doi:10.1111/j.1420-9101.2010.02074.x

Historical mutation rates predict susceptibility to radiationin Chernobyl birds
A. P. MØLLER* , J. ERRITZØEà, F. KARADAS§ & T. A. MOUSSEAU–*Laboratoire d’Ecologie, Systématique et Evolution, CNRS UMR 8079, Université Paris-Sud, Orsay Cedex, FranceCenter for Advanced Study, Oslo, NorwayàTaps Old Rectory, Christiansfeld, Denmark§Department of Animal Science, University of Yüzüncü Yil, Van, Turkey–Department of Biological Sciences, University of South Carolina, Columbia, SC, USA

Keywords:

Abstract

antioxidants;
birds;
extreme environmental perturbation;
mitochondrial DNA;
substitution rates.

Extreme environmental perturbations are rare, but may have important
evolutionary consequences. Responses to current perturbations may provide
important information about the ability of living organisms to cope withsimilar conditions in the evolutionary past. Radioactive contamination from
Chernobyl constitutes one such extreme perturbation, with significant but
highly variable impact on local population density and mutation rates ofdifferent species of animals and plants. We explicitly tested the hypothesis thatspecies with strong impacts of radiation on abundance were those with highrates of historical mutation accumulation as reflected by cytochrome b
mitochondrial DNA base-pair substitution rates during past environmental
perturbations. Using a dataset of 32 species of birds, we show higher historicalmitochondrial substitution rates in species with the strongest negative impactof local levels of radiation on local population density. These effects wererobust to different estimates of impact of radiation on abundance, weighting of
estimates of abundance by sample size, statistical control for similarity in the
response among species because of common phylogenetic descent, and effects
of population size and longevity. Therefore, species that respond strongly tothe impact of radiation from Chernobyl are also the species that in the past
have been most susceptible to factors that have caused high substitution rates
in mitochondrial DNA.

Introduction
Evolution on long time scales depends on the ability oforganisms to cope with extreme environmental perturbations (Hoffmann & Parsons, 1997). For example, mass
extinctions have occurred several times, leaving only asmall, but nonrandom sample of species to survive(Lawton & May, 1995). Mass extinctions during thePermian (e.g. Bowring et al., 1998; Raup & Sepkoski,1982) and the boundary between the Cretaceous and theTertiary (e.g. Alvarez et al., 1980; Jablonski, 1986)
constitute such events. The factors that contribute to
Correspondence: Anders P. Møller, Laboratoire d’Ecologie, Systématiqueet Evolution, CNRS UMR 8079, Université Paris-Sud, Bâtiment 362,
F-91405 Orsay Cedex, France.
Tel.: +33 1 69 15 56 88; fax: +33 1 69 15 56 96;
e-mail: anders.moller@u-psud.fr

such nonrandomness are of major importance for understanding macroevolutionary patterns, but also for understanding microevolution.
Extreme perturbations of the environment are rare
and hence difficult to study in situ. A prime example of an
extreme environmental perturbation is the impact of themeteors in Yucatan during the Cretaceous on climate andsubsequent biodiversity on Earth (Alvarez et al., 1980).
There are several other similar impacts. Volcanic eruptions such as those of Krakatoa and Mount St. Helens had
significant impact on global climate (Delmoral & Bliss,
1993; Zielinski, 1995), and previous volcanic eruptions
occasionally had even greater impacts (e.g. de Silva &Zielinski, 1998; Highwood & Stevenson, 2003). Suchextreme events may also have had random impacts on
the genomes of living organisms via population
bottlenecks followed by genetic drift, or the particularª 2010 THE AUTHORS. J. EVOL. BIOL.
JOURNAL COMPILATION ª 2010 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY

1

2

A. P. MØLLER ET AL.

physiology and ecology of certain species may haveproduced differences in the ability to cope with suchenvironmental perturbations.
The nuclear disaster at Chernobyl on 26 April 1986
resulted in the release of enormous amounts of radioactive material that contaminated thousands of square
kilometres across Europe (e.g. reviews in Shestopalov,
1996; Zakharov & Krysanov, 1996; Møller & Mousseau,
2006; Yablokov et al., 2009). We consider that Chernobyl
constitutes an extreme environmental perturbation thatmay provide important lessons for understanding ecological and evolutionary questions that are otherwise
difficult or impossible to study. Chernobyl has significantly increased mutation rates in plants and animals
including humans (reviews in Møller & Mousseau, 2006;
Yablokov et al., 2009). Severe negative ecological effects
such as reductions in abundance and biodiversity ofanimals including birds have also been documented(Møller & Mousseau, 2007b,c, 2008, 2009). This researchsuggests ecosystems have been significantly perturbed.
The underlying physiological mechanisms accounting forthese ecological effects have also been studied. The
physiological effects of radioactive contamination include
reductions in levels of antioxidants in humans andanimals (Bazhan, 1998; Ben-Amotz et al., 1998; Chaialo
et al., 1991; Ivaniota et al., 1998; Kumerova et al., 2000;
Lykholat & Chernaya, 1999; Neyfakh et al., 1998a,b).
Experimental studies involving humans have shown
improvements in health associated with beta carotene
supplementation (Ben-Amotz et al., 1998). It is also
possible that antioxidant levels, as affected by radiation,
have had negative effects on mutation rates; several
studies have indicated that the presence of high levels ofcarotenoids reduces mutation rates (reviews in Ferguson,
1994; Krinsky & Denek, 1982; Møller et al., 2000; Sies,
1993; Valko et al., 2004).
The objective of this study was to investigate whether
the short-term ecological response to radiation from
Chernobyl could be used to predict the accumulation of
mutations on an evolutionary time scale. The basis forthis objective is that species differ in susceptibility toenvironmental perturbations (e. g. Hoffmann & Parsons,
1991, 1997), including susceptibility to mutagens orability to repair DNA (reviews in Hoffmann & Parsons,
1991; Friedberg et al., 2006; Halligan & Keightley, 2009).
Likewise, species differ in susceptibility to the impact ofradiation from Chernobyl on abundance (Møller &Mousseau, 2007b) and mutation rates (reviews inKordyum & Sidorenko, 1996; Møller & Mousseau,
2006). Such interspecific differences in susceptibility toenvironmental perturbations could predict both shortterm ecological responses (such as changes in population
density) and long-term evolutionary responses (such as
DNA base-pair substitutions) if the underlying mechanisms were similar. For example, a specific life history
may render certain species particularly susceptible toshort-term environmental perturbations, but also toperturbations over time, with each major perturbation
increasing the rate of DNA base-pair substitutions.
Alternatively, differences in ecology may render certain
species particularly prone to population size fluctuations,with some species showing large fluctuations over time,
whereas others show smaller fluctuations over time, andthis difference might be expected to generate very
different patterns of DNA substitution rates through time
(Hartl & Clark, 1997). We tested the prediction that
ecological response in terms of reduction in population
density as a result of the extreme environmental perturbation caused by radioactive contamination from
Chernobyl was related to response to perturbations in
the evolutionary past as reflected by DNA base-pair
substitution rates. To this end, we used extensive census
data of breeding birds collected during 4 years of fieldwork in Ukraine and Belarus (Møller & Mousseau,
2007b, 2008) and an extensive database of DNA basepair substitution rates in the mitochondrial cytochrome bgene (Nabholz et al., 2008, 2009). A second objective wasto test whether the underlying mechanism for an
association between radiation and abundance and substitution rate, respectively, was based on interspecificdifferences in availability of antioxidants. To this end, weused an extensive database on concentrations of fatsoluble antioxidants (carotenoids, vitamin E) in the mainstorage organ, the liver (Møller et al., 2010). We controlled statistically for the potentially confounding effectsof two variables: population size and longevity. First,
common and widespread species could be better able tocope with adverse environmental conditions, and suchspecies may also fluctuate less in population size thanrare species with local distributions. Second, Nabholz
et al. (2008, 2009) suggested that longevity might berelated to substitution rates, with long-lived species being
especially affected by exposure to radioactive mutagens.
Thus, we tested the extent to which these two variables
could account for the association between reduction in
population density caused by radiation and DNA basepair substitution rate.

