Climate warming drives a temperate-zone lizard to its upper thermal limits, restricting activity, and increasing energetic costs

Climate warming drives a temperate-zone lizard to its upper thermal limits, restricting activity, and increasing energetic costs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Climate warming drives a temperate-zone lizard to its upper thermal limits, restricting activity, and increasing energetic costs Lisa I Doucette, Richard P Duncan, William S Osborne, Murray Evans, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2228630/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Jun, 2023 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract Ectotherms are considered vulnerable to climate change as many operate at close to their thermal maxima. Exposure to higher temperatures could reduce activity by forcing animals to shelter in thermal refugia to avoid exceeding lethal limits. While rising temperatures should reduce activity in tropical species, the situation is less clear for temperate-zone species where activity can be constrained by both low and high temperatures. Here, we measure the effects of natural variation in environmental temperatures on activity in a temperate grassland lizard and show that it is operating at its upper thermal limit in summer even when sheltering in thermal refuges. As air temperatures increased above 32°C, lizard activity declined markedly as individuals sought refuge in cool microhabitats while still incurring substantial metabolic costs. We estimate that warming over the last two decades has required these lizards to increase their energy intake by over 20% to offset metabolic losses caused by rising temperatures. Our results show that recent increases in temperature are sufficient to exceed the thermal and metabolic limits of temperate-zone lizards. Extended periods of high temperatures could place natural populations of ectotherms under significantly increased environmental stress and contribute to population declines and extinction. Biological sciences/Ecology/Climate change ecology Earth and environmental sciences/Ecology/Behavioural ecology Ectotherm microclimate refugia thermal tolerance activity window Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Human induced climate change is occurring rapidly 1 and challenging the ability of many species to respond in ways they have in the past 2 . Ectotherms are considered particularly vulnerable to the effects of climate change 3 because of low tolerance to high temperatures and limited ability to regulate their thermal physiology 4 . For most ectotherms, even limited exposure to temperatures beyond their thermal maxima can be fatal 5 , 6 forcing them to rely on thermal regulation through altered behaviour to avoid overheating during the warmest times 4 . As global temperatures rise, more species may reach the limits of their ability to avoid critically high temperatures at key points in their life cycle, leading to population declines and extinctions 7 – 9 . To counter high temperatures, ectotherms can alter their behaviour by seeking shelter in thermal refuges. However, spending more time in thermal refuges may come at a cost by decreasing the time available for other activities such as feeding and reproduction, potentially leading to lower rates of growth, survival and fecundity 2,10−12 and increasing risks of local extinction 10 , 13 . These effects could be amplified because reduced food intake has been shown to lower optimal and maximum temperatures for growth, potentially resulting in a catastrophic feedback whereby higher temperatures lead to accelerating metabolic costs by reducing activity times leading to reduced energy intake, which further lowers temperature tolerance 14 . Lizards are considered to be among the ectotherms most vulnerable to climate warming, particularly in the tropics 15 . Tropical species already experience high temperatures, operate near their critical thermal limits 5 , 7 , 8 , 10 , and have greater niche specialization than lizards from higher latitudes 13 , 15 , 16 - although montane tropical lizards may be an exception 17 . The impact of climate warming on temperate-zone lizards is less clear. Some lizards in the temperate-zone may be little affected by warming because they spend much of their time below their thermal optima 7 , 9 , with activity more likely restricted by low rather than high temperatures 4 , 18 , 19 . Climate warming could benefit lizards restricted by low temperatures by increasing their daily activity window, leading to fitness gains 18 – 22 . Alternatively, some temperate zone lizards could be vulnerable to climate warming if they operate in thermal environments close to key operational thresholds 23 , 24 with little capacity to increase those thresholds 23 . Studies predicting the effects of increasing temperature on lizards are often based on models that couple relationships between body temperature and performance, measured in the laboratory, with forecasted temperature changes used to predict likely outcomes in the field, often at broad spatial and temporal scales 13 , 18 , 22 , 25 . A difficulty with this approach is that models often use predicted changes in overall temperature as input; an approach which may not reflect what happens in the field where lizards can regulate their temperature by adjusting the amount of time they spend in different thermal microhabitats 26 – 30 . Previous field studies in which behavioural thermoregulation, involving individuals using refugia to stay within a preferred temperature range, have been largely limited to observations or intermittent measurements of lizard temperatures over several days 31 – 33 . Continuous quantitative data on the microhabitats occupied by lizards, coupled with fine scale temperature measurements within those microhabitats to assess thermal preferences, are rare 34 – 36 . This is because such data are particularly difficult to gather in the field requiring, as they do, intense study of single species and their thermal landscape. Nevertheless, gathering such information is critical to evaluating the extent to which behavioural thermoregulation can mitigate the impact of increasing temperatures on individual and population-level performance 21,22,27,30,37−39 . Here, we aim to better understand the potential risk of climate warming to temperate-zone lizards by examining how a small, diurnal lizard, the endangered Canberra grassland earless dragon ( Tympanocryptis lineata ), uses thermal microhabitats to behaviourally regulate its temperature. Tympanocryptis lineata is a grassland specialist that is confronted with wide daily and seasonal temperature fluctuations, experiencing summer ground temperatures as high as 70°C in exposed sites, and entering brumation in winter when temperatures fall below 0 °C. To escape temperature extremes, individuals commonly shelter in narrow, vertical burrows excavated in the soil by grassland arthropods, with burrows typically 1-2.4 cm wide and dug to a depth of 10–25 cm. These small burrows, along with the bases of dense tussock grasses, provide critical thermal refuges for the dragons. The small size of T. lineata (5–8 g; SVL 50–60 mm) makes them an ideal species to simultaneously record body temperature and environmental temperature using continuous temperature-sensitive radiotelemetry 40 . External temperature-sensitive transmitters and loggers have been shown to correlate well with skin temperatures (difference < 1 °C) for both ectotherms 34 – 36 , 41 , 42 and endotherms 43 – 47 and reliably predict body temperatures for small species (< 30 g) with differentials of less than < 2 °C 40 , 47 . In some cases, transmitters may heat or cool slightly faster than body temperature, but for an ectotherm less than 10 g the lag time between external and body temperature is less than a few minutes 40 , 48 . In this study, we address two questions that have been difficult to quantify in the field: (1) to what extent are individuals able to alter their use of microhabitats to buffer themselves from temperature extremes? and (2) as increasing temperatures extend the time individuals spend in thermal refuges, are activity windows restricted to the extent that energetic costs are likely to affect performance? Results We attached temperature sensitive transmitters to 28 adult T. lineata (17 males, 13 females) that were captured and released at four grassland sites during the breeding season (austral spring-summer) over two consecutive years (Oct-Feb 2012/13 and 2013/14). Laboratory tests in calibrated incubators showed that transmitter temperature (T trans ) was a good predictor of dragon skin (T skin ) and body temperature (T body ) (T skin =0.924T trans -0.013, R 2 = 0.939, P < 0.0001; T body =1.063T trans -3.20, R 2 = 0.947, P < 0.0001), with T skin measured using copper-constantan thermocouples attached to the dorsal surface of animals, and T body measured using thermocouples inserted 4 mm into the cloaca. In the field, the transmitters recorded the temperature (T trans ) of free-ranging individuals every 10 minutes for a total of 278 lizard-days and 19735 transmitter measurements during daylight hours. At the same time, we measured microclimate temperatures throughout these grasslands. We used shaded ground temperature (T shade ) as a baseline against which to compare temperature in two other microhabitats: exposed open ground (T sun ) and the temperature in arthropod burrows (T burrow ). Ground temperatures in the open sun can be extreme in these grasslands during late spring and summer, with an average daily maximum of 59°C and temperatures sometimes exceeding 70°C (Fig. 1 a). Shaded ground temperatures were cooler (average daily maximum 38°C), but even these could exceed 50°C. Burrows, in contrast, provided a more thermally buffered environment (Fig. 1 a): while shaded ground temperatures ranged from − 3 to 59°C, burrow temperatures ranged from 12 to 36.5°C (mean 23.2°C). Shuttling between microhabitats allowed individuals to stay between the critical maximum (38.3 to 42.8°C, mean = 40.6 ± 0.8°C) and minimum (8.3 to 11.5°C, mean = 9.9 ± 0.8°C) skin temperatures recorded in laboratory experiments (Fig. 1 b). Plotting dragon transmitter temperature (T trans ) as a function of shaded ground temperature (Fig. 1 b) revealed how dragons altered their use of thermal microhabitats as shaded ground temperature changed. Points above the 1:1 line were transmitters that were warmer than shaded ground, indicating dragons were in more open microhabitats, while points below the line indicate that dragons were occupying cooler microhabitats such as burrows. Across the range of shaded ground temperatures (-3 to 59°C), average transmitter temperature remained within the thermal tolerance limits of the species (skin temperature 9.9 to 40.6°C; Fig. 1 b) because individuals adjusted the amount of time they spent in different thermal microhabitats (Fig. 2 ). For example, when shaded ground temperature fell below the lower critical thermal limit for the species, average transmitter temperature remained above this limit because dragons spent more time in thermally buffered sites, such as burrows, particularly at night and during the coolest parts of the day (Fig. 2 ). As evidence for this, the distribution of transmitter temperatures closely matched the distribution of burrow temperatures when shaded ground temperature was less than 15°C (Fig. 3 ). At these temperatures, burrows were generally warmer than both shaded and open ground sites (Fig. 1 a, Fig. 3 ). At shaded ground temperatures between 15–25°C, the distribution of transmitter temperatures closely matched the distribution of open ground (T sun ) temperatures (Fig. 3 ), implying that dragons spent much of the time using open sites in this temperature range (see Fig. 2 ). As shaded ground temperature rose above 25°C, open ground temperatures steadily exceeded transmitter temperatures, implying that dragons increasingly avoided warmer open sunny sites at higher temperatures (Fig. 3 ). At shaded ground temperatures greater than 40°C, most transmitter temperatures were below this value, implying that dragons were avoiding shaded ground sites in favour of cooler microhabitats, such as burrows. The point at which shaded ground temperatures were sufficiently warm that dragons began to avoid this microhabitat is indicated by the sharp inflection in the average transmitter temperature curve when shaded ground temperature approached the upper thermal tolerance limit of the species (Fig. 1 b). At shaded ground temperatures above about 35°C, average transmitter temperature remained relatively constant, and below the thermal tolerance limit, as dragons increasingly sought refuge in cooler microhabitats. These data show that dragons sought refuge