Links between fear of humans, stress and survival support a non-random distribution of birds among urban and rural habitats.

Urban endocrine ecology aims to understand how organisms cope with new sources of stress and maintain allostatic load to thrive in an increasingly urbanized world. Recent research efforts have yielded controversial results based on short-term measures of stress, without exploring its fitness effects. We measured feather corticosterone (CORTf, reflecting the duration and amplitude of glucocorticoid secretion over several weeks) and subsequent annual survival in urban and rural burrowing owls. This species shows high individual consistency in fear of humans (i.e., flight initiation distance, FID), allowing us to hypothesize that individuals distribute among habitats according to their tolerance to human disturbance. FIDs were shorter in urban than in rural birds, but CORTf levels did not differ, nor were correlated to FIDs. Survival was twice as high in urban as in rural birds and links with CORTf varied between habitats: while a quadratic relationship supports stabilizing selection in urban birds, high predation rates may have masked CORTf-survival relationship in rural ones. These results evidence that urban life does not constitute an additional source of stress for urban individuals, as shown by their near identical CORTf values compared with rural conspecifics supporting the non-random distribution of individuals among habitats according to their behavioural phenotypes.

Large-scale spatial variation in feather corticosterone in invasive house sparrows (Passer domesticus) in Mexico is related to climate.

Ecologists frequently use physiological tools to understand how organisms cope with their surroundings but rarely at macroecological scales. This study describes spatial variation in corticosterone (CORT) levels in feathers of invasive house sparrows (Passer domesticus) across their range in Mexico and evaluates CORT-climate relationships with a focus on temperature and precipitation. Samples were collected from 49 sites across Mexico. Feather CORT (CORTf) was measured using methanol-based extraction and radioimmunoassay. Relationships between CORTf and spatial and climate variables were examined using simple linear regressions. Ordination was used on climate data, CORTf was plotted against the resulting axes, and univariate regression trees were used to identify important predictors of CORTf. Universal kriging interpolation was used to illustrate spatial variation in CORTf across Mexico. Correlations with ordination axes showed that high CORTf was associated with low precipitation during the rainy season and low dry season temperatures. Specifically, CORTf was negatively related to May precipitation and January and July minimum temperatures, and positively related to April deuterium excess and June minimum temperatures. CORTf was higher in second-year birds compared to after-hatch years and after-second years. House sparrows had higher CORTf levels in the hot, dry, north-central region of Mexico, and CORTf was negatively related to temperature and precipitation. House sparrows molt primarily from August-September but climate conditions throughout the year were important predictors of CORTf, suggesting that conditions outside of molt can carry over to influence energetics during feather growth. These data suggest that dry conditions are challenging for house sparrows in Mexico, supporting previous work showing that precipitation is an important predictor of broad-scale CORT variation. This work highlights the utility of CORTf for evaluating the influence of physiology on current avian range limits; furthermore, these data may allow us to predict future changes in species distributions.

Feather corticosterone reveals effect of moulting conditions in the autumn on subsequent reproductive output and survival in an Arctic migratory bird.

For birds, unpredictable environments during the energetically stressful times of moulting and breeding are expected to have negative fitness effects. Detecting those effects however, might be difficult if individuals modulate their physiology and/or behaviours in ways to minimize short-term fitness costs. Corticosterone in feathers (CORTf) is thought to provide information on total baseline and stress-induced CORT levels at moulting and is an integrated measure of hypothalamic-pituitary-adrenal activity during the time feathers are grown. We predicted that CORTf levels in northern common eider females would relate to subsequent body condition, reproductive success and survival, in a population of eiders nesting in the eastern Canadian Arctic during a capricious period marked by annual avian cholera outbreaks. We collected CORTf data from feathers grown during previous moult in autumn and data on phenology of subsequent reproduction and survival for 242 eider females over 5 years. Using path analyses, we detected a direct relationship between CORTf and arrival date and body condition the following year. CORTf also had negative indirect relationships with both eider reproductive success and survival of eiders during an avian cholera outbreak. This indirect effect was dramatic with a reduction of approximately 30% in subsequent survival of eiders during an avian cholera outbreak when mean CORTf increased by 1 standard deviation. This study highlights the importance of events or processes occurring during moult on subsequent expression of life-history traits and relation to individual fitness, and shows that information from non-destructive sampling of individuals can track carry-over effects across seasons.

