Rates of DNA change and phylogeny from the DNA sequences of the alcohol dehydrogenase gene for five closely related species of Hawaiian Drosophila

The sequence of 1.6 kb of DNA surrounding the alcohol dehydrogenase (Adh) gene from five species of the Planitibia subgroup of the Hawaiian picture-winged Drosophila, with estimated divergence times of 0.4-5.1 Myr, has been determined. The gene trees which were found by using the sequence divergence from different regions of the sequences are generally in accord with the phylogeny proposed for these species when chromosomal inversions and island of origin are used. One of the species (D. picticornis) appears to be more distant from the other species in this group than they are from a member of the Grimshawi group (D. affinidisjuncta) which is chromosomally more distant. Two of the species (D. differens and D. plantibia) show heterogeneity in the nucleotide changes in the Adh coding region, heterogeneity which is interpreted to be due to a gene conversion or recombination after hybridization between the two species. The minimal rate of nucleotide substitution of synonymous nucleotides and of nontranscribed nucleotides downstream from the coding region is estimated as 1.5 x 10(-8) and 1.1 x 10(-8) substitutions/nucleotide/year, respectively. This rate is two to three times the maximal rate estimated for mammalian synonymous substitutions.     

Isolation and characterization of the genomic region from Drosophila kuntzei containing the Adh and Adhr genes

The nucleotide sequences of the Adh and Adhr genes of Drosophila kuntzei were derived from combined overlapping sequences of clones isolated from a genomic library and from cloned PCR and inverse-PCR fragments. Only a proximal promoter was detected upstream of the Adh gene, indicating that D. kuntzei Adh is regulated by a one-promoter system. Further upstream of the Adh structural gene, an adult enhancer region (AAE) was found that contains most of the regulatory sequences described for AAEs of other Drosophila species. Analysis of the ADH protein showed an amino acid change from valine to threonine in the active site at position 189 which is also found in D. funebris but is otherwise unique among DROSOPHILA: This difference alone may be responsible for the very low ADH activity found in this species and may cause a difference in substrate usage pattern. Codon bias in Adh and Adhr was comparable and found to be very low compared with other species. Phylogenetic analysis showed that D. kuntzei is closest related to D. funebris and D. immigrans. The time of divergence between D. kuntzei and D. funebris was estimated to be 14.2-20.2 Myr and that between D. kuntzei-D. funebris and D. immigrans to be 30.8-44.0 Myr. An analysis of the genetic variation in the Adh gene and upstream sequences of four European strains showed that this gene was highly variable. Overall nucleotide diversity (pi) was 0.0139, which is two times higher than that in D. melanogaster.     

Pancreatic-Type Ribonuclease 1 Gene Duplications in Rat Species

Mammalian pancreatic-type ribonucleases (RNases) 1 represent single-copy genes in the genome of most investigated mammalian species, including Mus musculus and other murid rodents. However, in six species belonging to the genus Rattus and closely related taxa, several paralogous gene products were identified by Southern blotting and PCR amplifications of genomic sequences. Phylogenies of nucleotide and derived amino acid sequences were reconstructed by several procedures, with three Mus species as outgroup. Duplications of the RNase 1 occurred after the divergence of Niviventer cremoriventer and Leopoldamys edwardsi from the other investigated species. Four groups of paralogous genes could be identified from specific amino acid sequence features in each of them. Low ratios of nonsynonymous-to-synonymous substitutions and the paucity of pseudogene features suggest functional gene products. One of the RNase 1 genes of R. norvegicus is expressed in the pancreas. RNases 1 were isolated from pancreatic tissues of R. rattus and R. exulans and submitted to N-terminal amino acid sequence analysis. In R. rattus, the orthologue of the expressed gene of R. norvegicus was identified, but in R. exulans, two paralogous gene products were found. The gene encoding for one of these had not yet been found by PCR amplification of genomic DNA. A well-defined group of orthologous sequences found in five investigated species codes for very basic RNases. Northern blot analysis showed expression of messenger RNA for this RNase in the spleen of R. norvegicus, but the protein product could not be identified. Evolutionary rates of RNase 1, expressed as nucleotide substitutions per site per 10(3) million years (Myr), vary between 5 and 9 in the lines leading to Mus, Niviventer, and Lepoldamys (on the basis of an ancestral date of mouse/rat divergence of 12.2 Myr) and between 20 and 50 in the lines to the other sequences after divergence from Niviventer and Leopoldamys (5.5 Myr).     

