JOURNAL OF AVIAN BIOLOGY www.avianbiology.org Journal of Avian Biology 1 –––––––––––––––––––––––––––––––––––––––– © 2018 The Authors. Journal of Avian Biology © 2018 Nordic Society Oikos * Two last authors Subject Editor: Wolfgang Goymann Editor-in-Chief: Jan-Åke Nilsson Accepted 23 April 2018 2018: e01728 doi: 10.1111/jav.01728 Evidence that bird odour can encode social information that can be used in chemi- cal communication is growing, but is restricted to a few taxonomic groups. Among birds, diurnal raptors (i.e. birds from the Accipitriformes and Falconiformes order) have always been considered as mainly relying on their visual abilities. Although they seem to have a functional sense of smell, whether their odour can convey social infor- mation has yet to be determined. Combining gas-chromatography-mass-spectrometry (GCMS) and microsatellite data, we tested whether chemical compounds from preen gland secretions can encode sex, age, individuality, seasonal differences and genetic relatedness in the gregarious accipitriform black kite Milvus migrans . While no differ- ences in preen oil composition were found between age classes, an individual signature was detected. While a seasonal variation was found in both sexes, compounds differ between sexes in the non-breeding season. Finally, a significant correlation between chemical proximity and genetic proximity was detected in male–male dyads and male– female dyads but not in female–female dyads. Our study provides the first evidence in raptors that preen secretion can convey information that may potentially be used in individual recognition, reproductive synchronization and inbreeding avoidance, and suggests that raptors may rely upon their olfactory abilities more than previously thought. This study opens promising avenues for further studies in raptor chemical communication. Keywords: olfactory signature, preen oil, raptor Introduction Although chemical communication has been supposed to be the oldest and widespread way of communication in living organisms, birds have long been considered to rely primarily on visual and acoustic cues, rather than odour cues (Bradbury and Vehrencamp 2011). Nevertheless, birds possess a fully functional olfactory system Preen oil chemical composition encodes individuality, seasonal variation and kinship in black kites Milvus migrans Simon Potier, Malicia M. Besnard, David Schikorski, Bruno Buatois, Olivier Duriez, Marianne Gabirot, Sarah Leclaire* and Francesco Bonadonna* S. Potier (http://orcid.org/0000-0003-3156-7846) (sim.potier@gmail.com), M. M. Besnard, B. Buatois, O. Duriez, M. Gabirot, S. Leclaire (http://orcid. org/0000-0002-4579-5850) and F. Bonadonna, CEFE UMR 5175, CNRS – Univ. de Montpellier – Univ. Paul-Valéry Montpellier – EPHE, Montpellier, France. SP also at: Dept of Biology, Lund Univ., Lund, Sweden. SL also at: Laboratoire Evolution and Diversité Biologique UMR 5174, CNRS, Toulouse, France. – D. Schikorski, Laboratoire Labofarm, Loudeac, France. Article 2 (Steiger et al. 2008) and recent studies have suggested that birds use odour cues in various social contexts such as mate choice, mate synchronization or parental care (reviewed by Caro et al 2015). The main source of avian odour is probably the secretions of the preen gland (also called the uropygial gland) that birds spread onto their plumage during preening (Hagelin 2007, Mardon et al. 2011, Campagna et al. 2012). In domestic chicken Gallus gallus domesticus , while males preferred females with an intact uropygial gland over glan- dectomized females, anosmic males did not show any prefer- ences (Hirao et al. 2009), suggesting a crucial role for preen oil odour in sexual interactions in this species. In numerous avian species, the chemical composition of preen oil encodes information on various bird characteris- tics, such as sex, age, individuality, seasonal variation, spe- cies affiliation or genotype (Hagelin 2007, Mardon et al. 2010, Wyatt 2014, Caro et al. 2015, Gabirot et al. 2016, Krause et al. 2018). Although the biological meaning of these differences has rarely been investigated, a few stud- ies have shown that most of these odour cues