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: patamarerk.e@psu.ac.th

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

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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

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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.

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