Supporting Information

Murray et al. 10.1073/pnas.1208607109

SI Materials and Methods
Dissolved Inorganic Carbon Determinations. Dissolved inorganic

carbon (DIC) concentration for the 2005 Lake Vida brine (LVBr) was determined by infrared gas chromatography on acid-sparged samples following calibration with bicarbonate standards. DIC stable carbon isotope samples for 2005 LVBr were acidied with phosphoric acid (15 molL1) and the liberated CO2 was cryogenically separated on a vacuum extraction line, with DIC concentrations measured via digital manometer. Samples were analyzed on a dual inlet Thermo Finnigan Delta Plus XL mass spectrometer. Samples for DIC concentration and isotope analyses collected in 2010 were analyzed using a method closely following that of Salata et al. (1). In this approach, 100 L of LVBr were injected into evacuated 10-mL serum vials containing 100 L of concentrated H3PO4 (100%). After > 24 h of equilibration, vials were brought to atmospheric pressure with pure N2 and 150 L of the headspace was analyzed using a TraceGas System interfaced to an Isoprime mass spectrometer. Values are reported in notation relative to Vienna Pee Dee Belemnite (VPDB). The mass spectrometer has an analytical precision of 0.1. No mineral phase fractionation factors were applied. C-isotope values are reported with respect to the international standard, VPDB.
Inorganic Nitrogen and Sulfur Analyses. In addition to dissolved nutrient determinations described in the main text for samples collected in both expeditions, we determined oxidized intermediates of sulfur and isotopic compositions of N and O (from nitrate), N from ammonium, and S for the LVBr samples collected in 2010. The isotopic composition of nitrate was determined by steam distillation and microbial reduction method (2). The isotopic composition of ammonium was analyzed using the acidied disk method described by Holmes et al. (3). N and O isotope values determined using an Elementar Isoprime are reported with respect to the international standards Air and Vienna Standard Mean Ocean Water (VSMOW), respectively. Sulfur intermediate analyses were performed on samples preserved in a gas-tight glass syringe that was ash-frozen and kept at 78 C wrapped in foil until analysis on a Dionex Ion Chromatograph System (IC-2000) and were separated using an IonPac AS17-C column with KI eluent to separate different sulfur species. To determine sulde levels, samples were measured using CdCl2 precipitation and sulfur (34S) isotope values measured on a Finnigan Mat 252. Specically, LVBr was loaded into 60-mL syringes with 10 mL of 1.5 M CdCl2 solution to precipitate any free sulde as cadmium-sulde (CdS). LVBr syringe samples were rst ltered onto 0.4-m glass ber lters to trap any precipitated CdS and any particulates in the brine. Sulfate was recovered from the LVBr ltrate by bringing the brine sample to pH 3 and adding an excess of 0.2 M Ba(NO3)2 solution to precipitate dissolved sulfate as BaSO4. The BaSO4 was rinsed and centrifuged multiple times with ultrapure water to remove chlorine, and then dried overnight at 80 C before isotopic analysis. The ltered residue from LVBr samples was rst reacted with hot 6N HCl for 3 h in a N2-purged ask, following methods of Brchert and Pratt (4). Flowing N2 carried evolved H2S, from monosulde decomposition, through a buffer solution (0.1 M citrate solution adjusted to pH 4) into a 0.1 M AgNO3 solution where it was precipitated as Ag2S. The Ag2S precipitate was then ltered, rinsed, and dried onto a baked 0.22-m quartz-ber lter that was saved for isotopic analyses. Finally, the remaining acid-insoluble residue was reacted with mixture of 40 mL of 12 N HCl and 40 mL of 1.0 M chromous chloride (CrCl26H2O) in a N2-purged extraction ask and
Murray et al. www.pnas.org/cgi/content/short/1208607109

evolved H2S from any polysuldes was precipitated as Ag2S (see description above). No measurable sulde was recovered from either the mono- or polysulde extractions in any of the LVBr samples. Sulfur (34S) isotope values were measured (on a ViennaCanyon Diablo Troilite scale) using a Carlo Erba Elemental Analyzer (EA1110) coupled to a Finnigan Mat 252 isotope ratio mass spectrometer via a ConFlo II split interface in the Stable Isotope Research Facility at Indiana University. Analytical reproducibility was better than 0.13 (1 ) for standards and duplicate samples.
Elemental Analysis. All sample and standard preparation was performed under class 100 clean-room conditions using trace metal clean techniques. The 2005 LVBr samples were acidied to pH < 2 with ultra-pure nitric acid (HNO3), stored for > 2 wk, and diluted to a salinity of < 1 with 1% HNO3 (prepared with > 18 m ultrapure water) before analysis. Isobaric interferences were resolved using multiple resolution settings (nominally m/m 400, 4,000, and 10,000). The instrumental response was quantied, after normalization to an indium internal standard, using external standards (NIST traceable). Multiple method blanks and test standards (Environmental Protection Agency drinking water Quality Assurance Protocol solutions) were analyzed for quality control purposes. Brine samples collected in 2010 were analyzed for total and dissolved (<0.2 m, Steriltech silver membrane) elements. Triplicate total samples were prepared by nitric and hydrouoric acid digestion using a block heater and microwave digestion. Both total and dissolved samples were diluted with 1% HNO3 spiked with an indium internal standard. These samples were analyzed by high-resolution inductively coupled plasma mass spectrometry at the University of Tasmania under similar conditions to the 2005 samples with the exception that the guard electrode was deactivated to minimize isobaric interferences on some isotopes (e.g., 95Mo16O on 111Cd; following ref. 5). Quantitation of these samples was also by external calibration with NIST traceable standards (Gaithersburg, MD). The method accuracy was veried through the analysis of the NIST 1640 Trace Elements in Natural Water standard reference material. Samples analyzed by the total recoverable acid digestion and dissolved analytical methods were comparable. This result is likely because of the high fractional solubility of elements in the brine samples. Volatile Hydrocarbon and Dissolved Gas (N2O and H2) Analyses. The procedures for analysis of the 2010 LVBr samples included analyses to detect a broader spectrum of gases than for the 2005 samples (including hydrogen) in addition to a different approach for N2O determinations. A custom-designed gas chromatograph with FID/TCD detectors (analyses conducted at Microseeps) was used in determinations of CO2, CH4, C2H6, and C2H4. The detection sensitivity of CO2 was 0.11 mM and for hydrocarbons detected (CH4, C2H6, and C2H4) were 6.2, 0.83, and 0.89 nmolL1. The levels of C3H8, C3H6, C2H2, CO, C4H10 (iso and n-forms) were below detection limits (0.05 g/L1 for all, except 0.5 g/L1 for C2H2 and 1.0 mg/L1 for CO). The N2O concentration was determined based on the m/z 44 ion beam intensity produced within a mass spectrometer during isotopic analysis (Elementar Isoprime). Before introduction on to the mass spectrometer, N2O was puried using a Trace Gas system (Elementar) that involved quantitatively stripping of N2O from 100 mL of LVBr under He ow, water, and CO2 removal
1 of 14

