We report a study in Drosophila melanogaster of latitudinal clines for 23 SNPs embedded in 13 genes (Pgi, Gapdh1, UGPase, Pglym78, Pglym87, Eno, Men, Gdh, Sod, Pgk, Mdh1, TreS, Treh) representing various metabolic enzymes. Our samples are from 10 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,, 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.

Mitochondrial (mtDNA) and nuclear genes have to operate in a coordinated manner to maintain organismal function, and the regulation of this homeostasis presents a substantial source of potential epistatic (G × G) interactions. How these interactions shape the fitness landscape is poorly understood. Here we developed a novel mitonuclear epistasis model, using selected strains of the Drosophila Genetic Reference Panel (DGRP) and mitochondrial genomes from within Drosophila melanogaster and D. simulans to test the hypothesis that mtDNA × nDNA interactions influence fitness. In total we built 72 genotypes (12 nuclear backgrounds × 6 mtDNA haplotypes, with 3 from each species) to dissect the relationship between genotype and phenotype. Each genotype was assayed on four food environments. We found considerable variation in several phenotypes, including development time and egg-to-adult viability, and this variation was partitioned into genetic (G), environmental (E), and higher-order (G × G, G × E, and G × G × E) components. Food type had a significant impact on development time and also modified mitonuclear epistases, evidencing a broad spectrum of G × G × E across these genotypes. Nuclear background effects were substantial, followed by mtDNA effects and their G × G interaction. The species of mtDNA haplotype had negligible effects on phenotypic variation and there was no evidence that mtDNA variation has different effects on male and female fitness traits. Our results demonstrate that mitonuclear epistases are context dependent, suggesting the selective pressure acting on mitonuclear genotypes may vary with food environment in a genotype-specific manner.

In this report, we use synthetic, activity-variant alleles in Drosophila melanogaster to quantify interactions across the enzyme network that reduces nicotinamide adenine dinucleotide phosphate (NADP) to NADPH. We examine the effects of large-scale variation in isocitrate dehydrogenase (IDH) or glucose-6-phosphate dehydrogenase (G6PD) activity in a single genetic background and of smaller-scale variation in IDH, G6PD, and malic enzyme across 10 different genetic backgrounds. We find significant interactions among all three enzymes in adults; changes in the activity of any one source of a reduced cofactor generally result in changes in the other two, although the magnitude and directionality of change differs depending on the gene and the genetic background. Observed interactions are presumably through cellular mechanisms that maintain a homeostatic balance of NADPH/NADP, and the magnitude of change in response to modification of one source of reduced cofactor likely reflects the relative contribution of that enzyme to the cofactor pool. Our results suggest that malic enzyme makes the largest single contribution to the NADPH pool, consistent with the results from earlier experiments in larval D. melanogaster using naturally occurring alleles. The interactions between all three enzymes indicate functional interdependence and underscore the importance of examining enzymes as components of a network.

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 enzymespecific 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 to measure tethered flight performance. For most enzymes, we find that reduction to half of the normal activity has 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.I dentifying the causes of the differing patterns of molecular evolution among genes is a compelling problem in biology. In Drosophila and yeast, genome-wide studies have emphasized gene-specific variables, such as codon bias, expression level, dispensability, and connectedness, to establish correlations with the levels of purifying selection (1-7). One complex but unexplored cause of variation is the relative difference among enzymes that functional variation imparts to phenotype. When the phenotypic variation reflects the flux rate through a pathway, this relationship between enzyme activity and phenotype is defined by the level of flux control. For enzyme steps with little flux control, activity differences will have no impact on phenotypic variation and will have consequently no effect on phenotype-associated fitness. For this reason, the relative level of control at an enzyme step is expected to be a predictor of molecular evolution for a gene. The goal of this study is to evaluate the control of flight performance associated with activity variation for a number of enzymes of the glycolytic pathway.Flux control is expected to differ among enzymes as a result of pathway context, equilibrium status, and the presence or absence of steady-state conditions (8). The classical view of metabolic flux control proposes that unique steps, such as those under allosteric control and strongly displaced from equilibrium, control the pathway flux. These steps represent the textbook regulatory e...