Materials and methods
Study sites
Anders Pape Møller (APM) (wearing a radiation protection suit in the most contaminated areas) conducted
standard point counts during 29 May–9 June 2006, 1–11
June 2007, 29 May–5 June 2008 and 1–6 June 2009.
Each count lasted 5 min during which all birds seen orheard were identified and recorded according to standard
procedures (Møller, 1983; Bibby et al., 2005). We
emphasize that the use of the radiation protection suit
did not interfere with the censuses because the hood was
not used, allowing the same possibilities for recording
birds visually and from calls and songs among sites. For
the record, we note that this procedure is fully inª 2010 THE AUTHORS. J. EVOL. BIOL. doi:10.1111/j.1420-9101.2010.02074.x
JOURNAL COMPILATION ª 2010 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGYChernobyl and mitochondrial substitution rates

accordance with radiation protection procedures used in
the Chernobyl Exclusion Zone. Removal of the < 5% of
all census points for which the radiation protection suit
was used did not change any of the conclusions reported
here. The census was conducted within the Chernobyl
Exclusion Zone (or in areas adjacent on the southern andwestern borders) with a permit from the Ukrainian
authorities and in contaminated areas in southern

3

Belarus around Gomel (breeding seasons 2006–2009)(Fig. 1). A total of 254 points (breeding season 2006), 240
points (breeding season 2007), 237 points (breeding
season 2008) and 167 points (breeding season 2009), in
total 898 points, were located at ca. 100 -m intervals
within forested areas [excluding successional stages ofsecondary forest because of abandoned farming (theseareas are still almost exclusively open grassland)]. There

Fig. 1 Location of breeding bird census areas and levels of background radiation around Chernobyl. Partly developed from European
Union (1998).

ª 2010 THE AUTHORS. J. EVOL. BIOL. doi:10.1111/j.1420-9101.2010.02074.x
JOURNAL COMPILATION ª 2010 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY

4

A. P. MØLLER ET AL.

was no temporal and spatial nonindependence of census
data because the analyses only relied on the firstobservation from a given census point, and because
highly heterogeneous deposition of radioactive material
as a result of the peculiarities of the Chernobyl accident(Møller & Mousseau, 2007a; Shestopalov, 1996; Yablokov et al., 2009 and Fig. 1) have reduced the spatial
autocorrelation in background radiation to a negligible
level (see data and analyses in Møller & Mousseau,
2007a). In other words, by using neighbouring sites thatdiffer in level of background radiation, we can exclude
the possibility that differences in weather, human activity, soil type or any other potentially confounding factor
could have biased the analyses.
Bird census data
We censused birds at the end of May and the beginningof June when most individuals reach their annual
maximum of singing activity, making censuses of breeding birds highly reliable (Møller, 1983; Bibby et al.,2005). We directly tested the reliability of our counts
by letting two persons independently perform counts.
The degree of consistency was high for both species
richness and abundance (Møller & Mousseau, 2007a).
The number of individuals recorded is reported in the
Supporting Information.

curvilinear relationship with time of day, with highlevels of activity in the morning and to a lesser extent in
the evening (Møller, 1983; Bibby et al., 2005), we also
included time squared as an explanatory variable.
Measuring background radiation levels
We measured radiation levels in the field and crossvalidated these with measurements by the Ministry of
Emergencies, Kiev, Ukraine. We measured a, b, and cradiation at ground level at each census point after
having conducted the census (thus making the census
blindly with respect to radiation level) using a hand-held
dosimeter (Model: Inspector; SE International, Inc.,
Summertown, TN, USA). We measured levels several(2–3) times at each site and averaged the measurements.
Our data were validated with correlation against datafrom 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 & Mousseau,
2007a). Radiation levels vary greatly at a local scale asa result of heterogeneity in deposition of radioactive
material after the Chernobyl accident (Fig. 1; Shestopalov, 1996). Our measurements at the census points
ranged from 0.01 to 135.89 lSv h)1.
Impact of radiation on population density

Confounding habitat and weather variables
Bird abundance estimates can be affected by numerous
confounding variables (Møller, 1983; Bibby et al., 2005),
and, therefore, it is crucial to control such variables
statistically to assess the underlying relationship between
radiation and species richness and abundance. We
classified habitats (agricultural habitats with grasslandor 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 a distance of 50 m from theobservation point. Agricultural habitat included edges
between forest and open areas, and the agricultural
habitat variable thus also reflected the amount of edge
habitat between forest and open areas. Maximum heightof trees was estimated to the nearest 5 m, and soil typewas 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), and werecorded cloud cover at the start of each point count [tothe nearest eighth, range 0–1 during the censuses, mean
(SE) = 0.707 (0.011)], temperature [degrees Celsius,
range 12–25 C, mean (SE) = 20.477 (0.102) C], andwind force [Beaufort, range 0–4 during the censuses,
mean (SE) = 2.378 (0.039)]. For each census point, werecorded time of day when the count was started (to thenearest minute). Because bird activity may show a

