Journal of Insect Science: Vol. 13 | Article 51 Harris et al.

A test of the thermal melanism hypothesis in the wingless grass- hopper Phaulacridium vittatum

Rebecca M. Harris1a*, Peter McQuillan1b, Lesley Hughes2c

1School of Geography and Environmental Studies, University of Tasmania, Private Bag 78 Hobart, 7001, Australia 2Department of Biological Sciences, Macquarie University, North Ryde, NSW, 2109, Australia

Abstract Altitudinal clines in melanism are generally assumed to reflect the fitness benefits resulting from thermal differences between colour morphs, yet differences in thermal quality are not always dis- cernible. The intra-specific application of the thermal melanism hypothesis was tested in the wingless grasshopper Phaulacridium vittatum (Sjöstedt) (Orthoptera: Acrididae) first by measur- ing the thermal properties of the different colour morphs in the laboratory, and second by testing for differences in average reflectance and spectral characteristics of populations along 14 altitu- dinal gradients. Correlations between reflectance, body size, and climatic variables were also tested to investigate the underlying causes of clines in melanism. Melanism in P. vittatum repre- sents a gradation in colour rather than distinct colour morphs, with reflectance ranging from 2.49 to 5.65%. In unstriped grasshoppers, darker morphs warmed more rapidly than lighter morphs and reached a higher maximum temperature (lower temperature excess). In contrast, significant differences in thermal quality were not found between the colour morphs of striped grasshoppers. In support of the thermal melanism hypothesis, grasshoppers were, on average, darker at higher altitudes, there were differences in the spectral properties of brightness and chroma between high and low altitudes, and temperature variables were significant influences on the average reflec- tance of female grasshoppers. However, altitudinal gradients do not represent predictable variation in temperature, and the relationship between melanism and altitude was not consistent across all gradients. Grasshoppers generally became darker at altitudes above 800 m a.s.l., but on several gradients reflectance declined with altitude and then increased at the highest altitude.

Keywords: altitude, geographic variation, insects, Orthoptera, reflectance Abbreviations: PCA, principal components analysis Correspondence: a [email protected], b [email protected], c [email protected], *Corresponding author Received: 17 March 2012 Accepted: 31 December 2012 Copyright : This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unre- stricted use, provided that the paper is properly attributed. ISSN: 1536-2442 | Vol. 13, Number 51 Cite this paper as: Harris RM, McQuillan P, Hughes L. 2013. A test of the thermal melanism hypothesis in the wingless grasshopper Phaulacridium vittatum. Journal of Insect Science 13:51. Available online: http://www.insectscience.org/13.51

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Journal of Insect Science: Vol. 13 | Article 51 Harris et al.

Introduction lower temperatures are darker than those reared at higher temperatures. Melanism, the occurrence of darker pigmenta- tion in individuals within species or between Altitudinal gradients have traditionally been closely related species, is common in many used to test the thermal melanism hypothesis animal groups and well-documented in in- under natural conditions because they repre- sects. There are several hypotheses explaining sent steep gradients in temperature over why it has evolved, including mate attraction, relatively short distances, facilitating gene crypsis, and resistance to disease, desiccation, flow and minimising the influence of other co- and UV radiation (Majerus 1998). The hy- varying factors is (Körner 2007). pothesis that has attracted the most attention is the thermal melanism hypothesis, which states While it has been suggested that a positive that under conditions of low temperature, dark relationship between altitude and melanism individuals are at an advantage compared to has been well-demonstrated in a range of in- light individuals because they warm up more sects (True 2003; Clusella-Trullas et al. 2007), quickly at any given level of radiation much of the research has actually focused on (Clusella-Trullas et al. 2007; Clusella Trullas very few species, for example Colias butter- et al. 2007). This improved fitness is ex- flies (Watt 1968; Roland 1982; Ellers and pressed in higher proportions of dark Boggs 2002), or reports inter-specific compar- individuals being found in cold environments, isons (Watt 1968; Rapoport 1969; Kingsolver such as at high altitudes. 1983; Kingsolver and Watt 1983). Published papers that present tests of altitudinal clines in Increased fitness through thermal melanism melanism within species are in fact restricted has been demonstrated in several insect to one species of Homoptera (Halkka et al. groups, including butterflies (Roland 1982; 1980; Berry and Willmer 1986; Boucelham Ellers and Boggs 2004; Roland 2006), beetles and Raatikainen 1987), three species of Lepi- (Rhamhalinghan 1999), and grasshoppers doptera (Ellers and Boggs 2002; Karl et al. (Unsicker et al. 2008). Fitness consequences 2009; Karl et al. 2010), one species of Diptera of melanisation include increased activity (Munjal et al. 1997; Rajpurohit et al. 2008) (Roland 1982; Guppy 1986; de Jong et al. and one species of Orthoptera (Guerrucci and 1996; Ellers and Boggs 2004; Roland 2006), Voisin 1988). Further, very few of these stud- feeding and mating (Brakefield 1984b; ies have investigated change along continuous Parkash et al. 2008), fecundity and egg matu- altitudinal gradients. They report general cor- ration rates (Ellers and Boggs 2004), survival relations between altitude and colour at (Kingsolver 1995; Parkash et al. 2008), and unrelated sites (eg. Halkka et al. 1980; predator avoidance (Clusella-Trullas et al. Guerrucci and Voisin 1988) or compare the 2007). Additional support for the thermal extremes of altitude (Rajpurohit et al. 2008; melanism hypothesis is provided by laborato- Karl et al. 2010). Others are studies of ex- ry rearing experiments, in which insects, such treme habitats, such as alpine, arctic, or sub- as butterflies (Lewis 1985; Marriott and Antarctic habitats (Roland 1978; Mikhailov Holloway 1998; Karl et al. 2009) and hover- 2001; Davies et al. 2007). In this paper, dif- flies (Marriott and Holloway 1998), reared at ferences in melanism in temperate grasshoppers collected from several sites

