Received: 25 October 2016 | Revised: 12 January 2017 | Accepted: 28 January 2017 DOI: 10.1002/ece3.2836 ORIGINAL RESEARCH Conditions on the Mexican moulting grounds influence feather colour and carotenoids in Bullock’s orioles (Icterus bullockii) Kaitlin L. Sparrow1,2 | Kingsley K. Donkor2 | Nancy J. Flood1 | Peter P. Marra3 | Andrew G. Pillar1 | Matthew W. Reudink1 1 Department of Biological Sciences, Thompson Rivers University, Kamloops, BC, Canada 2 Department of Chemistry, Thompson Rivers University, Kamloops, BC, Canada 3 Migratory Bird Center, Smithsonian Conservation Biology Institute, Washington, DC, USA Abstract Carotenoid-­based plumage coloration plays a critical role for both inter-­ and intrasexual communication. Habitat and diet during molt can have important consequences for the development of the ornamental signals used in these contexts. When molt occurs away from the breeding grounds (e.g., pre-­alternate molt on the wintering grounds, or stopover molt), discerning the influence of habitat and diet can be particularly Correspondence Matthew W. Reudink, Department of Biological Sciences, Thompson Rivers University, Kamloops, BC, Canada. Email: mreudink@tru.ca ­important, as these effects may result in important carryover effects that influence Funding information Western Economic Diversification Canada; Natural Sciences and Engineering Research Council of Canada through USRA; Discovery Grants stopover site in the region affected by the Mexican monsoon climate pattern. This territory acquisition or mate choice in subsequent seasons. Several species of songbirds in western North America, including the Bullock’s oriole (Icterus bullockii), migrate from the breeding grounds to undergo a complete prebasic (post-­breeding) molt at a strategy appears to have evolved several times independently in response to the harsh, food-­limited late-­summer conditions in the arid West, which contrast strongly with the high productivity driven by heavy rains that is characteristic of the Mexican monsoon region. Within this region, individuals may be able to optimize plumage coloration by molting in favourable areas characterized by high resource abundance. We used stable isotope analysis (δ13C, δ15N) to ask whether the diet and molt habitat/location of Bullock’s orioles influenced their expression of carotenoid-­based plumage coloration as well as plumage carotenoid content and composition. Bullock’s orioles with lower feather δ15N values acquired more colorful plumage (orange-­shifted hue) but had feathers with lower total carotenoid concentration, lower zeaxanthin concentration, and marginally lower canthaxanthin and lutein concentration. Examining factors occurring throughout the annual cycle are critical for understanding evolutionary and ecological processes. Here, we demonstrate that conditions experienced during a stopover molt, occurring hundreds to thousands of kilometers from the breeding grounds, influence the production of ornamental plumage coloration, which may carryover to influence inter-­ and intrasexual signaling in subsequent seasons. KEYWORDS Bullock’s oriole, carotenoid, carryover effects, Icterus bullockii, Mexican monsoon region, molt-migration, plumage, stable isotope This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2017 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Ecology and Evolution. 2017;1–9.  www.ecolevol.org | 1 2 | SPARROW et al. 1 | INTRODUCTION in the acquisition of plumage coloration. The effects of habitat may Sexual selection is a strong evolutionary force that has been instru- insufficient amount of carotenoids being consumed and deposited, manifest directly, as molting in poor-­quality habitat can result in an mental in shaping the elaboration of ornamental traits and driving and indirectly, through malnourishment, exposure to parasites, high genetic divergence and speciation across a range of taxa. Individuals stress load, or other factors. Thus, both direct and/or indirect effects with larger, more elaborate, and more colorful ornamental traits tend may ultimately result in a need to utilize carotenoids elsewhere or limit to be favoured by sexual