Skin resistance to water gain and loss has changed in cane toads (Rhinella marina) during their Australian invasion

Abstract The water‐permeable skin of amphibians renders them highly sensitive to climatic conditions, and interspecific correlations between environmental moisture levels and rates of water exchange across the skin suggest that natural selection adapts hydroregulatory mechanisms to local challenges. How quickly can such mechanisms shift when a species encounters novel moisture regimes? Cutaneous resistance to water loss and gain in wild‐caught cane toads (Rhinella marina) from Brazil, USA (Hawai'i) and Australia exhibited strong geographic variation. Cutaneous resistance was low in native‐range (Brazilian) toads and in Hawai'ian populations (where toads were introduced in 1932) but significantly higher in toads from eastern Australia (where toads were introduced in 1935). Toads from recently invaded areas in western Australia exhibited cutaneous resistance to water loss similar to the native‐range populations, possibly because toads are restricted to moist sites within this highly arid landscape. Rates of rehydration exhibited significant but less extreme geographic variation, being higher in the native range than in invaded regions. Thus, in less than a century, cane toads invading areas that impose different climatic challenges have diverged in the capacity for hydroregulation.


| INTRODUC TI ON
The integument of most amphibians is highly permeable to water, restricting the activity of these animals to times and places where water is readily available or can be rapidly replenished (Buckley & Jetz, 2007;Lofts, 2012;Vitt & Caldwell, 2013;Wygoda, 1984).
Although an amphibian's behavior, physiology, and morphology all affect the rate at which it exchanges water with the environment (Lofts, 2012;Vitt & Caldwell, 2013), the skin's permeability to water ultimately limits the rate at which water can be gained or lost (Hillyard et al., 1998;McClanahan & Baldwin, 1969;Spotila & Berman, 1976;Tracy, 1975). Some amphibians exhibit "waterproofing" adaptations (such as the skin secretions of Litoria caerulea and Polypedates maculatus, and cocoons of Cyclorana australis) that allow them to survive in arid conditions (Christian & Parry, 1997;Lillywhite et al., 1997), but most amphibians instead exhibit high rates of water flux between the body and the external environment (Lofts, 2012;Spotila & Berman, 1976).
Interspecific correlations between skin permeability and environmental aridity suggest that a species' geographic distribution is constrained by its ability to maintain positive water balance (greater influx of water than efflux) under the conditions it encounters shift when a species encounters novel moisture regimes? Cutaneous resistance to water loss and gain in wild-caught cane toads (Rhinella marina) from Brazil, USA (Hawai'i) and Australia exhibited strong geographic variation. Cutaneous resistance was low in native-range (Brazilian) toads and in Hawai'ian populations (where toads were introduced in 1932) but significantly higher in toads from eastern Australia (where toads were introduced in 1935). Toads from recently invaded areas in western Australia exhibited cutaneous resistance to water loss similar to the native-range populations, possibly because toads are restricted to moist sites within this highly arid landscape. Rates of rehydration exhibited significant but less extreme geographic variation, being higher in the native range than in invaded regions. Thus, in less than a century, cane toads invading areas that impose different climatic challenges have diverged in the capacity for hydroregulation.

K E Y W O R D S
abiotic challenges, Anura, Bufo marinus, physiology, water balance (Seebacher & Franklin, 2011;Tingley et al., 2012). If so, we would predict a biological invasion from one hydric regime to another to impose selection on rates of desiccation and rehydration, and hence on skin permeability. The cane toad (Rhinella marina, formerly Bufo marinus; see Figure 1) provides an ideal model system with which to test that prediction. Native to relatively aseasonal, well-watered landscapes of South America, the species was translocated to much drier habitats on the Hawai'ian islands (Easteal, 1981;Ward-Fear et al., 2016) and then Australia (Easteal et al., 1985;Freeland & Martin, 1985).
Previous research has documented shifts in the species' morphology, physiology, and behavior coincident with translocation (e.g., Gruber et al., 2017;Hudson et al., 2016Hudson et al., , 2017McCann et al., 2014). We therefore quantified rates of evaporative water loss and gain in cane toads from populations within their native range (Brazil), their translocated range in Hawai'i (USA), and their current range in Australia to explore if skin permeability to water also has changed during translocation and range expansion. Specifically, we expected that colonization of seasonally arid environments in Australia would favor a shift toward higher cutaneous resistance to water flow.