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Journal of Insect Science: Vol. 13 | Article 51 Harris et al. along replicated gradients are described. Thus, (Pereboom and Biesmeijer 2003), butterflies trends in altitude and temperature, the exist- (Watt 1968, 1969), and grasshoppers ence of thresholds, and the influence of site or (Forsman 1997). However, it has also been habitat differences can be identified. suggested that the difference in body tempera- ture between dark and light individuals would Melanism has been quantified in a number of be too small to be of any ecological im- ways. In species that can be separated into dis- portance, or that other effects, such as tinct colour morphs, the representation of predator avoidance, would swamp any ther- colour morphs within populations has been moregulatory advantage (Digby 1955; compared (Brakefield 1984a; de Jong and Willmer and Unwin 1981). Brakefield 1998; Pereboom and Biesmeijer 2003). In other species, the area covered by The primary objective of this study was to test dark spots (Stewart and Dixon 1989), bands the intra-specific application of the thermal (Honek 1993), or scales (Lewis 1985; Guppy melanism hypothesis in the wingless grass- 1986) has been measured. However, these ap- hopper Phaulacridium vittatum (Sjöstedt) proaches are not possible in species where (Orthoptera: Acrididae). The hypothesis that melanism represents a gradation in colour. A there is no difference in the thermal properties few studies have used spectrometry to quanti- of the different colour morphs was tested us- fy average reflectance as a measure of ing laboratory experiments. Then, the melanism (e.g., Brakefield and Willmer hypothesis that the average reflectance and 1985). Here, the colour of grasshoppers along spectral characteristics of populations do not altitudinal gradients is described by quantify- differ along altitudinal gradients was tested. ing differences in spectral data. A spectrum is Furthermore, the underlying causes of clines a graphical representation of the reflection of in reflectance and body size was investigated light from a sample. As colour changes, the by considering the proportion of winged and slope of the curve, amplitude, and the number striped grasshoppers, and correlations between and position of peaks within the spectrum all reflectance, body size, and climatic variables change (Grill and Rush 2000). These spectral (temperature and rainfall). data can also be condensed into three colour components, namely brightness, chroma, and P. vittatum was used because it is a common, hue. Brightness is a measure of light intensity widely-distributed species that has variable across all wavelengths, chroma is a measure body size and is polymorphic for colour pat- of the purity of a colour, and hue is colour tern. If the thermal melanism hypothesis is (e.g., violet, blue, green, red) (Endler 1990; supported, the following would be expected: Grill and Rush 2000). darker individuals warm up and cool down more quickly and reach higher equilibrium The thermal melanism hypothesis rests on the temperatures than lighter ones; a greater pro- assumption that there is a relationship be- portion of dark individuals (i.e. lower tween reflectance, body size, and the reflectance) found at high altitude sites com- absorption of radiation. This has been demon- pared with low altitude populations; a strated under laboratory conditions in many negative correlation between melanism and insects, including beetles (Brakefield and body size; and a negative correlation between Willmer 1985; Stewart and Dixon 1989; de melanism and temperature (i.e., positive cor- Jong et al. 1996; Rhamhalinghan 1999), bees relation between reflectance and temperature).

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Journal of Insect Science: Vol. 13 | Article 51 Harris et al.

