, 20121065, published 20 March 201310 2013 J. R. Soc. Interface
?
Daniel B. Thomas, Cushla M. McGoverin, Kevin J. McGraw, Helen F. James and Odile Madden
?
pigments (spheniscins) in penguins
Vibrational spectroscopic analyses of unique yellow feather
?
?
Supplementary data
l 
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Research
Cite this article: Thomas DB, McGoverin CM,
McGraw KJ, James HF, Madden O. 2013
Vibrational spectroscopic analyses of unique
Received: 31 December 2012
Keywords:
plumage, Raman, spectroscopy,
Author for correspondence:
Vibrational spectroscopic analyses
where a range of compounds and structures can produce a diverse colour
palette. Feather colours, for example, span the visible spectrum and
 on March 27, 2013rsif.royalsocietypublishing.orgDownloaded from Electronic supplementary material is available
at http://dx.doi.org/10.1098/rsif.2012.1065 or
via http://rsif.royalsocietypublishing.org.Daniel B. Thomas
e-mail: thomas@si.eduSphenisciformes, spheniscinAccepted: 28 February 2013
Subject Areas:
chemical biology, biomaterialsyellow feather pigments (spheniscins) in
penguins. J R Soc Interface 10: 20121065.
http://dx.doi.org/10.1098/rsif.2012.1065mostly result from pigments in five chemical classes (carotenoids, melanins,
porphyrins, psittacofulvins and metal oxides). However, the pigment that
generates the yellow colour of penguin feathers appears to represent a
sixth, poorly characterized class of feather pigments. This pigment class,
here termed ?spheniscin?, is displayed by half of the living penguin
genera; the larger and richer colour displays of the pigment are highly attrac-
tive. Using Raman and mid-infrared spectroscopies, we analysed yellow
feathers from two penguin species (king penguin, Aptenodytes patagonicus;
macaroni penguin, Eudyptes chrysolophus) to further characterize spheniscin
pigments. The Raman spectrum of spheniscin is distinct from spectra of
other feather pigments and exhibits 17 distinctive spectral bands between
300 and 1700 cm21. Spectral bands from the yellow pigment are assigned
to aromatically bound carbon atoms, and to skeletal modes in an aromatic,
heterocyclic ring. It has been suggested that the penguin pigment is a pterin
compound; Raman spectra from yellow penguin feathers are broadly
consistent with previously reported pterin spectra, although we have not
matched it to any known compound. Raman spectroscopy can provide a
rapid and non-destructive method for surveying the distribution of different
classes of feather pigments in the avian family tree, and for correlating the
chemistry of spheniscin with compounds analysed elsewhere. We suggest
that the sixth class of feather pigments may have evolved in a stem-lineage
penguin and endowed modern penguins with a costly plumage trait that
appears to be chemically unique among birds.
1. Introduction
Biologists have long been fascinated with the variation in and vibrancy and evol-
ution of avian plumage colours. Birds achieve much of this plumage-colour
diversity using nanostructural mechanisms, as in the case of blues and iridescent
colour [1], as well as with five different classes of chemical pigment: indole
polymers (melanins: black, brown, grey, rufous and buff), tetraterpenoids (caro-
tenoids: yellow, orange, red andpurple), linear polyenals (psittacofulvins: yellow,
orange and red), porphyrins (red, brown and green) and metal oxides (rust-red)
[2?9]. However, the colour-producing pigmentary mechanisms for the vast
majority of bird species have not yet been analysed [10] and have only been
inferred from common ancestry or shared reflectance characteristics [11].
