Environment Or Kin: Whence Do Bees Obtain Acidophilic Bacteria?

Home , Enterococcus moraviensis, Eremococcus, Eremococcus coleocola, Halictus ligatus, Lactobacillus pontis

Molecular Ecology (2012) doi: 10.1111/j.1365-294X.2012.05496.x

Environment or kin: whence do bees obtain acidophilic bacteria?

QUINN S. MC FREDERICK,* WILLIAM T. WCISLO,† DOUGLAS R. TAYLOR,‡ HEATHER D. ISHAK,* SCOT E. DOWD§ and ULRICH G. MUELLER* *Section of Integrative Biology, University of Texas at Austin, 2401 Speedway Drive #C0930. Austin, TX 78712, USA, †Smithsonian Tropical Research Institute, Box 0843-03092, Balboa, Ancon, Republic of Panama, ‡Department of Biology, University of Virginia, Charlottesville, VA 22904-4328, USA, §MR DNA (Molecular Research), 503 Clovis Rd., Shallowater, TX 79363, USA

Abstract As honey bee populations decline, interest in pathogenic and mutualistic relationships between bees and microorganisms has increased. Honey bees and bumble bees appear to have a simple intestinal bacterial fauna that includes acidophilic bacteria. Here, we explore the hypothesis that sweat bees can acquire acidophilic bacteria from the environment. To quantify bacterial communities associated with two species of North American and one species of Neotropical sweat bees, we conducted 16S rDNA amplicon 454 pyrosequencing of bacteria associated with the bees, their brood cells and their nests. Lactobacillus spp. were the most abundant bacteria in many, but not all, of the samples. To determine whether bee-associated lactobacilli can also be found in the environment, we reconstructed the phylogenetic relationships of the genus Lactobacillus. Previously described groups that associate with Bombus and Apis appeared relatively specific to these genera. Close relatives of several bacteria that have been isolated from flowers, however, were isolated from bees. Additionally, all three sweat bee species associated with lactobacilli related to flower-associated lactobacilli. These data suggest that there may be at least two different means by which bees acquire putative probiotics. Some lactobacilli appear specific to corbiculate apids, possibly because they are largely maternally inherited (vertically transmitted). Other lactobacilli, however, may be regularly acquired from environmental sources such as flowers. Sweat bee–associated lactobacilli were found to be abundant in the pollen and frass inside the nests of halictids, suggesting that they could play a role in suppressing the growth of moulds and other spoilage organisms.

Keywords: 16S rDNA amplicon 454 pyrosequencing, acidophilic bacteria, insect diseases, insect symbiosis, Lactobacillus, phylogeny Received 8 July 2011; revision received 6 January 2012; accepted 13 January 2012

2011). On the opposite end of the spectrum, there is Introduction accumulating evidence for a worldwide association As Apis mellifera populations continue to decline, between Ap. mellifera and a core set of bacterial phylo- interest in the role of microbes in the health of pollina- types (Jeyaprakash et al. 2003; Mohr & Tebbe 2006, tors continues to increase. For example, several recent 2007; Babendreier et al. 2007; Cox-Foster et al. 2007; studies have highlighted the possible roles of pathogens Martinson et al. 2011). Similar associations have been in pollinator declines (Cox-Foster et al. 2007; Johnson reported between other corbiculate apids and bacteria, et al. 2009; Bromenshenk et al. 2010; Cameron et al. especially Bombus spp. (Mohr & Tebbe 2006; Koch & Schmid-Hempel 2011a; Martinson et al. 2011). Correspondence: Quinn S. McFrederick, Fax: +1 (512) 471-3878; Because speciﬁc bacteria are consistently associated E-mail: [email protected] with Ap. mellifera, it has been suggested that these bacteria

2012 Blackwell Publishing Ltd 2 Q. S. MCFREDERICK ET AL. are beneficial mutualists (Martinson et al. 2011). Experi- 2009; Vasquez et al. 2009), whereas several others have mental evidence of the benefits of core Apis bacteria on failed to detect L. kunkeei phylotypes (Jeyaprakash et al. bee health, however, is lacking, with the exception of the 2003; Babendreier et al. 2007; Cox-Foster et al. 2007; lactic acid bacteria (LAB). A mixture of Lactobacillus and Martinson et al. 2011). Although differences in method- Bifidobacterium phylotypes has been shown to decrease the ology across surveys may explain this variation, envi- infection rate of Ap. mellifera larvae that were exposed to ronmental acquisition of L. kunkeei is another possible the pathogen Paenibacillus larvae (Forsgren et al. 2010). In explanation. Additionally, LAB community composition addition, LAB have been isolated from the crop of appears to vary seasonally. Lactobacillus kunkeei was Ap. mellifera, freshly collected pollen and freshly fer- found to dominate the LAB microbiota of Ap. mellifera mented bee bread, which is the protein source for larval honey stomachs in both Sweden and the United States, Ap. mellifera (Vasquez & Olofsson 2009). The fermentative but LAB community membership and abundance was properties of LAB have been hypothesized to aid in the found to vary with foraging activity (Vasquez et al. conversion of pollen to bee bread and the protection of 2009). This response of the Ap. mellifera LAB commu- bee bread from spoilage (Vasquez & Olofsson 2009). It has nity to foraging may also be explained by environmen- also been hypothesized that Ap. mellifera add LAB to tal acquisition of a subset of the LAB microbiota. It is collected nectar and that LAB ferment nectar, possibly aid- therefore plausible that some LAB associated with ing in the conversion of nectar to honey (Olofsson & Ap. mellifera may be environmentally transmitted, while Vasquez 2008). Lastly, it has recently been shown that others are maternally inherited (vertically transmitted). bacteria associated with bumble bees can benefit their Here, we address the hypothesis that some bee- hosts. The microbiota of Bombus terrestris, which resembles associated, acidophilic bacteria are environmentally the Ap. mellifera microbiota, protects the bee hosts from acquired. First, we use a 16S rDNA amplicon survey to Crithidia bombi, a trypanosomatid gut parasite (Koch & study the microbiota of three species of halictid bees as Schmid-Hempel 2011b). well as the bacterial communities associated with the There exists, therefore, some evidence that Ap. mellif- contents of their nests. We use this information to era and Bombus spp. associate with beneficial bacteria. detect the presence of putative mutualists and patho- However, these bacterial phylotypes appear to be gen- gens and to explore bacterial community composition erally lacking from other bees. Martinson et al. (2011) and diversity. Next, we present a phylogeny of the lac- surveyed lineages across the bee phylogeny and found tobacilli, combining our 16S rDNA sequence data with the bacterial phylotypes associated with Ap. mellifera to publicly available sequence information. We find that be almost completely lacking from bees outside the cor- three of the four lactobacilli known from flowers have biculate apids. Instead, bees outside the corbiculate also been isolated from bees. Additionally, L. kunkeei apids exhibited no predictable associations, besides the has been isolated from flowers and Apis spp., and is presence of a common environmental bacterium, Burk- closely related to lactobacilli isolated from sweat bees. holderia cepacia, in most specimens. Why the corbiculate Together, these data suggest that the acquisition of apids are consistently associated with a small commu- putatively beneficial bacteria from the environment may nity of putative mutualists, whereas other bees are not, be more important than previously thought. is an open question. Martinson et al. (2011) suggested the hypothesis that bacteria associated with corbiculate Study organisms and methods apids are transferred vertically between generations and that eusociality facilitates this transmission, either The structure of halictid nests makes them particularly via trophallaxis (within nest oral-oral sharing of food) interesting for studies of bacterial communities. Halictid or via the swarming (nest fission) method of colony bees build brood cells that they line with the secretion foundation found in Ap. mellifera. of the Dufour’s gland, which creates a waterproof lining The fact that a core set of bacterial phylotypes are (Duffield et al. 1981). Adult females then provision the almost exclusively associated with corbiculate apids, brood cell with pollen and nectar, lay an egg on top of while consistent with the notion of vertical transmission the provisions and seal the cell with a plug (Michener of bacteria from mother to offspring, does not reject the 1974). This brood cell typically remains sealed until the hypothesis that beneficial bacteria can be acquired envi- brood ecloses into an adult, although some halictids ronmentally. Bacterial phylotypes other than the core open brood cells for inspection (Michener 1974). The set, for example, have been repeatedly detected in asso- newly eclosed adult bee breaks through the plug, and ciation with Ap. mellifera, but not consistently across all the cell is either abandoned or cleaned and reused. surveys. Several studies have detected Lactobacillus kun- Cells can also include microbes, mites, nematodes and keei in association with Ap. mellifera (Mohr & Tebbe springtails; they can therefore be thought of as minia- 2007; Olofsson & Vasquez 2008; Vasquez & Olofsson ture, ephemeral ecosystems (sensu Biani et al. 2009).

