Evolution of eutherian cytochrome c oxidase subunit II: heterogeneous rates of protein evolution and altered interaction with cytochrome c

Cytochrome c oxidase subunit II (COII), encoded by the mitochondrial genome, exhibits one of the most heterogeneous rates of amino acid replacement among placental mammals. Moreover, it has been demonstrated that cytochrome c oxidase has undergone a structural change in higher primates which has altered its physical interaction with cytochrome c. We collected a large data set of COII sequences from several orders of mammals with emphasis on primates, rodents, and artiodactyls. Using phylogenetic hypotheses based on data independent of the COII gene, we demonstrated that an increased number of amino acid replacements are concentrated among higher primates. Incorporating approximate divergence dates derived from the fossil record, we find that most of the change occurred independently along the New World monkey lineage and in a rapid burst before apes and Old World monkeys diverged. There is some evidence that Old World monkeys have undergone a faster rate of nonsynonymous substitution than have apes. Rates of substitution at four-fold degenerate sites in primates are relatively homogeneous, indicating that the rate heterogeneity is restricted to nondegenerate sites. Excluding the rate acceleration mentioned above, primates, rodents, and artiodactyls have remarkably similar nonsynonymous replacement rates. A different pattern is observed for transversions at four-fold degenerate sites, for which rodents exhibit a higher rate of replacement than do primates and artiodactyls. Finally, we hypothesize specific amino acid replacements which may account for much of the structural difference in cytochrome c oxidase between higher primates and other mammals.      

Different rates of substitution may produce different phylogenies of the eutherian mammals

Summary In an attempt to resolve some points of branching order in the phylogeny of the eutherian mammals, a phylogenetic analysis of 26 nuclear and 6 mitochondrial genes was undertaken using a maximum likelihood method on a constant rate stochastic model of molecular evolution. Seventeen of the nuclear genes gave a primates/artiodactyls grouping highest support whereas three of the mitochondrial genes found a rodents/artiodactyls grouping to be best supported. The primates/rodents grouping was never the best supported. On the assumption that rodents are indeed an outgroup to primates and artiodactyls and that the latter taxa diverged 70 million years ago, an estimation was made, for each gene, of the time of divergence of the rodent lineage. In most cases such estimates were beyond the limits set by present interpretations of the paleontological record as were many estimates of the divergence time of mouse and rat. These results suggest that, although there is locus variation, the divergent position of the rodent lineage may be an artifact of an elevated rate of nucleotide substitution in this order.     

The biochemical phylogeny of guinea-pigs and gundis, and the paraphyly of the order rodentia.

1. Molecular data indicate that caviomorphs (guinea-pig-like rodents) and myomorphs (rat-like rodents) are not monophyletic. 2. Rather, the evolutionary lineage leading to the guinea-pig may have branched off prior to the divergence among myomorphs, lagomorphs, primates, chiropterans, artiodactyls, and carnivores. 3. Thus, the guinea-pig lineage probably represents an ancient eutherian lineage, and should be conferred an independent ordinal status. 4. The gundis (Ctenodactylidae) also seem to have branched off before the divergence among myomorphs, primates, and artiodactyls, but after the divergence of the guinea-pig. 5. Therefore, the order Rodentia as defined at the present time is in all probability a paraphyletic group devoid of taxonomic validity. 6. Previous claims pertaining to large differences in the rate of molecular evolution between guinea-pigs and myomorphs may have been exaggerated in many cases as a result of the erroneous phylogenetic position attributed to the guinea-pig. 7. The average rate of amino acid replacement in the guinea-pig is comparable to that in the rat and the mouse. 8. Protein-coding genes of myomorphs and caviomorphs evole, on average, about two times faster than their counterparts in gundis and humans.     

Evolutionary conservation of 5' upstream sequence of nine genes between human and great apes

Nucleotide sequences of nine 5' upstream gene regions for human, chimpanzee, gorilla, and orangutan were determined. We estimated nucleotide differences (d) for each region between human and great apes. The overall d was 0.027 (ranged from 0.004 to 0.052). Rates of nucleotide substitution were estimated by using d and divergence times of human, chimpanzee, gorilla, and orangutan. The overall rate of nucleotide substitution between human and other hominoids was estimated to be 0.52-0.85 x 10(-9). This rate in 5' upstream regions was lower than that of synonymous sites, suggesting that 5' upstream regions have evolved under some functional constraints. Because lower rates have been reported for coding sequences in primates compared to rodents, we also estimated the rate (1.17-1.76 x 10(-9)) of nucleotide substitutions for the corresponding 5' upstream regions in rodents (mouse/rat comparison). Thus the primate rate was lower than rodent rate also for the 5' upstream regions.     

