Extensive amino acid polymorphism at the pgm locus is consistent with adaptive protein evolution in Drosophila melanogaster

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.     

Molecular Analysis of an Allozyme Cline: Alcohol Dehydrogenase in Drosophila Melanogaster on the East Coast of North America

Clines may either be selectively maintained or be the by-product of nonadaptive processes related to population structure and history. Drosophila melanogaster populations on the east coast of North America show a latitudinal cline in the frequencies of two common electrophoretically distinguishable alleles at the alcohol dehydrogenase locus (Adh), designated Adh-S and Adh-F. This cline may either be adaptive or an artifact of a possible recent dual founding of North American D. melanogaster populations in which frequencies of Adh alleles differed between founder populations. By means of a high resolution restriction-mapping technique, we studied the distribution of 113 haplotypes derived from 44 polymorphic DNA markers within the Adh region in 1533 individuals from 25 populations throughout the cline. We found significant clinal differentiation at the polymorphisms determining the mobility-difference causing amino acid replacement between Adh-F and Adh-S alleles. Hitchhiking was limited, despite extensive linkage disequilibrium, and other sites did not vary clinally. Such a pattern of differentiation implies that selection is responsible for the cline. To investigate whether selection acts only on the Adh-F/S site, we performed a ``selective equivalence'''' test under the assumption that all variability within the specified allelic class is selectively neutral. This revealed selective equivalence among Adh-S-bearing haplotypes, whose frequencies showed no differentiation throughout the cline, implying high levels of frequency-homogenizing gene flow. Geographical heterogeneity among Adh-F-bearing haplotypes implied the action of selection on one or more additional variants in linkage disequilibrium with Adh-F. In a further study of a subset of the data (n = 1076 from 18 populations), we found a combined insertion/deletion polymorphism, designated &1, located in the 5'' adult intron and in linkage disequilibrium with Adh-F, to show more marked clinal variation than Adh-F/S. Although the unequivocal identification of the precise target(s) of selection requires further study, we suggest that clinal selection may be acting epistatically on the Adh-F/S and &1 polymorphisms.     

From DNA to fitness differences: sequences and structures of adaptive variants of Colias phosphoglucose isomerase (PGI)

Colias eurytheme butterflies display extensive allozyme polymorphism in the enzyme phosphoglucose isomerase (PGI). Earlier studies on biochemical and fitness effects of these genotypes found evidence of strong natural selection maintaining this polymorphism in the wild. Here we analyze the molecular features of this polymorphism by sequencing multiple alleles and modeling their structures. PGI is a dimer with rotational symmetry. Each monomer provides a critical residue to the other monomer's catalytic center. Sequenced alleles differ at multiple amino acid positions, including cryptic charge-neutral variation, but most consistent differences among the electromorph alleles are at the charge-changing amino acid sites. Principal candidate sites of selection, identified by structural and functional analyses and by their variants' population frequencies, occur in interpenetrating loops across the interface between monomers, where they may alter subunit interactions and catalytic center geometry. Comparison to a second (and basal) species, Colias meadii, also polymorphic for PGI under natural selection, reveals one fixed amino acid difference between their PGIs, which is located in the interpenetrating loop and accompanies functional differences among their variants. We also study nucleotide variability among the PGI alleles, comparing these data to similar data from another glycolytic enzyme gene, glyceraldehyde-3-phosphate dehydrogenase. Despite extensive nonsynonymous and synonymous polymorphism at PGI in each species, the only base changes fixed between species are the two causing the amino acid replacement; this absence of synonymous fixation yields a significant McDonald-Kreitman test. Analyses of these data suggest historical population expansion. Positive peaks of Tajima's D statistic, representing regions of neutral "hitchhiking," are found around the principal candidate sites of selection. This study provides novel views of molecular-structural mechanisms, and beginnings of historical evidence, for a long-persistent balanced enzyme polymorphism at PGI in these and perhaps other species.     

Investigating latitudinal clines for life history and stress resistance traits in Drosophila simulans from eastern Australia

Latitudinal clines have been demonstrated for many quantitative traits in Drosophila and are assumed to be due to climatic selection. However, clinal studies are often performed in species of Drosophila that contain common cosmopolitan inversion polymorphisms that also show clinal patterns. These inversion polymorphisms may be responsible for much of the observed clinal variation. Here, we consider latitudinal clines for quantitative traits in Drosophila simulans from eastern Australia. Drosophila simulans does not contain cosmopolitan inversion polymorphisms, so allows the study of clinal selection on quantitative traits that are not confounded by associations with inversions. Body size showed a strong linear cline for both females and males. Starvation resistance exhibited a weak linear cline in females, whereas chill-coma recovery exhibited a significant nonlinear cline in females only. No clinal pattern was evident for development time, male chill-coma recovery, desiccation or heat resistance. We discuss these results with reference to the role inversion polymorphisms play in generating clines in quantitative traits of Drosophila.     

