Geographic variation in plasticity inEristalis arbustorum

Biological Journal of the Linnean Society (1998), 65: 215–229. With 6 figures Article ID: bj980243

Geographic variation in plasticity in Eristalis arbustorum MART M. OTTENHEIM∗, ANJA HENSELER AND PAUL M. BRAKEFIELD Institute of Evolutionary and Ecological Sciences, Section of Evolutionary Biology and Systematics, Leiden University, P.O. Box 9516, 2300 RA Leiden, The Netherlands Received 9 September 1997; accepted for publication 28 April 1998

To study the evolution of phenotypic plasticity in the field, six populations of the hoverfly Eristalis arbustorum were sampled along two parallel North–South transects over a maximum daily temperature gradient. Three populations were sampled per transect. Egg batches were collected and the offspring were reared in a split family set up over three different pupal temperature regimes in the laboratory to produce population reaction norms of colour pattern, pupal development time, wing length and thorax length. Wing length and colour pattern were corrected for body size. All four characters showed plasticity in response to rearing temperature and significant differences in height, slope and shape of the reaction norms were found. Only male colour pattern showed variation in reaction norms along the North–South gradient. Most other characters showed variation in reaction norms from West to East. The two populations lying in the middle of the transects were frequently different from the others. Within the populations, significant genotype-environment interactions were frequently found for wing length and colour pattern, indicating that genetic variation for plasticity was present. The results suggest that the populations may have evolved plastic responses to suit local environmental conditions.  1998 The Linnean Society of London

ADDITIONAL KEY WORDS:—colour pattern – development time – hoverflies – phenotypic plasticity – thorax length – Syrphidae – wing length. CONTENTS

Introduction . . . . . . . . Material and Methods . . . . Rearing of hoverflies . . . Measurements . . . . . Data manipulation and analysis Results . . . . . . . . . Corrected colour pattern . . Pupal development time . . Corrected wing length . . . Thorax length . . . . .

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∗ Correspondence to: M. M. Ottenheim. 0024–4066/98/100215+15 $30.00/0

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Discussion . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .

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INTRODUCTION

In the last decade a large number of studies on phenotypic plasticity have been reported (Gotthard & Nylin, 1995 and references therein). Many aspects of plasticity have been covered such as: genetics (e.g. Scheiner & Lyman, 1989, 1991; Hillesheim & Stearns, 1991; Windig, 1994), theoretical modelling (e.g. de Jong, 1990, 1995; van Tienderen & de Jong, 1994) and the adaptive implications of plasticity (Hart & Strathmann, 1994; Kingsolver, 1995, 1996). The nature of the phenotypic plasticity can be described graphically in terms of the norms of reaction (Schmalhausen, 1949). Variation among populations in phenotypic plasticity has also been studied (e.g. Miller & Fowler, 1993; Schmitt, 1993) although not in detail. However, studies covering geographic variation are rare (Conover & Present, 1990; Kudoh, Ishiguri & Kawano, 1995; Delpuech et al., 1995) except where it concerns geographic variation in diapausing insects (Danilevsky, Goryshin & Tyshchenko, 1970; Bradshaw, 1976; Bradford & Roff, 1995). Most of these studies report a correlation between climatic factors and geographic variation in plasticity. Such a correlation is an indication that natural selection shaped the reaction norm, at present or in the past (Endler, 1977). In contrast, remarkable resemblances between well separated populations have also been reported (Negus, Berger & Pinter, 1992). The colour pattern and the wing length of Eristalis arbustorum both vary in response to rearing temperature (Heal, 1981; Ottenheim, Waller & Holloway, 1995; Ottenheim, 1997). When the pupae are kept at relatively low temperatures the flies develop a smaller coloured pattern and longer wings. Flies reared at higher temperatures develop a larger coloured pattern and shorter wings. In one population, significant additive genetic variation was found under most rearing conditions and also in both characters significant genotype-environment interactions were present (Ottenheim et al., 1996; Ottenheim, 1997). This indicates that natural selection could shape the reaction norm and might have done so in the past. E. arbustorum hoverflies forage during the day to mature their reproductive organs (Gilbert, 1986). Males also actively search for females with which to mate. Therefore, the daytime temperature (indicated by the maximum temperature) is an important factor influencing activity and probably fitness. It has been suggested that the plasticity in colour pattern and wing length were means of adjusting the phenotype to prevailing day-time conditions (Ottenheim, 1997, Ottenheim, Wertheim & Brakefield, in press). The difference between the average daily temperature and the average maximum daily temperature is larger in the South of The Netherlands than in the North (Fig. 1). These geographic differences are consistent over the year. The average temperature a pupa experiences predicts the environment in which the adult is likely to live. A specific average temperature in the North of the Netherlands predicts a lower day-time temperature than that same average temperature in the South of The Netherlands. When the prediction of the environmental conditions changes or is different it is reasonable to assume that the response to the predicting factor also changes.

