DOI: 10.5061/dryad.w0vt4b8nw
title: Source data for: Antagonistic effects of intraspecific cooperation and interspecific competition on thermal performance

description: Understanding how climate-mediated biotic interactions shape thermal niche
 width is critical in an era of global change. Yet, most previous work on
 thermal niches has ignored detailed mechanistic information about the
 relationship between temperature and organismal performance, which can be
 described by a thermal performance curve. Here, we develop a model that
 predicts the width of thermal performance curves will be narrower in the
 presence of interspecific competitors, causing a species’ optimal breeding
 temperature to diverge from that of its competitor. We test this
 prediction in the Asian burying beetle Nicrophorus nepalensis, confirming
 that the divergence in actual and optimal breeding temperatures is the
 result of competition with their primary competitor, blowflies. However,
 we further show that intraspecific cooperation enables beetles to
 outcompete blowflies by recovering their optimal breeding temperature.
 Ultimately, linking abiotic factors and biotic interactions on niche width
 will be critical for understanding species-specific responses to climate
 change.
description: <b><i>Beetle collection and
 maintenance</i></b>  
 Lab experiments were conducted using <i>N.
 nepalensis</i> individuals from a laboratory-reared population. Our
 stable lab population was established in 2014 from 24 male and 24 female
 beetles caught near Meifeng, which is 2100m above sea level on Mt. Hehuan,
 Taiwan (24°5' N, 121°10'E).<i> </i>Since
 establishment, we have supplemented the lab strain with new individuals
 from the same location every one or two years to avoid inbreeding. We used
 hanging pitfall traps baited with rotting pork (mean ± SE: 100 ± 10 g) to
 collect adult beetles in the field. We checked traps and collected beetles
 on the fourth day after traps were set. In every generation, we
 established at least 20 families, approximately 600 individuals in total,
 to maintain the population within the lab. To ensure that beetles in the
 lab population were unrelated to each other, we always paired beetles
 collected from different traps. We then put one female and one male in a
 20 × 13 × 13 cm box with 10 cm of soil and a rat carcass (75 ± 7.5 g).
 Approximately two weeks after introducing adult beetles, all of the
 dispersing larvae that were ready to pupate from each breeding box were
 collected and allocated to a small, individual pupation box. After roughly
 45 days, beetles that emerged from pupae were housed individually in 320
 ml transparent plastic cups and fed once a week with superworms
 (<i>Zophobas morio</i>). All breeding experiments were
 conducted in walk-in growth chambers that imitated natural conditions
 where the lab population was collected at 2100 m on Mt. Hehuan.
 Temperature was set to daily cycles between 19℃ at noon and
 13℃ at midnight, and
 relative humidity was set to 83-100%. We completed all of the laboratory
 experiments within three generations.   <b><i>Breeding performance in
 the common garden experiment</i></b> To investigate breeding TPCs, we conducted
 solitary pairing experiments in six temperature conditions—8, 10, 12, 16,
 20 and 22ºC—in a common garden with no temperature variation in the lab.
 For each replicate, one male and one female were arbitrarily chosen from
 different nests to avoid inbreeding. We chose adult beetles that were
 sexually mature, roughly 2 to 3 weeks after their emergence. Each
 individual was weighed to the nearest 0.1 mg. We then placed the pair with
 a mouse carcass (75 ± 7.5 g) under each temperature condition in a
 transparent plastic container (21 × 13 × 13 cm with 10 cm of soil depth)
 for two weeks. Cases in which pairs fully buried the carcass and produced
 offspring were regarded as successful breeding attempts. Cases in which
 pairs failed to bury a carcass, or they buried it but did not produce
 offspring, were regarded as failed breeding attempts. The only instance
 (of 118 replicates) when beetles died during the experiment was excluded
 from analysis.  
 <b><i>Thermal regulation of locomotor performance
 </i></b> To determine the TPC
 for locomotor behaviors, we conducted a series of treadmill experiments
 under three temperature conditions—12, 16 and 20ºC—in a common garden with
 no temperature variation in the lab. We chose these temperatures because
 16ºC is the optimal performance temperature for reproduction, we wanted to
 further test whether this optimal temperature coincided with optimal
 physiological function. We set 72 replicates
 in total (12ºC: 25 replicates; 16ºC: 25 replicates; 20ºC: 22 replicates).
