Molecular Ecology (2006) 15, 623–637 doi: 10.1111/j.1365-294X.2006.02829.x © 2006 Blackwell Publishing Ltd Blackwell Publishing Ltd Detecting the signature of selection on immune genes in highly structured populations of wild sheep (Ovis dalli) K. WORLEY,* J. CAREY,† A. VEITCH ‡ and D. W. COLTMAN § *Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK, †Department of Environment, Government of Yukon, Whitehorse, Yukon, Canada Y1A 2C6, ‡Department of Resources, Wildlife & Economic Development, Sahtu Region, Government of the Northwest Territories, PO Box 130, Norman Wells, Northwest Territories, Canada X0E 0V0, §Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 Abstract 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 indi- cated 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 sub- stitution and trans-species polymorphism. The failure to detect balancing selection at micro- satellite loci linked to the MHC is likely the result of recombination between the markers and expressed gene regions. Keywords: major histocompatibility complex, microsatellite, neutrality tests, Ovis, selection Received 20 July 2005; revision received 13 October 2005; accepted 4 November 2005 Introduction Understanding processes responsible for the production and maintenance of genetic variation is a central aim in evolutionary biology. Levels of variation exhibiting deviation from neutral expectations of the mutation–drift equilibrium can result from both neutral demographic and selective processes. The task of separating these processes and identifying genes under the influence of selection can be challenging (Beaumont & Balding 2004). As a re- sult, efforts to identify genes affected by selection have concentrated on species with well-characterized genomes, such as Drosophila and humans (Wall et al . 2002; Glinka et al . 2003; Stajich & Hahn 2005), where many coding regions have been characterized and are available for study. It remains important to examine patterns of genetic variation in nonhuman natural populations to gain a more general understanding of how selection shapes genetic variation. For this to be possible we must advance current techniques available for identifying selection. Evidence for selection at the molecular level is most directly obtained by applying statistical tests of neutrality to patterns of sequence variation in coding regions (Tajima 1989; Kreitman & Akashi 1995; Fu 1997; Kreitman 2000; Nielsen 2001). However, it requires a great deal of gene isolation and de novo sequencing to apply this approach to multiple genes in nonmodel taxa. A solution lies in the use of neutral linked markers such as microsatellites which are also affected by selection through genetic hitch-hiking (Maynard Smith & Haigh 1974; Slatkin 1995). Recently, this process has been implemented in large-scale genome scans of many loci to identify regions of high linkage disequilib- rium reflecting the presence of selection (Payseur et al . 2002; Vigouroux et al . 2002; Kauer et al . 2003). As selection is locus specific, only those markers linked to genes under Correspondence: K. Worley, Fax: +44 (0)114 2220002; E-mail: k.worley@sheffield.ac.uk