Blue integumentary structural colours in dragonflies (Odonata) are not produced by incoherent Tyndall scattering
1 Department of Ecology and Evolutionary Biology, Yale University, PO Box
208105, New Haven, CT 06520, 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 12 August 2004
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Summary |
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Key words: structural colour, dragonfly, damselfly, coherent scattering, Tyndall scattering, Rayleigh scattering, pigment cells, Enallagma civile, Anax junius
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Introduction |
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Structural colours are common and broadly distributed in insects
(Fox, 1976;
Parker, 1999
). Most insect
structural colours are produced by periodic nanostructures in the cuticle
(Parker, 1999
) or the scales
of the wings (Ghiradella,
1991
). Exceptionally, the blue integumentary structural colours of
dragonflies and damselflies (Odonata) are produced by spherical nanostructures
within living epidermal cells that lie below the cuticle
(Vernon et al., 1974
;
Charles and Robinson,
1981
).
For nearly 80 years, since Mason
(1926), the non-iridescent
structural blue colours of dragonflies and damselflies (Odonata) have been
universally hypothesized to be produced by incoherent Tyndall or Rayleigh
scattering (Vernon et al.,
1974
; Fox, 1976
;
Charles and Robinson, 1981
;
Sternberg, 1996
;
Corbet, 1999
;
Parker, 1999
;
Srinivasarao, 1999
). Here, we
report an investigation of the anatomy and physics of structural colour
production in two distantly related odonates, in which we test whether these
colours are produced by incoherent or coherent scattering.
Incoherent and coherent scattering
Although mechanisms of structural colour production are often described as
quite diverse (Fox, 1976;
Parker, 1999
;
Srinivasarao, 1999
), almost
all of them can be productively understood as aspects of light scattering
(Prum and Torres,
2003a
,b
).
As light propagates through a medium, substantial scattering of light waves
occurs at the interfaces of materials with different refractive indices. Light
scattering from multiple objects or interfaces can be classified as either
incoherent or coherent (Prum and Torres,
2003a
,b
).
Tyndall and Rayleigh scattering refer to incoherent light scattering by
particles smaller than the wavelengths of visible light. Light scattering is
incoherent when the objects scattering the light are randomly distributed over
the spatial scale of visible light wavelengths. As a consequence of the
spatial independence of scatterers, scattered light waves will be random in
phase, and the phase relationships among scattered light waves can be ignored.
Thus, incoherent scattering can be described by the properties of the
individual scatterers alone size, refractive index of the scatterer
and refractive index of the surrounding medium. Traditionally, Tyndall
scattering has been used to refer to incoherent scattering by small particles
near the size of visible wavelengths, whereas Rayleigh scattering is used to
refer to incoherent scattering by all small particles down to the size of a
molecule (Young, 1982). Thus,
Rayleigh scattering includes Tyndall scattering. Tyndall scattering has been
more frequently used to refer to incoherent scattering in organisms because
the objects in organisms that are hypothesized to scatter light incoherently
are similar in size to wavelengths of visible light and are much larger than
individual molecules (e.g. Huxley,
1975
; Fox, 1976
).
However, we prefer to use Rayleigh scattering for all incoherent scattering in
organisms because most of the testable predictions about incoherent small
particle scattering are derived from Lord Rayleigh's work
(Young, 1982
). For example,
Rayleigh predicted that the magnitude of light scattering is inversely
proportional to the fourth power of the wavelength, producing blue colours, or
ultraviolet hues (Bohren,
1987
). Because the phases of each of the incoherently scattered
light waves are independent of one another, incoherently scattered colours do
not exhibit iridescence, or strong changes in hue, with angle of observation
or illumination.
Coherent light scattering occurs when spatial periodicity in the
distribution of the scattering objects results in nonrandom phase
relationships among scattered waves (Prum
and Torres, 2003a). Physical models of coherent scattering
describe the colour produced in terms of the differential reinforcement or
interference among the scattered light waves from multiple scattering
interfaces. Coherent scattering from different structural classes of
nanostructures has been described as a variety of different mechanisms
including constructive interference, reinforcement, diffraction and thin-film
interference, but all these phenomena share the common physical mechanism of
coherent scattering.
