Molecular Ecology (2011) 20, 2100–2110
doi: 10.1111/j.1365-294X.2011.05089.x
The molecular genetics of clinal variation: a case study
of ebony and thoracic trident pigmentation in Drosophila
melanogaster from eastern Australia
M A R I N A T E L O N I S - S C O T T , * A R Y A . H O F F M A N N * and C A R L A M . S G R Ò †
*Department of Genetics, and Centre for Environmental Stress & Adaptation Research, The University of Melbourne, Parkville,
Melbourne 3001, Australia, †School of Biological Sciences and Centre for Environmental Stress & Adaptation Research, Monash
University, Clayton, Melbourne 3800, Australia
Abstract
Widespread pigmentation diversity coupled with a well-defined genetic system of
melanin synthesis and patterning in Drosophila provides an excellent opportunity to
study phenotypes undergoing evolutionary change. Pigmentation variation is highly
correlated with different ecological variables and is thought to reflect adaptations to
different environments. Several studies have linked candidate genes from Drosophila
melanogaster to intra-population variation and interspecific morphological divergence,
but less clearly to variation among populations forming pigmentation clines. We
characterized a new thoracic trident pigmentation cline in D. melanogaster populations
from eastern Australia, and applied a candidate gene approach to explain the majority of
the geographically structured phenotypic variation. More melanized populations from
higher latitudes tended to express less ebony than their tropical counterparts, and an
independent artificial selection experiment confirmed this association. By partitioning
temperature dependent effects, we showed that the genetic differences underlying clinal
patterns for trident variation at 25 C do not explain the patterns observed at 16 C.
Changes in thoracic trident pigmentation could be a common evolutionary response to
climatically mediated environmental pressures. On the Australian east coast most of the
changes appear to be associated with regulatory divergence of the ebony gene but this
depends on temperature.
Keywords: clinal variation, Drosophila pigmentation, gene expression
Received 10 January 2011; revision received 15 February 2011; accepted 16 February 2011
Introduction
The striking array of animal body colouration and patterning is one of nature’s most conspicuous variations
on a theme. In insects, pigmentation diversity is a
prominent form of phenotypic variation and is widely
considered to be adaptive (reviewed Mani 1990; True
2003; Papa et al. 2008; Wittkopp & Beldade 2009). A
more melanized cuticle is associated with increased fitness under different biotic and abiotic stressors including predation, ultraviolet radiation, thermal stress,
aridity, and pathogen borne disease (reviewedWilson
Correspondence: Marina Telonis-Scott, Fax: +61 3 8344 5139;
E-mail: marinats@unimelb.edu.au
et al. 2001; True 2003; Rajpurohit et al. 2008a). Melanism is particularly well documented in Drosophila,
where the morphological diversification within and
among species is fast becoming one of the better characterized examples of evolution in action (Wittkopp &
Beldade 2009). Pigmentation varies continuously across
different geographies resulting in widespread, repeated
clines that likely reflect adaptations to spatially varying
selection pressures such as local climate rather than artefacts of demographic history (Hollocher et al. 2000;
Brisson et al. 2005; Pool & Aquadro 2007; Wittkopp
et al. 2010). Temperature and humidity are commonly
evoked selective forces for adaptive melanism; a darker
body surface is thought to more efficiently absorb solar
radiation, improve thermoregulation, and thicken the
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G E N E T I C S O F C L I N A L P I G M E N T A T I O N I N D R O S O P H I L A 2101
cuticle to stem water loss under low humidity
(reviewed True 2003; Clusella Trullas et al. 2007; Rajpurohit et al. 2008a). Darker abdominal pigmentation
correlates strongly with altitude in Drosophila melanogaster from sub-Sahara Africa (Pool & Aquadro 2007), and
forms parallel clines with desiccation resistance at high
latitude and altitude on the Indian subcontinent (Parkash et al. 2008a,c; Rajpurohit et al. 2008b). Abdominal
pigmentation varies by habitat in D. polymorpha, where
darker, more desiccation tolerant flies were associated
with a warm, arid environment (Brisson et al. 2005). In
contrast to D. melanogaster, the darkest abdomens in
species from the dunni subgroup along a latitudinal
cline were recorded closest to the equator, where species differentiation is likely shaped by complex natural
and sexual selection (Hollocher et al. 2000). The cline
for body colour in D. americana deviates from a latitudinal association to a longitudinal one, and is correlated
with relative humidity but not desiccation tolerance
(Wittkopp et al. 2010). The thermal melanism hypothesis may better explain patterns of local adaption in this
species, as darker individuals tend to occur in areas
with lower temperatures and solar radiation (Clusella
Trullas et al. 2007; Clusella-Trullas & Terblanche 2010).
Drosophila pigmentation variation is due to the differential localization and abundance of black, yellow and
brown pigments determined by the expression of core
structural genes including yellow, ebony and tan, first
identified in D. melanogaster (reviewed True 2003; Wittkopp et al. 2003; Wittkopp & Beldade 2009). The yellow
and ebony genes have opposite effects on integument
colour: yellow protein converts dopa to black dopa melanin while the ebony protein converts dopamine to N-balanyl dopamine (NBAD) to form yellowish sclerotin
and can suppress the action of yellow in forming black
pigmentation (Wittkopp et al. 2002a). Brown pigment is
produced by converting NBAD back to dopamine in a
reverse reaction by an NBAD hydrolase encoded by tan
(True et al. 2005). Polymorphisms affecting the function
of these genes are obvious candidates for intra- and
interspecific divergence (Wittkopp et al. 2002b, 2009;
Jeong et al. 2008; Rebeiz et al. 2009). The yellow protein
is spatiotemporally distributed to all cells with black
pigment and is dependent on tan activity to form black
melanin, and both genes contribute to divergent pigmentation patterns among Drosophila species (Wittkopp
et al. 2002a,b, 2009; Jeong et al. 2008). A loss of function
of ebony is also highly associated with melanism within
and between species including abdominal pigmentation
in D. melanogaster and body colour differences between
sister species D. novomexicana and D. americana (Pool &
Aquadro 2007; Rebeiz et al. 2009; Wittkopp et al. 2009).
In addition to the genetic differences underlying pigmentation variation, Drosophila populations exhibit a
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range of reaction norms for pigmentation in response to
developmental temperature (David et al. 1985, 1990;
Gibert et al. 2000). Repression of pigmentation enzymes
such as ebony underlie phenotypic plasticity for abdominal pigmentation in D. melanogaster, but at low temperatures these regulatory changes are modulated by
different mechanisms (Gibert et al. 2007; Rebeiz et al.
2009).
Low ebony expression also correlates with another
pigmentation polymorphism, the intensity of a ‘trident’
shaped melanic pattern on the thorax. Wittkopp et al.
(2002a) showed that ebony mutants exhibit an extreme
black trident pattern (with some yellow protein present),
while double ebony ⁄ yellow mutants express a trident
comprised of brown pigments. In D. melanogaster, while
ebony mRNA is present throughout development and in
young adults (Hovemann et al. 1998), expression levels
in non-pigmented thoraces are highest in the cells that
form the trident in ebony mutants and transformants
(Wittkopp et al. 2002a; Gibert et al. 2007). Takahashi
et al. (2007) mapped trident intensity differences to the
ebony locus in two field-derived inbred lines, where
reduced expression was responsible for the trident pattern in a Taiwanese line compared to a non-melanized
African line. Studies of natural populations suggest
that, like abdominal melanism, thoracic trident pigmentation is a highly plastic and likely adaptive trait. In D.
melanogaster, David et al. (1985) observed a steep transcontinental cline of increasing trident pigmentation
between 30 and 50 latitude, coinciding with cooler
temperate climates. Similarly to abdominal pigmentation, multiple ecological variables contribute to trident
variation in India (Munjal et al. 1997; Parkash & Munjal
1999), where over 90% of D. melanogaster thoracic trident variation could be predicted by both latitude and
altitude (Munjal et al. 1997). Cross-species clines further
support the adaptive significance of thoracic trident variation; D. simulans exhibits a cryptic latitudinal cline
despite reduced geographic variation compared to its
sibling species D. melanogaster (Capy et al. 1988). While
ebony is a compelling candidate for the genetic basis of
trident variation, this is largely limited to data from
mutant or isogenic lines with fixed phenotypes and
reduced phenotypic plasticity (Takahashi et al. 2007).
The contribution of expression variation at this locus to
clinally varying patterns of trident pigmentation
remains unclear.
To date, studies of the genetics of pigmentation diversity have mostly focused on within population or
between species comparisons, while the genetic factors
underlying clinal, intra-specific variation for the different
pigmentation traits are largely unexplored. The present
study addresses this disparity by examining the relationship between phenotypic variation for thoracic trident
2102 M . T E L O N I S - S C O T T , A . A . H O F F M A N N and C . M . S G R Ò
variation and expression of the candidate gene ebony
along a geographical gradient. Thoracic trident intensity
is a useful pigmentation metric in D. melanogaster that is
associated with similar geographic variables as abdominal pigmentation (David et al. 1985; Munjal et al. 1997).
Here, we link clinally varying trident variation in nature
with ebony expression variation in D. melanogaster collected from 18 sites along the east Australian coastline.
We show that increasing pigmentation is positively correlated with latitude and negatively correlated with ebony
expression. The ebony expression cline explained two
thirds of the pigmentation variation at 25 C and latitude
explained about 40% of the ebony expression variation
where the genetic differences for pigmentation intensity
among populations were clear. However these clines disappeared at 16 C where a plastic response to the low
rearing temperature resulted in a uniformly darker trident. Our results suggest that different mechanisms may
underlie these temperature-dependent plastic and
genetic phenotypes.
Materials and methods
Drosophila melanogaster populations
Australian east coast populations. Populations of D. melanogaster were collected from the Australian east coast in
April–June 2008 from 18 locations ranging from Innisfail
QLD (17.52S) to Sorrell TAS (43.15S) (Sgrò et al. 2010;
Table S1, Supporting Information). Each experimental
population was initiated from 30 isofemale lines following two generations of laboratory culture. The populations were maintained at 25 C under constant light at a
population size of at least 1000 flies prior to the assays.
Coffs Harbour mass breds and pigmentation selected
lines. For the ebony association study, an independent
population from the midpoint of the east coast transect
(Coffs Harbour, NSW, 30.37S) was collected in January
2010. Briefly, 60 females were brought into the laboratory and a mass bred population was established by
pooling 10 male and 10 virgin female F1 progeny from
each isofemale line derived from the field females (1200
flies). The mass bred population was allowed to freely
recombine at a population size of at least 1000 flies until
the experiment was initiated at F7 generation of laboratory culture. For the experiment, flies were reared at
25 C as described above. Light and dark trident lines
were established by selecting the 20 lightest and 20
darkest flies from 400 F7 Coffs Harbour mass bred
females. This was repeated three times, resulting in
three ‘light’ lines and three ‘dark’ lines. In addition,
two lines representing the ends of the Australian east
coast clinal distribution with darkest phenotype (Sth
Tasmania) and lightest phenotype (Innisfail, QLD) were
also selected at a lower intensity, where the 20 lightest
and 20 darkest flies were selected from 100 females.
One set of light and dark lines were established for
each of these cline end populations, bringing the total
number of lines selected to 10.
Thoracic trident pigmentation scoring
Australian east coast populations. David et al. (1985)
showed that two growth temperatures (17 C and 25 C)
were sufficient to characterize trident pigmentation variation in natural populations. Based on available controlled rearing environments, we chose 16 C and 25 C,
given that the response curves remain similar between
16 C and 17 C (David et al. 1985). Flies for the assays
were reared in temperature controlled rooms at 24 h constant light on standard dextrose medium in 500 mL glass
bottles. Density was standardized for two generations
prior to the assays by placing 25 mating pairs on food
and leaving them to oviposit for 2 and 6 days at 25 C
and 16 C respectively. Three bottles were set up for each
population to obtain three replicate measurements per
sex ⁄ temperature ⁄ population. Flies for phenotyping were
collected within 48 h of eclosion, and then separated by
sex into groups of 50 using aspiration without CO2. To
ease scoring, flies were matured for 20 days after transferring to fresh food every few days.