Though many classes of feather colourants are thought to be widespread in
birds (e.g. melanin across Aves, carotenoids across Passeriformes [12,13]), others
appear to be quite rare. Parrots are believed to be the only organisms in the
& 2013 The Author(s) Published by the Royal Society. All rights reserved.of unique yellow feather pigments
(spheniscins) in penguins
Daniel B. Thomas1,2, Cushla M. McGoverin3, Kevin J. McGraw4,
Helen F. James1 and Odile Madden2
1Department of Vertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington,
DC 20013, USA
2Museum Conservation Institute, Smithsonian Institution, Suitland, MD 20746, USA
3Department of Bioengineering, Temple University, Philadelphia, PA 19122, USA
4School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA
Many animals extract, synthesize and refine chemicals for colour display,
is
Mioc
Eudyptula minor
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proportional likelihood for
the display of spheniscin
unresolved likelihood
1
0
Palaeocene
65.5 50 25
Eocene Oligoceneworld that produce their unique class of feather pigments,
psittacofulvins [14]. Moreover, turacos (Cuculiformes; Muso-
phagidae) are considered to be the lone group to produce
their red and green copper-rich plumage compounds, turacin
and turacoverdin, respectively [15]. A recent study of yellow
feathers from penguins (Sphenisciformes) revealed a sixth
class of pigment that fluoresces in ultraviolet light [16]. The
solubility properties of the penguin pigment distinguish it
from other yellow feather colourants, and chromatography
and light-elemental analyses suggest the novel compound is
chemically similar to yellow and red pterin pigments [17],
which are common in amphibians, reptiles and butterflies
[18?20] but only previously found in the irides of birds [21].
The yellow feather pigment in penguins is potentially
unique to Sphenisciformes, and based on ancestral-state
reconstructions may have evolved de novo in a stem-penguin
lineage (figure 1). Furthermore, yellow plumage pigmentation
is a condition-dependent trait [28?30] and is important to the
sexual selection criteria of half the living penguin genera
[31?34]. Thus, further characterization of the chemical identity
of this pigment could provide clues into the costs, benefits and
evolution of these display traits.
We sought additional biochemical information on
yellow plumage pigments in penguins (hereafter named
spheniscins) using vibrational spectroscopic techniques. Such
Figure 1. Emperor and king penguins (Aptenodytes spp.), crested penguins (Eudyptes
animals known to display spheniscin pigments. If we consider losses and gains of s
ancestor shared by all living penguins did not have yellow feathers. Alternatively, if
appearances (i.e. an asymmetric model), then spheniscin probably evolved between 63
that the extinct penguin, Madrynornis mirandus, had a spheniscin-pigmented ances
reconstructions performed in MESQUITE v. 2.75 [22] (Mk1, equal rates model; Asymm. 2.
tary material. Figure shows results from asymmetric model. Not all gaviiform or proce
units of geological time; Gaviiformes and Procellariiformes branch lengths are from [2
formes, Procellariiformes and Sphenisciformes follows [26] as adapted by [25]. Inkaya
[27]. Pl., Pliocene; Q., Quaternary. (Online version in colour.)?Madrynornis mirandus
Eudyptes filholi
Eudyptes chrysocome
Eudyptes moseleyi
Eudyptes chrysolophus
Eudyptes schlegeli
Eudyptes sclateri
Eudyptes pachyrhynchus
Eudyptes robustus
ene Pl. Q.
0 Ma
Spheniscus magellanicus
Spheniscus demersus
Spheniscus humboldti
Spheniscus mendiculus
Megadyptes antipodesGaviiformes
Procellariiformes
Aptenodytes forsteri
Aptenodytes patagonicus
Pygoscelis antarctica
Pygoscelis papua
Pygoscelis adeliaetechniques include Raman and mid-infrared spectroscopies,
which describe atomic interactions within molecules and min-
erals. Raman and mid-infrared spectroscopies are versatile
methods for analysing chemical composition and molecular
structure. Notably, Veronelli et al. [35] determined chemical
characteristics of psittacofulvins from a Raman spectroscopic
study comparing parrot feather pigments and carotenoids,
and Mendes-Pinto recently used Raman spectral informa-
tion to link feather coloration with carotenoid-contortion [36].
Previous studies of spheniscin pigments have identified
chromatic, chromatographic retention-time and solubility prop-
erties [17]. Vibrational spectroscopic techniques were used here
to study functional groups within spheniscin, which could
potentially be elaborated into a chemical structure for the
penguin pigment.