in more thermally buffered environments, such as burrows, at both high and low shaded ground temperatures to maintain average temperature within their thermal limits. As such, key changes in the use of microhabitats in the field coincided with the upper and lower critical temperature thresholds of this species. Essential activities, such as feeding and finding mates, require dragons to be above-ground 49 , but high and low above-ground temperatures force dragons into thermal refuges such as burrows. Seeking refuge is a well-known activity in lizards to escape high temperatures 22 , 26 50 . However, we lack field data quantifying the extent to which high temperatures curtail above-ground activity, and hence the impact that high temperatures may have on the animal’s ability to forage above-ground to support their energetic demands. To understand the impact of a shortened activity window caused by high temperatures, we classified transmitter temperatures according to whether lizards were above or below shaded ground and burrow temperature for each transmitter temperature record. Transmitter temperatures below shaded ground temperature indicate the dragon was using a thermal refuge to escape heat, while transmitter temperatures above shaded ground temperature, but at or below burrow temperature, indicate that the dragon was using a refuge to escape cold. During daylight hours, above-ground activity was curtailed in the early morning and late evening as dragons sought refuge from cooler above-ground temperatures, particularly on days when the daily maximum air temperature was low (Fig. 4 ). On days when the maximum air temperature rose above 30°C, dragons increasingly sought thermal refuge from high temperatures during the middle of the day (Figs. 2 & 3 ). This resulted in a quadratic relationship between daily activity (the proportion of daylight hours dragons were active above-ground) and maximum daily air temperature (Fig. 5 a). Dragons spent, on average, more than half the day active when daily maximum air temperatures were between about 20–32°C. As the daily maximum temperature rose or fell beyond these values, dragons spent an increasing proportion of the day in thermal refuges. On days when the maximum air temperature approached 40°C, dragons spent about 80% of daylight hours in thermal refuges to avoid the heat of more open environments and to maintain their temperature within thermal tolerance limits. This pattern is similar to that projected for the widespread military dragon ( Ctenophorus isolepis ) 51 . To assess the implications of a shortened activity window on lizard energetics, we measured resting metabolic rates (RMR) for 12 post-absorptive individual T. lineata (6 males, 6 females) at five temperatures (20, 25, 30, 35 and 38°C) using flow-through respirometry 52 , 53 . Resting metabolic rates (RMR in ml O 2 g − 1 h − 1 ) were then used to calculate the average amount of energy expended by a dragon at rest in the field based on the recorded transmitter temperatures averaged on an hourly basis. We calculated the mean RMR of a dragon for each day that we had transmitter records by averaging the hourly RMR values and then plotted the daily mean as a function of maximum daily air temperature (Fig. 5 b). The mean RMR increased as daily maximum air temperature rose, as expected for an ectotherm, but started to level out once daily maximum temperature exceeded about 32°C. This is likely to occur because dragons used thermal refuges to escape the heat on warmer days (Figs. 1 b, 3 , 4 a) and thus limit their metabolic losses. Consequently, the mean RMR divided by the number of hours active increased sharply on days when the maximum temperature rose above 32°C (Fig. 5 c). We use the term energetic cost for the ratio: mean RMR / number of hours active. This ratio measures the amount of energy dragons must obtain per active hour by feeding to balance their daytime resting metabolic losses. While metabolic losses would also occur at night, dragons usually occupied cool night-time microhabitats, even on hot days (e.g. Figure 3 ), meaning nocturnal metabolic losses would be low. The resting energetic cost, and hence the energy dragons must obtain while active to offset this cost, increased about three-fold as daily maximum air temperature rose from 32 to 40°C (Fig. 5 c). Hence, while activity time was reduced on days with both low and high maximum temperatures, dragons incurred a substantially higher energetic cost on hot days because of greater metabolic losses at high temperatures coupled with reduced activity time in which to recoup those losses. Confining our calculations to daylight hours only and using resting metabolic rate as our measure of energy expenditure provides a conservative measure of total daily energy expenditure. If lizards are active, rates of energy metabolism are typically 1.5-3 times resting levels 54 , 55 . To examine changes in the energetic cost over time, we gathered data on daily maximum air temperature during breeding season (October-February) for the years 1941–2020 recorded at Canberra Airport, the nearest climate station to our study sites (all sites were within 7 km of the climate station and at the same altitude). For each day during this period, we estimated the proportion of daylight hours dragons were active and the mean energetic cost based on the maximum daily temperature and the relationships shown in Fig. 5 . We then calculated the mean maximum temperature, the mean activity time, and the mean energetic cost per day during the breeding season for each year (expressed as an anomaly from the mean for the period 1960–1990). Mean daily maximum temperature fluctuated around the mean from 1940–2000 but has increased in the period 2000 to 2020 to be on average more than 2°C greater than the mean, resulting in a decline in mean daily activity during summer of up to 4% (Fig. 6 ). The combination of hotter days and a shorter activity window caused the mean energetic cost for individual lizards to increase by an average of more than 20% during the same period (Fig. 6 ). This implies that between 2000–2020, increasing maximum daytime temperatures required dragons to increase their energy intake during their active period by more than 20%, relative to the 1960–1990 mean, to offset metabolic losses. Discussion Ectothermic activity in temperate regions is usually constrained by both low and high temperatures, meaning it is unclear whether warming will be beneficial (increase the activity window) or detrimental (decrease the activity window) to temperate-zone populations. We show that during the critical spring-summer months of the breeding season the activity window declines sharply for T. lineata as daily maximum temperature rises above 32°C, implying climate warming that resulted in a greater number of hotter days could negatively affect populations by forcing dragons to spend more time in thermal refuges, reducing the time available for essential activities such as foraging, feeding and finding mates. On hotter days, reduced activity coupled with greater metabolic losses incurs a substantial energetic cost. Between the years 2000–2020, we estimate that dragons had to increase their energy intake during their daily active period by more than 20% to offset metabolic losses owing to rising temperatures, even after accounting for behavioral adjustments to limit those losses by using thermal refuges. This estimate is conservative because we have used resting metabolic rate as our measure of energy expenditure and confined our calculations to daylight hours only. T. lineata will be active for significant portions of the day (not just resting) and will range widely to forage 56 . Given that activity will increase energy expenditure beyond that indicated by resting metabolic rate, and that dragons will continue to expend energy at night, the energetic impact of the observed increase in maximum daily temperature over the last 20 years (Fig. 5 ) will be even higher than our analysis reveals. Populations of T . lineata suffered widespread collapse between 2006-2010 57 . Our results suggest it is possible that an increase in the number of hot days in Canberra, with a sustained increase commencing around the year 2000 (Fig. 6 a), could have played a role in this population collapse by reducing activity times (Fig. 6 b) and increasing metabolic costs (Fig. 6 c) that may have been difficult to recoup. Sinervo et al 2 speculated that extinctions of lizard populations are likely if activity is restricted by 7 h or more per day, particularly during critical reproductive months. Our results show that T. lineata would exceed this threshold when daily maximum air temperatures rise above 35°C (Figs. 3 and 4 a) in the breeding season (October to February) when average day length is 13.5 h and T. lineata are active for less than 5.5 h. This is close to the daily maximum temperature where metabolic costs begin to rise sharply (Fig. 5 c), suggesting the 7 h activity restriction roughly corresponds to a critical threshold in energy expenditure for T . lineata . The microclimate temperatures we recorded were substantially higher than those obtained previously using an inanimate lizard model in temperate Australia 18 . Our data suggest that ectotherms at temperate-zone sites can reach dangerously high temperatures in open environments and may not be able to maintain optimal temperatures (30–35°C) in shaded environments 18 , 22 . In the scenarios modelled previously, lizards in full sun achieved body temperatures of around 40°C, and lizards thermoregulating by shuttling between sun and shade maintained body temperatures of up to 33°C 18 . Our results for T. lineata contrast sharply with these values, indicating that shaded microhabitats alone are insufficient to keep temperatures below critical thermal thresholds and that more thermally buffered refuges, such as burrows, are often required for survival (Figs. 1 and 2 ). Grasslands are exposed environments with the only shade available close to the ground, or below ground in burrows during periods of low vegetation cover, such as in droughts. Consequently, grassland lizards will be exposed to radiant and conductive heat at ground level that far exceeds the shaded temperatures 1 m above ground at which air temperatures are measured. It is therefore likely that lizards are exposed to much higher temperatures in grasslands, relative to more heavily vegetated environments with canopy shade, for a given air temperature. At warmer temperatures, we observed that individual T. lineata remained active by shuttling between open and shaded microhabitats, often moving into burrows to cool (Figs. 1 , 2 & 3 ). This shuttling behaviour is believed to be the key to avoiding temperature extremes and surviving climate warming in temperate environments 8 , 18 , 19 but has rarely been measured directly using body temperatures. The shuttling behavior we observed provides considerable capacity for T. lineata to manipulate body temperature, allowing lizards to remain above-ground on warm but not excessively hot days. However, this behavior depends on the availability of cooler microhabitats such as heavily shaded ground and burrows. Consequently, T. lineata is susceptible to the loss of these refuge habitats, including the removal of above-ground vegetation by burning, drought or overgrazing, and the loss of invertebrate species responsible for digging the burrows that dragons rely on for shelter 56 , 57 . Vegetation loss associated with drought, in addition to high temperatures, may have also contributed to the widespread population collapse in T. lineata from 2006-2010 57 . While it is possible that climate warming will extend the seasonal activity window for T. lineata , thereby shifting some activities to different times of the year, the extreme temperatures now experienced by these lizards in thermal refuges such as burrows (max 36.5°C) suggests there may no longer be safe refuges on extreme days when there is little above-ground vegetation. Our data offer strong empirical support for the proposition that high summer temperatures caused by climate warming may repeatedly exceed the thermal and metabolic limits of temperate-zone lizards. Extended periods of high temperatures and reduced activity times over longer periods could place natural populations of lizards under significantly increased levels of environmental stress and contribute to population decline and local extinction. Vulnerability to warming will be a function of the thermal requirements of a species, their ability to adapt both physiologically and behaviourally to increased temperatures 58 , and the availability of thermal refugia. Consequently, habitat degradation that alters the availability of thermal refugia will interact with climate warming to further imperil populations by reducing the opportunity for individuals to escape extreme heat while simultaneously increasing the need to increase their energy intake. Methods Animal ethics approval was granted by the University of Canberra’s Committee for Ethics in Animal Experimentation (CEAE 11–22 and CEAE 15 − 08) and all experiments were performed in accordance with the relevant guidelines and regulations as per the approval. Grassland Earless Dragons were handled and collected under permits from the ACT Government Territory and Municipal Services (Licence to Take LT2012604; Licence to Import LI2011594; LI2012737) and NSW Office of Environment and Heritage (Scientific Licence Section 132c SL100756). Study Species and Location We studied the Canberra Grassland Earless Dragon, Tympanocryptis lineata (formerly T. pinguicolla ) 59 , a small agamid now confined to sites near Canberra, Australia (36.31°S, 149.20°E; 580 m a.s.l). 