# Temperature has an overriding role compared to photoperiod in regulating the seasonal timing of winter moth egg hatching ## Description of the data and file structure Raw data belonging to the manuscript "Temperature has an overriding role compared to photoperiod in regulating the seasonal timing of winter moth egg hatching". We investigated whether photoperiod received at the egg stage influences the seasonal timing of egg hatching in the winter moth, both as a cue on its own and in interaction with temperature. In two split-brood experiments, we determined egg hatching date after giving eggs either an early or late season photoperiod treatment, with naturally changing day lengths shifted 2-4 weeks earlier or later compared to the actual calendar date. Temperature was kept constant in the first experiment, while the second experiment also incorporated two naturally changing temperature treatments – mimicking a cold and a warm year – to investigate the relative contribution of temperature and photoperiod. **NB: Treatment names consist of photoperiod treatment: (1) control [0 weeks shift], (2) very early season photoperiod [-4 weeks], (3) early season photoperiod [-2 weeks/K], (4) late season photoperiod [+2 weeks/L], and (5) very late season photoperiod [+4 weeks]; and temperature treatment: mimicking cold year 1999 [K] and warm year [W]** \--- Split-brood experiment 1: Photoperiod experiment 2013-2014 files \ Contains all experimental data collected from the photoperiod experiment (EggPhot2014), incl. for each sub-clutch (ClutchID) their origin (TubeID=female parent - 31 females in total - incl. catch info for this female parent i.e. Area, Site, Tree and NovemberDate=Catch date as julian dates with origin 2013-10-31); the treatment the ClutchID was assigned to (Treatment); the number of eggs in the sub-clutch (Eggs, at least 12); and when the sub-clutch hatched (D50Calc, i.e. julian dates with origin 2014-03-31). **NB: Median hatching date (D50Calc) per sub-clutch was only calculated if at least 10 eggs in the sub-clutch hatched.** \ Specifies the photoperiod treatment assignment for each ventilated box that eggs were kept in during the experiment. Needed to interpret the temperature data from the temperature loggers (see below). In total, 3 boxes per treatment (3x5 treatments = 15 boxes). \ Temperature logger data for boxes 1-5. \ Temperature logger data for boxes 6-10. \ Temperature logger data for boxes 11-15. Logger data per logger (LoggerName) measuring the Temperature for each ventilated box(LocationName/Location) for each measuring timepoint (Year, Month, Day, Date, Hour, Minute, Time), as obtained from the internal winter moth Access database (SysUser=who entered the data in the database, SysDate=when data entry happened, TempID and LocationID=database specific IDs). \--- Split-brood experiment 2: Photoperiod-temperature experiment 2000-2001 files \ Contains all experimental data collected from the photoperiod experiment (EggPhotTemp2001, ExperimentName=RN2001), with the same variables as described for above for . Treatment [KK]=Cold year and -2 weeks photoperiod; Treatment [WL]=Warm year and +2 weeks photoperiod. \ Temperature logger data for used incubators (indicated by Treatment name), with the same variables as described for above for . \--- Experimental photoperiod and temperature treatments \ Photoperiod treatments as used in the Photoperiod experiment (1 tab per treatment), giving the sunrise and sunset times in hours (HH:MM) for the calendar day the treatment was shifted to (date) for every day of the experiment (actual date). Note that this experiment started on January 15, while the same [-2 weeks] and [+2 weeks] treatments were used in the Photoperiod-temperature experiment, but starting earlier on December 12. \ \ Temperature treatments mimicked a specific year (Cold: 1973, Warm: 1999). For each day, a three-phase temperature cycle was used: 6 hours at the daily minimum temperature (h1), 12 hours at the mean of the daily maximum and the daily average (h7), and 6 hours at the daily average for each year (h19). ## Code/Software All code needed to reproduce the manuscript's data analysis using R are included.See the file for used R version and package versions. \--- Split-brood experiment 1 Script: <1_EggPhotoExp2014_analysis.R> R script to reproduce the analysis and visualization (incl. manuscript figures) of the 2014 winter moth egg photoperiod experiment: Does photoperiod received at the egg stage affect the seasonal timing of egg hatching? \--- Split-brood experiment 2 Script: <2_scripts/2_EggPhotoTempExp2001_analysis.R> R script to reproduce the analysis and visualization (incl. manuscript figures) of the 2001 winter moth egg photoperiod-temperature experiment: What is the relative contribution of photoperiod and temperature on the timing of egg hatching? \--- Visualization of experimental photoperiod and temperature treatments Script: <3_visualize-treatments.R> R script to visualize the photoperiod and temperature treatments as used in the two experiments, showing how they change over the season to mimic naturally changing environmental conditions. See for used R version and package versions.

To accurately predict species’ phenology under climate change, we need to gain a detailed mechanistic understanding of how different environmental cues interact to produce the seasonal timing response. In the winter moth (Operophtera brumata), seasonal timing of egg hatching is strongly affected by ambient temperature and has been under strong climate change-induced selection over the past 25 years. However, it is unclear whether photoperiod received at the egg stage also influences timing of egg hatching. Here, we investigated the relative contribution of photoperiod and temperature in regulating winter moth egg development using two split-brood experiments. We experimentally shifted the photoperiod eggs received by 2-4 weeks compared to the actual calendar date and measured the timing of egg hatching, both at a constant temperature and in combination with two naturally changing temperature treatments – mimicking a cold and a warm year. We found an eight-fold larger effect of temperature compared to photoperiod on egg development time. Moreover, the very small photoperiod effects we found were outweighed by both between- and within-clutch variation in egg development time. Thus, we conclude that photoperiod received at the egg stage does likely not play a substantial role in regulating the seasonal timing of egg hatching in the winter moth. These insights into the regulatory mechanism of seasonal timing could have important implications for predicting insect climate change adaptation, as we might expect different targets of selection depending on the relative contribution of different environmental cues.

We investigated whether photoperiod received at the egg stage influences the seasonal timing of egg hatching in the winter moth, both as a cue on its own and in interaction with temperature. In two split-brood experiments, we determined egg hatching date after giving eggs either an early or late season photoperiod treatment, with naturally changing day lengths shifted 2-4 weeks earlier or later compared to the actual calendar date. Temperature was kept constant in the first experiment, while the second experiment also incorporated two naturally changing temperature treatments – mimicking a cold and a warm year – to investigate the relative contribution of temperature and photoperiod. Eggs were collected from wild females (31 clutches and 20 clutches, for experiment 1 and 2 respectively), with each clutch of eggs split into sub-clutches and divided over the different photoperiod and photoperiod-temperature treatments. Raw egg hatching data (i.e. 2-3 observations per week of the number of freshly hatched caterpillars per sub-clutch) have been processed by calculating the median hatching date per sub-clutch (D50, i.e. the day at which 50% of the sub-clutch has hatched). D50 data were used for the statistical analyses and have been deposited here, together with the raw temperature logger data from the experiment.