Synchronizing feather-based measures of corticosterone and carotenoid-dependent signals: what relationships do we expect?

Carotenoids produce many of the red, orange and yellow signal traits of birds, and individuals must trade off utilizing carotenoids for physiological processes versus ornamentation. Proximate mechanisms regulating this trade-off are poorly understood, despite their importance for expression of color signals. Corticosterone (CORT) may play a significant mechanistic role in signal expression because it mobilizes energy substrates and influences foraging behavior. We used a unique feather-based approach to test whether CORT mediates expression of carotenoid-based coloration. First, we investigated relationships between levels of CORT from feathers (CORT(f)) and carotenoid-based plumage signals in common redpolls (Acanthis flammea). Then, we determined how the width of growth bars and probability of having fault bars on feathers varied with CORT(f), specifically whether these metrics reflected developmental costs of elevated CORT ("stress" hypothesis) or represented an individual's quality ("quality" hypothesis). CORT(f) correlated positively with the strength of carotenoid signals, but only in adult males. However, also in adult males, CORT(f) was positively related to width of feather growth bars and negatively with probability of having fault bars, providing support for the quality hypothesis. Overall, CORT(f) was lower in adult males than in females or young males, possibly due to dominance patterns. Our results indicate that CORT may indirectly benefit feather quality, potentially by mediating the expression of carotenoid signals. We place our sex-specific findings into a novel framework that proposes that the influences of CORT in mediating carotenoid-based plumage traits will depend on the extent to which carotenoids are traded off between competing functions.

Habitat quality affects early physiology and subsequent neuromotor development of juvenile black-capped chickadees.

In songbirds, the ability to learn and render the species-specific song is influenced by the development of both the song nuclei in the brain and the syrinx (bird's vocal apparatus) early in the bird's life. In black-capped chickadees (Poecille atricapillus), habitat quality is known to affect song structure, with birds in high-quality habitat (mature forest) having a higher song consistency than birds in low-quality habitat (young forest). Although this difference is suspected to stem from differences in development, the developmental status of juvenile birds in either habitat remains unexplored. In this study, we used ptilochronology and feather corticosterone to compare the conditional state of juvenile chickadees in young and mature forest during two distinct periods of song learning - the sensory phase, which occurs prior to settlement, and the sensorimotor phase, which occurs post-settlement. A sample of juvenile males was captured and euthanized several weeks prior to their first breeding season to compare the development of song center nuclei and syrinx in both habitats. The corticosterone levels of natally-grown feathers were greater among birds that settled in mature than young forests - as these feathers were grown pre-settlement, they reflect differences in physiology during the sensory phase. This difference in conditional state is reflected by differences in syrinx and song center nuclei development later during the sensorimotor phase - birds in young forest have smaller syrinx, and moderately-larger RA, than birds in mature forest. Those differences could be responsible for the difference in consistency in song structure observed across habitats. The difference in physiological state across habitats, combined with potential compounding effect of differences in winter resources between habitats, could influence the difference in syrinx and neural development seen in juvenile males during the early spring, and influence the male's ability to learn and render their species-specific song.

Can synchronizing feather-based measures of corticosterone and stable isotopes help us better understand habitat-physiology relationships?

