Functional Ecology 2014

doi: 10.1111/1365-2435.12283

Chronic exposure to low-dose radiation at Chernobyl
favours adaptation to oxidative stress in birds
n*,†1, Andrea Bonisoli-Alquati2, Shanna Jenkinson2, Ghanem Ghanem3,
Ismael Galva
Kazumasa Wakamatsu4, Timothy A. Mousseau2 and Anders P. Møller1
1

 matique et Evolution, CNRS UMR 8079, Universite
 Paris-Sud 11, Ba^timent 362, 91405
Laboratoire d’Ecologie, Syste
Orsay Cedex, France; 2Department of Biological Sciences, University of South Carolina, Columbia, SC 29208 USA;3
 rimentale (L.O.C.E.), Institut Jules Bordet, Universite
 Libre de Bruxelles,
Laboratoire d’Oncologie et de Chirurgie Expe ger-Bordet 1, 1000 Bruxelles, Belgium; and 4Department of Chemistry, Fujita Health University School of Healthrue HeSciences, Toyoake, Aichi 470-1192 Japan

Summary
1. Ionizing radiation produces oxidative stress, but organisms can adapt to their exposure withphysiological adaptive responses. However, the role of radioadaptive responses in wild populations remains poorly known.
2. At Chernobyl, studies of birds and other taxa including humans show that chronic exposure
to radiation depletes antioxidants and increases oxidative damage. Here, we present analysesof levels of the most important intracellular antioxidant (i.e. glutathione, GSH), its redox status, DNA damage and body condition in 16 species of birds exposed to radiation at Chernobyl. We use an approach that allows considering the individual bird as the sampling unit
while controlling for phylogenetic eﬀects, thus increasing the statistical power by avoiding theuse of species means as done for most previous comparative studies.
3. As a consequence, we found a pattern radically diﬀerent from previous studies in wild populations, showing that GSH levels and body condition increased, and oxidative stress and
DNA damage decreased, with increasing background radiation. Thus, when several species are
considered, the overall pattern indicates that birds are not negatively aﬀected by chronic exposure to radiation and may even obtain beneﬁcial hormetic eﬀects following an adaptive
response. Analysis of the phylogenetic signal supports the existence of adaptation in the studied traits, particularly in GSH levels and DNA damage.
4. We also show that, under equal levels of radiation, the birds that produce larger amountsof the pigment pheomelanin and lower amounts of eumelanin pay a cost in terms of decreased
GSH levels, increased oxidative stress and DNA damage, and poorer body condition. Radiation, however, diminished another potential cost of pheomelanin, namely its tendency to produce free radicals when exposed to radiation, because it induced a change towards the
production of less pro-oxidant forms of pheomelanin with higher benzothiazole-to-benzothiazine ratios, which may have facilitated the acclimation of birds to radiation exposure.
5. Our ﬁndings represent the ﬁrst evidence of adaptation to ionizing radiation in wild animals,
and conﬁrm that pheomelanin synthesis represents an evolutionary constraint under stressfulenvironmental conditions because it requires GSH consumption.
Key-words: adaptation, Chernobyl, ionizing radiation, oxidative stress, pheomelanin
Introduction
Ionizing radiation is composed of particles able to liberate
electrons from atoms or molecules and thus creates*Correspondence author. E-mail: galvan@ebd.csic.es†
Present address: Departamento de Ecologıa Evolutiva, Estaci
onBiol
ogica de Do~nana – CSIC, c/Americo Vespucio s/n, 41092
Sevilla, Spain.

partially reduced chemical species, the most common being
reactive oxygen species (ROS), which are involved in chain
reactions that are potentially damaging to cells (Riley
1994). Living organisms have evolved a diversity of antioxidant compounds that can eliminate these damaging eﬀects
by combating ROS, which are constantly produced in the
body by cellular metabolism. ROS activate cell signalling© 2014 The Authors. Functional Ecology © 2014 British Ecological Society

2 I. Galv
an et al.
pathways which may trigger adaptive responses (Vi~naet al. 2006), but when antioxidant levels are below thethresholds required to limit ROS production, it leads tostates of oxidative stress (Finkel & Holbrook 2000). Ionizing radiation is therefore an important source of oxidative
stress in cells (e.g. Simone et al. 2009). This means thationizing radiation can have profound eﬀects on the evolutionary ecology of organisms, as oxidative stress is the ultimate cause of the deterioration of phenotypes (i.e.
senescence) and the death of organisms, and it is thus considered a major determinant of the evolution of life-history
strategies (Dowling & Simmons 2009; Metcalfe & AlonsoAlvarez 2010; Galvan et al. 2012a).
However, most research on the biological eﬀects of ionizing radiation have been conducted with cells or withorganisms under laboratory conditions, which limits thecapacity to obtain information on consequences for the
ecology and evolution of organisms. Studies on wild populations are necessary to obtain a comprehensive view ofthe evolutionary consequences of ionizing radiation
because free-living populations may be limited or constrained in their ability to cope with eﬀects of ionizing
radiation. Natural background radioactivity levels show
extreme variation of several hundred-fold and haverecently been found to aﬀect mutational input and theexpression of certain phenotypic traits, but studies on natural radioactivity are still few and scattered (Galv
an &Alonso-Alvarez 2011; Møller & Mousseau 2013). Naturalradiation and radiation accidents like those produced at
the nuclear power plants of Chernobyl in 1986 and Fukushima in 2011 have had catastrophic environmental consequences, and the large levels of radioactivity released tothe environment represent involuntary experiments andgood opportunities for investigating the eﬀects of ionizing
radiation on wild populations of organisms. In Chernobyl,
several studies have reported signiﬁcant eﬀects of radiationon the abundance, distribution, life history and mutation
rates of plants and animals (Møller & Mousseau 2006),
and eﬀects on the abundance of animals have already been
detected in Fukushima (Møller et al. 2012, 2013). In particular, radioactivity from Chernobyl has been found to
produce oxidative stress by depleting antioxidants inhumans (e.g. Ivaniota, Dubchak & Tyshchenko 1998; Neyfakh, Alimbekova & Ivanenko 1998; Romanenko et al.
2000; Vartanian et al. 2004) and other animals (Møller,
Surai & Mousseau 2005; Møller, Karadasß & Mousseau2008). Radiation levels in Chernobyl have also been foundto covary with levels of cellular damage or dysfunctionthat may be mediated by oxidative damage (Sugg et al.
1996; Fenech, Perepetskaya & Mikhalevich 1997; Marozik
et al. 2007; Bonisoli-Alquati et al. 2010, 2011), and withother physiological consequences of oxidative stress suchas reductions in brain size (Møller et al. 2011) and theexpression of eye cataracts (Mousseau & Møller 2013).
There seems to be some consistency in reporting reductions in antioxidant levels and increases in oxidative damage in animals exposed to radioactive contamination (seestudies mentioned above). Some authors, however, havefound in humans that the levels of some antioxidants caneven increase at low doses of radiation, although high levels of radiation may deplete antioxidants (Ivanenko &Burlakova 2013), and a recovery of oxidative status can beproduced over time (Skesters et al. 2010). Indeed, the highdegree of radioactive contamination found in the region ofChernobyl and the relative long time (28 years) elapsed
since the accident make this an excellent scenario for investigating possible mechanisms of adaptation to ionizing
radiation in natural populations.
Radiation-induced adaptive responses have been well
documented for decades in a diversity of species including
humans through experiments in which cells or organisms
are exposed to low doses of radiation (priming or conditioning dose) before receiving a higher, challenging dose
(Olivieri, Bodycote & Wolﬀ 1984; Iyer & Lehnert 2002).
These studies have shown that chronic exposure to low,‘adapting’ doses of diﬀerent types of radiation increases
the resistance of cells against subsequent acute exposure tochallenging doses (Tapio & Jacob 2007). Correlative studies also report some evidence of radio-adaption in cells
from humans chronically exposed to low levels of radioactivity in Chernobyl (Tedeschi et al. 1995). The ultimate
mechanisms of the radio-adaptive response include complex patterns of cellular signalling and epigenetic changes
that would favour the transmission of the response to theoﬀspring (Kovalchuk et al. 2004). It seems that the starting point of the mechanism is not the direct eﬀect of ionizing radiation on cellular structures, but an induction by
ROS generated by radiation, which causes DNA damage(Tapio & Jacob 2007). As mentioned above, ROS generated by radiation is probably the cause of the observed
decreases in antioxidant levels and increases in oxidative
damage in humans and other animals from Chernobyl.
Thus, there is evidence of physiological costs of radiation
exposure in natural populations at Chernobyl, but not of
adaptation to it. Alternatively, however, organisms may
show adaptive responses to radiation at Chernobyl. This ispotentially plausible given the observed positive eﬀect of
low-dose radiation on some antioxidants (Ivanenko & Burlakova 2013), the recovery in the antioxidant status a longtime after exposure (Skesters et al. 2010), and the fact thatmany organisms in the Chernobyl region have been chronically exposed to low doses of radiation, conditions that
may favour radio-adaption (Tapio & Jacob 2007). Searching for adaptive responses to radiation in natural populations is of key importance as it can potentially determine
the capacity of species to evolve physiological adaptations
and thus diﬀerential susceptibilities to overcome environmental challenges such as those that occurred in Chernobyl and Fukushima (Somero 2010).
Adaptive responses to radiation in natural populations
at Chernobyl may not have been detected for several reasons. The large variability in radiation levels found in theentire Chernobyl zone represents a continuous environmental gradient, although there is a high temporal© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology

Adaptation of birds to ionizing radiation 3
consistency in the background radiation levels to which
individual organisms are exposed within their home ranges
at Chernobyl (see Materials and Methods), which may
limit the capacity to detect adaptive responses in natural
populations. In addition, it has been reported that therange of acute lethal doses of artiﬁcial ionizing radiation
varies greatly among taxa, which is for example considerably greater in plants than in higher vertebrates, and lower
in birds than in mammals (Newman & Unger 2003). This
among-taxa diﬀerence in susceptibility to radiation has
already been reported in animals from Fukushima andChernobyl regarding eﬀects on population abundance(Møller et al. 2013), and susceptibility to natural radioactivity also varies among taxa (Møller & Mousseau 2013).
Studies on the biological consequences of radioactivity at
Chernobyl have concentrated on a few taxa (Møller &Mousseau 2006), and in the particular case of studies thatreport antioxidant and oxidative damage levels, they are
all intraspeciﬁc and limited to humans, two species of birds
and one species of ﬁsh (see references cited above). Therefore, the among-taxa variability in susceptibility to radiation may represent an additional limitation in the capacity
to detect radio-adaptive responses. In fact, although theeﬀects of radioactivity on bird populations at Chernobyl
are negative overall, some species’ populations grow withincreasing radiation (Galvan, Mousseau & Møller 2011),
lending support to the potential role of an adaptive
response to chronic radiation exposure. Comparative studies may represent a solution for the two limitations mentioned above. Comparing several species that show
diﬀerent susceptibilities to radiation and that are subjected
to a range of radiation levels enhances the capacity todetect eﬀects. Comparative studies in which several phylogenetically distant species are investigated for antioxidant
status have to our knowledge never been conducted in natural populations at Chernobyl, but are clearly necessary
for developing insight into the potential role of radioadaption for the evolution of organisms.
The aim of this study was to investigate covariation
between levels of glutathione (GSH) and DNA damage
with levels of background radiation in wild populations ofseveral phylogenetically distant species of birds in the
Chernobyl region. We focus on GSH because it is one of
the antioxidants most susceptible to radiation (Riley 1994;
Ivaniota, Dubchak & Tyshchenko 1998; Neyfakh, Alimbekova & Ivanenko 1998; Ivanenko & Burlakova 2013),
the most important intracellular antioxidant, and its redox
status (GSH/GSSG) represents a relevant index of cellularoxidative stress (Wu et al. 2004). We consider DNA damage as measured by the comet assay, which quantiﬁes
strand breaks, as this is the most common damage to
DNA caused by ionizing radiation (e.g. Kovalchuk et al.
2004). In two species of birds from Chernobyl (the barnswallow Hirundo rustica and the great tit Parus major), circulating antioxidant levels have decreased and oxidative
damage has increased with radiation levels (Møller, Surai& Mousseau 2005; Møller, Karadasß & Mousseau 2008;

Bonisoli-Alquati et al. 2010, 2011). Thus, we predict thatthe same patterns should be found at the interspeciﬁc level
regarding GSH and GSH/GSSG if birds exposed to radioactive contamination show a general and consistent physiological cost mediated by radiation. Alternatively, if there
has been an adaptive response by birds to the chronic
exposure of background radiation at Chernobyl, radiation
should improve, at least up to certain level, the antioxidant
response of birds. This should in turn prevent ﬁnding adecrease in levels of GSH and GSH/GSSG and an increase
in DNA damage (which is probably caused by radiationinduced oxidative stress; Bonisoli-Alquati et al. 2010) andbody condition (which predicts the survival of birds; Møller & Szep 2001) with increasing radiation. Such an adaptive response may however be costly to maintain, and suchcosts may be reﬂected in the population trends of birds.
Therefore, we also analysed associations between theintensity of the physiological response and population
trends of the species of birds at Chernobyl (Galv
an, Mousseau & Møller 2011).
When searching for possible diﬀerential capacities of
species to adapt to chronic exposure to radiation, it is necessary to consider the production of melanins, the mostcommon animal pigments. We have previously found that
populations of species of birds expressing plumage colours
typically provided by pheomelanin, a polymer of benzothiazine and benzothiazole units that constitutes one of thetwo main types of melanin, are more susceptible to thenegative eﬀects of radiation at Chernobyl (Galv
an, Mousseau & Møller 2011). The hypothesized mechanism behind
this observation is that the sulphhydryl groups of cysteine
and GSH are incorporated into the pheomelanin structureduring its synthesis in melanocytes (Garcıa-Borr
on & Olivares S
anchez 2011; Ito et al. 2011a). Therefore, pheomelanin synthesis represents a consumption of an antioxidant
resource because GSH (which is also the main physiological reservoir of cysteine) can no longer exert its antioxidantrole once incorporated into the structure of the pigment,
which is then deposited in inert tegumentary structures
such as feathers and hair (Pavel, Smit & Pizinger 2011).
Thus, pheomelanin synthesis represents a physiologicalcost under exposure to environmental factors that produce
high levels of oxidative stress, as these conditions lead togreater demands of GSH for antioxidant protection(Galv
an, Ghanem & Møller 2012). However, it has neverbeen directly tested if pheomelanin production entails
GSH depletion and oxidative stress in organisms exposed
to ionizing radiation. This test is necessary to determine
why species producing large amounts of pheomelanin are
more susceptible to the eﬀects of radiation (Galv
an, Mousseau & Møller 2011). Therefore, we predict that underequal levels of background radiation birds with higher levels of pheomelanin in feathers should have lower levels of
GSH and higher oxidative stress and DNA damage thanbirds producing lower amounts of pheomelanin. In contrast, eumelanin, a polymer of 5,6-dihydroxyindole-2-carboxilic acid (DHICA) and 5,6-dihydroxyindole (DHI) units© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology

4 I. Galv
an et al.
that constitutes the other main type of melanin, is produced in the absence of cysteine and GSH (Garcıa-Borr
on& Olivares Sanchez 2011; Ito et al. 2011a) and protects cell
survival and decreases DNA damage under exposure toionizing radiation (Kinnaert et al. 2004). We thus predictthat the content of eumelanin in feathers should enhanceoxidative status and reduce DNA damage in birds exposed
to equal levels of background radiation.
Lastly, given that the two units of pheomelanin have different oxidation potentials, benzothiazine having a greater
reducing ability than benzothiazole, which is rather stabletowards oxidation (Wakamatsu, Ohtara & Ito 2009; Wakamatsu et al. 2012), we tested the possibility of a radiationmediated conversion of benzothiazine into benzothiazole.
Benzothiazole has a higher oxidation potential than benzothiazine, which makes the former produce more ROS when
exposed to energetic radiation (Takeuchi et al. 2004; Ye
et al. 2006). Thus, a conversion of benzothiazine into benzothiazole may protect birds at sites with radioactive contamination. We tested this by analysing the eﬀect ofbackground radiation on the ratio of thiazole-2,4,5-tricarboxylic acid (TTCA, a degradation product speciﬁc to the
benzothiazole moiety; see Methods) to 4-amino-3-hydroxyphenylalanine (4-AHP, a degradation product speciﬁc tothe benzothiazine moiety). We tested all predictions in wild
populations of birds that were sampled in several sitesaround Chernobyl with a large range of background radiation levels.

surements with those reported by the Ukrainian Ministry of
Emergencies. Once having ﬁnished a 5-min point count we measured radiation levels 2–3 times at ground level directly in the ﬁeld
at each point where we censused birds, using a hand-held dosimeter. We cross-validated our measurements against measurements
published by Shestopalov (1996), estimated at the midpoints of
the ranges published in the Chernobyl atlas. This analysis revealeda very strong positive relationship [linear regression on log-log
transformed data: F1,252 = 154649, R2 = 086, P < 00001, slope(SE) = 128 (010)], suggesting that our ﬁeld estimates of radiation
provided reliable measurements of levels of radiation among sites.
These measures of residential background radiation levels also
represent actual doses received by individual birds because background radiation levels and external and internal doses arestrongly positively correlated (T.A. Mousseau, A.P. Møller, D.
Tedeschi & A. Bonisoli-Alquati unpublished manuscript).
Repeatabilities in background radiation levels for the same individuals were estimated at three diﬀerent intervals: among captures
within the same day, among captures at the start and later on in
the season, and between years. All three repeatability estimates
were large and highly signiﬁcant (during the same day: r = 0997,
F26,45 = 104198,
P < 00001;
withinseason:r = 0985,
P < 00001;
among
years:
r = 0892,
F26,33 = 12940,
F60,61 = 1752, P < 00001). Repeatability estimates decreased withincreasing intervals between the captures. Still the repeatability ofbackground radiation was as high as 089 when based on estimatesobtained in subsequent years. Repeatabilities of this magnitude
are considered high by any yardstick (Becker 1984; Falconer &Mackay 1996). Individuals were almost always recaptured at thesame site as where they were ﬁrst captured. This is not surprising
given the high degree of site philopatry among birds during thebreeding season.

MEASUREMENT OF GLUTATHIONE LEVELS IN

Materials and methods

ERYTHROCYTESFIELD METHODS

We captured birds in mist nets at eight sites within and close to
the Chernobyl Exclusion Zone on May 25 through June 5, 2010
from four pairs of relatively uncontaminated and contaminatedsites (see Table S1 in Supporting Information). We used 35–50
mist nets 12 m long each for two consecutive days at each of thestudy site (i.e. one evening and one morning capture session ateach site). In addition, for capturing barn swallows Hirundo rustica we used mist nets deployed across the doors and windows ofbarns in farms, both within and just outside the Chernobyl Exclusion Zone. All birds were banded with a unique aluminium bandfor individual identiﬁcation and then sexed and aged according tostandard criteria, sampled for feathers, blood and sperm, andreleased. Blood samples were obtained by venipuncture at thewing artery with a sterilized needle and collected using heparinizedcapillary tubes, preserved in RNALater (only those for DNA
damage measurement; Ambion Life Technologies, Grand Island,
NY, USA) and kept on ice in the ﬁeld and stored at 4 °C upon
arrival in the laboratory. The birds belonged to 16 diﬀerent species (Fig. 1), although information on some variables was notavailable for some species (see below and Table S1).