Coherently scattering nanostructures with laminar or crystal-like
structures frequently exhibit iridescence. Recently, however, we have
identified a new class of coherently scattering nanostructures that do not
produce prominent iridescence, which we have termed quasiordered arrays (Prum
et al., 1998,
1999a
,b
,
2003
; Prum and Torres,
2003a
,b
,
2004
). Quasiordered arrays
have periodic spatial distribution of light-scattering interfaces at the
spatial scale of nearest neighbours, but they lack laminar or crystal-like
periodicity at larger spatial scales. Recent photonic analyses of
two-dimensional (2-D) quasiordered (= amorphous or disordered) photonic
crystals of parallel fibres demonstrate that quasiordered arrays can exhibit a
complete photonic band gap a range of light frequencies that cannot be
transmitted in any direction through the material
(Jin et al., 2001
). Since
photonic band gaps in light transmission are a consequence of coherent back
scattering of these untransmissable frequencies
(Joannopoulos et al., 1995
),
these analyses demonstrate independently that quasiordered arrays are capable
of coherently scattering light.
Mason (1923,
1926
) established the first
set of criteria for identifying incoherent Tyndall scattering in biological
tissues: (1) variation in refractive index, (2) particles smaller than 600 nm,
(3) scattered light blue and transmitted light red, (4) depth or shade of blue
dependent on particle size (larger produces whiter), (5) scattered light
polarized in plane normal to plane of incidence (but dependent on particle
size) and (6) intensity inversely proportional to the fourth power of the
wavelength. Mason's traditional criteria for identifying incoherent Tyndall
scattering do not include an examination of the critical assumption of spatial
independence of scatterers. Furthermore, Mason
(1923
,
1926
) did not appreciate the
potential of quasiordered arrays to produce non-iridescent structural colours
by coherent scattering. Consequently, since Mason
(1923
,
1926
), iridescence has
frequently been cited as a defining feature of coherent scattering
(interference, etc.), and many non-iridescent blue structural colours have
been indiscriminately attributed to incoherent Tyndall, Rayleigh scattering
(Fox, 1976
;
Herring, 1994
;
Parker, 1999
;
Srinivasarao, 1999
).
In previous research, we have tested and rejected the traditional
incoherent scattering explanations for the production of non-iridescent
structural colours in avian feather barbs (Prum et al.,
1998,
1999b
,
2003
), avian skin
(Prum et al., 1999a
;
Prum and Torres, 2003b
) and
mammalian skin (Prum and Torres,
2004
). We know of no instances in which a biological structural
colour attributed to incoherent Rayleigh or Tyndall scattering has been
critically tested by showing both the spatial independence of the scatterers
and the congruence between the reflectance spectrum and the inverse fourth
power prediction of Rayleigh.
Odonate structural colour
Odonates exhibit a variety of red, yellow, brown and black pigmentary
colours and a variety of structural colours, including pruinescence (e.g. many
libellulids), iridescence (e.g. Calopteryx maculata) and common
non-iridescent blue colours (Fig.
1; Gorb, 1995;
Corbet, 1999
;
Silsby, 2001
). The blue
integumentary structural colours of odonates were attributed to incoherent
Tyndall scattering by Mason
(1926
) after examination of an
Enallagma damselfly (Coenagrionidae). Mason
(1926
) did not measure whether
the reflectance spectra was congruent with the inverse fourth power prediction
of Rayleigh but he did note that the blue structural colour of
Enallagma appeared to lack the polarization predicted by Tyndall
scattering. Despite the lack of critical support, Mason
(1926
) concluded that the blue
colour was produced by Tyndall scattering by "fine particles enclosed in
a chitinous protein". The Tyndall scattering hypothesis has been cited
without question, without exception and without testing for the past seven
decades (Vernon et al., 1974
;
Fox, 1976
;
Charles and Robinson, 1981
;
Sternberg, 1996
;
Corbet, 1999
;
Parker, 1999
;
Srinivasarao, 1999
).
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The integumentary nanostructures that produce these structural colours were
first described by Vernon et al.
(1974) in a phylogenetically
broad sample of blue Australian odonates including aeshnids, amphipterygids
and coenagrionids. Vernon et al.
