Molecular and physiological innovations of butterfly eyes. In: Molecular Biology and Genetics of the Lepidoptera, eds. M. Goldsmith and F. Marec, CRC Press Inc., Bocaraton, Florida.

Color vision is a complex trait that can impact the survivorship of short-lived insects like the Lepidoptera. Within this order, the color vision systems are diverse and are best known among butterflies, which are classified into five families. Several recent reviews have focused on the eyes of
the basal papilionid (i.e., Papilio xuthus) and pierid butterfly (i.e., Pieris rapae) lineages (Stavenga and Arikawa 2006; Wakakuwa, Stavenga, and Arikawa 2007). Both of these groups have eyes that differ from each other and from the other butterfly families in terms of the copy number of the
opsin genes that encoded the visual pigments, their spatial expression pattern, and the distribution of lateral filtering pigments. Only one study to date has examined the visual pigments in a riodinid butterfly, Apodemia mormo (Frentiu et al. 2007). This chapter focuses on recent advances in our understanding of the unique visual system of lycaenid butterflies, with a special emphasis on the sexually dimorphic retina of Lycaena rubidus (Lycaeninae) and the color vision behavior of Polyommatus icarus (Polyommatinae). It is clear from character mapping of opsin genes and their expression patterns on a phylogeny of butterfly families that all butterfly eyes are derived from a much simpler eye that resembles the nymphalid eye (Briscoe 2008). Hence, to put the innovations
of the lycaenid butterfly visual system into an evolutionary framework, we begin by describing the much simpler visual system of nymphalid butterflies. We then trace the molecular changes in the opsin genes and their expression patterns, and the physiological changes in the visual receptors they
encode. Lastly, we discuss the potential behavioral outcomes of the unique eye design of lycaenids. In the course of the review, we mentioned some fertile areas of interest for future study.

Impact of duplicate gene copies on phylogenetic analysis and divergence time estimates in butterflies. BMC Evolutionary Biology journal. 9:99.

BACKGROUND: The increase in availability of genomic sequences for a wide range of organisms has revealed gene duplication to be a relatively common event. Encounters with duplicate gene copies have consequently become almost inevitable in the context of collecting gene sequences for inferring species trees. Here we examine the effect of incorporating duplicate gene copies evolving at different rates on tree reconstruction and time estimation of recent and deep divergences in butterflies. RESULTS: Sequences from ultraviolet-sensitive (UVRh), blue-sensitive (BRh), and long-wavelength sensitive (LWRh) opsins,EF-1 and COI were obtained from 27 taxa representing the five major butterfly families (5535 bp total). Both BRh and LWRh are present in multiple copies in some butterfly lineages and the different copies evolve at different rates. Regardless of the phylogenetic reconstruction method used, we found that analyses of combined data sets using either slower or faster evolving copies of duplicate genes resulted in a single topology in agreement with our current understanding of butterfly family relationships based on morphology and molecules. Interestingly, individual analyses of BRh and LWRh sequences also recovered these family-level relationships. Two different relaxed clock methods resulted in similar divergence time estimates at the shallower nodes in the tree, regardless of whether faster or slower evolving copies were used, with larger discrepancies observed at deeper nodes in the phylogeny. The time of divergence between the monarch butterfly Danaus plexippus and the queen D. gilippus (15.3-35.6 Mya) was found to be much older than the time of divergence between monarch co-mimic Limenitis archippus and red-spotted purple L. arthemis (4.7-13.6 Mya), and overlapping with the time of divergence of the co-mimetic passionflower butterflies Heliconius erato and H. melpomene (13.5-26.1 Mya). Our family-level results are congruent with recent estimates found in the literature and indicate an age of 84-113 million years for the divergence of all butterfly families. CONCLUSION: These results are consistent with diversification of the butterfly families following the radiation of angiosperms and suggest that some classes of opsin genes may be usefully employed for both phylogenetic reconstruction and divergence time estimation.

