Neutral evolution of the sex-determining gene transformer in Drosophila

The amino acid sequence of the transformer (tra) gene exhibits an extremely rapid rate of evolution among Drosophila species, although the gene performs a critical step in sex determination. These changes in amino acid sequence are the result of either natural selection or neutral evolution. To differentiate between selective and neutral causes of this evolutionary change, analyses of both intraspecific and interspecific patterns of molecular evolution of tra gene sequences are presented. Sequences of 31 tra alleles were obtained from Drosophila americana. Many replacement and silent nucleotide variants are present among the alleles; however, the distribution of this sequence variation is consistent with neutral evolution. Sequence evolution was also examined among six species representative of the genus Drosophila. For most lineages and most regions of the gene, both silent and replacement substitutions have accumulated in a constant, clock-like manner. In exon 3 of D. virilis and D. americana we find evidence for an elevated rate of nonsynonymous substitution, but no statistical support for a greater rate of nonsynonymous relative to synonymous substitutions. Both levels of analysis of the tra sequence suggest that, although the gene is evolving at a rapid pace, these changes are neutral in function.     

Scalable method to determine mutations that occur during adaptive evolution of Escherichia coli

Denaturing HPLC was used to determine mutations occurring during the adaptive evolution of Escherichia coli K-12. The strains were evolved over 700 generations on glycerol as the sole carbon source from a sub-optimal to an optimal growth rate. The mutations detected by direct sequencing of amplicons of the glycerol-phosphate regulon repressor (glpR) gene were a synonymous substitution Val20Val in two separately evolved strains. Non-synonymous substitutions, Val119Gly and Gly179Trp, were also observed in each of the two strains. This procedure can be scaled to determine genome-scale sequence variations that have occurred during adaptive evolution.     

Mammalian genes as molecular clocks?

In analyzing the silent nucleotide substitutions in some mammalian mitochondrial mRNA coding genes, we had found that the frequency of each of the four nucleotides in rat, mouse, and cow, but not in humans, is the same in the silent third codon position (Lanave C, Preparata G, Saccone C, Serio G (1984) J Mol Evol 20:86-93). Because our findings for these three species were compatible with a stationary Markov process for the evolution of nucleotide sequences, we applied such a model to calculate the effective evolutionary silent substitution rate (vs) and the divergence times among the species. In this paper we have analyzed the first and second codon positions in the same mammalian mitochondrial genes. We found that in the first and second codon positions the human mitochondrial genes satisfy the stationarity conditions. This has allowed us to use the stochastic model mentioned above to calculate the divergence times among mouse, rat, cow, and human. Furthermore, we have analyzed the silent substitution rate in one nuclear gene for these four mammals. We found that in this gene the effective silent substitution rate is about 3 times lower than in mitochondrial genes, and that humans are in this case stationary with respect to the other three mammals in the third codon position as well. Application of our Markov model to this latter gene yields divergence times consistent with our previous determinations.     

Mammalian genes as molecular clocks?

Summary In analyzing the silent nucleotide substitutions in some mammalian mitochondrial mRNA coding genes, we had found that the frequency of each of the four nucleotides in rat, mouse, and cow, but not in humans, is the same in the silent third codon position (Lanave C, Preparata G, Saccone C, Serio G (1984) J Mol Evol 20:86-93). Because our findings for these three species were compatible with a stationary Markov process for the evolution of nucleotide sequences, we applied such a model to calculate the effective evolutionary silent substitution rate (v_s) and the divergence times among the species. In this paper we have analyzed the first and second codon positions in the same mammalian mitochondrial genes. We found that in the first and second codon positions the human mitochondrial genes satisfy the stationarity conditions. This has allowed us to use the stochastic model mentioned above to calculate the divergence times among mouse, rat, cow, and human. Furthermore, we have analyzed the silent substitution rate in one nuclear gene for these four mammals. We found that in this gene the effective silent substitution rate is about 3 times lower than in mitochondrial genes, and that humans are in this case stationary with respect to the other three mammals in the third codon position as well. Application of our Markov model to this latter gene yields divergence times consistent with our previous determinations.     