2. Material and methods
2.1. Selected feathers
Raman and mid-infrared spectra were collected from feather
barbs and barbules of several species, including two penguin
species and several reference species in which the pigmentary
basis of coloration is known. Studied specimens included
three yellow crest feathers from a macaroni penguin (National
spp.) and the yellow-eyed penguin (Megadyptes antipodes) are the only living
pheniscin to be equally likely evolutionarily, then the most recent common
we consider a single evolution of spheniscin to be more likely than multiple
and 13 million years ago in an ancient penguin lineage. Both models suggest
tor. Results have been interpreted from maximum-likelihood ancestral-state
param., asymmetric model); MESQUITE output file in the electronic supplemen-
llariform taxa are shown. Sphenisciformes branch lengths are from [23,24], in
5] and have been scaled to geological time. The relationship between Gavii-
cu paracasensis included to represent stem-lineage penguins, silhouette from
wing covert from a male greater black-backed gull (Larus marinus;
USNM 638661); yellow on the rectrix of a female rose-ringed
2.5 ml Eppendorf tube following a method adapted from
feathers (figure 2). Raman spectra from the yellow feather
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of a female bokmakierie (Telophorus zeylonus; USNM 642574); a
rufous breast contour feather from a female American robin
(Turdus migratorius; USNM 623483). Additional black and brown
feathers were also analysed, including brown-orange feathers
from short-tailed albatross (Phoebastria albatrus; see the electronic
supplementary material).
2.2. Spectroscopy
Raman spectra were measured using excitation wavelengths
780 nm (dispersive) and 1064 nm (Fourier transform, FT). Disper-
sive Raman spectra were collected using a Nicolet Almega XR
spectrometer (Thermo Electron Corporation, Madison, WI,
USA) equipped with a 150 mW diode laser and a Peltier-
cooled CCD detector. Spectra were collected through a 50 or
100 Mplan apochromatic objective lens (Olympus, Melville,
NY, USA) and 50 or 100 mm pinhole aperture, in a BX51 confocal
microscope (Olympus). Each spectrum was a co-addition of
32 scans across 200?3400 cm21 (2.6?4.9 cm21 resolution), collected
at 1, 10 or 100 per cent laser power. Magnification, aperture and
laser powerwere optimized between analyses tominimize spectral
noise and to maximize spectral signal from samples with a range
of energy tolerances. FT-Raman spectra were collected using an
NXR FT-Raman module coupled to a 6700 FTIR spectrometer
(Thermo Electron Corporation). The Raman module is equipped
with a Nd : YVO4 laser, a CaF2 beam splitter and a Peltier-cooled
InGaAs detector. Spectra were collected using the 1 mm laser
spot of a microstage. Each spectrum was a co-addition of 1024
scans across 100?3700 cm21 (4 cm21 resolution), with laser
power set at 0.2, 1 or 1.4 W depending on the energy tolerance of
the feather barbs. Note that laser power at the sample surface
was not measured. Instrument control and data collection were
managed by OMNIC 7.2 (FT) and OMNIC 8.2 (dispersive)
(Thermo Fisher Scientific, Waltham, MA, USA). Raman instru-
ments are housed in the Museum Conservation Institute,
Smithsonian Institution, Suitland MD, USA.Museum of Natural History catalogue no. USNM 533533) and
three yellow-orange auricular and three yellow-orange breast
feathers from a king penguin (USNM 59243). The sex of each
penguin is unknown. The macaroni penguin crest feather was
entirely yellow, whereas the king penguin feather had a white
proximal end and became yellow-orange towards the distal
end. Permission for removing feathers from study skin speci-
mens (USNM 533533 and USNM 59243) was granted by the
Division of Birds, National Museum of Natural History, Smithso-
nian Institution. Chromatographic evidence from an earlier study
[17] had revealed a second, related pigment in the yellow feathers
of yellow-eyed (Megadyptes antipodes), rockhopper (Eudyptes chry-
socome) and Snares-crested (Eudyptes robustus) penguins [17].
Feathers from these other penguin species were not plucked for
analysis in the current study to reduce the impact of our work
on the study skin collection.
A comparative library of pigment spectra was constructed
from feathers of other birds for which feather colour had pre-
viously been established (summarized in [12,13,37]). A library
is useful for determining whether the spectrum of a compound
with an unknown structure (e.g. spheniscin) is distinct from spec-
tra of structurally resolved compounds. Spectral libraries may
also be useful for identifying functional groups in a studied
compound. Feather colours analysed in the current study inclu-
ded the red on a single remige from a female Livingstone?s
turaco (Tauraco corythaix livingstonii; USNM 636319); the turquoise
from a remige of a male lilac-breasted roller (Coracias caudata;
USNM 634477); rust coloration on the wing covert from a
female sandhill crane (Grus canadensis; USNM 641570); a whitebarbs of macaroni and king penguins contained identical
bands, in addition to b-keratin constituents, that were distinct
from the Raman spectra of other feather pigments (figure 3).
