DOI: 10.5061/dryad.5tb2rbp22
title: Data from: Environmental variation mediates the evolution of anticipatory parental effects

description: Theory maintains that when future environment is predictable, parents
 should adjust the phenotype of their offspring to match the anticipated
 environment. The plausibility of positive anticipatory parental effects is
 hotly debated and the experimental evidence for the evolution of such
 effects is currently lacking. We experimentally investigated the evolution
 of anticipatory maternal effects in a range of environments that differ
 drastically in how predictable they are. Populations of the nematode
 Caenorhabditis remanei, adapted to 20°C, were exposed to a novel
 temperature (25°C) for 30 generations with either positive or zero
 correlation between parent and offspring environment. We found that
 populations evolving in novel environments that were predictable across
 generations evolved a positive anticipatory maternal effect, since they
 required maternal exposure to 25°C to achieve maximum reproduction in that
 temperature. In contrast, populations evolving under zero environmental
 correlation had lost this anticipatory maternal effect. Similar but weaker
 patterns were found if instead rate-sensitive population growth was used
 as a fitness measure. These findings demonstrate that anticipatory
 parental effects evolve in response to environmental change so that
 ill-fitting parental effects can be rapidly lost. Evolution of positive
 anticipatory parental effects can aid population viability in rapidly
 changing but predictable environments. 
description: <strong>Experimental evolution</strong><br> As
 founder population, we used the wild-type SP8 strain of <em>C.
 remanei</em>, obtained from N. Timmermeyer at the Department of
 Biology, University of Tübingen, Germany. This strain was created by
 crossing three wild-type isolates of <em>C. remanei</em>
 (SB146, MY31, and PB206), harbour substantial standing genetic variation
 for life-history traits (Chen and Maklakov 2012; Lind et al. 2017), and
 has been lab-adapted to 20°C for 15 generations prior to setup of
 experimental evolution. <br> Experimental
 evolution was conducted for 30 generations in three climate cabinets; one
 set to 20°C, one to 25°C and one with a slowly increasing temperature (see
 below). Five experimental evolution regimes were used, and are summarized
 in Figure 1. Control 20°C was experiencing 20°C for 30 generations, and
 Warm 25°C was experiencing 25°C for 30 generations. Increased warming
 started in 20°C, the cabinet temperature was then raised by 0.1°C every
 2.13 day (rounded to whole days) to reach 25°C the last day of
 experimental evolution. Slow temperature cycles spend their first 10
 generations in 20°C, were then moved to the 25°C cabinet for 10
 generations, to finish the last 10 generations in the 20°C cabinet.
 Finally, the Fast temperature cycles regime were moved between 20°C and
 25°C every second generation, thus experiencing 14 temperature
 shifts.<br> Generation time in 20°C and 25°C was defined as the
 average difference in age between parents and offspring (Charlesworth
 1994) and was calculated from the life-table of age-specific reproduction
 and survival following (McGraw and Caswell 1996) with trial data from the
 SP8 lines, and was 4.0 days in 20°C and 3.4 days in 25°C. This resulted
 120 days of experimental evolution for Control 20°C, 114 days for Slow
 temperature cycles, 110 days for Increased warming and Fast temperature
 cycles and 101 days for Warm 25°C. For the two temperature cycle
 treatments, the worms spent shorter chronological time in 25°C than in
 20°C, because of the faster generation time in 25°C. This ensured equal
 exposure to the two temperatures over biological time. Since we had no
 data for generation time in the intermediate temperatures between 20°C and
 25°C, we did the simplifying assumption that the overall number of
 generations for the whole experiment would be similar in the Increased
 warming and Fast temperature cycle regimes. Therefore, the Increased
 warming regime was also run for 110 days, and the temperature increase was
 determined by the smallest temperature step the cabinet could be
 programmed (0.1°C). <br> Experimental evolution
 was conducted on 92 mm NGM-plates (Stiernagle 2006) and to combat
 infections the agar and bacterial LB also contained the antibiotics
 kanamycin and streptomycin, and the fungicide nystatin (Lionaki and
 Tavernarakis 2013; Lind et al. 2016). The plates were seeded with 2mL of
 the antibiotic resistant E. coli strain OP50-1 (pUC4K) obtained from J.
 Ewbank at the Centre d’Immunologie de Marseille- Luminy, France. To keep
 worm populations age-structured in overlapping generations, the
 populations were always kept in experimental growth face by cutting out a
 bit of agar containing 150 individuals of mixed ages and transferring this
 to freshly seeded plates. Transfer was conducted when needed (every 1-2
 day), always before food was finished. Six independent replicate
 populations of each experimental evolution regime were set up, resulting
 in a total of 30 populations. All populations were expanded for two
 generations and frozen after 30 generations.
 <strong>Phenotypic assays</strong><br> Before
 assays, worms were recover from freezing and grown 2 generations in common
 garden, each generation synchronized by bleaching, a standard procedure
 that kills all life-stages but eggs (Stiernagle 2006). The common garden
 temperature was 20°C or 25°C (see below). Phenotypic
 assays were performed to test for local adaptation to the experimental
 evolution regime and the evolution of adaptive maternal effects, and are
 summarized in figure 1. We therefore carried out three assays, by varying
 parental temperature (the 2 generations of common garden after defrosting
 the populations) and testing temperature for offspring. The 20°C - 20°C
 assay had both common garden and testing in 20°C. This is the environment
 the Control 20°C regime have experienced and tests for any cost of
 adaptation. Likewise, in the 25°C - 25°C assay both parents and testing
 worms experience 25°C, which is the selective environment for Warm 25°C
 and very close to the final environment for Increased warming. Finally,
 the 20°C - 25°C assay have 20°C as parental temperature, while the testing
 worms have their whole development in 25°C. This assay represents strong
 temperature fluctuations between generations, which is the selective
 environments for the Fast temperature cycle regime, and by comparing this
 assay to the 25°C - 25°C assay we can estimate the importance of maternal
 effects on fitness when adapting to a novel environment.
