Shifts in soil microorganisms in response to warming are consistent across a range of Antarctic environments

Supplementary Material

Etienne Yergeau, Stef Bokhorst, Sanghoon Kang, Jizhong Zhou, Charles W. Greer, Rien Aerts, George A. Kowalchuk

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Table S1: qPCR primers and conditions used

Target / Enzyme / Primers / Anneal. Tº. / Reading Tº / Standard / Standard curve R2 / Standard curve efficiency / Ct (gene copies1) NTC / Ref.
Ribosomal genes
Archaeal 16S / - / A517F/UA1204R / 55°C / 82°C / archaeal fosmid / 0.996 / 0.723 / 34.33 (1.21×103) / (Baker et al., 2003)
Bacterial 16S / - / Eub338/Eub518 / 53ºC / - / unidentified 16S clone / 0.999 / 0.927 / 27.92 (2.63×106) / (Fierer et al., 2005)
Fungal 18S / - / Fung5f/FF390r / 48ºC / - / unidentified 18S clone / 0.997 / 0.947 / 27.86 (4.50×105) / (Lueders et al., 2004)
Functional genes
Archaeal amoA / ammonia monooxygenase / amoA19F/CrenamoA616r48x / 50ºC / - / unidentified amoA clone / 0.999 / 0.960 / 30.13 (2.01×103) / (Leininger et al., 2006)
Bacterial amoA / ammonia monooxygenase / amoA-1F/amoA-2R-TC / 57°C / 81ºC / Nitrosomonas europaea / 0.994 / 0.863 / 29.67 (1.31×104) / (Nicolaisen and Ramsing, 2002)
chiA / Chitinase / GA1F/GA1R / 63ºC / - / Streptomyces sp. / 0.998 / 0.908 / 29.67 (5.78×104) / (Williamson et al., 2000)
mcrA / methyl-coenzyme M reductase / mcraF/mcraR / 52ºC / 81ºC / unidentified mrcA clone / 0.992 / 0.900 / 28.00 (4.14×104) / (Radl et al., 2007)
nirK / Cu-containing nitrite reductase / F1aCu/R3Cu / 57ºC / 85ºC / Pseudomonas sp. / 0.994 / 0.733 / 34.07 (7.44×104) / (Throbäck et al., 2004)
nirS / cd1-containing nitrite reductase / cd3aF/R3cd / 57°C / 83ºC / Pseudomonas fluorescens / 0.998 / 0.790 / 33.92 (4.76×106) / (Throbäck et al., 2004)
pmoA / particulate methane monooxygenase / mb661/A189 / 55ºC / 82ºC / Methylocystis parvus / 0.999 / 0.815 / 26.02 (2.75×105) / (Kolb et al., 2003)

1Gene copies in NTC: same calculations as for samples, so values are equivalent to gene copies per gram of soil

Information for standard curves is based on the average of 3-4 different runs.

