1
2
3
Supporting Information 2
Gene expression Figures and Tables
CD83 : Sex p= 0.657 ; Sex*Lake p= 0.638
-2.5
-1
-4
-3
-2
-5
-3
-6
-4
-5
-0.5
NKEF : Sex p= 0.058 ; Sex*Lake p= 0.029
-3.5
-3.5
-2.5
-2.5
-1.5
-1.5
-0.5
MX.1 : Sex p= 0.151 ; Sex*Lake p= 0.797
TCRbeta : Sex p= 0.295 ; Sex*Lake p= 0.54
-3.5
-4.0
-2.5
-3.0
-1.5
p40 : Sex p= 0.467 ; Sex*Lake p= 0.344
-5.0
4
5
6
7
8
9
10
11
12
13
14
15
IL1beta : Sex p= 0.408 ; Sex*Lake p= 0.11
-2
-1
IgM : Sex p= 0.088 ; Sex*Lake p= 0.161
-2.0
Log mean R0, unstandardized
0.0
0.5
-1.5
1.0
-0.5
1.5
ARP : Sex p= 0.644 ; Sex*Lake p= 0.789
LM
RO
GO
CO
MO
LM
RO
GO
CO
MO
SI 2 Figure S1. Sex and Lake and Sex*Lake interaction effects on unstandardized gene
expression levels of wild-caught fish. Symbol size and shape denote lake size and
watershed, respectively (larger symbols represent larger lakes within a watershed.. Blue
and red symbols distinguish means for males and females respectively. P-values for ANOVA
effects of sex and sex*lake interactions are listed above each panel along with the relevant
gene name. Only NKEF exhibited any sex-related effect, and it was weak enough not to be
retained following corrections for multiple comparisons. Consequently we do not examine
sex effects in depth in the remainder of this study.
1
IgM : p = 0.75068
b
a,b
a,b
-1.5
-2.5
-1.0
b
-1.5
-0.5
CD83 : p = 0.12059
-4.5
-2.0
-3.5
a
IL1beta : p = 0.00264
MX.1 : p = 0.00052
b
-3
-1.0
-2
b
a
a
-5
a
-6
p40 : p = 0.02986
-3.0
NKEF : p = 0.00549
b
a
a
b,c
b
-4.0
-1.0
-3.5
c
a
-2.0
a
a
-3.0
a
0.0
a
-2.0
-4
a
a,b
a
-3.0
-4.5
a
LM
RO
GO
CO
MO
TCRbeta : p = 7e-05
-1.0
b
-2.0
a
a,c
c
-3.0
c
16
17
18
19
20
21
22
23
24
25
26
LM
RO
GO
CO
MO
SI 2 Figure S2. Population means for Z-transformed log gene expression (rR0) in wildcaught fish, standardized by ARP, for each of seven immune genes (genes and p values for
ANOVA lake effects listed above each panel). Error bars indicate ±1 standard error
intervals. Lakes with the same letter next to their mean not significantly different. Letters
indicate lakes that are not significantly different in pairwise Tukey post-hoc tests. Lake
names are abbreviated as follows; LM: Little Mud Lake, RO: Roberts Lake, GO: Gosling Lake,
CO: Comida Lake, MO: Mohun lake. Smaller lakes are indicated with smaller symbols,
watersheds indicated by symbol type (circles: Amor watershed, triangle: Campbell River
watershed, diamonds: Mohun watershed). Higgins Lake is omitted because too few wild
fish were captured in August 2010.
2
CD83 : p = 0.0013
IL1beta : p = 0.00067
-4.0
-5
-4
-3.0
-3
-2.0
-2
-1.0
IgM : p = 0.29259
NKEF : p = 0.00448
-1.5
-1.5
-0.5
-0.5
MX.1 : p = 0.00014
-2.5
-2.5
Log mean R0, unstandardized
0.2
-1.5
0.6
-1.0
1.0
-0.5
0.0
1.4
ARP : p = 1e-05
TCRbeta : p = 7e-05
-3.0
-4.0
-2.0
-3.5
-1.0
-3.0
p40 : p = 0.00039
27
28
29
30
31
32
33
34
35
36
37
38
LM
RO
GO
CO
MO
LM
RO
GO
CO
MO
SI 2 Figure S3. Population means for absolute log-transformed gene expression (R0) in
wild-caught fish, for each of seven immune genes (genes and p values for ANOVA lake
effects listed above each panel). Error bars indicate ±1 standard error intervals. Letters
indicate lakes that are not significantly different in pairwise Tukey post-hoc tests. Lake
names are abbreviated as follows; LM: Little Mud Lake, RO: Roberts Lake, GO: Gosling Lake,
CO: Comida Lake, MO: Mohun lake. Smaller lakes are indicated with smaller symbols,
watersheds indicated by symbol type (circles: Amor watershed, triangle: Campbell River
watershed, diamonds: Mohun watershed). Higgins Lake is omitted because too few wild
fish were captured in August 2010.
