Bird population declines due to radiation
exposure at Chernobyl are stronger
in species with pheomelanin-based
coloration

Oecologia
ISSN 0029-8549
Volume 165
Number 4
Oecologia (2011) 165:827-835
DOI 10.1007/
s00442-010-1860-5

1 23

Author's personal copy
Oecologia (2011) 165:827–835
DOI 10.1007/s00442-010-1860-5

PHYSIOLOGICAL ECOLOGY - ORIGINAL PAPER

Bird population declines due to radiation exposure at Chernobyl
are stronger in species with pheomelanin-based coloration
Ismael Galván • Timothy A. Mousseau •Anders P. Møller

Received: 1 July 2010 / Accepted: 15 November 2010 / Published online: 7 December 2010
Ó Springer-Verlag 2010

Abstract Eumelanin and pheomelanin are the mostcommon pigments providing color to the integument ofvertebrates. While pheomelanogenesis requires high levelsof a key intracellular antioxidant (glutathione, GSH), eumelanogenesis is inhibited by GSH. This implies thatspecies that possess the molecular basis to produce large
amounts of pheomelanin might be more limited in copingwith environmental conditions that generate oxidative
stress than species that produce eumelanin. Exposure toionizing radiation produces free radicals and depletes
antioxidant resources. GSH is particularly susceptible toradiation, so that species with large proportions of pheomelanic integument may be limited by the availability of
GSH to combat oxidative stress and may thus suffer morefrom radiation effects. We tested this hypothesis in
97 species of birds censused in areas with varying levelsof radioactive contamination around Chernobyl. After
controlling for the effects of carotenoid-based color,body mass and similarity among taxa due to common

phylogenetic descent, the proportion of pheomelanic
plumage was strongly negatively related to the slope estimates of the relationship between abundance and radiationlevels, while no effect of eumelanic color proportion was
found. This represents the first report of an effect of theexpression of melanin-based coloration on the capacity toresist the effects of ionizing radiation. Population declines
were also stronger in species that exhibit carotenoid-based
coloration and have large body mass. The magnitude of
population declines had a relatively high phylogenetic
signal, indicating that certain groups of birds, especiallynon-corvid passeriforms, are particularly susceptible tosuffer from the effects of radioactive contamination due tophylogenetic inertia.
Keywords Eumelanin  Glutathione  Pheomelanin 
Plumage coloration  Radioactive contamination

Introduction
Communicated by Hannu Pöysä.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00442-010-1860-5) contains supplementarymaterial, which is available to authorized users.
I. Galván
Department of Evolutionary Ecology, Estación Biológica De
Doñana (CSIC), 41092 Sevilla, Spain
I. Galván (&)  A. P. Møller
Laboratoire d’Ecologie, Systématique et Evolution, CNRS UMR
8079 Université Paris-Sud 11, 91405 Orsay Cedex, France
e-mail: ismael.galvan@u-psud.fr
T. A. Mousseau
Department of Biological Sciences, University of South
Carolina, Columbia, SC 29208, USA

Melanin is the most common pigment of the vertebrate
integument. It is synthesized by animals from the aminoacid tyrosine in a process (i.e. melanogenesis) that is
intrinsically related to oxidative stress (i.e. the imbalance
between the production of reactive oxygen species and thestate of the antioxidant and repair machinery; Galván andAlonso-Alvarez 2008, 2009; Galván and Solano 2009;
Hõrak et al. 2010). This is because glutathione (GSH), a
tripeptide thiol found in virtually all animal cells thatfunctions as the main physiological reservoir of cysteine(Benedetto et al. 1981) and as the most important intracellular antioxidant (Anderson 1998; Wu et al. 2004),
exerts a direct influence on the process. This effect occurs
by directly inhibiting the action of tyrosinase (i.e. the123Author's personal copy
828

