Diversity and evolutionary patterns of bacterial gutassociates of corbiculate bees

HAUKE KOCH,*† DHARAM P. ABROL,‡ J ILIAN LI§ and PAUL SCHMID-HEMPEL*

*ETH Z€urich, Institute of Integrative Biology (IBZ), Universit€atsstrasse 16, CH-8092, Z€urich, Switzerland, †Ecology and

Evolutionary Biology, Yale University, 300 Heffernan Drive, West Haven, CT, 06516, USA, ‡Division of Entomology,

Sher-e-Kashmir University of Agricultural Sciences and Technology, Chatha, Jammu (J&K), 180009, India, §Key Laboratory of

Pollinating Insect Biology of the Ministry of Agriculture, Chinese Academy of Agricultural Science, Institute of Apicultural

Research, Xiangshan, Beijing 100093, China

Abstract

The animal gut is a habitat for diverse communities of microorganisms (microbiota).

Honeybees and bumblebees have recently been shown to harbour a distinct and spe-

cies poor microbiota, which may confer protection against parasites. Here, we investi-

gate diversity, host specificity and transmission mode of two of the most common, yet

poorly known, gut bacteria of honeybees and bumblebees: Snodgrassella alvi (Betapro-teobacteria) and Gilliamella apicola (Gammaproteobacteria). We analysed 16S rRNA

gene sequences of these bacteria from diverse bee host species across most of the hon-

eybee and bumblebee phylogenetic diversity from North America, Europe and Asia.

These focal bacteria were present in 92% of bumblebee species and all honeybee spe-

cies but were found to be absent in the two related corbiculate bee tribes, the stingless

bees (Meliponini) and orchid bees (Euglossini). Both Snodgrassella alvi and Gilliamel-la apicola phylogenies show significant topological congruence with the phylogeny of

their bee hosts, albeit with a considerable degree of putative host switches. Further-

more, we found that phylogenetic distances between Gilliamella apicola samples corre-

lated with the geographical distance between sampling locations. This tentatively

suggests that the environmental transmission rate, as set by geographical distance,

affects the distribution of G. apicola infections. We show experimentally that both bac-

terial taxa can be vertically transmitted from the mother colony to daughter queens,

and social contact with nest mates after emergence from the pupa greatly facilitates

this transmission. Therefore, sociality may play an important role in vertical transmis-

sion and opens up the potential for co-evolution or at least a close association of gut

bacteria with their hosts.

Keywords: co-evolution, microbiome, specificity, symbiont, transmission mode

Received 6 April 2012; revision received 5 December 2012; accepted 11 December 2012

Introduction

Bacteria are ubiquitous partners of animal hosts (e.g.

Hosokawa et al. 2006; Fraune & Bosch 2007; Ley et al.

2008) with fundamental importance for their ecology

and evolution (Fraune & Bosch 2010; Feldhaar 2011).

The intimacy of the relationship between hosts and bac-

teria may range from vertically transmitted, intracellu-

lar symbionts obligate for host survival and incapable

of surviving outside of host cells (Clark et al. 2000), to

transient relationships in which bacterial associates reg-

ularly grow in the environment outside of their hosts

(Gordon 2001). The outcome of these relationships may

be beneficial for both partners (mutualism), harmful for

the host (parasitism), or only beneficial for the bacteria

without affecting the host (commensalism). Of particu-

lar importance are the communities of microorganisms

(microbiota) found in the gut of almost all animals, with

crucial functions, for example, in digestion, immuneCorrespondence: Hauke Koch, Fax: (001) 203 737 3109;

E-mail: hauke.koch@yale.edu

© 2013 Blackwell Publishing Ltd

Molecular Ecology (2013) 22, 2028–2044 doi: 10.1111/mec.12209

system functioning and organ development (Dillon &

Dillon 2004; Ley et al. 2008; Zilber-Rosenberg & Rosen-

berg 2008; Fraune & Bosch 2010).

The ecological and evolutionary patterns and pro-

cesses shaping the association between these two par-

ties are a central, but poorly understood, research

question. At one end of the spectrum, the microbiota

may co-evolve with their hosts. In these associations,

the host immune system may actively shape the micro-

biota, and members of the microbiota in turn adapt to

different hosts and within-host environments and

potentially provide important functions to the host

(Ochman et al. 2010; Bevins & Salzman 2011; Frese et al.

2011; Brucker & Bordenstein 2012). In this case, gut

microbes will show a high degree of specificity, fre-

quent cospeciation with their host species and signs of

adaptation to different host environments at the level of

the genome (Falush et al. 2003; Hosokawa et al. 2006;

Kikuchi et al. 2009; Oh et al. 2010; Frese et al. 2011).

These microbes also tend to be vertically transmitted to

the offspring and incapable of growing in the environ-

ment outside of the host (Hosokawa et al. 2006; Sachs

et al. 2011). At the other end of the spectrum, gut

microbes may be environmentally (horizontally) trans-

mitted, short-term residents in the animal gut. In this

case, gut bacteria are expected to generally lack evi-

dence of codivergence (cospeciation) with their hosts,

show little or no host species specificity and may be

able to grow in the environment outside of the host

(Gordon 2001; Dunlap et al. 2007; Kikuchi et al. 2007,

2011; Sachs et al. 2011). In spite of the potential diver-

sity and ubiquitous nature of animal gut microbiotas,

few studies have so far examined these above-men-

tioned patterns in a phylogenetic comparative frame-

work of closely related host species.

Because of its accessibility and relative simplicity, the

gut microbiota of corbiculate bees presents a promising

study system to understand the host–microbiota interac-

tions in more detail. In fact, recent surveys have

described a relatively simple and distinct gut microbi-

ota for the corbiculate bee tribes Apini (honeybees) and

Bombini (bumblebees), with both host groups sharing

similar bacterial taxa (Mohr & Tebbe 2006; Koch &

Schmid-Hempel 2011a; Martinson et al. 2011). These

bacteria are apparently absent in solitary bee species,

which appear to possess a more variable, unspecific gut

microbiota (Martinson et al. 2011). Hence, it has been

speculated that sociality is an important factor that

might facilitate the vertical transmission of gut bacteria

and thus allows for co-evolution of host and gut micro-

biota (Martinson et al. 2011). Indeed, honeybee and

bumblebee workers raised in isolation after pupal emer-

gence have been shown to lack this specific gut microbi-

ota (Gilliam 1971; Koch & Schmid-Hempel 2011b;

Martinson et al. 2012), with potentially detrimental con-

sequences such as higher parasite susceptibility (Koch

& Schmid-Hempel 2011b, 2012). A comparative analysis

covering a larger number of species and individuals of

honeybees and bumble bees to assess the taxonomic

distribution and specificity of the members of the gut

microbiota is, however, still missing. Also absent are

studies on the microbiota of the other two related corbi-

culate bees tribes, the eusocial Meliponini (stingless

bees) and the mostly solitary Euglossini (orchid bees),

that could shed light on these issues.

In this study, we focused on two major bacterial taxa

from the bee gut microbiota, recently described as

Snodgrassella alvi (Betaproteobacteria) and Gilliamella api-

cola (Gammaproteobacteria) (Kwong & Moran in press).

These two species were previously found to be domi-

nant members of the gut microbiota of bumblebees

(Koch & Schmid-Hempel 2011a,b; Koch et al. 2012) and

honeybees (Jeyaprakash et al. 2003; Mohr & Tebbe 2006;

Babendreier et al. 2007; Martinson et al. 2011, 2012;

Moran et al. 2012). We screened a wide range of corbi-

culate bee species from Africa, Asia, Europe and North

America for these bacteria and analysed their bacterial

16S rRNA gene sequences. With these data, we investi-

gated the correlation between host phylogeny and geo-

graphical distances with the bacterial phylogeny. We

expect that if, on the one hand, the topology of the bac-

terial phylogeny shows significant congruence to the

host phylogeny, the hypothesis of an intimate co-evolu-

tionary relationship of hosts and gut microbiota would

gain ground. This intimate relationship could evolve

through a stable transmission across generations, poten-

tially mediated by social contact within honeybee and

bumblebee colonies [i.e. a prevalence of vertical trans-

mission to the colony’s offspring (Martinson et al.

