International Journal of Systematic and Evolutionary Microbiology (2016), 66, 1323–1329 DOI 10.1099/ijsem.0.000882

Apibacter adventoris gen. nov., sp. nov., a member of the phylum isolated from honey bees Waldan K. Kwong1,2 and Nancy A. Moran2

Correspondence 1Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT, USA Waldan K. Kwong 2Department of Integrative Biology, University of Texas, Austin, TX, USA [email protected]

Honey bees and bumble bees harbour a small, defined set of gut bacterial associates. Strains matching sequences from 16S rRNA gene surveys of bee gut microbiotas were isolated from two honey bee from East Asia. These isolates were mesophlic, non-pigmented,

catalase-positive and oxidase-negative. The major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH,

C16 : 0 and C16 : 0 3-OH. The DNA G+C content was 29–31 mol%. They had ,87 % 16S rRNA gene sequence identity to the closest relatives described. Phylogenetic reconstruction using 20 protein-coding genes showed that these bee-derived strains formed a highly supported monophyletic clade, sister to the clade containing species of the genera and within the family of the phylum Bacteroidetes. On the basis of phenotypic and genotypic characteristics, we propose placing these strains in a novel genus and species: adventoris gen. nov., sp. nov. The type strain of Apibacter adventoris is wkB301T (5NRRL B-65307T5NCIMB 14986T).

Bacteria from the phylum Bacteroidetes are often constitu- In July and August of 2014, samples of the Asian honey bee ents of animal microbiotas. In humans, members of the (Apis cerana) and the giant honey bee (Apis dorsata) were genera Prevotella and Bacteroides are among the most collected from Singapore and Kuala Lumpur, Malaysia numerically abundant gut symbionts, and appear to (Table S1, available in the online Supplementary Material). anchor different states of stability (enterotypes) within The entire intestinal tract was removed and crushed in the community (Arumugam et al. 2011). Honey bees and 19 % (v/v) glycerol and frozen. were recovered bumble bees have also been found to harbour members by plating out the frozen samples onto heart infusion of the phylum Bacteroidetes: culture-independent 16S agar (Hardy Diagnostics) supplemented with 5 % defibri- rRNA gene surveys of their gut communities have ident- nated sheep blood and incubating at 35 8Cin5%CO2. ified sequences falling within the order , After 2–3 days, small (,0.5 mm) white, non-haemolytic with .10 % divergence from described species. Although colonies appeared. Three isolates were chosen for further their prevalence appears sporadic compared with the core characterization: wkB180, wkB309 and wkB301T. bee gut microbiota (Babendreier et al., 2007; Koch & Strains wkB180, wkB309 and wkB301T grew on heart infusion Schmid-Hempel, 2011; Ahn et al., 2012; Moran et al., agar, brain heart infusion agar, trypticase soy agar, Columbia 2012; Lim et al., 2015), the bee Flavobacteriales sequences agar and lysogeny broth (LB) agar, but exhibited poor or no form a monophyletic clade, suggesting they are specific to growth on nutrient agar, MacConkey agar, Lactobacillus bee guts, or to other environments (e.g. plants, nectar, MRS agar or R2A agar (all media from BD Difco). No hive material) where they are prone to be ingested by growth was observed under anaerobic conditions (90 % N , bees. Here, we provide the first description of members 2 5% CO,5%H) or in ambient air. In liquid culture, of this novel group. 2 2 growth was observed in Insectagro DS2 (Corning), Columbia broth, trypticase soy broth, and LB (without NaCl), but not in Lactobacillus MRS. On solid media, the strains formed The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA smooth, round, pale/semi-translucent colonies. Strain T gene sequences of strains wkB301 , wkB309 and wkB180 are wkB309 exhibited rapidly spreading margins of growth KT149221, KT149220 and KT149222 respectively. Protein-coding extending from colony edges, consistent with gliding motility. genes from these strains are deposited under the accession numbers KT149223– KT149282. DNA was extracted as described by Powell et al. (2014) Four supplementary tables and one supplementary figure are available with the following modifications. RNase A was added with the online Supplementary Material. directly to cetyltrimethylammonium bromide (CTAB)