enzyme catalysing the first step of the process), by combating free radicals that stimulate the action of tyrosinase,and by increasing the ratio cysteine:dopaquinone (Galvánand Alonso-Alvarez 2009).
Vertebrates synthesise two main forms of melanin:
eumelanin and pheomelanin, the latter producing lighter
colors than the former. It is GSH levels that determine themelanin form that is synthesised, as melanogenesis caneither lead to the production of eumelanin when theactivity of tyrosinase is high and the ratio cysteine:dopaquinone low, or to the production of pheomelanin or evenan absence of melanin synthesis under opposing conditions(Ozeki et al. 1997; Galván and Alonso-Alvarez 2009).
Therefore, eumelanogenesis takes place when the levels of
GSH are low and pheomelanogenesis when the levels of
GSH are high, meaning that pheomelanogenesis proceedswith higher levels of endogenous oxidative stress as compared to eumelanogenesis (Galván and Solano 2009). The
evolutionary implication of this is that species in which
natural selection has favoured the development of pheomelanic traits may have a decreased capacity to combat
oxidative stress as compared to species with eumelanic
traits, as the maintenance of high GSH levels as requiredby pheomelanogenesis might be metabolically costly underadverse environmental conditions that generate oxidative
stress and thus use GSH resources (Galván and AlonsoAlvarez 2009; Galván and Solano 2009).
Ionising radiation produces free radicals and thus promotes the generation of oxidative stress and depletes antioxidant levels in animals (Riley 1994; Ivaniota et al. 1998;
Neyfakh et al. 1998). GSH is one of the most susceptible
antioxidants to radiation (Bump and Brown 1990; Navarro
et al. 1997; Vartanian et al. 2004). Therefore, species thathave the molecular basis to produce large amounts of pheomelanin may be more limited to use GSH to combat oxidative stress, and may thus be more susceptible to ionisingradiation than species in which melanogenesis has been
selectively directed towards the production of eumelanin.
We test this hypothesis by examining the response of bird
populations to radioactive contamination after the Chernobyl accident and relate responses to the proportion of euand pheomelanic-based plumage coloration exhibited by the
species.
Møller and Mousseau (2007a) previously showed in adataset of 57 bird species that the presence of melaninbased plumage coloration had no effect on population
density in relation to radiation. However, they did notseparately analyse colors generated by eumelanin fromthose generated by pheomelanin, and only prevalence (i.e.
presence vs absence) of melanin-based coloration wasmeasured. This may have obscured any relationship concerning pheomelanic species in their response to radiationlevels. Here, we use a larger dataset of 97 species of birds

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collected during 4 years that were censused during themain breeding season at points with varying levels ofradiation around Chernobyl, and quantitatively analyse theexpression of eu- and pheomelanic plumage coloration. We
predicted that the slope of the regression between abundance and background radiation should be negatively correlated with the proportion of plumage colored bypheomelanin, while no correlation should be observed forthe proportion of eumelanin-based plumage color. We also
predicted that the slope of the regression between abundance and radiation should be more negative in species
with plumage color patches generated by carotenoids, asthese pigments act as antioxidants and are also depleted byradiation (Møller and Mousseau 2007a).
Møller and Mousseau (2007a) found that declines inbird abundance with background radiation levels werestronger not only in species that exhibit carotenoid-based
coloration but also in species with long migration anddispersal distances, and large egg and body mass. Fromthese variables, body mass may also be related to theexpression of eu- and pheomelanic coloration, as the production of diverse plumage color patches has been found to
be constrained in large species (Galván et al. 2010). Thus,
body size may be related to both slope estimates of theregression between abundance and background radiation,and to the expression of melanin-based coloration, thereby
constituting a potentially confounding variable. Therefore,
we estimated the relationship between slope estimates andproportion of pheomelanic coloration while simultaneously
controlling for the effects of body mass.

