Mitochondrial DNA sequences of primates: Tempo and mode of evolution

Summary We cloned and sequenced a segment of mitochondrial DNA from human, chimpanzee, gorilla, orangutan, and gibbon. This segment is 896 bp in length, contains the genes for three transfer RNAs and parts of two proteins, and is homologous in all 5 primates. The 5 sequences differ from one another by base substitutions at 283 positions and by a deletion of one base pair. The sequence differences range from 9 to 19% among species, in agreement with estimates from cleavage map comparisons, thus confirming that the rate of mtDNA evolution in primates is 5 to 10 times higher than in nuclear DNA. The most striking new finding to emerge from these comparisons is that transitions greatly outnumber transversions. Ninety-two percent of the differences among the most closely related species (human, chimpanzee, and gorilla) are transitions. For pairs of species with longer divergence times, the observed percentage of transitions falls until, in the case of comparisons between primates and non-primates, it reaches a value of 45. The time dependence is probably due to obliteration of the record of transitions by multiple substitutions at the same nucleotide site. This finding illustrates the importance of choosing closely related species for analysis of the evolutionary process. The remarkable bias toward transitions in mtDNA evolution necessitates the revision of equations that correct for multiple substitutions at the same site. With revised equations, we calculated the incidence of silent and replacement substitutions in the two protein-coding genes. The silent substitution rate is 4 to 6 times higher than the replacement rate, indicating strong functional constraints at replacement sites. Moreover, the silent rate for these two genes is about 10% per million years, a value 10 times higher than the silent rate for the nuclear genes studied so far. In addition, the mean substitution rate in the three mitochondrial tRNA genes is at least 100 times higher than in nuclear tRNA genes. Finally, genealogical analysis of the sequence differences supports the view that the human lineage branched off only slightly before the gorilla and chimpanzee lineages diverged and strengthens the hypothesis that humans are more related to gorillas and chimpanzees than is the orangutan.Abbreviations mtDNA mitochondrial DNA - bp base pair - URF unidentified reading frame     

Biases of the estimates of DNA divergence obtained by the restriction enzyme technique

Summary A mathematical formula for the relationship between the average number of nucleotide substitutions per site and the proportion of shared restriction sites between two homologous nucleons is developed by taking into account the unequal rates of substitution among different pairs of nucleotides. Using this formula, the possible amount of bias of the estimate of the number of nucleotide substitutions obtained by the Upholt-Nei-Li formula for restriction site data is investigated. The results obtained indicate that the bias depends upon the nucleotides in the recognition sequence of the restriction enzyme used, the unequal rates of substitution among different nucleotides, and the unequal nucleotide frequencies, but the primary factor is the unequal rates of nucleotide substitution. The amount of bias is generally larger for four-base enzymes than for six-base enzymes. However, when many restriction enzymes are used for the study of DNA divergence, the bias is unlikely to be very large unless the rate of substitution greatly varies from nucleotide to nucleotide.     

Intraspecific nucleotide sequence differences in the major noncoding region of human mitochondrial DNA.

Nucleotide sequences of the major noncoding region of human mitochondrial DNA (mtDNA) from 95 human placentas have been determined. These sequences include at least a 482-bp-long region encompassing most of the D-loop-forming region. Comparisons of these sequences with those previously determined have revealed remarkable features of nucleotide substitutions and insertion/deletion events. The nucleotide diversity among the sequences is estimated as 1.45%, which is three- to fourfold higher than the corresponding value estimated from restriction-enzyme analysis of whole mtDNA genome. A hypervariable region has also been defined. In this 14-bp region, 17 different sequences were detected. More than 97% of the base changes are transitions. A significantly nonrandom distribution of nucleotide substitutions and sequence length variations were also noted. The phylogenetic analysis indicates that diversity among the negroids is much larger than that among the caucasoids or the mongoloids. In fact, part of the negroids first diverged from other humans in the phylogenetic tree. A striking finding in the phylogenetic analysis is that the mongoloids can be separated into two distinct groups. Divergence of part of the mongoloids follows the earliest divergence of part of the negroids. The remainder of the mongoloids subsequently diverged together with the caucasoids. This observation confirmed our earlier study, which clearly demonstrated, by the restriction-enzyme analysis, existence of two distinct groups in the Japanese.     