Methods and Materials Thermal properties of colour morphs Warming-up curves under known radiation P. vittatum is a common species of Acrididae, levels were used to test for differences in the widely distributed in open habitats in the cool, thermal qualities of the colour morphs of the temperate areas of eastern and southern Aus- wingless grasshopper. Twelve runs of four to tralia (-23° 36’ to -43° 06’ S latitude). It is six randomly chosen specimens were recorded restricted to higher elevations in the north, but (total n = 59). The results of a typical run are its altitudinal range extends from sea-level to shown in Figure 2. Thermocouples were in- 1500 m a.s.l. in cooler, more southerly loca- serted into the thorax of recently killed tions such as Tasmania (Key 1992). P. grasshoppers, and body temperature was rec- vittatum has an annual life cycle, overwinter- orded every ten seconds after a 375 W infra- ing in the egg stage. Hatchlings pass through red heat lamp, 52 cm above the insects, was five nymphal stages before emerging as turned on. The thermocouples were Type T adults. The earliest hatchlings may be ob- fine wire copper-constantan thermocouples served in the field in late spring, with adults (diameter of individual wire components 0.2 surviving into late autumn in warmer years mm) connected to a Campbell Scientific (Baker 2005). Adults can be macropterous, CR10X Datalogger (www.campbellsci.com) with functional wings, or brachypterous, inca- via an AM 1632 Multiplexer and using the pable of flight (referred to here as winged and LoggerNet 3.2 program and a 107TP refer- wingless, respectively). These forms occur ence temperature probe. The radiation level 2 together in almost all populations of P. vitta- was 565 W m , measured using a Campbell tum (Key 1992). The wingless form is most Scientific LI200X pyranometer with a spectral abundant in pastures, while the winged is the range of 400–1100 nm. The substrate on more abundant form in areas dominated by which the grasshoppers rested was sand glued shrubs, strips along forest margins, and in onto a cardboard base. The surface tempera- gardens (Clark 1967). ture of the substrate was measured using an exposed thermocouple positioned within 2 cm Females range in size from 12 to 20 mm long, of the grasshopper. After five minutes, a fan and males from 10 to 13 mm long (Baker was turned on to generate a wind speed of 4 -1 2005). Individuals range in colour from light, m/s , striking laterally across the body, and through to dark brown and black, and rarely, the temperature was logged for an additional 5 green. Individuals can be striped, with two minutes. Wind speed was measured using a white longitudinal stripes on the dorsal sur- Kestral 3000 pocket weather meter face, unstriped, or patterned, with very dark (www.kestrelmeters.com) positioned in the lateral surfaces on the pronotum and a light centre of the arena, 2 cm above the substrate. dorsal surface. All colours are manifested in The reflectivity of the ground surface was the winged and wingless forms, with the ex- measured by inverting the pyranometer and ception of the green morph and the very light expressing the value as a fraction of the up- patterned form, which are not found in winged right measurement. The colour, weight, and grasshoppers. The range of colour morphs can femur length of each specimen tested were be present within the same population and is recorded. set for an individual once it reaches the adult stage (Key 1992).

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Journal of Insect Science: Vol. 13 | Article 51 Harris et al.

found to be significantly different in females (Tukey’s q = 3.18; p < 0.05) and males (q =

3.31; p < 0.05), with the reflectance of the dark categories being significantly higher than

the light categories. A strong linear relation- ship existed between measured reflectance 2 2 and colour morph (r = -0.93 for males; r = - 0.75 for females), enabling the results of the

warming experiment to be related to the pat- terns found along the altitudinal gradients.

Sampling methods

Grasshoppers were collected along 14 altitu- dinal gradients in Tasmania. On each gradient

there were four sites, one in each of the fol- lowing altitudinal bands:

< 100 m, 200–250 m, 400–450 m, and 700– 800 m a.s.l. Where possible, a fifth site was

sampled at 800–1000 m a.s.l. Grasshoppers were collected from roadside verges and

clearings beside roads. A hand-held GPS was Figure 1. Average reflectance of the colour morphs of a) fe- used to record the latitude, longitude, and alti- male and b) male Phaulacridium vittatum based on a visual separation of colour morphs. Bars show ± 2 SE. High quality tude of each site. figures are available online. At each site, grasshoppers were collected by The relative warming and cooling rates were hand and a sweep net for 20 minutes. The estimated for each specimen as K = number of individuals collected varied across 0.5Texc/t1/2, where Texc is the temperature ex- sites, depending on their abundance. At sites cess, the maximum difference between where the grasshoppers were very abundant, ambient and body temperature that was approximately 20 individuals were collected, reached during heating (or cooling), and t1/2 is and an attempt was made to represent males the half time of the temperature change and females equally. The number of speci- (Pereboom and Biesmeijer 2003). mens measured at each site was therefore unequal across the sites, ranging from 1 to 25 To test whether colour codes could be as- with a mean number of 12.7 ± 0.56 (Table 1). signed by eye to colour morphs representing In total, 853 specimens were measured from significantly different thermal qualities, each the altitudinal gradients within Tasmania, rep- specimen was assigned to a light, medium, or resenting 414 females and 439 males. Both dark category within the striped and unstriped winged and wingless grasshoppers were in- groups. This visual separation into discrete cluded in the altitudinal analyses because colour morphs was checked against specimens wingless grasshoppers are often scarce at (n = 80) representing the full range of colours higher altitudes. in P. vittatum (Figure 1). The average reflec- tance of the light and dark colour morphs was