2011)]. We therefore experimentally tested the general

ability of Snodgrassella and Gilliamella to be vertically

transmitted to daughter queens. On the other hand, if

no match were found between host and bacterial phy-

logeny, codiversification between hosts and bacteria is

less likely, and bacteria might instead be frequently

transmitted horizontally between host species, for

example via environmental routes such as flowers (Dur-

rer & Schmid-Hempel 1994; McFrederick et al. 2012).

This hypothesis of ecologically driven host–bacterial

associations would be corroborated further by a correla-

tion between the geographical distance between sam-

pling locations and the genetic divergence in the

bacteria. In this case, the bacteria would also be rather

unspecific in their host range, but the environmental

transmission rate may be correlated with geographical

distance, such that individuals in closer proximity to

each other would share more similar strains (Gordon &

Lee 1999; Zamborsky & Nishiguchi 2011). Note that this

© 2013 Blackwell Publishing Ltd

EVOLUTION OF CORBICULATE BEE BACTERIA 2029

does not exclude the possibility that geographic dis-

tance correlates with host genetic differentiation and

the respective local adaptation of bacteria to their host

populations.

Materials and methods

Collection of samples

Wild bees were collected in pure ethanol in single 2-mL

plastic tubes and stored at �20 °C until further process-

ing (see Table S1, Supporting information for detailed

information on all specimens). We included all bumble-

bee subgenera and honeybee species that were available

to us. If possible, we sampled multiple species per sub-

genus and around five individuals per species to assess

specificity of bacterial strains to individual bee subgen-

era or species. Whenever possible, samples from multi-

ple locations for one host species were included.

However, studies have shown that the chances that sis-

ter workers are heavily over-represented in such field

samples is indeed rather small, and the relatedness

among sampled workers is in fact not different from

zero (e.g. Chapman et al. 2003).

To verify the transmission of microbiota of colonies

to their own daughter queens, we collected several

daughter queens of Bombus terrestris from within colo-

nies kept in the field in 2009 (near Ittingen abbey, TG,

NE Switzerland). The colonies were field-housed in

nesting boxes (Schwegler, Schorndorf/Germany) with

restricted entrance diameters, prohibiting daughter

queens from leaving the nest, while letting workers

pass through. We checked colonies once every week for

new daughter queens and froze them at �20 °C. The

daughter queens of B. terrestris normally spend the first

few days after pupal emergence within their mother

colony (Alford 1975). Therefore, our sampling protocol

allowed us to assess the microbiota acquired by daugh-

ter queens within their mother colony under natural

conditions, while ensuring that they could not have

been taken up outside of the nest.

DNA extraction, PCR, sequencing

Whole guts were removed from bee abdomens by sterile

dissection and crushed in DNA extraction buffer (see

Koch & Schmid-Hempel 2011a). DNA was extracted with

QIAGEN DNeasy Blood and Tissue Kit following the

procedure outlined by Koch & Schmid-Hempel (2011a)

including a predigestion with lysozyme for Gram-posi-

tive bacteria. Sufficient quality of the extracted DNA was

checked via a PCR with the universal 16S rRNA gene eu-

bacterial primers 27Mf (5′-AGA GTT TGA TCM TGG

CTC AG-3′) and 1492 Year (5′-ACG GYT ACC TTG TTA

CGA CTT-3′) with 1.5 lL DNA template in a total of

10 lL PCR volume. The PCR protocol consisted of 30

cycles of 94 °C (30 s), 51 °C (30 s) and 72 °C (1.5 min)

(Koch & Schmid-Hempel 2011a). PCR amplification suc-

cess was verified on a 1.5% agarose gel. Negative samples

were excluded from further analyses. Partial 16S rRNA

gene fragments were PCR amplified with the primer

pair Bprotf (5′-CAGC ACGGAGAGCTTGCTCTC-3′)/

Bprotr (5′-GCATACCGT GTTAAGCGACC-3′), specific

for Snodgrassella alvi and the primer pair Gprotf

(5′-GTATGGGGATCTGCCGA ATG-3′)/Gprotr (5′-AG-

CTATCTACTTCTGGTGCA-3′), specific for Gilliamella

apicola. We chose the specific primers based on an align-

ment of all 16S rRNA gene sequences obtained from

bumblebee guts in a previous study (Koch & Schmid-

Hempel 2011a) and included 16S rRNA gene sequences

of the target bacteria from Apis mellifera (Jeyaprakash

et al. 2003; Babendreier et al. 2007). Primer sequences

were then also cross-checked against 16S rRNA gene

sequences deposited in GenBank for their specificity for

the target bacterial taxa and their match with A. mellifera-

derived sequences. For both primer pairs, PCR condi-

tions were as follows: 35 cycles of 94 °C (30 s), 53 °C(30 s) and 72 °C (1.5 min). The PCR-mix consisted of

0.5 lL of each primer (10 lM), 5 lL 59 PCR buffer,

0.125 lL of each dNTP (50 lM), 0.75U of Taq polymerase,

15.35 lL of ultrapure water and 3 lL DNA template (in

total 25 lL). Samples yielding a single band of appropri-

ate size were sequenced directly from the PCR prod-

uct after incubation with exonuclease I and shrimp

alkaline phosphatase to remove unincorporated primers

and dNTPs. We conducted cycle sequencing in a volume

of 10 lL with 0.8 lL BigDye 3.1, 1.6 lL sequencing

buffer (ABI, Foster City, CA, USA), 0.16 lL primer

(10 lM), 4.94 lL ddH2O and 2.5 lL PCR product. In

addition to the PCR primers, we used the internal

primers F790 (5′-ATT AGA TAC CCT GGT AG-3′) and

R790 (5′-CTA CCA GGG TAT CTA AT-3′) for sequenc-

ing with a twofold coverage from both ends over the

whole amplicon length. Bprotf was replaced with Bprotf-

seq (5′-GAGTAATG CATCGGAACGTAC-3′) for the

sequencing reaction, as the former gave poor sequencing

results. We ran products on an ABI 3130xl capillary

sequencer.

Molecular cloning

All samples from Meliponini and Euglossini screened

negatively for the target bacteria with specific PCR

primers. As the construction of specific Snodgrassella

and Gilliamella PCR primers based on sequences of hon-

eybee and bumblebee bacteria could, however, have led

to a false-negative result for these bacteria in the

Meliponini and Euglossini samples, we selected two

© 2013 Blackwell Publishing Ltd

2030 H. KOCH ET AL.

specimens each of Melipona panamica, Meliponula bocan-

dei and Euglossa imperialis, as well as one specimen of

Eufrisea sp. and Eulaema sp. for the construction of indi-

vidual clone libraries with universal eubacterial 16S

rRNA gene primers. For this, we amplified the 16S

rRNA gene with the universal eubacterial primers 27Mf

and 1492 Year (see above) and cloned PCR products

with the pGEM-T Easy Vector kit (Promega) into

electrocompetent Escherichia coli cells. The molecular

cloning protocol was identical with the one described

by Koch & Schmid-Hempel (2011a). We picked 24

clones for each individual clone library and sequenced

inserts (sequencing protocol see above) with the primer

T7 (5′-TAATACGACTCACTATAGGG-3′). On the basis

of these partial sequences, we then selected different

inserts from each library and sequenced the complete

insert with the additional primers SP6

(5′-CTATTTAGGTGACACTATAG-3′) and F790/R790.