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.     

A Method for Calibrating Molecular Clocks and Its Application to Animal Mitochondrial DNA

A generalized least-squares procedure is introduced for the calibration of molecular clocks and applied to the complete mitochondrial DNA sequences of 13 animal species. The proposed technique accounts for both nonindependence and heteroscedasticity of molecular-distance data, problems that have not been taken into to account in such analyses in the past. When sequence-identity data are transformed to account for multiple substitutions/site, the molecular divergence scales linearly with time, but with substantially more variation in the substitution rate than expected under a Poisson model. Significant levels of divergence are predicted at zero divergence time for most loci, suggesting high levels of site-specific heterozygosity among mtDNA molecules establishing in sister taxa. For nearly all loci, the baseline heterozygosity is lower and the substitution rate is higher in mammals relative to other animals. There is considerable variation in the evolutionary rate among loci but no compelling evidence that the average rate of mtDNA evolution is elevated with respect to that of nuclear DNA. Using the observed patterns of interspecific divergence, empirical estimates are derived for the mean coalescence times of organelles colonizing sister taxa.     

Variation of mitochondrial DNA in Chinook salmon Oncorhynchus tschawytscha Walbaum populations from Kamchatka

The variation in mitochondrial DNA (mtDNA) structure among Chinook Salmon Oncorhynchus tschawytscha Walbaum populations from Kamchatka was inferred from restriction length polymorphism analysis using eight restriction endonucleases. The nucleotide sequence variation in three amplified mtDNA regions was examined at seven polymorphic restriction sites in 579 fish from 13 localities. Based on the frequencies of 11 combined haplotypes and the number of nucleotide substitutions, the among- and within-population variation was estimated. The heterogeneity test showed highly significant differences among all the populations. The estimated maximum time of independent divergence of the Asian Chinook salmon populations, whose differences was about 0.02% nucleotide substitutions, did not exceed 10000-20000 years. Apparently, the retreat of the late Pleistocene glacier triggered spreading, recolonization, and formation of the present-day pattern of the species subdivision into structural components.     

Variation of mitochondrial DNA in chinook salmon <Emphasis Type="Italic">Oncorhynchus tschawytscha</Emphasis> Walbaum populations from Kamchatka

The variation in mitochondrial DNA (mtDNA) structure among chinook salmon Oncorhynchus tschawytscha Walbaum populations from Kamchatka was inferred from restriction length polymorphism analysis using eight restriction endonucleases. The nucleotide sequence variation in three amplified mtDNA regions was examined at seven polymorphic restriction sites in 579 fish from 13 localities. Based on the frequencies of 11 combined haplotypes and the number of nucleotide substitutions, the among-and within-population variation was estimated. The heterogeneity test showed highly significant differences among all the populations. The estimated maximum time of independent divergence of the asian chinook salmon populations, whose differences was about 0.02% nucleotide substitutions, did not exceed 10 000–20 000 years. Apparently, the retreat of the late Pleistocene glacier triggered spreading, recolonization, and formation of the present-day pattern of the species subdivision into structural components.     

Analysis of nucleotide substitutions of mitochondrial DNAs in Drosophila melanogaster and its sibling species

To study the rate and pattern of nucleotide substitution in mitochondrial DNA (mtDNA), we cloned and sequenced a 975-bp segment of mtDNA from Drosophila melanogaster, D. simulans, and D. mauritiana containing the genes for three transfer RNAs and parts of two protein- coding genes, ND2 and COI. Statistical analysis of synonymous substitutions revealed a predominance of transitions over transversions among the three species, a finding differing from previous results obtained from a comparison of D. melanogaster and D. yakuba. The number of transitions observed was nearly the same for each species comparison, including D. yakuba, despite the differences in divergence times. However, transversions seemed to increase steadily with increasing divergence time. By contrast, nonsynonymous substitutions in the ND2 gene showed a predominance of transversions over transitions. Most transversions were between A and T and seemed to be due to some kind of mutational bias to which the A + T-rich mtDNA of Drosophila species may be subject. The overall rate of nucleotide substitution in Drosophila mtDNA appears to be slightly faster (approximately 1.4 times) than that of the Adh gene. This contrasts with the result obtained for mammals, in which the mtDNA evolves approximately 10 times faster than single-copy nuclear DNA. We have also shown that the start codon of the COI gene is GTGA in D. simulans and GTAA in D. mauritiana. These codons are different from that of D. melanogaster (ATAA).      

Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees

Examining the pattern of nucleotide substitution for the control region of mitochondrial DNA (mtDNA) in humans and chimpanzees, we developed a new mathematical method for estimating the number of transitional and transversional substitutions per site, as well as the total number of nucleotide substitutions. In this method, excess transitions, unequal nucleotide frequencies, and variation of substitution rate among different sites are all taken into account. Application of this method to human and chimpanzee data suggested that the transition/transversion ratio for the entire control region was approximately 15 and nearly the same for the two species. The 95% confidence interval of the age of the common ancestral mtDNA was estimated to be 80,000-480,000 years in humans and 0.57-2.72 Myr in common chimpanzees.      

Sequence Evolution of Drosophila Mitochondrial DNA

We have compared nucleotide sequences of corresponding segments of the mitochondrial DNA (mtDNA) molecules of Drosophila yakuba and Drosophila melanogaster, which contain the genes for six proteins and seven tRNAs. The overall frequency of substitution between the nucleotide sequences of these protein genes is 7.2%. As was found for mtDNAs from closely related mammals, most substitutions (86%) in Drosophila mitochondrial protein genes do not result in an amino acid replacement. However, the frequencies of transitions and transversions are approximately equal in Drosophila mtDNAs, which is in contrast to the vast excess of transitions over transversions in mammalian mtDNAs. In Drosophila mtDNAs the frequency of C----T substitutions per codon in the third position is 2.5 times greater among codons of two-codon families than among codons of four-codon families; this is contrary to the hypothesis that third position silent substitutions are neutral in regard to selection. In the third position of codons of four-codon families transversions are 4.6 times more frequent than transitions and A----T substitutions account for 86% of all transversions. Ninety-four percent of all codons in the Drosophila mtDNA segments analyzed end in A or T. However, as this alone cannot account for the observed high frequency of A----T substitutions there must be either a disproportionately high rate of A----T mutation in Drosophila mtDNA or selection bias for the products of A----T mutation. --Consideration of the frequencies of interchange of AGA and AGT codons in the corresponding D. yakuba and D. melanogaster mitochondrial protein genes provides strong support for the view that AGA specifies serine in the Drosophila mitochondrial genetic code.     

Rapid Rate of Control-Region Evolution in Pacific Butterflyfishes (Chaetodontidae)

Sequence differences in the tRNA-proline (tRNA^pro) end of the mitochondrial control-region of three species of Pacific butterflyfishes accumulated 33–43 times more rapidly than did changes within the mitochondrial cytochrome b gene (cytb). Rapid evolution in this region was accompanied by strong transition/transversion bias and large variation in the probability of a DNA substitution among sites. These substitution constraints placed an absolute ceiling on the magnitude of sequence divergence that could be detected between individuals. This divergence ``ceiling' was reached rapidly and led to a decay in the relative rate of control-region/cytb b evolution. A high rate of evolution in this section of the control-region of butterflyfishes stands in marked contrast to the patterns reported in some other fish lineages. Although the mechanism underlying rate variation remains unclear, all taxa with rapid evolution in the 5′-end of the control-region showed extreme transition biases. By contrast, in taxa with slower control-region evolution, transitions accumulated at nearly the same rate as transversions. More information is needed to understand the relationship between nucleotide bias and the rate of evolution in the 5′-end of the control-region. Despite strong constraints on sequence change, phylogenetic information was preserved in the group of recently differentiated species and supported the clustering of sequences into three major mtDNA groupings. Within these groups, very similar control-region sequences were widely distributed across the Pacific Ocean and were shared between recognized species, indicating a lack of mitochondrial sequence monophyly among species. Received: 30 June 1996 / Accepted: 15 May 1997     

