Supplementary Material
Amplicon library construction: We constructed amplicon libraries for bacterial 16S v3
through v5 domains (Bv3v5) and v4 through v6 domains (Bv6v4). The adapter and 16Sspecific sequences are shown in Table 1.
Each primer contains either of the A (5'-
CGTATCGCCTCCCTCGCGCCATCAG-3') or B (5'-CTATGCGCCTTGCCAGCCCGCTCAG-3') amplicon adapters followed by a 5 nt multiplex identifier (MID; barcode) and ends
with the 16S specific sequence. Bv6v4 amplicons are amplified with a pair of degenerate
primers (1064R and 518F) and the MID is present in both primers. Bv3v5 amplicons are
generated using a pool of two forward and three reverse primers and the MID is present in all
five oligonucleotides. All MIDs differ by at least two bases and contain no homopolymers.
The individual oligos are mixed in equal proportions to create F/R primer pools.
The
polymerase chain reaction mixture contains 1X Platinum HiFi Taq polymerase buffer, 1.6
units Platinum HiFi polymerase (Life Technologies, Carlsbad CA), 3.7 mM MgSO4, 200 uM
dNTPs (PurePeak polymerization mix, ThermoFisher, E. Providence RI), and 50 nM
combined primers. 5-25 ng of sample DNA is added to a master mix to a final volume of 100
ul and this is divided into three replicate 33 ul reactions. We include a no-template negative
control for each MID. Conditions are an initial denaturation at 94 oC for 3 minutes; 30 cycles
of 94oC for 30 seconds, 60oC for 45 seconds, and 72oC for 1 minute; and a final extension at
72oC for 2 minutes using an Applied Biosystems 2720 or 9700 cycler (Life Technologies).
The three replicates are pooled and we check 1 ul of the pool and the negative control on a
LabChip GX (Caliper, Hopkinton MA). We clean the reaction and remove products under
300 bp using Ampure beads at 0.75X volume (Beckman Coulter, Brea CA). The final
products are resuspended in 100 ul of 10mM Tris-EDTA, quantitated using PicoGreen
Quant-IT assay (LifeTechnologies), and assayed on a Bionanalyzer DNA1000 chip (Agilent
Technologies, Santa Clara, CA). We pool up to 40 amplicon libraries prior to emulsion PCR.
S-1
The emPCR, enrichment, and sequencing are done according to current Roche Titanium
amplicon sequencing protocols (Lib-A emPCR reagents, XLR sequencing reagents, two
region PicoTitre plate).
Image processing and signal calling are done using the Roche
amplicon-processing pipeline (version 2.53 for these datasets) with recursive phase correction
algorithm to maximize the number of long reads.
v3-v5
v4-v6
v6
pyrosequencing
clone
FISH
Table S1. Primers and probes used in the study.
Name
Sequence (5'-3')
Reference/Comment
Eub I
GCTGCCTCCCGTAGGAGT
(Daims et al., 1999)
Eub II
GCAGCCACCCGTAGGTGT
ibid
Eub III
GTCTGCCACCCGTAGGTGT
ibid
Arc 915
GTGCTCCCCCGCCAATTC
(Stahl and Amann, 1991)
Arc 344
TCGCGCCTGCTGCICCCCGT
(Raskin et al., 1994)
Aceto 125
TCCAAAGGGTAGGTTACCCA
(Gaidos et al., 2009)
Ssprill 572
TTCCAGCCTACGCATCCTTT
ibid
Scurvum 731 GTATCATCCCAGCAGATCGC
ibid
Palud 672
CCGCTACACAACACATTCCG
ibid
Non 338
ACTCCTACGGGAGGCAGC
(Amann et al., 1990)
23FLP (A)
GCGGATCCGCGGCCGCTGCAGAYC- (Barns et al., 1994)
TGGTYGATYCTGCC
27F (B)
AGAGTTTGATCCTGGCTCAG
(Lane, 1991)