Phylogenetic and statistic analyses

Raw sequences were aligned for each sample with Se-

quencher 5.0 (Gene Codes). Bases with ambiguous

sequence chromatogram peaks were coded with the

appropriate IUPAC nucleotide ambiguity code for fur-

ther analysis. We removed vector and primer sequences

and exported the consensus sequence. The sequences

were checked for chimaeras with Bellerophon (Huber

et al. 2004) and chimeric sequences were removed from

further analysis. We deposited all sequences in Gen-

Bank (Accession nos JQ389879–JQ390033 & JQ405085–

JQ405210, Table S1, Supporting information). We

searched the Ribosomal Database Project (RDP release

10, Cole et al. 2009) for the most similar type strains of

the sequences obtained from the clone libraries using

SeqMatch. We furthermore searched GenBank with

BLASTN for similar sequences with a particular focus

on sequences from bee or other insect associated bacte-

ria. For the analysis of Snodgrassella alvi and Gilliamella

apicola phylogenies, we incorporated a selection of hon-

eybee- and bumblebee-derived sequences from previous

studies (Jeyaprakash et al. 2003; Babendreier et al. 2007;

Olofsson & V�asquez 2008; Koch & Schmid-Hempel

2011a; Martinson et al. 2011; Disayathanoowat et al.

2012; V�asquez et al. 2012). To avoid a bias towards

studies and taxa with deep sampling of 16S, we hap-

hazardly selected a sequence for each bee species from

each study for each phylogenetic dataset. None of the

top hits for the sequences of Snodgrassella alvi and Gil-

liamella apicola were from environmental sources or

hosts other than honeybees or bumblebees, suggesting

these two bacteria to be specific to the latter two bee

host taxa (see also Kwong & Moran in press).

Sequences were aligned with ClustalW (Thompson et al.

1994) (http://align.genome.jp) with standard settings

for DNA (gap opening penalty 15, gap extension pen-

alty 6.66). We deposited the alignments in TreeBASE

(Sanderson et al. 1994; submission ID 13472). We deter-

mined an appropriate model of sequence evolution with

jModelTest 0.1 (Posada 2008), choosing the model with

the lowest Akaike information criterion. For all data

sets (Snodgrassella alvi, Gilliamella apicola, sequences from

clone libraries), we then calculated a maximum-likeli-

hood phylogenetic tree in PhyML 3.0 (Guindon et al.

2010, http://www.atgc-montpellier.fr/phyml/) using a

GTR+c+inv model. Branch support was assessed by

computing 500 bootstrap replicates from the original

matrices (GTR+c+inv model).

We assessed the correlation of the bacterial phyloge-

netic trees with the host phylogenetic tree and the geo-

graphical distance between sampling locations with

Mantel tests (Mantel 1967). For this analysis, we

excluded samples that had either unclear identifications

or originated from laboratory-kept hosts. We also

excluded the B. terrestris samples of daughter queens

sampled from within colonies in Switzerland as they

were sampled with a distinct protocol, and an inclusion

would have overrepresented this species in the analysis.

To run these tests, we first constructed distance matri-

ces for the bacterial phylogenies by computing the sum

of branch lengths between all pairs of taxa (patristic dis-

tance) in R (R Development Core Team 2011) using the

APE package (Paradis et al. 2004). We also calculated

the respective patristic distances for the host species,

using a phylogenetic tree generated from the alignment

of Hines (2008) deposited in TreeBASE (Sanderson et al.

1994; study ID 1927, alignment M2930). We pruned the

alignment of all bee taxa not examined in this study

and conducted a maximum-likelihood search in PhyML

3.0 (Guindon et al. 2010) with a GTR+c+inv model. The

obtained topology was largely identical to the one in

Hines (2008), but a few nodes differed (without boot-

strap support), presumably as a result of using a highly

pruned matrix (the analysis of Hines included 218 bum-

ble bee taxa). To avoid these potential artefacts due to

limited taxon sampling, we subsequently fixed the

topology to the well-supported topology of Hines

(2008). In addition, Apis cerana and Bombus tunicatus

were missing from the alignment of Hines (2008). We

therefore added mitochondrial 16S rRNA gene

sequences for both taxa from Cameron et al. (2007) and

Ram�ırez et al. (2010) to the alignment and fixed the

position of these two taxa with respect to the taxa

included in Hines (2008) according to the results of

Cameron et al. (2007) and Ram�ırez et al. (2010) (i.e. Apis

cerana as sister to Apis mellifera and Bombus tunicatus

as sister to Bombus patagiatus). We then ran a new

maximum-likelihood search in PhyML (GTR+c+inv

© 2013 Blackwell Publishing Ltd

EVOLUTION OF CORBICULATE BEE BACTERIA 2031

model) using these topological constraints and used the

result for further analyses.

Finally, we computed a geographic distance matrix

for all pairwise comparisons between sample locations

using the Geographic Distance Matrix Generator (Ersts

2012). For bacterial sequences downloaded from Gen-

Bank, we obtained geographical information from the

corresponding publications. We computed Mantel tests

in PASSaGE v2 (Rosenberg & Anderson 2011) using

10 000 replicates. We used partial Mantel tests (Legen-

dre & Troussellier 1988) to control for the possible inter-

dependence of bacteria phylogeny, host phylogeny and

geographical distance. As some sequences obtained

with the Gilliamella primer set fell outside the main

Gilliamella apicola clade and were closer to sequences

from other insects and the Gamma-2 phylotype of Mart-

inson et al. (2011), we analysed the Gilliamella phylogeny

separately excluding this distinct clade. These sequences

(mostly derived from the host subgenus Pyrobombus)

appear to belong to a novel clade within the Orbaceae

and may deserve recognition as a distinct genus.

To further test the congruence between the host and

bacterial trees, we performed cophylogeny reconstruc-

tions in Jane (version 4, Conow et al. 2010). We col-

lapsed branches of zero branch length in the bacterial

phylogenies and treated them as soft polytomies. In this

way, Jane will resolve the bacterial tree into bifurcations

while minimizing the overall cost of the cophylogeny

analysis. If several bacterial sequences from one host

species were present in the resulting polytomy, only

one representative bacterial sequence was maintained.

Furthermore, we collapsed monophyletic groups in the

bacterial phylogeny originating from a single host spe-

cies to one representative tip, to prevent the spurious

inference of duplication events at the tips of the tree.

The absence of a more detailed understanding of the

evolutionary interactions of the host and bacteria in our

system makes it difficult to choose a particular cost

matrix for the cophylogeny analysis. We therefore

evaluated two commonly used settings for the cost

matrix (Conow et al. 2010; Cruaud et al. 2012; Decelle

et al. 2012; Mendlov�a et al. 2012), which have been

implemented as default settings in Jane with a vertex-

based cost model: Setting 1 (cosp. = 0): assuming no

cost for cospeciation and cost = 1 for all other events

(cospeciation = 0, duplication = 1, duplication & host

switch = 2, loss = 1, failure to diverge = 1); Setting 2

(cosp. = 1): assuming cost = 1 for all events (cospecia-

tion = 1, duplication = 1, duplication & host switch = 2,

loss = 1, failure to diverge = 1). The genetic algorithm

was run with 100 generations and a population size of

300, with these settings the algorithm consistently found

solutions of identical costs. Significant matching of host

and bacterial phylogenies was assessed by computing

the costs of 500 replicates with random tip mapping

and comparing the resulting costs to the cost of the ori-

ginal associations.

