Journal of Thermal Biology 60 (2016) 237–245

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Journal of Thermal Biology

journal homepage: www.elsevier.com/locate/jtherbio

Thermal tolerance in the Andean toad Rhinella spinulosa (Anura:

Bufonidae) at three sites located along a latitudinal gradient in Chile
Nicza Alveal Riquelme a,b,n, Helen Díaz-Páez a, Juan Carlos Ortiz b
a
Departamento de Ciencias Básicas, Campus Los Ángeles, Universidad de Concepción, Casilla 341, Los Ángeles, Chile
b
Departamento de Zoología, Campus Concepción, Universidad de Concepción, Casilla 160-C, Concepción, Chile

art ic l e i nf o a b s t r a c t

Article history: Rhinella spinulosa is one of the anuran species with the greatest presence in Chile. This species mainly
Received 4 January 2016 inhabits mountain habitats and is distributed latitudinally along the western slope of the Andes Range.
Received in revised form These habitats undergo great temperature fluctuations, exerting pressure on the amphibian. To identify
15 July 2016
the physiological strategies and thermal behavior of this species, we analyzed the temperature variables
Accepted 18 July 2016
CTmin, CTmax, TTR, τheat, and τcool in individuals of three sites from a latitudinal gradient (22°S to 37°S).
Available online 21 July 2016
The amphibians were acclimated to 10 °C and 20 °C and fed ad libitum. The results indicate that the
Keywords: species has a high thermal tolerance range, with a mean of 38.14 7 1.34 °C, a critical thermal maxima of
Andean toad 34.6–41.4 °C, and a critical thermal minima of 2.6–0.8 °C, classifying the species as eurythermic. Fur-
Thermal biology
thermore, there were significant differences in CTmáx and TTR only in the northern site. The differences in
Latitudinal effect
thermal time constants between sites are due to the effects of size and body mass. For example, those
from the central site had larger size and greater thermal inertia; therefore, they warmed and cooled in a
slower manner.
The wide thermal limits determined in R. spinulosa confirm that it is a thermo-generalist species, a
characteristic that allows the species to survive in adverse microclimatic conditions. The level of plas-
ticity in critical temperatures seems ecologically relevant and supports the acclimatization of thermal
limits as an important factor for ectothermic animals to adapt to climate change.
& 2016 Elsevier Ltd. All rights reserved.

1. Introduction environments, including some that are extremely demanding such

as high altitudes, deserts, and mountains (Angilletta et al., 2002).
Amphibians have notable biological characteristics, including a Chile's geographical position with respect to high pressure
complex life cycle with an aquatic phase and a terrestrial phase, zones, the presence of the polar front, the influence of the ocean,
low dispersion capacity, varied mechanisms of respiration, and and the factors of latitude, altitude, and relief give the country a
high skin permeability (Duellman and Trueb, 1986; Jorgensen, highly heterogeneous environment (Ministerio del Medio Am-
1992; Miaud and Merilä, 2001). Due to these characteristics, they biente, 2011). The result of this is that populations of the same
have high sensitivity and a greater risk of extinction in changing species which are widely distributed latitudinally present pheno-
environmental conditions (Angilletta, 2009). The most important typic variations. Thus, natural environmental gradients have a
environmental variable for their subsistence is ambient tempera- strong influence on variation patterns within species (Mizera and
ture, since this affects fitness, survival, growth, and dispersion Meszéna, 2003). Phenotypic variations may be determined ge-
(Angilletta, 2009). The thermal environment that is produced by netically by local adaptation or by phenotypic plasticity due to
the combination of latitude and altitude in a given place de- environmental variation (Conover and Schultz, 1995; Loeschcke
termines the thermal range of a species, generating limitations for et al., 2000; Via and Lande, 1985).
the distribution of ectothermal organisms (Pough and Gans, 1982). An organism's thermal tolerance is positively correlated with
However, it has been reported that anurans have a high capacity to the temperatures of acclimation and acclimatization (Leroi et al.,
adapt, allowing them to inhabit a wide spectrum of thermal 1994); thus, when individuals are constantly submitted to higher
temperatures, they may modify their thermal tolerance to with-
n stand higher temperatures (Angilletta, 2009).
Corresponding author at: Departamento de Ciencias Básicas, Campus Los Án-
geles, Universidad de Concepción, Casilla 341, Los Ángeles, Chile. Geographical studies have demonstrated that organisms of
E-mail address: nicza7@gmail.com (N.A. Riquelme). tropical habitats and low elevations tolerate a smaller temperature