2012 Blackwell Publishing Ltd BEES AND ACIDOPHILIC BACTERIA 3

understory and exhibits trophallaxis (Wcislo et al. 2004; Collections Wcislo & Gonzalez 2006). We collected a solitary nest We sampled adults, brood, pollen provisions, frass, tun- (N 0909¢35.4¢¢ W 07950¢21.2¢¢) on 24 January 2011 and nel-wall scrapings and the nearby nesting substrate a social nest (one queen and one worker, N 0909¢40.7¢¢ from the nests of three species of sweat bees. For all col- W 07950¢26.7¢¢) on 3 February 2011. To collect samples, lections, we collected the samples directly into molecu- we carried the nests back to the laboratory and dis- lar-grade ethanol, with forceps that we flame-sterilized sected them inside a plastic container sanitized with between handling each sample. We carefully opened 10% bleach solution followed by 100% ethanol, kept on nests and brood cells to minimize contamination. its side and with a alcohol bulb burning in front of the First, we sampled the entire contents of a wild Aug- tub in order to minimize the entrance of airborne ochlora pura nest found in a decaying log at Blandy microbes into the samples. We were unable to amplify Experimental Farm in Boyce, Virginia (N 3903¢20.63¢¢ DNA from three samples from the social nest. Our sam- W7804¢22.48¢¢) on 14 August 2009. Au. pura is a soli- ples, however, still spanned developmental stages rang- tary halictid in the tribe Augochlorini (Michener 1974). ing from the pollen provision from an egg to the frass We dissected the nest in the field. For insect samples, from a pupa to an adult bee. We again collected sam- we screened the entire insect; therefore, these communi- ples from the tunnel walls of the nest and wood and ties represent both intestinal and surface microbes. We scrapings from different parts of the stick that did not sampled the adult mother of the nest along with three directly contact the nest, and exposed a vial of ethanol brood and their brood cell contents. Lastly, we haphaz- to the air as a negative control. We also measured the ardly sampled several portions of the tunnel walls from pH of the pollen provisions with pH paper (Hydrion, inside the nest, decayed wood that we haphazardly col- Micro Essential Laboratory) and pollen mixed 1:1 with lected from the rotten log that housed the nest (within nanopure water. 0.3 m of the nest), and opened a collecting vial to be exposed to air as a negative control. Bacterial tag-encoded FLX 454 pyrosequencing We also sampled the entire contents of a laboratory (bTEFAP) and bioinformatics nest of Halictus ligatus that was kept in an environmental chamber at the University of Virginia. Although Research and Testing Laboratories (Lubbock TX) H. ligatus is an obligate eusocial bee, workers collected conducted DNA extraction, PCR and sequencing using from flowers will initiate nests in a solitary manner. previously described protocols (Dowd et al. 2008; Sen The brood from these nests are usually entirely male, as et al. 2009; Ishak et al. 2011). We used the 28F (5¢GAGT most H. ligatus workers are not mated (Packer 1986). TTGATCNTGGCTCAG) and 519R (5¢GTNTTACNGCG The mother of this nest was a worker collected at GCKGCTG) primer pair, which amplifies the V1, V2 Blandy Experimental Farm from a floral head of Card- and V3 regions of the 16S rRNA gene. After sequenc- uus nuttans on 4 August 2009, who initiated nesting in ing, we removed low-quality sequence ends, tags and the laboratory on 19 August 2009. We built the nest primers. We then discarded any chimeric sequences as with autoclaved soil sandwiched between two glass detected by the program B2C2 (Gontcharova et al. 2010) plates, based on the design by Michener (1974). We dis- and denoised the remaining sequences. To assign reads sected the nest in a laminar flow hood on 13 September to phylotypes, we compared the sequences via BLAST 2009. We collected three pupae that ranged in age from against a curated database of 16S rDNA sequences that 12 to 15 days. We also collected a pollen provision that was compiled from the National Center for Biotechnol- the female had not oviposited on, and the remains of a ogy and maintained by the Medical Biofilm Research failed cell that had become infested with an unidenti- Institute. We assigned reads to the finest taxonomic fied fungus. We again collected tunnel-wall scrapings, level possible based on the following similarity criteria: soil from the surrounding substrate and a negative con- 96% and above for species, 94–96% for genus, 89–94% trol. The laboratory nest, although not exposed to soil for family, 85–89% for order, 80–85% for class and bacteria, was exposed to bacteria found on flowers 77–80% for phylum (Dowd et al. 2008; Sen et al. 2009; (mostly ruderal composites) that the adults foraged on Ishak et al. 2011). As an additional validation of our for nectar and pollen and to Helianthus annus pollen taxonomic assignments, we conducted a separate that we collected from garden grown plants and offered BLASTn search on all of our 454 sequences against the to the foraging bees in small dishes. SILVA database release 108 (Pruesse et al. 2007). To Lastly, we sampled the contents of two Megalopta ge- visualize the results, we processed the resulting BLAST nalis nests from Barro Colorado Island, Panama. M. ge- table with MEGAN4 (Huson et al. 2011). Finally, to nalis is a socially polymorphic, Neotropical sweat bee investigate the patterns of diversity, we calculated the that nests in decaying branches and sticks in the forest probability of an interspecific encounter (PIE), an

2012 Blackwell Publishing Ltd 4 Q. S. MCFREDERICK ET AL. estimator of the evenness of a community (Hurlbert SILVA database as a reference. As the SILVA database 1971). alignment results in many gaps (Ishak et al. 2011), we used the filter.seqs function in Mothur to remove these gaps. We then used a soft filter to remove insertions Firmicutes-specific reference database for lactobacilli that appeared in <10% of the samples. The resulting analyses alignment consisted of 2138 sequences with a length of The Medical Biofilm Research Institute’s 16S rDNA 431 bases. To create an approximate maximum- database does not fully encompass bacterial diversity likelihood tree for the Fast UniFrac analysis, we used for certain groups, and we found that many of the most the program FastTree (Price et al. 2010) under a general abundant bacteria were assigned to unknown taxo- time reversible (GTR) model of sequence evolution. nomic levels within the phylum Firmicutes (e.g. Bacilli, Lastly, to assess similarity in our sampled bacterial Lactobacillales, Lactobacillaceae). To gain a more communities, we used the resulting maximum-likeli- detailed understanding of where these samples fell hood phylogeny, a sample category mapping file and within the Firmicutes, we downloaded all of the 16S the sample ID mapping file as input for the Fast Uni- rDNA sequences that were labelled as belonging to the Frac analysis. In Fast UniFrac, we performed a phylum Firmicutes, were not an unidentified species weighted, normalized principal coordinate analyses and were publicly available from NCBI as of 3 October based on phylogenetic distances between the sampled 2011 (60027 sequences). We clustered sequences that communities. To determine whether some of the sam- were of 97% or greater sequence identity using CDhit ples were clustering because of the presence of abun- (Huang et al. 2010), and removed sequences that were dant Lactobacillus spp., we executed a second analysis shorter than 300 bases or longer than 2000 bases, which where we first removed Lactobacillus spp. reads, then resulted in a database of 6600 Firmicutes sequences. We ran the remaining sequences through the same Fast extracted reads from our 454 data set that we had UniFrac pipeline as described above. assigned to an unknown taxonomic position in the Fir- For the approximate maximum-likelihood phylogeny micutes with our previous BLAST search, and used of Lactobacillus, we first extracted sequence reads from BLASTn to compare these reads to the Firmicutes only our data set that were identified as Lactobacillus by our database. BLAST searches. We again clustered redundant sequences within host species using CDhit (Huang et al. 2010), with the sequence identity cut-off set to 97%.An Phylogenetic analyses input of 19844 sequences resulted in the clustering of 85 We conducted two separate phylogeny-based analyses: phylotypes with read length >350 nucleotides. a Fast UniFrac analysis (Hamady et al. 2010) on all of To represent lactobacilli that associate with other spe- the 454 sequence data and a maximum-likelihood anal- cies of bees, we obtained 16S rDNA sequences from ysis on amassed Lactobacillus sequences. For the Fast GenBank, mostly from previous studies of Apis mellifera UniFrac analysis, we used a pipeline previously (Jeyaprakash et al. 2003; Babendreier et al. 2007; Mohr described by Ishak et al. (2011) to analyse our 454 & Tebbe 2007; Olofsson & Vasquez 2008; Vasquez et al. sequence data. First, we used a custom Perl script to 2009; Martinson et al. 2011), as well as from Bombus select sequences that were 300 bases or longer. As the and other bees (Mohr & Tebbe 2007; Koch & Schmid- number of reads from each sample varied from 508 to Hempel 2011a; Martinson et al. 2011; Tajabadi et al. 11 027, we used another custom Perl script to randomly 2011; Disayathanoowat et al. 2012). Next, we down- sample 1000 reads from each sample. Six of our thirty- loaded 226 Lactobacillus full-length 16S rDNA sequences four samples had less than 1000 reads, so all of the from NCBI that were available as of 18 October 2011. reads greater than 300 bases long were used for those After removing sequences that shared 97% or greater samples (Q15-900 reads, Q16-894 reads, Q2A-694 reads, sequence identity and sequences from unidentified lac- GeSoL4Bc0019-564 reads, GeSoTuBc0019-508 reads, tobacilli, the alignment consisted of 100 known Lactoba- GeEuSuBc0029-995 reads). Next, to cluster redundant cillus sequences, 71 sequences from other studies of bee- sequences, we used CDhit (Huang et al. 2010), with the associated bacteria, 15 outgroup sequences and 85 of sequence identity cut-off set to 97%, as has been sug- our 454 generated sequences. To align these sequences, gested to avoid inflated diversity estimates caused by we used MUSCLE (Edgar 2004) to create an initial sequencing error (Kunin et al. 2010). In addition, we alignment. Although conserved segments of 16S rDNA used a custom Perl script to generate a sample ID map- aligned well, several variable sections were difficult to ping file that assigns the number of reads from each align. We therefore used the program SATe to simulta- sample to a specific CDhit cluster. We used Mothur neously estimate the alignment and phylogeny over 100 (Schloss et al. 2009) to align the sequences, with the iterations, using the general time reversible with invari-

