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First published online August 3, 2006
Journal of Experimental Biology 209, 3079-3090 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02360
Beauty in the eye of the beholder: the two blue opsins of lycaenid butterflies and the opsin gene-driven evolution of sexually dimorphic eyes
1 Comparative and Evolutionary Physiology Group, Department of Ecology and
Evolutionary Biology, 321 Steinhaus Hall, University of California, Irvine, CA
92697, USA
2 Department of Electrical Engineering, University of Washington, Seattle,
WA 98195-2500, USA
3 Functional Morphology Group, Department of Developmental Biology,
University of Erlangen-Nuremberg, 91058 Erlangen, Germany
* Author for correspondence (e-mail: abriscoe{at}uci.edu)
Accepted 5 June 2006
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Summary |
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max=360 nm), blue (B; max=437 nm and 500
nm, respectively) and long (LW; max=568 nm) wavelength
range. By combining in situ hybridization of cloned opsinencoding
cDNAs with epi-microspectrophotometry, we found that all four opsin mRNAs and
visual pigments are expressed in the eyes in a sex-specific manner. The male
dorsal eye, which contains only UV and B (max=437 nm)
visual pigments, indeed expresses two short wavelength opsin mRNAs,
UVRh and BRh1. The female dorsal eye, which also has the UV
and B (max=437 nm) visual pigments, also contains the LW
visual pigment, and likewise expresses UVRh, BRh1 and
LWRh mRNAs. Unexpectedly, in the female dorsal eye, we also found
BRh1 co-expressed with LWRh in the R3-8 photoreceptor cells.
The ventral eye of both sexes, on the other hand, contains all four visual
pigments and expresses all four opsin mRNAs in a non-overlapping fashion.
Surprisingly, we found that the 500 nm visual pigment is encoded by a
duplicate blue opsin gene, BRh2. Further, using molecular
phylogenetic methods we trace this novel blue opsin gene to a duplication
event at the base of the Polyommatine+Thecline+Lycaenine radiation. The blue
opsin gene duplication may help explain the blueness of blue lycaenid
butterflies.
Key words: eye evolution, sexual selection, visual pigment, color vision, butterfly, Lycaena rubidus
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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), there has been intense interest in understanding the
origins and evolution of sexually dimorphic traits. Butterflies offer
spectacular examples of sexual dimorphism associated with wing color variation
between males and females (sexual dichromatism). The use of wing color cues in
speciation (Silberglied, 1979)
and sexual selection (Obara,
1970) has long been recognized. Sympatric butterfly relatives that
look similar from the human perspective are readily distinguished, for
instance, by UVbased signals (Silberglied,
1979). Moreover, females have been shown to use species-specific
UV reflectance patterns to identify mates
(Silberglied and Taylor, 1973;
Rutowski, 1977;
Robertson and Monteiro, 2005).
Nonetheless, discussions of the evolution of butterfly wing colors in relation
to visually mediated components of courtship have typically been put forth in
the absence of information about sensory receptors.
Previous studies of both butterfly wing color cues and color vision have
focused on papilionid, pierid or nymphalid families
(Kelber et al., 2003). Little
attention, however, has been paid to the youngest of butterfly families, the
riodinids or the lycaenids, because they tend to be small and hard to
distinguish to the human eye. Lycaenids in particular comprise the second
largest of the butterfly families, with more than 4000 species named
worldwide, many of which are found in South America
(Johnson and Coates, 1999).
With a mounting abundance of physiological
(Eguchi et al., 1982;
Kinoshita et al., 1997),
molecular (see below) and behavioral data
(Zaccardi et al., 2006)
pointing to phenotypically variable butterfly visual systems, it seems
increasingly important to consider the evolution of butterfly wing color in
the context of the evolution of their eyes.
Opsins, together with a light-sensitive chromophore, form the visual
pigments expressed in the photoreceptor cells (R1-9) of the lepidopteran
compound eye. Most lepidopteran eyes contain a minimum of three visual
pigments with absorbances that peak at 
350 nm (UV), 440 nm (blue, B) and
530 nm (long wavelength, LW) (reviewed in
Briscoe and Chittka, 2001). In
the sphingid moth, the painted lady and monarch butterfly, these visual
pigments are encoded by paralogous UV, B and LW opsin genes, which were
present in the ancestor of all lepidopterans
(Briscoe et al., 2003;
White et al., 2003;
Sauman et al., 2005).
