Supplementary Information Material and Methods DNA Extraction from Lactobacillus cells Chromosomal DNA of lactobacilli was isolated using the Qiagen DNeasy Blood and Tissue Kit

(Qiagen) with slight modifications. Cells from three milliliters of an overnight culture (around 16

h) were harvested by centrifugation at 10,000 x g for 5 min. Cell pellets were resuspended in 1

ml wash buffer (20 mM Tris-HCl, 2 mM sodium EDTA, pH 8.0) and centrifuged again. Genomic

DNA was then extracted following the manufacturer’s instructions for gram-positive bacteria and

increasing the lysis incubation time at 37˚C to 60 min. DNA was stored frozen at -20°C.

Sequencing of the 16S rRNA gene

The 16S rRNA gene was amplified from chromosomal DNA using primers 8F and 1391R (SI

Table S4). PCR was performed using Takara TaqTM mix with 20 pmol primers following the

manufacturer’s instructions. The PCR program used is as follows: 94°C for 2 min, followed by

30 cycles of 94°C for 1 min, 55°C for 45s, and 72°C for 2 min, with a final extension period of 7

min at 72°C. PCR products were purified using the Qiaquick PCR Purification Kit (Qiagen) and

sequenced with primer 8F (SI Table S4) using a commercial sequencing provider (Eurofins

MWG Operon).

AFLP genotyping AFLP is an attractive approach as it provides a genome-wide comparison of strains, buffering

against the distorting effect of inter-species recombination at individual loci. AFLP was

performed with AFLP template preparation kit available from LI-COR Biosciences (Lincoln,

Nebraska, USA) with slight modifications to the manufacturer’s protocol. Briefly, 300 ng of

genomic DNA of L. reuteri strains was digested with EcoRI and MseI restriction enzymes prior

to ligation to EcoRI and MseI adaptors. The ligation mixtures were initially subjected to pre-

selective PCR amplification using primers shown in SI Table S4. Two individual selective PCR

reactions were performed for each strain using the primers EcoR1-A/Mse1-AG and EcoR1-

G/Mse1-CC, each with different overhanging nucleotides (SI Table S4). The EcoR1-A and

EcoR1-G primers were fluorescently labeled with IRDye700. PCR cycling conditions for the

selective amplification step was optimized and performed as follows: 94°C for 5 min, followed by

12 cycles of 94°C for 30s, 65°C for 30s, and 72°C for 1 min and then followed by 22 cycles of

94°C for 30s, 56°C for 30s, and 72°C for 1 min, with a final extension of 10 min at 72°C. PCR

products from the two individual selective PCR reactions per strain were resolved on denaturing

polyacrylamide gels with a LI-COR 4200 global analysis system using 41-cm plates, and KBPLUS

(3.7%) gel (LI-COR). Molecular size standards (50-700bp) and control strains were loaded in

every gel.

AFLP image analysis and PCA were performed using Bionumerics software Version 5.0

(Applied Maths, Kortrijk, Belgium). Bands were scored based on presence or absence and

binary files were exported for phylogenetic analysis. Composite datasets of the two

electropherograms of each strain were generated and used for both reconstruction of

phylogenetic trees and PCA. A total of 439 markers were scored based on presence or absence

of bands on electropherograms, as shown in SI Figure S1. The binary matrix was converted into

a distance matrix using Nei & Li distances in PAUP* 4.0b10 (Nei and Li, 1979). AFLP phylogeny

was reconstructed using the neighbor-joining (NJ) algorithm implemented in PAUP and

Lactobacillus coryniformis Li146:1 was used as an outgroup to root the L. reuteri phylogeny

(Saitou and Nei, 1987). The final tree was displayed using FigTree

(http://tree.bio.ed.ac.uk/software/figtree/).

MLSA genotyping Our MLSA scheme used seven different housekeeping genes: D-alanine-D-alanine ligase (ddl),

phosphoketolase (pkt), leucyl-tRNA synthetase (leuS), DNA gyrase B subunit (gyrB), D-alanine-

D-alanyl carrier protein ligase (dltA), RNA polymerase alpha subunit (rpoA) and recombinase

(recA). Most of these genes were previously used for MLSA in related organisms (de Las Rivas

et al., 2006; Diancourt et al., 2007; Rademaker et al., 2007). Each locus was amplified by PCR

using the Takara TaqTM with 40 pmol of primers shown in SI Table S4. PCR conditions for all

MLSA genes were as follows: 94°C for 2 min, followed by 30 cycles of 94°C for 30s, 55°C for

30s, and 72°C for 1 min, with a final extension period of 7 min at 72°C. PCR products were

purified and sequenced as described above for the sequencing of 16S rRNA genes. PCR

products were sequenced in both directions using the same pair of amplification primers.

