Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

Absorption

Deuteranomalous

Wavelength (nm)

Absorption

Protanomalous

Wavelength (nm)

S pigment and an L pigment, whereas deuteranomalous trichromats possess an S pigment and two spectrally distinct L pigments. Deuteranomalous defects are by far the most prevalent of any of the congenital color-vision defects, affecting nearly 5% of men. Individuals with deutan defects exhibit a reduction of sensitivity to colors in the green region of the spectrum, though the decrease in sensitivity is less pronounced than the long-wavelength depression in protans due to the manner in which the spectral sensitivities overlap (Figure 1). Deutans suffer similar hue-discrimination problems as the protans, but without the long-wavelength dimming. These discrimination errors are exploited in the design of color-vision tests; however, the real-world consequences of having certain red–green deficiency can be minimal. The perceptual consequences of having various color-vision defects are simulated in Figure 4. Interestingly, there is evidence that individuals with red–green defects may actually have an advantage when viewing camouflage, which is designed to blend into the environment, but this is done assuming a trichromatic visual system.

While the red–green color-vision defects can be behaviorally classified according to the cone subtype that is nonfunctional, the structural basis of the defects has been

unclear. Recent work using high-resolution, in vivo imaging of the human retina has shown that while conceptually one can think of a deutan retina containing only S and L cones, and a protan retina containing only S and M cones, the residual cone mosaic in red–green-deficient individuals can vary dramatically, depending on the genetic cause of the defect. Shown in Figure 5 are images of the cone mosaic from a normal trichromat and an individual with a red–green defect caused by an inactivating mutation in his M gene. Each circle is a single cone photoreceptor, and the dark spaces in the red–green-deficient retina represent cones that have degenerated or that are morphologically compromised. Thus, some individuals will have normal numbers of cones (though only two, instead of three types), while some will be structurally missing an entire cone class. The impact on vision in general is not yet known, and the genetic heterogeneity within the red–green defects remains to be reconciled with the mosaic phenotype.

Tritan Color-Vision Deficiencies

Tritan defects are an inherited autosomal dominant abnormality of S-cone function. These defects occur in

Figure 5 High-resolution images of the living retina obtained with adaptive optics. On the left is a retinal image from a patient with normal vision, at an eccentricity of approximately 1 temporal to fixation. Each circular structure is an individual cone photoreceptor. On the right is an image from a patient who is red–green color blind. Visible are numerous holes where cones have either died or degenerated, due to a mutation in one of the genes responsible for normal color vision. Despite the loss of nearly 33% of his functioning cones, this person has normal visual acuity. Scale bar ¼ 20 mm.

both males and females with equal frequency and are believed to be rare, affecting as few as 1 in 10 000 people. As the S-pigment gene is on chromosome 7 and humans are diploid, each S-cone photoreceptor expresses the S-cone pigment genes from both copies of chromosome 7. Consequently, a defect in one S-pigment gene can be sufficient to cause tritanopia, just as a mutation in one rhodopsin gene is sufficient to cause RP, a retinal degeneration that involves the degeneration of the rod photoreceptors.

Unlike the red–green defects, there is no genetic/pigment basis for a tritanomalous defect, as there is no spectral variation among functional S-cone pigments.

This disorder exhibits incomplete penetrance, meaning that individuals with the same underlying mutation manifest different degrees of color-vision impairment, even within a sibship. Mutations in the S-cone-opsin gene, which encodes the protein component of the S-cone photopigment, have been identified and they give rise to five different single amino acid substitutions that have only been found in affected individuals but not in unaffected control subjects (Figure 2(b)). Each substitution occurs at an amino acid position that lies in one of the transmembrane alpha helices, and is predicted to interfere with folding, processing, or stability of the encoded opsin. For example, one of the identified mutations corresponds to an amino acid position at which a substitution in the rod pigment, rhodopsin, causes autosomal dominant RP.

