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Canalised and plastic components of melanin-based colouration: a diet-manipulation experiment in house sparrows

Whether melanin-based plumage colouration accurately reflects a bird’s quality is still controversial. To better understand potential mechanisms behind the observed variation in plumage colouration, we shifted our attention from a high-level expression of colour to low-level physiological phenomena by targeting the microstructure and pigment content of the feather. In a well-studied model system, the house sparrow (Passer domesticus), we combined an experimental manipulation of birds’ physiological condition and availability of resources that are key to the production of the studied colouration (phenylalanine and tyrosine (PT). We found that feathers from sparrows fed with the control diet had noticeably lower values of brightness, suggesting a higher quality of the ornamental “blackness” in comparison to those sampled from birds fed with a PT-reduced diet. Electron paramagnetic resonance (EPR) spectroscopy detected higher melanin concentrations in samples from the control than the PT-reduced group. Our multi-level analysis excluded mechanisms such as barbule density and melanosomes’ distribution, clearly pointing to the finest-level proxy of colour: the concentration of melanin in melanosomes themselves. Despite melanins being manufactured by birds endogenously, the efficiency of melanogenesis can be noticeably limited by diet. As a result, the birds’ plumage colouration is affected, which may entail consequences in social signalling.


Supplementary methods
To better visualize the spectroscopic properties of two types of melanins found in birds, and verify the eumelanistic nature of sparrow's bib colouration, additional EPR measurements were performed using sparrow bib fathers and differently coloured chicken feather samples. The used feathers represented quantitative and qualitative variations in melanin content, and were compared with synthetic melanin standards and a paramagnetic reference.
A powder sample of 1,1-diphenyl-2-picrylhydrazyl (DPPH) was used as a paramagnetic reference, to determine the position of the feather melanin EPR signal, and indirectly -to determine the g-value. 3 small crystals of DPPH, approximately 2 mm in diameter each, were wrapped in parafilm (which did not disturb the free-radical signal) and placed inside the Wilmad finger quartz dewar. Additionally, powdered samples of dopa-auto-oxidation melanin and tyrosinase-catalysed cysteinyldopa melanin (both sealed in thin glass capillaries) served as synthetic equivalents of natural eumelanin and pheomelanin, respectively 1 .
Next, samples of bib feathers were selected (each from every experimental group) and measured again, followed by a sample from a random male sparrow's tail, characterized by a less pigmented, Kraków, Poland) were used as an amelanotic example and helped emphasize the difference in EPR signal quality in comparison to samples rich in melanin pigments 2,3 . Each EPR signal was averaged from 3 scans, lasting 160s each, and was recorded at room temperature, at X-band (9.26 GHz frequency), using the following parameters: magnetic field range 3240-3340 Gs, microwave power 1 mW, modulation frequency 100 kHz, modulation amplitude and time constant -1 Gs and 0,1 s .

Supplementary results
A statistically significant, positive, low to moderate (R-square 0.2955 -0.6367, p<0.05) linear relationships between mean EPR signal's amplitude values and its corresponding integral intensities were observed within each treatment group. At the same time, the values of EPR spectra linewidths remained relatively constant, and are unrelated to the shape of the recorded spectra. Therefore, the peak-to-peak height of the central feature of the spectra can be used as a sufficient approximation of the concentration of paramagnetic centres found in melanins 4 . The mean values of EPR amplitudes were used as a direct indicator of the melanisation level in feather samples.
Comparison between the EPR spectra of synthetic melanin analogues (DOPA melanin, cysteinyl-DOPA melanin) and differently coloured feather samples: black barbs and barbules of the Sussex chicken and sparrow's bib, reddish brown contour feathers of the Rhode Island Red, and control white samples of the Rhode Island White, helped visualize the spectroscopic differences between the presence of eumelanin and pheomelanin pigments, as well as between melanotic and amelanotic keratinous appendages (S2). These results further show that the EPR spectra recorded for sparrows black feathers are directly related to the paramagnetic centers found in melanins and not the free radicals formed in keratin.
The conducted spectroscopic analysis supports the assumption that the black patch located on the throat of a male house sparrow results from the presence of paramagnetic polymers -melanins 2 . The slightly asymmetric lineshape of EPR signals of similar linewidths (4.200 ± 0.063 [Gs]) and with no distinct hyperfine-splitting, resembles that of dopa melanins (S2), which are often used as a synthetic model of natural eumelanins. These features, along with the lack of the hyperfine splitting (typical for benzothiazine-based pheomelanins and their synthetic standard -cysteinyldopa melanin (S2)), strongly suggest that eumelanin is the dominant pigment responsible for the bib colouration. However, these results do not entirely exclude the presence of pheomelanins, which might be present in the investigated samples at a negligible level/below the detection level of the EPR spectrometer. Another possible explanation lies in the chemical structure of these polymers, namely the predominance of benzothiazole units, whose presence, compared to benzothiazine moieties, might result in a broadened signal and less pronounced splitting in the low-field part of the EPR spectrum [5][6][7] . DPPH provides a stable radical source with a well-defined g-value (2.0037 ± 0.0002). By assessing the position between the EPR signal of this free radical standard and the EPR spectra recorded for feathers samples and synthetic melanins, we were able to estimate the g-value of the paramagnetic centers detected in the sparrows bib and other feather samples, and prove that the recorded signals origin from the paramagnetic centers of natural melanins, embedded in the feathers keratin (S2).

SUPPLEMENTARY FIGURES AND TABLES
S1. Experimental timeline.

S2.
Samples of EPR spectra of 1,1-diphenyl-2-picrylhydrazyl (DPPH; used as a paramagnetic reference), synthetic standards of eumelanins (dopa melanin), synthetic standards of pheomelanins (cysteinyldopa melanin), black bib feathers of house sparrow, brownish tail feathers of house sparrow, chicken black feather as an example of feathers containing eumelanin (Sussex black), chicken mahogany feather as an example of feathers containing pheomelanin (Rhode Island Red red), chicken white feather as an example of amelanotic feather (Rhode Island White white).

S3
. Results of the model showing effects of body mass changes over the course of the experiment. The model included time, treatment (diet and injection) as fixed factors and the two-way interaction time * diet. Reference levels for fixed effects: time: before experiment; diet: control; injection: LPS.