Correction: CRISPR/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants.

[This corrects the article DOI: 10.1371/journal.pone.0261331.].

Short-wavelength-sensitive 1 (SWS1) opsin gene duplications and parallel visual pigment tuning support ultraviolet communication in damselfishes (Pomacentridae).

Damselfishes (Pomacentridae) are one of the most behaviourally diverse, colourful and species-rich reef fish families. One remarkable characteristic of damselfishes is their communication in ultraviolet (UV) light. Not only are they sensitive to UV, they are also prone to have UV-reflective colours and patterns enabling social signalling. Using more than 50 species, we aimed to uncover the evolutionary history of UV colour and UV vision in damselfishes. All damselfishes had UV-transmitting lenses, expressed the UV-sensitive SWS1 opsin gene, and most displayed UV-reflective patterns and colours. We find evidence for several tuning events across the radiation, and while SWS1 gene duplications are generally very rare among teleosts, our phylogenetic reconstructions uncovered two independent duplication events: one close to the base of the most species-rich clade in the subfamily Pomacentrinae, and one in a single Chromis species. Using amino acid comparisons, we found that known spectral tuning sites were altered several times in parallel across the damselfish radiation (through sequence change and duplication followed by sequence change), causing repeated shifts in peak spectral absorbance of around 10 nm. Pomacentrinae damselfishes expressed either one or both copies of SWS1, likely to further finetune UV-signal detection and differentiation. This highly advanced and modified UV vision among damselfishes, in particular the duplication of SWS1 among Pomacentrinae, might be seen as a key evolutionary innovation that facilitated the evolution of the exuberant variety of UV-reflectance traits and the diversification of this coral reef fish lineage.

Ultraviolet vision in anemonefish improves colour discrimination.

In many animals, ultraviolet (UV) vision guides navigation, foraging, and communication, but few studies have addressed the contribution of UV signals to colour vision, or measured UV discrimination thresholds using behavioural experiments. Here, we tested UV colour vision in an anemonefish (Amphiprion ocellaris) using a five-channel (RGB-V-UV) LED display. We first determined that the maximal sensitivity of the A. ocellaris UV cone was ∼386 nm using microspectrophotometry. Three additional cone spectral sensitivities had maxima at ∼497, 515 and ∼535 nm. We then behaviourally measured colour discrimination thresholds by training anemonefish to distinguish a coloured target pixel from grey distractor pixels of varying intensity. Thresholds were calculated for nine sets of colours with and without UV signals. Using a tetrachromatic vision model, we found that anemonefish were better (i.e. discrimination thresholds were lower) at discriminating colours when target pixels had higher UV chromatic contrast. These colours caused a greater stimulation of the UV cone relative to other cone types. These findings imply that a UV component of colour signals and cues improves their detectability, which likely increases the prominence of anemonefish body patterns for communication and the silhouette of zooplankton prey.

Higher ultraviolet skin reflectance signals submissiveness in the anemonefish, Amphiprion akindynos.

Ultraviolet (UV) vision is widespread among teleost fishes, of which many exhibit UV skin colors for communication. However, aside from its role in mate selection, few studies have examined the information UV signaling conveys in other socio-behavioral contexts. Anemonefishes (subfamily, Amphiprioninae) live in a fascinating dominance hierarchy, in which a large female and male dominate over non-breeding subordinates, and body size is the primary cue for dominance. The iconic orange and white bars of anemonefishes are highly UV-reflective, and their color vision is well tuned to perceive the chromatic contrast of skin, which we show here decreases in the amount of UV reflectance with increasing social rank. To test the function of their UV-skin signals, we compared the outcomes of staged contests over dominance between size-matched Barrier Reef anemonefish (Amphiprion akindynos) in aquarium chambers viewed under different UV-absorbing filters. Fish under UV-blocking filters were more likely to win contests, where fish under no-filter or neutral-density filter were more likely to submit. For contests between fish in no-filter and neutral density filter treatments, light treatment had no effect on contest outcome (win/lose). We also show that sub-adults were more aggressive toward smaller juveniles placed under a UV filter than a neutral density filter. Taken together, our results show that UV reflectance or UV contrast in anemonefish can modulate aggression and encode dominant and submissive cues, when changes in overall intensity are controlled for.

The brain structure and the neural network features of the diurnal cuttlefish Sepia plangon.