http://dx.doi.org/10.1016/j.jtherbio.2016.07.019
0306-4565/& 2016 Elsevier Ltd. All rights reserved.
238 N.A. Riquelme et al. / Journal of Thermal Biology 60 (2016) 237–245

range in comparison to those which inhabit higher latitudes and 68°12′53.73″W, elevation 2466 m), mean annual temperature of
altitudes. Individuals of higher altitudes and latitudes tend to 12.7 °C; Farellones, Metropolitana Region (33°21′27″S–70°18′14″W,
warm up more rapidly and cool down more slowly than organisms 2392 m), mean annual temperature 7.5 °C; Antuco, Biobío Región
of warmer habits (Addo-Bediako et al., 2000; Ghalambor et al., (37°28′32″S–71°19′10″W, 1409 m), mean annual temperature
2006; Tewksbury et al., 2008), allowing them to take better ad- 5.9 °C (Fig. 1). The three sites were slightly similar in environ-
vantage of the thermal resources at their disposition (Díaz et al., mental conditions, with mean maximum temperature of 22 °C and
1996; Clusella-Trullas et al., 2007). mean minima temperature of 2 °C; however, the northern site
R. spinulosa Wiegmann 1834 is an amphibian present in Chile stands out with both higher and lower temperatures (Table 1).
that has a wide geographic distribution range, from the Altiplano Toads were captured manually using a protocol for infectious
of Peru and Bolivia to the eastern and western slopes of the Andes disease control in amphibians in field studies (Lobos et al., 2011).
Range in Argentina and Chile (Cei, 1962). In Chile, it inhabits from We collected a total of 31 adult individuals, which were trans-
18° to 41°S (Correa et al., 2011); in zones near streams, lakes, and ported in conditioned cages to the Laboratorio de Ecofisiología de
high altitude wetlands, it can be found between 1000 and 4600 m Herpetozoos at the Universidad de Concepción Los Angeles cam-
(Cei, 1962; Veloso and Navarro, 1988). pus (Fig. 2) and quarantined before experimentation.
There are few studies on the dispersal capacity or habitat use of
this species. Previous research has shown that larvae and post- 2.2. Captivity and acclimation
metamorphics are diurnal, whereas adults are nocturnal, resting
under rocks during daytime (Cei, 1962; Lambrinos and Kleier, During the captivity period, groups composed of five in-
2003; Sanabria et al., 2015). It has also been documented that dividuals were placed in 40 cm wide, 42 cm long, and 35 cm high
adults are more terrestrial than aquatic forms (Cei, 1962), whereas aquaria. These were provided with conditions similar to those of
post-metamorphics are always found near water, at distances of their habitats; they were fed ad libitum with worms (Lumbricus
1 m to 15 m from a pond (Gallardo et al., 2011). terrestris) and larvae of Chilecomadia moorei and Tenebrio molitor.
Thermal studies indicate that this species employs different Toads were given individual marks using elastomers in the ventral
strategies of thermoregulation (Pearson and Bradford, 1976; and intermembrane areas of the hind extremities, using the pro-
Sinsch, 1989). It regulates its body temperature using heliothermal tocol of Northwest Marine Technology (2008). We used two ac-
behavior, briefly sunning itself in the mornings (Lambrinos and climation temperatures, 20 °C and 10 °C, which were the tem-
Kleier, 2003; Sinsch, 1991), and thigmothermal behavior, which peratures recorded in the field, and a 10:14 light:dark photoperiod.
helps to minimize evaporative water loss by using humid and
shaded areas in the afternoon (Sinsch, 1989). 2.3. Experiments and laboratory data collection
The wide distributional range of this species covers different
environmental conditions, in some cases of extreme character, Before each experiment, we measured the snout-vent length of
