Supporting information
Appendix 1. Supplementary methods
Animal maintenance – Sea water quality (temperature, salinity and dissolved oxygen) was
monitored and maintained during recovery, acclimation and experiments. Water temperature
was monitored using thermocouples (type T, gauge 24) and handheld thermometers (CHY
507, Taiwan; monitored daily) and recorded using I-button data loggers (DS1922L, ± 0.5°C,
Dallas Semiconductor Maxim, USA) protected in silicone capsules (SL-ACC06, Signatrol,
Tewkesbury, UK) and programmed to record every 5 min. Salinity and dissolved oxygen
were monitored every three days using salinity and dissolved oxygen meters (YSI 30 Salinity,
Conductivity and Temperature meter, Ohio, USA; DO-5510 Lutron Dissolved Oxygen
Meter, Taipei, Taiwan) during recovery, acclimations and slow-rate experiments. Sea water
was continuously aerated and fresh, filtered sea water at the required temperature was
provided every three days. Organisms were provided once with standardized amounts of food
(seaweed [Ulva spp.] or kelp [Durvillaea antarctica and Ecklonia maxima for experiments on
Marion Island and South Africa, respectively]) during recovery and acclimation and every 10
days during slow-rate experiments, and were maintained at a photoperiod of 12L: 12D. At no
point was water quality compromised owing to changes in temperature, dissolved oxygen and
salinity.
Identification - The identity of each individual was checked, following mortality or
conclusion of any aspect of the experiments using taxonomic keys (Day 1969; Griffiths 1976,
Kensley 1978; Branch et al. 1991), and by soliciting the input of taxonomic experts (C.
Griffiths at the Marine Research Institute, University of Cape Town, South Africa; N. Bruce
at the Queensland Museum, Australia). Sex of all individuals was determined following
standard methods (Griffiths 1976; Branch et al. 1991; Wilson 1991; Branch et al. 2007).
Given that both amphipods and isopods are brooding organisms and do not include a
dispersive planktonic larval stage, reduced gene flow between populations likely promotes
adaptation to local conditions (Teske et al. 2007; Sherman, Hunt & Ayre 2008). We found a
highly significant difference of CTmax between populations of Exosphaeroma laeviusculum
from Hangklip and Lamberts Bay (general linear model, population effect: rate of 0.1°C/min,
F1,
75=17.05,
P<0.001; rate of 0.5°C/min, F1,
75=21.12,
P<0.001) and therefore, these two
populations were included separately in later statistical analyses.
References
Branch, G.M., Griffiths, C.L., Branch, M.L. & Beckley, L.E. (2007) Two oceans: a guide to the
marine life of southern Africa. Struik Publishers, Cape Town.
Branch, M.L., Griffiths, C.L., Kensley, B. & Sieg, J. (1991) The benthic Crustacea of sub-Antarctic
Marion and Prince Edward Islands: illustrated keys to the species and results of the 1982 –
1989 University of Cape Town surveys. South African Journal of Antarctic Research, 21, 3-44.
Day, J.H. (1969) A guide to marine life on South African shores. Balkema AA, Cape Town.
Griffiths, C. (1976) Guide to the benthic marine amphipods of southern Africa. The Rustica Press,
Wynberg.
Kensley, B, (1978) Guide to the marine isopods of southern Africa. The Rustica Press, Wynberg.
Teske, P.R., Papadopoulos, I., Zardi, G.I., McQuaid, C.D., Edkins, M.T., Griffiths, C.L., Barker &
N.P. (2007) Implications of life history for genetic structure and migration rates of southern
African coastal invertebrates: planktonic, abbreviated and direct development. Marine Biology,
152, 697-711.
Sherman, C.D.H., Hunt, A. & Ayre, D.J. (2008) Is life history a barrier to dispersal? Contrasting
patterns of genetic differentiation along an oceanographically complex coast. Biological
Journal of the Linnean Society, 95, 106-116.
Wilson, G.D.F (1991) Functional morphology and evolution of isopod genitalia. Crustacean sexual
biology (eds R.T. Bauer & J.W. Martin), pp. 228-245. Columbia University Press, New York.
Appendix 2. Results of analyses testing for an effect of captivity on CT max and lipid
content
CTmax – In H. grandicornis, the CTmax of slow-rate controls was not significantly different
from the CTmax of field fresh individuals (Table S1). In this species, larger individuals had a
lower CTmax than smaller animals. In both E. laeviusculum populations there were significant
differences among the CTmax of slow-rate controls and field fresh individuals (Table S1). In
these populations, the mean CTmax of the 0.0001°C min-1 controls (the longest treatments)
differed most from that of field fresh individuals, however, this difference was < 1°C. Large
individuals of E. laeviusculum from Lamberts Bay had higher CTmax than small individuals.