Mitochondrial diversity in linguistic isolates of the Alps: a reappraisal

In Stenico et al. (1996) we reported unusually high levels of mitochondrial diversity in the Alps. In particular, two communities of Ladin speakers appeared the most extreme European mitochondrial outliers at that time. Recently, it has been observed that some rare nucleotide substitutions occur repeatedly among those sequences, raising the possibility of systematic sequencing errors. No biological material was left from the previous study, and hence we had to sample new individuals from the same communities. Here, we present the HVSI sequence variation, along with haplogroup assignment based on restriction fragment length polymorphism (RFLP), in 20 Ladin speakers of Colle Santa Lucia. None of the new sequences displays substitutions at the sites viewed as problematic. However, Ladins still show high levels of mtDNA diversity, both within their community and with respect to other Europeans, and they can still be considered one of the main European mitochondrial outliers.     

Rates of nuclear and cytoplasmic mitochondrial DNA sequence divergence in mammals

Differential rates of nucleotide substitution among different gene segments and between distinct evolutionary lineages is well documented among mitochondrial genes and is likely a consequence of locus-specific selective constraints that delimit mutational divergence over evolutionary time. We compared sequence variation of 18 homologous loci (15 coding genes and 3 parts of the control region) among 10 mammalian mitochondrial DNA genomes which allowed us to describe different mitochondrial evolutionary patterns and to produce an estimation of the relative order of gene divergence. The relative rates of divergence of mitochondrial DNA genes in the family Felidae were estimated by comparing their divergence from homologous counterpart genes included in nuclear mitochondrial DNA (Numt, pronounced "new might"), a genomic fossil that represents an ancient transfer of 7.9 kb of mitochondrial DNA to the nuclear genome of an ancestral species of the domestic cat (Felis catus). Phylogenetic analyses of mitochondrial (mtDNA) sequences with multiple outgroup species were conducted to date the ancestral node common to the Numt and the cytoplasmic (Cymt) mtDNA genes and to calibrate the rate of sequence divergence of mitochondrial genes relative to nuclear homologous counterparts. By setting the fastest substitution rate as strictly mutational, an empirical "selective retardation index" is computed to quantify the sum of all constraints, selective and otherwise, that limit sequence divergence of mitochondrial gene sequences over time.      

Genomewide comparison of DNA sequences between humans and chimpanzees

A total of 8,859 DNA sequences encompassing ~1.9 million base pairs of the chimpanzee genome were sequenced and compared to corresponding human DNA sequences. Although the average sequence difference is low (1.24%), the extent of changes is markedly different among sites and types of substitutions. Whereas ~15% of all CpG sites have experienced changes between humans and chimpanzees, owing to a 23-fold excess of transitions and a 7-fold excess of transversions, substitutions at other sites vary in frequency, between 0.1% and 0.5%. If the nucleotide diversity in the common ancestral species of humans and chimpanzees is assumed to have been about fourfold higher than in contemporary humans, all possible comparisons between autosomes and X and Y chromosomes result in estimates of the ratio between male and female mutation rates of ~3. Thus, the relative time spent in the male and female germlines may be a major determinant of the overall accumulation of nucleotide substitutions. However, since the extent of divergence differs significantly among autosomes, additional unknown factors must also influence the accumulation of substitutions in the human genome.     

Use of Microsatellite Locus Flanking Regions for Phylogenetic Analysis? A Preliminary Study of Sebastes subgenera

The systematics of the species-rich genus of Sebastes rockfish has not been resolved, and is unlikely to be resolved using morphological criteria. Using an alternative approach based on DNA sequence variation, we sampled the genome for random, presumably neutral, nuclear DNA sequences, by sequencing microsatellite flanking regions of 15 Sebastes species (representing 15 of 22 extant subgenera) and Hozukius emblemarius. We aligned sequences of flanking regions of eight of the 13 loci that amplified, which presumably are homologous. In aggregate, the aligned sequences included 848 bp, 53 bp of which were polymorphic. The base changes among species included 27 transitions, 20 transversions, three multiple substitutions, and three deletions or insertions. The flanking regions at different loci had different base substitution rates. Nucleotide divergence among Sebastes species ranged from 0.0012 to 0.0216, whereas nucleotide divergence between Sebastes and Hozukius species ranged from 0.0095 to 0.0240. We used maximum parsimony, neighbor-joining, and maximum likelihood methods to examine relationships among the species. H. emblemariusformed a separate group, and five of the western Pacific Ocean species of rockfish clustered together, suggesting that they may have shared an evolutionary history distinct from many North American species. The flanking regions of some microsatellite loci contain DNA sequence variation that distinguishes rockfish species and may prove useful for clarifying relationships among rockfish, or other closely related species.     