Cuttlefish are known for their rapid changes of appearance enabling camouflage and con-specific communication for mating or agonistic display. However, interpretation of their sophisticated behaviors and responsible brain areas is based on the better-studied squid brain atlas. Here we present the first detailed description of the neuroanatomical features of a tropical and diurnal cuttlefish, Sepia plangon, coupled with observations on ontogenetic changes in its visual and learning centers using a suite of MRI-based techniques and histology. We then make comparisons to a loliginid squid, treating it as a 'baseline', and also to other cuttlefish species to help construct a connectivity map of the cuttlefish brain. Differences in brain anatomy and the previously unknown neural connections associated with camouflage, motor control and chemosensory function are described. These findings link brain heterogeneity to ecological niches and lifestyle, feeding hypotheses and evolutionary history, and provide a timely, new technology update to older literature.

Long-wavelength-sensitive (lws) opsin gene expression, foraging and visual communication in coral reef fishes.

Coral reef fishes are diverse in ecology and behaviour and show remarkable colour variability. Investigating the visual pigment gene (opsin) expression in these fishes makes it possible to associate their visual genotype and phenotype (spectral sensitivities) to visual tasks, such as feeding strategy or conspecific detection. By studying all major damselfish clades (Pomacentridae) and representatives from five other coral reef fish families, we show that the long-wavelength-sensitive (lws) opsin is highly expressed in algivorous and less or not expressed in zooplanktivorous species. Lws is also upregulated in species with orange/red colours (reflectance >520 nm) and expression is highest in orange/red-coloured algivores. Visual models from the perspective of a typical damselfish indicate that sensitivity to longer wavelengths does enhance the ability to detect the red to far-red component of algae and orange/red-coloured conspecifics, possibly enabling social signalling. Character state reconstructions indicate that in the early evolutionary history of damselfishes, there was no lws expression and no orange/red coloration. Omnivory was most often the dominant state. Although herbivory was sometimes dominant, zooplanktivory was never dominant. Sensitivity to long wavelength (increased lws expression) only emerged in association with algivory but never with zooplanktivory. Higher lws expression is also exploited by social signalling in orange/red, which emerged after the transition to algivory. Although the relative timing of traits may deviate by different reconstructions and alternative explanations are possible, our results are consistent with sensory bias whereby social signals evolve as a correlated response to natural selection on sensory system properties in other contexts.

Developing and adult reef fish show rapid light-induced plasticity in their visual system.

The visual capabilities of fish are optimized for their ecology and light environment over evolutionary time. Similarly, fish vision can adapt to regular changes in light conditions within their lifetime, e.g., ontogenetic or seasonal variation. However, we do not fully understand how vision responds to irregular short-term changes in the light environment, e.g., algal blooms and light pollution. In this study, we investigated the effect of short-term exposure to unnatural light conditions on opsin gene expression and retinal cell densities in juvenile and adult diurnal reef fish (convict surgeonfish; Acanthurus triostegus). Results revealed phenotypic plasticity in the retina across ontogeny, particularly during development. The most substantial differences at both molecular and cellular levels were found under constant dim light, while constant bright light and simulated artificial light at night had a lesser effect. Under dim light, juveniles and adults increased absolute expression of the cone opsin genes, sws2a, rh2c and lws, within a few days and juveniles also decreased densities of cones, inner nuclear layer cells and ganglion cells. These changes potentially enhanced vision under the altered light conditions. Thus, our study suggests that plasticity mainly comes into play when conditions are extremely different to the species' natural light environment, i.e., a diurnal fish in "constant night". Finally, in a rescue experiment on adults, shifts in opsin expression were reverted within 24 h. Overall, our study showed rapid, reversible light-induced changes in the retina of A. triostegus, demonstrating phenotypic plasticity in the visual system of a reef fish throughout life.

Polarization vision mitigates visual noise from flickering light underwater.

In shallow water, downwelling light is refracted from surface waves onto the substrate creating bands of light that fluctuate in both time and space, known as caustics. This dynamic illumination can be a visual hindrance for animals in shallow underwater environments. Animals in such habitats may have evolved to use polarization vision for discriminating objects while ignoring the variations in illumination caused by caustics. To explore this possibility, crabs (Carcinus maenas) and cuttlefish (Sepia officinalis), both of which have polarization vision, were presented with moving stimuli overlaid with caustics. Dynamic caustics inhibited the detection of an intensity-based stimulus but not when these stimuli were polarized. This study is the first to demonstrate that polarization vision reduces the negative impacts that dynamic illumination can have on visual perception.