Lipid content – The slow-rate CTmax controls of H. hirtipalma and H. grandicornis had
higher lipid content than field fresh individuals (Table S2). In the E. laeviusculum
populations, the lipid content of slow-rate CTmax controls and field fresh individuals differed
significantly, with field fresh individuals having slightly lower lipid content than most control
groups. In the Hyale species and E. laeviusculum populations, there was a significant positive
relationship between lipid content and dry mass (Table S2). In E. laeviusculum from
Lamberts Bay, the increase in lipid content with dry mass was lower in field fresh individuals
(slope = 0.54 ± 0.12) than in the 0.0001 (0.61 ± 0.12), 0.0002 (0.86 ± 0.13), and 0.0007°C
min-1 (0.80 ± 0.13) control groups (significant Treatment x Dry mass interaction; Table S2).
Table S1. Results of general linear models testing for the effects of treatment (slow-rate CTmax controls versus field fresh), sex, mass and
interactions on the CTmax of H. grandicornis and E. laeviusculum populations. Only the significant parameters from the minimum adequate
model are presented. Different upper case letters indicate significant differences between treatment groups (significance set at p < 0.05).
Species
H. grandicornis
E. laeviusculum
E. laeviusculum
Collection site
Muizenberg
Mass
Hangklip
Treatment
0.0001°C min-1 controls
0.0002°C min-1 controls
0.0007°C min-1 controls
Field Fresh
Lamberts Bay
Treatment
0.0001°C min-1 controls
0.0002°C min-1 controls
0.0007°C min-1 controls
Field Fresh
Mass
Sample
size
15
15
15
15
Captivity
period
d.f.
P
1
5.847
0.023
3
14.572
< 0.001
58 days
50 days
19 days
3
15
15
15
15
F
5.049
5.725
-0.75 ± 0.31
39.03 ± 0.12
39.27 ± 0.12
39.86 ± 0.12
39.81 ± 0.12
A
A
B
B
38.93 ± 0.26
39.02 ± 0.26
39.37 ± 0.24
39.52 ± 0.24
9.35 ± 3.91
A
AB
BC
C
< 0.01
84 days
50 days
19 days
1
Estimate ±
s.e.
0.020
Table S2. Results of general linear models testing the effects of treatment (slow-rate CTmax controls versus field fresh), dry mass and
interactions on the lipid content (log10 transformed in cases where assumptions were not met) of Hyale species and E. laeviusculum populations.
Only the significant parameters from the minimum adequate model are presented. Different upper case letters indicate significant differences
between treatment groups (significance set at p < 0.05).
Species
H. hirtipalma
H. grandicornis
E. laeviusculum
E. laeviusculum
Collection site
Marion Island
Treatment
Controls
Field Fresh
Dry mass
Muizenberg
Treatment
Controls
Field Fresh
Dry mass
Hangklip
Treatment
0.0001°C min-1 controls
0.0002°C min-1 controls
0.0007°C min-1 controls
Field Fresh
Dry mass
Lamberts Bay
Treatment
0.0001°C min-1 controls
0.0002°C min-1 controls
0.0007°C min-1 controls
Field Fresh
Dry mass
Treatment x Dry mass
Sample size
Captivity period
d.f.
1
36
203
21
50
8
8
33
50
19
23
41
50
F
27.999
P
< 0.001
105 days
1
1790.080 < 0.001
1
5.034
1
667.626
< 0.001
3
7.170
< 0.001
58 days
50 days
19 days
1
207.593
< 0.001
3
4.772
< 0.01
84 days
50 days
19 days
32.259
5.777
-0.57 ± 0.12
-0.80 ± 0.12
1.07 ± 0.03
A
B
-1.18 ± 0.20
-1.05 ± 0.22
1.00 ± 0.04
A
B
0.0003 ± 0.0003
0.0005 ± 0.0003
0.00003 ± 0.0002
0.0007 ± 0.0001
0.17 ± 0.01
ABC
A
B
C
-3.30 ± 0.43
-2.28 ± 0.230
-2.51 ± 0.270
-3.48 ± 0.22
0.61 ± 0.11
AB
A
A
B
0.028
16 days
1
3
Estimate ± s.e.
< 0.001
< 0.001
Table S3: Mean (± SD) CTmax of field fresh organisms and those acclimated to low, average
and high sea water temperature at slow (0.0001, 0.0002 and 0.0007°C min-1) and fast (0.1 and
0.5°C min-1) rates of temperature change. FF denotes field fresh individuals. MI: Marion
Island, RSA: South Africa.
