Adaptation at Specific Loci. I. Natural Selection on Phosphoglucose Isomerase of Colias Butterflies: Biochemical and Population Aspects

ADAPTATION AT SPECIFIC LOCI. I. NATURAL SELECTION ON PHOSPHOGLUCOSE ISOMERASE OF COLIAS BUTTERFLIES: BIOCHEMICAL AND POPULATION ASPECTS

WARD B. WATT Departmeni of Biological Sciences, Stanford University, Stanford, California 94305* and The Rocky Mountain Biological Laboratory, Crested Butte, Colorado 81224 Manuscript received February 18, 1977

ABSTRACT

Electrophoretic variants of phosphoglucose isomerase (PGI) in Colias butterflies have been studied from field and laboratory viewpoints. The transmission pattern is that of a dimeric enzyme controlled by one structural gene locus. Populations usually harbor four to six allelic mobility classes. These mobility classes are shared among species complexes, though their frequencies differ widely. Preliminary Ferguson plot analysis of the variants has been carried out. Purified preparations of Colias PGI alleles are more effective in standardizing Ferguson plots than heterologous proteins, such as ferritin. Variation of Ferguson plot parameters is not an infallible guide to electrophoretically “cryptic alleles,” as one putative case proved to be due to non- allele-specific effects. S, M, and F mobility classes in two Colias semispecies show the same retardation coefficients in Ferguson plots. Adults early in the flight periods of their nonoverlapping generations show genotype frequencies in Hardy-Weinberg equilibrium, but heterozygote excess develops as the insects age. Simple directional selection and large-scale population mixing are unlikely to be causes of this, although several other selection modes remain possible. Identical-by-descent lines of the four frequent-todcommon alleles in C. eurytheme have been set up in culture, and enzyme has been purified from these for study of functional properties. Major differenecs in heat stability and in various kinetic parameters are found among the ten possible genotypes. In some cases, heterosis for kinetic parameters is seen; in other cases, opposing trends in kinetic function and heat stability create potential for net heterosis in function. Possible interpretations of these results in an adaptive metabolic context are discussed, and directions for further work are stated.

DAPTATION” is a central but troublesome concept in evolutionary biology. “AIt represents qualities OI phenotypic suitedness to environment which should be translatable into Darwinian fitness differentials, the selection coefficients of evolutionary population genetic theory. We do not yet know in any general terms how to perform this translation. One reason may be that only in a few cases have specific features of adaptations been traced in mechanistic terms back to their genetic basis as brought together by natural selection. AS the case of sickle-cell anemia in man (e.g.,ALLISON 1957; INGRAM1963) shows,

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Genetics 87: 177-194. September, 1977. 178 W. B. WATT crucial features of adaptation may occur at biochemical, physiological, and ecological levels. Therefore, we should like to study cases of adaptation at specific gene loci, preferably including as much allelic variability as possible for com- parative purposes, giving attention to a broad range of levels of organization. Such a body of data should give better general understanding of adaptive mechanisms and lead to a clearer concept of adaptation. The use of gel electrophoresis for surveying natural genetic variation in a variety of enzymes and organisms offers large opportunities for such work. If this variation were neutral to the action of selection, it would be uninformative concerning adaptive mechanisms. But the beginnings of study of functional differences among allotymes. in combination with field data on the same variants, suggest that much of this variation is selected upon (e.g., KOEHN,PEREZ and MERRITT1971; MERRITT1972; CLARKE1975). Important aspects of this variation might also result from epistatic interactions among loci, rather than locus- by-locus changes in properties (e.g.,SIMPSON 1964; FRANKLINand LEWONTIN 1970). The balance of importance between locus-specific properties and epistatic interactions will itself have to be sorted out empirically. The present work begins the study of variation in carbohydrate-metabolizing enzymes of Colias (“Sulfur”) butterflies. Work on the thermal physiology and ecology of adults (WATT1968) and larvae (SHERMANand WATT1973) of these insects suggested that study of thermal adaptations in energy-yielding biochemical processes might be valuable. Dietary input to adult Colias’ carbohydrate metabolism is a well-characterized mixture of simple sugars from flower nectar (WATT,HOCH and MILLS1974). Therefore, we have concentrated on the branch point region of metabolism concerned with initial carbohydrate intake, and storage or use. Phosphoglucose isomerase (PGI, E.C.5.3.1.9) has been studied first. Extensive PGI polymorphisms, detectable by gel electrophoresis, are found in Colias populations. The enzyme is present in large quantities in the insects, and can be purified in high yield. Thus the variants can be studied readily in the metabolic context as well as by field sampling. In choosing which functional properties of PGI variants to examine first, priority was given to possible action of thermal selective pressures. Sensitivity to heat denaturation was one obvious consideration, as was genotypic variation in thermal responses of kinetic parameters-but which parameters? Early com- parative biochemical work focused on the maximum activity of enzymes. More recently, HOCHACHKAand SOMERO(e.g., 1973) and others have emphasized substrate binding affinity, of which the Michaelis constant K, is a relative measure, though ultimately the actual binding constants themselves are of primary interest. The specific metabolic role of PGI had also to be thought of. This enzyme, which reversibly interconverts fructose-6-phosphate (F6P) and glucose- 6-phosphate (G6P), mediates among several reaction sequences: supply of F6P and G6P from nectar food via hexokinase; movement of resources to and from glycogen storage via a sequence leading to and from glycogen synthetase and phosphorylase, respectively; supply of resources to glycolysis via phosphofructokinase; or use of the pentose shunt in tissues which operate that pathway. SELECTION ON COLIAS PHOSPHOGLUCOSE ISOMERASE 179 PGI is thus quite literally a coupling enzyme among rate-limiting, frequently allosterically regulated reactions that allocate carbohydrates to different metabolic uses. EASTERBY( 1973) has analyzed the kinetic behavior of coupled enzyme sequences in some detail. His work first confirmed earlier conclusions that the maximum activity of coupling enzyme; in a pathway must be much greater than that of the “control-step” enzymes, if the latter are to be truly rate limiting and the pathway therefore responsive to metabolic regulation. Further, there is the matter of the response time of coupling steps to flux changes at rate-limiting steps preceding them. EASTERBYhas shown that the ratio V,,,/K, (which we may term the “coupling ratio”) of a coupling enzyme is inversely related to the “transient time” of that step, i.e., the time required for a change in flux into that step to be reflected in the flux out of it. (In his analysis, K,/V,,, = 7;57 is then the time required for flux exiting the step to reach 99% of its final value.) The transient time of a sequence of reactions is the sum of the individual step transients. Since the response time of resource mobilization in insects is as crucial as the level of response (see SACKTOR1970 and discussion below), the coupling ratios of Colias’ PGI allozymes at various temperatures are at least as important as their component parameters V,,, and &.

