Patterns of seasonal plasticity in evaporative water loss and preferred temperature in three geckos of the wet–dry tropics

Patterns of seasonal plasticity in evaporative water loss and preferred temperature in three geckos of the wet–dry tropics | 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 Research Article Patterns of seasonal plasticity in evaporative water loss and preferred temperature in three geckos of the wet–dry tropics Kimberley Day, Chava Weitzman, Kade Skelton, Angga Rachmansah, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5444175/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Seasonal physiological plasticity (acclimatisation) facilitates homeostasis in changing environments and has been studied extensively with respect to thermal biology and metabolism. Less is known about seasonal changes in evaporative water loss (EWL) in response to changing water availability and humidity. The wet–dry tropics of northern Australia experiences moderate seasonal temperature changes, but substantial changes in rainfall and humidity. We studied three gecko species ( Amalosia rhombifer , Heteronotia binoei and Hemidactylus frenatus ) in the wet and dry seasons with respect to their EWL, preferred body temperatures (T pref ), and their choice between a dry and humid refuge at and below T pref . EWL was significantly lower in the dry season (66% of wet season values). T pref for two of the species did not change seasonally, but A. rhombifer selected lower T pref during the warmer wet season. Given a choice of refugia, the humid refuge at low temperatures was never preferred over the warm microhabitat. When both refugia were at preferred temperature, only A. rhombifer showed a significant preference for the humid microhabitat. These results demonstrate that although thermoregulation is prioritised in the short term, hydroregulation (physiological plasticity in EWL) is adjusted in the longer term, with shifts occurring on a seasonal scale. However, previous studies suggest shifts in EWL may occur in response to prevailing weather conditions on an even shorter timescale. Before broad generalisations can be drawn about the phenomenon of EWL plasticity, measurements need to be taken from more species in different climatic regions at ecologically relevant timescales. Animal Physiology Acclimatisation seasonal plasticity evaporative water loss thermoregulation hydroregulation Figures Figure 1 Figure 2 Introduction Physiological plasticity is widespread in nature, maximising performance and survival in response to changing climate and ecological interactions across the year. For instance, plasticity can buffer against the negative effects of increasing temperatures, allowing for increasing heat tolerance in ectotherms to reduce the likelihood of overheating (Gunderson et al. 2017 ). Though commonly discussed in terms of seasonal temperature changes, flexibility in physiological processes may also be initiated, or optimised, by other environmental cues. Reductions in metabolic rate and preferred body temperature conserve energy and water during seasons when resource availability is limiting even if environmental temperatures are not (Christian et al. 1999a , 2023 ; Berg et al. 2017 ). Importantly, the extent or strength of plasticity may depend on the variability experienced by the organism (Muñoz & Bodensteiner 2019 ). A comparison of 41 bird species from the Central American wet tropics versus six species from a temperate site found greater seasonal change in the temperate species in thermal and metabolic measures (Pollock et al. 2019 ). Similarly, variability in resources in the seasonal (wet–dry) tropics may favour the evolution of physiological plasticity of ectotherms as compared to the relative stability of the aseasonal (wet) tropics (Christian et al., 1999a , 2023 ; Huey et al., 2012 ). Body temperature (T b ) influences all biological processes, including digestion, locomotion, reproduction, and growth and has been widely studied in reptiles and other ectotherms (Heatwole 1976 ; Huey 1982 ; Christian & Tracy 1981 ; Kearney & Predavec 2000 ; Navas et al. 2008 ; Chukwuka et al. 2020 ; Volkoff & Rønnestad 2020 , and see below). In seasonal climates, some species become dormant and forego thermoregulation, but other species thermoregulate to lower temperatures in winter (Seebacher 2005 ). The seasonality of the wet–dry tropics induces a significant acclimatisation response in some lizards, with thermal preference shifting towards lower T b in the cooler dry season compared to the warmer wet season (Christian et al. 1983 ; Christian & Bedford 1995 , 1996 ; Christian & Weavers 1996 ; Christian et al. 1999b ). However, rather than being a response to environmental temperatures per se , these examples of seasonal changes in preferred body temperature (T pref ) are likely mechanisms to conserve energy and water in response to the decrease in food and water resources in the dry season (Christian et al., 1999a , 2023 ; Berg et al. 2017 ). Energy budget analyses indicate that seasonal food availability is the driving force for physiological plasticity related to energy expenditure (T pref and metabolic rate) in several lizards from the seasonal tropics (Christian et al. 1996a , 1999a , 1999b ), and calculations indicate that frillneck lizards would starve in the dry season were it not for these physiological adaptations (Christian et al. 1996b ). Thus, physiological plasticity is likely to be essential for some species in environments with seasonal shortages of food coupled with high environmental temperatures (Christian et al. 2023 ). The species described above are diurnal and are thus exposed to more extreme thermal environments than nocturnal species. Nevertheless, geckos can thermoregulate by selecting microhabitats in their daytime refugia (Bustard 1967 ; Chukwuka et al. 2021 ; Kearney & Predavec 2000 ), and night-time habitats maintain some thermal heterogeneity, allowing nocturnal geckos to use warm microclimates to conductively thermoregulate while active (Autumn & DeNardo 1995 ; Nordberg & Schwarzkopf 2019 ). Nocturnal insects are substantially less abundant in the dry season compared to the wet season in the seasonal Australian tropics (Churchill 1994 ). However, the lower body temperatures typically experienced by nocturnal animals from this region (Nordberg & Schwarzkopf 2019 ) are similar to the daytime T b s of the diurnal species that have seasonally-reduced T pref values (Christian & Bedford 1995 ; Christian & Weavers 1996 ; Christian et al. 1996a , 1999b ), possibly obviating the need for further decreases in T b to conserve energy. Thus, it is difficult to predict whether or not nocturnal geckos would exhibit seasonal acclimatisation of T pref . Little is known about seasonal thermal acclimatisation in nocturnal species from the wet–dry tropics, but the gecko Oedura marmorata selected a lower T pref in the dry season compared to the wet (Christian et al. 1998 ). While thermoregulation is relatively well-studied, the importance of hydroregulation in reptiles has only recently become better understood (Grimm-Seyfarth et al. 2018 ; Kearney et al. 2018 ; Pirtle et al. 2019 ; Rozen-Rechels et al. 2019 ). In addition to the potentially lethal consequences of inadequate hydration, sublethal dehydration can result in negative physiological and ecological consequences (Pirtle et al. 2019 ; Rozen-Rechels et al. 2019 ). In particular, optimal thermoregulation and activity patterns can be disrupted, resulting in compromised performances related to foraging, predator avoidance, and reproductive success (Rozen-Rechels et al. 2019 ; 2021 ; Sannolo & Carretero 2019 ). Reptiles from wet or mesic environments typically have higher rates of evaporative water loss (EWL) compared to those from arid environments (Hillman & Gorman 1977 ; Dmi’el et al. 1997; Cox & Cox 2015 ), and within a climatic zone, animals occupying mesic microhabitats have higher rates of EWL than those from drier microhabitats (Belasen et al. 2017 ). The mechanisms behind these patterns are typically not known, but include the possibilities of genetic differences among populations, irreversible developmental phenotypic plasticity in response to environment, or reversible physiological plasticity in which an individual can change in response to environmental conditions (Dmi’el et al. 1997; Wilson and Franklin 2002 ; Cox & Cox 2015 ; While et al. 2018 ; Christian et al. 2023 ). An example of a reversible change in EWL is related to the morphological and metabolic consequences of pregnancy in a viviparous snake (Lourdais et al. 2017 ). Pregnant snakes have higher rates of EWL and select warmer and moister microhabitats, further supporting a pattern between EWL and microhabitat humidity, but in this case being driven by biological processes rather than by environmental conditions (Lourdais et al. 2017 ). Although EWL occurs across skin, eyes, and respiratory structures, cutaneous water loss is the largest component in reptiles (Bentley & Schmidt-Nielsen 1966 ; Cohen 1975 ; Shoemaker & Nagy 1977 ; Mautz 1982 ; Kobayashi et al. 1983 ), typically accounting for more than 70% of the total EWL (Blamires & Christian 1999 ). Physiological plasticity of EWL has been explored in lizards in the laboratory, with individuals exposed to humid conditions having higher rates of EWL than those acclimated to dry conditions (Kobayashi et al. 1983 ; Kattan & Lillywhite 1989 ; Rozen-Rechels et al. 2020 ; Weaver et al. 2022 , 2023 ). Seasonal changes in EWL have been documented in beetles exposed to semi-natural conditions (Cooper 1985 ) and, in arid adapted scorpions exposed to natural conditions, seasonal physiological plasticity in EWL has been mechanistically linked to seasonal changes in epicuticular biochemical composition (Toolson & Hadley 1979 ). We are only aware of a single published report of field acclimatisation in which lizards measured from a location during a wet season had higher rates of EWL than lizards measured from the same location during a dry time of the year (Blamires and Christian 1999 ). This study showed no significant seasonal differences in ocular or respiratory EWL, but there was a significant seasonal change in cutaneous EWL, which is consistent with measurements of skin permeability being higher in lizards acclimated to wet conditions as compared to those acclimated to dry conditions in the laboratory (Kobayashi et al. 1983 ; Kattan and Lillywhite 1989 ; Weaver et al. 2023 ). While there is growing evidence of seasonal thermal and metabolic plasticity in reptiles (Christian et al. 2023 ), the focus has been on diurnal species, despite differing ecological pressures on nocturnal versus diurnal habits. Before generalisations can be drawn about the pervasiveness and ecological drivers of acclimatisation, more information is needed from a range of climatic zones (Christian et al. 2023 ), and this is particularly true for nocturnal animals. Nocturnal geckos are informative in this regard because they can be compared to the better-studied lizard species from the same area, thus providing insight into both the prevalence of physiological plasticity and the ecological drivers. This study aims to quantify seasonal plasticity in thermal and hydric physiology of three common nocturnal gecko species at a site in the wet–dry tropics in Northern Australia. We focus the study on measuring acclimatisation in EWL and T pref in the wet and dry seasons. Given that water availability fluctuates more than temperature in the wet–dry tropics, we also test for the relative importance of behavioural hydroregulation versus thermoregulation, or the trade-offs between maintaining a preferred temperature or avoiding dehydrating microhabitats. We used this series of physiological and behavioural experiments to address three hypotheses, and although we compared across the species, our over-arching hypothesis was that the climatic conditions of the site was the driving force, and thus the three species would not differ. First, we hypothesised that geckos would decrease EWL to conserve water in the dry season (as per Blamires & Christian 1999 ). Second, we hypothesised that T pref would be lower in the dry season than in the wet as per other lizards in the seasonal tropics (Christian et al. 1983 ; 1998 ; 1999; 2003 ; Christian & Bedford 1995 ; 1996 ; Christian & Weavers 1996 ). Third, we hypothesised that, when provided options of a dry refuge at preferred temperature and a humid refuge at or below preferred temperature in the thermo-hydroregulation experiment, prioritisation of desiccation avoidance would emerge at increasing temperatures. That is, geckos would prioritise seeking preferred temperatures when the humid option was at low temperatures, but when offered a humid refuge at higher temperatures, the geckos would shift from a preference for warmth toward a preference for desiccation avoidance (as per Pintor et al. 2016 ). Thus, under this hypothesis, when both dry and humid refuge options are at preferred temperature, geckos would show a preference for the humid refuge, which allows both desiccation avoidance and thermoregulation. Methods Study species, collection site and husbandry We studied three widespread, common nocturnal gecko species in the wet–dry tropics of Darwin, Australia: zig-zag geckos ( Amalosia rhombifer ), Bynoe’s geckos ( Heteronotia binoei ) and Asian house geckos ( Hemidactylus frenatus ). A. rhombifer and H. binoei are endemic to Australia, occupying arboreal and predominantly terrestrial habitats, respectively. H. frenatus is a well-established invasive species from South Asia that became established in Darwin around 1960 (Hoskin 2011 ) and occupies arboreal habitats. The physiological ecology of A. rhombifer has not been studied previously. The T pref of H. binoei was 30.8°C as measured from individuals from the arid zone of South Australia (Kearney & Porter 2004 ). In H. frenatus , the thermal tolerance to high temperatures does not vary between Thailand and eastern Australia, although thermal tolerance to low temperatures is lower in cooler locations (Lapwong et al. 2021 ). We collected the adult native geckos in the early evening (19:00–22:00) from bushland and H. frenatus from both bushland and building walls at Charles Darwin University (CDU), Casuarina, Northern Territory, Australia (12°22’07”S, 130°51’58”E). The bushland consists of 5 ha of eucalypt-dominated savanna, adjacent to campus buildings, suburban housing and the Casuarina Coastal Reserve. Sampling was conducted during Darwin’s wet (November to April, with our experiments done from early January–early March) and dry (May to October, with our experiments done from early August–early September) seasons to align with climatic extremes of the wet–dry tropics. In Darwin, > 90% of the annual rainfall (mean = 1722.5 mm) occurs in the wet season, and mean monthly rainfall peaks in January at 429.8 mm and is lowest in July at 1.1 mm. There is some variability of humidity during the short transition periods between the wet and dry seasons, but for most of the wet season, humidity is consistently high, and during the dry season it is consistently low (Online Resource 1; climate statistics from Darwin Airport, 4.5 km south of the study site ; www.bom.gov.au/climate/ Accessed 3 October 2024). Mean minimum air temperature decreases by ~ 3 ˚C in the dry versus wet season, with less variability in maximum air temperatures (Online Resource 1). Geckos were returned to the wild after being run through the experiment(s) within a season, with new individuals captured each season. Measurements of EWL and T pref were taken within 48 h of capture. Individuals were fed every second day and provided water via a spray bottle daily. Many geckos in this study were used for two experiments; specifically, all A. rhombifer and wet season H. binoei in the EWL experiment were subsequently used in the thermal preference experiment, while wet season H. frenatus used the thermal preference experiment were also subjected to the thermo-hydroregulation experiment. Following the thermal experiment, lizards were housed in plastic terraria (38 × 23 × 12 cm) with a small hide and placed in a temperature-controlled room set to 28°C (mean 27.9 ± SD 0.11°C) with relative humidity of 40.0 ± 1.5% (vapor pressure deficit = 2.2 kPA), with an automated 12 h light-dark cycle until they were used in the thermo-hydroregulation