Deviations from this basic plan have been described in the most primitive of
butterfly families, the papilionids, in which two rounds of duplication of the
LW eye opsin gene have occurred (Briscoe,
1998; Kitamoto et al.,
1998; Briscoe,
2001); and in the pierids, in which a duplicate blue opsin gene
has been reported (Arikawa et al.,
2005). In all butterfly species studied, opsin mRNA expression in
the eye is similar between the sexes: the R1 and R2 photoreceptor cells
express either UV or B opsin mRNAs and the R3-9 photoreceptors express LW
opsin mRNAs.
Along with sexually dimorphic wings, sexually dimorphic eyes have likely
evolved multiple times in butterflies. To determine whether opsins have in
fact played a role in the evolution of sexually dimorphic eyes, we used a
combination of physiological, molecular and anatomical approaches to examine
the adult eye of the ruddy copper butterfly Lycaena rubidus
(Lycaenidae), a species with both sexually dimorphic wing coloration and
sexually dimorphic eyeshine. The small North American genus Lycaena
consists of 16 species in which males are often brilliantly colored iridescent
copper, blue, redorange or purple, while females are muted or predominantly
gray (Glassberg, 2001;
Pratt and Wright, 2002).
Females tend to mate only once (Gage et
al., 2002) so selection for correct mate choice is intense.
The opsins of L. rubidus are also of particular interest, because,
rather than the usual three visual pigments, the L. rubidus eye
contains four with peak absorbances (max values) at 360,
437, 500 and 568 nm that are distributed differentially both dorso-ventrally
and between the sexes (Bernard and
Remington, 1991). As duplicate LW opsin genes have been reported
only in the most ancient of butterfly families (Papilionidae), encoding 515,
520 and 575 nm visual pigments (Arikawa,
2003), the 500 and 568 nm visual pigments of the more derived
L. rubidus represent good candidates for being an independent LW
opsin gene duplication. We were therefore interested in investigating whether
parallel duplication of LW opsin genes had occurred between papilionid and
lycaenid butterflies.
After cloning the L. rubidus opsin-encoding cDNAs, we determined
by in situ hybridization both dorsal-ventral and sex-specific
differences in the opsin mRNA expression patterns in the eye. To our surprise,
we discovered that the BRh2 mRNA, which is exclusively expressed in
the ventral eye, encodes the 500 nm visual pigment. Rather than being the
result of a LW opsin gene duplication, it has actually evolved from a blue
opsin gene. We screened eye cDNA libraries from an additional ten butterfly
taxa and, using phylogenetic analyses, traced the gene duplication event to
the base of the Polyommatine+ Theclinine+Lycaenine radiation. Together, the
437 and 500 nm visual pigments may enhance color vision in the blue (400-500
nm) part of the visible light spectrum. We hypothesize that the blue opsin
gene duplication and the associated evolution of the 500 nm visual pigment may
be causally linked to the radiation of this family that is famous for blue
butterflies, including Nabokov's Blues (Tribe Polyommatini)
(Nabokov, 1945). While cues
from UV, LW and polarized light
(Silberglied, 1984;
Jiggins et al., 2001;
Fordyce et al., 2002;
Sweeney et al., 2003) have
been recognized as important social signals in butterfly communication, our
data suggest that blue cues in blue butterflies may provide an equally
important signal for visual communication.
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Materials and methods |
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;
Bernard, 1983b;
Briscoe et al., 2003;
Briscoe and Bernard, 2005).
Briefly, an intact Lycaena rubidus (Lycaenidae) (either female or
male) was mounted in a slotted plastic tube fixed to the goniometric stage,
then oriented to set the eye's direction of view to elevation 40° and
azimuth 15°. The microscope objective was a Leitz 8X/0.18P. The aperture
stop of the illuminator was set to create eyeshine in 20 ommatidia, then
focused on the deep pseudopupil for optimal collection of eyeshine and
reduction of stray light. After at least 1 h in the dark, the reflectance
spectrum of eyeshine was measured with a series of dim monochromatic
flashes.
Eyeshine photographs
Photographs of eyeshine were created by exchanging the photometer head for
a micro-photographic attachment. The illuminator slide was replaced with a
Leitz Mecablitz-III micro-flash. Film was Kodak ASA160 Daylight-Ektachrome.
The microscope was focused on the cornea with aperture stop fully open. After
several minutes of dark-adaptation, the shutter of the camera was opened long
enough for the eye to be flashed at full intensity by the Mecablitz flash.
Repeated photos from the same spot required several minutes of dark-adaptation
between flashes to ensure full recovery from pupillary responses prior to each
photo.
Tissue collection
Tissues used for total RNA extraction and cDNA synthesis were either
collected by the authors (Agriades glandon de Pruner, Colias
philodice Godart and Satyrium behrii Edwards) or provided as
gifts by the following individuals (Apodemia mormo Felder &
Felder, John Emmel; Basilarchia arthemis astyanax Fabricius, Austin
Platt; Bicyclus anynana Butler, Antónia Monteiro;
Heliconius melpomene Linnaeus, Larry Gilbert; Lycaena
rubidus Behr, Carol Boggs and Ward Watt; Nymphalis antiopa
Linnaeus, Peter Bryant; Polyommatus icarus Rottemburg, Almut Kelber).
Vanessa cardui Linnaeus were obtained from the Carolina Biological
Supply Co (Burlington, NC, USA). Adult L. rubidus used for in
situ hybridization were reared in the laboratory from wild-caught
caterpillars collected at Tioga Pass, Mono County, CA, USA or collected from
the Rocky Mountain Biological Laboratory, Gothic, CO, USA. Caterpillars were
fed Rumex crispus leaves until pupation. Adults were fed 20% sugar
water and sacrificed 1-2 days after eclosion for tissue fixation and
sectioning.
PCR, cloning and sequencing
Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA).
Adaptor-ligated double-stranded complementary DNA (cDNA) was synthesized from
total RNA using the Marathon cDNA Amplification Kit (BD BioSciences Clontech,
Mountain View, CA, USA). To obtain the complete cDNA sequences, 3'RACE
products were first amplified with a degenerate primer using ExTaq
DNA polymerase (TaKara Mirus Bio, Madison, WI, USA) under PCR conditions of 2
min at 94°C, then 35 cycles of 30 s at 94°C, 30 s at 50°C and 1
min at 68°C. Bands >500 bp were gel purified (Geneclean I Kit,
QBiogene, Irvine, CA, USA), then ligated into the pGEM T-easy vector (Promega,
Madison, WI, USA). The plasmids were prepared with the QIAprep Spin Miniprep
Kit (QIAGEN, Valencia, CA, USA). Clones were screened by EcoRI
digest. Clones were cycle sequenced using the Big Dye Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the core
sequencing facilities at the University of California, Irvine.
Multiplex PCR using opsin-specific primer pairs (supplementary material Table S1) was also performed on 3'RACE clones to identify other potential opsins that had not yet been sequenced as well as to eliminate the ones already sequenced. To obtain the 5'RACE products, gene-specific reverse primers and a touch-down PCR protocol with the BD Advantage Polymerase Kit (BD Biosciences, San Jose, CA, USA) was used as follows: 1 min at 95°C, 5 cycles of [30 s at 95°C, 1.5 min at 68°C], 5 cycles of [30 s at 95°C, 30 s at 65°C and 1.5 min at 68°C], 5 cycles of [30 s at 95°C, 30 s at 60°C and 1.5 min at 68°C], 25 cycles of [30 s at 95°C, 30 s at 55°C and 1.5 min at 68°C] and 10 min at 68°C.
Phylogenetic analysis of opsins
Cloned L. rubidus opsin sequences were aligned in MEGA 3.1
(Kumar et al., 2004) together
with 44 homologous insect opsin sequences downloaded from GenBank. Only
sequences with complete coding regions were used in the alignment. A
neighbor-joining tree sampled from 1000 bootstrap replicates was constructed
from the amino acid alignment with Poisson correction (MEGA 3.1). A total of
299 amino acid sites were used in the tree reconstruction.
Phylogenetic reconstructions of the lepidopteran blue opsins were performed
with PAUP* (Swofford,
2000) using the maximum likelihood method
(general-time-reversible, with a gamma substitution correction and a
proportion of invariant sites; model parameters estimated from the data).
Estimates of the proportion of invariant sites and gamma were then made using
this initial tree and used to run 500 ML bootstrap replicates in PhyML
(Guindon and Gascuel, 2003;
Guindon et al., 2005).
In situ hybridization
Butterfly heads were fixed in 4% phosphate-buffered formaldehyde for 2 h
and then immersed in a sucrose/1x PBS (phosphate buffered saline)
gradient of 10%-30% for 2 h at each step. The tissues were stored in 30%
sucrose at 4°C. Tissues were embedded in OCT freezing compound (Sakura
Finetek USA, Torrance, CA, USA) and cryostat-sectioned into 16 µm slices.
