MICROBIAL DIVERSITY IN INTESTINAL TRACTS OF ...

MICROBIAL DIVERSITY IN INTESTINAL TRACTS OF TWO SPECIES OF RODENTS IN MONTANE AREAS IN TAIWAN Hsiao-Pei Lu and Hon-Tsen Yu Institute of Zoology and Department of Life Science, National Taiwan University, Taipei, Taiwan, ROC.

Abstract Microorganisms living in mammalian guts have important effects on immune function, nutrient processing and other host activities. We analyzed the bacterial communities in three sections of the digestive tracts, including small intestines, caeca and large intestines of two montane species of rodents (Apodemus semotus and Eothenomys melanogaster) from the central mountain ranges in Taiwan. We constructed 16S rDNA clone libraries and identified the sequences through database searches. At the phylum level, the intestinal microflorae of A. semotus were composed of Firmicutes (43.26%), Proteobacteria (42.23%), Deinococcus-Thermus (9.84%), Verrucomicrobia (2.33%), Cyanobacteria (1.81%) and Actinobacteria (0.52%). The intestinal microflorae of E. melanogaster were composed of Proteobacteria (40.68%), Firmicutes

(32.70%),

Bacteroidetes

(22.05%),

Spirochaetes

(1.90%),

Actinobacteria (1.52%), TM7 (0.76%) and Deferribacteres (0.38%). We also found that the bacterial composition percentages between three gut sections of the same individual differed from one another. Moreover, while the SPF (specific pathogen free) mouse and human microflorae were dominated by Firmicutes and Bacteroidetes, the mouse and the vole in this study, respectively, have a large group of Proteobacteria which comprises a huge number of environmental bacteria. This discrepancy suggests that wild rodents have specific immune tolerance for some of Proteobacteria and these microbes may own unique metabolic traits to colonize the gut. This research offers an opportunity to know the commensal host-bacterial relationships in the guts of wild rodents.

1. Introduction The animal gut is the one of the most densely populated ecosystems on Earth (Savage, 1977). There are up to 1014 bacteria in the human intestinal tract, which is more than the total number of tissue cells in the entire body. Microorganisms have been traditionally identified through characterization of their morphological and physiological traits (Savage, 1986). Although the endogenous gastrointestinal microflorae have been studied in great detail by culture techniques, most of them cannot be cultured with available media. Microscopic counts on human feces suggest that 60 to 80% of the observable bacteria cannot be cultivated (Suau et al., 1999). The endogenous gastrointestinal microflorae play an important role in health and disease, yet this ecosystem remains mostly uncharacterized and its diversity poorly defined (Ramakrishna, 2007). Because only a limited number of species could be identified by using traditional culturing methods, investigators have begun to explore this ecosystem by using molecular techniques, such as polymerase chain reaction (PCR) and sequence analysis of cloned microbial small-subunit ribosomal RNA genes (16S rDNA) (Wilson & Blitchington, 1996; Wintzingerode et al., 1997). Remarkably, microorganisms living in mammalian guts have important effects on immune function, nutrient processing and other host activities. Studying the normal gut microflorae can improve our understanding of hostmicrobe interactions. Most uncultivable species become detectable when we use molecular techniques to investigate intestinal microbial populations (Zoetendal et al., 2006). However, a comprehensive enumeration of the gut microflorae has not yet been reported for wild mice or voles, even though rodents provide a good model for exploring the intestinal microbial ecology. This research offers an opportunity to know the commensal host-bacterial relationships in the guts of wild rodents.

2. Materials and methods Animals One Formosan field mouse (Apodemus semotus) and one Formosan black-bellied vole (Eothenomys melanogaster) from the central mountain ranges in Taiwan were employed for the study. The mouse was collected from Mt. Ho-Huan and the vole was collected from Mt. Ali. The animals were sacrificed one day after trapped and gut samples were frozen immediately in liquid nitrogen. DNA extraction Total DNA was extracted from small intestine, large intestine and caecum of each individual, respectively. Tissue samples (ca. 1 g) were placed in Eppendorf tubes with 500 μl lysozyme buffer (25 mM tris-HCl, 10 mM EDTA, 50 mM glucose, pH 8) and 50 μg lysozyme (20000 units/mg), followed by incubation at 370C with occasional agitation for 3 hr. At the beginning of the third hour, 100 μg proteinase K (60 units/mg), 100 μg RNase A (60 units/mg) and 500 μl lysis buffer (50 mM tris-HCl, 10 mM EDTA, 1% SDS, 100 mM NaCl, pH 8) were added to each tube, followed by gentle inversion. Freezethaw cycles of incubation at -800C for 60 min followed by 600C for 30 min were performed three times to release microbial DNA. The cell lysate was extracted with equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), rotated for 10 min to obtain the well-mixed milky solution and centrifuged (12000 rpm) for 10 min at room temperature. The top aqueous layer was placed in a new Eppendorf tube and 1 ml cold 100% ethanol (EtOH) was added. The tube was inverted to precipitate DNA and centrifuged for 5 min at 12000 rpm to pellet DNA. The pellet was re-suspended with 1 ml cold 70% EtOH and centrifuged for 2 min at 12000 rpm. Repeat again and pour off the supernatant. The pellet was air dried overnight or vacuum dried for 5 min with a MicroVacTM MV-100. The DNA was dissolved in 50 μl distilled water and stored at -200C.

