Reflection confocal laser scanning microscopy of eyespots in flagellated green algae

The reflection properties of different types of eyespots in three unicellular, flagellated green algae (Tetraselmis chui, Chlamydomonas eugametos, Hafniomonas reticulata) were investigated using confocal laser scanning microscopy in the epireflection mode. The eyespots differed with respect to the number of eyespot lipid globule layers and surface appearance (concave/convex). A strong reflection signal was observed in all eyespots, and a detailed quantitative analysis by optical xy (horizontal) and xz (vertical) sectioning was performed. By applying both sectioning capabilities, multi- and single/double-layered eyespots as well as concave and convex eyespot surfaces could be distinguished using living, immobilized cells. Focusing of the reflected light was only observed in eyespots with concave surfaces. In xz series of multi-layered eyespots at reduced laser intensities (0.01%), the intensity profiles of the reflection revealed a series of alternating maxima and minima with increasing reflection intensities toward the cell surface. At very low laser intensities (0.001%), multi-layered eyespots exhibited about twice the reflection intensity at the presumptive photoreceptor site compared to single/double-layered eyespots. Our results provide the first experimental evidence to support the proposal that multi-layered eyespots act as interference reflectors in photoaxis of green algae.     

The functional and phylogenetic significance of dinoflagellate eyespots

The eyespots or stigmas from five species of dinoflagellates fall into three distinct categories: independent eyespot which is not membrane bound; independent eyespot which is surrounded by three membranes; eyespot situated at the periphery of a chloroplast. In all cases the eyespot is situated behind the longitudinal groove or sulcus and there is a strand of microtubules between the eyespot and the cell covering or theca. In two cases the strand has been clearly shown to originate near the base of the longitudinal flagellum, which is the one passing over the eyespot and is also responsible for directional movement of the cell. The microtubular strand is presumed to play a part in the transmission of directional stimulation from eyespot to flagellum and a hypothesis is advanced to explain how this may be brought about. Phylogenetically, the structure of the various types of eyespots would link these dinoflagellates with euglenids and chrysophytes , and the diversity found in the dinoflagellates is probably a reflection of the diverse origin of chloroplasts in this group.     

Functional analysis of the eyespot in Chlamydomonas reinhardtii mutant ey 627, mt −

The function of the eyespot in phototaxis of the flagellate green alga Chlamydomonas reinhardtii Dangeard was studied using quantitative reflection confocal laser scanning microscopy and photoelectric measurements. The reflective properties of the eyespot and the photoreceptor current of the C. reinhardtii eyespot mutant ey 627, mt ^– were compared with those of Chlamydomonas strains possessing a well-developed eyespot. Under growth conditions in which strongly disorganized eyespots were observed in the mutant by electron microscopy, there was a significant reduction in the reflection intensity of the eyespot and in the amplitude ratio (500440 nm) of photoreceptor currents induced by flashes of 500- and 440-nm light in non-oriented cells. Photoelectrical responses of pre-oriented cells revealed that the latter effect is caused by an altered directional sensitivity of the antenna complex, whereas the functional state of the photoreceptor pigment is not strongly affected in mutant cells. Both the reflection intensity and the amplitude ratio of photoreceptor currents increased to the level of reference strains under conditions supporting the development of a well-organized eyespot in the mutant. Furthermore, incubation of the mutant with high concentrations of all-trans-retinal (10 M), independent of whether carotenoid biosynthesis was inhibited or not, was found to increase the reflection intensity of the eyespot. An increase in the rate of photoorientation of the mutant occurred concomitant with the increase in the reflective properties of the mutant eyespot. These observations demonstrate the importance of an intact eyespot for interference reflection and absorption of phototactically active light, and thus for the directional sensitivity of the eyespot apparatus.Abbreviations HSM high-salt medium This study was supported by the Deutsche Forschungsgemeinschaft. O. A. Sineshchekov was supported by a Research Fellowship from the Alexander von Humboldt Foundation. The authors wish to thank U. Powalowski (Botanisches Institut, Universität zu Köln) for help with electron microscopy.     