Sequences were entered into a local database that was established using MLSTdbNET (Jolley

et al., 2004).

Descriptive analyses of MLSA data were performed using DnaSP version 4.90.1 to determine

the fragment size, % G+C content, number of alleles, number of polymorphic nucleotide sites,

and gene diversity (Rozas et al., 2003). The START2 software was used to determine the

dN/dS ratios (Nei and Gojobori, 1986), generate in-frame concatenated sequences, and

conduct eBURST analysis (Feil et al., 2004; Jolley et al., 2001). For phylogenetic analysis, the

individual gene sequences and in-frame-concatenated sequences were aligned using ClustalW

implemented in MEGA4 software package (Tamura et al., 2007). Maximum likelihood (ML) trees

were reconstructed using the concatenated alignment of all seven MLSA loci, three loci that

showed little evidence of recombination (see below), as well as the alignments of individual

genes with GTR+I+G model of DNA evolution with 100 bootstrap replicates using PhyML

(Guindon and Gascuel, 2003; Guindon et al., 2005). In PhyML, the transition/transversion ratio,

proportion of invariable sites, and the gamma shape parameter were estimated, and the

subtree-pruning and regrafting (SPR) and nearest-neighbor interchange (NNI) tree topology

search methods were used. Lactobacillus vaginalis ATCC49540 was used as the outgroup to

root the L. reuteri phylogeny. Average nucleotide divergence within and between phylogenetic

lineages were determined using the JukesCantor correction in DNAsp version 4.90.1.

Test for recombination SplitsTree4 (version 4.10) was used to conduct phylogenetic network analysis based on the

neighbor-net algorithm, which incorporates both split-decomposition method and neighbor-

joining method (Huson, 1998). The split-decomposition method does not force the data into a

tree-like phylogeny allowing the detection of conflicting phylogenetic information. If there is a

conflicting phylogenetic signal, parallel edges between sequences are depicted. The pairwise-

homoplasy index (PHI) test implemented in the SplitsTree4 software was performed to identify

the extent of intragenic recombination (Bruen et al., 2006).

Competition experiment in ex-germ free (GF) mice Swiss Webster mice were reared germfree from stocks at the University of Nebraska gnotobiotic

facility. All animal care and procedures were performed with the approval of Institutional Animal

Care and Use Committee at UNL under protocol #0810056D. Overnight cultures of L. reuteri

strains were used to inoculate 10 ml of fresh mMRS media at 0.5%. Cultures were incubated for

14 h, what resulted in cell numbers between 4.65 x 108 and 1.68 x 109 cells/ml for individual

strains. Cells were recovered by centrifugation (4000 x g at room temperature), and pellets were

resuspended in 2.5 ml phosphate buffered saline (PBS, pH 6.0). Equal volumes of cell solutions

of each strain were combined. The mixture of bacterial cultures introduced into germfree

isolators with 30 minute decontamination, and each mouse was inoculated by rubbing the

culture onto the fur of the mice. Mice were housed in groups, but fecal samples of individual

mice were taken at days 1, 4, 8, and 11. Mice were sacrificed on day 11, and samples from the

forestomach epithelium and cecum were collected. All samples were homogenized and diluted

ten-fold in PBS (pH 6.0) and stored frozen at -80°C until DNA extractions were performed. Total

DNA was extracted from gut samples as described by Walter et al (Walter et al., 2001).

The strain composition in gut samples was determined by pyrosequencing of the partial leucyl-

tRNA synthetase (leuS) gene using a 454 GS-FLX sequencer (Roche). MLSA analysis revealed

the leuS gene to be most discriminatory for different strains, and all strains used in the

competition experiment with the exception of JCM 1081 and CP395 could be differentiated. An

internal region of the leuS gene was amplified by PCR using primers leuS-A and leuS-B

containing the A and B sequencing adaptors (454 Life Sciences) with individual barcodes on the

leuS-A primer for each time point in each animal (SI Table S4). The amplicons from all time

points of all animals were checked by agarose electrophoresis and mixed in equal volumes. The

pooled amplicons were sequenced by the Core of Applied Genomics and Ecology at the

University of Nebraska (http://cage.unl.edu/) using the 454 Roche sequencing A primer kit

following the standard FLX amplicon sequencing protocol (protocol available at

http://cage.unl.edu). The sequencing resulted in an average of 12,739 sequence tags per

sample. A local nucleotide database was established in Bioedit v7.0.9 for each sample, and the

blastn algorithm was used to determine the number of sequences that account for each

individual strain, using a threshold of 99% (http://www.mbio.ncsu.edu/BioEdit/bioedit.html).

