JOURNAL OF MORPHOLOGY 2 2 2 % - 7 2 (1994)

Structural Color Production by Constructive Reflection
From Ordered Collagen Arrays in a Bird
(Philepitta castanea: Eurylaimidae)
RICHARD 0. PRUM, RANDALL L.MORRISON,
AND GARY R. TEN EYCK

Department of Systematics and Ecology, and Museum of Natural History(R.O.P.,G.R.T.E.),and Department of Physiology and Cell Biology(R.L.M.),The University of Kansas, Lawrence, Kansas 66045-2454

ABSTRACT Ordered hexagonal arrays of parallel collagen fibers produce thebrilliant green structural color of the fleshy, supraorbital caruncles of male
Velvet Asity (Philepitta castanea; Aves: Eurylaimidae). The collagen arrays areorganized in larger macrofibrils that are packed irregularly within cone-shaped
papillae that cover the surface of the caruncle. The color of the caruncle
conforms closely to the wavelengths predicted by applying Bragg's Law of
constructive reflection to measurements of the size and spatial organization of
the collagen arrays. These observations constitute a novel mechanism ofstructural color production in animals. These collagen arrays are convergently
similar to the smaller, highly structured collagen arrays in the mammaliancornea, which exploit the same physical mechanism to produce optical transparency. @ 1994Wiley-Liss, Inc.
Structural colors of animals are producedby the interaction of light with organic andinorganic materials of different optical densities. The known mechanisms of structural
color production in animals include diffraction from colloids of liquids, solids, or gases,
and constructive reflection from ordered layers or films of mucus, keratin, chitin, melanin granules, or intracellular purine crystals(Fox, '76). In birds, the only documented
structural colors are produced by orderedlayers of melanin granules or air vacuoles
within the keratin of feathers (Durrer, '62,
'77, '84; Durrer and Villiger, '66; Lucas andStettenheim, '71; Dyck, '76, '87). However, structural color production in the dermis of birds has not been analyzed in detail.
Structural blue colors in bird skin have been
hypothesized to be produced by Tyndall scattering, but the anatomical basis of these colors has never been described (Rawles, '60:
223; Lucas and Stettenheim, '72:406, 410;
Fox, '76; Durrer, '84).
This investigation documents a novel
mechanism of structural color production
based on the optical properties of orderedcollagen fibers. Collagen is one of the mostabundant and structurally important proteins in animals, but it is not presently known
to contribute to production of any wave(11994 WILEY-LISS, INC.

length-specific structural color. However, theoptical properties of collagen do contribute toits functional role in many organisms. Whiteness can be produced by reflection of incident
light on unordered collagen fibers which varyin diameter and spacing. Structures such as
the sclera of the human eye and the dermis ofwhite axolotls appear white by this mechanism (Fox, '76; Vaezy and Clark, '91). Conversely, transparency of the vertebrate cornea is achieved by destructive interference
among reflections from small, uniformly sizedparallel collagen fibers in quasi-ordered arrays (Maurice, '57; Hart and Farrell, '69; Coxet al., '70; Benedek, '71; Vaezy and Clark,'91).
The novel structural color described here
is found in the Velvet Asity (Philepitta castanea; Aves: Eurylaimidae; formerly Philepittidae): a frugivorous, suboscine perching bird
in a small subfamily (Philepittinae) that is
endemic to the island of Madagascar (Amadon, '79; Langrand, '91; Prum, '93).We firstdescribe the color and anatomy of the caruncles of Philepitta castanea and then present a model for the production of the ob-

Randall L. Morrison is now at the Department of Biology, Hood
College, 401 Rosemont Avenue, Frederick,MD 21701.

