Paul S. Schmidt, Chen-Tseh Zhu, Jayatri Das, Mariska Batavia, Li Yang, and Walter F. Eanes. 2008 An amino acid polymorphism in the couch potato gene forms the basis for climatic adaptation in Drosophila melanogaster Proc. Natl. Acad. Sci. USA . 105: 1620716211 

Diapause is the classic adaptation to seasonality in arthropods, and its expression can result in extreme lifespan extension as well as enhanced resistance to environmental challenges. Little is known about the underlying evolutionary genetic architecture of diapause in any organism. Drosophila melanogaster exhibits a reproductive diapause that is variable within and among populations; the incidence of diapause increases with more temperate climates and has significant pleiotropic effects on a number of life history traits. Using quantitative trait mapping, we identified the RNA binding protein encoding gene couch potato (cpo) as a major genetic locus determining diapause phenotype in D. melanogaster and independently confirmed this ability to impact diapause expression through genetic complementation mapping. By sequencing this gene in samples from natural populations we demonstrated through linkage association that variation for the diapause phenotype is caused by a single Lys/Ile substitution in one of the six cpo transcripts. Complementation analyses confirmed that the identified amino acid variants are functionally distinct with respect to diapause expression, and the polymorphism also shows geographic variation that closely mirrors the known latitudinal cline in diapause incidence. Our results suggest that a naturally occurring amino acid polymorphism results in the variable expression of a diapause syndrome that is associated with the seasonal persistence of this model organism in temperate habitats. 

Jon M. Flowers, E. Sezgin, S. Kumagai, D. D. Duvernell, L. M. Matzkin, P. S. Schmidt, and W. F. Eanes. 2007. Adaptive evolution of metabolic pathways in Drosophila.   Mol. Biol. Evol. 24 : 1347-1354.

The adaptive significance of enzyme variation has been of central interest in population genetics, but how natural selection operates on enzymes in the larger context of biochemical pathways has not been broadly explored.    Here we use population genetic methods to identify the enzymatic steps subjected to adaptive selection in the pathways of central metabolism in Drosophila melanogaster and D. simulans.  We report polymorphism and divergence data for 17 genes that encode enzymes of five metabolic pathways that converge at glucose-6-phosphate (G6P).  Deviations from neutral expectations were observed at five of the 17 loci.   Of the 10 genes that encode the enzymes of glycolysis, only aldolase (Ald) deviated from neutrality.   The other four genes that were inconsistent with neutral evolution encode G6P branch point enzymes, which lie at the entry point to the pentose-phosphate (G6pd), glycogenic (Pgm), trehalose synthesis (Tps1), and gluconeogenic (G6pase) pathways. We reconcile these results with population genetics theory and existing data on metabolic regulation and propose that the incidence of adaptive selection in this system is related to the distribution of flux control.  The data provide additional examples of positive selection on enzymes in the central metabolic corridor in D. simulans and suggest that adaptive evolution of G6P branch point enzymes may be important for metabolic adaptation in Drosophila.

Walter F. Eanes,  Thomas J. S. Merritt, Jonathan M. Flowers, Seiji Kumagai,  Efe Sezgin, and Chen-Tseh Zhu. 2006. Flux control and excess capacity in the enzymes of glycolysis and their relationship to flight metabolism in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 103: 19413-19418.

An important question in evolutionary and physiological genetics is how the control of flux-base phenotypes is distributed across the enzymes in a pathway. This control is often related to enzyme-specific levels of activity that are reported to be in excess of that required for demand.  In glycolysis, metabolic control is frequently considered vested in classical regulatory enzymes, each strongly displaced from equilibrium. Yet, the contribution of individual steps to control is unclear. To assess enzyme-specific control in the glycolytic pathway, we used P-element excision-derived mutagenesis in Drosophila melanogaster, to generate full and partial knockouts of seven metabolic genes and measure tethered flight performance.  For most enzymes, we find that reductions to half normal activity have no measurable impact on wing beat frequency.  The enzymes catalyzing near-equilibrium reactions, phosphoglucose isomerase, phosphoglucomutase, and triosephosphate isomerase, fail to show any decline in flight performance even when activity levels are reduced to 17% or less. At reduced activities, the classic regulatory enzymes, hexokinase and glycogen phosphorylase, show significant drops in flight performance and are nearer to saturation. Our results show that flight performance is canalized or robust to the activity variation found in natural populations.  Furthermore, enzymes catalyzing near-equilibrium reactions show strong genetic dominance down to low levels of activity. This implies considerable excess enzyme capacity for these enzymes.