especially in populations inhabiting rather altitudinal environ- each individual (body size) with a digital caliper with 0.01 mm
ments with particularly severe physical and climatic conditions, precision and recorded the mass in a precision digital balance
low temperatures, regular frost, low atmospheric pressure, and (0.01 g) (Cei, 1962). Three replicates were used for the thermal
high solar radiation (Navas, 1999). These climatic factors pose a parameters described below.
challenging thermal environment; therefore, the species should
possess special physiological and behavioral characteristics to 2.3.1. Thermal tolerance
survive under these conditions (Beniston, 1994). Critical thermal maxima (CTmax) is the highest temperature
Moreover, previous studies have demonstrated that organisms that an organism can tolerate. This was calculated using the pro-
show high levels of variability in biological features among po- tocol of Sanabria and Quiroga (2011). Each individual was placed in
pulations that inhabit different ecological conditions (Mayr, 1963; a 2000 ml beaker with humid filter paper which was placed in a
Futuyma, 1998). This geographic variation provides the opportu- YCW-01 temperature-controlled bath and submitted to a gradual
nity to study factors which may cause divergence and differ- increase in temperature from the initial acclimation temperature.
entiation across populations. Specifically, comparing populations We recorded the dorsal temperature with a laser thermometer
exposed to environments generating different selection regimes every 30 s. The bath temperature increased approximately 1 °C
allows the exploration of the extent to which selective pressures every 30 s
result in adaptive responses and the geographic scale at which Critical thermal minima (CTmin) is the lowest temperature that
these phenomena take place (Gomez-Mestre and Tejedo, 2003). an organism can tolerate. This was estimated analogously to the
Common garden experiments can provide a more mechanistic maxima, submitting the organism to a gradual decrease in tem-
understanding of the causes of compositional and spatial variation perature in a freezer and recording the TC every two minutes
in response to warming. Such experiments are useful to determine (Sanabria and Quiroga, 2011). The experimental phase for CTmax
if geographically separated populations respond differently to and CTmin lasted until the individual lost the turning-over reflex;
warming and provide the opportunity to compare effects of their body temperatures were recorded as their CTmax and CTmin
warming on fitness (Pelini et al., 2012). Therefore, we use an ex- (Brooks and Sassman, 1965; Labra et al., 2001).
perimental approach to analyze the effect of temperature accli- Thermal Tolerance Range (TTR) is the range of temperature
mation and geographic origin on the thermal variables of R. spi- defined by the CTmax and CTmin of an organism (Cowles and Bo-
nulosa in a latitudinal gradient. gert, 1944). It was calculated as the difference between CTmin and
CTmax, (TTR¼ CTmax - CTmin).

2. Methodology 2.3.2. Thermal time constants

These constants were estimated using heating (Theat) and
2.1. Study area cooling (Tcool) rates, which are the times individuals need to warm
themselves from 15 °C to 35 °C and to cool from 35 °C to 15 °C,
Sampling was performed in spring and summer of 2012 and respectively (Vences et al., 2002). For Theat, we used a tempera-
2013. We collected from three sites in the western slope of the ture-regulated bath at a constant temperature of 43 °C; the in-
Andes Range in Chile: Katarpe, Antofagasta Region (22°53′22.26″S– dividual was first placed in a beaker with moist filter paper as
 N.A. Riquelme et al. / Journal of Thermal Biology 60 (2016) 237–245 239

Fig. 1. Sites studied of R. spinulosa in Chile.

