Journal of Thermal Biology 48 (2015) 36–44

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Journal of Thermal Biology

http://d0306-45

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n CorrEvolutivEdifício

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

Upper thermal tolerance plasticity in tropical amphibian species fromcontrasting habitats: Implications for warming impact prediction

Monique Nouailhetas Simon n, Pedro Leite Ribeiro, Carlos Arturo NavasDepartment of Physiology, Institute of Biosciences, University of São Paulo, Rua do Matão, travessa 14, CEP: 05508-090 São Paulo, Brazil

a r t i c l e i n f o

Article history:Received 21 August 2014Received in revised form25 November 2014Accepted 13 December 2014Available online 16 December 2014

Keywords:Acclimation scopeAnuran tadpolesAtlantic forestBrazilian CaatingaCTmax

Warming vulnerability

x.doi.org/10.1016/j.jtherbio.2014.12.00865/& 2014 Elsevier Ltd. All rights reserved.

viations: AF, Atlantic forest; CAA, Brazilian Caam; WT, Warming toleranceesponding author. Present adress: Departama, Instituto de Biociências, Universidade de SMinas Gerais, sala 4, CEP: 05508-090 São Pau

a b s t r a c t

Tropical ectothermic species are currently depicted as more vulnerable to increasing temperatures be-cause of the proximity between their upper thermal limits and environmental temperatures. Yet, theacclimatory capacity of thermal limits has rarely been measured in tropical species, even though they aregenerally predicted to be smaller than in temperate species. We compared critical thermal maximum(CTmax) and warming tolerance (WT: the difference between CTmax and maximum temperature, Tmax), aswell as CTmax acclimatory capacity of toad species from the Atlantic forest (AF) and the Brazilian Caatinga(CAA), a semi-arid habitat with high temperatures. Acclimation temperatures represented the meantemperatures of AF and CAA habitats, making estimates of CTmax and WT more ecologically realistic. CAAspecies mean CTmax was higher compared to AF species in both acclimation treatments. Clutches withinspecies, as well as between AF and CAA species, differed in CTmax plasticity and we discuss the potentialbiological meaning of these findings. We did not find a trade-off between absolute CTmax and CTmax

plasticity, indicating that species can have both high CTmax and high CTmax plasticity. Although CTmax washighly correlated to Tmax, CTmax plasticity was not related to Tmax or Tmax coefficients of variation. CAAspecies mean WT was lower than for AF species, but still very high for all species, diverging from otherstudies with tropical species. This might be partially related to over-estimation of vulnerability due tounder-appreciation of realistic acclimation treatments in CTmax estimation. Thus, some tropical speciesmight not be as vulnerable to warming as previously predicted if CTmax is considered as a shifting po-pulation parameter.

& 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The thermal breadths of animal species are typically expressedby the lower and upper thermal limits, widely known as CriticalThermal Minimum (CTmin) and Critical Thermal Maximum (CTmax;Moore, 1939; Huey and Stevenson, 1979). These tolerance limitsare likely to reflect organism thermal ecology, so that broaderthermal breadths are expected for populations/species exposed tohigh seasonal temperature variation, typical of higher latitudes,whereas tropical ectotherms are expected to express narrowerthermal ranges (Janzen, 1967; Levins, 1968). Recently, studies haveshown that upper thermal limits vary to a lesser degree than lowerlimits across latitude and altitude (Gaston and Chown, 1999;Addo–Bediako et al., 2000; Terblanche et al. 2006), and that sig-nificant phylogenetic inertia may account for CTmax variation when

tinga; CTmax, Critical Thermal

ento de Genética e Biologiaão Paulo, Rua do Matão, 277,lo, SP, Brazil.

a large number of species is considered (Kellermann et al., 2012).In addition, more complex models including rain patterns (e.g.,precipitation of the driest month) and behavior (e.g. basking ver-sus non-basking) can explain CTmax variation across a pool ofterrestrial ectotherms (e.g. Clusella-Trullas et al., 2011; Kellermannet al., 2012; Huey et al., 2009). In aquatic ectotherms, such asamphibian tadpoles, maximum environmental temperatures(Tmax) can partially explain geographic variation in CTmax (e.g.Duarte et al., 2012).

