A multi-isotope approach reveals seasonal variation in the reliance on marine resources, production of metabolic water, and ingestion of seawater by two species of coastal passerine to maintain water balance

Abstract

Tracing how free-ranging organisms interact with their environment to maintain water balance is a difficult topic to study for logistical and methodological reasons. We use a novel combination of triple-oxygen stable isotope analyses of water extracted from plasma (δ¹⁶O, δ¹⁷O, δ¹⁸O) and bulk tissue carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopes of feathers and blood to estimate the proportional contribution of marine resources, seawater, and metabolic water used by two species of unique songbirds (genus Cinclodes) to maintain their water balance in a seasonal coastal environment. We also assessed the physiological adjustments that these birds use to maintain their water balance. In agreement with previous work on these species, δ¹³C and δ¹⁵N data show that the coastal resident and invertivore C. nigrofumosus consumes a diet rich in marine resources, while the diet of migratory C. oustaleti shifts seasonally between marine (winter) to freshwater aquatic resources (summer). Triple-oxygen isotope analysis (Δ¹⁷O) of blood plasma, basal metabolic rate (BMR), and total evaporative water loss (TEWL) revealed that ~25% of the body water pool of both species originated from metabolic water, while the rest originated from a mix of seawater and fresh water. Δ¹⁷O measurements suggest that the contribution of metabolic water tends to increase in summer in C. nigrofumosus, which is coupled with a significant increase in BMR and TEWL. The two species had similar BMR and TEWL during the austral winter when they occur sympatrically in coastal environments. We also found a positive and significant association between the use of marine resources as measured by δ¹³C and δ¹⁵N values and the estimated δ¹⁸O values of ingested (pre-formed) water in both species, which indicates that Cinclodes do not directly drink seawater but rather passively ingest when consuming marine invertebrates. Finally, results obtained from physiological parameters and the isotope-based estimates of marine (food and water) resource use are consistent, supporting the use of the triple-oxygen isotopes to quantify the contribution of water sources to the total water balance of free-ranging birds.

Full text

Frontiers in Ecology and Evolution 01 frontiersin.org
TYPE Original Research
PUBLISHED 15 February 2023
DOI 10.3389/fevo.2023.1120271
A multi-isotope approach reveals
seasonal variation in the reliance on
marine resources, production of
metabolic water, and ingestion of
seawater by two species of coastal
passerine to maintain water balance
LucasNavarrete
1,2, NicoLübcker
3, FelipeAlvarez
1,2,
RobertoNespolo
2,4, 5, JuanCarlosSanchez-Hernandez
6,
KarinMaldonado
7, ZacharyD.Sharp
8, JohnP.Whiteman
9,
SethD.Newsome
3 and PabloSabat
1,2*
1 Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile, 2 Center
of Applied Ecology and Sustainability (CAPES), Pontificia Universidad Católica de Chile, Santiago, Chile,
3 Department of Biology, University of New Mexico, Albuquerque, NM, United States, 4 Instituto de Ciencias
Ambientales y Evolutivas, Universidad Austral de Chile, Valdivia, Chile, 5 Millennium Institute for Integrative
Biology (iBio), Santiago, Chile, 6 Laboratory of Ecotoxicology, University of Castilla-La Mancha, Toledo, Spain,
7 Departamento de Ciencias, Facultad de Artes Liberales, Universidad Adolfo Ibáñez, Santiago, Chile,
8 Department of Earth and Planetary Sciences, University of New Mexico, Albuquerque, NM, United States,
9 Department of Biological Sciences at Old Dominion University, Norfolk, VA, United States
Tracing how free-ranging organisms interact with their environment to maintain
water balance is a dicult topic to study for logistical and methodological reasons.
We use a novel combination of triple-oxygen stable isotope analyses of water
extracted from plasma (δ16O, δ17O, δ18O) and bulk tissue carbon (δ13C) and nitrogen
(δ15N) isotopes of feathers and blood to estimate the proportional contribution of
marine resources, seawater, and metabolic water used by two species of unique
songbirds (genus Cinclodes) to maintain their water balance in a seasonal coastal
environment. Wealso assessed the physiological adjustments that these birds use
to maintain their water balance. In agreement with previous work on these species,
δ13C and δ15N data show that the coastal resident and invertivore C. nigrofumosus
consumes a diet rich in marine resources, while the diet of migratory C. oustaleti
shifts seasonally between marine (winter) to freshwater aquatic resources (summer).
Triple-oxygen isotope analysis (Δ17O) of blood plasma, basal metabolic rate (BMR),
and total evaporative water loss (TEWL) revealed that ~25% of the body water pool
of both species originated from metabolic water, while the rest originated from a
mix of seawater and fresh water. Δ17O measurements suggest that the contribution
of metabolic water tends to increase in summer in C. nigrofumosus, which is
coupled with a significant increase in BMR and TEWL. The two species had similar
BMR and TEWL during the austral winter when they occur sympatrically in coastal
environments. Wealso found a positive and significant association between the use
of marine resources as measured by δ13C and δ15N values and the estimated δ18O
values of ingested (pre-formed) water in both species, which indicates that Cinclodes
do not directly drink seawater but rather passively ingest when consuming marine
invertebrates. Finally, results obtained from physiological parameters and the isotope-
based estimates of marine (food and water) resource use are consistent, supporting
the use of the triple-oxygen isotopes to quantify the contribution of water sources to
the total water balance of free-ranging birds.
OPEN ACCESS
EDITED BY
Jose A. Masero,
University of Extremadura,
Spain
REVIEWED BY
Luis Gerardo Herrera Montalvo,
Universidad Nacional Autónoma de México,
Mexico
Erick Gonzalez Medina,
University of Extremadura,
Spain
*CORRESPONDENCE
Pablo Sabat
psabat@uchile.cl
SPECIALTY SECTION
This article was submitted to
Ecophysiology,
a section of the journal
Frontiers in Ecology and Evolution
RECEIVED 09 December 2022
ACCEPTED 30 January 2023
PUBLISHED 15 February 2023
CITATION
Navarrete L, Lübcker N, Alvarez F, Nespolo R,
Sanchez-Hernandez JC, Maldonado K,
Sharp ZD, Whiteman JP, Newsome SD and
Sabat P (2023) A multi-isotope approach
reveals seasonal variation in the reliance on
marine resources, production of metabolic
water, and ingestion of seawater by two
species of coastal passerine to maintain water
balance.
