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.     

Adaptation at specific loci. VII. Natural selection,dispersal and the diversity of molecular-functional variation patterns among butterfly species complexes (Colias: Lepidoptera,Pieridae)

Natural genetic variants at the phosphoglucose isomerase, PGI, gene differ in spatial patterning of their polymorphism among species complexes of Colias butterflies in North America. In both lowland and alpine complexes, molecular-functional properties of the polymorphic genotypes can be used to predict genotype-specific adult flight performances and resulting large genotypic differences in adult fitness components. In the lowland species complex, there is striking uniformity of PGI polymorph frequencies at a number of sites across the American West; this fits with earlier findings of strong, similar differences in fitness components over this range. In an alpine complex, Colias meadii shows similar uniformity of PGI frequencies within habitat types, either montane steppe or alpine tundra, over several hundred kilometres in the absence of dispersal. At the same time, large shifts (10-20%) in frequency of the most common alleles occur between steppe and tundra populations, whether these are isolated or, as in some cases, are in contact and exchange many dispersing adults each generation. Data on male mating success of common C. meadii PGI genotypes in steppe and tundra show heterozygote advantage in both habitat types, with shifts in relative homozygote disadvantage between habitats which are consistent with observed frequency differences. Nonadaptive explanations for this situation are rejected, and alternative, thermal-ecology-based adaptive hypotheses are proposed for later experimental test. These findings show that strong local selection may dominate dispersal as an evolutionary agent, whether or not dispersal is present, and that selection may often be the major force promoting 'cohesion' of species over long distances. This case offers new opportunities for integrating studies of molecular structure and function with ecological aspects of natural selection in the wild, both within and among species.     

Adaptation at Specific Loci. V. Metabolically Adjacent Enzyme Loci May Have Very Distinct Experiences of Selective Pressures

The polymorphic phosphoglucomutase (PGM) and glucose-6-phosphate dehydrogenase (G6PD) loci have been studied in parallel to experimental work on the phosphoglucose isomerase (PGI) polymorphism in Colias butterflies. PGI, PGM and G6PD are also autosomal in Colias. PGM and G6PD are loosely linked (and represent the first identified autosomal linkage group in Colias); they assort independently from PGI. Recombination occurs in both sexes. Neither PGM nor G6PD shows large, consistent differences in flight capacity through the day among its genotypes, as PGI does. PGM shows some change of allele frequencies, and match to Hardy-Weinberg expectation, with air temperature in middle and latter parts of the season, but not early in the season. G6PD may show some heterozygote excess over Hardy-Weinberg expectation early in the day, but more testing is needed. No evidence for differential survivorship was seen at PGM or G6PD, in contrast to PGI. At the PGM and G6PD loci, male heterozygotes are advantaged in mating with females, but without the evidence of female choice which occurs for PGI. These effects are not correlated among the three loci. There is no assortative mating at G6PD (nor at PGI). There is minor positive assortative mating of PGM heterozygotes, but it is too weak to account for the PGM-genotype-specific male mating advantage. No trends of multilocus genotype frequencies involving PGI are seen. Certain PGM-G6PD two-locus genotypes are over-represented, and others under-represented, in wild adult samples, particularly among males and uniformly among successfully mating males. Our results emphasize that enzyme loci sharing a substrate need not have common experience of the existence or strength of natural selection, and suggest initial food-resource processing and allocation as a possible context for fitness-related effects of the PGM and G6PD polymorphisms.     