Thomas J. S. Merritt, Efe Sezgin, Chen-Tseh Zhu and Walter F. Eanes. 2006  Triglyceride pools, flight, and activity variation at the Gpdh locus in Drosophila melanogaster. Genetics 172: 293-304.. 

We have created a set of P-element excision-derived Gpdh alleles that generate a range of GPDH activity phenotypes ranging from full to zero activity. By placing these synthetic alleles in isogenic backgrounds we characterize the effects of minor and major activity variation on two different aspects of Gpdh function; the standing triglyceride pool and glycerol-3-phosphate shuttle-assisted flight. We observe small but statistically significant reductions in triglyceride content for adult Gpdh genotypes possessing 33 to 80% reductions from normal activity. These small differences scale to a notable proportion of the observed genetic variation in triglyceride content in natural populations.  Using a tethered fly assay to assess flight metabolism, we observed genotypes with 100 and 66% activity to exhibit no significant difference in wing beat frequency (WBF), while activity reductions from 60 to 10% show statistically significant reductions of about 7% in WBF.   These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection on the triglyceride pool.


Thomas J. S. Merritt, David Duvernell and Walter F. Eanes . 2005. Natural and synthetic alleles provide complementary insights into the nature of selection acting on the Men polymorphisms in Drosophila melanogaster. Genetics 171: 1707-1718.

Two malic enzyme alleles, Men113A and Men113G, occur at approximately equal frequency in North American populations of Drosophila melanogaster, while only Men113Aoccurs African populations.  We investigated of the population genetics, biochemical characteristics and selective potential of these alleles.  Comparable levels of nucleotide polymorphism in both alleles suggest that the Men113G allele is not recently derived, but we find no evidence in the DNA sequence data for selection maintaining the polymorphism.  Interestingly, the alleles differ in both Vmax and KM for the substrate malate.  Triglyceride concentration and isocitrate dehydrogenase (IDH) and glucose-6-phosphate dehydrogenase (G6PD) activities are negatively correlated with the in vivo activities of the Men alleles.  We examined the causality of the observed correlations using P-element excision derived knockout alleles of the Men gene and found significant changes in the maximum activities of both IDH and G6PD, but not triglyceride concentration, suggesting compensatory interactions between MEN, IDH and G6PD.  Additionally, we found significantly higher than expected levels of MEN activity in knockout heterozygotes which we attribute to transvection effects.  The distinct differences in biochemistry and physiology between the naturally occurring alleles and between the engineered alleles suggest the potential for selection on the Men locus.

Luciano M. Matzkin, Thomas J. S. Merritt, Chen-Tseh Zhu, and Walter F. Eanes. 2005. The structure and population genetics of the breakpoints associated with the cosmopolitan chromosomal inversion In(3R)Payne in Drosophila melanogaster. Genetics 170: 1143-1152

We report here the breakpoint structure and sequences of the Drosophila melanogaster cosmopolitan chromosomal inversion In(3R)P. Combining in situ hybridization to polytene chromosomes and long-range PCR, we have identified and sequenced the distal and proximal breakpoints. The breakpoints are not simple cut-and-paste structures; gene fragments and small duplications of DNA are associated with both breaks. The distal breakpoint breaks the tolkin (tok) gene and the proximal breakpoint breaks CG31279 and the tolloid (tld) gene.  Functional copies of all three genes are found at the opposite breakpoints.  We sequenced a representative sample of standard (St) and In(3R)P karyotypes for a 2 kb portion of the tok gene, as well as the same 2kb from the pseudogene tok fragment found at the distal breakpoint of In(3R)P chromosomes. The tok gene in St arrangements possesses levels of polymorphism typical of D. melanogaster genes.  The functional tok gene associated with In(3R)P shows little polymorphism.  Numerous single base changes, as well as deletions and duplications, are associated with the truncated copy of tok. The overall pattern of polymorphism is consistent with a recent origin of In(3R)P, on the order of Ne generations. The identification of these breakpoint sequences permits a simple PCR-based screen for In(3R)P.

