Consistency of Structural Color Across Molts: the Effects of Environmental Conditions and Stress on Feather Ultraviolet Reflectance Rebecca L

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AmericanOrnithology.org

Volume XX, 2019, pp. 1–17 DOI: 10.1093/auk/ukz019 RESEARCH ARTICLE Downloaded from https://academic.oup.com/auk/advance-article-abstract/doi/10.1093/auk/ukz019/5479445 by Cornell University Library user on 26 April 2019 Consistency of structural color across molts: The effects of environmental conditions and stress on feather ultraviolet reflectance Rebecca L. Windsor,1,2* Gordon A. Fox,2 and Reed Bowman1

1 Avian Ecology Laboratory, Archbold Biological Station, Venus, Florida, USA 2 Department of Integrative Biology, University of South Florida, Tampa, Florida, USA *Corresponding author: [email protected] Submission Date: 27 November, 2018; Editorial Acceptance Date: 21 February, 2019; Published April 25, 2019

ABSTRACT Plumage ornamentation is often considered a signal of fitness, condition, sex, or social status. This theory holds for species with structural UV color, which is influenced by a variety of factors such as environmental pressures during molt or heritability. However, little is known about the consistency of ornamentation and signaling across time in individuals with structural color. We compared juvenile and adult feathers in free-living Florida Scrub-Jays (Aphelocoma coerulescens) to measure UV color change within individuals across molts and to assess possible sources of change. We used multiple imputation to estimate missing data and a combination of pooled estimates and model averaging to infer which parameters explain observed variation in UV color. We also tested whether adult color or relative color change from juvenile to adult plumage predicted acquisition of breeding space. UV color was not consistent across annual molts, as adult feathers reflected significantly less light but greater proportions of UV light than juvenile feathers. Juvenile color was most affected by quality of natal environment and maternal effects whereas adult color was influenced by condition and juvenile color. Adults dosed with corticosterone produced feathers with less UV ornamentation compared to control adults. Feather color did not predict acquisition of breeding space in adults, but females that experienced reductions in UV color across molts were more likely to obtain breeding space, which may reflect sex-specific differences in reproductive strategies in Florida Scrub-Jays. Our evidence suggests that structural color acts as a signal of sex, age, and condition; but ornamentation is only weakly related to acquisition of breeding space and thus unlikely to be under strong sexual selection. Ornamentation may just be one factor among many, such as personality, social dominance, or position in social networks, that determine how jays interact and compete for breeding space. Keywords: Aphelocoma coerulescens, corticosterone, multiple imputation, plumage consistency, scrub-jay, structural color, ultraviolet

Consistencia del color estructural a lo largo de mudas sucesivas: Los efectos de las condiciones ambientales y del estrés en la reflectancia ultravioleta de las plumas

RESUMEN La ornamentación del plumaje es considerada a menudo una señal de adecuación biológica, de condición, de sexo o de estatus social. Esta teoría vale para las especies con color UV estructural, el cuál es influenciado por una variedad de factores como las presiones ambientales durante la muda o la heredabilidad. Sin embargo, poco se sabe sobre la consistencia de la ornamentación y la señalización a través del tiempo en individuos con color estructural. Comparamos plumas de juveniles y adultos libres de Aphelocoma coerulescens para medir cambios a nivel de individuo en el color UV a lo largo de mudas sucesivas y para evaluar posibles fuentes de cambio. Usamos imputación múltiple para estimar los datos faltantes y una combinación de estimaciones agrupadas y promedios de modelos para inferir qué parámetros explican la variación observada en el color UV. También evaluamos si el color del plumaje adulto o el cambio relativo de color del plumaje de juvenil a adulto predijeron la adquisición de espacio reproductivo. El color UV no fue consistente a lo largo de las sucesivas mudas anuales, ya que las plumas de los adultos reflejaron significativamente menos luz pero mayores proporciones de luz UV que las plumas de los juveniles. El color del juvenil estuvo más afectado por la calidad del ambiente de nacimiento y los efectos maternos mientras que el color del adulto estuvo influenciado por la condición y el color del juvenil. Los adultos dosificados con corticosterona produjeron plumas con menos ornamentación UV en comparación con los adultos control. El color de las plumas no predijo la adquisición de espacio reproductivo en los adultos, pero las hembras que experimentaron reducciones en el color UV a lo largo de las mudas tuvieron mayor probabilidad de obtener espacio reproductivo, lo que puede reflejar diferencias específicas de los sexos en las estrategias

reproductivas en A. coerulescens. Nuestra evidencia sugiere que el color estructural actúa como una señal de sexo, edad y condición, pero la ornamentación está solo débilmente relacionada con la adquisición de espacio reproductivo y por ende es poco probable que esté bajo fuerte selección sexual. La ornamentación puede ser simplemente un factor entre varios, como la personalidad, la dominancia social o la posición en las redes sociales, que determinen cómo A. coerulescens interactúa y compite por espacio reproductivo. Downloaded from https://academic.oup.com/auk/advance-article-abstract/doi/10.1093/auk/ukz019/5479445 by Cornell University Library user on 26 April 2019 Palabras clave: Aphelocoma coerulescens, color estructural, consistencia del plumaje, corticosterona, imputación múltiple, ultravioleta

INTRODUCTION of variation and consistency of ornamentation. Hegyi et al. (2015) observed consistency within individuals for Ornamental traits function as signals to conspecifics and carotenoid-based plumage in free-living Great Tits (Parus are often an important component in sexual selection major), but Adamík and Vaňáková (2011) noted consider- (Andersson and Iwasa 1996). However, elaborate orna- able change in ornament size throughout the season. For ments are costly to produce and maintain, and honest-sig- species with structural color, Chaine and Lyon (2015) found naling theory predicts that only high-quality individuals are moderate repeatability of Lark Bunting (Calamospiza mel- able to produce exaggerated ornamental displays (Zahavi anocorys) plumage across years; but in 2 experiments with 1975, Kodric-Brown and Brown 1984). In birds, a grow- free-living Blue Tits (Cyanistes caeruleus), UV reflectance ing body of literature has identified links between plumage within individuals differed across molts. When adults color and signaling (see review in Hill and McGraw 2006), received testosterone implants (Roberts et al. 2009) or had although little is known about the consistency of ornamen- their first clutches removed, forcing them to lay a second tation and signal content throughout individuals’ lifetimes. clutch (Doutrelant et al. 2012), UV reflectance changed Ornamentation for most avian species comes in the between molts and reflected observed differences in hor- form of biochrome pigments, which are continuously pro- mone levels and reproductive output, respectively. duced and deposited in feathers (McGraw 2006a, 2006b); Structural color is sensitive to several components, however, for species with structural plumage color, color including parasite infection (Doucet and Montgomerie results from the coherent scattering of light off feather 2003, Hill et al. 2005, Zirpoli et al. 2013), social environ- microstructures in the medullary layer (Shawkey et al. 2003, ment (Maia et al. 2012), habitat quality (Ferns and Hinsley Prum 2006). These nanostructures require precise con- 2007), stress (Henderson et al. 2013; but see Grindstaff struction and arrangement to produce color, and the num- et al. 2012), and possibly nutritional condition (McGraw ber of protein rods is correlated with feather UV chroma et al. 2002, Johnsen et al. 2003, Grindstaff et al. 2012; but (Shawkey et al. 2003). Birds can see in the UV spectrum see Peters et al. 2011, Henderson et al. 2013) and inter- and are able to differentiate between subtle variations in actions among these factors are possible. McGraw et al. UV color, where even small variations can produce visibly (2002) reported that nutrition influenced structural color- distinct colors (Derim-Oglu and Maximov 1994). Variation ation in Brown-Headed Cowbirds (Molothrus ater) under in structural plumage coloration results from differences in stressful conditions, whereas Peters et al. (2011) found no energy allocated to feather growth. Variation in environ- effects of nutrition on plumage in Blue Tits but did not test mental conditions during molt may result in differences in for a stressful interaction. Organisms respond directly to feather growth, with birds that grow feathers faster hav- environmental challenges through the stress response, and ing more ornamented feathers (Prum 2006, Siefferman glucocorticoids (corticosterone in birds, hereafter CORT) et al. 2008). Habitat quality, diet, social interactions, and break down proteins, altering feather nanostructures and parasite levels all can affect an individual’s energy level and structural color, potentially leading to interactive effects thus feather growth, making it an honest signal since birds between stress and environmental conditions. with large energy budgets can invest more in feather devel- In this study, we examine individual consistency of UV opment (Hamilton and Zuk 1982, Grubb 2006, Hill 2006, reflectance across molts and potential sources of variation in Grindstaff et al. 2012). a free-living population of Florida Scrub-Jays (Aphelocoma Most birds molt at least once a year, and variation in coerulescens). Florida Scrub-Jays are cooperative breed- feather growth implies that plumage quality (e.g., feather ers that have predominantly blue structural plumage and mass, color reflectance, saturation) may not be consistent are sexually dimorphic in the UV spectrum (Bridge et al. from year to year. Furthermore, adult plumage is often of 2007, Siefferman et al. 2008). UV chroma is correlated higher quality than juvenile plumage, and signals may differ with condition (Siefferman et al. 2008) and is associated between plumages of differing life stages (Prum 2006). To with dominance (Tringali and Bowman 2012) in wildland our knowledge, few studies have directly compared feath- juveniles, but is not related to condition and signaling in ers between molts within individuals to examine sources suburban scrub-jays (Tringali and Bowman 2015). Here,

we combine observational and experimental approaches they establish and defend permanent territories. Family to ask (1) does Florida Scrub-Jay UV reflectance change groups consist of a monogamous breeder pair and 0–6 across molts, and (2) how do environmental conditions nonbreeders, which are most often prebreeding offspring and experimentally delivered CORT interact to affect UV from previous seasons, that help with vigilance, territory Downloaded from https://academic.oup.com/auk/advance-article-abstract/doi/10.1093/auk/ukz019/5479445 by Cornell University Library user on 26 April 2019 reflectance? We examine consistency of UV color within defense, and nestling/fledgling provisioning (Woolfenden individuals by comparing their juvenile feathers collected and Fitzpatrick 1984, 1990). Young become nutritionally after preformative molt with their adult feathers collected independent at 80–90 days post-hatch and most remain after first pre-basic molt. Our analysis also focuses on vari- at home during their second year, but begin off-territory ation in UV color resulting from 4 categories of possible forays as early as 90 days, often in groups of juveniles from influences: (1) extrinsic factors including year and resident multiple territories. Mean age of first breeding is 2 or territory habitat composition; (2) social factors, includ- 3 years. ing group size and presence of a stepparent; (3) intrinsic Research was conducted at Archbold Biological Station, factors, including condition and endoparasite loads; and Highlands County, Florida (27.10°N, 81.21°W), where the (4) experimental factors, including administered doses of population of Florida Scrub-Jays has been studied since CORT. We expect UV reflectance will vary between molts 1969 (Woolfenden and Fitzpatrick 1984). As part of the and that considerable variation will be explained by extrin- long-term demography study, all birds are uniquely color- sic, intrinsic, social, and experimental factors. Specifically, banded and the entire population censused monthly, all we predict that jays in better nutritional condition, free nests are found, and all nestlings are banded on day 11. of endoparasite infection, living in large family groups on Fledglings that survive are recaptured once they reach high-quality territories will have more highly ornamented nutritional independence for additional banding, mea- feathers. We also predict that jays dosed with CORT will surements, and blood and juvenile feather collection after experience reductions in UV reflectance. preformative molt. We scanned blood samples to estimate Although UV color is associated with dominance in ju- endoparasite abundance (described below), and the 2 out- venile scrub-jays (Tringali and Bowman 2012), it does not ermost rectrices were collected for reflectance measure- predict adult reproductive success (Tringali et al. 2015), pos- ments. Florida Scrub-Jays frequently perform a “lateral sibly because the changes from juvenile to adult plumages display” during territorial encounters with one another, may vary among individuals. This suggests a third question: during which opposing birds circle one another on the Does adult color or the direction of color change from juve- ground, with tail feathers spread and tilted toward their nile to adult plumages predict future reproductive success? rivals (Woolfenden and Fitzpatrick 1996). This behavior We predict that highly ornamented birds or those with the suggests that tail color is an important signal during dom- greatest gains in UV ornamentation across molts will be inance displays and could affect competition for breeding more likely to transition to breeding than less-ornamented space. Our analyses are based on 3 annual cohorts of jays members of their cohorts. While sexual selection may not hatched in 2013, 2014, and 2015. explain UV sexual dimorphism in juvenile jays (Tringali and Bowman 2012), it may yet play a role in adult coloration if Corticosterone Administration breeders are more highly ornamented than nonbreeders. In the year following juvenile feather collection, we admin- Variation in the consistency of ornamentation across istered CORT to surviving individuals at the onset of their molts could have large implications on how studies eval- first pre-basic molt into adult plumage, starting in July. uate signaling and reproductive fitness, particularly for One-third of each annual cohort received doses of CORT, long-lived species. Many studies have shown that UV color one-third were provisioned undosed waxworms to control predicts condition, dominance, mate quality, behavior, re- for effects of waxworm supplementation, and one-third productive success, or hormone levels, but several of these were neither visited nor supplemented. Jays were randomly studies contradict one another, even within species. We assigned to each treatment group, ensuring that siblings hope to tease apart the interplay of social, environmental, within a family group received different treatments and and physiological effects on color by providing a compre- that male:female ratios were relatively even and consistent hensive modeling framework of UV color and change, thus between treatment groups. providing new insights into what structural color signals. Individuals were dosed with CORT (#C2505; Sigma Aldrich, St. Louis, Missouri, USA) without capture by METHODS provisioning them waxworms that had been injected with a solution of crystalline CORT suspended in peanut oil Study Species and Juvenile Feather Collection (Breuner et al. 1998, Schoech et al. 2007). CORT was dis- Florida Scrub-Jays are endemic residents of the fire-main- solved in heated peanut oil to a concentration of 0.8 mg tained oak scrub habitats of peninsular Florida where mL−1, and 25 µL were injected into each waxworm with a

100-µL Hamilton syringe, for a CORT dose of 20 µg wax- for inspection of plasma for endoparasites. We inspected worm−1. Sham waxworms were injected with 25 µL of the buffy coat layer and plasma portions of centrifuged peanut oil. Waxworms were prepped each morning prior capillary tubes under 20–40× magnification to count the to dosing and kept on ice to reduce their mobility and pre- number of microfilariae and trypanosomes following Downloaded from https://academic.oup.com/auk/advance-article-abstract/doi/10.1093/auk/ukz019/5479445 by Cornell University Library user on 26 April 2019 vent oil from seeping out. Robbins et al. (C. B. Robbins personal communication). We administered CORT-injected or sham-injected Microfilariae and trypanosomes continue to move within waxworms twice a day for 3 weeks beginning in mid-July or away from the buffy coat after being centrifuged, allow- when rectrices began growing during molt (Bancroft and ing for easier detection. A video of active microfilariae in Woolfenden 1982). The first dose was administered early in Florida Scrub-Jay plasma can be viewed online (https:// the morning (0630–0900 hours) and the second dose was www.youtube.com/watch?v=lFIF4WVwLd0). Only full administered either late morning (0900–1200 hours) or capillary tubes were scanned for parasites, and each tube early afternoon (1300–1500 hours) depending on weather was rotated and scanned 3 times. We examined 3.45 ± 1.51 or our ability to relocate individuals. This dosing method tubes per bird. This methodology increases parasite detec- has been validated in adult Florida Scrub-Jays in previous tion probability compared to traditional blood smears be- work: 20 µg doses increase plasma CORT concentrations cause larger volumes of blood can be searched (~5 µL for within 12 min, which return back to baseline levels within smears compared to ~65 µL per capillary tube), resulting an hour (Schoech et al. 2007). Thus, we ensured that the in fewer false negatives (C. B. Robbins personal communi- second dose was administered no less than 2 hr after the first cation). Additionally, this method allows for more accurate dose. Florida Scrub-Jays at Archbold Biological Station are estimations of parasite density because each capillary tube relatively tame, and doses were given by locating the target contains a known volume of blood compared to drops used groups, bringing them close by throwing peanuts, and then in blood smears. To minimize the effects of diel fluctua- tossing the appropriate treatment waxworm to the target tions on parasite levels (Boughton et al. 1938), birds were jay. Most individuals swallowed the waxworm upon re- trapped at 0700–1100 hours. All blood samples were kept ceiving it, but we observed those that flew off to ensure on ice until they could be centrifuged and processed in they were not caching or provisioning it to fledglings. If the lab. caching or provisioning occurred, we provided additional waxworms until the target individual ate it. Because most Habitat Mapping and Territory Acquisition of the jays dosed were helpers, it was not uncommon for Jay territories are mapped yearly during the breeding individuals to be off-territory foraying, thus we were not seasons at Archbold using playback, behavioral observa- always able to administer 2 doses to each bird every day. tions, and aerial maps (Woolfenden and Fitzpatrick 1984). Our dose administration was successful 87.5 ± 12.3% of the Territory delineations are then digitized in ArcGIS and time, and dosing amounts are further analyzed below. overlaid with mapped vegetation and fire history shape- files, which allowed us to assess habitat quality per resident Blood and Adult Feather Collection territory, per year. The following January and February (prior to the breeding As part of the monthly population censuses and breed- season), as birds entered their second full year and the one ing observations conducted for the long-term demog- in which about 50% of all helpers transition to breeders, raphy study, changes in the breeding pairs and creation we recaptured surviving individuals for measurements, of new territories are constantly monitored. We noted if blood sampling, and adult feather collection. These feath- any individuals in the 2013–2015 yearly cohorts acquired ers served as the second samples for each individual, allow- a breeder position. Birds that acquired a breeder position ing for pair-wise comparisons between juvenile and adult (i.e. defended a territory as the dominant bird of their plumages. Adults were trapped in the winter to minimize sex) were considered breeders for the study, regardless of the risk that structural plumage was affected by wear or whether they produced eggs that same year. Individuals bacteria (Shawkey et al. 2007, Avilés et al. 2008), and to en- that did not acquire breeding space were listed as helpers sure that our collection and UV measurements were close or denoted as missing. Breeding status was recorded at the to the time the birds were competing for breeding space. end of May since few individuals acquire breeding space Individuals were trapped using peanut-baited Potter after this point in the breeding season. traps. We collected 200–400 µL of blood (not exceeding 1% of an individual’s body mass) by veinipuncture of the Spectroscopy brachial vein to measure hematocrit and scan for endo- We quantified UV reflectance of the 2 outermost rec- parasites. Blood was collected with heparinized capillary trices with an Ocean Optics FLAME-S Spectrometer, a tubes, which were centrifuged in the lab at 3,000 rpm for DH-2000 deuterium halogen light source, and a bifur- 5 min to separate plasma from red blood cells and allow cated fiber optic probe (Ocean Optics, Dunedin, Florida,

USA) (Montgomerie 2006). Following previous reflectance Intrinsic factors. Three variables reflecting nutritional measurement protocols on Florida Scrub-Jays (Siefferman condition were included in our models of UV color: mass et al. 2008, Tringali and Bowman 2012), we used a rubber at time of capture, day 11 nestling mass standardized to mount fastened to the probe to maintain a 90° measuring mean mass for each brood, and blood hematocrit. Body Downloaded from https://academic.oup.com/auk/advance-article-abstract/doi/10.1093/auk/ukz019/5479445 by Cornell University Library user on 26 April 2019 angle to the feather at a distance of 0.5 cm. Integration time mass is positively correlated with feather growth bar width was set at 7 ms, 20 scans were averaged per reading, and (Grubb et al. 1998) and growth bar width is also positively boxcar smoothing was applied. We measured and averaged associated with UV chroma in scrub-jays (Siefferman et al. reflectance spectra at 3 points on each feather and summa- 2008), suggesting that larger birds may grow feathers faster rized reflectance by calculating 3 color variables that are and those feathers have increased ornamentation. Capture standard among studies on structural color: mean bright- mass and standardized nestling mass were highly corre- ness (mean of the summed percent of light reflected off lated, but we decided to maintain both variables in each Ʃ the feather between 300 and 700 nm, R300–700 / nw), UV color model to compare them as part of our exploratory chroma (ratio of reflectance in the UV range to the total analysis. Hematocrit, the proportion of red blood cells to Ʃ Ʃ reflectance, R300–400 / R300–700), and hue (wavelength total volume of blood, was measured once blood samples of peak reflectance, λRmax) (Andersson and Prager 2006, had been centrifuged. Data are conflicting across studies Montgomerie 2006, 2008). as to whether hematocrit accurately represents condition or energy expenditure and how that may affect plumage Color Model Independent Variable Selection (Fair et al. 2007), but some evidence suggests that hemato- Extrinsic factors. We included 2 characteristics of the crit positively covaries with ornamental tail length in Barn resident territory to indicate habitat quality in our mod- Swallows (Hirundo rustica) (Saino et al. 1997). els: total area of oak scrub and total area of habitat in We included 2 variables indicating endoparasite infec- optimal time since fire (TSF, 2–9 yr post-fire). Jays de- tion. Fifteen species of helminth worms have been docu- pend on oak scrub for nesting, acorns, and protection mented in Florida Scrub-Jays (Kinsella 1974) and recent from predators, and area of oak scrub is positively cor- work has focused on 2 focal hemotropic endoparasites: a related with overall territory size and juvenile body mass filarid worm (Aproctella sp.) and trypanosomes (species (Mumme et al. 2015), making it a good indicator of high- unknown) (C. B. Robbins personal communication). Thus, quality habitat. Furthermore, suitable habitat structure we included microfilariae and trypanosome infection for Florida Scrub-Jays is influenced by post-fire succes- (presence/absence) as independent categorical variables. sion, and population growth is lowest in very open habitat We chose to categorize infection status because many indi- (<2 yr post-fire) and in dense overgrown habitat (≥10 yr viduals had no infection of either parasite. post-fire) (Woolfenden and Fitzpatrick 1984, Breininger Experimental factors. Our experimental variable was and Schmalzer 1990). CORT treatment group (treatment, sham, and control), Social factors. We included 2 group composition param- which we included in models of adult color. CORT is neg- eters: family group size and presence of a same-sex steppar- atively correlated with body mass in Florida Scrub-Jays ent. Florida Scrub-Jays form strict dominance hierarchies (Schoech et al. 1997), suggesting that CORT and condition based on sex, age, and breeding status (Woolfenden and may interact and affect plumage. While some treatment Fitzpatrick 1977), and birds in large families may experi- and sham control birds did not receive every dose (dosing ence more frequent dominance interactions or face greater success = 87.5 ± 12.3%), dosing success was similar across competition for food. Because jays begin molting rectrices all 3 years and total dose amount was not correlated with in July (Bancroft and Woolfenden 1982), we used group color change when controlling for year. size from the July population census to best indicate group dynamics when individuals are growing their rectrices. We Statistical Analysis also included an interaction between area of oak scrub and All continuous variables were scaled and centered, and family group size because juvenile mass in territories with analyses were conducted with R 3.5.0 (R Development small areas of oak scrub is negatively influenced by a large Core Team 2018). To examine consistency of color, we cal- number of nonbreeders, presumably because they increase culated relative percent change in color within individuals competition for limited resources (Mumme et al. 2015). In and used paired t-tests to determine if juvenile color was cases where a breeder is replaced, helpers often receive ab- significantly different than adult color within individuals. normally high aggression levels from replacement breed- We used Pearson’s correlations to determine whether juve- ers, particularly from jays of the same sex (Woolfenden nile and adult color were correlated as a way to assess if and Fitzpatrick 1977, Goldstein et al. 1998). Presence of a an individual’s plumage color remains consistent through- same-sex stepparent within each focal individual’s group out its lifetime relative to its conspecifics, or if color is was also based on the July census. more plastic and responsive to environmental factors.

We compared relative change in color between males and be a stronger influence on color. Because jays are sexually females using Welch’s t-tests to determine if color change dimorphic in the UV spectrum (Siefferman et al. 2008), we differs between sexes. included sex as a categorical variable in all models as well. Because of occasional difficulty acquiring field samples Yearly effects were included as a categorical rather than Downloaded from https://academic.oup.com/auk/advance-article-abstract/doi/10.1093/auk/ukz019/5479445 by Cornell University Library user on 26 April 2019 and limited vegetation mapping, we found that 38.8% of random parameter because our study occurred over only the juvenile data and 29.5% of the adult data were miss- 3 yr (Bolker 2015). In models of adult color, we included ing select values in parasite counts, hematocrit, mass, juvenile color as an independent variable to investigate the area of oak scrub, or area of habitat with the optimal TSF. degree to which adult color is predicted by natal conditions Specifically, of the 209 juveniles included in our study, 70 vs. current environmental conditions. Despite the lack of (33.5%) did not have oak scrub or TSF habitat data, 15 evidence for sex-linked color inheritance in Florida Scrub- (7.2%) were missing endoparasite data, 8 (3.8%) did not Jays, maternal effects explain considerable variation in have hematocrit data, and 2 (1%) were missing mass data. juvenile color (Tringali et al. 2015). We were unable to in- Of the 78 adults included in our study, 21 (26.9%) were clude random variables in models that were averaged due missing oak scrub and TSF data, 2 (2.6%) were missing en- to limitations with the package (MAMI: Schomaker 2017) doparasite data, one (1.3%) was missing hematocrit data, we used, so Mother ID was included only in the pooled full and one (1.3%) was missing mass data. A few of these indi- models. viduals were missing both habitat and endoparasite data, While the subject of missing data and how to deal with it but most birds with missing data were only missing values is not new to ecologists (Nakagawa and Freckleton 2008), for one or two variables. While endoparasite, hematocrit, we recognize that multiple imputation is not yet common and mass data were missing randomly because of handling and also ran our 6 color models with only complete records issues or sampling error, habitat data were missing because for comparison (see Supplementary Material Tables S1– of a gap in our GIS vegetation map layer on the property. S4). The rigor of multiple imputation and the resulting Such missing data is not random, since it depends on an pooled estimates provide a more robust analysis of how unrelated variable (i.e. incomplete GIS coverage; Graham environmental variables may affect UV color than models 2009). The deletion of samples with missing data results in that would exclude ~35% of all individuals measured. The a loss of information and statistical power, and also intro- qualitative similarity between the conclusions drawn using duces potential bias in parameter estimates when data are imputation and those drawn by using only complete cases not missing at random (Nakagawa and Freckleton 2008, are notable and demonstrate the strength of these analyses. Graham 2009, Nakagawa 2015). To avoid removing so For analyses on color and breeding space acquisition, we many birds with missing data and the potential associated used GLMs (binomial distribution, logit link function) to biases from these deletions, we used multiple imputation test if adult color or the relative change in color from juve- by chained equations (MICE) to estimate missing values, nile to adult plumage predicted breeding fate (breeders vs. which were calculated using predictive mean matching helpers). Our initial models indicated a strong interaction (van Buuren and Groothuis-Oudshoorn 2011). We fol- between sex and UV color (see Supplementary Material lowed the recommendations of White et al. (2011) and Table S4), so we decided to run separate models for males created a number of iterated data sets that matched the and females to improve interpretation of these trends percentage of missing values (Nakagawa and Freckleton (Milliken and Johnson 1984). While we excluded missing 2008). birds (n = 7) from these analyses, we also ran multinomial Each color variable was modeled separately, resulting in models with missing birds included and all findings were 6 models total: 3 of juvenile color and 3 of adult color. We consistent with the binomial GLMs. approached the modeling from 2 exploratory directions due to limitations associated with ΔAIC model comparison for imputed datasets. First, we created full models RESULTS with all 10 independent variables and then pooled and averaged estimates across all imputed datasets according We measured 209 juveniles from the 2013–2015 cohorts, to Rubin’s Rules (Rubin 1987). Second, we performed post- of which 78 survived and were recaptured as adults. imputation linear model selection (Gaussian distribution) Most reflectance was in the UV-blue area of the spec- for each color model using AIC and model averaging– trum, and hue did not differ significantly between males based variable importance (VI), which sums the weights of and females as juveniles (difference between males and all candidate models that contain each particular variable females [Δ] = 4.96 nm, t204 = −1.34, P = 0.18) nor as adults

(Burnham and Anderson 2002, Schomaker and Heumann (Δ = 8.40 nm, t73 = −0.98, P = 0.33; Table 1). Females were 2014, Schomaker 2017). This VI measure ranges between brighter and reflected significantly more light than males

0 (least important) to 1 (most important), and we consid- at both ages (juvenile: Δ = 0.44%, t205 = −4.39, P < 0.001; ered any variable with a VI measure of 0.5 or greater to adult: Δ = 0.42%, t75 = −2.88, P = 0.005), but males reflected The Auk: Ornithological Advances XX:1–17, © 2019 American Ornithological Society R. L. Windsor, G. A. Fox, and R. Bowman UV color consistency across molts 7

TABLE 1. Mean ( ± SD) color measures of juvenile and adult Florida Scrub-Jay rectrices. Juvenile colors represent all captured individuals for the 2013–2015 cohorts (n = 209); adult colors represent surviving individuals recaptured after their first pre-basic molt (n = 78). Age Sex n Brightness (%) Chroma Hue (nm)

Juvenile Female 103 9.29 ± 0.68 0.251 ± 0.008 432.99 ± 27.86 Downloaded from https://academic.oup.com/auk/advance-article-abstract/doi/10.1093/auk/ukz019/5479445 by Cornell University Library user on 26 April 2019 Juvenile Male 106 8.85 ± 0.77 0.253 ± 0.008 428.04 ± 25.46 Adult Female 38 8.96 ± 0.65 0.260 ± 0.009 403.49 ± 40.61 Adult Male 40 8.54 ± 0.62 0.265 ± 0.009 395.09 ± 34.58

a significantly greater proportion of UV light than did females (juvenile: Δ = 0.002, t207 = 2.11, P = 0.04; adult:

Δ = 0.004, t76 = 2.22, P = 0.03; Table 1). As in other studies of UV plumage color (Shawkey et al. 2003, Siefferman et al. 2008), chroma was negatively correlated with hue (Pearson correlation r = −0.73, P < 0.001, n = 285). In contrast with Siefferman et al. (2008), we observed a negative correlation between chroma and brightness (r = −0.27, P < 0.001, n = 285). Brightness and hue were not correlated. For additional details on reflectance curves and relationships among color variables in Florida Scrub-Jays, see Siefferman et al. (2008) or Tringali and Bowman (2012).

Consistency of Color We recaptured 78 of 85 surviving individuals for adult feather collection; recapture rates did not differ between males (n = 40 of 44, 90.9%) and females (n = 38 of 41, 92.7%). Juvenile color was significantly different than adult color for all 3 color variables: adult feathers were significantly less bright than juvenile feathers (mean of FIGURE 1. Relative change in Florida Scrub-Jay rectrix color the differences between adults and juveniles [Δ] = 0.38%, within individuals for males and females. The dashed line t77 = 5.52, P < 0.001), with a greater proportion of light indicates zero change in color, or consistent UV reflectance across molt. Males and females experienced similar changes in color in the UV range (Δ = −0.009, t77 = −6.99, P < 0.001) and a significant shift toward shorter hues (Δ = 30.66 nm, across molts, although males experienced marginally significant increases in chroma compared to females (t68 = 1.85, P = 0.07). t77 = 6.02, P < 0.001; Figure 1). Males and females did not differ in their patterns of relative change from juvenile to adult plumage for any color component, except for Factors Influencing Variation in UV Color a trend for chroma to increase more in males (average We found that sex, year, and mother ID were strong pre- change = 4.68%) than in females (average change = 2.74%) dictors in all 3 models of juvenile color. Variation in juve-

(t68 = 1.85, P = 0.07). nile brightness was best explained by juvenile body mass, Brightness was positively correlated between juve- hematocrit, parasite infection, group size, and area of nile and adult plumages for all birds (t76 = 6.49, P < 0.001, habitat with optimal TSF (Table 2). Our juvenile bright- r = 0.60), both sexes (females: t36 = 4.16, P < 0.001, r = 0.57; ness model also identified area of oak scrub and the inter- males: t38 = 4.31, P < 0.001, r = 0.57), and all 3 yearly cohorts action between group size and scrub as minor influences

(2013: t35 = 4.58, P < 0.001, r = 0.61; 2014: t18 = 2.94, P = 0.01, on juvenile brightness, although these effects had lower r = 0.57; 2015: t19 = 2.45, P = 0.03, r = 0.49) (Figure 2). VI measures. Brighter juveniles were heavier, with greater Chroma was not correlated for all birds, but was correlated hematocrit and free of both microfilarid and trypanosome for females across all years (t36 = 2.12, P = 0.04, r = 0.33); infection, from large families and natal territories with less no correlation was found for males. Chroma (t35 = 3.41, habitat in the optimal TSF interval, but more oak scrub.