| Study species
Cane toads are large "true toads" (family Bufonidae) native to an extensive area of South America (Bessa-Silva et al., 2020;Zug & Zug, 1979), occurring in a variety of habitats including grassland, woodland, sand dunes, rainforest, mangroves, and anthropogenically disturbed areas. Whereas the dorsal skin is tough and leatherlike, the smoother ventral skin (especially the highly permeable pelvic patch) is responsible for most of the organism's water uptake (Parsons et al., 1993). The species' Latin name Rhinella marina, and one of its common names-the marine toad-come from its putative resistance to higher levels of salinity than are tolerated by most other anurans (Liggins & Grigg, 1985;Uchiyama & Konno, 2006;Wijethunga et al., 2016).
These terrestrial anurans were imported from French Guiana to Puerto Rico to control insect pests in sugar-cane plantations; from there, 150 toads were translocated to Hawai'i in 1932, and 101 descendants of the Hawai'ian toads were introduced to northeastern Australia in 1935 (Freeland & Martin, 1985;Lever, 2001;Shine, 2018). Both in Hawai'i and Australia, the toads now occupy xeric as well as mesic habitats. In Hawai'i, the toads were released in plantations on leeward as well as windward coasts, but are currently restricted to anthropogenically moistened areas (such as golf courses) in the drier regions (Ward-Fear et al., 2016). In Australia, toads were released in mesic coastal Queensland (QLD), but have since spread into seasonally arid habitats of Western Australia (WA) and the Northern Territory (NT), as well as colder montane habitats in New South Wales (NSW: for details see Feit et al., 2015;McCann et al., 2014;Newell, 2011;Shine, 2010;Tingley et al., 2012). Cane toads thus occupy a wider range of climatic conditions in Australia than in their native range (Tingley et al., 2014).

| Sampling locations
We collected adult toads (both males and females, ranging from 43 to 313 g; mean = 109.9 g ± 33.5 g SD) from locations in their native range (Brazil), in Hawai'i (USA), and in Australia (see Table 1). All toads were collected by hand at night, placed in damp cloth bags, and kept in a moist, cool environment. Following capture, toads were transported to local laboratory facilities for the experiments.

| Husbandry
After capture, we allowed the toads to acclimate in laboratory conditions for 2 weeks, fed them crickets and mealworms, and provided ad libitum access to water and shelter. The room was set to 25°C, with a 12:12 hr light cycle (for details of methods see Kosmala et al., 2017).
All animals were housed and tested in their respective countries of collection, in near-identical conditions. Prior to measurements, we emptied the toads' bladders by gently applying pressure to the abdomen until urine was released.

| Rates of evaporative water loss and cutaneous resistance
We used a closed system with a positive airflow, with silica gel cylinders at the intake and humidity probes at the outflow of the system (a similar system is described in detail in Young et al., 2005). Dry airflow (<1% relative humidity) was adjusted to 1 L/min, and we recorded baseline humidity values for the system for at least 20 min (after humidity values stabilized) prior to the toads being introduced to the system (average relative humidity 0.048% ± 0.064). Animals were held within an airtight cylindrical container of 1-L capacity. After a toad was placed into the container, we waited for it to adopt a waterconserving posture (WCP; with limbs folded beneath the body: see Withers et al., 1984), then recorded temperature and humidity of air that had passed across the toad's body for at least 20 min (as long as the toad remained in WCP). We then removed the animal from the container, and we continued to record temperature and humidity within the airflow chamber for another 20 min to ensure that baseline values had not changed. After it was removed from the airflow system, the toad was weighed and placed in a water tub in its home cage to rehydrate. We also measured rates of evaporative water loss of agar models (3% concentration) to measure the rate of evaporation from a free water surface (i.e., without cutaneous resistance), so that we could calculate individual cutaneous resistance to water loss (method validated by Spotila &Berman, 1976 andChristian et al., 2017). The agar models were made using molds taken from cane toads in WCP and were placed in the chambers such that the ventral portion was hidden (as is the case for a toad in WCP). Molds were made from 21 cane toads collected from the Darwin region, ranging in size from 9.5 to 365 g, and the water loss from the corresponding agar models was used to calculate total resistance, which corresponds to the boundary layer resistance of a toad of the same size. A regression equation was calculated for the relationship between toad mass and the total resistance of the agar models (resistance = 0.7222 + 0.0104 X mass; F 1,19 = 46.8, p < .001; r 2 = 71.1%), and this relationship was used to calculate the boundary layer resistance for each toad. The variability around the regression line was due, in part, to variability in the relationship between mass and surface area, resulting from differences in body condition. The mean boundary layer resistance was 1.9 s/cm (SD = 0.35), and the range was 1.4 (from the smallest toad) to 4.0 s/cm (from the largest toad).

