Ae,4 After a Suggestion by Remington,' Showed That the Day Length to Which the Immature Stages Are Exposed Controls the Adult Pigment

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ADAPTIVE SIGNIFICANCE OF PIGMENT POLYMORPHISMS IN COLIAS BUTTERFLIES, II. THERMOREGULATION AND PHOTOPERIODICALLY CONTROLLED MELANIN VARIATION IN COLIAS EURYTHEME By WARD B. WATT*

DEPARTMENT OF BIOLOGY, YALE UNIVERSITY Communicated by G. Evelyn Hutchinson, March 12, 1969 Abstract.-The butterfly Colias eurytheme requires body temperatures above 30'C for flight. When cold, it orients its exposed wing undersides to present maximum surface area to sunlight; when too warm, it orients for minimum exposure. Dark-winged color forms heat faster in sunlight than light ones. The seasonal color polymorphism of Colias appears to have been evolved to maximize solar heating in cold seasons and minimize overheating in warm seasons. Temperate zone organisms experience great seasonal variation in many environ- mental parameters. An organism limited in occurrence by one (or more) of these might adapt to this condition in several ways. It might restrict especially sensitive parts of its life cycle to favorable seasons or suspend activity altogether in unfavorable seasons. It might simply evolve broader tolerence to the limiting forces, or it might retain narrow tolerances but evolve some means of changing the limits of these from season to season. The latter might be done by establish- ment of a genetic polymorphism in the population, so that one morph would be maximally adapted to each season, and frequencies would cycle seasonally, as in certain Drosophila chromosomal polymorphisms.1 Alternatively, all individuals might show the same phenotype at one time but alter it from season to season to increase adaptation to each. Bearers of such a seasonal shift mechanism would not be selectively disfavored in some seasons as would, for example, a warm- adapted genetic-morph in a cool season. Such a seasonal shift is exhibited by the common "sulfur" butterflies, Colias eurytheme Boisduval and C. philodice Latreille. Summer broods of these closely related species2 display clear, unclouded orange or yellow color, due to pteridine pigments,3 on the hindwing undersides. Spring and fall broods show an intense darkening of this area due to replacement of the pteridines by black melanin. Ae,4 after a suggestion by Remington,' showed that the day length to which the immature stages are exposed controls the adult pigment. Long photoperiods cause larvae to produce summer-form adults, whereas sibling larvae reared under a short photoperiod produce dark spring-fall forms. In the normal adult Colias' resting position, the wings are folded over the back, exposing the undersides; thus, thorax and abdomen are closely covered by that part of the hindwing which is most darkened in the short-day form. This sug- gested that the dark form might absorb more solar energy than its light counterpart and thus be better adapted to the cool climate of spring and fall. Develop- ment of thermistor probes so small as to monitor Colias' temperatures without impeding their normal physical activity6 has made possible the test of this suggestion. 767 Downloaded by guest on September 27, 2021 768 ZOOLOGY: W. B. WATT PRoc. N. A. S.

