Introduction

Ionizing radiation is composed of particles able to liberate electrons from atoms or molecules and thus creates partially reduced chemical species, the most common being reactive oxygen species (ROS), which are involved in chain reactions that are potentially damaging to cells (Riley 1994). Living organisms have evolved a diversity of antioxidant compounds that can eliminate these damaging effects by combating ROS, which are constantly produced in the body by cellular metabolism. ROS activate cell signalling pathways which may trigger adaptive responses (Viña et al. 2006), but when antioxidant levels are below the thresholds required to limit ROS production, it leads to states of oxidative stress (Finkel & Holbrook 2000). Ionizing radiation is therefore an important source of oxidative stress in cells (e.g. Simone et al. 2009). This means that ionizing radiation can have profound effects on the evolutionary ecology of organisms, as oxidative stress is the ultimate cause of the deterioration of phenotypes (i.e. senescence) and the death of organisms, and it is thus considered a major determinant of the evolution of life‐history strategies (Dowling & Simmons 2009; Metcalfe & Alonso‐Alvarez 2010; Galván et al. 2012a).

However, most research on the biological effects of ionizing radiation have been conducted with cells or with organisms under laboratory conditions, which limits the capacity to obtain information on consequences for the ecology and evolution of organisms. Studies on wild populations are necessary to obtain a comprehensive view of the evolutionary consequences of ionizing radiation because free‐living populations may be limited or constrained in their ability to cope with effects of ionizing radiation. Natural background radioactivity levels show extreme variation of several hundred‐fold and have recently been found to affect mutational input and the expression of certain phenotypic traits, but studies on natural radioactivity are still few and scattered (Galván & Alonso‐Alvarez 2011; Møller & Mousseau 2013). Natural radiation and radiation accidents like those produced at the nuclear power plants of Chernobyl in 1986 and Fukushima in 2011 have had catastrophic environmental consequences, and the large levels of radioactivity released to the environment represent involuntary experiments and good opportunities for investigating the effects of ionizing radiation on wild populations of organisms. In Chernobyl, several studies have reported significant effects of radiation on the abundance, distribution, life history and mutation rates of plants and animals (Møller & Mousseau 2006), and effects on the abundance of animals have already been detected in Fukushima (Møller et al. 2012, 2013). In particular, radioactivity from Chernobyl has been found to produce oxidative stress by depleting antioxidants in humans (e.g. Ivaniota, Dubchak & Tyshchenko 1998; Neyfakh, Alimbekova & Ivanenko 1998; Romanenko et al. 2000; Vartanian et al. 2004) and other animals (Møller, Surai & Mousseau 2005; Møller, Karadaş & Mousseau 2008). Radiation levels in Chernobyl have also been found to covary with levels of cellular damage or dysfunction that may be mediated by oxidative damage (Sugg et al. 1996; Fenech, Perepetskaya & Mikhalevich 1997; Marozik et al. 2007; Bonisoli‐Alquati et al. 2010, 2011), and with other physiological consequences of oxidative stress such as reductions in brain size (Møller et al. 2011) and the expression of eye cataracts (Mousseau & Møller 2013).

There seems to be some consistency in reporting reductions in antioxidant levels and increases in oxidative damage in animals exposed to radioactive contamination (see studies mentioned above). Some authors, however, have found in humans that the levels of some antioxidants can even increase at low doses of radiation, although high levels of radiation may deplete antioxidants (Ivanenko & Burlakova 2013), and a recovery of oxidative status can be produced over time (Skesters et al. 2010). Indeed, the high degree of radioactive contamination found in the region of Chernobyl and the relative long time (28 years) elapsed since the accident make this an excellent scenario for investigating possible mechanisms of adaptation to ionizing radiation in natural populations.