MATERIALS AND METHODS

Members of three different species complexes of Colias have been studied so far. The alpine and subalpine Colias meadii Edwards has been studied at Cumberland Pass, Gunnison CO., Colorado, and on the Carson Divide, Hinsdale Co., Colorado. Colias alexandra Edwards, in a different species complex o€ montane and boreal grasslands, has been sampled at Brush Creek, Gunnison Co., Colorado. In the lowland North American species complex, two semispecies have been studied: C. philodics eriphyle Edwards, at the base of Crested Butte Mountain, Gunnison Co., Colorado, and C. eurytheme Boisduval, near Tracy, San Joaquin Co., California. The last named taxon is maintained in our laboratory in continuous culture, using vetch (Vicia) as larval food plant. Field sampled animals were captured with hand nets in strict order of nearest accessibility, to avoid collecting biases. A complication in the study of allozyme variants at the functional level is the possible occurrence of electrophoretically “cryptic” alleles which might differ in functional properties without detectable charge differences. To avoid confusion of results, this work has used stocks of C. eurytheme selected in the laboratory to contain only identical-by-descent copies of each of the four frequent-to-common PGZ alleles in this species : T,S,M,F. It will of course be important later to conduct an explicit search for such c‘~rypti~”alleles in the wild. Inbreeding depres- sion and related problems are minimized by the fact that these stocks are not isogenic. With a haploid chromosome number of 31 (MAEKI and REMINGTON1960), plus the presumption of recombination within linkage groups, very little of the genome need be affected to secure identity-by-descent of alleles at any one locus. Much variability remains, as shown by examining the Est-D locus (BURNS1975), which is still segregating 8-10 alleles in these stocks. Animals to be assayed are killed by section of the ventral nerve cord. Their abdomens are then removed and ground, using a Teflon-and-glass homogenizer, in 0.6 or 0.8 ml of a standard grinding buffer: 0.05 M TRIS, 0.1 M glucose, 0.1 M NH,CI, 0.001 M EDTA (ethylenediamine- tetraacetate), 0.001 M dithiothreitol, 0.02% sodium azide (antibacterial), pH 7.41 at 23”. Homogenates are placed in 1.5 ml plastic centrifuge tubes, cleared by 10 min top speed spin in a clinical centrifuge, then stored at 4”. Discontinuous polyacrylamide gel electrophoresis is carried out in Buchler or BioRad apparatus. For routine survey work, the TRIS-glycine buffer system of DAVIS(1964), using 180 W. B. WATT a 3% acrylamide stacking gel and an 8% separating gel, is used. Gels for Ferguson plot work (see below) use an imidazole buffer system as follows: separating gels are cast at the desired acrylamide percentage, using a “bis” concentration appropriate to maintain a constant crosslink percentage C = 0.4%, in 0.38 M imidazole, pH 7.9. Stacking gels are cast at 4% acrylamide in 0.05 M imidazole, pH 5.9. The electrode buffer is a solution of 0.002 M imidazole and 0.01 M asparagine, pH 7.6. JOHNSON (1975a,b) has introduced the Ferguson plot (see RODBARDand CHRAMBACH1971) as a criterion of electrophoretic allele identity. The logarithm of protein mobility, either as absolute value or normalized to a standard, is plotted against percent acrylamide from a series of gel runs. The resulting line’s extrapolated Y-intercept is related to gel-free electrophoretic mobility; the slope in large scale is related to molecular weight of the protein, and in small scale is reportedly sensitive to changes in protein conformation (JOHNSON1975a). We have used the imidazole buffer system for preliminary Ferguson plot study of Colias PGI. While the TRIS- glycine system runs faster, its higher pH causes the PGI allozymes to run in a more compressed pattern than that of the imidazole system, which is thus potentially more sensitive to small changes in electrophoretic behavior. For routine survey electrophoresis, horse heart myoglobin and/or horse spleen ferritin (Schwarz-Mann) are used as internal standard proteins (JOHNSON1975b). For Ferguson plots, a purified preparation of a fast-moving Colias PGI allele has been used as a second internal standard with ferritin. Spectrophotometric solution assay of PGI in the reaction direction of F6P conversion to G6P is done, using a Gilford 240 spectrophotometer, via a coupled enzyme