Complex archaea that bridge the gap between prokaryotes and eukaryotes

12 Pages·2015·0.93 MB·English

ARTICLE
doi:10.1038/nature14447
Complex archaea that bridge the gap
between prokaryotes and eukaryotes
AnjaSpang1*,JimmyH.Saw1*,SteffenL.Jørgensen2*,KatarzynaZaremba-Niedzwiedzka1*,JoranMartijn1,AndersE.Lind1,
RoelvanEijk1{,ChristaSchleper2,3,LionelGuy1,4&ThijsJ.G.Ettema1
The origin of the eukaryotic cell remains one of the most contentious puzzles in modern biology. Recent studies
have provided support for the emergence of the eukaryotic host cell from within the archaeal domain of life, but
theidentityandnatureoftheputativearchaealancestorremainasubjectofdebate.Herewedescribethediscovery
of ‘Lokiarchaeota’, a novel candidate archaeal phylum, which forms a monophyletic group with eukaryotes in
phylogenomicanalyses,andwhosegenomesencodeanexpandedrepertoireofeukaryoticsignatureproteinsthatare
suggestiveofsophisticatedmembraneremodellingcapabilities.Ourresultsprovidestrongsupportforhypothesesin
whichtheeukaryotichostevolvedfromabonafidearchaeon,anddemonstratethatmanycomponentsthatunderpin
eukaryote-specific features were already present in that ancestor. This provided the host with a rich genomic
‘starter-kit’tosupporttheincreaseinthecellularandgenomiccomplexitythatischaracteristicofeukaryotes.
Cellular life is currently classified into three domains: Bacteria, tantarchaealhomologuesofactin25andtubulin26,archaealcelldivision
Archaea and Eukarya. Whereas the cytological properties of proteins related to the eukaryotic endosomal sorting complexes
BacteriaandArchaeaarerelativelysimple,eukaryotesarecharacter- required for transport (ESCRT)-III complex27, and several informa-
izedbyahighdegreeofcellularcomplexity,whichishardtoreconcile tion-processingproteinsinvolvedintranscriptionandtranslation2,17,23.
giventhatmosthypothesesassumeaprokaryote-to-eukaryotetrans- These findingssuggest an archaeal ancestor of eukaryotes that might
ition1,2.Inthiscontext,itseemsparticularlydifficulttoaccountforthe have been more complex than the archaeal lineages identified thus
suggestedpresenceoftheendomembranesystem,thenuclearpores, far2,23,28.Yet,theabsenceofmissinglinksintheprokaryote-to-eukaryote
thespliceosome,theubiquitinproteindegradationsystem,theRNAi transitioncurrentlyprecludesdetailedpredictionsaboutthenatureand
machinery,thecytoskeletalmotorsandthephagocytoticmachinery timingofeventsthathavedriventheprocessofeukaryogenesis1,2,17,28.
inthelasteukaryoticcommonancestor(ref.3andreferencestherein). Herewedescribethediscoveryofanewarchaeallineagerelatedtothe
EversincetherecognitionofthearchaealdomainoflifebyCarlWoese TACKsuperphylumthatrepresentsthenearestrelativeofeukaryotesin
andco-workers4,5,Archaeahavefeaturedprominentlyinhypotheses phylogenomic analyses, and intriguingly, its genome encodes many
for the origin of eukaryotes, as eukaryotes and Archaea represented eukaryote-specificfeatures,providingauniqueinsightintheemergence
sister lineages in Woese’s ‘universal tree’5. The evolutionary link ofcellularcomplexityineukaryotes.
betweenArchaeaandeukaryoteswasfurtherreinforcedthroughstud-
ies of the transcription machinery6 and the first archaeal genomes7, GenomicexplorationofnewTACKarchaea
revealingthatmanygenes,includingthecoreofthegeneticinforma- While surveying microbial diversity in deep marine sediments influ-
tion-processingmachineriesofArchaea,weremoresimilartothoseof encedbyhydrothermalactivityfromtheArcticMid-OceanRidge,16S
eukaryotes8 rather than to Bacteria. During the early stages of the rRNA gene sequences belonging to uncultivated archaeal candidate
genomic era, it also became apparent that eukaryotic genomes were lineageswereidentifiedinagravitycore(GC14)sampledapproximately
chimaericbynature8,9,comprisinggenesofbotharchaealandbacterial 15km north-northwest of the active venting site Loki’s Castle29 at
origin,inadditiontogenesspecifictoeukaryotes.Yet,whereasmanyof 3283m below sea level (73.763167N, 8.464000E) (Fig. 1a)30,31.
the bacterial genes could be traced back to the alphaproteobacterial Subsequentphylogeneticanalysesofthesesequences,whichcomprised
progenitorofmitochondria,thenatureofthelineagefromwhichthe ,10% of the obtained 16S reads, revealed that they belonged to the
eukaryotic host evolved remained obscure1,10–13. This lineage might gammacladeoftheDeep-SeaArchaealGroup/MarineBenthicGroupB
eitherdescendfromacommonancestorsharedwithArchaea(follow- (hereafter referred to as DSAG)31–33 (Fig. 1b–d and Supplementary
ing Woese’s classical three-domains-of-life tree5), or have emerged Figs1and2),acladeproposedtobedeeply-branchingintheTACK
fromwithinthearchaealdomain(so-calledarchaealhostoreocyte-like superphylum23.DSAGconstitutesoneofthemostabundantandwidely
scenarios1,14–17).Recentphylogeneticanalysesofuniversalproteindata distributed archaeal groups in the deep marine biosphere, but so far
setshaveprovidedincreasingsupportformodelsinwhicheukaryotes noneofitsrepresentativeshavebeenculturedorsequenced31.
emergeassistertoorfromwithinthearchaeal‘TACK’superphylum18–22, Toobtaingenomicinformationforthisarchaeallineage,weapplied
a clade originally comprising the archaeal phyla Thaumarchaeota, deepmetagenomicsequencingtotheGC14sedimentsample,resulting
Aigarchaeota, Crenarchaeota and Korarchaeota23. In support of this inasmaller(LCGC14,8.6Gbp)andalarger,multiple-stranddisplace-
relationship, comparative genomics analyses have revealed several mentamplified(MDA)metagenomedataset(LCGC14AMP,56.6Gbp;
eukaryoticsignatureproteins(ESPs)24inTACKlineages,includingdis- Fig.2a;SupplementaryFig.3andSupplementaryTable1).Giventhe
1DepartmentofCellandMolecularBiology,ScienceforLifeLaboratory,UppsalaUniversity,SE-75123Uppsala,Sweden.2DepartmentofBiology,CentreforGeobiology,UniversityofBergen,N-5020
Bergen,Norway.3DivisionofArchaeaBiologyandEcogenomics,DepartmentofEcogenomicsandSystemsBiology,UniversityofVienna,A-1090Vienna,Austria.4DepartmentofMedicalBiochemistryand
Microbiology,UppsalaUniversity,SE-75123Uppsala,Sweden.{Presentaddress:GroningenInstituteforEvolutionaryLifeSciences,UniversityofGroningen,NL-9747AGGroningen,TheNetherlands.
*Theseauthorscontributedequallytothiswork.
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RESEARCH ARTICLE
a c Figure1|Identificationofanovelarchaeal
Thaumarchaeota lineage. a,Bathymetricmapofthesamplingsite
(1.2 / 8.9)
(GC14;redcircle)attheArcticMid-Ocean
Aigarchaeota (0 / 0)
Knipovich Ridge 0.1 7597 MCCrGen +a rCch3a (e0o.0ta3 (/0 0 /. 204)) TACK SaathcspsetreievlseaesfdmtvinereengnpttRrsoeiifdsteemg.nebit,c,ltroh1oce6baSiftarerladRcdtN1iiv5oAenkrsmaoimtfyftprihonliemcGorenCLs-op1bk4eaic.’stsBeivdCaerassotlne
DSAG (10.0 / 75.0) prokaryotictaxaandbarsontherightdepict
GC14 MHVG archaealdiversity.Numbersrefertooperational
550 m 89 100 Korarchaeota (0(0 /. 003) / 0.19) tHaxyodnrootmheicrmunalitVsefonrteGarcohugpr;oDuHp.VMEHGV-6G,D,Meeapr-isneea
HydrothermalVentEuryarchaeotaGroup6;
Mohns Ridge LCoaksit’lse EuryarDchPaAeNoNta ( 2(0.1 / /0 1)5.7) Mc,MBGa-xAimaunmd-lBik,eMlihaoroindepBheynlothgiecnGyroofutpheAaarcnhdaBea.l
3,500 m 16SrRNAreads(seeb),revealingthatDSAG
EU731915
b d HM998486 sequencesclusterdeeplyintheTACKsuper-
HM480258
Taxon abundance JN12G36Q19027603 phylum.Numbersbetweenbracketsindicate
0.8 0.2 0.1 0.0 57 53 DEUSJ6AQ8G31G 29L7Q3C839G92C9716448 rtoeltaatlivaendabaurcnhdaaenaclere(%ad)so,rfeesapcehctgivroeluyp.MreClaGtiv,eto
Other archaea 2 1 DSAG/MBG-B HHMM224444007405 MiscellaneousCrenarchaeotaGroup;MHVG,
All taxa CandidTaPhtleaOa undtUhmciventaioDrcsr miHlbcoahayVsnaccsE OeteiGfieotDere-tid61aas 391575657 15 9 211DMMGThHrHBoaVGVuuEGpm-GA Ca-r63chaeotaArchaeal taxa 75 EU7E3AU2Y07843K3215CD8HA49GG4QQ1BE21UDQ13U1765Q67908753720133382761614165838991268007110 γ liuMBinknodaeioitrlctii(shnarttoereiaondHpdgfyosptduhnhrpatoy)tpltobhothgereteelronmvDnyaagSloulsAfVetGs1oe6antoSbhtpoerGevRrDreaNoSt5uiAA0opnGg.aaedrlcen,teMscahlxsuaoeosxqwntiuemonrem..nucimces-
JN123687
Bacteroidetes 5 GQ926464 c,d,Scaleindicatesthenumberofsubstitutions
GQ926439
DSAG/MBG-B 1 EU732030 persite.
