bacterial communities in two antarctic ice cores analyzed ...2.3. dna extraction and...

Available online at www.sciencedirect.com

Polar Science 4 (2010) 215e227http://ees.elsevier.com/polar/

Bacterial communities in two Antarctic ice cores analyzedby 16S rRNA gene sequencing analysis

Takahiro Segawa a,b,*, Kazunari Ushida c, Hideki Narita d, Hiroshi Kanda b,Shiro Kohshima e

aTransdisciplinary Research Integration Center, 4-3-13 Toranomon, Minato-ku, Tokyo 105-0001, JapanbBiology Group, National Institute of Polar Research, 10-3, Midori-cho, Tachikawa-shi, Tokyo 190-8518, Japanc Laboratory of Animal Science, Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japand Institute of Low Temperature Science, Hokkaido Univ., Kita-19, Nishi-8, Kita-ku, Sapporo 060-0819, Japan

eWildlife Research Center of Kyoto University, JASSO Bldg. 3F, 2-24 Tanaka-Sekiden-cho, Sakyo-ku, Kyoto 606-8203, Japan

Received 31 January 2010; revised 16 April 2010; accepted 10 May 2010

Available online 21 May 2010

Abstract

Antarctic ice cores could preserve ancient airborne microorganisms.We examined bacteria in two Antarctic ice core samples, aninterglacial age sample from Mizuho Base and a glacial age sample from the Yamato Mountains, by 16S rRNA gene sequencinganalysis. Bacterial density, the number of bacterial OTUs and Simpson’s diversity index was larger in theMizuho sample than in theYamato sample. The 16S rDNA clone library from the Mizuho sample was dominated by the phylum Firmicutes, while the largepart of that from the Yamato sample was composed of the Gamma proteobacteria group. Major sources of these identified bacteriaestimated from their database records also differed between the samples: in the Mizuho sample bacterial species recorded fromanimals were higher than that of the Yamato sample, while in the Yamato sample bacteria from aquatic and snow-ice environmentswere higher than that of the Mizuho sample. The results suggest that these bacteria were past airborne bacteria that would vary indensity, diversity and species composition depending on global environmental change. Our results imply that bacteria in Antarcticice cores could be used as new environmental markers for past environmental studies.� 2010 Elsevier B.V. and NIPR. All rights reserved.

Keywords: Ice core; Antarctica; Bacteria; Cold environment; Glacier

1. Introduction

Analyses of ice cores have often been used as usedas a means to reconstruct past environments. However,analysis of the biological contents of ice cores has been

* Corresponding author. Biology Group, National Institute of Polar

Research, 10-3, Midori-cho, Tachikawa-shi, Tokyo 190-8518, Japan.

Tel.: þ81 42 512 0768; fax: þ81 42 528 3492.

E-mail address: [email protected] (T. Segawa).

1873-9652/$ - see front matter � 2010 Elsevier B.V. and NIPR. All rights

doi:10.1016/j.polar.2010.05.003

relatively rare. Recently, snow algae which grew in thesnow and ice on glaciers and subsequently stored in icecores have been used as environmental markers in theanalyses of ice cores from lower latitude regions suchas the Himalaya and Patagonia (Yoshimura et al., 2000;Kohshima et al., 2002; Shiraiwa et al., 2002; Nakazawaet al., 2004; Uetake et al., 2006).

In Antarctica, the possibility of microbial activity inthe ice sheet is very small due to the cold and dryenvironment. But, the ice cores from Antarctica could

reserved.

mailto:[email protected]

http://ees.elsevier.com/polar

Fig. 1. Location map of Antarctica. Position of drilling sites of the

Yamato core, the Mizuho core, Dome Fuji station and the Vostok

station are shown.

216 T. Segawa et al. / Polar Science 4 (2010) 215e227

preserve microorganisms from the ancient atmospheretrapped in the snow and ice of the ice sheet. Thebiomass and species composition of the microorganismin the ice cores could reflect the global environmentalconditions at that time. Thus, microorganisms inAntarctic ice cores could be useful to reconstruct pastenvironments. However, analysis of microorganisms inthe Antarctic ice is still very limited. Abyzov andothers (Abyzov et al., 1995) studied microorganisms inthe ice cores drilled at Vostok Station by microscopicdirect observation. Some recent studies on microor-ganisms in Antarctic ice cores (Bulat et al., 2004;Christner et al., 2001, 2006; Lavire et al., 2006)identified bacteria by 16S rRNA gene analysis.However, all these recent studies focused on extrem-obiosphere research to study bacteria living in thesubglacial lakes deep beneath the ice sheet.

In this study, we examined and compared bacterialdiversity in two Antarctic ice core samples, a Holocene(MIS1) interglacial age sample (2000e4000 years old)from Mizuho Base in Enderby Land and an MIS3glacial age sample (55,000e60,000 years old) from theYamato Mountains in Dronning Maud Land, by 16SrRNA gene sequencing analysis to evaluate thepotential use of these ice core bacteria in past envi-ronmental studies. Although the sample availabilitywas limited and the study was at a preliminary level,we found that bacterial groups and estimated sourcesof the bacteria in the ice cores were different betweenthe glacial and interglacial samples. Microorganismstrapped in ice cores can therefore be used as an indi-cator for the paleo-climate in addition to the tradi-tionally used chemical components i.e. isotopic ratios,because phylogenetic analyses on microorganisms canindicate their association with particular environmentssuch as soil, water, plants or animals. Therefore, ourresults demonstrate the potential of PCR-basedapproaches to the bacterial community identification asa new marker of past environmental conditions.

2. Materials and methods

2.1. Sampling procedures

Two ice core samples were obtained in 1983 byglaciologists of the 24th Japanese Antarctic ResearchExpedition. Although our ice cores were samples 20years ago, they have been carefully preserved at�20 �C in a plastic tube at the ice core storage facilityat the Institute of Low Temperature Science, HokkaidoUniversity, Japan. These samples did not suffer anymelting, nor showed any cracks that could have led to

a contamination. They were inspected with the utmostcare before conducting the analysis.