MEASUREMENT OF BACKGROUND RADIATION LEVELS

We measured background radiation levels at the exact capture
spot of each bird using a hand-held dosimeter (Model: Inspector,
SE International, Inc., Summertown, TN, USA). We have previously measured the level of background radiation in the ﬁeld inconnection with bird census studies and cross-validated these mea-

GSH was measured by HPLC following a modiﬁed procedure of
the technique developed by Araki & Sako (1987). Brieﬂy, 20 lLof red blood cell concentrate was lysed with 50 lL of a 100 mL
buﬀer solution containing 829 mg NH4Cl, 37 mg Na2EDTA/1 g
KHCO3. 50 lL of 10% TBP-tri-n-butylphosphine in dimethylformamide (DMF; reduction step = total glutathione) or 50 lL of
DMF (reduced GSH) was then added following incubation for
30 min at 4 °C. Then, proteins were precipitated by 10% chilled trichloroacetic acid containing 1 mM EDTA, vortexed vigorously andcentrifuged at 1000 g for 5 min at 4 °C. To 50–100 lL of the supernatant, 100–200 lL borate buﬀer (pH 95; 02 M) containing 4 mM
Na2EDTA and 100 lL of SBD-F (ammonium 7-ﬂuorobenzo2-oxa1, 3-diazole-4 sulphonate) 1 mg mL 1 in borate buﬀer were added.
The mixture was incubated for 60 min at 60 °C under constant agitation. The tubes were then cooled on ice, passed through a045 lm ﬁlter and 20 lL were separated on HPLC.
A Waters HPLC system (Waters Corporation, Milford, MA,
USA) was used with separation on a RP C18-ODS column(Chrompack Intersil, 15 cm 9 46 mm) using a phosphate buﬀer(pH: 60; 1/15 M) and methanol/H2O (50/50) as the eluent under agradient of 0–98% over 15 min with a ﬂow rate of 05 mL min 1.
Fluorescence was measured using an excitation wavelength of
385 nm and an emission of 515 nm. HPLC data were analysedwith the software MILLENNIUM 4.0 (Waters Corporation). We
obtained information on GSH and GSSG for 13 species of birds.

MEASUREMENT OF DNA DAMAGE LEVELS

We used red blood cells, which are nucleated in birds, for analysis
of genetic damage. We performed the comet assay using the© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology

Adaptation of birds to ionizing radiation 5
EV1

EV4

EV3

Lanius collurio
Parus major
Hirundo rustica
Phylloscopus sibilatrix
Sylvia atricapilla
Sylvia communis
Sylvia nisoria
Anthus trivialis

Fig. 1. Phylogenetic hypothesis for the species of birds used in the study. The sign ofthe scores of the species in the eigenvectors
obtained from phylogenetic vector regressions (PVR) made on the response variables is depicted by contrasting pairs of
diﬀerent colours. The ﬁrst phylogenetic
eigenvector (EV1) was selected for the
analysis of GSH levels and GSH:GSSG
ratio, the fourth eigenvector (EV4) was
selected for the analysis of DNA damage
scores, and the third eigenvector (EV3) was
selected for the analysis of body mass. Negative vs. positive eigenvector scores are represented by contrasting, respectively, redand blue branches (for EV1), yellow andblack branches (for EV4) and purple andgreen branches (for EV3).

protocol described by Singh et al. (1988) with modiﬁcations.
Avian red blood cells are highly susceptible to damage at alkali
labile sites, so performing electrophoresis at the recommended pHof >131 inﬂates damage estimates considerably. Because of theincreased sensitivity to alkaline conditions, we performed a modiﬁed comet assay with an alkaline unwinding step, but neutral electrophoresis conditions.
All steps were performed under incandescent light to prevent
additional DNA damage. Single-frosted slides (VWR, Radnor,
PA, USA) were prepared in advance by dipping the slides in 15%
normal melting-point agarose (BioRad, Hercules, CA, USA)
twice; the backs of the slides were then wiped clean and the slides
were allowed to dry for at least 24 h prior to use for the cometassay. Approximately, 5 lL of hemolymph in 50 lL of 19 PBS
was added to 450 lL of 1% low melting-point agarose (Amresco,
Solon, OH, USA) and 100 lL of the agarose mixture was immediately layered onto the prepared slides and covered with a glass
coverslip, then allowed to solidify for 5 min at 4 °C. The coverslip
was then removed and a second layer of 100 lL of low meltingpoint agarose was layered on top of the ﬁrst and covered again
with a coverslip, which was removed after 5 min. Two samples
were placed on each slide, with a total of four replicates for eachindividual. The slides were allowed to incubate for 1 h at 4 °C toallow the gel to fully solidify. The slides were then immersed in cold
lysis buﬀer (1% sodium sarcosinate, 25 M NaCl, 100 mM
Na2EDTA, 10 mM Tris, 1% Triton X-100, pH 10 with the Triton
X-100 added immediately prior to use) and kept for 1 h at 4 °C.
The slides were rinsed with cold ddH2O, and then immersed inalkali buﬀer (300 mM NaOH, 1 mM Na2EDTA, pH = 121) toallow the DNA to unwind for 30 min at 4 °C. Electrophoresis was
conducted using neutral buﬀer (300 mM sodium acetate, 100 mM
Tris, pH 10) for 30 min at 07 V cm 1 and 100 mA at 4 °C. We
rinsed the slides three times for 5 min each in a neutralization

Fringilla coelebs
Coccothraustes coccothraustes
Turdus viscivorus
Turdus philomelos
Turdus merula
Phoenicurus ochruros
Erithacus rubecula
Luscinia luscinia

buﬀer (04M Tris, pH 74) followed by 15 min in 70% ethanol fordrying. The slides were then placed in a darkened cupboard andallowed to dry overnight before storage in a dark slide box.
Slides were stained using a 1 : 10,000 dilution of SYBRâ Gold
(Trevigen Inc., Gaithersburg, MD, USA) and images were captured using METASYSTEMS METAFER 4 software (Metasystems
Group, INC, Newton, MA, USA), using a Zeiss Axioskop (Carl
Zeiss Microscopy, LLC, Thornwood, NY, USA) ﬁtted with anautomated slide stage. We captured 100–300 cells for each slide
analysed and used the CometScan module for automated analysis
at 209 magniﬁcation.
Standard comet parameters were automatically captured foreach cell, and percent DNA in tail was selected as the best parameter for analysis, which is one of the most widely used parameters
for analysis (Kumaravel et al. 2009). We calculated our estimate of
DNA damage based on the average percent of DNA in the tail ofeach comet from all cells measured on all side replicates for eachindividual. Distributions of tail damage within each individual areoften not normally distributed, so we also considered the median asa measure of central tendency, as well as the 75th percentile because
it is less sensitive to extreme values (Duez et al. 2003). We obtained
information on these variables for 14 species of birds.

MEASUREMENT OF MELANIN LEVELS IN FEATHERS

Feathers were collected from a total of 16 species, comprising 152
individual birds. A total of 10–20 feathers were plucked from thetwo most conspicuous colour patches of the plumage of birds, andstored in plastic bags in the dark until analyses were made. The
analyses of melanin content in feathers were thus made on mixtures of feathers from diﬀerent colour patches to get a generalindex of the melanin content in the plumage of the species.

© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology

6 I. Galv
an et al.
The microanalytical methods to quantify the amounts of eumelanin and pheomelanin were based on the formation and detection
by HPLC of speciﬁc degradation products, 4-AHP by reductivehydrolysis of pheomelanin with hydriodic acid (HI) (Wakamatsu,
Ito & Rees 2002) and pyrrole-2,3,5-tricarboxylic acid (PTCA) andTTCA by alkaline H2O2 oxidation of eumelanin and pheomelanin
respectively (Ito et al. 2011b). Thus, 4-AHP and TTCA are speciﬁc to pheomelanin and PTCA is speciﬁc to eumelanin. Feather
samples were ﬁrst homogenized with Ten-Broeck glass homogenizer at a concentration of 10 mg mL 1 water.
For 4-AHP analyses, 100 lL of sample homogenate was taken
in a 10 mL screw-capped conical test tube, to which 20 lL 50%
H3PO2 and 500 lL 57% HI were added. The tube was heated at
130 °C for 20 h, after which the mixture was cooled. An aliquot(100 lL) of each hydrolysate was transferred to a test tube andevaporated to dryness using a vacuum pump connected to a dry
ice-cooled vacuum trap and two ﬁlter ﬂasks containing NaOH pellets. The residue was dissolved in 200 lL 01 M HCl. An aliquot(10–20 lL) of each solution was analysed on the HPLC system.
For PTCA and TTCA analyses, 100 lL of sample homogenate
was taken in a 10 mL screw-capped conical test tube, to which
375 lL 1 M K2CO3 and 25 lL 30% H2O2 (ﬁnal concentration:15%) were added. The mixture was mixed vigorously at
25  1 °C for 20 h. The residual H2O2 was decomposed by adding 50 lL 10% Na2SO3 and the mixture was then acidiﬁed with
140 lL 6 M HCl. After vortex-mixing, the reaction mixture wascentrifuged at 4000 g for 1 min, and an aliquot (80 lL) of thesupernatant was directly injected into the HPLC system.
HI reductive hydrolysis products were analysed with an HPLC
system consisting of a JASCO 880-PU liquid chromatography, a
JASCO C18 column (JASCO Catecholpak; 46 9 150 mm; 7 lm
particle size) and an EICOM ECD-300 electrochemical detector(Eicom, Kyoto, Japan). The mobile phase used for analysis of 4AHP was 01 M sodium citrate buﬀer, pH 30, containing 1 mM
sodium octanesulphonate and 01 mM Na2EDTA: methanol, 98:2(v/v). Analyses were performed at 35 °C at a ﬂow rate of07 mL min 1. The electrochemical detector was set at +500 mV
vs. an Ag/AgCl reference electrode. A standard solution (10–20 lL) containing 500 ng each of 4-AHP and 3-AHP (3-amino-4hydroxyphenylalanine; 3-aminotyrosine from Sigma) in 1 mL
01 M HCl was injected every 10 samples.
H2O2 oxidation products were analysed with the HPLC system
consisting of a JASCO 880-PU liquid chromatography, a Shiseido
C18 column (Shiseido Capcell Pak MG; 46 9 250 mm; 5 lm particle size, Shiseido Co., Ltd., Tokyo, Japan) and a JASCO UV
detector. The mobile phase was 01 M potassium phosphate buﬀer,
pH 21: methanol, 99:1 (v/v). Analyses were performed at 45 °C at
a ﬂow rate of 07 mL min 1. Absorbance of the eluent was monitored at 269 nm. A standard solution (80 lL) containing 1 lg
each of PTCA, PDCA (pyrrole-2,3-dicarboxylic acid), TTCA and
TDCA (thiazole-2,3-dicarboxylic acid) in 1 mL water was injected
every 10 samples. We obtained information on these variables for
16 species of birds.

