Locally-adapted reproductive photoperiodism determines population vulnerability to climate change in burying beetles

Understanding how phenotypic traits vary among populations inhabiting different environments is critical for predicting a species’ vulnerability to climate change. Yet, little is known about the key functional traits that determine the distribution of populations and the main mechanisms—phenotypic plasticity vs. local adaptation—underlying intraspecific functional trait variation. Using the Asian burying beetle Nicrophorus nepalensis, we demonstrate that mountain ranges differing in elevation and latitude offer unique thermal environments in which two functional traits—thermal tolerance and reproductive photoperiodism—interact to shape breeding phenology. We show that populations on different mountain ranges maintain similar thermal tolerances, but differ in reproductive photoperiodism. Through common garden and reciprocal transplant experiments, we confirm that reproductive photoperiodism is locally adapted and not phenotypically plastic. Accordingly, year-round breeding populations on mountains of intermediate elevation are likely to be most susceptible to future warming because maladaptation occurs when beetles try to breed at warmer temperatures.


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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. We used the software ThermaCAM Researcher Professional 2.10 to obtain the thermal tolerance data of beetles.
All statistical analyses were performed in the R 3.0.2 statistical software package. Nicrophorus nepalensis from five populations: Mt. Hehaun, Taiwan, Mt. Lala, Taiwan, Wulai, Taiwan, Amami Oshima, Japan and Mt. Jiajin, China. We used the beetles which aged 2 to 3 weeks after emergence. No specific selection of individuals during field collection in each population but equal amounts of males and females were used in the breeding experiments.
To prevent type I and type II error, we set the significance level as 0.05, effect size as 0.8, and the statistical power as 0.8. We then got the sample size should be at least 25.
For the density survey, burying beetles were collected using hanging pitfall traps baited with rotting pork at each sites. The pitfall traps were checked in the morning on the fourth day of the experiment. Each beetle's pronotum was measured to the nearest 0.01 mm and sex was determined by the markings on the clypeus. The air temperature at every site was measured using iButton® devices that were placed approximately 120 cm above the ground within a T-shaped PVC pipe to prevent direct exposure to the sun For the reciprocal transplant experiments, we chose summer and winter in Mt. Hehuan and Wulai in Taiwan and the three sites on each mountain that the highest beetle abundance according in our field surveys. We conducted the experiments in which lab strains from either population were transplanted to the foreign mountain range (i.e. the lab strain individuals originating from the Wulai population were transplanted to Mt. Hehuan, and vice versa) and transplanted to native mountain range as the control (i.e. the lab strain individuals originating from the Wulai or Mt. Hehuan population were transplanted to Wulai or Mt. Hehuan, respectively). In each trial, we placed a male and a female with a mouse carcass (75 ± 7.5 g) in the breeding pot, which was covered by a gauze web and buried in the soil. A 75 g rat carcass was placed on the soil and covered with a 21 × 21 × 21 cm iron cage with a mesh size of 2 × 2 cm to prevent vertebrate scavengers from accessing the carcass. After two weeks, we would Jiajin population since these beetles can only breed under long-day conditions. All larvae from a pair of beetles were separated into the two photoperiodic conditions immediately at the time of dispersal. We used sexually mature beetles aged 2 to 3 weeks after emergence. For the ovary dissection experiments, we dissected and quantified ovarian weight at three-time points: on days 0, 7, and 14 after emergence. The general dissection protocol followed the method described by Wilson and Knollenberg. Briefly, we stored the beetles in ice for one hour to kill them. We began by measuring their body weight and cutting down their abdomens. Ovaries were then dissected in Ringer's solution. Next, we removed the spermatheca and accessory glands and immediately examined the wet weights of each organ. H.-Y. T. and Y.-M. F. conducted the lab experiments. For the thermal tolerance experiments, we first measured burying beetles' pronotum and transferred them to separate glass cups (200 mL, with lid) to calm down. This process usually took 1 hour at ambient temperature, which was the initial temperature of the thermal trail. The cup with the beetle was then submerged into either a 50°C or -10°C water bath to test its upper or lower thermal limit. Thermal limit was determined when an individual reached its critical temperature, lost coordinated leg movements, and could no longer remain in a roosting position. When this occurred, we immediately took them out of the cup to record their body temperature by a thermal camera (InfraCAM, FLIR Systems). Finally, we used ThermaCAM Researcher Pro 2.10 software (FLIR Systems) to capture the pronotum area of each individual as their core temperature of critical thermal maximum (CTmax) and minimum (  The result that beetles were dead during the experiments would be excluded to prevent the impact on the breeding result due to the beetles' death.
Both our field and lab experiments were based on large sample sizes but we did not repeat our experiments.
We randomly picked the beetles from different nests to conduct the breeding experiments in common garden, the ovary dissection experiments, thermal tolerance experiments and the transplant experiments.
While doing experiments, we picked the populations randomly to conduct all the experiments. We didn't expect any difference among populations while first conducting the breeding experiments and followed the protocol strictly. Thus, we gave them the equal environment and condition blindly. During data analysis, to prevent any bias toward the result, we randomly numbered our experimental samples regardless the treatment and test the relationship between number and the response vector.
Beetles samples were mostly collected in natural forest in all weather conditions.
Wulai in Taiwan We collect samples only once a month at the same location to minimized the disturbance frequency and impacted regions.
Nicrophorus nepalensis from five populations: Mt. Hehaun, Taiwan, Mt. Lala, Taiwan, Wulai, Taiwan, Amami Oshima, Japan and Mt. Jiajin, China. We used the hanging pitfall traps baited with rotting pork at each sites. The pitfall traps were checked in the morning on the fourth day of the experiment. After measuring beetle's pronotum and determining the sex by the markings on the clypeus, we released the beetles right after recording all the beetles' details. We only brought 2 males and 2 females of beetles in each plot back to the lab to make sure we didn't have a strong influence on the population density in the field. All wild-caught beetles were transported to the laboratory and allowed to reproduce in captivity.
Beetles were housed individually in 320 ml transparent plastic cups and fed superworms (Zophobas morio) weekly if they were kept for more than three days before the experiment. Beetles captured in the relatively short photoperiod season would be kept