Transmission experiments

Cocoons of daughter queens from five laboratory col-

onies of Bombus terrestris were removed from their

mother colonies and incubated at 30 °C, 60% rel.

humidity in sterile plastic boxes until emergence of

adults. Half of the emerged daughter queens were

then transferred to individual plastic boxes with auto-

claved cat litter and fed with filter sterilized (0.2 lmpore size) sugar water and heat-treated (85 °C,30 min) pollen. The other half of the daughter queens

were put back into their colony of origin for 3 days.

After this period, they were taken out of the colonies

and kept like the other half of the daughter queens

on sterilized food in individual boxes. Five daughter

queens of each group were frozen at �20 °C after

another 7 days. The remaining daughter queens were

mated with males and put into artificial hibernation

at 4 °C for 3 months. Males were obtained from iso-

lated pupae and had been kept on a sterile diet prior

to mating. After hibernation, daughter queens were

kept in individual boxes on a sterile diet as described

earlier. We had originally intended to let the daughter

queens lay eggs and found a colony. However, after

3 months only one queen had produced a small

amount of brood and, hence, we froze them at

�20 °C for analysis of their gut microbiota. DNA

extraction and specific PCRs for Snodgrassella alvi and

Gilliamella apicola for the daughter queens were identi-

cal to the ones described previously. We furthermore

extracted DNA from honey pot content and wax from

two laboratory colonies to screen for the presence of

the target bacteria in the nest environment.

Results

Phylogeny of Snodgrassella alvi, influence of hostphylogeny and geography

In total, we obtained partial 16S rRNA gene sequences

(approx. 1370 bp in length) from 126 different bee spec-

imens for Snodgrassella alvi. Together with the selection

of sequences from previous studies, this resulted in a

data set of 150 samples from 36 different corbiculate

bee host species. Within the tribe Bombini, we sampled

species from 12 of the 15 subgenera of the simplified

classification system by Williams et al. (2008). Our pri-

mer sets detected both Snodgrassella and Gilliamella in

92% of all sampled Bombus species and in all three

Apis species. In several cases, however, not all tested

© 2013 Blackwell Publishing Ltd

2032 H. KOCH ET AL.

individuals within an infected host species screened

positive. A summary of the presence of Snodgrassella

alvi and Gilliamella apicola in samples of all screened bee

species is provided in Table S2 (Supporting informa-

tion).

The phylogenetic tree of Snodgrassella alvi shows a

clear distinction between the sequences from the three

Apis species and the Bombus species (Fig. 1). On a finer

taxonomic scale, considerable separation can also be

observed for the different host species and subgenera.

For example, Apis mellifera bacterial strains from a wide

geographic range (including samples from Europe,

Asia, North America, and Africa) group together in a

monophyletic clade, different from Snodgrassella of other

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Fig. 1 Phylogenetic tree (maximum-likelihood) of 16S rRNA gene sequences from Snodgrassella alvi strains of different bee hosts. Sten-

oxybacter acetivorans (termite gut) was selected as the most closely related outgroup taxon for the root, and the root branch length

was shortened for display purposes. Branch labels are bootstrap values over 50%. Colours indicate different bumblebee subgenera

following Williams et al. (2008) (see legend) and different shades of grey represent different Apis species. Symbols code for geo-

graphic origin of each sample (see legend). Tip labels include species name and sample ID (see Table S1, Supporting information).

For further details, see Methods.

© 2013 Blackwell Publishing Ltd

EVOLUTION OF CORBICULATE BEE BACTERIA 2033

Apis species. The Bombus-associated Snodgrassella of the

host subgenera Thoracobombus, Subterraneobombus, Pyro-

bombus, Bombus s. str., Melanobombus and Orientalibom-

bus are nonrandomly distributed in the tree, and appear

to cluster to some extent according to the host subge-

nus. As an example, Snodgrassella strains from Bombus

(Subterraneobombus) appositus and B. (St.) melanurus fall

together in a well- supported clade despite having been

collected in the USA and India/Kyrgyzstan, respec-

tively, (with the exception of a single strain from B. (St.)

melanurus, which was closer to strains from B. (Th.) mes-

omelas). Some Snodgrassella also cluster according to host

species, for example for B. (Thoracobombus) mesomelas,

B. (Orientalibombus) haemorrhoidalis, B. (Melanobombus)

festivus or B. (Bombus) tunicatus (with the exception of a

single individual).

In some cases, however, less influence of the host

phylogeny can be observed. For example, Snodgrassella

from bumblebees of the subgenera Mendacibombus,

Psithyrus, Pyrobombus, Megabombus, Sibiricobombus, Mela-

nobombus and Kallobombus were each found in multiple

clades, showing weaker trends of host association.

Furthermore, a well-supported, mixed clade of Snodg-

rassella strains from B. (Melanobombus) lapidarius and

B. (Bombus) terrestris/patagiatus/tunicatus was found.

One sample of B. (Pyrobombus) jonellus and B. (Psithyrus)

bohemicus each were nested within Snodgrassella strains

from the subgenus Bombus sensu stricto.

A ‘tanglegram’ linking the tips of the host phylogeny

and the Snodgrassella phylogeny revealed some phyloge-

netic congruency, with clear exceptions in several lin-

eages (Fig. 2). We used partial Mantel tests to test the

relative influence of the host species phylogeny and

the geographical sampling location on the similarity of

the Snodgrassella 16S rRNA gene sequences. After control-

ling for geographical distance between samples, we

found a strong and highly significant positive correlation

between the Snodgrassella phylogeny and the host phy-

logeny (r = 0.680, two tailed P = 0.0001, 10 000 permuta-

tions, n = 132). In contrast, the correlation between the

Snodgrassella phylogeny and geographical distance after

controlling for the host phylogeny was not significant

with little of the variance explained (r = 0.057, P = 0.186).

The ratio of host switches to cospeciation events in

the cophylogenetic analysis with Jane 4 varied from 1.4

to 1.5 for setting 1 (cost = 0 for cospeciations, cost = 1

for all other events, see methods) to 1.7–2.2 for setting 2

(cost = 1 for all events) (Table 1, Figs S1 & S2, Support-

ing information, host n = 37 species, Snodgrassella

n = 80). For both event cost settings, the costs of the

cophylogeny analyses from randomly generated associ-

ations were markedly higher and did not overlap with

Md. defector

Ps. campestrisPs. barbutellus

Pr. flavescens

Mg. hortorum

Sb. asiaticus

Ml. lapidarius

Kl. soroeensisPr. melanopygusPr. impatiens

Pr. mixtusPr. jonellus

Pr. flavifrons

Pr. vosnesenskii

Pr. vandykeiAl. balteatusBo. lucorum

Bo. terrestris

Bo. tunicatus

Ps. bohemicus

Bo. sporadicus

Th. pascuorum

Th. impetuosusTh. remotus

Th. californicusTh. sonorus

Pr. hypnorum

St. melanurus

Th. mesomelas

St. appositus

Bo. patagiatus

Or. haemorrhoidalis

Ml. festivus

Ml. friseanusMl. rufofasciatus

Apis ceranaApis mellifera

Apis dorsata

Bee host Associations SnodgrassellaFig. 2 Tanglegram linking the bee host

phylogeny (left) to the 16S rRNA gene

phylogeny of Snodgrassella alvi (right)

from the bee gut. Associations (grey lines)

link bacterial sequences with the host

species they were obtained from. Host

names indicate bumblebee subgenera

(Williams et al. 2008; see legend Fig. 1),

tanglegram produced in TreeMap 3

(Charleston (2011), http://sites.google.

com/site/cophylogeny/software).

© 2013 Blackwell Publishing Ltd

2034 H. KOCH ET AL.

the cost of the original associations (Table 1, 500 repli-

cates, P < 0.002), thus indicating a significant, if not per-

fect, congruence between the Snodgrassella and host tree.