The lycaenid butterfly Polyommatus icarus uses a duplicated blue opsin to see green. Journal of Experimental Biology. 211. 361-369

The functional significance of gene duplication is rarely addressed at the level of animal behavior. Butterflies are excellent models in this regard because they can be trained and the use of their opsin-based visual pigments in color vision can be assessed. In the present study, we demonstrate that the lycaenid Polyommatus icarus uses its duplicate blue (B2) opsin, BRh2, in conjunction with its long-wavelength (LW) opsin, LWRh, to see color in the green part of the light spectrum extending up to 560 nm. This is in contrast to butterflies in the genus Papilio, which use duplicate LW opsins to discriminate colors in the long-wavelength range. We also found that P. icarus has a heterogeneously expressed red filtering pigment and red-reflecting ommatidia in the ventral eye region. In behavioural tests, the butterflies could not discriminate colors in the red range (570-640 nm). This finding is significant because we have previously found that the nymphalid butterfly Heliconius erato has filter-pigment mediated color vision in the long wavelength range. Our results suggest that lateral filtering pigments may not always influence color vision in insects.

Adaptive Evolution of Color Vision as Seen Through the Eyes of Butterflies. Proceedings of the National Academy of Sciences. 104: 8634-8640

Butterflies and primates are interesting for comparative color vision studies, because both have evolved middle- (M) and long-wavelength- (L) sensitive photopigments with overlapping absorbance spectrum maxima (lambda(max) values). Although positive selection is important for the maintenance of spectral variation within the primate pigments, it remains an open question whether it contributes similarly to the diversification of butterfly pigments. To examine this issue, we performed epimicrospectrophotometry on the eyes of five Limenitis butterfly species and found a 31-nm range of variation in the lambda(max) values of the L-sensitive photopigments (514-545 nm). We cloned partial Limenitis L opsin gene sequences and found a significant excess of replacement substitutions relative to polymorphisms among species. Mapping of these L photopigment lambda max values onto a phylogeny revealed two instances within Lepidoptera of convergently evolved L photopigment lineages whose lambda(max) values were blue-shifted. A codon-based maximum-likelihood analysis indicated that, associated with the two blue spectral shifts, four amino acid sites (Ile17Met, Ala64Ser, Asn70Ser, and Ser137Ala) have evolved substitutions in parallel and exhibit significant d(N)/d(S) > 1. Homology modeling of the full-length Limenitis arthemis astyanax L opsin placed all four substitutions within the chromophore-binding pocket. Strikingly, the Ser137Ala substitution is in the same position as a site that in primates is responsible for a 5- to 7-nm blue spectral shift our data.show that some of the same amino acid sites are under positive selection in the photopigments of both butterflies and primates, spanning an evolutionary distance > 500 million years.

Gene duplication is an evolutionary mechanism for expanding spectral diversity in the long‐wavelength photopigments of butterflies. Molecular Biology and Evolution. 24(9):1–13.

Butterfly long-wavelength (L) photopigments are interesting for comparative studies of adaptive evolution because of the tremendous phenotypic variation that exists in their wavelength of peak absorbance (lambda(max) value). Here we present a comprehensive survey of L photopigment variation by measuring lambda(max) in 12 nymphalid and I riodinid species using epi-microspectrophotometry. Together with previous data, we find that L photopigment lambda(max) varies from 510-565 nm in 22 nymphalids, with an even broader 505- to 600-nm range in riodinids. We then surveyed the L opsin genes for which lambda(max) values are available as well as from related taxa and found 2 instances of L opsin gene duplication within nymphalids, in Hermeuptychia hermes and Amathusia Phidippus, and 1 instance within riodinids, in the metalmark butterfly Apodemia mormo. Using maximum parsimony and maximum likelihood ancestral state reconstructions to map the evolution of spectral shifts within the L photopigments of nymphalids, we estimate the ancestral pigment had a lambda(max) = 540 nm +/- 10 nm standard error and that blueshifts in wavelength have occurred at least 4 times within the family. We used ancestral state reconstructions to investigate the importance of several amino acid substitutions (IIe17Met, Ala64Ser, Asn70Ser, and Ser137Ala) previously shown to have evolved under positive selection that are correlated with blue spectral shifts. These reconstructions suggest that the Ala64Ser substitution has indeed occurred along the newly identified blueshifted L photopigment lineages. Substitutions at the other 3 sites may also be involved in the functional diversification of L photopigments. Our data strongly suggest that there are limits to the evolution of L photopigment spectral shifts among species with only one L opsin gene and that opsin gene duplication broadens the potential range of lambda(max) values.

Beauty in the eye of the beholder: The two blue opsins of blue butterflies and the opsin gene-driven evolution of sexually dimorphic eyes. Journal of Experimental Biology. 209 (16): 3079-3090

Although previous investigations have shown that wing coloration is an important component of social signaling in butterflies, the contribution of opsin evolution to sexual wing color dichromatism and interspecific divergence remains largely unexplored. Here we report that the butterfly Lycaena rubidus has evolved sexually dimorphic eyes due to changes in the regulation of opsin expression patterns to match the contrasting life histories of males and females. The L. rubidus eye contains four visual pigments with peak sensitivities in the ultraviolet (UV; lambda(max)= 360 nm), blue (B; lambda(max)= 437 nm and 500 nm, respectively) and long (LW; lambda(max)= 568 nm) wavelength range. By combining in situ hybridization of cloned opsin-encoding 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 (lambda(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 (lambda(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.

Color discrimination in the red range with only one long-wavelength sensitive opsin. Journal of Experimental Biology. 209 (10): 1944-1955.

The basic precondition for color vision is the presence of at least two receptor types with different spectral sensitivities. The sensitivity of a receptor is mostly defined by the opsin-based visual pigment expressed in it. We show here, through behavioral experiments, that the nymphalid butterfly Heliconius erato, although it expresses short and medium wavelength opsins and only one long wavelength opsin, discriminates colors in the long-wavelength range (590 nm, 620 nm and 640 nm), whereas another nymphalid, Vanessa atalanta, despite having color vision, is unable to do so. In the eyes of H. erato we identified filtering pigments very close to the rhabdom which differ between ommatidia and produce the yellow and red ommatidial reflection seen under orthodromic illumination. The eyes of V. atalanta lack the filtering pigments, and reflect a homogeneous orange. We hypothesize that the filtering pigments found in the eyes of H. erato may shift the spectral sensitivity peak of the long wavelength receptors in some ommatidia towards longer wavelengths. The comparison of the signals between the two new receptor types makes color discrimination in the red range possible. To our knowledge, this is the first behavioral proof of color vision based on receptors expressing the same opsin.

A comparison between sites of growth, physiological performance and stress responses in transplanted Tridacna gigas. Aquaculture. 219: 815–828

The continuous increase of human activities in the tropical coastal zones threatens the water quality necessary for cultivating giant clams. In order to investigate the potential for disturbances, of natural and/or anthropogenic origin, to affect growth in giant clams, transplantation experiments were conducted in the north of the Philippines. After 6 months, there were significantly lower values for both wet weights (wwt) and shell lengths (SL) at the "disturbed sites", where the clams had experienced both natural turbidity and impacts from human settlement and fish pens, compared to the reference site. Differences between sites were also manifested as impaired physiological performance under identical light regimes and water quality in a laboratory experiment: gross production and respiration (P-g/R) ratios were lower, Rapid Light' Curves (RLC) showed lower maximal production at light saturation, chlorophyll (chl) a levels per cell was higher and the degree of epibionts increased with over 90% compared to controls. The suggested explanation to this is reduced light penetration as a result of both natural higher turbidity and nutrient loading brought about by, e.g. intensive fish aquaculture in the channel. Light penetration will be further reduced in the future with present development of fish pens in the channel. To assess tolerance to additional anthropogenic impacts, such as release of copper from antifouling treatment of fish cages, clams from different sites were exposed to a sublethal dose of copper (5 mug Cu2+ l(-1)) under laboratory conditions. Clams from all sites showed lowered P-g/R ratios when exposed to the metal, although the control clams displayed a larger percentage decrease than clams from the channel. This could be interpreted as higher tolerances in those clams already adapted to a stressful environment, or merely reflect an overall energy conservation through reduced metabolic activity. (C) 2003 Elsevier Science B.V. All rights reserved.