experiment. Evaporative Water Loss Gecko EWL was measured during the day in the wet and dry seasons for each species using a flow-through system (Blamires & Christian 1999 ; Young et al. 2005 ). Evaporative water loss components were plumbed in-line and housed in incubators (model MIR253, Sanyo and model XHC-25, IVYX Scientific) to maintain a nominal experimental temperature of 30°C (the exact air temperatures were measured by the probes). Five EWL lines were operated simultaneously. For each EWL line, air was drawn from the incubator through a silica gel drying column using a low flow sampler calibrated to 0.2 L min − 1 (model LFS-113, Gilian®). The dry air then passed through an experimental chamber housing the gecko, made from a modified 60 mL syringe (13.5 × 2.6 cm). A probe (model HMP 110, Vaisala™), housed in a plastic tube (30 mm diameter), recorded the relative humidity and air temperature downstream of the gecko. The output from the probes was recorded continuously on an Apple Macintosh computer using an ADInstruments PowerLab paired with LabChart software (model PL3508, ADInstruments Pty Ltd, Bella Vista, Australia), and EWL was calculated from the equations of Bernstein et al. ( 1977 ) for an open-flow system, in conjunction with calculations (List 1971 ) of saturation vapour density (needed to calculate the mass of water from the measurements of relative humidity). In brief, the mass flow of water from the animal: M w = V e (VD a – VD i ), where V e = experimental flow rate; VD a = water vapour density of the air in the experimental chamber with the animal (g cm − 3 ); and VD i = baseline water vapour density (g cm − 3 ). Relative humidity and temperature measurements were collected before (as baseline data) and after placing the gecko in the experimental chamber. Skin temperature was recorded from the dorsum of each gecko immediately after the experiment using an infrared thermometer (Traceable© mini, Thomas Scientific). Gecko EWL experiments generally lasted 30 min to 1 h to achieve a resting measure. A flat-line trace on the computer screen was indicative of a resting animal because movement resulted in increased water loss and an irregular trace. The lowest humidity reading over a 2 min period was taken during a rest period of at least 5 min duration. Generally, the readings were very stable while the animals were at rest. If an animal defecated or failed to rest during the experiment, it was re-run later that day. Baseline values were reconfirmed after each experiment. Total resistance (R) to water loss was calculated as: R = (VD s – VD a ) E c −1 , where R = total resistance to water loss (s cm − 1 ), VD s = the vapour density of the skin (taken as the saturation vapour density at the skin temperature, g cm − 3 ), VD a = water vapour density of the air in the experimental chamber with the animal (g cm − 3 ), and E c = the surface area-specific rate of water loss (g cm − 2 s − 1 ) (Spotila and Berman 1976 ). To calculate gecko surface area (SA), we used linear measurements of each gecko (Belasen et al. 2017 ; Chukwuka et al. 2020 ). First, SA of three life-like plastic toy lizard models was determined by covering each model in masking tape, colouring the outer tape with a marker, then carefully transferring the coloured tape to graph paper to obtain a direct measure of SA (Blamires & Christian 1999 ). Linear measurements were then taken from the same model and substituted into a range of geometric SA equations to determine which one most closely matched the direct measures of SA. Previously, Belasen et al. ( 2017 ) and Chukwuka et al. ( 2020 ) estimated that lizard SA was roughly equivalent to the body representing a cylinder and the tail a cone. Our estimates with lizard models found that the best geometric SA equation (as compared to the direct measure from the coloured tape, based on lowest average difference between estimate and direct SA) assumed that the torso (including the head, snout to vent), tail and legs were separate, single-ended cylinders. The length and greatest width of these components were used in calculating SA for each gecko by adding the SAs of the six single-ended cylinders corresponding to the six gecko body parts (Online Resource 2). Body measurements were collected after geckos completed the EWL trial. Alongside linear measurements, geckos were weighed to allow for a seasonal comparison of body condition (see Statistics below). Thermal Preference Seasonal thermal preference was measured by placing geckos in a thermal gradient of 20°C to 40°C and recording T b with a thermal camera (model 868, Testo SE & Co. KGaA, Titisee-Neustadt, Germany; Barroso et al. 2016 ; Nordberg & Schwarzkopf 2019 ; Sannolo & Carretero 2019 ). The thermal gradient consisted of an artificial crevice hide made from glazed porcelain tile (54 × 15 × 0.8 cm), supported 1.5 cm above the substrate by a terracotta spacer at each end, and a 50 W heat lamp at one end of the hide, suspended ~ 1 cm above the tile. Each thermal gradient was constructed in a glass aquarium (59 × 34 × 37 cm) with terracotta tiles (56 × 30 × 1.5 cm) as a substrate. Thermal gradients were assembled in controlled temperature rooms set to 19.5°C, with ten thermal gradients operated simultaneously. Individual geckos spent a total of approx. 48 h in the preferred temperature experiment, allowing an overnight period to explore the thermal gradient. Thermal images were collected over the following day and a half at hourly intervals during daylight hours, with a total of 12 thermal images collected from each gecko. Thermal images were processed manually using IR Soft thermal image analysis software (Testo SE & Co. KGaA, Titisee-Neustadt, Germany), with T b represented by a measurement from the gecko's dorsum (Nordberg & Schwarzkopf 2019 ) for each thermal image. For analysis, T pref data were reduced to a set point range defined by the central 50% of T b measures for each gecko (Christian & Weavers 1996 ). Thermo-Hydroregulation Geckos were given access to high and low-humidity refugia to determine preference for hydroregulation or thermoregulation across preferred and sub-optimal humidity and temperature combinations (Pintor et al. 2016 ). Briefly, they were given a choice between a warm (but dry) refuge and a cool (and humid) refuge, and, in one treatment, a choice between two warm refugia, with one being dry and the other humid. The temperature in the dry refuge remained constant at 32°C with a mean of 36.5% humidity (absolute humidity (AH) = 12.35 g m − 3 ; vapour pressure (VP) = 1.74 kPA; vapour pressure deficit (VPD) = 3.02 kPa) similar to values experienced in the late afternoon in the dry season in Darwin, NT), offering preferred temperature (based on the results of the thermal preference experiment, below) and relatively low humidity. While the dry refuge remained at a constant temperature, the ambient aquarium conditions and the humid refuge were adjusted to three temperature treatments (32, 27 and 22°C) and a high humidity of 99% in the humid refuge. The 32°C treatment is close to the T pref (see Results ), and the two lower temperatures are ecologically relevant nighttime temperatures (Online Resource 1). (At 32°C: AH = 33.49 g m − 3 , VP = 4.71 kPA, VPD = 0.05 kPa); at 27°C: AH = 25.52 g m − 3 , VP = 3.53 kPA, VPD = 0.04 kPa; at 22°C: AH = 19.24 g m − 3 , VP = 2.62 kPA, VPD = 0.03 kPa) We refer to these treatments as 32H/32D, 27H/32D, and 22H/32D throughout, indicating the humid and dry (constant) refuge temperatures. Temperatures in the humid refuge were varied between treatments by adjusting the room temperature, while the dry refuge temperature was maintained at 32°C independently using a heat mat. Thus, when geckos were not in either refuge, they would have experienced the treatment air temperature (22, 27 or 32°C) and an intermediate humidity, depending on their proximity to the humid and dry refugia. This experiment used the same setup from the thermal gradient, substituting the hide with two terracotta refugia (15 × 15 × 1.5 cm) at opposite ends of the aquarium acting as humid and dry refugia. High humidity was achieved by soaking the terracotta refuge in water to humidify the refuge. The second refuge was kept dry by drawing air from a drying column and pumping it into the refuge through aquarium tubing (4 mm) at 0.2 L min − 1 . Refuge conditions were monitored using hygro-thermometer (model 800027, Sper Scientific) probes placed under each refuge, with an electronic display at the back of the aquarium. Each treatment trial lasted 20 h, with an additional 4 h prior to data collection to allow conditions to stabilise. Six geckos were trialled in separate aquaria simultaneously, with treatments consecutively applied in the reverse order for each new set of geckos. Refuge selection was recorded using a webcam (Logitech) placed in front of the aquarium, positioned so that the gecko could be seen when occupying either refuge. An Apple MacBook running Evocam software (version 3.6.5, Evological©) was used to create a time-lapse recording refuge selection (interval 10 s, playback 10 frames s − 1 , 320 × 240 pixels) (Evosec GmbH & Co. KG, Germany). Time-lapse recordings were processed using QuickTime Player video playback software to determine time spent under each refuge for each temperature treatment. Statistics All analyses were run with R v4.3.1 in RStudio v2023.06.2 (RStudio Team 2023; R Core Team 2023 ). Where relevant, we used Wald’s tests to determine significance with the car package (Fox & Weisberg 2019) and pairwise posthoc contrasts in emmeans (Lenth 2023 ). Predictor variables for primary analyses of data from EWL and T pref experiments were species, season, sex, species×season, and sex×species unless otherwise stated. Posthoc tests of these analyses allow us to determine how species differ within each season and whether values differ between seasons within a species. We analysed EWL rates with a generalised linear model (GLM) with a log-link, with surface area as a covariate. Total resistance to EWL was also analysed with a GLM with a log-link, including gecko mass as a covariate. Resistance to EWL is an additional metric of water loss that incorporates skin temperature (Spotila & Berman 1976 ), and by measuring skin temperature we were able to evaluate the role of the skin in regulating EWL. An increase in evaporation from the skin would result in a decrease in skin temperature, due to the consequences of the latent heat of vaporisation across the skin. The relationship between resistance to EWL and skin temperature (T skin ) is shown in Table 4 in Young et al. ( 2005 ) with skin temperature being significantly dependent on skin resistance (t 23 = 4.19, p < 0.001, r 2 = 0.43). To determine if there was a seasonal change in cutaneous water loss (as a component of total EWL), we compared the differences between the air temperature in the experimental chamber (T chamber ) and gecko skin temperature (T sdiff = T chamber - T skin ) with an ANOVA. In addition to exploring seasonal EWL and seasonal T pref , we further used geckos collected for the EWL experiment to determine if the geckos differed in body condition between the two seasons. As a measure of body condition, we calculated the ratio of mass divided by SVL (Sion et al. 2021). To analyse body condition, we removed gravid females and analysed with an ANOVA. As six A. rhombifer (2 in dry season, 4 in wet season) were gravid, we separately analysed whether A. rhombifer females had EWL rates influenced by the interaction between season and gravid state with a log-link GLM including surface area as a covariate. Only two H. binoei were gravid at the time of sampling, which was too small a sample size to analyse. Preferred temperatures were analysed with a linear mixed effects model with the lme4 package (Bates et al. 2015 ). Gecko ID was included as a random factor because each individual had six readings included in the data. As six A. rhombifer (2 in dry season, 4 in wet season) were gravid, we separately analysed whether A. rhombifer females had thermal preferences influenced by the interaction between season and gravid state. To assess thermo-hydroregulation for each species, we used binomial generalised linear mixed effects models (GLMM) in the lme4 package (Bates et al. 2015 ) to test if the proportion of time spent in any refuge (as opposed to the open), and the proportion of refuge time spent in the humid refuge (as opposed to the dry), varied by treatment. Analyses were weighted by total time in the experiment, and total time spent in refugia (for the humid refuge analysis) and included gecko ID as a random variable. Geckos run in this experiment were mostly males (Table 1 ); the few female H. binoei used the refuges within the range of the males, and we performed no analyses based on sex for H. binoei or H. frenatus (all male). For A. rhombifer , analyses of refuge and humid refuge use included sex and the interaction between treatment and sex. Only one female A. rhombifer was gravid during this experiment, which performed within the range of other females of the species. In addition to our analyses of refuge and humid refuge use within species, we further analysed for differences between species, comparing the three species’ refuge use in the 32H/32D treatment with binomial GLMMs, with weights and random factor as above. Lastly, within each treatment for each species, we used tests of equal proportions to detect if time spent in the humid refuge indicated a preference for or against that option. In these tests, we compared values against 0.5 (50:50 per option, i.e., no preference), results of which would suggest trade-offs and prioritisation of water balance and temperature. Within a species and treatment, values significantly below 0.5 indicate a preference for the dry refuge, while values significantly above 0.5 suggest a humid refuge preference. Table 1 Sample sizes of three gecko species included in each physiological experiment. Values in parentheses indicate sample sizes per sex. Species Evaporative Water Loss (dry, wet) Preferred Temperature (dry, wet) Thermo-Hydroregulation Experiment A. rhombifer 9 (3F/6M), 16 (9F/7M) 9 (3F/6M), 16 (9F/7M) 9 (5F/4M) H. binoei 11 (4F/7M), 6 (6F) 10 (7F/3M), 6 (6F) 7 (2F/5M) H. frenatus 9 (9M), 10 (4F/6M) 10 (2F/8M), 9 (9M) 9 (9M) Results Seasonal Water Loss EWL differed significantly between both season and species (Fig. 1 a, Table 2 ), but not their interaction, nor was there a difference between males and females. Dry season EWL rates were significantly lower than wet season rates (65.5% of wet season rates, on average). A. rhombifer had significantly lower EWL than the other two species (48–54% of the others; p ≤ 0.001 each). Table 2 Results of GLMs (evaporative water loss, resistance to water loss), ANOVAs (T sdiff , body condition), and LMMs (preferred temperature) assessing physiological metrics in three gecko species. Bold denotes significant p-values. Response Predictor Stat, df P EWL Surface area 0.017, 1 0.9 Season 5.14, 1 0.02 Species 10.24, 2 0.006 Sex 0.02, 1 0.9 Season×Species 1.03, 2 0.6 Sex×Species 2.65, 2 0.3 Total Resistance to Water Loss Mass 3.13, 1 0.08 Season 24.96, 1 < 0.0001 Species 11.71, 2 0.003 Sex 0.97, 1 0.3 Season×Species 0.14, 2 0.9 Sex×Species 1.64, 2 0.4 T sdiff Season 24.66, (1,52) < 0.0001 Species 26.86, (2,52) < 0.0001 Sex 1.03, (1,52) 0.3 Season×Species 0.89, (2,52) 0.4 Sex×Species 0.97, (2,52) 0.4 Body Condition Season 0.02, (1,44) 0.9 Species 2.74, (2,44) 0.08 Sex 0.03, (1,44) 0.9 Season×Species 0.11, (2,44) 0.9 Sex×Species 1.00, (2,44) 0.4 Preferred Temperature Season 11.42, 1 0.0007 Species 7.28, 2 0.03 Sex 0.19, 1 0.7 Season×Species 18.90, 2 < 0.0001 Sex×Species 0.57, 2 0.8 As expected, results of total resistance to EWL were similar to those for EWL rates above (Table 2 ). Total resistance also differed between both season and species (Fig. 1 b), with A. rhombifer experiencing 183% the resistance of H. binoei (p = 0.0001) and marginally higher resistance than H. frenatus (142% the resistance of H. frenatus , p = 0.06). In contrast, H. binoei had 78% the resistance of H. frenatus (p = 0.3). On average, total resistance in the dry was 154% that in the wet, identifying the capacity for dramatic physiological changes between the seasons. Gecko skin temperatures in the EWL experiment ranged from 0.2–2.4 ˚C below T chamber , with greater difference from T chamber in the wet season by approx. 