We quantified the impact of radiation on population
density by relating the number of individuals per census
point to log10-transformed local background radiationlevels for each of the 885 census points, while simultaneously controlling statistically for the potentially confounding variables listed above and year as a factor (seeMøller & Mousseau (2007b) for further details). The
slope of the partial effect of radiation on abundance wassubsequently used as an estimate of the effect of radiation
on local population density. Slope estimates were highly
consistent among years (Møller & Mousseau, 2010),
showing that we could obtain similar information about
the impact of radiation on abundance in different years.
Population size
Population sizes were obtained from Hagemeijer & Blair(1997), who reported the total number of breeding pairs
in the Western Palearctic west of the Ural Mountains,obtained in a consistent way from national bird census
programmes in all countries. We used the arithmetic
mean of the minimum and maximum estimates.
Range size
We determined the global northernmost and southernmost latitude of the breeding and the wintering distributions, respectively, to the nearest tenth of a degree ofª 2010 THE AUTHORS. J. EVOL. BIOL. doi:10.1111/j.1420-9101.2010.02074.x
JOURNAL COMPILATION ª 2010 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGYChernobyl and mitochondrial substitution rates

all species. Breeding range was the northernmost minus
the southernmost breeding latitude, whereas wintering
range was the northernmost minus the southernmost
winter latitude. Information on breeding and wintering
ranges was obtained from Cramp & Perrins (1988–1994).
These estimates have previously been shown to provide
biologically meaningful measures of distribution (Gaston& Blackburn, 1996).
We also estimated breeding range in the Western
Palearctic and total breeding range as the area of the
shape bounded by the greatest span of latitude andlongitude of each species’ breeding range, as published in
Cramp & Perrins (1988–1994). To take account of thecurvature of the earth (which was assumed to be
spherical), this area was estimated by the equation
Area ¼ R2  ðLongitude1  Longitude2 Þ  ðsin ðLatitude1 Þ sin ðLatitude2 ÞÞ
where R are the radius of the earth (6366.2 km), andlatitude and longitude are expressed in radians.
In widespread species, Old and New World ranges were
calculated separately and subsequently summed. Thismethod overestimates true geographical range because
parts of the range that are unoccupied are also included,
although this error should be random with respect to thevariables of interest. Estimates of area were strongly
positively correlated with geographical range size as
calculated by counting one-degree grid cells overlain on
published distribution maps for a sample of 20 Palearctic
and Nearctic bird species (r = 0.87, P < 0.001), and withrange size as reported for a sample of 11 threatened
species (Stattersfield & Capper, 2000) (r = 0.98,
P < 0.001, based on log-transformed data). See Møller
et al. (2008) for further information.
Longevity
We estimated longevity using records of ringed birds
adjusted for sampling effort; this approach ahs been
proven to provide unbiased estimates (Møller, 2006). We
extracted information on maximum longevity of European species from EURING. Longevity records only
provide reliable information on maximum lifespan if
records are adjusted for sampling effort. Among the 120
species of common birds in Europe for which longevity
records were available for the present study, the total
number of recoveries and recaptures of banded birds
across Europe ranged from 110 to 187 764, with a total of
1 953 714 records (http://www.euring.org). Therefore,
we used total number of recoveries reported as a measureof variation in sampling effort.
Mitochondrial DNA substitution rates
Species-specific mtDNA base-pair substitution rates wereused as reported by Nabholz et al. (2008, 2009). Briefly,

5complete cytochrome b sequences for 1571 avian species
were aligned, and changes in the third base-pair of aminoacid encoding codons were used as a measure of genetic
divergence (Nabholz et al., 2009). Such changes in thethird base-pair of amino acid encoding codons are mostoften synonymous with respect to amino acid changes
and are thus not usually under selection. The third codon
position substitution rate provides a good approximationof synonymous substitution rate for two different reasons. First, the vast majority of the cytochrome bdivergence is synonymous (the ratio nonsynonymous ⁄ synonymous divergence is close to 2% (Nabholz
et al., 2008; Stanley & Harrison, 1999). Second, all thetransition substitutions (A<->G and T<->C) of the cytochrome b third codon position are synonymous, andmost of the substitution are transitions (the ratio transition ⁄ transversion being typical > 15 in the dataset).
Antioxidants in liver
JE received 660 specimens for taxidermy from Denmark,with most individuals originating from a radius of
100 km from Christiansfeld, Denmark, and he collecteda sample of fresh liver for biochemical analyses. Livers
were frozen immediately after collection and maintainedat )20 C until analysis. Any livers that were notabsolutely fresh were discarded from the present study.
For all specimens, JE also recorded date, year, site andcause of death upon receipt.
Vitamin E concentrations were determined using a
Shimadzu Prominence (Kyoto, Japan) full high performance liquid chromatography (HPLC) system (Sil-20A
Autosampler; LC-20AD solvent delivery system; RF-10
AXL Spectrofluorometeric detector, CBM-20Alite system
controller; Cto-100ASvp column oven) fitted with a
Spherisorb, type S30DS2, 3 lm C-18 reverse phase
HPLC column (15 cm · 4.6 mm; Phase Separations,Cambridge, UK). Chromatography was performed usinga mobile phase of methanol ⁄ water (97 : 3, v ⁄ v) at a flowrate of 1.05 mL min)1. Fluorescence detection of vitamin
E used excitation at 295 nm and emission at 330 nm.
Peaks of d-, c- and a-tocopherol were identified bycomparison with the retention time of standards of
tocopherols (Sigma, Poole, UK). All sampled livers wereanalysed for vitamin E concentration. Vitamin E wascalculated as the summed concentrations of d-, c- anda-tocopherol. Concentrations and not quantity of vitamin
E were used as the variable of interest in statisticalanalysis because concentration is the main factor in
determining physiological action of antioxidants at thelevel of tissues (Surai, 2002). The inter-assay coefficient ofvariation for a-tocopherol determination was 3.9% (Suraiet al., 1999).
Total carotenoid concentration of liver was determinedusing the same HPLC system with a diode array detector at
444 nm, fitted with a Waters Spherisorb type NH2 column(25 cm · 4.6 mm; Phase Separation) with a mobile phaseª 2010 THE AUTHORS. J. EVOL. BIOL. doi:10.1111/j.1420-9101.2010.02074.x
JOURNAL COMPILATION ª 2010 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY

6

A. P. MØLLER ET AL.

of methanol-distilled water (97 : 3), at a flow rate of
1.5 L min)1 as described by Hõrak et al. (2002). The HPLC
was calibrated using lutein standards (Sigma). All analytic
detections were performed at 30 C in column oven and aconstant heating, ventilating and air conditioning (HVAC)
controlled room temperature of 24 C.
Estimates of total carotenoids and vitamin E weresignificantly correlated among individuals of the same
species [carotenoids: F152,506 = 4.03, P < 0.0001, R(SE) = 0.37
(0.03);
vitaminE:
F152,506 = 1.65,
P < 0.0001, R (SE) = 0.10 (0.02)]. Møller et al. (2010)provide a full description of this dataset, but also show that
this dataset contains biologically relevant information.
Body mass
We recorded body mass from Cramp & Perrins (1988–
1994), using information from the breeding season andpreferably the estimate with largest sample size, if morethan a single estimate was available. All data are reported
in Appendix S1.
Statistical analyses
Radiation level, substitution rate, concentration of carotenoids and vitamin E and body mass were log10transformed, whereas ground coverage with farmland
and deciduous forest was square root arcsine-transformed (coniferous forest was not included as an
explanatory variable, as it simply represented the groundcoverage not attributed to farmland and deciduous
forest) to achieve approximately normal distributions.
None of the distributions deviated significantly fromnormal distributions after transformation. We also
included radiation level squared to account for nonlinear
relationships between species richness and abundance,
respectively, and radiation. We developed statisticalmodels to assess the relationship between species richness and abundance (response variables) and radiation(predictor variable), assuming a Poisson distribution,
after inclusion of the potentially confounding variables(additional predictor variables listed above), as implemented in the statistical software JMP (SAS Institute Inc.,
2000).
We quantified the relationship between abundance ofdifferent species and level of radiation by calculating theslope of the relationship between number of individuals
per census point and log10-transformed radiation for eachspecies. These slopes that are statistically heterogeneousamong species (Møller & Mousseau, 2007a) were used
for subsequent analyses, with each slope being weighted
by sample size to account for unequal intensity ofsampling.
We related the substitution rate to the slope of therelationship between abundance and radiation. Because
sample sizes differed considerably among species owing
to differences in abundance, we conducted a second

series of analyses that weighted estimates by sample size.
In order to avoid giving a very large weight to the mostabundant species, we used square root-transformed
sample size as a weight (Garamszegi & Møller, 2010).
Analyses using weights based on square root of sample
sizes, log10-transformed sample sizes or the standard
error of the slopes gave qualitatively similar conclusions.
Comparative analyses
Closely related species have more similar phenotypes
than species that are more distantly related, simply
because similarity among closely related species is likely
to be because of to such species sharing a recent common
ancestor. We controlled for similarity in phenotype
among species owing to common ancestry by calculating
standardized independent linear contrasts (Felsenstein,1985), using the program CAIC (Purvis & Rambaut,1995). We tested the statistical and evolutionary assumptions of the continuous comparative procedure (Garland
et al., 1992) by regressing absolute standardized contrasts
against their standard deviations. To reduce the consequent problem of heterogeneity of variance, (i) outliers(contrasts with Studentized residuals > 3) were excluded
from subsequent analyses (Jones & Purvis, 1997), and (ii)analyses were repeated with the independent variable
expressed in ranks. In neither case did these new
analyses change any of the conclusions.
The composite phylogeny used in the analyses was
mainly based on Sibley & Ahlquist (1990), combinedwith information from more recent sources (Hackett
et al., 2008; Jønsson & Fjeldså, 2006; Appendix S2).
Because information for the composite phylogeny camefrom different studies using different methods, consistentestimates of branch lengths were unavailable. Therefore,branch lengths were transformed assuming a gradualmodel of evolution with branch lengths being proportional to the number of species contained within a clade.
Results based on these branch lengths were compared tothose obtained using constant branch lengths (a punctuated model of evolution). Nowhere were results qualitatively different.
Regressions based on contrasts were forced throughthe origin since the comparative analyses assume that
there has been no evolutionary change in a character
when the predictor variable has not changed (Purvis &Rambaut, 1995).
We assessed relationships based on effect sizes according to the criteria listed by Cohen (1988) for small(Pearson r = 0.10, explaining 1% of the variance),
intermediate (9% of the variance) or large effects (25%of the variance).

Results
The combined abundance of all species of birds at census
points was negatively related to background radiationª 2010 THE AUTHORS. J. EVOL. BIOL. doi:10.1111/j.1420-9101.2010.02074.x
JOURNAL COMPILATION ª 2010 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGYChernobyl and mitochondrial substitution rates[F1,28672 = 28.52, P < 0.0001, slope (SE) = )0.0018(0.0003)], it differed among species (F31,28672 = 134.00,
P < 0.0001), and the effect of radiation differed among
species (F31,28672 = 13.83, P < 0.0001). Eleven of theseslopes were statistically significant, 10 negative and one
positive. Because there were significant differences inslope among species, it is meaningful to investigate
predictors of this heterogeneity. The substitution rate in
the sample of 32 species studied was on average 0.0480
changes in the third base-pair of amino acid encodingcodons for cytochrome b per site per million years(SE = 0.0072), range 0.0067–0.2020, differing significantly from zero (one-sample t-test, t31 = 6.66,
P < 0.0001).
Species more strongly affected by radiation had higher
substitution rates (Fig. 2), accounting for 17% of thevariance (Table 1), implying that mutation rates werehigher in species that were more strongly negatively
affected by radiation. Bootstrapped estimates of the slope
were )0.0303 (0.0124), based on 1000 bootstraps, almost
identical to the estimate reported in Table 1. Because theslope estimate was based on highly variable sample sizes
owing to differences in abundance, we used a second
model that weighted species-specific observations bysquare root-transformed sample size. This model wasagain highly significant, explaining 32% of the variance(Table 1). Using log-transformed sample size as a weighting factor provided an equally strong relationship[F1,30 = 10.44,
P = 0.0030,
slope(SE) = )0.051(0.016)]. Likewise, weighting by the standard error of
the slope estimate provided a qualitatively similar conclusion [F1,30 = 18.03, P = 0.0002, slope (SE) = )0.064(0.015)]. Inclusion of body mass as an additional predictor variable significantly improved the fit of the model[species-specific analyses: substitution rate partial
F1,29 = 14.25, P = 0.0007, slope (SE) = )0.078 (0.021),
0.08

Slope

0.04

0.00
c

c
c

–0.04

c
c

–0.08
c
–0.12
0.001

0.01

0.1

Substitution rate
Fig. 2 Slope of the relationship between population density ofdifferent species of breeding birds and radiation level around
Chernobyl in relation to mitochondrial substitution rate.

1

7

Table 1 Models relating slope of the relationship between population density of different species of birds and radiation around
Chernobyl and mitochondrial DNA substitution rate.