Acknowledgements

We are grateful to colleagues who provided strains: Rudi Vogel (Technische Universität

München, Germany), Gerald Tannock (University of Otago, New Zealand), Eiichi Satoh (Tokyo

University of Agriculture, Japan), Ricarda Engberg (Danish Institute of Agricultural Sciences,

Denmark), Gwen Allison (Australian National University, Australia), Filip Van Immerseel (Ghent

University, Belgium), Todd Klaenhammer (North Carolina State University, USA), Michael

Gänzle (University of Alberta, Canada), Martin Kalmokoff (Atlantic Food and Horticulture

Research Centre, Agriculture and Agri-Food Canada, Canada), James Versalovic (Baylor

College of Medicine, USA), Lin Tao (University of Missouri, USA), Eamonn Connolly (BioGaia

AB, Sweden), Katsunori Kimura (Meiji Dairies Corporation, Japan), Paul O’Toole (University

College Cork, Ireland), Gerald Blüml (Lactosan GmbH & Co. KG, Austria), Jennifer Spinler

(Baylor College of Medicine, USA), Wolfgang Souffrant (Universität Rostock, Germany), Jenni

Korhonen (University of Kuopio, Finland), Evelia Acedo Félix (Ciencias de los Alimentos,

Mexico), María Jesús Yebra (Consejo Superior de Investigaciones Cientificas, Spain), Yasuyuki

Takeda (Rakuno Gakuen University, Japan), Edna Tereza de Lima (São Paulo State University,

Brazil), and Kikuji Itoh (University of Tokyo, Japan).

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Table S1. Lactobacillus reuteri strains used in this study Strain Name

