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.     

Nucleotide diversity in gorillas

Comparison of the levels of nucleotide diversity in humans and apes may provide valuable information for inferring the demographic history of these species, the effect of social structure on genetic diversity, patterns of past migration, and signatures of past selection events. Previous DNA sequence data from both the mitochondrial and the nuclear genomes suggested a much higher level of nucleotide diversity in the African apes than in humans. Noting that the nuclear DNA data from the apes were very limited, we previously conducted a DNA polymorphism study in humans and another in chimpanzees and bonobos, using 50 DNA segments randomly chosen from the noncoding, nonrepetitive parts of the human genome. The data revealed that the nucleotide diversity (pi) in bonobos (0.077%) is actually lower than that in humans (0.087%) and that pi in chimpanzees (0.134%) is only 50% higher than that in humans. In the present study we sequenced the same 50 segments in 15 western lowland gorillas and estimated pi to be 0.158%. This is the highest value among the African apes but is only about two times higher than that in humans. Interestingly, available mtDNA sequence data also suggest a twofold higher nucleotide diversity in gorillas than in humans, but suggest a threefold higher nucleotide diversity in chimpanzees than in humans. The higher mtDNA diversity in chimpanzees might be due to the unique pattern in the evolution of chimpanzee mtDNA. From the nuclear DNA pi values, we estimated that the long-term effective population sizes of humans, bonobos, chimpanzees, and gorillas are, respectively, 10,400, 12,300, 21,300, and 25,200.     

Comparative nuclear and mitochondrial genome diversity in humans and chimpanzees

Restriction mapping and sequencing have shown that humans have substantially lower levels of mitochondrial genome diversity (d) than chimpanzees. In contrast, humans have substantially higher levels of heterozygosity (H) at protein-coding loci, suggesting a higher level of diversity in the nuclear genome. To investigate the discrepancy further, we sequenced a segment of the mitochondrial genome control region (CR) from 49 chimpanzees. The majority of these were from the Pan troglodytes versus subspecies, which was underrepresented in previous studies. We also estimated the average heterozygosity at 60 short tandem repeat (STR) loci in both species. For a total sample of 115 chimpanzees, d = 0.075 +/0 0.037, compared to 0.020 +/- 0.011 for a sample of 1,554 humans. The heterozygosity of human STR loci is significantly higher than that of chimpanzees. Thus, the higher level of nuclear genome diversity relative to mitochondrial genome diversity in humans is not restricted to protein-coding loci. It seems that humans, not chimpanzees, have an unusual d/H ratio, since the ratio in chimpanzees is similar to that in other catarrhines. This discrepancy in the relative levels of nuclear and mitochondrial genome diversity in the two species cannot be explained by differences in mutation rate. However, it may result from a combination of factors such as a difference in the extent of sex ratio disparity, the greater effect of population subdivision on mitochondrial than on nuclear genome diversity, a difference in the relative levels of male and female migration among subpopulations, diversifying selection acting to increase variation in the nuclear genome, and/or directional selection acting to reduce variation in the mitochondrial genome.      

DNA variation in a conifer,Cryptomeria japonica (Cupressaceae sensu lato)

We investigated the nucleotide variation of a conifer, Cryptomeria japonica, and the divergence between this species and its closest relative, Taxodium distichum, at seven nuclear loci (Acl5, Chi1, Ferr, GapC, HemA, Lcyb, and Pat). Samples of C. japonica were collected from three areas, Kantou-Toukai, Hokuriku, and Iwate. No apparent geographic differentiation was found among these samples. However, the frequency spectrum of the nucleotide polymorphism revealed excesses of intermediate-frequency variants, which suggests that the population was not panmictic and a constant size in the past. The average nucleotide diversity, pi, for silent sites was 0.00383. However, values of pi for silent sites vary among loci. Comparisons of polymorphism to divergence among loci (the HKA test) showed that the polymorphism at the Acl5 locus was significantly lower. We also observed a nearly significant excess of replacement polymorphisms at the Lcyb locus. These results suggested possibilities of natural selection acting at some of the loci. Intragenic recombination was detected only once at the Chi1 locus and was not detected at the other loci. The low level of population recombination rate, 4Nr, seemed to be due to both low level of recombination, r, and small population size, N.     