A fundamental difference between S cones and L/M cones is the potential for dominant negative interactions between normal and mutant opsins. This is because each S cone expresses both autosomal copies of the S-opsin gene, whereas L and M cones each only express one gene from the L/M array on the X chromosome. Rod photoreceptors also express a rhodopsin gene from two autosomes, and for rhodopsin mutations underlying autosomal dominant RP, dominant negative interactions lead to the death of the photoreceptors and ultimately the degeneration of the retina. Curiously, tritan defects have not been reported to cause progressive retinal degeneration and are only slightly

more rare than adRP; however, it has been suggested that the relative paucity of S cones compared to rods in the normal retina may be responsible for the absence of more general retinal degeneration. Nevertheless, the structural homology between the S-cone opsin and rhodopsin, taken together with the similar molecular mechanisms underlying the defects, suggest that S cones themselves degenerate in autosomal dominant tritan defects.

Blue-Cone Monochromacy

Blue-cone monochromacy (BCM) is a condition where both L- and M-cone function is absent. This disorder affects approximately 1 in 100 000 individuals. Since L and M cones comprise about 95% of the total cone population, these individuals have a rather severe visual impairment, including photophobia (light sensitivity), poor acuity, minimally detectable electroretinogram (ERG) responses, and diminished color discrimination. Any residual color vision in these individuals must be based solely on the S cones and rods. This means that under most conditions, they are monochromatic, though under mesopic (dim light; between photopic and scotopic levels) conditions they may gain some additional discrimination capacity.

As with the red–green defects, there are two main genetic causes of BCM, sometimes referred to as onestep or two-step mutations, though both lead to the absence of functional L and M cones. One-step mutations involve a deletion of essential cis-regulatory DNA elements needed for normal expression of the pigment genes. Two-step mutations involve a deletion of all but one of the X-chromosome visual pigment genes. This would normally confer red–green dichromacy (see above), but the one remaining gene contains a missense mutation. Due to the X-linked nature of the condition, female carriers are spared from a full manifestation of the associated defects, but they can show abnormal cone function on the ERG. One-step and two-step conditions may have important phenotypic differences in terms of the architecture of the cone mosaic in carriers. For the one-step mutations, the absence of an essential enhancer means that the cone photoreceptors cannot transcribe an opsin gene from the affected X chromosome. In contrast, for the two-step condition, there is a completely functional gene but the encoded opsin has a deleterious amino acid substitution, and the photoreceptor is expected to produce the mutant opsin. Depending on the nature of the mutation, it may either reduce or abolish proper folding of the encoded protein, or the gene may be transcribed but the message may be immediately targeted for degradation, and these may in turn ultimately affect the viability of the cone or its neighboring cells. While both conditions will likely compromise the viability of the cone, they may do so over different timescales.

Achromatopsia

Complete achromatopsia (i.e., rod monochromacy) is a congenital vision disorder in which all cone function is absent or severely diminished. It is typically characterized by an absolute lack of color discrimination, photophobia, reduced acuity, visual nystagmus, and a nondetectable cone ERG. Previously, exploration of this disease was limited to findings from histological and anatomical review and there was a debate surrounding whether the cones were absent, malformed, or merely minimally present. Recent work using in vivo cellular imaging, together with improved measures of cone function, suggest that not all achromats have the same cellular deficit, though in all cases their perception is dominated by the rod system and vision at normal light levels can be difficult if not impossible.

Rod monochromacy affects up to 1 in 30 000 people, and results from mutations in one of two components of the cone phototransduction cascade – transducin or the cyclic-nucleotide-gated (CNG) channel. Mutations in CNG alpha 3 (CNGA3) and CNG beta 3 (CNGB3), which encode the a- and b-subunit of the CNG channel, respectively, are by far the most common genetic cause of rod monochromacy, accounting for about 60–80% of the cases. A relatively small number of patients ( 5%) have a mutation in the GNAT2 gene, which encodes the a-subunit of the cone G-protein transducin. The current theory is that the genetic heterogeneity underlies the observed phenotypic variability in patients with this disease, and this likely explains the previous discrepancies in histological reports. However, a systematic linkage of genotype and phenotype has not been done. Recently, a gene therapy approach has been used to restore cone function in dog and mouse models of the disease; however, one would predict that the degree of remaining cone structure would be a predictor of whether a particular human achromat could benefit from similar therapeutic intervention.

