Animal Behaviour 83 (2012) 783e791

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Animal Behaviour
journal homepage: www.elsevier.com/locate/anbehav

Changes in the visual environment affect colour signal brightness and

shoaling behaviour in a freshwater fish
Jennifer L. Kelley*, Bree Phillips 1, Gabrielle H. Cummins 2, Julia Shand 3
Centre for Evolutionary Biology, Experimental & Regenerative Neurosciences, School of Animal Biology (M092), University of Western Australia, Perth, Australia

a r t i c l e i n f o
Aquatic organisms are exposed to highly variable light environments, which can affect the efficacy of colour
Article history: patterns that are used for communication or camouflage. Specifically, dissolved organic matter that is
Received 25 August 2011 common in turbid freshwater habitats tends to absorb short wavelength light causing a shift towards
Initial acceptance 4 November 2011 environments that are rich in long wavelengths (orange/red). We investigated how changes in the intensity
Final acceptance 13 December 2011 and wavelength of light affect colour pattern expression and shoaling behaviour in a colourful freshwater
Available online 26 January 2012 fish, the western rainbowfish, Melanotaenia australis. We used light filters to simulate environments rich
MS. number: 11-00676R in organic matter (yellow filters: long wavelength dominated, reduced luminance), habitats with
full-spectrum lighting but with reduced luminance (neutral-density filters) and a control in which no
Keywords: changes in the light environment occurred (no filters). We measured changes in the area and brightness of
adaptive coloration
colour patterns using digital photography and spectrometry and we evaluated the effect of lighting on fish
colour vision
social (shoaling) behaviour. Rainbowfish in the dissolved organic matter treatment showed an increase in
crypsis
Melanotaenia australis
the area and brightness of their colour patterns and individuals shoaled further apart than those in the
rainbowfish control group. The increased brightness of red colours in environments rich in organic matter could act to
shoaling enhance colour pattern conspicuousness, allowing individuals to maintain communication in altered
signalling visual environments. However, an understanding of the species’ visual system is required to determine
turbidity levels of contrast of the colour patterns with respect to variable background environments.
visual ecology Ó 2012 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Animal communication systems are a result of the combined
evolution of signal design and sensory perception (Endler 1992).
The environment has an important effect on this evolutionary
outcome because the efficacy of a signal depends on the trans-
mission properties of the medium (e.g. air/water) and the sensory
background (e.g. noise, visual complexity) against which the signal
is detected (Endler 1990). Selection must favour the signalling
system that is most effective in a particular environment, which, in
the case of sexually selected signals, can promote the processes of
reproductive isolation and speciation (Endler 1992; Endler & Basolo
1998; Boughman 2002).
Numerous studies have demonstrated that the colour patterns
of a species or population are often correlated with the composition
or luminance of light in the local habitat (Reimchen 1989;
* Correspondence: J. L. Kelley, Centre for Evolutionary Biology, Experimental &
Regenerative Neurosciences, School of Animal Biology (M092), University of
Western Australia, Crawley 6009, Perth, WA, Australia.
E-mail address: jennifer.kelley@uwa.edu.au (J. L. Kelley).
1
Present address: Department of Environment and Conservation, Great Southern
District, Katanning, WA, Australia.
2
Present address: Centre for Marine Futures, Oceans Institute, University of
Western Australia, Crawley, Perth, WA, Australia.
3
Deceased.
Seehausen et al. 1997; Boughman 2001; Gomez & Thery 2004;
Leal & Fleishman 2004; Cummings 2007; Stuart-Fox et al. 2007;
Morrongiello et al. 2010). However, signals that have evolved for
maximum efficacy in one environment may perform poorly in
another (Leal & Fleishman 2004), which should result in selection
for plasticity in signal production. While it is often assumed that
the relationship between signal variation and environmental
heterogeneity is a result of evolutionary adaptation, few
researchers have considered contextual plasticity in colour signal
expression (Ord et al. 2010).
Changes in the environmental conditions encountered during
signalling often cause animals to alter the intensity and/or duration
of their signalling behaviour. In visual communication systems,
males often perform their courtship displays at the time and/or
location at which their colour patterns will be perceived as most
conspicuous against the background environment (Endler 1987,
1991; Heindl & Winkler 2003; Schultz et al. 2008). In acoustic
systems, birds increase their dominant song frequency in noisy
urban environments (Slabbekoorn & Peet 2003; Mockford &
Marshall 2009) and whales respond to low-frequency sonar
emitted by the US navy by increasing the duration of their songs
(Miller et al. 2000). Although relationships between the environ-
ment and signal characteristics are best known for visual systems,

