1
Supplemental Figures and Tables:
2
3
Figure S1. Data on the visual system of Neolamprologus brichardi used to build visual
4
models
5
A) Relative opsin expression of N. brichardi determined by RNAseq. B) Normalized
6
transmittance of N. brichardi crystalline lens. C) Normalized absorbance of SWS1, RH2B and
7
RH2A. D) Estimated visual sensitivities incorporating the crystalline lens filtering medium. E)
8
Normalized downwelling and F) sidewelling irradiance at two depths. Increasing water depth
9
differentially reduces available longer light wavelengths. Refer to Figure 2.
10
11
Figure S1 (continued).
12
G) 95% confidence intervals of spectral reflectance in dominant and subordinate individuals.
13
Only horizontal facial stripe spectra do not overlap between dark and pale fish, other colors
14
overlap to various degrees. Colored curves refer to color patches in individuals with dark
15
horizontal stripe, while light grey curves refer to equivalent patches in individuals with pale
16
horizontal stripe. H) Chromatic contrast (mean ± SEM) is achieved by adjacency of color
17
elements and is not influenced by darkness of horizontal stripe. I) Achromatic contrast (mean ±
18
SEM) is achieved by paling of the horizontal stripe and has a differential influence on adjacent
19
and non-adjacent color elements. Refer to Figure 2.
20
21
Figure S2. Horizontal facial stripe provides reliable information on aggressive intent.
22
A) and B) Winners are larger and fight more aggressively than losers. Lines connect fish dyads.
23
C) and D) Principal components analysis of spectral data and signal experimental manipulation.
24
Principal Components 1 and 2 explain 96.5% of the variance (90.8% and 5.7%, respectively) and
25
clearly separate different colors. D) Zoom in at the melanistic area of the plot shows that
26
‘Eyeliner’ is similar to dark melanic stripes of dominant fish, while ‘Wound Snow’ is similar to
27
pale melanistic stripes of subordinates. See also Table S1 for clustering of Principal
28
Components. E), F) and G) Reaction norms of amount of aggression incurred by individuals with
29
out-of-equilibrium signals and controls. Retaliation costs are highest for individuals with
30
artificially darkened stripe (‘bluffers’, E and F), followed by individuals with paled stripe
31
(‘Trojans’, E and G), and then controls (i.e. reliable signalers, F and G), which were the ones that
32
received the lowest amount of aggression. Lines connect the same individual, tested twice with
33
two different treatments. Refer to Figure 4.
34
35
36
Table S1. Cluster analysis of principal components of spectral data
Clusters
Color patch
1
2
3
4 5
Eyeliner
0
1
0
0 0
Wound Snow
1
0
0
0 0
Vertical facial stripe (D)
0
10 0
0 0
Vertical facial stripe (P)
0
10 0
0 0
9
0
0 0
Horizontal facial stripe (P)
10 0
0
0 0
Lachrymal stripe (D)
8
2
0
0 0
Lachrymal stripe (P)
10 0
0
0 0
Head (D)
9
1
0
0 0
Head (P)
10 0
0
0 0
Blue (D)
0
0
10 0 0
Blue (P)
0
0
10 0 0
Branchiostegal (D)
0
0
0
0 10
Branchiostegal (P)
0
0
1
0 9
Yellow (D)
0
0
0
9 1
Yellow (P)
0
0
0
9 1
Horizontal facial stripe (D) 1
37
Five clusters were identified. ‘Eyeliner’ clusters together with black melanistic stripes (cluster 2),
38
while ‘Wound Snow’ clusters with pale horizontal stripe, lachrymal stripe and head (cluster 1).
39
D: dark. P: pale. Refer to Figures 4 and S2C.
40
Supplemental Experimental Procedures
41
Choice of species
42
The Princess of Burundi, Neolamprologus brichardi (Teleostei: Cichlidae), is a small (up to 8 cm
43
in standard length) fish native to Lake Tanganyika, eastern Africa. Together with N. pulcher, it
44
has emerged as a model system in studies on the evolution of cooperative breeding behavior [1].
45
Recently, substantial genomic and transcriptomic resources have become available for N.
46
brichardi [2]. Such resources make this species an excellent system for the study of speciation,
47
evolution of cooperative behavior and communication also from a genetic perspective.
48
Neolamprologus brichardi performs the complete range of behaviors observed in the wild under
49
lab conditions, which makes it an optimal species for behavioral studies [3]. Phylogenetic
50
relationships have been recently studied [4], which led some authors to synonymize it with N.
