Long-term Balancing Selection Research Articles

Long-Lived Dichotomous Lineages of the Proteasome Subunit Beta Type 8 (PSMB8) Gene Surviving More than 500 Million Years as Alleles or Paralogs

On an evolutionary time scale, polymorphic alleles are believed to have a short life, persisting at most tens of millions of years even under long-term balancing selection. Here, we report highly diverged trans-species dimorphism of the proteasome subunit beta type 8 (PSMB8) gene, which encodes a catalytic subunit of the immunoproteasome responsible for the generation of peptides presented by major histocompatibility complex (MHC) class I molecules, in lower teleosts including Cypriniformes (zebrafish and loach) and Salmoniformes (trout and salmon), whose last common ancestor dates to 300 Ma. Moreover, phylogenetic analyses indicated that these dimorphic alleles share lineages with two shark paralogous genes, suggesting that these two lineages have been maintained for more than 500 My either as alleles or as paralogs, and that conversion between alleles and paralogs has occurred at least once during vertebrate evolution. Two lineages termed PSMB8A and PSMB8F show an A(31)F substitution that would probably affect their cleaving specificity, and whereas the PSMB8A lineage has been retained by all analyzed jawed vertebrates, the PSMB8F lineage has been lost by most jawed vertebrates except for cartilaginous fish and basal teleosts. However, a possible functional equivalent of the PSMB8F lineage has been revived as alleles within the PSMB8A lineage at least twice during vertebrate evolution in the amphibian Xenopus and teleostean Oryzias species. Dynamic evolution of the PSMB8 polymorphism through long-term persistence, loss, and regaining of dimorphism and conversion between alleles and paralogs implies the presence of strong selective pressure for functional polymorphism of this gene.

Heterozygote advantage as a natural consequence of adaptation in diploids

Molecular adaptation is typically assumed to proceed by sequential fixation of beneficial mutations. In diploids, this picture presupposes that for most adaptive mutations, the homozygotes have a higher fitness than the heterozygotes. Here, we show that contrary to this expectation, a substantial proportion of adaptive mutations should display heterozygote advantage. This feature of adaptation in diploids emerges naturally from the primary importance of the fitness of heterozygotes for the invasion of new adaptive mutations. We formalize this result in the framework of Fisher's influential geometric model of adaptation. We find that in diploids, adaptation should often proceed through a succession of short-lived balanced states that maintain substantially higher levels of phenotypic and fitness variation in the population compared with classic adaptive walks. In fast-changing environments, this variation produces a diversity advantage that allows diploids to remain better adapted compared with haploids despite the disadvantage associated with the presence of unfit homozygotes. The short-lived balanced states arising during adaptive walks should be mostly invisible to current scans for long-term balancing selection. Instead, they should leave signatures of incomplete selective sweeps, which do appear to be common in many species. Our results also raise the possibility that balancing selection, as a natural consequence of frequent adaptation, might play a more prominent role among the forces maintaining genetic variation than is commonly recognized.

Long-Term Balancing Selection at the Antiviral Gene OAS1 in Central African Chimpanzees

Oligoadenylate synthetases (OAS) are interferon-induced enzymes that participate in the first line of defense against a wide range of viral infection in animals. Upon activation by viral double-stranded RNA, OAS synthesizes (2-5) oligoadenylates, which activate RNase L, leading to the nonspecific degradation of cellular and viral RNA. Some association studies in humans suggest that variation at one of the OAS genes, OAS1, could be influencing host susceptibility to viral infection. We assessed the diversity of OAS1 in hominoid primates with a focus on chimpanzees. We found that the OAS1 gene is extremely polymorphic in Central African chimpanzee and exhibits levels of silent and replacement diversity much higher than neutral regions of the chimpanzee genome. This level of variation strongly suggests that balancing selection is acting on OAS1, and indeed, this conclusion was validated by several tests of neutrality. We further demonstrated that balancing selection has been acting at this locus since the split between chimpanzees, humans, and gorillas (~8.6 Ma) and caused the persistence of two deeply divergent allelic lineages in Central African chimpanzees. These two groups of OAS1 alleles differ by a large number of amino acids (a.a.), including several a.a. putatively involved in RNA binding. It is therefore very likely that variation at the OAS1 locus affects the innate immune response of individuals to specific viral infection. Our data strongly suggest that interactions between viral RNA and OAS1 are responsible for the maintenance of ancestral polymorphisms at this locus for at least 13.2 My.

