Functional Ecology
Supporting information
This document contains supporting information for the manuscript, “How to assess Drosophila cold
tolerance: Chill coma temperature and lower lethal temperature are the best predictors of cold
distribution limits”. The supporting information includes a detailed supplemental discussion of
possible confounding effects of laboratory natural selection and inbreeding for the animals used in
the study as well as a discussion of the importance of population bias (i.e. the magnitude of interversus intra-species specific variance in cold tolerance). The supporting information also includes
information about fitting of different non-linear models, data from a phylogenetically controlled
analysis of the main data set using phylogenetic independent contrasts (PICs); a table summarizing
data used in the core analysis (presented as bar plots in the main manuscript); a statistical table
including a sequential Bonferroni correction; and a table summarizing the current knowledge of the
overwintering biology of the 14 species studied.
Contents
Appendix S1 - Supplementary Discussion ......................................................................................................... 2
Laboratory natural selection ......................................................................................................................... 2
Intra- and inter-species specific cold tolerance ............................................................................................. 2
Appendix S2 - Supplementary Results ............................................................................................................... 3
Table S1 – Non-linear models and phylogenetic independent contrasts. ..................................................... 4
Table S2 - Data on cold tolerance measures ................................................................................................. 5
Table S3 – Sequential Bonferroni corrected correlations. ............................................................................. 6
Table S4 - Overwintering strategies in Drosophila. ....................................................................................... 7
Supplementary figure ........................................................................................................................................ 9
Figure S1 – Relationships of phylogenetically independent contrasts........................................................... 9
1
How to assess Drosophila cold tolerance: Chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits
Jonas Lembcke Andersen, Tommaso Manenti, Jesper Givskov Sørensen, Heath Andrew MacMillan, Volker Loeschcke and Johannes Overgaard
Functional Ecology
Appendix S1 - Supplementary Discussion
Laboratory natural selection
Some of the species used in the present study have been maintained in laboratory culture for several
years, leading to the risk of both inbreeding and laboratory adaptation which could potentially
confine the assessment of species specific cold tolerance. Gilchrist, Huey and Partridge (1997)
studied the problem of laboratory adaptation and found that there was no significant difference in
cold survival of adult flies subjected to laboratory natural selection at 16.5 °C (for 10+ years), 25 °C
(for 9+ years) or 29°C (for 4+ years). This is a strong indication that the selection pressure is not very
high, and thus, does not cause large variations in cold tolerance among strains. Another study
working on inbreeding depression in 10 different species of Drosophila found that inbreeding had
only minor effects on the CTmin of the different species (an average change in critical thermal
minimum (CTmin) of only 0.35 ± 0.08 °C; Bechsgaard et al., 2013), the same study also demonstrated
that the effect of inbreeding on chill coma recovery time was generally modest. Moreover, in a
recent study comparing the CTmin of 95 species of Drosophila Kellerman et al. (2012a) demonstrated
that a subset of 19 species considered to be outbred had a similar (albeit slightly stronger)
correlation to environmental variables compared to those of the entire set of 95 species. In
combination, the information from previous studies demonstrates that there most likely is an effect
of inbreeding and, therefore, maybe from laboratory adaptation. However, it also shows that these
effects are minor in comparison to the large interspecific variance found in this and other studies (i.e.
the CTmin in this study spans from -1.7 to +7.5 °C, see also discussion in Kellermann et al., 2012)
Intra- and inter-species specific cold tolerance
The populations used in this study are unlikely to have all been collected at specific geographical
locations raising the concern that one species collected near its coldest range edge will display
considerable local adaptation compared to another species collected further from the edge. Such
sampling bias could skew the results of our interspecific comparison, particularly if the intraspecific
variation related to local adaptation is strong. In a previous study, the interspecific variability in CTmin
of D. melanogaster collected over a wide latitudinal range (16 to 44°S) changed significantly but, by
less than 1°C (Overgaard, Hoffmann & Kristensen 2011). Similarly, Hoffmann, Anderson and Hallas
(2002) found that populations of D. melanogaster collected from 9 to 44 °S along Australia’s coast
differed in survival following a 2 h cold shock (-2 °C). Survival increased from 25 to 60 % depending
on latitude. In line with this observation Kimura (2004) collected several species of Drosophila from 4
different latitudes in Japan and tested LTe50 after 1 to 3 laboratory generations. He found that LTe50
2
How to assess Drosophila cold tolerance: Chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits
Jonas Lembcke Andersen, Tommaso Manenti, Jesper Givskov Sørensen, Heath Andrew MacMillan, Volker Loeschcke and Johannes Overgaard
Functional Ecology
varied considerably (average ≈ 2.4 °C) when species were collected from positions more than 10 °
apart. In the present study the interspecific variability in LT50 was > 16 °C suggesting that the
interspecific variation is considerably larger than intraspecific variation recorded previously.