| Rates of rehydration
Following our measures of water flow, these animals were then used in studies to assess effects of desiccation on locomotor performance TA B L E 1 Collection sites of cane toads used for the current study, with data on year of cane toad introduction and annual rainfall  (Kosmala et al., 2017), which included experimental dehydration in dry air. Prior to all experiments, toads were kept in water for 2 hr to ensure full hydration. The toads were then weighed (after emptying the bladder: see above), placed in desiccating conditions, and reweighed frequently until they reached the desired reduction in mass.
Once it reached 70% of its initial body mass, the toad was weighed, placed in a container with ~0.5 mm depth of water, and reweighed every 2 min (after pat-drying with paper towel) for 14 min to measure rates of rehydration (McClanahan & Baldwin, 1969).

| Statistical analyses
Sites were grouped to form the populations as follows: BR = Alter

| RE SULTS
Preliminary analyses using site of collection as a factor in ANOVA revealed no significant differences in cutaneous resistance among sites within each broader location (country or state), and thus we combined sites within these broader locations for further analysis.
Likewise, cutaneous resistance and rehydration rate did not differ between sexes (both F < 1.55, both p > .22) and thus sex was excluded from further analyses.
There was a weak but statistically significant negative correlation between cutaneous resistance and rehydration rate (Spearman r = −0.19, p = .03).

| Geographic variation in cutaneous resistance
ANOVA with collecting location (country or state) as the factor showed significant geographic variation in cutaneous resistance (F 5,158 = 18.94, p < .001; Tukey post hoc tests identified two groups, with NSW and QLD similar to each other, and both significantly higher than the other four regions). Thus, cutaneous resistance to water loss was relatively low in cane toads from the native range and Hawai'i, high in relatively mesic areas of eastern Australia (NSW and QLD), but low in populations from seasonally arid sites within western Australia (NT and WA) (Table 2, Figure 3a).
There was a significant negative correlation between average cutaneous resistance at each site and the mean temperature re- correlation between average cutaneous resistance and average rainfall (Spearman r = −0.05, p = .88).

| Geographic variation in rates of rehydration
ANCOVA indicated that rehydration rates were positively related to body mass (F 1,124 = 17.65, p < .0001) and also significantly related to population of origin (F 5,124 = 3.70, p < .004). The interaction between mass and location was not significant (F 5,124 = 0.37, p = .87).
An ANOVA on the residuals of the regression of rehydration rates on body mass indicated that rehydration rates for Brazilian toads were higher than for all populations in either of the other two countries ( Figure 3b).
There were no significant correlations between average rehydration rate at each site and the mean temperature or rainfall (both Spearman r < 0.42, p > .18).

