Volume 36, Issue 11 p. 2756-2762
Open Access

Pigment molecular composition reveals significant information for visual communication

Ismael Galván

Corresponding Author

Ismael Galván

Department of Evolutionary Ecology, National Museum of Natural Sciences, CSIC, Madrid, Spain


Ismael Galván

Email: [email protected]

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First published: 06 September 2022
Handling Editor Caroline Isaksson



  1. Melanins are the most common pigments in vertebrates and, as such, fufill multiple adaptive functions, including honest signalling. This is the case of male pied flycatchers Ficedula hypoleuca, whose dorsal plumage, composed of black and grey feathers pigmented by eumelanin, is sexually selected by females regarding the proportion of black.
  2. However, the basis of such mating preferences and other associations with life-history traits are unknown.
  3. Here, I take the advantage of recent advances in Raman spectroscopy analysis to investigate the monomeric composition of eumelanin, constituted by 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) subunits, in male pied flycatchers.
  4. I found that plumage blackness (percentage of dorsal black feathers) increases with the DHICA:DHI ratio of the constituent black freathers, but not with that of grey feathers. The repeatability of DHICA:DHI measurements in black feathers is two times that in grey feathers.
  5. Eumelanin chemistry may thus constitute the basis of signal honesty in pied flycatchers, as females preferentially mate with males with higher relative DHICA feather contents and this may be related to the lower pro-oxidant effect of DHICA. Given the the ubiquitous nature of melanin-based pigmentation phenotypes, the monomeric composition of eumelanin should therefore be considered, instead of eumelanin as a whole, for a better understanding of how and why phenotypes are linked to life-history traits in animals.

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  1. Las melaninas son los pigmentos más comunes en vertebrados y, como tales, desempeñan múltiples funciones adaptativas, incluyendo la señalización honesta. Tal es el caso de los machos de papamoscas cerrojillo Ficedula hypoleuca, cuyo plumaje dorsal, compuesto de plumas negras y grises pigmentadas por eumelanina, es sexualmente seleccionado por las hembras en relación a la proporción de negro.
  2. Sin embargo, las bases de estas preferencias de emparejamiento y otras asociaciones con rasgos de historia vital son desconocidas.
  3. Aquí hago uso de recientes avances en el análisis por espectroscopía Raman para investigar la composición monomérica de la eumelanina, constituída por subunidades de 5,6-dihidroxindol (DHI) y 5,6-dihidroxindol-2-ácido carboxílico (DHICA), en machos de papamoscas cerrojillo.
  4. Encontré que la cantidad de negro en el plumaje (porcentaje de plumas dorsales negras) aumenta con el ratio DHICA:DHI de las plumas negras constituyentes, pero no con el de las plumas grises. La repetibilidad de las medidas de DHICA:DHI en las plumas negras es dos veces la de las medidas en las plumas grises.
  5. La química de la eumelanina puede por tanto constituir la base de la honestidad de señal en los papamoscas cerrojillos, ya que las hembras se emparejan preferentemente con machos con mayores contenidos relativos de DHICA en las plumas y esto podría estar relacionado con el menor efecto pro-oxidante de DHICA. Dada la naturaleza ubicua de los fenotipos de pigmentación generados por melaninas, la composición monomérica de la eumelanina debería por tanto ser considerada, en lugar de la eumelanina como un todo, para comprender mejor cómo y por qué los fenotipos se asocian a los rasgos de historia vital en animales.


Sexually selected traits often act as signals because of their ability to influence decisions of the recipients and thus affect their fitness (Hasson, 1997). Such fitness consequences are usually positive, leading to signal honesty (Searcy & Nowicki, 2005). Understanding why signals' recipients increase their fitness by perceiving the signals, which equals understanding the evolution of signals and honesty, can be achieved only through a comprehension of the mechanisms underlying signal production (i.e. the “black box”) (Dale, 2006). Production mechanisms may thus determine the precise information that recipients obtain when perceiving signalers, hence disentangling trait evolution.

Being the most common pigments in vertebrates, melanins confer an extraordinary richness of colours and patterns to different parts of the body of animals, consequently enabling the evolution of visual signals. This is particularly relevant in birds, in which diverse melanin-based plumage colours transfer information related to the processes underlying the chemistry of melanin synthesis in melanocytes (Galván & Solano, 2016). This chemical information has a high potential for the evolution of signal honesty, but is still not well understood (Guindre-Parker & Love, 2014). This limitation is in part given by the structural complexity of melanins, which are large insoluble polymers that make difficult their isolation from feathers and the direct analysis of their chemical diversity (Galván & Solano, 2016).