2012 Blackwell Publishing Ltd BEES AND ACIDOPHILIC BACTERIA 5 ant sites and gamma-distributed rate variation model of from a total of 125 005 analysis quality reads (for the sequence evolution. SATe outperforms two-step align- raw data, see Tables S1–S4, Supporting information). ment and phylogenetic estimation methods when work- These bacteria included putative mutualists, pathogens ing with genes with high variability and insertions and and commensals. Rarefaction curves indicated that the deletions, such as the 16S rRNA gene (Liu et al. 2009). majority of our sequencing was deep enough to charac- We then compared the resulting alignment to a struc- terize the bacterial communities, with the exception of tural model of the 16S rRNA small subunit (Cannone several samples taken from the substrate surrounding et al. 2002), which allowed us to manually refine the the nests (Figs S1–S4, Supporting information). conserved sections of the alignment in the program Bacteria from various taxonomic levels within the Mesquite (Maddison & Maddison 2010). phylum Firmicutes were abundant in all four bee nests, To determine the model of sequence evolution that although to a lesser degree in the Au. pura nest (Fig. 1). best fit our alignment, we used the program Modeltest We further scrutinized sequences that matched the phy- (Posada & Crandall 1998). We used the Akaike Informa- lum Firmicutes in our initial BLAST search with a section Criterion to select the most likely substitution ond BLAST search to a sequence database representing model (GTR + I + G) for approximate maximum-likeli- the full diversity of the Firmicutes. This second BLAST hood analyses. We conducted 20 independent search search revealed that the sequences shared ‡89% of their replicates and 100 bootstrap pseudoreplicates in the 16S rDNA sequence with lactic acid bacteria (LAB) program Garli (Zwickl 2006). from the genus Lactobacillus (Table 1.). For example, We additionally tested whether corbiculate apid and reads that shared 94.8% and 92.4% sequence identity halictid bees are more closely associated with flower- with Lactobacillus kunkeei were abundant in Au. pura associated lactobacilli than other lactobacilli. We first and M. genalis samples, respectively. Our additional used the best-scoring maximum-likelihood tree to calcu- BLAST results against the SILVA database and subse- late the patristic distances from corbiculate apid-associ- quent MEGAN4 analysis verified these data, also show- ated lactobacilli and halictid-associated lactobacilli to ing L. kunkeei as the most abundant bacterium from our flower-associated lactobacilli and all other lactobacilli. samples (Fig. S5, Supporting information). In general, After verifying that the distances were normally distrib- lactobacilli often occurred at high abundance in samples uted, we conducted two t-tests to determine whether (i) from the brood cells but not from the adult insects, with the distances from corbiculate apid-associated lactoba- the exception of the H. ligatus female (Fig. 1). cilli to flower-associated lactobacilli differ from the dis- Many of the other common bacteria also come from tances from corbiculate apid-associated lactobacilli to all acidophilic groups: members of the Acidobacteriaceae other lactobacilli and (ii) the distances from halictid- along with Saccharibacter, which is a member of the associated lactobacilli to flower-associated lactobacilli Acetobacteraceae (Fig. 1). Similar to the bee bread of differ from the distances from halictid-associated lacto- Apis mellifera (Anderson et al. 2011), the pollen provi- bacilli to all other lactobacilli. sions from three M. genalis brood cells exhibited acidic Lastly, we investigated divergence among paralogous pH (3.4, 3.4 and 3.7). In addition to acid-loving bacteria, copies of the 16S rRNA gene within lactobacilli ge- we detected endosymbiotic bacteria in the genera Wol- nomes in order to determine whether this might affect bachia and Cardinium (Zchori-Fein & Perlman 2004) our diversity analyses. We downloaded all of the anno- from several insect samples and at low abundance in tated 16S rDNA copies from 10 lactobacilli that had two frass samples (Fig. 1, Tables S1–S4, Supporting complete genome sequences available. We aligned the information). 16S rDNA copies from each genome separately, in the Some of the acidophilic bacteria that we detected in program Mesquite (Maddison & Maddison 2010), and association with sweat bees are closely related to bacte- determined the number of polymorphic sites per gen- ria that have also been detected at flowers. Bacteria ome. with greater than 96% sequence identity to Saccharibact- er floricola associated with the social M. genalis female (Table S3, Supporting information). S. floricola was Results described from pollen from Japanese flowers (Jojima et al. 2004) and is nested in the alpha-2 phylotype that Bacterial survey based on 16S rDNA amplicon forms part of the core Ap. mellifera microbiota (Martin- pyrosequencing son et al. 2011). Our SILVA BLAST searches also indi- Individual bees of the species Augochlora pura, Halictus cated that S. floricola was abundantly represented ligatus and Megalopta genalis and the contents of their (Fig. S5, Supporting information). Although uncom- nests hosted a rich diversity of microbes, with a total of mon, several M. genalis reads had 97–98% sequence 992 unique phylotypes identified across all samples identity with a Lactobacillus that is known from flowers,

2012 Blackwell Publishing Ltd 6 Q. S. MCFREDERICK ET AL.

Halictus ligatus Augochlora pura

80 60

60 50

40 30

20 20 Percent abundance Percent abundance 10

0 0 Female Female Pupa Frass pupa Lactobacillales Callow adult Lactobacillales Pupa Wolbachia sp. Frass Wolbachia sp. Frass pupa Mycobacterium sp. Pharate adult Mycobacterium sp. Pupa Clostridiales Clostridiales Pseudomonas sp. Frass Pseudomonas sp. Frass pupa Saccharibacter sp. Pupa Failed provision Saccharibacter sp. Acidobacteriaceae Frass Acidobacteriaceae Pollen provision Enterobacter sp. Enterobacter sp. Sample Tunnel Eubacteriaceae Phylotype Sample Tunnel Phylotype Substrate Eubacteriaceae Stenotrophomonas Substrate Stenotrophomonas

Megalopta genalis social nest Megalopta genalis solitary nest

80 80

60 60

40 40

20 20 Percent abundance Percent abundance

0 0 Female Female Pollen Egg Lactobacillales Larva Lactobacillales Wolbachia sp. Wolbachia sp. Larva Mycobacterium sp. Pollen Mycobacterium sp. Pollen Clostridiales Clostridiales Pseudomonas sp. Larva Pseudomonas sp. Frass pupa Saccharibacter sp. Frass Saccharibacter sp. Acidobacteriaceae Acidobacteriaceae Tunnel Enterobacter sp. Tunnel Enterobacter sp. Eubacteriaceae Sample Substrate Phylotype Sample Substrate Eubacteriaceae Phylotype Stenotrophomonas Stenotrophomonas

Fig. 1 Proportional abundance of the ten most commonly observed bacterial phylotypes from four sweat bee nests, as determined by BLAST searches against a database curated by the Medical Biofilm Research Institute. The first row, Firmicutes, includes the nested taxonomic levels Bacilli, Lactobacillales, Lactobacillaceae and Lactobacillus. Firmicute phylotypes all had top BLAST hits to Lac- tobacillus, but not all showed ‡97% sequence identity (Table 1). Rarefaction curves indicated that we sequenced deeply enough to accurately characterize most bacterial communities, with the exception of several samples taken from the substrate surrounding the nests (Figs S1–S4, Supporting information). For a heatmap version of this figure, see Fig. S7 (Supporting information).

Lactobacillus ozensis (Kawasaki et al. 2011). Finally, many dant in our samples. In a failed H. ligatus brood cell, of our reads had top BLAST hits to L. kunkeei (94.83% however, two unknown Paenibacillus phylotypes repre- and 92.43% average sequence identity for the Au. pura sented 11.7% and 5.7% of the bacterial sequences. and M. genalis associates, respectively), another species The microbiota of these miniature ecosystems exhibit known from ﬂowers (Table 1, Fig. S5, Supporting infor- interesting patterns of community composition and mation, Endo et al. 2011). diversity. For example, the surrounding substrate We also detected a putative pathogen: an unidentiﬁed showed the greatest richness and diversity from each Paenibacillus, a genus that includes a pathogen of honey nest, with the exception of the laboratory nest (Fig. 2). bees (Evans 2003). Paenibacillus was generally not abun- The soil that was used as the nesting substrate for the

2012 Blackwell Publishing Ltd BEES AND ACIDOPHILIC BACTERIA 7

Table 1 Top BLAST hit of sequences Average % % that were assigned to an unknown taxo- Host Top BLAST hit identity abundance nomic position in the phylum Firmi- cutes against a reference database Augochlora Lactobacillus kunkeei 94.83 13.33 compiled from Firmicutes sequences pura Lactobacillus keﬁri 89.72 0.22 from NCBI. Lactobacillus fructivorans 89.67 0.12 Lactobacillus lindneri 89.98 0.05 Halictus Lactobacillus fructivorans 89.93 41.52 ligatus Lactobacillus keﬁri 89.43 25.59 Lactobacillus lindneri 89.38 8.53 Lactobacillus homohiochii 90.20 0.81 Lactobacillus murinus 98.80 0.58 Lactobacillus intestinalis 96.81 0.10 Lactobacillus ozensis 90.21 0.01 Lactobacillus tucceti 90.42 0.01 Megalopta Lactobacillus kunkeei 92.43 73.14 genalis Lactobacillus casei 98.74 3.37 Lactobacillus rossiae 98.08 1.08 Lactobacillus crispatus 98.45 0.10 Lactobacillus ozensis 94.42 0.01 Lactobacillus plantarum 97.62 0.01 Lactobacillus sanfranciscensis 90.01 0.01

Average per cent identity represents the average sequence identity of those phylotypes that shared top hits to the same reference species. Per cent abundance represents the per cent abundance of a given phylotype within its respective host species, excluding the surrounding substrate samples. Only hits with a per cent abundance > 0.01% are shown.

H. ligatus nest had been autoclaved, so it is not surpris- ing that it exhibited some of the lowest phylotype rich- Frass callow adult

a ness. The H. ligatus soil sample, however, exhibited

r Callow adult u

p Frass pharate adult high evenness, indicating that no early colonizer was a

r Pharate adult o l Frass Pupa h able to dominate the soil environment. The M. genalis

c Pupa o

g Female

u nests exhibited lower evenness than the North American

A Tunnel Substrate bee nests, with the female from the social M. genalis nest exhibiting the lowest evenness of all samples (Fig. 2). Frass pupa Pupa The sweat bee–associated microbiota exhibit differing

s Frass pupa u

t Pupa community composition, as determined by our Fast

g i l Frass pupa UniFrac analysis (Fig. 3). The H. ligatus and Au. pura

s Pupa

u t

c Failed provision i

l microbiota clustered in different regions of principal

a Pollen provision no egg H Female coordinate 2, while several M. genalis samples were Tunnel Substrate more widely dispersed. The H. ligatus nest was a labo-

s ratory nest; therefore, comparisons should be viewed

i l

a Frass mature larva

n s

e Mature larva cautiously. However, Lactobacillus dominated several

e g

n Pollen young larva

t y

r H. ligatus and M. genalis samples that formed a cluster

p Young larva

t l

i Female

a o

g along coordinate 1 (Figs 2 and 3). When we ran the

s Tunnel e Substrate

M analysis with lactobacilli removed, these samples were

s i

l widely dispersed (Fig. 4), showing that the dominance

a Frass pupa

n s

e Pollen young larva of lactobacilli in these samples drove the clustering in

e g

n Young larva

l t

a Pollen from egg p

i Fig. 3.

o c

l Female

a s

g Tunnel

e Substrate M

0.5 0.7 0.9 50 150 250 Phylogeny of the genus Lactobacillus based on 16S Diversity (PIE) Phylotype richness rDNA

Fig. 2 Number of observed bacterial phylotypes and evenness Our phylogeny represents Lactobacillus from our 454- (PIE) for each sample. sequencing survey and publicly available Lactobacillus