Balancing Selection on Electrophoretic Variation of Phosphoglucose Isomerase in Two Species of Field Cricket: Gryllus Veletis and G. Pennsylvanicus

Two species of crickets, Gryllus veletis and G. pennsylvanicus, share six electrophoretic mobility classes for the enzyme phosphoglucose isomerase (PGI), despite evidence from other genetic markers that the two species are not closely related within eastern North American field crickets. Moreover, the frequencies of the two most common PGI electrophoretic classes (PGI-100 and PGI-65) covary in sympatric populations of these species in the eastern United States, suggesting that PGI may be subject to trans-specific balancing selection. To determine the molecular basis of the electrophoretic variation, we characterized the DNA sequence of the Pgi gene from 29 crickets (15 G. veletis and 14 G. pennsylvanicus). Amino acid substitutions that distinguish the electrophoretic classes are not the same in the two species, and there is no evidence that specific replacement substitutions represent trans-specific polymorphism. In particular, the amino acids that diagnose the PGI-65 allele relative to the PGI-100 allele differ both between G. veletis and G. pennsylvanicus and within G. pennsylvanicus. The heterogeneity among electrophoretic classes that covary in sympatric populations coupled with analysis of patterns of nucleotide variation suggest that Pgi is not evolving neutrally. Instead, the data are consistent with balancing selection operating on an emergent property of the PGI protein.     

Gd- allele distribution patterns in Azerbaijan. III. The identification of mutant forms of glucose-6-phosphate dehydrogenase

In 28 families with G6PD deficiency living in 3 settlements of Shekii district of Azerbaijan 11 G6PD variants of II and III classes differing by kinetic properties were identified according WHO program. 9 of them are characterized with the same electrophoretic mobility. Comparison of G6PD spectra in two subpopulations and in a mixed group permits to make a conclusion about existence of common and rare G6PD alleles in examined population. They distribute by gene drift supported by natural selection. Among 7 samples of G6PD with normal and increased activity two new variants of IV class -- Nukha and Bash-Kungut -- were found.     

The role of stress proteins in responses of a montane willow leaf beetle to environmental temperature variation

The heat shock response is a critical mechanism by which organisms buffer effects of variable and unpredictable environmental temperatures. Upregulation of heat shock proteins (Hsps) increases survival after exposure to stressful conditions in nature, although benefits of Hsp expression are often balanced by costs to growth and reproductive success. Hsp-assisted folding of variant polypeptides may prevent development of unfit phenotypes; thus, some differences in Hsp expression among natural populations of ectotherms may be due to interactions between enzyme variants (allozymes) and Hsps. In the Sierra willow leaf beetle Chrysomela aeneicollis, which lives in highly variable thermal habitats at the southern edge of their range in the Eastern Sierra Nevada, California, allele frequencies at the enzyme locus phosphoglucose isomerase (PGI) vary across a climatic latitudinal gradient. PGI allozymes differ in kinetic properties, and expression of a 70 kDa Hsp differs between populations, along elevation gradients, and among PGI genotypes. Differences in Hsp 70 expression among PGI genotypes correspond to differences in thermal tolerance and traits important for reproductive success, such as running speed, survival and fecundity. Thus, differential Hsp expression among genotypes may allow functionally important genetic variation to persist, allowing populations to respond effectively to environmental change.     

Temperature-dependent kinetic variation among phosphoglucose isomerase allozymes from the wing-polymorphic water strider, Limnoporus canaliculatus

Phosphoglucose isomerase (PGI) allozymes were isolated from the wing- polymorphic water strider, Limnoporus canaliculatus, and were characterized biochemically with respect to temperature-dependent kinetic and thermostability properties. At higher temperatures, the allozymes exhibited significant differences in Michaelis constant (Km) values for substrates of both the forward and reverse reaction directions. Results were consistent with expectations of adaptive kinetic differentiation based on the latitudinal variation of PGI allele frequencies. PGI genotypes also differed with regard to maximal velocity (Vmax)/Km ratios at higher temperatures. These differences were due primarily, if not exclusively, to allozyme-dependent variation in Km values. The allozymes also exhibited dramatic differences in thermostability. However, no thermostability differences were observed when the substrate analogue 6-phosphogluconate was present in the incubation medium. The data from this study, together with data from Mytilus edulis and Metridium senile on temperature-dependent kinetic variation among PGI allozymes, form a consistent picture of natural selection influencing the clinal variation of alleles at this locus in these three phylogenetically distant organisms. More definitive support of this hypothesis, however, must await additional studies on the physiological effects of the allozymic variation as well as direct measurements of fitness differences among the enzyme genotypes.      