Tolerance to temperature constitutes an appropriate tool toexplore the possible impacts of global warming because they re-late to geographic variation and to physiological sensitivity (e.g.Addo-Bediako et al., 2000; Deutsch et al., 2008; Huey et al., 2009;2012; Moritz et al., 2012; Kellermann et al., 2012; Duarte et al.,2012). Low latitude ectotherms may be more vulnerable towarming than temperate species because their upper thermallimits are currently closer to environmental temperatures(Deutsch et al., 2008; Huey et al., 2009; 2012). Testable hypothesesalong these lines require concrete parameters, and two metricshave been proposed (see Deutsch et al., 2008). The first is WarmingTolerance (WT), which is the difference between CTmax and mean

M.N. Simon et al. / Journal of Thermal Biology 48 (2015) 36–44 37

temperature; the second is Thermal Safety Margin (TSM), which isthe difference between optimal temperature (the temperaturemaximizing an ecologically relevant indicator of performance) andmean temperature. WT has also been regarded as the differencebetween CTmax and maximum temperatures (Tmax), under the ar-gument that CTmax is driven by extreme temperature events(Chown et al., 2010; Duarte et al., 2012). Note that these twometrics need to assume specific tolerances for populations orspecies under study, and therefore do not incorporate informationabout the capacity of individuals to adjust thermal tolerances (e.g.acclimation), even though this plastic adjustment constitutes a keymechanism when modelling the impact of global warming on theectothermic fauna (Stillman, 2003; Calosi et al., 2008; Somero,2010).

CTmax and WT vary among species and among environments(Deutsch et al., 2008, Duarte et al., 2012), but the individualplasticity of these parameters should also vary geographically(Janzen, 1967; Ghalambor et al., 2006; Bozinovic et al., 2011).However, data on the plasticity of thermal tolerances are stilllimited and empirical evidence leads to dubious patterns. For in-stance, in two well-studied groups, adult anuran amphibians(Brattstrom, 1968) and fruit flies (Overgaard et al., 2011; Cooperet al., 2012), tropical species/populations display comparableCTmax acclimatory capacity to temperate ones, diverging fromJanzen's predictions. Similarly, whereas Stillman (2003) reportsthat tropical porcelain-crab species that tolerate high tempera-tures have diminished acclimatory capacity, and therefore wouldbe more vulnerable to warming, Calosi et al. (2008) arrives to theopposite conclusion when studying a temperate clade of waterbeetles. Thus, generalizations on the geographical patterns ofthermal plasticity and its implication for species vulnerability towarming are not yet possible. As a step towards a better appre-ciation of this matter, we compared CTmax and its capacity foracclimation in tadpoles of Brazilian anurans from two thermallycontrasting environments, and discuss our findings in terms of theparameter WT. The species selected come from the Atlantic forest(AF) and the Caatinga (CAA), two tropical environments thatcontrast in climate. The CAA is a semi-arid habitat that presentssunlit ponds with higher pond temperatures and possibly morethermal variation in Tmax than the AF, which is characterized byforest ponds and streams with lower maximum temperatures. Thespecies chosen belong to four genera, two of which occur in bothhabitats (Rhinella and Physalaemus). We analyzed the plasticity ofCTmax for individuals exposed to contrasting experimental tem-peratures, within the range of actual field environmental values.We hypothesize that CAA species will display higher CTmaxplasticity and higher CTmax values than AF species. These hy-potheses are based on the expectation that CAA sites exhibithigher and more variable Tmax when compared to AF sites. Forsimilar reasons, and given the physiological challenge of adjustingCTmax to very high values, we expect CAA species to present lowerWT than AF species.

2. Material and methods

2.1. Study sites and environmental temperatures

We chose two tropical habitats that differ in temperature re-gimes and in anuran species composition to delineate ecologicallyrelevant acclimation treatments. Whereas the AF shares the mesiccharacteristics of tropical and subtropical humid forests, the Bra-zilian CAA is a tropical semi-arid biome that exhibit the highestindicators of solar radiation and mean annual temperature,alongside the lowest values of relative air humidity and annualprecipitation in the country (Reis, 1976). In addition, precipitation

is unpredictable and dry seasons may last several months (Nimer,1972). The richness of anuran species also contrasts between thesetwo environments: whereas some 50 species have been reportedfor the CAA with few endemics at time of publication (Trefaut-Rodrigues, 2003), the AF displays around 529 species with 85%endemism (Haddad et al., 2013). We consider the contrast be-tween these biomes as particularly informative for the CAA ap-parently emerged from a subtropical humid forest that wentthrough an aridization process initiated around 10,000 years ago(Bigarella and Andrade-Lima, 1982; Hartwig and Cartelle, 1996; deVivo, 1997; Carnaval, 2001). Specifically for aquatic thermal sites,the CAA presents extreme thermal peaks and thermal variation insunlit open-forest ponds, while the AF has several ponds andstream environments with much less extreme values. Therefore,the fauna that remained in the CAA should have thermal responsescompatible to this climate change.