Variable

Sum ofsquares d.f. F

Species
Mitochondrial DNA
0.711
substitution rate
Error
3.446
Mitochondrial DNA
0.089
substitution rate
weighted by sample size
Error
0.191

1

P

6.19 0.019

Slope (SE))0.030 (0.012)

30
1 13.97 0.0008 )0.069 (0.018)

30

Contrasts
Mitochondrial DNA
0.00066 1
substitution rate
Error
0.0049 30
Mitochondrial DNA
0.00441
substitution rate
weighted by sample size
Error
0.0220 30

4.19 0.048)0.031 (0.014)

5.59 0.025)0.036 (0.015)

with an additional effect of body mass: partial
F1,29 = 0.092, P = 0.035, slope (SE) = )0.014 (0.015);
contrasts: substitution rate partial F1,29 = 5.87, P = 0.022,
slope (SE) = )0.051 (0.021); body mass partial
F1,29 = 0.02, P = 0.89, slope (SE) = )0.002 (0.016)].
Two species had extreme slope values (Fig. 2), suggesting that a nonparametric analysis might be more
appropriate. A nonparametric Kendall rank-order correlation also revealed a highly significant negative relationship (Kendall s = )0.30, P = 0.018). Even exclusionof these two species produced a highly significant
negative relationship accounting for 42% of the variance[weighted analysis: F1,28 = 18.51, r2 = 0.40, P = 0.0002,
slope (SE) = )0.042 (0.010)].
Because previous studies have indicated that bird
species have reduced carotenoid levels in more contaminated areas, and that species with carotenoid-based
plumage have more suppressed population densities, weanalysed the independent effects of carotenoid levels andsubstitution rates on slopes. This analysis weighted bysample size showed a statistically significant effect of
substitution rate, but not of carotenoids on the slope(Table 2). Likewise, the analysis based on linear contrasts
showed a significant effect for substitution rate, but notfor carotenoids (Table 2). Vitamin E did not enter any ofthese models as a significant predictor.
We developed a statistical model with slope of therelationship between abundance and background radiation level as the response variable and substitution rate
and population size as predictors. Only the former
predictor showed a significant effect [species-specificanalysis weighted by sample size: partial F1,29 = 13.90,
r2 = 0.32, P = 0.0008, slope (SE) = )0.071 (0.019)],ª 2010 THE AUTHORS. J. EVOL. BIOL. doi:10.1111/j.1420-9101.2010.02074.x
JOURNAL COMPILATION ª 2010 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY

8

A. P. MØLLER ET AL.

Table 2 Models relating slope of the relationship between population density of different species of birds to radiation around
Chernobyl and mitochondrial DNA substitution rate and carotenoid
levels in the liver, weighted by square root-transformed sample sizes.

Variable
Species
Mitochondrial DNA
substitution rate
weighted by sample size
Carotenoids
Error

Sum ofsquares d.f. F

P

Slope (SE)

0.112

1

12.51 0.0024 )0.103 (0.029)

0.016
0.161

1
18

1.79 0.20

Contrasts
Mitochondrial DNA
0.010
substitution rate weighted
by sample size
Carotenoids
0.002
Error
0.026

)0.028 (0.021)

1

6.95 0.017)0.064 (0.024)

118

1.23 0.28)0.016 (0.015)

The model had the statistics F2,18 = 6.57, r2 = 0.42, P = 0.007 andF2,18 = 3.63, r2 = 0.17, P = 0.047.

whereas that was not the case for population size [partial
F1,29 = 0.28, r2 = 0.01, P = 0.60, slope (SE) = 0.005(0.010); contrast analysis weighted by sample size: partial
F1,29 = 5.93, r2 = 0.17, P = 0.021, slope (SE) = )0.049(0.020)], but not for population size [partial F1,29 = 1.35,
r2 = 0.04, P = 0.25, slope (SE) = )0.011 (0.009)]. A
similar conclusion was reached if range size was included
as an additional variable to account for the fact thatspecies with larger ranges obviously have larger population sizes [species-specific analysis weighted by sample
size: substitution rate partial F1,28 = 14.97, r2 = 0.35,
P = 0.0006, slope (SE) = )0.070 (0.018); population
size partial F1,28 = 1.99, r2 = 0.07, P = 0.17, slope(SE) = 0.016 (0.011); range size partial F1,28 = 3.42,
r2 = 0.11, P = 0.08, slope (SE) = )0.157 (0.085); contrast
analysis weighted by sample size: substitution rate partial
F1,28 = 5.64, r2 = 0.17, P = 0.025, slope (SE) = )0.046(0.019); population size partial F1,28 = 0.10, r2 = 0.00,
P = 0.75, slope (SE) = 0.004 (0.012); range size partial
F1,28 = 3.25, r2 = 0.10, P = 0.08, slope (SE) = )0.120(0.066)].
In a final series of analyses, we tested for an effect of
longevity, with sample size for longevity as an additionalvariable. There was still a strong relationship between
slope of the relationship between abundance and background radiation level as the response variable andsubstitution rate, longevity and sample size for longevity
as predictors, with only the former showing a significant
effect [species-specific analysis weighted by sample size
substitution rate partial F1,25 = 11.97, r2 = 0.32,
P = 0.0019, slope (SE) = )0.075 (0.022); contrast analysis weighted by sample size substitution rate partial
F1,25 = 5.01, r2 = 0.17, P = 0.034, slope (SE) = )0.041(0.018)], but not for longevity [species-specific analysis

weighted by sample size partial F1,25 = 0.36, r2 = 0.01,
P = 0.55, slope (SE) = )0.030 (0.050); contrast analysis
weighted by sample size partial F1,25 = 0.32, r2 = 0.01,
P = 0.58, slope (SE) = 0.017 (0.030)], or number ofrecoveries [species-specific analysis weighted by sample
size partial F1,25 = 0.83, r2 = 0.03, P = 0.37, slope(SE) = )0.009 (0.010); contrast analysis weighted bysample size partial F1,25 = 2.51, r2 = 0.09, P = 0.13, slope(SE) = )0.010 (0.006)].

Discussion
Here, we have shown that species of birds that haveseverely reduced population densities in contaminatedareas are the same species that have accumulated themost mutations over time as reflected by third base-pair
substitutions in codons of cytochrome b. We investigated a number of alternative explanations, but foundno evidence that any of these factors accounted for themain result. This major finding is consistent with our
hypothesis. Body size is an important predictor of
substitution rate (reviews in Nabholz et al., 2008,2009), but the effect of body size did not confound
any of these conclusions. These effects were similar
when the analyses were adjusted for interspecificdifferences in sampling effort. Likewise, comparative
analyses investigating the relationship after taking similarity among species owing to common phylogenetic
descent into account resulted in similar conclusions.
Because levels of antioxidants are significantly suppressed in birds in the most contaminated areas, we
conducted a second series of analyses relating substitution rates to both impact of radiation on abundance andantioxidant levels. However, the relationship between
substitution rate and impact of radiation on abundance
was independent of concentrations of antioxidants.
Finally, effects of population size and longevity did notconfound the conclusions, nor were such effects to be
expected for a neutral genetic marker. We interpretthese findings as being consistent with our hypothesisthat species differ in susceptibility to extreme environmental perturbations, and that such differences arereflected both on a short time scale as shown forabundance and radiation, but also on a long time scale
as for substitution rates. The underlying hypotheticalmechanism is that species differ in their ability to repair
DNA, which affects both DNA substitution rates andsusceptibility to radiation from Chernobyl.
Radiation from environmental contamination as aresult of the Chernobyl explosion has had important
negative effects on mutations, abundance and physiology of animals and plants (reviews in Zakharov &Krysanov, 1996; Møller & Mousseau, 2006; Yablokov
et al., 2009). In the present study, we investigated theimpact of radiation on abundance of common breeding
birds around Chernobyl in Ukraine and Belarus andrelated the impact of radiation on abundance to aª 2010 THE AUTHORS. J. EVOL. BIOL. doi:10.1111/j.1420-9101.2010.02074.x
JOURNAL COMPILATION ª 2010 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGYChernobyl and mitochondrial substitution rates