Front. Ecol. Evol. 11:1120271.
doi: 10.3389/fevo.2023.1120271
COPYRIGHT
© 2023 Navarrete, Lübcker, Alvarez, Nespolo,
Sanchez-Hernandez, Maldonado, Sharp,
Whiteman, Newsome and Sabat. This is an
open-access article distributed under the terms
of the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction
in other forums is permitted, provided the
original author(s) and the copyright owner(s)
are credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted which
does not comply with these terms.

Navarrete et al. 10.3389/fevo.2023.1120271
Frontiers in Ecology and Evolution 02 frontiersin.org
KEYWORDS
birds, metabolic water, metabolic rates, stable isotopes, Cinclodes
Introduction
Bird species face both predictable and unpredictable changes in
environmental conditions that impact food and water availability
(Maddocks and Geiser, 2000; Landes et al., 2020). For example, an
increase in ambient temperature and a decrease in the availability of
freshwater aects several aspects of avian physiology including rates of
energy expenditure, body mass, thermal tolerance, thermal conductance,
and evaporative water loss, all of which are directly linked to a bird’s
ability to maintain their water balance (Carmi etal., 1993; Sabat etal.,
2006a; Barceló etal., 2009; Sabat etal., 2009; Gerson and Guglielmo,
2011; Smith etal., 2017; McWhorter etal., 2018). Organisms living in
seasonal environments can adjust their morphology and physiology to
respond to predictable environmental changes, a phenomenon oen
referred to as acclimatization, a particular type of phenotypic plasticity.
It is increasingly important to explore the adaptive mechanisms behind
these adjustments and assess their impact on tness because of
unprecedented shis in environmental conditions resulting from
climate change, which will likely impact the amount and timing of
resource availability, especially water (Şekercioğlu etal., 2012; Khaliq
et al., 2014; Cooper et al., 2019; Whiteman et al., 2019; Huey and
Buckley, 2022).
Deserts and other xeric habitats are among the most challenging
environments for maintaining organismal water balance (Paces etal.,
2021; Cabello-Vergel etal., 2022). Despite the crucial importance of
water to survival, how animals deal with water scarcity has received less
attention than the consequences of reduced food availability (McKechnie
etal., 2016; Cooper etal., 2019; Gerson etal., 2019; Paces etal., 2021;
Cabello-Vergel etal., 2022). An organism’s water balance is a function
of the interplay between (1) physical environment and water availability,
(2) physiological and behavioral mechanisms for conserving water by
reducing the total evaporative water loss (TEWL) and/or thermal
conductance, and (3) the production of metabolic water (Bartholomew
and Cade, 1963; MacMillen, 1990; Gerson and Guglielmo, 2011;
Rutkowska etal., 2016; Albright etal., 2017). For example, some bird
species respond to dehydrating conditions by increasing their rates of
energy expenditure (e.g., basal metabolic rate, BMR), a response that is
commonly assumed to bethe cost of living in arid environments and/or
regularly consuming salty water (Arad etal., 1987; Gutiérrez etal., 2011;
Peña-Villalobos et al., 2013; Sabat et al., 2017). Such increases in
metabolic rate could bea mechanism for water production, reducing the
need for water conservation and the reliance on (pre-formed) drinking/
food water (see Peña-Villalobos et al., 2013; Sabat etal., 2017). is
hypothesis is supported by observations in captive rufous-collared
sparrows (Zonotrichia capensis), in which mass loss and an increase in
the mass-specic metabolic rates were associated with a higher
contribution of metabolic water to the body water pool (Navarrete etal.,
2021). No studies have examined this hypothesis in wild birds, and only
a handful have quantied the contribution of metabolic water to the
water budgets of free-ranging individuals (MacMillen, 1990; Williams
and Tieleman, 2001; Giulivi and Ramsey, 2015).
Coastal deserts are especially intriguing habitats because they do not
support large amounts of terrestrial productivity nor do they have
signicant sources of freshwater (Polis and Hurd, 1996), but can occur
adjacent to very productive nearshore marine ecosystems (Fariña etal.,
2008). Terrestrial animals can exploit abundant marine resources at the
cost of having to deal with high salt loads (Mahoney and Jehl, 1985;
Nyström and Pehrsson, 1988; Fariña etal., 2008). Salty foods can impose
signicant osmoregulatory challenges to songbirds (Order
Passeriformes), which lack functional salt glands (Shoemaker, 1972) and
have a reduced ability to concentrate urine (Goldstein and Skadhauge,
2000; Sabat, 2000). Worldwide, there are only a few passerine species
(genus Cinclodes) capable of living in arid coastal deserts while
consuming signicant amounts of salty marine prey, and several of them
are endemic to the central and northern coasts of Chile. Using stable
isotope analyses and osmometry to study three species of Cinclodes,
Sabat and del Río (2005) reported that the osmolality of stomach
contents increased as the proportion of marine diet (assessed by stable
isotope analysis) became more substantial. Typical salt concentrations
in the body uids of terrestrial and freshwater prey are 100–300 mOsm/
kg (Beyenbach, 2016), whereas some coastal Cinclodes consume prey
(e.g., mollusks and crustaceans) with salt concentrations of up to
800–1,100 mOsm/kg in their body uids (Schmidt-Nielsen, 1997).
Here, weuse multiple isotope tracers to explore seasonal variation
in diet and water balance of two species of endemic, South American
coastal passerines from the genus Cinclodes to investigate how seasonal
variation in habitat use, ambient temperature, and marine resource use
are related to how birds acquire (food/drinking versus metabolic) and
conserve water (TEWL), expend energy (BMR), and dissipate heat
(thermal conductance). C. nigrofumosus is year-round resident that
forages on marine invertebrates in intertidal environments, while its
sister species C. oustaleti also consumes invertebrates but migrates
seasonally between dry coastal habitats and high elevation freshwater
streams (Newsome etal., 2015; Rader etal., 2017; Tapia-Monsalve etal.,
2018). Weused carbon (δ
13
C) and nitrogen (δ
15
N) isotope analysis of
feathers and blood collected during the summer and winter to
characterize seasonal marine versus terrestrial resource use (Newsome
et al., 2007; Martínez del Rio et al., 2009). We then used a novel
methodological approach based on the measurements of the three stable
isotopes of oxygen in blood plasma to measure the proportion of the
body water pool that was derived from metabolic water (Whiteman
etal., 2019; Passey and Levin, 2021; Sabat etal., 2021). is method
utilizes natural dierences in the oxygen isotope composition of
preformed water versus atmospheric oxygen, which is the source of
oxygen for the formation of metabolic water in the mitochondria
(Whiteman etal., 2019). While several studies have focused on the
physiological adjustments these species use to reduce water loss, none
have identied seasonal shis in the use of dierent source(s) of water
(pre-formed versus metabolic) that is a critical to understanding water
balance in free-ranging birds (Navarro etal., 2018; Smit etal., 2019).