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Journal of Insect Science: Vol. 13 | Article 51 Harris et al. Reflectance the thorax, and one on the lateral surface of Reflectance is the ratio of reflected to incident the right femur. The average proportion of light, expressed here as a percentage. The re- light reflected at 5 nm intervals was calculated flectance of each specimen was measured for each individual. The mean of these meas- using an Ocean Optics USB2000 spectropho- urements was calculated across all tometer (Ocean Optics Incorporated, measurements for that individual to obtain an www.oceanoptics.com) with a PX-2 pulsed average. xenon light source. Measurements were taken at an angle of 45°. An attachment was used Body size that reduced the size of the light beam to less Body size was estimated using the length of than 2 mm in diameter, so that it was com- the right femur, which was measured using pletely covered by the body part being handheld vernier callipers (accurate to 0.02 measured. The spectrophotometer was con- mm). Femur length is closely correlated with nected to a PC running Ocean Optics body size and other size metrics in grasshop- OOIBase 32 v.1.0.2.0 software, with the inte- pers (Masaki 1967) and is more reliable than gration time set at 7 ms, and each body length, which can change as specimens measurement averaged 10 times by the soft- dry. ware (Bruce et al. 2005). All sample reflectance spectra were calculated relative to Climate Variables a Barium sulphate white standard, and a dark One of the main predictions of the thermal and white standard reference spectrum was melanism hypothesis is that reflectance should taken every 10 minutes during measurement be positively related to ambient temperature of samples. Measurements were taken in a and/or solar radiation. This hypothesis was dark room. The range of 300 to 700 nm was tested using temporally correspondent climate used, as this was the range of sensitivity of the data for each collecting site. Data were ex- machine. Ideally, reflectance would have been tracted from the Australian Water Availability measured over the wavelengths 290–2600 nm Project climate dataset (Jones et al. 2007). in order to incorporate the ultraviolet, visible, Annual maximum (Tmax) and minimum (Tmin) and infrared parts of the spectrum (Gates temperatures and annual rainfall for the year 1980; Nussear et al. 2000). However, very preceding each collection date were extracted few studies have objectively measured reflec- at a spatial resolution of 10 km. In this way, tance, and our measurements of visible the conditions prevailing from the laying of radiation represent a significant improvement the egg to adulthood were considered. on studies in which the continuum of colour has been simplified by arbitrarily separating Statistical Methods colours into discrete groups. Warming curves. To take size differences into account while testing for differences in All specimens were pinned and dried prior to relative warming and cooling rates (K), measurement. Willmer and Unwin (1981) maximum temperature excess (Texc), and half found less than 1% variability when they time of the temperature change (t1/2), a compared reflectance of freshly killed and multiple regression model was fitted with dried insects. The reflectance was measured at randomisation of modified residuals. The four different positions on each individual, predictor variables included were weight, two on the dorsal surface, one on the side of femur length, and colour code. Tests were

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Journal of Insect Science: Vol. 13 | Article 51 Harris et al. carried out using MiniTab Version 16.1.0 body size and reflectance was also investigat- (2010) and the REGRESSRESRAN macro ed. This method calculates the proportion of from Butler (2001). All probabilities given variance explained independently and jointly refer to those for two-sided randomizations. by each variable (Chevan and Sutherland 1991; MacNally 1996; Walsh and Mac Nally Spectral data. Principal Components Analy- 2005) eliminating spurious conclusions based sis (PCA) was used to condense the spectral on joint correlations with other independent data because it has been shown to be sensitive variables. Analyses were performed using the to slight differences in populations within the R public-domain statistical package (R Project same colour category (i.e., that are similar in for Statistical Computing release 1.9.0, hue) (Grill and Rush 2000). Data were first www.r-project.org). standardized to the area under the curve (Grill and Rush 2000) then grouped into 5 nm bins Randomisation was used to quantify relative and averaged, resulting in 81 summary values effect sizes associated with the partitioning by per spectrum. Eigenvalues were calculated estimating the z-score ([observed – mean from a correlation matrix, and PCA1, PCA2, {randomisations}] /SD{randomisations}) for and PCA3 were used as dependent variables each predictor variable, and the statistical sig- in further statistical analysis. One-way nificance based on the upper 95% confidence ANOVA was used to detect differences limit (z ≥ 1.65) (MacNally 2002). among means of components. Where differ- ences were significant in the ANOVA, Tukey- Results Kramer’s HSD post hoc comparisons were used to determine which altitude category dif- Warming curves fered significantly from the others. The typical warming and cooling curves showed that grasshoppers heated up rapidly Relationship between reflectance and cli- when exposed to radiation until they reached 2 mate. Hierarchical partitioning of R values an equilibrium temperature, and cooled-down was used to identify the climate variables with rapidly when wind was applied (Figure 2). the most independent influence on grasshop- per reflectance. The relationship between The trends predicted by biophysical theory, that smaller and darker grasshoppers warm up