measure of historical mutation rates as reflected byneutral third base-pair substitutions in codons of cytochrome b. The basis for this hypothesis is that species
differ in susceptibility to environmental perturbations,as shown by extensive ecological and physiologicalstudies (e.g. Hoffmann & Parsons, 1991, 1997; Bokony
et al., 2009). Likewise, both plants and animals differ insusceptibility to mutagens or efficiency of DNA repair(reviews in Hoffmann & Parsons, 1991; Friedberg et al.,2006; Halligan & Keightley, 2009). Similar effects havebeen reported based on research at Chernobyl. Speciesof plants and animals differ in susceptibility to theimpact of radiation from Chernobyl on abundance(Møller & Mousseau, 2007b) and mutation rates(reviews in Kordyum & Sidorenko, 1996; Møller &Mousseau, 2006). Such interspecific differences in susceptibility could predict both short-term ecologicalresponses (such as changes in population density) andlong-term evolutionary responses (such as DNA basepair substitutions) if the underlying mechanisms weresimilar. Here, we have shown that species of birds thathave severely reduced population densities in contaminated areas are the same species that have accumulated
the most mutations over time as reflected by third basepair substitutions in codons of cytochrome b. We
investigated a number of alternative explanations, but
found no evidence that any of these factors accounted
for the main result. This major finding is consistent with
our hypothesis.
There was a significant negative relationship between
the effects of background radiation on abundance andthird base-pair substitutions in codons of cytochrome bwith an intermediate to large effect size (sensu Cohen,1988), implying that mutation rates were higher in
species that were more adversely affected by radiation.
This conclusion remained unchanged by a number ofalternative analyses, exclusion of extreme data points,
and analyses corrected for possible phylogenetic biases.
Many factors are known to influence bird census results(Møller, 1983; Bibby et al., 2005), and we included thesepotentially confounding factors in the statistical analyses.
Obviously, we cannot be certain that some as yet
unknown confounding factor was included, although
this seems unlikely given the fact that standardized
national breeding bird census programmes have been
conducted in Europe since the 1960s, and that suchprogrammes are implemented in environmental monitoring programmes at national and continental scales.
These surprising findings imply that ecological effects ofradiation, as reflected by the reduced abundance of birds
in radioactively contaminated areas, provide important
information on the ability of individuals of different
species to cope with environmental perturbations in thepast. Numerous large environmental impacts haveoccurred on an evolutionary time scale, affecting the
distribution and abundance of taxa, but also their
extinction rates (e.g. Lawton & May, 1995). Mass

9extinctions such as those during the Permian (e.g.
Bowring et al., 1998; Raup & Sepkoski, 1982) and theboundary between the Cretaceous and the Tertiary havebeen linked to meteor impacts on Earth (e.g. Alvarez
et al., 1980; Jablonski, 1986). Here, we have shown that
long-term substitution rates during evolutionary time
can be used to make inferences about the impact of
extreme environmental perturbations during ecologicaltime. This also implies that responses of living organisms
to extreme perturbations such as the Chernobyl disastercan have important repercussions for basic evolutionary
and conservation research.
We investigated the underlying physiological mechanisms by analysing the relationship between antioxidants
and third base-pair substitutions in codons of cytochromeb. Studies of humans (Bazhan, 1998; Ben-Amotz et al.,
1998; Chaialo et al., 1991; Ivaniota et al., 1998; Kumerova et al., 2000; Lykholat & Chernaya, 1999; Neyfakh
et al., 1998a,b) and animals (Møller & Mousseau, 2007b;
Møller et al., 2005, 2008) have shown severely reduced
levels of antioxidants in individuals exposed to radiation,with effects being described for carotenoids and vitamin
E. Here, we found that bird species with strong impacts ofradiation on abundance also had reduced levels ofcarotenoids stored in the liver, but not of vitamin E,
independent of the relationship for substitution rates.
However, this apparent effect of carotenoids was not
maintained following corrections for phylogenetic biases,suggesting that the effect of carotenoids could not haveconfounded the relationship between substitution rateand effect of radiation on abundance.
Finally, we investigated the potential confounding
effects of population size and longevity on the conclusions, but found no evidence consistent with thesehypothetical effects. Thus, the conclusions were robust
and remained unaffected by any of the obvious potentially confounding variables.
In conclusion, we have found that neutral mutations
in cytochrome b accumulated during evolutionary time
were a predictor of the response of population density ofbirds to exposure to elevated background radiation from
Chernobyl.

Acknowledgments
Dr B. Nabholz generously provided access to his substitution data. We are grateful for logistic help during ourvisits to Ukraine and Belarus from O. Bondarenko,
M. Bondarkov, I. Chizhevsky, S. Gaschak, E. Konoplya,
A. Litvinchuk, G. Milinevski, A. M. Peklo, E. Pysanets,
E. Konoplya, V. Kudryashov and N. Saino. J. Waldron
provided comments. We received funding from the
University of South Carolina School of the Environment,
Bill Murray and the Samuel Freeman Charitable Trust,the National Science Foundation, NATO, the Fulbright
Program, CRDF and the National Geographic Society to
conduct our research.

ª 2010 THE AUTHORS. J. EVOL. BIOL. doi:10.1111/j.1420-9101.2010.02074.x
JOURNAL COMPILATION ª 2010 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY

10

A . P . M Ø LL E R E T A L .

