Showing papers in "Molecular Biology and Evolution in 1986"

Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions.

Abstract: Two simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions are presented. Although they give no weights to different types of codon substitutions, these methods give essentially the same results as those obtained by Miyata and Yasunaga's and by Li et al.'s methods. Computer simulation indicates that estimates of synonymous substitutions obtained by the two methods are quite accurate unless the number of nucleotide substitutions per site is very large. It is shown that all available methods tend to give an underestimate of the number of nonsynonymous substitutions when the number is large.

A comparison of the small ribosomal RNA genes from the mitochondrial DNA of the great apes and humans: sequence, structure, evolution, and phylogenetic implications.

Abstract: Restriction endonuclease fragments produced by EcoRI/AvaI or KpnI digestion and containing the small (12S) ribosomal RNA (rRNA) genes from the mitochondrial DNAs (mtDNAs) of the common chimpanzee, pygmy chimpanzee, gorilla, and orangutan were inserted into the plasmids pBR322 or pADD1. After species verification the inserted fragments were digested with SauIIIA, subcloned into M13mp7 vectors, and sequenced. The small rRNA gene sequences were compared with each other and with the published human sequence (Anderson et al. 1981). Substitutions were detected at 118 of the 955 nucleotide positions compared. Pairwise, the sequence differences ranged from 1% (between the chimpanzee species) to 9% (comparisons involving the orangutan); the proportion that were transitions ranged from 87% to 100%. Deletions and/or additions were noted at seven locations. With respect to evolutionary sequence lability, kinetic analysis indicated the presence of at least two classes of nucleotide positions; the more labile class occurs in sequences thought to form self-complementary duplexes (stems) in the mature rRNA. The high frequency of compensating substitutions, which maintain base-pairing within these sequences, corroborates their inferred structure. Phylogenetic inferences drawn from the sequence comparisons support the notion of an approximately equidistant relationship among chimpanzees, gorilla, and man, with the orangutan much less closely related. However, inference from a shared deletion suggests that the gorilla and the chimpanzees may be more closely related to one another than they are to man.

Estimating the reliability of evolutionary trees.

Abstract: Six protein sequences from the same 11 mammalian taxa were used to estimate the accuracy and reliability of phylogenetic trees using real, rather than simulated, data. A tree comparison metric was used to measure the increase in similarity of minimal trees as larger, randomly selected subsets of nucleotide positions were taken. The ratio of the observed to the expected number of incompatibilities for each nucleotide position (character) is a good predictor of the number of changes required at that position on the minimal (most-parsimonious) tree. This allows a higher weighting of nucleotide positions that have changed more slowly and should result in the minimal length tree converging to the correct tree as more sequences are obtained. An estimate was made of the smallest subset of trees that need to be considered to include the actual historical tree for a given set of data. It was concluded that it is possible to give a reasonable estimate of the reliability of the final tree, at least when several sequences are combined. With the present data, resolving the rodent-primate-lagomorph (rabbit) trichotomy is the least certain aspect of the final tree, followed then by establishing the position of dog. In our opinion, it is unreasonable to publish an evolutionary tree derived from sequence data without giving an idea of the reliability of the tree.

Mammalian phylogeny: comparison of morphological and molecular results.

Abstract: In an attempt to resolve the “bushy” part at the root of the eutherian tree, 182 nondental morphological characters from 100 species (79 extant and 21 extinct; 98 mammalian and 2 nonmammalian) were analyzed using two maximum-parsimony tree-building algorithms. Parallel analyses of 2,258 pairwise immunodiffusion comparisons with chicken antisera on 10 1 mammalian species and of amino acid sequence data of alpha and beta hemoglobins and other published protein sequences were also carried out. The morphological and molecular phylogenies agree in depicting the infraclass Eutheria as consisting of five major clades (thus resolving part of the “bush”). Rates of evolution were also found to be similar in the two types of phylogenies.