One of the samples was from near the Mizuho stationin Enderby Land and the other was from the southernpart of the Yamato Mountains, Dronning Maud Land,both of which are located in the East Antarctica (72�150

S, 46�320 E, 2700 m a.s.l. and 72�050 S, 35�110 E,2200 m a.s.l. respectively, Fig. 1). The estimated age ofthe ice core sample from Mizuho Base (98.00e99.50 mbelow surface) ranged from 2000 to 4000 years old, andthat from the Yamato Mountains sample(24.80e25.30 m below surface) ranged from 55,000 to60,000 years old (Moore et al., 2006). The age of theMizuho ice core sample was identified by diagnosticlayers of volcanic ash fallout from historically docu-mented eruptions and the pattern of delta 18Omeasurements (Dr. Hideki Narita, personal communi-cation). We could not compare glacial and interglacialsamples in the same ice core because we could not findgood ice samples without cracks for both periods in thesame ice core. The two ice core samples that weexamined were almost free from small cracks therebyminimizing the risk of contamination. Although the twoice cores were obtained from different location, theywere collected at almost the same latitude. In addition,the drilling locations of the two ice cores were relativelynear (about 300 km in distance), and at similar distancesfrom the coast (about 350 km and 250 km, respectively).Therefore, we compared these two ice core samples fortheir bacterial composition. Since both ice cores weredrilled without the aid of drilling fluid and the parts ofthe ice cores used for this study were ice without small

217T. Segawa et al. / Polar Science 4 (2010) 215e227

cracks, the possibility of contamination from the icecore surface during drilling operations and storageperiod is low.

The volume of each sample analyzed in this studywas approximately 1000 cm3. To avoid possiblecontamination from the outer surface of the ice core,the surface portion (1e2 cm thick) of the ice core wasremoved with a ceramic knife sterilized by MilliQwater, ethanol and UV exposure for 24 h before use.The remaining ice block was stored in a sterile plasticbag (Whirl-pak bags, Nasco, Fort Atkinson, WI,USA). This manipulation was done under asepticcondition in a cold room within a Class 100 Cleanbench (Sanyo, Japan). Christner et al. (2005) experi-mentally showed that contaminant bacterial cells onthe ice surface can be effectively removed byremoving surface ice by about 1 cm thick. We alsoconfirmed the effectiveness of our decontaminationstep by the experiment as follows. We made an iceblock of sterile MilliQ water with the size and shape(10 cm in diameter and 30 cm in length) just same asthe ice core samples and applied with culturedmedium of Escherichia coli JM109 (about30 ml � 107 cells/ml) on the surface. This E. coli wassuccessfully eliminated by the above mentionedremoval step, because no amplification of bacterial16S rDNA was observed by polymerase chain reac-tion (PCR) from the remaining ice block.

2.2. Bacterial concentrations

To determine the bacterial cells, the samples weremelted within a Class 100 Clean bench (Sanyo,Japan), enumeration of bacteria contained in icesamples was performed from 2% formalin-preservedthawed samples. Portions (2 ml) of thawed sampleswere filtered through 0.22 mm pore size filters (Mil-lipore Japan, Tokyo). Filters were stained for 15 minin the dark with SYBR Green I (Molecular Probes,CA) according to Noble and Fuhrman (1998). Stainedcells on the filters were counted using a Nikon Opti-phot 2 epifluorescent microscope at 1000� magnifi-cation. The total number of cocci and rods inrandomly selected 20 fields was determined, and thenumber of cells in a unit of volume (ml) wascalculated.

2.3. DNA extraction and PCR-amplification

All manipulations were conducted within a Class100 Clean bench (Sanyo, Japan). Another portion(approximately 1000 cm3) of ice block samples was

thawed and filtered through a sterile 0.22 mm filter unit(Nalgene, Tokyo). Membrane filter was transferred toa 15-ml sterile plastic tube (IWAKI, Tokyo) andsubjected to DNA extraction by the method of (Godonet al., 1997), with a FastPrep FP120 instrument afterbeing thoroughly washed with a solution A (1 ml of4 M guanidine thiocyanate in 0.1 M TriseHCl buffer(pH 7.5) mixed with 150 ml of 10% N-lauroylsarcosine).

A portion (2 ml) of bacterial DNA sample wassubjected to PCR-amplification of SSU rDNA withExTaq DNA polymerase (Takara, Kyoto, Japan)according to the manufacturer’s directions. The PCRprimers used for the amplification of V6 - V8 regionsof 16S rDNAwere 907F (Lane et al., 1985) and 1389R(Osborn et al., 2000). The PCR was performed in thefollowing temperature cycle; 3 min of initial denatur-ation at 95 �C followed by 30 thermal cycles (95 �C for30 s, 56 �C for 30 s, 72 �C for 45 s), and a finalextension at 72 �C for 10 min. The amplified productswere gel isolated and purified with a QIAquickTM GelExtraction Kit (QIAGEN, Hilden, Germany). Thesewere ligated into a pGEM-T vector (Promega, Madi-son, WI) according to the manufacturer’s directions.E. coli JM109 (TAKARA, Japan) was transformed bythe cloning vector, and the transformants were selectedby the blue-white selection on Luria-Bertani agarplates containing ampicillin (100 mg/ml), X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside; 1 mg/plate) and IPTG (isopropyl-beta-D-thiogalactopyrano-side; 2.38 mg/plate). The insertion of the appropriatesize DNA was determined by colony PCR-amplifica-tion with the universal primer sets (M13F and M13R)located on both sides of the cloning vector. The colonyPCR was performed in the following temperaturecycle; 3 min of initial denaturation at 94 �C followedby 30 thermal cycles (94 �C for 30 s, 54 �C for 30 s,72 �C for 60 s), and a final extension at 72 �C for10 min.