Development of dim-light vision in the nocturnal reef fish family Holocentridae. II: Retinal morphology.

Ontogenetic changes in the habitats and lifestyles of animals are often reflected in their visual systems. Coral reef fishes start life in the shallow open ocean but inhabit the reef as juveniles and adults. Alongside this change in habitat, some species also change lifestyles and become nocturnal. However, it is not fully understood how the visual systems of nocturnal reef fishes develop and adapt to these significant ecological shifts over their lives. Therefore, we used a histological approach to examine visual development in the nocturnal coral reef fish family, Holocentridae. We examined 7 representative species spanning both subfamilies, Holocentrinae (squirrelfishes) and Myripristinae (soldierfishes). Pre-settlement larvae showed strong adaptation for photopic vision with high cone densities and had also started to develop a multibank retina (i.e. multiple rod layers), with up to two rod banks present. At reef settlement, holocentrids showed greater adaptation for scotopic vision, with higher rod densities and higher summation of rods onto the ganglion cell layer. By adulthood, they had well-developed scotopic vision with a highly rod-dominated multibank retina comprising 5-17 rod banks and enhanced summation of rods onto the ganglion cell layer. Although the ecological demands of the two subfamilies were similar throughout their lives, their visual systems differed after settlement, with Myripristinae showing more pronounced adaptation for scotopic vision than Holocentrinae. Thus, it is likely that both ecology and phylogeny contribute to the development of the holocentrid visual system.

Development of dim-light vision in the nocturnal reef fish family Holocentridae. I: Retinal gene expression.

Developmental changes to the visual systems of animals are often associated with ecological shifts. Reef fishes experience a change in habitat between larval life in the shallow open ocean to juvenile and adult life on the reef. Some species also change their lifestyle over this period and become nocturnal. While these ecological transitions are well documented, little is known about the ontogeny of nocturnal reef fish vision. Here, we used transcriptomics to investigate visual development in 12 representative species from both subfamilies, Holocentrinae (squirrelfishes) and Myripristinae (soldierfishes), in the nocturnal coral reef fish family, Holocentridae. Results revealed that the visual systems of holocentrids are initially well adapted to photopic conditions with pre-settlement larvae having high levels of cone opsin gene expression and a broad cone opsin gene repertoire (8 genes). At reef settlement, holocentrids started to invest more in their scotopic visual system, and compared with adults, showed upregulation of genes involved in cell differentiation/proliferation. By adulthood, holocentrids had well developed scotopic vision with high levels of rod opsin gene expression, reduced cone opsin gene expression and repertoire (1-4 genes) and upregulated phototransduction genes. Finally, although the two subfamilies shared similar ecologies across development, their visual systems diverged after settlement, with Myripristinae investing more in scotopic vision than Holocentrinae. Hence, both ecology and phylogeny are likely to determine the development of the holocentrid visual system.

Seeing Picasso: an investigation into the visual system of the triggerfish Rhinecanthus aculeatus.

Vision is used by animals to find food and mates, avoid predators, defend resources and navigate through complex habitats. Behavioural experiments are essential for understanding animals' perception but are often challenging and time-consuming; therefore, using species that can be trained easily for complex tasks is advantageous. Picasso triggerfish, Rhinecanthus aculeatus, have been used in many behavioural studies investigating vision and navigation. However, little is known about the molecular and anatomical basis of their visual system. We addressed this knowledge gap here and behaviourally tested achromatic and chromatic acuity. In terms of visual opsins, R. aculeatus possessed one rod opsin gene (RH1) and at least nine cone opsins: one violet-sensitive SWS2B gene, seven duplicates of the blue-green-sensitive RH2 gene (RH2A, RH2B, RH2C1-5) and one red-sensitive LWS gene. However, only five cone opsins were expressed: SWS2B expression was consistent, while RH2A, RH2C-1 and RH2C-2 expression varied depending on whether fish were sampled from the field or aquaria. Levels of LWS expression were very low. Using fluorescence in situ hybridisation, we found SWS2B was expressed exclusively in single cones, whereas RH2A and RH2Cs were expressed in opposite double cone members. Anatomical resolution estimated from ganglion cell densities was 6.8 cycles per degree (cpd), which was significantly higher than values obtained from behavioural testing for black-and-white achromatic stimuli (3.9 cpd) and chromatic stimuli (1.7-1.8 cpd). These measures were twice as high as previously reported. This detailed information on their visual system will help inform future studies with this emerging focal species.