Host1 Provenance ST Allelic Profile Clonal

Complex2 MLSA AFLP

ddl pkt leuS gyrB dltA rpoA recA

1048 pig Europe 3 3 3 3 3 3 3 3 CC-3 IV A

1063 pig Europe 3 3 3 3 3 3 3 3 CC-3 IV A

1073 pig Europe 3 3 3 3 3 3 3 3 CC-3 IV A

173.5 pig Europe 3 3 3 3 3 3 3 3 CC-3 IV A

27.4 pig Europe 3 3 3 3 3 3 3 3 CC-3 IV A

atcc55739 rat ND 3 3 3 3 3 3 3 3 CC-3 IV A

atcc53608 pig Europe 3 3 3 3 3 3 3 3 CC-3 IV A

jw2015 pig North America 3 3 3 3 3 3 3 3 CC-3 IV A

jw2019 pig North America 3 3 3 3 3 3 3 3 CC-3 IV A

ks6 chicken Europe 3 3 3 3 3 3 3 3 CC-3 IV A

lem83 pig Europe 3 3 3 3 3 3 3 3 CC-3 IV A

lr85573 human (U) Europe 3 3 3 3 3 3 3 3 CC-3 IV A

tmw11294 Pig Europe 3 3 3 3 3 3 3 3 CC-3 IV A

10c2 Pig Australasia 5 3 5 3 3 3 3 3 CC-3 IV A

p97 Pig Europe 5 3 5 3 3 3 3 3 CC-3 IV A

pg3b Pig North America 5 3 5 3 3 3 3 3 CC-3 IV A

32 Pig South America 10 3 9 3 3 3 3 3 CC-3 IV A

676 Pig South America 12 3 3 3 3 9 3 3 CC-3 IV A

cp447 Pig Europe 12 3 3 3 3 9 3 3 CC-3 IV A

6s15 Pig Australasia 14 3 5 3 3 9 3 3 CC-3 IV A

cp415 Pig Europe 20 3 9 13 12 3 3 3 CC-3 IV A

lp16767 Pig North America 43 3 3 3 12 26 3 3 CC-3 IV A

lpa1 Pig North America 44 3 8 3 12 9 3 3 CC-3 IV A

tmw1146 Pig Europe 51 3 3 13 3 3 3 3 CC-3 IV A

tmw1137 Pig Europe 51 3 3 13 3 3 3 3 CC-3 IV A

100.93 Mouse Australasia 1 1 1 1 1 1 1 1 III B

ad23 Rat Europe 15 9 1 10 10 8 1 7 III B

dbc2 Mouse North America 25 10 15 16 14 1 11 8 III B

100-23 Rat Australasia 26 11 16 17 11 16 7 3 III B

dsm20053 human (F) ND 27 11 16 18 3 17 7 13 III B

ilc4 Mouse North America 32 15 1 23 1 21 7 7 CC-32/55 III B

mlc3 Mouse North America 48 22 1 31 22 27 15 19 III B

mouse56 Mouse Asia 50 24 25 33 24 1 11 18 III B

n2j Rat Europe 52 25 16 34 21 29 7 20 III B

r2lc Rat Europe 57 9 16 35 3 33 7 20 CC-53/57 III B

11283 Chicken North America 6 5 6 5 5 5 4 5 CC-6/41 VI Ci

11284 Chicken North America 6 5 6 5 5 5 4 5 CC-6/41 VI Ci

ke1 Chicken Europe 6 5 6 5 5 5 4 5 CC-6/41 VI Ci

ky21 Chicken Europe 6 5 6 5 5 5 4 5 CC-6/41 VI Ci

mf14c human (F) Europe 6 5 6 5 5 5 4 5 CC-6/41 VI Ci

mf23 human (F) Europe 6 5 6 5 5 5 4 5 CC-6/41 VI Ci

t1 Turkey North America 6 5 6 5 5 5 4 5 CC-6/41 VI Ci

1204 Chicken Europe 7 6 2 6 5 6 5 6 VI Ci

1366 Chicken Europe 7 6 2 6 5 6 5 6 VI Ci

atcc55730 human (B) South America 16 2 2 11 5 11 4 6 VI Ci

cf483a1 human (F) Europe 16 2 2 11 5 11 4 6 VI Ci

dsm17938 human (B) ND 16 2 2 11 5 11 4 6 VI Ci

m27u15 human (B) Africa 16 2 2 11 5 11 4 6 VI Ci

m45r2 human (B) Europe 16 2 2 11 5 11 4 6 VI Ci

m81r43 human (B) Asia 16 2 2 11 5 11 4 6 VI Ci

Table S1, continued mm344a human (B) Europe 16 2 2 11 5 11 4 6 VI Ci

mm361a human (B) Europe 16 2 2 11 5 11 4 6 VI Ci

mv362a human (V) Europe 16 2 2 11 5 11 4 6 VI Ci

mv41a human (V) Europe 16 2 2 11 5 11 4 6 VI Ci

nck1556 human (U) South America 16 2 2 11 5 11 4 6 VI Ci

t2 Turkey North America 16 2 2 11 5 11 4 6 VI Ci

t3 Turkey North America 16 2 2 11 5 11 4 6 VI Ci

csa9 Chicken North America 22 5 13 6 5 14 9 5 CC-22/23/39 VI Ci

