Diversification Pattern of the HMG and SOX Family Members During Evolution

From a database containing the published HMG protein sequences, we constructed an alignment of the HMG box functional domain based on sequence identity. Due to the large number of sequences (more than 250) and the short size of this domain, several data sets were used. This analysis reveals that the HMG box superfamily can be separated into two clearly defined subfamilies: (i) the SOX/MATA/TCF family, which clusters proteins able to bind to specific DNA sequences; and (ii) the HMG/UBF family, which clusters members which bind non specifically to DNA. The appearance and diversification of these subfamilies largely predate the split between the yeast and the metazoan lineages. Particular emphasis was placed on the analysis of the SOX subfamily. For the first time our analysis clearly identified the SOX subfamily as structured in six groups of genes named SOX5/6, SRY, SOX2/3, SOX14, SOX4/22, and SOX9/18. The validity of these gene clusters is confirmed by their functional characteristics and their sequences outside the HMG box. In sharp contrast, there are only a few robust branching patterns inside the UBF/HMG family, probably because of the much more ancient diversification of this family than the diversification of the SOX family. The only consistent groups that can be detected by our analysis are HMG box 1, vertebrate HMG box 2, insect SSRP, and plant HMG. The various UBF boxes cannot be clustered together and their diversification appears to be extremely ancient, probably before the appearance of metazoans. Received: 20 July 1998 / Accepted: 19 October 1998     

Fast Evolution of Interleukin-2 in Mammals and Positive Selection in Ruminants

Interleukin-2 (IL-2) is a cytokine involved in induction and regulation of the immune response in mammals. There have been numerous reports about the search for IL-2 in species other than mammals, and recently an IL-2-like gene has been isolated in chicken. Using PCR, we searched for IL-2 gene sequences in a wide variety of mammals, including marsupials and monotremes, as well as in birds. Although we can readily amplify IL-2 gene fragments in placental mammals, no amplification was obtained in other species. This is best explained by very high substitution rates. This suggest that strategies to isolate IL-2 homologous genes outside mammals should involve functional assays, as for the chicken gene, and not hybridization-based techniques. Nonsynonymous substitution rates are especially high in ruminants, due to positive selection acting on regions important in term of structure-function. We suggest that, although globally similar, the immune response of various mammals is not identical, mainly at the level of cytokine-mediated regulations. Received: 27 July 1999 / Accepted: 15 April 2000     

The Ghost of Selection Past: Rates of Evolution and Functional Divergence of Anciently Duplicated Genes

The duplication of genes and even complete genomes may be a prerequisite for major evolutionary transitions and the origin of evolutionary novelties. However, the evolutionary mechanisms of gene evolution and the origin of novel gene functions after gene duplication have been a subject of many debates. Recently, we compiled 26 groups of orthologous genes, which included one gene from human, mouse, and chicken, one or two genes from the tetraploid Xenopus and two genes from zebrafish. Comparative analysis and mapping data showed that these pairs of zebrafish genes were probably produced during a fish-specific genome duplication that occurred between 300 and 450 Mya, before the teleost radiation (Taylor et al. 2001). As discussed here, many of these retained duplicated genes code for DNA binding proteins. Different models have been developed to explain the retention of duplicated genes and in particular the subfunctionalization model of Force et al. (1999) could explain why so many developmental control genes have been retained. Other models are harder to reconcile with this particular set of duplicated genes. Most genes seem to have been subjected to strong purifying selection, keeping properties such as charge and polarity the same in both duplicates, although some evidence was found for positive Darwinian selection, in particular for Hox genes. However, since only the cumulative pattern of nucleotide substitutions can be studied, clear indications of positive Darwinian selection or neutrality may be hard to find for such anciently duplicated genes. Nevertheless, an increase in evolutionary rate in about half of the duplicated genes seems to suggest that either positive Darwinian selection has occurred or that functional constraints have been relaxed at one point in time during functional divergence. Received: 4 January 2001 / Accepted: 29 March 2001     

Molecular Characterization and Evolution of the Amylase Multigene Family of Drosophila ananassae