assay: F6P (0.002 M) and PGI are combined in the cuvette with 400 PM NADP and excess glucose-6-phosphate dehydrogenase (G6PdH) (Sigma Chemical Co.) in a final volume of 0.6 ml, and production of NADPH, is recorded at 340 nm. Standard buffer conditions are 0.01 M NH,Cl, 0.01 M MgCl,, 0.001 M EDTA, 0.05 M TRIS, 0.02% NaN,, pH 8.75 (optimum for Colias PGI) at appropriate experi- mental temperature, which is 30” for routine assays. Cuvette chamber temperature is controlled with a Haalre thermocirculator and chiller, and monitored with a thermistor probe in the blank cuvette. Sufficient G6PdH is used in all assays to maintain the transient time (5.r; see above, and EASTERBY1973) of the assay below 30 sec, thus maximizing accuracy; the amount used varies with temperature but is never less than 1 Enzyme Commission unit/ml in any experi- mental context. K, determinations are done using F6P concentrations of 25, 250, and 2500p~,with assays in triplicate at each concentration. The data are reduced as [FGP]/v us. [F6P] plots by the method of least squares, using a NOVA 2/10 computer. The determinations are themselves replicated three to five times, so that error statistics tabled refer to these replicated determina- lions rather than to within-plot variation. Individual plots invariably display linear regression coefficientssignificant beyond the P = 0.005 level. Visualization of PGI on gels is done in the standard buffer with a stain mixture composed of 0.002~F6P, 400 PM NADP, 0.8 Enzyme Commission units GGPdH/ml, 0.02 mg/ml phenazine methosulfate, 0.04 mgJml MTT dye. After 10-15 min incubation in the dark at 30°, gels are scored visually, or scanned at appropriate wavelengths, using the Gilford spectrophotometer with 2410s transport, for Ferguson plot analysis. Protein content of homogenates was routinely determined indirectly by use of the fact that protein content has a strong linear relationship to abdominal weight: the regression coefficient for mg protein (determined by a modified WADDELL’S(1957) method) on mg abdominal weight = 0.139, Fl,8 = 31.2, P < 0.001. Thus PGI activities as functions of genotype were normalized per mg abdominal weight. Assay of PGI preparations for protease activity was done by modifying the procedure of KUNITZ(1947) : preparations were incubated with 0.5% casein (Nutritional Biochemicals) in standard buffer conditions for 10 min at 30” (routine) or for 1 hr at 50” (to check on the heat denaturation experiments, see below) ; intact protein was precipitated with an equal volume of cold 10% trichloroacetic acid for 30 minutes; the precipitates were pelleted at top speed in a clinical centrifuge for 10 minutes; and the supernatants’ absorbance at 280 nm was read SELECTION ON COLIAS PHOSPHOGLUCOSE ISOMERASE 181 against blank controls in a Zeiss PMQ I1 spectrophotometer to measure peptide release by protease activity, if any. Details of Colias PGI purification and properties will be reported elsewhere, but procedures used in this work may be summarized: individual extracts of consistent genotype from the identical-by-descent lines are combined and centrifuged at 37,000 x g at 4' for 1 hr to pellet glycogen, which can otherwise interfere with protein fractionation. The supernatant is then concentrated with an Amicon ultrafilter and chromatographed at 4-6" through BioRad A-04m agarose molecular sieve gel, using Pharmacia and LDC columns, in a buffer of 0.05 M TRIS, 0.1 M NH,Cl, 0.001 M EDTA, 0.02% NaN,, pH 7.41/23". This results in a 12 to 15-fold purification in SO-SO% yield. The enzyme can be purified further by ion-exchange chromatography on DEAE-Sephadex A25, but this does not cause alteration of the kinetics of the enzyme as studied here, The molecular sieve step adequately removes phosphoglucomutase and other proteins which might bind PGI substrate or product, or otherwise interact with PGI parameters of interest. Therefore in the interest of time economy we have relied here on Colias PGI purified only through the molecular sieve. Such preparations contain no proteases detectable by the casein assay. They are stable in activity and electrophoretic phenotype for at least 8 months when stored at 4" in the standard buffer. Regression, analysis-of-variance, and contingency-table statistics were done following SOKAL and ROHLP(1969) and ROHLFand SORAL(1969), using programs written in BASIC for a NOVA 2/10 computer (Data-General Corp.).