Downloaded from www.microbiologyresearch.org by 000882 G 2016 IUMS Printed in Great Britain 1323 IP: 54.70.40.11 On: Sun, 04 Nov 2018 04:06:39 W. K. Kwong and N. A. Moran buffer prior to bead-beating, a final concentration of 0.5 % Ornithobacterium rhinotracheale, 2-mercaptoethanol was used, and the lysate was incubated and aquatile, respectively (Table S1). How- at 56 8C for 1 h instead of overnight. Samples were sub- ever, our strains could not be confidently placed in relation mitted to the Genome Sequencing and Analysis Facility to the clades encompassing the genera Empedobacter and at the University of Texas at Austin for Illumina MiSeq Chryseobacterium (clades E and C, respectively) using 16S 26300 bp library preparation and whole genome sequen- rRNA gene sequences alone (Fig. 1). To obtain better cing. Sequence reads were assembled with CLC Genomics phylogenetic resolution, we extracted and aligned 20 pro- Workbench 5.5 (CLC bio). Genome sizes and DNA tein sequences from the strains’ genomes and that of G+C content were inferred from draft assemblies. sequenced relatives to produce a tree based on 9746 total residues (Fig. 2). This tree strongly suggested that the bee Phylogenetic analysis based on 16S rRNA sequences gut strains are sister to clade C, and not to clade E. All phy- (1250 bp) showed that strains wkB180, wkB309 and logenetic analysis was done in MEGA 6 (Tamura et al. 2013). wkB301T clustered together and were only distantly related to previously described species (Fig. 1). Strain wkB301T We conducted further assays to characterize the phenotypic had 87.6, 87.5, 87.2 and 85.5 % 16S rRNA gene sequence traits of our strains. Scanning electron micrographs identity to the type strains of Empedobacter brevis, were taken to determine cell size and morphology.

LMG 18212T (AJ271010) 97 Chryseobacterium joosteii T 61 Chryseobacterium ureilyticum F-Fue-04IIIaaaa (AM232806) 92 T Chryseobacterium vrystaatense R-23566 (AJ871397) T 39 Chryseobacterium gleum ATCC 35910 (ACKQ01000057) T 0.2 NF802 (AM261868) 100 H9T (EF204446) 22 T 100 Chryseobacterium daecheongense CPW406 (AJ457206) T Chryseobacterium wanjuense R2A10-2 (DQ256729) 39 LMG 23089T (AM040439) 96 Chryseobacterium piscium 50 T Chryseobacterium indoltheticum LMG 4025 (AY468448) 51 T Chryseobacterium aquaticum 10-46 (AM748690) T Clade C 71 Chryseobacterium xinjiangense TSBY 67 (DQ166169) T 69 100 Epilithonimonas tenax EP105 (AF493696) T Epilithonimonas lactis H1 (NR_115989) T 100 normanense CCUG 46293 (AJ575430) T Cloacibacterium rupense R2A-16 (EU581834) T 77 88 zoohelcum ATCC 43767 (AGYA01000006) T 78 anatipestifer DSM 15868 (NR_074429) Flavobacteriaceae 90 8151T (NR_117970)

Riemerella columbipharyngis Bacteroidetes 100 T Soonwooa buanensis HM0024 (NR_116776) 51 T Elizabethkingia meningoseptica ATCC 13253 (ASAN01000081) 100 T 47 Elizabethkingia anophelis R26 (NR_116021) T Cruoricaptor ignavus IMMIB L-12475 (NR_108875) T Ornithobacterium rhinotracheale DSM 15997 (CP003283) 40 T Moheibacter sediminis M0116 (KF694750) T virosa DSM 16922 (CP002455) 80 85 T (KF694751) 51 100 Chishuiella changwenlii BY4 Clade E T 88 Empedobacter falsenii NF 993 (AM084341) T Empedobacter brevis LMG 4011 (AM177497) 100 Apibacter adventoris wkB180 (KT149222) Apis dorsata Apibacter adventoris wkB301T (KT149221) 100 100 uncultured clone HBG A2V3-2 (DQ837638) Apis mellifera uncultured clone HBG A2V3-4 (DQ837639) 100 sp. B1 (KJ741206) 100 Bacteroidetes 100 Apis cerana Bee-associated Apibacter adventoris wkB309 (KT149220)

85 uncultured clone BJ07179A4 (HM215037)

100 uncultured clone D08062F1 (HM215038) Bumble bee uncultured clone D08033D2 (HM215036) 97 uncultured clone Bt231.55A2 (JQ388908) Flavobacterium aquatile LMG 4008T (JRHH01000003) Prevotella melaninogenica ATCC 25845T (AY323525) Bacteroides fragilis NCTC 9343T (CR626927) Escherichia coli MG1655 (NC_000913)

Fig. 1. 16S rRNA gene phylogeny of strains wkB301T, wkB309, wkB180 and close relatives. Hosts of origin for bee-derived sequences or strains are highlighted. The tree was built with a maximum-likelihood algorithm using the Generalized Time Reversible substitution model with gamma-distributed rates and invariant sites. Escherichia coli MG1655 is the outgroup. Bootstrap percentages (based on 1000 replicates) are shown at nodes. Bar, 0.2 substitutions per site.

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T 100 Chryseobacterium indologenes NBRC 14944 (NZ_BAVL00000000) 99 T 0.2 Chryseobacterium gleum ATCC 35910 (NZ_ACKQ00000000) 100 Chryseobacterium taiwanense TPW19 (NZ_JWTA00000000) 99 Chryseobacterium vrystaatense LMG 22846T (NZ_JPRI00000000) 100 Epilithonimonas lactis H1T (NZ_JPLY00000000) Chryseobacterium haifense DSM 19056T (NZ_JASZ00000000) 73 100 Chryseobacterium antarcticum LMB 24720 (NZ_JPEP00000000) Clade C ATCC 13253T (NZ_BARD00000000) 100 Elizabethkingia meningoseptica 100 ATCC 33958 (NZ_JRFN00000000) 100 Elizabethkingia anophelis R26T (NZ_ANIW00000000) 100 Riemerella columbina DSM 16469T (NZ_ARFT00000000) 100 DSM 15868T (NC_014738) 100 ATCC 43767T (NZ_AGYA00000000) Apibacter adventoris wkB309 100 100 Apibacter adventoris wkB301T 100 Apibacter adventoris wkB180 Ornithobacterium rhinotracheale DSM 15997T (NC_018016) 100 98 DSM 16922T (NC_015144) T 100 Empedobacter brevis LMG 4011 (NZ_ARNT00000000) Clade E 100 Empedobacter falsenii 282 (NZ_JSYQ00000000) Flavobacterium aquatile LMG 4008T (NZ_JRHH00000000) T 100 Prevotella melaninogenica ATCC 25845 (NC_014371) Bacteroides fragilis NCTC 9343T (NC_003228) Escherichia coli MG1655 (NC_000913)

Fig. 2. Phylogenetic tree of strains wkB301T, wkB309, wkB180 and related taxa, based on 20 conserved proteins (encoded by genes alaS, atpA, dnaA, dnaN, ftsZ, fusA, groEL, gyrB, lepA, metK, nusG, pfkA, pyrG, recA, rplA, rplB, rpoB, rpsB, sdhA and secA). Accession numbers for these genes are listed in Table S2. Proteins were concatenated and aligned with MUSCLE and the tree was built with a maximum-likelihood algorithm using the Jones–Taylor–Thornton substitution model with gamma-distributed rates and invariant sites. Bootstrap percentages (1000 replicates) are shown at nodes. Bar, 0.2 substitutions per site.

Strains were grown on Columbia blood agar plates for (bioMe´rieux) according to manufacturer’s protocols. Cata- 48 h, then fixed with 2.5 % (v/v) glutaraldehyde in 0.1 M lase activity was tested by addition of cells directly to 3 % sodium cacodylate buffer (SCB) overnight at 4 8C. After H2O2 and observing for generation of gas. Presence of cyto- three rinses in 0.1 M SCB, they were post-fixed with 1 % chrome c oxidase was determined with OxiStrips (Hardy osmium tetroxide in 0.1 M SCB for 2 h. Cells were Diagnostics). Acetoin production was assayed by addition rinsed with water and dehydrated with increasing concen- of Voges–Proskauer reagents (Hardy Diagnostics) to tration of ethanol, followed by critical-point drying strains cultured in Methyl-Red Voges–Proskauer broth (Samdri-795; Tousimis). Cells were air-dried, mounted for 5–7 days. We tested for flexirubin-type pigments on scanning electron microscope (SEM) stubs and sputter using the KOH method as recommended by Bernardet coated with gold-palladium in a Cressington 108auto. et al. (2002). susceptibility tests against ampicil- Images were acquired on a Zeiss Sigma Field-Emission lin, chloramphenicol, gentamicin, kanamycin, spectinomy- SEM (University of Texas Southwestern Electron cin, streptomycin and tetracycline were performed with Microscopy Facility). Cells of strain wkB301T were MIC test strips (Table S3). Salt tolerance was tested by cul- approximately 2.060.3 mm in size and formed extensive tivation in LB with NaCl added in 0.5 % increments. filaments (Fig. 3a); when grown in liquid media, filamenta- Cellular fatty acid composition of strains wkB301T and tion was reduced. Strain wkB309 did not filament, but wkB309 was determined by fatty acid methyl ester instead consisted of short, rounded rods approximately (FAME) analysis (Sherlock MIS-MIDI; Microbial ID). 0.9–1.5 mm long and 0.4 mm wide (Fig. 3b). Strains were plated on 5 % sheep blood trypticase soy T Biochemical properties of strains wkB301 and wkB309 agar at 35 8C for 48 h under 3–5 % CO2, and cells from were determined with API 20 NE and API 50 CH kits the late-exponential phase portion were harvested for

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(a) will be needed to delineate the taxonomic status of strain wkB309 and other similar isolates. The ecological niche of Apibacter adventoris gen. nov., sp. nov. is not well understood, although current evidence suggests it is a bee gut specialist. Like other members of the bee gut microbiota, our strains did not readily grow in ambient air, but instead required a microaerobic atmo- sphere (Kwong & Moran, 2013; Engel et al., 2013). A smaller genome size compared with free-living relatives (Table 1), and the concomitant reduced functional capa- bilities, suggests Apibacter adventoris has a host-restricted, rather than a heterotrophic, free-living lifestyle. The limited 16S rRNA sequence data available thus far (Fig. 1) show clustering of sequences according to host species, indicat- (b) ing that different bee hosts may harbour their own special- ized species or strains of the genus Apibacter. The clustering observed is independent of geographical origin: the Apis dorsata-derived strains wkB180 and wkB301T are from Singapore and Malaysia, respectively, while the Apis cerana-derived strains wkB309 and Bacteroidetes sp. B1 are from Malaysia and South Korea, respectively. Greater sampling is thus needed to demonstrate the ecological ranges of various species and strains within the genus Api- bacter gen. nov., and the nature of their relationship with bee hosts.

Description of Apibacter gen. nov.