using chemical scrubbers (magnesium perchlorate and Carbosorb, respectively), then the gas was chromatographically separated on a Porapak Q column (6, 7). For the H2 and N2 determinations 150 mL of LVBr was introduced to evacuated glass bottles (300 mL) and equilibrated for 8 h at room temperature (8). Water was then removed by vacuum and sample gases introduced to an evacuated 3-mL gas sampling loop interfaced to a mass spectrometer (Elementar Isoprime) with magnesium perchlorate in-line for water vapor removal. H2 and N2 was then chromatographically puried using a molecular sieve 5 column under He ow and concentration determined from the m/z 2 ion beam intensity for the H2; concentrations were not determined for the N2 (9). The stable isotope values for N2 and H2 are in reference to air and VSMOW, respectively.
Dissolved Organic Carbon Methods. DOC in LVBr was quantied

using high temperature oxidation on a Shimadzu Total Organic Carbon (TOC) analyzer with a nonpurgeable organic carbon (NPOC) method (10). Carbohydrate concentration of the 2010 brine averaged over six replicates was measured after Dubois et al. (11) using pullulan (a soluble linear glucose polysaccharide; Sigma) as the standard. To characterize various fractions of DOC, LVBr was serially diluted to the point in which the last two samples were below the chloride interference threshold (12) based on anion chemistry data. Average values for the last two samples were reported. Fractions of dissolved organic carbon (DOC) were then isolated and characterized by acidifying two 1-L samples of LVBr to pH 2, diluting the LVBr (factor of 40) followed by a two-step ltration procedure. Diluted brine was partitioned into four fractions on XAD-8 and XAD-4 resins (13), separating fulvic (FA) and transphilic acid (TPIA) fractions, respectively followed by tangential ow ultra-ltration (TFUF; 1-kDa membrane) of the column efuent to concentrate the > 1 kDa fraction, which also results in a low molecular weight, permeate fraction (TFUF permeate). The FA, TPIA, and > 1 kDa fractions were analyzed with uorescence spectroscopy and freeze-dried for downstream analysis (14). 13C-DOC and radiocarbon (14C) ages of the six Lake Vida DOM fractions were prepared at the University of Colorado at Boulders radiocarbon laboratory (NSRL) and analyzed by accelerator mass spectrometry (AMS) at the University of Arizona AMS facility according to Stuiver and Polach (15).
Additional Microbial Activity Assays. Additional measures of heterotrophic bacterial growth were attempted via an evaluation of thymidine and acetate incorporation and respiration at 0 and 12 C with an ambient atmosphere headspace [incubating with 20 nM 3H-labeled thymidine for 54.5 h, extraction with cold 5% tricarboxylic acid (TCA), and ltering (16) and 10 mM 14C-labeled acetate for 65.5 h, extraction with cold 5% TCA, and ltering] in 2005. Although thymidine incorporation and acetate values were higher than formalin-killed controls, the difference was not signicant (MannWhitney test). Similarly, acetate respiration values were not different from formalin-killed controls. Thymidine incorporation assays conducted with the 2010 LVBr samples were run in parallel with the leucine incubations (17) under N2 atm at 0 and 13.5 C for 616 d, and again, signicant levels of incorporation indicating DNA biosynthesis were not detected. Microbial Cultivation. LVBr was transferred from anoxic bottles at the Desert Research Institute under an N2 atmosphere to an ambient atmosphere, where it was used to inoculate various media under aerobic or anoxic and cold (03 C) conditions. Halophilic broth (HB) and R2A (a freshwater heterotrophic media) did not produce evident growth after one month, whereas standard Marine Broth (DIFCO) yielded growing mixed cultures. Isolates were
Murray et al. www.pnas.org/cgi/content/short/1208607109

obtained by directly plating brine on Marine Agar (DIFCO) and incubating at 03 C. Colonies (appearing after 2 wk) were isolated through subsequent streaking (3 per colony). DNA was extracted from isolates following standard procedures (18), the small subunit (SSU) rRNA gene was PCR-amplied using bacterial primers (27F and 1492R), puried (Qiaquick; Qiagen), and sequenced at the Nevada Genomics Center, Reno, NV. Representatives were archived in 10% glycerol. Anaerobic enrichment media stock (10 mL per tube) was prepared using a hungate station in which LVBr was diluted deionized water (50% LVBr), degassed, and sterilized, and the headspace was lled with N2 (19). Fifteen millimolar malt extract (nal concentration) or 0.2% Casein extract (ICN Biomedicals) and 0.01 mL of Wollins Vitamin Solution (American Type Culture Collection) were injected into sealed Balch tubes just before inoculation. This media was inoculated with 1.1 mL anoxic brine and incubated in the absence of light at 2 C or 10 C for 9 mo. SSU rRNA gene sequences (Enrichment-D2, B11, C3, C6, C10, and C12) were obtained from a clone library (method described below) derived from DNA harvested from organisms grown with the 15 mM malt extract. SSU rRNA gene sequence derived from 2 C Enrichment-E3 was obtained from organisms grown in enrichment culture with 0.02% Casein at 10 C. DNA was extracted (DNeasy Blood and Tissue kit; Qiagen) from 2-mL enrichment culture aliquots that were concentrated to 30 L (Vivaspin; Sartorius).
Molecular Analyses. Cells were harvested in the eld in 2005 by ltering brine through 0.2-mm Sterivex lters (Millipore), then lters were stored frozen in either sucrose-Tris-EDTA buffer (500 mmol L1), 40 mmol L1, and 0.73 mol L1, respectively) until DNA extraction or RNAlater (Ambion, Inc.) for RNA. The results presented here were conducted with nucleic acids extracted from the 2005 LVBr following ref. 20. DNA was initially surveyed by PCR for amplication of the three domains of life. The primers used included: Archaea using Arch20F (20), 109F (21), and 958R (22); Bacteria using 27F and 1391R (23); and Eukarya using Euk1A and 1520R (24). A strong band in the bacterial amplication was detected, but no archaeal or eukaryal signals were observed. The bacterial SSU rRNA gene library was constructed using the the products of eight PCR reactions amplied with Bact27F and Univ1391R (25 cycles each) that were pooled then ligated into the PCR4TOPO vector (Invitrogen) and transformed into Escherichia coli cells in LB with 100 mg/mL1 ampicilin and kanamycin. Puried DNA (Millipore) with inserts of the correct size were sequenced bidirectionally on an AB 3100 automated sequencer (Applied Biosystems). Sequences were assembled using Sequencher (GeneCodes). Sequences were checked for chimeras using three on-line tools Bellepheron (25) (http:// greengenes.lbl.gov/cgi-bin/nph-bel3_interface.cgi), CheckChimera (26), and pintail (www.bioinformatics-toolkit.org/Web-Pintail/). Comparative phylogenetic analyses were performed with SSU rRNA gene sequence datasets from polar lakes and streams, brines, sea ice, and other sites with sequences in common to the LVBr community. Analyses presented here include 15 datasets where nearly full-length SSU rRNA gene sequences were available (Table S3). Datasets were downloaded from GenBank and aligned using the nearest alignment space termination alignment tool (27). Distance matrices were generated individually for each library, and DOTUR (28) was run to generate nonredundant datasets for each library with all sequences represented at a distance of 0.01. Overlapping regions between all libraries (460 sequences) provided an alignment with a minimum of 1,188 base pairs. Neighborjoining trees using pair-wise deletion were calculated in MEGA (v4) (29). UniFrac (30) was used to calculate statistical comparisons among the libraries. The evolutionary history of LVBr nearly full-length SSU rRNA gene sequences was inferred using the minimum evolution method
2 of 14