Materials and methods
Study sites
A.P.M. conducted standard point counts of breeding birds
during 29 May–9 June 2006, 1–11 June 2007, 29 May–5
June 2008 and 1–6 June 2009, with each count lasting
5 min during which birds seen or heard were identified andrecorded (Møller 1983; Bibby et al. 2005; Vořı́šek et al.
2010). A radiation protection suit was worn in the mostcontaminated parts of the Red Forest to keep dust particles
from contact with the body. The legs and torso, but not thehead, were covered by the radiation protection suit ensuring that observing and hearing birds were not affected by
the suit. The census was conducted within the Chernobyl
Exclusion Zone, Ukraine, or in areas adjacent on thesouthern and western borders with a permit from the
Ukrainian authorities, and in areas in Southern Belarus
around Gomel during the 2006–2009 breeding seasons.
The most distant census points were several hundred
kilometers apart [A total of 254 points (breeding season

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2006), 235 points (breeding season 2007), 237 points(breeding season 2008) and 159 points (breeding season2009) were located at ca. 100-m intervals within forested
areas (excluding successional stages of secondary forest
due to abandoned farming (these areas are still almost
exclusively open grassland)]. Only birds recorded less than adistance of 50 m from the observation point were included in
the census, thus avoiding the risk of inclusion of the same
individual multiple times. Most census points were sampledeach year to allow for analysis of species turnover. There wasno significant difference in radiation level at census points
among years (F3,894 = 1.25, P = 0.29).
We censused birds mainly during mornings before
1000 hours at the end of May and the beginning of June
when most individuals reach their annual maximum of
singing activity, making censuses of breeding birds highly
reliable (Møller 1983; Bibby et al. 2005; Vořı́šek et al.
2010). We directly tested the reliability of our counts byletting two persons independently perform counts. The
degree of consistency was high for both species richness
and abundance (Møller and Mousseau 2007a).

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Measuring background radiation levels
We measured radiation levels in the field and cross-validated these with measurements by the Ukrainian Ministryof Emergencies. We measured a, b, and c radiation at
ground level at each census point after having conducted
the census (thus making the census blind with respect toradiation level) using a hand-held dosimeter (Inspector; SEInternational, Summertown, TN, USA). We measured
levels 2–3 times at each site and averaged the measurements. Our data were validated with correlation against
data from the governmental measurements published by
Shestopalov (1996), estimated as the mid-point of theranges published, with analyses showing a high degree ofconsistency between methods (Møller and Mousseau
2007a). Radiation levels vary greatly at a local scale due toheterogeneity in deposition of radioactive material after the
Chernobyl accident (Shestopalov 1996). Our measurements
at the census points ranged from 0.01 to 135.89 mSv/h.
Background radiation levels for 90Sr and 137Cs are strongly
positively correlated with internal doses in Chernobyl birds(Gaschak et al. 2008).

Confounding habitat and weather variables
Melanin-based plumage coloration
Abundance estimates can be affected by numerous
confounding variables (Møller 1983; Bibby et al. 2005;
Vořı́šek et al. 2010), and, therefore, it is crucial to control
such variables statistically to assess the underlying relationship between radiation and abundance. We classified
habitats [agricultural habitats with grassland or shrub(either currently or previously cultivated), deciduous forest, or coniferous forest] and estimated to the nearest 10%
ground coverage by these different habitat types within adistance of 50 m from the observation point. Agricultural
habitat included edges between forest and open areas, andthe agricultural habitat variable thus also reflected theamount of edge habitat between forest and open areas.
Maximum height of trees was estimated to the nearest 5 m
and soil type was recorded as loam/clay or sand. The
presence of open water within a distance of 50 m was also
recorded. Weather conditions can affect animal activity
and hence census results (Møller 1983; Bibby et al. 2005;
Vořı́šek et al. 2010), and we recorded cloud cover at thestart of each point count (to the nearest eighth, range 0–1
during the censuses), temperature (°C, range 12–25°C), andwind force (Beaufort, range 0–4 during the censuses). For
each census point, we recorded time of day when the count
was started (to the nearest min). Because activity may
show a curvilinear relationship with time of day, forexample, with high levels of activity in the morning and toa lesser extent in the evening for birds (Møller 1983; Bibby
et al. 2005; Vořı́šek et al. 2010), we also included timesquared as an explanatory variable.