100
GaAmlpmhaapprrootteeoobbaacctteerriiaa 18 18 909090 α ββ21
0.0 0.1 0.2 0.6 0.1
deepercoverage,thelatterdatasetwasusedtoextractmarkergenesthat phylogeneticanalyseswereperformed,usingsophisticatedmodelsof
carry an evolutionary coherent phylogenetic signal (Supplementary molecularsequenceevolution.Byimplementingrelaxedassumptions
Tables2and3).Usingsinglegenephylogeniesofthesemarkers,contigs of homogeneous amino acid composition across sites or across
attributable to either one of the archaeal lineages present in the branches of the tree, these models are less sensitive to long-branch
LCGC14AMP metagenome (DSAG, DSAG-related, DPANN and attractionandotherphylogeneticartefacts.Bothmaximum-likelihood
Thaumarchaeota),couldbeextracted.Thesetaxon-specificcontigswere andBayesianinferenceanalysesofconcatenatedalignmentscompris-
usedastrainingsetsforsupervisedbinningofcontigspresentinboththe ing 36 conserved phylogenetic marker proteins20 (Supplementary
LCGC14andLCGC14AMPmetagenomes(SupplementaryFig.4).This Tables 2 and 3) revealed that the DSAG and DSAG-related archaea
approach resulted in the identification of two DSAG bins (from (hereafter referred to as ‘Lokiarchaeota’) represent a monophyletic,
LCGC14andLCGC14AMP,respectively)aswellasoneDSAG-related deeplybranchingcladeoftheTACKsuperphylum.Loki3represented
bin(binLoki2/3fromLCGC14AMP).WefocusedontheDSAGbin the deepest branch of the Lokiarchaeota, and Lokiarchaeum and
fromthenon-amplifieddatasettoavoidpotentialbiasesintroducedby Loki2 were inferred to be sister lineages with maximum support
MDA(seeMethods).Theanalysesofthelow-abundantDSAG-related (SupplementaryFig.7).Intriguingly,wheneukaryoteswereincluded
lineageswerebasedontheMDA-amplifiedLCGC14AMPdataset. inourphylogeneticanalyses,theywereconfidentlypositionedwithin
Afterremovalofsmall(,1kbp)andlow-coveragecontigs(Supple- theLokiarchaeota(posteriorprobability51;bootstrapsupport580;
mentaryFig.5),readsmappingtotheremainingDSAGbincontigswere Fig. 2b; Supplementary Figs 8 and 9), as the sister group of the
reassembledinto504contigs,yieldinga92%complete,1.4fold-redund- Loki3 lineage (Fig. 2b). Robust assessment of these phylogenetic
ant composite genome (‘Lokiarchaeum’) of 5.1Mbp, which encodes inferences (SupplementaryFigs10–14andSupplementaryTable5)
5,381proteincodinggenesaswellassinglecopiesofthe16Sand23S revealed strong support for the Lokiarchaeota–Eukarya affiliation
rRNAgenes(SupplementaryTable4andSupplementaryDiscussion1). (SupplementaryDiscussion2).
The DSAG-related bin (Loki2/3 from LCGC14AMP) was found to The proposednamingofthe Eukarya-affiliatedcandidate phylum
contain two low-abundant, distinct lineages, displaying slight but LokiarchaeotaandtheLokiarchaeumlineageismadeinreferencetothe
marked differences in GC content of 32.8 and 29.9%, allowing for samplinglocation,Loki’sCastle29,whichinturnwasnamedafterthe
separation into two distinct groups (Loki2 and Loki3) (Supple- Norsemythology’sshape-shiftingdeityLoki.Lokihasbeendescribed
mentary Fig. 6). Since these two lineages represent low-abundance as‘‘astaggeringlycomplex,confusing,andambivalentfigurewhohas
communitymembers,onlypartialgenomescouldberecovered.The beenthecatalystofcountlessunresolvedscholarlycontroversies’’34,in
Loki2/3contigsdidnotcontain16SrRNAgenes,renderingitimposs- analogytotheongoingdebatesontheoriginofeukaryotes.
ibletoattributethemtoanyoftheunculturedarchaeal16Sphylotypes
identifiedintheGC14sediments,suchasthelow-abundanceMarine PresenceofdiverseandabundantESPs
Hydrothermal Vent Group archaea (abundance ,0.05%; Fig. 1c). Asourphylogeneticanalysesstronglysupportacommonancestryof
However,phylogeneticmarkergeneswereextractedfortheselineages Lokiarchaeotaandeukaryotes,weinvestigatedthepresenceofputat-
aswell(21and34markersforLoki2andLoki3,respectively)sincetheir iveESPs24inthecompositeLokiarchaeumgenome.Theamountof
inclusionwaspotentiallyusefulinresolvingthephylogeneticplacement genomicdataobtainedfortheLoki2/3lineageswastoolowtoper-
oftheLokiarchaeumlineage. form detailed gene content analyses. A comparative taxonomic
assessment of theLokiarchaeum composite proteome revealed that
LokiarchaeotaandEukaryaaremonophyletic alargefraction(32%)ofitsproteinsdisplayednosignificantsimilarity
To determine the phylogenetic affiliation of Lokiarchaeum and the to any known protein, and that roughly as many proteins display
Loki2/Loki3 lineages, maximum-likelihood and Bayesian inference highestsimilarityto archaealandbacterialproteins(26%and29%,
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a proteome(175proteinsor3.3%)wasmostsimilartoeukaryoticpro-
LCGC14AMP LCGC14
(57 Gbp, amplified DNA) (8.6 Gbp, non-amplified DNA) teins(Fig.2c)andrevealedadominanceofproteins,whichineukar-
yotesareinvolvedinmembranedeformationandcellshapeformation
LCGC14AMP contigs LCGC14 contigs processes, including phagocytosis37 (Extended Data Table 1 and
(725 Mbp) (227 Mbp)
Supplementary Table 6). Several lines of evidence support that the
59 markers (arCOGs) Binning & Filtering presenceoftheseproteinsisnottheresultofpotentialcontaminating
Training sets eukaryoticsequencedata.First,genesencodingLokiarchaeumESPs
PVhiysluoagle sncerteice ntrineges for 6(c coantteiggso)ries Lokiarc(h5a.1e uMmb )contigs andotherproteinsmostsimilartoeukaryoteswerealwaysflankedby
prokaryoticgenes(SupplementaryFig.16),andmostwereencodedby
Binning LoLkCiaGrcCh1a4eAumM Pbin Lo5k9i amrcahrkaeerusm contigs that also contained archaeal signature genes. Second, ESP-
encodingcontigsdisplayedhigh(.203)readcoverage,whileeukar-
Loki2 (high GC) yotic sequences could not be detected in the LCGC14 dataset, and
Loki2/3 bin 21 markers Concatenated
phylogenies, representedonlyanegligiblefractionoftheLCGC14AMPmetagen-
ML (RAxML), ome.Furthermore,theamplicondatageneratedwithuniversal16S/
Split contigs based Loki3 (low GC) BI (Phylobayes)
on GC content 34 markers 18Sprimersdidnotrevealany18SrRNAgenesofeukaryoticorigin
(Fig. 1b). Third, phylogenetic analyses of several Lokiarchaeal ESPs
Legend Reads Contigs Genes Process/software Input/output
revealedtheiremergenceatthebaseofeukaryoticclades(seebelow),
b indicating that these proteins represent archaeal out-groups of the
Methanopyrus kandleri AV19
eukaryotic proteins rather than being truly eukaryotic in origin.
1 0.7 Euryarchaeota Fourth,Lokiarchaeumappearstocontainbonafidearchaealinforma-
100 tional processing machineries (Supplementary Discussion 4 and
0.98
0.7 DPANN SupplementaryTables7–9)and,irrespectiveofthesignificantamount
1 ofESPsinitsgenome,lacksmanyotherkeyeukaryoticfeatures.Finally,
0.7 100 Thermococcales
wecouldalsoidentifyhighlysimilarhomologuesoftheLokiarchaeal
1
1 100 Eukarya ESPsinarecentandindependentlygeneratedmarinesedimentmeta-
1 75 Loki3 (low GC) L genome derived from a sediment core sample off the Shimokita
810 1 Loki2 (high GC) okiarcha PtheenimnsiuclraoboifalJacpoamn,miunnwithy3i8c.hADsStAhGe fcuonmctpiorinsesanadsiegvnoifluictaionnt poafrtthoef
1 100 Lokiarchaeum eota LokiarchaealESPsholdrelevanceforunderstandingtheoriginofthe
94 eukaryoticcell,wereviewsomeofthekeyfindingsinmoredetailbelow.
