SUPPLEMENTAL METHODS AND RESULTS
Sequential comparison of conspecific male color by limnetic females
When limnetic females see two conspecific males in sequential trials, females
compare the coloration of the second male to the first and show greater preference for the
more colorful male [1]. To test if predators change this sequential preference for color in
conspecifics, we calculated the difference in preference between the two males (second
minus first male) and analyzed it in a model with coloration difference (second minus
first male), predator exposure, and their interaction.
There was no effect of predator exposure for limnetic females when sequentially
comparing coloration between first and second conspecific males (all limnetic female
interactions between difference in coloration and predator: all F1,36 < 0.49, p > 0.48).
SUPPLEMENTAL REFERENCES
1.
Kozak G.M., Head M.L., Lackey A.C., Boughman J.W. 2013 Sequential mate
choice and sexual isolation in threespine stickleback species. J. Evol. Biol. 26, 130-140.
Inspection
(a)
†
= control
= predator
Inspection
(b)
Inspection
(c)
First
Trial
Second
Trial
Supplemental Figure 1. Female inspection and
predator exposure.
Least-squared means + s.e. shown for female
inspection shown for trials with conspecific
mates and trials with heterospecific mates.
Benthic and limnetic female species combined.
Filled symbols indicate control exposure, open
symbols predator exposure. (a) Inspection of
conspecific males; (b) inspection of
heteospecific males; (c) inspection of
heterospecific males by females in the
achromatic data set (benthic females plus
limnetics females who saw dull males).
Significance of comparisons between trials
(within treatments) indicated immediately to
the right of symbols. † p < 0.10.
1
0.9
Follows
0.8
0.7
0.6
0.5
0.4
0.3
0.2
= control
= predator
0.1
0
0
2
4
6
8
10
12
Coloration
Supplemental Figure 2. Limnetic female preference for colorful males after exposure.
Follows per lead to heterospecific males in the second trial plotted against male
coloration (interaction between predator exposure, trial and male coloration F1,51.61 =
4.27). Untransformed values shown. Filled symbols/solid line indicate control
exposure, open symbols/dashed line predator exposure (predator-exposed β = -0.11 +
0.04, t37 = -2.5, p = 0.021; control-exposed: β = 0.071 + 0.05, t37 = 1.31, p = 0.21).
Conspecific Males
Log Latency
Predator
(trout,control)
Estimate
Trial (first,second)
Predator*Trial
Female species
Male Color
Female ID§
Asinroot Follows
Predator
(trout,control)
Trial (first,second)
Predator*Trial
Female species
Male Color
Female ID§
Asinroot Inspection
Predator
(trout,control)
Trial (first,second)
Predator*Trial
Female species
Male Color
Female ID§
All females
s.e.
ddf F1,ddf
Achromatic females
p Estimate
s.e.
ddf F1,ddf
Reused females removed
p Estimate
s.e.
ddf F1,ddf
p
0.44
0.38
49.31
1.37
0.25
0.15
0.44
32.40
0.11
0.74
0.44
0.38
41.39
0.87 0.36
0.01
0.30
0.30
0.01
0.73
0.29
0.59
0.44
0.12
0.85
50.20
50.24
50.48
98.90
0.01
0.27
0.47
0.01
0.97
0.61
0.5
0.96
0.05
0.04
0.20
0.01
0.44
0.39
0.77
0.47
0.15
0.67
35.17
33.26
32.73
65.99
0.02
0.01
0.19
0.01
0.9
0.95
0.67
0.97
0.01
0.30
0.30
0.01
0.73
0.29
0.59
0.44
0.12
0.85
42.19
42.16
41.46
80.98
0.10
0.01
0.05
0.13
0.75
0.92
0.82
0.71
0.09
0.01
0.03
0.20
0.05
0.10
0.10
0.05
0.11
0.12
0.02
0.32
49.54
50.07
50.11
50.52
82.50
0.72
0.02
0.09
2.62
3.65
0.4
0.9
0.76
0.11
0.06
0.08
0.01
0.06
0.23
0.05
0.10
0.13
0.08
0.15
0.14
0.03
0.32
32.61
34.69
33.11
32.89
57.84
0.33
0.03
0.18
2.80
1.93
0.57
0.86
0.68
0.1
0.17
0.06
0.04
0.10
0.12
0.06
0.10
0.11
0.06
0.11
0.14
0.03
0.32
41.63
42.07
42.05
41.68
64.08
0.25
0.53
0.69
0.72
6.00
0.62
0.47
0.41
0.4
0.02
0.02
0.17
0.13
0.28
0.05
0.10
0.13
0.09
0.18
0.15
0.04
0.31
49.35
50.17
50.21
50.55
96.96
0.03
3.50
0.54
3.71
1.48
0.86
0.07
0.47
0.06
0.23
0.12
0.19
0.09
0.30
0.04
0.08
0.16
0.13
0.25
0.16
0.05
0.28
32.44
35.11
33.22
32.77
65.65
0.62
2.13
0.12
3.32
0.73
0.43
0.15
0.73
0.08
0.4
0.05
0.25
0.09
0.14
0.03
0.11
0.14
0.09
0.19
0.17
0.04
0.32
41.46
42.15
42.12
41.52
76.72
0.14
6.78
0.23
0.65
0.40
0.71
0.01
0.63
0.42
0.53
Supplemental Table 1. Predator exposure and sexual selection within species. Results from linear mixed model with female identity
(ID) as a random factor for transformed latency (log), follows (arcsinroot), and inspection (arcsinroot). Estimate, standard error (s.e.),
denominator degrees of freedom (ddf) and F values shown for full data set (all females, N = 53 females), achromatic data set (only
limnetic females that saw dull males and benthic females: N = 36), and data set with reused (tested on a prior clutch) females removed
(N = 45). Significant effects (p < 0.05) shown in bold. § s.d. rather than s.e. shown.
Heterospecific Males
Log Latency
Estimate
Predator
(trout,control)
0.76
Trial
(first,second)
1.21
Predator*Trial
2.08
Female species
0.59
Male Color
0.14
Female ID§
0.34
Asinroot Follows
Predator
(trout,control)
0.18
Trial
(first,second)
0.15
Predator*Trial
0.10
Female species
0.28
Male Color
0.00
Female ID§
0.11
Asinroot Inspection
Predator
(trout,control)
0.16
Trial
(first,second)
0.17
Predator*Trial
0.17
Female species
0.26
Male Color
0.04
Female ID§
0.15
All females
s.e.
ddf F1,ddf
0.37
52.41
4.28
Achromatic females
p Estimate s.e. ddf F1,ddf
0.04
0.94 0.45 38.27
0.41
0.79
0.54
0.27
0.67
4.39
Reused females removed
p Estimate s.e. ddf F1,ddf
0.04
0.36 52.45 13.72 0.0005
0.66 52.34 9.88 0.003
0.42 54.57 1.99
0.16
0.19 101.30 0.53
0.47
0.58
1.56
1.53
0.57
0.06
0.45
0.11
52.59
2.58
0.11
0.26 0.12 38.52
4.53
0.04
0.07
0.13
0.12
0.03
0.33
52.25
52.15
54.74
98.28
4.98
0.62
5.01
0.01
0.03
0.44
0.03
0.93
0.11
0.37
0.38
0.04
0.11
39.68
39.12
49.48
67.99
1.65
7.52
7.28
0.95
0.21
0.009
0.01
0.33
0.15
52.56
1.13
0.29
0.22 0.16 38.37
1.80
0.19
0.11 52.33
0.22 52.22
0.17 54.80
0.05 103.46
0.39
2.28
0.64
2.35
0.58
