Structural colouration of avian skin: convergent evolution of coherently scattering dermal collagen arrays
1 Department of Ecology and Evolutionary Biology, and Natural History
Museum, Dyche Hall, University of Kansas, Lawrence, KS 66045-7561,
USA
2 Department of Mathematics, University of Kansas, Lawrence, KS 66045-2142,
USA
* Author for correspondence (e-mail: prum{at}ku.edu)
Accepted 4 April 2003
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Summary |
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Key words: structural colour, colour, collagen, integument, nanostructure, Fourier analysis, Aves
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Introduction |
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Accordingly, structural colour production mechanisms can be classified as
forms of either incoherent or coherent scattering
(van de Hulst, 1981;
Bohren and Huffman, 1983
).
Incoherent scattering occurs when individual light-scattering objects
differentially scatter visible wavelengths
(Fig. 1A). Incoherent
scattering models require that the light-scattering objects are spatially
independent (i.e. randomly distributed with respect to visible wavelengths) so
that the phase relationships of the scattered waves are random. Consequently,
incoherent scattering models ignore the phase relationships among the
scattered waves and describe colour production as the result of differential
scattering of wavelengths by the individual scatterers themselves
(van de Hulst, 1981
;
Bohren and Huffman, 1983
). By
contrast, coherent scattering occurs when the spatial distribution of
scatterers is non-random with respect to the wavelengths of visible light, so
that the phases of scattered waves are non-random
(Bohren and Huffman, 1983
).
Coherent scattering models describe colour production in terms of the phase
interactions among light waves scattered by multiple scatterers
(Fig. 1B). Scattered waves that
are out of phase destructively interfere and cancel one another, whereas
scattered waves that are in phase will constructively reinforce one another
and are coherently reflected. In incoherent scattering, colour is a function
of the properties of individual scatterers, whereas in coherent scattering
colour is determined by the spatial distribution of light-scattering
interfaces (Fig. 1).
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Incoherent scattering models include Rayleigh scattering (also erroneously
known as Tyndall scattering; see Young,
1982) and Mie scattering, which is a mathematically exact
description of light scattering by single particles that simplifies to the
Rayleigh scattering for small particle sizes
(van de Hulst, 1981
;
Bohren and Huffman, 1983
).
Examples of incoherent scattering include blue sky, blue smoke, blue ice and
blue snow. Coherent scattering encompasses various optical phenomena that can
also be described as diffraction, reinforcement and interference. Well-known
examples include the structural colours produced by brilliant iridescent
butterfly wing scales and avian feather barbules such as the peacock's tail
(Fox, 1976
;
Ghiradella, 1991
;
Parker, 1999
).
Coherent scattering often produces the phenomenon of iridescence a
prominent change in hue with angle of observation or illumination. Iridescence
occurs if changes in the angle of observation or illumination affect the mean
path length of scattered waves; such a change will affect the phase
relationships among the scattered waves and change which wavelengths are
constructively reinforced after scattering. Iridescence conditions are met
when the light-scattering objects are arranged in laminar or crystal-like
arrays. By contrast, incoherent scattering does not yield iridescence. In the
biological literature, at least since Mason
(1923), iridescence has been
often synonymized with coherent scattering (e.g.
Fox, 1976
;
Nassau, 1983
; Lee,
1991
,
1997
;
Herring, 1994
). Consequently,
all iridescent structural colours were hypothesized correctly to be due to
coherent scattering, but all non-iridescent structural colours were
erroneously hypothesized to be exclusively due to incoherent scattering (e.g.
Fox, 1976
;
Herring, 1994
).
Recently, however, it has been demonstrated that coherent light scattering
by quasi-ordered arrays of light scatterers can produce biological structural
colours that are not strongly iridescent (Prum et al.,
1998,
1999a
,b
).
Quasi-ordered arrays have unimodal distributions of size and spacing but lack
laminar or crystal-like organization at larger spatial scales that produce
iridescence. Quasi-ordered arrays have a similar organization to a bowl of
popcorn; each popped kernel is similar in size to its neighbour, and
centre-to-centre distances are quite similar, but beyond the spatial scale of
a single kernel there is no organization. An example of a colour-producing
nanostructure is the light-scattering air bubbles in the medullary keratin of
structurally coloured avian feather barbs; these airkeratin matrices
are sufficiently spatially ordered at the nanoscale level to produce the
observed hues by coherent scattering but are not ordered at larger spatial
scales (Prum et al., 1998
,
1999b
), so these colours are
not iridescent or are only weakly iridescent
(Osorio and Ham, 2002
).
In order to describe the spatial periodicity and to analyze the optical
properties of quasi-ordered biological arrays, we have developed an
application of the two-dimensional (2-D) Fourier transform to structural
colour production (Prum et al.,
1998,
1999a
,b
).
Based on transmission electron micrographs (TEMs) of the tissues, the method
permits the characterization of the spatial periodicity of the tissue in
multiple directions and the prediction of the hue, and potentially
iridescence, of its colour. This method is designed to test whether light
scatterers are spatially independent a fundamental assumption of
incoherent scattering models and whether the biological arrays are
appropriately nanostructured to produce the observed colours by coherent
scattering.
Structural colours of avian skin
Non-iridescent structural colours occur in the skin, bill (ramphotheca),
legs and feet (podotheca) of a broad diversity of birds from many avian orders
and families (Figs 2,
3). Auber
(1957) reported structurally
coloured skin in 19 avian families from 11 avian orders
(Table 1). Auber
(1957
) assumed that all blue or
green skin colours are structural rather than pigmentary because blue and
green pigments are unknown or very rare, respectively, in the avian integument
(Fox, 1976
). Using the same
conservative criterion, we have identified structurally coloured skin,
ramphotheca and podotheca in 129 avian genera in 50 families from 16 avian
orders (Table 1). Structurally
coloured skin is apparently present in more than 250 bird species, or roughly
more than 2.5% of avian biological species.
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Because they are noniridescent, structural colours of avian skin were long
hypothesized traditionally to be produced by Rayleigh (or Tyndall) scattering
(Camichel and Mandoul, 1901;
Mandoul, 1903
;
Tièche, 1906
;
Auber, 1957
;
Rawles, 1960
;
Lucas and Stettenheim, 1972
;
Fox, 1976
). The
light-scattering structures were variously hypothesized to be melanin
granules, biological colloids or turbid media of proteins, lipids, etc. in the
dermis. The Rayleigh scattering hypothesis was never actually tested with
either spectrophotometry to examine whether these structural colours
conform to the prediction of Rayleigh's inverse fourth power law or
with electron microscopy to examine whether the hypothesized
light-scattering objects were spatially independent. Green integumentary
colours were further hypothesized to be a combination of Rayleigh-scattering
blue and carotenoid yellow (e.g. Fox
1976
) but this was never confirmed.
Prum et al. (1994) were the
first to examine structurally coloured avian skin with electron microscopy. We
documented that the green and blue colours of the supraorbital caruncles of
the male velvet asity Philepitta castanea (Eurylaimidae) of
Madagascar are produced by coherent scattering from hexagonally organized
arrays of parallel collagen fibres in the dermis. Subsequently, Prum et al.
(1999a
) performed a
comparative analysis of the anatomy, nanostructure and structural colouration
of three species of the Malagasy asities. We applied the 2-D Fourier method to
asity caruncle collagen arrays to confirm the coherent scattering mechanism
and to describe the variations in hue produced by variations in collagen
nanostructure. We also documented that the crystal-like hexagonal
nanostructure of Philepitta is derived from the plesiomorphic,
quasi-ordered nanostructure of the sunbird asities Neodrepanis.