csf8 Chicken North America 23 5 14 15 5 14 9 5 CC-22/23/39 VI Ci

hw8 Chicken North America 23 5 14 15 5 14 9 5 CC-22/23/39 VI Ci

hwb7 Chicken North America 23 5 14 15 5 14 9 5 CC-22/23/39 VI Ci

d15 Chicken North America 24 2 2 6 5 15 10 12 VI Ci

hwh3 Chicken North America 31 14 2 22 5 11 9 5 VI Ci

jcm1081 Chicken Asia 33 2 18 24 17 22 9 6 CC-33/42 VI Ci

lb54 Chicken Asia 39 5 2 6 5 14 9 5 CC-22/23/39 VI Ci

lk146 Chicken Europe 39 5 2 6 5 14 9 5 CC-22/23/39 VI Ci

lk150 Chicken Europe 40 20 21 29 17 22 4 5 VI Ci

lk20 Chicken Europe 41 5 2 15 5 5 4 5 CC-6/41 VI Ci

lk94 Chicken Europe 42 2 18 24 17 22 4 6 CC-33/42 VI Ci

nck983 Chicken North America 54 5 21 36 5 31 9 5 VI Ci

nck985 Chicken North America 54 5 21 36 5 31 9 5 VI Ci

tu160 Turkey Europe 59 5 27 38 5 11 9 5 VI Ci

tu174 Turkey Europe 59 5 27 38 5 11 9 5 VI Ci

1013 Pig Europe 2 2 2 2 2 2 2 2 CC-19 VI Cii

104r Pig Europe 4 4 4 4 4 4 2 4 CC-47 II Cii

20.2 Pig Europe 9 2 8 8 7 2 2 2 CC-19 V Cii

3c6 Pig Australasia 11 2 8 2 8 2 2 2 CC-19 V Cii

4s17 Pig Australasia 11 2 8 2 8 2 2 2 CC-19 V Cii

cp395 Pig Europe 19 2 8 2 2 2 2 2 CC-19 V Cii

l461 Pig Europe 37 2 8 2 2 2 14 2 CC-19 V Cii

cf46g human (F) Europe 4 4 4 4 4 4 2 4 CC-47 II Ciii

cf62a human (F) Europe 4 4 4 4 4 4 2 4 CC-47 II Ciii

dsm20016t human (F) Europe 4 4 4 4 4 4 2 4 CC-47 II Ciii

fj1 human (O) Asia 4 4 4 4 4 4 2 4 CC-47 II Ciii

mm31a human (B) Europe 4 4 4 4 4 4 2 4 CC-47 II Ciii

mm41a human (B) Europe 4 4 4 4 4 4 2 4 CC-47 II Ciii

cf2a0 human (F) Europe 18 4 4 4 11 4 2 10 CC-47 II Ciii

me261 human (F) Asia 47 4 23 4 4 4 2 4 CC-47 II Ciii

sr11 human (S) Europe 58 4 23 4 4 4 2 21 CC-47 II Ciii

uga29 human (V) Africa 60 27 28 39 4 34 16 22 II Ciii

2010 Rat North America 8 7 7 7 6 7 6 3 II D

6799jm1 Mouse North America 13 8 10 9 9 10 8 8 CC-13/45 I D

lacto6798jm1 Mouse North America 13 8 10 9 9 10 8 8 CC-13/45 I D

lpuph1 Mouse North America 13 8 10 9 9 10 8 8 CC-13/45 I D

bmc1 Rat Europe 17 8 11 12 11 12 8 9 I D

bmc2 Rat Europe 17 8 11 12 11 12 8 9 I D

ml1 Mouse Europe 17 8 11 12 11 12 8 9 I D

cr Rat North America 21 8 12 14 13 13 2 11 I D

oneone Rat North America 21 8 12 14 13 13 2 11 I D

dsm20056 Rat ND 28 12 17 19 15 18 11 14 I D

fua3043 Rat North America 29 8 11 20 11 19 11 10 I D

fua3048 Rat North America 30 13 8 21 16 20 12 14 II D

Table S1, continued jw2016 Pig North America 34 16 19 25 18 23 3 4 II D

l16001 Mouse North America 35 18 10 26 9 10 3 15 I D

lpupjm1 Mouse North America 45 21 10 9 9 10 8 8 CC-13/45 I D

l16041 Mouse North America 36 17 10 27 19 24 13 16 III D

n2d Rat Europe 52 25 16 34 21 29 7 20 III D

n4i Rat Europe 53 9 16 35 25 30 7 20 CC-53/57 III D

number20 Mouse Australasia 55 15 1 1 1 8 7 7 CC-32/55 III D

mouse2 Mouse Asia 49 23 24 32 23 28 7 8 III E

r13 Mouse Asia 56 26 26 37 26 32 3 16 I E

rat19 Rat Asia 56 26 26 37 26 32 3 16 I E

l722 human (F) Europe 38 19 20 28 20 25 12 17 II E

lms11.1 human (F) North America 4 4 4 4 4 4 2 4 CC-47 II ND3

lms11.3 human (F) North America 4 4 4 4 4 4 2 4 CC-47 II ND