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R.O. PRUM ET AL.

sion electron micrographs. Equations for the
calculation of Bragg reflections from fibril
arrays were taken from Durrer's ('77, '84;
MATERIALS AND METHODS
Durrer and Villiger, '66) adaptation of Bragg's
These observations are based on caruncles Law for constructive reflection in matricesof five museum spirit specimens of Philepitta composed of materials of different optical
castanea which were examined using a dis- densities. Although the collagen arrays in
secting microscope, light microscopy, scan- Philepitta castanea are not perfectly orderedning electron microscopy (SEM), and trans- like a crystal lattice, Benedek ('71) and othmission electron microscopy (TEM). The ers (Hart and Farrell, '70) have demonmuseum specimens were collected in Mada- strated theoretically that Bragg wavelengths
gascar by S.M. Goodman in 1989, preserved constitute the only constructive reflectionsin the field in buffered 10% formalin, and from an array in which interfibril distances
later transferred to 70% ethanol for perma- are regular over the distance of the wavenent storage at the Field Museum of Natural lengths of incident light. These conditions
History (FMNH 345696-7,345708-10).Addi- are met by the collagen arrays in the cartional photographs were taken under the dis- uncles of Philepitta castanea, confirming thesection microscope of caruncle tissue from suitability of the application of Bragg's Law
three other specimens of P. castanea which to this system (see Discussion below).
were collected in 1993by S.M. Goodman, and
RESULTS
preserved in the field in 2.5% glutaraldehyde
Color
and anatomy
for 1 hour and stored in 0.1 M cacodylate
Female Philepitta castanea have largely
buffer (0.1 M sodium cacodylate, 0.0025 M
olive green plumage and lack supraorbital
calcium chloride, 4% sucrose).
Caruncle tissue from the museum speci- caruncles (Langrand, '91). Adult malemens was critical point dried in COz in prepa- Philepitta castanea have velvety black plumration for SEM. Specimens were then coated age with large, fleshy supraorbital facial carwith 30 nm of gold/palladium. Images and uncles (Langrand, '91) in the breeding seamicrographs were produced using a Philips son(September-February).
Inthe
501 on the 157.2 kV setting. For light micros- nonbreeding season (April-August), adultcopy, caruncle tissue was embedded in paraf- males have black plumage with olive-green
fin, cut in 10 km sections, and stained with barring, and lack well-developed, colorful carMasson's trichrome, which includes fast- uncles (Langrand, '91; S.M. Goodman, s.
green, a collagen-specific stain.
Zack, personal communication). The annual
Caruncle tissue observed by TEM was cycle of development of the caruncles andtaken from the cadual margin of the car- their structural color has not been docuuncle, which was bright green in life but mented. Further, male Philepitta castanea
violet to blue in the preserved museum speci- have delayed plumage maturation (S.M.
mens. Tissues were prepared for TEM using Goodman,personal communication),and firsta standard protocol (Hayat, '81; Morrison year males that are sexually mature haveand Frost-Mason, '91). Tissues were diced female-like plumage and lack completely deinto 1 mm2 pieces and placed in 2.5%glutar- veloped caruncles (e.g.,FMNH 345697). Early
aldehyde fixative for 1 hour at 4-6°C. They in development, the caruncles of immature
were then post-fixed in 2% osmium tetroxide males appear as uneven, whitish thickenings
for 1hour at 4-6"C, and then en bloc stained of the supraorbital skin.
with 2% uranyl acetate for 1 hour at room
The caruncles of adult males are feathertemperature. Tissue pieces were then dehy- less, fleshy excrescences of the dermis. Theydrated through an ethanol series and embed- are attached at their base to the dermis above
ded in Epon. Thin sections were cut and the eye. The caruncles are approximately 25
stained with 2% uranyl acetate and 0.4% lead mm long and 7 mm wide, and they extend
citrate. Specimens were viewed and photo- from the base of the bill t o behind and below
graphed in a Jeol JEM-1200 TEM. Fixation the eye (Fig. 1). The lateral surface of theof all caruncle tissues observed by TEM was fully developed caruncle is brilliant green
relatively poor due to the effects of formalin with bright blue immediately above the eyes
used to preserve the museum specimenswhen (Figs. 1, 2). The medial surface of the caruncle is black. In life, the color of the caroriginally collected in the field.
Measurements of fibril dimensions and uncle does not change with the angle of incispacing were made with calipers on transmis- dent light or the angle of observation (S.M.
served color based on the ultrastructure of
the caruncle.

