Laurie Araspin

Carla Wagener

Pablo Padilla

Anthony Herrel

John Measey

Shifts in the thermal dependence of locomotor performance across an altitudinal gradient in native populations of Xenopus laevis

Keywords: laevis, native populations, altitudes, temperature, locomotor performance, thermal performance curve

Few intraspecific studies exist on amphibians' performance depending on the elevation as few species are widely distributed along altitudes. Here, we characterized the locomotor performance of Xenopus laevis across a wide range of thermal environments along an altitudinal gradient and showed a decrease in the optimal temperature for exertion with altitude.

Introduction

Temperature is one of the dimensions of the ecological niche and consequently a characteristic of an animal's habitat [START_REF] Magnuson | Temperature as an ecological resource[END_REF]. Therefore, species distribution and abundance patterns are in large part driven by environmental temperature [START_REF] Clarke | Costs and consequences of evolutionary temperature adaptation[END_REF][START_REF] Hoffmann | Adaptation of Drosophila to temperature extremes: bringing together quantitative and molecular approaches[END_REF]. The thermal environment varies depending on geological times, seasons and days, and consequently all living beings are submitted to this selective pressure [START_REF] Brown | Toward a metabolic theory of ecology[END_REF]. The physiological processes of organisms are highly temperature dependent. Cellular responses to temperature are linked to the metabolism supplying ATP for cellular maintenance, membrane and protein synthesis [START_REF] Hulbert | Mechanisms underlying the cost of living in animals[END_REF]Gillooly 2001, Pörtner and[START_REF] Pörtner | Physiology and climate change[END_REF], and play a role in growth, reproduction, and locomotor performance. These processes have direct fitness consequences for living beings [START_REF] Johnston | Thermal plasticity of skeletal muscle phenotype in ectothermic vertebrates and its significance for locomotory behaviour[END_REF][START_REF] Guderley | Locomotor performance and muscle metabolic capacities: impact of temperature and energetic status[END_REF]). Local or regional differences in temperature related to climate lead to a variation in the spatial distribution of species, moreover depending on altitude as temperature tends to decrease with altitude [START_REF] Körner | The use of 'altitude' in ecological research[END_REF][START_REF] Montgomery | Variation in temperature with altitude and latitude[END_REF].

At high altitude the daily and seasonal variation between minimum and maximum mean environmental temperature can be extreme [START_REF] Jacobsen | Ecology of high altitude waters[END_REF]. Previous studies have investigated the impact of altitude on amphibians, including patterns of distribution depending on elevational gradients, their metabolic and locomotor physiology, their thermal safety margin, lifespan, plasticity of metamorphic traits, and their environmental adaptations [START_REF] Navas | Patterns of distribution of anurans in high Andean tropical elevations: Insights from integrating biogeography and evolutionary physiology[END_REF][START_REF] Navas | Implications of microhabitat selection and patterns of activity on the thermal ecology of high elevation Neotropical anurans[END_REF][START_REF] Sunday | Thermal-safety margins and the necessity of thermoregulatory behavior across latitude and elevation[END_REF][START_REF] Zhang | Amphibians live longer at higher altitudes but not at higher latitudes[END_REF][START_REF] Yu | Plasticity of metamorphic traits in a high-altitude toad: interactive effects of food level and temperature[END_REF][START_REF] Wang | Selection and environmental adaptation along a path to speciation in the Tibetan frog Nanorana parkeri[END_REF][START_REF] Wagener | Progeny of Xenopus laevis from altitudinal extremes display adaptive physiological performance[END_REF].

Here, we study Xenopus laevis, an exceptional model for investigating the impact of altitude as it ranges from sea level to over 3000 m above sea level (a.s.l.), an unusually large range within a single species. Xenopus laevis is a largely aquatic frog from sub-Saharan Africa, and inhabits a wide range of geographical and thermal environments [START_REF] Measey | Species account: Xenopus laevis (Daudin 1802). Atlas and red data book of the frogs of South Africa[END_REF][START_REF] Furman | Pan-African phylogeography of a model organism, the African clawed frog 'Xenopus laevis[END_REF]. In a previous study [START_REF] Araspin | Rapid shifts in the temperature dependence of locomotor performance in an invasive frog, Xenopus laevis, implications for conservation[END_REF] it was demonstrated that optimal temperatures for locomotion differed between native (South Africa) and invasive (France) populations of Xenopus laevis associated with differences in the thermal environment experienced by these populations. In its native range, this species displays an important phenotypic diversity which may suggests a plastic physiology [START_REF] Preez | Population-specific incidence of testicular ovarian follicles in Xenopus laevis from South Africa: A potential issue in endocrine testing[END_REF]. Recent studies on tadpoles suggest both an adaptive and plastic underpinning of population-level differences in physiology [START_REF] Wagener | Progeny of Xenopus laevis from altitudinal extremes display adaptive physiological performance[END_REF]Kruger et al., in press).

Xenopus laevis is invasive on four continents [START_REF] Measey | Ongoing invasions of the African clawed frog, Xenopus laevis: a global review[END_REF] and is a threat for local biota [START_REF] Courant | Assessing the impacts of the invasive frog, Xenopus laevis, on amphibians in western France[END_REF][START_REF] Kumschick | Impact assessment with different scoring tools: How well do alien amphibian assessments match?[END_REF]. Plasticity in its temperature tolerance may have been a key factor driving the invasive potential of this species. Furthermore, adaptation to new thermal environments may have facilitated survival in its invasive range.

The aim of the present study was to assess the thermal dependence of locomotor performance in adults from native populations across an altitudinal gradient and to identify potential differences among populations. We chose to measure exertion capacity (measured here as the distance or time swam until exhaustion) as it is fitness-relevant and a good proxy for dispersal capacity. We predict that populations from high altitudes and cold environments will perform relatively better at cold temperatures and conversely, populations from low altitude should perform better at warmer temperatures. We thus predict that the optimal temperatures for locomotor performance will decrease with increasing altitude. However, we do not expect changes in the upper thermal limit as it is determined by genetic and biochemical constraints and as behavioral thermoregulation is effective in avoiding critical maximal temperatures [START_REF] Blackburn | Evolutionary capacity of upper thermal limits: Beyond single trait assessments[END_REF][START_REF] Grigg | Conservatism of lizard thermal tolerances and body temperatures across evolutionary history and geography[END_REF][START_REF] Von | Divergence of thermal physiological traits in terrestrial breeding frogs along a tropical elevational gradient[END_REF][START_REF] Pintanel | Elevational and microclimatic drivers of thermal tolerance in Andean Pristimantis frogs[END_REF].

In contrast, the lower thermal limit is expected to decrease with altitude as observed in previous studies, at least partly as behavioral thermoregulation is less effective [START_REF] Bodensteiner | Thermal adaptation revisited: How conserved are thermal traits of reptiles and amphibians?[END_REF]. If so, then our results may have broader implications for our understanding of the evolution of thermal performance limits in ectothermic organisms and the ability of species to cope with variation in their current or future thermal environment.

Methods

Adult Xenopus laevis individuals were caught in three different areas in Kwa-Zulu Natal, eastern South Africa. Populations were sampled at different altitudes; Hluhluwe (60 m a.s.l.), N = 26, 13 females and 13 males; Dalton (1016 m a.s.l.), N = 25, 13 females and 12 males; near Phuthaditjhaba (1948 m a.s.l), N = 26, 13 females and 13 males. Individuals from a fourth population were caught in Lesotho at 3197 m a.s.l., N = 28, 14 females and 14 males.

Animals were captured at night in March 2021 using liver-baited funnel traps. Animals were transported to Stellenbosch University in South Africa. Upon arrival all individuals were pittagged allowing unique identification. Specimens were maintained in 72 L (60 x 30 x 40 cm) aquaria, 13 to 14 individuals per tank and separated by sex and the site of origin. Tanks contained rocks to provide hiding places. Animals were fed twice weekly with chicken heart.

The temperature of the water was maintained at 18°C. All protocols and animal welfare conditions were in accordance with the Research Ethics Committee for Animal care and use, protocol number ACU-2021-19215. In situ temperature data collection Temperature data loggers (HOBO MX2201, Onset Computer Corporation) were placed in ponds at a depth of approximately 60 cm at the sites of collection and recorded the ambient temperature every 30 minutes. Data from March 2021 to May 2021 were recorded and downloaded via the HOBO application (HOBOconnect, version 1.2.4 (25775)).