 The assays were initiated by synchronised egg-laying in the
 testing temperature by 40 females of each population. After 5h, females
 were killed by bleaching, and setup of L4 larvae was initiated 39h later
 (in 25°C) or 50h later (in 20°C), due to temperature-specific development
 time. The setup consisted of eight testing females per plate, together
 with the same number of background males from the SP8 line. Sex ratio was
 kept 1:1 throughout the experiment by adjusting the number of males to
 match the number of females present. Age-specific fecundity was measured
 by each day allowing the females 3h of egg-laying on an empty plate, where
 after the females were returned to a new plate (together with the males)
 and the number of hatched offspring on the egg-laying plate were killed
 with chloroform and counted two days later. The exact time the females
 were added to and removed from each plate was noted, and the number of
 offspring was corrected by exact number of minutes available for egg
 laying, and the number of females alive. Thus, we did not collect
 individual level data on total reproduction, but daily snapshot, in order
 to increase the number of individuals assayed and improve the reproduction
 estimate of each population. Daily reproduction was collected until
 reproduction had ceased. Four replicate plates of each population was set
 up, and for the 20°C - 20°C and 20°C - 25°C assays the replicates were
 evenly split between two climate cabinets per temperature, in order to
 separate cabinet and temperature effects. However, for logistical reasons,
 the 25°C - 25°C assay was reduced. We excluded the Slow temperature cycle
 treatment from this assay, and unfortunately we lost two Warm 25°C
 populations during common garden (due to overcrowding and subsequent
 starving, which is known to induce epigenetic effects, Rechavi et al.
 2014) and therefore these populations were excluded), leaving us with four
 replicate population of this treatment. This resulted in 30 replicate
 populations and 960 female worms for the 20°C - 20°C and 20°C - 25°C
 assays, and 23 replicate populations and 736 female worms for the 25°C -
 25°C assay. A small number of plates were excluded from analyses, because
 of accidents during egg-laying or chloroforming: six plates were removed
 from the 20°C - 20°C assay, four plates from the 20°C - 25°C assay and six
 plates from the 25°C - 25°C assay. <br>
 <strong>Statistical analyses</strong><br> The
 age-specific reproduction data was analysed as the total reproduction of
 each replicate plate, adjusted to the number of females present each day
 as well as rate-sensitive daily growth factor λ, which is equivalent to
 individual fitness (Brommer et al. 2002) but calculated per plate. λ
 encompasses the timing and number of offspring and is analogous to the
 intrinsic rate of population growth (Stearns 1992) and was calculated by
 constructing a population projection matrix for each plate, and then
 calculating the dominant eigenvalue from this matrix, following (McGraw
 and Caswell 1996). In the projection matrix, we set survival to 1 during
 the reproductive period, since any mortality during the early stages of
 the reproductive period (which is the most influential for λ) had
 non-natural causes (mainly worms climbing the wall of the plate where they
 dried out) and controlled for unequal number of worms by adjusting the
 reproductive value as described above. The first natural deaths in C.
 remanei are only observed long after the reproductive peak (Lind et al.
 2016, 2017). Since population size and age-structure was kept constant
 during the experimental evolution, rate sensitive fitness is maximised
 during experimental evolution and λ is therefore the most appropriate
 fitness measure for this study (Mylius and Diekmann 1995).
 <br> All statistical analyses were done using R 3.6.1, and
 models were implemented using the lme4 package (Bates et al. 2015).
 Significance tests were performed using the lmerTest package (Kuznetsova
 et al. 2017), and contrasts were analysed using the emmeans package. Total
 reproduction and daily growth factor λ in 20°C was analysed in separate
 mixed effect models with experimental evolution regime as fixed effect and
 population and cabinet as random effects. Both response variables were
 log-transformed before analysis. In 25°C, λ and total reproduction was
 analysed with experimental evolution regime and parental temperature as
 crossed fixed effects, and replicate population as random effect. Models
 were constructed with a random slope (parental temperature) in addition to
 random intercept. Since the 25°C - 25°C assay was conducted in only one
 cabinet, and moreover the Slow temperature cycle treatment was not run,
 the random effect of cabinet was excluded from the parental temperature
 models, as was the Slow temperature cycle treatment.
  
description: The data is in three files, one for the 20C-20C assay (called
 Rawdata20), one for the 20C-25C assay (called Rawdata25) and finally one
 for the 25C-25C assay (called Rawdata2525). *Selection:
 the experimental evolution regime *Line: the replicate
 population number within the regime (use new.Line instead for unique
 ID) *Plate: The replicate plate within the line (use
 new.Plate instead for unique ID) *Day: the adult day
 (age) of the worms *Cabinet: the climate cabinet the
 replicate plate was placed in (2 cabinets per temperature, except for the
 25C-25C assay where only one cabinet was used)
 *n.females: the number of females on the plate (started with 6
 females, sometimes females are lost by climbing the walls and drying out
 between the daily assays) *time start, time stop and
 timespan: the time females lay eggs on the plate (aimed for 3h, but
 differs sometimes a few minutes due to handling, this takes care of
 that.) *n eggs: number of eggs scored on a plate, laid
 by n females *n.eggs.3h: the avergae number of eggs per
 females over exactly 3h (this goes into analyses) *new
 Line: unique line ID *new Plate: unique plate
 ID