2.4. Nucleotide sequencing analyses

Sequence analysis was carried out on 59 randomlyselected clones from the Mizuho ice sample, and 56clones from the Yamato Mountains. Nucleotidesequencing was carried out with a Big Dye TMTerminator Cycle Sequencing Ready Reaction and anautomatic sequence analyzer (ABI 3100, AppliedBiosystems, Tokyo). Universal primers, M13F andM13R, were used as sequence primers. Chimericsequences were identified by using Chimera checkwith the Bellerophon (version 3) function on the


Greengenes web server (DeSantis et al., 2006).Homology searches were performed against the DDBJ/EMBL/GenBank nucleotide sequence database by thegapped FASTA program at the DDBJ web site (http://www.ddbj.nig.ac.jp/Welcome.html).

The obtained libraries were then analyzed using theMothur program (http://www.mothur.org/wiki/Main_Page) by comparing Operational taxonomic units(OTUs) on the basis of 98% similarity betweensequences as determined using the furthest neighboralgorithm in Mothur. A representative sequence waschosen from each OTU by a majority decision. Mothurdata were used to calculate a Simpson’s diversity index(D) and a Chao 1 richness estimate. The obtainedsequences were compared with those in the databases,and those displaying similarity higher than 98% withknown species were identified as correspondingspecies. Those displaying similarity lower than 98%with the sequences in the databases were considered asunknown taxa. The neighbor-joining method tree wasconstructed using Mega software, version 4 (Tamuraet al., 2007).

2.5. Nucleotide sequence accession numbers

The reported SSU rRNA sequences are listed withtheir respective GenBank accession numbers(AB555595 to AB555647).

3. Results

3.1. Total bacterial count

Direct counts by epifluoresence microscopyrevealed population densities of 2.4 � 104 and6.5 � 103 cells in 1 ml ice core melt water respectivelyfor the Mizuho ice core sample and the YamatoMountains ice core sample.

3.2. Identification of bacterial 16S rDNA in theAntarctic samples

Table 1 shows the list of bacterial species identifiedin the Antarctic ice core samples. Cloning analysisidentified 45 OTUs (operational taxonomic units) intotal from both ice core samples. Half (21 out of 45) ofthe OTUs were found to be closely related to sequencesof already isolated and identified organisms (>98.0%similarity). Our cloning analysis identified 33 and 20OTUs respectively for the Mizuho and Yamato ice coresamples. Eight OTUs were detected from both samples.The number of OTUs in theMizuho ice core sample was

larger than that of the Yamato ice core sample. FourteenOTUs from the Mizuho sample were identified asknown bacterial species, and 13 were identified asknown species in the Yamato sample. The remainders,19 and 7 clones, respectively for the Mizuho andYamato samples, were considered to be previouslyundescribed species, because these OTUs showed lowsimilarity (<98.0%) with already identified bacteria.The nonparametric abundance estimators Chao 1(Chao, 1984) were used to predict total richness at eachsite. The coverage (numbers of phylotypes/Chao1richness estimator) of the clone libraries was 48 and60% from the Mizuho and Yamato samples, respec-tively. Although clone numbers that we analyzed (59from Mizuho and 56 from Yamato) were not enough tocorrectly showOTU diversity in the samples, it providesan approximate measure of the difference in the OTUcomposition between the samples.

The Simpson’s reciprocal index of the Yamato icecore samples was 5.7. That was smaller than that ofMizuho ice core sample (24.1). So the numbers ofOTUs and biodiversity of the Mizuho ice core waslarger than that of the Yamato Mountains ice coresample. These were the bacteria belonging to classesAlpha-proteobacteria, Beta-proteobacteria, Delta-pro-teobacteria and Gamma-proteobacteria, phylum Bac-teroidetes, phylum Firmicutes, phylum Actinobacteria,phylum Cyanobacteria and phylum Deinococci.

Fig. 2 shows the prevalence of particular phyloge-netic groups detected in the ice core samples. Largeparts of the 16S rDNA clone library were composed ofbacteria belonging to the gamma proteobacteria (46%)in the ice sample from Yamato Mountains, whereas theFirmicutes (47%) was predominantly detected from theMizuho ice core sample. In the ice core sample fromYamato Mountains, most of OTUs were assignable toestablished bacterial species belonging to generaChryseobacterium, Clostridium, Kluyvera, Lactoba-cillus, Megasphaera, Methylobacterium, Moraxella,Propionibacterium, Succinivibrio, Streptococcus,Thauera, Thermus and Variovorax. In the ice coresample from the near Mizuho Base, most OTU wereassignable to established bacterial species belongingto genera Abiotrophia, Escherichia, Lactobacillus,Megasphaera, Prevotella, Propionibacterium, Staphy-lococcus, Streptococcus, Succinivibrio, Sutterella andVariovorax.

Among them, eight OTUs were equally detectedfrom both ice core samples. One was a psychrotrophicbacterium, Variovorax paradoxus (AY169432), 6enteric bacteria, Lactobacillus murinus (FJ348444),Megasphaera elsdenii (EU728750), Propionibacterium

http://www.ddbj.nig.ac.jp/Welcome.html

http://www.ddbj.nig.ac.jp/Welcome.html

http://www.mothur.org/wiki/Main_Page

http://www.mothur.org/wiki/Main_Page

Table 1

The list of bacterial species identified in the Antarctic ice core samples.

