Altitudinal Variation at Duplicated b-Globin Genes in Deer Mice: Effects of Selection, Recombination, and Gene Conversion

Spatially varying selection on a given polymorphism is expected to produce a localized peak in the between-population component of nucleotide diversity, and theory suggests that the chromosomal extent of elevated differentiation may be enhanced in cases where tandemly linked genes contribute to fitness variation. An intriguing example is provided by the tandemly duplicated b-globin genes of deer mice (Peromyscus maniculatus), which contribute to adaptive differentiation in blood–oxygen affinity between highand low-altitude populations. Remarkably, the two b-globin genes segregate the same pair of functionally distinct alleles due to a history of interparalog gene conversion and alleles of the same functional type are in perfect coupling-phase linkage disequilibrium (LD). Here we report a multilocus analysis of nucleotide polymorphism and LD in highland and lowland mice with different genetic backgrounds at the b-globin genes. The analysis of haplotype structure revealed a paradoxical pattern whereby perfect LD between the two b-globin paralogs (which are separated by 16.2 kb) is maintained in spite of the fact that LD within both paralogs decays to background levels over physical distances of less than 1 kb. The survey of nucleotide polymorphism revealed that elevated levels of altitudinal differentiation at each of the b-globin genes drop away quite rapidly in the external flanking regions (upstream of the 59 paralog and downstream of the 39 paralog), but the level of differentiation remains unexpectedly high across the intergenic region. Observed patterns of diversity and haplotype structure are difficult to reconcile with expectations of a two-locus selection model with multiplicative fitness. WHEN functionally distinct alleles are maintained as a balanced polymorphism, each of the associated genetic backgrounds accumulates its own set of neutral mutations. The partitioning of nucleotide variation between alternative allele classes results in elevated levels of diversity and linkage disequilibrium (LD), and this characteristic signature provides a possible means of identifying balanced polymorphisms in genomic survey data (Charlesworth 2006). The chromosomal extent of the elevated diversity and haplotype structure is jointly determined by the age of the selected polymorphism and the frequency of crossing over, as recombination provides a conduit for genetic exchange between alternative allele classes (Strobeck 1983; Hudson and Kaplan 1988; Kaplan et al. 1988; Schierup et al. 2000; Charlesworth et al. 2003; Nordborg and Innan 2003; Wiuf et al. 2004). In the case of spatially varying selection, alternative alleles will be present at different frequencies in locally adapted populations. This increases between-population components of diversity and LD relative to within-population components. Consequently, spatially varying selection on a given polymorphism is expected to produce a localized peak of between-population diversity, and the elevated differentiation at linked sites will decline as a function of genetic map distance from the selected site (Charlesworth et al. 1997; Feder and Nosil 2010). Over a broad range of population sizes and migration rates, recombination is expected to limit the peak of differentiation to a relatively narrow window spanning the selected polymorphism (Feder and Nosil 2010). The signature of selection Copyright © 2012 by the Genetics Society of America doi: 10.1534/genetics.111.134494 Manuscript received September 7, 2011; accepted for publication October 20, 2011 Supporting information is available online at http://www.genetics.org/content/ suppl/2011/10/31/genetics.111.134494.DC1. Sequence data from this article have been deposited in GenBank under accession nos. JN711139–JN711405 and EU204642. Present address: Department of Biochemistry and Molecular Biology, Mississippi State University, Mississippi State, MS 39762. Corresponding author: School of Biological Sciences, University of Nebraska, 320A Manter Hall, Lincoln, NE 68588. E-mail: [email protected] Genetics, Vol. 190, 203–216 January 2012 203 may be further eroded by gene conversion, which provides a second conduit for genetic exchange between selectively maintained allele classes. Peaks of differentiation may be most readily detectable in cases where epistatic selection maintains coadapted combinations of alleles at linked genes. This is because the selective elimination of recombinant chromosomes reduces the effective rate of crossing over between the selected loci (Ishii and Charlesworth 1977). In cases in which two or more linked genes jointly contribute to fitness variation, theoretical results suggest that it may be possible to detect the effects of epistatic selection by analyzing patterns of silent-site diversity in the intergenic region