Mammalian gene evolution: Nucleotide sequence divergence between mouse and rat

As a paradigm of mammalian gene evolution, the nature and extent of DNA sequence divergence between homologous protein-coding genes from mouse and rat have been investigated. The data set examined includes 363 genes totalling 411 kilobases, making this by far the largest comparison conducted between a single pair of species. Mouse and rat genes are on average 93.4% identical in nucleotide sequence and 93.9% identical in amino acid sequence. Individual genes vary substantially in the extent of nonsynonymous nucleotide substitution, as expected from protein evolution studies; here the variation is characterized. The extent of synonymous (or silent) substitution also varies considerably among genes, though the coefficient of variation is about four times smaller than for nonsynonymous substitutions. A small number of genes mapped to the X-chromosome have a slower rate of molecular evolution than average, as predicted if molecular evolution is male-driven. Base composition at silent sites varies from 33% to 95% G + C in different genes; mouse and rat homologues differ on average by only 1.7% in silent-site G + C, but it is shown that this is not necessarily due to any selective constraint on their base composition. Synonymous substitution rates and silent site base composition appear to be related (genes at intermediate G + C have on average higher rates), but the relationship is not as strong as in our earlier analyses. Rates of synonymous and nonsynonymous substitution are correlated, apparently because of an excess of substitutions involving adjacent pairs of nucleotides. Several factors suggest that synonymous codon usage in rodent genes is not subject to selection.     

Molecular evolution of recombination hotspots and highly recombining pseudoautosomal regions in hominoids

We examined the effects of recombination on the molecular evolution of noncoding regions in pseudoautosomal regions (PARs) and recombination hotspots in hominoids. The PAR-linked regions analyzed had on average longer branch lengths than those of the recombination hotspots. Moreover, contrary to previous observations, we found no correlation between recombination rate and silent site divergence in our data set and little change in the GC content during recent hominoid evolution. This suggests that the current rate of recombination is not a good indicator of the past rates of recombination for these highly recombining regions. Furthermore, human recombination hotspots show increased AT to GC substitutions in the human lineage, while no such pattern is detected for PAR-linked regions. Together, these observations suggest that recombination hotspots in hominoids are transient in the evolutionary time-scale. Interestingly, the 16p13.3 recombination hotspot locus violates a local molecular clock, though the locus appears to be noncoding and should evolve neutrally. We hypothesize that sudden changes in recombination rate have caused the changes in substitution rate at this locus.     

Experimental evolution of viruses: Microviridae as a model system

φX174 was developed as a model system for experimental studies of evolution because of its small genome size and ease of cultivation. It has been used extensively to address statistical questions about the dynamics of adaptive evolution. Molecular changes seen during experimental evolution of φX174 under a variety of conditions were compiled from 10 experiments comprising 58 lineages, where whole genomes were sequenced. A total of 667 substitutions was seen. Parallel evolution was rampant, with over 50 per cent of substitutions occurring at sites with three or more events. Comparisons of experimentally evolved sites to variation seen among wild phage suggest that at least some of the adaptive mechanisms seen in the laboratory are relevant to adaptation in nature. Elucidation of these mechanisms is aided by the availability of capsid and pro-capsid structures for φX174 and builds on years of genetic studies of the phage life history.     