We monitored sites that were used for reproduction by thespecies studied, as verified by the presence of larvae or eggs. All AFstudy sites were located at the Boracéia Ecological Station (28° 38′S, 45° 52′W), a forest reserve 110 km from the city of São Paulo, SEBrazil. CAA study sites were located near the municipality of An-gicos (5° 39′S, 36° 36′W), Rio Grande do Norte, NE Brazil. Withineach environment, we characterized the thermal heterogeneity ofwater bodies (e.g., waterfall, lake, pond, flooded area, weir or rockypools) used by anurans during the breeding season. To do this, wemeasured temperatures with data loggers (Ibutton, Maxim In-tegrated Products, California, USA) placed at the bottom of thewater bodies and set to register at one-hour intervals from aminimum of 3–4 days for more ephemeral pools in the CAA, up to4 weeks for permanent water bodies, such as lakes and weirs. Onaverage, we measured AF water body temperatures for 22710days and CAA temperatures for 1176 days. In total, we monitored17 and 18 sites in the CAA and AF localities, respectively, 6–12 foreach studied species (species shared several sites). From thesedata, we calculated means, maximum and minimum temperaturesfor each site used by the species and across all sites per species(attaining species-specific data). We also calculated an overalltemperature mean across all water bodies at both environments,which were the basis for the two experimental temperatureschosen to test acclimation scope. Finally, we calculated maximumtemperature coefficients of variation because this variable canrelate to CTmax plasticity, given that higher variability at the moststressful temperature may correlate with the plasticity in thermalresistance (Duarte et al., 2012).

2.2. Study species

We studied larvae of seven anuran species, four from the AF –

Rhinella icterica, Rhinella ornata, Physalaemus cuvieri and Hypsiboasfaber – and three from the semi-arid CAA – Rhinella granulosa,Physalaemus spp. (three species from CAA, see below) and Pleur-odema diplolistris. We preferred larvae over adults because the firsthave small body size granting quick isothermy with their aquaticenvironment (Lutterschmidt and Hutchison, 1997), thereforemaking thermal limits ecologically relevant and precise. Further-more, behavioral thermoregulation is probably impaired in larvaeas compared to adult individuals for two main reasons: first, theheat capacity and conductivity of water is high compared to air orsoil; second, aquatic tadpoles are unable to perform evaporativecooling under heat stress as adults (e.g. Lillywhite, 1971). Thus,selection on both upper resistance and its plasticity may bestronger in aquatic larvae than in terrestrial adult frogs (Feder andHofmann, 1999; Huey et al., 2012). We chose species based onavailability of eggs in the field and on their phylogenetic re-lationships, intending to consider closely related species acrossbiomes, particularly in the genera Physalaemus and Rhinella. In the

Hab

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Species

R. i

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a

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uvie

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aber

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losa

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A)

P. d

iplo

listri

s

10

20

30

40

HabitatAFCAA

Fig. 1. Species-specific thermal data. We monitored the thermal sites of eachspecies with data-loggers set to measure temperatures at one-hour intervals. Thetop and bottom temperatures of the bars indicate the mean maximum (Tmax) andmean minimum (Tmin) temperatures found for each species across all sites. Hor-izontal lines inside the bars are thermal means. It can be seen that species thermalenvironments within each habitat are more similar than thermal variables betweenhabitats.

Table 1Mean comparisons of environmental variables between Atlantic Forest (AF) andCaatinga (CAA) aquatic sites. Values shown are means7s.d. for AF and CAA sitesthat contained tadpoles of the studied species. We tested ean differences withseparate ANOVAs at a 0.05 significance level. F values and the corresponding de-grees of freedom are also shown, and values in bold indicate significant differences.Thab is the mean temperature, Tmax is the maximum temperature and CVTmax is theTmax coefficients of variation.

Thab (°C) Tmax (°C) CVTmax (°C)

AF 20.1þ0.24 30.8þ0.9 0.13þ0.33CAA 27.9þ0.35 35.0þ0.8 0.09þ0.007AF vs. CAA F1,33¼529.26;

Po0.001F1,33¼16.97;Po0.001

F1,5¼4.87;P¼0.07

M.N. Simon et al. / Journal of Thermal Biology 48 (2015) 36–4438

CAA locality, P. cuvieri is sympatric with P. albifrons and P. kruyeri(Rodrigues, MT pers. com.). Neither the eggs nor the larvae ofthese species can be distinguished from each other, and there areno phenological or alternative natural history traits that might aidto differentiate these species (e.g. Barreto and Andrade, 1995; Gallyand Zina, 2013). Hence, we refer to species in this genus as Phy-salaemus spp. (CAA), because we could not maintain larvae toadult stages and confirm species status. This restriction is notcritical in comparing species of the same genera across biomes, sowe included this species in the analysis and discussion of results.Given that we worked with a limited number of species, our dataare not adequate for phylogenetic analysis (Blomberg et al., 2003).However, since species within the same genera are more related toeach other than to any other species in the study, we incorporatedphylogenetic considerations in data analysis by using a topologybased on the tree retrieved from Pyron and Wiens (2011). In thecase of study species that were not analyzed by these authors, weused a closely related one to determine the topology.