Physiological mechanisms link the environment with population dynamics, and glucocorticoid hormones are of particular interest because they respond adaptively to environmental change and can influence vertebrate reproduction and fitness. We tested a novel approach of synchronizing feather-based measures of corticosterone (the primary avian glucocorticoid; CORTf) and ratios of stable isotopes (SIs) of C (δ(13)C) and N (δ(15)N) to provide information about environmental conditions and an integrated physiological response to those conditions over the same period of feather synthesis. Using a fragmented metapopulation of Dupont's larks Chersophilus duponti, an endangered steppe songbird, we analyzed interrelationships among CORTf, δ(13)C, δ(15)N, and the physical environment, including measures of habitat loss and fragmentation. CORTf was not related to any habitat variable measured directly. However, we detected a significant spatial structure to CORTf values and food availability, with greater similarity in both at smaller spatial scales. Using SIs as proxies for the local environment, we found CORTf was negatively related to δ(13)C. Values of CORTf, δ(13)C, and the relationship between the two were likely driven by variation in agricultural land use surrounding lark habitat patches. Our feather-based approach revealed that individual physiology was sensitive to environmental conditions (e.g., an interaction of food availability and variation in habitat) at a local scale, but not patch or landscape scales. Combining CORTf and SIs may be a promising tool because it can provide individual-based information about habitat, physiology, and their relationship during the same time period.

Birds of a feather: Neanderthal exploitation of raptors and corvids.

The hypothesis that Neanderthals exploited birds for the use of their feathers or claws as personal ornaments in symbolic behaviour is revolutionary as it assigns unprecedented cognitive abilities to these hominins. This inference, however, is based on modest faunal samples and thus may not represent a regular or systematic behaviour. Here we address this issue by looking for evidence of such behaviour across a large temporal and geographical framework. Our analyses try to answer four main questions: 1) does a Neanderthal to raptor-corvid connection exist at a large scale, thus avoiding associations that might be regarded as local in space or time?; 2) did Middle (associated with Neanderthals) and Upper Palaeolithic (associated with modern humans) sites contain a greater range of these species than Late Pleistocene paleontological sites?; 3) is there a taphonomic association between Neanderthals and corvids-raptors at Middle Palaeolithic sites on Gibraltar, specifically Gorham's, Vanguard and Ibex Caves? and; 4) was the extraction of wing feathers a local phenomenon exclusive to the Neanderthals at these sites or was it a geographically wider phenomenon?. We compiled a database of 1699 Pleistocene Palearctic sites based on fossil bird sites. We also compiled a taphonomical database from the Middle Palaeolithic assemblages of Gibraltar. We establish a clear, previously unknown and widespread, association between Neanderthals, raptors and corvids. We show that the association involved the direct intervention of Neanderthals on the bones of these birds, which we interpret as evidence of extraction of large flight feathers. The large number of bones, the variety of species processed and the different temporal periods when the behaviour is observed, indicate that this was a systematic, geographically and temporally broad, activity that the Neanderthals undertook. Our results, providing clear evidence that Neanderthal cognitive capacities were comparable to those of Modern Humans, constitute a major advance in the study of human evolution.

Feather corticosterone of a nestling seabird reveals consequences of sex-specific parental investment.

Offspring of long-lived species should face costs of parental trade-offs that vary with overall energetic demands encountered by parents during breeding. If sex differences exist in how parents make the trade-off, sex-specific differences may exist in the contribution of each parent to those costs. Adaptations of offspring facing such costs are not well understood, but the hormone corticosterone probably plays a role. We manipulated breeding effort in Cory's shearwaters (Calonectris diomedea) to increase costs to offspring and used an integrated measure of corticosterone from chick feathers to investigate how experimental variation in parental investment influences offspring physiology. Average foraging trip duration and foraging efficiency (FE) of breeding pairs were not related to chick corticosterone, but sex biases in FE were. Adult male investment was more strongly related to chick corticosterone than was female investment. Importantly, we show for the first time suppression of adrenocortical activity in nestling Procellariiform seabirds, and explain how our results indicate an adaptive mechanism invoked by chicks facing increased costs of parental trade-offs.