Electrophoretic detection of enzyme variation among Japanese red-coloured spider mites of the genus Tetranychus (Acari: Tetranychidae)

The genetic variation in phosphoglucoisomerase (PGI) and malate dehydrogenase (MDH) was studied in red-pigmented Japanese spider mites of the genus Tetranychus by means of poly-acrylamide gel electrophoresis. The analysis revealed (1) that Tetranychus kanzawai possesses five and three alleles for PGI and MDH, respectively and that PGI allele frequencies clearly differ between the Hokkaido and the Honshu populations (2) that Tetranychus urticae and Tetranychus pueraricola, two closely related species, have different alleles for PGI and (3) that two populations of Tetranychus piercei, a species which had only been found on the islands of Okinawa, were obtained from Honshu and that the PGI locus is fixed for different alleles in the two populations. The two enzyme systems are apparently useful not only for discrimination of spider mite species but also for the detection of intraspecific variation. © Rapid Science Ltd. 1998     

Isolation and genetic characterization of alcohol dehydrogenase thermostability variants occurring in natural populations of Drosophila melanogaster

Drosophila melanogaster collected from natural populations were examined fo thermostability variants within electrophoretic mobility classes of two enzymes. In alcohol dehydrogenases, two discrete forms of the "slow" allozyme and three discrete forms of the "fast" allozyme were revealed by postelectrophoretic treatments ranging from 15 sec at 40 C to 40 sec at 43 C. All variants have been mapped to within 0.7 unit of the Adh locus. Results of a geographic survey indicate that two alleles giving rise to fast-moderate and slow-moderate allozymes are common everywhere; other variants have a collective frequency ranging from 0% to 7%. In a test of the possibility that the rare Adh alleles could be generated by intragenic recombination between the two common alleles, electrophoresis and heat treatment of progeny recombinant for flanking markers of Adh revealed no new allozymes. Among 27 stocks containing slow alpha-glycerophosphate dehydrogenase allozymes and 109 fast stocks, heat treatments revealed no additional variation.     

Functional Effects of Pgi Allozymes in ESCHERICHIA COLI

Five alleles representing three electromorphs of phosphoglucose isomerase (PGI) have been transferred from natural isolates of E. coli into the genetic background of E. coli K12 and examined for their effect on growth rate in chemostats limited for glucose or fructose. With glucose limitation, all alleles are selectively neutral or nearly neutral within the limit of resolution of the technique, whether the genetic background is nonmutant or whether it contains a deletion of the locus of glucose-6-phosphate dehydrogenase, the enzyme that provides an alternative metabolic pathway for the substrate of PGI. With fructose limitation, one of the naturally occurring alleles has a small but reproducible detrimental effect on growth rate. A kinetic difference in this detrimental allozyme, apparently relating to an inhibition constant, has been observed in some, but not all, lots of substrate, and a similar difference has also been noted in one of the rare electromorphs that could not be transferred into E. coli K12. These results support a model of genetic variation in which the alleles are neutral or nearly neutral in the prevailing environment but have a potential for selection that can be expressed under the appropriate conditions of environment or genetic background. This hypothesis is discussed in the context of allozyme polymorphisms observed in other organisms.     

Is natural selection a plausible explanation for the distribution of Idh‐1 alleles in the cricket Allonemobius socius?