Table 1 2.4. Statistical analysis

Mean annual temperature, maximum temperature, and minimum temperature at
each of the analyzed sites of R. spinulosa.
We used SPSS version 13 and InfoStat/E version 2012 software
Mean annual mean maximum T° for mean minimum T° for (Di Rienzo et al., 2012) to determine if there were significant dif-
T° (°C) hotter period (°C) colder period (°C) ferences in acclimation temperature between sites in the thermal
parameters of R. spinulosa. We also used a non-parametric AN-
Northern 12.7 23.8  0,1
COVA in the sm ancova package in the R software (Bowman and
Central 7.5 21.2  3.3
Southern 5.9 21.4  1.5 Azzalini, 2014). Since the data did not fulfill the normality re-
quirements of parametric methods, we used the Kruskal-Wallace
test to examine the differences between sites, the Wilcoxon test
previously mentioned. We recorded the TC of the dorsum using a for thermal parameters at different acclimation temperatures, and
laser thermometer every thirty seconds until the body tempera- non-parametric linear regression and ANCOVA with mass and
ture reached 35 °C. For Tcool, we used a refrigerator with a constant
body size as covariates.
temperature of approximately 8 °C and individuals in beakers with
moist filter paper. The Tc was recorded every two minutes with a
laser thermometer until it reached 15 °C. Theat and Tcool were
transformed into thermal time constants (tau ¼ τ) for heating 3. Results
(τheat) and cooling (τcool), respectively. These values were derived
from the slope of ln (Tc  Ta) as a function of time, where Tc is body Populations of R. spinulosa showed significant differences in
temperature and Ta is ambient temperature [Ta (Theat): 43 °C; Ta body mass (H¼269.80; Po 0.0001) and body size (H ¼211.52;
(Tcool): 8 °C] and b ¼  0.4343/τ (Cossins and Bowler, 1987). We Po0.0001) among sites; animals from the center were sig-
performed a linear regression model to determine the heating and nificantly larger than those of northern and southern Chile, with a
cooling rate for each treatment acclimation (°C / min  1). mean length of 82.65 77.74 mm and mean mass of 76.677 8.08 g
240 N.A. Riquelme et al. / Journal of Thermal Biology 60 (2016) 237–245

Fig. 2. R. spinulosa from northern (A.), central (B.), and southern (C) Chile.