57 , 59 , 60 . The Köppen-Geiger climate classification system defines Canberra as “Cfb”: temperate, with no dry season, warm summers (12.5–27.1 °C) and cool winters (0.6–12.2 °C) (mean daily min-max temps; Australian Bureau of Meteorology (BOM) 1939–2008), although summer temperatures have risen in recent years (mean max 30.3 °C January 2008–2022). The maximum monthly summer temperature on record occurred in the last 5 years (Dec 2019 41.1 °C, Jan 2020 44°C, Feb 2020 42.7 °C). Temperatures reported by BOM are recorded at 1 m height in a shaded Stevenson box. Ground surface temperatures in the sun are much higher and can reach 70 °C (Fig. 1 ). Precipitation is relatively consistent throughout the year, with slightly more rainfall in spring (mean monthly 178 mm) and summer (168 mm) than autumn (141 mm) and winter (128 mm) (BOM 1939–2008). Frost is common in winter months. Temperature-Sensitive Radio-Telemetry We captured T. lineata at four sites in the Australian Capital Territory (35.3408° S, 149.1814° E.) and adjacent New South Wales (35.3737°S, 149.1940°E,.) using modified pitfall traps that mimic arthropod burrows the species uses naturally 61 . The habitat at all sites was natural temperate grassland characterised by Rytidosperma-Austrostipa open tussock grassland with no trees or shrubs and a history of livestock grazing with little to no fertilization or pasture improvement 56 , 62 . Few surface rocks exist and the primary refugia available for T. lineata are short, vertical burrows excavated by the Canberra Raspy Cricket ( Cooraboorama canberrae ) and wolf spiders (Lycosoidea). Following capture, we measured body mass (mean 6.1 g), snout-vent-length (mean 53.9 mm), and sexed dragons by inspecting for hemipenes. Individuals greater than 4.3 g were fitted with an external temperature-sensitive radio transmitter (model BD-2XT, Holohil Systems, Canada or model PIP31, Sirtrack, NZ) to record the temperature that individuals were experiencing, with the transmitter pulse rate varying as a function of temperature 45 , 46 Each transmitter was calibrated in a water bath to the nearest 0.1 ºC before attachment and transmitter pule rates were converted to temperatures (T trans ) using a quadratic polynomial function fitted to calibration data using least squares regression. The calibration was re-confirmed after transmitters were retrieved 46 . Transmitters (0.43–0.55 g) were attached to the dorsal base of the tail posterior to the vent opening with the flexible 10 to 15 cm thin whip antenna positioned to run parallel to the tail 63 . On average, transmitters represent less than 7% of the lizard’s body mass (range 4.7 to 9.1%; dragon mean mass 6.1 g; range 4.6 g to 9.1 g, n = 44) similar to those used in other comparable studies 63 , 64 , within the range of < 10% of animal body mass as recommended for small (< 30 g) non-flying animals 47 . Transmitter battery life varied from 5 to 40 d and individual lizards were tracked from 1 to 38 d (mean 14.5 d). We collected transmitter data using remote receiver/data logging stations comprising a three element Yagi antenna (Sirtrack, NZ) communicating with a receiver data logger (SRX_DL2, Lotek Wireless Inc, Canada). The data loggers were programmed to search for each lizard frequency and record transmitter pulse rates of each transmitter for 90 s every 10 min. The precise location of each lizard was confirmed several times each day using a handheld radio-telemetry device and a short wand antenna. Lizards were captured and tracked at four separate study sites. Microhabitat Temperatures We recorded environmental temperatures in the microhabitats available to T. lineata at each of the four study sites during the same period that transmitter temperatures were monitored. At each site, temperature data loggers (Thermochron iButtons®, Model DS1921G, ± 0.5°C, Maxim Integrated Inc., USA) were placed in microhabitats previously identified as used by T. lineata and in which T. lineata had been captured and tracked. The microhabitats measured were: 1) exposed open ground with vegetation cover less than 2 cm high (T sun ); 2) shaded open ground (T shade ); and 3) at the bottom of 15–20 cm deep burrows (T burrow ). iButtons used to measure open ground temperatures (T sun ) were placed in lengths of copper pipe (24 mm diameter, 55 mm length) that had been spray painted beige and terminated with 22 mm diameter plastic plugs 70. Shaded temperatures at ground level (T shade ) were measured using iButtons attached to the base of a wooden stake and covered to prevent radiant heating, and burrow temperatures (T burrow ) were measured using iButtons placed in handmade burrows (25 mm wide, 15–20 cm deep) formed by hammering a length of stainless-steel pipe into the ground. The depth of burrow at which the iButtons were placed approximated the depth of natural burrows used by T. lineata in the field (n = 42, mean = 16.6 cm depth, range = 10–27 cm). At each study site a minimum of 3 replicate sets of iButtons were installed. iButtons have been used extensively to sample habitat temperatures (e.g. Hubbart et al 2005; Doucette et al 2011; Brabyn et al 2014) and have been found to reliably record microhabitat thermal variability and predict temperatures experienced by small ectotherms (Vickers and Schwarzkopf 2015; Moore et al 2017). Maximum daily air temperatures (T air ) were taken at 1 m above ground in a Stevenson screen recorded at the Australian Bureau of Meteorology (BOM) Canberra Airport Site 070351. All four study sites were within a 7 km radius of the Canberra airport and at the same altitude. Respirometry We measured the resting metabolic rate (RMR) of 12 individual T. lineata (6 males, 6 females) from the captive breeding colony at the University of Canberra, Australia using open-flow respirometry to measure oxygen consumption 65 , 66 . Individuals were housed outdoors for several weeks before RMR measurements were conducted in November 2013. Respirometry was conducted on post-absorptive lizards during their rest phase (night) in darkened temperature-controlled cabinets. Individual body mass (± 0.01 g) was measured at the start and end of each trial and a linear rate of mass loss was assumed for calculation of mass-specific metabolic rate 47 . Oxygen consumption was measured using an oxygen analyzer (FOX, Sable Systems International Inc., USA), placed inside an insulated box in a temperature–controlled room at 19 ± 2 ºC. A sub-sampling design was used to keep the rate of flow of the sample air through the analyzer constant (63 ml min − 1 ) throughout the measurements. Outside air was pumped through silica gel to remove moisture while rotameters controlled the rate of airflow to the chambers. After passing through the chamber, excurrent air was dried again using silica gel and the flow rate of air was measured using a mass flow meter (Omega FMA-5606, USA). A chamber flow rate of 100 ml min − 1 was maintained throughout the experiments, which was sufficient to maintain the oxygen content in the excurrent air above 20%. The excurrent air from each chamber (2–3 chambers used) was sampled every three minutes, followed by three minutes sampling of a reference channel of dried outside air. Thus, one measurement for each lizard was obtained every 9 to 12 minutes 67 – 69 . Measurements of ambient temperature (T a ) were taken simultaneously to those of RMR via calibrated T-type thermocouples in the respirometry chambers. Data acquisition and processing were performed using software written by G. Körtner 70 . A respiratory quotient of 0.85 was assumed for all measurements and the rate of oxygen consumption was calculated using Eq. 3a of Withers 66 . RMRs were calculated for each individual as the average of the six consecutive lowest VO 2 values in resting individuals at each T a 71 . Chamber temperatures were set to 20, 25, 30, 35 and 38°C for a minimum of 2 hours. Only data from the final 75 min of testing once the chamber had reached the designated T a were considered in the analysis. Critical Thermal Limits All tests for thermal tolerance thresholds took place in an air-conditioned laboratory at 21°C. To prepare individuals for testing two T-type copper-constantan thermocouples (MicroDAQ) were attached to each dragon using clear surgical tape. One thermocouple was taped to the dorsal surface of the individual and a second thermocouple inserted several millimetres into the cloacal and taped in position. Dorsal T skin and cloacal temperatures (T body ) were recorded every minute. We used the panting threshold, the body temperature at which dragons started to pant open-mouthed and started to turn over to expose their lighter coloured ventral abdomen to the heat, as our measure of the dragon’s upper thermal tolerance. In Fig. 1 , we show the skin temperature recorded on lizards at this thermal maximum (CT max in Fig. 1 ). Individuals were placed in a plastic holding tank containing clay from the native grasslands under a 160 W self-ballasted mercury-vapour bulb and observed until they reached the panting threshold. The critical thermal minimum was determined using the same individual T. lineata as used for the CT max experiments. Each individual was placed in a dry 0.5 L plastic container submersed to the rim in ice water at 3.5 to 5°C. Once T body had reached 16°C individuals were gently flipped over onto their back and allowed to right themselves. CT min was considered to have occurred when individuals could no longer right themselves and became immobile. The CT min shown in Fig. 1 is the skin temperature recorded on animals at this lower thermal limit. Data Analysis Transmitter pulse rates were converted to temperatures (T trans ) using a quadratic polynomial function fitted to calibration data using least squares regression. Transmitter pulse rates (accuracy 0.1 beats per minute) were averaged across each 90 s recording period to obtain a single T trans for each individual every 10 min. Days with recordings that spanned less than 10 h, recordings outside daylight hours, and time periods with ≥ 120 min of missing data were excluded, resulting in a final dataset that comprised data from 28 lizards, 144 days, and 19735 transmitter temperature observations. Values of T trans were matched by time and location (study site) to the mean of the three microclimate temperature replicates for T sun , T shade, and T burrow at the respective site. This matching allowed us to determine the most likely microhabitat that individuals occupied at each recording time (the value of T sun , T shade, or T burrow that most closely matched T trans ). Dragons always remained within 100 m, and rarely moved more than 50 m, from the capture location creating small, uniform study areas. As the temperature differentials between each of these three microclimate types often exceeded 10 °C we could be confident in our inferences of dragon microclimate selection. We used the lizard transmitter and microhabitat temperatures, along with variation over time in the individual transmitter temperatures and signal strength 72 , to classify lizards as either active or inactive at each recording time. We scored an individual as active if T trans was greater than both T shade and T burrow during daylight hours (sunrise to sunset), indicating that the lizard was in the open and not seeking refuge (Fig. 2 ). Individuals were scored as inactive if T trans was below T shade for at least two consecutive measurements (20 minutes), implying lizards were in cooler refugia such as burrows, or if at least three consecutive measurements of T trans showed little variation in transmitter temperature (defined as a difference of < 0.5°C) or signal strength, indicating the lizard was stationary at one location for at least 30 minutes. Data were processed and analysed using R 3.2.1 (The R Foundation for Statistical Computing, Austria). Declarations