Riboprobes were generated from cloned opsin templates that were linearized
via PCR. Sense/anti-sense riboprobes were synthesized from 1 µg
template using the DIG RNA Labeling Kit (Roche Applied Science, Indianapolis,
IN, USA). The riboprobe yield was quantified by dot blot procedure. Sections
were immersed in hybridization buffer for 30 min at 60°C. Approximately
0.05 µg riboprobe per µl hybridization buffer was hybridized to the
histologic sections overnight at 60°C. Washing, detection and mounting
methods are as described (Briscoe et al.,
2003). Slides were viewed under an Axioskop microscope (Zeiss,
Thornwood, NY, USA) using bright field illumination. Digital photographs were
captured in an Axiocam digital camera (Zeiss) attached to the microscope.
Photographs were processed in Adobe Photoshop 7.0 for size, brightness and
contrast modification only.
Transmission electron microscopy
The tissue was dissected in 0.08 mol l-1 phosphate buffered 2%
glutaraldehyde/formaldehyde with 4% sucrose and fixed for 2 h at 4°C.
After washing in buffer solution and postfixation with 2% OsO4 at
4°C for 2 h, the specimens were dehydrated in ethanol and embedded in Epon
812 (Roth, Karlsruhe, Germany). For histological investigations of the
filtering pigments, semithin sections were cut and investigated with an
Axiophot microscope (Zeiss, Oberkochen, Germany) with differential
interference contrast. A F-View II-Camera (Soft Imaging System GmbH,
Münster, Germany) was used to collect images. Corresponding ultrathin
sections were cut, mounted on grids, and after staining with uranyl acetate
and lead citrate for 10 min each, they were examined with an EM 10 electron
microscope (Zeiss, Oberkochen, Germany). Images were taken with a ProScan Slow
Scan CCD camera (LEO Electron Microscopy, Oberkochen, Germany). All
photographs were processed in Adobe Photoshop 7.0 for size, brightness and
contrast modification only.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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), we used
light microscopy to examine the L. rubidus adult retina for the
presence or absence of non-opsin filtering pigments.
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We found two kinds of pigment granules, the first of which was the dark purple pupillary pigment present distally in R1-8 cells everywhere in the eye (data not shown) (Fig. 1A), including the dorsal rim area, that regulates the amount of light entering the eye. The second pigment type was a pink filtering pigment that was absent in dorsal eye ommatidia (Fig. 1B) and in the R5-8 cells of some, but not all, ventral eye ommatidia (Fig. 1C, arrowheads and arrow, respectively). There was no sex difference in the distribution of the two non-opsin filtering pigments.
Eyeshine and visual pigment distribution in dorsal eye
Butterfly eyeshine in L. rubidus, a species with sexually
dimorphic wing coloration (Fig.
2A,B), is produced by light reflected by multilayered tracheolar
mirrors (tapeta) at the base of each ommatidium
(Miller, 1979)
(Fig. 1A), following its
partial absorption by visual pigments in the rhabdom. The coloration of
eyeshine is not simply explained, as it depends on the absorbance spectra of
all rhodopsins contained with the rhabdom and the reflectance spectrum of the
tapetum. It may also be influenced by the presence of non-opsin filtering
pigments (Ribi, 1979;
Douglas and Marshall, 1999;
Stavenga, 2002a;
Briscoe and Bernard, 2005).
Furthermore, bright illumination can change the coloration of eyeshine by
photo-converting rhodopsins to spectrally shifted photoproducts.
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). Eyeshine of L. rubidus males is
predominantly green-orange, while that of females is blue-yellow
(Fig. 2C,D). Green-yellow
eyeshine has also been reported in dorsal eye of L. phlaeas of
unspecified sex (Stavenga,
2002b). To determine whether differences in bandwidth of the
tapetal reflectance spectra might account for this difference, we used
transmission electron microscopy to examine the ultrastructure of the tapeta
of male and female dorsal eye. We could not detect, however, any difference in
optical design of the tapetum (data not shown). As previously noted, Epon
sections of the eye also revealed an absence of the heterogeneously expressed
pink filtering pigments in the dorsal eye that was detected in the R5-8
photoreceptor cells of some ommatidia in the ventral eye of both sexes
(Fig. 1C). Therefore the dorsal
eyeshine pattern is not influenced by differences in tapetal structure or
non-opsin filtering pigments.