Table 2 although the northern population had greater values, these were
Means and standard deviation for the mass and body size at each of the analyzed not significantly different from the other two (Table 4).
sites of R. spinulosa. (Mean7 S.D.). Mean thermal time constants were different among the three
Northern Central Southern
sites, also showing an effect of acclimation temperature. The
northern site had a lower mean τheat (Table 3, Fig. 6), indicating
Mass (g) 277 15.14 76.677 8.08 53.677 9.40 that these toads warm up more quickly than those of the center
Size (mm) 56.63 713.57 82.65 7 7.74 74.94 7 5.44 and south. However, the ANCOVA analysis did not reveal sig-
nificant differences among sites as a function of body mass and
size or at different acclimation temperatures (Table 4). However,
(Table 2). There was a high correlation between body mass and
there was a trend of greater values of τheat when the acclimation
length (rs ¼0.846; P¼0.001), thus only one of these was used as a
temperature was 20 °C, that is, individuals took longer to warm.
covariate in further analyses. The TTR of the species was very wide
Through the calculation of the slope of a straight line fitted to
(more than 37 °C) (Table 3, Fig. 3). The northern population had a
the data, it was determined that R. spinulosa tended to warm up
larger TTR (Table 3) and showed an effect of acclimation tem-
more rapidly than cool down (Figs. 6 and 8). The species heated at
perature on TTR (Table 4). The CTmax showed high temperatures,
1.03 °C to 1.39 °C per minute and cooled at a rate of 0.95 °C to
above 36 °C, with a range of 34.6 °C 41.4 °C. The northern po- 1.18° C per minute. Temperature was then increased at a rate
pulation had higher CTmax than those from higher latitudes (Fig. 4, ΔT ¼1.2699 °C/min  1 at acclimation temperatures 10 °C and
Table 3); it was significantly different at both acclimation tem- ΔT ¼1.2020 °C/min  1 at acclimation temperatures 20 °C (Fig. 7),
peratures (Table 4). The central and southern populations were and average estimated CTmax ¼37 °C lasted 10 min
only significantly different after acclimation at 20 °C (Table 4); the The results for τcool showed that individuals from the north and
southern population had a greater dispersion of these values south cool off more rapidly than those from the center, especially
(Fig. 4, Table 3). when acclimated at 20 °C. The greater values of τcool for in-
Only the northern population showed a significant effect of dividuals from the center indicate that they require more time to
acclimation temperature on CTmax (Table 4). In this way, the CTmax cool (Fig. 8). Nonetheless, the ANCOVA analysis did not reveal
changes from 37.36 71.32 °C to 38.117 0.99 °C. Besides tolerating significant differences between sites. Only the northern and
high temperatures, the results indicate that adults of R. spinulosa southern populations showed differences in τcool due to acclima-
have a mean CTmin of less than 0 °C (Table 3, Fig. 5). However, by tion temperature (Table 4). In addition, the temperature was de-
comparing results of acclimation temperature between sites, creased at a rate ΔT ¼0.9834 °C/min  1 at acclimation

Table 3
Effects of location and acclimation temperature on the thermal parameters of R. spinulosa.

Northern (n¼ 10) Central (n ¼ 5) Southern (n ¼16)

10 °C 20 °C 10 °C 20 °C 10 °C 20 °C

CTmax(°C)
Mean 7 SD 37.36 7 1.32 38.117 0.99 36.39 7 1.04 36.277 0.50 36.707 0.77 36.80 7 0.92
(Rank) (35.20–39.80) (37.00/41.40) (34.60/38.40) (35.60/37.00) (35.40/38.80) (34.60/38.80)

CTmin(°C)
Mean 7 SD  1.017 0.52  1.167 0.65  0.89 7 0.47  1.03 7 0.32  0.93 7 0.46  0.89 70.71
(Rank) (  2.40/  0.20) (  2.60/0.00) (  2.40/  0.40) (  1.60/  0.60) (  2.60/0.00) (  2.40/0.80)

TTR
Mean 7 SD 38.377 1.41 39.277 1.22 37.29 7 0.94 37.29 7 0.63 37.63 7 0.93 37.64 7 1.06
Rank (36.00/41.60) (37.00/42.40) (35.20/38.80) (36.20/38.40) (36.40/40.20) (34.80 /40.40)

τheat (τ /min)
Mean 7 SD 6.43 7 2.66 7.107 3.83 9.09 7 3.43 9.57 72.96 8.04 72.88 8.717 2.87
Rank (2.40/13.11) (2.55/21.20) (5.46/16.67) (5.28/14.19) (4.28/13.70) (3.49/13.89)

τcool (τ /min)
Mean 7 SD 11.617 2.84 9.577 2.16 14.047 2.04 14.30 7 3.09 13.81 7 3.03 10.337 3.87
Rank (1.14/14.73) (6.48/14.89) (11.17/16.68) (9.98/17.96) (7.94/19.06) (4.01/16.88)

CTmax: Critical Thermal maximum, CTmin: Critical Thermal minimum, TTR: Thermal Tolerance Range, τheat: Time constant of heating, τcool: Time constant of cooling.
 N.A. Riquelme et al. / Journal of Thermal Biology 60 (2016) 237–245 241

Fig. 4. Critical Thermal maxima (CTmax) in degrees Celsius (°C) in adults of R. spi-
nulosa from northern, central, and southern sites in Chile. White: acclimated to
10 °C and gray: acclimated to 20 °C. Differences for each acclimation temperature
shown with *P o 0.05.

temperatures 10 °C and ΔT ¼1.1623 °C/min  1 at acclimation tem-

peratures 20 °C (Fig. 9).

4. Discussion

The thermal biology of Chilean amphibians has been little ex-

plored (Vidal et al., 2008), indicating that many of the aspects that
link and explain the presence of species in this country's diverse
habitats are little known. Although R. spinulosa is the species for
which there is the most thermal information, no previous research
Fig. 3. Thermal Tolerance Ranges (TTR) in degrees Celsius (°C) of R. spinulosa from has studied the influence of geographic origin and acclimation
the northern, central, and southern sites. A. acclimation to 10 °C and B. acclimation temperature on its thermal tolerance parameters.
to 20 °C. Differences for each site are shown with *P o0.05. According to Angilletta (2009), variations in an amphibian's

Table 4
Results of statistical analysis of the thermal parameters of R. spinulosa.

Thermal variable Acclimation temperature Wilcoxon

10 °C 20 °C

CTmáx H ¼6.57 H ¼ 37.73

PN-C ¼ 0.02* PN-C ¼1.8E-08** N: Z ¼  2.33, P ¼ 0.02*
PN-S ¼ 0.05* PN-S ¼ 1.8E-06** C: Z ¼  0.44, P ¼ 0.66
PC-S ¼ 0.42 PC-S ¼0.04* S: Z ¼  0.56, P ¼ 0.57

CTmin H ¼1.13 H ¼ 3.85

P ¼0.55 P ¼ 0.1419 N: Z ¼  0.98, P ¼0.32
C: Z ¼  1.20, P ¼ 0.22
S: Z ¼  0.66, P ¼0.50

TTR H ¼7.55 H ¼ 32.66

PN-C ¼ 0.02* PN-C ¼7.7E-07* N: Z ¼  2.33, P ¼ 0.02*
PN-S ¼ 0.02* PN-S ¼ 1.5E-06* C: Z ¼  0.44, P ¼ 0.66
PC-S ¼ 0.53 PC-S ¼0.21 S: Z ¼  0.56, P ¼ 0.57

τheat H ¼0.15 H ¼ 0.15

P ¼0.4123 P ¼ 0.2948 N: Z ¼  0.57, P ¼ 0.56
C: Z ¼  0.70, P ¼0.48
S: Z ¼  0.66, P ¼0.50

τcool H ¼0.15 H ¼ 0.15

P ¼0.4811 P ¼ 0.3368 N: Z ¼  2.64, P ¼ 0.00*
C: Z ¼  0.08, P ¼0.92
S: Z ¼  4.00, P ¼ 0.00*

CTmax: Critical Thermal maximum, CTmin: Critical Thermal minimum, TTR: Thermal Toler-
ance Range, τheat: Time constant of heating, τcool: Time constant of cooling, N: North, C:
Center and S: South. H and Z Critical values for statistical tests Kruskal Wallis - nonpara-
metric Ancova and Wilcoxon, respectively, P: probability value.
*
Significant.
**
Highly significant.
242 N.A. Riquelme et al. / Journal of Thermal Biology 60 (2016) 237–245

Fig. 5. Critical Thermal minima (CTmin) in degrees Celsius (°C) in adults of R. spi-
nulosa from northern, central, and southern sites in Chile. White: acclimated to Fig. 8. Time constant of cooling (τcool) in adults of R. spinulosa from northern,
10 °C and gray: acclimated to 20 °C. central, and southern sites in Chile. White: acclimated to 10 °C and gray: acclimated
to 20 °C. Differences for each acclimation temperature shown with *Po 0.05.

