Dichotomy of single-nucleotide polymorphism haplotypes in olfactory receptor genes and pseudogenes.

Substantial efforts are focused on identifying single-nucleotide polymorphisms (SNPs) throughout the human genome, particularly in coding regions (cSNPs), for both linkage disequilibrium and association studies. Less attention, however, has been directed to the clarification of evolutionary processes that are responsible for the variability in nucleotide diversity among different regions of the genome. We report here the population sequence diversity of genomic segments within a 450-kb cluster of olfactory receptor (OR) genes on human chromosome 17. We found a dichotomy in the pattern of nucleotide diversity between OR pseudogenes and introns on the one hand and the closely interspersed intact genes on the other. We suggest that weak positive selection is responsible for the observed patterns of genetic variation. This is inferred from a lower ratio of polymorphism to divergence in genes compared with pseudogenes or introns, high non-synonymous substitution rates in OR genes, and a small but significant overall reduction in variability in the entire OR gene cluster compared with other genomic regions. The dichotomy among functionally different segments within a short genomic distance requires high recombination rates within this OR cluster. Our work demonstrates the impact of weak positive selection on human nucleotide diversity, and has implications for the evolution of the olfactory repertoire.

Higher differentiation among subspecies of the house mouse (Mus musculus) in genomic regions with low recombination.

In the early stages of reproductive isolation, genomic regions of reduced recombination are expected to show greater levels of differentiation, either because gene flow between species is reduced in these regions or because the effects of selection at linked sites within species are enhanced in these regions. Here, we study the patterns of DNA sequence variation at 27 autosomal loci among populations of Mus musculus musculus, M. m. domesticus, and M. m. castaneus, three subspecies of house mice with collinear genomes. We found that some loci exhibit considerable shared variation among subspecies, while others exhibit fixed differences. We used an isolation-with-gene-flow model to estimate divergence times and effective population sizes (N(e) ) and to disentangle ancestral variation from gene flow. Estimates of divergence time indicate that all three subspecies diverged from one another within a very short period of time approximately 350,000 years ago. Overall, N(e) for each subspecies was associated with the degree of genetic differentiation: M. m. musculus had the smallest N(e) and the greatest proportion of monophyletic gene genealogies, while M. m. castaneus had the largest N(e) and the smallest proportion of monophyletic gene genealogies. M. m. domesticus and M. m. musculus were more differentiated from each other than either were from M. m. castaneus, consistent with greater reproductive isolation between M. m. domesticus and M. m. musculus. F(ST) was significantly greater at loci experiencing low recombination rates compared to loci experiencing high recombination rates in comparisons between M. m. castaneus and M. m. musculus or M. m. domesticus. These results provide evidence that genomic regions with less recombination show greater differentiation, even in the absence of chromosomal rearrangements.

Reduced introgression of the Y chromosome between subspecies of the European rabbit (Oryctolagus cuniculus) in the Iberian Peninsula.

The role of the Y chromosome in speciation is unclear. Hybrid zones provide natural arenas for studying speciation, as differential introgression of markers may reveal selection acting against incompatibilities. Two subspecies of the European rabbit (Oryctolagus cuniculus) form a hybrid zone in the Iberian Peninsula. Previous work on mitochondrial DNA (mtDNA), Y- and X-linked loci revealed the existence of two divergent lineages in the rabbit genome and that these lineages are largely subspecies-specific for mtDNA and two X-linked loci. Here we investigated the geographic distribution of the two Y chromosome lineages by genotyping two diagnostic single nucleotide polymorphisms in a sample of 353 male rabbits representing both subspecies, and found that Y chromosome lineages are also largely subspecies-specific. We then sequenced three autosomal loci and discovered considerable variation in levels of differentiation at these loci. Finally, we compared estimates of population differentiation between rabbit subspecies at 26 markers and found a surprising bimodal distribution of F(ST)values. The vast majority of loci showed little or no differentiation between rabbit subspecies while a few loci, including the SRY gene, showed little or no introgression across the hybrid zone. Estimates of population differentiation for the Y chromosome were surprisingly high given that there is male-biased dispersal in rabbits. Taken together, these data indicate that there is a clear dichotomy in the rabbit genome and that some loci remain highly differentiated despite extensive gene flow following secondary contact.

Single nucleotide polymorphisms and recombination rate in humans.

Levels of heterozygosity for single nucleotide polymorphisms vary by more than one order of magnitude in different regions of the human genome. Regional differences in the rate of recombination explain a substantial fraction of the variation in levels of nucleotide polymorphism, consistent with the widespread action of natural selection at the molecular level.