can be recog- nized by birds (Bonadonna and Nevitt 2004, Mardon and Bonadonna 2009) and influence social behaviour (Balthazart and Schoffeniels 1979, Hirao et al. 2009, Caspers et al 2015). For instance, kinship odours can be discriminated in Humbolt penguins Spheniscus humboldti (Coffin et al. 2011), European storm petrels Hydrobates pelagicus (Bonadonna and Sanz-Aguilar 2012) and zebra finches Taeniopygia guttata (Krause et al. 2012), and influence investment in reproduction in the latest species (Caspers et al. 2015). While the information conveyed in preen oil odours has been studied in several bird taxa, it has not yet been explored in Accipitriformes and Falconiformes (hereafter called rap- tors), who have always been considered to rely primarily on vision (Jones et al. 2007). Nevertheless, raptors have func- tional olfactory receptors (Zhan et al. 2013, Yang et al. 2015), and can use smell to forage (Houston 1986, 1988, Slater and Hauber 2017, Potier et al. unpubl.). In this study, we aimed to identify whether preen gland odours can convey different social information in the black kite Milvus migrans, a long-lived monogamous and gregarious raptor for which chemical communication may be important, particularly in a mating context (Hagelin 2007), such as inbreeding avoid- ance. Indeed, the genetic benefits of mate choice are crucial when choosing a single lifelong mate, even when extrapair paternity can be present, such as in black kites (Koga and Shiraishi 1994). Moreover, individuals of gregarious spe- cies interact frequently with conspecifics and individual and kinship recognition are expected to have evolved as they can promote social cohesiveness (Krebs and Dawkins 1984), as shown in mammals (Rosell and Nolet 1997, Whittle et al. 2000). Using gas chromatography-mass spectrometry (GC-MS) data, we tested whether preen oil chemical com- pounds vary with sex, age classes, season and individual iden- tity in black kites. Then by combining GC-MS data with microsatellite data, we tested whether preen oil odour reflects kinship, which might allow birds to avoid inbreeding (Zelano and Edwards 2002). Material and methods Study site and species Fieldwork was carried out in 2016 at the historical theme park ‘Le Grand Parc du Puy du Fou’ in France. Sampling was performed in the non-breeding (February) and breeding seasons (April) of the black kite (Del Hoyo and Elliott 1994). During the breeding season, adults were in reproductive con- dition, while, because juveniles were used in public represen- tations, their reproductive state/behaviour was interrupted. Samples were collected on 45 captive black kites including 22 males and 23 females from two age classes (26 juveniles and 19 adults). Birds that were not in immature plumage ( > 3 yr old) were considered as adults. All adults were born in the wild while all juveniles (offspring of the adults) were born in captivity. This study was conducted on captive birds that were housed in the same park, hence controlling for environ- mental factors, such as diet which is known to influence the chemical compounds in preen oil (Thomas et al. 2010). From October to mid-April, birds were all under the same diet (chicken chicks, common roach Rutilus rutilus , coypu Myocastor coypus and common quail Coturnix coturnix ). Odourant sample collection Preen secretions were sampled by gently squeezing the base of the uropygial gland and collecting the discharged waxy exudates in a glass capillary. The capillary was then inserted into an opaque 1.5 ml chromatographic glass vial (Macherey- Nagel & Co Ò , Düren, Germany) sealed with a Teflon Ò PTFE faced membrane. Clean nitrile gloves were worn at all time during sample collection. We stored all samples in the dark at –12 ° C from the day of collection to their arrival in the lab and at –20 ° C until chemical analyses. Overall, a total of 84 secretion samples were collected from 45 different birds (but it was impossible to collect secretions twice on 6 indi- viduals, so we had only one sample for those 6 individuals). Five ‘blank’ samples (i.e. an empty capillary inserted into a glass vial) were collected in the field to control