necessary to regulate experimental protocols. Rezende et al. (2011)

indicate that the development of lengthy experiments can affect
the metabolic conditions of individuals and cause thermal re-
sponses to be influenced, mainly at CTmax and CTmin. In this con-
text, it is important to emphasize that in our experiment, there
was variation in the time taken by each individual to achieve
critical temperatures, despite having maintained a similar level of
hydration for each individual. For amphibians, an increase in
temperature has been shown to increase metabolic rate (Pough,
1980; Rome et al., 1992). Thus, in the absence of compensatory
measures such as behavioral thermoregulation, warming the
thermal environment of an amphibian will cause an increase in
metabolic rate and overall energy expenditure. In turn, this creates
an ecological trade-off that may influence growth or other com-
Fig. 6. Time constant of heating (τheat) in adults of R. spinulosa from northern,
ponents of the energy budget that contribute to an individual's
central, and southern sites in Chile. White: acclimated to 10 °C and gray: acclimated fitness (Sears, 2005; DuRant et al., 2007). The results show that to
to 20 °C. incorporate the thermal tolerance of environmental effects (such
as climate change), experiments should be performed quickly and
thermal characteristics are related to the climatic characteristics of should include conditions resembling environmental tempera-
their habitats. In this sense, in order to determine the effect of tures. In this common garden experiment, we incorporated tem-
plastic responses to thermal acclimation or heritability, it is peratures similar to the environment via two-acclimation

Fig. 7. Heating rate (°C/min  1) in adults of R. spinulosa from northern, central, and southern sites in Chile. Acclimatized in a solid line at 10° and a dashed line at 20° C. A. and
D.: northern; B. and E.: central; and C. and F.: southern.
 N.A. Riquelme et al. / Journal of Thermal Biology 60 (2016) 237–245 243

Fig. 9. Cooling Rate (°C/min  1) in adults of R. spinulosa from northern, central, and southern sites of Chile. Acclimatized in a solid line at 10° and a dashed line at 20° C. A. and
D.: northern; B. and E.: central; and C. and F.: southern.

