George A Brusch
email: gbrusch@okstate.edu

Jean-François Le Galliard

Robin Viton

Rodrigo Gavira

Jean Clobert

Olivier Lourdais

Reproducing in a

Keywords: pregnancy, climate change, environmental constraints, hydration, temperature, reproductive output

Introduction

Organismal attempts to maintain homeostasis during accelerating shifts in climatic conditions involve both behavioral and physiological mechanisms [START_REF] Hoffmann | Low potential for climatic stress adaptation in a rainforest Drosophila species[END_REF][START_REF] Deutsch | Impacts of climate warming on terrestrial ectotherms across latitude[END_REF][START_REF] Dillon | Global metabolic impacts of recent climate warming[END_REF][START_REF] New | Four degrees and beyond: the potential for a global temperature increase of four degrees and its implications[END_REF]. The capacity or not to maintain a relatively stable internal environment may constrain the future survival of many species in this context [START_REF] Kearney | Mechanistic niche modelling: combining physiological and spatial data to predict species' ranges[END_REF][START_REF] Buckley | Can mechanism inform species' distribution models?[END_REF]. The physiological resistance to environmental stressors can vary significantly among populations and species [START_REF] Stillman | Acclimation capacity underlies susceptibility to climate change[END_REF][START_REF] Johansen | Increasing ocean temperature reduces the metabolic performance and swimming ability of coral reef damselfishes[END_REF][START_REF] Riddell | Plasticity reveals hidden resistance to extinction under climate change in the global hotspot of salamander diversity[END_REF]) and often involves resource-based trade-offs [START_REF] Zera | The physiology of life history trade-offs in animals[END_REF].

Understanding how multiple climatic stressors shape plastic responses and influence life-history strategies is therefore critical to predicting climate change effects [START_REF] Huey | Predicting organismal vulnerability to climate warming: roles of behaviour, physiology and adaptation[END_REF]). Yet, to a large degree, current studies provide little information on how concurrent environmental constraints will challenge organismal physiology and life history trade-offs.

Most of the previous research on animals' response to global climate change has understandably focused on the effects of changing temperatures [START_REF] Harvell | Climate warming and disease risks for terrestrial and marine biota[END_REF][START_REF] Portner | Physiology and climate change[END_REF][START_REF] Helmuth | Organismal climatology: analyzing environmental variability at scales relevant to physiological stress[END_REF], especially increases in mean daily temperature [START_REF] Thomas | Extinction risk from climate change[END_REF][START_REF] Parmesan | Ecological and Evolutionary Responses to Recent Climate Change[END_REF][START_REF] Musolin | Insects in a warmer world: ecological, physiological and life-history responses of true bugs (Heteroptera) to climate change[END_REF][START_REF] Huey | Can behavior douse the fire of climate warming?[END_REF]). This appears relevant for the majority of species, which are ectotherms, as their locomotion, growth, and reproduction are temperature-sensitive [START_REF] Tewksbury | Putting the heat on tropical animals[END_REF]). However, climate warming involves a characteristic set of changes in diurnal and geographic patterns of temperature, precipitation, and other atmospheric conditions [START_REF] Kingsolver | Impacts of global environmental change on animals[END_REF]Helmuth et al.2005). For example, nocturnal warming is greater than diurnal warming in most regions, leading to reduced variation in daily temperatures [START_REF] Dai | Effects of clouds, soil moisture, precipitation, and water vapor on diurnal temperature range[END_REF][START_REF] Davy | Diurnal asymmetry to the observed global warming[END_REF]. In fact, nearly all the climatic warming seen in the northern hemisphere over the past century is due to increased daily minima [START_REF] Vose | Maximum and minimum temperature trends for the globe: An update through 2004[END_REF]. Shifts in rainfall patterns and water availability are equally important to consider in terrestrial ecosystems as both are similarly affected by climate change [START_REF] Fung | Water availability in + 2 C and + 4 C worlds[END_REF][START_REF] Sanderson | Regional temperature and precipitation changes under high-end (≥ 4 C) global warming[END_REF]. Current and predicted climatic conditions thus include shifts in diurnal and nocturnal temperatures, precipitation patterns, and interactions between temperature and precipitation change [START_REF] Vose | Maximum and minimum temperature trends for the globe: An update through 2004[END_REF][START_REF] Adler | Relationships between global precipitation and surface temperature on interannual and longer timescales[END_REF][START_REF] Zhou | Spatiotemporal patterns of changes in maximum and minimum temperatures in multi-model simulations[END_REF]).

While it remains unclear how organisms will be influenced by these compounded changes, is the effects are likely to be significant [START_REF] Bonebrake | Tolerance adaptation and precipitation changes complicate latitudinal patterns of climate change impacts[END_REF]. Many terrestrial ectotherms are likely to be at risk from climate change as their body temperature depends on behavioral thermoregulation [START_REF] Huey | Can behavior douse the fire of climate warming?[END_REF][START_REF] Kearney | Mechanistic niche modelling: combining physiological and spatial data to predict species' ranges[END_REF].

However, most previous studies have primarily focused on changing average thermal conditions [START_REF] Deutsch | Impacts of climate warming on terrestrial ectotherms across latitude[END_REF][START_REF] Herrando-Pérez | Intraspecific variation in lizard heat tolerance alters estimates of climate impact[END_REF][START_REF] Verheyen | Temperature variation makes an ectotherm more sensitive to global warming unless thermal evolution occurs[END_REF], and it remains unclear how ectotherms will be affected by independent shifts in daytime and nighttime temperatures. For example, rising temperatures during the day may force ectotherms above their optimal temperature, leading to heat stress and reduced performance [START_REF] Niehaus | Predicting the physiological performance of ectotherms in fluctuating thermal environments[END_REF][START_REF] Stoks | Daily temperature variation and extreme high temperatures drive performance and biotic interactions in a warming world[END_REF], while rising temperatures at night may either result in enhanced performance [START_REF] Speights | Contrasting the potential effects of daytime versus nighttime warming on insects[END_REF] or increased energetic costs and parasite loads [START_REF] Rutschmann | Intense nocturnal warming alters growth strategies, colouration and parasite load in a diurnal lizard[END_REF].

