Molecular Ecology (2015) 24, 2916–2936
doi: 10.1111/mec.13198
INVITED REVIEWS AND SYNTHESES
Molecular tools and bumble bees: revealing hidden
details of ecology and evolution in a model system
S. HOLLIS WOODARD,*† JEFFREY D. LOZIER,‡ DAVID GOULSON,§ PAUL H. WILLIAMS,¶
J A M E S P . S T R A N G E * * and S H A L E N E J H A *
*Department of Integrative Biology, University of Texas, Austin, TX 78712, USA, †Department of Entomology, University of
California, Riverside, CA 92521, USA, ‡Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35401, USA,
§Evolution, Behaviour & Environment, School of Life Sciences, University of Sussex, Falmer, East Sussex BN1 9QG, UK,
¶Department of Life Sciences, Natural History Museum, London, SW7 5BD, UK, **USDA-ARS, Pollinating Insect Research
Unit, Utah State University, Logan, UT 84322, USA
Abstract
Bumble bees are a longstanding model system for studies on behaviour, ecology and
evolution, due to their well-studied social lifestyle, invaluable role as wild and managed pollinators, and ubiquity and diversity across temperate ecosystems. Yet despite
their importance, many aspects of bumble bee biology have remained enigmatic until
the rise of the genetic and, more recently, genomic eras. Here, we review and synthesize new insights into the ecology, evolution and behaviour of bumble bees that have
been gained using modern genetic and genomic techniques. Special emphasis is
placed on four areas of bumble bee biology: the evolution of eusociality in this group,
population-level processes, large-scale evolutionary relationships and patterns, and
immunity and resistance to pesticides. We close with a prospective on the future of
bumble bee genomics research, as this rapidly advancing field has the potential to further revolutionize our understanding of bumble bees, particularly in regard to adaptation and resilience. Worldwide, many bumble bee populations are in decline. As such,
throughout the review, connections are drawn between new molecular insights into
bumble bees and our understanding of the causal factors involved in their decline.
Ongoing and potential applications to bumble bee management and conservation are
also included to demonstrate how genetics- and genomics-enabled research aids in the
preservation of this threatened group.
Keywords: behavior/social evolution, ecology, genomics, insects, molecular evolution,
population genetics
Received 15 December 2014; revision received 7 April 2015; accepted 8 April 2015
Introduction
Bumble bees (Bombus spp.) are among the most recognizable, abundant and ecologically and economically important groups of pollinating insects. In natural plant–
pollinator communities, bumble bees are often considered keystone species because of their generalist pollination services, whereby they support plant community
diversity by visiting both rare and abundant plant species (Memmott et al. 2004; Goulson et al. 2008; Cusser &
Correspondence: Shalene Jha, Fax: 512-232-9529;
E-mail: [email protected]
Goodell 2013; Burkle et al. 2013; Brosi & Briggs 2013). In
temperate agroecosystems, bumble bees are one of the
most effective crop pollinators, frequently depositing
more pollen or setting higher fruit yields than managed
honey bees or other native bees in crop fields (Stubbs &
Drummond 2001; Kremen et al. 2002) and providing economically important pollination services in cultivated
crops, especially in greenhouses (Velthuis & van Doorn
2006). Unfortunately, despite the critical roles that bumble bees play in cultivated and natural systems, a number
of species are declining across the globe (Williams 1986;
Goulson et al. 2008; Williams & Osborne 2009; Cameron
et al. 2011; Bartomeus et al. 2013). Potential reasons for
© 2015 John Wiley & Sons Ltd
M O L E C U L A R T O O L S A N D B U M B L E B E E S 2917
bumble bee declines are numerous and include habitat
destruction and subsequent loss of nesting and food
resources, inbreeding in fragmented wild populations,
introduction of non-native parasites and impacts of pesticides, among other factors (reviewed in Goulson et al.
2008; Williams & Osborne 2009; Graystock et al. 2013).
The ecological and economic importance of bumble
bees, coupled with their recent declines, has led to a
growing interest in the investigation of bumble bee ecology, evolution and behaviour. There is a rich and longstanding history of bumble bee research (e.g. Sladen
1912; Alford 1975; Heinrich 2004). However, answers to
many questions about bumble bee ecology, evolution
and behaviour have remained elusive until the rise of the
modern molecular era. The rapid expansion of molecular
genetic and genomic techniques over the last few decades, in particular, has contributed to an enormous
expansion in our understanding of bumble bee biology
(Box 1). These molecular tools make it possible to study
aspects of bumble bee ecology and evolution that were
previously intractable, providing critical insights into
their basic biology, as well as management direction for
improved conservation of bumble bees and other pollinating insects. Although there have been numerous
reviews of factors associated with bumble bee decline, a
comprehensive review of the literature summarizing the
molecular ecology of Bombus is lacking. Here, we provide
a synthesis of some of the impressive insights into bumble bee biology that have been gained through the use of
genetic and genomic techniques, and we discuss future
avenues of research that are important for understanding
fundamental bee biology and developing wild bee conservation strategies. Our review is organized around the
biology of bumble bees and begins with an examination
of the evolution of sociality in this group. Next, we
expand to higher levels of biological organization and
explore findings related to populations and global phylogenetic and phylogeographic patterns. Finally, we examine the immune system and resistance to pesticides,
among the major factors involved in bumble bee decline,
and end with a prospective on the future of genomic
research in bumble bees. These four topics were chosen
for review because they are the ones in which substantial
headway in molecular research has been made, and
because they contain recent advances that are particularly relevant for the conservation of bumble bees.
Sociality
The evolution of eusociality
The evolution of eusociality represents one of the major
evolutionary transitions of life on Earth (Maynard
Smith & Szathmary 1995) and has occurred more times
© 2015 John Wiley & Sons Ltd
in the insects (> 11) than in any other group in the Animal Kingdom (Wilson & H€
olldobler 2005). Eusocial
insects live in groups with three defining characteristics:
a reproductive division of labour, cooperative brood
care and overlapping generations (Michener 1974). As
opposed to ‘highly’ eusocial species, such as honey
bees, which live in large perennial colonies, most bumble bees live a so-called ‘primitively’ eusocial lifestyle
(Michener 1974) wherein queens are solitary for part of
their annual colony cycle (Fig. 1A,C–E). The remainder
of the colony cycle comprises a social group with a single reproductive queen and her typically nonreproductive daughters (Fig. 1B) until late in the colony life
cycle, when new reproductive offspring are produced
(Fig. 1C). Recent studies of within-nest relatedness
using genetic markers have revealed surprising levels
of flexibility in the reproductive division of labour in
bumble bees. For example, workers (which are capable
of laying unfertilized eggs) may be responsible for a
small proportion of male production in the nest (Takahashi et al. 2010; Huth-Schwarz et al. 2011), and in many
species, workers may opportunistically sneak into unrelated nests to deposit eggs (Lopez-Vaamonde et al.
2004; O’Connor et al. 2013). Molecular data also reveal
that in some species, one queen may usurp another, so
that nests contain two or more cohorts of unrelated
workers (Paxton et al. 2001), or enter nests to dump
batches of eggs (O’Connor et al. 2013). Although different eusocial lineages may have undergone distinct
routes to eusociality (Woodard et al. 2011, 2014), primitively eusocial species serve as some of our best extant
models for understanding earlier stages in the evolution
of eusociality across insect species (Michener 1974).
The bee family with the majority of eusocial species,
Apidae, contains some of the most well-studied and
economically important bees, including the bumble
bees, honey bees and stingless bees (tribes Bombini,
Apini and Meliponini, respectively). Together with the
Neotropical orchid bees (Euglossini), these tribes form a
monophyletic group within the Apidae named the ‘corbiculate bees’ for the presence of a corbicula, a tibial
structure used for pollen collection. Early studies that
used behavioural, morphological and other phenotypic
traits for phylogenetic inference suggested that the
Apini and Meliponini are sister taxa, supporting a single evolution of eusociality in the corbiculate bees on
the branch leading to these tribes (Michener 1990).
However, molecular phylogenetic analyses support an
alternative phylogeny that suggests complex sociality in
this group either evolved twice independently
(Cameron & Mardulyn 2001) or only once at the base of
the group, followed by the loss of sociality in the Euglossini (Danforth et al. 2013). Comparative genomic
methods may ultimately shed light on whether
2918 S . H O L L I S W O O D A R D E T A L .
Box 1 The Bombus Molecular Toolkit
Classic molecular ecology tools
Population genetics research in bumble bees has thus far been dominated by microsatellite-based studies. Of 53
population genetics, phylogeographic or species delimitation studies in bumble bees from 1984 to 2014, 68% utilized microsatellites, 23% mitochondrial DNA sequences (mtDNA), 4% nuclear gene sequences, 13% allozymes,
one utilized restriction fragment length polymorphism (RFLP) and restriction site-associated DNA tag sequencing
(RADseq), and five incorporated multiple data types (Appendix S1). The dominance of microsatellites stems from
multiple published markers sets for Bombus, which together provide hundreds of candidate loci (Estoup et al. 1996;
Reber Funk et al. 2006; Stolle et al. 2009), many of which amplify across the genus. mtDNA sequences (primarily
from the ‘DNA barcoding’ region), the second most common data type, have largely been used for taxonomic and
phylogeographic studies, occasionally in conjunction with nuclear genes or microsatellites.
Next-generation sequencing approaches
Next-generation sequencing (NGS) approaches are rapidly revolutionizing the scale and resolution of population
genetics studies of nonmodel organisms (reviewed in Andrews & Luikart 2014). By offering massively parallel
DNA sequence data and flexible library preparation strategies, NGS technologies can be used to sequence increasingly large samples of individuals, allowing for population-level analyses of genomic data. Population genomic
methods have the potential to advance our understanding of several aspects of bumble bee biology, from precise
estimates of genetic diversity and gene flow, phylogeography, and speciation and admixture, to revealing the genomic targets associated with particular phenotypic traits, adaptation to complex environments, and disease susceptibility and resistance. Further, with increased domestication of bumble bees for pollination services, NGS data will
potentially provide a vast array of genomic markers for use in breeding and artificial selection.
Full genome sequences
The newly sequenced B. impatiens and B. terrestris genomes (~250 Mb per species; Sadd et al. 2015) will greatly
assist future bumble bee genomics research. These genomes reveal information that cannot be readily obtained
from other types of sequence data, such as the presence of 118 putative Bombus-specific orthologs, and a dramatic
expansion of a subfamily of bitter taste receptors in the gustatory receptor (GR) gene family. The strong synteny of
the two sequenced genomes, which are separated by ~18 million years, suggests that genome structure might be
relatively conserved in bumble bees. This degree of conservation will greatly facilitate alignment and annotation in
NGS studies of closely related species, although the similarity of these genomes to species with greater phylogenetic distance from the Pyrobombus and Bombus sensu stricto subgenera remains to be assessed. More bumble bee
genomes will almost certainly be available in the near future, as computational and economic barriers to wholegenome sequencing studies continue to fade (1000 Genomes Project Consortium et al. 2012; Soria-Carrasco et al.
2014).
Genome reduction and target enrichment methods
Many contemporary NGS approaches focus on reducing the proportion of the genome captured in a sequencing
library so that orthologous loci can be sequenced across many individuals and scaled to adjust the amount of
sequence data generated per individual. Among the most popular genome reduction approaches are RADseq
(Davey & Blaxter 2011) and target-capture enrichment strategies (Faircloth & Glenn 2012; Carstens et al. 2012).
These next-generation approaches are only beginning to be applied in bumble bees (Lozier 2014), but a number of
RADseq studies are underway, and the recent development of hymenopteran ‘ultraconserved elements’ (Faircloth
et al. 2015) provides a set of genomic markers that will probably prove useful in bees at multiple taxonomic levels.
eusociality was present, but lost, in the orchid bee lineage. These competing evolutionary scenarios have dramatically different implications for social plasticity and
evolution, and distinguishing between competing
phylogenetic hypotheses is critical for comparative studies examining potential mechanisms of social evolution.
The evolution of sterile worker castes is a defining
characteristic of eusociality, but is also an evolutionary
© 2015 John Wiley & Sons Ltd
2922 S . H O L L I S W O O D A R D E T A L .
Downstream of the initial signals that trigger caste
determination in bumble bees and honey bees, conserved components may play roles in social organiza€ppel
tion. For example, the transcription factor Kru
homolog 1 appears to be involved in the pheromonebased suppression of worker reproduction in both Apis
mellifera and B. terrestris (Grozinger et al. 2003; Shpigler
et al. 2010). Recent genomic studies have identified
many genes that are differentially expressed between
queen and worker bumble bees (Pereboom et al. 2005;
Li et al. 2010; Colgan et al. 2011; Woodard et al. 2013;
Amsalem et al. 2014; Woodard et al. 2014), which may
underlie phenotypic differences between the female
castes.
Population dynamics and dispersal
Genetic diversity, effective population size and nest
density
Sociality has profound consequences for understanding
how bumble bees will be impacted by habitat loss and
fragmentation. In particular, eusociality greatly reduces
effective population sizes because it is a system in
which the majority of the population is sterile (i.e.
workers), and haplodiploidy further reduces the effective population size by a fourth (Zayed 2004). In most
bumble bees, this is exacerbated by monandry, which
microsatellite-based studies suggest is a common feature across most bumble bee species (Estoup et al. 1995;
Schmid-Hempel & Schmid-Hempel 2000). The potential
for low effective-to-census population size ratios may
render bumble bees particularly susceptible to inbreeding, whereby genetic diversity in small and isolated
populations becomes eroded over time through accelerated drift. Low genetic diversity has another compounding effect on bumble bee population viability; it
increases the likelihood of diploid male and triploid
worker production. Individuals that are homozygous at
the sex determination locus or loci develop into diploid
males, which exhibit reduced fertility, in part because
they produce triploid offspring (Duchateau & Hoshiba
1994; Darvill et al. 2012). Diploid males can replace half
the worker force in affected colonies (Duchateau & Hoshiba 1994), and as males do little or no work in the
nest, colonies that produce them are likely to fail. This
can have compounding negative impacts on populations that are isolated, fragmented or otherwise low in
genetic diversity (Zayed et al. 2004; Zayed & Packer
2005), although research from invasion frontiers suggests that some populations can thrive even when
ploidy aberrations are present at high frequencies (Buttermore et al. 1998; Schmid-Hempel et al. 2007). Thus,
molecular tools are beginning to elucidate the mecha-
nisms of diploid and triploid production in bumble
bees, and these findings provide insight into how other
haplodiploid insect populations might be impacted by
inbreeding and decline in genetic diversity.
