|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online January 31, 2006
Journal of Experimental Biology 209, 748-765 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02051
Anatomically diverse butterfly scales all produce structural colours by coherent scattering
1 Department of Ecology and Evolutionary Biology, and Peabody Museum of
Natural History, Yale University, PO Box 208105, New Haven, Connecticut 06250,
USA
2 Department of Ecology and Evolutionary Biology, University of Kansas,
Lawrence, KS 66045, USA
3 Department of Mathematics, University of Kansas, Lawrence, KS 66045,
USA
* Author for correspondence (e-mail: richard.prum{at}yale.edu)
Accepted 20 December 2005
 |
Summary |
|---|
 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Key words: coherent scattering, structural colours, Fourier analysis, photonics, Lepidoptera, Callophrys, Celastrina, Morpho, Mitoura, Papilio, Parides, Parrhasius, Troides, Urania
|
Introduction |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
). In contrast, the
structural colours of organisms are produced by the physical interactions of
light waves with biological nanostructures that vary in refractive index
(Fox, 1976;
Nassau, 1983;
Parker, 1999;
Srinivasarao, 1999;
Prum and Torres, 2003a).
Structural colours are an important component of the phenotype of many animals
(Fox, 1976;
Herring, 1994;
Parker, 1999) and even some
plants (Lee, 1997).
The physical mechanisms of structural colour production are usually
described as being quite diverse (Fox,
1976; Nassau,
1983; Parker,
1999; Srinivasarao,
1999). A short list of commonly proposed mechanisms includes
interference, diffraction, reinforcement, multilayer reflection, Bragg
scattering, Rayleigh scattering, Tyndall scattering, Mie scattering, and more.
One major reason for this apparent mechanistic diversity is that the
traditional physical tools used to analyze structural colour production vary
with the anatomy of the colour producing nanostructure, i.e. its laminar,
crystal-like, or quasi-ordered organization
(Prum and Torres, 2003a).
However, the diversity of physical tools for a diversity of anatomical
organizations has reinforced the notion that the physical mechanisms of color
production are actually diverse as well. Traditionally, the physical mechanism
associated with each anatomy has been assigned based on the mathematical
method that has been used to analyze it. In this way, the biological
literature has drawn on a specific intellectual tradition within optics of
naming optical phenomena according to the historical, experimental conditions
in which each was first described (e.g.
Hecht, 1987). While convenient
in optical physics, this intellectual perspective has overshadowed the
appreciation of the overwhelming physical, mechanistic commonality that
underlyies the optical function of most colour-producing biological
nanostructures, despite their anatomical diversity.
Until recently, the biological literature on structural colour has obscured
the most fundamental physical distinction among all mechanisms of structural
colour production: incoherent vs coherent scattering (e.g.
Prum and Torres, 2003a;
Prum and Torres, 2003b;
Prum and Torres, 2004).
Incoherent scattering is the differential scattering of light wavelengths by
individual scatterers, and it is determined by the size, shape and refractive
index of individual scatterers without regard to the phase relationships among
multiple waves scattered by different objects
(van de Hulst, 1981;
Bohren and Huffman, 1983). In
contrast, coherent scattering is differential scattering of light wavelengths
from multiple objects, and it is determined by the phase relationships among
scattered light waves (Huxley,
1968; Benedek,
1971; Land, 1972;
Joannopoulos et al., 1995;
Prum and Torres, 2003a;
Prum and Torres, 2003b).
Incoherent light scattering requires that the light scattering objects are
spatially independent, or randomly distributed over spatial scales of the same
order of magnitude in size as the wavelengths of visible light. Spatial
independence insures that the phase relationships among scattered waves are
random, and can thus be ignored in the calculations of light scattering
(Bohren and Huffman, 1983).
Rayleigh and Tyndall scattering (Young,
1982; Prum and Torres,
2003a) describe incoherent scattering by particles the size of
visible light or smaller, and they predict the production of short wavelength
hues: blue, violet and ultraviolet.
Coherent scattering occurs when spatial variation in refractive index is periodic, resulting in predictable phase relationships among light waves scattered by different objects. Interference, reinforcement, diffraction, multilayer and thin-film reflection, and Bragg scattering are all forms of coherent scattering. A wide variety of different nanostructures with periodic spatial variation in refractive index over one, two or three dimensions, can result in coherent scattering.
Unlike incoherent scattering, coherent scattering can produce the
phenomenon of iridescence - a prominent change in hue or brilliance with angle
of observation or illumination - because changes in angle of observation and
illumination may affect the phase relationships among the scattered waves that
determine the hue. Consequently, since 1923, iridescence has often been
inaccurately synonymized with coherent scattering
(Mason, 1923), leading to the
indiscriminant assignment of noniridescent blue structural colours to
incoherent, Rayleigh or Tyndall scattering
(Fox, 1976;
Nassau, 1983;
Herring, 1994). Recently,
however, it has been shown that a previously unappreciated class of
nanostructures, called quasiordered arrays, can produce noniridescent or
weakly iridescent colours by coherent scattering alone
(Prum et al., 1998;
Prum et al., 1999a;
Prum et al., 1999b;
Prum and Torres, 2003a;
Prum and Torres, 2003b;
Prum and Torres, 2004). To
advance the understanding of the physics and evolution of organismal
structural colours, it is important to conduct comparative analyses of a
diversity nanostructures, and to investigate what they share in common and how
they differ.
Butterfly structural colours
Structural colours are an important component of the phenotype of many
butterflies and a few diurnal moths (Fig.
