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Pigmentation and behavior: Potential association through pleiotropic genes in

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Genes Genet. Syst. (2013) 88, p. 165–174

Pigmentation and behavior: potential association

through pleiotropic genes in Drosophila
Aya Takahashi1,2*
1
Department of Biological Sciences, Tokyo Metropolitan University, 1-1 Minamiosawa,
Hachioji 192-0397, Japan
2
Research Center for Genomics and Bioinformatics, Tokyo Metropolitan University,
1-1 Minamiosawa, Hachioji 192-0397, Japan

(Received 27 March 2013, accepted 27 May 2013)

The molecular basis of pigmentation variation within and among Drosophila

species is largely attributed to genes in melanin biosynthesis pathway, which
involves dopamine metabolism. Most of the genetic changes underlying pigmen-
tation variations reported to date are changes at the expression levels of the struc-
tural genes in the pathway. Within D. melanogaster, changes in cis-regulatory
regions of a gene, ebony, are responsible for the naturally occurring variation of
the body pigmentation intensity. This gene is also known to be expressed in glia,
and many visual and behavioral abnormalities of its mutants have been reported.
This implies that the gene has pleiotropic functions in the nervous systems. In
this review, current knowledge on pigmentation variation and melanin biosynthe-
sis pathway are summarized, with some focus on pleiotropic features of ebony and
other genes in the pathway. A potential association between pigmentation and
behavior through such pleiotropic genes is discussed in light of cis-regulatory
structure and pleiotropic mutations.