Conclusion

Inherited color-vision deficiencies range from mild difficulties discerning pale hues from gray to quite severe issues differentiating even the most saturated hues, and while most of these deficiencies are often categorized by the general misnomer color blindness, the defects are in fact quite different from one another and rarely indicate a condition in which the patient is truly blind to color. There is enormous genetic variation in all inherited color-vision defects, and it is becoming clear that this variation has consequences for the visual system that reach far beyond subtle differences in color perception/ discrimination.

See also: The Colorful Visual World of Butterflies; Cone Photoreceptor Cells: Soma and Synapse; Phototransduction: Adaptation in Cones; Phototransduction: Inactivation in Cones; Phototransduction: Phototransduction in Cones; Polarized-Light Vision in Land and Aquatic Animals; Primary Photoreceptor Degenerations: Retinitis Pigmentosa; Primary Photoreceptor Degenerations: Terminology; Rod and Cone Photoreceptor Cells: Outer Segment Membrane Renewal.

Further Reading

Birch, J. (1993). Diagnosis of Defective Colour Vision. New York: Oxford University Press.

Carroll, J., Choi, S. S., and Williams, D. R. (2008). In vivo imaging of the photoreceptor mosaic of a rod monochromat. Vision Research 48: 2564–2568.

Cruz-Coke, R. (1970). Color Blindness: An Evolutionary Approach. Springfield, IL: Charles C Thomas.

Dalton, J. (1798). Extraordinary facts relating to the vision of colours: With observations. Memoirs of the Literary and Philosophical Society of Manchester 5: 28–45.

Gegenfurtner, K. R. and Sharpe, L. T. (1999). Color Vision: From Genes to Perception. New York: Cambridge University Press.

Hess, R. F., Sharpe, L. T., and Nordby, K. (1990). Night Vision: Basic, Clinical, and Applied Aspects. New York: Cambridge University Press.

Mollon, J. D. (2003). The origins of modern color science.

In: Shevell, S. K. (eds.) The Science of Color, 2nd edn., pp. 1–39. New York: Elsevier.

Nathans, J., Piantanida, T. P., Eddy, R. L., Shows, T. B., and Hogness, D. S. (1986). Molecular genetics of inherited variation in human color vision. Science 232(4747): 203–210.

Pokorny, J. (1979). Congenital and Acquired Color Vision Defects. New York: Grune and Stratton.

Sharpe, L. T., Stockman, A., Ja¨gle, H., et al. (1998). Red, green, and red–green hybrid pigments in the human retina: Correlations between deduced protein sequences and psychophysically measured spectral sensitivities. Journal of Neuroscience 18(23): 10053–10069.

Glossary

Gene duplication – Duplication of a region of DNA containing a gene, thought to be one of the most powerful evolutionary mechanisms for diversification.

Filtering pigment – Molecules present in photoreceptor cells that act as spectral filters by absorbing particular wavelengths of light.

lmax – Wavelength of peak spectral absorbance of a visual pigment, measured in nm.

Neofunctionalization – The origin of a new gene function by mutation.

Phylogeny – A description of the evolutionary relationships among species inferred to have descended from a common ancestor. Positive selection – Process through which

advantageous mutations increase in frequency in a population.

Visual pigment – An opsin protein bound to a lightsensitive molecule, the chromophore, which is derived from retinal.

Introduction

Butterflies are some of the most colorful animals on the planet. The diversity of their wing colors and patterns has not only inspired human artistic expression but it has also made these animals prominent study systems in biology. For example, butterflies have become leading model animals for the study of evolution and development, particularly of how wings are patterned. Equally, our understanding of defensive signaling is based largely on butterflies, for example, mimicry in Heliconius species where two butterflies unpalatable to predators resemble one another in color and pattern. Butterflies also play an important ecologic and economic role by pollinating flowers and orchard crops, where they utilize vision among other senses to find sources of nectar. Understanding vision in butterflies therefore gives us a fascinating insight into how these animals might see their surroundings, their own wing colors, and may also inform us on their patterns of habitat use and pollination.