selection, as many of these traits act as hon- the ability of individuals to metabolically convert dietary carotenoids. est indicators of individual condition or quality and function in inter-­ Migratory birds vary greatly in the timing and location of their and intrasexual communication. Plumage coloration, in particular, has molt. Which feathers are replaced and when they are grown can vary garnered considerable attention from evolutionary biologists (Hill & among species and even within species across populations. In the arid McGraw, 2006) and a preponderance of work has demonstrated the West, several species, hereafter referred to as molt-­migrants, inter- importance of carotenoid coloration for social and sexual signaling. rupt fall migration to molt in the Mexican monsoon region, located Carotenoids are bright red, yellow, and orange dietary organic pig- in the southwestern United States and northwestern Mexico (Leu & ments that have a range of functions in animals, including producing Thompson, 2002; Pillar, Marra, Flood, & Reudink, 2015; Pyle et al., color, boosting immune response, and acting as antioxidants (García-­de 2009; Rohwer, Butler, Froehlich, Greenberg, & Marra, 2005). This strat- Blas, Mateo, Guzmán Bernardo, Martín-­Doimeadios, & Alonso-­Alvarez, egy likely evolved due to the extensive green-­up that occurs during 2014; Navara & Hill, 2003). To produce feather coloration, carotenoids the monsoon rains in late summer/early autumn, providing a greater can be deposited directly from the diet as yellow dietary carotenoids, amount of the food resources necessary for molt (Douglas, Maddox, or they can be modified prior to deposition in feathers. For example, lu- Howard, & Reyes, 1993; Higgins & Gochis, 2007; Rohwer & Manning, tein, zeaxanthin, β-­carotene, and β-­cryptoxanthin, the main dietary ca- 1990; Rohwer et al., 2005). The Mexican monsoon region covers a rotenoids, can be modified to the yellow carotenoids, 3’-­dehydrolutein, large geographic area and encompasses a range of habitats, suggesting canary xanthophyll A&B, or modified to red keto-­carotenoids such as that individuals may be able to optimize feather coloration by selecting α-­doradexanthin, astaxanthin, canthaxanthin, and adonirubin (McGraw, a high-­quality molt location. The challenge, however, is tracking habitat 2006). The main modifications to dietary carotenoids include oxidation use across seasons in animals moving hundreds or thousands of kilo- of hydroxyl groups to ketones, which produces modified yellow carot- meters between breeding, molting, and wintering areas. enoids, or the addition of ketones via oxidation, which produces red Because habitat can influence food and carotenoid availability, keto-­carotenoids. These metabolic conversions appear to be condition moving to areas with high food availability during molt could provide a dependent, indicating that, of all carotenoid-­based coloration, orange/ strong selective advantage. Although small migratory birds are difficult red feathers may be the most honest signals of individual quality and to track due to their size and the vast distances they travel, stable iso- condition (Hill, 2011; Hill & Johnson, 2012; Johnson & Hill, 2013; Hill, tope analysis has become a useful tool for linking different phases of 2014; but see Simons, Groothuis, & Verhulst, 2015). the annual cycle and determining diet and habitat use. Because stable While individual condition is essential for the expression of opti- isotopes in feathers are inert once the feathers are grown, analyzing mal coloration, individuals may also be constrained by habitat quality feather isotope values, in particular δ13C and δ15N, can provide infor- during molt. Several studies on great tits (Parus major) and Eurasian mation on diet and habitat use at the time of molt (Wassenaar, 2008; blue tits (Cyantistes caeruleus) have demonstrated the importance of see Methods for details). forest composition and quality at a local scale for influencing food/ Here, we ask whether conditions experienced during molt in the carotenoid availability and thus ultimately plumage coloration (e.g., Mexican monsoon region influence