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Supplementary SSCP analysis to the manuscript “Effect of the earthworms Lumbricus terrestris and
Aporrectodea caliginosa on bacterial diversity in soil” by Taras Nechitaylo et al.
Figure S2. Images of SSCP profiles generated with conventional primer set (Com1 and Com2-Ph) (A)
and the phylogenetic analysis of ribotypes detected from the SSCP profiles (B; bellow). The lanes and
the origin of ribotypes are marked as follows: RS - soil RNA; RL - L. terrestris cast RNA; RA A. caliginosa cast RNA; DS - soil DNA; DL - L. terrestris cast DNA; DA - A. caliginosa cast DNA; M marker. Different RNA- and DNA-derived profiles were generated during independent amplification
reactions from the corresponding samples. Numbers on the gel correspond to ribotypes on the
phylogenetic tree. Ribotypes from SSCP profiles are marked in bold; those detected on all profiles are
shown in gray boxes. Ribotypes detected in the same band are marked on the phylogenetic tree with
numbers and small letters. Sequences with identity >99.5% were taken as belonging to a single OTU
and are shown as one ID. Numbers at nodes indicate bootstrap values greater than 50%.
99
S-RS/RL-1b AM989555
S-RS/RL-1a AM989554
MITOCHONDRIA
mitochondrion Phytophthora infestans U17009
97
mitochondrion Saprolegnia ferax AY534144
uncultured bacterium AY274120
99
S-11a (AM989594, AM989595) not affiliated
S-DL-4, 10 AM989566
S-RS/RL-9/1a AM989592
S-DA-9/1b AM989584
S-6 (AM989573, AM989574)
S-DS-3 AM989563
56
Flavobacterium hibernum L39067
88 S-RL/DL/DA-2/1 (AM989559, AM989560)
53 Flavobacterium granuli AB180738
50
S-3 (AM989561, AM989562)
67 50 S-11b (AM989596, AM989597)
Flavobacterium limicola AB075232
89
S-RL/DL/DA-2 (AM989557, AM989558)
96
S-4a (AM989564, AM989565)
Bacteroidetes
99
S-5c (AM989571, AM989572)
S-10c (AM989590, AM989591)
99
Mesonia motionis DQ367409
S-RS/RL-9/1b AM989593
55
S-5b (AM989569, AM989570)
Adhaeribacter aquaticus AJ626894
83
S-DA-9/1a AM989583
99
98
Flexibacter flexilis AB078053
S-10b (AM989588, AM989589)
S-DA-9/1c AM989585
99
58
Flexibacter sancti AB078067
60
S-RS-14 AM989614
Leptothrix discophora L33974
82
Nitrosovibrio tenuis AY123803 Betaproteobacteria
S-DS-9c AM989581
56
S-RL-1c AM989556
99
74
Legionella pneumophila subsp. pascullei AF122885
Legionella longbeachae M36029
Cellvibrio fulvus AF448514
86
S-7 (AM989575, AM989576)
73
Pseudomonas aeruginosa Z76672
Gammaproteobacteria
98
S-RA-8 AM989577
51
Pseudomonas tolaasii AF255336
BACTERIA
S-9a (AM989578, AM989579)
50
Pseudomonas entomophila AY907566
74
Pseudomonas putida Z76667
95
Azospirillum brasilense AY324110
99
Azospirillum oryzae AB185396
S-DS-12b AM989606
S-RS-13b AM989610
99
S-DS/DL/DA-9b AM989580
99
Sphingobium chlorophenolicum X87164
59
Erythromicrobium ramosum AF465837
99
S-13a (AM989608, AM989609)
73
Terasakiella pusilla AB000482
Methylocystis sp. 5FB1 AJ868421
Alphaproteobacteria
64
92 Rhizobium rhizogenes AY626390
99 Rhizobium tropici AY166841
S-RL-13c AM989612
84
S-DS-9d AM989582
Mesorhizobium loti X67229
53
S-12b (AM989602, AM989603)
Paracoccus denitrificans Y16929
99
S-RL-13b AM989611
99 Sulfitobacter pontiacus AY159887
93
S-10a (AM989586, AM989587)
uncultured delta proteobacterium AY499971
Deltaproteobacteria
72
Pelobacter acetylenicus X70955
96
Pelobacter acidigallici X77216
89
S-DS-11c (AM989598, AM989599)
86
uncultured Gemmatimonadetes bacterium AY921858
99
Gemmatimonadetes
S-DS-12c AM989607
Gemmatimonas aurantiaca AB072735
S-12a (AM989600, AM989601)
75
Coriobacterium glomerans X79048
Acidimicrobium ferrooxidans U75647
66
Actinobacteria
Rhodococcus
opacus X80631
70
S-12c (AM989604, AM989604)
99
99 Nocardioides kribbensis AY835926
Acidobacterium capsulatum D26171
99 S-5a (AM989567, AM989568)
uncultured Acidobacteria bacterium AY921697
uncultured Acidobacteria bacterium AY281358 Acidobacteria
S-DS-11d
99
99 uncultured Acidobacteria bacterium AY281355
S-RL-13d AM989613 not affiliated
Methanobacterium aarhusense AY386124 ARCHAEA
75
99
0.05
Figure S2-B
A-RS/RL/RA-4 AM989617
A-RS/RL/RA-2 AM989616
A-RL/RA-9 AM989622
A-RS-9 AM989621
A-RS/RL/RA-6, 7 AM989619
A-RS-14 AM989627
A-RS/RL/RA-8 AM989620
A-RS/RL/RA-5 AM989618
A-RS/RL/RA-11 AM989625
A-RS/RL/RA-1, 3, 12 AM989615
A-RS/RA-10 AM989623
55
A-RS-13 AM989626
A-DA-1b, 5-7, 10c AM989673
73
A-RL-10 AM989624
Caulobacter leidyi AJ227812
Asticcacaulis excentricus AJ007800
65
63 Asticcacaulis biprosthecium AJ007799
Caulobacter subvibrioides CB81 M83797
Sphingomonas suberifaciens D13737
A-DL-9 AM989663
A-DS-15 AM989637
Sphingomonas oligophenolica AB018439
Sphingomonas echinoides AJ0124461
Sphingomonas wittichi AB021492
A-DL-12c AM989668
A-DS-29 AM989655
A-DL-10 AM989664
61
uncultured alpha proteobacterium AY921978
56
Blastomonas natatoria Y13774
A-DS-28 AM989654
uncultured alpha proteobacterium AY922004
A-DL-15, 16 AM989671
A-DS-23c AM989648
A-DS-24a AM989649
A-DS-6c 22b AM989632
Novosphingobium stygium U20775
Novosphingobium hassiacum AJ416411
89
A-DL-1, 2 AM989656
55
A-DL-5 AM989659
63
A-DL-14 AM989670
98
Amaricoccus veronensis U88043
A-DS-3, 6a, 16a AM989630, AM989899, AM989900
A-DL-7 AM989661
Brevundimonas nasdae AB071954
51
72
Brevundimonas vesicularis AY169433
83
A-DA-3 AM989675
Caulobacter fusiformis AJ227759
61
Caulobacter henricii AJ007805
81
A-DA-8, 9 AM989677
53
Caulobacter vibrioides AJ227755
92
A-DS-14 AM989636
A-DS-16c AM989638
A-DS-20b AM989642
Rhodoblastus sphagnicola AM040096
Beijerinckia mobilis AJ563932
78
Methylocella palustris AJ563927
69
A-DA-10a AM989678
A-DA-2 AM989674
A-DA-1a AM989672
Afipia felis AY513503
A-DS-22a AM989646
A-DS-2-5 AM989629
Aminobacter ciceronei AF034798
Rhizobium rhizogenes AY626390
A-DA-11 AM989680
98
A-DA-12 AM989681
Methylobacterium fujisawaense AB175634
98
Methylobacterium organophilum D32226
86
Caulobacterales
Rhizobiales
56
Sphingomonadales
unclassified
Sphingomonadaceae
A)
root
0.01
Figure S3. Phylogenetic analysis of Alphaproteobacteria (A) and outgrouping bacterial ribotypes (B;
bellow) from the SSCP profiles generated with Alphaproteobacteria-targeting primer set. Ribotypes
from SSCP profiles are marked in bold on the tree; their origins are marked as follows: RS - soil RNA;
RL - L. terrestris cast RNA; RA - A. caliginosa cast RNA; DS - soil DNA; DL - L. terrestris cast DNA; DA
- A. caliginosa cast DNA. Numbers on the gel (see manuscript) correspond to ribotypes on the
phylogenetic trees. Ribotypes amplified from RNA are shown in gray boxes; those detected in the same
band are marked on the phylogenetic tree with numbers and small letters. Sequences with identity
>99.5% were taken as belonging to a single OTU and are shown as one ID. Numbers at nodes
indicate bootstrap values greater than 50%.
