Unique clusters of Archaea in Salar de Huasco, an athalassohaline evaporitic basin of the Chilean

12 Pages·2010·0.22 MB·English

RESEARCH ARTICLE
UniqueclustersofArchaeainSalardeHuasco,anathalassohaline
evaporiticbasinoftheChileanAltiplano
CristinaDorador1,2,3,IrmaVila4,FranciscoRemonsellez5,JohannesF.Imhoff2&Karl-PaulWitzel1
1MaxPlanckInstituteforEvolutionaryBiology,Plo¨n,Germany;2Leibniz-InstituteofMarineSciences,Kiel,Germany;3DepartamentodeAcuicultura,
FacultaddeRecursosdelMar,UniversidaddeAntofagasta,Antofagasta,Chile;4DepartamentodeCienciasEcolo´gicas,FacultaddeCiencias,
UniversidaddeChile,Santiago,Chile;and5DepartamentodeIngenier´ıaQu´ımica,FacultaddeIngenier´ıayCienciasGeolo´gicas,UniversidadCato´licadel
Norte,Antofagasta,Chile
Correspondence:PresentAddress: Abstract
CristinaDorador,Departamentode
AnalysesofclonelibrariesfromwaterandsedimentsofdifferentsitesfromSalarde
Acuicultura,FacultaddeRecursosdelMar,
Huasco,ahigh-altitudeathalassohalinewetlandintheChileanAltiplano,revealed
UniversidaddeAntofagasta,Angamos601,
Casilla170,Antofagasta,Chile.Tel.:15655 the presence of five unique clusters of uncultured Archaea that have not been
657701;fax:[phone];e-mail: previouslyreportedorspecificallyassigned.Thesesequencesweredistantlyrelated
[email protected] (83–96% sequence identity) to a limited number of other clone sequences and
revealednoidentitytoculturedArchaea.TheabundanceofArchaeaandBacteria
Received19March2009;revised29March was estimated using qPCR and community composition was examined through
2010;accepted8April2010.
theconstructionofclonelibrariesofarchaeal16SrRNAgene.Archaeawerefound
Finalversionpublishedonline18May2010.
to be dominant over Bacteria in sediments from two saline sites (sites H4:
6.31(cid:2)104 and site H6: 1.37(cid:2)104mScm(cid:3)1) and in one of the water samples
DOI:10.1111/j.1574-6941.2010.00891.x
(freshwater from site H0: 607mScm(cid:3)1). Euryarchaeotal sequences were more
abundantthancrenarchaeotalsequences.Manyoftheclonesequences(52%)were
Editor:MichaelWagner
similartounculturedarchaealgroupsfoundinmarineecosystemshavingidentity
Y
Keywords values between 99% and 97%. A major fraction of the sequences (40%) were
G
archaealdiversity;16SrRNAgene;archaeal members of Methanobacteria, while others were included in the Marine Benthic
O amoAgene;AndeanAltiplano;Chile. Groups B and D, the Miscellaneous Crenarchaeotic Group, the Terrestrial
Miscellaneous Euryarchaeotal Group, Marine Group I and Halobacteria. The
L
O presence of uncultured archaeal groups in Salar de Huasco extends their known
distributionininlandwaters,providingnewcluesabouttheirpossiblefunctionin
C
theenvironment.
E

Y
G
pelagic deep-ocean picoplankton (Karner et al., 2001).
Introduction
O AmongtheEuryarchaeota,MarineGroupII(marineplank-
Archaea are widely distributed in both extreme (e.g. hot ton, anaerobic digestor), Marine Group III (marine sedi-
L
springs,hydrothermalvents,solfataras,saltlakes,sodalakes, ments, marine plankton) (DeLong, 1998), Marine Benthic
O
sewage digesters, rumen) and nonextreme (e.g. ocean Group D (MBGD, deep sea and salt marsh sediments)
I
B waters,lakes,soil)environments(Chabanetal.,2006).The (Vetriani et al., 1999) and the South Africa gold mine
O domainArchaeaconsistsoftwomajorphyla:Crenarchaeota euryarchaeoticgroup[SAGME-1,SAGME-2,sequencesalso
and Euryarchaeota. With the advent of molecular techni- included in the Terrestrial Miscellaneous Euryarchaeotal
R
ques, an immense number of 16S rRNA gene sequences of Group (TMEG); Takai et al., 2001] have been reported
C
‘uncultured Archaea’ have been retrieved in clone libraries frequently from terrestrial and marine environments (e.g.
MI fromdifferentenvironments(Schleperetal.,2005).Someof Inagakietal.,2003;Shaoetal.,2004;Sørensenetal.,2005;
these uncultured archaeal groups are known from marine Sørensen & Teske, 2006; Kendall et al., 2007). Among the
environments (DeLong, 1998; Vetriani et al., 1999; Takai Crenarchaeota,theMarineGroupI(MG-I),MarineBenthic
et al., 2001; Schleper et al., 2005; Teske & Sørensen, 2008) GroupA,BandMarineBenthicGroupC(MBGC,deepsea
and represent quantitatively important members of the sediments) were first reported from seawater (DeLong,
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292 C.Doradoretal.
1992;Fuhrmanetal.,1992;Vetrianietal.,1999),andmore
recently also from subsurface marine sediments (Sørensen
etal.,2004;Teske,2006).TheMiscellaneousCrenarchaeotic
Group (MCG) have a wider habitat range, which includes
terrestrialandmarine,hotandcold,surfaceandsubsurface
environments (Teske, 2006). Functional gene surveys and
pure culture studies have indicated that at least some
members of MG-I are aerobic and autotrophic ammonia
oxidizers(Francisetal.,2005;Ko¨nnekeetal.,2005).Studies
using reverse transcription have demonstrated that several
of these uncultured marine archaeal groups are metaboli-
cally active in deep subsurface sediments (Biddle et al.,
2006). Intensive studies of uncultured Archaea have been
conducted especially in marine environments, including
metagenomic analyses (e.g. Martin-Cuadrado et al., 2008).
