Water availability and environmental temperature correlate with geographic variation in water balance in common lizards

Abstract

Water-conservation strategies are well documented in species living in water-limited
environments, but physiological adaptations to water availability in temperate climate
environments are still relatively overlooked. Yet, temperate species are facing more
frequent and intense droughts as a result of climate change. Here, we examined
variation in field hydration state (plasma osmolality) and standardized evaporative
water loss rate (SEWL) of adult male and pregnant female common lizards (Zootoca
vivipara) from 13 natural populations with contrasting air temperature, air humidity, and
access to water. We found different patterns of geographic variation between sexes.
Overall, males were more dehydrated (i.e., higher osmolality) than pregnant females,
which likely comes from differences in field behaviour and water intake since SEWL
was similar between sexes. Plasma osmolality and SEWL rate were positively
correlated with environmental temperature in males, while plasma osmolality in
pregnant females did not correlate with environmental conditions, reproductive stage or
reproductive effort. The SEWL rate was significantly lower in populations without
access to free standing water, suggesting that lizards can adapt or adjust physiology to
cope with habitat dryness. Environmental humidity did not explain variation in water
balance. We suggest that geographic variation in water balance physiology and
behaviour should be taken account to better understand species range limits and
sensitivity to climate change.

Full text

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Oecologia
ISSN 0029-8549
Oecologia
DOI 10.1007/s00442-017-3973-6
Water availability and environmental
temperature correlate with geographic
variation in water balance in common
lizards
Andréaz Dupoué, Alexis Rutschmann,
Jean François Le Galliard, Donald
B.Miles, Jean Clobert, Dale F.DeNardo,
George A.Brusch, et al.

1 23
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Vol.:(0123456789)
1 3
Oecologia
DOI 10.1007/s00442-017-3973-6
PHYSIOLOGICAL ECOLOGY - ORIGINAL RESEARCH
Water availability andenvironmental temperature correlate
withgeographic variation inwater balance incommon lizards
AndréazDupoué1 · AlexisRutschmann2· JeanFrançoisLeGalliard1,3·
DonaldB.Miles4· JeanClobert2· DaleF.DeNardo5· GeorgeA.BruschIV5·
SandrineMeylan1,6
Received: 7 November 2016 / Accepted: 27 August 2017
© Springer-Verlag GmbH Germany 2017
temperature, air humidity, and access to water. We found dif-
ferent patterns of geographic variation between sexes. Over-
all, males were more dehydrated (i.e. higher osmolality) than
pregnant females, which likely comes from differences in
field behaviour and water intake since the rate of SEWL was
similar between sexes. Plasma osmolality and SEWL rate
were positively correlated with environmental temperature
in males, while plasma osmolality in pregnant females did
not correlate with environmental conditions, reproductive
stage or reproductive effort. The SEWL rate was signifi-
cantly lower in populations without access to free standing
water, suggesting that lizards can adapt or adjust physiology
to cope with habitat dryness. Environmental humidity did
not explain variation in water balance. We suggest that geo-
graphic variation in water balance physiology and behaviour
should be taken account to better understand species range
limits and sensitivity to climate change.
Keywords Ectotherm· Osmolality· Pregnancy·
Temperature· Water loss
Introduction
Water is a vital resource for animals that influences many
aspects of species’ functional traits including physiologi-
cal performance, such as locomotion and immunity, and
life history traits such as growth, reproduction and survival
(Whitehead etal. 1996; Lorenzon etal. 1999; Taylor etal.
2006; Marquis etal. 2008; Tingley etal. 2012; Moeller etal.
2013; Zylstra etal. 2013). The clarification of functional
responses related to water balance (i.e. the balance between
water intake and loss) is, therefore, critical in understanding
and predicting general ecological patterns such as distribu-
tion (Dunkin etal. 2013; Peterman and Semlitsch 2014),
Abstract Water conservation strategies are well docu-
mented in species living in water-limited environments,
but physiological adaptations to water availability in tem-
perate climate environments are still relatively overlooked.
Yet, temperate species are facing more frequent and intense
droughts as a result of climate change. Here, we exam-
ined variation in field hydration state (plasma osmolality)
and standardized evaporative water loss rate (SEWL) of
adult male and pregnant female common lizards (Zootoca
vivipara) from 13 natural populations with contrasting air
Communicated by Hannu J. Ylonen.
Electronic supplementary material The online version of
this article (http://doi.org/10.1007/s00442-017-3973-6) contains
supplementary material, which is available to authorized users.