Schmidt, P.S., Matzkin, L., Ippolito, M. L. and W.F. Eanes. 2005. Geographic variation in diapause incidence, life history traits, and climatic adaptation in Drosophila melanogaster. Evolution 59: 1721-1732..

 In Drosophila melanogaster, exposure of females to low temperature and shortened photoperiod can induce the expression of reproductive quiescence or diapause. Diapause expression has significant effects on life history profiles, including patterns of longevity, fecundity, and stress resistance.  When flies are collected from natural populations and exposed to diapause inducing cues in the laboratory, some lines undergo reproductive quiescence whereas others proceed with vitellogenesis and normal reproductive development.  We hypothesized that if diapause expression is associated with overwintering mechanisms and adaptation to temperate environments, the frequency of diapause incidence would exhibit a latitudinal cline among natural populations.  As stress resistance and reproductive traits are also clinal in this species, we also examined 1) how patterns of fecundity and longevity varied with geography, and 2) how stress resistance and associated traits differed constitutively between diapause and nondiapause lines.  Diapause incidence was shown to vary predictably with latitude, ranging from 35 to 90 percent among natural populations in the eastern U.S.  Survivorship under starvation stress differed between diapause and nondiapause lines; diapause phenotypes were also distinct for total body triacylglyceride content and the developmental distribution of oocytes in the ovary following stress exposure.  Patterns of longevity, fecundity, and ovariole number also varied with geography.  The data suggest that, for North American populations, diapause expression is functionally associated with overwintering mechanisms and that the genetic variance for diapause expression in D. melanogaster may reflect adaptation to variable climatic environments.

Efe Sezgin, David D. Duvernell, Luciano M. Matzkin, Yihao Duan, Chen-Tseh Zhu, Brian C. Verrelli and Walter F. Eanes. 2004 Single locus latitudinal clines in metabolic genes, derived alleles, and their relationship to temperate adaptation in Drosophila melanogaster. Genetics 168: 923-931.

 We report a study in Drosophila melanogaster of latitudinal clines for 23 SNPs embedded in thirteen genes (Pgi, Gapdh1, UGPase, Pglym78, Pglym87, Eno, Men, Gdh, Sod, Pgk, Mdh1, TreS, Treh) representing various metabolic enzymes. Our samples are from ten populations spanning latitude from southern Florida to northern Vermont. Three new clines with latitude were detected. These are the amino acid polymorphisms in the NAD-dependent glutamate dehydrogenase (Gdh) and trehalase (Treh) genes, and a silent site polymorphism in the UDP-glucose pyrophosphorylase gene (UGPase). The result, when combined with the overall incidence and pattern of reports for six other genes (Adh, Gpdh, Pgm, G6pd, 6Pgd, Hex-C), presents a picture of latitudinal clines in metabolic genes prevalent around the branch point of competing pathways. For six of the seven amino acid polymorphisms showing significant latitudinal clines in North America, the derived allele is the one increasing with latitude, suggesting temperate adaptation. This is consistent with a model of an Afrotropical ancestral species adapting to temperate climates through selection favoring new mutations.

Luciano M. Matzkin and Walter F. Eanes. 2003 Sequence variation of alcohol dehydrogenase (Adh) paralogs in cactophilic Drosophila. Genetics 163: 181-194. 