However, little is known about their function in the envir-
onment.Variousauthorshavedescribedtheoccurrenceand
abundanceofvariousarchaealgroupsininlandwaters(e.g.
Sørensenetal.,2005;Galandetal.,2006;Brie´eetal.,2007;
Auguet&Casamayor,2008;Jiangetal.,2008). Fig.1. MapindicatingthelocationofSalardeHuascoandfourstudy
SalardeHuascoislocatedintheChileanAltiplanoatan sites(H0,H1,H4andH6).Grayareasindicatethepresenceofpermanent
altitude of 3800m and exhibits salinity conditions from lagoons.
freshwatertosaturatedsaltwaters(Doradoretal.,2008a);it
is considered an athalassohaline system because its salt
composition is markedly different from that of seawater (201180S,681500W)(Fig.1).Watersampleswerecollectedin
(e.g. Oren, 2006). Abiotic conditions in the Altiplano, plastic sterile bottles of 1L from the surface and sediment
including low temperatures (mean annual temperature samplesweretakenwithapolycarbonatehandcorer(30cm
o51C),lowatmosphericpressure(40%lowerthanthatat length and 3cm diameter). The surface area of the Salar
sea level), high solar radiation (o1100Wm(cid:3)2), strong extends to c. 50km2, with open water representing only
variation in different environmental properties at daily, 2.5km2, and the water level can vary seasonally (Risacher
annual and interannual time scales and a negative water etal.,2003).ThepondsareshallowandlocatedalongaN–S
balance, shape the biological communities in these water aspectthroughtheSalar,forminganaturalsalinitygradient
bodies (Vila & Mu¨hlhauser, 1987). Considering the envir- withfreshwatersitesH0(stream)andH1(shallowlagoon)
onmental conditions of this inland aquatic system, we and the saline site H4 (shallow lagoon). Site H6 (shallow
expected to encounter a highly adapted archaeal commu- lagoon) exhibited lower salinity than site H4 because the
nity,including taxaadaptedto highsaltconcentrations. In water comes from a different freshwater stream (Risacher
the present study, we describe the composition and abun- et al., 1999). A summary of some key morphometric,
dance of archaeal assemblages in water and sediment physical and chemical characteristics of the sampling sites
samplesat, in terms ofsalt concentration, fourcontrasting ispresentedinTable1.Amoredetaileddescriptionoftheir
sites from Salar de Huasco using denaturing gradient gel propertieswasdescribedinDoradoretal.(2008a).
electrophoresis (DGGE), clone libraries of the 16S rRNA
gene and quantitative PCR (qPCR). In addition, the pre-
DNA extractionand PCRamplifications
sence of ammonia-oxidizing Crenarchaeota was examined
by cloning the ammonia monooxygenase gene amoA, re- EnvironmentalDNAwasextractedfromwaterandsediment
flecting the possible role of Archaea in the biogeochemical samplesfromeachsite.ForDNAextraction,watersamples
nitrogencycle. were filtered on-site in a 0.22-mL pore-size filter without
prefiltration, with the volume of filtered water ranging
between0.05Lforsalinesites(sitesH4andH6)and1Lfor
Materialsand methods
the freshwater site (site H0). For DNA extractions of
sediment samples, we used 600mg of homogenized sedi-
Site descriptionand sampling
ment. All DNA extractions were carried out using the
DuringJanuary2005(summer),twosampleswerecollected UltraClean Soil DNA isolation kit (MoBio Laboratories
per site at four different sites from the Salar de Huasco Inc.)accordingtothemanufacturer’sinstructions.
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ArchaealdiversityinSalardeHuasco 293
Table1. PhysicalandchemicalcharacteristicsofwatersamplesandmicrobialabundanceatthefoursitesinSalardeHuasco
Sites
Characteristics H0 H1 H4 H6
Location 2011503200,6815202500 2011600800,6815202900 2011704100,6815300000 2011904300,6815001900
Altitude(m) 3799 3795 3789 3789
Type Stream Lagoon Lagoon Lagoon
Conductivity(mScm(cid:3)1) 607 645 6.31(cid:2)104 1.37(cid:2)104
Totaldissolvedsalts(gL(cid:3)1) 0.42 0.46 64.93 9.38
Dissolvedoxygen(mgL(cid:3)1) 6.9 10.3 0 8.4
pH 7.7 8.7 8.2 8.8
Temperature(1C) 16.6 19 20.1 12
Hour 14:30 16:00 15:50 10:15
N-NO(cid:3)(mgL(cid:3)1) 55.5 53.5 60 30
3
P-PO43(cid:3)(mgL(cid:3)1) 40.3 20.3 3.91(cid:2)103 807
S-SO2(cid:3)(mgL(cid:3)1) 22.1 26.6 3990 141.2
4
Si(mgL(cid:3)1) 19.9 12.4 25.4 18.1
Hardness(mgL(cid:3)1) 162.5 130 3500 1000
Totalalkalinity(mM) 2 1.52 12 7
Chla(mgL(cid:3)1) 2.2 8.5 39 114.3
Cations Ca214Na14K14Mg21 Ca214Na14K14Mg21 Mg214Ca214K14Na1 Ca214Mg214Na14K1
Archaealabundancewater(cellsmL(cid:3)1) 69 174 1.68(cid:2)104 7.8(cid:2)104
Archaealabundancesediment(cellsg(cid:3)1) 3.24(cid:2)106 5.04(cid:2)107 3.6(cid:2)107 5.94(cid:2)106
Bacterialabundancewater(cellsmL(cid:3)1) 45 288 5.34(cid:2)105 2.7(cid:2)106
Bacterialabundancesediment(cellsg(cid:3)1) 2.16(cid:2)108 5.94(cid:2)108 1.08(cid:2)107 2.52(cid:2)106
Amplificationof16SrRNAarchaealgenewasperformed werecarriedoutwithPfupolymerase(Promega)following
using a nested PCR approach. Fragments of 1500bp were themanufacturer’sinstructions.