Fig. 3. Scanning electron micrographs of cells of strains Apibacter (a.pi.bac9ter. L. fem. n. apis bee; N.L. masc. n. wkB301T (a) and wkB309 (b). Bars, 1 mm. bacter a rod; N.L. masc. n. Apibacter a rod-shaped bacter- ium first isolated from bees). Cells are Gram-staining-negative, microaerophilic, meso- FAME analysis. Respiratory quinone and polar lipid anal- philic, salt-intolerant and rod-shaped. Positive for catalase ysis of strain wkB301T was performed on lyophilized cells and negative for oxidase. Flexirubin-type pigments are not by the Identification Service of the DSMZ, according to produced. The major fatty acids are iso-C15 : 0, iso-C17 : 0 the protocols of Tindall (1990) and Tindall et al. (2007) 3-OH, C16 : 0 and C16 : 0 3-OH. Menaquinone 6 (MK-6) is (Fig. S1), respectively. the only respiratory quinone detected. The major polar lipids include phosphatidylethanolamine, an unidentified T Differential characteristics of strains wkB301 and wkB309 lipid, two phospholipids, three aminolipids, a phospho- and those of their closest relatives are summarized in aminolipid and a glycolipid. The DNA G+C content is Tables 1 and S4. Compared with most species in clades 29–31 mol%. The genus belongs to the family Flavobacter- T E, C and the genus Ornithobacterium, strains wkB301 iaceae in the phylum Bacteroidetes. The type species is and wkB309 had distinctive fatty acid profiles (containing Apibacter adventoris. substantial C16 : 0 and C16 : 0 3-OH), low DNA G+C con- tent (29–31 mol%), and lack of flexirubin-type pigment production and cytochrome oxidase activity. Phylogenetic Description of Apibacter adventoris sp. nov. reconstruction showed that these strains comprise a diver- Apibacter adventoris (ad.ven.to9ris. L. gen. n. adventoris of a gent monophyletic lineage (Fig. 2). These data indicate that T visitor, of a guest, referring to the affiliation with the gut of strains wkB180 and wkB301 are representatives of a novel bee hosts). species and genus within the family Flavobacteriaceae of the phylum Bacteroidetes, for which we propose the name Api- In addition to the characteristics of its genus, the following bacter adventoris gen. nov., sp. nov. Additionally, at 95.8 % are displayed. Cells are approximately 2.0|0.3 mm in size, 16S rRNA gene sequence identity and 77.5 % average but may form longer filaments. Grows on heart infusion nucleotide identity (by the method of Goris et al., 2007) agar, brain heart infusion agar, trypticase soy agar and to wkB301T, strain wkB309 is sufficiently distinct to war- Columbia agar at 20–40 uC (optimum 30–35 uC) in 5 % rant eventual classification as a representative of its own CO2. No growth in anaerobic or normal atmospheric species within the genus Apibacter. Further characterization conditions. Grows in LB medium with 0–1.0 % NaCl