Elsewhere in animals, colour-producing, quasi-ordered collagen arrays have
only been described in the tapetum lucidum of the sheep eye
(Bellairs et al., 1975
).
With the exception of the asities (Eurylaimidae; Prum et al.,
1994,
1999a
), the incoherent
scattering hypothesis of structural colour production in avian skin has not
been tested. Here, we used fibre-optic spectrophotometry, light microscope
histology, TEM and 2-D Fourier analysis of TEM images to investigate
structurally coloured skin, ramphotheca and podotheca from 31 species of birds
from 17 families in 10 different orders
(Table 2). The sample includes
an ecologically diverse collection of birds with a wide variety of colours
from across avian phylogeny, from the paleognathes to the passerines (Figs
2,
3).
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Materials and methods |
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For light microscopy, specimens of structurally coloured skin from nine species were embedded in paraffin, cut into 10 µm sections and stained with Masson's trichrome, which includes the collagen-specific stain Fast Green. For TEM, skin and ramphotheca samples were placed in Karnovsky fixative (2.5% glutaraldehyde, 2.5% paraformaldehyde) for 2 h at 4°C. They were then post-fixed in 24% osmium tetroxide for 1.5 h. They were then stained with 2% aqueous uranyl acetate for 1 h. Tissue pieces were then dehydrated through an ethanol series and embedded in Eponate 12. They were sectioned with a diamond knife to a thickness of approximately 100 nm. Specimens were viewed with a transmission electron microscope (JEOL 12000 EXII; Peabody, MA, USA). TEM micrographs were taken with Polaroid negative film or were digitally captured using a Soft-Imaging Megaview II CCD camera (1024 pixelsx1200 pixels). Numerical analysis was conducted directly on the digital images or on the photograph negatives after scanning at a resolution of 300 d.p.i.
Spectrophotometry
If a substantial component of the original structural colour was preserved
after freezing or fixation, the reflectance spectra of the structurally
coloured tissues were measured using an S2000 fibre optic diode-array
spectrometer with a PX-2 pulsed xenon light source (Ocean Optics, Dunedin, FL,
USA). This spectrometer produces 2048 reflectance data points between 160 nm
and 865 nm (or 1520 data points in the range of 300800 nm) with a mean
error of 0.14 nm. Measurements were made with perpendicularly incident light
from approximately 23 mm away from the specimen for an illuminated
field of approximately 3 mm2 with 100 ms summation time. A
Spectralon diffuse reference standard from Ocean Optics was used as a white
standard, and the ambient light of a darkened room was used as a dark
reference. Percent reflectance (%R) was calculated by:
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2-D Fourier analysis
Coherent scattering of visible wavelengths is a consequence of nanoscale
spatial periodicity in refractive index of a tissue. Following a theory of
corneal transparency by Benedek
(1971), Prum et al.
(1998
,
1999a
,b
)
developed an application of the discrete Fourier 2-D transform to analyze the
periodicity and optical properties of structural coloured tissue. Discrete
Fourier analysis transforms a sample of data points into an equivalent sum of
component sine waves of different frequencies and amplitudes
(Briggs and Henson, 1995
). The
amplitudes of each Fourier component wave express the relative contribution of
that frequency of variation to the periodicity of the original data. The
variation in the squared amplitudes over all Fourier components is called the
Fourier power spectrum. The relative values of the different Fourier
components in the power spectrum express the comparative contribution of those
frequencies of variation to the original function. This application of Fourier
analysis is derived independently from electromagnetic optical theory
(Benedek, 1971
) and is distinct
from the traditional physical field of `Fourier optics', although they both
describe coherence among scattered light waves.
The digital or digitized TEM images were analyzed using the matrix algebra
program MATLAB (Version 5.2; MATLAB,
1992) on a Macintosh computer. The scale of each image (nm
pixel-1) was calculated from the number of pixels in the scale bar
of the micrograph. The largest available square portion of the array was then
selected for analysis; for most images, this area was 1024 pixels2,
but for a few images was as small as 600 pixels2. The mean
refractive index of each tissue was estimated by generating a two-partition
histogram of image darkness (i.e. the distribution of darker and lighter
pixels). The frequency distribution of darker and lighter pixels was used to
estimate the relative frequency of collagen and mucopolysaccaride in the image
and to calculate a weighted mean refractive index for the tissue. Previously,
we have used estimates of the refractive indices of collagen and the
mucopolysaccaride matrix between collagen fibres of 1.51 and 1.35,
respectively (Prum et al.,
1994
,
1999a
), taken from Maurice
(1984
). Recently, however,
more-refined methods have estimated the refractive indices of collagen and
mucopolysaccaride as 1.42 and 1.35, respectively
(Leonard and Meek, 1997
).
The numerical computation of the Fourier transform was done with the
well-established 2-D fast Fourier transform (FFT2) algorithm
(Briggs and Henson, 1995). We
calculated the 2-D Fourier power spectrum, or the distribution of the squares
of the Fourier coefficients. The 2-D Fourier power spectrum resolves the
spatial variation in refractive index in the tissue into its periodic
components in any direction from a given point. The 2-D Fourier power spectra
are expressed in spatial frequency (nm-1) by dividing the initial
spatial frequency values by the length of the matrix (pixels in the matrix
x nm pixel-1). Each value in the 2-D power spectrum reports
the magnitude of the periodicity in the original data of a specific spatial
frequency in a given direction from all points in the original image. The
spatial frequency and direction of any component in the power spectrum are
given by the length and direction, respectively, of a vector from the origin
to that point. The magnitude is depicted by the colour (from blue to red), but
the units are dimensionless values related to the total darkness of the
original digital images.
We calculated radial means of the power spectra using 100 spatial frequency bins, or annuli, between 0 nm-1 and 0.02 nm-1 and expressed them in % total Fourier power. Composite radial means were calculated from a sample of power spectra from multiple TEM images to provide an indication of the predominant spatial frequency of variation in refractive index in the tissue over all directions.
We also produced predicted reflectance spectra based on the 2-D Fourier power spectra, image scales and mean refractive indices. First, a radial mean of the % power was calculated for concentric bins, or annuli, of the power spectrum corresponding to 50 10 nm-wide wavelength intervals between 300 nm and 800 nm (covering the entire avian visible spectrum). The radial mean power values were expressed in % visible Fourier power by normalizing the total power values across all potentially visible spatial frequencies (i.e. potentially scattering light between 300 nm and 800 nm) to 1. The inverse of the spatial frequency means for each wavelength were then multiplied by twice the mean refractive index of the medium and expressed in terms of wavelength (nm). A few images depict oblique, elliptical sections of the cylindrical collagen fibres, which will bias the predicted hue towards longer wavelengths. In these cases, the radial mean was calculated from a single quadrant or from a custom radial section of the power spectrum. A composite predicted reflectance spectra for each tissue was produced by averaging the normalized predicted spectra from a sample of TEM images. The result is a theoretical prediction of the relative magnitude of coherent light scattering by the tissue that is based entirely on the spatial variation in refractive index of the tissue.
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Results |
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None of the reflectance spectra from the 14 species measured showed the
inverse fourth power relationship predicted by Rayleigh scattering. Each
reflectance spectrum revealed a discrete peak or a pair of peaks. The
lower-wavelength (i.e. left-hand) slopes of the spectra are not caused by
absorption (e.g. Finger,
1995), because the peaks are at substantially longer wavelengths
than the beginning of the absorption spectrum of collagen (approximately 290
nm). However, reflectance spectra from a number of diverse galliform species
exhibit dual peaks: one in the ultraviolet (approximately 350 nm) and another
in the blue portion of the spectrum (approximately 410 nm)
(Fig. 4A-C,E). The highly
repeated position of the dip in multiple reflectance spectra between the two
peaks at approximately 400 nm may indicate selective absorption of these
wavelengths by some unknown pigment or component of the epidermis. These
features of the reflectance spectra were not adequately explained by
Fourier-predicted reflectance spectra (see below) and currently require some
additional explanation.
None of these structural colours was iridescent, although under a dissection microscope, local (approximately 500 µm scale) variations in colour could be observed among different areas of the skin. In many samples, the hue could be changed or eliminated (i.e. turned white) by compression on the surface of the skin with forceps. Presumably, this occurs because of deformation of the colour-producing dermal collagen arrays.
Anatomy
Structurally coloured skin samples of nine species were examined with light
microscope histology (Fig. 5).
Most species had a thin epidermis between 12 µm and 50 µm thick. Below
the epidermis was a substantial colour-producing, dermal collagen layer that
varied, in most cases, between 200 µm and 500 µm in thickness (Figs
5,
6AC). In most species,
this collagen layer was underlain by a thick and continuous layer of melanin
granules parallel to the surface of the skin that completely covered the
deeper dermal tissue (Figs 5,
6C). One species, Procnias
nudicollis (Cotingidae), had a substantially thicker collagen layer of
5001000 µm with little or no melanin in the underlying dermis
(Fig. 5H). Even without
histological examination, it was easily observed that two species of
Neotropical antbirds, Gymnopithys leucapsis and Rhegmatorhina
melanosticta (Thamnophilidae), clearly lack any melanin deposition in the
skin (also confirmed by TEM) (not shown).
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The morphology of structurally coloured bird skin was quite distinct from that of uncoloured white skin and from that of bright red skin in the same or closely related species. For example, unpigmented white leg skin from P. nudicollis was less than 75 µm thick (Fig. 5G), or an order of magnitude thinner than the structurally coloured avian skin of P. nudicollis and other bird species. In the red lateral patches of the throat lappet of T. temminckii (Fig. 2E), Tragopan satyra and Tragopan caboti (Fig. 2F) and the red facial skin of the toucan Baillonius bailloni (Ramphastidae; not illustrated), the dermis showed abundant capillaries immediately below the epidermis and greatly reduced or no underlying melanin deposition (Fig. 5I). Purely structurally coloured bird skin also differs from carotenoid pigmented skin. In Ramphastos toco (Fig. 3C), the yellow facial skin has abundant carotenoid-containing lipid vacuoles within the epidermis and a complete lack of melanin deposition (Fig. 6F). The adjacent, structurally coloured, ultraviolet eye ring completely lacks epidermal lipid vacuoles and has abundant underlying melanosomes. Some tissues, including the yellow facial skin of R. toco, apparently combine both structural colouration and carotenoid pigmentation (see `Combined structural and pigmentary colours' below).
Nanostructure
Quasi-ordered arrays of parallel collagen fibres were observed in the
dermis of 30 of the 31 species examined
(Fig. 7). Collagen fibres were
identified by their circular cross-sections and by the distinctive collagen
banding pattern when viewed perpendicular to the fibre axes
(Fig. 6AC). In the
quasi-ordered arrays, the collagen fibres were similar in diameter and
interfibre distance but were not arranged in a crystal-like lattice or laminar
organization (Fig. 7). Only one
species, P. castanea (Fig.
7I), had collagen arrays organized in a highly regular,
crystal-like, hexagonal nanostructure
(Prum et al., 1994,
1999a
).
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The arrays of dermal collagen fibres were organized in larger,
lozenge-shaped structures, called macrofibrils by Prum et al.
(1994,
1999a
), which are apparently
produced by a single collagenoctye during development
(Fig. 6AC). These
collagen macrofibrils varied between 5 µm and 20 µm in diameter and were
20100 µm long (Figs
5,
6AC). The main axis of
most of these collagen fibres runs roughly parallel to the surface of the
skin, although there is substantial variation (Figs
5,
6AC).
No specimens exhibited any evidence of iridophores pigment cells
that contain arrays of purine or pterine crystals and produce structural
colours in the integument of fishes, amphibians and reptiles
(Bagnara and Hadley, 1973;
Fox, 1976
;
Bagnara, 1998
) and in the avian
iris (Ferris and Bagnara,
1972
; Oliphant et al.,
1992
; Bagnara,
1998
). Most samples showed no epidermal lipid vacuoles that may
contain carotenoid pigments (Lucas and
Stettenheim, 1972
; Menon and
Menon, 2000
), but combined structural and pigmentary colours in
T. caboti, Apaloderma aequatoriale, R. toco and D. concreta
are discussed below.
2-D Fourier analysis
Two-dimensional Fourier analysis was used to describe the spatial
periodicity of the variation in refractive index within the colour-producing
dermal collagen arrays. The 2-D Fourier power spectra of TEM images of
cross-sections of the collagen arrays exhibit circular rings of high magnitude
power values at intermediate spatial frequencies
(Fig. 8). Some power spectra
reveal a second high power ring of harmonic spatial frequencies at twice the
magnitude of the fundamental spatial frequency
(Fig. 8A,B,D,F,H,I). Radial
means of these power spectra indicate that these peak spatial frequencies vary
between 0.0049 nm-1 and 0.0097 nm-1 among the
differently coloured skin samples from different species
(Fig. 9;
Table 2; but see `Structural
colour production in antbird skin' below). This range of spatial frequencies
corresponds to centre-to-centre distances between neighbouring collagen fibres
of 110204 nm (Prum et al.,
1999a). This range of spatial frequency values should result in
coherently scattered colours within the visible spectrum of birds
(Fig. 9).
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The ring-shaped Fourier power distributions of most species demonstrate
that these collagen arrays are substantially nanostructured
(Fig. 8). Furthermore, the
predominant nanostructure is of the appropriate size to produce visible hues
by coherent scattering (Fig.
9). The circular rings in the power spectra further demonstrate
that the dermal collagen fibre arrays are quasi-ordered or equivalently
nanostructured in all directions perpendicular to the fibres. The circular
power spectra demonstrate that these collagen arrays lack the laminar or
crystal-like periodicity found in highly iridescent, coherently scattering
nanostructures. Thus, congruent with their appearance, ring-shaped 2-D Fourier
spectra predict that the collagen arrays should not be strongly iridescent.
There is also substantial variation in orientation of collagen arrays both in
angle to the epidermis and in fibre orientation at larger spatial scales (Figs
5,
6AC), which further
eliminates opportunities for iridescence at larger spatial scales. The unique,
hexagonal arrays of P. castanea
(Fig. 7I) produced power
spectra with a hexagonal distribution of high power values
(Fig. 8F;
Prum et al., 1999a). However,
P. castanea is also not iridescent because the many collagen arrays
in the dermis are arranged in many different angles and directions with
respect to the skin, eliminating the hexagonal order of the nanostructure
(Prum et al., 1999a
).
The demonstration of substantial nanostructure at these spatial scales (0.00490.0097 nm-1) directly falsifies a fundamental assumption of the incoherent scattering mechanisms, including Rayleigh (Tyndall) and Mie scattering (Figs 8, 9). The light-scattering collagen fibres in these tissues are not spatially independent (i.e. randomly distributed at the spatial scale of visible wavelengths) as the incoherent scattering models assume.