for potential contaminations. Sample preparation and compound extraction Chemical analyses were carried out at the Chemical Analysis for Ecology Platform (PACE), technical facilities of the LabEx CEMEB (CEntre Méditerranéen Environnement et Biodiversité, Montpellier, France). We extracted the organic compounds from preen secretions in 400 μl dichlorometh- ane (for analysis, Sigma-Aldrich Ò , Munich, Germany). We added to each vial 10 mg (5 μl of 2 mg μl –1 ) of n-methyl- Octadecanoate (retention index RI 2126, Sigma-Aldrich Ò , Munich, Germany) as an internal standard to ensure the qual- ity of the injection. Extracts were mixed (2200 turns min –1 ) during 1 min and then transferred into a microvolume insert in a second chromatographic vial using a 250 μl clean syringe (Evol Ò XR, SGE Analytical Science, Milton Keynes, UK). 1600048x, 2018, 7, Downloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1111/jav.01728 by University Of Florida, Wiley Online Library on [13/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3 Samples were analysed by gas chromatography and mass spectrometry, using a Shimadzu Quadrupole 2010 plus (Shimadzu Ò , Kyoto, Japan) equipped with a Shimadzu AOC-20i+s autosampler (Shimadzu Ò , Kyoto, Japan) and an Optima-5MS capillary column (30 m × 0.25 mm × 0.25 μm). Helium was used as the carrier gas at a constant pressure of 6.5 psi. We injected 4 μl of the extracted solu- tion with a 1:4 split from 0 to 2 min and then a 1:20 split. The injector was set at 250 ° C. The oven temperature was programmed as follow: 100 ° C for 3 min, 12 ° C min –1 from 100 ° C to 200 ° C, 6 ° C min –1 from 200 ° C to 320 ° C, and then 320 ° C for 4 min. The mass spectrometer was used in scan mode from 38 to 400 m z –1 , with 200 ° C and 280 ° C for ion source and transfer line respectively. To overcome saturation of some peaks, 5 samples were re-injected at 2 μl. In contrast, 18 samples that were too diluted (low GCMS signal) were concentrated under a nitrogen flux before reinjection. Chromatographic data processing Chemical data processing was carried out with the GC-MS Solution software ver. 2.71 (Shimadzu Ò , Kyoto, Japan). While the software automatically integrated large peaks, manual integration was performed for the smallest peaks. Because we could not control the amount of secretion collected and we did not know the identity of the compounds, we did not rely on the absolute abundance of chromatogram peaks; rather, we expressed each peak as the proportion of the peak area relative to the total area of the chromatogram. Genetic analyses We used diversity at microsatellite loci as a proxy of genome-wide diversity. Genetic analyses were conducted by the Labofarm laboratory (Loudéac, France). Due to the lack of specific microsatellite markers from black kites available in the literature, the panel of 55 microsatellite markers previously identified in phylogenetically related raptors species were firstly tested in few number of black kites. Among these markers, 15 were polymorphic in the black kite and thus selected to carry out individual genotyping (Table 1). Genomic DNA extractions were conducted from blood or eggshells using the QIAamp DNA MiniKit (QIAGEN), and from feathers using the Adiapure purification kit (Adiagene-BioX) following the manufacturer’s standard pro- tocols. The 15 selected microsatellite markers were amplified in 2 PCR multiplexes. PCR reactions were performed using the QIAGEN Multiplex PCR Kit in a 10 μl final volume containing 3 μl of genomic DNA diluted at 10 ng μl –1, 5 μl of QIAGEN’s multiplex master mix, 1 μl of QIAGEN’s Q-solution, and 1 μl of end-labelled primers mix at 10 μM each. Amplifications were carried out in a GeneAmp PCR System 2700 thermal cycler (Applied Biosystems). Cycling conditions were similar for the 2 PCR multiplexes: initial denaturation at 95 ° C for 15 min, followed by 35 cycles at 95 ° C for 40 s, 56 ° C for 90 s, and 72 ° C for 90 s, and a final extension at 60 ° C for 30 min. Amplified PCR frag- ments were then diluted and separated on an ABIPRISM 3130xl sequencer (Applied Biosystems) with GeneScan 500 Rox dye size standards. Allele size was