Summary
Polymorphisms of the juvenal plumage in birds have received little study in comparison to other plumages. We investigate a polymorphism in the juvenal plumage of the Yellow Warbler (Setophaga petechia) in southeastern Ontario, Canada. Yellow Warblers in juvenal plumage show either yellow or white plumage on their lower ventral surface of the neck, breast, and belly, and these differences have been speculated to be associated with sex. We tested the hypothesis that juvenal plumage color varies with sex by using blood samples to molecularly sex 10 yellow and 14 white nestlings. We found no relationship between plumage color and sex (yellow nestlings: 70% male, 30% female; white nestlings: 64% male, 36% female). Using the same 24 nestlings that we sexed, we measured feather color with a spectrometer and found that white and yellow morphs differ significantly in reflectance spectra of red and yellow chroma. Of 66 nestlings whose plumage color we visually inspected in the wild, 73% were white and 27% were yellow, showing that white nestlings are more common than yellow in our study population. We propose alternative explanations for the possible significance of yellow and white polymorphism in the juvenal plumage of the Yellow Warbler.
Methodology
Visual Inspection of Nestlings, and Blood and Feather Collection
During June and July 2010, we visually inspected 66 nestlings from a total of 21 nests at the Queen's University Biological Station in southeastern Ontario, Canada (44° 34' N, 76° 19' W, 135 m elevation). We inspected nestlings visually 6–9 days after hatching when juvenal feathers had erupted from their sheaths. We examined feathers from the upper breast, flank, and belly and categorized nestlings' color as either yellow or white. While there is variation within these two color morphs (e.g., pale yellow, dirty white; see Fig. 1), nestlings can be reliably classified into distinct yellow or white morphs. We collected feather samples from 60 nestlings by plucking 5–8 breast feathers from each individual, targeting feathers that were the most developed within the breast region. We used a subset of these samples to measure feather color with a spectrometer to quantify differences in plumage color. We collected blood samples from 59 of the same nestlings from which we had collected feathers and used a subset of these samples to genetically sex individuals whose feather color we had scored. After bachial venipuncture, we collected blood samples (∼75 μL) into heparinized microcapillary tubes. All feather and blood samples were collected under the appropriate Canadian Wildlife Service permit (CA 0199).
Measuring Color
To measure feather color in a subsample of 24 nestlings we used Ocean Optics (Dunedin, FL) equipment: a USB4000 UV-vis spectrometer, a PX-2 pulsed xenon lamp, a fiber-optic cable and probe (P400-2-UV-VIS), and SpectraSuite software. To measure feather color, we stacked all 5–8 breast feathers on top of each other, placed them on black velvet, and maintained them perpendicular to the probe. Feathers from a single nestling were all similar to each other in color, so stacking the fluffy, downlike plumes provided a more accurate measure of color because the black velvet was less likely to reduce reflectance measures. We recalibrated light and dark standards prior to measuring the color of all feather samples.
The yellow plumage of nestling Yellow Warblers is likely the result of a single carotenoid, lutein (McGraw et al. 2003), although other carotenoids are present in the diet (Partali et al. 1987, McGraw et al. 2003). We used two measures of spectral purity in our analyses of nestling color: red chroma (S1R), which calculates the proportion of total reflectance in the range 605–700 nm (S1R = R605–700/R300–700), and yellow chroma (S1Y), which calculates the proportion of total reflectance in the range 550–625 nm (S1Y = R550–625 /R300–700) (Endler 1990, Montgomerie 2006). These two color variables were highly correlated (r2 = 0.97), but we did not collapse them into a single principal component of color so that these data may be used for future comparative studies (Montgomerie 2006).
Molecular Sexing of Nestlings
We used a published method (Griffiths et al. 1998) to molecularly sex the same 24 nestlings (10 yellow, 14 white) whose color we measured with a spectrometer. In short, we extracted DNA from blood samples by using a Qiagen DNEasy tissue kit (Valencia, CA), following the manufacturer's instructions for nucleated blood cells. We then amplified fragments of sex-specific CHD-Z (present in males and females) and CHD-W (present in females only) genes with primers P2 and P8 (Griffiths et al. 1998), then ran these amplified fragments through a 2% agarose gel. Females are indicated by the presence of two visible bands on an agarose gel, representing fragments from both CHDW and CHD-Z genes. Males are indicated by a single band, representing fragments from the CHD-Z gene. To confirm the accuracy of this method for sexing Yellow Warblers, we sampled two adult males and two adult females whose sex was determined by gonadal inspection postmortem by VGR. All four adults of known sex were accurately sexed with the genetic material. We analyzed all samples of nestlings and adults in duplicate with 100% agreement of sex identification.