Long-Term Balancing Selection at the Blood Group-Related Gene B4galnt2 in the Genus Mus (Rodentia; Muridae)

Recent surveys of the human genome have highlighted the significance of balancing selection in relation to understanding the evolutionary origins of disease-associated variation. Cis-regulatory variation at the blood group-related glycosyltransferase B4galnt2 is associated with a phenotype in mice that closely resembles a common human bleeding disorder, von Willebrand disease. In this study, we have performed a survey of the 5' flanking region of the B4galnt2 gene in several Mus musculus subspecies and Mus spretus. Our results reveal a clear pattern of trans-species polymorphism and indicate that allele classes conferring alternative tissue-specific expression patterns have been maintained for >2.8 My in the genus Mus. Furthermore, analysis of B4galnt2 expression patterns revealed the presence of an additional functional class of alleles, supporting a role for gastrointestinal phenotypes in the long-term maintenance of expression variation at this gene.

A multifaceted genomics approach allows the isolation of the rice Pia‐blast resistance gene consisting of two adjacent NBS‐LRR protein genes

The Oryza sativa (rice) resistance gene Pia confers resistance to the blast fungus Magnaporthe oryzae carrying the AVR-Pia avirulence gene. To clone Pia, we employed a multifaceted genomics approach. First, we selected 12 R-gene analog (RGA) genes encoding nucleotide binding site-leucine rich repeats (NBS-LRRs) proteins from a region on chromosome 11 that shows linkage to Pia. By using seven rice accessions, we examined the association between Pia phenotypes and DNA polymorphisms in the 10 genes, which revealed three genes (Os11gRGA3-Os11gRGA5) exhibiting a perfect association with the Pia phenotypes. We also screened ethyl methane sulfonate (EMS)-treated mutant lines of the rice cultivar 'Sasanishiki' harboring Pia, and isolated two mutants that lost the Pia phenotype. DNA sequencing of Os11gRGA3-Os11gRGA5 from the two mutant lines identified independent mutations of major effects in Os11gRGA4. The wild-type 'Sasanishiki' allele of Os11gRGA4 (SasRGA4) complemented Pia function in both mutants, suggesting that SasRGA4 is necessary for Pia function. However, when the rice cultivar 'Himenomochi' lacking Pia was transfected with SasRGA4, the Pia phenotype was not recovered. An additional complementation study revealed that the two NBS-LRR-type R genes, SasRGA4 and SasRGA5, that are located next to each other and oriented in the opposite direction are necessary for Pia function. A population genetics analysis of SasRGA4 and SasRGA5 suggests that the two genes are under long-term balancing selection.

Using DNA Sequence Diversity to Test for Selection inSilene

It is beginning to be possible to test for the action of natural selection in nonmodel species, including in natural populations of plants, using DNA sequence diversity within species and divergence between related species. We suggest some questions of interest in plants in the genus Silene, where the evolution of unisexuals has occurred multiple times and related species suitable for use as outgroups in such comparisons are available. In the dioecious species Silene latifolia, the evolution of separate sexes raises many questions about selection, including the possibility of recent selective sweeps on the Y or other chromosomes. If recent selective sweeps have not occurred on the Y chromosome, one might be able to conclude that the low sequence diversity that is observed for Y-linked genes may be due to processes involved in genetic degeneration of Y chromosomes. In a dioecious species, it is also interesting to test whether sexual selection occurs (a question that has been difficult to study in plants and which could affect the effective population sizes of genes on the X and Y chromosomes and on the autosomes, which must be taken into account when testing for selection), and also whether selection occurs during pollination. Silene latifolia is also useful for testing selection in other Silene species, and we outline tests for long-term balancing selection in the mitochondrial genome associated with the presence of females in the gynodioecious species Silene vulgaris. The results of our tests for selection are preliminary, and there are important caveats associated with the conclusions. We therefore include discussion of some of the main difficulties and the kinds of data that should be helpful in refining the results in the future.