Accelerated rate of gene gain and loss in primates

The molecular changes responsible for the evolution of modern humans have primarily been discussed in terms of individual nucleotide substitutions in regulatory or protein coding sequences. However, rates of nucleotide substitution are slowed in primates, and thus humans and chimpanzees are highly similar at the nucleotide level. We find that a third source of molecular evolution, gene gain and loss, is accelerated in primates relative to other mammals. Using a novel method that allows estimation of rate heterogeneity among lineages, we find that the rate of gene turnover in humans is more than 2.5 times faster than in other mammals and may be due to both mutational and selective forces. By reconciling the gene trees for all of the gene families included in the analysis, we are able to independently verify the numbers of inferred duplications. We also use two methods based on the genome assembly of rhesus macaque to further verify our results. Our analyses identify several gene families that have expanded or contracted more rapidly than is expected even after accounting for an overall rate acceleration in primates, including brain-related families that have more than doubled in size in humans. Many of the families showing large expansions also show evidence for positive selection on their nucleotide sequences, suggesting that selection has been important in shaping copy-number differences among mammals. These findings may help explain why humans and chimpanzees show high similarity between orthologous nucleotides yet great morphological and behavioral differences.     

Genetic aspects in hominid evolution

Genomic comparison between apes and humans have made important contributions to our understanding of human evolution. The modern period of karyological comparisons between humans and other primates began about forty years ago and has been marked by a series of technical revolutions. In the 1960s pioneering genetic and chromosomal comparisons of human and great apes suggested, as had Darwin a century before, that our closest relative were the African apes. Early immunological analyses placed human/apes divergence at about five million year ago. Acceptance of man’s late divergence from the African apes was delayed by the scarcity of paleontological evidence coupled with a fallacious Asiatic origin hypothesis of the hominoids. Chromosome banding techniques in the seventies and high resolution methods in the eighties allowed a detailed comparison of the chromosomes between closely related primates and reinforced the hypothesis of an African origin for humans. It was clearly shown that humans were more closely related to African apes than to the orang-utan. The last decade has seen a vigorous integration of molecular and cytogenetic. This powerful combination promises to be quite fruitful because chromosomes can be compared directly at the DNA level. Fluorescentin situ hybridisation (FISH), chromosome painting, is a colourful technique for establishing chromosomal homology between species. Results obtained by FISH over the last ten years have resolved the cytogenetic problem of the homology between humans, apes, hylobates and Old World monkeys and defined the chromosomal syntenies and major translocations involved in the genome evolution of higher primates.     

Fixation of deleterious mutations at critical positions in human proteins

Deleterious mutations associated with human diseases are predominantly found in conserved positions and positions that are essential for the structure and/or function of proteins. However, these mutations are purged from the human population over time and prevented from being fixed. Contrary to this belief, here I show that high proportions of deleterious amino acid changing mutations are fixed at positions critical for the structure and/or function of proteins. Similarly, a high rate of fixation of deleterious mutations was observed in slow-evolving amino acid positions of human proteins. The fraction of deleterious substitutions was found to be two times higher in relatively conserved amino acid positions than in highly variable positions. This study also found fixation of a much higher proportion of radical amino acid changes in primates compared with rodents and artiodactyls in slow-evolving positions. Previous studies observed a higher proportion of nonsynonymous substitutions in humans compared with other mammals, which was taken as indirect evidence for the fixation of deleterious mutations in humans. However, the results of this investigation provide direct evidence for this prediction by suggesting that the excess nonsynonymous mutations fixed in humans are indeed deleterious in nature. Furthermore, these results suggest that studies on disease-associated mutations should consider that a significant fraction of such deleterious mutations has already been fixed in the human genome, and thus, the effects of new mutations at those amino acid positions may not necessarily be deleterious and might even result in reversion to benign phenotypes.     

