-
doi: 10.1098/rsif.2012.0191 published online 9 May 2012J. R.
Soc. Interface
Cao, Eric R. Dufresne and Richard O. PrumVinodkumar Saranathan,
Jason D. Forster, Heeso Noh, Seng-Fatt Liew, Simon G. J. Mochrie,
Hui angle X-ray scattering (SAXS) analysis of 230 bird
speciesnanostructures from avian feather barbs: a comparative small
Structure and optical function of amorphous photonic
Supplementary data
l
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"Data Supplement"
Referencesref-list-1http://rsif.royalsocietypublishing.org/content/early/2012/05/02/rsif.2012.0191.full.html#
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*Authors forichard.prum@Present addDepartmentOxford OX1OX1 3JA,
UK
Electronic sup10.1098/rsif.2
doi:10.1098/rsif.2012.0191Published online
Received 9 MAccepted 17 A
Structure and optical function ofamorphous photonic
nanostructures
from avian feather barbs: acomparative small angle X-ray
scattering (SAXS) analysis of 230bird species
Vinodkumar Saranathan1,5,*,, Jason D. Forster2,5, Heeso
Noh3,5,Seng-Fatt Liew3,5, Simon G. J. Mochrie35, Hui Cao35,
Eric R. Dufresne2,4,5 and Richard O. Prum1,5,6,*1Department of
Ecology and Evolutionary Biology and Peabody Museum of Natural
History,
2Department of Mechanical Engineering and Materials
Science,3Department of Applied Physics, 4Department of Physics, and
5Center for Research on
Interface Structures and Phenomena (CRISP), Yale University, New
Haven, CT 06520, USA6Donostia International Physics Center (DIPC),
20018 Donostia-San Sebastian, Spain
Non-iridescent structural colours of feathers are a diverse and
an important part of the phe-notype of many birds. These colours
are generally produced by three-dimensional, amorphous(or
quasi-ordered) spongy b-keratin and air nanostructures found in the
medullary cells offeather barbs. Two main classes of
three-dimensional barb nanostructures are known, charac-terized by
a tortuous network of air channels or a close packing of spheroidal
air cavities.Using synchrotron small angle X-ray scattering (SAXS)
and optical spectrophotometry, wecharacterized the nanostructure
and optical function of 297 distinctly coloured feathersfrom 230
species belonging to 163 genera in 51 avian families. The SAXS data
provided quan-titative diagnoses of the channel- and sphere-type
nanostructures, and confirmed the presenceof a predominant,
isotropic length scale of variation in refractive index that
produces strongreinforcement of a narrow band of scattered
wavelengths. The SAXS structural data identifieda new class of
rudimentary or weakly nanostructured feathers responsible for
slate-grey, andblue-grey structural colours. SAXS structural data
provided good predictions of the single-scattering peak of the
optical reflectance of the feathers. The SAXS structural
measurementsof channel- and sphere-type nanostructures are also
similar to experimental scattering datafrom synthetic soft matter
systems that self-assemble by phase separation. These results
furthersupport the hypothesis that colour-producing protein and air
nanostructures in feather barbsare probably self-assembled by
arrested phase separation of polymerizing b-keratin from
thecytoplasm of medullary cells. Such avian amorphous photonic
nanostructures with isotropicoptical properties may provide
biomimetic inspiration for photonic technology.
Keywords: biophotonics; organismal structural colours;
amorphousnanostructures; non-iridescence; single scattering;
self-assembly
1. INTRODUCTION
Structural colours are prevalent in nature, and gener-ally
produced by the selective scattering and
r correspondence ([email protected];yale.edu).
ress: Edward Grey Institute of Field Ornithology,of Zoology,
University of Oxford, South Parks Road,3PS, UK and Linacre College,
St Cross Road, Oxford.
plementary material is available at http://dx.doi.org/012.0191
or via http://rsif.royalsocietypublishing.org.
arch 2012pril 2012 1
reinforcement of specific bands of wavelengths from bio-photonic
nanostructures with variations in refractiveindex on the order of
visible wavelengths of light[15]. Like photonic crystals [6],
biophotonic nanostruc-tures vary in nanostructure in either one,
two or threedimensions (figure 1ac). However, they may alsovary in
whether they have long-range, crystallineperiodicity, or only
short-range (nearest neighbour),structural correlations [15]
(figure 1d f ). The latterreferred to as quasi-ordered or amorphous
biophoto-nic nanostructures are characterized by unimodal
This journal is q 2012 The Royal Society
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crystal-like(biophotoniccrystals)long-range order
(a)one-dimension two-dimension
spatial variation in refractive indexthree-dimension
(b) (c)
(d ) (e) ( f )
quasi-ordered(amorphous photonicbiomaterials)short-range
order
rang
e of
spa
tial p
erio
dici
ty
Figure 1. A classification of biophotonic nanostructural
diversity based on the dimensionality of spatial variation in
refractiveindex and its range of periodicity. (ac) One-, two- and
three-dimensional biological photonic crystals with long-range
periodicorder in refractive index modulations (after Joannopoulos
[6]). (d) A chirped lamellar stack, a one-dimensional
quasi-orderednanostructure with short-range spatial periodicity
(currently unknown in birds; after Parker [2]). (e) TEM cross
section of atwo-dimensional amorphous or quasi-ordered
nanostructure with short-range order comprising of parallel
collagen fibres in amucopolysaccharide matrix from the green tongue
of magnificent bird-of-paradise (Cicinnurus magnificus,
Paradisaeidae).( f ) TEM cross section of a three-dimensional
amorphous or quasi-ordered nanostructure of b-keratin and
spheroidal air vacuolesfrom the spongy medullary cells of the azure
blue crown feather barbs of male Blue-crowned Manakin (Lepidothrix
coronata,Pipridae) with short-range quasi-periodic order.
2 Avian amorphous photonic nanostructures V. Saranathan et
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distributions of scatterer size and inter-scattererspacing, and
a notable lack of any underlying period-icity beyond the span of a
few nearest neighbours(figure 2e,f ) [3,79].
Vivid, non-iridescent structural colours in bird plu-mages
(figure 2b,c) are taxonomically widespread andappear to have
evolved numerous independent timesduring the evolutionary history
of birds in many ecolo-gically diverse lineages [3,9]. They
constitute animportant component of the phenotype of manybirds, and
are frequently used in intraspecific communi-cation and camouflage
(reviewed in [10,11]). Becausethe optical properties of biophotonic
materials are inti-mately tied to the underlying nanostructures, a
precisemechanistic characterization of organismal structuralcolour
production is critical to study how such biologicalsignals
evolve.
The non-iridescent structural colours of avian featherbarbs are
generally produced by three-dimensional, quasi-ordered
nanostructures composed of b-keratin (refractiveindex, n 1.58+0.01;
[12]) and air in the medullarycells of feather barbs (reviewed in
[3]). The colours so pro-duced do not appreciably change in hue
with changes inangle of observation under natural lighting because
back-scattered light dominates under such conditions [13].Spongy
barb nanostructures have been directly examinedin only a relatively
small number of avian taxa (approx.28 species from 16 families
[3]), but typically known tooccur in one of two distinct
morphologies. The channel-type nanostructures are characterized by
a tortuous,interconnected bicontinuous network of air channels
andb-keratin bars of similar widths and shapes (figure
2e)[3,79,14]. The sphere-type nanostructures consist of
aquasi-ordered close packing of spheroidal air cavities thatare
separated by b-keratin walls and frequently intercon-nected by tiny
air passages (figure 2f ). However, DAlbaet al. [15] recently
discovered a unique two-dimensional,medullary, feather barb
nanostructure comprising bundles
J. R. Soc. Interface
of parallel, quasi-ordered,b-keratin nanofibres in air that
isresponsible for the non-iridescent blue colour of BluePenguin
(Eudyptula minor, Spheniscidae).
1.1. Small angle X-ray scattering (SAXS)
Previous research using Fourier analysis of two-dimensional
transmission electron microscopy (TEM)images of amorphous barb
nanostructures has documentedtheir isotropic quasi-order from their
ring-shaped two-dimensional Fourier power spectra [3,79,16].
Fourieranalyses of TEM images were sufficiently accurate to
falsifythe century-old, single particle (Tyndall or Rayleigh,
andMie) scattering hypotheses, which assumed that thecolour comes
from wavelength-dependent light scatteringproperties of isolated,
spatially uncorrelated scatterers[3,79,16]. But two-dimensional
Fourier power spectra ofTEMimages lack the resolution toaccount for
thevariationin reflectance features of these complex
three-dimensionalnanostructures [3,79,1720]. They also suffer from
arte-facts owing to EM sample shrinkage and others related
toanalysing a finite-thickness (approx. 90 nm), low-resol-ution,
two-dimensional slice of a three-dimensionalnanostructure, such as
aliasing, binning, etc. Nevertheless,most studies of avian
structural colours have used two-dimensional electron microscopy to
characterize theirunderlying three-dimensional biophotonic
nanostructures[3,79,14, 1719,2125]. However, fundamental
uncer-tainty remains about the exact organization of
thesethree-dimensional amorphous feather barbnanostructures.Shawkey
et al. [26] recently performed three-dimensionalelectron
tomographic reconstruction of the channel-typebarb nanostructure in
blue rump feathers of Eastern Blue-bird (Sialia sialis), and made a
reasonable prediction of theoptical reflectance from the azimuthal
average of the three-dimensional Fourier transform of the tomogram.
However,sample shrinkage and tomographic distortion limited
theaccuracy of structural and optical analyses [26,27].
