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1. Measuring the costs imposed by specific environmental challenges is difficult, because organisms adapt in ways that reduce those costs. Invasive species provide an opportunity to quantify environmental impacts before organisms can make adaptive changes.
2. The native range of cane toads (Rhinella marina) lies within the wet neotropics; although part of this range experiences seasonal drought, many of the places recently invaded by this large anuran species are much more arid.
3. Five years’ fieldwork from a seasonally arid site in the Australian wet–dry tropics shows strong seasonal shifts in the toads’ (i) population structure, reflecting seasonality in breeding and recruitment; (ii) adult morphology (secondary sexual characteristics in males); (iii) growth rates; (iv) energy balance; (v) spatial ecology (philopatry, dispersal rates) and (vi) adult mortality rates.
4. Some of these patterns accord with intuition: for example, wet-season conditions resulted in higher rates of growth, reproduction and movement, better body condition and more pronounced secondary sexual characteristics. However, seasonal patterns for other traits were non-intuitive: for example, neither hematocrit levels (reflecting hydration state) nor corticosterone levels (reflecting stress) showed significant seasonal variation, and mortality rates were higher in the wet season than the dry season.
5. The toads’ ability to flexibly adjust their behaviour and ecology to local hydric conditions has allowed them to thrive even under climatic conditions that preclude activity, feeding and reproduction for most of the year.
Even before Charles Darwin proposed a mechanism to explain the phenomenon, it was widely appreciated that organisms are well designed to surmount the specific abiotic challenges posed by their local environments (Williams 1966; Gould & Lewontin 1979). Since that time, extensive ecophysiological research has revealed abundant examples of complex and detailed fine-tuning of organismal physiology and behaviour in ways that enable organisms to deal with factors such as aridity and thermal extremes (e.g. Willmer, Stone & Johnston 2005; Hillman et al. 2009). Paradoxically, however, such adaptations make it difficult to measure the direct effects of those abiotic challenges on the organisms in question; they have adapted to avoid or tolerate the costs of exposure to the challenge in question, thus reducing their costs. Ideally, we need to measure such costs when organisms encounter the relevant challenges for the first time, before adaptive changes blunt the impact of the environmental force. Invasive species present an ideal opportunity for such studies. Understanding how taxa have flourished in places far removed (ecologically as well as geographically) from the places in which they evolved may clarify not only the costs of specific environmental challenges but also the determinants of invasion success (Romanuk et al. 2009), the eventual extent of the invaders’ spread (Urban et al. 2007; Kearney et al. 2008) and mismatches that may render the invader vulnerable to targeted control measures within its new range (Ward-Fear, Brown & Shine 2010; Florance et al. 2011).
Organisms that depend upon specific environmental features provide ideal systems with which to explore this issue. For example, amphibians are highly dependent upon water balance with the environment (Jørgensen 1997; Wells 2007). Many anuran species lose water at the same rate as does a free water surface (Lillywhite 2004, 2006; Young et al. 2005), a sensitivity that drives major ecological patterns such as spatial and temporal fluctuations in amphibian activity (Lillywhite & Mittal 1999). Anurans that evolve in dry regions exhibit a suite of behavioural, physiological and life-history traits that are adaptive to this hydric challenge: for example, they are inactive during dry periods, reduce rates of water loss by behavioural and physiological tactics and can initiate breeding rapidly if unpredictable rainfall events provide potential spawning sites (Tracy et al. 2007; Wells 2007; Hillman et al. 2009). Those distinctive characteristics suggest that dry environments impose strong selection on anurans; and hence, we would expect an anuran species from a wetter, less seasonal area to be ecologically and physiologically challenged by more extreme seasonal aridity.