STRUCTURAL COLOR PRODUCTION BY COLLAGEN ARRAYS

63

Fig. 1. Philepittu cmtunea. An adult male Velvet Asity showing supraorbital caruncle. (Photo courtesy of S. Zack).

Goodman, personal communication; R.O.
Prum, personal observation). The color of
the fleshy caruncle of adult male Philepitta
castanea has been described as “fluorescentgreen” (Langrand, ’91). Color photographsof caruncles of living specimens of P. castunea were compared to published color references and appeared closest to yellow-green of

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R.O. PRUM ET AL

Smithe ('75; Munsell 5.0 GY 8.0l10.0) andviridine yellow of Ridgway ('12; Munsell 1.0
GY 7.8/8.0). The color of the caruncle in life
is very similar to the color of caruncle tissuepreserved directly in glutaraldehyde (Figs. 1,2). Following initial fixation in buffered 10%
formalin and storage in 70% ethanol, thecaruncles appear violet blue or blue withbright iridescent highlights (Fig. 2). The museum specimens fixed in formalin have beenused for all the observations in this analysis.
The color of the adult supraorbital caruncle is produced by cone-shaped papillaethat cover its lateral surface (Figs. 1, 2, 3A).
The papillae have smooth surfaces, and arelargely perpendicular to the surface of theskin (Fig. 2,3A). Each papilla is composed ofa keratinized epidermal sheath and a capsulefilled with irregularly oriented, cylindricalstructures that stain densely with fast-green,a collagen-specific stain (Fig. 2). These collagen macrofibrils are hundreds of micrometers long and more than 50 p,m wide. Thebase of the papilla rests on the upper dermis,
which contains a dense layer of melanophores and unstructured collagen fibers (Fig.
2). The rest of the caruncle is composed oftypical dermal tissue with blood vessels,
nerves, and fine muscle fibers. In immature
males, the papillae are not well developed
and do not include large collagen macrofibrils.
Transmission electron microscopy reveals
that the cylindrical collagen macrofibrils in
the core of the papillae of adults are composed of highly ordered, hexagonal arrays ofparallel collagen fibers (Fig. 3B,C). The average diameter of the fibers is 98 nm (n = 60,
s.d. = 14) and the average spacing between
the centers of the fibers is 185 nm (n = 60,
s.d. = 22). In contrast, the collagen fibers
present in the white caruncles of an immature male are not organized in macrofibrils,
vary greatly in size, and do not form ordered
arrays (Fig. 3D).
Mechanisms of color production
The color of the caruncles of Philepitta
castanea is not produced by the keratinizedsheathes of the papillae, because the color
remains when the outer sheaths are removed(Fig. 2). Rather, the color emanates from thecollagenous structures inside of the papillae.
Tyndall scattering is an unlikely mechanism
for the production of the observed color, as
the collagen macrofibrils are much too large
to produce color by that mechanism (Fig. 2).
Structures that cause Tyndall scattering musthave a mean diameter of < 0.7 p,m (Fox, '76).
Furthermore, Tyndall scattering structures
would produce only a blue to violet color andwould require an additional pigment to produce the green observed in the caruncles ofliving Philepitta castanea (e.g., Fox, '76).No
pigment cells, however, occur in the core of
the papillae where the color is produced. The
hexagonal arrays in the collagen macrofibrils
are the only potential color-producingstructures inside the papillae.
The physical model for constructive reflection from a lattice of independent reflectors
is Bragg's Law (Bragg and Bragg, '151, which
has been adapted for application to constructive reflection by arrays of biological materials that differ in optical density (Durrer, '62,
'77; Durrer and Villiger, '66; Benedek, '71).
According to the Bragg model, light reflectedby fibrils in independent parallel planes of alattice travels different distances to any observation point, resulting in a shift in the phaseof light waves reflected from different planes.
The summation of these phase-shifted reflections results in destructive interference ofmost wavelengths, but produces constructive
reflection of light with a wavelength that isequal to the addition in path length (Fig. 4).
This wavelength, L, is given by the equation
for the Bragg reflection condition:
L = n2dsinn

where n is the refractive index of the medium
between the reflectors, d is the space latticedimension (the minimum distance between
parallel planes of reflection), and a is theangle of incident light (Bragg and Bragg, '15;

