key: cord-102734-ltuqoa2b
authors: Tsai, Hsiang-Yu; Rubenstein, Dustin R.; Chen, Bo-Fei; Liu, Mark; Chan, Shih-Fan; Chen, De-Pei; Sun, Syuan-Jyun; Yuan, Tzu-Neng; Shen, Sheng-Feng
title: Antagonistic Effects of Intraspecific Cooperation and Interspecific Competition on Thermal Performance
date: 2020-05-04
journal: bioRxiv
DOI: 10.1101/2020.05.03.075325
sha: 
doc_id: 102734
cord_uid: ltuqoa2b

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 a 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 blowflies. However, we further show that intraspecific cooperation enables beetles to outcompete blowflies by recovering their optimal breeding temperature. Ultimately, linking direct (abiotic factors) and indirect effects (biotic interactions) on niche width will be critical for understanding species-specific responses to climate change.

Recent anthropogenic climate warming makes understanding species vulnerability to changing climates one of the most pressing issues in modern biology. A cornerstone for understanding the distribution and associated ecological impacts of climate change on organismal fitness is the concept of the ecological niche, which describes a hyperspace with permissive conditions and requisite resources under which an organism, population, or species has positive fitness (Hutchinson 1957 2016), it has been developed largely independently from niche-based studies. Yet, characterizing the TPC is essentially a way to mechanistically quantify a species' thermal niche. Since the TPC describes the detailed relationship between temperature and fitness, the concept may actually be more informative than that of the thermal niche, which is typically defined as the range of temperatures over which organisms occur in nature (i.e. thermal niche width) (Huey & Stevenson 1979; Hillaert et al. 2015 Burying beetles (Silphidae, Nicrophorus) are ideal for investigating how social interactions influence both fundamental and realized TPCs because the potentially antagonistic effects of interspecific competition and intraspecific cooperation on the realized TPC can be studied simultaneously. Burying beetles rely on vertebrate carcasses for reproduction and often face intense intra-and interspecific competition for using these limiting resources (Pukowski 1933; Scott 1998; Rozen et al. 2008 Here, we extend classic ecological niche theory by introducing the concepts of fundamental and realized TPCs. We first construct a theoretical model by using a hypothetical TPC to predict how interspecific competition influences the width and optimal temperature of realized TPCs in order to provide a general understanding of the relationship between fundamental and realized TPCs. We then describe a series of laboratory and field experiments designed to test the predicted relationship between fundamental and realized TPCs in the Asian burying beetle Nicrophorus nepalensis ( Fig. 1) . We began our empirical work by measuring locomotor and breeding performance without interspecific competitors in the laboratory and field to determine N. nepalensis's fundamental TPC. We then quantified beetle breeding performance in the presence of interspecific competitors, mainly blowflies (Putman 1978; Putman 1983; Scott 1994; Sun et al. 2014) , to determine the beetle's realized TPC. Finally, we used a group size manipulation in the presence of interspecific competitors in the field to explicitly examine the role of intraspecific cooperation and interspecific competition on the beetle's realized TPC. Experimentally distinguishing between fundamental and realized TPCs will not only serve as a starting point for better understanding the relationships between direct and indirect drivers of organismal performance and fitness, but also for better predicting responses to climate change as the earth continues to warm.

We used hanging pitfall traps baited with rotting pork (mean ± SE: 100 ± 10 g) to collect adult beetles at Mt. Hehaun, Taiwan ranging from 500 m (121° 00′ E, 23° 98′ N) to 3200 m (121° 27′ E, 24° 13′ N) in 2018. We checked traps and collected beetles on the fourth day after traps were set. A maximum of two males and two females from each trap were brought back to the lab to reduce any influence of capture on the population structure in the field. In every generation, we established at least 20 families, approximately 600 individuals in total, to maintain population structure within the lab. To ensure that beetles in the lab strains were unrelated to each other, we used 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 with superworms (Zophobas morio) once a week. All breeding experiments were conducted in walk-in growth chambers that imitated natural conditions at 2100 m on Mt. Hehaun. 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.

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 (n = 20 replicates at each temperature). For each replicate, one wildtype male and one wildtype female were arbitrarily chosen from different lab strains 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.

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 set 72 replicates in total (12ºC: 25 replicates; 16ºC: 25 replicates; 20ºC: 22 replicates). For each replicate, we randomly picked one beetle from different lab strains and measured its weight and width of pronotum. 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 it 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 < 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.

Since our previous study showed that blowflies are the beetle's main interspecific 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 in 2014 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.

To investigate how cooperative behavior influences TPCs, we manipulated the group Each experiment was recorded by a digital video recorder (DVR) to determine whether N. nepalensis 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 and 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.

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. Environmental factors (elevation, daily minimum air temperature) were fitted as covariates of interest. 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. where is environmental temperature, * is the shape parameter describing the steepness of the curve at the lower end, )*+ is the optimal environmental temperature at which organisms have their highest performance, and 789 is the upper critical temperature. We assumed that performance becomes zero when > 789 .

Since environmental conditions also directly influence a species' average performance, we used a Gaussian function to describe the chance of encountering a particular temperature:

where | 7=8> represents the probability of getting given 7=8> , 7=8>

represents the mean environmental temperature, and describes the environmental temperature variability. We combined equations (1) and (2) We began by addressing how interspecific competition influences the realized TPC of a focal species, finding that when a low temperature specialist species (e.g. burying beetle) competes with a high temperature generalist species (e.g. blowfly), the optimal temperature of the realized TPC of the thermal specialist shifts towards a lower temperature and the width of the TPC decreases (Fig. 2b) . In other words, our model predicts that the optimal temperature of the realized TPC will decrease to below that of the optimal of fundamental TPC when a low temperature specialist competes with a high temperature generalist. To make the theoretical framework complete, we also explored the scenario of a high temperature specialist competing with a low temperature generalist. We found that if a high temperature specialist species competes with the low temperature generalist species (Fig. S1a) , the optimal temperature of the realized TPC of the thermal specialist shifts towards a higher temperature and the width of the realized TPC decreases (Fig. S1b) , which suggests that a shift in the optimal realized TPC away from the optimal temperature of the competing species is a general result.

We first explored N. nepalensis's fundamental TPC of breeding in a controlled lab environment. We found that the beetle's optimal breeding temperature or fundamental TPC-defined as the mean temperature at which breeding success was highest-was 15.6℃ (Fig. 3a , GLM, χ² 2 =26.61，p<0.001，n=118). To determine the physiological basis of this optimal TPC, we measured locomotion ability at different temperatures by performing a treadmill running experiment. We found that beetles had a greater likelihood of flying at 16℃ while running at a stable speed on the treadmill (Fig. 3b , GLM, χ² 2 =10.36，p<0.001，n=72). In other words, N. nepalensis took less energy to raise its body temperature enough to begin flying at 16℃ than at other temperatures ( Fig. 3c and d, GLM, χ² 2 =19.90，p<0.001，n=30).

Next, we investigated N. nepalensis's realized and fundamental breeding TPCs by studying breeding performance along an elevational gradient (1600 to 2800 m above sea level). As predicted by our model, in the presence of interspecific competitors (blowflies) in the wild, the optimal breeding temperature (i.e. the realized TPC) of N.

nepalensis was roughly 14.1℃, which is lower than the optimal temperature in the lab in the absence of blowflies (i.e. the fundamental TPC) (Fig. 4a , GLMM, χ² 2 =12.89，

p<0.001，n=343). Intriguingly, when excluding blowflies and removing the threat of intraspecific competition in the field, the optimal breeding temperature of N.

nepalensis increased to approximately 14.7℃ such that the realized TPC began to approach the fundamental TPC in the absence of blowflies, ultimately becoming broader than when in the presence of interspecific competitors (Fig. 4b, GLMM , χ² 2 =16.7，p<0.001，n=175). Thus, our experiment confirmed the causal relationship between interspecific competition and the shift in the realized TPC under natural conditions.