Thoracic trident pigmentation was scored by visual
examination using the four phenotypic classes (David
et al. 1985). Phenotypes were partitioned into the following categories: class 0 = no visible trident, class
1 = faint trident, class 2 = trident clearly marked and
class 3 = dark trident. Flies were lightly anesthetized
with CO2, examined under a dissecting microscope and
assigned a score based on the above classes. Pigmentation was quantified as the average score of 35 flies from
each population ⁄ sex ⁄ temperature combination, and
population means were taken from the grand average
of three scores (105 flies). In almost every case, 35 flies
from each replicate were examined over 3 days by a
single observer (35 · 3 replicates · 18 populations · 2
sexes · 2 temperatures = 7550 flies in total).
Coffs Harbour selected lines. F1 female progeny of light
and dark selected females were scored 20 days after
eclosion as described above, sample sizes ranging from
15 to 100 flies, (except for CH2 (dark), n = 2) for a total
of 446 flies.
Ebony gene expression assays
Fly rearing. Flies for the gene expression assays were
reared at 16 C and 25 C as described above. Density
2011 Blackwell Publishing Ltd
was controlled by allowing 25 mating pairs to oviposit
in food bottles for 2 h at 25 C and overnight at 16 C.
Imagos were collected within 1–)3 h post-eclosion to
capture ebony expression (Wittkopp et al. 2002a; Takahashi et al. 2007). Three replicates of 10 females (Australian east coast populations) or six females (selected
lines) per line were snap-frozen in liquid N2 and stored
at )70 C.
Thorax dissections and RNA extractions. To maintain the
narrow window of eclosion to detect ebony expression
among a large number of samples, thorax dissections
were performed on frozen flies. Thoraces (legs and
wings removed) were dissected from each line on ice
under a dissecting microscope and homogenized in
500 lL TRIzol Invitrogen reagent. RNA was
extracted following the manufacturer’s instructions,
purified using an RNeasy mini kit (QIAGEN) and
treated with TURBO DNA-free (Ambion) to remove
residual genomic DNA. The RNA was quantified
using the Take3 spectrophotometer (BioTek) and
integrity assessed with agarose gel electrophoresis.
Three separate RNA extractions were performed for
each line.
(three groups of 35 flies scored per combination) were
examined using the model
Yijkn ¼ l þ pi þ si þ tk þ psij þ ptjk þ stjk þ eijkn
where Yijkn is the mean pigmentation score for population p, sex s, temperature t, and replicate n; l is the
overall mean pigmentation score for population p; s is
the sex effect; t is the temperature effect; ps is for population-by-sex interaction; pt is for population-by-temperature interaction; st is for sex-by-temperature
interaction; and e is the error. All effects were considered fixed. A three-way interaction term of populationby-sex-by-temperature was initially fit and was not significant, and exclusion of this effect did not alter any
inferences. Latitudinal pigmentation patterns were
assessed via linear regressions with the overall average
pigmentation scores of each population as the data
points, and data were log transformed to improve the
fit to a normal distribution.
Ebony gene expression. The effects of population and
temperature on ebony expression were examined using
the model
Yijn ¼ l þ pi þ tj þ pi tj þ eijn
cDNA synthesis and real-time PCR. cDNA was synthesized from 500 ng total RNA using the following protocol in a 20 lL reaction volume: 2 lL 50 lM oligo-dT
primer and 4 lL 2.5 mM dNTPs were heated to 80 C
for 5 min then cooled on ice. 2 lL 10 · M-MuLV buffer
and 1 lL M-Mulv Reverse Transcriptase 200 U ⁄ lL was
added and the samples were incubated at 42 C for
60 min, then the enzyme was deactivated at 90 C for
10 min. The cDNA was diluted 1:10 in water. Real-time
PCR was performed on the LightCycler 480 (Roche)
using SYBR Green in a 2X universal buffer containing
the following: 50 mM MgCl2, 10 · Immolase Buffer,
2 · Roche High Resolution Melt, 25 mM dNTPs, Immolase Taq 5 U ⁄ lL. A typical 10 lL PCR reaction contained 5 lL universal buffer, 4 lL 1 lM primer mix and
1 lL cDNA. The ebony transcripts were amplified with
the following primers, forward: CTGGAATGTGCTGGTGGAG; reverse: AGACCCTTGGGCAGGTAGTT.
For each cDNA, three technical replicates were performed and ebony expression levels were normalized to
RpL11. As an initial PCR control, the 25 C east coast
populations were repeated and normalized to Rps20 to
ensure concordant results with RpL11.
Statistical analyses
Australian east coast population comparisons. For the east
coast population comparisons, the effects of population,
sex, and temperature on thoracic trident pigmentation
where Yijn is the mean normalized ebony expression relative to RpL11 for population p; temperature t and replicate n; l is the overall mean ebony expression; p is the
population effect; pt is the population-by-temperature
interaction; and e is the error. Patterns of pigmentation
and ebony expression were related to latitude via linear
regressions with the means of each population as the
data points for the east coast populations and the artificially selected populations. Pigmentation data were log
transformed to improve normality. All analyses were
performed using SAS software version 9.2 (SAS Institute,
Cary, NC, USA).