Table S2: Pyrosequencing primers used

Set # / Forward primer (5’-3’) / Reverse primer (5’-3’)
MID1-A / CGTATCGCCTCCCTCGCGCCATCAGACGAGTGCGTGAGTTTGATYMTGGCTC / CTATGCGCCTTGCCAGCCCGCTCAGATTACCGCGGCTGCTGGC
MID2-A / CGTATCGCCTCCCTCGCGCCATCAGACGCTCGACAGAGTTTGATYMTGGCTC / CTATGCGCCTTGCCAGCCCGCTCAGATTACCGCGGCTGCTGGC
MID3-A / CGTATCGCCTCCCTCGCGCCATCAGAGACGCACTCGAGTTTGATYMTGGCTC / CTATGCGCCTTGCCAGCCCGCTCAGATTACCGCGGCTGCTGGC
MID4-A / CGTATCGCCTCCCTCGCGCCATCAGAGCACTGTAGGAGTTTGATYMTGGCTC / CTATGCGCCTTGCCAGCCCGCTCAGATTACCGCGGCTGCTGGC
MID5-A / CGTATCGCCTCCCTCGCGCCATCAGATCAGACACGGAGTTTGATYMTGGCTC / CTATGCGCCTTGCCAGCCCGCTCAGATTACCGCGGCTGCTGGC
MID6-A / CGTATCGCCTCCCTCGCGCCATCAGATATCGCGAGGAGTTTGATYMTGGCTC / CTATGCGCCTTGCCAGCCCGCTCAGATTACCGCGGCTGCTGGC
MID7-A / CGTATCGCCTCCCTCGCGCCATCAGCGTGTCTCTAGAGTTTGATYMTGGCTC / CTATGCGCCTTGCCAGCCCGCTCAGATTACCGCGGCTGCTGGC
MID8-A / CGTATCGCCTCCCTCGCGCCATCAGCTCGCGTGTCGAGTTTGATYMTGGCTC / CTATGCGCCTTGCCAGCCCGCTCAGATTACCGCGGCTGCTGGC
MID9-A / CGTATCGCCTCCCTCGCGCCATCAGTAGTATCAGCGAGTTTGATYMTGGCTC / CTATGCGCCTTGCCAGCCCGCTCAGATTACCGCGGCTGCTGGC
MID1-B / CTATGCGCCTTGCCAGCCCGCTCAGACGAGTGCGTGAGTTTGATYMTGGCTC / CGTATCGCCTCCCTCGCGCCATCAGATTACCGCGGCTGCTGGC
MID2-B / CTATGCGCCTTGCCAGCCCGCTCAGACGCTCGACAGAGTTTGATYMTGGCTC / CGTATCGCCTCCCTCGCGCCATCAGATTACCGCGGCTGCTGGC
MID3-B / CTATGCGCCTTGCCAGCCCGCTCAGAGACGCACTCGAGTTTGATYMTGGCTC / CGTATCGCCTCCCTCGCGCCATCAGATTACCGCGGCTGCTGGC
MID4-B / CTATGCGCCTTGCCAGCCCGCTCAGAGCACTGTAGGAGTTTGATYMTGGCTC / CGTATCGCCTCCCTCGCGCCATCAGATTACCGCGGCTGCTGGC
MID5-B / CTATGCGCCTTGCCAGCCCGCTCAGATCAGACACGGAGTTTGATYMTGGCTC / CGTATCGCCTCCCTCGCGCCATCAGATTACCGCGGCTGCTGGC
MID6-B / CTATGCGCCTTGCCAGCCCGCTCAGATATCGCGAGGAGTTTGATYMTGGCTC / CGTATCGCCTCCCTCGCGCCATCAGATTACCGCGGCTGCTGGC
MID7-B / CTATGCGCCTTGCCAGCCCGCTCAGCGTGTCTCTAGAGTTTGATYMTGGCTC / CGTATCGCCTCCCTCGCGCCATCAGATTACCGCGGCTGCTGGC
MID8-B / CTATGCGCCTTGCCAGCCCGCTCAGCTCGCGTGTCGAGTTTGATYMTGGCTC / CGTATCGCCTCCCTCGCGCCATCAGATTACCGCGGCTGCTGGC
MID9-B / CTATGCGCCTTGCCAGCCCGCTCAGTAGTATCAGCGAGTTTGATYMTGGCTC / CGTATCGCCTCCCTCGCGCCATCAGATTACCGCGGCTGCTGGC

Pink: adapter, green: sequencing key, blue: multiplex identifier, red: sequence-specific primer.

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Table S3:Mean (standard deviation) bacterial phylum/classe relative abundance in 16S rRNA gene libraries for control and OTC plots at the Falkland, Signy and AnchorageIslands.

#seqs / Acido. / Actino. / Kted. / Verru. / Alpha / Others / uncl.
FI / Vegetated / Control / 13,991 / 28.0 (11.4) / 35.0 (7.2) / 0.6
(0.6) / 1.0
(0.6) / 27.6 (14.5) / 2.3 (0.9) / 5.5
(1.7)
OTC / 12,540 / 23.6 (7.8) / 37.3 (9.4) / 0.7
(0.5) / 1.2
(0.8) / 30.1 (14.6) / 2.3 (0.5) / 4.9
(1.3)
Fell-Field / Control / 3,569 / 29.8 (3.8) / 33.3 (5.3) / 0.0
(0.0) / 0.4
(0.2) / 19.4 (1.9) / 4.3 (0.8) / 12.9
(3.1)
OTC / 2,543 / 20.3 (10.3) / 46.7 (8.8) / 0.0
(0.1) / 0.4
(0.1) / 15.4 (3.4) / 4.3 (1.1) / 12.8
(2.8)
SI / Vegetated / Control / 13,452 / 39.2 (9.3) / 25.6 (4.0) / 5.3
(3.4) / 1.0
(1.6) / 16.5 (4.0) / 4.4 (2.0) / 8.0
(3.9)
OTC / 11,635 / 30.0 (12.3) / 29.0 (8.9) / 6.9
(6.7) / 1.5
(2.2) / 19.0 (3.8) / 5.0 (2.5) / 8.6
(5.0)
Fell-Field / Control / 7,196 / 20.9 (7.0) / 28.6 (6.7) / 6.8
(3.0) / 0.7
(0.7) / 16.7 (4.6) / 5.2 (1.5) / 21.1
(7.6)
OTC / 8,603 / 15.5 (3.6) / 31.9 (7.4) / 9.4
(6.1) / 0.7
(0.6) / 17.1 (4.1) / 4.5 (1.2) / 21.0
(6.3)
AI / Vegetated / Control / 6,116 / 18.4 (11.1) / 23.5 (1.0) / 11.9
(4.6) / 2.0
(1.5) / 19.2 (7.1) / 5.0 (0.9) / 19.9
(4.7)
OTC / 10,919 / 11.3 (7.9) / 17.1 (2.5) / 14.2
(13.4) / 2.5
(1.6) / 30.9 (12.4) / 6.4 (2.0) / 17.7
(3.3)
Fell-Field / Control / 5,615 / 10.2 (8.2) / 28.5 (11.7) / 21.6
(10.7) / 1.7
(1.6) / 12.0 (7.4) / 7.5 (5.6) / 18.4
(2.9)
OTC / 7,139 / 7.3 (3.4) / 32.0 (12.6) / 14.5
(11.2) / 2.1
(2.0) / 16.9 (7.7) / 9.7 (2.5) / 17.5
(4.1)
all / - / Control / 49,939 / 24.6 (12.9) / 29.4 (7.8) / 7.7
(9.0) / 1.2
(1.2) / 19.3 (10.1) / 4.6 (3.0) / 13.3
(7.5)
OTC / 53,379 / 18.3 (10.9) / 31.5 (11.6) / 7.6
(9.5) / 1.5
(1.5) / 22.9 (11.4) / 5.2 (3.0) / 12.9
(7.1)

