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8
Molecular Evolutionary Analyses of
Insect Societies
BRIELLE J. FISCHMAN,* S. HOLLIS WOODARD,* AND
GENE E. ROBINSON*†‡§¶
The social insects live in extraordinarily complex and cohesive societies, where
many individuals sacrifice their personal reproduction to become helpers in
the colony. Identifying adaptive molecular changes involved in eusocial
evolution in insects is important for understanding the mechanisms under-
lying transitions from solitary to social living, as well as the maintenance and
elaboration of social life. Here, we review recent advances made in this area
of research in several insect groups: the ants, bees, wasps, and termites. Draw-
ing from whole-genome comparisons, candidate gene approaches, and a
genome-scale comparative analysis of protein-coding sequence, we highlight
novel insights gained for five major biological processes: chemical signaling,
brain development and function, immunity, reproduction, and metabolism
and nutrition. Lastly, we make comparisons across these diverse approaches
and social insect lineages and discuss potential common themes of eusocial
evolution, as well as challenges and prospects for future research in the field.
T
he social insects are exemplars of cooperative group living.
Within their complex societies, there is a reproductive division
of labor in which only a small number of individuals reproduce,
whereas all other individuals belong to a functionally sterile worker
caste that specializes in tasks important for colony growth and develop -
*Program in Ecology, Evolution, and Conservation Biology, †Department of Entomology,
‡Institute for Genomic Biology, and §Neuroscience Program, University of Illinois, Urbana,
IL 61801. ¶ To whom correspondence should be addressed. E-mail: generobi@illinois.edu.
167
OCR for page 168
168 / Brielle J. Fischman et al.
ment (Wilson, 1971). Although there has been much theoretical research
on the evolutionary forces that may select for eusociality (Strassmann
and Queller, 2007; Nowak et al., 2010), less is known about the actual
molecular mechanisms involved in transitions from solitary to social
living and in the maintenance and elaboration of eusociality in insects
(C. R. Smith et al., 2008).
The social insects provide a powerful comparative framework for
investigating mechanisms involved in eusocial evolution. Eusociality
has arisen independently at least 12 times in the insects (Cameron and
Mardulyn, 2001; Brady et al., 2006; Hines et al., 2007; Cardinal et al.,
2010), and eusocial insects have all converged on the following three
characteristics: reproductive division of labor, cooperative brood care,
and overlapping generations (Michener, 1974). Additionally, despite shar-
ing this core set of traits, there are many differences among eusocial
lifestyles, which may be related to ecological, phylogenetic, or other fac -
tors specific to particular eusocial lineages (Wilson, 1971). By comparing
across social insect lineages, it is possible to both search for common
mechanisms of eusocial evolution and explore how eusociality evolves
under different conditions.
Analysis of adaptive evolution at the molecular level can yield great
insights into the mechanisms underlying the evolution of complex phe-
notypes, such as eusociality. Genomic sequence provides a molecular
record of how natural selection has shaped an organism’s evolutionary
history (Clark, 2006). Several methods have been developed for compar-
ing genes and genomes to identify molecular signatures of adaptation.
These methods were largely developed during the pregenomic era (Li,
1997) but gain enormous power when large genomic datasets are avail -
able, particularly for sets of closely related and phenotypically variable
species (Clark et al., 2003; Drosophila 12 Genomes Consortium, 2007).
For example, comparisons of primate genomes have identified adap-
tive genetic changes involved in the evolution of brain size in humans
(Pollard et al., 2006), and comparisons of drosophilid genomes have
shed light on the ecological pressures that shaped speciation in this
group (Clark et al., 2003).
Here, we review some of the first contributions of molecular evolu-
tionary research to our understanding of eusocial evolution in insects.
This research has focused on the most well-studied social insects, which
include several eusocial lineages within the order Hymenoptera, the
a nts, bees, and wasps, and the one eusocial lineage in the order Blat -
todea, the termites (Fig. 8.1). Some studies have performed targeted
molecular evolutionary analyses of candidate genes that have been par-
ticularly valuable in species for which large amounts of genomic sequence
are not yet available. Others have focused on comparative analyses of
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Molecular Evolutionary Analyses of Insect Societies / 169
Apini (honey bees) Bees
Euglossini (orchid bees)
Eusocial Bombini (bumble bees)
Meliponini (stingless bees)
Non-eusocial Additional tribes
Allodapini (allodapine bees)
Additional tribes
Additional tribes
Additional families
Halictini spp. (sweat bees)
Additional species
Halictini spp. (sweat bees)
Additional species
Additional species
Halictini spp. (sweat bees)
Additional species
Additional families
Additional families
Wasps
Polistinae
Vespinae
Additional subfamilies
Stenogastrinae
Additional subfamilies
Ants
Formicidae
Additional families
Termites
Termitidae
Additional orders
Additional orders
FIGURE 8.1 Cladogram showing the origins of eusociality in insects. Topology
and reconstruction of evolutions of eusociality are based on multiple studies
(Cameron and Mardulyn, 2001; Brady et al., 2006; Hines et al., 2007; Cardinal
pnas.1100301108 g01_pc
et al., 2010).
whole-genome sequence, which is currently available for six social insects,
the honey bee, Apis mellifera (Honeybee Genome Sequencing Consortium,
2006), plus five ant species (Bonasio et al., 2010; C. D. Smith et al., 2011;
C . R. Smith et al., 2011; Wurm et al., 2011), and for many solitary insects,
including three solitary hymenopterans in the parasitoid jewel wasp
g enus, N asonia ( Werren et al., 2010).
We also draw heavily from our own recent genome-scale study of pro-
tein-coding sequence evolution in bees (“bee molecular evolution study”).
This study analyzed ~3,600 genes from a set of 10 social and nonsocial
bee transcriptomes; these species encompass three independent origins
of eusociality (Woodard et al., 2011). Hundreds of genes were identi-
fied that exhibit a molecular signature of rapid evolution associated with
sociality, defined as a higher ratio of nonsynonymous-to-synonymous
nucleotide substitutions (dN/dS) in social relative to nonsocial bee lin-
eages (Woodard et al., 2011). Throughout this review, evidence for rapid
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170 / Brielle J. Fischman et al.
evolution is based on relative dN/dS, and positive selection is defined as
dN/dS > 1, unless otherwise specified.
