© 2020. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2020) 223, jeb223644. doi:10.1242/jeb.223644

RESEARCH ARTICLE The effect of ecological factors on eye morphology in the western , australis Thomas J. Lisney1,2, Shaun P. Collin1,2,3 and Jennifer L. Kelley4,*

ABSTRACT (Hughes, 1977; Land and Nilsson, 2012). Larger eyes can also Ecological factors such as spatial habitat complexity and diet can enhance visual sensitivity compared to smaller eyes. The size of the explain variation in visual morphology, but few studies have sought aperture of an eye (the pupil) dictates how much light can enter, and to determine whether visual specialisation can occur among that live in dim light conditions (e.g. nocturnal species and populations of the same species. We used a small Australian deep-sea animals), tend to have large eyes with large pupils freshwater (the western rainbowfish, )to (Warrant, 2004; Land and Nilsson, 2012). However, there are also determine whether populations showed variation in eye size and eye costs associated with having large eyes, such as the energetic cost of position, and whether this variation could be explained by manufacturing and maintaining the many millions of nerve cells environmental (light availability, turbidity) and ecological (predation within the retina, the hydrodynamic or aerodynamic costs incurred risk, habitat complexity, invertebrate abundance) variables. We by increased weight or drag associated with large eyes, and the cost investigated three aspects of eye morphology – (1) eye size relative of repairing damage to the eye (Hiller-Adams and Case, 1988; to body size, (2) pupil size relative to eye size and (3) eye position in Laughlin, 2001; Niven and Laughlin, 2008). Investment in the the head – for fish collected from 14 sites in a major river catchment in visual system relative to other body parts should therefore be northwest Western Australia. We found significant variation among specific to the visual requirements of a species (Laughlin, 2001; populations in all three measures of eye morphology, but no effect of Niven and Laughlin, 2008), and there is an evolutionary trend sex on eye size or eye position. Variation in eye diameter and eye towards large eyes in species that rely on vision (Walls, 1942; position was best explained by the level of habitat complexity. Howland et al., 2004; Lisney and Collin, 2007; Land and Specifically, fish occurring in habitats with low complexity (i.e. open Nilsson, 2012). ’ water) tended to have smaller, more dorsally located eyes than those Other aspects of eye morphology are also linked with an s occurring in more complex habitats (i.e. vegetation present). The size behaviour. For example, the diameter of the pupil or the cornea of the pupil relative to the size of the eye was most influenced by the (which sets the upper limit on the size of the pupil) relative to eye presence of surrounding rock formations; fish living in gorge habitats size is a consistent and useful predictor of activity patterns in a broad had significantly smaller pupils (relative to eye size) than those range of animals. Increasing the size of the pupil is an adaptation for occupying semi-gorge sites or open habitats. Our findings reveal that improving visual sensitivity; therefore, if all factors other than different ecological and environmental factors contribute to habitat- aperture size (e.g. eye size, corneal transmission) are held constant, specific visual specialisations within a species. an eye with a relatively larger aperture will have a greater light gathering capability than an eye with a proportionally smaller KEY WORDS: Eye position, Eye size, Freshwater fish, aperture (Kirk, 2004). In , lizards, birds and mammals, species Morphometrics, Predation risk, Pupil size that are crepuscular or nocturnal have relatively larger corneas/ pupils than diurnal species (Kirk, 2004; Hall and Ross, 2007; Hall, INTRODUCTION 2008; Schmitz and Wainwright, 2011; Veilleux and Lewis, 2011; The size, external morphology and position of the eyes can reveal a Lisney et al., 2012). Eye size, and the size of the aperture, can great deal about an animal’s behavioural ecology (Walls, 1942; therefore be used to indicate a species’ reliance on vision for Lythgoe, 1979). Many animals rely on vision for fundamental behavioural tasks, and its diurnal/nocturnal activity patterns. behaviours such as foraging, mate attraction and predator avoidance, The visual field is a key determinant of animal vision, because it and their visual capabilities are strongly linked with eye determines the volume of space that can be imaged upon the two morphology. For example, eye size has a major influence on retinas, and hence the amount of information that can be extracted at visual resolution, as a larger eye can house more photoreceptors and any one time (Martin, 2007, 2014). In vertebrates, the size and shape will have a longer focal length compared with a smaller eye of the visual field is predominantly determined by the position of the eyes in the head, along with other factors, such as the shape of the skull, depth of the eye socket and eye mobility (Collin and Shand, 1Oceans Graduate School, The University of Western Australia, 35 Stirling Highway, 2003). Animals with frontally positioned eyes, such as primates, Crawley, WA 6009, Australia. 2The Oceans Institute (M470), The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. 3School of Life cats and owls, have extensive binocular overlap in their frontal Sciences, La Trobe University, Bundoora, VIC 3086, Australia. 4School of Biological visual field, so the region of highest visual acuity projects forwards Sciences (M092), The University of Western Australia, 35 Stirling Highway, Crawley, (Hughes, 1977; Martin, 2007, 2014). In contrast, animals with WA 6009, Australia. laterally positioned eyes, such as rabbits and most birds, have *Author for correspondence ( [email protected]) extensive monocular visual fields, and a narrow region of binocular overlap, thus the regions of highest acuity project laterally (Hughes, T.J.L., 0000-0001-5149-3720; S.P.C., 0000-0001-6236-0771; J.L.K., 0000-0002- 8223-7241 1977; Martin, 2007, 2014). In fishes, the position of the eyes is related to habitat, with laterally positioned eyes associated with