An independent non-linear latitudinal cline for the sn-glycerol-3-phosphate (alpha- Gpdh ) polymorphism of Drosophila melanogaster from eastern Australia

Latitudinal variation of the polymorphic sn-glycerol-3-phosphate (alpha-Gpdh) locus in Drosophila melanogaster has been characterized on several continents; however, apparent clinal patterns are potentially confounded by linkage with an inversion, close associations with other genetic markers that vary clinally, and a tandem alpha-Gpdh pseudogene. Here we compare clinal patterns in alpha-Gpdh with those of other linked markers by testing field flies from eastern Australian locations collected in two separate years. The alpha-Gpdh variation exhibited a consistent non-linear cline reflecting an increase in the alpha-GpdhF allele at extreme latitudes. This pattern was not influenced by the In(2L)t inversion wherein this locus is located, nor was it influenced by the presence of the alpha-Gpdh pseudogene, whose presence was ubiquitous and highly variable among populations. The alpha-Gpdh pattern was also independent of a cline in allozyme frequencies at the alcohol dehydrogenase (Adh) locus, and two length polymorphisms in the Adh gene. These results suggest clinal selection at the alpha-Gpdh locus that is partially or wholly unrelated to linear climatic gradients along the eastern coast of Australia.     

Footprints of selection in wild populations of Bicyclus anynana along a latitudinal cline

One of the major questions in ecology and evolutionary biology is how variation in the genome enables species to adapt to divergent environments. Here, we study footprints of thermal selection in candidate genes in six wild populations of the afrotropical butterfly Bicyclus anynana sampled along a c. 3000 km latitudinal cline. We sequenced coding regions of 31 selected genes with known functions in metabolism, pigment production, development and heat shock responses. These include genes for which we expect a priori a role in thermal adaptation and, thus, varying selection pressures along a latitudinal cline, and genes we do not expect to vary clinally and can be used as controls. We identified amino acid substitution polymorphisms in 13 genes and tested these for clinal variation by correlation analysis of allele frequencies with latitude. In addition, we used two F_ST‐based outlier methods to identify loci with higher population differentiation than expected under neutral evolution, while accounting for potentially confounding effects of population structure and demographic history. Two metabolic enzymes of the glycolytic pathway, UGP and Treh, showed clinal variation. The same loci showed elevated population differentiation and were identified as significant outliers. We found no evidence of clines in the pigmentation genes, heat shock proteins and developmental genes. However, we identified outlier loci in more localized parts of the range in the pigmentation genes yellow and black. We discuss that the observed clinal variation and elevated population divergence in UGP and Treh may reflect adaptation to a geographic thermal gradient.     

Can Diversifying Selection Be Distinguished from History in Geographic Clines? A Population Genomic Study of Killifish (Fundulus heteroclitus)

A common geographical pattern of genetic variation is the one-dimensional cline. Clines may be maintained by diversifying selection across a geographical gradient but can also reflect historical processes such as allopatry followed by secondary contact. To identify loci that may be undergoing diversifying selection, we examined the distribution of geographical variation patterns across the range of the killifish (Fundulus heteroclitus) in 310 loci, including microsatellites, allozymes, and single nucleotide polymorphisms. We employed two approaches to detect loci under strong diversifying selection. First, we developed an automated method to identify clinal variation on a per-locus basis and examined the distribution of clines to detect those that exhibited signifcantly steeper slopes. Second, we employed a classic -outlier method as a complementary approach. We also assessed performance of these techniques using simulations. Overall, latitudinal clines were detected in nearly half of all loci genotyped (i.e., all eight microsatellite loci, 12 of 16 allozyme loci and 44% of the 285 SNPs). With the exception of few outlier loci (notably mtDNA and malate dehydrogenase), the positions and slopes of Fundulus clines were statistically indistinguishable. The high frequency of latitudinal clines across the genome indicates that secondary contact plays a central role in the historical demography of this species. Our simulation results indicate that accurately detecting diversifying selection using genome scans is extremely difficult in species with a strong signal of secondary contact; neutral evolution under this history produces clines as steep as those expected under selection. Based on these results, we propose that demographic history can explain all clinal patterns observed in F. heteroclitus without invoking natural selection to either establish or maintain the pattern we observe today.     

