PHYSIOLOGICAL ECOLOGY - ORIGINAL RESEARCH

Detrimental influence on performance of high temperatureincubation in a tropical reptile: is cooler better in the tropics?

Kris Bell • Simon Blomberg • Lin Schwarzkopf

Received: 5 April 2011 / Accepted: 21 June 2012 / Published online: 11 July 2012

� Springer-Verlag 2012

Abstract Global temperatures have risen over the last

century, and are forecast to continue rising. Ectotherms

may be particularly sensitive to changes in thermal

regimes, and tropical ectotherms are more likely than

temperate species to be influenced by changes in environ-

mental temperature, because they may have evolved nar-

row thermal tolerances. Keelback snakes (Tropidonophis

mairii) are tropical, oviparous reptiles. To quantify the

effects of temperature on the morphology and physiology

of hatchling keelbacks, clutches laid by wild-caught

females were split and incubated at three temperatures,

reflecting the average minimum, overall average and

average maximum temperatures recorded at our study site.

Upon hatching, the performance of neonates was examined

at all three incubation temperatures in a randomized order

over consecutive days. Hatchlings from the ‘hot’ treatment

had slower burst swim speeds and swam fewer laps than

hatchlings from the cooler incubation temperatures in all

three test temperatures, indicating a low thermal optimum

for incubation of this tropical species. There were no sig-

nificant interactions between test temperature and incuba-

tion temperature across performance variables, suggesting

phenotypic differences caused by incubation temperature

did not acclimate this species to post-hatching conditions.

Thus, keelback embryos appear evolutionarily adapted to

development at cooler temperatures (relative to what is

available in their habitat). The considerable reduction in

hatchling viability and performance associated with a

3.5 �C increase in incubation temperature, suggests climate

change may have significant population-level effects on

this species. However, the offspring of three mothers

exposed to the hottest incubation temperature were appar-

ently resilient to high temperature, suggesting that this

species may respond to selection imposed by thermal

regime.

Keywords Climate change � Developmental acclimation �Ectotherm � Performance � Phenotype

Introduction

Global temperatures have increased markedly since the

eighteenth century, and are predicted to rise at an

increasing rate over the next century (IPCC 2007). Tem-

perature has profound effects on the biological processes of

all animals (Birchard 2004). Ectotherms lack effective

physiological mechanisms to control body temperature,

and are therefore particularly sensitive to environmental

conditions (Bogert 1949). Because they have been exposed

to smaller variations in temperature over evolutionary time,

tropical ectotherms are likely to be more strongly influ-

enced by changes in environmental temperature than

temperate species (Huey et al. 2009; Sunday et al. 2010).

Embryos require appropriate levels of temperature, mois-

ture and gas exchange for successful development (Shine and

Thompson 2006). However, early stage embryos lack the

physiological and behavioural responses available to post-

hatching animals, suggesting that ectothermic organisms

may be most vulnerable to adverse thermal conditions at

Communicated by Mark Chappell.

K. Bell (&) � L. Schwarzkopf

School of Marine and Tropical Biology, James Cook University,

Townsville, QLD 4811, Australia

e-mail: kristianbell@gmail.com

S. Blomberg

School of Biological Sciences, The University of Queensland,

Brisbane, QLD 4072, Australia

123

Oecologia (2013) 171:83–91

DOI 10.1007/s00442-012-2409-6

early stages of development (Birchard 2004; Shine and

Thompson 2006). Thus, a warming climate has potentially

significant implications for embryogenesis in ectotherms.

Many oviparous ectotherms lay their eggs underground

(Booth 2006), presumably to provide protection from pre-

dation, and to avoid thermal and hydric extremes. The

thermal buffering properties of soil attenuate heat flux,

typically causing a complete absence of diel fluctuations in

temperature from depths of between 30 and 50 cm (Ack-

erman and Lott 2004). Despite little variation in tempera-

tures at even shallow soil depths, it is often implicitly

assumed that there are a wide range of thermal regimes

available, from which females can select suitable nest sites

(see Wilson 1998; Kolbe and Janzen 2002; Brown and

Shine 2004). Testing of this assumption is required if we

are to understand the potential susceptibility of oviparous

ectotherms to climate change.