So what do butterflies see? Can they see in color and if so, how do they use color vision? Here, what we currently know about color vision in the butterflies within an

evolutionary context has been reviewed. First, this article provides an overview of the butterfly group and its evolutionary history. Subsequently, a mechanistic account of how the structure of the butterfly eye makes color vision possible is provided, paying particular attention to the molecular basis of the visual pigments that enable this type of vision to occur. Next, is discussed the evolutionary processes that have led to a diversification of the visual systems present in this group that is unparalleled in other insects. Finally, the ecological significance of color vision in the butterflies is explored.

The Butterflies and Their Evolutionary

History

The butterflies (Rhopalocera) are a charismatic group of insects that includes an estimated 15 000 species. This is a recently evolved group within the order Lepidoptera, which includes the moths. The Rhopalocera comprises the true day-flying butterflies (Papilionoidea), the skippers (Hesperioidea), and a newly identified group of nocturnal butterflies (Hedyloidea) (Figure 1). Within the true butterflies (Papilionoidea), which are the subject of most studies, five families are recognized. The families are: the Papilionidae (including the swallowtails and birdwings), the Pieridae (including the whites and sulfurs), the Lycaenidae (the blues and coppers), the Nymphalidae (the brush-footed butterflies, including the famous monarch, the morphos, and frittilaries), and the Riodinidae (the metalmarks) (Figure 1).

The butterflies are thought to have originated on the ancient continent of Gondwana during the Cretaceous period, sometime prior to the extinction of dinosaurs at the Cretaceous–Tertiary (K/T) boundary and possibly concurrent with the radiation of the flowering plants. However, the paucity of fossil butterflies has led to scientific disagreement as to the exact date of when butterflies first appeared, with estimates of their age ranging from 150 to 70 Ma. New molecular approaches are helping resolve some of these controversies. Molecular phylogenies (i.e., reconstructions of the lineages of species using DNA sequence data) have indicated that the Nymphalidae and the Pieridae families were probably present at the K/T boundary, around 65 Ma, and in the case of the Pieriae, may date to 100 Ma. Definitive resolution of the age of the butterflies and timing of divergence of the major families, however, awaits the development of additional molecular markers.

Moths

Hesperiidae

Hedylidae

Riodinidae

Rhopalocera

Lycaenidae

Nymphalidae

Pieridae

Papilionoidea

Papilionidae

Figure 1 Evolutionary relationships of the major lineages in the Rhopalocera.

Color Vision and the Butterfly Eye

Most organisms detect light through the presence of photosensitive molecules located in specialized organs, such as eyes. However, only arthropods (the phylum that includes insects, spiders, and crustaceans) and vertebrates have developed eyes capable of true color vision. Color vision is the ability to discriminate between two visual stimuli based on their wavelength regardless of their relative intensities. Color vision also requires the presence of at least two photoreceptors with overlapping spectral ranges so that the same point in space can be compared.

Despite the observations that butterflies use colored flowers as food sources and that they employ color-based signaling via their wings, it was only relatively recently that color vision was demonstrated in butterflies using behavioral tests. Papilio butterflies were trained to associate rewards containing sucrose with particular colors. When butterflies were behaviorally tested, a majority of animals chose colors previously associated with food rewards among an array of colors regardless of their intensity.

Butterflies, like other insects, have compound eyes that contain thousands of units called ommatidia (Figure 2(a)). In butterflies, each ommatidium contains nine photoreceptor cells (cells R1 to R9) (Figure 2(b)), that is, cells that possess light-sensitive visual pigments that make color vision possible. The cell membranes of the photoreceptors are folded into microvilli (cell membrane projections) that form the rhabdomeres. The rhabdomeres of one

Retina

Lamina

Medulla

(a)

Cornea

Crystalline cone

Distal pigments

Rhabdom

Photoreceptor cell

Cell nuclei

(b)

Figure 2 Schematic diagram of the butterfly compound eye.

(a)Diagram of a longitudinal section through the eye showing the numerous repeated ommatidia comprising the butterfly retina as well as non photoreceptor regions (lamina and medulla).