Key words: dopamine, ebony, melanin biosynthesis pathway, pigmentation,

pleiotropy

ular basis of pigmentation in insects (True, 2003; Wittkopp

INTRODUCTION
Our goal has long been to understand the evolution of
ecologically relevant traits by integrating many different
biological areas such as ecology, genetics, developmental
biology, and neurobiology, i.e. from macro to micro
scale. Pigmentation is one of the suitable study materi-
als in this respect from both ecological and molecular
perspectives. Ecologically, pigmentation could have a
direct impact on individual fitness through factors such
as camouflage, mate attraction, and temperature adapta-
tion (reviewed in True, 2003; Gray and McKinnon, 2007;
Protas and Patel, 2008). From the molecular perspec-
tive, pigmentation variation within and among species is
widely observed and some causal genetic changes have
been identified in many organisms (e.g. Miyagi and Terai,
2013; Suzuki, 2013). Moreover, the pigmentation bio-
synthesis pathway is one of the well-studied metabolic
pathways in animals (reviewed in Sugumaran, 2002), and
its key players have been known for a long time in insects
(Wright, 1987).
Since there are many comprehensive reviews on molec-
Edited by Hiroshi Iwasaki
* Corresponding author. E-mail: ayat@tmu.ac.jp
et al., 2003; Wittkopp and Beldade, 2009; Protas and
Patel, 2008; Kronforst et al., 2012), I refer to them when
appropriate. Instead, the main focus of this review is on
the studies using naturally inhabiting model organism, D.
melanogaster, except where otherwise notified. I briefly
summarize our current knowledge on pigmentation vari-
ation, melanin biosynthesis pathway, and its role in bio-
genic amine metabolism. I also intend to look further
into the pleiotropic features of one of the genes in the
pathway, ebony, especially in relation to behavioral
aspects. Finally, I discuss about a potential association
between pigmentation and behavior in reference to the
structure and evolution of cis-regulatory regions of such
pleiotropic gene.
PIGMENTATION VARIATION IN DROSOPHILA
Various types of pigmentation variation within and
between closely related species are observed in many
insect taxa as well as in Drosophila species (reviewed in
True, 2003; Wittkopp et al., 2003; Wittkopp and Beldade,
2009; Kronforst et al., 2012). Among them, an intriguing
point one would notice is the existence of numerous
reports on closely related species pairs with contrasting
166 A. TAKAHASHI
appearances in terms of body pigmentation intensity.
Those cases include species pairs, D. ananassae - D.
pallidosa (Bock and Wheeler, 1972), D. yakuba - D.
santomea (Llopart et al., 2002), D. malerkotliana
malerkotliana - D. m. pallens (Ng et al., 2008), D. pseudo-
ananassae pseudoananassae - D. p. nigrens (Ng et al.,
2008), D. bipectinata - D. parabipectinata (Ng et al.,
2008), and D. novamexicana - D. americana (Wittkopp et
al., 2003). Actually, D. novamexicana is distinctively
pale compared to other D. virilis group species (Spicer,
1991). These species pairs have divergence time short
enough that ability to interbreed has allowed genetic
analyses and has helped narrowing down or to identify
responsive genetic changes underlying the pigmentation
differences (Llopart et al., 2002; Wittkopp et al., 2003,
2009; Carbone et al., 2005; Jeong et al., 2008; Ng et al.,
2008). Pigmentation divergence in these species within
short period of time suggests that pigmentation evolves