(31) and 1,000 bootstrapped trees. Evolutionary distances were computed using the maximum composite likelihood method (32) and are in units of the number of base substitutions per site, the complete deletion option was used. There were a total of 1,020 positions in the nal dataset. Neighbor-joining analysis (29) was used to generate the phylogenetic tree to characterize relatedness between crDNA and culture sequences, using MEGA4 based on 209 aligned positions, in which 1,000 bootstrap values were calculated. Four libraries with nearly full-length sequences and published evenness data [LVBr, Lake Vida ice cover at 4.8 and 15.9 m (LVI_4_8, LVI_15_9) and the surface outow of Blood Falls brine] were compared using weighted abundances to further investigate specic associations between these environments with similar microbial communities (and physical proximity). Neighbor-joining trees were calculated using complete deletion (144 unique sequences of 546 redundant sequences were compared with 1,255 bases) (30).
1. Salata GG, Roelke LA, Cifuentes LA (2000) A rapid and precise method for measuring stable carbon isotope ratios of dissolved inorganic carbon. Mar Chem 69(1-2):153161. 2. Casciotti KL, Bhlke JK, McIlvin MR, Mroczkowski SJ, Hannon JE (2007) Oxygen isotopes in nitrite: Analysis, calibration, and equilibration. Anal Chem 79(6):2427 2436. 3. Holmes R, McClelland J, Sigman D, Fry B, Peterson B (1998) Measuring 15N-NH4+ in marine, estaurine and fresh waters: An adaptation of the ammonia diffusion method for samples with low ammonium concentrations. Mar Chem 60(3-4):235243. 4. Brchert V, Pratt L (1996) Contemporaneous early diagenetic formation of organic and inorganic sulfur in estuarine sediments from St Andrew Bay, Florida, USA. Geochim Cosmochim Acta 60:2325 2332. 5. Townsend A (2000) The accurate determination of the rst row transition metals in water, urine, plant, tissue and rock samples by sector eld ICP-MS. J Anal At Spectrom 15(4):307314. 6. Sutka RL, Ostrom NE, Ostrom PH, Gandhi H, Breznak JA (2003) Nitrogen isotopomer site preference of N2O produced by Nitrosomonas europaea and Methylococcus capsulatus Bath. Rapid Commun Mass Spectrom 17(7):738745. 7. Sutka RL, et al. (2006) Distinguishing nitrous oxide production from nitrication and denitrication on the basis of isotopomer abundances. Appl Environ Microbiol 72(1): 638644. 8. Emerson S, Stump C, Wilbur D, Quay P (1999) Accurate measurement of O2, N2, and Ar gases in water and the solubility of N2. Mar Chem 64(4):337347. 9. Yang H, et al. (2012) Using gas chromatography/isotope ratio mass spectrometry to determine the fractionation factor for H2 production by hydrogenases. Rapid Commun Mass Spectrom 26(1):6168. 10. Suginura Y, Suzuki Y (1988) A high-temperature catalytic-oxidation method for the determination of non-volatile dissolved organic carbon in seawater by direct injection of a liquid sample. Mar Chem 24(2):105131. 11. Dubois M, Gilles K, Hamilton J, Reber P, Smith F (1956) Colorimetric methods for determination of sugars and related substances. Anal Chem 28(3):350356. 12. Aiken G (1992) Chloride interference in the analysis of dissolved organic carbon by the wet oxidation method. Environ Sci Technol 26(12):24352439. 13. Aiken G, McKnight DM, Thorn KA, Thurman EM (1992) Isolation of hydropholic arganicacids from water using nonionic macroporous resins. Org Geochem 18(4):567573. 14. Rees C, Jenkins WJ, Monster J (1978) The sulphur isotopic composition of ocean water sulphate. Geochim Cosmochim Acta 42(4):377381. 15. Stuiver M, Polach H (1977) Discussion: Reporting of 14C data. Radiocarbon 19(3): 355363. 16. Kirchman DL, Keil RG, Simon M, Welschmeyer NA (1993) Biomass and production of heterotrophic bacterioplankton in the oceanic subarctic Pacic. Deep Sea Res Part I Oceanogr Res Pap 40(5):967988. 17. Smith DC, Azam F (1992) A simple, economical method for measuring bacterial protein synthesis rates in seawater using 3H-leucine. Mar Microb Food Webs 6(2): 107114.