Drosophila ananassae is known to produce numerous alpha-amylase variants. We have cloned seven different Amy genes in an African strain homozygous for the AMY1,2,3,4 electrophoretic pattern. These genes are organized as two main clusters: the first one contains three intronless copies on the 2L chromosome arm, two of which are tandemly arranged. The other cluster, on the 3L arm, contains two intron-bearing copies. The amylase variants AMY1 and AMY2 have been assigned to the intronless cluster, and AMY3 and AMY4 to the second one. The divergence of coding sequences between clusters is moderate (6.1% in amino acids), but the flanking regions are very different, which could explain their differential regulation. Within each cluster, coding and noncoding regions are conserved. Two very divergent genes were also cloned, both on chromosome 3L, but very distant from each other and from the other genes. One is the Amyrel homologous (41% divergent), the second one, Amyc1 (21.6% divergent) is unknown outside the D. ananassae subgroup. These two genes have unknown functions. Received: 30 May 2000 / Accepted: 17 July 2000     

Isolation and Characterization of Five Actin cDNAs from the Cestode Diphyllobothrium dendriticum: A Phylogenetic Study of the Multigene Family

Five cDNAs (pDidact2–pDidact6), representing different actin genes, were isolated from a Diphyllobothrium dendriticum cDNA library, and the DNA as well as the putative amino acid sequences were determined. The corresponding Didact2 and Didact4 genes code for peptides 376 amino acids long, with molecular weights 41,772 and 41,744 Da, respectively, while the deduced Didact3 protein is 377 amino acids long and weighs 41,912 Da. The pDidact5 and -6 cDNAs lack nucleotides corresponding to three to six amino acids at the amino-terminus. Two of the five cDNAs contain the conventional AATAAA as the putative polyadenylation signal, one has the common variant ATTAAA, whereas the hexanucleotide AATAGA is found 15 and 18 nucleotides, respectively, upstream of the poly(A) site in two of the cDNAs. Phylogenetic studies including 102 actin protein sequences revealed that there are at least four different types of cestode actins. In this study three of these types were found to be expressed in the adult D. dendriticum tapeworm. Structurally the cestode actin groupings differ from each other to an extent seen only among the metazoan actins between the vertebrate muscle and cytoplasmic isoforms. In the phylogenetic trees constructed, cestode actins were seen to map to two different regions, one on the border of the metazoan actins and the other within this group. It is, however, difficult to say whether the cestode actins branched off early in the metazoan evolution or if this position in the phylogenetic tree only reflects upon differences in evolutionary rate. Received: 19 June 1996 / Accepted: 20 August 1996     

The Effect of Branch Length Variation on the Selection of Models of Molecular Evolution

Models of sequence evolution play an important role in molecular evolutionary studies. The use of inappropriate models of evolution may bias the results of the analysis and lead to erroneous conclusions. Several procedures for selecting the best-fit model of evolution for the data at hand have been proposed, like the likelihood ratio test (LRT) and the Akaike (AIC) and Bayesian (BIC) information criteria. The relative performance of these model-selecting algorithms has not yet been studied under a range of different model trees. In this study, the influence of branch length variation upon model selection is characterized. This is done by simulating sequence alignments under a known model of nucleotide substitution, and recording how often this true model is recovered by different model-fitting strategies. Results of this study agree with previous simulations and suggest that model selection is reasonably accurate. However, different model selection methods showed distinct levels of accuracy. Some LRT approaches showed better performance than the AIC or BIC information criteria. Within the LRTs, model selection is affected by the complexity of the initial model selected for the comparisons, and only slightly by the order in which different parameters are added to the model. A specific hierarchy of LRTs, which starts from a simple model of evolution, performed overall better than other possible LRT hierarchies, or than the AIC or BIC. Received: 2 October 2000 / Accepted: 4 January 2001     

The Molecular Clock Runs at Different Rates Among Closely Related Members of a Gene Family

The serum albumin gene family is composed of four members that have arisen by a series of duplications from a common ancestor. From sequence differences between members of the gene family, we infer that a gene duplication some 580 Myr ago gave rise to the vitamin D–binding protein (DBP) gene and a second lineage, which reduplicated about 295 Myr ago to give the albumin (ALB) gene and a common precursor to α-fetoprotein (AFP) and α-albumin (ALF). This precursor itself duplicated about 250 Myr ago, giving rise to the youngest family members, AFP and ALF. It should be possible to correlate these dates with the phylogenetic distribution of members of the gene family among different species. All four genes are found in mammals, but AFP and ALF are not found in amphibia, which diverged from reptiles about 360 Myr ago, before the divergence of the AFP-ALF progenitor from albumin. Although individual family members display an approximate clock-like evolution, there are significant deviations—the rates of divergence for AFP differ by a factor of 7, the rates for ALB differ by a factor of 2.1. Since the progenitor of this gene family itself arose by triplication of a smaller gene, the rates of evolution of individual domains were also calculated and were shown to vary within and between family members. The great variation in the rates of the molecular clock raises questions concerning whether it can be used to infer evolutionary time from contemporary sequence differences. Received: 28 February 1995 / Accepted: 6 October 1997     