* Andréaz Dupoué
andreaz.dupoue@gmail.com
1 CNRS UPMC, UMR 7618, iEES Paris, Université Pierre
et Marie Curie, Tours 44-45, 4 Place Jussieu, 75005Paris,
France
2 Station d’Ecologie Théorique et Expérimentale du CNRS à
Moulis, UMR 5321, 2 route du CNRS, 09200SaintGirons,
France
3 Département de biologie, Ecole normale supérieure,
PSL Research University, CNRS, UMS 3194, Centre
de recherche en écologie expérimentale et prédictive
(CEREEP-Ecotron IleDeFrance), 78 rue du château,
77140Saint-Pierre-lès-Nemours, France
4 Department ofBiological Sciences, Ohio University, Athens,
OH45701, USA
5 School ofLife Sciences, Arizona State University, Tempe,
AZ85287-4501, USA
6 ESPE de Paris, Université Sorbonne Paris IV, 10 rue Molitor,
75016Paris, France
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population dynamics (Foley etal. 2008; Marquis et al.
2008; McKechnie and Wolf 2010), or habitat use (Davis
and DeNardo 2009; Dunkin etal. 2013; Rozen-Rechels etal.
2015). Species’ water balance may be challenged as soon as
water availability is restricted and this is especially true in
terrestrial ectotherms due to their high physiological sensi-
tivity to climatic conditions (Deutsch etal. 2008; Kearney
etal. 2009).
Several studies have focused on flexible water conser-
vation strategies including changes in behavioural activity
(Lorenzon etal. 1999; Davis and DeNardo 2009; Hetem
etal. 2010), shifts in thermoregulatory strategies (Loren-
zon etal. 1999; Angilletta etal. 2010; Köhler etal. 2011),
metabolic depression (Kennett and Christian 1994; Tieleman
etal. 2002; Muir etal. 2007), or a combination of these.
These adjustments aim at lowering the rate of water loss
and limiting dehydration when individuals are exposed
to or colonize water-restricted environments (Moen etal.
2005). Among the functional traits linked with water bal-
ance, standardized evaporative water loss (i.e. evaporative
water loss at rest and in standardized conditions or SEWL)
is a common physiological measure to determine the water
balance regulation in intra- and inter-specific comparisons.
SEWL is critically influenced by surface area and skin per-
meability, which determine cutaneous loss of water, and by
metabolism and breathing activity, which determine ven-
tilatory water loss (Mautz 1982; Woods and Smith 2010).
According to previous comparative analyses, there is a
general relationship between the SEWL and habitat aridity,
where species and/or populations living in water-restricted
habitats or climates are characterised by lower SEWL (Tiele-
man etal. 2003; Williams etal. 2004; Moen etal. 2005;
Van Sant etal. 2012; Guillon et al. 2014; Cox and Cox
2015; Belasen etal. 2016). This relationship likely reflects
functional genetic adaptations and/or physiological accli-
mation to buffer the effects of more frequent heat stress,
lower air humidity and more restricted water availability on
water balance and energy expenditure (Webster etal. 1985;
Lillywhite 2006; Dupoué etal. 2015b). Yet, comparative
studies of SEWL across geographic gradients of tempera-
ture, humidity and free standing water availability are rare,
and whether these factors influence intra-specific variation
in water balance remain unclear. In addition, comparative
studies of SEWL have rarely examined concurrent varia-
tion in hydration state. If physiological components of the
water balance such as SEWL are adapted or adjusted to envi-
ronmental conditions, we expect that geographic variation
in SEWL will buffer environmental variation in hydration
state such that animals maintain to some extant more similar
hydration states across environments.
Within the same population, variation in physiological
state (e.g. breeding, moulting, or digesting) also impacts
the rate of water loss and the hydration state (e.g. Dupoué
etal. 2015a). During the reproductive season, this variation
is often closely related to sex and may generate sexual dif-
ferences in SEWL and hydration state. In particular, adult
females experience multiple changes specifically associated
with pregnancy or gravidity and involving their behaviour
(e.g. increased thermoregulatory precision; Lorioux etal.
2013; Shine 2006), physiology (e.g. higher metabolic rate;
Dupoué and Lourdais 2014; Schultz etal. 2008), and mor-
phology (e.g. greater physical burden; Miles etal. 2000; Le
Galliard etal. 2003). These changes can induce higher rates
of SEWL in adult females through increased rates of ventila-
tion and transpiration (Webster etal. 1985; Woods and Smith
2010; Dupoué etal. 2015b). Furthermore, offspring produc-
tion requires a considerable amount of water investment to
support vitellogenesis and/or embryonic development (Du
2004; Lourdais etal. 2015). Either of these two investments
may lead to a higher reliance of adult females on free stand-
ing water and water allocation trade-offs between mothers
and their offspring (Dupoué etal. 2015a). As a result, indi-
viduals may adjust their drinking behaviour to decrease the
level of risk induced by dehydration (Lourdais etal. 2015).
It is, however, still unclear if environmental conditions such
as air temperature, air humidity or the access to free standing
water could add with sexual differences and reproductive
state requirements in driving the regulation of water balance.
