Available via license: CC BY 4.0
Content may be subject to copyright.
An Acad Bras Cienc (2022) 94(3): e20210758 DOI 10.1590/0001-3765202220210758
Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences
Printed ISSN 0001-3765 I Online ISSN 1678-2690
www.scielo.br/aabc | www.fb.com/aabcjournal
An Acad Bras Cienc (2022) 94(3)
Running title: WINTERTIME
TALES: Liolaemus lineomaculatus
Academy Section: ANIMAL
SCIENCE
e20210758
2022
94
(3)
94(3)
DOI
10.1590/0001-3765202220210758
ANIMAL SCIENCE
Wintertime tales: How the lizard Liolaemus
lineomaculatus endures the temperate
cold climate of Patagonia, Argentina
NICOLÁS R. CECCHETTO, SUSANA M. MEDINA, FLORENCIA BAUDINO & NORA R.
IBARGÜENGOYTÍA
Abstract: In temperate, polar and montane environments, ectotherms must fi nd ways to
endure throughout the coldest months of the year. Lizards search for microsites where
temperatures remain warm or alter their biochemical balance to tolerate freezing or
avoid it by supercooling. We evaluated the cold hardiness and potential winter refuges
of two populations of Liolaemus lineomaculatus, from a temperate site (42°S) and a
cold site (50°S). We analysed the role of possible cryoprotectants by comparing a group
of cooled-down lizards with a control group of lizards that were not exposed to cold.
The populations of this study are not freeze tolerant and the biochemical analysis
showed no evidence of metabolites signifi cantly changing concentration after exposure
to cold. However, the species remained several hours at their Supercooling Point (SCP),
suggesting they can supercool. The analysis of potential winter refuges showed that
lizards using these potential refuges would spend almost no time at all at temperatures
close to or below their SCP. Furthermore, lizards from the cold site were able to survive
below 0°C temperatures with a lower SCP than lizards from the temperate site. Liolaemus
lineomaculatus developed physiological mechanisms that can help them survive when
temperatures drop sharply, even when lizards are in suitable shelters.
Key words: Cold hardiness, cryoprotectants, Liolaemus lineomaculatus, Patagonia, su-
percooling point, winter refuges.
INTRODUCTION
In temperate and cold habitats, ectotherms
such as lizards must spend at least half of their
lives coping with the challenges related to sub-
zero environmental temperatures and stressors
associated with overwintering (Williams et al.
2015). Even when environmental temperatures are
above 0°C, cold weather can still have a negative
effect on activity thresholds. Temperatures
below the Critical Thermal Minimum (CTMin)
(sensu Cowles & Bogert 1944) render the
animals unable to escape predators (Christian
& Tracy 1981) or forage to obtain resources for
overwintering, changing the dynamics of energy
storage (Tattersall et al. 2012).
Furthermore, in these harsh environments,
temperatures frequently reach negative
values and, when behavioural options (such
as burrowing) are insufficient, lizards can
respond by adopting one of two physiological
mechanisms: freeze tolerance or freeze
avoidance by supercooling. Freeze tolerance
is a mechanism where the lizard tolerates the
partial conversion of body fl uids into ice for a
variable amount of time, with high variation
among species and populations in the
resistance to a different percentage of frozen
body fluids, time frozen, and the number of
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 2 | 19
freezing and thawing episodes individuals can
tolerate (Voituron et al. 2002, Berman et al. 2016).
Meanwhile, by supercooling, the individual
“can remain unfrozen at temperatures below
the equilibrium crystallization temperature
of its body fluids” (Costanzo et al. 1995). This
mechanism involves less physiological stress
(Costanzo et al. 2008), but there is a risk of
spontaneous freezing at temperatures below
the equilibrium freezing point (Salt 1966),
with potentially lethal consequences (Storey
& Storey 1996). Despite their differences, both
freeze tolerance and freeze avoidance require
a stable temperature to improve the chances of
survival of the overwintering individuals (Pauli
et al. 2013). Moreover, these mechanisms involve
biochemical variables such as urea, glucose, and
lactate changing concentrations and increasing
osmolality (Costanzo et al. 2000, Grenot et al.
2000, Voituron et al. 2002), and the synthesis of
Anti-Freeze Proteins (AFPs), that help to avoid
freezing and recrystallization in supercooling
and freeze tolerance, respectively (Storey &
Storey 1986).
Populations of the same species living
in environments with different climates may
develop different cold hardiness capabilities,
even if the geographical separation (such as in
latitude or elevation) is not large; however, there
is not a clear pattern or correlation. For example,
the CTMin of a South American gecko (Homonota
darwinii) showed changes among populations
in correlation with cooler climates, although no
pattern was found regarding latitude (Weeks
& Espinoza 2013). Additionally, some studies
show an effect of latitude or elevation in cold
hardiness parameters of terrestrial ectotherms
such as CTMin (Sunday et al. 2011, Munoz et al. 2014,
Huang et al. 2006, Winne & Keck 2005). However,
there are also studies showing interpopulation
differences in cold hardiness capabilities that
could not be explained by winter severity
(Michels-Boyce & Zani 2015), differences that are
better explained by other factors (Voituron et
al. 2004, Costanzo et al. 2006, 2004, Spellerberg
1972), or even no interpopulation differences at
all (Gvozdík & Castilla 2001, Yang et al. 2008).
Nevertheless, climatic differences at the
landscape scale among populations may not be
representing accurately what lizards experience
in the microsite where they choose to spend the
winter.
Microsite selection is of paramount
importance in winter survival. Overwintering
animals heavily rely on thermally stable
structures that protect them from predators,
extreme weather variations, and other
disturbances (Williams et al. 2015, Kinlaw 1999,
Huey 1991). Furthermore, refuge availability
can have a larger impact on overwintering
than the thermal quality of the habitat as
a whole (Monasterio et al. 2009). A recent
potential refuge analysis showed that choosing
appropriate refuges might allow the lizard
Liolaemus pictus in the high elevation forest in
the north of Patagonia, Argentina, to endure the
cold environmental conditions without resorting
to physiological mechanisms such as freeze
tolerance or supercooling (Cecchetto et al. 2019).
Thus, unless lizards find a suitable winter refuge,
they would experience sub-zero environmental
temperatures during extended periods in the
steppes at the highlands and high latitudes of
Patagonia, Argentina, under a snowpack that
reaches a considerable depth (>1m).
In this study, we analysed the cold hardiness
by physiological and behavioural mechanisms
of a lizard, Liolaemus lineomaculatus
(Liolaemidae), a viviparous species with a broad
distribution from the high Andes in the north-
west of Patagonia, in Neuquén province (39°S),
at elevations up to 1800 m asl, to the lowlands
in Santa Cruz province (400 m asl 51°S; Cei 1988,
Scolaro 2005).
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 3 | 19
We propose that L. lineomaculatus,
living at higher latitude and elevation than L.
pictus, must have developed a cold hardiness
mechanism such as supercooling or freeze
tolerance to survive the coldest months of the
year in the steppes near the cities of Calafate
and Esquel. Additionally, we predict that after
experimental exposition to cold, individuals
of L. lineomaculatus will show a significant
increase in the concentration of at least one
of the selected biochemical variables (urea,
total proteins, glucose, lactate), previously
identified as cryoprotectants in other lizard
species (Costanzo et al. 2000, Grenot et al. 2000,
Voituron et al. 2002). Moreover, we also expect
to find differences between L. lineomaculatus
populations in the minimum temperatures
experienced throughout the year and in the
amounts of hours at sub-zero temperatures
within potential winter refuges or at surface
level. Furthermore, we hypothesize that these
two populations must have diverged in their
cold-hardiness capacities, varying with the
temperatures of the environment and thermal
quality of available refuges. From this hypothesis,
we predict that the L. lineomaculatus population
in the colder environment (Calafate) will show
a lower CTMin, a lower supercooling point, or
both, than the population located in the milder
environment in Esquel.