Acido: Acidobacteria; Actino: Actinobacteria; Kted: Ktedonobacteria (this is a class; not classified at the phylum level); Verru: Verrucomicrobia; Alpha: Alphaproteobacteria; uncl: unclassified

Others: All groups that represented less than 1% of the total reads (BRC1, Bacteroidetes, Chloroflexi, Cyanobacteria, Deinococcus-Thermus, Firmicutes, Gemmatimonadetes, Nitrospira, OD1, OP10, Planctomycetes, TM7, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria.

Table S4: Mean number of different variants detected on the GeoChip for selected genes of the N- and C-cycles for control and OTC plots at the Falkland (FI), Signy (SI) and Anchorage (AI) Islands.

amoA / cell. / chit. / laccase / nifH / nirK / nirS / nosZ / pmoA / urease
FI / V / Control / 2.1 (0.7) / 8.1 (9.9) / 6.6 (8.7) / 3.0 (5.0) / 8.0 (8.9) / 4.3 (3.5) / 1.7 (2.5) / 4.1 (3.1) / 3.1 (1.8) / 14.3 (15.5)
OTC / 0.4 (0.5) / 0.0 (0.0) / 1.8 (1.2) / 0.1 (0.4) / 0.8 (0.7) / 1.8 (1.3) / 0.1 (0.4) / 0.9 (0.8) / 0.9 (0.8) / 1.3 (1.0)
FF / Control / 4.3 (2.5) / 11.7 (4.2) / 7.7 (0.6) / 5.0 (1.7) / 14.0 (3.0) / 3.7 (0.6) / 2.3 (0.6) / 3.3 (0.6) / 4.0 (1.0) / 22.0 (7.5)
OTC / 0.0 (0.0) / 4.0 (2.8) / 0.5 (0.7) / 0.5 (0.7) / 0.0 (0.0) / 1.5 (2.1) / 0.0 (0.0) / 0.5 (0.7) / 1.0 (1.4) / 1.5 (2.1)
SI / V / Control / 1.5 (0.5) / 5.0 (3.5) / 3.8 (2.9) / 1.0 (1.5) / 5.0 (3.2) / 3.2 (1.0) / 1.0 (0.9) / 3.5 (1.4) / 2.8 (1.2) / 9.0 (5.8)
OTC / 1.6 (0.9) / 2.6 (1.1) / 1.4 (0.5) / 0.2 (0.4) / 1.6 (1.1) / 3.0 (0.0) / 0.2 (0.4) / 3.0 (0.0) / 3.0 (0.7) / 4.2 (0.8)
FF / Control / 5.4 (2.2) / 14.4 (6.6) / 7.6 (3.4) / 4.8 (3.9) / 10.4 (3.3) / 4.0 (1.0) / 2.6 (2.7) / 3.8 (1.1) / 5.8 (1.3) / 20.0 (7.7)
OTC / 1.5 (0.7) / 2.5 (2.1) / 1.0 (0.0) / 0.0 (0.0) / 1.5 (0.7) / 2.5 (0.7) / 0.0 (0.0) / 2.5 (2.1) / 2.0 (1.4) / 2.0 (1.4)
AI / V / Control / NA / NA / NA / NA / NA / NA / NA / NA / NA / NA
OTC / 1.0 (0.0) / 5.5 (5.6) / 2.5 (1.5) / 0.0 (0.6) / 2.0 (1.2) / 2.5 (0.6) / 0.0 (0.0) / 2.0 (1.2) / 3.5 (0.6) / 4.5 (2.1)
FF / Control / 1.7 (0.6) / 5.0 (1.7) / 1.3 (0.6) / 0.0 (0.0) / 1.7 (1.5) / 3.0 (0.0) / 1.0 (1.0) / 2.3 (0.6) / 1.7 (0.6) / 3.3 (2.1)
OTC / 1.2 (0.5) / 7.0 (2.8) / 3.8 (1.0) / 1.2 (0.5) / 1.8 (1.4) / 3.0 (0.0) / 0.6 (0.5) / 3.2 (0.5) / 3.2 (0.5) / 7.0 (1.4)
All / - / Control / 2.9 (2.0) / 8.7 (7.1) / 5.6 (5.3) / 2.8 (3.7) / 7.7 (6.3) / 3.7 (1.9) / 1.7 (1.9) / 3.6 (1.9) / 3.5 (1.8) / 13.8 (11.1)
OTC / 0.9 (0.8) / 3.0 (3.5) / 2.0 (1.4) / 0.4 (0.6) / 1.3 (1.1) / 2.4 (1.1) / 0.2 (0.4) / 2.0 (1.3) / 2.1 (1.3) / 3.4 (2.5)

