Patterns of natural selection at the Alcohol dehydrogenase gene of Drosophila americana

Similar outcomes are often observed in species exposed to similar selective regimes, but it is unclear how often the same mechanism of adaptive evolution is followed. Here we present an analysis of selection affecting sequence variation in the Alcohol dehydrogenase (Adh) gene of Drosophila americana, a species endemic to a large climate range that has been colonized by D. melanogaster. Unlike D. melanogaster, there is no evidence of selection on allozymes of ADH across the sampled range. This indicates that if there has been a similar adaptive response to climate in D. americana, it is not within the coding region of Adh. Instead, analyses of a combined dataset containing 86 alleles of Adh reveal purifying selection on the Adh gene, especially within its intron sequences. Frequency spectra of derived unpreferred variants at synonymous sites indicate that these sites are affected by weak purifying selection, but the deviation from neutrality is less drastic than observed for derived variants in noncoding introns. This contrast further supports the notion that noncoding sites in Drosophila are often subject to stronger selection pressures than synonymous sites.     

Positive and negative selection on noncoding DNA in Drosophila simulans

There is now a wealth of evidence that some of the most important regions of the genome are found outside those that encode proteins, and noncoding regions of the genome have been shown to be subject to substantial levels of selective constraint, particularly in Drosophila. Recent work has suggested that these regions may also have been subject to the action of positive selection, with large fractions of noncoding divergence having been driven to fixation by adaptive evolution. However, this work has focused on Drosophila melanogaster, which is thought to have experienced a reduction in effective population size (N(e)), and thus a reduction in the efficacy of selection, compared with its closest relative Drosophila simulans. Here, we examine patterns of evolution at several classes of noncoding DNA in D. simulans and find that all noncoding DNA is subject to the action of negative selection, indicated by reduced levels of polymorphism and divergence and a skew in the frequency spectrum toward rare variants. We find that the signature of negative selection on noncoding DNA and nonsynonymous sites is obscured to some extent by purifying selection acting on preferred to unpreferred synonymous codon mutations. We investigate the extent to which divergence in noncoding DNA is inferred to be the product of positive selection and to what extent these inferences depend on selection on synonymous sites and demography. Based on patterns of polymorphism and divergence for different classes of synonymous substitution, we find the divergence excess inferred in noncoding DNA and nonsynonymous sites in the D. simulans lineage difficult to reconcile with demographic explanations.     

Latitudinal Adh allozymic variation and ethanol tolerance in Indian populations of Drosophila melanogaster

Ten Indian geographical populations of D. melanogaster were assayed electrophoretically for Adh genic variation. The Indian geographical populations of D. melanogaster revealed significant clinal variation (3 % for 1 d? latitude) at Adh locus and Adh^F allelic frequency correlated significantly with increase in latitude. It was suggested that the abundance of secondary alcohols in the southern Indian tropical and humid environment might exert selective pressure favouring higher frequency of Adh^S allele. Patterns of ethanol utilization as well as ethanol tolerance were analyzed in larval and adult individuals of six geographical populations of D. melanogaster. Latitudinal variation in ethanol tolerance was observed in D. melanogaster populations from India. The parallel occurrence of latitudinal variation it Adh locus as well as ethanol tolerance in Indian geographical populations of D. melanogaster could be maintained by balancing natural selection varying spatially along the north-south axis of the Indian sub-continent.     