Recent studies have begun to document physiological and behavioral responses to changes in temperature and water availability [START_REF] Rozen-Rechels | When water interacts with temperature: Ecological and evolutionary implications of thermo-hydroregulation in terrestrial ectotherms[END_REF]Dupoué et al. 2020a) or to shifts in day and night temperatures [START_REF] Barton | Opposite effects of daytime and nighttime warming on top-down control of plant diversity[END_REF]. Reproduction is a particularly sensitive period that requires a considerable amount of energy and water investment as well as appropriate temperatures for the developing embryos. Reproductive mode may drive vulnerability to climate changes and viviparous species may be more susceptible because of substantial maternal investment during pregnancy [START_REF] Pincheira-Donoso | The evolution of viviparity opens opportunities for lizard radiation but drives it into a climatic cul-de-sac[END_REF]). In addition, viviparity generates parent-offspring conflicts for energetic resources [START_REF] Calow | The relationship between fecundity, phenology, and longevity: a systems approach[END_REF][START_REF] Doughty | etecting life history trade-offs: measuring energy stores in "capital" breeders reveals costs of reproduction[END_REF][START_REF] Reznick | Big houses, big cars, superfleas and the costs of reproduction[END_REF][START_REF] Wiley Kotiaho | Costs of sexual traits: a mismatch between theoretical considerations and empirical evidence[END_REF]) and hydric resources (Dupoue et al. 2015;[START_REF] Brusch | Reproduction alters hydration state but does not impact the positive effects of dehydration on innate immune function in children's pythons (Antaresia childreni)[END_REF][START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF][START_REF] Dupoué | Water restriction causes an intergenerational trade-off and delayed mother-offspring conflict in a viviparous lizard[END_REF]Brusch and DeNardo 2019;[START_REF] Brusch | Additive effects of temperature and water availability on pregnancy in a viviparous lizard[END_REF], which may be altered by climate change.

To address these questions, we manipulated day and night temperatures as well as water availability during gestation in female common lizards (Zootoca vivipara), a small cold-and wet-adapted and widespread viviparous lizard with a strictly diurnal activity. We applied ecologically relevant, daily thermal cycles mimicking behavioral thermoregulation during daytime and thermoconformity at night. Common lizards exhibit characteristics of mixed income and capital breeding [START_REF] Bleu | Reproductive allocation strategies: a long-term study on proximate factors and temporal adjustments in a viviparous lizard[END_REF]) and must make multiple physiological adjustments in response to climatic conditions during periods of heavy investment such as reproduction. We made the following three general hypotheses. First, warmer days would have a positive effect on reproductive output because of extended access to preferred temperature but would result in increased food intake to match the higher body-reserve mobilization, provided food availability is not limiting. Second, conversely, warmer nights would have a net negative impact on reproduction by increasing metabolic demands without providing thermal benefits for foraging [START_REF] Rutschmann | Intense nocturnal warming alters growth strategies, colouration and parasite load in a diurnal lizard[END_REF]. Third, water constraints would overall negate potential thermal benefits by negatively impacting maternal physiology [START_REF] Brusch | Additive effects of temperature and water availability on pregnancy in a viviparous lizard[END_REF].

Materials and methods

Study species

Zootoca vivipara are strictly diurnal, heliothermic lizards of small body size (adult snout-vent length, SVL 50-75 mm) with a very efficient thermoregulation behavior. Individuals are typically found in cool, mesic habitats such as bogs, heathland or wet meadows across northern Eurasia [START_REF] Dely | Lacerta vivipara Jacquin 1787-Waldeidechse[END_REF]. This species occurs in both viviparous and oviparous forms across Europe, but we considered a viviparous mode inhabiting populations from the Plateau de Millevache (Limousin) in France. Here, males copulate with females shortly after emerging in the spring with fertilization occurring in mid-late May. Pregnancy lasts 2-3 months, with parturition of 1-12 offspring occurring between mid-July and early August, depending on female body size and climatic conditions [START_REF] Foucart | Influence of reproductive mode on metabolic costs of reproduction: insight from the bimodal lizard Zootoca vivipara[END_REF][START_REF] Dupoué | Water restriction causes an intergenerational trade-off and delayed mother-offspring conflict in a viviparous lizard[END_REF]. After parturition, females replenish their reserves throughout the summer before entering over-wintering dormancy in late September [START_REF] Avery | Storage lipids in the lizard Lacerta vivipara: a quantitative study[END_REF][START_REF] Bauwens | The timing of reproduction in the lizard Lacerata vivipara: differences between individual females[END_REF].

Experimental design

Females used for this study were captured between 22-26 May (n = 115) or on 8 June (n = 20) 2019 from two different sites separated by less than 7 km. Pregnant females were captured by hand and reproductive status was inferred by the presence of recent male bite marks on the abdomen. We confirmed that all females captured were in the end stages of vitellogenesis or early pregnancy using ultrasonography (Sonosite MicroMaxx, Bothell, WA) and previously described methods (i.e., presence of spherical, echolucent follicles or uterine ova ~1 cm long; [START_REF] Gilman | Use of portable ultrasonography as a nondestructive method for estimating reproductive effort in lizards[END_REF]. Non-reproductive females were released at the site of capture within 24 h. All reproductive females were transported to the Centre d'Etudes Biologiques de Chizé, France. We determined body mass by placing each lizard into a plastic box on a tared balance (± 0.01 g) and SVL using digital calipers (± 0.1 mm), after which all ectoparasites were removed with forceps and females were treated with Frontline (Merial Inc. Duluth, GA, USA) to avoid a high burden of ectoparasites. Females were then housed individually with a 12L:12D light regimen in opaque containers (30 × 18 × 10 cm) with mesh tops securely fastened with elastic bands, filled with approximately 2 cm of moist peat, two different shelters, and ad libitum access to food (crickets -Acheta domestica) and water. During the 12 h light cycle, a subsurface heating element was provided below one end of each cage to create a thermal gradient (20° -40°C) and light was provided using LED rope lights (Ever Lighting, Chalu, China). Females were held in these conditions for 6-9 d prior to the start of the experiment after which we collected (in order) a blood sample, mass, scanned image, muscle measurement, and heart rate (see below) of all females. Three of the females failed to ovulate and one female needed veterinary assistance during parturition. These females were subsequently returned to their site of capture and not used for any analyses.

Experimental manipulations started on 3-5 or 12 June and lasted until 48 h postparturition for all females. We used a body condition index, based on standardized residuals from a linear regression using mass and SVL, to equally distribute lizards of similar size, condition, and date and location of capture and assign them to one of two treatments: with permanent access to water or water restricted (hereafter referred to "wet" or "dry", respectively) following previously described protocols [START_REF] Lorenzon | Effect of water constraint on growth rate, activity and body temperature of yearling common lizard (Lacerta vivipara)[END_REF][START_REF] Dupoué | Water restriction causes an intergenerational trade-off and delayed mother-offspring conflict in a viviparous lizard[END_REF]. Females in the wet treatment (n = 66) were provided with water ad libitum and water was sprayed (~20 mL) twice per day (morning [0800 -0900 h] and afternoon [1600 -1700 h]) on one wall of each container so that drops could be used for drinking up to 45 min after spraying. Females assigned to the dry group (n = 65) were only provided with water once per day (morning) by similarly spraying one wall of each container.

Lizards were further distributed into one of four temperature treatments based on body condition with a factorial manipulation of day temperature (hereafter "hot" or "cold day") and night temperature (hereafter "hot" or "cold night", see Figure 1 for details). During a portion of the day, females were either held at their preferred body temperature (31°C) from 10:00 to 18:59 (~9 hours, hot day) or from 10:00 to 13:59 (~4 hours, cold day). Overnight, females were either held at 22°C (~13-18 hours, hot night) or at 17°C (~11-16 hours, cold night). In between daytime and nighttime conditions, lizards spent 2 to 4 hours where temperatures were either gradually increasing or decreasing to avoid abrupt changes. Light regimens were similarly adjusted so that lights were on or off during day and night, respectively. Stable temperatures were maintained by housing females of similar treatment groups in climatic chambers (Vötsch VP 600, Balingen, Germany). The relative humidity in each chamber was adjusted depending on the temperature so that the water vapor deficit remained constant overnight (0.7 kPa at 17°C and 22°C) or for portions of the day (1.6 kPa at 31°C).

In the wild, pregnant females maintain relatively stable body temperatures during the day (29-33°C) by thermoregulating and are inactive during the night when their body temperatures decrease (15-20°C; Le [START_REF] Galliard | Timing of locomotor impairment and shift in thermal preferences during gravidity in a viviparous lizard[END_REF]Dupoué et al. 2017a). We chose environmental conditions that are within the range of variation seen between cold and some warm habitats or years excluding extreme situations (in the most endangered populations or the warmest years) when environmental temperatures exceed the preferred temperatures or maximum critical thermal limit. The daytime conditions include the range of variation between highland, cold habitats where females have restricted access to their preferred body temperatures during the day and lowland, warm habitats where females reach a maximum access to their preferred body temperatures [START_REF] Rutschmann | Climate and habitat interact to shape the thermal reaction norms of breeding phenology across lizard populations[END_REF]. The nighttime conditions also reflect the range of variation observed for soil surface temperatures during summer season in populations across an altitudinal gradient with a mean variation ranging between 8 and 15°C (Dupoué et al. 2017b) and a variation of upper quartiles ranging between 19 and 23°C (Dupoué and Rutschmann, unpublished data). Furthermore, the manipulation of water availability mirrors the geographic variation in water availability across populations, where some populations have permanent access to free standing water throughout the year (similar to our wet treatment group) whereas others have only temporary access to free standing water (dry treatment group). In the latter, water access is often restricted to morning mist and rainfall during the summer, dry season (Dupoué et al. 2017b). Additionally, the duration of our selected water restrictions was designed to fit with the entire length of the gestation period.

During the study, each female was provided with live crickets (0.80 ± 0.01 g) three times per week between 07:30 and 09:30, which were dusted with vitamin powder containing calcium and vitamin D3 (ReptiVite, Zoo Med Laboratories Inc, USA). During subsequent feedings any remaining crickets were removed and counted in order to estimate food consumption in the previous 48-72 hrs. Throughout the study, females were weighed twice per week, always before feedings between 07:00 and 08:00, and each cage was visually inspected twice per day for parturition. On the day of parturition, offspring were moved to separate containers while the same maternal variables from the start of the experiment were collected, after which females were returned to their respective treatment regimens for an additional 48 hrs in case of delayed parturition. Only one female in this study exhibited delayed parturition, which required veterinary assistance, and was removed from all analyses. Concomitantly, offspring from each female were quantified and weighed (± 1 mg) in the following categories: living, stillborn, or unfertilized ova. The ratio of living offspring to total litter size was used as a measure of reproductive success. We also recorded total litter size and litter mass (number and mass of living, stillborn, and unfertilized ova). Forty-eight hours after parturition, females were removed from the study, provided a standard thermal gradient (20° -40°C) and ad libitum access to food and water, before being released at their site of capture.

Blood sample collection and determination of plasma osmolality

We used 20 µL hematocrit tubes to collect a ~50 µL blood sample from the postorbital sinus of all reproductive females. Total time for capture, restraint, and blood collection did not exceed 4 min. We immediately centrifuged the blood samples at 4,000 g for 5 min at 20°C to separate plasma from blood cells, and then aliquoted plasma (~40 µL) into separate vials and froze at -30°C until we used them to measure plasma osmolality. We determined plasma osmolality in triplicate using a vapor pressure osmometer (±3 mOsm kg -1 ; model 5100C; Wescor Inc., Logan, Utah, USA). Prior to use, we calibrated the osmometer using sealed osmolality standards (100, 290, and 1000 mOsm kg -1 ) in accordance with factory recommendations. We ran samples in triplicate and used 290 mOsm kg -1 standards to check the osmometer for variation after every sample. If the standard varied more than the limits of the osmometer (±3 mOsm kg -1 ), the osmometer was recalibrated. If it continued to vary more than the limits, the osmometer head was cleaned and the machine was recalibrated before continuing duplicate analysis beginning with the last sample prior to calibration/cleaning to verify correct measurement of the sample.

Tail width, muscle width, and heart rate.