Recent advances in the application of noninvasive techniques such as Raman spectroscopy in the study of melanins open new opportunities to understand the signalling function of melanin-based plumage colouration. This is the case of eumelanin, the melanin form that gives rise to black, grey and brown plumage traits and that is composed by two monomers: 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (Galván & Wakamatsu, 2016). Up to now, the adaptive functions, including signalling, and evolution of eumelanin-based colours in birds and other animals have been investigated considering eumelanin as a whole (e.g. Caro & Mallarino, 2020; Eliason et al., 2019). However, the capacity of Raman spectroscopy for a direct determination of the monomeric composition of eumelanin in feathers and other integumentary structures without the need of destroying or invasively treating the samples has recently been demonstrated (Galván et al., 2018), which facilitates a more detailed study of eumelanin-based colours in animals.

The pied flycatcher Ficedula hypoleuca is a small passerine bird whose dorsal plumage is coloured by eumelanin, which pigments black and brown-grey feathers. The black:grey feather proportion is responsible for dorsal plumage colour variability in male pied flycatchers, which ranges from mostly brown-grey (grey hereafter) to almost fully black (Figure 1). This dorsal eumelanin-based colouration is a sexually selected trait, with blacker males achieving higher mating success than greyer males in at least some populations (Dale et al., 1999; Galván & Moreno, 2009) and affecting the attractiveness of other plumage traits in other populations (Sirkiä & Laaksonen, 2009), thus suggesting that it has evolved as an honest signal of quality. However, the signalling function of this trait, that is why females preferentially mate with blacker male pied flycatchers, is unknown. In fact, associations between dorsal plumage blackness and systemic markers of oxidative stress and damage have been shown to be absent (López-Arrabé et al., 2014; Moreno et al., 2011) or dependent on environmental factors such as temperature (Teerikorpi et al., 2019), which makes their interpretation difficult.

Details are in the caption following the image
Visual and chemical variability in the black plumage of male pied flycatchers. Images at the top of the figures show dorsal views of male pied flycatchers included in the study, showing variation in plumage blackness from 5 to 99% of black feathers in relation to grey feathers. Graphs at the bottom show Raman spectra of eumelanin in black feathers (black curves) and grey feathers (grey curves) obtained from all male pied flycatchers included in the study. Each curve represents the average Raman spectrum of four barbs and four barbules in two feathers of each bird. The graphs are separated by the median estimated DHICA:DHI ratio in black (20,829.41 a.u.) and grey feathers (20,582.40 a.u.) to show how the spectral shape changes with changing the DHICA:DHI ratio. Flycatcher images reproduced with permission from Pauliina Teerikorpi and Tiia Kärkkäinen.

Given that eumelanins composed of different proportions of DHI and DHICA give rise to different colours (black, grey and brown) in the feathers and hairs of different species of birds and mammals (Galván & Wakamatsu, 2016), the monomeric composition of eumelanin may also explain dorsal colour variability among male pied flycatchers. Such chemical explanation of intraspecific colour variation would provide insight into the signalling function of the trait, as the synthesis of DHI and DHICA differ in implications for oxidative stress and damage (Galván & Solano, 2015). Here, I use Raman spectroscopy to determine the eumelanin monomeric composition of dorsal feathers in male pied flycatchers and test its capacity to explain intraspecific plumage colour variation. The abovementioned ubiquitousness of melanin pigments in vertebrates makes that the implications of such association extend far beyond the specific case of the pied flycatcher, as the structure of eumelanin is the same in all organisms and its monomeric composition may thus have the potential to explain the life-history nexus of most existing pigmentation phenotypes.