RESULTS

Phenetics and genetics of Colias PGI variation: Six electrophoretic mobility classes of PGI bands have so far been found in Colias population samples; most contain four or more such variants. These occur singly or in triplet phenotype with an intermediate band, suggesting one-locus control of a dimeric enzyme with two homodimers and a centrally migrating heterodimer in heterozygotes. In lab colony breeding of C. eurytheme, now involving more than ten thousand progeny, and in less extensive breeding of C.p. eriphyle and C. meadii, the standard Mendelian segregations expected from this genetic model have always been found. Table 1 gives the mobilities of these variants in two electrophoretic buffer systems. Species share the same mobility classes even across broad taxonomic divisions, though they differ widely in frequency, as Table 2 shows. Figure 1 displays representative spectrophotometric scans of heterozygotes in imidazole gels. Study of allele identity with Ferguson plots: Coincidence of electrophoretic mobility at a single gel concentration is only a very coarse indicator of protein identity. JOHNSON(1975a) has advocated electrophoresis at a series of acrylamide

TABLE 1

Mobility, with reference io ferritin as standard protein, of Colias PGI allelic uariznts in 8% acrylamide gels in two buffer sysiems

Mobility ratios to ferritin Alleles in buffer system: T' T S M F F'

Imidazole-asparagine 0.09-0.14 0.27-0.33 0.44-0.53 ~ 0.65-0.72 0.87-0.92 1.01-1.04 TRIS-glycine 0.35-0.38 0.6CL0.63 0.75-0.79 0.91-0.95 1.14-1.17 1.33-1.36

See text for further details. 182 W. B. WATT TABLE 2 PGI allele frequencies for a variety of Colias populations and sampling times

73 T’ T S M F F’ C. alexandra 104. 0.09 0.24 0.58 0.09 - Brush Creek, Colo. 101 July 1975 C. meadii 88 0.59 0.33 0.01 - - Cumberland Pass, Colo. 5 August 1975 C. eurytheme 78 0.08 0.67 0.23 0.01 0.01 Patterson Pass, Ca. 19 October 1974 C.p. eriphyle Crested Butte Mtn., Colo. 25 June 1974 76 0.01 0.62 0.30 0.05 0.01 *I975 Brood I: 3 July 1975 62 0.03 0.58 0.31 0.08 - 11 July 1975 74 0.03 0.62 0.34 0.01 - 17 July 1975 88 0.05 0.67 0.27 0.01 - 23 July 1975 100 0.05 0.68 0.22 0.04. 0.01 *I975 Brood 11: 5 September 1375 170 0.10 0.57 0.26 0.05 0.01

The C. p. eriphyle data have been tested for homogeneity as follows: First, the 1974 sample, the first 1975 Brood I sample and the 1975 Brood I1 sample were compared; these are marginally different (x2= 10.9, df = 6, P - &IO), with the T allele contributing 85% of x2 (T’ was pooled with T,and P’ with F, for this analysis). Second, all six samples were compared; these differ significantly, again because of the T allele increase through 1975 Brood I (x2=26.8, df=15, 0.025 < P < 0.05). n = number of alleles sampled (twice the number of genotypes). * Ordinarily Brood I flies in June and Brood I1 in mid-August to mid-September. In 1975, owing to late snow and a cool summer, Brood 1’s flight was delayed a month, and Brood I1 three weeks, as compared to usual conditions.

concentrations (the Ferguson plot, see RODBARDand CHRAMBACH1971) as a means of detecting variants differing only in conformation or in subtle fractions of charge. We have done a preliminary study of this type, using four gel concentrations (7-10% acrylamide) ,with these issues in mind: (a) Can existing ways of standardizing Ferguson plots be improved on? (b) Is there evidence, at a preliminary resolution level, for “hidden allele” variation within PGI mobility classes? (c) Are there detectable differences between species in what appear, at standard gel concentration, to be “the same” mobility classes? Table 3 gives data on K,, the retardation coefficient or slope of the Ferguson plot, for S, M, and F alleles of C. p. eriphyle and the identical-by-descent C. eurytheme stocks. Previously (JOHNSON1975a) selected heterologous proteins have been used for internal standardization. The use of the F allele of PGI itself as internal standard increases precision beyond that afforded by ferritin, reducing the variance of K, by two to three fold. This is to be expected; alternative alleles of the same locus should respond more similarly to subtle changes in electro- SELECTION ON COLIAS PHOSPHOGLUCOSE ISOMERASE 183

6 5 4 3 2 1 0 cm Fer I - .4

-.3 8 -.2

-.I

LO 6 5 4 3 2 1 0 cm FIGURE1.-Representative spectrophotometric scans of Colias PGI heterozygotes at 532 nm (solid line) for MTT dye stain, and at 410 nm for internal standards ferritin (Fer) and myoglobin (Mb). F=front peak. Ocm=origin. (a) SM genotype; (b) MT genotype. Homo- dimers M,S,T, and heterodimers SM and MT marked accordingly.

phoretic pH, concentration of other migrating proteins, etc., than could heterologous proteins. One instance of striking “outlier” K, values was found: the S band of male C. p. eriphyle #2110 displayed a K, = 0.1534, more than two standard deviations from the mean of 0.1098 for this allele and population. #2110 was a PGI heterozygote. This allowed test of whether a “cryptic allele” was really involved; the K, values of the other bands in this individual were examined. The M homo- 184 W. B. WATT TABLE 3