REFLECTIVE PROPERTIES OF THE STIGMA IN MALE GAMETES OF ECTOCARPUS SILICULOSUS (PHAEOPHYCEAE) STUDIED BY CONFOCAL LASER SCANNING MICROSCOPY1

The reflection properties of the stigma in male gametes of Ectocarpus siliculosus (Dillw.) Lyngbye were investigated using confocal laser scanning microscopy in the epireflection contrast mode. The complex reflection pattern obtained after optical xy (horizontal) and xz (vertical) sectioning was consistent with stigma ultrastructure as revealed by serial thin sections. The intensity and pattern of the reflection signal varied with the orientation of the cell/stigma to the incident laser light. Maximal reflection occurred only in approximately normal orientation of the stigma to the light source. Focusing of reflected light from an elongated concave depression of the stigma on the region of the flagellar swelling was observed in xy and xz sections of living and fixed gametes. The results indicate the importance of mechanisms (focusing) other than quarter-wave interference reflection in signal amplification by the eyespot of flagellate algae.     

THE CYTOSKELETON OF THE NAKED GREEN FLAGELLATE SPERMATOZOPSIS SIMILIS (CHLOROPHYTA):FLAGELLAR AND BASAL BODY DEVELOPMENTAL CYCLE1

Flagellar and basal body development during cell division was studied in the biflagellate green alga Spermatozopsis similis Preisig et Melkonian by light microscopy of immobilized living cells, statistical analysis of flagellar lengths during the cell cycle, and electron microscopy of cells and isolated cytoskeletons. Interphase cells display two flagella of unequal/subequal length. An eyespot located in an anterior lobe of the chloroplast is connected to the basal body bearing the shorter flagellum by means of a five-stranded microtubular root. Until cell division, the two parental flagella attain the same length. During cell division, each cell forms two new flagella that grow to a length of 1.5 μm before they are distributed in a semiconservative fashion together with the parental flagella to the two progeny cells at cytokinesis. During the following interphase, the flagella newly formed during the preceding cell division grow to attain the same length as the parental flagella until the subsequent cell division. The shorter of the two flagella of a cell thus represents the developmentally younger flagellum, which transforms to the mature state during two consecutive cell cycles. Interphase cells display only two flagella-bearing basal bodies; two nascent basal bodies are formed during cell division and are connected to the microtubular d-roots of respective parental basal bodies with which the newly formed basal bodies are later distributed to the progeny cells. During segregation, basal body pairs shaft into the 11/5 o'clock direction, thus conserving the 1/7 o'clock configuration of basal body pairs of interphase cells. Prior to chloroplast and cell division, an eyespot is newly formed near the cell posterior in close association with a 1s microtubular root, while the parental eyespot is retained. During basal body segregation, eyespot-root connections for both the old and newly formed eyespots are presumably lost, and new associations of the eyespots with the 2s roots of the newly formed basal bodies are established during cytokinesis. The significance of this “eyespot-flagellar root developmental cycle” for the absolute orientation of the progeny cells is discussed.     

Eyespot behavior during the fertilization of gametes in Ulva arasakii Chihara (Ulvophyceae, Chlorophyta)

Behavior of the eyespots during the fertilization of Ulva arasakii Chihara was studied using field emission scanning electron microscopy (FE‐SEM). FE‐SEM enabled the visualization of the eyespot of biflagellate male and female gametes. The smaller male gamete has one protruded smaller (1.3 ± 0.15 μm× 1.0 ± 0.29 μm) eyespot and the larger female gamete has a larger (1.6 ± 0.2 μm× 1.1 ± 0.13 μm) one on a posterior position of the cell. The cell membrane over the eyespot region is relatively smooth compared to other parts of the cell body and exhibits hexagonal arranged lipid globules. Because the size of the cell and the morphology of the eyespot are different between male and female gametes, we could follow the fate of the eyespots during the fertilization. The initial cytoplasmic contact and fusion of the gametes takes place at their anterior end, slightly posterior to the flagellar base. The morphology of the fusing gametes followed two clearly distinguishable patterns. About half the gamete pairs lie side‐by‐side with their longitudinal axes nearly parallel, while the rest are oriented anti‐parallel to each other. In all cases, the larger female gamete fused along the same side as the eyespot, while the smaller male gamete fused along the side away from its eyespot. As fusion proceeds, the gamete pair is transformed into the quadriflagellate planozygote, in which the eyespots are positioned side‐by‐side on the region of cell fusion. These observations indicated that the opposite positioning of the eyespot relative to the cell fusion site in male and female gametes is important for the proper arrangement of the eyespots in the planozygote. The significance of this feature in advanced green algae is briefly discussed.     

Concerted evolution and developmental integration in modular butterfly wing patterns

Developing organisms are thought to be modular in organization so that traits in different modules evolve independently whereas traits within a module change in a concerted manner. The eyespot pattern in Bicyclus anynana butterflies provides an ideal system where morphological modularity can be dissected and different levels of genetic integration analyzed. Several lines of evidence show that all eyespots in an individual butterfly are genetically integrated, suggesting that the whole pattern, rather than the separate eyespots, should be considered as a single character. However, despite the strong genetic correlations between the two eyespots on the dorsal forewing of B. anynana, there is great potential for independent changes. Here we use laboratory lines selected in different directions for the size of those eyespots to study correlated responses in the whole eyespot pattern. We show clear changes in eyespot size across all wing surfaces, which depend on eyespot position along the anterior-posterior axis. There are also changes in the number of extra eyespots and in eyespot color composition but no changes in eyespot position relative to wing margin. Our analysis of eyespot pattern modularity is discussed in the light of what is known about the cellular and genetic mechanisms of eyespot formation and the great potential for evolutionary diversification in butterfly wing patterns.     

Eyespot-assembly mutants in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca(++)-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell.     

Visible sympathetic activity as a social signal in Anolis carolinensis: changes in aggression and plasma catecholamines

Darkening of postorbital skin in Anolis carolinensis occurs during stressful situations and is stimulated by sympathetic activation of beta(2)-adrenergic receptors via adrenal catecholamines. This eyespot forms more rapidly in dominant males during social interaction. Eyespot darkening (green to black) appears to function as a social signal communicating sympathetic activation and limiting aggressive interaction. To assess the value of the eyespot as a social signal, males were painted postorbitally with green, black, or red paint. Each male was exposed to a mirror following acclimation to the cage. The total number of aggressive displays toward the mirror image was greatest when eyespots were masked by green paint. In contrast, black or red artificial eyespots, regardless of size, inhibited biting behavior toward the mirror image. The most aggressive males, those who saw a reflected opponent with no eyespot (hidden with green paint), had significantly higher levels of all plasma catecholamines. These results suggest that A. carolinensis use information from the eyespot to assess their opponent's readiness to fight and thereby determine whether to be aggressive. Darkened eyespots are capable of inhibiting aggression, whereas aggressive displays from an opponent in the mirror without darkened eyespots do not. Darkened eyespots reflect rapid changes in plasma NE, DA, and Epi that may signal dominant social status.     

Color-pattern analysis of eyespots in butterfly wings: a critical examination of morphogen gradient models

Butterfly wing color patterns consist of many color-pattern elements such as eyespots. It is believed that eyespot patterns are determined by a concentration gradient of a single morphogen species released by diffusion from the prospective eyespot focus in conjunction with multiple thresholds in signal-receiving cells. As alternatives to this single-morphogen model, more flexible multiple-morphogen model and induction model can be proposed. However, the relevance of these conceptual models to actual eyespots has not been examined systematically. Here, representative eyespots from nymphalid butterflies were analyzed morphologically to determine if they are consistent with these models. Measurement of ring widths of serial eyespots from a single wing surface showed that the proportion of each ring in an eyespot is quite different among homologous rings of serial eyespots of different sizes. In asymmetric eyespots, each ring is distorted to varying degrees. In extreme cases, only a portion of rings is expressed remotely from the focus. Similarly, there are many eyespots where only certain rings are deleted, added, or expanded. In an unusual case, the central area of an eyespot is composed of multiple "miniature eyespots," but the overall macroscopic eyespot structure is maintained. These results indicate that each eyespot ring has independence and flexibility to a certain degree, which is less consistent with the single-morphogen model. Considering a "periodic eyespot", which has repeats of a set of rings, damage-induced eyespots in mutants, and a scale-size distribution pattern in an eyespot, the induction model is the least incompatible with the actual eyespot diversity.     

Generation of butterfly wing eyespot patterns: a model for morphological determination of eyespot and parafocal element

The determination of color patterns of butterfly wing eyespots has been explained by the morphogen concentration gradient model. The induction model has been proposed recently as a more realistic alternative, in which the eyespot-specifying signal does not depend entirely on focal activity. However, this model requires further elaboration and supporting evidence to be validated. Here, I examined various color patterns of nymphalid butterflies to propose the mechanics of the induction model. Based on cases in which an eyespot light ring is identical to the background in color, I propose that eyespots are fundamentally composed of dark rings and non-dark "background" spaces between them. In the induction model, the dark-ring-inducing signal that is released from a prospective eyespot focus (the primary organizing center) as a slow-moving wave effects both selfenhancement and peripheral induction of the dark-ring-inhibitory signal at the secondary organizing centers, resulting in an eyespot that has alternate dark and light rings. Moreover, there are cases in which an unseen "imaginary light ring" surrounds an eyespot proper and in which PFEs are integrated into the eyespot. It appears that PFEs constitute a periodic continuum of eyespot dark rings; thus, a background space between the eyespot and a PFE is mechanistically equivalent to eyespot light rings. The eyespot dark-ring-inducing signals and PFE-inducing signal are likely to be identical in quality, but released at different times from the same organizing center. Computer simulations based on the reaction-diffusion system support the feasibility of the induction model.     

The flagellar apparatus and cytoskeleton of the dinoflagellates

Summary Modern microscopical approaches have allowed more accurate investigations of the three-dimensional nature of the dinoflagellate flagellar apparatus (FA) and several other cytoskeletal protein complexes. Our presentation overviews the nature of the dinoflagellate FA and cytoskeleton in a number of taxa and compares them with those of other protists. As with other protists, the FA of the dinoflagellates can be characterized by the presence of fibrous and microtubular components. Our studies and others indicate that the dinoflagellate FA can be expected to possess a striated fibrous root on the basal body of the transverse flagellum and a multimembered microtubular root on the basal body of the longitudinal flagellum. Two other features that appear widespread in the group are the transverse striated root associated microtubule (tsrm) and the transverse microtubular root (tmr). The tsrm extends at least half the length of the transverse striated root while the tmr extends from the transverse basal body toward the exit aperture of the transverse flagellum. In most cases, the tmr gives rise to several cytoplasmic microtubules at a right angle. The apparent conserved nature of these roots leads us to the conclusion that the dinoflagellate FA can be compared to the FA of the cryptomonads, chrysophytes, and the ciliates for phylogenetic purposes. Of these groups, the chrysophytes possess an FA with the most structures in common with the dinoflagellates. Our immunomicroscopical investigations of the microtubular, actin and centrin components of the dinoflagellate cytoskeleton point to the comparative usefulness of these cytological features.Abbreviations aptb apical transverse microtubular band - FA flagellar apparatus - Imr longitudinal microtubular root - mls multilayered structure - tmr transverse microtubular root - tmre transverse microtubular root extension - tsr transverse striated fibrous root - tsrm transverse striated root associated microtubule     

Eyespot evolution: phylogenetic insights from Junonia and related butterfly genera (Nymphalidae: Junoniini)

SUMMARY Butterfly eyespots have been the focus of a number of developmental and evolutionary studies. However, a phylogenetic component has rarely been explicitly incorporated in these studies. In this study, I utilize a phylogeny to trace the evolution of eyespot number and position on the wing in a group of nymphalid butterflies, the subtribe Junoniini. These butterflies have two kinds of eyespot arrangements which I refer to as Serial and Individual . In the Serial arrangement, eyespots are placed in a series on compartments 1−6 (counting from the anterior wing margin). In the Individual arrangement, eyespots are isolated on specific compartments, ranging from 1 to 4 in number. This can be divided into four subtypes based on the number and positions of eyespots. I map the evolution of these five arrangements over a phylogeny of Junoniini reconstructed with ca. 3000 base pairs of sequence data from three genes. The results show that almost all arrangements have evolved at least twice, with multiple shifts between them by addition and deletion of eyespots. I propose a model involving genetic or developmental coupling between eyespots in specific compartments to explain these shifts. I discuss their evolution in light of existing knowledge about their development. I also discuss potential explanations for functional significance of the eyespot patterns found in the group. Differential selection for and against eyespots, both at different times over the phylogeny and in different regions, have driven the evolution of eyespot arrangements. The study throws open many questions about the adaptive significance of eyespots and the developmental underpinnings of the various arrangements.     

Carotenoids in the eyespot apparatus are required for triggering phototaxis in Euglena gracilis

Carotenoids are the most universal and most widespread pigments in nature. They have played pivotal roles in the evolution of photosensing mechanisms in microbes and of vision in animals. Several groups of phytoflagellates developed a photoreceptive organelle called the eyespot apparatus (EA) consisting of two separable components: the eyespot, a cluster of carotenoid‐rich globules that acts as a reflector device, and actual photoreceptors for photobehaviors. Unlike other algal eyespots, the eyespot of Euglenophyta lacks reflective properties and is generally considered to act as a shading device for the photoreceptor (paraflagellar body, PFB) for major photomovements. However, the function of the eyespot of Euglenophyta has not yet been fully proven. Here, we report that the blocking carotenoid biosynthesis in Euglena gracilis by suppressing the phytoene synthase gene (crtB) caused a defect in eyespot function resulting in a loss of phototaxis. Raman spectroscopy and transmission electron microscopy suggested that EgcrtB‐suppressed cells formed eyespot globules but had a defect in the accumulation of carotenoids in those packets. Motion analysis revealed the loss of phototaxis in EgcrtB‐suppressed cells: a defect in the initiation of turning movements immediately after a change in light direction, rather than a defect in the termination of cell turning at the appropriate position due to a loss of the shading effect on the PFB. This study revealed that carotenoids are essential for light perception by the EA for the initiation of phototactic movement by E. gracilis, suggesting one possible photosensory role of carotenoids in the EA for the phototaxis.     

Bird attacks on a butterfly with marginal eyespots and the role of prey concealment against the background

Small eyespots on butterflies have long been thought to deflect attacks, and birds are the presumptive drivers selecting for these patterns; however, evidence of this function is still ambiguous. Marginal eyespots typically consist of a UV‐reflective white pupil, surrounded by one black and one yellowish ring. We have recently shown that Cyanistes caeruleus (blue tits) attack such eyespots, but only under low light intensities with accentuated UV levels: the increased salience of the eyespots relative to the rest of the butterfly probably explains this result. Possibly the background against which the butterfly is concealed may deceive birds to make similar errors. We therefore presented speckled wood butterflies decorated with eyespots (or controls without eyespots) to C. caeruleus against two backgrounds: oak and birch bark. Our results show that: (1) eyespots, independent of background, were effective in deflecting attacks; (2) the time elapsed between a bird landing and the attack was interactively dependent on the background and whether the butterfly bore an eyespot; and (3) the speed at which a butterfly was attacked predicted the outcome, with faster birds being more prone to errors than slower birds. This underscores a speed–accuracy trade‐off in the predators, and that background plays a role in the defensive qualities of marginal eyespots. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 290–297.     

The ‘sparkle’ in fake eyes – the protective effect of mimic eyespots in lepidoptera

Many species of lepidoptera bear conspicuous circular patterns on their wings, known as eyespots, that are hypothesised to protect their bearers against predatory birds. In this study, we focus on a small but ubiquitous feature occurring naturally in lepidopteran eyespots, namely the so‐called ‘sparkle’. The ‘pupil’ in an eyespot is often highlighted by a ‘sparkle’, which is hypothesised to mimic a natural corneal total light reflection evident as a highlight, twinkle, or sparkle in the vertebrate eye. In a study exploring the presence of such sparkles, we found that 53% of lepidopteran eyespots exceeding 1 mm in diameter have a central, pinpoint‐like ‘sparkle’, 12% have a marginal, crescent‐shaped ‘sparkle’, 13% have a semi‐circular ‘sparkle’, and 22% have an intermediate semi‐circular to crescent‐shaped ‘sparkle’. In the lepidopterans’ natural resting position, the marginal ‘sparkles’ are positioned in the upper part of the eyespots’‘pupil’ and thus may create the illusion of a spherical eyeball. The ‘sparkles’ in lepidopteran eyespots do not only appear white to humans, but also reflect ultraviolet light. White and UV‐reflecting ‘sparkles’ also appear ‘white’ for UV‐sensitive viewers such as birds, and thus may effectively mimic the natural highlight in vertebrate eyes as an area of total light reflection. In field experiments using lepidopteran dummies baited with a mealworm, we show that the ‘sparkle’ is one of several components of eyespots eliciting a deterrent effect and that eyespots with a ‘sparkle’ in a natural position have a stronger deterrent effect than those with a ‘sparkle’ in an unnatural position. These findings support the eye mimicry hypothesis that better vertebrate eye mimicry improves the deterrent effect of eyespots.     