2.1 Study area, plumage colour assessment and feather collection

The study was conducted during May 2018 in a population of pied flycatchers breeding in nest-boxes in the Island of Ruissalo, Turku, Finland (ca. 60°25′60 N, 22°10′0 E). 51 adult males (≥2 years old) were captured in their nest-boxes during the period of egg laying, banded with a metal ring, and their body mass and tarsus length measured. The percentage of black feather colour (vs. grey colour) in the dorsal plumage of males was estimated by eye from 0% to 100% to the nearest 1% (Järvistö et al., 2015; Figure 1). The repeatability of this estimation has been reported as high (r = 0.88, F1,33 = 24.96, p < 0.001) on the basis of assessments made by two different observers (Teerikorpi et al., 2019). Among the 51 males included in this study, dorsal plumage blackness ranged from 5 to 99% (mean = 59.3%, SD = 33.7%; Figure 1). Plumage colour was also assessed using the Drost's scale, with values ranging from from 1 (darkest) to 7 (lightest), as other authors previously used this scale in the pied flycatcher (Dale et al., 1999).

After plumage colour assessment, three black feathers and three grey feathers were plucked from the back of males and stored at dark. Feathers were identified as black or grey considering only the exposed, main pigmented area that corresponds to the distal part of feathers, that is, not considering the basal part which is mostly covered by adjacent feathers and therefore does not contribute to the visible colour phenotype. In one bird with 5% plumage blackness, it was not possible to identify and collect feathers uniformly black coloured in the distal part, as only mixed coloured feathers were found. In one bird with 20%, another bird with 50% and 10 birds with ≥95% plumage blackness, it was not possible to identify and collect feathers uniformly grey coloured in the distal part.

Plumage colour assessment and feather sampling were conducted by Pauliina Teerikorpi and Tiia Kärkkäinen (Department of Biology, University of Turku) with permits from Finnish authorities cited elsewhere (Kärkkäinen et al., 2021).

2.2 Monomeric composition of feather eumelanin

The dorsal feathers of male pied flycatchers were analysed by Raman spectroscopy to quantify the relative content of the constituent monomers of eumelanin, that is, DHI and DHICA. I used a Thermo Fisher DXR confocal dispersive Raman microscope (Thermo Fisher Scientific, Madison, WI, USA) with a slit aperture of 25 μm to analyse the wavenumber range of 300–2500 cm−1. The excitation laser source was at 780 nm, and power was set at 0.5 mW. The spectra were obtained using a 100x confocal objective lens. The system was operated with Thermo Fisher OMNIC 8.1 software.

The Raman spectra of eumelanin were identified and collected on the basis of the three diagnostic bands of this pigment, located at 500, 1380 and 1580 cm−1 (Galván et al., 2013; Figure 1). I analysed two black feathers and two grey feathers from each male pied flycatcher (excepting those birds from which only black or grey feathers could be collected, see above). Two barbs and two barbules chosen at random were analysed in each feather. The average Raman spectra of eumelanin were calculated for each feather colour (black and grey) and each bird. A total of eight eumelanin Raman spectra were thus obtained per colour and bird (Figure 1).

The DHICA:DHI ratio in the eumelanin spectra of male pied flycatcher feathers was determined following Galván et al. (2018), who showed that the ratio increases with increasing Raman signal in the wavenumber ranges of 550–1200 and 1650–2300 cm−1. Therefore, I calculated the sum of Raman intensity in 550–1200 and 1650–2300 cm−1 in the eumelanin spectra of feathers, and used this value as an index of the proportion of DHICA and DHI. It must be noted that eumelanin is composed of only two monomers (DHICA and DHI) and thus variation in the DHICA:DHI ratio will be correlated with variation in the total content of eumelanin (i.e. DHICA + DHI). However, variation in the DHICA:DHI ratio has a relevance for the chemical structure of eumelanin that the total content cannot provide.

2.3 Statistical analyses

The capacity of eumelanin DHICA:DHI composition of black and grey feathers to predict the plumage blackness (percentage of dorsal black colour) of male pied flycatchers was assessed by means of Pearson's correlation tests. This was also used to test for an association between the percentage of plumage blackness and the Drost's scale. I also estimated the repeatability of eumelanin DHICA:DHI ratio measurements between the two black and grey feathers that were analysed per bird (Lessells & Boag, 1987).


The measure of dorsal plumage blackness in male pied flycatchers, calculated as the percentage of black covert feathers in the back (relative to grey feathers), was strongly and negatively correlated with the blackness value assigned on the basis of the Drost's scale (r = −0.90, n = 51, p < 0.0001). The assessment of plumage blackness is thus a consistent measure between methods, albeit more precise as it collects variation in blackness to the 1%.

