their size ranged between 0.5 and 1.3 kb (Appendix).

View larger version (34K): In this window In a new window Download PPT slide   Figure 1. Phylogenetic trees and summary of nucleotide variation in the regions studied. Regions have been named in alphabetical order starting in the region most proximal to the centromere. The neighbor-joining trees were built using Jukes-Cantor corrected distances and rooted using D. melanogaster. Only bootstrap values >80% are shown. Sequences belonging to the homogeneous and heterogeneous subsets are depicted as squares and circles, respectively. The lines previously found to be homogeneous in the G region (ROZAS et al. 2001 ) appear as solid, others appear as open. The D region includes two closely linked subregions that were pooled. The shaded values show the window identified by the sliding-window haplotype test. The bold values show the window identified by the single-locus haplotype tests. S, number of segregating sites; , nucleotide diversity; K, average nucleotide divergence between D. simulans and D. melanogaster corrected by the Jukes-Cantor method; ns, not significant. Superscript a indicates parameter settings were modified to consider sequences differing in up to one nucleotide position.

DNA sequencing strategy:
Oligonucleotides for PCR amplification and sequencing were designed on the available sequence of D. melanogaster (GenBank accession no. AE003772; ADAMS et al. 2000 ). Oligonucleotides and conditions for PCR amplification are available from the authors. After purification of PCR products with Microcon-PCR filter columns (Millipore, Bedford, MA), both strands were completely sequenced using internal primers whenever necessary. Sequencing reactions were performed with the ABI Prism BigDye Terminators v2.0 cycle sequencing kit (Applied Biosystems, Foster City, CA) following manufacturer's instructions. The sequencing reaction products were purified by ethanol precipitation and subsequently separated on an ABI PRISM 3700 automated DNA sequencer (Perkin-Elmer, Norwalk, CT). Sequences were assembled with the SeqEd 1.03 program (Applied Biosystems). Newly reported DNA sequences are deposited in the EMBL nucleotide sequence database under accession numbers AJ554064–AJ554203.

Data analysis for individual loci:
DNA sequences were multiply aligned using the Clustal X program (THOMPSON et al. 1997 ) and further edited with the MacClade 3.06 program (MADDISON and MADDISON 1992 ). Sites with alignment gaps were excluded from the analyses. The DnaSP 3.95 program (ROZAS and ROZAS 1999 ) was used to estimate nucleotide diversity () and the population recombination parameter (C = 2Nc, where N is the effective population size and c is the per-base recombination rate). It was also used to perform different tests of neutrality: Tajima's D test (TAJIMA 1989 ), haplotype diversity tests (DEPAULIS and VEUILLE 1998 ), and the Hudson-Kreitman-Aguadé (HKA) test (HUDSON et al. 1987 ). Regions departing from neutral equilibrium were also identified using the sliding-window haplotype test (ANDOLFATTO et al. 1999 ). The P values of Tajima's D and haplotype tests were estimated from 10,000 coalescent simulations assuming recombination and a fixed number of segregating sites. The Mega 2.1 program (KUMAR et al. 2001 ) was employed for phylogenetic reconstruction using the neighbor-joining algorithm; bootstrap values were based on 1000 replicates.

Multilocus tests of neutrality:
We built a multilocus statistic where O_i is the observed value in region i, E_i is the value expected under the null hypothesis, and n is the number of regions (n = 10 in our case). We determined whether the number of identical sequences or the haplotype diversity departs from the neutral model (i.e., mutation-drift equilibrium in a panmictic population). The expected values of identical haplotypes or of haplotype diversity for each region and the empirical distribution of were obtained by neutral coalescent simulations (1000 replicates) with recombination (C_M = 0.0368, obtained from the comparison of physical and genetic maps and assuming N = 2 x 10⁶ and c = 0.92 x 10^-8; ROZAS et al. 2001 ). Simulations were conditioned on the number of segregating sites. Random data sets of DNA fragments as long as the surveyed fragment (88 kb) were generated in the simulations. The P value of the test (two-tailed test) was obtained as the proportion of computer replicates with values more extreme than observed.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION APPENDIX LITERATURE CITED