Selection, recombination and demographic history in Drosophila miranda

Selection, recombination, and the demographic history of a species can all have profound effects on genomewide patterns of variability. To assess the impact of these forces in the genome of Drosophila miranda, we examine polymorphism and divergence patterns at 62 loci scattered across the genome. In accordance with recent findings in D. melanogaster, we find that noncoding DNA generally evolves more slowly than synonymous sites, that the distribution of polymorphism frequencies in noncoding DNA is significantly skewed toward rare variants relative to synonymous sites, and that long introns evolve significantly slower than short introns or synonymous sites. These observations suggest that most noncoding DNA is functionally constrained and evolving under purifying selection. However, in contrast to findings in the D. melanogaster species group, we find little evidence of adaptive evolution acting on either coding or noncoding sequences in D. miranda. Levels of linkage disequilibrium (LD) in D. miranda are comparable to those observed in D. melanogaster, but vary considerably among chromosomes. These patterns suggest a significantly lower rate of recombination on autosomes, possibly due to the presence of polymorphic autosomal inversions and/or differences in chromosome sizes. All chromosomes show significant departures from the standard neutral model, including too much heterogeneity in synonymous site polymorphism relative to divergence among loci and a general excess of rare synonymous polymorphisms. These departures from neutral equilibrium expectations are discussed in the context of nonequilibrium models of demography and selection.     

Nucleotide polymorphism in the Adh2 region of the wild rice Oryza rufipogon

DNA variation in the alcohol dehydrogenase (Adh2) region of the wild rice Oryza rufipogon and its related species was analyzed to clarify maintenance mechanisms of the DNA variation in these species. A dimorphic pattern was detected in the Adh2 region of O. rufipogon. The silent nucleotide diversity (π) in the Adh2 region in O. rufipogon was 0.011, which was higher than that of the Adh1 region in O. rufipogon. Especially, a high nucleotide diversity was detected at synonymous sites of the catalytic domain 1. Average nucleotide diversity at silent sites within each of the dimorphic sequence types of the Adh2 region was similar to that in the Adh1 region, indicating that the high level of silent polymorphism in the Adh2 region was caused by the difference between the dimorphic sequence types. On the other hand, the level of replacement polymorphism in the Adh2 region was as low as that in the Adh1 region. The neutrality test of Fu and Li indicated significantly negative deviation from the neutral mutation model for the replacement sites of the Adh2 region. This result suggests purifying selection on the replacement sites of the Adh2 region, as detected for the Adh1 region. Significant linkage disequilibria (16.4% of the tests) were detected between the Adh1 and Adh2 regions. Even when nonrandom association was tested for the strains belonging to one of the divergent sequence types of the Adh2 region, significant interlocus linkage disequilibria were detected. The close physical distance and/or epistasis between the two Adh regions could be invoked to explain these nonrandom associations.     

Patterns of selection on synonymous and nonsynonymous variants in Drosophila miranda

We have investigated patterns of within-species polymorphism and between-species divergence for synonymous and nonsynonymous variants at a set of autosomal and X-linked loci of Drosophila miranda. D. pseudoobscura and D. affinis were used for the between-species comparisons. The results suggest the action of purifying selection on nonsynonymous, polymorphic variants. Among synonymous polymorphisms, there is a significant excess of synonymous mutations from preferred to unpreferred codons and of GC to AT mutations. There was no excess of GC to AT mutations among polymorphisms at noncoding sites. This suggests that selection is acting to maintain the use of preferred codons. Indirect evidence suggests that biased gene conversion in favor of GC base pairs may also be operating. The joint intensity of selection and biased gene conversion, in terms of the product of effective population size and the sum of the selection and conversion coefficients, was estimated to be approximately 0.65.     

Complexity in evolved regulatory variation for alcohol dehydrogenase genes in HawaiianDrosophila

Summary The alcohol dehydrogenase gene (Adh gene) ofDrosophila affinidisjuncta is expressed at a higher level in the larval midgut and Malpighian tubules than the homologous gene fromDrosophila hawaiiensis. This study analyzed thecis-acting sequences responsible for these regulatory differences in larval tissues ofDrosophila melanogaster transformants. A series of 10 chimeric and deletedAdh genes was introduced into the germ line ofD. melanogaster, and tissue-specific expression levels were quantified by gel electrophoresis of tissue extracts. Sequences in the upstream region of the two genes had the strongest influence on enzyme production in the midgut and Malpighian tubules. Other sequence elements also showed effects, some of which were tissue specific. Most gene fragments displayed context-dependent effects, thus supporting the proposed model of polygenic regulation ofAdh gene expression.     