| D ISCUSS I ON
The rate at which an anuran loses water from its body is a critical aspect of its biology, restricting the times and places where it can be active (Hillyard et al., 1998;McCann et al., 2014;Seebacher & Franklin, 2011;Toledo & Jared, 1993;Young et al., 2005). We found What mechanisms underlie these shifts in hydroregulatory abilities of cane toads across northern Australia? First, the differences in cutaneous resistance might be the result of evolutionary changes that have occurred as the toads have adapted to the novel environments they have encountered during the westwards invasion of northern Australia. Another possibility is that, rather than genetic differences, the differences among populations are the result of developmentally plastic responses to local environments. A third possibility is that toads from eastern Australia may have exhibited an acute (temporary) physiological response that affected their cutaneous resistance during the period they were measured. Acute changes in water flux have been documented in toads in response to external agents (Dohm et al., 2001) and to pharmacological agents (biochemical blockers and stimulants) that affect cutaneous blood flow (Burggren & Vitalis, 2005;Hillyard et al., 1998). Acute changes to resistance can occur in frogs in response to environmental temperature (Buttemer & Thomas, 2003;Tracy et al., 2006). Because TA B L E 2 Sample sizes, body masses, rates of water loss, skin resistance, and rehydration rates of cane toads used in experimental protocols of the current study (mean we relied on measurements from field-caught animals, we cannot tease apart the degree to which geographic divergence was driven by evolved (heritable) changes versus developmentally plastic or acute responses to local environments. To distinguish among these possibilities, additional field sampling and studies on offspring raised under standardized conditions would be needed.
If the changes in resistance are an evolutionary response, then it could have occurred by one of two patterns: either the change to high cutaneous resistance occurred in Queensland but the trend reversed as the toads moved west over 80 years, or, alternatively, the westbound colonists retained the ancestral state (of low cutaneous resistance) while the toads from eastern Australia evolved high cutaneous resistance later, after the westwards spread of toads had already commenced. Two patterns suggest that the former scenario is more plausible. First, the toad invasion front expanded very slowly in the decades immediately post-translocation (Freeland & Martin, 1985;Kearney et al., 2008;Phillips et al., 2007;Urban et al., 2007), consistent with the time needed for adaptation of traits (such as cutaneous resistance) to deal with novel climatic challenges (e.g., Aikio et al., 2010;Kowarik, 1995 In contrast to cutaneous resistance, rates of rehydration showed a simpler pattern. The rates at which toads regain water have decreased over the course of the toad invasion, with native-range (Brazilian) animals taking up water more rapidly than did those from either Hawai'i or Australia. Thus, mesic conditions within the native range appear to have favored a system of both gaining and losing water rapidly. In contrast, more arid conditions in Hawai'i and Australia have resulted in toads exhibiting greater resistance to water loss in some invaded sites but not others; and a lower rate of rehydration relative to Brazilian toads.
The rate of hydration also depended on body mass, reflecting allometric effects. Relative to internal volume, larger toads have a relatively smaller surface area (across which they gain water) than do smaller conspecifics (McClanahan & Baldwin, 1969;Tracy, 1975;Withers et al., 1984).
Interspecific comparisons suggest that species of anurans exposed to desiccating conditions (e.g., arid or arboreal habitats) tend to exhibit higher cutaneous resistance to water loss (Spotila & Berman, 1976;Tingley et al., 2012;Titon & Gomes, 2017;Wygoda, 1984;Young et al., 2005). This pattern is consistent with the ancestral condition of low cutaneous resistance to evaporative water loss seen in toads from Brazil and the wetter site in Hawai'i.
Although the low resistance to water loss of toads from dry sites  (Figure 2; and see Kosmala et al., 2017 andMcCann et al., 2018 for climatic data). Among the 12 study sites, skin resistance was negatively correlated with average temperature. Thus, evaporative cooling (Borgnakke & Sonntag, 2016;Buttemer & Thomas, 2003;Tracy et al., 2006) might enable toads to deal with otherwise-lethal heat loads (Kosmala et al., 2020). Analogously, Gila monsters increase cutaneous and cloacal evaporative water loss in hot weather (DeNardo et al., 2004). A feedback between body temperature and rates of evaporative water loss (cooler cane toads lose water less rapidly: Malvin & Wood, 1991) might reduce the hydric costs of evaporative cooling.
Our results add two additional variables-rates of water gain and cutaneous resistance to water loss-to the list of functional traits that exhibit geographic variation within cane toads, including across different sites within their invaded range within Australia (e.g., Hudson et al., 2016;Kosmala et al., 2017;McCann et al., 2018). These data demonstrate that anurans can adjust fundamental aspects of their interactions with environmental factors rapidly, if the taxon in question is exposed to abiotic challenges that differ strongly from those which it experiences in the native range.

ACK N OWLED G EM ENTS
We thank Dr. Igor Kaefer, Seu Deco, and the Shine Lab for assistance with collecting toads. We thank Dr. Fernando Ribeiro-Gomes and Dr.
Bill Mautz for logistic assistance. We thank Melanie Elphick for editorial assistance and figure preparation. Collection and transport of toads in Brazil were approved by permit # 7015-1. Transport of toads in Hawai'i was approved by permit #EX-15-12. All procedures were approved by the University of Sydney Animal Ethics Committee (Approval #2014/703). Funding was provided by the Australian Research Council (FL120100074) and the Capes Foundation within the Ministry of Education, Brazil (grant #BEX /13734-13-0).

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data are available at Dryad https://doi.org/10.5061/dryad.ttdz0 8kwc.