Colour discrimination thresholds vary throughout colour space in a reef fish (Rhinecanthus aculeatus).

Animals use colour vision in a range of behaviours. Visual performance is limited by thresholds, which are set by noise in photoreceptors and subsequent neural processing. The receptor noise limited (RNL) model of colour discrimination is widely used for modelling colour vision and accounts well for experimental data from many species. In one of the most comprehensive tests yet of colour discrimination in a non-human species, we used Ishihara-style stimulus patterns to examine thresholds for 21 directions at five locations in colour space for the fish Rhinecanthus aculeatus. Thresholds matched RNL model predictions most closely for stimuli near the achromatic point, but exceeded predictions (indicating a decline in sensitivity) with distance from this point. Thresholds were also usually higher for saturation than for hue differences. These changes in colour threshold with colour space location and direction may give insight into photoreceptor non-linearities and post-receptoral mechanisms of colour vision in fish. Our results highlight the need for a cautious interpretation of the RNL model - especially for modelling colours that differ from one another in saturation (rather than hue), and for highly saturated colours distant from the achromatic point in colour space.

Comparative brain structure and visual processing in octopus from different habitats.

Octopods are masters of camouflage and solve complex tasks, and their cognitive ability is said to approach that of some small mammals. Despite intense interest and some research progress, much of our knowledge of octopus neuroanatomy and its links to behavior and ecology comes from one coastal species, the European common octopus, Octopus vulgaris. Octopod species are found in habitats including complex coral reefs and the relatively featureless mid-water. There they encounter different selection pressures, may be nocturnal or diurnal, and are mostly solitary or partially social. How these different ecologies and behavioral differences influence the octopus central nervous system (CNS) remains largely unknown. Here we present a phylogenetically informed comparison between diurnal and nocturnal coastal and a deep-sea species using brain imaging techniques. This study shows that characteristic neuroanatomical changes are linked to their habits and habitats. Enlargement and division of the optic lobe as well as structural foldings and complexity in the underlying CNS are linked to behavioral adaptation (diurnal versus nocturnal; social versus solitary) and ecological niche (reef versus deep sea), but phylogeny may play a part also. The difference between solitary and social life is mirrored within the brain including the formation of multiple compartments (gyri) in the vertical lobe, which is likened to the vertebrate cortex. These findings continue the case for convergence between cephalopod and vertebrate brain structure and function. Notably, within the current push toward comparisons of cognitive abilities, often with unashamed anthropomorphism at their root, these findings provide a firm grounding from which to work.

CRISPR/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants.

Genomic manipulation is a useful approach for elucidating the molecular pathways underlying aspects of development, physiology, and behaviour. However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR/Cas9 system in the false clown anemonefish, Amphiprion ocellaris. Microinjection of zygotes was used to demonstrate the successful use of our CRISPR/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene (RH2B) involved in vision, and Tyrosinase-producing gene (tyr) involved in the production of melanin. Analysis of the sequenced target gene regions in A. ocellaris embryos showed that uptake was as high as 73.3% of injected embryos. Further analysis of the subcloned mutant gene sequences combined with amplicon shotgun sequencing revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in F0 A. ocellaris embryos. Moreover, we clearly show a loss-of-function in tyr mutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful starting point to further explore the potential application of CRISPR/Cas9 in A. ocellaris, as a platform for studying gene function in anemonefishes and other reef fishes.

Thresholds of polarization vision in octopuses.