Genes identified in these studies are listed in Table 8.1. The insights
gained from these studies have implications for understanding how evo -
lutionary changes in the following five major biological processes might
be involved in the evolution of eusociality: chemical signaling, brain
development and function, immunity, reproduction, and metabolism and
nutrition. We discuss evidence and predictions for the putative functional
effects of identified molecular changes in these processes on social phe -
notypes. We also speculate on the potential adaptive significance of
these molecular changes and consider whether these changes evolved in
response to the origin, maintenance, or elaboration of eusociality, because
each case likely involved a distinct set of selective forces. For the pur-
poses of interpreting and synthesizing results across multiple studies,
we present each process separately, but it is important to recognize that
these biological processes may evolve in concert and that some molecular
TABLE 8.1 Genes Implicated in the Origin or Maintenance of Insect Society by
Molecular Evolutionary Research
Type of
Changea
Gene Function Evidence
Chemical signaling
Gland development Rapid evolution 1
decapentaplegic
( Bradley et al., 2003; i n eusocial bees
H arris et al., 2007) ( Woodard et al.,
2 011)
Gland development Rapid evolution 1
thickveins
( Bradley et al., 2003; i n eusocial bees
H arris et al., 2007) ( Woodard et al.,
2 011)
Gland development Rapid evolution 1
PDGF- and VEGF-
( Bradley et al., 2003; i n eusocial bees
related factor 1
H arris et al., 2007) ( Woodard et al.,
2 011)
OR (Wanner et al., Responds to main 2
AmOr11
2 007) c omponent of
q ueen honey bee
p heromone, 9-ODA
( Wanner et al., 2007)
β -Glycosidase-like Involved in 3
Neofem 2
(Korb et al., 2009; s ignaling queen
Weil et al., 2009) t ermite presence
(Korb et al., 2009;
Weil et al., 2009)
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Molecular Evolutionary Analyses of Insect Societies / 171
TABLE 8.1 Continued
Type of
Changea
Gene Function Evidence
Putative OBP (Keller Allelic variation 1,2
GP-9
and Ross, 1998; a ssociated with fire
Krieger and Ross, a nt queen number
2005; Gotzek et ( Keller and Ross,
a l., 2007; Leal and 1998; Krieger and
I shida, 2008; Gotzek Ross, 2005; Gotzek
a nd Ross, 2009) e t al., 2007; Leal and
I shida, 2008; Gotzek
a nd Ross, 2009)
Brain development
a nd function
cAMP/CREB Rapid evolution in 1
dunce
signaling pathways p rimitively eusocial
(Silva et al., 1998) b ees (Woodard et al.,
2 011)
CREB binding Rapid evolution in 1
nejire
p rotein (Silva et al., p rimitively eusocial
1 998) b ees (Woodard et al.,
2 011)
Immunity
Antimicrobial Positive selection in 1
defensin
p rotein (Viljakainen ants (Viljakainen and
a nd Pamilo, 2008) P amilo, 2008)
Antimicrobial Gene duplication, 1,2
termicin
p rotein (Bulmer and p ositive selection in
C rozier, 2004; Bulmer t ermites (Bulmer and
e t al., 2010) C rozier, 2004; Bulmer
e t al., 2010)
GNBP 1 and 2 Pattern recognition Gene duplication, 1,2
receptors (Bulmer p ositive selection in
a nd Crozier, 2006) t ermites (Bulmer and
C rozier, 2006)
Transcription factor, Positive selection in 1
relish
i nduces production t ermites (Bulmer and
o f antimicrobial C rozier, 2006)
peptides (Bulmer and
C rozier, 2006)
Reproduction
piRNA pathway Rapid evolution in 1
tudor
( Siomi et al., 2010) p rimitively eusocial
b ees (Woodard et al.,
2 011)
continued
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172 / Brielle J. Fischman et al.
TABLE 8.1 Continued
Type of
Changea
Gene Function Evidence
piRNA pathway Rapid evolution in 1
capsuleen
( Siomi et al., 2010) p rimitively eusocial
b ees (Woodard et al.,
2 011)
piRNA pathway Rapid evolution in 1
vasa
( Siomi et al., 2010) p rimitively eusocial
b ees (Woodard et al.,
2 011)
Sex determination Gene duplication, 1,2
csd
( Beye et al., 2003; p ositive selection in
H asselmann et al., honey bees (Beye et
2 008a,b) a l., 2003; Hasselmann
e t al., 2008a,b)
Metabolism and
n utrition
Main components of Gene family 2
MRJPs
royal jelly (Drapeau e xpansion, novel
e t al., 2006) f eeding-related
f unctions in honey
b ees (Drapeau et al.,
2 006)
Storage proteins Unique insertions in 1
Hex-1 and Hex-2
( Zhou et al., 2006, t ermites (Zhou et al.,
2 007) 2 006, 2007)
Key regulator of Rapid evolution 1
phosphofructokinase
glycolysis (Kunieda i n eusocial bees
e t al., 2006) ( Woodard et al.,
2 011)
Regulator of Rapid evolution 1
hexokinase
g lycolytic flux i n eusocial bees
( Kunieda et al., 2006) ( Woodard et al.,
2 011)
Regulator of Rapid evolution 1
pyruvate kinase
g lycolytic flux i n eusocial bees
( Kunieda et al., 2006) ( Woodard et al.,
2 011)
Note: Although many genes in this table are presumably involved in multiple biological
processes, they are classified in one of five processes with known links to insect sociality:
chemical signaling, brain development and function, immunity, reproduction, and me -
tabolism and nutrition.
aType of change: 1, protein coding sequence change; 2, novel gene; 3, change unknown.