On the day experimental manipulations began and at the end of parturition, we collected the following measurements between 07:30 and 08:30. First, ventral surfaces of all females were scanned at 600 dots per inch using a flatbed scanner (Canon Inc., Canoscan LiDE 400) to measure tail width at the 7 th sub-caudal scale using ImageJ2 [START_REF] Rueden | ImageJ2: ImageJ for the next generation of scientific image data[END_REF]. Next, we measured transverse muscle thickness of the right hind leg at the mid region of the upper limb segment (i.e., midpoint between joint and body on thigh). We used digital calipers (± 0.1 mm; Mitutoyo Corp., model 547-301) and took measurements in triplicate. Finally, females were allowed to acclimate undisturbed at 20°C for 1 hour. After which, we used ultrasonography (Sonosite MicroMaxx, Bothell,WA) to measure heart rate within 75 seconds of removing them from their cages and before any noticeable increase in heart rates due to handling.

Statistical analyses

We analyzed treatment effects on each morphological, physiological and reproductive output traits independently using model selection procedures in R software (R Development Core Team, version 3.5.2) with the packages 'MASS' [START_REF] Venables | Modern applied statistics with S-PLUS[END_REF], 'multcomp' (Hothorn et al. 2008), 'car' [START_REF] Fox | An R companion to applied regression[END_REF], and 'lmtest' [START_REF] Zeileis | Diagnostic checking in regression relationships[END_REF].

All initial models included additive and interactive effects of the three experimental treatments (categorical variables). We also controlled for inter-individual differences in size and reproductive investment among females because size indicates differences in condition, reproductive phenology, and/or age and because treatments may have stronger effects in females with a higher reproductive investment (Dupoué et al. 2020b). Water treatment started at or just prior to ovulation where final litter size is set in this lecithotrophic lizard [START_REF] Blackburn | Viviparous placentotrophy in reptiles and the parent-offspring conflict[END_REF]. Therefore, we did not expect any effect of experimental manipulations on total litter size. We first used relative body size (individual SVLmean SVL of all females) as a linear covariate instead of mass, especially because the body mass fluctuated considerably over the experimental period. To avoid collinearity issues, we further included in our initial models the effect of sizecontrolled fecundity or developed fecundity (standardized residuals from a linear regression of total litter size or developed litter size [i.e., number of living and stillborn offspring not including unfertilized ova] against SVL). Thus, our full models included fixed effects of day and night temperatures, water availability, and all interactions, additive effects of relative body size (RBS) and fecundity, and two-way interactions between water availability and fecundity and between water availability and RBS. These interactions were included because allocation trade-offs between mothers and embryos may change with differences in water availability [START_REF] Dezetter | Water deprivation compromises maternal physiology and reproductive success in a cold and wet adapted snake Vipera berus[END_REF]. Starting with these initial models, we used stepwise removal of insignificant coefficients as recommended for null hypothesis testing in manipulative studies [START_REF] Arnold | Uninformative parameters and model selection using Akaike's Information Criterion[END_REF][START_REF] Zuur | A protocol for data exploration to avoid common statistical problems[END_REF]. For models comparing change in a variable (muscle width, heart rate, etc.), we also included initial values as a covariate. To model post-parturition body condition (residuals from a linear regression using post-parturition mass and SVL), we used initial body condition as a covariate to control for initial differences in reserves between females. To compare differences in litter size and litter mass, we only included female RBS as a covariate. Most traits were fitted with linear models except reproductive success (proportion of number of living offspring/total offspring [i.e., number of living and stillborn offspring]) for which we used general linear models with a binomial distribution with overdispersion with maternal body size as a covariate (see Tables S1-11 for details of full and final models).

After comparing the combined effects of water availability, day, and night temperatures, we further used variance partitioning methods described by Anderson and Cribble [START_REF] Anderson | Partitioning the variation among spatial, temporal and environmental components in a multivariate data set[END_REF] to decompose our full response into orthogonal subsets. Using these subsets, we performed separate linear models to examine how day temperatures (hot or cold) influenced the relationship between plasma osmolality and heart rate with initial heart rate as a covariate. We also used orthogonal subsets and separate linear models to examine how water availability (± ad libitum access to water) influenced the relationships between dynamics of energetic reserves (change in tail width or change in muscle width) and parturition mass change and litter mass using maternal size as a covariate. A Tukey's HSD post hoc test was used to compare treatment groups. We checked all initial models to ensure the data met the assumptions for parametric testing and used transformations where necessary. Data are presented as mean ± SEM and differences were accepted as significant at the level of p < 0.05. Females at the start of the experiment had similar body mass (F7,123 = 0.53, p = 0.813), plasma osmolality (F7,123 = 1.11, p = 0.363), muscle width (F7,123 = 1.53, p = 0.164), heart rate (F7,123 = 0.98, p = 0.449), and tail width (F7,123 = 0.43, p = 0.883; Table S13) in the 8 treatment groups.

Results

Reproductive phenology, food consumption, and mass change

The length of gestation was negatively related to female size (F1,126 = 5.54, p = 0.020) and was further influenced by an interaction between day and night temperatures (F1,126 = 33.86, p < 0.001), where hot days or hot nights drastically reduced the duration of gestation but a combination of the two had a much smaller effect (Fig. 2A). After accounting for a positive effect of female size (F1,123 = 14.41, p < 0.001), the average mass of crickets consumed per day during gestation was influenced by an interaction between day and night temperatures (F1,123 = 50.37, p < 0.001) and by an interaction between day temperature and water availability (F1,123 = 6.34, p = 0.013). Overall, females exposed to hot days consumed high amounts of crickets per day (mean consumption of 0.27 ± 0.01g) regardless of night temperatures and water availability.

Conversely, females exposed to cold days consumed more if nights were hot compared to cold, independently from water availability (0.26 ± 0.01g versus 0.19 ± 0.01g, see Fig. 2B).

Dry conditions slightly reduced food intake in females exposed to cold days but had no detectable effect in females exposed to hot days (Fig. 2B).