Studies that integrate the physiological,
behavioural, and ecological responses related to
winter survival with the availability of potential
overwintering microsites in populations located
at different latitudes and elevations are relevant
to understand underlying processes of cold
hardiness, especially given the lack of studies
on this subject for species in the Southern
Hemisphere. While our previous work (Cecchetto
et al. 2019) focused on a single population of L.
pictus that showed mild cold hardiness, in this
study, we evaluate intraspecific differences in the
restraints and opportunities for two populations
located at the latitudinal and altitudinal extreme
of the distribution of one of the southernmost
species of Patagonian lizards.
MATERIALS AND METHODS
Study areas and capture methods
We captured adult males of L. lineomaculatus in
the steppes of Calafate, the cold site (50°15´ S,
71°29´ W; 450 m asl; February 2018, N= 20), and
on a mountain in Esquel, the temperate site (42°
49´ S, 71° 15´ W; 1800 m asl; March 2019; N=19),
in Argentina. Captures were made between the
end of summer and the beginning of autumn
considering that, at the selected locations, it is
a period of the year when air temperatures can
rapidly change and result in temperatures that
are close or below CTMin (Supplementary Material
- Figure S1). In addition, the carbohydrates used
as cryoprotectants by terrestrial animals are
synthesized almost exclusively from reserves
obtained during late summer and early autumn
feeding (Storey 1997). Therefore, captures
were made in the limit of the brumation of L.
lineomaculatus, which in the steppes at high
latitudes and elevations starts in mid-autumn
(May), and lasts until spring (September; Medina
et al. 2011).
In the steppes of Calafate, the typical terrain
is a plain, open field with frequent bushes
and tussocks, but almost no boulders or rocks
for lizards to hide under. In the high-Andean
steppes of Esquel, L. lineomaculatus can find
refuge under boulders, bushes, tussocks or in
the many abandoned burrows of small mammals
(such as rodents from the genus Ctenomys). In
a recent study we found that in Esquel, lizards
spent the majority (95%) of their hours of
activity in autumn, spring and the beginning of
summer within their thermal tolerance breadth
(i.e., at temperatures between their CTMin and
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 4 | 19
their CTMax), while in Calafate, during the same
months, lizards spent only 71% within their
thermal tolerance breadth (Cecchetto et al. 2020).
Lizards were captured by hand or loop, and we
measured body temperature (Tb) immediately
after capture, using a digital thermometer (±
0.1°C; Omega 871A, type K 9 thermocouple;
Stanford, CT) connected to a catheter probe
introduced about 1 cm inside the cloaca. We
handled individuals by the head and hips within
10 seconds of capture to avoid heat transfer.
Potential lizard refuges
To understand the challenges that the highlands
and tablelands of Patagonia represent for
the studied populations in the potential
refuges lizards use during the colder months,
we placed four lizard models in Calafate and
six lizard models in Esquel with thermistors,
connected to data loggers (HOBO TEMP®
H8, four-channel external data logger and its
thermistors) between March 2017 and January
2018. Temperature values were recorded for
these 11 months every 30 minutes. The models
were made of PVC pipes (1.5 cm diameter × 8 cm
length) which were then sealed at the ends with
silicone (Fastix®) and painted grey to mimic body
size, reflectance, thermodynamics, and shape
of lizard’s bodies. To determine if the model
was a good indicator of the temperature that a
non-thermoregulatory lizard would attain in the
environment or if corrections were needed, we
performed simultaneous trials for calibration in
two identical terraria using the PVC model in one
terrarium and a live lizard on the other. During
the trials, we moved the PVC model to mimic
the movements of the live lizard. Subsequently,
we regressed model temperature on lizard
body temperatures (Tb= 2.82 + 0.912 × Physical
model. Regression: Adjusted R2= 0.92; n= 2510;
Confidence Interval= 0.88 - 0.94), and amended
the values accordingly.
Following the calibration, we placed two
models (one at each location) on the ground,
partially covered but exposed to environmental
temperatures, as a reference point representing
temperatures typically experienced just outside
any type of refuge. We then selected the
potential refuges in which the species might
seek temporary shelter, to include the variety
of microenvironments at both sites (e.g., buried
~10-15 cm underground; beneath rocks; under
tussocks) and placed additional PVC models in
the potential refuges. These potential refuges
are speculative predictions of where lizards may
choose to spend the winter, based on what they
had available in the environment and burrows
used by them during activity season (from
September to March, when not in brumation,
personal observation). The natural history
information in the literature of species of similar
size with similar thermal ecology (Zootoca
vivipara, under a boulder -Fellenberg 1983-; or
buried 5-15 cm in the ground / under vegetation
-Berman et al. 2016-) was also considered. For
the potential refuges, we recorded the number
of periods or events when the temperature
dropped below 0°C and the duration of each
period (i.e., time until temperature raised again
above 0°C). In this way, if a potential refuge
spent 5 hours above 0°C, 2 hours at negative
temperature values and 3 hours later above
0°C, this would be registered as a single period
below 0°C that lasted for 2 hours.
In addition, to compare the “thermal quality”
of potential refuges, we applied the concept of
degree-days (sensu Lindsey & Newman 1956),
using as reference the value of 0°C (the melting
point of water at 1.01325 x 105 Pa). Degree-days
are the summation of temperature differences to
a reference value over time. In this way, degree-
days explain both the magnitude and duration
that lizards would experience temperatures
in relation to a reference chosen value. This
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 5 | 19
metric allows a direct comparison of thermal
regimes among different sites for many species
or species populations (Guisan & Hofer 2003,
Schwanz & Janzen 2008, Murphy et al. 2010,
Boyero et al. 2011, Graae et al. 2012, Mitchell et
al. 2012).
This reference value of 0°C allows
inferences about how much and for how long
L. lineomaculatus (that overwintered in the
selected potential refuges or in no shelter; i.e.,
surface-level model) would be subjected to
temperatures below the melting point of water.
We used degree-days to compare potential
refuges, and it was calculated as:
where HRDD0 is heating refuge degree-day
for 0°C, and Ti refers to registered temperature
values below 0°C (every 30 minutes).
Laboratory experiments and housing
conditions
We brought the lizards (N Calafate= 25 + N Esquel= 25)
to the laboratory where we measured snout-
vent length (SVL) and body mass (Table I) using
a digital calliper (± 0.02 mm) and an Ohaus
balance Scot Pro (± 0.01 g), respectively. Lizards
Table I. Descriptive data (Mean ± SD) of Scaled Mass Index (SMI) and critical minimum temperature (CTMin, °C) of
Liolaemus lineomaculatus from Calafate and Esquel. The hyphen symbol in CTMin (-), corresponds to absent data
(control individuals).
Calafate Esquel
CTMin (°C) Scaled Mass Index CTMin (°C) Scaled Mass Index
- 4.75 - 3.93
- 4.66 - 3.87
- 4.85 - 3.78
- 5.74 - 3.83
- 4.74 - 3.49
- 4.29 4.75 4.32
-4.93 4.54 3.39
- 4.40 6.21 3.61
- 4.83 5.22 4.32
- 4.75 5.44 4.41
5.79 4.08 5.87 4.61
4.86 5.21 4.19 4.28
2.23 4.62 5.27 3.82
4.17 5.61 5.58 4.05
3.81 4.55 5.19 3.00
5.86 4.66 5.11 3.86
5.36 4.93 3.97 5.59
2.77 5.16 4.83 3.90
3.09 5.11 5.59 3.77
5.01 4.79 4.81 3.68
Means (± SD) Means (± SD)
4.30 (1.29) 4.83 (0.4) 5.10 (0.61) 3.98 (0.54)
The hyphen symbol in CTMin (-), correspond to control individuals.
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 6 | 19
were brought to the laboratory in individual
cloth bags to minimize stress and were housed
in individual open-top terraria (15 × 20 × 20 cm)
at room temperature (day maximum ~20°C, night
minimum ~10°C) for a maximum of 48 hours
before the experiments, with a photophase of
approximately 12 hours. Within these terraria,
lizards were supplied with a refuge (a cardboard
cylinder ~10 cm length x 5 cm diameter). Lizards
were provided with water ad libitum, except
for 5 hours just prior to the experiments, to
avoid getting moisture on their body, which
could freeze at negative temperatures, risking
unwanted ice inoculation and freezing (to
further avoid this situation, we manually blotted
their skin dry with paper towels).
