1
Supplementary material
2
Methods
Phylogenetic tree analysis
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For phylogenetic analyses, 12S rRNA gene sequences of D. pulex, D. longicephala and 11
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other members of the subgenera Daphnia, Hyalodaphnia and Ctenodaphnia were downloaded
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(Supplementary Tab. 1) from GenBank. Sequences were aligned using MAFFT and the E-
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INS-I algorithm and cropped to a final length of 424 base pairs including gaps (alignment
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available from the authors on request). Phylogenetic trees were calculated using RAxML
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version 8 for Maximum Likelihood (ML) inferences, using the GTRCAT model and 10,000
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fast bootstraps to calculate branch support. Bootstrap values were mapped on the best ML
11
tree.
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Animal culture
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Daphnia pulex (clone R9 from Canada) and D. longicephala (clone Sonja from Lara Pond
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Australia) individuals were reared under constant conditions (20 °C and day : night cycle of
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12h : 12h) and taken from the culture collection of the Department of Animal Ecology,
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Evolution and Biodiversity, Ruhr-University Bochum, Germany for the performed
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experiments. All specimens were fed with the green algae Scenedesmus obliquus raised in the
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institute’s algae cultures under standardized conditions. Animals were age-synchronized in
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charcoal filtered tap water.
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Methods of classical nissl stainings
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Thyazines dyes used in Nissl staining identify the basophilic structure of the rough
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endoplasmatic reticulum (rER). Cells with a high rate of protein synthesis possess a large
1
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rER. Tissue was dehydrated in a graded series of 70% ethanol for 5 min and rinsed three
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times in deionized water for 1 min. Staining was performed for 15 min in cresyl violet
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solution (2.25 g cresyl violet, Chroma Münster, Germany in 100 ml aqua dest. and 1.5 ml
27
acetic acid) and differentiated in acidified deionized water (100 ml deionized water and 1
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drop acetic acid). Tissue was dehydrated in ethanol (70% 2*2 min, 96% 3*2 min, isopropyl
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alcohol 100% 2*2 min, roti®-Histol 3*3 min) and mounted in Entellan ® (Merck, Germany).
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Fluorescent Nissl stain and immunohistochemistry
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Tissues were rinsed using five steps of 10 min following two steps of 60 min. Specimens were
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stored at 4 °C overnight. Tissues were incubated at 5% normal goat serum (Dianova,
35
Germany) in PBS and 0.1% Triton X-100 (PBS-TX). Dopamine antibody (DOP 11s in rabbit;
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Acris Germany) was diluted 1:100 in PBS-TX and incubated for 48 h. After rinsing in PBS-
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TX for 24 h the secondary anti-body (anti-rabbit IgG-Cy3 conjugated in goat; Excitation/
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Emission (nm): 553⁄565; Dianova Germany) was incubated for 24 h. After another rinsing
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step of 24 h tissue was stained for 5 h in Nissl fluorescence by Neurofluor; Life Technologies
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(1:150 in PBS; Excitation/ Emission (nm): 500⁄525). After a final rinsing step (2 h) in PBS
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specimens were mounted on glass slides (Thermo Scientific) using VECTASHIELD ® (Vector
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Lab, Burlington) (see 18). Analysis was performed using a Zeiss LSM 510 (META software)
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confocal scanning microscope. Software supplied by Zeiss (LSM Image examiner rel. 4.2)
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was used to analyse, store and export pictures obtained. Figures were composed and labelled
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in Photoshop CS 4 (Adobe Systems, San Jose, CA). Saturation and brightness were optimized
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and no further editing was conducted.
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Quantitative Real-Time PCR
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Predator assay of Daphnia pulex
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Induction of D. pulex for gene expression analysis was performed in net cages by incubating
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age-synchronized adult females in 1 L glass beakers holding a nylon net cage containing 20
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Chaoborus sp. Larvae fed with 50 juvenile daphniids every 48 hours. Surrounding females
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and their offspring were in contact with Chaoborus-kairomones without being preyed upon.
