Supporting information
The supporting information includes detailed information on the methods used to estimate the
empirical measures of heat and cold tolerance in the 10 species of Drosophila and this section also
discuss the importance of the criteria used in the distribution models.

Detailed information on the location of the climate sites used for modeling the potential
impact of temperature on distribution and exposure to heat stress are presented in Fig. S1
and Table S1.

Species specific data of thermal tolerance, fitness and safety limits are presented in Figs. S2S6. Values for the estimated number of hours above the heat tolerance limit under current
and future conditions are also provided (Table S2).

Supporting information also includes tabled values of observed and predicted consequences
for distributions under current and future climate conditions using our predictive model
(Tables S3-S5).

Fig. S7 gives a graphical presentation of the importance of inclusion of plasticity of thermal
tolerance in the model

Fig. S8 graphically presents the outcome of a sensitivity analysis of some of the main
criteria used in models presented.

Information of the output of Two Way ANOVA analysis’ is found in Appendix S1.
Species used
The 5 tropically restricted species are Drosophila bipectinata, Drosophila birchii, Drosophila
bunnanda, Drosophila pseudoananassae and Drosophila sulfurigaster. The 5 widespread/temperate
species are Drosophila busckii, Drosophila hydei, Drosophila melanogaster, Drosophila repleta
and Drosophila simulans. Information on collection sites for these species and preferred habitat is
found in (Overgaard et al., 2011a). Species used to assess CTmin and CTmax were maintained on
cornmeal-yeast-dextrose medium at 12/12 light dark cycle for >10 generations before onset of
experiments. The relative humidity in the room was ~ 50% (but likely to be much higher in the vials
and bottles were the flies were maintained (not assessed)). Prior to experiments eggs were collected
and placed at controlled density (50 eggs pr 10 ml vial) and all 10 species were reared from egg to
1
adult under two acclimation regimes designed to reflect temperate and tropical thermal conditions,
respectively. These consisted of daily cycles with 6 hours spent at the minimum and maximum
temperatures separated by a gradual increase/decrease in temperature over the intervening 6-hour
period. The temperate conditions fluctuated between 13 and 25 ºC and tropical conditions
fluctuated between 25 and 29 ºC. Eggs from the species were set up at different times to
synchronize adult emergence within each acclimation treatment so that adults eclosed at the same
time. After eclosion, flies were aged at their respective acclimation regimes (∼50 adults of mixed
sex per vial with 10 mL of food). Two days before the experiments, the flies were briefly (<3 min)
sexed under CO2 anesthesia so that CTmin and CTmax of 3–8-dayold mated females could be tested.
These short exposures to CO2 are unlikely to affect performance (Nilson et al., 2006).
For all analysis in the main paper using CTmin and CTmax we employ the CTmin and CTmax obtained
from species acclimated to tropical conditions for the 5 tropical species while the thermal limits
from the widespread species are from animal’s acclimated to the more temperate conditions. The
use of different thermal limits obviously has an impact on the models output and thereby its ability
to predict the cold limits of distribution (see discussion below).
The same experimental populations of all 10 species were later transported to a different lab where
experiments investigating traits related to population growth were examined. Here the flies were
maintained on an oat- yeast-sucrose medium at 12:12h light:dark and ~ 50% RH in a temperature
room maintained at 20ºC. Before experiments eggs from each species were collected into vials with
7 mL of food (50 eggs per vial) and reared to adulthood. These emerging flies were subsequently
used to investigate reproductive activity, egg to adult viability and developmental speed.
For experiments of reproductive activity, egg to adult viability and developmental speed we
maintained parental flies at constant 20 ºC (∼50 adults of mixed sex per vial with 7 mL of food).
After 2-3 days the flies were sexed under brief CO2 anaesthesia and subsequently we daily
monitored female flies to see when egg laying activity was present. Two days after egg laying had
been observed we placed adults at different temperatures to monitor egg laying activity. At the
same time eggs were collected (20 eggs per vial) and these were also acutely placed at different
temperatures to evaluate egg to adult viability and developmental speed (see details below)
2
Knock down temperature under acute and gradual exposure to temperature stress
The data for knock down temperature are acquired from (Overgaard et al., 2011a) where both CTmin
and CTmax were quantified under acute and gradual exposure to thermal stress. As described
previously (Overgaard et al., 2011a), the thermal limit of heat and particularly cold knock down are
plastic in all the 10 species used here. Thus the temperature of acute cold knock down is decreased
considerably if temperature is lowered gradually (through rapid cold hardening) and is also lowered
by developmental acclimation at a lower temperature (CTmin is lower in the temperate acclimated
flies). In contrast there is little plasticity in CTmax (Overgaard et al., 2011a). For simplicity we only
use the thermal limits obtained using gradual stress exposure. Gradual environmental temperature
reflects a more realistic scenario than abrupt exposure (Terblanche et al., 2007; Overgaard &
Sørensen, 2008; Overgaard et al., 2011b) that allow time for physiological adjustments as well as
potential accumulation of thermal stress (Terblanche et al., 2007; Overgaard et al., 2012; Sørensen