(1974
) documented that the
structural colours are produced by light scattering from spherical objects
that are closely but irregularly packed within integumentary pigment cells
immediately below the cuticle. Apparently, the spatial distribution of the
light-scattering spheres met with the general expectations for an incoherently
scattering array, because the description of the anatomy of the structurally
coloured cells did not lead to a reevaluation of the Tyndall scattering
hypothesis. Subsequently, Charles and Robinson
(1981
) used scanning electron
microscopy to describe the same anatomical structures in a North American
damselfly, the familiar bluet, Enallagma civile (Coenagrionidae).
Neither Vernon et al. (1974
)
nor Charles and Robinson (1981
)
identified the material within the nanospheres. Even though the
colour-producing nanostructures are produced by pigment cells and may be
composed of pigment molecules, the colours produced are still structural.
Because many pigments also have high refractive indices, nanostructured arrays
of pigment granules are often involved in structural colour production: e.g.
pterines and purines in vertebrate iridiphores, and melanosomes in avian
feather barbules. Vernon et al.
(1974
) referred to the
colour-producing cells as chromatophores, a term that is usually reserved for
pigment cells involved in physiological colour changes. Here, we refer to
these cells more generally as pigment cells, since many species with this form
of structural colouration do not exhibit physiological colour change.
Here, we examine the anatomy and physics of the non-iridescent blue
structural colours of two distantly related North American odonates, a
damselfly and a dragonfly: the familiar bluet, Enallagma civile
(Coenagrionidae, Zygoptera; Fig.
1A), and the common green darner, Anax junius (Aeshnidae,
Anisoptera; Fig. 1B). We used
spectrophotometry to measure the reflectance spectra of the integument of
these odonates and transmission electron microscopy (TEM) to document the
anatomy of the cuticle and structural colour-producing cells. We then used 2-D
Fourier analysis of the TEM micrographs to characterize the spatial
periodicity of the colour-producing nanostructures, to test the spatial
independence of scatterers and to predict the reflectance spectrum produced by
coherent scattering from these nanostructures
(Prum and Torres, 2003a). Our
results falsify the spatial independence of the light scatterers that is
assumed by incoherent scattering mechanisms and provide some support for the
conclusion that the array of light-scattering spheres is appropriately
nanostructured to produce visible colours by coherent scattering.
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Materials and methods |
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Reflectance spectra
Reflectance spectra of living Enallagma and Anax were
measured with an Ocean Optics USB2000 fibre optic spectrophotometer and Dell
laptop computer. Reflectance was measured with normal incident light at 6 mm
distance from a 3 mm2 patch of the integument. The colour of
preserved specimens changed rapidly to a deep brown or black with no
measurable hue.
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), we have developed a
method of using the discrete 2-D Fourier transform to analyze the periodicity
and optical properties of structural coloured tissue and to predict its
reflectance spectrum due to coherent scattering (Prum et al.,
1998
,
1999a
,b
,
2003
; Prum and Torres,
2003a
,b
).
The digital TEM micrographs of the rapidly fixed specimens of Enallagma civile were analyzed using the matrix algebra program MATLAB (version 6.2; www.mathworks.com) on a Macintosh G4 computer. The scale of each image (nm pixel1) was calculated from the number of pixels in the scale bar of the micrograph. A 1024 pixels2 portion of each array was selected from each image for analysis. Because the molecular composition of the colour-producing nanospheres is unknown, we could not calculate an average refractive index of the nanostructure based on the frequency distribution of its components as in our previous applications of the method. However, we estimate the average refractive index of the material within the spheres necessary to produce congruence with the observed reflectance spectrum.
The Fourier transform was calculated with the 2-D fast Fourier transform
(FFT2) algorithm (Briggs and Henson,
1995). We then 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 spectrum was expressed in spatial frequency (nm1) by
dividing the initial spatial frequency values by the length of the matrix
(pixels in the matrix x nm pixel1).
We calculated radial averages of the power spectra using 100 spatial frequency bins, or annuli, between 0 and 0.02 nm1 and expressed them in terms of % total Fourier power. Composite radial averages were calculated from a sample of power spectra from five TEM images of the best preserved Enallagma sections to provide an indication of the predominant spatial frequency of variation in refractive index in the tissue over all directions.