Genomewide Patterns of Substitution in Adaptively Evolving Populations of the RNA Bacteriophage MS2

Experimental evolution of bacteriophage provides a powerful means of studying the genetics of adaptation, as every substitution contributing to adaptation can be identified and characterized. Here, I use experimental evolution of MS2, an RNA bacteriophage, to study its adaptive response to a novel environment. To this end, three lines of MS2 were adapted to rapid growth and lysis at cold temperature for a minimum of 50 phage generations and subjected to whole-genome sequencing. Using this system, I identified adaptive substitutions, monitored changes in frequency of adaptive mutations through the course of the experiment, and measured the effect on phage growth rate of each substitution. All three lines showed a substantial increase in fitness (a two- to threefold increase in growth rate) due to a modest number of substitutions (three to four). The data show some evidence that the substitutions occurring early in the experiment have larger beneficial effects than later ones, in accordance with the expected diminishing returns relationship between the fitness effects of a mutation and its order of substitution. Patterns of molecular evolution seen here—primarily a paucity of hitchhiking mutations—suggest an abundant supply of beneficial mutations in this system. Nevertheless, some beneficial mutations appear to have been lost, possibly due to accumulation of beneficial mutations on other genetic backgrounds, clonal interference, and negatively epistatic interactions with other beneficial mutations.     

Adaptive evolution after duplication of penaeidin antimicrobial peptides

Penaeidin antimicrobial peptides in penaeid shrimps are an important component of their innate immune system that provides immunity against infection caused by several gram-positive bacteria and filamentous fungal species. Despite the knowledge on the identification and characterization of these peptides in penaeid shrimps, little is known about the evolutionary pattern of these peptides and the underlying genetic mechanisms that maintain high sequence diversities in the penaeidin gene family. Based on the phylogenetic analyses and maximum likelihood-based codon substitution analyses, here we present the convincing evidence that multiple copies of penaeidins have evolved by gene duplication, and positive Darwinian selection (adaptive evolution) is the likely cause of accelerated rate of amino acid substitutions among these duplicated genes. While the average ratio of non-synonymous to synonymous substitutions (omega) for the entire coding region of both active domains is 0.9805, few codon sites showed significantly higher omega (3.73). The likelihood ratio tests that compare models incorporating positive selection (omega>1) at certain codon sites with models not incorporating positive selection (omega<1), failed to reject (p=0) the evidence of positive Darwinian selection. The rapid adaptive evolution of this gene family might be directed by the pathogens and the faster rate of amino acid substitutions in the N-terminal proline-rich and C-terminal cysteine-rich domains could be due to their direct involvement in the protection against pathogens. When the host expose to different habitats/environment an accelerated rate of amino acid substitutions in both the active domains may also be expected.     

On the rate of DNA sequence evolution inDrosophila

Summary Analysis of the rate of nucleotide substitution at silent sites inDrosophila genes reveals three main points. First, the silent rate varies (by a factor of two) among nuclear genes; it is inversely related to the degree of codon usage bias, and so selection among synonymous codons appears to constrain the rate of silent substitution in some genes. Second, mitochondrial genes may have evolved only as fast as nuclear genes with weak codon usage bias (and two times faster than nuclear genes with high codon usage bias); this is quite different from the situation in mammals where mitochondrial genes evolve approximately 5–10 times faster than nuclear genes. Third, the absolute rate of substitution at silent sites in nuclear genes inDrosophila is about three times hihger than the average silent rate in mammals.     

The McDonald-Kreitman test and slightly deleterious mutations

It is possible to estimate the proportion of substitutions that are due to adaptive evolution using the numbers of silent and nonsilent polymorphisms and substitutions in a McDonald and Kreitman-type analysis. Unfortunately, this estimate of adaptive evolution is biased downward by the segregation of slightly deleterious mutations. It has been suggested that 1 way to cope with the effects of these slightly deleterious mutations is to remove low-frequency polymorphisms from the analysis. We investigate the performance of this method theoretically. We show that although removing low-frequency polymorphisms does indeed reduce the bias in the estimate of adaptive evolution, the estimate is always downwardly biased, often to the extent that one would not be able to detect adaptive evolution, even if it existed. The method is reasonably satisfactory, only if the rate of adaptive evolution is high and the distribution of fitness effects for slightly deleterious mutations is very leptokurtic. Our analysis suggests that adaptive evolution could be quite prevalent in humans (>8%) and still not be detectable using current methodologies. Our analysis also suggests that the level of adaptive evolution has probably been underestimated, possibly substantially, in both bacteria and Drosophila.     