751F (A)
CCGACGGTGAGRGRYGAA
(Baker et al., 2003)
805R (U)
GACTACCCGGGTATCTAATCC
(Amann et al., 1995)
976R (A)
YCCGGCGTTGAMTCCAATT
(Baker et al., 2003)
1391R (B)
GACGGGCGGTGTGTRCA
(Barns et al., 1994)
1406R (A)
ACGGGCGGTGWGTRCAA
(Baker et al., 2003)
1544R (B)
CCCGGGATCCAAGCTTAGAAAGGA (Lane, 1991)
341F2
CCTACGGGNGGCWGCAG
this work
341F3
TCTACGGAAGGCTGCAG
this work
785F-a
GGATTAGATACCC
informatics landmark only
926R1
CCGTCAATTCNTTTRAGT
this work
926R3
CCGTCAATTTCTTTGAGT
this work
926R4
CCGTCTATTCCTTTGANT
this work
518F
CCAGCAGCYGCGGTAAN
this work
565F-a
TGGGCGTAAAG
informatics landmark only
1064R
CGACRRCCATGCANCACCT
(Huber et al., 2007)
967F
CAACGCGAAGAACCTTACC
(Huber et al., 2007)
1046R
CGACAGCCATGCANCACCT
(Huber et al., 2007)
B = Bacterial domain
A=Archaeal domain
U=Universal
S-2
v6
v4-v6
v3-v5
Table S2. Statistics of pyrosequencing libraries
Depth1
Filtered
Unique
Median
2
3
4
Sample
(meters) Reads
reads
sequences
length
A1
284
21034
21013
2682
416
A2
331
20549
20519
2774
415
A3
379
21975
21946
3221
415
B1
284
22059
22052
3366
416
B2
336
21508
21505
3290
416
B3
377
20798
20714
3094
416
B4
388
22970
22837
3243
415
Total lake
--- 150893 150586
51135
416
Outlet
0
19646
19644
2950
415
A1
284
24548
24420
2814
483
A2
331
23937
23518
4543
484
A3
379
18882
18487
2687
484
B1
284
18534
18402
1744
483
B2
336
19392
19364
2349
478
B3
377
21294
20801
2112
479
B4
388
23702
22957
2524
484
Total lake
--- 150289 147979
69729
484
Outlet
0
17916
17838
4931
486
B1
284
12197
10532
1180
98
B3
377
8143
6978
775
98
B4
388
13947
11628
1308
97
Kverkfjallalón
4
9872
8030
1089
97
Outlet
0
14910
13620
2677
100
West lake
412
9352
8984
1179
60
1
Below ice surface
2
After VAMPS pipeline processing
3
After removal of chimeras
4
After pre-clustering step (up to 2 differences for v3-v5 and 4 for v4-v6)
S-3
Sample
A1
A2
A3
B1
B2
B3
B4
Mean
% Var
pH
T(ºC)
5.75 (8.8)
5.81 (11.6)
5.45 (5.6)
5.19 (12.9)
5.25 (14.2)
5.22 (13.6)
5.21 (8.9)
-----
Table S3. East Skaftárketill lake geochemistry (concentrations in ppm)
CO2
SiO2
Na
K
Ca
Mg
Fe
Al
Sr
B
93.83 99.91 30.22 3.41 3.43 0.241 0.239 0.082
82.03 110.18 30.14 3.44 3.47 0.257 0.185 0.133
122.49 101.74 30.65 3.46 3.50 0.250 0.262 0.093
106.28 99.61 30.04 3.39 3.46 0.245 0.161 0.083
121.47 98.32 29.75 3.35 3.42 0.247 0.235 0.102
96.16 97.94 29.60 3.45 3.45 0.239 0.179 0.077
112.89 99.23 30.44 3.24 4.74 0.305 0.332 0.086
105.02 99.56 30.12 3.38 3.64 0.255 0.228 0.094
14.4
1.3
1.2 2.2 13.4
9.0 26.0 20.4
S-4
0.0064
0.0059
0.0063
0.0049
0.0049
0.0053
0.0089
0.0061
23.2
0.515
0.515
0.530
0.518
0.513
0.510
0.525
0.517
1.0
F
Cl
SO4
H2S
0.113
0.111
0.122
0.107
0.106
0.106
0.114
0.111
5.1
10.45
10.41
12.28
10.16
10.16
9.97
10.66
10.58
7.4
0.30
2.74
4.71
0.29
1.08
1.03
1.43
1.66
95.4
15.90
19.99
11.54
14.36
10.30
15.04
25.50
16.09
32.3
Table S4. Growth results for 3°C enrichments
Aerobic
Anaerobic
Sample
162-1
WO2 A-J C-J YPS Toga WN2
A1
+
+
+
+ +
+
A2
+
+
+
+
A3
+*
+
+ + +
+
B1
+
+
+
+

B2
+
+
+
+
+* +
B3
+
+ +
+
B4
+
+
+ + +
+
borehole1
jökulhlaup
+
+
+ + + +
+
Kverkfjallalón + +
+
+
+
+
+
1
Negative control
R2A medium used instead
Denotes strain isolated by serial dilution in liquid cultures at 3°C.