0003-3472/$38.00 Ó 2012 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.anbehav.2011.12.028
784 J. L. Kelley et al. / Animal Behaviour 83 (2012) 783e791
little attention has been paid to variation in luminance contrast,
which refers to differences in the amount of light reflected from an
object compared with the background (Wyszecki & Stiles 1982).
Animals such as reptiles, amphibians, cephalopods and fishes
have changeable coloration, allowing them to conceal their colora-
tion for predator defence (Hanlon 2007) and/or increase their
conspicuousness for communication with conspecifics (Stuart-Fox
et al. 2008; Doucet & Mennill 2010). Such changes in coloration
could also act to compensate for poor visual environments if, for
example, the brightness of colour patches can be increased to
enhance luminance contrast with the background. Examples of
changes in skin coloration and body pattern for background
matching are well known, particularly for cephalopods (Allen et al.
2010) and fishes (Whiteley et al. 2009; Clarke & Schluter 2011), but
the effects of environmental light (e.g. luminance and spectral
composition) on the brightness of colour patterns and their use as
behavioural communication signals is less clear. A notable exception
is the diurnal colour change of a small freshwater fish (the neon tetra,
Paracheirodon innesi), in which the lateral stripe is an iridescent blue-
green at noon but fades to violet at night (Lythgoe & Shand 1983).
Freshwater habitats present particularly spectrally diverse
environments because dissolved organic matter affects the scat-
tering, absorption and attenuation of light (Lythgoe 1979). While
suspended particulate matter (e.g. mineral particles, plankton and
detritus) causes wavelength-independent scattering and attenua-
tion of light, dissolved organic matter tends to absorb short
wavelengths of light (ultraviolet-blue) selectively, resulting in
a shift in the wavelength of maximum transmission of the water
towards longer wavelengths (reds; Kirk 1977; Bowling et al. 1986).
Waters that are eutrophic or highly turbid also have similar spectral
properties to those environments rich in dissolved organic matter
(Jerlov 1976; Bowling et al. 1986).
While a number of studies have reported a relationship between
the properties of the water and fish coloration (Reimchen 1989;
Seehausen et al. 1997; Boughman 2001; Fuller 2002; Morrongiello
et al. 2010), these have generally been field based making it diffi-
cult to disentangle the effects of selection and phenotypic plasticity.
Furthermore, increasing attention has been paid to the effects of
increased turbidity or eutrophication on mating behaviour and
sexual selection (Candolin et al. 2007; Wong et al. 2007); under-
standing the plasticity of the colour signals that underlie these
processes is important for predicting how aquatic communities
respond to anthropogenic impacts (Seehausen et al. 1997).
We adopted an experimental approach to test whether a col-
ourful freshwater fish, the western rainbowfish, Melanotaenia
australis, responds to changes in the light environment through
alterations in the expression of its colour patterns and/or shoaling
behaviour. The western rainbowfish is one of Western Australia’s
most ubiquitous freshwater fish and occupies a huge range of light
environments including clear springs, creeks, pools and lakes with
varying clarity and water colour (Allen et al. 2003). We simulated
environments that are rich in organic matter using coloured filters
that reduce both the availability of short wavelength light and
luminance (light intensity). Previous work with fish (black bream,
Acanthopagrus butcheri) has shown that these filters can induce
ontogenetic changes in the expression (i.e. sensitivity) of the visual
pigments (Shand et al. 2008). Neutral-density filters were used in
a second treatment group to reduce luminance without affecting
spectral composition. A third control group was not exposed to any
change in the light environment. Following 2 weeks in the treat-
ment tanks, the area and brightness of fish colour patterns were
measured using digital photography and spectrometry, respec-
tively, and compared among light treatments. We also examined
the combined effects of changes in the light environment and fish
colour pattern expression by observing the social (shoaling)
behaviour of fish in each of our treatment groups. Shoaling is the
primary social behaviour for most teleost fishes and provides the
basis for key skills including foraging behaviour (e.g. food detec-
tion), reproduction (e.g. group spawning) and predator defence
(e.g. vigilance, attack dilution; Pitcher & Parrish 1993). Further-
more, there is a body of experimental evidence linking both body
coloration (McRobert & Bradner 1998; Gomez-Laplaza 2009;
Rodgers et al. 2010, including our study species) and patterning
(Engeszer et al. 2004; Peichel 2004; Rosenthal & Ryan 2005) with
shoaling behaviour. If changes in the composition and/or lumi-
nance of light affect the expression (and conspicuousness) of colour
patterns then we would expect that shoaling behaviour, which is
influenced by these traits, would also be affected. Specifically, we
predicted that fish might compensate for poor signalling environ-
ments (i.e. reduced luminance and/or availability of short wave-
length light) by increasing the expression of their colour patterns
and adjusting their shoaling behaviour.
METHODS
Collection and Maintenance of Fish
The western rainbowfish belongs to the family Melanotaenidae,
a colourful group of freshwater fishes that is endemic to Australia
and Papua New Guinea. The colour patterns of western rainbowfish
consist of one or two dark mid-lateral stripes, several narrower red/
orange lateral stripes and two or three black sublateral stripes. There
is also a red/orange colour patch on the operculum (‘operculum
spot’) and the caudal fin can be colourless, yellow, orange or red.
Previous research with this species has shown that colour patterns
are highly variable among populations; variation in the area of
pattern (size of the cheek spot and number of lateral stripes) is
related to the level of predation risk while colour pattern brightness
appears to be related to environmental parameters (e.g. water
velocity, turbidity, vegetation, temperature; Young et al. 2010).
The rainbowfish used in these experiments were collected from



Wittenoom Gorge (22 14.8350 S, 118 19.420 E), part of the Fortescue
River catchment which runs through the Hamersley Range in the
Pilbara region of Western Australia. Fish used to evaluate the effect
of lighting on the area of colour patterns and shoaling behaviour
were collected in May 2006 for experiments in September 2007,
while those used to examine changes in colour pattern reflectance
were collected in January 2011 for use in May 2011. We expected
any effects of captivity on fish coloration (e.g. from diet, lighting
conditions) to be the same for both cohorts. Other fishes occupying
this habitat include predators such as spangled perch, Leiopother-
apon unicolor, and Fortescue grunters, Leiopotherapon aheneus, and
a catfish, Hyrtl’s tandan, Neosilurus hyrtlii. Like other freshwater
habitats in the Pilbara, for most of the year the river forms a series
of pools that become connected during summer rainfall events
(Beesley & Prince 2010). During the dry period, changes in water
quality result from the breakdown of organic matter and lack of
freshwater input or nutrient runoff increasing the algal content of
the water (in agricultural areas). During the wet season, more
dramatic changes in water quality can result as rainfall runoff leads
to high sedimentation and reduced water clarity.
Fish were maintained in aquaria (66  36 cm and 45 cm high) in
mixed-sex groups (approximately 1:1 sex ratio) and fed a diet of
commercial flake food at the University of Western Australia.
Aquarium lighting was provided by overhead fluorescent 30 W
bulbs on a 12:12 h light:dark cycle and the water temperature was
maintained at 26  1
C. As all experiments were performed
indoors no ultraviolet light was present. Rainbowfish are not
strongly sexually dimorphic but males often have brighter patterns
than females (Allen et al. 2003). In fishes, females often show