The total number of tested clutches for each species variedaccording to the availability of egg masses or strings and the oc-currence of rain cycles and subsequent breeding bouts. Wheneverpossible, we collected and tested more than one clutch per species.For AF species H. faber, we examined four clutches (109 tadpoles intotal), for R. icterica and P. cuvieri, two clutches each (53 tadpoleseach) and for R. ornata, just one clutch (31 tadpoles); for CAAspecies P. diplolistris, five clutches (147 tadpoles), for Physalaemusspp., three clutches (90 tadpoles) and for R. granulosa, just oneclutch (21 tadpoles). The larvae of the latter were difficult tomaintain in the laboratory and some had to be discarded due tofungus contamination. Because R. ornata and R. granulosa wererepresented in this study with only one clutch each, the CTmax

values of these species are preliminary. Accordingly, we excludedthese data from the formal statistical test of CTmax plasticity be-tween habitats, but maintained them in other analyzes in whichwe used mean CTmax values across clutches or correlations be-tween physiological and environmental data.

2.3. Acclimation scope tests

In this experiment, we proposed the use of 20 °C and 30 °C asbaseline thermal conditions for AF and CAA species, respectively,for these temperatures reflect typical environmental conditions(therefore providing baseline measures for CTmax in these species).The mean temperatures among species within habitats were verysimilar (AF: 20.170.24 °C and CAA: 27.970.35 °C), and thereforewe chose the AF and CAA overall means as the acclimation treat-ments. First we transferred field-collected egg strings and eggmasses to aerated 20 L plastic containers with aged tap water. Weplaced these containers inside a climatic room set at 18 °C andilluminated with fluorescent lamps on a 12 L:12 D photoperiod.Under their highest viable temperatures some CAA tadpoles canmetamorphose in one week (field observations), so we targetedthe slowest viable developmental rates to reduce ontogeneticdevelopment during experiments. Although 18 °C is not a commontemperature in the CAA habitat (see Section 3), we observed nodeaths due to developmental failure at this temperature and theacclimation period adopted (described below) was effective ineliciting acclimatory responses in the species. We fed larvae withboiled lettuce ad libitum after they became free swimmers (aroundstage 20, Gosner, 1960). We maintained this protocol until larvaereached Gosner stages 26–30. We did not use tadpoles aboveGosner stage 38 because they may exhibit comparatively reducedthermal tolerance (Sherman, 1980; Floyd, 1983).

We haphazardly collected up to 48 tadpoles from each eggstring or mass. Then, we randomly distributed them into twogroups of 24 individuals. We placed each of these two groups into

one of two 15 L temperature-controlled acclimation water con-tainers (20 °C or 30 °C). In the case of R. ornata and R. granulosa,we respectively assigned only 15 and 10 individuals to each ex-perimental temperature. The larvae were fed ad libitum withboiled lettuce. The acclimation period was three days and aimed toproduce physiological responses while preventing full metamor-phosis during experiments, an important consideration given thatthree days correspond to between one fourth and nearly half themean development time for CAA species such as R. granulosa (12–14 days) and P. diplolistris (6–9 days, field observations). On theother hand, acclimation of physiological function requires time forunderlying processes to occur, but anuran CTmax has been reportedto stabilize after 2–3 days of exposure to a new experimental re-gime (Hutchison, 1961; Brattstrom, 1968). This treatment was va-lidated and, as described in Section 3, was long enough to elicitthermal adjustment in the species tested.

2.4. Thermal limits: CTmax and WT

After the acclimation period, we placed each of two pairs oftadpoles from one temperature treatment inside each of two plasticcontainers with a netted bottom and a thermometer. We placedthese containers inside a temperature-controlled bath at the targettreatment temperature, and left tadpoles undisturbed for 10 min.Then, we gradually increased the water temperature targeting a1 °C/min change rate (measured change ranged from 0.93 to0.88 °C/min.). This rate is acute in comparison with field heatingrates, so it informs about baseline thermal limits and allows forample comparison with the literature, but does not inform aboutecological thermal limits, that is, those measured at ecologically

4042

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Fig. 2. Clutch thermal reaction norms for each species. We describe the thermal reaction norms by their intercepts and slopes. The variation in slopes shows that there isvariation in CTmax or WT plasticity inside species. R. icterica, H. faber and P. cuvieri are Atlantic forest species, whereas Physalaemus ssp. and P. diplolistris are Caatinga species.

