DOI: 10.5061/dryad.sbcc2frbx
title: Linking critical thermal maximum to mortality from thermal stress in a cold-water frog

description: Estimates of organismal thermal tolerance are frequently used to assess
 physiological risk from warming, yet the assumption that these estimates
 are predictive of mortality has been called into question. We tested this
 assumption in the cold water-specialist frog, Ascaphus montanus. For seven
 populations, we used dynamic experimental assays to measure tadpole
 critical thermal maximum (CTmax) and measured mortality from chronic
 thermal stress for three days at different temperatures. We tested the
 relationship between previously–estimated population CTmax and observed
 mortality, as well as the strength of CTmax as a predictor of mortality
 compared to local stream temperatures capturing varying timescales.
 Populations with higher CTmax experienced significantly less mortality in
 the warmest temperature treatment (25℃). We also found that population
 CTmax outperformed stream temperature metrics as the top predictor of
 observed mortality. These results demonstrate a clear link between CTmax
 and mortality from thermal stress, contributing evidence that CTmax is a
 relevant metric for physiological vulnerability assessments.
description: We sampled tadpoles to ensure that survival was the sole
 contributor to fitness during the experiments. For both dynamic and static
 experiments (detailed below), we used handheld fish nets to collect
 tadpoles from streams. Tadpoles were held in stream water in insulated
 containers during sampling with frequent water changes to maintain
 temperatures. We transported tadpoles to laboratory facilities (Fort
 Missoula, University of Montana) for experiments via the protocol outlined
 in [Essner et al., 2012. Herpetol. Rev. 43, 247–249].    
 <em>Dynamic CTmax Experiments
 (CTmax<sub>p</sub>)</em> See
 manuscript and references therein for full details of CTmax experiments.
 Briefly, we sampled 10–24 tadpoles from each of seven populations of
 <em>A</em>. <em>montanus</em> in Montana. We held
 tadpoles for three days at 8℃, a commonly-experienced temperature among
 populations, without food to reduce the effects of natal stream
 temperature and feeding. For CTmax experiments, tadpoles were moved to an
 experimental tank and held in individual mesh containers. They were given
 two minutes to habituate before temperature ramping at
 0.3℃min<sup>-1 </sup>began. CTmax was defined as the
 temperature at which tadpoles lost the ability to respond to tactile
 stimuli but fully recovered when placed in cooler temperatures. We used
 population median CTmax for analyses, hereafter referred to as
 CTmax<sub>p</sub>, representing previously-characterized
 CTmax. <em>Static Thermal Stress
 Experiments</em> <em>A</em>.
 <em>montanus</em> tadpoles overwinter in their natal streams
 for at least one year [Brown. 1975. Comp. Biochem. Physiol 50A, 397–405].
 To collect tadpoles for static thermal stress experiments, we returned two
 years later (3 July – 28 July, 2019) to substantially decrease the
 probability of sampling the same cohort. Since we permanently removed
 tadpoles from the population for CTmax<sub>p
 </sub>experiments, we did not resample the same individual. We
 collected ~60 tadpoles from each population (total n= 420).
 Tadpoles from each population were evenly and randomly assigned
 to one of five holding temperature treatments: 5℃, 10℃, 15℃, 20℃, 25℃.
 Temperatures were decided based on thermal regime data [Cicchino et al. In
 Press. Freshw. Biol]: 5℃ and 10 ℃ are commonly experienced temperatures;
 15℃ and 20℃ treatments are near maximum stream temperatures; 25℃ is
 greater than current maximum stream temperatures but is ecologically
 relevant given current rates of warming [Isaak et al. 2017. Water Resour.
 Res. 53, 9181–9205]. Tadpoles were held in the temperature treatments for
 three days and fed <em>ad libitum</em> by placing rocks
 collected from their stream in the holding tanks, from which they graze
 algae. We maintained oxygenation using flowing water and bubblers. After
 three days, we counted surviving tadpoles.
 <em>Local Stream Temperatures</em>
 We characterized stream temperatures experienced by the tadpole
 at three timescales: 1) immediate, 2) annual, and 3) long-term/
 multi-generational. To characterize the immediate and annual stream
 temperature metrics, we used quality-controlled [Dunham et al. 2005. USFS
 Tech. Rep. , 1–18] logged temperature data at 4-hour intervals from each
 stream (detailed in [Cicchino et al. In Press. Freshw. Biol]). Using Water
 Year 2018 data, we calculated “immediate thermal experience” by averaging
 maximum daily temperatures of the three days preceding sampling for the
 static mortality experiments. We calculated “annual thermal experience” by
 measuring the maximum temperature experienced in a year for each
 population. Lastly, we used modeled temperature data of 40-year averages
 of the mean temperature during the warmest month (August) for each stream
 (Isaak et al. 2016, 2017) to quantify “long-term thermal experience”.
 These metrics were uncorrelated with each other.
 <em>Data Analysis</em> Analyses
 were performed in R version 4.1.2 [R Core Team 2021]. Statistical
 significance was evaluated using α=0.05. To test differences in mortality
 among temperature treatments, we used a Fisher’s Exact Test for count
 data. Due to limited variation in mortality in other temperature
 treatments, we only investigated the relationship between the probability
 of mortality and CTmax<sub>p</sub> in the 25℃ treatments. For
 this and subsequent models, we used a logistic regression with a 2-column
 matrix of number of deaths and number survived as the response variable.
 To assess the strength of CTmax<sub>p</sub> as a predictor of
 mortality, we compared the performance of this model against ten other
 models: (1) null; each thermal experience metric modelling separately as
 (2–4) independent predictors, (5–7) additive predictors with
 CTmax<sub>p</sub>, and (8–10) interactive predictors with
 CTmax<sub>p</sub>. All models used a binomial distribution. We
 compared models using Akaike’s Information Criterion (Akaike, 1973)
 adjusted for sample size (AICc), AICc weights, and evidence ratios with
 the top model.
 <strong>Ethics</strong> All
 experimental protocols were approved by Colorado State University IACUC
 (16-6667AA) and the University of Montana IACUC (024-17WLDBS-042117).
 Collection was permitted by Montana Fish, Wildlife and Parks (Permit
 2017-060-W).
description: All analyses were performed using R version 4.1.2.