Prior to the development of molecular tools for population genetic analyses, bumble bee conservation and
management efforts were hindered by an inability to
accurately estimate effective population sizes. It is
relatively easy to count bumble bee workers, but a
more useful indicator of population size and status is
the number of colonies present in a given area.
Microsatellite markers (Estoup et al. 1995) made it possible to assess the number of genetic reproductive units
(i.e. colonies) within a landscape by assigning individual workers to colonies based on sibship analysis (Estoup et al. 1995; Darvill et al. 2004; Charman et al. 2010).
Several tools exist for sibship reconstruction in haplodiploid species; however, the method implemented in
COLONY (Jones & Wang 2010) is the most popular and
also probably the most accurate (Lepais et al. 2010) for
assessing colony membership in bumble bees. Having
established the number of colonies represented within a
sample, it is also possible to estimate the number of colonies present but not directly detected, to gain an overall estimate of population size (Goulson et al. 2010).
Results from molecular-based colony density measures have highlighted major differences in nesting densities between bumble bee species (Knight et al. 2005;
Goulson et al. 2010; Geib et al. 2015) and also across
landscapes (Knight et al. 2005; Goulson et al. 2010; Rao
& Strange 2012; Jha & Kremen 2013a), with estimated
nest densities for different UK bumble bee species ranging from 13 to 193 nests per km2 in the same landscape
(Darvill et al. 2004; Knight et al. 2005). Landscape composition can also contribute to differences in nesting
density. For example, wooded habitat (Goulson et al.
2010; Jha & Kremen 2013a) and mass-flowering crops
(Herrmann et al. 2007) can have positive influences,
whereas impervious cover (Jha & Kremen 2013b) can
negatively impact nesting density. By quantifying local
nesting densities through time, recent work has also
revealed that nest survivorship is highest in landscapes
with greater availability of resource-rich habitat, specifically flowering gardens (Goulson et al. 2010). Overall,
despite similarities in phenology, life cycle and body
size, the population dynamics and habitat preferences
of bumble bee species appear to be linked to local floral
and nesting resources, though distinct between species,
and therefore species-specific strategies may be best for
conserving bumble bees.
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Table 1 Continued
Gene
Function
Hexokinase A
Aldehyde dehydrogenase
Flight
Acetaldehyde metabolic
process
Oogenesis
Reproductive process
Sex determination
Oogenesis
Ecdysone receptor-mediated
signalling pathway
Axonogenesis
Glycolytic process
Visual learning
Dynein heavy chain 64C
bellwether
intersex
baiser
Nucleosome remodelling
factor – 38kD
unzipped
lethal (1) G0334
Formaldehyde
dehydrogenase
super sex combs
preli-like
vasa
Transcription elongation
factor SPT6
Transport and Golgi
organization
Cyclophilin 1
Src oncogene at 42A
dilute class unconventional
myosin
Dihydrolipoyl dehydrogenase
pathetic
Cyclin K
Eukaryotic initiation factor 4E
Pre-rRNA-processing protein
TSR1 homolog
pugilist
Elongation factor Tu
mitochondrial
Ubiquitin-specific protease 2
Transferrin 3
Locomotor rhythm
Dendrite morphogenesis
Oogenesis
Regulation of immune
response
Dendrite morphogenesis
Autophagic cell death
Axon guidance
Intracellular protein transport
Lipoamide metabolic process
Amino acid transport
Regulation of protein kinase
activity
Chromosome condensation
Neurogenesis
Oxidation–reduction process
Translation
Protein metabolic process
Response to biotic stimulus
Subset of protein-coding genes evolving more rapidly in Bombus impatiens and Bombus terrestris than in seven other nonBombus species, ranked in order of significance (all genes significant at FDR-adjusted P < 0.05). Gene identity based on orthology to Apis mellifera and D. melanogaster ; gene functions
are individual Gene Ontology (geneontology.org) biological process terms (D. melanogaster) chosen from among all associated
terms. Gene names follow FlyBase naming conventions. Table
modified from supplementary material in Woodard et al.
(2011).
into molecular distinctions between bumble bee workers. Some of the first studies of the molecular basis of
worker–worker division of labour in bumble bees have
focused on the foraging gene, which regulates feedingand foraging-related behaviours in a variety of vertebrate and invertebrate taxa (Fitzpatrick et al. 2005). In
line with its functional conservation across a variety
of organisms, there is some evidence that foraging is
© 2015 John Wiley & Sons Ltd
also involved in bumble bee worker division of labour
(Kodaira et al. 2009; Tobback et al. 2011) and its
expression may be socially regulated in queens (Woodard et al. 2013). The insulin signalling pathway is
another particularly compelling candidate for future
studies in this area, given its role in worker division
of labour in honey bees (Ament et al. 2008) and potential role in the regulation of brood-feeding behaviour
in B. terrestris (Woodard et al. 2014).
Early developmental fates
In honey bees, a single gene (complementary sex determiner; csd) initially controls sex determination [Honeybee Genome Sequencing Consortium (HGSC 2006)] and
appears to be under strong positive selection to maintain allelic diversity (Hasselmann & Beye 2004). The
primary sex determination locus in bumble bees
remains to be identified, as csd orthologs were not
found in the two recently sequenced bumble bee genomes (Sadd et al. 2015), supporting earlier evidence
that the gene is not present in bumble bees (Hasselmann et al. 2008). However, other genes in the sex
determination pathway (doublesex, transformer 2, fruitless
and transformer/feminizer) were located in the genomes,
suggesting that downstream of the initial signal, there
is conservation of the genetic architecture underlying
sex determination in bees and Drosophila. Elucidating
the genetic basis of sex determination in bumble bees is
a particularly exciting research frontier because it will
provide insights into how a single genome can give rise
to dramatically different male and female phenotypes
in this group.
In the later stages of bumble bee colony development,
most female-destined offspring will develop into queens
(Fig. 1C), which are morphologically very similar to
workers but are larger, differ markedly in their behaviour and physiology and are capable of mating and producing female offspring (R€
oseler & van Honk 1990). In
bumble bees, the initial switch between queen and
worker development occurs during larval development
and in some species is precipitated by the absence of
exposure to a queen pheromone (Ribeiro et al. 1999;
Pereboom 2000). In contrast, in honey bees the proximate cause of female caste determination is the ingestion of a worker glandular secretion called royal jelly,
which mediates caste determination in part by triggering changes in DNA methylation (Kucharski et al.
2008). Targeted examinations of the RJ-related complex
support the absence of an RJ-related caste determination substance in bumble bees, as there has been a large
expansion of this gene family in honey bees (Drapeau
et al. 2006) that does not appear to have occurred in
bumbles bees (Albert et al. 1999; Kupke et al. 2012).
2922 S . H O L L I S W O O D A R D E T A L .
Downstream of the initial signals that trigger caste
determination in bumble bees and honey bees, conserved components may play roles in social organiza€ppel
tion. For example, the transcription factor Kru
homolog 1 appears to be involved in the pheromonebased suppression of worker reproduction in both Apis
mellifera and B. terrestris (Grozinger et al. 2003; Shpigler
et al. 2010). Recent genomic studies have identified
many genes that are differentially expressed between
queen and worker bumble bees (Pereboom et al. 2005;
Li et al. 2010; Colgan et al. 2011; Woodard et al. 2013;
Amsalem et al. 2014; Woodard et al. 2014), which may
underlie phenotypic differences between the female
castes.