(b)Diagram of a longitudinal cross-section of an individual

ommatidium. Reproduced from Frentiu, F. D., et al. (2007). ã National Academy of Sciences USA.

ommatidium form a cylinder (the rhabdom) that acts as an optical waveguide for light passing through (Figure 2(b)). Light passes through the lens and is focused by the crystalline cone onto the rhabdom. When light propagates down the rhabdom, the majority of it is absorbed by the visual pigments in the photoreceptor cell membranes. Light that is not absorbed by the visual pigments is reflected back by the tapetum, a structure that sits at the base of the ommatidium and is found in most butterflies except papilionids and the pierid genus Anthocharis. In addition to visual pigments, butterflies also possess filtering pigments that surround the rhabdom and act as spectral filters by absorbing wavelengths of light and shifting the peak spectral sensitivity of their photoreceptors to longer wavelengths.

Key to color vision in both arthropods and vertebrates are the visual pigments and, most importantly, the presence of at least two spectrally distinct types of visual

pigments. Neural inputs from at least two types of photoreceptor cells that bear different visual pigments are required for color discrimination. A visual pigment comprises of an opsin protein bound to a light-sensitive molecule, the chromophore (Figure 3) that, in butterflies, is 11- cis-3-hydroxyretinal. Photons reaching the chromophore cause its photoisomerization and induce a conformational change in the opsin protein. In turn this activates a guanine nucleotide-binding protein (G-protein) that initiates the phototransduction cascade that converts light into signals to the brain through a series of biochemical reactions. The eyes of invertebrates employ fundamentally different phototransduction cascades than those of vertebrates.

The absorbance spectrum of a visual pigment depends on the interaction of the chromophore with critical amino acids in the opsin protein. By itself, the chromophore has a wavelength of maximum absorption (the lmax value) in the ultraviolet (UV) part of the light spectrum at approximately 380 nm. However, through the chromophore interacting with key amino acids in the binding pocket of the opsin protein, a diversity of visual pigment lmax values can be achieved, a phenomenon called spectral tuning. The sensitivities of different photoreceptors to light (Figure 4) are determined by the opsins that they express and any associated filtering pigments they may contain. The visual pigments of butterfly species sampled to date range in lmax from 340 to 600 nm (Table 1), although the exact opsin amino acids involved in producing this diversity remain to be elucidated.

The opsins that form the basis of visual pigments are ancient molecules that belong to the large G-protein- coupled receptor family. They have a seven transmembrane domain structure, with a diagnostic lysine residue in the seventh helix that binds to the chromophore. The opsin family predates the emergence of the major groups of animals present today. Phylogenetic reconstructions

Chromophore

Figure 3 Three-dimensional model of a visual pigment comprising a seven-transmembrane opsin protein and a chromophore (indicated by the arrow). The seven transmembrane domains are shown in different colors. Reproduced from Frentiu, F. D., et al. (2007). ã National Academy of Sciences USA.

suggest that the ancestral arthropod may have been able to see in color, including in the ultraviolet. Insect ultraviolet/blue (UV/B) and blue-green opsins originated early in arthropod evolution. Distinct insect UV and B opsins then evolved via duplication of the ancestral UV/B opsin and the long wavelength (LW) opsin evolved from a duplication of the ancestral blue-green opsin. The bluegreen opsin was however lost in most insects, with the exception of some flies (e.g., Drosophila). Insects such as bees, butterflies, and moths now possess at least three classes of visual pigment in their photoreceptors that enable them to see in the UV (UV, 300–400 nm), blue (B, 400–500 nm), and long wavelength (LW, 500–600 nm) parts of the light spectrum. However, compared to moths and bees, butterflies display an unusual diversification of their visual systems. In the following, I explore the diversification of butterfly eyes and the evolutionary processes that have produced it.

Spectral Heterogeneity of Butterfly Eyes

Although ommatidia are anatomically identical in structure, they are spectrally very heterogeneous: that is, they hold different complements of photoreceptors that express different visual pigments; different photoreceptors within an ommatidium are designated as R1-R9 and may express different pigments. Using molecular genetic techniques such as in situ hybridization to visualize patterns of opsin mRNA has allowed us to map the types of ommatidia present in the butterfly eye. Three types of ommatidia exist in the main retinas of butterflies, with all types of ommatidia expressing LW visual pigments but differing in the expression of the UV and B visual pigments.

