Interactions Between Heavy Metal Exposure and Blood Biochemistry in an Urban Population of the Black Swan (Cygnus atratus) in Australia.

There is growing recognition of the threat posed to wildlife by pollutants. Waterbirds are robust bioindicators of ecosystem health, and metal toxicity is a threat to these species in waterways worldwide. Urban waterbirds are likely to be at the highest risk of heavy metal exposure, but this issue has not been widely explored in Australia. Our aim was to estimate contemporary heavy metal exposure in a sedentary urban waterbird population: black swans (Cygnus atratus) inhabiting an inner-city wetland in one of Australia's largest cities, Melbourne. To investigate the physiological implications of legacy heavy metal exposure in these birds, we quantified blood biochemistry profiles and examined their relationships with metal concentrations in feathers. We caught 15 swans in 2021 and took feather samples to measure the concentration of eight heavy metals (chromium (Cr), manganese (Mn), iron (Fe), nickel (Ni), copper (Cu), zinc (Zn), lead (Pb), and mercury (Hg)), and blood samples to measure the concentration of 13 plasma analytes. Multivariate regression analysis revealed few associations between heavy metals and biochemistry markers, and no differences between sexes or age classes. This study presents a baseline dataset of these contaminants and blood biochemical profiles of swans at this wetland that can be used for future monitoring and is an important step toward a better understanding of the threat posed by heavy metals to Australian urban waterbirds.

High-resolution temporal wastewater treatment plant investigation to understand influent mass flux of per- and polyfluoroalkyl substances (PFAS).

This study aims to identify sources of per- and polyfluoroalkyl substances (PFAS) to wastewater treatment plants (WWTPs) and reveals previously undescribed variability in daily PFAS concentrations by measuring their occurrence in WWTP influent each hour over the course of a week. ∑50PFAS concentrations ranged between 89 ± 38 on Monday and 173 ± 110 ng L-1 on Friday, where perfluoroalkyl carboxylic acids (PFCAs), disubstituted phosphate esters (diPAPs), and perfluoroalkyl sulfonic acids (PFSAs) contributed the largest proportion to overall weekly concentrations 37%, 30%, and 17% respectively. Simultaneous pulse events of perfluorooctanesulfonic acid (PFOS; 400 ng L-1) and perfluoroheptanesulfonic acid (PFHpS; 18 ng L-1) indicate significant industrial or commercial waste discharge that persists for up to 3 h. The minimum number of hourly grab samples required to detect variation of PFOS and PFHpS concentrations are 7 and 9 samples respectively, indicating a high degree of variability in PFAS concentrations between days. Overall, the risk of sampling bias from grab samples is high given the variability in PFAS concentrations and more frequent sampling campaigns must be balanced against the cost of analysis carefully to avoid the mischaracterisation of mass flux to receiving surface waters.

Field-Based Distribution and Bioaccumulation Factors for Cyclic and Aliphatic Per- and Polyfluoroalkyl Substances (PFASs) in an Urban Sedentary Waterbird Population.

The field-based distribution and bioaccumulation factor (BAF) for per- and polyfluoroalkyl substances (PFASs) were determined in residential Black Swans (Cygnus atratus) from an urban lake (Melbourne, Australia). The concentrations of 46 aliphatic and cyclic PFASs were determined by HPLC-MS/MS in serum and excrement from swans, and water, sediment, aquatic macrophytes, soil, and grass samples in and around the lake. Elevated concentrations of ∑46PFASs were detected in serum (120 ng mL-1) and excrement (110 ng g-1 dw) were strongly related indicating a potential noninvasive sampling methodology. Environmental concentrations of PFASs were consistent with a highly impacted ecosystem and notably high concentrations of perfluoro-4-ethylcyclohexanesulfonate (PFECHS, 67584-42-3; C8HF15SO3) were detected in water (27 ng L-1) and swan serum (16 ng mL-1). In the absence of credible putative alternative sources of PFECHS input to the lake, we propose that the use of high-performance motorsport vehicles is a likely source of contamination to this ecosystem. The BAF of perfluorocarboxylic acids increased with each additional CF2 moiety from PFOA (15.7 L kg-1 ww) to PFDoDA (3615 L kg-1 ww). The BAF of PFECHS was estimated as 593 L kg-1 ww, which is lower compared with that of PFOS (1097 L kg-1 ww).

Evaluation and validation of methodologies for the extraction of per- and polyfluoroalkyl substances (PFASs) in serum of birds and mammals.