Phylogeny of Gilliamella apicola, influence of hostphylogeny and geography

We analysed a matrix of 165 partial 16S rRNA gene

sequences (approx. 1290 bp, 138 sequences new to this

study), from 41 different bee host species (12 out of the 15

subgenera (Williams et al. 2008) of the Bombini). In com-

parison with the tree of Snodgrassella alvi (Fig. 1), the tree

of Gilliamella apicola appeared to be less structured

according to host species and host subgenera (Fig. 3).

Importantly, some of the sequences obtained with our

primers from the bumblebee samples appeared to be

closer to (but distinct from) a separate group of A. mellif-

era bacteria previously referred to as Gamma-2 phylotype

and other insect associated bacteria (red branches in

Fig. 3, Martinson et al. 2011). The remaining sequences

belong to Gilliamella apicola as defined by Martinson et al.

(2012) and Kwong & Moran (in press). Gilliamella apicola

strains from the Apini are mostly distinct from the bum-

blebee strains; however, two strains from Apis cerana fall

together with two bumblebee Gilliamella strains with high

bootstrap support. For the Apis-derived strains of Gilliam-

ella apicola, the different Apis host species do not have

completely distinct bacterial strains, as for example one

A. cerana strain falls within a group of A. mellifera sam-

ples, and some A. mellifera samples are closer to A. dorsata

strains than to other A. mellifera strains.

For the Bombus species, Gilliamella strains from the

host species Bombus (Orientalibombus) haemorrhoidalis

and Bombus (Thoracobombus) californicus are clustered in

well-supported, distinct clades. Furthermore, there is

some clustering between samples from the subgenera

Thoracobombus, Pyrobombus and Melanobombus, all of

which, however, do not form exlusive monophyletic

groups. The Gilliamella apicola tree thus appears consid-

erably less structured according to host species and

phylogeny compared with that of Snodgrassella alvi. This

limited degree of specificity also becomes apparent in

the more ‘tangled’ associations of Gilliamella strains and

host species as shown in Fig. 4 compared with the tan-

glegram of Snodgrassella in (Fig. 2).

A partial Mantel test of the Gilliamella and host phy-

logenies, while controlling for geographic distances

between samples, shows a moderate and significantly

positive correlation (including all sequences (see Fig. 3):

r = 0.332, two tailed P = 0.0001, 10 000 replications,

n = 148; excluding phylotypes outside of the main

Gilliamella apicola clade: r = 0.549, P = 0.0001, n = 128).

A significant but weaker correlation between bacterial

phylogeny and geography, while controlling for host

phylogeny, was also found (all sequences: r = 0.142,

P = 0.0002, excluding phylotypes outside of main Gil-

liamella apicola clade: r = 0.1541, P = 0.0003). When com-

pared to Snodgrassella alvi, the correlation between host

and bacterial associate phylogeny was thus weaker for

Gilliamella apicola, while a stronger correlation of bacte-

rial phylogeny and geography was observed.

For the cophylonenetic analysis in Jane 4, the inferred

ratio of host switches to cospeciation events varied from

1.7 to 2.0 for setting 1 (cost = 0 for cospeciations,

cost = 1 for all other events) to 2.8–3.0 for setting 2

(cost = 1 for all events) (Table 1, Figs S3 & S4, Support-

ing information, host n = 34 species, Gilliamella n = 96).

These predicted ratios of host switches per cospeciation

event were thus slightly higher for Gilliamella than for

Snodgrassella. The costs of the cophylogeny analyses

from randomly generated associations were higher than

the original costs without overlap (Table 1, 500 repli-

cates, P < 0.002), suggesting a significant, though

certainly imperfect, congruence between the Gilliamella

and host trees.

Experimental transmission to daughter queens in themother colony

We detected both Snodgrassella alvi and Gilliamella apico-

la with specific 16S rRNA gene primers in the gut of

almost all of the daughter queens sampled within the

Table 1 Results for the cophylogeny analyses with Jane 4 for Snodgrassella and Gilliamella phylogenies mapped on the bee host tree.

Bacterium Scheme # cosp # d # hs # l Cost # hs/# cosp Rand. rep. cost % rep. < orig. cost

Snodgrassella cosp = 0 20–21 4 29–30 3 67 1.4–1.5 89 0 (~ P < 0.002)

Gilliamella cosp = 0 20–23 9–11 40–44 4–11 102 1.7–2.2 121 0 (~ P < 0.002)

Snodgrassella cosp = 1 16–18 5–6 31–32 1–2 87 1.7–2.0 98 0 (~P < 0.002)

Gilliamella cosp = 1 15–16 13–14 45 1 120 2.8–3.0 134 0 (~P < 0.002)

Scheme: cosp = 0: cost = 0 for cospeciation, cost = 1 for all other events, cosp = 1: cost = 1 for all events. # cosp = number of cospeci-

ation events, d = duplications, hs = host switches, l = losses, no ‘failure to diverge’ events were predicted. Cost = optimal cost for

each analysis. # hs/# cosp = ratio between the numbers of host-switching and cospeciation events. Rand. rep. cost = mean cost of

500 replicates with randomly mapped tips.% rep. < orig. cost = percentage of replicates with lower cost than original solution. See

Figs S1–S4 (Supporting information) for solutions of the analyses.

© 2013 Blackwell Publishing Ltd

EVOLUTION OF CORBICULATE BEE BACTERIA 2035

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Fig. 3 Phylogenetic tree (maximum-likelihood) of 16S rRNA gene sequences from Gilliamella apicola strains of different bee hosts.

Sequences outside the main Gilliamella apicola clade and closer to the Gamma-2 phylotype (sensu Martinson et al. 2011) are displayed

with dark red branches. Haemophilus paraphropha and Edwardsiella hoshinae were selected as most closely related outgroup taxa for the

root. Branch labels are bootstrap values over 50%. Colours indicate different bumblebee subgenera following Williams et al. (2008)

(see legend) and different shades of grey represent different Apis species. Symbols code for geographic origin of each sample (see leg-

end). Tip labels include species name and sample ID (see Table S1, Supporting information). For further details, see Methods.

© 2013 Blackwell Publishing Ltd

2036 H. KOCH ET AL.

field-placed B. terrestris colonies (Snodgrassella: 15/16

positive, Gilliamella: 16/16 positive). The 16S rRNA gene

sequences from Snodgrassella appeared to be distinct for

the four colonies, however, with overlap between

daughter queens from colonies 09.350 and 09.013 (Fig.

S5, Supporting information). The pattern was less clear

for Gilliamella sequences, with strains from all four colo-

nies being mixed in a phylogenetic tree, with only col-

ony 09.018 showing a distinct genotype for some of the

bacterial strains (Fig. S5, Supporting information). As

these bees remained inside of the colony environment

from eclosion until sampling, our findings indicate a

within-colony transfer of these elements of the gut mic-

robiota under natural conditions.

We further examined the transmission and mainte-

nance during hibernation of the target bacteria in labo-

ratory-reared daughter queens. Daughter queens that

had interacted as adults with other individuals within

the colony all tested positive for Snodgrassella (5/5) or

Gilliamella (5/5) before hibernation. After hibernation,

all surviving daughter queens tested positive for Snodg-

rassella (5/5), while only some of the daughter queens

harboured Gilliamella (2/5). In the group kept in perma-

nent isolation after pupal emergence, only very few

individuals tested positive for the presence of the target

bacteria (Snodgrassella: 1/5 before hibernation, 2/5 after

hibernation; Gilliamella: 1/5 before hibernation, 0/5 after

hibernation). Unfortunately, sample sizes after hiberna-

tion were rather low because out of the 40 daughter

queens originally put into hibernation an unusually low

number of only five individuals from each group (isola-

tion/colony contact) had survived. The three worker

offspring individuals produced by two of the colony

contact queens shared the infection pattern of their

mother queen with Snodgrassella being present and

Gilliamella absent. We also screened samples of honey

pot content and wax from two different laboratory colo-

nies with specific PCRs. Both honey pot samples and

both wax samples were positive for Gilliamella, while no

amplification was observed for Snodgrassella.