Population dynamics and dispersal
Genetic diversity, effective population size and nest
density
Sociality has profound consequences for understanding
how bumble bees will be impacted by habitat loss and
fragmentation. In particular, eusociality greatly reduces
effective population sizes because it is a system in
which the majority of the population is sterile (i.e.
workers), and haplodiploidy further reduces the effective population size by a fourth (Zayed 2004). In most
bumble bees, this is exacerbated by monandry, which
microsatellite-based studies suggest is a common feature across most bumble bee species (Estoup et al. 1995;
Schmid-Hempel & Schmid-Hempel 2000). The potential
for low effective-to-census population size ratios may
render bumble bees particularly susceptible to inbreeding, whereby genetic diversity in small and isolated
populations becomes eroded over time through accelerated drift. Low genetic diversity has another compounding effect on bumble bee population viability; it
increases the likelihood of diploid male and triploid
worker production. Individuals that are homozygous at
the sex determination locus or loci develop into diploid
males, which exhibit reduced fertility, in part because
they produce triploid offspring (Duchateau & Hoshiba
1994; Darvill et al. 2012). Diploid males can replace half
the worker force in affected colonies (Duchateau & Hoshiba 1994), and as males do little or no work in the
nest, colonies that produce them are likely to fail. This
can have compounding negative impacts on populations that are isolated, fragmented or otherwise low in
genetic diversity (Zayed et al. 2004; Zayed & Packer
2005), although research from invasion frontiers suggests that some populations can thrive even when
ploidy aberrations are present at high frequencies (Buttermore et al. 1998; Schmid-Hempel et al. 2007). Thus,
molecular tools are beginning to elucidate the mecha-
nisms of diploid and triploid production in bumble
bees, and these findings provide insight into how other
haplodiploid insect populations might be impacted by
inbreeding and decline in genetic diversity.
Prior to the development of molecular tools for population genetic analyses, bumble bee conservation and
management efforts were hindered by an inability to
accurately estimate effective population sizes. It is
relatively easy to count bumble bee workers, but a
more useful indicator of population size and status is
the number of colonies present in a given area.
Microsatellite markers (Estoup et al. 1995) made it possible to assess the number of genetic reproductive units
(i.e. colonies) within a landscape by assigning individual workers to colonies based on sibship analysis (Estoup et al. 1995; Darvill et al. 2004; Charman et al. 2010).
Several tools exist for sibship reconstruction in haplodiploid species; however, the method implemented in
COLONY (Jones & Wang 2010) is the most popular and
also probably the most accurate (Lepais et al. 2010) for
assessing colony membership in bumble bees. Having
established the number of colonies represented within a
sample, it is also possible to estimate the number of colonies present but not directly detected, to gain an overall estimate of population size (Goulson et al. 2010).
Results from molecular-based colony density measures have highlighted major differences in nesting densities between bumble bee species (Knight et al. 2005;
Goulson et al. 2010; Geib et al. 2015) and also across
landscapes (Knight et al. 2005; Goulson et al. 2010; Rao
& Strange 2012; Jha & Kremen 2013a), with estimated
nest densities for different UK bumble bee species ranging from 13 to 193 nests per km2 in the same landscape
(Darvill et al. 2004; Knight et al. 2005). Landscape composition can also contribute to differences in nesting
density. For example, wooded habitat (Goulson et al.
2010; Jha & Kremen 2013a) and mass-flowering crops
(Herrmann et al. 2007) can have positive influences,
whereas impervious cover (Jha & Kremen 2013b) can
negatively impact nesting density. By quantifying local
nesting densities through time, recent work has also
revealed that nest survivorship is highest in landscapes
with greater availability of resource-rich habitat, specifically flowering gardens (Goulson et al. 2010). Overall,
despite similarities in phenology, life cycle and body
size, the population dynamics and habitat preferences
of bumble bee species appear to be linked to local floral
and nesting resources, though distinct between species,
and therefore species-specific strategies may be best for
conserving bumble bees.
Inbreeding and bottlenecks have been detected in a
number of bumble bee species, with significant bottlenecks being more prevalent in island or isolated
populations (Ellis et al. 2006; Darvill et al. 2006; Schmid© 2015 John Wiley & Sons Ltd
M O L E C U L A R T O O L S A N D B U M B L E B E E S 2923
Hempel et al. 2007). High levels of inbreeding have also
been revealed by the presence of diploid males (Zayed
& Packer 2001; Souza et al. 2010), which have been documented in various bumble bee species (Duchateau &
Hoshiba 1994; Darvill et al. 2006; Ellis et al. 2006; Whidden & Owen 2011; Darvill et al. 2012), and may be particularly high in severely declining populations, such as
B. cryptarum florilegus in Japan (Takahashi et al. 2008).
Research on the prevalence of triploid bumble bees has
demonstrated relatively low levels of triploidy in wild
populations, with 2.7% of B. cryptarum florilegus workers
documented as triploid (Takahashi et al. 2008) and
between 0–8% of B. muscorum workers and 0-6% of
B. jonellus workers documented as triploid (Darvill et al.
2012).
Several studies have taken a comparative approach
to examine the role of low genetic diversity in Bombus
declines. Direct comparisons of genetic diversity in
common and recently declining bumble bee species,
most often using estimates of heterozygosity and allelic richness, have been aided by the large numbers of
microsatellite markers developed for bumble bees that
often amplify broadly across the genus (Box 1). Studies in Europe generally have found evidence of low
effective population sizes in rare and declining species,
particularly for insular populations (Ellis et al. 2006;
Goulson et al. 2008; Darvill et al. 2010; Charman et al.
2010). Likewise, in North America, microsatellite comparisons across stable and declining species have
revealed lower levels of genetic diversity in two oncewidespread species that are undergoing declines, when
compared to four stable species (Cameron et al. 2011;
Lozier et al. 2011). However, there may be limitations
to drawing inferences about genetic diversity from microsatellite data alone (Payseur et al. 2002; Haasl &
Payseur 2011) that can potentially be overcome by
using larger, genomic data sets (Hoffman et al. 2014).
A recent comparison of ~1 Mb of genomic DNA using
RAD tags (Box 1) in the stable North American species B. impatiens and the recently declining B. pensylvanicus showed comparable genomewide nucleotide
diversities for these two species in which microsatellites showed a diversity difference (Lozier 2014). Such
studies highlight the potential for discrepancies
between data sets for analysing population genetic
diversity, and underscore the importance of additional
research into these approaches as conservation genetics
incorporates more genomic methods (Allendorf et al.
2010).
Wild bumble bee populations with low levels of
genetic diversity may suffer from reduced population
growth and increased disease susceptibility. Multiple
generations of sib-mating in laboratory populations of
B. terrestris have been used to demonstrate the negative
© 2015 John Wiley & Sons Ltd
impacts of inbreeding on queen fecundity and colony
size (Beekman et al. 2000), and laboratory populations
of B. atratus with high levels of diploid male production
have been documented to exhibit slow colony growth
rates (Plowright & Pallett 1979). Furthermore, in a combined laboratory and field study comparing inbred colonies with diploid males and noninbred colonies,
colonies with diploid males had slower growth rates,
survived for a shorter time period in the field and produced significantly fewer offspring overall (Whitehorn
et al. 2009). In another study that manipulated genetic
diversity in B. terrestris colonies and exposed them to
parasitism, colonies with greater genetic diversity have
fewer parasites and higher rates of reproduction than
low-diversity colonies (Baer & Schmid-Hempel 1999).