Results
A latitudinal cline and phenotypic plasticity for
thoracic trident pigmentation in eastern Australia
We assessed thoracic trident pigmentation in male and
female D. melanogaster collected from 18 locations along
the Australian eastern seaboard. Pigmentation was measured as an average pigmentation score from 35 individuals per population ⁄ sex ⁄ temperature combination,
with all measurements repeated in triplicate. The means
and standard deviations of the grand average pigmentation score of 105 individuals for the 18 populations,
sexes, and two developmental temperatures (16 C and
25 The data were log transformed to linearize the effects of
population clustering and two outlying populations at
18.2S and 19.97S, although similar results were
obtained regardless of whether transformed or untransformed data were considered. A three-way ANOVA
showed significant differences in thoracic pigmentation
between populations, sexes and growth temperatures
(Table 1). Pigmentation was consistently darker across
the populations when reared at 16 C (Table S1, Supporting Information; Fig. 1), although there was a significant population by temperature interaction
(Table 1). While the sex term was significant, the two
sexes were highly correlated at both temperatures
(Pearson correlation r = 0.95, P < 0.0001 at both 25 C
and 16 C, Fig. 1), and neither the sex-by-temperature
nor sex-by-population interactions were significant
(Table 1). Linear regressions on the grand average pigmentation score of each population showed significant
latitudinal clines in both sexes and temperatures where
flies were darker with increasing latitude, although this
Table 1 Analysis of variance (ANOVA) on log transformed pigmentation scores in female and male D. melanogaster from the
Australian east coast
F
d.f. Mean squares
Population
17
3.54
Sex
1
0.98
Temperature
1 151.58
Population · Sex
17
0.18
Population · Temperature 17
2.25
Sex · Temperature
1
0.25
Error
156
0.13
Temperature dependent clinal expression of ebony. Next, we
explored the contribution of expression variation of
the candidate gene ebony to the clinal variation for trident pigmentation in the 18 east coast populations
using real-time PCR. Given the strong correlation
Table 2 Linear regressions testing for associations between
pigmentation and latitude in females and males at 25 C and
16 C
Trait
Female
pigmentation
Male
pigmentation
Female
pigmentation
Male
pigmentation
P value
25.52 <0.0001
7.12 0.0084
1091.13 <0.0001
1.31 0.1897
16.21 <0.0001
1.83 0.1783
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
2.5
Male mean pigmentation
R 2 = 0.904
2
1.5
R 2 = 0.907
1
0.5
0
0
0.5
1
1.5
2
2.5
Female mean pigmentation
Fig. 1 Thoracic trident pigmentation is highly correlated
between the sexes at both 16C (closed boxes) and 25C (open
boxes). Mean pigmentation is given as the grand average score
of 105 flies per population. The R2 shown is the fit of the linear
regression line, actual Pearson’s correlation r = 0.95.
R2
b (SE)
t
P value
0.52
0.10208 (0.02453)
4.16
0.0007
0.42
0.09768 (0.02863)
3.41
0.0036
0.25
0.01227 (0.00530)
2.32
0.034
0.24
0.01353 (0.00601)
2.25
0.038
(25 C)
(25 C)
(16 C)
(16 C)
Pigmentation scores are log transformed.
Mean log pigmentation
Source
association was much weaker at 16 C (Table 2; Fig. 2a)
compared to 25 C (Table 2; Fig. 2b).
G E N E T I C S O F C L I N A L P I G M E N T A T I O N I N D R O S O P H I L A 2105
Table 3 Analysis of variance (ANOVA) for ebony expression at
two temperatures in female D. melanogaster from the Australian
east coast
Source
d.f. Mean squares
Population
17
Temperature
1
Population · Temperature 1
Error
71
0.03
0.90
0.04
0.02
F
P value
1.57 0.0959
43.08 <0.0001
1.91 0.0269
between the sexes for pigmentation, only females were
used to examine gene expression patterns of the different populations. Two-way ANOVA showed no significant
differences between populations in ebony expression,
but a significant effect of temperature and a significant
population-by-temperature interaction was evident
(Table 3).
At 16 C, linear regression on the population means
showed no association of ebony expression and latitude
(Table 4; Fig. 3a) or pigmentation (Table 4; Fig. 4a).
In contrast, regression analysis revealed a significant
negative association between ebony expression and latitude at 25 C (Table 4; Fig. 3b), where ebony expression decreased with increasing latitude. As expected
from this clinal pattern, the phenotypic variation in
pigmentation at 25 C was significantly negatively
associated with variation in ebony expression, with linear regression explaining over 70% of the variation in
pigmentation where lower levels of ebony correspond
with a darker phenotype (Table 4; Fig. 4b). While the
results were consistent between log transformed and
untransformed pigmentation scores, the log transformation improved the fit of the regression line by
almost 30%.
Coffs Harbour association study. We independently corroborated the negative relationship between trident pigmentation and ebony expression using artificial selection
for divergent pigmentation phenotypes. The selection
lines were founded from three populations representing
the cline midpoint (Coffs Harbour, NSW) and endpoints (Innisfail, QLD and Sth Tasmania). Selection for
light and dark pigmentation for one generation resulted
in three ‘dark’ and three ‘light’ lines from a newly collected population from Coffs Harbour (collected in
2010), and one dark and light line each from Innisfail
and Sth Tasmania (collected in 2008) for a total of five
dark and five light lines. The more recently field
derived Coffs Harbour population had levels of pigmentation that were comparable to the earlier collection
(mean pigmentation score ± SE 2008 = 0.73 ± 0.02;
2010 = 0.78 ± 0.002). One generation of selecting the 5%
phenotypic extremes was sufficient to significantly alter
trident pigmentation in this population (Fig. 5a, paired
t-test light vs. dark; t = 9.37; d.f. = 4; P < 0.001). Innisfail flies were mostly non-melanic but did harbour
genetic variation for darker pigmentation at 25 C, evident in the marked increase in trident intensity in the
selected F1s, while the opposite was observed in the
darkest Tasmanian population with no change in either
direction (Fig. 5a).
Similar to the expression data for the clinal populations at 25 C described above, regression analyses
showed a significant, negative association between pigmentation and ebony expression relative to RpL11, with
over 65% of the variation in pigmentation explained by
a linear regression (Fig. 5b); once again, increasing pigmentation levels were associated with a strong reduction in ebony expression levels. The results were
consistent between log transformed and raw pigmentation data, although the transformation improved the fit
of the regression line by 20%. The darker lines always
expressed less ebony than the light lines, albeit in the
absence of a phenotypic shift in the dark Tasmanian
line, while the largest change in ebony expression was
associated with the largest phenotypic shift in the Innisfail lines. One caveat is that the cline end selected lines
were included to provide contrast and were not replicated in contrast to the Coffs Harbour lines.