3
NKEF
3
2.0
MX1
1.0
1.0
-1.5
-1
HI
LM
MO
CO
GO
p40
HI
LM
MO
CO
GO
IgM
HI
LM
MO
MO
HI
LM
MO
1.5
Roberts
0.0
means
c(0, 7)
0.5
Mohun
-1.5 -1.0 -0.5
0
means
-1
LM
GO
xvals
1.0
2
1
1.0
0.5
0.0
-0.5
HI
xvals
CO
TCR
xvals
xvals
1.5
xvals
GO
0.5
means
-0.5
-1.0
0
-1.0
GO
0.0
1
-0.5
means
0.0
means
2
0.5
1.5
1.0
0.5
0.0
-0.5
-1.0
CO
CO
39
IL1
-1.0
Z-standardized
relative expressionmeans
level, rR0
means
CD83
CO
GO
HI
xvals
LM
MO
Little Mud
Higgens
Gosling
Comida
CO
GO
Transplant destination
HI
LM
MO
xvals
c(0, 7)
40
SI 2 Figure S4 Effects of source population (color) and transplant destination on gene
41
expression for each immune gene examined (rR0). Group mean and standard error bars are
42
plotted. Statistical effects are summarized in Table 5. The vertical axis can be used to
43
estimate effect sizes, by representing the difference between population means in units of
44
standard deviations. Colors and symbols correspond to fish origin, using the same scheme
45
as Fig. 3 in the main text. Note that for some genes we have limited data on fish
46
transplanted into Little Mud (LM) cages, because of both low recapture rates and qPCR
47
failures (TCR).
48
4
350
300
Transplanted vs wild relatives
Transplanted vs wild residents
200
150
0
50
100
Frequency
250
Native caged vs wild relatives
0
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
1
2
3
Euclidian distance
SI 2 Figure S5. The distribution of pairwise multi-variate distances between fish, in gene
expression space (z-standardized log rR0 vales for the 7 immune genes). Pairwise
distances are grouped by three types of comparisons. Blue bars show the immune
expression differences between caged versus uncaged fish caught from the same native
lake (e.g., Mohun fish caged in Mohun, versus wild Mohun fish). Red bars indicate the
distances between caged fish transplanted to a foreign lake, and residents of that foreign
lake (e.g., Mohun fish caged in Comida, versus wild Comida fish). Green bars indicate the
distances between caged fish transplanted to a foreign lake, versus their wild relatives back
in their original lake (e.g., Mohun fish caged in Comida, versus wild Mohun fish). Mean
pairwise distances are indicated with correspondingly colored arrows. Significance of
differences in mean pairwise distance was evaluated by permuting the destination of caged
fish and recalculating the test statistic 10,000 times. These permutations confirmed that
fish are most similar to their wild relatives when they are kept in their original
environment (blue mean < red and green mean, P < 0.001). When fish are displaced to a
new lake (red and green bars), they are more similar to wild residents of their new
environment than to wild relatives back home (red mean < green mean, P < 0.001).
5
67
MX1
1
0.0
0
-0.5
-1.0
Native
Caged
Native
Caged
p40
-1.5
-1.0 -0.5
-0.5
0.0
0.5
1.0
0.5 1.0 1.5
1.5
NKEF
Native
Caged
Native
Caged
TCR
-0.5
0.5
0.0
0.5
1.0
1.0
1.5
IgM
68
69
70
71
72
73
74
75
76
77
78
79
-1.5
-0.5
-1.0
0.0
Z-standardized relative expression level, rR0
0.5
2
1.0
3
1.5
CD83
Native
Caged
Native
Caged
SI 2 Figure S6. Effects of caging on immune gene expression, showing mean (and standard
error) Z-standardized log rR0 . We focus on the three lakes for which we had sufficient
numbers of both wild-caught native fish and recaptured caged native fish (e.g. Comida fish
caged in Comida). Symbols: squares for Comida, circles for Little Mud, and triangles for
Mohun. CD83 was more strongly expressed in caged fish than their wild relatives just
outside the enclosures (F1,42 = 12.45, P = 0.001), with no significant lake or cage*lake effect
(both P > 0.1). MX1 was more strongly expressed in Little Mud caged versus wild fish, with
no cage effect in the other two lakes, resulting in no overall cage (F1,41 = 3.45, P = 0.07) or
lake (F1,41 = 0.40, P = 0.68) effects but a significant cage*lake interaction (F1,41 = 4.32, P =
0.019). NKEF was significantly more strongly expressed in both Little Mud and Mohun
caged fish, with no cage effect in Comida, resulting in significant effects of cage (F1,43 = 7.12,
6
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
P = 0.011) and a cage*lake interaction (F1,43 = P = 0.024). p40Phox was also more strongly
expressed in Little Mud caged fish than wild fish, but less strongly expressed in caged fish
in the other two lakes, resulting in a strong lake*cage interaction (F2,45 = 10.23, P = 0.0002)
but no cage or lake effects (F1,45 = 3.52, P = 0.07; F2,45 = 1.41, P = 0.25). IgM tended to be
more strongly expressed in caged fish in all three lakes although the effect was driven again
largely by Little Mud (cage F1,44 = 9.59, P = 0.003; lake F2,44 = 0.91, P = 0.41; cage*lake F2,44 =
2.73, P = 0.076). Finally TCR was more strongly expressed in Mohun caged than wild fish,
but there was no cage effect in Comida, leading to a significant cage*lake interaction (F1,40 =
11.98, P = 0.001). Note that the above effects, while significant, generally rely on especially
high expression of Little Mud fish caged in Little Mud. As we recovered only three native
caged fish in Little Mud, these results rest on a group (caged, Little Mud) with very low
sample size, and should therefore be treated as a provisional effect. However, even without
Little Mud we still see significant cage effects for CD83 (F1,31 = 4.96, P = 0.033), p40Phox
(F1,34 = 14.00, P = 0.0007), and a significant cage*lake interaction for TCR (lake F1,31 = 9.86,
P = 0.0037, cage*lake F1,31 = 10.69, P = 0.0026). In addition to effects of caging on gene
expression, we also observed lower body condition in caged native fish compared to their
wild relatives (ANOVA, cage effect F1,45 = 24.13, P < 0.0001; lake effect F2,45 = 1.45, P =
0.236; cage*lake F2,45 = 0.59, P = 0.559).