relatively fast in some species. Short time diversifica-
tion in abdominal pigmentation pattern is also well
described in D. dunni and D. cardini subgroups
(Hollocher et al., 2000a, 2000b; Brisson et al., 2006) and
in D. montium subgroup (Ohnishi and Watanabe, 1985).
Short time diversification in pigmentation pattern coin-
cides with that among populations from different locali-
ties in widely distributed Drosophila species such as D.
ananassae (McEvey et al., 1987; Tobari, 1993) and D.
elegans (Bock and Wheeler, 1972; Lemeunier et al., 1986;
Hirai and Kimura, 1997). The largest source of informa-
tion on within species variation in pigmentation is in the
cosmopolitan species, D. melanogaster. The thoracic and
dorsal pigmentation differences in typical dark and light
strains from a natural population are indicated in Fig.
1. As shown in Fig. 1A, pigmentation variation can be
easily observed in the trident-shaped area on the dorsal
side of a thoracic segment (Fig. 1A). The first report of
the trident polymorphism from a natural population was
Fig. 1. Pigmentation variation within Drosophila melanogaster. A typical dark (A) and light (B) colored individual reared at 20°C.
Differences in pigmentation intensity can be observed at thoracic trident (white arrows), abdominal segments (black arrows), and lat-
eral area of thoracic segment (black arrowheads).
by the pioneers of Drosophila geneticists, Morgan and
Bridges (1919), followed by Jacobs (1960, 1961), and
David et al. (1985). What is particularly noticeable
about the studies on pigmentation variation within this
species is that a clinal variation in pigmentation intensity
is repeatedly observed along altitude or latitude or both
(David et al., 1985; Munjal et al., 1997; Pool and Aquadro,
2007; Parkash et al., 2008a, 2008b, 2009; Telonis-Scott et
al., 2011). These observations of non-random distribu-
tion of the phenotype can be a circumstantial evidence
that supports environmental adaptation playing a role in
shaping the diverse patterns of body pigmentation.
Among many environmental factors, temperature is
thought to play a substantial role in adaptive changes of
the body pigmentation. Thermal budget (melanism)
hypothesis in ectoderms argues that a darker pigmenta-
tion is more adaptive in colder environments, where it can
help absorb heat from solar radiation, while a lighter pig-
mentation will prevent overheating in a warmer environ-
ment (see for review in Clusella Trullas et al., 2007). In
D. melanogaster, geographic distributions of pigmenta-
tion variation are consistent with this hypothesis (David
et al., 1985; Gibert et al., 1996, 2009; Munjal et al., 1997;
Pool and Aquadro, 2007; Telonis-Scott et al., 2011;
Takahashi and Takano-Shimizu, 2011). Also, phenotypic
plasticity of the pigmentation intensity by the developing
temperature is in the consistent direction with the
hypothesis, which is that the flies becomes darker when
grown at lower temperatures (David et al., 1985, 1990;
Das et al., 1994; Gibert et al., 1996, 2000, 2009; Munjal
et al., 1997; Gibert et al., 2007).
Nevertheless, there are contradictory views on whether
body color is effective in regulating body temperature in
Drosophila. Hirai and Kimura (1997) showed that
between black and brown morphs of D. elegans, the for-
mer had slightly but significantly higher body tempera-
ture (0.26°C) than the latter under irradiation in the
 Pigmentation and behavior in Drosophila 167