1) (Ghiradella,
1991; Nijhout,
1991; Ghiradella,
1998; Parker,
1999; Vukusic and Sambles,
2000; Vukusic et al.,
2000a). Structural colours of butterflies can function in many
ways from aposematic communication among species to mate choice within species
(e.g. Sweeney et al.,
2003).
|
;
Vukusic and Sambles, 2000;
Vukusic et al., 2000a).
Lepidopteran wing scales are arranged in a series of rows like shingles on a
house (Figs 2,
3). Typical butterfly scales
have a complex structure that is characterized by a series of longitudinal
ridges that are spanned by cross ribs, which sit on a basal lamellae that is
supported by columnar trabeculae (Downey
and Allyn, 1975; Ghiradella,
1985; Ghiradella,
1989; Ghiradella,
1991; Ghiradella,
1998). Structurally coloured scales exhibit a wide diversity of
specializations of the ridges, cross ribs and complex compartmentalization of
the basal region below `windows' formed by the ridges and cross ribs on the
surface of the scale (reviewed in
Ghiradella, 1974;
Ghiradella, 1985;
Ghiradella, 1991;
Ghiradella, 1998;
Vukusic and Sambles, 2000;
Vukusic et al., 2000a).
|
|
). Type
I scales include those with surface lamellae (or laminae) that function by
multilayer interference (Ghiradella,
1998; Vukusic et al.,
2000a) and/or by diffraction (e.g.
Kinoshita et al., 2002;
Yoshioka and Kinoshita, 2003).
Type II scales include those with internal structures that function by
multilayer interference. Type III scales include (a) internal nanostructures
forming 3D lattices that function by Bragg scattering or diffraction, and (b)
internal nanostructures that produce colour by incoherent Rayleigh/Tyndall
scattering.
The fundamental difficulty with this classification is that anatomical
criteria, i.e. superficial vs interior position within the scale, or
laminar vs crystal-like organization, have been combined with various
mechanistic/physical criteria, i.e. multilayer interference vs
diffraction vs Rayleigh/Tyndall scattering. This classification
proposes that the anatomical distinction between multilayer interference
scales (Types I and II) and all other mechanisms (Type III) is more
fundamental than the distinction between diffraction (Type IIIa) and
Rayleigh/Tyndall scattering (Type IIIb). Anatomical variations on incoherent
scattering are lumped with incoherent scattering, and separated from other
types of coherent scattering despite the fundamental mechanistic differences.
This conceptual obfuscation reflects a long intellectual tradition in the
study of biological structural colour production going back at least to 1976,
when Fox discussed Tyndall scattering and diffraction in one chapter and
iridescent colours in another (Fox,
1976).
To explore the physical commonalties shared among anatomically diverse
butterfly nanostructures, we examined thirteen different colors of
structurally coloured scales from twelve species of Lepidoptera from four
different families (Fig. 1;
Table 1) that represent all of
the previously recognized types of colour producing butterfly scales. These
species were chosen because the structural colours of these species (or their
very close relatives) have previously been studied and attributed to a
diversity of physical mechanisms including multilayer interference,
diffraction, Bragg scattering and Tyndall scattering
(Ghiradella, 1974;
Huxley, 1975;
Morris, 1975;
Allyn and Downey, 1976;
Ghiradella and Radigan, 1976;
Huxley, 1976;
Ghiradella, 1985;
Ghiradella, 1989;
Ghiradella, 1991;
Vukusic et al., 1999;
Vukusic and Sambles, 2000;
Vukusic et al., 2000a;
Vukusic et al., 2001a;
Kinoshita et al., 2002;
Yoshioka and Kinoshita, 2003).
We use the two dimensional (2D) Fourier transform of transmission electron
micrographs of the colour producing biological nanostructures in butterfly
scales to analyze the periodicity of spatial variation in refractive index and
predict the reflectance spectrum produced by coherent scattering from these
scales (Prum et al., 1998;
Prum et al., 1999a;
Prum et al., 1999b;
Prum and Torres, 2003a;
Prum and Torres, 2003b).
|
Our results document that this diversity of scales from all major
structural and optical classes all function by coherent scattering. Further,
we conclude that the blue colour of the scales of Papilio zalmoxis,
previously hypothesized to be produced by incoherent Tyndall scattering
(Huxley, 1976) is likely to be
a pigmentary colour. The conceptual unification of all lepidopteran structural
colour production as variation of a single physical mechanism will allow us to
understand better the evolution of the diversity of anatomy and the optical
properties that have fascinated all previous workers in the field.
|
Materials and methods |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Small (<cm2) samples of structural coloured butterfly wings
were taken from specimens in the Snow Entomology Collection of the University
of Kansas Museum of Natural History (Table
1). Most species were selected specifically because their
nanostructure and structural colours have been described before
(Ghiradella, 1974;
Huxley, 1975;
Morris, 1975;
Ghiradella and Radigan, 1976;
Huxley, 1976;
Ghiradella, 1985;
Ghiradella, 1989;
Ghiradella, 1991;
Vukusic et al., 1999;
Vukusic and Sambles, 2000;
Vukusic et al., 2000a;
Vukusic et al., 2001a). Images
in Fig. 1 are of specimens of
conspecific or closely related species from the Yale Peabody Museum Entomology
collection.