Data on retardation coefficients K,, slope of the Ferguson plot, of Colias PGI uarianis in imidazole-system gels of T=7-10%, C=0.4%

~ Uncorr.* S SM M SF F S M

10 6 9 2 2 I0 9 0.1098 0.1157 0.1134 0.1064 0.10671- 0.1125 0.1162 0.000346 0.000196 O.OW092 - - 0.000645 0.00025 0.0186 0.0140 0.0096 - 0.0254 0.0158

8 4 4 2 2 0.1079 0.1130 0.1089 0.1024 0.10671- 0.00016 0.000031 0.00004.5 - - 0.0127 0.0056 0.0067 - -

* Correction procedure using the FF standard is as follows: All data on mobility of the FF standard relative to ferritin (29 in the base sample used here) are processed for the entire sample, and the Ferguson plot resulting from the average of these data is taken as a “parametric” mobility of this standard. The data on field samples are then processed by the program FERGPLOT, generating uncorrected Ferguson plots for the FF standard and “sample” PGI bands for each individual studied. Finally, the program corrects the “sample” band mobilities by the reciprocal of the deviation of the FF internal standard from its “parametric” values, and produces corrected “sample” Ferguson plots on this basis. S, M, and F are homodimers of the alleles in question; SM and SF are heterodimers. ANOVA has been used to compare S, SM, M, and SF bands between the two semispecies. In all cases the K,s do not differ between semispecies; P values are all < 0.4, and P values > 0.5. +In all cases, the F band co-electrophoresed exactly in all gels with the I.B.D. FF standard purified from C. eurytheme. f Lines identical-by-descent for PGI alleles.

dimer and SM heterodimer of #2110 also showed high K, values, 0.1374 and 0.1432 respectively, more than 2 standard deviations from the means, 0.1 134 and 0.1 157, of their respective K, distributions. Since this extreme behavior extends to all PGI bands of this animal, it cannot be due to a “cryptic allele” of either mobility class, but must be the result of post-translation modification or some other nonspecific difference in the #2110 extract which even the FF internal standard does not adequately correct. Finally, there are no differences at the present level of resolution between corresponding allele mobility classes of C. p. eriphyle and the identical-by- descent C. eurytheme. Indeed, the two F alleles examined in C. p. eriphyle CO- electrophoresed exactly with the identical-by-descent FF standard at all acrylamide percentages, just as did the two individual C. eurytheme F bands run from the same colony line. Allele and genotype frequency analysis in wild populations: Initial sampling of several populations yielded some genotype arrays in close agreement with multinomial Hardy-Weinberg expectations, and others which displayed an excess of heterozygotes, but none with major heterozygote deficiencies. Table 4 presents these data. Colias populations studied here have nonoverlapping generations, and physical wear on the adults’ wings can be quantified to give a rough index SELECTION ON COLIAS PHOSPHOGLUCOSE ISOMERASE 185

0 186 W. B. WATT of age (WATTet al. 1977). By this criterion (Table 4), those samples close to Hardy-Weinberg equilibrium were composed of newly emerged adults, whereas those showing heterozygote excess contained older animals. This trend is significant by BURR’S(1960) procedures for the KENDALLrank correlation coefficient: n = 6, S = 14, T = 4-0.966, P = 0.0028 (see also KENDALL1946). Ideally, one would wish to do selection component analysis, such as that of CHRISTIANSEN and FRYDENBERG(1973), on this locus. However, the number of alleles, of genotypes. and of resulting mother-offspring combinations possible made this imprac- tical. The initial results would predict that repeated sampling of one brood through its flight season and examination of the following brood should reveal a steady increase of heterozygote excess as the first brood aged, with a return to Hardy-Weinberg expectation at the start of the next brood. Use of the arcsin- square root transformation on these sequential frequency data would allow regression analysis of trends found.

.I4 20 /

/Ci .I2 16 1. L Y b = ,5094 2.0246 /. F,,2 = 428.52, ,001 < P<.005 / I .io ,I l2 4 m E m fn W 0 2 .08 8% W W I- O y .06 4 N 0 LL W I- W .w I w .a2 0 Flight of 1975 Flight of 1975 Brood I Brood II r 1 I*