Morphometrics

Body dimensions were measured following [START_REF] Herrel | Intersexual differences in body shape and locomotor performance in the aquatic frog, Xenopus tropicalis: Sexual dimorphism in morphology and performance in Xenopus[END_REF]. The mass was measured with a digital scale (Ohaus, Brooklyn, NY, USA; precision ± 0.1 g) and snout-vent length (SVL) was measured using a pair of digital callipers (Mitutoya; precision ± 0.01 mm).

Critical temperatures

Individuals were placed by three in containers (18 x 30 x 10 cm) with some water to prevent dehydration and placed in an incubator (Sanyo MIR 154 Incubator, -10 to +60°C). Experiments started at 18°C (i.e., the housing temperature) from which the temperature was gradually decreased or increased at an average rate of 2°C each 45 minutes. When reaching 6°C the temperature was decreased at a rate of 1°C each 45 minutes for the determination of the critical minimum temperature. To determine the critical maximal temperature the temperature was increased at a rate of 1°C each 45 minutes from 27°C upwards. Animals were warmed up slowly as their thermal inertia was substantial and faster rates resulted in animals being cooler or warmer than the temperature of the incubator. Animals were inspected regularly and checked for lack of a righting response. As soon as animals were no longer able to turn over after placed on their back, we considered that temperature (measured with a thermocouple inserted into the cloaca) to be that animal's critical temperature.

Performance

Aquatic exertion tests were performed at 8°C, 12°C, 16°C, 19°C, 23°C and 27°C.

Individuals were placed by seven in containers (45x30x10 cm) with some water to prevent dehydration and left for 3 hours in an incubator set at the desired test temperature. The room was set at the test temperature. Before and after each performance trial body temperature was recorded using a K-type thermocouple. Measures of exertion were performed by chasing animals individually until exhaustion (defined as animals no longer being able to right themselves when put on their back) around a 4.43-meter-long circular track with an average water depth of 20cm. The total distance and time to exhaustion at the end of the trial were recorded. After each trial animals were left to recover at an average temperature of 20°C and then returned to their tank (water at 18°C). Animals were fed and left to rest for at least three days. Trials were repeated twice per individual and the maximal distance was retained for further analyses.

Statistical analyses

To fulfil assumptions of normality and homoscedasticity, all data were log10transformed before analyses. Analyses were performed using R (version i386 4.1.2; R core team 2021). To test for significant differences in the different locomotor performance traits (i.e., distance and time to exhaustion) among populations, we used a mixed model, with temperature, population, and sex as fixed effects, SVL as covariate, and frog ID as a random effect. To test for differences in SVL and mass among populations, two-way ANOVAs were run. Within populations (i.e., Hluhluwe, Dalton, Phuthaditjhaba, and Lesotho) we further tested for effects of temperature and sex on performance as well as their interactions. We used mass and SVL as covariates. To do so, we used a repeated measures multivariate analysis of variance (MANOVAs). Next, pairwise multiple comparisons tests (pairwise.t.test, "Stats" package version 3.4.2) with Bonferroni-Holm adjusted P-values were performed to test which temperatures differed from one another (temperature range: 8, 12, 16, 19, 23 and 27°C).

TukeyHSD tests were performed to test which populations differed from another depending on the tested temperature and to test for difference in critical temperatures among populations.

For each individual thermal performance curves [START_REF] Angilletta | The evolution of thermal physiology in ectotherms[END_REF] were built using the minimum convex polygon method [START_REF] Van Berkum | Evolutionary patterns of the thermal sensitivity of sprint speed in anolis lizards[END_REF]. Optimal temperatures as well as temperature breadths Tpb80 and Tpb95 were measured and analysed using univariate ANOVAs and Welch ANOVAs to test for differences among populations in locomotor performance (i.e., aquatic exertion).

To further assess the relationship between temperature and exertion capacity for each population we fitted a generalized additive model (GAM). We used sex and population as fixed factors and applied a smoothing parameter to SVL and temperature. Because the same individuals were used, we added individual identity as a random term. The smoothing parameter (k) was optimized based on the model adjusted R-squared values and using the functions of the "mgcv" R package (v1.8-35; Wood 2011). To compare the thermal optimum of each population, a GAM with the same parameters was also fitted for each individual and an ANOVA with population as factor was conducted on the predicted thermal optima, followed by a Tuckey HSD test to inspect significant differences within populations.

Results

Morphometric measurements

We observed significant differences in SVL and in mass between sexes for each population (Hluhluwe: F1,24 = 19.27, P <0.001; Dalton: F1,23 = 22.08, P <0.001; Phuthaditjhaba: F1,24 = 29.63, P <0.001; Lesotho: F1,26 = 106.7; P <0.001), with females being larger than males. Mass and SVL of individuals from all populations were correlated (r = 0.94 P < 0.001). We found a significant differences in mass between the Lesotho and Phuthaditjhaba populations (P = 0.001) and in SVL (P = 0.042), with males from the highaltitude Lesotho population being substantially smaller than males from the Phuthaditjhaba population (Supplementary Table 1).

In situ temperatures

Average temperatures between March and May 2021 at each site show a dramatic decrease with altitude, with an average temperature of 23.03°C for Hluhluwe (min: 17.24°C; max: 32.34°C), 17.19°C for Dalton (min: 7.59°C; max: 30.84°C), 17.18°C for Phuthaditjhaba (min: 9.39°C; max: 24.32°C) and 6.35°C for Lesotho (min: 2.96°C; 22.65°C). Minimum, maximum and average temperatures for each month between March and May are presented in Table 1.

Exertion

Temperature significantly affected the maximal distance swum in each population (Hluhluwe: F5,125 = 41.76, P < 0.0003; Dalton: F5,120 = 44.88, P < 0.0001; Phuthaditjhaba: F5,125 = 68.18, P < 0.0001; Lesotho: F5,135 = 48.25, P < 0.0001). The GAM model explains 87% of the variation in exertion and temperature has a non-linear significant effect on exertion within each population (Supplementary Figs. 1234; Supplementary Table 2). SVL also had a significant effect on the maximum distance swum in populations (Hluhluwe: F1,23 = 5.916, P = 0.02; Dalton: F1,22 = 18.29, P = 0.0003; Phuthaditjhaba: F1,23 = 15.94, P = 0.0005; Lesotho: F1,25 = 24.01, P < 0.0001). Maximal distance swum differed significantly among populations (Chisq = 16.97; P = 0.0007), and the interaction between population and temperature was significant (Chisq = 114.67, P < 0.0001) (Fig. 1). Time to exhaustion was also significantly affected by temperature (Chisq = 240.09, P < 0.0001) for each population (Fig. 2). Within populations, we also observed that temperature significantly affected the time to exhaustion (Hluhluwe: F5,125 = 26.73, P < 0.0001; Greytown: F5,120 = 44.78, P < 0.0001; Phuthaditjhaba: F5,125 = 90.7, P < 0.0001; Lesotho: F5,135 = 80.92; P < 0.0001) (Fig. 2, Table 2). The GAM model explained 85.7% of the variation in the time to exhaustion and temperature has a non-linear significant effect for each population, using same factors as for the maximal distance (Supplementary Table 3). Time to exhaustion of the Lesotho and Phuthaditjhaba populations was significantly shorter than the Hluhluwe population at 23 and 27°C (P < 0.04) and the Lesotho population exhibited a significantly shorter time to exhaustion than the Dalton population at 23 and 27°C (Table 2).

Post-hoc tests for maximal distance showed the significant differences depending on temperature within populations (Supplementary Tables 4567). For maximal distance significant differences depending on temperature among populations were observed (Table 8). There were significant differences in performance at cold temperatures (8 to 16°C) between the Hluhluwe population from low-elevation and the others. No difference was observed in the maximal distance among populations at 19°C. Conversely, at hotter temperatures (23 and 27°C), the performance of the Lesotho population from high elevation is significantly different from that of other populations. We further observed that the low-altitude Hluhluwe individuals were the least endurant at the lowest temperatures 8, 12 and 16°C. However, with increasing test temperatures (19, 23 and 27°C) the high-altitude Lesotho individuals swum shorter distances (Table 3).