M.N. Simon et al. / Journal of Thermal Biology 48 (2015) 36–44 39

relevant rates (Rezende et al., 2011; Ribeiro et al., 2012). We alter-nated the experimental treatments for each CTmax trial to grantsimilar acclimation times at both experimental temperatures.

Tadpoles tended to swim actively during the trials as watertemperature increased. Tadpoles which remained still were gently

stimulated to move by touching them with the thermometer. Inpreliminary tests we failed to detect unambiguously temperature-related muscular spasms, so we focused on the temperature atwhich the locomotor system of tadpoles becomes disorganized(Lowe and Vance, 1955). Accordingly, we ended experiments when

Table 2Inter-clutch variation in the intercepts and slopes of the thermal reaction norms ofspecies from the Atlantic Forest (AF) and the Caatinga (CAA). We obtained the in-tercepts and slopes by linear regressions of CTmax or WT values across the accli-mation treatments for tadpoles belonging to each clutch separately. These para-meters describe the clutches thermal reaction norms shown in Fig. 2. Since WT isdependent on CTmax, the reaction norm slopes are the same for both parameters,that is, plasticity in CTmax or WT is the same for each specific clutch. The tableshows that there is family variation in CTmax and WT plasticity within the species.The last lines in the table show the intercepts and slopes of the AF and CAA species,considering only the species with more than one clutch. The slopes significantlydiffer between habitats as described in the text.

Species Habitat Clutch Intercept (°C) Slope (°C)

CTmax WT

R. icferica AF 1 41.0 9.4 0.082 39.4 8.3 0.10

H. faber AF 1 40.5 8.9 0.092 41.5 9.9 0.063 40.6 9.0 0.104 42.3 10.7 0.01

P. cuvieri AF 1 37.5 8.0 0.152 35.9 6.4 0.20

Physalaemus ssp. CAA 1 36.9 2.9 0.242 38.0 4.0 0.233 38.7 4.7 0.18

P. diplolistris CAA 1 40.7 5.2 0.112 41.3 5.8 0.093 40.9 5.4 0.114 40.5 5.0 0.125 40.1 4.6 0.15

Habitat AF All 39.9 9.0 0.10CAA All 39.6 4.7 0.15

M.N. Simon et al. / Journal of Thermal Biology 48 (2015) 36–4440

tadpoles did not move after five consecutive taps. Immediatelyafter assessing CTmax, we transferred tadpoles to plastic cups withwater at ambient temperature. We validated the CTmax of in-dividuals that remained alive for 24 h after the test and discardedthe ones that died in this period. We calculated WT as the differ-ence between CTmax and Tmax, assuming that CTmax is driven byextreme temperature events (Chown et al., 2010; Duarte et al.,2012). Therefore, for any given species, and for the total length oftime sampled, Tmax was the mean of the highest temperaturesregistered among the sites sampled for each species. We presentthe CTmax and WT data under a reaction norm framework (Schi-litching and Pigliucci, 1998), with lines connecting the mean va-lues obtained at the two acclimation treatments for each speciesand also for each clutch within species. These lines correspond toreaction norms and are depicted by their intercepts and slopes(the last parameter equivalent to the degree of plasticity), obtainedby linear regressions.

2.5. Statistical analyses

In order to explain the variation in CTmax and WT in our ex-perimental design, we constructed a linear mixed-effects modelfor each of the dependent variables. Fixed factors were’Species’,nested within ‘Habitat’ (given that we had different species in eachhabitat, AF or CAA), and ‘Acclimation temperature’ (including allinteractions between these fixed factors). The random factor‘Clutch’, considered as a random draw of clutches from the naturalpopulation of clutches, was nested within ‘Species’. We used the‘lme4’ package (Bates et al., 2014) in R programming environmentand the function lmer to create the models, which strictly were:y�Habitat/Species�Acclimationþ(1|Species:Clutch); y beingeither CTmax or WT. As previously described, we excluded

one-clutch data from this test. The random effect of ‘Clutch’ wastested with a likelihood ratio test (LRT) between the full modelpresented above and a linear model with no random effect, withthe use of bootstrap methods from the ‘RLRsim’ package (Scheiplet al., 2008). The three-way interaction Habitat:Species:Acclima-tion from the full model explicitly tests the difference in CTmax

plasticity between AF and CAA species, i.e. whether the slopes ofthe reaction norms across acclimation temperatures differ be-tween habitats. Its significance was determined by comparing thefull model with a reduced model without the three-way interac-tion using a chi-square distribution test. We did not include bodysize as a covariate in our models because body size range wassmall, ontogenetic impacts could not be precisely measured andfinally because our sample sizes did not allow for full investigationof the impact of this variable (see for example Ribeiro et al., 2012).In addition, previous studies with amphibian larvae (Bury, 2008)and aquatic small organisms (e.g. Ospina and Mora, 2004; Anttilaet al., 2013) showed that body size variation was not related toCTmax variation; and trends generated by the data here presentedare unambiguous for the ontogenetic/mass ranges encompassed inthis study. We analyzed the relations between physiological andenvironmental variables with Pearson product correlations. Weadopted a significance level of 0.05 and performed all analysis andfigures in the R programming environment (R Core Team, 2014).

3. Results

3.1. Species-specific thermal data

The thermal data extracted for each species show that meantemperature and Tmax differ between AF and CAA habitats (Fig. 1and Table 1), whereas Tmax coefficients of variation are similarbetween the habitats (Table 1). Thermal data were comparablewithin habitats, although differences exist among the specific sitesoccupied by species, especially in Tmax. In the AF, P. cuvieri tadpolesoccurred in slightly cooler sites (mean Tmax of 29.575.0 °C) thanH. faber tadpoles (mean Tmax of 31.672.8 °C). In the CAA, Physa-laemus spp. sites presented lower Tmax (34.0þ3.2 °C) as comparedto R. granulosa sites (35.472.8) and P. diplolistris sites(35.572.9 °C).

3.2. Species CTmax, WT and CTmax plasticity

The random factor ‘Clutch’ partially explained CTmax and WTvariation (models with the random factor had a better fit to thedata than linear models with no random factor; CTmax:LRT¼225.94, Po0.001; WT: LRT¼122.0, Po0.001). The varianceexplained by ‘Clutch’ was 1.12 times higher than the residualvariance for CTmax, but was nearly half the residual variance forWT (for 16 clutches in total). The reaction norms for the inter-clutch variation in CTmax and WT can be seen in Fig. 2, and wedescribe their parameters in Table 2. AF and CAA species differedin their CTmax and WT plasticity (three-way interaction Habitat:Species:Acclimation was significant when compared to the re-duced model without this interaction; CTmax: χ2¼21.572,Po0.001; WT: χ2¼22.124, Po0.001). The reaction norms of theAF and CAA species (without R. ornata and R. granulosa) can beseen as the thick lines in Fig. 3A for CTmax and in Fig. 5A for WT(we present intercepts and slopes in the end of Table 2), as well asthe reaction norms of all the species in thinner lines. We show thespecies reaction norm slopes in a phylogenetic context in Fig. 3B.CAA species had higher CTmax values in both acclimation tem-peratures and lower WT values than AF species (Table 3). CTmax

was highly correlated with Tmax for the 30 °C treatment (r¼0.96,P¼0.005). This positive relationship with Tmax was marginally

Fig. 3. Species CTmax thermal reaction norms (A) and CTmax plasticity distribution in the species topology (B). (A) Thinner lines correspond to the thermal reaction norms ofeach species studied. The slopes of these lines are equivalent to the CTmax plasticity of each species. R. granulosa, Physalaemus ssp. and P. diplolistris are Caatinga (CAA)species, and the other four are Atlantic Forest (AF) species. Solid thick line is the CAA species thermal reaction norm (without R. granulosa data, which had only one clutch)while dashed thick line is the AF species norm (without R. ornata data, which also had only one clutch). The divergence between their slopes, that is, divergence in CTmax

plasticity between species of both habitats, was detected as significant in the mixed-effects model. (B) We show CTmax plasticity values for each species considering theirphylogenetic relatedness. The topology was based on the phylogenetic tree of Pyron and Wiens (2011). Dashed lines are for CAA species while solid lines are for AF species.

Table 3Mean7s.d. of CTmax and WT values of Atlantic Forest (AF) and Caatinga (CAA)species for each acclimation treatment. Mean and s.d. values were obtained fromfour species of the AF habitat and three species from the CAA habitat. Diff indicatesthe difference between CTmax and WT values of CAA and AF species at both ac-climation temperatures.

CTmax (°C) WT (°C)

20 °CAF 41.171.5 10.371.3CAA 42.770.5 7.870.3Diff 1.6 1.5

30 °CAF 42.570.5 11770.5CAA 44.370.1 9.370.8Diff 1.8 2.4

M.N. Simon et al. / Journal of Thermal Biology 48 (2015) 36–44 41

significant for CTmax obtained at 20 °C treatment (r¼0.72, P¼0.06;Fig. 4A). CTmax plasticity was not correlated with Tmax (r¼�0.13,P¼0.77; Fig. 4B) or with species-specific Tmax coefficients of var-iation (r¼0.2, P¼0.50; Fig. 4C). Yet, we detected a marginallynegative relationship between CTmax plasticity and CTmax at the20 °C treatment only (20 °C: r¼�0.74, P¼0.05; 30 °C: r¼�0.14,P¼0.74). Finally, WT absolute values were uncorrelated with CTmax

plasticity (20 °C: r¼�0.44, P¼0.32; 30 °C: r¼0.11, P¼0.80;Fig. 5B).

