Phenotypic plasticity in upper thermal limits is ... - Wiley Online Library

Functional Ecology 2011, 25, 661–670

doi: 10.1111/j.1365-2435.2010.01821.x

Phenotypic plasticity in upper thermal limits is weakly related to Drosophila species distributions Katherine A. Mitchell1,2,*,†, Carla M. Sgro`3 and Ary A. Hoffmann1,2,4 1

Centre for Environmental Stress & Adaptation Research, Bio21 Institute, Parkville, Vic. 3010, Australia; 2Department of Genetics, The University of Melbourne, Parkville, Vic. 3010, Australia; 3School of Biological Sciences, Monash University, Clayton, Melbourne, Vic. 3800, Australia; and 4Department of Zoology, The University of Melbourne, Parkville, VIc. 3010, Australia

Summary 1. Acclimation and hardening represent examples of phenotypic plasticity, the extent to which phenotypes produced by the same genotype vary under different environments. Widespread species are expected to differ in thermal plasticity from narrowly distributed tropical species, but this has rarely been tested particularly when species are reared under the same conditions. 2. We investigated acclimation and hardening responses of 11 widespread or tropically restricted Drosophila species from Australia using estimates of heat resistance where temperatures were increased suddenly (static measure) or slowly (ramping measure), and after controlling for phylogenetic relatedness. We predicted that restricted species would show little acclimation regardless of the method used, whilst widespread species would respond well after a hardening treatment (35 C for 1 h) particularly under ramping. 3. These predictions were partially supported. There was a tendency for the tropically restricted species to be less plastic than the widespread species, although variation among species within the two groups was generally greater than between the groups. For acclimation and stress resistance measured under ramping acclimation, there was an association between the southernmost latitude at which species were found (reflecting variability in climatic conditions they encountered) and knockdown resistance after controlling for phylogeny. There was also evidence of significant divergence from the ancestral state in the ramping trait, likely reflecting a history of direct or indirect selection for ramping knockdown resistance in Drosophila. 4. There was a significant negative association between basal resistance and hardening capacity for static acclimation in the widespread species, suggesting a limit to the extent that plastic responses vary independently of basal resistance. 5. The reduced plastic response in tropically restricted species and negative association between hardening and basal resistance suggest a limit to the effectiveness of plastic responses in changing upper thermal limits for countering increases in thermal stress under future climate change. Key-words: heat resistance, knockdown time, phylogenetic contrasts, ramping, static, widespread and restricted distributions

Introduction Some insect communities are under threat from global climate change due to the strong and direct influence of changing thermal environments on fitness traits such as survival, foraging, reproduction and development (Deutsch et al. 2008). Being small ectotherms, insects need to tolerate increasing air temperatures and decreasing humidity likely *Correspondence author. E-mail: [email protected] † Present address: Hoffmann Lab, Bio21 Institute, 30 Flemington Road, Parkville, Vic. 3052, Australia.

under climate change. The ability to adapt and tolerate these conditions will depend on the present levels of resistance of species as well as their potential to respond via plastic and adaptive changes (Chown et al. 2007; Hoffmann 2010). Tolerance is typically assessed in adult insects by exposing them to shifts in temperature and measuring the maximum temperature (CTmax) individuals can tolerate within a given time interval (Hoffmann, Sørensen & Loeschcke 2003b; Bowler 2005). While traditional methods for assessing adult thermal responses involve sudden temperature shifts to a high static level, these may not reflect the way upper thermal stress

2010 The Authors. Functional Ecology 2010 British Ecological Society

662 K. A. Mitchell et al. is experienced in nature, leading to inaccurate CTmax estimates. Terblanche et al. (2007) found CTmax of tsetse flies (Glossina pallipides) estimated using a slow rate of thermal increase (0Æ06 C min)1), which matched ambient changes likely to be experienced in nature, were much lower than those estimated using rapid temperature changes. Similar results were obtained when estimating the effects of adult acclimation on CTmax in Drosophila melanogaster and the Argentine ant, Linepithema humile (Chown et al. 2009). More recently, Mitchell & Hoffmann (2010) found that estimates of narrow-sense heritability (h2n) and the evolvability of upper thermal limits in D. melanogaster, which reflect the genetic capacity to adapt, were different under static and ramped temperature changes; under ramping, the h2n estimate could not be distinguished from 0, compared to values around 20% under static temperature stress. Nevertheless, there was a significant correlation between the static and ramping methods when values were compared across 11 Drosophila species (Mitchell & Hoffmann 2010), indicating partial overlap in the underlying mechanisms or correlated patterns of selection on these components despite the difference in estimated heritability. There is clearly a fundamental difference between measures of upper thermal limits, and these results highlight the importance of examining physiological traits using ecologically relevant methods when assessing adaptive potential. Acclimation and heat hardening represent examples of phenotypic plasticity, the amount by which the phenotype produced by the same genotype is changed under different environments (Bradshaw 1965). In the case of thermal responses, plasticity may protect species from an unfavourable thermal environment and provide buffering against the stress expected from climate change (West-Eberhard 2003; Chown & Terblanche 2007). If the plastic response has an additive genetic basis, levels of plasticity can evolve and directly contribute to evolutionary adaptation (West-Eberhard 2003; Ghalambor et al. 2007). Over time, selection may favour individuals constitutively expressing the plastic response, leading to a reduction in plasticity (Waddington 1952; Garland & Kelly 2006). Plasticity may also hinder selection by ‘shielding’ genotypes with lower underlying resistance from selection and thus preventing populations from evolving higher resistance (Bennett & Lenski 1999; Gabriel 2005; Ghalambor et al. 2007). It is uncertain whether species exposed to different thermal environments exhibit different levels of phenotypic plasticity as a consequence of adaptation (Spicer & Gaston 1999; Chown et al. 2007). It is often assumed that widespread species exhibit greater levels of phenotypic plasticity, allowing them to readily expand their ranges (i.e. Baker’s greater flexibility hypothesis; Baker 1965; Brattstrom 1968; Lynch & Gabriel 1987; Spicer & Gaston 1999; Stillman 2003; Pohlman, Nicotra & Murray 2005; Gaston et al. 2009; Calosi et al. 2010). In contrast, restricted tropical species are often assumed to have little plasticity due to the relatively stable environment that they inhabit (Levins 1968; Lynch & Gabriel 1987; Spicer & Gaston 1999). Plastic responses might be

reduced if there is a trade-off between plasticity and fitness (Dewitt, Sih & Wilson 1998; Liefting & Ellers 2008) or if the absence of selection for this trait leads to DNA decay in genes involved in plastic responses (Hoffmann & Willi 2008; Hoffmann 2010). Few studies have compared tropical and widespread taxa to test these ideas; where comparisons have been made, inconsistent patterns have often emerged (Levins 1968; Spicer & Gaston 1999; Stillman 2003; Pohlman, Nicotra & Murray 2005; Chown et al. 2007). For instance, Stillman (2003) found species of porcelain crabs with higher basal heat resistance had a lower potential for acclimation than those from cooler habitats. However, this contrasts with a phylogenetic analysis of upper thermal limits and phenotypic plasticity in diving beetles, where species with higher resistance had greater plasticity and wider geographic distributions (Calosi, Bilton & Spicer 2008). In general, variation in upper thermal limits is considerably lower and more difficult to predict than variation in lower thermal limits (Addo-Bediako, Chown & Gaston 2000; Chown 2001). There is a considerable body of literature investigating heat tolerance in Drosophila melanogaster and related species, including tests of plastic responses at different developmental stages in the laboratory and field (Feder & Krebs 1998; Hoffmann, Sørensen & Loeschcke 2003b; Kristensen, Loeschcke & Hoffmann 2007; Loeschcke & Hoffmann 2007; Sgro` et al. 2010). However, little attention has so far been paid to differences in plastic responses affecting CTmax, particularly when measured under gradual ramping. Here we compare 11 species of Drosophila from the east coast of Australia for plastic responses under static and ramping exposures. If tropical specialists from the wet tropics in Queensland are adapted to a relatively stable environment with maximum ambient temperatures that usually do not vary much from around 30 C (http://www.bom.gov.au), and that are lower than those found further south where conditions fluctuate (Hoffmann 2010), we might expect these species to exhibit a reduced ability to acclimate and to become heat hardened, regardless of how resistance is measured. We would then expect them to exhibit a lower hardening response than the more widespread species that can experience sharp changes in temperature in more temperate environments (e.g. Loeschcke & Hoffmann 2007). We were particularly interested in ramping rates of increase that reflect rates of temperature change experienced in temperate areas, and which can reduce upper limits in widespread insects such as D. melanogaster (Terblanche et al. 2007; Chown et al. 2009). Adults from each species were tested for upper thermal limits following acclimation for several days or a short period of hardening, and heat resistance was estimated as knockdown time under ramping or static temperatures. Because data from insect studies have found that upper thermal limits may be constrained by phylogeny (Addo-Bediako, Chown & Gaston 2000), which is often not considered in studies investigating plasticity in thermal limits (Angilletta 2009), we tested for phylogenetic signal and determined the level of differentiation in plasticity from the ancestral state as well as correcting for phylogeny in any trait comparisons.