References
Alvarez, L.W., Alvarez, W., Asaro, F. & Michel, H.W. 1980.
Extraterrestrial cause for the Cretaceous-Tertiary extinction:
experimental results and theoretical interpretation. Science
208: 1095–1108.
Bazhan, K.V. 1998. Lipid peroxidation and the antioxidant
system in subjects exposed to the influence of extreme factors.
Lik. Sprava 8: 47–50.
Ben-Amotz, A., Yatziv, S., Sela, M., Greenberg, S., Rachmilevich, B., Shwarzman, M. & Weshler, Z. 1998. Effect of naturalbeta-carotene supplementation in children exposed to radiation from the Chernobyl accident. Radiat. Environ. Biophys. 37:
187–193.
Bibby, C.J., Hill, D.A., Burgess, N.D. & Mustoe, S. 2005. Bird
Census Techniques. Academic Press, London, UK.
Bokony, V., Lendvai, A.Z., Liker, A., Angelier, F., Wingfield, J.C.
& Chastel, O. 2009. Corticosterone response and the value of
reproduction: are birds prudent parents? Am. Nat. 173: 589–
598.
Bowring, S.A., Erwin, D.H., Jin, Y.G., Martin, M.W., Davidek, K.
& Wang, W. 1998. U ⁄ Pb Zircon geochronology and tempo of
the end-Permian mass extinction. Science 280: 1039–1045.
Chaialo, P.P., Bereza, V.I. & Chobot’ko, G.M. 1991. Free-radical
processes and blood antioxidant systems in the late period
following acute radiation sickness. Med. Radiol. (Moscow) 36:
20–21.
Cohen, J. 1988. Statistical Power Analysis for the Behavioral Sciences,
2nd edn. Lawrence Erlbaum, Hillsdale, NJ.
Cramp, S. & Perrins, C. (eds) 1988–1994. The Birds of the Western
Palearctic, Vols. 1–9. Oxford University Press, Oxford, UK.
Delmoral, R. & Bliss, L.C. 1993. Mechanisms of primarysuccession: insights resulting from the eruption of Mount St.
Helens. Adv. Ecol. Res. 24: 1–66.
European Union 1998. Atlas of Caesium Deposition on Europe after
the Chernobyl Accident. EU Publication EUR 16733, Bruxelles,
Belgium.
Felsenstein, J. 1985. Phylogenies and the comparative method.
Am. Nat. 125: 1–15.
Ferguson, L.R. 1994. Antimutagens as cancer chemopreventive
agents in the diet. Mutat. Res. 307: 395–410.
Friedberg, E.C., Walker, G.C., Siede, W. & Wood, R.D. 2006.
DNA Repair and Mutagenesis. ASM Press, Washington, DC.
Garamszegi, L.Z. & Møller, A.P. 2010. Effects of sample size andintraspecific variation in phylogenetic comparative studies: ameta-analytic review. Biol. Rev. (in press).
Garland, T. Jr, Harvey, P.H. & Ives, A.R. 1992. Procedures for the
analysis of comparative data using phylogenetically independent contrasts. Syst. Biol. 41: 18–32.
Gaston, K.J. & Blackburn, T.M. 1996. Global scale macroecology:interactions between population size, geographic range sizeand body size in the Anseriformes. J. Anim. Ecol. 65: 701–714.
Hackett, S.J., Kimball, R.T., Reddy, S., Bowie, R.C.K., Braun,
E.L., Braun, M.J., Chojnowski, J.L., Cox, W.A., Han, K.-L.,
Harshman, J., Huddleton, C.J., Marks, B.D., Miglia, K.J.,
Moore, W.A., Sheldon, F.H., Steadman, D.W., Witt, C.C. &Yuri, T. 2008. A phylogenomic study of birds reveals theirevolutionary history. Science 320: 1763–1768.
Hagemeijer, W.J.M. & Blair, M.J. 1997. The EBCC Atlas ofEuropean Breeding Birds: Their Distribution and Abundance. T.
and A. D. Poyser, London, UK.

Halligan, D.L. & Keightley, P.D. 2009. Spontaneous mutation
accumulation studies in evolutionary genetics. Ann. Rev. Ecol.
Evol. Syst. 40: 151–172.
Hartl, D. & Clark, A.G. 1997. Principles of Population Genetics, 3rdedn. Sinauer, Sunderland, MA.
Highwood, E.J. & Stevenson, D.S. 2003. Atmospheric impact of
the 1783–1784 Laki eruption: Part II. Climate effect ofsulphate aerosol. Atmos. Chem. Phys. 3: 1177–1189.
Hoffmann, A.A. & Parsons, P.A. 1991. Evolutionary Genetics andEnvironmental Stress. Oxford University Press, Oxford, UK.
Hoffmann, A.A. & Parsons, P.A. 1997. Extreme Environmental
Change and Evolution. Cambridge University Press, Cambridge,UK.
Hõrak, P., Surai, P.F. & Møller, A.P. 2002. Fat-soluble
antioxidants in the eggs of great tits Parus major in relation
to breeding habitat and laying sequence. Avian Science 2: 123–
130.
Ivaniota, L., Dubchak, A.S. & Tyshchenko, V.K. 1998. Free
radical oxidation of lipids and antioxidant system of blood in
infertile women in a radioactive environment. Ukr. Biokhim.
Zh. 70: 132–135.
Jablonski, D. 1986. Background and mass extinctions: thealternation of macroevolutionary regimes. Science 231: 129–
133.
Jones, K.E. & Purvis, A. 1997. An optimum body size formammals? Comparative evidence from bats. Funct. Ecol. 11:
751–756.
Jønsson, K.A. & Fjeldså, J. 2006. A phylogenetic supertree ofoscine passerine birds (Aves: Passeri). Zool. Scripta 35: 149–
186.
Kordyum, E.L. & Sidorenko, P.G. 1996. The results of cytogenetic monitoring in angiosperm species of plants in the zone ofradionuclide contamination after the Chernobyl NPP accident.
Tsitol. Genet. 31: 39–46.
Krinsky, N.I. & Denek, S.M. 1982. Interaction of oxygen andoxy-radicals with carotenoids. J. Natl Cancer Inst. 69: 205–210.
Kumerova, A.O., Lece, A.G., Skesters, A.P., Orlikov, G.A.,
Seleznev, J.V. & Rainsford, K.D. 2000. Antioxidant defense
and trace element imbalance in patients with postradiation
syndrome: first report on phase I studies. Biol. Trace Elem. Res.
77: 1–12.
Lawton, J.H. & May, R.M. 1995. Extinctions. Oxford University
Press, Oxford, UK.
Lykholat, E.A. & Chernaya, V.I. 1999. Parameters of peroxidation and proteolysis in the organism of the liquidatorsof Chernobyl accident consequences. Ukr. Biokhim. Zh. 71:
82–85.
Møller, A.P. 1983. Methods for Monitoring Bird Populations in theNordic Countries. Nordic Council of Ministers, Oslo, Norway.
Møller, A.P. 2006. Sociality, age at first reproduction andsenescence: comparative analyses of birds. J. Evol. Biol. 19:
682–689.
Møller, A.P. & Mousseau, T.A. 2006. Biological consequences ofChernobyl: 20 years after the disaster. Trends Ecol. Evol. 21:
200–207.
Møller, A.P. & Mousseau, T.A. 2007a. Birds prefer to breed insites with low radioactivity in Chernobyl. Proc. R. Soc. Lond. B
274: 1443–1448.
Møller, A.P. & Mousseau, T.A. 2007b. Determinants of interspecific variation in population declines of birds from exposure
to radiation at Chernobyl. J. Appl. Ecol. 44: 909–919.