In this study, we examined geographic and sex-specific
sources of variation in water balance physiology in a wide-
spread, viviparous lizard (Z. vivipara) living in cool, wet
temperate environments. We sampled adult males and
females from 13 natural populations distributed across the
Massif Central mountain range in France. Sampling was
done during the same reproductive season at the end of the
mating period when females are undergoing pregnancy.
At the adult stage in this species, all females engage into
reproduction, and thereby we did not investigate the specific
cost of pregnancy. Instead, we checked how water balance
regulation may naturally differ between pregnant females
and males, and we further examined the influence of repro-
ductive stage and reproductive effort. In these populations,
rainfall intensity during the activity season has immediate
positive effects on offspring survival and delayed effects
on female reproductive performance (Marquis etal. 2008).
Past experiments on water restriction revealed that intense
water restriction during pregnancy can result in a dramatic
impairment of reproductive success (Dauphin-Villemant
and Xavier 1986). In contrast, limited restriction of water
availability leads to reduced activity and growth in yearlings
(Lorenzon etal. 1999) and has complex effects on reproduc-
tion in pregnant females (Lorenzon etal. 2001).
We focused on two functional traits related to water
balance, namely plasma osmolality (an indicator of hydra-
tion state in species lacking salt-glands; Peterson 2002)
and SEWL. We tested the influence of access to free water
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(availability of water in the environment), ambient tem-
perature, water vapour pressure in the air, and individual
reproductive investment on those physiological traits. It
is noteworthy that natural populations also differ in other
parameters including altitude and vegetation cover (Loren-
zon etal. 1999; Rutschmann etal. 2016), or slope orien-
tation and local wind speed conditions (pers. obs.), which
may influence water balance. However, we focused on the
environmental covariates that have previously been shown
to influence the regulation of water balance in laboratory
experiments. We hypothesized that lizards’ hydration state
and SEWL rate should differ according to environmental
conditions and physiological state (pregnancy). Specifically,
we tested three predictions. First, we expected pregnant
females to be more dehydrated than males due to a higher
rate of water loss during gestation (Webber etal. 2015; Dup-
oué etal. 2015b), and the investment of water into offspring
production (Dupoué etal. 2015a). Because of this, females
should also be more sensitive to environmental conditions
than males. Second, we predicted that lizards from sites with
lower access to free standing water should have lower SEWL
rates than lizard populations with greater water access to
maintain water homeostasis (i.e. similar plasma osmolality).
Third, because the rate of water loss directly positively cor-
relates with temperature and negatively with humidity (e.g.
Dupoué etal. 2015a), we predicted these effects to be miti-
gated so that lizards should have a similar hydration state;
that is, the SEWL rates would be lower in warmer climates
and lower in drier climate and osmolality would not cor-
relate either with temperature or humidity.
Materials andmethods
Study species, population descriptions, andcaptive
husbandry
The common lizard, Z. vivipara, is a small (adult snout–vent
length~50–75mm), widespread species in the family Lac-
ertidae that inhabits peat bogs and heathlands across north-
ern Eurasia. While the species has populations that are ovip-
arous and other populations that are viviparous, we limited
our study to 13 viviparous populations of the Massif Central
mountain range in south-central France. These populations
are located at the southern range limits for the viviparous
form of the species (Pilorge etal. 1983). Populations were
distributed along an elevational gradient and have differ-
ent water access and local climate conditions (TableS1, see
below). In these populations, males emerge in mid-April
while females emerge in early May. Males copulate with
females shortly after their emergence with fertilization
occurring in mid-late May. Pregnancy lasts 2–3months,
with parturition occurring between mid-July and early
August. Litter size varies from 1 to 12, and neonates do
not receive any post-natal parental care. After parturition,
females are lean and restore energy reserves before entering
into hibernation in late September.
At each locality, we recorded the presence or absence of
water sources available to the lizards (e.g. ponds, streams,
peat bogs), as well as temperature and humidity using two
temperature data loggers (Thermochron iButtons, Maxim
Integrated Products, Sunnyvale, CA, USA,±0.0625°C)
and one temperature–humidity data logger (Hygrochron
iButtons, Maxim Integrated Products, Sunnyvale, CA,
USA,±0.0625°C and 0.04% relative humidity—RH). Log-
gers were placed where we found most of lizards within
vegetation at ground level completely shaded to avoid the
effect of radiation. Because evaporative water loss depends
on water vapour density gradients (Mautz 1982), we used
water vapour density (in gm−3) as an index of “air humid-
ity” with the approximation of a stable barometric pressure
of 1013.25mbar (see details in Tieleman etal. 2002). Air
temperature and humidity were recorded every hour, and
we standardized the sampling period from 29th June to 17th
July to compare populations. These three weeks sampling
period was the best compromise we could achieve to charac-
terize accurately the differences in microclimatic conditions
during the active season among populations. Compared to
long-term meteorological data collected with nearby per-
manent stations that are difficult to extrapolate at high spa-
tial resolutions (Rutschmann etal. 2016), our data more
accurately reflect population characteristics and microcli-
matic conditions experiences by lizards. Over this sampling
period, we extracted the daily mean, minimum, and maxi-
mum temperatures (Tmean, Tmin, and Tmax, respectively) and
humidities (Hmean, Hmin, and Hmax) to assess the climate of
each population (TableS1).
