ORIGINAL ARTICLE
Molecular Evolution of the Odorant and Gustatory ReceptorGenes in Lepidopteran Insects: Implications for Their Adaptationand Speciation
Patamarerk Engsontia • Unitsa Sangket •
Wilaiwan Chotigeat • Chutamas Satasook
Received: 6 June 2013 / Accepted: 6 July 2014 / Published online: 20 July 2014
� Springer Science+Business Media New York 2014
Abstract Lepidoptera (comprised of butterflies and
moths) is one of the largest groups of insects, including
more than 160,000 described species. Chemoreception
plays important roles in the adaptation of these species to a
wide range of niches, e.g., plant hosts, egg-laying sites, and
mates. This study investigated the molecular evolution of
the lepidopteran odorant (Or) and gustatory receptor (Gr)
genes using recently identified genes from Bombyx mori,
Danaus plexippus, Heliconius melpomene, Plutella xylo-
stella, Heliothis virescens, Manduca sexta, Cydia pomo-
nella, and Spodoptera littoralis. A limited number of cases
of large lineage-specific gene expansion are observed
(except in the P. xylostella lineage), possibly due to
selection against tandem gene duplication. There has been
strong purifying selection during the evolution of both
lepidopteran odorant and gustatory genes, as shown by the
low x values estimated through CodeML analysis, ranging
from 0.0093 to 0.3926. However, purifying selection has
been relaxed on some amino acid sites in these receptors,
leading to sequence divergence, which is a precursor of
positive selection on these sequences. Signatures of posi-
tive selection were detected only in a few loci from the
lineage-specific analysis. Estimation of gene gains and
losses suggests that the common ancestor of the Lepidop-
tera had fewer Or genes compared to extant species and an
even more reduced number of Gr genes, particularly within
the bitter receptor clade. Multiple gene gains and a few
gene losses occurred during the evolution of Lepidoptera.
Gene family expansion may be associated with the adap-
tation of lepidopteran species to plant hosts, especially
after angiosperm radiation. Phylogenetic analysis of the
moth sex pheromone receptor genes suggested that chro-
mosomal translocations have occurred several times. New
sex pheromone receptors have arisen through tandem gene
duplication. Positive selection was detected at some amino
acid sites predicted to be in the extracellular and trans-
membrane regions of the newly duplicated genes, which
might be associated with the evolution of the new phero-
mone receptors.
Keywords Odorant receptor � Gustatory receptor �Molecular evolution � Lepidoptera � Chemoreception
Introduction
Moths and butterflies (Order Lepidoptera) are among the
largest groups of animals. More than 160,000 species have
been described in this group, and it is estimated to include
up to 500,000 species (Kristensen et al. 2007). These
species are important to humans in many ways, including
acting as significant pests in worldwide agricultural pro-
duction and storage and in major sources used for cloth
production (silk moths) (International Silkworm Genome
Consortium 2008). They also play important ecological
roles, such as serving as pollinators and primary consum-
ers. Chemoreception is crucial for their ecological success.
Electronic supplementary material The online version of thisarticle (doi:10.1007/s00239-014-9633-0) contains supplementarymaterial, which is available to authorized users.
P. Engsontia (&) � C. Satasook
Department of Biology, Faculty of Science, Prince of Songkla
University, Songkla 90112, Thailand
e-mail: [email protected]
U. Sangket � W. Chotigeat
The Center for Genomics and Bioinformatics Research,
Department of Molecular Biotechnology and Bioinformatics,
Faculty of Science, Prince of Songkla University,
Songkla 90112, Thailand
123
J Mol Evol (2014) 79:21–39
DOI 10.1007/s00239-014-9633-0
Male moths use their highly specialized olfactory sense to
detect female sex pheromones and locate their mates.
Female moths use both the olfactory and gustatory senses
in searching for egg-laying sites, while their larvae use
these senses for locating and discriminating among foods
(nutrients vs. toxins). Thus, chemosensory adaptation has
occurred in all species, allowing them to use different
niches (Thompson and Pellmyr 1991; Chapman 2003;
Renwick and Chew 1994). The aim of this study was to
investigate the molecular evolution of the lepidopteran
chemoreceptor genes. Our analysis was conducted to assess
the role of tandem gene duplication in determining the size
of the gene family, to investigate signatures of selective
forces on the evolution of receptor sequences and to esti-
mate the number of gene gains and losses during the
evolution of Lepidoptera. We further discussed how our
finding might be related to the adaptation and speciation of
the lepidopteran insects.
Lepidoptera have served as important models for che-
moreception studies (along with the important genetic
model Drosophila melanogaster). In fact, the first identi-
fied insect sex pheromone, bombykol, was found in Bom-
byx mori (Butenandt et al. 1959). Additionally, the odorant-
binding proteins, which are *15 kDa soluble proteins
found in the olfactory lymph of the insect olfactory sensilla
that carry odorant molecules to the odorant receptors on the
dendritic membrane of the olfactory receptor neurons, were
first identified in moths (Vogt and Riddiford 1981). In the
past decade, knowledge of the molecular basis of insect
chemoreception (both gustatory and olfactory) has
advanced greatly due to the availability of the insect gen-
ome databases. Such availability facilitates the identifica-
tion of members of the insect chemoreceptor gene
superfamily, including odorant receptor (Or) and gustatory
receptor (Gr) genes. In combination with novel molecular
techniques, multiple molecular and cellular bases for insect
olfactory-driven behaviors have been elucidated. Such
behaviors include host-seeking behavior in female mos-
quitoes (Hallem et al. 2004), the different responses of
male and female flies (negative and positive, respectively)
to a pheromone produced by males in D. melanogaster
(Kurtovic et al. 2007), the detection of female sex phero-
mones by male moths (Sakurai et al. 2011), and positive
chemotaxis behavior toward mulberry leaf volatiles in
B. mori larvae (Tanaka et al. 2009).
The insect chemoreceptor gene superfamily is ancient.
The origin of the gustatory receptor genes potentially dates
back to the origin of the arthropods, while the odorant
receptors presumably evolved later from the gustatory
receptor genes specifically in the insect lineage (Robertson
et al. 2003; Vieira and Rozas 2011). These genes are
similar in many respects. Both gene families encode 7
transmembrane domain proteins with an intracellular N
terminus and an extracellular C terminus (Benton et al.
2006; Zhang et al. 2011). These receptors are ligand-gated
cation channels. Ectopic expression studies in a heterolo-
gous cell system suggested that the insect gustatory
receptor can function independently (showing both ligand-
binding and ion channel functions) (Sato et al. 2011), while
the insect odorant receptors consist of heterodimers of a
ligand-binding receptor and a conserved olfactory core-
ceptor (Orco) that functions as an ion channel (Sato et al.
2008; Wicher et al. 2008). The expression and function of
these receptors are distinctly different. The Gr genes are
expressed in the gustatory neurons housed within the
gustatory sensilla (found on the mouth part-labium, max-
illary palps, antennae, and legs), and their axons project to
the suboesophageal ganglion. In contrast, the Or genes are
expressed in the olfactory neurons housed within the
olfactory sensilla (found mainly on the antenna), and their
axons project to the glomeruli in the antennal lobe (Vos-
shall 2000; Scott et al. 2001; Chyb 2004). However, it
should be noted that knowledge of the function of these
receptors is based solely on studies on the model insect
D. melanogaster. Whether this information can be gen-
eralized to the larger group of insects has yet to be con-
firmed. The gustatory receptors can respond to tastants
such as sugars, bitter substances, CO2, and some contact
pheromones, while the odorant receptors respond to vari-
ous volatile odorants and pheromonal molecules (e.g.,
Hallem and Carlson 2006; Montell 2009). Thus, the Or and
Gr gene families might have evolved under different types
of selection.
Increasing numbers of the Or and Gr genes have been
identified from many lepidopteran species. Genome dat-
abases have been used to identify 68 Or and 65 Gr genes in
B. mori (Wanner and Robertson 2008), 64 Or and 47 Gr
genes in Danaus plexippus (monarch butterfly) (Zhan et al.
2011), 79 Or and 25 Gr genes in Plutella xylostella (dia-
mondback moth) (You et al. 2013), and 70 Or and 73 Gr
genes in Heliconius melpomene (Heliconius genome con-
sortium 2012, Briscoe et al. 2013). Heliothis virescens
(tobacco budworm) presents at least 21 Or genes, which
has been identified through gene expression studies using
gene homology-based techniques (Krieger et al. 2002,
2004). Recently, next-generation DNA sequencing was
used to study the antennal transcriptomes of a number of
moth species, leading to the identification of 68 Or genes
from Manduca sexta (tobacco hornworm) (Grosse-Wilde
et al. 2011; Howlett et al. 2012), 39 Or genes from Cydia
pomonella (Codling moth) (Bengtsson et al. 2012), and 25
Or genes from Spodoptera littoralis (cotton leafworm)
(Legeai et al. 2011). Sex pheromone receptors have been
identified in a number of moths, including Ostrinia spp.
(Yasukochi et al. 2011; Leary et al. 2012). These receptor
sequences provide fruitful data for the investigation of
22 J Mol Evol (2014) 79:21–39
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molecular evolutionary processes and the selective pres-
sures acting on these gene families.
Lepidopteran sex pheromones are highly diverse in
terms of both their chemical components and ratios,
because species-specific pheromonal communication plays
an important role in the reproductive isolation of each
species (Ando and Yamakawa 2011). Multiple studies have
suggested that the insect desaturase multigene family has
played important roles in the evolution of female moth sex
pheromones. Possible mechanisms include ancient gene
duplication followed by subfunctionalization, the activa-
tion of nonfunctional gene transcripts, and the differential
regulation of the same gene in different moth species
(Knipple et al. 2002; Roelofs et al. 2002; Albre et al. 2012).
In Ostrinia, however, allelic variation of the fatty acyl
reductase gene causes divergence in female moth sex
pheromones (Lassance et al. 2010). A number of moth sex
pheromone receptors have been identified recently. Unlike
non-pheromone receptors, which are highly divergent in
their sequences, the sex pheromone receptors show a
higher degree of sequence conservation. Phylogenetic
analyses have suggested a monophyletic relationship of the
lepidopteran sex pheromone receptors (Miura et al. 2009;
Tanaka et al. 2009; Yasukochi et al. 2011; Grosse-Wilde
et al. 2011). Multiple ligand-receptor relationships have
been identified in lepidopteran insects, particularly between
female sex pheromones and pheromone receptors. In
B. mori, the receptors BmOR1 and BmOR3 respond spe-
cifically to bombykol (E10Z12-16:OH) and bombykal
(E10Z12-16:Ald), which are major and minor sex phero-
mone components, respectively (Sakurai et al. 2004,
Nakagawa et al. 2005). In Heliothis virescens, HvOR13
and HvOR6 respond to Z11-16:Ald and Z9-14:Ald, which
are also major and minor sex pheromone components,
respectively. HvOR16 responds to Z11-16:OH produced by
the pheromone glands of female H. virescens and by its
congener H. subflexa. This chemical can inhibit the
attraction of males at high doses. HvOR14 responds to
Z11-16:OAc, which is produced only by its congener H.
subflexa (as a sex pheromone), inhibiting the response of
H. virescens males (Wang et al. 2011; Grosse-Wilde et al.
2007; Baker 2009; Krieger et al. 2009; Vasquez et al.