Materials and Methods.-Experimental animals: Laboratory strains of C. eurytheme were used. Larvae were reared at 270C, with vetch (Vicia) as food plant. A photoperiod of 16 hr light:8 hr dark produced light forms, and one of 10 hr light:14 hr dark produced dark forms. Half the larvae of each brood were reared under each regime, so that the photoperiodic differences were studied against a background of genetic similarity. Assay of wing darkening: 16 hr and 10 hr Photoperiod forms are instantly distinguish- able to the eye. For quantitative assay of more continuous variation from intermediate photoperiods in field samples, light and dark scales in the four scale rows across the discal cell of the hindwing underside, immediately basal to the origin of vein Ml, were counted. Results were expressed as of dark scales out of the total. Thermistor instrumentation: Bead thermistors 0.25 mm in diameter, supported by a small rodlike body of epoxy resin, soldered to leads of 0.07-mm-diameter magnet wire 1/2-1 meter long, and monitored by a Wheatstone Bridge circuit calibrated between 00 and 50'C, were used to determine Colias' body temperatures. These probes were im- planted into the insects through the dorsal thoracic cuticle to a depth of 1-2 mm among the flight muscles. Insects so treated have a normal life expectancy, are able to fly, feed, or oviposit normally, and are not behaviorally disturbed in any respect relevant to this study. Evidence for this and details of the implantation technique appear elsewhere.6 Air temperature and solar heat load ("black-body temperature") were measured by sen- sors described elsewhere.6 Experimental procedures: Colias' flight as a function of body temperature was examined by recording the insects' temperatures and flight or inactivity every 20 seconds, while the insects were restrained only by the magnet wire probe leads. Studies in the field and in the laboratory used the sun and a battery of flood lamps, respectively, as energy sources. To guard against possible behavioral differences under the differing spectral conditions, the temperature ranges studied in the field and laboratory were overlapped extensively. Data were combined only after statistical confirmation, by methods described below, that the distributions were the same in the overlap zone. Alighted animals' orientation angles to sunlight around both yaw and roll axes were estimated visually in 15° increments. In "heat-seeking" orientation, both yaw and roll angles are > 60° (nearly perpendicular to sunlight). If either angle is <30° (nearly parallel to sunlight), the orientation is "heat-avoiding." Intermediate positions are "ambiguous." Perpendicular orientation maximizes, and parallel orientation minimizes, solar heating of Colias.6 For studies of cooling and solar heating rates, Colias, with wings closed over the back, were grasped by the wings with spring clips which were mounted on a rod clamped to a support stand. Clips were affixed sufficiently far from the insects' bodies to avoid their contributing to the insects' thoracic temperatures. This procedure caused no injury. A few insects struggled in the apparatus; these were not used in the experiments. Data analysis: Flight vs. body-temperature data were summed on 1-degree intervals for both color forms. A 2 X 2 table was formed for each such interval from flight and inactivity frequencies for each of the forms, and each table was tested for homogeneity of flight frequency by the x2 test.' Data of more than 1 interval were sometimes pooled to overcome statistical inaccuracies otherwise introduced by low numbers of observations. Heating rates of matched pairs of Colias were compared by tabulating the observed pairs of temperature-change values per measured time interval, and testing the locations of these paired series of values with the Wilcoxon Matched Pair Rank Order Test.8 9 Cooling rates for the same matched pairs of Colias were compared by plotting, for each insect studied, temperature change per sampled time interval as a function of the difference between body temperature Ti and environment temperature Te (approximated by air temperature) on that time interval. To each point scatter thus generated, a regression line was fitted by the method of least squares.7' 10 The regression coefficients were thus measures of the cooling rates. Significance tests of these coefficients and of differences between them were made by using Student's "t" or its normal approximation.'0 Spectrophotometry: Reflectance spectra of Colias wings were obtained in the wavelength Downloaded by guest on September 27, 2021 VOL. 63, 1969 ZOOLOGY: W. B. WATT 769 . ~a SD

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FiG. 1 Photoperiod forms (male) of Colias eurytheme. Top row: summer, long-photoperiod form. Bottom row: spring-fall, short-photoperiod form. Left: uppersides; right: undersides. ranges 350-750 nm (diffuse reflectance) and 600-2000 nm (specular reflectance) on a Perkin-Elmer 450 spectrophotometer. Also, attenuated total reflectance measurements were made over the range 830-7500 nm on a Perkin-Elmer 137G spectrophotometer. Downloaded by guest on September 27, 2021 770 ZOOLOGY: W. B. WATT PROC. N. A. S.