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Controls were incubated similarly but without Chaoborus larvae. Controls and predator-
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induced treatments were checked daily for first instar juveniles. As genes need to be activated
57
before trait expression, we investigated the first juvenile instar in D. pulex, which precedes the
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maximal formation of neck teeth in the second instar (23). Animals were collected, water was
59
removed and batches of 50 juveniles were preserved in 20 µl RNAlater (Qiagen) and stored at
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4 °C for 24 h and then at -40 °C until RNA extraction.
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RNA was extracted using the RNAqueous ®-Micro RNA Isolation kit (Ambion) according to
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the manufacturer’s protocol. Integrity of RNA was checked using the Experion RNA StdSens
63
Analysis kit (BioRad). The Turbo DNA-free (Ambion, Life Technologies) procedure was
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applied to 1 µg of each extraction. Subsequently, reverse transcription Omniscript RT kit
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(Qiagen) was performed. Samples were checked with PCR (G3PDH primer pair, see (27) for
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genomic DNA contamination. The cDNA was diluted tenfold with molgrade water and stored
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at -20 °C until further usage.
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qPCR conditions and data analysis
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PCR mix consisted of 2 µl cDNA (which is equivalent to approximately 10 ng cDNA), 10 µl
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of the F-415L Flash SYBR Green qPCR Kit (Finnzymes), primer concentrations of 300 nM
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of forward and reverse primers of reference genes (CAPON, Xbp, Tbp) and 100 nM (Ddc) as
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well as PCR-grade water up to a total volume of 20 µl. Reactions were performed in technical
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triplicates and a no-template control was included. Every gene was tested applying twelve
3
75
biological repeats. PCR reactions were performed using the DNA Engine Opticon 2-Color-
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Real-time-PCR Detection System (BioRad) and the following conditions: 10 min at 95 °C
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and 40 cycles of 95 °C for 15 s, followed by 60 °C for 1 min, finally at 55 °C. Amplification
78
specificity was verified based on the melting curve which was obtained by heating in steps of
79
0.3 °C from 60 °C to 95 °C.
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Gene expression analysis
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Ddc-gene expression analysis of induced versus control samples was performed using relative
83
expression software REST. This software analyses gene expression data based on CP values.
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We determined CP values and mean efficiencies acquiring LinReg PCR. Randomization tests
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were performed with 10,000 iterations to assess the significance. For D. longicephala, qPCR
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reactions did not reliably work despite several optimization steps (primer modification,
87
different primer dilutions). Therefore, down-stream analyses were only performed for D.
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pulex.
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Results
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Tab. S 1: Overview over the different 12S rRNA gene sequences used for the phylogenetic
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analyses.
Species
Accession number
Subgenus
Origin
Daphnia pulex
AY626357
Daphnia
Germany
Daphnia pulicaria
AY626355
Daphnia
Germany
Daphnia obtusa
AY626364
Daphnia
USA
Daphnia ambigua
AF523738
Daphnia
France
Daphnia galeata
HM161705
Hyalodaphnia
Russia
Daphnia cucullata
DCU34652
Hyalodaphnia
Germany
4
Daphnia curvirostris
EF375859
Hyalodaphnia
Germany
Daphnia laevis
DLU34734
Hyalodaphnia
USA
Daphnia longicephala AF064176
Ctenodaphnia
Australia
Daphnia magna
DMU34738
Ctenodaphnia
USA
Daphnia similis
AF277288
Ctenodaphnia
Spain
Daphnia angulata
AY921460
Ctenodaphnia
Australia
Daphnia lumholtzi
AY921466
Ctenodaphnia
Australia
92
93
94
95
96
Tab. S 2: One-way ANOVA on kairomone induced neck teeth expression in D. pulex in the
absence of neuroactive treatments. SS: Sum of Squares; d.f.: degree of freedom; MS: mean
squares: F: F-value, p: p-value
Chaoborus kairomone effect
SS
97
98
99
d.f.