We next estimated the visual pigment content of the male and female dorsal
eyes from experimental reflectance spectra using epi-microspectrophotometric
methods, as described (Bernard,
1982; Bernard,
1983a; Bernard,
1983b; Bernard and Remington,
1991; Briscoe et al.,
2003). A computational analysis of these spectra revealed two
short-wavelength visual pigments in the dorsal eyes of both sexes
(max=360 and 437 nm)
(Fig. 2E-H). In addition, the
female dorsal eye has an LW visual pigment (max=568) not
found in the male dorsal eye (Fig.
2E-H). The ventral eye of L. rubidus has been shown
previously to contain a fourth visual pigment (max=500 nm)
(Bernard and Remington, 1991).
Thus, the eyeshine and reflectance spectra data confirm the presence of three
visual pigments in L. rubidus, which are distributed in sexually
dimorphic patterns in dorsal eye.
Opsin sequences and phylogeny
We cloned the full-length cDNAs of four opsin genes, which, based upon
phylogenetic analyses (supplementary material Figs S1-S4), represent a UV
opsin (UVRh, AY587904), two B opsins (BRh1, AY587902; BRh2,
AY587903) and a LW opsin (LWRh, AY587901). The lack of a duplicate LW
opsin was unexpected. We note that the short wavelength opsins have a lysine
at residue 112 (UVRh) and a glutamate at residue 107 (BRh1 and BRh2), which
confer UVabsorbing (lysine) and blue-absorbing (glutamate) spectra
(Salcedo et al., 2003). The
situation in L. rubidus differs markedly from that of the nymphalid
butterflies Vanessa cardui or the monarch Danaus plexippus,
in which only three opsin mRNAs are expressed (UV, B, and LW) in the
photoreceptor cells of the adult eye
(Briscoe et al., 2003;
Sauman et al., 2005), or that
of the swallowtail butterfly, in which three duplicate LW opsin genes are
expressed (Briscoe, 1998;
Kitamoto et al., 1998). The
L. rubidus opsin expression pattern does, however, resemble the
situation in Pieris rapae, in which duplicate B opsin genes are also
expressed in the eye (Arikawa et al.,
2005) (but see below).
Sexually dimorphic expression of LWRh
Since our microspectrophotometric results indicate that the dorsal eye of
female L. rubidus contains a 568 nm visual pigment that is absent
from the dorsal eye of males (Fig.
2E-H), we examined the expression of the LWRh mRNA to
determine if it encodes this LW visual pigment. We found that the expression
of LWRh mRNA in the retina indeed differed between the sexes. In
males, LWRh was only expressed in photoreceptor cells R3 through R8
in all ommatidia in the ventral region of the eye, but the transcript was
totally absent in the dorsal area (Fig.
3A). In contrast, LWRh in females was expressed uniformly
in R3-8 of all ommatidia in both the dorsal and ventral regions of the eye
(Fig. 3B and inset). We note
that the expression of a LW opsin mRNA in the R3-8 photoreceptor cells is the
ancestral state for Lepidoptera (Briscoe
et al., 2003) (see below), so its absence in dorsal eye of males
is unprecedented.
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max=437 nm), identified through our
photochemical experiments in L. rubidus
(Fig. 2E-H), to the dorsal eye
of both males and females. In both sexes, BRh1 was abundantly
expressed in the R3-8 cells of the dorsal eye and, in addition, expressed in
the R1 and R2 cells of the ventral eye
(Fig. 3C,D). Unexpectedly, in
females, we found that the BRh1 mRNA was co-expressed with the
LWRh mRNA in R3-8 of all the ommatidia in the dorsal eye
(Fig. 3B,D insets). This
co-expression pattern was not found in males because of the absence of the
LWRh mRNA dorsally (see Fig.
3B). Of 177 ommatidia counted in the female dorsal eye, in which
adjacent tangential sections were stained alternately by LWRh and
BRh1 riboprobes, all of the R3-8 photoreceptor cells were labeled by
both riboprobes. This co-localization result, along with the results of our
microspectrophotometric experiments, indicate that each R3-8 photoreceptor
cell in female dorsal eye of L. rubidus expresses two different
visual pigments. This is a striking finding, because, to our knowledge, there
are no other reports of coexpression of a short and long wavelength-sensitive
opsin in butterflies.
Dorsal-ventral patterning of UVRh and BRh2 expression
To confirm the identity of the UV visual pigment
(max=360 nm) detected by microspectrophotometry in L.
rubidus dorsal eye (see Fig.
2E-H), we examined the distribution of the UVRh mRNA.
UVRh expression also showed variation in its dorsal-ventral
distribution across the retina in both males and females, as expression was
more abundant in the dorsal area (Fig.
