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Hydric balance and locomotor performance of an anuran (Rhinella marina) invading the Australian arid zone

R. Tingley , M. J. Greenlees and R. Shine

R. Tingley (reid.tingley@gmail.com), M. J. Greenlees and R. Shine, School of Biological Sciences A08, Univ. of Sydney, NSW 2006, Australia.

Invasive species often encounter environmental conditions well outside those found in their native geographic ranges, and thus provide ideal model systems with which to explore responses to novel abiotic challenges. Within Australia, the invasive cane toad Rhinella marina has colonized areas that are considerably more arid than those found within its native range. Has the colonization of these novel environments been accompanied by shifts in physiology and/or locomotor performance? We measured rates of evaporative water loss, water gain, and eff ects of desiccation on locomotor perfor-mance of cane toads from two invasion fronts: one mesic (the wet-dry tropics) and one arid (the semi-desert). Th e two populations diverged substantially. Contrary to intuition (but consistent with intra-specifi c comparisons between other toad populations from mesic vs arid areas), rates of evaporative water loss were lower (not higher) in toads from the mesic population. However, arid-zone toads gained water more rapidly through their ventral surfaces, and rates of water loss and gain were highly correlated within individual toads from the arid-zone population. Rates of water exchange in laboratory-acclimated toads from the semi-arid zone did not diff er from those of free-ranging conspecifi cs from the same population, suggesting that divergences between mesic and semi-arid toads refl ect genetic changes that have occurred during the spe-cies ’ Australian invasion. Mesic and semi-arid toads showed similar locomotor performance (endurance, distance per hop) when fully hydrated, but locomotor performance declined much more rapidly with desiccation in the mesic toads. Th us, within the short (decades-long) timespan of the cane toad ’ s Australian invasion, there has been substantial population divergence in the ability to withstand desiccating conditions. If we are to accurately predict the distributions (and hence impacts) of invading organisms, we will need to include adaptation potential in risk assessment schemes.

Oikos 121: 1959–1965, 2012 doi: 10.1111/j.1600-0706.2012.20422.x

© 2012 Th e Authors. Oikos © 2012 Nordic Society Oikos Subject Editor: Justin Travis. Accepted 7 February 2012

An extensive ecophysiological literature documents many examples of close covariation between an animal ’ s physi-ological abilities and the types of challenges that are imposed by the environments in which it lives (Willmer et al. 2005). For example, animals from hot, dry climates often have greater thermal tolerances, and a better ability to withstand desiccation, than do conspecifi cs from cooler and wetter areas (Casta ñ eda et al. 2004, Navas et al. 2004, Newton et al. 2010). Such a matching between environment and physiology clearly enhances organismal function. Many of these cases presumably refl ect adaptation over evolutionary timescales, but the amount of time required for a taxon to adjust its physiology to local abiotic challenges is unclear. In some cases, evolution can be rapid (Prentis et al. 2008, Whitney and Gabler 2008, Phillips et al. 2011) and in oth-ers, phenotypically plastic responses can enable organisms to function eff ectively after brief exposure to novel environ-mental challenges (Agrawal 2001, Kolbe et al. 2010).

Invasive species off er excellent opportunities to exam-ine adaptive changes over short timescales (Prentis et al. 2008, Whitney and Gabler 2008, Brown et al. 2011). If an introduced species encounters abiotic conditions outside those experienced in its native range, it may need to modify

its physiology and/or behaviour to confront the novel challenge. One of the most spectacular and intensively-monitored biological invasions involves cane toads Rhinella marina , large toxic anurans native to South and Central America, but now introduced to more than 40 countries worldwide (Lever 2001). Th e native-range of cane toads includes areas with seasonal precipitation, but some of the places where these toads now thrive in other parts of the world exhibit far more extreme hydric conditions (Brown et al. 2011). Most notably, cane toads in Austra-lia have penetrated deep into the semi-arid zone of cen-tral Queensland, an area with low and sporadic rainfall (Tingley and Shine 2011; Fig. 1). Comparisons of the cli-mates occupied by cane toads in their native and Australian ranges have demonstrated that the environmental condi-tions in central Queensland lie well outside of the toads ’ native realized niche (Tingley and M. Kearney unpubl.). To investigate whether this niche shift has been facilitated by shifts in physiology and/or locomotor performance, we measured rates of evaporative water loss, water uptake, and eff ects of desiccation on locomotor performance of cane toads from two Australian populations: one mesic (the wet-dry tropics) and one arid (the semi-desert).