Information on melanin-based plumage coloration for bird
species censused at Chernobyl was obtained by examining
illustrations in Cramp and Simmons (1977–1992) andCramp and Perrins (1993–1994). Eumelanic and pheomelanic traits are generally of distinctive colors, the former
being responsible for black and grey colors and the latterfor yellowish, reddish, chestnut and brown colors (Toral
et al. 2008). Eumelanin and pheomelanin normally occur
simultaneously in the tissues (Ozeki et al. 1997), but thedarker colors conferred by eumelanin (Toral et al. 2008)
make evident the lower content of this pigment in chestnut
and brown colors as compared to black and grey colors(Galván and Alonso-Alvarez 2009). Furthermore, many
bird species have feather melanin contents of high purity([90% of either eumelanin or pheomelanin; McGraw andWakamatsu 2004; J.J. Negro, personal communication).
Therefore, we considered that black and grey plumage
colors were predominantly generated by eumelanin, whilechestnut and brown colors were predominantly generated
by pheomelanin. We did not consider conspicuous yellow
or red colorations which are assumed to be generated byother pigments (i.e. carotenoids), unless chemically identified as melanin-based by Toral et al. (2008). Although
only a rough approximation to the real proportion of eumelanic and pheomelanic plumage, the assumption thatblack-grey colors are eumelanic and brown-chestnut colors
are pheomelanic should be adequate for comparative purposes (Owens and Hartley 1998). Thus, we quantified the123Author's personal copy
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proportion of melanic plumage parts by examining illustrations in Cramp and Simmons (1977–1992) and Crampand Perrins (1993–1994). Illustrations of both resting andflying adult birds in breeding plumage were examined. The
method used by Beauchamp and Heeb (2001) and Galván(2008) was followed to obtain estimates of the proportionof eu- and pheomelanic color present in the plumage ofeach species, assigning scores that ranged from 0 (totallack of melanic color) to 5 (all melanic). When a species
was sexually dichromatic regarding melanin-based coloration, eumelanic and pheomelanic scores were the averageobtained for males and females. When a species had different subspecies differing in extent or type of melaninbased coloration, we used the nominate subspecies. It must
be noted that eu- and pheomelanic color patches cancoexist in the same feathers, and thus the sum of both color
scores, in a species that presents both color types, is notalways necessarily limited to five, and higher values arealso possible. Information on eu- and pheomelanic plumage color scores for the species used in the study is provided in Online Resource 1.
Carotenoid-based plumage coloration
Prevalence of plumage patches colored by carotenoids wasdetermined by examining illustrations of adult breeding
birds in Cramp and Simmons (1977–1992) and Cramp andPerrins (1993–1994). We considered colors that were yellow, orange and red to be caused by carotenoids (Tella
et al. 2004; Olson and Owens 2005). Information on
carotenoid-based plumage color for the species used in the
study is provided in Online Resource 1.
Body mass
We extracted mean body mass of males and females during
the breeding season from Cramp and Simmons(1977–1992) and Cramp and Perrins (1993–1994) andLislevand et al. (2007). Body mass was estimated as themean value of the means for males and females. Body mass
values for the species used in the study are provided in
Online Resource 1.
Data analyses
We quantified the relationship between abundance of birds
and level of background radiation by estimating the coefficient of determination and by calculating the slope of therelationship between abundance and log10-transformed
background radiation, while including the potentiallyconfounding variables. Radiation level and abundancewere log10-transformed, while ground coverage withfarmland and deciduous forest was square root arcsine-