7
99
100
0.2
0.0
-0.4
Amor
101
102
103
104
-0.2
Body condition
0.0
-0.2
-0.4
Body condition
0.2
0.4
B) F = 6.55, P = 0.0118
0.4
A) F = 7.74, P = 0.0007
Campbell River Mohun
Destination watershed
benthic
limnetic
Native habitat
SI 2 Figure S7. Body condition varied as a function of destination watershed, and native
habitat. Condition was measured as the residuals of mass regressed on length, estimated
separately for each source population).
8
105
106
107
108
SI 2 Table S1. Loadings of ARP-PO-standardized expression of the seven immune genes on
four discriminant function axes (LD1 – LD4) separating the wild-caught stickleback from
five lakes (omitting Higgins, where too few wild fish were captured). Most of the variance is
explained by the first two axes (proportion of trace is provided for each LD axis).
Gene
LD1
LD2
LD3
LD4
CD83
0.4931
0.0264
0.2705
0.7830
IL1β
0.2649
0.5557 -0.8096 -0.4903
MX-1
0.7001
0.5319 -0.4164
0.4641
NKEF
0.3940
0.6024
0.8395 -0.5453
Phox
p40
0.1472
0.6982
0.2865 -0.2138
IgM
-0.0159 -0.6240
0.4185 -0.4877
TCRβ
-1.9307 -0.1356
0.3395
0.5206
Proportion
of trace
109
110
111
112
113
114
115
116
117
118
119
0.463
0.451
0.061
0.025
SI 2 Table S2. Loadings of ARP-PO-standardized expression of the seven immune genes on
the seven principal component axes (PC1-PC7) calculated for caged fish. Most of the
variance is explained by the first two axes (proportion of variance is provided for each PC
axis).
Gene
CD83
IL1β
MX-1
NKEF
p40Phox
IgM
TCRβ
Proportion
of variance
120
9
PC1
0.186
0.478
0.445
0.481
-0.012
0.309
0.461
PC2
0.488
-0.057
-0.195
-0.026
0.645
0.496
-0.240
PC3
0.788
-0.234
-0.067
0.101
-0.536
-0.144
-0.035
PC4
-0.252
0.062
-0.539
0.173
-0.463
0.628
-0.056
PC5
0.074
0.598
0.275
-0.321
-0.225
0.072
-0.635
PC6
0.018
-0.276
0.356
-0.678
-0.165
0.436
0.346
PC7
0.189
0.525
-0.517
-0.407
0.066
-0.218
0.450
0.502 0.224 0.107 0.075 0.036 0.035 0.022
SI 2 Table S3. Gene expression values of transplanted fish falls outside the distribution of expression values of their wild
relatives caged in their native lake. For each source population and gene, we determined the 95% quantile of ARP-normalized
expression, based on fish caged in their native lake (e.g., Comida Lake natives in Comida Lake). We then calculated the
percentage of fish moved from that lake to a foreign site, whose expression falls outside the 95% quantiles. This percentage of
extreme fish (% Extreme) was compared to a null expectation that 5% of fish should exhibit extreme values (χ2 and P-values
presented). Little Mud and Gosling Lakes are not examined because so few native fish were recaptured those lakes’ cages.
Gene
CD83
IL1β
MX-1
NKEF
p40Phox
IgM
TCRβ
10
Comida Lake
% extreme
20
95
25
90
70
60
70
Chisq
6.5
322.4
12.9
286.6
164.5
116
164.5
P
0.0052
<0.0001
0.0002
<0.0001
<0.0001
<0.0001
<0.0001
Higgins Lake
% extreme
66.7
33.3
100
66.7
66.7
66.7
33.3
Chisq
12.8
0.9
38.8
12.8
12.8
12.8
0.9
P
0.0002
0.1769
<0.0001
0.0002
0.0002
0.0002
0.1769
Mohun Lake
% extreme
80
30
20
30
80
30
70
Chisq
103.2
8.4
2.1
8.4
103.2
8.4
75.8
P
<0.0001
0.0019
0.0734
0.0019
<0.0001
0.0019
<0.0001