lr4020 Mouse North America 46 22 22 30 21 1 7 18 III ND

tmw1.1297 Pig Europe ND ND ND ND ND ND ND ND ND ND A

13S14 Pig Australasia ND ND ND ND ND ND ND ND ND ND A

JW2017 Pig North America ND ND ND ND ND ND ND ND ND ND A

1068 Pig Europe ND ND ND ND ND ND ND ND ND ND A

63/1 Pig Europe ND ND ND ND ND ND ND ND ND ND A

146/2 Pig Europe ND ND ND ND ND ND ND ND ND ND A

173/3 Pig Europe ND ND ND ND ND ND ND ND ND ND A

173/4 Pig Europe ND ND ND ND ND ND ND ND ND ND A

P26 Pig Europe ND ND ND ND ND ND ND ND ND ND A

1704 Pig South America ND ND ND ND ND ND ND ND ND ND A

N5D:1 Rat Europe ND ND ND ND ND ND ND ND ND ND B

me262 human (F) Asia ND ND ND ND ND ND ND ND ND ND B

Mouse 41 Mouse Asia ND ND ND ND ND ND ND ND ND ND B

Mouse 81 Mouse Asia ND ND ND ND ND ND ND ND ND ND B

DBC3 Mouse North America ND ND ND ND ND ND ND ND ND ND B

ILC3 Mouse North America ND ND ND ND ND ND ND ND ND ND B

MLC1A Mouse North America ND ND ND ND ND ND ND ND ND ND B

MLC1B Mouse North America ND ND ND ND ND ND ND ND ND ND B

MLC4 Mouse North America ND ND ND ND ND ND ND ND ND ND B

CS9 Chicken North America ND ND ND ND ND ND ND ND ND ND Ci

CSB7 Chicken North America ND ND ND ND ND ND ND ND ND ND Ci

LK159 Chicken Europe ND ND ND ND ND ND ND ND ND ND Ci

LK75 Chicken Europe ND ND ND ND ND ND ND ND ND ND Ci

LK139 Chicken Europe ND ND ND ND ND ND ND ND ND ND Ci

NCK984 Chicken North America ND ND ND ND ND ND ND ND ND ND Ci

SD2112 human (B) South America ND ND ND ND ND ND ND ND ND ND Ci

MV14-1a human (V) Europe ND ND ND ND ND ND ND ND ND ND Ci

MF7-J human (F) Europe ND ND ND ND ND ND ND ND ND ND Ci

L3B Chicken South America ND ND ND ND ND ND ND ND ND ND Ci

3S3 Pig Australasia ND ND ND ND ND ND ND ND ND ND Cii

MM2-3 human (B) Europe ND ND ND ND ND ND ND ND ND ND Ciii

CF2-7F human (F) Europe ND ND ND ND ND ND ND ND ND ND Ciii

CF15-6 human (F) Europe ND ND ND ND ND ND ND ND ND ND Ciii

SR14 human (S) Europe ND ND ND ND ND ND ND ND ND ND Ciii

FUA3041 Rat North America ND ND ND ND ND ND ND ND ND ND D

FUA3044 Rat North America ND ND ND ND ND ND ND ND ND ND D

JCM5869 Rat Australasia ND ND ND ND ND ND ND ND ND ND D

6798cm-1 Mouse North America ND ND ND ND ND ND ND ND ND ND D

Table S1, continued BMC3 Rat Europe ND ND ND ND ND ND ND ND ND ND D

L6799 Mouse North America ND ND ND ND ND ND ND ND ND ND D

L6800jm-1 Mouse North America ND ND ND ND ND ND ND ND ND ND D

Lacto1662 Mouse North America ND ND ND ND ND ND ND ND ND ND D

Rat 8 Rat Asia ND ND ND ND ND ND ND ND ND ND E

Rat 17 Rat Asia ND ND ND ND ND ND ND ND ND ND E

Mouse 20 Mouse Asia ND ND ND ND ND ND ND ND ND ND E

Mouse 76 Mouse Asia ND ND ND ND ND ND ND ND ND ND E

uga44-1 human (V) Africa ND ND ND ND ND ND ND ND ND ND E

1Isolation source for human strains: feces (F), stomach (S), breast milk (B), vagina (V), oral cavity (O) and

unknown (U). 2Clonal complexes (CCs) were inferred by eBURST analysis. 3ND, not determined

Table S2. Descriptive analysis of MLSA data

Locus Gene product

/description

DNA Coordinates

on DSM20016T

genome sequence1

Fragment

size (bp)