The extent of spectral heterogeneity of butterfly eyes differs among the major butterfly families. For example,

1 l max = 340 nm l max = 435 nm l max = 545 nm

Wavelength (nm)

Figure 4 Normalized absorbance spectra of visual pigments in the eye of the monarch, Danaus plexippus. Wavelengths of peak absorbance (lmax) for the three visual pigments are estimated to be 340, 435, and 545 nm. Peak absorbance values from Stalleicken et al. (2006) Journal of Comparative Physiology A 192: 321–331.

the eyes of Nymphalidae species are quite simple in terms of ommatidial heterogeneity. Studies of Nymphalidae species to date show that they have only three types of ommatidia in the main retina and one type in the dorsal rim area (DRA) of the eye, which is an eye region specialized for the detection of polarized skylight. All ommatidial types in the main retina express long wavelength visual pigments in their R3-R8 photoreceptor cells, but the expression of the UVand B opsins in the R1 and R2 cells is variable (the R9 cell expresses the LW opsin in the main retina and may express the UV opsin in the DRA). One ommatidial type contains one UV and one blue receptor, the second has two blue receptors, and the third has two UV receptors. In the DRA ommatidia, the R1-R8 cells express the UV opsin. This type of eye employs a straightforward, one-to-one relationship between the type of visual pigment expressed and spectral phenotype of the photoreceptor cell. It may also best represent the ancestral butterfly eye and it resembles the eyes of bees and moths.

By contrast, the eyes of other families of butterflies are much more diverse in their spectral complements.

For example, the butterfly Pieris rapae (family Pieridae) expresses four opsins but photoreceptors with seven different peak sensitivities have been identified, due to the presence of filtering pigments. Perhaps the most spectrally diverse butterfly eye studied so far belongs to the papilionid butterfly, the Japanese swallowtail,

Papilio xuthus. Papilio xuthus expresses five different opsins in the eye, has eight different types of photoreceptors, and employs tetrachromatic color vision. In these animals, both opsin gene duplications and pigments acting as spectral filters have led to spectral diversification of visual systems. Below, I consider the evolutionary mechanisms that have led to butterfly visual system diversity.

Diversification of Visual Pigments via Gene Duplication and Positive Selection

The eyes of some butterflies express a larger number of visual pigments than the three UV, B, and LW known to

be present in bees and moths. The diversity of visual pigments is primarily due to opsin gene duplications that have occurred independently in different butterfly families (Figure 5). Duplications of the B opsin gene have occurred independently in two of the five butterfly families: the Pieridae and the Lycanenidae, giving rise to four different visual pigments. In the Pieridae, as exemplified by the well-studied Pieris rapae (the cabbage white), duplicate B opsins have diversified into violet- (lmax ¼ 425 nm) and blue-absorbing (lmax ¼ 453 nm) visual pigments. In the Lycaenidae, duplication of the B opsin has also facilitated the emergence of two visual pigments. However, the peak wavelength sensitivities in species such as Polyommatus icarus are different from those found in the Pieridae, with one visual pigment absorbing in the blue (lmax ¼ 437 nm) and the other in the bluegreen (lmax ¼ 500 nm).

Duplications of the LW opsin gene have occurred independently in three butterfly families, also leading to diversification of visual pigments. In the Papilionidae, three LW opsins are now expressed in the eye, each the result of a round of gene duplication. In Papilio xuthus, these duplicated opsins now encode three different visual pigments with lmax values ranging from 515 to 575 nm. The LW opsin gene duplications have also occurred in two species of butterflies in the Nymphalidae and one in the Riodinidae families. In total, of more than 50 butterfly species studied to date, 7 LW opsin duplicates have been identified. The most red-shifted visual pigment known to date in the riodinid butterfly Apodemia mormo, with a lmax of 600 nm, has resulted from a gene duplication specific to the family Riodinidae. Molecular evidence has also

Butterfly ancestor

Figure 5 Evolution of visual pigment diversity in the butterflies and hypothesized complement of pigments in the ancestral butterfly eye. Circles indicate UV (gray), blue (blue), and long wavelength (orange) visual pigments. Diagonal lines along the branches of the phylogeny denote opsin duplications.

suggested that duplication was followed by elevated rates of amino acid evolution in one of the LW duplicates. However, not all opsin duplications have resulted in visual pigments expressed in the eye, with some expressed in the optic lobes and brain.