Abstract.  1. Allozyme alleles in natural populations have been proposed as either neutral markers of genetic diversity or the product of natural selection on enzyme function, as amino acid substitutions that change electrophoretic mobility may also alter enzyme performance. To address these possibilities, researchers have used both correlative analyses and empirical studies.
2. Here, geographically structured variation of the enzyme isocitrate dehydrogenase ( Idh- 1) in the striped ground cricket Allonemobius socius Scudder (Orthoptera: Gryllidae) is examined. The distributions of Idh- 1 alleles appear to be related to environmental gradients, as allele frequencies showed significant relationships with mean annual temperature and precipitation. Specifically, the slowest mobility allele was more frequent at colder temperatures, while the converse occurred for the fastest mobility allele.
3. An exploratory experiment was performed to examine fitness effects of possessing different Idh- 1 alleles at two temperatures to test the hypothesis that the geographic structure of this locus may reflect environmental adaptation. Results showed that a significant interaction between temperature and Idh- 1 genotype affected the number of eggs laid, with success of homozygous individuals matching environmental expectations.
4. The above results show that (1) variation in the frequency of Idh- 1 alleles is significantly related to environmental gradients in the eastern U.S.A. and (2) alternative alleles of Idh- 1 appear to influence the egg-laying ability of individuals differently depending on environmental temperature. Together, these results suggest that natural selection is a plausible mechanism underlying the distribution of Idh- 1 alleles in this species, although more detailed studies are needed.     

Isoallele Frequencies in Very Large Populations

The frequencies of electrophoretically distinguishable allelic forms of enzymes may be very different from the corresponding frequencies of structurally distinct forms, because many sequence variants may have identical electrophoretic charge. In large populations such frequencies will be determined largely by the number of amino acid sites that are free to vary. The number of distinguishable electrophoretic variants will remain fairly small. Beyond some limiting size, no further effect of population size on allele frequencies is expected, so isolated large populations will have closely similar allele frequencies if polymorphism is due largely to mutation and drift. The most common electrophoretic alleles are expected to be flanked by the next most common, with the rarer alleles increasingly distal. Neither strong selection nor mutation/drift interpretations of enzyme polymorphism are yet disproven, nor is any point between these extremes.     

Geographical variation in leucine aminopeptidase in Mytilus edulis L. From the Irish coasts

Phenotypic variation in leucine aminopeptidase has been demonstrated in Mytilus edulis L. from twelve localities in Ireland. It is proposed that one locus with three common and two rare alleles is responsible for one group of variants. The frequencies of the common alleles are significantly correlated with the relative amount of wave action (exposure) at the sites investigated. M. edulis seems to exhibit an above average amount of genetic variation thereby paralleling the variations in growth rate, shell morphology, filtration rate, and certain kinetic parameters previously recorded amongst individuals of this species. In view of the apparently close correlation between genotype and environmental conditions and since genotype in other species is known to control survival and growth rate, genetic studies of the kind described here could clearly have important implications for the commercial mussel fishery.     

Topological effects on the electrophoretic mobility of rigid rodlike DNA in polyacrylamide gels

The electrophoretic migration of rigid rodlike DNA structures with well defined topologies has been investigated in polyacrylamide (PA) hydrogels prepared by copolymerization of acrylamide and N, N'-methylenebisacrylamide. Previous studies have reported structural and dynamic characteristics of linear and branched DNA during electrophoresis in PA gels using a variety of experimental parameters. However, a thorough investigation aimed at establishing specific relationships between topological features of rigid rodlike DNA structures and their electrophoretic behavior is still needed. In order to study these topological effects on mobility, an intensive examination of the electrophoretic mobility of small linear and starlike DNA was performed. A series of model DNA structures with well-defined branched topologies were synthesized with varying molecular parameters, such as number of arms surrounding the branch point and arm length. The electrophoretic mobility of these structures was then contrasted with a series of data obtained using linear DNA of comparable molecular size. When large DNA stars (M >/= 60 bp) were compared with linear DNA of identical molecular weight, the Ferguson plots were quite different. However, small DNA stars (24-32 bp) and linear analogues had identical Ferguson plots. This indicates that a different motional mode or greater interaction with the gel exists for the larger DNA stars. When the total molecular weight of the DNA stars was held constant and the number of arms varied, the Ferguson plots for all the stars were identical. Additionally, a critical pore size was reached when the ratio of linear DNA mobility to star DNA mobility increased dramatically. Thus, while the incorporation of a single branch point can produce a large reduction in mobility, above a critical molecular size, the incorporation of additional branch points does not appear to provide further reduction in mobility. This finding is consistent with the transport properties of large synthetic star polymers, where a large reduction in their diffusion coefficient is observed when a single branch is added. When additional arms are incorporated, large synthetic stars do not display an appreciable further reduction in diffusion coefficient. The effect of arm length on mobility for rigid rod DNA stars was also studied. For four-arm DNA stars, the mobility was found to scale as an exponential function of the arm length. Finally, a recently proposed phenomenological model was used to successfully fit the mobility data for linear rigid rod DNA at various concentrations of PA.     

