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.     

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.     

Unraveling Selection in the Mitochondrial Genome of Drosophila

We examine mitochondrial DNA variation at the cytochrome b locus within and between three species of Drosophila to determine whether patterns of variation conform to the predictions of neutral molecular evolution. The entire 1137-bp cytochrome b locus was sequenced in 16 lines of Drosophila melanogaster, 18 lines of Drosophila simulans and 13 lines of Drosophila yakuba. Patterns of variation depart from neutrality by several test criteria. Analysis of the evolutionary clock hypothesis shows unequal rates of change along D. simulans lineages. A comparison within and between species of the ratio of amino acid replacement change to synonymous change reveals a relative excess of amino acid replacement polymorphism compared to the neutral prediction, suggestive of slightly deleterious or diversifying selection. There is evidence for excess homozygosity in our world wide sample of D. melanogaster and D. simulans alleles, as well as a reduction in the number of segregating sites in D. simulans, indicative of selective sweeps. Furthermore, a test of neutrality for codon usage shows the direction of mutations at third positions differs among different topological regions of the gene tree. The analyses indicate that molecular variation and evolution of mtDNA are governed by many of the same selective forces that have been shown to govern nuclear genome evolution and suggest caution be taken in the use of mtDNA as a ``neutral' molecular marker.     

Recombination, dominance and selection on amino acid polymorphism in the Drosophila genome: contrasting patterns on the X and fourth chromosomes

Surveys of nucleotide polymorphism and divergence indicate that the average selection coefficient on Drosophila proteins is weakly positive. Similar surveys in mitochondrial genomes and in the selfing plant Arabidopsis show that weak negative selection has operated. These differences have been attributed to the low recombination environment of mtDNA and Arabidopsis that has hindered adaptive evolution through the interference effects of linkage. We test this hypothesis with new sequence surveys of proteins lying in low recombination regions of the Drosophila genome. We surveyed >3800 bp across four proteins at the tip of the X chromosome and >3600 bp across four proteins on the fourth chromosome in 24 strains of D. melanogaster and 5 strains of D. simulans. This design seeks to study the interaction of selection and linkage by comparing silent and replacement variation in semihaploid (X chromosome) and diploid (fourth chromosome) environments lying in regions of low recombination. While the data do indicate very low rates of exchange, all four gametic phases were observed both at the tip of the X and across the fourth chromosome. Silent variation is very low at the tip of the X (thetaS = 0.0015) and on the fourth chromosome (thetaS = 0.0002), but the tip of the X shows a greater proportional loss of variation than the fourth shows relative to normal-recombination regions. In contrast, replacement polymorphism at the tip of the X is not reduced (thetaR = 0.00065, very close to the X chromosome average). MK and HKA tests both indicate a significant excess of amino acid polymorphism at the tip of the X relative to the fourth. Selection is significantly negative at the tip of the X (Nes = -1.53) and nonsignificantly positive on the fourth (Nes approximately 2.9), analogous to the difference between mtDNA (or Arabidopsis) and the Drosophila genome average. Our distal X data are distinct from regions of normal recombination where the X shows a deficiency of amino acid polymorphism relative to the autosomes, suggesting more efficient selection against recessive deleterious replacement mutations. We suggest that the excess amino acid polymorphism on the distal X relative to the fourth chromosome is due to (1) differences in the mutation rate for selected mutations on the distal X or (2) a greater relaxation of selection from stronger linkage-related interference effects on the distal X. This relaxation of selection is presumed to be greater in magnitude than the difference in efficiency of selection between X-linked vs. autosomal selection.     