Fig. 2. Philepittu castanea. Upper left: The surface ofa caruncle of an adult male viewed under a dissection
scope by reflected light. This specimen was fixed in 2.5%
glutaraldehyde in the field, and the colors preserved aresimilar to those of the tissue in life. Most of the caruncle
is green but a small portion that is usually hidden withina wrinkle above the eye is blue. Upper right: View of thecollagenous material inside a papilla from the caruncle ofan adult male (FMNH 345708) that was fixed in formalin
and transferred to ethanol, viewed by reflected light. The
keratin sheath that covers the papilla has been removed. Lower left:Light micrograph of a cross-section ofcaruncle papillae stained with Masson's trichrome stain.
Each papilla is composed of a keratinized sheath, a capsule filled with collagenous macrofibrils, and a basal layerof melanophores in the dermis. Collagen is stained with
fast-green and appears blue. The gap below the collagenmacrofibrils on the right is an artifact. Bar = 175 km.
Lower right: Light micrograph of the core of a single
papilla showing irregularly packed collagen macrofibrils.
Bar = 75 km. S, keratinized sheath; CM, collagen macrofibrils; M, melanophores; UC, normal, unordered collagen.

STRUCTURAL COLOR PRODUCTION BY COLLAGEN ARRAYS

Figure 265

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R.O.PRUM ET AL.

Fig. 3. PhzEepitta cmbuneu. A: Scanning electron micrograph of the surface of the caruncle of an adult male
(FMNH 345708). Surfaces of the caruncle papillae aresmooth. Bar, 200 pm. B: Transmission electron micrograph (TEM) of a cross-section of an array of collagen
fibrils in a collagen macrofibril. Bar = 100 nm. C: TEM
of a longitudinal section of collagen fibrils within a car-uncle papilla. Fibrils show the distinct banding pattern ofcollagen. Bar = 200 nm. D: TEM of a cross-section ofcollagen fibrils within the whitish caruncle of an immature male (FMNH 345697). Collagen fibrils varv ereatlv"
in diameter, and the interfibril spacing is irregular. Bar =100 nm, CF, collagen fibril.

Durrer, '66, '77, '84). I n a hexagonal array offibrils 185 nm apart, the primary family ofparallel planes has a space lattice dimensionof 160.5 nm (Fig. 5A). The potential angles ofincidence on these planes are limited by the
distribution and size of the fibrils (Durrer,'66, '77). In this hexagonal array, the potential angles of incidence range from 68-90"
and provide minimum and maximum values
for potential Bragg reflections (Fig. 5A). The
matrix between the collagen fibrils in the
caruncle and its refractive index is unknown;
however, Maurice ('57) determined the refractive index of the mucopolysaccharide between collagen fibrils in the mammalian cornea to be 1.35. We used this value as an
approximation of the actual refractive index.
Based on these values, the wavelengths ofpotential Bragg reflections from the collagenarrays in the caruncles of Philepitta castanea
range from 401 nm ( a = 68") to 433 nm(a= 900).These wavelengths range from violet to blue in color, and conform closely to theobserved color of the preserved caruncle tissue (Fig. 2 ) . Shallow angle Bragg reflections
could potentially occur at a few limited angles( =. 31"),but these reflections would be nonvisible ultraviolet wavelengths ( = 223 nm). Owing to the symmetry of the hexagonal array,
incident light at these shallow angles is also
incident on other planes of reflection thathave the same space lattice dimension andwill produce visible Bragg reflections in theabove wavelengths (Fig. 5B). The space lattice dimensions of other families of parallel
planes in the array are I 80 nm. These planes
have first order Bragg reflection wavelengths
entirely in the portion of the ultraviolet spectrum ( < 259 nm) that is not visible to birds
or humans, and reflections from these planes
should not contribute significantly to the observed color of the caruncle.