Isolation and partial characterization of the photoreceptive organelle for phototaxis of a flagellate green alga

We report on the isolation and purification of structurally intact eyespot apparatuses from the naked, biflagellate green alga Spermatozopsis similis. Two eyespot-enriched fractions, separated by sucrose gradient centrifugation, retained the typical reflective properties of eyespots in situ as demonstrated by reflection confocal laser scanning microscopy. Ultrastructurally, both fractions contained eyespot plates consisting of a single layer of lipid globules. Structurally intact eyespot apparatuses, including patches of plasma membrane and chloroplast envelope overlying the eyespot plate and a single thylakoid subtending the eyespot plate, were particularly enriched in one of the two fractions (fraction 2a). Measurement of several marker enzymes and chlorophyll content (less than 0.001% of total) established the absence of most other cell organelles from the eyespot fractions. The absorption spectra of the two fractions were dominated by carotenoids with an additional shoulder at 540 nm. Following extraction with organic solvents and sodium dodecyl sulfate polyacrylamide gel electrophoresis, several proteins were found to be considerably enriched in the two fractions. In addition to several proteins in the high Mr range, at least 4 polypeptides of 35, 29, 23, and 20 kDa are selectively enriched in fraction 2a with the 29 and 20 kDa proteins being the most prominent. The presence of glycoproteins in fraction 2a was demonstrated by binding of the mannose-specific lectin Galanthus nivalis agglutinin to several high molecular weight polypeptides. In addition, a hydrophobic component with abnormal electrophoretic mobility that reacts strongly with periodic acid-Schiff and thymol/sulfuric acid was prominent in both fractions. Mass isolation and purification of the intact phototactic apparatus of a flagellate green alga now greatly facilitates the biochemical and molecular characterization of the signal transduction chain involved in green algal phototaxis.     

Body size determines eyespot size and presence in coral reef fishes

Numerous organisms display conspicuous eyespots. These eye‐like patterns have been shown to effectively reduce predation by either deflecting strikes away from nonvital organs or by intimidating potential predators. While investigated extensively in terrestrial systems, determining what factors shape eyespot form in colorful coral reef fishes remains less well known. Using a broadscale approach we ask: How does the size of the eyespot relate to the actual eye, and at what size during ontogeny are eyespots acquired or lost? We utilized publicly available images to generate a dataset of 167 eyespot‐bearing reef fish species. We measured multiple features relating to the size of the fish, its eye, and the size of its eyespot. In reef fishes, the area of the eyespot closely matches that of the real eye; however, the eyespots “pupil” is nearly four times larger than the real pupil. Eyespots appear at about 20 mm standard length. However, there is a marked decrease in the presence of eyespots in fishes above 48 mm standard length; a size which is tightly correlated with significant decreases in documented mortality rates. Above 75–85 mm, the cost of eyespots appears to outweigh their benefit. Our results identify a “size window” for eyespots in coral reef fishes, which suggests that eyespot use is strictly body size‐dependent within this group.     

Conserved developmental processes and the formation of evolutionary novelties: examples from butterfly wings

The origin and diversification of evolutionary novelties-lineage-specific traits of new adaptive value-is one of the key issues in evolutionary developmental biology. However, comparative analysis of the genetic and developmental bases of such traits can be difficult when they have no obvious homologue in model organisms. The finding that the evolution of morphological novelties often involves the recruitment of pre-existing genes and/or gene networks offers the potential to overcome this challenge. Knowledge about shared developmental processes obtained from extensive studies in model organisms can then be used to understand the origin and diversification of lineage-specific structures. Here, we illustrate this approach in relation to eyespots on the wings of Bicyclus anynana butterflies. A number of spontaneous mutations isolated in the laboratory affect eyespots, lepidopteran-specific features, and also processes that are shared by most insects. We discuss how eyespot mutants with disturbed embryonic development may help elucidate the genetic pathways involved in eyespot formation, and how venation mutants with altered eyespot patterns might shed light on mechanisms of eyespot development.     

The "eyespot module" and eyespots as modules: development, evolution, and integration of a complex phenotype

Organisms are inherently modular, yet modules also evolve in response to selection for functional integration or functional specialization of traits. For serially repeated homologous traits, there is a clear expectation that selection on the function of individual traits will reduce the integration between traits and subdivide a single ancestral module. The eyespots on butterfly wings are one example of serially repeated morphological traits that share a common developmental mechanism but are subject to natural and sexual selection for divergent functions. Here, I test two hypotheses about the organization of the eyespot pattern into independent dorsal-ventral and anterior-posterior modules, using a graphical modeling technique to examine patterns of eyespot covariation among and within wing surfaces in the butterfly Bicyclus anynana. Although there is a hierarchical and complex pattern of integration among eyespots, the results show a surprising mismatch between patterns of eyespot integration and the developmental and evolutionary eyespot units identified in previous empirical studies. These results are discussed in light of the relationships between developmental, functional, and evolutionary modules, and they suggest that developmental sources of independent trait variation are often masked by developmental sources of trait integration.