Phylogeny of Meliponini and Euglossini bacteria

In our sample of two species of stingless bees and three

species of orchid bees, no positive amplification for the

targeted bacterial species Snodgrassella alvi and Gilliamel-

la apicola was observed with specific primers (Table S2,

Supporting information). A control PCR with universal

eubacterial 16S rRNA gene primers on the bee gut sam-

ples, however, resulted in strong amplification of a

Ml. lapidarius

Kl. soroeensis

Bo. terrestrisBo. tunicatus

Mg. hortorum

Pr. mixtus

Pr. impatiens

Md. defector

Ps. bohemicus

Al. balteatusPr. hypnorum

Bo. lucorumBo. patagiatus

Ps. campestris

Pr. jonellus

Pr. vosnesenskiiPr. melanopygusPr. caliginosus

Ps. fernaldae

Bo. sporadicus

A. ceranaA. dorsata

A. mellifera

Th. californicus

Cu. griseocollisMl. festivus

Ml. rufofasciatusMl. friseanus

Th. impetuosus

Th. pascuorumTh. remotus

Or. haemorrhoidalis

Th. mesomelas

St. melanurusSt. appositus

Mg. trifasciatus

Pr. bifarius

Pr. flavifronsPr. vandykei

Pr. flavescens

Host Associations GilliamellaFig. 4 Tanglegram linking the bee host

phylogeny (left) to the 16S rRNA gene

phylogeny of Gilliamella apicola from the

bee gut (right). Associations (grey lines)

link bacterial sequences with the host

species they were obtained from. Host

names indicate bee genus/subgenus (Wil-

liams et al. 2008; see legend Fig. 3).

© 2013 Blackwell Publishing Ltd

EVOLUTION OF CORBICULATE BEE BACTERIA 2037

product of the expected size (approx. 1.5 kb) indicating

sufficient quality of the DNA template. A negative

result with the specific 16S rRNA gene primers could

also have come from primer mismatches to Snodgrassella

and Gilliamella in the Meliponini and Euglossini. There-

fore, we cloned the amplification product of the PCR

with universal eubacterial 16S rRNA gene primers for a

selection of samples from both Euglossini and Melipo-

nini. A total of 24 randomly chosen clones were picked

and sequenced for each sample. After removing chime-

ric sequences, bacterial 16S rRNA gene sequences for a

total of 170 clones were obtained, of which 86 were

from the Meliponini and 84 from the Euglossini sam-

ples. None of the clones was similar to Snodgrassella alvi

or Gilliamella apicola from the Bombini and Apini. We

selected clones of distinct sequences from each clone

library for sequencing of the full vector insert.

From these tests, the phylogenetic position of the gut

bacteria of the Meliponini and Euglossini, in compari-

son with their closest described relatives and other pre-

viously sequenced bacteria from bees, is shown in

Fig. 5. In particular, all three Euglossini species had

high numbers of clones from the Acetobacteraceae,

forming a distinct clade related to other Acetobactera-

ceae previously found in flowers (Saccharibacter floricola,

see Jojima et al. 2004) and in other insects including

honeybees and a solitary bee (Colletidae: Caupolicana

yarrowi, around 3–4% divergent to alpha 2.2 sensu

Martinson et al. 2011). The Eulaema sp. sample also har-

boured a bacterium from the Enterobacteraceae close to

Yokenella regensburgi. Both Euglossa imperialis individuals

also had high numbers of clones of an unknown Alpha-

proteobacterium. These clones were closest to a bacte-

rium from the hemipteran Dactylopius opuntiae, but also

showed similarity to a bacterium of the isopod Porcelio

scaber (‘Candidatus Hepatincola porcellionum’) and a

bacterium from Daphnia magna.

Both species of Meliponini were also hosts to differ-

ent strains of Acetobacteraceae. In addition, a wide

variety of bacteria from the order Lactobacillales were

found, including strains close to the genera Streptococ-

cus, Leuconostoc, Aerococcus and Lactobacillus. Some of

these lactobacilli were closely related to bacteria previ-

ously found in honeybees.

Discussion

Snodgrassella alvi and Gilliamella apicola gut bacteriaof bumblebees and honeybees

We obtained and analysed 16S rRNA gene sequences of

bumblebees and honeybees covering most of the phylo-

genetic diversity of the hosts and spanning a wide geo-

graphical range over three continents (North America,

Europe and Asia). The vast majority of bumblebee spe-

cies (92%) and all honeybee species screened positive

for Gilliamella and Snodgrassella.

Our data reject a strict cospeciation of Snodgrassella

and Gilliamella with their bee hosts. The association

between these bacteria and bees appears to allow for a

considerable degree of host switching, contrasting with

the strict cospeciation pattern observed for some obli-

gate intracellular insect symbionts (Clark et al. 2000).

Both Mantel tests and reconciliation analyses suggest a

complex history with a number of possible host

switches. For example, we observed Snodgrassella geno-

types in bumblebees of the subgenus Mendacibombus

identical with genotypes in other distantly related spe-

cies [e.g. Bombus (Pyrobombus) flavescens]. These host

species have probably diverged more than 30 million

years ago (Hines 2008), which would lead to noticeable

16S rRNA gene sequence divergence between bacterial

strains in the case of strict cospeciation (Ochman et al.

1999). Nevertheless, for both Snodgrassella and Gilliamel-

la, we found a significant correlation between the 16S

rRNA gene bacterial phylogeny and the host phylog-

eny. The reconciliation analyses furthermore suggest

the possibility for a number of cospeciation events. In

contrast, geographic distance of the sampled bees corre-

lated only weakly with the Gilliamella tree and not at all

with the Snodgrassella tree. This suggests a close associa-

tion of both bacteria with their hosts, which has been

maintained over large geographical distances and long

periods of time.

Several mechanisms could underlie the topological

congruence of the host and Snodgrassella/Gilliamella

trees. For example, bacterial associates could be more or

less exclusively vertically transmitted (Clark et al. 2000;

Hosokawa et al. 2006). We provide experimental evi-

dence that vertical transmission from the mother colony

to young queens is possible, although the frequency of

horizontal transmission in the field remains to be deter-

mined. The presence of vertical transmission in this sys-

tem may allow for co-evolutionary dynamics in which

host and bacteria exert reciprocal selection on each

other. There is some evidence for a beneficial function

of Snodgrassella and Gilliamella in parasite defence for

Bombus terrestris (Koch & Schmid-Hempel 2011b, 2012),

but whether these members of the microbiota have fit-

ness effects on different honeybee and bumblebee

species remains to be investigated. In this light, it

remains possible that the bacteria have evolved host

specificity without exerting a selective pressure on the

host as commensalistic passengers in the bee gut. A

purely commensalistic relationship would, however, be

somewhat surprising given the high prevalence of both

bacteria across the different targeted bee species and

geographic region. Other explanations for nonrandom

© 2013 Blackwell Publishing Ltd

2038 H. KOCH ET AL.

topological matches between host and bacterial trees

should also be considered. For example, host-switching

event may predominantly occur between closely related

hosts, leading to a match of host and bacterial phylog-

eny in the absence of detectable cospeciation events

(De Vienne et al. 2007).