This study focuses on the population genetics of alcohol dehydrogenase (Adh) in cactophilic Drosophila. Drosophila mojavensis and D. arizonae utilize cactus hosts, and each host contains a characteristic mixture of alcohol compounds. In these Drosophila species there are two functional Adh loci, an adult form (Adh-2) and a larval and ovarian form (Adh-1). Overall, the greater level of variation segregating in D. arizonae than in D. mojavensis suggests a larger population size for D.arizonae. There are markedly different patterns of variation between the paralogs across both species. A 16-bp intron haplotype segregates in both species at Adh-2, apparently the product of an ancient gene conversion event between the paralogs, which suggests that there is selection for the maintenance of the intron structure possibly for the maintenance of pre-mRNA structure. We observe a pattern of variation consistent with adaptive protein evolution in the D. mojavensis lineage at Adh-1, suggesting that the cactus host shift that occurred in the divergence of D. mojavensis from D. arizonae had an effect on the evolution of the larval expressed paralog. Contrary to previous work we estimate a recent time for both the divergence of D. mojavensis and D. arizonae (2.4 0.7 MY) and the age of the gene duplication (3.95 0.45 MY). 

David D. Duvernell, Paul S. Schmidt, and Walter F. Eanes. 2003. Clines and adaptive evolution in the methuselah gene region in Drosophila melanogaster. Molecular Ecology 12: 1277-1285. 

In an effort to further characterize patterns of selection and adaptive evolution at the methuselah locus in Drosophila species, we extended an analysis of geographic variation to include single nucleotide polymorphisms (SNPs) in adjacent genes on either side of the mth locus, and examined the molecular variation in a neighboring methuselah paralog (mth2). An analysis of 13 SNPs spanning a region of nearly 19 Kb  surrounding the mth locus demonstrated that a clinal pattern associated with the most common mth haplotype does not extend to adjacent gene loci providing compelling evidence that the clinal pattern results from selection on as yet unidentified sites associated with the functional mth locus. Mth2 exhibited a significant pattern of adaptive divergence among D. melanogaster, D. simulans, and D. yakuba similar to that seen at mth. However, Ka/Ks ratios indicate a difference in levels of functional constraint at the two methuselah loci with mth2 exhibiting a five to six-fold reduction in levels of amino acid divergence relative to mth.

Brian C. Verrelli and Walter F. Eanes, 2001 The functional impact of Pgm amino acid polymorphism on glycogen content in Drosophila melanogaster. Genetics 159: 201-210.

Earlier studies of the common PGM allozymes in Drosophila melanogaster reported no in vitro activity differences.  However, our study of nucleotide variation observed that PGM allozymes are a heterogeneous mixture of amino acid polymorphisms.  In this study, we analyze ten PGM protein haplotypes with respect to PGM activity, thermostability, and adult glycogen content.  We find a two-fold difference in activity among PGM protein haplotypes that is associated with a three-fold difference in glycogen content.  The latitudinal clines for several Pgm amino acid polymorphisms show that high PGM activity, and apparently higher flux to glycogen synthesis, parallels the low activity clines at G6PD for reduced pentose shunt flux in northern latitudes.  This suggests that amino acid polymorphism is under selection at this branch point and may be favored for increased metabolic storage associated with stress resistance and adaptation to temperate regions.

Brian C. Verrelli and Walter F. Eanes. 2001 Clinal variation for amino acid polymorphisms at the Pgm locus in Drosophila melanogaster.  Genetics 157: 1649-1663.

Clinal variation is common for enzymes in the glycolytic pathway for Drosophila melanogaster and is generally accepted as an adaptive response to different climates. Although the enzyme phosphoglucomutase (PGM) possesses several allozyme polymorphisms, it is unique in that it had been reported to show no clinal variation. Our recent DNA sequence investigation of Pgm found extensive cryptic amino acid polymorphism segregating with the allozyme alleles. In this study, we characterize the geographic variation of Pgm amino acid polymorphisms at the nucleotide level along a latitudinal cline in the eastern United States. A survey of 15 SNPs across the Pgm gene finds significant clinal differentiation for the allozyme polymorphisms as well as for many of the cryptic amino acid polymorphisms. A test of independence shows that pervasive linkage disequilibrium across this gene region can explain many of the amino acid clines. A single Pgm haplotype defined by two amino acid polymorphisms shows the strongest correlation with latitude and the steepest change in allele frequency across the cline. We propose that clinal selection at Pgm may in part explain the extensive amino acid polymorphism at this locus and is consistent with a multilocus response to selection in the glycolytic pathway.

Brian C. Verrelli and Walter F. Eanes. 2000 Extensive amino acid polymorphism at the Pgm locus is consistent with adaptive protein evolution in Drosophila melanogaster . Genetics 156: 1737-175. 