Critical temperature, optimal temperature, and performance breadths

The minimum critical temperature (CTmin) differed significantly among populations (F3,36 = 11.26, P < 0.0001), particularly between Hluhluwe and the other populations (P < 0.03) and among the individuals from the Lesotho and Dalton populations (P = 0.04). As predicted, CTmin decreased with the increase in altitude (see Table 4, 5 andFig 1). The maximum critical temperature (CTmax) did not differ, however, among populations (F3,36 = 0.301, P = 0.82) (Table 4 and6). Temperature optima (Topt) differed significantly among populations irrespective of whether the convex polygon method (F3,101 = 8.85, P < 0.0001) or the GAM predictions were used (F3,101 = 22.68, P < 0.0001). Differences were marked among Lesotho and Dalton (P = 0.008) and Lesotho and Hluhluwe (P < 0.0001), and tended to differ among individuals from Lesotho and Phuthaditjhaba (P = 0.059). The high-altitude Lesotho population showed the lowest optimal temperature, followed by Phuthaditjhaba, Dalton, and the low-altitude Hluhluwe population (Table 4,Table 7, Fig. 1). There was no significant difference among populations in Tpb80 (F3,101 = 2.11, P = 0.09) and Tpb95 (F3,55.32 = 1.72, P = 0.18) (Table 4). However, there were significant differences in the upper and lower limits of the 80 and 95% performance breadth interval among populations (80% lower limit: F3,101 = 6.87, P <0.0002; 80% upper limit: F3,101 = 9.009, P <0.0001; 95% lower limit: F3,101 = 8.43, P <0.0001; 95% upper limit: F3,101 = 9.69, P <0.0001), indicating shifts in the thermal performance curves (Table 4 and Fig. 1). We observed a left-shift in the thermal performance curve of the high-elevation Lesotho population towards colder temperatures and a right-shift in the thermal performance curve of the low-elevation Hluhluwe population towards warmer temperatures (Table 4).

Discussion

Critical temperatures

Ectotherms are dependent on temperature to maintain their physiological functions, yet many species can behaviourally thermoregulate to avoid temperature extremes or to select preferred temperature ranges [START_REF] Sinclair | Can we predict ectotherm responses to climate change using thermal performance curves and body temperatures?[END_REF]. However, behavioural thermoregulation and mitigation strategies to avoid temperature extremes may be more difficult for aquatic animals like X. laevis as shallow waterbodies are more homogenous in temperature. Variation in thermal sensitivity depends on metabolic processes which are effective across a specific range of temperatures, referred to as an organism's thermal tolerance. Outside this range organisms cannot maintain activity and locomotion, or even survive [START_REF] Angilletta | Thermodynamic effects on organismal performance: Is hotter better?[END_REF][START_REF] Gillooly | Effects of size and temperature on metabolic rate[END_REF]). In our study, the maximal thermal limit (CTmax) did not differ significantly among populations from different altitudes, contrary to the minimal thermal limit (CTmin) (Table 5 and6). It is known that the maximal thermal limit is more limited by genetic or biochemical constraints [START_REF] Blackburn | Evolutionary capacity of upper thermal limits: Beyond single trait assessments[END_REF][START_REF] Grigg | Conservatism of lizard thermal tolerances and body temperatures across evolutionary history and geography[END_REF] and evolve less rapidly than the minimal thermal limit [START_REF] Von | Divergence of thermal physiological traits in terrestrial breeding frogs along a tropical elevational gradient[END_REF][START_REF] Pintanel | Elevational and microclimatic drivers of thermal tolerance in Andean Pristimantis frogs[END_REF]. Furthermore, hot temperatures can also be more easily behaviourally avoided than cold ones [START_REF] Bodensteiner | Thermal adaptation revisited: How conserved are thermal traits of reptiles and amphibians?[END_REF][START_REF] Muñoz | Evolutionary stasis and lability in thermal physiology in a group of tropical lizards[END_REF][START_REF] Muñoz | Basking behavior predicts the evolution of heat tolerance in Australian rainforest lizards[END_REF], which could explain why this did not differ significantly among populations. In contrast, with increasing altitude, CTmin decreased as observed in other studies on critical thermal limits depending on elevation [START_REF] Catenazzi | Thermal physiology, disease, and amphibian declines on the Eastern slopes of the Andes[END_REF][START_REF] Navas | Herpetological diversity along Andean elevational gradients: links with physiological ecology and evolutionary physiology[END_REF]. Our results show that adults from the populations examined conserved the same physiological upper thermal limit suggesting that part of the overall thermal ecology is conserved [START_REF] Sinervo | Erosion of lizard diversity by climate change and altered thermal niches[END_REF]. A study on tadpoles from parents collected at two of these sites (Phuthaditjhaba and Hluhluwe; [START_REF] Wagener | Progeny of Xenopus laevis from altitudinal extremes display adaptive physiological performance[END_REF] shows that their CTmin and CTmax vary in the same way, but they were lower (4.2 -2.5°C) and higher (37.0 -37.8°C), respectively, than for the adults measured here. This suggests that larvae have a wider thermal tolerance than adults in X. laevis [START_REF] Heatwole | Heat tolerances of tadpoles of two species of tropical anurans[END_REF]. Moreover, intraspecific variation in CTmax tends to differ less than interspecific variation at the adult stage [START_REF] Araújo | Heat freezes niche evolution[END_REF].

The low-altitude Hluhluwe population exhibits the highest CTmin and shows significant differences in CTmin compared to the other populations (Table 4).

Exertion performance

Thermal performance curves are expected to have the same general structure, with performance increasing with increasing temperature, reaching a maximum at the optimal temperature (Topt), and then quickly decreasing to reach zero performance are the critical minimal and maximal temperatures [START_REF] Angilletta | The evolution of thermal physiology in ectotherms[END_REF][START_REF] Schulte | Thermal performance curves, phenotypic plasticity, and the time scales of temperature exposure[END_REF]. We observed a similar overall structure of thermal performance curves in the four populations (see Fig. 1). Temperature significantly affected locomotor performance as expected. In general, amphibians exhibit an increase of their resting or basal metabolism (oxygen consumption) with increasing temperature [START_REF] Whitford | The effects of temperature on respiration in the Amphibia[END_REF][START_REF] Kreiman | Testing the metabolic homeostasis hypothesis in amphibians[END_REF]. Endurance capacity relies strongly on the cardiovascular system and on oxygen transport, which is extremely dependent on temperature [START_REF] Seymour | Behavioral thermoregulation by juvenile Green Toads, Bufo debilis[END_REF]. Exertion, as measured here, is also dependent on the cardiovascular system in addition to being dependent on muscle contractile properties. Individuals from populations from different altitudes consequently exhibited different optimal temperatures for locomotor performance (Table 4,Topt). The Lesotho population from the highest altitude (3197 m a.s.l.) has the lowest optimal temperature (16.64°C) and the Hluhluwe population from the lowest altitude (60 m a.s.l.) has the highest optimal temperature (21.11°C). Temperature optima in populations from middle altitudes Dalton (1016 m a.s.l.) and Phuthaditjhaba (1948 m a.s.l.) are intermediate (19.44°C and 18.8°C respectively). The optimal temperature for exertion performance of the populations tested here thus seems to be correlated with environmental temperatures depending on altitude with environmental temperatures decreasing with increasing altitude [START_REF] Körner | The use of 'altitude' in ecological research[END_REF]; Table 1).

Note that there is a mismatch between the mean environmental temperature and the optimal temperature for performance with frogs from Lesotho exhibiting a Topt (16.64°C) that is higher than the mean environmental temperature (6.35°C). However, a decrease in environmental temperature did result in a decrease in the optimal temperature. These results are concordant with the prediction that optimal temperature for fitness-relevant functions is set at the most commonly encountered temperature in the environment [START_REF] Huey | Evolution of resistance to high temperature in ectotherms[END_REF][START_REF] Gilchrist | Specialists and generalists in changing environments. I. Fitness landscapes of thermal sensitivity[END_REF][START_REF] Navas | Thermal relationships and exercise physiology in anuran amphibians: Integration and evolutionary implications[END_REF].

