DOI: 10.5061/dryad.73n5tb37j
title: Differential effects of foraging strategies on carotenoid-derived plumage color and individual quality in stripe-tailed yellow finches

description: Carotenoid-based plumage color is crucial in avian mate selection, often
 serving as an indicator of individual quality. To determine whether
 carotenoid-derived color can be a sign of individual condition and if
 there is a relationship between an individual’s condition and color
 production, it is necessary to identify how carotenoids are acquired by
 individuals and subsequently used by the organism. Our objective was to
 determine how carotenoid pigments are used in the stripe-tailed yellow
 finch Sicalis citrina, a species wherein females exhibit a light yellowish
 ventral color while males are bright yellow. By using carbon and nitrogen
 stable isotope analyses we were able to determine if these
 carotenoid-derived colors are a signal of individual condition in terms of
 physiological stress and body condition. Females with low δmode_comment13C
 values (≈ −18‰) indicating the consumption of C3 and C4 seeds, exhibited
 colors shifted toward longer wavelengths and better body condition, and
 those with such color shifts also had higher carotenoid concentrations. In
 contrast, brighter females had higher δ15N values, indicating greater
 consumption of arthropods. Males with more saturated ventral patches had
 higher carotenoid concentrations, while those with colors shifted toward
 shorter wavelengths or lower H/L ratios, i.e. less stress, exhibited high
 δ15N values, suggesting that they supplement their diet with arthropods
 (δ15N ≈ 5‰). Our results show that the carotenoids dynamics in
 stripe-tailed yellow finches differ between sexes and highlight how food
 sources impact condition, stress, and ornamentation. Our study indicates
 that sexual dimorphism extends beyond plumage color to deeper
 physiological and ecological differences.
description: We carried out fieldwork to capture stripe-tailed yellow finches
 in the Brasília National Park during two breeding seasons: 2018/2019, and
 2019/2020. We identified seven sites of greatest activity of individuals
 and set up three to five mist nets in the morning, totaling a minimum
 capture effort of 2268 h.m² per site. To maximize capture success, we
 changed the locations of mist nets daily, setting up the mist nets in the
 territories of the males we heard singing and/or displaying. We captured
 about 1 to 3 individuals at a time and as soon as they were captured, we
 (who were in hiding) removed them from mist nests. We banded all captured
 individuals with metal bands provided by CEMAVE (Brazilian National Center
 for Research and Conservation of Wild Birds), and a unique combination of
 colored plastic bands. We identified females as those individuals with
 incubation patches and males by their complete yellow plumages, sampling
 only these males since our focus is on carotenoid pigments. We collected
 five to ten ventral feathers to determine their colors and carotenoid
 type. We chose ventral feathers because this is the part of the body has
 the most saturated yellow color and which is most visible to other
 individuals during displays. Also, the ventral part is often one of the
 most important in social signaling (Shultz and Burns 2017). Feathers were
 attached by their calamus with tape to pieces of cardboard and wrapped
 with aluminum foil for protection from light and moisture. We also
 collected blood samples through brachial vein puncture, using a 0.45 × 13
 mm needle and non-heparinized capillary tubes. To assess physiological
 stress (described below), we used a drop of blood to produce blood smears
 on slides, which were fixed and stained with the rapid panopticon kit
 Instant Prov. We ensured that blood samples were collected immediately
 after biometric measurements and before any feather collection, minimizing
 the impact of handling stress on the analyses. To
 estimate the body condition, we measured tarsal length with Digimess®
 digital calipers (300 mm) and weighed all individuals with a precision
 spring balance (30 g). Finally, we collected one wing feather (first
 primary remige) to determine the relationship between diet, using the
 stable isotope protocol described below, and carotenoids color expression.
 These samples were stored in small paper bags. We handled all individuals
 for a maximum of five minutes to minimize their stress.