Received 18 February 2020; Accepted 12 April 2020 pelagic species, and dorsally positioned eyes associated with a Journal of Experimental Biology

1 RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb223644. doi:10.1242/jeb.223644 sedentary, benthic habit (Aleev, 1969; Hynes, 1970; Gatz, 1979; predation risk and whether the site was partly shaded by surrounding Watson and Balon, 1984; Motta et al., 1995; Frédérich et al., 2016). rock formations. Geometric morphometric analyses were used to Fishes that locate and attack prey from below often have dorsally evaluate eye position and head shape, while subsequent model oriented, upward-facing eyes (Pankhurst and Montgomery, 1989; selection allowed us to determine which of the environmental Warrant and Locket, 2004; Lisney and Collin, 2008), while benthic- variables best explained variation in eye size, pupil size and eye feeding fishes often also have dorsally positioned eyes, which may position in the head. facilitate predator detection when feeding in a head-down position (Bellwood et al., 2014). Thus, the relationship between the position MATERIALS AND METHODS of the eyes and an animal’s functional requirements requires a Sample sites and fish capture detailed understanding of a species’ behaviour and ecology. Adult western rainbowfish were sampled from 14 locations across Although much is known about the relationships between eye the Fortescue River catchment in the Pilbara region of northwest morphology and the behaviour and ecology of a species, far less is Western Australia in May–November 2013. The upper and lower known about how eye morphology varies within species. This is sub-catchments within our sampling region are considered to be surprising because a single species can occupy a wide variety of hydrologically isolated because they are separated by a geographic habitats, hence requiring different types of visual specialisation. barrier, the Goodiadarrie Hills (Skrzypek et al., 2013). We sampled Intraspecific differences in the size and morphology of compound a total of 312 individuals originating from seven sites in the upper eyes have been reported in insects, and these differences correlate Fortescue catchment (Coondiner Creek, Kalgan Creek and Weeli with variation in activity patterns among different strains or castes, Wolli Creek), two in the mid catchment (in Karijini National Park) and varies between the sexes (e.g. Roonwal and Bhanotar, 1977; and five in the lower catchment (Millstream National Park) Posnien et al., 2012; Streinzer et al., 2013). With regard to (Table S1; mean number of fish sampled per site=22.3, range=4– vertebrates, freshwater fish provide some interesting examples of 31). Our sampling design, and the number of fish captured, was intraspecific variation in eye morphology. For example, in the restricted due to the availability of freshwater habitats within this Atlantic molly (Poecilia mexicana), a species that has colonized semi-arid region. The Pilbara is characterised by hot summers caves in southern Mexico, there is a gradual reduction in eye size (24–40°C) and highly unpredictable rainfall, which mainly occurs from the chambers closest to the surface that receive some dim light, in the summer as a result of cyclonic activity (www.bom.gov.au). to the deepest chambers that are in constant darkness (Fontanier and For most of the year, creeks within this region therefore comprise a Tobler, 2009). In other freshwater fishes, such as red shiners series of discrete pools that are maintained by groundwater and (Cyprinella lutrensis) and the galaxid Aplochiton zebra, individuals become reconnected only during major flooding events (Beesley that live in turbid habitats have relatively larger eyes than their and Prince, 2010). counterparts living in clearer waters, presumably as an adaptation to Sampling was conducted between 10:00 h and 15:00 h, the time improve visual sensitivity (Lattuca et al., 2007; Dugas and Franssen, of day at which western rainbowfish tend to be most active, and to 2012). However, such studies that have examined within-species account for any difference in diurnal activity patterns between variation in eye morphology have typically considered the effect of sample sites. Fish were captured using seine nets (5 or 10 m length, a single environmental variable (usually light availability), rather 6 mm mesh size, depending on site) using multiple hauls (∼3–10 than the multiple and interacting ecological factors that will net captures) to remove all fish from the immediate sampling area determine visual performance. Additionally, previous work has (representing a surface area of approximately 150 m2). We also largely focused on eye diameter as a measure of eye morphology ensured that the region that was sampled was representative (e.g. and has not considered the size of the aperture (pupil) or the percentage cover of vegetation, etc.) of that particular habitat, and position of the eyes on the head, variables that can reveal important we used a similar level of fishing effort at each sample site. After information about the ecology, behaviour and activity patterns of a each seine haul, all fish were placed in lidded buckets (containing species within a given habitat. creek water and vegetation for cover) until the area had been In this study, we investigated three aspects of eye morphology – thoroughly sampled. After this time, we randomly selected adults (1) eye size relative to body size, (2) pupil size relative to eye size from the buckets, and photographed them out of the water on their and (3) eye position in the head – in a species of Australian right side using a digital camera (Olympus E-PL3, Olympus freshwater fish, the western rainbowfish [Melanotaenia australis Corporation, Tokyo, Japan) mounted on a tripod. Following the (Castelnau 1875)]. We chose this species because it occupies a large photography, fish were placed in a separate holding bucket, to avoid variety of freshwater habitats across northwest Western Australia, re-photographing the same individuals. We did not use anaesthesia including isolated springs and billabongs (pools), ephemeral during the photography because the procedure took only a few creeks, gorges and lakes (Allen et al., 2002). Previous studies seconds. Each image included a scale bar for subsequent image have shown that this species shows extensive variation in scaling. We determined the sex of each fish by placing them morphological characteristics, such as body shape and lateral line individually in a transparent container to examine the morphology morphology, in relation to environmental parameters (Young et al., of the dorsal and ventral fins. Males have pointed dorsal and ventral 2011a,b; Kelley et al., 2017; Spiller et al., 2017). However, no fins, while the fins of females are more rounded in shape (Allen previous study has specifically investigated variation in eye et al., 2002). Once the adults in the habitat had been photographed, morphology, even though these fish are considered to rely heavily all fish were returned to their natural habitat (including those that on vision for feeding, communication, sexual selection and predator were not photographed) at their location of capture. This study was detection (Brown and Warburton, 1997; Arnold, 2000; Brown, approved by the University of Western Australia Animal Ethics 2002, 2003; Hancox et al., 2010; Kelley et al., 2012). We collected Committee (protocol: RA/3/100/1176). western rainbowfish from 14 sites in a major river catchment and surveyed each site for a number of environmental and ecological Ecological habitat characterisation characteristics that may influence eye size and morphology, Each sample site was characterised according to a number of including turbidity, invertebrate abundance, habitat complexity, environmental and ecological characteristics (Table S1). This was Journal of Experimental Biology