Supercooling point (SCP) determination
SCP was determined to evaluate if lizards
from these populations are freeze tolerant.
Additionally, this experiment also provided
the supercooling point, understood as the
lowest temperature before a peak, indicating
the release of the latent heat of crystallization
(Costanzo et al. 2008).
Two small subsets of animals, one for each
population, were selected (NCalafate= 5; NEsquel= 6),
and SCPs were determined. We placed lizards
individually in dry plastic containers, positioned
them in a freezer at 18°C for 30 minutes (until
thermal stability was reached). We connected
the lizards to a TC-08 Data Acquisition Module
Omegas (8-Channel USB Thermocouple, ± 0.01°C)
by ultra-thin (1 mm) catheter thermocouples,
to register the body temperature and identify
the exothermic reaction of body-water freezing.
These thermocouples were fixed on the abdomen
and not inside the cloaca since thermocouples
placed in the cloaca at temperatures below
0°C can initiate unwanted freezing (Costanzo
et al. 2008). SCP determination consisted of
four stages. 1) lizards were cooled from 18°C
to 0°C at a stable rate of -0.5°C*hour-1 for 36
hours. 2) lizards continued cooling at a rate of
-0.25°C*hour-1 until an exothermic reaction was
reached (i.e., all lizards underwent crystallization
of body water). 3) the lowest temperature (i.e.,
the temperature of the last freezing exotherm)
was maintained for 12 hours to ensure full body
freezing. 4) finally, lizards were slowly thawed at
a rate of 2.5°C*hour-1 until they reached at least
the population’s CTMin and were taken out of
the freezer and their vital signs (i.e., breathing,
movement, righting response) were checked.
For these experiments and the following
cooling experiments, we used the lowest
possible rates in relation to what a lizard would
likely experience in the field (data from the lizard
PVC models) to minimize any harmful effect on
tissues from cooling too fast, but avoiding rates
slower than actual rates experienced in refugia.
Biochemical cooling experiments
The cooled-down group (NCalafate= 10; NEsquel= 10)
was placed individually in dry plastic containers
positioned in a freezer, while a control group
(NCalafate= 10; NEsquel= 9) was placed simultaneously
in the same conditions at room temperature
(20°C). Temperatures of the cooled-down group
were regulated by a control in the freezer that
allowed setting specific cooling rates and
times. We connected the lizards to a TC-08 Data
Acquisition Module Omegas (8-Channel USB
Thermocouple, ± 0.01°C) by ultra-thin (1 mm)
catheter thermocouples, to register the body
temperature in both groups during the cooling
experiment. A PVC pipe lizard model was set in
another plastic container and exposed to the
same temperature fluctuations as the lizards
from the cooled-down group.
We performed the experiment in three
stages, considering that lizards are normally
exposed to air temperature fluctuations with
smooth drops and extended periods hovering
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 7 | 19
near 0°C in this season, as seen in Zootoca
vivipara (Grenot et al. 2000, Costanzo et al. 2008).
In the fi rst stage (From 20°C to 0°C), we exposed
individuals to cold from the experimental
starting temperature (20°C) to 0°C for 6 hours,
at a rate of -3°C*hour-1. In the second stage
(Overnight), lizards stayed at approximately 0°C
for 12h. Finally, in the third stage (Below 0°C),
we dropped the freezer’s temperature at a rate
of approximately -0.75°C*hour-1 to ~-8°C for the
Calafate individuals and to ~-6°C for the Esquel
individuals. The fi nal values for this stage were
at fi rst chosen by observing the lowest value
obtained for each population in the analysis of
the environmental temperatures. However, since
values were below both populations’ SCP and
would have frozen lizards (and, given that they
were not able to survive freezing, most likely
killed them), we selected the closest value to
the SCP that the freezer could achieve (-8°C
and -6°C, respectively) and then maintained
that temperature for 6 hours. Lizards were
then warmed slowly at room temperature and
examined for biochemical changes.
When computing cooling rates for analysis,
in addition to raw temperature values, we used
an Adjusted Body Temperature (ATb) index to
standardize the temperature change, considering
that initial temperature values slightly varied
among individuals. This index illustrates the
temperature change independently from initial
values as follows:
where Tb is the body temperature at a given
time, and Tbi is the body temperature at the
beginning of the experiment.
At the beginning of the experiments, we
monitored lizards to determine the critical
minimum temperature (CTMin; Table I), defi ned
as the temperature at the lower extreme of
tolerance at which the animal cannot right
itself when placed on its back (i.e., the loss
of righting response sensu Doughty 1994). We
evaluated CTMin by quickly taking lizards out of
the containers and placing them on their back
as soon as they started reaching values of ~10°C.
If the animal was able to right itself, it was
placed back to continue cooling and the process
was repeated every 30-60 seconds or every
degree below the previous value, whichever
happened fi rst until the CTMin value was found.
To control for a potential effect of the handling
on the individuals, such as a release of glucose
caused by a sympathetic response, we handled
all individuals in the control group in the same
way as those in the treatment group.
At the end of the experiments, immediately
after the extraction of individuals from the
containers, we sacrifi ced lizards by decapitation,
and then, liver and heart samples of each
individual were individually stored in Eppendorf
tubes and kept in a freezer until they were
analysed the next day.
Milder complementary experiment for the
temperate site individuals
Lizards from the cooled-down group from the
temperate site, Esquel, were found dead after
the experiment, presumably from cold exposure
and not being able to supercool. Therefore, the
control group was divided in two (N cooled down=
6; N control= 3) and the experiment was repeated
using the same protocols but in the fi nal stage
(Below 0°C) we used a value 0.5°C higher than
the lowest SCP detected for this population
(-4°C, since the freezer’s controller panel,
didn’t allow for non-integer values) and the
temperature was maintained for less time (3
hours instead of 6), to ensure the survival of the
individuals (Table SII). It should be noted that,
given that these individuals were controls for
the previous experiment, they underwent sub-
zero temperatures only once.
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 8 | 19
Biochemical analysis
We analysed a liver and a heart sample per
individual. We homogenized each sample (liver
and heart separately) manually with a mortar,
diluted it with physiological saline (9% V/V) in
a 1:4 dilution, and then placed all samples in
Eppendorf tubes to be centrifuged at 3200 rpm
for 10 min. The material in each tube underwent
absorption spectroscopy with enzymatic assays,
to detect: urea, total proteins, and albumin.
We adapted the methodology implemented
in this study and made the selection of
the cryoprotectants analysed considering
biochemical variables found relevant in
previous studies of cold hardening in reptiles
(Costanzo et al. 2000, Grenot et al. 2000, Voituron
et al. 2002); for urea, glucose, and Anti-Freeze
Proteins (AFPs), respectively and in other taxa
(Storey & Storey 1986; for AFPs). We inferred the
presence of antifreeze proteins considering the
differences between total proteins and albumin
in the homogenate taking into account that an
increase in total proteins without a corresponding
increase in albumin would point to proteins
related to the cooling experiment (although not
necessarily AFPs). We determined all parameters
for the supernatant using a Shimadzu UV-1800
spectrometer (Shimadzu Inc., Kyoto, Japan) with
an absorption spectroscopy test with enzymatic
assays and chemical reagents (Wiener Lab,
Rosario, Argentina). The kits used were kinetic
urea UV AA, total proteins AA and albumin AA.
We previously reprogrammed the biochemical
kits methods in relation to proportions and
calibration values, to include sample values into
the standard calibration curve and to obtain
reliable results.
Given the lack of evidence for signifi cant
changes in the selected biochemical
components after the experiment from Calafate
and from a previous experiment with Liolaemus
pictus (Cecchetto et al. 2019), analyses for Esquel
individuals were focused only on glucose and
lactate, which could be obtained from a drop of
blood only.
Blood glucose and lactate
We measured glucose by taking a drop of blood
from the caudal vein near the cloaca, avoiding
the hemipenes, before and after the experiment,
using a glucometer (Accu-Chek® Performa Nano,
with a range of 10 mg/dL - 600 mg/dL) following
the methodology of Voituron et al. (2002).