RNAlater (Ambion) was added to brine samples ltered through either 0.2-mm or 47-mm Supor or Sterivex lters (Millipore), then stored at 4 C overnight, frozen at 20 C. RNA was extracted using a Totally RNA kit (Ambion) following the manufacturers instructions. Total RNA extracts were subjected to two rounds of DNase (Ambion) followed by purication with phenol:chloroform following standard procedures. PCR-denaturing gradient gel electrophoresis (DGGE) was used to compare reverse-transcribed SSU rRNA (crRNA) and SSU rRNA gene proles (33) in which crRNA was rst reverse-transcribed with superscript III using a 1:1 mixture of hexamers (300 ng/L1) and Univ1391R. Negative control PCR reactions were run with total RNA to check for DNA contamination in the RNA. One microliter of the crRNA product was used in PCR with GC-clamped primers GC358F and 517R (10 pmol each). A crRNA clone library was also prepared with the same cDNA (1 L) using primers 358F1391R to amplify the crRNA, which was then cloned into TOPO PCR4 vector (Invitrogen) following standard conditions. Sequencing proceeded as described above.
18. Sambrook J, Russell D (2001) Molecular Cloning: A Laboratory Manual (CSHL, New York), 3rd Ed. 19. Sowers KR, Schreier HJ, DasSarma S, Fleischmann EM (1995) Methanogens: Growth and identi cation. Archaea: A Laboratory Manual, Methanogens , eds Robb KRSFT, Schreier HJ, Place AR (Cold Spring Harbor Lab Press, Plainview, NY), pp 24 31. 20. Massana R, Murray AE, Preston CM, DeLong EF (1997) Vertical distribution and phylogenetic characterization of marine planktonic Archaea in the Santa Barbara Channel. Appl Environ Microbiol 63(1):5056. 21. Galand PE, Lovejoy C, Vincent WF (2006) Remarkably diverse and contrasting archaeal communities in a large arctic river and the coastal Arctic Ocean. Aquat Microb Ecol 44 (2):115126. 22. DeLong EF (1992) Archaea in coastal marine environments. Proc Natl Acad Sci USA 89 (12):56855689. 23. Lane DJ (1991) 16S/23S rRNA sequencing. Nucleic Acid Techniques in Bacterial Systematics, eds Stackebrandt E, Goodfellow M (Wiley, New York), pp 115175. 24. Dez B, Pedrs-Ali C, Marsh TL, Massana R (2001) Application of denaturing gradient gel electrophoresis (DGGE) to study the diversity of marine picoeukaryotic assemblages and comparison of DGGE with other molecular techniques. Appl Environ Microbiol 67 (7):29422951. 25. Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: A program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20(14):23172319. 26. Cole JR, et al.; Ribosomal Database Project (2003) The Ribosomal Database Project (RDP-II): Previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids Res 31(1):442443. 27. DeSantis TZ, Jr., et al. (2006) NAST: A multiple sequence alignment server for comparative analysis of 16S rRNA genes. Nucleic Acids Res 34(Web Server issue):W394-9. 28. Schloss PD, Handelsman J (2005) Introducing DOTUR, a computer program for dening operational taxonomic units and estimating species richness. Appl Environ Microbiol 71 (3):15011506. 29. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24(8):15961599. 30. Lozupone C, Hamady M, Knight R (2006) UniFrac An online tool for comparing microbial community diversity in a phylogenetic context. BMC Bioinformatics 7 (August):371. 31. Rzhetsky A, Nei M (1992) A simple method for estimating and testing minimum evolution trees. Mol Biol Evol 9(5):945967. 32. Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci USA 101(30):11030 11035. 33. Mosier AC, Murray AE, Fritsen CH (2007) Microbiota within the perennial ice cover of Lake Vida, Antarctica. FEMS Microbiol Ecol 59(2):274288.

Murray et al. www.pnas.org/cgi/content/short/1208607109

3 of 14

Fig. S1. (A) Map of Antarctica showing the location of the McMurdo Dry Valleys. (B) National Aeronautics and Space Administrations (NASA) Earth Observatory, Landsat 7 satellite image of a part of McMurdo Dry Valleys showing the location of Lake Vida (Vi) and Lake Vanda (Va), Fryxell (F), Hoare (H), Bonney (B), and Blood Falls (BF). The lled circle at the center of Lake Vida indicates the location of the borehole. The white rectangle indicates the area covered by the geologic map (Fig. S3). The location of sampling in 2005 and 2010 is indicated by a dot.

Murray et al. www.pnas.org/cgi/content/short/1208607109

4 of 14

Fig. S2. Geological description of Lake Vida basin and Victoria Valley. (A) View of the southern and southwestern shores of Lake Vida showing two large Ferrar dolerite sills (darker layers) cross cutting granite of the Orestes Pluton. The lake is 349 m above sea level; the marked summit is 1,450 m high. The arrow points to the dark dolerite-dominated Bull Drift. (B) View of the southern shore of Lake Vida showing two large Ferrar dolerite sills (darker layers) crosscutting granite (lighter in color). The marked summit is Mt. Cerberus (1,766 m). The arrow points to the camp site in the center of the lake. (C ) View toward the northeastern shore of Lake Vida. The darker layers are made of Ferrar dolerite the lighter layers are granite. The marked summit is above 1,200 m. The white arrow points to aeolian sand deposits dominated by Fe.Mg silicates and Ca.Fe.Mg silicates (pyroxenes). The black arrow points to camp in the center of Lake Vida. The rocks at the forefront are granite ventifacts.

Murray et al. www.pnas.org/cgi/content/short/1208607109

5 of 14

Fig. S3. Simplied geological map of the Victoria Valley around Lake Vida adapted from Turnbull et al. (1). The location of the area covered by this map is shown in Fig. S1. Adapted with permission from the Institute of Geological and Nuclear Science.

1. Turnbull IM, Allibone AH, Forsyth PJ, Heron DW (1994) Geology of the Bull Pass- St Johns Range area, southern Victoria Land, Antarctica, 1/50000. Institute of Geological & Nuclear Sciences Geological Map 14 (Institute of Geological & Nuclear Sciences Limited, Lower Hutt, New Zealand), 1 sheet and 52 pp.

Murray et al. www.pnas.org/cgi/content/short/1208607109

6 of 14

10

11

12

13

*
>

*
>

*
>

*
>

*
>

*
>

a < # # # < #

< #

< #

<

Fig. S4. PCR-DGGE analysis of Lake Vida DNA, reverse-transcribed RNA (crRNA), and cloned bacterial SSU rRNA gene sequences. The crRNA, an indicator of biologically active organisms, was PCR-amplied with the same primers as for the DNA (lane 5). Selected cloned SSU rRNA genes from Lake Vida brine were included in the analysis to assign identities to comigrating bands, (identied with symbols: lane 1 clone LVBR10cG04, an uncultivated Lentisphaera-related bacterium (same as LVBR10aD09, GQ167314.1); lane 2 clone LVBR10cE03, related to uncultivated Marinobacter clone ELB25-092, DQ015769.1, # (same as LVBR10aG09 GQ167320.1, and LVBR10cA05, GQ167307.1); lane 3 clone LVBR10cF06, a Sphaerochaeta-related strain, (same as LVBR10bB02, GQ167322.1); lane 11 clone LVBR10cD03, an uncultivated Psychrobacter-related bacterium, * (same as LVBR10cG07 GQ167313.1); lane 12 clone LVBR10cA08 (GQ167319.1), an uncultivated epsilonproteobacterium, >; and lane 13 clone LVBR10cA04, related to an uncultivated avobacterium, < (same as LVBR10cF01, GQ167312.1). DNA extracted from four brine samples was analyzed in independent lanes (6, 810). As a negative control for the RT-PCR, RNA that was not reverse-transcribed was amplied in the same manner as the other reactions to verify that amplication in the RT-PCR product was from cDNA and not contaminating DNA (lane 4). Lane 7 is empty. Bands excised and sequenced along with nearest neighboring sequence are indicated with letters: a, LVBR2ERNA-4, an uncultivated Sulfurovum-related epsilonproteobacterium, GQ167343; b, LVBR2B-2, uncultured Psychroexus bacterium, GQ167344.1.