 We arbitrarily selected individuals from different nests for replicates at
 each temperature. The beetles were brought to the experimental chamber one
 day before data collection began. Monofilament glued to the pronotum by UV
 glue attached each beetle to the treadmill, where it was allowed to walk
 at a stable speed of 1.5 m per min (see Figure 3). We turned off the
 treadmill if a beetle’s abdomen began to drag or if the beetle started to
 fly, both behaviors that indicated that the beetle could no longer walk.
 An individual was tested only once per day. After each experiment, beetles
 were returned to the transparent container with 3 cm of soil for recovery.
 We measured each beetle’s pronotum and
 the ambient temperature during running with a thermal imaging infrared
 camera (FLIR Systems, Inc., SC305; thermal sensitivity of &lt; 0.05ºC)
 at a resolution of 320*240 pixels. Pronotum temperature was measured at
 the center of the thorax and calculated as the average pronotum body
 temperature each minute until an individual dragged its abdomen or started
 flying. The ambient temperature was the average temperature of a 6 x 6 cm
 surface of the treadmill located near where the beetle was tested. The
 temperature difference was depicted by the difference between the beetle’s
 body and ambient temperatures.  
 <b><i>Breeding performance in the
 field</i></b> Since our previous study
 showed that blowflies are the beetle’s main interspecific competitor
 (Sun<i> et al.</i>
 2014), we conducted series of experiments in the
 field to investigate the breeding TPC with and without interspecific
 competition. In 2013 to 2016 (May-October), we investigated the natural
 pattern of <i>N. nepalensis</i> reproduction and its breeding
 success along an elevational gradient from 673m to 3422m on Mt. Hehuan in
 central Taiwan (24°11’ N, 121°17’ E) that encompasses broadleaf forests at
 lower elevations and mixed conifer-broadleaf forests at higher elevations.
 We chose 37 study sites, primarily in natural forests to avoid cultivated
 or open areas where temperatures are more variable (De Frenne<i> et al.</i>
 2019) and replicated each treatment at least 3
 times at each site. In each trial, a 75 g (± 7.5 g) rat carcass was placed
 on the soil to attract beetles and covered with a 21 × 21 × 21 cm (length
 x width x height) iron cage with 2 × 2 cm mesh to prevent vertebrate
 scavengers from accessing the carcass. We checked each carcass daily until
 it began to decay due to microbial activity (Payne 1965), was consumed
 by maggots or other insects, or was buried under the soil by beetles. If
 burying beetles completely buried the carcass, we checked the experiment
 after 14 days to determine if third-instar larvae appeared. Cases in which
 pairs produced third-instar larvae were regarded as successful breeding
 attempts. Cases in which pairs failed to produce larvae were regarded as
 failed breeding attempts. Breeding experiments without blowflies
 were conducted in the same experimental sites from 2014 to 2017, and 2019
 (May-October). The experimental design was the same as that described
 above, but we used screen mesh above the pots to also keep blowflies out.
 To record air temperature at every site, we placed iButton® devices
 approximately 120 cm above the ground within a T-shaped PVC pipe to
 prevent direct exposure to the sun but allow for air to circulate. One
 male and one female beetle that were reared in the lab were released into
 the pot to record fundamental breeding performance. After 14 days, we
 checked the pots to determine whether the burying beetles’ third-instar
 larvae appear. Cases in which pairs fully buried the carcass and produced
 larvae after 14 days were regarded as successful breeding attempts. Cases
 in which pairs failed to bury a carcass, or they buried it but did not
 produce larvae, were regarded as failed breeding attempts. Criteria of
 data exclusion was the same as the common garden experiments. The only 3
 instances (of 178 replicates) when beetles died during the experiment were
 excluded from analysis. <b><i>The influence of
 cooperation on thermal performance in the
 field</i></b> To investigate how cooperative behavior
 influences TPCs, we manipulated the group size of beetles in the field at
 38 sites along the elevational gradient. Our experimental device comprised
 a small plastic container (21 × 13 × 13 cm with 10 cm of soil) placed
 inside a large container (41 × 31 × 21.5 cm with 11 cm of soil). There
 were several holes on the small container’s side wall that allowed beetles
 to move freely between the two containers. A 2 x 2 cm iron mesh was placed
 around the top of the large container’s wall to let flies access the
 carcass but to keep out larger animals that might scavenge the carcass.