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transformed (coniferous forest was not included as an
explanatory variable, because it simply represents theground coverage not attributed to farmland and deciduous
forest). We also included radiation level squared to accountfor non-linear relationships between species richness andabundance, respectively, and radiation. Furthermore, population density often varies considerably among years.
Therefore, we included year as a factor in the analysis of
population density in relation to background radiationlevel. In any case, estimates of relationships between
abundance and background radiation levels were very
similar when including and excluding year as a factor(results not shown). It must be noted that the relationshipbetween abundance and background radiation level ishighly repeatable among years (Møller and Mousseau2010). These slopes were used for subsequent analyses.
Bird species are evolutionarily related as reflected byphylogeny, and, therefore, they should not be treated as
independent sample units (Felsenstein 1985; Harvey and
Purvis 1991). Thus, the effect of common ancestry amongtaxa can lead to an overestimation of degrees of freedom if
phylogenetic relationships are not taken into account. We
used phylogenetic eigenvector regression (PVR) to quantify the amount of phylogenetic signal and to correct for itin the analysis of the relationship between the expression ofmelanin-based coloration and bird responses to radiationlevels (Diniz-Filho et al. 1998). Diniz-Filho and Torres(2002) and Martins et al. (2002) have tested several comparative methods (Felsenstein’s independent contrasts,
autoregressive method, PVR, and phylogenetic generalised
least squares) and have found that PVR yields good statistical performance regardless of the details of the evolutionary mode used to generate the data and provides similar
results to other methods, with very good (i.e. low) error
types I and II. Moreover, PVR does not assume any a priori
evolutionary model (an advantage if the true evolutionarymodel is unknown, or if it is too complex) and has similar
statistical performance even under evolutionary processes
distinct from Brownian motion.
We first performed a principal coordinates analysis(PCORD) on the matrix of pairwise phylogenetic distances
between the 97 bird species (after a double-centre transformation). In a second step, we selected the first nineeigenvectors obtained by the broken-stick rule as the mostparsimonious accounting of the phylogenetic signal.
Eigenvectors extracted from double-centred phylogenetic
distance matrices are able to detect the main topologicalfeatures of the cladogram under different sample sizes or
number of taxa used in the analyses (Diniz-Filho et al.
1998). We found that the original matrix of phylogenetic
distances between the 97 bird species and the reproduced
matrix of distances estimated based on the first nineeigenvectors were very similar (Mantel test with 999

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randomised matrices to estimate significance: r = 0.840,
P \ 0.0001; test carried out using PopTools 3.2.3; Hood2010). These eigenvectors were used as additional predictor variables in a general linear model (see below) inorder to control for phylogeny.
The phylogenetic hypothesis (see Online Resource 2) was
taken from the species-level supertree constructed by Davis(2008), with additional information from other sources forsome species not covered by Davis (2008), Grosso et al.
(2006), Voelker et al. (2007), Alström et al. (2008) and thephylogeny compiled by Møller (2006). Since we used different phylogenies that employed different methods, we set all
branch lengths equal to unity in our compiled phylogeny, thus
assuming a speciational model of evolution.
We used a general linear model to regress the slope
estimates of the regression between bird abundance andradiation levels on the proportion of eu- and pheomelanic
plumage color, prevalence of carotenoid-based plumage
color, body mass (all log10-transformed) and the first nine
phylogenetic eigenvectors (EV1–EV9 hereafter). All predictor variables were added as covariates to the model.
Carotenoid-based plumage color score was added as adummy variable with two levels: no = 0, yes = 1. We
included the interaction between melanin- and carotenoidbased color scores to investigate whether its effect on slope
estimates differed from the effect of each color type separately. The phylogenetic signal [i.e. amount of variance(i.e. R2) exclusively explained by phylogeny] in slope
estimates was calculated by regressing this variable on
EV1–ssEV9. The models were weighted by number ofindividuals recorded to estimate the slope of the relationship between abundance and radiation, thereby placing
more weight on species with many individuals (slope
estimates were negatively related to sample size; r =-0.30, n = 97, P = 0.003). The assumption of normality
was checked by exploring the distribution of residuals.