We hypothesized that C. nigrofumosus and C. oustaleti used dierent
strategies to maintain water balance due to variation in their ecological
traits. We predicted that seasonal scarcity of freshwater and overall
higher consumption of marine resources by C. nigrofumosus would
result in higher osmoregulatory costs leading to elevated BMR and a
corresponding increase in metabolic water production to maintain their

Navarrete et al. 10.3389/fevo.2023.1120271
Frontiers in Ecology and Evolution 03 frontiersin.org
water balance during summer. Wealso expected that birds in summer
would exhibit lower TEWL and higher thermal conductance to reduce
water loss associated with evaporative cooling. In winter when the two
species occur in sympatry along the coast, wepredicted that migratory
C. oustaleti would rely less on salty marine resources than
C. nigrofumosus and instead consume more terrestrial invertebrates,
which provide a source of less salty (food) water. By extension this
would reduce the proportional contribution of metabolic water to their
body water pool.
Methods
Sample collection
Wild C. oustaleti (n = 11) and C. nigrofumosus (n = 9) were collected
at Bahia Mansa (32°14′22″S 71°30′54″W) on the central coast of Chile
in the austral winter (June 2021) when these species occur in sympatry.
We also collected seven individuals of C. nigrofumosus in summer
(January 2022) at the same locality. is study site has a Mediterranean
climate (mean annual precipitation = 396 mm) characterized by mild
dry summers (mean monthly precipitation = 2 mm, T
min
 = 13°C;
T
max
= 19) and cold rainy winters (mean monthly precipitation = 85 mm;
Tmin= 7°C; Tmax= 13) (di Castri and Hajek 1976). Weobserved no clear
signs of reproduction (e.g., brood patch) or active molting in
C. nigrofumosus captured during the summer. is species is not
sexually dimorphic so wecould not determine the sex of individuals
wecaptured. Weused mist nets and spring traps to capture birds, which
were caged individually in the dark aer capture to minimize stress.
Biometric parameters, cloacal temperature (Tb), and blood samples
were collected from each individual in the eld. Blood samples were
collected from the humeral vein using heparinized hematocrit
capillaries. Blood was maintained in coolers (~4°C) for <2 h and then
centrifuged at 10,000 rpm for 10 min to separate plasma from red blood
cells. e plasma was then stored frozen until cryogenic distillation
followed by oxygen isotope analysis. In addition, a subsample of whole
blood was dried on two glass microscope slides and then transferred to
microcentrifuge tubes and stored for δ13C and δ15N analysis.
Metabolic rates and total evaporative water
loss
Immediately aer capture, birds were transported to Algarrobo,
Chile ~15 min from the capture site for captive physiological
measurements. While in captivity, birds consumed mealworms and
water, which were available ad libitum. Wemeasured BMR (mL O2 h−1)
and total evaporative water loss (TEWL) in post-absorptive (fasted for
4-h), resting birds, during their inactive period between 21:00 and 07:00 h
using standard ow-through respirometry (Tapia-Monsalve etal., 2018).
Weremoved mealworms from the cages ~4 h before BMR measurements
started to ensure a post-absorptive state. Respirometry measurements for
BMR were performed on up to three birds per night. Measurements were
made at an ambient temperature (Ta) of 30.0 ± 0.5°C, which is within the
thermoneutral zone (TNZ), using an infrared O
2
-CO
2
analyzer equipped
with a hygrometer (FMS, Sable Systems®). All trials were conducted in
metallic metabolic chambers (volume 2,000 mL) that received air free of
water and CO2 removed via Drierite and CO2 absorbent, respectively, at
a ow of 800 mL/min (±1%). Inside these darkened metabolic chambers,
birds perched on a wire-mesh grid that allowed excreta to fall into a tray
containing mineral oil, thus trapping the water from this source. Oxygen
consumption was calculated according to the equation (Lighton, 2008):
VO
2
= FR × 60 × (F
i
O
2
−F
e
O
2
)/ (1 −F
i
O
2
), where FR is the ow rate in
mL min−1, and FiO2 and Fe O2 are the fractional concentrations of inow
and outow O
2
in the metabolic chamber, respectively. Wecalculated
absolute humidity (kg/m
3
) of air entering and leaving the chamber as
ρ =P/(T×R
w
), where P is water vapor pressure of the air in Pascal, T is
the dewpoint temperature in Kelvin and Rw is the gas constant for water
vapor (461.5 J/kg K, Lide, 2001). P was determined using the average
value of the vapor pressure of the air entering the empty chamber during
a baseline period of 15 min before and aer each experiment with a
dew-point hygrometer located in the FMS. TEWL was calculated as
TEWL = (Ve× ρout – Vi× ρin), where TEWL is in mg/mL, ρin and ρout are
the absolute humidity in kg/m
3
of the inlet air and the outlet air
respectively, V
i
is the ow rate of the air entering the chamber as given by
the mass ow controller (800 mL min-1), and Ve is the ow of exiting air.
V
e
was calculated following as: V
e
=V
i
– [VO
2
× (1–RQ)] + V
H2O
. V
in
and
VO
2
(mL min-1) are known, and weassumed a respiratory quotient (RQ)
of 0.71 (Sabat etal., 2006a,b). Output from the H
2
O (kPa) analyzer, the
oxygen analyzer (%), and the ow meter were digitalized using a
Universal Interface II (Sable Systems, Nevada, United States) and
recorded on a personal computer using EXPEDATA data acquisition
soware (Sable Systems, Nevada, UnitedStates). To estimate BMR and
TEWL, weaveraged O2 concentrations and water vapor pressures of the
excurrent air stream over a 20 min period aer steady state was reached,
which occurs aer 3 h in Cinclodes (Sabat etal., 2021). Weestimated the
metabolic water production (MWP) using the equivalence of 0.567 mL
H2O per liter O2 consumed (Schmidt-Nielsen, 1997) and calculated the
ratio between metabolic water production and water losses (MWP/
TEWL) for the 20 min period during which steady state was reached. e
ratio MWP/TEWL is interpreted as the ability of birds to rely on
metabolic water to maintain water balance.