Acknowledgements Funding was provided by an Australian Research Council (LP0776987) to SS, BG, AG, Tariq Ezaz, WO, and Don Fletcher and Conservation Research, Environment Planning and Sustainable Development Directorate, ACT. We thank Wendy Ruscoe and Jacqui Richardson for management of the lizard colony, and Emma Cook, Rod Pietsch, Samatha Vertucci, Brett Howland, Renee Brawata, Muhammad Rais, Don Fletcher and Emma Carlson for advice and field assistance. Fritz Geiser and Gerhard Körtner provided respirometry equipment and advice for metabolic measurements and Dan Noble provided helpful comments on an earlier draft of the manuscript. Contributions L.D., S.S., W.O., B.G. and A.G. conceived and designed the study. L.D. developed and oversaw all aspects of the field and laboratory data collection. R.D., L.D., B.G., and S.S. developed the analytical framework and analysed the data. All authors contributed to the discussion of the results and writing of the manuscript. Data Availability All data and code files related to this manuscript are available at https://zenodo.org/record/7305580#.Y2rEIHZBxaQ (DOI: 10.5281/zenodo.7305580). References Loarie, S. R. et al. The velocity of climate change. Nature 462 , 1052-U1111, doi:10.1038/nature08649 (2009). Sinervo, B. et al. Erosion of lizard diversity by climate change and altered thermal niches. Science 328 , 894-899, doi:10.1126/science.1184695 (2010). Chown, S. L. et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2228630","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":150781017,"identity":"5b081e2b-e154-4e63-8cfc-aecb9fe0ff32","order_by":0,"name":"Lisa I Doucette","email":"","orcid":"","institution":"University of Canberra","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lisa","middleName":"I","lastName":"Doucette","suffix":""},{"id":150781020,"identity":"addaf6bf-205c-424e-a9b6-7061da79b4fd","order_by":1,"name":"Richard P Duncan","email":"","orcid":"","institution":"University of Canberra","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Richard","middleName":"P","lastName":"Duncan","suffix":""},{"id":150781023,"identity":"ca994d6c-8db1-48ba-b8be-3da4eded3320","order_by":2,"name":"William S Osborne","email":"","orcid":"","institution":"University of Canberra","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"William","middleName":"S","lastName":"Osborne","suffix":""},{"id":150781026,"identity":"5c9cf8a3-f95e-4696-9a16-bc0fcaaae50d","order_by":3,"name":"Murray Evans","email":"","orcid":"","institution":"ACT Government","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Murray","middleName":"","lastName":"Evans","suffix":""},{"id":150781028,"identity":"bc8cd71b-3205-42b8-9ac2-53531034fbe0","order_by":4,"name":"Arthur Georges","email":"","orcid":"","institution":"University of Canberra","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Arthur","middleName":"","lastName":"Georges","suffix":""},{"id":150781030,"identity":"7aef5f5a-566c-47b4-84c8-3fd64269b3bb","order_by":5,"name":"Bernd Gruber","email":"","orcid":"","institution":"University of Canberra","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bernd","middleName":"","lastName":"Gruber","suffix":""},{"id":150781031,"identity":"64e74bcf-1f4b-4bb3-ab76-ad2f554120ff","order_by":6,"name":"Stephen D Sarre","email":"data:image/png;base64,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","orcid":"","institution":"University of Canberra","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Stephen","middleName":"D","lastName":"Sarre","suffix":""}],"badges":[],"createdAt":"2022-11-02 05:59:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2228630/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2228630/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-35087-7","type":"published","date":"2023-06-13T21:10:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":28958290,"identity":"c860b98b-7c9b-4916-ae81-592bc7ad3e38","added_by":"auto","created_at":"2022-11-11 15:56:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":583614,"visible":true,"origin":"","legend":"\u003cp\u003eEnvironmental temperatures matched to dates, times, and locations of transmitter temperatures (T\u003csub\u003etrans\u003c/sub\u003e) for \u003cem\u003eT. lineata\u003c/em\u003e recorded from Nov 2012 to Apr 2013 and Oct 2013 to Feb 2014. a) Temperatures (36,059 measurements over 144 days) recorded in burrows (T\u003csub\u003eburrow\u003c/sub\u003e = blue) and in copper pipe models placed in full sunlight (T\u003csub\u003esun\u003c/sub\u003e = red) in relation to shaded ground temperatures (T\u003csub\u003eshade\u003c/sub\u003e). The black line is a one-to-one comparison of shaded ground to other microhabitat temperatures. b) \u003cem\u003eT. lineata\u003c/em\u003e transmitter temperatures (T\u003csub\u003etrans\u003c/sub\u003e; 28 individuals, 144 days; 19,735 temperature measurements) plotted against shaded ground temperature. Grey circles are individual temperatures averaged across 90 seconds. Filled black circles are mean transmitter temperature for each degree of shaded ground temperature. The solid black line is the one-to-one line comparing T\u003csub\u003etrans\u003c/sub\u003e to T\u003csub\u003eshade\u003c/sub\u003e. The horizontal lines show \u003cem\u003eT. lineata\u003c/em\u003e skin temperature (CT\u003csub\u003emax\u003c/sub\u003e and CT\u003csub\u003emin\u003c/sub\u003e) at its upper and lower thermal limits (see Methods).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2228630/v1/183638703863976764f6af8a.png"},{"id":28958292,"identity":"595f214c-e838-4797-b6d4-7972a8b0672b","added_by":"auto","created_at":"2022-11-11 15:56:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22499,"visible":true,"origin":"","legend":"\u003cp\u003ePlot of \u003cem\u003eT. lineata\u003c/em\u003e transmitter temperatures (T\u003csub\u003etrans\u003c/sub\u003e) taken at 10 min intervals (circles) for one individual on one day, and microhabitat temperatures (T\u003csub\u003esun\u003c/sub\u003e = red line, T\u003csub\u003eshade\u003c/sub\u003e = orange line, and T\u003csub\u003eburrow\u003c/sub\u003e = blue line) at the same location for the same period. Filled circles indicate times when the individual was classified as active; open circles indicate times when the individual was classified as inactive. Dotted vertical lines show the times of sunrise and sunset. The dragon is in a warm refuge prior to sunrise, as indicated by T\u003csub\u003etrans\u003c/sub\u003e being greater than both T\u003csub\u003esun\u003c/sub\u003e and T\u003csub\u003eshade\u003c/sub\u003e, and remains inactive until ~1.5 hours after sunrise when T\u003csub\u003etrans\u003c/sub\u003e starts to track T\u003csub\u003esun\u003c/sub\u003e, indicating the dragon has moved into the open where it remains for over two hours. As T\u003csub\u003esun\u003c/sub\u003e rises above about 40°C, the dragon starts to shuttle between open and shaded sites before moving into a cool refuge (T\u003csub\u003etrans\u003c/sub\u003e \u0026lt;T\u003csub\u003eshade\u003c/sub\u003e) in the middle of the day where it remains inactive for several hours. It emerges again for a period in the late afternoon before becoming inactive again at around 1800 hours, where T\u003csub\u003etrans\u003c/sub\u003e starts to fall steadily in parallel with falling ambient temperatures (T\u003csub\u003esun\u003c/sub\u003e and T\u003csub\u003eshade\u003c/sub\u003e), with T\u003csub\u003etrans\u003c/sub\u003e levelling out around sunset at a value greater than T\u003csub\u003esun\u003c/sub\u003e and T\u003csub\u003eshade\u003c/sub\u003e, indicating the dragon was in a warmer refuge for the night. Note, that T\u003csub\u003eburrow\u003c/sub\u003e is the temperature at 150-200 mm depth in one burrow at the site, but the dragon could have taken refuge in another burrow and at a different depth, such that we don’t expect T\u003csub\u003etrans\u003c/sub\u003e to track T\u003csub\u003eburrow\u003c/sub\u003e precisely when the dragon is sheltering in a burrow.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2228630/v1/17ddc7212495ae3a4fde0378.png"},{"id":28959123,"identity":"1c107d1d-3383-46c6-ad69-ecbf678e4b2e","added_by":"auto","created_at":"2022-11-11 16:04:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":310213,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency distribution of transmitter and microhabitat temperatures as a function of shaded ground temperature grouped into 5°C bands (indicated by the numbers at the top right of each panel). The mean shaded-ground temperature (T\u003csub\u003eshade\u003c/sub\u003e) for each band is shown as the black vertical line. Blue shows the distribution of burrow temperatures (T\u003csub\u003eburrow\u003c/sub\u003e), red the distribution of full sun temperatures (T\u003csub\u003esun\u003c/sub\u003e), and black line/gray shading the distribution of transmitter temperatures (T\u003csub\u003etrans\u003c/sub\u003e) for each band of shaded-ground temperature.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2228630/v1/03f8ca3f4d1550ef4154b194.png"},{"id":28959560,"identity":"d473987c-84df-4407-89be-aa7882d5a5e0","added_by":"auto","created_at":"2022-11-11 16:12:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":147075,"visible":true,"origin":"","legend":"\u003cp\u003eProportion of time for each hour of the day between sunrise and sunset that \u003cem\u003eT. lineata\u003c/em\u003e were classed as active, with the days grouped by maximum daily air temperature into 5°C bands (indicated at the top of each panel, along with the number of lizard days, \u003cem\u003en\u003c/em\u003e, in each group). As maximum daily air temperature rises above 30°C, there is an increasingly marked period of inactivity around the middle of the day as dragons escaped the heat by sheltering in thermal refuges.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2228630/v1/1e5a5476bb40f0f09aae4a7f.png"},{"id":28959125,"identity":"c2ddcc59-3baa-42b3-bbbb-6d0427e933ce","added_by":"auto","created_at":"2022-11-11 16:04:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":718458,"visible":true,"origin":"","legend":"\u003cp\u003eA) The relationship between maximum daily air temperature and proportion of daylight hours active. The solid red line is a quadratic regression model fitted to the data, for which both the linear and quadratic terms were significant (P \u0026lt;0.0001, R\u003csup\u003e2\u003c/sup\u003e= 0.71). B) The relationship between maximum daily air temperature and mean hourly resting metabolic rate, with a spline (red line) fitted to indicate the trend. C) The relationship between maximum daily air temperature and mean energetic cost during each day (mean hourly resting metabolic rate / proportion of daylight hours active), with a spline (red line) fitted to indicate the trend. In each panel, each filled circle represents one day, with the size of the circle proportional to the number of dragons measured on that day (total of 278 dragon days).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2228630/v1/3ea82e43c94da82e3a7a699b.png"},{"id":28958288,"identity":"3d596b72-712f-4684-b87e-a1f0cab0fbe9","added_by":"auto","created_at":"2022-11-11 15:56:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1224137,"visible":true,"origin":"","legend":"\u003cp\u003eA) Mean maximum daily air temperature per year measured at the Canberra Airport for the period 1941-2020 and expressed as an anomaly from the mean for the period 1960-1990. B) The mean proportion of time dragons were active during daylight hours (activity window) for each year expressed as a proportion of the mean for the period 1960-1990. C) The mean energetic cost per year (mean hourly resting metabolic rate / proportion of daylight hours active) expressed as a proportion of the mean for the period 1960-1990. In each panel, blue columns indicate years below the 1960-1990 mean; red columns indicate years above the 1960-1990 mean; and the black line is a loess smoother fitted through the yearly values with span = 0.3.