The Rates of Molecular Evolution in Rodent and Primate Mitochondrial DNA

A higher rate of molecular evolution in rodents than in primates at synonymous sites and, to a lesser extent, at amino acid replacement sites has been reported previously for most nuclear genes examined. Thus in these genes the average ratio of amino acid replacement to synonymous substitution rates in rodents is lower than in primates, an observation at odds with the neutral model of molecular evolution. Under Ohta's mildly deleterious model of molecular evolution, these observations are seen as the consequence of the combined effects of a shorter generation time (driving a higher mutation rate) and a larger effective population size (resulting in more effective selection against mildly deleterious mutations) in rodents. The present study reports the results of a maximum-likelihood analysis of the ratio of amino acid replacements to synonymous substitutions for genes encoded in mitochondrial DNA (mtDNA) in these two lineages. A similar pattern is observed: in rodents this ratio is significantly lower than in primates, again consistent only with the mildly deleterious model. Interestingly the lineage-specific difference is much more pronounced in mtDNA-encoded than in nuclear-encoded proteins, an observation which is shown to run counter to expectation under Ohta's model. Finally, accepting certain fossil divergence dates, the lineage-specific difference in amino acid replacement-to-synonymous substitution ratio in mtDNA can be partitioned and is found to be entirely the consequence of a higher mutation rate in rodents. This conclusion is consistent with a replication-dependent model of mutation in mtDNA. Received: 24 September 1999 / Accepted: 18 September 2000     

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.     

Neutrality tests of conservative-radical amino acid changes in nuclear- and mitochondrially-encoded proteins

The neutralist-selectionist debate should not be viewed as a dichotomy but as a continuum. While the strictly neutral model suggests a neutralist-selectionist dichotomy, the nearly neutral model is a continuous model spanning strict neutrality through weak selection (Ns approximately 1) to deterministic selection (Ns>3). We illustrate these points with polymorphism and divergence data from a sample of 73 genes (31 mitochondrial, 36 nuclear genes from Drosophila, and six Arabidopsis data sets). In an earlier study we used the McDonald-Kreitman (MK) test to show that amino acid replacement polymorphism in animal mitochondrial genes and Arabidopsis genes show a consistent trend toward negative selection, whereas nuclear genes from Drosophila span a range from negative selection, through neutrality, to positive selection. Here we analyze a subset of these genes (13 Drosophila nuclear, ten mitochondrial, and six Arabidopsis nuclear) for polymorphism and divergence of conservative and radical amino acid replacements (a protein-based conservative-radical MK, or pMK, test). The distinct patterns of selection between the different genomes is not apparent with the pMK test. Different definitions of conservative and radical (based on amino acid polarity, volume or charge) give inconsistent results across genes. We suggest that segregating fitness difference between silent and replacement mutations are more visible to selection than are segregating fitness differences between conservative and radical amino acid mutations. New data on the variation among genes with different opportunities for positive and negative selection are as important to the continuum view of the neutralist-selectionist debate as is the distribution of selection coefficients within individual genes.     

Transpecific polymorphisms in an inversion linked esterase locus in Drosophila buzzatii

Nucleotide variation was studied in a 1.1 kb section of the coding region of an Esterase gene (Est-A) that maps in the center of the segments rearranged by polymorphic inversions in the cactophilic Drosophila buzzatii. We examine 30 homozygous second-chromosome lines differing in gene arrangement and three D. koepferae isofemale lines as outgroups. Our data show that Est-A is a highly polymorphic gene at both synonymous and replacement sites. Significant departures from homogeneity in the distribution of the ratio of silent polymorphism to divergence predicted by the neutral theory reveals a local excess of silent polymorphism. This is consistent with the presence of two apparent narrow peaks of elevated silent polymorphism surrounding nonconservative amino acid substitutions. These polymorphisms as well as others at synonymous and nonsynonymous sites are shared with D. koepferae. We suggest that the presence of shared nucleotide polymorphisms is probably due to interspecific gene flow and/or balancing selection acting on replacement variants and/or to a decreased probability of loss of ancestral polymorphisms caused by linkage to an adaptive inversion polymorphism. Recurrent mutation and persistence of neutral ancestral polymorphisms cannot, however, be ruled out. The analysis of the distribution of nucleotide variation among the three chromosomal arrangements sampled reveals that derived arrangements (J and JZ(3)) are less polymorphic than the ancestral ST, and that the widely distributed ST and J arrangements are genetically differentiated. However, a significant number of polymorphisms are shared between arrangements, suggesting frequent exchange either from gene conversion or from double crossovers in heterokaryotypes. Finally, our present results in combination with data of sequence variation at the breakpoints of inversion J suggest that this old gene arrangement has risen in frequency in relatively recent times.     