feather color and carotenoid Slagsvold & Lifjeld, 1985; Arriero & Fargallo, 2006; Eeva, Sillanpää, content/composition in the molt-­migrant, Bullock’s oriole (Icterus & Salminen, 2009; but see Ferns & Hinsley, 2008). Broader, regional bullockii). Recently, Pillar et al. (2015) used a combination of geolo- variation in habitat may also influence regional variation in plumage cators and stable hydrogen isotope analysis to confirm that Bullock’s coloration. For example, house finches (Haemorhous mexicanus) exhibit orioles interrupt their southward migration to stopover and molt in marked geographic variation in the color and extent of carotenoid-­ the Mexican monsoon region. The authors showed that there was based plumage coloration (Badyaev, Belloni, & Hill, 2012). Diet supple- extensive variation in feather hydrogen isotopes, indicating that molt mentation experiments revealed that the size of the carotenoid-­based might be occurring across a relatively large geographic area or range of patch resulted from genetic differences between the frontalis and habitats within the Mexican monsoon region. This finding is consistent griscomi subspecies, but that coloration per se resulted from differences with low ­reported molt location fidelity in Bullock’s orioles; Pyle et al. in carotenoid access during molt. In American redstarts (Setophaga ru- (2009) suggest that molt-­migrants, including Bullock’s orioles, track ticilla), males from more northern latitudes express higher red chroma resources during the molt period, which implies that individuals may values, which likely reflects higher feather carotenoid content (Norris, be able to optimize plumage coloration by selecting high-­quality molt Marra, Kyser, Ratcliffe, & Montgomerie, 2007); as for house finches, locations (i.e., areas where food, and perhaps even carotenoid-­rich this geographic variation in plumage coloration was attributed to food, is abundant). We predicted that Bullock’s orioles that molted in regional variation in diet and carotenoid availability. These studies high-­quality, wet habitats in the Mexican monsoon region (indicated demonstrate that molt location and habitat can play an important role by low δ13C values) would have more carotenoids (especially the red | 3 SPARROW et al. keto-­carotenoid canthaxanthin) deposited in their feathers than those 13 that molted in low-­quality, xeric habitats (high δ C values). However, 15 the relationship between feather carotenoids and δ N could be either 15 N is preferentially incorporated with increasing trophic level, resulting in greater δ15N values at higher trophic levels (Post, 2002; Poupin et al., 2011). Furthermore, δ15N values are negatively correlated with rainfall positive or negative, depending on the relative richness of dietary ca- and positively associated with temperature, so that δ15N level may be rotenoids and the specific biome in which the birds molt. indicative of the temperature and aridity of a biome (Craine et al., 2009; Sealy, van der Merwe, Lee-­Thorp, & Lanham, 1987). 2 | METHODS 2.1 | Study species Bullock’s orioles are songbirds that breed as far north as the southern For all feathers, analysis of stable-­carbon and stable-­nitrogen isotope ratios was performed at the Smithsonian Institution Isotope Mass Spectrometry Lab in Suitland, Maryland, USA. Feathers and claws were washed in a 2:1 chloroform-­methanol solution and allowed to dry. Approximately 0.30–0.40 mg of the distal tip of the feather interior of British Columbia, undergo prebasic molt in the Mexican mon- was sampled, excluding the rachis. Using a Thermo high-­temperature soon region, and overwinter from northern Mexico to Central America conversion elemental analyzer (TC/EA; Thermo Scientific, Waltham, (Leu & Thompson, 2002; Pillar et al., 2015; Pyle et al., 2009; Rohwer Massachusetts, USA) at 1350°C, samples were first pyrolyzed and se- et al., 2005). After second year (ASY) males exhibit bright orange plum- quentially analyzed by a Thermo Delta V Advantage isotope ratio mass age on their breast, belly, rump, and face (the crown, nape, back, scapu- spectrometer. We ran two in-­house standards (acetanilide and