Advances in analytical techniques have allowed greater detection of environmental contaminants from small volumes of sample. Four methodologies were evaluated for the extraction of 53 per- and polyfluoroalkyl substances (PFASs) from eight classes in 200 µL of avian and mammal serum. Spiked serums at four concentrations (0, 0.5, 5.0 and 25 ng mL-1) were prepared by protein precipitation (PPT), enhanced matrix removal (EMR), weak anion exchange (WAX), and hydrophilic-lipophilic balance (HLB) solid-phase extraction cartridges. The extract from each methodology was analysed by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS), and concentrations were compared with known concentrations in the spiked media. EMR performed the best overall, with 40 of 53 compounds effectively recovered at 5 ng mL-1. Furthermore, EMR was effective overall at concentrations ranging from 0.5 to 25 ng mL-1 for 39 out of 53. Similarly, PPT was effective for 35 of 53 compounds at all spiked serum concentrations. There was a negative correlation between internal standard recovery for compounds with increasing octanol-water coefficients (Kow) for WAX (R = - 0.65, p = 0.0043) and HLB (R = - 0.62, p = 0.0077) extractions, indicating methanol may not be a suitable solvent for long-chain PFAS extraction from protein-rich tissues. EMR and PPT represent fast and effective methodologies for the extraction of PFASs from low volumes of serum which allows greater accuracy and precision that can be applied to future human and wildlife biomonitoring programmes.

A baseline study of per- and polyfluoroalkyl substances (PFASs) in waterfowl from a remote Australian environment.

Elevated concentrations of PFASs in the liver may pose a toxicological risk to bird species and humans that consume them. This study aimed to determine concentrations of 43 per- and polyfluoroalkyl substances (PFASs) in livers (n = 80) of Australian Shelducks (Tadorna tadornoides), Pacific Black Ducks (Anas superciliosa), and Teals (Anas sp.), as well as water and sediment from a remote Australian environment. Maximum concentrations of PFBA (1.9 ng L-1), PFOA (1.7 ng L-1) and PFOS (0.99 ng L-1) in water were consistent with long-range atmospheric and oceanic transport. PFOS (30%) and PFNA (22%) were the most frequently detected PFASs in Australian Shelduck livers (0.31 ± 0.68 ng g-1 ww and 0.16 ± 0.15 ng g-1 ww respectively). Maximum concentrations of PFOS in Pacific Black Ducks (50%) and Teals (44%) was 2.4 ng g-1 ww and 5.3 ng g-1 ww respectively. While PFAS levels in birds from this remote environment were below current animal consumption guidelines, continued monitoring of this ecosystem is recommended to assess the human health risk of consumption of wild game.

Sex role similarity and sexual selection predict male and female song elaboration and dimorphism in fairy-wrens.

Historically, bird song complexity was thought to evolve primarily through sexual selection on males; yet, in many species, both sexes sing and selection pressure on both sexes may be broader. Previous research suggests competition for mates and resources during short, synchronous breeding seasons leads to more elaborate male songs at high, temperate latitudes. Furthermore, we expect male-female song structure and elaboration to be more similar at lower, tropical latitudes, where longer breeding seasons and year-round territoriality yield similar social selection pressures in both sexes. However, studies seldom take both types of selective pressures and sexes into account. We examined song in both sexes in 15 populations of nine-fairy-wren species (Maluridae), a Southern Hemisphere clade with female song. We compared song elaboration (in both sexes) and sexual song dimorphism to latitude and life-history variables tied to sexual and social selection pressures and sex roles. Our results suggest that song elaboration evolved in part due to sexual competition in males: male songs were longer than female songs in populations with low male survival and less male provisioning. Also, female songs evolved independently of male songs: female songs were slower paced than male songs, although only in less synchronously breeding populations. We also found male and female songs were more similar when parental care was more equal and when male survival was high, which provides strong evidence that sex role similarity correlates with male-female song similarity. Contrary to Northern Hemisphere latitudinal patterns, male and female songs were more similar at higher, temperate latitudes. These results suggest that selection on song can be sex specific, with male song elaboration favored in contexts with stronger sexual selection. At the same time, selection pressures associated with sex role similarity appear to favor sex role similarity in song structure.

Correlations between Per- and Polyfluoroalkyl Substances and Body Morphometrics in Fledgling Shearwaters Impacted by Plastic Consumption from a Remote Pacific Island.