Results
A total of 64 of the 97 species considered (66%) had
negative slope estimates for the regression between abundance and radiation levels, while 33 species (34%) had
positive slopes (Online Resource 1). Among the 97 slopes,
30 (30.9%) were statistically significant at the 5% level,
while 4.85 would be expected by chance (0.05 9 97).
Among negative slopes, 25 were significant, while only 5of the positive slopes were significant. The relationshipbetween eumelanic color score and slope estimates for the
regression between abundance and radiation levels was not
significant either alone (F1,84 = 0.05, P = 0.818) or when
including pheomelanic color score in the model(F1,83 = 2.96, P = 0.089). Since both color variables were831

strongly negatively correlated (b = -0.48, F1,95 = 19.15,
P \ 0.0001), eumelanic color score was not included insubsequent analyses to avoid problems of collinearity. Theinteraction between pheomelanin- and carotenoid-based
color scores was not significant (F1,83 = 2.50, P = 0.117),
so it was also removed from the model.
The full model was significant (F12,84 = 7.66, P \0.0001)
and explained 52.3% of variance in slope estimates. As predicted, the slope decreased with the proportion of pheomelanic plumage color (Table 1; Fig. 1), indicating that the effectof radioactive contamination on abundance was more negative in species that produce large amounts of pheomelanin.
The presence of carotenoid-based color was also negatively
related to slope estimates, as expected (Table 1). Body mass
also had a significant effect on slope estimates (Table 1).
Pheomelanic plumage color score was significantly related toslope estimates in the previous model (Table 1) even without
weighting by number of individuals (b = -0.03, F1,84 = 4.27,
P = 0.042).
The phylogenetic signal in slope estimates was significant(F9,87 = 5.83, P \ 0.0001) and relatively high (37.6%). The
influence of phylogeny on slope values was mainly reflected
through the significant positive effects of EV1 and EV4(especially EV1; Table 1). EV1 discriminated non-passerines and corvoid passerines (families Oriolidae, Laniidae
and Corvidae), on the positive part of the axis, from therest of passerines on the negative part. The negative part ofEV4 comprised the species of the non-passerine families
Phasianidae, Columbidae, Rallidae, Accipitridae, Ciconidae, Ardeidae, Charadriidae, Scolopacidae and Lariidae, andof the passerine families Hirundinidae, Sylviidae, Motacillidae, Passeridae, Emberizidae and Fringillidae.

Discussion
The proportion of pheomelanin-based plumage coloration
was negatively related to the slope of the regression
between abundance and radiation levels in 97 species ofbirds censused at Chernobyl, as predicted. This was independent of the presence of carotenoid-based coloration andbody mass, two factors that were also related to population
responses to radiation (Møller and Mousseau 2007a). Thus,
the negative effects of radiation exposure on bird populations (Møller and Mousseau 2007a, b, 2009) are stronger in
species that have the molecular basis to produce large
amounts of pheomelanin as reflected by their plumage
color. Thus, pheomelanic birds are constrained to adapt toand overcome environmental changes that increase environmental oxidative stress such as radioactive contamination following the Chernobyl accident. This is the firststudy reporting an effect of melanin-based coloration on
sensitivity to ionising radiation.

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Table 1 Results of the general linear model testing the effects of melanin- and carotenoid-based plumage coloration, body mass and phylogenetic effects on the slope of the regression between abundance and radioactive levels in 97 bird species at Chernobyl
Effectbdf

Sum of squares

FP

Pheomelanic-based plumage color score

20.063

1

0.80

6.29

0.014

Carotenoid-based plumage color score20.043

1

1.02

7.96

0.006

Body mass

20.064

1

1.00

7.85

0.006

EV1

0.014

1

1.69

13.21

<0.001

EV2-0.002

1

0.08

0.64

0.426

EV3

0.002

1

0.03

0.22

0.639

EV4
EV5

0.006
0.007

1
1

0.58
0.44

4.58
3.46

0.035
0.066

EV6

0.000

1

0.00

0.00

0.945

EV7

-0.001

1

0.00

0.01

0.924

EV8

0.004

1

0.15

1.16

0.283

EV9

-0.002

1

0.03

0.27

0.601

84

10.73

Error

For each variable, we show the fitted regression coefficient (b) and its corresponding significance. For carotenoid-based plumage color score theregression coefficient refers to a dummy variable with two levels: no = 0, yes = 1. Phylogenetic effects are computed from the first nineeigenvectors (EV1–EV9) obtained from a principal coordinates analysis applied to the matrix of pairwise phylogenetic distances between the 97
species. Significant effects are shown in bold
0.15
0.10
0.05

slope

0.00
-0.05
-0.10
-0.15
-0.20
-0.25
-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

log10 (pheomelanin-based color score + 1)