Evolution of Rh Blood Group Genes Have Experienced Gene Conversions and Positive Selection

There are two tightly linked loci (D and CE) for the human Rh blood group. Their gene products are membrane proteins having 12 transmembrane domains and form a complex with Rh50 glycoprotein on erythrocytes. We constructed phylogenetic networks of human and nonhuman primate Rh genes, and the network patterns suggested the occurrences of gene conversions. We therefore used a modified site-by-site reconstruction method by using two assumed gene trees and detected 9 or 11 converted regions. After eliminating the effect of gene conversions, we estimated numbers of nonsynonymous and synonymous substitutions for each branch of both trees. Whichever gene tree we selected the branch connecting hominoids and Old World monkeys showed significantly higher nonsynonymous than synonymous substitutions, an indication of positive selection. Many other branches also showed higher nonsynonymous than synonymous substitutions; this suggests that the Rh genes have experienced some kind of positive selection. Received: 16 March 1999 / Accepted: 17 June 1999     

Episodic Evolution of Protein Hormones in Mammals

Pituitary growth hormone (GH) and prolactin have been shown previously to display a pattern of evolution in which episodes of rapid change are imposed on a low underlying basal rate (near-stasis). This study was designed to explore whether a similar pattern is seen in the evolution of other protein hormones in mammals. Seven protein hormones were examined (with the common α-subunit of the glycoprotein hormones providing an additional polypeptide for analysis)—those for which sequences from at least four eutherian orders are available with a suitable non-eutherian outgroup. Six of these (GH, prolactin, insulin, parathyroid hormone, glycoprotein hormone α-subunit, and luteinizing hormone β-subunit) showed markedly variable evolutionary rates in each case with a pattern of a slow basal rate and bursts of rapid change, the precise positions of the bursts varying from protein to protein. Two protein hormones (follicle-stimulating hormone β-subunit and thyroid-stimulating hormone β-subunit) showed no significant rate variation. Based on the sequences currently available, and pooling data from all eight proteins, the phase of slow basal change occupied about 85% of the sampled evolutionary time, but most evolutionary change (about 62% of the substitutions accepted) occurred during the episodes of rapid change. It is concluded that, in mammals at least, a pattern of prolonged periods of near-stasis with occasional episodes of rapid change provides a better model of evolutionary change for protein hormones than the one of constant evolutionary rates that is commonly favored. The mechanisms underlying this episodic evolution are not yet clear, and it may be that they vary from one group to another; in some cases, positive selection appears to underlie bursts of rapid change. Where gene duplication is associated with a period of accelerated evolution this often occurs at the end rather than the beginning of the episode. To what extent the type of pattern seen for protein hormones can be extended to other proteins remains to be established. Received: 10 October 2000 / Accepted: 18 December 2000     

Molecular Evolution of the Myeloperoxidase Family

Animal myeloperoxidase and its relatives constitute a diverse protein family, which includes myeloperoxidase, eosinophil peroxidase, thyroid peroxidase, salivary peroxidase, lactoperoxidase, ovoperoxidase, peroxidasin, peroxinectin, cyclooxygenase, and others. The members of this protein family share a catalytic domain of about 500 amino acid residues in length, although some members have distinctive mosaic structures. To investigate the evolution of the protein family, we performed a comparative analysis of its members, using the amino acid sequences and the coordinate data available today. The results obtained in this study are as follows: (1) 60 amino acid sequences belonging to this family were collected by database searching. We found a new member of the myeloperoxidase family derived from a bacterium. This is the first report of a bacterial member of this family. (2) An unrooted phylogenetic tree of the family was constructed according to the alignment. Considering the branching pattern in the obtained phylogenetic tree, together with the mosaic features in the primary structures, 60 members of the myeloperoxidase family were classified into 16 subfamilies. (3) We found two molecular features that distinguish cyclooxygenase from the other members of the protein family. (4) Several structurally deviated segments were identified by a structural comparison between cyclooxygenase and myeloperoxidase. Some of the segments seemed to be associated with the functional and/or structural differences between the enzymes. Received: 25 January 2000 / Accepted: 19 July 2000     