0.6 ˚C (Fig. 1 c). The difference from T chamber also varied by species (Table 2 ), with A. rhombifer experiencing body temperatures closer to the chamber temperature than the other gecko species. This result is consistent with the significantly lower EWL (and higher resistance) by A. rhombifer because less evaporation results in less evaporative cooling (Young et al. 2005 ). T sdiff did not differ based on the other predictor variables. The seasonal shifts we found in EWL and resistance were likely not explained by food availability, as body condition did not differ between the seasons (Fig. 1 d, Table 2 ). Among female A. rhombifer , gravid state did not significantly predict EWL rates (gravidity: Χ 2 = 0.78, df = 1, p = 0.4; season×gravid: Χ 2 = 0.36, df = 1, p = 0.5). However, the data suggest that gravid females ( A. rhombifer and H. binoei ) may experience increased rates of evaporative water loss, though larger sample sizes are required to verify this. Thermal Preference T pref was significantly predicted by season, species and their interaction (Fig. 1 e, Table 2 ) but not sex. Unexpectedly, preferred temperatures were lower for A. rhombifer in the wet season than the dry season (2.7 ± 0.80 ˚C lower; p = 0.001), while H. frenatus had higher T pref in the wet compared to the dry (2.2 ± 0.91 ˚C higher; p = 0.02). H. binoei did not significantly change its T pref between the seasons (p = 0.2). In the dry season, A. rhombifer preferred temperatures were significantly greater than those of the other two species (p ≤ 0.04 each), while none of the species differed in the wet season (p > 0.06 each). Whether or not female A. rhombifer were gravid did not affect thermal preferences (gravidity: Χ 2 = 0.07, df = 1, p = 0.8; season×gravid: Χ 2 = 0.0002, df = 1, p = 1). Thermo-Hydroregulation A. rhombifer had reduced refuge use in increased treatment temperatures (Fig. 2 a, Table 3 ), with less time spent in refugia in the 32H/32D compared with the 22H/32D treatment (51% vs 69% use, respectively; p = 0.003). Proportion of refuge time spent in the humid refuge increased at higher treatment temperatures (Fig. 2 b), with significantly more time in the humid refuge at 32H/32D compared with the lower two treatments (49%, 48%, and 97% in increasing treatment temperature order; p < 0.0001 each). A. rhombifer was also the only species to prefer the humid refuge over the dry one, which occurred in the 32H/32D treatment (p = 0.046). Table 3 Results of GLMMs of refuge use in three species of geckos in a thermo-hydroregulation experiment. Dependent Species Predictor Χ 2 , df P Combined Refuge Use A. rhombifer Treatment 11.09, 2 0.004 Sex 3.74, 1 0.05 Treatment×Sex 1.25, 2 0.5 H. binoei Treatment 4.58, 2 0.1 H. frenatus Treatment 140.2, 2 < 0.0001 All, 32 Treatment Species 26.56, 2 < 0.0001 Humid Refuge Use A. rhombifer Treatment 51.40, 2 < 0.0001 Sex 2.44, 1 0.1 Treatment×Sex 0.85, 2 0.7 H. binoei Treatment 40.66, 2 < 0.0001 H. frenatus Treatment 32.63, 2 < 0.0001 All, 32 Treatment Species 2.07, 2 0.4 H. binoei regularly used refugia throughout the experiment (over 90% of time in each treatment, on average), with no significant differences among the treatments (Fig. 2 a, Table 3 ). H. binoei did, however, use the humid refuge in differing amounts among the three treatments (p < 0.002 each; Fig. 2 b), with increasing humid refuge use at increasing treatment temperatures (36%, 76%, 92% refuge time for 22H/32D, 27H/32D, and 32H/32D treatments, respectively). They never exhibited a preference for the humid or dry refuge (p > 0.4 each). In H. frenatus , refuge use and humid refuge use both differed among the treatments, with decreased overall refuge use in higher treatment temperatures (95%, 80%, 25%, respectively; Fig. 2 a), but an increase in proportion of refuge time spent in the humid option (< 1%, 6%, 60% in increasing treatment temperature order; Fig. 2 b). All posthoc tests among the treatments were significant (p ≤ 0.0003 each). H. frenatus only exhibited preference for the warm, dry refuge over the humid refuge in the 22H/32D treatment (p = 0.008). In the 32H/32D treatment, the gecko species differed in their refuge use (Fig. 2 a), with H. binoei using refuges significantly more than the other species (p ≤ 0.0002 each). The species did not differ in the proportion of refuge time that was spent in the humid refuge in this treatment (Fig. 2 b). Discussion In this study, we used a series of experiments to assess seasonal acclimatisation and trade-offs between thermal and hydric pressures in three gecko species in northern Australia. Supporting our first hypothesis, geckos had reduced water loss, and increased resistance to water loss, in the dry season compared with the wet season. The significant seasonal changes in EWL in these three nocturnal gecko species are consistent with the acclimatisation response found in a diurnal lizard from the same area in the wet–dry tropics (Blamires & Christian 1999 ). The significantly cooler skin temperatures of the geckos (relative to chamber air temperature) in the wet season indicates greater evaporative water loss and that the cutaneous component of total EWL changed seasonally. This is consistent with laboratory acclimation experiments (Kattan & Lillywhite 1989 ; Weaver et al. 2022 , 2023 ). High EWL in the wet season suggests that there is a cost to the maintenance of increased cutaneous resistance during the dry season (Weaver et al. 2023 ), which may be the energetic cost associated with lipid synthesis (Kattan & Lillywhite 1889). Although there are obvious advantages to conserving water during the dry season, the environmental factor(s) driving the seasonal change in physiology are not known. Seasonal reductions in the availability of food energy can elicit acclimatisation responses including metabolic depression (Christian et al. 1999a , 2023 ; Berg et al. 2017 ) and lower thermal preferences (Christian et al. 1983 ; Christian & Bedford 1995 , 1996 ; Christian & Weavers 1996 ) – both of which result in reduced energetic requirements in ectotherms. The limiting resource driving acclimatisation of EWL could be the overall availability of water (including water derived from food and drinking as well as atmospheric water), or it could simply be the availability of water in the air driving changes in skin structure. The fact that body condition did not decline in the dry season suggests that sufficient food (and associated water) is ingested during the dry season. Thus, it seems likely that the acclimatisation response resulting in lower EWL during the dry season is in response to low humidity (Weaver et al. 2023 ). Our second hypothesis, that T pref would be lower in the dry season, was not supported. Although two species exhibited non-significant trends toward decreased preferred temperatures in the dry season, the only significant indicator of acclimatisation suggested inverse acclimatisation in A. rhombifer which, contrary to our predictions, preferred warmer temperatures during the dry season. Studies have shown that nocturnal reptiles thermoregulate while active at night and while inactive in diurnal retreat sites and will bask opportunistically (Bustard 1967 ; Kearney & Predavec 2000 ; Nordberg & Schwarzkopf 2019 ). Furthermore, careful selection of diurnal retreat sites can allow the exploitation of microclimates and buffer environmental temperatures to maintain preferable T b while inactive (Webb & Shine 1998 ; Chukwuka et al. 2021 ). Aside from temperature, other environmental factors, such as resource availability, can influence preferred temperature (Smith et al. 2008 ; Abayarathna & Webb 2021 ; Christian et al. 2023 ). The lack of a significant shift in preferred temperature between seasons, as found in H. binoei and H. frenatus , has been observed in other reptile species (Hitchcock & McBrayer 2006 ; Smith et al. 2008 ; Christian et al. 2023 ), all of which either live in resource-rich environments with water or are nocturnal. The seasonal acclimatisation observed in A. rhombifer follows an inverse response similar to that identified in a range of physiological traits, including temperature preference, in other reptiles (Autumn & DeNardo 1995 ; Firth & Belan 1998 ; Berg et al. 2017 ). Inverse responses have been attributed to avoidance behaviours, where individuals seek relief from ambient environmental temperatures, evading thermal stress (Firth & Belan 1998 ). This inverse response may be tied to habitat use by A. rhombifer , where frequently perching on branches and shrubs may result in greater exposure to temperature fluctuations. However, sufficient thermal pressure to induce a response is unexpected if suitable retreat sites are available and utilised (Webb & Shine 1998 ; Chukwuka et al. 2021 ). Alternatively, a reduction in T pref could be a response to lower food availability (Brown & Griffin 2005 ; Gilbert & Miles 2016 ). However, the body condition of A. rhombifer was not different between seasons, and they have a lower T pref in the wet season when insect abundance is high (Churchill 1994 ). Thus, it seems unlikely that food availability explains the seasonal differences in T pref in A. rhombifer . In our experiment involving refugia with different thermal and hydric characteristics, we first examined the use of either refuge (as opposed to being elsewhere in the aquarium) as a function of temperature. Although the terrestrial Bynoe’s geckos spent most of their time in refugia regardless of temperature treatment, the two arboreal species increased refuge use at low temperatures, as would be expected given that ectotherms are more susceptible to predation at lower body temperatures (Christian & Tracy 1981 ). Considering periods when one or the other refuge was used, our experiment of preferences between humidity and warm thermal conditions supported our hypothesis, with a shift toward humid refuge use at higher temperatures. The geckos used the humid environment with more frequency as the temperature of that humid refuge increased, such that at suboptimal temperatures, the humid microhabitat was never preferred over the warm one. In fact, at low temperatures, H. frenatus preferred the warm, dry habitat, prioritising thermoregulation over hydroregulation. Another Australian lizard, Carlia rubrigularis , also prioritised thermal requirements by spending time in dry rather than slightly cooler wet environments, only preferring the wet habitat at temperatures closer to lizards’ preferred temperatures (Pintor et al. 2016 ). It is likely that short-term water limitations in well-hydrated lizards do not create a state of dehydration critical enough to be immediately addressed by the individual, highlighting the differing time scales that hydric and thermal stressors act on lizards. The interplay between thermoregulation and hydroregulation in natural systems is not well understood, and their associated behaviours may be at odds with each other (e.g., basking in low humidity versus sheltering in high humidity (Pirtle et al. 2019 ; Rozen-Rechels et al. 2019 ). Dehydration can influence activity patterns (Davis & DeNardo 2009 , 2010 ) and thermoregulation by reducing basking behaviours and thermoregulation precision, and lead to thermal depression (Ladyman & Bradshaw 2003 ; Kearney et al. 2018 ; Rozen-Rechels et al. 2020 ). However, dehydration in lizards generally occurs over a relatively long time-scale (Dupoué et al. 2020 ), while suboptimal thermal temperatures can have more immediate fitness impacts. High temperatures can result in death in a matter of minutes (Heatwole 1976 ), and less severe suboptimal temperatures can increase predation risk through reduced locomotor performance, as well as slow digestive rates (Christian & Tracy 1981 ; Waldschmidt & Tracy 1983 ). On the other hand, even eight days of water restriction only minimally increased use of a wet shelter by another small lizard ( Zootoca vivipara ; Chabaud et al. 2023 ). Although several days of dehydration elevates stress responses, it can also enhance some innate immune functions (Moeller et al. 2017 ; Brusch et al. 2019 ) and is unlikely to be lethal in most reptiles (Minnich 1982 ). Longer exposures to dry conditions, however, resulted in shifts in habitat selection to mitigate dehydration in vipers (Dezetter et al. 2023 ). Together, these studies demonstrate that thermoregulation and hydroregulation work on different time scales, with thermal requirements having greater importance on a short time scale, and hydric requirements being dealt with on a longer time scale, as evidenced by seasonal acclimatisation of EWL in the geckos of this study. During prolonged dry periods, some reptiles exhibit significantly decreased body conditions (Davis & DeNardo 2009 , 2010 ). Though water is not readily available during the dry season in the present study’s sampling site, no seasonal change in body condition suggests food availability is sufficient year-round. Although low compared to the wet season (Churchill 1994 ), measurements of dry season insect abundance have shown that significant numbers of flying insects are active in the first few hours after twilight, which overlaps with nocturnal gecko activity (Bustard 1967 ; Milne et al. 2005 ; Lei & Booth 2014 ). Although we do not have direct measurements of seasonal activity, the three species in this study were active throughout the year without obvious changes in habitat use or behaviour. This contrasts with some diurnal lizards, which show decreased levels of activity during dry periods (Christian et al. 1996a , 1996b , 1999b , 2003 ; Weaver et al. 2024 ), with notable exceptions being those that live near water (Christian & Weavers 1996 ). Mean minimum temperatures in the dry season are also within the thermal foraging range observed for H. frenatus , which, alongside the native nocturnal gecko Gehyra variegata , is as low as 18 ˚C (Bustard 1967 ; Lei & Booth 2014 ). Therefore, temperature is expected to have a negligible impact on foraging in this environment. Thus, the ability to thermoregulate, moderate environmental temperatures, and sufficient food availability throughout the year may lessen the advantages of seasonal physiological plasticity in thermal preference. The phenomenon of seasonal changes in EWL has implications for the effects of climate change and management decisions related to biodiversity conservation (Seebacher et al. 2015 ). Thus, it is important that we increase our understanding of the role of habitat variability, the mechanisms, the apparent costs, and the time required for physiological adjustments. The wet–dry tropics, in which humidity and the availability of water change more substantially across seasons than do environmental temperatures, may represent one end of a continuum of environments that favour EWL seasonal plasticity. Wet tropical climates may be at the opposite extreme (Huey et al. 2012 ; Christian et al. 2023 ). It is less clear whether or not temperate climates would be conducive to the evolution of EWL plasticity because winter inactivity may obviate the need for seasonal shifts in skin permeability. However, the discovery of an acclimation response after only 8 days (Weaver et al. 2022 , 2023 ) raises the possibility of short-term adjustments related to prevailing weather conditions as opposed to the months-long seasonal pattern we found in the seasonal tropics. Seasonal measurements, or even more frequent measurements, from additional species in a range of environments are required to answer these questions to provide a comprehensive understanding of plasticity in EWL. Declarations Conflict of Interest None to declare. Ethics approval All animal experiments were approved by the Charles Darwin University Animal Ethics Committee (permit A19005). Consent to participate Not applicable Consent for publication Not applicable Funding This research was supported by a grant from the Australian Research Council DP190102395. CLW was supported by the Australian Research Council grant DP210102176. Authors’ contributions KD, KS and KC conceived the ideas and designed the methodology. 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R Foundation for Statistical Computing. https://www.R-project.org/ R Studio Team (2023) RStudio: Integrated Development Environment for R. RStudio, PBC., Boston, MA, USA . http://www.rstudio.com/ Rozen-Rechels D, Dupoué A, Lourdais O, Chamaillé-Jammes S, Meylan S, Clobert J, Le Galliard JF (2019) When water interacts with temperature: Ecological and evolutionary implications of thermo-hydroregulation in terrestrial ectotherms. Ecol Evol 9:10029–10043. 