laboratory. However, measurements under natural

sunlight had shown that an insect as small as a fruitfly
(Diptera 3 mg) cannot keep its internal temperature
above ambient, therefore, body color would not make any
difference (Willmer and Unwin, 1981). So far, physiolog-
ical basis of the thermal budget (melanism) hypothesis in
Drosophila is still weak.
Regarding other environmental factors, a strong asso-
ciation of pigmentation intensity and desiccation resis-
tance has been reported within and among D.
melanogaster populations from India (Parkash et al.,
2008a, 2008b, 2009), which suggests that humidity
related adaptation may be important in some regions.
However, a study on longitudinal cline of pigmentation
variation in a different species, D. americana, did not find
any association between pigmentation and desiccation
resistance (Wittkopp et al., 2011; Clusella-Trullas and
Terblanche, 2011). Also, such association was not
detected among different strains of D. yakuba (Matute
and Harris, 2013). UV resistance is another ecological
factor that is thought to affect the adaptive changes in
pigmentation intensity (Jacobs, 1985; Majerus, 1998;
Matute and Harris, 2013), which awaits further investi-
gations.
MELANIN BIOSYNTHESIS PATHWAY
Adult pigments in Drosophila as well as in many other
insect species are synthesized by the acts of enzymes in
the melanin biosynthesis pathway in the developing epi-
dermis (reviewed in Wright, 1987; Wittkopp et al., 2003;
True, 2003; Wittkopp and Beldade, 2009; Kopp, 2009;
Anderson, 2010; Kronforst et al., 2012). The adult pig-
ment synthesis in Drosophila takes place during the first
several hours after eclosion by the activation of ecdysis
neuropeptide signaling pathway (Davis et al., 2007). The
color of the cuticle is determined by the proportions of dif-
ferent-colored melanin pigments produced during this
period, which is likely to be the outcome of the net balance
of those structural enzyme activities in the pathway.
The simplified schematic model showing pigment bio-
synthesis pathway is shown in Fig. 2. There are some
inconsistent views regarding the branches leading to the
final polymerized products (melanins), shown as broken
arrows in Fig. 2A. Some authors have hypothesized that
black melanin is derived from Dopa (Wittkopp et al.,
2002, 2003; True et al., 2005; Kronforst et al., 2012).
Whereas, others have suggested that at least part of the
black pigment comes from Dopamine (Wright, 1987;