For transmission electron microscopy (TEM), specimens were soaked in 100% ethanol for 24 h, and infiltrated with EMBED 812 (Electron Microscopy Services, Hatfield, PA, USA) for 24 h. Sections were cut approximately 100 nm thick, and stained with uranyl acetate and lead citrate and placed on formvar coated grids. Specimens were viewed with a JEOL EXII (JEOL USA, Peabody, MA, USA) transmission electron microscope. Digital micrographs were taken at various magnifications with a Soft-Imaging Megaview II CCD camera (Lakewood, CO, USA; 1024x1200 pixels).
Reflectance spectra
Reflectance spectra of the butterfly specimens were measured with an Ocean
Optics S2000 (Dunedin, FL, USA) fibre optic spectrophotometer and an Ocean
Optics deuterium-halogen light source, and a Dell laptop computer. The S2000
provides 2048 data points between 178 and 879 nm. Reflectance was measured
using normal incident light at 6 mm distance from a 3 mm2 patch of
the integument with a 300 µs integration time. Reflectance was calculated
in a standard fashion (e.g. Prum et al.,
1999a) using an Ocean Optics Spectralon white standard.
2D 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), we developed a method
of using the discrete Fourier 2D transform to analyze the periodicity and
optical properties of structural coloured tissue, and predict its reflectance
spectrum due to coherent scattering (Prum
et al., 1998; Prum et al.,
1999a; Prum et al.,
1999b; Prum et al.,
2003; Prum and Torres,
2003b; Prum and Torres,
2003a; Prum and Torres,
2004).
The digital TEM micrographs of structurally coloured butterfly scales were analyzed using the matrix algebra program MATLAB (Version 6.2; www.mathworks.com) on a Macintosh computer. The scale of each image (nm/pixel) was calculated from the number of pixels in the scale bar of the micrograph. A 1024 pixel2 portion of each array was selected from each image for analysis.
The average refractive index of the tissue in each image 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 chitin and air in the tissue, and to calculate a weighted average refractive index for the tissue using refractive indices of 1.54 for chitin and 1 for air.
The Fourier transforms were calculated with the 2D Fast Fourier Transform
(FFT2) algorithm (Briggs and Henson,
1995). We then calculated the 2D Fourier power spectrum, or the
distribution of the squares of the Fourier coefficients. The 2D Fourier power
spectra were expressed in spatial frequency (nm-1) by dividing the
initial spatial frequency values by the length of the matrix (pixels in the
matrixxnm/pixel). The 2D Fourier power spectrum resolves the spatial
variation in refractive index in the tissue into its periodic components in
any direction from a given point (Fig.
6).
|
|
Results |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
|
Reflectance spectra of almost all species revealed unimodal peak hues that correspond closely to the observed colours (Figs 8, 9, blue). Celastrina ladon showed the smallest wavelength peak reflectance of 375 nm (Fig. 9C), and Troides priamus exhibited the longest peak wavelength of 585 nm(Fig. 9B). The reflectance spectrum of the lycaenids in the sample (Celastrina ladon, Callophrys dumetorum, Mitoura gryneus and Parrhasius moctezuma) all showed blue or green peaks with gradually increasing reflectance above 600 nm(Fig. 9C-F). These long wavelength reflectances are very similar among species and may be produced by some unidentified pigment.
|
|
;
Morris, 1975;
Allyn and Downey, 1976;
Ghiradella and Radigan, 1976;
Ghiradella, 1985;
Ghiradella, 1989;
Ghiradella, 1991;
Ghiradella, 1998;
Vukusic et al., 1999;
Vukusic and Sambles, 2000;
Vukusic et al., 2000a;
Vukusic et al., 2001a;
Kinoshita et al., 2002;
Yoshioka and Kinoshita,
2003).
Troides brookiana (Papilionidae) and Morpho aega
(Nymphalidae) are characterized by complex, multilayer, laminar structures on
longitudinal scale ridges (Type I of
Vukusic et al., 2000a).
Troides brookiana has a unique arrangement of laminar outgrowths, or
microribs, of neighboring ridges that create a series of tubular air channels
between the laminae of each ridge and the laminae of adjacent ridges
(Fig. 4K,L). In Morpho
aega, the colour producing nanostructures are complex `pine tree-shaped'
elaborations of the longitudinal scale ridges
(Fig. 5I). As reported
previously from Morpho didius
(Vukusic et al., 1999;
Yoshioka and Kinoshita, 2003),
Morpho aega has a second class of `glass scales' with only a few
longitudinal ridges that are widely spaced on a thin basal lamina. These glass
scales function to diffuse the blue colour of the underlying scales
(Vukusic et al., 1999;
Yoshioka and Kinoshita,
2003).
|
|
). In Urania fulgens, the arrays of air spaces are
entirely flat (Fig. 4A-C).
However, the scales of Papilio ulysses
(Fig. 4D,E) and Parrhasius
moctezuma (Fig. 5H) have
concavities in the upper surfaces of the scales that distort the multilayers
of rectangular air cavities in the scale. Where adjacent concavities meet
beneath scale ridges, the multilayers of neighboring concavities intersect to
form a network of nearly square cavities that preserve the array dimensions of
each the intersecting layers (Figs
4D,E,
5H). In blue T.
urvillianus and green T. priamus, the upper surfaces of the
scales are covered with prominent, cone-shaped ridges, and the organization of
the air cavities within the body of the scales is substantially less regular
(Fig. 5A-C). In Celastrina
ladon, two or three layers of rectangular air cavities comprise almost
the entire body of the scale (Fig.
5D).