We produced predicted reflectance spectra for Enallagma civile based on the 2-D Fourier power spectra of the TEM micrographs, the image scales, estimated values of the average refractive index of the material and estimating the expansion of the arrays during preservation. First, a radial average of the % power was calculated for concentric radial bins, or annuli, of the power spectrum corresponding to fifty 10 nm-wide wavelength intervals between 300 and 800 nm (covering the light spectrum visible to insects). The radial average 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 and 800 nm) to 1. The inverse of the spatial frequency averages for each wavelength were then multiplied by twice the estimated average refractive index of the medium and expressed in terms of wavelength (nm). A composite predicted reflectance spectrum was produced by averaging the normalized predicted spectra from a sample of five TEM images of Enallagma civile. Values of the average refractive index and % expansion during tissue preparation were estimated by producing a reflectance spectrum congruent with the observed reflectance peaks.
Phylogenetic analysis
The distribution of non-iridescent blue integumentary structural colour was
estimated from a review of odonate diversity (by J.A.C.) and standard
references (Corbet, 1999;
Silsby, 2001
). The
phylogenetic pattern in the evolution of integumentary blue was estimated
using a recent and comprehensive phylogeny of the odonates
(Rehn, 2003
). The estimated
number of evolutionary events to describe that diversity was calculated using
MacClade 4 (Maddison and Maddison,
2000
).
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Results |
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The reflectance spectra of the blue portions of the integument of both Enallagma civile and male Anax junius revealed discrete peaks of 475 nm and 460 nm, respectively (Fig. 2). The reflectance spectra of both species lacked the inverse fourth power relationship predicted for incoherent Rayleigh scattering.
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Anatomy
Transmission electron micrographs of the integument of Enallagma
civile and Anax junius were entirely consistent with previous
anatomical descriptions of other blue odonates
(Vernon et al., 1974;
Charles and Robinson, 1981
). In
both Enallagma and Anax, the cuticle consists of chitin
58 µm thick (Fig.
3A). Immediately below the cuticle is a layer of box-shaped
pigment cells (Fig. 3A,B). In
Enallagma, the light-scattering nanospheres filled the distal
two-thirds of the pigment cells, creating a solid layer
510 µm
thick, and the ommochrome spheres were restricted to the basal third of the
cells (Fig. 3A,B). Vernon et
al. (1974
) identified these
ommochrome pigments as xanthommatin and dihydroxyxanthommatin in the pigment
cells of a broad diversity of blue odonates. Occasional cells showed cell
nuclei and other cellular organelles restricted to the basal third of the cell
volume (Fig. 3A). In all blue
Anax junius specimens observed, the ommochrome pigment granules were
restricted to the basal portions of the integumentary pigment cells, in the
state that produces structural blue colour and identical to
Enallagma. (During temperature-dependent colour change in
Anax, these ommochrome granules are capable of migrating upwards
amongst the light-scattering nanospheres; see Discussion.)
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The light-scattering nanospheres in the epidermal pigment cells were
200300 nm in diameter and packed immediately adjacent to one
another. As previously reported (Vernon et
al., 1974
; Charles and
Robinson, 1981
), the light-scattering spheres were enclosed within
pockets of the endoplasmic reticulum. Like Vernon et al.
(1974
), we observed occasional
`budding' of these spherical bodies from one another within a single pocket of
the endoplasmic reticulum (Fig.
3C). Many spheres showed a darkly staining central spot
(Fig. 3C,D). In the less well
preserved sections, spheres showed dark lines radiating from the centre of the
sphere (Fig. 4B,C; Vernon et al., 1974
). The
variation in position of the dark spots within the spheres indicates that each
sphere may have a linear structure, perhaps a fold of endoplasmic reticulum,
that runs through the centre of the sphere from pole to pole
(Fig. 3C,D). Vernon et al.
(1974
) did not identify the
material within these spheres but suggested that the spheres could be either
crystal or liquid filled. The radiating structures within the degrading
spheres could indicate a composition of biocrystalline or proteinaceous
materials. The nanospheres differ from the ommochrome granules in being
exclusively spherical and highly consistent in diameter. Further research is
required to describe the composition, structure and development of these
nanospheres.
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The green portions of the integument in Anax junius entirely lack underlying epidermal pigment cells. Rather, the green hue appears to be produced by a green pigment within the cuticle itself.