2012 Blackwell Publishing Ltd 8 Q. S. MCFREDERICK ET AL.

Several bee-associated Lactobacillus clades were

0.2 entirely or nearly bee specific. For example, the F-3 clade (sensu Babendreier et al. 2007) was composed of sequences from corbiculate bee isolates only (Fig. 5). The F-4 clade was also mostly specific to Apis and Bom- 0.1 Species Augochlora bus; however, an isolate from a spacecraft assembly Halictus clean room (accession number GQ129909, La Duc et al. Megalopta 2009) shared high sequence identity to F-4, as noted by 0.0 Type Martinson et al. (2011). We also found the F-5 clade to cell contents insect be mostly Bombus and Apis specific, with the exception substrate of isolates from Colletes inaequalis, a solitary bee (Fig. 5, –0.1 tunnel Martinson et al. 2011), and Anopheles stephensi (accession number FJ608053, Rani et al. 2009) with a top BLAST hit (98% sequence identity) to sequences from the F-5 Principal coordinate 2: 13.54% variation explained –0.2 clade. F-5 lactobacilli therefore appear to associate with insects other than corbiculate bees. Other bee-associated lactobacilli clustered with bacte- –0.3 –0.2 –0.1 0.0 0.1 0.2 ria isolated from flowers. For example, Lactobacillus kun- Principal coordinate 1: 40.87% variation explained keei and L. ozensis, both of which have been isolated Fig. 3 First two principal coordinates from an abundance- from flowers (Kawasaki et al. 2011; Endo et al. 2011), weighted UniFrac analysis of the bacterial communities from fell into a clade that also included lactobacilli isolated four sweat bee nests. from Augochlora pura, Megalopta genalis, Caupolicana yarrowi, Diadasia opuntiae and Apis mellifera (Fig. 5). The sis- ter clade to the L. kunkeei clade was composed of lactobacilli isolated from Halictus ligatus. Finally, L. floricola clustered with bacteria isolated from Ap. mellifera 0.2 and shared 99% sequence similarity with bacteria iso- Species lated from Agapostemon virescens. Augochlora Halictus The genetic distances within clades that include 0.1 Megalopta halictid-associated lactobacilli also support the floral Type transmission hypothesis. First, the genetic distance in the cell contents L. kunkeei clade (maximum ⁄ average patristic insect distance = 0.35 ⁄ 0.11) was in the range of the distances 0.0 substrate tunnel exhibited by the corbiculate apid-associated lactobacilli (maximum ⁄ average patristic distances: F4 clade = 0.54 ⁄ 0.25, F5 clade = 0.32 ⁄ 0.09, F3 clade = 0.14 ⁄ 0.08). Sec- –0.1 ond, halictid-associated lactobacilli exhibited signifi-

Principal coordinate 2: 18.18% variation explained cantly smaller patristic distances to flower-associated lactobacilli than to other lactobacilli (T = )16.8431, –0.2 –0.1 0.0 0.1 0.2 df = 370.08, P < 2.2e-16), while corbiculate apids did not Principal coordinate 1: 21.94% variation explained (T = 1.734, df = 249.859, P = 0.08416, Fig. S6, Supporting information). While these statistical tests support floral Fig. 4 First two principal coordinates from an abundance- weighted UniFrac analysis of the bacterial communities from transmission of halictid-associated lactobacilli, compari- four sweat bee nests, with phylotypes with top BLAST hits to sons based on phylogenetic tree topologies are difficult to Lactobacillus spp. removed. interpret, and future work is still necessary. Some bee-associated lactobacilli clustered with sequences. We found that bee-associated lactobacilli are bacteria associated with humans and other mammals. distributed across the Lactobacillus phylogeny. In gen- Several H. ligatus isolates clustered with L. murinus and eral, membership of the clades in our phylogeny are in L. animalis, both of which were originally isolated from agreement with other published 16S rDNA phylogenies rodent intestinal tracts. Several other isolates from of Lactobacillus (Canchaya et al. 2006; Martinson et al. M. genalis clustered with L. larvae, an associate of Ap. 2011). However, owing to the low bootstrap support for mellifera, and L. paracasei, which is thought to be a many of the clades, our phylogeny should not be used human probiotic (Gardiner et al. 2000). The shared to interpret deeper branching patterns in the lactobacilli. records of these lactobacilli from bees, rodents and

2012 Blackwell Publishing Ltd BEES AND ACIDOPHILIC BACTERIA 9 humans indicate that these bacteria likely occur in the transmission between bee lineages. Next, our sequence environment. data show that Lactobacillus spp. and S. floricola can be found at high abundance in association with sweat bees, but that there is intra- and internest variance in Within-genome 16S rDNA variation the presence and abundance of these bacteria (Fig. 1). Finally, our analysis of within-genome variation in par- Therefore, if Lactobacillus and S. floricola are only verti- alogous copies of the 16S rRNA gene from 10 lactobacil- cally transmitted, transmission must be incomplete. li genomes suggests that 16S rDNA serves as a reliable Although symbionts with imperfect vertical transmis- barcode for the genus. We found that the number of sion and no horizontal transmission can persist in a paralogous copies varied from 4 to 9 copies per gen- host population, for a symbiont with such imperfect ome, with a mode of five copies (Table S5, Supporting vertical transmission to persist, it would need to have information). Although there was some polymorphism very strong positive effects on host fecundity and ⁄ or present in the paralogous copies, the amount of poly- offspring survival (Fine 1975). While it is entirely plau- morphism did not exceed 1% within any of the sible that Lactobacillus spp. and S. floricola have such genomes (Table S5, Supporting information). strong effects, a more parsimonious explanation is that both positive effects on host fitness and environmental transmission play a role in the biology of bees. Discussion Our data largely agree with a previous study that Augochlora pura, Halictus ligatus and Megalopta genalis failed to detect the core gut microbiota of Ap. mellifera associate with putative beneficial and pathogenic bacte- outside the corbiculate apids (Martinson et al. 2011). ria, and some of the putatively beneficial bacteria are We did, however, detect one of the Ap. mellifera core possibly acquired by the bees from flowers. In addition gut microbes, S. floricola, in one M. genalis nest. Martin- to acquisition from flowers, our phylogeny agrees with son et al. (2011) found that Burkholderia cepacia, a wide- studies that suggest that some lactobacilli appear to be spread bacterium, was commonly isolated from bees specific to the corbiculate apids and may be vertically outside the corbiculate apids. While we detected transmitted (Koch & Schmid-Hempel 2011a; Martinson B. cepacia in association with H. ligatus, it was present et al. 2011). Bees may therefore acquire beneficial bacte- at very low abundance (<0.17%) and was absent from ria by at least two routes: one route from parent to off- the Au. pura and M. genalis nests. Instead, many of our spring (corbiculate bees) and a second route via flowers samples were dominated by lactobacilli that were and possibly other environmental sources. related to bacteria that Martinson et al. (2011) isolated Our analyses provide several lines of evidence sug- at low abundance from Halictus patellatus, C. yarrowi, gesting that transmission of bacteria from flowers to D. opuntiae and Hoplitis biscutellae and that were closely wild bees may be more important than previously related to L. kunkeei in our phylogenetic and BLAST thought. First, our 16S rDNA amplicon pyrosequencing analyses (Fig. 5, Table 1). The differences in abundance survey detected several bacterial phylotypes that either between our two studies may be explained by seasonal had at least 97% sequence identity to bacteria isolated variation in the presence of Lactobacillus at flowers. from flowers (Saccharibacter floricola and L. ozensis)or Alternatively, the variation in abundance between the had top BLAST hits at lower sequence identity to a bac- two studies may be explained by methodological differ- terium known to occur on flowers (L. kunkeei). Addi- ences; Martinson et al. (2011) surveyed abdomens while tional support comes from other studies documenting we surveyed entire bees and the contents of their nests. bacteria that have been isolated from bees and that are Our data agree with the findings of Martinson et al. also known to occur on flowers: L. kunkeei from Apis (2011), in that Lactobacillus was often found in low mellifera (Olofsson & Vasquez 2008); S. floricola from abundance or missing from our adult insect samples, Ap. mellifera, L. floricola from Agapostemon virescens with the exception of the H. ligatus mother (Fig. 1). (Martinson et al. 2011); L. floricola from Ap. mellifera Through our surveys of the contents of brood cells, (NCBI accession number HM534770); and L. ozensis however, we were able to determine that lactobacilli from Ap. mellifera (NCBI accession number HM534743). can be abundant in the nests of bees outside the corbi- The shared association of these bacteria with both bees culate apids. and flowers suggests that bees may be obtaining these Apis mellifera has been found to associate with Lacto- bacteria from flowers, although experimental confirma- bacillus kunkeei, and floral transmission would explain tion of this hypothesis is still necessary. Alternatively, why there has been variation in the presence and abun- bees may be the source of the lactobacilli and other aci- dance of Ap. mellifera-associated L. kunkeei across sur- dophilic bacteria found at flowers, but this would still veys, although variation in PCR parameters or other indicate that flowers are sites for potential horizontal methodological differences cannot be excluded. Floral