obtainedwithprimersAr4F(positions8–25)andUn1492R,
and then primers Ar3F (positions 7–26)–Ar9R (positions
DGGEanalysis
906–927)(Jurgensetal.,2000)wereusedtoamplifythe16S
rRNAgenefromArchaeawiththefirst-roundPCRproducts DGGEwasperformedaccordingtoCasamayoretal.(2000)
astemplatesinanestedPCR.Thereverseprimerused,Ar9R withPCRproductsofarchaeal16SrRNAgenegeneratedin
(Jurgens et al., 2000), exhibited mismatches with the un- a nested approach. Primers 344F (Raskin et al., 1994) and
cultured groups SAGMEG, Marine Hydrothermal Vent 915R (Stahl & Amann, 1991) were used to amplify the
Group(MHVG,Takai&Horikoshi,1999),AncientArchaeal archaeal 16S rRNA gene PCR product. The 344F primer
Group (AAG, Takai & Horikoshi, 1999) and Deep-Sea contained an additional 40nucleotide GC-rich sequence
Archaeal Group (DSAG, Inagaki et al., 2003) (Teske & (GCclamp)atits50-endinordertomaintainstablemelting
Sørensen, 2008), which can possibly underestimate the behavior during DGGE (Muyzer et al. 1993). The PCR
presence of these groups in environmental samples. Each conditions were as described aboveand PCRamplification
PCRreactioncontained10(cid:2)PCRbufferwith2mMMgCl was carried out using a touchdown protocol as follows:an
2
(Roche), 200mM dNTP mixture (Gibco), 1pmol of each initialdenaturingstepof5minat941C,20cyclesof30sat
primer,2.5UTaqpolymerase(Roche),10–100ng template 941C,45sat65–551C(decreasedby0.51Ceverycycle)and
DNA and water to a final volume of 50mL. The PCR 1.5minat721C,andthen10cyclesof30sat941C,45sat
conditions used were initial denaturation at 941C for 551Cand1.5minat721C.PCRproductswereappliedonto
5min, 35 cycles of denaturation (30s at 941C), annealing 7.5% polyacrylamide gels containing a linear gradient of
(45s at 551C) and extension (1.5min at 731C). Archaeal 30–60%denaturantwhere100%denaturantwasdefinedas
amoAgenefragmentswereamplifiedbyPCRusingprimers 7Mureaand40%formamide.DGGEwascarriedoutinthe
CrenAmo1F and CrenAmo1R (Ko¨nneke et al., 2005) and BioRad D Gene System (Bio-Rad Laboratories, Hercules,
thefollowingPCRconditions:initialdenaturationat941C CA)at601C,200V,for6h.GelswerestainedwiththeSYBR
for3min;30cyclesofdenaturation(30sat941C),anneal- Goldnucleicacidgelstain(MolecularProbes).Inorder to
ing(30sat561C)andextension(3minat721C);andafinal examine the relationships between communities in the
extension at 721C for 1min. For cloning, PCR reactions different samples, a matrix was constructed from the
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294 C.Doradoretal.
distribution pattern of the bands in different samples, and concentrationof0.25mM)andnuclease-freeH Oaddedtoa
2
cluster analyses (UPGMA), based on percent similarity totalof20mL.TheArchaeaamplificationprogramconsisted
betweenthesamples,wereconductedusingthemultivariate of an initial denaturing at 951C for 10min, and then 40
statisticalpackage(MSVP,version3.12d;KovachComputing cyclesof951Cfor35s,561Cfor40sand721Cfor20s.The
Services,Wales,UK). Bacteriaamplificationprogramconsistedofaninitialdena-
turing at 951C for 10min, and then 40 cycles of 951C for
Cloning and16SrRNAgene sequenceanalysis 30s,601Cfor30sand721Cfor20s.Forbothamplification
programs,thefluorescencemeasurementswererecordedat
Archaeal clone libraries were generated from water and
theendofeachextensionstepandthemeltingcurveswere
sediment samples collected from the four study sites.
measuredwith a ramp increasing thetemperaturefrom50
Samples for cloning (one sample per site) were selected
to 951C by 11C every 5s. All analyses were performed in
according to their richness detected by DGGE (data not
triplicateincludinganegativecontrol.
shown). Purified amplicons were cloned into pCR-Blunt
vector(Invitrogen)accordingtothemanufacturer’sinstruc-
Standard curves and data analysis
tions. Analysis of the inserts, sequencing and sequence
analysis were described previously (Dorador et al., 2008b). To generate bacterial and archaeal standard curves, we
Briefly, sequences were checked for chimeras, rarefaction extracted plasmid DNA from 16S rRNA gene clones con-
curves were constructed and nonparametric richness esti- taining Escherichia coli and clone Hua1-w87, respectively.
matorsS andS andtheShannon–Weaverdiversity The plasmid DNAwas quantified using gel electrophoresis
ACE Chao1
index were determined manually and via theweb interface with a DNA low-mass ladder (Promega). Standard curves
available at http://www.aslo.org/lomethods/free/2004/ forbacterialandarchaeal16SrRNAgeneswereconstructed
0114a.html(Kemp&Aller,2004). usingserialdilutionsoftheseplasmids’DNA,rangingfrom
850to0.0085pg,asatemplate.TheDNAconcentrationsin
Phylogenetic analysis ngmL(cid:3)1 were transformed to 16S rRNA gene copy
numbermL(cid:3)1, by means of the following transformation:
The phylogenetic affiliations of the archaeal sequences
first, we obtained the molecular weight of DNA (plasmid
obtained here wereestimated as described elsewhere (Dor-
1insert),assumingthat660istheaveragemolecularweight
ador et al., 2008b). Sequences were aligned using the
ofabasepair.WedividedAvogadro’snumber(6.02(cid:2)1023)
alignment tool of the ARB package (http://www.arb-home.