3E,F). The only visual pigment not detected in dorsal eye of
either sex was the 500 nm visual pigment that was previously detected by
epimicrospectrophotometry to be exclusively in the ventral eye of both sexes
(Bernard and Remington, 1991).
Consistent with the epi-microspectrophotometric result, we found BRh2
to be the only opsin transcript exclusively localized to ventral eye
(Fig. 3G,H). Therefore, we
conclude that BRh2 encodes the 500 nm visual pigment.
Non-overlapping expression of UVRh, BRh1 and BRh2
Unlike the unusual BRh1 mRNA expression in the outer R3-8
photoreceptor cells of the L. rubidus dorsal eye, the typical
lepidopteran expresses short wavelength opsin mRNAs only in the R1 and R2
cells (Kitamoto et al., 2000;
Briscoe et al., 2003;
White et al., 2003;
Sauman et al., 2005). Closer
inspection of UVRh, BRh1 and BRh2 expression in individual
ommatidia indicated that these transcripts are also present in the R1 and R2
photoreceptor cells of both sexes (Fig.
4). In the ventral and dorsal parts of the eye, adjacent sections
showed that the three opsin mRNAs have non-overlapping expression in the R1
and R2 photoreceptor cells. In the ventral retina, five different types of
ommatidia with respect to non-overlapping UVRh, BRh1 and
BRh2 expression in R1 and R2 photoreceptor cells can be seen
(Fig. 4A-C): ommatidia
containing UVRh-UVRh, UVRh-BRh1, UVRh-BRh2, BRh1-BRh1 and
BRh1-BRh2 (supplementary material Table S2). BRh2 mRNA
expression in both R1 and R2 was also observed in the ventral area, although
this ommatidial subtype was rare (data not shown). We note the finding of
BRh1 opsin mRNA in the ventral eye differs from the original report
(Bernard and Remington, 1991)
in which the 437 nm visual pigment was not found in ventral eye. Our finding
is, however, consistent with the report in the same paper of the presence of
P437 in the ventral eye of L. heteronea. The differences between our
finding and that of Bernard and Remington
(Bernard and Remington, 1991)
may be due to relative differences in visual pigment abundances between
species and also to differences in the sensitivity of the detection
methods.
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; Wakakuwa et al.,
2004), and have been shown to be important for butterfly color
vision (Zaccardi et al.,
2006).
In the dorsal eye, R1 and R2 photoreceptors expressed predominantly UVRh (Fig. 4D), while BRh2 expression was never observed (Fig. 4F). BRh1 mRNA was also expressed in R1 or R2 or both, but these ommatidial subtypes were rare (Fig. 4E, insets). Instead, BRh1 mRNA was predominantly expressed in R3-8 of all ommatidia in the dorsal eye (Fig. 4E, section taken from a different male individual). We did not examine opsin expression in the dorsal rim area (DRA) ommatidia.
Rhabdoms of the main retina are organized for color vision
In Drosophila (Wernet et al.,
2003) and monarch butterflies
(Sauman et al., 2005), the
ommatidia of the DRA have a unique pattern of opsin expression compared to the
main retina -this region contains ommatidia with rhabdoms specialized for
polarization vision (Labhart and Meyer,
1999; Stalleicken et al.,
2006). One possible explanation for the dramatically altered
pattern of opsin mRNA expression we identified in the dorsal part of the
L. rubidus eye is that it represents an expansion of the DRA. To test
this hypothesis, we examined the organization of the microvillous membranes
that form the rhabdoms in ultrathin sections of ommatidia from the DRA, the
dorsal eye, and the ventral eye using transmission electron microscopy
(supplementary material Fig. S6). We found that L. rubidus do indeed
have DRA ommatidia similar to those found in other butterflies
(Labhart and Meyer, 1999),
characterized by square-shaped rhabdoms that contain perpendicularly oriented
microvilli that are specialized for polarized sky light detection
(supplementary material Fig. S6A). The rhabdoms from ommatidia of both sexes
in the dorsal and ventral parts of the main retina, on the other hand, were
circular in shape (supplementary material Fig. S6B,C,D), consistent with the
idea that they are organized for detecting the spectral content of light (i.e.
colour of an object) (Stalleicken et al.,
2006). This architecture was verified by observing the
brush-shaped organization of the microvilli in longitudinal sections
(supplementary material Fig. S6E).