Discussion
Pigmentation diversity in Drosophila is widespread and
closely associated with ecological and climatic factors,
providing an ideal framework to study phenotypic
Table 4 Linear regressions testing for associations between ebony expression and pigmentation or latitude at 16 C and 25 C in
females from eastern Australia and from the artificial selection study at 25 C
Trait
16
25
16
25
25
C
C
C
C
C
pigmentation – east coast females
pigmentation – east coast females
latitude – east coast females
latitude – east coast females
pigmentation – selected females
Pigmentation scores are log transformed.
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R2
b (SE)
0.05
0.74
0.06
0.38
0.66
0.44660
)8.14868
0.00344
)0.00917
)17.46151
(0.45217)
(1.19786)
(0.00317)
(0.00296)
(4.43702)
t
P value
0.99
)6.80
1.08
)3.10
)3.94
0.3380
<0.0001
0.2945
0.0069
0.0043
ebony expression relative to RpL11
2106 M . T E L O N I S - S C O T T , A . A . H O F F M A N N and C . M . S G R Ò
0.7
Fig. 3 Temperature-dependent association of ebony expression and latitude in
females at (a) 16C where there no association and (b) at 25C where there is a
strong negative association. Average
pigmentation scores are log transformed
and linear regressions and R2 values are
shown.
(a)
0.6
0.5
0.4
0.3
0.2
0.1
0
15
20
25
30
35
40
45
50
45
50
ebony expression relative to RpL11
Latitude (oSouth)
0.7
(b)
0.6
0.5
0.4
0.3
R2 = 0.375
0.2
0.1
0
15
20
25
30
35
40
Latitude (oSouth)
Mean log pigmentation
0.9
(a)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.6
0.7
Fig. 4 Temperature-dependent association of ebony expression and pigmentation in females at (a) 16C where there
no association and (b) at 25C where
there is a strong negative association.
Average pigmentation scores are log
transformed and ebony expression is
shown relative to the housekeeping control RpL11 in newly eclosed females.
Linear regressions and R2 values are
shown.
Mean log pigmentation
ebony expression relative to RpL11
1
(b)
0.5
0
–0.5 0
–1
–1.5
–2
–2.5
–3
–3.5
–4
–4.5
0.1
0.2
0.3
0.4
0.5
R2 = 0.7431
ebony expression relative to RpL11
variation that is shaped by natural selection. Thoracic
trident pigmentation has been quantified in D. melanogaster across the world, but Australian sampling has previously been limited to two temperate populations (David
et al. 1985). We investigated this trait in multiple
populations sampled along the Australian east coast,
where, despite evidence of recent D. melangoaster colonization, clines for numerous traits spanning a wide
range of habitats have been reported, reflecting different
aspects of climatic adaptation (Hoffmann & Weeks
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G E N E T I C S O F C L I N A L P I G M E N T A T I O N I N D R O S O P H I L A 2107
3
(a)
Mean pigmentation
2.5
2
1.5
1
0.5
0
Innisfail
CH1
CH2
CH3
Sth TAS
Mean log pigmentation
1.5 (b)
1
0.5
0
–0.5
–1
R 2 = 0.659
–1.5
–2
–2.5
0
0.05
0.1
0.15
0.2
0.25
ebony expression relative to RpL11
Fig. 5 Pigmentation and ebony expression respond to selection
in females. (a) Mean pigmentation in five lines selected for
light pigmentation (open bars) and five lines selected for dark
pigmentation (closed bars). CH stands for Coffs Harbour and
error bars are + SE. (b) Negative association of ebony expression and pigmentation following selection for pigmentation
intensity (open markers = light selected lines, closed markers = dark selected lines, square markers = Innisfail, triangle
markers = Sth Tasmania and diamond markers = Coffs Harbour lines). Average pigmentation scores are log transformed
and ebony expression is shown relative to the housekeeping
control RpL11 in newly eclosed females. Linear regressions and
R2 values are shown.
2007). This includes morphological traits such as body
size and related metrics such as thorax length (e.g.
James et al. 1995; Azevedo et al. 1998; Arthur et al.
2008), life history traits related to fitness such as ovariole number and timing of egg production (Azevedo
et al. 1996; Mitrovski & Hoffmann 2001) and stress
adaptations such as thermotolerance (Hoffmann et al.
2002; Sgrò et al. 2010).
We report that thoracic pigmentation also shows significant plastic and genetically-based clinal variation
along the east coast of Australia. Consistent with earlier
work (David et al. 1985; Capy et al. 1988; Munjal et al.
1997; Parkash & Munjal 1999), the darkest flies tend to
2011 Blackwell Publishing Ltd
occur at higher latitudes, however differences in the
strength of the clinal patterns here and between other
studies are also evident. The association between latitude and pigmentation is weaker in the Australian
cline, particularly at 16 C, where latitude explained
just 24% of the pigmentation variation. At 25C,
latitude alone explained approximately 80% of the
variation from a world-wide sample (David et al. 1985),
and both latitude and altitude explained over 90% of
the variation in Indian populations of D. melanogaster
(Munjal et al. 1997; Parkash & Munjal 1999) in contrast
to an average of 47% in the current study.
Local adaptation to the unique climatic conditions
experienced on different continents is an obvious explanation for these differences. For example, relative
humidity correlates more strongly with latitude than
temperature on the Indian subcontinent, while seasonal
variation provides shifting climatic patterns according
to latitude that are different to seasonal patterns on
other continents such as Australia and Africa (Rajpurohit et al. 2008a). The impact of different climatic selection is evident for traits such as desiccation resistance,
which co-varies strongly with latitude and altitude in
Indian Drosophila species but shows no such relationship in Australian populations despite clinal variation
for other stress traits (Hoffmann et al. 2001; Arthur
et al. 2008). The association of pigmentation intensity
with survival to low humidity in Drosophila is largely
supported by data collected from India, where desiccation resistance forms parallel clines with body melanization (Rajpurohit et al. 2008a). This is particularly
evident along altitudinal gradients where conditions are
cooler and drier (Parkash et al. 2008a,b,c). In contrast,
we sampled D. melanogaster from a low elevation transect (<100 m) where geographic associations for desiccation tolerance are not apparent for either D. melanogaster
or its sibling species D. simulans (Hoffmann et al. 2001;
Arthur et al. 2008). This suggests that thermal adaptation is more likely to explain clinal patterns of trident
pigmentation diversity along the Australian east coast.
The ebony locus is located within the cosmopolitan
inversion In3R(P) that also shows clinal variation in
populations of D. melanogaster from eastern Australia
(Umina et al. 2005). Thus, while our results strongly
implicate the action of thermal selection in determining
the observed clinal patterns, further work is required to
determine the extent to which ebony itself is under
selection, or whether the clinal patterns are a by-product of direct selection acting on the inversion. Nonetheless, our results, and those of previous studies, confirm
the role of ebony in phenotypic variation in pigmentation (see below).
Clinal patterns may also result from neutral processes
such as genetic drift, or from population history
2108 M . T E L O N I S - S C O T T , A . A . H O F F M A N N and C . M . S G R Ò
(Kawecki & Ebert 2004; Hoffmann & Weeks 2007) and
it is important distinguish these factors from non-neutral processes. In the absence of spatially divergent
selection, gene flow is expected to largely erode genetic
differentiation for traits, thus patterns of phenotypic differentiation in populations connected by gene flow may
be due to natural selection for differences in environmental conditions (reveiwed in Kawecki & Ebert 2004).
Spatial genetic structure has been examined along the
Australian east coast (Gockel et al. 2001; Kennington
et al. 2003), and patterns of genetic variation show substantial, symmetrical gene flow following invasion of D.
melanogaster over 100 years ago. While gene flow does
not necessarily preclude trait differentiation due to
demographic history, i.e. levels of migration may be too
low or recent, migration between east coast D. melanogaster populations is extensive (Kennington et al. 2003).
Despite some weak isolation by distance along the cline,
Kennington et al. (2003) observed higher than expected
genetic homogeneity between sites up to 500 km apart,
including Tasmania and the mainland, while asymmetrical migration was rare and inconsistent with latitude.
Finally, reproducible trait patterns along different environmental gradients can provide evidence of natural
selection (Endler 1986). Worldwide clines, ample migration between sites, and the persistence of geographically
structured phenotypic variation under common garden
conditions argues strongly for a role of natural selection
in the maintenance of clinal trident pigmentation variation along the Australian east coast.
Elucidating the molecular targets of selection and
linking these to adaptive shifts in phenotypic traits is
arguably one of the biggest challenges in evolutionary
biology. Here, we link ebony expression variation to thoracic trident pigmentation variation in natural populations and demonstrate that this association is
temperature dependent. The strong negative association
between both pigmentation and latitude and ebony
expression at 25 C disappears when the developmental
temperature is shifted to 16 C. This corresponds to current data for abdominal pigmentation in D. melanogaster, where different mechanisms affecting ebony
expression appear to underlie genetic and plastic phenotypes. The ebony gene is a highly annotated, pleiotropic gene with major effects on pigmentation as well as
other traits including photoreception and phototaxis,
locomotion and reproduction (Kohn & Wittkopp 2007).
Transcriptional rather than protein modifications of
ebony appear to be important for adaptive pigmentation
traits, where regulatory differences are mediated by
modular enhancers that permit independent expression
patterns in different tissues of the developing fly (Wittkopp et al. 2003). In D. melanogaster, a partial selective
sweep near the cis-regulatory element (CRE) of the ebony
locus was found in the darkest abdomens in African
populations (Pool & Aquadro 2007) and at least five
non-coding mutations in this CRE underlie adaptive
abdominal melanism in a Ugandan population (Rebeiz
et al. 2009). Expression variation involving ebony and
tan explain pigmentation differences between closely
related species D. novomexicana (derived yellow body)
and D. americana (ancestral dark brown body), where
fixed non-coding alleles are associated with light pigmentation but are segregating in the more variable D.
americana (Wittkopp et al. 2009). At the plastic level,
variation for abdominal pigmentation may be achieved
by non-genetic temperature dependent mechanisms that
ultimately repress pigmentation enzymes including
ebony in a similar fashion to genetic differences between
mutants or populations and species (Gibert et al. 2007).
Gibert et al. (2007) found that temperature modulates
posterior abdominal pigmentation in female D. melanogaster by regulating a chromatin regulator network
which affects the regulation of pigmentation enzymes
and other characters such as sex comb development in
males, and suggest that phenotypic plasticity for pigmentation is a consequence of thermosensitivity of epigenetic mechanisms. While temperature dependent
modulation of ebony expression appears to shape
abdominal melanism in D. melanogaster, the mechanisms
underlying phenotypic plasticity for trident pigmentation require further investigation. If ebony is the target
pigmentation enzyme at lower temperatures where trident expression is highest, we would expect ebony
expression to be lower in the trident forming cells. We
did not observe this, but we did not directly quantify
ebony expression levels but normalized expression to a
‘housekeeping gene’ across the entire thorax to compare
large shifts in relative expression patterns between the
populations at each temperature. Moreover, ebony may
be temporally regulated at different developmental temperatures, and may require more detailed sampling to
capture transcriptional variation. We can conclude however, that in adult flies 1–3 h old, the genetic differences
that underlie the latitudinal patterns of ebony expression
at 25 C do not account for the patterns observed at
16 C.
Given that the heritable regulatory differences in
ebony expression along the cline manifest only at 25 C,
we independently confirmed that changes in ebony
expression evolve in concert with a darker trident phenotype at this temperature. Nonetheless, regulatory
divergence is an indirect indicator of natural selection
acting at the ebony locus and the ultimate confirmation
is to identify frequency shifts in causal polymorphisms
along the east coast cline. The obvious next step is to
focus on the ebony CRE to see if the phenotypic and
expression clines are matched by allelic clines. The lack
2011 Blackwell Publishing Ltd
G E N E T I C S O F C L I N A L P I G M E N T A T I O N I N D R O S O P H I L A 2109
of selection response for a light phenotype in the Tasmanian lines may be due to a history of stronger directional selection compared to the tropical populations,
and it will be interesting to see if this bears out in the
molecular data. In summary, while complex ecological
pressures and gene-by-environment interactions make it
difficult to universally explain thoracic trident pigmentation diversity, we show that on the Australian east
coast, latitude and differential ebony expression account
for the majority of clinal variation of a likely locally
adapted phenotype.