Predicted reflectance spectra based on the 2-D Fourier power spectra
correspond generally to the observed colours of the original tissues
(Fig. 10;
Table 2). Accurate quantitative
predictions of the peak wavelengths (max) of the
reflectance spectra of these tissue samples were limited by the variable
condition of the original specimens (e.g. frozen or fresh), by variable
collection circumstances, by different original fixatives (10% formalin
vs glutaraldehyde) and especially by degradation of nanostructure and
refractive index between the time of fixation and TEM examination (see
`Sources of error' below). The 2-D Fourier method, however, did provide
accurate predictions of tissue colours for those specimens that were fixed and
examined with TEM in a relatively short time period (less than six months;
Fig. 10). For example, the
predicted reflectance spectra match the measured reflectance spectra well for
T. temminckii, T. caboti, L. bulweri, Pilherodius pileatus, Syrigma
sibilatrix, Selenidera culik, R. toco and D. concreta. In
particular, the variation in colour between dark blue and light blue portions
of the caruncle shield of T. temminckii and T. caboti was
accurately predicted by the Fourier analysis of the nanostructures of the
collagen arrays from these parts of the tissues
(Fig. 10BD;
Table 2). Thus, the Fourier
analyses further support the conclusion that the hue of coherent scattering is
determined by mean collagen fibre size and interfibre spacing.
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Coherent scattering from dermal arrays produces a wide variety of structural hues in bird skin from ultraviolet to yellow. Although it has been hypothesized that integumentary green colours are produced by structural blue with carotenoid yellows, the green colours in the skin of S. culik, Selenidera reinwardtii, P. castanea, P. nudicollis and D. concreta and the yellow colour of the skin of Ceuthmochares aereus are purely structural. These tissues lacked any indication of carotenoid-containing lipid vacuoles in the integument, and Fourier analysis of their collagen arrays indicates that the fibres are appropriately nanostructured to create the observed hues by coherent scattering alone.
Combined structural and pigmentary colours
Several species examined had areas of yellow and orange skin that appear to
be produced by a combination of structural colouration and carotenoid
pigmentation: the orange facial and lappet skin of the pheasant T.
caboti (Fig. 2F), the
yellow facial skin of the trogon A. aequatoriale, the yellow facial
skin of R. toco and the yellowish portions of the circumorbital
caruncle of the Old World flycatcher D. concreta
(Fig. 3J). All four of these
samples showed substantial concentrations of lipid-filled cells in the
uppermost strata of the dermis (Fig.
6D,E). Pigments within these lipid vacuoles were not extracted or
identified but are presumed to be carotenoids.
T. caboti, R. toco and D. concreta had other purely structurally coloured areas of dark blue, light blue or green skin that completely lacked lipid-filled pigment cells that were immediately adjacent to the carotenoid pigmented areas. In all three species, the collagen arrays in these adjacent structurally coloured areas were appropriately nanostructured to produce the observed colours entirely by coherent scattering alone (Table 2). The dermis of the orange skin of T. caboti is thick with collagen arrays and the underlying dermis is highly melanized, indicating that this orange hue is not produced by capillary blood as in the paired, lateral, red patches of the facial lappet of all Tragopan species (Fig. 2E,F). Critically, in the yellow and orange areas of T. caboti, R. toco, A. aequitoriale and D. concreta tissue, the collagen arrays showed substantially larger collagen fibre diameters (Fig. 7F,K) and smaller peak spatial frequencies (0.00360061 nm-1; Figs 8I, 9D,H; Table 2), which should coherently scatter longer-wavelength colours (Fig. 10D,I; Table 2). Thus, it appears that in some avian taxa, longer-wavelength yellow and orange integumentary colours are produced by a combination of longer-wavelength structural colour and carotenoid pigmentation.
The importance of the structural component to the colours of the orange T. caboti (Fig. 2F; Table 2) and the yellow A. aequatoriale (Table 2) skins is further supported by the shapes of their reflectance spectra (Fig. 4F). Typically, carotenoid pigments produce a long wavelength plateau in their reflectance spectra. By contrast, the orange and yellow skins of T. caboti (Fig. 4F) and A. aequatoriale (data not shown) reveal a distinct reflectance peak at 600 nm and 530 nm, respectively, with a substantial drop off in reflectance at longer wavelengths beyond the peak. Furthermore, the reflectance peaks are generally congruent with the predicted reflectance spectra based on the Fourier analysis of the collagen arrays in this tissue (Fig. 10D; Table 2). Since carotenoid pigments typically do not fluoresce (i.e. they cannot emit wavelengths different from excitation wavelengths), reduction in the backscattering of longer wavelengths into the carotenoid pigment cells would reduce the emission of longer wavelengths by those pigments. Apparently, destructive interference of longer wavelengths by the quasi-ordered dermal collagen arrays limits backscattering of longer wavelength light from the dermis into the more superficial carotenoid pigment cells. This selective coherent scattering of mid-range wavelengths by dermal collagen evidently reduces the typical long wavelength reflectance of the carotenoid pigments and produces a colour whose brilliance is probably enhanced by the presence of the pigments but which has a reflectance spectrum that is distinctly more saturated at intermediate wavelengths than those of typical pigmentary colours. By contrast, the yellow skin of R. toco has a reflectance spectrum with a broad plateau in reflectance across all longer wavelengths (550750 nm; Table 2), which is typical of carotenoid pigments.
Structural colour production in antbird skin
The structurally coloured skin of one species of antbird (Thamnophilidae)
examined, Myrmeciza ferruginea, was typical in collagen nanostructure
and dermal melanization of the other bird species examined. However, two of
the three species examined from the Neotropical suboscine antbirds, G.
leucapsis and R. melanosticta (Thamnophilidae), had
conspicuously smaller nanostructures than all other species examined. Peak
spatial frequencies in these tissues 0.0105 nm-1 and 0.0136
nm-1, respectively correspond to fibre-to-fibre centre
distances of 6774 nm. The circumorbital tissues of G.
leucapsis and R. melanosticta are light blue in life
(Fig. 3G), but, unlike all
other samples, these tissues immediately lost their blue colour upon fixation
and became nearly transparent. Furthermore, both species lack any dermal
melanin. Dermal tissues of both species had prominent collagen fibre arrays,
but the fibre sizes were extremely small
(Fig. 7J) and the spatial
frequency rings were very large (0.0105 nm-1 and 0.0136
nm-1, respectively; Fig.
8G). These fibre arrays are outside the range of sizes that are
likely to make a visible colour by coherent scattering
(Fig. 9G). Similar spatial
frequencies in the collagen arrays of the human cornea create destructive
interference among all visible wavelengths, producing optical transparency and
coherent reinforcement of nonvisible light in the far ultraviolet
(Benedek, 1971;
Gisselberg et al., 1991
; Vaezy
and Clark, 1991
,
1993
). If G.
leucapsis and R. melanosticta produce colour by coherent
scattering from this nanostructure, it should be an extreme ultraviolet hue
rather than the observed light blue. But this cannot be determined in the
absence of reflectance spectrum of living specimens of these antbirds. Given
these substantial differences in anatomy and nanostructure from the other
coherently scattering tissues, it would be best to conclude that the mechanism
of integumentary structural colour production in Gymnopithys and
Rhegmatorhina antbirds remains to be established and requires further
investigation.