DATA ANALYSES

We analysed the relationships between the response variables(GSH level, GSH:GSSG ratio, DNA damage level and bodymass) and background radiation and melanin levels (predictor
variables, which also included wing chord in the case of the model
for body mass to account for variation in body condition independent of body size) by means of partial least squares regressions(hereafter PLSR; Carrascal, Galvan & Gordo 2009). This statistical tool is an extension of multiple regression analysis where associations are established with components extracted from predictor
variables that maximize the explained variance in the response
variable. These components are deﬁned as a linear combination ofpredictor variables, so the original multidimensionality is reduced
to a lower number of orthogonal components to detect structurein the relationships between predictor variables and between thesefactors and the response variable. The extracted components
account for successively lower proportions of original variance.
When multiple response variables are used, PLSR creates a synthetic response variable from the linear combination of the original response variables. Results obtained with PLSR are similar to
those from conventional multiple regression techniques. However,this method is extremely resilient to the eﬀects of sample size anddegree of correlation between predictor variables, which makes
PLSR especially useful when the sample size is small and in casesof severe multicollinearity (Carrascal, Galv
an & Gordo 2009).
There was a high degree of correlation among our predictor variables (Pearson’s correlation test: TTCA-4-AHP: r = 072,
N = 152, P < 00001; TTCA-PTCA: r = 018, N = 152,
P = 0023; PTCA-4-AHP: r = 019, N = 151, P = 0022; TTCAradiation: r = 046, N = 152, P < 00001; 4-AHP-radiation:r = 033, N = 151, P < 00001; PTCA-radiation: r = 035,
N = 152, P < 00001), which makes PLSR the most appropriate
analytical tool for our data. We also used PLSR to test the eﬀectof radiation on TTCA:4-AHP ratio, and to test for the eﬀects ofthe physiological responses found here on the population trendsof the species. To estimate the intensity of the physiologicalresponses, we obtained the studentized residuals of the regressions
between the response variables (i.e. GSH levels, GSH:GSSG ratio,
DNA damage score and body mass) and the scores of the PLSR
components described above, and population trends were calculated as the slope estimates of the relationship between abundance
and radiation levels (taken from Appendix 1 in Galv
an, Mousseau& Møller 2011). The latter test was made using the mean residualsof the regressions between the response variables and the scores of
the PLSR components per species, which were predictor variablesin PLSR models while slope estimates were the response variable.
The signiﬁcance of the extracted PLSR components was determined with two criteria. First, a cross-validation test of theparameter Q2 was carried out to determine if a component wassigniﬁcant. Then, we tested the signiﬁcance of the correlation coefﬁcient of the relationship between PLSR scores for the response
variable and PLSR component scores, thus determining if theamount of variance explained in the response variable was signiﬁcant. We also determined the contribution of predictors to the
PLSR model with two complementary criteria. First, we calculated the relative contribution of each variable to the derived
components by means of the square of the predictor weight,
considering that a predictor was important when it accounted
for more than 5% of the total variance in the response variable
explained by the PLSR component (i.e. square weight >005).
The second criterion consisted of testing the statistical signiﬁcance of the regression coeﬃcients of the predictors, thus determining the degree of correlation between the response variable
and these predictors. The latter test was made by bootstrapping
using 1000 replications. PLSR analyses were made with STATISTICA 8.0. (StatSoft, Inc., Tulsa, OK, USA) and TANAGRA 1.4(Rakotomalala 2005).
Bird species are evolutionarily related through their phylogenetic history, which can lead to an overestimation of degrees of
freedom if phylogenetic relationships are not taken into account(Felsenstein 1985). We used phylogenetic eigenvector regression(PVR) to correct for the eﬀect of common ancestry in the analysisof the relationship between the response variables and backgroundradiation and melanin levels (Diniz-Filho, De Sant’ana & Bini
1998). Diniz-Filho & Torres (2002) and Martins, Diniz-Filho &Housworth (2002) tested several comparative methods (Felsenstein’s independent contrasts, autoregressive method, PVR andphylogenetic generalized least squares) and found that PVR yields
good statistical performance regardless of the details of the evolutionary mode used to generate the data, and provides similar© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology

Adaptation of birds to ionizing radiation 7
results to other methods, with very good (i.e. low) type I and II
errors. Moreover, PVR does not assume any evolutionary model a
priori (an advantage if the true evolutionary model is unknown or
if it is complex), and it gives similar statistical performance evenfor evolutionary processes that are distinct from Brownian motion(i.e. evolutionary changes are added to values present at the previous node on a phylogenetic tree, thus creating similarity between
recently diverged lineages; e.g. Blomberg, Garland & Ives 2003).
PVR is based on the eigenfunction decomposition of phylogenetic
distance matrices, so that the phylogenetic relationships between
species can be translated into predictor variables (phylogenetic
eigenvectors) that capture phylogenetic eﬀects (Diniz-Filho, De
Sant’ana & Bini 1998).
To obtain the phylogenetic eigenvectors for the species of birds
included in the study, we used the PVR approach in the software
SAM 4.0 (Rangel, Diniz-Filho & Bini 2010) considering the species’ mean values of our response variables and of melanin levels
in feathers, thus considering correlations between variables whileobtaining the eigenvectors. The phylogenetic hypothesis used
(Fig. 1) was taken from the species-level supertree constructed by
Davis (2008), assuming all branch lengths being equal to unity.
SAM makes an approach to PVR that represents the only comparative method that can deal with non-normal variable distributions, thus being the most robust method to deviations fromnormality in the response variables (Dormann et al. 2007). However, PVR can ignore important phylogenetic information if traits
evolve under Brownian motion (Rohlf 2001), so we ﬁrst tested
the evolutionary model of our response variables by using phylogenetic signal-representation (PSR) curves (Diniz-Filho et al.
2011). PSR curves represent the amount of divergence in traits(measured by PVR’s R2) along the eigenvectors against the
cumulative eigenvalues of the eigenvectors. A linear relationshipbetween these parameters is indicative of Brownian motion, andnegative or positive deviations indicate that species resemble eachother less or more, respectively, than expected under Brownian
motion in an analogous way to Blomberg, Garland & Ives’s(2003) K-statistic (Diniz-Filho et al. 2011). Mean deviations fromthe PSR curve also represent a measure of phylogenetic signal(Diniz-Filho et al. 2011). We constructed PSR curves for our
response variables using the software PAM 0.9 (Phylogenetic
Analysis in Macroecology; T.F. Rangel & J.A.F. Diniz-Filho,
unpublished) considering the ﬁrst eight eigenvectors. Mean
deviations from the PSR curve were negative in the four response

variables, indicating that closely related species are less similar
regarding these traits than expected under Brownian motion evolution (Fig. 2). When traits follow this kind of nonlinear modelof evolution, only part of the eigenvectors can be used todescribe the phylogeny because not all eigenvectors are equally
useful for this aim (Diniz-Filho et al. 2011). Thus, from the phylogenetic eigenvectors generated with SAM, we selected thosethat reduced the largest amount of autocorrelation in the residuals below an arbitrarily deﬁned threshold for Moran’s I or its
statistical signiﬁcance, which is the most appropriate selection
method (Diniz-Filho et al. 2012).
Only one phylogenetic eigenvector was selected for the analysis of each response variable. The ﬁrst phylogenetic eigenvector(EV1) was selected for the analysis of GSH levels and GSH:
GSSG ratio, and discriminated birds from the families Turdidae,
Muscicapidae, Motacillidae and Fringilidae (positive scores)
from the rest of the phylogeny (negative scores) (Fig. 1). EV4
and EV3 were selected for the analyses of DNA damage andbody mass, respectively, and discriminated the genus Turdus(negative scores) from the rest of the phylogeny (positive scores)(Fig. 1).
PVR has the additional advantage that the extracted phylogenetic eigenvectors can be used as predictor variables in any otherstatistical linear model to correct for phylogenetic eﬀects on
response variables (Diniz-Filho, De Sant’ana & Bini 1998; Dormann et al. 2007; Diniz-Filho et al. 2011, 2012). In our case, thisfeature allowed us to use the individual bird as the sampling unit
in the PLSR models instead of the species, while controlling forthe eﬀect of common ancestry among groups of individual birds
that belong to the same species (Martins & Hansen 1996). Thispermits the analyses to include the entire variability in backgroundradiation levels, which would be highly reduced if the mean valuesof species were considered instead, which in turn would represent
a limitation to ﬁnding possible relationships between the response
variables and radiation levels as mentioned before (see Introduction). Thus, after obtaining the phylogenetic eigenvectors using
the species mean values of the variables as described above, weassigned the same eigenvector score of each species to all individuals belonging to that species, hence constituting predictor variables
that were added to the PLSR models. Therefore, we conducted
the analyses considering that all individuals of the same species
constitute a hard polytomy in the phylogeny (Purvis & Garland
1993), as they are all equally related to each other (in phylogenetic