In the current paper, we summarize data from field studies on an invasive anuran (the cane toad, Rhinella marina Linnaeus 1758) whose spread through Australia has taken it into areas with more arid conditions than occur in the species’ native range in South and Central America. How have cane toads responded to the divergence in rainfall regimes between their native range and their current invasion front in Australia? One way to answer that question would be to compare features of cane toad ecology in different places: for example, in the native range vs. the new range or in mesic vs. arid sites within the invaded range. However, any geographic differences might well reflect other aspects of local environments. A more direct comparison is possible: within the highly seasonal wet–dry tropics, we can compare the ecology of cane toads during the wet, putatively benign (ancestral) part of the year vs. the dry, putatively challenging (newly encountered) part of the year. Native frogs in this area show a wide range of adaptations to avoid or mitigate dry season stresses (Young et al. 2005), so how does an anuran that has evolved in relatively aseasonal hydric conditions deal with the seasonal advent of drought?
Although the cane toad is endemic to well-watered areas within the American tropics, some parts of its range exhibit seasonal rainfall regimes (Fig. 2). Adaptation to seasonally arid conditions may have been important in the evolutionary history of this clade (Zug & Zug 1979). The magnitude and duration of seasonal drought are, however, much greater in many of the areas to which this species has been translocated (Lever 2001; Kraus 2009). The most dramatic example comes from Australia, where toads were released in 1935. These animals were the descendants of specimens collected in French Guiana and thence introduced to Jamaica, then Puerto Rico and Oahu. In 1935, 101 toads were translocated from Oahu to Queensland, Australia, bred in captivity and their progeny (as toadlets) released into the wild (Lever 2001; Turvey 2009). The toad’s success in the island sites may reflect a broad similarity in rainfall regimes to its ancestral homeland (see Fig. 2 for a comparison of rainfall seasonality across these sites along the cane toads’ translocation history). Although seasonally arid conditions do occur within the cane toad’s native range, the dry seasons encountered in Australia are typically more prolonged and extreme. That seasonality becomes more marked as one moves west across the continent (as the toads have done during their invasion), so that toads in the Darwin area experience dry conditions for most of the year (80% of annual rainfall occurs within a 4-month period: December–March; see Figs 2 and 3).
Study Area and Climate
The Adelaide River floodplain is located 60 km southeast of Darwin in the Northern Territory of Australia (12°38′S, 131°19′E). It is a relatively flat, treeless plain formed by silt deposition from the Adelaide River. The study area is in the wet–dry tropics of northern Australia and experiences a highly seasonal cycle of rainfall and humidity. Temperatures are high year-round, with monthly average maxima above 30 °C in every month; minima range from 15 to 24 °C (Fig. 3). We divided the year into four three-month seasons based on precipitation: wet season (December–February), wet–dry transition season (March–May), dry season (June–August) and dry–wet transition season (September–November). These seasons also differ in mean minimum temperatures, but less so than for precipitation (Fig. 3).
Toads have been spreading across tropical Australia since 1936 and began to arrive at the eastern edge of our study site during February 2005. We immediately initiated an ongoing survey, mark–recapture and telemetry study on them. Data in the present study span the period from February 2005 to February 2010.
Surveys of Encounter Rate
Toads on roadways were counted from a vehicle and on foot, along two roads (3·0 and 6·4 km in length), passing through woodland and floodplain habitat. Road surveys were conducted, on average, 25 nights per month (range 10–31 nights) over 5 years.
Throughout each year, we hand-captured and marked toads from areas on or adjacent to the Adelaide River floodplain. Toads were held overnight in cloth bags, and in the next morning, these toads were measured for snout-to-urostyle length (SUL), head width, length of the right tibia and body mass. The three linear body measures were used as variables in a principal component analysis, and the first principal component (PC1), which described overall body size, was used in subsequent growth rate calculations. Toads were then individually marked by clipping the distal-most phalange of up to two toes per foot and then released at their point of capture the next evening. Sexes were differentiated based on skin texture and colour (males tend to be yellow rather than brown and to have spinose dorsal skin) and the presence of nuptial pads or release calls by males. Individuals smaller than 80 mm SUL lack secondary sexual characteristics and thus were classed as juveniles.