Sources of error
In acquiring this sample of structurally coloured skin from a diversity of
avian species from so many different sources, it was impossible to control for
variation in preservation methods and conditions. The diverse specimens
examined here were acquired over a period of eight years. Unfortunately, the
colours of many specimens changed or degraded substantially after the original
fixation during storage in cacodylate buffer at 4°C but before electron
microscopy. Often, colours decreased in intensity to a greyish hue. Others
also increased in wavelength with time. For example, the sample of light blue
ramphotheca from Oxyura jamaicensis (Anatidae) had a peak reflectance
of 465 nm in 1998 when the specimens were first thawed several days after
death and fixed (Fig. 4A), but
in 2002 at the time of the TEM observation the peak reflectance had changed to
590 nm. For this reason, accurate reflectance measurements could not be made
for all species (Table 2).
Furthermore, Fourier analysis of some tissues predicted reflectance peaks that
were of substantially longer wavelengths than the original colours. In the
case of Oxyura, however, the measured (590 nm) and predicted (580 nm)
hues after years of storage were closely correlated
(Table 2).
Apparently, the observed changes in hue are the result of changes in the
size of the colour-producing collagen arrays. Prum et al.
(1994) hypothesized that
shrinkage due to dehydration in 10% formalin and ethanol turned the green
caruncle tissue blue in P. castanea. Likewise, the increase in hue in
Oxyura is apparently a result of expansion in collagen array size
during storage in cacodylate buffer following fixation. Alternatively, any
changes in difference in refractive indices between the fixed collagen fibres
and the surrounding mucopolysaccaride matrix could result in degradation in
the magnitude of reflectance. For example, several samples (e.g. Sula
nebouxii and C. aereus) showed highly nanostructured collagen
under TEM that should have produced vivid structural colours, but these
preserved tissues were only dull grey in colour. Depending on the actual
direction of change in mean refractive index of the tissue, the degradation of
refractive indices could also contribute to increases or decreases in peak
wavelength of reflectance.
Evolution of structurally coloured avian skin
The existence of anatomically identical collagen nanostructures that
function by the same physical mechanism in many distantly related avian clades
is compelling evidence of extensive convergent evolution. Unfortunately,
higher level avian taxonomy is poorly understood, and there is no consensus
phylogeny of birds. Homology among the many instances of avian structurally
coloured skin would only be possible if these diverse genera were closely
related and basal within their clades. However, many of these avian genera are
members of diverse families in which there are many species that lack
structurally coloured skin (Table
1). Furthermore, there is no reason to hypothesize that genera
with structurally coloured skin are basal members of their families, that
families including many genera with structurally coloured skin are basal
within their orders or that families and orders with structurally coloured
skin are especially closely related to one another. Thus, the hypothesis of
homology among many or most instances of structurally coloured skin in birds
would require numerous evolutionary losses and would be wildly
unparsimonious.
Based on what is known about avian phylogeny, there is not a single
unambiguous instance of homology of structurally coloured skin between any two
avian families (Table 1). The
structural colours of the anhingas (Anhingidae) and the cormorants
(Phalacrocoracidae) come closest, but it is unlikely that the structurally
coloured species within these families are basal. There are a few examples of
diverse radiations of genera and species within families that potentially
share a single origin of structurally coloured skin. For example, the guans
and curassows (Cracidae), the pheasants (Phasianidae), the guineafowl
(Numididae), the herons (Ardeidae), the hornbills (Bucerotidae), the toucans
(Ramphastidae), the monarch flycatchers (Monarchidae) and the honeyeaters
(Meliphagidae) are clades with many genera and species that may share
homologous, structurally coloured skin. Several genera or clades of genera
have radiated with homologous structurally coloured skin patches: e.g.
Philepitta and Neodrepanis (Eurylaimidae) and
Dyaphorophyia (Platysteiridae). By contrast, other families have
probably had multiple evolutionary independent origins of this trait within
them. A phylogeny of cotingas (Cotingidae) indicates that the three species
with structurally coloured skin Perissocephalus tricolor, Procnias
nudicollis and Gymnoderus foetidus are each most closely
related to other species and genera that lack structurally coloured skin
(Prum et al., 2000). Likewise,
the bird of paradise genera Paradigalla, Cicinnurus and
Epimachus (Paradiseaidae) are not hypothesized to be most closely
related within the family (Frith and
Beehler, 1998
). Structurally coloured skin has probably had
multiple independent evolutionary origins within New World and Old World
cuckoos (Cuculidae; R. B. Payne, personal communication). Lastly, there are
numerous instances of single, phylogenetically isolated species or genera with
structurally coloured skin: e.g. Cariama (Cariamidae),
Opisthocomus hoazin (Opisthocomidae), Picathartes oreas
(Picathartidae), Lopoparadisea (Cnemophilidae), Cyphorhynus
phaeocephalus (Troglodytidae) and Leucopsar rothschildi
(Sturnidae) (Table 1).
A precise estimate of the number of evolutionary origins and losses of structurally coloured skin in Aves would require a well-resolved phylogeny with accurate relationships from the highest interordinal levels to interspecific and intergeneric levels within many families. Based on the distribution of this trait among and within families of birds (Table 1), however, it would be conservative to estimate 5065 evolutionarily independent origins of structurally coloured skin within extant birds.
Several instances of evolutionary radiation in structural colouration document that the hue of these structural colours can evolve rather easily in likely response to sexual and social selection (see Discussion). The collagen arrays of the yellow facial skin of C. aereus document that there are no physical constraints limiting the size of collagen fibre arrays and the colours they can produce. Four species observed T. caboti, A. aequatoriale, R. toco and D. concreta have evolved a combination of yellow and orange structural colours and carotenoid pigments. All species belong to diverse clades that have purely structurally coloured skin. Thus, it is possible in these clades that these combined structural pigmentary colours evolved from plesiomorphic structural colours with the derived addition of carotenoids.
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Discussion |
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Two-dimensional Fourier analysis of these colour-producing collagen arrays demonstrates that they are substantially nanostructured at the appropriate spatial scale to produce visible colours by coherent scattering (i.e. constructive interference). This quasi-ordered nanostructure is equivalent in all directions in the tissue perpendicular to the collagen fibres, which explains why these colours are not iridescent. In well-preserved tissues that are examined quickly, the 2-D Fourier analysis can provide an accurate prediction of the shape of the reflectance spectrum. No differences were found in anatomy or nanostructure among the structurally coloured skin, ramphotheca (Oxyura jamaicensis and Ramphastos vitellinus) or podotheca (Sula nebouxii).
The reflectance spectra of structurally coloured bird skin falsify the inverse fourth power prediction of the incoherent (Rayleigh) scattering hypothesis (Fig. 4). Furthermore, the ring-shaped maxima of the Fourier power spectra demonstrate directly that spatial variations in refractive index within these extracellular matrices are not spatially independent, as assumed by the incoherent scattering hypothesis (Figs 8, 9). After more than one century of unquestioned support, the Rayleigh (also known as Tyndall) scattering hypothesis has been falsified for a wide diversity of birds.
These results further discredit the common opinion among biologists that
coherent scattering, or interference, is synonymous with iridescence
(Mason, 1923;
Fox, 1976
;
Nassau, 1983
; Lee,
1991
,
1997
;
Herring, 1994
). Actually, both
iridescent and non-iridescent structural colours can be produced by coherent
scattering. Laminar and crystal-like arrays can produce iridescence.