Together, these studies demonstrate that there are fitness costs associated with inbreeding, diploid males
and the general loss of genetic diversity in bumble bee
populations. That said, there are several cases of bumble bee populations thriving despite massive bottlenecks, especially those associated with biological
invasions (Schmid-Hempel et al. 2007). Exploring the
relevance of genetic variation and underlying mechanisms of fitness costs at the individual level is an essential future direction for conservation genetics and may
be one of the greatest contributions of NGS methods
(Hoffman et al. 2014).
Dispersal, foraging and population structure
Molecular tools have also been used to examine relatedness between individual bumble bees and differentiation among populations, thereby enabling estimation of
patterns of Bombus dispersal and gene flow throughout
the landscape. The dispersal of reproductive individuals
(males and queens) is essential for maintaining gene
flow across populations and enabling new queens to
find suitable nesting habitat, and occurs at two separate
points in the bumble bee colony cycle: near the end of
the cycle when males and new queens disperse and
mate (Fig. 1C), and when mated queens disperse again
before and/or after hibernation (Fig. 1E) (Heinrich
2004; Goulson et al. 2008). Molecular studies have
revealed that bumble bee reproductives can disperse
much farther than worker bees typically forage, more
than 9 km for queens (Lepais et al. 2010; Jha & Kremen
2013a) and at least 2.6–9.9 km for males (Kraus et al.
2009). Even these large distances may be underestimates, however, as rates of dispersal where bumble
bees have been introduced appear to be much greater
(Hopkins 1914; Schmid-Hempel et al. 2013). Evidence of
extensive reproductive dispersal is illustrated in the relatively low levels of genetic structure seen in many (but
not all) continental populations. Although studies of
2924 S . H O L L I S W O O D A R D E T A L .
bumble bee population dynamics and gene flow have
found that rare or declining species in the UK, where
many of the most comprehensive studies have been
conducted, exhibit relatively high differentiation (Darvill et al. 2006; Ellis et al. 2006; Charman et al. 2010; Goulson et al. 2011), continental populations of these species
exhibit high gene flow at multiple spatial scales (Darvill
et al. 2006; Ellis et al. 2006; Goulson et al. 2011; Dreier
et al. 2014). Studies of several species across the continental United States (Lozier et al. 2011) have similarly
revealed relatively little genetic structure. However,
several species, including B. muscorum, B. hortorum,
B. bifarius and B. vosnesenskii, appear to exhibit dispersal
limitation across natural barriers, such as water bodies
(Goulson et al. 2011; Lozier et al. 2011; Jha & Kremen
2013b; Lozier et al. 2013; Jha 2015) and elevation gradients (Lozier et al. 2011, 2013), as well as across humanmodified landscapes at both regional (~200 km) and
continental (~1000 km) scales (Jha & Kremen 2013b; Jha
2015).
Spatial mapping of colony mates via molecular-based
colony assignment has also provided new insights into
the foraging ranges of worker bumble bees. For relatively short distances, bumble bee foraging patterns
have been successfully monitored using observational
methods such as small experimental arrays (Pyke 1982;
Chittka et al. 1997; Cresswell 2000), harmonic radar (Osborne et al. 1996) and radiotracking (Hagen et al. 2011).
However, molecular tools have created opportunities to
examine foraging patterns at larger spatial scales and
across multiple landscapes. Results from this research
have revealed that estimated maximum foraging distances are more extensive than those measured via radiotagging (Hagen et al. 2011) or direct observation of
marked workers (Osborne et al. 2008), ranging from
25 m to over 10 km (Chapman et al. 2003; Darvill et al.
2004; Knight et al. 2005; Charman et al. 2010; Rao &
Strange 2012; Jha & Kremen 2013a; Geib et al. 2015),
although as for sexual dispersal, determining the upper
limits of foraging distance is challenging. Foraging
distances can also vary dramatically depending on
landscape composition (Carvell et al. 2012; Jha &
Kremen 2013a), suggesting that bumble bees assess
resources at multiple spatial scales when making foraging decisions, and revealing the importance of landscape structure when managing for bumble bee
pollination services.
Phylogenetics and biogeography
Higher-level phylogenetic patterns
Over the past decade, molecular methods have also
revolutionized our understanding of Bombus evolution
by clarifying the pattern, timing and process of lineage
divergence across the genus and at different timescales
(Cameron et al. 2007; Hines 2008; Williams et al. 2012b;
Duennes et al. 2012; Lozier et al. 2013). Bumble bees
comprise about 250 species worldwide, assigned to 15
subgenera that reflect major monophyletic lineages
within Bombus (Williams 1998). Historically, estimating
relationships within the bumble bees has been hampered by difficulties in recognizing homologies in morphological characters. Molecular markers have helped
overcome such limitations, and in combination with
increased sampling compared to earlier studies (Koulianos & Schmid-Hempel 2000; Kawakita et al. 2004),
recent work has yielded a robust multilocus molecular
phylogeny comprising 238 of 250 recognized species
from all subgenera (Cameron et al. 2007). This molecular phylogeny provides a much-needed backbone for
studies requiring reliable estimates of relationships
among the major Bombus lineages. Taxonomically, the
Bombus phylogeny has enabled a simplified classification reducing the number of subgeneric names
(Williams et al. 2008) and identifying groups of
species with shared ecological and behavioural properties.
The incorporation of molecular data into the Bombus
phylogeny (Cameron et al. 2007) has improved our previous understanding of major evolutionary events in
the global biogeography and diversification of bumble
bee lineages (Williams 1985) (Fig. 2), especially by adding calibrated phylogenetic dating (Hines 2008). Diversification of the bumble bees began in the mountains of
Asia during a period of global cooling at the
Eocene–Oligocene boundary (30 mya), with repeated
colonizations of the Nearctic through Beringia during
cold periods (5–20 mya), and recent colonizations
(~7–15 mya) of the Neotropics. This history of global
colonization has produced greater contemporary species
diversity centred in the mountains of central China than
elsewhere in the world (An et al. 2014) and a decline in
species richness away from Asia through the Palearctic,
North America and into the comparably species-poor
South America (Fig. 2) (Williams 1985, 1996; Hines
2008).
In addition to clarifying dates of major biogeographic
events, one of the key insights for bumble bee conservation to emerge from the Bombus phylogeny is the pattern of broad phylogenetic correlations suggesting that
groups of related species may share susceptibilities to
population health threats. For example, North American
species belonging to the subgenera Bombus sensu stricto
(B. franklini, B. occidentalis, B. affinis) and Thoracobombus
(e.g. B. pensylvanicus, B. fervidus) show evidence of serious decline (Colla & Packer 2008; Grixti et al. 2009;
Cameron et al. 2011), whereas other lineages maintain
© 2015 John Wiley & Sons Ltd
M O L E C U L A R T O O L S A N D B U M B L E B E E S 2925
2 Major Colonization Periods
1st: ~5-20 Ma
2nd: ~5 Ma
Arctic
25 spp.