Since our previous study found that N. nepalensis will cooperate at carcasses to compete against blowflies, particularly in warmer environments (Sun et al. 2014) , we predicted that a group of N. nepalensis in a warm environment would have a better chance of expanding its realized TPC towards the fundamental TPC than would an individual pair. To test this prediction, we performed a group size manipulation to determine the realized TPCs of cooperative groups and solitary beetles. We found that N. nepalensis in cooperative groups had an optimal breeding temperature (i.e. realized TPC) of 15.6℃, which is identical to their optimal temperature from the fundamental TPC in the lab. In contrast, the optimal breeding temperature of solitary pairs was 14.1℃, similar to that of the realized TPC in the field (Fig. 5 , group size × temperature interaction, χ 2 2 = 6.78, p= 0.033, n = 328; for large groups, χ 2 2 = 9.49, p=0.009, n = 162; for small groups, χ 2 2 = 18.41, p<0.001, n = 166). These results suggest that beetles that cooperate are able to expand their realized TPCs such that they converge on their fundamental TPCs, whereas those do not cooperate have divergent realized and fundamental TPCs.

By combining the concepts of fundamental and realized niches from ecological niche theory with the that of the thermal performance curve (TPC), we found that a species' realized thermal performance curve is likely to change in time and space in response to biotic factors such as interspecific competition. Our theoretical model suggests that if thermal specialist species compete with thermal generalist species adapted to higher or lower temperatures, the optimal performance temperature of specialists will decrease or increase respectively. Our empirical results examining competition between burying beetles (thermal specialists) and blowflies (thermal generalists) for access to carcasses support this theoretical prediction, finding that blowflies force the beetle's optimal breeding temperature lower and the realized TPC narrower.

Intriguingly, our experiment also showed that cooperation in this facultatively social species not only enables beetles to overcome interspecific competition (Sun et al. The idea that interspecific competition will reduce the realized niche width of a species is well-accepted in ecology. However, our study further suggests that a more mechanistic understanding of how interspecific competitors affect the optimal temperature performance of species will be critical for understanding how climate change affects species' vulnerability. Since it is generally assumed in studies of macroecology and climate change that thermal performance is largely influenced by physiology, a single function is often used to describe a species' thermal performance curve (Sinclair et al. 2016 ). However, if biotic interactions are key to indirectly influencing the thermal performance of a species as we have shown here, the realized TPC of a species is likely to change in time and space and should not be described by a single function to represent the thermal performance of a species.

Integrating the idea of TPCs into the ecological niche concept helps bridge two rich, but largely independent, traditions of studying thermal adaptation. By simply recognizing the concept of realized TPCs, it becomes clear that we know little about how realized and fundamental TPCs differ in most species. We show that the realized TPC provides a way to quantify how temperature mediates species interactions, which also influence organismal fitness. Thus, the realized TPC extends the realized thermal niche concept, which only considers the temperature ranges in which a species can likely occur in N. nepalensis in warmer environments. Therefore, conserving high population densities, especially at lower elevations, will be crucial for N. nepalensis to compete against blowflies under increased climate warming. By examining the relationship between cooperative behavior and interspecific competition, our study thus helps understand the pressing issue of how habitat destruction affects the vulnerability of social organisms to climate change (Travis 2003) . When population density influences the likelihood of intraspecific cooperation in social species, habitat destruction will not only decrease habitat availability but also weaken a species' competitive ability against interspecific competitors, which in turn will lower the realized thermal performance of social organisms.

Our study has implications beyond interspecific competition in insects. Many classic studies of TPCs investigate how changes in body temperature influence physiological or behavioral performance (Chen et al. 2003; Zhang & Ji 2004) . Body temperature is often assumed to be the same as the environmental temperature in ectotherms.

However, accumulating evidence suggests that many ectotherms can at least partially regulate their own body temperature behaviorally or physiologically (Heinrich 1993 The concept of the TPC has received renewed interest because the earth has been warming rapidly for the past few decades. Yet, apparent gaps exist between studies of physiological function and those examining fitness consequences in changing environments. Our study shows that employing the concepts of fundamental and realized TPCs can help us predict the ecological impacts of climate change, especially because environmental change will likely reshuffle ecological communities and alter the strength of species interaction (Alexander et al. 2016 ). The importance of biotic interactions in shaping species distributions and community composition is intuitively obvious, yet historically has been difficult to quantify. We believe that the concept of realized TPCs can help fill this important knowledge gap and, ultimately, deepen our understanding of the ecological impact of climate change.

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