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Figure 1. The differences between the minimum monthly temperature and the maximum monthly temperature over the year for four weather stations close to the sampled populations (Figure 2). The climatic data was averaged over a 30 year period (1930–1960) (Μ) Gilze-Rijen, (Φ) De Bilt, (Ε) Valkenburg, (Β) De Kooy.

The aim of this study was to show a geographic variation in wing length and colour pattern plasticity of six populations of E. arbustorum. These populations were positioned along two parallel transects over a maximum daily temperature gradient in The Netherlands. We expect a cline in plasticity from North to South on the basis of the above arguments, with flies in the North having a smaller colour pattern and longer wings at a given rearing temperature.

MATERIAL AND METHODS

Two parallel transects were chosen from North to South through The Netherlands to cover a gradient in maximum daily temperature. Figure 2 shows the maximum daily temperatures isomers (data averaged over 30 years) for April. The pattern of the isomers is similar for all months between April and October, the flight period of E. arbustorum. On each transect, flies from three sites were collected and transported in a cooling box to the laboratory. The cooling box was used to reduce stress during transport.

Rearing of hoverflies Egg-batches were obtained following the procedure of Ottenheim and Holloway (1995). Populations and families were kept apart. Larvae were reared in a blended mixture of water, rabbit droppings and yeast, Saccharomyces cerevisiae siccum (20 g/l). The larvae were kept in small plastic trays (18×13×4.5 cm) which were separated into two equal-sized compartments and could be closed with a transparent ventilated lid. One compartment was filled with 100–150 ml medium. Larval densities never

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Figure 2. The two transects in The Netherlands on which collections are made. West transect: Schagen (North), Leiden (Middle), and Tilburg (South). East transect: Medemblik (North), Woerden (Middle) and den Bosch (South). The dotted lines are maximum daily temperature isomers of april averaged over 30 years.

exceeded 10 larvae per 100 ml medium to reduce competition over food. The trays were kept at 20±1°C at a 18/6 light/dark regime. The light conditions were chosen to ensure that larvae would not go into hibernation. Under these conditions, the first larvae will crawl from the medium to the second dry compartment of the tray after about 11 days and might pupate the following day. To make sure that the pupae spend all of their pupal stage at the experimental temperatures, the trays were transferred to the experimental temperatures 11 days after egg-hatching. Three temperatures were used to describe the reaction norms: 10°, 15°, and 20°C (each ±1°C). Pupae were collected daily and transferred to small transparent petri dishes (Ø= 5 cm) which were kept in the dark at the experimental temperature. Emerged adults were collected daily and kept at 20°C for 2 days to allow complete development of the abdominal colour pattern. This has no effect on the size of the coloured patches (M.M. Ottenheim, unpublished results). The flies were then transferred to a −20°C freezer after which they were dried and sexed.