Climatic selection on genes and traits after a 100 year-old invasion: a critical look at the temperate-tropical clines in Drosophila melanogaster from eastern Australia

Drosophila melanogaster invaded Australia around 100 years ago, most likely through a northern invasion. The wide range of climatic conditions in eastern Australia across which D. melanogaster is now found provides an opportunity for researchers to identify traits and genes that are associated with climatic adaptation. Allozyme studies indicate clinal patterns for at least four loci including a strong linear cline in Adh and a non-linear cline in alpha-Gpdh. Inversion clines were initially established from cytological studies but have now been validated with larger sample sizes using molecular markers for breakpoints. Recent collections indicate that some genetic markers (Adh and In(3R)Payne) have changed over the last 20 years reflecting continuing evolution. Heritable clines have been established for quantitative traits including wing length/area, thorax length and cold and heat resistance. A cline in egg size independent of body size and a weak cline in competitive ability have also been established. Postulated clinal patterns for resistance to desiccation and starvation have not been supported by extensive sampling. Experiments under laboratory and semi-natural conditions have suggested selective factors generating clinal patterns, particularly for reproductive patterns over winter. Attempts are being made to link clinal variation in traits to specific genes using QTL analysis and the candidate locus approach, and to identify the genetic architecture of trait variation along the cline. This is proving difficult because of inversion polymorphisms that generate disequilibrium among genes. Substantial gaps still remain in linking clines to field selection and understanding the genetic and physiological basis of the adaptive shifts. However D. melanogaster populations in eastern Australia remain an excellent resource for understanding past and future evolutionary responses to climate change.     

Diversifying selection underlies the origin of allozyme polymorphism at the phosphoglucose isomerase locus in Tigriopus californicus

The marine copepod Tigriopus californicus lives in intertidal rock pools along the Pacific coast, where it exhibits strong, temporally stable population genetic structure. Previous allozyme surveys have found high frequency private alleles among neighboring subpopulations, indicating that there is limited genetic exchange between populations. Here we evaluate the factors responsible for the diversification and maintenance of alleles at the phosphoglucose isomerase (Pgi) locus by evaluating patterns of nucleotide variation underlying previously identified allozyme polymorphism. Copepods were sampled from eleven sites throughout California and Baja California, revealing deep genetic structure among populations as well as genetic variability within populations. Evidence of recombination is limited to the sample from Pescadero and there is no support for linkage disequilibrium across the Pgi locus. Neutrality tests and codon-based models of substitution suggest the action of natural selection due to elevated non-synonymous substitutions at a small number of sites in Pgi. Two sites are identified as the charge-changing residues underlying allozyme polymorphisms in T. californicus. A reanalysis of allozyme variation at several focal populations, spanning a period of 26 years and over 200 generations, shows that Pgi alleles are maintained without notable frequency changes. Our data suggest that diversifying selection accounted for the origin of Pgi allozymes, while McDonald-Kreitman tests and the temporal stability of private allozyme alleles suggests that balancing selection may be involved in the maintenance of amino acid polymorphisms within populations.     

Digest: A clinal polymorphism and life‐history adaptations in Drosophila*

Does genetic variation in the insulin/insulin‐like growth factor signaling pathway (IIS) underlie latitudinal life‐history clines in North American Drosophila melanogaster? Durmaz et al. investigated how a clinally varying polymorphism in the IIS gene foxo affects fitness‐related traits by isolating the effects of alternative low and high latitude alleles. The phenotypic effects of the polymorphism—for example, on body size—matched those normally observed across the cline, suggesting that variation in IIS is important for clinal life‐history adaptation.     

Different selective pressures shape the molecular evolution of color vision in chimpanzee and human populations

A population genetic analysis of the long-wavelength opsin (OPN1LW, "red") color vision gene in a global sample of 236 human nucleotide sequences had previously discovered nine amino acid replacement single nucleotide polymorphisms, which were found at high frequencies in both African and non-African populations and associated with an unusual haplotype diversity. Although this pattern of nucleotide diversity is consistent with balancing selection, it has been argued that a recombination "hot spot" or gene conversion within and between X-linked color vision genes alone may explain these patterns. The current analysis investigates a closely related primate with trichromatism to determine whether color vision gene amino acid polymorphism and signatures of adaptive evolution are characteristic of humans alone. Our population sample of 56 chimpanzee (Pan troglodytes) OPN1LW sequences shows three singleton amino acid polymorphisms and no unusual recombination or linkage disequilibrium patterns across the approximately 5.5-kb region analyzed. Our comparative population genetic approach shows that the patterns of OPN1LW variation in humans and chimpanzees are consistent with positive and purifying selection within the two lineages, respectively. Although the complex role of color vision has been greatly documented in primate evolution in general, it is surprising that trichromatism has followed very different selective trajectories even between humans and our closest relatives.     