10.1002/ece3.5440 Rozen-Rechels D, Dupoué A, Meylan S, Qitout K, Decencière B, Agostini S, Le Galliard JF (2020) Acclimation to water restriction implies different paces for behavioral and physiological responses in a lizard species. Physiol Biochem Zool 93:160–174. 10.1086/707409 Rozen-Rechels D, Rutschmann A, Dupoué A, Blaimont P, Chauveau V, Miles DB, Guillon M, Richard M, Badiane A, Meylan S, Clobert J, Le Galliard JF (2021) Interaction of hydric and thermal conditions drive geographic variation in thermoregulation in a widespread lizard. Ecol Monogr 91:e01440. 10.1002/ecm.1440 Sannolo M, Carretero MA (2019) Dehydration constrains thermoregulation and space use in lizards. PLoS ONE 14(7):e0220384. 10.1371/journal.pone.0220384 Seebacher F (2005) A review of thermoregulation and physiological performance in reptiles: what is the role of phenotypic flexibility? J Comp Physiol B 175:453–461. 10.1007/s00360-005-0010-6 Seebacher F, White CR, Franklin CE (2015) Physiological plasticity increases resilience of ectothermic animals to climate change. Nat Clim Change 5:61–66. 10.1038/nclimate2457 Shoemaker V, Nagy KA (1977) Osmoregulation in amphibians and reptiles. Ann Rev Physiol 39:449–471. 10.1146/annurev.ph.39.030177.002313 Smith JG, Christian K, Green B (2008) Physiological ecology of the mangrove-dwelling varanid Varanus indicus . Physiol Biochem Zool 81(5):561–569. 10.1086/590372 Spotila JR, Berman EN (1976) Determination of skin resistance and the role of the skin in controlling water loss in amphibians and reptiles. Comp Biochem Phys A 55:407–411. 10.1016/0300-9629(76)90069-4 Toolson EC, Hadley NF (1979) Seasonal effects on cuticular permeability and epicuticular lipid composition in Centruroides sculpturatus Ewing 1928 (Scorpiones: Buthidae). J Comp Physiol 129:319–325. 10.1007/BF00686988 Volkoff H, Rønnestad I (2020) Effects of temperature on feeding and digestive processes in fish. Temperature 7:307–320. 10.1080/23328940.2020.1765950 Waldschmidt S, Tracy CR (1983) Interactions between a lizard and its thermal environment: implications for sprint performance and space utilization in the lizard Uta stansburiana . Ecology 64:476–484. 10.2307/1939967 Weaver SJ, Axsom IJ, Peria L, McIntyre T, Chung J, Telemeco RS, Westphal MF, Taylor EN (2024) Hydric physiology and ecology of a federally endangered desert lizard. Conserv Physiol 12:coae019. 10.1093/conphys/coae019 Weaver SJ, Edwards H, McIntyre T, Temple SM, Alexander Q, Behrens MC, Biedebach RE, Budwal SS, Carlson JE, Castagnoli JO, Fundingsland AD, Hart DV, Heaphy JS, Keller SW, Lucatero KI, Mills KH, Moallemi NM, Murguia AM, Navarro L, O’Brien E, Perez JK, Schauerman TJ, Stephens DM, Venturini MC, White CM, Taylor EN (2022) Cutaneous evaporative water loss in lizards is variable across body regions and plastic in response to humidity. Herpetologica 78:169–183. 10.1655/Herpetologica-D-21-00030.1 Weaver SJ, McIntyre T, van Rossum T, Telemeco RS, Taylor EN (2023) Hydration and evaporative water loss of lizards change in response to temperature and humidity acclimation. J Exp Biol 226:jeb246459. 10.1242/jeb.246459 Webb JK, Shine R (1998) Thermoregulation by a nocturnal elapid snake ( Hoplocephalus bungaroides ) in southeastern Australia. Physiol Zool 71:680–692. 10.1086/515979 While GM, Noble DW, Uller T, Warner DA, Riley JL, Du WG, Schwanz LE (2018) Patterns of developmental plasticity in response to incubation temperature in reptiles. J Exp Zool Part A 329:162–176. 10.1002/jez.2181 Wilson RS, Franklin CE (2002) Testing the beneficial acclimation hypothesis. Trends Ecol Evol 17:66–70. 10.1016/S0169-5347(01)02384-9 Young JE, Christian KA, Donnellan S, Tracy CR, Parry D (2005) Comparative analysis of cutaneous evaporative water loss in frogs demonstrates correlation with ecological habits. Physiol Biochem Zool 78(5):847–856. doi.org/10.1086/432152 Additional Declarations The authors declare no competing interests. Supplementary Files ESM1.xlsx ESM2Surfaceareacalculations.pdf ESM3.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5444175","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":377494226,"identity":"b7fe1a21-60e0-441a-b015-81c79515dc04","order_by":0,"name":"Kimberley Day","email":"","orcid":"","institution":"Charles Darwin University","correspondingAuthor":false,"prefix":"","firstName":"Kimberley","middleName":"","lastName":"Day","suffix":""},{"id":377498305,"identity":"d1c0980d-1175-4c4b-ad30-f69feef90f44","order_by":1,"name":"Chava Weitzman","email":"","orcid":"https://orcid.org/0000-0002-6103-1885","institution":"Charles Darwin University","correspondingAuthor":false,"prefix":"","firstName":"Chava","middleName":"","lastName":"Weitzman","suffix":""},{"id":377498630,"identity":"23f9fa8b-66fa-45e5-b29f-3e90f3436b36","order_by":2,"name":"Kade Skelton","email":"","orcid":"","institution":"Charles Darwin University","correspondingAuthor":false,"prefix":"","firstName":"Kade","middleName":"","lastName":"Skelton","suffix":""},{"id":377499111,"identity":"2adf8f1f-4694-4bd8-8475-b5675d07d2d1","order_by":3,"name":"Angga Rachmansah","email":"","orcid":"https://orcid.org/0000-0001-9851-754X","institution":"Charles Darwin University","correspondingAuthor":false,"prefix":"","firstName":"Angga","middleName":"","lastName":"Rachmansah","suffix":""},{"id":377499351,"identity":"d4741949-3cf9-4b22-9bf4-2021fc1fc5ba","order_by":4,"name":"Keith Christian","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-6135-1670","institution":"Charles Darwin University","correspondingAuthor":true,"prefix":"","firstName":"Keith","middleName":"","lastName":"Christian","suffix":""}],"badges":[],"createdAt":"2024-11-13 06:19:29","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-5444175/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5444175/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68974584,"identity":"f7735e15-741a-4eb5-b36a-547298aa7b4f","added_by":"auto","created_at":"2024-11-14 06:34:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":182947,"visible":true,"origin":"","legend":"\u003cp\u003eAcclimatisation in EWL and preferred temperatures in geckos, with no seasonal change in body condition. (a) EWL; (b) resistance to water loss; (c) T\u003csub\u003esdiff\u003c/sub\u003e (T\u003csub\u003echamber\u003c/sub\u003e - T\u003csub\u003eskin\u003c/sub\u003e); (d) body condition; and (e) preferred body temperatures. Points are raw values for individual geckos (one average value per gecko for preferred temperature) in the dry (open) and wet (filled) seasons. Horizontal lines denote emmeans estimates with 95% confidence intervals.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5444175/v1/b14cc08f456dd979a80deee0.png"},{"id":68974288,"identity":"f35d52a8-1f91-40b2-abc6-8c2fab362766","added_by":"auto","created_at":"2024-11-14 06:26:45","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":209252,"visible":true,"origin":"","legend":"\u003cp\u003eProportion of time geckos spent in refuges in three temperature treatments. In all three treatments the dry refuge was 32 ˚C. Ambient air and the humid refuge was 22, 27, or 32 ˚C (denoted as 22H/32D, 27H/32D, 32H/32D, respectively). (a) Proportion of total experiment time the geckos spent in either refuge. (b) Proportion of the total refuge time geckos spent in the humid refuge. Points are raw values for individual. Horizontal lines denote emmeans estimates with 95% confidence intervals from binomial GLMMs.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5444175/v1/36864c120c60c5f40ba08dae.jpeg"},{"id":68975873,"identity":"bb660c45-b27e-46fb-86fb-07b343d1019b","added_by":"auto","created_at":"2024-11-14 06:42:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1199682,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5444175/v1/01232c13-c56d-481a-88fa-d12f1fb249aa.pdf"},{"id":68974287,"identity":"cd741961-b0fa-4589-add3-58f022f0f27f","added_by":"auto","created_at":"2024-11-14 06:26:45","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15574,"visible":true,"origin":"","legend":"","description":"","filename":"ESM1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5444175/v1/57d9d654678c6001a5c53fb9.xlsx"},{"id":68974286,"identity":"14461407-6020-457f-9ea0-9bfdfd41064d","added_by":"auto","created_at":"2024-11-14 06:26:45","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":71515,"visible":true,"origin":"","legend":"","description":"","filename":"ESM2Surfaceareacalculations.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5444175/v1/654a911e5af24afec2d9c100.pdf"},{"id":68974290,"identity":"c4ffe33c-6172-4346-956c-cca77d09d91b","added_by":"auto","created_at":"2024-11-14 06:26:46","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":45718,"visible":true,"origin":"","legend":"","description":"","filename":"ESM3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5444175/v1/dc1350f19ace9205de6c4c85.xlsx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003ePatterns of seasonal plasticity in evaporative water loss and preferred temperature in three geckos of the wet–dry tropics\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePhysiological plasticity is widespread in nature, maximising performance and survival in response to changing climate and ecological interactions across the year. For instance, plasticity can buffer against the negative effects of increasing temperatures, allowing for increasing heat tolerance in ectotherms to reduce the likelihood of overheating (Gunderson et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Though commonly discussed in terms of seasonal temperature changes, flexibility in physiological processes may also be initiated, or optimised, by other environmental cues. Reductions in metabolic rate and preferred body temperature conserve energy and water during seasons when resource availability is limiting even if environmental temperatures are not (Christian et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999a\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Berg et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Importantly, the extent or strength of plasticity may depend on the variability experienced by the organism (Mu\u0026ntilde;oz \u0026amp; Bodensteiner \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A comparison of 41 bird species from the Central American wet tropics versus six species from a temperate site found greater seasonal change in the temperate species in thermal and metabolic measures (Pollock et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similarly, variability in resources in the seasonal (wet\u0026ndash;dry) tropics may favour the evolution of physiological plasticity of ectotherms as compared to the relative stability of the aseasonal (wet) tropics (Christian et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999a\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Huey et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBody temperature (T\u003csub\u003eb\u003c/sub\u003e) influences all biological processes, including digestion, locomotion, reproduction, and growth and has been widely studied in reptiles and other ectotherms (Heatwole \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Huey \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Christian \u0026amp; Tracy \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Kearney \u0026amp; Predavec \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Navas et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Chukwuka et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Volkoff \u0026amp; R\u0026oslash;nnestad \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, and see below). In seasonal climates, some species become dormant and forego thermoregulation, but other species thermoregulate to lower temperatures in winter (Seebacher \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The seasonality of the wet\u0026ndash;dry tropics induces a significant acclimatisation response in some lizards, with thermal preference shifting towards lower T\u003csub\u003eb\u003c/sub\u003e in the cooler dry season compared to the warmer wet season (Christian et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Christian \u0026amp; Bedford \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1995\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Christian \u0026amp; Weavers \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Christian et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999b\u003c/span\u003e). However, rather than being a response to environmental temperatures \u003cem\u003eper se\u003c/em\u003e, these examples of seasonal changes in preferred body temperature (T\u003csub\u003epref\u003c/sub\u003e) are likely mechanisms to conserve energy and water in response to the decrease in food and water resources in the dry season (Christian et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999a\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Berg et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Energy budget analyses indicate that seasonal food availability is the driving force for physiological plasticity related to energy expenditure (T\u003csub\u003epref\u003c/sub\u003e and metabolic rate) in several lizards from the seasonal tropics (Christian et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1996a\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999a\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999b\u003c/span\u003e), and calculations indicate that frillneck lizards would starve in the dry season were it not for these physiological adaptations (Christian et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1996b\u003c/span\u003e). Thus, physiological plasticity is likely to be essential for some species in environments with seasonal shortages of food coupled with high environmental temperatures (Christian et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe species described above are diurnal and are thus exposed to more extreme thermal environments than nocturnal species. Nevertheless, geckos can thermoregulate by selecting microhabitats in their daytime refugia (Bustard \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Chukwuka et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kearney \u0026amp; Predavec \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), and night-time habitats maintain some thermal heterogeneity, allowing nocturnal geckos to use warm microclimates to conductively thermoregulate while active (Autumn \u0026amp; DeNardo \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Nordberg \u0026amp; Schwarzkopf \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Nocturnal insects are substantially less abundant in the dry season compared to the wet season in the seasonal Australian tropics (Churchill \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). However, the lower body temperatures typically experienced by nocturnal animals from this region (Nordberg \u0026amp; Schwarzkopf \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) are similar to the daytime T\u003csub\u003eb\u003c/sub\u003es of the diurnal species that have seasonally-reduced T\u003csub\u003epref\u003c/sub\u003e values (Christian \u0026amp; Bedford \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Christian \u0026amp; Weavers \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Christian et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1996a\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999b\u003c/span\u003e), possibly obviating the need for further decreases in T\u003csub\u003eb\u003c/sub\u003e to conserve energy. Thus, it is difficult to predict whether or not nocturnal geckos would exhibit seasonal acclimatisation of T\u003csub\u003epref\u003c/sub\u003e. Little is known about seasonal thermal acclimatisation in nocturnal species from the wet\u0026ndash;dry tropics, but the gecko \u003cem\u003eOedura marmorata\u003c/em\u003e selected a lower T\u003csub\u003epref\u003c/sub\u003e in the dry season compared to the wet (Christian et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile thermoregulation is relatively well-studied, the importance of hydroregulation in reptiles has only recently become better understood (Grimm-Seyfarth et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kearney