0.14
0.43
0.13
0.45
0.09
0.04
0.56
0.14
0.13
0.52
0.03
8.39
5.43
0.47
0.86
0.006
0.02
0.07
0.14
0.14
0.04
0.33
0.12
0.24
0.19
0.06
0.36
39.97 13.89 0.0006
48.56 3.77
0.06
50.43 1.11
0.3
77.67 0.04
0.84
39.87
39.21
50.22
76.71
0.86 0.39 47.58
1.34
2.59
0.44
0.02
0.41
0.38
0.68
0.44
0.13
0.64
4.98
p
0.03
47.32 18.09 <0.0001
47.47 14.47 0.0004
48.31 1.01
0.32
92.14 0.03
0.85
0.22 0.12 47.74
3.29
0.08
0.15
0.15
0.26
0.02
0.11
47.14
47.28
48.45
88.28
4.87
1.06
3.64
0.25
0.03
0.31
0.06
0.62
0.22 0.16 47.73
1.84
0.18
0.21
0.03
0.24
0.01
0.18
3.92
0.02
1.68
0.03
0.05
0.89
0.2
0.87
0.07
0.14
0.13
0.03
0.34
0.11
0.22
0.18
0.05
0.43
47.18
47.33
48.47
91.93
Supplemental Table 2. Predator exposure and sexual isolation. Results from linear mixed model with female identity (ID) as a
random factor for transformed latency (log), follows (arcsinroot), and inspection (arcsinroot). Estimate, standard error (s.e.),
denominator degrees of freedom (ddf) and F values shown for full data set (all females, N = 55 females), achromatic data set (only
limnetic females that saw dull males and benthic females: N = 42), and data set with reused (tested on a prior clutch) females removed
(N = 50). Significant effects (p < 0.05) shown in bold. § s.d. rather than s.e. shown.
Conspecific males
Predator (trout,control)
Trial (first,second)
Predator*Trial
Female species
Male Color
Female ID§
Heterospecific males
Predator (trout,control)
Trial (first,second)
Predator*Trial
Female species
Male Color
Female ID§
All females
Estimate s.e.
Z
0.35 0.62 0.56
0.44 0.56 0.80
0.74 0.79 0.93
1.91 0.65 2.95
0.23 0.16 1.46
1.10 1.05
0.77
1.14
0.31
0.97
0.31
2.25
0.62
0.56
0.75
0.52
0.17
1.50
1.24
2.06
0.41
1.85
1.83
p
0.575
0.426
0.351
0.003
0.144
0.214
0.040
0.683
0.065
0.068
Achromatic females
Reused females removed
Estimate s.e.
Z
p Estimate s.e.
Z
p
0.07 0.76 0.09 0.931
0.42 0.67 0.63 0.532
0.71 0.67 1.06 0.290
0.41 0.61 0.68 0.499
0.93 0.96 0.97 0.333
0.40 0.85 0.47 0.641
2.02 0.71 2.84 0.005
1.38 0.69 1.99 0.047
0.20 0.19 1.04 0.298
0.13 0.17 0.76 0.448
1.07 1.03
0.93 0.97
1.00
1.83
1.56
2.92
0.94
0.86
0.67
0.94
0.89
0.28
3.20
1.17
2.73
1.67
3.26
3.33
1.79
0.244
0.006
0.095
0.001
0.001
0.53
1.53
0.79
0.66
0.26
3.39
0.69
0.64
0.82
0.59
0.18
1.84
0.78
2.40
0.96
1.13
1.41
0.436
0.017
0.335
0.257
0.157
Supplemental Table 3. Predator exposure and preference score. Preference score analyzed using ordinal
cumulative linear mixed models (logit link fitted with the adaptive Gauss-Hermite quadrature
approximation with 10 quadrature points). Estimate, standard error (s.e.), Z values for both sexual
selection (conspecific males) and sexual isolation (heterospecific males) analyses shown for all females
(Con N = 53 females; Het N=55 males), achromatic data set (only limnetic females that saw dull males
and benthic females: Con N = 36; Het N = 42), and data set with reused (tested on a prior clutch) females
removed (Con N = 45, Het N= 50). Significant effects (p < 0.05) shown in bold.
§ s.d. rather than s.e. shown.