The Effect of an Intronic Polymorphism on Alcohol Dehydrogenase Expression in Drosophila Melanogaster

Several lines of evidence indicate that natural selection controls the frequencies of an allozyme polymorphism at the alcohol dehydrogenase (Adh) locus in Drosophila melanogaster. However, because of associations among sequence polymorphisms in the Adh region, it is not clear whether selection acts directly (or solely) on the allozymic site. This problem has been approached by using in vitro mutagenesis to distinguish among the effects on Adh expression of individual polymorphisms. This study shows that a polymorphism within the first Adh intron ( &1) has a significant effect on the level of ADH protein. Like the allozyme, & shows a geographic cline in frequency, indicating that it may also be a target of natural selection. These results suggest that multisite selection models may be required to understand the evolutionary dynamics of individual loci.     

Rapid evolution of an adaptive cyanogenesis cline in introduced North American white clover (Trifolium repens L.)

White clover is polymorphic for cyanogenesis (HCN production after tissue damage), and this herbivore defence polymorphism has served as a classic model for studying adaptive variation. The cyanogenic phenotype requires two interacting biochemical components; the presence/absence of each component is controlled by a simple Mendelian gene (Ac/ac and Li/li). Climate-associated cyanogenesis clines occur in both native (Eurasian) and introduced populations worldwide, with cyanogenic plants predominating in warmer locations. Moreover, previous studies have suggested that epistatic selection may act within populations to maintain cyanogenic (AcLi) plants and acyanogenic plants that lack both components (acli plants) at the expense of plants possessing a single component (Acli and acLi plants). Here, we examine the roles of selection, gene flow and demography in the evolution of a latitudinal cyanogenesis cline in introduced North American populations. Using 1145 plants sampled across a 1650 km transect, we determine the distribution of cyanogenesis variation across the central United States and investigate whether clinal variation is adaptive or an artefact of population introduction history. We also test for the evidence of epistatic selection. We detect a clear latitudinal cline, with cyanogenesis frequencies increasing from 11% to 86% across the transect. Population structure analysis using nine microsatellite loci indicates that the cline is adaptive and not a by-product of demographic history. However, we find no evidence for epistatic selection within populations. Our results provide strong evidence for rapid adaptive evolution in these introduced populations, and they further suggest that the mechanisms maintaining adaptive variation may vary among populations of a species.     

A clinally varying promoter polymorphism associated with adaptive variation in wing size in Drosophila

Body size often shows adaptive clines in many ectotherms across altitude and latitude, but little is known about the genetic basis of these adaptive clines. Here we identify a polymorphism in the Dca (Drosophila cold acclimation) gene in Drosophila melanogaster that influences wing size, affects wing:thorax allometry and also controls a substantial proportion of the clinal wing‐size variation. A polymorphism in the promoter region of Dca had two common alleles showing strong reciprocal clinal variation in frequency with latitude along the east coast of Australia. The Dca‐237 allele increased towards the tropics where wing size is smaller. A within‐population association study highlighted that an increase in the frequency of this allele decreased wing size but did not influence thorax size. A manipulated increase in the level of expression of Dca achieved through UAS‐GAL4 was associated with a decrease in wing size but had no effect on thorax size. This was consistent with higher Dca expression levels in family lines with higher frequency of the Dca‐237 allele. Genetic variation in the promoter region of the Dca gene appears to influence adaptive size variation in the eastern Australian cline of Drosophila melanogaster and accounts for more than 10% of the genetic variation in size within and between populations.     

The functional impact of Pgm amino acid polymorphism on glycogen content in Drosophila melanogaster

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 10 PGM protein haplotypes with respect to PGM activity, thermostability, and adult glycogen content. We find a twofold difference in activity among PGM protein haplotypes that is associated with a threefold 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, parallel 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.     