Long-term balancing selection maintains trans-specific polymorphisms in the human TRIM5 gene

The human TRIM5 genes encodes a retroviral restriction factor (TRIM5α). Evolutionary analyses of this gene in mammals have revealed a complex and multifaceted scenario, suggesting that TRIM5 has been the target of exceptionally strong selective pressures, possibly exerted by recurrent waves of retroviral infections. TRIM5 displays inter-individual expression variability in humans and high levels of TRIM5 mRNA have been associated with a reduced risk of HIV-1 infection. We resequenced TRIM5 in chimpanzees and identified two polymorphisms in intron 1 that are shared with humans. Analysis of the gene region encompassing the two trans-specific variants in human populations identified exceptional nucleotide diversity levels and an excess of polymorphism compared to fixed divergence. Most tests rejected the null hypothesis of neutral evolution for this region and haplotype analysis revealed the presence of two deeply separated clades. Calculation of the time to the most recent common ancestor (TMRCA) for TRIM5 haplotypes yielded estimates ranging between 4 and 7 million years. Overall, these data indicate that long-term balancing selection, an extremely rare process outside MHC genes, has maintained trans-specific polymorphisms in the first intron of TRIM5. Bioinformatic analyses indicated that variants in intron 1 may affect transcription factor-binding sites and, therefore, TRIM5 transcriptional activity. Data herein confirm an extremely complex evolutionary history of TRIM5 genes in primates and open the possibility that regulatory variants in the gene modulate the susceptibility to HIV-1.

Nucleotide Polymorphism at a Gene (Pgi) under Balancing Selection in a Butterfly Metapopulation

The Glanville fritillary butterfly (Melitaea cinxia, Nymphalidae) has a large, well-studied metapopulation in the Aland Islands in Finland. Previous studies have found that the common allozyme genotypes at the phosphoglucose isomerase (PGI) locus are associated with individual variation in performance and fitness, with phenotypic data suggesting ongoing balancing selection via heterozygote advantage. Here, we analyze nucleotide polymorphism in the coding region of the Pgi gene. Pgi is exceptionally polymorphic, in contrast to three other metabolic genes (Mdh, Idh, and Gapdh) with low levels of polymorphism. Most of the variation is due to two common haplotype clades, which are highly divergent and exhibit extensive linkage disequilibrium. These two clades correspond to the two most common allozyme alleles previously studied. Molecular tests of selection and coalescence simulations indicate that patterns of nucleotide polymorphism depart from neutrality and are consistent with long-term balancing selection. The split between the two main haplotype clades is estimated to predate the last common ancestor of a clade of five extant Melitaea species. Comparative structural analysis of Pgi polymorphism in M. cinxia and the unrelated Colias eurytheme butterfly suggests a similar but not identical target of balancing selection. Our results indicate convergent evolution between these two species at both the phenotypic and molecular levels.

Targets of balancing selection in the human genome.

Balancing selection is potentially an important biological force for maintaining advantageous genetic diversity in populations, including variation that is responsible for long-term adaptation to the environment. By serving as a means to maintain genetic variation, it may be particularly relevant to maintaining phenotypic variation in natural populations. Nevertheless, its prevalence and specific targets in the human genome remain largely unknown. We have analyzed the patterns of diversity and divergence of 13,400 genes in two human populations using an unbiased single-nucleotide polymorphism data set, a genome-wide approach, and a method that incorporates demography in neutrality tests. We identified an unbiased catalog of genes with signatures of long-term balancing selection, which includes immunity genes as well as genes encoding keratins and membrane channels; the catalog also shows enrichment in functional categories involved in cellular structure. Patterns are mostly concordant in the two populations, with a small fraction of genes showing population-specific signatures of selection. Power considerations indicate that our findings represent a subset of all targets in the genome, suggesting that although balancing selection may not have an obvious impact on a large proportion of human genes, it is a key force affecting the evolution of a number of genes in humans.

The action of falciparum malaria on the human and chimpanzee genomes compared: Absence of evidence for a genomic signature of malaria at HBB and G6PD in three subspecies of chimpanzee

The historical association between Plasmodium and primates has meant that many Plasmodium species have coevolved with specific primate hosts. However, unlike humans that are infected by species such as P. falciparum that cause severe malaria, many non-human primates are infected by Plasmodium species that only cause mild disease. Here we investigate whether the genomic signatures of plasmodial infection found in humans are also present in chimpanzees. We find no evidence of the major deleterious mutations at HBB (β-globin) and G6PD in chimpanzees that confer resistance to malaria caused by P. falciparum nor evidence of long-term balancing selection at these loci . Our knowledge of malaria prevalence and pathogenesis in wild chimpanzees is severely limited, but it may be the case that β-globin and G6PD variation are not adaptive in chimpanzees because malaria is rare and/or less detrimental in this species. Alternatively, chimpanzees may utilise mechanisms that are different from those of humans to protect against malaria.