Nonetheless, the literature data suggest that intraspecific variation could affect our correlations if a
strong sampling bias was present (for example, if cold hardy species were generally collected from
particularly cold regions of their distribution and cold sensitive species were collected from
particularly warm regions). We did not deliberately sample species with any bias and looking up
collection sites for our species (where possible) we found that they were generally collected close to
the species average latitudinal distribution. D. montana was the most biased, collected ≈18 °N from
the average database latitude, whereas, the average difference for the remaining species was ≈5 °
latitude (spanning from 9.6 to 1.6 ° latitude).
Appendix S2 - Supplementary Results
We tested the correlations between cAMiT and the different measures of cold tolerance using the R²
values from fitting the data to different models to confirm whether correlations would improve or alter
our interpretations of the simple linear models (Table S1). When calculating the R² values for the
logarithmic, exponential and power functions, a positive constant was added to the data-columns
containing negative values (i.e. for CTmin we added 25 to remove negative values). Overall, we found that
the R² was very similar across the different models. Furthermore, the rank order of the different
measures did not change enough to alter the conclusions drawn from the simple linear models. Thus,
we conclude that for the sake of understanding and simplicity the linear model is the right choice.
To control for phylogenetic relationships in our analysis, we calculated phylogenetically independent
contrasts (PICs) using a previously published phylogeny containing all of the species used (Kellermann et
al., 2012). Extraneous species were trimmed from the tree and PICs of species trait means were
generated in R using the pic() function in the ape package (Paradis, Claude & Strimmer 2004).
Phylogenetically independent tests of relationships between cAMiT and each measure of cold tolerance
were conducted using linear regressions of PICs forced through the origin (Garland, Harvey & Ives 1992;
Fig. S1). Species traits were also tested for phylogenetic signal – a measure of the tendency for related
species to have similar trait values - by the K-statistic (Blomberg, Garland & Ives 2003; see Fig. S1 for Kand P-values). The PICs R²-values closely resemble those found by the linear model and the rank order
remained the same (except for switch between LTi50, 2 and 24 h). From this, we conclude that the
phylogenetic signal in our dataset is not large enough to change any on the conclusions drawn in this
paper.
3
How to assess Drosophila cold tolerance: Chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits
Jonas Lembcke Andersen, Tommaso Manenti, Jesper Givskov Sørensen, Heath Andrew MacMillan, Volker Loeschcke and Johannes Overgaard
Functional Ecology
Table S1 – Non-linear models and phylogenetic independent contrasts.
The R²-values of the linear and the 4 non-linear models are listed. Below the R² values are the ranks
(descending) of the values are presented to reveal if different models displace the rank of the
explanatory power of the different measures of cold tolerance. The averages (grey) and best fit of any
model (green) of the R² values are presented in bold under the data for the 5 fitted models along with a
descending rank, as above. The PICs are presented in bold in the bottom row along with their
descending rank, used to detect significant changes in explanatory power of the different measures
compared to each other.
Correlations to cAMiT
Correlation
CTmin LTe50-2 LTe50-24 LTi50-2 LTi50-24 CCRT SCP
Linear R²
0.634
0.641
0.470 0.511
0.508 0.373 0.247
rank
2
1
5
3
4
6
7
Logarithmic R²
0.631
0.650
0.501 0.527
0.525 0.387 0.259
rank
2
1
5
3
4
6
7
Exponential R²
0.634
0.644
0.504 0.632
0.586 0.437 0.251
rank
2
1
5
3
4
6
7
nd
2 order polynomial R² 0.634
0.659
0.519 0.614
0.618 0.417 0.317
rank
2
1
5
4
3
6
7
Power R²
0.633
0.653
0.515 0.639
0.598 0.461 0.263
rank
3
1
5
2
4
6
7
Average R²
0.633
0.649
0.505 0.584
0.567 0.415 0.267
average-rank
2.2
1.0
5.0
3.0
3.8
6.0
7.0
Best fit
0.634
0.659
0.519 0.639
0.618 0.461 0.317
rank
3
1
5
2
4
6
7
PIC R²
0.628
0.661
0.406 0.456
0.470 0.384 0.279
P-values
<0.001 <0.001
0.014 0.011
0.009 0.018 0.052
rank
2
1
5
4
3
6
7
cAMiT = coldest annual minimum temperature; CTmin = critical thermal minimum; LTe50 = 50% lethal
temperature; LTi50 = 50% lethal time at low temperature (in both cases, -2 and -24 = survival after 2 h and 24 h
of recovery, respectively); CCRT = chill coma recovery time and SCP = supercooling point.