We caught a total of 246 females
(mean ± SE, body mass (BM) = 4.84 ± 0.07 g,
snout–vent length (SVL)= 61.44±0.24 mm) and 135
males (BM= 3.47±0.06g, SVL=54.30±0.31mm)
between the 19th and 26th of June 2015. On the day of cap-
ture, lizards from 8 populations (males) and 12 populations
(females) were transferred to a field laboratory and housed
in individual terraria (18×12×12cm) with sterilized soil,
a shelter, and opportunities for thermoregulation to record
standardized water loss rates (TableS2, see below). During
captivity, we provided a 20–30°C thermal gradient for 6h
per day (09:00–12:00 and 14:00–17:00) using a 25W incan-
descent light bulb placed over one end of each terrarium. We
also provided water 3 times per day and fed lizards with 2
crickets (Acheta domesticus) every 2days. We recorded lit-
ter mass (i.e. the total mass of neonates) after parturition to
examine the influence of reproductive investment on female
water balance. Within 3days after parturition, we released
each female with her litter at her exact capture location. At
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the end of July, upon completion of experiments, we released
males at their exact capture locations.
Plasma osmolality
All lizards from all populations were bled in the field imme-
diately after capture (within 5min) using a standard proto-
col (Meylan etal. 2003). Blood samples (40–60µl whole
blood) were collected from the post-orbital sinus using 2–3
20µl microcapillary tubes. In the laboratory, blood sam-
ples were centrifuged for 5min (3000rpm), plasma was
separated from blood cells and kept frozen in airtight tubes
until used for subsequent analyses. Plasma osmolality was
then determined using a vapour pressure osmometer (model
5500, Wescor, Logan, UT, USA) and the protocol described
in Wright etal. (2013). Before analyses, plasma was diluted
(1:1) in reptile Ringer’s solution (300mOsmkg−1) prepared
following methods from Secor etal. (1994) so that plasma
osmolality could be determined from 10µl duplicates (intra-
individual variation: 3.9%). High osmolality values indicate
high dehydration.
Water loss estimations
On the day of capture, all lizards from a sub-set of 8 popu-
lations (males) and 12 populations (females, see TableS2)
were returned to the laboratory, weighed (BM1,±1mg) and
then maintained under constant temperature (23.5±0.1°C)
and humidity (14.6±0.1gm−3) without any access to water
or food. After 24h, all individuals were weighed again
(BM2,± 1mg), and we estimated the rate of water loss
(in mgh−1) using the loss of mass (BM2–BM1) over this
period. We used body mass loss as a proxy of total evapora-
tive water loss (i.e. the sum of ventilatory and cutaneous
evaporative water losses) because, in squamate reptiles,
variation in body mass is highly correlated with variation in
water loss (DeNardo etal. 2004; Moen etal. 2005; Dupoué
etal. 2015b). However, it is possible that the animals could
have lost some mass due to defecation and urination, and we
did not measure faeces and urine mass for technical reasons
related to husbandry priorities. The long period between the
two body mass measurements decreased the potential biases
related to small faeces production, since over 24h the loss of
body water are more likely to contribute to body mass loss.
Regardless, based on the previously reported mass of fae-
ces in this species (mean males: 37.9mg, females: 52.2mg;
González-Suárez etal. 2011), any defecation would have
represented a significant proportion of mass loss (males:
22.5%, females: 33.9%) and would have been detected. Thus,
upon reviewing the dataset, we excluded two females from
analyses because they showed extremely high mass losses
(655 and 1059mg) that are likely attributable to faeces or
egg loss.
Statistical analyses
All analyses were performed with R software (R Devel-
opment Core Team, version 3.2.0, http://cran.r-project.
org/). Initially, we used linear models to test the effects of
SVL, population, and sex and their interactions on plasma
osmolality and the rate of SEWL. Next, we investigated the
effect of water access and climatic conditions on plasma
osmolality and the rate of SEWL. We performed these lat-
ter analyses separately for each sex because females may
vary in rates of water loss due to the effects associated with
pregnancy and not to general sex-specific factors. We used
mixed-effects linear models (package nlme, Pinheiro etal.