determined using GeneMapper ver. 4.1 software system (Applied Biosystems, Life Technologies). Genetic relatedness between individuals was estimated using the relatedness index ‘identity’ ( R ID ) (Mathieu et al. 1990) calculated with the IDENTIX V1.1.5.0 software (Belkhir et al. 2002). The identity index has been validated as a good estimator of the consanguinity of offspring in cases where identical alleles are likely to be identical by descent (Belkhir et al. 2002). Statistical analyses All statistical analyses were performed with PRIMER ver. 6.1.12 software (Clarke and Gorley 2006) and the PERMANOVA+ ver. 1.0.2 package (Anderson et al. 2008), and with R 3.2.3 software and the ‘vegan’ (Oksanen et al. Table 1. List of selected markers used to calculate the genetic proximity between individuals. Locus Source species Number of alleles Alleles size (range) Reference Hal-01 Haliaeetus albicilla 6 121–163 Hailer et al. (2005) Hal-04 Haliaeetus albicilla 6 138–148 Hailer et al. (2005) Hal-09 Haliaeetus albicilla 4 138–158 Hailer et al. (2005) Hal-13 Haliaeetus albicilla 2 142–154 Hailer et al. (2005) Hvo-13 Haliaeetus vociferoides 3 226–240 Tingay et al. (2007) Hle-03 Haliaeetus leucocephalus 2 247–263 Tingay et al. (2007) IEAAAG04 Aquila heliaca 5 226–258 Busch et al. (2005) IEAAAG05 Aquila heliaca 3 127–139 Busch et al. (2005) IEAAAG13 Aquila heliaca 8 232–276 Busch et al. (2005) Hf-C5D4 Hieraaetus fasciatus 12 190–216 Mira et al. (2005) Aa04 Aquila adalberti 2 122–134 Martínez ‐ Cruz et al. (2002) Aa11 Aquila adalberti 2 251–255 Martínez ‐ Cruz et al. (2002) Aa49 Aquila adalberti 3 158–162 Martínez ‐ Cruz et al. (2002) Tgu06 Taeniopygia guttata 2 143–145 Olano-marin et al. (2010) GgaRBG18 Tyto alba 6 265–275 Klein et al. (2009) 1600048x, 2018, 7, Downloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1111/jav.01728 by University Of Florida, Wiley Online Library on [13/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4 2013), ‘indicspecies’ (De Caceres and Jansen 2016), and ‘ggplot2’ packages (Wickham and Chang 2014). To inves- tigate the relationship between the chemical profiles and the various environmental and individual factors, we used a robust distance-based multivariate approaches. Proportions of chemical compounds were square-root transformed to reduce the influence of peaks with low proportion and many zeros (Hellinger transformation) (Legendre and Gallagher 2001). Euclidean distances between every pairs of samples were calculated to create a distance matrix that formed the basis of ensuing analyses. To determine whether chemical composition varied with season, bird’s sex, and bird’s age, we used PERMANOVAs with 5000 permutations and type III sums of squares (SS) (Anderson 2001). Individual identity nested within sex and age was included as a random factor. We also tested the inter- actions with biological meaning (age × sex, age × season, sex × season). To determine the chemical compounds that were preferentially associated with a significant fixed effect, we used correlation indices (function ‘multipatt’ in R) (De Caceres and Jansen 2016) with the point biserial coefficient of association on untransformed proportions of chemical compounds. A Mantel test with Spearman rank correlation and 5000 permutations was used to test for an association between chemical distance and genetic distance in female–female (FF), male–male (MM) and male–female (MF) dyads in January and April 2016. Data deposition Data available from the Dryad Digital Repository: < http:// dx.doi.org/10.5061/dryad.90fq174 > (Potier et al. 2018). Results Black kites preen oil was mainly composed of unidenti- fied long-chained ketonic compounds ( > 19 carbon atoms; n 69 out of 86 detected compounds). One acid ester (Eicosanic acid methyl ester), and 3 sterols were also detected. The chemical profiles of adults and juveniles were not different (pseudo-F 1,43 1.08, p 0.33). An individual odour signature was detected across seasons (individual identity effect; pseudo-F 1.52, p 0.001, Fig. 1) as shown by the proximity of two points of same individual (same colour on Fig. 1). The chemical profile of an individual at a given season (e.g. breeding season) is closer to that of the other season (e.g. non-breeding season) than that of other individuals (Fig. 1). The chemical profiles varied with the interaction between sex and season (pseudo-F 1,40 3.71, p 0.001, Fig. 2). The chemical profiles of females and males differed in the non- breeding season (pairwise test: t 1,36 1.62, p 0.007, Fig. 2) while they did not differ in the breeding season (pairwise test: t 1,40 0.98, p 0.42, Fig. 2). Correlation index analyses shows that one sterol and 5 ketones with low abundance were higher in males than females in the non-breeding period. In addition, chemical variations between the non-breeding and the breeding seasons were observed in females (pairwise test: t 1,18 1.84, p 0.011, Fig. 2) and males (pairwise test: t 1,22 1.46, p 0.037, Fig. 2). In males, correlation indices show that one sterol decreased, while Eicosanoic acid methyl ester increased from the non-breeding to the breeding season. In females, the abundance of 8 ketones and 2 unidentified compounds was higher in the breeding season than in the non-breeding season. During the breeding season, chemical distances increased significantly with genetic distances in MM dyads (rho 0.27, p 0.005, n 22, Fig. 3), and MF dyads (rho 0.17, Figure 1. Partial constrained distance-based redundancy analysis plots (capscale function in R) in (a) females (n 34 samples), and (b) males (n 44 samples). Each colour represents a different individual. Plots are restricted to individuals from which two samples were col- lected. The period effect was partialled-out to better illustrate the separation. Triangles correspond to juveniles, while dots correspond to adults. 1600048x, 2018, 7, Downloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1111/jav.01728 by University Of Florida, Wiley Online Library on [13/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 5 p 0.035, n 43, Fig. 3), while no correlation between chemical and genetic distances was found in FF dyads (rho 0.13, p 0.12, n 21, Fig. 3). During the non- breeding season, chemical distances did not correlate with genetic distances in MM, FF and MF dyads (all p > 0.3). Discussion In this study, we provide the first evidence that preen oil com- pounds can encode information that may play key roles in the social ecology of raptors. We found that these compounds varied with season, with sex in the non-breeding season, and reflect individual identity across seasons in the black kite. In addition, preen oil compounds were found to reflect genetic relatedness in male–male and male–female dyads during the mating period. It is important to note that all these differ- ences cannot be accounted for diet differences as all black kites were fed with the same food. Despite high qualitative variations in the composition of preen secretions across avian species, the main com- ponents of preen secretions are usually monoester waxes (Campagna et al. 2012). Surprisingly, we detected only one ester in preen oil of black kites; rather, we detected a high diversity of long-chain ketones. Previous identification of the chemical composition of raptor preen secretions are scarce (Gamo and Saito 1971, Jacob and Poltz 1975) and none described ketones. The presence of short-chain methyl- ketones ( < C18) have been described in several passerines (Shaw et al. 2011, Amo et al. 2012, Soini et al. 2013). As captivity is known to only slightly affect bird chemical profile (Hagelin et al. 2003, Thomas et al. 2010), it is unlikely that captivity led to a wide shift in compound class (i.e. esters to ketones) in our study. However, variability in chemical pro- files between individuals might be greater in the wild than in captivity, and further studies on wild kites would help elu- cidate whether preen secretions convey similar information under more natural conditions. Sex but no age differences in chemical profiles We found that preen oil composition differs between sexes only in the non-breeding season, with males hav- ing higher abundance of one sterol and of a few ketonic compounds compared to females. In several bird species, preen oil composition varies with sex in the breeding sea- son (Whittaker et al. 2010, Leclaire et al. 2011, Amo et al. 2012, Jacob et al. 2014, Fischer et al. 2017). In many verte- brates, sex-differences in chemical profiles are mediated by steroid hormones (Wyatt 2014), for which sex variations are often higher during the breeding season (Wingfield et al. 1990). It is therefore striking to observe sex-differences in chemical profile only during the non-breeding season in black kites. However, sex recognition by smell outside breeding season may be important in this species. Wintering distribution and timing of migration often vary with sex in birds (Newton 2010), especially in raptors (Kjellén 1992, Mueller et al. 2000). Sex-differences in chemical profiles during the non-breeding season may thus act as a signal for females to group and/or to disperse. In several species, preen oil odour varies across ages and may reflect the ability to reproduce (Kolattukudy and Sawaya 1974, Sandilands et al. 2004, Shaw et al. 2011). In this study, we did not find differences in the composi- tion of preen oil secretions between juvenile and adult black kites. Hormonal levels are known to influence preen oil composition (Sandilands et al. 2004, Whelan et al. 2010, Whittaker et al. 2011). The lack of differences between adults and juveniles’ black kites may result from similar seasonal patterns in circulating sex-steroid hormones observed in young and adults (Blas and Hiraldo 2010), which may reflect the ability to reproduce during the first year (Del Hoyo and Elliot 1994). Preen oil compounds convey information about reproductive state? Female and male black kites showed a significant change in preen oil odour between the reproductive and the non-repro- ductive periods. This shift, however, is not similar between males and females. Because long- and short-chain ketones may have repellency property against insects (Innocent et al. 2008, Germinara et al. 2012), the shift in preen oil com- position may be useful for protection to ectoparasites. This Figure 2. Distance-based redundancy analysis plots showing separation between kites (n 84 samples) by period and sex. 1600048x, 2018, 7, Downloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1111/jav.01728 by University Of Florida, Wiley Online Library on [13/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6 may be of a great of importance for female black kites, which devote all their time maintaining a suitable nest environment until chick fledging (Bustamante and Hiraldo 1990). The shift in preen oil composition may also convey information about the ability to reproduce. Indeed, it has long been sug- gested that chemical compounds can encode female’s mating status and that males prefer the scent of unmated females (Thomas 2011). Evidence in birds has yet to be provided, but during the reproductive period, black kites display a peak of testosterone (Blas et al. 2010) which is known to stimulate the production of some volatile compounds in other birds such as the dark-eye juncos Junco hyemalis (Whittaker et al. 2011), reflecting probably the ability to reproduce. Seasonal changes in gland secretion composition may have other non- exclusive functions (Steiger et al. 2010), such as enhancing feather condition (Giraudeau et al. 2010) or colour (López- Rull et al. 2010) during the breeding period. Further experi- mental studies are needed to determine the adaptive function of the seasonal change in preen secretion chemical composi- tion in black kites. An individual odour signature As found in several bird species (Bonadonna et al. 2007, Mardon et al. 2010, Whittaker et al 2010, Leclaire et al. 2011), preen oil chemical composition carries an individ- ual signature in black kites. Individual olfactory signatures may be crucial for conspecific and partner recognition. After migration, most black kites return to their previous breeding site (Forero et al 1999), and thus share similar neighbours across years. Individual recognition may, there- fore, allow them to behave adaptively when they meet (Wyatt 2014). In addition, male and female raptors often migrate separately (Mueller et al. 2000) and partner rec- ognition may be essential to discriminate the mate at the return to the breeding site. Although Antarctic prions and blue petrels discriminate their partner from other conspecif- ics using odour cues (Bonadonna and Nevitt 2004, Mardon and Bonadonna 2009), no studies have clearly investigated odour-based individual recognition in other avian species. Understanding whether black kites do use the olfactory Figure 3. Relationship between genetic proximity and chemical proximity (as described by Euclidean distance) in (a) male–male dyads, (b) cross-sex dyads and (c) female–female dyads. Solid lines are linear model predicted values and SE. 1600048x, 2018, 7, Downloaded from https://nsojournals.onlinelibrary.wiley.com/doi/10.1111/jav.01728 by University Of Florida, Wiley Online Library on [13/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 7 individual signature to adjust their social behaviour requires further experiments. Encoding genetic relatedness through chemical profiles The existence of an individual signature may suggest a strong genetic determinism of preen oil composition. Accordingly, chemical and genetic proximities were positively correlated in male–male and male–female dyads during the breeding season. Correlations between genetic and chemical distances have been found in the song sparrow Melospiza melodia (Slade et al. 2016) and the black-legged kittiwake Rissa tridactyla (Leclaire et al. 2013, 2014), a bird that preferen- tially mate with unrelated individuals (Mulard et al. 2009). In addition, zebra finches (Krause et al. 2012), blue petrels Halobaena caerulea (Leclaire et al. 2017), European storm petrels (Bonadonna and Sanz-Aguilar 2012) and Humboldt penguins (Coffin et al. 2011) can discriminate genetic relat- edness based on odour cues. Odour-based kin recognition may allow individuals to avoid inbreeding by choosing non- kin mates (Bonadonna and Sanz-Aguilar 2012, Wyatt 2014) and might reduce intrasexual competition between kin, thereby increasing inclusive fitness. Correlations between genetic and chemical distances were not detected during win- ter when male–male competition or female mate choice may be less crucial. This seasonal dependent pattern was found in other vertebrates including the ring-tailed lemur Lemur catta (Charpentier et al. 2008) and the tree-spined stickleback Gasterosteus aculeatus (Milinski et al. 2010), and suggests that kinship signals may be costly to produce and individu- als should determine when to signal (Johansson and Jones 2007). Finally, this seasonal dependent pattern while indi- viduals signature was found across season was found suggests that chemical compounds involved for kinship signals and individual signature were different. Conclusion Our study provides the first evidence that, in raptors, chemi- cal compounds of preen secretion encode crucial informa- tion on individual traits, and suggests that olfaction may play an unsuspected role in raptor communication. Behavioural experiments are, however, needed to determine whether black kites can discriminate these differences in preen oil compositions. Acknowledgements – We thank N. De Villiers, L. Albert, J.-L. Liegeois and T. Bouchet of Le Grand Parc du Puy du Fou for allowing us to conduct experiments with their birds. We also thank H. Billaud, F. Blais, C. Gaborit, E. Challet, P. Bouffandeau, J. Barrier, A. Sahnoune, H. Cogny, J. Thomas, C. Leroy, A. Boyer, L. Deur and E. Antoine for their help with the fieldwork. Funding – SP was supported by a PhD fellowship from the Labex Cemeb and the Association Française des Parcs Zoologiques (AFdPZ). Especially, 13 raptor parks gave funding to AFdPZ for this study: Le Grand Parc du Puy du Fou, Le Rocher des Aigles, Les Ailes de l’Urga, Le Zoo d’Amnéville, La Volerie des Aigles, Le Donjon des Aigles, Le Bois des Aigles, Les Géants du Ciel, Le Zoo de la Bourbansais, Le Zoo de la boissière du Doré, Le Zoo de la Barben, Le Zoo du Pal, Le Parc des Oiseaux. SL was supported by the Agence Nationale de la Recherche Française (ANR grant ‘BactOdo’, no. ANR-13-PDOC-0002). DS was supported by the Labofarm laboratory. 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