The rate of molecular evolution of alpha-fetoprotein approaches that of pseudogenes

We conducted the present study in an attempt to correlate function with the rate of molecular evolution for serum albumin and alpha-fetoprotein. We found a high rate of silent substitution (between 5 X 10(-9) and 7 X 10(-9)/site/year) for both the albumin and alpha-fetoprotein genes, perhaps the highest so far reported for an expressed nuclear gene. The rates of effective substitution and amino acid changes were also very high, but in contrast to silent substitutions, they are higher for alpha-fetoprotein than for albumin by approximately 70%. For alpha-fetoprotein, the rate of effective substitution (1.5 X 10(-9)/site/year) may be approaching that for nonfunctional pseudogenes (about 3 X 10(-9)/site/year). Evolutionary divergence was also estimated at the amino acid level. It was found that the rate of change of alpha-fetoprotein (55% amino acids replaced in 100 Myr) approaches that of the fastest-evolving fibrinopeptides (92% amino acids replaced in 100 Myr). This high rate may indicate that alpha-fetoprotein can tolerate a great deal of molecular variation without its function being impaired in the process. Albumin evolves at a slower rate (39% amino acids replaced in 100 Myr), although still faster than either hemoglobin (17% amino acids replaced in 100 Myr) or cytochrome c (5% amino acids replaced in 100 Myr). The slower evolutionary rate may indicate that albumin has more refined functional specifications and hence can tolerate fewer mutational changes. The latter conclusion remains, however, to be reconciled with the condition of inherited analbuminemia, where a virtually complete absence of albumin produces surprisingly few symptoms.     

Profiles of adaptation in two similar viruses.

The related bacteriophages phiX174 and G4 were adapted to the inhibitory temperature of 44 degrees and monitored for nucleotide changes throughout the genome. Phage were evolved by serial transfer at low multiplicity of infection on rapidly dividing bacteria to select genotypes with the fastest rates of reproduction. Both phage showed overall greater fitness effects per substitution during the early stages of adaptation. The fitness of phiX174 improved from -0.7 to 5.6 doublings of phage concentration per generation. Five missense mutations were observed. The earliest two mutations accounted for 85% of the ultimate fitness gain. In contrast, G4 required adaptation to the intermediate temperature of 41.5 degrees before it could be maintained at 44 degrees. Its fitness at 44 degrees increased from -2.7 to 3.2, nearly the same net gain as in phiX174, but with three times the opportunity for adaptation. Seventeen mutations were observed in G4: 14 missense, 2 silent, and 1 intergenic. The first 3 missense substitutions accounted for over half the ultimate fitness increase. Although the expected pattern of periodic selective sweeps was the most common one for both phage, some mutations were lost after becoming frequent, and long-term polymorphism was observed. This study provides the greatest detail yet in combining fitness profiles with the underlying pattern of genetic changes, and the results support recent theories on the range of fitness effects of substitutions fixed during adaptation.     

Evolution of influenza virus genes

The nucleotide sequences of the eight different influenza A virus segments (genes) were compared among 14 different subtypes. These comparisons demonstrate the presence of molecular clocks in the viral genes; they accumulated both silent and amino acid-changing substitutions at approximately constant rates with respect to time during evolution. In addition, comparison of the rates of evolution among the eight viral genes, excluding the P2 gene, revealed a rapid and roughly equal rate of silent substitution for different genes. The P2 gene exception is explained as the result of recombination (reassortment) between distantly related strains. The rate of amino acid-changing substitution differs greatly from gene to gene. The rate of silent substitution was estimated to be 1.1 X 10(-2)/site/year on the average--that is, about 2 X 10(6) times higher than eukaryotic gene equivalents, which is remarkable. Strain A/USSR/90/77 was shown to evolve with a rate that is similar to those of other strains but to behave as if replication was frozen during a certain period (Nakajima et al. 1978). The frozen period was estimated to be 25 yr on the basis of the molecular clock. A similar analysis revealed another example of frozen replication--in this case, apparently for a period of about 9 yr- -in a duck strain, A/duck/Ontario/77.      

