Three hundred copepods were collected in April of 2018 from eastern Long Island Sound, Connecticut, USA (41.3°N, 72.0°W) and raised for one year (~12 generations) (14 degrees Celsius (°C), 400 microatmospheres (μatm) CO2, 30 ‰ salinity, 12:12 hours light:dark) as stock cultures to limit maternal effects (Falconer, 1989, Introduction to Quantitative Genetics). Three resulting stock cultures with >2,000 individuals each were combined and then split evenly into three groups for each of the four treatments. Groups were acclimatized within a generation to temperature (15°C or 13°C, 1°C per day) and pCO2 (1000 μatm, 100 μatm per day, OA treatments only). Groups seeded the F0 individuals for 7-10 days yielding ~15,000 eggs per treatment. Resulting F0 eggs and nauplii were combined for each treatment, redistributed among three replicate cultures, and returned to their respective experimental conditions. The experimental environmental conditions were: 1) Ambient control (AM): 13°C, 400 µatm CO2, pH = 8.2; 2) Ocean Acidification (OA): 13°C, 1000 µatm CO2, pH = 7.85; 3) Ocean Warming (OW): 15°C, 400 μatm CO2, pH = 8.2; 4) Combined warming and acidification (OWA): 15°C, 1000 μatm CO2, pH = 7.85. Copepods were fed equal proportions of the live phytoplankters Tetraselmis sp., Rhodomonas sp., and Thalassiosira weissflogii every 48-72 hours to achieve food-replete conditions (≥600 micrograms (μg) Carbon per liter (L)) (Feinberg and Dam, 1998. Marine Ecology Progress Series), deliberately raised under ambient conditions to avoid confounding effects of possible food quality changes.

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 = l­x / (lx-1) where lx represents the proportion of individuals on day x and lx - 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. Per capita EPR and HS are calculated as described in the preceding, with fecundity rates equalling 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). Full methods for all traits can be found in: deMayo, et al. 2023. Simultaneous warming and acidification limit population and reveal phenotype costs for a marine phenotype. Proc. R. Soc. B 290: 20231033. doi.org/10.1098/rspb.2023.1033