0 --Y/ : I &-.-. JUNE I II 21 31 10 20 30 9 1974 JULY AUG. e SEFT. 1975 -.02- FIGURE2.-Change of heterozygote frequencies with time in the Crested Butte Mountain population of Colias p. eriphyle in 1975. Excess of total heterozygote frequency over Hardy- Weiiiberg expectations, H, dots, and the arcsin-square root transformation of H, triangles, are plotted on the ordinate. Regression statistics on the transformed data are given in the figure. A sample from the end of Brood I in 1974 is plotted as a large dot for comparison. Note that Hardy-Weinberg expectations must be calculated separately €or each sample on the basis of allele frequencies in that sample, since these can change during selection. SELECTION ON COLIAS PHOSPHOGLUCOSE ISOMERASE 187 This was done in 1975 with the Skyland Campcrested Butte Mountain population of C. p. eriphyle. Figure 2 presents the data. From close fit to Hardy- Weinberg expectations in the earliest sample, heterozygote excess develops just as predicted through the first brood, and returns near to zero at the start of the second. This increase is significant by regression analysis of the transformed frequencies (0.001 < P < 0.005). The numbers of the most common heterozygote, SM, were sufficient to allow independent test: this genotype's frequency does increase significantly above Hardy-Weinberg expectation as the brood ages (b= 0.336 f 0.055; Fl,2= 37.2, 0.02 < P < 0.03). Some form of natural selection on PGI or closely linked loci must therefore be maintaining this polymorphism (see LEWONTINand COCKERHAM1959). Simple directional selection is not acting on genotypes lacking the T allele here, since neither S, M, nor F alleles increased in frequency across generations (Table 2), although the S allele appeared to increase within Brood I. The T allele did so increase, but it is hard to invoke simple directional selection involving the TT genotype, since none of these were found in Brood I at all. Heterozy- gote excess can be generated in multiple-allelic series by mixing of adjacent but distinct populations (LI, 1969; MILKMAN1975). Our sampling area is approxi- mately 2 hectares within a valley grassland 1.2 km wide and roughly 11 km in unobstructed length, over which C. p. miphyle is broadly distributed. Work in progress on this population's structure, in the manner of WATTet al. (1977), already shows that the insects' average dispersal in periods of 1-3 days is several hundred meters, which is several times the maximum dimensions of our genetic sampling area. The samples thus come from within one well-mixed neighborhood (WRIGHT1946; WATTet al. 1977), and geographic allele frequency heterogeneity on a scale at least up to 1 km would generate maximum heterozygote excess quite early in the flight period, in contrast to the gradual steady increase actually found. Very broad scale clinal mixing could still be responsible; preliminary sampling of other parts of the drainage has not revealed frequency differences consistent with this, but more work is needed for a final test. Opposed selective pressures arising from fine-grained environmental heterogeneity, spatial or temporal, or straightforward heterozygote advantage are compatible with these data. As to the issue of linked loci, functional study of the PGI genotypes should either provide a mechanistic rationale for the operation of selection on PGI itself, or in the absence of detectable functional differences, would suggest the importance of associative effects. Heat sensitivity diferences among genotypes: All ten genotype combinations of the four identical-by-descent C. eurytheme PGI alleles were assayed (at 30") at the beginning and end of an hour at 50" in the standard buffer conditions. (This temperature is very close to physiological experience of the animals; body temperatures as high as 47" have been recorded via chronic thermistor implants from live Colias in heat stress (WATT1968 and unpublished)). Table 5 shows the results. There is a strong relationship between electrophoretic mobility and heat resistance of homozygotes, the faster moving alleles being far more resistant. Heterozygotes display various patterns of partial dominance for heat resistance. Iaa W. B. WATT TABLE 5 Persistence of PGI activity after I hr at 50", pH 8.75, as a function of genotype

Average % activity Average % activity Genotype 7Z after treatment Genotype n after treatment TT 3 22.7 k 2.3 MF 3 89.4 +- 2.3 ST 3 55.3 +. 8.6 FF 4 94.1 +- 4.1 ss 3 72.3 +- 3.3 MT 3 36.3 C 5.7 SM 4 83.3 C 3 FT 3 71.0 f 5 MM 4 90.0 iz 4 SF 3 80.5 -C 5.6

Enzyme partially purified from identical-by-descent C. eurytheme lines. All assays done in triplicate before and after treatment. n = number of replicates. "Average deviations" are tabulated to indicate the dispersion of the data, but are not the basis of statistical testing. The arcsin- square root transform for percentages was used prior to analysis-of-variance; this ANOVA shows significance of differences: F,,,, = 80.7, P < 0.001.

Although no evidence of protease activity was routinely found in these partially purified preparations, a casein protease assay was carried out in duplicate on a TT preparation for one hour at 50°, to rule out the presence of residual protease activity that could have differential effects on PGI genotypes at this high temperature. No such activity was found. Maximum activity as a function of genotype: Total enzyme activity at 30" per mg abdominal weight (see MATERIALS AND METHODS for justification of this indirect specific activity measure) was next examined as a function of genotype. Table 6 presents data on fresh males; females vary more widely due to variability in abdominal egg mass, but are consistent with males as far as available data allow comparison. The apparent differences in activity are significant by analysis of variance. Various a priori comparisons among genotypes arise naturally from the field data and the Mendelian transmission pattern. We should be interested in differences among all homozygotes: this group comparison is highly significant

TABLE 6 Maximum activity per mg abdomen weight at 30" of uarious PGI genotypes in male Colias eurytheme, assayed in the direction of fructose-6-phosphate conversion to glucose-6-phosphate

Average Average Genotype n max. activity' S.D. Genotype n max. activity* S.D. TT 19 0.088 0.023 MF 17 0.077 0.015 ST 13 0.077 .0.021 FF 16 0.080 0.016 ss 13 0.062 0.010 MT 11 0.071 0.019 SM 11 0.077 0.019 FT 10 0.078 0.020 MM 12 0.062 0.013 SF 17 0.073 0.017