Bacterial 16S rDNA amplification A pair of primers 516 F (5' TGCCAGCAGCCGCGGTA 3') and 985 R (5' GTAAGGTTCTTCGCGTT 3') were used to amplify the bacterial 16S rDNA fragments (Nagashima et al., 2003). The PCR reaction mixture contained 100 ng DNA, 2.0 mM MgCl2, 0.4 mM dNTP, 0.4 µM each primer, 1X Ex TaqTM buffer and 1 U of Ex TaqTM polymerase (Takara). The thermal cycling conditions were initial denaturation at 940C for 3 min; followed by 25 cycles at 940C for 30 s, 560C for 30 s and 720C for 1 min; and final extension at 720C for 10 min. The concentration of amplified DNA was determined by electrophoresis on the 1% agarose gel (1 g agarose + 100 ml tris-Borate-EDTA buffer). Amplified products of the expected size (ca. 450 bp) were confirmed and excised from the gel to be recovered with QIAquick Gel Extraction Kits (QIAGEN). Cloning & Sequencing The purified products were ligated into the yT&A vector of the TA Cloning Kits (Yeastern) according to the manufacturer’s instructions. Ligated DNA was transformed into ECOS101 competent cells in the kits by heat shock at 42 0C for 45 s. The resulting clones were selected on Luria-Bertani medium with ampicillin (50 μg/ml), IPTG (isopropyl-β-D-thiogalactopyranoside) (0.1 M) and X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) (50 mg/ml). White colonies were picked from each clone libraries and screened for inserts by performing direct PCR using M13 forward and M13 reverse primers. Each insert DNA was obtained for sequencing with the TempliPhi DNA Amplification Kits (Amersham) following the manufacturer’s protocols. The partial fragments of 16S rDNA were sequenced with DY Enamic ET Dye Terminator Cycle Sequencing Kit (GE) and 109 aF primer. All reactions were done by a MegaBACE 1000 automated sequencer (GE).

Phylogenetic analysis Sequences were adjusted and trimmed with Sequencher program (Gene Code Corporation) to obtain sequences with high equal quality. Moreover, sequences were tested for possible chimeras with two online analyses, Bellerophon server (http://foo.maths.uq.edu.au/%7Ehuber/bellerophon.pl) and Chimera Check (http://35.8.164.52/cgis/chimera.cgi?su=SSU). Confirmed chimeric sequences were excluded from further analysis. Sequences were compared with those in the GenBank database by BLASTTM searches. Sequences with 99% similarity ( < 1% diversity ) were grouped into operational taxonomic units (OTUs). All sequences and their closest relatives were aligned with Clustal X program (Jeanmougin et al., 1998). Phylogenetic analysis was carried out using PAUP* 4.0 program (Swofford, 1993). The pairwise distances were analyzed with Kimura’s twoparameter correction (Kimura, 1980). The tree was generated from distance matrices by the neighbor-joining method. The stability of branches was checks by bootstrapping.

3. Results Phylogenetic analysis of the 16S rDNA sequences We obtained 386 sequences (89 sequences from the small intestine, 131 sequences from the caecum, and 166 sequences from the large intestine) from A. semotus and 263 sequences (93 sequences from the small intestine, 96 sequences from the caecum, and 74 sequences from the large intestine) from E. melanogaster in the final analysis. We used 99% similarity as a threshold to determine OTUs (Drancourt et al., 2000). In mouse, a total of 386 sequences were grouped into 67 OTUs and assigned to 6 phyla (subdivided into 9 classes) (Table 1). In vole, a total of 263 sequences were grouped into 98 OTUs and assigned to 7 phyla (subdivided into 13 classes) (Table 2). The reference sequence information and clone number of each OTU were also given in Table 1 and Table 2.