et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Pirtle et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Rozen-Rechels et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition to the potentially lethal consequences of inadequate hydration, sublethal dehydration can result in negative physiological and ecological consequences (Pirtle et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Rozen-Rechels et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In particular, optimal thermoregulation and activity patterns can be disrupted, resulting in compromised performances related to foraging, predator avoidance, and reproductive success (Rozen-Rechels et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sannolo \u0026amp; Carretero \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eReptiles from wet or mesic environments typically have higher rates of evaporative water loss (EWL) compared to those from arid environments (Hillman \u0026amp; Gorman \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Dmi\u0026rsquo;el et al. 1997; Cox \u0026amp; Cox \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and within a climatic zone, animals occupying mesic microhabitats have higher rates of EWL than those from drier microhabitats (Belasen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The mechanisms behind these patterns are typically not known, but include the possibilities of genetic differences among populations, irreversible developmental phenotypic plasticity in response to environment, or reversible physiological plasticity in which an individual can change in response to environmental conditions (Dmi\u0026rsquo;el et al. 1997; Wilson and Franklin \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Cox \u0026amp; Cox \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; While et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Christian et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). An example of a reversible change in EWL is related to the morphological and metabolic consequences of pregnancy in a viviparous snake (Lourdais et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Pregnant snakes have higher rates of EWL and select warmer and moister microhabitats, further supporting a pattern between EWL and microhabitat humidity, but in this case being driven by biological processes rather than by environmental conditions (Lourdais et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough EWL occurs across skin, eyes, and respiratory structures, cutaneous water loss is the largest component in reptiles (Bentley \u0026amp; Schmidt-Nielsen \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1966\u003c/span\u003e; Cohen \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Shoemaker \u0026amp; Nagy \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Mautz \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Kobayashi et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1983\u003c/span\u003e), typically accounting for more than 70% of the total EWL (Blamires \u0026amp; Christian \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Physiological plasticity of EWL has been explored in lizards in the laboratory, with individuals exposed to humid conditions having higher rates of EWL than those acclimated to dry conditions (Kobayashi et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Kattan \u0026amp; Lillywhite \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Rozen-Rechels et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Weaver et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Seasonal changes in EWL have been documented in beetles exposed to semi-natural conditions (Cooper \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1985\u003c/span\u003e) and, in arid adapted scorpions exposed to natural conditions, seasonal physiological plasticity in EWL has been mechanistically linked to seasonal changes in epicuticular biochemical composition (Toolson \u0026amp; Hadley \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). We are only aware of a single published report of field acclimatisation in which lizards measured from a location during a wet season had higher rates of EWL than lizards measured from the same location during a dry time of the year (Blamires and Christian \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). This study showed no significant seasonal differences in ocular or respiratory EWL, but there was a significant seasonal change in cutaneous EWL, which is consistent with measurements of skin permeability being higher in lizards acclimated to wet conditions as compared to those acclimated to dry conditions in the laboratory (Kobayashi et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Kattan and Lillywhite \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Weaver et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile there is growing evidence of seasonal thermal and metabolic plasticity in reptiles (Christian et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the focus has been on diurnal species, despite differing ecological pressures on nocturnal versus diurnal habits. Before generalisations can be drawn about the pervasiveness and ecological drivers of acclimatisation, more information is needed from a range of climatic zones (Christian et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and this is particularly true for nocturnal animals. Nocturnal geckos are informative in this regard because they can be compared to the better-studied lizard species from the same area, thus providing insight into both the prevalence of physiological plasticity and the ecological drivers. This study aims to quantify seasonal plasticity in thermal and hydric physiology of three common nocturnal gecko species at a site in the wet\u0026ndash;dry tropics in Northern Australia. We focus the study on measuring acclimatisation in EWL and T\u003csub\u003epref\u003c/sub\u003e in the wet and dry seasons. Given that water availability fluctuates more than temperature in the wet\u0026ndash;dry tropics, we also test for the relative importance of behavioural hydroregulation versus thermoregulation, or the trade-offs between maintaining a preferred temperature or avoiding dehydrating microhabitats. We used this series of physiological and behavioural experiments to address three hypotheses, and although we compared across the species, our over-arching hypothesis was that the climatic conditions of the site was the driving force, and thus the three species would not differ. First, we hypothesised that geckos would decrease EWL to conserve water in the dry season (as per Blamires \u0026amp; Christian \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Second, we hypothesised that T\u003csub\u003epref\u003c/sub\u003e would be lower in the dry season than in the wet as per other lizards in the seasonal tropics (Christian et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; 1999; \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Christian \u0026amp; Bedford \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Christian \u0026amp; Weavers \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Third, we hypothesised that, when provided options of a dry refuge at preferred temperature and a humid refuge at or below preferred temperature in the thermo-hydroregulation experiment, prioritisation of desiccation avoidance would emerge at increasing temperatures. That is, geckos would prioritise seeking preferred temperatures when the humid option was at low temperatures, but when offered a humid refuge at higher temperatures, the geckos would shift from a preference for warmth toward a preference for desiccation avoidance (as per Pintor et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Thus, under this hypothesis, when both dry and humid refuge options are at preferred temperature, geckos would show a preference for the humid refuge, which allows both desiccation avoidance and thermoregulation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy species, collection site and husbandry\u003c/h2\u003e \u003cp\u003eWe studied three widespread, common nocturnal gecko species in the wet\u0026ndash;dry tropics of Darwin, Australia: zig-zag geckos (\u003cem\u003eAmalosia rhombifer\u003c/em\u003e), Bynoe\u0026rsquo;s geckos (\u003cem\u003eHeteronotia binoei\u003c/em\u003e) and Asian house geckos (\u003cem\u003eHemidactylus frenatus\u003c/em\u003e). \u003cem\u003eA. rhombifer\u003c/em\u003e and \u003cem\u003eH. binoei\u003c/em\u003e are endemic to Australia, occupying arboreal and predominantly terrestrial habitats, respectively. \u003cem\u003eH. frenatus\u003c/em\u003e is a well-established invasive species from South Asia that became established in Darwin around 1960 (Hoskin \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and occupies arboreal habitats. The physiological ecology of \u003cem\u003eA. rhombifer\u003c/em\u003e has not been studied previously. The T\u003csub\u003epref\u003c/sub\u003e of \u003cem\u003eH. binoei\u003c/em\u003e was 30.8\u0026deg;C as measured from individuals from the arid zone of South Australia (Kearney \u0026amp; Porter \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In \u003cem\u003eH. frenatus\u003c/em\u003e, the thermal tolerance to high temperatures does not vary between Thailand and eastern Australia, although thermal tolerance to low temperatures is lower in cooler locations (Lapwong et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe collected the adult native geckos in the early evening (19:00\u0026ndash;22:00) from bushland and \u003cem\u003eH. frenatus\u003c/em\u003e from both bushland and building walls at Charles Darwin University (CDU), Casuarina, Northern Territory, Australia (12\u0026deg;22\u0026rsquo;07\u0026rdquo;S, 130\u0026deg;51\u0026rsquo;58\u0026rdquo;E). The bushland consists of 5 ha of eucalypt-dominated savanna, adjacent to campus buildings, suburban housing and the Casuarina Coastal Reserve. Sampling was conducted during Darwin\u0026rsquo;s wet (November to April, with our experiments done from early January\u0026ndash;early March) and dry (May to October, with our experiments done from early August\u0026ndash;early September) seasons to align with climatic extremes of the wet\u0026ndash;dry tropics. In Darwin, \u0026gt; 90% of the annual rainfall (mean\u0026thinsp;=\u0026thinsp;1722.5 mm) occurs in the wet season, and mean monthly rainfall peaks in January at 429.8 mm and is lowest in July at 1.1 mm. There is some variability of humidity during the short transition periods between the wet and dry seasons, but for most of the wet season, humidity is consistently high, and during the dry season it is consistently low (Online Resource 1; climate statistics from Darwin Airport, 4.5 km south of the study site ; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.bom.gov.au/climate/\" target=\"_blank\"\u003ewww.bom.gov.au/climate/\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.bom.gov.au/climate/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e Accessed 3 October 2024). Mean minimum air temperature decreases by ~\u0026thinsp;3 ˚C in the dry versus wet season, with less variability in maximum air temperatures (Online Resource 1). Geckos were returned to the wild after being run through the experiment(s) within a season, with new individuals captured each season.\u003c/p\u003e \u003cp\u003eMeasurements of EWL and T\u003csub\u003epref\u003c/sub\u003e were taken within 48 h of capture. Individuals were fed every second day and provided water via a spray bottle daily. Many geckos in this study were used for two experiments; specifically, all \u003cem\u003eA. rhombifer\u003c/em\u003e and wet season \u003cem\u003eH. binoei\u003c/em\u003e in the EWL experiment were subsequently used in the thermal preference experiment, while wet season \u003cem\u003eH. frenatus\u003c/em\u003e used the thermal preference experiment were also subjected to the thermo-hydroregulation experiment. Following the thermal experiment, lizards were housed in plastic terraria (38 \u0026times; 23 \u0026times; 12 cm) with a small hide and placed in a temperature-controlled room set to 28\u0026deg;C (mean 27.9 \u0026plusmn; SD 0.11\u0026deg;C) with relative humidity of 40.0 \u0026plusmn; 1.5% (vapor pressure deficit\u0026thinsp;=\u0026thinsp;2.2 kPA), with an automated 12 h light-dark cycle until they were used in the thermo-hydroregulation experiment.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEvaporative Water Loss\u003c/h3\u003e\n\u003cp\u003eGecko EWL was measured during the day in the wet and dry seasons for each species using a flow-through system (Blamires \u0026amp; Christian \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Young et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Evaporative water loss components were plumbed in-line and housed in incubators (model MIR253, Sanyo and model XHC-25, IVYX Scientific) to maintain a nominal experimental temperature of 30\u0026deg;C (the exact air temperatures were measured by the probes). Five EWL lines were operated simultaneously. For each EWL line, air was drawn from the incubator through a silica gel drying column using a low flow sampler calibrated to 0.2 L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (model LFS-113, Gilian\u0026reg;). The dry air then passed through an experimental chamber housing the gecko, made from a modified 60 mL syringe (13.5 \u0026times; 2.6 cm). A probe (model HMP 110, Vaisala\u0026trade;), housed in a plastic tube (30 mm diameter), recorded the relative humidity and air temperature downstream of the gecko. The output from the probes was recorded continuously on an Apple Macintosh computer using an ADInstruments PowerLab paired with LabChart software (model PL3508, ADInstruments Pty Ltd, Bella Vista, Australia), and EWL was calculated from the equations of Bernstein et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1977\u003c/span\u003e) for an open-flow system, in conjunction with calculations (List \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1971\u003c/span\u003e) of saturation vapour density (needed to calculate the mass of water from the measurements of relative humidity). In brief, the mass flow of water from the animal:\u003c/p\u003e \u003cp\u003eM\u003csub\u003ew\u003c/sub\u003e = V\u003csub\u003ee\u003c/sub\u003e (VD\u003csub\u003ea\u003c/sub\u003e \u0026ndash; VD\u003csub\u003ei\u003c/sub\u003e), where V\u003csub\u003ee\u003c/sub\u003e = experimental flow rate; VD\u003csub\u003ea\u003c/sub\u003e = water vapour density of the air in the experimental chamber with the animal (g cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e); and VD\u003csub\u003ei\u003c/sub\u003e = baseline water vapour density (g cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eRelative humidity and temperature measurements were collected before (as baseline data) and after placing the gecko in the experimental chamber. Skin temperature was recorded from the dorsum of each gecko immediately after the experiment using an infrared thermometer (Traceable\u0026copy; mini, Thomas Scientific). Gecko EWL experiments generally lasted 30 min to 1 h to achieve a resting measure. A flat-line trace on the computer screen was indicative of a resting animal because movement resulted in increased water loss and an irregular trace. The lowest humidity reading over a 2 min period was taken during a rest period of at least 5 min duration. Generally, the readings were very stable while the animals were at rest. If an animal defecated or failed to rest during the experiment, it was re-run later that day. Baseline values were reconfirmed after each experiment.