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Molecular Evolutionary Analyses of Insect Societies / 173
changes could potentially affect multiple processes. We end with a discus -
sion of future prospects and challenges for this young field.
CHEMICAL SIGNALING
Social insects use pheromones to coordinate the behavior and phys-
iology of colony members, such as directing the foraging activity of nest-
mates, reinforcing dominance status, and inhibiting ovary development
in workers (Le Conte and Hefetz, 2008). It is unknown whether chemical
signaling was important during the origins of eusociality, because other
mechanisms to mediate social interactions, such as physical interactions,
serve similar functions in some social insect societies (Wilson, 1971).
However, chemical signaling is certainly involved in the maintenance
and elaboration of eusociality because it is crucial for the coordination
and control of colony members. In humans, in whom vocalization is
a m ajor component of social communication, molecular signatures of
adaptation have been detected in genes underlying both the production
(Enard et al., 2002) and perception (Clark et al., 2003) of vocal signals.
Early studies in social insects suggest that analogous changes have
occurred in the molecular machinery underlying the production and
perception of chemical signals.
Gland Development
Our bee molecular evolution study identified ~200 genes evolving
more rapidly in social relative to nonsocial bee lineages (Woodard et al.,
2011). Gene ontology enrichment analysis revealed that this set of genes
was enriched for genes involved in gland development. This supports a
role for these genes in chemical signaling, because glands are the primary
organs involved in pheromone production in insects. Moreover, the evo -
lution of complex chemical signaling in the social insects has been asso-
ciated with the diversification of the gland repertoire (Wilson, 1971).
In other organisms, modular evolution, in which semiautonomous
genetic pathways evolve as a functional unit and are reused in multiple
c ontexts, appears to be a common evolutionary mechanism involved
in morphological diversification (Wagner et al., 2007). The sequence
changes identified in genes involved in gland development in social bees
may have caused modular changes to the gland development program,
resulting in functional changes to existing glands or the appearance of
entirely new glands. This is supported by the evidence that several of
these genes (decapentaplegic, thickveins, and PDGF- and VEGF-related factor
1) have specific roles in gland patterning during early development in
Drosophila (Bradley et al., 2003; Harris et al., 2007).
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Because diversification of gland function is a common characteristic
shared by all social insects, it would be fruitful to investigate the sequence
evolution and function of these genes in other social insect groups. It
is possible that molecular changes in the same or similar genes were
involved in gland evolution across other independent eusocial lineages.
Odorant Receptors
Given the diversity of chemical signals used by social insects, odor-
ant receptor genes (ORs) have been predicted to be important targets of
selection during eusocial evolution (Robertson and Wanner, 2006). Early
s upport for this prediction was found in the genome of the honey bee,
A. mellifera, which, at the time of its publication, contained the largest
n umber of ORs yet found in an insect genome (Honeybee Genome
Sequencing Consortium, 2006). However, as more insect genomes have
b een sequenced, it has been discovered that A. mellifera has an interme-
diate number of ORs, there is significant variation in OR number between
the five ant genomes (Bonasio et al., 2010; C. D. Smith et al., 2011; C. R.
S mith et al., 2011; Wurm et al., 2011), and several solitary insect genomes
have among the most ORs found in insects so far (Engsontia et al., 2008;
Robertson et al., 2010). Thus, the evidence no longer supports an associa-
tion between sociality and expansion of the OR repertoire. Furthermore,
studies in other organisms have revealed that ORs can function combi -
natorially and that bioinformatically predicted ORs may not all produce
functional proteins, which, together, suggest that the number of ORs in
a genome may not scale with the complexity of chemical communication
in a species (Nei et al., 2008).
As a result of their functional specificity, ORs are particularly good
targets for candidate gene studies, because the adaptive significance of
OR evolution may be easier to interpret than for genes with broader
functions (Nei et al., 2008). A functional genomics approach was used to
identify a novel OR in the A. mellifera genome, AmOr11, which responds
to the main component of the honey bee queen pheromone, ( E )-9-oxo-
2-decenoic acid (9-ODA) (Wanner et al., 2007). The queen pheromone
a ttracts workers to the queen, partially inhibits worker ovary devel -
opment, and acts as a sex pheromone, among other functions (Wanner
et al., 2007). The specific molecular characteristics of AmOr11 that are
involved in the perception of 9-ODA are not yet known, but it appears
that it arose early in Apis evolution (Plettner et al., 1997; Cruz-López et
al., 2005; Urbanová et al., 2008).
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Molecular Evolutionary Analyses of Insect Societies / 175
Termite Queen Pheromone
Neofem2 is the first gene discovered in termites that is involved in
signaling queen presence to workers. It was originally identified as being
up-regulated in female neotenic “replacement” reproductives relative
to other colony members in two species of Cryptotermes termites (Weil
et al., 2009). Knocking down Neofem2 in Cryptotermes secundus queens
using RNAi caused an increase in aggressive behavior among workers,
which is typically only exhibited under queenless conditions (Weil et al.,
2009). Based on sequence similarity, Neofem2 is most closely related to a
β-glycosidase expressed in the salivary glands of the termite Neotermes
koshunensis (Korb et al., 2009). β-glycosidases are enzymes that break
down polysaccharides; in wood-dwelling termites, such as N. koshunen-
sis and C. secundus, whose diet primarily consists of rotting bark, these
enzymes are important for breaking down cellulose (Tokuda et al., 2002).
It has thus been suggested that Neofem2 evolved from a wood-digesting
enzyme to pheromone (Korb et al., 2009). Supporting this speculation,
β-glycosidases exhibit pheromonal activity in other insects, including
the production of an egg recognition signal in another termite species
(Korb et al., 2009). The specific molecular changes that have occurred
in Neofem2 as it evolved this new social function remain to be discov-
ered. The story of Neofem2 highlights the importance of considering the
ecological context of social evolution in a given lineage, because the
origin of a social pheromone from a wood-digesting enzyme is almost
certainly a phenomenon specific to the wood-dwelling termites.