Korarchaeota
TACK
1 1 Potentialdynamicactincytoskeleton
98 Crenarchaeota Actinsrepresentkeystructuralproteinsofeukaryoticcellsandcom-
1
72 MCG prisefilamentsthatarecrucialforvariouscellularprocesses,including
1 0.99 1 cell division, motility, vesicle trafficking and phagocytosis39. The
99 Thaumarchaeota
0.25 100 Lokiarchaeum genome encodes five actin homologues that display
98 1
Aigarchaeota higher similarity to eukaryotic actins and actin-related proteins
100
(ARPs)thantocrenactins,agroupofarchaealactinhomologuesthat
c were recently shown to be involved in cell shape formation25,37,40
Cellular organisms
Lokiarchaeum Eukarya (SupplementaryTable6).Thisobservationwasconfirmedinaphylo-
Archaea geneticanalysisoftheLokiarchaealactinsthatalsoincludedhomo-
Euryarchaeota
Caldiarchaeum Crenarchaeota logues identified in a recently published marine sediment
Thaumarchaeota
Other archaea metagenome38 (up to 99% identity) as well as in the LCGC14 and
Korarchaeum Bacteria LCGC14AMP metagenomes (Fig. 3a and Supplementary Fig. 17).
Proteobacteria
Firmicutes Lokiarchaeal actins (‘Lokiactins’) comprise several distinct clusters,
Other bacteria
MCG Other someofwhichbranchatthebaseofdistinctiveeukaryoticactinand
No hits ARP clusters, albeit with weak support (Fig. 3a). Despite the poor
0.0 0.2 0.4 0.6 0.8 1.0 resolution of several deeper nodes in the actin tree, strong support
Normalized number of hits
is provided for a common ancestry of Lokiactins and eukaryotic
Figure2|Metagenomicreconstructionandphylogeneticanalysisof actins,indicatingthattheproliferationofactinsalreadyoccurredin
Lokiarchaeum. a,Schematicoverviewofthemetagenomicsapproach.BI, thearchaealancestorofeukaryotes.Notably,theLokiarchaeumgen-
Bayesianinference;ML,maximumlikelihood.b,Bayesianphylogenyof omealsoencodesseveralhypotheticalshortproteinscontaininggel-
concatenatedalignmentscomprising36conservedphylogeneticmarkerproteins solin-likedomainsthatsofarappeartobeabsentfrombacterialand
usingsophisticatedmodelsofproteinevolution(Methods),showingeukaryotes
anyotherarchaealgenomes(ExtendedDataTable1,Supplementary
branchingwithinLokiarchaeota.Numbersaboveandbelowbranchesreferto
Tables 6 and 10 and Supplementary Discussion 5). In eukaryotes,
Bayesianposteriorprobabilityandmaximum-likelihoodbootstrapsupport
these protein domains are part of the villin/gelsolin superfamily of
values,respectively.Posteriorprobabilityvaluesabove0.7andbootstrapsupport
valuesabove70areshown.Scaleindicatesthenumberofsubstitutionspersite. proteins, which comprise various key regulators of actin filament
c,PhylogeneticbreakdownoftheLokiarchaeumproteome,incomparisonwith assemblyanddisassembly41.Althoughthefunctionofthesehypothet-
proteomesofKorarchaeota,Aigarchaeota(Caldiarchaeum)andMiscellaneous icalgelsolin-domainproteinsremainstobeelucidated,itistempting
CrenarchaeotaGroup(MCG)archaea.Category‘Other’containsproteins tospeculatethatLokiarchaeumhasadynamicactincytoskeleton.
assignedtotherootofcellularorganisms,tovirusesandtounclassifiedproteins.
GenomicexpansionofsmallGTPases
respectively;Fig.2candSupplementaryFig.15),whichisinaccord- SmallGTPasesbelongingtotheRassuperfamilycompriseoneofthe
ance with recent findings that suggest major inter-domain gene largestproteinfamiliesineukaryotes,wheretheyareinvolvedinvarious
exchange between Bacteria and Archaea35,36 (Supplementary regulatoryprocesses,includingcytoskeletonremodelling,signaltrans-
Discussion 3). Most notably, a significant part of the predicted duction,nucleocytoplasmictransportandvesiculartrafficking42.Being
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a c
100 100 51100 1L0C0GC14AMPL LaCCnGGdC CL1o14k4AiAaMMrcPPh a ((2e5)u)m (4/1) LL(1Co1kG/i1aC)rc1h4aAeMuPm and 68 51 LokiarchL_o3k1ia9rL3co0hkaie3aur1cm5h4 a(24e5u)4m570 5(23 C8)6a5.C04al7d iMarecthhaaenuomca slduobcteorcrcaBEunuaser cuiyntmaeferrcirahn auaesn odta (19)
Actin and related sequences Lokiarchaeum (3)
Lokiarch_12880
Arp1 69 Lokiarchaeum (3)
96100 Arp2 Arp3 97 LokTiahrecrhmLaooekpuiahmrilc u(h4ma) esupm. ( 2(4))
LCGC14AMP_06532160 Lokiarchaeum (19)
LCGC14AMP_05736710 95 LokiarchL_o4k5ia4r2c0haeum (3)
100 LCGC14AMP and 99 Rab-family (7)
100 LCGC14AMP (2) Lokiarchaeum (5/1) 82 LokiaLrockhiaaercuhma e(2u)m (6)
b 0.4 100 83 LCGC14AMP/LokiarcChreaneuacmti n(11/2) 7987100998892 LokiaLrcohkaiaLBeroucakmhcRitaaa e(ern6rcui-)ahmf a_(5 3m(2)7il)1y1 R(07a)s-famRhilyo -(f5a)mily (7)
Nitrosopumilus maritimus SCM1 0 99 Lokiarchaeum (5) Bacteria and
Myxococcus xanthus DK1622 3 61 Euryarchaeota (12)
Lokiarchaeum (8)
KoCraarldchiaarecuhmae ucmryp stuobfitluermra OnePuFm8 1 2 93100 ASraf-rf1a-mfailmy i(l7y )(7)
170290521 Ca. Korarchaeum cryptofilum OPF8
TryApcPaMinydroeuoTstlbhiohpaaamrcnolaauofsAu lpbusnryirmaodurbusu caiismedre aiko r baboppnropssudeiohscluni eeltuderhi imio 9a ATnl2 iVI4aa7M61nn/9249aa 24 43111339 841097061100 100 97 EuryCarrAcehrnScaaLRheroEacbokehuetiaaaratya er((ac 55o(rh7))tcaa)h e(a2ue1moL)to La(k2o (i)a2kri)acrhcaheauemum (2 ()7)
Reticulomyxa filosa 200 96 EuEruyrayracrhcaheaoetoat a(9 ()77)
Saccharomyces cerevisiae S288c 34 503411226 Methanobacterium lacus
Giardia intestinalis ATCC 50581 23 98 9096 LokiarchLaoekuiamrc (h2a)eum (3)
Tetrahymena thermophila SB210 152 51 Bacteria (35)
Homo sapiens 453 Lokiarch_01230
170174596 Ca. Korarchaeum cryptofilum OPF8
Dictyostelium discoideum 153 499329248 Methanopyrus kandleri
Lokiarchaeum 92 74 EuEryuaryrcahrcaheaoetao t(a1 3(2)1)
Naegleria gruberi0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 217.80 2 0.4 87 73100 BBaaccteteriraia a (n4dB6 )aCcrteenraiar c(8h)aeota (10)
Per cent
Figure3|IdentificationandphylogenyofsmallGTPasesandactin archaealandbacterialspecies.NumbersrefertototalamountofsmallGTPases
orthologues. a,Maximum-likelihoodphylogenyof378alignedaminoacid perpredictedproteome.c,Maximum-likelihoodphylogenyof150aligned
residuesofactinhomologuesidentifiedinLokiarchaeumandinthe aminoacidresiduesofsmallRas-andArf-typeGTPases(IPR006689and
LCGC14AMPmetagenome,includingeukaryoticactins,ARP1–3homologues IPR001806)inalldomainsoflife.Numbersinbracketsrefertothenumberof
andcrenactins25.Consecutivenumbersinbracketsrefertothenumberof sequencesintherespectiveclades.a,c,Sequenceclusterscomprising
sequencesinarespectivecladefromLCGC14AMPandLokiarchaeum, Lokiarchaeumand/orLCGC14AMPsequences(red),eukaryotes(blue)and
respectively.b,RelativeamountofsmallGTPases(assignedtoIPR006689and Bacteria/Archaea(grey)havebeencollapsed.Bootstrapvaluesabove50are
IPR001806)intheLokiarchaeumgenomeincomparisonwithothereukaryotic, shown.Scaleindicatesthenumberofsubstitutionspersite.
key regulators of actin cytoskeleton dynamics, these small GTPases GTPase MglA43. Hence, the Lokiarchaeal roadblock/LC7 proteins
represent essential components for the process of phagocytosis in representpossiblecandidatesforalternativeGAPsinthisarchaeon.