We calculated the proportional change in
glucose or adjusted glucose change (ΔAGluc)
to account for the difference in glucose initial
values, given their uneven diet coming from the
fi eld, using the following formula:
Where Glucf was the glucose at the end of
the experiment and Gluci was the glucose at
the beginning. Initial and final glucose were
not analysed separately because the change in
glucose was already analysed as ΔAGluc. ΔAGluc
analyses the difference between initial and fi nal
glucose, accounting for individual differences in
initial values, which is why we found ΔAGluc as a
more relevant variable for this study.
We measured lactate by using another drop
of blood from the caudal vein near the cloaca
(from the same puncture made for the glucose
measurement), before and after the experiment,
using a lactometer (Lactate Scout+, SensLab
GmbH, Germany, with a range of 0.5 - 25.0 mM).
The small volumes of blood that we could
obtain from lizards without harming the animals
limited us to only one measurement on each
individual, one for glucose and one for lactate.
We also calculated the proportional change
for lactate, or adjusted lactate change (ΔALac)
to account for the difference in lactate initial
values, using the following formula:
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 9 | 19
Where Lacf was the lactate at the end of
the experiment and Laci at the beginning. Initial
and fi nal lactate were not analysed separately
because the change in lactate was already
analysed as ΔALac (in the same way as ΔAGluc).
Statistical analyses
We made comparisons of glucose (ΔAGluc) and
lactate (ΔALac) for each individual before and
after the cooling experiment using a paired
t-test, and comparisons for urea, total proteins,
and albumin using ANCOVA between control
and experimental groups, with the scaled
mass index (SMI) as a covariable. Comparisons
among potential refuges in degree-days were
performed with a χ2 test. In the case of Esquel,
where multiple measures were taken for control
individuals of the first experiment, a mixed
model was performed with the ‘lme4’ package
for R (Bates et al. 2015) to compare ΔAGluc and
ΔALac.
We analysed the variability in body sizes
and weights using scaled mass index (SMI),
calculated as:
Where Mi and SVLi are the mass and SVL of
the individual, SVL0 is the arithmetic mean SVL
of the population, and bSMA is the standardized
major axis slope from the regression of ln body
mass on ln SVL for the population (sensu Peig &
Green 2009). The bSMA exponent was calculated
using the package ‘lmodel2’ (Legendre 2014)
in R (R Core Team 2019). All the other analyses
were performed using the same software, with
the ‘nlme’ (Pinheiro et al. 2017) and ‘car’ (Fox
& Weisberg 2011) packages. The significance
threshold for p values was set at 0.05.
Captures were carried out with authorization
from the Wild Life Service of the Province of
Chubut (Permit # 0460/16 MP; Disposition
# 11/2016). We followed the ASIH/HL/SSAR
Guidelines for Use of Live Amphibians and
Reptiles as well as the regulations detailed in
Argentinean National Law #14346.
RESULTS
Body size (SVL), weight, and scaled mass index
(SMI)
Body size and body mass ranged from 50.13 to
60.74 mm and from 3.67 to 6.57 g for Calafate’s
individuals and ranged from 41.79 to 57.19 mm
and from 3.01 to 5.66 g for Esquel’s individuals.
There were no significant differences in the
SMI between control (mean= 4.80 ± 0.39) and
cooled-down individuals (mean= 4.87 ± 0.427)
from Calafate (ANOVA: F1; 18= 0.183; p= 0.674) or
between control (mean= 4.03 ± 0.31) and cooled-
down individuals (mean= 3.92 ± 0.70) from Esquel
(ANOVA: F1; 17= 0.054; p= 0.818).
Field body temperatures, thermal
microenvironments, and environmental
temperatures in the fi eld (degree-days)
Field body temperature of lizards was similar
between sites (Table SIII). The exposed lizard
PVC model (out of potential refuges) in Calafate
reached a minimum value of -8.91°C, while the
lowest temperature registered by lizard models
in potential refuges was -3.37°C (Figure S1). In
Esquel, the exposed model reached a minimum
value of -8.70°C, while the lowest temperature
registered by lizard models in potential refuges
was -6.58°C.
The PVC lizard models in potential refuges in
Calafate underwent 7 to 194 periods when they
registered consecutive temperatures below 0°C
that lasted between 1 and 3427 hours. Meanwhile,
in Esquel, lizard models underwent between 7
and 69 periods of temperatures below 0°C that
lasted between 1 and 103 hours (Table II).
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 10 | 19
In heating refuge degree-day for 0°C, the
refuge with fewer degree-days below 0°C in
Calafate was buried ~ 15 cm in the ground (9.15
degree-days); and the refuge with the most
degree-days below 0°C was buried ~ 10 cm in
the ground (108.78 degree-days). Meanwhile,
in Esquel, the refuge with fewer degree-days
below 0°C was buried ~ 10 cm in the ground
(3.34 degree-days); and the refuge with the most
degree-days below 0°C was placed under a
bush (21.95 degree-days; χ2 7 test= 1611,1; p< 0,001;
Figure 1).
Supercooling point (SCP) and Critical Thermal
Minimum (CTMin)
Lizards from Calafate showed freezing exotherms
at a mean temperature of -7.54 ± 0.49°C, while
the supercooling point for lizards from Esquel
was higher, at -5.80 ± 0.82°C (Table SI; ANOVA:
F1; 9=17.155; p=0.002). No lizard survived the slow
thaw after experiencing the exothermic freezing
reaction, neither from Calafate nor from Esquel.
Lizards showed a CTMin ranging from 2.23 to
5.86°C for Calafate’s individuals and from 3.97
to 6.21°C for individuals from Esquel. Calafate
individuals (mean= 4.30 ± 1.29) had lower CTMin
than Esquel individuals (mean= 5.10 ± 0.61;
ANOVA: F1; 23= 4.500; p= 0.004).
Table II. Comparison between data obtained from ten lizard models set in potential locations of Liolaemus
lineomaculatus for overwintering in Calafate and Esquel. Minimum, mean ± standard deviation (SD) and maximum
number of consecutive hours with temperatures below 0°C in a single sequence, number of times below zero, and
lowest recorded temperature.
Site Model Location Number of
temperatures
below 0°C
Mean (±SD)
(hours)
Minimum
and
maximum
(hours)1
Minimum and
maximum
temperatures
(°C)
Calafate
Exposed 177 21.5 (102.5) 1-1266 -8.91 to 44.89
Under a tussock (Mulinum
spinosum), near the roots 7 463.5 (1050.5) 9-2937 -1.97 to 31.93
Buried ~10 cm 13 276 (928) 2-3427 -3.37 to 32.76
Buried ~15 cm 194 3 (18.5) 1-257 -1.97 to 30.71
Esquel
Exposed 118 7.5 (7) 1-43 -8.70 to 48.37
Under a tussock (Mulinum
spinosum), near the roots 69 8 (8.5) 1-45 -6.58 to 39.32
Buried under a bush ~10 cm 25 13 (15) 1-72 -4.56 to 30.70
Buried under a rock of ~40 cm
diameter 23 9 (7) 1-21 -3.33 to 30.69
Buried ~10 cm 7 20.5 (34) 2-100 -1.61 to 30.90
Buried ~15 cm 7 26 (33) 8-103 -2.88 to 30.67
1Minimum and maximum number of consecutive hours with temperatures below 0°C in a single sequence.
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 11 | 19
Control and cooled-down individuals, before
and after the cooling experiments
During the cooling experiment, we did not detect
an exothermic reaction from any individual from
Calafate or Esquel and, after removing lizards
from the plastic containers, we found no ice
or evident sign of freezing (such as rigidity of
the animals or a change in the colour of their
skin). Additionally, individuals reacted seconds
after we took them out of the freezer (except
for lizards from the first Esquel experiment,
that were found dead), although in a seemingly
lethargic state, with slow movements.