General protein-9 in Fire Ants
General protein-9 (Gp-9) alleles are strongly associated with variation
in queen number in fire ants (genus Solenopsis). In monogynous (single
queen) colonies, all females are homozygous for B-type alleles and will
not tolerate the presence of multiple queens, whereas in polygynous (mul-
tiple queens) colonies, some individuals possess b-type alleles and do
accept multiple queens but only if those queens also possess the b-type
allele (Gotzek and Ross, 2009). Gp-9 has been called a “greenbeard gene”
(Keller and Ross, 1998), because workers carrying one allele favor queens
that share the same allele. Molecular phylogenetic analyses of Gp-9 both
within and across Solenopsis species have revealed that the b-like alleles
form a monophyletic clade, suggesting that monogyny was the ances -
tral condition in the genus and that polygyny arose once and has been
maintained through multiple speciation events (Krieger and Ross, 2005;
Gotzek et al., 2007).
At the protein sequence level, Gp-9 most closely resembles odorant-
binding proteins (OBPs), which are expressed in chemosensory sensilla
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lymph and bind and transport soluble odorants (Gotzek and Ross, 2009).
These results have led to the suggestion that Gp-9 is an OBP that plays a
role in pheromonal communication in fire ants (Gotzek and Ross, 2009).
However, Gp-9 is ubiquitously expressed in the hemolymph, suggesting
it may be involved in functions that are unrelated to chemosensation
(Leal and Ishida, 2008). In addition, Gp-9 is found in a genomic region
with a low recombination rate; therefore, other linked genes in the region
may potentially have more influence on the regulation of queen number
(Krieger and Ross, 2005; Gotzek and Ross, 2009). Gp-9 alleles are also
associated with variation in several life history traits in Solenopsis queens,
including body fat and dispersal behavior (Gotzek et al., 2007), suggest -
ing that Gp-9 either acts pleiotropically or with other genes in the region.
Although the function of Gp-9 is unresolved, molecular evolutionary
analyses suggest that this gene is evolving adaptively, implying that Gp-9
played an important role in fire ant evolution. A signature of positive
s election was detected in the branch leading to the b-like allele clade
(Krieger and Ross, 2005), suggesting that this allele had an adaptive
b enefit when it arose. In addition, all b - like alleles share the same
amino acid residues at three diagnostic codon positions, and two of
these positions show evidence of positive selection in Solenopsis invicta,
the species in which it has been best studied (Gotzek et al., 2007).
BRAIN DEVELOPMENT AND FUNCTION
Some of the most striking differences between social and solitary
insects are behavioral. Several social insect behaviors appear to be
truly novel, such as symbolic dance communication in honey bees and
slave making in ants (Wilson, 1971). Other behaviors exhibited by social
insects appear to be modified forms of behaviors performed by solitary
insects, for example, social foraging, which resembles nest provisioning
in solitary insects. It is likely that molecular changes affecting nervous
s ystem development and function were important in the evolution
o f social insect behaviors, but very little is currently known.
Brain Evolution in Primitively Eusocial Bees
Our bee molecular evolution study detected a strong signal of rapid
evolution in brain-related genes in primitively eusocial, but not highly
eusocial lineages across two independent origins of each lifestyle
(Woodard et al., 2011). Among these rapidly evolving genes were dunce
and nejire, two genes that mediate learning and memory in inver -
tebrates and vertebrates through cAMP/CREB signaling pathways
(Silva et al., 1998).
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Molecular Evolutionary Analyses of Insect Societies / 177
The detection of molecular changes in brain-related genes exclusively
in primitively eusocial bee lineages is perhaps surprising, given that this
f inding is not what may have been predicted by a prominent hypoth -
esis about the relationship between sociality and brain evolution in verte -
brates, the social brain hypothesis (SBH). Originally developed to explain
the evolution of the enlarged neocortex in many social vertebrates, the
SBH posits that the cognitive demands of social living are a strong selec -
tive force in brain evolution (Dunbar and Shultz, 2007). Given that highly
eusocial bee societies have larger colony sizes, greater social complexity,
and novel behaviors (i.e., dance communication in honey bees) relative
to primitively eusocial bees, one might have assumed that the cognitive
demands of social living are strongest in highly eusocial species and
lead to stronger selection on brain-related genes.
Unique features of insect sociality and the primitively eusocial life -
style may help to explain why selection on brain evolution appears to
have been stronger in the primitively eusocial bees. First, unlike in ver-
tebrate social evolution, where there has been an emphasis on increased
individual cognitive abilities, there appears to have been an emphasis
on increased connectedness among colony members in insect social
evolution, often accompanied by a reduction of individual behavioral
repertoires (Oster and Wilson, 1979; Gronenberg and Riveros, 2009).
Therefore, individual cognitive abilities may not be correlated with
group size in social insects, as has been found in vertebrates. There are
also several distinguishing features of the primitively eusocial bee life -
style that may have placed unique selective pressure on brain evolution
in these lineages. Social structure in primitively eusocial bee colonies is
typically maintained through fluid and dynamic dominance hierarchies
(Michener, 1974; O’Donnell et al., 2007), which can be an especially cog -
nitively challenging form of social interaction (O’Donnell et al., 2007;
Salvador and Costa, 2009). In addition, a primitively eusocial bee queen
is capable of behaving both solitarily, as she does during the colony-
founding phase of her life cycle, and socially, as she does once she has
reared her first brood of workers (Michener, 1974).
In both ants and wasps, which each evolved eusociality independent
of bees, there are some species in which queens exhibit a similar “solitary-
like” phase during colony founding and other species that found colonies
in swarms, like highly eusocial bees do (Wilson, 1971). A comparison of
brain-related genes and/or brain structure in ant and wasp species that
do or do not establish colonies solitarily may provide clues as to whether
this trait is a strong force in social insect brain evolution. One study in
paper wasps reported brain region volume differences between swarm
and independent-founding species, suggesting that these differences in
colony founding can affect brain evolution (Molina et al., 2009).