Variation in the Intergenic Spacer of Ribosomal DNA of Wild Wheat, Triticum dicoccoides. In Israel

Abstract: The variation in the intergenic spacer of ribosomal DNA (rDNA) of tetraploid wild emmer wheat, Triticum dicoccoides, in Israel was examined in 112 individual plants representing 12 populations tested earlier for allozymic variation encoded by 50 gene loci. The variation detected by means of restriction endonucleases results in part from variation in the number of 135-bp repeats that are in tandem array in the intergenic DNA. Intergenic spacers of genes within a single locus are relatively homogeneous, but populations of T. dicoccoides display a wide spectrum of rDNA spacer-length variation. Some populations are very homogeneous, whereas others are heterogeneous. The most homogeneous population consists of plants with spacerrDNA lengths that are the most common in T. dicoccoides in Israel. The lengths of specific regions within the spacer DNA are identical for loci on different chromosomes in the most homogeneous population. The allozymic and rDNA diversities within populations are highly and significantly intercorrelated, and both allozymic and rDNA diversities are significantly correlated with and predictable in terms of climatic variables.

Neighboring base effects on substitution rates in pseudogenes.

Abstract: Substitution rates in pseudogenes can be used to estimate the frequencies of different types of mutation on the assumption that pseudogenes are not subject to selective constraints. These rates are used here to investigate the effect of neighboring bases on mutation rates. There is a marked increase in the frequency of transitions, though not of transversions, from the doublet CG. There are also some smaller effects of neighboring bases on the frequencies of transitions from adenine and thymine. The results are used to predict dinucleotide frequencies in a stretch of DNA subject to no selective constraints and to investigate the possibility of non-randomness in the usage of stop codons.

Mitochondrial DNA evolution in the genus Equus.

Abstract: Employing mitochondrial DNA (mtDNA) restriction-endonuclease maps as the basis of comparison, we have investigated the evolutionary affinities of the seven species generally recognized as the genus Equus. Individual species' cleavage maps contained an average of 60 cleavage sites for 16 enzymes, of which 29 were invariant for all species. Based on an average divergence rate of 2%/Myr, the variation between species supports a divergence of extant lineages from a common ancestor approximately 3.9 Myr before the present. Comparisons of cleavage maps between Equus przewalskii (Mongolian wild horse) and E. caballus (domestic horse) yielded estimates of nucleotide sequence divergence ranging from 0.27% to 0.41%. This range was due to intraspecific variation, which was noted only for E. caballus. For pairwise comparisons within this family, estimates of sequence divergence ranged from 0% (E. hemionus onager vs. E. h. kulan) to 7.8% (E. przewalskii vs. E. h. onager). Trees constructed according to the parsimony principle, on the basis of 31 phylogenetically informative restriction sites, indicate that the three extant zebra species represent a monophyletic group with E. grevyi and E. burchelli antiquorum diverging most recently. The phylogenetic relationships of E. africanus and E. hemionus remain enigmatic on the basis of the mtDNA analysis, although a recent divergence is unsupported.

Natural selection and the molecular clock.

Abstract: This paper concludes that the statistical properties of protein evolution are compatible with a particular model of evolution by natural selection. The argument begins with a statistical description of the molecular clock based on a Poisson process with a randomly varying tick rate. If the time scale of the change of the tick rate of the molecular clock is assumed to be much less than the average time between substitutions, then it is shown that the substitution process must be episodic, with bursts of substitutions being separated by long periods of time with no substitutions. This analysis generalizes the recent work of Gillespie (1984~). The second part of the argument shows that a simple model of evolution by natural selectionone that incorporates a changing environment, the molecular landscape, and a simple form of epistasis -exhibits dynamics that are identical to those inferred from the statistical analysis. This leads to the conclusion that natural selection is a viable explanation for protein evolution. In addition, a correction formula for multiple substitutions is given that does not require that the substitution process be a Poisson process, and some comments on the inability of the neutral allele theory to account for the dynamics of the substitution process are presented.

Evolutionary history of the hybridogenetic hybrid frog Rana esculenta as deduced from mtDNA analyses.