To calculate the rates of toad growth within each season, we restricted analyses to recapture periods between 30 and 90 days. We modelled growth rates for linear measurements and body mass separately. Because the rate of growth over an interval of time typically is negatively related to body size at the beginning of that interval (i.e. larger animals grow more slowly), we included initial size in our measures of relative growth rate (residuals from regressions of growth rate in body length [PC1] on initial PC1 and growth rate in body mass on initial mass).
A total of 229 toads captured during the mark–recapture study were fitted with radiotransmitters (Holohil PD2, 5 g, <5% of toad body mass; see Brown et al. 2006 for detailed methods). Our analyses of seasonal differences in movement only used data from 214 individuals that were tracked for a minimum of 5 days each and only from the first full calendar month for any individuals that were tracked for >31 days. We located each radioequipped toad daily and recorded the GPS coordinates of its location. From these coordinates, we calculated the distance moved between consecutive days. We summed daily movements to calculate the total distance moved per toad and measured its final net displacement (straight line distance from the initial release point to the final location).
Fat Body Mass, Hematocrit and Corticosterone
In October 2008, we began collecting and euthanizing up to 20–30 toads per month from this study site for post-mortem examinations. A blood sample was collected from each toad via cardiac puncture within 3 min of capture, placed into a heparinized microcapillary tubule and refrigerated overnight. The next morning, we spun the tubules for 10 min at 14 000 g to measure hematocrit levels. Immediately after euthanasia, each toad was measured for SUL, tibia length, head width and mass, and the fat bodies were dissected out, patted dry and weighed to 0·001 g. We collected hematocrit data from 391 toads over 13 months and fat body data from 399 toads over 15 months. In addition, we analysed blood samples from 66 of these toads for corticosterone concentration using a commercial enzyme immunoassay kit (Corticosterone HS EIA; ImmunoDiagnostic Systems Ltd, Bolden, UK). These corticosterone assays were based on 5–35 samples from each of the four seasons.
To examine seasonal variation (except for radiotelemetry data), we calculated a mean monthly value for each variable during each year. This averaging resulted in up to five mean values for each month, one for each year of the study. Each month was assigned to one of the four three-month seasons, and we used one-way anovas to compare mean values among the four seasons. Because the movement measures calculated from telemetry data (e.g. total distance moved, final displacement) were dependent upon the number of moves made by the toad (which varied from 0 to 29), we used the number of moves as a covariate in the analyses of seasonal effects. Similarly, we used tracking duration as a covariate when looking at seasonal variation in the proportion of days during which an individual moved. We assessed residuals from all analyses for departures from the assumptions of normality and independence of errors. All analyses were carried out using jmp 7 (SAS 2007).
Rates of Encounter
Toads were not encountered in our surveys of roadways until November 2005, 9 months after they were known to be in the study area. We encountered toads on roadways about three times more often during the wet and wet–dry transition seasons than at other times of year (Table 1, Fig. 4a).
Table 1. Seasonal differences in individual and population variables of cane toads inhabiting a wet/dry tropical habitat in northern Australia
d.f. reflect analyses carried out on monthly mean values.
Significant if P < 0.05. PC, principal component; SUL, snout-to-urostyle length.
Encounter rate (toads per night)
Mean SUL (mm)
Sex ratio (proportion male)
Relative PC1 growth rate
Relative mass growth rate
Residual body mass
Fat body mass (%)
The average body sizes of captured toads varied among seasons (Fig. 4b), with maxima during the wet season and minima during the dry season. These seasonal differences were driven by changes in the proportion of juvenile toads (<80 mm SUL; Fig. 4c). The abundance of these small toads (3–6 months old, based on body sizes and growth rates) in the dry season suggests a concentration of metamorphosis late in the wet season.