4. Discussion

Our data show that CAA species display higher CTmax plasticitythan AF species. Yet, this result needs to be interpreted withcaution because: (1) The difference in slopes of the CAA and AFspecies reaction norms is only 0.05 °C, and with current data wecannot evaluate the ecological significance of this difference (al-though we found higher differences in slopes within species, seeTable 2); and (2) when including the one-clutch species R. gran-ulosa and R. ornata, the difference in CTmax plasticity drops to0.03°C. Also, most of the tropical anuran species that we studiedcan acclimate their CTmax independent of the habitat of origin,agreeing with other studies that did not find differences in upperthermal limits plasticity and latitude (Brattstrom, 1968; Overgaardet al. 2011, Cooper et al., 2012). Phylogeny seems not to influenceCTmax plasticity, a result that could be validated in the future withadditional species as it might not be a general pattern. For ex-ample, phylogenetic relatedness does account for some CTmax

variation in fruit flies (Kellermann et al., 2012), so its evolution isprobably influenced by the evolutionary history of given lineages.When considering the absolute mean change in CTmax across theacclimation treatments ([CTmax at 30 °C] – [CTmax at 20 °C]) insideeach habitat (AF: 1.4 °C and CAA: 1.6 °C), this overall plasticity ofCTmax approximates the difference in mean CTmax of speciesbetween habitats ([CTmax CAA]�[CTmax AF]: around 1.7 °C, seeTable 3). Therefore, this level of CTmax plasticity seems ecologi-cally relevant and supports the acclimation of thermal limits as animportant factor for ectothermic animals facing climate change

A B

C

Fig. 4. Relationships between species CTmax and Tmax (A); CTmax plasticity and Tmax (B); and CTmax plasticity and Tmax coefficients of variation (CVTmax) (C). (A) Circles areCTmax values of all the species at 20 °C (light gray) and 30 °C (dark gray) experimental treatments in relation to species sites Tmax. The correlation between CTmax and Tmax

was very high and significant at 30 °C (r¼0.96, P¼0.005) and is indicated by the solid line. At the 20 °C treatment, there was a marginally significant positive relationshipbetween CTmax and Tmax (r¼0.72, P¼0.06). (B) Triangles and diamonds are species CTmax plasticity values for AF species and CAA species, respectively, in relation to speciesTmax. We found no significant relationship between CTmax plasticity and Tmax. (C) There was also no relationship between species CTmax plasticity and CVTmax.

A B

Fig. 5. Species WT thermal reaction norms (A) and relationship between WT and CTmax plasticity (B). (A) WT was the difference between CTmax and Tmax of each species.Thinner lines correspond to the thermal reaction norms of each species studied. Solid thick line is the CAA species thermal reaction norm (without R. granulosa data, whichhad only one clutch) while dashed thick line is the AF species norm (without R. ornata data, which also had only one clutch). The divergence between their slopes, that is,divergence in WT plasticity between species from both habitats was significant in the mixed-effects model. (B) Circles are species WT values at 20 °C (light gray) and 30 °C(dark gray) experimental treatments in relation to species CTmax plasticity. There is no significant relationship between WT and CTmax plasticity.

M.N. Simon et al. / Journal of Thermal Biology 48 (2015) 36–4442

(Stillman, 2003; Calosi et al., 2008; Somero, 2010). Supportingother empirical findings (e.g. Calosi et al., 2010) our data does notsuggest any trade-off between CTmax and CTmax acclimation, thatis, species with high CTmax can also have high CTmax plasticity. Inour dataset, this is the case for R. granulosa and Physalaemus ssp.(CAA). Contrary to theoretical predictions that plasticity is drivenby environmental variation (e.g. Schlitching and Pigliucci, 1998),CTmax plasticity was not explained by species-specific variation in

extreme temperatures (Tmax coefficients of variation). Yet, thisdiscrepancy might be due to the similar Tmax variability betweenAF and CAA sites, which did not differ as much as we had originallyexpected (although CAA sites display smaller thermal amplitudes,see Fig. 1).