Cell.: cellulase; Chit.: chitinase; V: vegetated; FF: fell-field; NA: not available

Table S5: Mean abundance of bacterial and archaeal ammonia monooxygenase (amoA), chitinase (chiA), methyl-coenzyme A reductase (mcrA), nitrite reductase (nirK and nirS and particulate methane monooxygenase (pmoA) in real-time PCR assays for control and OTC plots at the Falkland (FI), Signy (SI) and Anchorage (AI) Islands.

Bact amoA / Arch amoA / chiA / mcrA / nirK / nirS / pmoA
105 copies g-1 soil FW / 103 copies g-1 soil FW / 105 copies g-1 soil FW / 104 copies g-1 soil FW / 107 copies g-1 soil FW / 107 copies g-1 soil FW / 105 copies g-1 soil FW
FI / V / Control / 0.8 (0.3) / 13.2 (12.9) / 20.8 (19.0) / 0.2 (0.2) / 5.4 (5.3) / 6.7 (4.3) / 2.9 (0.5)
OTC / 0.7 (0.4) / 7.6 (9.4) / 19.8 (21.5) / 0.3 (0.5) / 8.9 (13.4) / 6.1 (4.4) / 2.2 (0.5)
FF / Control / 1.0 (0.5) / 17.6 (17.8) / 284.7 (169.3) / 2.6 (0.7) / 33.2 (30.0) / 8.0 (3.3) / 2.4 (0.8)
OTC / 1.9 (1.6) / 24.6 (4.9) / 368.6 (87.7) / 3.0 (1.4) / 41.0 (19.9) / 8.9 (6.5) / 2.0 (0.3)
SI / V / Control / 0.4 (0.3) / 0.9 (1.6) / 0.2 (0.1) / 8.2 (5.8) / 0.1 (0.1) / 1.1 (0.7) / 0.2 (0.1)
OTC / 0.8 (0.6) / 0.1 (0.1) / 0.4 (0.3) / 8.0 (5.6) / 0.1 (0.1) / 2.6 (1.7) / 0.3 (0.2)
FF / Control / 3.6 (6.5) / 15.3 (8.7) / 3.9 (7.4) / 2.7 (1.7) / 5.6 (8.5) / 8.6 (9.8) / 1.4 (1.0)
OTC / 0.2 (0.2) / 6.4 (1.7) / 0.4 (0.4) / 0.9 (1.1) / 0.2 (0.2) / 2.2 (1.7) / 0.7 (0.4)
AI / V / Control / 3.5 (1.2) / 0.3 (0.5) / 3.2 (1.7) / 31.8 (31.5) / 0.1 (0.0) / 1.4 (1.2) / 1.1 (0.3)
OTC / 10.3 (6.4) / 2.8 (2.8) / 8.9 (5.6) / 198.5 (324.4) / 5.0 (NA) / 3.5 (2.5) / 2.0 (0.6)
FF / Control / 7.8 (3.1) / 14.4 (11.0) / 17.3 (10.9) / 4.9 (2.5) / NA / 11.6 (11.0) / 2.9 (0.8)
OTC / 11.5 (6.7) / 24.1 (31.5) / 25.4 (28.1) / 21.7 (25.4) / NA / 7.2 (5.0) / 2.6 (1.0)

Bact: bacterial; Arch: archaeal; V: vegetated; FF: fell-field; NA: not available

Fig. S1: Map of the sampling locations

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