 <em>Ornament color and carotenoids</em>
 First, we measured the ventral color of all individuals using the
 spectrophotometer Ocean Optics® USB4000 with pulsating xenon light source
 PX-2 (Ocean Optics, Dunedin, Florida), which enables reflectance in the
 ultraviolet spectrum. We obtained the measurements by positioning a single
 optical fiber at a 45° angle to the feathers and collected the
 backscattered radiation along the same axis (Pellegrino et al. 2020). We
 placed feather samples on a black, spectrally flat surface and recorded
 the average of three spectral measures (to the right of the rachis, above,
 and the left) using the OceanView® software from Ocean Optics
 (https://www.oceanoptics.com/software/oceanview/.). The spectra were
 interpolated at 1 nm intervals over the range of 300–700 nm. We used the R
 package ‘pavo’ (www.r-project.org, Maia et al. 2019) to smooth spectral
 curves and quantify the brightness, hue, and feather saturation. For this,
 we applied visual modeling considering the Blue Tit <em>Cyanistes
 caeruleus</em> visual system, and set the models assuming illuminant
 conditions for open habitats, where the stripe-tailed yellow finch breeds.
 We calculated brightness (‘luminance’), a metric related to the structural
 quality of the feathers, as the intensity of reflectance across the entire
 spectral range measured (300–700 nm). Saturation (‘r-achieved’), a more
 relevant metric for analyzing carotenoid concentration, was quantified as
 the relative distance of a given color (hue) from the achromatic origin
 (Stoddard and Prum 2008, Demko et al. 2020). Lastly, we determined the hue
 in the red-green-blue (RGB) and ultraviolet (UV) spectra (θ and Φ angles
 in radians). Higher RGB and UV hue values indicate a shift toward shorter
 wavelengths. Only hue in RGB and UV spectrum were highly correlated for
 both sexes (r<sub>p</sub> &gt; 0.70, p &lt;
 0.005;', Supporting information), therefore, hue in UV was
 disregarded from further analyses. We chose to keep the hue in RGB
 measurement because carotenoids are primarily expressed in this
 spectrum. Second, we used these same ventral samples to
 identify carotenoid types and estimated their concentration using ventral
 feathers of females and males with complete yellow plumage. We analyzed
 these samples in the Toxinology Lab of the University of Brasília, Brazil.
 We weighed samples on an analytical balance (0.0001 g) and placed them in
 Falcon tubes (15 ml) containing 3 ml of a solution composed of
 acetone:petroleum ether:didethyl ether (1:1:1). Subsequently, we subjected
 these samples to an cell disruptor for 3 cycles of 2 minutes each with an
 interval of 1 minute. After 48 hours at 5°C, each tube was centrifuged at
 12 000 RPM for 10 minutes. Then, we removed the solution and placed it in
 a new Falcon tube, which was subjected to vacuum drying in a speedvac
 system. After drying, the carotenes were resuspended in 400 μl of methanol
 and filtered through a 0.45 μm membrane for analysis in a high-performance
 chromatography system (HPLC) LC-10 (Shimadzu, Japan) with the analytical
 column C-18 KINETEX Phenomenex (0.,45 × 25 cm), oven at 35°C, mobile phase
 100% methanol, and a photodiode detector in the range of 300–600 nm. We
 identified the carotenoids present in the feather samples according to the
 comparison with the lutein, alpha-carotene, and beta-carotene standards of
 retention time and absorbance spectrum of 300–600 nm.
 We quantified the carotenoid concentration most well represented
 in the samples (lutein) by comparison with carotenoids that occur in
 cabbage. For this, we ground 50 g of cabbage and extracted the carotenoids
 separately with a 50 ml solution of acetone:petroleum ether:diethyl ether
 (1:1:1). The extract was divided into two 50 ml Falcon tubes and subjected
 to the cell disruptor for 3 cycles of 2 minutes each with an interval of 1
 minute. After 48 hours at 5°C, tubes were centrifuged at 12 000 RPM for 10
 minutes. We removed the extracts and conditioned them in new Falcon tubes
 (15 ml) which were then submitted to vacuum drying in a speedvac system.
 After drying, the carotenes were resuspended in 1 ml of methanol and
 filtered with a 0.45 μm membrane for analysis in an HPLC system. We
 collected and dried the lutein carotenoids separately in a vacuum system.
 These carotenoids were quantified according to Britton (1995).