"

YSTRUCTURAL COLOR PRODUCTION BY COLLAGEN ARRAYS

The Bragg model predicts that the change
in color from green to blue following fixationin 10% formalin and storage in 70% ethanol
was produced by the shrinking of the interfibril distances of the collagen arrays. The
distance between adjacent collagen fibrils
would have to be approximately 230 nm inlife (as opposed to 185 nm in the preserved
tissue of museum specimens) to produce thegreen color observed in nature by constructive reflections from the primary space lattice. This amount of tissue shrinkage (approximately 20%) is explicable and would be
expected as an artifact of dehydration during
fixation and preparation for microscopy(Hayat, '81; R.L. Morrison unpublished observations).
The Bragg reflection model is further corroborated by the observation that the whitish caruncle of a n immature male Philepitta
castanea is composed of collagen fibrils thatvary tremendously in size and interfibril distance (Fig. 3D). Irregular fibril size and interfibril distance on the scale of the wavelengthsof visible light will result in a lack of phasecorrelations among reflections from different
fibrils (Maurice, '57; Benedek, '71; Vaezy and
Clark, '91). As a result, most wavelengths oflight will be reflected by the tissue, and it willappear white.
DISCUSSION

The color of the preserved caruncles ofadult male PhiZepitta castuneu conforms wellto the wavelengths predicted by the application of the Bragg equation for constructive
reflection to the dimensions of collagen arrays in the caruncle papillae. The color produced by the caruncles of Philepitta castanea
is the summation of constructive reflections
from thousands of collagen arrays arranged
in macrofibrils within each papilla. Thechange in color of the caruncles from green toblue with preservation is accounted for byshrinking of the tissue due to the preservatives used. Wavelength-specific structural
color production is a n entirely novel function
for collagen in animals.
The first physical model for the productionof wavelength-specific colors by constructive
reflection from a n ordered lattice was developed by W.H. and W.L. Bragg ('15) who studied the interaction of X-rays and crystals.
Subsequent studies of arrays of melanin granules in a keratin matrix in bird feathers have
expanded the application of the Bragg model
to ordered arrays of fibrils in media of a different optical density (Durrer, '62, '77; Durrer

67

and Villiger, '66). Experimental and theoretical investigations of arrays of collagen fibrils
in mucopolysaccharide matrices in the vertebrate eye have also demonstrated that Bragg's
Law is a n appropriate model for analyzing
constructive reflection by regular arrays ofcollagen fibers (Maurice, '57; Benedek, '71).
Color production in Philepitta differs insome interesting ways from other known
mechanisms of reflective color production.
Many reflective structural colors are iridescent, or change hue with angle of observation. For example, iridescent bird feathers
produce color by constructive reflection fromsquare lattices of melanin granules that areoriented parallel to the surface of the feather
barb (Durrer, '62, '77, '84; Lucas and Stettenheim, '71; Dyck, '76). The color of thesefeathers varies with angle of observation because changes in the orientation of the observer alter the addition to the path length oflight waves reflected from the planes of melanin granules. In one genus of birds, the Ptilinopus pigeons, this color variation is eliminated by having curved arrays of melaninthat maintain the same space lattice dimension and path length addition from any pointof observation (Dyck, '87).
In contrast to iridescent structural colors,
two aspects of the structure of the caruncles
of Philepitta contribute to the creation of areflective color that is unchanged with theangle of incident light or the point of observation. First, each unit hexagon in a collagenarray includes three sets of parallel planes
with the appropriate space lattice dimension
for visible Bragg reflections (Fig. 5B). Eachset of planes intersects the others at 60"
angles. Light incident on a collagen arrayfrom any direction, except exactly parallelwith these planes of reflection, should produce visible Bragg reflections within a narrow band of wavelengths (approximately 30
nm wide) from one of these three sets ofplanes. Second, at a larger level of organization, the macrofibrils within the papillae arecomposed of numerous collagen arrays. They
are irregular in shape and orientation, andare packed together irregularly within thepapillae (Fig. 2). Light incident on the papillae from any angle will interact with thousands of collagen arrays within these irregularly packed macrofibrils. The reflected lightis the summation of the constructive reflections from different angles of incidence on
thousands of arrays.

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R.O. PRUM ET AL.

Out of PhasehH

Amplitude

Phase Shift

Destructive Interference
no reflected lightH

In Phase

Fig. 4. Philepittu custuneu. Diagram of the summation of independently reflected light waves. Reflected
waves that have traveled different distances, or path
lengths, may be out of phase, or at different stages in thecycle of the wave (upper left). The difference in path
length between the waves contributes to the phase shift,
the amount by which the two waves are out of phase.
Summation of two completely out-of-phase reflections of
the same wavelength and amplitude results in complete,

In the caruncles of Philepitta castanea, thesymmetrical structure of the hexagonal arrays and the irregular packing of the collagenmacrofibrils combine to produce a reflective
color that is noniridescent, uniform, and brilliant from any angle of observation. Changes
in the orientation of an observer t o the caruncle of Philepitta do not predictably alter
the angle of observation to the color producing arrays, because each array presents multiple opportunities for Bragg reflections fromdifferent directions, the arrays are so numerous, and the macrofibrils are irregularly oriented within the papillae.
Like many structures that produce colors
by either reflection or Tyndall scattering, thecaruncle papillae of Philepittu castuneu arebacked by a dense layer of melanophores(Fig. 2). Highly melanized dermal tissue eliminates reflections of white light that is transmitted entirely through the papillae. Irregular reflections from the tissue below thepapillae would be out of phase with the Bragg

Constructive Reflection

destructive interference; no light wave is propagated(upperright).Reflected waves that have traveled different path lengths may have shifted back in phase if thepath length addition is an integer multiple of the wavelength (lower left). Summation of two in-phase reflections will result in constructive reflection of a light wave
with the same wavelength and twice the amplitude (lower
right).A, wavelength.

Fig. 5. Philepittu caftaneu. A: Diagram of the Bragg
model of constructive reflection showing an array ofparallel collagen fibrils, the space lattice dimension of thearray (d), and the path length addition to reflected lighta t different angles of incident light (a),in cross-section.
Arrows indicate the paths of incident and reflected lightfrom adjacent fibrils in neighboring, parallel planes ofreflection. The planes of reflection and space lattice dimension are indicated by the shaded planes on the right. The
wavelength of Bragg reflections varies with the path
length addition which is determined by the angle of incident light (a)and the spatial conformation of the fibrils.
Values of a for this array vary from a minimum of = 68"(left) to a maximum of 90" (right).The primary spacelattice dimension in the preserved material is = 160.5
nm, and incident light on these planes should produce
visible Bragg reflections between violet (401 nm) andblue (433 nm) at a values from 68 to 90". B: Diagram of
the three sets of parallel planes with the maximum spacelattice di- mension ( = 160.5).Each set of planes (shownin different stippling) is parallel with two flat sides of thebasic, hexa- gonal unit cell of the array. Each set of planesintersects the others at a 60" angle. Incident light fromany angle except parallel with these planes will produce
visible Bragg reflections from one of these three setsof planes. a, angle of incident light; d, space latticedimension.

STRUCTURAL COLOR PRODUCTION BY COLLAGEN ARRAYS

Figure 569

70

R.O. PRUM ET AL.

