Multiple stressors linked to anthropogenic activities can influence how organisms adapt and evolve. So far, a consensus on how multiple stressors drive adaptive trajectories in natural populations has not been reached. Some meta-analysis reports show predominance of additive effects of stressors on ecological endpoints (e.g. fecundity, mortality), whereas others show synergistic effects more frequently. Moreover, it is unclear what mechanisms of adaptation underpin responses to complex environments.

Here, we use populations of the crustacean Daphnia magna resurrected from different times in the past to investigate mechanisms of adaptation to multiple stressors, and to understand how historical exposure to environmental stress shapes adaptive responses of modern populations. Using common garden experiments on resurrected modern and historical populations, we investigate: i) whether exposure to one stress results in higher tolerance to a second stressor; ii) the mechanisms of adaptation underpinning long-term evolution to multistress (genetic evolution, plasticity, evolution of plasticity); and iii) the interaction effects of multiple stressors on fitness (synergism, antagonism, additivity). We measure the combined impact of different levels of resource availability (algae) and biocides on fitness-linked life history traits and interpret these results in light of historical environmental exposures. We show that exposure to one stressor can alter tolerance to a second stressors and that the interaction effect depends on the severity of either stressor. We also show that mechanisms of adaptation underpinning phenotypic evolution significantly differ in single stress and multistress scenarios. These adaptive responses are driven largely by synergistic effects on fecundity and size at maturity, and additive effects on age at maturity. Exposure to multiple stressors shifts the trade-offs among fitness-linked life history traits, with a stronger effect on Daphnia populations when low resource availability and high biocide levels are experienced. Our study indicates that mitigation interventions based on single stress analysis may not capture realistic threats.

D. magna dormant embryos were revived from a sedimentary archive of Lake Ring, Denmark (55°57′51.83″N, 9°35′46.87″E) (Cuenca-Cambronero, Marshall, et al. 2018; Cuenca-Cambronero and Orsini 2018). Lake Ring has a well-known history of human impacts and experienced three main phases: eutrophication (EP) – 1960-1970 - high concentration of nutrients and low/no concentration of biocides, Pesticide (PP) – 1980- 1990 - high concentration of nutrients and biocides, and Clearwater (CWP) - >1999 - low concentration of nutrients and biocides (Cuenca - Cambronero et al. 2018; Cuenca - Cambronero and Orsini 2018). D. magna were isolated and hatched from each lake phase (hereafter referred to as populations EP, PP, and CWP), and maintained as isoclonal lines in standard laboratory conditions for several generations (16:8 light: dark regime, 10 ˚C and 0.4 mg Carbon/L of Chlorella vulgaris bi-weekly). From each time period, ten distinct genotypes were randomly selected to represent the population from that time to be used in this study (Fig. 1). The sample size per population was chosen based on previous results showing that 10 genotypes are representative of the local genetic diversity (Orsini et al. 2016). At the start of the experiment, the 30 genotypes were transferred to the following conditions for at least two generations to reduce interference from maternal effect and synchronize reproduction: 16:8 light: dark regime, 20 ˚C and fed ad libitum with 0.8 mg Carbon/L of Chlorella vulgaris daily; the growth medium (borehole water, collected from a deep well with stable microbiological composition and protected from rainwater) was renewed three times per week. After purging (grand)maternal effects, clonal replicates of the 30 genotypes were placed in common garden experiments to measure the effects of single and combined stressors on fitness. Due to the large number of genotypes and conditions tested, exposures were run in three batches. Juveniles of 24-48h from the second or following broods of the second generation were individually exposed to two food (algae) levels (0.2 mg C/L and 2.4 mg C/L of C. vulgaris) and two insecticide Carbaryl (Pestanatal) concentrations (4 µg/L and 8 µg/L), as well as to combinations of algae and Carbaryl at the two tested levels (Fig. 1). A control group was used throughout the exposures for posterior normalization of data and consisted of clonal lines of the same 30 genotypes kept in stress-free conditions (i.e. no Carbaryl and fed ad libitum 0.8 mg C/L of C. vulgaris daily). Fitness-linked life history traits were measured in the single and multiple stressor common garden experiments for the duration of each individual’s life cycle (i.e. until all experimental animals released their second brood). The life history traits measured were: i) fecundity, quantified as total number of offspring in first and second brood; ii) size at maturity, measured as the distance between the head and the base of the tail of adult Daphnia (mm); iii) age at maturity, age of release of first brood in the brood pouch (days); and iv) mortality, recorded as the day at which an animal went extinct in the course of the experiment spanning an individual’s life cycle. Experimental animals were observed daily.