B)
Alphaproteobacteria
A-DS-20a AM989641
A-DA-4a AM989676
66
A-DS-26 AM989652
A-DS-18a AM989639
Betaproteobacteria
A-DS-23b AM989647
Comamonas testosteroni AB064318
72
Simplicispira metamorpha Y18618
97
Archangium gephyra AJ233913
100
86
Myxococcus fulvus AJ233917
Anaeromyxobacter dehalogenans AF382398
A-DS-10 16b AM989633
A-DS-24b AM989650
A-DL-3 AM989657
100
53
Peredibacter starrii AF084852
Plesiocystis pacifica AB083432
A-DL-4 AM989658
A-DL-6 AM989660
89
A-DS-11, 19a AM989634
A-DS-1 AM989628
A-DL-8 AM989662
A-DS-21a AM989645
Deltaproteobacteria
A-DL-11 AM989665
67
88
uncultured delta proteobacterium AY622663
93
61
97
Haliangium ochraceum AB016470
Chondromyces apiculatus AJ233938
Polyangium cellulosum AJ316015
99
A-DS-21b AM989644
85 A-DL-12b AM989667
A-DS-12 AM989635
A-DL-12a AM989666
uncultured bacterium DQ067033
83
A-DS-18b AM989640
A-DS-20c AM989643
55
A-DL-13 AM989669
65
A-DS-6b 19b AM989631
A-DS-25c AM989651
A-DA-1 AM989679
99
Gammaproteobacteria
Stenotrophomonas maltophilia X95924
60
92 Stenotrophomonas rhizophila AJ293463
Flavobacterium johnsoniae AB078043
A-DS-27 AM989653
Bacteroidetes
100
99 Flavobacterium granuli AB180738
Methanobacterium aarhusense AY386124
0.05
Figure S3-B
56 B-DS/DL/DA-6a AM989693
84 Variovorax paradoxus AJ420329
51
B-DS/DL/DA-7b AM989696
B-DS/DL-8b AM989698
Endosymbiont of Acanthamoeba sp. AY549551
96
Comamonadaceae
Hylemonella gracilis AJ565423
Acidovorax delafieldii AF07876
Acidovorax avenae subsp. citrulli AF137506
94
85 B-DS/DA-1b AM989684
76 B-DS/DL/DA-5b AM989692
B-DS/DL/DA-8a AM989697
57
Ideonella dechloratans X72724
Schlegelella thermodepolymerans AY538709
60
unclassified
B-DS/DL/DA-9b AM989700
54
Aquabacterium citratiphilum AF035050
Burkholderiales
uncultured bacterium AY212711
B-DS/DL-5a AM989691
97
B-DS/DL-6b AM989694
78
uncultured betaproteobacterium AY921703
B-DS-4b AM989688
B-DS/DL/DA-4a AM989687
83
B-DL/DA-4/1 AM989689
99
unclassified
B-DL-10b AM989703
Betaproteobacteria
B-DL-10c AM989704
99
B-DS/DL-1a AM989683
B-DL/DA-4/2 AM989690
B-DS/DL/DA-2 AM989685
50
B-DS/DL/DA-3 AM989686
99
Burkholderia cepacia X87275
B-DS/DL/DA-10a AM989701
B-DS-10b AM989702
Nitrosomonadaceae
Nitrosospira briensis L35505
Nitrosolobus multiformis L35509
99
96 B-RS/RL/RA-1 AM989682
98
B-DS/DL/DA-12 AM989706
Alcaligenaceae
97
Alcaligenes faecalis E690 FN298276
Achromobacter xylosoxidans AF411021
94
B-DS/DL/DA-9a AM989699
Oxalobacter sp. p8E AJ496038
Massilia dura AY965998
B-DS/DL/DS-11 AM989705
Oxalobacteraceae
Duganella violaceinigra AY376163
56
B-DS/DL/DA-13 AM989707
Duganella zoogloeoides D14256
67
Azonexus fungiphilus AF011350
B-DS/DA-7a AM989695
91
63
99
root
0.02
Figure S4. Phylogenetic analysis of the ribotypes from the SSCP profiles generated with
Betaproteobacteria-targeting primer set. Ribotypes from SSCP profiles are marked in bold on the tree;
their origins are marked as follows: RS - soil RNA; RL - L. terrestris cast RNA; RA - A. caliginosa cast
RNA; DS - soil DNA; DL - L. terrestris cast DNA; DA - A. caliginosa cast DNA. Numbers on the gel (see
manuscript) correspond to ribotypes on the phylogenetic tree. Ribotypes amplified from RNA are shown
by a gray box. Ribotypes detected in the same band are marked on the phylogenetic tree with numbers
and small letters. Sequences with identity >99.5% were taken as belonging to a single OTU and are
shown as one ID. Numbers at nodes indicate bootstrap values greater than 50%.
A)
B)
73
98
root
D-DS-3 AM989851
D-DS-5c AM989855
88
D-DS-4 AM989852
65
D-DS-2 AM989850
D-DS-5a AM989853
65
100 D-DS-6, 7 AM989856
78
D-DA-1, 2 AM989848
D-DS-1 AM989849
99
98
uncultured Myxococcales bacterium DQ646302
Chondromyces apiculatus AJ233938
86
100 Polyangium thaxteri AJ233943
Nannocystis exedens AJ233946
Plesiocystis pacifica AB083432
100
D-DS-5b AM989854
Anaeromyxobacter dehalogenans AF382398
Cystobacter fuscus AJ233897
100
0.05
Figure S5. Images of SSCP profiles generated with Deltaproteobacteria-targeting primer set (A) and
the phylogenetic analysis of ribotypes from the TT-SSCP profiles (B). The lanes and the origin of
ribotypes are marked as follows: DS - soil DNA; DA - A. caliginosa cast DNA. Numbers on the gel
correspond to ribotypes on the phylogenetic tree. Ribotypes from SSCP profiles are marked in bold on
the tree; several ribotypes detected in the same band are marked on the phylogenetic tree with
numbers and small letters. Sequences with identity >99.5% were taken as belonging to a single OTU
and are shown as one ID. Numbers at nodes indicate bootstrap values greater than 50%.
A)
L-DS-8c AM989828
L-RS-9c AM989837
L-RA-5c AM989847
60
L-RL-1 AM989839
Bacillus sp. IDA3504 AJ544784
99 L-DS/RS-2b (AM989806, AM989807)
Bacillus megaterium AY030338
90 L-DS/RS-3a (AM989809, AM989809)
54
Bacillus licheniformis AF387514
94
L-RA-1 AM989840
99
98 Bacillus subtilis AY030331
L-DS-7b AM989825
L-DS-8b AM989827
94
L-RS-9b AM989836
52
L-RA-5b AM989846
99 L-DS-9 AM989829
97
L-RS-10 AM989838
87
Sporosarcina psychrophila D16277
Sporosarcina aquamarinus AF202056
52 Sporosarcina ureae AF202057
phylum Firmicutes
L-DS-7a AM989824
100
L-RA-4 AM989844
78
L-RS-8 AM989834
98
Paenibacillus mendelii AF537343
L-DS/RS-4b (AM989817, AM989818)
Paenibacillus kobensis D78471
99
78
63 Paenibacillus curdlanolyticus D78466
86
Paenibacillus larvae DQ079623
L-RS-7a AM989832
100
L-RA-3a AM989842
58
L-DS-6a AM989822
L-DS-6b AM989823
99
L-RS-7b AM989833
100
86
L-RA-3b AM989843
Dehalobacter restrictus U84497
L-DS/RS-3b (AM989811, AM989812)
67
Desulfosporosinus orientis Y11570
82
Desulfosporosinus auripigmenti AJ493051
100
62 Desulfosporosinus meridiei AF076247
Thermoactinomyces intermedius AF138734
L-RA-5a AM989845
100 L-DS-8a AM989826
70 L-RS-9a AM989835
Thermoactinomyces vulgaris AF138732
87
Polyangium vitellinum AJ233944
Nannocystis aggregans AJ233945
L-DS/RS-1 (AM989802, AM989803)
97
100
Chondromyces lanuginosus AJ233939
Chondromyces crocatus M94275
51
L-DS/RS-4c (AM989819, AM989820)
54
class Deltaproteobacteria
L-RS-6 AM989831
99
L-RA-2 AM989841
order Myxococcales
87
L-DS-5 AM989821
79
L-DS/RS-2a (AM989804, AM989805)
uncultured delta proteobacterium AY922176
L-RS-5 AM989830
65
L-DS/RS-3c (AM989813- AM989814)
64
99 L-DS/RS-4a (AM989815, AM989816)
0.05
B)
89
92
root
class Clostridia
class Bacilli
100
Figure S6. Images of SSCP profiles generated with Firmicutes-targeting primer set (A) and the
phylogenetic analysis of ribotypes from the TT-SSCP profiles (B). The lanes and the origin of ribotypes
are marked as follows: DS - soil DNA; RS - soil RNA; RL - L. terrestris cast RNA; RA - A. caliginosa
cast RNA. Numbers on the gel correspond to ribotypes on the phylogenetic tree. Ribotypes from SSCP
profiles are marked in bold on the tree; those amplified from RNA are shown in gray boxes. Several
ribotypes detected in the same band are marked on the phylogenetic tree with numbers and small
letters. Numbers at nodes indicate bootstrap values greater than 50%
A)
B)
V-DS/DL/DA-1b AM989858
uncultured bacterium AY212695
V-DS-7 AM989872
51
uncultured Verrucomicrobium clone Ac-EX-G12 AM266712
92
uncultured Verrucomicrobium clone Ef-EX-G10 AM266808
Verrucomicrobium spinosum X90515
95
V-DS/DL/DA-5c AM989868
V-DS/DL/DA-3b AM989863
65
uncultured Verrucomicrobium bacterium AJ132498
100
Chthoniobacter flavus AY388649
uncultured Verrucomicrobium bacterium EV101 AJ132479
97
V-DS/DL/DA-5b AM989867
99
uncultures Spartobacteria bacterium DQ088012
62
Akkermansia muciniphila AY271254
Candidatus Xiphinematobacter brevicolli AF217462
V-DL/DA-1/1 AM989860
93 V-DS/DL/DA-1c AM989859
98
uncultured bacterium AY212657
uncultured Verrucomicrobia clone Lt-GC-D6 AM266673
55