Murray et al. www.pnas.org/cgi/content/short/1208607109

7 of 14

Isolate Enrichment culture SSU rRNA gene clone SSU crRNA clone DGGE band clone
100

71 62 76 89

Isolate LVBR 79 Enrichment-D2 Enrichment-B11 LVBRrRNAA1 Enrichment-C3

Psychrobacter-group

Enrichment-C6 65 61 91 Enrichment-C10 LVBR10cG07

LVBR10cC08 69 49 46 Enrichment-E3 LVBR10cA05

*
Marinobacter-group

LVBRrRNAH2 Isolate LVBR 90

51

100 58 42

LVBRrRNAD1 LVBR10cE12 LVBRrRNAB2 LVBRrRNAF1

30 LVBR10aF06

36

LVBR10cF05 27 21 89 15 LVBR10aH12 DGGE-LVBR2B-2 LVBR10cE09 LVBR10aF03 17 42 64 34 LVBR10aA11 LVBR10cA11 LVBR10cD02 21 93 93 LVBR10cA08 99 57 LVBR10aG04 LVBR10aF05 13 20 34 LVBR10aA04 LVBR10cC03 52 LVBR10aH06 LVBR10cH06 34 78 100 LVBR10aC07 LVBR10aD09 LVBR10bA03 LVBR10cB02 LVBR10bB06 LVBR10aC08 LVBRrRNAG5 LVBR10cE02

*
Bacteriodetes-phylum

* *

Sulfurovum-group

DGGE-LVBR2ERNA-4

0.02

Fig. S5. Neighbor-joining phylogenetic tree of Lake Vida SSU rRNA and rRNA gene sequences derived from cultivated isolates, anaerobic enrichments, cloned crRNA, cloned DGGE bands (Fig. S4), and cloned rRNA gene sequences. Symbols indicate source of the sequence. The Marinobacter and Psychrobacter genera both were well-represented in cultivation-based, crRNA and PCR-amplication approaches suggesting that they are active in LVBr. An asterisk indicates sequence with comigrating crRNA-derived DGGE band.

Murray et al. www.pnas.org/cgi/content/short/1208607109

8 of 14

B
100.0

100.0 64.6

BF LVBR LVI 15 9 LVI 4 8

0.05
Fig. S6. LVBr microbial diversity and comparisons to other environmental SSU rRNA gene libraries. (A) Principle components analysis of 15 environmental SSU rRNA bacterial clone libraries based on sequence composition (richness, not evenness) of each library. Designations of libraries are as follows: Lake Vida Brine, LVBr; Lake Vida Ice, LVI; East and West Lobe Lake Bonney, ELB and WLB, respectively; oil sands tailings pond, OSTP; Romanian oil-contaminated soil, PIT; Canadian biodegraded oil reservoir, PL; Svalbard marine sediments, SVA; Blood Falls glacial outow, BF; Arctic cold springs Color Peak, CP and Gypsum Hill, GH; ARC-1 and ANT-1, Arctic sea ice Antarctic sea ice, respectively; ANT-P, seawater of Arthur Harbor (Antarctica); ACE-S, BURT-S, and ORG-S, sediments of Ace, Burton, and Organic lakes, respectively (Vestfold Hills, Antarctica); ABBB, BBBB, DBBB, and UBBB are Mediterranean deep anoxic brines of LAtalante, Bannock, Discovery, and Urania basins, respectively (see Table S3 for accompanying sample and library data). The rst and second principle components explain 10.53% and 8.04% of the variance, respectively. (B) Closely related libraries (BF, LVBr, and LVI surface at 4.8 m and deep ice at 15.9 m) were clustered with weighted abundances (evenness), branches were normalized to account for different rates and compared using the jackknife statistic. Values at the nodes are percentages of 1,000 iterations.

Murray et al. www.pnas.org/cgi/content/short/1208607109

9 of 14

Table S1. Geochemical data for Lake Vida, other dry valley and Vestfold Hills lakes and deep Mediterranean brines

Physiochemical or biological parameter n 77 23.31 S, 161 55.86 E 6.20 13.40 77439 S, 162209 E 6.25 0.21 0.4 0.3 77439 S, 77389 S, 162209 E 163 79 E 5.90 0.42 8.60 0.00 3.94 0.1 0.58 0.04 77379 S, 163 79 E 7.50 0.14 2.15 0.01 77329 S, 77439 S, 161339 E 161339 E 5.80 7.20 21.10 0.01 77 34 S, 161 11E 5.57 3.30 77 72 S, 162 26 E 6.20 5.20 34 16.56 N, 68 28.45 S, 19 58.56 E 78 11.10 E 6.55 NA 14.25 1.70 Don Juan Pond Bannok Basin Ace Lake (25 m) Deep Lake

Lake Vida 2010

Lake Vida 2010; 0.2 m fraction Lake Vida 2005 Lake Bonney (East) 25 m Lake Bonney (West) 35 m Lake Hoare (10 m) Lake Fryxell (15 m) Lake Vanda (68 m) Lake Joyce (33 m) Blood Falls (near mouth) 77 35 S, 160 15 E 7.40 10.917.2

Coordinates

77 23.31 S, 161 55.86 E 6.20 13.40

77 23.31 S, 161 55.86 E NA NA

3 NA NA NA 2 48.2 9.7 NA 0.31 329.2 1.71 0.10 1.64 0.07 0.15 0.01 1.38 0.08 0.42 5 2.68 0.09 1.51 0.35 7.20 0.95 NA NA 2.49 4.87