2012 Blackwell Publishing Ltd 10 Q. S. MCFREDERICK ET AL.

(a) AB033209 Lactobacillus algidus

0.90.9 FJ557241 Listeria rocourtiae AF466695 Bacillus coagulans NR 025863 Abiotrophia defectiva AB425924 Lactobacillus equi NR 026481 Eremococcus coleocola 0.760.76 NR 044710 Carnobacterium maltaromaticum NR 044706 Carnobacterium divergens 0.820.82 NR 024842 Streptococcus parasanguinis NC 013656 Lactococcus lactis AB362683 Lactobacillus satsumensis NR 028794 Enterococcus moraviensis EF533987 Enterococcus faecium AY735408 Enterococcus faecium AF028352 Clostridium innocuum NR 037082 Enterococcus hirae EU821345 Lactobacillus oeni AB033209 Lactobacillus algidus AB425924 Lactobacillus equi AB362683 Lactobacillus satsumensis EU821345 Lactobacillus oeni AB365978 Lactobacillus capillatus AB365978 Lactobacillus capillatus 0.640.64 M58824 Lactobacillus mali FJ157230 Lactobacillus mali AB162131 Lactobacillus nagelii AB242320 Lactobacillus mobilis 0.640.64 M58807 Lactobacillus animalis 0.64 0.950.95 0.640.64 EU161635 Lactobacillus murinus M58824 Lactobacillus mali F7SSZP003GEOFA frass F7SSZP003G8XXC pupa F7SSZP003FIY29 female F7SSZP003G0JMG female M58828 Lactobacillus ruminus FJ157230 Lactobacillus mali 1.001.00 HM534775 mellifera 0.610.61 0.530.53 HM534774 mellifera 0.530.53 HM534771 mellifera 0.980.98 AB186340 Lactobacillus salivarius 0.70.7 EU428010 mellifera AB162131 Lactobacillus nagelii 0.990.99 AB167386 Lactobacillus intermedius 1.01.0 AB001837 Lactobacillus aviariu G1OJGBH03GOLAZ social tunnel 0.910.91 AB282889 Lactobacillus similis AB159218 Lactobacillus paracollinoides AB242320 Lactobacillus mobilis AM113783 Lactobacillus malefermentans HM534765 mellifera M59295 Lactobacillus vermiform 0.840.84 AY363377 Lactobacillus rennanqilfy M58811 Lactobacillus buchneri FJ844993 Lactobacillus fermentum M58807 Lactobacillus animalis AB262732 Lactobacillus farraginis 0.990.99 AB362687 Lactobacillus faeni 0.950.95 HM534766 mellifera AM259118 Lactobacillus namurensis X61134 Lactobacillus brevis EU161635 Lactobacillus murinus M58810 Lactobacillus brevis CP000416 Lactobacillus brevis FJ915797 Lactobacillus plantarum 1.001.00 JN126052 Lactobacillus plantarum G1OJGBH03F8WFB social frass F7SSZP003GEOFA frass AM279758 Lactobacillus plantarum M58827 Lactobacillus plantarum FJ386491 Lactobacillus plantarum AB371714 Lactobacillus plantarum FN252881 Lactobacillus plantarum F7SSZP003G8XXC pupa AB362987 Pediococcus pentosaceus 1.001.00 X76330 Lactobacillus fructivorans 0.950.95 HM534764 mellifera X95421 Lactobacillus lindneri AB498047 Lactobacillus fructophile 0.920.92 X76327 Lactobacillus sanfranciscensis F7SSZP003FIY29 female X61132 Lactobacillus sanfranciscensis F7SSZP003FI5FN provision F7SSZP003HFPEY provision F7SSZP003G5ZTS provision F7SSZP003G3NEK female F7SSZP003G0JMG female F7SSZP003FJ41T female F7SSZP003FZDPQ female F7SSZP003HAPYQ provision F7SSZP003FVDOE female F7SSZP003FV2DK female M58828 Lactobacillus ruminus 0.530.53 F7SSZP003GB27S female 0.910.91 F7SSZP003FZBLV female 0.910.91 F7SSZP003FKU5E female F7SSZP003GM303 provision HM534775 mellifera F7SSZP003GSJYU provision 1.001.00 F7SSZP003G83HT female 1.001.00 F7SSZP003FQFVI female F7SSZP003G9G18 female F7SSZP003GVG3B female 0.610.61 HM534774 mellifera F7SSZP003FK90P provision 0.610.61 F7SSZP003FO813 provision F7SSZP003HB207 female F7SSZP003F6H96 female 0.530.53 F7SSZP003GLX8O provision F7SSZP003FP9MZ provision HM534771 mellifera F7SSZP003GUV9L female F7SSZP003GTPZN provision F7SSZP003GZRYO female F7SSZP003FNTFE provision 0.980.98 F7SSZP003GTXXJ failed provision 0.98 F7SSZP003GSQA4 female 0.980.98 0.910.91 AB186340 Lactobacillus salivarius F7SSZP003HHGTB female 0.930.93 F7SSZP003HB7B3 female F7SSZP003F33 female F7SSZP003HBKSR female 0.70.7 EU428010 mellifera F7SSZP003HHA6K failed provision 0.70.7 F7SSZP003GRNEB female 0.970.97 AB572588 Lactobacillus ozensis HM534743 mellifera HM109610 Caupolicana HM112623 Diadasia 0.990.99 HM112778 Diadasia 0.990.99 AB167386 Lactobacillus intermedius F348WMR04JDJG4 frass F348WMR04JGOIJ frass F348WMR04JBQ2M frass 1.01.0 F348WMR04IFQ7D frass 1.01.0 F348WMR04JKJ8J frass AB001837 Lactobacillus aviariu F348WMR04JKTXT frass HM109480 Caupolicana 0.980.98 HM112474 Diadasia 0.980.98 AB559820 Lactobacillus kunkeei AJ971903 mellifera G1OJGBH03GOLAZ social tunnel EU753691 kunkeei mellifera HM008721 mellifera 0.790.79 G1OJGBH03GX6IB social frass G1OJGBH03G5C2A social frass G1OJGBH03HAYWD social frass 0.910.91 AB282889 Lactobacillus similis G1OJGBH03GHBHU solitary pollen 0.910.91 G1OJGBH03FQASF solitary pollen G1OJGBH03FWMY4 solitary pollen G1OJGBH03FN2ZU social provision G1OJGBH03GQQ4M solitary larva G1OJGBH03F6ICY social larva AB159218 Lactobacillus paracollinoides G1OJGBH03G6R03 social pollen G1OJGBH03F1OGP social pollen G1OJGBH03GZEIB social frass G1OJGBH03GHVOY social adult G1OJGBH03FPP40 social pollen AM113783 Lactobacillus malefermentans G1OJGBH03HCW2S solitary pollen G1OJGBH03G4ZG5 social pollen G1OJGBH03F3HKE social pollen G1OJGBH03G5A0S social larva HM534765 mellifera G1OJGBH03FKN70 social frass G1OJGBH03F5LSE social frass G1OJGBH03G4BBA social frass G1OJGBH03G7UHR social frass G1OJGBH03GL6DL social frass 0.890.89 HM534763 mellifera M59295 Lactobacillus vermiform HM534760 mellifera G1OJGBH03G2VUK social frass G1OJGBH03G2OEC social frass EU555174 Lactobacillus sakei G1OJGBH03FYH6Y social frass 0.840.84 G1OJGBH03GO5Q0 social frass 0.840.84 AY363377 Lactobacillus rennanqilfy G1OJGBH03HCYM0 social frass AM920328 Lactobacillus rossiae AJ575744 Lactobacillus suebicus AB362691 Lactobacillus vaccinostercus 1.001.00 AF477498 Lactobacillus fermentum M58811 Lactobacillus buchneri NR 044659 Weissella kandleri 0.900.90 X76329 Lactobacillus pontis AF243177 Lactobacillus vaginalis HM534769 mellifera 0.910.91 AP007281 Lactobacillus reuteri FJ844993 Lactobacillus fermentum HM534768 mellifera 0.680.68 AY255802 Lactobacillus saerimneri 0.510.51 AY253658 Lactobacillus gastricus AM279150 Lactobacillus secaliphilus AF308147 Lactobacillus ingluviei AB262732 Lactobacillus farraginis 1.001.00 AF522394 Lactobacillus fermentum 0.990.99 EF535257 Lactobacillus fermentum 0.990.99 M58819 Lactobacillus fermentum GQ131191 Lactobacillus fermentum 0.530.53 AB268119 Lactobacillus composti 0.950.95 AB523781 Lactobacillus floricola AB362687 Lactobacillus faeni HM534770 mellifera 0.750.75 AJ417738 Lactobacillus letivazi 0.640.64 0.660.66 AJ496791 Lactobacillus versmoldensis 0.660.66 AJ576006 Lactobacillus tucceti HM534766 mellifera 0.920.92 M58804 Lactobacillus alimentarius 0.920.92 0.780.78 AY681134 Lactobacillus bobaliu s 0.840.84 AJ417499 Lactobacillus farciminis FJ645924 Lactobacillus crustorum 1.001.00 AB368915 Lactobacillus harbinensis Y19167 Lactobacillus perolens AM259118 Lactobacillus namurensis AY974809 Lactobacillus brevis HM534767 mellifera AB257864 Lactobacillus camelliae EU184020 Lactobacillus rhamnosus M23928 Lactobacillus casei X61134 Lactobacillus brevis AF000163 Lactobacillus manihotivorans 0.820.82 AB362679 Lactobacillus pantheris 0.570.57 M58831 Lactobacillus sharpeae GU138576 Lactobacillus guizhouensis G1OJGBH03FPFCW social larva AY773954 Lactobacillus paracasei M58810 Lactobacillus brevis AY667700 Lactobacillus larvae G1OJGBH03HE4SG social frass G1OJGBH03GD18U social frass G1OJGBH03GBVJ2 social frass G1OJGBH03FXRZZ social frass CP000416 Lactobacillus brevis G1OJGBH03HA4AJ social frass NR 024885 Paralactobacillus selangorensis 0.830.83 AJ576007 Lactobacillus rennini 0.590.59 DQ406862 Lactobacillus backi 0.880.88 M58809 Lactobacillus bifermentans FJ915797 Lactobacillus plantarum JN175330 Lactobacillus bifermentans 0.720.72 HM112055 mellifera HM113158 mellifera HM534810 mellifera 0.510.51 HM113222 mellifera JN126052 Lactobacillus plantarum 0.880.88 HM046576 cerana 1.001.00 EU753690 mellifera 1.001.00 DQ837632 mellifera HM113258 mellifera 1.001.00 DQ837631 mellifera DQ837630 mellifera G1OJGBH03F8WFB social frass HM215043 Bombus 0.670.67 HM215041 Bombus HM215042 Bombus HM534814 mellifera EU559601 Lactobacillus jensenii AM279758 Lactobacillus plantarum 0.860.86 M58801 Lactobacillus acetotolerans FR683099 Lactobacillus acetotolerans F7SSZP003GT0PR female F7SSZP003GZ6SM provision AY253657 Lactobacillus kalixensis M58827 Lactobacillus plantarum Y17361 Lactobacillus amylolyticus 0.910.91 AB290830 Lactobacillus equicursoris 0.970.97 M58823 Lactobacillus delbrueckii CP000156 Lactobacillus delbrueckii CP002338 Lactobacillus amylovorus M58802 Lactobacillus acidophilus FJ386491 Lactobacillus plantarum 0.810.81 AB186339 Lactobacillus kitasatonis EU419585 Lactobacillus helveticus EU878007 Lactobacillus acidophilus DQ317604 Lactobacillus crispatus EU559595 Lactobacillus crispatus AB371714 Lactobacillus plantarum G1OJGBH03GSTGE social pollen 0.520.52 AJ242969 Lactobacillus crispatus 1.001.00 AE017198 Lactobacillus johnsonii EU780913 Lactobacillus johnsonii mellifera 0.550.55 HM534808 mellifera FN252881 Lactobacillus plantarum 0.550.55 HM534806 mellifera HM534795 mellifera HM215048 Bombus AJ971929 Bombus HM534796 mellifera AB362987 Pediococcus pentosaceus HM534786 mellifera GU233458 dorsata GU233457 dorsata HM113257 mellifera HM113281 mellifera X76330 Lactobacillus fructivorans HM113326 mellifera 1.001.00 HM046579 cerana 1.001.00 DQ837635 mellifera DQ837634 mellifera HM534764 mellifera HM046568 mellifera AY370183 mellifera HM112010 mellifera 0.950.95 HM112866 Colletes 0.950.95 EU753698 mellifera HM113333 mellifera AY667698 Lactobacillus alvei X95421 Lactobacillus lindneri HM534805 mellifera DQ837637 mellifera HM113203 mellifera EU753697 mellifera AB498047 Lactobacillus fructophile DQ837636 mellifera AY667699 Lactobacillus insectis 0.920.92 X76327 Lactobacillus sanfranciscensis AY667701 Lactobacillus apis X61132 Lactobacillus sanfranciscensis F7SSZP003FI5FN provision F7SSZP003HFPEY provision F7SSZP003G5ZTS provision F7SSZP003G3NEK female F7SSZP003FJ41T female F7SSZP003FZDPQ female F7SSZP003HAPYQ provision F7SSZP003FVDOE female F7SSZP003FV2DK female 0.530.53 F7SSZP003GB27S female 0.910.91 F7SSZP003FZBLV female 0.910.91 F7SSZP003FKU5E female F7SSZP003GM303 provision F7SSZP003GSJYU provision F7SSZP003G83HT female F7SSZP003FQFVI female F7SSZP003G9G18 female F7SSZP003GVG3B female F7SSZP003FK90P provision F7SSZP003FO813 provision F7SSZP003HB207 female F7SSZP003F6H96 female F7SSZP003GLX8O provision F7SSZP003FP9MZ provision F7SSZP003GUV9L female F7SSZP003GTPZN provision F7SSZP003GZRYO female F7SSZP003FNTFE provision F7SSZP003GTXXJ failed provision 0.910.91 F7SSZP003GSQA4 female F7SSZP003HHGTB female 0.930.93 F7SSZP003HB7B3 female F7SSZP003F33 female F7SSZP003HBKSR female F7SSZP003HHA6K failed provision F7SSZP003GRNEB female 0.970.97 AB572588 Lactobacillus ozensis HM534743 mellifera HM109610 Caupolicana HM112623 Diadasia HM112778 Diadasia F348WMR04JDJG4 frass F348WMR04JGOIJ frass F348WMR04JBQ2M frass F348WMR04IFQ7D frass F348WMR04JKJ8J frass F348WMR04JKTXT frass HM109480 Caupolicana HM112474 Diadasia L. kunkeei Corbiculate apid 0.980.98 AB559820 Lactobacillus kunkeei AJ971903 mellifera EU753691 kunkeei mellifera HM008721 mellifera Flower 0.790.79 G1OJGBH03GX6IB social frass G1OJGBH03G5C2A social frass G1OJGBH03HAYWD social frass G1OJGBH03GHBHU solitary pollen Other bee G1OJGBH03FQASF solitary pollen G1OJGBH03FWMY4 solitary pollen G1OJGBH03FN2ZU social provision