\u003c/p\u003e \u003cp\u003eTotal resistance (R) to water loss was calculated as: R = (VD\u003csub\u003es\u003c/sub\u003e \u0026ndash; VD\u003csub\u003ea\u003c/sub\u003e) E\u003csub\u003ec\u003c/sub\u003e\u003csup\u003e\u0026minus;1\u003c/sup\u003e, where R\u0026thinsp;=\u0026thinsp;total resistance to water loss (s cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), VD\u003csub\u003es\u003c/sub\u003e = the vapour density of the skin (taken as the saturation vapour density at the skin temperature, g cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e), VD\u003csub\u003ea\u003c/sub\u003e = water vapour density of the air in the experimental chamber with the animal (g cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e), and E\u003csub\u003ec\u003c/sub\u003e = the surface area-specific rate of water loss (g cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Spotila and Berman \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e1976\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo calculate gecko surface area (SA), we used linear measurements of each gecko (Belasen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chukwuka et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). First, SA of three life-like plastic toy lizard models was determined by covering each model in masking tape, colouring the outer tape with a marker, then carefully transferring the coloured tape to graph paper to obtain a direct measure of SA (Blamires \u0026amp; Christian \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Linear measurements were then taken from the same model and substituted into a range of geometric SA equations to determine which one most closely matched the direct measures of SA. Previously, Belasen et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and Chukwuka et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) estimated that lizard SA was roughly equivalent to the body representing a cylinder and the tail a cone. Our estimates with lizard models found that the best geometric SA equation (as compared to the direct measure from the coloured tape, based on lowest average difference between estimate and direct SA) assumed that the torso (including the head, snout to vent), tail and legs were separate, single-ended cylinders. The length and greatest width of these components were used in calculating SA for each gecko by adding the SAs of the six single-ended cylinders corresponding to the six gecko body parts (Online Resource 2). Body measurements were collected after geckos completed the EWL trial. Alongside linear measurements, geckos were weighed to allow for a seasonal comparison of body condition (see \u003cem\u003eStatistics\u003c/em\u003e below).\u003c/p\u003e\n\u003ch3\u003eThermal Preference\u003c/h3\u003e\n\u003cp\u003eSeasonal thermal preference was measured by placing geckos in a thermal gradient of 20\u0026deg;C to 40\u0026deg;C and recording T\u003csub\u003eb\u003c/sub\u003e with a thermal camera (model 868, Testo SE \u0026amp; Co. KGaA, Titisee-Neustadt, Germany; Barroso et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Nordberg \u0026amp; Schwarzkopf \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sannolo \u0026amp; Carretero \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The thermal gradient consisted of an artificial crevice hide made from glazed porcelain tile (54 \u0026times; 15 \u0026times; 0.8 cm), supported 1.5 cm above the substrate by a terracotta spacer at each end, and a 50 W heat lamp at one end of the hide, suspended\u0026thinsp;~\u0026thinsp;1 cm above the tile. Each thermal gradient was constructed in a glass aquarium (59 \u0026times; 34 \u0026times; 37 cm) with terracotta tiles (56 \u0026times; 30 \u0026times; 1.5 cm) as a substrate. Thermal gradients were assembled in controlled temperature rooms set to 19.5\u0026deg;C, with ten thermal gradients operated simultaneously.\u003c/p\u003e \u003cp\u003eIndividual geckos spent a total of approx. 48 h in the preferred temperature experiment, allowing an overnight period to explore the thermal gradient. Thermal images were collected over the following day and a half at hourly intervals during daylight hours, with a total of 12 thermal images collected from each gecko. Thermal images were processed manually using IR Soft thermal image analysis software (Testo SE \u0026amp; Co. KGaA, Titisee-Neustadt, Germany), with T\u003csub\u003eb\u003c/sub\u003e represented by a measurement from the gecko's dorsum (Nordberg \u0026amp; Schwarzkopf \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) for each thermal image. For analysis, T\u003csub\u003epref\u003c/sub\u003e data were reduced to a set point range defined by the central 50% of T\u003csub\u003eb\u003c/sub\u003e measures for each gecko (Christian \u0026amp; Weavers \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eThermo-Hydroregulation\u003c/h3\u003e\n\u003cp\u003eGeckos were given access to high and low-humidity refugia to determine preference for hydroregulation or thermoregulation across preferred and sub-optimal humidity and temperature combinations (Pintor et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Briefly, they were given a choice between a warm (but dry) refuge and a cool (and humid) refuge, and, in one treatment, a choice between two warm refugia, with one being dry and the other humid. The temperature in the dry refuge remained constant at 32\u0026deg;C with a mean of 36.5% humidity (absolute humidity (AH)\u0026thinsp;=\u0026thinsp;12.35 g m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e; vapour pressure (VP)\u0026thinsp;=\u0026thinsp;1.74 kPA; vapour pressure deficit (VPD)\u0026thinsp;=\u0026thinsp;3.02 kPa) similar to values experienced in the late afternoon in the dry season in Darwin, NT), offering preferred temperature (based on the results of the thermal preference experiment, below) and relatively low humidity. While the dry refuge remained at a constant temperature, the ambient aquarium conditions and the humid refuge were adjusted to three temperature treatments (32, 27 and 22\u0026deg;C) and a high humidity of 99% in the humid refuge. The 32\u0026deg;C treatment is close to the T\u003csub\u003epref\u003c/sub\u003e (see \u003cem\u003eResults\u003c/em\u003e), and the two lower temperatures are ecologically relevant nighttime temperatures (Online Resource 1). (At 32\u0026deg;C: AH\u0026thinsp;=\u0026thinsp;33.49 g m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, VP\u0026thinsp;=\u0026thinsp;4.71 kPA, VPD\u0026thinsp;=\u0026thinsp;0.05 kPa); at 27\u0026deg;C: AH\u0026thinsp;=\u0026thinsp;25.52 g m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, VP\u0026thinsp;=\u0026thinsp;3.53 kPA, VPD\u0026thinsp;=\u0026thinsp;0.04 kPa; at 22\u0026deg;C: AH\u0026thinsp;=\u0026thinsp;19.24 g m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, VP\u0026thinsp;=\u0026thinsp;2.62 kPA, VPD\u0026thinsp;=\u0026thinsp;0.03 kPa) We refer to these treatments as 32H/32D, 27H/32D, and 22H/32D throughout, indicating the humid and dry (constant) refuge temperatures. Temperatures in the humid refuge were varied between treatments by adjusting the room temperature, while the dry refuge temperature was maintained at 32\u0026deg;C independently using a heat mat. Thus, when geckos were not in either refuge, they would have experienced the treatment air temperature (22, 27 or 32\u0026deg;C) and an intermediate humidity, depending on their proximity to the humid and dry refugia.\u003c/p\u003e \u003cp\u003eThis experiment used the same setup from the thermal gradient, substituting the hide with two terracotta refugia (15 \u0026times; 15 \u0026times; 1.5 cm) at opposite ends of the aquarium acting as humid and dry refugia. High humidity was achieved by soaking the terracotta refuge in water to humidify the refuge. The second refuge was kept dry by drawing air from a drying column and pumping it into the refuge through aquarium tubing (4 mm) at 0.2 L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Refuge conditions were monitored using hygro-thermometer (model 800027, Sper Scientific) probes placed under each refuge, with an electronic display at the back of the aquarium. Each treatment trial lasted 20 h, with an additional 4 h prior to data collection to allow conditions to stabilise. Six geckos were trialled in separate aquaria simultaneously, with treatments consecutively applied in the reverse order for each new set of geckos.\u003c/p\u003e \u003cp\u003eRefuge selection was recorded using a webcam (Logitech) placed in front of the aquarium, positioned so that the gecko could be seen when occupying either refuge. An Apple MacBook running Evocam software (version 3.6.5, Evological\u0026copy;) was used to create a time-lapse recording refuge selection (interval 10 s, playback 10 frames s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 320 \u0026times; 240 pixels) (Evosec GmbH \u0026amp; Co. KG, Germany). Time-lapse recordings were processed using QuickTime Player video playback software to determine time spent under each refuge for each temperature treatment.\u003c/p\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eAll analyses were run with R v4.3.1 in RStudio v2023.06.2 (RStudio Team 2023; R Core Team \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Where relevant, we used Wald\u0026rsquo;s tests to determine significance with the car package (Fox \u0026amp; Weisberg 2019) and pairwise posthoc contrasts in emmeans (Lenth \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Predictor variables for primary analyses of data from EWL and T\u003csub\u003epref\u003c/sub\u003e experiments were species, season, sex, species\u0026times;season, and sex\u0026times;species unless otherwise stated. Posthoc tests of these analyses allow us to determine how species differ within each season and whether values differ between seasons within a species.\u003c/p\u003e \u003cp\u003eWe analysed EWL rates with a generalised linear model (GLM) with a log-link, with surface area as a covariate. Total resistance to EWL was also analysed with a GLM with a log-link, including gecko mass as a covariate. Resistance to EWL is an additional metric of water loss that incorporates skin temperature (Spotila \u0026amp; Berman \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e1976\u003c/span\u003e), and by measuring skin temperature we were able to evaluate the role of the skin in regulating EWL. An increase in evaporation from the skin would result in a decrease in skin temperature, due to the consequences of the latent heat of vaporisation across the skin. The relationship between resistance to EWL and skin temperature (T\u003csub\u003eskin\u003c/sub\u003e) is shown in Table\u0026nbsp;4 in Young et al. (\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) with skin temperature being significantly dependent on skin resistance (t\u003csub\u003e23\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.19, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.43). To determine if there was a seasonal change in cutaneous water loss (as a component of total EWL), we compared the differences between the air temperature in the experimental chamber (T\u003csub\u003echamber\u003c/sub\u003e) and gecko skin temperature (T\u003csub\u003esdiff\u003c/sub\u003e = T\u003csub\u003echamber\u003c/sub\u003e - T\u003csub\u003eskin\u003c/sub\u003e) with an ANOVA. In addition to exploring seasonal EWL and seasonal T\u003csub\u003epref\u003c/sub\u003e, we further used geckos collected for the EWL experiment to determine if the geckos differed in body condition between the two seasons. As a measure of body condition, we calculated the ratio of mass divided by SVL (Sion et al. 2021). To analyse body condition, we removed gravid females and analysed with an ANOVA. As six \u003cem\u003eA. rhombifer\u003c/em\u003e (2 in dry season, 4 in wet season) were gravid, we separately analysed whether \u003cem\u003eA. rhombifer\u003c/em\u003e females had EWL rates influenced by the interaction between season and gravid state with a log-link GLM including surface area as a covariate. Only two \u003cem\u003eH. binoei\u003c/em\u003e were gravid at the time of sampling, which was too small a sample size to analyse.\u003c/p\u003e \u003cp\u003ePreferred temperatures were analysed with a linear mixed effects model with the lme4 package (Bates et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Gecko ID was included as a random factor because each individual had six readings included in the data. As six \u003cem\u003eA. rhombifer\u003c/em\u003e (2 in dry season, 4 in wet season) were gravid, we separately analysed whether \u003cem\u003eA. rhombifer\u003c/em\u003e females had thermal preferences influenced by the interaction between season and gravid state.\u003c/p\u003e \u003cp\u003eTo assess thermo-hydroregulation for each species, we used binomial generalised linear mixed effects models (GLMM) in the lme4 package (Bates et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) to test if the proportion of time spent in any refuge (as opposed to the open), and the proportion of refuge time spent in the humid refuge (as opposed to the dry), varied by treatment. Analyses were weighted by total time in the experiment, and total time spent in refugia (for the humid refuge analysis) and included gecko ID as a random variable. Geckos run in this experiment were mostly males (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e); the few female \u003cem\u003eH. binoei\u003c/em\u003e used the refuges within the range of the males, and we performed no analyses based on sex for \u003cem\u003eH. binoei\u003c/em\u003e or \u003cem\u003eH. frenatus\u003c/em\u003e (all male). For \u003cem\u003eA. rhombifer\u003c/em\u003e, analyses of refuge and humid refuge use included sex and the interaction between treatment and sex. Only one female \u003cem\u003eA. rhombifer\u003c/em\u003e was gravid during this experiment, which performed within the range of other females of the species. In addition to our analyses of refuge and humid refuge use within species, we further analysed for differences between species, comparing the three species\u0026rsquo; refuge use in the 32H/32D treatment with binomial GLMMs, with weights and random factor as above. Lastly, within each treatment for each species, we used tests of equal proportions to detect if time spent in the humid refuge indicated a preference for or against that option. In these tests, we compared values against 0.5 (50:50 per option, i.e., no preference), results of which would suggest trade-offs and prioritisation of water balance and temperature. Within a species and treatment, values significantly below 0.5 indicate a preference for the dry refuge, while values significantly above 0.5 suggest a humid refuge preference.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSample sizes of three gecko species included in each physiological experiment. Values in parentheses indicate sample sizes per sex.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEvaporative Water Loss (dry, wet)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePreferred Temperature (dry, wet)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThermo-Hydroregulation Experiment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. rhombifer\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9 (3F/6M), 16 (9F/7M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9 (3F/6M), 16 (9F/7M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (5F/4M)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. binoei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11 (4F/7M), 6 (6F)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (7F/3M), 6 (6F)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7 (2F/5M)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. frenatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9 (9M), 10 (4F/6M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (2F/8M), 9 (9M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (9M)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSeasonal Water Loss\u003c/h2\u003e \u003cp\u003eEWL differed significantly between both season and species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), but not their interaction, nor was there a difference between males and females. Dry season EWL rates were significantly lower than wet season rates (65.5% of wet season rates, on average). \u003cem\u003eA. rhombifer\u003c/em\u003e had significantly lower EWL than the other two species (48\u0026ndash;54% of the others; p\u0026thinsp;\u0026le;\u0026thinsp;0.001 each).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of GLMs (evaporative water loss, resistance to water loss), ANOVAs (T\u003csub\u003esdiff\u003c/sub\u003e, body condition), and LMMs (preferred temperature) assessing physiological metrics in three gecko species. \u003cb\u003eBold\u003c/b\u003e denotes significant p-values.