DNA evolution under weak selection

Some DNA data show patterns of variation not expected under the neutral theory. Here, the independent multicodon (IMC) model, a nearly neutral mutation model assuming no interaction among codons, was studied when population size changes using computer simulation. Patterns of variation expected under the model were investigated using statistics for the neutrality tests. The average dispersion index is more than one when population size changes slowly but it never becomes large. The diversity at linked silent site decreases when the strength of selection is intermediate and the reduction is larger when population size changes slowly. Tajima's (1989. Genetics 123, 585-595) D is generally negative. Rejections by the Tajima's test occur more frequently if population size changes quickly but the effect of selection is confounded with the size change itself in this case. If we apply the test of McDonald and Kreitman (1991. Nature 351, 652-654), the rejection is always in the direction of excess replacement polymorphisms. The rejection probability decreases as the rate of population size changes decreases. These results show that the predictions of the IMC model are consistent with the pattern observed in mitochondrial DNA data but not consistent with some data of nuclear DNA. Interaction among codons or variable selection would be necessary to explain such cases.     

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     

On the Evolution of Redundancy in Genetic Codes

We simulate a deterministic population genetic model for the coevolution of genetic codes and protein-coding genes. We use very simple assumptions about translation, mutation, and protein fitness to calculate mutation-selection equilibria of codon frequencies and fitness in a large asexual population with a given genetic code. We then compute the fitnesses of altered genetic codes that compete to invade the population by translating its genes with higher fitness. Codes and genes coevolve in a succession of stages, alternating between genetic equilibration and code invasion, from an initial wholly ambiguous coding state to a diversified frozen coding state. Our simulations almost always resulted in partially redundant frozen genetic codes. Also, the range of simulated physicochemical properties among encoded amino acids in frozen codes was always less than maximal. These results did not require the assumption of historical constraints on the number and type of amino acids available to codes nor on the complexity of proteins, stereochemical constraints on the translational apparatus, nor mechanistic constraints on genetic code change. Both the extent and timing of amino-acid diversification in genetic codes were strongly affected by the message mutation rate and strength of missense selection. Our results suggest that various omnipresent phenomena that distribute codons over sites with different selective requirements—such as the persistence of nonsynonymous mutations at equilibrium, the positive selection of the same codon in different types of sites, and translational ambiguity—predispose the evolution of redundancy and of reduced amino acid diversity in genetic codes. Received: 21 December 2000 / Accepted: 12 March 2001     

Evaluation of Sequence Variation and Selection in the Bindin Locus of the Red Sea Urchin, Strongylocentrotus franciscanus

Recent evidence suggests that gamete recognition proteins may be subjected to directed evolutionary pressure that enhances sequence variability. We evaluated whether diversity enhancing selection is operating on a marine invertebrate fertilization protein by examining the intraspecific DNA sequence variation of a 273-base pair region located at the 5′ end of the sperm bindin locus in 134 adult red sea urchins (Strongylocentrotus franciscanus). Bindin is a sperm recognition protein that mediates species-specific gamete interactions in sea urchins. The region of the bindin locus examined was found to be polymorphic with 14 alleles. Mean pairwise comparison of the 14 alleles indicates moderate sequence diversity (p-distance = 1.06). No evidence of diversity enhancing selection was found. It was not possible to reject the null hypothesis that the sequence variation observed in S. franciscanus bindin is a result of neutral evolution. Statistical evaluation of expected proportions of replacement and silent nucleotide substitutions, observed versus expected proportions of radical replacement substitutions, and conformance to the McDonald and Kreitman test of neutral evolution all indicate that random mutation followed by genetic drift created the polymorphisms observed in bindin. Observed frequencies were also highly similar to results expected for a neutrally evolving locus, suggesting that the polymorphism observed in the 5′ region of S. franciscanus bindin is a result of neutral evolution. Received: 19 June 1998 / Accepted: 2 August 2000     

Why are translationally sub-optimal synonymous codons used in Escherichia coli?