Bayesian analysis suggests that most amino acid replacements in Drosophila are driven by positive selection

One of the principal goals of population genetics is to understand the processes by which genetic variation within species (polymorphism) becomes converted into genetic differences between species (divergence). In this transformation, selective neutrality, near neutrality, and positive selection may each play a role, differing from one gene to the next. Synonymous nucleotide sites are often used as a uniform standard of comparison across genes on the grounds that synonymous sites are subject to relatively weak selective constraints and so may, to a first approximation, be regarded as neutral. Synonymous sites are also interdigitated with nonsynonymous sites and so are affected equally by genomic context and demographic factors. Hence a comparison of levels of polymorphism and divergence between synonymous sites and amino acid replacement sites in a gene is potentially informative about the magnitude of selective forces associated with amino acid replacements. We have analyzed 56 genes in which polymorphism data from D. simulans are compared with divergence from a reference strain of D. melanogaster. The framework of the analysis is Bayesian and assumes that the distribution of selective effects (Malthusian fitnesses) is Gaussian with a mean that differs for each gene. In such a model, the average scaled selection intensity (gamma = N(e)s) of amino acid replacements eligible to become polymorphic or fixed is -7.31, and the standard deviation of selective effects within each locus is 6.79 (assuming homoscedasticity across loci). For newly arising mutations of this type that occur in autosomal or X-linked genes, the average proportion of beneficial mutations is 19.7%. Among the amino acid polymorphisms in the sample, the expected average proportion of beneficial mutations is 47.7%, and among amino acid replacements that become fixed the average proportion of beneficial mutations is 94.3%. The average scaled selection intensity of fixed mutations is +5.1. The presence of positive selection is pervasive with the single exception of kl-5, a Y-linked fertility gene. We find no evidence that a significant fraction of fixed amino acid replacements is neutral or nearly neutral or that positive selection drives amino acid replacements at only a subset of the loci. These results are model dependent and we discuss possible modifications of the model that might allow more neutral and nearly neutral amino acid replacements to be fixed.     

Evolution of nested genes with special reference to cuticle proteins inDrosophila melanogaster

Summary One of the pupal cuticle protein (PCP) genes has been found within an intron of aDrosophila housekeeping gene (theGart locus) that encodes three enzymes involved in the purine pathway. This intronic gene has been described as a gene within a gene, and the gene is now called a “nested” gene. Because the intronic PCP gene has sequence similarity with the larval cuticle protein (LCP) gene, it may have been derived from one of the LCP genes or their ancestral gene. We have studied possible phylogenetic relationships among these five genes by comparing nucleotide sequences of four LCP genes with that of the PCP gene. The results obtained suggest that the PCP gene may have originated from an ancestral gene before duplication of the LCP genes occurred. Using the number of synonymous (silent) substitutions, we then estimated the divergence time between the PCP gene and the LCP genes to be about 70 million years (Myr). The divergence time estimated is much larger than that for the sibling species ofD. melanogaster (about 2.5 Myr), indicating that the “nested” gene structure can be seen not only inDrosophila melanogaster, but also in other distantly relatedDrosophila species.     

Geographic mitochondrial DNA variation in the rock hyrax, Procavia capensis.