 <em>Physiological stress</em> The
 blood smears were used to assess the physiological stress of individuals.
 White blood cell counts (heterophils, lymphocytes, monocytes, eosinophils,
 and basophils) were conducted by the commercial Santé Veterinary Lab,
 Brasília, DF. White blood cells, as part of the body’s immune defense
 system (Miesle 2011), have been used to study health parameters in several
 taxa (Dehnhard et al. 2011, Simons et al. 2012). Given that in the
 exploratory phase of our analyses, we found no eosinophils or basophils,
 that the number of monocytes was close to the normal range (0–3%) (as
 suggested by Miesle 2011), and that the numbers of heterophils,
 lymphocytes, and the heterophil/lymphocyte (H/L) ratio were highly
 correlated in females and males (r &gt; |0.95|; p &lt; 0.001), we
 chose to assess the levels of stress of individuals using the H/L ratio.
 The H/L ratio has been established as a robust measurement of
 physiological stress, such that an elevated H/L ratio implies a higher
 level of stress (Gross and Siegel 1983, Davis et al. 2008, Davis and Maney
 2018). The H/L ratio changes in response to external stressors that
 typically represent the risk of injury to individuals, which may include
 weather changes, social challenges, increased reproductive effort, or
 parasite infestation (Minias et al. 2019).
 <em>Body condition</em> Body
 condition results from accumulated energy in the individual's body as
 a result of food ingestion and can be an indicator of individual condition
 (Peig and Green 2009). A body condition index (BCI) was calculated for all
 individuals using the scaled mass index, due to its potential to remove
 covariance between body size and other body components (e.g. tarsal size)
 (Peig and Green 2009). We used the calculation provided by Peig and Green
 (2009):. <em>Stable isotope
 analysis</em> We used the nitrogen
 (<em>δ</em><sup>15</sup>N) and carbon
 (<em>δ</em><sup>13</sup>C) isotope ratio to
 estimate the food sources from which carotenoid pigments were extracted.
 Despite the high seasonality and dry conditions in the study area, which
 can limit plant transpiration and photosynthesis, leading to enrichments
 in <sup>15</sup>N and <sup>13</sup>C (Shipley and
 Matich, 2020), our systematic data collection (e.g. using the same feather
 sample) controls for potential isotopic differences due to water stress
 and maintains result consistency. Furthermore, our interpretations are
 grounded in an understanding of Cerrado dynamics and its isotopic
 fractionation, as detailed below.
 As <em>δ</em><sup>15</sup>N can be used
 to estimate trophic position, it may indicate the consumption of prey and
 assimilation of animal protein, with the smallest values indicating the
 consumption of seeds directly and the highest values suggesting prey
 consumption, in our specific system. In the Cerrado, the
 <em>δ</em><sup>15</sup>N basal of open areas with
 native and exotic grass vegetation is on average −1.70‰ and −1.86‰,
 respectively (Sena-Souza et al. 2023). Accounting for isotopic
 fractionation in the tissue,
 <em>δ</em><sup>15</sup>N close to these values
 indicates that individuals occupy a low position in the trophic chain,
 i.e. primary consumers. On the other hand, lower
 <em>δ</em><sup>13</sup>C values (average in −21‰)
 suggest that an individual´s food sources are Cerrado native
 C<sub>3</sub> grasses, while higher
 <em>δ</em><sup>13</sup>C values (average in −14‰)
 suggest that the food sources are native or exotic
 C<sub>4</sub> grasses, such as molasses grass (Sena-Souza et
 al. 2023, Navarro et al. 2023). Intermediate values suggest a mixed
 C<sub>3</sub> and C<sub>4</sub> diet (Zhao et al.
 2010, Andriollo et al. 2017, Navarro et al. 2023, Sena-Souza et al.