Abstract: mtDNA of the hybridogenetic hybrid frog Rana esculenta from Switzerland, Austria, and Poland was compared to mtDNA of the parental species R. ridibunda and R. lessonae using electrophoretic analysis of restriction enzyme fragments. Two mtDNA phenotypes, with 3.4% sequence divergence, are present in R. lessonae: type C is found in Poland, and type D is found in Switzerland. Rana ridibunda from Poland has either of two mtDNA phenotypes: type A is the typical ridibunda mtDNA, and type B is a lessonae mitochondrial genome, introgressed into R. ridibunda, that differs from type C mtDNA of R. lessonae by only 0.3%. Each of the three lessonae genomes differs from A, the typical ridibunda mtDNA, by -8%. All four types of mtDNA (A and B of R. ridibunda, C and D of R. lessonae) are found in R. esculenta. Of 62 R. esculenta from Poland, 58 had type C, three had type A, and one had type B mtDNA. All nine R. esculenta from Switzerland had type D mtDNA. All three R. esculenta from Austria, from a population in which males of R. esculenta are rare, had ridibunda mtDNA, two having type B and one having type A. Both field observations and studies of mating preference indicate that the primary hybridizations that produce R. esculenta are between R. ridibunda females and R. lessonae males; thereafter, R. esculenta lineages are usually maintained by matings of R. esculenta females with R. lessonae males. The presence of ridibunda mtDNA in the three R. esculenta sampled from Austria, its occasional presence in R. esculenta populations in Poland, and its absence from R. esculenta in Switzerland support both the direction of the original hybridization and the rarity of formation of new R. esculenta lineages. The preponderance of R. esculenta individuals with lessonae mtDNA in our samples from central Europe suggests that most lineages have gone through at least one mating between an R. lessonae female and an R. esculenta male. This reveals a greater reproductive role for R. esculenta males than their partial sterility and infrequent matings would suggest.

Evolutionary implication of heterogeneity of the nontranscribed spacer region of ribosomal DNA repeating units in various subspecies of Mus musculus.

Abstract: Genetic variability of the nontranscribed spacer (NTS) region within ribosomal DNA repeating units in the various subspecies of Mus musculus was determined. Mice belonging to several laboratory mouse strains were examined by means of Southern blot hybridization with a mouse ribosomal DNA probe. This probe encompasses the 3’ end of the 28s ribosomal RNA (rRNA) gene and the following spacer. Restriction enzyme digestions of the liver DNAs from various wild mice revealed that each of the subspecies has a unique pattern in the spacer encompassing a distance - 10 kb downstream from the ribosomal gene. These restriction patterns permit the classification of mouse subspecies and also provide insights into the origin of the laboratory mouse strains.

Distribution and conservation of mobile elements in the genus Drosophila.

Abstract: Essentially nothing is known of the origin, mode of transmission, and evolution of mobile elements within the genus Drosophila. To better understand the evolutionary history of these mobile elements, we examined the distribution and conservation of homologues to the P, I, gypsy, copia, and F elements in 34 Drosophila species from three subgenera. Probes specific for each element were prepared from D. melanogaster and hybridized to genomic DNA. Filters were washed under conditions of increasing stringency to estimate the similarity between D. melanogaster sequences and their homologues in other species. The I element homologues show the most limited distribution of all elements tested, being restricted to the melanogaster species group. The P elements are found in many members of the subgenus Sophophora but, with the notable exception of D. nasuta, are not found in the other two subgenera. Copia-, gypsy-, and F-element homologues are widespread in the genus, but their similarity to the D. melanogaster probe differs markedly between species. The distribution of copia and P elements and the conservation of the gypsy and P elements is inconsistent with a model that postulates a single ancient origin for each type of element followed by mating-dependent transmission. The data can be explained by horizontal transmission of mobile elements between reproductively isolated species.

Molecular evolution of pancreatic-type ribonucleases.

Abstract: Amino acid sequences of 39 mammalian ribonucleases have been used to construct trees by the maximum parsimony procedure. These trees are in fairly good agreement with the biological classification of the species involved. In the branching order of the six investigated eutherian mammalian orders, the edentates diverge first, followed, probably, by the primates. No definite conclusions can be drawn about the order of divergence of the perissodactyls, the rodents, and the group consisting of artiodactyls plus cetaceans. Nucleic acid sequences of part of the messenger RNAs of rat pancreatic and bovine seminal ribonuclease were compared. Both messengers have a second stop codon at position 129, which is in agreement with the addition of four residues at the C-terminus in several other ribonucleases. Turtle pancreatic ribonuclease and human angiogenin differ from each other and from the mammalian ribonucleases at 55%-70% of the amino acid positions; they share a number of structural features. Mammalian nonsecretory ribonucleases are homologous to the pancreatic ribonucleases in sequence regions where the active-site histidine residues are located.

Molecular evolution of bacteriophages: evidence of selection against the recognition sites of host restriction enzymes.