ª 2010 THE AUTHORS. J. EVOL. BIOL. doi:10.1111/j.1420-9101.2010.02074.x
JOURNAL COMPILATION ª 2010 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGYChernobyl and mitochondrial substitution rates

Møller, A.P. & Mousseau, T.A. 2007c. Species richness andabundance of birds in relation to radiation at Chernobyl. Biol.
Lett. 3: 483–486.
Møller, A.P. & Mousseau, T.A. 2008. Reduced abundanceof raptors in radioactively contaminated areas near Chernobyl.
J. Ornithol. 150: 239–246.
Møller, A.P. & Mousseau, T.A. 2009. Reduced abundance ofinsects and spiders linked to radiation at Chernobyl 20 years
after the accident. Biol. Lett. 5: 356–359.
Møller, A.P. & Mousseau, T.A. 2010. Efficiency of bio-indicatorsfor low-level radiation under field conditions. Ecol. Indic.
(in press).
Møller, A.P., Biard, C., Blount, J.D., Houston, D.C., Ninni, P.,
Saino, N. & Surai, P.F. 2000. Carotenoid-dependent signals:
indicators of foraging efficiency, immunocompetence ordetoxification ability? Poult. Avian Biol. Rev. 11: 137–159.
Møller, A.P., Surai, P.F. & Mousseau, T.A. 2005. Antioxidants,
radiation and mutation in barn swallows from Chernobyl.
Proc. R. Soc. Lond. B 272: 247–253.
Møller, A.P., Karadas, F. & Mousseau, T.A. 2008. Antioxidants ineggs of great tits Parus major from Chernobyl and hatching
success. J. Comp. Physiol. B 178: 735–743.
Møller, A.P., Erritzøe, J. & Karadas, F. 2010. Levels of antioxidants in rural and urban birds and their consequences.
Oecologia 163: 35–45.
Nabholz, B., Glemin, S. & Galtier, N. 2008. Strong variations ofmitochrondrial mutation rate across mammals: the longevity
hypothesis. Mol. Biol. Evol. 25: 120–130.
Nabholz, B., Glemin, S. & Galtier, N. 2009. The erratic
mitochondrial clock: variations of mutation rate, not population size, affect mtDNA diversity across birds and mammals.
BMC Evol. Biol. 9: 54.
Neyfakh, E.A., Alimbekova, A.I. & Ivanenko, G.F. 1998a.
Vitamin E and A deficiencies in children correlate withChernobyl radiation loads of their mothers. Biochemistry (Mosc)
63: 1138–1143.
Neyfakh, E.A., Alimbekova, A.I. & Ivanenko, G.F. 1998b.
Radiation-induced lipoperoxidative stress in children coupledwith deficit of essential antioxidants. Biochemistry (Mosc) 63:
977–987.
Purvis, A. & Rambaut, A. 1995. Comparative analysis byindependent contrasts (CAIC). Comp. Appl. Biosci. 11: 247–251.
Raup, D.M. & Sepkoski, J.J. 1982. Mass extinctions in themarine fossil record. Science 215: 1501–1503.
SAS Institute Inc. 2000. JMP. SAS Institute Inc., Cary, NC.
Shestopalov, V.M. 1996. Atlas of Chernobyl Exclusion Zone.
Ukrainian Academy of Science, Kiev, Ukraine.
Sibley, C.G. & Ahlquist, J.E. 1990. Phylogeny and Classification ofBirds, a Study in Molecular Evolution. Yale University Press, New
Haven, CN and London, UK.
Sies, H. 1993. Strategies of antioxidant defense. Eur. J. Biochem.
215: 213–219.
de Silva, S.L. & Zielinski, G.A. 1998. Global influence of AD 1600
eruption of Huaynaputina, Peru. Nature 393: 455–458.

11

Stanley, S.E. & Harrison, R.G. 1999. Cytochrome b evolution inbirds and mammals: an evaluation of the avian constraint
hypothesis. Mol. Biol. Evol. 16: 1575–1585.
Stattersfield, A.J. & Capper, D.A. 2000. Threatened Birds of theWorld. Lynx Ediciones, Barcelona, Spain.
Surai, P.F., MacPherson, A., Speake, B.K. & Sparks, N.H.C. 1999.
Designer egg evaluation in a controlled trial. Eur. J. Clin. Nutr.
54: 298–305.
Surai, P. 2002. Natural Antioxidants in Avian Nutrition andReproduction. Nottingham University Press, Nottingham, UK.
Valko, M., Izakovic, M., Mazur, M., Rhodes, C.J. & Telser, J.
2004. Role of oxygen radicals in DNA damage and cancer
incidence. Mol. Cell. Biochem. 266: 37–56.
Yablokov, A.V., Nesterenko, V.B. & Nesterenko, A.V. 2009.
Chernobyl: Consequences of the Catastrophe for People and Nature.
New York Academy of Sciences, New York, NY.
Zakharov, V.M. & Krysanov, E.Y. (eds) 1996. Consequences of theChernobyl Catastrophe: Environmental Health. Center for Russian
Environmental Policy, Moscow, Russia.
Zielinski, G.A. 1995. Stratospheric loading and optical depth
estimates of explosive volcanism over the last 2100 years
derived from the Greenalnd-ice-sheet-project-2 ice core.
J. Geophys. Res. Atmos. 100: 20937–20955.

Supporting information
Additional Supporting Information may be found in the
online version of this article:
Appendix S1 Summary statistics of DNA substitution
rate (divergence per site per million years), the slope ofabundance of different species of birds in relation to local
radiation level, after controlling statistically for confounding variables affecting abundance estimates, number of individuals censused at Chernobyl, population sizein Western Palearctic, range size in Western Palearctic(km2), longevity record (years), sample size for longevity
record, concentration of carotenoids and vitamin E in theliver (lg g)1) and body mass.
Appendix S2 Phylogenetic relationships among species.
As a service to our authors and readers, this journal
provides supporting information supplied by the authors.
Such materials are peer-reviewed and may be reorganized for online delivery, but are not copy-editedor typeset. Technical support issues arising from supporting information (other than missing files) should be
addressed to the authors.
Received 18 January 2010; revised 27 June 2010; accepted 7 July 2010
Dryad Digital Repository doi:10.5061/dryad.1750ª 2010 THE AUTHORS. J. EVOL. BIOL. doi:10.1111/j.1420-9101.2010.02074.x
JOURNAL COMPILATION ª 2010 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY