This article isprotected rights by All copyright. reserved. 10.1111/jeu.12388 doi: lead todifferences vers between this been and throughtypesetting, proofreadingwhich may thecopyediting, process, pagination This article undergone has for and buthas accepted been not review publication fullpeer the 18S, and the D1 Norway. Total RNA/cDNA amplifiedwas by haptophyte electron microscopy (SEM). composition, richnessand proportional abundanceby comparing two rRNA markersand scanning distribution by metabarcoding, improve and to the approach to study haptophytecommunity flagellates. Our aims were to investigatethe Oslofjorden hapto Haptophyta encompasses morethan 300 species of mostly marinepico ABSTRACT e Tele NorwayOslo, S. Gran Corresponding author b a *equal contribution Sandra Gran Metabarcoding and Scanning Electron M Haptophyte Diversity and V Gran Articletype : Original Article Accepted Date 07: Revised Date : 06 Received Date: 15

- Department of Biosciences, University of Oslo, Box PO 1066 Department Earthof Sciences, Uppsala University, Villavägen SE16, Acceptedmail: Article phonenumber: +47 - Stadniczeñko al. et - [email protected] Stadniczeñko, Department Biosciences,of University Oslo,of P. O. Box Blindern,1066 0316

- Stadniczeñko*

- - - Dec Sep Dec -

D2 regionD2 of the 28S rRNA. Taxonomy assignedwas using curated haptophyte

- - - 2016 2016 - 2016 --- 22 - 4884

Haptophyta D - a Samples werecollec 85 e , Luka Šupraha*

rtical Distribution Ex -

73 - 65

, FAXnumber: 0047 22 85 47 26 ion andthe of Pleasecite article Version Record. this as iversity and icroscopy b , Elianne Egge D. ted in August2013 at the Outer Oslofjorden, plored by 18S and 28S RibosomalRNA Gene

D istribution by - specific primerstargeting V4 the region of

a

Bl

and BenteEdvardsen phyte diversity and vertical indern M

- , 0316 Oslo, Norway 75236 Uppsala, Sweden etabarcoding -

and nanoplanktonic

a

This article isprotected Acceptedinvestigated haptophyte communitiesusing molecular methods species that commonly remain unnoticed in ecological surveys. Anumber studiesof specifically (Ed overcomesome theof limitations of microscopical analysis in investigating haptophytecommunities libraries and metabarcoding using high coccolithophore communities based methods Edvardsen 1999; Edvardsenal. et 2011) consuming whichhard lack and mineralized parts, body and can be only accurately identifiedthe by Morphological identifying haptophytes to a low taxonomiclevel is of greatimportance in ecological surveys. performance specificphysiological traits, which ultimately definetheir ecological and biogeochemical 1994; Granéli et al.2012) haptophytescan have strong a impact coastalon ecosystems th 1993; Robertson al. et 1994; Iglesias the c Haptophytesare major primary producersin open oceans flagella andhaptonema, a although secondary lostin a few members(e.g. Isochrysidales). structural features, notablypro the physiological and functional diversity group inhabits allseasand alsosome thrive infreshwater, exhibitinghigh a degreeof morphological, Articlenanoplanktonic flagellates THE Abu Keywords metabarcoding methodology. 28S to 18S rRNA gene sequences haptophytesof withoutcultured representatives,and to geneproportional abundances. coccolithophores novel lineages from family class.to Wealso recorded new speciesfor the area. supported and defined cladesconsisting of environmental sequences only, possiblyrepresenting as wellproportional as abundance.The rRNA18S dataset also contained OTUs assigned toe and Prymnesiaceaeto be thefamilies with highest number of Operational TaxonomicUnits (OTUs), referencedatabases an vardsen et al. 2016)

ndance p alcifying coccolithophores playa key role in the biogeochemical carbon cycle rotistdivision Haptophytaencompasses morethan 300 morphospecies of mostly pico With the advance of mole Beinglarge a and diversegroup, haptophytes commonlyexhibit species

; examination of organic scalesusing electron microscopy (EM) coccolithophores (Edva identification to the specieslevel particularly is difficult innon

allow by SEM rdsen and Paasche 1998; Langer et al.2006; Ridgwell al. et 2009)

for precise analysis of taxonomic composition and speciesabundance . d phylogeneticanalyses. Most importantly,Most molecular methods allow for detection rare of fragileor

. with metabarcoding showsa good correspond by copyright. All rights All bycopyright. reserved.

(Thomsen al.et

;

(Bollmann et al. 2002; Cros and Fortuño 2002; Youngal. et 2003) high

Our results contribute linkto morphological and molecular data and cular techniques,notably environmental sequencing of clone duction unmineralized of organic scales and possession of two - throughputsequencing - Rodríguez et al. 2002)

(Jordan and Chamberlain 1997) - . Onthe throughputsequencing it has (HTS), becomepossible to 1994; Jordan other hand, Both markergenes showed Chrysochromulinaceae

et al. et

( . In addition, blooms non of Liu et al. 2009; ; Oslofjorden;;

scanning electron microscopy (SEM)

2004; Edvard rough toxin production ( Liu

et al. et 2009 . Haptophytesshare common ence with the r 18S

phylogeny; richness. (Eikrem 1996; Eikrem and Andersen et al.1996 sen - calcifying groups,

; Bittner; etal. 2013; etal. Compar -

and even strain

(Holligan etal. . Therefore,

2016) -

calcifying ing (Moestrup

time

improve . R

The - of of ight

NA and -

- ) .

and - -

This article isprotected regions used for HTS metabarcoding, such asV4, the mayidentical be AcceptedrRNA gene may differ in onlya few basepairs between closely related species, and short variable useful marker for higher for numberThe of described haptophyt genedatabases Of both protists are metabolically active at the time collectionof (Stoecket al. 2007). DNA/RNA was In7.4). addition, compared to DNA, RNA isthought to moreaccurately picture which foundHaptophyta that wereunderrepresented in DNA compared to RNA surveys (average variability inrDNA copy numbers among taxonomic groups (Notet al. 2009). Massanaal. et (2015) than with DNA w community structure. However, Egge al.et (2013)found RNA that captured moreof diversity the al. et 2015) comparing DNA RNA vs templates as did find not any significantdifferences in diversity and it with the traditional SEM based approach reads from environmental samples than pyrosequencing in abundanceand biomass or cell number was also found the relativeOTU and major morphotypeabundances. Weak molecular results was observed in terms taxonomy,of butthe study found no strong correlation of andanalysis SEM) coccolithophoreof communities. A good match between the morphological and sequencing of clone Articleinvestigation. coccolithophore limitations inidentifying non investigations haptophyteon communitiesis difficult dueto the mentioned methodological complicates the estimation of th quantitative morphological analysis haptophyteof communities with molecular approaches al. 2001; Huse et al.2010; Haas et al. 2011) portion of ribotypes may still representchimeric sequences are many morphospecies still todescribed. be modern oceans largelywas underestimate a cultured and genetically characterised taxon. Thisindicated thathaptophyte the diversity inthe newof haptophyte sequences (OTUs) and new haptophyte lineages, which couldbe not assigned to based works of van derStaay al.et 2001; haptophyteswas provide theabout diversity and distribution ofgroup. the The major shiftin understandingdiversity the of Eggeet al.2015 Further, Previous metabarcoding studies haptophyt of However, despitethe rigorous processing dataof during analysis 454of 18S the and 28S proportional A study by 18S than18S 28S r Bittner et al. a, from s are arguablythe most appropriategroup of haptophytes for such comparative comparison differentof studies is complicated dueto use h

identify b ere lar ere a haptophyte mock community experiment. Since ) - , each themof furtherimproving the methodology and providing insights new libraries rRNA genes

cultured material and environmental clonelibraries ger cells werefavored. Using RNA may significantly reducethe bias due to (Youngal. et 2014)

d by the early clone ing Edvardsen etal by copyright. All rights All bycopyright. reserved. abundance coccolithophores.of

( - R ) approach) based on the rR 28S species in anenvironmental sample. However, calcifying speciesusing a morphological approach. 2013) NA e actuale speciesdiversity

thereare gene

and e speciese for whichare there reference sequences available are (96 vs 76) Egge et al. sequenc .