eukaryotes. Intriguingly, the analysis of Lokiarchaeal ESPs revealed
amultitudeofRas-superfamilyGTPases,comprisingnearly2%ofthe PresenceofaprimordialESCRTcomplex
Lokiarchaeal proteome (Fig. 3b). The relative amount of small In eukaryotes, the ESCRT machinery represents an essential com-
GTPasesintheLokiarchaeumgenomeiscomparabletothatobserved ponent of the multivesicular endosome pathway for lysosomal
in several unicellular eukaryotes, only being surpassed by the protist degradationofdamagedorsuperfluousproteins,anditplaysarole
Naegleriagruberi.Incontrast,bacterialandarchaealgenomesencode in several budding processes including cytokinesis, autophagy and
only few, if any, small GTPase homologues of the Ras superfamily viral budding44. The ESCRT machinery generally consists of the
(Fig. 3b). ESCRT-I–III subcomplexes, as well as associated subunits45. The
PhylogeneticanalysesoftheLokiarchaealsmallGTPasesrevealed analysis of the Lokiarchaeum genome revealed the presence of an
thattheserepresentseveraldistinctclusters,eachofwhichcomprises ESCRT gene cluster (Fig. 4a), as well as of several additional pro-
several GTPase sequences (Fig. 3c and Supplementary Fig. 18). teins homologous to components of the eukaryotic multivesicular
Althoughphylogeneticanalysesfailedtoresolvemostofthedeeper endosome pathway. For instance, Lokiarchaeum encodes divergent
SNF7domainproteinsoftheeukaryoticESCRT-IIIcomplex,which
nodes,severaloftheeukaryoticsmallGTPasefamiliesappeartoshare
appeartorepresentmembersoftheVps2/Vps24/Vps46andVps20/
acommonancestrywithLokiarchaealGTPases(Fig.3c),suggesting
Vps32/Vps60 families, respectively. A phylogenetic analysis of the
an archaeal origin of specific subgroups of the eukaryotic small
Lokiarchaeal SNF7 domain proteins revealed that these branch at
GTPases, followed by independent expansions in eukaryotes and
thebaseofthesetwoeukaryoticESCRT-IIIfamilieswithlowboot-
Lokiarchaeota.Thisscenariocontrastswithpreviousstudiesthathave
strap support (Fig. 4b and Supplementary Fig. 19), not only indi-
suggested that eukaryotic small GTPases were acquired from the cating that they might represent ancestral SNF7 copies, but also
alphaproteobacterialprogenitorofmitochondria37. suggestingthatthelasteukaryoticcommonancestoralreadyinher-
AlthoughgenesencodingcanonicaleukaryoticGTPase-activating ited two divergent SNF7-domain-encoding genes from its putative
proteins (GAPs) were absent in Lokiarchaeota, twelve roadblock/ archaealancestorratherthanasinglegene46.Furthermore,thegene
LC7-domain-containing proteins were identified (Supplementary cluster encodes an ATPase that displays closest resemblance to
Tables6and10).Whilesuchproteinshavebeenimplicatedindynein eukaryotic VPS4-type ATPases, including katanin, membrane scaf-
organizationineukaryotes,roadblock/LC7proteinMglBofthebac- foldprotein(MSP)andspastin(Fig.4candSupplementaryFig.20)
teriumMyxococcusxanthuswasshowntoactasaGAPofthesmall as well as hypothetical proteins that show significant similarity
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a b
EAP30-domain Vps25-like Vps4 Vps2/24/46 Vps20/32/60 96 LCGC14AMP_06867510
LCGC14AMP_06982930
Lokiarchaeum 61
7.5 kbp 8 kbp 8.5 kbp 9 kbp 9.5 kbp 10 kbp 10.5 kbp 11 kbp 11.5 kbp29 kbp 29.5 kbp 594999561 marine sediment metagenome
0.7 Lokiarch_37480 (Lokiarchaeum)
75
LCGC14AMP_08445690
96
LCGC14AMP_09186610
100
598791552 marine sediment metagenome
0 kbp 0.5 kbp 1 kbp 1.5 kbp 2 kbp 2.5 kbp 3 kbp 3.5 kbp 4 kbp 63 100 598849147 marine sediment metagenome
Loki 2/3 598763469 marine sediment metagenome
58
Vps25-like Vps4 Vps2/24/46 Vps20/32/60 EAP30-domain LCGC14AMP_06524000
c LCGC14AMP_08417280 (Loki2/3)
Archaeal Vps4 (group 3) (4) 90 Eukarya (Vps2) (24)
100 95 Cren-/thaum-/aigarchaeal Vps4 (group 1) (31) Eukarya (Vps24) (18)
100 Euryarchaeal Vps4 (group2) (15) Eukarya (Vps46) (18)
66 99 Eukarya (MSP) (4) 100 LCGC14AMP_09264370
95 Eukarya (spastin/fidgetin) (6) 92 93 Lokiarch_16760 (Lokiarchaeum)
89 100 Eukarya (Vps4) (4) LCGC14AMP_09392100
100 86 LCGC14AMP_10376800
95 Eukarya (katanin) (6)
595075673 marine sediment metagenome
100 595076476 marine sediment metagenome 97604350177 marine sediment metagenome
59
48 99 604295686 marine sediment metagenome 604262720 marine sediment metagenome
74 LCGC14AMP_06821030 93 96 598854014 marine sediment metagenome
LCGC14AMP_08417270 100 LCGC14AMP_08417290 (Loki2/3)
50 LCGC14AMP_01505270
604290277 marine sediment metagenome
100 100 Lokiarch_37470 (Lo kiarchaeum) 59 71 11834658E5u Tkeatrryaah y(Vmpesn2a0 t) h(1e1rm)ophila
LCGC14AMP_08445680 Eukarya (Vps32) (13)
0.3 52 595008158 marine sediment metagenome Eukarya (Vps60) (8)
92
LCGC14AMP_06982940 Cren-/Thaum-/Aigarchaeota (59)
Figure4|IdentificationofESCRTcomponentsintheLokiarchaeum phylogenyof388alignedaminoacidresiduesofAAA-typeVps4ATPases
genome. a,SchematicoverviewofESCRTgeneclustersidentifiedinLoki- includingrepresentativesforeachofthefourmajoreukaryoticsub-groups
archaeumandLoki2/3.Intensityofshadingbetweenhomologoussequencesis (membranescaffoldprotein(MSP),katanin,spastin/fidgetinandVps4)aswell
correlatedwithBLASTbitscore.b,Maximum-likelihoodphylogenyof207 ashomologuesidentifiedintheLokiarchaeumgenome,inLCGC14AMPand
alignedaminoacidresiduesofESCRT-IIIhomologuesidentifiedinLoki- insequencedarchaealgenomes.Bootstrapsupportvaluesbelow45arenot
archaeum,LCGC14AMPandotherarchaeallineages.Eukaryotichomologues shown.b,c,Scaleindicatesthenumberofsubstitutionspersite.Numbersin
includethetwodistantlyrelatedfamiliesVps2/24/46andVps20/32/60. bracketsrefertothenumberofsequencesintherespectiveclades.
Bootstrapsupportvaluesabove50areshown.c,Maximum-likelihood
to EAP30-domain-containing proteins (Vps36/22) and Vps25, finger/RING-domain-containing proteins, some of which might
respectively(Fig.4a;SupplementaryFigs21and22).Ineukaryotes, serveascandidatesforE3ubiquitinproteinligases(Supplementary
Vps22, Vps25 and Vps36 are components of the ESCRT-II com- Tables6and10).Severalofthesecomponentshavealsobeeniden-
plex, which comprises two to three of these proteins depending on tifiedinAigarchaeota49.
the eukaryotic species46. In addition, a protein domain analysis of
theLokiarchaeumproteomeidentifiedaVps28-likeprotein,acom- A‘complex’archaealancestorofEukarya
ponent of the eukaryotic ESCRT-I subcomplex that links the ubi- WehaveidentifiedandcharacterizedthegenomeofLokiarchaeota,a
quitinpathwaytovesiculartransportandwhich,apartfromVps28, novel, deeply rooting clade of the archaeal TACK superphylum,
comprises Vps23 and Vps37 (Extended Data Table 1 and whichinphylogenomicanalysesofuniversalproteinsformsamono-
Supplementary Fig. 23). The different subunits of the eukaryotic phyletic group with eukaryotes. While the obtained phylogenomic
ESCRT-I complex share similar two-helix core domains and have resolution testifies to a deep archaeal ancestry of eukaryotes, the
been suggested to have evolved from a single ancestral sequence47, Lokiarchaeumgenomecontentholdsvaluablecluesaboutthenature
which we now propose to be of archaeal origin. ofthearchaealancestorofeukaryotes,andabouttheprocessofeukar-
Finally,theLokiarchaeumproteomewasfoundtocontainhypo- yogenesis.ManyoftheESPspreviouslyidentifiedindifferentTACK
thetical proteins containing Longin-like domains, as well as several lineages are united in Lokiarchaeum, indicating that the patchy
proteins belonging to the BAR/IMD superfamily (Supplementary distribution of ESPs amongst archaea is most likely the result of
Tables 6 and 10), comprising curvature sensing protein families lineage-specificlosses2(Fig.5).Moreover,theLokiarchaeumgenome
involved in various aspects of vesicle/membrane trafficking or significantly expandsthetotalnumberofESPs inArchaea,lending
remodelling processes in eukaryotes. These findings suggest that supporttotheobservedphylogeneticaffiliationofLokiarchaeotaand
LokiarchaeumcontainsaprimordialversionofaeukaryoticESCRT eukaryotes.Finally,andimportantly,sequence-basedfunctionalpre-
vesicle trafficking complex. In eukaryotes, ubiquitylation of target dictionsforthesenewESPsindicateapredominanceofproteinsthat
proteinsrepresentsacriticalstepinESCRT-mediatedproteindegra- play pivotal roles in various membrane remodelling and vesicular
dation through the multivesicular endosome pathway44,48. The trafficking processes in eukaryotes. It is also noteworthy that
Lokiarchaeum genome contains a gene cluster that encodes several Lokiarchaeumappearstoencodethemost‘eukaryotic-like’ribosome
components required for a functional ubiquitin modifier system, identifiedinArchaeathusfar(SupplementaryDiscussion4),includ-
including homologues for ubiquitin-activating enzyme E1, ubiqui- ing a putative homologue of eukaryotic ribosomal protein L22e
tin-conjugatingenzymeE2,and26Sproteasomeregulatorysubunit (Fig.5;SupplementaryFig.24andSupplementaryTables7and8).
