DOI: 10.5061/dryad.zpc866t8t
title: Arboreality drives heat tolerance while elevation drives cold tolerance in tropical rainforest ants

description: Determining how species thermal limits correlate with climate is important
 for understanding biogeographic patterns and assessing vulnerability to
 climate change. Such analyses need to consider thermal gradients at
 multiple spatial scales. Here we relate thermal traits of rainforest ants
 to microclimate conditions from ground to canopy (microgeographic scale)
 along an elevation gradient (mesogeographic scale) and calculate warming
 tolerance in the Australian Wet Tropics Bioregion. We test the thermal
 adaptation and thermal niche asymmetry hypotheses to explain interspecific
 patterns of thermal tolerance at these two spatial scales. We tested
 CTmin, CTmax, and calculated CTrange using ramping assays for 74
 colonies of 40 ant species collected from terrestrial and
 arboreal habitats at lowland and upland elevation sites and recorded
 microclimatic conditions for one year. Within sites, arboreal ants were
 exposed to hotter microclimates and on average had a 4.2°C (95% CI: 2.7 –
 5.6°C) higher CTmax, and 5.3°C (95% CI: 3.5 – 7°C) broader CTrange than
 ground-dwelling ants. This pattern was consistent across the elevation
 gradient, whether it be the hotter lowlands or the cooler uplands. Across
 elevation, upland ants had significantly lower CTmin than lowland ants,
 whereas the change in CTmax was less pronounced, and CTrange did not
 change over elevation. Differential exposure to microclimates, due to
 localised niche preferences, drives divergence in CTmax while
 environmental temperatures along the elevation gradient drive divergence
 in CTmin. Our results suggest that both processes of thermal adaptation
 and thermal niche asymmetry are at play depending on the spatial scale of
 observation, and we discuss potential mechanisms underlying these
 patterns. Despite the broad thermal tolerance range of arboreal rainforest
 ants, lowland arboreal ants had the lowest warming tolerance and may be
 most vulnerable to climate change.
description: These datasets are ant thermal tolerance limits and environmental
 microhabitat temperatures from a tropical rainforest in the Australian Wet
 Tropics Bioregion. Ants were sampled and microclimate was recorded at
 ground and arboreal/canopy microhabitats at replicate trees at three sites
 along an elevation gradient, two lowland sites at 100 m asl, Carbine and
 Daintree, and a upland elevation site, 1200 m asl at Carbine. In each
 dataset, the latitude and longitude of sites are provided.  
 Leahy_Ant_Thermal_Limits.xlsx
 Across all sites, we considered all individuals of a
 species from a single tree to be from the same colony and ants from
 separate trees as from different colonies. Multiple colonies of each
 species were collected where possible. We tested 20 species represented by
 one colony and the remaining 20 species by 2–5 colonies (mean ± SE: 1.85 ±
 0.17). We aimed to test five individual worker ants from each colony. In
 some cases, there were more or less than five individuals available to
 test. Overall, the mean number tested per colony was 4.8 ± 0.3 SE. For the
 polymorphic genera <i>Pheidole</i> and
 <i>Camponotus</i> we tested minor workers only. Following
 thermal tolerance experiments (see below) ants were placed in ethanol and
 returned to the lab to confirm species identification and to measure body
 mass. Between 3–5 (mean ± SE: 4.1 ± 0.1) workers of each tested colony
 were oven dried for 24 hrs at 70°C using a Blue M Electric drying oven,
 and dry weight body mass was measured using a Satorius semi-microbalance
 scale with 0.01 mg accuracy. Voucher specimens were deposited in the ant
 collection held at CSIRO’s Tropical Ecosystems Research Centre in Darwin,
 Australia. We took measures to
 limit sources of variation from any potential seasonal or daily thermal
 trait plasticity. Ants from different sites were tested within a
 three-month period of one year during the dry season when the weather is
 more stable, and ants within sites were tested within a period of seven to
 ten days. In addition, ants were tested for thermal tolerance as soon as
 possible after collection to avoid experimental acclimation. Average time
 between collection in the field and beginning testing was 3 hrs 22 min (±
 SD 2 hrs) and the maximum time was 8 hrs and 47 min.