Our data suggests that variation in the plasticity of CTmax differsamong clutches within species. One likely explanation is the ex-istence of family variation in CTmax plasticity that responds to

M.N. Simon et al. / Journal of Thermal Biology 48 (2015) 36–44 43

natural selection and is heritable, although families may also beaffected by maternal effects or early environmental parameters.Thus, this issue would require a more intense investigation ofadditive variance in CTmax plasticity in amphibian species. Salmonspecies present family variation in upper thermal limits with asignificant genetic basis (Anttila et al., 2013), thus that may also betrue for amphibian tadpoles. CAA species seem to exhibit adap-tation to high temperatures, given the strong relationship betweenCTmax and Tmax and their absolute CTmax values, which figureamong the highest yet described for anurans (see Brattstrom,1968) and which are only comparable to those of some species ofthe Argentinian Chaco (Duarte et al., 2012) and to juveniles of theR. granulosa from the CAA (Navas et al., 2007). The previouslymentioned toads display diurnal activity, are exposed to very hightemperatures and exhibit mechanisms of enzyme stabilization athigh temperatures (Navas et al., 2007). Other studies have alsofound a correlation between CTmax and Tmax in anurans (Duarteet al., 2012), porcelain crabs (Stillman and Somero, 2000) and bi-valves (Compton et al., 2007); but not in insects or lizards (Addo-Bediako et al., 2002; Huey et al., 2009). Considering that amphi-bians, crabs and bivalves are all aquatic-related groups, it seemspossible that the relationship between CTmax and Tmax is moreimportant for aquatic ectotherms that are more restricted to be-havioral thermoregulation due to less micro-environmental ther-mal variation in aquatic versus aerial environments (Feder andHofmann, 1999; Huey et al., 2012).

CAA species are probably more vulnerable to warming than AFspecies, since their mean WT was lower. However, we report WTvalues for both the CAA and AF species that are remarkably highfor tropical species (as was the case for the sub-humid forest an-urans studied by Duarte et al. (2012)), even though WT did notrelate to CTmax plasticity. This finding highlights differences invulnerability to warming in animal tropical species, which are welldocumented with similar parameters for other animal lineages(Deutsch et al., 2008; Huey et al., 2009; Duarte et al., 2012).However, it should be noted that we used acute CTmax and WT inrelation to extreme temperatures, whereas some authors such asDeutsch et al. (2008) have used growth rates and ambient tem-peratures. Also, we must consider that we used fast ramping ratesto assess CTmax, and these WT values are probably overestimated(Rezende et al., 2011; Ribeiro et al., 2012). Finally, patterns be-tween AF and CAA species might become more complex with theinclusion of more species and more clutches per species. Still, thislower warming vulnerability found might be partially due to ouruse of more realistic acclimation treatments, giving WT measurescloser to what actually happens in the field.

5. Conclusions

Studies focusing on the plasticity of thermal limits (acclima-tion) and warming vulnerability remain rare even though the ac-climatory capacity of individuals representing diverse lineagesseems essential to fine tune predictions regarding warming impactand to give more realistic measures of both thermal tolerances(such as the “thermal range” in the case of Calosi et al., 2010) andwarming vulnerability (Clusella-Trullas and Chown, 2014). As weand other authors have shown, CTmax and WT assessment areplastic traits, and incorporating data derived from acclimationtreatments enhances predictions on warming vulnerability. Mea-suring CTmax potential for acclimation is important because thevulnerability of at least some species displaying high plasticitymight be over-estimated (Clusella-Trullas and Chown, 2014).

Acknowledgments

The authors would like to thank Francisco de Assis and familyfor providing house and field support at the CAA and the ad-ministration of the Boracéia Ecological Station (belonging to theMuseum of Zoology of USP) for allowing this work to be done.The authors also thank Dr. Miguel Tejedo and two anonymousreviewers for their helpful comments in improving the manu-script and Ricardo Nouailhetas Simon for revising the English. M.N.S also thanks Isabel Cristina Pereira for field help at the CAA.License for animal collection was given by ICMBio (SISBIO 12999).License for manipulating vertebrae larvae was given by the In-stitute of Biosciences Ethics Committee for using Vertebrate An-imals in Experiments (protocol 044/2007). This work was fi-nanced by the Fundação de Amparo à Pesquisa do Estado de SãoPaulo [FAPESP 2007/56856-0 to M.N.S.; 2007/57870-6 and 2008/57687-0 to C.A.N.].

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Monique Nouailhetas Simon: Masters degree in 2010at the Physiological Department of the University of SãoPaulo, currently a Ph.D. student at the Genetics andEvolutionary Biology Department of the University ofSão Paulo.

Pedro Leite Ribeiro: Ph.D. degree in 2009, Universityof São Paulo, Brazil, currently post-doctoral fellow atthe University of São Paulo, Brazil.

Carlos A. Navas: Ph.D. degree in 1995, University ofConnecticut, USA, currently Full Professor at the De-partment of Physiology, University of São Paulo, Brazil.