2016) in which population identity was included as a random
factor to account for repeated measurements within the same
population. Water access was treated as a categorical factor
while temperature metrics (i.e. Tmean, Tmin and Tmax) and
humidity metrics (i.e. Hmean, Hmin and Hmax) were treated as
linear and quadratic covariates to test for non-linear relation-
ships. We centred covariates by subtracting the mean from
each observation. Furthermore, we also estimated embryonic
development (ED) to account for the pregnancy stage: ED
was estimated as the number of days between capture date
and parturition date. We tested the influence of two esti-
mates of reproductive effort: the absolute reproductive effort
(ARE; estimated as the mass of all neonates) and the rela-
tive reproductive effort (RRE, derived from the linear rela-
tionship between litter mass and female size, F1,169=89.5,
p< 0.001, r2=0.35). We only present results based on
ARE since they were similar to those obtained from RRE
analyses.
Whenever we found significant variation in water bal-
ance indicators between populations, we further checked the
potential correlation with population characteristics. To do
so, we used a model selection approach using the Akaike
information criterion corrected for small sample size (AICc,
package AICcmodavg, Mazerolle 2016). We compared the
contribution of each environmental and reproductive vari-
able to the model, as well as additive models of each envi-
ronmental and reproductive variable, to a model including
only the random effect of the population (i.e. null model).
The best model was chosen as the one with the lowest AICc.
Models that have a difference of AICc lower than 2 have
comparable support of the data. In our analyses, one or more
models had a ΔAICc that was less than 2 when compared
to the best model. In the latter cases, we focused on the
model with the lowest number of parameters (k) and tested
the significance of covariates with likelihood ratio tests
(LRT). We did not record humidity measures in three popu-
lations due to logger failure; therefore, we first restricted the
analyses to a dataset without those populations (TableS3).
However, since model selection did not retain the influence
of humidity (TableS3), we tested the influence of water
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access, temperature, and reproductive investment on the full
dataset. Results were similar for the full and the restricted
data set. Finally, we used linear models to test the correlation
between plasma osmolality and the rate of water loss within
and between populations (Speakman etal. 2003). Results are
presented as mean±SE unless otherwise stated.
Results
Variation inwater balance amongpopulations
andbetweenthe sexes
Plasma osmolality and the rate of SEWL were not sig-
nificantly influenced by lizard SVL (F1,363 = 2.17,
p=0.142 and F1,332=0.52, p=0.473, respectively) but
differed significantly among populations (F12,366=2.47,
p= 0.004, and F11,333=6.88, p<0.001, respectively).
Moreover, osmolality was different between sexes (males:
311.1±3.1mOsmkg−1, females: 301.5±2.1mOsmkg−1,
F1,366=6.61, p=0.011), while the rate of SEWL was sim-
ilar between sexes (males: 6.73±0.38mgh−1, females:
6.48±0.20mgh−1, F1,333=0.37, p=0.544). Both osmolal-
ity and the rate of SEWL were impacted by the interaction
between population and sex (F12,354=2.88, p<0.001, and
F7,272=2.56, p=0.014, respectively), which indicates sex-
specific geographic patterns of water balance physiology.
Influence ofenvironmental conditions andindividual
state
In males, the two best models retained mean temperature
as the primary environmental factor influencing plasma
osmolality (Table1). Models for water loss in males
included significant effects of water access (no access ver-
sus access: β=3.66±1.42, t6,71=2.57, p=0.042, Fig.1a,
c) and minimum temperature. That is, males from popula-
tions without water access had a rate of SEWL rates that
were only about half that of males from population with
access to water (no access: 4.85±1.13mgh−1, access:
8.50±0.86mgh−1, Fig.1c) while remaining in similar
hydration states (no access: 302.7±7.7mOsmkg−1, access:
315.1±5.1mOsmkg−1; β=12.37±9.22, t11,104=1.34,
p=0.207, Fig.1a). In addition, plasma osmolality was posi-
tively correlated with mean temperature (β=10.67±4.85,
t11,104=2.20, p=0.050, Fig.2a), whereas the rate of SEWL
tended to be higher for populations with higher minimum
temperature (β=1.21 ± 0.50, t6,71=2.42, p= 0.052,
Fig.2b).
In females, there was more uncertainty among statistical
models. The best model (Table1) did not retain any influ-
ence of environmental conditions or reproduction for plasma
osmolality, but included an effect of water access on the
rate of SEWL (no access versus access: β=1.39±0.62,
t10,228=2.24, p=0.048, Fig.1b, d). None of the remain-
ing top-ranking models included significant covariates.