RPN11.Inaddition,severalhypotheticalproteinswithubiquitin-like Takentogether,ourdataindicatethatthearchaealancestorofeukar-
domains were identified in Lokiarchaeum, as well as diverse zinc- yoteswasevenmorecomplexthanpreviouslyinferred2andallowusto
00 MONTH 2015 | VOL 000 | NATURE | 5
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r-proteir-n pLr4ot1eier-n prSo2t5erie-n prSo3t0eire-n prLo1t3eire-n prLo3t8eieEnl oLn2g2aetTioopn ofiascoRtoNmr,eA r (apElsoUlFe) yb Ii1mqBeuirt(iacnsr ees (nyr)spat(coea)tri,) mnt sGuabulGienlssboliEn-SliCkeR Td-EI:oS mVCapiRsnT-2EsII8S:- lCiEkRAeT-PEII3S: 0 CVdRpoTs-EI2mISI5a:i- ClinVRkpTe-sSII2I/m: 2aVll4p /s4LG26oTc0n/Pg3ais2n/e-lB 6ieA0kxeR /pIdaonMsiDm-oaliinknse superfamily FedacPinouciiasgrlktatoocurarushicrrbsryheyeutoaddh5tetiieoceas|tislntTrr.rpciehoblhSeeefuycaotEchlinafooSedlPnmmiisfslaeoapinni.tslniesEddcexamiascocabaavahrtsrejeceoerEdhnrviSnacibaPeeedrywaicoiclshhfoaaaadatfnleeefptadcp-halseiwerhcsltittainiertcdheyduealaodagasfcerarsaEnoSdssP.
Eukarya whitecircles,respectively.aWhileeukaryotesand
Lokiarchaeota Lokiarchaeotacontainbonafideactins,other
archaeaencodethemoredistantlyrelatedCren-
Korarchaeota actins.bOnlyfewmembersoftheThaumarchaeota
Aigarchaeota TACK c(aorn-ttauibnudliinssta).nctTlyhraeulamte-d,Ahiogm-aonlodgssoomfetubulins
Thaumarchaeota Crenarchaeotacontaindistanthomologuesof
ESCRT-III(SNF7domainproteins).
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Res.39,3204–3223(2011). AuthorInformationSequencedatahavebeendepositedtotheNCBISequenceRead
50. Poole,A.M.&Gribaldo,S.Eukaryoticorigins:howandwhenwasthe ArchiveunderstudynumberSRP045692,whichincludes16rRNAreads(experiment
mitochondrionacquired?ColdSpringHarb.Perspect.Biol.6,a015990(2014). numberSRX872366).ProteinsequencesofLoki2/3weredepositedtoGenBankunder
accessionnumbersKP869578–KP869724.TheLokiarchaeumgenomebinandthe
SupplementaryInformationisavailableintheonlineversionofthepaper.
LCGC14metagenomeprojectshavebeendepositedatDDBJ/EMBL/GenBankunder
AcknowledgementsWededicatethispapertothememoryofRolfBernander.We theaccessionsJYIM00000000andLAZR00000000,respectively.Theversions
thankP.OffreandI.deBruijnfortechnicaladviceandusefuldiscussions,andA.Denny describedinthispaperareversionsJYIM01000000andLAZR01000000.Reprints
forimageprocessing.WealsoacknowledgethehelpfromchiefscientistR.B.Pedersen, andpermissionsinformationisavailableatwww.nature.com/reprints.Theauthors
thescientificpartyandtheentirecrewonboardtheNorwegianresearchvesselG.O. declarenocompetingfinancialinterests.Readersarewelcometocommentonthe
Sarsduringthesummer2010expedition.Allsequencingwasperformedbythe onlineversionofthepaper.Correspondenceandrequestsformaterialsshouldbe
NationalGenomicsInfrastructuresequencingplatformsattheScienceforLife addressedtoT.J.G.E.([email protected])orL.G.([email protected]).
00 MONTH 2015 | VOL 000 | NATURE | 7
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RESEARCH ARTICLE
METHODS (v8.0.22)63(GTRGAMMAmodelofnucleotidesubstitutionand100bootstraps).
TheresultingtreewasimportedintoiTOLonline64tocollapsemajorclades.
Nostatisticalmethodswereusedtopredeterminesamplesize.
PhylogeneticanalysisofDSAG-relatedOTU’s.All16SrRNAgenesequences
Samplingsiteandsampledescription.A2-mlonggravitycore(GC14)was
classifiedasDSAGbyJørgensenetal.52wereusedasqueriesinaBLASTsearch
retrievedfromtheArcticMid-OceanRidgeduringsummer2010(approximately (E,1025,identity.83%)againstallarchaealentriesintheSILVAdatabase
15kmnorth-northwestoftheactiveventingsiteLoki’sCastle;3283mbelowsea (release119)thatmetthefollowingcriteria:sequencelength.900bp,alignment
level;73.763167N,8.464000E)(Fig.1a).Samplesforgeochemistryandmicro- identity.70,alignmentquality.75andpintailquality.75andthequalityof
biologywerecollectedimmediatelyandeitherprocessedonboardorfrozenfor
recoveredsequenceswaschecked(forexample,using‘cut-head’and‘cut-tail’
lateranalysis.Uponportarrival,thecorewasstoredinsealedcorelinersat4uC information).Thenumberofsequencesinthedatasetwasreducedwhilekeeping
(coredepositoryfacility,UniversityofBergen,Norway).Comprehensivegeo- maximum diversity as follows. First, the retained 16S rRNA sequences were
chemicalandmicrobialcharacteristicsfromthisandadjacentsiteshavebeen alignedwithSINA(v1.2.11)65,usingallarchaeaintheSILVAdatabaseasref-
described elsewhere51,52. The core consists of hemipelagic-glaciomarine sedi- erence.ThealignmentwasmanuallycuratedwithSeaview(v4)66.Uponremoval
ments receiving episodic hydrothermal input. The oxygen penetration depth ofgaps,sequenceswereusedtocreateOTUswithUCLUST(v1.2.22)67(94%
wasestimatedto,50cmbelowseafloor(b.s.f.)andthecontentoforganiccarbon identitycut-offandthe‘–optimal’option).Allsequencesthatcorrespondedto
variedbetween0.6-1.3%.Whilenomeasurableamountsofmethaneorsulphide OTUseedswereselectedto representfullDSAGgeneticdiversityand,upon
couldbemeasured,highandfluctuatinglevelsofdissolvedironweredetected. addingarchaealoutgroupsequencesandthesingleampliconOTU,classified
Therelativeabundanceofbacterialandarchaeal16SrRNAgenecopynumbers asDSAG,thefinaldatasetwasalignedwithSINA(v1.2.11)asdescribedabove,
wasestimatedbyquantitativePCR(qPCR)previously52,indicatinghighabund- trimmedwithTrimAl(v1.4)(gapthresholdof50%)andsubjectedtoRAxML
anceoftheDSAGinseveraloftheinvestigatedsedimenthorizons,especiallyat phylogeneticanalyses(v7.2.8;GTRGAMMAsubstitutionmodel,100rapidboot-
75cmb.s.f.(upto40%ofthetotalprokaryoticpopulation;2.73106copiesper straps).Allinternalbrancheswith#40bootstrapsupportwerecollapsedwith
gramsediment).Thus,samplematerialfromhorizonat75cmb.s.f.wasusedfor Newick-Utilities(v1.6)68.TheresultingtreewasthenimportedintoiTOLonline64
alldownstreamanalysesincludingampliconandmetagenomelibraries. tocollapsemajorclades.
DNA extraction and genomic DNA amplification. To obtain sufficient Metagenomesequencingandassembly.
amountsofgenomicDNAforsequencinglibrarypreparation,newsamplemater- Librarypreparationandshotgunsequencing.Nexteralibraries(Illumina)were
ialwasobtainedfromthe75-cm-b.s.f.layerofgravitycoreGC14insummer2013. prepared according to the manufacturer’s instructions, using unamplified
After qPCR-based verification of high DSAG abundance in the re-sampled LCGC14(20ng)andamplifiedLCGC14AMP(50ng)asinputDNA.Sinceless
material,DNAwasextractedfrom7.5gsedimentusingtheFastDNAspinkit startingmaterialwasusedforthegenerationoftheunamplifiedlibrary,atotalof
forsoilinconjunctionwiththeFastPrep-24instrument(MPBiomedicals)fol- eightamplificationcycleswereusedinthePCRstepduringwhichtheIllumina
lowing manufacturer’s protocol, except for the addition of polyadenosine as barcodesandadapters(NextEraIndexkit)werefused,ratherthanthedefaultfive
describedinref.53.Theindividualextractionswerethenpooledandconcen- cycles.TheLCGC14andLCGC14AMPNextEralibrariesweresequencedwith
tratedtoafinalvolumeof50mlusingAmiconUltra-0.5filters(50.000NMWL) threeandtwolanes,respectively,ofHiSeq2500(Illumina),usingrapidmode
followingthemanufacturer’sprotocol(MerckMillipore).Duetolowyieldand setting, generating two 150-bp paired reads. These runs yielded 8.6Gbp and
presenceofinhibitors,2.73ngofthisgenomicDNAwasamplifiedusingthe 56.6Gbpofdatawithanaverageinsertsizeof620and350bpfortheLCGC14
REPLI-gultrafastminikit(Qiagen)accordingtothestandardprotocolforpuri- andLCGC14AMPNextEralibraries,respectively.
fiedgenomicDNA. Readpreprocessing.SeqPrep(v.b5efabc5f7,https://github.com/jstjohn/SeqPrep)
Ampliconsequencingandanalysisof16SrDNAphylogeneticanalyses.Toget was used to merge overlapping paired-end reads and to trim adapters, with
a better estimate of the microbial diversity of Loki’s Castle sediment core defaultsettings.Mergedreadsandnon-mergedpairsweretrimmedwithSickle
LCGC14, ‘universal’ primer pairs (A519F (59-CAGCMGCCGCGGTAA-39) (v.1.210,https://github.com/najoshi/sickle),using‘‘se’’and‘‘pe’’options,respect-
and U1391R (59-ACGGGCGGTGWGTRC-39)) were used to amplify a ively,anddefaultsettings.