urea) lars, and lores of males are black, and they possess a bold black eye-­line), for every 10 samples. All isotope ratios are reported in δ notation in while females of all ages have a pale yellow head and breast, with a throat units per mil (‰) relative to international standards PDB (carbon) and that is black, yellowish, or mixed yellow and black (females with throat air (nitrogen). Based on repeated measurements of standards, repeat- patches are hypothesized to be older individuals; Pyle, 1997; Jaramillo ability of carbon and nitrogen samples was ±0.2‰. & Burke, 1999). Second year (SY) males are similar to females, but also exhibit patches of ASY-­like plumage (Rising, P, & Poole, 1999). In this species, ASY males, who are more orange than SY males, have higher 2.4 | Color analysis reproductive success (Richardson & Burke, 1999; Williams, 1988), sug- Following Reudink, Marra, Boag, and Ratcliffe (2009), reflectance gesting plumage coloration has an important signaling function. spectrometry was performed on feathers using an Ocean Optics JAZ spectrometer (FL, USA) attached to a PX-­2 xenon pulsed light source 2.2 | Fieldwork to record reflectance spectra across the bird visual spectrum (300– 700 nm) from which the variables of hue, red chroma, and brightness Fieldwork was conducted in Kamloops, British Columbia, Canada were calculated using the R-­based color analysis program RCLR v2.8 (50.68 N 120.34 W). ASY male Bullock’s orioles were captured in (Montgomerie, 2008). Feathers were mounted on a minimally reflec- mistnets with the use of conspecific playback and decoys or by po- tive black background (<5% reflectance), and the probe was kept at a sitioning nets near oriole feeders. A single tail feather (R3) was col- 90° angle. Ten measurements were taken haphazardly (i.e., semiran- lected from each individual during the summers (May through July) of domly) across the widest region of the tail feather, avoiding the rachis 2012 (n = 18) and 2013 (n = 9). Identification of the sex and age class and feather edges. In between measurements for each bird, read- was made following descriptions provided by Rohwer and Manning ings from dark (sealed, black velvet lined box) and white (Spectralon; (1990) and Pyle (1997). Each bird was banded with a single Canadian Labsphere, NH, USA) standards were used for standardization. Hue, Wildlife Service-­issued aluminum band and a unique combination of which describes the color of the feather (i.e., yellow, orange, red), was three color bands for individual identification. calculated as arctan ([(R510-605 – R320-415)/R320-700]/[(R605-700 – R415510)/R320-700]). Red chroma, which describes the degree of color satu- 2.3 | Stable isotope analysis ration, was calculated as the amount of red light reflected relative to the overall reflectance: (R575-700)/(R300-700). Brightness was calculated To examine diet and habitat during molt, we looked at stable iso- as the mean amount of light reflected across all wavelengths: (mean topes of both carbon (δ13C) and nitrogen (δ15N). Carbon (δ13C) stable R300-700). isotope values can indicate habitat type due to differences in plant water stress and photosynthetic systems (Lajtha & Marshall, 1994). Specifically, low δ13C values indicate a diet containing high amounts of C3 plants (or prey that feeds on C3 plants) and plants experiencing 2.5 | Carotenoid analysis Carotenoid standards (Sigma-­Aldrich, Oakville, ON, Canada) were little water stress, while high δ13C values indicate diets with more C4 made by weighing out canthaxanthin, lutein, or zeaxanthin into a plants and/or greater water stress (Lajtha & Marshall, 1994). Differing volumetric flask and filling with a chloroform (CHCl3) and methanol 13 δ C values can also be indicative of marine versus terrestrial dietary (MeOH) solution (1:1, v/v). Standards were serial diluted, ranging from input (Lajtha & Marshall, 1994). 3 to 100 ppm. Nitrogen (δ15N) stable isotope values are often used in food web Feathers were blotched with hexane and allowed to dry. Once dry, studies, as they are associated with the trophic level of an organism. feathers were cut and weighed into glass vials. Antibumping granules 4 | SPARROW et al. were added along with acidic pyridine (three drops of HCl in 50 ml fixed effects, and individual as a random effect. All statistical analyses pyridine). The mixture was refluxed for 3 hr. Once cooled, the carot- were performed using JMP v12.0 (SAS 2012). enoids were transferred to hexane by adding hexane and mixing, then washing three times with water. The hexane layer was transferred to a new vial and the drying agent sodium sulfate was added. The extraction was evaporated to dryness with nitrogen gas, and then a known amount of CHCl3 and MeOH solution (1:1, v/v) was added. 3 | RESULTS 3.1 | Carotenoid concentration and composition The antioxidant butylated hydroxytoluene (BHT) was also added Average Total feather carotenoid concentration was 2.06 ± 0.86 μg/mg to prevent oxidation of the carotenoids (2 μl of 10 mg/ml BHT per SD for the 21 ASY Bullock’s orioles from which we were able to obtain sample). The extractions were kept in glass vials and stored in a dark carotenoid data. Lutein was the most abundant carotenoid measured in freezer until use. Bullock’s oriole feathers (0.99 ± 0.66 μg/mg SD), followed by zeaxan- Analysis was performed on an Agilent 1200 series HPLC sys- thin (0.76 ± 0.23 μg/mg SD) and canthaxanthin (0.31 ± 0.10 μg/mg SD). tem (Agilent Technologies, Mississauga, ON, Canada) coupled to an Agilent 6530 Accurate-­Mass Quadrupole Time-­of-­Flight (Q-­TOF) spectrometer, equipped with electrospray ionization (ESI) source (gas temperature, 300°C; drying gas, 8 L/min; nebulizer 35 psig; sheath 3.2 | Feather color, carotenoid concentration, and composition gas temperature, 350°C; sheath gas flow, 11 L/min; Vcap, 3500 V). We examined the relationship of (1) carotenoid content and (2) pro- Carotenoids were analyzed in positive ion mode, and mass spectra portion of each carotenoid to the plumage color measurements of were collected between 100 and 600 m/z. Samples of 5 μl of blanks, hue, red chroma, and brightness (Table 1). We observed no relation- extractions, and standards (3–100 ppm) were injected into the LC, with ship between any of the color variables and the concentration of any the flow rate set at 1.0 ml/min. Separation was achieved with a Luna carotenoid; however, the proportion of canthaxanthin was associated C8 (2) column (100 mm × 4.6 mm; 3 μm particle size; Agilent, Canada) with increased brightness and more orange-­shifted hue, while a higher kept at a constant temperature of 60 ± 0.2°C. The autosampler and col- proportion of lutein was associated with lower brightness and more umn were set for 60°C. Mobile phase (A) consisted of pure methanol yellow-­shifted hue. Zeaxanthin was positively associated with bright- and mobile phase (B) was composed of 70:30 v/v methanol with 0.1% ness (Table 1). ammonium acetate. Gradient elution was programed as follows: 95% B at time zero, 80% B at 10 min, 65% B at 15 min, 40% B at 20 min, 10% B at 24 min, and 95% B again at 25 min, with the effluent flowing into the Q-­TOF MS. 3.3 | Feather isotopes (δ13C, δ15N) and carotenoid concentration, composition, and plumage color Data were obtained by taking the area divided by the retention We asked whether carotenoid concentration or the proportion of can- time of the peak of interest. Using the data produced from the stan- thaxanthin, lutein, and zeaxanthin in the feathers was predicted by dards, a calibration curve was produced for each of canthaxanthin, lu- feather isotope values. δ13C was not associated with any measure of tein, and zeaxanthin. Calibration curves were used to determine the carotenoid concentration or composition; however, δ15N was signifi- given carotenoid content in the extraction sample, which in turn was cantly associated with total carotenoid composition and zeaxanthin used to determine the amount of carotenoid in μg per mg of feather extracted. We obtained carotenoid data for n = 21 of the 27 Bullock’s orioles captured in the field. 2.6 | Statistical analysis We used Pearson correlation to examine relationships between brightness, red chroma, and hue and (1) carotenoid concentration and (2) the proportion of each specific carotenoid relative to the total concentration of all three carotenoids. To examine whether carbon or nitrogen stable isotope values predicted either carotenoid content (concentration) or the proportion of each carotenoid, we constructed linear mixed models with carotenoid concentration or the proportion of each carotenoid as the response variable, δ13C, δ15N, and year as fixed effects, and individual as a random effect (due to repeated meas- T A B L E 1 Relationships between tail coloration and carotenoid concentration and composition (n = 21) Brightness Red chroma Hue Total carotenoid r = −.01 p = .67 r = .12 p = .61 r = .17 p = .45 Canthaxanthin r = .43 p = .05 r = −.16 p = .49 r = −.28 p = .21 Lutein r = −.29 p = .20 r = .20 p = .38 r = .32 p = .15 Zeaxanthin r = 28 p = .22 r = −.06 p = .80 r = −.15 p = .51 % Canthaxanthin r = .60 p = .004 r = −.37 p = .10 r = −.46 p = .04 % Lutein r = −.57 p = .007 r = .32 p = .16 r = .43 p = .05 % Zeaxanthin r = .55 p = .01 r = −.64 p = .21 r = −.42 p = .06 urements of three individuals in different years). We created a similar linear mixed model to examine whether carbon or nitrogen stable isotope values predicted feather color (hue, red chroma, brightness), using feather color as the response variable, δ13C, δ15N, and year as Bold values indicate significance at α = 0.05 | 5 SPARROW et al. concentration and marginally, but not significantly, associated with acquisition of an important ornamental signal in a songbird. Because canthaxanthin and lutein concentrations (Table 2; Figure 1). There of the importance of obtaining colorful plumage for both intra-­ and was also an effect of year for total carotenoid, canthaxanthin, and ze- intersexual communication, individuals should seek the highest quality, axanthin concentration. Next, we examined whether δ13C and δ15N most nutrient-­rich environment in which to molt in order to achieve were predictors of feather coloration. Neither brightness nor red optimal plumage coloration. Thus, while the molt-­migration strategy chroma was associated with feather isotope values; however, feathers may have evolved to compensate for low postbreeding food availabil- with low δ15N values had more orange-­shifted hue (Table 2; Figure 2). ity in the arid West (Rohwer et al., 2005), habitat selection by individuals within the general stopover region may also be critical for obtaining high-­quality plumage, which may then carryover to influence individual 4 | DISCUSSION success in subsequent seasons. To our knowledge, this study is the first to demonstrate that condi- stable isotope analysis, correlated with both feather color and carot- tions experienced during molt stopover, occurring hundreds to thou- enoid content; however, the nature of the links between environmen- sands of kilometers from the breeding grounds, correlate with the tal conditions, carotenoid intake, carotenoid metabolism, and color Environmental conditions on the molting grounds, as inferred by T A B L E 2 Relationships between feather isotopes (δ13C, δ15N) and carotenoid concentration and composition (top) and feather color (bottom) δ13C δ15N Year Est: 0.03 ± 0.09 Est: 0.24 ± 0.11 Est: −0.22 ± 0.09 F1,17 = 0.09 F1,17 = 4.60 F1,8.77 = 6.19 Carotenoid Total carotenoid Canthaxanthin Lutein Zeaxanthin % Canthaxanthin % Lutein % Zeaxanthin p = .77 p = .047 p = .035 Est: −0.004 ± 0.01 Est: 0.02 ± 0.01 Est: −0.04 ± 0.01 F1,17 = 0.18 F1,17 = 3.03 F1,5.29 = 17.47 p = .67 p = .10 p = .008 Est: 0.45 ± 1.89 Est: 0.15 ± 0.07 Est: −0.05 ± 0.07 F1,17 = 0.28 F1,17 = 2.73 F1,6.91 = 0.06 p = .61 p = .12 p = .46 Est: −0.007 ± 0.59 Est: 0.07 ± 0.03 Est: −0.12 ± 0.02 F1,17 = 0.09 F1,17 = 6.53 F1,13.86 = 31.89 p = .76 p = .02 p < .0001 Est: −0.002 ± 0.12 Est: −0.005 ± 0.006 Est: −0.01 ± 0.02 F1,16.97 = 0.25 F1,16.87 = 0.86 F1,4.37 = 0.42 p = .62 p = .37 p = .55 Est: 0.01 ± 0.34 Est: 0.01 ± 0.02 Est: 0.02 ± 0.02 F1,16.94 = 0.77 F1,16.99 = 0.77 F1,0.33 = 0.66 p = .39 p = .39 p = .71 Est: −0.007 ± 0.23 Est: 0.007 ± 0.01 Est: −0.02 ± 0.008 F1,17 = 0.73 F1,17 = 0.45 F1,8.46 = 4.15 p = .41 p = .51 p = .07 Est: −0.0005 ± 0.05 Est: −0.001 ± 0.002 Est: −0.02 ± 0.004 Color Brightness Red chroma Hue F1,21.33 = 0.07 