Plumage blackness was significantly and positively correlated with the DHICA:DHI ratio of eumelanin in black feathers (r = 0.36, n = 50, p = 0.011), indicating that feathers pigmented by eumelanin relatively richer in the DHICA monomer produce blacker, darker plumage colour phenotypes (Figure 2a). In contrast, plumage blackness was not correlated with the DHICA:DHI ratio of eumelanin in grey feathers (r = 0.12, n = 39, p = 0.474; Figure 2b). There were also notable differences between feather colours in the repeatability of DHICA:DHI ratio based on measurements in two feathers of the same bird. While DHICA:DHI ratio measurements were repeatable in both types of feathers, repeatability was moderate and notably higher in black feathers (r = 0.66, F48,49 = 4.88, p < 0.0001) than in grey feathers (r = 0.33, F37,38 = 2.02, p = 0.017). This was also observed when considering the four DHICA:DHI ratio measurements taken in every single feather, as this within-feather repeatability was low but highly significant and higher in black feathers (r = 0.28, F98,297 = 2.52, p < 0.0001) than in grey feathers (r = 0.21, F76,230 = 2.08, p < 0.0001).

Details are in the caption following the image
Eumelanin chemistry predictabiliy of black phenotype expression. Relationships between dorsal plumage blackness (percentage of black feathers) and eumelanin DHICA:DHI ratio, calculated from Raman spectra, of black feathers (a) and grey feathers (b). The lines are the best-fit lines.


These results show that the chemical composition of eumelanin, the pigment responsible for both black and grey feather colours, at the monomeric level explains variation in the resulting colour phenotype of male pied flycatchers. These birds become blacker, that is, with a higher proportion of black feathers relative to grey feathers in the back, as the relative content of the carboxylated subunit of eumelanin (DHICA) in black feathers increases. In interspecific comparisons, the species of birds with black plumage phenotypes are those with the highest contents of DHICA in feathers, as compared to species with grey or brown phenotypes (Galván & Wakamatsu, 2016). The results of male pied flycatchers therefore agree with the results of interspecific comparisons and indicate, for the first time, that intraspecific variation in colour phenotypes is produced by monomeric heterogeneity in the responsible pigment. The feathers of male pied flycatchers with a high proportion of black dorsal feathers may thus be considered as blacker than feathers of male pied flycatchers with fewer black dorsal feathers, though it is the proportion of black feathers the trait that is relevant for sexual selection (see below). Thus, it does not seem enough to characterize colour phenotypes as eumelanin-based to understand their signalling functions and more general evolutionary implications, as it has been made regarding animal melanin-based pigmentation to date (e.g. Caro & Mallarino, 2020; Eliason et al., 2019). This need extends beyond the case of the pied flycatcher to any animal whose body surface is pigmented by melanins, which are responsible for most pigmentation phenotypes.

Interestingly, I found a capacity of eumelanin DHICA:DHI ratio to predict the colour phenotype of male pied flycatchers only in black feathers, not in grey feathers. This has implications for the honest signalling of male pied flycatchers toward females, as black feathers are obviously responsible for the variation in the percentage of plumage blackness relative to grey feathers (Figure 1a). When eumelanin is synthesized in melanocytes, significant amounts of reactive oxygen species (ROS) are generated that make cells susceptible to oxidative stress and apoptosis. However, ROS amounts differ between the production of DHICA and DHI subunits (Galván & Solano, 2015). DHICA has a higher capacity to scavenge, and a lower susceptibility to generate ROS in melanocytes than DHI (Jiang et al., 2010; Micillo et al., 2016). This suggests that female pied flycatchers may show a preference to mate with males with higher percentages of black feathers (Dale et al., 1999; Galván & Moreno, 2009) because their higher relative DHICA contents in feathers make these males less susceptible to oxidative stress. This is likely, given the reported negative effects of oxidative stress on fitness-related traits in birds and other vertebrates (Mora et al., 2017; Pintus & Ros-Santaella, 2021). As male pied flycatchers become blacker with age (Galván & Moreno, 2009), this also means that the relative DHICA content of black feathers increases with age, providing a likely biochemical basis to understand why male mating success increases with age in this species (Lundberg & Alatalo, 2010).