Abstract: Restriction enzymes produced by bacteria serve as a defense against invading bacteriophages, and so phages without other protection would be expected to undergo selection to eliminate recognition sites for these enzymes from their genomes. The observed frequencies of all restriction sites in the genomes of all completely sequenced DNA phages (T7, lambda, phi X174, G4, M13, f1, fd, and IKe) have been compared to expected frequencies derived from trinucleotide frequencies. Attention was focused on 6-base palindromes since they comprise the typical recognition sites for type II restriction enzymes. All of these coliphages, with the exception of lambda and G4, exhibit significant avoidance of the particular sequences that are enterobacterial restriction sites. As expected, the sequenced fraction of the genome of phi 29, a Bacillus subtilis phage, lacks Bacillus restriction sites. By contrast, the RNA phage MS2, several viruses that infect eukaryotes (EBV, adenovirus, papilloma, and SV40), and three mitochondrial genomes (human, mouse, and cow) were found not to lack restriction sites. Because the particular palindromes avoided correspond closely with the recognition sites for host enzymes and because other viruses and small genomes do not show this avoidance, it is concluded that the effect indeed results from natural selection.

Polymorphism and evolution of influenza A virus genes.

Abstract: The nucleotide sequences of four genes of the influenza A virus (nonstructural protein, matrix protein, and a few subtypes of hemagglutinin and neuraminidase) are compiled for a large number of strains isolated from various locations and years, and the evolutionary relationship of the sequences is investigated. It is shown that all of these genes or subtypes are highly polymorphic and that the polymorphic sequences (alleles) are subject to rapid turnover in the population, their average age being much less than that of higher organisms. Phylogenetic analysis suggests that most polymorphic sequences within a subtype or a gene appeared during the last 80 years and that the divergence among the subtypes of hemagglutinin genes might have occurred during the last 300 years. The high degree of polymorphism in this RNA virus is caused by an extremely high rate of mutation, estimated to be 0.01/nucleotide site/year. Despite the high rate of mutation, most influenza virus genes are apparently subject to purifying selection, and the rate of nucleotide substitution is substantially lower than the mutation rate. There is considerable variation in the substitution rate among different genes, and the rate seems to be lower in nonhuman viral strains than in human strains. The difference might be responsible for the so-called freezing effect in some viral strains.

An analysis of replacement and synonymous changes in the rodent L1 repeat family.

Abstract: L1 is a family of long interspersed repetitive sequences in mammals that includes the BamHI family in rodents and the KpnI family in primates. Previous studies have shown that L1 repeats contain a long open reading frame and that the family evolves in concert. Working with 32 rodent elements for which DNA sequence is available, we used the distribution of replacement and synonymous changes to determine which L1 lineages had been expressing their reading frame. The evidence obtained is consistent with there having been a small number of L1 genes that have been expressing a functional protein. Much of the concerted evolution in L1 is accounted for by the tendency of these functioning L1 genes to continually create nonfunctional pseudogenes by reinsertion into the genome of sequences derived from their transcripts. The gain of new pseudogenes is balanced by the loss of old pseudogenes with a half-life of 2 Myr. Therefore, most of the observed L1 repeats are at a dead end with respect to either the expression of the L1 protein or the potential to elaborate further copies of themselves. However, the turnover of L1 pseudogenes is sufficient to constitute a vast flux of sequences into and then out of the flanking regions of all cellular genes. If the presence of flanking L1 pseudogenes affects the expression of other genes in even a subtle fashion, this process should represent a major source of genetic variation. A second level of concerted evolution occurs within the functional L1 sequences in a pattern that did not meet our expectations for selfish DNA. Also, in spite of the marked suppression of replacement relative to synonymous changes in functioning L1 genes, they evolve at an overall rate accelerated to the level of their own pseudogenes.

Pinniped phylogeny enlightened by molecular hybridizations using highly repetitive DNA

Abstract: Four non-cross-hybridizing, highly repetitive DNA components of the Weddell seal were cloned and used for Southern blot hybridizations in order to clarify pinniped phylogeny. Each of the components was present and possessed the identical fragment length in all pinnipeds, representing true seals, walrus, and sea lions. Three of the four components also hybridized to fragments of the same length in all mustelids except skunk. Limited hybridization also occurred to raccoon DNA. Hybridization to DNA of other terrestrial carnivores (polar bear, dog, cat) was barely recognizable. The fourth component was pinniped specific. The results are compatible with the mustelids and pinnipeds being sister groups. The findings also suggest that the pinnipeds are monophyletic, having separated from mustelid ancestors as one lineage that later differentiated into otariids, odobenids, and phocids. The hybridizations indicated clear differences between the skunk and other mustelids.