Previously classified as internal, diffraction arrays (Type IIIa of
Vukusic et al., 2000a),
Parides sesostris (Papilionidae), Callophrys dumetorum and
Mitoura gryneus (Lycaenidae) are all characterized by a complex
crystal-like array of spherical air cavities that are interconnected to one
another in a tetrahedral nanostructure (Figs
4H-J,
5E-G). Two-dimensional sections
through these nanostuctures reveal the extraordinary complexity of these air
bubble arrays. In addition, the entire dorsal surface of the Parides
sesostris scales is covered with a complex network of vertical ridges
that create large elliptical air spaces
(Fig. 4H). Although the
possible optical function of these superficial structures is unknown, the
structural colour is produced by the arrays in the body of the scale
(Ghiradella, 1985;
Ghiradella, 1991;
Vukusic and Sambles,
2003).
Papilio zalmoxis has been proposed to produce a blue colour by
Tyndall scattering (Huxley,
1976; Type IIIb of Vukusic et
al., 2000a). Papilio zalmoxis has a quite distinct
structure of tubular channels approximately 200 nm in diameter that run nearly
vertically from the dorsal surface of the scale to its basal lamina
(Fig. 4F,G) (Huxley, 1976;
Ghiradella, 1985;
Ghiradella, 1998). However,
the tubular channels are less than perfectly vertical as depicted by Huxley
(1976), and may meander
slightly horizontally (Fig.
4F).
Although only Morpho aega and Troides brookiana produced
structural colours primarily with laminar superficial scale ridges, as
previously recognized (Vukusic et al.,
2001a) other species also showed some type of periodic
ornamentation on the ridges of the scales, including Urania fulgens
(Fig. 4A), Papilio
ulysses (Fig. 4D),
Papilio zalmoxis (Fig.
4F), Troides urvillianus
(Fig. 5A) and Troides
priamus.
Fourier power spectra
The 2D Fourier analyses of the colour producing arrays from butterfly
scales reveal three general patterns of nanostructure
(Fig. 6). Regardless of whether
they are internal to the scale or formed by superficial scale ridges, laminar
arrays showed two points of high Fourier power values above and below the
origin, indicating that the predominant periodicity in these nanostructures
consists of intermediate spatial frequencies in the vertical direction: e.g.
Urania fulgens (Fig.
6A), Troides urvillianus
(Fig. 6F), Parrhasius
moctezuma (Fig. 6H) and
Morpho aega (Fig. 6I;
power spectrum is rotated 45° in orientation as were the arrays in the
original TEM).
Deviations from this simple 1D distribution of Fourier power reveal additional details about variations in laminar nanostructure. The Fourier power spectra of the laminar ridge structures of Troides urvillianus showed the typical vertical pair of dots indicating highly laminar nanostructure, but it also had high Fourier power values at a broad range of smaller spatial frequencies in the horizontal plane (Fig. 6E). These lateral Fourier power peaks document the periodicity of the larger spacing between neighboring ridges along the surface of the scale (Fig. 4K,L).
In Papilio ulysses, Fourier power spectra of TEMs taken from the
region of the scales immediately below the superficial ridges where the curved
multilayers of air cavities from adjacent concavities in the scale intersect
(Fig. 4E) reveal two prominent
directions of equivalent nanostructure separated by 
45°
(Fig. 6B). Similar results were
observed in power spectra from similar TEMs from Parrhasius moctezuma
(Fig. 5H). These power spectra
demonstrate that the laminar arrays within each scale concavity preserve a
consistent nanostructure despite the distortions from a simple plane and their
superimposition at the intersections of the concavities.
The less organized but generally laminar systems found in Troides urvillianus and T. priamus hecuba revealed a tendency toward a broader distribution of Fourier power in a ring of values in all directions of equivalent spatial frequency (e.g. Fig. 6F).
Crystal-like arrays of air cavities showed power spectra with hexagonal distribution of Fourier power values arranged in the directions of nearest neighbor cavities: e.g. Parides sesostris (Fig. 6D) and Callophrys dumetorum (Fig. 6G). These hexagonal power spectra show that periodicity is distributed along each of the lines of symmetry within the tetrahedral arrays.
TEM sections at different angles through the scales of Papilio zalmoxis showed an array of elliptical (Fig. 4F) or circular (4G) air spaces that were nearly vertical in orientation and which yielded either oval or circular distributions of Fourier power (Fig. 6C based on Fig. 4G). These results indicate that the tubular air cavities in Papilio zalmoxis are not spatially independent of one another over the spatial scale of visible light waves as assumed by the incoherent, Tyndall or Rayleigh scattering mechanisms.
Radial average of power spectra
Radial averages of the Fourier power spectra demonstrate that the peak
spatial frequencies of variation in refractive index within the structurally
coloured butterfly wing scales are within the range of values that would be
expected to produce visible colours by coherent scattering
(Fig. 7). Thus, the Fourier
power spectra indicate that the colour producing arrays in the butterfly
scales are appropriately nanostructured to produce a visible colour by
coherent scattering.
|
20 nm of error) were for Urania fulgens
blue (Fig. 8A), Parides
sesostris (Fig. 8E),
Troides brookiana (Fig.
8F), Troides urvillianus
(Fig. 9B), Celastrina
ladon (Fig. 9C),
Callophrys dumetorum (Fig.
9D) and Morpho aega
(Fig. 9G). These samples
include external laminar (Type I), internal laminar (Type II) and internal
crystal-like arrays (Type IIIa). More error (20-35 nm) existed in the
predicted peak hue of Urania fulgens green
(Fig. 8B), Papilio
ulysses (Fig. 8C),
Troides priamus (Fig.