Polarization vision is widespread in nature, mainly among invertebrates, and is used for a range of tasks including navigation, habitat localization and communication. In marine environments, some species such as those from the Crustacea and Cephalopoda that are principally monochromatic, have evolved to use this adaptation to discriminate objects across the whole visual field, an ability similar to our own use of colour vision. The performance of these polarization vision systems varies, and the few cephalopod species tested so far have notably acute thresholds of discrimination. However, most studies to date have used artificial sources of polarized light that produce levels of polarization much higher than found in nature. In this study, the ability of octopuses to detect polarization contrasts varying in angle of polarization (AoP) was investigated over a range of different degrees of linear polarization (DoLP) to better judge their visual ability in more ecologically relevant conditions. The 'just-noticeable-differences' (JND) of AoP contrasts varied consistently with DoLP. These JND thresholds could be largely explained by their 'polarization distance', a neurophysical model that effectively calculates the level of activity in opposing horizontally and vertically oriented polarization channels in the cephalopod visual system. Imaging polarimetry from the animals' natural environment was then used to illustrate the functional advantage that these polarization thresholds may confer in behaviourally relevant contexts.

The visual ecology of Holocentridae, a nocturnal coral reef fish family with a deep-sea-like multibank retina.

The visual systems of teleost fishes usually match their habitats and lifestyles. Since coral reefs are bright and colourful environments, the visual systems of their diurnal inhabitants have been more extensively studied than those of nocturnal species. In order to fill this knowledge gap, we conducted a detailed investigation of the visual system of the nocturnal reef fish family Holocentridae. Results showed that the visual system of holocentrids is well adapted to their nocturnal lifestyle with a rod-dominated retina. Surprisingly, rods in all species were arranged into 6-17 well-defined banks, a feature most commonly found in deep-sea fishes, that may increase the light sensitivity of the eye and/or allow colour discrimination in dim light. Holocentrids also have the potential for dichromatic colour vision during the day with the presence of at least two spectrally different cone types: single cones expressing the blue-sensitive SWS2A gene, and double cones expressing one or two green-sensitive RH2 genes. Some differences were observed between the two subfamilies, with Holocentrinae (squirrelfish) having a slightly more developed photopic visual system than Myripristinae (soldierfish). Moreover, retinal topography of both ganglion cells and cone photoreceptors showed specific patterns for each cell type, likely highlighting different visual demands at different times of the day, such as feeding. Overall, their well-developed scotopic visual systems and the ease of catching and maintaining holocentrids in aquaria, make them ideal models to investigate teleost dim-light vision and more particularly shed light on the function of the multibank retina and its potential for dim-light colour vision.

Does conspicuousness scale linearly with colour distance? A test using reef fish.

To be effective, animal colour signals must attract attention-and therefore need to be conspicuous. To understand the signal function, it is useful to evaluate their conspicuousness to relevant viewers under various environmental conditions, including when visual scenes are cluttered by objects of varying colour. A widely used metric of colour difference (ΔS) is based on the receptor noise limited (RNL) model, which was originally proposed to determine when two similar colours appear different from one another, termed the discrimination threshold (or just noticeable difference). Estimates of the perceptual distances between colours that exceed this threshold-termed 'suprathreshold' colour differences-often assume that a colour's conspicuousness scales linearly with colour distance, and that this scale is independent of the direction in colour space. Currently, there is little behavioural evidence to support these assumptions. This study evaluated the relationship between ΔS and conspicuousness in suprathreshold colours using an Ishihara-style test with a coral reef fish, Rhinecanthus aculeatus. As our measure of conspicuousness, we tested whether fish, when presented with two colourful targets, preferred to peck at the one with a greater ΔS - from the average distractor colour. We found the relationship between ΔS and conspicuousness followed-- a sigmoidal function, with high ΔS colours perceived as equally conspicuous. We found that the relationship between ΔS and conspicuousness varied across colour space (i.e. for different hues). The sigmoidal detectability curve was little affected by colour variation in the background or when colour distance was calculated using a model that does not incorporate receptor noise. These results suggest that the RNL model may provide accurate estimates for perceptual distance for small suprathreshold distance colours, even in complex viewing environments, but must be used with caution with perceptual distances exceeding- -10 ΔS.

More than noise: context-dependent luminance contrast discrimination in a coral reef fish (Rhinecanthus aculeatus).