Cytochromeb gene of marine mammals: Phylogeny and evolution

The DNA sequences of the mitochondrial cytochromeb gene of marine mammals (Cetacea, Pinnipedia, Sirenia) were compared with cytochromeb genes of terrestrial mammals including the semiaquatic hippopotamus. The comparison included 28 sequences, representing 22 families and 10 orders. The dugong (order Sirenia) sequence associated with that of the elephant, supporting the Tethytheria clade. The fin whale and dolphin (order Cetacea) sequences are more closely related to those of the artiodactyls, and the comparison suggests that the hippopotamus may be the extant artiodactyl species that is most closely related to the cetaceans. The seal sequence may be more closely related to those of artiodactyls, cetaceans, and perissodactyls than to tethytheres, rodents, lagomorphs, or primates. The cytochromeb proteins of mammals do not evolve at a uniform rate. Human and elephant cytochromeb amino acid sequences were found to evolve the most rapidly, while those of myomorph rodents evolved slowest. The cytochromeb of marine mammals evolves at an intermediate rate. The pattern of amino acid substitutions in marine mammals is similar to that of terrestrial mammals.     

Evolution of the Sry genes

Existing DNA sequence data on the Sry gene, the mammalian sex- determining locus in the Y chromosome, were analyzed for primates, rodents, and bovids. In all three taxonomic groups, the terminal sequences evolved faster than the HMG (high mobility group) boxes, and this applies both to synonymous (Ks) and nonsynonymous (Ka) nucleotide substitutions. Similar intragenic correlation between synonymous and nonsynonymous substitution rates was not found either in other mammalian genes that contain a conservative box (Sox, Msx) or in the MADS-box genes of plants. The rate of nonsynonymous substitutions exceeds significantly that of synonymous substitutions in the terminal Sry sequences of apes. We did not find good support for the hypothesis that the high evolutionary rate of Sry would be associated with a promiscuous mating system.      

Primate phylogeny: molecular evidence for a pongid clade excluding humans and a prosimian clade containing tarsiers

Unbiased readings of fossils are well known to contradict some of the popular molecular groupings among primates,particularly with regard to great apes and tarsiers.The molecular methodologies today are however flawed as they are based on a mistaken theoretical interpretation of the genetic equidistance phenomenon that originally started the field.An improved molecular method the ’slow clock’ was here developed based on the Maximum Genetic Diversity hypothesis,a more complete account of the unified changes in genotypes and phenotypes.The method makes use of only slow evolving sequences and requires no uncertain assumptions or mathematical corrections and hence is able to give definitive results.The findings indicate that humans are genetically more distant to orangutans than African apes are and separated from the pongid clade ～17.6 million years ago.Also,tarsiers are genetically closer to lorises than simian primates are.Finally,the fossil times for the radiation of mammals at the K/T boundary and for the Eutheria-Metatheria split in the Early Cretaceous were independently confirmed from molecular dating calibrated using the fossil split times of gorilla-orangutan,mouse-rat,and opossum-kangaroo.Therefore,the re-established primate phylogeny indicates a remarkable unity between molecules and fossils.     

Mammalian Male Mutation Bias: Impacts of Generation Time and Regional Variation in Substitution Rates

In mammals, males undergo a greater number of germline cell divisions compared with females. Thus, the male germline accumulates more DNA replication errors, which result in male mutation bias—a higher mutation rate for males than for females. The phenomenon of male mutation bias has been investigated mostly for rodents and primates, however, it has not been studied in detail for other mammalian orders. Here we sequenced and analyzed five introns of three genes (DBX/DBY, UTX/UTY, and ZFX/ZFY) homologous between X and Y chromosomes in several species of perissodactyls (horses and rhinos) and of primates. Male mutation bias was evident: substitution rate was higher for a Y chromosome intron than for its X chromosome homologue for all five intron pairs studied. Substitution rates varied regionally among introns sequenced on the same chromosome and this variation influenced male mutation bias inferred from each intron pair. Interestingly, we observed a positive correlation in substitution rates between homologous X and homologous Y introns as well as between orthologous primate and perissodactyl introns. The male-to-female mutation rate ratio estimated from concatenated sequences of five perissodactyl introns was 3.88 (95% CI = 2.90–6.07). Using the data generated here and estimates available in the literature, we compared male mutation bias among several mammalian orders. We conclude that male mutation bias is significantly higher for organisms with long generation times (primates, perissodactyls, and felids) than for organisms with short generation times (e.g., rodents) since the former undergo a greater number of male germline cell divisions. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Deborah Charlesworth]     