2 RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb223644. doi:10.1242/jeb.223644 aided by taking photographs of each habitat, and drawing detailed A maps to record the substrate, details of submerged vegetation and 1 cm rocks, surrounding riparian vegetation and overhanging vegetation or rock structures. We determined the amount of direct daylight each habitat was exposed to by examining the surrounding rock formations, such as whether sites were located in gorges or creek lines, and allocating each site a ‘gorge factor’ score between 0 and 2. Sites scoring 2 had high (>10 m) rock face on either side (i.e. sites Fork length in Karijini National Park) and were therefore only exposed to direct sunlight during midday hours. Sites scoring 1 had rock face on one BCD side (<10 m high; sites in Coondiner Creek), and therefore were Pupil diameter partially shaded for one half of the day, and sites scoring 0 were not located in gorges or creek lines and received direct sunlight throughout the day (sites in Millstream National Park). A habitat complexity score was used to represent the level of visual habitat Eye diameter structure present (Lostrom et al., 2015). Habitat structure, such as substrate and submerged vegetation, as well as debris such as snags, Fig. 1. An illustration of the eye morphology variables measured in this provide navigational obstacles and shelter, but also influences the study. (A) Image of western rainbowfish captured from Coondiner Creek (site HD1.5) in the Pilbara region of northwest Australia, illustrating fork length, light environment by creating shade. A habitat complexity score measured from the tip of the upper jaw to the fork in the tail. (B) Magnification of ranging from 1 to 5 was given for each habitat, where 1 was allocated the eye, showing the eye diameter and pupil diameter measurements. to open water habitats (e.g. large pools >1 km long) with gravel (C) Magnification of the head region, illustrating landmark placement of two substrates, while a score of 5 represented dense aquatic vegetation and fixed landmarks (white) and 10 semi-sliding landmarks (red). (D) An illustration submerged debris. Habitat complexity scores were allocated by two of the main features of the head described by the landmarks. independent observers, by referring to both the habitat maps and the photographs of each site. The scores were then collated and adjusted scaled photographs of each fish (Fig. 1) using ImageJ software (for one site) to reach a consensus. This procedure was performed (Schneider et al., 2012). The horizontal diameters of the eye and before the eye morphology data were collected. pupil were used to calculate a pupil/eye diameter ratio for each fish. Water velocity was measured using a flow meter (FP111; Global By using pupil size as a measure of the aperture of the eye in western Water, College Station, TX, USA) placed 10 cm below the surface rainbowfish, we assumed that this species does not exhibit any and averaged from three readings. We determined the predation significant pupil mobility, as is the case for the vast majority of bony pressure at each site using previous records of fish predator fishes (Douglas, 2018). Indeed, throughout our own experimental assemblages in the sampling areas (Young et al., 2011b), work with rainbowfish we have not observed any changes in pupil augmented by our own observations at the time of sampling. Fish size in response to factors such as changes in background illumination predators of rainbowfish have previously been categorised as ‘high or handling, for example. Nevertheless, we acknowledge that a very risk’ (e.g. barramundi, Lates calcarifer; western sooty grunter, small number of predominantly benthic bony fishes have been found Hephaestus jenkinsi)or‘low risk’ (e.g. spangled perch, to have mobile pupils (Douglas et al., 2002; Douglas, 2018), and so Leiopotherapon unicolor; barred grunters, Amniataba percoides) pupil size may not necessarily be an appropriate measure of the size of (Young et al., 2011b), and we used these classifications to assign the aperture of the eye in these species. each site a value of 0 (only low-risk predatory species present) or 1 We used geometric morphometric analyses (Zelditch et al., 2012) (where 1 or more high-risk predatory species have been recorded). to quantify variation in the position of the eye and shape of the head We sampled the total number of surface invertebrates by sweeping a of each fish. Images were scaled for size according to the scale bar in 250 µm mesh dip net over a 10 m length of the pool (Lostrom et al., each image before using the software program TPSDIG (version 2015). We collected three samples for each pool and calculated 2.17; available at http://life.bio.sunysb.edu/morph/) to assign the average number of surface invertebrates present. Benthic landmarks to each image. A total of 12 landmarks were placed on invertebrates were sampled using a 500 µm mesh D-net and each image: two landmarks were fixed and positioned on the tip of trampling the sediment over a 1 m2 area for 1 min. The contents of the snout and at the top of the operculum plate, while 10 were semi- the net were then passed through 2 mm and 500 µm steel mesh sliding landmarks and were assigned along the outline of the head sieves before counting all invertebrates present. The percentage and the back of the operculum (Fig. 1). The program TPSRELW cover of green filamentous macroalgae was also evaluated based on was subsequently used to generate a series of relative warps (RWs) on-site observations and used as a measure of food availability that describe overall changes in eye position and head shape, (rainbowfish are omnivorous). A water sample (30 ml, unfiltered) and centroids (measured as the squared distance of each landmark was collected at each site and kept cool and in the dark until from the mean or central position), which represent overall head subsequently analysed for turbidity in the laboratory. A turbidity size. The data supporting this study are publicly available at the meter (Hach 2100A; Hach, Loveland, CO, USA) was used to obtain University of Western Australia’s Research Repository (doi:10. three turbidity measures for each pool; the mean value was used in 26182/5e4b93d5b9a70). subsequent analyses. Statistical analyses Image analysis Eye size scales with body size in fish (Howland et al., 2004; Lisney In order to measure eye size relative to body size, and pupil size and Collin, 2007; Schmitz and Wainwright, 2011; Caves et al., relative to eye size, the horizontal diameters of the eye and the pupil, 2017). Therefore, we initially examined the relationship between along with fork length (FL; measured from the tip of the upper jaw body size (FL) and both eye diameter and the pupil/eye diameter to the fork in the tail), were measured to the nearest 0.1 mm from the ratio. There was a significant positive correlation between body size Journal of Experimental Biology