To estimate wet thermal conductance (C
w
), we also measured
metabolic rates of birds at a T
a
below the TNZ, on the day of capture,
between 8:00 and 17:00 h, as described above for BMR. Because the
“wet” thermal conductance is roughly constant in endotherms below
thermoneutrality (Nicol and Andersen, 2007; Rezende and Bacigalupe,
2015; Andreasson etal., 2020), for simplicity and logistic restrictions
wemeasured C
w
at 15.0 ± 0.5°C. C
w
was calculated as metabolic rate
(MR
15
) measured at 15°C using the equation MR/(Tb-Ta). In this case
water and food was available for birds until they were placed in the
metabolic chamber. Body mass was measured before the metabolic
measurements using an electronic balance (± 0.1 g) and cloacal body
temperature (Tb) was recorded with a thin Cole-Palmer copper-
constantan thermocouple attached to a Digisense thermometer (Model
92,800–15) within a minute aer the birds were removed from metabolic
chamber to minimize the eect of manipulation on the temperature
measurement (Nord and Folkow, 2019) We considered body
temperatures of ≤36°C as hypothermic (Swanson etal., 2012) and all
birds were normothermic at the end of 15°C or 30°C exposure trials.
Aer each respirometry measurement, the birds were provided food and
water ad libitum until their release.
Δ17O analysis
To estimate the contribution of metabolic water to the body water,
weused a method based on the measurement of Δ
17
O, which is the

Navarrete et al. 10.3389/fevo.2023.1120271
Frontiers in Ecology and Evolution 04 frontiersin.org
positive or negative deviation from the tight correlation that naturally
exists between values of δ
17
O and δ
18
O (Whiteman etal., 2019). e
premise of this method is that metabolic water and drinking/food water
together provide 80–99% of the body water of most animals (Bryant and
Froelich, 1995; Kohn, 1996), with the remaining contribution (1–20%)
resulting from condensation reactions that use bound oxygen from
dietary nutrients. Here, we ignore this latter contribution and
acknowledge that this induces uncertainty (Whiteman etal., 2019). Of
the two other sources, metabolic water is assumed to have a Δ17O value
of −0.44‰ reecting that of inhaled atmospheric oxygen (Whiteman
etal., 2019; Wostbrock etal., 2020), and drinking/food water is assumed
to have a Δ
17
O value of meteoric water with a mean Δ
17
O value of
~0.03‰ across a wide variety of potential sources (e.g., lakes, rivers,
precipitation; Sharp etal., 2018). While extensive evaporation lowers the
Δ
17
O values of the remaining water (Aron etal., 2021), recent studies
have suggested that in many biological applications assuming a xed
value of ~0.03‰ for meteoric water is reasonable (Whiteman etal.,
2019; Sabat etal., 2021). Under this assumption, a linear mixing model
can beused to calculate the proportional contribution from each source
(Whiteman etal., 2019). For example, an animal body water sample with
a Δ17O value of −0.44‰ represents pure metabolic water and a sample
with a Δ
17
O value of 0.03‰ represents pure meteoric water. is mixing
model is in the form: Δ
17
O
Body Water
= F
M
× (−0.44‰) + (1 – F
M
) × (0.030‰)
where F
M
represents the fractional contribution to body water from
metabolic water, and (1 – Fm) represents the contribution from
drinking/food water. In previous studies of captive sparrows (Zonotrichia
capensis) and mice (Peromyscus maniculatus) this equation accurately
predicted relative changes in Δ
17
O based on metabolic rate and drinking
water intake (Whiteman etal., 2019; Sabat etal., 2021).
We cryogenically distilled body water from blood plasma on a
vacuum line. Weuorinated 1.5 μL of the distilled water with BrF5 at
450°C for 15 min under a vacuum to evolve O2, which was puried via a
6 . × 1/8″ (1.83 m × 3.2 mm) 60/80 Mol Sieve 13X gas chromatograph
(GC) column and analyzed via dual-inlet on a ermo Scientic™ 253
Plus isotope ratio mass spectrometer (Sharp et al., 2018) against a
working reference O
2
gas at the University of New Mexico Center for
Stable Isotopes (UNM–CSI; Albuquerque). e measured values of δ
17
O
and δ
18
O were linearized (δ′
x
O = 1,000 × ln((δ
x
O/1000) + 1); x =
17
O or
18
O)
and then used to calculate Δ′
17
O (δ′
17
O – (0.528 × δ′
18
O); Whiteman etal.,
2019). Deviations from the normal linear mass-dependent fractionation
between δ′
17
O and δ′
18
O that is dened by an arbitrary reference line with
a slope (λ) of 0.528, are expressed as Δ′
17
O. Samples were corrected using
a one-point calibration based on intermittent measurements of an
in-house standard (NM2) calibrated against VSMOW-2.
In addition to using Δ
17
O values to understand reliance upon
metabolic water, weused the combination of FM values and δ18O values
of body water to calculate estimated δ
18
O values of ingested pre-formed
drinking/food water (δ
18
O
D + PF
) with the equation δ
18
O
DFW
= (δ
18
O
BW
-
(F
M
) × (δ
18
O
Air
)) / (1-F
M
) and assumed δ
18
O
Air
incorporated via
respiration was 19.4‰ due to the fractionation that occurs during
absorption of inhaled atmospheric oxygen. is fractionation depends
on the eciency of oxygen absorption (EO
2
; Epstein and Zeiri, 1988).
Although this eciency was not measured in our study species, previous
research suggests that an EO
2
of 0.4 is reasonable for small passerines
(Clemens, 1988; Arens and Cooper, 2005), which in humans produces
a fractionation of ~4.4‰ (Epstein and Zeiri, 1988). e estimated δ18O
of ingested water generally changes by <3‰ if youapply the plausible
range of fractionation values for absorbed oxygen (2–6‰) to equation
3, which is smaller than much of the naturally-occurring variation in
δ
18
O of potential water and, therefore, unlikely to aect our conclusions.