72.3 2.9 NA 2 2.96 NA NA 55

61.2 0.6

14.0 1.5

67.2 6.8

3.72 0.17

47.0 4.2

NA NA 0.33

NA NA NA

NA NA NA

1.44 0.28

Murray et al. www.pnas.org/cgi/content/short/1208607109

25 25 25 25 25 25 25 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 30.09 1.25 39.47 0.96 57.66 0.04 1.11 0.04 3,318 112 4074.31 19.09 2304.83 8.74 3.94 0.34 1.50 0.15 ND ND 0.06 0.01 82.81 2.77 60.64 4.83 35.1 0.44 0.46 0.02 664.87 22.52 1097.99 44.88 359.42 1.38 0.79 0.04 1914 60 1630.22 110.66 1684.54 8.51 4.42 0.18 58.43 2.27 30.77 2.60 47.4 0.80 0.76 0.06 NA NA NA NA 61 NA NA NA 188 216 137 0 NA NA 3.22 0.10 84.66 5.63 ND 4.27 0.03 10.41 0.14 94.31 1.96 1.5 0.47 NA NA 6 NA NA 650.48 2095.17 4.43 19.13 339.02 304.48 6.56 NA 6.70 117.00 NA NA 5.85 37.63 0.52 1.55 4.71 41.99 9.26 NA NA 4 NA NA NA NA 3080.03 587.23 6714.72 1197.05 1,375 11.74 NA 3.25 0.14 NA 63.34 19.89 NA 613.01 55.79 NA 0.68 0.05 55 NA 21.0 0.4 0.75 NA 315.00 NA NA NA 21.8 5,333 0.11 124.8 613.3 4,160 103.8 NA NA 322 NA NA 7.34 652 NA 11.7 85.9 538 0.687 NA 0.85 NA NA NA 59.9 4789.0 NA 108.4 514.3 2953.5 28.8 NA NA 260 NA NA NA NA 5 5 5 5 3,885 23.7 904 4.98 43 1.0 30 0.19 132.95 14.43 119.86 1.20 18.89 6.65 9.45 0.08 0.07 0.18 0.26 0.17 215.31 0.08 nd 4.55 0.54 0.01 0.10 295.26 23.48 0.25 ND 0.37 ND 0.16 28.66 1.85 241.00 1.67 167.50 104.11 ND 31 1.59 0.60 25.3 9.2 5,533 626 212.35 62.89 94 ND ND NA 3.35 NA NA 16 NA NA NA NA NA 0.1 0.1 1.2 0.05 2.9 0.2 3 3 3 3 3 1 1 3 3 3 3 3 3 3 10 11 0.9 0.1 0.5 0.03 ND 0.6 0.05 0.7 0.4 0.5 0.04 308 23 82 4 0.2 0.1 1 0.3 NA 0.9 0.4 NA 447 18 0.6 0.04 NA NA NA 0.02 0.8 NA 0.9 0.3 83 0.2 3 NA 0.2 NA 362 NA NA NA NA 0.007 1 NA 3 55 164 0.07 3 NA 0.5 NA 475 NA NA NA NA 0.00 0.00 NA 0.03 0.06 0.02 NA 0.02 NA ND NA NA NA NA NA NA 0.0001 ND NA 0.001 0.8 0.6 NA 0.03 NA NA NA 25 NA NA NA 5 ND ND NA 0.01 5 43 NA 0.01 NA ND NA 1 NA 0.9 NA NA ND ND NA ND 26 11 NA ND NA NA NA NA NA NA NA 11.00 0.00 0.01 NA 0.03 0.97 20.93 NA 0.06 NA 0.63 NA 12.21 NA NA NA NA NA NA NA NA 3450 NA NA NA NA NA NA NA NA NA NA 290 NA NA NA NA ND 5.00 NA NA NA NA NA 214 NA 3.3 NA NA NA 0.029 0.226 <0.008 2.69 0.039 NA 0.31 NA NA NA 28 NA NA NA NA 0.04 0.01 NA NA 0.14 0.03 NA NA NA NA NA 0.02 0.01 NA NA NA

64.7

8 8 8 8 8 8 8 8 9 6

Many

0.53 0.01 1.62 0.09 32.69 1.20 3,187 31 1.95 0.26 90.84 2.57 682.62 22.74 1954.62 65.00 66.34 4.29 20.34 0.13 NA 180210

pH Temperature (C) Carbon and Nitrogen DIC (mmolL1) DIC 13C (per mL) DOC (mmolL1) Major Ions (mmolL1) Li+ Br Ca+2 Cl F K+ Mg+2 Na+ SO4 -2 34S-SO42HCO3+CO2-2 Salinity (psu) Nutrients (molL1) NH4+-N NO2-N NO3-N PO4-P Metals (molL1) Al As Ba Cd Co Cr Cu Fe Mn Mo Ni P Pb S Sr U

3 2 3 2

3,600 190 27.3 0.7 1,120 130 3.42 0.03

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

2 0.3 0.7 0.09 0.7 0.04 0.005 0.003 0.7 0.05 0.06 0.06 0.07 0.03 256 12 83 3 0.7 0.3 1 0.1 14 2 ND 67,851 6,384 NA 0.7 0.09

0.7 0.1 0.6 0.1 ND# 0.005 0.0005 0.7 0.06 ND# 0.05 0.01 247 7 84 3 0.5 0.1 1 0.02 13 1 ND 66,147 4,724 NA 0.7 0.06

10 of 14

Table S1.

Cont.

Physiochemical or biological parameter n Don Juan Pond Bannok Basin Ace Lake (25 m) Deep Lake

Lake Vida 2010

Lake Vida 2010; 0.2 m fraction Lake Vida 2005 Lake Bonney (East) 25 m Lake Bonney (West) 35 m Lake Hoare (10 m) Lake Fryxell (15 m) Lake Vanda (68 m) Lake Joyce (33 m) Blood Falls (near mouth)

3 3 3

0.3 0.03 6 0.4 NA 10 8 NA 2 NA 0.6 NA 0.01 NA NA NA 0.04 NA NA NA 2.34 NA NA NA NA NA 0.39

0.3 0.02 7 0.6

NA NA

Murray et al. www.pnas.org/cgi/content/short/1208607109

0.00 NA NA NA NA NA NA NA NA NA NA NA 0.25 0.05 0.12 0.05 NA NA Suboxic NA NA NA NA 59 0.21 0.07 25 trace ND Anoxic NA NA NA NA 34.80 NA 0.02 0.02 0.14 0.08 NA 0.33 Suboxic NA NA NA NA 0.58 NA NA NA NA NA Oxic NA NA NA NA 0.02 NA NA NA 937.00 333 Anoxic NA NA NA NA 7.16 NA NA NA 328.00 NA Anoxic NA NA NA NA NA NA NA NA NA NA Anoxic NA NA NA NA NA NA NA NA 0.29 NA NA NA NA NA NA NA NA NA NA NA NA ND NA NA NA NA NA NA NA NA 2000.00 128 NA NA NA NA NA NA NA NA NA 7,100 4,240 Anoxic NA NA NA NA NA NA NA NA 0.00 NA NA NA NA NA NA 3 0.10 0.6 0.01 0.01 0.00 0.06 0.02 0.59 0.18 2.77 1.75 5.38 2.25 NA 0.06 NA 1.94 NA 48.5 24.6 13 1, 2, 4 5 2, 3, 6 1, 7, 8 1, 2, 9, 10 8, 11 12 13, 14 15 1618