Acknowledgements
We thank Belinda Van Heerwaarden and Jennifer Shirriffs for
technical support and three anonymous reviewers. This work
was supported by the Australian Research Council (ARC) and
the Commonwealth Environment Research Facility. AAH was
supported by an ARC Australian Laureate Fellowship.
References
Arthur AL, Weeks AR, Sgro CM (2008) Investigating
latitudinal clines for life history and stress resistance traits in
Drosophila simulans from eastern Australia. Journal of
Evolutionary Biology, 21, 1470–1479.
Azevedo RBR, French V, Partridge L (1996) Thermal evolution
of egg size in Drosophila melanogaster. Evolution, 50, 2338–
2345.
Azevedo RBR, James AC, McCabe J, Partridge L (1998)
Latitudinal variation of wing: thorax size ratio and wing –
aspect ratio in Drosophila melanogaster. Evolution, 52, 1353–
1362.
Brisson JA, De Toni DC, Duncan I, Templeton AR (2005)
Abdominal pigmentation variation in Drosophila polymorpha:
geographic in the trait, and underlying phylogeography.
Evolution, 59, 1046–1059.
Capy P, David JR, Robertson A (1988) Thoracic trident
pigmentation in natural populations of Drosophila simulans –
a comparision with Drosophila melanogaster. Heredity, 61, 263–
268.
Clusella Trullas S, van Wyk JH, Spotila JR (2007) Thermal
melanism in ectotherms. Journal of Thermal Biology, 32, 235–
245.
Clusella-Trullas S, Terblanche JS (2010) Local adaptation for
body color in Drosophila americana: commentry on Wittkopp
et al., Heredity, 2010; doi:10.1038/hdy.2010.90. Heredity Nov
10. [Epub ahead of print].
David JR, Capy P, Payant V, Tsakas S (1985) Thoracic trident
pigmentation in Drosophila melanogaster – differentiation of
geographical populations. Genetics Selection Evolution, 17,
211–223.
David JR, Capy P, Gauthier JP (1990) Abdominal pigmentation
and growth temperature in Drosophila melanogaster –
similarities and differences in the norms of reaction of
successive segments. Journal of Evolutionary Biology, 3, 429–
445.
Endler JA (1986) Natural Selection in the Wild. Princeton
University Press, Princeton, NJ.
2011 Blackwell Publishing Ltd
Gibert P, Moreteau B, David JR (2000) Developmental
constraints on an adaptive plasticity: reaction norms of
pigmentation in adult segments of Drosophila melanogaster.
Evolution & Development, 2, 249–260.
Gibert JM, Peronnet F, Schlotterer C (2007) Phenotypic
plasticity in Drosophila pigmentation caused by temperature
sensitivity of a chromatin regulator network. Plos Genetics, 3,
266–280.
Gockel J, Kennington WJ, Hoffmann A, Goldstein DB,
Partridge L (2001) Nonclinality of molecular variation
implicates selection in maintaining a morphological cline of
Drosophila melanogaster. Genetics, 158, 319–323.
Hoffmann AA, Weeks AR (2007) Climatic selection on genes
and traits after a 100 year-old invasion: a critical look at the
temperate–tropical clines in Drosophila melanogaster from
eastern Australia. Genetica, 129, 133–147.
Hoffmann AA, Hallas R, Sinclair C, Mitrovski P (2001) Levels
of variation in stress resistance in Drosophila among strains,
local populations, and geographic regions: patterns for
desiccation, starvation, cold resistance, and associated traits.
Evolution, 55, 1621–1630.
Hoffmann AA, Anderson A, Hallas R (2002) Opposing clines
for high and low temperature resistance in Drosophila
melanogaster. Ecology Letters, 5, 614–618.
Hollocher H, Hatcher JL, Dyreson EG (2000) Evolution of
abdominal pigmentation differences across species in the
Drosophila dunni subgroup. Evolution, 54, 2046–2056.
Hovemann BT, Ryseck RP, Walldorf U et al. (1998) The
Drosophila ebony gene is closely related to microbial peptide
synthetases and shows specific cuticle and nervous system
expression. Gene, 221, 1–9.
James AC, Azevedo RBR, Partridge L (1995) Cellular basis and
developmental timing in a size cline of Drosophila
melanogaster. Genetics, 140, 659–666.
Jeong S, Rebeiz M, Andolfatto P et al. (2008) The evolution of
gene regulation underlies a morphological difference
between two Drosophila sister species. Cell, 132, 783–793.
Kawecki TJ, Ebert D (2004) Conceptual issues in local
adaptation. Ecology Letters, 7, 1225–1241.
Kennington WJ, Gockel J, Partridge L (2003) Testing for
asymmetrical gene flow in a Drosophila melanogaster bodysize cline. Genetics, 165, 667–673.
Kohn MH, Wittkopp PJ (2007) Annotating ebony on the fly.
Molecular Ecology, 16, 2831–2833.
Mani GS (1990) Theoretical models of melanism in Biston
betularia – a review. Biological Journal of the Linnean Society,
39, 355–371.
Mitrovski P, Hoffmann AA (2001) Postponed reproduction as
an adaptation to winter conditions in Drosophila melanogaster:
evidence for clinal variation under semi-natural conditions.
