Asymmetric micro-evolutionary responses in a warming world: Heat-driven adaptation enhances metal tolerance, but not vice versa

We investigated how prior adaptation to either high temperature or copper (Cu) contamination influences subsequent tolerance to the other stressor in populations of the freshwater zooplanktonic rotifer Brachionus calyciflorus (Pallas 1766). Using an experimental evolution approach, we subjected populations to either gradually increasing Cu levels, elevated temperature, or control conditions over multiple generations. Subsequently, we conducted a common garden experiment to assess the effect of selection history on population tolerance. We found that heat-adapted populations exhibited increased tolerance to Cu, whereas Cu-adapted populations showed no enhanced tolerance to high temperatures.

Methods
We conducted a selection experiment followed by a common garden experiment. In the selection experiment, we exposed 9 genetically identical populations of the freshwater monogonont rotifer Brachionus calyciflorus s.s.to three treatments, i.e. a benign control treatment, a copper addition treatment, and a high-temperature treatment. All populations underwent six cycles (Cycles 1 to 6); during each cycle clonal population growth was followed by sexual reproduction and the formation of dormant propagules. In the copper addition treatment, copper levels were stepwise increased at the beginning of each cycle (from 30, 45, 50, 55, 57.5, 60 to 62.5 ug Cu/L in Cycles 1 to 6, respectively). In the temperature treatment, temperature levels were stepwise increased at the beginning of each cycle (from 24, 28, 30, 32, 35 and 35.5 °C, respectively). Dormant propagules produced during each cycle were stored. 
For the common garden experiment, we used propagules produced at the end of Cycle 6 to established four clonal lines from each population. In addition, we used six ancestral clones (i.e. randomly selected from the set of clones that were used to initiate the selection experiment with). Populations of all clonal lines were subjected to a multifactorial combination of a Cu and Heat treatment, i.e. Control conditions (22 °C, no Cu addition), a Cu treatment (22 °C, 62.5 µg Cu/L), a heat treatment (34 °C, no Cu addition) and exposure to a combination of Cu and heat (34 °C, 62.5 µg Cu/L). After an acclimation phase, demographic variables of experimental populations were monitored for 5 days.