Sex ratios of adult toads varied significantly among seasons (Table 1, Fig. 4d). Capture samples were male-biased during the wet season (against a null of 50% male, χ2 = 74·4 d.f. = 1, P <0·0001) but female-biased in all other seasons (wet–dry χ2 = 43·5, d.f. = 1, P <0·0001; dry χ2 = 159·0, d.f. = 1, P <0·0001; dry–wet χ2 = 11·6, d.f. = 1, P =0·0007: see Fig. 4d).
Toads grew more slowly during the dry season than in other times of year, in terms of both body length (Table 1, Fig. 5a) and body mass (Table 1, Fig. 5b).
Body condition varied significantly among seasons (Table 1), peaking during the transition between wet and dry seasons (Fig. 5c). Relative fat body mass also varied significantly among seasons (Table 1, Fig. 5d). Seasonal changes in relative fat body mass broadly mirrored changes in body condition, with both measures lowest during the dry–wet transition and increasing through the wet season to reach maximal levels during the wet–dry transition. However, the decrease in fat body mass during the dry season was more marked than the decrease in body condition.
Water Balance and Corticosterone Levels
Hematocrit levels did not vary significantly among seasons, mean values ranging only from 25 to 27% (Table 1, Fig. 6a).
Mean concentrations of corticosterone (from blood samples collected from free-ranging toads within 3 min of capture) did not differ among the four seasons (Table 1), albeit with a trend for lower values during drier times of year (Fig. 6b).
Cane toad movements differed substantially among seasons. Frequency of movement (proportion of days moved) tended to decrease as toads were radiotracked for longer periods. An ancova correcting for tracking duration indicated a significant difference in the frequency of movement among seasons (season F3,209 = 19·4, P <0·0001; tracking duration F1,209 = 67·8, P <0·0001: Fig. 7a). Differences in the frequency of movement among seasons closely mirrored the trends in encounter rates during road surveys, with higher values during the wet season and wet–dry period than in the other two seasons. After correcting for differences in the number of moves made, final displacement (season F3,209 = 5·34, P =0·0015; number of moves F1,209 = 10·51, P =0·0014: Fig. 7b) and total distance moved (season F3,209 = 7·37, P =0·0001; number of moves F1,209 = 52·33, P <0·0001: Fig. 7c) were highest during the dry–wet transition and the wet season, decreasing during the wet–dry transition and lowest during the dry season.
During wetter times of year, toads moved more often, moved further and displaced further from their initial location (Fig. 7). This tendency for dispersal during wetter periods is reflected in the proportion of capture samples made up of previously marked individuals (Table 1, Fig. 8a). During the wet season, only 12% of captures were of previously marked individuals; this proportion doubled during the dry season.
We radiotracked 229 individual toads for periods of 1–174 days, for a total of 4105 ‘toad days’ of data. Nineteen of these toads (8%) died from apparent predator attacks during the study: eight during the wet season, eight during the wet–dry transition, three during the dry season and none during the dry–wet transition (Fig. 8b). A likelihood ratio test of survival status (alive/dead) vs. season (treating each ‘toad day’ as an independent observation) indicated a significant difference in mortality rates among seasons (χ2 = 10·67, d.f. = 3, P =0·014).
Cane toads experience seasonal variation in precipitation within their native range; indeed, adaptations to deal with seasonally xeric conditions may have played a significant role in the evolution of the Rhinella marina group (Zug & Zug 1979). Nonetheless, that hydric seasonality is more pronounced over much of the toads’ introduced range than in its native range (Fig. 2); and in particular, the toads have encountered progressively more extreme seasonality in precipitation regimes in the course of their invasion through the Australian wet–dry tropics (Fig. 2). For example, the number of months per year with <100 mm of rainfall has more than doubled compared with that experienced in the native range (Fig. 2). The toads have dealt successfully with that challenge, continuing to move through this area at an accelerating pace (Phillips et al. 2007). The current study shows that seasonally dry conditions induce marked seasonal variation in aspects of the toads’ behaviour, physiology and demography, but that the long dry season does not compromise the toads’ ability to maintain hydric balance nor does it impose physiological stress. The toads’ ability to deal flexibly with relatively short-term, relatively mild hydric stress (presumably adaptive to conditions in their ancestral range) has enabled them to cope with longer and more severe drought within their invasive range.