2010 The Authors. Functional Ecology 2010 British Ecological Society, Functional Ecology, 25, 661–670

Plasticity is weakly related to Drosophila distributions

Materials and methods STOCKS AND SPECIES

Eleven Drosophila species were tested from the Sophophora and Drosophila subgenera. Details of original collection sites, maximal January temperatures (from http://www.bom.gov. au), latitudinal range and southernmost latitude (TaxoDros, http://www.taxodros.uzh.ch) can be found in Table 1. Mass bred populations were created by combining the offspring of a minimum of 15 isofemale lines collected from the field and maintained at a minimum of 1000 individuals per generation throughout the experiments. Flies were collected opportunistically from within their range due to differences in habitat preferences between the species (e.g. closed rainforest vs. open forest), however many species were collected from the same sites. Mass bred populations were maintained for a minimum of five generations before commencing experiments to control for environmental effects. The majority of the widespread species were collected from the southern end of their range, with the exception of D. busckii and D. repleta that were collected closer to the tropics. We have included all species in the analysis regardless of collection site because species differences are larger than within-species variation in thermal responses (Hoffmann, Sørensen & Loeschcke 2003b).

ACCLIMATION AND HARDENING

Adult females were 8-day post eclosion, reared at 25 C under constant light, and held with males to ensure they were mated. To control density and size during development, eggs were spotted on yeast-treacle medium (40 eggs per 50 mL medium). Four species, two tropically restricted (D. birchii and D. sulfurigaster) and two widespread (D. melanogaster and D. simulans), were examined in detail across multiple conditions to determine acclimation and hardening treatments that did not cause mortality before comparisons were made using all 11 species. Flies were acclimated for 36 h at 30 C with 24 h recovery at 25 C, or hardened at 34 C and 35 C for

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1 h with 6 h recovery at 25 C. Controls were maintained at a constant 25 C. The chosen 30 C acclimation temperature is close to the average maximum temperature experienced by the tropical species in the Wet Tropics, whilst the hardening temperature of 35 C is close to those known to induce heat shock response in D. melanogaster (Hoffmann, Sørensen & Loeschcke 2003b), but low enough to allow all species to survive. We acknowledge that these temperatures may be more stressful to some species than others, given variation in temperatures required for heat shock protein induction in different species of Drosophila (Krebs 1999). The hardening temperatures are above the threshold predicted to provide a significant fitness advantage for hardened D. melanogaster flies exposed to sudden hot conditions (Loeschcke & Hoffmann 2007). We predicted a reduced plasticity in the restricted species generally.

KNOCKDOWN TIME

This was estimated following the protocols outlined in Mitchell & Hoffmann (2010). Flies of all 11 species were placed in individual 5-mL vials with plastic caps that were held in a water bath with temperature controlled by a Ratek TH4 thermoregulator (Ratek Instruments, Melbourne). For the ramping measurement, water temperature was set at 28 C and increased at a rate of 0Æ06 C min)1 before plateauing at 38 C. This protocol follows the rate of thermal increase seen during stressful conditions in fruit orchards in inland southern Australia where Drosophila are found (A. A. Hoffmann, unpublished data). As the reduction in resistance estimates using ramping methods may be influenced by exposure time, we started the ramping stress at 28 C rather than 25 C and thereby reduced exposure times by around 45 min. The temperature was set at a constant 38 C for the static estimate. Knockdown was recorded as the time after immersion for individuals to lose muscle function and be unable to right themselves. Recent work has suggested a potential bias caused by desiccation and starvation when estimating upper thermal limits

Table 1. Species collection, maximum January temperature (http://www.bom.gov.au) and distribution information (obtained from TaxoDros database: http://www.taxodros.uzh.ch). Latitudinal range is the full latitudinal distribution of these species within their range, whereas southern latitude refers to species southern borders within Australia Collection details

Distribution (degrees)

Species

Latitude

Longitude

Max. temp (C)