,900bpfragmentofthe16SrRNAgenespresentinthenon-amplifiedgenomic Metagenomicassembly.Pre-processedpaired-endreadsandsinglereadswere
community DNA (extracted from LCGC14, 75cmb.s.f.) using the following assembledwithSPAdesv.3.0.069insingle-cellmode,totakeintoaccountthe
conditions:15minofheatactivationofpolymeraseat95uCand35cyclesof widelyvaryingcoverageofmetagenomicscontigsaswellastotrytoassemble
95uC(30s),54uC(45s),72uC(60s),followedbyfinalextensionat72uCfor contigswithlowcoverage.Thereadcorrectiontoolwasturnedonandkmers21,
7min.QiagenHotStarTaqDNApolymerasewasusedforthePCRreactions. 33, 55 and 77 were used. Mismatch correction was not performed on the
LCGC14AMPdataset.Contigsshorterthan1kbpwerediscarded.
Subsequently,PCRproductsofthecorrectsizewerepurifiedwithQiagenPCR
purificationkit,andquantifiedusingaNanodropND-3300fluorospectrometer Genepredictions.Proteincodinggenes(CDS)wereidentifiedwithprodigalv.
2.6070,usingthe‘meta’optionformetagenomes.RibosomalRNA(rRNA)genes
(ThermoScientific).CleanPCRproductswerethenusedasinputmaterialsfor
werecalledwithrnammerv.1.271,usingthearchaealmodelandsearchingforall
libraryconstructionusingTruSeqDNALTSamplePrepKit(Illumina)according
three rRNA subunits. Transfer RNA genes (tRNA) were identified with
tothemanufacturer’sinstructionsandappliedtosequencingwithanIllumina
tRNAscan-SEv.1.2372,usingthe‘–G’optionformetagenomesand‘–A’option
MiSeqinstrument.TheIlluminaMiSeqrunproducedtwo300-bppaired-end
fortheLokiarchaeumcompositegenome(seesubsequentparagraphs).Forthe
reads.RawMiSeqfastqsequencesweretreatedwithTrimmomatictool(v0.32)54
latter,theanalysiswasalsorunwithSPLITSX(noversionnumberavailable;
using the following options: TRAILING:20, MINLEN:235 and CROP:235, to
sourcecodedownloadedon14August2014)73todetecttRNAgenesthatare
remove trailing sequences below a phred quality score of 20 and to achieve
splitorthathavemultipleintrons.
uniformsequencelengthsfordownstreamclusteringprocesses.Remainingtraces
Proteinclustering.Archaea-specificclustersoforthologousgenes(arCOGs)74,
ofIlluminaadaptorsequenceswereremovedbySeqPrep(https://github.com/
basedon120archaealproteomes(hereaftercalledarCOGs2012),wereextended
jstjohn/SeqPrep) and by BLAST55 searches against NCBI Univec database.
withproteomesfrom45recentlysequencedorganisms,including31single-cell
Quality-filtered MiSeq reads were checked for correct orientation of the 16S
amplifiedgenomes(SAGs)(SupplementaryTable1).First,existingarCOGswere
rRNAsequenceinthepaired-endreadsandthosecontainingtheforwardprimer
attributed to the new proteomes: protein sequences in each of the 10,323
sequence(A519F)wereextractedforOTUclusteringwithUPARSEpipeline56,
arCOGs2012 were aligned with MAFFT L-INS-i v.7.130b61. Each alignment
settingaOTUcutoffthresholdto97%.Chimericsequenceswerefilteredoutby
was used as a query (-in_msa) to search the new proteomes using PSI-
the Uchime tool57 integrated in the UPARSE pipeline. Remaining chimeric BLAST55,ignoringthemastersequence,using1024asanE-valuecut-off,fixing
sequences,ifstillpresent,weremanuallycheckedandremoved.Abundancesof thedatabasesizeto108,gatheringatmost1,000sequences,andnotusingcom-
eachOTUwerecalculatedbymappingthechimaera-filteredOTUsagainstthe position-basedstatistics.Hitswerethensortedpersubjectproteinand,foreach
quality-filtered reads using the UPARSE pipeline. Using the mothur package subject, the highest-scoring query alignment was deemed the main arCOG.
(v1.33.2)58,representativesequencesforeachOTUwerealignedtogetherwith Wheneverapplicable,thenext-highest,non-overlappingqueryalignmentwas
theSilvaNR99release-11559alignmentfiletoclassifytheOTUs. deemedthesecondaryarCOG.Second,proteinswithoutarCOGattribution(sin-
Phylogenetic analysis of archaeal 16S rRNA gene sequences. Twenty-nine gletons)inboththeoriginalandextendedsetofproteomesweregathered,and
archaealOTUsidentifiedfromtheamplicondatawerealignedtogetherwith newarCOGs(arCOGs2014)werecreatedfromsymmetricalbesthits,usingthe
220 sequences representing the major clades in the archaeal 16S rRNA tree tools available in COG software suite, release 201204 (ref. 75). PSI-BLAST
accordingtothestudybyDurbinandTeske60.Atotalof249sequenceswere searcheswereperformedaccordingtotheCOGsoftwareinstructions.Lineage-
alignedwithMAFFTL-INS-i(v7.012b)61,trimmedwithTrimAl(v1.4)62,and specific expansions were identified with COGlse, using a job-description file
subjected to a maximum-likelihood phylogeny analysis using RAxML containingallpossiblepairsoforganismsthatdonotbelongtothesamephylum.
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ARTICLE RESEARCH
COGtriangleswasrunwithdefaultsettings,andyielded3,570newarCOGs.Of thefollowingcategories:Lokiarchaeum,Loki2/3(distantLokiarchaeaum-related
the325,405proteinsinthecombineddatasets(165proteomes),29,249(9%)had clades), Thaumarchaeota, DPANN, Diapherotrites, Mimivirus, Bacteria or
noarCOGattribution. unknown.Classificationwasbasedonphylogeneticplacement.Insomecases
AttributionofarCOGstometagenomesorcompositegenomesinthisstudy wherethephylogeneticplacementwasinconclusive,presenceonthesamecontig
wasperformedwithPSI-BLASTasdescribedabove,usingthearCOGs2014as ofanothergenealreadyclassifiedwasusedtoaidclassification.Thefactthat
queries. Lokiarchaeumistheonlycladeforwhichfourtosixdistinctbutcloselyrelated
Phylogeneticanalysesof‘taxonomicmarker’proteinsforbinningandcon- strainsarepresentinLCGC14AMPgreatlyaidedclassification.Inaminorityof
catenatedproteintrees. cases,somegeneswereclassifiedinacategorybutmarkedas‘putative’,astheir
Phylogenetic inference. Maximum-likelihood phylogenies were inferred with attributionwasslightlyambiguous.
RAxML8.0.963,calculating100non-parametricbootstraps.PROTGAMMALG Qualitycontrolofthetrainingset.Contigscontainingmarkersclassifiedinthe
andGTRGAMMAwereusedforaminoacidandnucleotidealignments,respect- firstsixcategoriesmentionedabovewereextractedfromtheassembly,andtheir
ively, unless otherwise stated. Bayesian inference phylogenies were calculated tetranucleotidefrequencies(TNF)werecalculated.Tothenassessthereliabilityof
with PhyloBayes MPI 1.5a76, using the CAT model and a GTR substitution theclassification,lineardiscriminantanalysis(LDA)wasperformedinR83with
matrix. Four chains were run, and runs were checked for convergence. packageMASS84,usingGCcontentandTNFasinputdata:halfofthecontigs
Wheneverconvergencewasnotreached,thetopologyofindividualchainswas belongingtoeachofthesixselectedcategorieswererandomlyselected(excluding
compared.Consensustreeswereobtainedwithbpcomp,usingallfourchainsand thecontigsmarkedas‘putative’),andusedtocalculateLDA(function‘lda’in
aburn-inofatleasthalfthegenerations.Toaddbootstrapsupportvaluestothe MASS)(SupplementaryFig.4).Basedonthis,classificationwaspredictedusing
Bayesianphylogenies,sumtrees.py(DendroPypackage77)wasused,withdefault theMASSfunctionpredict.lda.Incorrectpredictions(thatis,whentheprediction
settings,takingtheBayesianinferencetreeasaguidetreeandthe100bootstraps basedonLDAwasnotcongruentwiththeclassificationbasedonthephylogen-
asinput.Forconcatenatedphylogenies,amino-acidsequenceswerealignedagain etictrees)wererecorded.Theprocedurewasrepeated100times,andcontigsthat
withMAFFTL-INS-iindividuallyforeachcluster.Positionswith.50%gaps wereattributedtothewrongcategory30timesormoreweremanuallyreviewed
weretrimmedandalignmentswereconcatenated. andeventuallydiscardedfromthetrainingset(SupplementaryFig.4a).Contigs
Aminoacidbiasfiltering.Toassesstheeffectofaminoacidbiasonthephylo- markedasputativewereattributedtothecategoryifthepredictionwascongruent
genies,ax2filteringanalysiswasperformedontheconcatenatedalignment.Fora with the putative classification 90 times or more, or discarded otherwise. A
completedescription,seerefs78and79.Inbrief,aglobalx2scoreiscalculatedfor furthercycleofLDAcalculationandpredictionwasperformed,withnocontigs
theconcatenatedalignment,bysumming,foreachaminoacidandeachsequence, classifiedas‘putative’thistime(SupplementaryFig.4b).Tofurtherinvestigate
thenormalizedsquareddifferencebetweentheexpectedandobservedfrequency therobustnessofthemethod,werandomizedthecategoriesoftheinputand
oftheaminoacidinthisparticularsequenceanditsfrequencyexpectedfromthe performedthesameLDAcalculationandpredictionasabove,andassessedthe
wholealignment.Eachpositioninthealignmentisindividuallytrimmedandthe numberofincorrectpredictionsineachcase(SupplementaryFig.4c).Thistest
difference(Dx2)betweentheglobalx2scoreandthex2scorecalculatedonthe confirmedthatclassificationsbasedontreesweregenerallycongruentwithpre-
trimmedalignmentprovidesanestimationoftherelativecontributionofeach dictions based on LDA, significantly more often than just by chance
positiontotheglobalaminoacidcompositionheterogeneity.Positionsarethen (SupplementaryFig.4d–f).Thefinalsetofcontigswasusedasatrainingset
rankedbytheirDx2values,andthemostorleastbiasedsitesuptoathresholdare forphymmBL85(seebelow),andcomprised839kbpforLokiarchaeum,544kbp
removed. for Loki2/3, 521kbp for Thaumarchaeota, 646kbp for DPANN, 43kbp for
Treetopologytests.Tocomparehowwelldifferenttreesexplainedthealigned Diapherotritesand21kbpforMimivirus.