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2228630/v1/e2974e956f688f734f2e99ff.png"},{"id":44730965,"identity":"1c20094b-3219-4246-9a2e-397790c2bcee","added_by":"auto","created_at":"2023-10-16 21:38:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1987974,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2228630/v1/00161f04-67bb-4ecc-a53d-ab6efbc8e18b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Climate warming drives a temperate-zone lizard to its upper thermal limits, restricting activity, and increasing energetic costs","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman induced climate change is occurring rapidly\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and challenging the ability of many species to respond in ways they have in the past\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Ectotherms are considered particularly vulnerable to the effects of climate change\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e because of low tolerance to high temperatures and limited ability to regulate their thermal physiology\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. For most ectotherms, even limited exposure to temperatures beyond their thermal maxima can be fatal\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e forcing them to rely on thermal regulation through altered behaviour to avoid overheating during the warmest times\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. As global temperatures rise, more species may reach the limits of their ability to avoid critically high temperatures at key points in their life cycle, leading to population declines and extinctions\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo counter high temperatures, ectotherms can alter their behaviour by seeking shelter in thermal refuges. However, spending more time in thermal refuges may come at a cost by decreasing the time available for other activities such as feeding and reproduction, potentially leading to lower rates of growth, survival and fecundity\u003csup\u003e2,10\u0026minus;12\u003c/sup\u003e and increasing risks of local extinction\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. These effects could be amplified because reduced food intake has been shown to lower optimal and maximum temperatures for growth, potentially resulting in a catastrophic feedback whereby higher temperatures lead to accelerating metabolic costs by reducing activity times leading to reduced energy intake, which further lowers temperature tolerance\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLizards are considered to be among the ectotherms most vulnerable to climate warming, particularly in the tropics\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Tropical species already experience high temperatures, operate near their critical thermal limits\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and have greater niche specialization than lizards from higher latitudes\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e - although montane tropical lizards may be an exception\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The impact of climate warming on temperate-zone lizards is less clear. Some lizards in the temperate-zone may be little affected by warming because they spend much of their time below their thermal optima\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, with activity more likely restricted by low rather than high temperatures\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Climate warming could benefit lizards restricted by low temperatures by increasing their daily activity window, leading to fitness gains\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Alternatively, some temperate zone lizards could be vulnerable to climate warming if they operate in thermal environments close to key operational thresholds\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e with little capacity to increase those thresholds\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eStudies predicting the effects of increasing temperature on lizards are often based on models that couple relationships between body temperature and performance, measured in the laboratory, with forecasted temperature changes used to predict likely outcomes in the field, often at broad spatial and temporal scales\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. A difficulty with this approach is that models often use predicted changes in overall temperature as input; an approach which may not reflect what happens in the field where lizards can regulate their temperature by adjusting the amount of time they spend in different thermal microhabitats\u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Previous field studies in which behavioural thermoregulation, involving individuals using refugia to stay within a preferred temperature range, have been largely limited to observations or intermittent measurements of lizard temperatures over several days\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Continuous quantitative data on the microhabitats occupied by lizards, coupled with fine scale temperature measurements within those microhabitats to assess thermal preferences, are rare\u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This is because such data are particularly difficult to gather in the field requiring, as they do, intense study of single species and their thermal landscape. Nevertheless, gathering such information is critical to evaluating the extent to which behavioural thermoregulation can mitigate the impact of increasing temperatures on individual and population-level performance\u003csup\u003e21,22,27,30,37\u0026minus;39\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we aim to better understand the potential risk of climate warming to temperate-zone lizards by examining how a small, diurnal lizard, the endangered Canberra grassland earless dragon (\u003cem\u003eTympanocryptis lineata\u003c/em\u003e), uses thermal microhabitats to behaviourally regulate its temperature. \u003cem\u003eTympanocryptis lineata\u003c/em\u003e is a grassland specialist that is confronted with wide daily and seasonal temperature fluctuations, experiencing summer ground temperatures as high as 70\u0026deg;C in exposed sites, and entering brumation in winter when temperatures fall below 0 \u0026deg;C. To escape temperature extremes, individuals commonly shelter in narrow, vertical burrows excavated in the soil by grassland arthropods, with burrows typically 1-2.4 cm wide and dug to a depth of 10\u0026ndash;25 cm. These small burrows, along with the bases of dense tussock grasses, provide critical thermal refuges for the dragons.\u003c/p\u003e \u003cp\u003eThe small size of \u003cem\u003eT. lineata\u003c/em\u003e (5\u0026ndash;8 g; SVL 50\u0026ndash;60 mm) makes them an ideal species to simultaneously record body temperature and environmental temperature using continuous temperature-sensitive radiotelemetry\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. External temperature-sensitive transmitters and loggers have been shown to correlate well with skin temperatures (difference\u0026thinsp;\u0026lt;\u0026thinsp;1 \u0026deg;C) for both ectotherms\u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and endotherms\u003csup\u003e\u003cspan additionalcitationids=\"CR44 CR45 CR46\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e and reliably predict body temperatures for small species (\u0026lt;\u0026thinsp;30 g) with differentials of less than \u0026lt;\u0026thinsp;2 \u0026deg;C\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In some cases, transmitters may heat or cool slightly faster than body temperature, but for an ectotherm less than 10 g the lag time between external and body temperature is less than a few minutes\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we address two questions that have been difficult to quantify in the field: (1) to what extent are individuals able to alter their use of microhabitats to buffer themselves from temperature extremes? and (2) as increasing temperatures extend the time individuals spend in thermal refuges, are activity windows restricted to the extent that energetic costs are likely to affect performance?\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eWe attached temperature sensitive transmitters to 28 adult \u003cem\u003eT. lineata\u003c/em\u003e (17 males, 13 females) that were captured and released at four grassland sites during the breeding season (austral spring-summer) over two consecutive years (Oct-Feb 2012/13 and 2013/14). Laboratory tests in calibrated incubators showed that transmitter temperature (T\u003csub\u003etrans\u003c/sub\u003e) was a good predictor of dragon skin (T\u003csub\u003eskin\u003c/sub\u003e) and body temperature (T\u003csub\u003ebody\u003c/sub\u003e) (T\u003csub\u003eskin\u003c/sub\u003e=0.924T\u003csub\u003etrans\u003c/sub\u003e-0.013, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.939, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; T\u003csub\u003ebody\u003c/sub\u003e=1.063T\u003csub\u003etrans\u003c/sub\u003e-3.20, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.947, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with T\u003csub\u003eskin\u003c/sub\u003e measured using copper-constantan thermocouples attached to the dorsal surface of animals, and T\u003csub\u003ebody\u003c/sub\u003e measured using thermocouples inserted 4 mm into the cloaca.\u003c/p\u003e \u003cp\u003eIn the field, the transmitters recorded the temperature (T\u003csub\u003etrans\u003c/sub\u003e) of free-ranging individuals every 10 minutes for a total of 278 lizard-days and 19735 transmitter measurements during daylight hours. At the same time, we measured microclimate temperatures throughout these grasslands. We used shaded ground temperature (T\u003csub\u003eshade\u003c/sub\u003e) as a baseline against which to compare temperature in two other microhabitats: exposed open ground (T\u003csub\u003esun\u003c/sub\u003e) and the temperature in arthropod burrows (T\u003csub\u003eburrow\u003c/sub\u003e). Ground temperatures in the open sun can be extreme in these grasslands during late spring and summer, with an average daily maximum of 59\u0026deg;C and temperatures sometimes exceeding 70\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Shaded ground temperatures were cooler (average daily maximum 38\u0026deg;C), but even these could exceed 50\u0026deg;C. Burrows, in contrast, provided a more thermally buffered environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003ea): while shaded ground temperatures ranged from \u0026minus;\u0026thinsp;3 to 59\u0026deg;C, burrow temperatures ranged from 12 to 36.5\u0026deg;C (mean 23.2\u0026deg;C). Shuttling between microhabitats allowed individuals to stay between the critical maximum (38.3 to 42.8\u0026deg;C, mean\u0026thinsp;=\u0026thinsp;40.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u0026deg;C) and minimum (8.3 to 11.5\u0026deg;C, mean\u0026thinsp;=\u0026thinsp;9.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u0026deg;C) skin temperatures recorded in laboratory experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003ePlotting dragon transmitter temperature (T\u003csub\u003etrans\u003c/sub\u003e) as a function of shaded ground temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) revealed how dragons altered their use of thermal microhabitats as shaded ground temperature changed. Points above the 1:1 line were transmitters that were warmer than shaded ground, indicating dragons were in more open microhabitats, while points below the line indicate that dragons were occupying cooler microhabitats such as burrows. Across the range of shaded ground temperatures (-3 to 59\u0026deg;C), average transmitter temperature remained within the thermal tolerance limits of the species (skin temperature 9.9 to 40.6\u0026deg;C; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) because individuals adjusted the amount of time they spent in different thermal microhabitats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For example, when shaded ground temperature fell below the lower critical thermal limit for the species, average transmitter temperature remained above this limit because dragons spent more time in thermally buffered sites, such as burrows, particularly at night and during the coolest parts of the day (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As evidence for this, the distribution of transmitter temperatures closely matched the distribution of burrow temperatures when shaded ground temperature was less than 15\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At these temperatures, burrows were generally warmer than both shaded and open ground sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt shaded ground temperatures between 15\u0026ndash;25\u0026deg;C, the distribution of transmitter temperatures closely matched the distribution of open ground (T\u003csub\u003esun\u003c/sub\u003e) temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e), implying that dragons spent much of the time using open sites in this temperature range (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As shaded ground temperature rose above 25\u0026deg;C, open ground temperatures steadily exceeded transmitter temperatures, implying that dragons increasingly avoided warmer open sunny sites at higher temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At shaded ground temperatures greater than 40\u0026deg;C, most transmitter temperatures were below this value, implying that dragons were avoiding shaded ground sites in favour of cooler microhabitats, such as burrows. The point at which shaded ground temperatures were sufficiently warm that dragons began to avoid this microhabitat is indicated by the sharp inflection in the average transmitter temperature curve when shaded ground temperature approached the upper thermal tolerance limit of the species (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). At shaded ground temperatures above about 35\u0026deg;C, average transmitter temperature remained relatively constant, and below the thermal tolerance limit, as dragons increasingly sought refuge in cooler microhabitats. These data show that dragons sought refuge in more thermally buffered environments, such as burrows, at both high and low shaded ground temperatures to maintain average temperature within their thermal limits. As such, key changes in the use of microhabitats in the field coincided with the upper and lower critical temperature thresholds of this species.