Figure 1 showed the percentage of each group from three parts of gastrointestinal tracts of A. semotus. The overall intestinal microflorae were composed of Firmicutes (43.26%), Proteobacteria (42.23%), DeinococcusThermus (9.84%), Verrucomicrobia (2.33%), Cyanobacteria (1.81%) and Actinobacteria (0.52%). The phylum of Firmicutes was divided into 3 classes：Mollicutes (0.26%), Bacilli (3.11%), and Clostridia (39.89%). The phylum of Proteobacteria was divided into 2 classes：Gammaproteobacteria (17.88%) and Deltaproteobacteria (24.35%). Figure 2 showed the percentage of each group from three parts of gastrointestinal tracts of E. melanogaster. The intestinal microflorae were composed of Proteobacteria (40.68%), Firmicutes (32.70%), Bacteroidetes (22.05%), Spirochaetes (1.90%), Actinobacteria (1.52%), TM7 (0.76%) and Deferribacteres (0.38%). The phylum of Firmicutes was divided into 3 classes：Mollicutes (5.70%), Bacilli (0.76%), and Clostridia (26.24%). The phylum of Proteobacteria was divided into 5 classes：Alphaproteobacteria (7.98%),

Betaproteobacteria

(0.38%),

Gammaproteobacteria

(20.53%),

Deltaproteobacteria (10.65%), and Epsilonproteobacteria (1.14%). The phylogeny trees of 67 OTUs from A. semotus and 98 OTUs from E. melanogaster were given in Figure 3 and Figure 4, respectively. In mouse, most OTUs belonged to Clostridia (42 OTUs). The next dominant group was Deltaproteobacteria, containing 10 OTUs. The remaining 15 OTUs belonged to 7 classes, and each class contained only 1 to 4 OTUs, respectively (Figure 3). In vole, most OTUs also belonged to Clostridia (40 OTUs). But the next dominant group was Bacteroidetes, containing 28 OTUs. The remaining 30 OTUs belonged to 11 classes, and each class only contained 1 to 4 OTUs, respectively (Figure 4).

4. Discussion Compare the shared dominant groups in the clone libraries In this study, there were three phyla (Actinobacteria, Firmicutes, and Proteobacteria) shared between A. semotus and E. melanogaster. Only a few of sequences belonged to Actinobacteria and they just occurred in the mouse small intestine and the vole caecum. Both in mouse and in vole, Firmicutes and Proteobacteria were the dominant groups in the clone libraries; however, the bacterial composition of three gut sections of the same individual differed from one another. In mouse, over 60% of clone sequences from the small intestinal sample belonged to Proteobacteria, but less than 25% of clone sequences were identified as the members of Firmicutes. From the caecal sample, the percentage of Firmicutes and Proteobacteria were almost equal. By contrast, from the large intestinal sample, there were only 28% of clone sequences belonging to Proteobacteria, but more than 54% of clone sequences belonging to Firmicutes (Figure 1). In vole, there were 67% of clone sequences from the small intestinal sample belonging to Proteobacteria, but only 15% of clone sequences were identified as the members of Firmicutes. By contrast, from the caecal sample, there were only 21% of clone sequences belonging to Proteobacteria, but almost 50% of clone sequences belonging to Firmicutes. From the large intestinal sample, the percentage of Firmicutes and Proteobacteria were the same (Figure 2). Compare the clone sequences between mouse and vole The sequences belonging to Deinococcus-Thermus, Verrucomicrobia, and Cyanobacteria occurred only in the mouse. Although three parts of gut samples contained some sequences classified as Deinococcus-Thermus, the number of each sample was notable different. Verrucomicrobia occurred in all

gut samples, while Cyanobacteria were only detected in the caecal and large intestinal samples (Table 1). Compared with mouse, the phyla Bacteroidetes, Spirochaetes, TM7 and Deferribacteres occurred only in the vole. The clone libraries of the small intestine, caecum and the large intestine contained 18%, 20%, and 30% of sequences belonging to Bacteroidetes, respectively. Spirochaetes were both detected in the caecal and large intestinal samples, while TM7 only occurred in caecal sample and Deferribacteres only occurred in large intestinal sample (Table 2). Generally speaking, the intestinal microbial diversity of E. melanogaster was higher than A. semotus. In vole, we analyzed 263 sequences and got 98 OTUs. But in mouse, we analyzed 386 sequences and only got 67 OTUs. The most difference between the microflorae of these two species of rodents was the existence of Bacteroidetes or not. In vole, there were total 22% of 16S rDNA clones belonging to Bacteroidetes which contained 28 OTUs. But in mouse, there was none in this group. Besides, although both rodents had more than 40% of bacterial clones in Proteobacteria, the OTUs affiliated with Alphaproteobacteria, Betaproteobacteria, and Epsilonproteobacteria were only present in vole. However, the biggest OTU of mouse or vole intestinal microflora belonged to the same group：Gammaproteobacteria. Compared with the SPF mouse and human microflorae Ley et al have analyzed 5,088 bacterial 16S rDNA sequences from the distal intestinal microflorae of genetically obese ob/ob mice, lean ob/+ and wild-type siblings, and their ob/+ mothers. Although the majority species of each mouse are unique, their microflorae are dominated by Firmicutes and Bacteroidetes (Ley et al., 2005). In the specific pathogen free (SPF) mice, the majority of bacterial rDNA clones from the murine caecal and large intestines were affiliated with the taxa Bacteroidetes and Firmicutes, too (Kibe et al., 2004; Scupham et al., 2006).