G1OJGBH03GQQ4M solitary larva clade G1OJGBH03F6ICY social larva Augochlora pura G1OJGBH03G6R03 social pollen G1OJGBH03F1OGP social pollen G1OJGBH03GZEIB social frass G1OJGBH03GHVOY social adult Megalopta genalis G1OJGBH03FPP40 social pollen G1OJGBH03HCW2S solitary pollen G1OJGBH03G4ZG5 social pollen G1OJGBH03F3HKE social pollen Halictus ligatus G1OJGBH03G5A0S social larva G1OJGBH03FKN70 social frass G1OJGBH03F5LSE social frass G1OJGBH03G4BBA social frass G1OJGBH03G7UHR social frass G1OJGBH03GL6DL social frass

0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.0

2012 Blackwell Publishing Ltd BEES AND ACIDOPHILIC BACTERIA 11

0.90.9 FJ557241 Listeria rocourtiae AF466695 Bacillus coagulans NR 025863 Abiotrophia defectiva NR 026481 Eremococcus coleocola 0.760.76 NR 044710 Carnobacterium maltaromaticum NR 044706 Carnobacterium divergens 0.820.82 NR 024842 Streptococcus parasanguinis NC 013656 Lactococcus lactis NR 028794 Enterococcus moraviensis EF533987 Enterococcus faecium AY735408 Enterococcus faecium AF028352 Clostridium innocuum NR 037082 Enterococcus hirae AB033209 Lactobacillus algidus (b) AB425924 Lactobacillus equi AB362683 Lactobacillus satsumensis EU821345 Lactobacillus oeni AB365978 Lactobacillus capillatus 0.640.64 M58824 Lactobacillus mali FJ157230 Lactobacillus mali AB162131 Lactobacillus nagelii AB242320 Lactobacillus mobilis 0.950.95 M58807 Lactobacillus animalis 0.950.95 EU161635 Lactobacillus murinus F7SSZP003GEOFA frass F7SSZP003G8XXC pupa F7SSZP003FIY29 female F7SSZP003G0JMG female M58828 Lactobacillus ruminus 1.001.00 HM534775 mellifera 0.610.61 0.530.53 HM534774 mellifera 0.530.53 HM534771 mellifera 0.980.98 AB186340 Lactobacillus salivarius 0.70.7 EU428010 mellifera 0.990.99 AB167386 Lactobacillus intermedius 1.01.0 AB001837 Lactobacillus aviariu 0.890.89 G1OJGBH03GOLAZ social tunnel 0.890.89 HM534763 mellifera 0.910.91 AB282889 Lactobacillus similis AB159218 Lactobacillus paracollinoides AM113783 Lactobacillus malefermentans HM534765 mellifera M59295 Lactobacillus vermiform HM534760 mellifera 0.840.84 AY363377 Lactobacillus rennanqilfy M58811 Lactobacillus buchneri FJ844993 Lactobacillus fermentum 0.990.99 AB262732 Lactobacillus farraginis AB362687 Lactobacillus faeni HM534766 mellifera AM259118 Lactobacillus namurensis G1OJGBH03G2VUK social frass X61134 Lactobacillus brevis M58810 Lactobacillus brevis CP000416 Lactobacillus brevis FJ915797 Lactobacillus plantarum 1.001.00 JN126052 Lactobacillus plantarum G1OJGBH03G2OEC social frass G1OJGBH03F8WFB social frass AM279758 Lactobacillus plantarum M58827 Lactobacillus plantarum FJ386491 Lactobacillus plantarum AB371714 Lactobacillus plantarum EU555174 Lactobacillus sakei FN252881 Lactobacillus plantarum AB362987 Pediococcus pentosaceus 1.001.00 X76330 Lactobacillus fructivorans 0.950.95 HM534764 mellifera X95421 Lactobacillus lindneri AB498047 Lactobacillus fructophile G1OJGBH03FYH6Y social frass 0.920.92 0.920.92 X76327 Lactobacillus sanfranciscensis X61132 Lactobacillus sanfranciscensis F7SSZP003FI5FN provision F7SSZP003HFPEY provision F7SSZP003G5ZTS provision G1OJGBH03GO5Q0 social frass F7SSZP003G3NEK female F7SSZP003FJ41T female F7SSZP003FZDPQ female F7SSZP003HAPYQ provision F7SSZP003FVDOE female G1OJGBH03HCYM0 social frass F7SSZP003FV2DK female 0.530.53 F7SSZP003GB27S female 0.910.91 F7SSZP003FZBLV female 0.910.91 F7SSZP003FKU5E female F7SSZP003GM303 provision AM920328 Lactobacillus rossiae F7SSZP003GSJYU provision F7SSZP003G83HT female F7SSZP003FQFVI female F7SSZP003G9G18 female F7SSZP003GVG3B female AJ575744 Lactobacillus suebicus F7SSZP003FK90P provision F7SSZP003FO813 provision F7SSZP003HB207 female F7SSZP003F6H96 female F7SSZP003GLX8O provision F7SSZP003FP9MZ provision AB362691 Lactobacillus vaccinostercus F7SSZP003GUV9L female F7SSZP003GTPZN provision F7SSZP003GZRYO female 1.001.00 F7SSZP003FNTFE provision 1.001.00 F7SSZP003GTXXJ failed provision AF477498 Lactobacillus fermentum 0.910.91 F7SSZP003GSQA4 female 0.910.91 F7SSZP003HHGTB female 0.930.93 F7SSZP003HB7B3 female F7SSZP003F33 female F7SSZP003HBKSR female NR 044659 Weissella kandleri F7SSZP003HHA6K failed provision F7SSZP003GRNEB female 0.970.97 AB572588 Lactobacillus ozensis HM534743 mellifera HM109610 Caupolicana 0.900.90 X76329 Lactobacillus pontis HM112623 Diadasia 0.900.90 HM112778 Diadasia F348WMR04JDJG4 frass F348WMR04JGOIJ frass F348WMR04JBQ2M frass F348WMR04IFQ7D frass AF243177 Lactobacillus vaginalis F348WMR04JKJ8J frass F348WMR04JKTXT frass HM109480 Caupolicana 0.980.98 HM112474 Diadasia HM534769 mellifera 0.980.98 AB559820 Lactobacillus kunkeei AJ971903 mellifera EU753691 kunkeei mellifera HM008721 mellifera 0.910.91 0.790.79 G1OJGBH03GX6IB social frass 0.910.91 G1OJGBH03G5C2A social frass AP007281 Lactobacillus reuteri G1OJGBH03HAYWD social frass G1OJGBH03GHBHU solitary pollen G1OJGBH03FQASF solitary pollen G1OJGBH03FWMY4 solitary pollen HM534768 mellifera G1OJGBH03FN2ZU social provision G1OJGBH03GQQ4M solitary larva G1OJGBH03F6ICY social larva G1OJGBH03G6R03 social pollen G1OJGBH03F1OGP social pollen G1OJGBH03GZEIB social frass G1OJGBH03GHVOY social adult 0.680.68 AY255802 Lactobacillus saerimneri G1OJGBH03FPP40 social pollen 0.680.68 G1OJGBH03HCW2S solitary pollen G1OJGBH03G4ZG5 social pollen G1OJGBH03F3HKE social pollen G1OJGBH03G5A0S social larva 0.510.51 AY253658 Lactobacillus gastricus G1OJGBH03FKN70 social frass 0.510.51 G1OJGBH03F5LSE social frass G1OJGBH03G4BBA social frass G1OJGBH03G7UHR social frass G1OJGBH03GL6DL social frass AM279150 Lactobacillus secaliphilus 0.890.89 HM534763 mellifera HM534760 mellifera G1OJGBH03G2VUK social frass G1OJGBH03G2OEC social frass EU555174 Lactobacillus sakei AF308147 Lactobacillus ingluviei G1OJGBH03FYH6Y social frass G1OJGBH03GO5Q0 social frass G1OJGBH03HCYM0 social frass AM920328 Lactobacillus rossiae AJ575744 Lactobacillus suebicus AB362691 Lactobacillus vaccinostercus 1.001.00 AF522394 Lactobacillus fermentum 1.001.00 AF477498 Lactobacillus fermentum NR 044659 Weissella kandleri 0.900.90 X76329 Lactobacillus pontis AF243177 Lactobacillus vaginalis HM534769 mellifera EF535257 Lactobacillus fermentum 0.910.91 AP007281 Lactobacillus reuteri HM534768 mellifera 0.680.68 AY255802 Lactobacillus saerimneri 0.510.51 AY253658 Lactobacillus gastricus AM279150 Lactobacillus secaliphilus M58819 Lactobacillus fermentum AF308147 Lactobacillus ingluviei 1.001.00 AF522394 Lactobacillus fermentum EF535257 Lactobacillus fermentum M58819 Lactobacillus fermentum GQ131191 Lactobacillus fermentum 0.530.53 AB268119 Lactobacillus composti GQ131191 Lactobacillus fermentum 0.950.95 