temperatures. The central and southern sites had well-defined TTR and CTmax
However, the three populations of R. spinulosa studied did not independent of the environmental conditions to which they were
vary in all thermal parameters. The significant differences due to exposed, which is similar to results for the congeneric species R.
geographic origin and acclimation temperature were mostly for arenarum of the Desierto del Monte in Argentina (Sanabria et al.,
the northern population. This population had greater values of 2012). Therefore, this may be a characteristic of the genus. Besides
CTmax and RTT; its capacity to endure higher temperatures is tolerating temperatures above 36 °C, R. spinulosa may withstand
probably a local adaptation to the selective pressures of its habitat temperatures below 0 °C, which they also face as larvae (Navas
(Navas, 1999). Climatic records in sites of northern Chile, such as et al., 2010; Sanabria et al., 2015). This tolerance to low tempera-
San Pedro de Atacama, show the most extreme indexes of ultra- ture allows the species to inhabit and be active at greater altitudes
violet radiation, reaching extreme values during the entire year and latitudes where low temperatures dominate, which may be
(Ecofysvalgesta, 2009). These sites also present the highest T ° reflected in a high capacity for thermal adaptation (Navas, 1999;
average maximum for hotter period (Table 1). This exposition Smith and Smith, 2007). It must be emphasized that acclimation
produces lasting changes in thermal sensitivity and also partially temperature did not affect CTmin as would be expected for a plastic
determines the resistance to extreme temperatures (Leroi et al., character (lower acclimation temperature did not generate greater
1994; Sinclair and Roberts, 2005). The northern population also tolerance to low temperatures), thus it would be interesting to
showed greater phenotypic plasticity, with a positive relationship evaluate the genetic base of this characteristic in future studies.
between acclimation temperature and critical temperatures. In Correa et al. (2010) presented two highly divergent lineages of
aquatic ectotherms, such as amphibian tadpoles, maximum en- R. spinulosa, one from northern Chile and the other from southern
vironmental temperatures (Tmax) can partially explain geographic Chile, each differing in phylogeographic structure. Within the
variation in CTmax (Simon et al., 2015). Northern individuals tol- southern lineage, a group of populations located in the southeast
erate extreme high and low temperatures, but also take less time part of the Salar de Atacama showed the highest genetic diver-
to reach these temperatures. However, the absence of this pattern gence. Correa et al. (2010) explained this pattern by proposing a
in other populations prevents us from affirming that the thermal model of peripatric differentiation for these populations. There-
response of the species is directly related to acclimation tem- fore, it would be interesting to evaluate the population genetic
perature. For this reason, a plausible explanation is that the var- structure of this lineage, considering whether habitat features can
iation determined in TCMax and TCmin for the northern site, where shed light on the conditions that molded the genetic structure of
both Temperature Criticals differ from the other populations these amphibian populations (Gallardo et al., 2011).
analyzed, is a response to environmental conditions of northern Our results are consistent with the description for R. spinulosa
Chile, with both extreme high and low temperatures. Although as a thermos-generalist species, with ecological, behavioral, and
differences in the thermal tolerances of R. spinulosa are only dis- physiological plasticity (Sanabria et al., 2015). The wide TTR of R.
played in the northern population, it is probable that the heating spinulosa suggests that it is a eurythermic species capable of tol-
and cooling rates are affected by the temperature of acclimation. erating a wide range of temperatures that allow it to inhabit areas
Furthermore, this may also be an effect of dehydration produced in where there are strong fluctuations in environmental temperature
both cases. (Addo-Bediako et al., 2000; Ghalambor et al., 2006; Tewksbury
Additionally, there is information indicating that desiccation et al., 2008). These heterogeneous characteristics of the environ-
potentially overshadows thermal acclimation effects as a result of ment produce more generalist organisms (Stillman, 2003) with a
individuals’ short-term responses to changing temperatures, and high capacity for acclimation in comparison to organisms from low
more importantly, that a drop in CTmax with slower heating rate latitudes and altitudes (Calosi et al., 2008; Gilman et al., 2006);
does not necessarily rule out the ‘beneficial acclimation hypoth- this characteristic would favor the species in global climate change
esis’ (Rezende et al., 2011). (Ghalambor et al., 2006; Stillman, 2003). Temperature tolerance
244 N.A. Riquelme et al. / Journal of Thermal Biology 60 (2016) 237–245

constitutes an appropriate tool to explore the possible impacts of and physiological plasticity, allowing it to survive in adverse mi-
global warming due to the fact that it is related to geographic croclimatic conditions. Therefore, this level of CTmax plasticity
variation and to physiological sensitivity (Simon et al., 2015). Low seems ecologically relevant and supports the acclimation of ther-
latitude ectotherms may be more vulnerable to warming than mal limits as an important factor for ectothermic animals facing
temperate species as their upper thermal limits are currently climate change (Simon et al., 2015).
closer to environmental temperatures (Huey et al., 2012).
The thermal time constants of the species also favor its survival
in the environments it inhabits; our results indicate that in- Acknowledgements
dividuals from the three sites studied warm up more quickly and
cool down more slowly (Clusella-Trullas et al., 2007; Díaz et al., The authors thank the Servicio Agrícola y Ganadero (SAG) for
1996), showing a high correlation between acclimation tempera- providing collection permit No. 5496/2013, CONICYT Fellowship
ture and thermal time constants. Similar results have been re- 2120671 and the VRID project 213.413.010-1.0. Lafayette Eaton and
ported for species that inhabit greater altitudes and latitudes Ian Scott translated and provided criticism of the manuscript. We
(Angilletta, 2009). also Marcela Vidal, Claudio Correa Angelo Sandoval, Evelyn Cortés,
The differences in τheat and τcool between sites are a function of Pabla Hernández, Cristian Sáez and Juan Leiva.
body mass and size; these variables significantly affect the ther-
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