Downloaded from www.microbiologyresearch.org by 1326 International Journal of Systematic and Evolutionary Microbiology 66 IP: 54.70.40.11 On: Sun, 04 Nov 2018 04:06:39 Apibacter adventoris gen. nov., sp. nov. were ) ) )|| C. gleum d 2 + V V 2 2 + 5 + + + + + + + ( ND ND ( ( V water, and V soils, hospitals Diseased humans, , variable between strains; V O. rhinotracheale , negative; -glucopyranoside, amygdalin, arbutin, ) ) ) ) ) ) D 2 - D 2 + + + + + 2 2 2 2 2 tract a + ( ND ND NDNDND V V ( ( ( ( ( V V V V V V , Positive; Avian respiratory + -sorbitol. No data for D 222 2 2 2 -mannopyranoside, methyl +++ +++ + + + + water D (multiple strains). Flavobacteriaceae - a -mannitol and D Diseased humans, C. gleum -sorbose, methyl ) L -rhamnose, dulcitol, inositol, cellobiose or raffinose, but these were negative in the other strains. 222 22 22 + + L (multiple strains); 5, Apis cerana ( -xylopyranoside, Asian honey bee D - b -fucose, potassium gluconate, potassium 2-ketogluconate or potassium 5-ketogluconate, but these were negative in the O. rhinotracheale D ) ;4, T -xylose, methyl , wkB309 and closely related members of the family L -tagatose, T D 22 22 22 2 22 2 22 22 222 222 2222 222 222 222 +++ +++ + + + ++ ++ , but this was negative in the other strains. CCUG 7320 , no data. All strains were negative for acid production from Apis dorsata ( -lyxose, ND D -arabinose, L C. gleum were available for acid production from -fucose in L Empedobacter brevis ), majority negative; O. rhinotracheale 2 ( V ; 2, wkB309; 3, T C content (mol%) 29.0 30.6 32.7 37–39 36–39 + Differential characteristics of strains wkB301 -Arabinose -Ribose -Xylose -Adonitol -Galactose -Glucose -Fructose -Glucosidase (aesculin) -Galactosidase (PNPG) D D D Catalase Cytochrome oxidase Indole production b MacConkey agar b Gelatinase Nutrient agar Nitrate reduction Urease D D D D Arginine dihydrolase Glycerol ), majority positive; + Flexirubin pigment production Growth on: Gliding motility Genome size§DNA G 2.7 Mb 2.3 Mb 3.79 Mb 2.40 Mb 5.57 Mb CharacteristicEnzymic 1 activity: 2Acid production from: 3* 4 Isolated from: Giant honey bee ( available for acid production from erythritol, inulin, melezitose, xylitol, gentiobiose, turanose, No data for acid production from other strains. No data for Table 1. Taxa: 1, wkB301 V

Downloaded from www.microbiologyresearch.org by http://ijs.microbiologyresearch.org 1327 IP: 54.70.40.11 On: Sun, 04 Nov 2018 04:06:39 W. K. Kwong and N. A. Moran c 9 v 3-OH (35.0 %), d 17 : 1 2 2 2 5 + + ND ND ND 17 : 0 (22.0 %), (20.0 %) 15 : 0 iso-C iso-C iso-C 3-OH ) ) ) ) ) (57.4 %), D 2 + + + + ( ND NDNDND ND ND ND ( ( ( ( 15 : 0 17 : 0 V V V V V C 15 : 0 (20.2 %), iso- 3-OH (8.1 %) iso-C iso-C (2002, 2006). 3-OH and/or (15.5 %) c c (24.5 %), 7 et al. 6 2 2 + ++ + 17 : 0 v v (17.6 %), 15 : 0 16 : 1 16 : 1 iso-C C C iso-C 3-OH 3-OH 16 : 0 22 22 + 15 : 0 (21.6 %), iso- 17 : 0 (13.8 %) C (22.2 %), 16 : 0 iso-C C (15.4 %), C 16 : 0 3-OH (2003), for two strains. Additional data from Bernardet 16 : 0 et al. (1994), where 21 and 443 strains were tested, respectively. 22 222 2222 22 222 222 2 22 + ++ + (23.2 %), iso- 17 : 0 (11.0 %) C et al. 15 : 0 3-OH (18.0 %), C (15.1 %), C (1997) and Vandamme (1984), for 12 strains, and Hugo (2014). et al. et al. et al. cont. -Acetylglucosamine -Mannose -Arabitol -Arabitol Maltose Sucrose Trehalose Starch D L Lactose D N Salicin Glycogen Characteristic 1Major cellular fatty acids iso-C 2 3* 4 Data from van Empel Data from Holmes Data from Zhang Table 1. D d §Size information from NCBI||ONPG genomes. assay. *