Females from populations without access to water lost
almost 25% less water than did females from populations
with access to water (no access: 5.49±0.50mgh−1, access:
6.88±0.37mgh−1, Fig.1d) yet remained in similar hydra-
tion states (no access: 304.2± 5.7mOsmkg−1, access:
301.3±4.0mOsmkg−1, β=−2.87±6.92, t11,213=−0.41,
p= 0.686, Fig.1b). Model comparisons also retained a
slight non-linear relationship between the rate of SEWL
and the minimal temperature (water loss~ Tmin+T2
min:
β (Tmin) = 0.23 ± 0.23, t9,228 = 1.03, p = 0.328, β
(T2
min)=0.21±0.11, t9,228=1.85, p=0.098). We did not
find any influence of reproductive advancement (ED) or
reproductive investment (ARE, and RRE) on either plasma
osmolality (Fig.3a, b, c) or the rate of SEWL (Fig.3d, e,
f) (Table1).
Relationship betweenosmolality andSEWL
In both sexes, osmolality was not correlated with the rate
of SEWL either among or within populations (TableS4).
Discussion
In this study, we investigated variation in water balance (i.e.
hydration state and water loss) in wild populations of a wide-
spread lizard species (Z. vivipara) that differ in their access
to water and in local climate conditions. Males were more
dehydrated than females, whereas the rate of SEWL was
similar between sexes. In addition, the rate of SEWL was
higher in individuals from populations with access to water,
which is consistent with our second prediction. Finally and
contrary to our last prediction, plasma osmolality and the
rate of SEWL were positively correlated with environmen-
tal temperature in males, yet there was no correlation with
environmental humidity.
Sex differences in dehydration rate and/or the rate of
SEWL have been previously documented in other spe-
cies including humans (Stachenfeld etal. 2001; Cryan and
Wolf 2003; Weldon etal. 2013). In this study, we expected
pregnant females to be more dehydrated than males due to
physiological changes associated with pregnancy (Dupoué
etal. 2015a). In particular, pregnant or gravid females have
higher metabolic rates and higher transpiration rates caused
by body distension (Schultz etal. 2008; Dupoué and Lour-
dais 2014; Webber etal. 2015; Dupoué etal. 2015b), which
should increase the rate of SEWL (Mautz 1982; Woods and
Smith 2010). Furthermore, developing embryos also need
water for somatic growth (Du 2004; Lourdais etal. 2015),
and water allocation to embryonic development can impair
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Table 1 AICc-based model
selection comparing the
influence of water access,
climatic variables and
reproductive performance on
plasma osmolality and on the
rate of standardized evaporative
water loss (SEWL) in males
and pregnant female common
lizards (Z. vivipara)
Models are fitted on all sampled populations in which air humidity was not always available. Models were
built with each environmental covariate alone (linear or non-linear) and in addition with reproductive per-
formance in pregnant females. Population was treated as a random factor to account for intra-population
Physiological measure Sex Variable kAICc ΔAICc wiLog likelihood
Osmolality Males Tmean 4 1135.93 0.00 0.38 −563.79
Tmean+T2
mean 5 1137.80 1.87 0.15 −563.63
Null 3 1137.94 2.01 0.14 −565.87
Water access 4 1138.42 2.49 0.11 −565.03
Tmax 4 1139.06 3.13 0.08 −565.35
Tmax+T2
max 5 1139.82 3.89 0.05 −564.64
Tmin 4 1140.08 4.15 0.05 −565.86
Tmin+T2
min 5 1140.34 4.42 0.04 −564.90
Females Null 3 2241.33 0.00 0.19 −1117.61
ARE 4 2241.84 0.52 0.14 −1116.83
Tmin 4 2242.39 1.07 0.11 −1117.11
Tmean 4 2242.89 1.56 0.08 −1117.35
Water access 4 2243.23 1.90 0.07 −1117.52
ED 4 2243.32 2.00 0.07 −1117.57
Tmax 4 2243.39 2.07 0.07 −1117.61
Tmin+T2
min 5 2244.44 3.11 0.04 −1117.08
ED+Tmin 5 2244.45 3.13 0.04 −1117.09
ED+Tmin+ARE 6 2244.55 3.23 0.04 −1116.08
Tmean+T2
mean 5 2244.96 3.63 0.03 −1117.34
ED+Tmean 5 2244.98 3.66 0.03 −1117.35
ED+Tmean+ARE 6 2245.16 3.84 0.03 −1116.39
Tmax+T2
max 5 2245.18 3.86 0.03 −1117.46
ED+Tmax 5 2245.41 4.08 0.02 −1117.57
ED+Tmax+ARE 6 2246.04 4.71 0.02 −1116.83
SEWL Males Water access 4 431.38 0.00 0.31 −211.42
Tmin 4 431.82 0.45 0.25 −211.64
Tmax 4 433.27 1.90 0.12 −212.37
Tmin+T2
min 5 433.46 2.08 0.11 −211.32
Null 3 434.11 2.73 0.08 −213.89
Tmax+T2
max 5 434.41 3.03 0.07 −211.79
Tmean 4 435.22 3.84 0.05 −213.34
Tmean+T2
mean 5 437.42 6.04 0.02 −213.30
Females Water access 4 1208.61 0.00 0.22 −600.22
Tmin+Tmin
25 1208.94 0.33 0.18 −599.34
Tmin 4 1209.87 1.26 0.12 −600.85
ED+Tmin 5 1210.00 1.39 0.11 −599.87
Null 3 1210.69 2.07 0.08 −602.29
ED 4 1211.54 2.93 0.05 −601.68
Tmax 4 1211.59 2.98 0.05 −601.71
ED+Tmin+ARE 6 1211.94 3.33 0.04 −599.79
ED+Tmax 5 1212.26 3.65 0.04 −601.00
ARE 4 1212.71 4.10 0.03 −602.27
Tmean 4 1212.75 4.14 0.03 −602.29
Tmax+T2
max 5 1213.54 4.93 0.02 −601.64
ED+Tmean 5 1213.57 4.96 0.02 −601.66
ED+Tmax+ARE 6 1214.33 5.72 0.01 −600.98
Tmean+T2
mean 5 1214.84 6.23 0.01 −602.29
ED+Tmean+ARE 6 1215.62 7.01 0.01 −601.63
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female hydration state (Dupoué etal. 2015a). Yet, we found
no difference in mean SEWL between sexes and males
were slightly more dehydrated on average than females.