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(a) (b) (c)
(d) (e) ( f )
(g) (h) (i)
Figure 2. Diversity of non-iridescent feather barb structural
colours in birds and morphology of their underlying
three-dimensionalamorphous photonic nanostructures with short-range
quasi-periodic order. (a) Female Silver-breasted Broadbill
(Serilophus lunatus,Eurylaimidae). (b) Male Eastern Bluebird (S.
sialis, Turdidae). (c) Male Plum-throated Cotinga (Cotinga maynana,
Cotingidae).(d) SEM image of a rudimentary nanostructure with a
very thin layer (1 mm or less) of a disordered network of spongy
b-keratinbars present at the periphery of the medullary barb cells
from the pale blue-grey primary coverts of S. lunatus, (e) TEM
image of achannel-type b-keratin and air nanostructure from royal
blue back contour feather barbs of S. sialis. ( f ) TEM image of a
sphere-type b-keratin and air nanostructure from the dark turquoise
blue back contour feather barbs of C. maynana. (g i)
Representativetwo-dimensional small-angle X-ray scattering (SAXS)
diffraction patterns for the rudimentary, channel- and sphere-type
featherbarb nanostructures in (d f ), respectively. The SAXS
patterns for both channel- and sphere-type nanostructures exhibit
ring-like features that demonstrate the isotropy and short-range
spatial periodicity of these nanostructures, whereas the
rudimentarybarb nanostructure shows a diffuse, disc-like pattern.
The false colour encoding corresponds to the logarithm of the X-ray
scatteringintensity. Scale bars: (d) 250 nm; (e,f) 500 nm; (g i)
0.05 nm21. Photo credits: (a) Yiwen Yiwen (image in the public
domain);(b) Ken Thomas (image in the public domain); and (c) Thomas
Valqui (reproduced with permission).
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Here, we use synchrotron small angle X-ray scattering(SAXS) to
quantitatively characterize the nanostructureand optical function
of a large sample of structurallycoloured feathers with spongy barb
nanostructures,from across the phylogeny of birds. We examine
thenanostructure and optical properties of 297 distinc-tly coloured
feathers from 230 species belonging to163 genera in 51 avian
families (see electronic sup-plementary material, table S1). SAXS
is a precisionstructural tool routinely used in material science
todirectly measure bulk structural correlations in com-plex
nanostructured morphologies [2831]. SAXSenables a direct
experimental measurement of the two-dimensional projection of the
three-dimensional Fouriertransform of the scattering structure
(figure 3) withessentially no sample preparation, allowing for
rapidthroughput inconceivable with electron microscopymethods
[29,31,32]. The azimuthal average of theSAXS pattern gives the
X-ray scattering intensity as a
J. R. Soc. Interface
function of q, the scattering wavevector, or spatialfrequency of
variation in electron density (which is aproxy for variation in
refractive index). The SAXSpatterns resolve spatial correlations of
dimensions 2p/qthat range from a few tens to several hundred
nano-metres (figures 35). X-rays also interact only weaklywith soft
biological tissues because of the relatively lowelectron density of
biological media [2831,35]. Hence,SAXS provides single scattering
data that are highlysuited to quantitatively predict the
interactions ofvisible light with the nanostructure without
artefactsresulting from multiple scattering. Recently, we
appliedSAXS to a few species with non-iridescent featherbarb
structural coloursEastern Bluebird (S. sialis),Purple-throated
Cotinga (Cotinga maynana), BlueCotinga (Cotinga cotinga), Asian
Fairy Bluebird(Irena puella), Indian Roller (Coracias
benghalensis)and Blue Penguin (Eudyptula minor)and
successfullymodelled the directional light scattering properties
of
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101
q (nm1)
Porod asymptote (q4)
M. caerulescensA. clypeataG. victoria (unstructured)
H. mustelina (control)
101
A. laminirostris
scal
ed s
catte
ring
inte
nsity
visible q
(a) (b)
visible q
Figure 4. SAXS structural diagnosis ofweaklystructured,
controland unstructured feather barbs. (a) Representative
azimuthalSAXS profiles for the rudimentary sphere-type
nanostructure(structured*, electronic supplementary material, table
S2) inA. laminirostris (Ramphastidae), and the rudimentary
channel-type nanostructures (structured, see electronic
supplementarymaterial, table S2) in Melanotis caerulescens
(Mimidae) andAnas clypeata (Anatidae) as well as unstructured
feather barbsfrom Goura victoria (Columbidae) and Hylocichla
mustelina(Turdidae). The azimuthal profiles are normalized to one
alongthe intensity axis for ease of comparison. (b) The
azimuthal
inte
nsity
I(q
)
kks
ki
q
q
synchrotron X-rays
Figure 3. Experimental schematic for SAXS experiments onfeather
barb nanostructures. A small (approx. 50 mm2)sample of the distal
pennaceous portion of the feather vane isshown affixed to cover a 3
mm diameter hole on an aluminiumblock, which is then mounted in a
plane perpendicular to theincident X-ray beam. The two-dimensional
SAXS diffractionpatterns for both channel- and sphere-type
nanostructures exhi-bit ring-like features. Exploiting the circular
symmetry of theSAXS diffraction patterns, the scattering intensity
(I) is azi-muthally averaged as a function of q to obtain
scatteringprofiles, where the peaks correspond to the rings
observed inthe respective two-dimensional diffraction patterns. The
scatter-ing wavevector q measures the momentum transfer or
themagnitude and direction of the scattering of incident photons(ki
into ks) as a result of constructive interference from
structuralcorrelations of size 2p/q within the nanostructure.
4 Avian amorphous photonic nanostructures V. Saranathan et
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their underlying amorphous photonic
nanostructures[13,15,3638].
SAXS profiles for 18 weakly structured (blue lines), five
control(grey lines) and 16 unstructured feather barbs (black lines)
on asemi-log scale. The azimuthal profiles are vertically
displacedalong the intensity axis for clarity. The azimuthal
scatteringprofiles of the control feathers, many purple, magenta
andbright white feathers as well as several marginally
blue-grey(black lines) feathers did not deviate from Porods Law
even atlow q (,0.04 nm21). Thus, these feathers do not possess
anyunderlying barb nanostructure, ruling out any contribution
ofconstructive interference to their observed colours.The
azimuthalSAXS profiles from feathers with mainly slaty blue-black
to palegreyish-blue colours show slight to moderate deviations
fromPorods Law at low q, with these features resembling a
shoulderrather than a peak. Nevertheless, the spatial correlations
thatthese feather barbs do possess appear to be at the
appropriatelength scales to be able to produce visible structural
coloursthrough interference. (a,b) The thick horizontal line
indicatesthe range of spatial frequencies relevant for avian
visiblestructural colour production.
1.2. Self-assembly by phase separation
Macro-molecular self-assembly through phase separationis a
fundamental property of soft condensed matter sys-tems [28]. The
stability of a molecular mixture isdetermined by its temperature,
the strength of intermole-cular interactions (x) and the relative
volume fractions ofthe component materials. At lower temperatures
andintermediate volume fractions, a mixture may becomeunstable and
can proceed to unmix by one of two funda-mental physical processes
[28]. Phase separation of acompletely unstable mixture can proceed
via spinodaldecomposition (SD), which usually produces a
character-istic morphology of interconnected bicontinuous
channels[39,40]. The observed fractal-like patterns or motifs
beginat small length scales and spontaneously coarsen orthicken
over time roughly maintaining the same averageshape in a
scale-independent fashion (self-similarity). Bycontrast, a
meta-stable mixture can unmix throughnucleation-and-growth, which
proceeds via the develop-ment and subsequent coarsening of
spherical droplets ofthe minority component [41,42]. Unlike SD,
however,nucleation requires the crossing of an activation
barrier.If nucleation is fast and growth, relatively slow,
nearlyidentical (or monodisperse) spheres can form [43,44].For
simplicity, we have summarized here the
J. R. Soc. Interface
morphologies observed during the classical phase separ-ation of
a simple binary fluid mixture. However, phaseseparation
phenomenology should be modified to includenonlinear viscoelastic
mechanisms when the two nascentphases have distinct rheological
(i.e. mechanical) pro-perties, such as in mixtures of a
network-forming orpolymerizing component and a fluid [4547]. In
this scen-ario, polymerizing proteins may form networks that
resist
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(a) (b) (c) (d )
1 2 3 1 2 3103
102
1
10
1 2 3 4
37
Porod asymptote (q4)amorphous PS spheres(C. maynana)(Ta.
larvata)
1 2 3 4
scal
ed in
tens
ity I
(q)/
I(q p
k)
101
103
102
1
10
101
scaled q (q/qpk) scaled q (q/qpk)
spinodal polymer blendsI. puellaS. sialisP. iris
Porod asymptote (q4)
Figure 5. SAXS structural diagnosis of amorphous photonic
nanostructures in feather barbs. (a) and (c) depict,
respectively,representative normalized azimuthal SAXS profiles for
channel-type nanostructures from I. puella (Irenidae), S. sialis
(Turdidae),and Pitta iris (Pittidae) and sphere-type nanostructures
from C. maynana (Cotingidae) and Tangara larvata (Thraupidae) on
aloglog scale exhibiting clearly distinguishable structural
differences. The azimuthal profiles are scaled to compare across
differ-ent colours and nanostructural sizes. (b) and (d) show,
respectively, scaled azimuthal SAXS profiles for 159 channel- and
96sphere-type nanostructures on a semi-log scale. The colour of
each profile is coded to the approximate colour of the
correspondingfeathers based on its primary optical peak hue (pure
UV colours shown in black). These azimuthal profiles are vertically
displacedalong the y-axis for clarity. In addition to the primary
peak, the channel-type nanostructures (a,b) either have a weak to a
pro-nounced shoulder at approximately twice the dominant spatial
frequency, 2*qpk or lack any other significant feature, while
thesphere-type nanostructures (c,d) exhibit one or more pronounced
higher-order scattering peaks in addition to the primary peakat
ratios of approximately
p3 and
p7 times qpk. The grey dashed lines in all figures plot the
scaled experimental scattering profiles
from two polymer mixtures undergoing spinodal decomposition
[33,34] (a,b) and an amorphous film of self-assembled
colloidalpolymer spheres (c,d). The vertical lines at 1,2,3 (a,b)
and at 1,
p3 and
p7 (c,d) are visual guides for the expected positional
ratios for the SAXS peaks based on experimental observations of
classical spinodal and nucleated, close-packed spheremorphologies,
respectively.