4 4 4 3 4 4

86.6 5.9 NA NA NA ND 0.05 0.01 ND 8,860 190 10.47 0.02 0.0027 0.0005 0.0052 0.0004

0.147 0.025

V Zn Gasses and volitiles (molL1) N2O MeSH DMS DMSO H2S CH4 Oxygen CO2 H2 Ethane Ethene Microbiological characteristics Stained particles > 0.2 m 106 mL1 Stained particles 0.2 m 106 mL1 Reference

61.4 15.9

Nutrient, ion, purgeable organic carbon, purgeable organic nitrogen, and microbial cell counts for McMurdo Dry Valley Lakes represent averages of two dates sampled in the 20032004 season; cell counts were averages of three samples (www.mcmlter.org/). SDs shown (). DIC data (19) and N2O data (20) are from the literature. NA, not analyzed; ND not detected. # represents high blank values.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

17. 18. 19. 20.

Lee PA, et al. (2004) Thermodynamic constraints on microbially mediated processes in lakes of the McMurdo Dry Valleys, Antarctica. Geomicrobiol J 21(3):221237. Lee PA, et al. (2004) Elevated levels of dimethylated-sulfur compounds in Lake Bonney, a poorly ventilated Antarctic lake. Limnol Oceanogr 49(4):10441055. Knoepe JL, Doran PT, Kenig F, Lyons WB, Galchenko VF (2009) Particulate organic and dissolved inorganic carbon stable isotopic compositions in Taylor Valley lakes, Antarctica: The effect of legacy. Hydrobiologia 632(1):139156. Ward BB, Granger J, Maldonado MT, Wells ML (2003) What limits bacterial production in the suboxic region of permanently ice-covered Lake Bonney, Antarctica? Aquat Microb Ecol 31(1):3347. Green WJ, Ferdelman TG, Gardner TJ, Varner LC, Angle MP (1986) The residence time of 8 trace-metals in a closed-basin Antarctic lake - Lake Hoare. Hydrobiologia 134(2):249255. Green WJ, et al. (1989) Geochemical processes in the Lake Fryxell basin (Victoria Land, Antarctica). Hydrobiologia 172(1):129148. Green WJ, Ferdelman TG, Caneld DE (1989) Metal dynamics in Lake Vanda (Wright Valley, Antarctica). Chem Geol 76(1-2):8594. Webster JG (1994) Trace metal behavior in oxic and anoxic Ca-Cl brines of the Wright Valley drainage, Antarctica. Chem Geol 112(3-4):255274. Roberts EC, Priscu JC, Wolf C, Lyons WB, Laybourn-Parry J (2004) The distribution of microplankton in the McMurdo Dry Valley Lakes, Antarctica: response to ecosystem legacy or present-day climatic controls? Polar Biol 27(4):238249. Shacat JA, Green WJ, Decarlo EH, Newell S (2004) The geochemistry of Lake Joyce, McMurdo Dry Valleys, Antarctica. Aquat Geochem 10(3-4):325352. Samarkin VA, et al. (2010) Abiotic nitrous oxide emission from the hypersaline Don Juan Pond in Antarctica. Nat Geosci 3(5):341344. Mikucki JA, et al. (2009) A contemporary microbially maintained subglacial ferrous ocean Science 324(5925):397400. Delange GJ, et al. (1990) Composition of anoxic brines in the Tyro and Bannock basins, Eastern Mediterranean. Mar Chem 31(1-3):6388. Henneke E, Delange GJ (1990) The distribution of DOC and POC in the water column and brines of the Tyro and Bannock basins. Mar Chem 31(1-3):113122. Mancuso CA, Franzmann PD, Burton HR, Nichols PD (1990) Microbila community structure and biomass estimates of a methanogenic Antarctic lake ecosystem as determined by phospholipid analyses. Microb Ecol 19(1):7395. Barker RJ (1981) Physical and chemical parameters of Deep lake, Vestfold Hills, Antarctica. in Australian National Antarctic Research Expeditions Scientic Reports, Series 5B, Limnology (Australian Government Publication Service, Canberra, Australia), p 73. Bowman JP, McCammon SA, Rea SM, McMeekin TA (2000) The microbial composition of three limnologically disparate hypersaline Antarctic lakes. FEMS Microbiol Lett 183(1):8188. Ferris JM, Burton HR (1988) The annual cycle of heat -content content and mechanical stability of hypersaline Depp Lake, Vestfolds Hills, Antarctica. Hydrobiologia 165(1):115128. Neumann K, Lyons WB, Priscu JC, Desmarais DJ, Welch KA (2004) The carbon isotopic composition of dissolved inorganic carbon in perennially ice-covered Antarctic lakes: Searching for a biogenic signature. Annals of Glaciology 39:518524. Priscu JC (1997) The biogeochemistry of nitrous oxide in permanently ice-covered lakes of the McMurdo Dry Valleys, Antarctica. Global Change Biology 3(4):301315.

11 of 14

Table S2. Characterization of LVBr dissolved organic material fractions

DOM fraction Filtered water 1 Filtered water 2 LV-FA 1 LV-FA 2 LV-TPIA 1 LVTPIA 2 LV > 1 kDa 1 LV > 1 kDa 2 TFUF permeate 1 TFUF permeate 2 Neutrals % Carbon 9.3 10 7.4 7.9 48.6 54.2 24.4 25.8 36.5 FI 1.78 1.79 1.56 1.59 1.79 1.84 1.46 1.58 1.78 1.75 13C-DOC 17.7 19.5 17.9 16.8 15.3 14.6
14

C Fraction modern 0.6226 0.6021 0.6118 0.5964 0.6922 0.6399

14

C Fraction modern error 0.0011 0.0012 0.0012 0.0011 0.0013 0.0015

14

C age (y BP) 3,805 4,075 3,945 4,150 2,955 3,585

14

C age error (y BP) 15 20 20 20 15 20

Initial determinations were conducted with 0.45 m ltered brine before column chromatography and TFUF. DOC was partitioned into FA, and TPIA fractions, followed by TFUF of the column efuent which yielded a > 1 kDa retentate fraction and a < 1 kDa permeate fraction. SUVA254 is the carbon normalized UV absorbance at 254 nm, which is an indicator of aromaticity. The percent carbon of each fraction was determined by mass balance using dissolved organic materials (DOM) concentrations and fraction volumes. The uorescence index (FI) is the ratio of the emission intensities at 470 nm and 520 nm when excited at 370 nm and is an indicator of DOM origin. 14C data were derived using the prepared DOM fractions.