more quickly and darker ones reach higher equilibrium temperatures, were generally

demonstrated, but the differences were not always significant (Table 2). In unstriped

grasshoppers, darker morphs warmed more rapidly than the lightest and reached a higher

maximum temperature (lower temperature excess) (Table 3). After weight was accounted

for, colour was significantly negatively related to the maximum temperature excess reached

(Table 2). Although the relative warming rate Figure 2. Results of a typical warming run. Ambient tempera- was not significantly different between colour ture is shown by the thick line, and body temperatures of individual Phaulacridium vittatum from a range of colour morphs morphs, the half time of the temperature are indicated by the dashed (males) and full (female) lines. High quality figures are available online. Journal of Insect Science | http://www.insectscience.org 7

Journal of Insect Science: Vol. 13 | Article 51 Harris et al.

change (t1/2) was significantly different, with altitude, the darker the animals. Over all gra- darker morphs warming to equilibrium tem- dients, the proportion of winged grasshoppers perature more quickly. Weight was a and unstriped morphs increased at higher alti- significant factor in striped grasshoppers, with tudes (Figure 3). Body size remained constant smaller grasshoppers demonstrating higher with altitude (ρ = 0.03, p > 0.05). temperature excesses. Colour was not a signif- icant factor in this group after weight was Males, when analysed separately from fe- accounted for. Cooling rates were not signifi- males, did not show a significant relationship cantly related to colour code or size in striped between reflectance and size (ρ = 0.040, p > or unstriped grasshoppers. 0.05). However, they did show a marginally significant (ρ = -0.091; p = 0.05) negative cor- Patterns in reflectance and body size along relation between reflectance and altitude. The altitudinal gradients average reflectance of females showed a sig- The average reflectance values for P. vittatum nificant negative correlation with altitude (ρ = ranged between 2.49 and 5.65% (Table 4). -0.114; p < 0.05), but not femur length (ρ = - Females (n = 414) were significantly lighter 0.027, p > 0.05). These correlations were than males (n = 439) (F1,851 = 56.94, p < strengthened if sites above 900 m were ex- 0.0001). Striped grasshoppers (n = 203) were cluded. Femur length and altitude were not slightly lighter than unstriped (n = 650) (F1,851 significantly correlated in either males (ρ = - = 8.45, p < 0.005). Winged grasshoppers (n = 0.031, p > 0.05) or females (ρ = 0.045, p > 150) were, on average, significantly darker 0.05). (F1,851 = 14.00, p < 0.0001) and larger (F1,851 = 8.15, p < 0.005) than wingless grasshoppers (n On average, grasshoppers collected close to = 703). sea level were lighter than those collected be- tween 800 and 900 m a.s.l. (Figure 4). This Overall, average reflectance was negatively relationship was significant in male grasshop- correlated with femur length (Spearman’s ρ = pers, but not female (Tukey’s q = 2.96, p < -0.206, p < 0.01) and negatively correlated 0.05). The reflectance then increased at sites with altitude (ρ = -0.104; p < 0.01). So, larger above 900 m a.s.l. to close to the same level as grasshopper were darker, and the higher the at low altitude sites.

Figure 3. Percentage of winged (black bars) and striped (hatched bars) Phaulacridium vittatum, and mean femur length Figure 4. Mean average reflectance of female (open bars) and (mm) (open bars) within altitudinal bands. High quality figures male (hatched bars) Phaulacridium vittatum within altitudinal are available online. bands. Bars show ± 2 SE. High quality figures are available online. Journal of Insect Science | http://www.insectscience.org 8

Journal of Insect Science: Vol. 13 | Article 51 Harris et al.