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coarsening while the solvent lacks such dynamic elas-ticity.
Such viscoelastic processes lack self-similarity inthe coarsening
of domains, and are also hypothesized todynamically self-arrest
either owing to the cross-linkingof networks or from the
evaporation of solvent duringphase separation [4547].
Prum et al. [25] used TEM of serial sections to observethe
development of channel-type nanostructure in thegrowing feather
germ of parrot feathers. They foundthat these amorphous
intracellular nanostructuresdevelop spontaneously without any
underlying biologicaltemplate or prepattern of cytoskeletal fibres
or mem-branes and thus evidently self-assembled. Furthermore,they
pointed out that these nanostructures bear a quali-tative
similarity to morphologies self-assembled duringSD. Later, based on
the SAXS data from two species,Dufresne et al. [38] hypothesized
that both channel andsphere nanostructures in birds could be
self-assembledby arrested phase separation of filamentous
b-keratinprotein from the cellular cytoplasm. Further, they
[38]proposed that the two classes of nanostructures, channel-and
sphere-types, could possibly be self-assembled by SDand
nucleation-and-growth mechanisms, respectively.
Here, we further test the hypothesis that
constructiveinterference of light scattered by
three-dimensional
J. R. Soc. Interface
quasi-ordered photonic nanostructures is responsible forthe
non-iridescent plumage structural colours found indiverse avian
lineages [3,8,9]. We also quantitativelycompare the SAXS data from
hundreds of feathernanostructures with experimental scattering data
fromself-assembled, synthetic soft condensed matter systems.
Using single scattering theory [8,13,16,48], we pre-dict the
optical reflectance of each nanostructure fromthe SAXS structural
data and compare it with normalincidence optical measurements, and
in addition per-form angle-resolved spectrophotometry on a subset
ofthe feathers. Further, we quantitatively explore thedifferences
in the nanostructure and optical functionof channel- and
sphere-type barb nanostructures.
2. MATERIAL AND METHODS
2.1. Taxon sampling
We surveyed the birds of the world to identify all avianfamilies
and genera with probable non-iridescent structu-rally coloured barb
colours (usually blues, violets, greens,etc.) from museum specimens
and published illustrations(see electronic supplementary material,
table S1).We cross-checked target species for the presence of
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non-iridescent structural colours (peaked reflectance pro-files)
by visual inspection and optical spectrophotometryof museum skins.
We sampled across the gamut of knownstructural hues including the
near ultraviolet (visible tobirds but not to humans), violet, blue,
cyan and green,as well as saturated (peaked) yellowred hues
whenthey co-occur on birds with obvious barb structuralcoloration
(blues and greens). We included feathersamples of multiple plumage
patches with different col-ours from the same species as well as
differentlycoloured, but homologous patches from both sexes
ofsexually dichromatic species (see electronic supplemen-tary
material, table S1). We included in the samplesome species with
ambiguous bluegrey (e.g. Polioptilacaerulea, Pachyptila vittata)
and dull slate-coloured(e.g. Brachypteryx montana, Rhyacornis
fuliginosus)feathers (see electronic supplementary material,table
S1). We also sampled five control feathers witheu- and
phaeo-melanin, carotenoid pigments, and unpig-mented matte white
colour, chosen from the avian generaCorvus, Hylocichla, Carduelis,
Saltator and Larus thatshould in theory lack any structural
colour-producingbarb nanostructure (see electronic
supplementarymaterial, table S2). In order to assess the
variability ofbarb structural colour within and among individuals
ofa single species, we assayed feathers from multiplestudy skins of
S. sialis and C. maynana (see theelectronic supplementary
material).
In total, including controls, we examined the nano-structure of
297 distinctly coloured feathers from 230species belonging to 163
genera in 51 avian families(see electronic supplementary material,
table S1).Feathers were obtained from study skins of the taxaof
interest from Yale Peabody Museum of Natural His-tory (New Haven,
CT, USA), University of KansasNatural History Museum and
Biodiversity ResearchCenter (Lawrence, KS, USA), American Museum
ofNatural History (New York, NY, USA), Natural His-tory Museum at
the Academy of Natural Sciences(Philadelphia, PA, USA), Harvard
UniversityMuseum of Comparative Zoology (Cambridge, MA,USA) and
University of Oxford Natural HistoryMuseum (Oxford, UK) (see
electronic supplementarymaterial, table S1). Immature individuals
or specimenswith obvious fading or degradation were avoided.
2.2. Small angle X-ray scattering
For SAXS data collection, small (approx. 50 mm2)samples of the
distal pennaceous portion of the feathervanes were affixed to an
aluminium block using SuperGlue (DuPont, Wilmington, DE, USA) over
a 3 mmdiameter hole. The block was mounted in a plane
per-pendicular to the incident X-ray beam as shown inthe
experimental schematic (figure 3). Pinhole SAXSdata on two to three
individual barbs per feathersample were collected in transmission
geometry, atbeamline 8-ID-I of the Advanced Photon Source,Argonne
National Laboratories (Chicago, IL, USA).We used a 15 mm (Horiz.
Vert.) beam (1.68 A,7.35 keV, 50 0.2 s exposures, sample-detector
dis-tance 3.56 m, flux 2.7 109 photons s21). Beam sizewas minimized
to sample as few spongy medullary
J. R. Soc. Interface
cells as possible (they are typically approx. 1015 mm3 but vary
with taxon [49]). The azimuthallyaveraged scattering profiles were
calculated from theCCD-collected two-dimensional SAXS speckle
diffrac-tion patterns using the freely available Matlab-implemented
software, XPCSGUI, developed by beamline8-ID
(http://8id.xor.aps.anl.gov/UserInfo/Analysis/)at 200 equal
q-partitions, and with customized masksto filter out the beam stop
[15,38,50]. SAXS data fromthe feathers of Cittura cyanotis and the
yellow throatof Psarisomus dalhousiae (see electronic
supplementarymaterial, tables S1 and S2) were collected using a7.35
keV beam (1.68 A, 50 mm horizontal 50 mm ver-tical, 9.24 m camera
length, 50 0.1 s exposures) on aPilatus2M detector at beamline I22
of the DiamondLight Source, Didcot, UK.
Biomimetic amorphous samples that closely mimicquasi-ordered
arrays of a nucleation-and-growth struc-ture were prepared by drop
casting a 50 : 50 bidispersemixture of 258 and 286 nm diameter
polystyrene (PS)spheres [51]. SAXS measurements of
biomimeticsamples were carried out by sandwiching the samplein an
aluminium sample holder between two pieces of0.0025-inch (approx.
63.5 mm) thick adhesive Kaptontape, purchased from McMaster-Carr
(Catalogue no.7648A33). Light scattering data for phase-separating
spi-nodal morphologies were obtained from Takenaka &Hashimoto
[33] and Hayashi et al. [34].
2.3. Normal incidence and angle-resolvedspectrophotometry
Normal incidence reflectance measurements were madefrom two or
three different locations within eachsampled plumage patch and
averaged. Whenever poss-ible, the same museum study skins that were
sampledfor the SAXS structural assays were used for the
corre-sponding reflectance measurements. For 77 plumagepatches, the
original study skins were locally unavail-able for
spectrophotometry, and reflectance wasmeasured from the individual
feathers collected forSAXS assays (see electronic supplementary
material,table S1). These feather-based reflectance measure-ments
were essentially identical to those measuredfrom other museum
specimens of the same species. Allmeasurements were made in
relative darkness using anOcean Optics S2000 (Dunedin, FL, USA)
fibre opticspectrophotometer and an Ocean Optics DH-2000-BAL
deuteriumhalogen light source, following stan-dard procedure [79].
The S2000 provides 2048 datapoints between 178 and 879 nm. In order
to shieldany ambient light and control the irradiance, the
bifur-cated fibre-optic cable was inserted into a probe
holder.Reflectance was measured using normally incident lightat a
distance of approximately 6 mm from approxi-mately a 3 mm2
illuminated patch of the integumentwith a 500 ms integration time
and calibrated usingan Ocean Optics Spectralon matte white
reflectancestandard and with a matte black velvet cloth asdark
reference.
We also conducted angle-resolved spectrophotometryin diffuse
scattering geometry [13,36,37] on a smallerset of 22 individual
feather samples with both channel-
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and sphere-type nanostructures, to differentiatebetween double
scattering versus pigmentary origin ofthe short-wavelength
secondary reflectance featuresusing the effects of angular
dispersion, as well as to esti-mate the nanostructural parameters
independent of theSAXS data. The bird feathers were mounted
horizon-tally so that their axes were perpendicular to therotation
axis of a goniometer. Collimated white lightfrom an Optics
DH-2000-BAL deuteriumhalogenlight source was incident on the sample
at normal inci-dence and with a spot size of approximately 1 mm.The
scattered light was collected by a lens and focusedonto an optical
fibre connected to a spectrometer(Ocean Optics S2000). The spectral
resolution was1.5 nm and the angular resolution, determined
mainlyby the collection angle of the lens, was about 58.To measure
the scattered light, we fixed the feathersample and rotated only
the detection arm, in whichcase, the illumination angle remained
constant whilethe angle of observation changed. The measured
spectraof scattered light were normalized by the incident
sourcelight spectrum after dark subtraction.
2.4. Electron microscopy
We followed standard specimen embedding proceduresfor TEM [79].
For scanning electron microscopy(SEM), longitudinally and
cross-sectionally fracturedfeather barb samples were gold-coated
and studied on aHitachi SU-70 and a Philips XL 30 environmental
SEMat a range of tilt angles.