Species
Exosphaeroma gigas
Exosphaeroma gigas
Exosphaeroma gigas
Exosphaeroma gigas
Exosphaeroma gigas
Exosphaeroma gigas
Exosphaeroma gigas
Exosphaeroma gigas
Exosphaeroma gigas
Exosphaeroma gigas
Exosphaeroma gigas
Exosphaeroma gigas
Exosphaeroma gigas
Exosphaeroma gigas
Exosphaeroma gigas
Exosphaeroma gigas
Exosphaeroma gigas
H. hirtipalma
H. hirtipalma
H. hirtipalma
H. hirtipalma
H. hirtipalma
H. hirtipalma
H. hirtipalma
H. hirtipalma
H. hirtipalma
H. hirtipalma
H. hirtipalma
H. hirtipalma
H. hirtipalma
H. hirtipalma
H. hirtipalma
H. hirtipalma
H. hirtipalma
H. grandicornis
H. grandicornis
H. grandicornis
H. grandicornis
H. grandicornis
H. grandicornis
H. grandicornis
H. grandicornis
H. grandicornis
H. grandicornis
H. grandicornis
Collection site
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Trypot Beach, MI
Muizenberg, RSA
Muizenberg, RSA
Muizenberg, RSA
Muizenberg, RSA
Muizenberg, RSA
Muizenberg, RSA
Muizenberg, RSA
Muizenberg, RSA
Muizenberg, RSA
Muizenberg, RSA
Muizenberg, RSA
Rate
0.0001
0.0001
0.0001
0.0002
0.0002
0.0002
0.0007
0.0007
0.0007
0.1
0.1
0.1
0.1
0.5
0.5
0.5
0.5
0.0001
0.0001
0.0001
0.0002
0.0002
0.0002
0.0007
0.0007
0.0007
0.1
0.1
0.1
0.1
0.5
0.5
0.5
0.5
0.0001
0.0001
0.0002
0.0002
0.0007
0.0007
0.0007
0.1
0.1
0.1
0.1
Acc
3
7
11
3
7
11
3
7
11
3
7
11
FF
3
7
11
FF
3
7
11
3
7
11
3
7
11
3
7
11
FF
3
7
11
FF
11
15
11
15
11
15
19
11
15
19
FF
Mean ± SD
21.3 ± 2.5
22.2 ± 1.8
21.0 ± 1.8
24.4 ± 0.6
24.5 ± 0.4
23.7 ± 1.6
27.0 ± 0.5
27.3 ± 0.0
27.0 ± 0.5
29.7 ± 1.5
31.2 ± 0.9
32.3 ± 0.6
31.9 ± 0.3
29.9 ± 0.8
30.0 ± 1.6
33.9 ± 1.3
30.9 ± 1.2
18.2 ± 2.0
18.5 ± 1.3
19.1 ± 0.7
20.0 ± 1.1
20.6 ± 0.5
20.1 ± 1.1
23.0 ± 0.7
22.5 ± 1.3
23.3 ± 0.4
25.4 ± 2.5
27.4 ± 0.6
27.3 ± 0.7
28.4 ± 1.2
28.0 ± 1.2
30.0 ± 1.3
29.5± 1.9
28.6 ± 1.2
16.5 ± 1.2
16.4 ± 1.1
16.9 ± 2.2
17.4 ± 1.7
20.1 ± 4.9
20.0 ± 4.2
21.0 ± 5.1
34.6 ± 0.5
35.3 ± 0.5
34.6 ± 2.2
34.8 ± 0.5
n
19
10
13
15
12
14
18
18
20
15
15
15
15
15
15
15
30
19
28
18
26
23
27
32
18
29
9
10
10
20
15
15
15
25
13
12
14
11
18
14
12
13
11
11
14
H. grandicornis
H. grandicornis
H. grandicornis
H. grandicornis
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma laeviusculum
Exosphaeroma antikraussi
Exosphaeroma antikraussi
Exosphaeroma antikraussi
Exosphaeroma antikraussi
Exosphaeroma antikraussi
Exosphaeroma antikraussi
Exosphaeroma antikraussi
Exosphaeroma antikraussi
Muizenberg, RSA
Muizenberg, RSA
Muizenberg, RSA
Muizenberg, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Lamberts Bay, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
Hangklip, RSA
0.5
0.5
0.5
0.5
0.0001
0.0001
0.0001
0.0002
0.0002
0.0002
0.0007
0.0007
0.0007
0.1
0.1
0.1
0.1
0.5
0.5
0.5
0.5
0.0001
0.0001
0.0001
0.0002
0.0002
0.0002
0.0007
0.0007
0.0007
0.1
0.1
0.1
0.1
0.5
0.5
0.5
0.5
0.1
0.1
0.1
0.1
0.5
0.5
0.5
0.5
11
15
19
FF
11
15
19
11
15
19
11
15
19
11
15
19
FF
11
15
19
FF
11
15
19
11
15
19
11
15
19
11
15
19
FF
11
15
19
FF
11
15
19
FF
11
15
19
FF
36.5 ± 0.8
36.8 ± 1.0
37.2 ± 0.7
35.7 ± 0.5
21.7 ± 1.2
20.4 ± 1.5
20.9 ± 1.2
27.2 ± 2.2
25.9 ± 3.3
25.4 ± 2.9
32.3 ± 0.7
32.0 ± 0.9