Females, regardless of treatment, gained mass (1.78 ± 0.05 g) up until parturition. Mass increase per unit time was positively influenced by body size (F1,122 = 54.19, p < 0.001) and by an interaction between water availability and litter size (F1,122 = 4.61, p = 0.034). Litter size did not covary with relative mass gain in females in wet conditions (β = 0.001, SE < 0.01, p = 0.066, R 2 adj = 0.037) but positively covaried in females in dry conditions (β = 0.003, SE < 0.01, p < 0.001, R 2 adj = 0.165). We further found an interaction between day and night temperatures (F1,122 = 4.66, p = 0.033) that mirrored results on the length of gestation: hot days or nights led to increased daily mass gains relative to the cold controls, while a combination of the two did not have an additive effect (Fig. 2C; Table S12). Post-parturition body condition was positively influenced by initial body condition at the start of the experiment (F1,125 = 32.57, p < 0.001). In addition, females either exposed to hot nights or held in dry conditions had lower post-parturition body conditions (F1,125 = 5.87, p = 0.017; F1,125 = 3.95, p = 0.049, respectively; Table S11).

Maternal physiology

Significant day temperature by water availability interactions influenced plasma osmolality (F1,126 = 4.47, p = 0.036; initial osmolality: F1,126 = 9.65, p = 0.002) and muscle width (F1,125 = 11.46, p = 0.001; initial muscle width values: F1,125 = 61.84, p < 0.001) changes throughout the experiment. Females exposed to hot days had larger increases in osmolality when conditions were dry (40.88 ± 1.44 mOsm kg -1 ) compared to when conditions were wet (13.41 ± 0.76 mOsm kg -1 ), but both groups lost the same amount of muscle during pregnancy (both -0.12 ± 0.02 mm).

Similarly, females exposed to cold and dry days had higher osmolality (27.91 ± 0.93 mOsm kg -1 ) compared to those with ad libitum access to water (4.97 ± 0.62 mOsm kg -1 ). However, water restricted cold day females lost a higher amount of muscle (-0.18 ± 0.02 mm) when compared to cold day females in wet conditions (-0.04 ± 0.02 mm; Fig. 3A,B). We found no main effects or interactions (all p > 0.05) when comparing night temperatures between the treatment groups (Table S4).

When comparing changes in heart rate and accounting for initial heart rate (F1,125 = 63.22, p < 0.001), females in wet conditions had relatively small increases in heart rate (4.31 ± 1.08 bpm) compared to females in dry conditions (13.20 ± 1.19 bpm; water availability: F1,125 = 44.87, p < 0.001). Irrespective of water treatment, heart rate increases were slightly higher in females exposed to cold days and hot nights or hot nights and cold days (9.43 ± 1.61 bpm and 9.05 ± 1.28 bpm, respectively) and lower in females exposed to hot days and nights or cold days and nights (8.14 ± 2.35 bpm and 8.25 ± 1.72 bpm, respectively; day × night temperature interaction: F1,125 = 4.01, p = 0.047; see Fig. 3C).

Overall, females gained tail width throughout gestation (0.09 ± 0.05 mm), however, when comparing across water treatments, these increases were driven by females in wet conditions (0.19 ± 0.08 mm) while females in dry conditions tended to lose tail width (-0.02 ± 0.07 mm).

After including the effects of initial tail width (F1,126 = 59.88, p < 0.001), a significant interaction between water availability and fecundity (F1,126 = 4.54, p = 0.035) influenced changes in tail width. In females in wet conditions, fecundity negatively covaried with tail width change (β = -0.057, SE = 0.03, p = 0.041, R 2 adj = 0.438), but not in females in dry conditions (β = 0.031, SE = 0.03, p = 0.368, R 2 adj = 0.269; Fig. 4). 0.616; Fig. S1A) and with litter mass (β = -1.106, SE = 0.37, p = 0.004, R 2 adj = 0.471; Fig. S1C) in females provided in wet conditions but not in those in dry conditions (both p > 0.05; Fig. S1B,D). Similarly, tail width changes also negatively covaried with both parturition mass loss (β = -0.244, SE = 0.11, p = 0.031, R 2 adj = 0.514; Fig. S2A) and litter mass (β = -0.205, SE = 0.07, p = 0.003, R 2 adj = 0.476; Fig. S2C) in females in wet conditions but not in those in dry conditions (both p > 0.05; Fig. S2B,D).

Discussion

Our study independently manipulated day and nighttime temperatures as well as water availability to examine how concurrent environmental conditions impact an organism's ability to maintain homeostasis when faced with multiple abiotic constraints during reproduction. Overall,

(1) hot day or night treatments put reproductive females in the "fast lane", decreasing gestation length and increasing resource demands per unit time;

(2) hot day treatments, coupled with water restrictions, increased dehydration during reproduction; (3) water restrictions affected females' ability to allocate resources but had no impact on reproductive output; and (4) longer access to preferred temperature (hot days) was beneficial to current reproductive output and success whereas hot nights had the opposite effect.

High temperatures put females in the "fast lane"

As expected, hot days or hot nights drastically decreased pregnancy duration, but the effects of higher temperatures were not additive because the combination of hot days and hot nights did not significantly shorten pregnancy durations even though mean daily temperature was the highest in this treatment group (Fig. 1 and2A). This suggests that females exposed to hot days and nights were at or near a phenological limit (~28 days in our study) for embryonic development (Olsson and Shine 1997a;Olsson and Shine 1997b;[START_REF] Lourdais | Protein catabolism in pregnant snakes (Epicrates cenchria maurus Boidae) compromises musculature and performance after reproduction[END_REF]. Reductions in gestation length can have important implications on offspring fitness and survival [START_REF] Chamaillé-Jammes | Global warming and positive fitness response in mountain populations of common lizards Lacerta vivipara[END_REF][START_REF] Warner | Fitness of juvenile lizards depends on seasonal timing of hatching, not offspring body size[END_REF][START_REF] While | Long-lasting effects of among-but not within-litter timing of birth in a viviparous lizard[END_REF], and, as suggested by our study, require tremendous increases in resource acquisition per unit time. Daytime and nighttime temperatures both influenced the mass of crickets consumed per unit time and contrasting temperature regimes had distinct effects on energetic intake, possibly due to temperature-dependent changes in foraging and digestive efficiencies as well as the temporal dissociation of these two mechanisms influencing net energy gain. Body temperatures in ectotherms impact feeding behaviors and digestive physiology, where lizards exposed to higher temperatures drastically increase the quantity of food consumed. However, gut passage time ultimately limits the ability to continuously consume food [START_REF] Angilletta | Thermal and physiological constraints on energy assimilation in a widespread lizard (Sceloporus undulatus)[END_REF][START_REF] Levy | Diminishing returns limit energetic costs of climate change[END_REF]. In juxtaposition, colder temperatures decrease feeding behaviors while also reducing gut-throughput rates [START_REF] Van Damme | The thermal dependence of feeding behaviour, food consumption and gut-passage time in the lizard Lacerta vivipara Jacquin[END_REF]. Irrespective of nighttime temperatures, females in our study exposed to hot days were potentially consuming the physiological "maximum" amount of food. However, females exposed to cold days may have been behaviorally limited in their food consumption and were further reliant on nighttime temperatures to influence their digestive efficiencies (Fig. 2B).