Murray et al. www.pnas.org/cgi/content/short/1208607109

12 of 14

Table S3. List of studies used for rRNAgene library comparisons

No. of Library No. of sequence clones Salinity/ designation OTUs in library conductivity Temp C LVBR ELB-16 ELB-19 ELB-25 WLB-13 WLB-16 BF LVI-48 LVI-15_9 ORG-S BUR-S ACE-S ARK-I_1 ANT-I SVA GH CP OSTP PL PIT ABBB BBBB DBBB UBBB ANT-P 32 14 20 4 9 16 16 62 34 11 51 37 40 16 68 46 30 42 53 99 41 43 49 17 52 155 95 114 144 28 57 81 135 181 NA NA NA 106 103 353 155 174 NA NA NA NA NA NA NA NA 188 psu 1 S/m 10 S/m 11 S/m 1 S/m 8 S/m NA NA NA 21 psu 43 psu 35 psu NA NA NA 7.9 psu 15.5 psu NA 3 psu NA 358 psu 322 psu 470 psu 237 psu 33.5 psu 13.4 6 5.5 4 3 0 NA 25 11.6 6 - -8 1.8 2.5 NA NA 3 6.3 5.7 NA 20 NA 13.9 14.7 14.5 16.7 1

Source Anoxic brine

Location

H 2.87 2.7 2.6 2.0 2.1 2.4 1.75 NA NA 1.01 NA NA 1.09 0.84 NA 3.17 2.16 NA NA NA 3.26 3.2 3.5 2.17 N/A

S Chao1 S Ace Coverage % Source 82 32 38 27 15 104 30 133 48 32 NA NA NA NA NA 71 50 NA NA NA NA NA NA NA NA 112 40 40 20 24 52 27 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 63 83 91 NA NA 74 85 72 84 91 NA NA NA NA NA NA NA NA Present study 1 1 1 1 1 2 3 3 4 5 5 6 6 7 8 8 9 10 10 10 10 11

Lake Vida, Victoria Valley Lake water East Lake Bonney, column Taylor Valley Lake water East Lake Bonney, column Taylor Valley Lake water East Lake Bonney, column Taylor Valley Lake water West Lake Bonney, column Taylor Valley Lake water West Lake Bonney, column Taylor Valley Glacier ice Blood Falls, Taylor Valley Lake ice Lake Vida, Victoria Valley Lake ice Lake Vida, Victoria Valley Lake sediment Organic Lake, Vestfold Hills Lake sediment Burton Lake, Vestfold Hills Lake sediment Ace Lake, Vestfold Hills Sea ice Arctic sea ice Sea ice Antarctic sea ice, Weddell Sea Marine sediment Svalbard Spring water Gypsum Hill, Canadian Arctic Spring water Color Peak, Canadian Arctic Oil sand tailings Canadian arctic pond Biodegraded oil Canadian arctic reservoir Oil contaminated Romania soil Deep anoxic LAtalante Basin, brine Mediterreanean Deep anoxic Bannok Basin, brine Mediterranean Deep anoxic Discovery Basin, brine Mediterreanean Deep anoxic Urania Basin, brine Mediterranean Seawater Arthur Harbor, Antarctic Peninsula

Select environmental and diversity-related parameters are shown. NA, not analyzed. GenBank accession numbers for studies not published include OSTP: EF420191.1EF420233.1; EU517535.1EU517561.1. and PIT: DQ366084.1DQ378273.1.

Murray et al. www.pnas.org/cgi/content/short/1208607109

13 of 14

1. Glatz RE, Lepp PW, Ward BB, Francis CA (2006) Planktonic microbial community composition across steep physical/chemical gradients in permanently ice-covered Lake Bonney, Antarctica. Geobiology 4(5):5367. 2. Mikucki JA, Priscu JC (2007) Bacterial diversity associated with Blood Falls, a subglacial outow from the Taylor Glacier, Antarctica. Appl Environ Microbiol 73(12):40294039. 3. Mosier AC, Murray AE, Fritsen CH (2007) Microbiota within the perennial ice cover of Lake Vida, Antarctica. FEMS Microbiol Ecol 59(2):274288. 4. Bowman JP, McCammon SA, Rea SM, McMeekin TA (2000) The microbial composition of three limnologically disparate hypersaline Antarctic lakes. FEMS Microbiol Lett 183(1):8188. 5. Bowman JP, Rea SM, McCammon SA, McMeekin TA (2000) Diversity and community structure within anoxic sediment from marine salinity meromictic lakes and a coastal meromictic marine basin, Vestfold Hilds, Eastern Antarctica. Environ Microbiol 2(2):227237. 6. Brinkmeyer R, et al. (2003) Diversity and structure of bacterial communities in Arctic versus Antarctic pack ice. Appl Environ Microbiol 69(11):66106619. 7. Ravenschlag K, Sahm K, Pernthaler J, Amann R (1999) High bacterial diversity in permanently cold marine sediments. Appl Environ Microbiol 65(9):39823989. 8. Perreault NN, Andersen DT, Pollard WH, Greer CW, Whyte LG (2007) Characterization of the prokaryotic diversity in cold saline perennial springs of the Canadian high Arctic. Appl Environ Microbiol 73(5):15321543. 9. Grabowski A, Blanchet D, Jeanthon C (2005) Characterization of long-chain fatty-acid-degrading syntrophic associations from a biodegraded oil reservoir. Res Microbiol 156(7): 814821. 10. van der Wielen PW, et al.; BioDeep Scientic Party (2005) The enigma of prokaryotic life in deep hypersaline anoxic basins. Science 307(5706):121123. 11. Murray AE, Grzymski JJ (2007) Diversity and genomics of Antarctic marine micro-organisms. Philos Trans R Soc Lond B Biol Sci 362(1488):22592271.

Murray et al. www.pnas.org/cgi/content/short/1208607109

14 of 14