Conditional neutrality at two adjacent NBS‐LRR disease resistance loci in natural populations of Arabidopsis lyrata

We examined patterns of nucleotide diversity at a genomic region containing two linked candidate disease resistance (NBS-LRR) genes in seven populations of the outcrossing plant Arabidopsis lyrata. In comparison with two adjacent control genes and neutral reference genes across the genome, the NBS-LRR genes exhibited elevated nonsynonymous variation and a large number of major-effect polymorphisms causing early stop codons and/or frameshift mutations. In contrast, analysis of synonymous diversity provided no evidence that the region was subject to long-term balancing selection or recent selective sweeps in any of the seven populations surveyed. Also in contrast with earlier surveys of one of these R genes, there was no evidence that the resistance genes or the major-effect mutations were subject to elevated differentiation between populations. We suggest that conditional neutrality in the absence of the corresponding pathogen, rather than long-term balancing selection or local adaptation, may in some circumstances be a significant cause of elevated functional polymorphism at R genes. In contrast with the R genes, analysis of diversity and differentiation at the flanking FERONIA locus showed high population divergence, suggesting local adaptation on this locus controlling male-female signalling during fertilization.

The evolution of TEP1, an exceptionally polymorphic immunity gene in Anopheles gambiae

BackgroundHost-parasite coevolution can result in balancing selection, which maintains genetic variation in the susceptibility of hosts to parasites. It has been suggested that variation in a thioester-containing protein called TEP1 (AGAP010815) may alter the ability of Anopheles mosquitoes to transmit Plasmodium parasites, and high divergence between alleles of this gene suggests the possible action of long-term balancing selection. We studied whether TEP1 is a case of an ancient balanced polymorphism in an animal immune system.ResultsWe found evidence that the high divergence between TEP1 alleles is the product of genetic exchange between TEP1 and other TEP loci, i.e. gene conversion. Additionally, some TEP1 alleles showed unexpectedly low variability.ConclusionThe TEP1 gene appears to be a chimera produced from at least two other TEP loci, and the divergence between TEP1 alleles is probably not caused by long-term balancing selection, but is instead due to two independent gene conversion events from one of these other genes. Nevertheless, TEP1 still shows evidence of natural selection, in particular there appears to have been recent changes in the frequency of alleles that has diminished polymorphism within each allelic class. Although the selective force driving this dynamic was not identified, given that susceptibility to Plasmodium parasites is known to be associated with allelic variation in TEP1, these changes in allele frequencies could alter the vectoring capacity of populations.

Disentangling the effects of recombination, selection, and demography on the genetic variation at a major histocompatibility complex class II gene in the alpine chamois

The major histocompatibility complex (MHC) harbours some of the most polymorphic loci in vertebrate genomes. MHC genes are thought to be subject to some form of balancing selection, most likely pathogen-mediated selection. Hence, MHC genes are excellent candidates for exploring adaptive processes. In this study, we investigated the genetic variation at exon 2 of the DRB class II MHC locus in 191 alpine chamois (Rupicapra rupicapra) from 10 populations in the eastern Alps of Italy. In particular, we were interested in distinguishing and estimating the relative impact of selective and demographic factors, while taking into account the confounding effect of recombination. The extremely high d(n)/d(s) ratio and the presence of trans-species polymorphisms suggest that a strong long-term balancing selection effect has been operating at this locus throughout the evolutionary history of this species. We analysed patterns of genetic variation within and between populations, and the mitochondrial D-loop polymorphism patterns were analysed to provide a baseline indicator of the effects of demographic processes. These analyses showed that (i) the chamois experienced a demographic decline in the last 5000-30 000 years, most likely related to the postglacial elevation in temperature; (ii) this demographic process can explain the results of neutrality tests applied to MHC variation within populations, but cannot justify the much weaker divergence between populations implied by MHC as opposed to mitochondrial DNA; (iii) similar sets of divergent alleles are probably maintained with similar frequencies by balancing selection in different populations, and this mechanism is also operating in small isolated populations, which are strongly affected by drift.