PGM plays a central role in the glycolytic pathway at the branch point leading to glycogen metabolism and is highly polymorphic in allozyme studies of many species. We have characterized the nucleotide diversity across the Pgm gene in Drosophila melanogaster and D. simulans to investigate the role that protein polymorphism plays at this crucial metabolic branch point shared with several other enzymes. Although D. melanogaster and D. simulans share common allozyme mobility alleles, we find these allozymes are the result of many different amino acid changes at the nucleotide level. In addition, specific allozyme classes within species contain several amino acid changes, which may explain the absence of latitudinal clines for PGM allozyme alleles, the lack of association of PGM allozymes with the cosmopolitan In(3L)P inversion, and the failure to detect differences between PGM allozymes in functional studies. We find a significant excess of amino acid polymorphisms within D. melanogaster when compared to the complete absence of fixed replacements with D. simulans. There is also strong linkage disequilibrium across the 2354 bp of the Pgm locus, which may be explained by a specific amino acid haplotype that is high in frequency yet contains an excess of singleton polymorphisms. Like G6pd, Pgm shows strong evidence for a branch point enzyme that exhibits adaptive protein evolution.

Paul S. Schmidt, David D. Duvernell, and Walter F. Eanes. 2000. Adaptive evolution of a candidate gene for aging in Drosophila Proc. Natl. Acad. Sci. USA 97: 10861-10865.

Examination of the phenotypic effects of specific mutations has been extensively used to identify candidate genes affecting traits of interest. However, such analyses do not reveal anything about the evolutionary forces acting at these loci, or whether standing allelic variation contributes to phenotypic variance in natural populations. The Drosophila gene methuselah (mth) has been proposed as having major effects on organismal stress response and longevity phenotype. Here, we examine patterns of polymorphism and divergence at mth in population level samples of Drosophila melanogaster, D. simulans, and D. yakuba. Mth has experienced an unusually high level of adaptive amino acid divergence concentrated in the intra- and extracellular loop domains of the receptor protein, suggesting the historical action of positive selection on those regions of the molecule that modulate signal transduction. Further analysis of single nucleotide polymorphisms (SNPs) in D. melanogaster provided evidence for contemporary and spatially variable selection at the mth locus. In ten surveyed populations, the most common mth haplotype exhibited a 40% cline in frequency that coincided with population level differences in multiple life-history traits including lifespan. This clinal pattern was not associated with any particular SNP in the coding region, indicating that selection is operating at a closely linked site that may be involved in gene expression. Together, these consistently nonneutral patterns of inter- and intraspecific variation suggest adaptive evolution of a signal transduction pathway that may modulate lifespan in nature.

David D. Duvernell and Walter F. Eanes, 2000 Contrasting molecular population genetics of four hexokinases in Drosophila melanogaster, D. simulans and D. yakuba. Genetics 156: 1191-1201. 

As part of a larger study contrasting patterns of variation in regulatory and nonregulatory enzymes of the central metabolic pathways we have examined the molecular variation in four uncharacterized hexokinase genes unique to muscle, fat body, and testis in Drosophila melanogaster, D. simulans, and D. yakuba. Earlier isoenzyme studies had designated these genes as Hex-A, Hex-C, and Hex-t. There are two tightly linked testes-specific genes designated here as Hex-t1 and Hex-t2. Substantial and concordant differences across species are seen in levels of both amino acid and silent polymorphism. The flight muscle form Hex-A is the most conserved followed by the fat body hexokinase Hex-C and testis-specific hexokinases Hex-t1 and Hex-t2. While constraints acting at the amino acid level are expected, the silent polymorphisms follow this pattern as well. All genes are in regions of normal recombination, therefore hitchhiking and background selection are not likely causes of interlocus differences. In D. melanogaster latitudinal clines are seen for amino acid polymorphisms at the Hex-C and Hex-t2 loci. There is evidence for accelerated amino acid substitution in Hex-t1 that has lost residues known to be associated with glucose and glucose-6-phosphate binding. D. simulans shows substantial linkage phase structuring that suggests historical population subdivision. (see Data Set).