Performance breadths

Similar to the differences in temperature optima among populations, we observed directional shifts in the 80% thermal performance curve for the populations from the two extreme altitudes (Lesotho: 3197 m and Hluhluwe: 60 m a.s.l.). For example, the thermal performance curve of the Lesotho population shows a left-shift of about 3.7 degrees compared to the Hluhluwe population (see Fig. 1,Table 4, Tpb80 interval). Consequently, the population from the highest altitude, in Lesotho, appears better adapted to lower temperatures for aquatic exertion. It is known that adaptation to cold environment is predicted to entail the loss of performance in warmer environments [START_REF] Bennett | An experimental test of evolutionary trade-offs during temperature adaptation[END_REF], as is effectively observed for the Lesotho population. On the other hand, the Hluhluwe population from the lowest altitude appears better adapted to higher temperatures. Overall, differences in thermal sensitivity of locomotor performance among populations tend to be mainly driven by two populations, Hluhluwe and Lesotho. However, the breadth of the 80% thermal performance interval did not differ among populations, showing that the overall shape of the performance curve did not evolve. These results can suggest a level of intrinsic constraint which appears to limit the ability in X. laevis adults to broaden the temperature performance curve.

Generally, species with broad distribution ranges express phenotypic differences among local populations, following predictable patterns along spatial and environmental gradients [START_REF] Chevin | Adaptation to marginal habitats by evolution of increased phenotypic plasticity[END_REF]. Thus, thermal performance curves may shift to become locally adapted to environmental conditions such as temperature [START_REF] Narum | Thermal adaptation and acclimation of ectotherms from differing aquatic climates[END_REF].

Environmental gradients exert a major effect on patterns of intraspecific variation [START_REF] Mizera | Spatial niche packing, character displacement and adaptive speciation along an environmental gradient[END_REF] and coupled with natural selection in the long term may produce differences between populations resulting in local adaptation [START_REF] Olsson | Thermal environment, survival and local adaptation in the common frog, Rana temporaria[END_REF]. Local adaptation is a process of natural selection whereby resident populations evolve higher relative fitness in their local habitat than populations originating elsewhere [START_REF] Sexton | Evolution of ecological niche breadth[END_REF][START_REF] Schmid | The role of phenotypic plasticity on population differentiation[END_REF]. The Xenopus laevis populations included in our study show different phenotypes in locomotor performance depending on temperature and altitude [START_REF] Arnold | Morphology, Performance and Fitness[END_REF].

These may increase fitness in a given thermal environment as individuals and populations from cold environments and high altitudes perform relatively better at cold temperatures. The thermal performance curves from these populations thus show patterns in thermal physiology that covary with the thermal environment and the altitudinal gradient. Indeed, in situ temperature data from the collected sites show a thermal gradient in average temperature depending on the altitude ranging from 6.35°C to 23.03°C. The average temperatures from March to May are 23.03°C in Hluhluwe (60m a.s.l.), 17.19°C in Dalton (1016m a.s.l.), 17.18°C in Phuthaditjhaba (1948m a.s.l.) and 6.35°C in Lesotho (3197m a.s.l.) (Table 1).

In order to understand the processes that underlie the observed differences in optimal temperatures for locomotion in Xenopus laevis, future studies may benefit from using common garden experiments to tease apart the role of phenotypic plasticity versus genetic adaptation. Even if the responses are plastic, this may promote future adaptation by allowing populations to perform well in novel environments [START_REF] Crispo | The Baldwin effect and genetic assimilation: Revisiting two mechanisms of evolutionary change mediated by phenotypic plasticity[END_REF][START_REF] Ghalambor | Adaptive versus nonadaptive phenotypic plasticity and the potential for contemporary adaptation in new environments[END_REF] and may be one of the critical traits driving the invasion potential of the species. Moreover, genomic and transcriptomic approaches coupled to the study of phenotypic diversity would provide better insights into the processes underlying thermal adaptation in these organisms.

The broad thermal tolerance and ability to adapt to different thermal environments may explain the invasive nature of this species and its presence on four continents [START_REF] Furman | Pan-African phylogeography of a model organism, the African clawed frog 'Xenopus laevis[END_REF][START_REF] Measey | Ongoing invasions of the African clawed frog, Xenopus laevis: a global review[END_REF] comprising areas outside of its native climate envelope [START_REF] Rödder | Global realized niche divergence in the African clawed frog Xenopus laevis[END_REF]. Including physiological data on locomotor performance depending on temperature in species distribution models can provide better and more biologically informed insights into the potential future spread of this species under different scenarios of climate change [START_REF] Coulin | Linking thermo-tolerances of the highly invasive ant, Wasmannia auropunctata, to its current and potential distribution[END_REF][START_REF] Gamliel | Incorporating physiology into species distribution models moderates the projected impact of warming on selected Mediterranean marine species[END_REF]Ginal et al., in press). Finally, our results suggest that whereas optimal temperatures for locomotion show population-level differences, the shape of the performance curves did not. This may hint at a more general mechanism of the evolution of thermal dependence of locomotion, yet remains to be tested in other species.

A.H., J.M. and L.A. conceived the study. J.M., C.W. and L.A. collected the animals in the field. L.A. collected the data. L.A. and A.H. and P.P. analysed the data. L.A. and A.H. wrote the article and all the authors contributed to and approved the final version. Table 1. Average in situ temperatures on the sites of collection (March to May 2021). Figure 2. Temperature-performance curves illustrating the impact of temperature on maximum time to exhaustion for X. laevis populations.

Tables

Alti

Fig. 1 Fig. 2

 12 Figure legends

Figure 1 .

 1 Figure 1. Temperature-performance curves illustrating the impact of temperature on maximum distance swam for X. laevis populations.

Table 2 .

 2 Time to exhaustion (in s) at different temperatures for the different populations.

		Hluhluwe	Dalton	Phuthaditjhaba	Lesotho
		(60 m)	(1016 m)	(1948 m)	(3197 m)
		(N = 26)	(N = 25)	(N = 26)	(N = 28)
	8°C	205.48 ± 5.94	210.60 ± 5.52	193.27 ± 5.76	206.57 ± 7.25
	12°C	221.63 ± 13.20 239.74 ± 11.97 224.28 ± 8.13	239.25 ± 9.02
	16°C	232.59 ± 16.76 269.08 ± 15.19 244.78 ± 7.45	251.70 ± 13.34
	19°C	273.48 ± 16.63 265.93 ± 16.22 239.19 ± 11.03	229.84 ± 13.09
	23°C	271.96 ± 21.69 249.60 ± 16.72 210.35 ± 6.56	191.87 ± 12.03
	27°C	149.35 ± 9.94	130.34 ± 6.39	110.31 ± 5.29	108.47 ± 6.34
	Table entries are means ± standard errors.

Table 3 .

 3 Aquatic exertion (distance covered, in cm) for the tested populations at different temperatures.

		Hluhluwe	Dalton	Phuthaditjhaba	Lesotho
		(60 m)	(1016 m)	(1948 m)	(3197 m)
		(N = 26)	(N = 25)	(N = 26)	(N = 28)
	8°C	4104.69 ±	5135.16 ±	5023.11 ±	5185.28 ±
		155.73	178.75	193.32	207.04
	12°C	5445.23 ±	6621.48 ±	6614.42 ±	6616.86 ±
		304.77	224.61	256.80	245.88
	16°C	6709.92 ±	8076.68 ±	7118.57 ±	7293.61 ±
		377.99	447.04	281.20	340.72
	19°C	7943.57 ±	8548.12 ±	7948.65 ±	7190.25 ±
		473.86	429.74	347.70	403.43
	23°C	8468.46 ±	8110.12 ±	7228.08 ±	6250.54 ±
		510.92	402.60	270.40	377.82
	27°C	4951.11 ±	4797.76 ±	4534.23 ±	3665.39 ±
		280.75	267.14	241.95	200.39
	Table entries are means ± standard errors.

Table 6 .

 6 Results of Tukey post-hoc tests testing for differences in CTmax among populations.

Acknowledgements J.M., C.W. and L.A. would like to thank the DSI-NRF Centre of Excellence for Invasion Biology and Stellenbosch University. Capture and experimental permits were provided by Ezemvelo KZN Wildlife, permit No: OP7/2021; the Lesotho Ministry of Tourism, Environment and Culture MTEC/NES/PRO/2.1; and the Animal ethics clearance application ACU-2020-19215 in Research Ethics, Animal Care and Use.

Author contributions

Table 4. Critical temperatures and optimal temperatures for the populations of X. laevis tested. 10.64 ± 0.79 11.5 -22.2 4.51 ± 0.65 14.8 -19.3 Critical minimum and maximum temperatures (CTmin and CTmax), optimal temperature (Topt), 80 and 95% thermal performance breadths (Tpb80 and Tpb95) and their interval.