The effects of climate change on ectotherms could

potentially be mitigated by acclimation. Several studies have

found that hatchlings prefer, or respond positively to,

ambient temperatures corresponding to temperatures expe-

rienced during development (Blouin-Demers et al. 2000;

Bronikowski 2000; Geister and Fischer 2007), suggesting

that acclimation may pre-adapt individuals to post-hatching

conditions (the Beneficial Acclimation Hypothesis; Leroi

et al. 1994). The majority of existing studies that examine the

plasticity of ectotherms to incubation regime test offspring at

only one temperature (see review by Deeming 2004). Such

an approach prevents the accurate assessment of differential

temperature-performance relationships (temperature reac-

tion norms), because it excludes the effects of phenotypic

acclimation (Seebacher 2005; Deere and Chown 2006).

Plasticity in the ecological and life history traits of

snakes make them good ‘model’ organisms in which to

examine available nest temperatures, offspring responses

to incubation temperature, and offspring acclimation to

incubation temperature (Shine and Bonnet 2000). How-

ever, many past studies do not base incubation temperature

treatments on ‘natural’ temperatures (Burger et al. 1987;

Gutzke and Packard 1987; Lin et al. 2005; Ji et al. 2007;

Du and Ji 2008). To determine the availability of suitable

nest sites to oviparous reptiles in a wetland ecosystem in

north-eastern Australia, we measured the magnitude of

thermal heterogeneity at different soil depths, by measuring

soil temperature profiles. We then used these temperature

profiles, together with current projections for climate over

the next century, as a basis to create appropriate incubation

treatments. We examined the morphological and pheno-

typic responses of hatchling keelback snakes (Tropidono-

phis mairii) to incubation temperatures. Evidence of

acclimation responses were examined by incubating eggs

from each clutch at three temperatures, then testing the

performance of all hatchlings at the three test temperatures.

Materials and methods

Study species

The freshwater keelback snake (Tropidonophis mairii,

Gray 1841) is a small, natricine colubrid that occurs in

wetland and woodland habitats throughout tropical Aus-

tralia (Cogger 1986) (Fig. 1). They are oviparous, and

natural nests occur in soil cracks and burrows at depths of

between 10 and 20 cm (Webb et al. 2001; Brown and Shine

2004).

Data collection

Soil temperature

To determine the range of temperatures available to keel-

backs in nest sites, 24 iButton� temperature data loggers

were deployed on 12th February 2010 at the Townsville

Town Common Conservation Park, northern Queensland

(19�12028.6900S, 146�44025.1700E). Locations for data log-

gers were based on randomly generated vectors (using

S-PLUS to generate values between -15 and 15, repre-

senting distances in metres for north/south and east/west

bearings) from catch locations of gravid female keelbacks.

At each location, we deployed iButtons� at depths that

reflected the range in which natural nests have been

observed (10–20 cm), as well as the temperature taken just

under the soil’s surface (2 cm depth) for comparison. The

iButtons� recorded temperatures hourly for a minimum of

2 weeks. The loggers were relocated on 3 March and 17

March 2010, thus providing temperature profiles for 24

locations adjacent to capture points, at a time of year when

eggs were likely to be in the soil. The proximity of vege-

tation, water and shade were recorded for each location.