MS
F
p
Intercept
48026.496
1
48026.496
222.348158
<0.001
Kairomone
24731.263
2
12365.632
57.249137
<0.001
Error
45575.330
211
215,99682
Tab. S 3: Tukey-Kramer post hoc test on neck teeth expression in D. pulex in the absence of
neuroactive substances d.f.: degree of freedom; MS: mean squares: F: F-value, p: p-value
Tukey-Kramer post hoc Test; arcsin (neck teeth expression)
Error: Between MS = 216.00, d.f. = 211.00
Kairomone (larvae/ L)
0
0
<0.001
<0.001
5
10
100
101
102
103
5
10
<0.001
<0.001
<0.001
<0.001
Tab. S 4: One-way ANOVA on kairomone induced crest height/ body length ratio in D.
longicephala in the absence of neuroactive substances. SS: Sum of Squares; d.f.: degree of
freedom; MS: mean squares: F: F-value, p: p-value
Notonecta kairomone effect
SS
d.f.
Intercept
0.8023
Kairomone
0.0112
0.008
Error
MS
1
F
p
0.803
4023.9368
<0.001
2
0.005
29.315
<0.001
42
0.0002
104
105
106
107
108
109
110
5
111
112
113
114
Tab. S 5: Tukey Kramer post hoc test on crest height/ body length ratio in D. longicephala in
the absence of neuroactive substances. d.f.: degree of freedom; MS: mean squares: F: F-value,
p: p-value
Tukey-Kramer post hoc test; crest height/ body length
Error: Between MS = 0.00019, df = 42.00
Kairomone (%)
0
0
<0.001
<0.001
25
75
115
116
117
118
119
120
121
25
75
<0.001
<0.001
n.s.
n.s.
Tab. S 6 One-way ANOVA on neck teeth expression in D. pulex in the absence of Chaoborus
kairomones for: control group (without neuroactive substances), 15 µm dopamine, 5 nM
physostigmine, and dopamine + physostigmine. SS: Sum of Squares; d.f.: degree of freedom;
MS: mean squares: F: F-value, p: p-value
Kairomone 0 Larvae/ L
122
123
124
125
126
SS
d.f.
MS
F
p
Intercept
773.767
1
773.768
13.677
<0.001
arcsin (neck teeth)
1290.500
3
430.167
7.603
<0.001
Error
12050.556
213
56.575
Tab S 7: One-way ANOVA on crest height/ body length ratio in D. longicephala in the
absence of Notonecta kairomone for control group without neuroactive substances, 15 µm
dopamine, 5 nM physostigmine, and dopamine + physostigmine. SS: Sum of Squares; d.f.:
degree of freedom; MS: mean squares: F: F-value, p: p-value
Kairomone 0% Notonecta
127
128
129
130
131
SS
d.f.
MS
F
p
Intercept
0.551
1
0.551
2975.205
<0.001
Crest height/ body length
0.003
3
0.001
6.222
<0. 01
Error
0.007
36
0.0001
Tab. S 8: Tukey-Kramer post hoc test on neck teeth expression in D. pulex in the absence of
Chaoborus kairomones for control group without neuroactive substances (0), 15 µm
dopamine (DA), 5 nM physostigmine (PHY), and dopamine + physostigmine (DA+PHY).