Homogenates of identical-by-descent lines used throughout. n = number of individuals of each genotype assayed. ANOVA for overall significance: F,,,,, = 3.1, 0.001 < P <0.005. These data were taken at pH 8.0. These genotypes all show the same pH-us.-activity curve in the range pH 8 to pH 9; at 30", pH 8.75 activity is 1.06 & 0.04 times that at pH 8.0. * Units of activity are E.C. units/mg abdominal weight. SELECTION ON COLIAS PHOSPHOGLUCOSE ISOMERASE 189 (F3,129= 8.7; P < 0.001). The various pairwise allele combinations should be examined for heterozygote advantage (ABvs. AA f BB comparison), dominance (AAvs. AB 4- BB; BB vs. AB f AA),or heterozygote intermediacy (AAvs. AB vs. BB). Thus for alleles S and T we find no heterozygote advantage (Fl,120< 0.01, 0.75 < P),but strong evidence of intermediacy (TT vs. ST vs. SS,F2,,,, = 8.6, P < 0.001). Heterozygote advantage is seen in the case of SM vs. SS 4- MM (Fl,129= 6.06,O.Ol < P < 0.02). To summarize the wearying number of remaining comparisons, F is dominant to both M and T with respect to heterozygote activity, while SF and MT show heterozygote intermediacy. These activity differences could be due in principle to: (a) differing levels of nonspecific inhibitors in the homogenates; (b) genotype-specific inhibitors in the homogenates; (c) genotype-specific differential synthesis of PGI; (d) actual differences in the catalytic rate constants of the PGI allozymes. Alternative (a) is ruled out by the fact that the activity of mixtures of different genotype homogenates is strictly additive over a broad range of mixing proportions. To evaluate (b), we consider first that in these assays 5 microliters of homogenate was added to 0.6 ml of reaction mixture, so that the putative inhibitors would have to be effective at very low concentrations. Also, they would have to bind PGI so tightly as to resist removal by the molecular sieve gel in purification, or alternatively be of the same molecular weight as PGI, since otherwise we should have found apparent activation by the molecular sieve of the more severely "inhibited genotype preparations by the molecular sieve. NO such "genotype-specific yield" has been found. Discrimination of the remaining alternatives awaits measurement of catalytic rate constants using completely purified enzyme. As Figure 3 shows for representative cases, Colias PGI genotypes do not differ widely in their change of maximum activity with temperature over the range 10"-30". The ratio of activity at 30" to that at 10" is 5.00 4 0.29. A greater spread of activities is seen at 40". Substrate binding and coupling effectiveness as functions of genotype and temperature: Table 7 presents values of K, for F6P for all ten genotypes at 10" and 30". Values at 30" are much smaller than at lo", showing an increase in binding effectiveness with temperature; this is a clear case of what HOCHACHKA and SOMERO(1973) have termed "negative thermal modulation." There are significant differences among genotypes at 10", but not at 30". F-containing genotypes, except for FT, are disadvantaged in binding at lo", whereas MT is notably dective at this temperature. By combining the results on maximum activities and their thermal variation with these lL data, we arrive at the figures for coupling ratios of the ten genotypes presented in Table 7. Variations in the component parameters interact to produce a complicated pattern of coupling ratio values, with the relative stand- ings of the genotypes shifting markedly with temperature in some cases, such as MT and most F-containing genotypes. 190 W. B. WATT

9- . .TT I ASS +SM + 8- ST 0 MM 'FF s 0 MT 7- 0

T ("C) FIGURE3.-Change of maximum velocity (ordinate) with temperature (abscissa) for various PGI genotypes of Colias. All are normalized to an arbitrary value of 5 at 30" for ease of comparison. All ten genotypes studied fall within the ranges of the seven genotypes shown at each temperature in comparison to 30". Enzyme partially purified from identical-by-descent C. eurytheme lines.

DISCUSSION The possible predominance of associative selective effects on Colias PGI, while not yet ruled out definitively, seems now implausible. It would in the first place require parallel behavior of unknown linked loci in different species complexes at considerable phyletic remove. Second, the functional differences among PGI allozymes already found are consistent with the field data. For example, the heat-sensitive T mobility class is at high frequency in cold-habitat tundra populations of C. meadii, where overheating of adults is quite rare (see WATT1968). C. alexandra, flying in the warmest part of the Colorado summer, should and SELECTION ON COLIAS PHOSPHOGLUCOSE ISOMERASE 191 TABLE 7 Fructose-6-phosphateK, values and coupling ratios at 10" and 30" at pH 8.75 for the ten Colias PGI genotypes

Genotypes: TT ST ss SM MM MF FF MT SF FT Km (PM) 10" 124+8 11628 112211 11Ok11 12328 13727 130+5 913Z9 1323Z11 105-1-12 n 3 3 3 4 4 3 54 4 4 ANOVA F9,27= 12.5; P Q 0.001 30" 66-1-9 72k6 76k7 68k2 763~15 743Z2 65-1-5 65-1-8 76-1-8 70+10 n 4 3 3 3 4 3 43 3 3 ANOVA F,,,, = 1.56; 0.1 < P < 0.25 Coupling ratio index* 10" 1.50 1.41 1.18 1.48 1.06 1.18 1.30 1.65 1.18 1.58 30" 14.1 11.4 8.6 11.9 8.6 10.7 13.1 11.5 10.2 11.9