a stronger shoaling tendency than males (because of differences in
reproductive potential between the sexes, Magurran et al. 1992);
thus we decided to focus on females only for our experiments.
Simulating Differences in the Light Environment
We used acetate filters to simulate changes in the light envi-
ronment because these affect the wavelength composition and
luminance of light entering the water without altering the prop-
erties of the water (e.g. light scattering from suspended particles).
Environments rich in organic matter (‘yellow filter’ treatment
group) were simulated by placing two layers of yellow acetate filter
(‘764 Sun Colour Straw’, Lee Filters) around the front, back and top
of each tank. Like natural dissolved organic matter, these filters
acted to reduce the intensity and availability of short wavelength
light (transmittance between 400 and 550 nm ¼ 30e80%, see
Shand et al. 2008). The ‘neutral-filter’ treatments were set up in the
same way but using two layers of neutral-density filter (0.3ND
filter, Lee Filters; approximately 50% transmission 400e700 nm);
these acted to reduce luminance without affecting wavelength
composition. This allowed us to disentangle the effects of changes
in light wavelength (colour) and luminance (light brightness) on
fish colour patterns and social behaviour. A final treatment group
(‘control’) was set up in the same way without any filters and using
a transparent glass lid.
The absolute irradiance (total downwelling light measured over


180 ) was measured for each treatment group using a spectrometer
(see below and Fig. 1a). Measures of absolute irradiance in a natural
3.5E.07
(a)
3.0E.07
2.5E.07
Absolute irradiance (µmoles photons/m2 per s per nm)
2.0E.07
1.5E.07
1.0E.07
0.5E.07
0 over 2 weeks (15 days), after which they were rephotographed
400 500
0.008 (b)
0.007
0.006
0.005
0.004
0.003
0.002
0.001
0 area of pigmentation on the body (excluding fins) that lay between
300 400 500
Wavelength (nm)
Figure 1. (a) Absolute irradiance (mmoles photons/m2 per s per nm) measured in the
three treatment groups: orange filter (dotted line), neutral filter (solid black line)
and control (grey line; no filter) treatment tanks. (b) Absolute irradiance (mmoles
photons/m2 per s per nm) measured in the upper (dashed line) and lower (solid line)
reaches of the Fortescue River. Note that the spectral composition of light at the
downstream site (containing more dissolved organic matter) is shifted towards longer
wavelengths compared with the upstream (clear spring) site.
J. L. Kelley et al. / Animal Behaviour 83 (2012) 783e791 785
habitat of the western rainbowfish are shown in Fig. 1b for
comparison. These measures were made at the collection site of the
fish (Fortescue River upstream, a relatively clear water site) and
a downstream site of the same river (containing greater amounts of