d.f.: degree of freedom; MS: mean squares: F: F-value, p: p-value
Tukey-Kramer post hoc test; arcsin (neck teeth expression)
Error: Between MS = 56.575, df = 213.00
Kairomone= 0 larvae/ L
0
PHY
DA
DA+ PHY
0
PHY
0.253
0.253
1
0.445
0.0001
0.092
DA
1
DA+PHY
0.0001
0.445
0.092
0.005
0.005
132
133
134
135
136
137
6
138
139
140
141
142
143
144
Tab. S 9: Tukey-Kramer post hoc test on crest height/ body length ratio in D. longicephala in
the absence of Notonecta kairomone for control group without neuroactive substances (0),
15µm dopamine (DA), 5 nM physostigmine (PHY), and dopamine + physostigmine
(DA+PHY). d.f.: degree of freedom; MS: mean squares: F: F-value, p: p-value
Tukey-Kramer post hoc test; crest height/ body length
Error: Between MS = 0.00019, d.f. = 36.000
Kairomone= 0 %
0
0
145
146
147
148
149
150
151
PHY
DA
DA+PHY
0.023
0.04
0.002
0.818
0.999
PHY
0.023
DA
0.04
0.818
DA+ PHY
0.002
0.999
0.725
0.725
Tab. S 10: One-way ANOVA on neck teeth expression in D. pulex in the presence of a low
Chaoborus kairomone concentration for control group without neuro-active substances,
15 µm dopamine, 5 nM physostigmine, and dopamine + physostigmine. SS: Sum of Squares;
d.f.: degree of freedom; MS: mean squares: F: F-value, p: p-value
Kairomone 5 Larvae/ L
Intercept
SS
d.f.
MS
F
p
153,044.737
1
15,3044.737
523.319
<0.01
4.816
<0.01
arcsin (neck teeth)
4,225.796
3
1,408.598
Error
91,844.649
314
292.410
152
153
154
155
156
157
Tab. S 11 One-way ANOVA on neck teeth expression in D. pulex in the presence of a high
Chaoborus kairomone concentration for control group without neuro-active substances, 15
µm dopamine, 5 nM physostigmine, and dopamine + physostigmine. SS: Sum of Squares;
d.f.: degree of freedom; MS: mean squares: F: F-value, p: p-value
158
Kairomone 10 Larvae/ L
SS
d.f.
MS
F
p
219,645.128
1
219,645.128
724.394
<0.001
arcsin (neck teeth)
3,779.057
3
1,259.686
4.154
<0.001
Error
58,519.981
193
303.212
Intercept
159
160
161
162
163
164
Tab. S: 12 One-way ANOVA on crest height/ body length ratio in D. longicephala in the
presence of a 25% Notonecta kairomone for control group without neuro-active substances,
15 µm dopamine, 5 nM physostigmine, and dopamine + physostigmine. SS: Sum of Squares;
d.f.: degree of freedom; MS: mean squares: F: F-value, p: p-value
Kairomone 25% Notonecta
Intercept
SS
d.f.
MS
F
p
1.409
1
1.409
2293.283
<0.01
7
Crest height/ body length
0.005
3
0.002
Error
0.037
60
0.001
2.771
<0.05
165
166
167
168
169
170
171
Tab. S 13 One-way ANOVA on crest height/ body length ratio in D. longicephala in the
presence of a 75% Notonecta kairomone for control group without neuro-active substances,
15µm dopamine, 5 nM physostigmine, and dopamine + physostigmine. d.f.: degree of
freedom; MS: mean squares: F: F-value, p: p-value
Kairomone 75 % Notonecta
SS
Degr. of F
MS
F
p
Intercept
2.174
1
2.174
3207.610
<0.001
Crest height/ body length
0.013
3
0.004
6.285
<0.001
Error
0.048
71
0.001
172
173
174
175
176
Tab. S 14: Tukey-Kramer post hoc test on neck teeth expression in D. pulex in the presence
of a low Chaoborus kairomone concentration for control group without neuro-active
substances (0), 15µm dopamine (DA), 5 nM physostigmine (PHY), and dopamine +
physostigmine (DA+PHY). d.f.: degree of freedom; MS: mean squares: F: F-value, p: p-value