Quasi-ordered arrays, however, are sufficiently nanostructured to produce
vivid structural colours but are not appropriately ordered at larger spatial
scales to create strong iridescence (Prum et al.,
1998
,
1999a
,b
).
A simple review of previous citations of the Rayleigh scattering mechanism in
biological systems indicates that this mechanism has never been satisfactorily
demonstrated with the reflectance spectra that conform to the Rayleigh's
predicted inverse fourth power law or with evidence of the spatial
independence of light-scattering objects
(Mason, 1923
;
Fox, 1976
;
Nassau, 1983
;
Herring, 1994
;
Parker, 1999
). Given the
historic lack of understanding of coherent scattering by quasi-ordered arrays,
experimental re-evaluation of all proposed biological examples of Rayleigh
scattering is required.
Our results demonstrate that nanostructured collagen in the avian dermis can be combined with carotenoid pigments to produce vivid integumentary colours. Furthermore, in some species, coherent scattering of yellow and orange wavelengths by nanostructured dermal collagen results in a brilliant, pigment-enhanced hue that is more saturated than typically carotenoid pigment colours. Exploiting a similar phenomenon, artists often coat canvases with a preliminary layer of brilliantly white gesso to enhance the colour of their pigments. In some birds, however, a specific, underlying, coherently scattered hue serves to actually alter the colour of the superficial pigments by limiting long wavelength scattering. In the future, the presence of carotenoid pigments in the skin should not be sufficient to conclude that an integumentary colour is purely pigmentary, because there remains the possibility that carotenoid hues may be enhanced or altered by an underlying structural component. Specifically, the presence of integumentary carotenoid pigmentation with a discrete peak in the reflectance spectrum may indicate a structural component. This phenomenon should be looked for in birds, other reptiles, amphibians and fishes.
Two of the three genera of Neotropical antbirds (Thamnophilidae) provide the only exception among the structurally coloured bird skins examined. The dermal collagen arrays in these genera are so small that they should be optically transparent or should produce an extremely ultraviolet colour rather than the observed light blue hues. Additional investigation with better specimens and reflectance spectra from life are required to further investigate the mechanism of colour production in these genera. Furthermore, the double peaks in some galliform reflectance spectra (Fig. 4AC,E) were not predicted by the Fourier analysis of the collagen nanostructure and may indicate the existence of some selective absorption of light at approximately 400 nm in the epidermis of these species.
Primitively, fishes, amphibians and reptiles produce integumentary
structural colours with iridophores, specialized pigment cells in the dermis
that contain guanine or pterine crystals
(Bagnara and Hadley, 1973;
Bagnara, 1998
), but iridophores
are absent from structurally coloured bird skin. These results confirm the
conclusion that birds, like mammals, have evolutionarily lost integumentary
iridophores (Oliphant et al.,
1992
; Bagnara,
1998
), although birds retain iridophores as an important mechanism
of structural colour production in the iris
(Oehme, 1969
;
Ferris and Bagnara, 1972
;
Oliphant, 1981
,
1987a
,b
;
Oliphant et al., 1992
;
Oliphant and Hudon, 1993
).
In at least two lineages Oxyura (Anatidae) and the
Malagasy asities (Eurylaimidae) (Figs
2B,
3D,E) structurally
coloured integumentary ornaments are seasonally variable. In Oxyura,
males develop blue ramphotheca colouration during the breeding season
(AprilJuly) but have black bills during the rest of the year
(Hays and Habermann, 1969). In
asities, the structurally coloured facial caruncles develop in the breeding
season and atrophy completely (including the dermal melanosomes and muscles)
during the rest of the year (Prum and
Razafindratsita, 1997
; Prum et
al., 1999a
). All other structural colours in avian skin are
apparently permanent once developed. One bird not examined here shows change
in the colour of the facial caruncle during ontogeny. In the blue-faced
honeyeater Entomyzon cyanotis (Meliphagidae), adults have blue facial
skin but immature individuals have greenish facial skin. Apparently, the
ontogeny of colour in Entomyzon is characterized by a reduction in
collagen nanostructure, which creates a shift in hue. The head and neck of
wild turkey Meleagris gallipavo can change rapidly from white to blue
(Schorger, 1966
; A. Krakauer,
personal communication). This rapid colour change may occur by mobilization of
melanosomes within melanocytes in the dermis in response to hormonal cues, as
in amphibians and other reptiles (Bagnara
and Hadley, 1973
; Bagnara,
1998
), but this hypothesis has not been examined.
The 2-D Fourier method provides a new tool for the analysis of
nanostructure and optical function in biological tissues (Prum et al.,
1998,
1999a
,b
).
The method is effective in testing alternative physical hypotheses of
structural colour production. Under the best conditions that limit shrinkage
or degradation of extracellular matrix nanostructure, the method provides an
accurate prediction of the reflectance spectra of structurally coloured skin.
Future research may be able to use the method to analyze the nanostructural
basis of behaviourally relevant variation in structurally coloured hues within
populations and species.
Evolution of avian structurally coloured skin
Two fundamental evolutionary questions are: how have these structurally
coloured collagen arrays evolved and why has convergent evolution of these
arrays been so frequent? Collagen is a ubiquitous and abundant extracellular
matrix molecule in connective tissues of metazoan animals. Despite its
diversity in molecular sequence, all collagens form self-assembled,
triple-helical fibres that are composed of collagen polypeptides and are
surrounded by a mucopolysaccaride matrix. Structural colour production by
arrays of collagen fibres requires the appropriate specification of two
components of collagen nanostructure that are already intrinsic to collagen
itself fibre diameter and interfibre spacing. Furthermore, the
functional contribution of collagen to the elasticity and support of the
integument ensures that a substantial component of dermal collagen fibres will
be arranged parallel to the surface of the skin (D. Homberger, personal
communication).
Thus, the collagenous extracellular matrix of the skin provides an inherent nanostructure that is very near to the appropriate spatial frequency and orientation to produce visible hues. Selection of integumentary collagen for a colour production function requires more rigid specification of preexisting features of this extracellular matrix. Latent genetic variation in the nanostructure of integumentary collagen may occasionally create heritable, visible variations in reflectance that could become subject to subsequent natural, sexual or social selection for structural colour production. We proposed that the frequent, convergent exaptation of integumentary collagen for a novel colour production function in birds has been fostered by the nature and function of integumentary collagen itself. Interestingly, the broad visual sensitivity of birds to near-ultraviolet light would permit them to observe optical consequences of a broader class of latent variations in integumentary collagen nanostructure and may create a broader set of opportunities for the evolution of nanostructured, colour-producing collagen from plesiomorphic collagen in the skin.
At larger anatomical scales, the evolution of structural colour production
by integumentary collagen also requires the development of a sufficient number
of light-scattering arrays to produce an observable colour. The reflectance
(R; i.e. the proportion of ambient light scattered at a single
interface between two materials of different refractive indices) is calculated
by the Fresnel equation to be:
![]() | (2) |
The evolution of integumentary structural colouration also requires the
development of a physical mechanism to prevent incoherent scattering of white
light by deeper tissues that underlie the superficial colour-producing
nanostructures. In almost all the species examined, colour-producing collagen
arrays are underlain by a thick layer of melanin granules
(Fig. 5). The anatomical
association between integumentary structural colouration and melanin
deposition is so strong that the melanin granules were frequently hypothesized
to produce the colour themselves by Rayleigh scattering
(Mandoul, 1903;
Rawles, 1960
;
Hays and Habermann, 1969
;
Fox, 1976
). Several authors
have remarked that the disappearance of the structural colour upon removal of
the melanin layer supports the conclusion that melanin actually produces the
colour (e.g. Hays and Habermann,
1969
). Actually, the functional role of underlying melanin is to
absorb any light transmitted completely through the array and to prevent
incoherent scattering of white light from the deeper tissues, which are not
nanostructured for colour production. A functional alternative to an
underlying melanin barrier is to have enough light-scattering objects in the
array so that virtually all the incident light is coherently scattered in the
appropriate hue. Thus, the approximately 5000 light-scattering collagen fibres
in a dermal cross-section in P. nudicollis, which lacks dermal
melanin (Fig. 5H), approaches
the theoretical level of total reflection of incident light that would render
any underlying melanin layer unnecessary.