sequencedata,approximatelyunbiasedtests80wereperformedonconcatenated BinningusingPhymmBL.PhymmBLversion4.085wasrunseparatelyforbinning
as well as single-gene alignments. Two maximum-likelihood hypothesis trees thecontigslargerthan1kbpfrombothLCGC14AMPandLCGC14.Astraining
weretestedagainstthealignments.Thefirstone,showingLokiarchaeotagroup- sets,allprokaryoticgenomespublishedinGenBank(retrievedon2014-03-04,
ingwitheukaryotes,wasobtainedfromtheconcatenationof36markers,shown 2716genomes)werecomplementedwiththe60newlysequencedgenomesused
inFig.2b.Thesecondwasobtainedfromtheconcatenationofthe21ribosomal toconstitutethearCOGsetthatwasabsentfromGenBank(SupplementaryTable
proteins present in the previous set, and shows Korarchaeota grouping with 1),andthesixtrainingsets(Lokiarchaeum,Loki2/3,Thaumarchaeota,DPANN,
eukaryotes.Forindividualgenetrees,thetaxamissinginthealignmentwerealso DiapherotritesandMimivirus)obtainedfromLCGC14AMPasdescribedabove.
prunedfromthehypothesistreesusingtheutilitynw_prunefromtheNewick ReassemblyofLokiarchaeumbin.IntheLCGC14assembly,3,165contigs(18.6
Utilitiespackage68.Foreachalignmenttested,per-sitemaximumlikelihoodwas Mbpintotal)werepredictedtobelongtotheLokiarchaeumgenus,indicatinga
calculatedforbothhypothesistreeswithRaxML8.0.9,usingtheoption‘–fG’,and largedegreeofmicrodiversity.Inordertoreduceredundancy,contigsetswere
the PROTGAMMALG model. CONSEL 0.2081 was then used to perform constituted,withincreasinglow-coveragecut-offs(from1to1003,witha13
approximatelyunbiasedtests,usingdefaultsettings. increment).Thecompletenessandredundancyofeachsetwasthenestimated
Identificationoftaxonomicmarkers.Areferencesetof59highlyconserved,low- usingthemicompletescript(manuscriptinpreparation).Inbrief,micomplete
orsingle-copygeneswereusedbothastaxonomicmarkersinthebinningprocess bases its predictions on the presence or absence of a set of single-copy pan-
andforconcatenatedphylogenies(SupplementaryTable2).Fifty-sevenofthese, orthologs,inthiscase162markersdefinedinref.86.Toavoidoveremphasizing
whichwereshowntobepronetoveryfewornohorizontalgenetransferswere thepresenceofmarkersthatareoftenveryclosetoeachother(forexample,
takenfromref.79.TwofurtherarCOGs(arCOG04256andarCOG04267,sub- ribosomalproteins),eachmarkerreceivesaweightcoefficientbasedonthedis-
unitsA0andA9oftheDNA-directedRNApolymerase,respectively)wereadded tancebetweenthismarkeranditsclosestneighboursbothupstreamanddown-
totheset(seeSupplementaryInformationandSupplementaryTable2foralist stream,averagedoverarepresentativesetof70Archaea(setdescribedinref.79).
overwhicharCOGisincludedineachphylogeny). Completenessisthefractionofweightedmarkerspresent,andisthusconstrained
Unlessotherwisestated,alltreesincludedthesamesetof101referencegen- between0(nomarkerpresent)and1(allmarkerspresent).Redundancyiscal-
omes:58archaealgenomesselected79fromthe120analysedbyWolfetal.74;21 culatedasthetotalnumberofcopiesofweightedmarkerspresentdividedbythe
selectedfromthe45newlysequencedorganismsthatwerealsousedforcluster- numberofweightedmarkerspresent,andisthusalwaysgreaterthanone,where
ing,someofthemalreadyanalysedinGuyetal.82;twogroupsofthreeclosely onewouldmeanthatallmarkerspresentaresinglecopy.Thesetwonumbers
relatedSAGswerepooledtoprovidemorecompleteproteomes;tenbacteriaand werecalculatedforeachcontigset,andacut-offof243representedthebest
teneukaryotes,asinGuyetal.82(SupplementaryTable1).Toremoveparalogues compromisebetweencompleteness(0.89)andredundancy(1.67)(Loki243set,
andobtainsetswithatmostonehomologuepergenome,membersofeachofthe SupplementaryFig.5a).
selectedarCOGswerealignedwithMAFFTL-INS-iandamaximum-likelihood To obtain a better assembly with longer contigs with only reads from
phylogenywasinferredwithRAxML,underaPROTCATLGmodelwith100 Lokiarchaeum,readsbelongingtoLokiarchaeumcontigswerereassembledas
slowbootstraps.Previouslyremovedparalogues79werenotincluded.Treeswere follows.ReadsfromtheLCGC14dataset,correctedbySPAdes,weremapped
thenvisuallyinspectedandparaloguesremovedusingthesameguidelinesasin againstthewholeLCGC14assemblywithbwa-mem87,andreadsthatmatched
ref.79.Thisset,includingatmostonecopyofeachofthe59referencearCOGsin contigsintheLoki243setwereextracted.Forpaired-endreads,bothreadswere
101genomes,ishereafterreferredtoas‘59ref’. retainedifatleastonereadmatchedtheLoki243set.Theseextractedreadswere
Binning. assembledwithSPAdesasabove,butwithoutthesingle-cellmodeandwithout
Training set. After arCOG attribution (see above), genes from LCGC14AMP readcorrection.Again,completenessandcoveragewereassessedforsetsofcon-
belongingtotherespectivearCOGswereaddedtothe59refset.Sequenceswere tigswithincreasinglow-coveragecut-offs,andathresholdof203coveragewas
alignedandindividualtreeswerebuiltforeacharCOG,asdescribedabove.Trees foundtogivethebestcompromisebetweencompleteness(0.92)andredundancy
werethenvisuallyinspectedandsequencesfromLCGC14AMPwereclassifiedin (1.44)(SupplementaryFig.5b).Theselected504contigs,hereafterreferredtoas
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RESEARCH ARTICLE
‘Lokiarchaeum’,represented5.14Mbpofsequence.TheN50andN90ofthis ‘Other’category.ResultsareshowninFig.2b.Usingthesameparameters,func-
assemblywere15.4and5kbp,respectively. tionalCOGcategorieswereassignedtotheLokiarchaealproteometogetinsights
Annotation and contamination assessment of Lokiarchaeum genome bin. into the functional and taxonomic affiliation of the Lokiarchaeal proteome
AnnotationofallpredictedopenreadingframesoftheLokiarchaeumgenome (SupplementaryFig.15).
binwasdoneusingprokka88,usingaconcatenationofthethreekingdom-specific Phylogenetic analyses of selected eukaryotic signature proteins (ESPs).