The outputs of these instruments were converted to relative absorbed intensity spectra as described elsewhere.6 Experiments.-Variation in thefield and laboratory: The photoperiodic control of Colias underside color is illustrated by the following. Offspring of one female were reared at 270C under 16 hours light/day or 10 hours light/day. Mlean dark-scale percentages in the hindwing underside sampling area (see above) for the resulting adults (N = 5 in each group) are: 16-hour males, 12 per cent; 16-hour females, 14 per cent; 10-hour males, 34 per cent; 10-hour females, 35 per cent. Figure 1 illustrates light and dark-form males. Variation in wild populations is illustrated by a sample of specimens collected unselectively by Mr. Bryant Mlather near Jackson, Mississippi. Dark-scale percentages of the specimens are plotted against their capture dates in Figure 2. AfIidsummer specimens reach values 50 as low as 5 per cent dark scales, while

40-.a short-day winter ones attain 35-40

Li 30 per cent. <3: * . uOrientation to sunlight: Figure 3 , 20 shows Colias' orientation to sunlight :10-* E as a function of body temperature. Below the heat-seeking per- MAR.APR- SLY 350C, JAN 'FEB 'MAR APR MAY LUN JUL A 'G SEP CC NC DFC pendicularorientationpredominates; above 380C, heat-avoiding parallel FIG. 2.-Occurrence of dark and light photo- orientation predominates. periodic forms of C. eurytheme through the year. The per cent dark scaling of each individual's hind- Flight dependence on temperature: wing underside is plotted against its capture date. C. eurytheme might possibly have

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20 0 20 40 60 0 20 40 60 20 620 00'01d6'o6'oNumber of cbservctiors Number of observatiors FIG. 4.-Flight activity versus body tem- FIG. 3.-Orientation of resting Colias to perature in long-day and short-day forms solar radiation as a function of thoracic (16 hr and 10 hr) of C. eurytheme (males temperature. Types of orientation are de- only). White bar: flight. Black bar: in- fined in the text. activity. Downloaded by guest on September 27, 2021 VOL. 63,1969 ZOOLOGY: W. B. WATT 771

TABLE 1. Statistical tests for identity of flight distributions presented in Figure 4 for male photoperiodic forms of Colias eurytheme. Temperature interval, IC X2 P 29-31 0.1169 0.70 < P < 0.80 31-32 Data identical, no test 32-33 0.1197 0.70 < P < 0.80 33-34 0.2930 0.50 < P < 0.70 34-35 0.8360 0.30 < P < 0.50 35-36 0.1025 0.70 < P < 0.80 36-37 0.3215 0.50 < P < 0.70 37-39 0.0754 0.70 < P < 0.80 39-41 0.1735 0.50 < P < 0.70 Contingency tables 2 X 2 were formed on the indicated body temperature intervals from flight and inactivity frequencies of long- and short-day forms. P = probability of obtaining the same or greater heterogeneity in the data by chance alone. No significant heterogeneity was found. In other words, the photoperiod forms do not differ in their temperature preferenda for flight. No flight occurred below the lowest interval noted here. adapted to cold seasons by lowering, under photoperiodic control, the range of body temperature that it prefers for activity. Figure 4 plots flight activity against body temperature for both color forms (males only; data on females are similar and are omitted here). Tests of these data show in Table 1 that the spring- fall form does not fly at lower body temperatures than the summer form. This harmonizes with the previous finding that different Colias species, from habitats differing widely in temperature, show the same body temperature optimum for activity (about 35-38oC).6 Heating of Colias: Colias of the same sex and size, one of each color form, were placed 5 cm apart in the restraint device described above and exposed perpendicular to sunlight as their temperatures were monitored. Sample plots of heating-rate tests of such size-matched pairs are shown in Figure 5. The bulky raw data are not presented here but are available from the author on request. Results of Wilcoxon tests of the data appear in Table 2, showing that dark forms heat significantly faster in sunlight than light forms. (Average heating rates are presented for illustration, though they are not directly related to the statistical tests.) Figure 5b shows that the heating advantage of the dark forms persists after death (the insects of this trial had been dead 14 days). Thus, light-cued

FIG. 5.-Solar heating of size- a))35 matched pairs of light- and dark-form C. eurytheme. Filled triangles: dark member of each pair. Filled circles: ( 30 light member of each pair. Filled w - squares: ambient air temperature. ' Open circles: black-body temperature, < 25 f measure of radiant heat load. Black w bar: duration of exposure to sunlight. t (a) Males, pair 1 of Tables 2 and 3; , 20 (b) females, dead 14 days before heat- o ing experiment; (c) per cent flight mc.o_ activity vs. body temperature, derived '5 --> - from Fig. 4 for comparison with (a) - and (b). ° Time in min. Downloaded by guest on September 27, 2021 772 ZOOLOGY: W. B. WATTPPROC. N. A. S.