Fig. 1 The keelback snake (Tropidonophis mairii)

84 Oecologia (2013) 171:83–91

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Incubation

Thirty-six gravid female keelbacks were captured during

nightly surveys between 14 January and 25 February 2010

in the Townsville Town Common Conservation Park. The

adults were maintained in ventilated 37 9 30 9 20 cm

(L 9 W 9 H) plastic bins, provided with water ad libitum,

a 650-ml hide containing a substrate of vermiculite and

water (1:1 by mass), and a ‘heat mat’ for thermoregulation,

and allowed to lay eggs. Containers were checked twice

daily for eggs. Immediately following discovery, eggs were

weighed and measured (maximum length and width in mm

using callipers) before being placed in separate 300-ml

plastic tubs containing 100 ml of vermiculite substrate

moistened with 10 ml of water, and covered in plastic cling

film to prevent excessive water loss. Eggs were then ran-

domly assigned, in a split-clutch design, to one of three

temperature treatments (24.9 �C ± 0.7, 26.6 �C ± 0.9,

and 30.1 �C ± 0.5) until they hatched. Temperatures

within each treatment fluctuated by the same magnitude as

those recorded at the study site. The coolest temperature

treatment (hereafter referred to as ‘cold’) reflected the

average minimum daily temperature, at a 20-cm depth, of

the coldest iButton location (24.8 �C ± 0.8). The inter-

mediate temperature incubation treatment (‘mid’) reflected

the average daily temperature, at a depth of 20 cm, across

all iButton locations (26.2 �C ± 1.0). The ‘hot’ incubation

treatment reflected the average maximum daily tempera-

ture, at 20-cm depth, of the warmest iButton location

(29.7 �C ± 0.7). The hot treatment also reflected the pre-

dicted average temperature at a depth of 20 cm (or current

‘mid’ treatment) by 2090–2099, given current best esti-

mates of climate change (IPCC 2007). Humidity in all

three rooms was held constant at approximately 80 %.

Eggs were checked daily and were re-arranged within each

room every 2 days to avoid position effects. Following

oviposition, females were released at their point of capture.

Upon hatching, neonates were weighed, measured

[snout-vent length (SVL), tail length, head width and

length] to the nearest millimetre using callipers, sexed by

eversion of hemipenes and checked for the presence of

ventral scale asymmetries, which can indicate develop-

mental instability (Shine et al. 2005; Lowenborg et al.

2010). Once measured, neonates were placed in ventilated

650-ml containers, provided with water ad libitum and

randomly assigned to one of the three temperature treat-

ments. Following an acclimation period of 24 h, hatchlings

were tested (see below) before being transferred to the next

(randomly assigned) treatment room and allowed to accli-

mate for a further 24 h, so that each snake was tested over

3 consecutive days in each temperature treatment. Once

testing was complete, the neonates were held in conditions

similar to those described for gravid females above, until a

first slough had been completed, and then they were

released at the point of capture of the mother.

The effects of incubation treatment on hatchlings were

assessed over a range of performance measures. Locomotor

ability was assessed using video-recorded swim trials.

Keelbacks live near water and often swim to escape pre-

dation and to forage for food (Webb et al. 2001). Snakes

were placed in a circular swim chamber (outer diameter,

120 cm; inner diameter, 80 cm) filled with water to a depth

of 4 cm. The water used in swim trials was left to stand in

each treatment room for a minimum of 24 h prior to test-

ing, and water temperatures were measured immediately

prior to swim trials. The hatchlings were encouraged to

swim by gently tapping their tails with a test tube brush.

Each snake was allowed to swim for 5 min, and the number

of times they were tapped, the number of laps completed

and the time of the first escape attempt (whereby hatchlings

attempted to crawl up the sides of the chamber) were

recorded. The fastest time taken by each hatchling to cover

a 1/8th section (47 cm) of the circuit was measured by

examination of the videos, and used as a measure of burst

speed.

Data analysis

Incubation

There were significant correlations between SVL, tail

length, and head length/width of hatchlings, so a principal

components analysis (PCA) was conducted on the covari-

ance matrix to reduce the dimensionality of morphological

traits. PC1 and PC2 were used as dependent variables in

unbalanced, mixed-effects ANOVAs, with clutch-of-origin

as a random, block effect and incubation treatment as a

fixed effect. Body mass scaled differently from length and

width measurements, and was therefore not included in the

PCA. Variables that violated the assumptions of ANOVA

were log-transformed prior to analysis.