2011). In Ostrinia nubilaris (European corn borer), Onu-
bOR6 and OnubOR5 respond to Z11-14:OAc and E11-
14:OAc, respectively (Wanner et al. 2010; Miura et al.
2009, 2010). It should be noted that in natural populations,
there are two strains of O. nubilaris (the E and Z strains),
which are reproductively isolated. Females of the Z strain
produce a 3:97 mixture of (E)- and (Z)-11-14OAc, while
females of the E strain produce a 99:1 E/Z blend. Thus, the
males of the E and Z strains exhibit different DNA
sequences of the OnubOR6 and OnubOR5 genes (Smadja
and Butlin 2009; Leary et al. 2012). Some male moths and
butterflies have also been shown to produce and secrete sex
pheromones from hairpencil glands (e.g., Baker et al. 1981;
Teal and Tumlinson 1989; Jacquin et al. 1991), suggesting
that male-produced sex pheromone also plays important
roles in sexual copulation at a short range and is therefore
relevant to the evolution of the lepidopteran chemoreceptor
gene family. However, male-produced sex pheromones and
their roles in the evolution of the Lepidoptera have been
investigated to a much lesser extent, and the odorant
receptors for male-produced sex pheromone have not been
identified. Hence, knowledge about the molecular mecha-
nisms underlying the evolution of new sex pheromones and
sex pheromone receptors is still limited.
Analyses of molecular evolution have suggested that
natural selection has played important roles in the evolu-
tion of the insect chemoreceptor gene family and may be
associated with the adaptation and speciation of the insects.
For example, a high level of differentiation in chemore-
ceptor genes is observed among host races of the pea aphid,
Acyrthosiphon pisum. A test of positive selection con-
ducted related to the lineage-specific expansion Or and Gr
genes revealed multiple positively selected sites (Smadja
et al. 2009, 2012). In Drosophila spp., host-specific and
endemic species show higher rates of Gr gene loss, and
their orthologous genes (both Or and Gr genes) have
evolved under relaxed purifying selection (McBride and
Arguello 2007; Gardiner et al. 2008; Guo and Kim 2007).
Here, we report an analysis of the molecular evolution of
the lepidopteran Or and Gr gene family using recently
identified genes. We discussed how our findings shed light on
the adaptation and speciation of the lepidopteran insects.
Materials and Methods
Identification of Plutella xylostella Or and Gr Genes
You et al. (2013) identified 79 Or and 25 Gr genes from the
reference genome of Plutella xylostella. We manually
annotated these gene families again trying to discover some
genes that might be missing from a previous study, espe-
cially the Gr genes. In brief, we conducted tBLASTn
searches iteratively against the P. xylostella genome (ver-
sion 2) (http://iae.fafu.edu.cn/DBM/blast.php) using B.
mori and H. melpomene Or and Gr genes as input
sequences (Tanaka et al. 2009; Wanner and Robertson
2008; Heliconius genome consortium 2012; Briscoe et al.
2013). Multiple hits were identified and many of which
were new genes. We used GeneWise (http://www.ebi.ac.
uk/Tools/Wise2/) to predict intron–exon boundaries in our
gene models (input protein sequences = exons identified in
the tBLASTn searches; input DNA sequences = scaffold
nucleotides). We aligned translated protein sequences from
J Mol Evol (2014) 79:21–39 23
123
our new gene models with their orthologous proteins from
B. mori and H. melpomene and re-check for possible errors.
Regions that do not show any conserved residues were
corrected by finding sequences containing conserved resi-
dues from other translational frame. The P. xylostella Gr
genes were defined by the conserved C-terminus motif
TYhhhhhQF, where h is any hydrophobic amino acid,
which is shared among the insect Gr genes (Robertson
et al. 2003).
Odorant and Gustatory Receptor Sequences
We studied Or and Gr gene family from all lepidopteran
species that have been identified by the time that we started
the analyses. However, we did not include species that
have only their sex pheromone receptor genes identified
such as Ostrinia spp., Mythimna separata, Epiphyas post-
vittana, and Diaphania indica (Sakurai et al. 2011).
To examine the phylogenetic relationships of the lepi-
dopteran insect odorant receptors, the amino acid sequences
of 423 ORs (including the conserved olfactory coreceptors)
that have previously been identified from 8 lepidopteran
species (discarding sequences shorter than 100 amino acids
because they are gene fragments that cannot be confidently
aligned with other receptors) were included in the analysis:
65 BmORs (B. mori—Tanaka et al. 2009), 39 CpomORs (C.
pomonella—Bengtsson et al. 2012), 64 MsORs (M. sexta—
Grosse-Wilde et al. 2011), 20 HvORs (H. virescens—Krie-
ger et al. 2004), 25 SlitORs (S. littoralis—Legeai et al. 2011),
52 DpORs (D. plexippus—Zhan et al. 2011), 92 PxORs (P.
xylostella—this study), and 65 HmORs (H. melpomene—
Heliconius genome consortium 2012). However, we edited
the gene models of some DpOr genes (14 of the 64 genes
identified Supplementary data 1) to cover the missing N and/
or C termini of the translated sequences and to correct
reading frameshifts in some exons of the gene models, such
that the alignment of the receptor proteins was significantly
improved. A phylogenetic tree of the lepidopteran gustatory
receptors was generated from 66 BmGR, 42 DpGR, 64
PxGR, and 73 HmGR protein sequences (Wanner and
Robertson 2008; Zhan et al. 2011; Briscoe et al. 2013),
excluding protein fragments similar to a phylogenetic ana-
lysis of lepidopteran ORs.
For the analysis of the molecular evolution of the sex
pheromone receptors, 19 sex pheromone receptors from 3
moth species (B. mori, H. virescens and M. sexta) and the
monarch butterfly (D. plexippus) were analyzed. We chose
only the sex pheromone receptors with known chromosomal
locations so that their evolutionary history (e.g., gene
duplication and translocation) could be studied. The loca-
tions of BmOr2, BmOr3, BmOr4, BmOr5, BmOr7, and
BmOr9 (B. mori) were identified using the SilkMap tool from
the Silkworm Genome Database: SilkDB (www.silkdb.org/
silksoft/silkmap.html). We also extended the search for the
entire family of BmOr and BmGr genes and constructed a
map on the chromosomes of B. mori. The location of DpOr1b
was identified using the BLAST tool in the latest genome
assembly (v3) from MonarchBase (http://monarchbase.
umassmed.edu/blast.html). HvOr6, HvOr14, HvOr15, and
HvOr16 are located on H. virescens chromosome 27,
whereas HvOr11 and HvOr13 are located on chromosome 3
and the Z sex chromosome, respectively, according to Gould
et al. (2010). OnubOr5a, OnubOr5c, OnubOr7a, and Onu-
bOr8 are located on the O. nubilaris Z chromosome, whereas
OnubOr1 and OnubOr3 are located in a cluster on an
unknown autosomal chromosome (Yasukochi et al. 2011).
Intron Analysis
We used intron structures as characters to investigate
evolution of these gene families. We studied introns
structures of Or and Gr genes from B. mori and D.
plexippus as they have genomic tool for determining intron
structures. Locations and phases of introns in the BmOr,
DpOr, and DpGr genes were determined using GeneWise
(http://www.ebi.ac.uk/Tools/Wise2/). Results were com-
pared with previous studies (Robertson et al. 2003; Wanner
and Robertson 2008; Kent and Robertson 2009).
Phylogenetic Analyses
The amino acid sequences of 423 ORs (or 255 GRs) were
aligned using MUSCLE (http://www.ebi.ac.uk/Tools/msa/
muscle), with some manual adjustments being performed
using BioEdit (http://www.mbio.ncsu.edu/bioedit/bioedit.
html). Sites with more than 90 % gaps were excluded from
the analysis. Phylogenetic analyses were conducted using
three methods. The maximum likelihood method was
performed with the Jones, Taylor, Thornton (JTT) substi-
tution model and a gamma distribution using PhyML
(http://www.atgc-montpellier.fr/phyml/) (Guindon et al.
2010). The distance method was applied using PROTDIST
in the PHYLIP 3.69 package (Felsenstein 1989), and a
neighbor-joining tree was generated. Bayesian analysis was
conducted using MrBayes v3.1 (Ronquist and Huelsenbeck
2003) with the JTT substitution model, four chains, one
million generations, and two runs. Trees were sampled
every 100 generations, discarding a burn-in of 250,000
generations. Branch supports were calculated using the
approximate likelihood ratio test (aLRT) for the ML tree,
500 uncorrected distance bootstrap replications for the NJ
tree (1,000 replications for Gr tree) and Bayesian posterior
probabilities (PPS) for the Bayesian tree. The ML tree was
presented using the tree-visualizing program, FigTree
(http://tree.bio.ed.ac.uk/software/figtree/). The support for
major branches is shown only on the branches that existed
24 J Mol Evol (2014) 79:21–39
123
in all trees generated via the three methods. The branch
supports (at least 2 out of the 3) must also be considered as
highly confident, i.e.,[70 % for the bootstrap analysis and
[90 % for aLRT and PPS. The Or tree was rooted with the
outgroup (6 conserved olfactory coreceptor genes), while
the Gr tree was mid-point rooted.
The phylogenetic tree for the sex pheromone receptors
was generated in a similar manner. The amino acid
sequences of 19 sex pheromone receptors described pre-
viously and 4 conserved coreceptors (Dple\Orco, Bmor\-
Orco, Hvir\Orco and Onub\Orco) were aligned using
MUSCLE. The ML tree was produced using PhyML with
the JTT model and a gamma distribution. Branch supports
were obtained using 1,000 bootstrap replications and are
shown on the nodes if the values were above 68 %. The
tree was rooted with 4 conserved olfactory coreceptors.