% G+C

Content

No. of

alleles

No. of

polymorphic

sites

Gene

diversity

(Hd)

dN/dS

ratio

Phi-

test2

ddl D-alanine-d-

alanine ligase

530489-531119 (+) 633 38.7 27 62 0.881 0.009 0.02753

pkt Phosphoketolase 1747597-1747032(-) 567 44.0 28 62 0.923 0.160 0.3880

leuS Leucyl-tRNA

synthetase

1342592-1342042 (-) 554 41.6 39 78 0.933 0.025 0.00013

gyrB DNA gyrase B

subunit

5423-5946 (+) 526 41.9 26 55 0.853 0.006 0.00003

dltA D-alanine-

activating enzyme

289730-289199 (-) 534 39.9 34 88 0.930 0.021 0.00003

rpoA RNA polymerase

alpha subunit

1517154-1516656 (-) 499 39.4 16 16 0.854 0.038 0.9870

recA Recombinase 583686-584211 (+) 528 39.1 22 37 0.883 0.003 0.1374

1Accession number for DSM20016T: NC_009513 2P-value obtained from Phi-Test for recombination as implemented in the SplitsTree4 software 3Recombination has been identified as significant

Table S3. Average nucleotide divergence within and between clusters1

Lineages

I

(Rodents

2)

II

(Human)

III

(Rodents 1)

IV

(Pig 1)

V

(Pig 2)

VI

(Poultry/

Human)

I (Rodents 2) 1.53% 2.82% 3.09% 3.26% 2.04% 3.26%

II (Human) 0.87% 3.11% 2.47% 3.08% 4.62%

III (Rodents 1) 1.72% 2.04% 3.02% 4.13%

IV (Pig 1) 0.04% 3.07% 4.41%

V (Pig 2) 0.33% 2.38%

VI (Poultry/

Human) 0.66%

1All lineages are inferred from ClonalFrame analyses. All divergence data was determined using the

Jukes Cantor correction in DNAsp Version 4.90.1.

Table S4. PCR primers used in this study

Application Gene Primer Name Primer sequence 5' to 3' PCR

Fragment size (bp)1

AFLP Pre-amplification

Pre-EcoR1 GACTGCGTACCAATTC ND2

Pre-Mse1 GATGAGTCCTGAGTAA ND

AFLP Selective amplification

Primer pair 1 EcoR1-A GACTGCGTACCAATTCA ND

Mse1-AG GATGAGTCCTGAGTAAAG ND

Primer pair 2 EcoR1-G GACTGCGTACCAATTCG ND

Mse1-CC GATGAGTCCTGAGTAACC ND

16S rRNA sequencing

16S rRNA 8F AGAGTTTGATCCTGGCTCAG 1383 (700)

1391R GACGGGCGGTGWGTRCA

MLSA

ddl ddl-F ATTTCTTCTTCCCTGTTATCC 738 (633)

ddl-R TTCGTTCAAATTCTTGTAATCC

pkt pkt-F CACGAAGAAATGGCTAAGAC 689 (567)

pkt-R GTTGCGAAGAATCCGTGAC

leuS leuS-F TACGACGCGGGCAGATAC 810 (554)

leuS-R ATAGAGATCAACTGGTGACC

gyrB gyrB-F AGAATTCCATTATGAAGGTGG 677 (526)

gyrB-R TTTCAACATTCAAGATCTTTCC

dltA dltA-F TTGTCGATCATCAACAGCTTG 753 (534)

dltA-R CAGTTCGGTAAGCAGGCAC

rpoA rpoA-F CGGTTATGGAACCACTCTC 571 (499)

rpoA-R AGCHGTTTCTGTTAAATCAAC

recA recA-F TGAAAGTTCTGGTAAGACTAC 580 (528)

recA-R CTTTTTAGCATTTTCACGACC

Primers for 454 pyrosequencing‡

leuS leuS-A gcctccctcgcgccatcagNNNNNNNNCGGAAGGAACAGCCATTAC 233

leuS leuS-B gccttgccagcccgctcagTACGACGCGGGCAGATAC

1 Length of nucleotide sequence used for the MLSA analysis is shown in brackets 2 ND, not determined ‡ For pyrosequencing, the uncapitalized nucleotides are the adapter/sequencing primer and N represents

nucleotides in the 8-base barcode

  

Figure S1 AFLP electropherograms of 165 Lactobacillus reuteri strains that were generated from selective

amplification of primer pair 1 (EcoR1-A/Mse1-AG) and primer pair 2 (EcoR1-G/Mse1-CC). AFLP

images analysis was performed using Bionumerics software. A neighbor-joining dendrogram

based on AFLP similarity coefficients (Dice) was reconstructed in Bionumerics. Although the

dendrograms in this figure and in Figure 1A were reconstructed using a different distance

method, host specific clusters were obtained.