Patterns of DNA variability at X-linked loci in Mus domesticus.

Introns of four X-linked genes (Hprt, Plp, Glra2, and Amg) were sequenced to provide an estimate of nucleotide diversity at nuclear genes within the house mouse and to test the neutral prediction that the ratio of intraspecific polymorphism to interspecific divergence is the same for different loci. Hprt and Plp lie in a region of the X chromosome that experiences relatively low recombination rates, while Glra2 and Amg lie near the telomere of the X chromosome, a region that experiences higher recombination rates. A total of 6022 bases were sequenced in each of 10 Mus domesticus and one M. caroli. Average nucleotide diversity (pi) for introns within M. domesticus was quite low (pi = 0.078%). However, there was substantial variation in the level of heterozygosity among loci. The two telomeric loci, Glra2 and Amg, had higher ratios of polymorphism to divergence than the two loci experiencing lower recombination rates. These results are consistent with the hypothesis that heterozygosity is reduced in regions with lower rates of recombination, although sampling of additional genes is needed to establish whether there is a general correlation between heterozygosity and recombination rate as in Drosophila melanogaster.

Microsatellite variation and recombination rate in the human genome.

Background (purifying) selection on deleterious mutations is expected to remove linked neutral mutations from a population, resulting in a positive correlation between recombination rate and levels of neutral genetic variation, even for markers with high mutation rates. We tested this prediction of the background selection model by comparing recombination rate and levels of microsatellite polymorphism in humans. Published data for 28 unrelated Europeans were used to estimate microsatellite polymorphism (number of alleles, heterozygosity, and variance in allele size) for loci throughout the genome. Recombination rates were estimated from comparisons of genetic and physical maps. First, we analyzed 61 loci from chromosome 22, using the complete sequence of this chromosome to provide exact physical locations. These 61 microsatellites showed no correlation between levels of variation and recombination rate. We then used radiation-hybrid and cytogenetic maps to calculate recombination rates throughout the genome. Recombination rates varied by more than one order of magnitude, and most chromosomes showed significant suppression of recombination near the centromere. Genome-wide analyses provided no evidence for a strong positive correlation between recombination rate and polymorphism, although analyses of loci with at least 20 repeats suggested a weak positive correlation. Comparisons of microsatellites in lowest-recombination and highest-recombination regions also revealed no difference in levels of polymorphism. Together, these results indicate that background selection is not a major determinant of microsatellite variation in humans.

Estimate of the mutation rate per nucleotide in humans.

Many previous estimates of the mutation rate in humans have relied on screens of visible mutants. We investigated the rate and pattern of mutations at the nucleotide level by comparing pseudogenes in humans and chimpanzees to (i) provide an estimate of the average mutation rate per nucleotide, (ii) assess heterogeneity of mutation rate at different sites and for different types of mutations, (iii) test the hypothesis that the X chromosome has a lower mutation rate than autosomes, and (iv) estimate the deleterious mutation rate. Eighteen processed pseudogenes were sequenced, including 12 on autosomes and 6 on the X chromosome. The average mutation rate was estimated to be approximately 2.5 x 10(-8) mutations per nucleotide site or 175 mutations per diploid genome per generation. Rates of mutation for both transitions and transversions at CpG dinucleotides are one order of magnitude higher than mutation rates at other sites. Single nucleotide substitutions are 10 times more frequent than length mutations. Comparison of rates of evolution for X-linked and autosomal pseudogenes suggests that the male mutation rate is 4 times the female mutation rate, but provides no evidence for a reduction in mutation rate that is specific to the X chromosome. Using conservative calculations of the proportion of the genome subject to purifying selection, we estimate that the genomic deleterious mutation rate (U) is at least 3. This high rate is difficult to reconcile with multiplicative fitness effects of individual mutations and suggests that synergistic epistasis among harmful mutations may be common.

Deleterious mutations at the mitochondrial ND3 gene in South American marsh rats (Holochilus).

Statistical analyses of DNA sequences have revealed patterns of nonneutral evolution in mitochondrial DNA of mice, humans, and Drosophila. Here we report patterns of mitochondrial sequence evolution in South American marsh rats (genus Holochilus). We sequenced the complete mitochondrial ND3 gene in 82 Holochilus brasiliensis and 21 H. vulpinus to test the neutral prediction that the ratio of nonsynonymous to synonymous nucleotide changes is the same within and between species. Within H. brasiliensis we observed a greater number of amino acid polymorphisms than expected based on interspecific comparisons. This contingency table analysis suggests that many amino acid polymorphisms are mildly deleterious. Several tests of the frequency distribution also revealed departures from a neutral, equilibrium model, and these departures were observed for both nonsynonymous and synonymous sites. In general, an excess of rare sites was observed, consistent with either a recent selective sweep or with populations not at mutation-drift equilibrium.