Estimation of Gene Gains and Losses
We used BadiRate (Librado et al. 2012) to estimate the
number of Or and Gr gene gains and losses during the
evolution of Lepidopteran insects. We conducted the ana-
lysis only on 4 lepidopteran species, B. mori, P. xylosstella,
H. melpomene, and D. plexippus, because the estimation
required complete sets of the gene families which are only
available in species that have genome databases.
We inferred the Or (or Gr) orthologous groups based on
reciprocal best hits within and between gene family of each
lepidopteran species using the OrthoMCL software (infla-
tion of 1.5 and e value threshold of 10-5) (Li et al. 2003).
These data were used to construct a family size file [a
matrix (column = species, row = orthologous group)
showing the number of genes from each lepidopteran
species found in each orthologous group]. The phyloge-
netic tree of the 4 lepidopteran species with branch lengths
reflecting divergence times was inferred from Wahlberg
et al. (2013). We used the same method (BDI-FR-CML)
that was applied to estimate the odorant-binding protein
and chemosensory protein genes gains and losses (Vieira
and Rozas 2011). Briefly, for each orthologous group, the
number of genes in each internal node was inferred using
those numbers in extant species and the phylogenetic
branch lengths. Gene gains and losses in each phylogenetic
branch were then estimated using maximum likelihood
under the BDI stochastic model (Hahn et al. 2005)
assuming that each branch has its specific turnover rates.
The estimation for Gr genes was performed in a similar
manner to that of the Or genes.
Molecular Evolution Analysis
Two models in the CodeML program from PAML package
version 4.6 (Yang 2007) were employed to study the
different types of selective pressures acting on the evolu-
tion of the lepidopteran Or and Gr genes. The ‘‘site-specific
model’’ was applied to the orthologous/paralogous genes
(Figs. 1, 2) to test whether there has been variation of
selective pressures at different amino acid sites and which
sites might have evolved under positive selection. The
‘‘branch-site’’ model was applied specifically to the evo-
lution of the sex pheromone receptors to test whether there
are amino acid sites that evolved under positive selection in
a specific sex pheromone receptor lineage. To prepare data
for these analyses, alignments of DNA sequences on the
branch of interest were prepared using their protein align-
ment as a guide with the PAL2NAL program (http://www.
bork.embl.de/pal2nal/) (see Supplementary data 2 for
example of the protein alignment). The maximum likeli-
hood trees of these DNA alignments were built using
PhyML under default parameters for DNA trees.
The program estimated the ratio of the normalized
nonsynonymous (dN) to the synonymous (dS) substitution
rate via the maximum likelihood method. The x value
infers the mode of evolution, with x[ 1 being considered
evidence of positive selection for amino acid replacement,
whereas x\ 1 indicates purifying selection, and x = 1
indicates neutral selection. The details of the models (M0,
M3, M7, M8, M8a) employed under the ‘‘site-specific
model’’ were described in previous reports (Yang and
Nielsen 2002; Yang et al. 2000; Swanson et al. 2003).
Briefly, M0 uses one x ratio for all sites, and M3 includes
three classes of sites with a different x ratio for each site
class. If the data fit model M3 better than M0, it indicates
the variation of selective pressures on the sites within
receptor proteins. M7 includes eight classes of sites with
eight x ratios in the range of 0–1, taken from the beta
distribution (‘‘beta’’ neutral model). M8 is similar to M7
but uses an additional site class, with x ratios varying from
0 to[1. If the data fit M8 significantly better than the M7,
there is evidence that some sites in the receptor proteins
evolved under positive selection. The M8a model (similar
to M8, but with x1 is fixed at 1) was also compared to M8
to reduce false-positive detection because this test is con-
sidered more stringent than M8 vs. M7. We conducted the
M7 vs. M8 test only on the clades with mean dS value from
all branches in the range of 0.5 \ dS \ 1 to avoid satura-
tion of dS because CodeML is powerful and reliable for
detecting positive selection at this sequence divergence
(Yang 2007; Anisimova et al. 2001).
The phylogenetic relationships and chromosomal loca-
tions of the sex pheromone receptors revealed that inde-
pendent gene duplication events had occurred in different
chromosomal regions in each moth species. We labeled the
branch of interest as the foreground branch and the
remaining branches as background branches (Fig. 6). To
test whether positive selection had acted on these newly
J Mol Evol (2014) 79:21–39 25
123
duplicated genes, we implemented a ‘‘branch-site model’’
to explore changes in x for a set of sites on a foreground
branch. The alternative model in which some sites on the
foreground branch were allowed to change to a value of
x[ 1 was compared with the null model of neutral evo-
lution. Likelihood ratio tests (LRTs) were used for com-
parisons between models, and significant results were
determined using v2 tests.
In cases where LRT was significant, the Bayes empirical
Bayes (BEB) procedure was used to identify sites of
positive selection within the amino acid sequences (Yang
et al. 2005). The sites of positive selection were then
mapped onto the receptor topology predicted by
TOPCONS (http://topcons.cbr.su.se). Diagrams represent-
ing the 2D structures of the odorant receptors were gen-
erated using TOPO2 (http://www.sacs.ucsf.edu/TOPO2).
Results
Plutella xylostella Or and Gr Gene Family
You et al. (2013) previously identified 79 Or and 25 Gr
genes in Plutella xylostella. We performed multiple itera-
tions of tBlastn searches and discovered additional Or and
Gr genes (also correcting some former gene models),
Fig. 1 Phylogenetic relationships of the olfactory receptor genes
from 8 lepidopteran species: Bombyx mori (BmOR), Heliothis
virescens (HvOR), Manduca sexta (MsOR), Cydia pomonella
(CpomOR), Spodoptera littoralis (accession number), Danaus plexip-
pus (DpOR), Heloconius melpomene (HmOR), and Plutella xylostella
(PxOR). Trees were constructed from amino acid sequences using the
maximum likelihood, Bayesian analysis, and neighbor-joining meth-
ods; the Bayesian analysis tree is shown here. The tree was rooted
with conserved olfactory coreceptor (Orco) genes. A test of positive
selection was conducted on the lineage highlighted in gray. Branch
supports are shown on each node (a, b, c: a = aLRT, b = 10,000
quartet maximum likelihood puzzling steps and c = 500 bootstrap
replications; all shown as a %), but only if [90 % for the aLRT and
quartet maximum likelihood puzzling steps and [70 % for the
bootstrap support analysis, and their topologies obtained from all
three building methods must be in agreement. Possible orthologous
and paralogous groups discussed in the text are highlighted in gray
26 J Mol Evol (2014) 79:21–39
123
resulting in a total of 95 PxOr genes and 69 PxGr genes
(Supplementary data 1). Our gene models include intact
genes (sequences containing start and stop codons with all
exons intact), partial genes (missing some exons or the 50
and 30 terminus), and some pseudogenes (containing a stop
codon or frameshift mutation in the sequence). Among the
95 PxOr genes, there were 67 intact genes, 26 partial
genes, and 2 pseudogenes, whereas among the 69 PxGr
genes, there were 43 intact genes, 21 partial genes, and 5
pseudogenes. The average lengths of P. xylostella odorant
and gustatory receptor proteins (from intact genes) are 393
and 387 amino acids, respectively. Most of the identified
genes were found as singletons on each DNA scaffold.
However, multiple tandem gene duplications were also
observed in both Or and Gr genes, e.g., 15 genes (PxOr66–
PxOr80) on Scaffold 402 spanning a 71 kb region, 13
Fig. 2 Phylogenetic relationships of the gustatory receptor genes
from Bombyx mori (BmGR), Plutella xylostella (PxGR), Heliconius
melpomene (HmGR), and Danaus plexippus (DpGR). Trees were
constructed from amino acid sequences using the maximum likeli-
hood, Bayesian analysis, and neighbor-joining methods; the Bayesian
analysis tree is shown here. The tree was rooted at the mid-point. A
test of positive selection was conducted on the lineage highlighted in
gray, including putative CO2 receptor and sugar receptor clades.
Branch supports are shown using the same method as in Fig. 1 but
c = 1000 bootstrap replications
J Mol Evol (2014) 79:21–39 27
123
genes (PxGr17–PxGr29) on scaffold 45 spanning a 54 kb
region, 8 genes (PxOr28–PxOr35) on scaffold 156 span-
ning a 52 kb region, and 7 genes (PxGr10–PxGr16) on
scaffold 74 spanning a 49 kb regions.
Phylogenetic Analyses
The lepidopteran Or and Gr sequences were observed to be
highly divergent, sharing sequence identities ranging from
\10 to [95 %. However, the phylogenetic tree of the
lepidopteran ORs (Fig. 1) revealed many orthologous
groups shared among 3–8 species, including the highly
conserved olfactory coreceptor Orco genes and the female
sex pheromone receptor genes that are specifically
expressed in the male antennae. Most of the orthologous
groups contained genes from each species in nearly a 1-to-
1 ratio, suggesting that the lineage-specific expansion of
these lepidopteran Or genes was constrained by purifying
selection. On the other hand, genes from P. xylostella
showed lineage-specific expansion, e.g., Group 8 (14
genes) and Group 9 (22 genes).
Monophyletic relationship of female sex pheromone
receptor genes was formerly reported in previous studies
(e.g., Miura et al. 2009; Tanaka et al. 2009; Yasukochi
et al. 2011; Grosse-Wilde et al. 2011). This clade in our
tree, however, has low branch support. Interestingly, 22
lineage-specific PxOr genes (Group 9) are in this clade.
According to Sun et al. (2013), some of these genes
(PxOr13, PxOr46, PxOr47, PxOr48) were formally iden-
tified as female sex pheromone receptors (PxylOr6,
PxylOr5, PxylPr3, PxylOr4, respectively). Future func-
tional analysis will be essential to confirm if other genes in
this clade also function as sex pheromone receptors in
P. xylostella. These orthologous/paralogous relationships
could be considered confident as inferred from the obtained
branch support. However, the deeper relationships between
each group showed low branch support in most cases (or
were not in agreement between the different phylogenetic
methods), suggesting that the divergence of each ortholo-
gous group might have occurred long ago and predated the
common ancestor of these Lepidoptera.
Many orthologous groups of Gr genes shared between
B. mori, P. xylostella, H. melpomene, and D. plexippus
were also observed in the phylogenetic tree of lepidopteran
Gr genes (Fig. 2). Genes in some orthologous groups
showing a high degree of sequence homology may share
the same function, such as serving as CO2, sugar, and
fructose receptors. We observed more cases of lineage-
specific expansion ([5 genes) in the lepidopteran Gr genes,
especially in the putative bitter receptor clades such as
Group 8 (12 PxGrs), Group 10 (8 PxGrs), and Group 14
(10 HmGrs). Thus, it is possible that the purifying selection
acting on the lineage-specific expansion of Gr genes might
be weaker compared to that on Or genes. However, if
genes from additional lepidopteran species were to be
included in the analysis, more lineage-specific expansion
might be found (similar to what was described in P. xy-
lostella), and the differences between Or and Gr genes
could be less pronounced.
Gene Duplication and Chromosomal Map
Gene duplication by means of unequal crossing over results
in tandem gene repeats or clusters of related Or genes on
chromosomes (Robertson and Wanner 2006). To obtain a
better understanding of gene family expansion in Lepi-
doptera, the locations of BmOr and BmGr genes on the
chromosomes of B. mori were investigated. We found that
the chromosomal locations of the genes were correlated
with the information deduced from the phylogenetic ana-
lysis. The genes were distributed across almost all 28
chromosomes, excluding chromosomes 2, 4, 11, 14, and 24
(Fig. 3), suggesting that these genes diverged quite some
time ago. We defined a group of genes located next to each
other within a 106 base pairs (100 Kb) region as a gene
cluster. Genes in each cluster shared common ancestor as
seen from the phylogenetic tree. Most of the gene locations
were singletons (67 and 58 % for BmOr and BmGr,
respectively). The largest expansion of the BmOr genes in a
cluster included only 4 genes, followed by 3 and 2 genes
(found 4, 4, and 7 times, respectively), whereas that of the
BmGr genes included 9 genes, followed by 8, 5, 4, 3, and 2
genes (found 1, 1, 1, 3, 1, and 4 times, respectively;
Fig. 3b). This result is similar to the Or and Gr map on the
chromosomes of H. melpomene, where these genes are also
distributed as singletons and small clusters of genes
(Briscoe et al. 2013).
Estimation of Gene Gains and Losses
We further investigated how the Or and Gr genes might
have been gained and lost during the evolution of B. mori,
P. xylostella, D. plexippus, and H. melpomene. The num-
bers of Or (or Gr) genes on each node represent the esti-
mated number of genes in the common ancestors, and the
Fig. 3 Distribution of the chemoreceptor genes on the chromosomes
of B. mori. a Locations of the BmOr and BmGr genes on the
chromosome linkages (Chr. 1–28). The map was constructed using
the SilkMap tool from the Silkworm Genome Database: SilkDB
(www.silkdb.org/silksoft/silkmap.html), which determined the posi-
tion of each gene as the number of base pairs from the p telomere. The
chromosomal representations were adapted from SilkDB. A gene
cluster was defined as a group of genes whose members are located
within 105 base pairs of the adjacent genes. b The graph shows the
number of cases in which gene clusters containing different numbers
of genes were found on the map
c
28 J Mol Evol (2014) 79:21–39
123
J Mol Evol (2014) 79:21–39 29
123
numbers of gene gains and losses are shown on the bran-
ches (upper and lower, respectively) (Fig. 4). A similar
trend was observed for both Or and Gr gene evolution. The
common ancestor of these Lepidoptera might have fewer
numbers of genes: 47 Or and 13 Gr genes (&66 and
&20 % of average number of Or and Gr genes in extant
species, respectively). New genes then arose over time,
with only a few genes being lost. Multiple gene gains were
observed on the branches leading to each lepidopteran
species, e.g., 26 and 39 Gr genes on the branches leading to
D. plexippus and H. melpomene, respectively. The greatest
number of gene gains was observed on the branch from the
most common ancestor to P. xylostella (54 and 50 for Or
and Gr genes, respectively) possibly because it showed the
longest divergence time. However, fewer gene gains and
losses were observed on the branches from the common
ancestor to the more recent common ancestors. This is
most likely due to their short divergence times.
Analysis of Intron Evolution
Differences in intron patterns provide important informa-
tion on the evolution of gene families. Here, we observed
multiple changes in the patterns of the introns situated in
lepidopteran chemoreceptor genes. The Or genes from B.
mori and D. plexippus shared introns 1, 2, 3, and 4 near the
30 end as shown in Fig. 5 (phase 2, 0, 0, and 0, respec-
tively). These observed introns have also been reported in
the D. melanogaster and T. castaneum Or genes (Robert-
son et al. 2003; unpublished result). These introns might
have been shared since the origin of insect odorant receptor
gene family. We found introns (A–F) which were shared
between BmOr and DpOr genes and multiple idiosyncratic
introns that were independently gained or lost in the first
half region of the genes.
As presented in Fig. 5 (top bar), shared introns #1, #2,
and #3 (phase 0, 0 and 1, respectively) were found in most
DpGr genes in the putative bitter receptor clade similar to
what have been reported in 55 bitter receptor genes in B.
mori (Wanner and Robertson 2008). Interestingly, these
introns are not present in the Gr genes of D. melanogaster
and T. castaneum (Wanner and Robertson 2008). In con-
trast, two shared phase 0 introns near the C terminus in
gustatory receptor genes of D. melanogaster, A. mellifera,
and T. castaneum (corresponding to shared introns called
#2 and #3 in Robertson et al. (2003) and r and s in Kent and
Robertson (2009)) were missing in all of these lepidopteran
Gr genes in the bitter receptor clade but still retained in
sugar receptor genes of D. plexippus and B. mori (intron t
and u as shown in Fig. 5, top bar). From this information, it
could be assumed that the common ancestor of Lepidoptera
had sugar receptor genes that shared introns structures with
other insect gustatory receptor genes, but their bitter
receptor genes had intron structure that is unique to Lepi-
dopteran lineage.
There is a clade of intronless Gr genes (Fig. 2, Group 8)
with members from all 4 lepidopteran species (PxGr17-
PxGr29, HmGr22-HmGr26, HmGr53, BmGr53, and
DpGr33), suggesting that the common ancestor of these
intronless genes was found in the common ancestor of
these Lepidoptera, possibly due to the retrotransposition
and that there has been independent lineage-specific
expansion in the lineage specific to H. melpomene and P.
xylostella.
Selective Pressures on the Lepidopteran Or and Gr
Genes
We calculated the ratio of normalized nonsynonymous to
synonymous substitution rates (x, or dN/dS) for the protein-
coding sequences from different gene clades. Among the
Or genes, the conserved Orco gene clade (Group 1), PxOr
lineage-specific expansion clade (Group 9) and multiple
orthologous/paralogous groups (Group 2–8) were tested
(Fig. 1). For the Gr genes, possible orthologous/paralogous
groups (Group 1–14) including putative sugar and CO2
receptor clades were studied (Fig. 2). The results are shown
in Table 1. In general, the dN/dS ratios estimated from
model M0 (assuming the same selective pressures on all
Fig. 4 Estimation of Or and Gr gene gains and losses during the
evolution of lepidopteran species. The phylogenetic tree with
estimated divergence times (million year ago) was inferred from
Wahlberg et al. (2013). The numbers at the tree termini are the
numbers of genes found in each species, and the numbers at the tree
nodes are the numbers of genes in their most recent common
ancestors. The numbers of gene gains and losses are shown above and
below the branches, respectively
30 J Mol Evol (2014) 79:21–39
123
amino acid sites) were low in all clades, ranging from
0.0093 to 0.3926, suggesting the existence of strong puri-
fying selection. However, the comparison between models
M0 and M3 provided strong evidence of variation in
selective pressures at different amino acid sites in all Or
and Gr clades (P values \10-4), indicating that purifying
selection has been relaxed at some amino acid sites. We
further compared models M7 and M8 for clades that
showing 0.5 \ dS \ 1 to investigate whether some amino
acid positions actually evolved under positive selection.
Only Group 8 of the Or genes presented evidence of
positive selection (P = 0.004) with 4 positively selected
sites (PSSs). For the Gr genes, there were 3 groups
showing some evidence of positive selection (Group 5,
Group 12, and Group 14; P = 0.001, 0.0004 and
2.4 9 10-5, respectively) with 16, 8, and 11 PSSs,
respectively. Most of these PSSs only show 50 % PPS
confidence in the BEB procedure. Thus, they have weak
statistical support. The tests between two models (M8 vs.
M8a) resulted in a corrected P value of 1 for all groups,
suggesting a lack of power in detecting positive selection,
and we did not report the results here.
Strong purifying selection is expected to have occurred
during the evolution of sex pheromone receptors because
mutations in these receptor sequences could decrease male
performance in detecting the call of female sex phero-
mones. However, purifying selection alone cannot explain
the vast diversity of lepidopteran sex pheromones. We
constructed a phylogenetic tree of sex pheromone receptors
using known chromosomal regions from 4 lepidopteran
species (Fig. 6). In B. mori, H. virescens and O. nubilaris,
pheromone receptor genes are found on both the sex
chromosomes and autosomal chromosomes (Gould et al.
2010; Yasukochi et al. 2011). Z chromosomes are highly
conserved among Lepidoptera, possibly dating back to the
common ancestor of Lepidoptera and Trichoptera (Sahara
et al. 2012). However, the orthologous genes on the sex
(Z) chromosomes were not grouped on the same phylo-
genetic branch. This suggests that multiple gene translo-
cations have occurred between sex and autosomal
chromosomes, resulting in the complex relationships
between genes and chromosomal locations. Gene duplica-
tions in species-specific lineages were observed for all
species (e.g., three OnubOr genes on the sex chromo-
somes—branch #3, and four HvOr genes on chromosome
27—branch #7). These findings suggest that multiple
molecular evolutionary processes have operated on the
pheromone receptor genes. We used a ‘‘branch-site-spe-
cific’’ model to estimate the number of positively selected
sites in a specific lineage (branches #1–#7). A number of
positively selected sites (PSSs) were detected on all of the
tested branches (Table 2). However, only branches #3, #2,
and #7 presented significant results after Bonferroni cor-
rection (P = 1.7 9 10-4, 0.0039 and 0.0022, respec-
tively). To avoid false-positive detection, we only focused
on the result showing strong statistical support, i.e., that for
branch #3. Approximately 12 % (50/422, PPs = 50 %) of
the full-length receptor protein evolved under positive
selection. However, this percentage was reduced to 3 %
(12/422) or 0.5 % (2/422) when considering the sites with
higher statistical confident (PPs = 90 and 95 %,
respectively).
The predicted PSSs were plotted onto the predicted
topology of the odorant receptor proteins (Fig. 7). The pro-
portion of PSSs among the amino acid residues was the
highest in the extracellular region (&19 %, 12/63), followed
Fig. 5 Approximate locations of introns (above the lines) and their
phases (below the lines) in the B. mori and D. plexippus olfactory
receptor genes and D. plexippus gustatory receptor genes (BmOR,
DpOR, and DpGR, respectively). Their positions are shown relative
to a scale of the average receptor protein size (number of amino
acids), excluding the large insertions or deletions in some receptors.
The ancestral introns near the 30 end that are shared among most of
the Or genes (intron 1, 2, 3, and 4) are shown (*). The black arrows
indicate other introns (A–F) shared between the Or genes from two
lepidopteran species. The shared introns (#1, #2, and #3) that are
found in most of the DpGr genes are indicated
J Mol Evol (2014) 79:21–39 31
123
by the transmembrane region (&12.4 %, 18/145) and the
intracellular region (&12.10 %, 23/190). These proportions,
however, did not differ significantly from a random
distribution of sites across classes (v2 test, P = 0.35). The 2
PSSs with the highest statistical confidence were located in
outer loop 2 and transmembrane domain 4.
Table 1 Tests of positive selection on the chemoreceptor gene clades
Clade na dN/dSb Mean dS 2Dlc
M0 vs. M3 M7 vs. M8
1. Olfactory receptor gene
Group1 (conserved
olfactory coreceptor)
7 0.02329 0.979 248.594788** (P \ 10-4) N/A
Group2 6 0.06825 1.909 165.4043** (P \ 10-4) N/A
Group3 7 0.01684 7.883 248.983902** (P \ 10-4) N/A
Group4 6 0.11962 1.198 110.811316** (P \ 10-4) N/A
Group5 11 0.08004 1.428 491.296248** (P \ 10-4) N/A
Group6 16 0.09604 1.233 620.58625** (P \ 10-4) N/A
Group7 11 0.01849 7.058 301.295216** (P \ 10-4) N/A
Group8 15 0.16376 0.612 272.08387** (P \ 10-4) 11.1095 (P = 0.004)*
Group9 192 0.16465 0.296 481.16493** (P \ 10-4) N/A
2. Gustatory receptor genes
Group1 (putative
sugar receptor clade)
5 0.0546 2.416 877.337606** (P \ 10-4) N/A
Group2 (putative
CO2 receptor clade)
12 0.0093 7.689 160.145058** (P \ 10-4) N/A
Group3 9 0.31429 0.410 137.811492** (P \ 10-4) N/A
Group4 6 0.1128 2.207 129.09214** (P \ 10-4) N/A
Group5 6 0.31636 0.622 310.776922** (P \ 10-4) 13.798978 (P = 0.001)*
Group6 12 0.28885 0.828 208.804808** (P \ 10-4) 3.022322 (P = 0.221)
Group7 14 0.18964 1.001 461.339978** (P \ 10-4) N/A
Group8 19 0.16928 0.762 469.033396** (P \ 10-4) 3.455604 (P = 0.178)
Group9 11 0.2729 0.570 138.05415** (P \ 10-4) 3.159042 (P = 0.206)
Group10 8 0.39265 0.318 156.0981** (P \ 10-4) N/A
Group11 9 0.21201 0.738 225.363638** (P \ 10-4) 1.773724 (P = 0.412)
Group12 6 0.23433 0.664 207.60114** (P \ 10-4) 15.600984 (P = 0.0004)*
Group13 6 0.2222 0.775 130.356838** (P \ 10-4) 0
Group14 9 0.27944 0.634 248.795312** (P \ 10-4) 21.24359 (P = 2.4E - 05)**
Genes Parameter estimated under M8 model Positively selected sites (PSSs) from Bayes
empirical Bayes (BEB) analysis
Or genes Group8 p0 = 0.97817, p = 1.34291, q = 6.40687,
p1 = 0.02183, x = 1.44346
106K 117I 131Q 228Q
Gr genes Group5 p0 = 0.99999, p = 0.45627, q = 0.47348,
p1 = 0.00001, x = 1.00000
1A 48Y 51V 52L 55G 58V 124S 128F 133S 137A
146H 176S 204D 218V 229S 257H
Group12 p0 = 0.93418, p = 0.75665, q = 1.99983,
p1 = 0.06582, x = 5.30838
56M 66L 77P 80L 85L 150G 152D 156V
Group14 p0 = 0.79341, p = 1.65218, q = 5.59969,
p1 = 0.20659, x = 1.16918
51K 53E 78E 126F 127F 130R 131H 134V 206T
208L 212I
Log likelihood values and parameters were estimated under different site models. PSSs in bold show 90 % posterior probability confidence.
Other PSSs show 50 % posterior probability confidence
N/A not tested because mean dS [ 1 or \ 0.5
* Significant within the 1 % interval after Bonferroni correction; **Significant within the 0.1 % interval after Bonferroni correctiona Number of genes testedb dN/dS estimated under M0c Likelihood ratio test
32 J Mol Evol (2014) 79:21–39
123
Discussion
Tandem Gene Duplication and Size of the Lepidoptera
Or and Gr Gene Family
The numbers of Or genes identified in 8 species of Lepi-
doptera (21–96 genes) were relatively low compared to
other insect orders. For example, 157–400 Or genes are
found in Hymenoptera (Wanner et al. 2007; Smith et al.
2011a, b; Robertson et al. 2010), 57–340 in Coleoptera
(Engsontia et al. 2008; Mitchell et al. 2012) and 60–158 in
Diptera (Nozawa and Nei 2007; Pelletier et al. 2010). The
number of glomeruli reported in lepidopteran species ran-
ges from 59 to 67 (e.g., see the review in Schachtner et al.
2005). Because the number of Or genes per glomerulus is
generally accepted to show an approximately 1:1 ratio
(Vosshall et al. 2000; Gao et al. 2000; Ramaekers et al.
2005), it might be plausible to assume that the number of
Or genes estimated for each lepidopteran species would
also be approximately 60–70. It is important to note,
however, that this assumption might not hold true if more
Lepidopteran species are investigated (as seen in
P. xylostella).
The relatively small number of Or genes found in
Lepidoptera cannot be explained by the genome sizes of
these species. In Drosophila spp., the number of Or genes
is positively correlated with genome size (Gardiner et al.
2008), though this is not true when data from other insects
are included. The ants Linepithema humile and Pogono-
myrmex barbatus and the wasp Nasonia vitripennis show a
much larger number of Or genes than B. mori (300–400 vs.
68 genes), although their genome sizes are smaller
(250–300 vs. 450 Mb) (Smith et al. 2011a, b).
Repeated tandem gene duplications are responsible for
the great expansion of the Or gene family as reported in
insects which have large Or gene family, e.g., a tandem
array of 60 Or genes in A. mellifera (Robertson and
Wanner 2006). The chromosomal map of BmOr and BmGr
genes showed that only a few clusters of genes (containing
more than 5 genes) have arisen from tandem gene dupli-
cation, similar to the results obtained in H. melpomene
(Briscoe et al. 2013). However, we observed more cases of
Fig. 6 Phylogenetic relationships of female sex pheromone receptor
genes in relation to their chromosomal location. The maximum
likelihood tree was constructed from the amino acid sequences of the
19 female sex pheromone receptor genes from 4 lepidopteran species
(DpOR: D. plexippus, BmOR: B. mori, HvOR: H. virescens, and
OnubOR: O. nubilaris) and 4 conserved olfactory coreceptor genes
which were used for rooting. Branch supports higher than 68 %
(1,000 bootstrap replications) are shown on the nodes. The location
and topological order of the Or genes on either the sex (Z) chromo-
somes (black bars) or autosomal chromosomes (white bars) for HvOR
and OnubOR were based on previous studies (Gould et al. 2010 and
Yasukochi et al. 2011, respectively), whereas those for BmOR and
DpOR were identified from genome databases. Independent gene
duplication events in different chromosomal regions that occurred
during the evolution of the female sex pheromone receptors genes are
highlighted with numbers (#1–#7)
J Mol Evol (2014) 79:21–39 33
123
lineage-specific expansion ([5 genes) in the lepidopteran
Gr genes from the phylogenetic analyses (Fig. 2).
These data suggest that there were selective pressures
limiting the expansion of the lepidopteran Or gene family,
possibly after the number of genes reached an optimum
number. It has been suggested that in insects, newly
duplicated Or genes might have difficulty finding space for
expression because gene regulation is precise and strict
(Ramdya and Benton 2010). This might also be the case for
the Lepidoptera as there is evidence of differential
expression between sexes, developmental stages, sensilla
types, and olfactory neurons for B. mori Or genes (Sakurai
et al. 2004; Wanner et al. 2007; Anderson et al. 2009;
Tanaka et al. 2009). Factors that control the development
of the lepidopteran olfactory sensilla, such as proneural
genes, might also be involved in the limited number of
olfactory receptor neurons and glomeruli, which in turn
limits the number of Or genes. However, Gr genes show
broader expression sites (e.g., in the antennae, mouthparts,
legs, wings, and ovipositors), suggesting that regulatory
control of the expression of Gr genes is less strict, which
might be the reason that lineage-specific expansion is
greater in the lepidopteran Gr genes compared to the Or
genes.