et al., 2013).
CTmax was scored by placing individual flies in empty 5 ml glass vials subsequently
submerged in a water bath pre-set at 23 ºC. The temperature of the water bath was then gradually
increased from 23 to 43 ºC at a rate of 0.1 ºC/min and the critical thermal maximum (CTmax) was
recorded as the temperature at which the flies were knocked down by heat and became unable to
move any body part. A similar approach was used to assess CTmin. Here we recorded CTmin while
gradually cooling the flies from 23 to 0⁰C at a rate of 0.1⁰C/min. For all ramping knock down tests
we aimed at testing 20 flies from each species and each acclimation group.
Egg to adult viability and developmental speed
Egg to adult viability was measured in all 10 species under exposure to constant temperatures at 11,
15, 19, 23, 27, 31 and 33 ºC, respectively. For each species we collected 5 replicate vials with 20
eggs that were left to develop under the 7 different constant temperature regimes. Egg to adult
viability (developmental success) was assessed by counting the number of emerging adults and
calculating the % of developmental success relative to the 20 eggs provided originally. The
emergence of adults was registered daily and developmental speed for each vial was recorded as
1/(average developmental duration of flies from vial) for each species.
Fecundity
3
The capacity for egg production was measured in all 10 species under exposure to constant
temperatures at 11, 15, 19, 23, 27, 31 and 33 ºC, respectively. Each species was raised under low
density (50 eggs per 7 ml vial) at 20 ºC and male and female flies were separated 2-3 days after
eclosion. The flies remained at 20 ºC until 2 days after egg-laying activity was first observed (4-8
days after eclosion depending on species). 35 pairs (1 male and 1 female) were then placed in vials
with plastic spoons containing yeast covered media and randomly assigned to the 7 experimental
temperatures (5 pairs per species per temperature). Capacity for egg production was assessed over
three successive days by counting the number of eggs produced per pair daily.
Population growth/fitness estimate
The measures of fecundity, developmental time and egg to adult viability were combined to
calculate an estimate of the potential for population growth within each species. This measure of
composite fitness was performed by summing the relative fractional score of each of these three
variables. Thus the species specific “composite fitness” at 11 oC was calculated as:
(fecundity at 11 oC/max fecundity)* (egg to adult viability at 11 oC/max egg to adult viability)*
(developmental speed at 11 oC/max developmental speed).
Justification/exploration of cold tolerance assumptions
An explicit aim of the present study was to investigate the predictive power of simple distribution
models using easily obtained empirical data from simple measures of thermal capacity and thermal
tolerance of insects. Such a simple approach may include some weaknesses related to the simplicity
of the measures of thermal limits and how they are used in the models; however these weaknesses
may be outweighed by the ease of use and applicability. In the main paper we presented the output
of our model using a specific set of adult thermal limits and threshold limits for population growth
(presented in the main paper). Thus one model is considering tolerance of the adult stage while the
other is considering the ability of larval and pupal stages to complete development. This may be a
simplified view as thermal tolerance of larval and pupal stages are also important components of
thermal adaptation (Coyne et al., 1983; Feder et al., 1996; Gibbs et al., 2003; Hoffmann et al.,
2003; Strachan et al., 2011). However, the adult stage also endures extreme temperature events and
it is in this stage that the flies survive winter and sensitivity of adults to thermal extremes could
4
therefore potentially constrain distribution limits. (Kimura, 1988; Izquierdo, 1991; Hoffmann et al.,
2003; Hoffmann, 2010)
The models including CTmin and CTmax do incorporate different plasticity of widespread and
tropical species. Below we demonstrate and discuss how the model will be affected by altering this
set of assumptions.
Specifically we address
1. What cold limit would the model predict if we assumed all flies to experience the same
developmental acclimation (all 10 species receiving either temperate or tropical acclimation
during development) (See Fig. S7)
2. What cold limit would the model predict if the arbitrary threshold of 480 h/year below CTmin
is changed to be more or less conservative threshold? (See Fig. S8a)
3. What cold limit would the model predict if we altered the arbitrary fitness limit of 20% of
optimal for sustainable populations? (See Fig. S8b)
Most insects display a considerable phenotypic plasticity with regard to thermal limits (Hoffmann
et al., 2003; Chown & Nicolson, 2004; Chown & Terblanche, 2006), and particularly cold limits are
often altered considerably by environmental cues of different lengths (e.g.(Overgaard et al., 2011b;
Colinet & Hoffmann, 2012)). Temperate/widespread species are often expected to have greater
acclimation potential than their tropical counterparts (Janzen, 1967; Levins, 1968; Ghalambor et al.,
2006; Angilletta, 2009) but empirical measurements do not always support this pattern (Brattstrom,
1968, 1970). For the 10 species used here we have previously shown that widespread and tropical
species have similar acclimation potential (Overgaard et al., 2011b). Widespread and temperate
species do, nevertheless, experience different environmental temperatures and it is therefore
important to incorporate the potential effect of acclimation. In our analysis we did this by assuming
that widespread species have been exposed to developmental acclimation at temperate conditions
(average of 19ºC) while data from tropical are from flies reared at warm condition (average of