by this molecular weight to estimate the abundance of
de), a maximum likelihood analysis in the program PHYML moleculesper1g.Finally,thisvaluewasdividedby1(cid:2)109
(Guindonetal.,2005)withtheGTRsubstitutionmodeland
to obtain the gene copy number per ng of DNA. The
100 bootstrap resamplings. Trees were edited using MEGA4
calibration curves were generated using the OPTICON MON-
(Tamura et al., 2007). Sequences with similarities 497%
ITORTMver.3.1.32software(Bio-RadLaboratories),andfor
were considered to represent the same phylotype (Stack-
each standard, the concentration was plotted against the
ebrandt&Goebel,1994).
cyclenumber,andthevalueatwhichthefluorescencesignal
increasedabovethethresholdvaluewasthecyclethreshold
Nucleotide sequenceaccession numbers
(C value). The bacterial and archaeal standard curve
t
The nucleotide sequences from this study are available in showed an efficiency of 1.23 (R2 value of 0.995) and 0.91
GenBank (http://www.ncbi.nlm.nih.gov) under accession (R2valueof0.999),respectively.
numbers EU481526–EU481630 for 16S rRNA gene se-
quences and FJ839431–FJ839434 for archaeal amoA Results
sequences.
MicrobialabundanceinSalar deHuasco and
qPCR quantification of16SrRNAgenes
qPCR was conducted using SYBR-Green PCR Master Mix Thetotalnumbersofmicrobialcellsinwaterandsediment
(Biotools)inanMJMini-Opticonthermocycler(Bio-Rad). samplesofsitesH0andH1werecalculatedbyqPCRresults
Primers for Archaea (ARCH349F and ARCH806R) and assumingonecopyof16SrRNAgenepercellandwere101
Bacteria (UBactF and UBactR) (Takai & Horikoshi, 2000; and 102cellsmL(cid:3)1 and 106–108cellsg(cid:3)1, respectively. For
Nadkarni et al., 2002) were used for the quantification of sites H4 and H6, these values fluctuated between 104 and
total archaeal and bacterial 16S rRNA genes, respectively. 106cellsmL(cid:3)1 for water and 106 and 107cellsg(cid:3)1 for sedi-
Thereactionmixturecontained10mLofSYBR-GreenPCR ment (Table 1). The estimated abundance of Bacteria
Master Mix (Biotools), 1mL of template DNA ((cid:5)10ng), andArchaeainwatersandsedimentsfromthefoursitesof
1mL of the corresponding oligonucleotide primers (final Salar de Huasco is shown in Fig. 2. There was a clear
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ArchaealdiversityinSalardeHuasco 295
MG-I) and represented a minor fraction in the sediments.
Euryarchaeota dominated in the sediments and were the
exclusive archaeal representatives in water, except site H0,
althoughdifferentgroupsdominatedinthedifferentwater
samples.Allsedimentshadaclearlydifferentcompositionas
comparedwiththecorrespondingwatersamples(Fig.3and
Fig.S4).
Crenarchaeota identified by clone sequences represented
14% of all clones and belonged to different phylogenetic
groups.AllclonesfromH0-w(fourphylotypes)andasingle
phylotype from H0-swereaffiliated to MG-I (Fig. S4)and
clustered in Group I.1b and Group I.1a (DeLong, 1998;
Fig.2. RelativeabundanceofBacteriaandArchaeainSalardeHuasco
Schleper et al., 2005), respectively. This cluster includes
atthesamplingsites,determinedbyqPCR.
sequences from soils, sediments, freshwater and subsurface
sediments. Sequences from H0-w were 97% similar to an
differentiation between water and sediment samples unculturedcrenarchaeonretrievedfromaradioactivether-
throughout the salinity gradient from site H0 (freshwater mal spring in the Austrian Alps (Weidler et al., 2007) and
stream)toH4(salinelagoon)(Fig.2). withthemoderatelythermophiliccrenarchaeoteCandidatus
Nitrososphaeragargensis(Hatzenpichleretal.,2008).Repre-
Construction of16SrRNAgene clone libraries sentatives of the MCG were found in sediments from sites
and estimation ofarchaeal richness H0, H1 and H6 (four phylotypes). Clone QLW1200-A33
from Qinghai Lake (Jiang et al., 2008) exhibited 97%
Clone libraries of 16S rRNA genes were constructed from
sequenceidentitywiththephylotypesHua6-s39andHua6-
sites H0, H1, H4 and H6. In total, 137 clones from water
s29. Also, the MBGB was found only in sediment samples
samplesand197fromsedimentswereobtained.Rarefaction
(H0-s,H4-s,H6-s).DatabasescontainsequencesofMGBG
curvesfrom thefour sites showedsaturation at low phylo-
retrieved from subseafloor sediments, lake sediments and
typenumbers,from4to11inwatersamplesandfrom10to
hydrothermalvents(Fig.4andFig.S4).
32 phylotypes in sediment samples (Supporting Informa-
Most of the clone sequences from water and sediment
tion, Fig. S1). The richness estimators S and S
ACE Chao1 samples were affiliated to various groups of Euryarchaeota
(Chao, 1984; Chao, 1987) provided estimates of the total
(Figs 3 and 4): Halobacteria, Methanosarcinales, Methano-
numberofphylotypesrangingfrom5to44(S )and4to
ACE microbiales, MBGD, TMEG and a group of unidentified
26(S )inwatersamplesandfrom23to107and18to91
Chao1 Euryarchaeota (7% of the clones). Methanogenic Archaea
insedimentsamples(Table2).TheShannon–Weaverdiver-
were most frequently detected in Salar de Huasco (40% of
sityindexindicatedahigherarchaealdiversityinsediments
theclones)(Fig.3).
(1.4–3.0) compared with the water samples (0.7–1.7). The
Halobacteria were represented by a single phylotype
numberofDGGEbandsrangedbetween4and16inwater
Hua6-w15, the closest cultured relative (89% sequence
samplesandbetween9and10insedimentsamples,which
identity) of which was Halorubrum lipolyticum (Cui et al.,
arelowerthantherichnessestimatedbyclonelibraries,and
2006)isolatedfromAibisaltlakeinXin-Jiang,China.
did not show differences between water and sediment
Methanosarcinalesrepresented25%ofallclonesandwere
samples.Nevertheless,thecompositionoftheDGGEbands
foundinsamplesH1-w,H0-sandH1-s(Fig.3andFig.S4).
showsonecluster thatlargelyconsistsofsedimentsamples
The most frequent clone sequence from H1 (phylotype
but also includes the water sample from site H6 (Fig. S2).
Hua1-s2) was 97% similar to Methanosaeta concilii (Eggen
The pattern of bands from water samples collected from
et al., 1989), while the most abundant clone from H0
different sites within the Salar de Huasco showed no clear
(phylotypeHua0-s82)was98%similartoMethanomethylo-
grouping, indicating that they were only distantly related.
voranshollandica.Thismethanogenwasisolatedpreviously
Forexample,watersamplesfromH4andH1were o60%,
from freshwater sediment and utilizes dimethyl sulfide as a
andthewatersamplefromH0was o50%similartowater
carbonandenergysource(Lomansetal.,1999).CloneHua0-
samplesfromalltheothersites.
s95 was 94% similar to Methanolobus oregonensis, isolated
fromanoxic,subsurfacesedimentsofasaline,alkalineaquifer
Phylogenetic analysis ofarchaeal communities
nearAlkaliLakeinOregon,USA(Liuetal.,1990).
inwaterand sediment
Methanomicrobiales were represented by 16% of the
Crenarchaeota were absent from the water samples (the clones. Members of this group were found in sediment
exception was site H0 with clones exclusively belonging to samples from the sites H0, H1 and H4, but a single clone
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296 C.Doradoretal.
Table2. Numberofclonesandphylotypes,richnessestimatorsS andS ,numberofbandsinDGGEandShannondiversity(H0)inthelibrariesof
ACE Chao1
16SrRNAgenefromwaterandsediment
Numberofclones Numberofphylotypes Predictedvalue Predictedvalue Shannondiversity Numberofbands
Site Clonelibrary inlibrary observed ofS ofS index(H0) inDGGE
ACE Chao1
H0 Water 27 4 5 4 0.7 16
H1 Water 56 6 8 7 1.1 4
H4 Water 33 11 44 26 1.6 10
H6 Water 21 8 17 10 1.7 7
H0 Sediment 64 23 34 30 2.8 10
H1 Sediment 35 13 23 18 2.1 10
H4 Sediment 45 10 36 32 1.4 9
H6 Sediment 53 32 107 91 3 9
Fig.3. Compositionofclonelibrariesof16S
rRNAgenefromwaterandsedimentsamples.
Affiliationofthephylogeneticgroupsisindicated
forEuryarchaeota(E)andCrenarchaeota(C).
Groupdesignations:MBGD,MarineBenthic
GroupD;MBGB,MarineBenthicGroupB
(Vetrianietal.,1999);TMEG,Terrestrial
MiscellaneousEuryarchaeotalGroup(Takaietal.,
2001);MG-I,MarineGroupI(DeLong,1998);
MCG,MiscellaneousCrenarchaeoticGroup
(Teske,2006).
(Hua1-w46) was also retrieved from the water of site H1 clones.Thisgroupwasclearlypredominantinthewaterof
(Fig.S4).CloneHua4-s2wasthemostabundantclonefrom site H4, but was absent from other water samples (Fig. 3).
thesedimentsampleofsiteH4andexhibited95%sequence Clone Hua4-w4 had 89% sequence identity to the clone
identitytothecloneMH1492_4Fretrievedfromaminero- ArcA08 previously retrieved from a hypersaline endoeva-
trophicfen(Cadillo-Quirozetal.,2008).Thisclonealsohad poritic microbial mat in Israel and was reported as a
94% sequence identity to the Candidatus Methanoregula member of the ‘Halophilic Cluster 2’ (Sørensen et al.,
boonei, an acidiphilic methanogen isolated from an acidic 2005). In the sediments, TMEG was absent from H1, but
peat bog (Bra¨uer et al., 2006). Clone Hua1-s38 exhibited present in the other sites, representing 11 phylotypes, with
96% sequence identity to Methanolinea tarda (Sakai et al., Hua0-s78 being closely related (97% sequence identity) to
2007),amethanogenisolatedfromricepaddyfieldsthatisa the clone WCHD3-02, previously found in a hydrocarbon
memberoftheRiceClusterI. andchlorinatedsolvent-contaminatedaquifer(Dojkaetal.,
TheMBGDwasrepresentedby21%ofallclonesandwas 1998).
foundinsitesH1,H4andH6(Fig.3andFig.S4),withmost Unidentified Euryarchaeota that formed novel, and to
clone sequences obtained from site H6 (81% of the clones date, unreported groups, which did not cluster with pre-
fromwaterand53%fromthesediment).Sequencesofthis viously described groups, for example archaeal DGGE
clusterwerereportedpreviouslyfromsubsurfacesediments, sequences previously reported from other wetlands of
marinesediments,waterandsedimentsofQinghaiLakeand northern Chile (Demergasso et al., 2004) and that repre-
variousotherhabitats.TheabundantclonesHua1-w90and sented 7% of all clones, were found in six out of eight
Hua6-w21exhibited91%and97%sequenceidentitytothe samples (Fig. 3). They formed five different groups (Fig. 4
clone BCMS-5 described from prawn farm sediments in andFig.S4).GroupHua-1consistedoftwophylotypesfrom
China(Shaoetal.,2004). sediment samples of site H0 exhibiting a low identity
The TMEG was first established by clones from South (83–84%) to their closest relative, the clone SBAK-mid-46
African gold mines (SAGMA; Takai et al., 2001), but also retrievedfrommarinesedimentsoftheSkanBayinAlaska,
included sequences retrieved from subsurface and marine classified as ‘other Euryarchaeota’ (Kendall et al., 2007).