9B), Mitoura gryneus
(Fig. 9E) and Parrhasius
moctezuma (Fig. 9F). Yet,
in all cases, the predicted reflectance spectra showed prominent peak hues
within the visible range, indicating that these colours are produced by
coherent scattering. In most instances, the 2D Fourier power spectra did not predict the detailed shape of the butterfly scale reflectance spectra (exceptions include Figs 8A,E, 9A). This could be due the limited samples of scales examined and micrographs taken in this broad survey. Also, the 2D Fourier power spectra from complex 3D organizations can gave a distorted view of the overall nanostructure. Images of sections through a plane of fusions among adjacent air cavities could indicate an increased size of the air cavities and result in exaggerating the size of the overall periodicity (e.g. Figs 4I, 5E).
The Fourier predicted reflectance spectrum for Papilio zalmoxis
features a prominent peak at 740 nm, which would be expected for coherent
scattering from a complex array of larger air cavities (200 nm in
diameter) (Fig. 8D). This peak
is completely unrelated to the measured peak hue of 474 nm for directly
incident light (Fig. 8D).
However, it is congruent with the yellow luster that P. zalmoxis
shows at a shallow angle of view (Huxley,
1976). At shallow angles, light waves should coherently scatter
efficiently from the vertical air channels within the scales. The Fourier
analysis of P. zalmoxis indicates that the shorter visible
wavelengths of light that are scattered by neighboring air cavities will be
non-randomly out of phase with each other, and will cancel out upon
scattering. Therefore, the observed periodicity falsifies the underlying
assumption of the hypothesis that incoherent Tyndall scattering contributes to
the production of this blue colour
(Huxley, 1976) (see
Discussion).
|
Discussion |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The Fourier predicted reflectance peaks corresponded to within 15 nm of the measured reflectance peaks for the majority of species examined, including those with colour producing nanostructures from all previously recognized, major classes of nanostructure: multilayer external lamina, multilayer internal lamina, and internal crystal-like nanostructures (Types I, II and IIIa).
We examined blue and green scales from the same individual of Urania fulgens (Type I; Figs 1A, 2A-C, 4A-C, 8A,B), and blue and green scales from the two closely related species Troides urvillianus and T. priamus (Type II; Figs 1F,G, 2H,I, 5A-C, 9A,B). In both cases, the Fourier analyses correctly predicted the differences in hue between the conspecific samples. Interestingly, in both instances, the nanostructures producing the longer wavelengths are characterized by both smaller spatial frequencies of periodicity (i.e. larger lattice spacing), and smaller air cavity size (Figs 4B,C, 5B,C). Along with the increase in array dimensions, the reduction in cavity size will raise the volume fraction of chitin in the array, raising the average refractive index of the tissue, and further contributing to a longer wavelength coherent scattering. Additional comparative studies are required to determine if this pattern is generalizable to other lepidopterans.
While exploiting the common physical mechanism of coherent scattering,
Lepidoptera have evolved extraordinary anatomical diversity and complexity in
nanostructure. Many of these anatomical variations create additional optical
effects beyond hue itself, including iridescence, highly or partially
polarized reflections, and colour mixing effects
(Ghiradella, 1985;
Ghiradella, 1991;
Vukusic et al., 1999;
Vukusic et al., 2000a;
Vukusic et al., 2000b;
Vukusic et al., 2001a;
Vukusic et al., 2001b;
Vukusic et al., 2002).
Papilio zalmoxis
Papilio zalmoxis is the only lepidopteran that has been
specifically hypothesized to produce structural colour by incoherent, Tyndall
scattering (Huxley, 1976).
Although Huxley's Tyndall scattering hypothesis for P. zalmoxis was
not questioned in subsequent literature (e.g.
Nijhout, 1991;
Ghiradella, 1998;
Parker, 1999;
Vukusic et al., 2000a), Huxley
actually expressed substantial uncertainty in his original paper. His complex
final description of the blue colour in male Papilio zalmoxis was
`A basic Tyndall scattering spectrum... is modified by thin film action of
the basement lamella and by strong pigmentary absorption in the violet and
u.v.' Further, he conceded that his proposed mechanism `would be
complicated by multiple scattering and by the partial irregularities of the
alveolar arrays'.
The Fourier analyses conducted here provides the analysis of multiple
scattering that was missing from Huxley's analysis
(Huxley, 1976). Our results
imply that the incoherent Tyndall component of Huxley's proposed mechanism
does not occur. The vertical tubular cavities within scales are open at the
top, and really inappropriate in shape for vertical light scattering. Light
scattering can occur only from light incident on the sides or the bottom of
these cavities, and not on the top surfaces. Therefore, the critical
dimensions of the cavities are the neighbor-to-neighbor distances between
cavities and not the vertical height of the tubes. TEM cross-sections of the
scales produce a diversity of profiles of the tubular cavities from highly
elliptical to perfectly circular, depending apparently on the angle of the
cross sections and the variation in the orientation of the tubular cavities
(Fig. 4F-G). The majority of
light scattering must be produced by the sides of the tubes. The Fourier power
spectra of these tubular spaces document a distinct and repeatable
nanoperiodicity at a spatial scale appropriate for the coherent scattering of
a longer wavelength visible colour (Figs
6C,
8D). This result can be easily
understood given the large (>200 nm) diameter of the spaces and the
additional distance between nearest neighbors
(Fig. 4G). Light scattering
from these tubular air cavities will be coherent scattering of longer visible
wavelengths, not incoherent scattering of smaller visible wavelengths. Smaller
visible wavelengths scattered by neighboring cavities will be predictably out
of phase, resulting in destructive interference among these wavelengths. This
result is inconsistent with the hypothesis of incoherent scattering by these
nanostructures. Huxley remarked that Papilio zalmoxis produce a
yellow luster when viewed at `grazing angles'
(Huxley, 1976). This easily
observed yellow hue is clearly structural since it disappears with the
application of a high refractive index fluid (e.g. isopropanol). Apparently,
this yellow color is coherently scattered light from neighboring
nanocavities.