Conflict Among Individual Mitochondrial Proteins in Resolving the Phylogeny of Eutherian Orders

The phylogenetic relationship among primates, ferungulates (artiodactyls + cetaceans + perissodactyls + carnivores), and rodents was examined using proteins encoded by the H strand of mtDNA, with marsupials and monotremes as the outgroup. Trees estimated from individual proteins were compared in detail with the tree estimated from all 12 proteins (either concatenated or summing up log-likelihood scores for each gene). Although the overall evidence strongly suggests ((primates, ferungulates), rodents), the ND1 data clearly support another tree, ((primates, rodents), ferungulates). To clarify whether this contradiction is due to (1) a stochastic (sampling) error; (2) minor model-based errors (e.g., ignoring site rate variability), or (3) convergent and parallel evolution (specifically between either primates and rodents or ferungulates and the outgroup), the ND1 genes from many additional species of primates, rodents, other eutherian orders, and the outgroup (marsupials + monotremes) were sequenced. The phylogenetic analyses were extensive and aimed to eliminate the following artifacts as possible causes of the aberrant result: base composition biases, unequal site substitution rates, or the cumulative effects of both. Neither more sophisticated evolutionary analyses nor the addition of species changed the previous conclusion. That is, the statistical support for grouping rodents and primates to the exclusion of all other taxa fluctuates upward or downward in quite a tight range centered near 95% confidence. These results and a site-by-site examination of the sequences clearly suggest that convergent or parallel evolution has occurred in ND1 between primates and rodents and/or between ferungulates and the outgroup. While the primate/rodent grouping is strange, ND1 also throws some interesting light on the relationships of some eutherian orders, marsupials, and montremes. In these parts of the tree, ND1 shows no apparent tendency for unexplained convergences. Received: 5 December 1997 / Accepted: 24 February 1998     

Phylogenetic Analysis of Vertebrate Lactate Dehydrogenase (LDH) Multigene Families

In this paper we analyzed 49 lactate dehydrogenase (LDH) sequences, mostly from vertebrates. The amino acid sequence differences were found to be larger for a human–killifish pair than a human–lamprey pair. This indicates that some protein sequence convergence may occur and reduce the sequence differences in distantly related species. We also examined transitions and transversions separately for several species pairs and found that the transitions tend to be saturated in the distantly related species pair, while transversions are increasing. We conclude that transversions maintain a conservative rate through the evolutionary time. Kimura's two-parameter model for multiple-hit correction on transversions only was used to derive a distance measure and then construct a neighbor-joining (NJ) tree. Three findings were revealed from the NJ tree: (i) the branching order of the tree is consistent with the common branch pattern of major vertebrates; (ii) Ldh-A and Ldh-B genes were duplicated near the origin of vertebrates; and (iii) Ldh-C and Ldh-A in mammals were produced by an independent gene duplication in early mammalian history. Furthermore, a relative rate test showed that mammalian Ldh-C evolved more rapidly than mammalian Ldh-A. Under a two-rate model, this duplication event was calibrated to be approximately 247 million years ago (mya), dating back to the Triassic period. Other gene duplication events were also discovered in Xenopus, the first duplication occurring approximately 60–70 mya in both Ldh-A and Ldh-B, followed by another recent gene duplication event, approximately 20 mya, in Ldh-B. Received: 5 October 2001 / Accepted: 24 October 2001     

Molecular Evolution of Cytochrome c Oxidase Subunit IV: Evidence for Positive Selection in Simian Primates

Cytochrome c oxidase (COX) is a multi-subunit enzyme complex that catalyzes the final step of electron transfer through the respiratory chain on the mitochondrial inner membrane. Up to 13 subunits encoded by both the mitochondrial (subunits I, II, and III) and nuclear genomes occur in eukaryotic organisms ranging from yeast to human. Previously, we observed a high number of amino acid replacements in the human COX IV subunit compared to mouse, rat, and cow orthologues. Here we examined COX IV evolution in the two groups of anthropoid primates, the catarrhines (hominoids, cercopithecoids) and platyrrhines (ceboids), as well as one prosimian primate (lorisiform), by sequencing PCR-amplified portions of functional COX4 genes from genomic DNAs. Phylogenetic analysis of the COX4 sequence data revealed that accelerated nonsynonymous substitution rates were evident in the early evolution of both catarrhines and, to a lesser extent, platyrrhines. These accelerated rates were followed later by decelerated rates, suggesting that positive selection for adaptive amino acid replacement became purifying selection, preserving replacements that had occurred. The evidence for positive selection was especially pronounced along the catarrhine lineage to hominoids in which the nonsynonymous rate was first faster than the synonymous rate, then later much slower. The rates of three types of ``neutral DNA' nucleotide substitutions (synonymous substitutions, pseudogene nucleotide substitutions, and intron nucleotide substitutions) are similar and are consistent with previous observations of a slower rate of such substitutions in the nuclear genomes of hominoids than in the nuclear genomes of other primate and mammalian lineages. Received: 22 May 1996 / Accepted: 24 November 1996     