Bacterial

division

cluster number Accession

number

No. of

clones

Closest GenBank relative/Accession number %

similarity

Actinobacteria Miz-cluster19 AB555614 1 Uncultured bacterium clone FFCH3263/EU134558 98

Miz-cluster28 AB555623 1 Propionibacterium acnes/EF592610 100

Bacteroidetes Miz-cluster5 AB555600 1 Uncultured bacterium clone p-1273-a5/AF371904 99

Miz-cluster6 AB555601 1 Uncultured bacterium clone p-251-o5/AF371913 99

Miz-cluster8 AB555603 1 Uncultured bacterium clone Lq-C2-7/AY816531 95

Miz-cluster10 AB555605 2 Uncultured bacterium clone calf32_6wks_grp5_D01/GQ448180 99

Miz-cluster12 AB555607 2 Uncultured bacterium clone p-1297-a5/AF371872 97

Miz-cluster15 AB555610 1 Uncultured bacterium clone RRH_aaa04d04/EU475009 94

Miz-cluster17 AB555612 1 Prevotella sp. DJF_LS17/EU728743 100

Miz-cluster21 AB555616 1 Uncultured equine intestinal eubacterium sp. clone CW07/AJ408077 95

Miz-cluster22 AB555617 1 Uncultured bacterium clone RHSD_aaa02f06/EU778050 96

Miz-cluster23 AB555618 2 Uncultured bacterium clone p-1273-a5/AF371904 96

Miz-cluster27 AB555622 1 Uncultured bacterium clone calf32_2wks_grp1_A01/GQ448041 91

Miz-cluster29 AB555624 1 Uncultured bacterium clone TS6_a04b03/FJ371366 99

Miz-cluster31 AB555626 2 Uncultured bacterium clone VWP_aaa03a06/EU475121 99

Cyanobacteria Miz-cluster3 AB555598 1 Uncultured cyanobacterium clone 0M1_H8/DQ513851 100

Firmicutes Miz-cluster0 AB555595 8 Megasphaera elsdenii/EU728750 100

Miz-cluster2 AB555597 1 Uncultured bacterium clone SMB105/AM183062 100

Miz-cluster4 AB555599 5 Uncultured bacterium clone R-8224/FJ881232 100

Miz-cluster7 AB555602 7 Streptococcus alactolyticus/EU728776 100

Miz-cluster11 AB555606 1 Uncultured bacterium clone fcr21/AY438442 99

Miz-cluster16 AB555611 1 Abiotrophia defectiva strain 99383068/AY879306 100

Miz-cluster18 AB555613 1 Staphylococcus sp. SIIA 2055/FJ492844 100

Miz-cluster20 AB555615 1 Unidentified microorganism clone Cal18/AJ241722 100

Miz-cluster26 AB555621 1 Uncultured bacterium clone F146/AM500849 99

Miz-cluster30 AB555625 1 Lactobacillus acidophilus strain KLDS 1.0738/EU626023 100

Miz-cluster32 AB555627 1 Uncultured bacterium clone B843/DQ325956 99

a-proteobacteria Miz-cluster9 AB555604 2 Uncultured alpha proteobacterium clone G03_MO03/EF220873 99

Miz-cluster13 AB555608 1 Uncultured bacterium clone calf32_2wks_grp1_C10/GQ448009 99

Miz-cluster24 AB555619 1 Uncultured bacterium clone DP7.2.69/FJ612263 99

g-proteobacteria Miz-cluster1 AB555596 4 Uncultured bacterium clone RT_aai10h09/EU459690 99

Miz-cluster14 AB555609 1 Escherichia coli (EHEC Strain ATCC43895)/Z83205 99

d-proteobacteria Miz-cluster25 AB555620 2 Uncultured bacterium clone COL_aai15g07/EU460169 99

Actinobacteria SY-cluster1 AB555629 3 Propionibacterium acnes/EF592610 100

Bacteroidetes SY-cluster6 AB555634 1 Uncultured bacterium clone HY2_e09/EU776101 92

SY-cluster10 AB555638 1 Uncultured bacterium clone RRH_aaa04d04/EU475009 94

SY-cluster11 AB555639 1 Uncultured bacterium clone RRH_aaa03g11/EU474928 98

SY-cluster12 AB555640 1 Uncultured bacterium clone ML_aaj26b12/EU461645 99

SY-cluster16 AB555644 1 Uncultured bacterium clone CR98-35-34/AF428916 99

SY-cluster18 AB555646 1 Uncultured bacterium clone calf32_2wks_grp3_E03/GQ448838 93

Deinococci SY-cluster14 AB555642 1 Thermus sp. dNBae-1X/EU680808 100

Firmicutes SY-cluster0 AB555628 1 Uncultured bacterium clone C2-66/GQ896811 99

SY-cluster2 AB555630 2 Streptococcus alactolyticus/EU728776 99

SY-cluster4 AB555632 8 Megasphaera elsdenii/EU728750 100

SY-cluster13 AB555641 1 Uncultured bacterium clone A1-197/GQ897503 99

SY-cluster17 AB555645 1 Uncultured bacterium clone SMC120/AM183092 99

SY-cluster19 AB555647 1 Uncultured bacterium clone R-8224/FJ881232 99

a-proteobacteria SY-cluster9 AB555637 2 Methylobacterium sp. C1FA3/GQ228576 100

b-proteobacteria SY-cluster8 AB555636 1 Uncultured bacterium clone A1-G3_M13R/GU083406 100

SY-cluster15 AB555643 3 Uncultured bacterium clone DP7.2.69/FJ612263 98

g-proteobacteria SY-cluster3 AB555631 22 Bacterium A2(2009)/GQ398339 100

SY-cluster5 AB555633 2 Kluyvera cryocrescens/Y07651 99

SY-cluster7 AB555635 2 Uncultured bacterium clone RT_aai10h09/EU459690 99

* Miz: Mizuho ice core samples.