Huxley's hypothesis was weakly supported by his own data
(Huxley, 1976). The
reflectance spectra of Papilio zalmoxis after extraction of a
fluorescent pigment (provisionally identified as a kynurenine) shows only the
slightest of increases in scattering of short wavelengths and also increases
at longer wavelengths above 550 nm (fig. 10 in
Huxley, 1976). The reflectance
spectrum of the pigment extracted scales does not conform at all to the
Rayleigh scattering prediction that the magnitude of scattering will be
inversely proportional to the fourth power of the wavelength
(van de Hulst, 1981;
Young, 1982;
Bohren and Huffman, 1983), as
Huxley (1976) proposed.
Rather, the resulting reflectance spectra had entirely lost the blue hue and
are essentially colorless (fig. 10 in
Huxley, 1976). Furthermore,
the emission spectrum of the fluorescent pigment matches the reflectance
spectrum of the scales almost exactly (fig. 12 in
Huxley, 1976). Thus, Huxley's
data strongly support the conclusion that the blue of Papilio
zalmoxis is essentially a pigmentary colour.
In conclusion, the hypothesis of incoherent Tyndall scattering by
Papilio zalmoxis is not supported by our Fourier analyses or by
Huxley's own data. The blue colour of Papilio zalmoxis is produced by
a fluorescent pigment. Apparently, the function of the tubular nanostructure
within the scale is to provide internal surfaces for the deposition of the
pigment (Huxley, 1976), and to
coherently scatter incident light horizontally into the fluorescent pigment
molecules on adjacent alveolar surfaces and increase the brilliance of the
pigment. Interestingly, in addition to coherently scattering a long wavelength
visible color at the 740 nm, the tubular nanostructure of P.
zalxmoxis should also coherently scatter wavelengths that are one half
this size, or approximately 370 nm. This wavelength is very close to the
excitation maximum of the kyurenine pigment extracted from P.
zalmoxis (Huxley,
1976)
Recently, Vukusic and Hooper
(2005) came to a similar
conclusion for a closely related species, Papilio nireus, which has
apparently identical anatomy and nanostructure. Vukusic and Hooper concluded
also that this nanostructure is designed to coherently scatter light
wavelengths in the horizontal plane to increase the flourescence of the blue
pigment. Based on a photonic analysis of an idealized crystal-like array, they
propose that the nanostructure is tuned to coherently scatter wavelengths that
are near to the observed reflectance peak. However, our analysis implies that
this nanostructure should produce a peak reflectance at much longer visible
wavelengths. The predominant spatial frequency we had documented
(Fig. 6C) is replicated exactly
in Fourier analysis presented in the supplementary materials by Vukusic and
Hooper (fig. S4 in Vukusic and Hooper,
2005). Our prediction is also congruent with the yellow luster
observed at shallow angles in both P. zalmoxis and P.
nireus, which is clearly a structural color (see above). So far, the
analysis of Vukusic and Hooper
(2005) provides no explanation
of the origin of this yellow structural color. Further research may be
necessary to establish the source of this disparity.
|
; Prum et al.,
1999b; Prum et al.,
2003), avian skin (Prum et
al., 1999a; Prum et al., 2003b), mammalian skin
(Prum and Torres, 2004),
odonate integument (Prum et al.,
2004), and now butterflies. We know of no examples of incoherent
scattering in organisms that have been supported by both reflectance spectra
showing congruence with Rayleigh's inverse fourth power law, and with evidence
that the light scatterers are spatial independent. Thus, all proposed examples
of organismal incoherent scattering require further testing.
Iridescence
Fourier analyses of scale nanostructures provide insights into how
iridescence - strong directionality in color - can be created or suppressed by
variation in nanostructure. Laminar arrays from butterfly scales displayed
single Fourier power peaks at intermediate spatial frequencies above and below
the origin (Fig. 6); these
peaks document that periodicity in array nanostructure is not equivalent in
all directions. This condition will produce strong directionality in back
scattering, resulting in iridescence, or prominent changes in hue with angle
of observation and illumination (Prum and
Torres, 2003a). Interestingly, Wickam et al. (2005) have recently
documented that laminar nanostructures from the ridge lamellae and tilted
microribs are more highly iridescent than are scales with horizontal
microribs.
Hexagonal, crystal-like arrays of air bubbles also provide some nanostructural opportunities for iridescence, but this iridescence is often reduced by variation at larger spatial scales in the orientation of the array among multiple cells of the butterfly scale (e.g. Figs 4H, 5E). The underlying opportunities for iridescence in these butterfly scales can still be observed in the occasional, sparkling, opalescent highlights of red and gold produced by the crystal-like nanostructures of green Callophrys dumetorum and Mitoura gryneus scales (Fig. 3B,C). The dense and complex ridges on the scales of Parides sesostris (Fig. 4H) may be to diffuse the structural colour produced by the underlying nanostructure uniformly over many directions.