Bold values indicate significant differences.

Supplementary figures

Suppl. Fig. 1 Temperature-performance curves predicted using GAM models illustrating the non-linear effect of temperature on aquatic endurance for the X. laevis population from Hluhluwe (60m a.s.l.). Note that negative values can predicted values beyond the critical thermal limits, illustrating the limits of this approach beyond the estimate of the optimal temperature.

Suppl. Fig. 2 Temperature-performance curves predicted using GAM models illustrating the non-linear effect of temperature on aquatic endurance for the X. laevis population from Dalton (1016m a.s.l.). Note that negative values can be predicted values beyond the critical thermal limits, illustrating the limits of this approach beyond the estimate of the optimal temperature.

Suppl. Fig. 3 Temperature-performance curves predicted using GAM models illustrating the non-linear effect of temperature on aquatic endurance for the X. laevis population from Phuthaditjhaba (1948m a.s.l.). Note that negative values can be predicted values beyond the critical thermal limits, illustrating the limits of this approach beyond the estimate of the optimal temperature. Suppl. Fig. 4 Temperature-performance curves predicted using GAM models illustrating the non-linear effect of temperature on aquatic endurance for the X. laevis population from Lesotho (3197m a.s.l.). Note that negative values can be predicted values beyond the critical thermal limits, illustrating the limits of this approach beyond the estimate of the optimal temperature.


Arnaud Viola

Raphaël Chattot

Vincent Martin

Galina Tsirlina

Jaysen Nelayah

Jakub Drnec

Frédéric Maillard

Hydrogen Trapping in Palladium Nanoparticles Revealed by Electrochemical, X-ray Scattering, and Spectrometric Measurements

Keywords: palladium nanoparticles, hydrogen trapping, hydrogen absorption, hydrogen embrittlement, wide-angle X-ray scattering

come

INTRODUCTION

Due to the unique ability of palladium (Pd) to reversibly insert (absorb) and de-insert (desorb) large quantities of hydrogen (H) at room temperature and low pressure, the palladium-hydrogen (Pd-H) system has been widely studied. This unique property makes Pd and its alloys attractive materials for several applications including hydrogen storage [START_REF] Dekura | Hydrogen in Palladium and Storage Properties of Related Nanomaterials: Size, Shape, Alloying, and Metal-Organic Framework Coating Effects[END_REF] , separation and purification [START_REF] Rahimpour | Palladium Membranes Applications in Reaction Systems for Hydrogen Separation and Purification: A Review[END_REF][START_REF] Hatlevik | Palladium and Palladium Alloy Membranes for Hydrogen Separation and Production: History, Fabrication Strategies, and Current Performance[END_REF] , sensing [START_REF] Behzadi Pour | Hydrogen Sensors: Palladium-Based Electrode[END_REF][START_REF] Ndaya | Recent Advances in Palladium Nanoparticles-Based Hydrogen Sensors for Leak Detection[END_REF] and (electro)catalysis [START_REF] Joudeh | Synthesis Methods and Applications of Palladium Nanoparticles: A Review[END_REF][START_REF] Saldan | Chemical Synthesis and Application of Palladium Nanoparticles[END_REF] . H atoms can be inserted into Pd by applying a H2 pressure.

The α phase (PdHx-α), corresponding to a PdHx solid solution with slightly expanded lattice constant, first forms at low H2 pressure (< 0.01 atm). The β phase (PdHx-β), corresponding to a non-stoichiometric Pd hydride phase where H atoms occupy the octahedral lattice sites (forming a NaCl structure), forms at higher H2 pressure. H atoms can also be inserted into Pd electrochemically, as the electrode potential (equivalent of the pressure in electrochemistry) is a measure of effective H2 fugacity under equilibrium conditions. Similar to what was found in the gas phase, in electrochemistry, H is first dissolved in the α phase followed by formation of the β phase, and the two phases were shown to coexist in bulk Pd for intermediate values of x [START_REF] Flanagan | Electrode Potentials of the Palladium + Hydrogen System[END_REF] . At room temperature and under equilibrium, the phase transition occurs at potentials of ca. 55-60 mV in reversible hydrogen electrode (RHE) scale.

In situ techniques [START_REF] Landers | Dynamics and Hysteresis of Hydrogen Intercalation and Deintercalation in Palladium Electrodes: A Multimodal In Situ X-Ray Diffraction, Coulometry, and Computational Study[END_REF][START_REF] Chattot | Electrochemical Strain Dynamics in Noble Metal Nanocatalysts[END_REF][START_REF] Benck | Producing High Concentrations of Hydrogen in Palladium via Electrochemical Insertion from Aqueous and Solid Electrolytes[END_REF] have been frequently used to provide information concerning lattice expansion/contraction of the Pd-H system. It is well documented that H insertion and formation of β hydrides cause linear expansion of the Pd lattice, adversely affecting the stability of bulk Pd samples (foils, wires, thin films), a phenomenon that is known at the macroscopic level as H embrittlement. The Pd lattice irreversibly expands upon repeated H insertion/de-insertion cycles [START_REF] Auer | The Kinetics of Hydrogen Absorption in Palladium (α-and β-Phase) and Palladium-Silver-Alloys[END_REF] , resulting in the formation of microstructural defects, such as dislocations [START_REF] Ulvestad | Three-Dimensional Imaging of Dislocation Dynamics during the Hydriding Phase Transformation[END_REF][START_REF] Zhang | Hydrogen Absorption of Palladium Thin Films Observed by In Situ Transmission Electron Microscopy with an Environmental Cell[END_REF] , and modifying the rate of α↔β phase transitions [START_REF] Zhang | Hydrogen Absorption of Palladium Thin Films Observed by In Situ Transmission Electron Microscopy with an Environmental Cell[END_REF][START_REF] Luo | Supersaturation and Hydride Formation in the Dilute Phase of Pd-H and Pd-Mn-H Alloys[END_REF][START_REF] Flanagan | The Effect of Lattice Defects on Hydrogen Solubility in Palladium[END_REF] . Cycling also fragilizes the texture (distribution of crystallographic orientations) [START_REF] Cabrera | Structural Changes Induced by Hydrogen Absorption in Palladium and Palladium-Ruthenium Alloys[END_REF] and changes the dimensions (length and thickness) [START_REF] Krause | On Palladium-Hydrogen[END_REF] of bulk Pd materials while irreversible cracks and delamination were reported for Pd thin film samples [START_REF] Benck | Producing High Concentrations of Hydrogen in Palladium via Electrochemical Insertion from Aqueous and Solid Electrolytes[END_REF][START_REF] Lee | Hysteresis Behavior of Electrical Resistance in Pd Thin Films during the Process of Absorption and Desorption of Hydrogen Gas[END_REF] .

The behavior of Pd nanocrystallites differs. Lee et al. first reported that irreversible mechanical deformation of Pd films is suppressed in the films thinner than 20 nm [START_REF] Lee | Hysteresis Behavior of Electrical Resistance in Pd Thin Films during the Process of Absorption and Desorption of Hydrogen Gas[END_REF] . Moreover, if dislocations easily appear in bulk Pd materials, they are unlikely to form in Pd nanoparticles (NPs) smaller than 300 nm, as recently shown by Uvelstad et al. [START_REF] Ulvestad | Three-Dimensional Imaging of Dislocation Dynamics during the Hydriding Phase Transformation[END_REF] . According to Vogel et al. [START_REF] Vogel | Palladium Nanoparticles "Breathe" Hydrogen; a Surgical View with X-Ray Diffraction[END_REF] , H can be reversibly inserted/de-inserted in/from 4 to 6 nm Pd NPs with no modification of the crystal structure. On the contrary, non-closing pressure-composition-isotherm curves and more expanded lattice constant after H de-insertion have been frequently reported for Pd nanomaterials after H insertion/deinsertion experiments [START_REF] Bardhan | Uncovering the Intrinsic Size Dependence of Hydriding Phase Transformations in Nanocrystals[END_REF][START_REF] Nützenadel | Critical Size and Surface Effect of the Hydrogen Interaction of Palladium Clusters[END_REF][START_REF] Jose | Nature of Hydrogen Atom Trapped inside Palladium Lattice[END_REF][START_REF] Sachs | Solubility of Hydrogen in Single-Sized Palladium Clusters[END_REF][START_REF] Pundt | Hydrogen Sorption in Elastically Soft Stabilized Pd-Clusters[END_REF][START_REF] Kobayashi | On the Nature of Strong Hydrogen Atom Trapping inside Pd Nanoparticles[END_REF] . Recently, Liu et al. [START_REF] Liu | Size-Dependent Hydrogen Trapping in Palladium Nanoparticles[END_REF]