2016 for comprehensivelist studiesof d inprevious microscopic investigations, and that there morethan 6 . Thelack studiesof combining qualitative and to assess if thisis

aimed at comparing the molecular (environmental - library studies

es fromes (Edvardsen ( 2015 es (Bittneret al.2013) and protists (Massana

0 by 0 different0 a, b clone

and abundance Egge et al. (2013)

or amplificationor artefacts NA ) or noor correlation between relative OTU (Moon

. Thesestudies id et al. an

- libraries, there is a needto compare gene appropriate

2016) - reference van der Staay al. et 2000; Moon with the morpholo HTS (Bittner et al.2013; Edvardsen

which makes

. Conducting such (Edvardsen et al.2016) generates much more

of differentof marker genes.

in haptophytes using 454 method for studying the

sequence entified anabundance - Therefore, reads ) , as well as HTS (Speksnijder et , a significant 18S 18S s gical (LM

available in a more read

the18

ratio - . S - This article isprotected ribos the of amplification AcceptedHigh the with transcription (Macherey II NucleoSpin a RNA following using conducted were samples filtered of by protocol modified pyrosequencing 454 and PCR extraction, RNA RNA extraction, PCR and 454 and 40 m)described as in measurements wereperformed sampleson collected at 700 fluorometer sensor (TurnerDesigns, Sunnyvale,CA, USA) attached to a rosette. Nutrients conductivity nitrogen and stored at sizefractions: nanoplankton (3 with a MilliporeTripod unit andperistaltic a pump for maximum 4 through 3 Niskin bottlesand pre Skagerrak Auguston 21 throughputsequencing werecollected at the OF2 station ( Water samples from subsur Sampling MATERIALS AND METHODS Articlemicroscopy? composition of the coccolithophore community compare between representativein 18S results with the two markers composition and that havenot previously been r proportional microscopy. alsoWe aim at testing and improving the approach to study haptophyte diversity and vertical distribution by using haptophytes same samples. richnessand taxonomiccomposition of complementary views of may occur recently diverged species the al. et 2016) D1 - D2 aimThe of studyour was to investigatethe Oslofjorden haptophyte community diversity

- , which µm and 0.8 ha . T -

.

tempera abundance ve he 28S To our knowledge,no study

therefore proportional will rRNA gene

ture r - - Egge et al et Egge overestimate speciesrichness µm size pore polycarbonate (PC)filters (142 filtered through a 45 R - , 80 NA

. addressedWe thefollowing questions I)Do we find novel taxa speciesor 2013. Twenty L seawaterof from each sampling depth w environmental haptophyte communities, itimportant is to compare

omal 18S V4 RNA gene region we used the forward 528Flong and reverse and 528Flong forward the used we region gene RNA V4 18S omal - (Liu et al. 2009; Bittner et al. 2013) by copyright. All rights All bycopyright. reserved. Egge al. et been suggested to be

depth device(CTD, Falmouth Scientific Inc., Cataumet, USA) MA, and TD °C until

- gene face (1face Fidelity 1st Strand cDNA Synthesis Kit (Agilent, Santa Clara, CA). For PCR For CA). Clara, Santa (Agilent, Kit Synthesis cDNA Strand 1st Fidelity

18 and 28S r - pyrosequencing abundanceby depth and sizefraction? - ecorded in the Oslofjorden? II) 45 . has more variable regio

- (2015a) HTS defined clades?How V) does qualitative the and quantitative

µm)and picoplankton (0.8 RNA extraction m) and m) deep chlorophyll maximum (DCM, 8 (

- 2015 with two ae, üe, emn) n cnetd o DA y reverse by cDNA to converted and Germany) Düren, Nagel, 18S and 28S metabarcoding datasets obtained from . Briefly, RNA was isol was RNA Briefly, . R b NA

has so farsuch done rigorous a comparison µm nylon Subsequently,mesh. an in ) .

genes RNA more . Water column profiling was carried out using a

markers supp (Liu et al. 2009) ? appropriate I V) ns eight Can weplace 28S OTUs withoutcul a

59.19 than 18S, and , -

although 3 different depths (1, 2, 4, 8, Is theredifference a in the community ated from 1 from ated

µm). Filters were frozenfast in liquid N, lemented with scanning electron 0 0 min. Thisprocedureyielded two - barcodes HTS mm Millipore) was performed 10.69 .

As thesetwo markers III) Do we obtainsame the

some intra and scanning electron

regions theof 28S such as

E) inE) theouter Oslofjorden,

m and 8 and m for

m) for m) high distinguishing - linefiltration ere - species variation

m filter samples filter m collected with

for

12, 16, 20 -

offer the

and tured -

This article isprotected blast pipeline Phylogenies wereperform Phylogenetic a smallestsample size. sequenceidentity. Lifeportal ( query sequences, and our asOTUs subjectsequences. al., et 2015a,b) OTUs to the OTUs obtainedHTS by samplesof from thesame station taken intwo earlier yea one twoor reads across all samples)were removed beforefurther analysis. To compare our 18S ( sequences (seedescription rRNA genedatabase from this study, based on rRNA genedatabase by taxonomic assignation haptophyta OTUs were extracted with the‘get.lineage’ 18S OTUs assignation to phylum againstthe full Protist Ribosomal Data Base (PR2, haptophytere spurious diversity rRNA needed clustering at a highersimilarity level clustered our 18S OTUs at 99% beTo able to compareour 18S Sequenceclustering was done Additional chimera check donewas using the ‘chimera.uchime’ shorter than 365 pr chimeras wereidentified and removed using Perseus in AmpliconNoise. Sequences were denoised using AmpliconNoise v.1.6.0 Processing and analyses ofpyrosequencing raw data (tot gene marker and fraction size depth, samples). each for analysed were replicates technical ( Oslo of University Biosciences, of Department the at (NSC) Centre GS 454 the with sequenced and Switzerland) respectively). 28S and 18S 53 and (55 temperature annealing the of exception the with 2013, Egge in as same the was from pair primer 1 LSU the used we describedprimersPRYM01+7in https://figshare.com/acc

Accepted Articleocessing donewas in Mothur v. 1.36.1 .

Curated haptophyte (del Campo, pers.

, and againstthe SILVA reference LSU www.lifeportal.uio.no ads, t , ran we mega nalyses

bp

and found

Toable be to comparebetween samples, they wererarefied (subsampled) to the he ‘classify.seqs’ and with homopolymers > 8

of haptophyteof reads was

Edvardsen al. et ount/home#/projects/11914 by copyright. All rights All bycopyright. reserved.

ed following EUKREF RAxML comm 18S and18S 28S r in FileS1

97% BLAST h apio lbay a poesd s ecie b Rce (Base Roche by described as processed was library amplicon The using the‘cluster’ rRNA similarity Egge et al et Egge ). un to bereasonable, a which we usedheretoo

Detection an of OTU from the previous study is defined as ≥

.) (Morgulis et al. 2008) - )

command was usedto performfirst a OTU taxonomical results to previous studies (i.e. . for a morereliable taxonomic assignation readsof than by Bittner et al. (2013) al. et Bittner Both databases with alignmentsare available at

e ( . (Schloss et al. 2009) 2016) ference alignments of the referencedatabases described Bittner al.et (2013) .

(201

Bittner al.(2013)et - database ( L Ttnu sy Titanium FLX 3 Hapt done -

bp command )

to balancebetween speciesdetection and whereas for amplification of the 28S theamplificationfor whereas of ) . wereremoved using the ‘trim.seqs’ ophyta Single

by blast againstby blast the curated Haptophyta 18S mega - - EPA in QIIME command.

(v. 123)

with the OTUs of the previous study as .

- (Evolutionary Placement Algorithm) with the averageneighbour algorithm. tons andtons double BLAST runwas theon University of Oslo - Amplification protocol for bot for protocol Amplification PiP , unless otherwisestated.

showed that - comman ) tm t h Nrein Sequencing Norwegian the at stem (Quince al. et 2009)

and a curated Haptophyta 28S for the 28S , consisting of 184 unique Sequencesimilarity

Egge et al.2015 www.sequencing.uio.no Furtherbioinformatic Guillou al., et 2013) d, with defaultsettings. the more variable 28S - rRNA tons (OTUs with only . To removenon OTU . Putative a, b

f s andfirst a Sequences

igshare - D1 . ) command.

The we - h markers h D2

l f 16 of al for the rs -

).

region

⁰ 99% (Egge

C Two

for l , This article isprotected and standard deviation theon normal transformation, for each of the 16 samples. The Mean datasets determinethe significance between pseudo species Pielou’ eachFor Statistical analyses literature coccolithophores between 1 analysing 2.07 analysedof seawater at1 depthm and 4.15 ml eachon filter. Using 600 fields of viewcovered 6.89 mm same number of fields of view (600 for 1 andm DCM samples, for 300 othersamples) was analysed analysis theof coccolithophorecommunity was conducted following piece filterof was mounted analuminium on stub and sputter dried inoven at50 filter (0.8 (0. samples)and 300 ml (1 sample),m was filtered under weak vacuum onto the polycarbonate filter samp Water samples were collected at depths8 (1, 4,2, 8, 12, 16, 20 and 40 using m) 5 SEM analysis newi rRNA28S gene OTUs on our RAxML referencetrees, wherethe alignmentsand thetree Abel computer cluster. Finally, the program the two reference alignments the sequences. Gilles etal. 2011) between 5 polylepis Prymnesiophyceae have a six Ahomopolymer(position 751 Geneious were removed using ‘filter_alignment.py’ inQIIME. aligned abovewere created with MAFFT G

Accepted 8 Article - µm Cyclopore, 25 ck format werethe input files. lers. Aknown volume from each sample, ranging between 250 (2, 4, 12, 16, and 20 40 m s

againstthe reference i evenness index (J

and Sisthetotal number species.of Student’s one sample t (Pielou 1966) 454 - 18S

µm Whatman) to ensurean even distribution of material. After the filtration, filters were (Cros and v.7.1.9 , - 045 6

-

bp rR pyrosequencing ized data to test replicate reproducibility – - Maxi NA 1 . Too short longor homopolymersis a common error with 454 pyrosequencing (e.g.