51
pulcher. The two species differ in their facial pigmentation patterns and are thought to behave
52
similarly. We adopt the pre-synonymy taxonomy because it highlights the differences in facial
53
pigmentation pattern, which are the focus of our study. This should not create taxonomic
54
confusion and favors the accumulation of clear information for each of the pigmentation
55
phenotypes. Like most other species of the Tanganyikan cichlid tribe Lamprologini, N. brichardi
56
is sexually monochromatic, i.e. coloration patterns are identical between males and females [5].
57
The facial pigmentation of N. brichardi consists of two black melanic stripes, arranged in a
58
horizontal T-shape, surrounded by structural blue coloration, yellow pigmentation elements and
59
a white branchiostegal membrane. Another less conspicuous stripe is present in the pre-orbital
60
(lachrymal) area. The species has a beige body with fine orange elements in the posterior half
61
and white-fringed fins (see Figure 2 in main text).
62
Substantial data on life history and behavioral traits have been documented for these
63
species. The dominant, breeding couple has the peculiarity of being aided by up to 25
64
subordinate helpers in their tasks, and the social group is organized in a strict linear hierarchy [6–
65
8]. As a consequence of cooperative breeding and colony life, individuals repeatedly and
66
regularly interact [9] and it is known that vision plays a role in individual recognition of mates,
67
kin and neighbors [3, 10, 11]. Social groups of N. brichardi can be found on coastal rocky
68
substrates of Lake Tanganyika, usually between 3–50 m deep. The rocky substrate provides a
69
territory with shelters and breeding grounds where adhesive eggs are spawned [12, 13]. The
70
breeding male is always the largest individual of the group, usually followed by the breeding
71
female and subordinate helpers are the smallest [1]. Groups aggressively defend their territory
72
and dominant females have dominance behavior similar to dominant males, show high
73
testosterone levels and brain arginine vasotocin expression (a neuropeptide involved in
74
vertebrate territorial, reproductive and social behaviors) [14]. Group hierarchy is based on size, is
75
relatively stable over time and fish adopt one of two queuing strategies for breeding. Fish either
76
queue and breed in their natal group, or they disperse to a new group where they queue and breed
77
[1]. The mean distance between adjacent territories is 1.6 m and territories are clustered into
78
colonies [15]. These life history and behavioral traits create conditions for repeated interactions
79
among individuals. Most of them involve submissive behaviors, followed by aggressive
80
behaviors and only then territory maintenance (such as digging) and broodcare [16].
81
82
Study animals
83
Neolamprologus brichardi were raised and kept under controlled captive conditions at the
84
Zoological Institute, University of Basel, Switzerland, on a 12:12 h light:dark regime, in tanks
85
with about 1.5 cm of sand, a foam filter, a heater and terracotta flowerpots as shelters. Fish were
86
fed a combination of newly hatched Artemia nauplii, commercial flakes and frozen cichlid food
87
once or twice a day. All experiments were authorized by the Cantonal Veterinary Office, Basel,
88
Switzerland (permit numbers 2317 & 2356).
89
90
Color reflectance spectra
91
Spectral reflectance measurements of N. brichardi facial patterns were taken using a USB4000
92
spectrophotometer (Ocean Optics Inc.) and DH-2000-DUV Mikropack deuterium-halogen light
93
source, connected to a laptop computer running Ocean Optics SpectraSuite software. Twenty
94
individuals were tranquilized using a solution of KOI MED® Sleep (KOI&BONSAI, 0.5% v/v
95
2-Phenoxyethanol) before being transferred to a shallow tray filled with sufficient water to fully
96
cover the fish. Because tranquilizing the fish before measuring their spectral reflectance may
97
induce a short term darkening of their skin pigmentation we took care to measure reflectance
98
after original conditions were re-established (~15 seconds). Spectral reflectance of various facial
99
color patches (Figures 2A, 2B, S1G) was measured with a 200 µm bifurcated optic UV⁄visible
100
fiber. The bare end of the fiber was held at a 45º angle to prevent specular reflectance. A
101
Spectralon 99% white reflectance standard was used to calibrate the percentage of light reflected
102
at each wavelength from 350–750 nm. At least ten measurements per facial pattern per
103
individual were taken and subsequently averaged. Spectra were assessed based on the
104
wavelength at which light was reflected and the shape of the reflectance curves, and classified
105
into previously established categories of reef fish colors [17].
106
107
108
Visual system
109
To characterize the visual system of N. brichardi, we used published quantitative opsin data [2,
110
18] and amino acid sequences from eye RNAseq data [2] done on our stock of N. brichardi, and
111
collected new ocular transmission measurements from wild specimens. Our N. brichardi express
112
the UV-sensitive SWS1, and the two green-sensitive RH2A and RH2B opsin genes, which is a
113
common opsin expression palette in cichlid species, including the ones from Lake Malawi
114
(Figure S1) [19]. On comparisons of amino acid sequences of these three genes to the sequences
115
of their Lake Malawi relatives we found that there are only minor differences between species. In
116
particular, N. brichardi and Metriaclima (Maylandia) zebra show amino acid similarity of 95.4%
117
(± 1.1%, SEM) at SWS1, 98% (± 0.7%, SEM) at RH2Aα and 97.7% (± 0.8%, SEM) at RH2B
118
(similarities calculated in MEGA6) [20]. We measured ocular media transmission of the whole
119
eye (by cutting a window in the back), cornea, and the lens from wild caught N. brichardi (Cape
120
Kachese, Zambia, Lake Tanganyika; n = 3) to gain an understanding of the physical light
121
filtering properties of the eye, following previously established protocols [19, 21]. Light from a
122
pulsed xenon light source (Jaz-PX, Ocean Optics Inc.) was directed through the pinhole using a
123
400 µm optical fiber and the ocular media and collected by a 100 µm optical fiber attached to a
124
Jaz spectrometer (Ocean Optics Inc.). A Spectralon 99% white standard was used as a reflection
125
standard. At least three measurements per media were taken and subsequently averaged. Spectra
126
were thereafter normalized using their maximum transmission and the wavelength at which 50%
127
transmission (T50) was reached was determined within the 300–750 nm interval [19, 21]. We
128
found the lens to be the limiting light transmission medium of the N. brichardi eye with a T50
129
cut-off value of 359 nm (Figure S1). Based on our molecular assessment we followed Dalton et
130
al. [22], and used opsin absorbance spectra (λmax = 368 nm for short wavelength (SWS), λmax =
131
488 nm for mid wavelength (MWS), λmax = 533 nm for long wavelength (LWS) [23, 24] from M.
132
(M.) zebra, a rock-dwelling cichlid species from lake Malawi, to reconstruct the visual
133
sensitivities of N. brichardi (Figure S1). We then incorporated our lens transmission
134
measurements to create a template of the visual system of N. brichardi to model how N.
135
brichardi perceives color (Figure S1).
136
137
Underwater light environment in Lake Tanganyika
138
We took measurements of the natural ambient light under which the fish color patterns evolved.
139
Measurements were taken at Isanga Bay (Zambia, Lake Tanganyika) in September 2011 at
140
depths of 3 m and 7 m (Figure S1). Illumination was measured using a USB2000 spectrometer
141
attached to a PALM-SPEC computer running native software (Ocean Optics), enclosed in an
142
underwater housing (Wills Camera Housings, Victoria, Australia). We used a shortened (60 cm)
143
1000 µm UV/visible optical-fiber with a cosine corrector to provide an 180º hemisphere to
144
measure both, downwelling (by pointing the fiber upwards) and sidewelling light (pointing the
145
fiber horizontally into the middle or towards the shore of the lake). However, there was no
146
significant difference in our overall conclusion when using either of the measurements.
147
148
Visual modeling
149
We used a theoretical visual model [25, 26] to quantify the chromatic (‘color’ or ‘hue’) and
150
achromatic (‘intensity’ or ‘luminance’) color contrasts between the facial pattern elements using
151
the N. brichardi visual system and assuming ambient light conditions as measured from their
152
natural habitat (see above). The chromatic model calculates the color distance (ΔS) within the
153
visual space of the fish, where low values of ΔS denote similar colors and high values of ΔS
154
indicate chromatically different colors. When calculating ‘hue’ distances between color patches,
155
luminosity is disregarded within the model, the colors are assumed to be encoded by an
156
opponency mechanism based on the sensitivities of the fish visual system, and chromatic
157
discrimination is thought to be limited by photoreceptor noise determined by the relative
158
proportion of each photoreceptor [25, 26]. The receptor quantum catch (qi) in the photoreceptor
159
cell of type i is calculated as
160
qi = ∫ Ri(λ)S(λ)I(λ)dλ
161
where λ denotes the wavelength, Ri(λ) the spectral sensitivity of the photoreceptor cell, S(λ) the
162
spectral reflectance of the color patch, I(λ) the illumination spectrum entering the eye and
163
integration is over the range of 350–750 nm (equation 1 of Vorobyev & Osorio [25]).