The control individuals from Calafate
showed negative values of adjusted glucose
change (ΔAGluc: mean= -22.7 %) and individuals
that were cooled down showed positive values
(mean= 8.9%; ANOVA: F1; 18= 126.24; p < 0.001). For
adjusted lactate change, no differences were
found between control (mean= -33.35 %) and
cooled-down individuals (mean= 0.29 %; ΔALac:
ANOVA, F1; 18=1.79; p=0.20). A comparison of ΔAGluc
between control groups of both populations
showed no signifi cant differences (ANOVA, F1; 17=
0.31; p= 0.59).
Control individuals from the Esquel
experiment showed negative values of ΔAGluc
(mean= -19.49 %) and individuals that were
cooled down showed positive values (mean=
26.68%; χ224 test= 54.39; p< 0.001). In the “milder
complementary experiment”, control individuals
showed negative values of ΔAGluc as well (mean=
-36.64 %; N= 3) and cooled-down individuals
positive values (mean= 32.96%; N= 6). For ΔALac,
control individuals showed signifi cantly lower
increases (mean= 12.16 %) than cooled-down
individuals (mean= 387.28 %; χ224 test= 31.99; p<
0.001). Meanwhile, in the “milder complementary
experiment”, control individuals showed
negative values of ΔALac (mean= -37.54 %, N=
3) and cooled-down individuals an increase
(mean= 201.83%, N= 6; Figure 2, Table SII).
The urea, total proteins, and albumin, which
were measured only after the experiments from
Calafate, did not show signifi cant differences
Figure 1. Thermal quality of the potential winter refuges (degree-days) in Calafate (light grey) and Esquel (dark
grey). Values for degree-days below 0°C are represented for each potential refuge.
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 12 | 19
between controls and cooled-down individuals
(Table III).
DISCUSSION
Where to spend the winter seems to be crucial
in how Liolaemus lizards are coping with the
dangers of low temperatures in Patagonia.
Populations of L. lineomaculatus from this
study were not able to tolerate freezing but
survived cold exposure with low supercooling
points comparing with other reptile species of
similar environments (-7.5°C for lizards from
the cold site, Calafate, and -5.8°C for lizards
from the temperate site, Esquel). Results from
the biochemical analyses showed increases
in concentration only in glucose after cold
exposure, in possible association with cold
hardiness mechanisms. However, the increase
in concentration is probably not enough to
elevate osmolality in an ecologically significant
way, considering similar experiments (Costanzo
et al. 1991) with external glucose loading, where
survival was increased when concentrations
in plasma reached over 50 μmol*ml-1 (in our
experiments, values ranged between ~5 and ~15
μmol*ml-1). Lizards could spend a very short time
at temperatures near or below their population’s
SCP in any of the potential refuges analysed in
this study. Furthermore, there was a correlation
Figure 2. Results from the
Adjusted Glucose (ΔAGluc) and
Adjusted Lactate (ΔALac) analyses
corresponding to the Calafate
and Esquel cooling experiments.
Values for a) individuals from
Calafate (N cooled down= 10; N control=
10) for ΔAGluc and ΔALac; and
b) individuals from Esquel
(N cooled down= 10; N control= 9), for
ΔAGluc and ΔALac from the first
experiment (1-2) and the milder
complementary experiment (3-4;
N cooled down= 6; Ncontrol= 3). Median
(black horizontal line) and mean
(rhombs) are represented in all
groups. The middle 50% of values
are inside each box; whiskers
represent upper and lower
quartiles.
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 13 | 19
between cold hardiness and severity of weather
or thermal quality of potential refuges, as we
expected. Lizards from Calafate, where the PVC
models were exposed to cold temperatures or
more frequent cold spells showed a lower mean
CTMin and were able to supercool with a lower
SCP than lizards from Esquel. Furthermore,
lizards from Esquel did not survive exposure
to temperatures near their SCP, even when
they did survive the exposure to temperatures
below 0°C for several hours. It is very likely
that L. lineomaculatus relies mostly on use of
appropriate refuges rather than physiological
mechanisms to overwinter in Patagonia, as
does L. pictus (Cecchetto et al. 2019), although
probably with a higher capacity to endure a cold
climate, reaching a lower SCP (-5°C; Cecchetto
2021) and lower CTMin (6.9°C, Kubisch et al. 2011).
The actual thermal regime experienced
by ectotherms may be more heterogeneous
than predicted by only latitude or elevation
(Ficetola et al. 2018), but results from the
potential-refuges showed that lizards living in
Esquel are more likely than those in Calafate
to find and use microhabitats that are better
thermally buffered (e.g., under rocks or within
rock crevices). There was variation between
populations in critical thermal minimum (CTMin):
individuals from Calafate had a lower CTMin than
individuals from Esquel. This is consistent with
several studies showing CTMin for ectotherms,
varying across environments with different cold
Table III. Comparison of biochemical variables between control (n = 10 for both Calafate and Esquel) and cooled
down individuals (n = 10 for Calafate and n = 9 for Esquel) of Liolaemus lineomaculatus. In the case of urea, values
include the significant covariable SMI (F1; 10 = 10.356; p = 0.001). All means are expressed in g/L. Analyses were
performed as ANCOVAS.
Site Variables F p Control means (±SD) Cooled down means (±SD)
Calafate
Urea (heart) n = 20 0.065 0.802 0.11 (0.07) 0.12 (0.11)
Urea (liver) n = 20 0.031 0.862 0.12 (0.08) 0.12 (0.08)
Total proteins (heart) n = 20 0.225 0.641 18.50 (8.69) 20.28 (8.10)
Total proteins (liver) n = 20 3.526 0.077 132.05 (54.15) 172.81 (42.18)
Albumin (heart) n = 20 0.564 0.463 10.94 (1.84) 9.96 (3.69)
Albumin (liver) n = 20 1.959 0.181 45.5 (6.09) 49.56 (10.64)
Initial glucose n = 20 - - 1.97 (0.22) 1.88 (0.22)
Final glucose n = 20 - - 1.52 (0.29) 2.31 (0.26)
Initial lactate n = 20 - - 0.41 (0.28) 0.32 (0.18)
Final lactate n = 20 - - 0.31 (0.16) 0.16 (0.07)
Esquel
Initial glucose n = 19 - - 1.85 (0.30) 1.71 (0.44)
Final glucose n = 19 - - 1.46 (0.14) 2.09 (0.38)*
Initial lactate n = 19 - - 0.27 (0.08) 0.48 (0.19)
Final lactate n = 19 - - 0.29 (0.19) 1.70 (0.23)*
All means are expressed in g/L. Analyses were performed as ANCOVAS with SMI as a covariable, if significant. Initial and final
glucose were not analyzed because the change in glucose was analyzed as ΔAGluc (%), and the same was the case for lactate,
analyzed as ΔALac (%).
*These values were obtained from lizards found dead after the experiment.
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 14 | 19
regimes (Hoffmann et al. 2002, Huang & Tu 2008,
Moritz et al. 2012, Clusella-Trullas & Chown 2014),
suggesting that CTMin is physiologically relevant
and could directly affect survival at cold and
temperate habitats (but see also Winne & Keck
2005, Du 2006, Yang et al. 2008).
The CTMin values of both populations of L.
lineomaculatus were among the lowest values
recorded for liolaemids from Patagonia (Bonino
et al. 2015, Kubisch et al. 2016), which is not
surprising given that this species is one of the
southernmost lizard species in Argentina.
The values of supercooling points (SCP)
obtained from these Liolaemus lineomaculatus
populations fall in the range of other lizards
such as Uta stansburiana (varying among
populations, between -7°C and -10°C , Michels-
Boyce & Zani 2015), Eulamprus tympanum and
E. kosciuskoi (-6.5°C and -8.5°C, respectively;
Spellerberg 1972), and Podarcis muralis (-5°C;
Claussen et al. 1990). Furthermore, we also
found variation between populations in
supercooling points: lizards from the cold site,
Calafate, showed a mean value lower than
lizards from the temperate site, Esquel. Notably,
only the Calafate population was able to survive
for several (12) hours at near SCP temperature.