Fig. 2. Simplified schematic models of the proposed biosynthetic pathways in Drosophila. (A) Pathways for producing cuticular pig-
ments, redrawn from figures in Wright (1987), Walter et al. (1996), Wittkopp et al. (2002, 2003), True et al. (2005), Gibert et al. (2007),
Kopp (2009), Riedel et al. (2011), and Kronforst et al. (2012). Some inconsistent views exist about whether the majority of the black
melanin is produced from dopa or dopamine (pathways indicated by red broken arrows). (B) Pathways for histamine recycling in the
visual system; redrawn from Borycz et al. (2002, 2012), True et al. (2005) and Gavin et al. (2007). Core enzymes in the pathways are
indicated in blue. Broken arrows indicate paths leading to the final products (colored pigments). NBAD; N-beta-alanyl dopamine,
NBAH; N-beta-alanyl histamine, TH; tyrosine hydroxylase, DDC; dopa decarboxylase.
168 A. TAKAHASHI
Walter et al., 1996; Gibert et al., 2007). This issue is
related to the role of Yellow required for the production
of black melanin. Recent studies actually support that
this enzyme acts downstream of Dopamine as well as that
of Dopa suggesting the latter view (Gibert et al., 2007;
Riedel et al., 2011). During the immune response, the
black melanin produced through DDC activity seems to
be the predominant melanization pathway, although a
minor path leading to black melanin from Dopa is also
implicated (Nappi and Christensen, 2005; Nappi, 2010).
Most of the genetic changes underlying pigmentation
variations reported to date are changes at the expression
levels of those structural genes (i.e. ebony, yellow, tan,
reviewed in Wittkopp et al., 2003; Wittkopp and Beldade,
2009; Protas and Patel, 2008; Kronforst et al., 2012).
Changing the cis-regulatory sequences is perhaps the
simplest way to adjust the relative activity levels of those
enzymes. Thus, this simplicity may be a contributing
factor that has enabled fast evolutionary changes of body
pigmentation seen in some species, in spite of the fact
that many structural genes are highly conserved even
among different insect taxa (Kayser, 1985).
It should be added that many recent studies have iden-
tified factors outside the core members of the melanin bio-
synthesis pathway. These factors include transcription
factors such as Abdominal-B, bric-à-bric (Kopp et al.,
2000; Gibert et al., 2007; Williams et al., 2008), and
optomotor-blind (Kopp and Duncan, 1997), a global chro-
matin regulator network affected by Hsp83 (Gibert et al.,
2007), and a cell growth pathway activated by Rheb
(Zitserman et al., 2012). Also there is a recent study on
microRNA, miR-8, which is required for the proper pig-
mentation patterning of the adult cuticle (Kennel et al.,
2012). Thus, pigmentation is a reflectant of many
related developmental systems in flies.
PIGMENTATION VARIATION IN D.
MELANOGASTER
In D. melanogaster, the widely accepted prediction had
been that the commonly observed variation in thoracic
trident pigmentation intensity is caused by ebony, whose
mutants show dark trident pattern on their thoracic seg-
ments (Jacobs, 1960; Wittkopp et al., 2002). Some direct
evidences linking this gene to the thoracic or abdominal
pigmentation variation in natural populations were put
forward by an associated linkage pattern in the 5’
upstream region of this gene (Pool and Aquadro, 2007), a
genetic mapping result using two wild-derived dark and
light strains (Takahashi et al., 2007), and quantitative
comparisons of the transcription levels induced by cis-
regulatory elements from strains with different pigmen-
tation intensities (Rebeiz et al., 2009). The lower expres-
sion of this gene in developing epidermis is associated
with darker pigmentation, and the higher expression with
lighter pigmentation (Takahashi et al., 2007; Rebeiz et
al., 2009). This association is reported in populations
from Africa (Rebeiz et al., 2009), Australia (Telonis-Scott
et al., 2011), and southern Japan (Takahashi and Takano-
Shimizu, 2011).
The summary of cis-regulatory polymorphisms and
their association with the pigmentation phenotype are
described in Fig. 3. Rebeiz et al. (2009) have performed
a precise mutagenesis assay on the cis-regulatory region
of ebony, and showed that at least five changes surround-
ing the core enhancer region have altered transcriptional
activity of this gene in African populations. In Iriomote
(southern Japan) population, 19 pigmentation-associated
sites in the 5 prime region of this gene were identified
(Takahashi and Takano-Shimizu, 2011). Interestingly,
those sites that had complete association with the pheno-
type in the Iriomote population were not associated with
the phenotype in populations from Uganda and Kenya,
and the 5 nucleotide sites that have caused expression
level changes in abdomens of African flies were either not
segregating or not associated with the pigmentation phe-
notype in the flies from the Japanese population (Fig. 3;
Takahashi and Takano-Shimizu, 2011). These compari-
sons suggest that there are many potential cis-regulatory
mutations that can alter transcriptional activity, and that
different mutations could lead to similar phenotypic vari-
ation in different populations within this species.
Other than the structural gene, ebony, multiple alleles
at a transcription factor bric-à-bric locus, are also likely
to contribute to the natural variation in pigmentation
pattern in this species (Kopp et al., 2003). This tran-
scription factor plays a role in shaping the sexually
dimorphic abdominal pigmentation pattern (Kopp et al.,
2000) and mainly affects the width of the dark pigment
band on the female posterior abdominal segments,
whereas the ebony locus affects the overall abdominal pig-
mentation intensity outside the band (Rebeiz et al.,
2009). It is plausible that close investigations of other
structural genes in the melanin biosynthesis pathway or
other transcription factor genes acting as pre-pattern for-
mation may be involved in the natural pigmentation vari-
ation to some extent.
Another interesting phenomenon is that in Iriomote
population from southern Japan, Light-type enhancer
haplotype is strongly linked to a cosmopolitan inversion,
In(3R)Payne (Fig. 3; Takahashi and Takano-Shimizu,
2011), which is predominant in warmer climatic regions
in both hemispheres (Mettler et al., 1977; Knibb et al.,
1981; Knibb, 1982; Inoue and Igarashi, 1994; Umina et
al., 2005). The latitudinal clines of genetic and pheno-
typic variation, including In(3R)Payne along the eastern
Australian coast, have been extensively studied (reviewed
in Hoffmann and Weeks, 2007), and recently, Telonis-
Scott et al. (2011) have characterized the clines of
pigmentation intensity and ebony expression level that
 Pigmentation and behavior in Drosophila 169