P = 0.002, r = 0.50) and hue (t35 = 2.05, P = 0.05, r = 0.33) Variation in juvenile chroma was best explained by juvenile were significantly correlated for individuals in the 2013 co- mass and an interaction between group size and area of hort, but not for birds in the 2014 and 2015 cohorts. We oak scrub (Table 3). Hematocrit, trypanosome infection, observed no correlations between juvenile and adult hue and area of optimal TSF were also maintained in the juve- for either sex. nile chroma model but had smaller estimates and lower VI

The Auk: Ornithological Advances XX:1–17, © 2019 American Ornithological Society 8 UV color consistency across molts R. L. Windsor, G. A. Fox, and R. Bowman Downloaded from https://academic.oup.com/auk/advance-article-abstract/doi/10.1093/auk/ukz019/5479445 by Cornell University Library user on 26 April 2019

FIGURE 2. Juvenile vs. adult rectrix color in male and female Florida Scrub-Jays. Each point represents one individual (males n = 40, females n = 38). The dashed line indicates a 1:1 ratio where juvenile color is identical to adult color within individuals, and the solid and dotted lines represent significant correlations for males and females, respectively. Juvenile and adult brightness were highly correlated for both males and females, but chroma measures were only correlated in females. Overall, individuals experienced increases in chroma across molt and corresponding shifts to shorter hues. measures. Variation in juvenile hue was best explained by parasite infection, area of habitat with optimal TSF, group brood-standardized nestling mass, an interaction between size, and its interaction with area of scrub were selected group size and area of oak scrub, and area of optimal TSF in the models, but had smaller estimates and lower VI (Table 4). Because chroma and hue covary, similar factors measures (Tables 3–4). Adults that were more highly orna- influenced them: more highly ornamented juveniles with mented, with greater chroma and shorter hues, were more greater chroma and shorter hues were lighter and from highly ornamented as juveniles, larger than their brood large families, in natal territories with little oak scrub habi- mates, and resided in territories with more oak scrub habitat but more area in the optimal TSF interval. Individuals tat. More ornamented adults also tended to have greater with greater chroma also tended to have trypanosome hematocrit, parasite infections, and resident territories infections and less hematocrit, though these effects were with larger families and more optimal TSF habitat, though not as prominent in the models. Juvenile models run on these effects were not as prominent in the models. CORT data with complete cases (n = 128) showed similar results, treatment also influenced adult chroma. We observed but also suggested that optimal TSF may play a greater role lower chroma in birds that received doses of CORT com- in juvenile brightness than indicated in multiple imputa- pared with birds from control and sham groups. We found tion (see Supplementary Material Tables S1–S3). Overall, no evidence of an effect of presence of stepparents on model estimates from both imputed and complete case adult feather color. Adult models run on data with com- datasets were of similar magnitude, resulting in similar plete cases (n = 55) showed similar results, but suggested conclusions. that CORT treatment may have a greater effect on adult Fewer independent variables were selected and main- chroma and hue than indicated in the imputed models (see tained in our models of adult color compared with our Supplementary Material Tables S1–S3). Overall, model models of juvenile color (Tables 2–4). Year was a strong estimates from both imputed and complete case datasets predictor of adult chroma and hue, but not brightness. were of similar magnitude, resulting in similar conclusions. Mother ID was not a predictor of adult color, although juvenile color was positively related to adult color for all 3 adult Effects of Color on Acquisition of Breeding Space models. In addition, variation in adult brightness was best We monitored breeding fate for 71 of the 78 adults we explained by adult body mass, microfilarid infection, area recaptured for feather collection. Seven individuals disap- of habitat with optimal TSF, and area of oak scrub, which peared from the study population before the May breeding was maintained in the model but had a lower VI measure. season cutoff, and these individuals were removed from bi- Brighter adults tended to be the brightest juveniles, were nomial GLMs of breeding fate. The proportions of males lighter and infected with microfilarids, and resided in ter- and females acquiring breeding space did not differ (males: ritories with more habitat with optimal TSF and more 25 of 38, 65.8%; females: 20 of 33, 60.6%) and in both sexes, oak scrub (Table 2). Models for adult chroma and hue more acquired breeding space than remained as helpers. were again similar, best explained by brood-standardized Neither adult brightness, chroma, nor hue was associated nestling mass and area of oak scrub habitat. Hematocrit, with acquisition of breeding space for either sex. Within

The Auk: Ornithological Advances XX:1–17, © 2019 American Ornithological Society R. L. Windsor, G. A. Fox, and R. Bowman UV color consistency across molts 9 — — — — — Downloaded from https://academic.oup.com/auk/advance-article-abstract/doi/10.1093/auk/ukz019/5479445 by Cornell University Library user on 26 April 2019 0.43 0.07 0.18 0.8 0.24 0.3 0.37 0.31 0.5 1 0.36 0.28 0.66 0.39 1 0.18 0.47 0.64 0.54 0.97 0.93 0.93 0.99 0.36 1 Variable importance Variable — — — — — — — — — — — 95% CI 0.81 0.46 to 0.82 0.16 to 0.66 0.08 to 0.29 0.04 to 0.38 0.09 to 0.19 −0.14 to 0.46 −0.02 to 0.31 −0.51 to 0.15 −0.11 to −0.36 to −0.001 −0.36 to 0.21 −0.19 to −1.13 to −0.47 −1.13 to −0.88 to −0.16 −0.88 to 0.10 −0.17 to 0.12 −0.11 to 0.09 −0.29 to 0.16 −0.09 to 0.58 −0.15 to −0.46 −1.02 to Model selection — — — — — — — — — — — 0.03 0.22 0.64 0.02 0.01 0.007 0.03 0.49 0.37 0.17 0.24 0.22 −0.10 −0.18 −0.80 −0.52 −0.03 −0.10 −0.74 Not included Not included Estimate P 0.41 0.67 0.95 0.50 0.98 0.62 0.16 0.09 0.99 0.47 0.55 0.09 0.77 0.28 0.10 0.001* 0.92 0.97 0.002* 0.33 0.72 0.09 0.20 0.002* 0.03* 0.002* 0.42 0.001* 0.14 <0.001* <0.001* <0.001* 95% CI 0.80 0.44 to 0.57 0.16 to 0.78 0.18 to 0.58 0.03 to 0.30 0.07 to 0.39 0.10 to 0.60 −0.25 to 0.55 −0.35 to 0.25 −0.27 to 0.35 −0.17 to 0.56 −0.54 to 0.33 −0.55 to 0.14 −0.85 to 0.43 −0.03 to 0.49 −0.49 to 0.31 −0.14 to 0.48 −0.26 to 0.06 −0.80 to 0.16 −0.22 to 0.29 −0.09 to 0.03 −0.41 to 0.56 −0.51 to 0.47 −0.45 to −1.18 to −0.53 −1.18 to −0.90 to −0.20 −0.90 to 0.07 −0.20 to 0.20 −0.14 to 0.03 −0.37 to 0.24 −0.05 to 0.07 −0.16 to 0.61 −0.09 to −0.98 to −0.44 −0.98 to Pooled full model Pooled 0.18 0.10 0.09 0.007 0.20 0.002 0.08 0.11 0.62 0.10 0.03 0.009 0.36 0.03 0.09 0.48 0.30 0.19 0.24 0.26 −0.008 −0.11 −0.36 −0.37 −0.03 −0.19 −0.85 −0.55 −0.07 −0.17 −0.05 −0.71 Estimate Variable Intercept Intercept Hematocrit Hematocrit Group size * Area of oak scrub * Area size Group (treatment) treatment CORT (sham) treatment CORT Area of oak scrub (ha) Area (2014) Year (2015) Year Mother ID (ha) TSF of optimal Area (yes) Stepparent size Group Trypanosome infection (non-infected) infection Trypanosome Microfilarid infection (non-infected) color Juvenile Standardized nestling mass (g) Standardized Adult mass (g) Adult Sex (male) (2015) Year Mother ID (2014) Year Group size * Area of oak scrub * Area size Group Area of oak scrub (ha) Area (ha) TSF of optimal Area size Group Trypanosome infection (non-infected) infection Trypanosome Microfilarid infection (non-infected) Standardized nestling mass (g) Standardized Juvenile mass (g) Juvenile Sex (male) Linear mixed model (Gaussian distribution) selection results for parameters that explain juvenile and adult brightness. Variables were centered and scaled prior centered were Variables and adult brightness. explain juvenile that parameters distribution) selection model (Gaussian for results Linear mixed TABLE 2. TABLE Adult Juvenile Age to multiple imputation and modeling. Variable importance measures were calculated during model selection. calculated Mother included in the pooled full model as a random importance ID was were measures Variable and modeling. multiple imputation to not calculated. and CI were estimates not chosen during with dashes since model selection denoted are Variables with an asterisk. denoted are parameters Significant variable. and 78 adults. 209 juveniles was Sample size

The Auk: Ornithological Advances XX:1–17, © 2019 American Ornithological Society 10 UV color consistency across molts R. L. Windsor, G. A. Fox, and R. Bowman — — — Downloaded from https://academic.oup.com/auk/advance-article-abstract/doi/10.1093/auk/ukz019/5479445 by Cornell University Library user on 26 April 2019 — — 0.99 0.87 0.43 0.56 0.34 0.46 0.99 0.47 0.7 0.55 1 0.89 0.42 0.91 0.45 0.9 0.29 0.54 0.43 0.29 0.41 0.63 0.2 0.53 1 Variable importance Variable — — — — 95% CI 0.91 0.30 to 0.39 0.09 to 0.86 0.07 to 0.42 0.05 to 0.42 0.06 to 1.74 0.82 to 0.57 −0.15 to −0.35 to −0.04 −0.35 to 0.09 −0.23 to 0.25 −0.47 to 0.26 −0.12 to 0.15 −0.21 to 0.08 −0.31 to −0.90 to −0.12 −0.90 to −1.10 to −0.36 −1.10 to 0.10 −0.82 to 0.13 −0.18 to 0.11 −0.18 to 0.20 −0.66 to 0.09 −0.08 to 0.23 −0.15 to 0.36 −0.18 to 0.13 −0.17 to 0.29 −0.49 to 0.33 −0.26 to 0.24 −0.60 to Model selection — — — — 0.21 0.60 0.07 0.24 0.46 0.24 0.24 0.005 0.04 0.09 0.04 1.28 −0.20 −0.07 −0.11 −0.03 −0.11 −0.51 −0.73 −0.36 −0.03 −0.03 −0.23 −0.02 −0.10 −0.18 Estimate Not included Not included P 0.37 0.02* 0.28 0.12 0.56 0.16 0.41 0.03* 0.75 0.21 0.03* 0.003* 0.10 0.004* 0.18 0.002* 0.27 0.007* 0.96 0.25 0.95 0.52 0.45 0.71 0.14 0.35 0.58 0.33 0.98 <0.001* <0.001* <0.001* 95% CI 0.89 0.32 to 0.34 0.02 to 0.68 0.34 to 1.00 0.19 to 0.47 0.11 to 0.40 0.06 to 1.76 0.84 to 0.56 −0.21 to −0.34 to −0.03 −0.34 to 0.05 −0.19 to 0.03 −0.22 to 0.38 −0.21 to 0.09 −0.57 to 0.30 −0.12 to 0.17 −0.24 to 0.06 −0.25 to −0.79 to −0.04 −0.79 to −0.87 to −0.18 −0.87 to 0.08 −0.89 to 0.06 −0.34 to 0.08 −0.28 to 0.40 −0.42 to 0.14 −0.55 to 0.22 −0.20 to 0.33 −0.17 to 0.36 −0.16 to 0.37 −0.55 to 0.05 −0.38 to 0.21 −0.57 to 0.29 −0.51 to 0.62 −0.21 to 0.59 −0.58 to Pooled full model Pooled 0.61 0.09 0.18 0.09 0.51 0.60 0.29 0.23 0.007 0.08 0.10 0.21 1.30 0.007 0.18 −0.19 −0.07 −0.10 −0.24 −0.03 −0.10 −0.41 −0.52 −0.40 −0.14 −0.10 −0.01 −0.20 −0.09 −0.16 −0.18 −0.11 Estimate Intercept Sex (male) mass (g) Juvenile Standardized nestling mass (g) Standardized Hematocrit Microfilarid infection (non-infected) (non-infected) infection Trypanosome size Group (ha) TSF of optimal Area of oak scrub (ha) Area Group size * Area of oak scrub * Area size Group (2014) Year (2015) Year Mother ID Intercept Sex (male) mass (g) Adult Standardized nestling mass (g) Standardized Hematocrit color Juvenile Microfilarid infection (non-infected) (non-infected) infection Trypanosome size Group (yes) Stepparent (ha) TSF of optimal Area of oak scrub (ha) Area of oak scrub * Area size Group CORT treatment (treatment) treatment CORT (2014) Year (sham) treatment CORT (2015) Year Mother ID Variable Linear mixed model (Gaussian distribution) selection results for parameters that explain juvenile and adult chroma. Variables were centered and scaled prior to and scaled prior to centered were Variables and adult chroma. explain juvenile that parameters distribution) model (Gaussian selection for results Linear mixed TABLE 3. TABLE Juvenile Adult multiple imputation and modeling. Variable importance measures were calculated during model selection. calculated Motherthe pooled full model as a random included in importance ID was were measures Variable and modeling. imputation multiple not calculated. and CI were estimates not chosen during with dashes since model selection denoted are Variables with an asterisk. denoted are parameters Significant variable. and 78 adults. 209 juveniles was Sample size Age

The Auk: Ornithological Advances XX:1–17, © 2019 American Ornithological Society R. L. Windsor, G. A. Fox, and R. Bowman UV color consistency across molts 11 1 Downloaded from https://academic.oup.com/auk/advance-article-abstract/doi/10.1093/auk/ukz019/5479445 by Cornell University Library user on 26 April 2019 — — — — — 0.3 0.91 0.54 0.94 0.32 0.34 0.29 0.99 0.87 0.66 0.48 0.73 0.39 0.29 0.88 0.28 0.59 0.38 0.41 0.36 0.36 0.46 0.11 0.28 Variable importance Variable 95% CI — — — — — — — — 0.55 0.03 to 0.31 0.04 to −0.71 to −0.09 −0.71 to 0.27 −0.09 to −0.39 to −0.11 −0.39 to 0.13 −0.14 to 0.26 −0.11 to 0.09 −0.69 to 0.35 −0.31 to 0.56 −0.26 to 0.22 −0.29 to −0.44 to −0.06 −0.44 to 0.38 −0.04 to 0.18 −0.15 to 0.50 −0.26 to 0.06 −0.06 to 0.12 −0.16 to 0.10 −0.12 to 0.08 −0.07 to 0.85 −0.06 to −1.62 to −0.73 −1.62 to −0.41 to −0.002 −0.41 to Model selection — — — — — — — — 0.29 0.09 0.18 0.07 0.02 0.15 0.17 0.01 0.12 0.006 0.40 −0.40 −0.25 −0.21 −0.009 −0.30 −0.04 −0.25 −0.003 −0.02 −0.01 −1.18 Estimate Not included Not included P 0.17 0.003* 0.17 0.006* 0.30 0.82 0.46 0.02* 0.10 0.71 0.25 0.02* 0.60 0.66 0.26 0.66 0.006* 0.91 0.08 0.47 0.42 0.74 0.83 0.41 0.78 0.20 0.48 0.64 0.08 1.00 <0.001* <0.001* 95% CI 0.29 0.05 to 0.59 0.25 to 0.62 −0.11 to −0.71 to −0.14 −0.71 to 0.26 −0.05 to 0.19 −0.06 to 0.32 −0.25 to 0.44 −0.20 to −0.35 to −0.04 −0.35 to 0.03 −0.40 to 0.18 −0.26 to 0.25 −0.07 to −0.81 to −0.07 −0.81 to 0.25 −0.43 to 0.64 −0.40 to 0.19 −0.70 to 0.27 −0.17 to −0.47 to −0.08 −0.47 to 0.18 −0.20 to 0.37 −0.02 to 0.60 −0.28 to 0.53 −0.22 to 0.19 −0.27 to 0.43 −0.53 to 0.14 −0.34 to 0.22 −0.30 to 0.39 −0.08 to 0.57 −0.27 to 0.33 −0.54 to 0.88 −0.04 to −1.60 to −0.63 −1.60 to 0.43 −0.43 to Pooled full model Pooled 0.11 0.17 0.07 0.03 0.12 0.09 0.42 0.12 0.05 0.18 0.16 0.15 0.15 0.15 0.42 0.00 0.26 −0.42 −0.19 −0.18 −0.04 −0.44 −0.09 −0.25 −0.28 −0.01 −0.04 −0.05 −0.10 −0.04 −0.10 −1.12 Estimate Intercept Variable Sex (male) Juvenile mass (g) Juvenile Standardized nestling mass (g) Standardized Hematocrit Microfilarid infection (non-infected) Trypanosome infection (non-infected) infection Trypanosome size Group (ha) TSF of optimal Area Area of oak scrub (ha) Area Group size * Area of oak scrub * Area size Group (2014) Year (2015) Year Mother ID Intercept Sex (male) Adult mass (g) Adult Standardized nestling mass (g) Standardized Hematocrit color Juvenile Microfilarid infection (non-infected) Trypanosome infection (non-infected) infection Trypanosome size Group (yes) Stepparent (ha) TSF of optimal Area Area of oak scrub (ha) Area Group size * Area of oak scrub * Area size Group CORT treatment (treatment) treatment CORT (sham) treatment CORT (2014) Year (2015) Year Mother ID Linear mixed model (Gaussian distribution) selection results for parameters that explain juvenile and adult hue. Variables were centered and scaled prior to multiple and scaled prior to centered were Variables and adult hue. explain juvenile that parameters distribution) model (Gaussian selection for results Linear mixed TABLE 4. TABLE Juvenile imputation and modeling. Variable importance measures were calculated during model selection. calculated variable. Mother included in the pooled full model as a random importance ID was were measures Variable and modeling. imputation Sample not calculated. and CI were estimates not chosen during with dashes since model selection denoted are Variables with an asterisk. denoted are parameters Significant and 78 adults. 209 juveniles was size Age Adult

TABLE 5. Generalized linear model (binomial distribution, logit link function) of relative change in color variables within individuals that predict breeding fate. Males and females were assessed with separate models because of the significant interactions between sex and color variables (Supplementary Material Table S4). Significant predictors are denoted with asterisks. Sample size was 38 males and 33 females that remained on the study site until their third year. Downloaded from https://academic.oup.com/auk/advance-article-abstract/doi/10.1093/auk/ukz019/5479445 by Cornell University Library user on 26 April 2019 Sex Parameter Estimate SE Z P Males Intercept −0.14 0.52 −0.27 0.79 Change in brightness 0.03 0.05 0.60 0.55 Change in chroma −0.10 0.11 −0.88 0.38 Change in hue −0.01 0.06 −0.15 0.88 Females Intercept −0.34 0.53 −0.64 0.52 Change in brightness 0.16 0.08 2.02 0.04* Change in chroma 0.41 0.21 1.99 0.05* Change in hue 0.15 0.07 2.09 0.04*

FIGURE 3. Relative change in Florida Scrub-Jay rectrix color for individuals that became breeders vs. individuals that remained helpers. In males, the change in color from juvenile to adult plumage did not predict acquisition of breeding space. Female breeding space acquisition was significantly predicted by change in all 3 color variables (brightness: Z = 2.02, P = 0.04; chroma: Z = 1.99, P = 0.04; hue: Z = 2.09, P = 0.04). Significance is indicated with asterisks. sexes, breeders and helpers had similar feather color. In a variety of influences. Within individuals, juvenile plumage males, the change in color from juvenile to adult plum- was significantly different from adult plumage, and these dif- age did not predict acquisition of breeding space; however, ferences were consistent for both males and females (Figure females that became breeders showed significant reduc- 1). Within individuals, adult feathers reflected significantly tions in brightness (mean change ± SD = −4.83 ± 6.48%, less total light than juvenile feathers but had greater pro- Z = 2.02, P = 0.04) and chroma (2.35 ± 3.53, Z = 1.99, portions of UV light with peak reflectance at shorter wave- P = 0.05), and their hue was significantly shifted toward lengths (Table 1). For most avian species, adults are more shorter UV wavelengths (−5.99 ± 11.6 nm, Z = 2.09, highly ornamented with higher-quality plumage than juve- P = 0.04) relative to helpers (Table 5). Helpers showed little niles (Prum 2006, Gill 2007), and our findings are consistent change in brightness (−1.25 ± 6.51%), chroma (3.16 ± 4.39), with these trends. UV chroma is positively correlated with or hue (−3.83 ± 11.9 nm) (Figure 3). the number of circular keratin rods within the spongy medullary layer in Eastern Bluebird (Sialia sialis) feather barbs DISCUSSION (Shawkey et al. 2003), and thus, Florida Scrub-Jays with greater UV ornamentation as adults likely had higher-qual- Consistency of Color ity feathers with more keratin rods in their barbs. In Florida Scrub-Jays, UV plumage color is a plastic trait, Despite being more highly ornamented, adult feathers changing between juvenile and adult plumages as a result of reflected less total light than juvenile feathers, suggesting

that brightness is not associated with ornamentation in dominance, which likely determines how jays perceive and this species as are chroma and hue. Unlike chroma and interact with one another. hue, brightness is not influenced by feather barb nan- Despite significant differences in reflectance between ostructure (Shawkey et al. 2003), suggesting that other juvenile and adult plumage, some measures of structural Downloaded from https://academic.oup.com/auk/advance-article-abstract/doi/10.1093/auk/ukz019/5479445 by Cornell University Library user on 26 April 2019 feather structures, such as thickness of the spongy layer color were correlated between juvenile and adult plum- (Andersson 1999), may contribute to variation in UV re- ages (Figure 2). Brightness was consistent across molts; flectance. We propose 2 possible explanations that would the brightest juveniles tended to be the brightest adults. In clarify our observations of greater brightness in juveniles: contrast, juvenile and adult chroma were correlated only bacterial degradation and signal evolution via parental in females, and hue was not correlated between plumages preference. Juveniles could be exposed to more kerati- for either sex. This suggests that chroma and hue may be nolytic bacteria in the nest or during the post-fledging more likely to be affected by extrinsic and intrinsic factors period, or may not be as experienced with preening and than is brightness. Florida Scrub-Jays tend to occupy the plumage maintenance, and both scenarios would result in same territory for the first few years of life, thus typically greater bacterial loads on feathers, thus increasing bright- experiencing similar environmental conditions, but other ness (Shawkey et al. 2007). In contrast, UV reflectance factors may have greater influences on ornamentation than in fledglings may have evolved as a signal in response to early-life conditions, thus keeping it an honest signal. parental preference (Tanner and Richner 2008). Great Tit nestlings treated with UV blockers were fed less fre- Factors Influencing Variation in Feather UV Color quently than control nestlings; thus selection may favor Several factors explained variation in Florida Scrub-Jay bright fledglings if it attracts more attention from provi- feather reflectance, but at different stages of life. Juvenile sioning parents. color seemed more strongly affected by the quality of their Regardless of the mechanism responsible, the disparity natal environment and maternal effects, likely because they in brightness between juvenile and adult plumage suggests are still dependent on their families for food and protection that brightness could function as a signal of age in Florida while they are growing their rectrices. Mass and hemato- Scrub-Jays, consistent with the observations of Bridge crit had strong effects on juvenile color and both are prob- et al. (2007) in other Aphelocoma jays. As juveniles begin ably influenced by the quality of their parents and/or their foraying and searching for breeding vacancies their second natal habitat (Simon et al. 2005, Sakaluk et al. 2014). This is year, perhaps their brighter feathers help signal their age supported by the strong interaction between family group and thus their status as prebreeders to adults, helping them size and area of oak scrub, where additional helpers on avoid aggression. Although brightness does not appear to territories with little scrub and limited resources become be a component of ornamentation in this species, the dif- competitors for food, rather than adding to food available ferences in brightness between males and females in both for recently independent young (Mumme et al. 2015). age groups suggests otherwise. In this and other stud- In contrast to juveniles, adults are nutritionally inde- ies, both adult and juvenile males are less bright but have pendent during their pre-basic molt, and thus the influence greater chroma and more UV-shifted hues than females of additional stressors, such as competition for breeding (Bridge et al. 2007, Tringali and Bowman 2012; Table 1). space, might have stronger effects. For adults, juvenile color In addition to chroma ornamentation, some other species and adult body condition, both mass and endoparasite in- that appear sexually monomorphic in the visible light spec- fection, were the biggest influences on color. Maternal trum are dimorphic in brightness in the UV spectrum as effects are reduced, but there could be some carryover well (Eastern Bluebird: Siefferman and Hill 2005, Blue Tit: since maternal effects are strong on juvenile color and ju- Peters et al. 2011). We propose that multiple components venile color predicts adult color. The interaction between of UV color indicate both age and sex, which are important group size and oak scrub was important in adults, but less factors in Florida Scrub-Jay dominance hierarchies, where than for juveniles, suggesting that adult Florida Scrub-Jays males always dominate females, and older birds domi- may be able to better cope with costs associated with com- nate younger birds (Woolfenden and Fitzpatrick 1977). petition in small territories or short-term resource limi- Furthermore, experimental reduction in UV chroma in tation. Most importantly, experimentally induced stress, tail feathers resulted in a decrease in dominance in both through CORT treatment, significantly reduced chroma, male and female juvenile jays (Tringali and Bowman 2012), lending support for our hypothesis of a causal link be- yet no measure of UV color predicted dominance. Thus, tween CORT and structural color (Henderson et al. 2013). plumage reflectance may indicate sex and age, which are Corticosterone inhibits feather growth and protein deposi- important predictors of dominance, but may not act as a tion (Romero et al. 2005, DesRochers et al. 2008), which in signal of dominance itself. Rather, it is the combination of turn affects feather nanostructure. Feather nanostructure UV color and other phenotypes, such as personality and most strongly influences variation in UV color (Shawkey