dissolved organic matter; 21 17.5480 S, 116 08.6720 E) in May 2010. In
the experiment, tank treatments were randomly distributed
spatially to account for lighting variations among aquaria; however,
we also measured the total absolute irradiance of light in each tank
so that this could be included as part of our analysis. Brown card-
board was placed on the sides of all tanks to prevent fish in adjacent
treatment tanks from interacting with one another. Each treatment
tank contained a layer of brown gravel, an airstone and a piece of
artificial plant. Sample sizes used in each part of the experiment are
given in the relevant sections below.
Effect of Treatment on Colour Pattern Area
Prior to being assigned to their treatments, each female was
lightly anaesthetized in clove oil (80 mg/litre) and photographed
against a white standard using a digital camera (Olympus Mju
1030SW). Lighting was provided by two bench lamps containing
broad-spectrum fluorescent tubes (2  11 W each). It was not
possible to set the white balance on our camera manually so we
used the factory setting for photography under fluorescence
lighting. We also included a code for each individual’s photograph
such that later analysis (see below) could be performed blind of
treatment group. Anaesthesia is known to affect the brightness of
fish colour patterns (Gray et al. 2011) and we have noticed that
clove oil causes a progressive darkening of the skin, particularly the
dorsal surface. The Animal Ethics Committee (AEC) of Western
Australia currently advises the use of clove oil for fish anaesthesia;
nevertheless, colour patterns should be affected equally across all
treatment groups as each fish spent the same period of time in
clove oil (<1 min). Each fish was allowed to recover in conditioned
(Armour Coat) aerated water before being assigned to a treatment
group. To avoid stress associated with testing fish in isolation,
females were placed in their treatment tanks in groups of four,
giving a total of 72 fish allocated to this part of the experiment
(N ¼ 24 fish/treatment). However, data from two females were
missing because of photography error, giving a final sample size of
70 individuals (yellow filter: N ¼ 23; neutral filter: N ¼ 24; control:
N ¼ 23). Fish were maintained in their treatment groups for just
600 700
before taking part in the shoaling trials.
Each fish photograph was imported into Image J, version 1.44
(http://rsb.info.nih.gov/ij/index.html) and calibrated for size
according to the ruler photographed with each image. Images were
first split into separate RGB (red, green, blue) colour channels, each
displayed as an 8-bit greyscale image, to reveal different compo-
nents of the colour pattern (Stevens et al. 2007). A preliminary
inspection revealed that the green channel appeared to show most
detail of the colour patterns, while the blue channel accentuated
the orange coloration such as the lateral stripe and operculum spot.
We therefore used the green and blue channels for different
elements of our subsequent analyses. We selected a threshold value
of 60 for the green channel and for each fish we calculated the total
600 700
the values of 0 and 60. In the blue channel, we selected a threshold
of 10 before measuring the area of the operculum spot and the area
comprising the orange lateral stripes (i.e. total area of these
patterns with pixel intensities between 0 and 10). Thus we used the
photographs to measure the shape and size of the colour patterns,
while spectrometry (see below) was used to evaluate the colour
and brightness of these patches. We chose these regions of color-
ation based on previous studies showing population variation in
786 J. L. Kelley et al. / Animal Behaviour 83 (2012) 783e791
these traits (Young et al. 2010). We also measured the standard
body length (SL) of each fish to determine the relationship between
colour patterns and body length.
Effect of Treatment on Shoaling Behaviour
Shoaling trials were performed in a circular observation area
(diameter ¼ 70 cm, depth ¼ 7 cm), which had black sides, and
a white base marked with 5 cm grid lines. Lighting was provided by
two fluorescent bench lamps (2  11 W bulbs each) positioned on
each side of the arena. The shallow water allowed us to observe
shoaling behaviour in two dimensions, which is advantageous for
evaluating interindividual shoaling distance. We covered the top of
the shoaling arena with a filter, such that fish were tested under the
same lighting treatments that they had experienced during the
previous 15-day period. Thus yellow-filter fish were tested with
two layers of yellow filter, neutral-filter fish were tested with two
layers of neutral filter and control fish were tested with transparent
acetate. Glass was too heavy for the apparatus and differences in
the transmittance of glass and acetate occur in the ultraviolet
wavelengths, which were absent during the experiment.
Pairs of females originating from the same treatment group
were placed in a circular shoaling arena and left to acclimate for
10 min. We used pairs of females rather than groups of four to
increase the number of replicates that were performed. Although
this could lead to the development of social relationships (e.g.
dominance interactions) that might not be observed in larger
natural shoals, this was unlikely to occur during the short time
frame in which the fish were associated (15 min) but could have
occurred during the 15-day treatment. Furthermore, simple pair-
wise interactions between individuals can provide reliable indica-
tors of the spatial organization of larger (e.g. 10 and 30 fish) social
groups (Katz et al. 2011). We used all 72 females that had been
allocated to the light treatments, to give a sample size of 36 pairs of
fish (N ¼ 12 pairs per treatment). Following the acclimation period,
we recorded the shoaling behaviour of the fish for a period of 5 min
using a digital video camera (Canon MVX150). Following each trial,
fish were returned to stock aquaria where they played no further
part in these experiments. Videos were imported into the program
Zoom Player and the distance between shoaling fish analysed using
the program Pesci (M. Martinelli, University of Padova, Italy).
Distance was calibrated using the markings on the bottom of the
arena and readings were taken at 30 s intervals to give a total of 10
readings, which were averaged for each pair of fish sampled.
Effect of Treatment on Colour Pattern Brightness
Following the methods described above, a separate group of
female fish was randomly assigned to yellow-filter, neutral-filter or
control treatment groups, where they were maintained for a period
of 15 days in groups of four. A total of 64 females were used, none of
which took part in the previous experiment. Twelve individuals
were removed and placed in stock aquaria because they were
behaving aggressively or were subjected to aggression from other
fish (see Ethical Note, below); this gave us a sample size of 52
females for the spectrometry (yellow filter: N ¼ 17; neutral filter:
N ¼ 16; control: N ¼ 19). Fish were removed from their treatment
tanks in their groups of four, assigned an identification code (such
that subsequent data analysis was performed blind of treatment)
and transferred to a dark room for the spectrometry.
Fish were anaesthetized using the methods described above
before being placed on a matt black wedge, allowing the body to be
positioned horizontally. The wedge was placed on a photography
stage that allowed fine movement in the vertical plane. Laboratory