4
How to assess Drosophila cold tolerance: Chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits
Jonas Lembcke Andersen, Tommaso Manenti, Jesper Givskov Sørensen, Heath Andrew MacMillan, Volker Loeschcke and Johannes Overgaard
Functional Ecology
Table S2 - Data on cold tolerance measures
All measured variables for all species of Drosophila used for correlations (presented as histograms in
Fig. 1). All values are presented as mean ± SE.
CTmin = critical thermal minimum; LTe50 = 50% lethal temperature; LTi50 = 50% lethal time at low
temperature (in both cases, 2h and 24h = survival after 2 h and 24 h of recovery, respectively); CCRT =
chill coma recovery time and SCP = supercooling point. “N, flies” = the number of flies used to acquire
the measurement. “N, vials” = number of vials of 8-11 flies, used to acquire the measurement, in total.
Species
CTmin
(°C)
D. montana
-1.7 ± 0.2
D. sordidula
D. obscura
N,
flies
2h LTe50
(°C)
24 h LTe50
(°C)
N,
vials
2 h LTi50
(h)
24 h LTi50
(h)
N,
vials
CCRT
(s)
N,
flies
SCP
(°C)
N,
flies
11
-13.2 ± 0.3
-13.2 ± 0.3
9
> 624 **
> 624 **
15
238 ± 4
11
-21.4 ± 0.3
11
1.9 ± 0.1
11
-10.0 *
-10.0 *
9
374.0 ± 10.8
366.5 ± 11.1
10
511 ± 71
11
-20.9 ± 0.5
12
-0.4 ± 0.1
13
-13.0 ± 0.1
-13.0 ± 0.1
10
251.5 ± 12.0
251.5 ± 12.0
11
983 ± 118
12
-21.9 ± 0.3
16
D. melanogaster
2.6 ± 0.1
18
-8.1 ± 0.2
-5.7 ± 0.3
11
26.1 ± 1.6
24.1 ± 1.7
7
476 ± 26
14
-20.0 ± 0.5
16
D. persimillis
0.2 ± 0.2
12
-12.1 ± 0.5
-12.1 ± 0.5
10
407.8 ± 13.1
386.8 ± 13.3
9
694 ± 45
12
-22.8 ± 0.7
9
D. simulans
4.6 ± 0.1
14
-4.5 *
-4.5 *
9
28.5 ± 1.3
16.9 ± 0.7
10
790 ± 67
14
-20.0 ± 0.4
16
D. mojavensis
0.3 ± 0.0
15
-8.5 *
-8.5 *
7
145.2 *
145.3 *
8
485 ± 20
11
-15.8 ± 0.7
15
D. mercatorum
4.9 ± 0.1
15
-5.4 ± 0.2
3.2 ± 0.7
10
15.5 ± 0.7
5.3 ± 0.9
10
731 ± 79
15
-16.3 ± 0.3
16
D. kikkawai
5.4 ± 0.1
15
-3.1 ± 0.5
0.1 ± 0.5
9
6.9 ± 0.2
2.3 ± 0.3
9
712 ± 47
12
-18.2 ± 0.3
16
D. bipectinata
5.9 ± 0.1
20
-4.5 ± 0.3
-2.9 ± 0.2
8
3.6 ± 0.2
2.5 ± 0.3
7
897 ± 103
15
-18.9 ± 0.4
16
D. birchii
6.6 ± 0.1
15
-3.3 ± 0.2
-2.4 ± 0.3
8
3*
2.5 ± 0.2
8
937 ± 52
14
-19.6 ± 0.5
16
D. formosana
3.1 ± 0.1
14
-4.5 *
-4.0 ± 0.2
8
2.8 ± 0.1
2.5 ± 0.2
7
1113 ± 94
13
-16.8 ± 0.8
16
Z. tuberculatus
3.4 ± 0.1
10
-6.5 ± 0.5
-5.5 ± 0.4
11
34.3 ± 3.4
24.8 ± 2.6
7
545 ± 38
10
-19.5 ± 0.4
16
D. equinoxialis
7.5 ± 0.2
10
-4.6 ± 0.3
-2.6 ± 0.3
8
3.7 ± 0.2
3.1 ± 0.3
7
867 ± 68
10
-20.3 ± 0.3
16
* Test was unable to determine true standard error, see text for details.