 2023)<em>.</em> We collected the first
 primary feather of both females and males. These feather samples reveal
 the isotopic values corresponding to the beginning of the breeding season
 of stripe-tailed yellow finches, since they molt prior to breeding
 (unpublished data, de-Carvalho unpubl.). We cleaned these samples by
 leaving them submerged for 30 minutes in a 2:1 solution of chloroform and
 methanol to remove dust particles and oil residues (Paritte and Kelly
 2009). After this step, feathers were oven-dried at 50°C for 48 hours, the
 distal part of the vane was cut, weighed (~ 0.8–2.0 mg), encapsulated in
 tin capsules, and sent for isotope-ratio mass spectrometry analysis. The
 nitrogen and carbon isotope ratios were determined by combustion using an
 elemental analyzer (Carlo Erba, CHN-1100) coupled to a Thermo Finnigan
 Delta Plus mass spectrometer at the Laboratory of Isotope Ecology of the
 Centro de Energia Nuclear na Agricultura (CENA/Universidade de São
 Paulo), Piracicaba, SP, Brazil. The results are expressed in delta
 notation (<em>δ</em>), in parts per thousand (‰), based on
 internationally recognized standards. We used the following equation:
 <em>δ</em><sup>15</sup>N or
 <em>δ</em><sup>13</sup>C (‰) = (Rsample –
 Rstandard)/Rstandard × 1.000), where Rsample and Rstandard represent the
 heavy/light isotope molar ratio of the sample and standard, respectively.
 The standard used for nitrogen analysis was atmospheric air
 (<sup>15</sup>N:<sup>14</sup>N ratio = 0.0036765)
 and the standard used for carbon analysis was Vienna Pee Dee Belemite
 (Vienna PDB; <sup>13</sup>C:<sup>12</sup>C ratio =
 0.01118). Internal standards (tropical soil and sugarcane leaves) are
 routinely interspersed with target samples during analyses. The Llong-term
 analytical error for the internal standards is 0.2‰ for both
 <em>δ</em><sup>15</sup>N and
 <em>δ</em><sup>13</sup>C.
description: # Differential effects of foraging strategies on carotenoid-derived
 plumage color and individual quality in stripe-tailed yellow finches
 [https://doi.org/10.5061/dryad.73n5tb37j](https://doi.org/10.5061/dryad.73n5tb37j) ## Description of the data and file structure Our objective was to determine how carotenoid pigments are used in the stripe-tailed yellow finch *Sicalis citrina*, a species wherein females exhibit a light yellowish ventral color while males are bright yellow. By using carbon and nitrogen stable isotope analyses we were able to determine if these carotenoid-derived colors are a signal of individual condition in terms of physiological stress and body condition. Our data is available in CSV format, where each column represents a variable and each row contains the data of a capture. ### Files and variables #### File: data\_for\_JAVBIO.csv ##### Variables * ID: individual idetification * sex: Sex * carot type: Types of carotenoids identified: * L: Lutein * A: Alfa-caroten * B: Beta-caroten * lutein (ug/g): Concentrations of lutein * area: Code of the area where the individual was captured. * lat long: Geographic coordinates in decimal degrees (latitude and longitude). * H/L: Heterophil/lymphocyte (H/L) ratio * date: Capture date * SMI.rob: Body condition index according Peig and Green 2009.  * d15N: Nitrogen isotopic ratio. * d13C: Carbon isotopic ratio. * u_ventre s_ventre m_ventre l_ventre u.r_ventre s.r_ventre m.r_ventre l.r_ventre x_ventre y_ventre z_ventre h.theta_ventre h.phi_ventre r.vec_ventre r.max_ventre r.achieved_ventre lum_ventre B1_v B2_v B3_v S1U_v S1V_v S1B_v S1G_v S1Y_v S1R_v S2_v S3_v S4_v S5_v S6_v S7_v S8_v S9_v S10_v H1_v H2_v H3_v H4_v H5_v: colorimetic variables according to ‘pavo’ package ([www.r-project.org](http://www.r-project.org), Maia et al. 2019) **Note:** The cells containing "NA" indicate cases where it was not possible to obtain a value for the corresponding variable. **References** Maia, R., Gruson, H., Endler, J. A. and White, T. E. 2019. pavo 2: new tools for the spectral and spatial analysis of colour in R. – Methods Ecol. Evol. 10: 1097–1107. Peig, J. and Green, A. J. 2009. New perspectives for estimating body condition from mass/length data: the scaled mass index as an alternative method. – Oikos 118: 1883–1891.