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that contribute to a charge change, and they fall on the external surface
of the predicted protein structure, suggesting that these sites may
i nteract with a fungal membrane receptor (Bulmer and Crozier, 2004).
A different study of 13 Nasutitermes termite species also found evi-
dence that gene duplication and positive selection are involved in termite
immune gene evolution (Bulmer and Crozier, 2006). This study focused
on genes encoding Gram-negative bacterial-binding protein 1 and 2
( GNBP1 and GNBP2), which are thought to have duplicated early in
termite evolution, and the transcription factor relish, which induces
production of antimicrobial peptides in Drosophila. All three genes show
evidence of positive selection, with relish showing the strongest signal.
Four of the five positively selected sites in relish are in a “spacer” region
of the protein that is cleaved by the caspase Dredd. This cleavage is
thought to activate relish by generating a DNA-binding Rel homology
domain that translocates to the nucleus and binds to promoters of target
genes (Stoven et al., 2003). Analysis of the Drosophila simulans ortholog
also found positive selection in this spacer region (Bulmer and Crozier,
2006). It was hypothesized that microbial pathogens may be targeting this
region of relish to prevent its activation, sparking an evolutionary arms
race as relish evolves counterresponses to maintain its normal function
(Bulmer and Crozier, 2006). Another study found evidence of positive
selection in termicin but not in GNBP2 in two Reticulitermes termite spe-
cies, a genus distantly related to the Nasutitermes genus (Bulmer et al.,
2010). This study used a population genetics approach to analyze intra -
specific polymorphism and interspecific divergence in coding sequence,
and results indicated that termicin underwent a selective sweep driven
by positive selection for beneficial amino acid changes.
REPRODUCTION
In many insect societies, queens are highly reproductive individu -
als, whereas workers perform almost no reproduction activity. Worker
sterility is achieved through a variety of morphological, behavioral, and
physiological mechanisms in social insects (Wilson, 1971). For example,
in many social species, workers lack spermatheca for sperm storage.
In addition, ovary development is tightly regulated by social cues, and
queens and workers typically have grossly over- and underdeveloped
ovaries, respectively, relative to solitary insects (Wilson, 1971). Social-
ity also has strong implications for reproductive behavior, particularly
for mating frequency, which can affect genetic variation among colony
members.
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Molecular Evolutionary Analyses of Insect Societies / 181
Ovary Development in Primitively Eusocial Bees
Our bee molecular evolution study identified some genes involved
in ovary development evolving most rapidly in primitively eusocial bees
(Woodard et al., 2011). Although both highly and primitively eusocial bee
societies have a strong reproductive division of labor, the reproductive
differences between queen and worker in primitively eusocial species
are less extreme, and ovary development appears to be more sensitive
to social cues in primitively eusocial species (Wilson, 1971). Perhaps the
molecular changes in ovary development-related genes found only in the
primitively eusocial lineages underlie some of the unique characteristics
of the reproductive biology of this eusocial lifestyle.
Several genes (i.e., tudor, capsuleen, vasa) evolving rapidly in one or
both of the primitively eusocial bee lineages interact together in the
PIWI RNA (piRNA) pathway. The piRNA pathway is expressed only in
gametic tissue, and it is involved in regulating gametic cell division and
differentiation (Siomi et al., 2010). Functional PIWI genes have recently
been discovered in A. mellifera (Liao et al., 2010), suggesting that the
piRNA pathway is present and functional in bees. These genes are par -
ticularly good candidates for further study, because the tissue specificity
of the piRNA pathway suggests that selection on these genes is specifi-
cally directed at changes related to reproductive processes, in contrast
t o genes with broader ranges of tissue expression, where the functional
target of selection is harder to infer. Additional ovary development-
related genes unrelated to the piRNA pathway also showed a signature of
rapid evolution in these primitively eusocial bees (Woodard et al., 2011).
Sex Determination and complementary sex determiner in Honey
Bees
More is known about the evolution of complementary sex determiner
(csd) in honey bees than probably any other gene in the social insects.
The story of csd involves the origin of entirely new genes and pathways,
as well as a classic example of balancing selection. Sex determination in
honey bees is based on genotype at the csd locus; individuals heterozy-
gous at the csd locus develop into females, whereas hemizygous individu-
als develop into males (Beye et al., 2003). Sex in many Hymenoptera is
probably determined through a similar single-locus system of comple -
mentary sex determination (Cook, 1993), but csd is the first and only locus
that has been discovered thus far. The genomic region containing csd was
first identified through mapping (Beye et al., 2003), and the function of the
gene was confirmed by RNAi, which showed that reducing csd expression
in genetically female eggs results in male-like development (Hasselmann
et al., 2008a). Complementary sex determination not only regulates sex
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determination but influences many aspects of social insect biology that are
influenced by kinship and degrees of relatedness, including kin selection
and the genetic composition of colonies, which are important for division
of labor and colony immunity (C. R. Smith et al., 2008).
The csd gene appears to be a honey bee–specific gene because it has
been found in multiple Apis species (Hasselmann et al., 2008b) but not
outside of the genus (Hasselmann et al., 2008a). The gene likely evolved
through the duplication of an adjacent gene, feminizer (fem). The csd
and fem genes are similar (>70%) in amino acid sequence, and both are
serine/arginine-rich proteins, a class of proteins involved in RNA splic -
ing (Hasselmann et al., 2008a). Both genes share two major domains,
b ut c sd h as an additional hypervariable region located between these
other domains (Hasselmann et al., 2008a). The fem gene has been found
in several non-honey bee species and in Nasonia wasps, but not in any
additional insect species, suggesting that it evolved sometime before the
split between the hymenopteran superfamilies Apoidea and Chalcidoidia
~140 Mya but after the split from Drosophila ~300 Mya (Hasselmann et al.,
2008a). The fem gene shares some functional and sequence similarities to
transformer (tra), a gene involved in sex determination in Drosophila, and
it perhaps evolved from an ancestral form of tra common to fly and bee
lineages (Beye et al., 2003; Hasselmann et al., 2008a). RNAi experiments
were used to show that csd acts upstream of fem in the sex determination
pathway. Genetically female embryos treated with f em R NAi develop
m ale heads, and RNAi knockdowns of csd cause male-specific fem
splicing, suggesting that csd is involved in fem splicing (Hasselmann et
al., 2008a).