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and eye diameter (r311=0.90, P<0.001), but no relationship between region (Fig. 2) shows that the collection locations were clustered into the pupil/eye diameter ratio and body size (r311=−0.03, P=0.62). three discrete regions (sub-catchments). We therefore accounted Therefore, we used the residuals of the regression relationship for this in our analyses by entering the term ‘sub-catchment’ (three between body size and eye diameter in all further analyses. Use of levels) as a random effect in all models. We then considered a the residuals allows us to determine whether fish have a larger priori which of the ecological and environmental variables might (positive residuals) or smaller (negative residuals) eye relative to influence eye morphology (Table S2); these included gorge factor their body size. The residuals of eye diameter were not correlated (three levels), predation risk (two levels), habitat complexity (ordered with the pupil/eye diameter ratio (r311=−0.05, P=0.36). factor with 5-levels), invertebrate abundance (benthic+surface invertebrates: a covariate) and turbidity (a covariate). Prior to Testing for variation in eye size and position among collection sites running the linear mixed models, we performed data exploration, Our first set of analyses used ANOVA/MANCOVA to test whether following the methods outlined in Zuur et al. (2010) to explore the there was any variation among sample sites, or any variation structure of our data, to examine relationships among the variables, attributable to sex, in our measures of eye size and morphology. We and to test the validity and assumptions of our models. Thus, in then conducted a further set of tests, using linear mixed models, to addition to examining the plots of the residuals versus the fitted examine the role of the ecological and environmental predictors in values, we constructed conditional plots to verify homogeneity of explaining variation in eye morphology (while controlling for each of the factors, and histograms (for each factor) to investigate sampling design). For the ANOVA/MANCOVA tests, we tested the skewness in the data. We found that some of the environmental effect of sex (entered as a fixed effect) because western rainbowfish characteristics were correlated: surface water velocity was negatively are sexually dimorphic (Allen et al., 2002; Lostrom et al., 2015) and correlated with both turbidity (r311=−0.16, P=0.005) and filamentous because sexual dimorphism in eye size has been reported in a macroalgae cover (r311=−0.41, P<0.001), and the level of number of fish species (Echeverria, 1986; Cooper et al., 2011; filamentous macroalgae was positively correlated with habitat Webster et al., 2011; Dugas and Franssen, 2012; Záhorská et al., complexity (Spearman’s rank test: ρ=0.60, n=312, P<0.001). To 2013). We tested for an effect of collection site (entered as a fixed avoid potential issues associated with collinearity, we dropped the factor), sex and the site by sex interaction on the eye diameter covariates surface water velocity and filamentous macroalgae as these residuals and the pupil/eye diameter ratio using ANOVA. We used were deemed less biologically relevant for visual tasks than turbidity MANCOVA, with centroid entered as the covariate to account for and habitat complexity. One site (Kalgan) had unexpectedly higher body size, to investigate the effect of these factors on the first five turbidity values than the rest, and only four fish were captured RWs (RW1–RW5) describing variation in eye position in the head. (Table S1), increasing the probability that these samples are not Centroid and sex were retained in subsequent models if they had a representative of the population. We chose to exclude these samples significant effect in the MANCOVA. from our turbidity analyses; we confirmed that removal of these four fish had no effect on the model outcomes for either the turbidity Testing the relative importance of the ecological and environmental analyses, or on the modelling for each of our dependent variables. We variables on eye size and position also noted that treating habitat complexity as a fixed effect (low We determined the importance of the ecological and environmental complexity=scores 1–2; high complexity=scores 3–5) had no variables on RW1 (which accounts for the most variation in eye size/ outcome on the model rankings. Our model selection approach shape; 34.5%) using linear mixed models. These models allowed us also included fitting a null model, which contained only the to evaluate the importance of the predictors while controlling for the intercept, and we compared the top-fitting model with the null spatial component of the sampling design. A map of the sampling model using the likelihood ratio (LR) test. We used Akaike’s