Long-Term Balancing Selection at the West Nile Virus Resistance Gene, Oas1b, Maintains Transspecific Polymorphisms in the House Mouse

Oligoadenylate synthetases (OASs) are interferon-inducible enzymes that participate in the first line of defense against a wide range of viral infection. Recent studies have determined that Oas1b, a member of the OAS gene family in the house mouse (Mus musculus), provides specific protection against flavivirus infection (e.g., West Nile virus, dengue fever virus, and yellow fever virus). We characterized the nucleotide sequence variation in coding and noncoding regions of the Oas1b gene for a large number of wild-derived strains of M. musculus and related species. Our sequence analyses determined that this gene is one of the most polymorphic genes ever described in any mammal. The level of variation in noncoding regions of Oas1b is an order of magnitude higher than the level reported for other regions of the mouse genome and is significantly different from the level of intraspecific variation expected under neutrality. Furthermore, a phylogenetic analysis of intronic sequences demonstrated that Oas1b alleles are ancient and that their divergence predates several speciation events, resulting in transspecific polymorphisms. The amino acid sequence of Oas1b is also extremely variable, with 1 out of 7 amino acid positions being polymorphic within M. musculus. Oas1b alleles are comparatively more divergent at synonymous positions than most autosomal genes and the ratio of nonsynonymous to synonymous substitution is remarkably high, suggesting that positive selection has been acting on Oas1b. The ancestry of Oas1b polymorphisms and the high level of amino acid polymorphisms strongly suggest that the allelic variation at Oas1b has been maintained in mouse populations by long-term balancing selection.

Directional and balancing selection in human beta-defensins

BackgroundIn primates, infection is an important force driving gene evolution, and this is reflected in the importance of infectious disease in human morbidity today. The beta-defensins are key components of the innate immune system, with antimicrobial and cell signalling roles, but also reproductive functions. Here we examine evolution of beta-defensins in catarrhine primates and variation within different human populations.ResultsWe show that five beta-defensin genes that do not show copy number variation in humans show evidence of positive selection in catarrhine primates, and identify specific codons that have been under selective pressure. Direct haplotyping of DEFB127 in humans suggests long-term balancing selection: there are two highly diverged haplotype clades carrying different variants of a codon that, in primates, is positively selected. For DEFB132, we show that extensive diversity, including a four-state amino acid polymorphism (valine, isoleucine, alanine and threonine at position 93), is present in hunter-gatherer populations, both African and non-African, but not found in samples from agricultural populations.ConclusionSome, but not all, beta-defensin genes show positive selection in catarrhine primates. There is suggestive evidence of different selective pressures on these genes in humans, but the nature of the selective pressure remains unclear and is likely to differ between populations.

Complex Signatures of Selection and Gene Conversion in the Duplicated Globin Genes of House Mice

Results of electrophoretic surveys have suggested that hemoglobin polymorphism may be maintained by balancing selection in natural populations of house mice, Mus musculus. Here we report a survey of nucleotide variation in the adult globin genes of house mice from South America. We surveyed nucleotide polymorphism in two closely linked alpha-globin paralogs and two closely linked beta-globin paralogs to test whether patterns of variation are consistent with a model of long-term balancing selection. Surprisingly high levels of nucleotide polymorphism at the two beta-globin paralogs were attributable to the segregation of two highly divergent haplotypes, Hbbs (which carries two identical beta-globin paralogs) and Hbbd (which carries two functionally divergent beta-globin paralogs). Interparalog gene conversion on the Hbbs haplotype has produced a highly unusual situation in which the two paralogs are more similar to one another than either one is to its allelic counterpart on the Hbbd haplotype. Levels of nucleotide polymorphism and linkage disequilibrium at the two beta-globin paralogs suggest a complex history of diversity-enhancing selection that may be responsible for long-term maintenance of alternative protein alleles. The alternative two-locus beta-globin haplotypes are associated with pronounced differences in intraerythrocyte glutathione and nitric oxide metabolism, suggesting a possible mechanism for selection on hemoglobin function.

Non-neutral evolution of the major histocompatibility complex class II gene DRB1 in the sac-winged bat Saccopteryx bilineata

The immune genes of the major histocompatibility complex (MHC) are classical examples for high levels of genetic diversity and non-neutral evolution. This is particularly true for the regions containing the antigen-binding sites as, for instance, in the exon 2 of the MHC class II gene DRB. We surveyed, for the first time in the order Chiroptera, the genetic diversity within this exon in the sac-winged bat Saccopteryx bilineata. We detected 11 alleles among 85 bats, of which 79 were sampled in one population. Pairwise comparisons revealed that interallelic sequence differences ranged between 3 and 22%, although nucleotide substitutions were not evenly distributed along the exon sequence. This was most probably the result of intragenic recombination. High levels of sequence divergence and significantly more nonsynonymous than synonymous substitutions (dN/dS>1) suggest long-term balancing selection. Thus, the data are consistent with the hypothesis that recombination gives rise to new alleles at the DRB locus of the sac-winged bat, and these are maintained in the population through balancing selection. In this respect, the sac-winged bat closely resembles other mammalian species.