Evidence for interspecific transfer of the transposable element mariner betweenDrosophila andZaprionus

Summary The transposable element mariner occurs widely in themelanogaster species group ofDrosophila. However, in drosophilids outside of themelanogaster species group, sequences showing strong DNA hybridization with mariner are found only in the genusZaprionus. the mariner sequence obtained fromZaprionus tuberculatus is 97% identical with that fromDrosophila mauritiana, a member of themelanogaster species subgroup, whereas a mariner sequence isolated fromDrosophila tsacasi is only 92% identical with that fromD. mauritiana. BecauseD. tsacasi is much more closely related toD. mauritiana than isZaprionus, the presence of mariner inZaprionus may result from horizontal transfer. In order to confirm lack of a close phylogenetic relationship between the genusZaprionus and themelanogaster species group, we compared the alcohol dehydrogenase (Adh) sequences among these species. The results show that the coding region of Adh is only 82% identical betweenZ. tuberculatus andD. mauritiana, as compared with 90% identical betweenD. tsacasi andD. mauritiana. Furthermore, the mariner gene phylogeny obtained by maximum likelihood and maximum parsimony analyses is discordant with the species phylogeny estimated by using the Adh genes. The only inconsistency in the mariner gene phylogeny is in the placement of theZaprionus mariner sequence, which clusters with mariner fromDrosophila teissieri andDrosophila yakuba in themelanogaster species subgroup. These results strongly suggest horizontal transfer.     

Adaptive significance of the action of the Drosophila melanogaster alcohol dehydrogenase locus through the heat shock protein system

Four Drosophila melanogaster strains, each homozygous for one of the two major ADH aliozymes, Fast and Slow (Adh ^F1, Adh ^S , Adh ^F2 and Adh ^S2) were used to study the interaction of the Adh locus with ethanol and temperature. The separate and especially the combined effects of these two parameters allow the conclusion that the Adh locus of D. melanogaster intervenes in the adaptation process through the heat shock protein system.     

Sequence and evolution of the Drosophila pseudoobscura glycerol-3-phosphate dehydrogenase locus

The Gpdh genomic region has been cloned and sequenced in Drosophila pseudoobscura. A total of 6.8 kb of sequence was obtained, encompassing all eight exons of the gene. The exons have been aligned with the sequence from D. melanogaster, and the rates of synonymous and nonsynonymous substitution have been compared to those of other genes sequenced in these two species. Gpdh has the lowest rate of nonsynonymous substitution yet seen in genes sequenced in both D. pseudoobscura and D. melanogaster. No insertion/deletion events were observed, and the overall architecture of the gene (i.e., intron sites, etc.) is conserved. An interesting amino acid reversal was noted between the D. melanogaster Fast allele and the D. pseudoobscura gene.     

MOLECULAR ANALYSIS OF THE ALLELES OF ALCOHOL DEHYDROGENASE ALONG A CLINE IN DROSOPHILA MELANOGASTER. I. MAINE,NORTH CAROLINA,AND FLORIDA

Clinal variation in natural populations is often assumed to be due to the operation of natural selection. However, for many clines there exist plausible neutralist explanations which suggest that aspects of population structure maintain differences among subpopulations for particular traits. We used a restriction-mapping technique to investigate the contributions of population subdivision and selection to the maintenance of the allozyme polymorphism at the alcohol dehydrogenase (Adh) locus of Drosophila melanogaster. Digestions of genomic DNAs from 270 lines of flies by seven enzymes reveal 15–20% of all possible nucleotide substitutions and virtually all of the insertion/deletion variation in a 2.7-kilobase region containing the Adh structural locus. Analysis of large samples from each of three populations along the east coast of the United States provides evidence of founder effects in the most northerly population. Although there are signs of population differentiation among the samples, similarities between two of the populations indicate that migration among populations is extensive and strengthen the argument that natural selection plays a role in maintaining the cline.     