Natural selection favors certain synonymous codons which aid translation in Escherichia coli, yet codons not favored by translational selection persist. We use the frequency distributions of synonymous polymorphisms to test three hypotheses for the existence of translationally sub-optimal codons: (1) selection is a relatively weak force, so there is a balance between mutation, selection, and drift; (2) at some sites there is no selection on codon usage, so some synonymous sites are unaffected by translational selection; and (3) translationally sub-optimal codons are favored by alternative selection pressures at certain synonymous sites. We find that when all the data is considered, model 1 is supported and both models 2 and 3 are rejected as sole explanations for the existence of translationally sub-optimal codons. However, we find evidence in favor of both models 2 and 3 when the data is partitioned between groups of amino acids and between regions of the genes. Thus, all three mechanisms appear to contribute to the existence of translationally sub-optimal codons in E. coli. Received: 18 July 2000 / Accepted: 17 April 2001     

Evolutionary Lability of Context-Dependent Codon Bias in Bacteria

In bacteria, synonymous codon usage can be considerably affected by base composition at neighboring sites. Such context-dependent biases may be caused by either selection against specific nucleotide motifs or context-dependent mutation biases. Here we consider the evolutionary conservation of context-dependent codon bias across 11 completely sequenced bacterial genomes. In particular, we focus on two contextual biases previously identified in Escherichia coli; the avoidance of out-of-frame stop codons and AGG motifs. By identifying homologues of E. coli genes, we also investigate the effect of gene expression level in Haemophilus influenzae and Mycoplasma genitalium. We find that while context-dependent codon biases are widespread in bacteria, few are conserved across all species considered. Avoidance of out-of-frame stop codons does not apply to all stop codons or amino acids in E. coli, does not hold for different species, does not increase with gene expression level, and is not relaxed in Mycoplasma spp., in which the canonical stop codon, TGA, is recognized as tryptophan. Avoidance of AGG motifs shows some evolutionary conservation and increases with gene expression level in E. coli, suggestive of the action of selection, but the cause of the bias differs between species. These results demonstrate that strong context-dependent forces, both selective and mutational, operate on synonymous codon usage but that these differ considerably between genomes. Received: 6 May 1999 / Accepted: 29 October 1999     

Expected Relationship Between the Silent Substitution Rate and the GC Content: Implications for the Evolution of Isochores

The relationship between the silent substitution rate (K _s) and the GC content along the genome is a focal point of the debate about the origin of the isochore structure in vertebrates. Recent estimation of the silent substitution rate showed a positive correlation between K _s and GC content, in contradiction with the predictions of both the regional mutation bias model and the selection or biased gene conversion model. The aim of this paper is to help resolve this contradiction between theoretical studies and data. We analyzed the relationship between K _s and GC content under (1) uniform mutation bias, (2) a regional mutation bias, and (3) mutation bias and selection. We report that an increase in K _s with GC content is expected under mutation bias because of either nonequilibrium of the isochore structure or an increasing mutation rate from AT toward GC nucleotides in GC-richer isochores. We show by simulations that CpG deamination tends to increase the mutation rate with GC content in a regional mutation bias model. We also demonstrate that the relationship between K _s and GC under the selectionist or biased gene conversion model is positive under weak selection if the mutation selection equilibrium GC frequency is less than 0.5. Received: 28 March 2001 / Accepted: 16 May 2001     

The Evolution of Codon Preferences in Drosophila: A Maximum-Likelihood Approach to Parameter Estimation and Hypothesis Testing

Synonymous codon usage in related species may differ as a result of variation in mutation biases, differences in the overall strength and efficiency of selection, and shifts in codon preference—the selective hierarchy of codons within and between amino acids. We have developed a maximum-likelihood method to employ explicit population genetic models to analyze the evolution of parameters determining codon usage. The method is applied to twofold degenerate amino acids in 50 orthologous genes from D. melanogaster and D. virilis. We find that D. virilis has significantly reduced selection on codon usage for all amino acids, but the data are incompatible with a simple model in which there is a single difference in the long-term N _e, or overall strength of selection, between the two species, indicating shifts in codon preference. The strength of selection acting on codon usage in D. melanogaster is estimated to be |N _e s|≈ 0.4 for most CT-ending twofold degenerate amino acids, but 1.7 times greater for cysteine and 1.4 times greater for AG-ending codons. In D. virilis, the strength of selection acting on codon usage for most amino acids is only half that acting in D. melanogaster but is considerably greater than half for cysteine, perhaps indicating the dual selection pressures of translational efficiency and accuracy. Selection coefficients in orthologues are highly correlated (ρ= 0.46), but a number of genes deviate significantly from this relationship. Received: 20 December 1998 / Accepted: 17 February 1999     