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResponse\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePredictor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStat, df\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEWL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurface area\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.017, 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeason\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.14, 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.02\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.24, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.006\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02, 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeason\u0026times;Species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.03, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex\u0026times;Species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.65, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Resistance to Water Loss\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMass\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.13, 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeason\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24.96, 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.71, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.003\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.97, 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeason\u0026times;Species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.14, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex\u0026times;Species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.64, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT\u003csub\u003esdiff\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeason\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24.66, (1,52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26.86, (2,52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.03, (1,52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeason\u0026times;Species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.89, (2,52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex\u0026times;Species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.97, (2,52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBody Condition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeason\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02, (1,44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.74, (2,44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03, (1,44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeason\u0026times;Species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.11, (2,44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex\u0026times;Species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.00, (2,44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePreferred Temperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeason\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.42, 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.0007\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.28, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.03\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.19, 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeason\u0026times;Species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18.90, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex\u0026times;Species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.57, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAs expected, results of total resistance to EWL were similar to those for EWL rates above (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Total resistance also differed between both season and species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), with \u003cem\u003eA. rhombifer\u003c/em\u003e experiencing 183% the resistance of \u003cem\u003eH. binoei\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0001) and marginally higher resistance than \u003cem\u003eH. frenatus\u003c/em\u003e (142% the resistance of \u003cem\u003eH. frenatus\u003c/em\u003e, p\u0026thinsp;=\u0026thinsp;0.06). In contrast, \u003cem\u003eH. binoei\u003c/em\u003e had 78% the resistance of \u003cem\u003eH. frenatus\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.3). On average, total resistance in the dry was 154% that in the wet, identifying the capacity for dramatic physiological changes between the seasons.\u003c/p\u003e \u003cp\u003eGecko skin temperatures in the EWL experiment ranged from 0.2\u0026ndash;2.4 ˚C below T\u003csub\u003echamber\u003c/sub\u003e, with greater difference from T\u003csub\u003echamber\u003c/sub\u003e in the wet season by approx. 0.6 ˚C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The difference from T\u003csub\u003echamber\u003c/sub\u003e also varied by species (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), with \u003cem\u003eA. rhombifer\u003c/em\u003e experiencing body temperatures closer to the chamber temperature than the other gecko species. This result is consistent with the significantly lower EWL (and higher resistance) by \u003cem\u003eA. rhombifer\u003c/em\u003e because less evaporation results in less evaporative cooling (Young et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). T\u003csub\u003esdiff\u003c/sub\u003e did not differ based on the other predictor variables.\u003c/p\u003e \u003cp\u003eThe seasonal shifts we found in EWL and resistance were likely not explained by food availability, as body condition did not differ between the seasons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Among female \u003cem\u003eA. rhombifer\u003c/em\u003e, gravid state did not significantly predict EWL rates (gravidity: Χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.78, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.4; season\u0026times;gravid: Χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.36, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.5). However, the data suggest that gravid females (\u003cem\u003eA. rhombifer\u003c/em\u003e and \u003cem\u003eH. binoei\u003c/em\u003e) may experience increased rates of evaporative water loss, though larger sample sizes are required to verify this.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThermal Preference\u003c/h3\u003e\n\u003cp\u003eT\u003csub\u003epref\u003c/sub\u003e was significantly predicted by season, species and their interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) but not sex. Unexpectedly, preferred temperatures were lower for \u003cem\u003eA. rhombifer\u003c/em\u003e in the wet season than the dry season (2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80 ˚C lower; p\u0026thinsp;=\u0026thinsp;0.001), while \u003cem\u003eH. frenatus\u003c/em\u003e had higher T\u003csub\u003epref\u003c/sub\u003e in the wet compared to the dry (2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91 ˚C higher; p\u0026thinsp;=\u0026thinsp;0.02). \u003cem\u003eH. binoei\u003c/em\u003e did not significantly change its T\u003csub\u003epref\u003c/sub\u003e between the seasons (p\u0026thinsp;=\u0026thinsp;0.2). In the dry season, \u003cem\u003eA. rhombifer\u003c/em\u003e preferred temperatures were significantly greater than those of the other two species (p\u0026thinsp;\u0026le;\u0026thinsp;0.04 each), while none of the species differed in the wet season (p\u0026thinsp;\u0026gt;\u0026thinsp;0.06 each).\u003c/p\u003e \u003cp\u003eWhether or not female \u003cem\u003eA. rhombifer\u003c/em\u003e were gravid did not affect thermal preferences (gravidity: Χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.07, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.8; season\u0026times;gravid: Χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.0002, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;1).\u003c/p\u003e \u003cp\u003e \u003cem\u003eThermo-Hydroregulation\u003c/em\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eA. rhombifer\u003c/em\u003e had reduced refuge use in increased treatment temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), with less time spent in refugia in the 32H/32D compared with the 22H/32D treatment (51% vs 69% use, respectively; p\u0026thinsp;=\u0026thinsp;0.003). Proportion of refuge time spent in the humid refuge increased at higher treatment temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), with significantly more time in the humid refuge at 32H/32D compared with the lower two treatments (49%, 48%, and 97% in increasing treatment temperature order; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 each). \u003cem\u003eA. rhombifer\u003c/em\u003e was also the only species to prefer the humid refuge over the dry one, which occurred in the 32H/32D treatment (p\u0026thinsp;=\u0026thinsp;0.046).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of GLMMs of refuge use in three species of geckos in a thermo-hydroregulation experiment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDependent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePredictor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eΧ\u003c/em\u003e \u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003edf\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e\u003cem\u003eCombined Refuge Use\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eA. rhombifer\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.09, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.74, 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreatment\u0026times;Sex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.25, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eH. binoei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.58, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eH. frenatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e140.2, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAll, 32 Treatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26.56, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e\u003cem\u003eHumid Refuge Use\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eA. rhombifer\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e51.40, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.44, 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreatment\u0026times;Sex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.85, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eH. binoei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40.66, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eH. frenatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e32.63, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAll, 32 Treatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.07, 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eH. binoei\u003c/em\u003e regularly used refugia throughout the experiment (over 90% of time in each treatment, on average), with no significant differences among the treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). \u003cem\u003eH. binoei\u003c/em\u003e did, however, use the humid refuge in differing amounts among the three treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.002 each; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), with increasing humid refuge use at increasing treatment temperatures (36%, 76%, 92% refuge time for 22H/32D, 27H/32D, and 32H/32D treatments, respectively). They never exhibited a preference for the humid or dry refuge (p\u0026thinsp;\u0026gt;\u0026thinsp;0.4 each).\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eH. frenatus\u003c/em\u003e, refuge use and humid refuge use both differed among the treatments, with decreased overall refuge use in higher treatment temperatures (95%, 80%, 25%, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), but an increase in proportion of refuge time spent in the humid option (\u0026lt;\u0026thinsp;1%, 6%, 60% in increasing treatment temperature order; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). All posthoc tests among the treatments were significant (p\u0026thinsp;\u0026le;\u0026thinsp;0.0003 each). \u003cem\u003eH. frenatus\u003c/em\u003e only exhibited preference for the warm, dry refuge over the humid refuge in the 22H/32D treatment (p\u0026thinsp;=\u0026thinsp;0.008).\u003c/p\u003e \u003cp\u003eIn the 32H/32D treatment, the gecko species differed in their refuge use (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), with \u003cem\u003eH. binoei\u003c/em\u003e using refuges significantly more than the other species (p\u0026thinsp;\u0026le;\u0026thinsp;0.0002 each). The species did not differ in the proportion of refuge time that was spent in the humid refuge in this treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we used a series of experiments to assess seasonal acclimatisation and trade-offs between thermal and hydric pressures in three gecko species in northern Australia. Supporting our first hypothesis, geckos had reduced water loss, and increased resistance to water loss, in the dry season compared with the wet season. The significant seasonal changes in EWL in these three nocturnal gecko species are consistent with the acclimatisation response found in a diurnal lizard from the same area in the wet\u0026ndash;dry tropics (Blamires \u0026amp; Christian \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The significantly cooler skin temperatures of the geckos (relative to chamber air temperature) in the wet season indicates greater evaporative water loss and that the cutaneous component of total EWL changed seasonally. This is consistent with laboratory acclimation experiments (Kattan \u0026amp; Lillywhite \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Weaver et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). High EWL in the wet season suggests that there is a cost to the maintenance of increased cutaneous resistance during the dry season (Weaver et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which may be the energetic cost associated with lipid synthesis (Kattan \u0026amp; Lillywhite 1889). Although there are obvious advantages to conserving water during the dry season, the environmental factor(s) driving the seasonal change in physiology are not known. Seasonal reductions in the availability of food energy can elicit acclimatisation responses including metabolic depression (Christian et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999a\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Berg et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and lower thermal preferences (Christian et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Christian \u0026amp; Bedford \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1995\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Christian \u0026amp; Weavers \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) \u0026ndash; both of which result in reduced energetic requirements in ectotherms. The limiting resource driving acclimatisation of EWL could be the overall availability of water (including water derived from food and drinking as well as atmospheric water), or it could simply be the availability of water in the air driving changes in skin structure. The fact that body condition did not decline in the dry season suggests that sufficient food (and associated water) is ingested during the dry season. Thus, it seems likely that the acclimatisation response resulting in lower EWL during the dry season is in response to low humidity (Weaver et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur second hypothesis, that T\u003csub\u003epref\u003c/sub\u003e would be lower in the dry season, was not supported. Although two species exhibited non-significant trends toward decreased preferred temperatures in the dry season, the only significant indicator of acclimatisation suggested inverse acclimatisation in \u003cem\u003eA. rhombifer\u003c/em\u003e which, contrary to our predictions, preferred warmer temperatures during the dry season. Studies have shown that nocturnal reptiles thermoregulate while active at night and while inactive in diurnal retreat sites and will bask opportunistically (Bustard \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Kearney \u0026amp; Predavec \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Nordberg \u0026amp; Schwarzkopf \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Furthermore, careful selection of diurnal retreat sites can allow the exploitation of microclimates and buffer environmental temperatures to maintain preferable T\u003csub\u003eb\u003c/sub\u003e while inactive (Webb \u0026amp; Shine \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Chukwuka et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Aside from temperature, other environmental factors, such as resource availability, can influence preferred temperature (Smith et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Abayarathna \u0026amp; Webb \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Christian et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The lack of a significant shift in preferred temperature between seasons, as found in \u003cem\u003eH. binoei\u003c/em\u003e and \u003cem\u003eH. frenatus\u003c/em\u003e, has been observed in other reptile species (Hitchcock \u0026amp; McBrayer \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Smith et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Christian et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), all of which either live in resource-rich environments with water or are nocturnal.