Proceedings of the Royal Society of London Series B-Biological
Sciences, 268, 2163–2168.
Munjal AK, Karan D, Gibert P et al. (1997) Thoracic trident
pigmentation in Drosophila melanogaster: latitudinal and
altitudinal clines in Indian populations. Genetics Selection
Evolution, 29, 601–610.
Papa R, Martin A, Reed RD (2008) Genomic hotspots of
adaptation in butterfly wing pattern evolution. Current
Opinion in Genetics & Development, 18, 559–564.
Parkash R, Munjal AK (1999) Phenotypic variability of thoracic
pigmentation in Indian populations of Drosophila
2110 M . T E L O N I S - S C O T T , A . A . H O F F M A N N and C . M . S G R Ò
melanogaster. Journal of Zoological Systematics and Evolutionary
Research, 37, 133–140.
Parkash R, Rajpurohit S, Ramniwas S (2008a) Changes in body
melanisation and desiccation resistance in highland vs.
lowland populations of D. melanogaster. Journal of Insect
Physiology, 54, 1050–1056.
Parkash R, Ramniwas S, Rajpurohit S, Sharma V (2008b)
Variations in body melanization impact desiccation
resistance in Drosophila immigrans from Western Himalayas.
Journal of Zoology, 276, 219–227.
Parkash R, Sharma V, Kalra B (2008c) Climatic adaptations of
body melanisation in Drosophila melanogaster from Western
Himalayas. Fly, 2, 111–117.
Pool JE, Aquadro CF (2007) The genetic basis of adaptive
pigmentation variation in Drosophila melanogaster. Molecular
Ecology, 16, 2844–2851.
Rajpurohit S, Parkash R, Ramniwas S (2008a) Body
melanization and its adaptive role in thermoregulation and
tolerance against desiccating conditions in drosophilids.
Entomological Research, 38, 49–60.
Rajpurohit S, Parkash R, Ramniwas S, Singh S (2008b) Variations
in body melanisation, ovariole number and fecundity in
highland and lowland populations of Drosophila melanogaster
from the Indian subcontinent. Insect Science, 15, 553–561.
Rebeiz M, Pool JE, Kassner VA, Aquadro CF, Carroll SB
(2009) Stepwise modification of a modular enhancer underlies
adaptation in a Drosophila population. Science, 326, 1663–1667.
Sgrò CM, Overgaard J, Kristensen TN et al. (2010) A
comprehensive assessment of geographic variation in heat
tolerance and hardening capacity in populations of
Drosophila melanogaster from eastern Australia. Journal of
Evolutionary Biology, 23, 2484–2493.
Takahashi A, Takahashi K, Ueda R, Takano-Shimizu T (2007)
Natural variation of ebony gene controlling thoracic
pigmentation in Drosophila melanogaster. Genetics, 177, 1233–
1237.
True JR (2003) Insect melanism: the molecules matter. Trends in
Ecology & Evolution, 18, 640–647.
True JR, Yeh SD, Hovemann BT et al. (2005) Drosophila tan
encodes a novel hydrolase required in pigmentation and
vision. Plos Genetics, 1, 551–562.
Umina PA, Weeks AR, Kearney MR, McKechnie SW,
Hoffmann AA (2005) A rapid shift in a classic clinal pattern
in Drosophila reflecting climate change. Science, 308, 691–693.
Wilson K, Cotter SC, Reeson AF, Pell JK (2001) Melanism and
disease resistance in insects. Ecology Letters, 4, 637–649.
Wittkopp PJ, Beldade P (2009) Development and evolution of
insect pigmentation: genetic mechanisms and the potential
consequences of pleiotropy. Seminars in Cell & Developmental
Biology, 20, 65–71.
Wittkopp PJ, True JR, Carroll SB (2002a) Reciprocal functions
of the Drosophila Yellow and Ebony proteins in the
development and evolution of pigment patterns.
Development, 129, 1849–1858.
Wittkopp PJ, Vaccaro K, Carroll SB (2002b) Evolution of yellow
gene regulation and pigmentation in Drosophila. Current
Biology, 12, 1547–1556.
Wittkopp PJ, Carroll SB, Kopp A (2003) Evolution in black and
white: genetic control of pigment patterns in Drosophila.
Trends in Genetics, 19, 495–504.
Wittkopp PJ, Stewart EE, Arnold LL et al. (2009) Intraspecific
polymorphism to interspecific divergence: genetics of
pigmentation in Drosophila. Science, 326, 540–544.
Wittkopp PJ, Smith-Winberry G, Arnold LL et al. (2010) Local
adaptation for body color in Drosophila americana. Heredity Jul
7 [Epub ahead of print].
The Hoffmann and Sgrò laboratories utilise a variety of organisms to investigate the genetic basis of adaptation to environmental change, with a particular focus on adaptation to
climatic stresses. M.T.-S. is a post-doctoral researcher based in
the Hoffmann lab and is interested in molecular adaptation
along environmental gradients and the role of post-transcriptional mechanisms in phenotypic plasticity for stress resistance
in Drosophila. A.H. FAA is an Australian Laureate Fellow with
broad research interests in addition to stress adaptation including developing new approaches for pollutant biomonitoring,
using genetic tools to control pest species including microorganism control for mosquitoes and other pests. C.S. uses a
combination of techniques including field studies of phenotypic divergence, experimental evolution, quantitative genetics
and genomics to examine how organisms adapt to changing
environmental conditions. She is also interested in exploring
how evolutionary processes can be explicitly incorporated into
biodiversity conservation and management.
Data Accessibility
The raw thoracic pigmentation scores and normalized ebony
expression data from the Australian east coast lines and average pigmentation scores and normalized ebony expression data
for the selected lines are deposited at Dryad doi:10.5061/
dryad.8768.
Supporting information
Additional supporting information may be found in the online
version of this article.
Table S1 Average pigmentation scores of female and male D.
melanogaster at two temperatures from different geographic
locations from the Australian east coast.
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