Table 2 Test of positive selection (site and branch-site models): Moth sex pheromone receptor genes (Branch numbers (#1–#7) referring to
Fig. 6)
Site model na dN/dSb Mean dS 2Dlc
M0 vs. M3 M7 vs. M8
Lepidopteran sex pheromone receptor genes 19 0.1818 0.673 1075.64304** (P \ 10-4) 2.041478 (P = 0.360)
Branch-site
model
#Branch
H0 lnL
versus
H1 lnL
df 2Dlc and
P value
Parameter Estimated
under H1
Positively Selected Sites (PSSs)
#1 -21,871.33
-21,868.72
1 5.22
P = 0.0223
p0 = 0.633, p1 = 0.350,
p2a = 0.011, p2b = 0.006,
x1 = 1.000, x2 = 224.021
207Y 235C
#2 -21,867.09
-21,862.93
1 8.32
P = 0.0039**
p0 = 0.595, p1 = 0.324,
p2a = 0.052, p2b = 0.028,
x1 = 1.000, x2 = 79.879
31R 34M 98I 157Y 159I 160F 171 N 204A 216D
240C 326T 347C 368P 374Y
#3 -21,855.20
-21,848.17
1 14.06
P = 1.7 9 10-4
p0 = 0.509, p1 = 0.281,
p2a = 0.136, p2b = 0.075,
x1 = 1.000, x2 = 89.283
14L 17R 18E 23A 27F 37P 65A 98I 103L 104G 106L
109I 115Q 130V 134M 140G 141P 143Y 161A 168A
181V 184V 203F 205L 208K 211G 216D 217P 220Y223S 229V 239V 254I 257V 258Y 289E 301D 305N
329L 332L 338A 345L 349S 358G 375G 386M 390E
395P 397S 423Y 424K 426S 438S
#4 -21,870.07
-21,868.09
1 3.96
P = 0.0466
p0 = 0.606, p1 = 0.327,
p2a = 0.044, p2b = 0.024,
x1 = 1.000, x2 = 9.164
17R 109I 144K 155Y 178F 200T 261R 298E 315C 390E
#5 -21,871.37
-21,869.71
1 3.32
P = 0.0684
p0 = 0.634, p1 = 0.345,
p2a = 0.014, p2b = 0.008,
x1 = 1.000, x2 = 223.062
54E 230Y 448F
#6 21,871.24
-21,868.57
1 5.34
P = 0.0208
p0 = 0.626, p1 = 0.340,
p2a = 0.022, p2b = 0.012,
x1 =
94M 160F 224T 413T
#7 -21,870.87
-21,866.17
1 9.4
P = 0.0022**
1.000, x2 = 397.756
p0 = 0.622,
p1 = 0.336,
p2a = 0.028, p2b = 0.015,
x1 = 1.000, x2 = 20.604
28K 88E 100F 224T 303E 405C
PSSs in bold show 90 % posterior probability confidence. PSSs that are underlined show 95 % posterior probability confidence. Other PSSs show
50 % posterior probability confidence
*Significant within the 0.1 % interval after Bonferroni correction; **Significant within the 5 % interval after Bonferroni correction
34 J Mol Evol (2014) 79:21–39
123
Gains and Losses of Lepidopteran Or and Gr Genes
The results obtained through the estimation of gene gains
and losses (Fig. 4) suggest that the common ancestor of
these Lepidoptera might have presented only a few genes
from these families (47 Or and 13 Gr genes) and that size
of the gene families subsequently expanded greatly due to
multiple gene gains and few gene losses during the evo-
lution of these species. The expansion of the Gr gene
family can be readily observed in the expanded putative
bitter receptor clade. An increasing number of genes might
be associated with angiosperm radiation (&100 MYA), as
observed in the large number of gene gains on branches
leading to each lepidopteran species. New Or genes might
facilitate the adaptation of lepidopteran insects to use new
plant odorants as clues for finding food, habitats, and egg-
laying sites, whereas new Gr genes might facilitate the
detection of host plant tastants or toxic secondary metab-
olites for the discrimination of suitable hosts. The number
of lepidopteran chemoreceptor genes that exist today
(53–93 Or genes and 58–73 Gr genes) may remain func-
tionally stable, partly because this number of genes might
be sufficient for detecting a set of odorants or tastants (such
as common plant chemicals) that is essential for survival
and reproduction. Conversely, the expansion of the gene
family might be constrained by the strict gene regulations
as discussed previously.
The pattern of introns in lepidopteran Gr genes supports
the hypothesis that there were few Gr genes in the common
ancestor of Lepidoptera. As mentioned previously, the
lepidopteran Gr genes in bitter receptor clade shared
introns #1, #2, and #3 (Fig. 5), which appeared to be
unique to the Lepidoptera (Wanner and Robertson 2008).
In contrast, the shared introns of insect gustatory receptor
genes were retained in the few sugar receptor genes. A
parsimonious way of explaining this phenomenon is that
the common ancestor of Lepidoptera might have lost most
of its Gr genes in bitter receptor clade with the remaining
genes showing a new intron structure. Host specialization
and endemism are believed to be responsible for the high
rates of Gr gene losses in some Drosophila species (e.g., D.
sechellia and D. erecta) (McBride and Arguello 2007;
Gardiner et al. 2008). A. mellifera has only 10 Gr genes,
which is believed to be due to its nurturing behavior and
mutualism with some plant species (Robertson and Wanner
2006). We therefore hypothesized that the common
ancestor of these Lepidoptera that existed long time ago
before the angiosperm radiation might be a highly host-
specialist species and/or show mutualism with certain plant
species.
Selective Pressures on the Evolution Lepidopteran Or
and Gr Genes
Our evolutionary analysis suggested that both the lepi-
dopteran Or and Gr gene families evolved under strong
purifying selection, though the relaxation of purifying
selection at some amino acid sites has caused divergent
sequences. Mutation at some amino acid sites that are not
involved in ligand-receptor specificity may not alter odor-
ant/gustatory response profile of the receptors. Mutation
could also be functionally redundant as other receptors,
which have similar response profile, can still function.
These processes allow mutant genes to maintain in the gene
pool. Positive selection was detected at very few loci
(1 group for Or gene and 3 groups for Gr genes—Table 1).
Members of these groups are mainly lineage-specific
expansion genes (Group 5: 11 PxGr, Group 12: 6 BmGr,
Group 14: 9 HmGr), suggesting that diversifying selection
on these genes might be associated with chemosensory
response specific to different ecological contexts of each
Lepidopteran species. It is important to note that these
predicted positively selected sites were not discovered
under the more stringent test (M8 vs. M8a), and the pos-
sibility that some, if not all, of these sites resulted from
false-positive detection therefore cannot be excluded.
The monophyletic relationships of the male sex phero-
mone receptors, the conserved amino acid sequences in
Fig. 7 Predicted topology and positively selected sites of the sex
pheromone receptor proteins. The receptor proteins contain 7
transmembrane domains and exhibit an intracellular N terminus,
which are typical characteristics of insect olfactory receptors. Each
circle or square represents an amino acid residue. Gray circles
represent conserved amino acid sites shared among members of the
OR clades. Black squares represent amino acids that evolved under
positive selection (P [ 50 %); * and ** indicate P [ 90 and 95 %,
respectively
J Mol Evol (2014) 79:21–39 35
123
their receptor proteins, and the gene regulatory control of
male-biased expression suggest that sex pheromone com-
munication using sex pheromone receptors is an ancient
phenomenon, present since the common ancestor of Lepi-
doptera. During lepidopteran evolution, multiple changes
occurred within the sex pheromone receptor genes, such as
chromosomal translocation, but their functions have
remained intact. We observed gene duplications in sex-
specific lineage leading to new sex pheromone receptor
genes. Molecular evolutionary analyses suggest that over-
all, the sex pheromone receptors also evolved under strong
negative selection (x = 0.1818, Table 2). However, we
detected multiple signatures of positive selection at some
amino acid sites in newly duplicated genes compared to the
other sex pheromone receptors in the tree (Figs. 6, 7;
Table 2). We hypothesized that positive selection sites on
the sex pheromone receptors may be associated with sex
pheromone response specificity. A recent study supports
our hypothesis. Leary et al. (2012) conducted CodeML
analysis and detected positive selection on some amino
acid sites in the odorant receptors of Asian corn borer moth
(Ostrinia furnacalis). These sites play a crucial role in the
response specificity to Asian corn borer sex pheromone
components, as confirmed through mutagenesis techniques
and ectopic cellular recoding.
How positive selection might operate on the newly
duplicated sex pheromone receptors is still unknown. A
recent study showed that in the wasp N. vitripennis, mutant
individuals that produce new sex pheromone compounds
exist in the population, although the receivers cannot dis-
tinguish them. This finding is important because it suggests
that genes encoding new sex pheromones could persist in
the gene pool long enough for the receivers to evolve the
new pheromone receptors to detect them (Niehuis et al.
2013). Diversifying of new sex pheromone receptors might
be accelerated by genetic drift. Female moths with muta-
tions in their sex pheromone component could become
dominant in a new population due to a founder effect. New
pheromone receptors might arise via gene duplication in
some male moths, and positive selection would favor
changes in receptor sequences allowing the male moths to
detect the new sex pheromones, in turn securing their
reproductive success. The origination of new forms of
pheromonal communication could lead to speciation by
means of species recognition (Smadja and Butlin 2009).
Conclusion
The common ancestor of the Lepidoptera may have har-
bored only a few Or and Gr genes, as demonstrated via the
estimation of gene gains and losses and the interpretation
of the unique pattern of lepidopteran Gr genes. The number
of genes has increased greatly during the evolution of these
Lepidoptera but is still relatively small compared to other
insect groups. This is possibly due to the limited cases of
tandem gene duplication (tandem array of [5 genes).
Newly duplicated genes may have faced the problem of
finding space for expression under strict gene regulatory
control, especially for the Or genes. This might explain
why the lineage-specific expansion of lepidopteran Gr
genes tends to be greater than that of Or genes as the
spatial expression of Gr genes is broader in many sensory
organs. The divergence of receptor sequences is due to the
relaxation of purifying selection on amino acid sites, which
might be a precursor for further positive selection. Positive
selection was detected only on a few loci from lineage-
specific expansion clade which might be associated with
chemosensory response specific to different ecological
contexts of each Lepidopteran species. New lepidopteran
sex pheromone receptors have arisen through gene dupli-
cation. We found signatures of positive selection at some
amino acid sites in sex pheromone receptors, which might
play an important role in the speciation of lepidopteran
species.
Acknowledgments We would like to thank Hugh Robertson from
the University of Illinois, Urbana Champaign, for the BmGr DNA
sequences; Shui Zhan and Stephen Reppert from the University of
Massachusetts Medical School, for the DpOR and DpGR protein
sequences. Fillipe Vieira and Julio Rozas from the University of
Barcelona for suggestion for the use of BadiRate. We thank the editor
and the three anonymous reviewers for giving constructive sugges-
tions for the improvement of this paper. This study was financially
supported by the Graduate School and the Department of Biology,
Faculty of Science, Prince of Songkla University.
References
Albre J, Lienard MA, Sirey TM, Schmidt S, Tooman LK, Carraher C,
Greenwood DR, Lofstedt C, Newcomb RD (2012) Sex phero-
mone evolution is associated with differential regulation of the
same desaturase gene in two genera of leafroller moths. PLoS
Genet 8(1):e1002489
Anderson AR, Wanner KW, Trowell SC, Warr CG, Jaquin-Joly E,
Zagatti P, Robertson H, Newcomb RD (2009) Molecular basis of
female-specific odorant responses in Bombyx mori. Insect
Biochem Mol Biol 39(3):189–197
Ando T, Yamakawa R (2011) Analyses of lepidopteran sex pheromones
by mass spectrometry. Trends Anal Chem 30(7):990–1002
Anisimova M, Bielawski JP, Yang Z (2001) Accuracy and power of
the likelihood ratio test in detecting adaptive molecular evolu-
tion. Mol Biol Evol 18(8):1585–1592
Baker TC (2009) Nearest neural neighbors: Moth sex pheromone
receptors HR11 and HR13. Chem Senses 34(6):465–468
Baker TC, Nishida R, Roelofs WL (1981) Close-range attraction of
female oriental fruit moths to herbal scent of male hairpencils.