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Methods

Study locations

We compared cane toads from two recent invasion fronts in Australia that lie within and outside of the species ’ native realized niche, respectively: 1) the wet-dry tropics of the Northern Territory (Middle Point, 50 km east of Darwin), and 2) semi-arid Queensland (Whitehill Station, 30 km southwest of Longreach). Toads colonized these areas during the wet seasons of 2004/2005 and 2006/2007, respectively. Mean monthly temperatures are similar in the two popula-tions, but precipitation regimes are vastly diff erent (median precipitation from 1961 – 1990, semi-arid Queensland: 437 mm, wet-dry tropics: 1422 mm, � www.bom.gov.au � ; see Brown et al. (2011) and Tingley and Shine (2011) for more detailed descriptions of the study sites).

Population-level differences in rates of water loss and uptake

In April and May 2010, 64 adult toads ( � 86.4 mm snout-urostyle length; 32 males, 32 females) were collected at each study site (total n � 128) and transported to shaded outdoor enclosures (0.6 � 0.4 � 0.4 m) nearby. Within each enclo-sure, toads were provided with dry substrate and a large pool of water. Animals were not fed for three days after capture so that defecation did not aff ect the results of dehydration trials. On the fourth day, toads were randomly allocated to one of three hydration treatments: 100% (n � 40), 90% (n � 40) or 80% (n � 48) of their fully hydrated mass (determined by weighing toads after they had been submerged in 1 cm of dis-tilled water for one hour: Titon et al. 2010). Toads from the 100% hydration treatment were used in locomotor tests only. Toads allocated to the 90% and 80% treatments were placed in wind tunnels made from plastic guttering (0.3 � 0.15 � 0.1 m) with a small electric fan attached to one end. While in the wind tunnels, toads were weighed every 30 min until they reached their target hydration level (i.e. 90% or 80%). Th e proportion of time spent in the water conserving

posture was determined by inspecting toads every 15 min. Each animal was used in only a single dehydration trial.

Rates of evaporative water loss were calculated by divid-ing the slopes of regressions of body masses over time by each toad ’ s surface area (calculated using an empirical equa-tion based on body mass: McClanahan and Baldwin 1969). Estimated surface areas were corrected to account for the fact that the water conserving posture exposes 2/3 of a toad ’ s surface to the air (Withers et al. 1982, Titon et al. 2010). Only toads from the 80% treatment ( n � 24 from each pop-ulation) were used in analyses of water loss rates.

To determine whether rates of water uptake diff ered between toads from the semi-arid and mesic populations, dehydrated individuals from the 80% hydration treatment were placed in plastic containers fi lled with 1 cm of distilled water for one hour (Titon et al. 2010). Every fi ve minutes, toads were removed from the containers, dried with paper towel, and weighed. Rates of water uptake were calculated from the slopes of regressions of body masses versus time.

To obtain independent estimates of rates of water exchange, we also used a vapometer to measure transepider-mal evaporative water loss (TEWL) across the dorsal and ventral surfaces of 25 toads from each population (Lillywhite et al. 2009). Eight readings were taken on each surface, and these readings were averaged to produce a single measure-ment for each surface of each animal.

To determine whether rates of TEWL were phenotypically labile, we measured the dorsal and ventral skin permeabil-ity of 18 adult toads (9 males, 9 females) from the semi-arid population that had been kept under ‘ mesic ’ thermal and hydric conditions for 13 months. Th ese captive toads were housed in similar enclosures to those used in our fi eld trials. Enclosures contained a large pool of water and were kept in a temperature-controlled room set to 29 ° C with a 12 h light cycle. Toads were fed crickets twice weekly.

Eff ects of population of origin on rates of evapora-tive water loss in wind tunnels were analyzed using an ANCOVA, with the proportion of time spent in the water conserving posture as a covariate. Diff erences in rates of water uptake between populations were tested with a t-test. Population-level diff erences in rates of dorsal and ventral TEWL were tested using non-parametric Kruskal – Wallis tests. To control for body-mass eff ects, we used the resid-uals from a regression of body-mass versus TEWL rates as response variables in these analyses. Mann – Whitney U-tests (with Bonferonni adjustments) were used for post hoc comparisons.