Palearctic
124 spp.
Reverse dispersal
~4 Ma
W.
Nearctic
46 spp.
E.
Nearctic
33 spp.
Early Divergence
24-50 Ma
Oriental
116 spp.
Reverse dispersal ~1 Ma
S.
Nearctic
21 spp.
N.
Neotropical
11 spp.
3 Major Colonization Periods:
1st: 7-15 Ma
2nd: <7 Ma
W.
3rd: ~3 Ma
Neotropical
*
1990s
Japanese
26 spp.
Sumatran
6 spp.
20 spp.
19821998
*
E.
Neotropical
11 spp.
S.
Neotropical
5 spp.
*
1992
18851906
*
Fig. 2 Global patterns of Bombus species diversity and major biogeographic events in the evolutionary history of bumble bee colonization and diversification (red lines and text), including recent introductions and invasions (blue *s and text). Species numbers and
biogeographic region groupings follow Williams (1998), with some recent unpublished modifications (see http://nhm.ac.uk/bombus
for ongoing updates). Major biogeographic events follow Williams (1985) and Hines (2008), with dates representing best current estimates from calibrated phylogenetic analysis (Hines 2008). Dates for recent invasions are reviewed in Goulson (2010).
robust and widespread populations, possibly from
shared susceptibility to specific factors contributing to
decline, including certain pathogens (Cameron et al.
2011). Similar patterns of decline are also evident for
other lineages outside of North America (Goulson et al.
2008; Williams & Osborne 2009). Although phylogenetic
correlation does not necessarily point to a causative
role, comparative analyses of phylogenetic trait correlations may reveal mechanistic insights that may differentiate ‘winners and losers’ with respect to many
conservation threats, including a genetic basis for pathogen resistance and susceptibility, floral specificity, or
phenology (Williams 2005; Williams et al. 2007; Goulson
et al. 2008; Williams & Osborne 2009; Williams et al.
2010).
At the species level: phylogeographic and population
genetic patterns
Phylogenetic relationships among major Bombus lineages are now well resolved, but patterns of diversification at the species level are much less clear, due in
part to the extraordinary phenotypic variation within
and among species. Bumble bees have a complex history of taxonomic confusion at the species level, with
over 2800 names ascribed to fewer than 250 currently
recognized species (Williams 1998). Across the globe,
bumble bees can be grouped into 24 major colour pattern complexes (Williams 2007), with an average of
about three distinguishable colour patterns per species.
© 2015 John Wiley & Sons Ltd
Bumble bees thus present an interesting challenge in
that colour patterns converge among species, while at
the same time intraspecific populations exhibit divergent coloration in different geographic regions (Vogt
1909; Tkalc
u 1968). M€
ullerian mimicry is likely to be a
major force influencing these patterns (Plowright &
Owen 1980), but other factors may include crypsis and
thermoregulation in relation to particular habitats (Williams 2007).
Clarifying relationships within Bombus at shallower
timescales is thus an area of active research, including
studies aimed at species identification and discovery,
phylogeography, and identifying the conservation status of cryptic species. Traditional approaches involving
DNA sequencing from one or more gene regions have
enabled assessments of diversity and species status, in
particular for the many morphologically cryptic groups
(Carolan et al. 2012; Williams et al. 2012b; Duennes et al.
2012; Hines & Williams 2012) and in geographic regions
where taxonomy is poorly understood (An et al. 2014).
‘DNA barcoding’ with cytochrome c oxidase I (COI)
mitochondrial DNA sequences has become a popular
tool for bee taxonomy when combined with explicit
species discovery criteria, especially in groups where
identification is challenging due to morphological
homogeneity (Sheffield et al. 2009; Carolan et al. 2012;
Williams et al. 2012a; An et al. 2014). Although some
constraints exist (Meyer & Paulay 2005; Cameron et al.
2006; Rubinoff 2006; Magnacca & Brown 2010), the ease
of sequencing COI makes barcoding useful for bumble
2926 S . H O L L I S W O O D A R D E T A L .
bee ecology and biodiversity surveys in regions with
poorly known or cryptic species diversity, provided
that representative sampling and accurate reference
sequences are available.
Where recent divergence may have occurred in large
populations, which is probably typical for many insects
such as bumble bees, multilocus approaches are preferable for phylogeographic and speciation analyses (Fujita
et al. 2012). Single-locus analyses can be affected by
problems such as incomplete lineage sorting in large
populations (Maddison 1997; Degnan & Rosenberg
2006; Edwards 2009), and often cannot reveal complexities such as divergence times in the presence of admixture or population size changes, or recent speciation
events where divergence is heterogeneous throughout
the genome, both of which are increasingly recognized
as important in the speciation process (Pinho & Hey
2010; Seehausen et al. 2014). Many studies interested in
understanding the evolutionary forces underlying
diversification in bumble bees have increasingly relied
on multiple genetic markers, including nuclear gene
sequences (Duennes et al. 2012; Hines & Williams 2012)
or microsatellites (Duennes et al. 2012; Lozier et al.
2013), and genomewide data (Lozier 2014) will soon
become commonplace. Next-generation phylogeographic approaches (Hickerson et al. 2010) using model-based
methods that explicitly incorporate parameters such as
divergence times, population sizes and gene flow will
contribute to a more complete understanding of Bombus
population history, including questions related to adaptation and demography.
A common theme to emerge from studies on bumble bee species boundaries is that cryptic lineage
diversity is likely to be greater than is currently recognized (Williams et al. 2012b; Hines & Williams 2012).
As a recent example, phylogeographic analysis of the
Mesoamerican ephippiatus complex has revealed multiple lineages that appear genetically isolated across
multiple genetic markers (Duennes et al. 2012). Similar
patterns are apparent in the highly colour-polymorphic
trifasciatus complex in South-East Asia, where multilocus analysis also revealed a complex of divergent colour pattern-associated lineages that probably reflect
multiple species (Hines & Williams 2012). When species discovery is an objective of molecular studies, rigorous methods such as general mixed Yule/coalescent
models are available (Pons et al. 2006; Fujita et al.
2012) and have recently been applied in the subgenus
Bombus sensu stricto, a group that has long been suspected of including cryptic species (Williams et al.
2012b).
The small number of bumble bee phylogeographic
studies conducted to date suggests several themes
about Bombus diversity, among the most common of
which is that species (or major lineages within species)
show substantial genetic cohesiveness at broad geographic scales. Although declines in population density
and genetic diversity have been documented for a
number of bumble bee species over contemporary time
periods at the local scale (as described above), many
species have extensive ranges and large overall effective population sizes, so drift at the timescale relevant
for speciation is probably low. Substantial barriers to
dispersal thus seem crucial for the emergence of phylogeographic isolation. Several landscape features do
contribute to reproductive isolation, however, including mountain ranges and deserts, which are factors of
key importance in promoting vicariance (Williams
1996; Widmer & Schmid-Hempel 1999; Williams et al.
2011; Duennes et al. 2012; Hines & Williams 2012; Lozier et al. 2013). Mountains probably played a crucial
role in the early diversification of Bombus lineages as
well as divergence at more recent timescales (Williams
1985; Hines 2008), as bumble bee species richness is
greatest in montane regions (Williams 1998), and population structure within species is also greater for
those sampled in mountainous regions than from more
homogeneous landscapes (Lozier et al. 2011). For highelevation specialists, low-elevation areas act as substantial barriers to gene flow, maintaining phylogeographic
differentiation (Lozier et al. 2011, 2013; Duennes et al.