These trends are based on all the sites com- bined, but they were not apparent within all

gradients. Of the 14 gradients, six did not show any significant difference in average

reflectance between sites along the gradient (p > 0.05). On six of the remaining eight gradi-

ents, grasshoppers were generally darker at higher altitudes. While the transition was

gradual along one gradient, on the others it occurred more sharply above a threshold,

which occurred at different altitudes on differ- ent gradients (e.g. 400 m a.s.l. on one and 800

m a.s.l. in two other gradients) (Figure 5a). It was only on three of these gradients that

grasshoppers became lighter at the highest altitude (Figure 5b). The remaining two gradi-

ents showed significant differences that appeared to be site specific (Figure 5c). The

differences in reflectance along individual gradients were not related to the number of

winged or striped grasshoppers.

Climatic correlates The climate variables that explained a signifi-

cant amount of the variability in average female reflectance were Tmax (23%) and Tmin

(20%), which were positively correlated with reflectance (Figure 3.6a). Altitude also inde-

pendently explained 32% of the variability. The negative joint contribution of altitude,

femur length, and Tmax indicates that the ma- jority of the relationships between these

variables and the other predictors were sup- pressive and not additive (Chevan and

Sutherland, 1991). The very high value for the joint contribution of Tmin indicates that this

variable was strongly collinear with annual temperature and altitude.

In contrast to the females, rainfall was the on-

ly variable that explained a significant amount of the variability in average male reflectance Figure 5. Average reflectance along gradients that showed sig- nificant differences between altitudes. a) Gradients on which (57.3%) (Figure 6b). Although altitude ex- Phaulacridium vittatum were darker at high altitudes compared with low altitudes, b) gradients on which P. vittatum became lighter at the highest altitude, c) gradients with site specific pat- terns. Bars show ± 2 SE. High quality figures are available online.

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Figure 7. Component loadings from the principle components analysis. PCA1 dotted line, PCA2 fine black line, PCA3 solid black line. High quality figures are available online.

interpreted as chroma, or the degree to which colour is comprised of neutral white and grey

light, because the loadings were strongest at the low and high end of the spectra. Chroma is

a function of how rapidly intensity changes with wavelength (Endler 1990). Spectra with

steeper maximum slopes and greater differ- ences among parts of the spectra will appear

Figure 6. Percentage of variance in the average reflectance of a) to have more chroma than those with more female and b) male Phaulacridium vittatum explained independent- gradual, smaller changes. The component ly (open bars) and jointly (striped bars) by the climatic variables. Significant independent correlates are indicated by an asterisk. loadings for PCA3 related to reflectance be- High quality figures are available online. tween 500 and 650 nm, where the slope was greatest; this was interpreted as hue. The hue plained 21.8% of the variability, it was not a of a spectrum is a function of its shape, and is statistically significant contribution. directly related to the regions of the spectrum where slope is highest, which usually occur in Differences in spectra the middle of a spectrum (Endler 1990; Grill The first three principle components explained and Rush 2000). This component only ex- approximately 86.95% of the variance in sam- plained 7.07% of the variation because all ple reflectance. The principle components can grasshoppers were similar in colour. be interpreted by looking at the strength of the loadings across the range of wavelengths ana- The means of all principle components were lysed and the shape of the spectral curve significantly different between the (Endler 1990; Grill and Rush 2000) (Figure altitudinal bands (PCA1: F6,846 = 5.05; p < 7). Following Grill and Rush (2000), principle 0.0001; PCA2: F6,846 = 12.56; p < 0.0001; component 1 (PCA1), which explained PCA3: F6,846 = 2.21; p < 0.05). Pair-wise 47.03% of the variance, was interpreted as comparisons of PCA1 showed that brightness being related to the brightness, or overall light was significantly higher at the lowest altitude intensity of the sample, because the compo- (<50 m a.s.l) compared with the altitudinal nent loadings were relatively even across the bands 700–800 m a.s.l. and 800–900 m a.s.l. range of wavelengths measured. PCA2 ex- (Tukey’s q = 2.96, p < 0.05). There was no plained 32.86% of the variance, and was difference between this lowest altitude and the