Restriction-fragment-length polymorphisms in mitochondrial DNA (mtDNA) were used to evaluate geographic population genetic structure in the rock hyrax, Procavia capensis, a species which occurs widely, though restricted to rocky habitat, throughout South Africa. Ten restriction endonucleases were employed to assay mtDNAs from 55 specimens representing 10 localities. Haplotypes showed strong geographic patterning, and estimates of nucleotide sequence divergence indicate two major clades thought to be dispersing along separate routes. The divergence time of approximately 2 Myr between clades is relatively high for intraspecific variation. We speculate that the marked genetic break distinguishing the northwestern populations from those constituting the south/central clade may be indicative of two species in what has conventionally been regarded as P. capensis.     

Transpecific polymorphisms in an inversion linked esterase locus in Drosophila buzzatii

Nucleotide variation was studied in a 1.1 kb section of the coding region of an Esterase gene (Est-A) that maps in the center of the segments rearranged by polymorphic inversions in the cactophilic Drosophila buzzatii. We examine 30 homozygous second-chromosome lines differing in gene arrangement and three D. koepferae isofemale lines as outgroups. Our data show that Est-A is a highly polymorphic gene at both synonymous and replacement sites. Significant departures from homogeneity in the distribution of the ratio of silent polymorphism to divergence predicted by the neutral theory reveals a local excess of silent polymorphism. This is consistent with the presence of two apparent narrow peaks of elevated silent polymorphism surrounding nonconservative amino acid substitutions. These polymorphisms as well as others at synonymous and nonsynonymous sites are shared with D. koepferae. We suggest that the presence of shared nucleotide polymorphisms is probably due to interspecific gene flow and/or balancing selection acting on replacement variants and/or to a decreased probability of loss of ancestral polymorphisms caused by linkage to an adaptive inversion polymorphism. Recurrent mutation and persistence of neutral ancestral polymorphisms cannot, however, be ruled out. The analysis of the distribution of nucleotide variation among the three chromosomal arrangements sampled reveals that derived arrangements (J and JZ(3)) are less polymorphic than the ancestral ST, and that the widely distributed ST and J arrangements are genetically differentiated. However, a significant number of polymorphisms are shared between arrangements, suggesting frequent exchange either from gene conversion or from double crossovers in heterokaryotypes. Finally, our present results in combination with data of sequence variation at the breakpoints of inversion J suggest that this old gene arrangement has risen in frequency in relatively recent times.     

Rates of Nucleotide Substitution in Angiosperm Mitochondrial DNA Sequences and Dates of Divergence Between Brassica and Other Angiosperm Lineages

We obtained 16 nucleotide sequences (∼1400 bp each) of the first intron of the mitochondrial (mt) gene for NADH subunit 4 (nad4) from 10 species of Brassicaceae. Using these new sequences and five published sequences from GenBank, we constructed a phylogenetic tree of the Brassicaceae species under study and showed that the rate of nucleotide substitution in the first intron of nad4 is very low, about 0.16–0.23 × 10⁻⁹ substitution per site per year, which is about half of the silent rate in exons of nad4. The ratios of substitution rates in this intron, ITS, and IGS are approximately 1:23:73, where ITS is the nuclear intergenic spacer between 18S and 25S rRNA genes and IGS is the intergenic spacer of 5S rRNA genes. A segment (335 bp) in the first intron of nad4 in Brassicaceae species that is absent in wheat was considered as a nonfunctional sequence and used to estimate the neutral rate (the rate of mutation) in mtDNA to be 0.5–0.7 × 10⁻⁹ substitution per site per year, which is about three times higher than the substitution rate in the rest of the first intron of nad4. We estimated that the dates of divergence are 170–235 million years (Myr) for the monocot–dicot split, 112–156 Myr for the Brassicaceae–Lettuce split, 14.5–20.4 Myr for the Brassica–Arabidopsis split, and 14.5–20.4 Myr for the Arabidopsis–Arabideae split. Received: 14 July 1998 / Accepted: 1 October 1998     

Mutations in the Drosophila hook gene inhibit endocytosis of the boss transmembrane ligand into multivesicular bodies