Classification and Phylogenetic Analysis of the cAMP-Dependent Protein Kinase Regulatory Subunit Family

The members of the PKA regulatory subunit family (PKA-R family) were analyzed by multiple sequence alignment and clustering based on phylogenetic tree construction. According to the phylogenetic trees generated from multiple sequence alignment of the complete sequences, the PKA-R family was divided into four subfamilies (types I to IV). Members of each subfamily were exclusively from animals (types I and II), fungi (type III), and alveolates (type IV). Application of the same methodology to the cAMP-binding domains, and subsequently to the region delimited by β-strands 6 and 7 of the crystal structures of bovine RIα and rat RIIβ (the phosphate-binding cassette; PBC), proved that this highly conserved region was enough to classify unequivocally the members of the PKA-R family. A single signature sequence, F–G–E–[LIV]–A–L–[LIMV]–x(3)–[PV]–R–[ANQV]–A, corresponding to the PBC was identified which is characteristic of the PKA-R family and is sufficient to distinguish it from other members of the cyclic nucleotide-binding protein superfamily. Specific determinants for the A and B domains of each R-subunit type were also identified. Conserved residues defining the signature motif are important for interaction with cAMP or for positioning the residues that directly interact with cAMP. Conversely, residues that define subfamilies or domain types are not conserved and are mostly located on the loop that connects α-helix B′ and β strand 7. Received: 2 November 2000/Accepted: 14 June 2001     

Positive Selection Scanning Reveals Decoupling of Enzymatic Activities of Carbamoyl Phosphate Synthetase in Helicobacter pylori

In an effort to detect factors which may be under positive selection, a survey for such genes in two pathogenic strains of Helicobacter pylori (J99 and 26695) was performed. Based on an analysis of synonymous and nonsynonymous substitutions, we identified 19 candidate genes under positive selection. A search for homologues with known crystallographic structures revealed Escherichia coli carbomoyl phosphate synthetase as a homologue of H. pylori carbamoyl phosphate synthetase. Carbamoyl phosphate synthetase as isolated from E. coli is a heterodimeric enzyme that possesses two different but coupled functionalities and is involved in the first committed step in the separate biosynthetic pathways for arginine and pyrimidine nucleotides. In this study, we provide evidence indicating that one of these functionalities appears to be under selective pressure. Reports from previously published site-directed mutagenesis studies point to a decoupling of amidotransferase and synthetase activities. Implications of these findings for a metabolic enzyme under positive selection are discussed in terms of the mechanisms of H. pylori pathogenesis. Received: 11 June 2001 / Accepted: 12 September 2001     

Sequence Variation in the Gene Encoding the Nonstructural 3 Protein of Hepatitis C Virus: Evidence for Immune Selection

To determine whether the persistent nature of hepatitis C infection is related to the emergence of antigenic variants driven by immune selection, we examined the sequence heterogeneity in a portion of the hepatitis C virus (HCV) nonstructural 3 (NS3) gene of a patient infected over the course of more than 2 years. By PCR amplification, cloning, and sequencing, we observed several variable and conserved regions in the NS3 segment of the HCV genome. All variable regions had higher ratios of nonsynonymous/synonymous mutations and encompassed immunodominant epitopes, and their locations were not essential to maintain the known function of HCV RNA helicase. In contrast, the regions that are critical for HCV RNA helicase activity were found to be conserved with lower heterogeneity or lower ratios of nonsynonymous/synonymous mutations, and none except one of these regions was encoded within immunodominant epitopes. Our results are consistent with immune selection of viral variants at the epitope and molecular levels that may enable HCV to evade host defenses over time. Plotting the relatedness of sequence variants revealed a star topology suggesting that a wild-type HCV sequence is maintained, unlike HIV. Received: 2 November 2000 / Accepted: 1 October 2001     

Episodic Evolution of Protein Hormones: Molecular Evolution of Pituitary Prolactin