Heterogeneity in rates of recombination across the mouse genome.

If loci are randomly distributed on a physical map, the density of markers on a genetic map will be inversely proportional to recombination rate. First, proposed by Mary Lyon, we have used this idea to estimate recombination rates from the Drosophila melanogaster linkage map. These results were compared with results of two other studies that estimated regional recombination rates in D. melanogaster using both physical and genetic maps. The three methods were largely concordant in identifying large-scale genomic patterns of recombination. The marker density method was then applied to the Mus musculus microsatellite linkage map. The distribution of microsatellites provided evidence for heterogeneity in recombination rates. Centromeric regions for several mouse chromosomes had significantly greater numbers of markers than expected, suggesting that recombination rates were lower in these regions. In contrast, most telomeric regions contained significantly fewer markers than expected. This indicates that recombination rates are elevated at the telomeres of many mouse chromosomes and is consistent with a comparison of the genetic and cytogenetic maps in these regions. The density of markers on a genetic map may provide a generally useful way to estimate regional recombination rates in species for which genetic, but not physical, maps are available.

Contrasting evolutionary histories of two introns of the duchenne muscular dystrophy gene, Dmd, in humans.

The Duchenne muscular dystrophy (Dmd) locus lies in a region of the X chromosome that experiences a high rate of recombination and is thus expected to be relatively unaffected by the effects of selection on nearby genes. To provide a picture of nucleotide variability at a high-recombination locus in humans, we sequenced 5. 4 kb from two introns of Dmd in a worldwide sample of 41 alleles from Africa, Asia, Europe, and the Americas. These same regions were also sequenced in one common chimpanzee and one orangutan. Dramatically different patterns of genetic variation were observed at these two introns, which are separated by >500 kb of DNA. Nucleotide diversity at intron 44 pi = 0.141% was more than four times higher than nucleotide diversity at intron 7 pi = 0.034% despite similar levels of divergence for these two regions. Intron 7 exhibited significant linkage disequilibrium extending over 10 kb and also showed a significant excess of rare polymorphisms. In contrast, intron 44 exhibited little linkage disequilibrium and no skew in the frequency distribution of segregating sites. Intron 7 was much more variable in Africa than in other continents, while intron 44 displayed similar levels of variability in different geographic regions. Comparison of intraspecific polymorphism to interspecific divergence using the HKA test revealed a significant reduction in variability at intron 7 relative to intron 44, and this effect was most pronounced in the non-African samples. These results are best explained by positive directional selection acting at or near intron 7 and demonstrate that even genes in regions of high recombination may be influenced by selection at linked sites.

Nonneutral mitochondrial DNA variation in humans and chimpanzees.

We sequenced the NADH dehydrogenase subunit 3 (ND3) gene from a sample of 61 humans, five common chimpanzees, and one gorilla to test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution. Within humans and within chimpanzees, the ratio of replacement to silent nucleotide substitutions was higher than observed in comparisons between species, contrary to neutral expectations. To test the generality of this result, we reanalyzed published human RFLP data from the entire mitochondrial genome. Gains of restriction sites relative to a known human mtDNA sequence were used to infer unambiguous nucleotide substitutions. We also compared the complete mtDNA sequences of three humans. Both the RFLP data and the sequence data reveal a higher ratio of replacement to silent nucleotide substitutions within humans than is seen between species. This pattern is observed at most or all human mitochondrial genes and is inconsistent with a strictly neutral model. These data suggest that many mitochondrial protein polymorphisms are slightly deleterious, consistent with studies of human mitochondrial diseases.

DNA variability and recombination rates at X-linked loci in humans.

We sequenced 11,365 bp from introns of seven X-linked genes in 10 humans, one chimpanzee, and one orangutan to (i) provide an average estimate of nucleotide diversity (pi) in humans, (ii) investigate whether there is variation in pi among loci, (iii) compare ratios of polymorphism to divergence among loci, and (iv) provide a preliminary test of the hypothesis that heterozygosity is positively correlated with the local rate of recombination. The average value for pi was low 0.063%, SE = 0.036%, about one order of magnitude smaller than for Drosophila melanogaster, the species for which the best data are available. Among loci, pi varied by over one order of magnitude. Statistical tests of neutrality based on ratios of polymorphism to divergence or based on the frequency spectrum of variation within humans failed to reject a neutral, equilibrium model. However, there was a positive correlation between heterozygosity and rate of recombination, suggesting that the joint effects of selection and linkage are important in shaping patterns of nucleotide variation in humans.