sediments. Sequences of TMEG comprised 17% of all Group Hua-2 was represented by a single phylotype
(cid:4)c2010FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol73(2010)291–302
PublishedbyBlackwellPublishingLtd.Allrightsreserved
ArchaealdiversityinSalardeHuasco 297
Fig.4. Phylogenetictreebasedonpartial16S
rRNAgenesequences((cid:5)800bp)ofphylotypesof
Archaeainwatercalculatedbymaximumlikelihood
analysis.Groupdesignations:MG-I,MarineGroupI
(DeLong,1998);MCG,Miscellaneous
CrenarchaeoticGroup(Teske,2006);MHVG,
MarineHydrothermalVentGroup(Takai&
Horikoshi,1999);AAG,AncientArchaealGroup
(Takai&Horikoshi,1999);MBGB,MarineBenthic
GroupB(Vetrianietal.,1999);MBGD,Marine
BenthicGroupD;TMEG,TerrestrialMiscellaneous
EuryarchaeotalGroup(Takaietal.,2001);Hua-1,
Hua-2,Hua-3,Hua-4,Hua-5,Unidentified
Euryarchaeotaclusters.Thescalebarrepresentsa
10%nucleotidesequencedifference.Aquifex
pyrophilus(M83548)wasusedasanoutgroup.
GroupsingrayincludesequencesfromSalarde
Huasco.Groupsinblacklargelyconsistof
sequencesfromSalardeHuasco.FigureS4shows
thedetailedtree.
Hua6-s43,whichexhibited83%sequenceidentitytoaclone w51 was 99% similar to the clone QLS1-399-A8 retrieved
ofthesamelibrary(SBAK-shallow-04)classifiedas‘unaffi- from sediment samples of Qinghai Lake in China (Jiang,
liatedEuryarchaeota’(Kendalletal.,2007).GroupHua-3was 2009).
formed by phylotypes Hua1-w46 and Hua1-s26 together
with three sequences retrieved previously from wastewater
Discussion
sludge, lake sediments and an anaerobic reactor (Fig. S4).
Group Hua-4 is represented by the largest number of
Noveltyofthearchaeal sequences
unidentified sequences and was found in H4-w, H6-w,
H0-s,H1-sandH6-s.Additionalsequencesincludedinthis Alargeportionofthesequenceswerenotcloselyrelatedto
groupwererecordedfrommarinesediments,fromasulfur any cultured archaeon, and 24% of the sequences had
spring and from petroleum-contaminated soil. The two similarities lower than 98% to their closest relatives. Inter-
clones from marine sediments (Skan Bay, Alaska) were estingly, the rest of the sequences had clear phylogenetic
similar to two different phylotypes of this study. The affiliations, including previously described clusters for un-
phylotypesequenceHua6-s37was96%similartotheclone cultured Archaea. We described fivegroups of unidentified
SBAK-shallow-06classifiedasMBGD(Kendalletal.,2007). Euryarchaeota, which exclusively contained representatives
Group Hua-5 included a single phylotype (Hua6-s27) that from Salar de Huasco (Hua-1, Hua-2 and Hua-5), and of
had90%sequenceidentitytocloneMKCSB-C12,whichwas twodistinctgroupscontainingrepresentativesfromSalarde
the closest relative and was retrieved from mangrove soil Huasco (Hua-3 and Hua-4), together with single clone
(Yanetal.,2006). sequencesfromothersources(Fig.4andFig.S4).Important
clusters of other groups (Halobacteria, Methanomicrobiales
andTMEG)includingrepresentativesfromSalardeHuasco
Evidence ofammonia-oxidizingArchaea(AOA)
wereonlydistantlyrelatedtothesequencesavailableinthe
inSalar deHuasco
databases.
ToevaluateapossibleroleofArchaeainthenitrogencycleof Inprokaryotictaxonomy,anidentitythresholdof97%is
Salar de Huasco, we tested for the presence of AOA in all generallyacceptedinordertoseparatespecies(Stackebrandt
samples. PCR products of archaeal amoA, the gene coding &Goebel,1994)andthisthresholdhasevenbeenincreased
forammoniamonooxygenasesubunitA,wereonlyobtained to 498%(Stackebrandt&Ebers,2006).Consequently,the
from the water of site H0. A clone library produced from five clusters described for uncultured Euryarchaeota from
these products consisted of four phylotypes that were Salar de Huasco exhibited similarities lower than 96% to
affiliated to sequences retrieved from sediment and water their closest relatives, highlighting the novelty of the
samples(Table3).Threephylotypesexhibitedhighidentity sequencesretrievedfromthislocation.Theseresultsjustify
(97–99%)totwoclonesofaclonelibraryconstructedfrom considering Salar de Huasco as a unique archaeal habitat
water samples from a drinking water distribution system and a suitable location for further, more detailed studies.
