1
Tiago and Veríssimo, Supplementary data
“Caldanaerocella colombiensis” (AY464940)
Geosporobacter subterrenus (DQ643978)
53
0.10
uncultured bacterium clone Y36 (AF407679)
uncultured Gram-positive bacterium (AJ810556)
uncultured bacterium clone CVCloAm3Ph95-OTU32 (AM778027)
uncultured bacterium clone CVCloAm3Ph34-OTU32 (AM778012)
uncultured low G+C Gram-positive bacterium clone DR546BH1103001SAD18 (DQ234642)
DGGE band CVA Lane18CL70 (FN401545)
DGGE band CVA Lane20CL79 (FN401552)
uncultured bacterium clone CVCloAm3Ph72-OTU33 (AM778019)
uncultured low G+C Gram-positive bacterium clone DR546BH1103001SAD28 (DQ234647)
96
69
uncultured bacterium clone: SMG133 (AM930313)
uncultured bacterium clone SMQ127 (AM930342)
uncultured bacterium clone: SMG133 (FN667367)
uncultured bacterium clone: SHBZ1630 (EU638482)
89
69
50
uncultured bacterium clone CVCloAm2Ph136-OTU22 (AM777955)
uncultured bacterium clone CVCloAm2Ph138-OTU22 (AM777957)
uncultured bacterium clone CVCloAm2Ph137-OTU22 (AM777956)
77 uncultured bacterium clone CVCloAm2Ph97-OTU22 (AM777980)
uncultured bacterium clone CVCloAm2Ph130-OTU22 (AM777953)
94
uncultured bacterium clone CVCloAm2Ph80-OTU23 (AM777977)
uncultured bacterium clone CVCloAm2Ph50-OTU37 (AM777975)
DGGE band CVA Lane21CL81 (FN401554 )
uncultured bacterium clone CVCloAm3Ph15-OTU35 (AM778006)
uncultured bacterium clone B11-15_GoMY (AB476673)
62
uncultured bacterium clone ANTXXIII_706-4_Bac71 FN429799)
DGGE band CVA Lane12Cl46 (FN401534)
63
DGGE band CVA Lane13CL49 (FN401537)
DGGE band CVA Lane19CL74 (FN401547)
uncultured bacterium clone CVCloAm3Ph87-OTU34 (AM778023)
76
uncultured bacterium clone CVCloAm3Ph98-OTU34 (AM778028)
DGGE band CVA Lane19CL73 (FN401546)
uncultured bacterium clone dr36g (AY540845)
92
uncultured bacterium clone KL441HWDS3.4 (DQ191818)
57
iron-reducing bacterium enrichment culture clone HN-HFO75 (FJ269100)
uncultured Anaerobranca sp. clone SRB2 (DQ069229)
uncultured bacterium clone SGNY0002 (EU730978)
uncultured bacterium clone SGNY0215 (EU731004)
92
70
81
90
82
92
78
92
52
56
78
Candidatus “Desulforudis audaxviator” MP104C (CP000860)
uncultured bacterium clone CVCloAm2Ph32-OTU25 (AM777970)
uncultured bacterium clone CVCloAm2Ph29-OTU25 (AM777967)
uncultured bacterium clone CVCloAm3Ph1-OTU25 (AM778000)
uncultured bacterium clone CVCloAm3Ph39-OTU25 (AM778013)
uncultured bacterium clone CVCloAm2Ph30-OTU27 (AM777969)
uncultured bacterium clone EV818BHEB6102702SAFF1 (DQ256295)
uncultured bacterium clone BE325FW032701CTS_hole1-2 (DQ088771)
uncultured bacterium clone CVCloAm2Ph18-OTU26 (AM777962)
DGGE band CVA Lane12Cl48 (FN401536)
uncultured bacterium clone CVCloAm2Ph49-OTU20 (AM777974)
DGGE band CVA Lane12Cl47 (FN401535)
DGGE band CVA Lane12CL24a (FN401533)
uncultured bacterium clone CVCloAm2Ph12-OTU21 (AM777952)
uncultured bacterium clone DR9IPCB16SCT8 (AY604055)
uncultured bacterium clone TTMF18 (AY741692)
uncultured bacterium TTMF141 (AY741691)
uncultured bacterium clone CVCloAm2Ph48-OTU29 (AM777973)
uncultured bacterium clone TTMF70 (AY741706)
Cryptanaerobacter phenolicus strain LR7.2 (NR_025757)
uncultured Moorella group bacterium clone HS07Ba14 (EU016419)
57
71
uncultured bacterium clone: DR938CH110701SACH36 (DQ230961)
uncultured bacterium clone CVCloAm3Ph10-OTU24 (AM778001)
88
uncultured bacterium clone MiJ7 (EU645123)
uncultured bacterium clone BE325FW032701CTS_hole1-77 (DQ088758)
DGGE band CVA Lane15CL58 (FN401540)
uncultured bacterium clone CVCloAm2Ph135-OTU42 (AM777954)
56
uncultured bacterium clone CVCloAm1Ph79-OTU38 (AM777998)
59
uncultured bacterium clone CVCloAm3Ph44-OTU41 (AM778015)
86
uncultured bacterium clone CVCloAm3Ph60-OTU40 (AM778017)
uncultured bacterium clone CVCloAm2Ph84-OTU40 (AM777978)
87
uncultured bacterium clone CVCloAm3Ph74-OTU43 (AM778020)
uncultured bacterium clone CVCloAm2Ph109-OTU43 (AM777951)
89
uncultured Gram-positive bacterium (AJ431345)
65
uncultured low G+C Gram-positive bacterium clone ML-A-7 (DQ206408)
Dethiobacter alkaliphilus (EF422412)
uncultured low G+C Gram-positive bacterium clone ML635J-4 (AF507887)
76
uncultured bacterium clone F4-B15 (FJ206817)
87
uncultured bacterium clone BE325FW032701CTS_hole1-13 (DQ088768)
77
65 uncultured bacterium clone CVCloAm2Ph23-OTU30 (AM777965)
uncultured bacterium clone CVCloAm2Ph25-OTU30 (AM777966)
89
uncultured bacterium clone CVCloAm3Ph84-OTU30 (AM778022)
53
uncultured bacterium clone BE325FW032701CTS_hole1-10 (DQ088769)
67
DGGE band CVA Lane17CL68 (FN401544 )
98
uncultured bacterium clone CVCloAm1Ph72-OTU31 (AM777996)
53
uncultured bacterium clone CVCloAm2Ph42-OTU36 (AM777972)
74
uncultured bacterium clone CVCloAm2Ph103-OTU39 (AM777948)
91
81
74
83
1
2
uncultured bacterium clone N05May-64 (EU442937)
uncultured low G+C Gram-positive bacterium clone ML635J-16 (AF507885)
uncultured bacterium clone BE325FW032701CTS_hole1-69 (DQ088759)
DGGE band CVA Lane21CL84 (FN401555)
uncultured bacterium clone CVCloAm2Ph102-OTU28 (AM777947)
uncultured bacterium clone BE325FW032701CTS_hole1-27 (DQ088764)
uncultured bacterium clone BE325FW032701CTS_hole1-1 (DQ088772)
Supplementary Figure S1
Clostridia
2
Tiago and Veríssimo, Supplementary data
3
4
5
0.10
DGGE band CVA Lane17Cl67 (FN401543)
77
92
uncultured bacterium clone: De2104 (HQ184020)
DGGE band CVA Lane5Cl18 (FN401566)
90
uncultured candidate division OP11 bacterium clone Bol26 (AY193135)
75
78
86
99
84
90
92
98
59
71
74
99
67
99
93
DGGE band CVA Lane2Cl7 (FN401561)
uncultured bacterium clone CVCloAm2Ph140-OTU17 (AM777958)
uncultured earthworm intestine bacterium clone wi14 (AY154482)
uncultured bacterium clone HTH (DQ497748)
uncultured Acidobacteria bacterium clone 1B5 (AM902634)
6
7
8
9
10
11
12
13
14
15
16
17
Acidobacteria
uncultured bacterium clone CVCloAm2Ph22-OTU44 (AM777964)
uncultured bacterium clone CVCloAm2Ph99-OTU44 (AM777981)
uncultured bacterium clone CVCloAm2Ph156-OTU44 (AM777960)
uncultured bacterium clone CVCloAm2Ph115-OTU44 (AM777984)
uncultured actinobacterium clone NRB9 (HM041926)
uncultured bacterium clone: SMD-B10 (AB478002)
uncultured bacterium clone Er-LLAYS-23 (EU542498)
Propionibacterium acnes strain ES_MS23c (EU888516)
88
uncultured bacterium clone SD04E02 (FM875678)
98
DGGE band CVA Lane16CL64 (FN401541)
99
uncultured Propionibacterium sp. isolate DPEU18 (EF127670)
uncultured bacterium clone 21_1 (FJ390446)
uncultured bacterium clone CVCloAm2Ph8-OTU45 (AM777976)
uncultured bacterium clone CVCloAm2Ph19-OTU45 (AM777963)
uncultured bacterium clone LD_Ba_TopB18 (EU644259)
Actinobacteria
99
uncultured bacterium clone p8a18ok (FJ479391)
54
unclassified bacteria
uncultured candidate division OD1 bacterium clone MS11-3-D12 (AM712350)
uncultured bacterium clone: ORI-860-02-P_S221-223 (JN123504)
uncultured Chlamydiales bacterium clone:KB19 (AB074958)
uncultured bacterium clone ORSFC2_a11 (EF393413)
uncultured bacterium a2b013 (AF419661)
uncultured bacterium clone D14R15C85 (FM956798)
uncultured bacterium clone Ax3_115 (EU762760)
99
94
DGGE band CVA Lane8Cl29 (FN401571)
DGGE band CVA Lane8Cl32 (FN401572)
Gram-positive bacterium strain SOGA31 (AJ244807)
Bacterium strain: Ellin6529 (HM748677)
Supplementary Figure S1 (cont)
Chloroflexi
3
Tiago and Veríssimo, Supplementary data
50
uncultured bacterium clone CVCloAm3Ph92-OTU1 (AM778025)
uncultured bacterium clone pGXAR39 (DQ266903)
uncultured bacterium clone CVCloAm3Ph23-OTU1 (AM778009)
0.10
uncultured bacterium clone CVCloAm1Ph57-OTU1 (AM777993)
uncultured bacterium clone CVCloAm3Ph105-OTU1 (AM778002)
55 uncultured bacterium clone CVCloAm2Ph132-OTU1 (AM777986)
79 uncultured bacterium clone CVCloAm1Ph58-OTU1 (AM777994)
62 uncultured bacterium clone CVCloAm2Ph147-OTU1 (AM777959)
65
uncultured bacterium clone CVCloAm3Ph113-OTU1 (AM778004)
70 uncultured soil bacterium clone FL9cT72-43 (AM884739)
87 uncultured Burkholderiales bacterium clone 88B-2E (EU073801)
uncultured bacterium clone CVCloAm2Ph144-OTU5 (AM777988)
DGGE band CVA Lane14CL53 (FN401538)
DGGE band CVA Lane20CL80 (FN401553 )
Betaproteobacteria
51
DGGE band CVA Lane14CL56 (FN401539 )
uncultured bacterium clone CVCloAm2Ph92-OTU2 (AM777979)
73
DGGE band CVA Lane10Cl40 (FN401530)
63 Hydrogenophaga flava (AB021420)
96 Hydrogenophaga pseudoflava (AF078770)
97
Hydrogenophaga atypica (AJ585992)
uncultured bacterium clone CVCloAm2Ph106-OTU3 (AM777983)
53
uncultured bacterium clone CVCloAm2Ph121-OTU3 (AM777985)
uncultured bacterium clone CVCloAm2Ph141-OTU3 (AM777987)
phenanthrene-degrading bacterium 90 (AY177371)
53
Methylibium subsaxonicum (AM774413)
97 Janthinobacterium lividum (Y08846)
uncultured bacterium clone CVCloAm2Ph108-OTU4 (AM777950)
Janthinobacterium agaricidamnosum (Y08845)
55 DGGE band CVA Lane16CL63 (FN401542 )
64
uncultured bacterium clone HY1_b11_2 (EU776115)
50
DGGE band CVA Lane23CL91 (FN401558 )
Pseudomonas umsongensis strain DPNP-6 (FJ592169)
95 Pseudomonas fluorescens (AJ308308)
Gammaproteobacteria
69
Pseudomonas sp. Strain Pb1 (DQ898312)
90 DGGE band CVA Lane7Cl28 (FN401570 )
84
uncultured gamma proteobacterium clone 357 (AB252880)
Acinetobacter calcoaceticus (EU834256)
90
Acinetobacter johnsonii (EU594557)
Legionella geestiana (Z49723)
uncultured bacterium clone CVCloAm2Ph3-OTU7 (AM777968)
80
uncultured bacterium clone EV818BHEB5102702SAS80 (DQ256358)
Silanimonas lenta (AY557615)
uncultured Xanthomonadaceae bacterium clone DR938CH110701SACH44 (DQ230964)
53
uncultured bacterium clone CVCloAm3Ph43-OTU6 (AM778014)
60 Ochrobactrum anthropi (U88444)
Ochrobactrum tritici (AY429607)
Alphaproteobacteria
uncultured bacterium clone CVCloAm2Ph36-OTU8 (AM777971)
97
Truepera radiovictrix (DQ022076)
uncultured endolithic bacterium clone 2B_WNS (AB374367)
75
uncultured bacterium strain AKIW1046 (DQ129340)
uncultured bacterium clone CVCloAm1Ph74-OTU9 (AM777997)
uncultured bacterium clone CVCloAm1Ph47-OTU9 (AM777992)
Deinococci
uncultured bacterium strain6 (EU017377)
98 Thermus thermophilus (X07998)
uncultured endolithic bacterium clone 2B_WNS (AB374367)
uncultured bacterium clone CVCloAm2Ph160-OTU10 (AM777961)
18
19
20
21
22
23
24
25
26
Suplementary Figure S2
4
Tiago and Veríssimo, Supplementary data
DGGE band CVA Lane1CL1a (FN401548 )
uncultured bacterium clone CVCloAm3Ph93-OTU16 (AM778026)
DGGE band CVA Lane3Cl11 (FN401563)
DGGE band CVA Lane5Cl20 (FN401568)
uncultured bacterium clone CVCloAm3Ph22-OTU15 (AM778008)
DGGE band CVA Lane2Cl5 (FN401559)
50 DGGE band CVA Lane1Cl3 (FN401550)
61 DGGE band CVA Lane1Cl4 (FN401551)
uncultured bacterium clone CVCloAm3Ph79-OTU11 (AM778021)
uncultured bacterium clone CVCloAm3Ph5-OTU11 (AM778016)
99
uncultured bacterium clone 11S_02e05 (FJ382718)
DGGE band CVA Lane2Cl6 (FN401560)
DGGE band CVA Lane4Cl13 (FN401565)
DGGE band CVA Lane5Cl19 (FN401567)
DGGE band CVA Lane2Cl8 (FN401562)
DGGE band CVA Lane22CL85 (FN401556)
91 uncultured eubacterium OCG1 (AB047111)
DGGE band CVA Lane9Cl34 (FN40157 )
uncultured bacterium clone CVCloAm3Ph9-OTU12 (AM778024)
75 DGGE band CVA Lane1Cl2 (FN401549)
74 DGGE band CVA Lane22CL88 (FN401557)
DGGE band CVA Lane3Cl12 (FN401564)
99 DGGE band CVA Lane6Cl23 (FN401569)
uncultured bacterium clone CVCloAm3Ph31-OTU13 (AM778011)
uncultured bacterium clone MiJ37 (EU645147)
uncultured Sphingobacteria bacterium clone A1870 (EU283537)
63
55
55
Bacteroidetes
uncultured Sphingobacteria bacterium clone A2187 (EU283539)
uncultured bacterium clone ambient_alkaline-9 (GU454992)
uncultured Bacteroidetes bacterium clone 02N1A240 (DQ330406)
99 uncultured Sphingobacteria bacterium clone A845 (EU283534)
52
uncultured Sphingobacteria bacterium clone A1503 (EU283512)
uncultured Bacteroidetes bacterium clone ML635J-44 (AF507864)
uncultured bacterium clone HDBW-WB41 (AB237704)
82
88
uncultured bacterium clone G3DCM-198 (EU037344)
uncultured Bacteroidetes bacterium clone ML623J-20 (AF507861)
uncultured bacterium clone CVCloAm3Ph114-OTU14 (AM778005)
94
uncultured bacterium clone NED5C1 (EF445243)
uncultured bacterium clone 1103200819980 (EU844042)
uncultured bacterium clone Saki_aaj64a06 (EU475765)
88 uncultured bacterium clone CVCloAm3Ph64-OTU18 (AM778018)
82 uncultured bacterium clone CVCloAm1Ph2-OTU18 (AM777989)
uncultured bacterium clone CVCloAm2Ph1-OTU18 (AM777982)
DGGE band CVA Lane11Cl44 (FN401532)
89
DGGE band CVA Lane11Cl41 (FN401531)
56
uncultured bacterium clone CVCloAm3Ph2-OTU19 (AM778007)
68
uncultured bacterium clone MD2896-B96 (EU385709)
uncultured bacterium clone 2WB_25 (EU574661)
uncultured Nitrospirae bacterium clone S15A-MN131 (AJ534688)
0.10
50
27
28
29
30
31
Suplementary Figure S2 (cont)
Nitrospira
5
Tiago and Veríssimo, Supplementary data
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 7+ OTU3a (FN690957)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 7 OTU3a (FN690956)
unclassified
uncultured archaeon partial 16S rRNA gene, clone CVA-3 phyl 3 gr7 OTU3a (FN690970)
94 uncultured archaeon partial 16S rRNA gene, DGGE band CVA 4+ OTU3a (FN690952)
group 2
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 8 OTU3a (FN690958)
88
uncultured archaeon clone MKCSB-A2 (DQ363753)
uncultured crenarchaeote clone LP30MA92 (FJ901994)
99
96 uncultured archaeon clone Fapm1aA55 (AB213062)
uncultured archaeon clone Fapm1aA55 (AB213057)
uncultured archaeon clone TVG8AR06 (GQ848376)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 9+ OTU4a(FN690961)
uncultured crenarchaeote clone MLSB_30m_2E_A (EF420185)
75
uncultured archaeon partial 16S rRNA gene, clone CVA-3 phyl 37 gr1Dtot OTU4a (FN690972)
uncultured archaeon clone MLSB_30m_6G_A (EF420189)
uncultured archaeon clone ss016a (AJ969779)
77
uncultured archaeon clone ASC37 (AB161337)
uncultured archaeon clone A7 (GU322882)
anaerobic methanogenic archaeon ET1-8 (AJ244284)
anaerobic methanogenic archaeon ET1-10 (AJ244286)
Geogemma indica strain 296 (DQ492260)
Pyrodictium occultum strain PL-19 (NR_025933)
0.10
0.10
97
33
34
35
36
37
38
uncultured archaeon partial 16S rRNA gene, clone CVA-2,3 phyl grBtot OTU5a (FN690968)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 6 OTU5a (FN690955)
uncultured archaeon clone DR9IPCA16SCT1 (AY604063)
ANME-1a
uncultured archaeon clone MS149BH1062003_10 (DQ354742)
87
uncultured euryarchaeote clone pIta-HW-4 (AB301860)
uncultured archaeon clone A163H03 (FJ455963)
uncultured archaeon clone Arch125 (FN428824)
82
Methanosaeta harundinacea (AY970347)
Methanomethylovorans hollandica (AY260433)
uncultured archaeon clone Ele_A_14 (EF444662)
uncultured archaeon clone Thp_A_72 (EF444631)
unclassified
71
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 3+ OTU6a (FN690950)
group 3
uncultured archaeon clone CP-A127 (DQ521194)
uncultured archaeon clone MD3057C-58 (GQ994159)
Methanothermococcus okinawensis strain IH1 (AB057722)
Methanococcus aeolicus strain Mic6c09 (AB546256)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 13 OTU1a (FN690944)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 13a OTU1a (FN690945)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 12 OTU1a (FN690942)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 13+ OTU1a (FN690946)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 13a+ OTU1a (FN690943)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 12+ OTU1a (FN690941)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 11 OTU1a (FN690939)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 4 OTU1a (FN690951)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 11+ OTU1a (FN690940)
uncultured archaeon partial 16S rRNA gene, clone CVA-3 phyl 43 grAc OTU1a (FN690974)
uncultured archaeon partial 16S rRNA gene, clone CVA-3 phyl grAftot OTU1a (FN690977)
uncultured archaeon partial 16S rRNA gene, clone CVA-3 phyl grAatot OTU1a (FN690976)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 5+ OTU1a (FN690954)
SAGMEG 2
uncultured archaeon partial 16S rRNA gene, clone CVA-2 phyl 24 grAk OTU1a (FN690962)
uncultured archaeon partial 16S rRNA gene, clone CVA-3 phyl 40 grAd OTU1a (FN690973)
uncultured archaeon partial 16S rRNA gene, clone CVA-2 phyl grAe OTU1a (FN690967)
83 uncultured archaeon partial 16S rRNA gene, clone CVA-3 phyl 35 grAm OTU1a (FN690971)
uncultured archaeon partial 16S rRNA gene, clone CVA-2 phyl 36 grAy OTU1a (FN690964)
uncultured archaeon partial 16S rRNA gene, clone CVA-3 phyl 20 grAj OTU1a (FN690969)
81
uncultured archaeon partial 16S rRNA gene, clone CVA-2 phyl 66 grAh OTU1a (FN690966)
uncultured archaeon clone NO27FW100501SAB190 (DQ230938)
uncultured archaeon clone NO27FW100501SAB175 (DQ230939)
96
uncultured archaeon clone EV818BHEB6102702SAFF101 (DQ256304)
uncultured archaeon SAGMA-12 (AB050242)
uncultured archaeon clone MP104-1109-a19 (DQ088781)
uncultured archaeon SAGMA-U (AB050226)
uncultured archaeon SAGMA-H (AB050212)
89
uncultured archaeon partial 16S rRNA gene, clone CVA-3 phyl 65 gr5tot OTU2a (FN690975)
uncultured archaeon partial 16S rRNA gene, clone CVA-2 phyl 51 gr5tot OTU2a (FN690965)
uncultured archaeon partial 16S rRNA gene, clone CVA-2 phyl 27 gr1Etot OTU2a (FN690963)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 10+ OTU2a (FN690938)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 9 OTU2a (FN690960)
unclassified
65 uncultured archaeon partial 16S rRNA gene, DGGE band CVA 5 OTU2a (FN690953)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 10 OTU2a (FN690937)
group 1
52 uncultured archaeon partial 16S rRNA gene, DGGE band CVA 8+ OTU2a (FN690959)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 14+ OTU2a (FN690948)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 14 OTU2a (FN690947)
uncultured archaeon partial 16S rRNA gene, DGGE band CVA 1_2 OTU2a (FN690949)
99
uncultured archaeon clone FeOrig_A_7 (GQ356919)
80
uncultured archaeon clone TWP6-42 (GQ410922)
uncultured archaeon clone MD3059V-68 (GQ927645)
uncultured archaeon clone HDBW-WA14 (AB237747)
73
Suplementary Figure S3
Phylum Euryarchaeota
32
Class Thermoprotei
6
Tiago and Veríssimo, Supplementary data
50
a)
45
40
number of OTU's
35
30
25
20
15
10
5
0
0
100
200
300
number of clones screened
400
500
600
b)
cutoff value
Number of BP-OTUs
500
0.01
0.02
400
0.03
0.04
300
0.05
0.06
200
0.07
0.08
100
0.09
0.1
0
0
2000
4000
6000
8000
Number of bacterial pyrosequences
350
cutoff value
0.01
300
0.02
Number of AP-OTUs
250
0.03
0.04
200
0.05
150
0.06
0.07
100
0.08
50
0.09
0.1
0
0
39
40
41
42
1000
2000
3000
Number of archaeal pyrosequences
4000
5000
Suplementary Figure S4
7
Tiago and Veríssimo, Supplementary data
CVA-accC- clone:36 (HE686926)
49
61
28
53
58
20
81
54
32
85
11
41
50
0.05
43
CVA-accC- clone:7 (HE686933)
CVA-accC- clone:25 (HE686925)
52
CVA-accC-Cluster-1
CVA-accC- clone:22 (HE686924)
100
CVA-accC- clone:17 (HE686922)
CVA-accC- clone:15 (HE686921)
Dehalococcoides sp. GT (YP_003461973)
Ornithinibacillus sp. TW25 (ZP_08784876)
Geobacillus kaustophilus HTA426 (YP_147452)
Anoxybacillus flavithermus WK1 (YP_002314550)
100
Geobacillus sp. WCH70 (YP_002949846)
81
Geobacillus sp. Y4.1MC1 (ZP_05374279)
100
92
Geobacillus thermoglucosidasius C56-YS93 (ZP_06809961)
Archaeoglobus profundus DSM 5631 (YP_003399787)
98
Ferroglobus placidus DSM 10642 (YP_003434777)
CVA-accC- clone:19 (HE686923)
CVA-accC- clone:14 (HE686920)
100
CVA-accC-Cluster-2
70 CVA-accC- clone:40 (HE686929)
CVA-accC- clone:47 (HE686932)
Methanobacterium sp. SWAN-1 (YP_004520289)
99
Methanobrevibacter smithii DSM 2375 (ZP_03607571)
Methanohalobium evestigatum Z-7303 (YP_003726235)
Methanocaldococcus vulcanius M7 (YP_003247216)
Methanococcus voltae A3 (YP_003708349)
Bacteroides salanitronis DSM 18170 (YP_004257877)
100
Bacteroides sp. 2_1_16 (ZP_06094446)
Wolinella succinogenes DSM 1740 (NP_907271)
Cyanothece sp. PCC 8802 (YP_003137206)
98
Synechococcus elongatus PCC 6301 (YP_170887)
Gloeobacter violaceus PCC 7421 (NP_924958)
Chlorobium tepidum TLS (NP_661063)
Halothermothrix orenii H 168 (YP_002508378)
Acetohalobium arabaticum DSM 5501 (YP_003828313)
100 Leptospirillum rubarum (mine drainage metagenome) (EAY55884)
Leptospirillum sp. Group II '5-way CG' (mine drainage metagenome) (EDZ39228)
uncultured Geobacter sp. clone: BEMACCC (ABR01040)
CVA-accC- clone:41 (HE686930)
62
CVA-accC-Cluster-4
100
clone: 25.05.2004. 9m depth (CAI85103)
Thermodesulfovibrio yellowstonii DSM 11347 (YP_002248245)
Aquifex aeolicus VF5 (NP_214014)
Novosphingobium sp. PP1Y (YP_004534660)
Mariprofundus ferrooxydans PV-1 (ZP_01453567)
Phenylobacterium zucineum HLK1 (YP_002131863)
90
Oceanicaulis sp. HTCC2633 (ZP_00957838)
82
CVA-accC-clone:42 (HE686931)
CVA-accC-clone:39 (HE686927)
100
CVA-accC-Cluster-3
98
CVA-accC-clone:4 (HE686928)
44
45
46
47
48
49
50
Supplementary figure S5
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
Pathway / Target gene
16S rRNA Domain Bacteria
Primers
Nucleotide sequence (5′–3′)
Bac 27F
GAG TTT GAT CCT GGC TCA G
Bac 1525R
AGA AAG GAG GTG ATC CAG CC
16S rRNA Domain Archaea
Arc 21F
TTC CGG TTG ATC CYG CCG GA
Arc 958R
YCC GGC GTT GAM TCC AAT T
18S rRNA
EukA
AAC CTG GTT GAT CCT GCC AGT
EukB
TGA TCC TTC TGC AGG TTC ACC TAC
26S rRNA gene D1/D2 domain
NL1
GCA TAT CAA TAA GCG GAG GAA AAG
NL4
GGT CCG TGT TTC AAG ACG G
ITS-5.8S rDNA region
ITS 1
GTC GTA ACA AGG TTT CCG TAG GTG
ITS 4
TCC TCC GCT TAT TGA TAT GC
16S rRNA Domain Bacteria (DGGE)
DGGE GC clamped 341f*
CCT ACG GGR GGC AGC AG
DGGE 534r
ATT ACC GCG GCT GCT GG
16S rRNA Domain Archaea (DGGE)
Parch519f
CAG CCG CCG CGG TAA
DGGE GC clamped Arch915*
GTG CTC CCC CGC CAA TTC CT
18S rRNA (DGGE)
Euk1A
CTG GTT GAT CCT GCC AG
Euk516r-GCb*
ACC AGA CTT GCC CTC C
*GC clamp to be added on 5' terminal - CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG G
16S rRNA Domain Bacteria (Pyrosequencing)
Gray28F
TTT GAT CNT GGCT CAG
Gray519r
GTN TTA CNG CGG CKG CTG
16S rRNA Domain Archaea (Pyrosequencing)
Arch 21F
TTC CGG TTG. ATC CYG CCG GA
Arch 534r
ATT ACC GCG GCT GCT GG
References
Rainey et al. , 1996
Vetriani et al ., 1999
Behnke et al ., 2006
O’Donnell, 1993
White et al ., 1990
Sørensen et al ., 2005
Coolen et al ., 2004
Díez et al ., 2001
Dowd et al ,. 2008
Brazelton et al ., 2006
Sánchez et al ., 2007
Expected amplicon sizes
~1500 bp
~900 bp
~1100 bp
~740 bp
550 - 900 bp
200 bp
400 bp
560 bp
~500 bp
~500 bp
Supplementary Table S1 Primer sets used for detection of genes encoding 16S rRNA SSU for both Domains Bacteria and Archaea and genes encoding key enzymes of CO2 metabolic pathways,
sulphur and methane cycles.
Tiago and Veríssimo, Supplementary data
8
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
methane monooxygenase
pmoA
MCR
methyl coenzymeM reductase
GGT GGT GTM GGA TTC ACA CAR TAY GCW ACA GC
TTC ATT GCR TAG TTW GGR TAG TT
GGN GAC TGG GAC TTC TGG
CCG GMG CAA CGT CYT TAC C
MCRF
MCRreverse primer
A 189
mb661
GCG CCA ACY GGR CCR TA
Methane cycle
TGG CAG ATC ATG ATY MAY GG
aps1F
aps4R
GCT GGA GAT GGA GCY TCY TCW ATT A
aprA
GCT GAT GCT ATA CAT CCW GGW TAY GG
OOR67F
ACAC254f
CTT GCA GCC TGT NGG MAK NCC NGT
TTC TTC GCT GGG TAY CCN ATH ACN CC
oorA 345R
ACAC720r
CIG GIT GYG GIG AAA CIC C
RTC ICT TYC ICC IAR ACC
CCI ATR TCR TAI GCC CAI CCR TC
GAT CAG GTC CTT CAG NCC NTT CCC
porA 900F
porA 1101R
nifJ 2933R
ATA GTT KGG SCC ACC TCT TC
nifJ 2459F
TGG ACM ATG GTD GCY GGK GGT
aclB 892F
aclB 1204R
TCG AAC TTG ATT TCT TTC CA
TTC TGG CTG GGB GGH GAY TTY ATY AAR AAY GAC
GACGR TGG TAR TG
CCG TGR CCR GCV
GAC TTC ACC AAA GAC GAC GA
Nucleotide sequence (5′–3′)
APS reductase
Sulpur cyle
biotin carboxylase
accC
3HP and 3-HP/4-HB cyles
2-oxoglutarate:ferredoxin oxidoreductase
form oor A
pyruvate:ferredoxin oxidoreductase
form nifJ
pyruvate:ferredoxin oxidoreductase
form por
ATP citrate lyase beta subunit
aclB
rTCA cycle
cbbM F
formII RubisCO
cbbM
cbbM R
cbbL F
cbbL R
cbbL
Primers
CBB cycle
form I RubisCO
Pathway / Target gene
470 bp
480 bp
396 bp
460 bp
278 bp
474 bp
201 bp
312 bp
328 bp
711 bp
Expected amplicon sizes
Costello and Lidstrom, 1999
Luton et al ., 2002
Blazejak et al ., 2006
Auguet et al ., 2008
Campbell and Cary, 2004
Campbell and Cary, 2004
Campbell and Cary, 2004
Campbell et al. , 2003
Campbell and Cary, 2004
Campbell and Cary, 2004
References
Supplementary Table S1 (cont.) Primer sets used for detection of genes encoding 16S rRNA SSU for both Domains Bacteria and Archaea and genes encoding key enzymes of CO2 metabolic
pathways, sulphur and methane cycles.
Tiago and Veríssimo, Supplementary data
9
10
Tiago and Veríssimo, Supplementary data
99
100
101
Supplementary Table S2 Chemical
composition, temperature and pH of the
a
Cabeço de Vide aquifer
102
103
Chemical
Oxygen
Carbon dioxide
Anions
Concentration (mM)
<0.05
<0.068
Chloride, Cl-
104
1.482 ± 0.100b
Sulfate, SO4 2-
105
0.030 ± 0.011
Carbonate, CO3
Nitrate, NO3
106
2-
-
≤0.008
-
≤0.008
Fluoride, F
-
Hydroxyl, OH
107
Silicate, H3 SiO4
1.708 ± 0.186
-
0.087 ± 0.008
-
108
Sulfide, SH
Cations
Potassium, K
111
112
117
118
119
120
121
122
≤0.008
0.576 ± 0.029
2+
2+
≤0.008
≤0.008
+
Lithium, Li
Silicon dioxide, SiO2
≤0.008
0.0383 ± 0.042
a
Results of chemical analysis performed three
times a year, between 1991 and 2011, by a
recognized state laboratory and provided by M.
Fontaínhas. The pH of the water was 11.4 ± 0.16,
and the temperature was 20.5 ± 0.8ºC.
b
116
+
+
Magnesium, Mg
Iron, Fe
115
0.117 ± 0.009
Ammonium, NH4
Calcium, Ca2
114
2.263 ± 0.158
+
110
113
0.025 ± 0.005
Sodium, Na+
109
0.108 ± 0.057
The the mean ± standard deviation is presented.
11
Tiago and Veríssimo, Supplementary data
123
124
125
126
127
128
129
130
Supplementary Table 3 Statistical analysis results
a) Values of coverage, Shannon index and Shannon equitability determined from the clone library and
pyrosequence analyses results
Domain Bacteria
Domain Archaea
Clone library Pyrosequencing Clone library Pyrosequencing
Coverage
89.8%
97.7%
98.1%
99.4%
Shannon index (H)
2.28
3.5
0.74
1.6
H max
3.81
5.2
1.61
3.4
Shannon equitability
0.60
0.67
0.46
0.48
b) Sørensen similarity index applied to the DGGE profiles obtained for samples 2 to 7.
vs
Samples
2
3
4
5
6
a
2
1.00
0.90
0.90
0.90
3
4
5
6
7
131
132
133
a
134
135
136
137
138
139
140
141
142
143
144
145
146
b
0.85
0.86
0.75
0.86
0.86
0.86
0.89
1.00
0.75
0.90
0.89
0.75
0.75
b
0.90
1.00
0.89
0.67
0.90
1.00
1.00
0.75
7
0.90
0.90
1.00
1.00
1.00
-
, upper right values concern Domain Bacteria values, , lower left concern Domain Archaea values.
12
Tiago and Veríssimo, Supplementary data
147
Supplementary Data References
148
Auguet, J.C., Borrego, C.M., Baneras, L., and Casamayor, E.O. (2008) Fingerprinting the
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genetic diversity of the biotin carboxylase gene (accC) in aquatic ecosystems as a potential
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marker for studies of carbon dioxide assimilation in the dark. Environ. Microbiol. 10, 2527-
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2536.
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Behnke, A., Bunge, J., Barger, K., Breiner, H-W., Alla, V., and Stoeck, T. (2006)
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Microeukaryote Community Patterns along an O2/H2S Gradient in a Supersulfidic Anoxic
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Fjord (Framvaren, Norway). Appl Environ Microbiol 72: 3626-3636.
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Blazejak, A., Kuever, J., Erséus, C., Amann, R., and Dubilier, N. (2006) Phylogeny of 16S
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rRNA, ribulose 1,5-bisphosphate carboxylase/oxygenase, and adenosine 5-phosphosulfate
157
reductase genes from gamma and alphaproteobacterial symbionts in gutless marine worms
158
(Oligochaeta) from Bermuda and the Bahamas. Appl Environ Microbiol 72:5527-5536.
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Brazelton, W.J., Schrenk, M.O., Kelley, D.S., and Baross, J.A. (2006) Methane- and Sulfur-
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Metabolizing Microbial Communities Dominate the Lost City Hydrothermal Field Ecosystem.
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Appl Environ Microbiol 72:6257–6270.
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Campbell, B.J. and and Cary, S.C. (2004) Abundance of reverse tricarboxylic acid cycle genes
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in free-living microorganisms at deep-sea hydrothermal vents. Appl Environ Microbiol
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Campbell, B.J., Stein, J.L., and Cary, S.C. (2003) Evidence of chemolithoautotrophy in the
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bacterial community associated with Alvinella pompejana, a hydrothermal vent polychaete.
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Sinninghe Damst´e, J.S. (2004) Evolution of the methane cycle in Ace Lake (Antarctica) during
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Díez, B., Pedrós-Alió, C., Marsh, T.L., and Massana, R. (2001) Application of denaturing
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