Male-Biased Mutation Rates Revealed from Z and W Chromosome-Linked ATP Synthase α-Subunit (ATP5A1) Sequences in Birds

Whether the mutation rate differs between sexes has been a matter of discussion for years. Molecular analyses of mammals have indicated that males mutate more often than females, as manifested by the faster rate of neutral sequence evolution on the Y chromosome than on the X chromosome. However, these observations can as well be interpreted as specific reduction of the X chromosome mutation rate, which would be adaptive because of reducing the number of slightly deleterious recessive mutations exposed in hemizygote males. Recently, data from birds have suggested that vertebrate mutation rates may indeed be male-biased. In birds, females are the heterogametic sex (ZW), and analyses of the Z-linked CHD1Z gene have shown that it evolves faster than its W-linked and thus female-specific homologue, CHD1W. We have now studied the second avian gene known to exist in a copy on the nonrecombining regions of both the Z and the W chromosome, viz., the ATP synthase α-subunit (ATP5A1). In independent comparisons of three pairs of bird species from divergent lineages, intron sequences of the Z-linked copy (ATP5A1Z) were consistently found to evolve faster than the W-linked copy (ATP5A1W). From these data we calculated male-to-female mutation rate ratios (α) of 1.8, 2.3, and 5.0 in Galliform, Anseriform, and Ciconiiform lineages, respectively. Therefore, this study provides independent support for a male-biased mutation rate in birds. Received: 15 July 1999 / Accepted: 5 January 2000     

The Impact of Message Mutation on the Fitness of a Genetic Code

The Standard Genetic Code is organized such that similar codons encode similar amino acids. One explanation suggested that the Standard Code is the result of natural selection to reduce the fitness ``load' that derives from the mutation and mistranslation of protein-coding genes. We review the arguments against the mutational load-minimizing hypothesis and argue that they need to be reassessed. We review recent analyses of the organization of the Standard Code and conclude that under cautious interpretation they support the mutational load-minimizing hypothesis. We then present a deterministic asexual model with which we study the mode of selection for load minimization. In this model, individual fitness is determined by a protein phenotype resulting from the translation of a mutable set of protein-coding genes. We show that an equilibrium fitness may be associated with a population with the same genetic code and that genetic codes that assign similar codons to similar amino acids have a higher fitness. We also show that the number of mutant codons in each individual at equilibrium, which determines the strength of selection for load minimization, reflects a long-term evolutionary balance between mutations in messages and selection on proteins, rather than the number of mutations that occur in a single generation, as has been assumed by previous authors. We thereby establish that selection for mutational load minimization acts at the level of an individual in a single generation. We conclude with comments on the shortcomings and advantages of load minimization over other hypotheses for the origin of the Standard Code. Received: 4 April 2001 / Accepted: 22 October 2001     

Fluctuating Mutation Bias and the Evolution of Base Composition in Drosophila

The idea that the pattern of point mutation in Drosophila has remained constant during the evolution of the genus has recently been challenged. A study of the nucleotide composition focused on the Drosophila saltans group has evidenced unsuspected nucleotide composition differences among lineages. Compositional differences are associated with an accelerated rate of amino acid replacement in functionally less constrained regions. Here we reassess this issue from a different perspective. Adopting a maximum-likelihood estimation approach, we focus on the different predictions that mutation and selection make about the nonsynonymous-to-synonymous rate ratio. We investigate two gene regions, alcohol dehydrogenase (Adh) and xanthine dehydrogenase (Xdh), using a balanced data set that comprises representatives from the melangaster, obscura, saltans, and willistoni groups. We also consider representatives of the Hawaiian picture-winged group. These Hawaiian species are known to have experienced repeated bottlenecks and are included as a reference for comparison. Our results confirm patterns previously detected. The branch ancestral to the fast-evolving willistoni/saltans lineage, where most of the change in GC content has occurred, exhibits an excess of synonymous substitutions. The shift in mutation bias has affected the extent of the rate variation among sites in Xdh. Received: 4 May 1999 / Accepted: 26 July 1999     