31.9 ± 0.8
38.4 ± 0.5
38.5 ± 0.3
38.3 ± 0.3
38.1 ± 0.3
39.0 ± 0.6
39.3 ± 0.3
39.7 ± 0.8
39.8 ± 0.2
22.7 ± 2.5
21.2 ± 1.7
19.2 ± 3.5
28.8± 1.8
29.3 ± 1.1
21.7 ± 3.6
32.5 ± 0.7
32.1 ± 1.3
29.8 ± 3.8
38.4 ± 0.2
38.8 ± 0.3
38.8 ± 0.2
38.8 ± 0.2
39.6 ± 0.4
39.8 ± 0.6
40.3 ± 0.8
40.0 ± 0.5
37.2 ± 0.8
37.5 ± 0.4
37.6 ± 0.6
37.4 ± 0.4
37.4 ± 0.7
38.4 ± 1.2
38.8 ± 0.5
38.6 ± 0.9
14
15
14
14
20
16
20
22
16
24
23
16
30
15
13
14
15
15
15
15
15
27
17
29
31
20
27
36
28
26
14
15
14
15
14
15
12
15
12
13
12
15
13
14
13
12
Table S4. Species’ warming tolerances (CTmax – mean habitat temperature) at all rates of temperature change. CTmax data from acclimation at
the mean sea water temperature from the locality of origin (7°C for Marion Island species and 15°C for South African species) were used. To
calculate warming tolerances, relevant microsite temperatures were used depending on the level of exposure in the intertidal zone (submerged,
semi-exposed and exposed).
Species
Collection site
H. hirtipalma
Marion Island
E. gigas
Marion Island
H. grandicornis Muizenberg
E. laeviusculum Hangklip
E. laeviusculum Lamberts Bay
E. antikraussi
Hangklip
Exposure
Submerged
Semi-exposed
Exposed
Submerged
Semi-exposed
Exposed
Submerged
Semi-exposed
Exposed
Submerged
Semi-exposed
Exposed
Submerged
Semi-exposed
Exposed
Submerged
Semi-exposed
Exposed
Warming tolerances (°C)
0.5°C/min
0.1°C/min
24.3
21.7
24.6
22.0
0.0007°C/min
16.8
17.1
0.0002°C/min
14.8
15.2
0.0001°C/min
12.8
13.1
24.3
24.6
25.5
25.8
21.6
21.9
18.8
19.1
16.5
16.8
20.1
19.3
17.9
22.6
21.8
20.4
24.1
23.9
21
21.6
20.8
19.4
18.6
17.8
16.4
21.7
20.9
19.5
23.1
22.9
20
20.8
20.0
18.6
3.3
2.5
1.1
15.3
14.5
13.1
16.4
16.2
13.3
0.7
-0.1
-1.5
9.2
8.4
7.0
13.6
13.4
10.5
-0.3
-1.1
-2.5
3.7
2.9
1.5
5.5
5.3
2.4
Figure S1. Schematic of the experimental design followed for each species on Marion Island (MI) and South Africa (SA). After recovery,
individuals were acclimated at low, average and high temperatures, followed by critical thermal maximum (CTmax) protocols at one of 5 rates of
temperature change. After CTmax, individuals were placed at average acclimation temperatures and checked one hour later for potential recovery.
Controls were maintained at three temperatures and CTmax and lipid content obtained at the end of experimental periods. Control data were
compared with CTmax and lipid content of field fresh individuals.
Figure S2. Slow-rate experimental set-up which consisted of a jacketed Perspex tank
containing filtered and aerated sea water. Individuals were placed in containers (honey jars
with coarse mesh sides) allowing the responses of individuals to be checked while not
preventing the flow of sea water through the containers. The temperature of the sea water
inside the tank was controlled by a water bath (Grant Instruments GP 200-R4, Cambridge,
UK) that pumped and circulated freshwater into the outer jacket of the tank. The temperature
of each trial was monitored throughout the day and was increased daily at 09h30am to
maintain the rate of temperature change required for the experiments.