2.48 ml of seawater. Number cellsof counted for each analysed rangedsample , and p , . To avoid inflated OTUrichness, we truncated this Ahomopolymer to 5 122 using fields 600 of view and 76 was determined to the highest possible level using the standard taxonomic

gene ⁰

Fortuno, 2002; Jordan al.,et 2004; Young et al., 2003) C. Beforethe analysis underZeiss a Supra35 mum likelihood analyses (RAxML v.8.0.26; - .

mm diameter, Whatman)that placedwas on cellulos

OTU proportional abundances werenormalized by performingsquare a root ositions that did of of thedifferences )

(Egge al. et 2015a) ’ by copyright. All rights All bycopyright. reserved. =

H’/lnS) werecalculated, where alignments sample Shannonsample with -

substitution model INS - i

not align well were (

using ‘align_seqs.py’ http://mafft.cbrc.jp/alignment/server/

were computed and added as dotted lines. - , but raxmlHPC replicates diversitiesfor the two different marker - Wiener’s speciesdiversity index (H’ at DCM. Using 300 fields of view covered 3.45 mm i n our Thealignment was - 570 using 300 fields of view. Taxonomy of - -

PTHREADS GTR difference (Bland

. Differencesbetween replicates wereplotted 18S 2

of of the filterarea, corresponding to 4.90 ml +

– datasetthis hom edited CATwith 100 bootstraps p 756 inreferencesequence AJ004866 i

. G - isproportion the individualsof of coated with gold. Quantitative Stamatakis, - aps and hypervariable positions VP scanning electron microscope, a - SSE3 . All known members of - (Bollmann al. et testwas then conducted to

checked manually was runwas to place - Altman) method usedwas .

2006 e nie o polymer varied trate membranes ). )

- was performedwas on L NiskinL water Reads were

=

- run the on UiO 2002) Σ p - filesin our 18S and i (ln

bp inall in . The p i )) and)) 2 P. P. ,

This article isprotected haptophytespecies based on microscopy and 156 OTUs obtainedHTS, by estimating species, have the 28S rRNA dataset contained 432 OTUs singletons and double rRNA28S haptophyte reads. Finally, afterclustering atand 99% 97%similarity and removal of Subsequent removal of chimeric reads discarded reads wereassigned to haptophytes(9 yielded (< with similar numbers readsof of Denoising initial of reads and removal of putativechimaeras using AmpliconNoisegenerated a total Haptophyta richness fresh water inflowfrom land. above20 Them. silicate concentratio µM) in1 Concentration dissolved of inorganic nitrogen ([NO 8 (an estimatefor relativephytoplankton biomass) increased with depth, reaching maximum valuesat uppershallow mixed the surfaceand gradually decreased down to 80 m stabilising °C. to 6.8 densityThe plotindicates a increased by depth down to 90 m wherestabilised it at34.6 ThePSU. temperature was °C 18.5 at influencehaptophyte speciescomposition. At the surface( Figur Hydrography assess microscopy coccolithophoreson alsowas performed sampleson taken a However, thesestudies used only 18S rRNA asa marker and only included onedepth. Electron community haspreviously been describedhigh by throughputsequencing referencedatabases to determine their taxonomical assignation. The Oslofjorden haptophyt phylogeneticplacement calle the entirerange, we collected samplesbetween and 0.8 45 µm divided into two sizefractions here been morphologically describedfound are inthe sizefraction between 2 community amplified and18S 28S distribution and to improvethe approach to haptophyte study diversity by Our aims were investigate to the Oslofjorden haptophyte co RESULTS AND DISCUSSION -

10 (deepm, chlorophyll maximum, DCM). Nutrient concentratio 365 bp)reads and reads with homopolymers 8> bp from the pooled within Accepted120 Article d picoplankton (0.8 e

, 1 shows1 environmental variables at the samplingOF2 site the at day of the 282 112 The haptophyteThe richness intheSkagerrak areais relatively high. Todate,total a of 85 - 20 m. Also20 m. phosphate (PO

semi amplicon reads theof V4 18S rRNA genean , in 958

theOslofjorden two at dif -

quantitativecapacity of of reads -

la - rRNA/cDNA genes with haptophyte singletons, the 18S rRNA datasetcontained 215 OTUs yer in 0 -

(

3 3 µm)and nanoplankton (3 of 18S rRNA and

RAxML by copyright. All rights All bycopyright. reserved.

among the replicates within each marker - 2 2 m with a pycnocline pronouncedmost at 2 - EPA 4 3 - ) ) concentrations) werelow (0.26 n peak

of OTUs and curated 18S and 28S 5 .5%)compared to ferent depths. Most of the haptophytespecies that have metabarcoding 30

(29 ed at 3 m (up to 3.34 µM), , 981 , 751 reads)

7 reads of 28S rRNA. Of those, , 3 454 - - ] + [NO 45 µm). We used d

(

data. 6.6 38 , respe - 30 specificprimers to identify the summer 0 , %) theof 18S rRNA and 2 795 - - mmunity diversity and vertical ]) was ]) was 1 1 m) the salinity was 23.7 andPSU ,

892

ctively. of theof D1

of of the rRNA28S reads (9 ns by depthsns by areshown near -

maximum 0.29 µM) ineuphotic the zone probably originated from

(Table S1). Removal shortof rRNA genesequence the detection limit ( - t eight differentdepths to 40 µm. In order to cover - D2 region of 28S rRNA, (Egge al. et 2015a metabarcoding - (104 8 m.fluorescence The - sampling 112 markerdataset - likelihood , 345 re , 399 288

18S rRNA ( that may 0.9 a ds)

. We 9 in Fig. %) of . 0 7 , e

while - b) %). %). s 0.29

.

a 2. 2. the

This article isprotected accounted for 0.9 cladesHAP3, HAP4, and sequenceswithout belong Syracosphaeraceae(12 proportion of 30 referencesequence. environmental seque class Prymnesiophyceae, represented by only (42) and Phaeocystaceae(27) (Fig Chrysochromulinaceaehosted the highest number of (52,OTUs 24.2%), followed by Prymnes taxonomic levels from class to family. Within the class Prymnesiophyceae,family the 18S rRN perOTU are marked to the right) didOTUs notfall within a supported clade and are shown individually inthe tree are marked to the rightof thecollapsed and supported clades with bootstrap values> in Fig. Phylogenetictrees Taxonomic composition and may impossible to removeall such errors (e.g. techniquessuch as 454 and notfully also reproducible, which is why wedid noti GenBank very stringent manual OTU filtering step (manual editing homopolymers of and chimera check to synthesis and PCR amplification protocols wereidentical; however, Egge al.et partdue be to differencesin bioinformatic the filtering procedure. The RNA extraction, cDNA higherrichness in this study (TableS2a). were not detected (i.e. < (20 2015a) thanday theonly previous study applying HTS on monthly samples from two years observed with microscopy in thisa considerably higherhaptophyte diversity (215 OTUs)in Oslofjorden the than has previously been currently comprises 312 morphologicallydescribed species (Edvardsen etal. 2016). Werecovered been recorded from theSkagerrak area

Acceptedmost abundantOTUs Article 15a) theoreticall

3. 3. ed to OTUs that couldbe not assigned furtherthan Prymnesiophyceae. OTUs that matched . In our study, both 1

A. The higherThe number of only only 1 The taxonomic assignationbased is on thesetrees. - sequencesby BLAST). Thisstringent filteringnot is feasible with a highernumber of OTUs, At 99% proportional reads

m depthm was analysed. In total 104 theof OTUs recovered from the DCM in this study y % of the total reads remove genuinephylotypes. sequence inferred from (33

four and two nces withoutclade a name. The five %) %) followed%) by are well known to

in Table1. Chrysochromulinaceae was the family with thehighest

99%similar to any OTU) in

seven read abundancewithin each major clade . Phaeocystaceaerepresented 9 by copyright. All rights All bycopyright. reserved.

similarity, wedetected OTUsrepresenting m andm deep chlorophyll maximum weresampled, whereas in proportional

18S inOTUs this study compared to Eggeet al.

in

. 18S and18S 28S referencesequences,

. 3a Clade OTUs respectively. A total of 33 OTUs wereplaced outside the rea. We also recovered ahigher 18S richnessOTU oneon single . , TableS3

Noelaerhabdaceae (18%), (Egge et al.2015a and re

HAP3, nine Huseet al. 2010) introducesequencing errors and chimeras, and itnear is

abundance phylogeneticplacement to any supported clade

). Noelaerhabdaceae and Syracosphaeraceae,were

in HAP4 a Egge al. et (2015a

ncludeit in this study. PCR and HTS

% of% the remainingdid OTUs notcluster with any . On the other hand, too stringentfiltering Thenumber OTUsof nd 12 were matching haptophyte

ferences therein) Prymnesiaceae

reads is shown in TableS3 with OTUsour ), which contributed to a 18 . A total

supported ( 2015a ( 2015a

of 3.1of

(1 (number readsof

within each clade . Haptophyta 5%) and added (Egge al. et )

clades at

) may also in

% of the

50%. Some include ,

and the of ,

Egge al. et are show

iaceae d a reads n This article isprotected Karlson 1994; Dahl and Johannessen 1998; Lekve al.et 2006) as the most abundan resembled this group. Members of Chrysochromulinaceae and Prymnesiaceae beenhave reported species of beveryspecies rich inthe Skagerrak and Kattega consistent distribution 1 of were proportionally Chrysochromulinaceae, Prymnesiaceae, andHAP3 HAP4 werealso represented with followed by Phaeocystaceaeand Calcihaptophycidae ( ( genus between ~500 Phaeocystis clustered within thereference sequences of proportion (15%) could not be proportion (1 Syracosphaeraceae gene(See Fig represent the clades withouta cultured and sequenced representativeas definedthe by 18S rRNA PRY formed clades without affiliation to any referencesequences from cultures ( in each theof Phaeocystaceae(49) and Syrac Chrysochromulinaceaewas the diversemost family with 134 OTUs, followed by 28S rRNA. rarest OTUs Syracosphaeraceaesp. (OTU_ were nested within Accepted2015b) Article - Previous microscopy surveys havealso reported Prymnesiaceaeand Chrysochromulinaceaeto I foundWe three Highest proportion of reads ( LSU2, PRY n samples Chrysochromulina

The 10The abundantmost OTUs we found that Prymnesiaceae and Chrysochromulinaceae werethe most OTU

Chrysochromulina After clustering 97% at similarity we obtained OTUs within with sp. (OTU_ (<10 reads) . 7

five - 3 - %) %) alsowas found for 1000 sequences. Thirty percent of the 280 rarestOTUs (<10 reads)belonged to the from the Oslofjorden LSU3 and PRY b Egge et al. (2015 8S 8S ).

remaining clades with cultured representativ rRNA represented thesecond abundantmost family (17%)(Table S Chrysochromulina

28S OTUs with morethan 1500 reads each,constituting the t L non 003)( r most abundantmost groups inthe

OTUsand proportional abundanceof different the groups, results our (Table S2b). epresented by copyright. All rights All bycopyright. reserved. - calcifying Table 2 ( - S sensu lato LSU4),and only

osphaeraceae (38) 003). They constituted 47 assigned furtherthan Prymnesiophyceae. b). 34

%) , S2b, Fig. S2

Prymnesiaceae

haptophyte 0.5 collected sp. (OTU_ was ), and scales of 20 undescribed forms, which morphologically represent in a % theof reads (Fig Emiliania huxleyi

found in 3 Syracosphaera pulchra - three 4 OTUs4 in 2009 the and 2010 latesummer samples. in S ). The 12 next mostabundant OTUs contained t. groups inJune

001), August (Fig Jensen

werenot placed in any clade. These may possibly Chrysochromulinaceae followed by Phaeocystaceae(11. se . 3b, deVargas etal. 2007) ll samples (Fig. Emiliania huxleyi

sample % of% the final reads (Table1 - ,

( Syracosphaerace Septe TableS3 . 1998)

S2). .

es. A major part ofOTUs the (2 s –

mber mber 2009 and 2010, .

September recorded Thus, both in terms of taxonomic )

. (OTU_ Ten lessor

1 S1a). firstThe three 18S OTUs 2

(OTU_

supported clades. L , while c. , and Phaeocystaceae 001 and OTU_ . here called (Kuylenstierna and 18 The novel lineages 3 0 S % of thetotal. These

002) and OTUswere detected Prymnesiaceae( morphological 3 ). Asimilar - rich groups, , S2a 5 Eggeet al. %). Ahigh%).

PRY ). The L

002) and - LSU1, 2

are 126 %) 91 ), This article isprotected composition and abundances,plots the get consistently linear indicating high similarity. The had TR similar taxonomic that largerdisagreement 4 similar for all pair filter in two and performing extraction, PCR and pyrosequencing separa and filtering replication, a Reproducibility of andPCR 454 sequencing for and18S 52%for rRNA28S gene). (28 for 18S and and 53 for A28S). considerable numberof OTUs werefound in onlyone sample and18S 28S samplesare presented separately. Only ~13%of the OTUs werepresent in and 42 OTUs wereremoved respectively. In theVenn diagram (Fig After subsampling Sample comparisons (e.g. isthere large a diversity of haptophytesthat remains to be cultured and DNA sequencedetermined with DNA sequences readsor from environmental s characterised several morphological forms havebeen recognised thatare not yetin culture and genetically sequences was Phaeocystaceae and Chrysochromulinaceae OTUmost 2015a sequences Tergestiella adriatica Oslofjorden. Haptophyta identical to OTUsfrom majority of these(62 OTUs)were present with ≥ 10 reads. Out the of of OTUsour were cultured species, haptophyte presentin had ≥ sequenceidentity Detection cultured a of species an or environmental sequenceis defined here as ≥ W Novel taxa orrecords for the area b, Accepted Article performede taxonomical assignation of OTUsblast by against the Haptophyta

S1).Bland The Liu etal. 2009; Bittneral. et 2013; deVargas al.et 2015)

99% match to a cultured species, whereas ). Thus

- the rich groups, the group with the highest proportion of OTUs matching environmental classified as

- clade

PiP (Robasky etal., 2014) , it may represent a For instance, s (Jensen 1998; Medlin and Zingone 2007) Haptophyta experimental errors

database. proportional (Tabl s

- inphylogenetic the tree (Fig. 3) ≥ to one of the

Altman plot to t of samples (R

99%identical to any OTUrecover

(95.8%), was 99.7%identical to nestingan OTU withinclade a of environmental he lowest number of reads (9148 and 1577 for e S4e

Eggeet al.2015a Calcihaptophycidaein s

by copyright. All rights All bycopyright. reserved.

OT betwe - a This suggests that these 26 OTUs have only been detected inthe PiP database (Table S4) , b U

) abundance for the different

_S . (Fig

18S 18S or 28S Comparing 18Sour OTUsto 2 en TR werefound among the rareOTUs. With increasing coccolithophore 07

> 0.9, p . In studyour we tested technical replicates (TR),dividing each

. can ariseduring sample and library preparation or sequencing

2, 2, whoseclosest S3)performed for

and atthe same time<

< . HTS

rRNA 0.001)presenting Egge et al. (2015 studies 47 OTUs . Of the432 28S OTUs species that hasnotyet been sequenced. the Of . Thirty amples, supports previous studies showing that had ≥ match inmatch ed in Egge al. et 2015a (Table , respectively normalised . Our result ha

- 99% ve six major taxonomicgroups

(Table S4 Egge et al.(2015a, b)

often been criticized lack for of . (16%) did not similar the

a) match to an environmental sequence

99% identical to any sequencein the (cf. OTU113, Fig

Haptophyta , OTU abundancesOTU clearly showed .

that several OTUshad best match . Of the 215 18S OTUs, only 5

) unique) and shared OTUs theof se proportional a 18S 18S and 28S rRNA genes), four ) . Within both o , 68 OTUs, tely. The wereTR highly

47

nest within had ≥ - PiPdatabase -

. PiP databa PiP 26 26 were≥

9

we found that68

abundances (Fig 4 in 4 99% 7 S

(Fig % match to a 2 a f these groups, Egge et al.

any and S4a or or ≥ . 4a, all samples

specific

99%

was se. 97% 4b) ). The 20

. (60%

.

This article isprotected and HAP4, w 1 at findings nanoplankton found the at DCM differences also occur (Shi al. et 2009) composition according to depth: inthe Mediterranean Sea Comparing depths and size fractions. 18S theor rRNA28S gene. revealed that the majority haptophyteof species can be number OTUsof and sequences abundances arefound between representatives(Clade clades28S (PRY 18S representatives. askedWe whether wecould place 28S O dataset therea are number defined of clades (from class 28S marker,due to the sp. haptophyteknown family and therefore was assigned to either Prymnesiophyceaesp. or Haptophyta the 28S dataset. were only detected in the samples.18S In contrast, HelicosphaeraceaeOTUs wereonly observed in may prevent elongation Isochrysidales (0.5%,F Phaeocystaceae, whereas in28S the dataset members Noelaerhabdaceae of werealmost missing abundantfamilies in the 18S dataset wereNoelaerhabdaceae, all samples followed by Prymnesiaceae, both innumberof OTUs and read proportion. The next most in18S the samples (Table S1). represents intra using same resolution or higherthan 18 metabarcoding studies haptophytes.of Comparing markers. (2015) the importance TRof in abundantmost groups. However, many of the rare OTUs appear

Acceptedere Article The 18SThe markerprovided more a accurateand extensiveassessment of species identit -

m and 102 only inthe DCM. defined clades. We compared our an 18S

only found at1 28S .

ig. Sixty We (Bittner al. et 2013)

haptophyte

4 and

s b) - uggestthat do TR not bring considerable extra information to studies focusing on three 18S OTUs were present

1 1 m and theLSU1 forward primer, one This is likely dueto mismatchesbetween

Coccolithus - - and inthe Red Sea LSU2, Re

or interspecificor variation. The number of detectedOTUs higherwas in the 28S t

in samples contained spectively

picoplankton (0.8 There are no previous studies comparing 18S and 28S rRNA geneas markersin m - on specificprimers (Fig. 6) - - LSU3 and D,

lowernumber of (Table S . OTUsbelonging families to the Isochrysidaceae recovering

theNorwegian coast. highestThe number of OTUs in our datasets was by copyright. All rights All bycopyright. reserved. Clade

sp. were only present intheDCM sample .

4% and 4% 15.5 In the 18S dataset, m - 2 E+F, CladeE+F, B3 OTU a – S. ) LSU4) that may represent 18S defined cladeswithout culture . Contrarily, those belonging to Clades Prymnesiales B3, D, F E, ( lowabundant OTUs. Thisagreeswith Man Compared to 18S rRNA we s that Haptophyta communities may havedifferent species fewer - 3 3 µm)samples bothfor markers, and pooled Herewe wanted toexamine ifthe 28S marker gives the - 28S referencesequences. Aharonovichal. et 2010) %

clustered with at of 18S and 28S OTUs OTUs than DCM at d 28S phylogenetictrees (Fig. . However,it is notunderstood ifthis diversity

- both depths (Table B5, respectively). Although differences inboth of embers of Chrysochromulinaceae dominated in whi ch occurs at the end3’ ofprimer, the which the 28S sequence of members of assigned to a -

to genus TUs withoutcultured a representativein Is

(Bittner al. et 2013) OTUs could notbe assigned toany ochrysis

Syracosphaeraceaeand , foundhigh a

corresponding with previous ed the

S2 . We -

In addition, for the reference18S level in only oneTR (Fig

a

sp. markers,both rR within 18S rRNA marker ), whereas 46 were found only defined aimed

and ) withoutcultured

and Braarudosphaeraceae findings by

3) and identified three er Tergestiella adriatica to examineif th

clade haptophytediversity , inSouth the Pacific pico with either t Massana etal. NA datasets .

S1), indicatingS1), -

y than the and (Table e s the e han he

This article isprotected an opposite diversity patterndepth for by the two markers. mismatch NoelaerhabdaceaeThe ( foundwas for the 28S samples. regarding depth, the 18S DCM sample was more diversethan the evenness than the nano the samedepth 18S the samples descriptions. S methodology used similar among the technical index ( ( Fig. Diversity Syracosphaeraceaeare known to be However, explanation can be that d were found inboth size fractions, although almostall known sp huxleyi O nanoplankton. contained28S largernumber of OTUs inthe mediterranea Umbilicosphaera OTUs, 88 were only found 1 at sp inpico the with previous findings by observed thatthe picopla for DCM) DCM were equal and thus lightavailability phosphorus (Malinverno al.et 2003) differencein proportional abundancesdepth by (Fig. 4b). OTUs S Emili 18S t 2 TUs assignedTUs to . wereunique in the nano Accepted Article a

). S 1 The ania huxleyi - of of each ) were) used to calculate theShannon´s diversity and Pilou’s evenness indexes. test: = t 0.29, df= 3, p 18S t

and The 28SThe dataset contained 171 OTUspresent 1 1 m

in the 28S datasets

and evenness. could explain the differences found incommunity structure. highest num

somehaploid stages in the primers (discussed above). Thisdifferenceis probably - fraction

-

Umbilicosphaera and lightavailability. nanoplankton seems to harbour the lowestdiversity. Whenpooling thesamples test: t = 0.05, df 3, = p

taxonomic group whereas imilar The uniqueThe , seewe the samepattern, that the pico sp. were uniquely found at 1

Chrysotila stipitata, as the abundant)most in this study and

findings wereobtained by ber of OTUs was found

Heli

OTUs Tables with OTUs from -

differences incommunities by depth can be explained by temperature, f

uring filtration cells breakand ribosomespass through thefilter pores raction, resulting inhighe B n by copyright. All rights All bycopyright. reserved. OTU forOTU thepicoplankton ittner al.(2013)et hadkton higher number of OTUs cosphaera

, - replicates

- fraction the nano DCM samples appear value =value 0.7 sp. occurred in very lowread proportion

m and 131 theat DCM. OTUs of coccolithophores was similar In the data18S set we observed is

wereonly found inthenanoplankton samples

O adequatefor obtainingbeta robust - value = 0.97, and 28S t n samplingour date,temperature and phosphate at 1 Chrysochromulina rotalis proportional

<3µm insize.

Coccolithus braarudii, Coronosphaera mediterranea, Emiliania sp. wereunique thefor DCM sample (Fig. 9, and9, 28S t representing

and differences insal among 6). Theseresults suggestthat the m picopla . in DCM samples for both markers.

OTUsnested within Clades B3, andE Fwereexclusive m, and Massanaal. et (2015)

abundances persamp

all samples

r (i.e - n

- diversity test: t = 0.88, df = p 3, kton size fraction at Coccolithusbraarudii

a high Emiliania huxleyi

- both depths (Tables fraction had both higherrichness OTU and As described in previous studi - test: =t

, less than best

proportion Tergestiella adriatica was in the picoplankton

- (Fig. 4a) fraction compared to the ecies are largerthan µm.An 3

inity few diversethan the However, rest.

matching the nanoplankton, corresponding 1 m clustered with s - 0.37, df = 3, p

in the data,28S due to a

Noelaerhabdaceae OTUs . (23.7 forPSU 1 -

diversity and taxonomic When looking single at . one, whereas the opposite Comparing the leand OTU (not shown, see Therehowever is aclear ) and) somemembers of

of of - s reason

, value 0.44)= the total Chrysotila stipitata . Coronosphaera S2 As for the data,18S the

. e b

Coccolithophores tabarcoding

). Of the remaining The Helicosphaera . However, -

value =value 0.74) size why we observe and

m andm 29.1 PSU reads (Fig. proportion of Shannon index fractions, Coccolithus es

and Pilou's

m and

from and were 4 . sp.

we ). for .

This article isprotected Haptophyta sequences Braarudosphaeara bigelow reference a homopolymereregion. cultures this of species (e.g. abundant assignedOTU to samples, andSEM and Syracospaherales cf. sequences level identification regionD2 environmental sequencing of richness the coccolithophore species 2015 to the number OTUsof assigned to the Rhabdosphaeraceae families. Also, the number of coccolithophore OTUs in studyour corresponded failed to accountfor thehigh level of Rhabdosphaeraceae ( Syracosphaeraceae(3). r 18S holococcolithophoretaxa (2 species) and Rhabdosphaeraceae(2 spe thewas speciesmost number 28S of detectedalso with rRNA metabarcoding 14, with a detected at the the number of morphotypes by 12 3 numbered 22 coccolithophore species, two of which co phasesand combination coccospheres(intermediate life detected 26 distinctcoccolithophore morphotypes(from diversitySpecies estimation Comparison of SEM and Accepted Article). Our observations expandchecklist the of species detected intheScandinavian waters by and6,

number of speciesidentified inthe a R , and 8 ) NA

at The to Due the lownumber referenceof sequences obtained from cultures Syracosphaeof numberThe of

using metabarcoding data Emiliania huxleyi

. Agood n OTUs the same of of the 28S are n from

increasein speciesnumber observed from 9 Syracosphaeraceae Pi

OTUs based 28S on P the

rRNA - atthese two depths generated a total 2 of were mostly ass two depths usedfor the method comparison ( eeded to obtainbetter a link betweenand OTU species database morphologicallyverified picked cells correspondence 28S r , metabarcoding

but also overestimat location

than theregionV4 of the rRNA18S gene OTUs from - 8 rich ) 18S rRNA R

The and Calyptrosphaeraceae (6) richness NA metabarcoding incertae sedis

Using rRNA 28S

by copyright. All rights All bycopyright. reserved. ( , representedwas by the typeA morphotypein SEM counts. family at the two depths with eight S E. huxleyi i . Taxonomic analysis coccolithophoreof community atusi OF2 i yracosphaera . Onthe other hand, 28S r , for , for which EU106795 geneas marker

clonelibraries. This c were obtained theat species level. The most abundantspecies inour

igned to Noelaerhabdaceae (4) family 51 , generating highernumber coccolithophoreof comparedOTUs to R OTUs rRNA . The number. The of 18S NA

(Eikrem 1999; Egge al. et 2015 b to 6

etween SEM and (OTU_S002) richness OTUs showed highest

). Onebase differed, . Theincrease in

is represented wit obtained SEM analysis. Similar resultsshowing an overestimation of the OTU subclass Calcihaptophycidae(29) 9 (Edvardsen ing

for from generated pulchra

diversity were obtained instudy the of analyses of

within theSyracosphaeraceae and 1 1 m _

the was similwas S

by phylogeny and 033

ould to DCM et al. six 28S r (Liu et al. 2009

of of (Table metabarcoding Coronosphaera mediterranea clustered 1 1 m

rRNA

uld notprecisely be OTUs coccolithophore bedu B. bigelowii

coccolithophore - 2016) h onlytwo cultured species in cycleforms), the complete taxonomic list eight R ar to the number of observed species, but 9

. In the SEM analysis, Syracosphaeraceae (8 species) to DCM (14 species). The NA markerseemed to overestimate the

as the OTU_S002 had Asinstead 5 of in 6 coccolithophoreOTUs base richness of 4 OTUs increased from 1 match e toe faster evolutionaryrates

) ,

species,followed by various subsurface( . ,

giving , Rhabdosphaeraceae (2)and

depths). When corrected lifefor was only few only direct matchesbetwe with a ) . The total number speciesof . ing the ) 99.7%similar to sequences of

. More cies). On the other hand, the a

a clade was observed in higherresolution

Syracosphaeraceae(

based on OTU diversity in communities 1 Youngal. et

culture the study by identified (Fig.

28S rRNA28S m) including

and this gene d

3 DCM ( ), neither), of E. huxleyi

to reference

by depth reference The most (

d theon 18S the 2014)

23 (Eggeet al. for species

ng SEM 8 m 7, 7, Table

. in theD1 and the

38

) using ) was en r ) - , ales was cycle

- - This article isprotected contribution (44 coccolithophore Table ina peak abundance at 4 (2.8m x 10 estimation. Abundance 2015) boundary(66 million years a detection of metabarcoding referencesequences were available for species and Rhabdosphaeraceae, where 28S this study. issueThe was especially pronounced in highlydiverse families such as Syracosphaeraceae butdidallow not for detailed species level identification for most theof coccolit cells (from cultures pickedor cells) resulted in overall assignmentgood OTUsof to the family level, in shown number availableof referencesequences obtained from taxonomically verified material, as was suchtaxa, as coccolithophores other than Coccolithales and Isochrysidales only, suchClades as E and F for and18S Clade P representatives. placed within the observed in the SEM analysis. members of Calcidiscaceae ( Tergestiella adriatica 18S survey. Thoseincluded studyour matched the sequences coccolithophoreof species by with both markers Helladosphaera holococcolithophoretaxa (both markersfor provided OTUs species, Similarly, a sequence C. mediter references. OTUs Syracosphaeraceae which

Accepted ArticleSEM. However, ,

whichidentical is for thetwo species)

. were 3

were ). The community was dominated by Overall, the taxonomic assignment using The Algirosphaera robusta Young etal. (2014) likely ranea

number OTUsof w assigned to this family The observed in theSEM analysis.

Haptophyta P a very rarespecies

highlysimilar

combined with represent

OTU sp.) obtained from cultured material. detectedWe - was cell Most 70%)in thedeeper layers. Syracos Calcihaptophycidae

no ( , and noneplacedwas OTU_S017, and18S _L098 was abundancein the top 4 m layerand decreasing inabundanceand relative

present

(OTU_S078 OTUs

of theseof were placed in defined cladesconsisting environmentalof sequences

Coccolithusbraarudi The quantitativeanalysis coccolithophoreof community using revealed SEM by copyright. All rights All bycopyright. reserved. i different P clustered - OTU . go) until go) it was recently described in modern plankton database

to thisspecies’ referencesequence, confirming the findingSEM by in samples our This lack referenceof sequences from morphologically characterized Finally,number a OTUsof (pooled in category “Other” in Table ere

in both 18S and 28S a curated

placed close to Tergestiella adriatica _ , 18S

, L224 placed within

exhibit Syracosphaera 5 rRNA

). In addition, molecular analysis confirmed with cells L cladebu contained only two refe ) and) Pleurochrysidaceae(OTU_L222), none of which were OTU_L053, L233 reference ing close

Only two 18S rRNA OTUs , Helladosphaera markeryielded high number Helicosphaera carteri/wallichii

. - Calyptrosphaera sphaeroidea Emiliania huxleyi 1 (

The OTU ) decreasing) gradually towards the different degree

t couldbe not C. mediterranea -

RY to leve Rhabdosphaeraceae, represented by

metabarcodin OTUs _

-

t species or members of Syracosphaeraceae. L

he two species phaeraceaeshowed anincreasein contribution LSU3 sequencedatabase rRNA gene 137 l taxonomic assignment. The power of , that consideredwas to be extinct after K/Pg

clustering with ), , L .

Coccolithuspelagicus/braarudi

350, 28S) sp. Finally,number a OTUsof detected in placed to a clade with cultured

, accounting for over 90% of

that s rence sequences for

reference

g of similarity with thetwo 28S

is strongly constrained for some

were not observed in theSEM (

Fig OTUs clustered culturereference indicating that and . the

s 3a). 3a). was illustrated by our

of OTUs, but two only also database assigned to

and 28S , (OTU_L377 S. pulchra

by the relatively low However

detected within hophore OTUs in 40 depthm the presence of the s rRNA (Hagino et al. . Both markers

, reference C. sphaeroidea

onecultured , 28S 38 this

for for ( cultured 28S rRNA OTU the ) and) species (Fig. 4 _

were S .

075

S4

,

, This article isprotected samples (e.g. Clades HAP Acceptedtaxonomically assigned to a majorcladeconsisting of sequencesfrom cultures or o with updated taxonomy verifiedphylogeny. by With this genesome haptophyte couldtaxa notbe thereforebe the preferred template. that need requires reversetranscription into cDNA seem to be morerepresented in the total RNA than DNA In thisstudy we usedRNA as templatebecause we wereinterested in living cells haptophytes of that Methodolog availability of absoluteabu morphology main advantages of the SEM technique; a high degreeof taxonomic precision, well besuitable representation Noelaerhabdacof observed thesame marked underrepresentation compared to SEM counts. OTUs showed weakcorrespondence between SEM countsand proportional study was the first one using quantitative SEM analysis to testthe usage HTSof for coccolithophore quantitative analysis of coccolithophore communities for many years (Eggeet al.2013) amountof RNA extracted from each species varies with size, g ArticleCalyptrosphaeraceae, notablyat 1 bothcounts, 18 Rhabdosphaeraceae 28S rR rRNA OTUs. the LSU1 primerpair, underestimated the from 16% at the 1 depthm to Syracosphaeraceae were thesecond abundantmost group, in Noelaerhabdaceae accounted for 72%at the inSEM describing abundance readsof between the markers compared to the DCM. profile Braarudosphaeraceae and holococcolith taxa werepresent in lowernumbers (up to 28%) inlayers below 8 (Young al. et 2014) . Itis importantt Here Using HTSfor quantitative analysis haptophyteof communities is challenging a astask, the quantitativeThe analysis using

toidentified be and removed

for

- However, the trends inthe ical based taxonomy ofgroup the abundance estimation. wecontribute with a curated rRNA28S genereference database based on cultures proportional S

considerations

. and proportional Scanning electron microscopy hasbeenstandard a method for qualitative and proportional

2 and showed high relati 8 o noteo that the coccolithophoreabundance was slightly lowerat

S , largely owing to poor -

3),due to a constrained reference database. We observed, however,a rRNA by copyright. All rights All bycopyright. reserved. ndancedata, were allconfirmed in this study. NAfollowed OTUs same the pattern as the SEM counts. abundanceestimation

m depth,m while other families, such as Rhabdosphaeraceae, 61

abundance theof coccolithophore families. The OTUsbelonging to metabarcoding

% at% the DCM. On the other hand, the 2 eae iseae likely related to primer mismatch, abundance Noelaerhabdaceaeof

m depth.m Environmental sequencing of

metabarcoding . In monitoring surveys total protistof community DNA could proportional which (Fig. (Cros and Fortuño 2002; Young al. et 2003)

ve contribution ve (34%) taxonomicallyof unidentified 28S 1 m depth and decreased to

8) showedsignificant a contribution of may introduce additional chimeras (Egge al. et 2013) representation . The 18S rR

if primers abundance Syracosphaeracof

showed largevariation inthe prop (Massana et al

NA showed overall withoutany mismatches

ortional creasing in rowth rate and is likely

of Noelaerhabdaceae sequences. We clone , likely due to misma (Bollmann al. et 2002) abundance of 28S . 2015). 8S rR 8S

libraries has previously proportional 28 and

In % at % the DCM. NA all

this themarker

However, RNA similar trends to along the vertical highly

study, the low - nly environmental eae eae and

establis Unlike t proportional group

abundance are used and tches with 1 1 m rRNA gene could still hed, heSEM - .

specific This .

The This article isprotected the Abel cluster atthe University of Oslo within bioinformatics protocols and Tom and crew boardon R/V TrygveBraarud and EE J This research was ACKNOWLEDG picoplankton, even in a diversity thatremains todescribed be both morphologicallyand genetically, especially in the are 3µm smalleror (Edvardsen et al.2016). We concludefrom this that therea is largehaptophyte contained more OTUs thannanoplankton, the even if a only fe sample more than depth one toreveal the full diversity. Further, the picoplankton sizefraction by 18S rRNA significantlywas higherat DCM than at subsurface. Thisshows that isthere need a to by SEM. speciesThe composition differed significantly at the two depths and thediversity revealed Oslofjorden and Skagerrak. Sixcoccolithophore species wererecorded forfirst the time in the area match with an environmental sequ hadpreviously not been recorded in the area,showing the that majority of the OTUshad best a A tenth Conclusions that also will confirm the linkbetween 18S and 28S rRNA cladeswithout cultured alsoWe suggestthe construction a of curated concatenated 18S and rRNA28S reference database, sequences bothof t and specificity of metabarcoding future studies should use modified 28S primers withoutthis mismatch. could accountfor the differencein re better assign to major clades. The observed mismatch Isochrysidales to with the primerLSU1 pair coccolithophore species, the 18S rRNA genehas more referencesequences definedof cladesand studies. the smaller samplevolume is practical that to examineresult inthat some species are overlooked. routine monitoring of coccolithophore communities or surveys highwith a number the method is timeconsuming and requires taxonomic expertise, meaningit that is not suitablefor studies focusing on the mostabundant groups, butimportant is inrecovering low abundantOTUs. technicalfor replicates in metabarcoding and found thatit do notadd considerable information in percentage of OTUs and reads for the divermost considerably higherrichness using the rRNA28S than Both18S. markersshowed, however,the same Accepted orijntje Article ) QIIME

, and theUniversity of Oslo strategicfunding (to SGS and BE) of theof OTUs with both markers matchedcultured a species. We Some th of While the 28S rRNA geneseems to better distingui H enderiks

suggestthat 28S can bea useful complementto 18S in haptophyte metabarcoding

on the on Biocluster atthe Institutde Ciències del ( Mar MENTS supported , HAPTODIV project 190307 to BEand and EE, MICROPOLA e possibledrawbacks theof SEM method were also observed. Most importantly, he 28S rRNAand 18S genes are produced from cultures and isolated single cells.

relatively well by copyright. All rights All bycopyright. reserved. by the Research Council Norwayof (FRIMEDBIO project 197823 protistcommunities

Andersen for statistical suggestions enceand were recorded for thefirst timefor the Outer lativeabundance estimatesbetween markers. Therefore, - studied areafor phytoplankton diversity, Skagerrak.as the

for assistance during field work,Ramiro Logares for adviceon .

seand abundantfamilies.

we recommendwe that more reference sh between closely related

w ofdescribed the haptophytespecies CSIC Morethan half .