clamps were used to position two optical fibres at 90 to one


another, with each fibre at 45 to the fish’s body. One of these fibres,
the ‘read’ fibre (400 nm), was fitted with a collimating lens focused
to sample an area of 1 mm diameter. The read fibre was connected
to a USB4000 spectrometer (Ocean Optics, Inc., Dunedin, FL, U.S.A.),
configured to sample light wavelengths within the range of
172e881 nm. The other optical fibre (600 nm) was connected to
a light source (200e1700 nm; Mini-DT-1000, Analytical Instru-
ment Systems, Inc., Flemington, NJ, U.S.A.). Prior to each fish being
measured, we ensured that the probes were in alignment and took
measurements of white (WS-1, Ocean Optics, Inc.: 100% reflec-
tance) and dark standards, ensuring that the height of the stage was
optimal for maximum reflectance. The reflectance measures for
each fish were therefore performed in relation to these light/dark
standards, with the software (Spectrasuite, Ocean Optics, Inc.) set to
average across three sample spectra.
Fish were oriented with their right side down such that the
illuminating fibre was positioned to provide light from the anterior
direction of the fish while the read fibre was positioned to collect
light from the posterior end of the fish. Ideally, illumination would
be provided from the direction of ambient light in nature (i.e.
dorsally, Endler 1990) but this proved difficult with the curvature of
the fish’s body. We sampled along the predominant mid-lateral
orange stripe (posterioreanterior direction, along 1/3 of body),
sampling approximately every one or two scales to give 5e10
spectral samples per individual. The fish was moved up and down
in the vertical plane to find the peak reflectance for each reading.
Following spectrometry, fish were allowed to recover in condi-
tioned, aerated water before being returned to stock aquaria where
they played no further part in the experiment. For each sampling
point, 3648 spectral readings were obtained across the range
172e881 nm. These data were reduced to the interval 300e700 nm
using moving averages across 2 nm intervals (every 10 readings) to
give 259 data points for each reflectance measure (i.e. a measure of
reflectance for each of the 259 different wavelengths). We then
calculated a mean reflectance spectrum for each individual fish
and subsequent statistical analyses (see below) are based on each
individual’s mean reflectance spectrum.
For the irradiance data, a 600 nm optical fibre was fitted with
a cosine collector (CC-3-UV, Ocean Optics, Inc.) and oriented
vertically (to collect downwelling light) in each treatment tank.
Absolute irradiance measures (in mmoles of photons/nm) were
performed with reference to a calibration lamp (LS-1-CAL-220,
300e1050 nm; Ocean Optics, Inc.) and measured in Spectrasuite.
Total absolute irradiance (in photons/cm2 per s) was calculated for
each treatment tank by integrating irradiance over the wavelength
range 400e700 nm (no ultraviolet light, 300e400 nm, was present
in our laboratory experiment).
Statistical Analysis
We used general linear mixed models (GLMMs) to test the effect
of treatment (yellow filter, neutral-density filter or control) on our
three response variables: the total area of pigmentation, the area
comprising the lateral orange stripes and the area of pigmentation
in the operculum patch (all measured following the 15 days in
treatment tanks). We first confirmed that there were no differences
in patterns between groups before they were assigned to their
treatments. Individual tanks (N ¼ 6 per treatment) were allocated
to each treatment; we therefore entered tank nested within
treatment as a random factor. Thus the degrees of freedom reflect
the number of tanks rather than the number of individual fish. We
also entered ‘standard body length’ as a covariate to determine its
effect on the dependent variables. Although our response variables
were weakly correlated with one another (correlation coefficients
ranging from 0.22 to 0.37), we chose to analyse them separately
 J. L. Kelley et al. / Animal Behaviour 83 (2012) 783e791 787

because a preliminary principal components analysis (PCA) showed Table 1

that they were equally loaded in different directions, making the Results of general linear mixed models, testing the effect of light treatment (three
levels) and standard body length (SL) on the three pattern (response) variables
results difficult to interpret. All statistical analyses were performed
in JMP version 9.0 (SAS Institute Inc., Cary, NC, U.S.A.). Response variable Fixed effect df F P
GLMMs were also used to test for the effect of treatment on colour Total area of pigmentation Treatment 2, 13.8 1.93 0.182
pattern brightness. We first used PCA to reduce the 259 wavelength SL 1, 64.1 17.01 <0.0001
Area of striped pigmentation Treatment 2, 15.1 4.02 0.040
measures to two principal components; these typically describe the
SL 1, 65.8 5.82 0.019
brightness (PC1 if loaded equally across all wavelengths) and hue Area of operculum pigmentation Treatment 2, 13.3 0.94 0.413
(PC2) of colour patterns, respectively (Cuthill et al. 1999). In the SL 1, 62.9 0.11 0.737
absence of any information on the visual sensitivity of the study Tank was nested in treatment (six tanks per treatment) and entered as a random
species, PCA is a better alternative than the ‘segment classification’ factor. Significant effects are shown in bold.
method, which is unsuitable for species with more than three
photoreceptor types (Endler 1990); preliminary work with the
western rainbowfish has revealed five photoreceptor classes so far RESULTS
J.L. Kelley, N.S. Hart, J. Shand, S.P. Collin & D.M. Hunt, unpublished
data). We accounted for differences in ambient lighting (caused by Effect of Treatment on Colour Pattern Area
tank position) in our analysis by entering the total absolute irradiance
for each tank as a random factor. This was particularly important We confirmed that there was no difference between individuals
because, despite our attempts to equalize luminance across the in the total area of pigmentation (F2, 68 ¼ 0.065, P ¼ 0.937), the area
treatments, control tanks had higher total absolute irradiance than of stripe pigmentation (F2, 68 ¼ 0.50, P ¼ 0.611) or the area of
neutral tanks (control mean ¼ 6.16e13 photons/cm2 per s, range operculum pigmentation (F2, 68 ¼ 0.15, P ¼ 0.866) before fish were
3.11e13 photons/cm2 per s, neutral mean ¼ 4.49e13 photons/cm2 per assigned to their treatment groups. Treatment had no effect on the
s, range 1.1e13 photons/cm2 per s; ANOVA: F2, 13 ¼ 4.95, P ¼ 0.025; total area of pigmentation but a significant effect on the area of
post hoc Tukey test: P < 0.05). However, there was no significant striped pigmentation (Table 1, Fig. 2); a post hoc Tukey test revealed
difference between the total absolute irradiance in the neutral-filter that fish in the yellow-filter group displayed a greater area of stripe
and yellow-filter treatments (P < 0.05). A total of 52 reflectance pigmentation than those in the control group (Fig. 2; P < 0.05).
spectra (based on a mean value for each individual) were used in the There was no effect of treatment on the area of operculum
final analysis. Fish body length (SL) data were only collected for pigmentation (P > 0.05; Table 1). Both the total area of pigmenta-
a subset of 18 fish; these data were therefore used to investigate the tion (t68 ¼ 4.15, P < 0.001) and area of striped pigmentation
relationship between colour (hue and brightness) and body size. (t68 ¼ 2.22, P ¼ 0.030) showed a positive linear relationship with
We tested for the effects of treatment on shoaling behaviour fish standard body length (Table 1).
using ANCOVA to control for variation in body length, which can
influence shoaling behaviour in fishes (Ranta et al. 1993). As we did Effect of Treatment on Colour Pattern Brightness
not determine which individuals were selected for the shoaling
trials we used the variance in body length (variance in standard PC1 showed strong positive loadings across all wavelengths
length for the four individual fish in each treatment tank) as a co- (300e700 nm; loadings: 0.80e1.0), whereas PC2 was weakly
variate in the analysis. positively loaded across the range 350e500 nm (loadings: 0.3e0.5)
and weakly negatively loaded from 550 to 700 nm (loadings: 0.1
Ethical Note to 0.23). The two principal components that were extracted (PC1
and PC2) explained 84.1% and 10.5% of the variation in our data,
This work was performed in accordance with the Australian respectively. For PC1, which is usually correlated with mean
Code of Practice for the Care and Use of Animals for Scientific reflectance (or brightness) there was a significant effect of treat-
Purposes (2004) and approved by the Animal Ethics Committee of ment group (F2, 19.5 ¼ 3.98, P ¼ 0.036); a post hoc Tukey test
the University of Western Australia (licence RA/3/100/691). In the revealed a significant difference between the control and yellow-
field, fish were captured using a 3 m seine net and adults placed in filter groups at P < 0.05 (Figs 3, 4a).
sealed plastic bags for transport by air to the University. The
transport bags contained river water, a small amount of vegetation,
Armour Coat (for skin protection) and zeolite minerals (for
10
ammonia absorption) and were packed into polystyrene crates.
During the 15-day laboratory experiment (light treatments),
8
Stripe area (mm2)