Mitochondrial DNA variation and the evolution of Robertsonian chromosomal races of house mice, Mus domesticus.

The house mouse, Mus domesticus, includes many distinct Robertsonian (Rb) chromosomal races with diploid numbers from 2n = 22 to 2n = 38. Although these races are highly differentiated karyotypically, they are otherwise indistinguishable from standard karyotype (i.e., 2n = 40) mice, and consequently their evolutionary histories are not well understood. We have examined mitochondrial DNA (mtDNA) sequence variation from the control region and the ND3 gene region among 56 M. domesticus from Western Europe, including 15 Rb populations and 13 standard karyotype populations, and two individuals of the sister species, Mus musculus. mtDNA exhibited an average sequence divergence of 0.84% within M. domesticus and 3.4% between M. domesticus and M. musculus. The transition/transversion bias for the regions sequenced is 5.7:1, and the overall rate of sequence evolution is approximately 10% divergence per million years. The amount of mtDNA variation was as great among different Rb races as among different populations of standard karyotype mice, suggesting that different Rb races do not derive from a single recent maternal lineage. Phylogenetic analysis of the mtDNA sequences resulted in a parsimony tree which contained six major clades. Each of these clades contained both Rb and standard karyotype mice, consistent with the hypothesis that Rb races have arisen independently multiple times. Discordance between phylogeny and geography was attributable to ancestral polymorphism as a consequence of the recent colonization of Western Europe by mice. Two major mtDNA lineages were geographically localized and contained both Rb and standard karyotype mice. The age of these lineages suggests that mice have moved into Europe only within the last 10,000 years and that Rb populations in different geographic regions arose during this time.

Occurrence and treatment of the mange mite Notoedres muris in marsh rats from South America.

The mange mite Notoedres muris is reported from a new host, the marsh rat (Holochilus brasiliensis) from Argentina. The infection involved alopecia and encrustations on the ears and face, and was treated successfully by a subcutaneous injection of ivermectin. The new record suggests that Notoedres muris has become self-maintaining within this marsh rat population.

The frequency of multiple paternity suggests that sperm competition is common in house mice (Mus domesticus).

Sexual selection is an important force driving the evolution of morphological and genetic traits. To determine the importance of male-male, postcopulatory sexual selection in natural populations of house mice, we estimated the frequency of multiple paternity, defined as the frequency with which a pregnant female carried a litter fertilized by more than one male. By genotyping eight microsatellite markers from 1095 mice, we found evidence of multiple paternity from 33 of 143. Evidence for multiple paternity was especially strong for 29 of these litters. Multiple paternity was significantly more common in higher-density vs. lower-density populations. Any estimate of multiple paternity will be an underestimate of the frequency of multiple mating, defined as the frequency with which a female mates with more than a single male during a single oestrus cycle. We used computer simulations to estimate the frequency of multiple mating, incorporating observed reductions in heterozygosity and levels of allele sharing among mother and father. These simulations indicated that multiple mating is common, occurring in at least 20% of all oestrus cycles. The exact estimate depends on the competitive skew among males, a parameter for which we currently have no data from natural populations. This study suggests that sperm competition is an important aspect of postcopulatory sexual selection in house mice.

Local adaptation in the rock pocket mouse (Chaetodipus intermedius): natural selection and phylogenetic history of populations.

Elucidating the causes of population divergence is a central goal of evolutionary biology. Rock pocket mice, Chaeotdipus intermedius, are an ideal system in which to study intraspecific phenotypic divergence because of the extensive color variation observed within this species. Here, we investigate whether phenotypic variation in color is correlated with local environmental conditions or with phylogenetic history. First, we quantified variation in pelage color (n=107 mice) and habitat color (n=51 rocks) using a spectrophotometer, and showed that there was a correlation between pelage color and habitat color across 14 sampled populations (R2=0.43). Analyses of mtDNA sequences from these same individuals revealed strong population structure in this species across its range, where most variation (63%) was partitioned between five geographic regions. Using Mantel tests, we show that there is no correlation between color variation and mtDNA phylogeny, suggesting that pelage coloration has evolved rapidly. At a finer geographical scale, high levels of gene flow between neighboring melanic and light populations suggest the selection acting on color must be quite strong to maintain habitat-specific phenotypic distributions. Finally, we raise the possibility that, in some cases, migration between populations of pocket mice inhabiting different lava flows may be responsible for similar melanic phenotypes in different populations. Together, the results suggest that color variation can evolve very rapidly over small geographic scales and that gene flow can both hinder and promote local adaptation.