Pre-existing melanin deposition in the skin may enhance the likelihood of subsequent evolution of structural colouration within a lineage by making the optical effects of chance variation in superficial collagen nanostructure immediately more visible. Many bird genera with structurally coloured skin have close relatives with melanin-pigmented facial skin: e.g. cormorants (Phalacrocoracidae), ducks (Anatidae), avocets (Recurvirostridae) and honeyeaters (Meliphagidae). Likewise, plesiomorphic bare skin may also foster the evolution of structurally coloured skin, since variations in integumentary nanostructure would be immediately observable and potentially subject to selection. The featherless eye ring is a good example of a broadly distributed bare skin, and, not surprisingly, the most common position for structural colours is around the eyes (Figs 2, 3).
There is at least one instance of evolution of integumentary structural colour by artificial selection in a domestic breed of chicken (Gallus gallus). Silkie chickens were first bred in China more than a thousand years ago. Silkies have many associated novel phenotypic features that evolved by artificial selection during domestication. These include feather abnormalities, polydactyly, highly melanized skin, albino feathers and deep blue to turquoise earlobes. It is likely that the original mutation in Silkie that produced extreme dermal melanization, unique in Gallus, created the opportunity for subsequent artificial selection for structurally coloured blue earlobes.
Function of integumentary structural colours
Little is known about the function of structural coloured skin in the lives
of birds. Structurally coloured ornaments feature prominently in the courtship
displays of some polygynous species. During the courtship displays of the
polygynous male tragopans (Tragopan; Phasianidae), the throat lappet
is extended over the breast by the shunting of blood, exposing complex patches
of structural ultraviolet, structural light blue and pigmentary blood red
hues; simultaneously, a pair of structurally coloured light blue horns is
erected from the sides of the crown by the contraction of helical or
cylindrically arranged muscles fibres
(Murie, 1872; R. O. Prum,
personal observation). The elongate, ultraviolet coloured facial caruncles of
Bulwer's pheasant Lophura bulweri (Phasianidae) are erected by a
combination of vascular and muscular mechanisms during its courtship displays
(Fig. 2D;
Schneider, 1938
). In the
velvet asity Philepitta castanea (Eurylaimidae), the two structurally
coloured green and blue supraorbital caruncles are erected by muscular
contraction to form two brilliant planes that intersect above the bill like a
V-shaped, tricorner hat (Prum and
Razafindratsita, 1997
). Thus, in some lineages (e.g. Phasianidae,
Eurylaimidae, Cotingidae, Paradisaeidae and Cnemophilidae), the evolution and
radiation of sexually dimorphic skin structural colours is associated with
polygyny and consequent sexual selection. However, there are as many instances
of sexually monomorphic skin structural colours (Ardeidae, Cariamidae,
Bucerotidae, Ramphastidae, Meliphagidae, Monarchidae, etc.). Apparently, in
other instances, integumentary structural colours have evolved in both sexes
for other social communication purposes.
Although birds with structurally coloured skin are phylogenetically and
ecologically diverse, the trait appears to have evolved most frequently in
interior humid rainforest birds. For example, nearly all species with
structurally coloured skin within the Casuariiformes, Galliformes,
Opisthocomiformes, Cuculiformes, Trogoniformes, Coraciiformes, Piciformes and
Passeriformes are found in tropical forests and woodlands, mostly in lowland
tropical forests. Since tropical forest species account for a large proportion
of all birds, it is ultimately necessary to test phylogenetically whether the
observed frequency of structurally coloured skin among tropical forest birds
is higher than random. However, the striking and complete conformity to this
pattern within Passeriformes, which is diverse in both tropical and temperate
habitats, provides strong support for the pattern. Although not predicted by
previous sensory drive theory (Endler,
1993), the quality of ambient light within tropical forest
habitats may foster convergent evolution of communication signals in the
smaller wavelength portion of the visible spectrum, in which vertebrate
pigments are unavailable. Vorobyev and Osorio
(1998
) have hypothesized that
receptor noise is an important determinant of colour perception thresholds.
Their model of tetrachromatic bird vision predicts that ultraviolet and blue
sensitivity should rise in habitats where ultraviolet and blue wavelengths are
rare (Vorobyev and Osorio,
1998
). Endler
(1993
) has shown that smaller
visible wavelengths are under-represented in tropical forest interiors under
sun. Thus, smaller wavelength structural colours may have evolved more
frequently in tropical forest and woodland birds in response to natural or
sexual selection for signals that exploit increased visual sensitivity to
these wavelengths in the ambient light environment.
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Acknowledgments |
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References |
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---|
Auber, L. (1957). The distribution of structural colors and unusual pigments in the Class Aves. Ibis 99,463 -476.
Bagnara, J. T. (1998). Comparative anatomy and physiology of pigment cells in nonmammalian tissues. In The Pigmentary System Physiology and Pathophysiology (ed. J. J. Nordlund, R. E. Boissy, V. J. Hearing, R. A. King and J. P. Ortonne), pp.9 -40. Oxford: Oxford University Press.
Bagnara, J. T. and Hadley, M. E. (1973). Chromatophores and Color Change. New Jersey: Prentice Hall.
Bellairs, R., Harkness, M. L. and Harkness, R. D. (1975). The structure of the tapetum of the eye of the sheep. Cell Tissue Res. 157,73 -91.[Medline]
Benedek, G. B. (1971). Theory of transparency of the eye. Appl. Optics 10,459 -473.
Bohren, C. F. and Huffman, D. R. (1983). Absorption and Scattering of Light by Small Particles. New York: John Wiley & Sons.
Briggs, W. L. and Henson, V. E. (1995). The DFT. Philadelphia: Society for Industrial and Applied Mathematics.
Burkhardt, D. (1989). UV vision: a bird's eye view of feathers. J. Comp. Physiol. A 164,787 -796.
Camichel, C and Mandoul, H. (1901). Des colorations bleue et verte de la peau des Vertébrés. Compte Rendu Seances Acad. Sci. 133,826 -828.
Derim-Oglu, E. N. (1994). Small passerines can discriminate ultraviolet surface colours. Vision Res. 34,1535 -1539.[CrossRef][Medline]
Endler, J. A. (1993). The color of light in forests and its implications. Ecol. Monogr. 61, 1-27.
Ferris, W. and Bagnara, J. T. (1972). Reflecting pigment cells in the dove iris. In Pigmentation: Its Genesis and Biological Control (ed. V. Riley), pp.181 -192. New York: Appleton-Century-Crofts.
Finger, E. (1995). Visible and UV coloration in birds: Mie scattering as the basis of color production in many bird feathers. Naturwissenschaften 82,570 -573.[CrossRef]
Fox, D. L. (1976). Animal Biochromes and Structural Colors. Berkeley: University of California Press.