164
Illumination was set as measured at a depth of 7 m and coming from above (no difference was
165
found when using spot tests and illumination at 3 m). In the absence of retinal morphological and
166
physiological data for our study species, we based the relative proportion of cone receptors on
167
studies from other cichlid fishes, which frequently have a square pattern [27–29] with a ratio of
168
1:2:2 (SWS:MWS:LWS), which is consistent with RNAseq data for N. brichardi (Figure S1).
169
The weber fraction (ω) was set to assume a 0.05 LWS noise threshold, which is a conservative
170
approach representing approximately half the sensitivity of the human LWS cone system [30]. In
171
addition to chromatic contrast we also calculated achromatic ‘luminance’ contrast as a second
172
property of the visual signal. Long wavelength receptors are thought to be responsible when
173
perceiving differences in luminance (for discussion see Marshall et al. [31]) and we therefore
174
used the differences in the natural logarithm quantum catch (Q) of the long wavelength receptor
175
(L) to calculate luminance differences between color patches using
176
ΔL = ln(QLpatchX) – ln(QLpatchY).
177
We predict that the more ΔS increases above the threshold of 1 JND (just noticeable
178
difference), the more distinguishable colors become from one another, which might be especially
179
important for long-range signals where intervening water and particles start to blur colors [31].
180
181
Resource contest experiment
182
A total of 40 N. brichardi (20 males and 20 females) originating from several stock tanks were
183
sexed (by examination of the genital papilla), measured (standard length [SL], taken as the
184
distance between the tip of the snout and the insertion of caudal fin rays) and weighed (body
185
mass [BM], taken after one day fasting). Female SL was 5.41 ± 0.55 cm (mean ± standard
186
deviation) and BM was 4.30 ± 1.34 g. Male SL was 5.62 ± 0.56 cm and BM 4.67 ± 1.48 g. To
187
control for daily variation in behaviors, all territorial dyadic combats were conducted between
188
11AM and 1PM [32]. Combats were performed in an aquarium (60 × 30 × 30 cm) divided in the
189
middle of the longer side into two equal compartments by a removable opaque grey plastic
190
barrier. The conditions in both compartments were the same: each had a filter, a heater, ca. 2 cm
191
of sand and a quarter of a terracotta flowerpot (12 cm in diameter, 10 cm long) adjacent to the
192
barrier. Due to the social nature of these fish, small opposite-sexed conspecifics (one per
193
compartment) were introduced in transparent plastic bottles to encourage territory establishment
194
of the focal fish. Dyads of fish were matched by sex, SL (Mann-Whitney U test, V = 233, P =
195
0.27) and BM (V = 316, P = 0.21). Test fish were caught from their tanks of origin and released
196
in one of the two compartments to establish their territory in the quarter flowerpot for three days.
197
The compartment was randomly selected. Fish were fed commercial flakes or frozen cichlid food
198
twice a day and at least one hour before starting the trial to control for effects of feeding regime
199
[33]. Following this acclimation period the visual barrier was removed, merging the territories of
200
the two fish, and merging the flowerpot shelter into one. This procedure guarantees that both fish
201
have simultaneous ownership over a territory and that they cannot divide this resource after the
202
barrier has been removed. Neolamprologus brichardi is a highly territorial species and
203
immediately starts to combat for ownership of the shelter [9, 34]. To avoid disturbances by the
204
experimenter, the interactions of the fish were videotaped with a Sony HDR XR 550VE
205
camcorder. After each trial ended, fish were moved to separate holding nets in their original
206
tanks. (see end of file)
207
Intensity of the horizontal facial stripe (pale or dark) was recorded at the beginning and
208
end of experiments. Outcome and behaviors of the 20 min combats were recorded via video
209
analysis (for a detailed ethogram of the species, modified after Sopinka et al. [35] and Taves et
210
al. [34], see below). The winner was declared as the fish from which the loser fled three times
211
without counter-strike or constantly held a submissive posture [9, 36]. Alternatively, a fish was
212
declared winner if it owned the flowerpot in the end of the combat (i.e. the most valuable
213
resource of the territory). Behaviors of both fish were counted and fit into four categories of
214
recorded behaviors due to their very diverse behavioral repertoire (see below), which is
215
consistent with other studies of Neolamprologus [7, 34, 35]. A fighting ability index for each fish
216
was calculated by subtracting the total of submissive behaviors from the sum of territorial,
217
display and contact aggressive behaviors (called dominance index by Aubin-Horth et al. [14]).