investigated H trapping into carbon-supported

Pd NPs with sizes between 1.4 and 6.0 nm. They reported an increasing loss of H sorption capacity with decreasing Pd NPs size. Density functional theory (DFT) and tight-binding calculations supported the experimental findings, and revealed that H atoms are trapped in the subsurface interstitial sites. However, this study was limited to a single sorption-desorption cycle in the gas phase. Campesi et al. [START_REF] Campesi | Hydrogen Storage Properties of Pd Nanoparticle/Carbon Template Composites[END_REF] observed that the total H sorption capacity of 2 nm Pd clusters/porous carbon composite at 5 atm H2 markedly decreases after the first insertion/de-insertion cycle and in a less pronounced manner later on, yielding H:Pd atomic ratios of 0.78, 0.55 and 0.49 for the first, second and third cycles, respectively. Using unsupported Pd NPs as a catalyst for ammonia-borane hydrolysis, Jose et al. [START_REF] Jose | Nature of Hydrogen Atom Trapped inside Palladium Lattice[END_REF] reported that some H atoms remain trapped in the Pd lattice even after the reaction, leading to expanded Pd lattice constant, more pronounced after 10 cycles than after the first one. In contrast, Rather et al. [START_REF] Rather | Hyperstoichiometric Hydrogen Storage in Monodispersed Palladium Nanoparticles[END_REF] reported stable H storage capacities (measured at 25°C and 1.7 MPa H2 pressure) for ca. 13 and 8 nm Pd NPs after up to 8 H insertion/de-insertion cycles.

Importantly, the samples were outgassed under vacuum at 200°C between successive cycles, which may account for the apparent stability of their H sorption capacity.

With the motivation of providing comprehensive evidence of H trapping process in Pd NPs and of clarifying the influence of this process on their long-term H sorption capacity, we combined here in situ wide-angle X-ray scattering (WAXS), in situ inductively-coupled plasma mass spectrometry (ICP-MS), identical-location transmission electron microscopy (IL-TEM) and coulometric quantification of the amount of H inserted and de-inserted in/from Pd NPs. The results show continuous expansion of the Pd lattice during repeated H insertion/de-insertion cycles, resulting in a progressive loss of their reversible H sorption capacity and in appearance of delay in lattice relaxation during H de-insertion.

METHODS

Details about materials, methods, and experimental techniques are presented in Supplementary

Information.

RESULTS & DISCUSSION

Figure 1a shows characteristic low-and medium-magnification transmission electron microscopy (TEM) images of the commercial carbon-supported Pd catalyst (Pd/C) used in this study. The Pd NPs are isolated and homogeneously distributed onto the Vulcan XC72 support. Their numberaveraged mean NP size is 𝑑 𝑛 ̅̅̅ = 3.6 ± 0.7 𝑛𝑚 (see Figure S1 in Supporting Information for more details). The Pd content amounts to 17.6 wt.%, as determined by microwave-assisted digestion of a given mass of catalyst in aqua regia, dilution and analysis by ICP-MS. Figure 1b displays a typical cyclic voltammogram (CV) of the Pd/C sample recorded between 0.0 and 0.6 V in Ar-saturated 0.1 M HClO4 (Note that all electrode potentials are referred to the reversible hydrogen electrode, RHE). In agreement with literature, peaks 1 and 1' can be ascribed completely or partly to the adsorption and desorption of underpotentially-deposited H, and peaks 2 and 2' to the insertion (absorption)/de-insertion (desorption) of H mostly into/from the PdHx-β phase [START_REF] Łukaszewski | The Method of Limited Volume Electrodes as a Tool for Hydrogen Electrosorption Studies in Palladium and Its Alloys[END_REF] .

The PdHx-α phase contribution can be distributed between these two features. For lower potentials, the formation of molecular H2 (dissolved in solution) proceeds simultaneously to the insertion of H ("tail" 3). rate of 10 mV s -1 (starting from 0.4 V by a cathodic scan). The measurements were performed using a Pd loading of 30 µgPd cm -2 geo and a temperature of 25 °C.

To track the changes in structure of the Pd/C NPs during the H insertion/de-insertion process, we performed in situ WAXS experiments at the European Radiation Synchrotron Facility (ESRF, Grenoble) using a home-made electrochemical flow cell, previously introduced in reference [START_REF] Chattot | Electrochemical Strain Dynamics in Noble Metal Nanocatalysts[END_REF] .

A WAXS pattern measured at E = 0.40 V in Ar-saturated 0. and Lander et al. [START_REF] Landers | Dynamics and Hysteresis of Hydrogen Intercalation and Deintercalation in Palladium Electrodes: A Multimodal In Situ X-Ray Diffraction, Coulometry, and Computational Study[END_REF] , respectively. This value slightly exceeds 3.889 Å found recently for the same catalyst after cycling in alkaline solution [START_REF] Chattot | Electrochemical Strain Dynamics in Noble Metal Nanocatalysts[END_REF] , and also the values for Pd powder available in the ICDD database (e.g., 3.8874 and 3.8898 Å). This can result from 0.006 Å uncertainty of calibration, as discussed in Supporting Information. with cycle number (0.028 V for the 1 st and 0.052 V for the 50 th cycles). During the 50 th cycle, it starts when less than a half of newly inserted H penetrates to the lattice, i.e. a lot of H undergoes sorption into already expanded Pd. At anodic scans, the appearance of two-phase region (the end of orange segments, and the start of green segments) shifts to more positive potentials. In the 1 st cycle, PdHx-α phase structural signatures appear when only a small portion of H is de-inserted (at 0.037 V), when in the 50 th cycle about a half of H is de-inserted before the lattice starts to relax (at 0.085 V). Moreover, lattice relaxation in the 50 th cycle corresponding to the appearance of Pd metal takes place only at ca. 0.20 V, and results in still extended PdHx-α phase lattice.

The described behavior results in a pronounced widening of the potential region in which PdHx-α and PdHx-β phase lattices coexist (Figure 3e). These structural changes are pronounced initially and reach a pseudo-plateau after ca. 10 cycles (Figure 3b-d-e). The absence of correlation between the charge spent for H insertion/de-insertion and lattice expansion-relaxation events becomes more pronounced with cycling, and the loss of correlation is accompanied by the development of lattice microstrain (Figure S5).

The comparison between the H:Pd ratios measured/estimated from electrochemical and X-ray measurements is also enlightening. Using the linear calibration between the H:Pd ratio and the PdHx-β lattice parameter reported by Benck et al . [START_REF] Benck | Producing High Concentrations of Hydrogen in Palladium via Electrochemical Insertion from Aqueous and Solid Electrolytes[END_REF] , the Pd lattice parameter in the PdHx-β phase was converted into a H:Pd ratio, denoted H:PdWAXS. As illustrated by Figure 3b, the H:PdWAXS ratio at 0.025 V was independent of the number of CV cycles (0.49 ± 0.035), suggesting that the same quantity of H was inserted in the Pd/C NPs at this potential. This seems to be in contradiction with the 19 % decrease of the H:PdRDE ratio experimentally measured at 0.025 V (Figure 3a). As the H:PdRDE is precisely determined by measuring the H insertion/de-insertion charge and the mass of Pd deposited on the working electrode (ICP-MS measurement, see Supporting Information),

while the H:PdWAXS is estimated from the variation of the PdHx-β lattice parameter, the difference between the H:PdWAXS and H:PdRDE ratios may arise from two reasons: either the absence of correlation between lattice expansion and reversibly inserted H amount under less equilibrium conditions, or the detachment or the dissolution of Pd nanocrystallites during the 50 CVs.