A total of 340 segregating sites (nucleotide diversity = 0.0157) and 45 insertion/deletion polymorphisms were detected across the sequenced regions in the 14 lines from Maputo (Mozambique; Fig 1). The number of segregating sites in each region was rather high and ranged from 18 to 53. The high recombination rate of the genomic fragment surveyed (see Introduction) is apparent in the polymorphism data. A minimum number of 48 recombination events (R_M) were detected in the sample using the four-gamete rule method (HUDSON and KAPLAN 1985 ). This is clearly an underestimate since we did not obtain a continuous sequence along the chromosome. The minimum C value (C_L) compatible (at 5% level) with the observed R_M value constitutes a lower bound of the population recombination parameter (ROZAS et al. 2001 ). The C_L value estimated from the data using coalescent simulations (HUDSON 1983 , HUDSON 1990 ) was 0.0284. This value obtained from an underestimated R_M is not very different from the more realistic C_M value (C_M = 0.0368; ROZAS et al. 2001 ). Recombination has, therefore, played an important role in the history of the 88-kb genomic fragment analyzed.

Despite extensive recombination, nucleotide variation was highly structured along an extended genomic area encompassing rp49 and the three paralogous genes (Fig 1). The clustering pattern in phylogenetic trees is very similar, in many regions, to that previously found: a cluster of identical or nearly identical sequences within a set of highly diverged and heterogeneous sequences. Two features of the data (Fig 1) are consistent with a selective sweep. First, the unusual haplotype structure does not extend across the entire fragment. The affected area is surprisingly large, but flanked by boundary regions with no appreciable haplotype structure (A, I, and J). Second, the number of DNA sequences within the homogeneous subset decays gradually on both sides of the most structured stretch (E and F). Remarkably, some of the lines that form part of the homogeneous subset in a particular region belong to the heterogeneous subset in the surrounding regions. This indicates that recombination has disrupted the association between regions on either side of the most structured stretch.

Positive selection is also inferred from a nonuniform deviation from neutral expectations along the entire fragment, as revealed by several neutrality tests. In all the tests, probability values were obtained by coalescent-Monte Carlo simulations assuming recombination (HUDSON 1983 , HUDSON 1990 ; ROZAS and ROZAS 1999 ). We first tested for either an excess of identical sequences or a reduction in haplotype diversity in individual regions (DEPAULIS and VEUILLE 1998 ; ROZAS et al. 2001 ). The analyses showed a significant or marginally significant excess of identical sequences and a significant reduction in haplotype diversity along an 24-kb stretch covering the regions with strong haplotype structure (C, D, E, F, and G; Fig 1). However, the footprint of selection likely spans >38 kb, since the flanking regions B and H still show a detectable haplotype structure (Fig 1) with a number of identical sequences higher than expected (Fig 2). To test whether the observed heterogeneity along the entire fragment occurred simply by chance, we used a coalescent-based approach to take into account stochastic variation in the number of identical sequences or in the haplotype diversity along the entire fragment. For this analysis, we generated random samples of 14 sequences of 88 kb with 4282 segregating sites (those resulting from scaling up the number observed in the sequenced regions) to test for spatial structure across the surveyed fragment using the multilocus -statistic (see MATERIALS AND METHODS). Both the overall high number of identical sequences observed and the overall low haplotype diversity are clearly incompatible with the neutral model (P < 0.005 in both cases). The results from the multilocus tests were corroborated using a different method, the sliding-window haplotype test (ANDOLFATTO et al. 1999 ). This analysis takes into account stochastic variation across the fragment to identify which sequence subset holds the minimum number of haplotypes. The test supported a nonuniform haplotype structure along the entire fragment due to a stretch covering regions E, F, and G, although only the F and G subset remained significant after correction for multiple tests and window sizes (P = 0.011; P = 0.049 after correction). This latter test is conservative in our case, since the contribution of recombination in nonsequenced regions was ignored.