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Measurements Before the flies were pinned and set (see Ottenheim et al., 1995) they were relaxed by soaking them in water with a drop of detergent. Up to six flies per sex per temperature per family were measured. These flies were randomly chosen from all flies produced by a given family reared at one of the experimental temperatures. Thorax and wing length were measured to the nearest 0.1 unit using a Wild binocular microscope with a micrometer fitted in one of the oculars, at 12× magnification. Thorax length was measured along the dorsal surface from behind the head to the front edge of the scutellum. As a measure of wing length, the distance between the cross vein ‘h’ and the most distal point of the vein ‘R4+5’ was taken (for terminology see, Stubbs & Falk, 1983). The colour patch size and tergite II size were also measured with a Wild microscope using a 25× magnification. In one of the oculars a 20×20 grid (0.15 mm2 per square) was fitted and projected over the abdomen of the fly. The abdomen was positioned in the left top corner of the grid. All squares were counted that were filled with 50% or more of the yellow/ dark yellow pattern. The size of tergite II was similarly measured. The measurements were transformed to mm and mm2 before analysis. Replicate measurements of both characters were taken by Mart Ottenheim and Anja Henseler. The measurements were highly repeatable (r>95%). The colour intensity was not measured (Ottenheim et al., 1996)

Data manipulation and analysis To correct for body size the residuals were calculated around the linear regression lines for wing length on thorax length, and colour patch size on tergite II size of all six populations combined. The mean was restored by adding the overall mean of the original characters (wing length and colour patch size) to the residuals (Bookstein et al., 1985). Hereafter, these calculated characters will be called ‘corrected wing length’ and ‘corrected colour pattern’, respectively. To analyse the cline, a three factor analysis of variance (ANOVA) was performed using the GLM procedure in Minitab 9.1 with transect site (North, Middle, South), transect (West, East), and rearing temperature (10, 15, 20°C) as factors. The genotype-environment interaction was calculated separately for each population by performing a two factor ANOVA with rearing temperature and family as factors. Only those families that had representatives at all temperatures could be used.

RESULTS

Table 1 summarizes corrected colour pattern, pupal development time, corrected wing length, and thorax length for males and females at the three rearing temperatures. Rearing temperature (F2,2700=1.63, P>0.001) and sex (F1,2700=3.85, P>0.001) had a significant influence on corrected colour pattern. At lower temperatures smaller colour patches were developed. Males had larger coloured patches than females. The response to rearing temperature was not the same for males and females (interaction:F2,2700=15.1, P<0.001). Pupal development was longer at lower

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T 1. Corrected colour pattern, pupal development time, corrected wing length, and thorax length (all ± SE) for all populations combined in each of the three rearing temperatures. n=number of measured files n

Male

n

Corrected colour pattern (mm2) 10° 486 3.03 ± 0.034 15° 428 4.14 ± 0.032 20° 424 4.92 ± 0.030

Female

501 1.59 ± 0.030 435 2.52 ± 0.031 432 3.27 ± 0.029

Pupal development time (days) 10° 485 29.3 ± 0.15 15° 419 16.1 ± 0.13 20° 414 9.33 ± 0.079 Corrected wing length (mm) 10° 432 7.99 ± 0.012 15° 373 7.84 ± 0.015 20° 370 7.76 ± 0.016 Thorax length (mm) 10° 432 3.59 ± 0.014 15° 373 3.70 ± 0.016 20° 370 3.68 ± 0.015

501 29.6 ± 0.16 431 15.9 ± 0.099 419 9.18 ± 0.059 476 8.24 ± 0.014 388 8.06 ± 0.016 367 7.92 ± 0.017 476 3.56 ± 0.013 388 3.62 ± 0.014 367 3.74 ± 0.016

T 2. Analysis of variance F values for corrected colour pattern with transect site (North–South), transect (West–East), and rearing temperature as factors for males and females separately. The degrees of freedom (df ) are given for all factors and interactions with an error df for males and females separately Male Factor North–South (N–S) West–East (W–E) Temperature N–S∗W–E N–S∗Temperature W–E∗Temperature N–S∗W–E∗Temperature Error

Female

df

F

df

F

2 1 2 2 4 2 4 1320

3.85∗ 8.24∗ 885.36∗∗ 2.11N.S. 0.94N.S. 0.94N.S. 1.08N.S.