artificial plants were used to provide refuge for the fish. Fish were
monitored twice daily for signs of aggression, which included
6
nipping and chasing behaviours. Fish that were aggressive towards
others or were subjected to aggression (i.e. showed signs of fin
4
damage) were removed and isolated (for recovery) before being
returned to stock aquaria. A total of 12 fish were excluded from our 2
experiments because of aggressive behaviours and we did not
observe any other adverse behaviour in our treatment tanks. 0
During the photography and spectrometry we minimized the time Y N C
fish spent in anaesthesia (which was <1 min and approximately Treatment group
3 min, respectively) and ensured rapid recovery by placing fish in
aerated water after the procedure. Fish did not show any signs of ill Figure 2. Differences in the mean 1 SE area of pigment (mm2) comprising the two
lateral orange stripes (revealed by thresholding digital images, see Methods) in rain-
health during the photography or spectrometry and no deaths bowfish assigned to the yellow-filter (Y), neutral-filter (N) and control (no filter, C)
occurred as a result of these procedures. At the end of the experi- treatment groups. Sample sizes for the Y, N and C treatment groups were 23, 24 and 23
ments, all fish were returned to stock aquaria. females, respectively.
788 J. L. Kelley et al. / Animal Behaviour 83 (2012) 783e791

There was also an effect of treatment on PC2 (typically correlated 20

(a)
with hue; F2, 19.5 ¼ 4.06, P ¼ 0.034; Fig. 4b); a post hoc Tukey test
revealed a significant different between the yellow- and neutral-

Least – squares mean (pc1)

15
filter groups (P < 0.05). Thus fish in the yellow-filter treatment
with positive PC2 scores tended to have colours with high reflec-
10
tance at short wavelengths (violet-blue: 400e500 nm) and low
reflectance at long wavelengths (yellow-orange: >550 nm) while
5
the reverse was true for fish in the neutral-filter treatment (high
reflectance at long wavelengths, low reflectance at short wave-
lengths). However, PC2 accounted for only 10.5% of the variation in 0
our data and the average spectrum for each treatment group (Fig. 3)
suggests changes in brightness rather than hue. The random effect of –5
total absolute irradiance had a significant effect in our models (PC1:
F13, 36 ¼ 3.54, P ¼ 0.001; PC2: F13, 36 ¼ 7.60, P < 0.001). There was no –10
relationship between body length (SL) and PC1 (t16 ¼ 0.24, P ¼ 0.81) Y N C
or PC2 (t16 ¼ 0.94, P ¼ 0.36).
6
(b)
Effect of Treatment on Shoaling Behaviour

Least – squares mean (pc2)

4
We found a significant effect of treatment on shoaling behaviour
(F2, 32 ¼ 3.43, P ¼ 0.045; Fig. 5) but no effect of the covariate (tank 2
variance in body length: F1, 32 ¼ 0.48, P ¼ 0.49). Following removal
of the covariate from our model (treatment: F2, 33 ¼ 3.87, 0
P ¼ 0.031), a post hoc Tukey test revealed a significant difference
between the yellow-filter treatment and both the neutral-filter and –2
control treatment groups (P < 0.05; Fig. 5). Fish in the yellow
treatment group retained an average distance of 22.8 þ 3.1 mm –4
(mean þ SE) from their shoaling partner while those in the neutral
and control groups remained closer together (15.0 þ 1.5 mm and
–6
14.5 þ 2.2 mm, respectively). Y N C