Tukey-Kramer post hoc Test; arcsin (neck teeth expression)
Error: Between MS = 290.39 df = 309.00
Kairomone= 5 Larvae/ L
0
PHY
DA
DA+ PHY
0
PHY
0.04
DA
<0.001
0.97
0.04
<0.001
0.64
0.97
0.95
0.04
DA+PHY
0.04
0.95
0.64
177
178
179
180
181
Tab. S 15: Tukey-Kramer post hoc test on neck teeth expression in D. pulex in the presence
of a high Chaoborus kairomone concentration for control group without neuro-active
substances (0), 15µm dopamine (DA), 5 nM physostigmine (PHY), and dopamine +
physostigmine (DA+PHY). d.f.: degree of freedom; MS: mean squares: F: F-value, p: p-value
Tukey-Kramer post hoc Test; arcsin (neck teeth expression)
Error: Between MS = 303.21, df = 193.00
Kairomone=10 larvae/ L
0
PHY
DA
0.833
0
PHY
0.833
DA
0.554
0.987
DA+ PHY
0.003
0.089
DA+PHY
0.554
0.003
0.987
0.089
0.127
0.127
182
183
184
185
186
Tab. S 16: Tukey-Kramer post hoc test on crest height/ body length in D. longicephala in the
presence of 25% Notonecta kairomone for control group without neuro-active substances (0),
15 µm dopamine (DA), 5 nM physostigmine (PHY), and dopamine + physostigmine
(DA+PHY). d.f.: degree of freedom; MS: mean squares: F: F-value, p: p-value
Kairomone= 25%
Tukey-Kramer post hoc test; crest height/ body length
Error: Between MS = 0.00061, df = 60.000
0
PHY
DA
DA+PHY
8
0.199
0
187
188
189
190
191
192
PHY
0.199
DA
0.472
0.998
DA+ PHY
0.030
0.600
0.472
0.030
0.998
0.600
0.663
0.663
Tab. S 17: Tukey-Kramer post hoc test on crest height/ body length in D. longicephala in the
presence of 75% Notonecta kairomone for control group without neuro-active substances (0),
15µm dopamine (DA), 5 nM physostigmine (PHY), and dopamine + physostigmine
(DA+PHY). d.f.: degree of freedom; MS: mean squares: F: F-value, p: p-value
Tukey-Kramer post hoc test; crest height/ body length
Error: Between MS = 0.00068, df = 71.000
Kairomone= 75 % 0
0
PHY
DA
DA+ PHY
PHY
0.033
0.033
0.004
<0.001
DA
0.004
0.914
0.914
0.888
DA+PHY
0.001
0.888
0.999
0.999
193
194
Hyalodaphnia
ata
al
.g
ll
ucu
D. c
D
ea
cur
viro
stri
D. la ev is
ta
D.
s
Daphnia
D.
pu
lex
R9
99
98
D.
l
pu
ex
65
li c a
D. pu
G2
r ia
99
92
10
Ctenodaphnia
94
10
ag
.m
D
D.
am
bi
gu
a
D. a
ngu
lata
is
ep ha
D . lo ng ic
na
il
sim
66
0
44
a
10
0
D. ob
tu s
Daphnia lumholtzi
D.
0
100
0.06 subst. per site
la
195
9
196
Fig. S 1: Unrooted Maximum Likelihood 12S rDNA phylogenetic tree of Daphnia species
197
from the three subgenera Daphnia (blue lines), Hyalodaphnia (black lines), and Ctenodaphnia
198
(red lines). Daphnia pulex (blue) and Daphnia longicephala (red) occur in the two
199
phylogenetically distinct subgenera Daphnia and Ctenodaphnia. For the calculation of the
200
tree and accession numbers see Tab. S 1.
201
202
Fig. S 2: Inducible morphological defences in D. pulex and D. longicephala. (A) Image of
203
Daphnia pulex control morph (left) and an induced morph (right). (B) Image of Daphnia
204
longicephala control morph (left) an induced morph (right).
205
206
10
207
Fig. S 3: Bulged cells in Daphnia spec. A: Nissl fluorescent bulged cell in D. pulex with an
208
intensely stained and distinct nucleus (yellow arrow). Scale = 20 µm. B: Comparable bulged
209
cell in D. longicephala stained using classical Nissl method. Nucleus is characteristically
210
marked (yellow arrow). Scale = 10 µm.
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