Rapidly evolving mitochondrial genome and directional selection in mitochondrial genes in the parasitic wasp nasonia (hymenoptera: pteromalidae)

We sequenced the nearly complete mtDNA of 3 species of parasitic wasps, Nasonia vitripennis (2 strains), Nasonia giraulti, and Nasonia longicornis, including all 13 protein-coding genes and the 2 rRNAs, and found unusual patterns of mitochondrial evolution. The Nasonia mtDNA has a unique gene order compared with other insect mtDNAs due to multiple rearrangements. The mtDNAs of these wasps also show nucleotide substitution rates over 30 times faster than nuclear protein-coding genes, indicating among the highest substitution rates found in animal mitochondria (normally <10 times faster). A McDonald and Kreitman test shows that the between-species frequency of fixed replacement sites relative to silent sites is significantly higher compared with within-species polymorphisms in 2 mitochondrial genes of Nasonia, atp6 and atp8, indicating directional selection. Consistent with this interpretation, the Ka/Ks (nonsynonymous/synonymous substitution rates) ratios are higher between species than within species. In contrast, cox1 shows a signature of purifying selection for amino acid sequence conservation, although rates of amino acid substitutions are still higher than for comparable insects. The mitochondrial-encoded polypeptides atp6 and atp8 both occur in F0F1ATP synthase of the electron transport chain. Because malfunction in this fundamental protein severely affects fitness, we suggest that the accelerated accumulation of replacements is due to beneficial mutations necessary to compensate mild-deleterious mutations fixed by random genetic drift or Wolbachia sweeps in the fast evolving mitochondria of Nasonia. We further propose that relatively high rates of amino acid substitution in some mitochondrial genes can be driven by a "Compensation-Draft Feedback"; increased fixation of mildly deleterious mutations results in selection for compensatory mutations, which lead to fixation of additional deleterious mutations in nonrecombining mitochondrial genomes, thus accelerating the process of amino acid substitutions.     

Polymorphism and divergence at the prune locus in Drosophila melanogaster and D. simulans

The prune locus of Drosophila melanogaster lies at the tip of the X chromosome, in a region of reduced recombination in which nearby loci show reduced variation relative to evolutionary divergence from D. simulans. DNA sequencing of prune alleles from D. melanogaster and D. simulans reveals extremely low variation in D. melanogaster but greater variation in D. simulans. Divergence between the two species is not reduced. This pattern may be explained by either positive selection leading to hitchhiking of neutral variation or background selection against deleterious mutations. The pattern of silent versus replacement polymorphism and divergence at prune is consistent with either a model of weakly deleterious selection against amino acid substitutions or balancing selection.      

Correlated evolutionary divergence of egg size and a mitochondrial protein across the Isthmus of Panama

An explicit assumption of studies that employ a mitochondrial DNA (mtDNA) molecular clock is that mtDNA evolves independently of morphology. Here we report a very strong correlation between egg size divergence and cytochrome c oxidase-1 (CO1) amino acid sequence divergence among sister species of bivalve molluscs separated by the Central American Isthmus (i.e., "geminate" species). Analyses of the molecular data reveal that CO1 sequences likely did not diverge as a function of time or evolve in response to positive natural selection. Given that an excess of CO1 amino acid polymorphism exists within species (as expected if most mutations are only slightly deleterious), a third hypothesis is that reductions in effective population size could simultaneously increase the fixation rate of nearly neutral mtDNA polymorphisms and in some way also facilitate egg size evolution. The remarkable strength of the relationship between egg size and CO1 amino acid sequence demonstrates that, even in the absence of an obvious functional relationship or clock-like evolution, the amounts of molecular and morphological change can be tightly correlated, and therefore may reflect common processes. Accordingly, the assumption that the evolutionary divergence of molecules and morphology are independent must always be carefully examined.     

Departure from neutrality at the mitochondrial NADH dehydrogenase subunit 2 gene in humans,but not in chimpanzees.