2.5. Parametrization of small angle X-rayscattering structural
data and opticalreflectance spectra
The two-dimensional SAXS diffraction patterns forboth channel-
and sphere-type nanostructures exhibitring-like features (figure
2h,i). Exploiting the circularsymmetry of the SAXS diffraction
patterns, we azi-muthally integrated them using the XPCSGUIpackage
after masking out the beam stop pixels, toobtain profiles of the
scattered intensity as a functionof scattering wavevector, I(q), at
200 equal q-partitionsor spatial frequency bins (figures 35). The
peaks in theazimuthal profiles correspond to the rings observed
inthe respective two-dimensional diffraction patterns(figure 3).
The azimuthally averaged profiles weredeconvolved, or peak-fitted,
to estimate the peak qvalue, intensity, and the full-width at
half-maximum(FWHM) of the scattering peaks, using the freely
avail-able peak-fitting software, Fityk (v. 0.8.2; [52]) on
aWindows platform. We used a Porod background (q24
dependence; see 3 and figures 45) and the split-Pearson VII
function with a LevenbergMarquardtleast square method to fit all
the observed scatter-ing features (peaks and shoulders) present in
theazimuthal profiles. The Pearson VII function is a com-bination
of Gaussian and Lorentzian (Cauchy) typepeak profiles that is
generally used to closely approxi-mate X-ray scattering peaks
[53,54]. The split-PearsonVII accommodates any asymmetry in peak
shapes.
J. R. Soc. Interface
The unprocessed optical reflectance measurementswere also
similarly deconvolved to estimate the relevantoptical peak
parameters such as wavelength of peak reflec-tance, intensity and
FWHM of the reflectance peak usingFityk. The FWHM characterizes the
saturation of optical(reflectance) signals [55]. We used a constant
backgroundand a Gaussian or a split Gaussian function with a
Leven-bergMarquardt least square method to fit all theobserved
spectral features (peaks and shoulders) presentin the reflectance
profiles. The split Gaussian functionwas used for asymmetrical peak
profiles.
2.6. Small angle X-ray scattering single-scattering reflectance
predictions
We used the azimuthally averaged SAXS structuralspectra to
directly predict the optical reflectance spec-tra of the respective
amorphous barb nanostructuresusing single-scattering theory by
mapping the SAXSintensity from wavevector or spatial frequency (q)
towavelength (l) space [8,13,16,48,50]. This result followsfrom
Braggs Law, for under normal incidence of lightand back-scattering
geometry (the angle between inci-dence and observation, u 08), the
scatteringwavevector (q) and the wavelength of light (l) aresimply
related as
l 2 2pq
navg; 2:1
where 2p/q is the average inter-scatterer or nearest-neighbour
spacing D, and navg is the average or effectiverefractive index of
the nanostructure [13].
2.7. Statistical analyses
Regression analyses were performed using the statisticstoolbox
of Matlab 2008a (The MathWorks Inc., Natick,MA, USA) and MINITAB
statistical software, release 16(Minitab Inc., State College, PA,
USA) running on aWindows platform. One-way ANOVA and generallinear
model tests for the statistical difference of theslopes and
intercepts of two regression lines [56] wereall performed in
MINITAB. The p-value for statistical sig-nificance was set at
0.05.
3. RESULTS
3.1. Comparative structural diagnoses of featherbarb photonic
nanostructures
Of the 297 feathers assayed in this study, the
azimuthalscattering profiles of the control feathers (n 5)did not
deviate from Porods Law even at low q(,0.04 nm21) (figure 4, and
electronic supplementarymaterial, table S2). The scattering
profiles of some feath-ers with longer-wavelength reflectance,
including deeppurple and magenta (n 9), bright whites (n 3) andsome
blue-grey feathers (n 4) did not deviate fromPorods Law as well
(figure 4 and electronic supplemen-tary material, table S2). Porods
Law (I(q)/ q24) is thenull expectation for the scattering from an
unstructuredmaterial characterized by sharp interfaces or edges
separ-ating two media [31]. Thus, these 21 feathers do not
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slope = 1.87, R2 = 0.94 (channels)slope = 1.69, R2 = 0.97
(spheres)
slope = 2 (spinodal)slope = 3 (close-packed spheres)
0.025 0.030 0.035 0.040
0.040
0.050
0.060
0.070
0.080
0.090
first SAXS peak position, qpk1 (nm1)
seco
nd S
AX
S pe
ak p
ositi
on, q
pk2
(nm
1 )
Figure 6. Regression plot of the first and second SAXS peaksof
channel- (open triangles) and sphere-type (shaded circles)amorphous
barb nanostructures. The colour of each triangleor circle is coded
to the approximate colour of the correspond-ing feather (UV colours
in black). The thin vertical andhorizontal lines at each data point
indicate the standarderror of the mean (s.e.m). The solid blue and
green lineswith corresponding slopes of 2 and
p3 indicate the expected
positional ratios for the second SAXS peak based on
exper-imental observations of spinodal and nucleated,
close-packedsphere morphologies, respectively. The solid and dashed
greylines, respectively, indicate the 95% confidence interval ofthe
regressions.
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possess any underlying barb structure at optically
relevantlength scales ruling out any contribution of
constructiveinterference to their observed colours.
The SAXS data from a further 18 out of 297 feathers(see
electronic supplementary material, table S2) withslaty blue-black
to pale greyish-blue colours, such asthe primary coverts of the
Silver-breasted Broadbill(Serilophus lunatus, Eurylaimidae; figures
2a and 4)exhibited diffuse, disc-like two-dimensional SAXS
dif-fraction patterns (figure 2g). Their correspondingazimuthal
averages showed slight to moderate devi-ations from Porods Law at
low q; these featuresresembled a shoulder rather than a peak
(figure 4).Nevertheless, the spatial correlations of these
featherbarbs appear to be at the appropriate length scales
toproduce visible structural colours through
constructiveinterference (figure 4). For 16 of these 18 feathers,
theprimary scattering feature could not be estimatedwith
peak-fitting procedures (called structured, seeelectronic
supplementary material, table S2).
We examined some of these slaty, blue-grey featherswith
distinctive disc-like SAXS patterns using SEM.SEM images from two
structured species, Serilophuslunatus (Eurylaimidae) and Melanotis
caerulescens(Mimidae), revealed a very restricted and thin layer(1
mm or less) of a disordered channel-like network ofspongy b-keratin
bars present at the periphery of themedullary barb cells (figure 2d
and electronic supplemen-tary material, S1c). All of these 16
structured speciesare closely related to other species known to
possess thechannel-type medullary barb nanostructures (figure 5band
electronic supplementary material, table S2). Thetwo species for
which the peaks could be estimated(called structured*, see
electronic supplementarymaterial, table S2) were the toucans
Andigena laminiros-tris and Pteroglossus viridis (Ramphastidae).
Othertoucans have sphere-type nanostructures (figure 5d andsee
electronic supplementary material, table S2). SEMimages of A.
laminirostris revealed a very thin layer(1 mm or less) of hollow
spheroidal concavities of highlyvariable sizes and shapes (highly
polydisperse) in thespongy b-keratin at the periphery of the
medullary cells(see electronic supplementary material, figure
S1d).Thus, these blue-grey and slate-grey feathers characterizedby
diffuse, disc-like SAXS patterns possessed rudimentaryand highly
disordered versions of channel- (structured)and sphere-type
(structured*) nanostructures, found intheir close relatives.
The two-dimensional SAXS diffraction patterns ofrest of the barb
nanostructures assayed exhibit ring-likefeatures that demonstrate
strong nanostructural isotropyand short-range spatial periodicity
(figure 2h,i). Theazimuthally averaged scattering profiles display
peaksthat correspond to the rings observed in the
respectivetwo-dimensional diffraction patterns (figure 3). For
com-parison of feathers across different structural
colours,nanostructural dimensions and scattering intensities,we
normalized all azimuthal SAXS profiles by the pri-mary peak spatial
frequency (qpk) and intensity (I(qpk))(figure 5). In the high q
region (q . 0.1 nm21), theSAXS profiles follow Porods Law. However,
at low andintermediate q values (q , 0.1 nm21), the azimuthalSAXS
profiles of most barbs exhibit clearly
J. R. Soc. Interface
distinguishable features that can be used to identifythe barb
nanostructures.
Of the remaining 258 feathers, 218 were readily clas-sifiable
into the two known classes of three-dimensionalbarb nanostructures
based on the features (observedversus expected number and relative
positions ofpeaks) of their SAXS patterns. Many feathers thatlacked
any higher-order scattering features besides theprimary structural
correlation peak (n 48) or exhib-ited a low intensity second-order
shoulder atapproximately twice the dominant spatial
frequency(1.879+ 0.005; n 95) were identified as the channel-type
(figures 5a,b and 6). Both the structure factorscalculated from TEM
images of channel-type nano-structures [25], as well as
experimental scattering datafrom other interconnected bicontinuous
network nano-structures [33,34] support these conclusions (see
3.2).By contrast, many other feathers (n 75) diagnosti-cally
exhibited two or more pronounced, higher-orderpeaks indicative of
spherical form-factor scatteringfringes [25,57] at ratios of
approximately
p3 (1.693+
0.004; n 75), p7 (2.498+ 0.010; n 75) and p11(3.475+ 0.063; n
11) and were recognized as thesphere-type (figures 5c,d and 6). The
secondaryshoulder at approximately 2*qpk in channel-type
nano-structures is comparatively much broader in width andweaker in
intensity relative to the primary peak than isthe second-order peak
(approx.
p3*qpk) from sphere-
type nanostructures (see electronic supplementarymaterial,
figure S2). We further corroborated many ofthese SAXS structural
assignments of barb nanostruc-tures based on previously published
(see [3] andreferences therein) and our own TEM and SEMimages for
39 channel- and 27 sphere-type barb
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nanostructures (see electronic supplementary material,table
S2).