We investigated the concentrations of 45 per- and polyfluoroalkyl substances (PFASs) in fledgling flesh-footed shearwater (Ardenna carneipes; n = 33) and wedge-tailed shearwater (A. pacifica; n = 9) livers via liquid chromatography-tandem mass spectrometry and their relationship to body morphometrics and ingested plastic mass recorded in 2019 on Lord Howe Island (NSW, Australia). Sixteen PFASs were detected, of which perfluorooctanesulfonate (PFOS) was the dominant compound, detected in 100% of birds (1.34-13.4 ng/g wet wt). Long-chain perfluorocarboxylic acids, including perfluorodecanoic acid (PFDA; <0.04-0.79 ng/g wet wt) and perfluorotridecanoic acid (PFTrDA; <0.05-1.6 ng/g wet wt) were detected in >50% of birds. There was a positive correlation between PFDA and PFTrDA concentrations and wing chord length (Rs = 0.36, p = 0.0204; Rs = 0.44, p = 0.0037, respectively), and between PFDA concentrations and total body mass (Rs = 0.33, p = 0.032), suggesting that these compounds may impact shearwater fledgling morphometrics. Plastic was present in the intestinal tract of 79% of individuals (<7.6 g), although there was no correlation between PFAS concentrations and plastic mass, indicating that ingested plastic is not the likely primary exposure source. The widespread occurrence of PFASs in fledgling marine birds from a relatively pristine location in the Southern Hemisphere suggests that further studies in adult shearwaters and other marine birds are warranted to investigate whether there are any long-term physiological effects on bird species. Environ Toxicol Chem 2021;40:799-810. © 2020 SETAC.

Urban noise restricts, fragments, and lightens sleep in Australian magpies.

Urban areas are inherently noisy, and this noise can disrupt biological processes as diverse as communication, migration, and reproduction. We investigated how exposure to urban noise affects sleep, a process critical to optimal biological functioning, in Australian magpies (Cracticus tibicen). Eight magpies experimentally exposed to noise in captivity for 24-h spent more time awake, and less time in non-rapid eye movement (non-REM) and REM sleep at night than under quiet conditions. Sleep was also fragmented, with more frequent interruptions by wakefulness, shorter sleep episode durations, and less intense non-REM sleep. REM sleep was particularly sensitive to urban noise. Following exposure to noise, magpies recovered lost sleep by engaging in more, and more intense, non-REM sleep. In contrast, REM sleep showed no rebound. This might indicate a long-term cost to REM sleep loss mediated by noise, or contest hypotheses regarding the functional value of this state. Overall, urban noise has extensive, disruptive impacts on sleep composition, architecture, and intensity in magpies. Future work should consider whether noise-induced sleep restriction and fragmentation have long-term consequences.

White and Amber Light at Night Disrupt Sleep Physiology in Birds.

Artificial light at night can disrupt sleep in humans [1-4] and other animals [5-10]. A key mechanism for light to affect sleep is via non-visual photoreceptors that are most sensitive to short-wavelength (blue) light [11]. To minimize effects of artificial light on sleep, many electronic devices shift from white (blue-rich) to amber (blue-reduced) light in the evening. Switching outdoor lighting from white to amber might also benefit wildlife [12]. However, whether these two colors of light affect sleep similarly in different animals remains poorly understood. Here we show, by measuring brain activity, that both white and amber lighting disrupt sleep in birds but that the magnitude of these effects differs between species. When experimentally exposed to light at night at intensities typical of urban areas, domestic pigeons (Columba livia) and wild-caught Australian magpies (Cracticus tibicen tyrannica) slept less, favored non-rapid eye movement (NREM) sleep over REM sleep, slept less intensely, and had more fragmented sleep compared to when lights were switched off. In pigeons, these disruptive effects on sleep were similar for white and amber lighting. For magpies, however, amber light had less impact on sleep. Our results demonstrate that amber lighting can minimize sleep disruption in some birds but that this benefit may not be universal. VIDEO ABSTRACT.

Conspicuous Plumage Does Not Increase Predation Risk: A Continent-Wide Test Using Model Songbirds.