Food consumption was also influenced by daytime temperatures and water availability,

where animals with limited access to preferred temperatures did not consume as much relative crickets especially when water was restricted. After a meal is ingested, a significant amount of fluid is initially secreted into the lumen of the gastro-intestinal tract which is essential for digestion, absorption, and motility [START_REF] Skoczylas | Physiology of the digestive tract[END_REF]. While the vast majority of those fluids are eventually reabsorbed in the large intestine, the initial hydric cost of ingesting a meal may have forced pregnant females in dry conditions to forgo meal consumption. Previous studies in other species of reptile have similarly found that animals can experience negative water balance after consuming prey [START_REF] Beaupre | Field metabolic rate, water flux, and energy budgets of mottled rock rattlesnakes, Crotalus lepidus, from two populations[END_REF][START_REF] Wright | Meal consumption is ineffective at maintaining or correcting water balance in a desert lizard, Heloderma suspectum[END_REF][START_REF] Murphy | Rattlesnakes must drink: meal consumption does not improve hydration state[END_REF]. In addition, it is not clear if lower temperatures during daytime decreased drinking behaviors as is seen with feeding in this species [START_REF] Van Damme | The thermal dependence of feeding behaviour, food consumption and gut-passage time in the lizard Lacerta vivipara Jacquin[END_REF]. Interestingly, within temperature treatments, females in wet or dry conditions gained similar relative mass per day, suggesting that females in wet conditions either drank less or females in dry conditions were able to reduce evaporative water loss. Temperatures were imposed by climatic chambers to eliminate the possibility of behavioral thermoregulation, but differences in transcutaneous or ocular water loss rates may have occurred due to changes in behavioral hydroregulation or plasticity of the skin barrier to transepidermal water loss [START_REF] Dupoué | Intergenerational trade-off for water may induce a mother-offspring conflict in favour of embryos in a viviparous snake[END_REF][START_REF] Refsnider | Developmental plasticity of thermal ecology traits in reptiles: Trends, potential benefits, and research needs[END_REF][START_REF] Dezetter | Water deprivation compromises maternal physiology and reproductive success in a cold and wet adapted snake Vipera berus[END_REF].

High temperatures and water restrictions increase physiological costs of reproduction

Limited hydric resources for pregnant females should result in higher maternal-offspring conflicts for water [START_REF] Speakman | Oxidative stress as a cost of reproduction: Beyond the simplistic trade-off model[END_REF]Dupoue et al. 2015) and burden mothers with increased plasma osmolality [START_REF] Dupoué | Water restriction causes an intergenerational trade-off and delayed mother-offspring conflict in a viviparous lizard[END_REF]) and higher muscle catabolism [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF]. Our results confirm this and further suggest that daytime temperatures can exacerbate these conflicts and increase some of the physiological costs of reproduction. First, females exposed to high temperatures during the day had greater increases in plasma osmolality during gestation compared to those exposed to colder daytime temperatures. When considering water restrictions, these increases in plasma osmolality were further pronounced and highest in hot day females in dry conditions. Second, all females experienced muscle loss during the experiment and losses were highest in females in dry conditions, especially if they had limited access to preferred temperatures during the daytime. Muscle wasting during pregnancy is common in reptiles [START_REF] Lourdais | Protein catabolism in pregnant snakes (Epicrates cenchria maurus Boidae) compromises musculature and performance after reproduction[END_REF][START_REF] Lourdais | Structural and performance costs of reproduction in a pure capital breeder, the Children's python Antaresia childreni[END_REF][START_REF] Lorioux | Are glucocorticoids good indicators of pregnancy constraints in a capital breeder?[END_REF]) and skeletal muscle has been shown to be an important substrate for the reallocation of hydric resources when demands are high and intake is limited [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF][START_REF] Brusch | Additive effects of temperature and water availability on pregnancy in a viviparous lizard[END_REF][START_REF] Dezetter | Water deprivation compromises maternal physiology and reproductive success in a cold and wet adapted snake Vipera berus[END_REF].

Because proteins are a much lower energetic source compared to lipids and their oxidation provides five times more water [START_REF] Jenni | Fuel supply and metabolic constraints in migrating birds[END_REF], increased muscle catabolism likely supported the hydric costs of reproduction in our study. Third, females in wet conditions throughout gestation had better post-parturition body conditions compared to those in dry conditions. Maternal condition after giving birth can have major impacts on their future reproduction [START_REF] Bleu | Reproductive allocation strategies: a long-term study on proximate factors and temporal adjustments in a viviparous lizard[END_REF], and a female with a lower body condition and smaller muscles might not have the ability to capture prey, avoid predators, and survive until the next year.

In reptiles, cardiac function is maximized when individuals are maintained in their preferred temperature ranges allowing the animal to optimize oxygen consumption to meet metabolic demands [START_REF] Lillywhite | Resting and maximal heart rates in ectothermic vertebrates[END_REF]. In viviparous lizards, oxygen consumption drastically increases during later stages of pregnancy to support the increased metabolic demands of developing offspring [START_REF] Demarco | Physiological cost of pregnancy in a viviparous lizard (Sceloporus jarrovi)[END_REF][START_REF] Demarco | Metabolic rates of female viviparous lizards (Sceloporus jarrovi) throughout the reproductive cycle: do pregnant lizards adhere to standard allometry?[END_REF][START_REF] Foucart | Influence of reproductive mode on metabolic costs of reproduction: insight from the bimodal lizard Zootoca vivipara[END_REF].