Seasonality in precipitation is a major constraint on anuran activity in the Australian wet–dry tropics. Some native anurans spend the long dry season virtually inactive within shelter sites (Tracy et al. 2007). Other native frogs remain active during the dry season, but restrict their activity to areas where they have access to moisture (Young et al. 2005). Toads adopt this latter strategy and (although lacking the drought resistance physiological adaptations of native species: Lillywhite 2004, 2006; Tracy et al. 2008) can be found on the roads at all times of year (Fig. 4a). Radiotracking studies confirm that lower encounter rates during the dry season reflect lower activity levels: in drier seasons, toads moved shorter distances and moved less often (Fig. 7). Cane toads also modify the types of refugia they use on a seasonal basis, based on moisture-retaining qualities (Schwarzkopf & Alford 1996; Seebacher & Alford 2002). Toads use burrows as refugia more often during the dry season and tend to reuse the same burrow day after day at that time of year (Schwarzkopf & Alford 1996); levels of movement and dispersal also fall during dry conditions (Seebacher & Alford 1999; Schwarzkopf & Alford 2002). These patterns are similar to the ones that we found in the wet–dry tropics: if the surrounding landscape is dry, toads appear unwilling to disperse away from sources of moisture. Widespread rainfall renders the entire landscape available for incursion by toads.
Because our cane toads were sedentary during the dry season, recapture rates were high at this time of year (Fig. 8a). Low activity levels both of the toads and of their insect prey (possibly imposed/enforced by low temperatures and humidity) reduced opportunities for toad feeding, thereby reducing rates of growth in body length and body mass (Fig. 5a,b), as well as energy stores and thus body condition (Fig. 5c,d). Interestingly, body condition and fat bodies were both low during the dry–wet transition season despite high rates of growth; the toads appear to channel energy directly into growth rather than storage at this time of year. This balance then shifts, as energy allocation to storage increases late in the wet season – with the result that the toads accumulate body reserves prior to the challenging dry season ahead.
Poor body condition, low-humidity constraints on activity and a reduced availability of waterbodies all may favour a reduction in reproductive effort during drier times of the year. The apparent seasonal shift in adult sex ratios (Fig. 4d) is a consequence of this reproductive seasonality: the sex of male toads in this area can be reliably determined from external morphology (colour, spinosity, nuptial pads) and behaviour (release calls) only during the (wet season) reproductive period. Outside that period, males progressively lose their secondary sexual characteristics as they become reproductively quiescent and become increasingly difficult to differentiate from females based on external features (Zug, Lindgren & Pippet 1975; Crystal Kelehear unpublished data). Seasonal shifts in mean body sizes and the proportion of juveniles within the population (Fig. 4b,c) also reflect the restriction of breeding activity to hydrically favourable conditions.
The simplistic interpretation of these patterns would be that cane toads are poorly suited to the xeric conditions that apply for most of the year in the Australian wet–dry tropics. However, our data suggest a more nuanced scenario. During prolonged periods of low humidity, the toads modify both their activity levels and the nature of those activities, such that they are less active, feed less often, grow less rapidly and cease reproducing. However, they do not appear to be under direct hydric stress, in that hematocrit levels remain as high during the dry season as during the wet season (Fig. 6a). Hematocrit levels of Rhinella marina are relatively resistant to moderate desiccation (up to 15% of body mass: Hillman, Zygmunt & Baustian 1987; Malvin & Wood 1991) and do not increase proportionally with dehydration until water loss exceeds 24% of body mass (Hillman, Zygmunt & Baustian 1987). Nonetheless, experimental studies show a consistent correlation between hydric status and hematocrit in toads (Konno et al. 2005), and our data suggest that cane toads are not dehydrated during the dry season at our study site.