We used linear mixed-effects models (LMM) and gen-

eralised linear mixed-effects models (GLMM) to analyse

snake performance. Clutch of origin and snake ID (nested

within clutch) were included as random factors. Incubation

treatment and test temperature treatment (and their inter-

action) were included as fixed effects, and PC1 was

included as a covariate. For the number of times tapped, a

Poisson GLMM with a square-root link function was used.

LMMs were used for burst speed and number of laps. Plots

of the (weighted) residuals versus the fitted values were

examined to check for heteroskedasticity, and normal

quantile–quantile plots were examined to check for

departures from normality of the residuals. Analyses were

performed in R (R Development Core Team 2012), using

the lme4 package (Bates et al. 2011). p values for the fixed

Oecologia (2013) 171:83–91 85

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effects in LMMs were calculated using Markov Chain

Monte Carlo (MCMC) methods, using the languageR

package for R (Baayen 2011). Significance of the fixed

effects in GLMM was tested using likelihood ratio Chi-

square tests. Post hoc comparisons of fixed effects were

conducted using the package multcomp for R (Hothorn

et al. 2008). Significance of random effects were assessed

using likelihood ratio Chi-square tests.

Results

Soil temperature

The average temperature recorded across all 24 sites and

depths between February and May 2010 was 26.2 �C (±2.3

SE). There was a significant difference in temperature (t test,

t(12989) = 14.36, p \ 0.001) and variance (F test, F(10318,

10788) = 7.29, p \ 0.001) among sites exposed to the sun and

those in the shade. Shaded sites (26.1 �C ± 1.1) were cooler

and exhibited less variation in temperature than sites exposed

to the sun (26.5 �C ± 3.1). Temperature and variation dif-

fered significantly among 2, 10 and 20 cm depths (means:

Kruskall–Wallis, v2ð2Þ ¼ 158:7, p \ 0.001; Variances:

Levene’s Homogeneity of Variance test(2,35351) = 2,112.3,

p \ 0.001). Temperatures 2 cm underground (26.5 �C ±

3.5) were warmer and varied more than at 10 cm

(25.9 �C ± 1.3) and 20 cm (26.2 �C ± 1.0) (Fig. 2).

Incubation

Egg weight, width and length were not significantly different

among incubation treatments [egg weight, mean = 3.0 g

(cold, mid, hot), F(2,390) = 0.02, p = 0.98; egg width,

mean = 25.7 mm (cold), 25.8 mm (mid), 26.1 mm (hot),

F(2,390) = 0.37, p = 0.70; egg length, mean = 14.0 mm

(cold), 14.1 mm (mid), 14.0 mm (hot), F(2,390) = 0.71,

p = 0.49], so the random allocation of eggs among treat-

ments was effective. Across all treatments, the number of

female hatchlings (103) did not differ significantly from

males (112) (Binomial test, n = 215, p = 0.59). Also,

there were no significant differences in sex ratios among

incubation treatments (Pearson, v2ð2Þ ¼ 2:80, p = 0.25).

After hatching, hot-incubated snakes took significantly

longer (11.4 days ± 0.9) to slough than snakes from

mid (7.2 days ± 0.3) or cold (7.5 days ± 0.3) incubation

treatments (Kruskall–Wallis, v2ð2Þ ¼ 6:65, p = 0.036;

Table 1).

Hatching success

Incubation time was inversely related to incubation tem-

perature, with neonates from the hottest incubation tem-

perature hatching earliest (41.4 ± 1.4 days), followed by

mid (51.0 ± 2.3 days) then cold (60.7 ± 2.2 days)

(ANOVA, F(2,189) = 3,253.6 p \ 0.001). Hatching success

decreased significantly with increasing incubation temper-

ature (v2ð2Þ ¼ 107:8, p \ 0.001), with a hatching success

rate of 79.2 and 72.9 % in cold and mid incubation tem-

peratures respectively, dropping to a success rate of 21.4 %

in the hottest temperature treatment. The probability of a

neonate hatching successfully from the hottest incubation

temperature was not distributed randomly among mothers,

with significant clustering in hatching success amongst

clutches (three mothers had an average hatching success of

85 %, compared to a hatching success of 14 % on average

for all other mothers, Poisson, v2ð4Þ ¼ 22:5, p \ 0.001).