218
Factors determining the outcome of resource contests were identified with linear mixed
219
models with ‘body mass’ and ‘fighting ability’ (the difference between aggressive and
220
submissive behaviors) as response and ‘outcome’, ‘sex’ and their interaction as explanatory
221
variables. We fit a generalized linear mixed model (GLMM) with binomial error distribution,
222
logit link function and ‘pair’ as random effect, to test whether outcome is significantly associated
223
to the intensity of the facial stripe at the beginning or at the end of the contest. For this analysis
224
we used the R package lme4 [37].
225
226
Ethogram of behavior repertoire of Neolamprologus brichardi
Category
Behaviors
Description
Contact aggression
Bite
Focal fish bites another fish
Chase
Focal fish follows another fish
Displace
Focal fish swims towards another fish, forcing it to move
Mouth-lock*
Two fish lock jaws and push against each other
Ram
Focal fish hits another fish with its head, but jaws remain closed
Aggressive posture
Focal fish lowers its head towards another fish and shows the
Display aggression
side of its body with spread fins
Submission
Head shake
Fish tosses its head from left to right
Puffed throat
Fish opens operculum and lower jaw cavity
Bitten
Focal fish gets bitten by another fish
Flee
Focal fish swims away from another fish
Submissive posture
Focal fish has a (nearly) vertical position, with the head directing
upwards
Submissive display
Focal fish is positioned with a submissive posture accompanied
by a quivering caudal fin
Territoriality
Body digging
Focal fish quivers its body on the substrate and moves sand
Digging
Focal fish takes sand in its mouth, sometimes swims to a
different area and spits it out
Lookout
Focal fish observes another fish from its shelter
Hover
Focal fish defends brood chamber, inhibit other fish from
entering
Cleaning
227
* both fish get the score
Focal fish removes algae from shelter by nibbling on them
228
To determine which color elements changed in chromatic or achromatic contrast between
229
dominant and subordinate fish, pairwise Mann-Whitney U tests were calculated for each
230
comparison. False discovery rate (FDR) was applied to correct for multiple testing. To detect
231
overall differences between adjacent and non-adjacent color patches in chromatic and achromatic
232
contrasts in territorial and non-territorial fish, we ran linear mixed-effects models (LMM) with
233
the R package nlme [38]. As we measured several color patches per fish and then used them in
234
different comparisons, all adjacent and all non-adjacent chromatic or achromatic contrasts were
235
averaged per individual. ‘Individual’ was then used as random effect. Shapiro tests confirmed
236
normality of the residuals. As achromatic contrast deviated from normality, it was square-root
237
transformed. First, to test whether the facial color pattern is conspicuous to the fish eye, we
238
compared chromatic and achromatic contrasts between adjacent and non-adjacent color patches
239
of dominant fish (i.e. fish with dark horizontal stripes, which is the state in which the phenotype
240
is normally expressed). In a second step we further analyzed subordinate fish (i.e. fish with pale
241
horizontal stripes) to investigate how changes in facial stripe intensity affected the phenotype.
242
We ran mixed models with ‘adjacency’, ‘stripe intensity’ and their interaction as fixed effects
243
and ‘individual’ as random effect.
244
245
Facial stripe out-of-equilibrium manipulation and standard mirror image stimulation
246
experiment
247
Standard mirror image stimulation (MIS) experiments were used to determine if N. brichardi are
248
able to recognize and punish unreliable signaling by measuring the response of each individual to
249
its image. MIS provides instantaneous feedback without some of the confounding factors
250
resulting from using other individuals as stimuli [39]. Cichlids, including Neolamprologus, are
251
known to react aggressively towards their mirror images [3, 36, 40]. Additionally, it has been
252
shown that N. pulcher behaves similarly towards mirror images as towards conspecifics [41].