Fig. 2. Phylogenetic signal-representation(PSR) curves for the response variables
analysed in the study constructed with theresults of eight phylogenetic vector regressions (PVR) sequentially increasing thenumber of eigenvectors. R2 indicates theamount of variance in the response variables that is explained by the phylogenetic
eigenvectors, and is represented against the
eigenvalues of the eigenvectors used in
the PVR models expressed as proportion of
the trace. The 45° dashed line represents
the expected pattern under Brownian
motion. Inserts are the mean values of thediﬀerence between R2 and eigenvalue,
which is indicative of the phylogenetic signal in the traits. DNA damage refers to themean percentage DNA in the comet tail as
measured by means of the comet assay.
© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology

8 I. Galv
an et al.
terms) and are separated by the same phylogenetic distance fromthe other groups of conspeciﬁc individuals.
Although we have found GSH:GSSG ratios <1 (which indicate
more oxidized than reduced glutathione and thus an impaired oxidative status) in other studies with birds (own observations), in
this study we found GSH:GSSG ratios below 1 in several of ourbird samples (see Results), so to determine if these cases were notnormal values potentially aﬀecting results, we repeated the analyses excluding samples with the lowest ratios (lower than or equal
to 05). Furthermore, the sex of birds was added as a predictor
variable to all PLSR models, but it never contributed importantly
to them (i.e. accounting for more than 5% of the total variance inthe response variable explained by the model), so it was removed
from the analyses. We also included radiation level squared as apredictor to account for nonlinear relationships between the
response variables and radiation, but it also was not an important
predictor in any model. Lastly, to determine if results considering
several species of birds diﬀer from previous intraspeciﬁc studies on
eﬀects of radiation on antioxidant and oxidative damage levels at
Chernobyl (Møller, Surai & Mousseau 2005; Møller, Karadasß &Mousseau 2008; Bonisoli-Alquati et al. 2010, 2011), we also show
the results of analyses considering data from the barn swallow
only, as only this species had sample sizes suﬃcient for robust statistical power in intraspeciﬁc tests, and it was one of the two species considered in the previous studies.
The data used for this study are archived in the Dryad Digital
Repository (Galvan et al. 2014).

Results
EFFECT OF RADIATION ON GLUTATHIONE LEVELS

The mean (SE) GSH level in birds was
62176  4438 ng mg 1 pellet, and ranged from 2389 to
259820 ng mg 1. The mean redox status of GSH, represented by the GSH:GSSG ratio, was 180  014, and ranged from 003 to 847.
The PLSR model for GSH levels resulted in a signiﬁcantcomponent that explained 192% (P < 00001) of the variance in this variable. The eﬀect of background radiationlevel was positive, indicating that GSH levels increase withradiation. Other important predictors were the markers forpheomelanin content of feathers (TTCA and 4-AHP),

which had a negative eﬀect on GSH levels (Table 1). This
indicates that under equal levels of background radiation
the birds that produce more pheomelanin have lower levelsof GSH. The phylogenetic eigenvector (EV1) was also an
important predictor, but the eumelanin content of feathers
was not (Table 1). All important predictors except TTCA
were also signiﬁcant (Table 1). GSH levels were signiﬁcantly positively correlated with the PLSR component(r = 044, N = 120, P < 00001; Fig. 3a). Results did notchange when samples with GSH:GSSG ratios lower than05 (N = 20) were excluded, as one signiﬁcant PLSR component was obtained explaining 175% (P < 00001) ofvariance in GSH levels with the same important predictors
as the model with all data (predictor weights: radiation:
053, TTCA = 028, 4-AHP = 055, EV1 = 056) andshowing the absence of contribution of the eumelanin content of feathers (PTCA’s predictor weight = 012). When
only data for the barn swallow (N = 56) were considered,no signiﬁcant PLSR component was obtained.
The PLSR model for the redox status of GSH also
resulted in a signiﬁcant component that explained 131%(P < 00001) of variance in the GSH:GSSG ratio. The
eﬀect of background radiation level was positive (Table 1),
which means that oxidative stress in the cells of birds
decreased as radiation increased. As in the model for GSH
levels, the eﬀect of pheomelanin content in feathers was
negative, and the eumelanin content of feathers was also
an important predictor with a positive eﬀect on redox status (Table 1). Thus, under equal levels of radiation, thebirds that produce more pheomelanin and less eumelaninhave higher levels of oxidative stress. EV1 was the mostimportant predictor, accounting for 414% of the total
variance in the GSH:GSSG ratio of birds explained by themodel. Radiation level and EV1 were also signiﬁcant predictors (Table 1). The GSH:GSSG ratio was signiﬁcantly
positively correlated with the PLSR component (r = 036,
N = 118, P < 00001; Fig. 3b). Again, results did notchange when samples with GSH:GSSG ratios lower than

Table 1. Results of partial least squares regression (PLSR) models explaining the relationship between four response variables (GSH levels, GSH:GSSG ratio, DNA damage score and body mass) and pheomelanin (TTCA and 4-AHP) and eumelanin (PTCA) content offeathers, radiation levels at the capture sites and a phylogenetic eigenvector in birds from Chernobyl. Phylogenetic eigenvectors wereselected from phylogenetic vector regressions (PVR) made with the response variables and pheomelanin and eumelanin levels considering
the phylogeny of the species of birds. The DNA damage score is a synthetic response variable built in the PLSR, composed by the mean,
median and 75th percentile percentage DNA in the comet tail as measured by means of the comet assay. The model for body mass
includes wing chord as a predictor, thus actually analysing variation in the body condition of birds. Predictor weights (i.e. the contributionof each predictor variable to the PLSR component) and percentage of variance in the response variables explained by the PLSR models
are shown

log10 TTCA (ng mg 1 feather)log10 4-AHP (ng mg 1 feather)log10 PTCA (ng mg 1 feather)log10 Radiation (lSv h 1)
Phylogenetic eigenvector scorelog10 Wing chord (cm)% variance explained

log10 GSH (ng mg 1 blood)

log10 GSH:GSSG ratio

DNA damage scorelog10 Body mass (g)027043***
018
059***
060***
–
192

032
025
028
058**
064***–
131045***
026*
040*
065***
038
–
120

027***
024***
007
030***
077***
042630Predictor weights explaining more than 5% of the total variance are marked in bold. Asterisks indicate predictors whose regression coeﬃcients are signiﬁcant: *P < 005; **P < 001; ***P < 0001.
© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology

Adaptation of birds to ionizing radiation 9
(a)

(b)

(c)

(d)

Fig. 3. Relationship between the scores of partial least-squares regression (PLSR) components and (a) GSH levels, (b) GSH:GSSG ratios,(c) DNA damage scores and (d) body mass in birds from Chernobyl. PLSR component scores represent the position of sampling units(i.e. individual birds) along an axis composed of the predictor variables pheomelanin content of feathers (measured as TTCA and 4-AHP
levels), eumelanin content of feathers (measured as PTCA levels), and radiation levels at capture site and phylogenetic eﬀects (accounted
for as the scores of a selected eigenvector from phylogenetic vector regression (PVR) models). The names of predictors (excluding the phylogenetic eigenvector and also wing chord in (d) for the sake of simplicity) below the PLSR components indicate which side of the axes
increased with increasing values. In (c), DNA damage score is a synthetic response variable built in the PLSR, composed of the mean,
median and 75th percentile percentage DNA in the comet tail as measured by means of the comet assay. Results did not change when thetwo outlying points at the bottom of the graph were removed from the analysis (see text). In (d), wing chord is included as a predictor sothat variation in body mass actually reﬂects variation in body condition. The lines are the regression lines. Colour codes: solid black: Anthus trivialis; hollow black: Coccothraustes coccothraustes; solid red: Erithacus rubecula; hollow red: Fringilla coelebs; solid blue: Hirundo
rustica; hollow blue: Lanius collurio; solid light green: Luscinia luscinia; hollow light green: Parus major; solid pink: Sylvia atricapilla; hollow pink: Sylvia communis; solid dark green: Sylvia nisoria; hollow dark green: Turdus merula; solid grey: Turdus philomelos; hollow grey:
Phoenicurus ochruros; solid orange: Phylloscopus sibilatrix; hollow orange: Turdus viscivorus.

05 were excluded, as one signiﬁcant PLSR component was
obtained explaining 76% (P = 0005) of variance in the
GSH:GSSG ratio in which only the eumelanin content offeathers was no longer an important predictor (predictor
weights: radiation: 058, TTCA = 038, 4-AHP = 029,
PTCA = 019, EV1 = 062), and no signiﬁcant PLSR component was obtained when only data for the barn swallow(N = 56) were considered.
EFFECT OF RADIATION ON DNA DAMAGE

The mean, median and 75th percentile of percent DNA in
tail were response variables in a PLSR model, which created a synthetic response variable from the linear combina-

tion of the three measures. These measures were all
positively related to the synthetic response (response loadings for mean = 059, median = 055, 75th percentile = 059), which thus represent a general index of DNA
damage. A signiﬁcant PLSR component explaining 120%(P < 0001) of the variance in DNA damage was obtained.
The eﬀect of radiation was negative (Table 1), indicatingthat DNA damage decreased with increasing backgroundradiation. DNA damage increased with increasing pheomelanin content of feathers and decreased with increasing
eumelanin content (Table 1). EV4 was an additional
important predictor (Table 1). All predictors accounted
for more than 5% of the total variance explained by thiscomponent, and all were signiﬁcant except EV4 (Table 1).

© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology

10 I. Galv
an et al.
The synthetic index of DNA damage was signiﬁcantly positively correlated with the PLSR component (r = 035,
N = 112, P < 0001; Fig. 3c). When two outlying points(see Fig. 3c) were removed from the analysis, a signiﬁcant
PLSR component explaining 143% of the variance in
DNA damage was obtained that included the same predictors than the model including those points. When GSH
level was added to the model as a predictor variable, it
constituted an important predictor accounting for 168%of the total variance in DNA damage explained by themodel (122%, P < 0001), with a negative eﬀect (predictor
weight = 041) on this trait. Thus, DNA damage
decreased with increasing GSH levels in cells.
When using data on barn swallows only (N = 53), a signiﬁcant PLSR component was obtained that explained
64% (P = 0067) of the variance in DNA damage. The
eﬀect of radiation, as well as that of pheomelanin content
in feathers, was positive (predictor weights: radiation = 047, TTCA = 071, 4-AHP = 049), while the eumelanin content was not an important predictor (PTCA’s
predictor weight = 019). This indicates that, when only
barn swallows were considered and contrary to the patternfound for all species, background radiation levels andpheomelanin production increased DNA damage. It must
be emphasized, however, that the variance in backgroundradiation levels to which barn swallows were exposed(084) was signiﬁcantly (506-fold) lower as compared to
the variance considering all species (42512; Levene’s test:
F1,163 = 4478, P < 00001). Additionally, the maximum
level of radiation to which barn swallows were exposed(290 lSv h 1) was 32-fold reduced as compared to the
maximum level in the entire data set of species(9290 lSv h 1).

of the total variance in body mass explained by the model(635%; predictor weight = 018). No signiﬁcant PLSR
components were obtained when only data for the barnswallow (N = 59) were considered.
EFFECT OF RADIATION ON PHEOMELANINS

When the eﬀect of radiation on the TTCA:4-AHP ratio
was analysed, a signiﬁcant PLSR component that
explained 65% of variance (P = 0002) was obtained. The
model showed that, as predicted, the eﬀect of radiation
was signiﬁcant (P < 0001) and positive and accounted for>5% of the variance explained by the component(weight = 081). The phylogenetic eigenvector also
accounted for >5% of the variance (weight = 058), but
it was not signiﬁcantly related to the TTCA:4-AHP ratio(P = 0061).
PHYLOGENETIC SIGNAL IN ANTIOXIDANT STATUS,OXIDATIVE DAMAGE AND BODY CONDITION

Mean deviations from the 45° line in the PSR curves were
negative for all response variables (Fig. 2), indicating thatthe species considered diﬀered more than expected under
Brownian motion regarding these traits. However, themagnitude of deviations diﬀered between traits, being relatively large for GSH and DNA damage ( 0224 and0270 respectively) and small for GSH:GSSG ratio andbody mass ( 0077 and0063 respectively) (Fig. 2).
Thus, there was considerably more phylogenetic signal in
the levels of reduced GSH than in oxidative stress levels
represented by the redox status of GSH.
EFFECT OF PHYSIOLOGICAL RESPONSES ON THE

EFFECT OF RADIATION ON BODY CONDITIONPOPULATION TRENDS OF SPECIESThe PLSR model for body mass resulted in three signiﬁcant components regarding Q2, but only the ﬁrst two components explained signiﬁcant amounts of variance in thatvariable (component 1: 630%, P < 00001; component 2:
102%, P < 00001; component 3: 13%, P = 0162). We
only selected the ﬁrst component because the information
generated by the second component was redundant withthe ﬁrst component. The eﬀect of radiation was positive,
indicating that the body condition of birds increased withincreasing background radiation, but eumelanin content
was not an important predictor (Table 1). The eﬀect ofpheomelanin content in feathers was negative (Table 1),
indicating that under equal levels of radiation birds producing more pheomelanin were in poorer condition. EV3
and wing chord were additional important predictors. All
important predictors except wing chord were also signiﬁcant (Table 1). Body mass was signiﬁcantly positively correlated with the PLSR component (r = 079, N = 152,
P < 00001; Fig. 3d). When GSH level was added to themodel as a predictor variable, it tended to covary positively with body mass, but it accounted for less than 5%

The PLSR model with the mean residuals per species for
GSH levels, GSH:GSSG ratio, DNA damage score andbody mass as predictors of the population trends of thespecies resulted in a component that explained a marginally signiﬁcant proportion of the variance in the slope estimates (27%, P = 0047), although the component was not
signiﬁcant (Q2 = 019). Thus, the physiological responses
against radiation did not have negative consequences forthe population trends of the species.

Discussion
EFFECTS OF IONIZING RADIATION ON OXIDATIVESTATUS, DNA DAMAGE AND BODY CONDITION

Birds improve their antioxidant levels and body conditionand decrease their oxidative stress levels and DNA damage
with increasing background radiation to which they are
exposed at Chernobyl. Ionizing radiation creates ROS,
depletes antioxidant levels and thus induces oxidative stressin cells, but as in any toxic compound, the magnitude of© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology

Adaptation of birds to ionizing radiation 11
these eﬀects is largely dependent on the magnitude of thedoses (Riley 1994). This also means that the dose of radiation determines the capacity of organisms to adapt to their
exposure (Tapio & Jacob 2007). In our study area around
Chernobyl, birds are exposed to background radiation levels ranging from 002 to 9290 lSv h 1, the mean value
being 1624 lSv h 1. Thus, radiation levels are remarkably
high in some sites, but most sites have low radioactivity,
albeit signiﬁcant as compared to non-contaminated control
sites in the neighbourhood of Chernobyl. Furthermore, theaccident at the nuclear power plant at Chernobyl took
place 28 years ago, which has caused chronic exposure to
low-dose radiation across many generations. These conditions should favour individual responses of physiologicalplasticity to achieve adaptation or ‘acclimation’ to these
new environmental conditions, and variation in these
responses may be aﬀected by evolution (Woods & Harrison2002). These conditions are also known to particularly
favour physiological adaptation of organisms to ionizing
radiation (Tapio & Jacob 2007). Our study provides evidence that birds have physiologically adapted to chronic
exposure to radiation at Chernobyl, as radiation levels did
not negatively aﬀect their oxidative status, DNA integrityor physical condition.
Our analyses are for obvious reasons entirely correlational, implying that we cannot make strong inferences
about causation. Likewise, we cannot assume that
unknown variables may have not aﬀected our analyses andconclusions. We ﬁnd the latter assumption unlikely toapply because we included a range of variables that were
known to correlate with our response variables. Our study
sites were generally unaﬀected by human disturbance,
which we can dismiss as a potentially confounding variable. We also consider food abundance or quality to be an
unlikely confounding variable since animals generally are
distributed across resource gradients in an ideal free fashion. The distance among the study sites is short and allsites can be reached by ﬂying birds in less than an hour.
Hence, resource abundance per capita should be similar
across environments diﬀering in level of background radiation. This is also supported by little or no eﬀect of background radiation on success or condition of nestling birds
in sites diﬀering in background radiation level at Chernobyl (Møller et al. 2005; Møller, Karadasß & Mousseau2008). Hence, it is likely that the mechanisms that we have
hypothesized according to our review of the literature area reliable cause of the ﬁndings reported here.
The analysis of phylogenetic signals in the studied traits
supports the existence of physiological adaptation in birds.
In fact, deviations from the expected Brownian motion
model of evolution were negative for all the response variables (except body mass, which had a deviation close tozero as expected for interspeciﬁc variation in body size),
and as these deviation values can be interpreted in an analogous way to Blomberg, Garland & Ives (2003) K-statistic(Diniz-Filho et al. 2011), with negative values indicative ofadaptation in at least some of the species considered(Blomberg, Garland & Ives 2003). Interestingly, the deviation value was large for GSH levels, but low for the redox
status of GSH, which suggests adaptation in the levels of
the most important intracellular antioxidant (i.e. GSH),
but not in oxidative stress levels. Thus, GSH levels seem to
be more labile than its redox status, and the physiologically plastic response of birds to radiation would be mediated by reduced GSH and not by its oxidation rate. This
makes sense, as birds may be able to mount adaptive
responses by varying GSH synthesis, but not its susceptibility to oxidation. This is congruent with the view of antioxidants having the capacity to inﬂuence the evolution oflife-history strategies in birds (Galv
an et al. 2012a). Similarly, the large deviation value found for DNA damage
suggests that birds develop physiological adaptations to
reduce this physiological cost. To our knowledge, this represents the ﬁrst evidence of adaptation to ionizing radiation in wild populations of animals.
These results contrast with previous intraspeciﬁc studies
on two species of birds (Møller, Surai & Mousseau 2005;
Møller, Karadasß & Mousseau 2008; Bonisoli-Alquati et al.
2010, 2011) and also in humans and one species of ﬁsh(Sugg et al. 1996; Fenech, Perepetskaya & Mikhalevich
1997; Ivaniota, Dubchak & Tyshchenko 1998; Neyfakh,
Alimbekova & Ivanenko 1998; Romanenko et al. 2000;
Vartanian et al. 2004; Marozik et al. 2007), showing thatantioxidant levels decrease and oxidative damage increases
with radiation at Chernobyl. However, this apparent contradiction may just be the consequence of diﬀerent taxonomic scales in the analyses. There is large variation
among taxa in susceptibility to the eﬀects of ionizing radiation (Newman & Unger 2003; Møller & Mousseau 2013;
Møller et al. 2013), and in our study area there is hightemporal consistency in background radiation levels towhich individual birds are exposed (see Materials andMethods). Thus, studies that focus on single species may
have limited capacity to detect adaptive responses to radiation. Indeed, when we restricted our analyses to the species
with the largest sample size (i.e. the barn swallow), we
found that, as previously reported (Møller, Surai & Mousseau 2005; Bonisoli-Alquati et al. 2010, 2011), DNA damage increased with radiation levels. But both the range andmaximum level of background radiation for barn swallows
was considerably reduced as compared to the values
observed considering all species (a 506-fold increase in variance and a 32-fold increase in maximum level). It is actually expected that species diﬀer in their capacity to adapt
to changing environmental conditions (Somero 2010),
which may explain why, although the eﬀect of radiationon the population trends of birds in our study area at
Chernobyl is overall negative, the populations of several
species appear to be positively aﬀected by radiation(Galv
an, Mousseau & Møller 2011). We have previously
reported that background radiation negatively aﬀects thesurvival of several species of birds in our study area
(Møller et al. 2012), which also contradicts the results
shown here. This is probably also explained by the© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology

12 I. Galv
an et al.
diﬀerential adaptive capacities mentioned above, as here
we sampled surviving individuals and therefore only thosethat actually achieved adaptation to radiation. Therefore,
our study stresses the importance of comparative studies
to increase the amplitude of environmental conditions andpotential responses to them, which thus increases thecapacity to detect physiological adaptations.
Our results do not only show that GSH levels and body
condition of birds were not negatively aﬀected by background radiation, but that these traits even increased withradiation levels. One explanation may be that birds were
responding to an oxidative challenge by transiently
increasing the levels of antioxidants. However, this may bevalid for acute exposure to ionizing radiation (Kovalchuk
et al. 2007; Dauer et al. 2010), but not for chronic exposure as experienced by birds at Chernobyl. Furthermore,this could not explain the positive eﬀect on body conditionof birds, which actually suggests that exposure to radiation
may increase survival of birds (Møller & Szep 2001). Additionally, and more importantly, background radiation levels covaried negatively with oxidative stress (GSH:GSSG
ratio) and DNA damage levels, which can neither be
explained by the eﬀect of a transient exposure to radiation
as shown in mice in which the GSH:GSSG ratio decreases
after an acute exposure to X-ray radiation (Navarro et al.
1997). The positive eﬀect of radiation on oxidative stressand DNA damage levels further supports the view thatbirds can beneﬁt from chronic exposure to radiation, andthe fact that these two diﬀerent measures of physiologicaldamage show the same pattern of covariation with radiation levels demonstrates congruence in this interpretation.
We did not ﬁnd an eﬀect of the intensity of this physiological response on the population trends of the species, whichsuggests that birds do not pay a cost of maintaining such a
response in the long term. The explanation for the overall
beneﬁcial eﬀects of radiation found here may be that birds
mount an adaptive physiological response (Dimova, Bryant & Chankova 2008) that results in individuals overcoming the initial challenge of ionizing radiation and achieving
an improved antioxidant status, DNA integrity and body
condition, which may be related to radiation hormesis(Luckey & Lawrence 2006). Albeit surprising, these results
agree with recent ﬁndings in Drosophila that had been
exposed to X rays as instar larvae, in which irradiation
reduced the frequency of somatic mutations that may
result from DNA damage but increased the frequency inmutants deﬁcient in DNA repair (Koana, Takahashi &Tsujimura 2012). This suggests that low-dose radiation can
activate DNA repair genes (Koana, Takahashi & Tsujimura 2012). Indeed, this may also explain a similar eﬀect
found in developing red-legged partidges Alectoris rufa
that reduced their levels of oxidative damage after being
chronically exposed to a pro-oxidant compound (Galv
an& Alonso-Alvarez 2009). Similarly, GSH levels in plasmaof humans chronically exposed to radiation at Chernobyl
increases only under low doses of radiation (Ivanenko &Burlakova 2013). In accordance with the adaptive natureof these plastic responses, it has been shown that a chronic
exposure to low-dose c radiation can lead to a prolongation of life span (Ina & Sakai 2004).
The plastic responses that can lead to adaptation toradiation exposure may be found in a broad diversity oforganisms, as exempliﬁed by studies carried out in several
taxa (Dauer et al. 2010). For example, grasshoppers
chronically exposed to low levels of radiation at Chernobyl
have been reported to have lower DNA damage levels(measured as levels of 8-hydroxydeoxyguanosine) after anacute challenging irradiation than grasshoppers that had
not previously been exposed to radiation (Mortensen2013). In mice, the damaging eﬀects (prevalence of thymic
lymphomas) of a challenging X-ray irradiation were considerably reduced by previous low-dose irradiation, andthis reduction was even greater when the mice had been
continuously irradiated with c-rays in the long term (morethan 1 year) (Ina et al. 2005). Chronically irradiated micealso showed greater body mass (as found here in birds)
and immune activity than controls (Ina et al. 2005). Protective eﬀects of low ‘adapting’ doses of radiation before achallenging dose have also been reported for natural radioactivity levels in three species of ungulates (Ulsh et al.
2004). Radio-adaptive responses are also observed inhumans. Lymphocytes from inhabitants of Ramsar, Iran,one of the world’s places with the highest natural radioactivity levels, exposed to background radiation throughout
life show lower frequency of chromosome aberrations thanpersons exposed to negligible radiation levels (Ghiassi-Nejad et al. 2002), although DNA damage measured by thecomet assay has been reported to be considerably greaterin lymphocytes of Ramsar inhabitants than in persons
exposed to normal background radiation, the repair rate ishigher in the former only if exposure to radiation was relatively low (Masoomi et al. 2006). In Chernobyl, lymphocytes of people chronically exposed to low doses fromfallout did not show evidence of radio-adaptation regarding frequency of chromosome and chromatid aberrations
after a challenging c-ray irradiation (Padovani et al. 1995),
but adaptation was shown to occur after a challenge witha glycopeptide that causes double strand DNA breaks(Tedeschi et al. 1995). There are several other studies
reporting evidence of adaptation in humans occupationally
exposed to X- and c-rays (Tapio & Jacob 2007).
The hypothesized physiological adaptive responses that
may explain our results could be transferred from adultbirds to their oﬀspring, thus being transmitted across generations producing the patterns that we observed (i.e. anadaptive maternal eﬀect; Mousseau & Fox 1998). This is
likely for radio-adaptive responses, as illustrated by Kovalchuk et al.’s (2004) report of adaptation of plants to radiation around Chernobyl. These authors demonstrated thatthe progeny of plants that had been chronically exposed toradiation (although they basically only compared one irradiated site with one non-irradiated site, so results should
be taken with caution as unknown factors diﬀerent fromradiation may also account for these eﬀects) was more

© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology

Adaptation of birds to ionizing radiation 13
resistant to mutagens, showing a higher expression of genesthat control the main enzymatic antioxidants and DNA-repair
for several generations. They also determined that genome
stabilization, measured as homologous recombinationlevels, was higher in plants collected from contaminatedsites at Chernobyl than those from control sites. The global
genome DNA of two generation of plants grown at laboratory conditions from seeds collected in contaminated sites
was also considerably hypermethylated in comparison tocontrol plants (Kovalchuk et al. 2004). As genome stabilization prevents reshuﬄing of the hereditary material andmethylation is one of the main epigenetic mechanisms, theirresults represent important cues about the mechanisms that
permit the inheritance of radio-adaptive responses. Studies
on human cells also show similar mechanisms. Thus, lymphoblastoid cells exhibiting adaptive response after receiving a low dose of radiation before a challenging dose show
an up-regulation of protein synthesis genes and down-regulation of metabolic and signal transduction genes (Coleman
et al. 2005), and ROS production in ﬁbroblasts increases
with increasing radiation dose and this leads to changes inmiRNA expression (Simone et al. 2009). Therefore, epigenetic mechanisms such as DNA methylation and miRNA
expression could be key in the inheritance of the adaptive
response to ionizing radiation, and may explain why we canobserve physiological adaptation in some birds 28 years
after the nuclear power plant accident at Chernobyl.
PHYLOGENETIC INERTIA IN OXIDATIVE STATUS, DNA
DAMAGE AND BODY CONDITION

Physiological adaptation to low doses of ionizing radiationis thus possible, and our study suggests that it may haveimportant evolutionary implications because physiologicalplasticity that allows variation in GSH and DNA damagelevels seem to diﬀer across species of birds as indicated bynegative phylogenetic signals (Blomberg, Garland & Ives
2003; Diniz-Filho et al. 2011). This variability could
favour the role of natural selection (Woods & Harrison2002). Furthermore, phylogenetic eigenvectors were important predictors of variation in GSH levels, GSH redox status, DNA damage and body condition, supporting theinterpretation of interspeciﬁc variation in capacity tomount radio-adaptive responses. In EV1, the phylogenetic
eigenvector selected to account for phylogenetic eﬀects inthe analyses of GSH levels and redox status showed thatbirds from the families Turdidae, Muscicapidae, Motacillidae and Fringilidae were positioned at the positive part of
the axis while the rest of the phylogeny (i.e. families Lanidae, Paridae, Sylviidae and Hirundinidae) were positioned
at the negative part. The eﬀect of EV1 on GSH levels and
GSH:GSSG ratio was positive, meaning that species thatbelong to the families Lanidae, Paridae, Sylviidae and Hirundinidae are phylogenetically constrained to increase
their GSH levels and decrease oxidative stress, thus being
particularly limited to express plastic adaptive responses to
ROS exposure. In fact, the two species of birds in whichradiation at Chernobyl has been reported to deplete antioxidant levels and increase oxidative damage (the barnswallow and the great tit) belong to these families (Møller,
Surai & Mousseau 2005; Møller, Karadasß & Mousseau
2008; Bonisoli-Alquati et al. 2010, 2011). EV4 and EV3,
the eigenvectors selected for the analyses of DNA damage
and body condition, were negatively related to these variables, and Turdus species were positioned at the negative
part of these axes. As Turdus species were included in thepositive part of EV1, this suggests that phylogenetic inertia
causes the birds that belong to this genus to obtain a large
beneﬁt from radiation exposure in terms of increased GSH
levels and body condition and decreased oxidative stress
but also pay a cost in terms of increased DNA damage.
This may have important conservation implications thatshould be considered in bird populations exposed to radioactive contamination or other pro-oxidant agents.
INFLUENCE OF MELANIN PRODUCTION ON IONIZINGRADIATION EFFECTS

Production of pheomelanin, one of the two main types ofthe most abundant pigments in animals, represents a physiological cost under stressful environmental conditions. We
found that under equal levels of background radiation
birds that produce larger amounts of pheomelanin havelower levels of GSH, higher oxidative stress and higher levels of DNA damage, and they are in poorer condition thanbirds that produce lower levels of this pigment. We havepreviously found that the population trends of species ofbirds that show a higher expression of plumage colours
typically conferred by pheomelanin are more negatively
aﬀected by radiation exposure at Chernobyl than populations of species with lower expression of these colours(Galv
an, Mousseau & Møller 2011), the hypothesizedmechanism behind that (i.e. consumption of GSH duringpheomelanogenesis) thus being consistent with the resultsof this study. Other studies of wild populations of animals
also show that the expression of pheomelanin-based traits
may limit the development of costly physiological processes or viability under adverse, stressful environmental
conditions. Across species of birds, the expression of pheomelanin-based colour is negatively associated with brainsize, whose production requires high GSH levels (Galv
an& Møller 2011), and positively related to the prevalence of
cataract, which GSH critically contributes to prevent(Galv
an et al. 2012b). Western bluebirds Sialia mexicana
with a greater expression of pheomelanic breast plumage
coloration have been reported to be more likely to die ofan epidemic (Keyser & Sieﬀerman 2005). Tawny owls Strix
aluco belonging to the pheomelanic morph have lower viability during adverse environmental conditions than conspeciﬁcs belonging to the eumelanic morph (Karell et al.
2011). Similar eﬀects can also be found in mammals, as
shown by a positive association between the degree of pelage pheomelanization and lipid oxidative damage in wildboars (Galv
an, Alonso-Alvarez & Negro 2012).

© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology

14 I. Galv
an et al.
Our study now suggests that the mechanism behind theobserved patterns between the production of pheomelaninand costly physiological processes or stressful environmental conditions is as previously hypothesized, that is, theincorporation of sulphhydryl groups from cysteine or
GSH into the pheomelanogenesis pathway (Garcıa-Borr
on& Olivares Sanchez 2011; Ito et al. 2011a), which represents a consumption of cysteine/GSH and thus a decreasein antioxidant capacity (Galvan, Ghanem & Møller 2011;
Pavel, Smit & Pizinger 2011; Galvan, Ghanem & Møller
2012). Accordingly, Simone et al. (2009) showed thathuman ﬁbroblasts exposed to ionizing radiation suﬀeredfrom less radiation-mediated ROS production if theyreceived a previous treatment with cysteine (which isdepleted during pheomelanin production in melanocytes).
Recently, it has been reported that pheomelanin production per se is a physiological cost that enhances melanoma
development in mice (Mitra et al. 2012), and the consumption of cysteine during pheomelanogenesis has been attributed to such an eﬀect (Morgan, Lo & Fisher 2013). Our
results indicate that the consumption of GSH by pheomelanin production is accompanied by increased oxidative
stress, DNA damage and decreased body condition inbirds. This suggests that pheomelanin synthesis has profound implications for the physiological plasticity oforganisms. Therefore, pheomelanogenesis represents animportant physiological cost and thus a constraint toadaptation to stressful environmental conditions.
Eumelanogenesis occurs in the absence of or below a
threshold level of sulphhydryl groups in melanocytes(Garcıa-Borr
on & Olivares Sanchez 2011; Ito et al.
2011a). Accordingly, we found that, in contrast to pheomelanin, eumelanin levels in feathers did not aﬀect GSH
levels but were positively related to the GSH:GSSG ratio
and negatively related to DNA damage levels. This means
that, under equal levels of background radiation, the birds
that produced more eumelanin suﬀered less oxidative
stress and DNA damage than birds producing less eumelanin. This is consistent with the previously observed protective eﬀect of eumelanin against DNA damage in humanmelanoma cells, which also show increased survival by
producing this pigment (Kinnaert et al. 2004). The black
pigments of fungi are also protective and enhance growthunder exposure to ionizing radiation (Dadachova et al.
2007).
Interestingly, we also found that radiation had an eﬀect
on the structure of pheomelanin produced by birds. We
predicted that ionizing radiation may degrade the benzothiazine moiety of pheomelanin (here represented by 4AHP) to a benzothiazole moiety (represented by TTCA)
because of the higher oxidation potential of the former(Wakamatsu, Ohtara & Ito 2009). It is known that UVA
radiation produces pheomelanin radicals and solvated electrons, which induces a reduction of molecular oxygen tosuperoxide anions, thus making pheomelanin phototoxic(Takeuchi et al. 2004; Ye et al. 2006): another physiological cost of this pigment. However, this ROS production

might be reduced in the benzothiazole moiety of pheomelanin as compared to the benzothiazine moiety due to therather stable nature of the former, so pheomelanins withhigher relative contents of benzothiazoles are less prooxidant under radiation exposure (Wakamatsu, Ohtara & Ito2009). Photodamage of pheomelanin, which actuallyoccurs in natural hair (Wakamatsu et al. 2012), may therefore have beneﬁcial biological eﬀects as suggested byincreases in the production of melanin free radicals under
low UVA doses but large decreases at high doses (Fern
andez et al. 2012). The conversion of benzothiazine to benzothiazole had never been reported for an eﬀect other than
UV radiation, but our results indicate that the TTCA:4AHP ratio in the feathers of birds increases with background radiation, suggesting that ionizing radiation also
induces a change in the structure of pheomelanin. Thechange towards the production of forms of pheomelaninmore stable to oxidation may actually have facilitated theacclimation of birds to ionizing radiation despite the GSH
consumption during pheomelanin production (see above).
Although feathers are inert structures when mature, melanins are synthesized in melanocytes located in the feather
follicles before being transferred to feathers (Yoshihara
et al. 2011), and these melanocytes can be aﬀected by ionizing radiation.

Conclusions
The inclusion of several species of birds in the analysis ofeﬀects of ionizing radiation on oxidative status, DNAdamage and body condition allowed us to detect a pattern of covariation that diﬀers from studies that focus on
single species, probably because this permits inclusion ofa greater range of variation in radiation levels to whichbirds are exposed and a greater variability in susceptibility
to radiation. We considered the individual bird as the
sampling unit instead of mean values per species for thestudied variables, thus controlling for the eﬀect of common ancestry among individuals that belong to the same
species. This analytical approach, which was made byconsidering individuals as polytomies at the tips of thephylogeny, probably also increased our capacity to discern a general pattern. We thus found that GSH levels
and body condition increased, and oxidative stress and
DNA damage decreased, with increasing backgroundradiation levels to which birds were exposed at Chernobyl. This suggests that birds have the capacity to adapt
to chronic exposure to ionizing radiation, which may haveresulted in a hormetic response. Our analysis of phylogenetic signal in the studied traits supports the existence ofadaptation. Epigenetic mechanisms, as shown in otherorganisms, may favour such an inherited radio-adaptive
response, which may lead to the pattern observed
24 years after the nuclear power plant accident at
Chernobyl. Under equal levels of radiation, the birds that
produce more pheomelanin have lower GSH levels,
higher oxidative stress, more DNA damage and poorer© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology

Adaptation of birds to ionizing radiation 15
condition, while those that produce more eumelanin are
better protected against oxidative stress and DNA damage. A radiation-induced change towards the productionof more stable forms of pheomelanin may have facilitatedthe acclimation of birds to radiation exposure despite thecost of pheomelanin production.
Therefore, birds have the capacity to adapt to chronic
exposure to low-dose ionizing radiation, although thiscapacity varies across species and is particularly reduced inthose producing larger amounts of pheomelanin and thosethat are phylogenetically constrained to mount plastic
responses for GSH levels. Our study thus stresses theimportance of incorporating a multi-species approach toinvestigate the biological eﬀects of ionizing radiation as
conclusions derived from single-species studies may not
represent general trends in taxonomic terms. However, it isnecessary to be cautious and understand that the positiveeﬀects of radiation exposure that we are reporting here
represent an overall pattern, which is very useful for inferring evolutionary implications that should be viewed at theﬁne scale for biodiversity conservation purposes because
the capacity to develop adaptive responses depends on thespecies, and intraspeciﬁc variation is also possible (Luckey& Lawrence 2006). Indeed, the eﬀects of radiation at Chernobyl on the populations of organisms, and for birds inparticular, have been negative overall (Møller & Mousseau
2006; Møller et al. 2012), which does not preclude positiveeﬀects for populations of some species (Galvan, Mousseau& Møller 2011) or physiological adaptations in the surviving individuals (this study). Thus, any conclusion about
biological eﬀects of ionizing radiation is likely to beneﬁt
from an integrated approach that considers intra- andinterspeciﬁc studies as well as physiological and population
studies.

Acknowledgements
We thank Luis M. Carrascal for his discussions and suggestions about statistical analyses. I.G. was supported by a Marie Curie Intra-European Fellowship (PIEF-GA-2009-252145) within the 7th European Community
Framework Programme. K.W. was partly supported by a Japan Society for
the Promotion of Science (JSPS) grant (No. 21500358, and No. 24500450).
We gratefully acknowledge logistic support and help in Ukraine by I. Chiz
hevsky, E. Flensted-Jensen, W. Mardal, G. Milinevski and W.C. Arestrup.
The University of South Carolina College of Arts and Sciences, the University of South Carolina Oﬃce of Research, the Fulbright Programme, andthe Samuel Freeman Charitable Trust provided ﬁnancial support for components of this study.

Data accessibility
Data deposited in the Dryad repository http://dx.doi.org/10.5061/dryad.
rb5hr.

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Received 17 January 2014; accepted 16 April 2014
Handling Editor: Jonathan Blount

Supporting Information
Additional Supporting information may be found in the online
version of this article:
Table S1. Sample sizes for diﬀerent species and sites.

© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology