This article isprotected rights by All copyright. reserved. 10.1111/mec.15068doi: lead todifferences version between this andthe ofPleasec Version Record. been and throughtypesetting, proofreadingwhich may thecopyediting, process, pagination This article undergone has for and buthas accepted been not review publication fullpeer Running Delineation title: of ESTUsofmarineBacteroidetes Article :Article type Original CRISTINADR. DÍEZ Delineation of ecologically distinct units of marine Bacteroidetes Bacteroidetes marine units of distinct ecologically of Delineation 2 1 8 7 6 5 4 3

Department of Marine Biology andOceanography, Institut deCiències del Mar (CSIC), 08003 Departamento de Biología deSistemas Instituto Español de Oceanografía, Department Biology, of Culture and Pathology Marine of Species, School Biotechnology of andBiomolecular Sciences, Life Sciences Department, Natural History Museum, SW7 5BD London, UK.

AcceptedCentre for Marine Ecosystems Research, School of Science. Edith Cowan University, Joondalup, Article Instituto de Oceanografía y Cambio Global, IOCAG, Universidad de las Palmas deGranCanaria, Barcelona, Catalunya, Spain. WA, Australia Spain. ULPGC, Científico Parque Tecnológico Marino de Taliarte Spain Sal,de la CSIC. 12595 Ribera de Cabanes, Castellón, Spain University New of South Wales, Sydney, NSW, Australia Cristina DíezCristina Ferrera

1 ,5 , Marta Sebastián -

Vives in the Northwestern Northwestern in the - VIVES (OrcidVIVES : ID 0000

1 ,2 , Shaun

1, 6

, Carlos Pedrós Nielsen

Centro Oceanográfico de Málaga . Centro Nacional de Biotecnología, CNB 3 Acinas , Pablo Sánchez - 0002 Mediterranean Sea Mediterranean - 1 Alió *

Centre for Marine Bio - 1772 1, 7 , M. Gasol Josep

- 1 , s/n

7092) , Oswaldo

. 35214, Gran Canaria, Spain.

Instituto deAcuicultura Torre ,

29640 Fuengirola, Málaga, Palenzuela - 1, Innovation, The

8

ite this articleite this as and - CSIC, Madrid,

Silvia G. Silvia 4 , Isabel,

This article isprotected rights by All copyright. reserved. Accepted which are in forward step a constitutes work This suggest analyses iso no are there from ESTU A months. clades marine NS2b ESTUs recurring ESTUs of depths different at ESTUs among overlap no almost them delimiting sequencing amplicon Article delineated were clades Bacteroidetes the and phylogeny from libraries w group, this within units meaningful understood. poorly is structure population phyla dominant the of one is Bacteroidetes Abstract 230 9555. E author: *Corresponding Owenweeksia that there is a high degree of niche speciali niche of degree high a is there that with identif

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drastic change towards the predominance of of predominance the towards change drastic the n the in NS5 marine clade marine NS5

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players inorganicplayers matter NS5 h evrnetl rfrne f oe f them. of some of preference environmental the alo studies associated ng n NS2b and -

mail most of the year the of most ) , we describe we ,

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ecological was typically present typically was hs ETs to ESTUs these , aie clade marine . Muricauda e y considering By

constructed disentangling d

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s s Seasonally . i deep In . n h N Mediterranean NW the in some ation within these closely related clades. related closely these within ation

, Tel (+34) 93 230 8565; Fax (+34) 93 (+34) Fax 8565; 230 93 (+34) Tel , ng determine er full near bact

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s tl calnig bcue the because challenging, still is soitd wit associated

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ae y studies by made yet we know little about little know we yet

2007), and 2007), et al. et Substantial

2011). However, linking phylogenetic clusters to distinct distinct to clusters phylogenetic linking However, 2011). Polz mn 20; Alonso 2000; hman,

the model rges n eciig h dvriy f class of diversity the describing in progress et al et relationship between function and phylogeny and betweenfunction relationship

phylogenetic defining with with peit ta pyoeei cutr likely clusters phylogenetic that predicts )

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global microbial Moore

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This article isprotected rights by All copyright. reserved. Acceptedsequences populations. aimed of goal the With 2016). Synechococcus which patterns distribution spatial coworkers phenotypes Synechococcus isolates cultured of divisions observed previously partitioned of delineation studies of number Articlepioneering segmentation contrast In gel orgradient by rather electrophoresis) the were 2003 certai in diversity their into insights detailed obtain to attempt construction. library Pereira Alonso NS; clades marine (i.e. Sea North the in species uncultured ; either either

Chen

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of , studies

limited by the low resolution of the fingerprinting approach fingerprinting the of resolution low the by limited used metagenomic reads of a single a of reads metagenomic used

et al. et bacterial bacterial and geography (Rocap and

W samples obtained obtained samples 2010), but this was done using general bacterial primers primers bacterial general using done was this but 2010), of clades into ecologically significant groups (ecotypes) (ecotypes) groups significant ecologically into clades of

hs eoye wt nwr ehius Fr instance, For techniques. newer with ecotypes these into several genetically distinct new clades associated with particular depths, particular with associated clades new distinct genetically several into no e Ecological new into cntutd rfrne re f h na full near the of tree reference a constructed e

2008; Mühling 2008; investigat

have ( Moore groups f A oue on focused ew ing t al. et

uh as such additional

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1998 divided et al. et ly ecotype

the the

2008; Díez 2008; otatn evrnet t ietf habitat identify to environments contrasting infcn Txnmc nt (SU; Farrant (ESTUs; Units Taxonomic Significant ;

2002; Ferris 2002; tde ue Bacteroidetes used studies et Saln 199 Scanlan & West C

well yanobacteria low bnac ad yais ad on and dynamics, and abundance - defined populations of populations defined number of clones functional - Vives et al. et ,

et al. et have

2003). 2003). gene marker (i.e. (i.e. marker gene

2013 9 eevd oe attention more received ). hbtt ( habitats n

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ly denaturing denaturing ) to obtain obtain to ) ;

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t al. et clone t al et ,

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This article isprotected rights by All copyright. reserved. Accepted (Millipore), asin Massana described a in concentrated and phenol with extracted acids nucleic Microbial flash 8.0), pH sucrose, M 0.75 EDTA, Tris mM lysis(50 buffer ml 1.8 with were saturated filters, μm 0.22 The pump. (Whatman) prefilters 200 20 (September samples Both surface 2008 September to 2006 September 2°48’E 41°40’N, (BBMO, strategies. sampling different two with taken samples seawater from collected was Bacterioplankton Article Collection Sample Methods Material and the field defining allowed clades Bacteroidetes tree. phylogenetic Units the with them clades

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The temporal a temporal The - with ), which could), which befurther associatedwith specific environmental parameters. mesh - cell western western First two a deep sample (2,000 m depth) depth) (2,000 m a sample deep , and sequentially filtered through filtered sequentially and , s

th

ly large were were were lysed were

eie a 9% dniy ee nldd y asmn it or pre our into parsimony by included were identity 99% at defined to 23 to ,

surface water surface

Clustering based on the on based Clustering Mediterranean and DNA extraction - scale scale , taken taken

rd nd spatial distribution of the defined of the spatialdistribution nd and 0.22 μm Sterivex filters (Durapore, Millipore) using a peristaltic peristaltic a using Millipore) (Durapore, filters Sterivex μm 0.22 and , 2007). ,

Illumina with lysozyme, proteinase K and sodium dodecyl sulfate, and the the and sulfate, dodecyl sodium and K proteinase lysozyme, with ; a

board Gasol About samples were samples Sea

amplicon 16S rRNA gene rRNA 16S amplicon the t al. et et al. ) . Representative sequences of sequences Representative . . -

frozen in liquid N liquid in frozen ESTUs (i.e. subgroups within clades that clades within subgroups (i.e. ESTUs 8 S

RV RV - econd 10 L of seawater of L 10

(1997). 2016 obtained temporal and spatial and temporal ‘García del Cid’ during during Cid’ del ‘García taken ly a 20 μm 20 a ) ,

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at the Blanes Bay Microbial Observatory Bay Microbial Blanes the at

from compared - 2

mesh, ,

a and kept at kept and an an clades were

Aio Utaetiua Unit Ultracentrifugal Amicon n two offshore tag GF/A or polycarbonate 3 μm μm 3 polycarbonate or GF/A

- in situ in s ( s this coastal location at the the at location coastal this year distribution patterns of the the of patterns distribution w as 16S 16S Operational Taxonomic Operational

- assessed by assessed comparing location esnl study seasonal 80 °C until extraction.until 80°C the the

prefiltered through a through prefiltered iT ag ‘Modivus’ s)

- 111 km datasets from datasets

HCl, 40 mM 40 HCl, co

- - occur i occur existen

cruise cruise away ( from n t .

This article isprotected rights by All copyright. reserved. Accepted1996) Sange and library each from selected expected were size the containingfollowing inserts manufacturer’s of Clones instructions. the clone The Generation of Methods Acinas in described as amplicons heteroduplex of occurrence the minimize to PCR reconditioning a (D primer forward the of issues latter The S2) (Table primer reverse with out carried was amplification DNA Articlethese libraries from were samples obtained depth) DNA using constructed were libraries Summer t along c seasonal (Díez two with seasonality demonstrated using study previous a In Bacteroidetes - , Vives ,

he 2 he et al. et eerd o s h Srae omnt ‘’ n te ep omnt ‘’ Nt that Note ‘D’. community Deep the and ‘S’ community Surface the as to referred 968F - Fall S

ommunities, using pooled monthly samples monthly pooled using ommunities, is , 1

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t al. et

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‘SF’

designed ( distinct t al. et Bacteroidetes community structure at the BBMO displayed a marked a displayed BBMO the at structure community Bacteroidetes : Manz Manz

e

denaturing gradient gel electrophoresis ( electrophoresis gel gradient denaturing (see Table S1 for details). for S1 Table (see ight samples ight e ul a Bacteroidetes a built We

et al. et

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et al. et

on : 1 taken at the end at oftaken summer. frad primer forward a 49

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n Summer in

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was fingerprinting, we we fingerprinting, R NA

modified periods taken taken periods

(Manz gene elve

phylum -

Spring in and et al. et with

3’) the the a .

This article isprotected rights by All copyright. reserved. Accepted1953 of function a as OTUs of (number clusters sequence with rRNA 16S full near the Operation Clone alignment manually was (Pruesse dataset subunit small the of Alignment Project) Database (Ribosomal (Pruesse SINA classifier the with (Altschul Information Biotechnology for Centre National database, ArticleThe Classification GenBank numbers accession KP887163 under a had sequences representative some analyses. the software conducted was electropherograms software in , Chao ), ribosomal

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relatives . ). eune similarity sequence

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classified Trimmed and RDPand - off unique curves Good Good . The The . using ,

the for for n .

This article isprotected rights by All copyright. reserved. application for data ecological and values 10 with The S case, this In ( habitat projected same the sharing strains related of (groups ‘populations’ cohesive ecologically and categories) ‘ learn to model Markov clustering OTU simplistic than species existing of knowledge without and off cut similarity depth) and populations season (i.e. branch each with associated ‘ (Hunt AdaptML model evolutionary full near the with built was tree phylogenetic reference A of‘ Identification 1901) 1973) (Hill index Summer populations F

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software) Winter )

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t combined we categories, environmental our define To and and and &

projected sequences t

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m that using the interactive Tree of Life web web Life of Tree interactive the using

and thus and Table Surface ‘ Surface

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was set at 0.05 at set was to length 16S rRNA sequences rRNA 16S length dpM identifies AdaptML

identify putative putative identify S , and offers a better approach approach better a offers and , 1) and hence were considered were hence and 1)

were tagged as tagged were The The S ’

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Model predictions Model

D kept at default default at kept clades ’ S sequences. S ) he temporal he

categories. ecological

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Jaccard (named

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This article isprotected rights by All copyright. reserved. SequenceunderID Bioproject Archive Read PRJNA495818 both kept. from were sequences clades predefined Bacteroidetes our of limits the within located were that sequences Only rRNA 16S our in included then were OTUs candidate default using v.2.6.0 BLASTN by similarity >99% with OTUs recruit to years 5 from samples (64 identity identity. 99% at defined the dataset HOTMIX The against theSILVA123 database as reference. were V3 the and samples, HOTMIX the supplementary sequencin and amplification extraction, DNA samples of from BBMO 2014; aboard HOTMIX from generated Tag OTUs of abundance To Generation of

Accepted Article samples from samples

add (16S iT (16S

clustered into OTUs at 99% at OTUs into clustered

al S Table

distribution and ecological context context ecological and distribution sd n the in used ag

S s) s) eptember 2006 eptember high 3 material ) sequen w the or the tr ape collected samples ater -

throughput sequencingthroughput (

( rm ufc waters surface from NW NW

defined at 99% 99% at defined R/V R/V covers the whole Mediterranean whole the covers . Briefly, . c atriee clone Bacteroidetes ing

region The Blanes Bay dataset included 33,005 OTUs defined at 99% 99% at defined OTUs 33,005 included dataset Bay Blanes The amet d Gamboa de Sarmiento datasets from the North the from datasets to

December 2011 December t only only he V4 he monthly sampling monthly cut - V4 region was amplified for the BBMO series. BBMO the for amplified was region V4 - T datasets HTS off, (65 samples from 9 locations) 9 from samples (65 cut - V5 region of the 16S 16S the of region V5 - and were and off n a in

either either ) irr construction library HTS

from two different two from

cn e on i the in found be can g to time

(Table S4) (Table )

these data n pig 04 ( 2014 spring in taxonomically annotated using BLAST using annotated taxonomically ). - uig h trans the during - western Mediterranean Sea Mediterranean western h series station station series Our clone Our ave ave

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clades en umte t submitted been , . , which which ,

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April includ gene was amplified was gene ‐

Mediterranean cruise cruise Mediterranean r

RNA gene RNA sequences were used used were sequences ehd S2 Methods 27 ing parameters. The The parameters. o the Genbank Genbank the o . monthly th

Details of the the of Details

14,421 OTUs 14,421 o May to . using using These were These the 2 years 2 the Sequences Sequences

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f the of at ARB. ARB. 29 the for th N - ,

This article isprotected rights by All copyright. reserved. M chlorophyll data these which to parameters environmental The 2018 parameters ordination to lines contour depth or temperature linear para environmental with clustering, studied factors environmental the to similarly 2013). an 5% of level alpha an with SIMPROF using Bray the using compared sample each for The up torepresentESTU abundance the was cluster a to belonging OTU each of abundance relative The together. clustered ESTU 2015) methods iT OTUs recruited clustering by determined were ESTUs EcologicallyDefining

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modelling The and i mplemented in mplemented ,

result to a and covariates covariates and contribution slnt, rai cro concentration carbon organic salinity, , within each clade each within aiae the validate . A eeaie adtv model additive generalised A . Stacked barplots were used to visualize ESTUs. visualize to used were barplots Stacked ing

groups were termed ‘Environmental Groups’ ‘Environmental termed were groups Significant

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urtis similarity coefficient and clustered and coefficient similarity urtis each were associations, i.e. OTUs with similar distribution patterns were were patterns distribution similar with OTUs i.e. associations, h mtos sd o ban hs variables these obtain to used methods the ,

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to s

relative vrad n h ordination the on overlaid the Bacteroidetes the relative . PCoA ordination was used to visualize sample visualize to used was ordination PCoA s 99 emttos (Somerfield permutations 999 d Scrucca Scrucca .

Ordinations, overlaying of environmental environmental of overlaying Ordinations, abundances using model using abundances ( abundances of OTUs clustered in clustered OTUs of abundances GAM ,

from both amplicon 16S 16S amplicon both from n tmeaue n h Bae Bay Blanes the in temperature and were

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further The

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using distance based based distance using

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This article isprotected rights by All copyright. reserved. sample this within diverse most the was library by followed richness operon ribosomal similarity c three the (Fig. libraries all in definition OTU 99% by asymptotes approached nt) 10 (~ divergence 1% only within diversity sequence distinct s Cluster number (Table ofsequences WS sequences Chimeric sequences. 646 of dataset final chimeras, putative as flagged were 55 which of obtained, were sequences 701 to primers overlapping three The Diversity Results duringHOTMIX arecruise inMartínez the described Romera Accepted(Table libraries the among 94% to 80 from ranged overage Article lowest

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99% had D ’ ), a s This article isprotected rights by All copyright. reserved. communities H habitat 1 categories environmental Using smaller fraction among thesurface (12% environments ofthe clon a represented and sequences), clone the of (55% environments deep in overrepresented were ‘Unclassified’. as to referred here are they and any by level family to than further classified not were sequences Le Formosa, contained NS7 and latter Cryomorpha sequences) sequences), (28 Sphingobacteriia sequences), S by sequences the of Classification hit BLAST closest their to T d Taxonomic (Table the by followed OTUs, of 36% ) he Accepted H Habitat . Article m 16S rRNA gene rRNA 16S . , the majority of sequences belonged to uncultured uncultured to belonged sequences of majority the ,

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7 (6 ) . .

This article isprotected rights by All copyright. reserved. Acceptedto A (named each ESTUs 4 into divided (Fig. ESTUs independent with dataset sequencing inc not did that clades for assessed be not S8 Table OTUs, 3 to 0 included C20 and C18 C11, C8, C7, C6, (C5, study scale temporal the from OTUs many match not did environments deep to confidentlyrelate OTUs the of tree phylogenetic rRNA 16S the in clades OTUs 183 these, Bacteroidetes total the of 42% to correspondedwhich dataset), the in (1,380 318 western Articleidentity patterns Ecologically infer to order In Using sequences environments waters deep in sequences only habitat specific the of one for preference deep. and winter

OTUs were recovered were OTUs distinct distribution patterns, likely occupying different ecological niches, were split into were niches,split ecological distribution likelydifferentdistinctoccupying patterns, W the distributionthe of ) obtained from from obtained )

inter of eierna Sea Mediterranean

mainly in shallow water mainly inshallow the -

S outside

( rn sqecs n surface in sequences pring Bacteroidetes e.g

d The algorithm identifi algorithm The (20.6% of sequences) sequences) of (20.6% .

to ( C i.e. six i.e.

1 the previous the of 7,

) h bac lmt, but limits, branch the two C Bacteroidetes clades .

2 S2A, S2A,

with

O clades). and 1 (see clades nly a few few a nly amplicons 16S 16S amplicons >99% similarity to our clones from all Bacteroidetes OTUs OTUs Bacteroidetes all from clones our to similarity >99% Significant al S Table aeil n M and Material ; Bacteroidetes C

Fig.

previously delineated previously Clades 22

cl ed 24 ed soitd o H to associated 1 D s could be placed within within placed be could B 8 ades

), C17 in C17 ), (e.g. (Table S8) (Table . o instance, For ). , ) lude any OTU OTU any lude . displaying more than one than more displaying

C Units clades were were 5

to d to RA gene rRNA clades

- clades it C ethods). 8 efine ESTUs

to a done was (ESTUs) more , from

. 3 ESTUs 3

most C This was a stringent step that left half left that step stringent a was This .

C 4 and 4 As expected, clone sequences related sequencesclonerelated expected, As ).

, using using , osse of consisted distributed broadly In Abundance and distribution could distribution and Abundance

from the BBMO high BBMO the from H of which of c iT

, lades lades B

the temporal temporal the to

ag C , we

present

and C and 12 data s counts of counts ensure the limits of limits the C analysed associated to associated

16, presented 2, ed coherent OTU cluster cluster OTU coherent sets sets a mix of SF and WS and SF of mix a

that C

C

3, only 1 and 21 OTUs (at (at OTUs dataset

from sequences C

the h distribution the across 9 and and 9 predefined our

S remarkable a

H OTUs - ummer throughput throughput the

C C (BBMO)

C different includ 2 were 22 the 24 in 24 . From . North

were - 99% F all ed to - ,

This article isprotected rights by All copyright. reserved. g Environmental revealed community the of abundances relative The distribution Temporal rangerelativeabundances, ofrare from to ones abundant (seeFig iT diversity. meaningful these sampling analysis phylogenetic (i.e. ESTUs were samples bathypelagic to assigned clades OTUs included clones In consistency homogenous sample. per abundances their of clustering on based each, ESTUs 2

Acceptedag Article the spatial the uig u clo our using s maintained clades ,

in the phylogenetic tree. phylogenetic the in (18.2% rm the from i five n those OTUs those n rgn ie saoa and seasonal (i.e. origin )

( indicate WS were examined for each monthly sample. monthly each for examined were C Table S Table distribution distribution

dataset 7, Environmental Environmental f atriee sequences Bacteroidetes of

op icuig ige ape wr not were samples single including roups as C community 1,978 15, one s e eune, e bevd ht hs mthd Ts cover OTUs matched these that observed we sequences, ne 8

Similarly . ). that that (HOTMIX) divided Interestingly, when recruiting OTUs from the two two the from OTUs recruiting when Interestingly, C patterns

ESTU 17 and 17 represented 32.6% of all Bacteroidetes sequences Bacteroidetes all of 32.6% represented Ts lsiid s atriee i the in Bacteroidetes as classified OTUs pattern for all OTUs OTUs all for pattern these , and and , . Others were split in split were Others . Bacteroidetes clusters Bacteroidetes

temporal of Bacteroidetes ESTUs provide a finer estimate of estimate finer a provide ESTUs In this In C ,

, 23 some some

C 2 identified spatial) 19 ; 5

Fig. S2B and and S2B Fig.

o SF to case, only two only case, OTUs were recovered were OTUs

clade Groups Groups ) ,

were were h icuin of inclusion the

s had homogenous distributions distributions homogenous had s community were not split split not were SU ( ESTUs

ESTUs lsee b similarity by clustered confidently to

Table S Table in

2

Differential clades

ESTUs several clade several

with

), likely because we also included included also we because likely ),

8 considered. but ). did not include not did relative epc to respect lcd ihn u predefined our within placed

ih 9 iiaiyt our to similarity 99% > with each Thus, although Thus, . S3 letter Bacteroidetes d

istribution of the ESTUs ESTUs the of istribution ). dataset (i.e. s , which were which ,

abundance values for for values abundance A C

C

h large The was still added for added still was display lades

h Bacteroidetes the . 16 and 16 . (E 16S 16S From these, From

Th any OTUs any t G of OTUs OTUs of our sequences e Fg 2A) Fig. ; ecological RA gene rRNA ed C

based based st 24), or or 24), previous previous

a wide wide a group ing 147 ( and and C o

ly n 4 3 a .

This article isprotected rights by All copyright. reserved. the winter mix. mesopelagic winter. after ESTUs in increase Finally, S4A). (Fig pattern opposite an showed with negatively and 16A ESTUs While 16A ESTUs group, limits undetectable to decreased 15A ESTU but 3) (EtG months next the in abundantremained 14A ESTU changes. abundance relevant show not did and months previous the in present already was group) marine NS4 the within (also DOC with negatively and Chl with correlated positively was abundance whose group), marine NS4 the to (belonging largest The abundances. their varied ESTUs many EtG4 to EtG5 from transition the in 2A), (Fig. ESTUs of distribution individual the at Looking May of and June from samples to correlated ( of concentration the within assemblages bacterial the of patterns ( PCoA E

Accepted Article t G 4 )

) included 25 samples mostly samples 25 included Fg 2C) (Fig.

waters (see below), and probably reached the surface at detectable levels during during levels detectable at surface the reached probably and below), (see waters t ld 2 see t b a br a be to seemed 24 Clade the transition transition the emperature and DOC and emperature

several yearsseveral . 22D and 22D - - eepstvl creae ih eprtr n DOC and temperature with correlated positively were D D E Chl h cnetain of concentration the

t abundances decreased notably, but ESTUs 17A abundance increased. increased. abundance 17A ESTUs but notably, decreased abundances G fre b summer by formed 5

a

NO , concentration period from period 24A

(Fig 3 - - B, but a decrease in ESTU 22C, which becamewhichundetectable22C, ESTU in decrease a but B,

n S, as Si, and .

from a 2

concentr

B concentration ).

late spring late

winter n tempera and Chl Chl E nh f h N2 aie ru dmnn in dominant group marine NS2 the of anch ation t indicated G - within within - a 4 seem 4 fall fall spring samples spring

NO , in ,

, salinity, NO salinity, , to whereas certain samples 2 early the NS2 marine group group marine NS2 the ture (Fig. (Fig. ture ed

- ad NO and , by the the by

increased notably. This ESTU This notably. increased to

change occurred for ESTU 14A 14A ESTU for occurred change

summer months. Within the NS5 marine NS5 the Within months. be ma be icue assemblages included , E t G

rnia coordinate principal (Fig. 2A (Fig. 1, 3 2 3 - i ) Hwvr ET 15A ESTU However, A). -

te SU 7 ad C and A 17 ESTU the , nly driven by temperature by driven nly and NO and E , t particularly from particularly G 2 and 2 - B ) 3 . - The distribution The

we observed we concentrations, E concentration t G 3 included 3

analyses mostly mostly April,

14A

an , , This article isprotected rights by All copyright. reserved. group) marine NS5 the of Cryomorphaceae 8A ESTUs were waters deep of specific and abundant were that ESTUs Other ESTUs related dominance the by characterized was ESTUs individual of Distribution among theminthe deep the of any than other each to similar samples 4 samples (Fig Fig similarity, samples) single with groups Similarly, distributionSpatial related inabundance. ESTUsdropped when 2 EtG in on later increased group, marine NS5 this to belonging ESTUs other The 16C. ESTU for abundance in increase new a showed EtG4, the in decrease a after E and The community Formosa 3 In(roughlyApril). from case, thelargest this changeinvolved Clade March to 21(assignedto EtG to 4 EtG from occurred communities Bacteroidetes the in change drastic Another AcceptedC) Article . seven E t s G5 G , linked to linked , , hc bomd n ac ad otiue u to up contributed and March in bloomed which ), 3 , decreased radically in numbers in EtG3. EtG3. in numbers in radically decreased , the ESTUs forming ESTUs included mesopelagic samples, and mesopelagicsamples,included

in EtG3, in .

4 s .

patial samples patial A

n deep in 4 - ) A) B , higher higher PCoA ESTU ESTU )

. patterns

that that with ESTUs 21B and D showing higher abundances than 21A and C. and 21A than abundances higher showing D and 21B ESTUs with niomna s Environmental - water

were correlated plot (Fig.plot pH, O pH, 13A the NS2 marine group, that were in general abundant over abundant general in were that group, marine NS2 the

and of Bacteroide based on the on based could be could

samples ( 2 Leeuwenhoekiella , ESTU

and TOC and 4 C)

mostly , grouped in 7 groupedin patial . 19A

with - distribution of the identified ESTUs identified the of distribution tes . water samples water

Samples from shallow water shallow from Samples

G ( ESTUs high salinit high Muricauda E roups roups ESTU 7C, followed by ESTUs 7A and and 7A ESTUs by followed 7C, ESTU s G - related

from 4 to 7 representedsurfaceDCM 7 and to 4 from E

nvironmental E ld 16 Clade s G

ies and nutrient concentrationsies and nutrient ) - , as indicated by the short distance distance short the by indicated as , 1 and and 1 , related ESTU

o te S mrn group) marine NS5 the (of ) E 7 o te Bacteroide the of 47% . 18A 18A s spatial n eoeai samples, mesopelagic In G 2 included included 2 (a deep wate deep (a s

G were of roups Owenweeksia

(clustered by (clustered overall (unclassified

deep (excluding Formosa r branch r -

water

E more more (Fig t 7 G4 tes B. - - , .

This article isprotected rights by All copyright. reserved. Accepted community architectures(Acinas next from those as such to struggle and resolution typicallylack sequencing technologies sequences, gene marker short from constructed identify to order in sequences, full near including libraries clone from constructed was treereference fine the characterise To bringing the ecology into novel insights ofthisgroup. perform ocean (e.g. groups bacterial compari in explored poorly been has clades Bacteroidetes of partition ecological and phylogeny their on based results Our ArticleDiscussion deep 21A (ESTUs group (i.e. clade marine NS5 to related ESTUs different temperature factors other group) marine NS2b of branch ESTU

- ( water ESTUs

Formosa s and play and

7 ed A

-

C a comprehensive study to to study comprehensive a , indicate that Bacteroidetes clades can be divided into ecologically distinct units distinct ecologically into divided be can clades Bacteroidetes that indicate

and TOC and was still abundant but ESTU but abundant still was such - ing 15A related

key functions in functions key (Fig - as C

C ) and 14A), and

aiiy n nutrients and salinity . (Fig. . yanobacteria

3 All these ESTUs presented opposite opposite presented ESTUs these All B - cl pyoeei dvriy f atriee, hg quality high a Bacteroidetes, of diversity phylogenetic scale ) . 3

B also in their in also ) . . followed by ESTUs 23A ESTUs by followed

consistently et al. ESTU This For shallower water shallower For global ) d )

2004 espite being one of the most abundant groups in the in groups abundant most the of one being espite increase increase

relative abundance in different environments. different in abundance relative carbon s a

24 ; ad eaiey orltd with correlated negatively and ,

24B upre cae. hlgntc re ta are that trees Phylogenetic clades. supported Singer Singer A - B biogeochemical cycling biogeochemical the

a pstvl creae wt d with correlated positively was ESTUs 16A ESTUs became

et a

s , the assemblages were dominated by dominated were assemblages the , nweg of knowledge

l . -

C 2016)

predominant (NS2 marine group) marine (NS2 correlations than those of those than correlations -

B confidently

and -

length 16S rRNA gen rRNA 16S length aie Bacteroidetes marine 17A

(from a deep water water deep a (from .

- In this work, we we work, this In dete C ),

oxygen NS4 marine NS4 ct fine ct son , - and ESTU ESTU and generation ph and epth to

- pH, , other scale scale

The the the e

This article isprotected rights by All copyright. reserved. richness largest the presented library dept sequencing sufficient suggesting plateau, diversity their reached libraries all that indicated curves accumulation library clone Saturated 2011). histories evolutionary delineatio species on impact small relatively a generally has heterogeneity al. is bacteria diverge sequence of degree the common, relatively is genes rRNA 16S paralogous (Acinas two either have 40% ca. but operons, 15 to 1 from contain usually genomes Bacterial analysis community in error systematic of source a represent differentiation(Acinas phylogenies, bacterioplankton within a grouped sequences with purposes comparative delineation OTU for used similarity sequence rRNA 16S 99% 98.2 to 96% isolates prokaryotic identical the include Some

Accepted Article 2004a for species demarcation species for mandatory

-

Bacteroidetes rrn 99% of 16S rRNA sequence similarity ( similarity sequence rRNA 16S of 99% ;

rather definition of the of definition Pei

operons within a genome (Crosby & Criddle 2003). Criddle & (Crosby genome a within operons t al. et

t al. et low, with the vast majority showing < 1% nucleotide differences (Acinas differences nucleotide 1% < showing majority vast the with low, ehdlgcl considerations methodological

2004b suggested an suggested bove

n bacteria in al. et 00. t a b asmd ht intra that assumed be can It 2010). species

Epj & lz 2018 Plaza & Espejo ; the

threshold for species for threshold 2004 te mcoil clg studies. ecology microbial other

( Richter & Richter and . 9 sqec smlrt, niaig hg microdiversity high a indicating similarity, sequence 99% a

M average nucleotide identity nucleotide average ). (e.g. (e.g. t has it icrodiversity icrodiversity On the otherm hand,the On

o Bceodts populations Bacteroidetes for one Vnam, 200 Vandamme, & Coenye Rosselló en suggested been wt ti apoc. h S ( SF The approach. this with h

discrimination operon rRNA the on relying study any in Kim - Móra, 2009 Móra, ). Although microheterogeneity between between microheterogeneity Although ). lses r cmol osre in observed commonly are clusters et al. et

ultiple non ultiple that

2014).

) in this work is appropriate for appropriate is work this in (ANI) threshold range of of range threshold (ANI) Moreover, Moreover, . a be may – , and the coexistence of non of coexistence the and ,

eoi 1S RA gene rRNA 16S genomic ( These values corresponded values These

rsy Cide 2003 Criddle & Crosby Comparative genomics of genomics Comparative

Therefore, the cut the Therefore, - 3; identicaloperons rRNA i cnrs wt the with contrast in ,

nee Gerwick, & Engene ennfl nt of units meaningful n and inference of inference and n t o hrs f the of thirds wo S ummer only - nce in in nce one or or one off of of off - F 94 all) all) et ) - - .

This article isprotected rights by All copyright. reserved. (Santinelli gradients be could al (Blümel studies DGGE in gene rRNA 16S the using Bacteroidetes 2017 depths different samples. surface The understand some ofthe counts clades information physiological which sequences segregated ‘ 2013 groups uncultured Adapt the using applied successfully been has of Delineation ( for observations previous with agreement in libraries, ( communities bacterial whole of trend common habitats Pommier Gilbert

Accepted 2013 . Article Bacteroidetes b ;

) Chimetto described here are difficult to determine. to difficult are here described .

based . pasbe yohss for hypothesis plausible A ). The notable occurrence of d of occurrence notable The ’ t al. et

related to related and 24 24 and et al. retrieved in this study represent uncultured clusters uncultured represent study this in retrieved by

n luia 16S Illumina on depth but depth 2012 ecologically distinct distinct ecologically , 2010

( Tonon the

ecological n h sail rnet hr ws lot o vra aog ESTUs among overlap no almost was there transect spatial the In ( assemblages from assemblages Connor Connor ). However, the deep library contained more diversity than the surface surface the than diversity more contained library deep the However, ). different types of types different water was well stratified in the H the in stratified well was water t al. et ;

Sunagawa

group et al. et showed some mixing some showed

et al. et 02 Hde 2002 Hedges 2002;

c distribution and the distribution 2015). lades lades

2010 RA gene rRNA et al s lacking is populations, based on different gene different on based populations,

associated epth In our study, t study, our In deep . ; organic matter, which typically change through depth depth through change typically which matter, organic

such

2015 Biller - specific specific

waters were waters ad hs h evrnetl rfrne o the of preferences environmental the thus and , ). ih priinn i ta depth that is partitioning niche

et al. et ML

iT between with with ;

ag Nonetheless, ir enr 2002) Benner ,

clades had previously been suggested for for suggested been previously had clades algorithm relation parameters. environmental with s 2012; he model identified model he hc so dvriy ek i winter in peaks diversity show which the soitd ih u cae hle to helped clades our with associated marine OTMIX se substantially different from different substantially seasons

Szab

habitats. habitats. with

bacterioplankton bacterioplankton the use of use the o (7

.

. cruise et al. et

6 R Most

% of clone sequences) clone of % both bacterial isolates and isolates bacterial both et al. et ecent findings of a clear clear a of findings ecent The The

; 2013

marine Bacteroidetes marine Martínez

three

2007 - c Bacteroidetes based phylogenies, based lades ; -

specific clusters clusters specific Shepheard

; non

Díez communities communities ee clearly were - Pérez - redundant - Vives those of those et al. et OTU OTU et al. et

for for

et at

This article isprotected rights by All copyright. reserved. blooms phytoplankton to responding groups dominant the of one are phylotypes group marine included occurred April (EtG 4), The 2013). temporal multi presented Bacteroidetes Seasonally, different preferences,onlydepth also not season. niche but with related with further and analyses dist abundance of distributions, depth different with clades three distinguished approximation al was NS2b clade marine This niches. narrower to adapted specialists of suite complex a by constituted likely is whole, a as generalistseeming despite which NS2b, clade marine large the on findingsour with tune in is Pereira (Bauer proteins and polysaccharides different of degradation for clades & (Bernadet 200 Flavobacteriia Nakagawa, class the of genera cultured among varies macromolecules (Bergauer uptake matter organic weight abundanc column water the in Favobacteria of stratification depth

Accepted Article. 2007), two

Specifically, et al., et

longest stable periods occurred from July to October (EtG 5) and from and 5) (EtG October to July from occurred periods stable longest Formosa o TonB of e

from patterns and ribotypes related to this group appeared in all our libraries. A first group phylogenetic allappeared ourlibraries. ribotypes and relatedin tothis

s 2012

with a notable with anotable

ac t April to March 6) erae considerably. decreased

, - found in found

related ; n g and

five periods with periods five - Fernández eedn tasotr (BT) wih ly rl i high in role a play which (TBDTs), transporters dependent enomic data also point to specialization of distinct distinct of specialization to point also data enomic

and ribution within these clades confirmed the presence of ESTUs with ESTUswith presenceof these within the ribution clades confirmed other bacterial phyla phyla bacterial other transition co from described first the the . - Gómez Gómez Groups that increased that Groups NS5 marine group, while ESTU while group, marine NS5 fairly from from et al et - rvos tde have studies Previous annual dynamic of recurring clusters, similarly to similarly clusters, recurring of dynamic annual

stable ESTU assemblages were identified were assemblages ESTU stable . October toNovember.October , 2013 , t al. et (Vergin ;

astal North Sea surface waters (Alonso (Alonso waters surface Sea North astal

Bennke 2017). their

et al. et et al. et abundance In fact, the use of complex complex of use the fact, In were

2013, Restrepo 2013,

s related to NS2b and NS4 NS4 and NS2b to related s shown 2016 Likewise, eeld y h relative the by revealed

). et al. et ln ti transition this along

This that

drastic changesdrastic 2006

f specialization f November to to November lavobac lavobacterial

- ( Ortiz Ortiz - Teeling, molecular ;

Gómez

(EtGs) et al. et te rial rial et et et - .

This article isprotected rights by All copyright. reserved. bacteria th of evolution and distribution identif isolated previously genera to associated taxonomically isolates, bacterial were including samples, water deep and surface both from as identification of diversity u conclusion, In environments in deep in Bacteroidetes of variability temporal the investigate further to interesting be would environmental changing particularly further We substrates. available to adaptation their driving repertories gene distinct display likely most t Units, these of content gene functional the on information have not do we Although recognizable contain p similar (Teeling bloom the after and during and organicmatter, algal the of consumption progressive the to related others, and clades, marine NS3a clades,namely al. ea

Acceptedphylogenetic Article 2012 k

n March in y

. increase

and 2016, Buchan Buchan 2016, and oe physico some and observed that o that observed

structure structure More . Ulvibacter Ulvibacter

clades supported the of sing ecologically

the gene repertories that allow the uptake and use of specific biopolymers biopolymers specific of use anduptake the allow that repertoriesgene the Formosa light ofthe presentedhere.light observations taxa

‘Units’ ‘Units’

fine importantly of of - chemical

rvnig hi peoyi description phenotypic their preventing marine Bacteroide marine - spp. ther cl phylogenies scale et al. et

- however (ESTUs) related , significant taxonomic taxonomic significant t e various subgroups of this of subgroups various e Tenacibaculum emporal

their spatial and temporaltheir spatial niche

2014 w showed we , variables

n NS5 and

t al. et conditions ). that respond differently respond that h m the -

specific specific a of consists response This

06 Unfried 2016, t ht ol be could that es

ost we have successfully outlined outlined successfully have we , .

aie clade marine Formosa

that The examination of the specific distr specific the of examination The abundant ESTUs seem ESTUs (e.g. temperature, temperature, (e.g. hs gop ae o hmgnos but homogeneous not are groups these nt (ESTUs) units

,

Polaribacter

t al. et

ESTUs abundant group of heterotrophic of group abundant cig as acting ebr atr h chlorophyll the after members ed

to environmental conditions environmental to

partitioning to 08. e lo eetd a detected also We 2018). .

could could specialize in response to response in specialize

. Despite succession of succession inorganic inorganic ESTUs, of number A drivers spp o b rltd to related be not . , NS9 ,

, and , that , extent the nutrients) shaping the the shaping

, , NS4, NS5, NS4, , hence e could we particular - derived ibution ibution

the hey .

of ir It .

This article isprotected rights by All copyright. reserved. Altsc Alonso Wa Alonso C, Acinas SG Acinas Sarma SG, LA, KlepacAcinas Marcelino SG, Acinas Klepac SG, References Ministry ofEconomy CDV ULPGC (MICINN) ( grant by supported was work This data, Arístegui, J. analyses these in used samples the collecting thank We Acknowledgements Accepted ArticleCTM201 P ws upre by supported was PS . hul SF, Gish W, Millerhul SF, GishW, Myers W, Lipman (1990)alignment Basic EW, DJ search local and 3535. Mediterranean waters. coastal bacterialgroups theuptake to oflow Flavobacteriaplankton. inmarine Bacteroidetes populations. fluorescence FM, (2015) Salazar G, JM Validation Gasol of catalysed a new reporter deposition 8966 constructed sample. thesame from artifacts fromrRNA 16S comparisonclone insights oflibraries two andbias: 186 rRNA16S sequences rrn ingenomes operons. multiple with bacterialcomplex community. tool (BLAST).tool - Sáez L,Sáez the contributions (2007)in JM Seasonal Gasol of variations different Fnnil upr ws rvdd y hD JAE Ph.D. by provided was support Financial .

, 2629 5 r Fred Dr. , . – -

M. Ferrera Ferrera 70340 8969.

M. MS rneckeAmann PernthalerF, (2007) R, Highglobal diversity J and local of Coll, Coll, – Álvarez

2635. was supported was

- - - in situ R erick Rupavtarm R, Klepac R, Rupavtarm Ceraj V,Hunt D Ceraj , ) Journal of MolecularJournal Biology of

Sarmento Sarmento V. V. & Competitivity.&

to JMG from the Spanish Ministry of of Ministry Spanish the from JMG to

,

oa fr hosting for Cohan

Balagué X.A. Alvarez X.A. hybridization probe forhybridization theaccurate quantificationof marine project

by a by Environmental Microbiology M Zamanillo M. , , - Díez Ceraj V, Polz V, Polz CerajMFand (2004b) redundancy Divergence of Applied and Environmental MicrobiologyApplied andEnvironmental s Nature et al.

Viera y Clavijo contract funded by the ACIISI and the the and ACIISI the by funded contract Clavijo y Viera AG ( MAGGY Malaspinomics

- - Salgado Environmental Microbiology Vives FornI, C, Ruiz - Applied and Environmental MicrobiologyApplied andEnvironmental Ceraj V, Polz M(2005) PCR Polz V, Ceraj

- (2004 molecular , CDV 430 ; E. Garcés for the BBMO sequencing analyses; sequencing BBMO the for Garcés E. ; , V , a , 551 ) Fine)

CTM2017

, in . n te lns a smln team sampling Bay Blanes the and 215 Vieitiez and Vieitiez - – i laboratory his weight northwestern compoundsin

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Journal OfBacteriology - 15461 - cec ad Innovation and Science R) R) at ,

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This article isprotected rights by All copyright. reserved. AcceptedCohan FM, ofbacterial Asystematicsunits (2007) Perry for Ediscovering fundamental the Cohan FM aconceptual of (2006) union bacterial Towards andoperational systematics, Cohan are FM (2002) species? bacterial What CoenyeIntragenomicT, Vandamme(2003) P heterogeneity 16S ribosomal between multiple TononLA,Chimetto BS Silva APB de O,Moreira Chen X,ZengN (2008) Y,Jiao Characterization of Chen X,ZengN rDNA (2006) evaluation Y,Jiao Developmentprime and 16S ofspecific LinChao A,Colwell RK, C Carlson Morris CA, Parsons R R, LeCleirBuchan GonzA, GulvikCA, GR, Imhoff Blümel (2007) JF M,Süling J, Depth Article Mosier AC,Biller Wells GF, SJ, Francis Global (2012) Aquatic Biodiversity CA of BernardetY (2006) tothe family Nakagawa Anintroduction JF, BergauerK, Fernàndez BensonM, Clark K,Karsch DA, Cavanaugh BennkeKappelma K, Krüger CM, Benner and R(2002) reactivity. composition InChemical & Hansell CarlsonDA (eds) CA Bauer M,Kube H M,Teeling Ashelford AJ, Weightman(2006) NA, ScreeningJones FryNew KE, JC, AJ Chuzhanova ecology, and evolution. 49. genomes. MicrobiologyRNALetters, operons bacterialFEMS insequenced Ocean.Southwest Atlantic structure and ofvibriosbenthic pelagic in the alonga gradient habitats in latitudinal Journal ofMicrobiology andBiotechnology structureBering inthe bycluster Sea for marine speciesminimum richnessestimators. journal the euphotic and mesopelagicofthe zones northwestern Sargasso Sea. Microbiology func oligotrophic eastern Mediterranean Sea. 252. Ammonia Prokaryotes. 3 Dworkin Flakow Stackebran M, KH, E,Schleifer Rosenberg S, Academy theUnited of America States of ofSciences revealedAtlantic watercolumn as by MG (2013) GenBank Environmental Microbiology Bacteroidetes Biogeochemistry organic marine dissolved Elsevier, matter. of pp. 59 organic matter. Bacteroidetes“ Chimeras. Lar RecentSoftware thatMost Shows diversity. Series B,Biological sciences

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, 283 Curr Biol Curr - (2018) Organic matter processing by microbial throughout communities the Oxidizing Archaea by Habitat. isPartitioned Applied andEnvironmental Microbiology Cytophaga

, from contrasting two inthe sites North open Atlantic. ocean rd 12 – Gramella forsetii Gramella Environmental Microbiology Environmental

295. edn.,pp. 455 vol7.Springer,New York, . Nucleic Acids. Nucleic Research, , - Guerra Pachiadaki A,Garcia R, Sprenger JAL, Stepanauskas RR, 686 ,

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This article isprotected rights by All copyright. reserved. Accepted Article InformationAdditional Supporting may be fou Information Supporting same color. Samples are color fitted with Bray to according samples of analysis point. sampling library Deep the for ‘D’ and point, sampling library Surface the bars) (grey Modiv cruise the from HOTMIX.Samples groups environmental as color described samples, of not Distribution were samples single of Clusters are Numbers Groups Environmental similarity. (A) Figure

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This article isprotected rights by All copyright. reserved. Accepted Article

This article isprotected rights by All copyright. reserved. Accepted Article

This article isprotected rights by All copyright. reserved. Accepted Article

This article isprotected rights by All copyright. reserved. Accepted Article