Fig. 3. Summary of the cis-regulatory polymorphisms of ebony associated with pigmentation phenotype in populations
from Iriomote (southern Japan) and Africa. Positions of Light-type specific SNPs and Indels in Iriomote population are
indicated by red arrowheads and red arrows, respectively (data from Takahashi and Takano-Shimizu, 2011). The region
resides within an inversion, In(3R)Payne, with which thoracic pigmentation intensity has a strong association in this
population. Note that multiple mutations locate within the ~0.9 kb epidermis enhancer element indicated by the blue
bar, and are different from the 5 nucleotide sites indicated by black arrowheads that have caused expression level changes
of ebony in abdomens of African flies (Rebeiz et al., 2009).

parallels the cline of In(3R)Payne frequency. A common
association of In(3R)Payne with the lighter body pheno-
type in this species awaits further investigations.
NEURONAL FUNCTION AND BEHAVIORAL
EFFECT OF EBONY IN D. MELANOGASTER
Visual defects are among the well-described phenotypes
of ebony mutants. It has been known from the early
years that the electroretinograms of the mutants are
abnormal, lacking the lamina potentials (Hotta and Benzer,
1969; Heisenberg, 1971). Phototactic and optomotor
responses were also compared and it was shown that those
responses are reduced in ebony flies, whose functional def-
icit in retinula cells 1–6 was suspected (Heisenberg,
1972). Kyriacou et al. (1978) even concluded that e11
homozygotes were effectively blind.
The molecular function of Ebony in the visual system
is now known to be related to the histamine recycling
pathway. It is suggested that Ebony functions as an
enzyme which conjugates histamine to beta-alanine in the
glia close to histamine release sites of photoreceptor cells
in the lamina (Borycz et al., 2002; Richardt et al., 2002,
2003; True et al., 2005; Wagner et al., 2007; Gavin et al.,
2007). This reaction terminates the action of the hista-
mine released into the synaptic cleft in response to light
by removing it by glial cells, where it is converted into
carcinine (beta-alanyl histamine) by Ebony (see Fig. 2B
for a simplified model).
Thus, Ebony is involved in at least two different bio-
genic amines, dopamine and histamine, metabolism path-
ways (Fig. 2). Moreover, tests on in vitro catalytic ability
of Drosophila Ebony for other biogenic amines revealed
that this enzyme accepts a variety of amines for beta-
alanyl conjugation (Richardt et al., 2003). Still unknown
functional properties of this enzyme may exist in vivo.
Another apparent phenotype of the ebony mutant is a
defect on circadian rhythm. The rhythm defect was first
170 A. TAKAHASHI
reported in tyrosine aminotransferase activity cycle (Tau-
ber and Hardeland, 1977). Later on, Newby and Jackson
(1991) found that 5 of the tested ebony mutants show
abnormal locomotor activity rhythms in both constant
dark and in light:dark cycles. They also measured eclo-
sion rhythms of the mutants, which was unaffected.
Two microarray studies indicated that ebony transcript
has a rhythmic expression in head with a peak in the
morning hours (Claridge-Chang et al., 2001; Ueda et al.,
2002). Suh and Jackson (2007) elucidated the role of
ebony on locomotor rhythmicity in their experiments.
They showed that ebony is expressed in a discrete popu-
lation of glial cells in larval and adult brains and that
glial expression of Ebony rescues the altered circadian
behavior of the ebony null mutant, e1. It is interesting
and yet still rather puzzling that a high constitutive
expression of Ebony without rhythmicity can rescue the
aberrant locomotor rhythm in e1.
These authors also provided two evidences that support
the ebony function in dopaminergic signaling. First, the
rhythmic glial expression takes place in cells that are in
close proximity to dopaminergic and serotonergic neurons
of the larval and adult brains. Second, a genetic inter-
action exists between ebony and the dopamine trans-
porter gene, DAT. The study emphasizes the importance
of glia –neuron communication in coordinating fly behav-
ior.
Rather ambiguous phenotypes of the ebony mutants are
those related to the mating behavior. There are reports
on the mutants’ disadvantages on mating success, which
are the predictable consequences of their visual deficits
(Rendel, 1951; Kyriacou et al., 1978; Kyriacou, 1981).
Interestingly, most of these studies suggest that mutant
males mate more efficiently in the dark or in diminished
light conditions. Jacobs (1960, 1961) used wild-derived
dark and light flies collected in North Carolina, referring
to them as ebony and light flies, respectively, and
reported observation in consistent with the laboratory
derived ebony mutants. It is noteworthy that Jacobs
(1960) observed non-random mating combination of his
ebony and light strains such as that the frequency of
mated pairs between light females and ebony males was
substantially low (13 out of 369 mated pairs). This
observation implies that there could be some variation in
mate preference associated with the pigmentation varia-
tion segregating in a natural population.
Also, there are numbers of descriptions from the earlier
studies reporting unusual sexual behavior of ebony
mutants that may not be related to visual factors such as
less frequent wing extention (Jacobs, 1960), frequent
courtship abortion (Crossley and Zuill, 1970), and
changes in acoustic parameters of the courtship song
(Kyriacou et al., 1978). However, there have not been