Denotes strain isolated as above and ribotyped.
*Denotes clone library constructed from enrichment.
S-5
Table S5. OTUs from 60°and 80°C enrichments of east Skaftárketill lake sample B4
Clones GenBank acc.
Closest match (% identity)
Environment
1
EU299434.1 Uncultured Pedomicrobium clone
soil
GASP KC1W2 (96)
1
AY684097.1 Uncultured Thermoanaerobacteriaceae hot spring
clone MRE50b20 (85)
38
AF255600.1 Uncultured bacterium SRI-280 (99)
Iceland hot spring
3
AF2555602.1 Nitrospira sp. SRI-237 (98)
Iceland hot spring
5
EY409943.1 Caloramator australicus strain KCTC
Great Artesian
5601 (98)
Basin of Australia
1
X97693.1
Pedomicrobium australicum (96)
Great Artesian
Basin of Australia
1
AP009389.1 Pelotomaculum thermopropionicum SI anaerobic sludge
(96)
reactor
1
FJ748765.1
Thermoanaerobacter sp. RH0806 (80) hot spring
S-6
v6 (10%)
v4-v6 (3%)
v3-v5 (3%)
Table S6. Estimated community richness and evenness from pyrosequence libraries (95% confidence intervals)
Locus Sample Sobs
ACE
Chao1
Shannon H
Simpson D
ACE*
A1
63 794 (565,1127)
228 (130,470)
1.15 (1.14,1.16) 0.379 (0.375,0.382) 73  24
A2
56 762 (564,1035)
223 (117,513)
1.00 (0.99,1.02) 0.491 (0.485,0.498) 74  28
A3
63 340 (253,466)
230 (124,520)
1.14 (1.12,1.15) 0.427 (0.422,0.431) 72  17
B1
74 627 (493,804)
215 (135,401)
1.49 (1.48,1.50) 0.287 (0.284,0.291) 89  22
B2
64 192 (144,267)
126 (88,222)
1.44 (1.42,1.45) 0.310 (0.306,0.313) 69  13
B3
96 590 (463,763)
273 (179,475)
1.51 (1.49,1.52) 0.305 (0.301,0.309) 107  22
B4
92 382 (282,535)
223 (156,358)
1.53 (1.51,1.54) 0.285 (0.282,0.288) 99  18
summed 321 2384 (2053,2777) 1140 (857,1572) 1.39 (1.38,1.40) 0.325 (0.324,0.326) ND
hlaup1
156 330 (253,468)
534 (445,649)
0.38 (0.36,0.40) 0.901 (0.895,0.907) ND
A1
329 2338 (2041,2686) 1183 (889,1632) 1.48 (1.46,1.50) 0.325 (0.322,0.329) 356  32
A2
575 3029 (2721,3381) 1583 (1323,1932) 1.64 (1.62,1.66) 0.337 (0.332,0.341) 616  34
A3
400 2638 (2326,3001) 1447 (1113,1938) 1.76 (1.74,1.79) 0.285 (0.281,0.289) 421  28
B1
208 1972 (1685,2314) 747 (537,1118)
1.53 (1.51,1.55) 0.333 (0.328,0.338) 228  31
B2
267 1555 (1330,1827) 759 (584,1029)
1.59 (1.57,1.61) 0.324 (0.319,0.330) 283  24
B3
337 2630 (2278,3046) 1150 (881,1552) 1.72 (1.70,1.74) 0.316 (0.311,0.321) 363  31
B4
410 2835 (2506,3217) 1378 (1081,1808) 1.79 (1.77,1.81) 0.263 (0.260,0.267) 448  40
summed 1500 5695 (5352,6068) 3255 (2960,3608) 1.76 (1.75,1.77) 0.283 (0.282,0.285) ND
hlaup1
725 2038 (1863,2240) 1523 (1333,1771) 2.02 (1.98,2.06) 0.462 (0.453,0.471) ND
B1
82 189 (152,245)
121 (99,173)
1.25 (1.23,1.28) 0.434 (0.426,0.443) ND
B3
73 224 (174,299)
137 (100,222)
1.36 (1.33,1.40) 0.376 (0.369,0.385) ND
B4
90 294 (230,387)
184 (133,295)
1.09 (1.07,1.12) 0.485 (0.477,0.493) ND
summed 154 364 (304,449)
253 (208,335)
1.24 (1.21,1.25) 0.437 (0.432,0.442) ND
1
hlaup
445 834 (760,926)
703 (619,826)
3.13 (3.09,3.16) 0.156 (0.151,0.162) ND
2
Kverk
67 200 (155,269)
110 (85,167)
0.69 (0.65,0.72) 0.762 (0.750,0.774) ND
3
west
402 1467 (1293,1674) 983 (807,1237)
1.85 (1.80,1.90) 0.422 (0.411,0.434) ND
1
2
3
jökulhlaup sample
Kverkfjallalón subaerial lake
west Skaftárketill subglacial lake
S-7
Supplementary Figures
Figure S1. Temperature profiles in the west (grey) and east (black) Skaftárkatlar lakes with
depths reported relative to the ice-lake interface. The east lake is shallower than the west
lake at the borehole locations. Dotted and solid lines are for profiles beneath boreholes A
and B, respectively, in the east lake. The dashed line is the maximum density temperature of
pure water. Circles mark sampling depths, with temperatures chosen to illustrate from which
borehole the samples were obtained.
S-8
Figure S2. Concentrations of major dissolved solids in the west (1 sample) and east (7
samples) Skaftárkatlar lakes. Two major constituents, CO2 (averaging 1300 and 105 ppm in
west and mean east lakes, respectively) and H2S (averaging 37 and 16 ppm) are not shown
because they may vary due to degassing during sample retrieval. The dendogram was
constructed by single linkage of euclidian distances between samples.
S-9
Figure S3
Unrooted ARB tree of isolates from east Skaftárketill lake, the Kverkfjallalón
subaerial lake, and a sample of a jökulhlaup in the Skaftá river. For the Skaftárketill isolates,
the sample name and enrichment conditions are suffixed.
S-10
Figure S4. Rank abundance of OTUs (3% clustering criterion) in v4-v6 and v3-v5
pyrosequencing libraries from east lake samples. The dashed line is an approximation to the
v4-v6 trends that is reproduced in the v3-v5 plot to aid comparison.
S-11
Figure S5. Rarefaction curves for v3-v5 and v4-v6 pyrosequence libraries generated from
samples of the east Skaftárkatlar lake. Note the log-log scale. OTUs are defined by a 3%
average difference cutoff.
S-12
Figure S6. Fractional abundance of v3-v5 and v4-v6 reads assigned to the same taxonomic
groups. Squares are assignments of reads based on common taxonomic affiliations at the
genus level. Diamonds are assignments based on a clustering of representative sequences
from independently determined v3-v5 and v4-v6 OTUs (cutoff of 11%). The dashed line is
equality, and the dot-dashed lines are 2-sigma standard deviations due to Poisson statistics
alone.
S-13
Figure S7. Unrooted UPGMA trees (left) and majority consensus trees from 1000 bootstrap
replicates (right) based on Bray-Curtis distances between individual v3-v5 libraries (top) and
v4-v6 libraries.
S-14
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S-15