** SY: Yamato ice core samples.


0.05

100

100

100

93

100

100

100

100

99

99

67

88

100

100

100

100

65

SY-cluster0 (1

AY234442 Bacterium Ellin5

Miz-cluster19 (1 clone)_AB

EF59

SY-cluster4 (

Miz-cluster11 (1 clone )_

Miz-c

SY-cl

FJ492844 Staphyloc

Miz-cluster18 (1 clo

Miz-clus

Miz-cluster0

EU728750 M

AB034191 Anaerovibr

EU626023 Lactoba

Miz-cluster30 (1 cl

SY-cluster1

X77844 Clos

X68178

SY-cluster1

Miz-clu

Miz-cluster20 (1 clone

GQ422722 Selenomo

FJ348444 Lactobacillus murinu

Miz-cluster32 (1

EU728736 Lachn

Miz-cluster7 (7 clon

AY879306 Abiotrop

Miz-cluster16 (1 clo

SY-cluster19 (1 clone)_AB55

Miz-cluster4 (5 clones)_AB55

EU728776 Streptococ

SY-cluster2 (2 clon

100

Fig. 3. Neighbor-joining tree showing the phylogenetic relationship of the

their nearest cultured relatives based on GenBank 16S rRNA gene sequence

the number of clones within each cluster is shown. Bootstrap values generat

at the nodes. Bootstrap values greater than 60% are shown.

Fig. 2. Distribution of OTU sequences from Mizuho ice core and

from Yamato ice core in major phylogenetic groups.


acnes (EF592610), Succinivibrio dextrinosolvens(Y17600), Streptococcus alactolyticus (EU728776)and Uncultured bacterium clone p-251-o5 (AF371913)were also detected from the two ice core samples. Theremainder one cluster was considered to be unknownspecies, because the OTU showed low similarity withalready identified bacteria.

Fig. 3 and Fig. 4 shows the phylogenetic analysis ofsmall subunit rRNA sequences amplified from DNA inthe ice core samples. Miz-cluster-9 was 3.0% differentfrom Sphingomonas sp. Enf2 (DQ339610), SY-cluster-17 was 3.4% different from Clostridium butyricum(X68178), SY-cluster0 was 4.2% different fromBacterium AN1045 (DQ286651), and Miz-cluster-23

clone)_AB555628

0

555614

2610 Propionibacterium acnes

8 clones)_AB555632

AB555606

luster28 (1 clone)_AB555623

uster1 (3 clones)_AB555629

occus sp. SIIA 2055

ne) _AB555613

ter2 (1 clone)_AB555597

(8 clones)_AB555595

egasphaera elsdenii

io lipolytica

cillus acidophilus strain KLDS 1.0738

one)_AB555625

3 (1 clone) _AB555641

tridium celatum strain DSM 1785

Clostridium butyricum NCIMB8082

7 (1 clone)_AB555645

ster26 (1 clone)_AB555621

)_AB555615

nas sputigena clone K168

s strain M-8

clone)_AB555627

ospiraceae bacterium DJF CR52

es )_AB555602

hia defectiva strain 99

ne)_AB555611

5647

5599

cus alactolyticus

es)_AB555630

Firmicutes

Actinobacteria

Firmicutes and Actinobacteria detected in the ice core samples and

s. One representative clone sequence within each cluster is shown and

ed from 1000 replicates using the neighbor-joining method are shown

99

100

100

100

91

98

67

82

94

100

100

64

81

60

80

73

100

100

99

100

100

86

98

100

98

100

100

100

60

100

62

100

100

87

100

100

76

100

87

EF601823 Chryseobacterium sp. HX31

EU680808 Thermus sp. dNBae-1X

SY-cluster3 (22 clones)_AB555631

GQ284472 Moraxella osloensis strain PCWCW3

CP000252 Syntrophus aciditrophicus SB

Y17600 Succinivibrio dextrinosolvens

EU728713 Prevotella sp. DJF B116

Z83205 E.coli EHEC Strain ATCC43895

Y07651 Kluyvera cryocrescens

DQ339610 Sphingomonas sp. Enf2

GQ228576 Methylobacterium sp. C1FA3

GQ861460 Variovorax sp. LZA10

AF218619 Prevotella ruminicola strain TC2-28

AY550998 Prevotella sp. oral clone IDR-CEC-0093

AB244774 Prevotella stercorea strain CB35

GQ422736 Prevotella sp. oral taxon 302 strainF0020

AY207061 Prevotellaceae bacterium P4P 62

EU850616 Thauera sp. Q4

AJ566849 Sutterella stercoricanis CCUG 47620

AF040719 Prevotella sp. RZ

EU728743 Prevotella sp. DJF LS17




Miz-cluster22 (1 clone)_AB555617










Miz-cluster14 (1 clone) _AB555609








Miz-cluster12 (2 clones) _AB555607






Miz-cluster13 (1 clone)-AB555608




AF218620 Prevotella ruminicola strain TF2-5

0.05

Bacteroidetes

Deinococci

proteobacteria

Fig. 4. Neighbor-joining tree showing the phylogenetic relationship of the Proteobacteria, Bacteroidetes and Deinococci detected in the ice core

samples and their nearest cultured relatives based on GenBank 16S rRNA gene sequences. One representative clone sequence within each cluster

is shown and the number of clones within each cluster is shown. Bootstrap values generated from 1000 replicates using the neighbor-joining

method are shown at the nodes. Bootstrap values greater than 60% are shown.


was 4.8% different from Prevotellaceae bacteriumP4P_62 P1 (AY207061), suggesting that these clusterscould originate from novel species. Several OTUs wereeither distinct from any sequence in the databases (e.g.,Miz-cluster-8) or were closely related to environmentalsequences detected only by 16S rRNA gene amplifi-cation (e.g. Miz-cluster-10, Miz-cluster-12,Miz-cluster-20, Miz-cluster-25, Miz-cluster-26, andMiz-cluster-31). The difference in sequences between

these OTUs and characterized isolates was greater than5%, suggesting that they are sufficiently different fromknown organisms to be representatives of new taxa.

3.3. Source of the bacteria estimated from the recordsin the database

Thirty-six OTUs were found to be closely related tosequences of already detected microorganisms in the


database (>98.0% similarity). The source of thesebacteria was estimated from the records in the database(including descriptions of uncultured bacteria). Table 2shows the source of bacteria suggested from databaserecords for each OTU detected. As shown in Table 2,the OTUs detected in this study belonged to broadrange of bacteria from different habitats such as soil

Table 2

List of bacterial species distribution based on the source environment of the

Cluster No. Soil Sea

water

Fresh

water

Thermophilic

env.