Nucleotide sequence of the genes for tryptophan synthase in Pseudomonas aeruginosa.

Abstract: We have determined the DNA sequence of the two adjacent genes for the a and p chains of tryptophan synthase in Pseudomonas aeruginosa, along with 34 S-flanking and 799 3’-flanking base pairs. The gene order is trpBA as predicted from earlier genetic studies, and the two cistrons overlap by 4 bp; a ribosome binding site for the second gene is evident in the coding sequence of the first gene. We have also determined the location of three large deletions eliminating portions of each gene. A detailed comparison of the deduced P. aeruginosa amino acid sequence with those published for E. coli, Bacillus subtilis, and Saccharomycks cerevisiae shows much similarity throughout the p and most of the a subunit. Most of the residues implicated by chemical modification or mutation as being critical for enzymatic activity are conserved, along with many others, suggesting that three-dimensional structure has remained largely constant during evolution. We also report the construction of a recombinant plasmid that overproduces a slightly modified a subunit from P. aeruginosa that can form a functionally effective multimer with normal

Temporal scaling of molecular evolution in primates and other mammals.

Abstract: Molecular clocks are routinely tested for linearity using a relative rate test and routinely calibrated against the geological time scale using a single or average paleontologically determined time of divergence between living taxa. The relative rate test is a test of parallel rate equality, not a test of rate constancy. Temporal scaling provides a test of rates, where scaling coefficients of 1.0 (isochrony) represent stochastic rate constancy. The fossil record of primates and other mammals is now known in sufficient detail to provide several independent divergence times for major taxonomic groups. Molecular difference should scale negatively or isochronically (scaling coefficients less than 1.0) with divergence time: where two or more divergence times are available, molecular difference appears to scale positively (scaling coefficient greater than 1.0). A minimum of four divergence times are required for adequate statistical power in testing the linear model: scaling is significantly nonlinear and positive in six of 11 published investigations meeting this criterion. All groups studied show some slowdown in rates of molecular change over Cenozoic time. The break from constant or increasing rates during the Mesozoic to decreasing rates during the Cenozoic appears to coincide with extraordinary diversification of placental mammals at the beginning of this era. High rates of selectively neutral molecular change may be concentrated in such discrete events of evolutionary diversification.

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme.

Abstract: We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a lkb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3’ untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.

Concerted transpositions of mobile genetic elements coupled with fitness changes in Drosophila melanogaster.

Abstract: In an inbred low-activity (LA) strain of Drosophila melanogaster with a low level of fitness and a complex of inadaptive characters, in situ hybridization reveals an invariant pattern of distribution of three copia-like elements (mdg-1, mdg-3, and copia). Rare, spontaneous, multiple transpositions of mobile elements in the LA strain were shown to be coupled with a drastic increase of fitness. A changed pattern of various types of mobile elements was also observed on selecting the LA strain for higher fitness. High-fitness strains show transpositions of mobile elements to definite chromosomal sites (“hot spots”). Concerted changes in the location of three different mobile elements were found to be coupled with an increase of fitness. The mdg-1 distribution patterns were also examined in two low-fitness strains independently selected from the high-fitness ones. Fitness decrease was accompanied by mdg-I excision from the hot spots of their location usually detected in the highfitness strains. The results suggest the existence of a system of adaptive transpositions of mobile elements that takes part in fitness control.

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution.

Abstract: In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha-globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.

Micro-complement fixation: a quantitative estimator of protein evolution.

Abstract: The quantitative immunological technique of micro-complement fixation (MC'F) has been routinely used during the past decade to assess evolutionary relationships among living vertebrate species. The large data base that has been generated, along with the excellent correlations between immunologically measured genetic distances and paleontologically derived estimates of divergence times, have formed the basis for the albumin molecular clock. Immunological distance (ID) involves a logarithmic transformation of experimentally measured antibody concentrations. The justification for this transformation has rested entirely on empirical correlations. Consequently, several other transformations have been proposed as giving better fits to particular data sets. We derive, from first principles, the relationship between ID and the amino acid sequence replacements (AAR) between compared albumins. ID is shown to be a linear estimator of AAR. This ID-AAR relationship is based on a proposed process of antibody assortment and exclusion. We present experimental data confirming that such an antibody assortment-exclusion process occurs in MC'F. This process can explain both the high sensitivity and the quantitative phylogenetic nature of the MC'F assay. The assortment-exclusion process also predicts a divergence limit beyond which MC'F data no longer provide robust phylogenetic data.