Radiation‐induced adaptive responses have been well documented for decades in a diversity of species including humans through experiments in which cells or organisms are exposed to low doses of radiation (priming or conditioning dose) before receiving a higher, challenging dose (Olivieri, Bodycote & Wolff 1984; Iyer & Lehnert 2002). These studies have shown that chronic exposure to low, ‘adapting’ doses of different types of radiation increases the resistance of cells against subsequent acute exposure to challenging doses (Tapio & Jacob 2007). Correlative studies also report some evidence of radio‐adaption in cells from humans chronically exposed to low levels of radioactivity in Chernobyl (Tedeschi et al. 1995). The ultimate mechanisms of the radio‐adaptive response include complex patterns of cellular signalling and epigenetic changes that would favour the transmission of the response to the offspring (Kovalchuk et al. 2004). It seems that the starting point of the mechanism is not the direct effect of ionizing radiation on cellular structures, but an induction by ROS generated by radiation, which causes DNA damage (Tapio & Jacob 2007). As mentioned above, ROS generated by radiation is probably the cause of the observed decreases in antioxidant levels and increases in oxidative damage in humans and other animals from Chernobyl. Thus, there is evidence of physiological costs of radiation exposure in natural populations at Chernobyl, but not of adaptation to it. Alternatively, however, organisms may show adaptive responses to radiation at Chernobyl. This is potentially plausible given the observed positive effect of low‐dose radiation on some antioxidants (Ivanenko & Burlakova 2013), the recovery in the antioxidant status a long time after exposure (Skesters et al. 2010), and the fact that many organisms in the Chernobyl region have been chronically exposed to low doses of radiation, conditions that may favour radio‐adaption (Tapio & Jacob 2007). Searching for adaptive responses to radiation in natural populations is of key importance as it can potentially determine the capacity of species to evolve physiological adaptations and thus differential susceptibilities to overcome environmental challenges such as those that occurred in Chernobyl and Fukushima (Somero 2010).

Adaptive responses to radiation in natural populations at Chernobyl may not have been detected for several reasons. The large variability in radiation levels found in the entire Chernobyl zone represents a continuous environmental gradient, although there is a high temporal consistency in the background radiation levels to which individual organisms are exposed within their home ranges at Chernobyl (see 2), which may limit the capacity to detect adaptive responses in natural populations. In addition, it has been reported that the range of acute lethal doses of artificial ionizing radiation varies greatly among taxa, which is for example considerably greater in plants than in higher vertebrates, and lower in birds than in mammals (Newman & Unger 2003). This among‐taxa difference in susceptibility to radiation has already been reported in animals from Fukushima and Chernobyl regarding effects on population abundance (Møller et al. 2013), and susceptibility to natural radioactivity also varies among taxa (Møller & Mousseau 2013). Studies on the biological consequences of radioactivity at Chernobyl have concentrated on a few taxa (Møller & Mousseau 2006), and in the particular case of studies that report antioxidant and oxidative damage levels, they are all intraspecific and limited to humans, two species of birds and one species of fish (see references cited above). Therefore, the among‐taxa variability in susceptibility to radiation may represent an additional limitation in the capacity to detect radio‐adaptive responses. In fact, although the effects of radioactivity on bird populations at Chernobyl are negative overall, some species’ populations grow with increasing radiation (Galván, Mousseau & Møller 2011), lending support to the potential role of an adaptive response to chronic radiation exposure. Comparative studies may represent a solution for the two limitations mentioned above. Comparing several species that show different susceptibilities to radiation and that are subjected to a range of radiation levels enhances the capacity to detect effects. Comparative studies in which several phylogenetically distant species are investigated for antioxidant status have to our knowledge never been conducted in natural populations at Chernobyl, but are clearly necessary for developing insight into the potential role of radio‐adaption for the evolution of organisms.