For the remaining 40 feathers, we provisionally ident-ified the
nanostructures as the structural interpretationof their SAXS data
was less straightforward and/or insome feathers, the azimuthal
profiles were comparativelynoisier, i.e. jagged (see figure 5 and
electronic supple-mentary material, table S2). The azimuthal
SAXSprofiles of 21 such feathers (sphere*, see electronic
sup-plementary material, table S2) exhibited only a
broadsecond-order peak between 1.604 and 1.737 times qpk,but
consistent with the distribution for unambiguoussphere-type
morphologies (see electronic supplementarymaterial, figure S2). The
scattering intensities of thesecond-order peaks from most of these
feathers werealso considerably higher than the mean intensity of
thechannel-type second-order shoulders (see electronicsupplementary
material, figure S2). By contrast, the azi-muthal profiles of eight
feathers were noisy with onlythe primary scattering peak present,
consistent with thechannel morphology, while a further 11 feathers
exhibitedsecond-order shoulders but at smaller positional
ratios(1.7471.796) than expected (channel*, see
electronicsupplementary material, table S2). These
tentativenanostructural assignments were validated based on
EMimages (nine channel* and five sphere*) and/or theunambiguously
identified nanostructure present in otherstructurally coloured
plumage patches on the samespecies or in a few cases, indirectly
assumed from thatin closely related taxa within the same genus (see
elec-tronic supplementary material, figure S2 and table S2).We also
evaluated the relatively noisy barb morphologiespresent in Myiomela
leucura (Turdidae), Chiroxiphia cau-data (Pipridae) and Euneornis
campestris (Thraupidae),using SEM. SEM images of M. leucura
revealed a spindlychannel morphology with anastomosing networks
ofb-keratin bars of variable thickness (see electronic
sup-plementary material, figure S1a). SEM images ofC. caudata and
E. campestris revealed sphere-type nanos-tructures with a greater
degree of polydispersity in thesize and the shape of the air
spheres than in typicalsphere nanostructures found in their close
relatives (seeelectronic supplementary material, figure S1b,f ).
Thesenoisier nanostructures appear to be more variable
(poly-disperse) versions of the types of nanostructures foundin
their closest, structurally coloured relatives.
3.2. Structural comparisons of amorphous barbnanostructures and
synthetic soft mattersystems
We compared the normalized azimuthal scattering pro-files of the
unambiguously diagnosed instances ofchannel (n 143) and sphere (n
75) barb nanostruc-ture to experimental light-scattering data from
polymermixtures in early and late stages of SD [33,34], and
aself-assembled, amorphous film of colloidal polymer(PS) spheres,
mimicking a quasi-ordered nucleation-and-growth nanostructure
(figure 5ad).
The shape of the scattering profile of a classical spi-nodal
mixture is scale independent; the overall structureof spinodal
morphologies are universal even though thespecific structure in a
phase-separating sample may
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differ locally [39,40], but experimentally, there is
sub-stantial variation between early and late stages of SDat
intermediate and high q [33,34,58,59]. The exper-imental spinodal
polymer profiles [39,40] provide areasonable fit to the normalized
channel-type scatteringprofiles from feather barbs at low q (figure
5a), eventhough the width (FWHM) of the primary SAXSpeak of
channel-type nanostructures narrows as thedominant length scale of
the nanostructure increases,i.e. for those producing longer
wavelength colours(figure 5b and electronic supplementary
material,figure S3a). At high q, the polymer spinodal mor-phologies
lack any higher-order feature at the earlystage or exhibit a
second-order shoulder at approxi-mately 2 or approximately 3qpk at
the late stage[33,34]. Similarly, the channel-type barb
nanostructureseither lack or exhibit a shoulder or second-order
maxi-mum at approximately twice the peak spatialperiodicity (mean
1.87) (figures 5a,b and 6). However,the positions of the secondary
shoulder from the channelnanostructures are probably underestimated
becausesuch shallow (broad and low intensity, see electronic
sup-plementary material, figure S2) features are difficult
toprecisely estimate through curve-fitting procedures.
The SAXS scattering profile from a film of self-assembled,
quasi-ordered, colloidal polymer spheresreveals a series of
higher-order form factor diffraction(fringes) peaks at similar
relative positions (
p3 andp
7) to those seen in azimuthal profiles of
sphere-typenanostructures (figure 5c,d). The width (FWHM) ofthe
primary SAXS peak of sphere-type nanostructuresis also in good
agreement with that of the self-assembled,amorphous PS spheres.
(The X-ray scattering from anarray of solid spheres is
indistinguishable to that fromits inverse structureair spheres in
solidand thereforethis direct comparison is valid according to
Babinetstheorem [31].) Although the PS spheres are self-assembled
into an amorphous structure, the spheresthemselves were not
synthesized in situ by a nuclea-tion-and-growth process; however,
the scattering profileof a three-dimensional amorphous array of
spheresgrown by a nucleation-and-growth mechanism shouldbe similar
[57]. The number and strength (intensityand width) of the
higher-order peaks in the sphere-typenanostructures are
sample-specific, and reflect thedegree of quasi-periodic or
nearest-neighbour order andsphere size monodispersity.
3.3. Comparative structural properties ofamorphous barb
nanostructures
We examined the width (FWHM) of the primary SAXSpeak, Dq, to
quantitatively characterize the extent ofspatial periodicity in the
channel and sphere classes ofamorphous nanostructures (n 255;
structured andstructured* were excluded; see electronic
supplementarymaterial, table S2).
The spatial coherence length, j, is given by 2p/Dq.For ordered
systems, j describes the crystallinedomain size, whereas in
quasi-ordered or amorphoussystems, the coherence length (after
scaling by the cor-responding peak spatial periodicity, j/D) can
provide ameasure of the extent of short-range nearest-neighbour
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order [13,60]. For both classes of amorphous barbnanostructures,
j is only a few times the dominantlength-scale of spatial
correlations, D (2p/qpk), reflect-ing the very local nature of
spatial order in thesesystems [13]. However, the mean structural
FWHM(Dq) of sphere nanostructures is significantly smallercompared
with channels (one-way ANOVA, F 26.82, p , 0.001, n 255), even
after scaling by thecorresponding peak spatial frequency of
structural cor-relations, qpk (one-way ANOVA, F 28.51, p , 0.001,n
255), suggesting that sphere-type nanostructuresare more ordered,
or have a larger coherence lengththan channel-type nanostructures
(see electronicsupplementary material, figure S3).
In addition, the FWHM of the primary SAXS peakincreases
significantly with the dominant spatial fre-quency of structural
correlations, qpk, for both channel-(r2 0.22, p , 0.001) and
sphere-type (r2 0.29, p ,0.001) nanostructures (see electronic
supplementarymaterial, figure S3a,b). However, the statistical
signifi-cance of this relationship persists only for
channel-typenanostructure (r2 0.067, p 0.001), after scaling
theFWHM by the corresponding qpk (see electronic sup-plementary
material, figure S3c,d). In other words,channel-type nanostructures
with larger size scale ofspatial periodicity (i.e. D) producing
longer wavelengthcolours have a smaller structural (SAXS) peak
width,and consequently a larger coherence length or
greatershort-range order. In contrast, the sphere-type
nanostruc-tures appear to be nearly scale invariant as revealed
bythe same relative widths of the structural peaks acrossall length
scales (see electronic supplementary material,figure S3d). These
structural differences between channeland sphere morphologies are
perhaps a result of their dis-similar processes of phase separation
and arrest duringintracellular self-assembly, which are also
probablyaffected by the subsequent desiccation of medullarybarb
cells in different ways.
3.4. Comparative optical function of amorphousbarb
nanostructures
The slaty blue-black to pale greyish-blue feathers
withrudimentary (structured and structured*) barbnanostructures
generally exhibited a broad, low inten-sity, sperm-whale-shaped
reflectance profile with agradually decreasing reflectance at
longer wavelengthsand a more rapid decline at shorter wavelengths.
Thepeak parameters from the optical reflectance of thesefeathers
could not all be consistently estimated (seeelectronic
supplementary material, table S2) and aretherefore excluded from
further optical analyses. Never-theless, the SAXS results
demonstrate that thesefeathers are sufficiently nanostructured at
the appropri-ate length scales to produce the observed colours
viaconstructive interference (figure 4).
The spectral peaks in the optical reflectance measure-ments of
the structurally coloured feathers with channel-and sphere-type
nanostructures characterized in thisstudy varied from 343.83 to
639.37 nm (n 255; seeelectronic supplementary material, table S2).
Manyfeathers, particularly royal (medium) blue to turquoise(light)
blue ones, with either class of barb nanostructure
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(n 86), exhibited a characteristic bimodal reflectanceprofile
with an additional peak in the ultraviolet (UV)/violet distinct
from the primary reflectance peak in thevisible (400700 nm) region
(figure 7df,mo, andelectronic supplementary material, table S2).
These sec-ondary (short wavelength) peaks are qualitatively
quitedifferent from the relatively low intensity UV pig-mentary
(carotenoid) transmittance peaks seen innanostructured feather
barbs with cortical pigmentation,for instance, in structural greens
(figure 7gi,q). The dipbetween the two peaks in the former are
relatively shal-lower and the secondary peak is nearly of the
sameamplitude or higher than the primary single-scatteringpeak
(figure 7). Unlike the UV peaks in spongy barbnanostructures with
cortical pigments, these secondarypeaks are of structural origin
owing to the double scatter-ing of light and not explained by the
higher-orderstructural correlations in the X-ray scattering
data[36,37]. Diagnostically, the double scattering peaksoccur at
nearly constant relative spectral ratio to oneanother (1/
p2) as expected from optical theory [36,37]
(criterion 1). Moreover, double scattering peaks arealso
depolarized (criterion 2) and exhibit reverse angulardispersion
(see electronic supplementary material, figureS4bf ) that is
specifically predicted by optical theory(criterion 3) [36,37],
whereas the static spectral featuresthat are produced by pigmentary
absorption are not(see electronic supplementary material, figure
S4g,h).We have described the complete mechanistic basis ofthe
double scattering phenomenon in detail elsewhere[36,37]. We
tentatively identified 58 of these 86 feathersas double scattering
candidates based on criterion 1alone (see electronic supplementary
material, table S2),while we were able to unambiguously document
doublescattering in the remaining 28 feathers based on criterion2
and/or 3 (see electronic supplementary material, figureS4bf ).