We investigated whether rates of water loss were corre-lated with rates of water uptake and population of origin using an ANCOVA. Because the proportion of time spent in the water conserving posture was signifi cantly correlated with rates of water loss, we used the residuals from a regres-sion of amount of time spent in the water conserving posture versus water loss rate as a response variable in the ANCOVA. We also used an ANCOVA to determine whether vapometer measurements of TEWL across the dorsal surfaces of toads could be predicted from ventral readings after accounting for population membership. To control for negative eff ects of body mass on vapometer measurements, we used the residuals from a regression of TEWL rates versus body mass for both dorsal and ventral readings.

Figure 1. Staged photograph of a cane toad Rhinella marina in Longreach, Queensland, Australia. Cane toads inhabit drier con-ditions in Longreach than in the wet-dry topics of the Northern Territory. Photo by Richard Shine.

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Assessing effects of hydration status on locomotor performance

To determine whether hydration status infl uenced loco-motor performance, toads from the three hydration treat-ments (100%, 90% or 80% of fully hydrated mass; n � 120) were raced along an indoor raceway (5 � 0.3 � 0.3 m) cov-ered with artifi cial turf. Because toads are nocturnal, all racing trials were conducted in the evening. Toads were encouraged to hop down the racetrack by gently tapping them on the urostyle. Racing trials ceased when a toad remained inactive despite being tapped 10 times (Llewelyn et al. 2010). All trials were conducted at a consistent tem-perature (mean � SE � 24.8 ° C � 0.0668).

Eff ects of hydration treatment and population of origin on the endurance (log-total distance travelled), log-number of hops per meter, and hopping speed (average time taken to cover 1 m) were tested using two-way ANOVAs. Toads that failed to hop were assigned the slowest recorded speed (similar results were obtained when stationary toads were removed from the analyses). Multiple comparisons were investigated with Tukey ’ s honestly sig-nifi cant diff erence tests. Although cane toads from invasion front populations have greater endurance than toads from long-established populations (Llewelyn et al. 2010), our results control for this eff ect by comparing populations with similar invasion histories (i.e. time since invasion). Mean body sizes (snout-urostyle lengths) and tibia lengths did not diff er among hydration treatments or populations, suggesting that the observed shifts in locomotor perfor-mance also were not due to morphological characteris-tics that can infl uence the speed of toads (Llewelyn et al. 2010). All statistical analyses were conducted in R 2.12.0 (R Development Core Team).

Results

When placed in wind tunnels, cane toads from a mesic region of Australia lost water at a slower rate than did con-specifi cs from the semi-arid zone (Fig. 2A; population: F 1,45 � 23.8, p � 0.0001; proportion of time spent in water conserving posture: F 1,45 � 42.6, p � 0.0001). However, rates of water uptake through the ventral surfaces of toads showed the opposite pattern: when dehydrated, toads from the semi-arid population gained water more rapidly than did toads from the mesic population (Fig. 2B; t 42.4 � 2.11, p � 0.0405). Vapometer measurements of TEWL rates across the dorsal and ventral surfaces of toads provided qualitatively similar results with respect to rates of water loss (Fig. 3A; χ 2 � 40.5, DF � 2, p � 0.0001) and water uptake (Fig. 3B; χ 2 � 36.5, DF � 2, p � 0.0001). Free-ranging toads from the mesic-population had lower rates of dor-sal and ventral water exchange than did free-ranging and laboratory-acclimated toads from the semi-arid zone, but dorsal and ventral readings of free-ranging and laboratory-acclimated toads from the semi-arid population were statis-tically indistinguishable.

Overall, rates of water uptake predicted rates of water loss within individual toads (Table 1). However, this relation-ship was modifi ed by an interaction between water uptake

rate and population of origin. Rates of water loss and uptake were positively correlated within the semi-arid popula-tion, but not within the mesic population (Table 1). After accounting for population-eff ects, vapometer measurements of dorsal skin permeability were positively correlated with ventral readings, but there was no evidence for an inter-action between ventral readings and population of origin (Table 2). Qualitatively similar results were obtained when free-ranging and laboratory-acclimated toads from the semi-arid population were combined.

