1384Mol. Biol. Evol. 17(9):1384–1395. 2000q2000 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038A 39-kb Sequence Around a Blackbird Mhc Class II Gene: Ghost ofSelection Past and Songbird Genome ArchitectureScott V. Edwards,* Joe Gasper,* Daniel Garrigan,*1Duane Martindale,† and Ben F. Koop†*Department of Zoology, University of Washington; and †Center for Environmental Health, Department of Biology,University of Victoria, British Columbia, CanadaTo gain an understanding of the evolution and genomic context of avian major histocompatibility complex (Mhc)genes, we sequenced a 38.8-kb Mhc-bearing cosmid insert from a red-winged blackbird (Agelaius phoeniceus). TheDNA sequence, the longest yet retrieved from a bird other than a chicken, provides a detailed view of the processof gene duplication, divergence, and degeneration (‘‘birth and death’’) in the avian Mhc, as well as a glimpse intomajor noncoding features of a songbird genome. The peptide-binding region (PBR) of the single Mhc class II Bgene in this region, Agph-DAB2, is almost devoid of polymorphism, and a still-segregating single-base-pair deletionand other features suggest that it is nonfunctional. Agph-DAB2 is estimated to have diverged about 40 MYA froma previously characterized and highly polymorphic blackbird Mhc gene, Aph-DAB1, and is therefore younger thanmost mammalian Mhc paralogs and arose relatively late in avian evolution. Despite its nonfunctionality, Agph-DAB2 shows very high levels of nonsynonymous divergence from Agph-DAB1 and from reconstructed ancestralsequences in antigen-binding PBR codons—a strong indication of a period of adaptive divergence preceding lossof function. We also found that the region sequenced contains very few other unambiguous genes, a partial Mhc-class II gene fragment, and a paucity of simple-sequence and other repeats. Thus, this sequence exhibits some ofthe genomic streamlining expected for avian as compared with mammalian genomes, but is not as densely packedwith functional genes as is the chicken Mhc.IntroductionThe genomes of various vertebrate groups are char-acterized by differences in size, gene and repeat density,and isochore composition. We expect these features to bereflected in the genomic structure of multigene familiesof these groups. For example, avian genomes are;50%smaller than those of mammals (Tiersch and Wachtel1991), and chicken genomes are depauperate in simplesequence repeats (Primmer et al. 1997), have higherGC% contents (Bernardi, Hughes, and Mouchiroud1997), higher gene densities (McQueen et al. 1996), andsmaller introns (Hughes and Hughes 1995) than do thoseof mammals. These genomic differences between birdsand mammals have been suggested to have their originin lineage-specific selection for small cell and genomesize imposed by flight and its associated metabolic andbehavioral demands (Tiersch and Wachtel 1991; Hughes1995) or possibly other unknown selective agents.The major histocompatibility complex (Mhc)ofvertebrates, a region containing the most polymorphicgenes in the vertebrate genome (Edwards and Hedrick1998), many of which have functions in defense againstpathogens, appears to reflect some of these trends. Forexample, the chicken Mhc (;100 kb) is orders of mag-nitude smaller than the Mhc of mice and humans (;4Mb) (Trowsdale 1995). It has long been known thatchicken Mhc genes possess much smaller introns thanthose of mammalian Mhc genes (Kaufman, Salamonsen,and Flajnik 1991), and it was recently shown that, atKey words: Mhc, microsatellites, introns, CpG islands, balancingselection.1Present address: Department of Zoology, Arizona StateUniversity.Address for correspondence and reprints: Scott V. Edwards, De-partment of Zoology, University of Washington, Box 351800, Seattle,Washington 98195. E-mail: [email protected] gene per 5 kb, the chicken Mhc (B complex) ismuch more gene dense than the class I or II regions ofmammals (Kaufman et al. 1999b). These differencessuggest that the chicken Mhc may have responded to thesame selective pressures as the rest of the avian genome.The smaller number of Mhc genes in the ‘‘minimalessential’’ chicken Mhc is thought to focus parasite-me-diated selection adaptively on a few target genes, there-by resulting in associations between specific haplotypesand disease resistance that are stronger than those ob-served in mammals (Kaufman 1995). In addition, thespecific organization and tight linkage of genes in thechicken Mhc has been suggested to facilitate coevolu-tion of functionally associated protein products, such asMhc class I and peptide transporters (TAP) (Kaufman etal. 1999a). However, we know little about the genomicorganization of Mhcs in birds other than the chickenwith which to test the generality of these structural fea-tures. Coding sequences of Mhc genes in songbirds andgame birds suggest that the long-term pattern of class IIgene evolution in birds is characterized by higher ratesof concerted evolution, or more recent postspeciationduplications of genes, than are found in mammals (Ed-wards, Wakeland, and Potts 1995; Edwards et al. 1999;Wittzel et al. 1999). However, some songbirds exhibit agreater complexity of class II genes on Southern blotsthan do chickens (Edwards, Nusser, and Gasper 2000),and a recent molecular analysis of class I genes in thegreat reed warbler (Acrocephalus arundinaceus) sug-gested a much greater number of expressed class I genesin this songbird than in chickens (Westerdahl, Wittzell,and von Schantz 1999). Thus, it is not clear the extentto which evolutionary trends and genomic organizationof chicken Mhc genes will represent those of songbirdsand other avian lineages.Understanding in detail the long-term evolution ofthe Mhc in birds requires appropriate phylogenetic sam-Sequence of an Mhc-Bearing Cosmid in Blackbirds 1385pling at both the root and the tips of the avian tree (Ed-wards et al. 1999). It has recently been proposed on thebasis of complete mitochondrial genome sequences thatperching birds (Passeriformes) may represent a basal lin-eages within birds, perhaps the sister group to all otherbirds (Ha¨rlid and Arnason 1999; but see Groth and Bar-rowclough 1999; van Tuinen, Sibley, and Hedges 2000).Thus, pending clarification of the phylogenetic place-ment of perching birds, it would be useful to gain insightinto Mhc structure in this lineage. We recently charac-terized Mhc class II B genes at the genomic level in