Evolutionary distance estimation under heterogeneous substitution pattern among lineages

Most of the sophisticated methods to estimate evolutionary divergence between DNA sequences assume that the two sequences have evolved with the same pattern of nucleotide substitution after their divergence from their most recent common ancestor (homogeneity assumption). If this assumption is violated, the evolutionary distance estimated will be biased, which may result in biased estimates of divergence times and substitution rates, and may lead to erroneous branching patterns in the inferred phylogenies. Here we present a simple modification for existing distance estimation methods to relax the assumption of the substitution pattern homogeneity among lineages when analyzing DNA and protein sequences. Results from computer simulations and empirical data analyses for human and mouse genes are presented to demonstrate that the proposed modification reduces the estimation bias considerably and that the modified method performs much better than the LogDet methods, which do not require the homogeneity assumption in estimating the number of substitutions per site. We also discuss the relationship of the substitution and mutation rate estimates when the substitution pattern is not the same in the lineages leading to the two sequences compared.     

A novel mitochondrial intergenic spacer reflecting population structure of Pacific oyster

Nucleotide sequence divergence in a novel major mitochondrial DNA intergenic spacer (IGS) of Pacific oyster Crassostrea gigas was analyzed for 29 cultured individuals within the Goseong population (Korea). A total of 7 variable sites were detected within the IGS, and the relative frequency of nucleotide alteration was determined to be 1.16%;. All alterations were due to a single nucleotide substitution, and 5 transitions and 2 transversions were observed. Among 29 specimens, only 8 haplotypes could be identified, and 6 of the haplotypes were unique to particular specimens. Pairwise genetic diversity of all 8 haplotypes was calculated to be 0.412+/-0.134 from multiple sequence substitutions based on the two-parameter model. The phylogenetic tree obtained for these haplotypes according to the neighbor-joining method illustrated a single cluster of linkages, which comprised 5 haplotypes associated with 23 specimens, while the other 3 haplotypes associated with 6 specimens were scattered. The results indicate that the IGS is higher polymorphic and thus more suitable as a genetic marker for population structure analysis of Pacific oyster than the mtDNA coding regions, such as cytochrome c oxidase I and 16S ribosomal RNA genes.     

Estimation of the Transition/Transversion Rate Bias and Species Sampling

The transition/transversion (ti/tv) rate ratios are estimated by pairwise sequence comparison and joint likelihood analysis using mitochondrial cytochrome b genes of 28 primate species, representing both the Strepsirrhini (lemurs and lories) and the Anthropoidea (monkeys, apes, and humans). Pairwise comparison reveals a strong negative correlation between estimates of the ti/tv ratio and the sequence distance, even when both are corrected for multiple substitutions. The maximum-likelihood estimate of the ti/tv ratio changes with the species included in the analysis. The ti/tv bias within the lemuriform taxa is found to be as strong as in the anthropoids, in contradiction to an earlier study which sampled only one lemuriform. Simulations show the surprising result that both the pairwise correction method and the joint likelihood analysis tend to overcorrect for multiple substitutions and overestimate the ti/tv ratio, especially at low sequence divergence. The bias, however, is not large enough to account for the observed patterns. Nucleotide frequency biases, variation of substitution rates among sites, and different evolutionary dynamics at the three codon positions can be ruled out as possible causes. The likelihood-ratio test suggests that the ti/tv rate ratios may be variable among evolutionary lineages. Without any biological evidence for such a variation, however, we are left with no plausible explanations for the observed patterns other than a possible saturation effect due to the unrealistic nature of the model assumed. Received: 1 October 1997 / Accepted: 29 September 1998