Using multilocus sequence data to assess population structure, natural selection, and linkage disequilibrium in wild tomatoes

We employed a multilocus approach to examine the effects of population subdivision and natural selection on DNA polymorphism in 2 closely related wild tomato species (Solanum peruvianum and Solanum chilense), using sequence data for 8 nuclear loci from populations across much of the species' range. Both species exhibit substantial levels of nucleotide variation. The species-wide level of silent nucleotide diversity is 18% higher in S. peruvianum (pi(sil) approximately 2.50%) than in S. chilense (pi(sil) approximately 2.12%). One of the loci deviates from neutral expectations, showing a clinal pattern of nucleotide diversity and haplotype structure in S. chilense. This geographic pattern of variation is suggestive of an incomplete (ongoing) selective sweep, but neutral explanations cannot be entirely dismissed. Both wild tomato species exhibit moderate levels of population differentiation (average F(ST) approximately 0.20). Interestingly, the pooled samples (across different demes) exhibit more negative Tajima's D and Fu and Li's D values; this marked excess of low-frequency polymorphism can only be explained by population (or range) expansion and is unlikely to be due to population structure per se. We thus propose that population structure and population/range expansion are among the most important evolutionary forces shaping patterns of nucleotide diversity within and among demes in these wild tomatoes. Patterns of population differentiation may also be impacted by soil seed banks and historical associations mediated by climatic cycles. Intragenic linkage disequilibrium (LD) decays very rapidly with physical distance, suggesting high recombination rates and effective population sizes in both species. The rapid decline of LD seems very promising for future association studies with the purpose of mapping functional variation in wild tomatoes.     

Intraspecific nuclear DNA variation in Drosophila

We have summarized and analyzed all available nuclear DNA sequence polymorphism studies for three species of Drosophila, D. melanogaster (24 loci), D. simulans (12 loci), and D. pseudoobscura (5 loci). Our major findings are: (1) The average nucleotide heterozygosity ranges from about 0.4% to 2% depending upon species and function of the region, i.e., coding or noncoding. (2) Compared to D. simulans and D. pseudoobscura (which are about equally variable), D. melanogaster displays a low degree of DNA polymorphism. (3) Noncoding introns and 3' and 5' flanking DNA shows less polymorphism than silent sites within coding DNA. (4) X-linked genes are less variable than autosomal genes. (5) Transition (Ts) and transversion (Tv) polymorphisms are about equally frequent in non-coding DNA and at fourfold degenerate sites in coding DNA while Ts polymorphisms outnumber Tv polymorphisms by about 2:1 in total coding DNA. The increased Ts polymorphism in coding regions is likely due to the structure of the genetic code: silent changes are more often Ts's than are replacement substitutions. (6) The proportion of replacement polymorphisms is significantly higher in D. melanogaster than in D. simulans. (7) The level of variation in coding DNA and the adjacent noncoding DNA is significantly correlated indicating regional effects, most notably recombination. (8) Surprisingly, the level of polymorphism at silent coding sites in D. melanogaster is positively correlated with degree of codon usage bias. (9) Three proposed tests of the neutral theory of DNA polymorphisms have been performed on the data: Tajima's test, the HKA test, and the McDonald-Kreitman test. About half of the loci fail to conform to the expectations of neutral theory by one of the tests. We conclude that many variables are affecting levels of DNA polymorphism in Drosophila, from properties of nucleotides to population history and, perhaps, mating structure. No simple, all encompassing explanation satisfactorily accounts for the data.      

Effective population size and tests of neutrality at cytoplasmic genes in Arabidopsis.