The signalling role of black feathers proposed here is also consistent with the difference in repeatability of eumelanin DHICA:DHI ratio measurements within male pied flycatchers, as repeatability in black feathers is two times that of grey feathers. Only traits that are expressed consistently within individuals can respond to (sexual) selection (Boake, 1989), thus female pied flycatchers can rely on the information perceived on the black feathers of males for mating decisions, but not on information perceived on the grey feathers. Although the eumelanin DHICA:DHI ratio predicted plumage blackness variation in males, the correlation coefficient (0.36) indicated that a high proportion of variance remains unexplained. This may be the reason why different studies have not found clear associations between plumage blackness and oxidative stress markers in male pied flycatchers (López-Arrabé et al., 2014; Moreno et al., 2011; Teerikorpi et al., 2019) and other species with eumelanin-based mating signals (Henschen et al., 2018), as the eumelanin DHICA:DHI ratio of black feathers may be a trait that more directly reveals the physiological status of birds.

The variance in plumage blackness that remains unexplained by the eumelanin DHICA:DHI ratio may also open other possibilities. Indeed, it has previously been highlighted the relevance for ecology of a chemical observation: once DHICA and DHI (which are orthodiphenols, i.e. o-diphenols) are formed they are further oxidized to the corresponding orthoquinones (o-quinones): 5,6-indolequinone (IQ) in the case of DHI, and indole-2-carboxylic acid-5,6-quinone (IQCA) in the case of DHICA (Galván & Solano, 2015). IQCA and IQ are thus present in the eumelanin polymers, albeit in small quantities, together with DHICA and DHI. Noninvasive methods, such as Raman spectroscopy, that allow an easy determination of o-quinones in biological samples pigmented by eumelanin have still not been developed, but it could be speculated that the variance in plumage blackness that cannot explain the DHICA:DHI ratio may be at least partly explained by the IQCA:IQ ratio in feathers. This should be investigated in the future to obtain a more complete view of the molecular regulators of pigment phenotypic traits in animals.

The implications of these findings are not limited to the signalling content of plumage colour, but are also relevant to other life-history traits in pied flycatchers and other species pigmented by eumelanin. Thus, male plumage blackness has been related to tonic immobility and dispersal propensity in pied flycatchers, but the authors have proposed that these associations may be explained by pleiotropic effects in the genes involved in melanin synthesis (Camacho et al., 2018). As such pleiotropic effects remain untested and undefined in pied flycatchers and most birds, the results shown here indicate that, in contrast, associations of plumage colour with life-history traits are more likely dependent on the chemistry of eumelanin at the monomeric level.

Additionally, simplistic determinations of animal pigmentation, such as the quantification of pigment expression with a few scores after the visualization of animal images, may underestimate associations with life-history traits due to a limited capacity to collect variation in the mechanistic regulator of pigmentation. In the last decade, there has been a high interest in oxidative stress as an important mediator of life-history strategies and evolutionary trade-offs in organisms (Metcalfe & Alonso-Alvarez, 2010). The physiological ability to avoid oxidative stress actually equals the ability to adapt to the environment (Storey, 1996). The biosynthesis of melanins in cells is related to oxidative stress (see above), hence the relevance of studying melanin-based phenotypes in ecological studies. However, the association between melanin-based phenotypes and oxidative stress is still not fully understood. This is exemplified by a recent comparative study neglecting any association between pigmentation and oxidative status in birds after comparing many species whose pigment phenotypic expression was assessed by asigning scores from 0 to 5 obtained from the examination of book plates (Marton et al., 2022). While these methods for determining phenotypic expression have been useful in the past, the present study stresses that the ecological implications of pigmentation will be unveiled only by the study of pigment traits at the molecular level. This will be particularly relevant for interspecific studies. The present study thus shows the potential of determining phenotypic expression by considering the monomeric composition of pigments (eumelanin); thus, it may help in solving the question of how (and why) melanin-based phenotypes are associated to life-history strategies in animals.


I thank Marina García Guerra for her help with the analysis of Raman spectra, and Pauliina Teerikorpi and Tiia Kärkkäinen for collecting feathers from the birds. I declare no competing or financial interests.


    Ismael Galván is an Associate Editor of Functional Ecology, but took no part in the peer review and decision-making processes for this paper.


    Data deposited in the Dryad Digital Repository https://doi.org/10.5061/dryad.q573n5tmx, (Galván, 2022).