Latitudinal range

Southern latitude

Drosophila melanogaster D. simulans D. hydei D. replete D. busckii D. serrata D. birchii D. bunnanda D. bipectinata D. pseudoananassae D. sulfurigaster

3747¢30¢¢S 3747¢30¢¢S 3747¢30¢¢S 1922¢28¢¢S 1922¢28¢¢S 2104¢11¢¢S 1716¢45¢¢S 1811¢45¢¢S 1710¢59¢¢S 1710¢59¢¢S 1710¢59¢¢S

14526¢05¢¢E 14526¢05¢¢E 14526¢05¢¢E 14642¢23¢¢E 14642¢23¢¢E 14838¢13¢¢E 14537¢56¢¢E 14552¢07¢¢E 14553¢05¢¢E 14553¢05¢¢E 14553¢05¢¢E

26Æ30 26Æ30 26Æ30 31Æ30 31Æ30 29Æ40 29Æ00 31Æ50 31Æ40 31Æ40 31Æ40

129Æ40 118Æ51 112Æ32 101Æ75 119Æ31 38Æ70 33Æ37 7Æ68 65Æ62 67Æ40 67Æ80

43Æ20 41Æ50 38Æ00 38Æ00 38Æ00 34Æ30 22Æ80 19Æ40 19Æ10 19Æ10 27Æ30


664 K. A. Mitchell et al. using slow rates of temperature increase (Rezende, Tejedo & Santos 2010). Two of the species included in our study (D. birchii and D. bunnanda) are known to have poor desiccation tolerance and may suffer from this bias (Hoffmann et al. 2003a; Kellerman et al. 2006; Kellermann et al. 2009). However these species do not exhibit particularly low levels of heat resistance under ramping compared to static assays (see Fig. 2, Mitchell & Hoffmann 2010). The species least tolerant to starvation (D. birchii) does not experience mortality until held for more than 45 h without food (Griffiths, Schiffer & Hoffmann 2005), so we suspect that variation in starvation resistance is also not of concern in the ramping test.

STATISTICAL ANALYSIS

To remove block effects and skewness in the knockdown time measures, estimates were standardized to normal deviates following log10 transformation. Reaction norms were investigated using a general linear model (GLM) in SPSS (ver. 16) including species and treatment as fixed factors. The two traits (ramping and static knockdown time), along with the two treatment categories (acclimation and hardening vs. controls) were analysed separately. As the Type II error rate could be inflated through multiple comparisons, q-values were calculated using q-value (Storey 2002) for R (R Development Core Team 2010) to determine the false discovery rate (Benjamini & Hochberg 1995) of our analysis. The relationship between distribution and plastic response was also examined by regressing southern distribution limits (as latitudes) against basal and hardened or acclimated resistance levels, in a manner similar to Calosi et al. (2010). Hardening or acclimation capacity was investigated by linear regression of basal resistance (knockdown time at 25 C) against the hardening ⁄ acclimation response (i.e. hardened ⁄ acclimated minus basal resistance) for both types of knockdown trait. If acclimation ⁄ hardening capacity was not independent of innate resistance levels, we would expect those with higher basal knockdown resistance to have a relatively lower hardening capacity (a negative correlation between basal and absolute hardening capacity). Phylogenetically independent contrasts (PICs) (Garland, Harvey & Ives 1992) were used to determine if there was an effect of common ancestry on knockdown time estimates. The procedure followed Mitchell & Hoffmann (2010). Briefly, a phylogenetic tree was created with two mtDNA and two nuclear gene sequences for all 11 species. These data were then used by PDAP:PDTree software (ver. 1Æ14, Midford, Garland & Maddison 2005) for Mesquite (ver. 2Æ6, Maddison & Maddison 2009) to calculate PICs. The least squares regressions created using this analysis were then compared to those obtained from only the standardized values. The phylogenetic contrasts analysis using PDAP:PDTree software also provides a value of the ancestral state, calculated as the weighted mean of the branch tip values (Garland, Harvey & Ives 1992). With these estimates we examined the amount of differentiation from the ancestral state by removing the ancestral value

from the branch tips and performing a GLM analysis, including the same factors as the previous GLM analysis. One trait and treatment combination (ramping hardening) exhibited a significant lack of fit of tip data to branch lengths following examination of absolute values of PICs against their standard deviation (Garland, Harvey & Ives 1992), which was corrected by exponentially transforming branch lengths for these contrasts.