The scales of some laminar butterfly nanostructures reduce iridescence by
having concavities in the scale surface that distort the planar orientation of
their multilayer nanostructures (e.g. Papilio ulysses, and
Parrhasius moctezuma; Figs
4D,
5J). A 2D Fourier analysis of a
single scale concavity from Papilio ulysses shows an arc of high
power spectrum values at a broad range of angles for a single prominent
spatial frequency (Fig. 10).
This arc-shaped power spectrum indicates that the concavity expands the
directions over which the scale nanoperiodicity is equivalent, and creates a
wider range of directions over which directly backscattered light will
coherently scatter the same peak hue. The result is a uniformity of colour
with angle of observation under general omnidirectional, natural lighting, and
a reduction in the iridescence produced by a laminar nanostructure. The scale
concavities of Papilio ulysses and Parrhasius moctezuma are
functionally analogous and strikingly similar to the iridescence reducing
convexities of the laminar arrays of melanin granules in feather barbules of
some fruit pigeons (Dyck,
1987) and cuckoos (Durrer and
Villiger, 1970), which show similar arc-shaped power spectra
(Prum, 2006).
Likewise, the deviations from a uniformly laminar orientation in the
nanostructure of T. priamus across multiple ridges is also likely to
function in expanding the angles over which the primary hue is observed
(Fig. 5A). This relaxed laminar
organization approaches the non-iridescent, quasiordered nanostructures at
larger spatial scales (Prum et al.,
1998; Prum et al.,
1999a; Prum et al.,
1999b; Prum and Torres,
2003a; Prum and Torres,
2003b; Prum and Torres,
2004). Butterflies with laminar arrays can also reduce iridescence
by morphological adjustments at larger spatial scales. For example Urania
fulgens, Papilio ulysses, Troides urvillianus, T. priamus and
Parrhasius moctezuma all have prominently curved scales, which will
contribute to the same optical effect (Figs
2A-E,H,I,
3D). The effects of these
multiple methods of reducing iridescence can easily be observed by comparing
the laminar but weakly iridescent Papilio ulysses, Urania fulgens, Troides
urvillianus, T. priamus and Parrhasius moctezuma to the highly
iridescent Morpho aega, which maintains both planar, laminar
nanostructures and perfectly flat scales
(Fig. 3F). These structural
variations further underscore how anatomical variation can contribute to
variation in optical function within a common physical mechanism.
Physics and evolution of butterfly structural colours
Although the mechanisms of structural colour production of butterflies and
other organisms have been traditionally viewed as mechanistically diverse,
this apparent diversity of optical phenomena is more productively understood
as derived variations of coherent scattering, rather than as distinct
phenomena based on different optical mechanisms. Although physically
sophisticated readers may find this mechanistic unification to be trivial,
ascribing different traditional optical categories to diverse biological
anatomies has created substantial intellectual costs in the study of the
evolution of biological nanostructure and optical function. For example, it
has never been previously recognized that the common coherent scattering
mechanism supports the likelihood that butterfly nanostructures have evolved
among anatomical/optical classes while consistently retaining a structural
color production function. As in avian feathers and skin
(Prum et al., 1999b;
Prum and Torres, 2003a;
Prum, 2006), it appears that
many butterfly clades have probably diversified evolutionarily among different
anatomical classes that have been previously classified as mechanistically
distinct, and have traditionally required different, incompatible mathematical
tools to analyze their optical function. Traditional optical analyses of these
diverse structures would require distinct mathematical methods even if the
ancestral forms had consistently maintained a coherently scattering optical
function throughout their evolutionary history. Thus, following traditional
methods, it would be impossible to analyze an evolutionary transition between
a crystal-like array and a multilayer thin film, or a diffraction grating and
a Bragg scatterer.
Traditional classifications of butterfly scale nanostructures may have created conceptual obstacles to understanding the evolution of nanoscale diversity. In contrast, understanding the common physical mechanism behind the morphological diversity provides insights into how the startling variety of nanostructures and optical functions could have evolved into one another as elaborations of a common physical mechanism.
A physicist may conveniently adopt the most appropriate tool for a given physical situation, but a biologist interested in the evolution of nanostructure and optical function must adopt an analytical tool that can span the multiple classes of nanostructural organization of the evolutionary histories of the organisms. Organismal evolution presents unique demands that have not been addressed by traditional optical techniques. In short, evolutionary biology may demand new physical approaches and methods.
These results provide an important insight into how diversity in
nanostructure and optical function may have evolved in lepidopterans through
selection on novelties in optical function. For example, the Fourier power
spectra (Fig. 6B) of the
intersections of the concave multilayers of air bubbles in Papilio
ulysses (Fig. 4E) demonstrate how internal laminar (Type II) and internal crystal-like (Type
IIIa) nanostructures can intergrade into one another. These scale concavities
function and have likely evolved by selection to reduce iridescence (see
above, Fig. 10), and have
produced the periodic establishment across the scale of an intermediate form
of nanostructure between laminar and crystal-like arrays (Figs
4E,
6B). It is easy to imagine how
selection on optical function could lead to an evolutionary transition between
these two types of nanostructures. Furthermore, the deeper concavities of
Papilio palinurus that produce optical colour-mixing of yellow and
blue (Vukusic et al., 2000b;
Vukusic et al., 2001a) likely
evolved as derived elaborations of the type of iridescence reducing
concavities found in Papilio ulysses.
As previously recognized (Vukusic et
al., 2001a), many of the species with colour producing, laminar
nanostructures within the body of the scales also have superficial ridges with
periodic ornamentation that quite likely also function in coherent scattering,
e.g. Urania fulgens (Fig.