99 uncultured Verrucomicrobia clone Lt-EX-D10 AM266673
V-DS/DL/DA-2a AM989861
99
uncultured bacterium U81738
V-DS/DL/DA-5a AM989866
V-DS/DL/DA-6a AM989869
58
Opitutus terrae AJ229246
V-DS/DL/DA-9a AM989874
100
uncultured Verrucomicrobia bacterium AY622244
V-DS/DL/DA-4 AM989865
90
V-DS/DL/DA-1a, 3a, 9b AM989857
V-DS/DL/DA-6b AM989870
Planctomyces brasiliensis X81949
Blastopirellula marina X62912
74
56
Pirellula staleyi X81946
82
Gemmata obscuriglobus AJ231191
V-DS/DL/DA-2b AM989862
52
V-DS/DL-3c AM989864
Planctomyces limnophilus X62911
Planctomyces maris X62910
64
Parastreptomyces abscessus DQ000673
V-DS/DL/DA-8 AM989873
100
95 Streptomyces canescens Z76684
65
Streptomyces speibonae AF452714
Streptomyces albus AJ621602
V-DS/DL/DA-7a AM989871
Streptomyces virginiae D85121
72
99 Streptomyces lateritius AF454764
98
Planctomycetes
52
Verrucomicrobia
80
Actinobacteria
root
0.05
Figure S7. Images of SSCP profiles generated with Verrucomicrobia-targeting primer set (A) and the
phylogenetic analysis of ribotypes from the TT-SSCP profiles (B). The lanes and the origin of ribotypes
are marked as follows: DS - soil DNA; DL - L. terrestris cast DNA; DA - A. caliginosa cast DNA; M marker. Numbers on the gel correspond to ribotypes on the phylogenetic tree. Ribotypes from SSCP
profiles are marked in bold on the tree; several ribotypes detected in the same band are marked on the
phylogenetic tree with numbers and small letters. Sequences with identity >99.5% were taken as
belonging to as a single OTU and are shown as one ID. Numbers at nodes indicate bootstrap values
greater than 50%.
A)
B)
P-DS/DL/DA-1 AM989875
P-DS/DL/DA-21 AM989892
uuncultured planctomycete AB116422
Planctomyces brasiliensis X81949
P-DS/DL/DA-12,
19a AM989885
75
Planctomyces sp. X81954
99
Planctomyces maris X62910
P-DS/DL/DA-17 AM989888
P-DS/DL/DA-3 AM989877
P-DS/DL/DA-14-16 AM989887
uncultured sludge bacterium A40 AF234736
P-DS/DL/DA-6 AM989879
P-DS/DL/DA-9 AM989882
99
uncultured planctomycete AB116406
85
Pirellula staleyi X81946
P-DS/DL/DA-11a AM989883
P-DS/DL/DA-4, 5 AM989878
P-DS/DL/DA-20 AM989891
P-DS/DL/DA-7
AM989880
91
Gemmata obscuriglobus AJ231191
91
P-DS/DL/DA-13 AM989886
P-DS/DL/DA-8, 10 AM989881
P-DS/DL/DA-2 AM989876
P-DS/DL/DA-18 AM989889
Isosphaera pallida AJ231195
P-DL/DA-11b AM989884
P-DS/DL/DA-22 AM989923
P-DS/DL/DA-19b AM989890
Isosphaera sp. X81959
59
95
57
57
68
52
65
83
98
root
71
70
50
59
83
0.05
Figure S8. Images of SSCP profiles generated with Planctomycetes-targeting primer set (A) and the
phylogenetic analysis of ribotypes from the TT-SSCP profiles (B). The lanes and the origin of ribotypes
are marked as follows: DS - soil DNA; DL - L. terrestris cast DNA; DA - A. caliginosa cast DNA.
Numbers on the gel correspond to ribotypes on the phylogenetic tree. Ribotypes from SSCP profiles are
marked in bold on the tree; several ribotypes detected in the same band are marked on the
phylogenetic tree with numbers and small letters. Sequences with identity >99.5% were taken as
belonging to a single OTU and are shown as one ID. Numbers at nodes indicate bootstrap values
greater than 50%.