(VanderWielen etal.,2009).The fourth phylotypeHua0- The novelty of sequences and clusters has been reported
FEMSMicrobiolEcol73(2010)291–302 (cid:4)c2010FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
298 C.Doradoretal.
Table3. Sequence identity of Crenarchaeota amoA sequences from Salar de Huasco compared with Nitrosopumilus maritimus (NM, accession
numbersDQ085098andAAZ38768)andthefirsthitintheBLASTsearchfornucleotideandaminoacidsequences
FirsthitinBLAST
Similaritywith
Clone Type Similarity(%) Clonename Habitat NM(%)
Hua0-w20 Nucleotide 97 ClonePLANTBARRSF-IIOTU3(EU852699) Drinkingwaterdistributionsystem 89
Hua0-w51 Nucleotide 99 CloneQLS1-399-A8(EU197155) Sediment,QinghaiLake,Tibet,China 74
Hua0-w79 Nucleotide 97 ClonePLANTBARRSF-IIOTU3(EU852699) Drinkingwaterdistributionsystem 89
Hua0-w92 Nucleotide 99 ClonePLANTBARDNOTU5(EU852705) Drinkingwaterdistributionsystem 90
Hua0-w20 Protein 99 ClonePLANTAARDNOTU4(ACF71541) Drinkingwaterdistributionsystem 94
Hua0-w51 Protein 100 CloneQLS1-399-A6(ABZ01900) Sediment,QinghaiLake,Tibet,China 82
Hua0-w79 Protein 100 ClonePLANTAARDNOTU4(ACF71541) Drinkingwaterdistributionsystem 94
Hua0-w92 Protein 100 ClonePLANTBARDNOTU5(ACF71557) Drinkingwaterdistributionsystem 94
recently for Cyanobacteria in Salar de Huasco (Dorador (dry season) (Dorador, 2007). However, only a single
et al., 2008a,b), for Bacteria in Laguna Tebenquiche at the phylotypewasreportedasamemberoftheHalobacteriain
SalardeAtacama(Demergassoetal.,2008)andforBacteroi- thepresentstudy(Figs3and4,andFig.S4).Astheprimers
detes in different saline evaporitic basins of northern Chile and amplification conditions used were the same in both
(Dorador et al., 2009). Along with the current work, this studies, this suggests that these differencesreflect temporal
highlightsthatwetlandslocatedinnorthernChilerepresent climaticvariations(e.g.variablesaltconcentration).
potentialreservoirsofundescribedmicroorganisms,atrend The presence of uncultured archaeal groups in Salar de
probably due to the unusual physicochemical conditions Huascoisaninterestingfinding,becausethesegroupswere
andgeographicalisolationofthewetlands,aswellasalack primarily described from marine environments as an im-
of knowledge regarding the microbial diversity in these portant component of the deep-ocean picoplankton (De-
environments. Long, 1998). Nevertheless, recently, uncultured marine
archaeal groups were also reported from Qinghai Lake, an
athalassohaline lake located in the Tibetan plateau, and in
Communitycomposition ofArchaeainSalar de
othernonmarinesalineenvironments(Sørensenetal.,2005;
Huasco
Yanetal.,2006;Jiangetal.,2008),extendingthedistribution
Salar de Huasco could be considered as a moderately ofthesegroups.
athalassohalinewetland(Table1),comparableinitsphylo-
geneticgroupswithotherhigh-altitudecoldaquaticsystems
Crenarchaeota
(e.g.TibetanLakes:Dongetal.,2006;Jiangetal.,2008)or
coldsalinelakes(e.g.Antarcticlakes:Karretal.,2006).The RecoveredsequencesrelatedtoCrenarchaeotawereaffiliated
abundance and composition of archaeal communities is tomarineunculturedarchaealgroupswithasyetunknown
likely to be driven by the availability of water, and hence, functionsintheenvironment.Theonlymesophilecultured
dissolution or concentration effects. The dominance of representative of MG-I, Nitrosopumilus maritimus (Group
Archaea over Bacteria has been reported in habitats with 1.1a), grows by aerobicallyoxidizing ammonia to nitrite, a
NaClconcentrationsclosetosaturation,whereHalobacteria metabolic activity previously unknown for Archaea
arethedominantaerobicheterotrophs(Oren,2002).How- (Ko¨nneke et al., 2005). Also, members of the MG-I have
ever, the levels of Archaea detected in this study (summer been reported to demonstrate autotrophic carbon fixation
sampling),includingthesedimentsofthemostsalinesites, whilesomecanbemixotrophic(Ingallsetal.,2006).Inthis
were not associated with halophilic Archaea, but with context,itisinterestingthatwehavefoundMG-Imembers
uncultured marine archaeal groups and methanogens. The in the water of site H0, where the amoA gene was also
presenceofextremelyhalophilicArchaeawouldbepredicted detected (Table 3), although the majorityof the 16S rRNA
onlyforsiteswithsaltconcentrationshigherthan150gL(cid:3)1 gene sequences were not similar (o90%) to those of
(Ventosa et al., 1998). Such situations can be found in Nitrosopumilus sequences, currently the only mesophile
particularwithinthedryseasonwhenelevatedsaltconcen- representative known to oxidize ammonia. Recently, two
trations occur due to strong evaporation. Previous studies newthermophilicmembersofMG-I(Group1.1b)thatcan
haddescribedelevatedproportionsofhalophilicArchaeain oxidize ammonia have been described. The moderately
watersamplescollectedfrombothSalardeHuasco(siteH1) thermophilic(461C)CandidatusN.gargensis(Hatzenpich-
and Salar de Ascota´n, from northern Chile during winter ler et al., 2008) enriched from hot spring microbial mats
(cid:4)c2010FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol73(2010)291–302
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ArchaealdiversityinSalardeHuasco 299
exhibitedahighersequenceidentity(97%)thanN.mariti- (Fig.3).Contrastingly,insedimentsamples,Methanomicro-
mus with Salar de Huasco sequences, while the other biales were the most abundant group (77% of the se-
thermophilic (741C) Candidatus Nitrosocaldus yellowstonii quences). These contrasting differences in the water
(delaTorreetal.,2008)retrievedfromhotspringsediments chemistry can help in future studies to understand the
showedlowersimilarity(o90%). functional role of uncultured Archaea and the interaction
MembersoftheMBGBandMCGhavebeenproposedas with methanogens and sulfate-reducing bacteria. Also, it is
the major oxidizers of methane without assimilation of interesting to note the low identity of the methanogenic
methane-derived carbon in subsurface sediments off Peru clonesequenceswiththedescribedspecies,highlightingthe
(Biddle etal., 2006).The presenceofMBGB (sediments of potentialdiscoveryofnewspeciesofmethanogenicArchaea.