Cytoplasmic genomes typically lack recombination, implying that genetic hitch-hiking could be a predominant force structuring nucleotide polymorphism in the chloroplast and mitochondria. We test this hypothesis by analysing nucleotide polymorphism data at 28 loci across the chloroplast and mitochondria of the outcrossing plant Arabidopsis lyrata, and compare patterns with multiple nuclear loci, and the highly selfing Arabidopsis thaliana. The maximum likelihood estimate of the ratio of effective population size at cytoplasmic relative to nuclear genes in A. lyrata does not depart from the neutral expectation of 0.5. Similarly, the ratio of effective size in A. thaliana is close to unity, the neutral expectation for a highly selfing species. The results are thus consistent with neutral organelle polymorphism in these species or with comparable effects of hitch-hiking in both cytoplasmic and nuclear genes, in contrast to the results of recent studies on gynodioecious taxa. The four-gamete test and composite likelihood estimation provide evidence for very low levels of recombination in the organelles of A. lyrata, although permutation tests do not suggest that adjacent polymorphic sites are more closely linked than more distant sites across the two genomes, suggesting that mutation hotspots or very low rates of gene conversion could explain the data.     

Human-chimpanzee DNA sequence variation in the four major genes of the renin angiotensin system

The renin angiotensin system (RAS) is involved in blood pressure control and water/sodium metabolism. The genes encoding the proteins of this system are candidate genes for essential hypertension. The RAS involves four main molecules: angiotensinogen, renin, angiotensin I-converting enzyme, and the angiotensin II type 1 receptor (encoded by the genes AGT, REN, DCP1, and AGTR1, respectively). We performed a molecular screening over 17,037 bp of the coding and 5' and 3' untranslated regions of these genes, from three to six common chimpanzees. We identified 44 single-nucleotide polymorphisms (SNPs) in chimpanzee samples, including 18 coding-region SNPs, 5 of which led to an amino acid replacement. We observed common and different features at various sites (synonymous, nonsynonymous, and noncoding) within and between the four chimpanzee genes: (1) the nucleotide diversity at noncoding sites was similar; (2) the nucleotide diversity at nonsynonymous sites was low, probably reflecting purifying selection, except for the AGT gene; (3) the nucleotide diversity at synonymous sites, which was dependent on the G+C content at the third position of the codon, was high, except for the AGTR1 gene. Comparison of the chimpanzee SNPs with those previously reported for humans identified 119 sites with fixed differences (including 62 coding sites, 17 of which resulted in amino acid differences between the species). Analysis of polymorphism within species and divergence between species shed light on the evolutionary constraints on these genes. In particular, comparison of the pattern of mutation at polymorphic and fixed sites between humans and chimpanzees suggested that the high G+C content of the DCP1 gene was maintained by positive selection at its silent sites. Finally, we propose 68 ancestral alleles for the human RAS genes and discuss the implications for their use in future hypertension-susceptibility association studies.     

The relationship of nucleotide polymorphism, recombination rate and selection in wild tomato species

We analyzed the effects of mating system and recombination rate on single nucleotide polymorphisms using 14 single-copy nuclear loci from single populations of five species of wild tomatoes (Solanum section Lycopersicon). The taxa investigated comprise two self-compatible (SC) and three self-incompatible (SI) species. The observed reduction in nucleotide diversity in the SC populations compared to the SI populations is much stronger than expected under the neutral effects of the mating system on effective population size. Importantly, outgroup sequences available for 11 of the 14 loci yield strong positive correlations between silent nucleotide diversity and silent divergence, indicative of marked among-locus differences in mutation rates and/or selective constraints. Furthermore, using a physical estimate of local recombination rates, we find that silent nucleotide diversity (but not divergence) is positively correlated with recombination rate in two of the SI species. However, this correlation is not nearly as strong as in other well-characterized species (in particular, Drosophila). We propose that nucleotide diversity in Lycopersicon is dominated mainly by differences in neutral mutation rates and/or selective constraints among loci, demographic processes (such as population subdivision), and background selection. In addition, we hypothesize that the soil seed bank plays an important role in the maintenance of the large genetic diversity in the SI species (in particular L. peruvianum).     