2 1 2 2 4 2 4 1350

2.06N.S. 13.66∗∗ 803.06∗∗ 0.84N.S. 1.01N.S. 0.03N.S. 1.78N.S.

N.S.=not significant, ∗=P<0.05, ∗∗=P<0.01, ∗∗∗=P<0.001

temperatures (F2,2663=14.0, P<0.001). The sexes responded similarly to rearing temperature (F1,2663=0.06, P>0.05, N.S.) and there was no significant interaction (F2,2663=2.73, P>0.05, N.S.). At lower temperatures shorter thoraces were developed (F2,2400=97.4, P<0.001) but with relatively longer wings (F2,2400=167.4, P<0.001). Females had longer wings corrected for thorax length than males (F2,2400=333.3, P<0.001) and on average shorter thoraces (F2,2400=27.4, P<0.001). Neither interaction was significant (Corrected wing length, F2,2400=2.41, P>0.05, N.S.; thorax length, F2,2400=2.33, P>0.05, N.S.). Corrected colour pattern The main factor influencing the corrected colour pattern was the rearing temperature (Table 2). for both sexes the populations on the West transect were

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Figure 3. Average corrected colour pattern (mm2) for males and females along the West and East transects for each experimental temperature. (Μ) North, (Ε) Middle, (Φ) South. (A) Male - West; (B) Male - East; (C) Female - West; (D) Female - East.

significantly darker over all experimental temperatures than flies from populations on the East transect. Furthermore, when studied closely, Figure 3 shows a tendency for males to have relatively larger colour patches in the North than in the South. In the West transect the Northern flies have larger colour patches at all rearing temperatures than the other populations while in the East transect the Tilburg flies have the smallest colour patches. However, this effect cannot be found in females. None of the interactions were significant. The two factor ANOVAs per population also showed a strong rearing temperature effect and significant family effects (Table 3). Five out of twelve family–environment interactions were significant when calculated for the populations separately. This suggests that there is genetic variation in the height of the reaction norm within each population and in some populations also the degree of plasticity significantly varies between families. Pupal development time The most important factor influencing pupal development time was rearing temperature (Table 4). The geographic distribution did not have any significant effect. However, in both sexes the interaction between rearing temperature and

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T 3. Analysis of variance F values for corrected colour pattern with rearing temperature and family as factors for each population and for each sex separately. The degrees of freedom (df ) are given for each factor and interaction Males

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Schagen Temperature Family Family∗Temperature

2,114 9,114 18,114

69.90∗∗∗ 2.25∗ 1.45N.S.

2,107 9,107 18,107

119.66∗∗∗ 7.74∗∗∗ 1.71∗

Leiden Temperature Family Family∗Temperature

2,161 10,161 20,161

162.47∗∗∗ 2.31∗ 1.43N.S.

2,175 10,175 20,175

149.72∗∗∗ 5.72∗∗∗ 1.13N.S.

Tilburg Temperature Family Family∗Temperature

2,139 12,139 24,139

94.42∗∗∗ 3.28∗∗∗ 1.44N.S.

2,144 13,144 26,144

112.77∗∗∗ 4.57∗∗∗ 1.27N.S.

Medemblik Temperature Family Family∗Temperature

2,180 13,180 26,180

161.08∗∗∗ 3.39∗∗∗ 1.41N.S.

2,182 14,182 28,182

165.91∗∗∗ 3.09∗∗∗ 2.95∗∗∗

Woerden Temperature Family Family∗Temperature

2,159 16,159 32,159

140.73∗∗∗ 4.43∗∗∗ 1.92∗∗

2,158 15,158 30,158

111.14∗∗∗ 2.72∗ 0.92N.S.