When compared to Snodgrassella alvi, it emerged that

the association of Gilliamella apicola appears to be less

specific. While we still detect a significant correlation

between host and bacterial associate phylogeny, with

some clades of Gilliamella exclusively restricted to cer-

tain host species (e.g. Bombus haemorrhoidalis, Bombus

0.2

EU034639 Gluconobacter oxydans

GU125609 Lactobacillus sakei

GQ853368 Alphaproteobacterium (Dactylopius opuntiae)

JQ389885 Melipona panamica (B10.043)

AB681877 Yokenella regensburgeri

FJ025127 Sinorhizobium meliloti

EF187245 Lactobacillus (A. mellifera)

JQ389893 Melipona panamica (B10.042)

HM534760 Lactobacillus (Melipona beecheii)

EU096234 Gluconobacter morbifer (Drosophila)

EF187239 Lactobacillus (A. mellifera)

HM534763 Lactobacillus (Melipona beecheii)

HM113169 Lactobacillus (A. mellifera)

NR_024819 Saccharibacter floricola (flower)

HM534761 Lactobacillus (Trigona sp.)

JN392909 Alphaproteobacterium (Daphnia magna)

HQ425688 Aerococcus viridans

AY370183 Lactobacillus (A. mellifera)

HM534779 Lactobacillus (A. cerana)

JQ389891 Meliponula bocandei (B10.046)

Y11374 Lactobacillus kunkeei

HM534811 Lactobacillus (A. florea)

AY189806 “Ca. Hepatincola porcellionum”

AB499842 Acetobacter pasteurianusAB513363 Aceteobacteraceae (flower)

AY773947 Lactobacillus acidophilus

AF009487 Streptococcus suis (pig)

JQ389880 Euglossa imperialis (B10.023)

JQ389890 Melipona panamica (B10.043)

JQ389892 Melipona panamica (B10.043)

HM108484 Acetobacteraceae (A. dorsata)

EF187244 Lactobacillus (A. mellifera)

AB678443 Gluconobacter frateurii

X95421 Lactobacillus lindneri

JQ389894 Meliponula bocandei (B10.046)

AJ971850 Acetobacteraceae (A. mellifera)

DQ068792 Enterobacteriaceae (Myrmeleon)

JN644571 Aerococcus (Culex quinquefasciatus)

HM112107 Lactobacillus (A. mellifera)

JQ389879 Eulaema sp. (B10.049)

HM534814 Lactobacillus (Trigona sp.)

JQ389889 Melipona panamica (B10.042)

AM157443 Streptococcus (human)

JQ389884 Eulaema sp. (B10.049)

HM046576 Lactobacillus (A. cerana)

DQ837625 Acetobacteraceae (A. mellifera)

AB680295 Streptococcus equinus (horse)

AJ419836 Acetobacter peroxydans

HM534764 Lactobacillus (A. koschevnikovi)

AB362721 Leuconostoc citreum

DQ860016 Streptococcus (anchovi)

HM109591 Acetobacteraceae (Caupolicana yarrowi)

AB682268 Klebsiella oxytoca

JQ389887 Meliponula bocandei (B10.046)

AB429371 Lactobacillus kefiri

HM534762 Lactobacillus (Meliponula bocandei)

HM215048 Lactobacillus (B. terrestris)

HM534813 Lactobacillus (Meliponula bocandei)

HM534806 Lactobacillus (Melipona beecheii)

U11014 Rickettsia bellii

JQ389888 Melipona panamica (B10.042)

JQ389883 Eufrisea sp. (B10.050)

JQ389881 Euglossa imperialis (B10.022)

AB326299 Leuconostoc pseudomesenteroides

JQ389895 Melipona panamica (B10.043)

HM112629 Leuconostoc (Diadasia opuntiae)

JQ389882 Euglossa imperialis (B10.022)

EU096229 Acetobacter pomorum (Drosophila)

EF179826 Enterobacteriaceae (Anopheles)

NR_042194 Lactobacillus tucceti

JQ389886 Meliponula bocandei (B10.047)

AB680259 Aerococcus urinaeequi

53

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100

Fig. 5 Phylogenetic tree of bacterial 16S rRNA gene sequences from stingless bees and orchid bees. Tip label colours indicate isola-

tion source: from the gut of stingless bees (green), orchid bees (blue), honey bees (red), other bee species (purple) and bacterial type

strains/isolates from nonbee hosts (black). Tip labels include GenBank Accession nos, host species and bee specimen sample num-

bers for sequences obtained from clone libraries in this study. The tree was computed using maximum-likelihood and was midpoint

rooted; branch labels indicate bootstrap values over 50% (for further details, see Methods).

© 2013 Blackwell Publishing Ltd

EVOLUTION OF CORBICULATE BEE BACTERIA 2039

californicus), inferred host switches appear to be more

common both within honeybees and bumblebees. Fur-

thermore, the influence of geographical sampling loca-

tion appears to be stronger for these bacteria and,

hence, horizontal transfer of these bacteria through

environmental sources may be more common (see

‘Transmission mode’ section of the Discussion).

Potentially, clades within the Snodgrassella and Gil-

liamella tree may also differ in their host specificity,

with some clades apparently present in a number of

distantly related host species, while others seem highly

specialized to distinct hosts. Therefore, considerable

variation may exist in the evolutionary dynamics within

both bacterial taxa, leading to a complex pattern from

frequent host switches to specialized interactions.

Some limitations are associated with the approach of

direct sequencing of 16S rRNA gene fragments in our

study. For example, the 16S rRNA gene is highly con-

served, whereas bumblebees are a relatively recently

diverged group with extant species probably sharing

their last common ancestor only about 35 million years

ago, and speciation within the different subgenera

occurring mostly within the last 15 million years (Hines

2008). The 16S rRNA gene alone may furthermore con-

siderably underestimate the true genetic strain diversity

in the bee gut (Engel et al. 2012). Therefore, an

approach including additional molecular markers such

as multilocus sequence typing (MLST) may be needed

to resolve potential specificity among gut bacteria of

recently diverged hosts (Falush et al. 2003; Oh et al.

2010; Kwong & Moran in press). However, as we never-

theless find strong evidence for a correlation between

host and bacteria phylogeny even at the level of low

resolution with the 16S rRNA gene, this pattern can be

assumed to be robust. As an additional caveat, our

approach of directly sequencing PCR products obtained

with specific primers may bias our analysis towards the

majority strain present in a particular sample, while

rarer strains will be overlooked. Our approach can thus

not exclude the existence of additional rare strains with

different patterns of codiversification. In Apis mellifera,

several strains of Snodgrassella and Gilliamella with dis-

tinct 16S rRNA gene sequences can be present in one

individual (Moran et al. 2012), although individuals in

general harboured a single dominant phylotype. Gener-

ally, in our study, directly sequenced PCR products

gave clear chromatograms of good quality. In some

cases, however, ambiguous bases were detected and

coded as such in the phylogenetic analysis, indicating

the presence of different strains in one individual (0.2%

ambiguous bases for Gilliamella, 0.06% for Snodgrassella).

In addition, queens of single colonies also had multiple,

nonmonophyletic strains (Fig. S5), pointing to within-

colony strain diversity. The extent of strain variation of

Snodgrassella and Gilliamella at the scale of individual

bees thus remains to be investigated.

Transmission mode

We show that both Snodgrassella and Gilliamella are

transmitted to daughter queens within the mother col-

ony in the field. Furthermore, we show that in a labora-

tory environment, social contact of daughter queens

after pupal emergence increases the chances of transmis-

sion of these bacteria and that bacteria can persist in the

daughter queens during hibernation. Similarly, Martin-

son et al. (2012) showed that in honeybees these bacteria

are acquired by newly emerged workers within the col-

ony environment. A comparison of the two bacterial

taxa appears to be in line with the findings of the

across-host species comparison in this study, which

showed a higher degree of specificity for Snodgrassella.