any rigorous experiments done to comfirm whether these
observed mating phenotypes of the mutants are replicable
using transgenic flies in a controlled genetic background.
Also, since it is well established that dopamine level
affects many different aspects of mating behavior
(Neckameyer, 1998; Marican et al., 2004; Liu et al, 2008,
2009; Wicker-Thomas and Hamann, 2008), further inves-
tigations are awaited to see whether these behavior phe-
notypes are the indirect consequences of the abnormal
neurotransmitter abundances in specific tissues or from
other unknown factors.
OTHER PLEIOTROPIC FUNCTIONS OF THE
ENZYMES IN DOPAMINE BIOSYNTHESIS
PATHWAY
Dopamine has another pleiotropic function in trachea
development, and the expression of its metabolic pathway
genes such as ebony, Punch, and Catsup in the developing
trachea system of embryo has been reported (Hsouna et
al., 2007; Perez et al., 2010). Also, there are series of
studies by Neckameyer and colleagues showed that
manipulation of dopamine levels affects many other devel-
opmental processes such as that in ovary (Neckameyer,
1996) and sensory tissue development (Neckameyer et al.,
2001), where the genes in its biosynthesis pathway may
also be playing a critical role.
The melanization reaction observed upon injury or on
the surface of pathogens and parasites occurs as a part of
the innate immune response (see review in Lemaitre and
Hoffmann, 2007). And this response also involves phe-
noloxidase and some enzymes such as Ddc in the dop-
amine biosynthesis pathway (Fig. 2C and reviews: Nappi
and Christensen, 2005; Nappi, 2010). It has also been
suggested that N-beta-alanyldopamine has an antimicro-
bial activity in vitro, and the activation of its synthase,
Ebony, was observed in the epidermis of the medfly,
Ceratitis capitata, and the yellow mealworm, Tenebrio
molitor, upon bacterial infection (Schachter et al., 2007).
PLEIOTROPY AND CIS-REGULATORY
STRUCTURE
I have described many pleiotropic functions of the genes
involved in the melanin biosynthesis pathway. Those
pleiotropic features can provoke conflicting natural selec-
tion pressures from various functional demands. It has
been argued that the conflict can be partly resolved by
possessing modularly structured cis-regulatory elements
that allow evolutionary modifications of specific expres-
sion patterns without affecting others. Indeed, such
modularly restricted cis-regulatory evolution has been
repeatedly demonstrated in genes involved in pigmenta-
tion pattern variations among fruitfly species (Gompel et
al., 2005; Prud’homme et al., 2006; Jeong et al., 2008).
However, those fixed changes among species may repre-
sent only a small portion of mutations, which have limited
 Pigmentation and behavior in Drosophila
effect on other functions (Stern and Orgogozo, 2008;
Gompel and Prud’homme, 2009). But even within spe-
cies, Rebeiz et al., (2009) have shown that changes in the
cis-regulatory region of ebony that affects pigmentation
has no effect on its expression level in other tissues, such
as wing, haltere, larval mouth hooks, larval anterior spir-
acles, and larval brain. These results imply modularly
restricted non-pleiotropic effects of regulatory mutations.
The generality of this conclusion and the possible scenar-
ios of the regulatory evolution (i.e. Monteiro and Podlaha,
2009) await further investigations.
PERSPECTIVES
Recent studies have shown that the major causal
genetic differences underlying naturally occurring pig-
mentation variation in D. melanogaster lie in the cis-
regulatory region of ebony (Pool and Aquadro, 2007;
Takahashi et al., 2007; Rebeiz et al., 2009; Telonis-Scott
et al., 2011; Takahashi and Takano-Shimizu, 2011). This
leads us to a question why ebony, among other structural
genes in the melanin biosynthesis pathway, has been sub-
jected to frequent changes or possibly the target of natu-
ral selection in this species. Systematic comparisons
among cases in other species (i.e. Kopp, 2009) may yield
some answers to the evolvability of the gene network com-
ponents in the pathway.
There is another evolutionary question about how those
pleiotropic features of the pathway has evolved and what
was the most ancestral function. Rigorous genomic
screenings and phylogenetic analyses of the gene mem-
bers of the pathway may give some answers to this ques-
tion.
The modularly structured cis-regulatory elements of a
pleiotropic gene seem to be an effective strategy in resolv-
ing the conflicting natural selection acting on the gene
from different functional demands. However, there
must be at least some cases where a simple modular
arrangement is not feasible. In these cases, some muta-
tions may affect the efficiencies of multiple functional reg-
ulatory elements, which would bring about changes in
multiple pleiotropic characters such as pigmentation and
behavior. Since within-population variation harbors
neutral and slightly deleterious changes, a wider spec-
trum of mutational effect should be observed than when
looking at only the fixed sites between species. Further
investigations of the extent of such pleiotropic mutational
effect within population may shed light on some new fea-
tures of the genetic processes leading to species diver-
gence due to associated evolutionary changes of the
pleiotropic functions.
A. T. is supported by Grant-in-Aid for Scientific Research on
Innovative Areas from the Ministry of Education, Culture,
Sports, Science and Technology of Japan.
171
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