Eckburg et al. have analyzed 13,355 prokaryotic 16S rDNA sequences from multiple colonic mucosal sites and faces of healthy humans. A total of 395 bacterial phylotypes were identified. A majority of the bacterial sequences corresponded to uncultivated species and novel microorganisms. Most of the inferred organisms were members of the Firmicutes and Bacteroidetes phyla (Eckburg et al., 2005). Other studies also indicate that about 75% of fecal bacteria can be characterized, and belong to the group Clostridium and the group Bacteroides (Ramakrishna, 2007). Based on the previous studies, the mouse and human microflorae are similar at the division lever, with Firmicutes and Bacteroidetes dominating. However, the gut samples from the mouse and the vole in this study, respectively, have a large of 16S rDNA clones belonging to Proteobacteria which comprises a huge number of environmental bacteria. This dissimilarity suggests that wild rodents have specific immune tolerance for some members of Proteobacteria and these microbes may own unique metabolic traits to colonize the gut (Schiffrin & Blum, 2002).

5. Conclusion We constructed the bacterial 16S rDNA clone libraries from three parts of gastrointestinal tracts of two species of rodents and compared the microbial composition between different samples. This research offers an opportunity to further our understanding of the intestinal microflorae of wild rodents. Those kinds of researches provide a solid scientific basis for the effective use and development of probiotics (Marchesi & Shanahan, 2007). In the future, studies are needed to characterize the rules controlling microbial diversity in the gastrointestinal tracts. Multiple factors regulate the population number of these bacteria, including gastric acidity, intestinal transit, dietary factors, antibiotic use, and bacterial interactions with other bacteria and with the host epithelium (Ramakrishna, 2007). Much more extensive work would be required if the microflorae diversity reflect differences in diet, age, or genotype of each host individual (Bennegadi-Laurent et al., 2003).

6. Reference Bennegadi-Laurent N., Fonty G., Liliane M., Thierry G., and Dominique L. (2003). Effects of age and dietary fibre level on caecal microbial communities of conventional and specific pathogen-free rabbits. Microbial Ecology in Health & Disease 15: 23-32. Drancourt M., Bollet C., Carlioz A., Martelin R., Gayral J. P., and Raoult D. (2000). 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. Journal of Clinical Microbiology 38: 3623-3630. Eckburg P. B., Bik E. M., Bernstein C. N., Purdom E., Dethlefsen L., Sargent M., Gill S. R., Nelson K. E., and Relman D. A. (2005). Diversity of the human intestinal microbial flora. Science 308: 1635-1638. Jeanmougin F., Thompson J. D., Gouy M., Higgins D. G., and Gibson T. J. (1998). Multiple sequence alignment with Clustal x. Trends in Biochemical Sciences 23: 403-405. Kibe R., Sakamoto M., Hayashi H., Yokota H., and Benno Y. (2004). Maturation of the murine cecal microbiota as revealed by terminal restriction fragment length polymorphism and 16S rRNA gene clone libraries. FEMS Microbiology Letters 235: 139-146. Kimura M. (1980). A Simple Method for Estimating Evolutionary Rates of Base Substitutions through Comparative Studies of NucleotideSequences. Journal of Molecular Evolution 16: 111-120. Ley R. E., Backhed F., Turnbaugh P., Lozupone C. A., Knight R. D., and Gordon J. I. (2005). Obesity alters gut microbial ecology. Proceedings of the National Academy of Sciences of the United States of America 102: 11070-11075. Marchesi J., and Shanahan F. (2007). The normal intestinal microbiota. Current Opinion in Infectious Diseases 20: 508-513. Nagashima K., Hisada T., Sato M., and Mochizuki J. (2003). Application of new primer-enzyme combinations to terminal restriction fragment