A Phylogenetic Perspective on Sequence Evolution in Microsatellite Loci

We examined the evolution of the repeat regions of three noncoding microsatellite loci in 58 species of the Polistinae, a subfamily of wasps that diverged over 140 million years ago. A phylogenetic approach allows two new kinds of approaches to studying microsatellite evolution: character mapping and comparative analysis. The basic repeat structure of the loci was highly conserved, but was often punctuated with imperfections that appear to be phylogenetically informative. Repeat numbers evolved more rapidly than other changes in the repeat region. Changes in number of repeats among species seem consistent with the stepwise mutation model, which is based on slippage during replication as the main source of mutations. Changes in repeat numbers can occur even when there are very few tandem repeats but longer repeats, especially perfect repeats led to greater rates of evolutionary change. Species phylogenetically closer to the one from which we identified the loci had longer stretches of uninterrupted repeats and more different motifs, but not longer total repeat regions. The number of perfect repeats increased more often than it decreased. However, there was no evidence that some species have consistently greater numbers of repeats across loci than other species have, once ascertainment bias is eliminated. We also found no evidence for a population size effect posited by one form of the directionality hypothesis. Overall, phylogenetic variation in repeat regions can be explained by adding neutral evolution to what is already known about the mutation process. The life cycle of microsatellites appears to reflect a balance between growth by slippage and degradation by an essentially irreversible accumulation of imperfections. Received: 13 April 1999 / Accepted: 8 September 1999     

Codon Usage in Plastid Genes Is Correlated with Context, Position Within the Gene, and Amino Acid Content

Highly expressed plastid genes display codon adaptation, which is defined as a bias toward a set of codons which are complementary to abundant tRNAs. This type of adaptation is similar to what is observed in highly expressed Escherichia coli genes and is probably the result of selection to increase translation efficiency. In the current work, the codon adaptation of plastid genes is studied with regard to three specific features that have been observed in E. coli and which may influence translation efficiency. These features are (1) a relatively low codon adaptation at the 5′ end of highly expressed genes, (2) an influence of neighboring codons on codon usage at a particular site (codon context), and (3) a correlation between the level of codon adaptation of a gene and its amino acid content. All three features are found in plastid genes. First, highly expressed plastid genes have a noticeable decrease in codon adaptation over the first 10–20 codons. Second, for the twofold degenerate NNY codon groups, highly expressed genes have an overall bias toward the NNC codon, but this is not observed when the 3′ neighboring base is a G. At these sites highly expressed genes are biased toward NNT instead of NNC. Third, plastid genes that have higher codon adaptations also tend to have an increased usage of amino acids with a high G + C content at the first two codon positions and GNN codons in particular. The correlation between codon adaptation and amino acid content exists separately for both cytosolic and membrane proteins and is not related to any obvious functional property. It is suggested that at certain sites selection discriminates between nonsynonymous codons based on translational, not functional, differences, with the result that the amino acid sequence of highly expressed proteins is partially influenced by selection for increased translation efficiency. Received: 21 July 1999 / Accepted: 5 November 1999     

A Survey of the Molecular Evolutionary Dynamics of Twenty-Five Multigene Families from Four Grass Taxa