reflections from the collagen lattices andwould dim the color of the constructive reflections (Lucas and Stettenheim, '71; Fox, '76;
Fujii et al., '89).
The brilliance of the color of the caruncles
of Philepitta castanea may be related to thenumber of reflective arrays within each papilla. In bright green species of Ptilinopus
pigeons, Dyck ('87) has shown a linear relationship between the peak percent reflectance of light incident on the feathers and thenumber of ordered layers of the constructively reflecting melanin granules within thefeather barbules. A maximum of over 60%
reflectance was observed in Ptilinopus victor
which has 20 layers of melanin granules. In
comparison, any cross-section of a papilla ofPhilepitta castanea intersects dozens of collagen macrofibrils; each macrofibril ( 50 p,min diameter) includes hundreds of reflective
planes of collagen fibers ( - 160 nm apart).
This enormous number of independent reflective planes within the papillae apparently
contributes to the brilliance of the color produced by the caruncle. Spectral reflectancemeasurements from the caruncles of Philepitta castanea should be made to quantify theefficiency of this color production mechanism.
The production of wavelength-specificstructural color is a unique and unexpectedfunction for collagen. However, investigations of the mammalian cornea demonstratethat corneal collagen fibrils produce transparency by essentially the same physical mechanism. The cornea of mammals is composed ofparallel fibrils of collagen (20-30 n m in diameter) in a quasi-ordered array with a n average center-to-center fibril spacing of 60 nm(Maurice, '84).Maurice ('57) was the first to
hypothesize that corneal collagen fibrils behave like a crystal lattice, and that corneal
transparency is the result of destructive interference among correlated, phase-shifted reflections from different collagen fibrils. The
corneal collagen fibrils and interfibril spacing
are so small that the only resulting reflections would be Bragg wavelengths in thenonvisible ultraviolet spectrum (Maurice,'57).
Subsequent theoretical physical models of
the optical properties of arrays of parallel
fibrils by Benedek ('71) and others (Hart andFarrell, '69) demonstrate that the Bragg reflection phenomena can occur even when thearray of fibrils is not a perfect lattice.
Benedek's models demonstrate that constructive reflections a t Bragg frequencies should-

occur when predictable spatial variation in
optical density occurs over distances comparable to the wavelength of incident light. In
specific, an array of fibers will scatter orreflect wavelengths related to the Fouriercomponents of the spatial variation in opticaldensity. Experimental work by Vaezy and
Clark ('91) has confirmed Benedek's model oftransparency for the human cornea. The Fourier components of the spatial variation in
optical density of the cornea are very low a t
all wavelengths related to visible light, butthere was a large peak in fourier components
a t 80 nm, close to the interfiber distance andresulting in constructive reflections in thenonvisible ultraviolet spectrum. In comparison to the cornea, the mammalian sclera
appears white, and is composed of collagen
fibrils that are highly variable in diameter(40-200 nm) and larger interfibril spacing(-250 nm; Maurice, '84; Vaezy and Clark,'91). The Fourier components for spatial
variation in the reflective index of the humansclera are high across all wavelengths with apeak in the 300-400 nm range corresponding
to frequencies of visible light (Vaezy and
Clark, '91). These irregularly organizedscleral fibrils do not produce predictable correlations in path length addition among reflections from different fibrils, resulting innearly complete reflection of incident lightand the white color (Maurice, '57; Benedek,'71; Vaezy and Clark, '91).
Theoretical and experimental results fromthe mammalian eye support our applicationof Bragg's Law to the analysis of constructive
reflection from collagen lattices in a bird.
Although the collagen arrays in the caruncles
of Philepitta do not constitute a perfect lattice, the fibril size and interfibril spacing are
regular and highly predictable over distances
greater than all wavelengths of visible light(Fig. 3B). Under these conditions, the arrayshould produce Bragg reflections at the observed wavelengths (Benedek, '71). Furthermore, the irregularly sized and spaced collagen fibrils in the whitish caruncle of a n
immature male Philepitta castanea apparently function like the similarly structured
collagen of the mammalian sclera. Observations of immature males confirm that theregularity in size and spacing of collagen in
the adult caruncles is responsible for thevivid green structural color.
The vertebrate eye has evolved throughnatural selection for visual perception (Williams, '92). The supraorbital caruncle of