Linkage of the alcohol dehydrogenase structural genes in pearl millet (Pennisetum typhoides)

Pearl millet produces three ADH isozymes, Sets I, II, and III. Naturally occurring ADH electrophoretic variants affecting Sets I and II isozymes but not III have been previously described. Analysis of such variants led to the identification of the Adh1 structural gene. The existence of a second Adh structural gene was inferred from dissociation-reassociation studies of Set II. In the present report, a naturally occurring variant affecting the electrophoretic mobility of Sets III and II but not Set I is described. Analysis of this variant confirms the existence of a second structural gene, Adh2. Crosses utilizing this Adh2 marker reveal a dissimilarity with maize and other plants such as sunflower and narrow-leafed lupins. Adh1 and Adh2 of pearl millet do not segregate independently; indeed, no recombinants have been observed. This is the first major difference encountered in an otherwise remarkably similar genetic and environmental control of the ADH isozymes in maize and millet. The organization of the Adh genes of pearl millet may reflect a more primitive arrangement than that of maize.This work was supported by a PHS National Research Service Award Training Grant in Genetics to the Biology Department of the University of Oregon.     

Alcohol dehydrogenase polymorphism in populations of Drosophila melanogaster. II. Relation between ADH activity and adult mortality

Eight Drosophila melanogaster strains, seven homozygous for Adh ^F alleles and one for an Adh-null mutant, were compared for ADH activity in males and adult mortality on ethanol-supplemented food. The strains differed considerably in these qualities. A positive correlation was found between ADH activity and ld ₅₀. The relevance of this finding is discussed in relation to the differential selection acting on Adh genotypes kept on ethanol-supplemented food.     

Positive Selection on Nucleotide Substitutions and Indels in Accessory Gland Proteins of the Drosophila pseudoobscura Subgroup

Genes encoding reproductive proteins often diverge rapidly due to positive selection on nucleotide substitutions. While this general pattern is well established, the extent to which specific reproductive genes experience similar selection in different clades has been little explored, nor have possible targets of positive selection other than nucleotide substitutions, such as indels, received much attention. Here, we inspect for the signature of positive selection in the genes encoding five accessory gland proteins (Acps) (Acp26Aa, Acp32CD, Acp53Ea, Acp62F, and Acp70A) originally described from Drosophila melanogaster but with recognizable orthologues in the D. pseudoobscura subgroup. We compare patterns of selection within the D. psuedoobscura subgroup to those in the D. melanogaster subgroup. Similar patterns of positive selection were found in Acp26Aa and Acp62F in the two subgroups, while Acp53Ea and Acp70A experienced purifying selection in both subgroups. These proteins have thus remained targets for similar types of selection over long (>21-MY) periods of time. We also found several indel substitutions and polymorphisms in Acp26Aa and Acp32CD. These indels occur in the same regions as positively selected nucleotide substitutions for Acp26Aa in the D. pseudoobscura subgroup but not in the D. melanogaster subgroup. Rates of indel substitution within Acp26Aa in the D. pseudoobscura subgroup were up to several times those in noncoding regions of the Drosophila genome. This suggests that indel substitutions may be under positive selection and may play a key role in the divergence of some Acps. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Willis Swanson]     

A biochemical genetic study of alcohol dehydrogenase isozymes of the medfly,Ceratitis capitata wied