We surveyed the molecular evolutionary characteristics of 25 plant gene families, with the goal of better understanding general processes in plant gene family evolution. The survey was based on 247 GenBank sequences representing four grass species (maize, rice, wheat, and barley). For each gene family, orthology and paralogy relationships were uncertain. Recognizing this uncertainty, we characterized the molecular evolution of each gene family in four ways. First, we calculated the ratio of nonsynonymous to synonymous substitutions (d _N/d _S) both on branches of gene phylogenies and across codons. Our results indicated that the d _N/d _S ratio was statistically heterogeneous across branches in 17 of 25 (68%) gene families. The vast majority of d _N/d _S estimates were <<1.0, suggestive of selective constraint on amino acid replacements, and no estimates were >1.0, either across phylogenetic lineages or across codons. Second, we tested separately for nonsynonymous and synonymous molecular clocks. Sixty-eight percent of gene families rejected a nonsynonymous molecular clock, and 52% of gene families rejected a synonymous molecular clock. Thus, most gene families in this study deviated from clock-like evolution at either synonymous or nonsynonymous sites. Third, we calculated the effective number of codons and the proportion of G+C synonymous sites for each sequence in each gene family. One or both quantities vary significantly within 18 of 25 gene families. Finally, we tested for gene conversion, and only six gene families provided evidence of gene conversion events. Altogether, evolution for these 25 gene families is marked by selective constraint that varies among gene family members, a lack of molecular clock at both synonymous and nonsynonymous sites, and substantial variation in codon usage. Received: 25 May 2000 / Accepted: 16 October 2000     

Variable Immune-Driven Natural Selection in the Attachment (G) Glycoprotein of Respiratory Syncytial Virus (RSV)

A maximum-likelihood analysis of selection pressures acting on the attachment (G) glycoprotein gene of respiratory syncytial virus (RSV) from humans (HRSV) and bovines (BRSV) is presented. Six positively selected sites were identified in both group A and group B of HRSV, although only one site was common between them, while no positively selected sites were detected in BRSV. All positively selected sites were located within the ectodomain of the G protein and showed some association with positions of immunoglobulin (Ig) epitopes and sites of O-glycosylation. These results suggest that immune (antibody)-driven natural selection is an important determinant of RSV evolution and that this selection pressure differs among strains. The passage histories of RSV strains were also shown to affect the distribution of positively selected sites, particularly in HRSV B, and should be considered whenever retrospective analysis of adaptive evolution is undertaken. Received: 15 August 2000 / Accepted: 2 November 2000     

Selection and Methionine Accumulation in the Fat Body Protein 2 Gene (FBP2), a Duplicate of the Drosophila Alcohol Dehydrogenase (ADH) Gene

The Drosophila fat body protein 2 gene (Fbp2) is an ancient duplication of the alcohol dehydrogenase gene (Adh) which encodes a protein that differs substantially from ADH in its methionine content. In D. melanogaster, there is one methionine in ADH, while there are 51 (20% of all amino acids) in FBP2. Methionine is involved in 46% of amino acid replacements when Fbp2 DNA sequences are compared between D. melanogaster and D. pseudoobscura. Methionine accumulation does not affect conserved residues of the ADH-ADH^r-FBP2 multigene family. The multigene family has evolved by replacement of mildly hydrophobic amino acids by methionine with no apparent reversion. Its short-term evolution was compared between two Drosophila species, while its long-term evolution was compared between two genera belonging respectively to acalyptrate and calyptrate Diptera, Drosophila and Sarcophaga. The pattern of nucleotide substitution was consistent with an independent accumulation of methionines at the Fbp2 locus in each lineage. Under a steady-state model, the rate of methionine accumulation was constant in the lineage leading to Drosophila, and was twice as fast as that in the calyptrate lineage. Substitution rates were consistent with a slight positive selective advantage for each methionine change in about one-half of amino acid sites in Drosophila. This shows that selection can potentially account for a large proportion of amino acid replacements in the molecular evolution of proteins. Received: 12 December 1994 / Accepted: 15 April 1996     

The Codon-Degeneracy Model of Molecular Evolution

Mitochondrial genetic codons can be categorized by four patterns of nucleotide-site degeneracy based on varying combinations of twofold- or nondegenerate sites at first codon positions and twofold- or fourfold-degenerate sites at third codon positions. Herein, a model of molecular evolution is introduced that uses these patterns to calculate expected substitution frequencies for each codon position and substitution type relative to overall number of synonymous or nonsynonymous substitutions. Regions of the pocket gopher cytochrome oxidase subunit I (COI) and cytochrome b (cyt-b) genes are analyzed using this model. Chi-square distributions are used to produce relative goodness-of-fit (GF) scores for measuring the difference between substitution frequencies predicted by the codon-degeneracy model (CDM), and frequencies inferred using a well-supported phylogenetic tree of closely related species. The GF scores for expected and observed synonymous (GF_syn= 0.429, p= 0.807) and nonsynonymous (GF_ns= 2.309, p= 0.679) substitution frequencies resulted in a failure to reject the CDM as a null hypothesis for the molecular evolution of COI and cyt-b in pocket gophers. Alternative tree topologies and calculations of transition bias for these data result in higher GF scores. Received: 25 March 1999 / Accepted: 17 September 1999     