In our small sample, we observed distinct genotypes of

Snodgrassella strains in daughter queens from different

colonies, while this pattern was not apparent for Gilliam-

ella strains. Furthermore, hibernating daughter queens

may be more likely to lose Gilliamella than Snodgrassella

during hibernation. While all daughter queens in our

laboratory sample that had spent time in their mother

colony after emergence harboured both Snodgrassella

and Gilliamella, some of the daughter queens screened

after hibernation lacked Gilliamella, while Snodgrassella

were still present in all individuals. The detection of Gil-

liamella in the content of nectar pots and on wax surfaces

within the nest and their growth on standard culture

media, in contrast to Snodgrassella (Olofsson & V�asquez

2008; Koch & Schmid-Hempel 2011b), further suggests

that Gilliamella is less intimately associated with their

hosts and present in the hive environment.

Taken together, these results suggest that both Snodg-

rassella and Gilliamella can be transmitted ‘vertically’

from the mother colony to the young gynes. Gilliamella,

however, may be transmitted horizontally outside of the

nest more frequently than Snodgrassella. Potentially, the

horizontal transmission could occur on flowers. Recent

studies have shown a considerable diversity of bumble-

bee-transmitted yeasts in flowers (Brysch-Herzberg

2004; Herrera et al. 2008). In a study focusing on lactoba-

cilli associated with bees (McFrederick et al. 2012), flow-

ers were suggested as transmission sites especially for

Lactobacillus spp. of halictid bees. Bacterial communities

in flowers have so far, however, been very poorly stud-

ied, and the few available surveys did not report the

bacteria that we focused on in this study (Junker et al.

2011; Fridman et al. 2012). Bees may furthermore acquire

bacteria horizontally through direct contact with other

bee species. For example, we detected a Snodgrassella

strain from the cuckoo bumblebee Bombus (Psithyrus)

© 2013 Blackwell Publishing Ltd

2040 H. KOCH ET AL.

bohemicus, nested within bacterial strains of its host spe-

cies, that is, members of the subgenus Bombus s.str. As

B. bohemicus parasitizes nests of B. lucorum (Alford 1975)

without producing a worker caste on its own, it may

potentially have acquired bacteria from its host colony.

In conclusion, the relative importance of horizontal

and vertical transmission in the field for the two

groups of target bacteria thus remains to be investigated

further.

Bacteria in Euglossini and Meliponini

Only few studies have previously looked at microor-

ganisms of stingless bees (Meliponini). Rosa et al. (2003)

found a variety of yeasts, Gilliam et al. (1985, 1990)

described several Bacillus spp., and V�asquez et al. (2012)

focussed on lactic acid bacteria associated with stingless

bees. There has not been, to our knowledge, any study

of microorganisms associated with orchid bees (Euglos-

sini) to date.

In our survey of two stingless bee and three orchid

bee species, we detected none of the Snodgrassella alvi or

Gilliamella apicola bacteria commonly found in bumble-

bees and honeybees. This suggests that these microbial

associates may be either rare or absent in the Euglossini

and Meliponini and are potentially unique to the Apini

and Bombini.

Bacteria from the Acetobacteraceae appear to be com-

mon members of the gut microbiota of the Euglossini

and Meliponini. Acetic acid bacteria have previously

been found to be common in the guts of insects relying

on a sugar-based diet, including honeybees, fruit flies,

and mealy bugs (Crotti et al. 2010). They also occur in

flowers (Jojima et al. 2004), suggesting the possibility of

environmental transmission to bees through their food

source. In addition to members of the Acetobacteraceae,

the orchid bee Euglossa imperialis harboured a novel Al-

phaproteobacterium, so far not recorded from bees, and

related to the isopod associated bacterium ‘Candidatus

Hepatincola porcellionum’ (Rickettsiales) (Wang et al.

2004).

The stingless bee gut microbiota furthermore contains

a variety of lactic acid bacteria (Lactobacillales), includ-

ing undescribed members of the genera Lactobacillus,

Leuconostoc, Streptococcus and Aerococcus. In this respect,

the stingless bee microbiota appears to be similar to the

microbiota of honeybees and to a lesser degree also

bumblebees (Olofsson & V�asquez 2008, 2009; Koch &

Schmid-Hempel 2011a; Martinson et al. 2011; V�asquez

et al. 2012). In honeybees, these lactic acid bacteria have

been suggested to play a beneficial role in protection

against pathogens (Forsgren et al. 2010; V�asquez et al.

2012). In line with the findings of V�asquez et al. (2012),

lactic acid bacteria may in conclusion present hitherto

neglected but important associates of stingless bees.

Acknowledgements

G. Cisarovsky, D.W. Roubik, B. Sadd, R. Schmid-Hempel,

E. Stolle, E. Tesch and M. Tognazzo provided help with collect-

ing bee samples (see Table S1, Supporting information). S. Jost,

M. Berchtold and E. Karaus helped with molecular cloning

and sequencing. T. Stadler gave helpful advice on statistical

analyses. Sequence data were generated in the Genetic Diver-

sity Centre (GDC) of ETH Zurich. The study was supported by

grants of the Swiss SNF (grant no. 31003A-116057 to PSH &

no. 140157 to HK), an ERC Advanced Grant (no. 268853 to

PSH), and a Sino-Swiss Science and Technology Cooperation

Agreement (EG13-032009 and 2009DFA32600 to PSH and JL).

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H.K. and P.S.-H. designed research, H.K. performed

laboratory experiments, P.S.-H., H.K., J.L. and D.P.A.

organized collection trips and contributed bee speci-

mens, H.K. analysed data and wrote the paper, H.K.,

P.S.-H. and J.L. contributed to revisions of the original

manuscript.

Data accessibility

DNA sequences: GenBank Accessions JQ389879–

JQ390033 & JQ405085–JQ405210. Alignment of 16S

rRNA gene phylogenies: Treebank submission ID 13472.

Sample information (sample ID, species identification,

sampling locality, collector, corresponding GenBank

Accession nos) in Table S1 (Supporting information).

Supporting information

Additional supporting information may be found in the online ver-

sion of this article.

Fig. S1 Result of reconciliation analysis with Jane (Conow et al.

2010) for Snodgrassella alvi and bee host phylogenies for the

event cost setting cosp. = 0.

© 2013 Blackwell Publishing Ltd

EVOLUTION OF CORBICULATE BEE BACTERIA 2043

Fig. S2 Result of reconciliation analysis for Snodgrassella alvi

and bee host phylogenies for the event cost setting cosp. = 1.

Fig. S3 Result of reconciliation analysis for Gilliamella apicola

and bee host phylogenies for the event cost setting cosp. = 0.

Fig. S4 Result of reconciliation analysis for Gilliamella apicola

and bee host phylogenies for the event cost setting cosp. = 1.

Fig. S5 Unrooted phylogenetic trees (maximum-likelihood) of

16S rRNA gene sequences of Snodgrassella alvi and Gilliamella

apicola strains of the gut from recently emerged (1–7 days old)

young queens collected inside four different Bombus terrestris

field colonies.

Table S1 List of all honey bee and bumble bee specimens

with bee species identification, sample ID, sampling location/

country, sampling date, coordinates in decimal degrees (DD),

collector (BS: Ben Sadd, DPA: Dharam P. Abrol, DWR: David

W. Roubik, ES: Eckart Stolle, HK: Hauke Koch, JL: Jilian Li,

MT: Martina Tognazzo, RSH: Regula Schmid-Hempel) and

GenBank Accession nos of 16S rRNA gene sequences for

Snodgrassella and Gilliamella.

Table S2 Results of the screen of all corbiculate bee species

samples (Table S1, Supporting information) with specific 16S

rRNA gene primers for the target bacteria Snodgrassella alvi

and Gilliamella apicola.

© 2013 Blackwell Publishing Ltd

2044 H. KOCH ET AL.