length polymorphism profiling of bacterial Populations in human Feces. Applied and Environmental Microbiology 69: 1251-1262. Ramakrishna B. S. (2007). The normal bacterial flora of the human intestine and its regulation. Journal of Clinical Gastroenterology 41: S2-S6. Savage D. C. (1977). Microbial Ecology of Gastrointestinal-Tract. Annual Review of Microbiology 31: 107-133. Savage D. C. (1986). Gastrointestinal Microflora in Mammalian Nutrition. Annual Review of Nutrition 6: 155-178. Schiffrin E. J., and Blum S. (2002). Interactions between the microbiota and the intestinal mucosa. European Journal of Clinical Nutrition 56: S60S64. Scupham A. J., Presley L. L., Wei B., Bent E., Griffith N., McPherson M., Zhu F. L., Oluwadara O., Rao N., Braun J., and Borneman J. (2006). Abundant and diverse fungal microbiota in the murine intestine. Applied and Environmental Microbiology 72: 793-801. Suau A., Bonnet R., Sutren M., Godon J. J., Gibson G. R., Collins M. D., and Dore J. (1999). Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Applied and Environmental Microbiology 65: 4799-4807. Swofford D. L. (1993). Paup - a Computer-Program for Phylogenetic Inference Using Maximum Parsimony. Journal of General Physiology 102: A9-A9. Wilson K. H., and Blitchington R. B. (1996). Human colonic biota studied by ribosomal DNA sequence analysis. Applied and Environmental Microbiology 62: 2273-2278. Wintzingerode F. V., Gobel U. B., and Stackebrandt E. (1997). Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. Fems Microbiology Reviews 21: 213-229. Zoetendal E. G., Vaughan E. E., and de Vos W. M. (2006). A microbial world within us. Molecular Microbiology 59: 1639-1650.

Table 1. The intestinal microflorae of Apodemus semotus Phylum

Class

Group

Libraries

Reference sequence

Accession no. Similarity % Clones no.

Verrucomicrobia Actinobacteria

Verrucomicrobiae Actinobacteria

OTU_10

S2C4L3

Uncultured bacterium

AY986278

100

9

OTU_50

S1

Corynebacterium pseudogenitalium

AJ439348

100

1

OTU_52

S1

Human intestinal bacterium

AY310748

93

1

OTU_14

C5L2

Uncultured cyanobacterium

AY304018

95

7

OTU_02

S10C1L26

Meiothermus silvanus

Y13599

100

37

OTU_51

S1

Meiothermus silvanus

Y13599

100

1

OTU_65

L1

uncultured Mollicutes bacterium

AB218347

96

1

OTU_15

S4L3

Streptococcus uberis

AB023576

96

7

OTU_37

S3

Anoxybacillus flavithermus

AY672762

100

3

OTU_66

S1

Streptococcus uberis

AB023576

96

1

OTU_67

L1

Lactobacillus intestinalis

AJ306299

100

1

OTU_16

L6

Uncultured Clostridiales bacterium

AB234467

90

6

OTU_49

L2

Uncultured Clostridiales bacterium

AB234467

90

2

OTU_63

L1

Unidentified rumen bacterium

AB009208

94

1

OTU_64

S1

Candidatus Arthromitus

D86305

98

1

OTU_09

C8L3


DQ014910

99

11

OTU_11

C2L5


DQ014992

98

7

OTU_12

C1L6


DQ015401

99

7

OTU_20

C3L2

Clostridium orbiscindens

AY730665

97

5

OTU_23

C4


AY730665

97

4

OTU_26

C2L2


DQ015401

98

4

Cyanobacteria Deinococcus-Thermus Firmicutes

Cyanobacteria Deinococci Mollicutes Bacilli

Clostridia

OTU_27

L4


DQ015402

98

4

OTU_28

L3


DQ014825

99

3

OTU_29

C1L2


AY730665

97

3

OTU_36

C3

Ruminococcus bromii

X85099

96

3

OTU_40

L2


DQ014992

97

2

OTU_41

L2


DQ015401

98

2

OTU_48

L2


DQ015614

99

2

OTU_59

C1


DQ014588

99

1

OTU_60

C1


AY992487

99

1

OTU_61

L1

Anaerotruncus colihominis

DQ002932

95

1

OTU_62

L1


AY916378

99

1

OTU_06

C8L6


AY992805

97

14

OTU_07

C6L8


AY994018

98

14

OTU_13

S2C2L3

Clostridium sp.

AF157053

98

7

OTU_17

C1L5


AY991865

98

6

OTU_19

S5L1

Clostridium sp.

AF157053

97

6

OTU_22

C3L2


AY993232

98

5

OTU_24

C4


AY993337

100

4

OTU_25

L4

Clostridium sp.

AY960574

94

4

OTU_34

S2L1


AY993706

99

3

OTU_35

L3


DQ015606

99

3

OTU_38

C1L1

Butyrate-producing bacterium

AJ270488

96

2

OTU_39

L2


DQ015316

96

2

OTU_42

C1L1


DQ015511

98

2

Proteobacteria

Deltaproteobacteria

Gammaproteobacteria

OTU_43

C1L1


AY991749

98

2

OTU_44

S1C1


AY993686

97

2

OTU_45

S1C1


AY992550

98

2

OTU_54

C1


AY305314

95

1

OTU_55

C1


AY305315

95

1

OTU_56

C1


AY991749

97

1

OTU_57

L1


AY991750

97

1

OTU_58

L1


DQ014892

98

1

OTU_03

S8C13L6

Uncultured delta proteobacterium

AB192287

93

27

OTU_04

S1C14L6

Desulfovibrio sp.

AJ251630

96

21

OTU_05

S1C13L1

Uncultured delta proteobacterium

AB192287

92

15

OTU_08

S1C4L8

Desulfovibrio desulfuricans

DQ092636

96

13

OTU_18

C4L2

Desulfovibrio desulfuricans

DQ092636

93

6

OTU_30

C1L2

Bilophila wadsworthia

L35148

94

3

OTU_31

C3

Desulfovibrio fairfieldensis

U42221

95

3

OTU_32

S1C1L1

Desulfomonas pigra

AF192152

92

3

OTU_46

S1C1

Desulfovibrio sp.

AJ251630

94

2

OTU_53

L1

Bilophila wadsworthia

L35148

91

1

OTU_01

S37C7L15

Shigella boydii

AY696681

100

59

OTU_21

C2L3


AY591501

91

5

OTU_33

S2L1

Shigella boydii

AY696681

99

3

OTU_47

S2

Yersinia aleksiciae

AJ627597

100

2

Table 2. The intestinal microflorae of Eothenomys melanogaster Phylum

Class

Group

Actinobacteria

Actinobacteria

OTU_14

C2

subsurface water clone EV818CFSSAHH49

DQ336995

100

2

OTU_48

C1

mouse cecum clone SWPT15_aaa04f07

EF098514

91

1

OTU_49

C1


EF098514

92

1

AB192001

84

7

Bacteroidetes

Bacteroidetes

OTU_28

Libraries Reference sequence

S3C1L3 termite gut homogenate clone M1PL1-23

Accession no. Similarity % clone no.

OTU_29

S1C1

UASB bioreactor wastewater clone E13

AY426457

85

2

OTU_30

S1L2

termite gut homogenate clone MgMjW-42

AB234442

84

3

AB015525

85

8

OTU_31

S3C3L2 deep-sea sediment clone BD1-16

OTU_32

C2

mouse cecum clone SWPT1_aaa04a04

EF098962

96

2

OTU_33

C1L3

mouse cecum clone SWPT14_aaa01b07

EF098075

93

4

OTU_34

S1L1


EF100100

93

2

OTU_35

S4C1


EF099015

93

5

OTU_36

C3L1

rat feces clone rc2-30(4)

AY239409

95

4

OTU_37

C2L1

mouse cecum clone aab22h06

DQ815831

93

3

OTU_43

C1


EF096785

94

1

OTU_46

C1

mouse cecum clone SWPT16_aaa04c08

EF098745

83

1

OTU_47

C1

mouse cecum clone SWPT13_aaa04e03

EF097034

89

1

OTU_52

C1

mouse cecum clone SWPT13_aaa04g02

EF097052

92

1

OTU_60

C1


EF099724

94

1

OTU_66

L1

rat feces clone R-1292

DQ777921

94

1

OTU_67

L1


DQ777909

95

1

OTU_68

L1


EF099101

93

1

OTU_69

L1


EF098962

84

1

OTU_72

L1

mouse cecum clone SWPT13_aaa04d06

EF097026

91

1

OTU_73

L1


EF098397

92

1

OTU_74

L1


EF099015

93

1

OTU_77

L1


EF098762

93

1

OTU_78

L1


DQ777907

94

1

OTU_82

S1

termite gut homogenate clone BOf6-16

AB288906

88

1

OTU_92

S1


DQ815831

93

1

OTU_93

S1


EF099041

92

1

OTU_96

S1


EF099648

92

1

Deferribacteres

Deferribacteres

OTU_76

L1

swine intestine clone p-2881-6C5

AF371927

91

1

Firmicutes

Mollicutes

OTU_16

L15

Mycoplasma penetrans str. GTU-54-6A1

L10839

97

15

Bacilli

OTU_81

L1

mouse cecum clone aab47e08

DQ815398

97

1

Bacilli

OTU_97

S1


EF099666

97

1

Clostridia

OTU_15

C3

mouse cecum clone aab22c03

DQ815781

95

3

Clostridia

OTU_17

C3


EF097743

90

3

Clostridia

OTU_18

C2


EF098757

90

2

Clostridia

OTU_19

C1L1

mouse cecum clone SWPT12_aaa02h08

EF097960

93

2

Clostridia

OTU_20

C1L1

swine intestine clone p-1529-b5

AF371637

98

2

Clostridia

OTU_21

C2

mouse cecum clone aab24f01

DQ815940

97

2

Clostridia

OTU_22

S3

swine intestine clone p-5278-2Wa3

AF371938

96

3

Clostridia

OTU_23

AB198630

89

4

Clostridia

OTU_24

C3

dairy cow rumen clone NED5G11

EF445284

92

3

Clostridia

OTU_25

C2

mouse cecum clone aab41b10

DQ815469

98

2

Clostridia

OTU_26

S1C3L3 mouse cecum clone SWPT15_aaa01g07

EF098372

93

7

Clostridia

OTU_38

C7


DQ815849

98

7

Clostridia

OTU_40

C2


DQ815966

97

2

S1C2L1 termite gut fluid clone RsC01-059

Clostridia

OTU_41

C1


DQ815849

99

1

Clostridia

OTU_42

C1


EF097728

96

1

Clostridia

OTU_44

C1

termite gut wall fraction clone RsW01-038

AB198472

94

1

Clostridia

OTU_45

C1

swine intestine clone p-5459-2Wb5

AF371947

96

1

Clostridia

OTU_50

C1

rat feces clone R-1274(2)

DQ777951

94

1

Clostridia

OTU_51

C1

Selenomonas infelix str. ATCC 43532

AF287802

86

1

Clostridia

OTU_53

C1


DQ815479

94

1

Clostridia

OTU_56

C1


EF098164

97

1

Clostridia

OTU_57

C1

termite gut clone Rs-041

AB100480

95

1

Clostridia

OTU_58

C1

swine intestine clone p-30-a5

AF371665

95

1

Clostridia

OTU_59

C1


DQ777919

99

1

Clostridia

OTU_61

C1


DQ815544

98

1

Clostridia

OTU_62

C1


EF099106

96

1

Clostridia

OTU_63

C1

mouse cecum clone aab42d08

DQ815537

92

1

Clostridia

OTU_64

C1


EF098919

93

1

Clostridia

OTU_65

L1


EF097914

97

1

Clostridia

OTU_71

L1

termite gut homogenate clone RsTz-65

AB192175

90

1

Clostridia

OTU_79

L1

swine intestine clone p-1528-b5

AF371777

89

1

Clostridia

OTU_80

L1


DQ815430

97

1

Clostridia

OTU_83

S1


EF097083

91

1

Clostridia

OTU_85

S1

rabbit cecum clone NED2E8

EF445193

96

1

Clostridia

OTU_86

S1


EF099955

96

1

Clostridia

OTU_89

S1

mouse cecum clone obob2_aaa01d07

EF096351

96

1

Clostridia

OTU_90

S1


DQ815443

89

1

Clostridia

OTU_91

S1


EF099813

93

1

Clostridia

OTU_94

S1


DQ815777

98

1

Clostridia

OTU_98

S1


EF098932

94

1

Genera_incertae_sedis_TM7 Genera_incertae_sedis_TM7 OTU_27 OTU_08 Proteobacteria Alphaproteobacteria

C2


EF097659

100

2

S4

forest soil clone FAC17

DQ451456

83

4

OTU_09

S10

ferromanganous micronodule clone MND8

AF292999

83

10

OTU_10

S6

ferromanganous micronodule clone MND8

AF292999

84

6

OTU_87

S1

carbon tetrachloride contaminated soil clone

DQ248306

87

1

Betaproteobacteria

OTU_55

C1

subsurface water clone EV818CFSSAHH51

DQ336997

96

1

Gammaproteobacteria

OTU_13 S33C2L18 termite gut wall fraction clone RsW01-004

AB198445

91

53

OTU_84

DQ009674.2

89

1

EF098228

91

10


EF098228

90

2

Deltaproteobacteria

Spirochaetes

OTU_01

S1

termite gut homogenate clone RSB1

S3C5S2 mouse cecum clone SWPT14_aaa03c12

OTU_02

C1L1

OTU_03

C2


DQ815453

90

2

OTU_04

C2


EF098228

91

2

OTU_05

C1L1


DQ815453

91

2

OTU_06

C2


EF098228

90

2

OTU_07

C2L1


EF098228

91

3

OTU_54

C1


DQ815586

91

1

OTU_70

L1

U42221

89

1

OTU_75

L1

Desulfovibrio fairfieldensis mouse cecum clone SWPT14_aaa03c12

EF098228

91

1

OTU_88

S1


EF098228

91

1

OTU_95

S1


DQ815453

92

1

Epsilonproteobacteria

OTU_11

S2C1


EF097950

96

3

Spirochaetes

OTU_12

C1L1

Treponema zioleckii str. kT

DQ065758

89

2

OTU_39

C3

Brachyspira hyodysenteriae str. 174-92

U14931

97

3

Relative abundance of sequences

Percentage of clones

100%

Class Deltaproteobacteria Gammaproteobacteria

80%

Mollicutes 60%

Clostridia

40%

Bacilli Deinococci

20%

Verrucomicrobiae near Cyanobacteria Actinobacteria

0% S

C

L

Clone libraries of gut samples Figure 1. The percentage of each bacterial class of clone libraries from the small intestine, caecum and large intestine of Apodemus semotus

Relative abundance of sequences Class

Percentage of clones

100%

Genera_incertae_sedis_TM7 Spirochaetes Deferribacteres Bacteroidetes Mollicutes Clostridia Bacilli Epsilonproteobacteria Deltaproteobacteria Gammaproteobacteria Betaproteobacteria Alphaproteobacteria Actinobacteria