TABLE 2. Heating properties of seasonal forms of C. eurytheme. Average heating rate, Pair (IC/min) n Tn P (1)l0hr e 3.6 vs. 19 188 0.001 16 hrci' 2.6 (2)10hrc3' 3.2 vs. 19 172 0.001 16hrc? 2.6 (3) lOhr 9 3.8 vs. 10 50'/2 0.005 16hr 9 3.2 (4) 10 hr 9 2.4 vs. 6 21 0.001 16hr 9 1.8 (5) lOhr 9 3.3 vs. 12 66 0.013 16 hr 9 2.9 Wilcoxon tests, and averages, of heating rates in sunlight of size-matched pairs of C. eurytheme, one dark form (10-hr photoperiod) and one light form (16-hr photoperiod) in each pair. P = probability of obtaining a Wilcoxon score, T. > that observed by chance alone. n = number of pairs of rate values tested in each case. In all cases the dark form heated significantly faster than the light form. physiological changes cannot be a major cause of the heating difference. Colias have never been seen warming themselves by muscular activity, as do some other Lepidoptera,11 12 nor have any ever given evidence for use of an externally in- visible metabolic heating process. Since mass and surface area variables were eliminated by the size matching, the only remaining possible causes for the heat- TABLE 3. Cooling properties of seasonal forms of C. eurytheme. Coefficient Difference of Regression Significance Coefficients Pair coefficient n t* P n t* P (1) l0hrc? 0.331 18 6.939 <0.001 vs. 36 0.393 0.50 < P < 0.70 16bhrcP 0.304 18 6.319 <0.001 (2) 10hrcdi 0.232 31 7.606 <0.001 vs. 62 3.367 P < 0.01 16hr ci 0.183 31 5.440 <0.001 (3) 10hr 9 0.301 21 7.977 <0.001 vs. 42 0.462 0.50 < P < 0.70 16hr 9 0.275 21 6.575 <0.001 (4) 10 hr 9 0.198 12 6.646 <0.001 vs. 24 0.887 0.30 < P < 0.50 16 hr 9 0.236 12 7.732 <0.001 (5) 10 hr 9 0.313 10 5.704 <0.001 vs. 20 0.132 0.80 < P < 0.90 16 hr 9 0.300 10 3.736 <0.01 Regression coefficients, and tests thereof, of temperature loss (AT/30 sec) on the difference between insect and environment temperatures (Ti - Te) for size-matched pairs of Coliaz, one member of each pair light (16 hr) and one dark (10 hr); same pairs as in Table 2. The regression coefficient thus measures cooling rate. In no case does a light form cool faster, i.e., have a significantly greater regression coefficient, than a dark form. * For n > 30, t was approximated by c, the normal deviate. Downloaded by guest on September 27, 2021 VOL. 63,1969 ZOOLOGY: W. B. WATT 773

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crypticFIG. 6.-Cooling~darkgdfernebewe ~rate ~vs. insect-environment ~ lghfrs temperature difference (Ti - TO) for matched pair 3 (Table 3) of dark- and light-form Colio2. ing rate differences are (a) greater energy absorption by dark forms and (b) some cryptic cooling difference between forms. Cooling of Colias: After each sun exposure during heating tests, experimental insects were shaded and their cooling was recorded. Selected cooling plots appear in Figure 6 and all statistics in Table 3. In no case does a light form cool faster than its dark fellow. (One dark insect cooled faster than its light fellow. This was by far the darkest individual studied, and the result may have been due to an increased rate of radiative heat loss with darkening, which reached detect- able levels in this insect but in no other.) The net heating rate advantage of dark forms over light must result from greater energy absorption by dark forms. Spectrophotometry: Relative absorbed intensity spectra were determined from 400 to 1500 nm for the hindwing undersides of light and dark C. eurytheme. This spectral region contains 85-90 per cent of all direct solar energy reaching the ground."3, 14 The quantitative impact in the field was estimated by multiplying these spectra by the intensity spectrum of solar energy at sea level and solar zenith (air mass 1).13, 14 Under these conditions, the dark insect tested would absorb 0.62 cal/min/cm2 compared to 0.39 cal/min/cm2 for the light form tested. Downloaded by guest on September 27, 2021 774 ZOOLOGY: W. B. WATT PROC. N. A. S.