Transmembrane ligands can be internalized across cell boundaries into receptor-expressing cells. In the developing Drosophila eye imaginal disc, the bride of sevenless transmembrane protein (boss) is expressed on the surface of R8 cells. After internalization into neighboring R7 cells, the boss protein accumulates in multivesicular bodies. In a search for genes that affect this cell-type-specific pattern of boss endocytosis, we found that mutations in the hook gene inhibit the accumulation of boss in multivesicular bodies of R7 cells. In addition, hook flies exhibit pleiotropic phenotypes including abnormal bristle morphology and eye degeneration. The wild-type-pattern of boss endocytosis was restored in hook mutants by a genomic rescue fragment containing the hook gene or by a hook cDNA expressed in R7 cells under control of a sevenless (sev) enhancer. The hook gene encodes a novel cytoplasmic protein of 679 amino acids with a central coiled-coil domain of some 200 amino acids. Truncated, epitope-tagged hook proteins coimmunoprecipitated the full-length protein, indicating dimerization mediated by the coiled-coil domain. The hook protein localizes to vesicular structures that are part of the endocytic compartment. The requirement of the hook protein in R7 cells for the accumulation of boss protein in multivesicular bodies, and the localization of the hook protein to endocytic vesicles indicate that the hook gene encodes a novel component of the endocytic compartment that plays an important role in the endocytosis of transmembrane ligands or their transport to multivesicular bodies.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

The molecular clock ticks regularly in muroid rodents and hamsters

Summary Extensive DNA sequence data are used to compare the rates of nucleotide substitution in the mouse, rat, and hamster lineages. A relative rate test using hamster sequences as references shows that the rates of synonymous and nonsynonymous substitution in the mouse and rat lineages are nearly equal and a test using human sequences as references shows that the rates in the mouse, rat, and hamster lineages are also nearly equal. Under the assumptions that the guinea pig lineage and the myomorph (mouse, rat, and hamster) lineage diverged 70–100 million years (Myr) ago and that the rate of nucleotide substitution has been constant in all these lineages since their divergence, the date of the mouse-rat split is estimated to be between 20 and 29 Myr ago, which is considerably older than the date ( 12 Myr) suggested by available rodent fossils and considerably younger than the date ( 35 Myr) suggested by Wilson and colleagues. The murid-hamster split is estimated to be 1.6 times older than the mouse-rat split.     

Patterns of nucleotide polymorphism and divergence in the odorant-binding protein genes OS-E and OS-F: analysis in the melanogaster species subgroup of Drosophila

The Olfactory Specific-E and -F genes (OS-E and OS-F) belong to the odorant-binding protein gene family, which includes the general odorant-binding proteins and the pheromone-binding proteins. In Drosophila melanogaster, these genes are arranged in tandem in a genomic region near the centromere of chromosome arm 3R. We examined the pattern of DNA sequence variation in an approximately 7-kb genomic region encompassing the two OS genes in four species of the melanogaster subgroup of Drosophila and in a population sample of D. melanogaster. We found that both the OS-E and the OS-F gene are present in all surveyed species. Nucleotide divergence estimates would support that the two genes are functional, although they diverge in their functional constraint. The pattern of nucleotide variation in D. melanogaster also differed between genes. Variation in the OS-E gene region exhibited an unusual and distinctive pattern: (i) a relatively high number of fixed amino acid replacements in the encoded protein and (ii) a peak of nucleotide polymorphism around the OS-E gene. These results are unlikely under the neutral model and suggest the action of natural selection in the evolution of the two odorant-binding protein genes.     