80% 60%

40% 20%

0 %

S

C

L

Clone libraries of gut samples Figure 2. The percentage of each bacterial class of clone libraries from the small intestine, caecum and large intestine of Eothenomys melanogaster

100

Meiothermus silvanus (Y13599) OTU 02 OTU 51

Deinococci 100

100 100

97

0.1

Uncultured bacterium (AY986278) OTU 10 Uncultured bacterium (AY571501) OTU 21

Uncultured cyanobacterium (AY304018) OTU 14

Yersinia aleksiciae (AJ627597) OTU 47 100 Shigella boydii (AY696681) OTU 01 OTU 33 OTU 53 Uncultured delta proteobacterium (AB192287) OTU 04 OTU 31 OTU 08 OTU 46 OTU 05 100 OTU 32 OTU 03 OTU 18 Desulfomonas pigra (AF192152) Desulfovibrio desulfuricans (DQ092636) Desulfovibrio sp. (AJ251630) Desulfovibrio fairfieldensis (U42221) Bilophila wadsworthia (L35148) OTU 30 Corynebacterium pseudogenitalium (AJ439348) 100 OTU 50 Human intestinal bacterium (AY310748) OTU 52 Streptococcus uberis (AB023576) OTU 15 OTU 66 100 Lactobacillus intestinalis (AJ306299) OTU 67 Anoxybacillus flavithermus (AY672762) 96 OTU 37 Uncultured Mollicutes bacterium (AB218347) OTU 65 Uncultured Clostridiales bacterium (AB234467) OTU 16 100 OTU 49 Candidatus Arthromitus (D86305) OTU 64 Unidentified rumen bacterium (AB009208) OTU 63 Uncultured bacterium (AY916378) OTU 62 Ruminococcus bromii (X85099) OTU 36 Uncultured bacterium (DQ015614) OTU 48 Anaerotruncus colihominis (DQ002932) OTU 61 OTU 29 100 Clostridium orbiscindens (AY730665) OTU 23 OTU 20 Uncultured bacterium (DQ014910) OTU 09 Uncultured bacterium (AY992487) OTU 60 Uncultured bacterium (DQ014992) Uncultured bacterium (DQ015401) OTU 11 OTU 12 OTU 26 OTU 27 OTU 40 OTU 41 91 Uncultured bacterium (DQ014825) OTU 28 Uncultured bacterium (DQ014588) OTU 59 Uncultured bacterium (AY993706) OTU 34 Clostridium sp. (AY960574) OTU 25 Butyrate-producing bacterium (AJ270488) OTU 38 OTU 19 Clostridium sp. (AF157053) OTU 13 100 Uncultured bacterium (AY993337) OTU 24 OTU 54 Butyrate-producing bacterium (AY305314) OTU 55 Uncultured bacterium (DQ015511) OTU 42 Uncultured bacterium (DQ014892) OTU 58 OTU 57 Uncultured bacterium (AY991749) OTU 43 OTU 56 Uncultured bacterium (AY992805) OTU 06 Uncultured bacterium (AY993686) OTU 44 Uncultured bacterium (AY992550) OTU 45 Uncultured bacterium (AY994018) OTU 07 Uncultured bacterium (AY991865) OTU 17 Uncultured bacterium (AY993232) OTU 22 Uncultured bacterium (DQ015316) OTU 39 Uncultured bacterium (DQ015606) OTU 35

Cyanobacteria Verrucomicrobiae Gammaproteobacteria

Deltaproteobacteria

Actinobacteria Bacilli Mollicutes

Clostridia

Figure 3. Neighbor-joining tree of 67 OTUs of Apodemus semotus based on the 16S rDNA sequences.

OTU_27

Genera_incertae_sedis_TM7

Bacteroidetes (total 28 OTUs)

99

OTU_16

50

Mollicutes

OTU_97 99

Bacilli

OTU_81

Clostridia (total 40 OTUs)

50

OTU_76

Deferribacteres

OTU_02 OTU_01 68

OTU_03 OTU_95 OTU_88 OTU_05 100

Deltaproteobacteria

OTU_04 OTU_06 OTU_07 OTU_54 OTU_75 OTU_70 100

OTU_10 OTU_09

62

OTU_08 100

Alphaproteobacteria

OTU_87

OTU_55

Betaproteobacteria

OTU_84 96

OTU_13

Gammaproteobacteria

OTU_11

Epsilonproteobacteria

OTU_14

93

OTU_49 100

Actinobacteria

OTU_48 OTU_12 OTU_39

Spirochaetes

0.05

Figure 4. Neighbor-joining tree of 98 OTUs of Eothenomys melanogaster based on the 16S rDNA sequences.