Prey behaviour across antipredator adaptation types: how does growth trajectory influence learning of predators?

Despite the fact that the ability of animals to avoid being consumed by predators is influenced by their behaviour, morphology and life history, very few studies have attempted to integrate prey responses across these adaptation types. Here, our goal was to address the link between life-history traits (size and growth trajectory) of tadpoles and behavioural responses to predators. Specifically, we wanted to determine whether information learned about predators was influenced by prey growth trajectory before and after learning. We manipulated the size/growth trajectory of tadpoles by raising them under different temperatures. Tadpoles raised on a slow-growth trajectory (under cold conditions) and taught to recognize a salamander subsequently showed stronger responses after 2 weeks than tadpoles that were raised on a fast-growth trajectory (under warm conditions). When we account for the effect of size (r (2)=0.22) on the responses of prey to predator cues, we find that the growth trajectory pre-learning but not post-learning influences the learned responses of the tadpoles. The differences in responses to predators may reflect differential memory associated with the predator. To our knowledge, this is the first study that has attempted to link life-history traits (size and growth rate) with learning of predators. In order to gain a comprehensive understanding of antipredator responses of prey animals, we call for additional integrative studies that examine prey anti-predator responses across adaptation types.

Does environmental enrichment reduce stress? An integrated measure of corticosterone from feathers provides a novel perspective.

Enrichment is widely used as tool for managing fearfulness, undesirable behaviors, and stress in captive animals, and for studying exploration and personality. Inconsistencies in previous studies of physiological and behavioral responses to enrichment led us to hypothesize that enrichment and its removal are stressful environmental changes to which the hormone corticosterone and fearfulness, activity, and exploration behaviors ought to be sensitive. We conducted two experiments with a captive population of wild-caught Clark's nutcrackers (Nucifraga columbiana) to assess responses to short- (10-d) and long-term (3-mo) enrichment, their removal, and the influence of novelty, within the same animal. Variation in an integrated measure of corticosterone from feathers, combined with video recordings of behaviors, suggests that how individuals perceive enrichment and its removal depends on the duration of exposure. Short- and long-term enrichment elicited different physiological responses, with the former acting as a stressor and birds exhibiting acclimation to the latter. Non-novel enrichment evoked the strongest corticosterone responses of all the treatments, suggesting that the second exposure to the same objects acted as a physiological cue, and that acclimation was overridden by negative past experience. Birds showed weak behavioral responses that were not related to corticosterone. By demonstrating that an integrated measure of glucocorticoid physiology varies significantly with changes to enrichment in the absence of agonistic interactions, our study sheds light on potential mechanisms driving physiological and behavioral responses to environmental change.

Flaws and pitfalls in the chemical analysis of feathers: bad news-good news for avian chemoecology and toxicology.

Ecologists have frequently used biochemical assays as proxies for processes or phenomena too difficult to explore by traditional means of investigation. Feathers have been subjected to a number of chemical analyses to study such things as their elemental composition, contaminants, and hormones. The reliance on standard methodology of using concentrations to express quantities of chemical substances is seriously problematic because it creates artifacts by ignoring the physiology of feathers. Some elements and compounds are incorporated into the feather as part of the very building blocks of the keratin. However, others that are less functionally important to feathers (but not necessarily to the bird) enter the developing cells in proportion to their abundance in the bloodstream; in other words, feathers are merely receptacles, and deposition of chemicals is time dependent. In the latter case, one that applies to much of the work done on feather chemistry, data expressed as concentrations are meaningless because the varying mass across the feather alters concentrations in a way that has no biological significance. I discuss this problem and various pitfalls in the chemical analysis of feathers, and offer solutions that ultimately will offer a better understanding of the mechanisms influencing feather composition and, thus, the ecological patterns and processes they were meant to study.

Linking predator risk and uncertainty to adaptive forgetting: a theoretical framework and empirical test using tadpoles.