Achromatic (luminance) vision is used by animals to perceive motion, pattern, space and texture. Luminance contrast sensitivity thresholds are often poorly characterised for individual species and are applied across a diverse range of perceptual contexts using over-simplified assumptions of an animal's visual system. Such thresholds are often estimated using the receptor noise limited model (RNL). However, the suitability of the RNL model to describe luminance contrast perception remains poorly tested. Here, we investigated context-dependent luminance discrimination using triggerfish (Rhinecanthus aculeatus) presented with large achromatic stimuli (spots) against uniform achromatic backgrounds of varying absolute and relative contrasts. 'Dark' and 'bright' spots were presented against relatively dark and bright backgrounds. We found significant differences in luminance discrimination thresholds across treatments. When measured using Michelson contrast, thresholds for bright spots on a bright background were significantly higher than for other scenarios, and the lowest threshold was found when dark spots were presented on dark backgrounds. Thresholds expressed in Weber contrast revealed lower thresholds for spots darker than their backgrounds, which is consistent with the literature. The RNL model was unable to estimate threshold scaling across scenarios as predicted by the Weber-Fechner law, highlighting limitations in the current use of the RNL model to quantify luminance contrast perception. Our study confirms that luminance contrast discrimination thresholds are context dependent and should therefore be interpreted with caution.

Dynamic Courtship Signals and Mate Preferences in Sepia plangon.

Communication in cuttlefish includes rapid changes in skin coloration and texture, body posture and movements, and potentially polarized signals. The dynamic displays are fundamental for mate choice and agonistic behavior. We analyzed the reproductive behavior of the mourning cuttlefish Sepia plangon in the laboratory. Mate preference was analyzed via choice assays (n = 33) under three sex ratios, 1 male (M): 1 female (F), 2M:1F, and 1M:2F. We evaluated the effect of modifying polarized light from the arms stripes and ambient light with polarized and unpolarized barriers between the cuttlefish. Additionally, to assess whether a particular trait was a determinant for mating, we used 3D printed cuttlefish dummies. The dummies had different sets of visual signals: two sizes (60 or 90 mm mantle length), raised or dropped arms, high or low contrast body coloration, and polarized or unpolarized filters to simulate the arms stripes. Frequency and duration (s) of courtship displays, mating, and agonistic behaviors were analyzed with GLM and ANOVAs. The behaviors, body patterns, and their components were integrated into an ethogram to describe the reproductive behavior of S. plangon. We identified 18 body patterns, 57 body patterns components, and three reproductive behaviors (mating, courtship, and mate guarding). Only sex ratio had a significant effect on courtship frequency, and the male courtship success rate was 80%. Five small (ML < 80 mm) males showed the dual-lateral display to access mates while avoiding fights with large males; this behavior is characteristic of male "sneaker" cuttlefish. Winner males showed up to 17 body patterns and 33 components, whereas loser males only showed 12 patterns and 24 components. We identified 32 combinations of body patterns and components that tended to occur in a specific order and were relevant for mating success in males. Cuttlefish were visually aware of the 3D-printed dummies; however, they did not start mating or agonistic behavior toward the dummies. Our findings suggest that in S. plangon, the dynamic courtship displays with specific sequences of visual signals, and the sex ratio are critical for mate choice and mating success.

Visual system diversity in coral reef fishes.

Coral reefs are one of the most species rich and colourful habitats on earth and for many coral reef teleosts, vision is central to their survival and reproduction. The diversity of reef fish visual systems arises from variations in ocular and retinal anatomy, neural processing and, perhaps most easily revealed by, the peak spectral absorbance of visual pigments. This review examines the interplay between retinal morphology and light environment across a number of reef fish species, but mainly focusses on visual adaptations at the molecular level (i.e. visual pigment structure). Generally, visual pigments tend to match the overall light environment or micro-habitat, with fish inhabiting greener, inshore waters possessing longer wavelength-shifted visual pigments than open water blue-shifted species. In marine fishes, particularly those that live on the reef, most species have between two (likely dichromatic) to four (possible tetrachromatic) cone spectral sensitivities and a single rod for crepuscular vision; however, most are trichromatic with three spectral sensitivities. In addition to variation in spectral sensitivity number, spectral placement of the absorbance maximum (λmax) also has a surprising degree of variability. Variation in ocular and retinal anatomy is also observed at several levels in reef fishes but is best represented by differences in arrangement, density and distribution of neural cell types across the retina (i.e. retinal topography). Here, we focus on the seven reef fish families most comprehensively studied to date to examine and compare how behaviour, environment, activity period, ontogeny and phylogeny might interact to generate the exceptional diversity in visual system design that we observe.