Figure S2 Phylogenetic analysis of 116 strains of Lactobacillus reuteri based on the concatenated

sequences of (A) all seven MLSA loci, and (B) the three MLSA loci that were not significantly

affected by recombination according to the PHI test. Both maximum-likelihood (ML) trees were

reconstructed using PhyML with GTR+I+G model of nucleotide substitution and 100 bootstrap

replicates. Lactobacillus vaginalis ATCC49540 was used as the outgroup. Only bootstrap values

above 50% are shown at the nodes. In (A), clonal complexes (CC) as identified by eBURST that

contain more than 4 strains are shown. Isolates from rats are labeled with a circle, and isolates

from turkey are labeled with a triangle.

Figure S3 Maximum likelihood (ML) trees of the in-frame concatenated MLSA sequence (all seven loci)

and the individual genes (ddl, pkt, leuS, gyrB, dltA, rpoA, recA) were constructed in PhyML

using the 60 unique STs detected for L. reuteri as input. Trees were reconstructed using the

GTR+I+G model of nucleotide substitution. L. vaginalis ATCC49540 was used as the outgroup.

Figure S4 Phylogenetic-network analysis for all individual loci of the 60 unique STs. The split-

decomposition graph was obtained using the neighbor-net algorithm, implemented with the

SplitsTree4 program. Conflicting phylogenetic signals are visualized by the net-like structure in

the center. Loci that showed significant evidence of recombination by PHI statistical test are

marked with an asterisk (*).

100

999897969594939291908988878685848382818079

M81R43

MV141a

M45R2

MV41a

SD2112

NCK1556

ATCC55730

MM361a

M27U15

T2

MF7J

CF483A1

DSM17938P

MM344A

MV362A

T3

HW8

HWB7

CSF8

CSB7

CS9

1204

CSA9

LB54

JCM1081

tu160

HWH3

L3B

tu174

NCK984

NCK985

NCK983

LK20

LK159

LK75

LK146

LK150

LK139

LK94

1366

D15

MF14C

T1

Ke1

Ky21

11284

11283

MF23

CP395

1013

4S17

3C6

3S3

104R

CF156

MM31A

CF27F

CF62a

MM23

MM41a

DSM20016T

CF46g

FJ1

CF2A0

Uga29

SR14

ME261

SR11

Mouse76

202

L461

6S15

TMW1.1294

P97

P26

Ks6

Lr85573

1068

1048

LEM83

JW2017

TMW1.137

TMW1.1297

CP415

LpA1

TMW1.146

13S14

1073

Lp16767

JW2015

JW2019

10C2

ATCC55739

1063

ATCC53608

32

676

1704

PG3B

146.2

173.5

173.3

173.4

63.1

27.4

CP447

ME262

DSM17509

N2D

N2J

DSM20053

N5D1

R2LC

JCM5869

N4I

2010

JW2016

BMC3

BMC2

BMC1

CR

OneOne

L16041

L16001

FUA3044

ML1

L6799

6799jm1

6798cm1

L6800jm1

Lacto6798jm1

Lacto1662

Lpupjm1

Lpuph1

DSM20056

FUA3048

FUA3043

FUA3041

R13

#20

100.93

AD23

Mouse41

Mouse56

MLC1A

ILC4

MLC3

MLC1B

ILC3

DBC2

MLC4

DBC3

Mouse81

Mouse20

Mouse2

Rat8

Rat19

Rat17

Uga441

L722

Li1461

Primer pair 1 Primer pair 2

RodentsPorcineHuman Poultry

Host

Figure S1

0.02

52

72

80

100

90

74

100

64

97

66

B

0.04

CC-6/41

CC-19

CC-22/23/39

CC-22/23/39

CC-19

CC-3

CC-47

V (Pig 2)

IV(Pig 1)

II(Human)

CC-13/45

VI(Poultry/Human)

III(Rodents 1)

I(Rodents 2)

98

99

100

94

94

100

74

100

78

67

63

64

66

A

ST-16

RodentsPorcineHuman Poultry

Host

Figure S2

All 7 Loci ddl

pkt leuS

RodentsPorcineHuman Poultry

Host

0.04

0.02 0.03

0.02

gyrB dltA

rpoA recA

0.03

0.002 0.01

0.02

Figu

re S3

ddl*

pkt

gyrB*

dltA*

rpoA

recAleuS*

0.001

0.0010.01

0.01

0.0010.001

0.01

Figure S4