F1,22.91 = 0.44 F1,21.61 = 41.18 p = .80 p = .51 p < .0001 Est: −0.004 ± 0.003 Est: −0.004 ± 0.003 Est: 0.01 ± 0.006 F1,22.71 = 2.31 F1,22.98 = 1.24 F1,17.92 = 3.92 p = .14 p = .28 p = .06 Est: 0.0006 ± 0.13 Est: 0.01 ± 0.006 Est: 0.14 ± 0.01 F1,19.62 = 0.01 F1,22.55 = 5.55 F1,21.61 = 176.31 p = .91 p = .03 p < .0001 Bold values indicate significance at α = 0.05 6 | SPARROW et al. F I G U R E 2 Lower δ15N values in tail feathers were associated with more orange-­shifted hue values (associated with lower trophic levels, higher rainfall, and lower temperatures) were associated with more orange-­shifted hue. This seemingly contradictory finding may indicate that dietary carotenoids were not limited on the molting grounds in any habitat, but that individuals with diets (e.g., high fruit intake relative to insects) or residing in habitats that resulted in lower δ15N values were better able to metabolically convert dietary carotenoids into red keto-­carotenoids (McGraw, 2006), perhaps because they were in better condition. Future work is clearly needed to disentangle these complex patterns. We also detected significant year effects; the oriole feathers collected in 2013 (which had been molted in 2012) had lower δ15N values, higher deposits of carotenoids and expressed more orange-­ shifted hue than those collected in 2012 (molted in 2011). One possibility is that birds sampled in 2013 were more reliant on fruit in their diet or molted in cooler, wetter environments. However, September 2011 experienced substantially higher rainfall during the monsoon season than 2012 (14.2 cm compared to 0.97 cm; Tuscon, AZ, USA, http://www.wrh.noaa.gov/twc/monsoon/monsoon.php), and thus, one might predict that the high rainfall year (2011) should have resulted in the opposite pattern. Critically though, the Mexican monsoon region encompasses a vast area and molt-­migrant birds appear to have low molt site fidelity. For example, Chambers et al. (2011) found that molt-­migrants in southeastern Arizona were more likely to be detected in riparian areas in a dry monsoon season, suggesting these birds track habitat and resource availability. Regardless, across years, low δ15N values were associated with more orange-­shifted hue, but lower carotenoid content. Based on the extensive literature on F I G U R E 1 Tail feather δ15N values were positively associated with (a) total carotenoid concentration and (c) zeaxanthin concentration and were marginally, but not significantly, associated with (b) lutein and (d) canthaxanthin concentration American redstarts, overwintering in cooler, wetter habitats is associated with better condition and lower stress level (Marra, Hobson, & Holmes, 1998; Angelier et al. 2009), as well as higher interannual survival (Studds and Marra 2005). One possibility is that carotenoids may not be limited during molt, but rather, orioles molting in cooler, expression will require further investigation. Individuals with higher wetter habitats might be in better condition, enabling them to make total carotenoid and zeaxanthin concentrations had high δ15N values, costly metabolic conversions and produce more colorful plumage (Hill, which are associated with eating at higher trophic levels, low rainfall, 2011, 2014; Hill & Johnson, 2012; Johnson & Hill, 2013). and high temperatures, suggesting that one or more of these factors Carryover effects (events occurring during one season that affect may be associated with carotenoid acquisition. However, lower δ15N performance in a following season) can drive differences in fitness | 7 SPARROW et al. through their impact on survival and reproduction (Harrison, Blount, America; our results suggest that individuals may be able to opti- Inger, Norris, & Bearhop, 2011). For example, in the American red- mize plumage coloration through the selection of high-­quality hab- start, acquisition of a high-­quality winter territory results in earlier itat within this region. Habitat selection and conditions during molt departure from the tropical wintering grounds (Reudink, Marra, may therefore represent an important, but largely unstudied, carry- Kyser, et al., 2009), earlier arrival on the