2012). For other species, high-elevation habitats may
act as dispersal barriers, as in the case of B. pascuorum,
where the Alps maintain differentiation between historically isolated populations in an otherwise homogenous species (Widmer & Schmid-Hempel 1999). This
study points to the importance of glacial and postglacial history, as is also apparent in the closely related
species B. subterraneus and B. distinguendus, which
show widely differing degrees of COI structuring
around the Northern Hemisphere, likely related to different degrees of population fragmentation by glaciations at different latitudes (Williams et al. 2011).
Finally, studies of island–mainland populations demonstrate that bodies of water also act as important biogeographic mechanisms of reproductive isolation,
even when mainland populations show weak structure
over equivalent scales of separation (Estoup et al. 1996;
Goulson 2010; Lozier et al. 2011; Jha 2015). Landscape
genetic models incorporating bathymetric data as geneflow resistance layers point to historical isolation from
changing sea depths as a factor in relatively recent
(<10 000 years) allopatric isolation of B. hortorum on
the Western Isles of Scotland (Goulson et al. 2011).
Together, these examples highlight the utility of an
integrative, spatially explicit phylogeographic approach
for revealing underlying factors facilitating diversity in
Bombus.
© 2015 John Wiley & Sons Ltd
M O L E C U L A R T O O L S A N D B U M B L E B E E S 2927
Phylogeographic approaches for studying Bombusassociated invasions
Anthropogenic transport of bumble bees and their associated biota have dramatically impacted ecosystems
throughout the globe, with potentially severe negative
consequences for native biodiversity (Goulson 2003,
2010). Phylogeographic and population genetic methods
are of tremendous utility for understanding the genetic
and ecological processes associated with biological invasion, including the geographic origins, number and size
of introductions, and the potential for admixture with
native species (Estoup et al. 1996; Roderick & Navajas
2003; Lozier et al. 2009; Estoup & Guillemaud 2010; Lye
et al. 2011). This information can then be used to identify and regulate introduction vectors or to target mechanisms to reduce the impact of non-native organisms.
There is a long history of transporting bumble bees
internationally for pollination services. In the late 19th
century, British bumble bees were introduced into New
Zealand for clover pollination, resulting in the establishment and spread of four species (Hopkins 1914).
Subsequent introductions and establishments have
occurred in Tasmania, Japan and South America (Macfarlane & Gurr 1995; Stout & Goulson 2000; Matsumura
et al. 2004; Schmid-Hempel et al. 2013). The New Zealand Bombus introduction provides an excellent example
of the utility of genetic data and statistical phylogeographic methods for studying invasions with relatively
well-understood histories. Lye et al. (2011) used microsatellite markers and approximate Bayesian computation to examine this introduction and found that the
four species have successfully established healthy populations despite massive genetic bottlenecks of as few as
two mated individuals, suggesting that populations can
thrive following introductions despite very low genetic
diversity (Schmid-Hempel et al. 2007). As the commercial Bombus pollination service industry grows, it seems
likely that more complex invasions may occur, including with multiple introductions and re-introductions,
and methods such as those employed in this study will
be valuable for untangling these scenarios (Estoup &
Guillemaud 2010).
Phylogeographic analysis of native fauna under consideration for domestication can also be beneficial,
given the potential for hybridization between native
species and introduced species and the increasingly
common usage of Bombus species for pollination outside
of their native ranges. Establishing the nature of cryptic
diversity is particularly pressing for bumble bee domestication candidates and their close relatives (Williams
et al. 2012b; Duennes et al. 2012). For the many morphologically homogenous Bombus species, molecular methods should be routinely implemented in the
© 2015 John Wiley & Sons Ltd
domestication process, in particular for classifying evolutionarily distinct lineage diversity (e.g. ecologically or
evolutionarily significant units; ESUs) within currently
conspecific populations. In particular for widespread
bumble bee species, populations inhabiting different
habitats or geographic regions may have distinct evolutionary histories and harbour unique subsets of locally
adapted alleles (Chittka et al. 2004). Preventing the
disruption of locally adapted allele frequencies through
the introduction of bees from one part of a species’
range into another should thus be considered in the
domestication of new species (Williams et al. 2012b; a;
Duennes et al. 2012) and will require both accurate taxonomy and assessments of gene flow. Hybridization
between closely related species that do not naturally cooccur is also a concern (Kanbe et al. 2008). Multilocus
population genetic methods can distinguish the genotypes of wild bumble bees from greenhouse escapees
even within species (Kraus et al. 2011), allowing for
monitoring of interspecific admixture between native
and managed Bombus.
Introduced bumble bees may also negatively impact
native ecosystems through co-introduction of non-native
pathogens and parasites, the so-called pathogen spillover effect. Phylogeographic analyses of parasites may
thus provide a unique perspective on the consequences
of Bombus introductions. In Japan, mtDNA analysis of
Locustacarus buchneri tracheal mites demonstrated the
great potential for introduced parasites to spread
between introduced and native bumble bees (Goka et al.
2001, 2006). In North America, Nosema bombi has been
suggested as a possible factor in the rapid decline of
several related groups of Bombus species (Thorp &
Shepherd 2005; Goulson et al. 2008; Cameron et al.
2011; Cordes et al. 2012). Specifically, researchers have
speculated that movement of bumble bees between
rearing facilities in Europe and North America during
development of the commercial Bombus pollination
industry may have led to the accidental introduction of
novel N. bombi strains into wild North American populations. Although N. bombi appears to be more prevalent in some declining species (Cameron et al. 2011), a
direct test of the hypothesis is lacking and could benefit
from global population genomic comparisons of
N. bombi isolates. Sequences from the small ribosomal
subunit are largely identical between North American
and European N. bombi isolates, but lack of variability
in this region hampers interpretation (O’Mahoney et al.
2007; Cameron et al. 2011; Cordes et al. 2012).
In South America, a similar collapse of a once common bumble bee, B. dahlbomii, also appears to have
been influenced by pathogens spread from the establishment of the non-native B. terrestris, which was introduced to the continent in 1996 (Morales et al. 2013;
2928 S . H O L L I S W O O D A R D E T A L .
Schmid-Hempel et al. 2013). The trypanosomatid Crithidia bombi shows similar population genetic structure at
microsatellite loci as the introduced host bumble bees
in South America, suggesting that it spread with the
invading host (Schmid-Hempel et al. 2013). A second
parasite, the protozoa Apicystis bombi, has also been
implicated as a possible factor in the decline of B. dahlbomii. Molecular screening of Bombus in Argentina found
this pathogen to be absent from samples prior to the
introduction of B. terrestris, and A. bombi infections that
are genetically similar to those found throughout European Bombus are now found in introduced Bombus
species and native South American B. dahlbomii (Arbetman et al. 2013; Maharramov et al. 2013). Phylogeographic studies that track introduced bumble bees and
their associated parasites will probably increase in
importance in the near future, becoming crucial components of efforts to regulate interregional transport of
bumble bees for commercial pollination.
Immunity and resistance
Bumble bees have been developed extensively as a
model system for experimental examinations of how
immunity and host–parasite interactions function in
social systems. In addition to Nosema, Crithidia, Apicystis
and parasitic mites, bumble bees face additional disease
threats, such as deformed wing virus (Genersch et al.