Den Bosch Temperature Family Family∗Temperature

2,194 17,194 34,194

181.43∗∗∗ 2.45∗∗ 1.66∗

2,180 15,180 30,180

171.48∗∗∗ 6.79∗∗∗ 2.25∗

N.S.=not significant, ∗=P<0.05, ∗∗=P<0.01, ∗∗∗=P<0.001

T 4. Analysis of variance F values for pupal development time with transect site (North–South), transect (West–East), and rearing temperature as factors for males and females separately. The degrees of freedom (df ) are given for all factors and interactions with an error df for males and females separately Male Factor North–South (N–S) West–East (W–E) Temperature N–S∗W–E N–S∗Temperature W–E∗Temperature N–S∗W–E∗Temperature Error

Female

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2 1 2 2 4 2 4 1300

0.03N.S. 0.04N.S. 6099.98∗∗∗ 0.59N.S. 6.64∗∗∗ 1.47N.S. 1.10N.S.

2 1 2 2 4 2 4 1333

0.76N.S. 0.02N.S. 8019.05∗∗∗ 1.69N.S. 8.25∗∗∗ 3.33∗ 3.35∗

N.S.=not significant, ∗=P<0.05, ∗∗=P<0.01, ∗∗∗=P<0.001

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Figure 4. Average pupal development time (days) for males and females along the West and East transects for each experimental temperature. (Μ) North, (Ε) Middle, (Φ) South. A–D as per Fig. 3.

transect site was significant. As can been seen from Figure 4 this was primarily an effect of the two middle populations from Leiden and Woerden. The reaction norms of these two populations were steeper than those of the four other populations. In females the interaction between transect and rearing temperature was significant. The response of the populations on the West transect to the rearing temperature was greater than that of the populations on the East transect. The three-way interaction was also significant in females, indicating that the response to rearing temperature from North to South is not the same along the two transects. Corrected wing length There was a clear difference in corrected wing length among the transect sites in response to rearing temperature (Fig. 5). Both sexes from the populations of Leiden and Woerden have significantly longer wings than the flies from the other populations (Table 5). In females there was also an effect from West to East, where females from the West transect had shorter wings over all experimental temperatures. For both sexes the transect site–transect interaction was significant, probably because the differences among the populations from North to South were larger along the West transect than along the East transect (see Fig. 5). The significant transect–temperature

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Figure 5. Average corrected wing length (mm, see text) for males and females along the West and East transects for each experimental temperature. (Μ) North, (Ε) Middle, (Φ) South. A–D as per Fig. 3.

T 5. Analysis of variance F values for corrected wing length with transect site (North–South), transect (West–East), and rearing temperature (temperature) as factors for males and females separately. The degrees of freedom (df ) are given for all factors and interactions with an error df for males and females separately Male

Female

Factor df North–South (N–S) West–East (W–E) Temperature N–S∗W–E N–S∗Temperature W–E∗Temperature N–S∗W–E∗Temperature Error

2 1 2 2 4 2 4 1157

F 24.39∗∗∗ 0.77N.S. 74.15∗∗∗ 4.37∗ 1.88N.S. 2.27N.S. 1.43N.S.

df 2 1 2 2 4 2 4 1213

N.S.=not significant, ∗=P<0.05, ∗∗=P<0.01, ∗∗∗=P<0.001

F 27.04∗∗∗ 8.06∗ 105.61∗∗∗ 3.35∗ 1.76N.S. 4.37∗ 0.58N.S.

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T 6. Analysis of variance F values for corrected wing length with rearing temperature and family as factors for each population and for each sex separately. The degrees of freedom (df ) are given for each factor and interaction Male df Schagen Temperature Family Family∗Temperature Leiden Temperature Family Family∗Temperature Tilburg Temperature Family Family∗Temperature Medemblik Temperature Family Family∗Temperature Woerden Temperature Family Family∗Temperature Den Bosch Temperature Family Family∗Temperature

Female F

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2,95 9,95 18,95

14.14∗∗∗ 6.63∗ 2.29∗

2,90 9,90 18,90

12.17∗∗∗ 12.19∗∗ 1.10N.S.