We observed 17 distinct bands in the spheniscin spectrum;
the five most intense bands occurred at 1577 (vs), 1285 (s),
683 (m), 1469 (m) and 1351 (m) cm21. The band with the
highest wavenumber position occurred at 1700 cm21. The
feather extracts fluoresced at 780 and 1064 nm excitation
and did not produce informative Raman spectra. In contrast,McGraw et al. [17]. The solution turned yellow and the feather
remained yellow. Note that the yellow pigment was very
weakly soluble in 0.5, 5 and 13 M NH4OH, and insoluble
in acetone, acetonitrile, ethyl acetate, methanol, toluene and a
50 : 50 mixture of acetonitrile and methanol, and that the insolu-
bility in volatile solutions and hydrolysis of keratin in strong
basic solutions has complicated our efforts to collect a mass spec-
trum. The feather was removed from the tube, and 1 M HCl was
added dropwise to the feather extract solution. A white precipi-
tate formed at neutral pH (confirmed with pH indicator strips),
which was concentrated by centrifugation, separated from the
supernatant and evaporated on a glass slide. Raman spectra
were collected from the residue according the method outlined
above. Infrared absorption spectra were collected using a Nicolet
6700 FTIR spectrometer (Thermo Electron Corporation) with an
ATR accessory (Golden Gate, Specac Ltd, Orpington, UK). The
residue was compressed against a 1 mm2 diamond window for
data collection. Each spectrum was a co-addition of 64 scans
across 100?3700 cm21 at 4 cm21 resolution. The infrared spec-
trometer is housed in the Museum Conservation Institute,
Smithsonian Institution, Suitland, MD, USA.
The effect of pH on the Raman spectrum of the yellow pig-
ment was studied by immersing barbs from a king penguin
feather (USNM 59243) in water or in aqueous acid. The pH of
100 ml distilled water was adjusted with 0.5 M HCl and 0.1 M
NaOH; pH was monitored with an Accumet AB15 pH meter
(Fisher Scientific, Pittsburgh, PA, USA). The pH meter was cali-
brated using tris (hydroxymethyl) amino methane (pH ? 10.4)
and potassium biphthalate (pH ? 4). A single aliquot (0.5 ml)
was removed from the stock volume at pH 7, 5, 3, 2 and 1.
One feather barb was immersed in each aliquot for 30 min
and removed immediately in order to collect Raman spectra
with the dispersive instrument (100% laser power, 16 scans,
50 objective, 100 mm aperture).
3. Results
Raman spectra support the chemical disparity between
known feather pigments and the yellow pigment in penguinFourier transform infrared (FTIR) spectra of feathers were col-
lected using a Perkin Elmer Spotlight 400 IR imaging system with
an attenuated total reflectance (ATR) imaging attachment (Perkin
Elmer, Waltham, MA, USA). A 100  100 mm area was imaged
usinga 6.25 mmpixel; the spectrumof eachpixelwasthe co-addition
of 32 scans recordedwith 4 cm21 resolution from 750 to 4000 cm21.
Ten spectra recorded from feather barbule regions within the
100  100 mm area were averaged for each sample. The infrared
spectrometer is housed in the Department of Bioengineering,
Temple University, Philadelphia, PA, USA.