Fourier analysis
Two-dimensional Fourier analysis of TEM micrographs of the light-scattering
nanospheres produced power spectra with a ring-shaped distribution of peak
Fourier power at intermediate spatial frequencies
(Fig. 5A). The ring-shaped
Fourier power distribution shows that the arrays are highly nanostructured at
intermediate spatial frequencies and equivalently periodic in all directions.
The composite radial average power spectra from a sample of five micrographs
of Enallagma civile shows a peak spatial frequency at 0.00307
nm1 (Fig.
5B). This peak spatial frequency is of the same order of magnitude
as the wavelengths of visible light waves, which indicates substantial
nanostructure at the appropriate spatial scale to produce visible colours
(Fig. 5B). This spatial
frequency corresponds to an average centre-to-centre distance between
neighbouring spheres of 322 nm(Prum et al.,
1998,
1999a
; Prum and Torres,
2003a
,b
).
This substantial nanostructure (Fig.
5A,B) falsifies a fundamental and critical assumption of all
incoherent scattering models, including Tyndall, Rayleigh and Mie
scattering.
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Although the arrays are appropriately nanostructured to produce visible
light by coherent scattering, predicting the peak hue due to coherent
scattering from the arrays of nanospheres in Enallagma is subject to
substantial error. Assuming a minimal average refractive index in the cells of
1.35 (typical of cellular cytoplasm), the 2-D Fourier power spectra of
Enallagma civile predict a peak reflectance of 796 nm, which is at
the long wavelength extreme of the visible spectrum and 40% longer than
the wavelength of the observed reflectance peak. With a more realistic average
refractive index of 1.55 (a value typical of protein), the nanostructure would
have had to expand by
50% during preparation for TEM and imaging to
produce the observed peak hue of 475 nm
(Fig. 5C). Consequently, the
nanospheres in life could have been distributed with an average
centre-to-centre distance of 153 nm (see Discussion for problems with this
explanation).
An alternative explanation may be that substantial light scattering occurs at both the periphery of the spherical nanostructures and at the dark (i.e. electron-dense) structures within the spheres. Many of the light-scattering spheres show a conspicuous dark spot (Figs 3, 4). This spot could be an indication of a molecular structure that organizes the material within the spheres and perhaps contributes to their size. If this internal structure has a distinct refractive index (e.g. a fold of endoplasmic recticular membrane that runs from pole to pole within the spheres), then light scattering could occur at both the peripheral boundaries and the centre of the spheres. This would essentially double the fundamental spatial frequency of variation in refractive index and produce exact congruence with the observed reflectance spectrum, assuming an average refractive index of 1.55 (Fig. 5C).
Phylogenetic analysis
Non-iridescent, blue, integumentary structural colours are known in at
least 14 families within all major clades of odonates
(Table 1). Based on the most
recent and comprehensive phylogenetic hypothesis for the odonates
(Rehn, 2003), a parsimony
analysis of the phylogenetic distribution of non-iridescent integumentary blue
colour indicates that at least 11 historically independent evolutionary
derivations (with up to three losses) of this mode of structural colouration
have occurred within the odonates (Fig.
6). Given the lack of resolved phylogenies within most odonate
families, there may have been additional independent origins and losses within
large clades that have a diversity of species with and without structural
colour (e.g. Aeshnidae, Chlorocyphidae, Coenagrionidae). Structural
colouration from quasiordered nanostructures within pigment cells has evolved
numerous times within odonates. Including the results from the present study
and those of Vernon et al.
(1974
) and Charles and
Robinson (1981
), the anatomy of
the structural colour-producing pigment cells of odonates has been described
from Aeshnidae, Diphlebiinae, Sympecmatinae and Coenagrionidae. According to
this hypothesis of character evolution, each of these four clades represents
an evolutionarily independent origin of this form of structural colouration
(Fig. 6, boldface). These
evolutionarily independent instances show remarkable parallelism in anatomy
and structure.