2,143 9,143 18,143

6.75∗ 7.62∗∗∗ 1.51N.S.

2,153 9,153 18,153

20.94∗∗∗ 9.26∗∗∗ 2.27∗

2,110 12,110 24,110

27.39∗∗∗ 6.63∗∗∗ 1.88∗

2,121 13,121 26,121

22.45∗∗∗ 2.96∗ 2.05∗

2,162 13,162 26,162

20.83∗∗∗ 6.68∗∗∗ 1.49N.S.

2,165 13,165 26,165

37.40∗∗∗ 5.52∗∗∗ 2.21∗

2,136 17,136 34,136

4.72∗ 4.05∗∗∗ 1.94∗

2,160 15,160 30,160

9.32∗∗∗ 5.03∗∗∗ 1.44N.S.

2,164 15,164 30,164

23.49∗∗∗ 7.00∗∗∗ 2.67∗∗∗

2,146 13,146 26,146

39.38∗∗∗ 5.75∗∗∗ 2.56∗∗∗

N.S.=not significant, ∗=P<0.05, ∗∗=P<0.01, ∗∗∗=P<0.001

interaction in females is consistent with the observation that the reaction norms in the West were on average less steep and/or more concave than those in the East where they tended to be more linear and/or more convex. On a qualitative basis these findings also seem to apply to the males, but the differences were not statistically significant. For corrected wing length also all populations showed significant rearing temperature and family effects (Table 6). The family–environment interactions for corrected wing length were significant in eight out of twelve calculations. Again, this suggests genetic variation for the height and shape of the reaction norm. Thorax length Thoraces were significantly longer at higher temperatures than at lower temperatures (Fig. 6, Table 7). In males a significant difference was found between the two transects. Thoraces of the males from the West transect were on average larger over all rearing temperatures than of those from the East transect. Both sexes showed a significant transect site–temperature interaction and transect–temperature interaction. DISCUSSION

The aim of this study was to show geographic variation in plasticity and to correlate this with a North–South maximum daily temperature gradient. We did

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Thorax length

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Figure 6. Average thorax length (mm) for males and females along the West and East transects for each experimental temperature. (Μ) North, (Ε) Middle (Φ) South. A–D as per Fig. 3.

T 7. Analysis of variance F values for thorax length with transect site (North–South), transect (West–East), and rearing temperature as factors for males and females separately. The degrees of freedom (df ) are given for all factors and interactions with an error df for males and females separately Male Factor North–South (N–S) West–East (W–E) Temperature N–S∗W–E N–S∗Temperature W–E∗Temperature N–S∗W–E∗Temperature Error

df 2 1 2 2 4 2 4 1157

Female F

df N.S.

2.20 4.72∗ 65.81∗∗∗ 0.80N.S. 3.67∗∗ 6.07∗∗ 1.81N.S.

2 1 2 2 4 2 4 1213

F 2.65N.S. 0.85N.S. 42.53∗∗∗ 2.18N.S. 7.29∗∗∗ 9.05∗∗ 1.75N.S.

N.S.=not significant, ∗=P<0.05, ∗∗=P<0.01, ∗∗∗=P<0.001

find differences in plasticity between the populations but not from North to South as expected. The two middle populations differed from the other four populations. Depending on the character, they were different in height (corrected colour pattern