Tempo and mode of concerted evolution in the L1 repeat family of mice

A 300-bp DNA sequence has been determined for 30 (10 from each of three species of mice) random isolates of a subset of the long interspersed repeat family L1. From these data we conclude that members of the L1 family are evolving in concert at the DNA sequence level in Mus domesticus, Mus caroli, and Mus platythrix. The mechanism responsible for this phenomenon may be either duplicative transposition, gene conversion, or a combination of the two. The amount of intraspecies divergence averages 4.4%, although between species base substitutions accumulate at the rate of approximately 0.85%/Myr to a maximum divergence of 9.1% between M. platythrix and both M. domesticus and M. caroli. Parsimony analysis reveals that the M. platythrix L1 family has evolved into a distinct clade in the 10-12 Myr since M. platythrix last shared a common ancestor with M. domesticus and M. caroli. The parsimony tree also provides a means to derive the average half-life of L1 sequences in the genome. The rates of gain and loss of individual copies of L1 were estimated to be approximately equal, such that approximately one-half of them turn over every 3.3 Myr.      

Analysis of nucleotide substitutions of mitochondrial DNAs in Drosophila melanogaster and its sibling species

To study the rate and pattern of nucleotide substitution in mitochondrial DNA (mtDNA), we cloned and sequenced a 975-bp segment of mtDNA from Drosophila melanogaster, D. simulans, and D. mauritiana containing the genes for three transfer RNAs and parts of two protein- coding genes, ND2 and COI. Statistical analysis of synonymous substitutions revealed a predominance of transitions over transversions among the three species, a finding differing from previous results obtained from a comparison of D. melanogaster and D. yakuba. The number of transitions observed was nearly the same for each species comparison, including D. yakuba, despite the differences in divergence times. However, transversions seemed to increase steadily with increasing divergence time. By contrast, nonsynonymous substitutions in the ND2 gene showed a predominance of transversions over transitions. Most transversions were between A and T and seemed to be due to some kind of mutational bias to which the A + T-rich mtDNA of Drosophila species may be subject. The overall rate of nucleotide substitution in Drosophila mtDNA appears to be slightly faster (approximately 1.4 times) than that of the Adh gene. This contrasts with the result obtained for mammals, in which the mtDNA evolves approximately 10 times faster than single-copy nuclear DNA. We have also shown that the start codon of the COI gene is GTGA in D. simulans and GTAA in D. mauritiana. These codons are different from that of D. melanogaster (ATAA).      

Molecular evolution of nitrate reductase genes

To understand the evolutionary mechanisms and relationships of nitrate reductases (NRs), the nucleotide sequences encoding 19 nitrate reductase (NR) genes from 16 species of fungi, algae, and higher plants were analyzed. The NR genes examined show substantial sequence similarity, particularly within functional domains, and large variations in GC content at the third codon position and intron number. The intron positions were different between the fungi and plants, but conserved within these groups. The overall and nonsynonymous substitution rates among fungi, algae, and higher plants were estimated to be 4.33 × 10⁻¹⁰ and 3.29 × 10⁻¹⁰ substitutions per site per year. The three functional domains of NR genes evolved at about one-third of the rate of the N-terminal and the two hinge regions connecting the functional domains. Relative rate tests suggested that the nonsynonymous substitution rates were constant among different lineages, while the overall nucleotide substitution rates varied between some lineages. The phylogenetic trees based on NR genes correspond well with the phylogeny of the organisms determined from systematics and other molecular studies. Based on the nonsynonymous substitution rate, the divergence time of monocots and dicots was estimated to be about 340 Myr when the fungi–plant or algae–higher plant divergence times were used as reference points and 191 Myr when the rice–barley divergence time was used as a reference point. These two estimates are consistent with other estimates of divergence times based on these reference points. The lack of consistency between these two values appears to be due to the uncertainty of the reference times. Received: 10 April 1995 / Accepted: 10 September 1995     

Paleo-demography of the Drosophila melanogaster subgroup: application of the maximum likelihood method