δ13C and δ15N analysis
We used δ13C and δ15N analysis to estimate the relative contribution
of marine and terrestrial prey to the diet of Cinclodes species (Martínez
del Rio etal., 2009). In general, baseline δ
13
C and δ
15
N values are higher
in marine than terrestrial food webs (Martínez del Rio etal., 2009). For
C. nigrofumosus, seasonal comparisons were made via analysis of whole
blood collected in summer and winter, a tissue that integrates dietary
resources assimilated during ~1–2 months prior to capture (Martínez
del Rio etal., 2009). For C. oustaleti, seasonal comparisons were made
by comparing the isotopic composition of two tissues: a primary feather
(P1) reecting dietary resources assimilated during the molting period
that occurs during the austral summer in Cinclodes (Bertolero and
Zavalaga, 2003), and blood representing the winter during which they
were captured. Wechose not to correct for tissue-specic discrimination
between feathers and whole blood, which in a controlled experiment on
passerine Dendroica coronata consuming a diet containing 97% insect
were ~ 2.1‰ for δ13C and ca. 0.8 ‰ for δ15N (Pearson etal., 2003); these
values are small in comparison to observed dierences between
potential marine and terrestrial food resources available at the study
sites (Martínez del Rio etal., 2009).
Approximately 0.5–0.6 mg of dried whole blood or feather was
weighed into tin capsules, and carbon (δ
13
C) and nitrogen (δ
15
N)
isotope values were measured on a Costech 4,010 elemental analyzer
coupled to a ermo Scientic Delta V Plus isotope ratio mass
spectrometer at UNM–CSI. Isotope values are reported using standard
delta (δ) notation in parts per thousand or per mil (‰) as: δX = (R
sample
/
Rstandard–1) where Rsample and Rstandard are the ratios of the heavy to light
isotope of the sample (e.g.,
15
N/
14
N) and the reference, respectively. e
internationally accepted references are Vienna PeeDee Belemnite
(VPDB) for δ
13
C and atmospheric N
2
(AIR) for δ
15
N. Within-run
precision (SD) for both δ
13
C and δ
15
N was estimated via analysis of
three proteinaceous internal reference materials and measured to
be≤0.2‰ for both isotope systems.
Statistical analysis
Because body mass did not dier between seasons, aer checking
normality (Shapiro–Wilk) and homoscedasticity (Levene), weused a
Student t-test to compare mean BMR, TEWL, MWP/TEWL, and
thermal conductance between seasons. Because some isotopic data sets
(δ
15
N) did not meet the assumptions of normality, wedecided to use
non-parametric test to compare data. A Mann–Whitney U-test was
used to compare δ
13
C and δ
15
N values between seasons for the same
tissue between species and a Wilcoxon W test to compare δ
13
C and δ
15
N
values of feathers and blood collected from the same species to provide
a seasonal comparison. To compare physiological data between species
in winter weused ANCOVA with body mass as a covariate. Weused a
Pearson product–moment correlation matric to explore the relationship
between and diet and body water Δ
17
O using all dataset. JAMOVI
soware (Version 2.3., Jamovi project 2022) was used for all statistical
analyses. Following Mu etal. (2021), in this paper we expressed
statistical results in the language of evidence instead of the use of
arbitrary value of p thresholds (e.g., p= 0.05). is approach translates
approximate ranges of p-values into specic language, although the
boundaries of such ranges should not beunderstood as hard thresholds:
p< 0.01 as “strong evidence,” 0.01 <p< 0.05 as “moderate evidence,” and
0.05 < p 0.10 as “weak evidence”; for further details see M u 
etal. (2021).

Navarrete et al. 10.3389/fevo.2023.1120271
Frontiers in Ecology and Evolution 05 frontiersin.org
Results
Physiological capacities
For C. nigrofumosus, wefound no evidence that m
b
diered between
seasons (t8 = −1.22, p = 0.243; all results described here are presented in
Table1). Wefound strong evidence that its summer whole organism
BMR was ~19% higher (t
8
= −3.03, p = 0.009), and moderate evidence
that their TEWL was ~29% higher (t8 = −2.36, p = 0.034), than winter.
ere was no evidence that estimated water balance at 30°C (MWP/
TEWL) diered between seasons in C. nigrofumosus (t
8
 = 1.26;
p= 0.230), but wet thermal conductance increased ~18% in summer
compared with winter in this species (t
8
= −2.48; p= 0.026). A linear
regression of all data from both species collected in winter provided
strong evidence that log BMR (F1, 18= 57.58; r2= 0.76; p= 0.001) and log
TEWL (F1, 18= 20.54; r2= 0.53; p< 0.001) were positively correlated with
log body mass. Aer removing the eect of body mass, there was no
evidence of dierence between species in whole-organismal BMR (F1,
17= 0.019, p= 0.89) and TEWL (F1, 17= 1.53, p= 0.232). e ratio MWP/
TEWL was also similar between species (t
8
= 0.022; p = 0.983). Finally,
there was strong evidence that Cw was ~46% higher in C. nigrofumosus
than C. oustaleti (t8 = −7.08, p < 0.001).
Isotopic niches
ere was no evidence of a dierence between summer and winter
δ
13
C (Mann–Whitney U = 17, p= 1.0) and δ
15
N (U= 7.5, p= 0.12) values
of C. nigrofumosus (Table2). In contrast, there was strong to moderate
evidence that values of δ
13
C and δ
15
N diered between summer and
winter in C. oustaleti as feather δ
13
C values representing summer were
4‰ higher (Wilcoxon W = −28, p= 0.015) and δ
15
N values were 13‰
higher (W= −28, p= 0.05) than isotope values for blood representing
winter (Table2). ere was strong evidence that d
13
C and d
15
N values of
feathers representing summer foraging diered between species (Mann–
Whitney U= 0.0, p< 0.001; Table2). ere was no evidence that δ
13
C
values from blood collected in winter diered between species (Mann–
Whitney U= 11.0, p= 0.11), and moderate evidence of a dierence for
δ15N (U = 6, p= 0.04) (Table2). ere was no evidence that δ13C values
from blood collected in winter diered between species (Mann–
Whitney U = 11.0, p = 0.11), and moderate evidence of a dierence for
δ15N (U= 6, p= 0.04) (Table2).
Metabolic and drinking/food water
ere was little or no evidence that values of Δ
17
O diered
between summer and winter for C. nigrofumosus (t
7
= −1.76, p= 0.122,
Figure1). Using equation 2, this dierence would translate to seasonal
contributions from metabolic water to body water (Fm) of 24.8% in
summer versus 20.7% in winter. ere was no evidence that values of
Δ17O (and hence Fm) diered between species in winter (t12= 0.652,
p= 0.527) (Figure1). ere was also no evidence that the mean δ18O
values of plasma diered between winter and summer for
C. nigrofumosus (−1.4 ± 0.7‰ and −1.6 ± 0.5‰ respectively;
t
7
 = 0.277, p = 0.790), or between C. oustaleti (−3.5 ± 1.1‰) and
C. nigrofumosus (using pooled data from summer and winter:
−1.5 ± 1.3‰; t17= 1.57, p= 0.135). e mean estimated δ18O value of
the combined drinking/food water ingested by C. nigrofumosus did
not dier between seasons (winter −6.5 ± 2.4‰, summer −8.1 ± 0.8‰;
t
7
= 1.41, p= 0.203). However, wefound moderate evidence that the
drinking/food water ingested by C. oustaleti (δ
18
O = −9.7 ± 2.6‰) was
more negative than for C. nigrofumosus in winter (−6.5 ± 2.4‰,
t12= 2.18, p= 0.05) (Figure2).
Using the whole dataset (i.e., both species and seasons), there was
moderate evidence that the estimated isotopic value of drinking water
TABLE1 Mean (±SD) physiological and biochemical variables measured in
C. nigrofumosus captured in summer and winter and for C. oustaleti
captured in winter.
C. nigrofumosus C. oustaleti
Summer Winter Winter
n7 9 11
Body mass (g)73.67 ± 2.6 70.5 ± 6.3*25.5 ± 1.8*
BMR (mL O2
h −1)
179.6 ± 18.2a145.3 ± 25.2b*66.1 ± 21.2*
BMR (mL O2
h−1g−1)
2.4 ± 0.2a2.06 ± 0.3 b2.6 ± 0.9
TEWL (mg
H2O h−1)
363.9 ± 72.3a255.8 ± 102.9b*113.9 ± 48.1*
TEWL (mg
H2O h−1 g−1)
5.0 ± 1.1a3.7 ± 1.5b4.5 ± 2.0
Cw (cal h °C−1)39.4 ± 6.3a32.2 ± 5.3 b*17.4 ± 4.0*
Tb (°C) 40.9 ± 0.3 41.3 ± 0.4 40.7 ± 0.4
MWP (mg
H2O h−1)
101.8 ± 10.3a82.4 ± 14.3b*37.5 ± 12.0*
MWP/TEWL 0.28 ± 0.07 0.32 ± 0.13 0.33 ± 0.12
Letters denote signicant dierences between seasons, and asterisks denote signicant
dierences between species in winter.
TABLE2 Mean (±SD) δ13C and δ15N values of C. nigrofumosus and C. oustaleti tissues collected from a coastal locality from central Chile.
C. nigrofumosus C. oustaleti
Tissue Feathers Blood Feathers Blood
Season Summer (12) Winter (5) Summer (7) Summer (11) Winter (8)
δ13C−13.3 ± 0.6a−15.0 ± 1.5 −15.0 ± 0.8 −20.1 ± 0.7*,b −16.2 ± 0.6
δ15N19.1 ± 1.3a17.2 ± 0.5a17.8 ± 1.0 3.2 ± 0.7*b16.2 ± 0.4b
Season (winter or summer) denotes the period of the annual life cycle reected in each tissue. For migratory C. oustal eti, the isotopic composition of feathers represents dietary inputs during the
(previous) summer when they forage in streams at high elevations, while blood integrates dietary information during the season of collection (winter). For resident C. nigrofumosus, seasonal
variation in diet was assessed by comparing the isotopic composition of blood collected in winter and summer; data for feathers is shown for comparison to C. oustaleti. Asterisks denote dierences
between seasons for each tissue within species, and dierent letters denote dierences between species for the specic season and tissue.

Navarrete et al. 10.3389/fevo.2023.1120271
Frontiers in Ecology and Evolution 06 frontiersin.org
(δ
18
O
DW
) was dierent between species (t
17
= 2.18, p= 0.03) and that
d
18
O
DW
was positively correlated with the δ
13
C and δ
15
N values of tissues
(Supplementary Table S1 for statistical details). When data were
analyzed separately for each species, there was moderate evidence that
δ
18
O
DW
was positively correlated with blood δ
15
N in C. oustaleti
(r
2
 = 0.714, p = 0.034) but no evidence of a relationship in
C. nigrofumosus (r
2
 = 0.018 p = 0.7330) (Figure 3). ere was no
evidence of a relationship between F
M
and δ
15
N in either species
(r
2
 = 0.439, p = 0.15 and r
2
 = 0.02, p = 0.73 for C. oustaleti and
C. nigrofumosus respectively). Finally, when weanalyzed the whole
dataset wefound very strong evidence that Δ17O correlated positively
with δ18O values of plasma (r2= 0.54, p< 0.001).”
Discussion
e main objective of our study was to evaluate the integrated eect
of seasonal variation on selected physiological and ecological traits of
passerine birds living in a dry coastal environment. We explored
whether the interaction between a suite of physiological variables––
thermoregulation, osmoregulation, and water balance—varies
seasonally in two closely related passerine species that dier in their
consumption of marine versus terrestrial resources. Our results suggest
that C. nigrofumosus and C. oustaleti vary in their reliance on marine
resources (Table 1): δ
13
C and δ
15
N values of blood and feathers
conrmed the coastal resident C. nigrofumosus consumes a diet rich in
marine invertebrates, while the diet of migratory C. oustaleti shis
seasonally between marine (winter) and freshwater/terrestrial (summer)
resources indicative of their migration from wintering in marine
intertidal habitats to stream habitats at high elevations during the
summer in central Chile (Martínez del Rio etal., 2009; Newsome etal.,
2015; Tapia-Monsalve etal., 2018). Triple oxygen isotope analysis of
blood plasma revealed that a similar proportion of the body water pool
of both species originated from metabolic water, and the contribution
of metabolic water tended to increase in summer in C. nigrofumosus in
concert with increases in BMR and decreases in TEWL and C
w
. In the
following sections, weexplore the causes and consequences of the
seasonal variation in physiological variables and the contribution of
dierent water sources to the total water balance of each species.
Physiological parameters linked to energy
and water budget
In passerines, the intake of moderately salty water (~400 mOsm/kg
NaCl) tends to increase urine osmolality and BMR (Peña-Villalobos
etal., 2014; Sabat etal., 2017). e observed seasonal increase in BMR of
C. nigrofumosus, however, does not appear to beassociated with an
increased osmotic challenge as δ
13
C and δ
15
N data show this species
consumed a high but similar proportion of marine resources between
seasons. Previous studies suggest that the osmoregulatory physiology of
Cinclodes is inuenced by both ecological (diet composition) and
A
B
FIGURE1
(A) Mean (±SE) Δ’17O values of body water cryogenically distilled from
blood plasma and (B) the estimated proportion of metabolic water to
the total body water pool in two species of Cinclodes inhabiting a
coastal environment in central Chile.
FIGURE2
Estimated δ18O values (mean ± SE) of ingested pre-formed (drinking/
food) water in two species of Cinclodes inhabiting a coastal
environment in central Chile.
FIGURE3
Positive and significant linear correlation between blood δ15N values
and the estimated δ18O of ingested water based on Δ17O for two
species of Cinclodes inhabiting a coastal environment in central Chile.
The dotted line represents the relationship for data pooled across both
species, while the solid line represents the relationship for only C.
oustaleti.

Navarrete et al. 10.3389/fevo.2023.1120271
Frontiers in Ecology and Evolution 07 frontiersin.org
environmental (climate) factors in a complex fashion (Sabat and del Río,
2005; Sabat etal., 2006a,b). For example, isotopic data for C. nigrofumosus
from another locality in central Chile (Los Molles, 32°14’22’S
71°30’54’W) suggested a greater consumption of marine resources and
an increase in plasma concentration during winter compared to summer;
however, urine was more concentrated in the (hot and dry) summer than
in the (cold and rainy) winter (Sabat and del Río, 2005). is pattern
suggests that the eect of salt intake on osmoregulatory physiology in
C. nigrofumosus depends on environmental temperature and availability
of meteoric water. Furthermore, the mechanistic link between salt intake,
energy expenditure, and the role of metabolic water in maintaining water
balance is an intriguing topic that requires further attention.
Several studies have investigated seasonal changes in BMR and
other measures of metabolic rate (e.g., RMR or FMR) in response to
environment temperature, but results have revealed a noticeable
dierence in the magnitude of the response to thermal (i.e., seasonal)
acclimatization (Arens and Cooper, 2005; Cavieres and Sabat, 2008;
Noakes and McKechnie, 2020; Swanson etal., 2020) and the mechanisms
for the global pattern of BMR acclimatization is poorly understood. It is
generally believed that BMR in free-ranging birds is primarily driven by
temperature, although it is possible that other abiotic and biotic factors
such as photoperiod, reproduction, and body condition may
be important (Daan et al., 1990; Chastel et al., 2003; Vézina and
Williams, 2003; Zheng etal., 2008; McNab, 2009; Vezina and Salvante,
2010). Our results reveal considerable exibility in the thermal
physiology of C. nigrofumosus, as wefound a ~ 20% decrease in BMR
and thermal conductance and a ~ 30% decrease in TEWL in winter
relative to summer, but no seasonal change in body mass. Reduced
thermal conductance may enable a seasonal decline in BMR because
heat is more eectively retained in winter (Speakman and Król, 2010;
Rezende and Bacigalupe, 2015; Nord and Nilsson, 2019). is
combination of trends suggests that the intake/storage of dietary energy
and loss of heat to the environment were reduced in concert (Novoa
etal., 1994; Cooper etal., 2019). Under such a scenario, the contribution
of metabolic water to the body water pool would decrease. Our results
contrast with the typical acclimatization response of birds from higher
latitudes (McKechnie et al., 2015; Noakes and McKechnie, 2020;
Swanson etal., 2022) and supports the idea that changes in BMR is not
related to enhancing cold tolerance in areas where birds face milder
winter minimum temperatures and more modest
thermoregulatory demands.
Reproduction may also inuence BMR and TEWL because
behaviors such as nest building, courtship/mating, and parental care in
addition to synthesizing eggs are costly and inuence energy budgets
(Wiersma etal., 2004; Mainwaring and Hartley, 2013; Williams, 2018).
e inuence of reproduction on resting rates of energy expenditure
(including BMR) in free-ranging birds, however, is controversial
(Nilsson, 2002; Chastel etal., 2003; Welcker etal., 2015). While seasonal
increases in BMR can beexplained by reproductive demands, such
changes in metabolic activity may also bean adaptive response to
increase metabolic water production (MacMillen, 1990; Navarrete etal.,
2021). is hypothesis is consistent with results of both eld- and
lab-based studies that report increases in mass-specic BMR in free-
ranging desert birds during the summer (Smit and McKechnie, 2010;
McKechnie etal., 2015) and in captive sparrows (Z. capensis) who
responded to water restriction by losing mass and increasing their mass-
specic BMR. is hypothesis is also supported by the trend reported
here showing a seasonal increase in the contribution of metabolic water
to the body water pool of C. nigrofumosus in summer (see below).
Overall, it is important to note that whole-organism metabolic rate can
beaected by essentially any change in morphology or physiology, so
changes in traits such as BMR can be consistent with multiple,
non-exclusive mechanistic explanations.
Triple oxygen analysis: Water budget and
water sources
Δ
17
O results suggest that the contribution of metabolic water to the total
water budget in C. nigrofumosus was slightly higher in summer (~25%) than
in winter (~21%), in agreement with expectations based on dierences in
BMR between seasons. Both estimates are slightly lower to previously
reported
17
O-based estimates for C. nigrofumosus (~28%) sampled from
another locality ~200 km to the north of our eld site (Sabat etal., 2021). For
C. oustaleti, estimates of the metabolic water contribution (23%) were nearly
identical to those reported for this species from the more northern locality
(Sabat etal., 2021). Overall, these
17
O-based estimates for the importance of
metabolic water in Cinclodes in the eld agree with those based on (1)
respirometry under controlled conditions in the lab, where MWP/TEWL
ratios vary from 28 to 33% (Table2), and (2) doubly-labeled water (DLW)
administered to free-ranging zebra nches (Taeniopygia guttata), an arid-
adapted passerine (Cooper etal., 2019).
Given the importance of marine resources for both species in the
winter, estimated δ18O values of preformed (drinking/food) water was
expected to beclose to that of seawater (0‰); however, mean (±SD)
values were lower and signicantly diered between C. oustaleti
(−9.7 ± 2.6‰) and C. nigrofumosus (−6.5 ± 2.4‰). e mean δ
18
O value
for C. oustaleti is nearly identical to that measured inlocal meteoric and
tap waters (−9.7 ± 0.5‰, n = 3). Acknowledging that the end-member
δ
18
O value for local meteoric waters is poorly constrained at present, a
two-source mixing model shows that seawater contributes ~0–45%
and ~ 24–66% of the total water ingested by C. oustaleti and
C. nigrofumosus, respectively. ese estimates dier from those obtained
from a limited number of C. oustaleti (n = 3) and C. nigrofumosus (n = 3)
individuals sampled at a more arid locality 200 km to the north of our
study site, where ~48–100% of ingested water was sourced from seawater
(Sabat etal., 2021). is dierence could reect lower terrestrial primary
productivity at the more northern location, where higher consumption
of marine prey would result in increased intake of seawater (Sabat etal.,
2006b). Lastly, the positive correlation reported here between tissue δ
15
N
and δ
18
O of the blood plasma (Figure 3; Supplementary Table S1)
supports the hypothesis that Cinclodes do not directly drink seawater,
but passively ingest it when consuming intertidal invertebrates (Sabat
etal., 2021). Future studies that evaluate the importance of seawater in
Cinclodes along a latitudinal gradient in aridity are crucial to establish
the relative importance of ecological (resource use) and environmental
(temperature and/or humidity) factors that inuence water balance in
this unique group of passerines.
A recent meta-analysis of eld metabolic rate (FMR) and eld water
ux (FWF) collected from a diverse set of birds reported that seabirds
had a higher FMR than terrestrial species, and granivores had a lower
FMR than other functional groups (Song and Beissinger, 2020). Similarly,
seabirds and terrestrial birds inhabiting regions with higher rainfall had
higher FWF. Because the proportion of metabolic water in the total body
water pool is dependent on both metabolic rate and water intake, both
variables must beconsidered to understand water balance. Using data for
species with both FMR and FWF data (n = 59) and assuming 0.567mL
of metabolic water is produced per liter O2 consumed (Sabat etal., 2021),
the average proportion of metabolic water to the body water pool for
terrestrial birds and seabirds is 22 and 16%, respectively. e estimated

Navarrete et al. 10.3389/fevo.2023.1120271
Frontiers in Ecology and Evolution 08 frontiersin.org
proportion of the total water pool derived from metabolic water for
Cinclodes (~21–25%) is slightly higher than terrestrial birds and in the
upper range for seabirds. ese results suggest that Cinclodes is more
dependent on metabolic water than other terrestrial birds but cannot
solely rely on seawater to maintain water balance, which likely is the
result of limitations imposed by renal function. e maximum
concentrating capacity of Cinclodes urine rarely exceeds 100 mOsm/kg,
which is the concentration of seawater (Sabat etal., 2004), while seabird
salt gland secretions can bemore than twice this concentration (Sabat,
2000). Finally, it is important to note that the DLW-based estimates of
FMR and FWF for free-ranging birds are typically lower than those for
captive birds studied in the laboratory under controlled conditions
(Bartholomew and Cade, 1963; MacMillen, 1990). Overall, the
17O-based method agrees with more direct methods using DLW (eld)
or respirometry (lab), which conrms the usefulness of using triple
oxygen isotope measurements to estimate the water balance in the eld.
Potential caveats
Although our Δ
17
O-based estimates of the contribution of metabolic
water to the total body water pool of Cinclodes are consistent with
patterns in other physiological measurements (BMR, TEWL) and
previous studies using other methods (DLW), it is important to
recognize that Equation 2 is a simplication and includes assumptions
that have not yet been fully explored. While meteoric waters collected
in a wide variety of environmental contexts have a mean Δ
17
O (±SD)
value of 0.03 ± 0.02‰ (Sharp et al., 2018), a more comprehensive
understanding of how Δ
17
O values of drinking and food water available
to animals in dierent environmental contexts is needed to rene this
approach in eld-based studies of water balance. For instance, extensive
evaporation reduces the Δ’17O values of the residual water (Aron etal.,
2021; Passey and Levin, 2021), a process that could impact meteoric
waters or organism body water via evaporative water loss in animals
living in arid environments. e possibility of fractionation eects on
Δ17O could becaptured in a ux-based model, rather than the mixing-
model approach described by Equation 2. Future studies should consider
these complexities and build on the nascent applications of this method
(Pack etal., 2013; Whiteman etal., 2019; Sabat etal., 2021).
Conclusion
Our study revealed that the seasonal acclimatization response of
C. nigrofumosus is not the typical response of birds from more mesic
environments at higher latitudes. e higher BMR observed in summer
could beassociated with a higher intake of marine prey/seawater or with
the energetic costs of reproduction, which may lead to an increase in the
contribution of metabolic water to the body water pool. Triple oxygen
isotope analysis suggests that the contribution of metabolic water is
~23% of the total water budget in both Cinclodes species, with a slight
increase in summer relative to winter for C. nigrofumosus, concomitant
with the observed seasonal increase in BMR. ese results agree with
more direct methods for estimating the proportional contribution of
metabolic water to the body water pool based on DLW, conrming the
usefulness of Δ
17
O to examine the water balance of free-ranging birds.
Finally, water use strategies also diered between species with seawater
contributing 24–66% and 0–45% of the pre-formed water ingested by
C. nigrofumosus and C. oustaleti respectively, highlighting the
importance of seawater in maintaining water balance in this unique
group of passerines.
Data availability statement
e raw data supporting the conclusions of this article will bemade
available by the authors, without undue reservation.
Ethics statement
e animal study was reviewed and approved by the animal study
was reviewed and all protocols were approved by the institutional
Animal Care Committee of the University of Chile (CICUA), and
National Research and Development Agency (ANID).
Author contributions
PS, SN, and JW: designed research. LN, NL, FA, and PS: performed
research. LN and PS: analyzed data. PS, SN, RN, JS-H, KM, ZS, NL, and
JW: wrote the paper. All co-authors edited the paper. All authors
contributed to the article and approved the submitted version.
Funding
is work was funded by ANID PIA/BASAL FB0002, ANID/
CONICYT FONDECYT Regular Nº 1200386, and National Science
Foundation grants to SN (IOS-1941903) and JW (IOS-1941853).
Acknowledgments
We thank to Andrés Sazo and its invaluable eldwork assistance.
Conflict of interest
e authors declare that the research was conducted in the absence
of any commercial or nancial relationships that could beconstrued as
a potential conict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their aliated organizations,
or those of the publisher, the editors and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
e Supplementary material for this article can befound online at:
https://www.frontiersin.org/articles/10.3389/fevo.2023.1120271/
full#supplementary-material

Navarrete et al. 10.3389/fevo.2023.1120271
Frontiers in Ecology and Evolution 09 frontiersin.org
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