Mitochondrial-nuclear interactions and accelerated compensatory evolution: evidence from the primate cytochrome C oxidase complex

Accelerated rates of mitochondrial protein evolution have been proposed to reflect Darwinian coadaptation for efficient energy production for mammalian flight and brain activity. However, several features of mammalian mtDNA (absence of recombination, small effective population size, and high mutation rate) promote genome degradation through the accumulation of weakly deleterious mutations. Here, we present evidence for "compensatory" adaptive substitutions in nuclear DNA- (nDNA) encoded mitochondrial proteins to prevent fitness decline in primate mitochondrial protein complexes. We show that high mutation rate and small effective population size, key features of primate mitochondrial genomes, can accelerate compensatory adaptive evolution in nDNA-encoded genes. We combine phylogenetic information and the 3D structure of the cytochrome c oxidase (COX) complex to test for accelerated compensatory changes among interacting sites. Physical interactions among mtDNA- and nDNA-encoded components are critical in COX evolution; amino acids in close physical proximity in the 3D structure show a strong tendency for correlated evolution among lineages. Only nuclear-encoded components of COX show evidence for positive selection and adaptive nDNA-encoded changes tend to follow mtDNA-encoded amino acid changes at nearby sites in the 3D structure. This bias in the temporal order of substitutions supports compensatory weak selection as a major factor in accelerated primate COX evolution.     

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     

Substitution rates in a new Silene latifolia sex-linked gene, SlssX/Y

Dioecious white campion Silene latifolia has sex chromosomal sex determination, with homogametic (XX) females and heterogametic (XY) males. This species has become popular in studies of sex chromosome evolution. However, the lack of genes isolated from the X and Y chromosomes of this species is a major obstacle for such studies. Here, I report the isolation of a new sex-linked gene, Slss, with strong homology to spermidine synthase genes of other species. The new gene has homologous intact copies on the X and Y chromosomes (SlssX and SlssY, respectively). Synonymous divergence between the SlssX and SlssY genes is 4.7%, and nonsynonymous divergence is 1.4%. Isolation of a homologous gene from nondioecious S. vulgaris provided a root to the gene tree and allowed the estimation of the silent and replacement substitution rates along the SlssX and SlssY lineages. Interestingly, the Y-linked gene has higher synonymous and nonsynonymous substitution rates. The elevated synonymous rate in the SlssY gene, compared with SlssX, confirms our previous suggestion that the S. latifolia Y chromosome has a higher mutation rate, compared with the X chromosome. When differences in silent substitution rate are taken into account, the Y-linked gene still demonstrates significantly faster accumulation of nonsynonymous substitutions, which is consistent with the theoretical prediction of relaxed purifying selection in Y-linked genes, leading to the accumulation of nonsynonymous substitutions and genetic degeneration of the Y-linked genes.     

Local changes in GC/AT substitution biases and in crossover frequencies on Drosophila chromosomes