27ºC).
As illustrated in Fig S7, the widespread species do have higher cold tolerance (lower CTmin)
regardless of acclimation treatment, but the difference in cold tolerance between tropical and
5
widespread species becomes larger if widespread species are considered to have developed at lower
temperature than their tropical counterparts. Inclusion of acclimation therefore increases the
predictive power of the estimated cold limit and it is likely that further acclimation could have
improved the fit of the model even more.
For the main paper we chose an arbitrary cold tolerance limit that allowed for time (480 hours/year)
below the critically lower limit. Although the estimate of chill coma temperature used in the present
study includes appropriate cold acclimation of both temperate and tropical species, the minimum
temperature of the environment is lower than the CTmin as measured in chill coma assays (Fig. 1a).
Drosophilids, like many other chill sensitive insects, can however, endure some time below the
functional cold limit without incurring long term injury (Sinclair & Roberts, 2005; Chown &
Terblanche, 2006; Hoffmann, 2010; Macmillan & Sinclair, 2011).
The present study uses developmental acclimation at an average of 27 and 18 ºC, for tropical and
temperate species respectively. These acclimation regimes are higher than the average tropical
temperatures and particularly larger than the temperatures under winter conditions in the temperate
areas. It is therefore likely that we have underestimated the full acclimation potential from
developmental acclimation (Gibert & Huey, 2001; Hoffmann, 2010). Moreover we did not
introduce long term adult cold acclimation although this has been shown to be additive to that of
developmental acclimation and rapid cold hardening in D. melanogaster (Colinet & Hoffmann,
2012). The present study also neglect the putative positive influence of reproductive diapauses
which is found in some Drosophila species (Czajka & Lee, 1990; Schmidt & Paaby, 2008).
Repeated cold or heat exposures may in some cases be more detrimental than beneficial as seen for
example for freeze tolerant insects that have lower survival (Marshall & Sinclair, 2011) or in
drosophila where repeated cold exposure can disrupt reproductive capacity (Marshall & Sinclair,
2010) and repeated heat exposure can cause damage to neural function (Wang et al., 2007).
Nevertheless several studies have shown that periodic periods of heating above the CTmin may act
against cumulative chill damage in drosophila and other insects (Petavy et al., 2001; Colinet et al.,
2006; Marshall & Sinclair, 2010), and in our analysis the time spend below the lower thresholds is
periodic (during multiple cold nights) rather than the consequence of a continued cold exposure.
Thus, in terms of survival these different factors will all tend to underestimate the realized cold
tolerance in the field. This will particularly be the case in southern regions of the species
6
distribution and we therefore employed a cold limit allowing for considerable time below our
assessment of CTmin. The 480 hour period was arbitrary but meant to reflect the fact that in
temperate Australia winter extends across 3-4 months with temperatures normally hovering around
minimum temperatures for several hours every night. For the same reasons it is likely that cold
tolerance of widespread species is underestimated when compared to those of tropical species
because the environmental cues initiating both acclimation and diapause will be more prominent for
the southern (cold exposed) populations of the widespread species. As illustrated in Fig. S8a we get
the same qualitative relation between the modeled and observed latitude even if the 480 limit is
decreased or increased by 50% (240 or 720 h). The model therefore predicts the overall pattern of
distribution regardless of the limit used, but does not do this perfectly (as would be indicated by a
slope of 1) which may in part be due to the incomplete induction of cold tolerance of particularly
southern species.
To evaluate the predictive power of our composite fitness estimate we employ an arbitrary
minimum “fitness” value of 20%. The composite fitness at a given geographical location is
calculated using hourly temperature estimates and then calculating the average of the corresponding
fitness from the temperature/fitness relationship. Thus a value of 100% would represent a situation
where the species is constantly at the optimal temperature for population growth. A value of 0 %
would represent a situation where the species never experiences temperatures permissible for
population growth. A value of 50% could in principle represent that a species is constantly
experiencing a fixed temperature giving 50% of optimal conditions or in the other extreme that it
experiences optimal conditions half of the year and unsustainable conditions the other half. The
value of 20% was chosen as it is evident that no species experience optimal conditions all the time
and all species are also known to tolerate exposures to temperatures above or below those
permissible for population growth (i.e. they can experience periods of 0% fitness for some time and
still recover to ensure the population when conditions become more favorable). Species residing in
temperate conditions may for example experience one half of the year where the scope for
population growth is reasonable (above 50%) and one half where there is no or very little scope for
population growth. The value of 20% is arbitrary, but as illustrated in Fig S8b this value has no
influence on the overall conclusion. Thus decreasing the cutoff value to 10% or increasing it to 30%
gives the same pattern where the observed critical latitude does not correlate with the modeled
critical latitude (Fig. S8b).
7
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