Frith, C. B. and Beehler, B. M. (1998). The Birds of Paradise. Oxford: Oxford University Press.
Ghiradella, H. (1991). Light and colour on the wing: structural colours in butterflies and moths. Appl. Optics 30,3492 -3500.
Gisselberg, M., Clark, J. I., Vaezy, S. and Osgood, T. (1991). A quantitative evaluation of Fourier components in transparent and opaque calf cornea. Am. J. Anat. 191,408 -418.[Medline]
Hart, N. S. (2001). The visual ecology of avian photoreceptors. Prog. Ret. Eye Res. 20,675 -703.[CrossRef][Medline]
Hays, H. and Habermann, H. (1969). Note on bill color of the ruddy duck, Oxyura jamaicensis rubida. Auk 86,765 -766.
Hecht, E. (1987). Optics. Reading, MA: Addison-Wesley Publishing.
Herring, P. J. (1994). Reflective systems in aquatic animals. Comp. Biochem. Physiol. A 109,513 -546.[CrossRef]
Huxley, A. F. (1968). A theoretical treatment of the reflexion of light by multilayer structures. J. Exp. Biol. 48,227 -245.
Jacobs, G. H. (1992). Ultraviolet vision in vertebrates. Am. Zool. 32,544 -554.
Land, M. F. (1972). The physics and biology of animal reflectors. Prog. Biophys. Mol. Biol. 24, 77-106.
Lee, D. W. (1991). Ultrastructural basis and function of iridescent blue colour of fruits in Elaeocarpus. Nature 349,260 -262.[CrossRef]
Lee, D. W. (1997). Iridescent Blue Plants. Am. Sci. 85,56 -63.
Leonard, D. W. and Meek, K. M. (1997). Refractive indices of the collagen fibrils and extrafibrillar material of the corneal stroma. Biophys. J. 72,1382 -1387.[Abstract]
Lucas, A. M. and Stettenheim, P. R. (1972). Avian Anatomy Integument. Washington, DC: U.S. Department of Agriculture.
Mandoul, H. (1903). Recherches sur les colorations tégumentaires. Annal Sci Natur B. Zool. 8 Ser., 18,225 -463.
Mason, C. W. (1923). Structural colors of feathers. I. J. Phys. Chem. 27,201 -251.
MATLAB (1992). MATLAB Reference Guide. Natick: The Mathworks, Inc.
Maurice, D. M. (1984). The cornea and sclera. In The Eye (ed. H. Davson), pp.1 -158. New York: Academic Press.
Menon, G. K. and Menon, J. (2000). Avian epidermal lipids: functional considerations and relationship to feathering. Am. Zool. 40,540 -552.
Murie, J. (1872). Cranial appendages and wattles of the horned tragopan. Proc. Zool. Soc. Lond. 1872,730 -736.
Nassau, K. (1983). The Physics and Chemistry of Color. New York: John Wiley & Sons.
Neville, A. C. (1975). Biology of the Arthropod Cuticle. New York: Springer-Verlag.
Neville, A. C. (1993). Biology of Fibrous Composites. Cambridge: Cambridge University Press.
Oehme, H. (1969). Vergleichende Untersuchungen über die Farbung der Vogeliris. Biologische Zentralblatt 88,3 -35.
Oliphant, L. W. (1981). Crystalline pteridines in the stromal pigment cells of the iris of the great horned owl. Cell Tissue Res. 217,387 -395.[Medline]
Oliphant, L. W. (1987a). Observations on the pigmentation of the pigeon iris. Pigment Cell Res. 1, 202-208.[Medline]
Oliphant, L. W. (1987b). Pteridines and purines as major pigments of the avian iris. Pigment Cell Res. 1, 129-131.[Medline]
Oliphant, L. W. and Hudon, J. (1993). Pteridines as reflecting pigments and components of reflecting organelles in vertebrates. Pigment Cell Res. 6, 205-208.[Medline]
Oliphant, L. W., Hudon, J. and Bagnara, J. T. (1992). Pigment cell refugia in homeotherms the unique evolutionary position of the iris. Pigment Cell Res. 5,367 -371.[Medline]
Osorio, D. and Ham, A. D. (2002). Spectral reflectance and directional properties of structural coloration in bird plumage. J. Exp. Biol. 205,2017 -2027.[Medline]
Parker, A. R. (1999). Invertebrate structural colours. In Functional Morphology of the Invertebrate Skeleton (ed. E. Savazzi). pp. 65-90. London: John Wiley & Sons.
Prum, R. O., Morrison, R. L. and Ten Eyck, G. R. (1994). Structural color production by constructive reflection from ordered collagen arrays in a bird (Philepitta castanea: Eurylaimidae). J. Morphol. 222, 61-72.
Prum, R. O. and Razafindratsita, V. R. (1997). Lek behavior and natural history of the velvet asity Philepitta castanea (Eurylaimidae). Wilson Bull. 109,371 -392.
Prum, R. O., Rice, N. H., Mobley, J. A. and Dimmick, W. W. (2000). A preliminary phylogenetic hypothesis for the cotingas (Cotingidae) based on mitochondrial DNA. Auk 117,236 -241.
Prum, R. O., Torres, R. H., Kovach, C., Williamson, S. and
Goodman, S. M. (1999a). Coherent light scattering by
nanostructured collagen arrays in the caruncles of the Malagasy asities
(Eurylaimidae: Aves). J. Exp. Biol.
202,3507
-3522.
Prum, R. O., Torres, R. H., Williamson, S. and Dyck, J. (1998). Coherent light scattering by blue feather barbs. Nature 396,28 -29.[CrossRef]
Prum, R. O., Torres, R. H., Williamson, S. and Dyck, J. (1999b). Two-dimensional Fourier analysis of the spongy medullary keratin of structurally coloured feather barbs. Proc. R. Soc. Lond. Ser. B. Biol. Sci. 266,13 -22.[CrossRef]
Rawles, M. E. (1960). The integumentary system. In Biology and Comparative Physiology of Birds, vol.1 (ed. A. J. Marshall), pp.189 -240. New York: Academic Press.
Schneider, A. (1938). Bau und erektion der hautlappen von Lobiophasis bulweri Sharpe. J. Ornithol. 86,5 -8.
Schorger, A. W. (1966). The Wild Turkey; Its History and Domestication. Norman, OK: University of Oklahoma Press.
Srinivasarao, M. (1999). Nano-optics in the biological world: beetles, butterflies, birds, and moths. Chem. Rev. 99,1935 -1961.[CrossRef][Medline]
Tièche, M. (1906). Über benigne melanome ("Chromatophore") der Haut "blaue Naevi". Virch. Arch. Pathol. Anat. Physiol. 186,216 -229.
Vaezy, S. and Clark, J. I. (1991). A quantitative analysis of transparency in the human sclera and cornea using Fourier methods. J. Microsc. 163, 85-94.[Medline]
Vaezy, S. and Clark, J. I. (1993). Quantitative analysis of the microstructure of the human cornea and sclera using 2-D Fourier methods. J. Microsc. 175, 93-99.
van de Hulst, H. C. (1981). Light Scattering by Small Particles. New York: Dover.
Vorobyev, M and Osorio, D. (1998). Receptor noise as a determinant of colour thresholds. Proc. R. Soc. Lond. Ser. B. Biol. Sci. 265,351 -358.[CrossRef][Medline]
Young, A. T. (1982). Rayleigh Scattering. Phys. Today 35,42 -48.
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