Fig. 2. Map of the sample sites (scale bar=50 km) showing that locations are clustered into three sub-catchments: the upper (orange), mid (green) and lower (blue) regions of the Fortescue River in northwest Western Australia (inset). The yellow symbol represents the location of a geographic barrier (the Goodiadarrie Hills) that isolates the upper and lower sub-catchments. Images obtained from GoogleMaps. Journal of Experimental Biology

4 RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb223644. doi:10.1242/jeb.223644 information criterion for smaller sample sizes (AICc) to evaluate with ΔAICc<2 were considered equal best models (Symonds and the fit of each model, where models with lower AICc values are Moussalli, 2011). All statistical analyses were performed using the considered more parsimonious (Symonds and Moussalli, 2011). software program R (https://www.r-project.org/) using the software Models with a change in AICc (ΔAICc) >10 were excluded from the packages lme4 (Bates et al., 2015) and AICcmodavg’ (https://CRAN. model set, those with 6<ΔAICc<10 were considered unlikely and those R-project.org/package=AICcmodavg).

A

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Fig. 3. Variation in eye morphology over different geographic sampling scales. Mean (±1 s.e.m.) values for (A) eye diameter residuals, (B) the pupil/eye diameter ratio and (C) RW1 attributable to collection site (left panels) and catchment (right panels). Collection site codes and sample sizes are given in Table S1 and are grouped according to catchment (orange, lower, N=128; blue, mid, N=51; green, upper, N=133). Journal of Experimental Biology

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RESULTS centroid size (r311=−0.10, P=0.067). These findings suggest that Eye size and pupil/eye diameter negative allometry is associated with eye and head morphology and The ANOVAs revealed a significant effect of collection site on both all subsequent models thus included centroid as a covariate. the eye diameter residuals (F13,284=16.8, P<0.001) and the pupil/ The MANOVAs revealed an overall effect of site (F13,296=8.02, eye diameter ratio (F13,284=14.4, P<0.001; Fig. 3; Table S3), but no P<0.001) and centroid (F1,196=23.6, P<0.001) on the combined effect of sex on either of these parameters (eye diameter residuals: RWs (RW1–RW5), but no effect of sex (F1,296=0.78, P=0.57). The F1,284=2.24, P=0.14; pupil/eye diameter: F1,284=3.47, P=0.064) linear mixed models and subsequent model selection procedure and no significant site×sex interaction (eye diameter residuals: were performed only for RW1, which explained the greatest F13,284=0.44, P=0.95; pupil/diameter size: F13,284=0.56, P=0.89). proportion of variation in shape (34.5%), and excluded the factor The sex factor was therefore not included in subsequent models. sex. The linear mixed models revealed that habitat complexity was The model selection analyses (Table 1) revealed that habitat the top-fitting model (ΔAICc<2) in explaining variation in RW1 complexity was the top model (ΔAICc<2) for the residuals of eye (Table 1). The model containing the effect of habitat complexity diameter and was significantly better than the null model that was significantly better than the null model that contained only the contained only the random intercept (LR test: χ2=47.1, d.f.=4, random intercept and the centroid (LR test: χ2=59.3, d.f.=5, P<0.001; Table 1). The residuals of eye diameter differed according P<0.001; Table 1). Fish from sites with a habitat complexity score to habitat structure such that fish occurring in habitats with the of 2 (low complexity) had significantly lower RW1 scores ( post hoc lowest complexity score (i.e. 1=open water) tended to have smaller Tukey tests; P<0.01; Fig. 4B), meaning that they had more dorsally eyes than those occurring in habitats with higher complexity scores located eyes, less sloped heads and broader operculum plates than (i.e. dense vegetation; Fig. 4A). Variation in the pupil/eye diameter those from sites with higher levels of habitat complexity. ratio was best explained by the gorge factor (comparison to null model: LR test: χ2=27.21, d.f.=2, P<0.001; Table 1). Fish DISCUSSION occupying gorge habitats had significantly smaller pupil/eye Investigations of intraspecific variation in eye morphology are diameter ratios than those occupying half gorge sites or open relatively rare, but have the potential to reveal how environmental habitats (Table S3; Fig. 5). and ecological variables contribute to visual specialisations. In this study, we investigated eye morphology in a single species of Australian Eye position freshwater fish, the western rainbowfish, which is found in a range of The first five RW scores (RW1–RW5) explained 85.8% of the total habitats with diverse ecological and environmental characteristics variation in shape (RW1=34.5%, RW2=20.9%, RW3=15.1%, (Allen et al., 2002; Lostrom et al., 2015). We found significant RW4=8.5%, RW5=6.7%). Visualization of the relative warps variation in all three aspects of eye morphology that we investigated – using ordination plots (Fig. 6) revealed that negative RW1 scores eye size relative to body size, pupil size relative to eye size, and the were associated with an elevated eye position, reduced slope of the position of the eye in the head – among individuals collected from head and broadening of the operculum plate relative to positive different habitats in a single river catchment. Our modelling approach, scores. RW2 described the overall bend of the head (upwards tilt for which allowed us to evaluate the relative role of the environmental and negative values), while RW3 accounted for the overall widening of ecological variables, revealed that the size of the eye and pupil, and the the posterior region of the head (broader for negative scores). Note position of the eye in the head, are related to two habitat components: that RW1–RW3 were correlated with the pupil/eye diameter ratio the structural complexity of the habitat and the presence of surrounding (RW1: t310=2.50, P=0.013; RW2: t310=2.57; RW3: t310=−2.37, rock formations (i.e. gorges). Our findings reveal that multiple, P=0.018). Centroid size was significantly negatively correlated with interacting factors influence eye morphology in western rainbowfish, RW1 (r311=−0.31, P<0.001) and RW3 (r311=−0.15, P<0.010), and suggesting that visual systems are not only species-specific, but there was a negative trend for an association between RW2 and dependent on an individual’s habitat and behavioural tasks.