Results In the four species comparison, the restricted species had lower resistance across both traits (static and ramping knockdown time) and treatments (acclimated and hardened) compared to the widespread species, and the rank order across treatments was usually D. melanogaster > D. simulans > D. birchii > D. sulfurigaster (Fig. 1). The GLM analysis (Table 2) revealed significant effects of species for each trait and treatment, and both ramping and static knockdown time after hardening had significant treatment · species interactions. As D. melanogaster has been previously shown to be an outlier in phenotypic plasticity assays (Kellett, Hoffmann & McKechnie 2005), we also analysed species separately to compare treatment effects when hardening was applied. Drosophila birchii and D. sulfurigaster displayed significant negative responses to hardening prior to the ramping estimate (Fig. 1b), whereas there was a significant positive effect of hardening in the static resistance assay in D. melanogaster (* in Fig. 1d). The 34 C hardening pre-treatment produced similar results to the 35 C hardening pre-treatment but the effects were less pronounced. The 30 C acclimation treatment did not influence heat resistance of these four species regardless of the stress method used (Fig. 1a, c). When all 11 species were examined before phylogenetic correction, the GLM analysis (Table 3) revealed the same effect of species and treatment as reported for the four species comparison. There were significant species effects for all traits and treatments, while treatment had a significant effect on the ramping ⁄ hardening combination. To investigate the relationship between distribution and plastic responses in the 11 species, the association between the southern distribution limit and basal or acclimated ⁄ hardened resistance was considered (Fig. 2a–f). Before accounting for phylogeny, three combinations showed no significant overall relationship between southern latitude and resistance: static ⁄ basal resistance (Fig. 2b), ramping ⁄ acclimation (Fig. 2c) and static ⁄ hardening (Fig. 2f). All other overall regressions were significant, with ramping ⁄ basal (Fig. 2a), static ⁄ acclimation (Fig. 2d) and ramping hardening (Fig. 2e) showing a significant linear relationship with southern latitude. Once phylogeny was taken into account, only the overall linear relationship between ramping ⁄ basal and ramping ⁄ hardening and southern latitude remained significant (Table 4). When the widespread and restricted species were analysed separately, there were significant associations between basal and acclimated resistance and southern latitude in the restricted species for some trait ⁄ treatment combinations


Plasticity is weakly related to Drosophila distributions

(a)

(b)

(c)

(d)

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Fig. 1. Reaction norms of four species of Drosophila for ramping and static knockdown time after treatment by acclimation at 30 C for 36 h or hardening at 34 C or 35 C for 1 h with recovery. Controls were provided at 25 C. The unbroken lines indicate reaction norms for widespread species, whilst the dashed lines are tropically restricted. * indicate significant treatment effects for that particular species and treatment.

Table 2. General linear model of knockdown time using the ramping and static knockdown time method for four Drosophila species after acclimation and hardening pre treatments

Trait

Factor

Mean square df

Ramping Acclimation Species 0Æ66 Treatment 0Æ001 Species * treatment 0Æ001 Error 0Æ001 Hardening Species 0Æ85 Treatment 0Æ07 Species * treatment 0Æ02 Error 0Æ01 Static Acclimation Species 13Æ77 Treatment 0Æ08 Species * treatment 0Æ08 Error 0Æ15 Hardening Species 48Æ58 Treatment 0Æ17 Species * treatment 0Æ72 Error 0Æ19

F

3 159Æ81** 1 0Æ001 3 0Æ50 123 3 113Æ99** 2 9Æ57** 6 2Æ93** 182 3 93Æ82** 1 0Æ55 3 0Æ55 107 3 254Æ74** 2 0Æ91 6 3Æ77** 206

Table 3. General linear model analysis of ramping and static knockdown time for widespread and restricted Drosophila species after acclimation and hardening pretreatments

Trait

Factor

Mean square df

Ramping Acclimation Species 7Æ68 Treatment 0Æ57 Species * treatment 0Æ82 Error 0Æ62 Hardening Species 7Æ91 Treatment 16Æ63 Species * treatment 2Æ69 Error 0Æ65 Static Acclimation Species 19Æ55 Treatment 3Æ58 Species * treatment 0Æ34 Error 0Æ56 Hardening Species 14Æ58 Treatment 0Æ95 Species * treatment 0Æ84 Error 0Æ34