The toads’ ability to maintain homeostasis reflects the success of behavioural modifications (remaining close to a moist retreat site and foregoing activity during dry conditions; Schwarzkopf & Alford 1996). If toads are able to locate a reliable source of moisture during extended dry periods, then mortality as a direct result of hydric stress is likely to be rare, because the toads can maintain water balance by abandoning activities that would challenge their hydration state. However, that tactic relies upon a continuous moisture supply. Reynolds & Christian (2009) documented significant desiccation in cane toads found in a drying creekbed in the late dry season. Within our study area, most toads spend the dry season close to permanent water sources and thus may experience such hydric challenges only rarely.
Consistent with the idea that toads cope successfully with the long dry season by down-regulating activity levels, baseline corticosterone levels did not vary significantly among seasons, and indeed, levels of the ‘stress hormone’ tended to be lowest during the driest times of year. Increased activity and reproduction during the wet season may invoke more stress than that of a sedentary lifestyle. More active toads also may be more vulnerable to predation, with rates of mortality highest during the putatively ‘favourable’ wet season (Fig. 8b). Toads are most vulnerable to predators when they are moving, especially when they are far from their usual shelter sites (Phillips et al. 2007, 2008). Predators also may be more active during the wet season.
In summary, intuition suggests that the prolonged arid conditions experienced every year in the Australian wet–dry tropics would pose a formidable challenge to an invasive anuran from a less seasonal environment. Anurans are highly dependent upon substrate moisture to maintain hydric balance, and most native anurans in the wet–dry tropics show seasonal inactivity to avoid the harshly desiccating dry season (Young et al. 2005; Tracy et al. 2007). Contrary to that scenario, invasive cane toads have thrived in the wet–dry tropics, by flexibly modifying their behaviour (amount, location and type of activity) to wait out the long periods when the broader landscape is hostile. That flexibility (perhaps reflecting adaptations to dealing with briefer dry periods in their native range) has enabled the toads to persist in a landscape that provides favourable conditions for feeding and reproduction only briefly.
More generally, the cane toads’ invasion of seasonally arid areas in Australia provides a powerful example of the ways in which a capacity for behavioural flexibility can enable an organism to avoid physiologically stressful conditions and thus thrive in an environment that provides ‘unsuitable’ conditions over most of the landscape for most of the year. Although many organisms surmount environmental challenges in this way (Pigliucci 2001; West-Eberhard 2003), invasive species offer some of the best opportunities to explore the role of behavioural plasticity in dealing with environmental challenges. In cases where the conditions encountered within the newly colonised range fall well outside those typical of the species’ ancestral range (such as the cane toad in Australia), we can look both at the organisms’ immediate response (mediated through plasticity) and its longer-term adjustments (mediated through genetically based shifts). Thus, for example, it would be of great interest to compare the traits we have measured in toads that are in the process of colonising a seasonally arid site, to the same variables in conspecifics in long-colonised areas where the animals have had decades to adapt to seasonally arid conditions. Plasticity may be so effective in meeting the novel challenges that there is little or no longer-term adaptive shift, or alternatively, challenges initially met by plasticity may subsequently be dealt with by genetically canalised solutions (genetic assimilation: see Aubret & Shine 2009). The plethora of invasive taxa, in diverse ecosystems across the globe, provides abundant material with which to explore the ways in which organisms respond to novel environmental pressures.
We thank Beatrice Hill Farm for access to the study area, the Northern Territory Land Corporation for housing, Christine Rioux for laboratory assistance, Team Bufo for discussion and comments and the Australian Research Council for funding. The research was carried out under permits issued by the Northern Territory Parks and Wildlife Commission and with approval of the University of Sydney Animal Care and Ethics Committee (L04/4-2009/3/4999).