To determine whether the changes in the lattice structure are accompanied by changes in size or morphology of the Pd/C nanoparticles, we used identical-location aberration-corrected (highresolution) transmission electron microscopy (IL-(HR)TEM). Five different regions of the Pd/C sample were imaged before/after 1,000 square potential cycles between 0.400 and 0.100 V (10 s at each potential), Subsequently, 1,000 square potential cycles between 0.400 and 0.025 V were performed (Figure 4), followed by 1,000 square potential cycles between 0.400 and 0.000 V (see To precisely estimate the amount of Pd being dissolved from the Pd/C NPs during the 50 CVs, we used an electrochemical flow cell coupled in situ to an ICP-MS (see Refs. [START_REF] Chattot | Break-In Bad: On the Conditioning of Fuel Cell Nanoalloy Catalysts[END_REF][START_REF] Abbou | Manipulating the Corrosion Resistance of SnO2 Aerogels through Doping for Efficient and Durable Oxygen Evolution Reaction Electrocatalysis in Acidic Media[END_REF] and Supporting Information for more details). Figure 5 shows time-and potential-resolved Pd concentration profiles during the break-in procedure (composed of 400 cycles between 0.100 and 0.500 V at 500 mV s -1 ) or 25 cycles between 0.025 and 0.600 V at 5 mV s -1 . The in situ ICP-MS measurements show that the Pd concentration signal amounts to 10 ng L -1 maximum during the break-in procedure. The 25 cycles at 5 mV s -1 allow a better resolution in time and potential. The S7) and that (ii) no measurable current associated to H desorption was detected after a few seconds during the coulometric H deinsertion experiment (see Figure S3a). We thus conclude that H de-insertion kinetics does not undergo substantial changes during the 50 CV cycles, and that the progressive increase of the PdHx- phase lattice parameter after the 50 consecutive cycles, the observed H trapping and the microstrain development are mutually interrelated events Finally, to confirm that H atoms remain trapped into the Pd NPs, we also designed an additional experiment, leaning on the work of Liu et al. [START_REF] Liu | Size-Dependent Hydrogen Trapping in Palladium Nanoparticles[END_REF] . Briefly, a thin-film of Pd/C catalyst was electrochemically characterized before (fresh catalyst) and after 50 CVs between 0.025 and 0.600 V (aged catalyst). In line with what was found in Figure 3e, the H de-insertion charge decreased during the 50 CVs (Figure 6a), translating into a drop of the H:PdRDE ratio from 0.43  0.02 to 0.35  0.02 (Figure 6b). After this second characterization, the thin-film rotating disk electrode was removed from the electrochemical cell, rinsed with MQ-grade water and vacuumpumped for 10 h at 150°C to desorb the trapped H, and a characterization CV was recorded in the same experimental conditions as initially. The near-perfect superposition of the initial and the final CVs, and the associated increase of the quantity of de-inserted H are unequivocal evidences that H atoms were trapped in the Pd/C material during the 50 CV cycles. The results presented in this study therefore suggest that, similarly to what was observed during catalytic reactions [START_REF] Jose | Nature of Hydrogen Atom Trapped inside Palladium Lattice[END_REF] , H atoms may trap during electrochemical insertion/de-insertion at low enough RHE potentials. Let us however stress that trapping as studied in this work does not corresponds to complete equilibration of Pd-H system, first of all because of unavoidably slow phase transition [START_REF] Lewis | The Palladium-Hydrogen System: Structures near Phase Transition and Critical Points[END_REF] . In frames of the time scale available for in situ structural characterization, we solidly documented that lattice expansion and reversible H sorption are not completely correlated: lattice parameter hysteresis loop is shifting to more positive potentials than corresponding charge hysteresis (compare Figures 3b andS3). This factor of finite characterization time appears also for H trapping analysis based on irreversible lattice expansion [START_REF] Liu | Size-Dependent Hydrogen Trapping in Palladium Nanoparticles[END_REF] (see additional remarks in Supplementary Information after Figure S3).

One can assume that the expansion itself provides the traps, i.e. new defective positions supporting stronger bonding and, correspondingly, slower de-insertion of H. This trapped H can be formally assigned to PdHx-α phase despite its specific sorption energy, as it is stable in the lattice just at typical PdHx-α phase pressures/potentials, and could be finally extracted under equilibrium conditions. The amount of this trapped H can be much higher as compared to PdHx-α phase solid solution in ideal Pd lattice. Namely, H:Pd values up to ~0.06 were reported for thin Pd films [START_REF] Bucur | Effect of Trapping on the Solubility and Diffusivity of Hydrogen in Palladium (α-Phase)[END_REF] , and >0.03 for dispersed electrodeposited Pd 37 . Our estimate of the quantity of trapped H agrees well with these quantities. The principal aspect of our observation is generation of the traps by H inserted in subsequent cycles, in contrast to native defects typical for some Pd materials.

The results reported here are important for a fundamental understanding of structure -catalytic

Figure 1 .

 1 Figure 1. Microscopic and electrochemical characterization of the Pd/C nanocatalyst used in

Figure 2 .

 2 Figure 2. Time-resolved monitoring of insertion and de-insertion of H atoms into the Pd/C

Figure 3 .

 3 Figure 3. Monitoring the hydrogen-induced phase transformation of Pd/C nanoparticles via the

Figure S6 )

 S6 Figure S6). The IL-TEM images presented in Figure4and FigureS6suggest that the Pd NPs

Figure 4 .

 4 Figure 4. Identical-location aberration-corrected transmission electron microscopy images

  dissolution of Pd initiates during the the very beginning of the anodic potential sweep, and reaches a maximum of 10 ng L -1 (equivalent to about 4 ngPd g -1 Pd) at ca. 0.05-0.10 V. Dissolution of Pd during H-deinsertion/desorption (positive-going potential sweeps) is a striking previously unreported result. Overall, the mass of Pd dissolved during the break-in and the subsequent 25 cycles was estimated to be inferior to 0.2 ng representing 0.003  0.001 % of the total Pd mass loaded in the thin-film electrode. We can thus reliably discard any influence of the dissolution of Pd on the change in H insertion/de-insertion properties.

Figure 5 .

 5 Figure 5. In situ monitoring of the amount of Pd being dissolved during successive cyclic

Figure 6 .

 6 Figure 6. Electrochemical evidence of H trapping into Pd/C nanoparticles. a) Cyclic

  activity relationships on Pd-based nanomaterials. Indeed, according to the d-band theory pioneered by Hammer and Norskov, trapping of H atoms into the Pd lattice drastically changes its electronic states, broadening its d-band, and resulting in a negative shift of the d-band center. The latter in turn will lead to a change in the catalytic properties of Pd. For example, PdH0.43 NPs were shown to be more active for methanol oxidation reaction compared to the pure metal Pd counterparts[START_REF] Zhao | Synthesis of Stable Shape-Controlled Catalytically Active β-Palladium Hydride[END_REF] .Schmidt et al. have also recently shown that expansion of the Pd lattice leads to weaker adsorption energy of adsorbed H, enhancing the hydrogen evolution reaction rate in acidic medium[START_REF] Schmidt | Elucidation of Structure-Activity Relations in Proton Electroreduction at Pd Surfaces: Theoretical and Experimental Study[END_REF] .CONCLUSIONSIn summary, our results shed fundamental light into the loss of H sorption capacity of Pd/C nanoparticles during repeated H insertion/de-insertion cycles. By combining electrochemistry and wide-angle X-ray scattering, we demonstrated that H atoms get progressively trapped into the Pd lattice. H trapping progressively expands the Pd lattice, leading to stabilization of the PdHx-β lattice. The 3.6 nm Pd nanoparticles investigated in this study lost 19 % of their initial H sorption capacity during successive 50 H insertion/de-insertion cycles between 0.025 and 0.600 V at a potential sweep rate of 5 mV s -1 . The trapped H atoms can be desorbed by vacuum-pumping and heating to 150°C.

  It displays the expected reflections for the face-centered cubic structure of Pd. The Pd lattice parameter obtained from Rietveld refinement analysis of the WAXS pattern was 3.8945 Å, matching with reported values for Pd films of 3.892 Å and ~3.897 Å obtained byBenck et al. 11

	1 M HClO4 is represented in
	Figure 2a.

  ). This relative irreversible increase (~0.4 %) of the lattice parameter after 50 cycles agrees with the relative increase of nearest Pd-Pd distance of 0.73 % and 0.36 % reported by Liu et al. for 2 and 6 nm Pd particles, respectively, after a single H sorption/desorption cycle 27 .

	appearance (starting point of green segments) becomes more positive with cycle number (0.056 V
	and 0.106 V for the 1 st and 50 th cycles, respectively). Moreover, starting already from the 5 th cycle,
	this potential corresponds to very low charge spent for H sorption. Assuming the hypothesis of

Let us also stress that Rietveld refinement analysis of the WAXS patterns measured at 0.40 V indicates that the microstrain values (Figure

S5

) increase with the number of H insertion/deinsertion cycles (from 93 to 129 %% for the Pd/C catalyst before and after 50 CVs, respectively), suggesting that strain progressively and heterogeneously develops within the Pd/C NPs during repeated H insertion/de-insertion cycles.