Phylogeny and DNA-DNA hybridization.

Abstract: Resolving the phylogeny of the hominoid primates is a test case for molecular systematic methods because, by most genetic measures, humans, chimpanzees, and gorillas are very closely related. Recently, Sibley and Ahlquist (1984) applied DNADNA hybridization techniques to infer the hominoid phylogenetic tree with humans and chimpanzees closest. Templeton (1983, 1985) argues rather that the chimpanzee and gorilla are closest relatives and criticizes the DNA-DNA hybridization methodology in general as well as Sibley and Ahlquist’s analysis in particular. As part of this criticism, Templeton introduces the delta Q-test, a nonparametric test ofphylogenetic relationships that is based on distance data. Applied to the DNA-DNA hybridization data, this test favors Sibley and Ahlquist’s phylogeny over his own-but, according to Templeton ( 1985), not significantly. Not only does this test not support Templeton’s favored phylogeny over Sibley and Ahlquist’s, but it can be shown that, contrary to his claim, the delta Q-test lacks sufficient power to discriminate between these phylogenies. Templeton does raise the valid criticism that intraspecific variation needs to be documented. This criticism also applies to most molecular systematic studies, because, with few exceptions, only one individual per species is examined. Although not reported as such, Sibley and Ahlquist’s original ( 1984) and new (personal communication) data include information on intraspecific variability. While Sibley and Ahlquist did not measure delta TsOH values among pairs of individuals within species, they did measure several individuals of a species against one individual of another. For example, averaged reciprocal delta TsoH (or one-way values where reciprocals are not available) vary between one human and three chimpanzee individuals by 0.23 degrees C (N = 64), between one human and four gorilla individuals by 0.53 degrees C (N = 69), and among two chimpanzees and three gorillas by 0.18 degrees C (N = 69). Eliminating the one-way reciprocal values reduces the delta TsOH range between the one human and three gorillas to 0.27 degrees C. These ranges are less than the smallest measurement in the data set, between species of Pan (delta TsoH + SE of 0.7 f 0.1 degrees C). Human intraspecific single-copy DNA variation has been estimated at 0. l%, approximately equivalent to 0.1 degrees C (R. J. Britten 1986). While we still need direct intraspecific delta TsOH measurements, the data already gathered suggest that variation between individuals within a species did not confound the hominoid intergenic measurements. Templeton (1985) refers to two types of error that can affect any data set. The first type, experimental error, is effectively dealt with by controlling experimental conditions and by repeating each measurement many times, both of which Sibley and Ahlquist (1984) do. The second type of error, stochastic variation, arises from the nature of the evolutionary process itself. There is little question that, over short time periods, the stochastic variations in the evolution of single genes could be significant and confound phylogenetic reconstruction. But Templeton’s comments on stochasticity as applied to Sibley and Ahlquist’s data do not take into account the great number of

Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state.

Abstract: The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta-glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta-glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.

Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: I. Sequence of trpG encoding the glutamine amidotransferase subunit.

Abstract: We have determined the DNA sequence of the distal 148 codons of trpE and all of trpG in Pseudomonas aeruginosa. These genes encode, respectively, the large and small (glutamine amidotransferase) subunits of anthranilate synthase, the first enzyme in the tryptophan synthetic pathway. The sequenced region of trpE is homologous with the distal portion of E. coli and Bacillus subtilis trpE, whereas the trpG sequence is homologous to the glutamine amidotransferase subunit genes of a number of bacterial and fungal anthranilate synthases. The two coding sequences overlap by 23 bp. Codon usage in these Pseudomonas genes shows a marked preference for codons ending in G or C, thereby resembling that of trpB, trpA, and several other chromosomal loci from this species and others with a high G + C content in their DNA. The deduced amino acid sequence for the P. aeruginosa trpG gene product differs to a surprising extent from the directly determined amino acid sequence of the glutamine amidotransferase subunit of P. putida anthranilate synthase (Kawamura et al. 1978). This suggests that these two proteins are encoded by loci that duplicated much earlier in the phylogeny of these organisms but have recently assumed the same function. We have also determined 490 bp of DNA sequence distal to trpG but have not ascertained the function of this segment, though it is rich in dyad symmetries.

Doubled Duplication of the Structural Gene for Cytosolic Phosphoglucose Isomerase in the Dactylis glomerata L. Polyploid Complex

Abstract: Evolution of genes that specify cytosolic phosphoglucose isomerases (PGIs) has been studied in a natural intraspecific polyploid complex (Dactylis glomerata L.). Analyses of genetic control, polymorphism, and gene expression have been carried out in plants collected from numerous populations that belong to 14 diploid subspecies originating in the middle or at the end of the Tertiary period and to three tetraploid subspecies that very likely developed during the last Ice Age. The results show that in each one of the 17 Dactylis subspecies, cytosolic PGIs are specified by two tightly but not completely linked polymorphic loci. Moreover, these show considerable structural similarity since the numerous subunits that each locus specifies associate with those coded by the other locus and most of the dimeric enzymes formed by subunits specified at each locus show exactly the same electrophoretic mobilities. Consequently, one of the two genes is considered to have originated from the other by a process of gene duplication (very likely a tandem one). In this particular case, the duplicated gene has not been distinguished from the ancestral one. The PGI duplication is present in the whole Dactylis monospecific genus but has never been found in the other genera of the same tribe. Since the fixation of a particular gene duplication is considered to be a rare or even unique event, it might have occurred in the primitive forms of diploid Dactylis. Tetraploids show a doubled duplication consequent on the polyploidization process of diploids that already possessed the PGI duplication. The reasons why this duplication has been conserved with the full expression of all the genes throughout evolution of this polyploid complex are examined separately for the two ploidy levels. In diploids, the main reasons could be gene recombination that slows down the rate of gene silencing, unregulated enzyme synthesis, and also differences in enzyme properties. These may confer a selective advantage when distinct alleles can be present, because of the gene duplication, in a single individual. The quite recent origin of the tetraploids and their autopolyploid genetic structure-which does not show f&d heterozygosity and, consequently, possible negative heterodimeric interactions between homeoalleles such as would be the case in allopolyploids-could explain why full expression of the cytosolic PGIs’ doubled duplication is maintained in tetraploid Dactylis.

Paenungulata: a comparison of the hemoglobin sequences from elephant, hyrax, and manatee.

Abstract: Inspection of the amino acid differences among hemoglobin sequences of a wide range of mammalian species suggested that at alpha 19, alpha 110, alpha 111, beta 23, beta 44, and beta 56, synapomorphies group manatee (Trichechus inungius, Sirenia), Indian and African elephant (Elephas maximus and Loxodonta africana, Proboscidea), and rock hyrax (Procavia habessinica, Hyracoidea) into a monophyletic clade. Results obtained by parsimony analysis provide evidence for this grouping--and thus support for the genealogical validity of Simpson's superorder Paenungulata, which contains as the extant orders Proboscidea, Sirenia, and Hyracoidea. All of the 39 most, or nearly most, parsimonious of 10,395 trees constructed from a tandemly combined alpha- and beta-hemoglobin sequence for 103 vertebrate species (of which 79 were mammals from 16 extant orders), depicted Paenungulata as one of the most anciently separated branches of Eutheria. It was found on examining thousands of alternative trees that to not group Proboscidea, Hyracoidea, and Sirenia in a monophyletic clade required at least four additional substitutions.

Nucleotide sequence analysis of the lemur beta-globin gene family: evidence for major rate fluctuations in globin polypeptide evolution.

Abstract: Lemur beta-related globin genes have been isolated and sequenced. Orthology of prosimian and human epsilon-, gamma-, and beta-related globin genes was established by dot-matrix analysis. All of these lemur globin genes potentially encode functional beta-related globin polypeptides, though precisely when the gamma-globin gene is expressed remains unknown. The organization of the 18-kb brown lemur beta-globin gene cluster (5' epsilon-gamma-[psi eta-delta]-beta 3') is consistent with its evolution by contraction via unequal crossing-over from the putative ancestral mammalian beta-globin gene cluster (5' epsilon-gamma-eta-delta-beta 3'). The dwarf lemur nonadult globin genes are arranged as in the brown lemur. Similar levels of synonymous (silent) nucleotide substitutions and noncoding DNA sequence differences have accumulated between species in all of these genes, suggesting a uniform rate of noncoding DNA divergence throughout primate beta-globin gene clusters. These differences are comparable with those observed in the nonfunctional psi eta pseudogene and have therefore accumulated at the presumably maximal neutral rate. In contrast, nonsynonymous (replacement) nucleotide substitutions show a significant heterogeneity in distribution for both the same gene in different lineages and different genes in the same lineage. These major fluctuations in replacement but not silent substitution rates cannot be attributed to changes in mutation rate, suggesting that changes in the rate of globin polypeptide evolution in primates is not governed solely by variable mutation rates.

A Comparison of Phylogenies Based on Structural and Tissue-Expressional Differences of Enzymes in a Family of Telecost Fishes (Salmoniformes: Umbridae)

Abstract: Differences in tissue patterns of enzyme-locus expression were used to infer the extent of divergence among species. Enzyme activities, at 27 loci in six tissues of 15 individuals in four species of Umbridae (Salmoniformes), were estimated on the basis of starch-gel electrophoresis of tissue extracts subjected to twofold serial dilutions and subsequent histochemical staining to determine the visual endpoints. Some locus-tissue expressions diverged markedly among species, while other locustissue expressions were conserved. Differences in activity for a given enzyme among tissues, as well as for different enzymes within the same tissue, were sufficiently independent to permit each locus-tissue expression of a species to be treated as a separate character. Statistically significant differences in levels of tissue-enzyme activities among species were then used to construct a phylogeny. The phylogeny constructed using tissue-enzyme expressional differences was similar to that based on enzyme structural differences (genetic distances) and to one of the morphologically based phylogenies. This congruence suggests that species differences in tissue patterns of enzyme-locus expression can be used to test a variety of systematic and evolutionary hypotheses.

Further comments on the statistical analysis of DNA-DNA hybridization data.

Abstract: Saitou ( 1986) and Ruvolo and Smith ( 1986) have criticized the delta Q-test that I proposed (Templeton 1985) for testing the ability of DNA-DNA hybridization data to discriminate between two alternative phylogenies. The criticisms fall into two basic categories: (1) those concerning the statistical properties of the delta Q-test and (2) those claiming that DNA-DNA hybridization data are superior to alternative types of molecular data. Saitou’s critique concerns the statistical properties of the delta Q-test, and his first criticism is that the null distribution does not include the hierarchical structure that is commonly found in genetic-distance data. This criticism reappears in his discussion of adding a hypothetical chimpanzee species. This hypothetical example uses the fact that the power of the delta Q-test depends on the number of informative taxa contained in the sample. By itself, the dependency of power on the number of informative taxa is a reasonable and desirable property of the test, but Saitou points out that the power can be affected in peculiar ways because of the hierarchical relationships among the informative taxa. Fortunately, it is very easy to incorporate hierarchical structure into the null distribution of the delta Q-test to eliminate these difficulties. In the original draft of the delta Q-test paper, I derived the probability distribution of delta Q in two different fashionsone (that which was ultimately published) in which no hierarchical structure enters into the null distribution and another that accomplishes the derivation by randomly permuting the taxa (not individual distance entries). These two different definitions of the null distribution were suggested by Pielou (1979) and, following her definitions, I referred to them respectively as “primary randomness” and “secondary randomness.” As emphasized by Pielou ( 1979), secondary randomness preserves the hierarchical structure present in the original distance matrix. Unfortunately, the discussion of secondary randomness was deleted from the published version because the conclusion-namely, that the null hypothesis imputing no discrimination could not be rejected-reached concerning the Sibley and Ahlquist (1984) data was the same under either definition of randomness. Hence, the reviewers felt that nothing new was added and that the paper should be restricted to the simpler definition of randomness. I therefore welcome Saitou’s criticism since it affords an opportunity to reintroduce Pielou’s concept of secondary randomness for the delta Q-statistic. Saitou’s next criticism is that the delta Q-test is inadequate for testing phylogeny A versus phylogeny B (using the same labels as in Saitou’s fig. 1) because one cannot accept phylogeny A at the 5% level unless d43 is less than ddl or d42. Saitou feels that the dependency on this inequality makes the delta Q-test inadequate because this inequality has a “high probability” of occurring even if phylogeny A is true. However, one should keep in mind that the purpose of the delta Q-test is not to provide confirmation that phylogeny A is true but rather to see whether the distance data can discriminate between phylogenies A and B, neither of which is known to be true a priori. Any event that has different probabilities under the two phylogenies is informative