Heat tolerance responses to different thermal selection scenarios in the invasive fly, Drosophila subobscura. Luis E. Castañeda*, Angélica Jaramillo & Andres Mesas Instituto de Ciencias Ambientales y Evolutivas, Universidad Austral de Chile, Chile *E-mail: lecastane@gmail.com Funded by FONDECYT 1140066 Result 2. Correlated response of knockdown time Introduction Most of the evidence for low adaptive potential of heat tolerance comes from slow ramping assays. However, these assays increase the amount of experimental noise (e.g. resource depletion, stochasticity and measurement error), which might be explaining the low adaptive potential estimates for heat tolerance. In the present work, we evaluated the effect of the ramping rate on the evolutionary response of the heat tolerance in Drosophila subobscura. To accomplish this goals, artificial selection on heat tolerance was performed using slow (chronic thermal stress) and fast (acute thermal stress) ramping selection protocols. We also evaluated correlated responses of other heat tolerance estimates measured at four static assays. Using these data, we estimated the thermal death time (TDT) curves for each selection group. TDT curves (Fig. 1) give a more complete and reliable estimate of heat tolerance, and also an unified measurement of chronic and acute heat tolerance (Rezende et al. 2014). LRT: 2(2) = 1.33 p = 0.51 LRT: 2(2) = 25.66 p < 0.0001 Acute-Control: p < 0.001 Chronic-Control: p = 0.001 Acute-Chronic: p = 0.60 LRT: 2(2) = 52.69 Chronic-Control: p < 0.001 Acute-Chronic: p = 0.03 LRT: 2(2) = 4.59 p = 0.10 We found correlated responses of knockdown time for acute and chronic selection protocols. We found significant effect of selection protocol, except at 35 and 38ºC. Selected lines resisted for more time stressful temperatures than control lines. Interestingly, only at 37ºC, acute-selected lines showed longer knockdown times than chronic-selected lines. Figure 1. Thermal tolerance landscapes Thermal tolerance varies with the intensity and duration of a stress, and can be represented as a bivariate plot for a given survival probability (50% in the left panels) or as a landscape describing how survival probability is affected by both factors (below). CTmax z Result 3. Correlated response of TDT curves CTmax Selection: F2,12 = 6.0, p = 0.02 Sex: F1,12 = 0.5, p = 0.59 Interaction: F2,12 = 2.4, p = 0.13 Predicted CTmax for selected lines Predicted CTmax for control lines Thermal sensitivity (Z) Selection: F2,12 = 1.1, p = 0.37 Sex: F1,12 = 0.2, p = 0.66 Left: Correlated responses of CTmax for acute and chronic thermal selection. Significant differences in CTmax were for selected lines, but no for thermal sensitivity (Z). Non-differences were found between acute and chronic thermal selection. Below-left: Possible outcomes of changes in heat tolerance according to Castañeda et al. (2015): (A) only CTmax; (B) both CTmax and Z; and (C) only Z. Below-right: Correlated response of TDT in selected lines was only related to a change in CTmax as described in case w1. Methodology 1) Twelve D. subobscura lines were established and assigned to one of the selection protocols: Fast ramping (3), Control-Fast ramping (x3), Slow ramping (x3), and Control-Slow ramping (x3). 2) Selection was performed measuring heat tolerance with two ramping rates: 0.4 ºC/min (fast  30 min) and 0.08 ºC/min (slow  120 min). Flies within the 30% upper of knockdown temperature values were selected as parents of the next generation. For control lines parents were selected at random. 3) After 17 generations of artificial selection and 6 of relaxed selection, knockdown time was estimated at 35, 36, 37 and 38ºC for each selection protocol. Survival analyses at each temperature were performed to compare differences among selection protocols. 4) Using the TDT approach (Fig.1), we estimated the critical thermal limit (CTmax) and the thermal sensitivity (Z). These values were compared among selection protocols and sexes. Conclusions Heat tolerance evolved in an artificial selection experiment for knockdown temperature, increasing almost 1ºC regarding to control lines after 16 generations of selection. Evolutionary response of knockdown temperature was significantly higher for lines selected in acute than those selected in chronic thermal stress, suggesting that “ecologically relevant” protocols (i.e. slow ramping assays) are underestimating the evolutionary response of heat tolerance. Detection of differences in knockdown time among selection protocols depends on the static temperature assayed, which could unmasks correlated responses of thermal-related traits. TDT curves offer a straightforward approach for the study of thermal tolerance because unified heat resistance to chronic and stressful temperatures. Result 1: Evolutionary response of heat tolerance We found that evolutionary response of knockdown temperature was higher for flies selected in the acute than those selected in the chronic selection protocol. This result suggests that experimental noise related to long, slow ramping assays could be constrained evolutionary change of heat tolerance in lab conditions. For further reading Rezende, EL, LE Castañeda & M Santos (2014) Tolerance landscapes in thermal ecology. Functional Ecology 28: 799-809. Castañeda LE, EL Rezende & M Santos (2015) Heat tolerance in Drosophila subobscura along a latitudinal gradient: contrasting patterns between plastic and genetic responses. Evolution 69: 2721-2734.