Functional hemizygosity in the human genome: direct estimate from twelve erythrocyte enzyme loci

Summary Cord blood samples from 2020 unrelated newborns were screened for levels of enzyme activity for twelve enzymes. The level of enzymatic activity for 100 determinations were consistent with the existence of an enzyme-deficiency allele. The frequency of deficiency alleles in the Black population (0.0071) was four times higher (after removal of the G6PD^*A^- variant) than in the Caucasian sample (0.0016). These frequencies are approximately double the frequency of rare electrophoretic mobility variants at similar loci in the same population. Given the number of functionally important loci in the human genome, these enzyme deficiency variants could constitute a significant health burden.     

Adaptation at Specific Loci. II. Demographic and Biochemical Elements in the Maintenance of the Colias Pgi Polymorphism

Demographically oriented sampling in the wild and biochemical study of allozymes in the laboratory have been used to probe maintenance of the phosphoglucose isomerase polymorphism of Colias butterflies.—The several alleles at this locus show negative or no covariation among their frequencies in the wild. This rules out Wahlund effects as a cause of observations of heterozygote excess at this locus in broods that fly as single cohorts. Unusually heavy mortality among adults, due to drought stress or other causes, can preclude manifestation of differential survivorship among phosphoglucose isomerase genotypes. In broods composed of overlapping cohorts, heterozygote deficiency, apparently due to Wahlund effects in time as cohorts of different survivorship experience mix, can be found. Heterozygotes at this locus fly under a broader range of weather conditions than other genotypes.—Previously detected kinetic differentiation among the genotypes extends in greater magnitude to the glycolytic reaction direction, as well as to a broader range of test conditions than examined before. The heterozygote 3/4 is strikingly heterotic for several measures of kinetic functional effectiveness. Other heterozygotes are sometimes heterotic, more often intermediate (but not exactly so, nor additive in any sense) in properties between homozygotes.—Predictions are made from the biochemical analysis and from the insects' thermal ecology concerning distributions of the genotypes in the wild. Some agree with facts already established. Others are tested and confirmed from data already on hand. Still others are to be tested as reported in an accompanying paper.—All available evidence points to a combination of heterozygote advantage and fluctuating-environment selection as responsible for maintaining this polymorphism. There is considerable evidence for the operation of protein-structural constraint on the range of adaptations possible at this locus.     

An Experimental Test of Adaptive Introgression in Locally Adapted Populations of Splash Pool Copepods

As species struggle to keep pace with the rapidly warming climate, adaptive introgression of beneficial alleles from closely related species or populations provides a possible avenue for rapid adaptation. We investigate the potential for adaptive introgression in the copepod, Tigriopus californicus, by hybridizing two populations with divergent heat tolerance limits. We subjected hybrids to strong heat selection for 15 generations followed by whole-genome resequencing. Utilizing a hybridize evolve and resequence (HER) technique, we can identify loci responding to heat selection via a change in allele frequency. We successfully increased the heat tolerance (measured as LT₅₀) in selected lines, which was coupled with higher frequencies of alleles from the southern (heat tolerant) population. These repeatable changes in allele frequencies occurred on all 12 chromosomes across all independent selected lines, providing evidence that heat tolerance is polygenic. These loci contained genes with lower protein-coding sequence divergence than the genome-wide average, indicating that these loci are highly conserved between the two populations. In addition, these loci were enriched in genes that changed expression patterns between selected and control lines in response to a nonlethal heat shock. Therefore, we hypothesize that the mechanism of heat tolerance divergence is explained by differential gene expression of highly conserved genes. The HER approach offers a unique solution to identifying genetic variants contributing to polygenic traits, especially variants that might be missed through other population genomic approaches.     