The forces shaping female plumage color have long been debated but remain unresolved. Females may benefit from conspicuous colors but are also expected to suffer costs. Predation is one potential cost, but few studies have explicitly investigated the relationship between predation risk and coloration. The fairy-wrens show pronounced variation in female coloration and reside in a wide variety of habitats across Australasia. Species with more conspicuous females are found in denser habitats, suggesting that conspicuousness in open habitat increases vulnerability to predators. To test this, we measured attack rates on 3-D-printed models mimicking conspicuously colored males and females and dull females in eight different fairy-wren habitats across Australia. Attack rates were higher in open habitats and at higher latitudes. Contrary to our predictions, dull female models were attacked at similar rates to the conspicuous models. Further, the probability of attack in open habitats increased more for both types of female models than for the conspicuous male model. Across models, the degree of contrast (chromatic and achromatic) to environmental backgrounds was unrelated to predation rate. These findings do not support the long-standing hypothesis that conspicuous plumage, in isolation, is costly due to increased attraction of predators. Our results indicate that conspicuousness interacts with other factors in driving the evolution of plumage coloration.

Exploring mechanisms and origins of reduced dispersal in island Komodo dragons.

Loss of dispersal typifies island biotas, but the selective processes driving this phenomenon remain contentious. This is because selection via, both indirect (e.g. relaxed selection or island syndromes) and direct (e.g. natural selection or spatial sorting) processes may be involved, and no study has yet convincingly distinguished between these alternatives. Here, we combined observational and experimental analyses of an island lizard, the Komodo dragon (Varanus komodoensis, the world's largest lizard), to provide evidence for the actions of multiple processes that could contribute to island dispersal loss. In the Komodo dragon, concordant results from telemetry, simulations, experimental translocations, mark-recapture, and gene flow studies indicated that despite impressive physical and sensory capabilities for long-distance movement, Komodo dragons exhibited near complete dispersal restriction: individuals rarely moved beyond the valleys they were born/captured in. Importantly, lizard site-fidelity was insensitive to common agents of dispersal evolution (i.e. indices of risk for inbreeding, kin and intraspecific competition, and low habitat quality) that consequently reduced survival of resident individuals. We suggest that direct selection restricts movement capacity (e.g. via benefits of spatial philopatry and increased costs of dispersal) alongside use of dispersal-compensating traits (e.g. intraspecific niche partitioning) to constrain dispersal in island species.

Reflection of near-infrared light confers thermal protection in birds.

Biologists have focused their attention on the optical functions of light reflected at ultraviolet and human-visible wavelengths. However, most radiant energy in sunlight occurs in 'unseen' near-infrared (NIR) wavelengths. The capacity to reflect solar radiation at NIR wavelengths may enable animals to control heat gain and remain within their critical thermal limits. Here, using a continent-wide phylogenetic analysis of Australian birds, we show that species occupying hot, arid environments reflect more radiant energy in NIR wavelengths than species in thermally benign environments, even when controlling for variation in visible colour. Biophysical models confirm that smaller species gain a greater advantage from high NIR reflectivity in hot, arid environments, reducing water loss from compensatory evaporative cooling by up to 2% body mass per hour. These results highlight the importance of NIR reflectivity for thermal protection, which may become increasingly critical as the frequency of extreme climatic events increases.

The microstructure of white feathers predicts their visible and near-infrared reflectance properties.

Research on the optical properties of animal integuments, including fur, feather, skin and cuticle, has focussed almost exclusively on animal-visible wavelengths within the narrow range of 300-700 nm. By contrast, the near-infrared (NIR) portion of direct sunlight, spanning 700-2600 nm, has been largely ignored despite its potentially important thermal consequences. We quantified variation in visible and NIR reflectance and transmission for white body contour feathers of 50 bird species, and examined how well they are predicted by feather macro- and micro-structural morphology. Both visible and NIR reflectance of the feathers varied substantially across species. Larger, thicker, and sparser feathers that are characteristic of larger species, and feathers with rounder barbs and more closely spaced barbules, had high average reflectance, particularly within avian-visible wavelengths (300-700 nm). Feathers with rounder barbs and more closely situated barbules also had high average reflectance, particularly for NIR wavelengths. Barb roundness and barbule density were the only predictors of NIR reflectance after accounting for variation in visible reflectance and body size. Our results highlight the potential for adaptive variation in NIR reflectance mediated by feather structure, which may inform the design of functional materials to control light and heat.

Impacts of artificial light at night on sleep: A review and prospectus.

