Copepods were collected in June of 2016 from Esker Point Beach in Groton, Connecticut, USA (41.320725°N, 72.001643°W) and raised for at least three generations as stock cultures prior to the start of transgenerational experiments to limit maternal effects (Falconer, 1989. Introduction to Quantitative Genetics). Stock cultures were split evenly into eight groups of 160 females and 80 males. Four of these eight groups were acclimatized to high temperature at 1 degree Celsius per day and used to seed the two high temperature treatments (OW and OWA). The other four groups remained at ambient temperature and were used to seed the ambient and acidification treatments. After temperature acclimatization, groups of stock cultures seeded the parental (F0) individuals for two days. Stock culture groups yielded an average of 7,173 eggs per group to produce approximately 57,000 parental (F0) eggs. Resulting parental eggs and N1 nauplii were acclimated to one of four experimental treatments over the entire F0 generation. The four lines of the copepods were established with four replicates of each condition. The target (actual ± standard deviation) conditions were: ambient temperature = 18 degrees Celsius (°C) (18 ± 0.34, N = 330), ambient pCO2 = 400 μatm (379 ± 36, N = 18; pH = 8.26 ± 0.1, N = 330); high temperature = 22°C (22 ± 0.81, N = 336), and high pCO2 = 2000 µatm (2301 ± 215, N = 18; pH = 7.55 ± 0.08, N = 330). Copepods were fed every 48-72 hours at food-replete concentrations (≥800 micrograms (μg) Carbon per liter (L)) consisting of equal proportions of the phytoplankters Tetraselmis sp., Rhodomonas sp., and Thalassiosira weissflogii, deliberately raised under ambient conditions for the entire length of the experiment to avoid confounding effects of possible changes in food quality due to the different temperature and CO2 among treatments.

The population net reproductive rate, λ, was calculated as the dominant eigenvalue of an assembled projected age-structured Leslie matrix constructed from survival and fecundity data (Caswell, H. 2001. Matrix Population Models: Construction, Analysis, and Interpretation). Briefly, day-specific probabilities of survival are calculated from day-specific survival as Px = l_x /(l_x−1) where l_x represents the proportion of individuals on day x and l_x - 1 represents the proportion of individuals on day x − 1. Probabilities of survival on day 1 are assumed to be 100%, or a value of 1.0. EPR was calculated as (E_u+E_h)/t where E_u represents unhatched eggs, E_h represents hatched eggs (nauplii) and t represents egg-laying time. HS was calculated as E_h/(E_u+E_h). Fecundity rates equal the product of EPR and HS. Because only females produce offspring, total fecundity rates must be scaled to the sex ratio (proportion of females to males). To account for differences in individual development time for each treatment, fecundity rates are assigned to all days after the first matured adult is observed. We assume that surviving individuals represented by the survival experiments are equally as likely to experience any of the fecundity values observed in EPR experiments. Therefore, each mate-pair fecundity rate was paired with each survival beaker to construct a matrix. This yields a maximum of 120 matrices per treatment per generation (3 survival beakers × 4 replicate cultures × 10 mate pairs). Relative measures of each value are calculated as the trait value divided by the mean value of that trait. Standardized measures of each value are calculated as the trait value minus the mean trait value and divided by the standard deviation. The target (actual ± standard deviation) conditions were as follows: ambient (AM) temperature = 18 °C (18 ± 0.34, N = 330), AM pCO2= 400 μatm (379 ± 36, N = 18; pH = 8.26 ± 0.1, N = 330); high temperature = 22 °C (22 ± 0.81, N = 336); and high pCO2= 2,000 µatm (2,301 ± 215, N = 18; pH = 7.55 ± 0.08, N = 330). AM target levels represented extant conditions for this species in northeast Atlantic estuaries. Full methods can be found in Dam, et al. 2021 Nature Climate Change. doi: 10.1038/s41558-021-01131-5.