Species extinction rates are many times greater than the direst predictions made two decades ago by environmentalists, largely because of human impact. Major concerns are associated with the predicted higher recurrence and severity of extreme events, such as heat waves. Although tolerance to these extreme events is instrumental to species survival, little is known whether and how it evolves in natural populations, and to what extent it is affected by other environmental stressors. Here, we study physiological and molecular mechanisms of thermal tolerance over evolutionary times in multifarious environments. Using the practice of ‘resurrection ecology’ on the keystone grazer Daphnia magna, we quantified genetic and plastic differences in physiological and molecular traits linked to thermal tolerance in historical and modern genotypes of the same population. This population experienced an increase in average temperature and occurrence of heat waves, in addition to dramatic changes in water chemistry, over five decades. On genotypes resurrected across the five decades, we measured plastic and genetic differences in CTmax, body size, Hb content and differential expression of four heat shock proteins after exposure to temperature as single stress and in combination with food levels and insecticide loads. We observed evolution of the critical thermal maximum and plastic response in body size, HSP expression and Hb content over time in a warming only scenario. Molecular and physiological responses to extreme temperature in multifarious environments were not predictable from the response to warming alone. Underestimating the effect of multiple stressors on thermal tolerance can lead to wrong estimates of species evolvability and persistence.

Total haemoglobin contentTotal haemoglobin content measured in µmol/L in the 30 genotypes of Daphnia magna studied here at the control temperature (20C) and after exposure to hyper-thermal stress (30C). Genotype ID, population ID, average Hb content across three replicates and log2 fold change of total Hb content between the two experimental temperatures are shown. The Hb data are From Cambronero et al. 2017.Cambronero_etal_Hb.xlsxCTmax and body sizeCTmax and body size measured on the 30 genotypes resurrected from Lake Ring. Data were collected after exposure to temperature by itself (CGE1) and in combination with either food levels (CGE2) or insecticide Carbaryl loads (CGE3). The experimental temperatures are: 18C and 24C. The two nutrient levels are: 0.2 mg C/L and 2.4 mg C/L. The two concentrations of Carbaryl are: 4 µg/L and 10 µg/L. Empty cells are missing data. The data on body size are from Cambronero et al., in review in Scientific Reports.Cambronero_etal_CTmax_bodysize.xlsxHSPs expressionHSPs log2 fold change normalized gene expression of four heat shock proteins (HSP20, HSP60, HSP70, HSP90). The expression of these proteins was measured on genotypes exposed to temperature ramping (TR) treatment immediately after the completion of the common garden experiments conducted by Cambronero et al. (in review) and on the same genotypes not exposed to TR. These included exposure to temperature as single stress (Warming), temperature combined with food limitation (Warming and food limitation), and temperature combined with the insecticide Carabryl (Warming and insecticide). Empty cells indicate missing values.Cambronero_etal_HSP expression.csv