0.950.95 AB523781 Lactobacillus floricola HM534770 mellifera 0.750.75 AJ417738 Lactobacillus letivazi 0.640.64 0.660.66 AJ496791 Lactobacillus versmoldensis 0.530.53 0.660.66 AJ576006 Lactobacillus tucceti 0.53 AB268119 Lactobacillus composti 0.920.92 M58804 Lactobacillus alimentarius 0.530.53 0.920.92 0.780.78 AY681134 Lactobacillus bobaliu s 0.840.84 AJ417499 Lactobacillus farciminis 0.950.95 FJ645924 Lactobacillus crustorum AB523781 Lactobacillus floricola 1.001.00 AB368915 Lactobacillus harbinensis 0.950.95 1.001.00 Y19167 Lactobacillus perolens AY974809 Lactobacillus brevis HM534767 mellifera AB257864 Lactobacillus camelliae HM534770 mellifera EU184020 Lactobacillus rhamnosus M23928 Lactobacillus casei AF000163 Lactobacillus manihotivorans 0.820.82 AB362679 Lactobacillus pantheris 0.570.57 M58831 Lactobacillus sharpeae GU138576 Lactobacillus guizhouensis 0.750.75 AJ417738 Lactobacillus letivazi G1OJGBH03FPFCW social larva 0.750.75 AY773954 Lactobacillus paracasei AY667700 Lactobacillus larvae G1OJGBH03HE4SG social frass 0.640.64 G1OJGBH03GD18U social frass 0.640.64 G1OJGBH03GBVJ2 social frass AJ496791 Lactobacillus versmoldensis G1OJGBH03FXRZZ social frass G1OJGBH03HA4AJ social frass 0.660.66 NR 024885 Paralactobacillus selangorensis 0.66 0.830.83 0.660.66 AJ576007 Lactobacillus rennini 0.590.59 DQ406862 Lactobacillus backi AJ576006 Lactobacillus tucceti 0.880.88 M58809 Lactobacillus bifermentans JN175330 Lactobacillus bifermentans 0.720.72 HM112055 mellifera HM113158 mellifera HM534810 mellifera 0.920.92 M58804 Lactobacillus alimentarius 0.510.51 HM113222 mellifera 0.92 0.880.88 HM046576 cerana 0.920.92 0.880.88 EU753690 mellifera DQ837632 mellifera HM113258 mellifera 0.780.78 AY681134 Lactobacillus bobaliu s 1.001.00 DQ837631 mellifera 0.780.78 DQ837630 mellifera HM215043 Bombus 0.670.67 HM215041 Bombus 0.840.84 HM215042 Bombus 0.840.84 HM534814 mellifera AJ417499 Lactobacillus farciminis EU559601 Lactobacillus jensenii 0.860.86 M58801 Lactobacillus acetotolerans FR683099 Lactobacillus acetotolerans F7SSZP003GT0PR female F7SSZP003GZ6SM provision FJ645924 Lactobacillus crustorum AY253657 Lactobacillus kalixensis Y17361 Lactobacillus amylolyticus 0.910.91 AB290830 Lactobacillus equicursoris 0.970.97 M58823 Lactobacillus delbrueckii CP000156 Lactobacillus delbrueckii 1.001.00 AB368915 Lactobacillus harbinensis CP002338 Lactobacillus amylovorus 1.001.00 M58802 Lactobacillus acidophilus 0.810.81 0.810.81 AB186339 Lactobacillus kitasatonis EU419585 Lactobacillus helveticus EU878007 Lactobacillus acidophilus Y19167 Lactobacillus perolens DQ317604 Lactobacillus crispatus EU559595 Lactobacillus crispatus G1OJGBH03GSTGE social pollen 0.520.52 AJ242969 Lactobacillus crispatus 1.001.00 AE017198 Lactobacillus johnsonii EU780913 Lactobacillus johnsonii mellifera AY974809 Lactobacillus brevis HM534808 mellifera 0.550.55 0.550.55 HM534806 mellifera HM534795 mellifera HM215048 Bombus HM534767 mellifera AJ971929 Bombus HM534796 mellifera HM534786 mellifera GU233458 dorsata GU233457 dorsata HM113257 mellifera AB257864 Lactobacillus camelliae HM113281 mellifera HM113326 mellifera HM046579 cerana DQ837635 mellifera DQ837634 mellifera EU184020 Lactobacillus rhamnosus HM046568 mellifera AY370183 mellifera HM112010 mellifera HM112866 Colletes EU753698 mellifera HM113333 mellifera M23928 Lactobacillus casei AY667698 Lactobacillus alvei HM534805 mellifera DQ837637 mellifera HM113203 mellifera EU753697 mellifera AF000163 Lactobacillus manihotivorans DQ837636 mellifera AY667699 Lactobacillus insectis 0.820.82 AB362679 Lactobacillus pantheris AY667701 Lactobacillus apis 0.570.57 M58831 Lactobacillus sharpeae GU138576 Lactobacillus guizhouensis G1OJGBH03FPFCW social larva AY773954 Lactobacillus paracasei AY667700 Lactobacillus larvae G1OJGBH03HE4SG social frass G1OJGBH03GD18U social frass G1OJGBH03GBVJ2 social frass G1OJGBH03FXRZZ social frass G1OJGBH03HA4AJ social frass NR 024885 Paralactobacillus selangorensis 0.830.83 AJ576007 Lactobacillus rennini 0.590.59 DQ406862 Lactobacillus backi 0.880.88 M58809 Lactobacillus bifermentans JN175330 Lactobacillus bifermentans 0.720.72 HM112055 mellifera HM113158 mellifera HM534810 mellifera 0.510.51 HM113222 mellifera 0.880.88 HM046576 cerana F4 EU753690 mellifera DQ837632 mellifera HM113258 mellifera 1.001.00 DQ837631 mellifera DQ837630 mellifera HM215043 Bombus 0.670.67 HM215041 Bombus F3 HM215042 Bombus HM534814 mellifera EU559601 Lactobacillus jensenii 0.860.86 M58801 Lactobacillus acetotolerans FR683099 Lactobacillus acetotolerans F7SSZP003GT0PR female F7SSZP003GZ6SM provision AY253657 Lactobacillus kalixensis Y17361 Lactobacillus amylolyticus 0.910.91 AB290830 Lactobacillus equicursoris 0.970.97 M58823 Lactobacillus delbrueckii CP000156 Lactobacillus delbrueckii CP002338 Lactobacillus amylovorus 0.810.81 M58802 Lactobacillus acidophilus 0.810.81 AB186339 Lactobacillus kitasatonis EU419585 Lactobacillus helveticus EU878007 Lactobacillus acidophilus DQ317604 Lactobacillus crispatus EU559595 Lactobacillus crispatus G1OJGBH03GSTGE social pollen 0.520.52 AJ242969 Lactobacillus crispatus 1.001.00 AE017198 Lactobacillus johnsonii EU780913 Lactobacillus johnsonii mellifera HM534808 mellifera Corbiculate apid 0.550.55 HM534806 mellifera HM534795 mellifera HM215048 Bombus AJ971929 Bombus HM534796 mellifera Flower HM534786 mellifera GU233458 dorsata GU233457 dorsata HM113257 mellifera Other bee HM113281 mellifera HM113326 mellifera HM046579 cerana DQ837635 mellifera Megalopta genalis DQ837634 mellifera HM046568 mellifera F5 AY370183 mellifera HM112010 mellifera Halictus ligatus HM112866 Colletes EU753698 mellifera HM113333 mellifera AY667698 Lactobacillus alvei HM534805 mellifera DQ837637 mellifera HM113203 mellifera EU753697 mellifera DQ837636 mellifera AY667699 Lactobacillus insectis AY667701 Lactobacillus apis

1.0 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.0

Fig. 5 Best-scoring Lactobacillus 16s rDNA tree of 20 maximum-likelihood search replicates, with bootstrap values from 100 pseudoreplicates placed on branches with greater than 50% support. Corbiculate apid-associated sequences are in boldface, orange font; ﬂower-associated sequences are in boldface, pink font; other bee-associated sequences are in boldface, blue font; Augochlora pura sequences are in italicized, light green font; Megalopta genalis sequences are in italicized dark green font; and Halictus ligatus sequences are in italicized, brown font. Fig. 5a,b represent the respective outlined portion of the larger tree, as indicated by the smaller complete tree.