View larger version (17K): In this window In a new window Download PPT slide   Figure 2. Observed vs. expected number of identical sequences for the 10 regions surveyed. The expected values for each region were obtained by neutral coalescent simulations (10,000 replicates) with recombination. The regions are ordered as in Fig 1.

We also used Tajima's D statistic to examine the fit of the observed frequency spectrum of polymorphic sites to that expected under neutrality. No significant departure from selective neutrality was found in any individual region or in the fragment as a whole (Fig 1). However, 8 out of the 10 regions show negative D values indicating an excess of singleton variants. Although all singletons are derived (as inferred from the D. melanogaster outgroup sequence), many occur in the heterogeneous subset of sequences. The negative Tajima's D values are thus not caused by new mutations occurring after the selective sweep, but may be due, for example, to historical events predating the sweep. On the other hand, comparison of polymorphism and divergence levels between the region(s) studied and the vermilion locus (BEGUN and AQUADRO 1995 ) by means of the HKA test (HUDSON et al. 1987 ; BEGUN and AQUADRO 1991 ) also failed to detect any deviation from predictions of the neutral model (results not shown).

The haplotype tests clearly indicate that natural selection has acted recently and strongly. The homogeneous subset was most likely a formerly rare haplotype driven to an intermediate frequency due to its linkage to a positively selected variant. This is consistent with its phylogenetically derived position (Fig 1) and the high number of fixed derived nucleotide variants in this subset relative to the heterogeneous subset (21 out of 24 along regions B–H). Selection would favor the same mutation in the whole distribution area of the species, since the same haplotype was found at intermediate frequency in the populations surveyed for rp49 and the three paralogous genes (PARSCH et al. 2001 ; ROZAS et al. 2001 ). However, in no region within the structured genomic domain has the fixation of a single haplotype been detected, indicating that the sweep is partial. The selected haplotype could be on its way either to fixation or to reach some equilibrium frequency. Alternatively, it could have already reached an equilibrium frequency and thus reflect a very recently established balanced polymorphism. This could explain the lack of significance of Tajima's D and HKA neutrality tests, since these tests are unlikely to detect departures from neutrality under the above selective scenarios (HUDSON et al. 1994 ).

The surprisingly large genomic distance over which selection is likely to exert a strong effect on linked variation (at least 24–38 kb of a highly recombining region) implies a high selection coefficient. From these distances, we estimated the selection coefficient s to be 0.011 or higher (d x c/0.01, where d is half the length of the affected fragment; KAPLAN et al. 1989 ). Although this estimation method assumes a complete selective sweep, the true s value for the partial sweep case should not be very different, since the extent of the hitchhiking effect is mostly established when the frequency of the selected allele is still low (STEPHAN et al. 1992 ). The high estimated selection coefficient also points to the difficulty of identifying the specific site under selection. An approximate estimate of the most likely location of the selected site could be given by the narrow 3-kb interval defined by the sliding-window haplotype test. However, valleys of depleted variability may be rather asymmetrical around the site of the beneficial mutation (KIM and STEPHAN 2002 ), which implies that the window defined by this criterion may provide rather imprecise information about the location of the target of selection.

Our observation of a single major haplotype depleted of variation strongly supports a partial selective sweep (HUDSON et al. 1994 ). The presence of this intermediate-frequency haplotype and of substantial recombination since the selective event is difficult to reconcile with the alternative selective hypotheses proposed to explain partitions of nucleotide variation into distinct sets of haplotypes (KIRBY and STEPHAN 1995 , KIRBY and STEPHAN 1996 ; WANG et al. 2002 ; ZUROVCOVA and AYALA 2002 ). Indeed, if the homogeneous and heterogeneous subsets at intermediate frequency were due to an old balanced polymorphism, they should harbor comparable levels of variation. Alternatively, if they were maintained by epistatic selection favoring particular combinations of variants, both subsets should show reduced levels of variation, and the number of recombinants should be low. Finally, if the pattern observed were due to the presence of two adaptive mutations preventing each other's fixation ("traffic hypothesis"; KIRBY and STEPHAN 1996 ), the number of recombinants should also be low.