I present here evidence of remarkable local changes in GC/AT substitution biases and in crossover frequencies on Drosophila chromosomes. The substitution pattern at 10 loci in the telomeric region of the X chromosome was studied for four species of the Drosophila melanogaster species subgroup. Drosophila orena and Drosophila erecta are clearly the most closely related species pair (the erecta complex) among the four species studied; however, the overall data at the 10 loci revealed a clear dichotomy in the silent substitution patterns between the AT-biased- substitution melanogaster and erecta lineages and the GC-biased-substitution yakuba and orena lineages, suggesting two or more independent changes in GC/AT substitution biases. More importantly, the results indicated a between- loci heterogeneity in GC/AT substitution bias in this small region independently in the yakuba and orena lineages. Indeed, silent substitutions in the orena lineage were significantly biased toward G and C at the consecutive yellow, lethal of scute, and asense loci, but they were significantly biased toward A and T at sta. The substitution bias toward G and C was centered in different areas in yakuba (significantly biased at EG:165H7.3, EG:171D11.2, and suppressor of sable). The similar silent substitution patterns in coding and noncoding regions, furthermore, suggested mutational biases as a cause of the substitution biases. On the other hand, previous study reveals that Drosophila yakuba has about 20-fold higher crossover frequencies in the telomeric region of the X chromosome than does D. melanogaster; this study revealed that the total genetic map length of the yakuba X chromosome was only about 1.5 times as large as that of melanogaster and that the map length of the X-telomeric y-sta region did not differ between Drosophila yakuba and D. erecta. Taken together, the data strongly suggested that an approximately 20- fold reduction in the X-telomeric crossover frequencies occurred in the ancestral population of D. melanogaster after the melanogaster-yakuba divergence but before the melanogaster-simulans divergence.     

Gnarled-trunk evolutionary model of influenza A virus hemagglutinin

Human influenza A viruses undergo antigenic changes with gradual accumulation of amino acid substitutions on the hemagglutinin (HA) molecule. A strong antigenic mismatch between vaccine and epidemic strains often requires the replacement of influenza vaccines worldwide. To establish a practical model enabling us to predict the future direction of the influenza virus evolution, relative distances of amino acid sequences among past epidemic strains were analyzed by multidimensional scaling (MDS). We found that human influenza viruses have evolved along a gnarled evolutionary pathway with an approximately constant curvature in the MDS-constructed 3D space. The gnarled pathway indicated that evolution on the trunk favored multiple substitutions at the same amino acid positions on HA. The constant curvature was reasonably explained by assuming that the rate of amino acid substitutions varied from one position to another according to a gamma distribution. Furthermore, we utilized the estimated parameters of the gamma distribution to predict the amino acid substitutions on HA in subsequent years. Retrospective prediction tests for 12 years from 1997 to 2009 showed that 70% of actual amino acid substitutions were correctly predicted, and that 45% of predicted amino acid substitutions have been actually observed. Although it remains unsolved how to predict the exact timing of antigenic changes, the present results suggest that our model may have the potential to recognize emerging epidemic strains.     

A maximum likelihood method for analyzing pseudogene evolution: implications for silent site evolution in humans and rodents.

We present a new likelihood method for detecting constrained evolution at synonymous sites and other forms of nonneutral evolution in putative pseudogenes. The model is applicable whenever the DNA sequence is available from a protein-coding functional gene, a pseudogene derived from the protein-coding gene, and an orthologous functional copy of the gene. Two nested likelihood ratio tests are developed to test the hypotheses that (1) the putative pseudogene has equal rates of silent and replacement substitutions; and (2) the rate of synonymous substitution in the functional gene equals the rate of substitution in the pseudogene. The method is applied to a data set containing 74 human processed-pseudogene loci, 25 mouse processed-pseudogene loci, and 22 rat processed-pseudogene loci. Using the informatics resources of the Human Genome Project, we localized 67 of the human-pseudogene pairs in the genome and estimated the GC content of a large surrounding genomic region for each. We find that, for pseudogenes deposited in GC regions similar to those of their paralogs, the assumption of equal rates of silent and replacement site evolution in the pseudogene is upheld; in these cases, the rate of silent site evolution in the functional genes is approximately 70% the rate of evolution in the pseudogene. On the other hand, for pseudogenes located in genomic regions of much lower GC than their functional gene, we see a sharp increase in the rate of silent site substitutions, leading to a large rate of rejection for the pseudogene equality likelihood ratio test.