Clutches in the hottest incubation temperature were more

likely to have many successful hatchings or none at all, and

significantly less likely to produce intermediate numbers of

viable offspring than would be expected if hatching success

was random (Fig. 3).

Morphology

The number and proportion of ventral scale asymmetries

differed significantly among incubation temperatures

(v2ð2Þ ¼ 110:6, p \ 0.001), with the hottest treatment show-

ing the highest prevalence (85.7 %, 24/28), followed by cold

(6.9 %, 7/101), then mid (6.4 %, 6/94). Similarly, the

occurrence of other developmental abnormalities (typically

post-cloacal kinking of the spine) was significantly different

among incubation treatments (v2ð2Þ ¼ 111:1, p \ 0.001).

71.4 % (20/28) of hot incubated snakes displayed someFig. 2 Average daily soil temperatures at the study site at different

soil depths

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visible form of developmental abnormality, compared to just

3.2 % (3/94) and 3.0 % (3/101) of mid- and cold incubated

snakes respectively.

PC1 (56.2 %) and PC2 (19.6 %) explained 75.8 % of

the total variance among morphological variables (Fig. 4).

The morphological variables with the highest loadings on

PC1 were tail length (0.81) and SVL (0.79). Head width

(0.79) had a considerably larger loading on PC2 than all

other variables, thus PC1 and PC2 appear to reflect body

‘size’ and body ‘shape’ respectively. Body size (PC1)

differed significantly among incubation treatments, after

correcting for maternal effects (ANOVA, F(2,189) = 34.9,

p \ 0.001) (Fig. 3). Hatchlings from the hot incubation

treatment were significantly smaller than both cold and mid

incubation treatments, though cold and mid incubation

temperatures did not differ from one another. Body shape

(PC2), particularly head width, was also significantly dif-

ferent among the three treatments (ANOVA, F(2,189) =

10.9, p \ 0.001). Cold and hot incubated hatchlings had a

slimmer build than hatchlings from the mid incubation

temperature.

Hatchlings from the hot incubation treatment (2.46 g ±

0.30) weighed significantly less than hatchlings from the

mid (2.68 g ± 0.44) and cold treatments (2.75 g ± 0.44)

(ANOVA, F(2,188) = 9.0, p \ 0.001).

Performance

Burst speed was significantly influenced by both incubation

temperature (LMM, F(2,x) = 82.75, MCMC p \ 0.0001

and the temperature at which trials were conducted (LMM,

F(2,x) = 13.01, MCMC p = 0.0004) (Fig. 5a). The inter-

action between incubation temperature and trial tempera-

ture was not significant (LMM, F(4,x) = 0.58, MCMC

p = 0.7742), after correcting for body size, maternal

effects and ID effects.

Body size was significantly positively related to burst

speed (slope = 1.756, t(x) = 2.852, MCMC p = 0.0011).

The proportion of variance associated with maternal effects

was only 7 %, and this was not significantly different from

zero (LMM, LR v2ð1Þ ¼ 2:6915, p = 0.1009). The propor-

tion of variance explained by individual snake ID was

higher and significant at 28 % (LMM, LR v2ð1Þ ¼ 15:676,

p \ 0.0001). After averaging over body size and the

incubation 9 test interaction, the hot incubation treatment

had significantly lower burst speeds than either the cold

treatment (Tukey HSD test, z = 6.469, p \ 0.0001) or the

mid treatment (Tukey HSD, z = 5.187, p \ 0.0001). The

Cold treatment was not significantly different from the mid

treatment (Tukey test, z = -1.234, p = 0.433).