2.3. Pigment extractions and pH tests
A single, yellow crest feather from a macaroni penguin
(USNM 533533) was available for destructive analysis. The
feather was soaked in 1 ml of 0.5 M NaOH for 30 min in a
rsif.roya
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were characteristic of a porphyrin, carotenoid and psittacoful-
vin, respectively [35,38]. Rufous-brown barbs in the robin
4. Discussion
carotenoid
porphyrin
psittacofulvin
phaeomelanin
ar
bi
tra
ry
 in
te
ns
ity
iron oxide on eumelanin
eumelanin
b-keratin
200 500 100
0
150
0
200
0
Raman shift (wavenumber, cm?1)
250
0
300
0
340
0
Figure 2. Raman spectra from feather pigments and an unpigmented
feather. Five chemical classes have previously been recognized as feather
pigments: porphyrins (Livingstone?s turaco), tetraterpenoids (carotenoid, bok-
makierie), linear polyenals ( psittacofulvin, rose-ringed parakeet), metal oxides
(iron oxide, sandhill crane) and indole polymers (eumelanin, lilac-breasted
roller; phaeomelanin, American robin). Spectra from known pigments and
unpigmented b-keratin (greater black-backed gull) differ from the spectrum
recovered from yellow penguin feathers (?spheniscin?), which represents a
sixth class of feather pigment. Images of a Knysna turaco and kelp gull
are shown; other bird images match the respective feathers. The porphyrin
spectrum is from extracted pigment; all other analyses were in situ. Sphenis-
cin, porphyrin, carotenoid, psittacofulvin and b-keratin spectra were
measured at 780 nm; melanin-containing spectra were measured at
1064 nm. ?Spheniscin?, carotenoid and b-keratin spectra have been baseline
corrected. Unmanipulated spectra are available in the electronic supplemen-
tary material. King penguin photo by Liam Quinn and sandhill crane photo by
Tom Friedel. Other photos by D.B.T. (Online version in colour.)
lsocietypublishing.org
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10:20121065The distinct Raman spectrummeasured in yellow feathers from
both king andmacaroni penguins differentiates this colour from
the five known classes of avian plumage pigmentation. Sphe-
niscin spectral bands were most intense in the yellow feather
tips, and absent in the white, calamus-proximal regions of the
barbs. Indeed, the Raman spectrum of the white feather barbs
only contained bands attributed to b-keratin, and the relative
intensity of the spheniscin bands increased with yellow color-
ation. The relationship between colour saturation and band
intensities shows the ?spheniscin? spectrum to be the spectral
response from the yellow colourant and that the colour is due
to a pigment and not a structural manipulation of keratin: the
b-keratin spectral bands lost relative intensity and did not
shift band position, as the spheniscin bands became stronger.
Each spheniscin spectrum collected from the king andmacaroni
penguin feathers showed the same bands at the same relative
intensities, which indicates a single compound or a strictly
conserved ratio between multiple compounds.
4.1. Functional groups
Raman data provide indications of the functional groups
present within spheniscin, and permit comparison of its spec-
trum to that of known compounds. The most prominent band
in the spheniscin spectrum occurs at 1577 cm21. Very strong
bands between 1500 and 1650 cm21 have been attributed
elsewhere to carbon atoms bound with double bonds
(C?C) in aromatic (i.e. graphene) or conjugated (i.e. polyace-
tylene) systems [35,42]. The relative intensities of bands
attributed to C?C stretching are stronger in polymers (i.e.feather fluoresced at both 780 and 1064 nm excitation;
780 nm excitation induced a sinusoidal disturbance of the base-
line between 200 and approximately 1600 nm for all brown
feather pigments, i.e. a spectral artefact (see the electronic sup-
plementarymaterial). Similarly, the turquoise and black feather
regions from the roller feather intensely fluoresced under both
wavelengths (including spectral artefact at 780 nm excitation).
Spectral consistency between the eumelanin-bearing colour
regions of the same feather, and comparison with other black
feather barbs and barbules (see the electronic supplementary
material), confirm this is a diagnostic fluorescent response for
eumelanin. Spectra from the white gull feather showed only
bands from b-keratin [39].
Barbs from yellow penguin feathers that were exposed to
pH 7, 5, 3 and 2 for 30 min retained the spheniscin spectrum
(figure 4), though the barb exposed to pH 1 for 30 min yielded
a different spectrum. In this case, a new band appeared at
1608 cm21, and the prominent 1577 cm21 band lost relative
intensity and shifted to 1574 cm21. Bands at 1469, 1351,
1285 and 683 cm21 shifted to 1471, 1341, 1291 and 693 cm21,
respectively. Prominent pigment bands were associated
with yellow colour saturation (figure 4). Bands attributed to
b-keratin did not shift after exposure to pH 1.