Hydration status and population of origin had strong interactive eff ects on endurance, and the distance covered per hop (Table 3). When dehydrated to 80% of their fully hydrated mass, toads from the mesic invasion front had less endurance (Fig. 4A), and travelled in shorter hops (Fig. 4B), than did conspecifi cs from all other treatment-population combinations (all p � 0.01). Mesic-area toads from the 90% hydration treatment also had less endurance than did toads from all other treatments and populations (all p � 0.05), with the exception of semi-arid toads that were 80% hydrated (p � 0.827).

Hopping speeds diff ered among hydration treatments, and populations, but there was no evidence for an inter-action between these two variables (Table 3). Toads that were 80% hydrated were slower than conspecifi cs from the other two hydration treatments (all p � 0.0001), and mesic-area toads were slower than those from the semi-arid zone (Fig. 4C; p � 0.001).

Discussion

At least 35 species of toads inhabit semi-arid regions world-wide (Prates and Navas 2009), and these species possess a

Figure 2. Rates of water loss (A) and water uptake (B) in fi eld-collected cane toads Rhinella marina from mesic and semi-arid invasion fronts in Australia. Boxes represent medians and quartiles, whiskers extend to the most extreme observations that are no more than 1.5 times the interquartile range from the box, and observa-tions which lie outside of this range are shown as points.

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Table 2. Rates of water loss from individual cane toads Rhinella marin a as a function of water uptake rates and population of origin (free-ranging semi-arid, acclimated semi-arid, or free-ranging mesic). Unlike the results shown in Table 1, water loss and uptake rates shown here were derived from vapometer measurements.

Variable Test statistic p

Across populationswater uptake F 1,66 � 84.2 � 0.0001

Within populationswater uptake F 1,62 � 91.3 � 0.0001population F 2,62 � 3.35 0.0415water uptake � population F 2,62 � 1.41 0.251

Figure 3. Mass-corrected vapometer estimates of rates of water exchange across the dorsal (A) and ventral (B) surfaces of cane toads Rhinella marina from mesic and semi-arid invasion fronts in Australia. Toads were measured directly after being captured ( ‘ Free– ranging ’ ), or after being kept in captivity under stable thermal and hydric conditions for 13 months ( ‘ Acclimated ’ ). Treatments labeled with diff erent letters diff er from one another at α � 0.05 (after correcting for multiple comparisons). Boxes represent medians and quartiles, whiskers extend to the most extreme observations that are no more than 1.5 times the interquartile range from the box, and observations which lie outside of this range are shown as points.

range of morphological, physiological, and behavioural mechanisms to cope with desiccating conditions (reviewed by Bentley 1966, J ø rgensen 1997). Traditionally, research-ers have envisioned such adaptations as having evolved over long time scales (thousands to millions of years; Stockwell et al. 2003). In contrast, our results demonstrate that the colonization of semi-arid Australia by invasive cane toads has resulted in shifts in both their physiological and locomotor performance in a matter of decades.

If cane toads in Australia are adapting to more arid con-ditions, what shifts would we expect to see in their hydric balance and locomotor performance? Intra-specifi c com-parisons of rates of water uptake in semi-desert versus mesic toads showed that toads from the semi-desert are capable of absorbing water more rapidly through their ventral surfaces. Open pools of water are rare features in arid landscapes, and thus the ability to utilize available surface water is critical

for maintaining hydric balance in arid-zone anurans (McClanahan and Baldwin 1969, Walker and Whitford 1970). Under such conditions, possessing a highly perme-able ventral surface may provide a strong selective advantage for semi-arid toads. Both inter- and intra-specifi c compari-sons have demonstrated higher rates of water uptake in spe-cies and populations that inhabit arid or semi-arid regions (Bentley et al. 1958, Warburg 1965, 1967, Main 1968, Fair 1970, Walker and Whitford 1970, Navas et al. 2004, but see Th orson 1955, Claussen 1969, Canziani and Cannata 1980). Th us, our data on invasive cane toads show the same type of diff erence between arid and mesic areas as has been described in other amphibian species inhabiting these two types of habitats within their native ranges.

Paradoxically, however, arid-zone cane toads lost water more quickly through their dorsal surfaces than did mesic-zone toads. Published comparisons between amphibians that diff er in their degree of terrestriality have generally found the opposite pattern (Warburg 1965, 1967, Canziani and Cannata 1980, de Andrade and Abe 1997, Young et al. Table 1. Rates of water loss from individual cane toads Rhinella

marina as a function of water uptake rates and population of origin (semi-arid or mesic). Water loss and uptake rates were derived from whole-animal measurements.