This indicates that factors other than breeding state per se
may be responsible for the observed sexual differences in
water balance. Indeed, despite higher water demands caused
by pregnancy in females compared to males, behavioural
factors may contribute to buffer sexual differences in field
evaporative water loss or water balance. For instance, preg-
nant females of Z. vivipara select lower temperature and stay
relatively inactive in the field (Van Damme etal. 1986; Le
Galliard etal. 2003). These differences in thermoregulation
and activity might reduce water loss and thus dehydration in
females. Besides, the regulation of hydration state can also
be adjusted by water intake. Pregnancy may be associated
to an increase drinking behaviour caused by a decrease in
the osmotic threshold of thirst (Cheung and Lafayette 2013;
Lourdais etal. 2015). Together, these behavioural adjust-
ments of thermoregulation and water intake might be par-
ticularly relevant to better understand the functional regula-
tion of the water balance.
When investigating the effects of water access, we found
that individuals from habitats with permanent access to
water had, in general, higher SEWL rates compared to liz-
ards from populations without access to water. Interestingly,
hydration state was not different between those populations
suggesting that a lower rate of water loss might compensate
for lower water availability to maintain hydration state, and
therefore, physiological homeostasis. That is, individuals
from water-restricted populations may remain normosmotic
by having a lower rate of water loss, either via acclimation
or genetic adaptations to the drier environment, for example
through reduced ventilatory rate or reduced peripheral perfu-
sion (Tieleman etal. 2003; Williams etal. 2004; Moen etal.
2005; Van Sant etal. 2012; Guillon etal. 2014; Cox and Cox
2015; Belasen etal. 2016). Yet, contrary to our last set of
predictions, we found that water balance indicators did not
correlate with air humidity. This suggests that the access to
free-standing water is a better descriptor of the rate of water
loss than environmental humidity, which was relatively high
in sampled areas of dense vegetation used by lizards in all
populations.
variation and non-independence. See text for details
k number of parameters, ΔAICc difference with AICc of the best model, wi model likelihood
Boldfaced characters are included for significant variables according to LRT tests
Table 1 (continued)
Fig. 1 Effects of the access
to water in natural popula-
tions of common lizards on the
indicators of water balance a, b
plasma osmolality, and c, d the
rate of standardized evapora-
tive water loss (SEWL) in
males (left panel) and females
(right panels). Points represent
mean±SE and significant
effects of water access are
symbolised: *p<0.05, n.s. non-
significant
Osmolality
(
mOsm.kg
−1)
290 300 310 320
an.s. bn.s.
SEWL
(
mg.h
−1)
No water access Water access
46810
c*
No water access Water access
d*
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In addition, the SEWL rate was slightly and positively
correlated with environmental temperature in males,
whereas it tended to increase non-linearly with tempera-
ture in females. Our best statistical models for females
included correlations with minimum daily temperature
instead of mean or maximum values. Although the AICc
suggested some uncertainty among the best statistical mod-
els, and therefore, point to the need of further studies with
a larger sample size, this result may reflect the ecological
relevance of minimal temperatures for ectotherms. Dur-
ing summer season, lizards are exposed to minimal tem-
peratures measured on the ground since they stay inac-
tive in shelter very close from the surface such as inside
grass tufts, shallow crevices in soil and rocks, and dead
trunk cavities (pers. obs.). Therefore, lizards must endure
minimal conditions for a relatively long period inside their
night shelters, which are likely closed from surface (e.g.