2006; Li et al. 2011; Evison et al. 2012), black cell queen
virus (Peng et al. 2011) and Israeli acute paralysis virus
(Singh et al. 2010), which may be spreading from managed honey bee populations. Improved beekeeping and
land management practices may help reduce these
threats, but it is important to functionally characterize
the bumble bee immune system in order to understand
how they may respond to ongoing and emerging
threats to their health and viability.
Prior to the application of genomic methods, a number of components of bumble bee immunity had been
identified experimentally, including immune priming,
specificity and trans-generational immunity (Sadd et al.
2005; Sadd & Schmid-Hempel 2007); constitutive immunity (Brown et al. 2003); and costs of immune system
activation on survival (Moret & Schmid-Hempel 2000)
and learning (Riddell & Mallon 2006). However, genomic data have made several novel contributions to our
understanding of bumble bee immunity. First, these
data suggest that bumble bees possess all of the major
immune pathways found in insects; however, as has
been found in honey bees [Honeybee Genome Sequencing Consortium (HGSC 2006); Claudianos et al. 2006)],
ants and wasps (Werren et al. 2010; Bonasio et al. 2010;
Wurm et al. 2011), they have a reduced number of
immune-related genes relative to dipterans (Barribeau
et al. 2015; Sadd et al. 2015). To achieve complex immunity, bumble bees may rely more heavily on other
immune mechanisms, such as RNAi-mediated immunity, the components of which are present in the
sequenced bumble bee genomes (Barribeau et al. 2015;
Sadd et al. 2015) or through gut microbe-mediated
immune responses (Koch & Schmid-Hempel 2011; Koch
et al. 2012). Lastly, gene expression studies highlight the
dynamic nature of the bumble bee immune system, by
suggesting that immune genes may be expressed constitutively or chronically (Xu et al. 2013), or upregulated
on shorter timescales in response to immune challenges
(Kim et al. 2006; Schl€
uns et al. 2010; Erler et al. 2011;
Barribeau et al. 2014).
Xenobiotic detoxification enzymes, including glutathione-S-transferases, carboxyl/cholinesterases and cytochrome p450 monooxygenases, are among the immunityrelated gene families that are depauperate in the
sequenced bumble bee genomes (Sadd et al. 2015) and
transcriptomes (Xu et al. 2013). These detoxification
enzymes are particularly important for ameliorating deleterious effects of pesticides, and their reduced gene
number in bees may be partially responsible for the sensitivity of bees to certain classes of pesticides (Claudianos
et al. 2006). There is overwhelming evidence that several
commonly used systemic insecticides, such as neonicotinoids (Whitehorn et al. 2012; Goulson 2013), pyrethroids
(Gill et al. 2012) and spinosad (Morandin et al. 2005), are
harmful to bumble bees at field realistic doses and that
effects may be either dramatic, such as a severe (85%)
decline in queen production (Whitehorn et al. 2012) or
sublethal and detrimental, for example via the impairment of learning and foraging (Gill et al. 2012; Feltham
et al. 2014). Although there has been a recent movement
within the USA and EU to restrict the use of these neonicotinoid pesticides, they are still widely available on the
market. Major areas of future genomic research include
the elucidation of the molecular basis of pesticide susceptibility and resistance in bumble bees, and the incorporation of gene expression-related tests for pesticide risk
assessments, which will be capable of detecting subtle
negative impacts of pesticides that current methods are
not able to detect.
Conclusion
The study of bumble bee ecology, evolution and behaviour has been greatly advanced by the application of
molecular tools. Genetic and genomic approaches have
been particularly fruitful and have revealed insights
regarding social evolution, the determination of sexes
and division of labour, the impacts of geography on
population decline and speciation events, and the structure and function of immune responses, among other
© 2015 John Wiley & Sons Ltd
M O L E C U L A R T O O L S A N D B U M B L E B E E S 2929
topics. These insights are most applicable to bumble
bees but also shed light on ecological and evolutionary
processes in other bees, and more broadly in insects.
The following are three key, broad areas that lie at the
forefront of bumble bee research.
Evolution of unique traits in bumble bees
Many types of genomic changes are likely to have been
involved in the evolution of bumble bees, including
nucleotide substitutions in protein-coding and
noncoding regulatory sequences, gene gain and loss,
and genomic rearrangements (Clark et al. 2007; Fischman et al. 2011). Full genome sequences for more bumble bee species, and more closely related outgroup
species, will allow us to identify molecular changes that
are specific to bumble bees and may underlie unique
phenotypic characteristics of this lineage (Clark et al.
2003; Boffelli et al. 2004). Genomic comparisons between
bumble bee species are also important for identifying
the molecular basis of variation within the genus, such
as the evolutionary transition to a socially parasitic lifestyle in the subgenus Psithyrus. The age of the divergence of the corbiculate bee tribes (>70 mya) probably
precludes obtaining high-quality sequence data from
preserved specimens from the lineage that gave rise to
bumble bees (P€a€abo et al. 2004). However, more modern
museum specimens may yield sequence data that can
be used to reconstruct ‘ancient DNA’ sequences for
intermediate forms of bumble bees (Maebe et al. 2013;
Lozier & Cameron 2009), although the degradation of
nucleic acids in museum-preserved specimens (Strange
et al. 2009) requires the development of protocols to
overcome these challenges.
Connecting genotype to phenotype
A more thorough understanding of how bumble bee
genes and genomes function is needed to draw inferences about the adaptive significance of molecular
changes in bumble bees. At present, gene functional
information in bumble bees is typically inferred from orthology to Drosophila or Apis genes and lacks experimental validation in bumble bees. We anticipate dramatic
developments in this area of research in the near future,
as methods for examining gene function (e.g. RNAi-mediated knockdowns) will be greatly enabled by the recent
publication of two bumble bee genome sequences (Sadd
et al. 2015) and transcriptomic data (Woodard et al. 2011;
Xu et al. 2013). These whole-genome sequences also serve
as a reference for identifying mechanisms of gene regulation in bumble bees (e.g. epigenetics, alternative splicing,
microRNAs) and for understanding how genes interact
to mediate complex, multilocus traits.
© 2015 John Wiley & Sons Ltd
Resilience and adaptation in Bombus populations
The massive numbers of genetic markers generated by
next-generation sequencing methods make it possible to
distinguish between neutral processes, such as drift,
and the footprints of recent, local adaptation in wild
populations (Schoville et al. 2012). These population
genomic data will facilitate genome association scans to
answer questions such as how biotic and abiotic factors,
and processes such as domestication, continue to shape
bumble bee evolution and how selection and local
adaptation might be influenced by the large ranges of
many bumble bee species. We believe that population
genomics-based approaches to compare declining and
nondeclining bumble bee species will be especially
promising for providing molecular clues as to why
some bumble bee populations appear to be stable,
whereas others are in decline.
Acknowledgements
We thank A. Bourke and two anonymous reviewers for their
insightful reviews and S. Barribeau and B.M. Sadd for sharing
early versions of their manuscripts. This work was supported
by a USDA-NIFA Postdoctoral Fellowship awarded to S.H.W.
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Supporting information
Additional supporting information may be found in the online
version of this article.
Appendix S1 Types of molecular data used for Bombus population genetic studies.
© 2015 John Wiley & Sons Ltd