** All flies were still alive at the termination of the experiment, see text.
5
How to assess Drosophila cold tolerance: Chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits
Jonas Lembcke Andersen, Tommaso Manenti, Jesper Givskov Sørensen, Heath Andrew MacMillan, Volker Loeschcke and Johannes Overgaard
Functional Ecology
Table S3 – Sequential Bonferroni corrected correlations.
Coefficients of determination (R²-values; lower left) and sequential Bonferroni corrected P-values (upper
right) from correlations among measures of cold tolerance and environmental variables from 14
Drosophila species. aLat = average |latitude| of species collection sites; cAMiT = coldest annual
minimum temperature; aAMiT = average annual minimum temperature; CTmin = critical thermal
minimum; LTe50 = temperature of 50% mortality to a 2 h exposure; LTi50 = exposure time at -2 °C causing
50% mortality. In both cases mortality is assessed after 2 h and 24 h of recovery, respectively; SCP =
Supercooling point. Light grey are non-significant correlations (P > 0.002) while dark grey are significant
correlations (P < 0.002). Non-significant correlations’ R²-values are written in italic, not bold and in
parenthesis.
aLat
CTmin
2h
LTe50
2h
LTe50
2h
LTi50
24h
LTi50
CCRT
SCP
<0.001
<0.001
<0.001
<0.001
0.001
0.004
0.003
0.184
0.046
<0.001
0.001
0.001
0.005
0.006
0.006
0.020
0.071
<0.001
<0.001
0.001
0.001
0.001
0.037
0.045
<0.001
<0.001
0.004
0.003
0.065
0.266
<0.001
<0.001
<0.001
0.080
0.027
0.001
<0.001
0.181
0.012
<0.001
0.354
0.039
0.368
0.041
cAMiT
0.866
aAMiT
0.916
0.964
CTmin
0.699
0.633
0.697
2h
LTe50
0.741
0.641
0.744
0.831
24h
LTe50
0.619
(0.490)
0.615
0.744
0.820
2h
LTi50
(0.550)
(0.511)
0.666
(0.553)
0.753
0.676
24h
LTi50
(0.557)
(0.508)
0.669
(0.561)
0.762
0.691
0.998
CCRT
(0.142)
(0.373)
(0.314)
(0.256)
(0.233)
(0.144)
(0.078)
(0.074)
SCP
0.291
(0.247)
0.295
(0.102)
0.346
0.419
0.334
0.328
P-Values
R²-values
aLat
cAMiT aAMiT
0.623
(0.021)
6
How to assess Drosophila cold tolerance: Chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits
Jonas Lembcke Andersen, Tommaso Manenti, Jesper Givskov Sørensen, Heath Andrew MacMillan, Volker Loeschcke and Johannes Overgaard
Functional Ecology
Table S4 - Overwintering strategies in Drosophila.
Information on overwintering biology of the Drosophila species used in this study. The information
obtained through personal communications is written in italics and the person who provided the
information is written in the References column. Note that the personal communication is not based on
experimental observations. The empty cells implies that we have been unable to acquire information on
those particular species, however, Z. tuberculates and D. equanoxialis, formosana, kikkawai and
mercatorum are all tropical species and are likely to continue to reproduce throughout the winter.
Species
D. montana
D. sordidula
D. obscura
D. melanogaster
Winter biology
Adult overwinter, photoperiodic
diapause, almost univoltine in Finland,
multivoltine in Hokkaido.
Adults overwinter, multivoltine in
Hokkaido.
Adults overwinter, photoperiodic
diapause, partially bivoltine in Finland,
3-4 generations in England.
Adults, with or without reproductive
diapause.