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Journal of Insect Science: Vol. 13 | Article 51 Harris et al. highest (> 900 m a.s.l.). Chroma at the highest males and females. In support of the thermal altitude was significantly higher than at all melanism hypothesis, maximum and mini- other altitudes (q = 2.96, p < 0.05). There mum temperature were found to be significant were no significant pair-wise comparisons in independent influences on the average reflec- hue. tance of female grasshoppers. That altitude is a significant independent correlate suggests Discussion that there is another variable that covaries with altitude, influencing reflectance. If this The thermal melanism hypothesis rests on the were UV radiation, which increases by 3 to assumption that dark individuals are at an ad- 7% with 300 m a.s.l. of altitude (Pfeifer et al. vantage under colder conditions compared to 2006), it would further support the thermal light individuals, because they warm more melanism hypothesis. Analyses of spectral quickly and reach higher equilibrium tempera- differences along the altitudinal gradients tures (Majerus 1998). In P. vittatum, suggest this is the case. Patterns in chroma melanism represents a gradation in colour ra- were found to be significantly different at the ther than distinct colour morphs, with highest altitudes (> 900 m a.s.l.) compared to reflectance ranging from 2.49 to 5.65%. This all other altitudes. While chroma in insects range in reflectance, although small in magni- has been linked to camouflage and predation tude, is sufficient to lead to differences in the avoidance, it has also been shown to improve thermal qualities of light and dark colour UV protection (Endler 1993). morphs and maintain altitudinal clines in mel- anism, although habitat differences may In contrast to females, the average reflectance weaken the relationship. of males was not significantly influenced by temperature variables. This result is consistent Support for the thermal melanism with a broader scale study of reflectance and hypothesis body size along a latitudinal gradient (Harris The magnitude of the differences in thermal et al. 2012). Along the altitudinal gradients, qualities between the extreme colour morphs rainfall was the only variable with a statisti- of unstriped grasshoppers is sufficient to af- cally significant influence on reflectance. fect the thermal balance of these morphs. For Parkash et al. (2009) found that melanism in example, the maximum temperature excess of Drosophila in Australia was related to hu- the lightest unstriped colour morph ranged midity, which supports the melanism- from 33.14 to 42.42 C, while that of the desiccation hypothesis rather than thermal darkest unstriped morph ranged from 22.58 to melanism. Alternatively, and possibly more 32.42 C. At low altitudes, where higher sur- likely, rainfall could be reflecting cloudiness face temperatures are common, dark or radiation levels, which play a primary role grasshoppers are therefore disadvantaged be- in maintaining body temperature in basking cause they will spend more time thermo- ectotherms. regulating than lighter grasshoppers, and less time on activities such as feeding and mating. In the striped morphs, the absence of a signifi- cant relationship between melanism and Generally, grasshoppers were darker at higher thermal characteristics indicates that the exist- altitudes, and there was a significant negative ence of stripes is driven by some factor other correlation between reflectance and altitude in

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Journal of Insect Science: Vol. 13 | Article 51 Harris et al. than thermal conditions, such as predator 2000). If this were the case, it would be neces- avoidance (Forsman and Appelqvist 1999). sary to measure reflectance across the full spectral range to represent the differences be- Habitat differences tween the colour morphs. Nevertheless, since By comparing melanism along many gradi- the relationship between reflectance in the in- ents, different patterns and thresholds above frared and the visible spectra is most likely to which reflectance declined were identified. be consistent within the species, and the cur- Along several gradients, differences in mela- rent purpose was to compare between sites at nism became apparent above 800 m a.s.l., different altitudes, the reflectance measure- while in one the decline occurred above 400 ments are an improvement on visual m a.s.l. On several gradients, grasshoppers allocation to colour morphs. became darker up to approximately 800 m a.s.l., and then lightened again above 900 m Additionally, site differences had a strong in- a.s.l. to levels similar to the lowest altitude fluence on melanism, as has been populations. These sites were more open habi- demonstrated in other insect groups (Halkka tats, where convective cooling due to higher et al. 1975; Boucelham and Raatikainen wind speeds would rapidly reduce any thermal 1984). The assumption when studying altitu- advantage to darker animals (de Jong et al. dinal gradients is that populations are exposed 1996). to predictable variation in temperature, but this is rarely the case. Habitat differences Along several of the gradients there was no would strongly influence the thermal qualities correlation between average reflectance and of a site, so that the microclimatic tempera- altitude. Several factors could be acting to re- tures experienced by a grasshopper would duce the strength of the altitudinal clines in differ widely across sites. Where high altitude melanism. First, the distance covered in these sites represent marginal habitats, the strength gradients was substantially less than that typi- of the relationship between size, reflectance, cal of altitudinal studies in the northern and altitude will change. Population density, hemisphere, which often extend from sea- habitat patchiness, and connectedness will al- level to 2000 m a.s.l. However, evidence of so affect gene flow and the potential for local thermal melanism has been found elsewhere adaptation (Hanski and Meyke 2005). Fur- along short gradients, for example in subant- thermore, the quality of the light within the arctic beetles along a gradient covering only different habitats would differ with changes to 50–600 m a.s.l. (Davies et al. 2007). vegetation structure, aspect, and so on. Endler (1993) characterised four major light habitats Second, measurements of reflectance may be based on forest structure, and showed how the an imperfect predictor of the total reflectivity differences in the light spectra could have of the different colour morphs. The visible wide ranging effects on visibility, signalling, part of the spectrum, between 400 and 750 and microhabitat choice in a range of animals. nm, represents less than half of the incident Although average reflectance was not signifi- solar radiation (Gates 1980). The near- and cantly different at the highest altitudes, there long-wave infrared region can represent a were differences in chroma, possibly reflect- large proportion of the total reflectivity of an ing such differences. animal, and may represent a different propor- tion in different colour morphs (Nussear et al.