Hundreds of studies have examined how prey animals assess their risk of predation. These studies work from the basic tennet that prey need to continually balance the conflicting demands of predator avoidance with activities such as foraging and reproduction. The information that animals gain regarding local predation risk is most often learned. Yet, the concept of 'memory' in the context of predation remains virtually unexplored. Here, our goal was (i) to determine if the memory window associated with predator recognition is fixed or flexible and, if it is flexible, (ii) to identify which factors affect the length of this window and in which ways. We performed an experiment on larval wood frogs, Rana sylvatica, to test whether the risk posed by, and the uncertainty associated with, the predator would affect the length of the tadpoles' memory window. We found that as the risk associated with the predator increases, tadpoles retained predator-related information for longer. Moreover, if the uncertainty about predator-related information increases, then prey use this information for a shorter period. We also present a theoretical framework aiming at highlighting both intrinsic and extrinsic factors that could affect the memory window of information use by prey individuals.

Variation in immune function, body condition, and feather corticosterone in nestling tree swallows (Tachycineta bicolor) on reclaimed wetlands in the Athabasca oil sands, Alberta, Canada.

In the Athabasca oil sands region of northern Alberta, mining companies are evaluating reclamation using constructed wetlands for integration of tailings. From May to July 2008, reproductive performance of 40 breeding pairs of tree swallows (Tachycineta bicolor), plus growth and survival of nestlings, was measured on three reclaimed wetlands on two oil sands leases. A subset of nestlings was examined for i) feather corticosterone levels, ii) delayed-type hypersensitivity response, and iii) innate immune function. Nestlings on one of two wetlands created with oil sands process affected material (OSPM) were heavier and had greater wing-lengths, and mounted a stronger delayed-type hypersensitivity response compared those on the reference wetland. Corticosterone was significantly higher in male nestlings on one of two OSPM-containing wetland compared to the reference wetland. Body condition of 12-day-old female nestlings was inversely related to feather corticosterone. Under ideal weather conditions, reclaimed wetlands can support healthy populations of aerially-insectivorous birds.

Tracking stress: localisation, deposition and stability of corticosterone in feathers.

How animals cope with stressors is an important determinant of their well being and fitness. Understanding what environmental perturbations are perceived as stressors, and quantifying how they are responded to, how often they occur and the negative consequences of exposure to glucocorticoids, has been problematic and limited to short-term physiological measures. By contrast, the quantification of corticosterone (CORT) in feathers represents a long-term, integrated measure of hypothalamic-pituitary-adrenal activity. In the present study, we show that by understanding how the hormone is deposited in feathers, in combination with specific sampling protocols, one can identify localised patterns of CORT deposition that reveal different temporal patterns of a bird's response to stressors. CORT in feathers appears to be stable over time, is resistant to heat exposure and is useful in determining both the overall exposure of the bird to the hormone over days or weeks, as well as identifying discrete, punctuated, stressful events. Variation in feather CORT can also be examined among individuals of a population at one point in time, as well as over years by using museum specimens. The ability to track stress over time allows for new questions to be asked about the health and ecology of birds and their environment.

Porphyrins and pheomelanins contribute to the reddish juvenal plumage of black-shouldered kites.

Porphyrins are a widespread group of pigments in nature, but, contrary to melanins and carotenoids, their occurrence as plumage colorants seems to be anecdotal and their function, if any, is unknown. Using thin-layer chromatography and high pressure liquid chromatography, we have found coproporphyrin III, the same porphyrin type previously reported in owls, in the plumage of nestling black-shouldered kites (Elanus caeruleus). The first plumage grown at the nest in this species includes reddish-brown contour feathers in the upperparts, and particularly in the breast area, which fade during the weeks-long post-fledging period to become either gray or white consistent with the definitive adult plumage. In these reddish feathers, we have also found small amounts of pheomelanins and traces of eumelanin. The contribution of each pigment to the final colour perceived by birds or other animals is unknown. In white and grey feathers of the same species no porphyrin was found, and only traces of eumelanin were detected in the grey ones. The fact that the reddish feathers are only found in the juvenal plumage, when individuals are vulnerable in an open nest, leads us to hypothesize a camouflage role for this ephemeral plumage. As porphyrins are involved, although not exclusively, we can for the first time ascribe them a function in the plumage of birds.