We We thankc .

) in) Barcelona, and Amplicon To improvethe resolution We also testedneed the R project N aptain SindreHolm

representatives oise oise runwas

of samples. And of of the 18S OTUs 225956 Mothur on

to

to BE

.

This article isprotected Egge Edvardsen Edvardsen Edvardsen deVargas, C.,Audic, S.,Henry,N., Decelle,J., Mahé, F.,Logares, R., Lara, Berney, E., C., LeBescot, N., deVarg Dahl Cros Bo Bittner Andersen LITERATURE CITED

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L. L. – This article isprotected replicates Fig in eachOTU. number OTUsof presented ineachclade. Supportvalues are inferred us Fig Oslofjorden OF2 station on the (P), and total phos Fig dottedwith a line. OF2 21on August 2013. Thedeep Fig. FIGURE LEGENDS Young Young Thomsen Stoeck,T. A.Zuendorf, H.Breiner,A. W. Stamatakis, A. 2006. RAxML Speksnijder Shi . . . Accepted Article , Cryptogam. Algol. phylosp extant coccolithophore taxonomy. Press, Oxford. 51: phytoplankton. doi: microbesin environmental eukaryoteclone libraries. doi:10.1093/bioinformatics/btl446. thousands taxaof and mixed m s Microvariation Ocean. r

epresentatives equences. 3. 2. 1. X. L., X. L., Marie 4 .

, , Maximum likelihood (RAxML) Haptophyte phylogeny for profilesDepth of temperature,salinity, density and fluorescence at outer O Concentrations (µM) dissolved of inorganic silicate (SiO

10 J. R., Liu J. R.,Geisen Haptophyte taxonomic Haptophyte , .1007/s00248

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- doi:10.7872/crya.v35.iss4.2014.353. G. A., DeJong b Brosou ed by PCR and

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This article isprotected were removed after subsampling. Taxonomic assignations arebased phylogenetic on placement. Table S2b. removed aftersubsampling. Taxonomic assignations arebased phylogeneticon placement. Table S2a innumber the of uniquesequences (OTUs, operational taxonomic units)along theanalysis pr Table S1 and 8 depthsm wereused thefor method comparison. Fig represented as dotted lines. differences (± replicates for OTU Fig blue(the three most abundantin darkblue). Red indicates rare taxa. Fig reads wererecorded). samples and replicates. Fig reads wererecorded). samples and replicates. Fig SUPPORTING INFORMATION and number rea of Fig representing a transition phase,HOL phase,COMB Abbreviations usedfor speciesdetected in morethan life one Fig linessplit thesingle samples from thepooled ones insizeand depth. the OF2 station. continuedThe linesplits the two different markers´datasets, whereas the dotted Fig forOTUs the four set samplesof studied at OF2 station. Fig. 5. . . . Accepted. . . . Article.

S2. S1b. S1a. 8. 7. 6. S4. S3.

Proportional Scanning electron micrographs coccolithophoreof morphotypes detected in thisstudy. Shann Four

Vertical distribution coccolithophore of families observed at the OF2 station. Sa Mean Rank

. Total number of reads at the beginning and end of the bioinformatics analysis and changes Heat map showing Heat .

Haptophyte V4 rRNA18S OTUs recorded inSkagerrak the inAugust 2013. Red OTUs were Haptophyte D1 - way Venn diagram illustratingnumber the uniqueof and shared haptophyte18S and 28S - on diversityon index and Pielou´sevenness index for all18S and 28S samples studied at abundance forcurves 18S (a) and 28S rRNA g -

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combination coccosphere with both heterococcoliths a ds obtained using 18S and 28S rR proportional abundanceof coccolithophorefamili

Proportional Proportional

(

0.83 standard deviation of thedifference for and18S 28S respectively)are Bland by copyright. All rights All bycopyright. reserved. - D2 D2 28S rRNA OTUsrecorded intheSkagerrak

proportional proportional

- abundances in the 18S an Altman

read abundance scaledwas by color (white indicatesthat no read abundance scaledwas by color (white indicatesthat no –

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plotshowing level of agreementbetween technical abundances all of 18S rRNA geneOTUs thefor different abundances all of 28S rRNA geneOTUs thefor different NA markersat 1 and 8 m depth

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This article isprotected S1File sequencesfrom cultured strains. b) culture. seque environmental ≥ from numbers The Hapto in present sequence any to similar 99% < is time same the at but a,b), (2015 al. et Egge Haptowith any 99% < & OTU study). 2015a al. et Egge the from sequences novel or sequencing, Sanger by obtained sequences environmental species, cultured either represent may (these Oslofjorden from samples OF Haptophyta the in species cultured a from sequence a with match BLAST 99% Haptophyta the in sequence” Hapto 99% Haptophyta the in sequence group. each Table S4 rRNA genes. Table S3.

-

Accepted of consisting database, reference haptophyta 28S Articlethe to OTUs 28S of matching over Overview OTUs: Number of OTUs that have OTUsthat Number of OTUs: . Description of how the

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a) Total and proportional read abundances and OTUs within each major cladefor 18S and 28S -

Overviewover i_N: ubr f Ts ht have that OTUs of Number PiP_ENV: ≥ 99% any sequence: Number of OTUs that have that OTUs of Number sequence: any 99% nces in the Haptophyta the in nces 9 Hapto 99% by copyright. All rights All bycopyright. reserved. matching of matching OTUs of 18S to otherdatabases. Total Total: OTUsnumberof in - - PiP sequence: PiP N PiP database, or an OTU from Oslofjorden from Egge et al. 2015a. 2015a. al. et Egge from Oslofjorden from OTU an or database, PiP - i database. PiP haptophyte

- i_N and PiP_ENV ≥ 99% BLAST match with an OTU previ OTUan BLASTwith match99% 28S referencedatabase was created. - ≥ PiP database may also come from species that exist in exist that species from come also may database PiP umber from 99%match anOTU ≥ have of thatto OTUs 9 Hapto 99% ≥ 9 Hapto 99% ≥ 9 BAT ac wt a “environmenta an with match BLAST 99% - i_UT Nme f Tsta have that OTUs of Number PiP_CULT:

- i_UT a nt d u, because up, add not may PiP_CULT ≥ 99% BLAST match with either any either with match BLAST 99% ously obtained by HTS of by obtained ously -

PiP database. PiP ≥ 99% OF 99% ≥ ≥ - 99% PiP. ≥ ≥ - l This article isprotected Table 1 OTU_S021 OTU_S020 OTU_ OTU_S018 OTU_S017 OTU_S016 OTU_S015 OTU_S014 OTU_S013 OTU_S012 OTU_S011 OTU_S010 OTU_S009 OTU_S008 OTU_S007 OTU_S006 OTU_S005 OTU_S004 OTU_S003 OTU_S002 OTU_S001

Accepted Article OTU ID S019 . List of the 30 most abundant

reads (N) 10,500 18,849 19,473 1,037 1,284 1,378 1,679 2,132 2,148 2,334 2,592 2,650 2,757 2,784 3,126 3,349 4,231 4,234 5,186 Total 866 871

by copyright. All rights All bycopyright. reserved. reads (%) 10.06 18.06 18.66 Total 2.24 2.48 2.54 2.64 2.67 3.00 3.21 4.05 4.06 4.97 0.83 0.83 0.99 1.23 1.32 1.61 2.04 2.06

haptophyteV4 18S rRNA OTUsdetected. Thetaxonomic assignment was based phylogeneticon placement. T subsampling (N) otal reads after 11,413 13,715 1,235 1,690 1,258 1,614 1,928 1,963 2,255 1, 2,293 2,847 3,252 3,364 3,893 6,669 640 632 775 927 810 976

Total reads after subsampling (%)

15.5 18.74 0.87 0.86 1.06 1.27 1.11 1.69 2.31 1.72 2.21 2.63 2.68 3.08 2.70 3.13 3.89 4.44 4.60 5.32 9.11 9

Only DCM Depth Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both

Only nano fraction Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Size

Phaeocystaceae Chrysochromulinaceae B4 PrymnesialesClade B3 Phaeocystaceae Calyptrosphaeraceae Prymnesiaceae B4 PrymnesialesClade B3 Syracosphaeraceae Phaeocystaceae Prymnes Prymnesiaceae Chrysochromulinaceae Chrysochromulinaceae Chrysochromulinaceae Chrysochromulinaceae Phaeocystaceae Prymnesiaceae Prymnesiaceae Syracosphaeraceae Noelaerhabdaceae Chrysochromulinaceae - - B5 B5

Group iophyceae

- -

Phaeocystis cordata Chrysochromulina PrymnesialesClade B4 Phaeocystis sphaeroidea Calyptrosphaera Prymnesium polylepis Pry Syracosphaeraceae Phaeocystis Prymnesiophyceae Prymnesiaceae Chrysochromulina Chrysochromulina Chrysochromulina Chrysochromulina Phaeocystis Haptol Prymnesium Syracosphaeraceae Emiliania huxleyi Chrysochromulina Lowest taxonomic level possible to determine mnesialesClade B4 ina

This article isprotected + OTU_S030 OTU_S029 OTU_S028 OTU_S027 OTU_S026 OTU_S025 OTU_S024 OTU_S023 OTU_S022 Accepted Article

364 443 449 479 633 722 765 778 797

by copyright. All rights All bycopyright. reserved. 0.35 0.42 0.43 0.46 0.61 0.69 0.73 0.75 0.76