and in the external flanking regions (Kelly 2000; Kelly and Wade 2000; Barton and Navarro 2002; Navarro and Barton 2002; Chevin et al. 2009; Takahasi 2009). An empirical test of these predictions requires a system where allelic polymorphism at linked genes is associated with fitness variation under natural conditions. The tandemly duplicated globin genes of the deer mouse (Peromyscus maniculatus) are well suited to this purpose, as genetic variation in hemoglobin function mediates the adaptive fine tuning of blood–oxygen affinity in mice that are native to different elevational zones (Snyder 1981, 1985; Snyder et al. 1982, 1988; Chappell and Snyder 1984; Chappell et al. 1988; Storz 2007; Storz et al. 2007a, 2009, 2010a; Storz and Kelly 2008; Storz and Moriyama 2008). The two tandemly duplicated b-globin genes of these mice exhibit especially intriguing patterns of altitudinal variation and haplotype structure (Storz et al. 2009). These two genes, HBB-T1 and HBB-T2, encode the b-chain subunits of adult hemoglobin and are separated by 16.2 kb of noncoding DNA on chromosome 1 (Hoffmann et al. 2008a). In highland and lowland populations of deer mice from Colorado, the HBB-T1 and HBB-T2 genes segregate the same pair of functionally distinct alleles due to a history of interparalog gene conversion (Storz et al. 2009, 2010a). At both HBB-T1 and HBB-T2, the two alternative alleles are distinguished by four amino acid substitutions: 62 (Ala/Gly), 72 (Gly/Ser), 128 (Ser/ Ala), and 135 (Ala/Ser). The d1 allele class is defined by the four-site amino acid combination 62Gly/72Gly/128Ala/ 135Ala, and the d0 allele class is defined by the alternative four-site combination 62Ala/72Ser/128Ser/135Ser. TheHBB-T1 andHBB-T2 genes exhibit striking haplotype structure, as alleles of the same functional type are in perfect coupling-phase LD: one chromosome harbors the d1 allele at both paralogs, and the alternative chromosome harbors the d0 allele at both paralogs. The alternative two-locus haplotypes also exhibit pronounced frequency differences between elevational zones: the d1d1 haplotype is nearly fixed at high altitude, and the alternative d0d0 haplotype predominates at low altitude (Storz et al. 2009, 2010a). In previous surveys, all sampled mice were d0d0/d0d0 double homozygotes, d0d0/d1d1 double heterozygotes, or d1d1/d1d1 double homozygotes. Recombinant d0d1 or d1d0 chromosomes were never observed in highland or lowland population samples, but it is possible that such haplotypes would be recovered by additional sampling at intermediate elevations. A multilocus analysis of nucleotide diversity and LD indicated that the altitudinal differences in HBB haplotype frequencies reflect a history of divergent selection between highland and lowland populations (Storz et al. 2009). Moreover, functional experiments revealed that the two-locus HBB haplotype that predominates at high altitude (d1d1) is associated with increased hemoglobin–oxygen affinity, which helps safeguard arterial oxygen saturation under conditions of severe hypoxia (Storz et al. 2009, 2010a,b). Allelic differences in hemoglobin–oxygen affinity are caused by differences in intrinsic oxygen-binding affinity, as well as differences in the sensitivity to allosteric cofactors that stabilize the low-affinity, deoxygenated conformation of the hemoglobin tetramer. Thus, the altitudinal patterns of allele frequency variation can be related to fitness-related differences in hemoglobin function (reviewed by Storz and Wheat 2010). The objectives of this study were (i) to characterize the altitudinal pattern of allele frequency variation and haplotype structure at the HBB genes; (ii) to determine whether the elevated level of altitudinal differentiation at the HBB genes extends to closely linked loci; and (iii) to gain insight into the nature of selection that may be responsible for maintaining the two-locus haplotype structure at the HBB genes. To accomplish these objectives, we analyzed allele frequency variation at the HBB genes in mice sampled across a steep altitudinal gradient, and we conducted a multilocus analysis of nucleotide polymorphism and LD in mice with different genetic backgrounds at the HBB genes. Using two sets of mice that were alternative double homozygotes at the two HBB paralogs (d1d1/d1d1 and d0d0/d0d0), we compared levels and patterns of nucleotide variation at a total of 16 autosomal loci: the two divergently selected HBB genes, eight additional linked loci that span the b-globin gene cluster on chromosome 1, and six unlinked autosomal loci. Finally, we conducted a forward-time simulation study tailored to the design of the empirical population genetic analysis. We used the simulation results to assess whether the observed LD between the two HBB paralogs could be maintained by spatially varying selection that acts independently on each gene (multiplicative fitness effects). Materials and Methods