Positive and negative selection on noncoding DNA close to protein-coding genes in wild house mice

During the past two decades, evidence has accumulated of adaptive evolution within protein-coding genes in a variety of species. However, with the exception of Drosophila and humans, little is known about the extent of adaptive evolution in noncoding DNA. Here, we study regions upstream and downstream of protein-coding genes in the house mouse Mus musculus castaneus, a species that has a much larger effective population size (N(e)) than humans. We analyze polymorphism data for 78 genes from 15 wild-caught M. m. castaneus individuals and divergence to a closely related species, Mus famulus. We find high levels of nucleotide diversity and moderate levels of selective constraint in upstream and downstream regions compared with nonsynonymous sites of protein-coding genes. From the polymorphism data, we estimate the distribution of fitness effects (DFE) of new mutations and infer that most new mutations in upstream and downstream regions behave as effectively neutral and that only a small fraction is strongly negatively selected. We also estimate the fraction of substitutions that have been driven to fixation by positive selection (α) and the ratio of adaptive to neutral divergence (ω(α)). We find that α for upstream and downstream regions (～ 10%) is much lower than α for nonsynonymous sites (～ 50%). However, ω(α) estimates are very similar for nonsynonymous sites (～ 10%) and upstream and downstream regions (～ 5%). We conclude that negative selection operating in upstream and downstream regions of M. m. castaneus is weak and that the low values of α for upstream and downstream regions relative to nonsynonymous sites are most likely due to the presence of a higher proportion of neutrally evolving sites and not due to lower absolute rates of adaptive substitution.     

Genetic variation and population structure of a threatened timber tree <Emphasis Type="Italic">Dalbergia cochinchinensis</Emphasis> in Cambodia

Dalbergia cochinchinensis Pierre ex Laness. (Fabaceae) is a commercially important tree in Southeast Asia. Although this species is under legal protections, illegal logging and disorderly developments have reduced its populations, and the conservation of this species is currently of much concern. In this study, we determined nucleotide sequences at six chloroplasts and ten nuclear loci in four populations of D. cochinchinensis in Cambodia, followed by population genetic analyses. The average silent nucleotide diversity over the nuclear loci, excluding one with an exceptionally high value, was 0.0057 in the entire population, and the mean F _ST across the nuclear loci between each population pair was between 0.135 and 0.467. Thus, the nucleotide diversity in the studied populations was not low compared with that in other tree species, and the level of population differentiation was high. Neutrality test statistics indicated a recent reduction of population size and a subdivision of the population within this species. The divergence times and migration rates were estimated with a likelihood-based method assuming the isolation with migration model. Based on the results, the three populations split 68,000–138,000 years ago, possibly corresponding to the start of the last glacial period, and the level of gene flow among the populations was very low thereafter. Moreover, after the split, population sizes were reduced considerably. Notably, the nucleotide diversity in an insertion sequence in a noncoding region of nuclear C4H was much higher than the mean nucleotide diversity in silent sites across other nuclear genes, indicating that the region was affected by selection.     

Genetic variation within and among populations of Arabidopsis thaliana.

We investigated levels of nucleotide polymorphism within and among populations of the highly self-fertilizing Brassicaceous species, Arabidopsis thaliana. Four-cutter RFLP data were collected at one mitochondrial and three nuclear loci from 115 isolines representing 11 worldwide population collections, as well as from seven commonly used ecotypes. The collections include multiple populations from North America and Eurasia, as well as two pairs of collections from locally proximate sites, and thus allow a hierarchical geographic analysis of polymorphism. We found no variation at the mitochondrial locus Nad5 and very low levels of intrapopulation nucleotide diversity at Adh, Dhs1, and Gpa1. Interpopulation nucleotide diversity was also consistently low among the loci, averaging 0.0014. gst, a measure of population differentiation, was estimated to be 0.643. Interestingly, we found no association between geographical distance between populations and genetic distance. Most haplotypes have a worldwide distribution, suggesting a recent expansion of the species or long-distance gene flow. The low level of polymorphism found in this study is consistent with theoretical models of neutral mutations and background selection in highly self-fertilizing species.     