253
The test setup consisted of an aquarium (40 × 25 × 25 cm) with a 2.84 mm-thick plane glass
254
mirror (25 × 25 cm), placed inside the tank, behind a terracotta flowerpot arch (10 cm in
255
diameter; 3 cm wide) at a sidewall. Using a flowerpot arch instead of a closed flowerpot
256
guaranteed that the fish could see their reflection at all times, including inside the shelter,
257
avoiding the generation of impossible reflection angles that could confuse them. At the
258
beginning the arch and the mirror were hidden behind an opaque grey plastic barrier. After
259
removal of the opaque barrier, the mirror image should reflect a conspecific territory owner to
260
the test fish. This setup further addresses common limitations faced when presenting
261
manipulated individuals to focal dominant, territorial individuals [42]. In our setup, the focal fish
262
act as intruders and test the repellent effect of the manipulated signals in individuals of the same
263
size they perceive as territory owners. (see end of file)
264
A total of 49 N. brichardi (25 males and 24 females) originating from several stock tanks
265
were sexed, measured (SL), and weighed (BM) as in the resource contest experiment (above).
266
Female SL was 5.90 ± 0.74 cm (mean ± standard deviation) and BM was 5.19 ± 2.21 g. Male SL
267
was 5.92 ± 0.84 cm and BM 5.20 ± 2.62 g. Before fish were tested, they were separated from
268
their social group for two days and kept in a pre-test tank (40 × 25 × 25 cm), covered on all four
269
sides to minimize disturbance. This tank contained a flowerpot arch placed adjacent to one wall
270
so fish learned to use this as a shelter instead of a closed flowerpot. After this isolation period of
271
two days, during which they became territorial, fish were gently netted out of the pre-test tank,
272
partially anesthetized with KOI MED® Sleep (KOI&BONSAI, 0.5% v/v 2-Phenoxyethanol) and
273
the horizontal facial stripe was manipulated randomly in one of three different ways:
274
1. Darkened facial stripe: The facial stripe was brushed with black waterproof eyeliner
275
(Collection 2000, USA). To control for the paling treatment (see below), wound snow and
276
wound spray (KOI MED®) were applied in the head region above the facial stripes.
277
2. Paled facial stripe: To control for the darkening treatment, the facial stripe was first
278
painted with the black waterproof eyeliner (Collection 2000, USA) and then covered up with
279
‘Wound Snow’ and ‘Wound Spray’ (KOI MED®).
280
281
3. Control sham-manipulation: Same treatment as 2. applied in the head region above the
facial stripe as in 1., so the facial stripe was left un-manipulated.
282
Spectral reflectance measurements show that treatments result in the desired effect of
283
extreme darkening and paling that was similar to non-manipulated horizontal stripes. A principal
284
components analysis (PCA) of spectral reflectance data clearly groups black ‘eyeliner’ with dark
285
melanistic stripes (typical of dominant fish) and groups ‘Wound Snow’ with pale horizontal
286
stripe, lachrymal stripe and head (typical of subordinate fish) (Figure S2). A cluster analysis
287
conducted with R package mclust [43] confirms this visual assessment (Table S1).
288
After facial stripe manipulation fish were released into the test tank compartment without
289
the flowerpot arch and mirror, and allowed to recover for 5 min from anesthesia and treatment.
290
For motivational purposes and to ease acclimation to new surroundings, fish were fed a little
291
amount of newly hatched Artemia nauplii. After the recovery period, the opaque barrier was
292
removed and the fish could interact with its mirror image. To control for daily variation in
293
behaviors, all experiments were conducted between 9AM and 11:30AM [32]. To control for
294
individual effects of aggression levels between behavioral types [44], fish were tested twice with
295
two different treatments. Order of treatment was randomized. All aggressive (display and
296
contact) and submissive behaviors towards the mirror image were counted during a period of 2.5
297
min from a video recording (Sony camcorder, see above), starting after the removal of the
298
opaque barrier.
299
We fit a linear mixed model with ‘aggressive bouts’ as response and ‘treatment’, ‘sex’
300
and their interaction as explanatory variables. To normalize the residuals, ‘aggressive bouts’ was
301
square-root transformed. As fish were tested twice, ‘individual’ was added as a random effect.
302
Tukey’s HSD post-hoc analysis was performed to test for differences among treatment levels.
303
304
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Schematic representation of behavior experimental setups. In the territory resource contest
418
setup (upper row), fish are visually separated and allowed to establish their territory in the
419
terracotta flowerpot for three days (a), after which the opaque divider is removed and
420
territories/shelters merged (b) and individuals are allowed to fight over non-divisible territorial
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resource for 20 minutes. Observer could see the fish without being seen to control for dangerous
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levels of aggression. In the standard mirror image stimulation setup (bottom row), fish with
423
manipulated signals are introduced to a bare side of the tank with no shelters (c). After opaque
424
divider is removed (d), individuals can see a shelter and a fish holding a territory next to it (i.e.
425
their mirror image) and are allowed to interact with it.