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), where
papilionid and pierid butterflies represent the most basal lineages, and
riodinid and lycaenid the most derived. Because the two pairs of B opsin genes
identified from pierid (Pieris rapae)
(Arikawa et al., 2005) and
lycaenid butterflies (this study) are from distantly related families, we were
interested in testing the hypothesis that the blue opsin genes of pierids and
lycaenids evolved independently. We therefore screened eye-specific cDNA
libraries from an additional ten butterfly taxa from four families
(supplementary material Table S3).
We cloned a total of 14 full-length blue opsin-encoding cDNAs from these
ten additional taxa, including homologues of both BRh1 and
BRh2 in all surveyed lycaenid subfamilies. Only one blue opsin cDNA
was detected in each of the seven species of nymphalid surveyed.
Neighbor-joining and maximum likelihood analyses using all three nucleotide
positions unambiguously indicated that the blue opsin genes of L.
rubidus evolved independently of the pierid duplicate blue opsin genes
(Fig. 5). To our knowledge, no
other insects besides pierid and lycaenid butterflies have two blue opsin
genes. Our finding of independent duplication events is quite consistent with
the very different max values (425 nm and 453 nm) of the
pierid blue visual pigments (Arikawa et
al., 2005) compared to the lycaenid (max=437 nm
and 500 nm, respectively). Our results also indicate (bootstrap support=100%)
that the L. rubidus blue opsin gene duplication event occurred before
the radiation of the coppers, hairstreaks and blues.
(Lycaeninae+Theclinae+Polyommatinae)
(Eliot, 1973).
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;
Sauman et al., 2005;
Zaccardi et al., 2006). This
pattern is also found in the moth Manduca sexta
(White et al., 2003), and
likely represents the ancestral lepidopteran eye plan. L. rubidus
differs from nymphalids and moths, in having a duplicate B opsin gene
(Fig. 6, Step 1). A duplicated
gene can have two fates; either it will have the same function as the
ancestral copy, or it will evolve a new function (neofunctionalization)
(Force et al., 1999). In
L. rubidus, the visual pigment encoded by the B opsin gene copy (B2)
has evolved an extremely redshifted (500 nm) peak absorbance due to positive
selection on the amino acid coding region
(Fig. 6, Step 2)
(neofunctionalization). The LW opsin also has an unusually redshifted peak
absorbance (568 nm) compared to the typical lepidopteran absorbance (520-530
nm) (White et al., 2003;
Vanhoutte and Stavenga, 2004; Briscoe and
Bernard, 2005) and probably evolved in a coordinated fashion with
the 500 nm visual pigment. The duplicated B2 opsin BRh2 found only in
the ventral retina of both sexes is expressed in a nonoverlapping fashion with
UVRh and BRh1 in the R1 and R2 photoreceptor cells
(Fig. 6, Step 3)
(subfunctionalization) (Force et al.,
1999). Alteration in the regulation of BRh1 (B1) spatial
expression to include co-expression with LWRh (LW) in the outer R3-8
photoreceptor cells of dorsal eye of both sexes probably occurred next
(Fig. 6, Step 4). Finally, the
expression of LWRh opsin was suppressed in the dorsal eye of males
only (Fig. 6, Step 5), perhaps
due to strong selection for male-male recognition or the recognition of
conspecific females (Bernard and Remington,
1991). |
Discussion |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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) probably use their dorsal eye for dichromatic color vision
and detection of flickering moving objects, such as airborne males. Strong
male-male interactions seem to be a common theme among lycaenids (e.g.
Polyommatus icarus) (Lundren,
1977). UV-reflecting `flickering' wing patterns have also been
proposed as a stimulus useful in mating displays
(Meyer-Rochow, 1991).
A dorsal eye, which has predominantly UV and B receptors, seems to be a
common phenomenon in male insects having sexually dimorphic mates. Such a
pattern is found in the male honeybee
(Muri and Jones, 1983;
Menzel et al., 1991;
Velarde et al., 2005),
dragonfly Sympetrum (Labhart and
Nilsson, 1995), mayfly Atalophlebia
(Horridge and McLean, 1982),
Musca domestica (Hardie,
1986) and the bibionid fly Bibio marci
(Burkhart and De LaMotte,
1972; Zeil, 1983).
These data suggest that a sexually dimorphic eye may represent the ancestral
state in insects. However, other more basal butterflies that have been
examined, such as the swallowtail, the painted lady, the heliconian and the
monarch butterfly, do not have sexually dimorphic patterns of opsin mRNA
expression (Kitamoto et al.,
1998; Briscoe et al.,
2003; Sauman et al.,
2005; Zaccardi et al.,
2006). Rather, our data suggest that sexually dimorphic male eyes,
expressing predominantly UV and B opsins, may have evolved independently in
insects.