Variable Test statistic p

Across populationswater uptake F 1,45 � 7.22 0.0101

Within populationswater uptake F 1,43 � 10.9 � 0.01population F 1,43 � 18.0 � 0.001water uptake � population F 1,43 � 7.17 0.0105

Semi-arid populationwater uptake F 1,22 � 8.89 � 0.01

Mesic populationwater uptake F 1,21 � 1.17 0.291

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rates of water loss and uptake were highly correlated within our arid-zone population but not within our mesic-zone population. Within the arid-zone population, then, the individual toads that lost water more rapidly also had higher rates of water uptake. Further research could usefully exploreskin morphology in Australian cane toads, to evaluate the link between geography, skin morphology, and hydric balance.

A cane toad ’ s hydration status aff ected its locomotor performance, as has been shown previously in other anuran species (Schmajuk and Segura 1982, Moore and Gatten 1989, Titon et al. 2010). However, in our study, hydra-tion status had a more marked eff ect on the locomotor performance of cane toads from the mesic population. Mesic and semi-arid toads demonstrated similar perfor-mance when fully hydrated, but when dehydrated to 80% of their initial mass, mesic-area toads had less endurance, and travelled in shorter hops than did their arid-zone conspecifi cs. Semi-arid toads lost water rapidly from their dorsal surfaces, and thus the ability to travel long dis-tances when dehydrated may have clear fi tness advantages in a strongly desiccating environment. In contrast, mesic climates provide abundant moist retreat sites where toads can rehydrate (dry-season conditions experienced by our mesic-site toads do not aff ect the toads ’ hydration levels or impose physiological stress: Brown et al. 2011). Compa-rable studies of population-level diff erences in the eff ects of dehydration on the locomotor performance of anurans are rare; however, Titon et al. (2010) reported a lower sensitiv-ity of locomotor performance to dehydration in the savan-na-dwelling Rhinella schneideri than in two congeners that occupy rainforest habitats. As for water-uptake rates, then, the attributes of desert-invading populations of cane toads resemble those seen in anurans that have evolved in rela-tively arid regions.

Several potential mechanisms could be responsible for the population-level diff erences in physiology and behav-iour that we have observed, and these mechanisms are not mutually exclusive. First, the cane toad ’ s successful inva-sion of arid Australia could be due to phenotypic plasticity (direct induction by local environmental factors, without requiring allelic substitutions). Higher levels of such plastic-ity in invasive species can facilitate invasion success (Parker et al. 2003, Sol et al. 2005, 2008, Wilson et al. 2009, Nyamukondiwa et al. 2010, Amiel et al. 2011). For exam-ple, plasticity in cold tolerance has facilitated the cane toad ’ s invasion of colder regions of southeastern Australia (Kolbe et al. 2010). However, we are unaware of any robust evidence of temporal shifts in traits such as water permeabil-ity of the skin among anurans; indeed, rates of evaporative water loss tend to be consistent year-round even in spe-cies that inhabit regions with highly seasonal precipitationregimes (Young et al. 2005). Comparable rates of dorsal and ventral skin permeability in free-ranging and laboratory-acclimated cane toads from the semi-arid zone further sug-gest that the observed diff erences between mesic and semi-arid populations were not due to phenotypic plasticity.

Alternatively (or additionally), population-level diver-gences in physiology and locomotor performance could refl ect genetic changes during the cane toads ’ Australian inva-sion. Cane toads are prolifi c breeders, and attain high densi-ties in Australia (Lampo and De Leo 1998). Th ese factors

2005) or no pattern at all (Th orson 1955, Claussen 1969, Seymour and Lee 1974, Buttemer 1990, Titon et al. 2010). However, diff erences between studies with respect to experi-mental conditions (e.g. temperature, humidity, wind speed), and the types of habitats examined, largely confound meaningful comparisons between studies (Warburg 1971). Intriguingly, though, Brazilian Rhinella granulosa showed the same pattern as did our cane toads: individuals from a mesic region (the Atlantic Forest) had higher cutaneous resistance to evaporative water loss than did conspecifi cs from a semi-arid population (Prates and Navas 2009). Th is divergence appears to refl ect the presence of thin, smooth, highly vascularised skin in semi-arid toads (Navas et al. 2004). In turn, thinner skin may benefi t arid-zone anurans if the average benefi t of accelerating water uptake is greater than the benefi t of retarding water loss (Warburg 1967, Prates and Navas 2009). Within individual cane toads,

Table 3. Effects of hydration status and population of origin (semi-arid or mesic) on endurance, the distance covered per hop, and hopping speed of cane toads Rhinella marina from two invasion fronts in Australia.