FTIR-ATR spectra of each feather were dominated by bands
attributed to b-keratin [40] and do not inform about pigments
(see the electronic supplementarymaterial). TheFTIR-ATRspec-
tra of the feather extracts were identical to b-keratin, which
suggests that dissolution of the pigment in NaOH co-occurs
with hydrolysis of keratin [41] and will require a subsequent
separation step for isolating the pigment in future studies.
t (wave
1700
nguin p
nsities
and m
er was
ine corr
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capable of Fermi resonance [46], suggesting strong conjugation
oraromaticity in spheniscin.Note that the n3 band typical of con-
jugated chains (C?C stretch, approx. 1155 cm21) is absent from
Raman shif
316
441
200 500 1000 1500
500
544
683
723
812no
rm
al
iz
ed
 in
te
ns
ity
 (a
rb.
 
u
n
its
)
1203
1177
1351
1306
1285 1469
1526
1577
1071
Figure 3. Raman spectra of the yellow penguin pigment and b-keratin. Pe
penguin feather barbs (i.e. with yellow saturation). Changes in relative inte
b-keratin spectral bands. The macaroni penguin feather is yellow throughout
with a minor contribution from b-keratin. The greater black-backed gull feath
spectrum. Spectra were collected at 780 nm excitation and have been basel
material. (Online version in colour.)the spheniscin spectrum, and the single band between 1515 and
1540 cm21 is weak instead of dominant (i.e. conjugated n1(C?C
stretch), approx. 1520 cm21) [35]: hence, aromatic C?C stretch-
ing is a more likely assignment for the 1577 cm21 band than
carbon stretching in a conjugated chain. Furthermore, any ali-
phatic C?H stretching bands that would typically occur
around 2900 cm21 are very weak (i.e. obscured by b-keratin
bands) or absent from spheniscin. Weak aliphatic C?H stretch-
ing bands can also signify a molecule structured around an
aromatic ring [44,47].
Several possible assignments are available for the second,
fourth and fifth strongest bands (1285, 1469 and 1351 cm21,
respectively). Assignment options include C?H bending
(1288 cm21), cyclic amide stretching (lactam; 1466 cm21)
and C?C stretching (1351 cm21) [48,49], all of which are
common functionalities in yellow pigments (e.g. bilirubin in
vertebrates, riboflavin in plants and bacteria, xanthopterin in
micro-organisms, insects and vertebrates [50?52]). Bands
between 1285 and 1469 cm21 have also been attributed to
C?N stretching, Fermi resonance in a heterocyclic aromatic
ring and aromatic ring breathing [50,53,54]. The absence of a
spheniscin band around 1670 cm21 suggests that assignments
for 1351and1469 cm21 areunlikely tobeamide II or III stretches
(b-keratin amide I occurs at 1672 cm21) [55]. The third strongest
band (683 cm21) occurs in the fingerprint region. Such low
wavenumber bands typically indicate stretching between
heavier atoms (e.g. Si?O) or skeletal modes of organic systems,
such as pyrimidine ring breathing [56,57]. Indeed, a strong band
around 683 cm21 is found in many pterins [49].4.2. Pterin hypothesis
McGraw et al. [17] noted that the penguin pigment shared solu-
bility, light-absorption and retention-time properties with
king penguin (Aptenodytes patagonicus)
contour feather yellow
number, cm?1)
yellow
white
white
2000 2500 3000 3400
macaroni penguin (Eudyptes chrysolophus)
crest feather
greater black-backed gull (Larus marinus)
wing covert feather
igment spectral bands become more intense towards the distal tips of king
allowed bands in the penguin pigment spectrum to be differentiated from
ostly gives a ?spheniscin? spectrum (i.e. dominated by the penguin pigment),
unpigmented and does not contain the 17 bands identified in the spheniscin
ected. Unmanipulated spectra are available in the electronic supplementarypterin compounds, which can be produced endogenously in
animals (discussed in [37]). Pterins have not been reported
from feathers, although they are abundant in nature; pterins
occur in the eyes of some birds and fruit flies, in the integu-
ments of butterflies, amphibians and reptiles [18?21], and
perform physiological roles within many animals (e.g. folic
acid, an essential vitamin that many animals obtain from
their diet [58]). Pterins are heterocyclic compounds that tauto-
merize such that each pterin has several structural isomers.