Physiological stress mediates the honesty of social signals.

BACKGROUND: Extravagant ornaments used as social signals evolved to advertise their bearers' quality. The Immunocompetence Handicap Hypothesis proposes that testosterone-dependent ornaments reliably signal health and parasite resistance; however, empirical studies have shown mixed support. Alternatively, immune function and parasite resistance may be indirectly or directly related to glucocorticoid stress hormones. We propose that an understanding of the interplay between the individual and its environment, particularly how they cope with stressors, is crucial for understanding the honesty of social signals. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed corticosterone deposited in growing feathers as an integrated measure of hypothalamic-pituitary-adrenal activity in a wild territorial bird, the red grouse Lagopus lagopus scoticus. We manipulated two key, interrelated components, parasites and testosterone, which influence both ornamentation and fitness. Birds were initially purged of parasites, and later challenged with parasites or not, while at the same time being given testosterone or control implants, using a factorial experimental design. At the treatment level, testosterone enhanced ornamentation, while parasites reduced it, but only in males not implanted with testosterone. Among individuals, the degree to which both parasites and testosterone had an effect was strongly dependent on the amount of corticosterone in the feather grown during the experiment. The more stressors birds had experienced (i.e., higher corticosterone), the more parasites developed, and the less testosterone enhanced ornamentation. CONCLUSIONS/SIGNIFICANCE: With this unique focus on the individual, and a novel, integrative, measure of response to stressors, we show that ornamentation is ultimately a product of the cumulative physiological response to environmental challenges. These findings lead toward a more realistic concept of honesty in signaling as well as a broader discussion of the concept of stress.

Immunological development in nestling American kestrels Falco sparverius: post-hatching ontogeny of the antibody response.

Avian research involving examination of immune function or testing of immunocompetence in wild birds has been based upon information on Galliforms, (chicken and quail) even though they are precocial, whereas most wild species with which ecologists, biologists and toxicologists work are altricial; blind, naked and completely dependent at hatching. Here we begin to address this gap in knowledge, offering insight into the early, post-hatching, humoral immune response in an altricial bird, the American kestrel (Falco sparverius). Over two breeding seasons, nestling kestrels were immunized with a non-pathogenic antigen, dinitrophenol keyhole limpet hemocyanin (DNP-KLH), between 3 and 9 days post-hatching and boostered 6 days later. Background levels, primary and secondary immune responses were measured using an enzyme linked immunosorbent assay. The specificity of our laboratory produced rabbit, anti-kestrel antibody was determined using a double immunodiffusion assay. Results showed the rabbit antiserum to have specific anti-kestrel IgG activity. Birds as young as three days old could successfully mount an antibody response, the magnitude of which increased with age at first vaccination. Early immunization did not compromise growth rate, nor did it affect the maximum secondary response. Comparatively, adult kestrels immunized during the same season and following the same protocol, had antibody levels four times higher than those of the nestlings.

Effects of inhalation exposure to a binary mixture of benzene and toluene on vitamin a status and humoral and cell-mediated immunity in wild and captive American kestrels.