Table 1 Effects of incubation treatment on hatching success, mor-

phology, behaviour and performance in hatchling keelback snakes

(Tropidonophis mairii)

Variable Incubation temperature, mean (SD)

Cold

(n = 106)

Mid

(n = 94)

Hot

(n = 27)

Sex ratio (M:F) 44:57 46:44 9:15

Time to first shed

(days)

7.5 (0.3) 7.2 (0.3) 11.4 (0.9)*

Hatchability

Incubation time

(days)

60.7 (2.2)* 51.0 (2.3)* 41.4 (1.4)*

Hatching success

(%)

79.2 72.9 21.4*

Residual egg

weight (g)

0.69 (0.23) 0.61 (0.28) 0.75 (0.39)

Morphology

Mass (g) 2.7 (0.4) 2.7 (0.4) 2.5 (0.3)*

SVL (cm) 15.6 (1.0) 15.3 (0.8) 14.7 (0.9)*

Tail length (cm) 4.4 (0.5) 4.4 (0.5) 4.0 (0.4)*

Head width (mm) 4.6 (0.1)* 4.6 (0.2) 4.5 (0.2)

Head length (mm) 9.7 (0.3) 9.6 (0.3) 9.4 (0.4)

Ventral scale

asymmetries (%)

6.9 6.4 85.7*

Spinal deformities

(%)

3.0 3.2 71.4*

Performance

Burst speed (cm/s) 22.0 (7.1) 19.9 (6.4) 8.0 (6.1)*

Number laps 5.4 (0.1) 5.45 (0.1) 2.3 (0.3)*

Number taps 12.0 (0.6) 13.7 (0.6) 6.9 (1.2)*

* Statistically different subgroup

Fig. 3 Frequencies of observed and expected hatchling numbers

from clutches incubated in the warmest temperature treatment. Note

that many more clutches had no hatchlings than expected in the hot

treatment

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An additional measure of swim speed, the number of

laps completed in 5 min, also showed significant differ-

ences with incubation temperature (F(2,x) = 113.94,

MCMC p \ 0.0001) and performance test temperature

(F(2,x) = 8.211, MCMC p \ 0.0001), with no significant

interaction between the two factors (F(4,x) = 1.952, MCMC

p = 0.16159). See Fig. 5b. Again, there was a significant

effect of body size, with larger individuals swimming further

(slope = 0.3180, tx = 3.543, MCMC p \ 0.0001). Mater-

nal and individual components of variance were 7.6 %

(LLMM, LR v2ð1Þ ¼ 8:524, p = 0.0035), and 16.6 %

(LLMM, LR v2ð1Þ ¼ 17:983, p \ 0.0001), respectively. Post

hoc comparisons of means (averaged over the fixed factor

interaction and the body size covariate) revealed that the hot

incubated animals swam fewer laps than either the cold

(Tukey HSD, z = -11.941, p \ 0.0001) or mid temperature

(Tukey HSD, z = 12.301, p \ 0.0001) incubated animals,

but that the cold and mid temperature incubation treatments

were not significantly different (Tukey HSD, z = 0.452,

p = 0.89). Post hoc analysis of the performance temperature

treatments showed that there was a significant difference

between hot and cold running temperatures (Tukey HSD,

z = 2.919, p = 0.0098), but not between hot and mid

(Tukey HSD, z = -1.967, p = 0.121) or between cold and

mid (Tukey HSD, z = 0.951, p = 0.608), indicating that the

effect of performance temperature on the number of laps

swum in 5 min is less than the effect of incubation temper-

ature on this response variable.

Willingness to swim, as measured by the number of taps

required during testing (Brown and Shine 2002), showed a

significant interaction between incubation temperature and

performance temperature (LR v2ð4Þ ¼ 11:586, p = 0.0207;

see Fig. 5c). Post hoc tests for the incubation temperature

effect (averaged over the interaction term and body size

covariate) revealed a significant difference between both

hot and cold incubation treatments (Tukey HSD, z =

-4.017 p \ 0.001) and hot and mid incubation treatments

(Tukey HSD, z = -5.120, p \ 0.001) but not mid and cold

incubation treatments (Tukey HSD, z = 1.757, p = 0.177).