A concerted effort is under way to analyze, at the genetic, biochemical, and molecular level, theAdh gene system in the medflyCeratitis capitata, an important agricultural pest. The isoelectric focusing (IEF) pattern of alcohol dehydrogenase (ADH) of the medfly demonstrates the presence of two well-differentiated, genetically independent dimeric proteins, called ADH-1 and ADH-2. These proteins do not exhibit interlocus heterodimeric isozymes, and the genes are not controlled coordinately during development,Adh ₁ andAdh ₂ being expressed mainly in muscle or in fat body and ovary, respectively. From the intensity of the IEF isozyme patterns, primary alcohols are judged to be better substrates than secondary alcohols, in contrast withDrosophila melanogaster ADH, and ethanol is probably the most efficient substrate for both sets of isozymes. The isoelectric points of ADH-1 (pI=5.4) and ADH-2 (pI=8.6) are different fromD. melanogaster ADH (pI=7.6), but the medfly ADH-1 has a native molecular weight (approx. 58 kD) close to that ofD. melanogaster. A population survey of samples both from laboratory strains and from wild geographically different populations showed that theAdh ₁ locus is more polymorphic thanAdh ₂. The most variable populations are from Africa, the supposed source area of the species. Further, a case of selection at theAdh ₁ locus under laboratory conditions is reported. The hypothesis ofAdh gene duplication and the degree of similarity between medfly andDrosophila ADH are also discussed. This research was supported mainly by National Research Council of Italy, Special Project RAISA, Sub-project No. 2, Paper No. 342. Grants from the International Atomic Energy Agency, Vienna, Austria, from European Communities Commission, Second R & D Programme, “Science and Technology for Development,” and from the Italian Ministry of University and Scientific Research and Technology (“Funds 40%”) also supported this work. This paper was written when the senior author was on leave of absence at the IMBB, Crete, Greece; he was financially supported by an ECC Senior Fellowship.     

Length polymorphism in the threonine-glycine-encoding repeat region of theperiod gene inDrosophila

Summary Single-fly polymerase chain reaction amplification and direct DNA sequencing revealed high levels of length polymorphism in the threonine-glycine encoding repeat region of theperiod (per) gene in natural populations ofDrosophila melanogaster. DNA comparison of two alleles of identical lengths gave a high number of synonymous substitutions suggesting an ancient time of separation. However detailed examination of the sequences of different Thr-Gly length variants indicated that this divergence could be understood in terms of four deletion/insertion events. InDrosophila pseudoobscura a length polymorphism is observed in a five-amino acid degenerate repeat, which corresponds tomelanogaster's Thr-Gly domain. In spite of the differences betweenD. melanogaster andD. pseudoobscura in the amino acid sequence of the repeats, the predicted secondary structures suggest evolutionary and mechanistic constraints on theper protein of these two species.     

Perturbation of gene frequencies in a natural population of Drosophila melanogaster: evidence for selection at the Adh locus

The cellar population of Drosophila melanogaster at the Chateau Tahbilk Winery (Victoria, Australia) was perturbed for alcohol dehydrogenase (Adh) gene frequencies. Phenol oxidase (Phox) frequencies were also perturbed and monitored as a control. Subsequent gene frequency changes, together with information on population structure, indicated that selection acted on the chromosome regions of both loci. Adh gene frequencies returned to preperturbation levels in a predictable manner. A model in which the relative fitness of Adh phenotypes was determined by temperature-dependent specific activities of enzymes of Adh genotypes adequately accounts for the rate of gene frequency change at this locus. Thus temperature behaves as a selective agent in modulating Adh gene frequencies in this cellar environment.     

The alcohol dehydrogenase alleloenzymes AdhS and AdhF from the fruitfly Drosophila melanogaster: An enzymatic rate assay to determine the active-site concentration

A rapid and reproducible enzymatic rate assay for the quantitative determination of the concentration of active sites is presented for the alleloenzymes Adh^S and Adh^F from Drosophila melanogaster. Using this procedure the turnover numbers as catalytic-center activities were found to be 12.2 sec^–1 for Adh^F and 3.4 sec^–1 for Adh^S with secondary alcohols. This showed a slower dissociation of the coenzyme from the binary enzyme-NADH complex with Adh^S and hence a stronger binding of NADH to this alleloenzyme. With ethanol, the catalytic-center activity was 1.4 sec^–1 for Adh^S and 2.8 sec^–1 for Adh^F, and hence the single amino acid mutation distinguishing the two alleloenzymes also affected hydride transfer.