The csd gene has been subject to rigorous population genetic analysis.
Because homozygous males do not reproduce, it was predicted that there
would be strong negative frequency-dependent selection at the csd locus
(Hasselmann et al., 2008b). This prediction has been upheld, because at
least 15 different csd alleles have been found in natural populations around
the world in three different Apis species (Hasselmann et al., 2008b) and
t he gene has accumulated 10- to 13-fold more mutations than the rest
of the genome (Hasselmann et al., 2008b). Pairwise nonsynonymous dif-
ferences between alleles are highest in exons 6 and 7 (Hasselmann et al.,
2008b), suggesting that this region is a target of positive selection, and is
therefore presumably functionally important. Six fixed amino acid dif-
ferences between csd and fem are located in the coiled-coil domain, which
is important in protein binding (Hasselmann et al., 2008a). Strong positive
selection was detected on the branch right after the split between the two
genes, suggesting that positive selection played a role in their diversifica -
tion (Hasselmann et al., 2008a).
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Molecular Evolutionary Analyses of Insect Societies / 183
METABOLISM AND NUTRITION
Transcriptomic analyses have shown that nutritional and metabolic
pathways play an important role in queen-worker caste determination
in every eusocial insect lineage thus far studied and also contribute
to worker-worker division of labor in many species (C. R. Smith et al.,
2008). Given these fundamental connections to eusociality, nutritional
and metabolic pathways are well studied in social insects and several
molecular evolutionary studies have identified changes associated with
their function.
Major Royal Jelly Proteins
The evolution of the Major Royal Jelly Proteins (MRJPs) in honey bees
is an excellent example of novel genes playing an integral role in the
social biology of a species. In the honey bee, A. mellifera, the develop-
mental fate of female larvae is determined by the amount of royal jelly
they consume (Kamakura, 2011). Royal jelly is a protein- and lipid-rich
substance secreted from the hypopharyngeal glands of brood-feeding
“nurse” bees and fed to larvae, which triggers endocrine and epigenetic
events that lead to the development of either a worker or a queen (Lyko
et al., 2010; Kamakura, 2011). The main components of royal jelly are
t he MRJPs. The A. mellifera genome contains 10 mrjp genes, encoding 9
MRJPs (one mrjp is a pseudogene). These genes are arranged in tandem
in the genome, have high sequence similarity (~60%) to one another,
and have a conserved intron/exon structure, suggesting that they are a
fairly young gene family (Drapeau et al., 2006). There is evidence that
mrjp genes are also present in other Apis species (Drapeau et al., 2006;
Yu et al., 2010).
The mrjp gene family in A. mellifera appears to have evolved via a
gene duplication event from a member of the yellow gene family. The clus-
ter of mrjp genes in the A. mellifera genome is flanked by members of the
yellow gene family, and one of the flanking yellow genes, yellow-e3, shares
the characteristic intron/exon structure of the mrjp genes, suggesting
that it is their progenitor (Drapeau et al., 2006). Members of the y ellow
gene family are involved in pigmentation, reproductive physiology, and
courtship behavior in insects (Ferguson et al., 2011).
The use of mrjp genes for larval feeding appears to be a derived
social trait that is unique to honey bees. Although mrjp-like genes have
been found in other social and nonsocial Hymenoptera species, evi -
dence suggests that the yellow gene family is prone to duplication and
that the mrjp-like genes in non-Apis species evolved independently of
Apis (Werren et al., 2010; C. D. Smith et al., 2011). Furthermore, there
is no evidence of a food-related role for any mrjp-like or yellow-like gene
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outside of Apis (Ferguson et al., 2011). Because many other social insect
species manipulate larval nutrition for the purposes of caste determi -
nation without the use of specialized glandular secretions (Webster and
Peng, 1988), the evolution of the mrjp genes in honey bees appears to be
associated with the elaboration of eusociality and may have been cor -
related with or dependent on other evolutionary changes, such as
changes in gland function.
Hexamerins
The work done on the termite hexamerins is another excellent exam -
ple of linking genetic changes to protein function and social phenotype.
In the lower termites, workers may develop into either reproductives or
soldiers, depending on a number of social and environmental cues, and
differentiation into the soldier caste is induced by high juvenile hor-
mone (JH) titers (Zhou et al., 2007). RNAi studies in the termite Reticu-
litermes flavipes have shown that two hexamerin genes, Hex-1 and Hex-2,
are involved in the regulation of this caste determination (Zhou et al.,
2006). In many insects, hexamerins act as storage proteins that sequester
substances from the diet and release them when food is scarce or inacces-
sible, such as during early development (Zhou et al., 2007). It has been
hypothesized that Hex-1 a nd H ex-2 w ork together to regulate caste
d ifferentiation in termites via direct interactions with JH (Zhou et al.,
2006); however, elucidating the specific molecular mechanisms involved
in JH action is a difficult challenge in insects in general (Riddiford, 2008).
Molecular evolutionary studies of Hex-1 and Hex-2 provide clues
as to how these genes may interact with JH. Relative to 100+ known
Hex genes in other insects, both termite Hex genes have distinctive
insertions in their coding regions; the unique insertion in Hex-1 con-
tains a prenylation motif with a proposed function in JH binding, and
the unique insertion in Hex-2 shares sequence similarities to the well-
characterized blowfly (Calliphora vicina) hexamerin receptor (Drapeau et
al., 2006). Consistent with these predicted functions, follow-up experi -
ments demonstrated that the Hex-1 protein has strong binding affinity
for JH and the Hex-2 protein shows strong membrane affinity, as would
be expected for a receptor protein (Zhou et al., 2006).