Science 214(4527):1359–1361
Bengtsson JM, Trona F, Montagne N, Anfora G, Ignell R, Witzgall P,
Jacquin-Joly E (2012) Putative chemosensory receptors of the
36 J Mol Evol (2014) 79:21–39
123
codling moth, Cydia pomonella, identified by antennal tran-
scriptome analysis. PLoS ONE 7(2):e31620
Benton R, Sachse S, Michnick SW, Vosshall LB (2006) Atypical
membrane topology and heteromeric function of Drosophila
odorant receptors in vivo. PLoS Biol 4(2):e20
Briscoe AD, Macias-Munoz A, Kozak KM, Walters JR, Yuan F,
Jamie GA, Martin SH, Dasmahapatra KK, Ferguson LC, Mallet
J, Jacquin-Joly E, Jiggins CD (2013) Female behaviour drives
expression and evolution of gustatory receptors in butterflies.
PLoS Genet 9:e1003620
Butenandt VA, Beckmann R, Stamm D, Hecker E (1959) Uber den
Sexual-Lockstoff des Seidenspinners Bombyx mori. Reindarstel-
lung und Konstitution. Z Naturforsch 14:283–284
Chapman RF (2003) Contact chemoreception in feeding by phytoph-
agous insects. Annu Rev Entomol 48(1):455–484
Chyb S (2004) Drosophila gustatory receptors: from gene identifica-
tion to functional expression. J Insect Physiol 50(6):469–477
Engsontia P, Sanderson AP, Cobb M, Walden KKO, Robertson HM,
Brown S (2008) The red flour beetle’s large nose: an expanded
odorant receptor gene family in Tribolium castaneum. Insect
Biochem Mol Biol 38(4):387–397
Felsenstein J (1989) PHYLIP—Phylogeny Inference Package (Ver-
sion 3.2). Cladistics 5:164–166
Gao Q, Yuan B, Chess A (2000) Convergent projections of
Drosophila olfactory neurons to specific glomeruli in the
antennal lobe. Nat Neurosci 3(8):780–785
Gardiner A, Barker D, Butlin RK, Jordan WC, Ritchie MG (2008)
Drosophila chemoreceptor gene evolution: selection, specializa-
tion and genome size. Mol Ecol 17(7):1648–1657
Gould F, Estock M, Hillier NK, Powell B, Groot AT, Ward CM,
Emerson JL, Schal C, Vickers NJ (2010) Sexual isolation of
male moths explained by a single pheromone response QTL
containing four receptor genes. Proc Natl Acad Sci USA 107(19):
8660–8665
Grosse-Wilde E, Gohl T, Bouche E, Breer H, Krieger J (2007)
Candidate pheromone receptors provide the basis for the
response of distinct antennal neurons to pheromonal compounds.
Eur J Neurosci 25(8):2364–2373
Grosse-Wilde E, Kuebler LS, Bucks S, Vogel H, Wicher D, Hansson
BS (2011) Antennal transcriptome of Manduca sexta. Proc Natl
Acad Sci USA 108(18):7449–7454
Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W,
Gascuel O (2010) New algorithms and methods to estimate
maximum-likelihood phylogenies: assessing the performance of
PhyML 3.0. Syst Biol 59(3):307–321
Guo S, Kim J (2007) Molecular evolution of Drosophila odorant
receptor genes. Mol Biol Evol 24(5):1198–1207
Hahn MW, De Bie T, Stajich JE, Nguyen C, Cristianini N (2005)
Estimating the tempo and mode of gene family evolution from
comparative genomic data. Genome Res 15(8):1153–1160
Hallem EA, Carlson JR (2006) Coding of odors by a receptor
repertoire. Cell 125(1):143–160
Hallem EA, Nicole Fox A, Zwiebel LJ, Carlson JR (2004) Olfaction:
mosquito receptor for human-sweat odorant. Nature 427(6971):
212–213
Heliconius Genome Consortium (2012) Butterfly genome reveals
promiscuous exchange of mimicry adaptations among species.
Nature 487(7405):94–98
Howlett N, Dauber K, Shukla A, Morton B, Glendinning J, Brent E,
Gleason C, Islam F, Izquierdo D, Sanghavi S, Afroz A, Aslam A,
Barbaro M, Blutstein R, Borovka M, Desire B, Elikhis A, Fan Q,
Hoffman K, Huang A, Keefe D, Lopatin S, Miller S, Patel P,
Rizzini D, Robinson A, Rokins K, Turlik A, Mansfield J (2012)
Identification of chemosensory receptor genes in Manduca sexta
and knockdown by RNA interference. BMC Genom 13(1):211
Jacquin E, Nagnan P, Frerot B (1991) Identification of hairpencil
secretion from male Mamestra brassicae (L.) (Lepidoptera:
Noctuidae) and electroantennogram studies. J Chem Ecol
17(1):239–246
Kent L, Robertson H (2009) Evolution of the sugar receptors in
insects. BMC Evol Biol 9:41
Knipple DC, Rosenfield CL, Nielsen R, You KM, Jeong SE (2002)
Evolution of the integral membrane desaturase gene family in
moths and flies. Genetics 162(4):1737–1752
Krieger J, Raming K, Dewer YME, Bette S, Conzelmann S, Breer H
(2002) A divergent gene family encoding candidate olfactory
receptors of the moth Heliothis virescens. Eur J Neurosci 16(4):
619–628
Krieger J, Grosse-Wilde E, Gohl T, Dewer YME, Raming K, Breer H
(2004) Genes encoding candidate pheromone receptors in a moth
(Heliothis virescens). Proc Natl Acad Sci USA 101(32):11845–
11850
Krieger J, Gondesen I, Forstner M, Gohl T, Dewer Y, Breer H (2009)
HR11 and HR13 receptor-expressing neurons are housedtogether in pheromone-responsive sensilla trichodea of male
Heliothis virescens. Chem Senses 34(6):469–477
Kristensen NP, Scoble MJ, Karsholt O (2007) Lepidoptera phylogeny
and systematics: the state of inventorying moth and butterfly
diversity. Zootaxa 1668:699–747
Kurtovic A, Widmer A, Dickson BJ (2007) A single class of olfactory
neurons mediates behavioural responses to a Drosophila sex
pheromone. Nature 446(7135):542–546
Lassance J-M, Groot AT, Lienard MA, Antony B, Borgwardt C,
Andersson F, Hedenstrom E, Heckel DG, Lofstedt C (2010)
Allelic variation in a fatty-acyl reductase gene causes divergence
in moth sex pheromones. Nature 466(7305):486–489
Leary GP, Allen JE, Bunger PL, Luginbill JB, Linn CE, Macallister
IE, Kavanaugh MP, Wanner KW (2012) Single mutation to a sex
pheromone receptor provides adaptive specificity between
closely related moth species. Proc Natl Acad Sci USA 109(35):
14081–14086
Legeai F, Malpel S, Montagne N, Monsempes C, Cousserans F, Merlin
C, Francois M-C, Maibeche-Coisne M, Gavory F, Poulain J,
Jacquin-Joly E (2011) An expressed sequence tag collection from
the male antennae of the Noctuid moth Spodoptera littoralis: a
resource for olfactory and pheromone detection research. BMC
Genom 12(1):86
Li L, Stoeckert CJ Jr, Roos DS (2003) OrthoMCL: identification of
ortholog groups for eukaryotic genomes. Genome Res 13(9):
2178–2189
Librado P, Vieira FG, Rozas J (2012) BadiRate: estimating family
turnover rates by likelihood-based methods. Bioinformatics
28(2):279–281
McBride CS, Arguello JR (2007) Five drosophila genomes reveal
nonneutral evolution and the signature of host specialization in
the chemoreceptor superfamily. Genetics 177(3):1395–1416
Mitchell RF, Hughes DT, Luetje CW, Millar JG, Soriano-Agaton F,
Hanks LM, Robertson HM (2012) Sequencing and characteriz-
ing odorant receptors of the cerambycid beetle Megacyllene
caryae. Insect Biochem Mol Biol 42(7):499–505
Miura N, Nakagawa T, Tatsuki S, Touhara K, Ishikawa Y (2009) A
male-specific odorant receptor conserved through the evolution
of sex pheromones in Ostrinia moth species. Int J Biol Sci
5(4):319–330
Miura N, Nakagawa T, Touhara K, Ishikawa Y (2010) Broadly and
narrowly tuned odorant receptors are involved in female sex
pheromone reception in Ostrinia moths. Insect Biochem Mol
Biol 40(1):64–73
Montell C (2009) A taste of the Drosophila gustatory receptors. Curr
Opin Neurobiol 19(4):345–353
J Mol Evol (2014) 79:21–39 37
123
Nakagawa T, Sakurai T, Nishioka T, Touhara K (2005) Insect sex-
pheromone signals mediated by specific combinations of olfac-
tory receptors. Science 307(5715):1638–1642
Niehuis O, Buellesbach J, Gibson JD, Pothmann D, Hanner C, Mutti
NS, Judson AK, Gadau J, Ruther J, Schmitt T (2013) Behav-
ioural and genetic analyses of Nasonia shed light on the
evolution of sex pheromones. Nature 494(7437):345–348
Nozawa M, Nei M (2007) Evolutionary dynamics of olfactory
receptor genes in Drosophila species. Proc Natl Acad Sci USA
104:7122–7127
Pelletier J, Hughes DT, Luetje CW, Leal WS (2010) An odorant
receptor from the southern house mosquito Culex pipiens quin-
quefasciatus sensitive to oviposition attractants. PLoS ONE
5(4):e10090
Ramaekers A, Magnenat E, Marin EC, Gendre N, Jefferis GSXE, Luo
L, Stocker RF (2005) Glomerular maps without cellular redun-
dancy at successive levels of the Drosophila larval olfactory
circuit. Curr Biol 15(11):982–992
Ramdya P, Benton R (2010) Evolving olfactory systems on the fly.
Trends Genet 26(7):307–316
Renwick JAA, Chew FS (1994) Oviposition behavior in Lepidoptera.