Here, we mainly consider the primary opticalreflectance peak, which
originates from the single scatter-ing of incident light whereby
each incident photon isscattered only once before it exits the
nanostructure [13].
Many feathers (n 77) producing structural greensand longer
wavelength hues with reflectance peaksabove 500 nm have distinct
spectral indications of thepresence of carotenoid or psittacofulvin
pigments in theouter b-keratin cortex of the barb, the
mechanisticbasis of which are well established for many
species[3,14,61]. Unlike a typical sigmoidal reflectance profileof
carotenoid pigmented barbs that plateau at higherwavelengths [10],
the reflectance from these featherswere distinctly peaked or
saturated, but with a relativelysharp cessation of the reflectance
intensity on theshort-wavelength side of the peak (figure 7gi,pr
andelectronic supplementary material, figure S4g,h). Inaddition,
the reflectance spectra of these feathersshowed a minor UV
transmittance peak at approximately350 nm and/or several low
intensity sub-peaks at inter-mediate wavelengths [3,14,61]. In
contrast to doublescattering feathers, these pigmentary spectral
featureswere angle-independent (see electronic
supplementarymaterial, figure S4g,h). Therefore, we
conservativelyexcluded these nanostructures for analyses involving
thesaturation or widths (FWHM) of the primary reflectancepeaks, as
the short and middle wavelength pigmentary
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(d)
(g) (h) (i)
( j) (k) (l)
(m)
(p) (q) (r)
(n) (o)
(e) ( f )
Figure 7. Single-scattering SAXS reflectance predictions for the
primary optical peaks of channel (a i) and sphere-type (
jr)amorphous barb nanostructures. SAXS single-scattering
reflectance predictions (black lines) and measured normal
incidencereflectance curves (coloured lines) for (a) UV (black)
belly feather barbs of Charmosyna papou (Psittacidae), (b) violet
primaryfeather barbs of Acryllium vulturinum (Psittacidae), (c)
royal blue rump feather barbs of S. sialis (Turdidae), (d) sky blue
rumpfeather barbs of Alcedo atthis (Alcedinidae), (e) deep azure
blue back feather barbs of Irena puella (Irenidae), ( f ) electric
bluewing covert feather barbs of Pitta maxima (Pittidae), (g)
emerald green back feather barbs of Ailuroedus buccoides
(Ptilonor-hynchidae), (h) emerald green back feather barbs of
Charmosyna papou (Psittacidae), (i) emerald green back feather
barbs ofCalyptomena whitehadi (Eurylaimidae), ( j) deep blue throat
feather barbs of Tangara chilensis (Thraupidae), (k) royal bluewing
covert feather barbs of Wetmorethraupis sterrhopteron (Thraupidae),
(l ) violet scapular feather barbs of Conirostrum albi-frons
(Thraupidae), (m) dark turquoise blue back feather barbs of C.
maynana (Cotingidae), (n) sky blue back feather barbs ofmale
Tersina viridis (Thraupidae), (o) azure blue rump feather barbs of
Lepidothrix serena (Pipridae), ( p) golden yellow crownfeather
barbs of Lepidothrix vilasboasi (Pipridae), (q) electric green back
feather barbs of Chloronis riefferii (Thraupidae), (r)golden crown
feather barbs of Tangara larvata (Thraupidae). The colour of the
measured reflectance curves is approximatelycoded to the colour of
the feather barbs based on the spectral position of the primary
reflectance peak.
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0.025 0.030 0.035 0.040
0.010
0.012
0.014
0.016
0.018
0.020(a) (b)
SAXS peak spatial frequency, qpk (nm
1)SAXS peak spatial frequency,
qpk (nm1)
0.020 0.030 0.0400.008
0.010
0.012
0.014
0.016
0.018
0.020
navg = 1.201; R2
= 0.77
f = 34%navg = 1.265; R
2 = 0.84
f = 46%
optic
al p
eak
spat
ial f
requ
ency
,k p
k (n
m
1 )
Figure 8. Regression plots of the primary optical peak hue from
normal incidence reflectance measurements expressed as peakspatial
frequency (kpk 2p/lpk) against the dominant spatial frequency of
structural correlations (qpk) measured using SAXSfor (a) channel-
(shaded triangles) and (b) sphere-type (shaded circles)
nanostructures. For both nanostructural classes, thesize of the
nanostructural periodicity measured by SAXS strongly predicts, i.e.
scales with the measured primary peak hue,demonstrating that the
underlying barb nanostructures are tuned to produce the observed
structural colours. The inverse oftwice the slope of the regression
yields navg, the average or effective refractive index (and hence
f, the keratin volume fraction)for each class of nanostructure. The
estimated navg and f for sphere nanostructures on the whole (1.265,
46%) is significantlyhigher than that for channel morphologies
(1.201, 34%) and congruent with predictions of the phase separation
hypothesis.The colour of each triangle or circle is coded to the
approximate colour of the corresponding feather (UV colours in
black).The vertical and horizontal lines at each data point
indicate the standard error of the mean (s.e.m).
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absorption could lead to an underestimation of theiractual
widths (see figure 7gi,pr and electronic sup-plementary material,
figure S4g,h). However, thespectral position of the reflectance
peak or hue (lpk) isrelatively unaffected by the pigmentary
absorption andthe inclusion or exclusion of these data here did not
sig-nificantly alter the results.
3.4.1. Single-scattering optical predictions of amorphousbarb
nanostructuresBased on single scattering theory, we applied
BraggsLaw (equation (2.1)) to the azimuthal SAXS profiles toobtain
the single scattering optical reflectance predic-tions of amorphous
barb nanostructures [38,50]. Fromequation (2.1), for a given
nanostructure, the predictedpeak hue depends on the size of the
nanostructuremeasured by SAXS (D) and the average or
effectiverefractive index of the nanostructure (navg), while
thepredicted optical saturation or peak width depends onthe spatial
coherence length, j (2p/Dq) alone. Althoughwe predicted the optical
reflectance curves for all 255barb nanostructures, here we present
these results onlyfor a small subset of feathers, owing to space
limitations(figure 7). We summarize below the goodness of fit
ofSAXS single scattering optical predictions to normal-incidence
reflectance measurements, based on pairwiseregressions of peak hue
and saturation of the reflectancemeasurements and reflectance
predictions for bothchannel- and sphere-type nanostructures.
There is a strong positive correlation between themeasured
primary optical peak hue (expressed in spatialfrequency, k 2p/lpk)
and the dominant spatialfrequency of structural correlations, i.e.
the primarySAXS peak wavevector (qpk) for both channel and
J. R. Soc. Interface
sphere-type nanostructures (figure 8a,b). For bothclasses of
barb nanostructure, the size of the nanostruc-tural periodicity
scales strongly with primary peak hue,demonstrating that the
underlying barb nanostructuresare tuned to produce the observed
structural colours.The correlation is stronger for sphere-type (r2
0.84)than for channel-type nanostructures (r2 0.77).
Thisrelationship persists even for those barb
nanostructuresproducing longer wavelength colours (peaking
atapprox. 550 nm and higher) that probably involvecortical
pigments.
Although the average inter-scatterer spacing D or thedominant
length scale of nanostructural periodicity canbe directly measured
using SAXS, there is no directmethod to measure the average or
effective refractiveindex of the amorphous barb nanostructure,
navg. Weused two independent methods to estimate navg: (i)
bycorrelating normal incidence optical measurements withSAXS
structural data using equation (2.1) and (ii)from angle-resolved
optical reflectance measurements.
First, we use the regression relationship in figure 8 toestimate
the average or effective refractive index (navg),and hence the
filling or volume fraction (f) of b-keratinfor each class of
nanostructure as a whole, using the Max-wellGarnett effective
medium approximation [51]. Theinverse of twice the slope of the
regression yields navg (seeequation (2.1)). The estimated global
navg for spherenanostructures (1.265; 46% f) is significantly
largerthan that for channel morphologies (1.201; 34% f; one-way
ANOVA, F 31.45, p , 0.001, n 255). Thisresult is congruent with
predictions of the phase separ-ation hypothesis, since, under
similar thermodynamicconditions (kBT/x), nucleation-and-growth
shouldoccur at higher volume fractions of keratin (hence navg)
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compared with SD [25,38]. Further, for each of the 255barb
nanostructures, we calculated its navg by substitut-ing the values
of qpk from SAXS profiles and lpk fromnormal-incidence optical
reflectance data in equation(2.1) (see electronic supplementary
material, table S2).The navg estimated thus is significantly
positively corre-lated with the peak optical hue, lpk for both
channels(r2 0.44, p , 0.001) and spheres (r2 0.17, p ,0.001) (see
electronic supplementary material, figureS5a,b). The increase in
navg with lpk suggests that pro-duction of a longer wavelength hue
by either class ofnanostructure involves increases in both the
lengthscale of spatial periodicity (D) (figure 8) as well as
thekeratin volume fraction (navg), instead of independentlyvarying
one parameter or the other. The scatter in theplot probably
reflects variation in the length scale ofspatial periodicity and
keratin volume fraction (hencenavg) among different species with
similar structurallycoloured plumages.