To gain further insights into the Pd nanostructure -H sorption capacity relationship, we compared the information provided by electrochemistry and WAXS measurements. The potential regions where the PdHx-α or PdHx-β lattices are individually observable or co-exist are represented in blue, orange and green, respectively in Figure

3c

. At cathodic scans, the potential of PdHx-β phase accumulating trapped H, one can assign this shift to participation of both trapped and newly inserted H in PdHx-β phase formation. The end of green segments/start of orange segments demonstrates the same shift: lattice expansion corresponding to pure PdHx-β phase starts earlier

  Taken all together, the results then confirm that a fraction of H atoms remains trapped in Pd lattice at least in the timescale of our experiment, i.e. the rate of H insertion is essentially faster than the H de-insertion rate. This hypothesis is in line with the single-cycle results of Liu et al. for Pd NPs during gas phase H sorption 27 . The authors reported that smaller Pd NPs are more prone to trap H, with the loss of H sorption capacity amounting to ca. 26 % for 1.4 and 2 nm Pd/C NPs. In this work, the amount of trapped H atoms approaches 19 % for 3.6 nm Pd/C NPs, in quantitative agreement with the values of 26 % and 9 % reported by Liu et al. for 2 and 6 nm Pd/C NPs, respectively. Complementary in situ WAXS and electrochemical experiments revealed that (i) the Pd lattice parameter measured at E = 0.40 V after the 50 CVs remains constant for more than 5 minutes (see Figure

ACKNOWLEDGEMENTS

This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement HERMES N o 952184. The authors thank ESRF for the provision of the beamtime at ID31 beamline. GT is grateful to Pause program for support.

ASSOCIATED CONTENT

This information is available free of charge on the ACS Publications website at https://pubs.acs.org/XXXXX.

Supporting Information

Details and methods about the electrocatalyst used, electrochemical measurements, hydrogen loading quantification, in situ synchrotron wide-angle X-ray scattering measurements, Rietveld refinement, in situ ICP-MS measurements, TEM imaging.

AUTHOR INFORMATION

Corresponding Author

Notes

The authors declare no competing financial interest.

TOC Graphic

By combining in situ X-ray scattering and electrochemistry, we provide evidence of continuous expansion of the lattice of 3.6 nm carbon-supported Pd nanoparticles during repeated H insertion/de-insertion cycles, resulting in a progressive loss of their reversible H sorption capacity.


The PDMS is a database system that manages the personal data of a user under their control, based on a paradigm inversion for the management of personal data: instead of externalizing the personal data of the user to the services performing the processing, it is the processing code that comes to execute on the side of the PDMS close to the data in a controlled execution environment. To exemplify this trend, let's consider a scenario where companies incentivize their employees for environmentally friendly behavior by offering a green bonus based on the number of commutes made by bike. GPS traces are collected from a reliable service and processed locally by the user's PDMS. The result is then delivered to the employer with proof of compliance. Several similar scenarios are realistic, e.g., patients providing statistics to hospitals, power meters, etc. This scenario is challenging since it calls for extensiveness to develop and deploy ad hoc code, and security to assure PDMS users that their detailed personal data is not disclosed to third parties outside the sphere of control of their PDMS.

To solve this tension between extensiveness and security, we proposed in [START_REF] Anciaux | Personal Data Management Systems: The security and functionality standpoint[END_REF] a three-layer logical architecture where a minimal Secure Core implementing basic operations on personal data is extended with Isolated Data Tasks themselves accessed by Applications on which no security assumption is made (see Figure 1, left). The objective is to control the flow of raw personal data from the Core to the outside, such that only expected results are declassified to untrusted applications or third parties.

The right part of Figure 1 gives details on the concrete implementation of an ES-PDMS, with some examples of data tasks to answer the above scenario. Properties 1 to 5 are generic security properties that must be properly orchestrated to strengthen the security of our architecture. Security properties 6 to 8 were designed for the specific ES-PDMS context and were introduced in [START_REF] Carpentier | Data Leakage Mitigation of User-Defined Functions on Secure Personal Data Management Systems[END_REF] and demonstrated in [L2].

Figure 1: ES-PDMS logical architecture and a concrete implementation [L2].

This is a first step, and many challenges remain, depending on the type of processing and the manipulated data that must be properly protected:

• Personal data of a single PDMS user. To properly address this use case, presented above, it is necessary to consider processing functions that handle large volumes of personal data, such as aggregation functions. These functions are defined by a third-party application called App, and their code is evaluated in the PDMS environment. However, the PDMS user cannot fully trust the App. Therefore, it is essential to focus on execution mechanisms that mitigate information leakage through the successive processing of these functions. As preliminary results, we introduce in [START_REF] Carpentier | Data Leakage Mitigation of User-Defined Functions on Secure Personal Data Management Systems[END_REF] new execution strategies based on partitioning and replay of processing in different stateless Intel SGX enclaves, in order to guarantee an upper bound on data leakage through legitimate results, for certain categories of aggregation functions typical of PDMS use cases, and with a reasonable performance overhead.

• Personal data of a community of PDMS users. Individuals can cross data within large communities of users, e.g., to compute statistics for epidemiological studies or to train a machine learning model for recommendation systems or automatic classification of user data.

While, in the previous case, we can reasonably assume that the user will not attempt to attack the confidentiality of their own data, this is no longer the case as soon as we assume decentralized computations on PDMSs that will manipulate the data of other users. It is therefore necessary to consider implementation strategies that limit data leakage in case of a breach of the (hardware) security of the PDMS. As a first possible scenario, we consider in [START_REF] Javet | Edgelet Computing: Pushing Query Processing and Liability at the Extreme Edge of the Network[END_REF] a threat model that we call the "Wolf in the sheepfold", in which (a small number of) PDMSs have been instrumented by their owner and thus operate in the so-called "sealed glass proof" mode, without confidentiality (but still as expected, with integrity guarantees). Other more difficult models are to be considered, where the communications between PDMS can be analyzed and where the nodes could be corrupted also in integrity or in greater number.

• Personal data of third parties. Some cases of computation on PDMS require the exploitation of potentially sensitive third-party data (e.g., a machine learning model derived from a personal dataset or an IP-protected dataset). Integrating or storing third-party data in the application during deployment would, compared to the two cases above, raise additional security issues (the same set of sensitive data must be protected in all PDMSs running the application) with update and performance difficulties after deployment. Therefore, it is necessary to allow for the application to dynamically retrieve third-party data when running on the PDMS side, which represents a new potential leakage channel for PDMS data and requires new solutions to regulate PDMS data flows at runtime. Since the initial paper [START_REF] Anciaux | Personal Data Management Systems: The security and functionality standpoint[END_REF] on the extensive and secure PDMS architecture, the PETRUS project has obtained interesting research results and realized some demonstrators [L2, L3] which underscores the potential of the PDMS approach, fostered by the development of TEEs.

Links:

  Extensive and Secure Personal Data Management Systems by Nicolas Anciaux and Luc Bouganim (Inria, UVSQ and Univ. Paris-Saclay) Personal Data Management Systems (PDMS) are emerging to provide individuals with appropriate tools to collect, manage and make accessible their personal data to applications. At the same time, the deployment of Trusted Execution Environments (TEE) both on the cloud (Intel SGX processors) and on smartphones (ARM TrustZone) opens new architectural perspectives for these PDMS. The PETRUS project [L1] proposes new solutions for PDMS, allowing third-party applications to exploit the individual's personal data while guaranteeing individuals the legitimacy of uses and privacy. A first proof of concept implementation of this new architecture has just been developed on Intel SGX processor.

  Core / Isolated Data Tasks P2: Secure communications (TLS) P3: Core as proxy P4: End-to-end access control P5: End-to-end attestations P6: Limited result size P7: Stateless Data Tasks P8