Historical Selection, Amino Acid Polymorphism and Lineage-Specific Divergence at the G6pd Locus in Drosophila Melanogaster and D. Simulans

The nucleotide diversity across 1705 bp of the G6pd gene is studied in 50 Drosophila melanogaster and 12 D. simulans lines. Our earlier report contrasted intraspecific polymorphism and interspecific differences at silent and replacement sites in these species. This report expands the number of European and African lines and examines the pattern of polymorphism with respect to the common A/B allozymes. In D. melanogaster the silent nucleotide diversity varies 2.8-fold across localities. The B allele sequences are two- to fourfold more variable than the derived A allele, and differences between allozymes are twice as among B alleles. There is strong linkage disequilibrium across the G6pd region. In both species the level of silent polymorphism increases from the 5' to 3' ends, while there is no comparable pattern in level of silent site divergence or fixation. The neutral model is not rejected in either species. Using D. yakuba as an outgroup, the D. melanogaster lineage shows a twofold greater rate of silent fixation, but less than half the rate of amino acid replacement. Lineage-specific differences in mutation fixation are inconsistent with neutral expectations and suggest the interaction of species-specific population size differences with both weakly advantageous and deleterious selection.     

Common chimpanzees have greater diversity than humans at two of the three highly polymorphic MHC class I genes

 MHC class I polymorphism improves the defense of vertebrate species against viruses and other intracellular pathogens. To see how polymorphism at the same class I genes can evolve in different species we compared the MHC-A, MHC-B, and MHC-C loci of common chimpanzees and humans. Diversity in 23 Patr-A, 32 Patr-B, and 18 Patr-C alleles obtained from study of 48 chimpanzees was compared to diversity in 66 HLA-A, 149 HLA-B, and 41 HLA-C alleles obtained from a study of over 1 million humans. At each locus, alleles group hierarchically into families and then lineages. No alleles or families are shared by the two species, commonality being seen only at the lineage level. The overall nucleotide sequence diversity of MHC class I is estimated to be greater for modern chimpanzees than humans. Considering the numbers of lineages, families, and alleles, Patr-B and Patr-C have greater diversity than the HLA-B and HLA-C, respectively. In contrast, Patr-A has less polymorphism than HLA-A, due to the absence of A2 lineage alleles. The results are consistent with ancestral humans having passed through a narrower population bottleneck than chimpanzees, and with pathogen-mediated selection having favored either preservation of A2 lineage alleles on the human line and/or their extinction on the chimpanzee line. Received: 8 December 1999 / Accepted: 30 December 1999     

High level of functional polymorphism indicates a unique role of natural selection at human immune system loci

Several studies have shown that immune system proteins have on average a higher rate of amino acid evolution between different species of mammals than do most other proteins. To test whether immune-system-expressed loci show a correspondingly elevated rate of within-species nonsynonymous (amino acid altering) polymorphism, we examined gene diversity (heterozygosity) at 4,911 single nucleotide polymorphism (SNP) sites at 481 protein-coding loci. At loci with nonimmune functions, gene diversity at nonsynonymous SNP sites was typically lower than that at silent SNP sites (those not altering the amino acid sequence) in the same gene, a pattern that is an evidence of purifying selection acting to eliminate slightly deleterious variants. However, this pattern was not seen at nonsynonymous SNPs causing conservative amino acid replacements in immune system proteins, indicating that the latter are subject to a reduced level of functional constraint. Similarly, immune system genes showed higher gene diversities in their 5′ noncoding regions than did other proteins. These results identified certain immune system loci that are likely to be subject to balancing selection that acts to maintain polymorphism in either coding or regulatory regions. Electronic Supplementary Material Supplementary material is available for this article at .     

Nuclear gene genealogies reveal historical,demographic and selective factors associated with speciation in field crickets

Population genetics theory predicts that genetic drift should eliminate shared polymorphism, leading to monophyly or exclusivity of populations, when the elapsed time between lineage-splitting events is large relative to effective population size. We examined patterns of nucleotide variation in introns at four nuclear loci to relate processes affecting the history of genes to patterns of divergence among natural populations and species. Ancestral polymorphisms were shared among three recognized species, Gryllus firmus, G. pennsylvanicus, and G. ovisopis, and genealogical patterns suggest that successive speciation events occurred recently and rapidly relative to effective population size. High levels of shared polymorphism among these morphologically, behaviorally, and ecologically distinct species indicate that only a small fraction of the genome needs to become differentiated for speciation to occur. Among the four nuclear gene loci there was a 10-fold range in nucleotide diversity, and patterns of polymorphism and divergence suggest that natural selection has acted to maintain or eliminate variation at some loci. While nuclear gene genealogies may have limited applications in phylogeography or other approaches dependent on population monophyly, they provide important insights into the historical, demographic, and selective forces that shape speciation.     