Sno

ice

Miz-cluster0, SY-cluster4 e e e e e

Miz-cluster1, SY-cluster7 e e e e eMiz-cluster5 e e e e e

Miz-cluster6, SY-cluster11 e e e e e


Miz-cluster11 e e e e e


Miz-cluster13 e e e e eMiz-cluster16 e e e e e







SY-cluster0 e e e e e

SY-cluster12 e e e e eSY-cluster13 e e e e e

SY-cluster14 e e e B e

Miz-cluster2 B e B e e

Miz-cluster4, SY-cluster19 B e e e eMiz-cluster14 B e e e e

Miz-cluster18 B B B B e

Miz-cluster19 B e e e e

Miz-cluster24, SY-cluster15 B B B e BMiz-cluster28, SY-cluster1 B e B B e

Miz-cluster3 B e B e e

Miz-cluster9 B e e e eSY-cluster16 e e B e e

SY-cluster3 B B B B B

SY-cluster5 B B B e B

SY-cluster8 B e B e eSY-cluster9 B B B e B





Miz-cluster27 e e e e eSY-cluster6 e e e e e



bacteria, thermophilic bacteria, and enteric bacteria.We classified their possible sources as soil (13 OTUs),fresh water environments (10 OTUs), seawater envi-ronments (5 OTUs), thermophilic environments (4OTUs), snow and ice environments (4 OTUs), atmo-sphere (4 OTUs), plant (2 OTUs), animal (25 OTUs)and human (15 OTUs). About half (18 of 36) of the

closest relatives.

w and Air Plant Animal Human Unknown Environment

e e B B e

Intestinal

disease germ

e e B e ee e B e e

e e B e e

e e B e ee e B e e

e e B e e

e e B e e

e e B B ee e e B e

e e B e e

e e B e e

e e B e ee e B e e

e e B e e

e e B B e

e e B e ee e B B e

e e e B e

e e B B ee e B B e

e e e e e Thermo

B e B B e

Various

e e B e ee e B B e

B e B B e

e e e e e

e B e e eB e B B e

e e e e e

e e e e ee e e B e

e e e B e

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e e e e eB e e e e

e e e e B

Unknown

e e e e Be e e e B

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e e e e B

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e e e e B


OTUs were found to be closely related to bacteriarecorded only from single environment. Thirteen OTUswere recorded only from animal, 2 OTUs were recor-ded only from human, 2 OTUs were recorded onlyfrom soil and 1 OTU was recorded only from ther-mophilic environment. The remainders, 18 OTUs, wererecorded from multiple environments.

Thirteen OTUs were close to bacteria isolated fromsoil environments (Table 2). For example, Miz-cluster-28 and SY-cluster-1 were close to ActinobacteriumPI_GH2.1.C2 (AY162041, 100% similarity) isolatedfrom tropical rain forest soil in Ghana (Zengler et al.,2002). SY-cluster-9 was close to Methylobacteriumsp. HN2006B (AM231910, 100% similarity) isolatedfrom agriculture soil in Malaysia (Huyop F.Z,Unpublished).

Ten OTUs were close to bacteria isolated from freshwater environments. For example, SY-cluster-3 wasclose to Moraxella osloensis (AJ505859, 99% simi-larity) isolated from lake water in Germany (Probianet al., 2003).

Five OTUs were close to bacteria isolated fromseawater. For example, SY-cluster-3 was close touncultured gamma proteobacterium clone SBET_132.(AJ630697, 98% similarity) isolated from North Seasurface water (Sekar et al., 2004).

Four OTUs were recorded from thermophilic envi-ronment. For example, SY-cluster-14 was close tobacteria detected only from thermal environmentsThermus sp. RH-0401 (AY731822, 99% similarity)detected from the Rehai geothermal region in Teng-chong, Yunnan, China (Lin et al., 2005), and Thermusbrockianus (Y18409, 98% similarity) from Yellow-stone National Park, USA (Chung et al., 2000).

Four OTUs were close to bacteria isolated fromsnow and ice environments. SY-cluster-9 was close toMethylobacterium sp. G296-5 (AF395035, 99% simi-larity) isolated from Lake Vostok accretion ice(Christner et al., 2001) and Methylobacterium sp. zf-IVRht11 (DQ223682, 99% similarity, Zhang,S. et al.unpublished) isolated from ice cores of East RongbukGlacier near Mt. Everest. Miz-cluster-24 and SY-cluster-15 were close to V. paradoxus (AY169432, 99%similarity) isolated from a Greenland ice core sample(Sheridan et al., 2003), and Variovorax sp. Tibet-S862(DQ177491, 98% similarity, unpublished) isolatedfrom Qinghai-Tibet Plateau permafrost, and Glacierbacterium FJI16 (AY315173, 98% similarity) isolatedfrom a glacier in New Zealand (Foght et al., 2004).This clone was identical to the bacterial species (100%similarity) that was reported to grow in the mountainsnow of Teteyama mountains, Japan (Segawa et al.,

2005). SY-cluster-3 was close to Moraxella sp. I(DQ497238, 99% similarity) isolated from Arctic snowsample in Spitsbergen (Amato et al., 2007). SY-cluster-5 was close to Serratia sp. HHS4 (AJ846269, 99%similarity, Shivaji,S. and Chaturvedi,P. unpublished)isolated from Hamta glacier in Indian Himalaya.

Two OTUs were close to the bacteria isolated fromthe surface of living plants. SY-cluster-5 was close toKlebsiella sp. P2 (AB114634, 98% similarity, Meun-chang, S. et al., unpublished) isolated from a leaf ofpineapple and to Enterobacteriaceae bacterium A2JM(AY338233, 98% similarity) isolated from beansprouts (Rasch et al., 2005). Miz-cluster-24 and SY-cluster-15 were close to V. paradoxus strain HOT25(AY738651, 98% similarity) detected from a freshtomato leaf (Flagan and Leadbetter, 2006).