Examination of Allelic Variation at the Hexokinase Loci of DROSOPHILA PSEUDOOBSCURA and D. PERSIMILIS by Different Methods

Recently a number of electrophoretic techniques have been applied to reveal the presence of additional genetic variation among the electrophoretic mobility classes of the highly polymorphic xanthine dehydrogenase (XDH ) and esterase-5 (est-5) loci. We examined the hexokinase loci of Drosophila pseudoobscura and D. persimilis using a variety of techniques to determine whether further allelic variation could be revealed for these much less polymorphic loci and to analyze the nature of the known variation at the hexokinase-1 (hex-1) locus. The following studies were conducted: 135 strains of the two species from six localities were examined with buffer pH ranging from 5.5 to 10.0; 40 strains of D. pseudoobscura and 9 strains of D. persimilis from Mather were studied using starch gel concentrations ranging from 8.5 to 15.5% and were examined for differences in heat stability and reactivity to the thiol reagent pCMSA; strains were also tested for susceptibility to urea denaturation and differences in relative activities. Major findings of the work are: (1) No additional allelic variation could be detected at any of the hexokinase loci by applying these techniques. The finding of abundant hidden genetic variation in XDH and est-5 does not extend to all enzyme loci. (2) Evidence from studies using pCMSA indicates that the hex-1 alleles 0.6, 0.8, 1.0 and 1.2 of the two species form a series of unit charge steps. Since the 0.94 allele of D. persimilis has mobility intermediate between 0.8 and 1.0, it is argued that routine electrophoretic techniques are sensitive to at least some conservative amino acid substitutions. (3) Strong correlations were found at the hex-1 locus between low allelic frequency, reduced relative activity and reduced stability to heat and urea denaturation. Since the three sibling species, D. pseudoobscura, D. persimilis and D. miranda, all appear to share a common high frequency allele (1.0) at that locus, these findings are taken as evidence that the observed allelic frequencies are a result of directional selection and mutation, rather than any form of balancing selection.     

Genetic differentiation between alpine and lowland populations of a butterfly is related to PGI enzyme genotype

Understanding the ecological process of population differentiation and identifying the molecular changes that contribute to adaptation is a central aspect of evolutionary biology. In this study we analyzed the geographic variation in allozyme allele frequencies (based on 15 enzyme systems representing 18 loci) across 18 populations of the butterfly Lycaena tityrus from different altitudes. Population genetic analyses showed that within population genetic diversity (e.g. mean number of alleles per loci: 1.8; expected heterozygosity: 12%) was within the typical value range for the Lepidoptera. The populations of L. tityrus investigated showed a remarkable genetic differentiation (F_ST: 0.065), being clearly divided into an alpine (high-altitude) and a non-alpine (low-altitude) cluster. This differentiation was almost entirely caused by variation at a single enzyme locus, PGI. Although the involvement of historical events cannot be ruled out, several lines of evidence suggest that the specific pattern of allozyme (and in this case particularly PGI) variation found is caused by thermal selection. For example, genetic variation was highly locus-specific rather than relatively uniform, as should be expected for effects of natural selection. Further, the PGI 2–2 genotype dominating in alpine (in contrast to low-altitude) populations is known to exhibit increased cold stress resistance and relatively long development times typical of alpine populations. Thus, PGI (or possibly a closely linked gene) is an obvious target for thermal selection in L. tityrus . This study exemplifies how allozyme analyses can be used to detect candidate loci likely to be under selection.