\u003c/p\u003e \u003cp\u003eEssential activities, such as feeding and finding mates, require dragons to be above-ground\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, but high and low above-ground temperatures force dragons into thermal refuges such as burrows. Seeking refuge is a well-known activity in lizards to escape high temperatures \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e 50\u003c/sup\u003e. However, we lack field data quantifying the extent to which high temperatures curtail above-ground activity, and hence the impact that high temperatures may have on the animal\u0026rsquo;s ability to forage above-ground to support their energetic demands. To understand the impact of a shortened activity window caused by high temperatures, we classified transmitter temperatures according to whether lizards were above or below shaded ground and burrow temperature for each transmitter temperature record. Transmitter temperatures below shaded ground temperature indicate the dragon was using a thermal refuge to escape heat, while transmitter temperatures above shaded ground temperature, but at or below burrow temperature, indicate that the dragon was using a refuge to escape cold. During daylight hours, above-ground activity was curtailed in the early morning and late evening as dragons sought refuge from cooler above-ground temperatures, particularly on days when the daily maximum air temperature was low (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e). On days when the maximum air temperature rose above 30\u0026deg;C, dragons increasingly sought thermal refuge from high temperatures during the middle of the day (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This resulted in a quadratic relationship between daily activity (the proportion of daylight hours dragons were active above-ground) and maximum daily air temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Dragons spent, on average, more than half the day active when daily maximum air temperatures were between about 20\u0026ndash;32\u0026deg;C. As the daily maximum temperature rose or fell beyond these values, dragons spent an increasing proportion of the day in thermal refuges. On days when the maximum air temperature approached 40\u0026deg;C, dragons spent about 80% of daylight hours in thermal refuges to avoid the heat of more open environments and to maintain their temperature within thermal tolerance limits. This pattern is similar to that projected for the widespread military dragon (\u003cem\u003eCtenophorus isolepis\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo assess the implications of a shortened activity window on lizard energetics, we measured resting metabolic rates (RMR) for 12 post-absorptive individual \u003cem\u003eT. lineata\u003c/em\u003e (6 males, 6 females) at five temperatures (20, 25, 30, 35 and 38\u0026deg;C) using flow-through respirometry\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Resting metabolic rates (RMR in ml O\u003csub\u003e2\u003c/sub\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were then used to calculate the average amount of energy expended by a dragon at rest in the field based on the recorded transmitter temperatures averaged on an hourly basis. We calculated the mean RMR of a dragon for each day that we had transmitter records by averaging the hourly RMR values and then plotted the daily mean as a function of maximum daily air temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The mean RMR increased as daily maximum air temperature rose, as expected for an ectotherm, but started to level out once daily maximum temperature exceeded about 32\u0026deg;C. This is likely to occur because dragons used thermal refuges to escape the heat on warmer days (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) and thus limit their metabolic losses. Consequently, the mean RMR divided by the number of hours active increased sharply on days when the maximum temperature rose above 32\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). We use the term energetic cost for the ratio: mean RMR / number of hours active. This ratio measures the amount of energy dragons must obtain per active hour by feeding to balance their daytime resting metabolic losses. While metabolic losses would also occur at night, dragons usually occupied cool night-time microhabitats, even on hot days (e.g. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e), meaning nocturnal metabolic losses would be low.\u003c/p\u003e \u003cp\u003eThe resting energetic cost, and hence the energy dragons must obtain while active to offset this cost, increased about three-fold as daily maximum air temperature rose from 32 to 40\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Hence, while activity time was reduced on days with both low and high maximum temperatures, dragons incurred a substantially higher energetic cost on hot days because of greater metabolic losses at high temperatures coupled with reduced activity time in which to recoup those losses. Confining our calculations to daylight hours only and using resting metabolic rate as our measure of energy expenditure provides a conservative measure of total daily energy expenditure. If lizards are active, rates of energy metabolism are typically 1.5-3 times resting levels\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo examine changes in the energetic cost over time, we gathered data on daily maximum air temperature during breeding season (October-February) for the years 1941\u0026ndash;2020 recorded at Canberra Airport, the nearest climate station to our study sites (all sites were within 7 km of the climate station and at the same altitude). For each day during this period, we estimated the proportion of daylight hours dragons were active and the mean energetic cost based on the maximum daily temperature and the relationships shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e. We then calculated the mean maximum temperature, the mean activity time, and the mean energetic cost per day during the breeding season for each year (expressed as an anomaly from the mean for the period 1960\u0026ndash;1990). Mean daily maximum temperature fluctuated around the mean from 1940\u0026ndash;2000 but has increased in the period 2000 to 2020 to be on average more than 2\u0026deg;C greater than the mean, resulting in a decline in mean daily activity during summer of up to 4% (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The combination of hotter days and a shorter activity window caused the mean energetic cost for individual lizards to increase by an average of more than 20% during the same period (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This implies that between 2000\u0026ndash;2020, increasing maximum daytime temperatures required dragons to increase their energy intake during their active period by more than 20%, relative to the 1960\u0026ndash;1990 mean, to offset metabolic losses.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEctothermic activity in temperate regions is usually constrained by both low and high temperatures, meaning it is unclear whether warming will be beneficial (increase the activity window) or detrimental (decrease the activity window) to temperate-zone populations. We show that during the critical spring-summer months of the breeding season the activity window declines sharply for \u003cem\u003eT. lineata\u003c/em\u003e as daily maximum temperature rises above 32\u0026deg;C, implying climate warming that resulted in a greater number of hotter days could negatively affect populations by forcing dragons to spend more time in thermal refuges, reducing the time available for essential activities such as foraging, feeding and finding mates. On hotter days, reduced activity coupled with greater metabolic losses incurs a substantial energetic cost. Between the years 2000\u0026ndash;2020, we estimate that dragons had to increase their energy intake during their daily active period by more than 20% to offset metabolic losses owing to rising temperatures, even after accounting for behavioral adjustments to limit those losses by using thermal refuges. This estimate is conservative because we have used resting metabolic rate as our measure of energy expenditure and confined our calculations to daylight hours only. \u003cem\u003eT. lineata\u003c/em\u003e will be active for significant portions of the day (not just resting) and will range widely to forage\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Given that activity will increase energy expenditure beyond that indicated by resting metabolic rate, and that dragons will continue to expend energy at night, the energetic impact of the observed increase in maximum daily temperature over the last 20 years (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e) will be even higher than our analysis reveals. Populations of \u003cem\u003eT\u003c/em\u003e. \u003cem\u003elineata\u003c/em\u003e suffered widespread collapse between 2006-2010\u003csup\u003e57\u003c/sup\u003e. Our results suggest it is possible that an increase in the number of hot days in Canberra, with a sustained increase commencing around the year 2000 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), could have played a role in this population collapse by reducing activity times (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) and increasing metabolic costs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003ec) that may have been difficult to recoup.\u003c/p\u003e \u003cp\u003eSinervo et al \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e speculated that extinctions of lizard populations are likely if activity is restricted by 7 h or more per day, particularly during critical reproductive months. Our results show that \u003cem\u003eT. lineata\u003c/em\u003e would exceed this threshold when daily maximum air temperatures rise above 35\u0026deg;C (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) in the breeding season (October to February) when average day length is 13.5 h and \u003cem\u003eT. lineata\u003c/em\u003e are active for less than 5.5 h. This is close to the daily maximum temperature where metabolic costs begin to rise sharply (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), suggesting the 7 h activity restriction roughly corresponds to a critical threshold in energy expenditure for \u003cem\u003eT\u003c/em\u003e. \u003cem\u003elineata\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe microclimate temperatures we recorded were substantially higher than those obtained previously using an inanimate lizard model in temperate Australia\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Our data suggest that ectotherms at temperate-zone sites can reach dangerously high temperatures in open environments and may not be able to maintain optimal temperatures (30\u0026ndash;35\u0026deg;C) in shaded environments\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In the scenarios modelled previously, lizards in full sun achieved body temperatures of around 40\u0026deg;C, and lizards thermoregulating by shuttling between sun and shade maintained body temperatures of up to 33\u0026deg;C\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Our results for \u003cem\u003eT. lineata\u003c/em\u003e contrast sharply with these values, indicating that shaded microhabitats alone are insufficient to keep temperatures below critical thermal thresholds and that more thermally buffered refuges, such as burrows, are often required for survival (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Grasslands are exposed environments with the only shade available close to the ground, or below ground in burrows during periods of low vegetation cover, such as in droughts. Consequently, grassland lizards will be exposed to radiant and conductive heat at ground level that far exceeds the shaded temperatures 1 m above ground at which air temperatures are measured. It is therefore likely that lizards are exposed to much higher temperatures in grasslands, relative to more heavily vegetated environments with canopy shade, for a given air temperature.