The general structure of a pterin includes two six-membered
rings and keto-, imine, amine, hydroxyl functional groups
(dependent on the tautomer; see the electronic supplementary
material). A ?pterin-specific? arrangement of functional groups
should provide a ?pterin-diagnostic? Raman spectrum.
We tested the hypothesis that the yellow feather pigment is
a pterin by comparing the spheniscin spectrum to published
Raman spectra for pterins, which are summarized as follows.
Moore et al. [49] studied three pterin tautomers that varied
by pyrazine oxidation state. The tallest band in biopterin,
7,8 dihydrobiopterin and 5,6,7,8 tetrahydrobiopterin spectra
was a pteridine skeletal mode, which is a pyrimidine ring
out of plane deformation (694.2, 705.7 and 690.9 cm21, respect-
ively). Other low wavenumber bands attributed to skeletal
modes included pyrimidine out of plane bending (539.5,
538.2 and 535.5 cm21, respectively) and pyrazine in plane
deformation (509.3, 513.9 and 515.2 cm21, respectively).
Lower wavenumber bands linked to skeletal modes exhi-
bited a range of relative intensities and band positions,
particularly pyrazine ring quadrant stretching (1480.9, 1472.0,
low
low
low
ite
ite
ite
1000
(wave
vels. Ban
ent bec
(when c
e in the
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stretching (1582.2, 1589.8 and 1574.4 cm21, respectively).
In contrast to these spectrally variable tautomers, surface-
enhanced Raman spectra of two related pterins with different
pyrazine oxidation states showed identical band positions
with only slight differences in relative intensities [59]. Both
6-acetyl-7,7-dimethyl-7,8-dihydropterin and 6-acetyl-7,7-
dimethyl-7,8-dihydropterin featured strong bands around
1479 and 1567 cm21. Vibrational modes were not assigned,
but they may be analogous to the quadrant stretches described
king penguin (Aptenodytes patagonicus)
contour feather
king penguin (Aptenodytes patagonicus)
contour feather barb
yel
yel
yel
wh
wh
wh
greater black-backed gull (Larus marinus)
wing covert feather 693
683
200 400 600 800
n
o
rm
al
iz
ed
 in
te
ns
ity
 (a
rb.
 
u
n
its
)
Raman shift 
Figure 4. Raman spectra of king penguin feather barbs exposed to different pH le
new band appeared at 1608 cm21. Raman spectral bands of the pH-altered pigm
yellow saturation).b-keratin bands did not shift position in the pH-altered feather
excitation and have been baseline corrected. Unmanipulated spectra are availablbyMoore et al. [49], which would indicate a strong alkyl group
influence on pteridine skeletal modes. Indeed, the Raman spec-
tra of folic acid [60] and xanthopterin [51] differ substantially
between 1470?1480 cm21 and 1570?1590 cm21, both from
one another and from the previously discussed pterins.
We sought unambiguous pterin marker bands for compari-
son with spheniscin; the band positions and relative intensities
of high wavenumber pteridine skeletal modes may be too vari-
able for this purpose, but the low wavenumber skeletal modes
might be useful. For example, pyrimidine and pyrazine defor-
mations produce an intense band between 640 and 710 cm21.
For biopterins, the pyrimidine skeletal mode is constrained
between 690 and 710 cm21 and the pyrazine skeletal mode
occurs between 640 and 670 cm21 [49]. Other pterins, such as
folic acid [60] andxanthopterin [51], have skeletalmodesbetween
these two regions. Hence, pterins appear to have a strong band
between 640 and 710 cm21 that is due to deformation in the pter-
idine skeleton. Here, we interpret the 683 cm21 band in the
spheniscin spectrum as deformation in an aromatic, nitrogenous
heterocyclic ring, which is consistent with a pteridine skeleton
(following similar assignments in [49,51,60]). Bands between
500 and 540 cm21 have also been attributed to skeletal modes
in pterins [49], and the presence of these bands in the spheniscin
spectrum is further evidence for an aromatic, heterocyclic ring.
The spheniscin spectrum does not match any published spec-
trum, however. Aromatic and nitrogen-bearing heterocyclicrings are features of several chemical classes, including pterins,
tetrapyrroles and flavins, for example. Hence we cannot
unambiguously describe spheniscin as a pterin.