Table 1. Model selection procedure used to test which of the linear mixed models (testing each of the environmental predictors) best explains variation in the residuals of eye diameter, pupil/eye size ratio and variation in relative warp 1 (RW1) Response variable Effect k AIC ΔAICc Estimate±s.e. tR2 Residuals of eye diameter Habitat complexity 7 304.5 0.00 Complexity (1): 0.09±0.05 3.55 0.11 Complexity (2): −0.11±0.02 −5.57 Complexity (3): 0.02±0.02 0.76 Complexity (4): −0.02±0.02 −0.97 Null model 4 −264.4 40.07 – –– Pupil/eye size Gorge score 5 −1519.8 0.00 Gorge (H): −1.24e−3±1.34 e−2 −0.09 0.13 Gorge (O): 0.017±0.013 1.31 Null model 3 −1496.7 23.09 – –– RW1 Habitat complexity+centroid 8 −1217.9 0.00 Complexity (1): 5.16e−3±5.82 e−3 0.89 0.17 Complexity (2): 2.83e−3±4.89e−3 0.58 Complexity (3): −1.80e−2±5.20e−3 −3.47 Complexity (4): 1.87e−2±4.90e−3 3.82 Centroid: −3.33e−4±4.96e−5 −6.71 Null model 3 −1169.1 48.86 ––– Centroid was entered as a covariate for the RW1 model and sub-catchment was a random effect in all models. k, number of parameters in the model; AIC, Akaike’s information criterion; ΔAICc, change in AICc. Only models with ΔAICc<10 are shown, along with the null model containing a random intercept. Habitat complexity score ranges from open water habitats (score=1) to sites with dense vegetation (score=5). The gorge score classified sites as open with no shading rock formations (O, score=0), half gorge sites (H, score=1) or gorge sites (score=2). Estimates of the fixed effects and t are also shown, along with the variance 2 explained by the fixed effects (marginal R ). Journal of Experimental Biology

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A ** *** *** 0.50 *** *

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*** 0 0.45

–0.1

Mean eye diameter residuals 0.40

–0.2 Pupil/eye diameter ratio

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0.35 B *** Gorge Half gorge Open ** Gorge score *** Fig. 5. Histograms showing the relationship between exposure to direct ** sunlight in gorge sites (shaded by rock >10 m high), semi-gorge sites (surrounding rock <10 m high) and open habitats (no shading rock 0 formation) and the mean (±1 s.e.m.) pupil/eye diameter ratio. Note the scale on the y-axis; significant paired comparisons are indicated with asterisks ( post hoc Tukey tests: ***P<0.001, *P<0.05). Sample sizes are N=51 (gorge sites), 69 (semi-gorge sites) and 192 (open sites).

Mean RW1 –0.02 (Kirk, 1994). Complex habitats may therefore favour increased visual sensitivity, which can (along with other visual adaptations) be attained by increasing the diameter of the eye (Land and Nilsson, 2012). A larger eye can also increase spatial resolution, which may be –0.04 advantageous for visually mediated behaviours such as prey 12345 detection and navigation in structurally complex environments Habitat complexity score (Hughes, 1977). A recent comparative study of 159 species of ray- finned fishes found that a large eye size was associated with Fig. 4. The relationship between habitat complexity and eye morphology. increased visual acuity, and that species in complex habitats tended Histograms of the relationship between habitat complexity score [ranging from open water habitats (score=1) to sites with dense vegetation (score=5)] and the to have higher visual acuity than expected after accounting for eye mean (±1 s.e.m.) residuals of (A) eye diameter and size relative to body size (Caves et al., 2017). A positive relationship (B) relative warp 1 (RW1). Asterisks indicate significant paired comparisons between eye size and visual acuity is also supported by a study of ( post hoc Tukey tests; ***P<0.001, **P<0.01). Note that the boxplot marked closely related cichlids (from the same clade) that found that a *** in A is significantly different to all other plots (P<0.001). Sample sizes for each species from highly structured rock habitats (Asprotilapia leptura) habitat complexity value are N=41, 29, 125, 72 and 45 for habitat complexity had higher visual acuity (measured behaviourally) than species from values of 1, 2, 3, 4 and 5, respectively. sandy habitats (Xenotilapia flavipinnis) and intermediate sandy/ rocky habitats (X. spiloptera) (Dobberfuhl et al., 2005). Visual Habitat complexity and eye size and position acuity can also be evaluated by mapping retinal topography, and in Our finding that rainbowfish eye size is linked to the structural coral reef fishes, differences in the number and density of ganglion complexity of the habitat is supported by other studies showing that fish cells are linked with a species’ habitat (Collin and Pettigrew, from structurally complex habitats tend to have larger eyes than those 1988a,b). Specifically, the sampling region of the visual field is from less complex habitats (Dobberfuhl et al., 2005; Willacker et al., specialised depending on whether the species views a broken horizon 2010). Larger eyes may be associated with more complex habitats or an unobstructed horizon, resulting in species-specific variation in because these environments favour increased visual sensitivity and/or the visual acuity (Collin and Pettigrew, 1988a,b, 1989). Collectively, need for higher spatial resolution (Land and Nilsson, 2012). Complex these previous studies lead us to predict that rainbowfish in structured benthic habitats may have reduced light availability owing to habitats have relatively large eyes in order to increase visual acuity attenuation of light with depth (Lythgoe, 1979), and because of and to enhance behavioural performance in these environments. It shading from macrophytes or other submerged structures (e.g. tree would therefore be interesting to assess visual acuity (either branches). Further reductions in light, along with shifts in spectral behaviourally or from retinal structure) and examine corresponding composition, can occur if complex habitats also contain high amounts visual behaviours (e.g. foraging tasks) in western rainbowfish to of suspended sediments, phytoplankton and dissolved organic matter determine whether there are any population differences. Journal of Experimental Biology