Variable cold hardiness has also been reported
for Zootoca vivipara, which not only showed
different temperatures of crystallization in
populations from different habitats (Voituron
et al. 2004, Berman et al. 2016), but also
the possibility to alternate between freeze
tolerance and supercooling (Grenot et al. 2000,
Voituron et al. 2002). Interestingly, in the case
of Uta stansburiana, among the 12 populations
sampled, there was no correlation between
winter harshness and supercooling points
(Michels-Boyce & Zani 2015). Future endeavours
could focus on sampling more populations of L.
lineomaculatus to determine if the association
between thermal quality of the sampling sites
and cold hardiness found in the present study
persists as a trend related to environmental
restraints.
Consistent with results obtained for L.
pictus (Cecchetto et al. 2019), we did not find
any significant differences between control and
cooled-down individuals for urea, total proteins,
or albumin. Urea has been associated with cold
hardiness by increasing the plasma osmolality
of some reptiles such as hatchlings of Chrysemys
picta (Costanzo et al. 2000) and some amphibians
such as Lithobates sylvaticus (Costanzo &
Lee 2005), but the evidence suggests that it
is not directly involved in the cold hardiness
mechanisms of L. lineomaculatus. On the other
hand, the search for antifreeze proteins (AFPs)
in the blood of ectotherms, other than fish, has
not yet yielded positive results. Researchers
have tried to find AFPs in freeze-tolerant wood
frogs (Lithobates sylvaticus; Wolanczyk et al.
1990), turtle hatchlings (Chrysemys picta; Storey
et al. 1991 and Chelydra serpentine; Costanzo
et al. 2000), and the European common lizard,
Zootoca vivipara (Voituron et al. 2002), without
success. Given the scarce information regarding
AFPs in liolaemids, the search for potential
proteins related to cold hardiness is worthy of
research. Nevertheless, we found no evidence of
AFPs as part of the mechanisms used in L. pictus
(Cecchetto et al. 2019) or L. lineomaculatus to
survive winter in Patagonia.
Cooled-down individuals of L. lineomaculatus
from both Calafate and Esquel showed an
increase in blood glucose during experiments,
while control individuals showed a general
decrease. Lactate, on the other hand, did not
present such a clear pattern. Final lactate
concentration in individuals from Calafate was
also almost 10 times less than that in individuals
from Esquel, which could be due to mechanisms
regulating the acid-base homeostasis that could
not begin in the Esquel population since those
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 15 | 19
individuals died from the cold. Additional work
with larger sampling would be necessary to fully
understand the cold hardiness in this species.
However, in individuals from Esquel, there was an
increase in lactate for cooled-down individuals
and a decrease in controls. While it is tempting
to associate the glucose increase in cooled-down
individuals with a cold hardiness response, the
small concentration of this increase suggests that
glucose for this species may not be specifically
associated with colligative cryoprotection, in the
same way as Voituron et al. (2002) concluded
for Zootoca vivipara (where concentrations
reached ~25 μmol*ml-1, while values obtained
in our experiments ranged between ~5 and ~15
μmol*ml-1). Furthermore, the increase in lactate
in cooled-down individuals from Esquel could
be indicating that the contribution of glucose
in elevating osmolality may be secondary
to its role in anaerobic energy metabolism.
The role of glucose as a metabolic fuel in
anaerobic metabolism during periods where low
temperature slows or halts oxygen circulation
is well known (Calderon et al. 2009, Sinclair et
al. 2013), especially for organs like the brain,
which relies on glucose derived from the liver
glycogenolysis during anoxia (Clark & Miller 1973).
Thus, we consider that the role of glucose in L.
lineomaculatus at cold temperatures is mainly
related to maintaining metabolism despite cold-
induced anoxia and perhaps protecting cells by
limiting cell dehydration.
Vegetation structure and land topography
can cause big differences in soil temperature
and snow disappearance over short distances
(Ford et al. 2013). Here, we explored the thermal
quality of potential refuges for lizards in small
areas of each sampling site, representing
a sample of the options individuals might
choose every year when winter comes. Previous
analyses from potential refuges for L. pictus
showed alternatives where lizards could spend
most, if not all winter above 0°C (Cecchetto et
al. 2019). Liolaemus lineomaculatus inhabits
colder environments than L. pictus (at higher
elevations or latitudes); it is, therefore, unlikely
that it could spend most winter above 0°C. Even
though the thermal quality of potential refuges
varied greatly at each site, and between Calafate
and Esquel, potential refuges rarely remained at
temperatures near or below each population’s
SCPs. We found that, despite being buried at ~10
-15 cm, lizard models were well buffered from air
temperatures at the selected potential refuges,
which is consistent with previous works that
found that 10 cm of soil caused significant thermal
buffering where below-ground raiding species
were collected (Baudier et al. 2015). It should
be pointed out that, while the homogeneity of
the environment allowed us to cover the most
representative microenvironments with few PVC
models, the relatively low number of models
used in this study did not allow for replicate
measurements of each potential refuge at each
site. Appropriate refuge selection is most likely
what allows individuals of L. lineomaculatus to
survive the winters without heavily investing
resources in costly physiological mechanisms,
preserving those resources for the sporadic
heavy winter spells. In this viviparous species,
saving energy can be vital, considering that
females give birth to 3-4 individuals between
late summer and the beginning of autumn and
post-partum females start brumation in early
autumn (Medina & Ibargüengoytía 2010).
Liolaemus lineomaculatus occupies
locations with harsher cold climates than L. pictus
in the highlands and high latitudes of Patagonia
and, unlike L. pictus, this species seems to be able
to supercool. This ability to supercool appears
to be related to the cold regime of the location,
varying between populations, although further
studies are needed to determine if it is a result
of adaptation or plasticity. In our study, we could
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 16 | 19
not find evidence of biochemical metabolites
that explain the endurance of L. lineomaculatus
to live in one of the coldest environments for
Liolaemidae, except for a small increase in
glucose. While potential refuges analysis for L.
lineomaculatus revealed that suitable refuge
selection must be key in the survival of lizards
in the winters of Patagonia, perhaps even more
so than any physiological mechanism. However,
the threat of reduced snow deposition caused
by global warming might force lizards to rely on
plastic physiological and behavioural responses
to survive the winter at the risk of depleting
energy reserves. This work provides information
and results on the physiological and ecological
aspects of the question: “How is a 15-cm lizard
able to endure the cold in the highlands and
high latitudes of Patagonia, Argentina?” However,
further work to discover what is going on under
the snow with ectotherms in temperate and cold
environments is needed.
Acknowledgments
Thanks to F. Duran for his help in the field, capturing
lizards. The group would also like to thank S. Taussig for
his very helpful suggestions on the methodology, and
M. Molina, G. Reiner, and M. Langenheim for patiently
helping us with the biochemical assays. J. Krenz
reviewed the manuscript. This study was conducted
with research grants from Fondo para la Investigación
Científica y Tecnológica (PICT-2017-0553), and Consejo
Nacional de Investigaciones Científicas y Técnicas
(PIP-11220120100676).
REFERENCES
BATES D, MÄCHLER M, BOLKER B & WALKER S. 2015. Fitting
Linear Mixed-Effects Models Using lme4. J Stat Soft 67(1):
1-48.
BAUDIER KM, MUDD AE, ERICKSON SC & O’DONNELL S. 2015.
Microhabitat and body size effects on heat tolerance:
Implications for responses to climate change (army ants:
Formicidae, Ecitoninae). J Anim Ecol 84(5): 1322-1330.
BERMAN DI, BULAKHOVA NA, ALFIMOV AV & MESHCHERYAKOVA
EN. 2016. How the most northern lizard, Zootoca vivipara,
overwinters in Siberia. Polar Biol 39(12): 2411-2425.
BONINO MF, MORENO AZÓCAR DL, SCHULTE JA & CRUZ FB. 2015.
Climate change and lizards: changing species’ geographic
ranges in Patagonia. Reg Environ Change 15(6): 1121-1132.
BOYERO L ET AL. 2011. A global experiment suggests
climate warming will not accelerate litter decomposition
in streams but might reduce carbon sequestration. Ecol
Lett 14(3): 289-294.
CALDERON S, HOLMSTRUP M, WESTH P & OVERGAARD J. 2009.
Dual roles of glucose in the freeze-tolerant earthworm
Dendrobaena octaedra: cryoprotection and fuel for
metabolism. J Exp Biol 212: 859-866.
CECCHETTO NR. 2021. ¿Cómo sobreviven los lagartos de
Patagonia al frío extremo? El caso de Liolaemus pictus
y Liolaemus lineomaculatus. PhD thesis, Universidad
Nacional del Comahue. (Unpublished).
CECCHETTO NR, MEDINA SM & IBARGÜENGOYTÍA NR.
2020. Running performance with emphasis on low
temperatures in a Patagonian lizard, Liolaemus
lineomaculatus. Sci Rep 10(1): 1-13.
CECCHETTO NR, MEDINA SM, TAUSSIG S & IBARGÜENGOYTÍA
NR. 2019. The lizard abides: cold hardiness and winter
refuges of Liolaemus pictus argentinus in Patagonia,
Argentina. Can J Zool 782: 773-782.
CEI JM. 1988. Reptiles del centro, centro-oeste y sur
de la Argentina. Herpetofauna de las zonas áridas y
semiáridas. Torino: Museo Regionale di Scienze Naturali.
CHRISTIAN KA & TRACY CR. 1981. The effect of the thermal
environment on the ability of hatchling Galapagos land
iguanas to avoid predation during dispersal. Oecologia
49(2): 218-223.
CLARK VM & MILLER AT. 1973. Studies on anaerobic
metabolism in the fresh-water turtle (Pseudemys scripta
elegans). Comp Biochem Physiol A Physiol 44(1): 55-62.
CLAUSSEN DL, TOWNSLEY MD & BAUSCH RG. 1990.
Supercooling and freeze-tolerance in the European wall
lizard, Podarcis muralis, with a revisional history of the
discovery of freeze-tolerance in vertebrates. J Comp
Physiol B 160(2): 137-143.
CLUSELLA-TRULLAS S & CHOWN SL. 2014. Lizard thermal trait
variation at multiple scales: A review. J Comp Physiol B
184(1): 5-21.
COSTANZO JP, BAKER PJ & LEE RE. 2006. Physiological
responses to freezing in hatchlings of freeze-tolerant
and -intolerant turtles. J Comp Physiol B 176(7): 697-707.
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 17 | 19
COSTANZO JP, DINKELACKER SA, IVERSON JB & LEE RJ. 2004.
Physiological ecology of overwintering in the hatchling
painted turtle: multiple-scale variation in response to
environmental stress. Physiol Biochem Zool 77(1): 74-99.
COSTANZO JP & LEE RE. 2005. Cryoprotection by urea
in a terrestrially hibernating frog. J Exp Biol 208(21):
4079-4089.
COSTANZO JP, LEE RJ, DEVRIES AL, WANG T & LAYNE JR. 1995.
Survival mechanisms of vertebrate at subfreezing
temperatures: applications in cryomedicine. Fed Am Soc
Exp Biol J 9(5): 351-358.
COSTANZO JP, LEE RE & ULTSCH GR. 2008. Physiological
ecology of overwintering in hatchling turtles. J Exp Zool
Part Ecol Genet Physiol 309(6): 297-379.
COSTANZO JP, LEE RE & WRIGHT MF. 1991. Glucose loading
prevents freezing injury in rapidly cooled wood frogs. Am
J Physiol 261: 1549-1553.
COSTANZO JP, LITZGUS JD, IVERSON JB & LEE RJ. 2000. Seasonal
changes in physiology and development of cold
hardiness in the hatchling painted turtle Chrysemys
picta. J Exp Biol 203: 3459-3470.
COWLES RB & BOGERT CM. 1944. A preliminary study of the
thermal requirements of desert reptiles. Bull Am Mus
Nat Hist 83(5): 261-296.
DOUGHTY P. 1994. Critical thermal minima of garter snakes
(Thamnophis) depend on species and body size. Copeia
1994(2): 537-540.
DU WG. 2006. Preferred body temperature and thermal
tolerance of the northern grass lizard Takydromus
septentrionalis from localities with different longitudes.
Acta Zool Siniica 52(3): 478-482.
FELLENBERG G. 1983. Ergenzende Mitteilungen zur Biologie
der Waldeidechse (Lacerta vivipara) in Sudwestfalen.
Nat Heim 43(2): 40-45.
FICETOLA GF, LUNGHI E, CANEDOLI C, PADOA-SCHIOPPA E, PENNATI
R & MANENTI R. 2018. Differences between microhabitat
and broad-scale patterns of niche evolution in terrestrial
salamanders. Sci Rep 8(1): 1-12.
FORD KR, ETTINGER AK, LUNDQUIST JD, RALEIGH MS & HILLE
RIS LAMBERS J. 2013. Spatial Heterogeneity in Ecologically
Important Climate Variables at Coarse and Fine Scales
in a High-Snow Mountain Landscape. PLoS ONE 8(6):
e65008.
FOX J & WEISBERG S. 2011. An R Companion to applied
regression. Second ed. Thousand Oaks: Sage, CA.
GRAAE BJ ET AL. 2012. On the use of weather data in
ecological studies along altitudinal and latitudinal
gradients. Oikos 121(1): 3-19.
GRENOT CJ, GARCIN L, DAO J, HÉROLD JP, FAHYS B & TSÉRÉ-PAGÈS
H. 2000. How does the European common lizard, Lacerta
vivipara, survive the cold of winter? Comp Biochem
Physiol - Mol Integr Physiol 127(1): 71-80.
GUISAN A & HOFER U. 2003. Predicting reptile distributions
at the mesoscale: Relation to climate and topography. J
Biogeogr 30(8): 1233-1243.
GVOZDÍK L & CASTILLA AM. 2001. A comparative study of
preferred body temperatures and critical thermal
tolerance limits among populations of Zootoca vivipara
(Squamata: Lacertidae) along an altitudinal gradient. J
Herpetol 35(3): 486-492.
HOFFMANN AA, ANDERSON A & HALLAS R. 2002. Opposing
clines for high and low temperature resistance in
Drosophila melanogaster. Ecol Lett 5(5): 614-618.
HUANG SP, HSU Y & TU MC. 2006. Thermal tolerance and
altitudinal distribution of two Sphenomorphus lizards in
Taiwan. J Therm Biol 31(5): 378-385.
HUANG SP & TU MC. 2008. Cold tolerance and altitudinal
distribution of Takydromus lizards in Taiwan. Zool Stud
47(4): 438-444.
HUEY RB. 1991. Physiological consequences of habitat
selection. Am Nat 137: S91-S115.
KINLAW A. 1999. A review of burrowing by semi-fossorial
vertebrates in arid environments. J Arid Environ 41(2):
127-145.
KUBISCH EL, FERNÁNDEZ JB & IBARGÜENGOYTÍA NR. 2016.
Vulnerability to climate warming of Liolaemus pictus
(Squamata, Liolaemidae), a lizard from the cold
temperate climate in Patagonia, Argentina. J Comp
Physiol B 186(2): 243-253.
LEGENDRE P. 2014. lmodel2: Model II Regression.
LINDSEY AA & NEWMAN JE. 1956. Use of official wather
data in spring time: temperature analysis of an Indiana
phenological record. Ecology 37(4): 812-823.
MEDINA SM & IBARGÜENGOYTÍA NR. 2010. How do viviparous
and oviparous lizards reproduce in Patagonia? A
comparative study of three species of Liolaemus. J Arid
Environ 74(9): 1024-1032.
MEDINA SM, SCOLARO A, MÉNDEZ-DE LA CRUZ F &
IBARGÜENGOYTÍA NR. 2011. Thermal relationships between
body temperature and environment conditions set upper
distributional limits on oviparous species. J Therm Biol
36(8): 527-534.
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 18 | 19
MICHELS-BOYCE M & ZANI PA. 2015. Lack of supercooling
evolution related to winter severity in a lizard. J Therm
Biol 53: 72-79.
MITCHELL N, HIPSEY M, ARNALL S, MCGRATH G, TAREQUE H,
KUCHLING G, VOGWILL R, SIVAPALAN M, PORTER W & KEARNEY M.
2012. Linking Eco-Energetics and Eco-Hydrology to Select
Sites for the Assisted Colonization of Australia’s Rarest
Reptile. Biology 2(1): 1-25.
MONASTERIO C, SALVADOR A, IRAETA P & DÍAZ JA. 2009. The
effects of thermal biology and refuge availability on the
restricted distribution of an alpine lizard. J Biogeogr
36(9): 1673-1684.
MORITZ C, LANGHAM G, KEARNEY M, KROCKENBERGER
A, VANDERWAL J & WILLIAMS S. 2012. Integrating
phylogeography and physiology reveals divergence of
thermal traits between central and peripheral lineages
of tropical rainforest lizards. Philos Trans R Soc B Biol Sci
367(1596): 1680-1687.
MUNOZ MM, STIMOLA MA, ALGAR AC, CONOVER A, RODRIGUEZ AJ,
LANDESTOY MA, BAKKEN GS & LOSOS JB. 2014. Evolutionary
stasis and lability in thermal physiology in a group of
tropical lizards. Proc R Soc B Biol Sci 281(1778): 20132433.
MURPHY MA, EVANS JS & STORFER A. 2010. Quantifying Bufo
boreas connectivity in Yellowstone National Park with
landscape genetics. Ecology 91(1): 252-261.
PAULI JN, ZUCKERBERG B, WHITEMAN JP & PORTER W. 2013. The
subnivium: A deteriorating seasonal refugium. Front Ecol
Environ 11(5): 260-267.
PEIG J & GREEN AJ. 2009. New perspectives for estimating
body condition from mass/length data: The scaled mass
index as an alternative method. Oikos 118(12): 1883-1891.
PINHEIRO J, BATES D, DEBROY S, SARKAR D & R CORE TEAM. 2017.
NLME: Linear and nonlinear mixed effects models.
R CORE TEAM. 2019. R: A Language and Environment for
Statistical Computing.
SALT RW. 1966. Factors influencing nucleation in
supercooled insects. Can J Zool 44(1): 117-133.
SCHWANZ LE & JANZEN FJ. 2008. Climate change and
temperature-dependent sex determination: can
individual plasticity in nesting phenology prevent
extreme sex ratios? Physiol Biochem Zool 81(6): 826-834.
SCOLARO JA. 2005. Reptiles Patagónicos Sur: una guía de
campo. Argentina: Universidad Nacional de la Patagonia,
Trelew.
SINCLAIR BJ, STINZIANO JR, WILLIAMS CM, MACMILLAN HA,
MARSHALL KE & STOREY KB. 2013. Real-time measurement
of metabolic rate during freezing and thawing of the
wood frog, Rana sylvatica: implications for overwinter
energy use. J Exp Biol 216(2): 292-302.
SPELLERBERG IF. 1972. Temperature tolerances of
Southeast Australian reptiles examined in relation to
reptile thermoregulatory behaviour and distribution.
Oecologia 9(1): 23-46.
STOREY KB. 1997. Organic solutes in freezing tolerance.
Comparative Biochemistry and Physiology Part A:
Physiology 117(3): 319-326.
STOREY KB, MCDONALD DG, DUMAN JG & STOREY JM. 1991.
Blood chemistry and ice nucleating activity in hatchling
painted turtles. Cryo-Lett 12(6): 351-358.
STOREY KB & STOREY JM. 1986. Freeze tolerance and
intolerance as strategies of winter survival in terrestrially
hibernating amphibians. Comp Biochem Physiol 83(4):
613-617.
STOREY KB & STOREY JM. 1996. Natural freezing survival in
animals. Annu Rev Ecol Syst 27(1996): 365-386.
SUNDAY JM, BATES AE & DULVY NK. 2011. Global analysis of
thermal tolerance and latitude in ectotherms. Proc R Soc
B Biol Sci 278(1713): 1823-1830.
TATTERSALL GJ, SINCLAIR BJ, WITHERS PC, FIELDS PA, SEEBACHER
F, COOPER CE & MALONEY SK. 2012. Coping with thermal
challenges: Physiological adaptations to environmental
temperatures. Compr Physiol 2(3): 2151-2202.
VOITURON Y, HEULIN B & SURGET-GROBA Y. 2004. Comparison
of the cold hardiness capacities of the oviparous and
viviparous forms of Lacerta vivipara. J Exp Zool Part A -
Comp Exp Biol 301(4): 367-373.
VOITURON Y, STOREY JM, GRENOT C & STOREY KB. 2002. Freezing
survival, body ice content and blood composition of
the freeze-tolerant European common lizard, Lacerta
vivipara. J Comp Physiol B 172(1): 71-76.
WEEKS DM & ESPINOZA RE. 2013. Lizards on ice: Comparative
thermal tolerances of the world’s southernmost gecko. J
Therm Biol 38(5): 225-232.
WILLIAMS CM, HENRY HAL & SINCLAIR BJ. 2015. Cold truths:
How winter drives responses of terrestrial organisms to
climate change. Biol Rev 90(1): 214-235.
WINNE CT & KECK MB. 2005. Intraspecific differences in
thermal tolerance of the diamondback watersnake
(Nerodia rhombifer): Effects of ontogeny, latitude, and
sex. Comp Biochem Physiol - Mol Integr Physiol 140(1):
141-149.
WOLANCZYK JANP, STOREY KB & BAUST JG. 1990. Ice Nucleating
Activity in the Blood of the Freeze-Tolerant Frog ,Rana
sylvatica. Cryobiology 27: 328-335.
NICOLÁS R. CECCHETTO et al. WINTERTIME TALES: Liolaemus lineomaculatus
An Acad Bras Cienc (2022) 94(3) e20210758 19 | 19
YANG J, SUN Y-Y, AN H & JI X. 2008. Northern grass lizards
(Takydromus septentrionalis) from different populations
do not differ in thermal preference and thermal tolerance
when acclimated under identical thermal conditions. J
Comp Physiol B 178(3): 343-349.
SUPPLEMENTARY MATERIAL
Tables SI, SII, SIII
Figure S1
How to cite
CECCHETTO NR, MEDINA SM, BAUDINO F & IBARGÜENGOYTÍA NR. 2022.
Wintertime tales: How the lizard Liolaemus lineomaculatus endures the
temperate cold climate of Patagonia, Argentina. An Acad Bras Cienc 94:
e20210758. DOI 10.1590/0001-3765202220210758.
Manuscript received on May 19, 2021;
accepted for publication on October 1, 2021
NICOLÁS R. CECCHETTO1
https://orcid.org/0000-0002-7004-424X
SUSANA M. MEDINA2
https://orcid.org/0000-0003-1381-7063
FLORENCIA BAUDINO3
https://orcid.org/0000-0001-5256-9432
NORA R. IBARGÜENGOYTÍA1
https://orcid.org/0000-0002-1959-2718
1Instituto de Investigaciones en Biodiversidad y
Medioambiente, Consejo Nacional de Investigaciones
Científicas y Técnicas (INIBIOMA, CONICET-Universidad
Nacional del Comahue), Ecophysiology and Life History
of Reptiles: Research Laboratory, Quintral 1250, 8400
San Carlos de Bariloche, Río Negro, Argentina
2Consejo Nacional de Investigaciones Científicas y
Técnicas (CIEMEP-CONICET),Centro de Investigación
Esquel de Montaña y Estepa Patagónica, Gral.
Roca 780, 9200 Esquel, Chubut, Argentina
3Instituto de Investigaciones en Biodiversidad y
Medioambiente, Consejo Nacional de Investigaciones
Científicas y Técnicas (INIBIOMA, CONICET-Universidad
Nacional del Comahue), Laboratorio Ecotono, Quintral
1250, 8400 San Carlos de Bariloche, Río Negro, Argentina
Correspondence to: Nicolás R. Cecchetto
E-mail: nrcecchetto@comahue-conicet.gob.ar
Author contributions
N.R.C., S.M.M. and N.R.I. were involved in the conception and
design of the study, captured the lizards, and performed the
experiments with F.B.; N.R.C., S.M.M., and N.R.I. performed the
data analyses. N.R.C. wrote the manuscript, and F.B., S.M.M., and
N.R.I. revised the manuscript.