Log Latency
Predator
(trout,control)
Trial (first,second)
Predator*trial
Male color
Male color*trial
Male color*predator
Male
color*predator*trial
Female ID§
Asinroot Follows
Predator
Trial
Predator*trial
Male color
Male color*trial
Male color*predator
Color*predator*trial
Female ID§
Asinroot Inspection
Predator
Trial
Predator*trial
Male color
Male color*trial
Male color*predator
Color*predator*trial
Female ID§
Preference Score
Predator
Trial
Predator*trial
Male color
Male color*trial
Male color*predator
Color*predator*trial
Female ID§
Conspecific Males
Estimate
s.e.
ddf F1,ddf
Heterospecific Males
p Estimate
s.e.
ddf F1,ddf
p
0.59
0.55
36.36
0.89
0.35
0.49
0.63
36.33
0.56
0.46
0.03
0.59
0.34
1.21
0.44
0.45
0.37
0.64
0.54
0.51
34.97
61.94
34.97
65.20
61.94
0.18
0.03
0.28
3.17
0.05
0.67
0.87
0.6
0.08
0.82
1.95
0.38
1.66
0.71
0.03
0.62
0.55
0.85
0.71
0.71
36.03
69.24
36.03
66.5
69.24
6.85
0.01
3.8
2.74
0.04
0.01
0.94
0.06
0.1
0.85
1.05
1.00
0.75
1.00
65.20
1.90
0.17
0.13
0.23
0.91
0.48
66.5
0.02
0.89
0.07
0.05
0.04
0.12
0.10
0.02
0.08
0.10
0.12
0.08
0.07
0.11
0.10
0.10
0.14
0.31
37.09
34.48
47.83
34.48
50.26
47.83
50.26
0.01
0.03
5.79
1.26
0.86
0.06
0.3
0.94
0.86
0.02
0.27
0.36
0.81
0.59
0.07
0.15
0.06
0.09
0.09
0.03
0.41
0.10
0.15
0.12
0.12
0.16
0.15
0.16
0.19
0.31
36.99
34.89
63.75
34.89
51.61
63.75
51.61
0.74
1.78
0.05
0.29
1.22
4.73
4.27
0.39
0.19
0.82
0.59
0.27
0.03
0.04
0.12
0.26
0.10
0.11
0.29
0.09
0.19
0.13
0.16
0.12
0.10
0.17
0.15
0.14
0.21
0.35
36.68
34.74
55.68
34.74
58.92
55.68
58.92
0.17
5.52
0.61
0.44
3.25
0.01
0.79
0.68
0.02
0.44
0.51
0.08
0.94
0.38
0.40493
0.06137
0.03575
0.34284
0.05354
0.14199
0.26667
0.1923
0.236
0.192
0.194
0.265
0.249
0.251
0.312
0.439
36.88
35.16
66.85
35.16
55.14
66.85
55.14
1.46
3.08
0.00
1.67
0.25
0.00
0.70
0.24
0.09
0.96
0.20
0.62
0.96
0.41
0.71
0.20
0.48
0.55
0.61
0.56
0.26
3.58
0.95
0.76
0.65
1.05
0.96
0.88
1.28
1.89
0.75
0.27
0.75
0.53
0.64
0.63
0.20
0.46
0.79
0.46
0.60
0.52
0.53
0.84
1.11
0.77
0.17
0.65
0.85
0.28
1.46
4.06
0.98
0.73
0.73
0.99
0.98
1.02
1.23
2.02
1.14
1.05
0.23
0.65
0.86
0.27
1.19
0.25
0.29
0.82
0.51
0.39
0.79
0.24
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Supplemental Table 4. Predator exposure and color preference in limnetics. Results for limnetics with
conspecific males (N = 40) and heterospecific males (N = 39) from mixed model with female identity as a
random factor and all interactions with male coloration included. Estimates, standard error (s.e.),
denominator degrees of freedom (ddf) and F values shown for transformed latency (log), follows
(arcsinroot), and inspection (arcsinroot). Z values from cumulative mixed model with female identity as a
random factor shown for preference score (see Supplemental Table 3). Significant effects (p < 0.05)
shown in bold. § s.d. rather than s.e. shown.