H0,H4andH6)andMCG(sedimentsofH0,H1andH6) The presence of methanogens (e.g. Methanosarcinales)
confirms the cosmopolitan distribution of this group. and benthic Archaea (MBGD) in water samples from site
Becauseoftheirpresenceunderdiversegeochemicalcondi- H1, which exhibited dissolved oxygen concentrations of
tions,itisnotpossibleatpresenttocorrelatetheirpresence 10mgL(cid:3)1,isintriguing.Thewaterlevelsindifferentsitesof
toparticularenvironmentalconditions. Salar de Huasco are shallow (few cm) and the typically
strongafternoonwindsintheAltiplanocanresultinawell-
mixed water column, and therefore the presence of ana-
Euryarchaeota erobicandbenthicmicroorganismsinwatersamples.Atsite
Sequences of MBGD were particularly abundant in water H6,methanogenswereabsentfromclonelibraries,andthe
most abundant group in water and sediment samples was
andsedimentsamplesfromsiteH6andalsowatersamples
MBGD.Thefunctionalroleofthisgroupisnotyetknown.
from H1. This group (equivalent to Marine Group III, as
Recently, it has been demonstrated that microorganisms
defined by DeLong, 1998) has been reported as being
that perform anaerobic oxidation of methane (AOM) can
metabolically active in subsurface sediments (e.g. Sørensen
use alternative electron acceptors to sulfate, including Mn
& Teske, 2006), and recently, a metagenomic analysis of
and Fe. It has been shown experimentally that the MBGD
bathypelagic plankton revealed that members of Marine
representsthemostabundantgroupinanaerobicsediments
GroupIIIEuryarchaeotamightalsoutilizetheoxidationof
ammonia to gain energy (Martin-Cuadrado et al., 2008). treatedwithdifferentelectronacceptors,indicatingapoten-
tialroleinmethanecycling(Bealetal.,2009).Interestingly,
TheTMEGgroupwasalsoreportedasbeingmetabolically
siteH6receiveswaterfromastreamdifferentfromtheother
activeinsubsurfacesediments(e.g.Sørensen&Teske,2006),
sites of Salar de Huasco (Risacher et al., 1999) and has
butaspecificphysiologicalfunctionofthisgrouphasnotyet
contrasting water chemistry (Fig. S3), indicating that the
beenfound.
chemical conditions of the water (Table 1) might favor
the development of members of the MBGD instead of
RoleofArchaeainhigh-altitudesalinewetlands:
themethanogensrecordedfromothersites.
methanogenesis and evidence ofarchaeal
Nitrogenlimitationhasbeenreportedinseveralwetlands
ammonia oxidizers
intheAltiplanoandhasalsobeenreportedforthesitesH1
MethanogenicArchaeadominatedthe16SrRNAgeneclone and H4 in Salar de Huasco (Dorador et al., 2008a). The
librariesofsedimentsamplesfromsitesH0,H1andH4,and studyoftheN-cycleinthissystemhasincludedanalysisof
probably represents an indicator of elevated methanogenic enrichment culturesofammonia-oxidizingbacteria (AOB)
activity. In sediments, the sequences clustered with four thataredominatedbyNitrosomonas(Doradoretal.,2008a).
generaofMethanosarcinales:Methanosarcina,Methanosaeta Here,wereportedAOAsequencesfromsiteH0fromSalar
(aceticlastic methanogens), Methanomethylovorans and de Huasco, indicating the potential role of this archaeal
Methanolobus(methylotrophicorganisms),highlightingthe group in the ammonia oxidation. AOA has also been
diverse substrates used by methanogens in these environ- reportedasbeingmoreabundantthanAOBinothersaline
ments.Consideringthehighsulfateconcentrationsreported mountain lakes (Qinghai Lake: Jiang et al., 2009). Specific
inSalardeHuasco(Risacheretal.,1999),sulfatereduction studiesrelatedtothefunctionalroleofunculturedArchaea
couldbeexpected,especiallyatthesalinesitesH4andH6, (includingAOA)arepromisinginSalardeHuascobecause
where the presence of 16S rRNA gene sequences related to oftheexceptionalenvironmentalconditions.
sulfate-reducing bacteria has been reported previously
(Dorador,2007).SiteH4wasanoxicatthetimeofsampling
Acknowledgements
(Table1)andexhibitedthehighestsulfateconcentrationin
water (4gL(cid:3)1, almost double that of seawater, for example We thank Carolina Vargas for help with sample collection.
Holmer&Storkholm,2001);undertheseconditions,almost We are grateful to Conny Burghardt and Annika Busekow
all sequences fromwater samples matched into the TMEG for technical assistance. Special thanks are due to Chris
FEMSMicrobiolEcol73(2010)291–302 (cid:4)c2010FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
300 C.Doradoretal.
HarrodforEnglishcorrectionsandstatisticalhelpandtwo DemergassoC,EscuderoL,CasamayorEO,ChongG,Balague´V
anonymousreviewers,whohelpedtoimprovethequalityof &Pedro´s-Alio´ C(2008)Noveltyandspatio-temporal
themanuscript.C.D.wassupportedbyadoctoralfellowship heterogeneityinthebacterialdiversityofhypersalineLake
fromtheGermanAcademicExchangeService(DAAD). Tebenquiche(SalardeAtacama).Extremophiles12:491–504.
DojkaMA,HugenholtzP,HaackSK&PaceNR(1998)Microbial
diversityinahydrocarbon-andchlorinated-solvent-
contaminatedaquiferundergoingintrinsicbioremediation.
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