Since amorphous feather barb nanostructures havethe same angular
dispersion for specular reflection anddiffuse scattering peaks,
they both share a commonphysical origin, and under directional
lighting con-ditions, the reflectance peak depends only on theangle
between incidence and observation [13,36,37].Exploiting this, we
measured the angle-resolved diffusescattering spectra of barb
nanostructures for a smallsubset of channel- (n 11) and sphere-type
(n 11)nanostructures. We estimated navg independent of theSAXS data
by analysing the angular dispersion of theprimary optical peak
using a modified form of BraggsLaw (equation (2.1)), taking into
account the reducedangle with respect to the normal at which light
travelsinside the barb nanostructure (i.e. Snells Law):
lpku 2Dn2avg sin2u1=2; 3:2
where the primary optical peak position, lpk, varieswith u, the
angle between incidence and observation.A plot of l2pk against
sin
2u yields navg (p
y-intercept)but also D (0.5/
pslope) [62,63] (see electronic sup-
plementary material, figure S6a). The correspondingvalues of D
(see electronic supplementary material,figure S6b) and navg (see
electronic supplementarymaterial, figure S6c) obtained from these
two indepen-dent methods are consistent and agree to be within
7percent of each other. Unlike photonic crystals, however,
theoptical diffuse scattering and specular reflection intensitiesof
amorphous barb nanostructures falls off rapidly atshallower angles
[13] and so the SAXS and normalincidence optical characterization
(method 1) of theamorphous barb photonic nanostructures is
probablymore accurate.
We obtained measured and predicted optical band-widths for both
channel- and sphere-type nanostructuresby scaling the saturation
(FWHM) of the measured reflec-tance and the width (FWHM) of the
corresponding singlescattering azimuthal SAXS profiles by the
respective peakhue (lpk) and peak spatial frequency (qpk), in order
tocompare across feathers of different colours and nanos-tructural
length scales (feathers with cortical pigmentswere excluded because
pigmentary absorption can leadto an underestimation of the true
peak widths). The
J. R. Soc. Interface
bandwidth of the primary optical peak is positively corre-lated
with the scaled widths of the primary SAXS peakfor both channel (r2
0.47) and sphere-type (r2 0.71)nanostructures (see electronic
supplementary material,figure S7a,b). This result indicates that
the width of theprimary single scattering SAXS peak reasonably
predictsthe optical saturation of the nanostructure. Concordantwith
the structural results (see 3.3), nanostructureswith larger size
scales of spatial periodicity (i.e. D) gener-ally make more
saturated colours (smaller FWHM).Variations in the inter-scatterer
spacing, D (whichincrease the SAXS peak width thereby decreasing
thecoherence length, j), result in broader, less
saturatedstructural colours. The measured optical bandwidth
isconsistently larger than the single-scattering
structuralprediction for both nanostructural classes (see
electronicsupplementary material, figure S7a,b), probably becauseof
multiple scattering [36].
4. DISCUSSION
We have characterized the nanostructure and optical prop-erties
of hundreds of structurally coloured feathersencompassing the gamut
of non-iridescent structuralhues from diverse taxa across the
phylogeny of birdsusing a combination of SAXS, electron
microscopy,normal incidence and angle resolved
spectrophotometry.The SAXS structural information enabled
quantitativediagnoses of the channel and sphere-type
nanostructures,and documented the presence of a predominant,
isotropic,short-ranged order. The nanostructural variation in
refrac-tive index is of the appropriate length scales to
producestrong reinforcement of a narrow band of
scatteredwavelengths.Noisyand ambiguous cases of structural
diag-noses from SAXS data were corroborated by EM
data.Additionally, we have identified a previously unknownclass of
slaty blue-black to blue-grey structural coloursthat are produced
by rudimentary or highly variable ver-sions of channel- and
sphere-type nanostructures.Overall, the SAXS results represent a
substantial improve-ment over Fourier analyses of EM [3,79,1719]
andthree-dimensional electron tomography data [26].
SAXS structural data also provided good predictionsof the
primary, single-scattering peaks in optical reflec-tance
measurements. Both the spectral position andshape (FWHM) of the
peaks in the azimuthal SAXSprofiles were highly correlated with
those of thecorresponding primary peak of optical
reflectancemeasurements (figure 8 and electronic
supplementarymaterial, figure S7). The discrepancies between
theoptical measurements and structural predictionsespecially for
short wavelength peaks (lpk , 450 nm;electronic supplementary
material, figure S7) barbscan be explained in part by the multiple
scattering oflight, since scattering (and multiple scattering) is
stron-ger at shorter wavelengths of light, which could result
insignificant broadening of the optical reflectance peaks.
We have also documented quantitative differences inthe nature of
structural colour production by sphere-and channel-type
nanostructures. On average, the coloursgenerated by sphere-type
nanostructures are significantlymore saturated (smaller FWHM) than
those produced by
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channel-type, and this has a strong nanostructuralbasis (see
electronic supplementary material, figure S3).However, the FWHM of
the primary optical peaks pro-duced by both types of nanostructures
decreases(i.e. saturation increases) with increasing peak hue
(i.e.longer wavelength colours), reflecting the underlyingincrease
in short-range quasi-periodic order within thenanostructure with
increasing nanostructural size (seeelectronic supplementary
material, figures S3 and S7).
4.1. Development of amorphous feather barbnanostructures
The development of the channel-type amorphous featherbarb
nanostructure in Blue-and-Yellow Macaw proceedsin the spongy
medullary cells in the telling absence ofany precursor biological
template or pre-pattern createdby cytoskeleton, organelles or
membranes [25]. Duringfeather cell maturation, capillary forces
owing to thedrying of the spongy cells apparently drive higher
mol-ecular weight materials to the cells periphery, resultingin
dense peripheral aggregations of granular materialsand a large,
electron-lucid cytoplasmic volume in thecentre of the cell. The
channel-type nanostructurearises spontaneously from within the
peripheral regionsof dense, granular cytoplasmic material, coarsens
overtime and grows to fill the volume of the cell [25]. Wehave
hypothesized that this self-assembly processoccurs by phase
separation, possibly regulated by therates of b-keratin expression
and polymerization[25,38]. How could phase separation stop at the
correctsize to be able to produce the appropriate colour?Phase
separation could be arrested by either mechanicaljamming or a
glass-transition in the b-keratin proteinphase [25,38], thus
determining the characteristiclength scale of the nanostructure.
Phase separation hasbeen studied in detail in other protein
solutions such aslysozyme, etc. [45,64]. These hypotheses are
ultimatelytestable with experimental analyses of the
self-assemblyof b-keratin polymers. Upon barb cell death, the
cyto-plasm dries out completely, and is replaced by airresulting in
the final keratin-and-air amorphous photonicnanostructure.
The morphological similarities in the previouslypublished EM
images [3,25] and experimental X-rayscattering data reported here
(figure 5ad) forchannel- and sphere-type amorphous barb
nanostruc-tures and synthetic self-assembled soft matter systemsis
congruent with the hypothesis that avian barb nanos-tructures
probably self-assemble via arrested phaseseparation of polymerizing
b-keratin from the cellularcytoplasm, as suggested earlier for a
few avian species[25,38]. Although the channel- and sphere-type
barbnanostructures, respectively, appear to be similar
tomorphologies observed during classical phase separ-ation via SD
and nucleation-and-growth, to concludethat they indeed develop via
phase separation,let alone assign a particular mode of phase
separa-tion using just morphology is not straightforward[4547]. The
lack of a perfect agreement between chan-nel-type barb
nanostructures and classical spinodalmorphologies perhaps suggests
that there may beimportant differences between a biological soft
matter
J. R. Soc. Interface
system and a simple binary fluid de-mixing, perhapsinvolving
some viscoelastic phase separation processes[4547]. Nevertheless,
careful observations of b-keratinself-assembly in developing
feathers, together within vitro investigations are necessary to
pinpoint theprecise mechanisms of their self-assembly.
4.2. Double scattering of light by amorphousfeather barb
nanostructures
The phenomena of double scattering and cortically pig-mentation
of spongy nanostructures are distinguishableby their starkly
differing angular dispersion (see elec-tronic supplementary
material, figure S4bh),predictable spectral position of the
relatively strongdouble-scattering peak in relation to the
primarypeak, polarization dependence [36,37], and by the lim-ited
classes of available pigments in birds [10]. Wehave documented the
widespread occurrence of doublescattering in the optical function
of amorphous featherbarb nanostructures (figure 7d f,mo and
electronicsupplementary material, table S2). Many such huessuch as
turquoise (light) blue (e.g. in male Cotingaspp., figure 7m and
male Tersina viridis) include twostrong and distinct spectral
peaks, one in the UV(visible to birds but not to humans) and the
other ingreen. To birds, these hues are distinct colours
stimulat-ing non-adjacent cone types (i.e. UV and medium-wavelength
spectral sensitivities) in the avian retinathat will be perceived
by birds as distinct colours[65,66]. Thus, the double scattering
spectral featuresprobably contribute significantly to the colours
ofnon-iridescent structural plumages perceived by birds,given that
most birds can see in the UV/deep violet[11,67,68]. This suggests
that the double scatteringfrom amorphous barb nanostructures
constitutes asource of rich UV signals in birds [69].
The occurrence of double scattering in the opticalreflectances
of amorphous barb nanostructures is prob-ably underestimated here,
since at shorter wavelengths,it is much harder to separately
estimate the double scat-tering peak from the primary peak and the
sensitivity ofthe spectrophotometer steeply decreases. In fact,
manyreflectance profiles with a short-wavelength primarypeak but
without an obvious double-scattering peakhave a distinct shoulder
on the short-wavelength sideof the reflectance spectrum (e.g. many
Malurus spp.and tanagers, see figure 7j,k).
4.3. Structurepigment interactions
The combination of spongy medullary barb photonicnanostructure
and carotenoid or psittacofulvin pig-ments is well known to produce
longer wavelengthcolours that cannot be produced by pigments
alone,such as structural greens [3,14,61]. Structural analysesof
these feather barbs demonstrate that the underlyingbarb
nanostructures are larger in spatial periodicitythan those
producing purely structural hues (such asUV, violet and blue).