D. persimilis
Adult diapause or quiescence
D. simulans
D. mojavensis
Adult diapause or quiescence
Probably; All life stages, no evidence of
winter diapause
D. mercatorum
D. kikkawai
D. bipectinata
D. birchii
D. formosana
Z. tuberculates
D. equanoxialis
Probably; Adult no diapause
Adult no diapause
Probably; All life stages
Probably; Adult no diapause
References
Baker (1975), Lumme (1978); Turner (1981);
Ashburner et al. (1983); Hoikkala and
Suvanto (1999)
Watabe and Beppu (1977); Ichijo, Kimura &
Minami 1980 (1980)
Basden (1954); Begon (1975, 1976, 1978);
Lumme (1978); Goto et al. (1999)
Izquierdo (1991); Williams and Sokolowksi
(1993); Mitrovski and Hoffmann (2001);
Boulétreau-Merle, Fouillet & Varaldi (2003)
Ashburner, Carson & Thompson Jr.
(1983)Vol. 3d pp. 171-220
Boulétreau-Merle et al. (2003)
A. Gibbs (pers. comm.)
M.T. Kimura (pers. comm.)
Hirai et al. (2000)
A. Hoffmann (pers. comm.)
M.T. Kimura (pers. comm.)
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Baker, W.K. (1975) Linkage disequilibrium over space and time in natural populations of Drosophila
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Basden, E.B. (1954) Diapause in Drosophila (diptera: Drosophilidae). Proceedings of the Royal Entomological
Society of London. Series A, General Entomology, 29, 114–118.
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How to assess Drosophila cold tolerance: Chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits
Jonas Lembcke Andersen, Tommaso Manenti, Jesper Givskov Sørensen, Heath Andrew MacMillan, Volker Loeschcke and Johannes Overgaard
Functional Ecology
Begon, M. (1975) The relationships of Drosophila obscura fallén and D. subobscura collin to naturallyoccurring fruits. Oecologia, 20, 255–277.
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Hoikkala, A. & Suvanto, L. (1999) Male Courtship Song Frequency as an Indicator of Male Mating Success in
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How to assess Drosophila cold tolerance: Chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits
Jonas Lembcke Andersen, Tommaso Manenti, Jesper Givskov Sørensen, Heath Andrew MacMillan, Volker Loeschcke and Johannes Overgaard
Functional Ecology
Supplementary figure
Figure S1 – Relationships of phylogenetically independent contrasts
cAMiT = coldest annual minimum temperature; CTmin = critical thermal minimum (A); CCRT = chill coma recovery
time (B); LTe50 = 50% lethal temperature (C-D); LTi50 = 50% lethal time at low temperature (in both cases, 2h and
24h = survival after 2 h and 24 h of recovery, respectively; E-F); and SCP = supercooling point (G).
The phylogenetically independent tests of relationships between cAMiT and each measure of cold
tolerance, the P- and R²-values of the correlations are shown in black (Garland et al., 1992). The strength
of the phylogenetic signal – a measure of the tendency for related species to have similar trait values –
is shown in grey P- and K-values (Blomberg et al., 2003).
9
How to assess Drosophila cold tolerance: Chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits
Jonas Lembcke Andersen, Tommaso Manenti, Jesper Givskov Sørensen, Heath Andrew MacMillan, Volker Loeschcke and Johannes Overgaard
Functional Ecology
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Phylogenetically Independent Contrasts. Systematic Biology, 41, 18–32.
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Temperatures. Physiological Zoology, 70, 403–414.
Hoffmann, A.A., Anderson, A. & Hallas, R. (2002) Opposing clines for high and low temperature resistance in
Drosophila melanogaster. Ecology Letters, 5, 614–618.
Kellermann, V., Loeschcke, V., Hoffmann, A.A., Kristensen, T.N., Fløjgaard, C., David, J.R., Svenning, J.-C. &
Overgaard, J. (2012) Phylogenetic Constraints in Key Functional Traits Behind Species’ Climate
Niches: Patterns of Desiccation and Cold Resistance Across 95 Drosophila Species. Evolution, 66,
3377–3389.
Kimura, M.T. (2004) Cold and heat tolerance of drosophilid flies with reference to their latitudinal
distributions. Oecologia, 140, 442–449.
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thermal resistance in Drosophila melanogaster using ecologically relevant assays. Journal of
Thermal Biology, 36, 409–416.
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How to assess Drosophila cold tolerance: Chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits
Jonas Lembcke Andersen, Tommaso Manenti, Jesper Givskov Sørensen, Heath Andrew MacMillan, Volker Loeschcke and Johannes Overgaard