proteindatabasesshippedwithprokkaasthemaindatabase,predictingtRNA Selection of ESCRT-III homologues. For the ESCRT-III phylogeny, eukaryotic
and rRNA as above. Furthermore, proteins were compared to sequences in ESCRT-IIIhomologuesasdescribedinMakarovaetal.97(comprisingthefamilies
NCBI’s non-redundant database and RefSeq using BLAST55 and results were Vps60/Vps20/Vps32andVps46/2/24),aswellasarchaealESCRT-IIIhomolo-
inspectedusingMEGAN89.Additionally,anInterProScan590(whichintegrates guesbelongingtoarCOG00452,arCOG00453andarCOG00454familiespresent
acollectionofproteinsignaturedatabases suchasBlastProDom, FPrintScan, in Crenarchaeota, Thaumarchaeota and Aigarchaeota were extracted from
HMMPIR, HMMPfam, HMMSmart, HMMTigr, ProfileScan, HAMAP, GenBank.ThemoredistantlyrelatedSNF7-likearCOGfamilies(arCOG09747,
PatternScan, SuperFamily, SignalPHMM, TMHMM, HMMPanther, Gene3D, arCOG09749andarCOG07402)97presentinafeweuryarchaealspecieswerenot
PhobiusandCoils)wasperformedandthegenomewasviewedandanalysed includedinthealignment.Subsequently,respectivearCOGswereretrievedfrom
inMAGE91.Selectedgenesofinterestfortheevolutionoftheeukaryoticcelland/ boththeLCGC14AMPmetagenomeandLokiarchaeumfinalbin(seesectionon
or subjected to phylogenetic analyses were checked manually and annotated arCOGattribution).TheESCRToperonpresentonaLoki2/3contigrevealedthe
according to their protein domains/signatures based on PSI-BLAST55 results, presence of an additional ESCRT-III homologue (most similar to eukaryotic
arCOGattributions(SupplementaryTables6–10)aswellasproteinstructure Vps20/32/60sequences), whichwasnot attributedtoan archaealCOG.This
predictions using Phyre292. To check for the presence of particular genes of homologuewasusedasanadditionalquerytoretrievehighlysimilarsequences
interest,suchasspecificeukaryoticribosomalproteins,oreukaryoticribosomal fromtheLCGC14AMPmetagenomeaswellastheLokiarchaeumfinalbinusing
proteinL41ewhichhasbeendetectedinseveralEuryarchaeota93,existingalign- blastp.Finally,eachofthetwodifferentSNF7-familyproteins,whicharepartof
mentsfromarCOGsand/orKOGsweredownloadedfromeggNOG94andusedin theESCRToperonsofLokiarchaeumandLoki2/3,respectively,wereusedas
PSI-BLASTsearchesasqueryagainsttheLokiarchaealcompositegenome. queriestosearchpublishedmetagenomes(NCBI)withblastp.Highlysimilar
Severalcontrolswereperformedtoconfirmtheabsenceofobviouscontami- sequences(coverage.70%;identity.40%)wereretrievedandincludedinthe
nantsinthefinalLokiarchaeumbin.Mostimportantly,allcontigscontaining phylogenyaswell.
ESPsdiscussedinthemanuscriptweremanuallyinspectedtoverifythatthese SelectionofVps4homologues.ArchaealsequencesassignedtoarCOG01307(cell
actuallybelongtoLokiarchaeum,by:(1)inspectingneighbouringgenesforthe division ATPase of the AAA1 class, ESCRT system component) as well as
presenceofarchaealmarkers;(2)byqueryingallproteinspresentoncontigs eukaryoticVps4homologues,includingafewproteinsofthecdc48subfamily,
containingESPsagainsttheLCGC17metagenometocheckwhetherhighlysim- wereretrievedfromGenBank.Thelatterproteinfamilyservedasoutgroup,as
ilarhomologuescouldbefoundseveraltimesinthesample(generallybetween described in Makarova et al.97 Sequences assigned to arCOG01307 were also
3–7copies)accountingforthedifferent,highlyrelatedLokiarchaeotastrains;and extractedfromLCGC14AMPmetagenomeaswellasfromtheLokiarchaeum
(3)byqueryingthesameproteinsagainstenvironmentalmetagenomespublicly bin, and sequences highly similar to the Vps4 of Lokiarchaeum were
availableatNCBI,controllingthatmostofthemhadhighlysimilarhomologues retrievedfrompublishedmetagenomes(coverage.60%;identity.50%).The
inanoceansedimentmetagenome95,butnotinanyothermetagenome.Thislast LCGC14AMPmetagenomecontainedalargeamountofsequencesassignedto
checkwasbasedonourfindingthatallESPsofLokiarchaeumhadhighlysimilar arCOG01307,includinghitstoVps4homologuesofThaumarchaeota.However,
homologuesinthismarinesedimentmetagenome(forexample,upto98%for ATPasesthat,basedonphylogeneticanalyses,turnedouttobeunrelatedtoVps4
Lokiactins)indicatingthatcloselyrelatedgenomesofmembersofLokiarchaeota wereremovedfromtheanalysis.Basedontheinitialphylogenythatincludedall
arepresent,whichisinaccordancewiththefindingthatDSAGrepresentsan ofthesesequences,onlythoseLCGC14AMPVps4homologuesthatclustered
abundantgroupinthesesub-seafloorsediments96. withtheVps4homologueoftheLokiarchaeumbinwereselectedtoavoidthe
Finally,proteinscomprisinginformationalprocessingmachinerieswerealso inclusionoffalsepositives.
investigatedusingMEGAN89.Theabsenceofbacterialinformationalprocessing SelectionofEAP30-domain(Vps22/36-like)andVps25homologues.EAP30and
proteinsindicatedthatthereisnobacterialcontaminationinthefinalbin(see Vps25homologueshavesofarnotbeendetectedinArchaeaandthustherespect-
SupplementaryDiscussion3). ive sequences present in Lokiarchaeum (Extended Data Table 1 and
Identificationoftaxonomicmarkersinthebins.ForLokiarchaeum,arCOG SupplementaryTable6)havenotbeenassignedtoanarCOGfamily.Thus,only
attributionwasperformedasdescribedabove,andtaxonomicmarkerswereiden- Lokiarchaeum homologues, as well as selected representative eukaryotic
tifiedbytheirarCOGattribution.Wheneverthereweretwocopiesofthesame sequences spanning the eukaryotic diversity that were retrieved from the
marker,thecopylocatedonthecontigwiththehighestcoveragewasselected. GenBank database were included in these phylogenetic reconstructions.
ForLoki2/3,thecategoryhadtwocopiesof19outof36markerspresent,with Putative EAP30- and Vps25-like homologues were discovered in the
divergentphylogeneticplacement.AclearGCcontentdifferencecouldalsobe Lokiarchaeum genome since they are part of the ESCRT operon present on
observedbetweenthecopies,and,withasingleexception,thetwosetsofcopies contig119.Thesesequenceswereusedasqueriestoalsoretrievehomologues
werenotoverlapping(SupplementaryFig.6).Theexceptionwasdiscardedand fromtheLCGC14AMPmetagenome(Ecut-off,0.1;qcoverage,85)aswellas
theremainingtwo-copymarkersweredividedintotwobins,Loki2(highGC, frommetagenomesdepositedatNCBI.
rangingbetween32.2–37.3%)andLoki3(lowGC,rangingbetween27.7–30.7%). SelectionofsmallGTPasefamilyhomologues(IPR006689andIPR001806).The
Single-copymarkerswithaGCcontentfallingintotherangeofeitherofLoki2or investigationoftheLokiarchaeumproteomerevealedlargenumbersofproteins
Loki3wereattributedtothecorrespondingbin,theothercopieswerediscarded. homologoustosmallGTPasesoftheRasandArffamilies.Inordertoreliably
Loki2(highGC,average32.8%)consistedof21markersandLoki3(lowGC, identifyallputativesmallGTPasesintheLokiarchaeumbin,anInterProscan90,98
average29.9%)of34markers. was performed and all proteins assigned to IPR006689 (Ras type of small
Taxonomic affiliation of the Lokiarchaeum proteome. To estimate how GTPases) and IPR001806 (Arf/Sar type of small GTPases) were extracted.
Lokiarchaeum relates to its closest relatives, its proteome was aligned to Subsequently,archaealreferencesequencesbelongingtotheseIPRfamilieswere
NCBI’snon-redundantdatabaseusingblastp,withanEthresholdof0.001.To retrieved from GenBank. Eukaryotic and bacterial reference sequences were
provide a way to compare results, the complete proteomes of ‘Candidatus selectedbasedonapreviousstudybyDongetal.99thatinvestigatedthephylo-
Korarchaeum cryptofilum’ OPF8, ‘Candidatus Caldiarchaeum subterraneum’ geneticrelationshipsofmembersoftheRassuperfamily.Duetothelargenumber
andtheincompleteproteomeofSCGCAB-539-E09,solerepresentativeofthe ofGTPasehomologuesintheLokiarchaeumbin,andthedifficultyassigning
MiscellaneousCrenarchaeotalgroup(MCG)weresimilarlyanalysed.Theresults theseproteinstoaparticulartaxon,itwasdecidednottoanalyseallGTPase
oftheblastswerefilteredtoremoveself-hitsandhitstoorganismsbelongingto homologuespresentinmetagenomes.UponinspectionoftheMAFFTL-INS-i
thesamephylum.In thecaseoftheMCGrepresentative,onlyself-hitswere alignment,partialsequencesandextremelydivergenthomologueswereremoved.
removed.FilteredresultswerethenanalysedwithMEGAN5.4.0.Lastcommon Selectionofactinhomologues.Sofar,theonlyactin-relatedproteinsdetectedina
ancestorparametersweresetasfollows:MinScore,50;MaxExpected,0.01;Top fewmembersofthearchaeabelongtoarCOG05583andhavebeenreferredtoas
Percent,5;MinSupport,1;MinComplexity,0.0.Foreachresult,brancheswere crenactins100. Three proteins encoded by the Lokiarchaeum genome were
uncollapsed at the level below super-kingdom. Profiles were compared using assignedtothisarCOG,andablastpsearchagainstRefSeqrevealedthatthese
Absolute counts, and the results were exported and further analysed in R. proteinsaremorecloselyrelatedtobonafideactinsofEukaryotesthantoarchaeal
Categoriestowhichlessthan100hitswereattributedinLokiarchaeumwere crenactins.Inordertoidentifyadditionalfull-lengthactinhomologues,blastp
groupedunderthe‘OtherArchaea’or‘OtherBacteria’categories.Hitsto‘root’, (E-value cut-off,10210) searches were performed against the Lokiarchaeum
viruses,unclassifiedsequencesandhitsnotassignedweregroupedunderthe genomeaswellastheLCGC14AMPmetagenome,usingthisLokiarchaeumactin
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