DOI: 10.5061/dryad.q83bk3jht
title: Evolutionary mechanisms underpinning fitness response to multiple stressors in Daphnia

description: 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.
description: <i>D. magna </i>dormant<i> </i>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) - &gt;1999 - low concentration of
 nutrients and biocides (Cuenca - Cambronero et al. 2018; Cuenca -
 Cambronero and Orsini 2018). <i>D. magna </i>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 <i>Chlorella vulgaris </i>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 <i>ad
 libitum</i> with 0.8 mg Carbon/L of <i>Chlorella
 vulgaris </i>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 <i>C. vulgaris</i>) 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 <i>fed ad libitum </i>0.8 mg C/L of <i>C.
 vulgaris</i> 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 <i>Daphnia</i> (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.