 To measure thermal limits, we
 used a custom digital dry bath with 20 individual wells set into an
 insulated aluminum block above a Peltier plate that was programmed to heat
 and cool at a set rate. Each single ant was placed in a 1.5 ml
 microcentrifuge vial capped with a firm piece of foam to prevent it from
 hiding in the lid space above the thermal block. We tested several species
 at one time and individual workers of different species were randomly
 placed in one of the 20 well positions to prevent any systematic biases.
 We reserved one random well position for a temperature test vial without
 an ant, which had a microcentrifuge vial with a thermocouple wire inserted
 and capped with firm foam. This ensured we were recording the temperature
 that an ant would experience inside the vial. Following Kaspari et al.
 (2015), we used a protocol that ramped temperatures down to
 CT<sub>min</sub>, allowed ants to recover, and then ramped
 upwards to CT<sub>max</sub>. Kaspari et al. (2015) did not
 find any evidence that ramping down before ramping upwards systematically
 biases the final estimate of CT<sub>max</sub>. We placed ants
 in the thermal block for 15 min at 20°C to allow the resumption of normal
 behaviors. To test CT<sub>min</sub> we ramped temperature down
 at 0.5°C per minute. This ramping rate was chosen as a careful balance
 between a slow ramping rate that is more likely to be experienced in
 nature and a faster ramping rate that avoids the potentially confounding
 effect of desiccation associated with long experimental times (Rezende et
 al. 2011). On ramping down, the ants were checked at 15 °C, 12°C, and then
 every 1°C until there was loss of muscle coordination or absence of any
 movement after flicking the vial, indicating CT<sub>min</sub>
 (Lutterschmidt and Hutchison 1997). Upon reaching
 CT<sub>min</sub> ants were removed from the experiment and
 left to acclimate back to room temperature. Once all ants were removed,
 they were left for 15 min, after which they were checked for survival and
 normal movement. Ants that did not recover were replaced by a worker from
 the same colony for the CT<sub>max</sub> assay (15 out of 380
 ants tested, 4% of tests). Ants were then acclimated for another 15 min at
 20°C in the thermal block before temperature was ramped up. Temperature
 was raised 0.5°C per minute and ants were checked at every 2°C increment
 between 30–40°C and then every 1°C from 40°C until
 CT<sub>max</sub> was reached. CT<sub>max</sub> was
 determined as above for CT<sub>min</sub> and ants were checked
 for recovery after 15 min. Ants that did not recover from
 CT<sub>max</sub> were excluded from the analysis as they had
 reached a lethal rather than critical thermal limit. We calculated
 CT<sub>range</sub> (CT<sub>max</sub> –
 CT<sub>min</sub>) for individuals which recovered from both
 CT<sub>min</sub> and CT<sub>max</sub> assays.
 Excluding failed tests, we analysed the test results for 40 species for
 each thermal trait with 355 individuals of 74 colonies for
 CT<sub>min</sub>, 277 individuals of 73 colonies for
 CT<sub>max</sub>, and 269 individuals of 73 colonies for
 CT<sub>range.</sub> The
 dataset provided is the colony average (averaged over the individuals
 tested) CT limits and body mass, and includes the species vertical niche
 (ground or arboreal) and site of capture. 
 Leahy_2019-2020_Microclimate_Temperature.txt We recorded ambient temperature at 30 min intervals using HOBO Pro v2 (U23-001) temperature data loggers at ground (~0.5 m) and canopy (~20 m) vertical habitat at 100 m asl and 1200 m asl Carbine sites.  Loggers were placed for one year from March 2019 – March 2020 in the same year as ant sampling. In the dataset provided, we have calculated daily minimum, daily maximum, and daily mean temperature for each vertical habitat and elevation site.
description: Leahy_Ant_Thermal_Limits.xlsx
 There are missing values for one colony for CTmax and CTrange
 indicated by NA. Models in the manuscript were run for each thermal trait
 separately such that there were 74 colonies included in the model for
 CTmin, and 73 colonies included in the models for CTmax and
 CTrange.