To test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution, nucleotide sequences were determined for the 1041 bp of the NADH dehydrogenase subunit 2 (ND2) gene in 20 geographically diverse humans and 20 common chimpanzees. Contingency tests of neutrality were performed using four mutational categories for the ND2 molecule: synonymous and nonsynonymous mutations in the transmembrane regions, and synonymous and nonsynonymous mutations in the surface regions. The following three topological mutational categories were also used: intraspecific tips, intraspecific interiors, and interspecific fixed differences. The analyses reveal a significantly greater number of nonsynonymous polymorphisms within human transmembrane regions than expected based on interspecific comparisons, and they are inconsistent with a neutral equilibrium model. This pattern of excess nonsynonymous polymorphism is not seen within chimpanzees. Statistical tests of neutrality, such as TAJIMA''s D test, and the D and F tests proposed by FU and LI, indicate an excess of low frequency polymorphisms in the human data, but not in the chimpanzee data. This is consistent with recent directional selection, a population bottleneck or background selection of slightly deleterious mutations in human mtDNA samples. The analyses further support the idea that mitochondrial genome evolution is governed by selective forces that have the potential to affect its use as a "neutral" marker in evolutionary and population genetic studies.     

Molecular population genetics of Xdh and the evolution of base composition in Drosophila

Few loci have been measured for DNA polymorphism and divergence in several species. Here we report such data from the protein-coding region of xanthine dehydrogenase (Xdh) in 22 species of Drosophila. Many of our samples were from closely related species, allowing us to confidently assign substitutions to individual lineages. Surprisingly, Xdh appears to be fixing more A/T mutations than G/C mutations in most lineages, leading to evolution of higher A/T content in the recent past. We found no compelling evidence for selection on protein variation, though some aspects of the data support the notion that a significant fraction of amino acid polymorphisms are slightly deleterious. Finally, we found no convincing evidence that levels of silent heterozygosity are associated with rates of protein evolution.     

Molecular population genetics of the male and female mitochondrial DNA molecules of the California sea mussel, Mytilus californianus

The presence of two gender-associated mitochondrial genomes in marine mussels provides a unique opportunity to investigate the dynamics of mtDNA evolution without complications inherent in interspecific comparisons. Here, we assess the relative importance of selection, mutation, and differential constraint in shaping the patterns of polymorphism within and divergence between the male (M) and female (F) mitochondrial genomes of the California sea mussel, Mytilus californianus. Partial sequences were obtained from homologous regions of four genes (nad2, cox1, atp6, and nad5) totaling 2307 bp in length. The M and F mtDNA molecules of M. californianus exhibited extensive levels of nucleotide polymorphism and were more highly diverged than observed in other mytilids (overall Tamura-Nei distances >40%). Consistent with previous studies, the M molecule had significantly higher levels of silent and replacement polymorphism relative to F. Both genomes possessed large numbers of singleton and low-frequency mutations that gave rise to significantly negative Tajima's D values. Mutation-rate scalars estimated for silent and replacement mutations were elevated in the M genome but were not sufficient to account for its higher level of polymorphism. McDonald-Kreitman tests were highly significant at all loci due to excess numbers of fixed replacement mutations between molecules. Strong purifying selection was evident in both genomes in keeping the majority of replacement mutations at low population frequencies but appeared to be slightly relaxed in M. Our results suggest that a reduction in selective constraint acting on the M genome remains the best explanation for its greater levels of polymorphism and faster rate of evolution.     

Recombination enhances protein adaptation in Drosophila melanogaster

Evolutionary theory predicts that the rate and level of adaptation will be enhanced in sexual relative to asexual genomes because sexual recombination facilitates the elimination of deleterious mutations and the fixation of beneficial ones by natural selection. To date, the most compelling evidence for this prediction comes from experimental evolution studies and from loci completely lacking recombination, such as those on Y chromosomes, which often show reduced adaptation and even degeneration. Here, by analyzing replacement and silent DNA polymorphism and divergence at 98 loci, I show that recombination increases the efficacy of protein adaptation throughout the genome of the fruit fly Drosophila melanogaster. Genes residing in genomic regions with reduced recombination rates suffer a greater load of segregating, mildly deleterious mutations and fix fewer beneficial mutations than genes residing in regions with higher recombination rates. These findings suggest that the capacity to respond to natural selection varies with recombination rate across the genome, consistent with theory on the evolutionary advantages of sex and recombination.     