Twenty-five OTUs were close to bacteria detectedfrom animals. Most of them were from digestive tractof animals such as pigs and mice. Thirteen OTUs wereclose to bacteria isolated only from animal intestine.Other 12 OTUs were close to bacteria isolated not onlyfrom animals but also from other environments, suchas soil and aquatic environments.

Fifteen OTUs were close to bacteria detected fromhumans. For example, Abiotrophia defectiva strain99383068 (AY879306) and Uncultured bacteriumclone Eldhufec298 (AY920173) isolated from humanblood and human skin. However, only 2 OTUs (Miz-cluster-16 and SY-cluster-0) were close to bacteriarecorded only from humans.

The abundance of bacterial species distributionbased on the source environment of the closest rela-tives in the database is as follows. Soil: 36% (13/36),fresh water 28% (10/36), sea water 14% (5/36), snowand ice: 11% (4/36), thermophilic environments: 11%(4/36), atmosphere: 11% (4/36), plants: 6% (2/36),animals: 69% (25/36) and humans: 42% (15/36). Themajor part of the bacteria had been detected fromanimals or humans, and the next majority was detectedfrom the soil and fresh water environments.

The abundance and number of bacterial speciesestimated to come from each source environmentdiffered between the Yamato ice core sample and theMizuho ice core sample (Fig. 5). Although animalbacteria were dominant in both ice core samples, theabundance and number of the bacterial species recor-ded from animals were much higher in Mizuho sample(81%, 22 OTU) than in Yamato sample (56%, 9 OTU).In particular, bacteria recorded only from animals wereremarkably higher in the Mizuho sample (48%, 13OTU) than in the Yamato sample (19%, 3 OTU). Incontrast, the abundance of the bacterial species

Fig. 5. Abundance of bacterial species distribution based on the

source environment of the closest relatives in the database.


recorded from the following sources other than animalswere higher in the Yamato sample than in the Mizuhosample: the soil (Yamato: 44%, 7 OTU; Mizuho: 33%,9 OTU), the fresh water (Yamato: 44%, 7 OTU;Mizuho: 19%, 5 OTU), the sea water (Yamato: 25%, 4OTU; Mizuho: 7%, 2 OTU), the thermophilic envi-ronment (Yamato: 19%, 3 OTU; Mizuho: 7%, 2 OTU)the snow and ice environment (Yamato: 25%, 4 OTU;Mizuho: 4%, 1 OTU) and plants (Yamato: 13%, 2OTU; Mizuho: 4%, 1 OTU) (Fig. 5). Especially, thenumber of bacterial species recorded from the freshwater environment and those from the snow and iceenvironment was remarkably larger in the Yamatosample than in the Mizuho sample.

4. Discussion

4.1. Origin of the bacteria detected from the ice coresamples

A large part of the bacteria detected from the icecore samples in this study was interpreted as ancientairborne bacteria trapped and preserved in the snowand ice of the ice sheet because it contained bacteriafrom various environments. Though Antarctic indige-nous bacteria could be present in the ice cores, some ofthem were likely to have been transported by longdistance air circulation from outside of Antarctica.Twelve OTUs detected in the samples were identifiedto be bacterial species which have been detected onlyfrom various habitats in temperate and/or tropicalregions, such as the soil of tropical rain forest, Germanlake water, North Sea surface water, Chinese and NorthAmerican thermophilic environments and the surfaceof living tropical plant. These bacteria were quiteunlikely to come from Antarctica and its vicinities. Inparticular, those derived from the surface of vascularplants (SY-cluster-5, Miz-cluster-24, and SY-cluster-15) are most likely transported from terrestrial

ecosystems outside of Antarctica because there arealmost no vascular plants in Antarctica. Animalbacteria detected from the samples were also thoughtto be airborne bacteria transported over a long distancebecause the animal habitats nearest to the drilling sites,in the coastal region of Antarctica, are located morethan 250e350 km away. As we discuss later, thebacterial species composition of the samples domi-nated by animal bacteria, especially that of Mizuhosample, is probably due to the location of the drillingsites being relatively close to the costal region.

Four OTUs (Miz-cluster24, SY-cluster15, SY-cluster-3, SY-cluster-5, SY-cluster-9) were identified tobe bacterial species which have been isolated fromvarious snow and ice environments in the world, suchas Lake Vostok accretion ice in Antarctica, Himalayanglacier ice, Greenland ice core samples and Arcticsnow samples, respectively. These four bacteria alsocould be airborne bacteria transported from the snowand ice environments in Antarctica and/or from thosein various parts of the world. However, it is possiblethat they are surviving in the ice sheet because theyseem to be pre-adapted to this cold environment(Carpenter et al., 2000).

Bacteria detected from the ice core samples whichhave been previously recorded from human intestinesand skin surfaces could be contaminants introducedduring the sampling and/or analyzing procedure. Thesebacteria, however, could be also airborne bacteria fromvarious environments because most of these bacterialspecies have been reported not only from humans butalso from other environments, such as soil, fresh waterand air. Only two OTUs were identified to be speciesrecorded only from humans. Many animal and humanintestinal bacteria were also detected from Greenlandice core samples by 16S rDNA analysis with welldesigned decontamination processes (Sheridan et al.,2003). So, we estimated that most of these bacteriawere also airborne bacteria.