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A B Fig. 6. Variation in head morphology and eye position explained by the first three relative warps. (A,B) RW1, (C,D) RW2 and (E,F) RW3 account for 34.5%, 20.9% and 15.1%, respectively, of the total variation in shape. Warp scores (negative scores: A, C, E; positive scores: B, D, F) were generated in the software program TPSRELW and visualised using ordination plots. The outline shape of the head and operculum have been added to aid interpretation.

C D

E F

In this study, the structural complexity of the habitat also We similarly anticipate that the differences in head shape and eye influenced the position of the eye in the head, as well as the slope of position in the present study will relate to variation in behaviour in the head and the width of the operculum plates. Specifically, rainbowfish; in complex habitats, more ventrally located eyes may rainbowfish from complex habitats tended to have more ventrally facilitate detection of predators or prey that are located below the located eyes, along with more sloped heads and narrower operculum fish. Although habitat complexity was the most important predictor plates compared with those from less complex habitats. Most studies of variation in eye size and eye position in this study, it is important that have reported a link between habitat complexity and to acknowledge that other habitat variables (including correlates of morphology in fishes have focused on the divergence between habitat complexity, such as water flow and macroalgal cover) may two classic morphotypes – benthic/littoral morphs that feed on also underlie our results. Controlled laboratory experiments are invertebrates in sediments or on macrophytes, and limnetic forms clearly necessary to establish cause and effect. We also that feed mainly on zooplankton in the open water (Schluter and acknowledge that our findings may be influenced by sampling McPhail, 1993). This results in a strong relationship between trophic bias, if, for example, the spatial and temporal activity patterns, morphology and diet (e.g. Svanbäck and Eklov, 2002, 2003), which habitat preferences and survival of fish are differentially affected by may also determine the location of the eyes in the head. In an individual’s eye morphology. sticklebacks (Gasterosteus aculeatus), for example, benthic forms have more dorsally located eyes than limnetic forms (Behm et al., Light availability and pupil size 2010), which could contribute to their increased foraging success on Because light intensities in shaded habitats can be several orders of susbtrates relative to limnetic forms (Bentzen and McPhail, 1984). magnitude lower than those in open habitats (Ovington and Journal of Experimental Biology

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Madgewick, 1955; Endler, 1993; Fleishman et al., 1997), we Acknowledgements predicted that fish in the shaded gorge habitats would have larger We would like to thank an anonymous reviewer and Mateo Santon for comments on the manuscript that greatly improved the scope and quality of our work. We are eyes with relatively larger pupils, in order to increase the light- grateful to S. Lostrom, P. Grierson, J. Evans and P. Davies for advice and support gathering capabilities of the eyes. For example, nocturnal reef fishes and to S. Luccitti, S. Wild, A. Storey, J. Delaney, J. Iles, A. Siebers, K. Bowler and have relatively larger eyes and larger, more rounded pupils than G. Skrzypek for assistance in the field. We would like to acknowledge the rangers at diurnal reef species (Pankhurst, 1989; Schmitz and Wainwright, Karijini and Millstream National Parks for information and access to sampling sites. 2011). However, our results contradict this pattern as we found that fish from sites surrounded by rock formation (i.e. reduced light Competing interests The authors declare no competing or financial interests. availability) had relatively smaller pupils for a given eye diameter than those from semi-gorge or open sites. A more detailed consideration of Author contributions eye morphology, for example, incorporating the diameter of the lens Conceptualization: T.J.L., J.L.K.; Methodology: T.J.L., J.L.K.; Formal analysis: and the shape of the pupil (e.g. the optical ratio), might reveal subtle T.J.L., J.L.K.; Writing - original draft: T.J.L.; Writing - review & editing: T.J.L., S.P.C., morphological adaptations for enhanced visual sensitivity (Schmitz J.L.K.; Visualization: J.L.K.; Funding acquisition: S.P.C., J.L.K. and Motani, 2010). Future studies could also determine whether Funding individuals from the dimmer gorge habitats possess retinal adaptations This work was supported by the Australian Research Council [LP120200002 to for improving visual sensitivity, such as larger photoreceptors, a S.P.C., FT180100491 to J.L.K.] and an Endeavour Research Fellowship from the higher proportion of rod photoreceptors or higher photoreceptor to Department of Education of the Australian Government [T.J.L.]. retinal ganglion cell summation ratios (Marshall, 1979; Wagner, 1990; Land and Nilsson, 2012), and whether these adaptations are Data availability Supporting data are publicly available at the University of Western Australia’s associated with improved visual performance. Research Repository (doi:10.26182/5e4b93d5b9a70).