Previous studies have shown that pituitary growth hormone displays an episodic pattern of evolution, with a slow underlying evolutionary rate and occasional sustained bursts of rapid change. The present study establishes that pituitary prolactin shows a similar pattern. During much of tetrapod evolution the sequence of prolactin has been strongly conserved, showing a slow basal rate of change (approx 0.27 × 10⁹ substitutions/amino acid site/year). This rate has increased substantially (∼12- to 38-fold) on at least four occasions during eutherian evolution, during the evolution of primates, artiodactyls, rodents, and elephants. That these increases are real and not a consequence of inadvertant comparison of paralogous genes is shown (for at least the first three groups) by the fact that they are confined to mature protein coding sequence and not apparent in sequences coding for signal peptides or when synonymous substitutions are examined. Sequences of teleost prolactins differ markedly from those of tetrapods and lungfish, but during the course of teleost evolution the rate of change of prolactin has been less variable than that of growth hormone. It is concluded that the evolutionary pattern seen for prolactin shows long periods of near-stasis interrupted by occasional bursts of rapid change, resembling the pattern seen for growth hormone in general but not in detail. The most likely basis for these bursts appears to be adaptive evolution though the biological changes involved are relatively small. Received: 31 August 1999 / Accepted: 9 February 2000     

Evolution of the PP2C Family in Caenorhabditis: Rapid Divergence of the Sex-Determining Protein FEM-2

To investigate the causes and functional significance of rapid sex-determining protein evolution we compared three Caenorhabditis elegans genes encoding members of the protein phosphatase 2C (PP2C) family with their orthologs from another Caenorhabditis species (strain CB5161). One of the genes encodes FEM-2, a sex-determining protein, while the others have no known sex-determining role. FEM-2's PP2C domain was found to be more diverged than the other PP2C domains, supporting the notion that sex-determining proteins are subjected to selective pressures that allow for or cause rapid divergence. Comparison of the positions of amino acid substitutions in FEM-2 with a solved three-dimensional structure suggests that the catalytic face of the protein is highly conserved among C. elegans, CB5161, and another closely related species C. briggsae. However, the non-conserved regions of FEM-2 cannot be said to lack functional importance, since fem-2 transgenes from the other species were unable to rescue the germ-line defect caused by a C. elegans fem-2 mutation. To test whether fem-2 functions as a sex-determining gene in the other Caenorhabditis species we used RNA-mediated interference (RNAi). fem-2 (RNAi) in C. elegans and C. briggsae caused germ-line feminization, but had no noticeable effect in CB5161. Thus the function of fem-2 in CB5161 remains uncertain. Received: 11 April 2001 / Accepted: 6 August 2001     

Molecular Phylogenetic Characterization of Streptomyces Protease Inhibitor Family

We previously found that proteinaceous protease inhibitors homologous to Streptomyces subtilisin inhibitor (SSI) are widely produced by various Streptomyces species, and we designated them ``SSI-like proteins' (Taguchi S, Kikuchi H, Suzuki M, Kojima S, Terabe M, Miura K, Nakase T, Momose H [1993] Appl Environ Microbiol 59:4338–4341). In this study, SSI-like proteins from five strains of the genus Streptoverticillium were purified and sequenced, and molecular phylogenetic trees were constructed on the basis of the determined amino acid sequences together with those determined previously for Streptomyces species. The phylogenetic trees showed that SSI-like proteins from Streptoverticillium species are phylogenetically included in Streptomyces SSI-like proteins but form a monophyletic group as a distinct lineage within the Streptomyces proteins. This provides an alternative phylogenetic framework to the previous one based on partial small ribosomal RNA sequences, and it may indicate that the phylogenetic affiliation of the genus Streptoverticillium should be revised. The phylogenetic trees also suggested that SSI-like proteins possessing arginine or methionine at the P1 site, the major reactive center site toward target proteases, arose multiple times on independent lineages from ancestral proteins possessing lysine at the P1 site. Most of the codon changes at the P1 site inferred to have occurred during the evolution of SSI-like proteins are consistent with those inferred from the extremely high G + C content of Streptomyces genomes. The inferred minimum number of amino acid replacements at the P1 site was nearly equal to the average number for all the variable sites. It thus appears that positive Darwinian selection, which has been postulated to account for accelerated rates of amino acid replacement at the major reaction center site of mammalian protease inhibitors, may not have dictated the evolution of the bacterial SSI-like proteins. Received: 23 August 1996 / Accepted: 20 November 1996