Bacterial density and diversity in the Mizuho andYamato ice core samples are larger than those reportedin previous studies on Antarctic ice cores, though westill have very limited data to compare with our results.Christner et al. (2006) who examined bacteria in theVostok ice core reported that cell density in the glacialice (171 me3,537 m depth) above lake Vostok rangedfrom 3.4 � 101 to 3.8 � 102 cells ml�1, a smaller valuethan our results (6.5 � 103 and 2.4 � 104 cells ml�1).They detected only 10 bacterial OTUs in the deepVostok ice core (3622 m depth), while our cloninganalysis identified 33 and 20 OTUs respectively for theMizuho and Yamato ice core samples. This difference


between the Vostok ice core and our ice cores is prob-ably due to the difference in the location of the drillingsite. Both of the ice core samples that we examined werecollected at the drilling site located near the coast (about250e350 km), a possible local source of airbornebacteria, while the drilling site of the Vostok ice corewas located in central part of the Antarctic ice sheet>1300 km from the coast. The bacterial speciescomposition of our samples are dominated by animalbacteria which also suggests that distance from the coastis a factor, because marine mammals and seabirds suchas Antarctic petrels and snow petrels living in the ice-free areas near the cost (Marchant and Higgins, 1990)are a possible source of these animal bacteria. Thisdifference could be also affected by the difference in themethods of analysis. In the analysis of the previousstudies on the Vostok ice core, bacterial DNA wasdirectly amplified by PCRwithout DNA extraction stepsusing very small amount of melted ice core samples(from 10 to 67 mL), while we amplified the DNAextracted from 1 L of melted ice.

4.2. Comparison between the Yamato sample (glacialage) and the Mizuho sample (interglacial age)

Bacterial cell density and biodiversity was higher inthe Mizuho ice core sample than in the Yamato ice coresample. This difference is mainly caused by thedifference in the number of animal bacteria. TheMizuho sample contained much more species ofanimal bacteria (22/27) than the Yamato sample (9/16).In contrast, the abundance of bacterial species esti-mated to come from other environments, such as thesoil, fresh water, sea water, thermophilic environments,snow and ice environments and plants, were higher inthe Yamato sample than in the Mizuho sample. Thesedifferences resulted in the differences in dominanttaxonomical groups of bacteria observed between thesamples. The Phylum Firmicutes, dominant in theMizuho sample, contained many animal bacteria, whilegamma Proteobacteria group, dominant in Yamatosamples, contained many bacteria from other envi-ronments (Table 2). Eight OTUs were detected fromboth samples, but we could not find an environmentalcommonality among the OTUs.

These differences observed between the Mizuhosample and the Yamato sample can be explained by thedifference in the environmental conditions between theHolocene MIS1 interglacial age and MIS3 glacial ageice. Bacterial density and species composition of theMizuho sample dominated by animal bacteria suggeststhat influx of animal bacteria from the coast area, the

local animal habitats, increased at the drilling site inthe interglacial age due to the retreat of the ice sheetand sea ice surrounding it. In contrast, the bacterialcomposition of the Yamato sample containing morebacterial species estimated to be transported overa long distance suggests that transportation of bacteriaby the atmospheric circulation and/or bacteria supplyfrom distant source areas increased in the glacialperiod. Such environmental conditions in the glacialperiod have been also inferred from studies on mineralparticles in Antarctic ice cores. The density of themineral particles in Antarctic ice cores has beenreported to increase in the glacial period, suggestingthat particle transportation increased by enhancedatmospheric circulation (Petit et al., 1999) and/or theexpansion of potential source areas of the particles, forexample dry areas around Patagonia, which increaseddue to the sea-level change and climate aridification inthe glacial period (Wolff et al., 2006).

Yao et al. (2006) who analyzed microorganisms inthe ice core from the Malan ice cap, China showed thatmicroorganisms concentrations tended to be negativelycorrelated with the temperature, and more microor-ganisms were associated with colder periods, sug-gesting increased particle transportation by wind inthe colder period. The Yamato sample (glacier agesample) contained more bacterial species (4 OTUs)estimated to come from snow and ice environmentsthan the Mizuho sample (interglacial age sample, 1OTU), though the total species number (20 OTUs) inthe Yamato sample was lower than that of the Mizuhosample (33 OTUs). It is possible that this was causedby increased bacterial supply from the cold environ-ments that expanded in the glacial age. Yao et al.(2006) also reported that number of bacteria isolatedby low temperature was much higher in the samples ofcold periods, suggesting that supply of cold adaptedbacteria from the cold environments increased in thecold period.

It is possible that the differences in bacterialcommunities observed between the Mizuho sampleand Yamato sample are a product of the differences inenvironmental conditions between the drilling sites.However, the Mizuho sample contained more bacterialcells dominated by more animal bacteria than Yamatosample, even though the drilling site of Mizuho sampleis higher in altitude (2700 m a.s.l.) and located furtherfrom the coast (350 km) than the drilling site of theYamato sample (2200 m a.s.l., 250 km). The factsuggests that the difference in bacterial communityobserved between these samples resulted not from thedifference in the location of the drilling site rather from


the difference in the age of accumulation. Thus, ourresults suggest that the bacterial groups present and theinferred source of the bacteria in the ice cores weredifferent between glacial and interglacial samplesreflecting the difference in environmental conditions.The microorganisms contained in the past Antarcticatmosphere and preserved in the ice core would changetherefore reflecting changes in environmental condi-tions of the source areas of these microorganisms overtime. Therefore, our results imply that bacteria inAntarctic ice cores could be used as new environmentalmarkers for past environmental studies. We can oftenestimate the source environment of the microorganismsusing database records, while it is generally difficultin geochemical tracers such as chemical ions andisotopic ratios. It is a potential advantage of usingmicrobes in ice cores as a new tool to reconstructpaleo-environments.

Acknowledgements

We thank Dr. Kuniko Kawai and Dr. Koji Fujimurafor their advice on the technique of PCR and phylo-genetic analysis. This research was partly supported bya grant-in-aid for young scientists (20710019) andgrants-in-aid for Basic Research B (No.16310004 andNo.19310020) from the Ministry of Education,Culture, Sports, Science, and Technology of Japan andby a grand from the Transdisciplinary Research Inte-gration Center (TRIC), Research Organization ofInformation and systems.

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