Hexamerins also exhibit novel social functions in other social insect
species, suggesting that they may be particularly prone to social co-
option. Evidence in honey bees (Martins et al., 2010) and Polistes wasps
(J. H. Hunt et al., 2010) suggests that hexamerins may be important in
caste determination in these social insect lineages, and in ants, hexam -
erins appear to be have been important in the evolution of elaborated
life history characteristics (Wheeler and Buck, 1995).
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Molecular Evolutionary Analyses of Insect Societies / 185
JH, Insulin, and Vitellogenin Axis
In the highly eusocial honey bee, A. mellifera, the JH and insulin/insulin-
like growth factor-1 (IIS) signaling pathways, as well as the yolk protein
precursor vitellogenin (Vg), interact with one another and function in
novel ways that are important in multiple social contexts. JH does not
function as a gonadotropin in adult honey bees as it does in most
insects; instead, it plays a strong role in caste determination and worker
division of labor (Robinson and Vargo, 1997). The IIS signaling pathway
interacts with JH and is also involved in worker division of labor. Forag -
ers exhibit higher expression of genes in the IIS pathway in the brain rela -
tive to nurses, and down-regulating IIS signaling delays the age-related
transition from nursing to foraging (Ament et al., 2008). This represents
a reversal of the traditional positive relationship between high nutrition
and IIS signaling, because foragers are nutritionally deprived relative to
nurses (Ament et al., 2008). Vg also shows novel social functions in honey
bees. It is highly expressed in some workers, although they are largely
nonreproductive; it may be used by nurses in the synthesis of royal jelly
(Amdam et al., 2003); and it functions as an antioxidant that may be
involved in promoting longevity in queen bees (Corona et al., 2007).
The molecular changes underlying these novel functions of JH,
IIS, and Vg are unknown, but insights from solitary insects may pro -
vide clues as to what these changes may be. The relationship between
genetic variation and regulation of JH titers has been particularly well
studied in crickets and butterflies (Zera et al., 2007), molecular evolu -
tion and function of the IIS pathway have been investigated across the
complete genomes of 12 Drosophila species (Alvarez-Ponce et al., 2009;
Grönke et al., 2010), and insect Vgs and their receptors are well charac -
terized at the molecular level (Sappington and Raikhel, 1998).
Carbohydrate Metabolism
Several studies in bees suggest that the evolution of the highly euso -
cial lifestyle involved molecular changes in genes related to carbohy -
drate metabolism. Our bee molecular evolution study revealed that
genes involved in carbohydrate metabolism are evolving more rapidly
in eusocial relative to noneusocial bee lineages and are evolving most
rapidly in highly eusocial lineages (Woodard et al., 2011). In particular,
15 genes encoding glycolytic enzymes showed evidence of rapid evolu-
tion in eusocial lineages, including enzymes that play a key regulatory
role (e.g., phosphofructokinase) or are involved in glycolytic flux (e.g., hexo-
kinase, pyruvate kinase) (Kunieda et al., 2006). Analysis of protein sequence
evolution of genes with queen-biased brain gene expression in A. mel-
lifera found that queen-biased genes involved in metabolism, includ -
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ing carbohydrate metabolism, were among the most rapidly evolving
(based on branch lengths in phylogenetic trees inferred from protein
sequence) relative to orthologs from several solitary insects (B. G. Hunt
et al., 2010). Comparative analysis of the genome sequences of A. mellifera,
D. melanogaster, and A. gambiae suggest that there may also have been bee-
specific changes in gene copy number for carbohydrate-metabolizing
genes (Kunieda et al., 2006). Given that carbohydrate metabolism is
such a fundamental “housekeeping” process, it is not immediately clear
why there has been unique selective pressure on these processes in highly
eusocial bee lineages. Here, we offer three speculative hypotheses.
First, increases in the flight demands of highly eusocial bees may
have placed strong selective pressure on increasing efficiency of glyco -
lytic enzymes, because carbohydrates are the main fuel for flight in bees
(Suarez et al., 2005). The individual foraging activity of highly eusocial
bee workers appears to be higher than for solitary bees (Roubik, 1992),
although, to the best of our knowledge, no direct comparisons of highly
and primitively eusocial bee foraging activity have been performed.
Second, highly eusocial bees are unique in relying exclusively on
a diet of modified stored sugars (i.e., honey) for long periods of time.
Nest thermoregulation during winter months is completely reliant on
honey stores as a fuel source to sustain workers, who shiver to pro -
duce metabolic heat to maintain optimal hive temperature (Southwick
and Heldmaier, 1987). Perhaps these differences in diet have placed
some novel selective pressure on glycolytic enzymes in highly eusocial
lineages.
Third, perhaps the greatly extended life span of queens in highly
eusocial species evolved through changes in metabolism-related genes,
including those involved in carbohydrate metabolism. A connection
b etween reduced metabolic rate and increased life span has been
shown in many species (Finkel and Holbrook, 2000). In the honey bee,
A. mellifera, queens exhibit an age-related reduction in IIS signaling
(Corona et al., 2007) that regulates carbohydrate metabolism. If the
m olecular changes in carbohydrate metabolism genes in highly euso -
cial bees were attributable to selection for extended queen life span, it
c an be predicted that similar molecular changes may also be found
in independent social insect lineages that also exhibit extended queen
life spans (Wilson, 1971).
PROSPECTS AND CHALLENGES
Recent work on molecular evolutionary changes in social insects has
identified specific genes, molecular pathways, and biological processes
that appear to have been shaped by natural selection. Some of these
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Molecular Evolutionary Analyses of Insect Societies / 187
changes can be plausibly associated with the origins, maintenance, or
elaboration of eusociality, albeit speculatively.
Two insights emerge from this review. First, it appears that there have
been unique genetic changes in different social insect lineages, suggesting
that the multiple independent occurrences of eusociality have involved
multiple molecular routes. These differences may reflect distinct ecologi-
cal or other constraints for each lineage. For example, the evolution of
a q ueen pheromone in termites from a wood-digesting enzyme seems
fitting, given that many termite societies live in rotting wood (Korb et
al., 2009).
Second, genetic changes also have occurred in similar biological
functions across diverse species of social insects. This supports the con -
cept of a genetic toolkit for eusociality (Toth and Robinson, 2009). This
c oncept is reasonable, because despite the striking diversity among
social insect species, they all have converged on a similar suite of traits,
which are the defining characteristics of eusociality (Michener, 1974). Pre -
vious research suggesting components of a genetic toolkit for eusociality
h as focused on genes and molecular pathways that are associated both
with solitary and related social behaviors in insects, for example, the
foraging gene, which is involved in feeding behavior in Drosophila and
a variety of other solitary organisms, and social foraging behavior in
honey bees and ants (Toth and Robinson, 2009). Transcriptomic studies
have also identified shared sets of genes whose expression patterns are
associated with division of labor in independent social insect lineages
(Toth et al., 2010).
The molecular evolutionary studies we reviewed identify biological
processes and specific genes that may be excellent systems in which to
investigate the concept of a genetic toolkit for eusociality further. Among
the most promising are the following:
(i) Hexamerins. As discussed above, hexamerins have been shown to be
involved in queen physiology and other social traits in a variety of social
insects, and the work on Hex-1 and Hex-2 in termites demonstrates how
hexamerin sequence evolution can be studied and linked to social traits.
(ii) Gland development genes. The rapidly evolving gland development
g enes identified in our bee molecular evolution study (Woodard et
al., 2011) are also good candidates for further study, because the gene
functions are relatively well characterized, and gland diversification is
a universal phenomenon in social insect evolution.
(iii) Brain-related genes. The rapidly evolving brain-related genes iden-
tified in primitively eusocial lineages in our bee molecular evolution
study (Woodard et al., 2011) are prime candidates for further study in
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primitively eusocial bees, as well as in ant and wasp species that share
the primitively eusocial bee lifestyle feature of solitary nest-founding.
The molecular changes and biological processes highlighted in this
review are currently the most well studied in social insects. There are
almost certainly other equally important types of molecular changes
and biological processes associated with social insect evolution that have
not yet been discovered, perhaps because of the limited range of taxa
subjected to these types of analyses thus far. This gap in our knowledge is
largely attributable to a lack of genomic resources, especially for closely
related social and nonsocial species. For example, some types of genetic
changes, such as chromosomal rearrangements and patterns of DNA
methylation, are not possible to study with only fragments of the genome.
In addition, the identification of truly novel genes is limited by the
small sample size of available genomes and less well-developed forward
genetic analyses in social insects relative to model genetic organisms.
As these limitations are overcome, it should be possible to search more
broadly for different types of genetic changes associated with the evolu -
tion of eusocial traits. These analyses can be guided by several theoretical
models that have been proposed to predict the types of genetic changes
that are most important in social evolution (Nonacs and Kapheim,
2007; Linksvayer and Wade, 2009; Johnson and Linksvayer, 2010).
Whole-genome scans for molecular signatures of adaptive evolu-
tion specific to social insects will be particularly useful for generating new
hypotheses and implicating new biological processes in social insect
evolution. Candidate gene approaches across a broad sample of social
and nonsocial insects will allow for greater accuracy in reconstructing
the phylogenetic history of molecular changes and testing their asso -
ciations with social evolution. Once specific sequence changes are
identified, functional analyses are necessary to determine their effect on
protein-, organismal-, and group-level phenotypes, as well as the adap -
tive significance of the phenotype change (Dean and Thornton, 2007).
This leads us to raise one important caveat for most molecular evolu-
tionary studies in the social insects: the lack of species-specific information
about gene function. As is often the case, gene function in this chapter is
typically inferred from orthology to the fruit fly D. melanogaster, which
shared a common ancestor with eusocial insect lineages over 300 Mya
(Honeybee Genome Sequencing Consortium, 2006). Although gene func -
tion for molecular processes is generally highly conserved over evolution-
ary time, when interpreting findings, it is important to consider the pos -
sibility that a particular gene has evolved a novel function. Furthermore,
many genes have multiple functions; thus, the target of selection can be
difficult to infer solely from identifying molecular evolutionary changes.
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Molecular Evolutionary Analyses of Insect Societies / 189
Experimental approaches to determining gene function in social insects,
via RNAi and transgenesis, will strengthen the interpretation of molecu -
lar evolutionary findings. Additional challenges arise in determining the
adaptive or ecological significance of molecular changes, even when their
functional significance is understood (Feder and Mitchell-Olds, 2003).
Despite these challenges, molecular evolutionary analysis of social
insect societies holds promise for testing venerable theories of social
evolution using genomic data. Multiple evolutionary scenarios have been
proposed as potential routes to group living in insects. These include
the composition of incipient social groups, such as associations between
mothers and offspring (the “subsocial” route) or between related and
unrelated individuals of the same generation (“semisocial” route)
(Michener, 1974); mechanisms through which altruism is achieved, such
as kin selection (Strassmann and Queller, 2007); parental manipula-
tion of offspring or voluntary helpers at the nest (Charnov, 1978);
and necessary preadaptations for social living, such as a monogamous
mating system (Hughes et al., 2008) or progressive provisioning of off -
spring (Nowak et al., 2010). Wedding this rich theory with genome-scale
molecular evolutionary analysis and functional experimentation holds the
promise of finally answering the compelling question of how eusociality
evolved in insects.
ACKNOWLEDGMENTS
We thank members of the G.E.R. laboratory for comments that
improved this manuscript. Our bee molecular evolution study was sup -
ported by 454 Life Sciences (Roche Diagnostics Corporation) via the
Roche 1GB contest, National Science Foundation Grant DEB07–43154 (to
G.E.R., Primary Investigator), National Science Foundation Predoctoral
Fellowship NSF DGE 07–15088 FLW (to B.J.F.), and National Institutes
of Health–University of Illinois Sensory Neuroscience Training Grant
PHS2T32DC006612 (to S.H.W. and A. S. Feng, Primary Investigator).
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