Here, heart rate increases were largest in females held in dry conditions and the difference was more extreme in females exposed to hot days and hot nights (Fig. 3C). Furthermore, our exploration of increased hydric demands using orthogonal subsets revealed that changes in heart rate were positively related to changes in plasma osmolality. Increases in plasma osmolality may be due to decreased water volume, increased solutes (mainly sodium and chloride, [START_REF] Burg | Intracellular organic osmolytes: function and regulation[END_REF], or both, and we cannot tell if our results were due to hypovolemia or hypernatremia. Regardless, mild levels of dehydration and decreased blood volume typically increase heart rate, while clinical levels of dehydration elicit decreased heart rates in reptiles [START_REF] Mitchell | Therapeutics. Reptile medicine and surgery[END_REF]. Many species of reptile can tolerate wide ranges of plasma osmolality [START_REF] Minnich | The use of water[END_REF]) and even the most dehydrated female in our study was within the range of previously reported osmolality from wild populations (Dupoué et al. 2020a). This suggests that females in our study were at sub-clinical levels of dehydration as the result of water restrictions, which led to higher heart rates. Lizards are able to increase heart rates to meet metabolic demands [START_REF] Dawson | Physiological responses to temperature in the lizard Crotaphytus collaris[END_REF][START_REF] Licht | Effects of temperature on heart rates of lizards during rest and activity[END_REF]) and to rapidly gain heat during periods of intermittent basking [START_REF] Bartholomew | Physiological control of body temperature[END_REF][START_REF] Seebacher | A review of thermoregulation and physiological performance in reptiles: what is the role of phenotypic flexibility?[END_REF]. While females exposed to hot days and hot nights expectedly had larger increases in heart rates, ours is the first study to similarly link dehydration with increased heart rate.

Water restrictions affect allocation of resources but not reproductive investment

The tail is an essential site for protein and lipid storage in many species of lizards [START_REF] Bateman | To cut a long tail short: a review of lizard caudal autotomy studies carried out over the last 20 years[END_REF], including Z. vivipara where tail volume and energy content fluctuate seasonally throughout the year [START_REF] Avery | Storage lipids in the lizard Lacerta vivipara: a quantitative study[END_REF]. As intermediates on the capital-income breeding spectrum, Z.

vivipara can accumulate vast lipid reserves after parturition, prior to hibernation [START_REF] Avery | Clutch size and reproductive effort in the lizard Lacerta vivipara Jacquin[END_REF], but are also able to assimilate nutrients during pregnancy [START_REF] Bleu | Reproductive allocation strategies: a long-term study on proximate factors and temporal adjustments in a viviparous lizard[END_REF]. Previous studies have found that higher temperatures, compounded by dehydration, reduce tail reserves in gestating lizards [START_REF] Brusch | Additive effects of temperature and water availability on pregnancy in a viviparous lizard[END_REF]. Our results align with this and further suggest a potential trade-off between tail reserves and fecundity, where larger female with small litter size are able to assimilate and invest nutrients into tail reserves but only if water is readily available (Fig. 4A).

In contrast, water restricted females may not have had this option, and were required to use all available resources to immediately fuel their reproductive efforts, regardless of fecundity (Fig. 4B). Similarly, females in wet conditions were able to use both stored lipid (in the tail) and protein (in the leg muscle) resources to fuel reproductive investment, while those in dry conditions could not (Fig. S1 andS2). Such dynamical changes in tail and leg muscles may be relevant for future survival and reproduction. Together, these findings suggest that water restrictions during pregnancy may negatively affect a female's ability to allocate resources between current and future reproduction unless post-partum females are able to rapidly make up the deficit.

High nighttime temperatures negatively impact reproductive output

Total litter size was not influenced by climatic conditions during gestation, which is not entirely surprising given that our treatments started at the tail-end of vitellogenesis or start of pregnancy, after the number of ova is typically determined in lizards (see Price 2017 for review). However, females held in hot nights had lower mass loss during parturition, significantly lighter litters, and reduced number of live neonates per litter when compared to females held in cold nights. While females exposed to hot days had higher parturition mass loss compared to females exposed to cold days, daytime temperatures had no effect on litter mass. These results suggest that pronounced downstream effects of nighttime temperatures on reproductive effort. During nonreproductive periods of the year, Z. vivipara experimentally exposed to warmer night conditions direct more resources towards growth at the expense of body condition [START_REF] Rutschmann | Intense nocturnal warming alters growth strategies, colouration and parasite load in a diurnal lizard[END_REF].

Females in our experiment who were exposed to hot nights may have adjusted their allocation strategies as has been previously documented in viviparous lizards facing resource constraints [START_REF] Shine | Can pregnant lizards adjust their offspring phenotypes to environmental conditions?[END_REF][START_REF] Wapstra | Maternal basking opportunity affects juvenile phenotype in a viviparous lizard[END_REF][START_REF] Bleu | Reproductive allocation strategies: a long-term study on proximate factors and temporal adjustments in a viviparous lizard[END_REF]. Interestingly, water deprivation had no effect on reproductive output and the number of live neonates. Water availability in the field has been shown to influence growth, reproduction, and reproductive output [START_REF] Lorenzon | Effect of water constraint on growth rate, activity and body temperature of yearling common lizard (Lacerta vivipara)[END_REF][START_REF] Galliard | Ontogenic sources of variation in sexual size dimorphism in a viviparous lizard[END_REF][START_REF] Marquis | Intergenerational effects of climate generate cohort variation in lizard reproductive performance[END_REF]), but our study supports other laboratory findings that suggest that mothers primarily pay the cost during maternal-offspring conflicts for hydric resources (Dupoue et al. 2015;[START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF][START_REF] Dupoué | Water restriction causes an intergenerational trade-off and delayed mother-offspring conflict in a viviparous lizard[END_REF]). However, the exploration of more cryptic traits beyond litter mass and number of offspring has also found evidence of a water-based mother-offspring intergenerational trade-off, where both mothers and offspring are similarly impacted by water restrictions (Brusch and DeNardo 2019;Brusch et al. 2019).