The species divergence times and demographic histories of Drosophila melanogaster and its three sibling species, D. mauritiana, D. simulans, and D. yakuba, were investigated using a maximum likelihood (ML) method. Thirty-nine orthologous loci for these four species were retrieved from DDBJ/EMBL/GenBank database. Both autosomal and X-linked loci were used in this study. A significant degree of rate heterogeneity across loci was observed for each pair of species. Most loci have the GC content greater than 50% at the third codon position. The codon usage bias in Drosophila loci is considered to result in the high GC content and the heterogenous rates across loci. The chi-square, G, and Fisher's exact tests indicated that data sets with 11, 23, and 9 pairs of DNA sequences for the comparison of D. melanogaster with D. mauritiana, D. simulans, and D. yakuba, respectively, retain homogeneous rates across loci. We applied the ML method to these data sets to estimate the DNA sequence divergences before and after speciation of each species pair along with their standard deviations. Using 1.6 x 10(-8) as the rate of nucleotide substitutions per silent site per year, our results indicate that the D. melanogaster lineage split from D. yakuba approximately 5.1 +/- 0.8 million years ago (mya), D. mauritiana 2.7 +/- 0.4 mya, and D. simulans 2.3 +/- 0.3 mya. It implies that D. melanogaster became distinct from D. mauritiana and D. simulans at approximately the same time and from D. yakuba no earlier than 10 mya. The effective ancestral population size of D. melanogaster appears to be stable over evolutionary time. Assuming 10 generations per year for Drosophila, the effective population size in the ancestral lineage immediately prior to the time of species divergence is approximately 3 x 10(6), which is close to that estimated for the extant D. melanogaster population. The D. melanogaster did not encounter any obvious bottleneck during the past 10 million years.     

Positive and negative selection on the human genome.

The distinction between deleterious, neutral, and adaptive mutations is a fundamental problem in the study of molecular evolution. Two significant quantities are the fraction of DNA variation in natural populations that is deleterious and destined to be eliminated and the fraction of fixed differences between species driven by positive Darwinian selection. We estimate these quantities using the large number of human genes for which there are polymorphism and divergence data. The fraction of amino acid mutations that is neutral is estimated to be 0.20 from the ratio of common amino acid (A) to synonymous (S) single nucleotide polymorphisms (SNPs) at frequencies of > or =15%. Among the 80% of amino acid mutations that are deleterious at least 20% of them are only slightly deleterious and often attain frequencies of 1-10%. We estimate that these slightly deleterious mutations comprise at least 3% of amino acid SNPs in the average individual or at least 300 per diploid genome. This estimate is not sensitive to human population history. The A/S ratio of fixed differences is greater than that of common SNPs and suggests that a large fraction of protein divergence is adaptive and driven by positive Darwinian selection.     

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Multilocus analysis of nucleotide variation and speciation in Oryza officinalis and its close relatives

Nucleotide variation in 10 unlinked nuclear genes was investigated in species-wide samples of Oryza officinalis and its close relatives (Oryza eichingeri and Oryza rhizomatis). Average estimates of nucleotide diversity were the lowest in O. rhizomatis ((sil) = 0.0038) and the highest in O. eichingeri ((sil) = 0.0057) that is disjunctly distributed in Africa and Sri Lanka. These wild rice species appeared to harbor relatively low levels of nucleotide variation relative to other plant species because the diversity level of O. eichingeri is only 23-46% of those in Zea species and 35% of that in Arabidopsis thaliana. The lower nucleotide diversity in these Oryza species could be best explained by their smaller historic effective population sizes. The speciation model test indicated that O. officinalis and its close relatives might have undergone a process of population contraction since divergence from their ancestor. Incongruent topologies among 10 gene trees, particularly regarding the positions of O. eichingeri and O. rhizomatis accessions might be attributed to lineage sorting arising from ancient polymorphism and hybridization/introgression between the Sri Lankan O. eichingeri and O. rhizomatis. However, the null hypothesis of the isolation model was not rejected for any contrast between taxa, which suggested that no subsequent gene flow shaped the present patterns of nucleotide variation since their divergence and that introgression was not pervasive in this group of species. Our molecular dating provides an approximate divergence time of 0.37 Myr between 2 geographical races of O. eichingeri, much more recent compared with the times of other speciation events in this group (0.63-0.68 Myr). A long-distance dispersal from West Africa to Sri Lanka was more likely to play a role in the disjunct distribution of O. eichingeri.