Natural cycles of light and darkness govern the timing of most aspects of animal behavior and physiology. Artificial light at night (ALAN)-a recent and pervasive form of pollution-can mask natural photoperiodic cues and interfere with biological rhythms. One such rhythm vulnerable to perturbation is the sleep-wake cycle. ALAN may greatly influence sleep in humans and wildlife, particularly in animals that sleep predominantly at night. There has been some recent evidence for impacts of ALAN on sleep, but critical questions remain. Some of these can be addressed by adopting approaches already entrenched in sleep research. In this paper, we review the current evidence for impacts of ALAN on sleep, highlight gaps in our understanding, and suggest opportunities for future research.

Multiple hypotheses explain variation in extra-pair paternity at different levels in a single bird family.

Extra-pair paternity (EPP), where offspring are sired by a male other than the social male, varies enormously both within and among species. Trying to explain this variation has proved difficult because the majority of the interspecific variation is phylogenetically based. Ideally, variation in EPP should be investigated in closely related species, but clades with sufficient variation are rare. We present a comprehensive multifactorial test to explain variation in EPP among individuals in 20 populations of nine species over 89 years from a single bird family (Maluridae). Females had higher EPP in the presence of more helpers, more neighbours or if paired incestuously. Furthermore, higher EPP occurred in years with many incestuous pairs, populations with many helpers and species with high male density or in which males provide less care. Altogether, these variables accounted for 48% of the total and 89% of the interspecific and interpopulation variation in EPP. These findings indicate why consistent patterns in EPP have been so challenging to detect and suggest that a single predictor is unlikely to account for the enormous variation in EPP across levels of analysis. Nevertheless, it also shows that existing hypotheses can explain the variation in EPP well and that the density of males in particular is a good predictor to explain variation in EPP among species when a large part of the confounding effect of phylogeny is excluded.

Habitat structure is linked to the evolution of plumage colour in female, but not male, fairy-wrens.

BACKGROUND: Both natural and sexual selection may drive the evolution of plumage colouration in birds. This can lead to great variation in plumage not only across species, but also between sexes within species. Australasian fairy-wrens are famous for their brightly coloured males, which exhibit colours ranging from bright blue to red and black. Female plumage in fairy wrens (and in general) has been rarely studied, but it can also be highly variable, including both bright and cryptic plumages. We use a comparative framework to explore the basis for this variation, and test the possibility that female fairy-wrens experience selection for cryptic plumage when they occupy more exposed habitats that offer little concealment from predators. We use spectral measurements of plumage for species and subspecies of Australasian fairy-wrens. RESULTS: We show that female colouration (contrast against background) is strongly correlated with vegetation cover: females in open habitats show less contrast to background colours than females in closed habitats, while male colouration is not associated with habitat type. CONCLUSIONS: Female plumage appears to be under stronger natural selection than male plumage in fairy-wrens, providing an example of how selection may act differently on males and females of the same species.

Personality and innate immune defenses in a wild bird: Evidence for the pace-of-life hypothesis.

We tested the two main evolutionary hypotheses for an association between immunity and personality. The risk-of-parasitism hypothesis predicts that more proactive (bold, exploratory, risk-taking) individuals have more vigorous immune defenses because of increased risk of parasite exposure. In contrast, the pace-of-life hypothesis argues that proactive behavioral styles are associated with shorter lifespans and reduced investment in immune function. Mechanistically, associations between immunity and personality can arise because personality differences are often associated with differences in condition and stress responsiveness, both of which are intricately linked with immunity. Here we investigate the association between personality (measured as proactive exploration of a novel environment) and three indices of innate immune function (the non-specific first line of defense against parasites) in wild superb fairy-wrens Malurus cyaneus. We also quantified body condition, hemoparasites (none detected), chronic stress (heterophil:lymphocyte ratio) and circulating corticosterone levels at the end of the behavioral test (CORT, in a subset of birds). We found that fast explorers had lower titers of natural antibodies. This result is consistent with the pace-of-life hypothesis, and with the previously documented higher mortality of fast explorers in this species. There was no interactive effect of exploration score and duration in captivity on immune indices. This suggests that personality-related differences in stress responsiveness did not underlie differences in immunity, even though behavioral style did modulate the effect of captivity on CORT. Taken together these results suggest reduced constitutive investment in innate immune function in more proactive individuals.

MHC class II ß exon 2 variation in pardalotes (Pardalotidae) is shaped by selection, recombination and gene conversion.