10 1 10 334 10 1 10 268 10 1 10 401 10 1 9 296

F 12Æ40** 0Æ92 1Æ33 12Æ22** 25Æ70** 4Æ15 35Æ08** 6Æ43 0Æ61 42Æ38** 2Æ76 2Æ43

**Significant at a = 0Æ05 after false discovery rate analysis.


(Fig. 2a–c), but not for the static ⁄ acclimation combination (Fig. 2d) or any of the hardened treatments (Fig. 2e, f). Regressions for the widespread species were not significant for any trait ⁄ treatment combination (Fig. 2a–f ). The regressions for the restricted species tended to have high R2 values and the regression lines were all negative reflecting a

higher resistance level in the more tropically restricted species. The influence of acclimation or hardening on knockdown resistance across the 11 species can be inferred from the analyses of basal resistance and acclimation ⁄ hardening capacity. Each treatment and estimate method induced different levels


666 K. A. Mitchell et al.

(a)

(b)

(c)

(d)

(e)

(f) Fig. 2. Regression analysis of distribution, i.e. southern latitude ( south) vs. knockdown time of basal and acclimated ⁄ hardened resistance in Drosophila species. Filled circles represent tropically restricted species; open squares, widespread species. Regression lines indicate significant linear regression results. Dashed lines indicate significant regressions prior to PIC analysis, whole lines, significant before and after PIC analysis.

of plasticity in the species, as seen in the number of data points above or below the acclimation ⁄ hardening capacity value of 0 (indicative of no difference between basal and treatment resistance; Fig. 3a–d). The static ⁄ acclimation combination provided the greatest positive plastic response, with only two species with negative acclimation capacity (Fig. 3b). The ramping ⁄ hardening combination was least conducive to improving plasticity, with only a few widespread species showing increased plasticity after the treatment (Fig. 3c). Static ⁄ hardening produced the greatest spread in plastic responses, with even numbers of widespread and restricted species falling above and below the y = 0 line (Fig. 3d), a trend similar to the ramping ⁄ acclimation combination, only with a slight bias towards negative acclimation capacity (Fig. 3a). To determine if basal resistance levels predicted acclimation or hardening capacity, we conducted regression analyses across the 11 species (Table 5). With or without phylogenetic correction, the overall regression analyses suggested little association between basal resistance and hardening ⁄ acclimation capacity except for a significant positive association

between basal and hardening capacity for the static ⁄ hardening combination following PIC analysis (Fig. 3d). For the widespread species there was a highly significant negative relationship between basal resistance and acclimation capacity for the static ⁄ acclimation combination (Fig. 3b), otherwise there were no significant trends within the widespread or restricted groups when analysed separately. These results suggest no strong association between basal resistance and hardening or acclimation effects except in the case of acclimation and hardening response tested using the static heat knockdown assay. The positive regression of the level of divergence from the ancestral state vs. southern latitude over all species was significant and linear for ramping knockdown resistance (Fig. 4a). While there was a positive trend between southern latitude and divergence from the ancestral state over all species for static heat knockdown, this was not significant (Fig. 4b). There were no significant trends between divergence from the ancestral state vs. southern latitude within the widespread and restricted species for ramping heat knockdown, but a significant negative relationship between divergence and


Plasticity is weakly related to Drosophila distributions Table 4. Results of regression analysis of species southern most latitude (as a proxy for distribution) and knockdown resistance measured using ramping and static methods following a 30 C acclimation, 35 C hardening treatment or 25 C control (basal). F ratios have one degree of freedom (df) for group, and error df as indicated. Regression analyses that did not have sufficient power (i.e. 0Æ5 or higher) are not presented Trait

Treatment

Group

R2

F (df error)

Ramping

Basal

Overall PIC Widespread Restricted Overall PIC Restricted Overall PIC Overall PIC Restricted Overall PIC Widespread Overall PIC Widespread Restricted

0Æ47 0Æ38 0Æ37 0Æ89 0Æ15 0Æ15 0Æ86 0Æ51 0Æ42 0Æ29 0Æ15 0Æ97 0Æ48 0Æ31 0Æ22 0Æ29 0Æ15 0Æ36 0Æ66

8Æ11** (9) 5Æ48** (9) 2Æ34 (4) 24Æ33** (3) 1Æ57 (9) 1Æ54 (9) 18Æ80** (3) 9Æ48** (9) 6Æ41** (9) 3Æ70 (9) 1Æ59 (9) 83Æ36** (3) 8Æ14** (9) 4Æ06 (9) 1Æ13 (4) 3Æ68 (9) 1Æ62 (9) 2Æ22 (4) 5Æ87 (3)

Acclimation

Hardening Static

Basal

Acclimation

Hardening


southern distribution within the restricted species was evident (Fig. 4b).

Discussion Plastic responses are considered important in countering stressful conditions and ameliorating the strong directional

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Table 5. Regression analysis of basal and acclimation ⁄ hardening capacity for ramping and static knockdown time. F ratios have one degree of freedom (df) for group, and error df as indicated. Regression analyses that did not have sufficient power (i.e. 0Æ5 or higher) are not presented Trait

Treatment

Group

R2

F (d.f. error)

Ramping

Acclimation

Overall PIC Widespread Restricted Overall PIC Overall PIC Widespread Restricted Overall PIC

0Æ09 0Æ21 0Æ40 0Æ38 0Æ05 0Æ11 0Æ22 0Æ08 0Æ64 0Æ58 0Æ13 0Æ36

0Æ87 (9) 2Æ42 (9) 2Æ64 (4) 1Æ83 (3) 0Æ48 (9) 1Æ07 (9) 2Æ54 (9) 0Æ81 (9) 7Æ14** (4) 4Æ21 (3) 1Æ36 (9) 5Æ01** (9)

Hardening Static

Acclimation

Hardening


selection expected under climate change (West-Eberhard 2003; Chown et al. 2007; Ghalambor et al. 2007; Deutsch et al. 2008; Angilletta 2009). Differences in the level of plasticity between species groups are predicted to have evolved under past selection, with widely distributed species expected to have higher levels of plasticity than species with restricted distributions particularly if the latter come from thermally stable environments (Baker 1965; Dewitt, Sih & Wilson 1998; Spicer & Gaston 1999). Loss of thermal plasticity in insects from relatively more stable environments may come about because of potential costs of maintaining plasticity or loss of function due to DNA decay (Hoffmann 2010). Based on these expectations, we predicted that restricted tropical Drosophila species would exhibit less plasticity than widespread species,

(a)

(b)

(c)

(d)

Fig. 3. Regression of basal knockdown resistance and acclimation ⁄ hardening capacity in 11 Drosophila species. Filled circles, tropically restricted; open squares, widespread species. Dotted line indicates acclimation ⁄ hardening capacity of zero, indicative of no plastic response following treatment. 2010 The Authors. Functional Ecology 2010 British Ecological Society, Functional Ecology, 25, 661–670

668 K. A. Mitchell et al. (a)

(b)

Fig. 4. Regression analysis of distribution, i.e. southernmost latitude and the mean difference from ancestral state for the ramping and static knockdown estimates of all 11 Drosophila species examined. Filled circles, tropically restricted; open squares, widespread species. Ancestral state was calculated as the mean of the weighted branch tips in the phylogenetic contrast analysis in PDAP:PDTree software (ver. 1.14, Midford, Garland & Maddison 2005) for Mesquite (ver. 2.6, Maddison & Maddison 2009).

particularly when measured using the ramping estimate. We also expected that widespread species would be relatively more resistant than tropical species after a hardening pretreatment, particularly when the static method of stress was applied and resistance improved, such as after the induction of heat-shock proteins. In the initial comparison of four species, the 30 C acclimation treatment did not improve resistance in the restricted or widespread species regardless of the method used to assess resistance. Resistance was only increased following hardening in D. melanogaster when measured with the static estimate, consistent with previous studies showing a hardening effect in this species (Hoffmann & Watson 1993; McColl, Hoffmann & McKechnie 1996; Dahlgaard et al. 1998; Hoffmann, Sørensen & Loeschcke 2003b; Kellett, Hoffmann & McKechnie 2005). However, in the comparison of all 11 species, there

was no clear association between acclimation ⁄ hardening capacity and distribution (Fig. 3), in contrast to our predictions. There was limited evidence for greater plasticity in more widespread species, however there was substantial variation in resistance within the widespread or restricted species groupings. Within the restricted group, two are endemic to the Wet Tropics rainforest region of northern Queensland and Papua New Guinea (D. birchii and D. bunnanda, Schiffer & McEvey 2006), whereas the other species can be found across the Asia region (Bock 1976) even though they are restricted to the tropics. Our findings are consistent with other studies showing no clear association between plasticity and the widespread ⁄ restricted nature of species distributions (Spicer & Gaston 1999; Pohlman, Nicotra & Murray 2005). Although we did not find evidence of consistent differences in plastic responses related to species distributions, we did confirm species differences in heat resistance under ramping and static knockdown assays (Kellett, Hoffmann & McKechnie 2005; Mitchell & Hoffmann 2010). A ramping rate of 0Æ06 C min)1 is likely to reflect sudden temperature increases in nature in temperate areas (A. A. Hoffmann, unpublished data) and there was a significant regression of southern latitudinal distribution on this trait as well as an ancestral difference regardless of treatment. Although tropical flies experience warmer conditions on average than temperate species, it is possible that widespread species have been selected to respond to the sudden increases in temperature, with higher resulting maxima, that are common to temperate (rather then tropical) areas of their range. This is evident in the significant, negative association between distribution and both resistance estimates in the basal and acclimated treatments in the restricted species together with the significantly greater divergence from ancestral resistance in the static knockdown trait. Whilst tropical insects may appear to be more at risk from climate change when responses to constant temperatures are compared (e.g. Deutsch et al. 2008), they experience extremes less regularly than populations in temperate environments, and therefore the higher thermal extremes experienced by southern populations of widespread species may place these at a higher relative risk in southern latitudes (Hoffmann 2010). The phylogenetic signal we detected in regressions between southern distributions and knockdown resistance in all but two traits suggests that thermal responses are phylogenetically constrained, as previously noted for a much broader group of ectotherms by Addo-Bediako, Chown & Gaston (2000). Plastic responses may also be constrained by a tradeoff between plasticity and basal levels of resistance. We did find a significant negative association between static basal resistance and acclimation capacity in the widespread species but this pattern was not evident overall in our comparisons. In contrast to this, we also found a significant positive association between static basal resistance and hardening capacity, but only after accounting for phylogeny. Kellett, Hoffmann & McKechnie (2005) and Calosi et al. (2010) previously noted a positive association between basal resistance and the extent of the hardening response (i.e. hardening


Plasticity is weakly related to Drosophila distributions capacity), however Stillman (2003) found the reverse pattern and this issue needs to be investigated across a wider cross section of taxa. Together with the limited narrow-sense heritability estimate for ramping upper thermal limit in D. melanogaster (Mitchell & Hoffmann 2010), Drosophila species may have reached a limit in their ability to further increase levels of heat resistance, a result in agreement with other physiological traits in some Drosophila species (e.g. desiccation resistance, Kellermann et al. 2009). We have assumed that the populations tested are representative of each species; population variation in plastic responses and basal resistance levels within species were ignored. Previous studies have shown variation among D. melanogaster populations for heat resistance along the eastern Australian cline (Hoffmann, Anderson & Hallas 2002) but not in D. simulans (Arthur, Weeks & Sgro` 2008) and D. birchii (Griffiths, Schiffer & Hoffmann 2005). Differences among conspecific populations in plastic responses to heat stress have not been previously detected (Hoffmann & Watson 1993) except for a weak trend across multiple populations in D. melanogaster (Sgro` et al. 2010), highlighting the fact that any differences among conspecific populations are likely to be smaller than those detected among species. It is possible that the adult traits we examined in this study may not be under direct selection. Further studies should consider the less mobile immature life history stages (Feder, Blair & Figueras 1997; Spicer & Gaston 1999; Bowler & Terblanche 2008). Drosophila egg and larval stages of development are also exposed to thermal stress in nature (Feder, Blair & Figueras 1997) and selection for heat tolerance and plasticity at these stages may be even stronger than in adults due to the reduced ability of eggs and larvae to behaviourally avoid thermal extremes. Differences in habitat between tropical rainforest and temperate regions should also be considered. Rainforest fruit used by tropical Drosophila species will tend to be located in shady conditions, whereas fruit in other environments like commercial orchards will often be exposed to sunlight and can reach high temperatures (Feder, Blair & Figueras 1997). These selection pressures might lead to differences in plastic responses among species. However at the moment we do not have clear evidence that restricted tropical species have lower levels of plasticity for heat responses, despite variation among species in basal levels of resistance.

Acknowledgements The authors would like to thank Lauren B. Carrington, Belinda van Heerwaarden for comments on earlier drafts and Vanessa Kellermann and Sarah De Garis for assistance with species collection and maintenance. We would also like to thank Piero Calosi and two anonymous reviewers whose comments improved this manuscript. This research was supported by grants and fellowships from the Australian Research Council and Commonwealth Environmental Research Facility.

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