559 520 276 287 298 276 577 579 557

0.76 0.71 0.38 0.39 0.41 0.38 0.79 0 0.76 .79

Only DCM Only 1m Both Both Both Both Both Both Both

Both Both Both Both Both Both Both Both Both

Phaeocystaceae Chrysochromulinaceae Prymnesi Prymnesiaceae Calcihaptophycidae B4 PrymnesialesClade B3 B4 PrymnesialesClade B3 B4 PrymnesialesClade B3 Rhabdosphaeraceae - - - B5 B5 B5

ophyceae

- - -

Phaeocystis globosa Chrysochromulina Prymnesiophyceae Prymnesium Calcihaptophycidae PrymnesialesClade B4 PrymnesialesClade B4 PrymnesialesClade B4 Algirosphaera robusta

This article isprotected OT OTU_L020 OTU_L019 OTU_L018 OTU_L017 OTU_L016 OTU_L015 OTU_L014 OTU_L013 OTU_L012 OTU_L011 OTU_L010 OTU_L009 OTU_L008 OTU_L007 OTU_L006 OTU_L005 OTU_L004 OTU_L003 OTU_L002 OTU_L001 Table 2. OTU OTU ID

AcceptedU_L021 Article

List List theof 30 most abundanthaptophyte D1

reads Total 1,555 1,830 1,980 (N) 412 426 467 468 491 516 538 5 576 597 645 648 659 676 710 782 862 957 60

reads (%) by copyright. All rights All bycopyright. reserved. Total 1.38 1.43 1.57 1.57 1.65 1.73 1.81 1.88 1.94 2.01 2.17 2.18 2.22 2.27 2.39 2.63 2.90 3.22 5.23 6.15 6.66

subsampling Total reads after 186 180 188 160 198 234 234 257 257 232 224 269 319 299 318 339 365 494 679 685 740 (N)

- D2 D2 28S rRNA OTUsdetected. subsampling Total reads after 1.47 1.43 1.49 1.27 1.57 1.85 1.85 2.04 2.04 1.84 1.78 2.13 2.53 2.37 2.52 2.69 2.89 3.92 5.38 5.43 5.87

(%)

Only DCM Depth Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both

The taxonomicThe assignment basedwas phylogeneticon placement.

fraction Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Size

Prymnesiaceae Prymnesiophyceae Phaeocystaceae Syracosphaeraceae Prymnesiaceae Chrysochromulinaceae Prymnesiaceae Chrysochromulinaceae Prymnesiaceae Prymnesiophyceae Chrysochromulinaceae Phaeocystaceae Prymnesiophyceae Chrysochromulinaceae Chrysochromulinaceae Prymnesiaceae Prymnesiaceae Chryso Phaeocystaceae Syracosphaeraceae Syracosphaeraceae chromulinaceae Group

Haptolina CladePRY Phaeocystis Syracosphaera pulchra Prymnesium Chrysochromulina camella Prymnesium kappa Chrysochromulina Prymnesium polylepis CladePRY Chrysochromulina camella Phaeoc CladePRY throndsenii/ C. campanulifera Chrysochromulina Chrysochr Haptolina /fragaria Haptolina ericina/hirta Chrysochromulina acantha Phaeocystis Syracosphaera pulchra Syracosphaera pulchra Lowest taxonomic level possible to determine ystis

omulina - - - LSU2 (Clade D?) LSU2 (Clade D?) LSU3 (CladeE

- F?)

AcceptedThis article isprotected Article OTU_L030 OTU_L029 OTU_L028 OTU_L027 OTU_L026 OTU_L025 OTU_L024 OTU_L023 OTU_L022

291 305 327 339 351 352 355 367 406

by copyright. All rights All bycopyright. reserved. 0.98 1.03 1.10 1.14 1.18 1.18 1.19 1.23 1.36

133 117 157 140 130 155 178 137 174

1.23 1.41 1.09 1.38 1.05 0.93 1.24 1.11 1.03

Both Both Both Both Both Both Both Both Both

Both Both Both Both Both Both Both Both Both

Chrysochromulinaceae Coccolitha Syracosphaeraceae Prymnesiaceae Chrysochromulinaceae Chrysochromulinaceae Chrysochromulinaceae Rhabdosphaeraceae Chrysochromulinaceae les

Chrysochromulina simplex Chrysochromulina simplex Chrysochromulina Algirosph Chrysochromulina throndsenii Chrysochromulina simplex Coccolithaceae Syracosphaera pulchra Dicrateria rotunda aera robusta

Syracosphaeraceae Rhabdosphaeraceae Noelaerhabdace Species Syracosphaeraan Ophiasterminimus Ophiasterhydroideus Calciopappuscaudatus Rhabdosphaeraxiphos Algirosphaerarobusta Algirosphaerarobusta Acanthoicaquattrospina Acanthoicaquattrospina Emiliania huxleyi

AcceptedThis article isprotected Articlethe method comparison. Table 3 . Species list coccolithophoresof detected by SEM and their abundances (cells L ae

TYPE A TYPE

thos

a

a HOL

HOL a

a

by copyright. All rights All bycopyright. reserved. (Lohmann 1912) Janin,1912) 1987(Lohmann & Oates,1983 Manton Lohmann,1903) (Lohmann 1913 & Ramsfjell,Gaarder 1954 1954)& Fert (Deflandre Norris, 1984 1913) (Schiller Deflandre,1952 1902) (Lohmann Norris, 1984 2000et al. Cros 1903 Lohmann, & Westbroek, Young 1991 Author

1 m 1 602 7 214,07

- 1 ) at theat ) sampled depths. Columns ingrey markstations the used for 2 m 2 482 2,410 1 237,13

964 8 253,51 m 4

1,446 6,266 241 241 2,410 0 170,86 m 8

402 2,009 1,205 402 2,009 7 140,22 m 12

964 9,157 482 964 482 1,928 99,769 m 16

482 5,784 482 964 80,972 m 20

964 5,302 1,928 482 16,387 m 40

Papposphaeraceae (holococcoliths) Calyptrosphaeraceae Corisphaerastrigilis Calyptrosphaerasphaeroidea Pappomonasflabellifera Papposphaeralepida Syracosphaeraossa Syracosphaera nodosa Syracosphaera nodosa Syracosphaeranodosa Syracosphaeramolischii Syracosphaeramarginaporata Syracosphaerahalldalii Syracosphaeracorolla Syracosphaeraborealis

AcceptedThis article isprotected Article

a a

a

HOL COMB a

a

a

by copyright. All rights All bycopyright. reserved. Gaarder, 1962Gaarder, 1913 Schiller & Oates,1975 Manton 1972 Tangen, Loeblich 1968& Tappan, (Lecal1966) study This study This 1941Kamptner, 2003 Young, 1993Knappertsbusch, 1994 Jordan, Gaarder in & HasleGaarder ex 1971 1966 Lecal, 1977 & McIntyre, Okada

1,607 602 201 7,230 803

482 482 964 7,712 1,446

964 1,928 13,977 1,928 482 964

723 2,410 1,687 1,687 1,44 7,953 46,992 5,784 241

6

804 4,822 804 1,607 402 1,607 4,420 23,706 4,822 402 804

1,446 4,820 482 964 4,338 15,423 9,639 482

482 3,374 482 1,928 2,892 18,797 10,121 1,446 964

482 964 482 7,712 1,928

Braarudosphaeraceae taxa Undetermined Braarudosphaerabigelowii HETUndetermined HOLUndetermined This article isprotected unclear tree placement. Taxa marke and Isochrysidaceae families well as OTUsas placed within clades Calcihaptophycidae, Coccolithales, Zygodiscales, Clade E,C species per family obtained using SEM data includes species from 8analysed d Table 4. a

Accepted Article previouslyTaxa not

Number of OTUs and morphospecies within coccolithophore families obtained using the twomolecular markers and SEM.the The nu

a a

reported from the Oslofjorden area.

Rhabdosphaeraceae Pontosphaeraceae Helicosphaeraceae Coccolithaceae Calcidiscacea Noelaerhabdaceae

by copyright. All rights All bycopyright. reserved. (Gran & Braarud 1935)Deflandre,(Gran 1947 d as “Other”d as in the SEM data represent two unidentified morphotypes with anunclear taxonomy. e

1 m 18S rRNA (OTUs) 2 0 0 0 0 1

DCM

1 0 0 1 0 4

Total 2 0 0 1 0 4

1 m

28S rRNA (OTUs) 5 0 0 1 1 6

epths. *Category “Other” includes OTUs placed within the Watznaueriaceae

201 DCM 8 0 1 0 0 2

Total 8 1 1 1 1 6

1 m 1 0 0 0 0 1

SEM (morphospecies) DCM 2 0 0 0 0 1

Total 1,446 2 0 0 0 0 1

Total (OF2) 8,840

3 0 0 0 0 1

lade Fand OTUs with an

5,784 964 482

mber of 3,856

AcceptedThis article isprotected Article Other* Braarudosphaeraceae Pleurochrysidaceae Calyptrosphaeraceae Papposphaeraceae Syracosphaeraceae

by copyright. All rights All bycopyright. reserved.

Sum:

13 6 2 0 1 0 1

23 13 1 0 2 1 0

29 17 0 2 2 0 1

18 51 15 4 0 0 1

36 69 17 5 0 0 0

38 87 25 0 0 1 6

0 0 5 8 0 1 0

14 0 1 0 2 0 8

14 0 1 0 2 0 8

22 11 2 1 0 2 2

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