 Small, non-cooperative groups contained one male and one female, whereas
 large, cooperative groups contained three males and three females (Chen<i> et al.</i>
 2020; Liu<i> et
 al.</i> 2020). We captured the local beetles
 using the same hanging pitfall traps described above, and then conducted
 two group size treatments at each site. Based on our previous work
 exploring the natural pattern of arrival times, we released the marked
 beetles into the experimental device 1, 2 and 3 days after the trials
 began at elevations of 1700–2000 m (low), 2000–2400 m (intermediate) and
 2400–2800 m (high), respectively (for details, see (Sun<i> et al.</i>
 2014). Each experiment was
 recorded by a digital video recorder (DVR) to determine whether
 <i>N. nepalensis</i> successfully buried the carcass. We
 placed the same temperature measurement device as described above at every
 site. Cases in which beetles buried the carcass completely<i>
 </i>and<i> </i>produced larvae after 14 days after were
 regarded as successful breeding attempts. Cases in which beetles failed to
 produce larvae were regarded as failed breeding attempts.
   <b><i>Data
 analysis</i></b> We used generalized linear mixed models
 (GLMMs) with binomial error structure to compare thermal performance
 curves among treatments (with/without interspecific competitors;
 with/without intraspecific cooperation) in the field. The outcome of
 breeding success (1 = success, 0 = failure) was fitted as a binomial
 response term to test for differences in the probability of breeding
 successfully. The variables of interest (i.e. mean daily temperature, type
 of experimental treatment) were fitted as fixed factors. Other
 environmental factors (elevation, daily minimum air temperature) were
 fitted to test the generality of the results. However, since elevation,
 daily minimum air temperature, and mean air temperature were highly
 correlated, we only included mean daily temperature in the final model. We
 also modeled the potential nonlinear effects of the environmental factors
 by fitting a quadratic regression model and compared the model fit with
 the linear model. Thus, the thermal performance curves (TPC) were
 determined statistically by the GLMM. To account for repeated sampling in
 the same plot, we set the field plot ID as a random factor (coded as
 1|plot ID) in the R package lme4 (Bates<i> et
 al.</i> 2014). We also included year as a
 random factor to account for sampling at different time points. (See
 Source Code File 1 for further details) We used a
 general linear models (GLMs) to determine the rates of <i>N.
 nepalensis</i> breeding and locomotor performance in the lab. The
 outcome (1 = success, 0 = failure) was fitted as a binomial response term
 to test the difference in the probabilities of interest (burial or
 non-burial) under different temperatures. For locomotor performance, the
 outcome (1 = flying, 0 = not flying) was fitted as a binomial response
 term to test for a difference in the probability of interest (flying or
 not) under different temperatures conditions. The relationship of
 temperature difference (body temperature minus ambient temperature) was
 fitted as a Gaussian response to different temperature conditions. (See
 Source Code File 1 for further details) Finally, the optimal temperature
 (<i>T<sub>optimal</sub></i>) of the TPC was
 calculated by taking the derivative of the regression line that described
 the relationship between temperature and the likelihood of breeding
 successfully. In other words,
 <i>T<sub>optimal</sub></i> was estimated from the
 unimodal statistical model of the TPC. To estimate TPC breadth, we
 calculated the 95% confidence interval of the regression line. The
 boundaries of the TPC were the points that there was not significant
 difference between regression lines and zero. All statistical analyses
 were performed in the R v3.0.2 statistical software package (Team. 2018). (See Source
 Code File 1 for further details)
description: Beetles died during the experiment were excluded from
 analysis. We have marked that in the dataset.