Variable Test statistic p

Endurancepopulation F 1,114 � 24.9 � 0.0001hydration status F 2,114 � 23.5 � 0.0001population � hydration status F 2,114 � 9.65 � 0.001

Number of hops per meterpopulation F 1,112 � 1.05 0.308hydration status F 2,112 � 16.5 � 0.0001population � hydration status F 2,112 � 4.71 0.0109

Speedpopulation F 1,114 � 14.2 � 0.001hydration status F 2,114 � 21.3 � 0.0001population � hydration status F 2,114 � 1.55 0.216

Figure 4. Eff ects of hydration status on endurance (A), the distance covered per hop (B), and hopping speed (C) of cane toads Rhinella marina from mesic and semi-arid invasion fronts in Australia.

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Amiel, J. J. et al. 2011. Smart moves: eff ects of relative brain size on establishment success of invasive amphibians and reptiles. – PLoS One 6: e18277.

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Lever, C. 2001. Th e cane toad. Th e history and ecology of a successful colonist. – Westbury Academic and Scientifi c Publishing.

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Llewelyn, J. et al. 2010. Locomotor performance in an invasive species: cane toads from the invasion front have greater endur-ance, but not speed, compared to conspecifi cs from a long-colonised area. – Oecologia 162: 343 – 348.

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could have helped promote genetic diff erentiation between populations by increasing the probability that advanta-geous mutations developed in novel environments (Urban et al. 2007). Common-garden transplants of cane toads in northern Australia have documented genetically-based changes in the dispersal abilities of toads at the vanguard of the invasion front (Phillips et al. 2011). Similar studies could usefully examine the genetic basis (if any) of geographic divergences in cane toad behaviour and physiology.

Lastly, the physiological and locomotor shifts that we observed may be unrelated to the climatic conditions experi-enced by toads at the two invasion fronts. Th at is, the two populations may have diverged for non-adaptive reasons (e.g. genetic drift, founder eff ects), or as a response to some unknown selective pressures unrelated to hydric balance. Th e primary objections to this interpretation are that 1) our fi ndings largely accord with a priori predictions regarding the direction of the observed diff erences (based on intra- and inter-specifi c correlations between physiology, behavior, and habitat in congeneric species), and 2) the diff erences that we measured involve traits that are likely to have strong fi tness consequences. Future work could clarify the mechanisms responsible for our results by exploring acclimation and genetic eff ects in additional populations.

Th e invasion process can impose strong selective pressures on the phenotypes of invading organisms (Lee et al. 2007). Invasive species thus provide ideal model systems to explore how organisms overcome novel environmental challenges – and especially, to clarify the early stages of such responses. However, the ability of invaders to adapt rapidly to envi-ronmental challenges also has important implications for managing the spread of invasive species: if we ignore that adaptability, we will underestimate the full extent of their invasion potential. Most risk assessment schemes envision invasive species as fi xed entities (Whitney and Gabler 2008), but recent research has shown that many invasive species are capable of mounting rapid adaptive responses to novel envi-ronments (Stockwell et al. 2003, Westley 2011). Our results add to this growing body of evidence. Within 75 years of their arrival in Australia, invasive cane toads have adopted many of the same physiological and behavioural mechanisms that allow their congeners to survive semi-arid conditions in their native ranges. Including adaptation potential in risk assessment schemes will therefore be critical if we are to make robust predictions of the future distributions of invad-ing organisms.

Acknowledgements – Th e Emmott Family graciously provided accommodation and supplies during the course of fi eldwork. RT was funded by a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship, an Endeavour International Postgraduate Research Scholarship, and a Univ. of Sydney International Postgraduate Award. Additional funding was provided by the federal government ’ s Caring for our Country initiative, and by the Australian Research Council. Experiments were approved by the Univ. of Sydney Animal Care and Ethics Committee (protocol no. L04/7 -2010/1/5362).

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