Thus, hatchlings from the hot incubation treatment were

more willing to swim than hatchlings from both mid and

cold incubation treatments. An equivalent analysis of the

performance treatments revealed no significant difference

between the mid and cold treatments (Tukey HSD,

z = 0.415, p = 0.91) but differences between the hot and

cold treatments (Tukey HSD, z = 6.614, p \ 0.0001) and

between hot and mid treatments (Tukey HSD, z = 6.198,

p \ 0.0001). Hence, the hot performance temperature

required less taps than either the mid or cold performance

temperature treatments.

Discussion

Clearly, keelback snakes show high levels of phenotypic

plasticity in response to thermal regime; incubation

Fig. 4 Principal components

analysis plotting body ‘size’

(PC1) and body ‘shape’ (PC2)

in hatchling keelback snakes

from hot (circles), mid

(squares) and cold (crosses)

incubation treatments. Darkbold markers indicate

centroids ± standard errors

88 Oecologia (2013) 171:83–91

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temperature significantly affected 12 of the 15 traits

examined. The temperature at which hatchlings were

incubated had a stronger influence on performance than did

test temperature across all performance measures, includ-

ing locomotor ability and willingness to swim.

Performance was ‘‘best’’, i.e., offspring had fewer abnor-

malities, were larger, and could swim faster and for longer,

if they had experienced cold and mid temperatures as

embryos, suggesting that development is evolutionarily

adapted to occur at these temperatures. Apparent adapta-

tion to cooler temperatures is consistent with a surprisingly

low optimal incubation temperature in this tropical reptile

(Brown and Shine 2006). The only characteristic that was

not ‘‘best’’, or of apparently higher fitness, in offspring

incubated in the two cooler treatments was willingness to

swim. Smaller, slower offspring from warmer temperatures

swam more readily when coaxed compared to offspring

from other treatments. This may be due to covariation

among physiological traits, such that high speed is a trade-

off against higher endurance (or willingness to swim)

(Vanhooydonck et al. 2001). However, many studies dis-

pute the existence of significant trade-offs between speed

and endurance (Huey et al. 1990; Secor et al. 1992, 1995;

Brodie and Garland 1993; Goodman et al. 2007). Alter-

natively, perhaps increased willingness to swim is a

behavioural adaptation that mitigates smaller size and

slower swimming speeds. For all other characters, both

behavioural and morphological, cooler temperatures

appeared to produce ‘fitter’ offspring.

In our study, individuals exposed to cooler incubation

temperatures (similar to cooler ranges of temperatures

available in the wild) showed higher performance in all

incubation temperatures. The lack of interaction between

incubation temperatures and test temperatures indicates

that developmental acclimation did not pre-adapt individ-

uals to post-hatching environments. Cooler incubation

temperatures are often better in terms of hatchling fitness in

ectotherms (Partridge et al. 1995; Atkinson and Sibly 1997;

Shine 1999; Stillwell and Fox 2005; Brown and Shine

2006). However, our study does not really support the

‘cooler is better’ hypothesis of thermal acclimation,

because we tested only the range of temperatures actually

available to female snakes in the wild. Thus, we did not

demonstrate that colder temperatures in general produce

fitter hatchlings, rather we showed that, within the range of

temperatures available to females, cooler temperatures

were better for successful embryonic development. These

results are, therefore, also consistent with the local adap-

tation hypothesis of thermal acclimation (Caley and

Schwarzkopf 2004), because females apparently select cool

nest sites, at least in other regions of Australia (Brown and

Shine 2006).

Preference for relatively cool nest sites may increase the

sensitivity of T. mairii to global increases in environmental

temperatures. The 3.4 �C difference in average soil tem-

peratures between the hottest and coldest sites (at a depth

of 20 cm) suggests that the availability of suitable nest sites

may be limited, even in relatively complex environments.

Fig. 5 Incubation treatment and test temperature effects on the

performance of hatchling keelback snakes. a, b Measures of

locomotor speed, c willingness to swim (note: more taps indicate

snakes were less willing). The x axis represents the temperature at

which snakes were tested. The y axis represents the means of the

dependent variables. Hatchlings incubated at the cold temperature

treatment are indicated by the dotted black line, mid temperature by

the grey line and hot by the solid black line. Vertical bars standard

errors

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Global average surface temperatures are forecast to rise by

up to 4 �C by 2100 (IPCC 2007). Should surface temper-

atures increase as predicted, then not one of the 24 sites at

which soil temperature profiles were recorded would pro-

vide optimal conditions for development of T. mairii.

However, despite the apparently small variation in soil

temperature, gravid keelbacks may still be able to locate

cool nest sites, and therefore they may be able to com-

pensate behaviourally for the influences of higher envi-

ronmental temperatures. Reptiles may adjust behaviour to

select thermally appropriate sites that enhance their fitness,

and expand or alter their geographic range (Huey et al.

2003). For example, grass snakes (Natrix natrix) have

extended their range in Europe into high latitudes by taking

behavioural advantage of heat sources such as manure piles

(Lowenburg 2010). Alternatively, compensation may occur

through temporal shifts in the breeding season, such as

laying eggs earlier in the spring. However, the importance

of moisture to the viability of eggs, and the correlation of

moisture levels with season, may restrict the extent to

which keelbacks could alter the timing of breeding (Brown

and Shine 2004). Given the detrimental impacts on

hatchling viability of relatively minor differences in incu-

bation temperature, further investigation into the presence

of cooler microclimates may be critical in predicting how

this species responds to climate change.

Another way that snake populations may respond to

global temperature increases is evolutionarily (Bradshaw

and Holzapfel 2006; Kearney et al. 2009). Although all the

clutches were highly sensitive to incubation temperature,

inconsistent hatching success among dams in the hot

incubation treatment suggests there may be genetic dif-

ferences in the thermal tolerance of individuals within

populations. The presence of apparently ‘heat tolerant’

dams suggests this population may be resilient to some of

the impacts of climate change. Identifying the genetic basis

for these differences may provide insight into the extent of

resilience in other populations of keelbacks, and poten-

tially, other species of snake.

While we have described the likely short-term conse-

quences of increased incubation temperature, little is known

about how specific phenotypic changes lead to differences in

post-hatching survival and, ultimately, fitness. Survival can

be negatively, positively or uncorrelated to incubation tem-

peratures within turtles, lizards and crocodiles (Deeming

2004). Limited evidence from mark–recapture studies on

keelbacks suggests that large sizes at hatching enhance sur-

vival (Brown and Shine 2004). Despite a lack of information

on long-term compensation effects, and the relationship

between specific traits and survivorship, a relatively small

increase in incubation temperature can considerably reduce

viability and performance of hatchling keelbacks. Hot-

incubated hatchlings had a lower hatching success rate, were

smaller and had more developmental abnormalities than

hatchlings from the mid and cold incubation treatments.

Climate change is predicted to cause local reptile extinctions

of 39 % worldwide by 2080 (Sinervo et al. 2010). Further

studies examining the links between phenotype and fitness,

the availability of suitable nest sites and the mechanisms

behind the high variability in phenotypic response to incu-

bation temperature, are critical to understanding how ovip-

arous reptiles will respond to a changing climate.

Acknowledgments We thank Teresea Lambrick, Gus McNab, Ross

Gottlieb, Joel Voiselle, John Llewelyn and Matthew Crowther for

their assistance in the field and laboratory. The work was carried out

under a permit from the Environmental Protection Agency and

Queensland Parks and Wildlife Service (WITK06346309), and James

Cook University Animal Ethics Committee (A1469). This research

was supported financially by a grant from the National Climate

Change Adaptation Research Facility.

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