breeding grounds (Marra over effect influencing sexual selection in the subsequent breeding et al., 1998; Norris, Marra, Kyser, Sherry, & Ratcliffe, 2004; Reudink, season. Marra, Kyser, et al., 2009), and consequently, positively influences reproductive success (Norris et al., 2004; Reudink, Marra, Kyser, et al., 2009). While the literature on carryover effects has grown rapidly in AC KNOW L ED G M ENTS recent years (Harrison et al., 2011), very few studies have examined We thank D. Pouw and G. Whitworth for support with the LC/MS, the potential carryover effects of conditions experienced during molt C. France for isotopic analysis, and S. Joly and O. Greaves for help to subsequent seasons, despite the fact that any such effects may with data collection. Funding was provided by Western Economic substantially influence the process of sexual selection. Saino, Szep, Diversification Canada (KKD), and the Natural Sciences and Ambrosini, Romano, and Møller (2004) showed that trans-­Saharan Engineering Research Council of Canada through USRA (KLS) and migratory barn swallows (Hirundo rustica), which undergo a complete Discovery Grants (MWR, KKD). The authors also thank Thompson molt on their wintering grounds in sub-­Saharan Africa, returned to Rivers University’s Departments of Biological Sciences and Chemistry the breeding grounds with shorter tail ornaments following years for funding and support. K. McGraw graciously provided standards with high normalized difference vegetation index (NDVI) values, an and both K. McGraw and J. Hudon provided helpful advice on carot- index indicative of low primary productivity. This finding is particu- enoid analysis. Research for this study was conducted under MWR’s larly important because tail ornament length is directly linked to re- Canadian Master Banding Permit 10834, Canadian Federal Scientific production (Saino et al., 2004). Similarly, ornament production has Collection Permits BC-­12-­0022 and BC-­13-­0002, and TRU Animal been linked to over-­wintering conditions in both pied (Ficedula hy- Use and Protocol 100095. poleuca) and collared flycatchers (Ficedula albicollis), which also molt in sub-­Saharan Africa (Garant, Sheldon, & Gustafsson, 2004; Järvistö, Calhim, Schuett, & Laaksonen, 2016). In North America, yellow warblers (Setophaga petechia) undergo a pre-­alternate molt of body feath- CO NFL I C T O F I NT ER ES T None declared. ers on the wintering grounds; birds overwintering in higher-­quality habitat (inferred via stable isotope analysis) produced more colorful feathers (higher chroma), which are important for mate choice during breeding (Jones, Drake, & Green, 2014). Though American redstarts molt near the breeding grounds, Reudink et al. (2015) demonstrated DATA ACC ES S I B I L I T Y Once published, data for this paper will be available from DRYAD (datadryad.org). that over an 11-­year period, the amount of rainfall experienced during the postbreeding molt was associated with carotenoid-­based tail coloration in the subsequent season. In this species, coloration appears to have important signaling functions during both the breeding (Reudink, Marra, Boag, et al. (2009)) and nonbreeding seasons (Reudink, Studds, Kyser, Marra, & Ratcliffe, 2009; but see Tonra et al., 2014). The limited number of studies examining the potential for carryover effects to influence sexual selection by affecting ornament production is perhaps surprising given the immense body of literature on the function and evolution of plumage coloration. Our study thus represents an important line of evidence demonstrating that conditions experienced during stopover molt may have important carryover effects, which have the potential to influence mate choice and the process of sexual selection. A large body of literature has clearly demonstrated the importance of carotenoid-­based plumage coloration for inter and intrasexual communication. Here, we demonstrate that in Bullock’s orioles, plumage coloration, which is directly linked to carotenoid content, is associated with conditions experienced during molt in the Mexican monsoon region. 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