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Journal of Insect Science: Vol. 13 | Article 51 Harris et al. These observations are not based on quanti- higher preferred temperature, the cline in mel- fied assessments of the differences between anism would be maintained even when the the sites. To do so without the need for exten- thermal qualities are only slightly different. sive structural measurements of the Samietz et al. (2005) found that grasshoppers vegetation, the ambient light spectra at each from high altitude sites spent significantly site could be measured. Differences in the more time actively increasing their body tem- proportion of direct to diffuse sunlight could peratures through basking in sunnier be measured, as well as the quality of availa- positions. In support of this, dark unstriped ble light under different conditions. These individuals of P. vittatum do have a higher measurements would encapsulate many char- preferred temperature and maintain higher live acteristics of the site, including the extent and body temperatures than light morphs (R.M. makeup of leaves, bark, and substrate type, Harris, unpublished data). The contribution of which determine the spectra of the diffuse behaviour to the maintenance of altitudinal light sources, as well as the angle of the sun clines in melanism deserves further study. (Endler 1993). This approach would provide information on the quality of the available Conclusions light under different conditions, and could Many tests of the thermal melanism hypothe- easily be translated into available tempera- sis have focussed on species with very distinct tures. colour morphs representing a wide range in melanism. We have tested the thermal mela- Finally, clinal variation in body size and mel- nism hypothesis on a species in which anism may be less important if behavioural melanism represents a gradation in colour, and modifications are the principle adaptations for in which the range in reflectance is small thermoregulation, and these modifications (2.49 to 5.65%). Patterns in melanism and may not be consistent across habitat types. In spectral characteristics along altitudinal gradi- patchy environments, where body temperature ents in P. vittatum broadly support the thermal can be maintained easily by shuttling, behav- melanism hypothesis, although site-specific iour is the least costly method of factors also contribute to the expression of the thermoregulation, so changes to melanism or colour polymorphism. size are less necessary (Angilletta et al. 2006). Acknowledgements In contrast, the thermal advantage of mela- nism is likely to be greatest at open sites with Thank you to Mariella Herberstein (Macquar- high radiation levels. At these sites, behav- ie University) for the use of the spectrometer. ioural thermoregulation will magnify the R.M. Harris was the recipient of an Australian effect of slight thermal differences between Postgraduate Award, and the study was par- the colour morphs and maintain the altitudinal tially funded by an Australia and New clines. The cumulative effect of small thermal Zealand Banking Group Holsworth Wildlife differences is likely to have an impact on ac- Research Endowment. tivity times and duration. This will be further reinforced where the colour morphs have dif- References ferent behavioural and physiological adaptations. For example, if dark grasshoppers Angilletta MJ, Bennett AF, Guderley H, at high altitudes bask for longer to achieve a Navas CA, Seebacher F, Wilson RS. 2006.

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Table 1. Number of Phaulacridium vittatum sampled from each Table 2. F-ratios and significance of predictor variables in multi- altitudinal band (m a.s.l.) on gradients 1–14. ple regression. Significant predictors are indicated in bold.

Table 4. Average reflectance and femur length of Phaulacridium vittatum.

Table 3. Mean values (± SE) of warming parameters for the

different colour morphs of Phaulacridium vittatum.

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Article Linker Click highlighted text for a new search on that item. ISSN: 1536-2442 Title: Journal of Insect Science: Paving Additional Title the way to the future of scientific Information publishing Publishing Body: Library of the University of Arizona Country: United States Status: Active Start Year: 2001 Frequency: Annual Document Type: Journal; Academic/Scholarly Refereed: Yes Abstracted/Indexed: Yes Media: Online - full text Language: Text in English Price: Free (effective 2011) Subject: BIOLOGY - ENTOMOLOGY Dewey #: 595.7 LC#: QL461 CODEN: JISTCY Special Features: Statistics, Illustrations Editor(s): Henry H Hagedorn (Editor-in-Chief) E-Mail: [email protected] URL: http://www.insectscience.org/ Description: Covers biology of insects and their agricultural and medical impact.

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