Sequence variation in human succinate dehydrogenase genes: evidence for long-term balancing selection on SDHA.

BackgroundBalancing selection operating for long evolutionary periods at a locus is characterized by the maintenance of distinct alleles because of a heterozygote or rare-allele advantage. The loci under balancing selection are distinguished by their unusually high polymorphism levels. In this report, we provide statistical and comparative genetic evidence suggesting that the SDHA gene is under long-term balancing selection. SDHA encodes the major catalytical subunit (flavoprotein, Fp) of the succinate dehydrogenase enzyme complex (SDH; mitochondrial complex II). The inhibition of Fp by homozygous SDHA mutations or by 3-nitropropionic acid poisoning causes central nervous system pathologies. In contrast, heterozygous mutations in SDHB, SDHC, and SDHD, the other SDH subunit genes, cause hereditary paraganglioma (PGL) tumors, which show constitutive activation of pathways induced by oxygen deprivation (hypoxia).ResultsWe sequenced the four SDH subunit genes (10.8 kb) in 24 African American and 24 European American samples. We also sequenced the SDHA gene (2.8 kb) in 18 chimpanzees. Increased nucleotide diversity distinguished the human SDHA gene from its chimpanzee ortholog and from the PGL genes. Sequence analysis uncovered two common SDHA missense variants and refuted the previous suggestions that these variants originate from different genetic loci. Two highly dissimilar SDHA haplotype clusters were present in intermediate frequencies in both racial groups. The SDHA variation pattern showed statistically significant deviations from neutrality by the Tajima, Fu and Li, Hudson-Kreitman-Aguadé, and Depaulis haplotype number tests. Empirically, the elevated values of the nucleotide diversity (% π = 0.231) and the Tajima statistics (D = 1.954) in the SDHA gene were comparable with the most outstanding cases for balancing selection in the African American population.ConclusionThe SDHA gene has a strong signature of balancing selection. The SDHA variants that have increased in frequency during human evolution might, by influencing the regulation of cellular oxygen homeostasis, confer protection against certain environmental toxins or pathogens that are prevalent in Africa.

Balancing selection and the evolution of functional polymorphism in Old World monkey TRIM5α

Retroviral restriction factor TRIM5alpha exhibits a high degree of sequence variation among primate species. It has been proposed that this diversity is the cumulative result of ancient, lineage-specific episodes of positive selection. Here, we describe the contribution of within-species variation to the evolution of TRIM5alpha. Sampling within two geographically distinct Old World monkey species revealed extensive polymorphism, including individual polymorphisms that predate speciation (shared polymorphism). In some instances, alleles were more closely related to orthologues of other species than to one another. Both silent and nonsynonymous changes clustered in two domains. Functional assays revealed consequences of polymorphism, including differential restriction of a small panel of retroviruses by very similar alleles. Together, these features indicate that the primate TRIM5alpha locus has evolved under balancing selection. Except for the MHC there are few, if any, examples of long-term balancing selection in primates. Our results suggest a complex evolutionary scenario, in which fixation of lineage-specific adaptations is superimposed on a subset of critical polymorphisms that predate speciation events and have been maintained by balancing selection for millions of years.

Scan of Human Genome Reveals No New Loci Under Ancient Balancing Selection

There has been much speculation as to what role balancing selection has played in evolution. In an attempt to identify regions, such as HLA, at which polymorphism has been maintained in the human population for millions of years, we scanned the human genome for regions of high SNP density. We found 16 regions that, outside of HLA and ABO, are the most highly polymorphic regions yet described; however, evidence for balancing selection at these sites is notably lacking--indeed, whole-genome simulations indicate that our findings are expected under neutrality. We propose that (i) because it is rarely stable, long-term balancing selection is an evolutionary oddity, and (ii) when a balanced polymorphism is ancient in origin, the requirements for detection by means of SNP data alone will rarely be met.