On the Scaling of Tooth Size in Mammals

We must establish the allometric regularities of functionalscalingin interspecific, "mouse-to elephant" plots in orderto provide criteria for the recognition of special adaptationsunrelated to the requirements of size. The qualitative literaturesuggests that postcanine tooth areas of herbivorous mammalsshould increase with positive allometry in such plots. Thispositive allometry might reflect the demands of metabolism orthe ecological strategies of large vs. small hervivores embodiedin Levins' concept of environmental grain. Plots of postcaninearea vs. body size display the expected postive allometry inall groups studied: hystricomorph rodents, suine artiodactyls(pigs, peccaries, and hippos), cervoid artiodactyls (deer, s.l),and four groups of primates considered separately (lemuroids,ceboids, cercopithecoids, and great apes). Sketchy data foraustralopithecines also indicate positive allometry and therelatively larger cheek teeth of robust forms may only reflecttheir larger body size and not the dietary differences so oftenadvocated. Phyletic dwarfs of large herbivores display negativeallometry (relatively larger cheek teeth in dwarfs) in oppositionto the interspecific trend.     

A comparison of primate, carnivoran and rodent limb bone cross-sectional properties: are primates really unique?

The cross-sectional properties of mammalian limb bones provide an important source of information about their loading history and locomotor adaptations. It has been suggested, for instance, that the cross-sectional strength of primate limb bones differs from that of other mammals as a consequence of living in a complex arboreal environment (Kimura, 1991, 1995). In order to test this hypothesis more rigorously, we have investigated cross-sectional properties in samples of humeri and femora of 71 primate species, 30 carnivorans and 59 rodents. Primates differ from carnivorans and rodents in having limb bones with greater cross-sectional strength than mammals of similar mass. This might imply that primates have stronger bones than carnivorans and rodents. However, primates also have longer proximal limb bones than other mammals. When cross-sectional dimensions are regressed against bone length, primates appear to have more gracile bones than other mammals. These two seemingly contradictory findings can be reconciled by recognizing that most limb bones experience bending as a predominant loading regime. After regressing cross-sectional strength against the product of body mass and bone length, a product which should be proportional to the bending moments applied to the limb, primates are found to overlap considerably with carnivorans and rodents. Consequently, primate humeri and femora are similar to those of nonprimates in their resistance to bending. Comparisons between arboreal and terrestrial species within the orders show that the bones of arboreal carnivorans have greater cross-sectional properties than those of terrestrial carnivorans, thus supporting Kimura's general notion. However, no differences were found between arboreal and terrestrial rodents. Among primates, the only significant difference was in humeral bending rigidity, which is higher in the terrestrial species. In summary, arboreal and terrestrial species do not show consistent differences in long bone reinforcement, and Kimura's conclusions must be modified to take into account the interaction of bone length and cross-sectional geometry.     

HIGHER-LEVEL RELATIONSHIPS OF THE RECENT EUTHERIAN ORDERS: MORPHOLOGICAL EVIDENCE

Abstract— Diverse morphological evidence from both living and fossil taxa suggests several higher-level groupings of the Recent orders of eutherian mammals. The strongest hypotheses closely relate rodents and lagomorphs within Glires, proboscideans and sirenians within Tethytheria, hyracoids and tethytheres within Paenungulata, chiropterans and dermopterans, and pholidotans and edentates. Somewhat weaker evidence supports groupings of Glires with macroscelideans, primates and tree-shrews with bats and flying lemurs (Archonta), and all Eutheria excluding pangolins and edentates (Epitheria). There is some tenuous evidence for the monophyly of all modern ungulate orders (including cetaceans), and for the division between artiodactyls and other ungulates. Rather than providing only a confusing and unresolved picture of higher eutherian relationships, comparative morphology and paleontology offer some compelling hypotheses that comprise a framework for studies of macromolecular traits.     