Tmin>4h,~03:00–08:00h). Instead, maximal conditions
experienced during activity, daytime last for a shorter dura-
tion (e.g. Tmax<1h,~15:00h) and can be avoided through
microhabitat selection (Davis and DeNardo 2009; Guillon
etal. 2014). Rate of water loss increases with temperature
due to lower skin resistance and higher metabolic rate of
reptiles at higher body temperatures (Webster etal. 1985;
Lillywhite 2006; Dupoué etal. 2015b). To buffer this bio-
physical relationship, we would have expected a negative
correlation between the rate of SEWL and environmental
temperature, so that osmolality would have not correlated
with temperature. Instead, we observed a positive correla-
tion between water loss and air temperature and between
osmolality and temperature in males.
We hypothesize the geographic variation in water access
and temperature was due to permanent and consistent dif-
ferences among populations related to altitude, slope orien-
tation and habitat type (pers. obs.). Thus, we propose that
geographic differences in the rate of SEWL may reflect local
acclimation and/or adaptations to prevailing environmen-
tal conditions. For example, natural selection related to the
water balance could favour plastic and/or genetic changes
in the properties of the skin barrier, likely resulting from
changes in lipid composition, organization and/or mobiliza-
tion among populations (Kattan and Lillywhite 1989; Lil-
lywhite 2006). In uricotelic species (e.g., squamate reptiles
and birds), transcutaneous water loss is the main avenue for
water loss (Kattan and Lillywhite 1989; Lillywhite 2006;
Williams etal. 2012). The keratin–lipids complex (sand-
wich-like layers) localized in the stratum corneum (i.e. the
outer layer of the epidermis) constitute the main barrier lim-
iting transcutaneous water loss (Bouwstra etal. 2003; Lil-
lywhite 2006; Champagne etal. 2012). The permeability of
this water barrier can be adjusted by modifying its thickness
(quantity of lipids), the proportion of the different lipids with
specific polarity (e.g. cholesterol, fatty acids, phospholipids
and ceramides), and/or their geometry (Lillywhite 2006;
Williams etal. 2012). Further investigations are needed to
quantify the contribution of geographic differences in skin
water permeability and distinguish whether these differences
are caused by plastic responses or genetic adaptations to
short- or long-term exposure to climatic conditions.
Although comparative studies cannot determine the
causes of relationships, ecological comparisons across
geographical distributions such as this one provide use-
ful opportunities to understand and predict how spe-
cies may respond to climatic conditions (Somero 2011;
Rezende and Diniz-Filho 2012). Water is an essential yet
relatively overlooked resource in ecological studies. As
demonstrated here, water balance may be affected by local
climate conditions and water availability, and therefore,
geographic variation in the water balance strategies should
be integrated into global change studies (Todgham and
Tmean (°C)
Osmolality (mOsm.kg−1)
a
18.2 18.8 19.4 20 20.6
200 250 300 350 400
T
min
(°C)
SEWL (mg.h−1)
b
8.5 9.5 10.5 11.5 12.5
0510 15
Fig. 2 Positive relationships between indicators of water balance and
thermal conditions in males. Trend lines are included for significant
(solid line) or marginal (dashed line) correlations between a plasma
osmolality and mean temperature (p = 0.050, r2=0.10) and b the
rate of standardized evaporative water loss (SEWL) and minimal tem-
perature (p=0.052, r2=0.25)
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Stillman 2013). For instance, further studies should spe-
cifically examine the relative contributions of physiologi-
cal adaptation, acclimation, and behavioural mitigation in
adjusting the rate of water loss to minimize dehydration.
Such information is essential to improve the accuracy of
models that predict species’ responses to climate change
and to promote effective conservation measures (Wikelski
and Cooke 2006; Cooke etal. 2013).
Acknowledgements We thank Pauline Blaimont, Pauline Dufour,
Laurène Duhalde, Amélie Faure, Julia Rense, and Qiang Wu for their
help with fieldwork. We also thank Clotilde Biard for lending us some
of the loggers. We are grateful to the ‘Office Nationale des Forêts’,
the ‘Parc National des Cévennes’, and the regions Auvergne, Rhône
Alpes and Languedoc Roussillon for allowing us to sample lizards.
This study was funded by the Centre National de la Recherche Sci-
entifique (CNRS) the Agence Nationale de la Recherche (ANR-13-
JSV7-0011-01 to SM) and the National Science Foundation (NSF-
EF1241848 to DBM).
Author contribution statement AD, AR, JFLG, DBM, JC, and SM
conceived the ideas and designed methodology; AD, AR, JFLG, DBM,
JC, and SM captured lizards; AD and AR collected water loss data;
AD, GAB, and DD collected osmolality data; AD analysed the data;
AD and AR led the writing of the manuscript. All authors contributed
critically to the drafts and gave final approval for publication.
Compliance with ethical standards
Conflict of interest The authors declare no competing or financial
interests.
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