The high levels of polymorphism and allelic diversity which characterise genes in the major histocompatibility complex (MHC) are thought to be generated and maintained through the combined effects of different evolutionary processes. Here, we characterised exon 2 of the MHC class II ß genes in two congeneric passerine species, the spotted (Pardalotus punctatus) and striated pardalote (Pardalotus striatus). We estimated the levels of allelic diversity and tested for signatures of recombination, gene conversion and balancing selection to determine if these processes have influenced MHC variation in the two species. Both species showed high levels of polymorphism and allelic diversity, as well as evidence of multiple gene loci and putative pseudogenes based on the presence of stop codons. We found higher levels of MHC diversity in the striated pardalote than the spotted pardalote, based on the levels of individual heterozygosity, sequence divergence and number of polymorphic sites. The observed differences may reflect variable selection pressure on the species, resulting from differences in patterns of movement among populations. We identified strong signatures of historical balancing selection, recombination and gene conversion at the sequence level, indicating that MHC variation in the two species has been shaped by a combination of processes.

New data from basal Australian songbird lineages show that complex structure of MHC class II ß genes has early evolutionary origins within passerines.

BACKGROUND: The major histocompatibility complex (MHC) plays a crucial role in the adaptive immune system and has been extensively studied across vertebrate taxa. Although the function of MHC genes appears to be conserved across taxa, there is great variation in the number and organisation of these genes. Among avian species, for instance, there are notable differences in MHC structure between passerine and non-passerine lineages: passerines typically have a high number of highly polymorphic MHC paralogs whereas non-passerines have fewer loci and lower levels of polymorphism. Although the occurrence of highly polymorphic MHC paralogs in passerines is well documented, their evolutionary origins are relatively unexplored. The majority of studies have focussed on the more derived passerine lineages and there is very little empirical information on the diversity of the MHC in basal passerine lineages. We undertook a study of MHC diversity and evolutionary relationships across seven species from four families (Climacteridae, Maluridae, Pardalotidae, Meliphagidae) that comprise a prominent component of the basal passerine lineages. We aimed to determine if highly polymorphic MHC paralogs have an early evolutionary origin within passerines or are a more derived feature of the infraorder Passerida. RESULTS: We identified 177 alleles of the MHC class II ß exon 2 in seven basal passerine species, with variation in numbers of alleles across individuals and species. Overall, we found evidence of multiple gene loci, pseudoalleles, trans-species polymorphism and high allelic diversity in these basal lineages. Phylogenetic reconstruction of avian lineages based on MHC class II ß exon 2 sequences strongly supported the monophyletic grouping of basal and derived passerine species. CONCLUSIONS: Our study provides evidence of a large number of highly polymorphic MHC paralogs in seven basal passerine species, with strong similarities to the MHC described in more derived passerine lineages rather than the simpler MHC in non-passerine lineages. These findings indicate an early evolutionary origin of highly polymorphic MHC paralogs in passerines and shed light on the evolutionary forces shaping the avian MHC.

Variation at the DRD4 locus is associated with wariness and local site selection in urban black swans.

BACKGROUND: Interactions between wildlife and humans are increasing. Urban animals are often less wary of humans than their non-urban counterparts, which could be explained by habituation, adaptation or local site selection. Under local site selection, individuals that are less tolerant of humans are less likely to settle in urban areas. However, there is little evidence for such temperament-based site selection, and even less is known about its underlying genetic basis. We tested whether site selection in urban and non-urban habitats by black swans (Cygnus atratus) was associated with polymorphisms in two genes linked to fear in animals, the dopamine receptor D4 (DRD4) and serotonin transporter (SERT) genes. RESULTS: Wariness in swans was highly repeatable between disturbance events (repeatability = 0.61) and non-urban swans initiated escape from humans earlier than urban swans. We found no inter-individual variation in the SERT gene, but identified five DRD4 genotypes and an association between DRD4 genotype and wariness. Individuals possessing the most common DRD4 genotype were less wary than individuals possessing rarer genotypes. As predicted by the local site selection hypothesis, genotypes associated with wary behaviour were over three times more frequent at the non-urban site. This resulted in moderate population differentiation at DRD4 (FST = 0.080), despite the sites being separated by only 30 km, a short distance for this highly-mobile species. Low population differentiation at neutrally-selected microsatellite loci and the likely occasional migration of swans between the populations reduces the likelihood of local site adaptations. CONCLUSION: Our results suggest that wariness in swans is partly genetically-determined and that wary swans settle in less-disturbed areas. More generally, our findings suggest that site-specific management strategies may be necessary that consider the temperament of local animals.