Molecular evolution of the phytochrome gene family in sorghum: changing rates of synonymous and replacement evolution

The photoreceptor phytochromes, encoded by a small gene family, are responsible for controlling the expression of a number of light-responsive genes and photomorphogenic events, including agronomically important phenotypes such as flowering time and shade-avoidance behavior. The understanding and control of flowering time are particularly important goals in sorghum cultivar development for diverse environments, and naturally occurring variation in the phytochrome genes might prove useful in breeding programs. Also of interest is whether variation observed at the phytochrome loci in domesticated sorghum, or in particular races, is a result of human selection. Population genetic studies can reveal evidence of such selection in patterns of polymorphism and divergence. In this study we report a population genetic analysis of the PHY gene family in Sorghum bicolor (L.) Moench in a diverse panel including both cultivated and wild accessions. We show that the level of nucleotide variation in all gene family members is about half the average for this species, consistent with purifying selection acting on these loci. However, the rate of amino acid substitution is accelerated at PHYC compared to the other two loci. In comparisons to a closely related sorghum species, PHYC shows a pattern of intermediate frequency amino acid changes that differ from the patterns observed in comparisons across longer evolutionary distances. There is also a departure from expected patterns of polymorphism and divergence at synonymous sites in PHYC, although the data do not fit a simple model of directional or diversifying selection. Cultivated sorghum has a level of variation similar to that of wild relatives (ssp. verticilliflorum), but many polymorphisms are subspecies-specific, including several amino acid variants.     

Ecological genomics of Anopheles gambiae along a latitudinal cline: a population-resequencing approach

The association between fitness-related phenotypic traits and an environmental gradient offers one of the best opportunities to study the interplay between natural selection and migration. In cases in which specific genetic variants also show such clinal patterns, it may be possible to uncover the mutations responsible for local adaptation. The malaria vector, Anopheles gambiae, is associated with a latitudinal cline in aridity in Cameroon; a large inversion on chromosome 2L of this mosquito shows large differences in frequency along this cline, with high frequencies of the inverted karyotype present in northern, more arid populations and an almost complete absence of the inverted arrangement in southern populations. Here we use a genome resequencing approach to investigate patterns of population divergence along the cline. By sequencing pools of individuals from both ends of the cline as well as in the center of the cline- where the inversion is present in intermediate frequency- we demonstrate almost complete panmixia across collinear parts of the genome and high levels of differentiation in inverted parts of the genome. Sequencing of separate pools of each inversion arrangement in the center of the cline reveals large amounts of gene flux (i.e., gene conversion and double crossovers) even within inverted regions, especially away from the inversion breakpoints. The interplay between natural selection, migration, and gene flux allows us to identify several candidate genes responsible for the match between inversion frequency and environmental variables. These results, coupled with similar conclusions from studies of clinal variation in Drosophila, point to a number of important biological functions associated with local environmental adaptation.     

Latitudinal allozymic variability in natural populations ofZaprionus indianus from India

Twelve natural populations ofZ. indianus from India, collected along lines of latitude, were analyzed electrophoretically and the geographical pattern of variation was compared at 15 allozyme loci. Except forMdh-2, all the loci revealed extensive polymorphism. The allele frequency variation revealed a statistically significant correlation as well as regression coefficients with the latitude. The populations revealed extensive clinal variation at all the polymorphic loci, which depicted moderate to higherF _ST values. Thus, the observed genetic divergence in colonizing populations ofZ. indianus shows the role of natural selection that varies spatially along the north-south axis of the Indian subcontinent.Financial assistance from UGC, New Delhi, is gratefully acknowledged.     

Properties of allelic variants of phosphoglucomutase from the sea anemone Metridium senile

The phosphoglucomutase (Pgm) locus from populations of the sea anemone Metridium senile has three alleles in natural populations from the northeastern coast of North America. Two of the alleles exhibit clinal variation north of Cape Cod, suggesting a possible association of allele frequency with environmental temperature. This clinal pattern is reproducible and stable over at least brief periods of time. The allozymes encoded by each of the six Pgm genotypes have been partially purified and characterized. The symmetrical pH optimum for Vmax is pH 7.5; the apparent Km (Kmapp) of glucose-1-phosphate declines monotonically as the pH increases from 6.5 to 8.5. There are no pronounced differences in heat stabilities of PGM produced by various genotypes, nor are there significant differences in specific activities. There are no differences in the sensitivity of Vmax to temperature. Kmapp values are very low for all genotypes, ranging from about 2 to 12 microM, depending upon the temperature. Kmapp of glucose-1-phosphate declines as the temperature is raised for all genotypes, whether the pH is held constant or allowed to vary with the temperature. Under certain conditions, there are small significant differences among genotypes in Kappm values, but there is no systematic pattern to these differences. The present data provide no biochemical explanation for the maintenance of the Pgm cline by selection for functional differences under different thermal regimes.