4.3. pH effect
The most intense band in the Raman spectrum of a pterin
molecule is rarely attributed to C?C stretching, but band
positions and intensities shift with tautomerization; different
pH environments can alter the ratio of tautomers [49,51,52,
1291
1285 1351 1469 1577
1341 1471 1574 1608
pH 1
pH 1
pH 1
pH 2
pH 3
pH 5
pH 7
1200 1400 1600 1800
number, cm?1)
d positions in the yellow penguin pigment shifted between pH 1 and pH 2, and a
ame more intense towards the distal tips of king penguin feather barbs (i.e. with
ompared with an untreated, white gull feather). Spectra were collected at 780 nm
electronic supplementary material. (Online version in colour.)60,61]. King penguin feather barbswere exposed to dilute hydro-
chloric acid to observe any changes in Raman spectra (figure 4).
We observed a shift from the spheniscin spectrum at pH 2, to a
new spectrum at pH 1, including major changes in the C?C
stretch region. An interpretation of the shifted spectrum could
involveprotonation of nitrogen atoms in an aromatic heterocyclic
ring, which would affect ring conjugation and thus change the
C?C stretching environment. Here, the pH influence on the
spheniscin spectrum allows us to confidently assign the
1577 cm21 band as aromatic C?C stretching. A similar pH
effect has been observed with pterins [49,62,63] and other aro-
matic compounds such as flavins and imidazoles; [47,64]), all
of which contain nitrogen-bearing heterocyclic rings. While this
effect is not diagnostic for pterins, it does indicate aromatic func-
tionality and excludes spheniscin from being a completely
aliphatic molecule like palmitic acid; note that aliphatic C?H
stretching around 2900 cm21 was not observed in spheniscin.
4.4. Other compounds
We considered whether the spheniscin spectrum could be better
matched to other yellow compounds in nature. Most of the com-
pounds we examined were spectrally inconsistent with
spheniscin. Flavone derivatives, such as quercitin, tend to have
amedium to strong band at 1000+ 10 cm21, that has been ident-
ified asC?C stretching in each of the three benzinoid rings, and a
strong band at 1600+ 10 cm21, owing to stretching within the
phenyl ring [65]. Curcuminoids (e.g. turmeric) and xanthonoids
a
c a
a
ps
i
tre
be
s
nd
minent band between 1610 and 1620 cm attributed to C?C
stretching [72,73].
haem C contains nitrogenous heterocyclic aromatic rings,
crested (Eudyptes) and yellow-eyed (Megadyptes) penguins
ot
[1
b
b
a
el
yg
nt
on
of displaying the penguin pigment.
Previous chemical analyses described fluorescence, chro-
881 Die
tudien 5
882 Die
gleich. P
he deve
ull. Mus
rsif.royalsocietypublishing.org
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SocInterface
10:20121065
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 on March 27, 2013rsif.royalsocietypublishing.orgDownloaded from which give distinct skeletal stretching modes. Detailed
comparisons are currently limited, however, because most
Raman spectra of cytochrome c? and Fe-porphyrins have
been reported from experiments using ultraviolet excitation
wavelengths, which gave resonance Raman spectra [75,77?
79]. Relative intensities can vary between resonant and non-
resonant spectra from the same compound. Haem-group
porphyrins should be considered potential candidates in
future investigations of the yellow penguin pigment, especially
considering the precedence of porphyrins as feather colourants
[37]. Both pterin and porphyrin pigments are endogenously
synthesized by birds [37], which is consistent with the display
of yellow pigments by both captive and wild birds that
have taxonomically disparate diets (wild diets summarized
by [80]). Furthermore, we collected X-ray fluorescence spectra
from a king penguin feather (yellow, spheniscin) and a
turaco feather (red, copper porphyrin). X-ray spectral evidence
for copper was readily apparent in the turaco feather, but a
metal centre could not be identified in the spectrum from the
penguin feather. Further research could evaluate whether
spheniscin is a free-base porphyrin (i.e. without ametal centre).
5. Conclusions
Much of the interest to date in penguin plumage colours has
either been on black-and-white countershading [81] or on the
communication role of yellow plumage [29,31,33,34,82,83].
For example, the yellow feathers in great (Aptenodytes),
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