\u003c/p\u003e \u003cp\u003eThe seasonal acclimatisation observed in \u003cem\u003eA. rhombifer\u003c/em\u003e follows an inverse response similar to that identified in a range of physiological traits, including temperature preference, in other reptiles (Autumn \u0026amp; DeNardo \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Firth \u0026amp; Belan \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Berg et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Inverse responses have been attributed to avoidance behaviours, where individuals seek relief from ambient environmental temperatures, evading thermal stress (Firth \u0026amp; Belan \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). This inverse response may be tied to habitat use by \u003cem\u003eA. rhombifer\u003c/em\u003e, where frequently perching on branches and shrubs may result in greater exposure to temperature fluctuations. However, sufficient thermal pressure to induce a response is unexpected if suitable retreat sites are available and utilised (Webb \u0026amp; Shine \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Chukwuka et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Alternatively, a reduction in T\u003csub\u003epref\u003c/sub\u003e could be a response to lower food availability (Brown \u0026amp; Griffin \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Gilbert \u0026amp; Miles \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, the body condition of \u003cem\u003eA. rhombifer\u003c/em\u003e was not different between seasons, and they have a lower T\u003csub\u003epref\u003c/sub\u003e in the wet season when insect abundance is high (Churchill \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Thus, it seems unlikely that food availability explains the seasonal differences in T\u003csub\u003epref\u003c/sub\u003e in \u003cem\u003eA. rhombifer\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn our experiment involving refugia with different thermal and hydric characteristics, we first examined the use of either refuge (as opposed to being elsewhere in the aquarium) as a function of temperature. Although the terrestrial Bynoe\u0026rsquo;s geckos spent most of their time in refugia regardless of temperature treatment, the two arboreal species increased refuge use at low temperatures, as would be expected given that ectotherms are more susceptible to predation at lower body temperatures (Christian \u0026amp; Tracy \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). Considering periods when one or the other refuge was used, our experiment of preferences between humidity and warm thermal conditions supported our hypothesis, with a shift toward humid refuge use at higher temperatures. The geckos used the humid environment with more frequency as the temperature of that humid refuge increased, such that at suboptimal temperatures, the humid microhabitat was never preferred over the warm one. In fact, at low temperatures, \u003cem\u003eH. frenatus\u003c/em\u003e preferred the warm, dry habitat, prioritising thermoregulation over hydroregulation. Another Australian lizard, \u003cem\u003eCarlia rubrigularis\u003c/em\u003e, also prioritised thermal requirements by spending time in dry rather than slightly cooler wet environments, only preferring the wet habitat at temperatures closer to lizards\u0026rsquo; preferred temperatures (Pintor et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It is likely that short-term water limitations in well-hydrated lizards do not create a state of dehydration critical enough to be immediately addressed by the individual, highlighting the differing time scales that hydric and thermal stressors act on lizards.\u003c/p\u003e \u003cp\u003eThe interplay between thermoregulation and hydroregulation in natural systems is not well understood, and their associated behaviours may be at odds with each other (e.g., basking in low humidity versus sheltering in high humidity (Pirtle et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Rozen-Rechels et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Dehydration can influence activity patterns (Davis \u0026amp; DeNardo \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and thermoregulation by reducing basking behaviours and thermoregulation precision, and lead to thermal depression (Ladyman \u0026amp; Bradshaw \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Kearney et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rozen-Rechels et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, dehydration in lizards generally occurs over a relatively long time-scale (Dupou\u0026eacute; et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), while suboptimal thermal temperatures can have more immediate fitness impacts. High temperatures can result in death in a matter of minutes (Heatwole \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1976\u003c/span\u003e), and less severe suboptimal temperatures can increase predation risk through reduced locomotor performance, as well as slow digestive rates (Christian \u0026amp; Tracy \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Waldschmidt \u0026amp; Tracy \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). On the other hand, even eight days of water restriction only minimally increased use of a wet shelter by another small lizard (\u003cem\u003eZootoca vivipara\u003c/em\u003e; Chabaud et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although several days of dehydration elevates stress responses, it can also enhance some innate immune functions (Moeller et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Brusch et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and is unlikely to be lethal in most reptiles (Minnich \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). Longer exposures to dry conditions, however, resulted in shifts in habitat selection to mitigate dehydration in vipers (Dezetter et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Together, these studies demonstrate that thermoregulation and hydroregulation work on different time scales, with thermal requirements having greater importance on a short time scale, and hydric requirements being dealt with on a longer time scale, as evidenced by seasonal acclimatisation of EWL in the geckos of this study.\u003c/p\u003e \u003cp\u003eDuring prolonged dry periods, some reptiles exhibit significantly decreased body conditions (Davis \u0026amp; DeNardo \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Though water is not readily available during the dry season in the present study\u0026rsquo;s sampling site, no seasonal change in body condition suggests food availability is sufficient year-round. Although low compared to the wet season (Churchill \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), measurements of dry season insect abundance have shown that significant numbers of flying insects are active in the first few hours after twilight, which overlaps with nocturnal gecko activity (Bustard \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Milne et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Lei \u0026amp; Booth \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although we do not have direct measurements of seasonal activity, the three species in this study were active throughout the year without obvious changes in habitat use or behaviour. This contrasts with some diurnal lizards, which show decreased levels of activity during dry periods (Christian et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1996a\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1996b\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999b\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Weaver et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), with notable exceptions being those that live near water (Christian \u0026amp; Weavers \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Mean minimum temperatures in the dry season are also within the thermal foraging range observed for \u003cem\u003eH. frenatus\u003c/em\u003e, which, alongside the native nocturnal gecko \u003cem\u003eGehyra variegata\u003c/em\u003e, is as low as 18 ˚C (Bustard \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Lei \u0026amp; Booth \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, temperature is expected to have a negligible impact on foraging in this environment. Thus, the ability to thermoregulate, moderate environmental temperatures, and sufficient food availability throughout the year may lessen the advantages of seasonal physiological plasticity in thermal preference.\u003c/p\u003e \u003cp\u003eThe phenomenon of seasonal changes in EWL has implications for the effects of climate change and management decisions related to biodiversity conservation (Seebacher et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Thus, it is important that we increase our understanding of the role of habitat variability, the mechanisms, the apparent costs, and the time required for physiological adjustments. The wet\u0026ndash;dry tropics, in which humidity and the availability of water change more substantially across seasons than do environmental temperatures, may represent one end of a continuum of environments that favour EWL seasonal plasticity. Wet tropical climates may be at the opposite extreme (Huey et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Christian et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It is less clear whether or not temperate climates would be conducive to the evolution of EWL plasticity because winter inactivity may obviate the need for seasonal shifts in skin permeability. However, the discovery of an acclimation response after only 8 days (Weaver et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) raises the possibility of short-term adjustments related to prevailing weather conditions as opposed to the months-long seasonal pattern we found in the seasonal tropics. Seasonal measurements, or even more frequent measurements, from additional species in a range of environments are required to answer these questions to provide a comprehensive understanding of plasticity in EWL.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eNone to declare.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics approval\u003c/strong\u003e \u003cp\u003eAll animal experiments were approved by the Charles Darwin University Animal Ethics Committee (permit A19005).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to participate\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by a grant from the Australian Research Council DP190102395. CLW was supported by the Australian Research Council grant DP210102176.\u003c/p\u003e\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e \u003cp\u003eKD, KS and KC conceived the ideas and designed the methodology. KD, KS, KC, and AR collected the data. CLW analysed the data. KD, CLW and KC wrote the draft of the manuscript. All authors collected geckos and gave final approval for publication.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe respectfully acknowledge the Larrakia people, the traditional owners of the land where this work was undertaken. All sampling was conducted under permit 64816 from the Northern Territory Parks and Wildlife Commission.\u003c/p\u003e\u003ch2\u003eAvailability of data and material\u003c/h2\u003e \u003cp\u003eThe raw data for this manuscript are in Online Resource 3.\u003c/p\u003e\u003ch2\u003eCode availability\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbayarathna T, Webb JK (2021) Do Incubation Temperatures Affect the Preferred Body Temperatures of Hatchling Velvet Geckos? Front Ecol Evol 9:727602. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fevo.2021.727602\u003c/span\u003e\u003cspan address=\"10.3389/fevo.2021.727602\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAutumn K, DeNardo DF (1995) Behavioral thermoregulation increases growth rate in a nocturnal lizard. 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Physiol Biochem Zool 78(5):847\u0026ndash;856. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.1086/432152\u003c/span\u003e\u003cspan address=\"10.1086/432152\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"20210db0-3dfc-4efe-bf82-dfc2f506779a","identifier":"10.13039/501100000923","name":"Australian Research Council","awardNumber":"DP190102395 and DP210102176","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Charles Darwin University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Acclimatisation, seasonal plasticity, evaporative water loss, thermoregulation, hydroregulation","lastPublishedDoi":"10.21203/rs.3.rs-5444175/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5444175/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSeasonal physiological plasticity (acclimatisation) facilitates homeostasis in changing environments and has been studied extensively with respect to thermal biology and metabolism. Less is known about seasonal changes in evaporative water loss (EWL) in response to changing water availability and humidity. The wet\u0026ndash;dry tropics of northern Australia experiences moderate seasonal temperature changes, but substantial changes in rainfall and humidity. We studied three gecko species (\u003cem\u003eAmalosia rhombifer\u003c/em\u003e, \u003cem\u003eHeteronotia binoei\u003c/em\u003e and \u003cem\u003eHemidactylus frenatus\u003c/em\u003e) in the wet and dry seasons with respect to their EWL, preferred body temperatures (T\u003csub\u003epref\u003c/sub\u003e), and their choice between a dry and humid refuge at and below T\u003csub\u003epref\u003c/sub\u003e. EWL was significantly lower in the dry season (66% of wet season values). T\u003csub\u003epref\u003c/sub\u003e for two of the species did not change seasonally, but \u003cem\u003eA. rhombifer\u003c/em\u003e selected lower T\u003csub\u003epref\u003c/sub\u003e during the warmer wet season. Given a choice of refugia, the humid refuge at low temperatures was never preferred over the warm microhabitat. When both refugia were at preferred temperature, only \u003cem\u003eA. rhombifer\u003c/em\u003e showed a significant preference for the humid microhabitat. These results demonstrate that although thermoregulation is prioritised in the short term, hydroregulation (physiological plasticity in EWL) is adjusted in the longer term, with shifts occurring on a seasonal scale. However, previous studies suggest shifts in EWL may occur in response to prevailing weather conditions on an even shorter timescale. Before broad generalisations can be drawn about the phenomenon of EWL plasticity, measurements need to be taken from more species in different climatic regions at ecologically relevant timescales.\u003c/p\u003e","manuscriptTitle":"Patterns of seasonal plasticity in evaporative water loss and preferred temperature in three geckos of the wet–dry tropics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-14 06:26:41","doi":"10.21203/rs.3.rs-5444175/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"14d0cf98-a1cf-4fdc-a04e-9444f7fe1580","owner":[],"postedDate":"November 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":40190004,"name":"Animal Physiology"}],"tags":[],"updatedAt":"2024-11-14T06:26:41+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-14 06:26:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5444175","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5444175","identity":"rs-5444175","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}