\u003c/p\u003e \u003cp\u003eAt warmer temperatures, we observed that individual \u003cem\u003eT. lineata\u003c/em\u003e remained active by shuttling between open and shaded microhabitats, often moving into burrows to cool (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This shuttling behaviour is believed to be the key to avoiding temperature extremes and surviving climate warming in temperate environments\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e but has rarely been measured directly using body temperatures. The shuttling behavior we observed provides considerable capacity for \u003cem\u003eT. lineata\u003c/em\u003e to manipulate body temperature, allowing lizards to remain above-ground on warm but not excessively hot days. However, this behavior depends on the availability of cooler microhabitats such as heavily shaded ground and burrows. Consequently, \u003cem\u003eT. lineata\u003c/em\u003e is susceptible to the loss of these refuge habitats, including the removal of above-ground vegetation by burning, drought or overgrazing, and the loss of invertebrate species responsible for digging the burrows that dragons rely on for shelter\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Vegetation loss associated with drought, in addition to high temperatures, may have also contributed to the widespread population collapse in \u003cem\u003eT. lineata\u003c/em\u003e from 2006-2010\u003csup\u003e57\u003c/sup\u003e. While it is possible that climate warming will extend the seasonal activity window for \u003cem\u003eT. lineata\u003c/em\u003e, thereby shifting some activities to different times of the year, the extreme temperatures now experienced by these lizards in thermal refuges such as burrows (max 36.5\u0026deg;C) suggests there may no longer be safe refuges on extreme days when there is little above-ground vegetation.\u003c/p\u003e \u003cp\u003eOur data offer strong empirical support for the proposition that high summer temperatures caused by climate warming may repeatedly exceed the thermal and metabolic limits of temperate-zone lizards. Extended periods of high temperatures and reduced activity times over longer periods could place natural populations of lizards under significantly increased levels of environmental stress and contribute to population decline and local extinction. Vulnerability to warming will be a function of the thermal requirements of a species, their ability to adapt both physiologically and behaviourally to increased temperatures\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, and the availability of thermal refugia. Consequently, habitat degradation that alters the availability of thermal refugia will interact with climate warming to further imperil populations by reducing the opportunity for individuals to escape extreme heat while simultaneously increasing the need to increase their energy intake.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eAnimal ethics approval was granted by the University of Canberra\u0026rsquo;s Committee for Ethics in Animal Experimentation (CEAE 11\u0026ndash;22 and CEAE 15\u0026thinsp;\u0026minus;\u0026thinsp;08) and all experiments were performed in accordance with the relevant guidelines and regulations as per the approval. Grassland Earless Dragons were handled and collected under permits from the ACT Government Territory and Municipal Services (Licence to Take LT2012604; Licence to Import LI2011594; LI2012737) and NSW Office of Environment and Heritage (Scientific Licence Section 132c SL100756).\u003c/p\u003e\n\u003ch2\u003eStudy Species and Location\u003c/h2\u003e\n\u003cp\u003eWe studied the Canberra Grassland Earless Dragon, \u003cem\u003eTympanocryptis lineata\u003c/em\u003e (formerly \u003cem\u003eT. pinguicolla\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, a small agamid now confined to sites near Canberra, Australia (36.31\u0026deg;S, 149.20\u0026deg;E; 580 m a.s.l).\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. The K\u0026ouml;ppen-Geiger climate classification system defines Canberra as \u0026ldquo;Cfb\u0026rdquo;: temperate, with no dry season, warm summers (12.5\u0026ndash;27.1 \u0026deg;C) and cool winters (0.6\u0026ndash;12.2 \u0026deg;C) (mean daily min-max temps; Australian Bureau of Meteorology (BOM) 1939\u0026ndash;2008), although summer temperatures have risen in recent years (mean max 30.3 \u0026deg;C January 2008\u0026ndash;2022). The maximum monthly summer temperature on record occurred in the last 5 years (Dec 2019 41.1 \u0026deg;C, Jan 2020 44\u0026deg;C, Feb 2020 42.7 \u0026deg;C). Temperatures reported by BOM are recorded at 1 m height in a shaded Stevenson box. Ground surface temperatures in the sun are much higher and can reach 70 \u0026deg;C (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Precipitation is relatively consistent throughout the year, with slightly more rainfall in spring (mean monthly 178 mm) and summer (168 mm) than autumn (141 mm) and winter (128 mm) (BOM 1939\u0026ndash;2008). Frost is common in winter months.\u003c/p\u003e\n\u003ch2\u003eTemperature-Sensitive Radio-Telemetry\u003c/h2\u003e\n\u003cp\u003eWe captured \u003cem\u003eT. lineata\u003c/em\u003e at four sites in the Australian Capital Territory (35.3408\u0026deg; S, 149.1814\u0026deg; E.) and adjacent New South Wales (35.3737\u0026deg;S, 149.1940\u0026deg;E,.) using modified pitfall traps that mimic arthropod burrows the species uses naturally\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. The habitat at all sites was natural temperate grassland characterised by \u003cem\u003eRytidosperma-Austrostipa\u003c/em\u003e open tussock grassland with no trees or shrubs and a history of livestock grazing with little to no fertilization or pasture improvement\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Few surface rocks exist and the primary refugia available for \u003cem\u003eT. lineata\u003c/em\u003e are short, vertical burrows excavated by the Canberra Raspy Cricket (\u003cem\u003eCooraboorama canberrae\u003c/em\u003e) and wolf spiders (Lycosoidea).\u003c/p\u003e\n\u003cp\u003eFollowing capture, we measured body mass (mean 6.1 g), snout-vent-length (mean 53.9 mm), and sexed dragons by inspecting for hemipenes. Individuals greater than 4.3 g were fitted with an external temperature-sensitive radio transmitter (model BD-2XT, Holohil Systems, Canada or model PIP31, Sirtrack, NZ) to record the temperature that individuals were experiencing, with the transmitter pulse rate varying as a function of temperature\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e Each transmitter was calibrated in a water bath to the nearest 0.1 \u0026ordm;C before attachment and transmitter pule rates were converted to temperatures (T\u003csub\u003etrans\u003c/sub\u003e) using a quadratic polynomial function fitted to calibration data using least squares regression. The calibration was re-confirmed after transmitters were retrieved\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Transmitters (0.43\u0026ndash;0.55 g) were attached to the dorsal base of the tail posterior to the vent opening with the flexible 10 to 15 cm thin whip antenna positioned to run parallel to the tail\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. On average, transmitters represent less than 7% of the lizard\u0026rsquo;s body mass (range 4.7 to 9.1%; dragon mean mass 6.1 g; range 4.6 g to 9.1 g, n\u0026thinsp;=\u0026thinsp;44) similar to those used in other comparable studies\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, within the range of \u0026lt;\u0026thinsp;10% of animal body mass as recommended for small (\u0026lt;\u0026thinsp;30 g) non-flying animals\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Transmitter battery life varied from 5 to 40 d and individual lizards were tracked from 1 to 38 d (mean 14.5 d). We collected transmitter data using remote receiver/data logging stations comprising a three element Yagi antenna (Sirtrack, NZ) communicating with a receiver data logger (SRX_DL2, Lotek Wireless Inc, Canada). The data loggers were programmed to search for each lizard frequency and record transmitter pulse rates of each transmitter for 90 s every 10 min. The precise location of each lizard was confirmed several times each day using a handheld radio-telemetry device and a short wand antenna. Lizards were captured and tracked at four separate study sites.\u003c/p\u003e\n\u003ch2\u003eMicrohabitat Temperatures\u003c/h2\u003e\n\u003cp\u003eWe recorded environmental temperatures in the microhabitats available to \u003cem\u003eT. lineata\u003c/em\u003e at each of the four study sites during the same period that transmitter temperatures were monitored. At each site, temperature data loggers (Thermochron iButtons\u0026reg;, Model DS1921G, \u0026plusmn;\u0026thinsp;0.5\u0026deg;C, Maxim Integrated Inc., USA) were placed in microhabitats previously identified as used by \u003cem\u003eT. lineata\u003c/em\u003e and in which \u003cem\u003eT. lineata\u003c/em\u003e had been captured and tracked. The microhabitats measured were: 1) exposed open ground with vegetation cover less than 2 cm high (T\u003csub\u003esun\u003c/sub\u003e); 2) shaded open ground (T\u003csub\u003eshade\u003c/sub\u003e); and 3) at the bottom of 15\u0026ndash;20 cm deep burrows (T\u003csub\u003eburrow\u003c/sub\u003e). iButtons used to measure open ground temperatures (T\u003csub\u003esun\u003c/sub\u003e) were placed in lengths of copper pipe (24 mm diameter, 55 mm length) that had been spray painted beige and terminated with 22 mm diameter plastic plugs 70.\u003c/p\u003e\n\u003cp\u003eShaded temperatures at ground level (T\u003csub\u003eshade\u003c/sub\u003e) were measured using iButtons attached to the base of a wooden stake and covered to prevent radiant heating, and burrow temperatures (T\u003csub\u003eburrow\u003c/sub\u003e) were measured using iButtons placed in handmade burrows (25 mm wide, 15\u0026ndash;20 cm deep) formed by hammering a length of stainless-steel pipe into the ground. The depth of burrow at which the iButtons were placed approximated the depth of natural burrows used by \u003cem\u003eT. lineata\u003c/em\u003e in the field (n\u0026thinsp;=\u0026thinsp;42, mean\u0026thinsp;=\u0026thinsp;16.6 cm depth, range\u0026thinsp;=\u0026thinsp;10\u0026ndash;27 cm). At each study site a minimum of 3 replicate sets of iButtons were installed. iButtons have been used extensively to sample habitat temperatures (e.g. Hubbart et al 2005; Doucette et al 2011; Brabyn et al 2014) and have been found to reliably record microhabitat thermal variability and predict temperatures experienced by small ectotherms (Vickers and Schwarzkopf 2015; Moore et al 2017). Maximum daily air temperatures (T\u003csub\u003eair\u003c/sub\u003e) were taken at 1 m above ground in a Stevenson screen recorded at the Australian Bureau of Meteorology (BOM) Canberra Airport Site 070351. All four study sites were within a 7 km radius of the Canberra airport and at the same altitude.\u003c/p\u003e\n\u003ch2\u003eRespirometry\u003c/h2\u003e\n\u003cp\u003eWe measured the resting metabolic rate (RMR) of 12 individual \u003cem\u003eT. lineata\u003c/em\u003e (6 males, 6 females) from the captive breeding colony at the University of Canberra, Australia using open-flow respirometry to measure oxygen consumption\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Individuals were housed outdoors for several weeks before RMR measurements were conducted in November 2013. Respirometry was conducted on post-absorptive lizards during their rest phase (night) in darkened temperature-controlled cabinets. Individual body mass (\u0026plusmn;\u0026thinsp;0.01 g) was measured at the start and end of each trial and a linear rate of mass loss was assumed for calculation of mass-specific metabolic rate\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Oxygen consumption was measured using an oxygen analyzer (FOX, Sable Systems International Inc., USA), placed inside an insulated box in a temperature\u0026ndash;controlled room at 19\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026ordm;C. A sub-sampling design was used to keep the rate of flow of the sample air through the analyzer constant (63 ml min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) throughout the measurements. Outside air was pumped through silica gel to remove moisture while rotameters controlled the rate of airflow to the chambers. After passing through the chamber, excurrent air was dried again using silica gel and the flow rate of air was measured using a mass flow meter (Omega FMA-5606, USA). A chamber flow rate of 100 ml min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was maintained throughout the experiments, which was sufficient to maintain the oxygen content in the excurrent air above 20%. The excurrent air from each chamber (2\u0026ndash;3 chambers used) was sampled every three minutes, followed by three minutes sampling of a reference channel of dried outside air. Thus, one measurement for each lizard was obtained every 9 to 12 minutes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Measurements of ambient temperature (T\u003csub\u003ea\u003c/sub\u003e) were taken simultaneously to those of RMR via calibrated T-type thermocouples in the respirometry chambers. Data acquisition and processing were performed using software written by G. K\u0026ouml;rtner\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. A respiratory quotient of 0.85 was assumed for all measurements and the rate of oxygen consumption was calculated using Eq.