et al. 2003, Siefferman et al. 2008), thus increased lev- 3). UV brightness is correlated with reproductive fitness in els of stress during molt likely prevent birds from grow- other avian species (Hill 1988, Avilés et al. 2008), and we ing high-quality feathers and reduces ornamentation at a acknowledge that the component of fitness that we meas- nanostructural scale. ured, acquisition of breeding space, although appropriate Downloaded from https://academic.oup.com/auk/advance-article-abstract/doi/10.1093/auk/ukz019/5479445 by Cornell University Library user on 26 April 2019 We found support for our hypothesis that UV color acts given the relationship between juvenile dominance and as a signal for nutritional condition, as all juvenile and color, might not have the same relationship to UV color adult color variables were explained by mass, either brood- as other fitness components, such as the total number standardized nestling mass or body mass at time of cap- of fledglings produced. However, Florida Scrub-Jays are ture. Our findings corroborate those of some other studies a long-lived species and lifetime reproductive success on avian structural plumage (McGraw et al. 2002, Johnsen is correlated with age at first breeding (Woolfenden and et al. 2003, Grindstaff et al. 2012). Our results may also Fitzpatrick 1984); thus early acquisition of breeding space suggest why other studies have documented a lack of sup- is expected to be a rather substantial component of repro- port for the link between condition and UV color (Peters ductive fitness. et al. 2011, Henderson et al. 2013). In Florida Scrub-Jays, Unlike UV color in adult plumage, the relative change condition has a large effect on color, but so do other indi- in UV color from juvenile to adult plumage was a signifi- vidual attributes, as well as environmental conditions, so cant predictor of acquisition of breeding space, but only in perhaps the lack of evidence for condition dependence in females (Table 5). Females that experienced greater reduc- these studies is masked by other factors. tions in brightness and chroma and shifts toward shorter We observed strong yearly effects within each model of hues were more likely to obtain breeding space than juvenile and adult color, with birds hatched in 2015 hav- females who experienced very little relative change across ing substantially greater ornamentation as adults than molts (Figure 3). This seems to suggest that females who the other 2 cohorts. This may have been a result of the had less energy to devote to feather growth as adults were strong El Niño event in 2015–2016. This season was wet- more likely to acquire breeding space. Twice each year, ter than average, which could have increased or affected immediately before and after the breeding season, non- the diversity of food resources, a factor for which we did breeding birds make extensive forays from their natal ter- not account. Humidity also causes swelling of the keratin ritories (Woolfenden and Fitzpatrick 1984), presumably in cortex of feathers, resulting in short-term, reversible shifts search of breeding opportunities. Such forays vary greatly in structural iridescent color (Eliason and Shawkey 2010). among individuals, some traveling great distances, others Climate patterns, including both rainfall and temperature, frequently staying home (A. Tringali personal communica- were correlated with plumage ornamentation in American tion). The benefits of such forays are obvious, but they also Redstarts (Setophaga ruticilla) and Collared Flycatchers carry some costs, such as energy use. The post-breeding (Ficedula albicollis) (Hegyi et al. 2007, Reudink et al. 2015), foray period coincides with the first pre-basic molt, and and future studies of plumage ornamentation should in- it is likely that females making more frequent and longer clude year or climate parameters to account for this vari- forays may divert energy from molt and produce less-orna- ation. These broad-scale environmental parameters often mented feathers. However, their increased forays improve result in unidirectional change in all individuals across a their probability of obtaining breeding space the following population (Garant et al. 2004), and are important to in- spring. We are unsure as to why breeder females experi- clude, but do not reveal the effects of small-scale environ- enced shifts to shorter hues since they experienced reduc- mental conditions that highlight the multidirectional ways tions in chroma and the 2 color variables negatively covary that individuals in a population can change. and are based on feather structure (Shawkey et al. 2003); however, we observed the greatest variation in hue across Effects of Color on Acquisition of Breeding Space individuals, so perhaps any patterns in hue were masked. Our results suggest that UV reflectance is unlikely to be In contrast to females, the relative change in color in under strong sexual selection. Contrary to our prediction, males had no effect on acquisition of breeding space. no measure of adult rectrix color for any of our 3 cohorts This may reflect sex-specific dispersal strategies to max- was a significant predictor of breeding. Since UV reflect- imize lifetime reproductive success. Because of a rigid ance is associated with dominance in juveniles (Tringali social dominance hierarchy in which males dominate and Bowman 2012), we had predicted that the brighter females (Woolfenden and Fitzpatrick 1977), males may individuals with greater proportions of chroma would be acquire breeding space via inheritance or budding, where more likely to obtain breeding positions; thus we expected the dominant male nonbreeder acquires a small portion differences in UV color between breeders and helpers. of his natal territory (Woolfenden and Fitzpatrick 1984). However, breeders and helpers had similar measures of Inheritance and budding account for 48% of territory ac- brightness, chroma, and hue when separated by sex (Figure quisition in males (Woolfenden and Fitzpatrick 1990).

In contrast, most females acquire space by replacing lost advice on measuring feather color, S. Schoech, T. Small, breeders, thus extensive forays may be a more efficient E. Elderbrock, and B. Jones for help with CORT administra- strategy for females than for males. Furthermore, females tion protocol, and S. Pruett, A. Tringali, and countless interns that first breed at 2 yr old produce more breeding offspring for their help with field data collection. Downloaded from https://academic.oup.com/auk/advance-article-abstract/doi/10.1093/auk/ukz019/5479445 by Cornell University Library user on 26 April 2019 than females that breed at age 3 or older; whereas in males, Funding statement: Financial support was provided by a Sigma Xi Grant-in-Aid of Research (G201510151641890), birds that first breed at 2 or 3 have similar lifetime repro- the Florida Ornithological Society Helen G. and Allan ductive success. In both sexes, birds that first breed at 1 yr D. Cruickshank Research Award (2015), graduate student do poorly, probably because of a lack of experience; how- funding from the University of South Florida, and a grant ever, females do much better as 1-year-olds than do males. from the National Science Foundation (DEB 1257628). Thus, strong selection may exist for females to breed early, Ethics statement: All procedures were conducted under whereas males may have an advantage if they delay their permits from the U.S. Geological Survey (banding and age at first breeding (A. Tringali personal communication; Endangered Species permits 07732, TE824723-9) and U.S. Fish R. Bowman personal observation), potentially leading to and Wildlife Service (CORT dosing, TE117769-7). Our work females expending more energy by conducting more ex- was approved by the University of South Florida Institutional tensive forays earlier in life than males. This could lead to Animal Care and Use Committee (W IS00000947). the patterns we observed of females obtaining breeding Author contributions: R.L.W., G.F., and R.B. formulated the questions, design, and methodology for this project. space having a greater reduction in feather ornamentation R.L.W. and R.B. collected data and conducted the research. than those that remained helpers and the absence of such G.F. and R.B. supervised research. R.L.W., G.F., and R.B. ana- a pattern in males. lyzed the data and wrote the paper.

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