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Discussion |
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Although the arrays of light-scattering spheres are appropriately nanostructured to produce visible colours by coherent scattering, there are still substantial uncertainties about the precise mechanism of colour production in these arrays. The predicted reflectance spectrum based on the Fourier power spectra produces a substantial overestimate of the wavelength of peak reflectance. Uncertainty about refractive indices of the composite materials is unlikely to contribute to this error because even assuming the lowest reasonable average refractive index (1.35 for cytoplasm) produces a 40% overestimate in the peak wavelength. Alternatively, assuming a 50% expansion in the size of the spheres and an average refractive index of 1.55 produces an excellent fit to the observed reflectance spectrum (Fig. 4C). More specifically, a doubling of the peak spatial frequency, or a division of the array into more spheres with half the diameter of the observed spheres, would produce the observed reflectance spectrum.
The 50% expansion in the size of the spheres during preparation is not
reasonable. The spherical structures are of unknown composition within pockets
of the endoplasmic reticulum of the pigment cells. Because more than
two-thirds of the `volume' of these pigment cells consists of nanospheres that
are outside the cellular cytoplasm, these unusual pigment cells may be
particularly susceptible to perturbation during fixation. During fixation, all
specimens rapidly lost structural colour and turned black, indicating that
whatever produced the structural colour was perturbed by fixation itself.
(This perturbation was not caused by migration of ommochrome granules, which
remained in their basal position within the pigment cells after fixation; Figs
3A,B,
4A.) However, the size of the
nanospheres in the fixed pigment cells of Enallagma, Anax and the
other species described by Vernon et al.
(1974) did not vary
extensively. Furthermore, if the nanospheres expanded
50% during
fixation, then the array would have required twice the number of spheres
before preservation to retain the same volume and would double the predominant
spatial frequency. Such a big change in nanostructure during fixation would
probably create more massive perturbation of pigment cell structure than is
documented in these images (Figs
3,
4).
Alternatively, light scattering may be occurring at both the periphery of the spheres and the centres of the spheres. If the spot within many of the light-scattering spheres is an indication of an internal structure of a different refractive index (e.g. a central fold of endoplasmic reticulum) running through the centre of the spheres, then this structure could also cause light scattering. This would exactly double the peak spatial frequency of variation in refractive index and would produce a reflectance spectrum that is exactly congruent with the observed reflectance spectrum (Fig. 5C). The variable position of the spot indicates that it is produced by a linear structure running from pole to pole through the nanospheres (e.g. imagine a jar of pitted olives; Fig. 3C,D). Because these polar structures are not oriented in a single direction within the cells (Fig. 3C,D), their orientation is not periodic in any particular planes, and the coherently scattered reflections would lack iridescence or polarization.
The chemical composition, refractive index and structure of the material
within the light-scattering spheres in odonate pigment cells needs to be
investigated further. Understanding the composition of the spheres would
provide a further test of the coherent scattering hypothesis by establishing
whether the refractive index of the spheres is high enough to produce the
observed magnitude of reflectance. Our investigation gives only indirect
evidence on the identity of these spheres. In the more degraded preparations,
the spheres also showed a series of electron-dense lines radiating from the
central spot (Fig. 4B,C; also
reported by Vernon et al.,
1974), but these lines are absent in the better preserved, quickly
fixed samples of Enallagma (Fig.
3C,D). Vernon et al.
(1974
) suggested that these
radiating lines may indicate a crystalline structure within the spheres,
perhaps like spherical versions of the purine crystals of vertebrate
iridophores. However, these lines may also be an indication of how material
degrades rather than representing its intact structure. Vernon et al.
(1974
) have further shown that
the development of the light-scattering nanospheres in teneral
Austrolestes dragonflies (Lestidae) is similar to the ommochrome
pigment granules. Both are produced by deposition of materials into pockets of
the endoplasmic reticulum. Vernon et al.
(1974
) documented occasional
anomalous or chimeric structures that were developmentally and anatomically
intermediate between typical ommochrome granules and the light-scattering
nanospheres. One section of Enallagma civile observed here documents
the interconnections between neighbouring light-scattering spheres, indicating
that these aggregations of spheres are complexly organized like pearls on a
string (Fig. 3C).
Additional research is required to determine the mechanism of colour production in these odonate pigment cells. Most importantly, the biochemical composition and structure of the light-scattering spheres within these odonate pigment cells needs to documented and it needs to be determined whether they change substantially in size or have an internal light-scattering structure within them.