The aim of this study was to investigate covariation between levels of glutathione (GSH) and DNA damage with levels of background radiation in wild populations of several phylogenetically distant species of birds in the Chernobyl region. We focus on GSH because it is one of the antioxidants most susceptible to radiation (Riley 1994; Ivaniota, Dubchak & Tyshchenko 1998; Neyfakh, Alimbekova & Ivanenko 1998; Ivanenko & Burlakova 2013), the most important intracellular antioxidant, and its redox status (GSH/GSSG) represents a relevant index of cellular oxidative stress (Wu et al. 2004). We consider DNA damage as measured by the comet assay, which quantifies strand breaks, as this is the most common damage to DNA caused by ionizing radiation (e.g. Kovalchuk et al. 2004). In two species of birds from Chernobyl (the barn swallow Hirundo rustica and the great tit Parus major), circulating antioxidant levels have decreased and oxidative damage has increased with radiation levels (Møller, Surai & Mousseau 2005; Møller, Karadaş & Mousseau 2008; Bonisoli‐Alquati et al. 2010, 2011). Thus, we predict that the same patterns should be found at the interspecific level regarding GSH and GSH/GSSG if birds exposed to radioactive contamination show a general and consistent physiological cost mediated by radiation. Alternatively, if there has been an adaptive response by birds to the chronic exposure of background radiation at Chernobyl, radiation should improve, at least up to certain level, the antioxidant response of birds. This should in turn prevent finding a decrease in levels of GSH and GSH/GSSG and an increase in DNA damage (which is probably caused by radiation‐induced oxidative stress; Bonisoli‐Alquati et al. 2010) and body condition (which predicts the survival of birds; Møller & Szep 2001) with increasing radiation. Such an adaptive response may however be costly to maintain, and such costs may be reflected in the population trends of birds. Therefore, we also analysed associations between the intensity of the physiological response and population trends of the species of birds at Chernobyl (Galván, Mousseau & Møller 2011).

When searching for possible differential capacities of species to adapt to chronic exposure to radiation, it is necessary to consider the production of melanins, the most common animal pigments. We have previously found that populations of species of birds expressing plumage colours typically provided by pheomelanin, a polymer of benzothiazine and benzothiazole units that constitutes one of the two main types of melanin, are more susceptible to the negative effects of radiation at Chernobyl (Galván, Mousseau & Møller 2011). The hypothesized mechanism behind this observation is that the sulphhydryl groups of cysteine and GSH are incorporated into the pheomelanin structure during its synthesis in melanocytes (García‐Borrón & Olivares Sánchez 2011; Ito et al. 2011a). Therefore, pheomelanin synthesis represents a consumption of an antioxidant resource because GSH (which is also the main physiological reservoir of cysteine) can no longer exert its antioxidant role once incorporated into the structure of the pigment, which is then deposited in inert tegumentary structures such as feathers and hair (Pavel, Smit & Pizinger 2011). Thus, pheomelanin synthesis represents a physiological cost under exposure to environmental factors that produce high levels of oxidative stress, as these conditions lead to greater demands of GSH for antioxidant protection (Galván, Ghanem & Møller 2012). However, it has never been directly tested if pheomelanin production entails GSH depletion and oxidative stress in organisms exposed to ionizing radiation. This test is necessary to determine why species producing large amounts of pheomelanin are more susceptible to the effects of radiation (Galván, Mousseau & Møller 2011). Therefore, we predict that under equal levels of background radiation birds with higher levels of pheomelanin in feathers should have lower levels of GSH and higher oxidative stress and DNA damage than birds producing lower amounts of pheomelanin. In contrast, eumelanin, a polymer of 5,6‐dihydroxyindole‐2‐carboxilic acid (DHICA) and 5,6‐dihydroxyindole (DHI) units that constitutes the other main type of melanin, is produced in the absence of cysteine and GSH (García‐Borrón & Olivares Sánchez 2011; Ito et al. 2011a) and protects cell survival and decreases DNA damage under exposure to ionizing radiation (Kinnaert et al. 2004). We thus predict that the content of eumelanin in feathers should enhance oxidative status and reduce DNA damage in birds exposed to equal levels of background radiation.

Lastly, given that the two units of pheomelanin have different oxidation potentials, benzothiazine having a greater reducing ability than benzothiazole, which is rather stable towards oxidation (Wakamatsu, Ohtara & Ito 2009; Wakamatsu et al. 2012), we tested the possibility of a radiation‐mediated conversion of benzothiazine into benzothiazole. Benzothiazole has a higher oxidation potential than benzothiazine, which makes the former produce more ROS when exposed to energetic radiation (Takeuchi et al. 2004; Ye et al. 2006). Thus, a conversion of benzothiazine into benzothiazole may protect birds at sites with radioactive contamination. We tested this by analysing the effect of background radiation on the ratio of thiazole‐2,4,5‐tricarboxylic acid (TTCA, a degradation product specific to the benzothiazole moiety; see Methods) to 4‐amino‐3‐hydroxyphenylalanine (4‐AHP, a degradation product specific to the benzothiazine moiety). We tested all predictions in wild populations of birds that were sampled in several sites around Chernobyl with a large range of background radiation levels.