STRUCTURAL COLOR PRODUCTION BY COLLAGEN ARRAYS

Philepitta castanea is a secondary sexual
structure that has probably evolved throughsexual selection (e.g., Darwin, 1871). Naturaland sexual selection on the optical propertiesof these two very different structures haslead to convergent evolution in ultrastructure of their collagen fibrils. Each structure
exploits the same physical mechanism toachieve its own distinct optical quality, transparency on the one hand, and vivid, multidirectional color production on the other.
Much remains to be learned about the function, development, and evolution of collagenous color-producing arrays. We have madeobservations of the caruncles of a single immature Philepitta castanea, and additional
observations should be made of the development of the caruncle in young males. Furthermore, nothing is known about the process ofannual caruncle development and atrophy inadult males, and additional observations
should be made of the annual variation incaruncle size, color, and ultrastructure. Unfortunately, material of these poorly known
rain forest birds is very rare.
Nothing is known about the possible distribution of this color production mechanism inother birds. However, all three other speciesof asities have colorful facial caruncles (Langrand, ’911, and the caruncles of these species also differ in color and morphology from
Philepitta castanea. Philepitta schlegeli hasa larger caruncle that is “pearly light green
below and in front of the eyes, blue above theeyes, and turquoise behind the eyes” (Langrand, ’93:249). From observations of museum study skins, the surface of the caruncle
in schlegeli appears to be covered with broad,round, bubble-like tubercles instead of thesmall, erect papillae as in castanea. In Neodrepanis coruscuns, the caruncle is light blue
above the eye and ultramarine blue below
with a turquoise eye ring (Langrand, ’93:250). The surface of the caruncles of both
species of Neodrepanis are smooth, and entirely lack papillae or tubercles (R.O. Prum,
personal observation). The caruncles of otherasities are homologous to those of Philepitta
castanea (Raikow, ’87; Prum, ’93), and wewould predict that the vivid blues, greens,and purple colors of the caruncles of otherasities are produced by collagen arrays. However, the color and morphology of the caruncles of these other species are different
from those of Philepitta castanea, indicatingthat various anatomical aspects of this color
production mechanism have continued to
evolve since its origin.

71

The constructive reflection model predicts
that variation in hue in different parts of asingle caruncle and among the caruncles ofdifferent species of asities is a result of differences in the space lattice dimensions of thecollagen arrays. Future investigations should
focus on testing this critical prediction. Observations of other species of asities should provide additional opportunities to examine theassociation between collagen ultrastructure
and color production, and to investigate theevolution of this novel color production
mechanism.
More generally, detailed anatomical investigations of structural color production in theavian dermis are necessary. The repeatedsuggestions in the literature that the vivid
blues and greens of the facial skin of the
cassowaries (Casuariidae), peafowls (Numididae), and toucans (Ramphastidae) are produced by Tyndall scattering are entirely unsupported by any direct observations (Rawles,’60:223;Lucas and Stettenheim, ’72:406,410;
Fox, ’76). No detailed studies of structural
color production in the avian dermis havebeen published previously (Lucas and Stettenheim, ’72:406, 410; Fox, ’76; Durrer, ’84).
The preoccupation of Ornithologists with thebrilliant feathers of birds have resulted in an
entire literature on structural color production in these dermal derivatives, but may
have contributed to the dearth of information about color production in the avian skin.
ACKNOWLEDGMENTS

The Direction des Eaux et For&tsand theMinistere de 1’Enseignement Superieur of
the Republic of Madagascar kindly granted
permission to S.M. Goodman of the Field
Museum of Natural History to collect specimens of Philepitta castanea. S.M. Lanyon of
the Field Museum of Natural History lent
the specimens to us for the research. S.M.
Goodman, S. Zack, and H. Rakotomananagave valuable comments on the natural history of Philepitta castanea. s. Zack kindly
gave permission for the use of an originalcolor photograph (Fig. 1A). B. Cutler prepared the SEMs. The manuscript was improved by comments from A. Brush, S.K.
Frost-Mason, M. Popovici, L. Trueb, and W.
Yelon. The research was supported by grants
from the National Science Foundation, theUniversity of Kansas Graduate Teaching
Fund and Graduate Research Fund to R.O.
Prum; from the Wesley Foundation to S.K.
Frost-Mason; and from the National Science
Foundation to Linda Trueb.

72

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J,