Synonymous Codon Usage, Accuracy of Translation, and Gene Length in Caenorhabditis elegans

In many unicellular organisms, invertebrates, and plants, synonymous codon usage biases result from a coadaptation between codon usage and tRNAs abundance to optimize the efficiency of protein synthesis. However, it remains unclear whether natural selection acts at the level of the speed or the accuracy of mRNAs translation. Here we show that codon usage can improve the fidelity of protein synthesis in multicellular species. As predicted by the model of selection for translational accuracy, we find that the frequency of codons optimal for translation is significantly higher at codons encoding for conserved amino acids than at codons encoding for nonconserved amino acids in 548 genes compared between Caenorhabditis elegans and Homo sapiens. Although this model predicts that codon bias correlates positively with gene length, a negative correlation between codon bias and gene length has been observed in eukaryotes. This suggests that selection for fidelity of protein synthesis is not the main factor responsible for codon biases. The relationship between codon bias and gene length remains unexplained. Exploring the differences in gene expression process in eukaryotes and prokaryotes should provide new insights to understand this key question of codon usage. Received: 18 June 2000 / Accepted: 10 November 2000     

The Genetic Code: What Is It Good For? An Analysis of the Effects of Selection Pressures on Genetic Codes

How did the ``universal' genetic code arise? Several hypotheses have been put forward, and the code has been analyzed extensively by authors looking for clues to selection pressures that might have acted during its evolution. But this approach has been ineffective. Although an impressive number of properties has been attributed to the universal code, it has been impossible to determine whether selection on any of these properties was important in the code's evolution or whether the observed properties arose as a consequence of selection on some other characteristic. Therefore we turned the question around and asked, what would a genetic code look like if it had evolved in response to various different selection pressures? To address this question, we constructed a genetic algorithm. We found first that selecting on a particular measure yields codes that are similar to each other. Second, we found that the universal code is far from minimized with respect to the effects of mutations (or translation errors) on the amino acid compositions of proteins. Finally, we found that the codes that most closely resembled real codes were those generated by selecting on aspects of the code's structure, not those generated by selecting to minimize the effects of amino acid substitutions on proteins. This suggests that the universal genetic code has been selected for a particular structure—a structure that confers an important flexibility on the evolution of genes and proteins—and that the particular assignments of amino acids to codons are secondary. Received: 29 December 1998 / Accepted: 8 July 1999     

A theoretical method for evaluating the relative importance of positive selection and neutral drift from observed base changes

To evaluate the relative importance of positive selection and neutral drift from the nucleotide base changes observed in the homologous alignment of genes, a theoretical equation of base changes is formulated by including both the influence of selection and the base substitutions due to mutations. Under the assumption that the average rate of base substitutions estimated from synonymous changes is the ``true' mutation rate applicable at all positions, this method is applied to the vertebrate globin gene family, and evaluates the departures of base change rates from the ``true' mutation rate at the first and second codon positions as a consequence of preferential selection for the conservation of important function. In addition to the strong effect of selection on the amino acid residues in the internal region mostly common to myoglobin and hemoglobin chains, the distinctive directions of selective parameter values are seen at sites on the globin surface, distinguishing the subunit contact residues of hemoglobins from the polar residues on the surface of myoglobins. Moreover, this effect of selection distinguishing between the myoglobin and hemoglobin chain genes becomes weaker in cold-blooded vertebrates, especially in fish, strongly suggesting the possibility that the clear distinction between these globins is a result of selection out of the changes regarded as neutral ones in an ancestor of vertebrates. Thus, the present method may also serve to investigate the homology of many other proteins from the aspect of molecular evolution, mainly focusing on the evolution of their biological functions. Received: 2 January 1996 / Accepted: 20 February 1997