Conclusion

Recent work examining how invertebrate (Speights and Barton, 2019;Higashi et al. 2020) and vertebrate (Clarke and Zani, 2012;[START_REF] Rutschmann | Intense nocturnal warming alters growth strategies, colouration and parasite load in a diurnal lizard[END_REF] ectotherms will be impacted by increasing nocturnal temperatures has generally found that abnormally warmer nights may have a net negative effect on components of lifetime reproductive success. Climate change will also continue to alter rainfall patterns and disrupt water availability for a plethora of organisms around the globe [START_REF] Fung | Water availability in + 2 C and + 4 C worlds[END_REF][START_REF] Sanderson | Regional temperature and precipitation changes under high-end (≥ 4 C) global warming[END_REF]. Ours is the first study to examine the combined effects of day and nighttime temperatures, and water restrictions in a vertebrate. By using pregnant females experiencing a crucial life-history stage marked by high physiological demands, we were able to broaden our understanding of the ways in which organisms may or may not be able to physiologically adjust to abiotic shifts including future climate conditions.

Overall, our results suggest that high nighttime temperatures and, to some extent, reduced water availability further increase the burden on individuals already constrained by heavy resource investment during pregnancy. Negative effects of higher nighttime temperatures may provide a

Figure 1 -

 1 Figure 1 -Hourly temperature treatments of reproductive Zootoca vivipara throughout the majority of pregnancy. Females were exposed to either hot day and hot night (dashed grey line; n = 34), hot day and cold night (solid blue line; n = 33), cold day and hot night (solid grey line; n = 33), or cold day and cold night (dashed blue line; n = 31). Average daily temperatures were highest in the hot day and night treatment (26°C), intermediate and similar in the hot day cold night and cold day hot night treatments (23.8°C and 24.2°C), and lowest in the cold day cold night treatment (20.9°C). Arrows indicate when water was sprayed on the sides of individual cages each morning (wet and dry treatments) and afternoon (wet treatment only). See text for full details. Final treatment groups were: hot day and hot night wet (n = 17) or dry (n = 17), hot-day and cold-night wet (n = 17) or dry (n = 16), cold-day and hot-night wet (n = 17) or dry (n = 16), and cold day and cold night wet (n = 15) or dry (n = 16).

Figure 2 -

 2 Figure 2 -Median and quartile distribution of (A) the length of gestation (duration from start of the experiment to parturition), (B) the mass of crickets consumed per day and (C) the mass increase per day of female Zootoca vivipara in wet (W; n = 66) or dry (D; n =65) conditions and exposed to hot day and night (HH; n = 34), hot day and cold night (HC; n = 33), cold day and hot night (CH; n = 33), or cold day and night (CC; n = 31) throughout the majority of pregnancy. Boxplots of the distribution of data are displayed whereas circles represent individual females.

Figure 3 -

 3 Figure 3 -Median and quartile distribution of change from start of experiment until parturition for (A) plasma osmolality, (B) muscle width and (C) heart rate of female Zootoca vivipara held in wet (W; n = 66) or dry (D; n =65) conditions and exposed to hot day and night (HH; n = 34), hot day and cold night (HC; n = 33), cold day and hot night (CH; n = 33), or cold day and night (CC; n = 31) throughout the majority of pregnancy. Boxplots of the distribution of data are displayed whereas circles represent individual females.

Figure 4 -

 4 Figure 4 -Relationships between change in tail width and relative fecundity (residual of litter size against female body size) in female Zootoca vivipara held in wet (n = 66) or dry (n =65) conditions throughout the majority of pregnancy. A line of best fit with gray-shaded confidence interval is included for significant relationships (p < 0.05; see text for details). Closed circles represent individual females.

mechanistic explanation of recent observations that warm-edge populations are vulnerable to hot and dry summer conditions and that these vulnerabilities may trigger demographic declines (Dupoué et al. 2017a(Dupoué et al. , 2018)). Future research should examine potential inter-generational impacts of these same climatic variables and explore how offspring might be similarly affected by maternal physiological condition during pregnancy.

Reproductive output

Mass loss at parturition increased with female size (F1,127 = 111.05, p < 0.001) and was influenced by day (F1,127 = 4.17, p = 0.043) and night temperatures (F1,127 = 14.46, p < 0.001) but not by their interaction (p > 0.05). Females exposed to hot days lost slightly more mass (1.89 ± 0.09 g) compared to females in cold days (1.71 ± 0.10 g), whereas females in hot nights lost less (1.62 ± 0.73 g) compared to females in cold nights (2.00 ± 0.81 g). Similarly, after correcting for female size (F1,128 = 99.44, p < 0.001), females exposed to hot nights had a smaller litter mass (0.86 ± 0.05 g) compared to females in cold nights (0.97 ± 0.05 g; F1,128 = 99.44, p < 0.001). We also found a significant effect of nighttime temperatures (F1,130 = 5.70, p = 0.020) on reproductive success, where females exposed to hot nights had a 26% higher chance of producing stillborn or unfertilized ova at parturition. We found no significant effect of daytime temperatures or water availability on reproductive success or litter mass (both p > 0.05; Table S9, S14). Total litter size was not influenced by day-or nighttime temperatures, or water availability (all p > 0.05; Table S14).

Orthogonal subset analyses

We used orthogonal subset analyses to independently assess the effect of hydric state on heart rate and explore the relationships between maternal dynamics of energetic reserves and their reproductive output. Plasma osmolality changes positively covaried with heart rate changes in females exposed to hot days (β = 0.325, SE = 0.07, p < 0.001, R 2 adj = 0.416) as well as in females exposed to cold days (β = 0.293, SE = 0.71, p < 0.001, R 2 adj = 0.441). Furthermore, muscle width changes negatively covaried with parturition mass loss (β = -2.634, SE = 0.55, p < 0.001, R 2 adj =

Conflict of Interest:

No competing interests declared.

Data availability:

The datasets supporting this article can be accessed at https://doi.org/10.5281/zenodo.6394479

Ethics: This study was performed in accordance with laws relative to capture, transport, and experiments on Zootoca vivipara (DREAL permit #13016_19042018) and all procedures were approved by an independent ethical committee (Apafis #2018060111033048_v5).