In addition to gene duplication, a mechanism that generates spectral diversity in butterflies is positive selection on single opsin genes, whereby novel mutations that enhance an organism’s fitness spread through a population and may replace other genetic variants. The most striking example of this process to date comes from the North American butterfly genus Limenitis. Butterflies in this group have radiated across the North American continent from a European ancestor during the past 3–4 My. The genus is best known for species that display wing color pattern mimicry such as the viceroy Limenitis archippus, which mimics the monarch

Danaus plexippus, and L. arthemis astyanax, which mimics the more toxic pipevine swallowtail Battus philenor. An unusual diversity in lmax values of the LW visual pigment has been found in this group of butterflies, suggesting that their visual systems had diversified in tandem with wing color patterns (Figure 6). Reconstruction of phylogenetic relationships within the genus indicated that a shift in spectral sensitivity towards the blue part of the light spectrum had occurred (Figure 6). Wavelength sensitivities of LW visual pigments had diversified from an ancestral lmax of 545 nm in

L. arthemis astyanax to a lmax of 515 nm in L. archippus archippus.

Using several molecular evolutionary analyses, the signature of positive selection was found at several amino acid sites in Limenitis LW opsins. The Limenitis opsin protein was modeled against the bovine rhodopsin crystal structure (homology modeling) in order to visualize where the amino acid sites were located. The results indicated that some of the amino acids found to be under positive selection were located in the chro- mophore-binding pocket, strongly suggesting that they interact with the chromophore to determine spectral sensitivities. The same amino acids were found to change in parallel in butterfly species that were distantly evolutionarily related to the Limenitis genus but that also showed a shift in lmax in the same direction (Figure 6). Interestingly, one of the amino acid sites under positive selection in butterflies is evolutionarily homologous to an amino acid site in the cone opsin of humans that is responsible for a 5–7 nm shift to the blue part of the light spectrum and which is under balancing selection in New World monkeys. These findings suggest a common molecular basis for spectral shifts in insects and vertebrates, a feature that has been retained across more than 540 My of evolution. However, opsin amino acid sites suggested by molecular evolutionary analyses to be involved in the spectral tuning of butterfly visual pigments need to be tested and functionally characterized experimentally.

Diversification of Photoreceptor Types via Filtering Pigments

Some butterflies possess photoreceptor cells containing filtering pigments that coat the rhabdom (Figure 1), which are red, orange, and yellow in color. Filtering pigments act as spectral filters by absorbing particular wavelengths of light traveling down the rhabdom. Whilst the breadth of wavelength sensitivity of a photoreceptor is determined by the opsin it expresses, by absorbing shorter wavelengths of light, filtering pigments shift the lmax of some photoreceptors towards longer wavelengths. The color discrimination abilities of butterflies in a particular wavelength range are thus enhanced by comparing neural inputs from two types of photoreceptors that express the same opsin but have differing peak spectral sensitivities (lmax) due to the presence or absence of filtering pigments.

Filtering pigments play a role in the diversification of the peak spectral sensitivity of photoreceptors in the ommatidia

of some butterflies. The same visual pigment, in conjunction with filtering pigments, is used to expand photoreceptor sensitivities in Heliconius butterflies. For example, in Heliconius erato, two types of LW photoreceptors express the same LW visual pigment but differ in peak spectral sensitivity as a result of the presence of a red-filtering pigment. This expanded wavelength discrimination in the LW range is used in foraging behavior. In Pieris rapae, filtering pigments expressed in conjunction with the same LWopsin are used to produce two additional photoreceptor types that enhance wavelength discrimination in the red part of the light spectrum, although the behavioral and ecological reasons for this pattern are unclear.

Currently very little is known about the genetic basis, molecular identity, evolution, and ecological significance of butterfly filtering pigments. Both Pieris and Papilio butterflies have lateral filtering pigments suggesting that filtering pigments are an evolutionarily old feature of the butterfly eye. Some of the pigments used in butterfly wing