Contrasting rates of nucleotide substitution in the X-Linked and Y-Linked zinc finger genes

We have sequenced the entire exon (1,180 bp) encoding the zinc finger domain of the X-linked and Y-linked zinc finger genes (ZFX and ZFY, respectively) in the orangutan, the baboon, the squirrel monkey, and the rat; a total of 9,442 by were sequenced. The ratio of the rates of synonymous substitution in the ZFY and ZFX genes is estimated to be 2.1 in primates. This is close to the ratio of 2.3 estimated from primate ZFY and ZFX intron sequences and supports the view that the male-to-female ratio of mutation rate in humans is considerably higher than 1 but not extremely large. The ratio of synonymous substitution rates in ZFY and ZFX is estimated to be 1.3 in the rat lineage but 4.2 in the mouse lineage. The former is close to the estimate (1.4) from introns. The much higher ratio in the mouse lineage (not statistically significant) might have arisen from relaxation of selective constraints. The synonymous divergence between mouse and rat ZFX is considerably lower than that between mouse and rat autosomal genes, agreeing with previous observations and providing some evidence for stronger selective constraints on synonymous changes in X-linked genes than in autosomal genes. At the protein level ZFX has been highly conserved in all placental mammals studied while ZFY has been well conserved in primates and foxes but has evolved rapidly in mice and rats, possibly due to relaxation of functional constraints as a result of the development of X-inactivation of ZFX in rodents. The long persistence of the ZFY-ZFX gene pair in mammals provides some insight into the process of degeneration of Y-linked genes.Correspondence to: W.-H. Li     

The molecular taxonomy and evolution of the guinea pig.

On the basis of 18 protein sequences totaling 2,413 aligned amino acid sites, it is suggested that the guinea pigs and the myomorphs (rat-like rodents) are not monophyletic. Rather, the evolutionary lineage leading to the guinea pig seems to have branched off prior to the divergence among myomorphs, lagomorphs, primates, chiropterans, artiodactyls, and carnivores. It is suggested therefore that the Caviomorpha (guinea pig-like rodents) and possibly the Hystricomorpha (porcupine-like rodents) should be elevated in taxonomic rank and conferred an ordinal status distinct from the Rodentia. This suggestion calls for a reevaluation of the morphological evolution of guinea pigs and further molecular studies on the possibility of paraphyly of the order Rodentia. If the monophyly of rodents holds, it must be concluded that the pattern of molecular evolution in many guinea pig genes has been extremely unusual and that the causes for this pattern should be sought. It is also suggested that claims of large differences in the rate of molecular evolution between guinea pigs and myomorphs may have been exaggerated in many cases as a result of an erroneous phylogenetic position for the guinea pig. The average rate of amino acid replacement in the guinea pig seems to be comparable to that in the rat and the mouse. However, the data indicate that myomorph and caviomorph genes evolve, on average, about two times faster than their human counterparts. Finally, our analysis provides evidence against the hypothesis that the gundi (an African rodent) represents the most ancient rodent lineage.     

Synonymous and nonsynonymous rate variation in nuclear genes of mammals

A maximum likelihood approach was used to estimate the synonymous and nonsynonymous substitution rates in 48 nuclear genes from primates, artiodactyls, and rodents. A codon-substitution model was assumed, which accounts for the genetic code structure, transition/transversion bias, and base frequency biases at codon positions. Likelihood ratio tests were applied to test the constancy of nonsynonymous to synonymous rate ratios among branches (evolutionary lineages). It is found that at 22 of the 48 nuclear loci examined, the nonsynonymous/synonymous rate ratio varies significantly across branches of the tree. The result provides strong evidence against a strictly neutral model of molecular evolution. Our likelihood estimates of synonymous and nonsynonymous rates differ considerably from previous results obtained from approximate pairwise sequence comparisons. The differences between the methods are explored by detailed analyses of data from several genes. Transition/transversion rate bias and codon frequency biases are found to have significant effects on the estimation of synonymous and nonsynonymous rates, and approximate methods do not adequately account for those factors. The likelihood approach is preferable, even for pairwise sequence comparison, because more-realistic models about the mutation and substitution processes can be incorporated in the analysis. Received: 17 May 1997 / Accepted: 28 September 1997     

Higher rates of amino acid substitution in rodents than in humans.

An analysis of 54 protein sequences from humans and rodents (mice or rats), with the chicken as an outgroup, indicates that, from the common ancestor of primates and rodents, 35 of the proteins have evolved faster in the lineage to mouse or rat (rodent lineage) whereas only 12 proteins have evolved faster in the lineage to humans (human lineage). The average rate of amino acid substitution is significantly faster in the rodent lineage than in the human lineage. In addition, the average rate of insertion/deletion is also faster in rodents than in humans and there is a positive correlation between the rate of amino acid substitution and the rate of insertion/deletion in a protein sequence.