We have considered the alternative hypothesis that the unusual haplotype structure was due to the recent admixture of two differentiated populations, one of them devoid of variation (ROZAS et al. 2001 ). The results of haplotype and multilocus tests do not support this possibility, since they indicate that the haplotype structure is restricted to a specific region of the studied fragment. Coalescent simulations also show that demographic factors are unlikely to generate the pattern observed here (our unpublished results). Furthermore, the similar pattern of variation detected in the rp49 region in the African population studied here and in a European population (ROZAS et al. 2001 ) indicates that the sweep predates the out-of-Africa expansion of D. simulans (LACHAISE et al. 1984 ). Adaptive hitchhiking can thus overwhelm the effect of putative demographic events associated with the species expansion.

Recent studies in Drosophila have yielded promising results in the struggle to understand whether natural selection plays a substantial role in protein evolution, where adaptive amino acid substitutions appear to occur at a remarkably high rate (FAY et al. 2002 ; SMITH and EYRE-WALKER 2002 ). The results of our study, taken in combination with those on protein evolution, provide evidence not only that positive selection can be common, but also that its distinctive footprint can extend across very large segments even in highly recombining regions. The more general significance of our findings is that they support the hypothesis (GILLESPIE 1991 ) that the signature of selection may be important at the genome-wide level and more common than usually accepted. Thus, neutral variation could be shaped to a great extent by the hitchhiking effects associated with positive Darwinian selection, rather than just by genetic drift.

	ACKNOWLEDGMENTS

We thank S. Ramos-Onsins for sharing his unpublished coalescent-simulation program, G. Blasco for technical assistance, and W. Stephan for critical comments on the manuscript. We also thank Serveis Científico-Tècnics from Universitat de Barcelona for automated sequencing facilities and Centre de Supercomputació de Catalunya (CESCA) for computer facilities. This work was supported by grants BMC2001-2906 from Comisión Interdepartamental de Ciencia y Tecnología (CICyT) and 2001SGR-101 from Comissió Interdepartamental de Recerca i Innovació Tecnològica (CIRIT), Catalonia, Spain, to M.A.

Manuscript received February 6, 2003; Accepted for publication June 6, 2003.

	APPENDIX

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION APPENDIX LITERATURE CITED

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION APPENDIX LITERATURE CITED

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				A. J. Vilella, A. Blanco-Garcia, S. Hutter, and J. Rozas VariScan: Analysis of evolutionary patterns from large-scale DNA sequence polymorphism data Bioinformatics, June 1, 2005; 21(11): 2791 - 2793. [Abstract] [Full Text] [PDF]

				T. A. Schlenke and D. J. Begun Linkage Disequilibrium and Recent Selection at Three Immunity Receptor Loci in Drosophila simulans Genetics, April 1, 2005; 169(4): 2013 - 2022. [Abstract] [Full Text] [PDF]

				B. P. Lazzaro Elevated Polymorphism and Divergence in the Class C Scavenger Receptors of Drosophila melanogaster and D. simulans Genetics, April 1, 2005; 169(4): 2023 - 2034. [Abstract] [Full Text] [PDF]

				C. D. Meiklejohn, Y. Kim, D. L. Hartl, and J. Parsch Identification of a Locus Under Complex Positive Selection in Drosophila simulans by Haplotype Mapping and Composite-Likelihood Estimation Genetics, September 1, 2004; 168(1): 265 - 279. [Abstract] [Full Text] [PDF]

				Y. Kim and R. Nielsen Linkage Disequilibrium as a Signature of Selective Sweeps Genetics, July 1, 2004; 167(3): 1513 - 1524. [Abstract] [Full Text] [PDF]