2012 Blackwell Publishing Ltd 12 Q. S. MCFREDERICK ET AL. transmission, for example, could explain why L. kunkeei and growth of pathogens is suppressed (Lindgren & was absent in an Ap. mellifera hive surveyed in January, Dobrogosz 1990). L. sanfranciscensis, for example, is a time in winter when little forage is available (Martin- thought to suppress fungi through the secretion of a son et al. 2011), while L. kunkeei was abundant in sur- variety of acids, thus prolonging the shelf life of sour- veys of actively foraging colonies (Olofsson & Vasquez dough breads (Corsetti et al. 1998). We found that the 2008). The floral transmission hypothesis was rejected pH of the three pollen samples from M. genalis was by Olofsson & Vasquez (2008), as they sampled several quite low (3.4–3.7); however, whether the acidic provi- species of flowers, honey and specimens of Ap. mellifera sions is a cause or a consequence of Lactobacillus pres- and detected L. kunkeei from the bees and honey only. ence remains to be tested. Additionally, some sweat The hypothesis deserves revisiting, however, as L. kun- bee–associated lactobacilli are related to L. sanfranciscen- keei has since been reported from azalea, narcissus, cos- sis and may inhibit mould growth on pollen provisions mos, morning glory and crape myrtle flowers (Endo and frass inside of bee brood cells. However, further et al. 2009, 2011). work is necessary to determine whether fermentative Besides acidophilic bacteria, we found several other properties are conserved in bee-associated lactobacilli, abundant bacteria associated with the sweat bees in our as bacterial phenotype can be unpredictable from phyl- study. Wolbachia has been previously detected in sweat otype (Schloss & Westcott 2011). bees (Werren & Windsor 2000), was present in all three It has been hypothesized that bacteria associated with species and, besides very low levels detected in frass Apis and Bombus represent a co-evolutionary relation- and pollen, was found only in the insect samples ship (Martinson et al. 2011); however, evidence of reci- (Fig. 1). Another endosymbiotic bacterium that is procal selection is currently lacking. Our phylogeny known to distort the sex ratios of its host, Cardinium largely agrees with the finding that some lactobacilli are (Zchori-Fein & Perlman 2004), was found in high abun- specific to the corbiculate apids, a condition conducive dance in a couple of samples from the M. genalis soli- to co-evolution. We found that the F-3 clade was com- tary nest (Table S3, Supporting information). How pletely Apis- and Bombus-specific, which is consistent these endosymbiotic bacteria interact with their bee with suggestions that these lactobacilli may be vertically hosts remains unknown. Bacteria that are commonly transmitted (Koch & Schmid-Hempel 2011a; Martinson found in the soil, such as Clostridiales and Pseudomonas et al. 2011). Clades F-4 and F-5 appear to be mostly, but (Janssen 2006), were both found in two of the four not entirely, associated with the corbiculate apids. The nests, indicating that environmental bacteria can colo- isolation of F-4 and F-5 sequences from a clean room, a nize bee nests. solitary bee and a mosquito suggests that more compre- If future studies show that sweat bee–associated lac- hensive sampling may change future views of any of tobacilli or other acidophilic bacteria provide benefits to these relationships. their hosts, environmental transmission has some inter- Our data suggest promising lines of future research. esting implications, because association with acidophilic It has been hypothesized that social structure affects the bacteria is not guaranteed. Through secretion of acids, acquisition of beneficial bacteria by bees (Koch & Sch- lactobacilli and other acidophilic bacteria may provide mid-Hempel 2011a; Martinson et al. 2011), and the first protection of the pollen provisions of bees such as evidence supporting this hypothesis has recently been M. genalis, H. ligatus and Au. pura from exploitation by published. Exposure of laboratory-reared bumble bees other bacteria and fungi. If so, variation in the presence to their nest mate’s faeces allowed for the establishment of these bacteria may explain some variation in wild of bacteria that protected the bees from the parasite Cri- bee health. Floral nectar pH varies widely, with thidia bombi, indicating that social contacts may be extremes values from 3 to 10, although most nectars important in intracolony transmission (Koch & Schmid- appear to be slightly acidic (Baker & Baker 1983). There Hempel 2011b). Although low replication means that may be variation in the presence of acidophilic bacteria our data should not be overinterpreted, Lactobacillus in flowers, related to variation in nectar pH, or vice consistently occurred in samples from social nests and versa. Broad surveys of bacteria associated with flowers patchily occurred in samples from solitary nests and experiments testing for the transmission of putative (Fig. 1). M. genalis is known to exhibit trophallaxis mutualists from flowers to bees would further elucidate (Wcislo & Gonzalez 2006), which may help to maintain these relationships. If flowers do vary in their suitability the presence of lactobacilli in social nests (Martinson as reservoirs of acidophilic bacteria, planting those et al. 2011); however, trophallaxis has not been reported flowers that best provide acidophilic bacteria to wild in H. ligatus. More work teasing out the role of environ- bees may be an applied outcome of such surveys. mental and social transmission of beneficial bacteria is The fermentation of human foods by LAB does not needed. Additionally, understanding how microbes affect the food’s nutritional value, but rather spoilage affect bee health is of timely importance. For example,

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software for describing and comparing microbial communities. Supporting information Applied and Environmental Microbiology, 75, 7537–7541. Sen R, Ishak HD, Estrada D et al. (2009) Generalized Additional supporting information may be found in the online antifungal activity and 454-screening of Pseudonocardia and version of this article. Amycolatopsis bacteria in nests of fungus-growing ants. Fig S1 Rarefaction curves for Augochlora pura samples. Proceedings of the National Academy of Sciences, 106, 17805– 17810. Fig S2 Rarefaction curves for Halictus ligatus samples. Tajabadi N, Mardan M, Abdul Manap MY et al. (2011) Fig S3 Rarefaction curves for Megalopta genalis social nest sam- Detection and identification of Lactobacillus bacteria found in ples. the honey stomach of the giant honeybee Apis dorsata. Apidologie, 42, 642–649. Fig S4 Rarefaction curves for Megalopta genalis solitary nest Vasquez A, Olofsson TC (2009) The lactic acid bacteria samples. involved in the production of bee pollen and bee bread. Fig S5 Results of BLAST searches of 16S rDNA amplicons Journal of Apicultural Research, 48, 189–195. against the SILVA database, as visualized in MEGAN4. Vasquez A, Olofsson TC, Sammataro D, Hartfelder K (2009) A scientific note on the lactic acid bacterial flora in honeybees Fig S6 Average patristic distance from bee-associated lactoba- in the USA: a comparison with bees from Sweden. cilli to flower-associated lactobacilli and from bee-associated Apidologie, 40, 26–28. lactobacilli to all other lactobacilli, excluding flower-associated Wcislo WT, Gonzalez VH (2006) Social and ecological contexts lactobacilli. Error bars are 95% confidence intervals. of trophallaxis in facultatively social sweat bees, Megalopta genalis and M. ecuadoria (Hymenoptera, Halictidae). Insectes Fig S7 Natural log of the proportional abundance of the ten Sociaux, 53, 220–225. most commonly observed bacterial phylotypes from four sweat Wcislo WT, Arneson L, Roesch K et al. (2004) The evolution of bee nests. nocturnal behaviour in sweat bees, Megalopta genalis and Table S1 Halictus ligatus. Percent abundance of phylotypes M. ecuadoria (Hymenoptera: Halictidae): an escape from per sample according to BLAST matches at >96% for species, competitors and enemies? Biological Journal of the Linnean 94% >96% for genus, 89% >94% for family, 85% >89% for Society, 83, 377–387. order, 80% >85% for class and 77% >80% for phylum. Werren JH, Windsor DM (2000) Wolbachia infection frequencies in insects: evidence of a global equilibrium? Proceedings of the Table S2 Augochlora pura. Percent abundance of phylotypes Royal Society of London. Series B: Biological Sciences, 267, 1277– per sample according to BLAST matches at >96% for species, 1285. 94% >96% for genus, 89% >94% for family, 85% >89% for Zchori-Fein E, Perlman SJ (2004) Distribution of the bacterial order, 80% >85% for class and 77% >80% for phylum. symbiont Cardinium in arthropods. Molecular Ecology, 13, Table S3 Megalopta genalis social nest. Percent abundance of 2009–2016. phylotypes per sample according to BLAST matches at >96% Zwickl DJ (2006) Genetic Algorithm Approaches for the for species, 94% >96% for genus, 89% >94% for family, Phylogenetic Analysis of Large Biological Sequence Datasets under 85% >89% for order, 80% >85% for class and 77% >80% the Maximum Likelihood Criterion. The University of Texas, for phylum. Austin, Texas. Table S4 Megalopta genalis solitary nest. Percent abundance of phylotypes per sample according to BLAST matches at >96% Q.S.M. is interested in how interactions with microbes, other for species, 94% >96% for genus, 89% >94% for family, organisms and the environment affect wild bees. W.T.W. stu- 85% >89% for order, 80% >85% for class and 77% >80% dies evolution and behavior in social insects, mainly at the for phylum. cusp of sociality. D.R.T. studies how evolution is influenced by Table S5 Within genome variation of 16S copies. population structure and multi-level selection, mostly in plant reproductive systems. H.D.I. is interested in how coevolution Data S1 Alignment of 16S rDNA from lactobacilli. Sequences and symbioses shape the ecology of insects. S.E.D. is interested were both obtained from NCBI GenBank and generated for this in microbial ecology in diverse environments. U.G.M. studies study. the ecology and evolution of cooperative and antagonistic interactions. Data S2 Alignment of 16S rDNA from sweat bee-associated bacteria, used in FastTree and Fast UniFrac analyses.

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting information supplied by the Data accessibility authors. Any queries (other than missing material) should be 16S rDNA amplicon reads: available on NCBI’s sequence read directed to the corresponding author for the article. archive (SRA), accession number SRP009083. Phylogenetic data: http://purl.org/phylo/treebase/phylows/study/TB2:S12120, also available as online supplemental material.

2012 Blackwell Publishing Ltd