We have now tested and rejected the traditional hypothesis of incoherent
Rayleigh or Tyndall scattering for structural colours from avian feather barbs
(Prum et al., 1998,
1999b
,
2003
), avian skin (Prum et
al., 1999a
, 2003b), mammalian
skin (Prum and Torres, 2004
)
and the odonate integument (present study). We know of no hypothesized
instance of biological colour production by incoherent scattering that has
been verified by examining whether the scattering objects are spatially
independent and whether the reflectance spectrum conforms to Rayleigh's
inverse fourth power law. The historical lack of appreciation of coherent
scattering by quasiordered arrays has led to the erroneous association of
non-iridescent blue colours with incoherent scattering. Additional putative
examples of incoherent scattering in various vertebrates and invertebrates
need to be tested critically to identify the actual anatomical and physical
conditions under which it may occur in organisms.
Evolution of nanostructured arrays
Here, we document that two of the most distantly related odonate clades
aeshnid dragonflies and coenagrionid damselflies have
essentially identical structural colour-producing pigment cells. Previous work
by Vernon et al. (1974)
further documents that two additional clades of structurally coloured odonates
lestid and amphipterygid damselflies have identical
colour-producing pigment cell anatomy.
A comparative phylogenetic analysis of the distribution of non-iridescent, blue, integumentary structural colour in odonates indicates that this anatomy has evolved 1114 times independently. This estimate is quite conservative, since the available phylogenies do not yet resolve the genera and species within the several diverse clades that include large numbers of structurally coloured and nonstructurally coloured taxa (e.g. Aeshnidae, Chlorocyphidae, Coenagrionidae). This feature could have evolved convergently additional times within several of these groups.
Like many odonates with non-iridescent, blue, integumentary structural
colours (Sternberg, 1996;
Corbet, 1999
), Anax
junius is capable of temperature-dependent colour change from blackish,
at low body temperatures, to blue at higher body temperatures
(Corbet, 1999
). Vernon et al.
(1974
) have shown that
temperature-dependent colour change in other odonates occurs by vertical
migration of the ommochrome spheres from the basal third of pigment cells
during the high-temperature blue condition to become uniformly distributed
among the light-scattering spheres of the entire cell area. There, the
ommochrome granules absorb sufficient incident light to prevent the creation
of the blue structural colour. Congruent with Vernon et al.
(1974
), our observations
indicate that there are no major anatomical differences between pigment cells
of odonates that have or lack the capacity for temperature-dependent colour
change. Taxa with temperature-dependent colour change appear to differ only in
the capacity of the ommochrome pigment vesicles to migrate among the
light-scattering nanospheres in physiological response to basal body
temperature (Sternberg, 1996
).
Nothing is known about the mechanism of ommochrome granule motility during
physiological colour change but it may be facilitated by cytoskeletal
filaments.
Most insects' colours are restricted to the cuticle. The evolution of
structural colour production by living pigment cells in the epidermis below
the cuticle in numerous independent lineages of odonates has also fostered the
convergent evolution of temperature-dependent colour change in many odonate
clades (e.g. Aeshnidae, Lestidae, Megapodagrionidae, Coenagrionidae;
Sternberg, 1996;
Corbet, 1999
).
It has been hypothesized that green odonate colours are produced by a combination of structural Tyndall blue and a cuticular pigmentary yellow. However, the green integument of Anax junius completely lacks the subcuticular pigment cells that produce the structural colour. This green colour appears to be produced exclusively by a green cuticular pigment.
Odonates are a highly visual order of insects, and the colour of the
integument is known to function in mate choice and intraspecific communication
in many species (reviewed in Corbet,
1999). It appears likely that the combination of advanced visual
perception and epidermal, or subcuticular, pigmentary cells has fostered the
independent evolution of structural colouration in many lineages of odonates.
The sexual dimorphism of structural colouration in many lineages suggests that
sexual selection and mate choice may have played an important role in the
evolution and diversification of these colours. More detailed work is needed
on the physical mechanisms of colour signalling, development and heritability
of structural colouration in odonates in order to understand whether these
colours may function as honest indicators of condition. The temperature
dependency of structural colour in many lineages would provide a mechanism for
such signalling to evolve, but it is uncertain whether information on such an
ephemeral aspect of condition as body temperature would be of utility in mate
choice. Future studies on odonate behaviour should incorporate understanding
of the physics of structural colouration in investigations of its function and
evolution.
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