4A), Papilio ulysses
(Fig. 4D), Papilio
zalmoxis (Fig. 4F) and
Troides urvillianus (Fig.
5A). These anatomical intermediates between traditional Type I and
Type II scales indicate how these two classes of nanostructure are
functionally continuous. Furthermore, vivid structural colours are ubiquitous
in the genus Troides (Papilionidae). It is very likely that the
extraordinarily different nanostructures of Troides brookiana, T.
urvillianus and T. priamus have evolved from a structurally
coloured common ancestor through persistent selection on optical function.
Thus, recognizing that ridge lamellae (Type I) and interior air cavities (Type
II and IIIa) both function by the same coherent scattering mechanism makes it
easier to conceptualize the evolution of nanostructural diversity exhibited by
butterfly clades.
Even within the small sample of species analyzed, there is evidence of extraordinarily detailed, convergent evolution in butterfly scale optical nanostructures among the four distantly related lepidopteran clades. The crystal-like arrays of air spheres in the scales of the papilionid Parides sesostris (Fig. 4H-J) are strikingly similar to those in the lycaenids Callophrys dumetorum and Mitoura gryneus (Fig. 5E-G). Further, the laminar arrays with iridescence reducing concavities of the papilionid Papilio ulysses (Fig. 4D,E) are extremely similar to those of the lycaenid Parrhasius moctezuma (Fig. 5H). Natural and sexual selection on the optical properties of structurally coloured butterfly scales has produced identical anatomical solutions in phylogenetically independent lineages.
Future analyses of structural colour evolution in butterflies should
investigate optical function of wing scales in a phylogenetic context, to
document the patterns of origin, maintanence, diversification and convergence.
Prum et al. (2004) have begun
these analyses in odonates. Recently, Wickham et al.
(2005) presented a
phylogenetic analysis of the evolution of structurally colored butterfly
scales with surface multi-layers (Type I). Unfortunately, the small, biased
sample that they analyzed included too little taxonomic or structural
diversity to be meaningful. The color-producing multilayer nanostructures
found in various species of nymphalids and papilionids are not homologous, but
the limited sample of species analyzed by Wickham et al.
(2005) ensures that they will
be. (Imagine a phylogenetic study of the evolution of red hair in mammals that
analyzed only species with red hair.) Their exclusive focus on the evolution
of superficial multilayer scales, to the exclusion of other classes of color
producing nanostructures that are found in close relatives of the species
sampled (e.g. within Troides), further documents the conceptual
limitations created by traditional categories of optical mechanism.
Ghiradella (Ghiradella,
1974; Ghiradella and Radigan,
1976; Ghiradella and Radigan, 1985; Ghiradella and Radigan, 1989;
Ghiradella and Radigan, 1991; Ghiradella and Radigan, 1998) has pioneered
studies of the development of the colour producing nanostructures in butterfly
scales. These fascinating investigations document that intricate optical
nanostructures structures develop in a variety of different mechanisms, even
within a single family (Ghiradella,
1989; Ghiradella,
1998). Functional, developmental and phylogenetic studies of a
diversity of structurally coloured butterfly species within a major clade
(e.g. Lycaenidae, Papilionidae, Nymphalidae) would provide exciting new data
on the dynamics of structural colour evolution in lepidopterans. Previous
research has provided advanced mathematical models
(Nijhout, 1991) and detailed
molecular mechanisms of wing pattern determination in butterflies
(Carrol et al., 1994;
Brunetti et al., 2001). Recent
work has also examined the developmental correlation between scale structure
and pigmentation (Janssen et al.,
2001; Otaki and Yamamoto,
2004). None of this research has yet focused on the development of
structural colouration patterning with its combination of extreme anatomical
modifications of scales and complex distribution on the wing surfaces. For
example, Urania fulgens shows variation in both wing pattern and
structural colour that could be a new model species for this research program.
Another premier experimental system for investigation of these processes could
be Precis octavia (Nymphalidae) with an exceptional environmentally
induced polyphenism in which homologous scales vary between structural blue or
pigmentary orange or black (Nijhout,
1991).
Structural white
After focusing on the physics of production of wavelength specific
structural colors in butterflies, it is appropriate to comment that broad
spectrum white reflectance is also a common and important optical property of
the scales of many butterflies. White is produced by incoherent scattering
from unpigmented chitin of butterfly scales. The magnitude of scattering can
be enhanced by the specific derived structures within various parts of the
scale (e.g. Stavenga et al.,
2004).
Polarized signals
We did not analyze polarization of the colors of these butterflies.
Recently, Sweeney et al.
(2003) have shown that
polarized structural colors function in intersexual communication in
Heliconius cydno. Although only structural colors can be polarized,
not all structural colors are polarized. Many coherently scattering
nanostructures will not produce polarized reflections. In brief, polarized
colors are produced by materials with periodic variation in refractive index
in one or two dimensions. In butterflies, this corresponds to species that
have ornamented outer ribs of the scales (Type I). However, not all
nanostructures will produce polarized reflections. In particular,
nanostructures with periodic 3D variation in refractive index are unlikely to
produce strongly and predicatably polarized signals.
Uses and limits of the Fourier method
In a series of papers, we have developed a method using 2D Fourier analyses
to study the physical mechanisms of structural colour production in organisms
(Prum and Torres, 2003a).
Originally, the method was developed to analyze colour production by
quasiordered arrays of light scattering objects that could not be analyzed
using traditional thin-film optics or Bragg scattering methods