Fig. 1 Relationship between pheomelanin-based plumage color scoreand slope estimate of the regression between abundance and radiationlevels in 97 species of birds at Chernobyl. The size of the data points
increases logarithmically with the number of individuals used toestimate the slopes for illustrative purposes only. The line is theregression line

The increased susceptibility of pheomelanic species toradioactive contamination might be due to the physiological costs of meeting the GSH demands of pheomelanogenesis (Ozeki et al. 1997; Galván and Alonso-Alvarez
2009; Galván and Solano 2009), as this probably limits
levels of GSH available to combat free radicals. Indeed,
GSH is one of the antioxidants most susceptible to ionisingradiation (Bump and Brown 1990; Navarro et al. 1997;
Vartanian et al. 2004). Although GSH is endogenously
synthesised by organisms, its production depends on the123

availability of the amino acid cysteine and its precursor
methionine (Lu 1999). Thus, although GSH levels areunder genetic control (Soltaninassab et al. 2000), they arealso influenced by environmental factors.
Similarly, the hypothesised trade-off between protection
against free radicals and coloration likely also explains thenegative slopes of the regression between abundance andradiation that were found in species with carotenoid-based
plumage coloration, as carotenoids that are used to color
the integument can no longer fulfil their role as circulating
antioxidants (reviewed in Møller et al. 2000). This latter
result reaffirms the previous findings by Møller andMousseau (2007a) that had been detected with a smaller
sample of species.
Since GSH, which is a key intracellular antioxidant,
inhibits the synthesis of eumelanin and directs the process
toward the synthesis of pheomelanin, eumelanogenesis
requires lower antioxidant levels to proceed than pheomelanogenesis (Galván and Alonso-Alvarez 2008, 2009;
Galván and Solano 2009; Hõrak et al. 2010). This meansthat eumelanin synthesis should more readily proceed thanpheomelanin synthesis under environmental conditions thatgenerate oxidative stress such as radiation exposure at
Chernobyl (see Almasi et al. 2008; Dauwe and Eens 2008
and Roulin et al. 2008 for intraspecific studies suggesting
positive selection on eumelanism by different factors thatgenerate oxidative stress). However, the slope of theregression between abundance and radiation was not significantly related to the proportion of plumage colored byeumelanin, even when eu- and pheomelanic color scores
were negatively correlated. The reason for this is probably