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(optimum, 0–0.5 %), pH 6.0–8.0. Positive for Voges–Pros- Edited by M. Dworkin, S. Falkow, E. Rosenberg, K.-H. Schleifer & kauer reaction and aesculinase (b-glucosidase). Positive for E. Stackebrandt. New York: Springer. nitrate reduction (strain wkB309 is negative). Negative for Engel, P., James, R. R., Koga, R., Kwong, W. K., McFrederick, Q. & arginine dihydrolase, b-galactosidase (strain wkB309 is Moran, N. A. (2013). Standard methods for research on Apis 52 positive), indole production, gelatinase and urease. Acid mellifera gut symbionts. J Apic Res , 1–24. is produced from D-adonitol, arabitol and glycerol (strain Goris, J., Konstantinidis, K. T., Klappenbach, J. A., Coenye, T., Vandamme, P. & Tiedje, J. M. (2007). DNA-DNA hybridization wkB309 is negative), and D-glucose, D-fructose and D-man- values and their relationship to whole-genome sequence similarities. nose. Resistant to the aminoglycosides kanamycin and gen- Int J Syst Evol Microbiol 57, 81–91. tamicin. Fatty acid composition of the type strain, T Holmes, B., Owen, R. J., Steigerwalt, A. G. & Brenner, D. J. (1984). wkB301 , is as follows: iso-C15 : 0 (23.2 %), iso-C17 : 0 Flavobacterium gleum, a new species found in human clinical 3-OH (18.0 %), C16 : 0 (15.1 %), C16 : 0 3-OH (11.0 %), specimens. Int J Syst Bacteriol 34, 21–25. C16 : 1v7c and/or C16 : 1v6c (9.6 %), iso-C15 : 0 3-OH v Hugo, C. J., Segers, P., Hoste, B., Vancanneyt, M. & Kersters, K. (4.5 %), C18 : 2 6,9c and/or anteiso-C18 : 0 (4.0 %), (2003). Chryseobacterium joostei sp. nov., isolated from the dairy v C18 : 1 9c (2.5 %), iso-C17 : 0 (1.8 %) and anteiso-C15 : 0 environment. Int J Syst Evol Microbiol 53, 771–777. (1.5 %). Koch, H. & Schmid-Hempel, P. (2011). Bacterial communities in The type strain is wkB301T (5NRRL B-65307T5NCIMB central European bumblebees: low diversity and high specificity. Microb Ecol 62, 121–133. 14986T), which was isolated from the gut of an adult worker Apis dorsata collected from Kuala Lumpur, Kwong, W. K. & Moran, N. A. (2013). Cultivation and characterization + of the gut symbionts of honey bees and bumble bees: description of Malaysia. The DNA G C content of the type strain is Snodgrassella alvi gen. nov., sp. nov., a member of the family 29.0 mol%. Neisseriaceae of the Betaproteobacteria, and Gilliamella apicola gen. nov., sp. nov., a member of Orbaceae fam. nov., Orbales ord. nov., a sister taxon to the order ‘Enterobacteriales’ of the Acknowledgements Gammaproteobacteria. Int J Syst Evol Microbiol 63, 2008–2018. We thank Gary Kong-Wah Sing and Eunice Soh for their assistance in Lim, H. C., Chu, C. C., Seufferheld, M. J. & Cameron, S. A. (2015). collecting bee specimens, and Luis Medina for strain cultivation. This Deep sequencing and ecological characterization of gut microbial work was supported by Yale University, the Yale University Depart- communities of diverse bumble bee species. PLoS One 10, e0118566. ment of Ecology and Evolutionary Biology Chair’s Fund, Sigma Xi Moran, N. A., Hansen, A. K., Powell, J. E. & Sabree, Z. L. (2012). Grants-in-Aid of Research, and a Canadian Natural Sciences and Engin- Distinctive gut microbiota of honey bees assessed using deep eering Research Council Postgraduate Scholarship (to W. K. K.), and sampling from individual worker bees. PLoS One 7, e36393. the US National Science Foundation Dimensions of Biodiversity Powell, J. E., Martinson, V. G., Urban-Mead, K. & Moran, N. A. (2014). awards 1046153 and 1415604 (to N. A. M.). Routes of acquisition of the gut microbiota of the honey bee Apis mellifera. Appl Environ Microbiol 80, 7378–7387. Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013). 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