Annu Rev Entomol 39(1):377–400
Robertson HM, Wanner KW (2006) The chemoreceptor superfamily
in the honey bee, Apis mellifera: expansion of the odorant, but
not gustatory, receptor family. Genome Res 16(11):1395–1403
Robertson HM, Warr CG, Carlson JR (2003) Molecular evolution of the
insect chemoreceptor gene superfamily in Drosophila melano-
gaster. Proc Natl Acad Sci USA 100(Suppl 2):14537–14542
Robertson HM, Gadau J, Wanner KW (2010) The insect chemore-
ceptor superfamily of the parasitoid jewel wasp Nasonia
vitripennis. Insect Mol Biol 19:121–136
Roelofs WL, Liu W, Hao G, Jiao H, Rooney AP, Linn CE (2002)
Evolution of moth sex pheromones via ancestral genes. Proc Natl
Acad Sci USA 99(21):13621–13626
Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic
inference under mixed models. Bioinformatics 19(12):1572–
1574
Sahara K, Yoshido A, Traut W (2012) Sex chromosome evolution in
moths and butterflies. Chromosome Res 20(1):83–94
Sakurai T, Nakagawa T, Mitsuno H, Mori H, Endo Y, Tanoue S,
Yasukochi Y, Touhara K, Nishioka T (2004) Identification and
functional characterization of a sex pheromone receptor in the
silkmoth Bombyx mori. Proc Natl Acad Sci USA 101(47):16653–
16658
Sakurai T, Mitsuno H, Haupt SS, Uchino K, Yokohari F, Nishioka T,
Kobayashi I, Sezutsu H, Tamura T, Kanzaki R (2011) A Single
sex pheromone receptor determines chemical response specific-
ity of sexual behavior in the silkmoth Bombyx mori. PLoS Genet
7:e1002115
Sato K, Pellegrino M, Nakagawa T, Nakagawa T, Vosshall LB,
Touhara K (2008) Insect olfactory receptors are heteromeric
ligand-gated ion channels. Nature 452(7190):1002–1006
Sato K, Tanaka K, Touhara K (2011) Sugar-regulated cation channel
formed by an insect gustatory receptor. Proc Natl Acad Sci USA
108(28):11680–11685
Schachtner J, Schmidt M, Homberg U (2005) Organization and
evolutionary trends of primary olfactory brain centers in
Tetraconata (Crustacea ? Hexapoda). Arthropod Struct Dev
34(3):257–299
Scott K, Brady R Jr, Cravchik A, Morozov P, Rzhetsky A, Zuker C, Axel
R (2001) A chemosensory gene family encoding candidate
gustatory and olfactory receptors in Drosophila. Cell 104(5):661–
673
Smadja C, Butlin RK (2009) On the scent of speciation: the
chemosensory system and its role in premating isolation. Heredity
102(1):77–97
Smadja C, Shi P, Butlin RK, Robertson HM (2009) Large gene family
expansions and adaptive evolution for odorant and gustatory
receptors in the pea aphid, Acyrthosiphon pisum. Mol Biol Evol
26(9):2073–2086
Smadja CM, Canback B, Vitalis R, Gautier M, Ferrari J, Zhou JJ,
Butlin RK (2012) Large-scale candidate gene scan reveals the
role of chemoreceptor genes in host plant specialization and
speciation in the pea aphid. Evolution 66(9):2723–2738
Smith CD, Zimin A, Holt C, Abouheif E, Benton R, Cash E, Croset V,
Currie CR, Elhaik E, Elsik CG, Fave MJ, Fernandes V, Gadau J,
Gibson JD, Graur D, Grubbs KJ, Hagen DE, Helmkampf M,
Holley JA, Hu H, Viniegra AS, Johnson BR, Johnson RM, Khila
A, Kim JW, Laird J, Mathis KA, Moeller JA, Munoz-Torres MC,
Murphy MC, Nakamura R, Nigam S, Overson RP, Placek JE,
Rajakumar R, Reese JT, Robertson HM, Smith CR, Suarez AV,
Suen G, Suhr EL, Tao S, Torres CW, van Wilgenburg E,
Viljakainen L, Walden KK, Wild AL, Yandell M, Yorke JA,
Tsutsui ND (2011a) Draft genome of the globally widespread
and invasive Argentine ant (Linepithema humile). Proc Natl
Acad Sci USA 108(14):5673–5678
Smith CR, Smith CD, Robertson HM, Helmkampf M, Zimin A,
Yandell M, Holt C, Hu H, Abouheif E, Benton R, Cash E, Croset
V, Currie CR, Elhaik E, Elsik CG, Fave MJ, Fernandes V,
Gibson JD, Graur D, Gronenberg W, Grubbs KJ, Hagen DE,
Viniegra AS, Johnson BR, Johnson RM, Khila A, Kim JW,
Mathis KA, Munoz-Torres MC, Murphy MC, Mustard JA,
Nakamura R, Niehuis O, Nigam S, Overson RP, Placek JE,
Rajakumar R, Reese JT, Suen G, Tao S, Torres CW, Tsutsui ND,
Viljakainen L, Wolschin F, Gadau J (2011b) Draft genome of the
red harvester ant Pogonomyrmex barbatus. Proc Natl Acad Sci
USA 108(14):5667–5672
Sun M, Liu Y, Walker WB, Liu C, Lin K, Gu S, Zhang Y, Zhou J,
Wang G (2013) Identification and characterization of pheromone
receptors and interplay between receptors and pheromone
binding proteins in the diamondback moth. PLoS ONE 8:e62098
Swanson WJ, Nielsen R, Yang Q (2003) Pervasive adaptive evolution
in mammalian fertilization proteins. Mol Biol Evol 20(1):18–20
Tanaka K, Uda Y, Ono Y, Nakagawa T, Suwa M, Yamaoka R, Touhara
K (2009) Highly selective tuning of a silkworm olfactory receptor
to a key mulberry leaf volatile. Curr Biol 19(11):881–890
Teal PE, Tumlinson JH (1989) Isolation, identification, and biosyn-
thesis of compounds produced by male hairpencil glands of
Heliothis virescens (F.) (Lepidoptera: Noctuidae). J Chem Ecol
15(1):413–427
The International Silkworm Genome C (2008) The genome of a
lepidopteran model insect, the silkworm Bombyx mori. Insect
Biochem Mol Biol 38(12):1036–1045
Thompson JN, Pellmyr O (1991) Evolution of oviposition behavior
and host preference in Lepidoptera. Annu Rev Entomol 36(1):
65–89
Vasquez GM, Fischer P, Grozinger CM, Gould F (2011) Differential
expression of odorant receptor genes involved in the sexual
isolation of two Heliothis moths. Insect Mol Biol 20(1):115–124
Vieira FG, Rozas J (2011) Comparative genomics of the odorant-
binding and chemosensory protein gene families across the
Arthropoda: origin and evolutionary history of the chemosensory
system. Genome Biol Evol 3:476–490
Vogt RG, Riddiford LM (1981) Pheromone binding and inactivation
by moth antennae. Nature 293(5828):161–163
Vosshall LB (2000) Olfaction in Drosophila. Curr Opin Neurobiol
10(4):498–503
Vosshall LB, Wong AM, Axel R (2000) An olfactory sensory map in
the fly brain. Cell 102(2):147–159
Wahlberg N, Wheat CW, Pena C (2013) Timing and patterns in the
taxonomic diversification of Lepidoptera (butterflies and moths).
PLoS ONE 8:e80875
38 J Mol Evol (2014) 79:21–39
123
Wang G, Vasquez GM, Schal C, Zwiebel LJ, Gould F (2011)
Functional characterization of pheromone receptors in the tobacco
budworm Heliothis virescens. Insect Mol Biol 20(1):125–133
Wanner KW, Robertson HM (2008) The gustatory receptor family in
the silkworm moth Bombyx mori is characterized by a large
expansion of a single lineage of putative bitter receptors. Insect
Mol Biol 17(6):621–629
Wanner KW, Anderson AR, Trowell SC, Theilmann DA, Robertson
HM, Newcomb RD (2007) Female-biased expression of odou-
rant receptor genes in the adult antennae of the silkworm. Insect
Mol Biol 16(1):107–119
Wanner KW, Nichols AS, Allen JE, Bunger PL, Garczynski SF, Linn
CE Jr, Robertson HM, Luetje CW (2010) Sex pheromone
receptor specificity in the European corn borer moth, Ostrinia
nubilalis. PLoS ONE 5(1):e8685
Wicher D, Schafer R, Bauernfeind R, Stensmyr MC, Heller R,
Heinemann SH, Hansson BS (2008) Drosophila odorant recep-
tors are both ligand-gated and cyclic-nucleotide-activated cation
channels. Nature 452(7190):1007–1011
Yang Z (2007) PAML 4: Phylogenetic analysis by maximum
likelihood. Mol Biol Evol 24(8):1586–1591
Yang Z, Nielsen R (2002) Codon-Substitution Models for Detecting
Molecular Adaptation at Individual Sites Along Specific Lin-
eages. Mol Biol Evol 19(6):908–917
Yang Z, Nielsen R, Goldman N, Pedersen A-MK (2000) Codon-
substitution models for heterogeneous selection pressure at
amino acid sites. Genetics 155(1):431–449
Yang Z, Wong WSW, Nielsen R (2005) Bayes empirical bayes
inference of amino acid sites under positive selection. Mol Biol
Evol 22(4):1107–1118
Yasukochi Y, Miura N, Nakano R, Sahara K, Ishikawa Y (2011) Sex-
linked pheromone receptor genes of the European corn borer,
Ostrinia nubilalis, are in tandem arrays. PLoS ONE 6(4):e18843
You M, Yue Z, He W, Yang X, Yang G, Xie M, Zhan D, Baxter SW,
Vasseur L, Gurr GM, Douglas CJ, Bai J, Wang P, Cui K, Huang
S, Li X, Zhou Q, Wu Z, Chen Q, Liu C, Wang B, Li X, Xu X, Lu
C, Hu M, Davey JW, Smith SM, Chen M, Xia X, Tang W, Ke F,
Zheng D, Hu Y, Song F, You Y, Ma X, Peng L, Zheng Y, Liang
Y, Chen Y, Yu L, Zhang Y, Liu Y, Li G, Fang L, Li J, Zhou X,
Luo Y, Gou C, Wang J, Wang J, Yang H, Wang J (2013) A
heterozygous moth genome provides insights into herbivory and
detoxification. Nat Genet 45(2):220–225
Zhan S, Merlin C, Boore Jeffrey L, Reppert Steven M (2011) The
monarch butterfly genome yields insights into long-distance
migration. Cell 147(5):1171–1185
Zhang H-J, Anderson AR, Trowell SC, Luo AR, Xiang Z-H, Xia Q-Y
(2011) Topological and functional characterization of an insect
gustatory receptor. PLoS ONE 6:e24111
J Mol Evol (2014) 79:21–39 39
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