A multilocus sequence survey in Arabidopsis thaliana reveals a genome-wide departure from a neutral model of DNA sequence polymorphism

The simultaneous analysis of multiple genomic loci is a powerful approach to studying the effects of population history and natural selection on patterns of genetic variation of a species. By surveying nucleotide sequence polymorphism at 334 randomly distributed genomic regions in 12 accessions of Arabidopsis thaliana, we examined whether a standard neutral model of nucleotide sequence polymorphism is consistent with observed data. The average nucleotide diversity was 0.0071 for total sites and 0.0083 for silent sites. Although levels of diversity are variable among loci, no correlation with local recombination rate was observed, but polymorphism levels were correlated for physically linked loci (<250 kb). We found that observed distributions of Tajima's D- and D/D(min)- and of Fu and Li's D-, D*- and F-, F*-statistics differed significantly from the expected distributions under a standard neutral model due to an excess of rare polymorphisms and high variances. Observed and expected distributions of Fay and Wu's H were not different, suggesting that demographic processes and not selection at multiple loci are responsible for the deviation from a neutral model. Maximum-likelihood comparisons of alternative demographic models like logistic population growth, glacial refugia, or past bottlenecks did not produce parameter estimates that were more consistent with observed patterns. However, exclusion of highly polymorphic "outlier loci" resulted in a fit to the logistic growth model. Various tests of neutrality revealed a set of candidate loci that may evolve under selection.     

Modern African ape populations as genetic and demographic models of the last common ancestor of humans, chimpanzees, and gorillas

In order to fully understand human evolutionary history through the use of molecular data, it is essential to include our closest relatives as a comparison. We provide here estimates of nucleotide diversity and effective population size of modern African ape species using data from several independent noncoding nuclear loci, and use these estimates to make predictions about the nature of the ancestral population that eventually gave rise to the living species of African apes, including humans. Chimpanzees, bonobos, and gorillas possess two to three times more nucleotide diversity than modern humans. We hypothesize that the last common ancestor (LCA) of these species had an effective population size more similar to modern apes than modern humans. In addition, estimated dates for the divergence of the Homo, Pan, and Gorilla lineages suggest that the LCA may have had stronger geographic structuring to its mtDNA than its nuclear DNA, perhaps indicative of strong female philopatry or a dispersal system analogous to gorillas, where females disperse only short distances from their natal group. Synthesizing different classes of data, and the inferences drawn from them, allows us to predict some of the genetic and demographic properties of the LCA of humans, chimpanzees, and gorillas.     

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.     

Neutral and Non-Neutral Evolution of Drosophila Mitochondrial DNA

To test hypotheses of neutral evolution of mitochondrial DNA (mtDNA), nucleotide sequences were determined for 1515 base pairs of the NADH dehydrogenase subunit 5 (ND5) gene in the mitochondrial DNA of 29 lines of Drosophila melanogaster and 9 lines of its sibling species Drosophila simulans. In contrast to the patterns for nuclear genes, where D. melanogaster generally exhibits much less nucleotide polymorphism, the number of segregating sites was slightly higher in a global sample of nine ND5 sequences in D. melanogaster (s = 8) than in the nine lines of D. simulans (s = 6). When compared to variation at nuclear loci, the mtDNA variation in D. melanogaster does not depart from neutral expectations. The ND5 sequences in D. simulans, however, show fewer than half the number of variable sites expected under neutrality when compared to sequences from the period locus. While this reduction in variation is not significant at the 5% level, HKA tests with published restriction data for mtDNA in D. simulans do show a significant reduction of variation suggesting a selective sweep of variation in the mtDNA in this species. Tests of neutral evolution based on the ratios of synonymous and replacement polymorphism and divergence are generally consistent with neutral expectations, although a significant excess of amino acid polymorphism within both species is localized in one region of the protein. The rate of mtDNA evolution has been faster in D. melanogaster than in D. simulans and the population structure of mtDNA is distinct in these species. The data reveal how different rates of mtDNA evolution between species and different histories of neutral and adaptive evolution within species can compromise historical inferences in population and evolutionary biology.