\u0026nbsp;3a of Withers\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. RMRs were calculated for each individual as the average of the six consecutive lowest VO\u003csub\u003e2\u003c/sub\u003e values in resting individuals at each T\u003csub\u003ea\u003c/sub\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Chamber temperatures were set to 20, 25, 30, 35 and 38\u0026deg;C for a minimum of 2 hours. Only data from the final 75 min of testing once the chamber had reached the designated T\u003csub\u003ea\u003c/sub\u003e were considered in the analysis.\u003c/p\u003e\n\u003ch2\u003eCritical Thermal Limits\u003c/h2\u003e\n\u003cp\u003eAll tests for thermal tolerance thresholds took place in an air-conditioned laboratory at 21\u0026deg;C. To prepare individuals for testing two T-type copper-constantan thermocouples (MicroDAQ) were attached to each dragon using clear surgical tape. One thermocouple was taped to the dorsal surface of the individual and a second thermocouple inserted several millimetres into the cloacal and taped in position. Dorsal T\u003csub\u003eskin\u003c/sub\u003e and cloacal temperatures (T\u003csub\u003ebody\u003c/sub\u003e) were recorded every minute.\u003c/p\u003e\n\u003cp\u003eWe used the panting threshold, the body temperature at which dragons started to pant open-mouthed and started to turn over to expose their lighter coloured ventral abdomen to the heat, as our measure of the dragon\u0026rsquo;s upper thermal tolerance. In Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, we show the skin temperature recorded on lizards at this thermal maximum (CT\u003csub\u003emax\u003c/sub\u003e in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Individuals were placed in a plastic holding tank containing clay from the native grasslands under a 160 W self-ballasted mercury-vapour bulb and observed until they reached the panting threshold.\u003c/p\u003e\n\u003cp\u003eThe critical thermal minimum was determined using the same individual \u003cem\u003eT. lineata\u003c/em\u003e as used for the CT\u003csub\u003emax\u003c/sub\u003e experiments. Each individual was placed in a dry 0.5 L plastic container submersed to the rim in ice water at 3.5 to 5\u0026deg;C. Once T\u003csub\u003ebody\u003c/sub\u003e had reached 16\u0026deg;C individuals were gently flipped over onto their back and allowed to right themselves. CT\u003csub\u003emin\u003c/sub\u003e was considered to have occurred when individuals could no longer right themselves and became immobile. The CT\u003csub\u003emin\u003c/sub\u003e shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e is the skin temperature recorded on animals at this lower thermal limit.\u003c/p\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003eData Analysis\u003c/h2\u003e\n \u003cp\u003eTransmitter pulse rates were converted to temperatures (T\u003csub\u003etrans\u003c/sub\u003e) using a quadratic polynomial function fitted to calibration data using least squares regression. Transmitter pulse rates (accuracy 0.1 beats per minute) were averaged across each 90 s recording period to obtain a single T\u003csub\u003etrans\u003c/sub\u003e for each individual every 10 min. Days with recordings that spanned less than 10 h, recordings outside daylight hours, and time periods with \u0026ge;\u0026thinsp;120 min of missing data were excluded, resulting in a final dataset that comprised data from 28 lizards, 144 days, and 19735 transmitter temperature observations. Values of T\u003csub\u003etrans\u003c/sub\u003e were matched by time and location (study site) to the mean of the three microclimate temperature replicates for T\u003csub\u003esun\u003c/sub\u003e, T\u003csub\u003eshade,\u003c/sub\u003e and T\u003csub\u003eburrow\u003c/sub\u003e at the respective site. This matching allowed us to determine the most likely microhabitat that individuals occupied at each recording time (the value of T\u003csub\u003esun\u003c/sub\u003e, T\u003csub\u003eshade,\u003c/sub\u003e or T\u003csub\u003eburrow\u003c/sub\u003e that most closely matched T\u003csub\u003etrans\u003c/sub\u003e). Dragons always remained within 100 m, and rarely moved more than 50 m, from the capture location creating small, uniform study areas. As the temperature differentials between each of these three microclimate types often exceeded 10 \u0026deg;C we could be confident in our inferences of dragon microclimate selection. We used the lizard transmitter and microhabitat temperatures, along with variation over time in the individual transmitter temperatures and signal strength\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e, to classify lizards as either active or inactive at each recording time. We scored an individual as active if T\u003csub\u003etrans\u003c/sub\u003e was greater than both T\u003csub\u003eshade\u003c/sub\u003e and T\u003csub\u003eburrow\u003c/sub\u003e during daylight hours (sunrise to sunset), indicating that the lizard was in the open and not seeking refuge (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Individuals were scored as inactive if T\u003csub\u003etrans\u003c/sub\u003e was below T\u003csub\u003eshade\u003c/sub\u003e for at least two consecutive measurements (20 minutes), implying lizards were in cooler refugia such as burrows, or if at least three consecutive measurements of T\u003csub\u003etrans\u003c/sub\u003e showed little variation in transmitter temperature (defined as a difference of \u0026lt;\u0026thinsp;0.5\u0026deg;C) or signal strength, indicating the lizard was stationary at one location for at least 30 minutes. Data were processed and analysed using R 3.2.1 (The R Foundation for Statistical Computing, Austria).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding was provided by an Australian Research Council (LP0776987) to SS, BG, AG, Tariq Ezaz, WO, and Don Fletcher and Conservation Research, Environment Planning and Sustainable Development Directorate, ACT. We thank Wendy Ruscoe and Jacqui Richardson for management of the lizard colony, and Emma Cook, Rod Pietsch, Samatha Vertucci, Brett Howland, Renee Brawata, Muhammad Rais, Don Fletcher and Emma Carlson for advice and field assistance. Fritz Geiser and Gerhard K\u0026ouml;rtner provided respirometry equipment and advice for metabolic measurements and Dan Noble provided helpful comments on an earlier draft of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.D., S.S., W.O., B.G. and A.G. conceived and designed the study. L.D. developed and oversaw all aspects of the field and laboratory data collection. R.D., L.D., B.G., and S.S. developed the analytical framework and analysed the data. All authors contributed to the discussion of the results and writing of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data and code files related to this manuscript are available at https://zenodo.org/record/7305580#.Y2rEIHZBxaQ (DOI: 10.5281/zenodo.7305580).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLoarie, S. R.\u003cem\u003e et al.\u003c/em\u003e The velocity of climate change. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e462\u003c/strong\u003e, 1052-U1111, doi:10.1038/nature08649 (2009).\u003c/li\u003e\n\u003cli\u003eSinervo, B.\u003cem\u003e et al.\u003c/em\u003e Erosion of lizard diversity by climate change and altered thermal niches. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e328\u003c/strong\u003e, 894-899, doi:10.1126/science.1184695 (2010).\u003c/li\u003e\n\u003cli\u003eChown, S. 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Heart rate as a predictor of metabolic rate in heterothermic bats. \u003cem\u003eThe Journal of Experimental Biology\u003c/em\u003e \u003cstrong\u003e217\u003c/strong\u003e, 1519-1524, doi:10.1242/jeb.098970 (2014).\u003c/li\u003e\n\u003cli\u003eCurrie, S. E., Kortner, G. \u0026amp; Geiser, F. Heart rate as a predictor of metabolic rate in heterothermic bats. \u003cem\u003eJournal of Experimental Biology\u003c/em\u003e \u003cstrong\u003e217\u003c/strong\u003e, 1519-1524, doi:10.1242/jeb.098970 (2014).\u003c/li\u003e\n\u003cli\u003eAdelman, J. S., Cordoba-Cordoba, S., Spoelstra, K., Wikelski, M. \u0026amp; Hau, M. Radiotelemetry reveals variation in fever and sickness behaviours with latitude in a free-living passerine. \u003cem\u003eFunctional Ecology\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 813-823, doi:10.1111/j.1365-2435.2010.01702.x (2010).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ectotherm, microclimate, refugia, thermal tolerance, activity window","lastPublishedDoi":"10.21203/rs.3.rs-2228630/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2228630/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEctotherms are considered vulnerable to climate change as many operate at close to their thermal maxima. Exposure to higher temperatures could reduce activity by forcing animals to shelter in thermal refugia to avoid exceeding lethal limits. While rising temperatures should reduce activity in tropical species, the situation is less clear for temperate-zone species where activity can be constrained by both low and high temperatures. Here, we measure the effects of natural variation in environmental temperatures on activity in a temperate grassland lizard and show that it is operating at its upper thermal limit in summer even when sheltering in thermal refuges. As air temperatures increased above 32\u0026deg;C, lizard activity declined markedly as individuals sought refuge in cool microhabitats while still incurring substantial metabolic costs. We estimate that warming over the last two decades has required these lizards to increase their energy intake by over 20% to offset metabolic losses caused by rising temperatures. Our results show that recent increases in temperature are sufficient to exceed the thermal and metabolic limits of temperate-zone lizards. Extended periods of high temperatures could place natural populations of ectotherms under significantly increased environmental stress and contribute to population declines and extinction.\u003c/p\u003e","manuscriptTitle":"Climate warming drives a temperate-zone lizard to its upper thermal limits, restricting activity, and increasing energetic costs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-11 15:56:06","doi":"10.21203/rs.3.rs-2228630/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-11-22T07:15:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-11-15T23:20:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"026681e2-b44d-4604-a598-1c8e82223df9","date":"2022-11-09T21:49:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-11-09T21:32:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-11-09T21:27:31+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-11-09T15:00:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-11-09T14:54:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-11-02T05:56:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4a7fa3cb-cce3-40de-86ec-699273d45e68","owner":[],"postedDate":"November 11th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":16844691,"name":"Biological sciences/Ecology/Climate change ecology"},{"id":16844692,"name":"Earth and environmental sciences/Ecology/Behavioural ecology"}],"tags":[],"updatedAt":"2023-10-16T21:21:02+00:00","versionOfRecord":{"articleIdentity":"rs-2228630","link":"https://doi.org/10.1038/s41598-023-35087-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-06-13 21:10:35","publishedOnDateReadable":"June 13th, 2023"},"versionCreatedAt":"2022-11-11 15:56:06","video":"","vorDoi":"10.1038/s41598-023-35087-7","vorDoiUrl":"https://doi.org/10.1038/s41598-023-35087-7","workflowStages":[]},"version":"v1","identity":"rs-2228630","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2228630","identity":"rs-2228630","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}