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related to the fact that, while the synthesis of pheomelanin(for which GSH is a substrate) is traded against otherprocesses that demand GSH resources to combat free
radicals, the synthesis of eumelanin does not necessarily
improve the performance of these processes. In otherwords, pheomelanin seems to act as a limiting factor, while
eumelanin does not improve the antioxidant machinery.
The fact that species with a large proportion of pheomelanic plumage were particularly affected by radiation,
while species with a large proportion of eumelanic
plumage were unaffected, suggests that the characteristics
of melanin-based plumage coloration may be changing
the competitive interactions between species inhabiting theChernobyl area. This is because the negative effect on theabundance of pheomelanic species may favour other species that thrive or even occupy the niche space that may
have been abandoned or modified in width by pheomelanic
species (Ricklefs 2010). Indeed, 34% of the species considered here had a positive slope estimate for the regression
between abundance and radiation level, indicating thatsome species were favoured or little affected by radioactive
contamination. Although the activity of free radicals at
certain levels is essential for development of certain
physiological processes, particularly those related toimmunity (Knight 2000), to our knowledge there are no
published reports of positive effects of free radicals generated by radioactive contamination in any living organism, certainly because the levels of free radicals that thisproduces are far higher than those needed for physiologicalprocesses (Knight 2000). Therefore, it is probable that thepositive slope estimates for the regression between abundance and radiation levels that we obtained for some species of birds are due to these species benefiting fromrelaxation of competitive interactions with other species
that suffer more strongly from the effects of radiation, anda possible occupancy of new niche space by the former
species.
If species with positive slope estimates benefit fromrelaxation of competition with species that are negatively
affected by radiation, it should be expected that the former
are closely phylogenetically related to the latter. This isbecause the most intense competitive interactions occur
between closely related species, since phylogenetic similarity confers ecological similarity and thus favours niche
overlap (Elton 1946; Jackson 1981). In our case, the 33
species with positive slopes were widely distributed acrossthe phylogeny (see Online Resources 1 and 2), implyingthat there was no clear phylogenetic signal in the sign ofslope estimates. Furthermore, with the exception of piedand collared flycatchers (Ficedula hypoleuca and F. albicollis) and Savi’s and river warblers (Locustella luscinioides and L. fluviatilis), there were no congeneric sistergroups in our phylogeny in which both species had positive833

slopes (see Online Resource 2). Thus, the closest relativesof species with positive slopes were species with negative
slopes. This may support the argument that species thathave been favoured by radioactive contamination havebeen so due to relaxation of competition with phylogenetically similar species that were negatively affected byradiation.
Interestingly, we found a relatively strong phylogenetic
signal in the slope estimates between abundance andradiation levels. This means that there are phylogenetic
constraints in the capacity of species to mount an adaptive
response that counteracts the negative effects of radioactive
contamination. This constraint was especially marked in
non-corvoid passeriform birds, which corresponds to theEurasian expansion of passerines (Davis 2008). Thus,
independently of other factors that affect slope estimates(e.g. plumage coloration and body mass), collectively, thisgroup of related species appear to be particularly sensitive
to radioactive contaminants.
Finally, we found that population declines were particularly pronounced in large-sized species, in contrast to theresults of Møller and Mousseau (2007a), who found apositive effect of body mass on slope estimates. These
conflicting results may be due to the larger number of
species considered in the present study, and the inclusion ofa new variable (i.e. pheomelanin-based coloration) that is
intrinsically related to oxidative stress. Møller and Mousseau (2007a) argued that population declines were more
pronounced in small-sized species because their higher
metabolic rates may require more antioxidant resources,
which would make them more sensitive to radiation.
However, smaller species may also have evolved stronger
antioxidant and repair mechanisms to counteract theirhigher metabolic rates (Holmes et al. 2001), which may
explain our results. Additionally, Møller and Mousseau(2007a) indicated that large-sized species often possesslarger home ranges, which may increase the heterogeneity
in radiation levels of the areas they encounter and reduce
their average exposure to contaminants. For this reason,large-sized species may also have a greater propensity toencounter highly contaminated areas by chance that could
be a critical factor in determining genetic and physiologicalconsequences of radiation. Individual level dosimetry
would be needed to disentangle these two hypotheses.
Responses to radioactive contaminants may seemextreme and perhaps even of little relevance to biology
under natural conditions. However, natural levels of radiation vary by more than two orders of magnitude (e.g.
Bristow 1978; Sanderson et al. 2001), suggesting that theeffects reported here may even be discernible elsewhere.
We also note that species with strongly reduced abundancein contaminated areas are more susceptible to mutagens as
reflected by their higher historical DNA substitution rates

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(Møller et al. 2010). Thus, the effects reported here may
also have implications for understanding natural variationin mutation accumulation.
In conclusion, bird species that have a large proportionof their plumage colored by pheomelanin, have large bodymass, or belong to non-corvoid passeriform families, display particularly large population declines in radioactively
contaminated areas. These results should be valuable forconservation purposes in areas affected by radioactive
contamination.

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