Benzene and toluene are representative volatile organic compounds (VOC) released during production, storage, and transportation associated with the oil and gas industry and are chemicals of concern, as they are released in greater and possibly more biologically significant concentrations than other compounds. Most studies of air pollution in high oil and gas activity areas have neglected to consider risks to birds, including top-level predators. Birds can be used as highly sensitive monitors of air quality and since the avian respiratory tract is physiologically different from a rodent respiratory tract, effects of gases cannot be safely extrapolated from rodent studies. Wild and captive male American kestrels were exposed for approximately 1 h daily for 28 d to high (rodent lowest-observed-adverse-effect level [LOAEL] of 10 ppm and 80 ppm, respectively) or environmentally relevant (0.1 ppm and 0.8 ppm, respectively) levels of benzene and toluene. Altered immune responses characteristic of those seen in mammalian exposures were evident in kestrels. A decreased cell-mediated immunity, measured by delayed-type hypersensitivity testing, was evident in all exposed birds. There was no effect on humoral immunity. Plasma retinol levels as measured by high-performance liquid chromatography (HPLC) analysis were decreased in wild and captive kestrels exposed to the rodent LOAEL for combined benzene and toluene. This study indicates that American kestrels are sensitive to combined benzene and toluene. The study also illustrates the need for reference concentrations for airborne pollutants to be calculated, including sensitive endpoints specific to birds. Based on these findings, future studies need to include immune endpoints to determine the possible increased susceptibility of birds to inhaled toxicants.

Cell-mediated immune activation rapidly decreases plasma carotenoids but does not affect oxidative stress in red-legged partridges (Alectoris rufa).

In animals yellow-orange-red sexual traits pigmented by carotenoids have been suggested to act as signals of current health. Because carotenoids have important physiological functions, individuals might trade-off allocating these pigments to self-maintenance versus coloration. Carotenoids may act as scavengers of free radicals that are released during an immune response. Here, we experimentally assessed whether a local cell-mediated immune response affects circulating carotenoids, antioxidant status, oxidative damage and the expression of a carotenoid-based trait. Male red-legged partridges (Alectoris rufa) were subcutaneously injected with phytohaemagglutinin (PHA) or with phosphate buffer solution (controls). The effect of the treatment on circulating carotenoids, total plasma antioxidant status (TAS), lipid oxidative damage in erythrocytes (TBARS) and ornamentation was assessed. Immune challenge induced a 13% decrease in circulating carotenoids within 24 h. However, this treatment did not affect TAS, TBARS or coloration. Coloration, circulating carotenoids and cell-mediated immune response were positively correlated, but these were not related to TAS or TBARS. Carotenoids were only weakly related to TAS after controlling for the effect of uric acid levels. These results suggest that carotenoid-based ornaments may honestly indicate immunocompetence but probably not antioxidant capacity in this species, and that carotenoids might be relatively weak antioxidants in the plasma. Furthermore, even a relatively harmless and locally elicited immune challenge had important effects on circulating carotenoids, but this effect did not appear to be associated with oxidative stress. Alternative mechanisms linking carotenoids to immunity (not necessarily relying on the use of these pigments as antioxidants) should be considered in future studies on birds.

Positive assortative pairing by plumage colour in Spanish imperial eagles.

Intraspecific variation in plumage may have a functional significance in mate choice. As a result, breeding birds are often paired assortatively with respect to colour. However, whether this within-pair correlation is a result of homotypic preference whereby individuals pick a mate that looks like themselves, or directional preference whereby all individuals have the same phenotypic preference, is often unknown. Using data collected between 1989 and 2006, we describe intraspecific variation in the striking white feathers on the leading edge of the wing, lesser coverts and mantle of 144 Spanish imperial eagles (Aquila adalberti), one of the most endangered birds of prey in the world. Females had, on average, more white than males, and pairs mated in a positive assortative fashion. Coloration was not related to age of the bird, food supply (i.e., territory quality) or breeding productivity. Our results are most consistent with the process of homotypic-preference assortative mating, and this may be a result of sexual imprinting, and function as a mechanism to optimize the degree of outbreeding. This pattern of mate selection may explain the rapid evolutionary divergence of A. adalberti from the continental population of imperial eagle A. heliaca.