Geographic patterns of chromosomal variation in South American marsh rats, Holochilus brasiliensis and H. vulpinus.

Karyotypes were prepared from 146 individuals, representing nine populations evenly spaced along a 2,000-km north-south transect in Paraguay and Argentina, to determine the nature, extent, and pattern of chromosomal variation in Holochilus brasiliensis chacarius and H. vulpinus. Two distinct patterns of chromosomal variation characterized these two species. In H. brasiliensis, the diploid number (2n) ranged from 48 to 56 and the nombre fondamental (NF) from 57 to 63. Four classes of chromosomal variation were found in populations of H. brasiliensis: whole-arm Robertsonian (Rb) translocations, including Rb changes with monobrachial homology, variation in the number and kind of supernumerary (B) chromosomes, centromeric rearrangements (putative pericentric inversions), and variation in the amount of euchromatin. The amount of structural variation was uniformly high in all populations of H. brasiliensis sampled, and all rearrangements appeared to be in Hardy-Weinberg proportions, corroborating the hypothesis that chromosomal rearrangements are not strongly underdominant in this species. In H. vulpinus, 2n ranged from 35 to 39 and NF from 57 to 61. Two classes of variation were found in this species: variation in the number, but not the kind, of supernumerary chromosomes and variation in the amount of euchromatin.

Meiotic studies of Robertsonian polymorphisms in the South American marsh rat, Holichilus brasiliensis.

Meiosis was studied in male South American marsh rats (1) to help clarify the mechanisms that allow unusually high levels of Robertsonian (Rb) polymorphisms to be maintained in wild populations of these animals and (2) to test competing assumptions in two distinct models of chromosomal speciation. In both simple Rb heterozygotes and Rb heterozygotes with monobrachial homology, no univalency was observed in prophase I or metaphase I. Rates of nondisjunction were uniformly low (less than 10%) and did not differ significantly among any of the animals studied, regardless of karyotype and in contrast to the frequency of nondisjunction in other mammalian species. Robertsonian heterozygotes exhibited significantly more chiasmata than did homozygotes, largely owing to an increase in the number of terminally located chiasmata. There was a significant bias favoring the transmission of two acrocentrics over the single metacentric for some Rb rearrangements in the heterozygous state. In addition, the frequency of sex-chromosome univalency increased with increasing Rb heterozygosity, although the ratio of X- and Y-bearing secondary spermatocytes did not differ significantly from 1:1, and no secondary spermatocytes were observed that were nullisomic or disomic for an X or Y chromosome.

Deleterious mutations in animal mitochondrial DNA.

A simple neutral model predicts that the ratio of non-synonymous to synonymous fixed differences between species will be the same as the ratio of non-synonymous to synonymous polymorphisms within species. This prediction is tested with existing mitochondrial datasets from 25 animal species. In slightly over half of the studies, the ratio of replacement to silent polymorphisms within species is significantly greater than the ratio of replacement to silent fixed differences between species. These observations are best explained by a substantial number of mildly deleterious amino acid mutations that contribute to heterozygosity but rarely become fixed.

Y chromosome variation of mice and men.

DNA sequences from the nonrecombining portion of the Y chromosome were compared with autosomal and X-linked sequences from mice and humans to test the neutral prediction that ratios of polymorphism to divergence are the same for different genes. Intraspecific variation within Mus domesticus was compared with divergence between M. domesticus and Mus caroli for Sry, a region 5' to Sry, and four X-linked genes, Hprt, Plp, Amg, and Glra2. None of these comparisons revealed significantly reduced variation on the Y chromosome. Intraspecific variation within humans was compared with divergence between humans and chimpanzees for three Y-linked loci (Zfy, the YAP region, and the Sry region), seven X-linked loci (Il2rg, Plp, Hprt, Gk, Ids, Pdhal, and Dmd), and the beta-globin locus on chromosome 11. In these comparisons, the observed level of variation on the human Y chromosome was slightly lower than expected, but was significantly lower in only one case (Sry region vs. Dmd). These results suggest that the levels of variability on the Y chromosome in mice and humans are close to expected values given the effective population size and mutation rates for these loci. There is at most only a modest reduction in variability that may be attributed to natural selection (either genetic hitchhiking or background selection).