The situation in females is different. Our microspectrophotometric data
indicate the dorsal eye of females expresses UV, B and LW opsins that,
together with the chromophore, produce the 360, 437 and 568 nm visual
pigments, respectively. Six outer photoreceptor cells (R3-8) double-labeled by
LWRh and BRh1 riboprobes, strongly indicate that these
photoreceptors contain two visual pigments. Although the co-expression of two
LW visual pigments in one photoreceptor cell has been observed in other
arthropods (Sakamoto et al.,
1996; Kitamoto et al.,
1998), to our knowledge, L. rubidus is the first insect
species to have two visual pigments of both short and long wavelength spectral
types co-expressed in the same photoreceptor cells. Assuming that both visual
pigments are involved in phototransduction, the co-expression of LWRh
and BRh1 in a single photoreceptor cell would indicate that the
receptors have a broad sensitivity from the violet to orange-red spectrum
(350-650 nm). Intracellular recordings of the spectral sensitivity of a single
photoreceptor cell co-expressing these visual pigments are required to confirm
this point. Together with UV receptors, the L. rubidus female is
outfitted with a receptor type in the dorsal area that would in principle
provide trichromatic color vision over a broader part of the spectrum, as
compared to the male.
Sexual dimorphic butterfly eyes have likely evolved independently multiple
times through a variety of physiological mechanisms. In the case of the small
white cabbage butterfly, Pieris rapae (Pieridae)
(Arikawa et al., 2005), three
short wavelength receptors were found, sensitive to ultraviolet
(max=360 nm), violet (max=425 nm) and
blue (max=453 nm) light, and each expressing a unique
opsin. A spectral filtering pigment was found co-expressed with the blue opsin
only in males, producing a uniquely narrow blue receptor, highlighting the
changes in the spatial expression patterns of non-opsin filtering pigments as
a mechanism for producing a sexually dimorphic retina. The situation in
pierids, however, differs completely from what we have found in lycaenids with
respect to both the physiological basis of the sexual dimorphism and the fate
of the duplicate B opsin genes.
The presence of a fourth visual pigment mRNA BRh2, together with
UVRh and BRh1, furnishes Lycaena with six
ommatidial subtypes in the ventral eye area
(Fig. 4), twice the number
found in any other lepidopteran (Arikawa,
2003; Briscoe et al.,
2003; White et al.,
2003; Sauman et al.,
2005; Zaccardi et al.,
2006). Even in Pieris rapae
(Arikawa et al., 2005), which
as noted above has independently evolved a violet receptor from a duplicate
blue opsin gene, only three ommatidial types have been reported: those
expressing UV-UV, UV-B, and V-V opsin mRNAs. The Lycaena ommatidial
subtypes are heterogeneously distributed in the ventral region, suggesting
that with appropriate neuronal wiring, there may be good spectral
discrimination (i.e. blue color vision) in this part of the eye; a hypothesis
that can be tested using behavioral experiments.
Conclusions
The eye design of L. rubidus is exceptional. We have shown that
the novel sex-specific distributions of opsin mRNAs do not resemble that of
any other lepidopteran studied. In the female eye, the co-expression of
BRh1, encoding a bluesensitive visual pigment
(max=437 nm), and LWRh, encoding a long
wavelength-sensitive (max=568 nm) visual pigment, provides
an exception to the one-receptor, one-cell rule. We speculate that the
expression of the LWRh transcript has been suppressed in males due to
strong selection for male-male and conspecific mate recognition
(Fig. 6). Visual signals appear
to play a major role in the interspecific social interactions of lycaenid
butterflies, as evidenced by behavioral observations and the unparalleled
diversity of wing colors among sympatric species
(Lukhtanov et al., 2005). We
suggest that the molecular evolution of the 500 nm visual pigment and the
novel ommatidial subtypes have likely enhanced color vision in the short
wavelength part of the spectrum and have provided a mechanism for the rapid
evolution of wing color in the largest of lycaenid subfamilies
(Polyommatinae+Theclinae+ Lycaeninae)
(Johnson and Coates,
1999).
max
|
Acknowledgments |
|---|
|
Footnotes |
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|
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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+I model with estimated gamma shape
parameter=0.574 and proportion of invariant sites=0.1474. GenBank accession
numbers for sequences are provided in supplementary material Table S3. Inset:
Dorsal wing of a male Polyommatus icarus, one of the lycaenids
surveyed.