Discussion.-Figure 5 presents, in addition to the Colias heating plots, a plot of flight vs. body temperature, derived from Figure 4, for comparison with the heating curves. This comparison quickly shows that under low habitat temperature the dark, short-day, cold-season Colias will reach the high body temperatures needed for flight a much greater part of the time than will its light, long-day, summer counterpart. Since Colias cannot feed or reproduce without active flight,6 '5 the energy-absorbing dark coloration clearly entails adaptive advantage in the cold. In warm summer conditions, on the other hand, the lesser-absorbing light form must be more adaptive, as it will have less tendency to overheat; even if body temperatures do not become lethally high, overheated Colias spend most of their time in the forced inactivity of heat avoidance to the neglect of reproduc- tion.6 Other selective forces may conceivably act on this pigment system, but clearly the pressure to maintain optimum body temperature under seasonally different habitat temperatures has been significant in the evolution of Colias' photoperiodic pigment variation. Future work may quantitate this selective pressure. Biochemical work begun on Colias pigment synthesis6 should clarify the physiological control of the pigment pattern. This may further advance understanding of this seasonally varying system's evolution to its present state, wherein one genotype produces, on cue, whichever alternative phenotype is most seasonally adaptive. I thank Drs. C. L. Remington and T. L. Poulson for encouragement and advice in the course of this work, and Dr. Wyatt W. Anderson for critically reading this manuscript. * Predoctoral fellow of the National Science Foundation, U. S. Public Health Service, and Yale University. Present address: Department of Biological Sciences, Stanford University, Stanford, California 94305. This work was drawn from a thesis submitted to the Faculty of the Graduate School of Yale University in candidacy for the degree of Doctor of Philosophy. 1 Dobzhansky, Th., in Insect Polymorphism (London: Royal Entomological Society, 1961), pp. 30-42. 2 Klots, A. B., A Field Guide to the Butterflies (Boston: Houghton Mifflin Co., 1951). 3Watt, W. B., Nature, 201, 1326 (1964). 4Ae, S. A., Lepid. News, 11, 207 (1957). 6 Remington, C. L., Advan. Genet., 6, 403 (1954). 6 Watt, W. B., Evolution, 22, 437 (1968). 7Fisher, R. A., Statistical Methodsfor Research Workers (New York: Hafner and Co., 1958), 13th ed. 8 Owen, D. B., Handbook of Statistical Tables (Reading, Mass.: Addison-Wesley, 1962). 9 Bradley, J. V., Distribution-Free Statistical Tests (Washington: U.S. Dept. of Commerce, Office of Technical Services, 1960). 10Mather, K., Statistical Analysis in Biology (London: Methuen and Co., 1951), 4th ed. 11 Dotterweich, H., Zool. Jahrb. Abt. Allg. Zool. Physiol. Tiere, 44, 399 (1928). 12 Adams, P. A., and J. E. Heath, Nature, 201, 20 (1964). 13 Moon, P., J. Franklin Inst., 230, 583 (1940). 14Withrow, R. B., and A. P. Withrow, in Radiation Biology (New York: McGraw-Hill and Co., 1956), vol. 3, pp. 125-128. 16 Stern, V. M., and R. F. Smith, Hilgardia, 29, 411 (1960). 16 Watt, W. B., J. Biol. Chem., 242, 565 (1967). Downloaded by guest on September 27, 2021