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and corrected wing length) and slope (pupal development time) of the reaction norm. Only for the corrected colour pattern in males could a North–South geographic variation be observed; male flies from the North population tended to have larger colour patches over all rearing temperatures than the flies from the South population. A more consistent geographic variation was found from West to East. In some characters the height of the reaction norm was different (corrected colour pattern, female corrected wing, and female thorax length) and sometimes the shape of the reaction norm varied (corrected wing length). These results suggest that an environmental factor varies from West to East and that the populations are genetically differentiated to suit the local environmental conditions. Other studies also reported differences in reaction norms between populations. Conover and Present (1990) found that the reaction norms of growth rate to temperature of Northern populations, Atlantic silversides (Menidia menidia) were steeper than the reaction norms displayed by the Southern populations. They could only speculate about the mechanisms behind this difference. Kudoh et al., (1995) reasoned that the differences they found in life-history trait reaction norms between populations of the Crucifer Cardamine flexuosa, were due to geographic variation in climatic conditions. Delpuech et al. (1995) reported differences in height of the reaction norm of ovaria size to rearing temperature in Drosophila melanogaster. Isofemale lines from two populations in the Congo had on average fewer ovarioles than those from a single French population. Some debate about these results is possible since Delpuech et al. did not correct ovaria size for overall body size and this character is also known to vary geographically. Drosophila flies from colder latitudes are larger than flies from warmer latitudes when reared at the same temperature ( James, Azevedo & Partridge, 1995). Another method to study relative wing length is to calculate the wing–thorax ratio (e.g. David et al., 1994). In E. arbustorum, this ratio is still strongly related to the thorax length (all populations combined, males, r=−0.677, n=1175; females, r=−0.642, n=1231) Thus flies with a larger thorax length have relatively shorter wings. Therefore, one could argue that the observed differences in corrected wing length between the west and the east populations are caused by genetically determined differences in body size. The thoraces of males tend to be larger on the West transect. The above argument would then predict that the male flies along the West transect would have relatively smaller wings. However, no significant differences in corrected wing length were found for males. Females along the West transect have relatively short wings but the thorax length was similar along both transects, indicating that the observed differences in corrected wing length could not primarily be caused by the morphometric relationships between thorax length and wing length and that selection could have shaped the reaction norm. Previous experiments showed a strong correlation between abdominal colour pattern and pupal development time (Ottenheim et al., 1995). This led to the hypothesis that the size of the colour pattern is not influenced by the pupal rearing temperature per se, but may be dependent on the pupal development time. A strong correlation was also found in the present study between these two characters (all populations combined, male r=0.757, n=1318; female r=−0.725, n=1351). The results on pupal development time (Fig. 4, Table 4) showed that the slope of the reaction norms significantly differed among the populations along the two parallel transects with steeper slopes for the two middle populations. When the colour pattern is directly influenced by the pupal development time we would expect that

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the reaction norms of colour pattern should also differ in slope. However, there was no evidence for such difference. This could indicate that the colour pattern is not solely determined by pupal development time but also by temperature. Alternatively, the flies may respond differently to the same factor (pupal development time). For wing length and colour pattern in many populations significant genotypeenvironment interactions were found, indicating that genetic variation for plasticity was present. It is noteworthy that this interaction was significant in only one out of the four possible genotype–environment interactions for the Leiden populations. Two previous studies with flies from the Leiden did find significant genotype–environment interactions for both characters in both sexes (Ottenheim et al., 1995; submitted). Both these studies used six instead of three temperatures and all flies of a family were measured. It is possible that the present experiment did not allow us to detect all genotype–environment interactions for wing length and colour pattern in all populations. The question arises why so little evidence for North–South geographic variation was found. Perhaps the geographic gradient was not sufficient to be able to detect a parallel variation in plasticity. Sampling a larger transect spanning a larger environmental-gradient might overcome this problem. However, it is possible that there is a cline from West to East and this might be a climatic variable such as wind speed. The wind comes on average in the Netherlands from the South-West and the wind speed is less in the East of The Netherlands than in the West. However, the two Southern populations were chosen because of their similar climatic conditions and wind speed does not vary between these populations. Still, a clear difference can be found in plasticity. Alternatively, ecological variables may be of importance. For example, the flowers on which the flies forage may be flowering at different times of the day. Finally, this West–East variation might have arisen due to nonrandom sampling of the populations, although sampling was always done carefully to exclude such an effect.

ACKNOWLEDGEMENTS

We would like to thank Hans Roskam and two anonymous referees for their constructive comments on the paper. The research reported here forms part of a Ph.D project funded by Leiden University.

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