DISCUSSION
While links between the light environment and a species’ colour
patterns are often considered in the context of signal evolution, we
have provided evidence that changes in the brightness of colour
patches can also arise through alterations in the light environment.
We found that a reduction in the relative availability of short
wavelength light caused an increase in the brightness (reflectance)
of colour patterns. Fish responded to the combined effects of
changes in their coloration and the ambient light environment by
altering their shoaling behaviour; fish in waters with reduced
45
40
35
Mean % reflectance
Figure 4. Least-squares means  1 SE from general linear mixed models used to test
the effect of treatment (yellow filter, Y, N ¼ 17; neutral filter, N, N ¼ 16; control group,
C, N ¼ 19) on the response variables (a) PC1 and (b) PC2. PC1 represents brightness
(achromatic reflectance) while PC2 represents variation in the relative reflectance of
the lateral stripe at short (350e500 nm) versus long (550e700 nm) wavelengths;
positive PC2 scores suggest colour pigments that reflect strongly at short wavelengths
(e.g. blues) while negative PC2 scores represent patches that are more reflective at
longer wavelengths (e.g. oranges).
relative availability of short wavelength light had greater interin-
dividual shoaling distances (shoaled further apart) than those in
the low luminance (neutral filter) or control groups. Our findings
suggest that alterations in the light environment affect the area and
brightness of colour patterns, which could subsequently influence
their use as behavioural signals.

30 30
25
Mean distance (mm)