Turbidity, predation risk and invertebrate abundance Supplementary information Turbidity can significantly hinder vision underwater, causing a Supplementary information available online at reduction in visual resolution (Wells, 1969), a reduced visual range http://jeb.biologists.org/lookup/doi/10.1242/jeb.223644.supplemental and decreased target contrast (Lythgoe, 1979; Utne-Palm, 2002). In References contrast to other studies with fishes (Kotrschal et al., 1991; Huber Aleev, Y. G. (1969). Function and Gross Morphology in Fish (translated from and Rylander, 1992; Huber et al., 1997; Lisney and Collin, 2007; Russian, 1969). Jerusalem: Israel Program for Scientific Translations. Caves et al., 2017), we found no evidence that fish in turbid habitats Allen, G. R., Midgley, S. H. and Allen, M. (2002). Field Guide to the Freshwater have smaller eyes. In addition to investing in non-visual senses, such Fishes of Australia. Perth: Western Australian Museum. Arnold, A. E. (2000). Kin recognition in rainbowfish (Melanotaenia eachamensis): as chemoreception (Kotrschal et al., 1998), this finding may be due sex, sibs and shoaling. Behav. Ecol. Sociobiol. 48, 385-391. doi:10.1007/ to the fact that turbidity values were generally similar at all sites at s002650000253 the time of sampling. Although many fishes rely on vision for Beesley, L. S. and Prince, J. (2010). Fish community structure in an intermittent predator detection and evasion (Chivers et al., 2001; Fischer et al., river: the importance of environmental stability, landscape factors and within-pool habitat descriptors. Mar. Freshw. Res. 61, 605-614. doi:10.1071/MF09137 2017), we also found no relationship between the position of the Bates, D., Mäechler, M., Bolker, B. and Walker, S. (2015). Fitting linear mixed- eyes and predation risk. One explanation for this is our broad effects models using lme4. J. Stat. Soft. 67, 1-48. doi:10.18637/jss.v067.i01 characterisation of risk (i.e. presence/absence of predator species) Behm, J. E., Ives, A. R. and Boughman, J. W. (2010). Breakdown in postmating rather than using a more stringent measure, such as predator density isolation and the collapse of a species pair through hybridization. Am. Nat. 175, 11-26. doi:10.1086/648559 or diel activity patterns. Finally, because large eyes are associated Bellwood, D. R., Goatley, C. H. R., Brandl, S. J. and Bellwood, O. (2014). Fifty with zooplanktivory and insectivory in fishes (Motta et al., 1995; million years of herbivory on coral reefs: fossils, fish and functional innovations. Huber et al., 1997; Behm et al., 2010), we predicted a relationship Proc. R. Soc. B 281, 20133046. doi:10.1098/rspb.2013.3046 between invertebrate abundance and eye size. However, as Bentzen, P. and McPhail, J. D. (1984). Ecology and evolution of sympatric suggested by these previous studies (Motta et al., 1995; Huber sticklebacks (Gasterosteus): specialization for alternative trophic niches in the Enos Lake species pair. Can. J. Zool. 62, 2280-2286. doi:10.1139/z84-331 et al., 1997; Behm et al., 2010), habitat use is probably a more Brown, C. (2003). Habitat-predator association and avoidance in rainbowfish important contributor to morphological variation in eye size in (Melanotaenia spp.). Ecol. Freshw. Fish 13, 118-126. doi:10.1034/j.1600-0633. rainbowfish than diet, even though the two are clearly linked. 2003.00007.x Brown, C. (2002). Do female rainbowfish (Melanotaenia spp.) prefer to shoal with familiar individuals under predation pressure? J. Ethol. 20, 89-94. doi:10.1007/ Conclusions s10164-002-0059-6 A variety of environmental and ecological factors have been Brown, C. and Warburton, K. (1997). Predator recognition and anti-predator invoked to explain interspecific variation in eye size and shape, but responses in the rainbowfish Melanotaenia eachamensis. Behav. Ecol. Sociobiol. few studies have examined variation in the visual system of a single 41, 61-68. doi:10.1007/s002650050364 species. Our findings suggest that individuals vary in the degree to Caves, E. M., Sutton, T. T. and Johnsen, S. (2017). Visual acuity in ray-finned fishes correlates with eye size and habitat. J. Exp. Biol. 220, 1586-1596. doi:10. which they invest in their visual system, which we predict will 1242/jeb.151183 translate to differences in visual sensitivity and visual acuity among Chivers, D. P., Mirza, R. S., Bryer, P. J. and Kiesecker, J. M. (2001). Threat- populations, with corresponding implications for behavioural sensitive predator avoidance by slimy sculpins: understanding the importance of performance. The extent to which the sensory systems of fishes visual versus chemical information. Can. J. Zool. 79, 867-873. doi:10.1139/z01-049 Collin, S. P. and Hart, N. S. (2015). Vision and photoentrainment in fishes: The can be adapted to specific ecological and environmental conditions effects of natural and anthropogenic perturbation. Integrat. Zool. 10, 15-28. doi:10. means that they may also be highly susceptible to rapid changes 1111/1749-4877.12093 caused by human activity (Collin and Hart, 2015; Kelley et al., Collin, S. P. and Pettigrew, J. D. (1988a). Retinal topography in reef I: 2018). Such impacts may be particularly significant in species that some species with well-developed areae but poorly-developed streaks. Brain Behav. Evol. 31, 269-282. doi:10.1159/000116594 exhibit habitat-specific variation in eye morphology because visual Collin, S. P. and Pettigrew, J. D. (1988b). Retinal topography in some reef teleosts specialisations may no longer enhance fitness and may be II: some species with prominent horizonatal streaks and high-density areae. 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