Neutrality tests on mtDNA: unusual results from nematodes

McDonald-Kreitman tests of neutrality on mitochondrial DNA (mtDNA) of butterflies, Drosophila, and a variety of vertebrates usually show excess (over the neutral expectation) intraspecific polymorphism at nonsilent sites. These results are of great interest because they are the opposite of what is usually found for nuclear genes, in which the neutral pattern or evidence of adaptive divergence between species is usually observed. However, only vertebrates and insects have been tested so far, so it is not clear whether this intriguing pattern is typical for mtDNA in all taxa. Here I tested three pairs of nematode species and found that they all show a deficit of replacement polymorphism. Taken at face value, this result suggests that adaptive evolution proceeds more efficiently in nematode mtDNA than in mtDNA of vertebrates or insects. An alternate explanation is that the nematode pattern is an artifact of silent-site saturation that results from the rapid and composition-biased way in which nematode mtDNA evolves. Further studies are needed to distinguish between these two hypotheses.     

The frequency distribution of nucleotide variation in Drosophila simulans.

Patterns of codon bias in Drosophila suggest that silent mutations can be classified into two types: unpreferred (slightly deleterious) and preferred (slightly beneficial). Results of previous analyses of polymorphism and divergence in Drosophila simulans were interpreted as supporting a mutation-selection-drift model in which slightly deleterious, silent mutants make significantly greater contributions to polymorphism than to divergence. Frequencies of unpreferred polymorphisms were inferred to be lower than frequencies of other silent polymorphisms. Here, I analyzed additional D. simulans data to reevaluate the support for these ideas. I found that D. simulans has fixed more unpreferred than preferred mutations, suggesting that this lineage has not been at mutation-selection-drift equilibrium at silent sites. Frequencies of polarized unpreferred polymorphisms are not skewed toward rare alleles. However, frequencies of unpolarized unpreferred codons are lower in high-bias genes than in low-bias genes. This supports the idea that unpreferred codons are borderline deleterious mutations. Purifying selection on silent sites appears to be stronger at twofold-degenerate codons than at fourfold-degenerate codons. Finally, I found that X-linked polymorphisms occur at a higher average frequency than polymorphisms on chromosome arm 3R, even though an average X-linked site is significantly less likely to be polymorphic than an average site on 3R. This result supports a previous analysis of D. simulans indicating different population genetics of X-linked versus autosomal mutations.     

High proportions of deleterious polymorphisms in constrained human genes

Previous studies on human mitochondrial genomes showed that the ratio of intra-specific diversities at nonsynonymous-to-synonymous positions was two to ten times higher than the ratio of interspecific divergences at these positions, suggesting an excess of slightly deleterious nonsynonymous polymorphisms. However, such an overabundance of nonsynonymous single nucleotide polymorphisms (SNPs) was not found in human nuclear genomes. Here, genome-wide estimates using >14,000 human-chimp nuclear genes and 1 million SNPs from four human genomes showed a significant proportion of deleterious nonsynonymous SNPs (～ 15%). Importantly, this study reveals a negative correlation between the magnitude of selection pressure and the proportion of deleterious SNPs on human genes. The proportion of deleterious amino acid replacement polymorphisms is 3.5 times higher in genes under high purifying selection compared with that in less constrained genes (28% vs. 8%). These results are explained by differences in the extent of contribution of mildly deleterious mutations to diversity and substitution.     

Molecular population genetics of male accessory gland proteins in Drosophila

Drosophila seminal proteins have an unusually high rate of molecular sequence evolution, suggesting either a high rate of neutral substitution or rapid adaptive evolution. To further quantify patterns of polymorphism and divergence in genes encoding seminal proteins, also called accessory gland proteins (Acp's), we conducted a sequencing survey of 10 Acp genes in samples of Drosophila melanogaster and D. simulans (Acp29AB, Acp32CD, Acp33A, Acp36DE, Acp53Ea, Acp62F, Acp63F, Acp76A, Acp95EF, and Acp98AB). Mean heterozygosity at replacement sites in D. simulans was 0.0074 for Acp genes and 0.0013 for a set of 19 non-Acp genes, and mean melanogaster-simulans divergence at replacement sites was 0.0497 for Acp genes and 0.0107 at non-Acp genes. The elevated divergence of Acp genes is thus accompanied by elevated within-species polymorphism. In addition to the already-reported departures of Acp26A, Acp29AB, and Acp70A from neutrality, our data reject neutrality at Acp29AB and Acp36DE in the direction of excess replacements in interspecific comparisons.