Indeed, the underlying spongymedullary keratin nanostructure in
each case wastuned to the appropriate length scale to produce
theobserved reflectance peak by constructive interference
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alone (figure 8 and electronic supplementary material,table S2).
Therefore, contrary to prevalent simplisticnotions of colour mixing
(structural blue pigmentaryyellow green) [70], the peaked or
saturated green col-ours in feather barbs are produced by a
combination ofmedullary barb nanostructures tuned to produce
thoselonger wavelength colours and the absorption of someportions
of the shorter wavelength double-scatteringpeak and the
intermediate wavelengths between thetwo peaks. Similar
structurepigment interactionshave been proposed in saturated yellow
(Ramphastostoco, Ramphastidae) and orange (Tragopan
caboti,Phasianidae) colours in avian skin [3,71].
In feathers with structurepigment interactions, theincident
light first passes through the pigmented outercortex layer of the
barbs, a portion of which is absorbedby the pigments, and the rest
is transmitted to theunderlying medullary layer of nanostructure.
The pig-ment acts like a band-pass filter, i.e. only the range
ofwavelengths that is selectively transmitted by thepigments in the
cortex can be scattered by the nano-structure underneath. However,
the nanostructure istuned to constructively reinforce and scatter
only a por-tion of the transmitted pigmentary reflectance, with
thelonger wavelengths getting destructively interfered andthereby
reducing the typical broadband longer wave-length reflectance of
the pigments, just like in avianskin [3,71]. The light scattered by
the nanostructureonce again passes through the pigmented cortex on
itsway out of the feather barb. The result is a uniquelysaturated,
brighter longer wavelength colour that isspectrally quite distinct
from both the superficial pig-ments and the nanostructure below
(figure 7).Furthermore, just as in avian skin [71], there appears
tobe no intrinsic constraints to the production of
longer-wavelength structural colours in feather barbs by
con-structive interference from amorphous nanostructures(figure 8).
However, given the propensity for doublescattering owing to the
amorphous nature of spongybarb nanostructures [36,37] (figure
7df,mo and elec-tronic supplementary material, table S2), pure
(highlysaturated) non-iridescent long-wavelength structuralhues are
unlikely to occur in feather barbs [72].
The deposition of spectrally absorbing pigments super-ficially
in the barb cortex can result in the attenuation ofthe double
scattering short-wavelength structural peak inthe optical
reflectance (see above), resulting in more satu-rated
long-wavelength hues (e.g. purer structural greensand yellows as in
many tanager feathers). However, a por-tion of the short-wavelength
double-scattering peak can bereinforced, if the transmission
spectra of the depositedpigments have an overlapping but relatively
less intenseUV peak (characteristic of carotenoids like lutein,
forinstance; figure 7gi,p,q) or not (as in the case of
manytanagers; figure 7r). In bird species with both non-irides-cent
blue and structural green plumage patches, therespective absence or
presence of cortical pigments inthe barb appears to be the general
mechanism by whichplumage reflectance differences arise between
blue andgreen patches of sexually monomorphic species
(e.g.Corythaeola cristata, Musophagidae; Merops viridis,Meropidae;
Eumomota superciliaris, Momotidae;Aulacorynchus prasinus,
Ramphastidae; Forpus
J. R. Soc. Interface
xanthopterygius, Psittacidae; Calyptomena hosii,Eurylaimidae;
Vireolanius pulchellus, Vireonidae;Erythrura trichroa, Estrildidae;
Chlorophonia occipita-lis, Fringillidae; Tangara chilensis,
Thraupidae; seeelectronic supplementary material, table S2),
andbetween homologous blue (usually in males) and green(usually in
females) patches in the opposite sexes ofsexually dichromatic
species (e.g. Lepidothrix spp.,Pipridae; Cyanerpes cyaneus, Te.
viridis, Thraupidae;see electronic supplementary material, table
S2).
In the optical reflectance of a further 21 (out of 255)feather
barbs examined (see electronic supplementarymaterial, table S2), a
longer wavelength pigmentaryreflectance plateau from apparently
modified keto-carotenoids [73] or phaeo-melanins complements
ashorter wavelength (violet to blue) single or doublestructural
peaks, producing distinct extra-spectral col-ours including vivid
shades of violets and purples toblue hues with hints of lilac (e.g.
Acryllium vulturinum,Numididae, figure 7b; C. maynana, figure 7m;
Maluruscoronatus, Maluridae; Sitta oenochlamys, Sittidae).Thus,
complex interactions between structural and pig-mentary mechanisms
produces hues that areunavailable to either modes of colour
production, butadditional research is required to understand
thenature of these interactions more precisely.
4.4. Evolution of non-iridescent barb structuralcolours
Non-iridescent, structural colour-producing three-dimen-sional
amorphous barb nanostructures appear to haveindependently evolved
at least 44 times within 41 familiesacross Aves, conservatively
assuming a single evol-utionary origin within each family examined,
with anadditional five families possessing species with
marginalbarb structural coloration (see electronic
supplementarymaterial, table S2). However, there appears to be
mul-tiple origins of non-iridescent barb structural colourwithin
some families (see electronic supplementarymaterial, table S2). An
accurate estimate of the numberof evolutionary gains and losses of
barb structural coloursin extant birds requires a well-resolved
phylogeny of allbirds. Therefore, a formal, detailed
macro-evolutionaryanalysis of barb structural colours will have to
await sig-nificant progress in unravelling the evolutionary tree
ofbirds. Nevertheless, the phylogenetic distribution ofphotonic
barb nanostructures at the inter-ordinal levelis distinctly
non-random. The channel nanostructures(n 36) occur more frequently
than the spheres (n 8)among both passerine (perching) and
non-passerinebirds (electronic supplementary material, table S2).
Foreach avian family with barb structural colours, we haveexamined
multiple species in different genera and multipleplumage patches
within at least one species. In nearlyevery instance, all specimens
sampled within a familyshared the same class of nanostructure.
However, weidentified a few instances in which the
nanostructuresvaried within a single avian family. Within the
tanagers(Thraupidae), Cyanicterus cyanicterus, Diglossa cyanus(see
electronic supplementary material, figure S1e) andXenodacnis
petersi possess channel-type nanostructurewhile the rest of the
tanagers examined have spheres.
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But these three species are closely related members of adistinct
branch of the family [74], and this couldrepresent a genuinely
independent evolutionary origin ofcolour-producing nanostructure
within the family. Simi-larly within the cardinals and grosbeaks
(Cardinalidae),all taxa examined appear to possess a channel-type
nano-structure except Cyanoloxia glaucocaerulea, which hasthe
sphere-type morphology, based on SAXS data andSEM images (see
electronic supplementary material,figure S1g,h).
4.5. Biomimicry lessons for amorphousphotonics
Recently, there has been a flurry of interest in the
opticalproperties, design and synthesis of amorphous
nanos-tructures at optical length scales, as they possess
bothisotropic optical properties (non-iridescence) and
omni-directional photonic bandgaps at high refractive indexcontrast
[7580], unlike conventional, angle-dependentphotonic crystals [6].
In contrast to amorphous opalnanostructures of synthesized
dielectric spheres featuredin contemporary engineering approaches
[75,7880], thespongy feather barbs possess an amorphous
inverse-opal nanostructure, which is likely to possess better
opti-cal properties than their opal counterparts, by analogyto
photonic crystals [6]. Indeed, the optical properties offeather
barbs currently surpass those of engineeredamorphous colloidal
photonic materials [51]. Amorphousphotonic nanostructures that are
probably self-assembled in bird feather barbs with pronounced
isotro-pic short-range order could therefore provide a
useful,tunable biotemplate for positive cast replication
ordielectric infiltration. A thorough understanding of thephysics
and development of organismal structuralcolour that have evolved
over millions of years of selec-tion for a consistent optical
function may thus guidebio-inspired technological innovations
[5,51,8183].
This work was supported with seed funding from the YaleNSF-MRSEC
(DMR 1119826) and NSF grants to R.O.P.(DBI-DBI-0078376), H.C.
(PHY-0957680) and E.R.D.(CAREER CBET-0547294) as well as Yale
University fundsto V.S. and R.O.P. R.O.P. would like to
acknowledgesupport of the Ikerbasque Science Fellowship and
theDonostia International Physics Center. We thank twoanonymous
reviewers for their helpful comments, ThomasValqui for his kind
permission to use his photograph ofC. maynana and Kristof Zyskowski
for help with birdtaxonomy. We are grateful to the Yale Peabody
Museum ofNatural History, the University of Kansas Natural
HistoryMuseum and Biodiversity Research Center, the AmericanMuseum
of Natural History (Paul Sweet), the NaturalHistory Museum at the
Academy of Natural Sciences (NateRice), the Harvard University
Museum of ComparativeZoology (Jeremiah Trimble and Scott Edwards),
and theUniversity of Oxford Natural History Museum
(MalgosiaNowak-Kemp) for the feather samples. Tim Quinn obtainedTEM
images of some bird feather barbs. SAXS data on birdfeathers were
collected with the help of Alec Sandy andSuresh Narayanan at beam
line 8-ID-I of the AdvancedPhoton Source at Argonne National Labs,
and supported bythe US Department of Energy, Office of Science,
Office ofBasic Energy Sciences, under Contract No.
DE-AC02-06CH11357. We thank Nick Terrill and Tobias Richter forhelp
with SAXS data collection at beamline I22 of the
J. R. Soc. Interface
Diamond Light Source (sm6905-1) that contributed to someof the
results presented here. V.S. initiated, designed andperformed the
research with R.O.P.; J.D.F. prepared thebiomimetic bidisperse PS
sphere films; H.N. and S.-F.L.collected angle-resolved data on a
few feathers; V.S.analysed and discussed the data with all authors;
and V.S.wrote the manuscript with E.R.D and R.O.P.
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