Evidence for changing symbiotic algae in juvenile tridacnids. Journal of Experimental Marine Biology and Ecology 241:207-221

This study investigated the effects of different clonal strains of Symbiodinium sp. (zooxanthellae) on clam growth and survival, while monitoring the persistence of the induced symbioses in outdoor tanks and in the held using allozyme and random amplified polymorphic DNA (RAPD) analyses. Aposymbiotic clam larvae that were inoculated with homologous zooxanthellae (cultured or freshly isolated from the same host species) or heterologous zooxanthellae (cultured from different host species) had significantly-different survival rates at harvest (3 months post-spawning) with small growth differences. The improved survival rates in juvenile Hippopus hippopus (heterologous infection) and Tridacna gigas (homologous infection) were maintained through 3 months onshore and 3 months offshore. However, isozyme and RAPD analyses of re-isolated zooxanthellae after 3, 6, and 9 months revealed a high genetic diversity of symbionts (ca. 99% variation in 200 re-isolates) from individual hosts, within and between treatments. Furthermore, the genetic patterns of the re-isolated algae following clonal culture were different from those of the introduced clones, which, in contrast, retained their unique genetic patterns over many culture generations in the laboratory. These results demonstrate that the subsequently-established symbiont populations in juvenile clams were not clonal. The allozyme and RAPD techniques detected individual genetic differences in clam symbionts, but not differences between algal taxa. The presence of significant survival trends suggests possible differences between subsequently-established dominant symbiont taxa, which were uncultureable or undetectable using these genetic markers. The implications of this symbiont diversity in giant clams are discussed. (C) 1999 Elsevier Science B.V. All rights reserved.

Genetic variation in Symbiodinium isolates from giant clams based on random-amplified-polymorphic DNA (RAPD) patterns. Marine Biology. 136:829- 836

We have compared the random-amplified-polymorphic DNA (RAPD) patterns of Symbiodinium isolates from seven species of giant clams to investigate the large genetic variation that we previously reported for this group of dinoflagellate symbionts using allozyme analysis. Comparisons of 163 RAPD characters by unweighted pair-group arithmetic-average cluster analysis (UPGMA) corroborate our previous findings that giant clams associate with a large number of genetically distinguishable algal symbionts, and that the isolates from a single Tridacna gigas individual form a group of closely related algae. However, the overall topology of the UPGMA tree constructed from RAPD data differs From that of the previous allozyme data, indicating that the combined data we have collected to date are insufficient to accurately infer phylogenetic affiliations between the isolates studied. Comparisons of our data set with those published for strains of Gymnodinium catenatum, a toxic dinoflagellate with a sexual life stage, shows that our isolates are even more diverse. Algal isolates from giant clams have a level of RAPD variation comparable to organisms that are able to undergo sexual recombination. This study demonstrates the sensitivity of the RAPD technique in detecting genetic diversity in this group of algae, and highlights the need for more comparative data for the major clades of Symbiodinium.

Allozyme electrophoresis as a tool for distinguishing different zooxanthellae symbiotic with giant clams. Proceedings of the Royal Society of London B. 265:1949-1956

http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1689488&blobtype=pdf

The taxonomy of zooxanthellae in marine invertebrate symbioses is not well understood owing mainly to their lack of reliable morphological differences. Nevertheless, previous work using protein and DNA electrophoreses has set the stage for advancing our taxonomic understanding of cnidarian zooxanthellae. Here we present the use of allozymes as genetic markers for distinguishing algal isolates from tridacnid hosts. Zooxanthellae from seven Tridacna and Hippopus species were isolated and maintained in axenic clonal cultures over many generations. Of 16 enzyme systems, alpha- and beta-esterase (EST), esterase-F (EST-F), glucose phosphate isomerase (GPI), and malate dehydrogenase (MDH) were found suitable polymorphic markers of genetic differences among clonal cultures. Of 39 clonal isolates, 97% were found to be genetically distinguishable. This high extent of genetic variation in zooxanthellae within and between clam species was unexpected, and is difficult to explain based solely on the general notion of asexual reproduction in symbiotic zooxanthellae. Our results are also consistent with the occurrence of sexual reproduction in clam zooxanthellae. The close genetic similarity of the symbionts of Tridacna gigas, the largest and fastest-growing clam species, and the difficulty of initiating their clonal cultures in the given nutrient medium, compared with the symbionts of other clam species, are further indicative of possibly distinct alg al symbionts in T. gigas. These findings are discussed in light of current taxonomic understanding of these organisms.