Enzyme partially purified from identical-by-descent C. eurytheme lines. ANOVA values for significance of differences among K, values are given in the table. The genotypes are closely similar at 30", far more different at 10". * The coupling ratio index is calculated as follows: maximum activities are calculated, correct- ing for pH (see caption of Table 6) and temperature (see text) as appropriate, for each genotype at pH 8.75, 10" and 30", then multiplied by 104 to eliminate unwieldly decimal expression and divided by the K,values as tabulated. For interpretation of coupling ratios, see the text and EASTERBY(1973). It should be noted that EASTERBY'Suse of the parameter "VmBx"actually refers to total activity present as used here.

does have higher frequencies of the relatively heat-resistant M mobility class than does C. p. eriphyle, whose two broods fly before and after C. alexandra in distinctly cooler weather. The possibilities for favorable heterozygote function seen in the laboratory are consistent with observed heterozygote increase with time in the field. Of course, the work needed to quantify fitness differences among Colias PGI genotypes has only begun. More kinetics work, especially focusing on the reaction direction of G6P to F6P conversion, will be needed. Study of fluxes through the PGI step in uiuo in relation to genotype and to varying environmental conditions will be needed. This can be done by combining isotope techniques with the methods of SACKTORand WORMSER-SHAVITT(1966) for studying transients of intermediary metabolism in flying insects. Various artificial selection experiments suggest themselves, as well as additional field sampling work. The task of interpreting in vitro results in the in vivo context is eased somewhat by the fact that glycolysis in general, and the PGI reaction in particular, operates in the cytosol rather than in complex interaction with subcellular ultrastructure. Indeed, HESS(1 973) reports successful calculation of in viuo flux through several glycolytic steps, including PGI, from in uitro kinetic data. The complexity of functional differences found among these genotypes suggests that selection on them could be equally complex. SM has a heterotic advantage in maximum activity and in coupling ratio at both high and low 192 W. B. WATT temperatures, as well as fairly high heat stability. MT is strikingly heterotic for K, and coupling ratio at low temperature, but not so at 30°, and this genotype would also suffer from the near-complete dominance of TT’s heat sensitivity. There is the potential for net heterosis over time in the case of ST, in that the higher heat stability of SS and the higher maximum activity and coupling ratio of TT are both partially dominant. BERGER(1976) suggested that heterozygotes might in general be more catalytically efficient, thus requiring less cost of enzyme synthesis to maintain a given activity level. While such a mechanism is perfectly plausible, Colias PGI heterozygotes are not uniformly heterotic in functional properties, and activity levels are not being regulated in this case, but vary extensively with genotype. JOHNSON( 1974) suggested that naturally selected polymorphisms should be concentrated at those enzyme loci which regulate flux through pathways. While his argument may hold in many cases, it probably does not apply to PGI. PGI’s reaction is freely reversible, and its activity in Colias is seven- to ten-fold greater than those of surrounding enzymes in the glycogen-glycolysis-pentose shunt branch point, such as phosphoglucomutase, hexokinase, or glucose-6-phosphate dehydrogenase (WATT,unpublished data). It is not known to display allosteric modulation of its activity. It is, in short, a very unlikely candidate for a regula- tory role in the usual sense. What seems more likely, especially in context of EASTERBY’S(1973) results, is that selection would act to prevent PGI from delaying or interfering with controlled flux changes in surrounding rate-limiting steps (glycogen phosphorylase, phosphofructokinase, etc.), and would do so by maximizing its coupling effectiveness. Small insects at rest shut down their energy processing metabolism almost completely (review: SACKTOR1970), and must restart it in a very short time at the next onset of activity. A short transient time for the PGI step would then be crucial in allowing the speedy mobilization of energy resources. The finding that shifts in Ferguson plot behavior can occur which are neither PGI-allele-specific nor controlled away by a homologous PGI standard under- scores the need for extreme care in the search for electrophoretically cryptic variation. Post-translational modification may itself vary greatly among individuals in a heritable way. Electrophoresis and heat sensitivity studies alike (e.g., BERNSTEIN,THROCKMORTON and HUBBY 1973) are subject to a variety of protein-protein and other interactions which may be extremely specific to particular proteins and/or to particular regions of electrophoresis gels. Subtle shifts in mobility may readily be due to differential screening or binding effects of variants segregating at other protein loci, especially when one considers that electrophoretic conditions in a concentrated whole-homogenate separation are very far from those of infinite-dilution, ideal chemical conditions. The use of heterozygotes as test subjects in this work, in combination with homologous internal standards, affords increased control of these alternatives.

I thank ERIKWHITEHORN for superlative technical assistance, RALPH and LOISWATT for help in stock maintenance, and DAVIDBEAUFAIT, LAWRENCE GALL, DIANEHENNEBERGER, JANE HAYES,HANNE SMITH, MAUREEN STANTON, ALICE WATT, and JEAN WATTfor help in catching SELECTION ON COLIAS PHOSPHOGLUCOSE ISOMERASE 193 field samples of Colias. This work benefitted from stimulating commentary by WILLIAMBAKER, FRANCESCHEW, FREDDY CHRISTIANSEN, MARCUS FELDMAN, CLAY SASSAMAN, JOHANNA SCHMITT, ROBERTSIMONI and LEE SNYDER.Support came from the National Scien'ce Foundation (BMS 74 22243, DEB 75 23458).

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