25
20
20
15
15
10
5 10

0 5
300 400 500 600 700
Wavelength (nm) 0
Y N C
Figure 3. Mean percentage reflectance of the upper orange lateral stripe of fish Treatment group
assigned to the yellow-filter (solid line), neutral-filter (dashed line) and control (dotted
line) treatment groups. Standard errors surrounding the mean are shown in grey (1 Figure 5. Effect of yellow (Y), neutral (N) and control (C) treatment groups (N ¼ 12
SE). Sample sizes are 17, 16 and 21 fish for the yellow-filter, neutral-filter and control pairs per treatment) on the mean interindividual distance between shoaling females
groups, respectively. (error bars represent 1 SE).
 J. L. Kelley et al. / Animal Behaviour 83 (2012) 783e791
Previous studies have investigated how differences in the light
environment contribute to variation in the hue (colour) and
brightness of animal colour pattern signals. For example, bird
species from closed habitats that are characterized by a predomi-
nance of long wavelength light tend to exhibit colours that are rich
in long wavelengths (McNaught & Owens 2002), which should
serve to enhance chromatic contrast against background light
(Endler 1990). Colour patch brightness has also been shown to vary
across species; birds in open canopy habitats rich in blue and UV
light tend to exhibit brighter coloration (higher reflectance relative
to incident light) than those occupying understory environments
that are rich in green and orange light; these differences in color-
ation may allow birds to appear cryptic in their respective natural
environments (Gomez & Thery 2004). In dwarf chameleons, Bra-
dypodion spp., the pattern is similar with males in closed habitats
exhibiting less bright coloration than those in open habitats, but
their colour patches allow for greater brightness contrast against
the background in these closed habitats (Stuart-Fox et al. 2007).
These studies illustrate the importance of considering both
ambient light and the (chromatic/luminance) contrast provided by
the visual background (Endler 1990; Stuart-Fox et al. 2007).
Field studies with other species of fish have also reported
a correlation between the light environment and red body colora-
tion. For example, populations of southern pygmy perch, Nanno-
perca australis, tend to have redder coloration in habitats that are
rich in long wavelengths (550e700 nm; Morrongiello et al. 2010)
and in red shiners, Cyprinella lutrensis, males from turbid habitats
have brighter caudal fins during the breeding season than those
from less turbid waters (Dugas & Franssen 2011). In contrast,
sticklebacks, Gasterosteus aculeatus, from stained waters that have
low transmittance in the 350d450 nm range have less red throat
pigmentation than those from clearer waters or tend to be replaced
by melanic (black) males that may be more conspicuous in these
environments (Reimchen 1989). The ventral red throat pouches in
sticklebacks from stained habitats are unlikely to be perceived as
conspicuous because the long wavelength-shifted upwelling light
and the high reflectance of ventral surfaces minimize contrast with
the background (Reimchen 1989).
In mid-water-dwelling fishes, such as rainbowfishes, individuals
will often be viewed against the background provided by the water
column, primarily consisting of sidewelling light. Colour patches
will be perceived as brighter if they reflect wavelengths that are
relatively more abundant in the downwelling light than the
sidewelling light (Endler 1990); thus red colour patterns should be
most conspicuous in habitats that are rich in dissolved organic
matter (Lythgoe 1979; Levine et al. 1980). The enhanced brightness
of red colour patterns observed in our reduced light intensity and
red-shifted (yellow filter) light environments should act to increase
the brightness of the red-coloured stripes, providing enhanced
(luminance) contrast against a reduced light intensity background.
However, the visual background provided by the surrounding skin
pigmentation (which is silvery and highly reflective in the ultravi-
olet (300e400 nm) wavelength range) is also important and, in this
case, the increased brightness of the red stripes in red-shifted light
environments would produce less luminance contrast against the
silvery skin (assuming the reflectance of these patterns remained
unchanged), reducing the overall conspicuousness of the entire
colour patterns. Information on the visual sensitivity of rainbowfish
would allow us to reveal whether the observed colour pattern
changes facilitate enhanced conspicuousness or crypsis of the
colour pattern components (see concluding paragraph also). We
acknowledge that the spectral composition of the fluorescent
lighting used in this study is very different to the spectral charac-
teristics of light in the species’ natural habitat (Fig. 1). None the less,
other studies conducted under similar (artificial) lighting
789
conditions have demonstrated changes in the optomotor response
(Kroger et al. 2003) and in the expression of opsin genes (which
determine the sensitivity of the visual pigments; Shand et al. 2008)
in response to changes in the light environment.
In this study, the red lateral stripes that were measured are
located in the mid- to mid-ventral posterior regions of the body and
are therefore less likely to be affected by upwelling light than
ventral colour patterns. Although we understand little of the
function of colour patterns in rainbowfishes, studies with other
species have revealed that lateral longitudinal stripes can serve as
important cues in shoaling behaviour (Peichel 2004; Rosenthal &
Ryan 2005). These stripes must appear conspicuous against the
background of the fish’s skin if they are to serve a communicative
function. For example, Long & Houde (1989) examined female
preference for males when viewed behind orange filters (peak
transmittance 600e690 nm range) and found that the female’s
usual preference for orange diminished under orange light,
because of a lack of contrast of orange spots with the
surrounding skin.
Several studies have examined the relationship between
colour patterns and the visual sensitivity of the receiver; for
example in guppies, Poecilia reticulata, there is no relationship
between male coloration and water colour, but females from
waters with greater relative long wavelength transmittance show
a stronger preference for orange and black males (Endler & Houde
1995). In studies with sticklebacks, males inhabiting waters
characterized by the dominance of long wavelength light tend to
exhibit less red coloration which is linked to the females’ reduced
sensitivity to red in these populations (Boughman 2001). The light
environment has also been shown to influence mating behaviour
in guppies with females responding more to males observed
under simulated woodland shade environments (which stimu-
lates cones sensitive to short and medium wavelengths) than
under forest shade or early/late environments (which stimulate
cones sensitive to long and mediumelong wavelengths, respec-
tively; Gamble et al. 2003).
Our observation that shoaling behaviour was only modified in
the yellow-filter treatment group (reduced relative availability of
short wavelength light) could suggest that the increase in area and/
or brightness of colour patterns was sufficient for these fish to
interact over greater distances, but not sufficient to alter the
behaviour of fish in the other (neutral and control) groups. The
colour pattern area and brightness of fish in the neutral group
appeared to be intermediate to those in the control and yellow-
filter groups, providing some evidence for this. An alternative
explanation for the differences in shoaling behaviour between
treatment groups is that changes in the wavelength composition of
the light stimulated some other change in the behaviour of female
rainbowfish. Changes in shoaling density (i.e. interindividual
shoaling distances) are often associated with the trade-off between
foraging efficiency and predation risk with an increase in shoal
density facilitating increased protection from predators (e.g. risk
dilution) at the cost of reduced foraging efficiency (because of
competition among group members; Pitcher & Parrish 1993). Shifts
in the properties of light that are consistent with diurnal variations
may induce behavioural shifts, or light conditions that are consis-
tent with high turbidity may be associated with a reduction in
predation risk and an increased motivation to feed (reviewed in
Utne-Palm 2002).
We acknowledge that linking shoaling behaviour and fish
coloration to environmental light, rather than addressing a more
specific behaviour, such as female choice for male colour patterns,
makes it difficult to predict how these variables might interact
to influence animal fitness. However, many studies have linked
red/orange coloration to fitness traits in freshwater fishes; for
790 J. L. Kelley et al. / Animal Behaviour 83 (2012) 783e791
example, by selecting males with high levels of red/orange color-
ation female sticklebacks avoid parasitized males (and may
produce offspring with increased capacity for parasite resistance;
Milinski & Bakker 1990) and female guppies produce offspring with
enhanced antipredator skills (i.e. ‘good genes’ models of sexual
selection; Evans et al. 2004). We hope our work will stimulate other
researchers to consider how environmentally mediated colour
pattern plasticity may influence evolutionary processes such as
sexual selection.
We cannot predict whether the increased reflectance (bright-
ness) of rainbowfish colour patterns serves to increase conspicu-
ousness against the background (light) environment without
considering the visual sensitivity of the rainbowfish (or its preda-
tors). While we are in the process of describing the visual system of
the western rainbowfish, a member of the same genus, Melano-
taenia maccullochi, has six spectral classes of cone with maximum
sensitivities ranging from the ultraviolet (lmax ¼ 377 nm) to
yellows/oranges (lmax ¼ 577 nm; Reckel et al. 2002), which
provides a wide spectral range for visually mediated behaviours.
With data on the sensitivity of the photoreceptors in each cone
class for M. australis, it would be interesting to use visual models to
determine whether the increased colour pattern reflectance acts to
maintain communication in a poor signalling environment. These
types of models could be used to determine the distances over
which individuals can maintain shoaling associations in variable
light environments. Such an approach would allow deeper insights
into how changes in the aquatic light environment affect the
physiology, social behaviour and ecology of fishes.
Acknowledgments
We thank Suzanna Chan for her help with data collection and
Rick Roberts, Husnan Ziadi and Cameron Duggin for practical
assistance. Thanks also to Jon Evans for use of his spectrometer,
Dale Roberts for access to laboratory facilities and Bob Black for
statistical advice. Justin Marshall and Martin Stevens provided
advice on measuring fish coloration. We are especially grateful to
John Endler for numerous discussions about rainbowfish and for
suggesting the spectrometry part of this study. Nathan Hart, John
Endler and the anonymous referees kindly provided excellent
comments that greatly improved the manuscript. J.L.K. is grateful to
the University of Western Australia for a Postdoctoral Research
Fellowship.
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